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use of antiepileptic drugs ( aeds ) during pregnancy presents the dilemma of minimising the risk of seizure and avoiding adverse effects in the unborn child .
various studies revealed that prenatal exposure of aeds imparts serious effects on infants [ 13 ] .
further , prenatal exposures to aeds were also reported to produce cognitive impairment [ 4 , 5 ] .
janz and fuchs reviewed that exposure to aed during pregnancy increases the risk of miscarriage and stillbirth rate .
however , little attention was paid to the cellular effects of aeds during postnatal development and in adulthood .
lamotrigine ( ltg ) , a phenyltriazine derivative , is one of the most widely used second - generation antiepileptic agents used for both partial and generalised seizures . in pregnant women ,
ltg is reported to produce side effects such as rash , mania , memory and cognitive problems , mood changes , runny nose , cough , nausea , indigestion , abdominal pain , weight loss , vaginitis , and leukopenia and retardation in development of fetus [ 810 ] .
recently , exposure to ltg during pregnancy was shown to impart adverse outcome within different developmental domains .
on the other hand its use in pregnancy is associated with risk of seizure deterioration , because its clearance is accelerated in pregnancy .
this reveals the need of additional information on the therapeutic window of ltg or its possible toxic effects in pregnancy .
prenatal exposure to ltg ( 520 mg / kg / day ) alone and in combination with mx-801 , phenobarbital , or phenytoin resulted in cell death in the neonatal rat brain .
but , katz et al . showed no such effects in the same corresponding dose .
thus the effects of ltg during pregnancy , offspring development , and behaviour are still needed to be studied .
the present study was undertaken to investigate the effects of prenatal exposure of ltg on postnatal development and its impact on offspring behaviour in rats .
ltg was a kind gift from m / s sun pharmaceutical pvt . ltd . ,
trizol reagent was purchased from sigma aldrich , usa . unless mentioned , all other chemicals and reagents were of analytical grade .
animals were housed in polypropylene cages in a well - ventilated room ( air cycles : 1215 air changes / min , recycle ratio : 70 : 30 ) under an ambient temperature of 23 2c and 4065% relative humidity , with an artificial photoperiod of 12 h light / dark cycle .
they were provided with rodent feed ( provimi animal nutrition india pvt . ltd . ) and purified water ad libitum .
animals were acclimatized for a period of 7 days to the laboratory conditions prior to initiation of the experiment
. guidelines of guide for the care and use of laboratory animals ( institute of laboratory animal resources , national academic press 1996 , nih publication number 85 - 23 , revised 1996 ) were strictly followed throughout the study .
institutional animal ethics committee ( iaec ) , sri ramachandra university , chennai , india , approved the study protocol ( iaec / xiv / sru/99/2008 ) .
following acclimatization , female rats were introduced with proven breeder during night time ( 18:00 ) and were isolated during day hours ( 09:00 ) in the ratio of 3 : 1 , respectively , for copulation .
vaginal smear test was performed for each female rat every 24 h and the pregnancy were confirmed by the existence of diestrum stage for three consecutive days .
the day of confirmation of pregnancy was designated as gnd 3 and the pregnant rat was isolated into a separate cage .
the dose of ltg was arrived from the maximum recommended human dose ( 500 mg ) in the management of epilepsy ( http://www.drugs.com/ ) .
pregnant rats were randomized into 4 groups ( 6 in each ) based on stratified body weight .
group - i received vehicle ( 0.5% cmc ; 10 ml / kg , p.o . ) ; group - ii , iii , and iv received ltg at 11.5 , 23 , and 46 mg / kg , p.o .
, respectively ( three - dose regimen was selected to investigate the dose dependent response ) .
body weight gain was measured on postnatal days once in a week till the study termination .
the day of occurrence for pinna detachment , incisor eruption , eye opening , vaginal opening , and testes descent was also recorded .
offspring were allowed to mature for 90 days with free access to food and water ad libitum .
elevated plus maze was constructed of black painted wood with two open arms ( 50 10 1 cm ) and two closed arms ( 50 10 40 cm ) extending from a common central platform ( 10 10 cm ) and elevated to 45 cm above floor level .
experiments were carried out in a sound - attenuated , temperature - controlled room and illuminated by two 60 w fluorescent light .
on pnd 91 , rats were individually placed in the center of the maze facing open arm .
number of entries into open and closed and time spent in open and closed arms were recorded for a period of 5 min [ 1619 ] .
open field apparatus consisted of a plywood floor ( 96 96 cm ) with high walls .
the entire apparatus was painted black except for 6 mm thick white lines which divided the floor into 16 squares .
on pnd 91 , each animal was placed at one corner of the apparatus and observed for next 5 min .
the number of squares crossed , immobility period ( in seconds ) , and number of rearing and grooming were recorded .
radial arm maze was made of black painted wood finished with a polyurethane coating and consisted of 8 arms with a center platform of 60 cm in diameter .
a disposable plastic cup was placed at each end of the arm to reinforce the sweet smash and all arms were baited with either a food cup or an empty cup .
the offspring were food - restricted to 8590% and maintained for the duration of training and testing period .
the maze task was divided into three phases : adaptation , training , and retention phases . in adaptation phase ,
the rats were allowed to explore the radial arm maze by 3 5 min trials . during this period ,
all of the arms of the maze were baited with the food so that the rats received experience in gaining a feed reinforcement by completely traversing an arm and reaching into the food cup .
time taken by the animal to move from the starting point ( lag period ) , number of working memory errors , and time taken by the animal to munch the food in all arms were recorded . during the training period , four
the trial was terminated when the rat has entered and eaten from all the four baited arms . at the end of the three trials of each day ,
lag time , number of reference memory errors , working memory errors , correct entries , and total time taken by the animal to munch the food were recorded .
after behavioural assessment , all the experimental animals were euthanized using co2 exposure and cortical brain structures were collected for gaba - a and gaba - b mrna expression .
briefly , the total rna was extracted using trizol reagent ( sigma , usa ) .
after homogenization , the tubes were incubated for 10 min and centrifuged at 1000 rpm for 5 min .
200 l of chloroform was added to the supernatant , allowed to incubate for 5 min at room temperature , and centrifuged at 12000 rcf for 20 min .
then 500 l of isopropyl alcohol was added to the supernatant to precipitate total rna and centrifuged at 12000 rcf for 15 min following the incubation period of 10 min .
supernatant was decanted carefully and pellet was washed thrice with 75% alcohol and centrifuged at 12000 rcf for 15 min and the pellet was dried .
the pellet was resuspended in rnase - free water and stored in 80c until use .
the isolated rna was allowed to undergo reverse transcription and polymerization reaction to get cdna using rt - pcr master cycler gradient ( genet bio , korea ) .
the gene expression was analyzed using the bands formed in agarose gel electrophoresis , captured using gel documentation unit ( vilber laumar , germany ) and quantified by bio i d software .
primers sequence used were as follows : gaba - a : sense , 5-aag gac cca tga cag tgc tc-3 ; antisense , 5-ggc tcc ctt gtc cac tca ta-3. gaba - b : sense , 5-gct gga tgg tta cca cat ag-3 ; antisense 5-ggt cac agg agc agt gat g-3 and -actin : sense , 5-tgc tgt ccc tgt atg cct ct-3 ; antisense , 5-agg tct tta cgg atg tca acg-3 .
mean differences between the groups were analysed by one way anova followed by tukey 's multiple comparison as post - hoc test .
administration of ltg , at 46 mg / kg , p.o , produced severe signs of toxicity such as hyperesthesia , vocalisation , recumbency , vaginal bleeding , nasal discharge , and finally death of the dam between gnd 15 and 17 .
a significant decrease in body weight , food , and water consumption was observed in ltg ( 11.5 and 23 mg / kg , p.o ) administered dam on gnd 14 ( f(3,20 ) = 5.81 , p <
0.01 ; f(3,20 ) = 43.11 , p < 0.01 and f(3,20 ) = 15.51 , p < 0.01 , resp . ) and 21 ( f(2,15 ) = 6.74 , p < 0.01 ; f(2,15 ) = 55.23 , p < 0.01 and f(2,15 ) = 13.08 , p < 0.01 , resp . ) when compared to control ( table 1 ) . ltg ( 11.5 and 23 mg / kg , p.o ) significantly increased ( f(2,15 ) = 23.83 , p < 0.01 ) the gestational period when compared to control rats . no significant difference in litter size between the groups was observed .
however , a nonsignificant decrease in pups viability index was observed in ltg administered group when compared to control group ( table 2 ) .
ltg ( 11.5 and 23 mg / kg , p.o ) produced a significant delay in incisor eruption in both male ( f(2,15 ) = 6.96 , p < 0.05 ) and female ( f(2,9 ) = 6.36 , p < 0.05 ) offspring when compared to control offspring . a similar observation was recorded in vaginal opening in female offspring ( f(2,9 ) = 286.5 , p < 0.01 ) .
there were no significant differences in the day of pinna detachment , eye opening , and testes decent observed between ltg and control offspring ( table 3 ) .
no significant difference in body weight was observed in both male and female offspring in comparison to control offspring throughout the study ( figures 1 and 2 ) .
ltg male showed no significant difference in number of entries and time spent between open and closed arms .
however , ltg female ( 23 mg / kg , p.o ) showed a significant increase in number of entries ( f(2,9 ) = 5.14 , p < 0.05 ) and time spent ( f(2,9 ) = 58.57 , p < 0.01 ) in open arms and decreased number of entries ( f(2,9 ) = 20.88 , p < 0.01 ) and time spent ( f(2,9 ) = 58.57 , p < 0.01 ) in closed arms when compared to control rats ( table 4 ) .
no significant difference was observed in number of squares crossed , immobility period , and between the experimental groups .
however , a significant ( f(2,9 ) = 4.07 , p < 0.05 ) increase in rearing behaviour was observed in ltg ( 23 mg / kg , p.o ) female offspring when compared to control group ( table 5 ) .
there was no significant difference in lag period , number of reference and working memory errors , number of correct entries , and total time taken by the animal to munch the food recorded between the experimental groups ( table 6 ) .
mrna expression of gaba - a was found to be significantly ( f(2,3 ) = 17.07 , p < 0.05 ) downregulated in ltg female offspring ( 23 mg / kg , p.o ) when compared to counterparts ; whereas , no difference in gaba - b expression was observed between the experimental groups ( figure 3 ) .
use of antiepileptic drugs , such as ltg and levetiracetam , during pregnancy has become challenging these days as they have toxic effects on the developing embryo .
administration of ltg in the form of single therapy or polytherapy is at a high risk of developing signs and symptoms of fetal toxicity .
the present study demonstrates that prenatal exposure of ltg in rats produced severe signs of toxicity in dam and gender differential behavior in female offspring .
various studies demonstrated that prenatal ltg exposure at a dose of 250 mg / kg ( half the human equivalent dose ) increases stillbirth and postnatal death of the offspring [ 25 , 26 ] . in the current study ,
gestational ltg exposure at 46 mg / kg produced severe toxic signs such as hyperesthesia , vocalisation , recumbency , vaginal bleeding , nasal discharge , and finally death of the dam .
. showed that lamotrigine administration increases the follicle stimulating hormone ( fsh ) and luteinizing hormone ( lh ) which in turn stimulated estrogen secretion .
increased estrogen secretion results in maturation of graaffian follicle followed by ovulation which leads to embryo detachment .
this may be the possible reason for the observed fetal death in the present study .
in addition , ltg induces the secretion of parathyroid hormone ( pth ) thereby modulating calcium homeostasis leading to osteoporosis .
it is well known that osteogenesis of the embryo occurs at gnd 1521 in rats . increased serum calcium levels by pth reduce the fetal osteogenesis during embryonic development which could also be one of the possible factors for observed fetal death at gnd 1517 in ltg high dose administered rats . decreased body weight gain and feed and water intake of dam treated with ltg revealing its possible maternal toxic effects .
further , delayed vaginal opening and incisors eruption in offspring of ltg lower doses group might also be due to its maternal toxic effects .
on the other hand , it is reported that ltg administration along with the estrogen - containing hormonal contraceptives reduces the serum ltg concentration .
hence the dose has to be increased when administered with such contraceptives to maintain its level of action .
in addition , increased estrogen level induces fsh and secretes progesterone and testosterone by feedback control process thereby lengthening the gestational period .
although progesterone and testosterone levels were not measured in dams , the increased gestational length observed in our study of rats who administered ltg at lower doses may be due to impaired luteolysis and increased progesterone levels .
earlier studies have shown that blockade of gaba - a receptor in rat brain induces hyperexcitability / anxiolytic behaviour in elevated plus maze [ 30 , 31 ] .
further , estrogen suppresses gaba - a receptor expression thereby slowing down the gaba mediated inhibition . in the present study , the observed decrease in gaba - a expression and hyperactivation behaviour ( as evidenced by the elevated plus maze and open field test ) may be due to the overactivation of g - protein - coupled receptor 30 ( gpr30 ) by estrogen hormone in the female offspring . put together , the observations showed that the anxiolytic behaviour in female offspring but not the male may be due to the defect in gaba - a expression and also the alterations in gpr30 mediated estrogen secretion .
in conclusion , the present study demonstrates the potential untoward effects of ltg in pregnant rats and also its influence on the postnatal development and gender based differential behavioural effects in offspring .
our lab is further investigating the mechanisms and reason for these untoward effects so as to utilise the therapeutic benefits of ltg in a safer way . | use of antiepileptic drugs ( aeds ) in pregnancy warrants various side effects and also deleterious effects on fetal development .
the present study was carried out to assess the effects of prenatal exposure to lamotrigine ( ltg ) on postnatal development and behavioural alterations of offspring .
adult male and female sprague dawley rats weighing 150180 g b. wt . were allowed to copulate and pregnancy was confirmed by vaginal cytology .
pregnant rats were treated with ltg ( 11.5 , 23 , and 46 mg / kg , p.o ) from gestational day 3 ( gnd 3 ) and this treatment continued till postnatal day 11 ( pnd 11 ) .
offspring were separated from their dam on day 21 following parturition .
ltg , at 46 mg / kg , p.o , produced severe clinical signs of toxicity leading to death of dam between gnd 15 and 17 .
ltg , at 11.5 and 23 mg / kg , p.o , showed significant alterations in offspring 's incisors eruption and vaginal opening when compared to age matched controls .
ltg ( 23 mg / kg , p.o ) exposed female offspring expressed hyperactive behaviour and decreased gaba - a receptor expression when compared to control rats .
these results reveal that prenatal exposure to ltg may impart differential postnatal behavioural alterations between male and female rats which paves way for further investigations . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion |
a hemangioma can rarely be pedunculated ; as a result , it may undergo torsion and infarction , which can make it symptomatic .
we report the case of a 45-year - old woman with acute abdominal pain due to torsion of a giant pedunculated hepatic hemangioma around its vascular stalk .
pedunculated hemangioma of the liver is an uncommon benign tumor , a rare differential diagnosis for a mass located in the upper abdomen .
all incidentally detected pedunculated hemangiomas must be surgically managed , as these have a tendency to become torsioned , and there is also a risk of malignancy or rupture .
hemangioma is the most common benign tumor of the liver , with an autopsy prevalence of 0.4% - 20% .
women are more often affected , at a ratio of 5:1 - 6:1 ( 1 , 2 ) . hemangiomas can occur at any age , are usually solitary , and are found more commonly in the right lobe , especially in the posterior segment ( 3 ) .
liver hemangiomas are asymptomatic in most cases , and are commonly detected incidentally by imaging modalities such as ultrasonography , ct , or mri ( 4 ) .
hepatic hemangiomas are usually < 3 cm in diameter , and those measuring > 4 cm are classified as giant ( 5 ) .
giant hemangiomas can become large enough to cause symptoms due to mass effect on the abdominal viscera , abdominal pain , discomfort , hemorrhage , jaundice , and nausea ( 5 - 8 ) .
exophytic forms of hepatic hemangiomas , especially pedunculated forms , are extremely rare , with only a few cases reported in the literature ( 9 , 10 ) .
this case study is a report of a patient with a giant pedunculated hepatic hemangioma , who was referred to the emergency ward with acute abdominal pain due to torsion of the hemangioma around its vascular stalk .
a 45-year - old woman with acute abdominal pain presented to the emergency ward of a private general hospital in babol , northern iran , on may 4 , 2015 .
her pain had originated in the epigastrium and had an incremental trend for two days prior to her admission .
she had also experienced nausea , vomiting , and loss of appetite during that time . the patient had a history of chronic abdominal discomfort and upper gastrointestinal bleeding ( ugib ) due to gastritis .
she also reported that she had taken oral contraceptive pills ( ocps ) for approximately 5 years , many years earlier . on examination ,
her vital signs were all normal : blood pressure 125/75 mmhg , pulse rate 78 beats / min , respiratory rate 18 breaths / min , and oral temperature 37.3c .
there was a mild generalized tenderness , as well as severe tenderness in the upper part of the abdomen , prominently the epigastrium , but there was no rebound tenderness or guarding .
complete blood count , basic metabolic profile , coagulation tests , and liver function tests were all within normal limits .
there were no abnormal findings on the upright chest film and abdominal x - ray .
ultrasonography revealed a 92 42 72 mm solid ovoid mass with exophytic features , originating from the anterior border of the left lobe of the liver .
abdominopelvic ct scan with contrast media demonstrated a 100 64 mm lobulated oval mass showing central necrosis in the anterior aspect of the abdomen , suggesting a mesenteric mass lesion , such as gastrointestinal stromal tumor ( gist ) , as the first differential diagnosis . due to her past history of ugib
, she underwent esophagogastroduodenoscopy by a gastroenterologist , and no mass or ulcer was observed . however , there was mass effect , most probably gastric compression due to an extragastric mass lesion , with a remote possibility of a submucosal lesion .
, she had a low - grade fever and decreased hemoglobin ( from 10 to 8.3 g / dl ) . according to the clinical and paraclinical findings , the unknown etiology of the mass , the possibility of malignancy , and the risks of bleeding and hemoperitoneum
, we decided to perform surgical management . in the operating room , under general anesthesia ( ga ) and with full monitoring in the supine position ,
a large , multilobulated , dark red mass was discovered attached to the anterior edge of the left lobe of the liver by a vascular pedicle .
macroscopically , it measured 10.5 8.8 7 cm and weighed 316 g. the histopathological analysis confirmed a huge pedunculated hepatic hemangioma , with marked vascular congestion and focal hemorrhagic infarct , with no evidence of atypia .
she was discharged from the hospital three days after surgery ( figures 1 - 3 ) .
cavernous hemangioma is the most common benign tumor of the liver , probably with a congenital origin , and there is no potential for malignant transformation ( 1 ) .
it predominates in females 5:1 to 6:1 , and is most common from age 30 to 50 years .
it is usually discovered incidentally on imaging modalities such as ultrasonography , ct , and mri ( 2 ) .
these tumors can measure from a few millimeters up to 20 cm in size , and are commonly classified as giant if they measure > 4 cm in diameter ( 1 ) .
a pedunculated hemangioma can extend beyond the border of the liver ( 2 ) , but giant pedunculated hemangiomas , as in the present case , are very rare ( 11 - 13 ) .
a good illustration of this is our patient , who experienced abdominal distention and a mass , abdominal pain , early satiety , nausea , vomiting , icterus , and thrombocytopenia ( 1 , 6 , 10 , 14 ) .
the first reported case of a giant pedunculated hepatic hemangioma was by ellis et al . in 1985 ( 3 ) .
another study reported a total of 24 cases of pedunculated , exophytic hemangiomas , which was consistent with the existing literature up to the year 2008 ( 2 , 9 ) .
pedunculated hepatic hemangiomas are sometimes mistaken for other pedunculated tumors , such as hepatocellular carcinoma , hamartoma , focal nodular hyperplasia , and adenoma ( 3 ) .
our patient had a history of ocp use , which could increase the incidence of adenoma .
however , if the hemangioma is large or giant and pedunculated , it can cause mass effect , abdominal symptoms , and a potential risk of rupture ( 2 ) . very rarely , these hemangiomas can spontaneously rupture with intra - abdominal hemorrhage , which may also occur due to blunt trauma , creating acute abdominal symptoms . for this condition , surgery is the best treatment choice , especially for a giant symptomatic hemangioma with an uncertain diagnosis .
a giant pedunculated hemangioma may undergo torsion around its pedicle , as in our case , and can become infracted , thereby becoming symptomatic .
pain is the most frequent symptom , which is due to infarction or pressure on the adjacent organs ( 1 ) .
torsion of a giant pedunculated hemangioma is extremely rare , and had not been reported more than once in the literature up to the year 2010 .
as the majority of hemangiomas are asymptomatic , they do not require treatment . in symptomatic cases ,
the indications for surgical intervention include a palpable mass , rapid growth , thrombocytopenia , and rupture with intraperitoneal bleeding pain or epigastric discomfort , as demonstrated in our patient ( 4 ) . in the present case , we decided to perform surgical treatment after considering the symptoms , the large diameter of the mass , the inability to exclude malignancy , and the risk of rupture of the lesion .
if surgery is contraindicated , other treatment modalities , such as radiation therapy , hepatic artery ligation , and arterial embolization can be used ( 10 ) . in conclusion ,
pedunculated hemangiomas of the liver are uncommon benign tumors that should be included in the differential diagnosis of any mass located in the upper abdomen .
imaging modalities , such as ultrasound , ct , mri , radionuclide scintigraphy , and angiography , may be used to diagnose these tumors .
this case report and other similar studies indicate that all incidentally detected pedunculated hemangiomas must be surgically managed , as they have a tendency to become torsioned .
in conclusion , pedunculated hemangiomas of the liver are uncommon benign tumors that should be included in the differential diagnosis of any mass located in the upper abdomen .
imaging modalities , such as ultrasound , ct , mri , radionuclide scintigraphy , and angiography , may be used to diagnose these tumors . this case report and other similar studies
indicate that all incidentally detected pedunculated hemangiomas must be surgically managed , as they have a tendency to become torsioned . | introductionhemangioma is the most common benign tumor of the liver .
most cases are asymptomatic and do not require treatment .
a hemangioma can rarely be pedunculated ; as a result , it may undergo torsion and infarction , which can make it symptomatic.case presentationwe report the case of a 45-year - old woman with acute abdominal pain due to torsion of a giant pedunculated hepatic hemangioma around its vascular stalk.conclusionspedunculated hemangioma of the liver is an uncommon benign tumor , a rare differential diagnosis for a mass located in the upper abdomen .
all incidentally detected pedunculated hemangiomas must be surgically managed , as these have a tendency to become torsioned , and there is also a risk of malignancy or rupture . | Introduction
Case Presentation
Conclusions
1. Introduction
2. Case Presentation
3. Discussion
3.1. Conclusions |
many studies have examined the effects and treatment of post - stroke depression ( psd).1 patients with left - sided basal ganglia lesions have shown a significantly higher frequency and severity of depression compared with patients with right - sided basal ganglia or thalamic lesions.2 emotional lability described as emotionalism , pathological laughing , crying and emotional incontinence ( ei ) are common complications in stroke patients .
the prevalence of ei has been reported to be 15%20%.3 a previous study showed that ei was frequent in patients with small lenticulocapsular strokes , and was often associated with lesions affecting the dorsal rather than ventral part of the globus pallidus.4 infarcts of the corona radiata are common in the regions supplied by the small blood vessels of the middle cerebral artery ( mca ) .
thus , ischemic injury involving the internal capsule and basal ganglia seems to be associated with emotional disorders as a result of a stroke .
the pathogenesis of post - stroke emotional disorders can involve damage to the biogenic amine pathways , such as serotonin projections that play an important role in the modulation of mood.2 controlled studies of cases with post - stroke emotional disorders ( ei and psd ) have examined the effectiveness and tolerability of selective serotonin reuptake inhibitors ( ssris ) such as citalopram , fluoxetine , and paroxetine.57 many patients with ei have shown a rapid response to low - dose ssri treatment . based on evidence of intolerance or poor treatment response to ssris , it has been reported that a change in lamotrigine dosage has been effective.8 a rapid response to mirtazapine was observed in a study where subjects with both ei and psd failed to respond to ssris.9 moreover , in a double - blind study using nortriptyline for ei , the non - psd group showed greater improvements in ei compared with the psd group.10 as a result , it appears that improvements in psd are not directly associated with improvements in ei . to the current authors knowledge
, studies on the treatment of complex emotional disorders such as comorbid ei and psd following stroke did not include the use of adjunctive therapy .
the current study reports two cases of emotional disorders following corona radiata infarct that required differential treatment , thereby suggesting that low - dose aripiprazole adjunctive therapy could be effective in some patients with complex emotional disorders .
a 60-year - old woman being treated for hypertension developed dysarthria , right hemiplegia , and tingling . as a result
, a diagnosis of acute phase ischemic stroke in the perforating arteries of the mca was made .
evidence of a single infarct of the left corona radiata was observed on magnetic resonance imaging ( mri ) taken the next day ( figure 1 ) .
the patient had no history of psychiatric illness such as depression on presentation , and there was no history of psychiatric treatment .
although this distressed her caregivers , it was not initially recognized as a psychiatric problem . as a result ,
she had been crying for no reason more than ten times a day , but it was also observed that occasionally she could not stop laughing .
she described the crying spells as lasting several minutes and stated that she could not resist them .
a depressed mood and symptoms consistent with a mood disorder were dismissed following medical examination .
the patient had a hamilton rating scale for depression ( hrsd17)11 score of 10 points out of a possible 50 ( 10/50 ) . to measure the severity of the ei
, the current study evaluated the patient using the pathological laughter and crying scale ( placs).10 a pretreatment placs score of 21/27 points suggested moderate disability .
paroxetine 10 mg daily , an ssri whose efficacy has been supported by previous treatment case reports of ei , was administered . at 2 week follow - up ,
the subject reported that her crying spells had significantly decreased after initiation of paroxetine , and her placs score had decreased from 21 points to 10 points .
after 4 weeks of treatment , her placs score had decreased to 4/27 . at 8 weeks follow - up , she described that her crying spells had ceased .
she subsequently had a relapse of crying spells 2 months later accompanied by a placs score of 19/27 .
treatment with paroxetine 10 mg was restarted and ei was rapidly alleviated . within 4 weeks , the patient s placs score decreased to 2/27 .
medication with low - dose paroxetine was continued for 3 months and then treatment was stopped with no signs of recurrence of ei . a 67-year - old man with a history of depression since
age 50 presenting with symptoms of dysarthria and left hemiplegia was admitted to the stroke care unit .
an mri showed an infarct of the right corona radiata as well as infarcts in the caudate nucleus , lentiform nucleus , and right posterior temporal lobe ( figure 2 ) due to occlusion of the mca . an mca stem ( m1 ) occlusion was observed on brain angiography .
he showed improvements in blood flow to the mca after treatment based on acute stroke treatment guidelines ; after 30 days , he was transferred to a rehabilitation hospital .
three months after his stroke , he was crying for no reason several times a day and complained of loss of interest and a tendency to be pessimistic .
an hrsd17 score of 18/50 suggested that the patient had developed mild to moderate depression and a placs score of 20/27 suggested moderate disability . a score of 28 on the mini mental state examination ( mmse)12 did not suggest significant cognitive impairment . as a result
, he was diagnosed with comorbid psd and ei and was started on paroxetine 10 mg .
the placs score decreased to 10/27 during the first 4 weeks and hrsd17 decreased to 13 .
the left hemiplegia did not improve , even 9 months after his stroke , and he subsequently received superficial temporal artery - to - middle cerebral artery bypass surgery after assessment by cerebral angiography .
he was transferred again to the rehabilitation hospital , where his clinical symptoms improved after surgery .
his mmse score did not decrease but his placs and hrsd17 scores did not decrease .
in addition , unstable emotional thinking , pessimistic irritability , and aggressive behavior were observed .
therefore , with the informed consent of the patient and family , adjunctive therapy with low - dose aripiprazole 3 mg / day was started . within 4 weeks , uncontrolled crying spells stopped .
aripiprazole was increased to 6 mg / day over 2 weeks , and after 2 more weeks , his depressive symptoms , irritability , and aggressive behavior were relieved , with a hrds17 score of 7 and sustained clinical improvement in ei .
psd and ei remained in remission ( placs 0 ; hrds17 4 ) after 2 months of adjunctive therapy with aripiprazole .
a 60-year - old woman being treated for hypertension developed dysarthria , right hemiplegia , and tingling . as a result
, a diagnosis of acute phase ischemic stroke in the perforating arteries of the mca was made .
evidence of a single infarct of the left corona radiata was observed on magnetic resonance imaging ( mri ) taken the next day ( figure 1 ) .
the patient had no history of psychiatric illness such as depression on presentation , and there was no history of psychiatric treatment .
although this distressed her caregivers , it was not initially recognized as a psychiatric problem . as a result ,
she had been crying for no reason more than ten times a day , but it was also observed that occasionally she could not stop laughing .
she described the crying spells as lasting several minutes and stated that she could not resist them .
a depressed mood and symptoms consistent with a mood disorder were dismissed following medical examination .
the patient had a hamilton rating scale for depression ( hrsd17)11 score of 10 points out of a possible 50 ( 10/50 ) . to measure the severity of the ei ,
the current study evaluated the patient using the pathological laughter and crying scale ( placs).10 a pretreatment placs score of 21/27 points suggested moderate disability .
paroxetine 10 mg daily , an ssri whose efficacy has been supported by previous treatment case reports of ei , was administered . at 2 week follow - up , the subject reported that her crying spells had significantly decreased after initiation of paroxetine , and her placs score had decreased from 21 points to 10 points .
after 4 weeks of treatment , her placs score had decreased to 4/27 . at 8 weeks follow - up , she described that her crying spells had ceased .
she subsequently had a relapse of crying spells 2 months later accompanied by a placs score of 19/27 .
treatment with paroxetine 10 mg was restarted and ei was rapidly alleviated . within 4 weeks , the patient s placs score decreased to 2/27 .
medication with low - dose paroxetine was continued for 3 months and then treatment was stopped with no signs of recurrence of ei .
a 67-year - old man with a history of depression since age 50 presenting with symptoms of dysarthria and left hemiplegia was admitted to the stroke care unit .
an mri showed an infarct of the right corona radiata as well as infarcts in the caudate nucleus , lentiform nucleus , and right posterior temporal lobe ( figure 2 ) due to occlusion of the mca . an mca stem ( m1 ) occlusion was observed on brain angiography .
he showed improvements in blood flow to the mca after treatment based on acute stroke treatment guidelines ; after 30 days , he was transferred to a rehabilitation hospital .
three months after his stroke , he was crying for no reason several times a day and complained of loss of interest and a tendency to be pessimistic .
an hrsd17 score of 18/50 suggested that the patient had developed mild to moderate depression and a placs score of 20/27 suggested moderate disability . a score of 28 on the mini mental state examination ( mmse)12 did not suggest significant cognitive impairment . as a result
, he was diagnosed with comorbid psd and ei and was started on paroxetine 10 mg .
the placs score decreased to 10/27 during the first 4 weeks and hrsd17 decreased to 13 .
the left hemiplegia did not improve , even 9 months after his stroke , and he subsequently received superficial temporal artery - to - middle cerebral artery bypass surgery after assessment by cerebral angiography .
he was transferred again to the rehabilitation hospital , where his clinical symptoms improved after surgery .
his mmse score did not decrease but his placs and hrsd17 scores did not decrease .
in addition , unstable emotional thinking , pessimistic irritability , and aggressive behavior were observed .
therefore , with the informed consent of the patient and family , adjunctive therapy with low - dose aripiprazole 3 mg / day was started . within 4 weeks ,
aripiprazole was increased to 6 mg / day over 2 weeks , and after 2 more weeks , his depressive symptoms , irritability , and aggressive behavior were relieved , with a hrds17 score of 7 and sustained clinical improvement in ei .
psd and ei remained in remission ( placs 0 ; hrds17 4 ) after 2 months of adjunctive therapy with aripiprazole .
case 1 showed post - stroke ei after a single lesion infarct of the left corona radiata who responded rapidly to low - dose ssri .
case 2 showed both post - stroke psd and ei after multiple lesions , including a right corona radiata infarct . in case 2 , ssri treatment showed partial efficacy for the post - stroke ei but was ineffective for the depressive symptoms of psd .
adjunctive therapy with low - dose aripiprazole was beneficial for the treatment of post - stroke ei and psd .
these results suggest that the course of post - stroke emotional disorders and prognosis for successful treatment after infarction of the mca can be different in subjects with ei or with a combination of ei and psd . in a previous report on the association between psd and corona radiata infarction ,
vataja et al13 reported that in psd patients , the brain infarct often significantly affected the basal ganglia , mainly the caudate , putamen and pallidum , as well as the posterior corona radiata .
they also reported that the frequency of depression was greater in these subjects , with larger infarct size at the sites .
however , a clear relationship between specific stroke lesion and development of ei was not established .
previous studies have reported that small lenticulocapsular strokes of the basal ganglia in the right anterior region were closely related to ei.1416 patients with lenticulocapsular stroke more often develop ei than depression.4 the risk factors associated with psd are : history of depression , increased stroke severity , and post - stroke cognitive or physical impairment.17 however , ei does not appear to be related to the presence of motor or general disability.4 most ei patients also have psd .
ei was found to be significantly correlated with mood score and post - stroke depression , as well as with lesion size , activities of daily living ( adl ) scores , and intellectual impairment , but not with lesion location , history of stroke , or depression.18 another study found that a past history of depression and cortical lesions were independent predictors of ei.19 psd patients with ei had more severe depressive symptoms compared with patients without comorbid ei.3 therefore , it is thought that the pathologies of post - stroke ei and psd are partially linked .
these previous reports suggest a possible explanation for the two cases in the current study .
case 1 with no history of depression appeared to have pure ei due to a single small corona radiata infarction that was disrupting serotonergic ascending projections . in case 2
, there was a possibility that the activity of other monoamines and serotonin was reduced by multiple infarctions , including the corona radiata , and that history of depression and physical impairment also played an important role in the appearance of both psd and ei .
starkstein et al20 reported that psd following mca lesions was of significantly longer duration than psd following brainstem and/or cerebellar infarcts .
moreover , adl were more significantly impaired in patients without psd than in patients with psd in the mca group.20 thus , the delays in recovery from psd and recovery from physical impairment were interrelated in case 2 .
the most interesting finding in these case reports was that low - dose aripiprazole adjunctive therapy could be effective for post - stroke emotional disorders that were not improving with ssris alone .
the efficacy of antidepressant treatment for psd and ei is well - established , and ssris are the first - line drugs of choice .
many pure ei cases showed a rapid response to low - dose ssri treatment as in case 1 .
andersen et al21 investigated the correlation between severity of post - stroke pathological crying with lesion size and location in twelve ei patients .
patients with the most severe ei had relatively large bilateral pontine lesions without lesions in the hemispheres , and clinically least - affected patients had mainly unilateral large subcortical lesions .
therefore , the authors concluded that the destruction of serotonergic raphe nuclei or their ascending serotonergic hemispheric projections could be underlying causes of ei.6 this hypothesis is supported by positron emission tomography ( pet)-mapping results of high - binding serotonin 5-ht1a receptors in the raphe nuclei of patients with post - stroke ei.22 in addition , polymorphisms in the serotonin transporter 5-htt gene have been shown to be associated with a prevalence of post - stroke ei , with the 5-htt gene - linked promoter region 5-httlpr genotype conferring increased susceptibility to ei.23 there was rapid improvement in ei symptoms with antidepressant administration but psd did not improve .
the effectiveness of ssris for psd has not been completely established but a more significant effect of nortriptyline compared to fluoxetine has been reported.1 a recent study reported on the efficacy and tolerability of adjunctive aripiprazole in patients with treatment - resistant major depression and partial response to antidepressant treatment.24 the authors stated that aripiprazole is a second generation antipsychotic with pharmacological properties distinct from other antipsychotics .
it acts as a partial agonist at dopamine d2 and d3 and serotonin 5-ht1a receptors , and as an antagonist at 5-ht2a receptors .
activity at these receptors is shared by a number of antidepressant agents , giving aripiprazole a pharmacological rationale for use in depression.24 herrmann et al reported that atypical antipsychotics can be associated with an increased risk of stroke in the elderly.25 us food and drug administration approved revisions to the safety labeling for aripiprazole warn of the risk of cerebrovascular adverse events , including stroke , associated with its use in elderly patients with dementia - related psychosis . in recognition of these findings , the current study added a low dose of aripiprazole to the case with post - stroke emotional disorder without dementia that had not improved with ssri treatment , expecting that its dopamine and serotonin agonism would target the symptoms of irritability and aggressive behavior .
severity of depression , other psychopathologies , and neurobiological factors appear to contribute to irritability and aggressive behavior in stroke patients .
moreover , post - stroke irritable and aggressive patients with higher hrsd17 scores responded more favorably to antidepressants .
although depressed mood was more likely to occur in subjects with emotionalism , most of them were not depressed.26 calvert et al27 reported that post - stroke ei was associated with simple depression , irritability , and ideas of reference among 14 possible psychiatric symptoms derived by aggregating scores from individual items of using a short form of the present state examination ( pse ) administered during the interview .
post - stroke emotional disorder can have a negative impact on a patient s participation in the rehabilitation process and associated rehabilitation outcomes and be a cause of caregiver stress.2 accordingly , early medical assessment for psd and appropriate treatment can lead to improved physical and cognitive recovery and decreased mortality .
it is well known that serotonergic and noradrenergic fibers that originate from the brainstem nuclei and innervate the limbic system , prefrontal cortex , and associated structures are involved in the regulation of mood .
the effectiveness of a combination of modafinil , a dopamine agonist , and aripiprazole in a young adult psd case with aggressive behavior and emotional lability has been reported.28 in addition to monoamines , the cholinergic system , through nicotinic acetylcholine receptors , has recently been suggested to be involved in the etiology of major depressive disorders .
these pathways can be disrupted by the stroke lesion , which can result in depression and contribute to the complexity of the disorder.29 the first - line treatment for psd are antidepressants but there are exceptions .
dementia was not a factor in the current study , but the risks of using second generation antipsychotics in elderly patients should have been considered .
adjunctive therapy with aripiprazole to minimize sedation can be effective in antidepressant refractory post - stroke emotional disorders , particularly those associated with irritability and aggression .
further studies are required to clarify the efficacy of antipsychotic drugs in complex post - stroke emotional disorders . | many emotional disturbances such as post - stroke depression ( psd ) and emotional incontinence ( ei ) commonly occur following cerebrovascular events .
the efficacy of antidepressants for these conditions has been established but their comorbid treatment has not been well characterized . in the current study ,
the authors describe two cases of post - stroke emotional dysregulation ; one case with ei ; and the other with ei complicated by psd .
the authors describe their differential responses to treatment .
case 1 developed ei after an infarct due to occlusion of the penetrating branches of the left middle cerebral artery ( mca ) . case 2 developed both psd and ei after right mca stem occlusion .
both patients were initially treated with the selective serotonin reuptake inhibitor ( ssri ) paroxetine .
case 1 reacted promptly to ssri treatment . however , case 2 had only a partial response to paroxetine , even after many months of treatment .
adjunctive therapy with low - dose aripiprazole was eventually added , resulting in complete improvement of both ei and psd after 2 additional months of treatment .
thus , case 2 required a different treatment strategy than case 1 .
these findings suggest that aripiprazole adjunctive therapy could be effective for some complex post - stroke emotional disorders . | Introduction
Case 1
Case 2
Discussion |
38 cases ( 13.92% ) had varied symptoms of anxiety , irritability , sleeplessness , agitation , unprovoked aggressive behavior , and mild depression following the detection of hiv positivity table 1 .
hiv seropositive status and psychological symptoms a detailed analysis of the seropositive cases from jan 2010 to aug 2010 revealed that out of 1977 cases 74 ( 3.74% ) were found positive and almost all the cases had symptoms of anxiety , irritability ,
depressive symptoms , and agitation [ table 2 ] . a closer look of the positive cases revealed that the rate of positivity was higher in females than males .
a total of 6135 who were subjected to pre - test counseling from jan 2008 to aug 2010 . a total of 5688 ( 92.71% ) underwent tests for hiv status [ table 1 ] . out of these ,
significance of the test was explained to the cases and if they found positive what steps they ultimately take to get themselves treated with antiretroviral therapy , testing of family members , and the universal precautions that they have to resort to were highlighted . 273 ( 4.80% )
cases were found positive for hiv by western blot , 246 ( 90.10% ) cases were put on art , and 1 case ( 0.37% ) was detected to have developed had stage ii [ figures 14 ] .
mri brain showing hyper intense areas in frontal and temporo - parietal areas mri brain showing hyper intense areas in internal capsule and thalamus mri brain showing subcortical lesions in frontal and temporal regions mri brain showing subcortical lesions in frontal and temporal regions 38 cases ( 13.92% ) had varied psychiatric symptoms of anxiety , irritability , sleeplessness , agitation , unprovoked aggressive behavior , and mild depression following the detection of hiv positivity .
neuropsychiatric manifestations following detection of hiv virus has been described since the earliest reports on aids .
it is now recognized that neuropsychiatric signs and symptoms can result from direct effects of hiv on the brain and cns or from indirect effects such as opportunistic infections or tumors associated with immunosuppression , cerebrovascular disease , systemic toxicity , and complications of antiretroviral therapy .
as understanding of the broad range of neuropsychiatric manifestations of hiv has grown , new classification and diagnostic criteria has replaced the earlier more inexact terms such as hiv encephalopathy and adc .
hiv virus enters the cns early in the course of illness and induces neural injury / inflammation .
although hiv does not productively infect neurons , replication of the perivascular macrophages and microglia alters the usual functioning through cascade of neurotoxic molecular events .
histologically major changes are seen in the subcortical areas of the brain and include pallor and gliosis ; multinucleated giant cell encephalitis , and vacuolar myelopathy [ ref figures 14 vide infra of the had stage ii case ] .
hiv rna may be present throughout the cns and hiv - associated neuropathies are consequently observed in many brain regions including white matter tracts , neo - cortex , basal ganglia , and limbic system . the preferential impact of hiv infection on fronto - striatal circuits results in prototypical pattern of deficits , including slowed information processing efficiency , executive dysfunction , and deficient episodic memory encoding and retrieval .
clinically , impaired ability to concentrate , increased forgetfulness , difficulty in reading , increased difficulty in performing complex tasks especially dual tasks or divided attention paradigm , deficits in working memory , verbal and spatial working memory process , apathy , reduced spontaneity , inertia , irritability , lack of initiative , and social withdrawal . among the motor deficits are unsteady gait , poor balance , tremor , and difficulty with rapid alternating movements . increased tone and
although had is the initial aids defining illness in 3% of hiv + ve patients , it is a clinical dilemma in undiagnosed hiv cases in the early stages .
the hiv patients can hope for a better living with the easy availability of drugs made available by the government .
education of the ill effects of the hiv remains the mainstay with regard to control of this dreaded infection . | background : considerable clinical research has been conducted to increase our knowledge in understanding the underlying neuropathological significance of hiv viral infection.aim:to find the incidence of hiv associated dementia in a suburban part of india.materials and methods:6135 prospective cases from january 2008 to august 2010 were subjected to pretest counseling .
those willing were tested for hiv status using western blot test.results:5688 ( 92.71% ) underwent for detection of hiv.273 ( 4.8% ) were tested positive .
246 out of these ( 90.10% ) were put on art .
1 ( 0.37% ) was detected to have had stage ii.38 cases ( 18.92% ) had varied psychiatric symptoms.conclusion:haart has considerably reduced morbidity in hiv infection . | MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
lung fibroblasts play an important role in the repair processes following lung injury and contribute to the pathogenesis of chronic obstructive pulmonary disease ( copd ) . impaired
repair function of lung fibroblasts could result in emphysema , while overproduction of extracellular matrix by fibroblasts may lead to peribronchial fibrosis , both of which are major pathologic characteristics of copd [ 1 , 2 ] .
fibroblast - mediated collagen gel contraction is an in vitro model of tissue remodeling and is widely used to investigate repair functions of fibroblasts from a variety of tissues including lung and skin [ 3 , 4 ] .
for example , tgf-1 and pdgf are potent stimulators of fibroblast - mediated collagen gel contraction , while prostaglandin e2 ( pge2 ) and -adrenergic agonists are inhibitors of this process [ 58 ] .
the inhibitory effect of -agonist exposure on fibroblast - mediated collagen gel contraction is based on increased intracellular camp levels .
lung fibroblasts are also important sources of pge2 , which modulates the level of intracellular camp levels and thus modulates repair functions through autocrine and paracrine pathways [ 2 , 9 ] .
pdes comprise 11 related gene families that have been classified according to their amino acid sequences and substrate specificities . among the pde families , the pde4 family , which includes four isoforms named pde4a , -4b , -4c , and -4d , specifically hydrolyzes camp [ 10 , 11 ] .
several studies have shown that upregulation of pde4 activity reduces cell sensitivity to the camp - elevating reagents such as -agonist [ 10 , 1214 ] .
we have shown that pge2 exposure augments pde4 activity and induces functional homologous and heterologous attenuation of camp - mediated inhibition of fibroblast chemotaxis .
similarly , pde4 activity induced by pge2 is involved in the desensitization of -mimetics in human myometrium during the late stages of pregnancy . moreover
, salbutamol also upregulates pde4 activity and induces a heterologous desensitization of u937 cells to pge2 .
thus , we hypothesize that endogenous and exogenous pge2 may reduce sensitivity of the lung fibroblasts to long - acting -agonist through upregulating pde4 . we have previously reported that -adrenergic agonists and pde4 inhibitors can modulate fibroblast - mediated collagen gel contraction through the camp / pka pathway [ 7 , 17 ]
. the synergistic effect of -agonist and pde4 inhibitor on fibroblast - mediated collagen gel contraction and its mechanism , however , has not been investigated . in the current paper , we first studied the effect of varying concentrations of salmeterol plus cilomilast or vice versa on lung fibroblast - mediated collagen gel contraction . secondly , we studied the role of endogenous and exogenous pge2 in regulating the synergistic effect of salmeterol and cilomilast on fibroblast - mediated collagen gel contraction .
salmeterol and cilomilast were kindly provided by glaxosmithkline ( upper marrion , pa , usa ) .
anti - pde4 antibodies were purchased from abcam ( pde4a : cat # : ab14607 ; pde4b : cat # : ab14611 ; pde4c : cat # : ab97356 ; pde4d : cat # : ab14613 , abcam , inc . , cambridge , ma , usa ) .
native type i collagen , rat tail tendon collagen ( rttc ) , was extracted from rat tail tendon as described previously .
human fetal lung fibroblast ( hfl-1 ) was purchased from the american type culture collection ( atcc , rockville , md , usa ) .
cells were cultured in dmem supplemented with 10% fcs , penicillin / streptomycin , and fungizone .
gels were prepared using a previously published method . briefly , distilled water , 4x concentrated dmem and rttc were mixed so that the final mixture resulted in a physiological ionic strength , that is , 1x dmem , and a ph of 7.4 .
cells were suspended with serum - free dmem ( sf - dmem ) at a density of 10 cells / ml and then mixed with the rttc solution mentioned above so that the final cell density in the collagen solution was 4 10 cells / ml and the final concentration of collagen was 0.75 mg / ml . for pretreatment with pge2 or indomethacin , fibroblast monolayers were refed with sf dmem supplemented with or without pge2 or indomethacin and then incubated for 24 hours prior to harvest .
aliquots of the mixture of fibroblasts and collagen were then cast into each well of a 24-well tissue culture plate ( 0.5 ml / well ) .
after 20 minutes for gelation at room temperature , the gels were released into 60 mm tissue culture dishes ( three gels per each dish ) , which contained 5 ml of freshly prepared sf - dmem with or without salmeterol or cilomilast as indicated .
the gels were then incubated at 37c in a 5% co2 atmosphere for 25 days as indicated , and the area of each gel was measured with an optomax v image analyzer ( optomax , burlington , ma , usa ) daily . data were expressed as the percentage of the size compared to original gel size .
immunoblotting for pde4a , -4b , -4c , -4d and for -actin was carried out with whole cell lysates obtained from the cells pretreated with either pge2 or indomethacin for 24 hrs .
cells were then treated with sf - dmem supplemented with ethanol ( 1 : 1000 dilution as reagent dissolvent ) , pge2 ( 10 m ) , or indomethacin ( 10 m ) .
after 24 hrs of culturing , cells were washed once with cold pbs and then treated with cell lysis buffer ( 35 mm tris .
hcl , ph 7.4 , 0.4 mm egta , 10 mm mgcl2 , and 1 : 1000 of proteinase inhibitor cocktail ) .
lysates were briefly sonicated on ice and centrifuged at 10,000 g for 5 min at 4c .
page - sds ( 10% ) gels were prepared , and 10 g / lane of total protein was loaded .
the resolved proteins were transferred to pvdf membrane and incubated with primary antibodies at 1 : 500 dilution .
after incubation with hrp - conjugated 2nd antibodies , images were visualized with lumigen ps-3 ( beckman , southfield , mi , usa ) and quantified with a typhon scanner ( amersham pharmacia biotech , little chalfont , buckinghamshire , england , uk ) .
all experiments were performed on three separate occasions , each of which included triplicate gels for each condition .
statistical comparisons of multi - group data were analyzed by analysis of variance ( anova ) followed by bonferroni 's ( two - way ) posttest correction using prism4 software .
human lung fibroblast- ( hfl-1- ) mediated collagen gel contraction , an in vitro model of tissue remodeling , was used to investigate the effect of a camp - elevating agent on fibroblast tissue repair functions .
the long - acting -agonist salmeterol inhibited fibroblast - mediated collagen gel contraction in a concentration - dependent manner ( 10 ~ 10 m ; p < 0.05 at concentrations of 10 and 10 m compared to control , figure 1(a ) ) .
by contrast , a pde4 inhibitor , cilomilast , had no effect on fibroblast - mediated collagen gel contraction when it was added alone at concentrations of 10 , 10 , and 10 m ( figure 1(b ) ) .
when salmeterol ( 10 m ) and cilomilast ( 10 m ) , concentrations that were without effect when added alone , were added together , fibroblast - mediated collagen gel contraction was significantly inhibited from day 1 through day 5 ( p < 0.01 , figure 1(c ) ) .
next , we examined the combination of varying concentrations of salmeterol plus one concentration of cilomilast , or vice versa , and the resultant effect on collagen gel contraction . in the presence of cilomilast
( 10 m ) , lower concentrations of salmeterol ( 10 and 10 m ) significantly ( 10 m , p < 0.05 ; 10 m , p < 0.01 ) inhibited fibroblast - mediated collagen gel contraction compared to salmeterol alone ( figure 2(a ) ) .
while cilomilast alone at concentrations of 10 ~ 10 m had no effect on fibroblast - mediated collagen gel contraction , in the presence of a low concentration of salmeterol ( 10 m ) cilomilast at all three concentrations tested ( 10 , 10 , and 10 m ) significantly inhibited collagen gel contraction ( p < 0.01 , figure 2(b ) ) . since human lung fibroblasts release pge2 and endogenous pge2 may modulate the effect of -agonists by modulating the camp signaling pathway , hfl-1 cells were pretreated with indomethacin before being cast into the collagen gels .
after the pretreatment with indomethacin ( 10 m ) for 24 hours , the inhibitory effect of salmeterol on fibroblast - mediated collagen gel contraction was further significantly potentiated ( p < 0.05 , figure 3(a ) ) .
by contrast , there was a slight reduction of contraction in the presence of cilomilast that did not reach statistical significance ( figure 3(b ) ) .
in addition , indomethacin pretreatment augmented the inhibitory effect of salmeterol and cilomilast added together ( p < 0.01 , figure 3(c ) ) . since the indomethacin pretreatment suggested that endogenous pge2 could attenuate the effect of salmeterol and salmeterol plus cilomilast , we next assessed the effect of exogenous pge2 in modulating salmeterol and cilomilast inhibition of fibroblast - mediated collagen gel contraction . to accomplish this
, hfl-1 cells were pretreated with pge2 ( 10 m ) for 24 hrs prior to being cast into collagen gels and exposed to salmeterol and/or cilomilast .
pge2-pretreated cells contracted gels more than the control did cells ( figure 4(a ) ) . by contrast to indomethacin pretreatment , which potentiated salmeterol inhibition of collagen gel contraction , pge2 pretreatment of lung fibroblasts resulted in loss of salmeterol inhibition of fibroblast - mediated collagen gel contraction ( figure 4(a ) ) . by contrast , pge2-pretreatment potentiated the effect of cilomilast when added alone , and after pge2-pretreatment , the inhibition of fibroblast - mediated collagen gel contraction by cilomilast was statistically significant ( p < 0.05 , figure 4(b ) ) . in cells preexposed to pge2 , salmeterol plus cilomilast
also significantly inhibited collagen gel contraction ( p < 0.01 , figure 4(c ) ) . to investigate the mechanism by which endogenous and exogenous pge2 modulate long - acting -agonist ( salmeterol ) and pde4 inhibitor ( cilomilast ) inhibition of fibroblast - mediated collagen gel contraction
, hfl-1 cells were pretreated with 10 m indomethacin or 10 m pge2 for 24 hrs .
whole cell lysate proteins were then extracted and evaluated by immunoblotting for pde4 isoforms ( pde4a , pde4b , pde4c , and pde4d ) with -actin utilized as a loading control . as shown in figure 5 , pde4a , -4b , and -4c were expressed by hfl-1 cells , while the pde4d isoform was not expressed ( figure 5(a ) ) .
furthermore , pge2 significantly stimulated pde4a and -4c ( p < 0.05 ) expression , while pde4b was not affected ( figure 5(b ) ) .
indomethacin , by contrast , inhibited pde4c expression ( p < 0.05 ) , but it did not affect pde4a or -4b .
fibroblast - mediated collagen gel contraction is widely used as an in vitro model of tissue remodeling . in the current study , using this in vitro model of tissue remodeling , we have demonstrated a synergistic effect of the long - acting -agonist salmeterol and the pde4 inhibitor cilomilast in inhibiting fibroblast - mediated collagen gel contraction .
in addition , we have demonstrated that this interaction is modulated by both endogenous and exogenous pge2 .
finally , we have demonstrated that pge2 modulates the expression of several pde4 isoforms , providing a mechanism for regulating the synergistic interaction of salmeterol and cilomilast .
consistent with a previous report , the long - acting -agonist , salmeterol , inhibited fibroblast - mediated collagen gel contraction at concentrations of 10 and 10 m. the pde4 inhibitor , cilomilast alone , did not affect fibroblast - mediated collagen gel contraction at concentrations of 10 , 10 , and 10 m. however , when added together , 10 m salmeterol and cilomilast ( 10 , 10 , and 10 m ) significantly inhibited collagen gel contraction .
cilomilast , by blocking camp degradation , could permit accumulation of camp concentrations sufficient to inhibit gel contraction in response to concentrations of salmeterol that could not achieve such concentrations alone .
consistent with this , 10 m cilomilast also potentiated the actions of higher concentrations of salmeterol ( 10 and 10 m ) in inhibiting collagen gel contraction .
several studies have demonstrated that agents that increase camp can induce pde activity [ 14 , 2228 ] .
consistent with these studies , pretreatment of the fibroblasts with 10 m indomethacin for 24 hrs prior to being cast into collagen gels resulted in further inhibition of collagen gel contraction by salmeterol added alone , consistent loss of pde activity . in contrast , pretreatment with indomethacin did not affect contraction in the presence of cilomilast .
this contrasts with the effect of pretreatment of the fibroblasts with 10 m pge2 .
the mechanism of this effect of pge2 pretreatment is not defined , but it resembles the results of michalski who observed augmentation of chemotaxis following pge2 pretreatment . pge2
pretreatment also changed the response of the fibroblasts to salmeterol and cilomilast added alone . by contrast to control cells , following pge2 pretreatment , cilomilast added alone significantly inhibited gel contraction while salmeterol was without significant effect .
this is consistent with the previously observed increase in pde activity observed in a variety of cell types in response to increases in camp [ 14 , 2228 ] . also consistent with such a mechanism , we observed that pde4c was significantly decreased when the cells were pretreated with indomethacin , while , by contrast , pde4a and pde4c were significantly increased when the cells were pretreated with pge2 .
recently , a pde4 inhibitor has been approved for use in selected copd patients to reduce exacerbation risk .
long - acting -agonists have the potential to modulate fibroblast tissue remodeling through inhibiting fibroblast proliferation , differentiation , and collagen production through mechanisms that depend on increasing intracellular camp
. phosphodiesterases ( pdes ) , especially pde4 , may also modulate fibroblast - mediated tissue remodeling by increasing intracellular camp levels . in this regard
, we have previously reported that -agonists and pde4 inhibitors could inhibit human lung fibroblast - mediated collagen gel contraction , a widely used model of the tissue contraction that characterizes tissue remodeling [ 7 , 17 ] .
synergy between a -agonist and a pde4 inhibitor on fibroblast - mediated collagen gel contraction , however , has not been previously demonstrated .
we have previously reported that cilomilast alone at the concentrations of 10 ~ 10 m inhibited fibroblast migration and collagen gel contraction , while the same concentrations of cilomilast did not alter fibroblast - mediated collagen gel contraction in the current study .
these differing results are likely due to different experimental designs . in the previous report on cilomilast modulation of fibroblast - mediated collagen gel contraction , 0.5%
fcs was added to the medium in which gels were floated . in the current study , however , there was no serum in the medium in which gels were floated .
while the effect of serum is not clear , the current study clearly shows that pge2 can modulate cilomilast responsiveness .
cells pretreated with pge2 were responsive to cilomilast inhibition , which is consistent with pge2 induction of pde expression .
consistent with this resulting from the induction of pde4 , the synergistic effect of cilomilast added together with salmeterol was also observed in pge2 pretreated cells .
lung fibroblasts synthesize and release pge2 , and the current study also provides evidence that endogenous pge2 can modulate -agonist signaling by affecting pde4 isoform expression .
consistent with previous reports , hfl-1 cells expressed predominantly pde4a , -4b , and -4c but not pde4d .
interestingly , indomethacin significantly inhibited pde4c expression , while pge2 significantly stimulated pde4a and -4c .
these findings indicate that pge2 , by augmenting pde4 expression , may accelerate camp degradation .
consistent with our findings , it has also been reported that pge2 upregulates pde4 activity through induction of enzyme synthesis in the myometrium , and by this mechanism pge2 lessens the responsiveness of myometrium to -mimetic activation [ 16 , 22 ] .
taken together , these results suggest that pde4 is upregulated by both endogenous and exogenous pge2 . in the presence of low levels of pde4 ,
when pde4 levels are augmented , however , -agonist becomes less effective since pde4 results in degradation of any camp generated .
this loss of responsiveness to -agonist may be particularly important in an inflammatory milieu such as that which exists in copd where pge levels are high [ 3234 ] . when added together ,
this suggests that the synergy between pde4 inhibition and -agonists may be particularly important in copd where high levels of pge are present . in summary
, the current paper demonstrates synergy between a long - acting -agonist ( salmeterol ) and a pde4 inhibitor ( cilomilast ) in their ability to inhibit fibroblast - mediated collagen gel contraction , a widely used model of tissue remodeling .
through such an action , these agents , which are currently in use to treat copd , could also alter the tissue remodeling present in copd .
in addition , the current study demonstrates that pde4 expression in lung fibroblasts is induced by pge2 suggesting that this synergy may be particularly important in inflammatory diseases such as copd where pge levels are high . | in the current study , we investigated the effect of a long - acting -agonist ( salmeterol ) and a phosphodiesterase 4 ( pde4 ) inhibitor ( cilomilast ) on human lung fibroblast - mediated collagen gel contraction .
higher concentrations of salmeterol ( 107 and 106 m ) inhibited fibroblast - mediated collagen gel contraction . no effect was observed with cilomilast alone ( up to 105 m ) . in the presence of 108 m salmeterol ,
however , cilomilast could significantly inhibit fibroblast - mediated collagen gel contraction in a concentration - dependent manner ( 107~105 m ) .
blockade of endogenous pge2 by indomethacin further potentiated the inhibitory effect of salmeterol on fibroblast - mediated collagen gel contraction , but it did not affect cilomilast 's effect .
pretreatment with pge2 abolished the inhibitory effect of salmeterol , but it potentiated the inhibitory effect of cilomilast on fibroblast - mediated collagen gel contraction . finally , indomethacin slightly inhibited pde4c expression , while pge2 stimulated the expression of pde4a and -4c in human lung fibroblasts .
these findings suggest that long - acting -agonist and pde4 inhibitor have a synergistic effect in regulating fibroblast tissue repair functions and that pge2 can modulate the effect of -agonist and pde4 inhibitor at least in part through the mechanism of regulating pde4 expression . | 1. Introduction
2. Materials and Methods
3. Result
4. Discussion |
worldwide prevalence of diabetes has been recently increased over two - fold and is estimated to affect 439 million people by the year 2030 ( 1 , 2 ) .
diabetes is a complex metabolic disease characterized by high blood glucose level resulting from defects in insulin secretion or action ( 2 ) . 1
, 25-dihydroxyvitamin d3 ( vit d ) , the most potent metabolite of vitamin d , plays an important role in calcium and phosphate homeostasis , also vital for a number of other biological activities including immunomodulation and normal release of insulin from cells ( 3 - 5 ) .
pancreatic islets have both vitamin d receptors and vitamin d - dependent calcium - binding proteins , suggesting a role for vitamin d in insulin secretion ( 6 , 7 ) .
it has been reported that vitamin d deficiency impairs insulin synthesis and secretion in both human and animal models and its replenishment improves cell function and glucose tolerance ( 4 , 8 , 9 ) .
have shown improvement in glucose metabolism and insulin secretion in vitamin d - deficient rats following a single subcutaneous injection of vitamin d ( 10 ) .
in addition , it has been reported that de novo insulin synthesis is reduced in isolated islets in vitamin d - deficient rats and insulin biosynthetic capacity can be restored in vitro by injection of vitamin d ( 9 ) .
vitamin d deficiency may be related to type 2 diabetes , cardiovascular disease and cancer ( 11 , 12 ) .
subjects with type 2 diabetes have lower circulating vitamin d levels compared to healthy controls ( 3 , 13 ) .
furthermore , epidemiological studies have shown that vitamin d receptor restriction site polymorphisms are associated with genetic susceptibility to type 1 diabetes in different populations , while vitamin d supplementation in early childhood is associated with a reduced risk of type 1 diabetes ( 14 ) .
nikooyeh et al . have reported that daily consumption of vitamin d improves glycemic status in patients with type 2 diabetes ( 15 ) .
interventional studies investigating vitamin d for treatment of type 2 diabetes have shown negligible effects , and the results available in humans have been contradictory ( 15 ) .
previous studies have reported that high glucose concentrations significantly decreased total insulin secretion from cells ( 16 ) .
the effects of coincubation and preincubation of vitamin d with high glucose concentration on insulin secretion from islets have not been fully understood yet .
therefore , the aim of this study was to determine the effects of vitamin d on insulin release from isolated islets of rats .
adult male wistar rats ( two months old , weighing 200 - 250 grams ) were obtained from the laboratory animal house of the research institute for endocrine sciences , shahid beheshti university of medical sciences and housed in an animal room at 22 3c , relative humidity of 50 6% , an inverse 12 : 12 hour light / dark cycle .
rats had free access to standard rat chow ( pars co. , tehran ) and tap water during the study .
all experimental procedures and rat care and handling were performed in accordance with guidelines provided by the local ethics committee of the research institute for endocrine sciences , shahid beheshti university of medical sciences .
animals were anesthetized by intraperitoneal injection of ketamine / xylazine ( 50 mg / kg and 10 mg / kg ) and the abdomen was exposed , and 0.5 mg / ml of collagenase p ( roche , cat .
# 1213 , germany ) in 10 ml cold hanks balanced salt solution ( hbss ) [ ph = 7.4 ; containing kcl , 5.36 ; cacl2 , 1.26 ; nacl , 136 ; mgso4 7 h2o , 0.8 ; na2hpo4 2 h2o , 0.33 ; nahco3 , 4.16 ; kh2 po4 , 0.44 ) all in mm ( merck , germany ) and gassed with 95% o2 , 5% co2 for five minutes at the beginning ] was injected into the common bile duct ; the expanded pancreas was detached , transmitted to a 50 ml falcon container and placed in a water bath ( 37c ) for 15 minutes . at the end of digestion , 30 ml cold hbss was added and the tube was shaken for one minute .
tube contents were filtered through a 500 m plastic mesh to discard any undigested tissue .
after three washes with cold hbss , islets were selected under a stereomicroscope ( kyowa , sdz - tr - pl japan ) ; a group of 10 islets were incubated in a cell strainer containing 4 ml of rpmi 1640 medium containing 10% fetal bovine serum and 1% of penicillin / streptomycin ( 17 , 18 ) .
insulin release was assessed in response to vitamin d ( d1530 , sigma ) following 24 and 48 hours coincubation of islets with vitamin d ( 0.1 , 1 , and 10 nm ) and glucose ( 5.6 , 11.1 and 16.7 mm ) .
in addition , islets were preincubated with vitamin d for 24 and 48 hours and glucose - stimulated insulin secretion ( gsis ) was assessed for one hour in the presence of 5.6 and 16.7 mm glucose . for in vitro assessment of insulin secretion , batches of eight islets were transferred into 1.5 ml plastic cups , and 1 ml of krebs ringer solution [ ( ph = 7.4 ) ; nacl , 115 ; kcl , 5 ; mgcl2 6 h2o , 1 ; cacl2 , 2.5 ; nahco3 , 24 ( merck , germany ) ; hepes , 16 ( sigma , usa ) all in mm ] and 5 g / dl bsa ( fluka , usa ) with different glucose concentrations ( 5.6 and 16.7 mm ) were added into the cups , incubated for 60 minutes in 37c water bath and gassed with 95% o2 , 5% co2 for five minutes at the beginning .
intra and inter - assay coefficients of variations for insulin measurements were 6.1% and 9.7% , respectively .
all data were expressed as mean sem and analyzed using graphpad prism software ( version 5.00 for windows , graphpad software , san diego california usa ) .
two - way analysis of variance ( anova ) and bonferroni post - hoc test were used to compare serum insulin concentrations in different levels of glucose and vitamin d. p value below 0.05 was considered statistically significant .
all data were expressed as mean sem and analyzed using graphpad prism software ( version 5.00 for windows , graphpad software , san diego california usa ) .
two - way analysis of variance ( anova ) and bonferroni post - hoc test were used to compare serum insulin concentrations in different levels of glucose and vitamin d. p value below 0.05 was considered statistically significant .
effects of coincubation of vitamin d with glucose on insulin release from islets were shown in figure 1 . as shown ,
coincubation of islets with 10 nm of vitamin d and 11.1 mm glucose increased insulin release , while 1 and 10 nm vitamin d with 16.7 mm glucose decreased insulin release .
vitamin d had no effect on insulin release from islets during 24 hours coincubation with 5.6 mm glucose or 48 hours coincubation with 5.6 , 11.1 , and 16.7 mm glucose .
effects of 5.6 and 16.7 mm glucose on gsis in islets , following 24 and 48 hours preincubation with vitamin d were shown in figure 2 . as seen , 24 and 48 hours preincubation with 10 nm of vitamin d and 48 hours preincubation with 1 and 10 nm of vitamin d increased gsis in the presence of 16.7 mm glucose .
insulin release in response to 24-hours ( a ) and 48-hours ( b ) incubation with different concentrations of vitamin d in presence of 5.6 , 11.1 , and 16.7 mm glucose . * : p < 0.05 compared to control ( vitamin d= 0 ) .
insulin release in response to 5.6 and 16.7 mm glucose for a duration of one hour in islets from normal rats following 24-houes ( a ) and 48-hours ( b ) preincubation with vitamin d. * : p < 0.05 compared to control ( vitamin d = 0 ) ; : p < 0.05 compared to vitamin d 1 nm .
preincubation of vitamin d with glucose for 24 or 48 hours increased gsis from islets only at high doses of vitamin d and glucose .
in addition , when vitamin d and glucose were coincubated for 24 hours , insulin release was increased with 10 nm vitamin d and 11.1 mm glucose ; whereas , insulin release decreased with 1 and 10 nm vitamin d and 16.7 glucose .
these results were consistent with those studies reporting an association between vitamin d and insulin release from cells ( 19 - 21 ) .
preincubation with vitamin d increased gsis in response to 16.7 mm glucose , findings in agreement with the results of billaudel et al . who reported that gsis is eliminated by vitamin d deficiency in rats ; however , six hours preincubation with 0.001 - 1000 nm vitamin d had a stimulatory effect on gsis in response to 8.3 mm glucose ( 8) .
furthermore , it has been reported that 24 hours preincubation with 10 nm vitamin d increases gsis in the presence of 8.3 mm glucose in vitamin d - deficient rats ( 22 ) .
tanaka et al . have also reported that insulin secretion induced by 16.7 mm glucose in vitamin d deficient rats was only 35% of that of control rats , but was restored to that of the controls in vitamin d - treated rats ( 23 ) .
have also reported that insulin secretion is destroyed in vitamin d deficient rats and restored by 1 , 25-(oh)2d3 ( 24 ) .
results of this study showed that vitamin d affects insulin secretion only in the presence of high glucose level , whereas it does not affect insulin secretion in presence of 5.6 mm glucose .
vitamin d enhances sensitivity of cells to glucose , and has been suggested that the effect of vitamin d on insulin secretion may be atp - dependent ( 4 , 22 ) .
have shown that coincubation of 0.1 nm and 1 nm vitamin d with 10 mm glucose did not increase insulin release in medium for up to 180 hours ( 25 ) .
faure et al . have confirmed that coincubation of 0.001 nm vitamin d with 8.3 mm glucose did not affect insulin secretion ( 22 ) .
chan et al . have reported that in vitro addition of vitamin d had no effect on insulin secretion from normal or vitamin d deficient rats ( 26 ) .
in addition , two human studies found no change in insulin release following vitamin d supplementation in adults with either obesity or insulin resistance ( 19 - 21 ) .
contradictory to our results , billaudel et al . have reported that coincubation of 0.001 - 1000 nm of vitamin d with 8.3 mm glucose had an incremental dose - dependent effect on insulin secretion ( 8) .
moreover , demden et al . have demonstrated that coincubation of 10 nm vitamin d with 10 mm glucose increased insulin secretion 2.5 fold in a cell medium ( 25 ) . to date , no study has investigated the effect of vitamin d on insulin secretion in the presence of 16.7 mm glucose in short duration ( up to 24 hours ) . lower insulin secretion has been reported in the presence of high glucose concentrations compared to low glucose concentrations in cell medium .
in addition , high glucose concentrations significantly decrease insulin secretion in cultured cells ; these data indicates that exposure to high glucose levels induces a glucotoxic situation , which could impair insulin secretion in response to glucose ( 16 ) .
contrary to the finding of our study , demden et al . have demonstrated that coincubation of 10 nm vitamin d with 20 mm glucose increased insulin secretion in a cell medium ( 25 ) , but suggested a significantly increased insulin release in islets following 96 hours coincubation or longer
. the exact mechanisms of vitamin d effect on insulin secretion in the presence of glucose are not yet fully understood and need further studies , but it has been suggested that vitamin d directly increases insulin secretion in cells ( 25 ) .
faure et al . have reported that vitamin d enhances ca entry or ca mobilization in cells , which could amplify insulin secretion in the presence of glucose ( 22 ) .
have also shown that vitamin d requires a delay to increase insulin secretion from cells ; this delay varies from 6 hours pre - incubation in vitro , 3 hours to 20 hours in vivo and 48 hours in culture , which can indicates the genomic effects of vitamin d ( 9 ) .
in conclusion , effects of coincubation of islets with vitamin d and glucose depend on glucose concentration .
vitamin d significantly decreased insulin secretion in the presence of 16.7 mm glucose , but increased insulin secretion in the presence of 11.1 mm glucose .
in addition , this study showed that preincubation of islets with vitamin d increased gsis at high levels of glucose ( 16.7 mm ) , while no effect was observed at low levels . | background : vitamin d ( vit d ) affects glucose metabolism . receptors of vitamin d have been identified in cells and studies show that vitamin d deficiency reduces glucose - stimulated insulin secretion ( gsis).objectives : the aim of this study was to examine the effect of vitamin d on insulin release from isolated islets of rats.materials and methods : islets were isolated from male wistar rats , weighing 200 - 250 grams , using the collagenase digestion method .
insulin release was assessed following 24 and 48 hours coincubation of islets with vitamin d ( 0.1 , 1 and 10 nm ) and glucose ( 5.6 , 11.1 and 16.7 mm ) .
in addition , islets were preincubated with vitamin d for 24 and 48 hours and gsis was assessed for one hour in the presence of 5.6 and 16.7 mm glucose.results:coincubation of islets with vitamin d ( 10 nm ) and 11.1 mm glucose increased islet insulin release ( 37.27 3.75 vs. 24.64 2.83 ng / islet/24 hours ; p < 0.05 ) , while vitamin d ( 1 and 10 nm ) decreased insulin release in the presence of 16.7 mm glucose ( 21.14 3.58 and 18.65 3.84 vs. 37.71 4.63 ng/ islet/24 hours ; p < 0.05 ) .
islets preincubation with vitamin d ( 1 and 10 nm ) increased gsis in the presence of 16.7 mm glucose ( 4.39 0.73 and 4.39 0.63 vs. 2.07 0.43 ng / islet/1 hour ; p < 0.05).conclusions : preincubation of islets with vitamin d increased gsis but decreased insulin release in coincubation with high levels of glucose .
insulin secretion from cells in the presence of glucose seems to be related to the dosage of vitamin d and duration of preincubation . | 1. Background
2. Objectives
3. Materials and Methods
3.1. Statistical Analysis
4. Results
5. Discussion |
necrobiosis lipoidica ( nl ) is a disease with clinical features that are seldom misinterpreted .
a 67-year - old woman was admitted to our department with a well - defined , persistent plaque on her right shin ( fig
the old lesion , which was located on the anterior surface of the shin , was characterized by sharply defined , slightly elevated , red - bluish borders and a yellow - orange , atrophic , porcelain - like center .
both lesions were not painful , but slightly pruritic and there was no history of recent trauma or infection .
the patient had a history of type 2 diabetes mellitus ( dm ) since 2000 .
blood glucose levels had been poorly controlled ever since , with hba1c reaching 7.3% , but with no apparent complications of dm .
although clinically the shin lesion was compatible with nl , the arm lesion was not typical of any dermatosis .
therefore , both lesions were biopsied to identify their nature and rule out sarcoidosis or granuloma annulare .
histology revealed identical features despite the dissimilar clinical presentation . a granulomatous dermatitis with foci of degenerated collagen surrounded by histiocytes , some of them multinucleated ( fig .
3 ) , as well as thickened collagen bundles arranged horizontally in the dermis are all typical findings of nl . the patient was treated with 0.1% topical tacrolimus ointment twice daily for 8 weeks and once daily for 8 weeks .
a significant improvement and no further lesions were observed after 1 year of follow - up .
it more commonly occurs in women than in men with a ratio of 3:1 [ 1 , 2 , 3 ] .
the average age of onset is 3040 years [ 1 , 2 , 3 ] , although it has been frequently reported in older age groups and seldom in children .
the lesions are distributed on the anterior and lateral surfaces of the lower legs . however , there are other sites that can rarely be affected , such as upper extremities [ 1 , 2 ] , trunk [ 1 , 3 ] , face ( even periorbitally ) , scalp [ 1 , 3 ] and penis [ 3 , 4 ] .
however , diabetic microangiopathy , immune - complex vasculitis , and collagen abnormalities are some potential underlying causes .
nakajima et al . assert that both hyperlipidemia and venous reflux , in addition to other pathogenic factors , can trigger tissue damage in the lower legs and lead to the onset of nl . in constrast ,
refute the hypothesis that nl is a manifestation of microvascular ischemic disease of the skin and conclude that the increased blood flow seen in nl lesions suggests an ongoing inflammatory process . despite the debates on pathogenesis ,
7590% of patients with nl have or will develop dm [ 2 , 3 ] .
in contrast , nl is present only in 0.33% of the diabetic population [ 1 , 2 , 3 ] .
it is most commonly seen in patients with type 1 dm , but may also occur in type 2 dm . whether glucose control and treatment of dm improve or
have no effect on nl lesions is still a controversy [ 1 , 2 ] .
nl has also been associated with other diseases such as sarcoidosis , rheumatoid arthritis , autoimmune thyroid disease and inflammatory bowel disease .
ulceration is the most frequent and hard to treat complication of nl in 2533% of patients [ 1 , 2 , 3 ] .
additionally , squamous cell carcinoma developing in areas of nl has also been reported [ 1 , 2 , 3 ] .
although there are plenty therapeutic options for nl , its treatment is still challenging and sometimes ineffective [ 1 , 2 ] .
first - line treatment includes corticosteroids , either topically or intralesionally , and sometimes systemically [ 1 , 2 , 3 , 8 ] . according to some reports ,
other therapeutic options are antiplatelet agents , cyclosporine [ 1 , 2 , 3 , 8 ] , thalidomide [ 3 , 8 ] , clofazimine , anti - tnf agents [ 3 , 8 ] , fumaric acid esters [ 2 , 8 ] , puva [ 2 , 3 , 8 ] , photodynamic therapy [ 8 , 9 ] , hydroxychloroquine and tacrolimus [ 3 , 11 , 12 , 13 ] .
we report a case of non - ulcerated , recent lesions of nl well managed with topical tacrolimus 0.1% ointment twice daily for 8 weeks and once daily for 8 weeks . despite the limited experience with topical tacrolimus treatment in nl
tacrolimus induces anti - inflammatory and immunomodulatory effects with suppression of the granulomatous infiltrate in granuloma annulare and nl , including the reduction of chemotactic activity of fibroblasts and the inhibition of collagen synthesis [ 11 , 12 , 13 ] .
subsequently , tacrolimus is thought to be effective at the early inflammatory stages of nl .
making a diagnosis of nl of the lower legs is not difficult ; however , a high index of suspicion regarding nl lesions with atypical clinical presentation on different body sites is advised in order to avoid misdiagnosis , wrong treatment decisions and ulceration .
additionally , it appears that topical tacrolimus treatment is an effective therapeutic option in patients with recent , non - ulcerated nl lesions . | we present a case of necrobiosis lipoidica ( nl ) with atypical early lesions and good response to topical tacrolimus .
nl is a disease with clinical features that are seldom misinterpreted .
often histology just confirms the clinician 's diagnosis . only in rare cases , the clinical presentation and
the involved body sites may be misleading .
a 67-year - old diabetic woman was admitted to our department with a well - defined , persistent plaque on her left arm and on her right shin .
histologic examination of both lesions revealed features of nl despite the dissimilar clinical presentation .
the patient was treated with 0.1% topical tacrolimus ointment twice daily for 8 weeks and once daily for 8 weeks .
a significant improvement and no further lesions were observed after 1 year of follow - up .
a high index of suspicion regarding nl lesions with atypical clinical presentation on different body sites is advised in order to avoid misdiagnosis , wrong treatment decisions and ulceration .
additionally , it appears that topical tacrolimus treatment is an effective therapeutic option in patients with recent , non - ulcerated nl lesions . | Introduction
Case Report
Discussion
Disclosure Statement |
the call to action on diabetes by the international diabetes federation was based on a clear message that diabetes is everyone 's business.(1 ) we see tremendous efforts in this space both from the government , as well as from non - governmental organizations , researchers , academicians , and corporate sector .
the ministry of health and family welfare , government of india , launched the national program on prevention and control of diabetes , cardiovascular diseases and stroke ( npdcs ) in 2008.(2 ) the non - communicable diseases ( ncd ) cell of government of india supervises and monitors the implementation of the program at various levels .
mcgm is dedicated to ncd program in mumbai providing facilities for diagnosis , treatment , follow - up and referrals in 55 dispensaries , 18 peripheral hospitals and three teaching institutes .
mcgm has complemented the ncd program with innovative initiatives for educating , screening , and tracking , such as patient database & tracking system , school program , workplace intervention survey , community camps , extensive iec and public - private partnerships . in a city like mumbai , which hosts over 1.1 million people with diabetes,(3 )
the way forward is an integrated care model that can harness the expertise of different sectors like the government , private sector and community . in this communication
, we describe the successful implementation of one such integrated public - private partnership model .
mcgm public health department has a specific cell working on ncd , with a strong diabetes component .
mcgm has 55 diabetes clinics with facilities of testing , consultation , treatment and diet counseling .
the primary health care system is well - linked to the secondary and tertiary system for referrals .
2013 saw the opening of a new chapter in the ncd program , when mcgm partnered with the private sector in a transparent and positive way to fight this problem . through this partnership , eli lilly , a us - headquartered pharmaceutical company , brought to the table its expertise in patient education .
this partnership aims to strengthen the capacity of diabetes clinics of mcgm across the city and special diabetes outpatient department clinics in peripheral and major hospitals .
eli lilly supported mcgm to build the capacity of primary workforce by transferring skills and tools for better management of diabetes .
this was done through an educational tool called diabetes conversation map ( dcm ) , created by healthy interactions , in collaboration with international diabetes federation , and sponsored by eli lilly .
this tool uses interactive group participation to empower people with diabetes to become actively involved in managing their ailment.(4 ) this group learning experience is facilitated by trained diabetes educators , who have the necessary skill and expertise to co - ordinate education among a group of health workers , caregivers or patients as the case may be using different types of dcms .
fifty - five medical officers of mcgm diabetes dispensaries were sensitized and 28 amos located at health posts of mcgm were trained in partnership with eli lilly , in use of dcm .
capacity building training on dcm by certified master trainer was preceded by training on clinical spectrum and management protocol by experts from medical colleges in mumbai . following the training ,
a systematic implementation plan was chalked out for conducting the dcm sessions in the dispensaries . trained amos under the supervision of medical officers took sessions in diabetes dispensaries . during initial sessions
altogether , amos from these 28 health posts conducted 168 sessions in attached 25 dispensaries ( some dispensaries were attached to more than one health post ) and 1616 beneficiaries availed the sessions over just six months ( july - dec 2013 ) . although , no quantitative measurement of impact was done during implementation of this model , general feedback obtained on a regular basis from health providers is that dcm helps clear misconceptions among patients in an interactive way and helps improve compliance of patients .
it helps save time of medical officers , who can effectively educate multiple patients in a single session regarding the basics of the disease , its outcome , complications and importance of lifestyle modification and treatment adherence .
the dcm sessions also act as a forum for patients to interact with other individuals facing similar problems and they learn through mutual interaction , eventually making life easier for them .
these sessions bring out the faiths , beliefs , and cultural influences of patients and their relatives , which otherwise are unknown to the concerned health provider , and may act as hindrances .
the health care provider can tailor his / her health care delivery accordingly for better treatment outcomes .
patients who are educated through dcms tend to accept lifestyle modification better than one - way health talks , as this is a more visual and interactive medium . with this encouraging feedback ,
26 additional amos located at health posts attached to remaining diabetes clinics , have been trained on dcm , who will now roll it out to the patients and their care givers .
fifty - five medical officers of mcgm diabetes dispensaries were sensitized and 28 amos located at health posts of mcgm were trained in partnership with eli lilly , in use of dcm .
capacity building training on dcm by certified master trainer was preceded by training on clinical spectrum and management protocol by experts from medical colleges in mumbai . following the training ,
a systematic implementation plan was chalked out for conducting the dcm sessions in the dispensaries . trained amos under the supervision of medical officers took sessions in diabetes dispensaries . during initial sessions
altogether , amos from these 28 health posts conducted 168 sessions in attached 25 dispensaries ( some dispensaries were attached to more than one health post ) and 1616 beneficiaries availed the sessions over just six months ( july - dec 2013 ) . although , no quantitative measurement of impact was done during implementation of this model , general feedback obtained on a regular basis from health providers is that dcm helps clear misconceptions among patients in an interactive way and helps improve compliance of patients .
it helps save time of medical officers , who can effectively educate multiple patients in a single session regarding the basics of the disease , its outcome , complications and importance of lifestyle modification and treatment adherence .
the dcm sessions also act as a forum for patients to interact with other individuals facing similar problems and they learn through mutual interaction , eventually making life easier for them .
these sessions bring out the faiths , beliefs , and cultural influences of patients and their relatives , which otherwise are unknown to the concerned health provider , and may act as hindrances .
the health care provider can tailor his / her health care delivery accordingly for better treatment outcomes .
patients who are educated through dcms tend to accept lifestyle modification better than one - way health talks , as this is a more visual and interactive medium . with this encouraging feedback ,
26 additional amos located at health posts attached to remaining diabetes clinics , have been trained on dcm , who will now roll it out to the patients and their care givers .
there is enough evidence to show how education of patients positively impacts the disease outcomes.(56 ) physicians strive to help patients embrace their treatment plan .
however , the burden of patients in a routine outpatient department hardly leaves physicians with sufficient time to engage in individual discussion around education and counseling .
thus , many queries of these patients remain unanswered either due to lack of time or their hesitation to ask questions to the doctor .
moreover , the patients decisions are often driven by other sources of information , including friends , relatives and advocates of alternative remedies .
thus , it is necessary to empower them to take their own decisions and of course , right decisions , when medical help is not around .
although overcoming these tensions in the patient journey is a mammoth task , dcm does help to some extent by engaging people in a meaningful conversation about diabetes . through dcm
, information is delivered in an engaging atmosphere through peer - to - peer discussions , and patients find it easier to apply this knowledge to real - life situations , thus taking small , meaningful steps towards managing their condition better .
the success of this program was possible due to an effective collaboration of a public organization that were rightly committed for improving the situation of diabetes patients and a private organization who had the tools and resources to cater to the needs of diabetes patients . at this time
, there is no statistical analysis to prove the positive outcome of this collaboration or the education tool used .
there was no randomized controlled trial conducted to prove effectiveness or impact of the tool or the model .
nonetheless , any effort in this direction to educate patients on self - management goes a long way and the clinical benefit can not be underestimated .
moreover , although , the impact of the tool was not studied in this model , there are other reports of positive impact with this tool.(7 ) while the world is now discussing how the private sector can get engaged and share stakes in the business of community health , this is a small step in that direction and testament to the transformative potential of innovative thinking .
no single sector has all the answers to reverse the path to this national catastrophe . | contextrising number of diabetes cases in india calls for collaboration between the public and private sectors.aims:municipal corporation of greater mumbai ( mcgm ) partnered with eli lilly and company ( india ) [ eli lilly ] to strengthen the capacity of their diabetes clinics.materials and methodsmedical officers , dispensaries and assistant medical officers ( amos ) located at attached health posts were trained on an educational tool , diabetes conversation map ( dcm ) by a master trainer .
this tool was then used to educate patients and caregivers visiting the mcgm diabetes clinics.resultstwenty-eight centers conducted 168 sessions , and 1616 beneficiaries availed the education over six months .
general feedback from health providers was that dcm helps clear misconceptions among patients and caregivers in an interactive way and also improves compliance of patients.conclusionsthis communication highlights a unique public - private partnership where the sincere efforts of public sector organization ( mcgm ) were complemented by the educational expertise lent by a private firm . | Introduction
Materials and Methods
Results
Implementation of the Education Model
Discussion |
ellis - van creveld ( evc ) syndrome , also called chondroectodermal dysplasia , mesoectodermal dysplasia , or chondrodystrophy , is a rare autosomal recessive congenital disorder [ 1 , 2 ] .
its reported incidence in amish population of pennsylvania , usa , is one in 1,500,000 live births and in non - amish population , the incidence is seven in 1,000,000 live births . in iran , however , the incidence of this syndrome has not been previously reported .
evc was first reported by mcintosh in 1933 and was subsequently described in detail by ellis and van creveld in 1940 .
evc ( omim # 225500 ) , is caused by a mutation in the evc1 gene or a mutation in a non - homologous gene , evc2 , which is located close to evc gene in a head - to - head configuration [ 4 , 5 ] .
parental consanguinity has been observed in about 30% of the cases [ 1 , 5 , 6 ] .
this syndrome is characterized by a variable spectrum of clinical findings , among which chondrodystrophy , polydactyly , ectodermal dysplasia , and congenital cardiac anomalies are the most common [ 5 , 7 ] . about half of the children suffering from evc syndrome fail to survive because of respiratory problems , and the rest need different procedures to manage their disease .
oral manifestations of evc syndrome include hyperplastic frenula , absence of mucobuccal fold , gingival hypertrophy , hypodontia , dystrophic philtrum , and cleft palate [ 2 , 8 , 9 ] .
the maxillary and mandibular alveolar processes may present notching or submucosal clefts [ 8 , 9 ] .
uncommon oral and dental diseases have helped physicians to diagnose this syndrome aptly and refer the patients to potentially prevent additional complications .
a two - and - a - half year old female presented to the department of pediatric dentistry in isfahan university of medical sciences in april 2012 , with the chief complaint of delayed tooth eruption .
she had no teeth , and exhibited hypertrophy of the labio - gingival frenulum ( figure 1 ) .
all the primary incisors , canines , and first molars , as confirmed by radiographs , were congenitally missing ( figure 2 )
. she did not exhibit gingival hypertrophy , and had missing teeth in the gingival area .
the rest of her physical examination was significant for polydactyly ( hexadactyly ) of both hands , and dystrophic nails ( figure 3 ) .
her head morphology and facial appearance were normal , except for hidrotic and fine hair .
she had post - axial polydactyly with fusion of the fifth and the sixth metacarpal bones and disproportionately shorter distal phalangeal bones ( acromelia ) ( figure 5 ) .
she was referred to a pediatric hospital and was examined for anomalies in other organ systems .
specifically , she was examined for bony abnormalities , as well as defects in her kidneys , heart and lungs .
ellis - van creveld ( evc ) syndrome is a rare autosomal recessive congenital disorder , characterized by chondrodystrophy , polydactyly , ectodermal dysplasia , and congenital cardiac anomalies .
patients may present with growth failure of the proximal tibial epiphysis and the tibial segment may be disproportionately shorter than the femoral segment , or fibula may be shorter than tibia .
oral manifestations include hyperplastic frenula , absence of mucobuccal fold , gingival hypertrophy , hypodontia , dystrophic philtrum , and cleft palate [ 2 , 8 , 9 ] .
the differential diagnosis of evc includes weyers syndrome , mckusick - kaufman syndrome and jeune syndrome .
mkk syndrome , which is a recessively inherited disorder , can be distinguished from evc by its characteristic presence of hydrometrocolpos . in weyers syndrome ,
ectodermal disturbances are similar to evc , but unlike evc , thoracic dysplasia , congenital heart diseases and dwarfism are absent in weyers syndrome .
jeune syndrome , an inherited form of dwarfism , is characterized by short limbs , similar to those observed in evc .
however , patients suffering from jeune syndrome do not exhibit fingernail hypoplasia , which is commonly observed in patients with evc [ 8 , 10 , 11 ] .
patients with evc should be managed through a multidisciplinary approach . particularly during the neonatal period ,
our patient remained undiagnosed , because she had a functional murmur ; which was disregarded .
management of bone deformities requires orthopedic follow - ups and oral manifestations need intense professional attention .
prognosis is coupled to the respiratory problems during the first few months of life that are attributable to thoracic narrowness and heart defects .
treatment should be started as early as possible in order to attain the best possible clinical outcome .
patients may require gingivectomy , frenectomy , and amputation of extra digits under sedation or general anesthesia .
dental care after eruption of teeth includes oral hygiene instructions , dietary counseling and effective plaque control .
definitive management throughout adulthood includes dental implants and prosthetic rehabilitation to compensate for congenital oligodontia .
although evc syndrome has been reported to be quite rare , our case which was detected under normal conditions can be alarming for dental care providers . due to diverse physical problems that such patients may suffer from , and the restrictions they may have for the medications that they take , it is imperative to pay utmost attention while examining or screening these patients for consequent referrals . | chondroectodermal dysplasia ( ellis - van creveld syndrome ) is a rare autosomal recessive congenital abnormality .
this syndrome is characterized by a spectrum of clinical findings , among which chondrodystrophy , polydactyly , ectodermal dysplasia , and congenital cardiac anomalies are the most common .
it is imperative to not overlook the cardiac complications in patients with this syndrome during dental procedures .
the case presented here , although quite rare , was detected under normal conditions and can be alarming for dental care providers.clinical reports outline the classical and unusual oral and dental manifestations , which help health care providers diagnose chondroectodermal dysplasia , and refer patients with this syndrome to appropriate health care professionals to receive treatment to prevent further cardiac complications and bone deformities . | INTRODUCTION
CASE REPORT
DISCUSSION |
the golden triad for a successful outcome in posterior urethral anastomosis ( pua ) has been defined as complete excision of scarred tissue , a lateral fixation of healthy urethral end mucosa , and the creation of a tension - free anastomosis . even in patients with unfavorable conditions , such as a stricture gap that exceeds 3 cm , a previously failed repair , associated perineal fistulas , rectourethral fistulas , periurethral cavities , false passages , or an open bladder , the aforementioned factors are key to a successful urethral reconstruction .
however , these complex conditions may require removal of a vast amount of tissue , which creates a large dead space .
in such situations , additional methods are required to overcome the difficulties that arise . a gracilis muscle flap ( gmf ) has been widely used in reconstructive surgical procedures such as rectourethral fistula repair because the gmf is long enough to reach the perineum and is endowed with a good blood supply from well - vascularized muscle .
thus , the gmf was introduced to manage urethral end - to - end anastomosis and the perianastomotic dead space by wrapping the urethral anastomosis and filling the perianastomotic dead space .
the gmf likely supplements the blood supply to the impaired vascularity of an anastomosis and prevents the compression of the urethral anastomosis by a perianastomotic hematoma .
we previously reported that a gmf can be useful in patients with a stricture longer than 3 cm and in patients who have previously undergone perineal urethroplasty .
although we confirmed its therapeutic effects , whether to apply a gmf to all urethroplasties remains debatable because its benefits have only been demonstrated in a limited number of cases , and a gmf necessitates another long incision of the thigh .
therefore , better evidence is required to determine the indications for the use of a gmf .
the objective of our study was to evaluate the preoperative clinical factors that affect surgical outcome to determine who will benefit from the use of a gmf in pua .
after acquiring approval from the cha bundang medical center institutional review board , we reviewed the medical records of 202 patients who underwent urethral reconstruction for a traumatic urethral injury between february 2001 and june 2011 .
patients aged 18 years who had undergone a delayed pua with the use of a gmf owing to posterior urethral injury were evaluated ; pua patients with neurogenic issues that affected voiding were excluded .
a successful outcome was defined as meeting the following criteria : 1 ) peak urinary flow rate greater than 15 ml / s at 3 and 12 months postoperatively , 2 ) no evidence of stricture recurrence on retrograde urethrogram or cystourethroscopy at 3 months postoperatively , and 3 ) no obstructive urinary symptoms for at least 12 months postoperatively .
patients were divided into two groups according to whether they experienced a successful surgical outcome .
the length of the urethral defect and patency of the anterior urethra was assessed by voiding cystourethrography with retrograde urethrography .
the bladder neck and length of urethral defect were assessed by urethroscopy and antegrade cystourethroscopy through the suprapubic cystostomy tract .
all patients were placed in a lithotomy position and underwent an inverted y - shaped perineal incision .
the distal urethral end was identified by retrograde passage of a metallic urethral sound and the proximal urethral end was identified by antegrade passage of a metallic urethral sound through the suprapubic cystostomy tract . after determining the extent of the urethral defect , all fibrotic tissues of the urethral defect including any periurethral scar tissue were completely excised . to avoid tension on the suture site ,
urethral end - to - end anastomosis was performed by using a progressive perineal approach for midline separation of the proximal corporal bodies , inferior pubectomy , and supracorporal urethral rerouting .
the proximal and distal ends of the urethra were spatulated and an anastomosis between the two ends was performed over a 16-fr silicon urethral catheter using 4 - 0 or 5 - 0 vicryl sutures .
the skin incision was made parallel to the long axis of the gracilis muscle of the left thigh .
the gracilis muscle was dissected from the medial aspect of the left thigh and released from its insertion .
the gmf was rotated , and its distal end was brought to the perineal area through a subcutaneous tunnel .
the muscle was then wrapped around the anastomosized urethra , and the perineal defect was filled with the rotated gmf .
a suprapubic catheter was placed for urinary diversion , and two suction drains were placed in the retropubic space .
the urethral catheter was removed 3 weeks postoperatively if no extravasation was visualized on retrograde urethrography .
the suprapubic catheter was removed if the patient voided in the same manner as before the urethral injury .
if any symptoms of obstruction or poor urinary flow were present , retrograde urethrography was performed to confirm the urethral stricture .
age , body mass index ( bmi ) , a history of previous surgery , the cause of the urethral injury , incidence of pelvic bone injury , incidence of bladder injury , incidence of rectal injury , the urethral lengthening procedure , the length of urethral defect , and the time interval between the original urethral injury and the pua or between a previous urethroplasty and the pua were all evaluated for their influence on surgical outcome .
the age , bmi , time interval , and urethral defect length of patients whose surgical outcome was successful were compared with those values in patients whose surgical outcome was unsuccessful by t - test .
the number of patients who had undergone a previous urethroplasty ; the cause of the urethral injury ; the incidence of pelvic bone injury , bladder injury , or rectal injury ; and the number of patients who had undergone each urethral lengthening procedure were compared between patients with a successful surgical outcome and those with an unsuccessful outcome by using the chi - square test and fisher exact test . to identify the preoperative clinical factors that affected surgical outcome , univariate and multivariate logistic regression analyses were performed .
regression analysis results are shown as the odds ratio ( or ) and 95% confidence interval ( ci ) .
statistical analyses were performed by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) .
after acquiring approval from the cha bundang medical center institutional review board , we reviewed the medical records of 202 patients who underwent urethral reconstruction for a traumatic urethral injury between february 2001 and june 2011 .
patients aged 18 years who had undergone a delayed pua with the use of a gmf owing to posterior urethral injury were evaluated ; pua patients with neurogenic issues that affected voiding were excluded .
a successful outcome was defined as meeting the following criteria : 1 ) peak urinary flow rate greater than 15 ml / s at 3 and 12 months postoperatively , 2 ) no evidence of stricture recurrence on retrograde urethrogram or cystourethroscopy at 3 months postoperatively , and 3 ) no obstructive urinary symptoms for at least 12 months postoperatively .
patients were divided into two groups according to whether they experienced a successful surgical outcome .
the length of the urethral defect and patency of the anterior urethra was assessed by voiding cystourethrography with retrograde urethrography .
the bladder neck and length of urethral defect were assessed by urethroscopy and antegrade cystourethroscopy through the suprapubic cystostomy tract .
all patients were placed in a lithotomy position and underwent an inverted y - shaped perineal incision .
the distal urethral end was identified by retrograde passage of a metallic urethral sound and the proximal urethral end was identified by antegrade passage of a metallic urethral sound through the suprapubic cystostomy tract . after determining the extent of the urethral defect , all fibrotic tissues of the urethral defect including any periurethral scar tissue were completely excised . to avoid tension on the suture site ,
urethral end - to - end anastomosis was performed by using a progressive perineal approach for midline separation of the proximal corporal bodies , inferior pubectomy , and supracorporal urethral rerouting .
the proximal and distal ends of the urethra were spatulated and an anastomosis between the two ends was performed over a 16-fr silicon urethral catheter using 4 - 0 or 5 - 0 vicryl sutures .
the skin incision was made parallel to the long axis of the gracilis muscle of the left thigh .
the gracilis muscle was dissected from the medial aspect of the left thigh and released from its insertion .
the gmf was rotated , and its distal end was brought to the perineal area through a subcutaneous tunnel .
the muscle was then wrapped around the anastomosized urethra , and the perineal defect was filled with the rotated gmf . a suprapubic catheter was placed for urinary diversion , and two suction drains were placed in the retropubic space .
the urethral catheter was removed 3 weeks postoperatively if no extravasation was visualized on retrograde urethrography .
the suprapubic catheter was removed if the patient voided in the same manner as before the urethral injury .
if any symptoms of obstruction or poor urinary flow were present , retrograde urethrography was performed to confirm the urethral stricture .
age , body mass index ( bmi ) , a history of previous surgery , the cause of the urethral injury , incidence of pelvic bone injury , incidence of bladder injury , incidence of rectal injury , the urethral lengthening procedure , the length of urethral defect , and the time interval between the original urethral injury and the pua or between a previous urethroplasty and the pua were all evaluated for their influence on surgical outcome .
the age , bmi , time interval , and urethral defect length of patients whose surgical outcome was successful were compared with those values in patients whose surgical outcome was unsuccessful by t - test .
the number of patients who had undergone a previous urethroplasty ; the cause of the urethral injury ; the incidence of pelvic bone injury , bladder injury , or rectal injury ; and the number of patients who had undergone each urethral lengthening procedure were compared between patients with a successful surgical outcome and those with an unsuccessful outcome by using the chi - square test and fisher exact test . to identify the preoperative clinical factors that affected surgical outcome , univariate and multivariate logistic regression analyses were performed .
regression analysis results are shown as the odds ratio ( or ) and 95% confidence interval ( ci ) .
statistical analyses were performed by using ibm spss ver . 19.0 ( ibm co. , armonk , ny , usa ) .
forty - nine patients underwent a delayed pua using a gmf to treat a posterior urethral injury ( table 1 ) .
the mean age of the 49 patients was 37.213.5 years ( range , 19 to 68 years ) and the mean follow - up duration was 43.428.0 months ( range , 12 to 126 months ) .
the urethral defect was significantly shorter in patients with a successful outcome than in patients with an unsuccessful outcome ( p=0.010 ) ( table 1 ) .
there were significant differences between the two outcome groups in terms of surgical history ( p=0.036 ) and pelvic bone injury ( p=0.012 ) .
the two groups did not differ in terms of age , bmi , time interval , cause of the urethral injury , incidence of bladder injury , incidence of rectal injury , or the urethral lengthening procedure .
table 2 shows the results of the univariate and multivariate logistic regression analyses of the effect of preoperative clinical factors on surgical outcome .
multivariate logistic regression analysis revealed that only previous surgical history ( or , 0.218 ; 95% ci , 0.050 to 0.947 ; p=0.042 ) was a significant preoperative clinical factor in predicting the outcome of a delayed pua using a gmf .
table 3 shows the success rates versus the urethral defect length in patients who underwent a delayed pua using a gmf . as the urethral defect length increased , the success rate decreased . in cases in which the urethral defect was shorter than 4 cm
however , when the urethral defect was longer than 4 cm but shorter than 6 cm , the success rate fell below 40% .
no successful outcome was observed in any case in which the urethral defect was longer than 6 cm .
table 4 shows success rates according to the urethral lengthening procedure in patients who underwent a delayed pua using a gmf .
as the urethral lengthening procedure progressed , the success rate also decreased . among patients treated with urethral mobilization , corporal separation , and inferior pubectomy ,
the success rate was 48.3% . among patients treated with an additional urethral rerouting procedure , the success rate was 21.4% .
the gmf has been used previously to repair urethral strictures , in which case an epilated gmf was used , and to reconstruct the urinary sphincter in the treatment of post - prostatectomy incontinence . in these two different trials , reconstruction using a gmf was deemed feasible ; both techniques take advantage of the good blood supply to the gmf . on the basis of these results
, we previously demonstrated the therapeutic effects of using a gmf in recurrent complete posterior urethral stricture .
however , the therapeutic benefit of the gmf in pua is unproven in the treatment of other conditions , such as in cases without prior surgery or in the treatment of relatively short urethral strictures . rather than providing a benefit , the wrapping of the gmf around the anastomosed urethra may actually increase the tension on it . to identify the therapeutic effect of the gmf in pua
, a randomized controlled study is required ; before that type of study can be carried out , the selection criteria for use of a gmf must be defined .
therefore , we evaluated the preoperative clinical factors affecting surgical outcome to define the proper indications for this technique . in our study , only a history of previous surgical treatment significantly affected the surgical outcome in pua with a gmf .
patients without prior urethroplasty had a better chance of a successful pua with a gmf than did patients with a prior history . the success rate in patients who had not undergone prior urethroplasty was 55.1% compared with 25.0% in patients who had undergone a prior urethroplasty .
similarly , a previous failed urethroplasty was reported to significantly decrease the success rate of subsequent anastomotic urethroplasty .
a failed urethroplasty can cause widespread fibrosis , ischemia from impaired vascularity , and shortening of the urethra so that its length is inadequate for mobilization , thus deteriorating the patient 's surgical situation .
in contrast , one study indicated that repeat urethroplasty is feasible with good surgical results even in patients with failed previous operations .
however , the mean stricture length in this study was relatively short and the number of patients was too low to confirm the result . in another report in which repeat surgery was used to successfully treat urethral stricture after an initial failed urethroplasty , the authors suggested that various stricture management techniques including a transpubic approach should be performed .
therefore , we believe that previous urethroplasty history will adversely affect the surgical outcome . because mobilization of the anterior urethra can provide an additional 4.5 cm of elastic lengthening , and 2 cm of this length is used to trim and spatulate the two urethral ends , a urethral defect length exceeding 3 cm usually demands a progressive perineal approach to achieve a tension - free anastomosis [ 12 - 14 ] . nevertheless , this process may increase the dead space around the urethra and result in the formation of a hematoma owing to the increased chance of bleeding . a perianastomotic hematoma and a dissection of the periurethral tissue for mobilization may deteriorate the blood supply of an anastomosis and consequently result in an unsuccessful surgery .
however , the length of the urethral stricture does not predict the surgical outcome in treating traumatic posterior urethral strictures . in our study ,
the longer the urethral defect , the lower the success rate ( table 3 ) .
the urethral defect length seemed to affect surgical outcome , but the effect was not significant .
we suppose that the urethral lengthening procedure ensured a tension - free anastomosis , or that a gmf may have helped to overcome the large perianastomotic dead space caused by the long urethral defect . on the other hand , wrapping of the gmf around the anastomosed urethra
if the perianastomotic dead space is small because of a short urethral defect , there is less need to fill the dead space , or worse , tension on the anastomosed urethra may increase .
we believe that the gmf may be beneficial in most cases because perianastomotic dead space is created to a certain extent during any urethral mobilization .
although the urethral lengthening procedure did not significantly affect surgical outcome , the success rate became lower with more aggressive procedures ( table 4 ) .
in particular , the success rate was very low in cases of pua with urethral rerouting .
this technique is thought to have little benefit in pua and we agree that its benefit is limited . taken together , the preoperative clinical factors that predict a successful pua with the use of a gmf do not differ from those predicting the success of any pua .
we previously showed the therapeutic effects of using a gmf to treat recurrent complete posterior urethral stricture . however , pua with a gmf is more likely to be successful in patients without prior operative manipulation . in this study ,
the overall success rate was 42.9% , which is lower than previously reported success rates in excess of 90% [ 1 - 2,12 ] .
this discrepancy may result from the inclusion of more complex cases in our study group , such as the inclusion of more cases with a long urethral defect , a history of previous urethroplasty , and pelvic bone injury .
moreover , we did not intend to evaluate the contribution of a gmf in the success rate of pua but to evaluate the factors that predict a successful pua with a gmf . instead of the gmf , the pedicled omentum or the scrotal dartos muscle
have been used to wrap the urethral anastomosis and fill the perianastomotic dead space . in particular
a pedicled omental flap is supple and absorbs inflammatory debris , but bowel herniation has been reported with its use . in this respect ,
a gmf will be a more suitable option for obliterating the perianastomotic dead space in a perineal pua .
our study was limited by a patient group comprising rather difficult cases that were all thought to be characterized by a large dead space .
we use a gmf as frequently as possible if the defect size around the urethral anastomosis is considered large or hematoma formation is feared .
however , we believe that our results are valid concerning the use of a gmf because the gmf will be used primarily in these more complex cases .
future studies that include predictive imaging to preoperatively estimate the amount of fibrotic tissue to be removed and the size of the dead space around the anastomosed urethra should be performed ; these measurements are usually made during surgery . magnetic resonance imaging has been reported to aid in delineating the precise site and density of scar tissue in posterior urethral distraction defects .
the preoperative measurement of the amount of fibrotic tissue and the dead space size by imaging can be used preoperatively to indicate when a gmf should be used .
the results of our study demonstrate that a prior history of urethroplasty is a significant preoperative clinical factor in determining the surgical outcome of pua with a gmf .
pua with a gmf in patients who have not previously undergone surgery is more likely to be successful than that in patients with a prior history .
this observation may aid in choosing patients who will benefit from a pua with a gmf . | purposewe evaluated the preoperative clinical factors that affect the surgical outcome of posterior urethral anastomosis ( pua ) with a gracilis muscle flap ( gmf ) to determine which factors predict benefit from the use of the gmf.materials and methodsthis was a retrospective analysis of 49 patients who underwent a delayed pua with a gmf .
a successful clinical outcome was defined as achieving a peak urinary flow rate greater than 15 ml / s at 3 and 12 months postoperatively without evidence of stricture recurrence on a retrograde urethrogram or cystourethroscopy at 3 months postoperatively .
multiple clinical factors were evaluated by use of univariate and multivariate analyses.resultsthe outcome of 21 of 49 patients ( 42.9% ) was deemed successful .
the mean age of the 49 patients was 37.213.5 years and the mean follow - up duration was 43.428.0 months .
the length of the urethral defect was significantly shorter in patients with a successful outcome than in patients with an unsuccessful outcome ( p=0.010 ) .
the outcome differed significantly depending on whether the patients had a previously successful urethroplasty ( p=0.036 ) or whether they had suffered a pelvic bone injury ( p=0.012 ) .
multivariate logistic regression analyses revealed that a previous urethroplasty was the only preoperative clinical factor that significantly affected the surgical outcome in pua with a gmf ( odds ratio , 0.218 ; 95% confidence interval , 0.050 to 0.947 ; p=0.042).conclusionsa history of previous urethroplasty is a preoperative clinical factor that significantly affects the surgical outcome in pua with a gmf ; the procedure is more likely to be successful in patients who have not previously undergone urethroplasty . | INTRODUCTION
MATERIALS AND METHODS
1. Patients
2. Preoperative and operative procedures
3. Follow-up and data analysis
RESULTS
DISCUSSION
CONCLUSIONS |
attempts to define life have touched nearly every scientific discipline and have been a long - standing subject in philosophy .
even when limited to the realm of biology , the definitions of life fall short of universal agreement and remain a widely debated subject .
the practice of reductionism , whereby life is considered solely from the perspective of the genetic material , also generates controversy and complications .
many of these complications have been appreciated recently as a result of the large - scale application of whole - genome sequencing to microbes .
the specific issue we discuss here is an attempt to define the minimum number of genes or functions necessary to support cellular life .
the appeal of this subject lies largely in the fact that it represents a fundamental question in biology .
we have approached the concept of a ' minimal genome ' in a manner not unlike that taken by physicists interested in understanding the nature of the universe : we have relied on an underlying assumption or guiding force that states that a simple set of rules must exist .
the difficulty lies in finding the proper way to strip away the outer layers of complexity in order to uncover what is truly general and therefore satisfactorily describes all things .
it is our belief that some set of basic parameters and principles are common to all cellular life on this planet . on the surface , especially in the age of genomics
, it would appear that we will indeed be able to draw inferences and define commonalities .
the advent of whole - genome shotgun sequencing has changed biology in ways that we will not fully appreciate for many years to come .
advances in functional genomics , structural genomics and computational biology have also contributed greatly to our ability to make ' sense ' of the huge quantities of dna sequence data being generated .
as we begin to apply these methodologies to the question of the minimal genome , we are faced with new perspectives which emphasize that , like any good scientific endeavor , the more knowledge we acquire , the less equipped we feel to answer our original question .
over the years , a number of experiments have been undertaken to approximate the number of essential genes within various model organisms .
most often this was done through the generation of a set of mutations that were subsequently classified with respect to lethality . through extrapolation ,
one derives an estimate of the number of genes or the amount of dna required to support life .
one of the difficulties in these early studies was that the data generated were analyzed and interpreted in the absence of complete genomic sequence , and therefore without our current awareness of the relatively large amount of potential functional redundancy in most microbial genomes examined to date .
paralogs arise through gene duplication events , not by vertical descent . in some cases ,
paralogous gene families within a genome are represented by simple gene pairs , whereas other families have many individual members with varied chromosomal arrangements .
it is assumed that maintenance of most paralogous genes in a microbial genome is due to the increased adaptive potential the organism derives from their presence .
but the existence of paralogous genes in genomes greatly hampers the interpretation of genome - scale mutagenesis experiments . on the basis of sequence analysis alone ,
we are not currently able to distinguish whether two or more members of a paralogous gene family have identical or non - redundant functions .
this ambiguity forces investigators to take care to discriminate between dispensable genes and dispensable functions .
this perception was initially based on their extreme fastidiousness : growth of these microbes in the laboratory requires the addition of many extracts and complex nutrient sources .
the validity of the minimalist viewpoint would have to await advances in dna technology , such as pulsed - field gel electrophoresis ( pfge ) . among the mycoplasmas ,
initial estimates , based on pfge of chromosomal restriction fragments , indicated that the m. genitalium genome was approximately 600 kilobases ( kb ) in size .
this result was quite startling and was sufficient to attract the collective attention of clyde hutchison and one of us ( s.n.p . ) at the university of north carolina , chapel hill , and craig venter and the other of us ( c.m.f . ) at the institute for genomic research . in both instances , our instincts were to sequence the genomic dna of this curious microbe , to satisfy an urge to know what genes are present within its freakishly small genome . ultimately , this curiosity provided the motivation for determining the complete sequence of the 580 kb m. genitalium genome ; it encodes a mere 480 proteins .
the completion of the m. genitalium sequence was followed by the determination of the complete sequence of m. genitalium 's closest relative , m. pneumoniae , just one year later .
the m. pneumoniae genome has approximately 685 identified open reading frames ( orfs ) . with the recent report of the ureaplasma urealyticum genomic sequence , and the expected completion of three other mycoplasma species in the next year or two ,
the high density of sequences from the mycoplasma lineage will provide great potential for understanding the reductive evolution of genomes .
the evolution of mycoplasma genomes is thought to have occurred through a series of dna deletions from larger gram - positive ancestors .
the fact that mycoplasmas are predominantly found in association with humans , plants or animals suggests that their diminutive genome size is a direct reflection of their parasitic lifestyle .
given our current level of sampling of microbes , the smallest genomes among free - living organisms appear to be those of pathogens .
it is not known precisely what selective pressures generate minimal genomes , but it is conceivable that an organism begins the process through the acquisition of new functions that enable it to parasitize a host cell .
this newly acquired ability has a dramatic impact on the selective value of many genes present in the genome . under relaxed selection
the numerous genes required to synthesize all 20 amino acids , and to synthesize cell wall components , as well as genes encoding enzymes of the citric acid cycle and the majority of all other biosynthetic genes have been lost in these two organisms , presumably because they have evolved in such a way as to acquire these products from their host in vivo .
ultimately , a definition of a minimal set of genes required to sustain cellular life requires an agreed - upon definition of what is living and what is not .
gene loss in m. genitalium has come at some expense in terms of fitness in the laboratory setting .
( m. genitalium is able to grow in culture in a cell - free environment and therefore meets the definition of free - living . )
this is evident in terms of doubling time when comparing m. pneumoniae ( 680 orfs ) to m. genitalium ( 480 orfs ) .
the doubling time of wild - type m. pneumoniae in culture is approximately 6 hours compared to about 12 hours for m. genitalium
. experiments currently being performed in our laboratory aimed at introducing a saturating number of mutations into the m. genitalium chromosome indicate that the fitness of the cultures is gradually reduced upon successive rounds of mutagenesis ( unpublished observations ) .
it is anticipated that at some point the ability to maintain active cultures will become severely challenged . in many ways
it seems plausible , if not likely , that the fitness reductions observed through loss of gene function could be compensated for through changes in the growth media ( provided by an omniscient microbial physiologist ) .
this point emphasizes the intimate relationship of environment and a minimal genome definition , and the difficulty of ever defining a minimal genome .
a minimal cell would require an ' ideal ' environment , free of any selective pressure .
it may be instructive to consider all genomes as being comprised of two types of genes .
the first set includes those that confer the core set of functions required for basic cellular processes . by definition
for example , it may be safely assumed that all cells require an ability to replicate their dna , produce message through some basic transcription machinery and translate that message to produce protein .
in addition , all cells will have some requirements for energy production and transport of raw materials across the cell membrane and maintenance of cellular homeostasis .
it is well established that some features of a genome are selectively neutral and others maladaptive .
if we ignore these cases , the remaining genes in any genome are presumably those that confer a specific selective advantage to an organism within the environmental niche it occupies .
these functions may be dedicated to immune - response avoidance , expanded metabolic and biosynthetic potential , or stress - management skills , to name a few .
it is assumed that the number of core functions required by any cell is relatively similar .
we know that eubacterial genomes range in size from less than 0.6 mb to greater than 9 mb .
the number of accessory or adaptive proteins maintained in various genomes is therefore highly variable and in fact indicates a lot about the environment and selective pressures each organism faces .
according to current evolutionary thinking , the environmental niche occupied by a microbe affects the set of genes retained in its genome .
this idea gives rise to the possibility that one day we will be able to derive information about the microenvironment that an organism lives in through an examination of its gene complement .
it is meaningless to refer to a gene as being essential or dispensable without an accompanying statement that defines the environment or growth conditions in which the categorization was made . for all of these reasons , discussion of minimal genomes often becomes a matter of semantics and definitions .
microbial genomes are no longer seen as static entities evolving through the accumulation of point mutations and infrequent chromosomal rearrangements .
gene acquisition and gene loss may be far more prevalent in microbes than was previously appreciated .
once resident in this new environment , the selective value of each gene in the genome is necessarily redefined and may allow the loss of previously useful genes .
gene loss is much more likely to be a one - way street in the course of genome evolution . despite the presumed prevalence of horizontal gene transfer in nature ,
the number and types of functions that have evolved in the history of cellular life is far greater than the number of functions ' sampled ' by any single organism .
mycoplasmas have dispensed with many metabolic functions and have retained very few genes required for transcription , repair and recombination , for example .
the loss of any given gene from a genome may limit the number and types of losses that are tolerated in the future : gene loss has consequences for the future evolution of the remaining genes in the genome .
for example , it is known that the repertoire of transporters in mycoplasmas is fewer than in most microbes .
it has been speculated that the reduction of transporters has been compensated for by a broadened substrate specificity of the retained transporters .
it is also possible that m. genitalium and m. pneumoniae have been able to shed many of the genes involved in transcriptional regulation by shifting the burden of regulation toward translational processes ( and s.n.p .
an examination of the genes present in the m. genitalium genome can fool one into thinking that the organism has found the minimal solutions for basic cellular needs .
this is of course very unlikely to be true for every pathway and every functional category within the cell .
it is anticipated that future genome - sequencing efforts and functional characterization of genes will identify novel and more economical solutions for various cellular functions compared to those present in the genome of m. genitalium . on the basis of these arguments ,
it is evident that an experimentally defined minimal genome is lineage - specific , stating little , if anything , about what a minimal gene set might be for another phylogenetic lineage .
this study derived a minimal genome by comparing what at the time were the only two fully sequenced microbial genomes ( haemophilus influenzae and m. genitalium ) .
an all - against - all whole - genome comparison was performed in order to identify genes held in common by both organisms .
the reasoning behind this comparison was simple but powerful and starts with a straightforward assumption : genes conserved across large phylogenetic distances are likely to be essential .
thus , if one compares two genomes that have diverged from a common ancestor a very long time ago ( 1,500 million years ago in this example ) , the genes in common will be highly enriched for those that carry out the most basic cellular functions common to all organisms .
the application of this idea resulted in the definition of a minimal gene set consisting of about 250 genes . while the starting assumptions of this analysis appear reasonable , the two organisms in question are both human parasites and so may have too much in common , with regard to environmental selective pressures since the time of divergence , to be representative of genomes as a whole .
it was the speculation of at least these authors that the results would also be improved if one were to repeat the analysis at a later time when more microbial genomes could be sampled , representing a more diverse phytogeny ( see below for further discussion of this issue ) .
soon after the report of the m. pneumoniae genome sequence , a comparison of the m. pneumoniae and m. genitalium genomes was performed .
a comparison of amino - acid sequences among orthologs in m. pneumoniae and m. genitalium reveals an average of 65% identity , suggesting a much larger distance between the two genomes than was previously thought .
approximately six major chromosomal rearrangements together with many tens of chromosomal deletions account for the large - scale differences between the two genomes .
these facts also support the idea that there is substantial evolutionary distance between the two genomes .
the overall conservation of gene order ( synteny ) is remarkably high over very large distances in many cases , however ; this observation is consistent with the notion that the two species are very close relatives .
an attempt to explain this apparent dichotomy - divergence of individual sequences but close relationship of gene order - has been provided by the possibility that mycoplasmas have a significantly increased mutation rate relative to most bacterial species ; this is presumably due to the loss of several dna - mismatch repair systems and mutator loci .
virtually every gene in the m. genitalium genome has an ortholog in the m. pneumoniae genome .
given that these genomes were formed through reductive evolution from ancestors with larger genomes , it may be reasonable to assume that some , if not most , of the differences between the two genomes reflect gene loss in m. genitalium that occurred after divergence from their common ancestor .
it is therefore notable , if not downright bizarre , that the net gene loss that has taken place in these genomes - after speciation - is unidirectional .
this is counter to expectations . given that the two organisms reside in different host sites ( the lung versus the urogenital tract ) , and are presumably under different selective pressures , one would expect gene loss to have occurred differentially in the two genomes : some genes , lost from m. genitalium , would be present in m. pneumoniae , and vice versa .
perhaps the asymmetry of gene loss indicates that the environments these two human parasites occupy are substantially different .
it is conceivable that m. genitalium has adapted to a new environment with a much reduced selective pressure compared to m. pneumoniae .
one way to explain the gene - content differences of these genomes is to speculate that m. genitalium has acquired the status of an intracellular pathogen .
the recent report of a third complete mycoplasma genome sequence , that of u. urealyticum ( 0.75 mb ) , has allowed further comparative genomics of minimalism .
the unusual genomic relationship of m. genitalium and m. pneumoniae does not apply to the 613-orf genome of u. urealyticum . in this case , the findings are more as expected : unequal gene loss has occurred between u. urealyticum and the other two mycoplasma genomes .
phylogenetically , u. urealyticum is more distantly related to m. genitalium and m. pneumoniae than the latter two are to each other .
a comparison of the more distantly related genomes has allowed greater insights into mycoplasma evolution to be made .
only 324 genes are shared between u. urealyticum and m. genitalium ; and m. genitalium has some genes ( 74 ) that are absent from u. urealyticum . on the surface , it might be tempting to assume that these 74 genes are dispensable in m. genitalium .
but as the authors point out , 10 of these 74 encode functions involved in energy production , in this case glycolysis .
since u. urealyticum has evolved a unique means of generating atp from urea hydrolysis , there has been a wholesale substitution of glycolytic genes for this system .
another notable feature of the u. urealyticum genome sequence is the apparent lack of the chaperone proteins groes and groel , as well as the cytokinesis protein ftsz .
it is unclear whether these functions are truly absent from this mycoplasma , or alternatively whether the functions are being carried out by proteins with sequences unlike previously annotated proteins of the same function .
irrespective of which possibility is more likely , this finding emphasizes a strong predisposition held by almost all molecular biologists , namely to define expectations on the basis of what is clear and evident in the most well - studied model microorganisms , escherichia coli , bacillus subtilis and saccharomyces cerevisiae .
the wealth of knowledge that these model systems have brought to our understanding of cellular mechanisms and general principles is undisputed .
perhaps for the first time , since the advances in genome sequencing we are perceiving that although these models are very useful , they are not in every case representative .
this statement should not be thought of as blasphemous , but rather as an obvious point , considering the large number of specialized functions that have evolved in microbes .
whole - genome analysis and gene annotation is intimately linked to those precious few genes for which actual functional experiments have been conducted in the laboratory .
it is still true that most functional assignments that have a foundation in laboratory experiments are limited to the great model systems .
our experimental approach for defining a minimal genome identified , on a rather large - scale , transposon - insertion events in m. genitalium and m. pneumoniae that , due to their location on the chromosome and presence in viable cells , are likely to have disrupted the activity of a gene function that could be considered dispensable to the cell . despite the technical difficulties associated with working with these organisms , and the lack of genetic and reverse - genetic tools , the fact that natural selection had already been working on them in a natural ' minimal genome project ' for millions of years made them a very attractive model system .
it is fair to say that before initiating this study we probably did not have a strong set of expectations as to the number of genes that would be dispensable in m. genitalium .
it would not have been completely surprising if the genome of m. genitalium harbored only a few dispensable functions or several dozen .
the fact that we were approaching a question for which little insight or predictive power was available made all the more important the use of good control experiments : we needed to gain confidence that the transposon insertions were capable of reliably identifying dispensable genes .
we made use of the fact that the m. genitalium genome is a proper subset of the m. pneumoniae genome as a rationale for the assumption that the additional 210 genes present in m. pneumoniae should be dispensable in the laboratory setting .
we considered the m. genitalium genome as containing two types of genes , essential and dispensable , whereas the m. pneumoniae genome contains three types of genes : shared ( in m. genitalium and m. pneumoniae ) essential genes , shared dispensable genes and dispensable pneumoniae - specific genes .
we predicted that the proportion of recovered gene - disruption events should be greater in the pneumoniae - specific portion of the genome than in the shared portion of the genome .
we did in fact recover approximately six times the frequency of insertions from the pneumoniae - specific portion of the m. pneumoniae genome .
we used statistical analyses based on the poisson distribution and extrapolation of our data set to estimate that between 180 and 215 dispensable genes exist in the m. genitalium genome .
this somewhat startling result raises several interesting questions . how are we to interpret the findings
it is important to mention first that an examination of the m. genitalium genome reveals a lack of any recently evolved paralogous gene families .
unfortunately , as stated before , it is not possible to be certain whether existing genes with paralogous relationships within the m. genitalium genome carry out identical functions .
the accuracy of the assumption that there are no redundant functions in the m. genitalium genome determines the certainty with which we are able to interpret our disruption data .
the natural next question to ask is whether the catalog of dispensable functions identified in ' a one gene at a time ' study could be tolerated in combination , and if so , to what extent ? as mentioned before , we have initiated a saturation mutagenesis of the m. genitalium genome .
while mutagenesis is still ongoing , it appears unlikely that any cell in the population will be recovered harboring more than 100 functionally null genes ( s.n.p .
there are two reasonable explanations for this apparent discrepancy between the ' gene by gene ' approach and the combinatorial approach .
first , some genes in the m. genitalium genome do carry out redundant or partially redundant functions , so while single - gene disruptions are tolerated , the combination of two disruptions is not . the second , and probably more important , explanation is that dispensable genes , especially in m. genitalium , are not likely to be lost without some discrete reduction in the overall fitness level of the organism . in this regard , it maybe appropriate to consider dispensable genes to be associated with a quantifiable fitness contribution to the cell in any given environment . if one considers the combination of genes in the wild - type m. genitalium genome as providing a global fitness value above some minimal threshold for survival , then ultimately the minimal genome will be obtained by sequential disruption of genes that individually contribute the least to the fitness of the cell .
viewed in this manner , it seems that minimal genomes are more an interesting mathematical problem or , worse yet , a matter for an accountant 's spreadsheet , than an issue that can be defined experimentally .
we would argue that it is perhaps more important to attempt to grasp the quantitative selective value that any gene contributes to the fitness of the organism than to attempt to define a minimal gene set .
what do the transposon results tell us about the overall architecture of gene functions in a genome ? it is assumed that the activity of individual enzymes within the cell has an impact on other pathways and processes , giving rise to a view that the cell is an intricate sensing system that is responding not only to the outside environment but also to its own actions .
this ability to modulate is especially important to bacteria , where waste is not considered a virtue .
but the fact that we were able to disrupt so many gene functions in a minimal cell suggests something subtle about the nature of gene functions within genomes : it suggests that there is in fact a lack of interconnectivity among gene functions .
it appears that pathways evolve which in many cases show interdependency , and epistasis is a well - defined genetic idea that speaks directly to the functional interdependency of genes .
but among dispensable functions , our ability to recover a living organism bearing disruptions of nearly one - third of its gene complement lends support to the view that interdependency beyond epistatic relationships may be uncommon . if we examine gene disruption data in terms of the functional roles of the genes in the cell , we are able to gain some additional insights about minimal genomes .
the group of genes for which we recovered by far the largest number of disruptive insertions is the group of unknown function ( figure 1 ) .
this is in contrast to the number of insertions tolerated in genes involved in translation , for example .
the functional group for which the highest proportion of putative disruptions were identified was the genes encoding cell - envelope proteins .
a precise interpretation of this result is not possible without extensive experimentation , but it may serve to highlight the previously mentioned idea that the definition of the dispensability of a gene is dependent on the environment . it is conceivable that the group of cell - envelope proteins plays a significant role in the context of a host infection but is of relatively little value to the cell in laboratory growth media . by examining the data shown in figure 1 , we can see that not all functional roles in m. genitalium are equally dispensable ; some functional systems are closer to minimal than others .
one can obtain a visual sense of the functional distribution of genes in a minimal cell by examining the relative proportions of genes in each functional group ; it is interesting that nearly two - thirds of the cell is dedicated to the translation of message into protein and genes for which we do not yet know the function or cellular role .
the genes of mycoplasma genitalium categorized according to function and whether or not they were disrupted by transposon mutagenesis .
the fact that an estimated one third of the essential set of genes in this minimal genome are of undefined function is an important result that has at least two potential interpretations .
first , it draws dramatically into question a basic assumption held by many biologists that the fundamental mechanisms and functions underlying cellular life have for the most part been identified and well characterized .
if approximately 100 genes in the simplest functioning cell are of unknown function and are essential to basic cellular processes , this assumption becomes quite dubious .
an alternative perspective is that many of the 100 genes of unknown function in the minimal gene set encode well - characterized functions in e. coli , but are simply not recognizable as orthologs in m. genitalium .
it is likely that both explanations are at least partly valid , but the conclusion remains the same : we have much work to do before we can claim to have a clear understanding of even the simplest cell and its functions .
since the time of the work on the comparison of the h. influenzae and m. genitalium genomes , many new microbial genomes have been sequenced , representing many lineages of eubacteria , archaebacteria and eukaryotes . in theory ,
this additional data set should allow the comparative approach to more precisely define a minimal gene set .
recently , 21 completed genomes were compared , this time employing the more sensitive gene - comparison tool clusters of orthologous groups of proteins ( cogs ) to find orthologs common to all genomes .
cogs are defined by a grouping of at least three proteins from distantly related organisms that are more similar to each other than to any other gene in their respective genomes . in this way , cogs define orthology groups and ancestral relationships .
the added sensitivity derived from the cogs analysis arises because cogs are not dependent on blast cutoff scores and arbitrary sequence - relatedness criteria ; genes with relatively unimpressive sequence identity can be placed into an orthology group .
first , 55 - 83% of proteins encoded by bacterial and archaeal genomes can be placed into discrete cogs , suggesting that many genes present in bacteria and archaea are highly conserved .
very few genes appear to be universally , or nearly universally , conserved in all 21 organisms analyzed , however , and it was this criterion that was used in the original analysis for defining the essential gene set of a cell . in this expanded study
clearly , this number of genes is insufficient to serve as a satisfactory answer to the question ' what is the minimal number of functions required for cellular life ? ' was there something wrong with the starting set of assumptions made in the comparative approach ? among the possible interpretations of this troubling result ,
one is that there is something fundamentally faulty about cogs analysis , which is unlikely .
a second possibility is that many genes and functions arose a very long time ago .
these ' ancient ' genes would therefore have diverged to such a great extent that they are unrecognizable by any sequence - relatedness algorithms . a third , and extremely interesting
, possibility is that many gene functions have evolved independently more than once since the beginning of cellular life on the planet .
the term that has been used to describe this occurrence is non - orthologous gene displacements ( nods ) .
there is scattered evidence for this idea for various genes found in nature , but the cogs analysis with 21 genomes places a potential magnitude on the phenomenon .
the implications of this idea , if correct , are significant but beyond the scope of this article . despite the arguments put forward here to highlight the complications and potential inappropriateness associated with attempts to define minimal genomes , it is clearly instructive to try .
the analogy we would make is with the knowledge we gain from sequencing microbial genomes . we learn a lot by sequencing each one , but far more by comparing the similarities and differences of many genomes .
similarly , we have learned much about genome evolution and the gene functions required for cellular existence - and hopefully will continue to do so - and anticipate a future ability to compare the results of similar experiments from models other than ' the minimal genome ' .
the discussion here highlights the importance of generating functional data for genes in organisms from diverse lineages
it is unlikely that we will exploit this resource efficiently until many more microbial model organisms are developed and studied at a similar level to e. coli or yeast .
we are forging ahead in genomics at unprecedented speed , with too few frames of reference , and there is a risk of becoming lost in a sea of sequence data without a functional framework , unless we ensure that functional analysis does not lag too far behind .
we believe that the concept of the minimal genome is a useful tool in attempting to organize our thoughts about gene function - even though we may never , in practice , be able to reach a definition of a minimal gene set that is applicable to all types of organism . | what is the minimum number of genes or functions necessary to support cellular life ?
the concept of a ' minimal genome ' has become popular , but is it a useful concept , and if so , what might a minimal genome encode ?
we argue that the concept may be useful , even though the goal of defining a general minimal genome may never be attained . | None
A model organism with a minimal genome?
Can the minimal genome be defined?
How does a minimal genome arise?
The theoretical minimal genome
An experimental approach
Comparing many more genomes |
opening of the asylum in hadzi sinan tekke in sarajevo in the eighteenth century practically meant the beginning of an organized health services in bosnia and herzegovina ( 1 ) .
the first hospital was the one in sarajevo , named vakuf hospital , built in year 1866 .
austro - hungarian administration in our country begins to raise the health care level for population in all fields .
a new era of health services employed specialized medical personnel from austria , hungary , czech republic , italy and other countries , carried out a number of health care reforms , adopting legal acts etc .
( 1 , 2 , 3 ) following the development of infectology in b&h can perceive the overall presentation of the health development in bosnia and herzegovina following the rule of the ottoman empire as the oldest form of organized documented history of our region ( 5 waqf hospitals were founded during the time of sheriff topal osman pasha governing in the period since 1861 to 1869 ( sarajevo , tuzla , mostar , banja luka and travnik ) and during the austro - hungarian period ( in bosnia and herzegovina was established state hospital in sarajevo , 6 regional , 9 district and 22 municipal hospitals for the population of 1,890.440 . in 1921st
the state hospital in sarajevo constantly recruited personnel for other institutions and services in b&h , including infectology services in health institutions in b&h , in which the most significant changes , the fastest and best quality development of health care in b&h occurred .
monitoring the development of infectology is possible through a number of legal acts , records and decisions in medical documents and exhibits which are kept in the fund of the state archives archives of the federation of b&h in sarajevo , among which are worth mentioning ( 1 ) : measures to prevent the appearance of cholera and measures to be taken during the epidemic in bosnia and herzegovina from 1886 .
founding of quarantine for those returning from the hajj to mecca and medina in 1891 .
construction of barracks for the acceptance of patients in case of cholera or other epidemics in 1892 .
report of the police directorate in sarajevo on the status of communicable diseases in november and december of 1917 .
( 1 ) according to official data from 1875 the overall mortality of the population in b&h amounted to 24.4 per thousand , infant mortality was 261.8 per thousand .
poor social , economic and health situation has favored the occurrence of several large outbreaks of infectious diseases cholera ( 1876 , 1895 , 1912 . ) , smallpox ( 1880 , 1888 , 1889 , 1890 , 1901 . ) , tuberculosis ( 1908 mortality rate was 12.5% of the total mortality rate ) , then in year 1901 in b&h was recorded 214 cases of leprosy , and 351 case of trachoma , and a large number of cases of other diseases such as pellagra , scurvy , etc .. during their rule b&h has recorded a total of 23 000 cases of syphilis .
it should be noted that of all infectious diseases the most attention has been given to venereal diseases , because they were responsible for as much as 17.52% of patients who were treated in hospitals during the the austro - hungarian rule ( 1 ) . although these diseases are now separated and processed at the dermatology clinic
sector for venereal diseases , it should be noted that , in the past , they were treated as infectious diseases , as well as typhoid fewer , tuberculosis , cholera .
the fact is that the major part in history of infectology development can be followed through observing frequency of syphilis , leprosy , tuberculosis and ways of their treatment .
also , there were widespread variety of endemic and pandemic for the simple reason that it was an insufficient number of healthcare facilities and insufficient qualified health personnel ( 1 ) .
for example , in the state hospital in sarajevo ( founded on july 1 , 1894 , with 4 divisions : internal , surgery , gynecology and obstetrics and dermatosiphylitic with a total of 204 beds ) , there was no pavilion for infectious diseases , so for purposes of this in 1895 they built a shack by the system of doctor koch with 22 beds , enclosed with separate entrance and completely isolated in a remote area of 40 m from the rest of the hospital .
this ward serve for the required isolation of patients suffering from acute infectious diseases : scarlet fever , diphtheria , cholera , intestinal typhus and other infectious diseases .
they built one new floors due to increased demand ( for a total of 67 beds ) .
tuberculosis patients were initially placed in the same room with the other internal ( or infectious ) patients .
the tbc pavilion was built in 1923 , for better care and long - term treatment with generous food , as well as for isolation of patients with open tb from healthy family members .
initially , the department was managed by foreign doctors , nun s nurses and paramedics . in 1897
for patients suffering from leprosy , in 1897 a special leprosaria was built and put into function , only one in europe in that period , which caused the attention of european scientists , so in 1903 in sarajevo , was organized the german - bosnian congress of dermatovenereologists . in the treatment of infectious diseases for which was not known etiologic agents , but only the clinical features , the popular form of symptomatic therapy were in form of balmcoddle , as those against children rashes , and various types of crusts and scabs itch .
recipes for some of these drugs among the people of this region are still in use as an effective means of treatment .
this popular treatment is developed in families , so they were known families humo in mostar and family kalajdzic , which kept the secret of recipes for their balms . in 1897 the state government decides on free treatment of favus , leprosy , trachoma , and venereal diseases and these diseases will be treated in third class hospitals , for free .
researches were performed on infectious diseases on the state hospital patients with even certain fundamental knowledge about syphilis , leprosy and favus in world literature , it originated from the experience of physicians in our region .
for example , doctor geza kobler ( manager of the state hospital ) did research on typhoid fever , and doctor leopold gluck on erythema multiform exudative .
notwithstanding the increase in personnel , the country was still ravaged by epidemic of cholera , typhoid fever , recurrent , malaria , etc .
after the first world war in 1918 , b&h was faced with even greater poverty , and doctors who were mostly foreigners leave the country and outbreaks of communicable diseases ravages leaving serious consequences .
in addition , economic and cultural backwardness and evident poverty of the population ( that are favorable for bond related ) are often caused outbreaks of infectious diseases , their repertoire was inexhaustible .
the highest mark in the creation of nuclei of infectology department from this period in sarajevo and bosnia - herzegovina gave a head doctor hamdija karamehmedovic md ( trebinje , 1883sarajevo , 1968 ) , extremely versatile and educated physician , but also the organizer of health services in b&h ( 2 , 3 ) .
as the best student of the vienna medical school in 1909 ( c - ring winner for his success during the studies ) is coming to sarajevo and start to work 1910 as a doctor in state hospital . according to the established principle he practiced in all departments of the state hospital , but he focused on organizing infectology service , because he recognized the importance of preventing and treating infectious diseases which covered a large percentage of the overall pathology . in 1918 with the title of chief physician , he established department of infectious diseases .
he spoke seven languages and followed all available medical literature , had excellent organizational skills and in 1914 , as a member of the health council of the provincial government in sarajevo proposed law on health care institutions and gave the idea to establish the sarajevo school of medicine .
if gavrilo princip did not make a shot and the first world war started the idea of doctor karamehmedovic would be realized .
his organizing skills enabled him the position of minister of health of the kingdom of yugoslavia in year 1921 .
after the dismissal of this function , he returns to the position of head of the department of infectious diseases , state hospital in sarajevo from 1924 to 1931 and he became governor of the state hospital .
beside other great doctors , andrija stampar and milan jovanovic - batuta he remained the most noted figure in medical science and profession in the first five decades of the 20 century .
unfortunately , seen as all great men , ended his career as a librarian in the library of the central public health institute in sarajevo .
he left an indelible mark there too , because alongside the professor sacir sikiri , he translated from arabic , turkish and persian languages over 40 major papers that were used in medical education also in the later period .
the best - known work which was translated by these two giants is a famous excerpt from ibn sina s avicenna s code of medicine , called .
it presents the basic principles of diagnosis and treatment of the golden period of arab medicine .
after chief physician hamdija karamehmedovic department of infectious diseases ( which was expanded and adapted ) was governed by chief physician ivo pehovac ( leading it until he died from typhus ) , then chief physician asaf sarac , who was the first chief of clinic of infectious diseases , founded in 1946 , when on the 16 ofnovember officially opened the first medical faculty in bosnia and herzegovina ( sarajevo 1946 , in tuzla 1976 , in banja luka 1978 , in mostar 1994 , foca in 1997 . ) . in 1923 within the general state hospital of sarajevo was founded the fixed bacteriologic station . as part of this and hygienic bureau
the period between two world wars and during the second world war was rather terse with data on the development of infectious diseases in sarajevo and b&h . with the establishment of the medical faculty in sarajevo in 1946 infectology
as a health care branch is experiencing an expansion in professional and academic terms , so the clinic of infectious diseases of university hospital clinic in sarajevo ( renamed the general state hospital to university hospital clinic ) becomes the basis for teaching the subject of infectious diseases .
first chief became a professor blagoje djordjevic , md , phd ( in the period from 1949 to 1970 ) , and after him this clinic led by : professor mohammed teftedarija , md , phd ( 1971 to 1983 ) , professor dragan djordjevic , md , phd ( 1984 to 1990 ) , professor sadzida telalbasic , md , phd ( from 1991 to 2001 ) , professor sajma krkic - dautovic , md , phd ( 2005 - 2008 ) , and from 2009 to the present clinic is managed by professor nada koluder - cimic , md , phd .
the first heads of institutes and clinics for infectious diseases on the medical faculties in tuzla and banja luka were : professor sava sibalic , md , phd ( clinic in tuzla was founded in 1976 ) , professor dragan djordjevic , md , phd ( clinic in banja luka , founded in 1978 ) , professor ilija kuzman , md , phd ( clinic in mostar , founded in 1994 ) .
clinic of infectious diseases in sarajevo has experienced many radical organizational and functional changes , from its founding as a special clinic in the university hospital in sarajevo in 1946 , especially in the organization of the university clinical center , 1974 and the clinical center of sarajevo university , 1992 at the clinic numerous professional and skillful staff worked , who also performed as a regular activity of the infectious diseases of the tertiary level , but also the academic part the training of students of biomedical engineering , university of sarajevo .
too much space would be required to describe all the important events and participants in the work and the provision of hospital and other forms of health care in the field of infectious diseases .
let s look at the latest and most impressive ones . since its establishment , the capacity of infectology clinic in sarajevo was increased . upon completion of the second world war , with the increasing number of beds and number of specialists and other staff , the organization of clinic changed .
admission triage clinics was formed and besides it , also three divisions with 6 sections .
first division ( sections i-1 , i-2 ) hospitalized patients of intestinal infectious diseases , tropical diseases , hemorrhagic fever , vague febrile illness and some rash children s diseases ( scarlet fever , immunization ) .
second division ( sections i-3 , i-4 ) hospitalized patients from infectious hepatitis , other infectious diseases of the liver and urinary tract .
third division ( sections i-5 , i-6 ) treated patients suffering from neuro - infectious and respiratory diseases . later in 1976
the modern intensive care units and intensive care ward has been founded with two apparatuses for directed mechanical ventilation , defibrillator , aspirator , noninvasive monitoring , oxygenation , chest x - ray machine , and transportable machine for plasmopheresis .
doctors and more nurses are educated in the infectology intensive therapy in ljubljana , zagreb , belgrade , and the first head of the listed department , ass .
was a counseling ceter for infectious intestinal disease with cabinet for rectoscopy and fecal diagnosis .
doctors of infectious disease with a subspecialty in gastroenterology in the seventies were the educators of endoscopic skills for internists and gastroenterologists of the future .
within infectious clinics operated biochemistry laboratory , which founded the graduate engineer on chemistry , franko cetinic , as a professor .
it is from this laboratory , in the early 70 s , that he formed the central biochemistry laboratory for the entire hospital , which now operates under the clinical center of the sarajevo university .
in addition , the department acted within the bacteriological laboratory , which was responsible for the entire hospital . later , inside the infectious clinic was focused on the diagnosis of anaerobic infections , led by professor marija miladinovic md phd . in nineties it merged with the institute of microbiology . from previous laboratory , which belonged to infections diseases clinic , the others are to act microhematocitological laboratory with a reduced number of laboratory technicians , who have made cytological analysis of csf in neurological and neurosurgical clinic .
since 1985 within this laboratory works also the referral laboratory for the diagnosis of viral hepatitis and hiv / aids , which leads untill the war chief physician dubravka ungar md phd , and during the war ass .
professor danko lupi md phd until the decision of the director general that complete laboratory with high medical personnel move and merge with the institute of microbiology .
infectious disease clinic in sarajevo was the first in the former yugoslavia which formed the out - patient ambulatory infectology service that extends to three ambulances but organizationally within the hospital , where specialists in infectious diseases 5 - 7 of them worked .
work was performed in clinics , but the service also organized home visits where possible . during the war , because of reduced capacity and staff , the clinic was reorganized .
the departments where abolished , there were only two working departments , intensive and semi intensive unit care , and clinic ( one located at the clinic and the other two in primary health care centers center - vrazova
after the war , these clinics are canceled , but long acting infectological clinic in primary health care center
stari grad was , but due to chronic lack of personnel or the inability to organize work , this was also abolished . after the war department of clinic
is reorganized several times , the last time in 2008 and as such rganization exist today .
the medical profession requires immediate professional training , so the subspecialists where recruited . at the beginning of the aggression on b&h at the clinic for infectious diseases
were 34 doctors , 26 specialists in infectious diseases , 7 subspecialist ( gastroenterologist , hepatologist , two clinical pharmacologists , immunologist , cardiologist , medical informatics ) , 14 chief physicians , 6 residents , 2 general practitioners on training , 19 senior nurses , 86 secondary nurses , 10 administrative employees and 24 unskilled workers .
the clinic department had two professors , three assistant professors , a total of 6 doctors of medical sciences , 5 masters of medical sciences , 2 senior assistants , and 6 assistants .
some of them went to the side of the aggressor , the other in exile , the third was retired . at the beginning of the war in the title of a specialist clinic
received three doctors exiled from other medical centers , three are residents , 3 pre - war residents , who have a specialist examination in the war .
two infectious disease specialists go to the clinic for infectious diseases : ass professor bakir kapetanovic md phd , 1993passed the specialist exam in internal medicine and moves cardiology part of clinic and chief physician zlatko cardaklija md msc moved to the ministry of health of sarajevo canton .
combat operations in surrounded sarajevo , lack of energy , water , food , removing the waste have led to two major outbreaks of infectious intestinal diseases , including highly malignant form of unexpected dysentery and viral hepatitis , and extremely increased number of patients with hemorrhagic fever among the soldiers of the army of b&h .
due to limited space in the new hospital building , at the time of the epidemic , the clinic outsourced some parts of the premises of the clinic for endocrinology and the psychiatric clinic . as a result of
a lack of necessary clinical space decreased the length of hospitalization of these patients , which resulted in the occurrence and recurrence of protrached forms of intestinal infections , especially dysentery , infectious hepatitis , and botulism . in the war of unexpected infectious disease
was first encountered in one child with a malignant form of diphtheria , tetanus , but the expected occurrence is absent due to proper immunization before the war , and wartime .
actualized as posttraumatic / postoperative meningitis with polymicrobial flora , including gram - negative bacteria , other bacterial meningitis , sepsis , osteomyelitis of long bones , spine , skin infections and soft tissue infections , nosocomial infections , herpetic encephalitis , polyradiculoneuritis , leukoencephalitis , aids .
so the staff has been working under difficult circumstances , unseen till then and not experienced , but those who remained at the clinic and the city , with great efforts and sacrifices were conducted its core business , and more . at the beginning of the war , ass .
professor danko lupi md phd being wounded by shrapnel in action of withdrawing from primary health care center ilidza colonoscope machine .
alija drnda , md , nurse dusanka babic risks their lives saving from the jarcedoli one of our staff .
jasna topalovic - cetkovic , md and nurse mirsada kucinovic go through the famous tunnel in butmir to retrieve the donated medicines for the clinic . in the winter of 1993/1994 rounds with the aggressor destroys the lateral position of the wall on the second floor and springs into the room so called ten , but fortunately there was no room in the patients .
springs into medical rounds , which are driven into the floor , but not exploding . in june 1995 , citing a shell into the so - called six , the order goes through a series of rooms full of patients .
the consequence was three deaths of patients , major trauma for the present and virtually incapacitated the second floor for the reception of patients . during the war ,
staff trough self education bring us a doctor guides for diagnosis and treatment of patients . at the clinic until the end of 1993 operates a library by master lybrarian jelena koprivica .
establishment of the institute for scientific research fused to the remaining staff together with the valuable library fund in which we were known .
our 3 specialist trainee pass the exam , they receive the three doctors on specialization , promote two chief physicians , participates in the work of two naval symposium , evaluate the war results , diagnostic and therapeutic approaches from which to extract the essence of the first congress of surgeons .
after the war , the clinic was in late 1997 restored with the help of the european community humanitarian office echo cooperazione internazionale movimondo . since the beginning of the war in 1992
was introduced systematization of the clinics that simplify the organization of the clinic in four departments , which has been retained to 2004 . that automatically implies the impossibility of restoring the frame .
the period since 1996 2004 is once again characterized by a large dissipation of the frame so that the total work is organized with only 11 specialists . at the same time work at the clinic
is hampered by reducing the working space , the elimination of outpatient clinics in the old city , omer maslic and ilidza , the equipment was obsolete , and newer related to work on hepatitis and hiv , stripped and connected to the central laboratory of the clinical center .
infectious disease doctors inaugural conference organized by the b&h association of infectious diseases specialist in 1994 , which continued organized activity of infectology profession .
after the last war , new personnel changes started in terms of arrivals of refugees , from other medical centers , specialization , and retirement , to other jobs in the city and abroad .
the main activity of the clinic is the professional , scientific and educational fields of infectious diseases .
clinic performs admission , diagnosis and treatment of patients with infectious diseases through the hospital , then , outpatient clinic , cabinet , and advisory services .
clinic for infectious diseases have 7 departments with the appropriate wards , and counseling for sexually transmitted diseases , counseling for antibiotics , counseling for prevention of nosocomial diseases , counseling for the infection of pregnant women and prevention of congenital anomalies , cabinet of intestinal infection with endoscopy , section for hepatology and diagnostics , neuroinfections cabinet , cabinet for aids and immunodeficient states and cytological laboratory .
today the clinic have 21 doctors , 18 infectious diseases specialist , 3 residants , 6 chief nurses and 34 nurses , 1 administrative worker and 14 unskilled workers .
chair consists of one doctor of medical sciences , associate professor , 3 masters of medical sciences , a senior assistant , three assistants and one associate assistant .
professional teaching staff of the clinic for infectious diseases are : professor nada - koluder cimic md phd , infectious disease and clinical pharmacologist , head of the clinic and the department of infectious diseases ; chief physician alija drnda md msc , infectious diseases specialist , department head , chief physician snjezana mehanic md msc , infectious disease specialist , the head of department ; chief physician fikret pinjo md , infectious diseases specialist , department head , chief physician vesna hadziosmanovic md , infectious diseases specialist , department head , chief physician planinka muratovic md , infectious diseases specialist , department head , chief physician merdina ferhatovic md , infectious diseases specialist , department head , enra lukovac md , infectious diseases specialist , department head , chief physician jasna topalovic - cetkovic md , infectious diseases specialist , chief physicial akif osmic md , infectious disease specialist , nermin mostarac md , infectious disease specialist , meliha hadzovic - cengic md , infectious disease specialist , belma gazibera md , infectious disease specialist , ilhama jusufi huric md , infectious disease , rusmir baljic md msc , infectious disease specialist , refet gojak md , infectious disease specialist , amir hadzic md , infectious disease specialist , velida mulabdic md , infectious disease specialist , aida begic ahmetspahic
md , resident , alma sejtarija memisevic md , resident ; selma hasimbegovic ibrahimovic md , resident .
in addition to insufficiency in a highly educated staff , the clinic have chronic lack in nursing and senior staff .
the process of the clinic work depends on the epidemiological situation on the field . inside the building ,
space in the low ground was converted into a vcct center , counseling centers and day hospitals .
now we have concentrated on equipping intensive care units with related equipment including the two apparatus for artificial respiration , which we suspended its work in the intensive therapy internist and a parallel is to educate young staff .
infectious diseases care as an organized health care dates back to the austro - hungarian period , when a part of the provincial hospital in sarajevo formed a separate department of infectious diseases .
thanks to the capable and qualified teaching staff , also later in this discipline with the importance of health care of patients with infectious diseases , in sarajevo and other cities of bosnia and herzegovina has experienced expansion of infectious diseases and the growing importance and quality .
infectious disease specialists are very quick to organize their professional association and exchange of knowledge and experience has contributed substantially to the above assertion . in last war , in 1994 was founded the association of infectious diseases of bosnia and herzegovina and in 1997 was organized the first congress of the association of the infectious disease specialist with international participation .
true , such an association has existed since 1959 in the framework of the association of b&h doctors , but as the infection section , led by teachers and associates of the clinic for infectious diseases in sarajevo . since 1972 until 1991 , the interrepublic and intersectional meetings were held with the infectious disease association of macedonia . in the framework of the yugoslav association of infectious diseases was held five conferences on infectious diseases , of which the first was held in sarajevo in 1971 .
this brief overview of the development of infectious diseases as a medical profession in our area from late 19 century to the end of the 20 century shows the determination and modernity of doctors in our area to raise the level of population health of these diseases are the most common and fastest passed and ravaged the population .
once an infectious disease is under control , we can say that the whole demographic picture is changing for the better , which is not so obvious and dramatic case with other illnesses .
improving the health of the population in the area of infectious diseases significantly helped infectology clinics at clinical centers in bosnia and herzegovina and infectious disease departments within the hospitals in b&h .
association of the infectious disease specialists is a significant scientific and professional activity coordinator and the environment in which infectious disease specialists are able to share their knowledge and experience from practice , and the ability to publish these experiences and make them available to the wider scientific community in and outside b&h . | summaryconflict of interest : none declaredthere is a relatively low amount of historical data about development of infectology in bosnia and herzegovina , but that does not mean that these medical disciplines did nt have important events and actors that are important for the development of this medical discipline in b&h . in this review
we intent to show your several characteristic events and important persons , which left a lasting impression in the development of infectology service in bih and especially in sarajevo .
development center for this discipline was in sarajevo .
experts on infectious diseases were sent from sarajevo to other health centers to organize new ministry there .
infectology as an organized health care dates back to the austro - hungarian period , when a part of the state hospital in sarajevo formed a separate department of infectious diseases .
thanks to the competent professional and later teaching staff in this discipline and the importance of health care of patients with infectious diseases , in sarajevo and other cities in bosnia , infectious diseases care has experienced expansion and increasing importance and quality.infectious disease specialists were very quick in organizing their professional association and with exchange of knowledge and experience have contributed substantially to the above assertion .
the association of infectious diseases of bosnia and herzegovina was founded during the aggression on bih , in 1994 and in 1997 they organized the first scientific congress of the association of the infectious disease specialist with international participation .
improving the health of the population in the area of infectious diseases was significantly helped by infectious diseases clinics at clinical centers in bih and departments for infectious diseases within the hospitals in b&h .
association of the infectious disease is a significant coordinator for scientific and professional activities , but also the environment in which infectious disease specialists are able to share their knowledge and experiences . | 1. THE BEGINNINGS OF THE DEVELOPMENT OF INFECTOLOGY IN BOSNIA AND HERZEGOVINA
2. A BRIEF HISTORICAL OVERVIEW OF DEVELOPMENT OF THE CLINIC OF INFECTIOUS DISEASES IN SARAJEVO
3. PROFESSIONAL TEACHERS AND INFECTIOUS CLINIC IN SARAJEVO
4. THE ORGANIZATION OF INFECTIOUS CLINIC DURING THE AGGRESSION ON B&H (1992-1995)
5. INFECTIOUS CLINIC, CLINICAL CENTER UNIVERSITY OF SARAJEVO TODAY
6. CONCLUSION |
it is a common disease in tropical and subtropical regions with a worldwide prevalence of 2%7%.1 although historically described as a degenerative condition , it is more closely associated with inflammation and progressive fibrovascular proliferation.2 a pterygium commonly grows from the nasal side of the bulbar conjunctiva within the palpebral fissure and is usually associated with ultraviolet light exposure ( eg , sunlight ) , dry weather , and dust .
symptoms of pterygium include persistent redness , foreign body sensation , tearing , and dry and itchy eyes . in advanced cases ,
the pterygium may affect vision through obscuring the optical center and inducing astigmatism and corneal scarring.3 the pathogenesis of pterygium includes inflammation , fibrovascular proliferation , and angiogenesis , which is responsible for pterygium formation and progression.4 several studies confirmed that increased levels of vascular endothelial growth factor ( vegf ) , basic fibroblast growth factor ( bfgf ) , transforming growth factor - beta ( tgf- ) , and platelet - derived growth factor secreted by fibroblasts and inflammatory cells are associated with the primary formation and recurrence of pterygia.5 lee et al6 confirmed that vegf is selectively elaborated by two cells , t - helper and type 2 lymphocytes .
formation of new blood vessels from preexisting vasculature is called angiogenesis , and it is involved in a large number of physiological processes , such as growth and differentiation , ovulation , and would healing . it may also be involved in pathological conditions , such as neoplasia and proliferative eye diseases , such as proliferative diabetic retinopathy and neovascular glaucoma , which cause severe visual loss.7 new vascularization is associated with the activation of cell - derived angiogenic factors with synthesis of extracellular matrix responsible for anchorage of migrating endothelium.8 the surgical treatment of pterygium focuses on the excision and prevention of recurrence.9 recurrent pterygia are more aggressive and dangerous than primary ones because the underlying cornea may be thinner , the extensive proliferation adversely affects visual acuity , and further recurrence after the second surgery is common.10 the recurrence is mainly due to accelerated fibroblastic proliferation produced by the trauma of operation much in the same way as the production of keloid tissue .
fibroblastic proliferation and invasion adequately explain the clinical appearance and behavior of a pterygium with some histological support.2 mitomycin c ( mmc ) is an alkylating agent with cytotoxic effects , which inhibits dna synthesis and is widely used in ophthalmology .
mmc leads to the death of cells caused by the inability to repair the genotoxic injury caused by alkylation .
it acts against all cells regardless of the cell cycle and even acts in cells that are not synthesizing dna .
inhibition of dna synthesis leads to inhibition of mitoses , especially when mmc comes into contact with cells that are in the late g1 and early s phases of the cell cycle.1113 the topical application of mmc following the excision of pterygium can reduce the rate of recurrence .
however , its apoptotic effects can cause serious complications such as scleral thinning and ischemia.14 with subconjunctival injection of mmc , the epithelial and scleral toxicities can be diminished .
the subconjunctival route also allows exact dose delivery , which is equivalent to approximately one drop of 0.2 mg / ml of mmc , rather than the inexact and substantially higher dosing with sponge delivery during ocular surgery.15 bevacizumab ( avastin ; genentech , inc , san francisco , ca , usa ) is a humanized monoclonal antibody to vegf used by intravenous route and approved mainly for the treatment of colorectal cancer.16 various clinical studies across the world applied bevacizumab for intravitreal administration and confirmed its safety and efficacy in wet age - related macular degeneration and macularedema.17 in patients with impending recurrent pterygium , marked regression of limbal - conjunctival neovessels and long - standing delayed recurrence have been reported with the use of topical bevacizumab.18 we thought of coupling the antifibrotic effect of mmc with the antivascular effect of bevacizumab in order to obtain a lower recurrence rate than that obtained by either of the two drugs alone with reduction of mmc concentration to decrease the complications .
hence , this study aimed to determine the safety , efficacy , and histological effects of subconjunctival combined injection of both bevacizumab and mmc 1 month before the excision of the primary pterygium using the bare sclera technique .
this prospective study was conducted at the department of ophthalmology of sohag university hospital , egypt , in the period between january and june 2013 .
twenty eyes of 20 patients with primary pterygia measuring 4.5 mm in the surface area were included in this study .
the exclusion criteria included patients with other ocular diseases , eg , iritis , herpetic keratitis , glaucoma , and cataract or previous ocular surgery , including previous pterygium surgery , as well as any predisposing condition to ulceration or poor wound healing , such as dry eye or atopic keratoconjunctivitis .
pregnant or lactating women and patients with a history of strokes or thrombus elsewhere in the body were also excluded .
this study followed the tenets of the declaration of helsinki , and an approval was obtained from the ethics committee of sohag faculty of medicine .
a written informed consent was signed by every patient after full explanation of the technique and the possible complications of the procedure . a complete ocular examination , including measurement of the uncorrected and best corrected visual acuity and refractive state , and a photographic documentation of the pterygia were performed for each patient before surgery .
the surface area of the pterygium in square millimeters was calculated by multiplying the height of the pterygium by half its base , with the height measured as the distance from the apex to the limbus and the base measured at the limbus .
one month before the surgery , a subpterygium injection of 0.1 ml of mmc ( 0.1 mg / ml ) with 0.1 ml of avastin ( 1.25 mg/0.1 ml ) using an insulin syringe under topical anesthesia was done ( figure 1 ) .
the mmc was freshly prepared on the day of injection using sterile distilled water as a diluent to achieve a concentration of 0.1 mg / ml . all patients were operated by the first author .
the findings were compared with the histological features of pterygia of two patients who did not have preoperative injection of avastin or mmc and rather had conjunctival autografts to prevent recurrence .
these two patients used as control for histological comparison had the same exclusion criteria as the preinjected patients .
fixed tissues were processed for preparation of serial paraffin sections of 5 m thickness that were subjected to19
hematoxylin and eosin staining for general histological examination.light green staining for collagen fibersimmunohistochemical staining for cd31 antibody to assess the microvessel density by avidin biotin peroxidase complex method .
they were immersed in citrate buffer ( 0.1 ml , ph 0.6 ) and put in the microwave for 12 min .
cd31 monoclonal antibody ( dilution 1/50 ; dako ) was applied overnight at 4c , and then , the process was completed with the addition of secondary antibody .
positive control slides were included in all cases , as well as negative control ( omitting the step of primary antibody ) .
light green staining for collagen fibers immunohistochemical staining for cd31 antibody to assess the microvessel density by avidin
they were immersed in citrate buffer ( 0.1 ml , ph 0.6 ) and put in the microwave for 12 min .
cd31 monoclonal antibody ( dilution 1/50 ; dako ) was applied overnight at 4c , and then , the process was completed with the addition of secondary antibody .
positive control slides were included in all cases , as well as negative control ( omitting the step of primary antibody ) .
all measurements were taken using the image analyzer ( leica q 500 mc program ; leica , wetzlar , germany ) in the department of histology , sohag faculty of medicine , sohag university .
examinations were performed under five high - power fields per five different sections in each specimen .
microvessel density was evaluated by cd31 staining of vascular endothelial cells as described by aspiotis et al.20 histological statistical analyses were performed using a paired t - test ( spss program , version 17 ; ibm corporation , somers , ny , usa ) , and the results are represented as mean standard error .
postoperative topical antibiotic ( moxifloxacin 0.5% ) was administered four times daily for 1 week , and steroid ( prednisolone acetate 0.5% ) was administered four times daily with gradual withdrawal for 6 weeks .
all patients were examined after 1 day , 1 week , 1 month , 3 months , and then every 6 months for 2 years .
the postoperative recurrence was classified into the following four grades:9
grade 1 : no vessels at the surgical site with healthy normal conjunctiva.grade 2 : fine episcleral vessels at the surgical site l with healthy cojunctiva.grade 3 : fibrovascular tissue localized to the area of excision not extending to the cornea ; however , the fibrovascular proliferation impending actual recurrence.grade 4 : a true recurrence with fibrovascular tissue covering the excision area and invading the cornea .
grade 3 : fibrovascular tissue localized to the area of excision not extending to the cornea ; however , the fibrovascular proliferation impending actual recurrence .
grade 4 : a true recurrence with fibrovascular tissue covering the excision area and invading the cornea . in this study
, we considered the first and second grades as no recurrence , while the third grade was considered as an early recurrence and the fourth grade as recurrent pterygium .
all measurements were taken using the image analyzer ( leica q 500 mc program ; leica , wetzlar , germany ) in the department of histology , sohag faculty of medicine , sohag university .
examinations were performed under five high - power fields per five different sections in each specimen .
microvessel density was evaluated by cd31 staining of vascular endothelial cells as described by aspiotis et al.20 histological statistical analyses were performed using a paired t - test ( spss program , version 17 ; ibm corporation , somers , ny , usa ) , and the results are represented as mean standard error .
postoperative topical antibiotic ( moxifloxacin 0.5% ) was administered four times daily for 1 week , and steroid ( prednisolone acetate 0.5% ) was administered four times daily with gradual withdrawal for 6 weeks .
all patients were examined after 1 day , 1 week , 1 month , 3 months , and then every 6 months for 2 years .
the postoperative recurrence was classified into the following four grades:9
grade 1 : no vessels at the surgical site with healthy normal conjunctiva.grade 2 : fine episcleral vessels at the surgical site l with healthy cojunctiva.grade 3 : fibrovascular tissue localized to the area of excision not extending to the cornea ; however , the fibrovascular proliferation impending actual recurrence.grade 4 : a true recurrence with fibrovascular tissue covering the excision area and invading the cornea .
grade 3 : fibrovascular tissue localized to the area of excision not extending to the cornea ; however , the fibrovascular proliferation impending actual recurrence .
grade 4 : a true recurrence with fibrovascular tissue covering the excision area and invading the cornea . in this study
, we considered the first and second grades as no recurrence , while the third grade was considered as an early recurrence and the fourth grade as recurrent pterygium .
this study included 20 eyes of 20 patients with primary pterygia , including 13 males ( 65% of 20 patients ) and 7 females ( 35% of 20 patients ) with the age ranging from 44 to 68 years ( mean of 582.8 years ) .
the affected eye was right in 12 eyes ( 60% of 20 patients ) and left in 8 eyes ( 40% of 20 patients ) , and the follow - up period ranged from 25 to 36 months with a mean of 303.4 months .
the preoperative spherical refractive error ranged between 4.75 and + 3.25 d ( mean 1.20.6 d ) , while cylindrical error ranged between 4.00 and + 5.50 d ( mean + 3.31.3 d ) .
the surface area of the pterygium ranged from 5 to 12.5 mm with a mean of 82.1 mm .
the follow - up period ranged from 26 to 36 months with a mean of 30.43.6 months .
table 1 shows the preoperative data of 20 patients involved in our study , while table 2 shows the data of the two cases used as control for histological comparison . on the first postoperative day , all patients had corneal epithelial defects , which healed completely within 1 week with no corneal fluorescein staining .
diffuse punctate epithelial erosions with excessive photophobia was seen in one eye ( 5% ) that resolved with topical lubricants within 1 month postoperatively .
subconjunctival hemorrhage during sublesional injection occurred in one case ( 5% ) , persisted for 2 weeks , and resolved spontaneously . as regards
the postoperative recurrence ( figure 2 ) ,
of the 19 eyes , 12 eyes ( 60% of 20 patients ) with no recurrence were classified as first grade and 7 eyes ( 35% of 20 patients ) were classified as second grade.one eye ( 5% of 20 patients ) was classified as third stage ( early recurrence ) : this case with early recurrence was the same one that had subconjunctival hemorrhage during preoperative injection .
this patient was followed up for 33 months without progression from the third to the fourth stage.no cases were classified as fourth stage of recurrence.the two patients used as control for histological comparison were followed up for 24 months , with no recurrence detected and classified as grade 1 .
of the 19 eyes , 12 eyes ( 60% of 20 patients ) with no recurrence were classified as first grade and 7 eyes ( 35% of 20 patients ) were classified as second grade .
one eye ( 5% of 20 patients ) was classified as third stage ( early recurrence ) : this case with early recurrence was the same one that had subconjunctival hemorrhage during preoperative injection .
this patient was followed up for 33 months without progression from the third to the fourth stage .
the two patients used as control for histological comparison were followed up for 24 months , with no recurrence detected and classified as grade 1 .
there were no serious complications , such as scleral thinning or melting , corneal decompensation , postoperative iritis , cataract , and glaucoma , detected in any patient during the follow - up period .
some areas of hyaloid degeneration of collagen fibers and numerous nuclei of fibroblast cells were seen ( figures 35 ) .
using cd31 staining , more intense angiogenic activity was observed in the conjunctival tissue , especially at the subepithelial area ( figure 6 ) .
the preinjected pterygia sections had an apparent decrease in the number of goblet and fibroblast cells with less inflammatory cellular infiltration and absence of neovascularization ( figures 7 and 8) .
cd31 staining also revealed an apparent decrease in the positive immunostained cells ( figure 9 ) .
there was also a significant increase in the mean of area percentage of collagen fibers in the treated pterygia ( 30.510.9 ) compared to that in the untreated group ( 16.071.2 ) , as well as a significant decrease in microvessel density in the injected pterygia ( 10.571.4 ) compared to that in the noninjected ones ( 21.831.8 ) ( p0.05 ) ( figure 10 ) .
pterygium is a benign fibrovascular growth of the conjunctiva over the cornea . due to high prevalence in the world , especially in our tropical sunny and dusty countries with high recurrence rate
, pterygium has always been a major problem for ophthalmologists.21 in this study , we tried to overcome this problem by the use of two famous drugs widely used in ophthalmology .
the first drug is mmc with well - known antifibroblastic activity , which has been used intraoperatively since 1988 as an adjunctive therapy during pterygium excision to prevent recurrence.22 the second drug is bevacizumab with anti - vegf activity to reduce the vascularization , which is the main cause of recurrence.23 the strength of the current study is using a combination of bevacizumab and mmc subconjunctivally before the bare sclera excision of primary pterygium .
one eye ( 4% ) was complicated by subconjunctival hemorrhage that resolved within 2 weeks . another eye ( 4% )
was complicated by punctate epithelial erosions that resolved within 1 month with lubricants and steroids .
as regards the recurrence rate , there was only one case ( 4% ) with fibrovascular proliferation at the site of excision without crossing the limbus into the cornea .
this is considered as no recurrence that appeared after 13 months , increased slightly within 33 months , and occurred in the same eye that was complicated by intraoperative subconjunctival hemorrhage , which may suggest that the coagulated blood enhanced the fibroblastic activity . in this study
, we decreased the concentration of mmc to avoid serious ophthalmological complications as we used 0.1/ml , which is lower than the concentration used in most of the previous studies , ranging from 0.2 to 0.15/ml .
we also applied both drugs through the subconjunctival route to control the dose and decrease the effect on the ocular surface .
the lack of recurrence is probably due to the use of combination of two drugs with synergistic effect , while the low concentration of mmc ( 0.1 mg / ml ) decreased the incidence of complications . in order to increase its efficacy and
decrease its epithelial toxicity , donnenfeld et al15 injected mmc in the head of pterygium 1 month before pterygium excision .
they delivered mmc subconjunctivally to minimize the exposure to the ocular surface and reduce the epithelial stem cell toxicity associated with topical application .
they used 0.1 ml of 0.15 mg / ml concentration because this concentration is slightly higher than the therapeutic window but well below the toxic level associated with cell death .
they included 36 eyes in their study and reported recurrence in two patients ( 6% ) after a mean follow - up period of 24.4 months .
the route of administration of the drug matches with our study ; however , the concentration of mmc in our study was reduced by 50% to avoid complications .
the recurrence rate is less in the current study in comparison to theirs ; however , the number of cases is less in our study .
ghoneim et al24 compared the efficacy of a preoperative subconjunctival injection of 0.15 mg / ml of mmc 1 day before the bare sclera excision with an intraoperative application of mmc . in their study , the rates of recurrence were 5.7% in the previously injected pterygia and 8.57% in topical application after 1 year of follow - up .
the complications in their study were scleral thinning that occurred in one eye in each group and resolved by 5 months postoperatively .
in contrast to our study , no recurrence or scleral thinning occurred with longer follow - up period due to the synergistic effect of both drugs and the decreased concentration of mmc .
vegf has been shown to be increased in the pterygium tissue and may be strongly involved in its pathogenesis,25,26 and hence an anti - vegf therapy may be used for inducing regression of blood vessels and size of pterygium and prevention of recurrence.27 teng et al28 were the first to report a case of subconjunctival injection of bevacizumab in primary pterygium .
they injected 1.25/0.05 ml of bevacizumab subconjunctivally into an inflamed primary pterygium at the limbus .
although the signs and symptoms of inflammation and neovascularization regressed rapidly , the effect was short term , lasting ~2 weeks .
the transient effect was likely related to the limited bioavailability of the drug in the setting of continued vegf expression . in the current study , we surgically removed the pterygia after injection using the bare scleral technique as we thought that it was a radical solution to the problem .
shenasi et al29 in their study after subconjunctival bevacizumab injection immediately after the excision of primary pterygium found that the recurrence rate was lower in these patients . in the current study
, we thought that 1 month before surgery allowed good time for drug application and tissue preparation than intraoperative use . in their randomized clinical trial , razeghinejad and banifatemi30
the patients were randomized into the following two groups : group 1 received a total of 7.5 mg subconjunctival bevacizumab ( 5 mg/0.2 ml on the day of surgery and 2.5 mg/0.1 ml on the fourth day after surgery ) and group 2 received subconjunctival balanced salt solution in the same manner .
after 6 months , four patients in group 1 versus eight patients in group 2 ( p=0.17 ) had fibrovascular tissue crossing the limbus .
they concluded that the frequency of fibrovascular tissue crossing the limbus in the bevacizumab group was half that in the bss group ; however , the difference failed to reach a statistically significant level .
the concentration of bivacizumab used was higher than that used in our study , with higher recurrence rate , which may be due to the use of mmc in our study .
in contrast to our study and most of the previous studies on the use of bivacizumab in pterygium surgery , maha et al31 and mohamed et al32 found that a subconjunctival injection of 1.25 mg/0.05 ml of bevacizumab at the end of the surgery did not result in a statistically significant difference in the recurrence rates or postoperative conjunctival erythema with discomfort , lacrimation , photophobia , or healing of corneal epithelial defects after pterygium excision .
the histological examination of noninjected patients showed that the covering epithelium containing numerous goblet cells collected in the form of intraepithelial glands .
this might be due to the exposure to ultraviolet rays and dust particles in the air , which are the main predisposing factors for pterygium .
the stroma showed inflammatory mononuclear cellular infiltration with dilated congested blood vessels and areas of degeneration of collagenous fibers .
similar findings were confirmed ultrastructurally by chang et al33 after subconjunctival injection of 0.1 ml of 0.15 mg / ml of mmc 1 month before the excision of pterygium . in this study , the presence of abundant cd31-positive microvessels in noninjected pterygium was consistent with the previous reports of aspiotis et al.20 they considered that neoangiogenesis is an important pathogenic factor of pterygium .
previous studies added that inflammatory cells in the stroma of pterygium secrete proinflammatory cytokines that lead to extracellular matrix remodeling and angiogenesis .
growth factors with potent angiogenic activity have been found to be secreted from fibroblasts and inflammatory pterygium cells.5 in the current study , the usage of mmc and bevacizumab led to a significant decrease in cd31-positive cells and an apparent decrease in the inflammatory cellular infiltration and the number of fibroblasts and goblet cells . there was a significant increase in the area percentage of collagen fibers .
this could be secondary to the anti - vegf action of bevacizumab and the antifibroblastic activity of mmc.22,34 all the previous studies focused on the use of only one of these drugs to decrease the recurrence of pterygium .
to the best of our knowledge , this study is the first to inject a combination of bevacizumab and mmc subconjunctivally before bare sclera excision of the primary pterygium .
the combined subconjunctival injection of bevacizumab and mmc 1 month before bare sclera excision of the primary pterygium is a safe and effective procedure that can further decrease the rate of recurrence without increasing the rate of complications .
however , larger scale studies with longer follow - up periods are needed to prove this . | purposethe aim of this study was to detect the clinical and histological effects of preoperative subconjunctival injection of both bevacizumab and mitomycin c ( mmc ) 1 month before the surgical excision of primary pterygium using a bare sclera technique.patients and methodsa total of 20 patients with primary pterygium underwent subconjunctival combined injection of 0.1 ml of mmc ( 0.1 mg / ml ) and 0.1 ml of bevacizumab ( 1.25 mg/0.1 ml ) 1 month before bare sclera excision of the pterygium .
the excised pterygium tissues were examined histologically and immunohistologically by cd31 staining , and the patients were followed up clinically for at least 2 years .
the excised pterygia of two patients without preoperative injection were used for histological comparison.resultsclinically , there were no intraoperative or postoperative complications .
no recurrence was noted during the follow - up period .
histologically , the previously injected pterygia showed a decreased number of epithelial cells and stromal fibroblasts .
the latter were rounded or oval and swollen rather than spindle shaped , and some were degenerating or apoptotic .
collagen and elastic fibers were degenerated , distorted , and decreased in density , while blood capillaries were obliterated .
there was a significant decrease in cd31-positive cells in previously injected pterygia.conclusionpreoperative subpterygium combined injection of bevacizumab and mmc is safe and effective in reducing the postoperative recurrence of primary pterygium .
histological and immunohistological changes in the form of decreased fibrovascular activity and degeneration of the extracellular matrix and nerve axons were noted . | Introduction
Patients and methods
Morphometric and statistical analyses
Results
Discussion
Conclusion |
there are two main mechanisms ensuring the efficient transfer of amyloidogenic proteins
from the cytosolic or extracellular milieu to neuronal plasma membranes .
the first one is
the reduction of dimensionality ( from 3d to 2d ) that occurs when a soluble protein
dissolved in the bulk solvent binds to a membrane surface ( ref .
the underlying idea is that the membrane 2d space can
concentrate proteins far more efficiently than the 3d space of the bulk solvent . as a
consequence ,
the average distance between two proteins is shorter in 2d than in 3d , and
this effect is more pronounced for higher protein concentrations ( ref .
this phenomenon is particularly important for amyloidogenic
proteins that tend to self - aggregate after a conformational transition ( fig .
2 ) . thus , the simple fact of the concentration of
monomers in a restricted membrane area is , by itself , a crucial step in the complex
process of amyloid oligomerisation / aggregation .
figure 2neuronal membranes as concentration platforms and chaperones for
amyloidogenic monomers : key role of sphingolipid
( a ) amyloidogenic monomers have a low affinity for the
liquid - disordered ( ld ) phase of plasma membranes enriched in phosphatidylcholine .
( b ) by contrast , the monomers have a high affinity for
sphingolipid cholesterol domains in the liquid - ordered ( lo ) phase . in this
case , monomers are not only concentrated on the membrane surface , but also undergo a
major conformational change induced by the sphingolipids .
this property is referred
to as the chaperone effect of sphingolipids on amyloidogenic proteins .
neuronal membranes as concentration platforms and chaperones for
amyloidogenic monomers : key role of sphingolipid cholesterol
domains .
( a ) amyloidogenic monomers have a low affinity for the
liquid - disordered ( ld ) phase of plasma membranes enriched in phosphatidylcholine .
( b ) by contrast , the monomers have a high affinity for
sphingolipid cholesterol domains in the liquid - ordered ( lo ) phase . in this
case , monomers are not only concentrated on the membrane surface , but also undergo a
major conformational change induced by the sphingolipids .
this property is referred
to as the chaperone effect of sphingolipids on amyloidogenic proteins . the second mechanism explaining the affinity of amyloidogenic proteins for neuronal
membranes is lipid specificity .
converging data obtained from various laboratories with a
broad range of experimental approaches have shown that amyloidogenic proteins interact
with the lipids found in the lo phase that is , sphingolipids and cholesterol
( fig .
alzheimer a peptides
recognise several glycosphingolipids , including neutral species such as asialo - gm1 or
galactosylceramide ( galcer ) as well as gangliosides such as gm1 ( refs 26 , 27 ,
28 , 29 ) ; -synuclein binds to gangliosides gm1 and gm3 ( refs 30 , 31 ) ; prp
interacts with sphingomyelin , galcer , gm1 and gm3 ( refs 27 , 32 , 33 ) and is associated with sphingolipid signalling platforms ( ref .
overall , this high affinity for
sphingolipids determines the concentration of amyloid proteins in lipid raft areas of the
extracellular leaflet of the plasma membrane .
several amyloidogenic proteins can also bind
to negatively charged glycerophospholipids ( refs 35 , 36 ) , which are enriched in the
cytoplasmic leaflet of the plasma membrane ( ref .
this type of interaction has been extensively studied for
-synuclein , which recognises anionic phosphatidylserine and phosphatidylglycerol
( refs 36 , 37 , 38 , 39 ) . in this review ,
we restrict our analysis to the role of cholesterol and
sphingolipids in the interaction of amyloidogenic proteins and membranes .
as we will see ,
these lipids play a more active role in the conformation , oligomerisation and aggregation
of amyloidogenic proteins than just acting as a concentration platform .
that the same amyloidogenic protein could interact with several distinct sphingolipids
( refs 26 , 27 , 31 ) could be viewed as paradoxical
and difficult to reconcile with a real specificity of interaction .
a biochemical analysis
of sphingolipid structures is necessary to get a clearer view on this issue .
sphingolipids
are formed by the condensation of a fatty acid chain with a sphingoid base ( which is
generally sphingosine although five different sphingoid bases have been detected in
mammalian cells ) .
the biochemical diversity of sphingolipids is first generated by the
numerous fatty acids ( more than 20 , varying in chain length , degree of saturation and
degree of hydroxylation ) that can be attached to the sphingoid base to form a ceramide
( ref .
sphingolipids are then classified into
sphingomyelin and glycosphingolipids , according to the biochemical nature of the polar
part attached to ceramide : phosphorylcholine for sphingomyelin , and a glycan moiety for
glycosphingolipids .
several hundred different carbohydrate structures have been described
in glycosphingolipids , with various combinations of neutral sugars , sulfated sugars and
sialic acids ( ref .
this is
the case for the glycosphingolipids galcer , globotriaosylceramide ( gb3 ) and
gm3 , which have a distinct sugar moiety but a common galactosyl residue with the same
orientation ( fig .
c ) . as a logical
consequence of this common structural feature , a protein that recognises one of these
glycosphingolipids ( e.g. galcer ) , through binding to this galactose residue , might also
interact with the other two ( in this case , gb3 and gm3 ) .
this means that the
biochemical code governing glycosphingolipid protein interactions is degenerate .
an example of this striking property is the v3 loop domain of the human immunodeficiency
virus 1 ( hiv-1 ) surface - envelope glycoprotein gp120 , which was shown to recognise first
galcer ( refs 41 , 42 , 43 ) , and then , several
years later , both gb3 and gm3 ( refs 44 , 45 ) .
however , there is a unique
molecular mechanism that accounts for the interaction of each of these glycosphingolipids
with gp120 ( ref .
figure 3glycosphingolipids and amyloidogenic proteins : a common mechanism of
interaction ? despite their high level of biochemical diversity , some
glycosphingolipids share a common galactosyl ring with an orientation compatible
with the establishment of stacking ch- interactions between the sugar and
an aromatic residue .
such galactosyl rings are indicated in shaded circles in the
sugar moiety of ganglioside gm3 ( a ) , globotriaosylceramide gb3 ( b ) and
galactosylceramide galcer ( c ) . in these cases
, the apolar side of the galactosyl
ring fits particularly well with the aromatic - ring side chain of an aromatic residue
( phe , in this case ) exposed at the surface of an amyloid protein ( d ) . the electronic
cloud of the system stacks onto the ch groups of the galactosyl ring .
this interaction is favoured by the common geometric structure of both rings , which
allows a coordinated binding process between ch groups and electrons .
cholesterol , which has a strong affinity for sphingolipids , can further improve the
accessibility of the galactosyl group through fine tuning of glycosphingolipid
conformation ( e ) .
this effect involves the establishment of an h - bond network
between the oh group of cholesterol , the nh group of the sphingolipid and the oxygen
atom of the glycosidic bond linking the ceramide and the sugar part of the
sphingolipid .
galcer - nfa , galcer with a nonhydroxylated fatty acid in the ceramide
moiety .
glycosphingolipids and amyloidogenic proteins : a common mechanism of
interaction ? despite their high level of biochemical diversity , some
glycosphingolipids share a common galactosyl ring with an orientation compatible
with the establishment of stacking ch- interactions between the sugar and
an aromatic residue .
such galactosyl rings are indicated in shaded circles in the
sugar moiety of ganglioside gm3 ( a ) , globotriaosylceramide gb3 ( b ) and
galactosylceramide galcer ( c ) . in these cases ,
the apolar side of the galactosyl
ring fits particularly well with the aromatic - ring side chain of an aromatic residue
( phe , in this case ) exposed at the surface of an amyloid protein ( d ) . the electronic
cloud of the system stacks onto the ch groups of the galactosyl ring .
this interaction is favoured by the common geometric structure of both rings , which
allows a coordinated binding process between ch groups and electrons .
cholesterol , which has a strong affinity for sphingolipids , can further improve the
accessibility of the galactosyl group through fine tuning of glycosphingolipid
conformation ( e ) .
this effect involves the establishment of an h - bond network
between the oh group of cholesterol , the nh group of the sphingolipid and the oxygen
atom of the glycosidic bond linking the ceramide and the sugar part of the
sphingolipid .
galcer - nfa , galcer with a nonhydroxylated fatty acid in the ceramide
moiety .
let us first consider the way in which proteins bind to galcer , which is both the major
lipid of myelin sheaths ( ref .
47 ) and the
simplest possible brain - expressed glycosphingolipid , consisting of a ceramide linked to a
single galactosyl group ( i.e. a ceramide monohexoside ) .
most of the galcer molecule is
dipped into the membrane , so that the galactosyl moiety is the principal region of the
glycolipid available for protein binding . in this respect
, there is a functional
similarity between proteins that recognise galcer and galactose - specific lectins .
however ,
the local concentration of galactosyl groups in a nanodomain of galcer is extremely high
compared with the maximal concentration of free galactose in biological fluids ( another
illustration of the reduction - in - dimensionality concept discussed above ) .
moreover , the
conformation of the galactose head group in the glycolipid is restricted by the ceramide
backbone , which allows only limited motion of the sugar ( ref .
by contrast , galactose in water solution is free to adopt a wide
range of conformations . finally ,
the most polar part of ceramide , which includes both
amide and hydroxyl ( oh ) groups , as well as the -glycosidic bond linking ceramide
to galactose , can also participate in the binding reaction and further stabilise the
galcer protein complex . taken together , these biochemical properties of the
galactosyl head group of galcer
protein interactions are
distinct from galactose lectin interactions ( ref . 49 ) . at the molecular level , numerous proteins that bind to galcer
have a solvent - exposed
aromatic residue that provides a complementary surface for a stacking interaction with the
galactose ring ( fig .
3d ) . as a result of the
distribution of the oh groups linked to the pyranosyl ring of galactose
, this sugar has
two chemically distinct faces : one apolar with hydrocarbyl ( ch ) groups and the other polar
with oh groups .
therefore , the apolar face of galactose is particularly suited for a
stacking interaction with an aromatic structure ( ref .
50 ) . this type of interaction between ch groups and a -electron cloud
system has been described and referred to as ch interaction by nishio
and co - workers ( ref .
the
ch stacking mechanism is involved in the interaction between
glycosphingolipids and several amyloidogenic proteins , including prp ( ref .
, the apolar side of the central galactosyl group has a similar
orientation to that in galcer ( fig .
3a c ) ; thus , galcer - binding proteins are predicted to also bind to
gb3 and gm3 through ch stacking .
note that the
conformation of the sugar moiety of glycosphingolipids is restricted by a network of
h - bonds ( ref .
52 ) that involves either an oh
group in the acyl chain of the ceramide ( intramolecular network ) or the oh group of
cholesterol ( intermolecular network , fig .
3e ) , as
further discussed below ( see the section the outside and inside effects of
cholesterol ) .
the notion that unrelated proteins , displaying a solvent - accessible aromatic residue in a
hairpin domain , might bind these glycosphingolipids through a similar stacking mechanism
has motivated the search for v3-like domains in various proteins , including amyloidogenic
proteins ( ref .
this is the concept of the
glycosphingolipid - binding domain or , more generally , of the sphingolipid - binding domain
( ref .
such domains are characterised by the
presence , in a flexible region of the surface of a protein , of an aromatic residue with
its side chain oriented towards the solvent , as illustrated in figure 4a with the superimposition of the glycosphingolipid - binding
domains of a and -synuclein ( ref .
the recognition of the glycosphingolipid by this region of the protein is
thought to proceed through an induced - fit mechanism involving the conformational mobility
of the aromatic side chain and a concomitant adjustment of the orientation of the
galactosyl head group ( fig .
( a )
amyloidogenic proteins share a common sphingolipid - binding domain , which can be
shown by superimposing the structure of micelle - bound alzheimer a peptide
[ pdb i d : 1ba4 , in red ] ( ref .
150 ) onto the
structure of micelle - bound -synuclein [ pdb i d : 1xq8 , in blue ] ( ref . 142 ) .
the motif corresponds to a
helix turn helix structure displaying an aromatic residue ( tyr10
for a ; tyr39 for -synuclein ) oriented towards the solvent and
located at a similar position in the loop .
the location of glu residues associated
with glu to lys mutations in genetic forms of alzheimer and parkinson diseases is
indicated ( glu22 for a and glu46 for -synuclein , respectively ) .
the amino acid sequence of both proteins ( upper panel ) show very little homology ,
apart from the above - mentioned tyr residues ( asterisk ) and a common val residue
( val52 for -synuclein and val24 for a ) .
( b ) due to the high
conformational plasticity of amyloidogenic proteins , the sphingolipid - binding domain
can adopt several distinct conformations , as shown for the core amyloid - forming
motif of amylin [ nfgailss octapeptide , pdb i d : 1kuw ] ( ref .
the -carbon chain of the peptide is coloured in
blue , and the phe residue in red .
twenty conformers obtained by nuclear magnetic
resonance ( nmr ) analysis are superposed . once bound to the glycosphingolipid ( gsl ,
coloured in green )
53 obtained with lactosylceramide
( laccer ) . a common sphingolipid - binding domain in amyloidogenic proteins .
( a )
amyloidogenic proteins share a common sphingolipid - binding domain , which can be
shown by superimposing the structure of micelle - bound alzheimer a peptide
[ pdb i d : 1ba4 , in red ] ( ref .
150 ) onto the
structure of micelle - bound -synuclein [ pdb i d : 1xq8 , in blue ] ( ref .
the motif corresponds to a
helix turn helix structure displaying an aromatic residue ( tyr10
for a ; tyr39 for -synuclein ) oriented towards the solvent and
located at a similar position in the loop .
the location of glu residues associated
with glu to lys mutations in genetic forms of alzheimer and parkinson diseases is
indicated ( glu22 for a and glu46 for -synuclein , respectively ) .
the amino acid sequence of both proteins ( upper panel ) show very little homology ,
apart from the above - mentioned tyr residues ( asterisk ) and a common val residue
( val52 for -synuclein and val24 for a ) .
( b ) due to the high
conformational plasticity of amyloidogenic proteins , the sphingolipid - binding domain
can adopt several distinct conformations , as shown for the core amyloid - forming
motif of amylin [ nfgailss octapeptide , pdb i d : 1kuw ] ( ref .
68 ) . the -carbon chain of the peptide is coloured in
blue , and the phe residue in red .
twenty conformers obtained by nuclear magnetic
resonance ( nmr ) analysis are superposed . once bound to the glycosphingolipid ( gsl ,
coloured in green )
53 obtained with lactosylceramide
( laccer ) . in summary , a glycosphingolipid - binding domain is a flexible domain containing
turn - inducing ( gly , pro ) , basic ( arg , lys , his ) and aromatic residues ( phe , tyr , trp )
( refs 27 , 54 , 55 , 56 , 57 ) .
the critical involvement of
aromatic residues in the interaction between proteins and glycosphingolipids has been
convincingly demonstrated by using synthetic peptides in which these residues were
replaced by ala or leu ( refs 53 , 54 , 55 ) :
correspondingly , the interaction with the glycosphingolipid has been dramatically
decreased or even totally lost .
reciprocally , conservative aromatic substitutions ( e.g.
phe to trp ) did not affect binding to glycosphingolipids ( ref .
glycosphingolipid interactions driven by
ch stacking of a sugar and aromatic residue ( refs 52 , 58 ) .
one important consequence of protein sphingolipid interactions is that the
conformation of the protein can be significantly affected on binding to sphingolipids
( fig .
this chaperone effect of
sphingolipids is a direct consequence of the unique modalities of
protein sphingolipid interactions ( ref .
as soon as binding begins , the structure of the sphingolipid changes and the
protein gradually adapts its shape to this moving target .
a stable anchoring of the
protein to the sphingolipids can be obtained by optimising protein
the
flexibility of the sphingolipid - binding domain is particularly well suited to trigger
structural rearrangements in the protein ( ref .
. this unexpected consequence of protein sphingolipid interactions
has been extensively studied with hiv-1 .
briefly , on binding to raft glycosphingolipids
through its v3 domain , hiv-1 gp120 undergoes a series of conformational changes that are
required for virus fusion ( refs 19 , 45 ) .
because the sphingolipid - binding domain is
evolutionarily conserved ( it has been found in virus , bacteria , insect and mammalian
proteins ) , it is likely that sphingolipids can modulate the conformation and regulate the
function of a wide range of proteins ( refs 52 ,
53 , 54 , 55 , 56 , 57 ) .
how could this concept of a sphingolipid - mediated chaperone effect apply to
membrane - bound amyloids ?
seminal work by matsuzaki , yanagasiwa and co - workers has shown
that lipid rafts act as surface catalysts able to accelerate the aggregation of
a ( refs 29 , 59 , 60 , 61 , 62 ,
63 ) .
most interestingly , gm1-bound a
has been identified as a pre - amyloid form acting as an endogenous seed for the formation
of neurotoxic amyloid fibrils in alzheimer disease ( refs 29 , 61 , 62 , 63 ) .
these data have not
only strengthened the role of lipid rafts as preferential binding sites for unstructured
amyloidogenic proteins on neuronal membranes but have also suggested that these membrane
domains behave as conformational catalysts for amyloid formation . in other words , lipid
rafts can control the conformation of membrane - bound amyloid through a chaperone effect
( refs 49 , 59 , 64 ) . yet
, conflicting data have
suggested that binding of a to gm1 induces a conformational transition from
random coil to a protective -helical structure ( ref .
30 ) , or to a fibrillation - prone -structure
( refs 61 , 66 ) .
the structure of amyloidogenic proteins bound to gm1 depends on several
physicochemical parameters , including ph , membrane fluidity , gm1-carrier lipid ratios ,
protein concentration and the absence or presence of cholesterol , which could explain the
discrepancies reported in the literature . in any case
, the available data suggest that
lipid rafts could ( 1 ) modulate the conformational changes of unstructured monomers from
the unfolded state to more - ordered or structures , and ( 2 ) control
the balance between and structures , which in turn determine the
oligomerisation / aggregation status of amyloidogenic proteins .
59 ) have shown that the
oligomerisation and aggregation of a peptides occur in ganglioside - enriched
domains of the plasma membrane .
a possible sequence of events could be as follows : ( 1 )
soluble a monomers bind to the sugar head group of gangliosides in
cholesterol sphingolipid - enriched domains such as lipid rafts ; ( 2 ) on binding to
gangliosides , a is constrained to adopt an -helical structure ; ( 3 ) as
the protein density on the membrane increases , a undergoes a conformational
transition from an -helix - rich structure to a -strand - rich one ; and
( 4 ) the ganglioside - bound a complex with acquired secondary structures serves as
a seed for amyloid fibril formation .
indeed , a fibrils with high toxicity have
been successfully generated in an acellular system using gm1-containing raft - like
liposomes ( ref .
67 ) . altogether , these data
strongly support the concept that the ganglioside cluster can act as a chaperone able to
lower the activation energy for the conformational changes of a ( ref .
this scenario is also consistent with the
involvement of the glycosphingolipid - binding domain in membrane
unstructured amyloidogenic proteins can adopt a wide range of conformations in water and
in biological membranes ( fig .
the
conformational flexibility of the amyloid core peptide of amylin ( nfgailss ) has been
studied by nuclear magnetic resonance ( nmr ) ( ref .
, this peptide can adopt three
distinct classes of conformations owing to the wide flexibility of the peptide chain
( fig .
4b ) . correspondingly , the unique phe
residue of the motif has three main possible orientations ( ref .
53 ) . on binding to a glycosphingolipid bearing an appropriately
oriented galactosyl group ( lactosylceramide in fig
.
4b ) , the phe residue stacks onto the sugar so that only one of the numerous peptide
conformations is selected and locked .
this illustrates the potency of ch stacking interactions to
stabilise a thermodynamically unstable conformation at a minimal energetic cost , just like
a wedge can efficiently block a car on a sloping street ( ref .
this simple but efficient mechanism could allow raft
glycosphingolipids to act as molecular chaperones inducing -helix - rich
structures in amyloidogenic proteins ( fig .
the stabilisation of an -helix structure is beneficial , because
this can prevent protein misfolding and aggregation ( ref .
49 ) . however , the -helix conformation could also facilitate
membrane insertion and channel / pore formation , leading to dramatic perturbations of
membrane permeability , as shown for -synuclein ( ref .
in addition , the chaperone effect of glycosphingolipids is not
irreversible . changing the physicochemical properties of the raft
protein
complex , for example through oxidative damage to the proteins or lipids ( refs 70 , 71 ) ,
could theoretically induce the dissociation of the amyloid peptides from
glycosphingolipids such as galcer .
then , the sudden exposure of aromatic side chains would
allow a tight packing ( through interactions ) between
adjacent helices .
this would lead to the formation of a dimer , a key step in the
generation of amyloid fibrils ( refs 72 , 73 ) , which is thought to proceed through an ordered
polymerisation of structures ( ref .
when bound to glycosphingolipids ( gsls ) through the
sphingolipid - binding domain , the aromatic residues of two vicinal amyloidogenic
proteins can not interact with each other and the protein is forced to adopt an
-helix conformation .
the molecular mechanism of this interaction is the
ch stacking between the galactosyl ring of galcer and the
aromatic residue of the protein .
the -helix structure of amyloidogenic
proteins is not only present in membrane - bound monomers ( as illustrated in fig .
2 , right panel ) , but also in oligomeric
-synuclein channels ( fig .
the
-amyloid aggregation process is triggered when the
glycosphingolipid protein complex is destabilised , resulting in the
release of the protein from its glycosphingolipid chaperone ( a ) . in this new
glycosphingolipid - free environment
, the aromatic residues of two vicinal
amyloidogenic proteins can interact together ( b ) , inducing a drastic conformational
change from an initial -helix to a -strand structure .
amyloid
aggregation then results from the -driven assembly of
aromatic side chains ( for a review , see ref .
79 ) , forming amyloid dimers that in turn associate to form amyloid fibrils
( as illustrated in fig .
when bound to glycosphingolipids ( gsls ) through the
sphingolipid - binding domain , the aromatic residues of two vicinal amyloidogenic
proteins can not interact with each other and the protein is forced to adopt an
-helix conformation .
the molecular mechanism of this interaction is the
ch stacking between the galactosyl ring of galcer and the
aromatic residue of the protein .
the -helix structure of amyloidogenic
proteins is not only present in membrane - bound monomers ( as illustrated in fig .
2 , right panel ) , but also in oligomeric
-synuclein channels ( fig .
the
-amyloid aggregation process is triggered when the
glycosphingolipid protein complex is destabilised , resulting in the
release of the protein from its glycosphingolipid chaperone ( a ) . in this new
glycosphingolipid - free environment
, the aromatic residues of two vicinal
amyloidogenic proteins can interact together ( b ) , inducing a drastic conformational
change from an initial -helix to a -strand structure .
amyloid
aggregation then results from the -driven assembly of
aromatic side chains ( for a review , see ref .
79 ) , forming amyloid dimers that in turn associate to form amyloid fibrils
( as illustrated in fig .
, we would like to underscore ( 1 ) the key role of aromatic residues
in the self - assembly of amyloid fibrils and ( 2 ) the chaperone effect of raft
glycosphingolipids that could induce otherwise thermodynamically unstable -helix
structures of amyloid fibril - forming proteins . indeed , most amyloidogenic proteins
( including a and prp ) have an amino acid sequence that is expected to form
but not structures ( ref .
-helix/-strand discordant. lipid rafts can
prevent those proteins from adopting the conformation they prefer , stabilising their
structure in a forced conformation .
this stabilisation
can occur in the early biosynthetic pathway as demonstrated for prp ( ref .
inhibition of sphingolipid biosynthesis
logically increases prp misfolding into disease - associated structures ( ref .
this mechanism seems to also apply for
several neurotransmitter receptors ( e.g. the nicotinic acetylcholine receptor ) whose
transport and assembly in the plasma membrane require the assistance of sphingolipids
( ref .
mutations in amyloidogenic proteins
that are associated with inherited forms of neurodegenerative diseases have been shown to
directly affect sphingolipid binding .
this is the case for the
creutzfeldt jakob - linked e200k mutation of prp , which affects
prp sphingomyelin interactions ( ref .
, by providing a complementary surface for amyloidogenic proteins , cellular
glycosphingolipids might constitutively stabilise their conformation and
inhibit their aggregation , as recently demonstrated for amylin ( ref .
protein
interactions ( refs 18 , 50 ) would then trigger amyloid growth
and deposition ( refs 49 , 79 ) .
to what extent glycosphingolipids could also stimulate
-strand - mediated amyloid aggregation ( ref .
the mutual
effect of gm1 and a on their respective conformations is likely to displace the
equilibrium of the reaction towards the formation of amyloid aggregates , as observed in
vitro ( ref .
finally , it should be noted that noncovalent interactions distinct from
ch stacking might also contribute to sphingolipid
81 ) , h - bonds and electrostatic bridges ( especially for sulfated
glycosphingolipids , gangliosides and sphingomyelin ) ( ref . 82 ) .
indeed , in addition to aromatic side chains ( tyr10 ) , several
aliphatic ( ref .
81 ) and basic residues such as
his13 have been implicated in gm1a interactions ( ref .
anionic phospholipids such as
phosphatidylserine , which bind to -synuclein through electrostatic interactions
( ref .
84 ) , could , in the cytoplasmic
leaflet of the plasma membrane , have a role equivalent to the one played by gangliosides
in the external leaflet .
high cholesterol has been identified as a major risk factor for both alzheimer ( ref .
85 ) and parkinson ( ref . 86 ) diseases , and dysregulation of cholesterol homeostasis is a
hallmark of neurodegenerative diseases ( ref .
unfortunately , the effects of cholesterol on amyloid fibrillogenesis and toxicity are not
well understood and the results reported so far are controversial ( ref .
89 ) and stimulates its insertion into phospholipid
monolayers ( ref . 90 ) .
92 ) or
decreases ( refs 93 , 94 ) a polymerisation is still uncertain .
moreover , the
generation of a peptides through app proteolysis occurs within lipid rafts and
is sensitive to inhibitors of cholesterol biosynthesis ( ref .
95 ) , so that the involvement of cholesterol homeostasis in alzheimer
disease can not be simply ascribed to the regulation of a fibrillogenesis . in the case of parkinson disease , data obtained with both cultured neuronal cells and
transgenic mice have shown that the cholesterol - depleting agent
methyl--cyclodextrin decreased the level of -synuclein in membrane
fractions ( ref .
96 ) . moreover , metabolic
inhibition of cholesterol biosynthesis with statins reduced the levels of
-synuclein accumulation in neuronal membranes , whereas cholesterol
supplementation of cultured neurons increased -synuclein aggregation ( ref .
consistent with these studies , it was recently
reported that lovastatin treatment of -synuclein transgenic mice was associated
with a marked reduction of -synuclein aggregation and abrogation of neuronal
pathology ( ref .
this suggests that treatment
with cholesterol - lowering agents might be beneficial for patients with parkinson disease .
it has been suggested that oxidised cholesterol metabolites could accelerate
-synuclein aggregation ( ref .
71 ) , which
provides a mechanistic link between oxidative stress and parkinson disease .
yet the
molecular mechanisms by which cholesterol and/or its metabolites could affect the
oligomerisation / aggregation status of amyloidogenic proteins have not been fully
deciphered .
first , cholesterol has a major impact on the conformation of sphingolipids . the apolar
part of sphingolipids ( i.e. the most important part of the ceramide ) interacts with the
smooth face of cholesterol , whereas the oh group of cholesterol is accessible to the polar
part of the sphingolipid ( ref .
this oh group is involved in an h - bond network that restricts the
conformation of the sugar moiety in a parallel orientation with respect to the membrane .
this effect is particularly important when the ceramide contains a nonhydroxylated fatty
acid ( nfa , fig .
because this conformation of
the sphingolipid is particularly suited for a sphingolipid - binding domain , cholesterol
usually accelerates protein binding to sphingolipid ( ref .
by contrast , when the sphingolipid contains a
hydroxylated fatty acid ( hfa ) , the oh group of cholesterol is excluded from the h - bond
network and it can not exert its conformational effect on the sphingolipid . in this case ,
cholesterol does not improve but rather perturbs the organisation of sphingolipids and can
even inhibit glycosphingolipid binding to amyloidogenic proteins ( ref .
thus , according to the distribution of hfas
versus nfas in sphingolipids , an increase of membrane cholesterol can lead to opposite
effects on sphingolipid - mediated amyloidogenic protein binding and aggregation . in any
case , these effects are due to a fine tuning of sphingolipid conformation induced by
cholesterol , and do not involve any kind of physical interaction between amyloidogenic
proteins and cholesterol .
by contrast , the second mechanism involves a direct interaction between the amyloidogenic
protein and cholesterol .
therefore , this effect requires the insertion of a part of the
amyloid protein in the membrane ; otherwise the protein would not be in physical contact
with cholesterol .
100 )
have shown that a peptide aggregation on the bilayer surface requires a
sphingomyelin - rich environment but can occur in the absence of cholesterol .
this is in
line with our hypothesis that sphingolipids are fully responsible for the initial binding
of amyloidogenic proteins to lipid rafts , and that cholesterol is not directly involved at
this stage .
however , at the second step the presence of cholesterol may greatly facilitate
peptide insertion into the bilayer .
the preferential interaction of amyloidogenic proteins
with lipid raft domains ensures that cholesterol is indeed present underneath the
sphingolipids that have attracted the unstructured monomer ( fig .
once inserted in the membrane , the amyloidogenic protein
will immediately find cholesterol and interact with its free side that is , the
face that is not in contact with sphingolipids ( for a detailed explanation of cholesterol
topology , see ref .
figure 6lipid - dependent formation of an -synuclein oligomeric
channel . unfolded -synuclein monomers ( a ) are attracted by the
polar heads of glycosphingolipids and concentrated on the surface of
sphingolipid cholesterol membrane domains ( b ) .
sphingolipids , complexed
with cholesterol , induce a conformational change ( unstructured to -helix
transition ) of -synuclein .
the newly formed -helices can insert
into the plasma membrane ( c ) where they can further oligomerise under the control of
cholesterol molecules ( blue arrows ) ( d ) and eventually form an oligomeric ion
channel .
such channels are thought to disturb membrane permeability to calcium ions
( red arrows ) , resulting in neuronal dysfunction and toxicity .
the minus signs ( in
red ) refer to the negative charges of -synuclein .
unfolded -synuclein monomers ( a ) are attracted by the
polar heads of glycosphingolipids and concentrated on the surface of
sphingolipid cholesterol membrane domains ( b ) .
sphingolipids , complexed
with cholesterol , induce a conformational change ( unstructured to -helix
transition ) of -synuclein .
the newly formed -helices can insert
into the plasma membrane ( c ) where they can further oligomerise under the control of
cholesterol molecules ( blue arrows ) ( d ) and eventually form an oligomeric ion
channel .
such channels are thought to disturb membrane permeability to calcium ions
( red arrows ) , resulting in neuronal dysfunction and toxicity .
the minus signs ( in
red ) refer to the negative charges of -synuclein . by stimulating the oligomerisation of membrane - inserted amyloidogenic proteins ,
cholesterol could facilitate the formation of a pore - like assembly displaying ion channel
properties ( fig .
10 ) have hypothesised that amyloid pore / channels
provide the most direct pathway for inducing neurodegenerative effects , through loss of
ionic homeostasis increasing cell calcium to toxic levels .
the structure of amyloid pores ,
which have been observed by atomic force microscopy ( refs 9 , 10 ) , remains to be experimentally
elucidated at the atomic level .
computational models have suggested that most amyloid
pores are probably formed by a complex assembly of -rich protofibrils or
oligomers ( ref .
130 ) , as shown in figure 1 . however , in the case of
-synuclein , tsigelny et al .
101 )
have elegantly modelled a realistic oligomeric channel formed by the assembly of
-helical monomers .
the possible events leading to the formation of an
-synuclein oligomeric channel under the dual control of sphingolipids and
cholesterol are summarised in figure 6 .
there is a
striking similarity between -synuclein ( refs 69 , 31 ) and colicin e1 , a bacterial
toxin that also inserts into anionic areas of the plasma membrane , forming channel - like
structures consisting of an -helix bundle ( ref .
moreover , it has been hypothesised that amyloid pores might be
similar to -barrel pore - forming bacterial toxins ( ref .
103 ) . since cholesterol controls the oligomerisation and insertion
of these bacterial toxins ( ref .
104 ) in the
target membrane , it is likely that it is also required for the assembly of amyloid pores .
how could cholesterol control the oligomerisation process of membrane - inserted
amyloidogenic proteins and facilitate the formation of amyloid pores ?
the recent
high - resolution crystal structure of an engineered human adrenergic 2 receptor ,
suggesting a molecular mechanism by which cholesterol mediates receptor dimerisation ( ref .
on the basis of these structural data , one could hypothesise that
cholesterol binds to membrane - embedded fragments of amyloidogenic proteins , facilitates
their recruitment and coordinates their oligomerisation .
overall , this would mean that
amyloid pore formation and ion channel function are under the control of both
sphingolipids and cholesterol , in agreement with recent data obtained in model membranes
with -synuclein ( ref .
finally , it has been reported that
spherical amyloid oligomers or protofibrils can also increase membrane conductance without
forming ion channels or pores ( ref .
it is
not known if cholesterol and/or sphingolipids are involved in this nonspecific
permeabilising activity .
the involvement of gangliosides in several neurodegenerative diseases is not totally
surprising if one considers that these sphingolipids are critical for neuronal integrity and
plasticity ( ref .
since the probability of acquiring these
diseases gradually increases with age , it is important to evaluate how ganglioside
expression varies during the lifetime of individuals , and whether these diseases can alter
this process ( ref .
the
differentiation of pc12 cells into neuron - like cells caused a marked increase in both
gangliosides and cholesterol , and thereby greatly potentiated the accumulation and
cytotoxicity of a ( ref .
gm1 seems
to control the oligomerisation and aggregation process of a and
-synuclein ( refs 29 , 30 , 31 ) .
gm3
has been shown to control -synuclein channel formation and to correct the
channelopathy induced in planar lipid membranes by the parkinson - disease - linked e46k mutant
of the protein ( ref .
gm3
is a minor ganglioside , which , in marked contrast with the major ganglioside species gm1 and
gd1a , shows a regular increase with age ( ref .
the expression of gm3 is highly regulated during brain development ( ref . 113 ) , and this ganglioside has been implicated in the
regulation of neuronal cell death ( ref .
114 ) .
moreover , the homozygous loss - of - function mutation of gm3 synthase , which totally suppresses
the expression of gm3 and all gm3-derived gangliosides , has been linked to an infantile
symptomatic epilepsy syndrome ( ref .
these data indicate that ganglioside gm3 probably plays a more important role in
brain physiology and pathology than its low expression levels in adult brain could have
suggested in the past .
several studies support the view that deregulation of lipid metabolism is an important
feature of neurodegenerative diseases .
the three main lipid categories
glycerophospholipids , sphingolipids and cholesterol are affected ( refs 116 , 117 ,
118 , 119 , 120 , 121 ) .
a large body of data has established a link between cholesterol
homeostasis , apolipoprotein e polymorphism and app processing ( ref .
correspondingly , cholesterol - lowering strategies with statins
have acquired potential therapeutic importance in treating alzheimer disease ( ref .
statins have also proved to be efficient in
decreasing -synuclein aggregation and neuronal toxicity in animal models of
parkinson disease ( ref .
specific changes in
ganglioside content have been detected in the brains of patients with alzheimer disease
( refs 116 , 125 ) : a decrease or even loss of the major gangliosides gm1 , gd1a , gd1b and gt1b ,
and an increase in gm2 , gm3 and gd3 .
similar alterations of ganglioside expression were
observed in the brains of transgenic mouse models of alzheimer disease ( ref .
moreover , the balance between nfa and hfa ceramides is altered in animal models ( ref .
interestingly , the authors of the latter study
showed that there is a gender - dependent accumulation of ceramides in the cerebral cortex :
female mice exhibited a strong increase in hfa species , and males in nfa species .
this
observation could be linked to the increased risk of women versus men for developing
alzheimer disease ( ref .
129 ) . because cholesterol
can exert distinct effects on amembrane interactions according to the
nfa : hfa content of brain sphingolipids ( ref .
52 ) ,
it will be interesting to determine how cholesterol impacts on
aglycosphingolipid interaction in men and women suffering from
alzheimer disease .
how amyloidogenic proteins kill neurons is still a mystery . in particular , as recently
discussed by butterfield and lashuel ( ref .
130 ) ,
there remains a knowledge gap regarding the molecular - level details by which
amyloid - forming proteins act on the membrane and induce membrane
permeabilization. deciphering the complex interplay between amyloidogenic
proteins and membrane lipids , especially sphingolipids and cholesterol , will certainly help
to achieve a clearer view on this enigma .
several milestones have already been reached ,
which have inspired an important part of today 's research efforts worldwide . despite their
lack of amino acid sequence homology ,
amyloidogenic proteins share a number of intriguing
properties : ( 1 ) an important conformational plasticity , allowing the same protein to remain
unordered or to form highly ordered and structures ( refs 4 , 5 ) ; ( 2 )
self - oligomerising capacities that revealed unexpected common antigenic properties ( ref .
7 ) ; ( 3 ) the ability to form pore - like structures
with ion channel properties ( refs 9 , 10 ) ; ( 4 ) self - aggregating activity leading to
fibrillation and plaque deposition ( ref .
131 ) ; and
( 5 ) common structural motifs allowing specific interactions with sphingolipids ( ref .
49 ) and cholesterol ( yet uncharacterised ) in lipid raft
domains of the plasma membrane .
it is intriguing that hiv-1 , which interacts with
glycosphingolipids through the v3 domain of its surface glycoprotein gp120 , can induce major
neurological disabilities , identified as hiv-1-associated dementia ( ref .
110 ) . in this respect , it is also worth noting that
the protein cln3 , involved in batten disease ( the juvenile form of neuronal ceroid
lipofuscinosis ) , also binds to galcer through a v3-like sphingolipid - binding domain ( ref .
this neural disease is caused by a
mutation in this domain , namely e295k ( ref .
133 ) ,
which is analogous to mutations e22k in a , e46k in -synuclein and e200k
in prp , all associated with inherited forms of neurodegenerative disease and located in the
sphingolipid - binding domains of these proteins . at least for two of these proteins ( prp and
-synuclein ) , the substitution of an anionic glutamic acid side chain by a cationic
lysine resulted in altered binding to membrane sphingolipids ( refs 27 , 31 ) .
this further
emphasises the key role of the sphingolipid - binding domain in neuronal diseases .
future studies will be necessary to better evaluate the neurotoxicity of the various forms
of amyloidogenic proteins , including monomers , oligomers and aggregates , and to understand
how these different species cooperate to accelerate ( or slow down ) the onset of
neurodegenerative symptoms . this will help to decide on a rational basis which therapeutic
strategy should be used at the different stages of the diseases .
this will be possible with
the finding of nontoxic drugs specifically affecting amyloid channels ( ref .
135 ) . combining molecular ( refs 31 , 52 ) , biophysical ( refs
14 , 31 ,
69 , 136 , 137 ) , cellular ( refs 93 , 114 ,
138 ) and animal ( refs 126 , 128 , 139 ) studies will allow a better characterisation of
the sphingolipids that regulate amyloid oligomerisation / aggregation .
biophysical properties
of the membrane such as membrane curvature should be taken into consideration ( ref .
130 ) . indeed , depending on the surface curvature of
the model membrane ,
interestingly , gm1 and gm3 are
concentrated in membrane areas that markedly differ in membrane curvature ( ref .
143 ) , so that on binding to distinct gangliosides , the
same amyloid protein could adopt distinct conformations
. this could be the case for
-synuclein , which recognises both gm1 ( ref .
antiganglioside
antibodies could interfere with normal ganglioside function and could thus play a role in
disease pathogenesis , as anti - gm1 antibodies probably do in some parkinson patients ( refs
110 , 144 ) .
careful determinations of the titres of these antibodies in various biological
fluids , including cerebrospinal fluid , will be particularly informative
. beneficial effects
of ganglioside supplementation have been reported in animal models of parkinson disease
( ref .
enzymes involved in glycosphingolipid metabolism might represent targets that inhibit both
the production and amyloid aggregation of alzheimer a peptides ( ref .
correspondingly , lipid raft disruption has been
shown to protect neurons against amyloid oligomer toxicity in vitro ( ref .
we need to better understand how cholesterol
interacts with amyloidogenic proteins , regulates their supramolecular structures and is
involved in the pathophysiology of neurodegenerative diseases ( refs 86 , 87 ) .
we should also
determine how mutations of amyloidogenic proteins affect their interaction with neural
membranes ( refs 27 , 69 , 148 ) .
a decisive
breakthrough will be to understand why neurodegenerative diseases involve specific brain
areas , and how local lipid composition may account for such geographic restrictions ( refs
127 , 135 ) .
finally , we have to identify the environmental molecules ( such as food
contaminants , pesticides and mycotoxins ) that could modulate amyloid formation through
direct binding to amyloid proteins and could represent important risk factors for
neurodegenerative diseases ( ref . | alzheimer , parkinson and other neurodegenerative diseases involve a series of brain
proteins , referred to as amyloidogenic proteins , with exceptional
conformational plasticity and a high propensity for self - aggregation .
although the
mechanisms by which amyloidogenic proteins kill neural cells are not fully understood , a
common feature is the concentration of unstructured amyloidogenic monomers on
bidimensional membrane lattices .
membrane - bound monomers undergo a series of
lipid - dependent conformational changes , leading to the formation of oligomers of varying
toxicity rich in -sheet structures ( annular pores , amyloid fibrils ) or in
-helix structures ( transmembrane channels ) .
condensed membrane nano- or
microdomains formed by sphingolipids and cholesterol are privileged sites for the binding
and oligomerisation of amyloidogenic proteins . by controlling the balance between
unstructured monomers and or conformers ( the chaperone effect )
,
sphingolipids can either inhibit or stimulate the oligomerisation of amyloidogenic
proteins .
cholesterol has a dual role : regulation of protein
sphingolipid
interactions through a fine tuning of sphingolipid conformation ( indirect effect ) , and
facilitation of pore ( or channel ) formation through direct binding to amyloidogenic
proteins . deciphering this complex network of molecular interactions in the context of
age- and disease - related evolution of brain lipid expression will help understanding of
how amyloidogenic proteins induce neural toxicity and will stimulate the development of
innovative therapies for neurodegenerative diseases . | Mechanisms of concentration of amyloidogenic proteins on neuronal membranes
Molecular mechanisms of sphingolipidamyloidogenic-protein interactions
Glycosphingolipids exert a chaperone effect on amyloidogenic proteins
The outside and inside effects of cholesterol
Changes in lipid content of the brain: what impact on neurodegenerative diseases?
Perspectives |
atherosclerosis , a progressive inflammatory disease , results in the development of plaques within the arterial walls that may contain intracellular and extracellular lipids , a variety of inflammatory cells , extracellular matrix , smooth muscle cells , fibroblasts , and calcification [ 14 ] .
acute coronary syndrome ( acs ) and stroke are typically associated with the development of vulnerable plaques .
a vulnerable plaque is characterized by an accumulation of macrophages and macrophage - derived foam cells , degradation of the extracellular matrix , and the formation of a necrotic core , neoangiogenesis , intraplaque hemorrhage , and a thin fibrous cap [ 3 , 57 ] .
thus , the prevention of a heart attack or stroke would benefit from the early detection of the biological processes resulting in the inflammation and neoangiogenesis associated with atherosclerotic lesions .
the noninvasive detection of atherosclerotic plaque has been clinically limited mostly to the measurements of arterial stenosis in selected arterial beds [ 811 ] .
such measurements have been shown to be poor predictors of clinical outcomes , thus stimulating further research into the biology of atherosclerotic lesions and development of new biomarkers with a better predictive value for the formation of vulnerable plaques [ 1217 ] .
inflammation of arterial walls is initially triggered by the accumulation of low - density lipoprotein ( ldl ) particles in the vessel wall and their subsequent oxidative and enzymatic modification followed by the activation of adhesion molecules and recruitment of t lymphocytes and monocytes .
activated monocytes differentiate into macrophages , which accumulate modified ldl particles , evolving into foam cells [ 18 , 19 ] . as atherosclerosis progresses ,
endothelial cells , macrophages , and foam cells die , releasing matrix metalloproteinases and other intracellular enzymes such as cathepsins [ 2022 ] .
the released proteases cleave the extracellular matrix destabilizing the plaque core and the fibrous cap [ 23 , 24 ] .
transformation of monocytes into macrophages and eventually into foam cells is controlled by expression of extracellular receptors including v3 integrin .
another destabilizing factor of atherosclerotic lesions is angiogenesis , which is associated with v3 integrin expression , and it is involved in cell - cell and cell - matrix adhesion .
the formation of new vessels originates from adventitial vasa vasorum and results in immature , fragile , and leaky vasculature , triggering intraplaque hemorrhage .
molecular imaging biomarkers detecting metalloproteinase and cathepsin protease activity have been used by radiolabel - based and optical imaging modalities for the detection of inflammation in animal models of atherosclerosis and tissues from human atherosclerotic lesions [ 9 , 30 ] .
cathepsin activatable , optical imaging agents based on cleavage of l - lysine sequences have been successfully applied in the characterization of atherosclerotic lesions in mouse and rabbit models of atherosclerosis and in the detection of cathepsin protease activity in human atherosclerotic plaques obtained at carotid endarterectomy [ 3136 ] . using these agents , fluorescence molecular tomography ( fmt ) and computed tomography ( ct )
have been applied to quantitatively evaluate the levels of inflammation in the mouse apoe models of atherosclerosis .
furthermore , agents with v3 integrin targeted ligand have been employed to detect inflammation as well as angiogenesis in atherosclerotic lesions of animal models using a broad spectrum of imaging modalities [ 3843 ] . here
we describe the development and evaluation of new cathepsin activatable agents and compare them to the new v3 integrin targeting agent in a longitudinal study of atherosclerotic apoe mice undergoing prophylactic treatment with the anti - inflammatory therapeutic ezetimibe [ 44 , 45 ] using fmt .
hek293-v3 cells were lifted with trypsin - edta and washed four times with serum - free mem .
cells ( 25,000 cells / well ) were added to microtiter wells coated with vitronectin or fibronectin and allowed to attach for 2 hours at 37c in a humidified incubator in the absence or presence of increasing concentrations of integrisense or parent compound .
similar experiments were carried out with hek293 cells stably transfected with v5 , iib3 , or 51 .
the attached cells were quantified by colorimetric detection of hexosaminidase enzymatic activity in a vmax plate reader ( molecular devices , menlo park , ca ) .
the number of attached cells was quantified using a standard curve for each cell line assayed and expressed as a mean value of triplicate samples .
ten - week old apoe female mice ( n = 184 total : 20 for in vivo - ex vivo signal correlation study , 44 for longitudinal study , 120 for treatment study ; taconic farms , inc , hudson , ny ) fed on a 0.15% [ wt / wt ] cholesterol diet which included 9% of their caloric intake from fat ( research diets , inc , new brunswick , nj ) were used as an animal model of atherosclerosis . in the longitudinal study , c57bl/6 mice (
n = 14 total ; taconic farms , inc , hudson , ny ) fed on a normal chow diet served as controls . in the treatment study
, apoe mice were fed the diet described above with or without cholesterol absorption inhibitor ezetimibe ( 7 mg / kg / d ) .
all experiments were approved by our institutional animal care and use committee ( iacuc ) .
aged apoe mice ( 8 to 10 months on diet ) , which developed atherosclerotic lesions , were injected with prosense 750 ( 5 nmol per animal ) , prosense fast 750 ( 8 nmol per animal ) , or integrisense 750 ( 4 nmol per animal , all from perkin elmer , boston , ma ) via tail vein 24 hr or 48 hr before fmt imaging , respectively .
images were acquired by fmt2500 ( perkin elmer ) at 750 nm wavelength . the resulting 3d dataset was reconstructed and the region of interest ( roi ) was defined within the chest area ( figure 2 ) using the vendor 's software ( truequant , perkin elmer ) , and the threshold
after in vivo imaging , animals were euthanized with co2 , and their aortas were excised .
fluorescence reflectance images of the dissected arteries ( carotid arteries with aortic arch and thoracic part of descending aorta ) were then acquired with the fmt2500 . to verify the in vivo fmt signal noninvasively
, the mice were imaged with a ge explore locus ultra ( micro ct , ge ) after fmt acquisition using a ct bed designed to hold the fmt animal cassette therefore ensuring the animal remained in the same position .
the fmt dataset was exported in dicom format and then co - registered to the ct dataset using amira 5.2 ( visage imaging , san diego , ca ) .
after probe - injected mice were euthanized , their aortas were excised and terminal blood samples were collected . the aortas were minced with fine scissors and dissociated into single cells by incubating with liberase th and tm ( roche applied science , indianapolis , in ) at 37c for 1.5 hours .
the resulting cell suspensions were then passed through 40 m cell strainer ( bd biosciences , bedford , ma ) and washed with pbs .
the blood samples were incubated in ack buffer ( cambrex , walkersville , md ) to remove blood cells and then washed with pbs . for multicolor flow cytometry analysis of lymphoid cells , myeloid cells ( including monocytes / macrophages and neutrophils ) and other cells ( including stromal cells of the aortic wall ) , cells were incubated with a cocktail of mabs ( bd biosciences , san francisco , ca ) against t cells ( cd90-fitc),b cells ( b220-fitc ) , nk cells ( cd49b - fitc , nk1.1-fitc ) , and myeloid cells ( cd11b - pe ) at 4c for 30 min and then analyzed on an lsrii ( bd biosciences , san francisco , ca ) .
ashland , or ) . to demonstrate co - localization of macrophages and the integrisense 680 and prosense 680 probes ,
ten micron - thick cryosections were prepared and fixed in ice - cold acetone / ethanol ( 3 : 1 ) for 10 min . and washed in pbs .
nonspecific binding was blocked by incubation with 5.0% nonimmune goat serum for 30 min followed by incubation with the rat anti - mouse cd68 antibody ( 5 g / ml ; mca-1957 , abd serotec , raleigh , nc ) for 1 hr at room temperature .
sections were washed in pbs and incubated with alexafluor 488 goat anti - rat igg ( 4 g / ml ; molecular probes , eugene , or ) for 1 hr and again washed in pbs .
sections were mounted using a fluorescent mount medium with or without dapi counterstain and examined with a nikon e-1000 microscope equipped with the appropriate band - pass filters .
for the longitudinal study , both apoe ( n = 15 for each agent ) and c57bl/6 ( n = 9 for prosense , n = 5 for integrisense ) mice were injected with prosense750 or integrisense750 and imaged with the fmt2500 as described above .
the scans were conducted at the times indicated in the figures . for the treatment study , prosense ,
catb fast agent or prosense fast ( 8 nmol per animal , perkin elmer ) were injected into apoe mice ( both treated ; n = 15 for each agent , and untreated ;
n = 15 for each agent ) via tail vein 24 hr before imaging .
integrisense ( 2 nmol per animal , perkin elmer ) injected mice were images 48 hr after injection .
the imaging acquisition was carried out at the time points as depicted in figures 7 and 8 .
after apoe mice were euthanized , their aortas were excised and fixed in 10% buffered formalin ( figure 6(a ) ) .
all vessels were embedded into paraffin blocks , sectioned at 4 m , and placed onto adhesive - coated slides .
slides were stained with hematoxylin and eosin to demonstrate general morphology and immunostained with mabs against the f4/80 antigen ( 1 : 100 , ebioscience , inc . , san diego , ca ) .
slides were subsequently incubated with a peroxidase - conjugated anti - mouse igg antibody and 33-diaminobenzidine ( dab ) chromogen to identify macrophages within the plaque regions and counterstained with hematoxylin ( figure 6(c ) ) .
the stained slides were analyzed using the ariol image analysis platform ( genetix , san jose , ca ) for macrophage quantification .
regions of interest were drawn to include only the plaque area and appropriate size , and color classifiers were applied to specifically identify the dab stained ( brown ) macrophages , which were then counted .
terminal serum samples ( n = 12 ) were collected at the end of the treatment study , and the hdl - c and total cholesterol levels were determined by following the manufacturer 's protocol of hdl - cholesterol e kit ( wako chemicals , richmond , va ) with and without adding precipitating reagent , respectively .
terminal plasma samples ( n = 12 ) were collected at the end of the treatment study and assayed for and ezetimibe - glucuronide using aria lc - ms / ms with a standard calibration range from 5 to 10,000 nm and a quantification limit of 5 nm .
data are presented as mean sem . for statistical analysis , we used anova followed by bonferroni t - test for multiple comparisons .
the correlation between the in vivo and ex vivo signal was evaluated using linear regression analysis .
structural schematics , spectral characteristics , and the activation selectivity of the cathepsin agents and a chemical structure of v3 integrin agent used in our experiments are depicted in figure 1 .
the cathepsin activated agent prosense ( figures 1(a ) and 1(b ) ) , used in the detection of atherosclerotic lesions before [ 3437 ] , is based on ~400 kda polylysine macromolecules with covalently attached fluorophores .
the newly developed cathepsin agents prosense fast and cat b fast are based on low molecular weight substrate peptides with fluorochromes attached to the c- and n - termini of the peptide resulting in 95% of the fluorescence quenching and a hypsochromic ( blue ) shift of about 60 nm in the absorption spectra ( figures 1(c ) and 1(d ) ) .
the quenching of the fluorophores in the prosense agent is achieved by tight packing of the polymer coil , which slows the activation of this agent .
thus , the full activation of the prosense agent requires 24 hours while the newly designed peptide - based agents are fully activated within 6 to 8 hours ( data no shown ) .
the selectivity of the agents for activation by 24 hours incubation with different cathepsins is shown in figure 1(e ) .
both prosense and prosense fast agents were activated by most of the evaluated cathepsins , while the cat b fast agent was activated mostly by cathepsin b ( figure 1(e ) ) .
the v3 targeted agent , integrisense , is based on a low molecular weight peptidomimetic ligand of v3 integrin , which was chemically modified to covalently attach the vivotag ( nirf ) ( figure 1(f ) ) .
the affinity of the agent for activated v3 integrin was determined to be ~ 0.5 nm and the agent is highly selective for v3 ( table 1 ) . to assess the severity of atherosclerotic disease , we focused on the aortic arch and the proximal portion of the descending aorta .
since there is limited anatomical information to guide in vivo fmt analysis , we compared fluorescent signal within the roi drawn to capture the entire volume of the chest region with the signal measured within the roi drawn on the excised arteries ( for details see methods ) to ensure the accuracy of the in vivo quantification ( figures 2(a)2(f ) ) . the total amount of the probes ( pmol ) detected in vivo correlated well with the fluorescence ( counts / energy ) measured ex vivo for all evaluated agents ( figures 2(b ) , 2(d ) , and 2(f ) ) .
this suggests that the in vivo quantification reflects the actual signal from the area of interest and can therefore be used to measure the inflammation non - invasively using fluorescence of these imaging agents . to further identify the location of the signal , we co - registered the fmt signal with ct images based on fiducial landmarks that are incorporated into the animal holder and are identified by both fmt and ct .
using anatomical detail from the ct image combined with fmt molecular information , we determined that the majority of the fluorescent signal was in the aortic root , arch , and the proximal portion of the descending aorta of animals injected with cathepsin - activated ( figures 3(a ) and 3(b ) or v3-targeted agents ( figures 3(c ) and 3(d ) ) . to validate the cellular source of the fluorescence signal in vivo , we performed flow cytometry of single cell suspensions generated from digested murine aortas .
monocytes / macrophages were identified as the dominant cellular signal source for all cathepsin activated agents tested .
we observed a similar labeling profile in the blood ; however , the signal intensity was 10 times lower compared to the aortic signal .
fluorescence signal from the v3 targeted agent , integrisense , was found not only in monocytes / macrophages but also in lymphoid and stromal cells .
the signal from integrisense was higher in monocytes / macrophages than in lymphoid cells in the blood ( figure 4 ) .
the microscopic fluorescence distribution of both cathepsin activated and v3 targeted agents was compared to immunoreactive staining for macrophages cd68 ( figure 5 ) .
there was good colocalization of fluorescence from the optical agents with immunofluorescent cd68 signal , while some of v3 agent was found located in the adventitia .
macrophages have been shown to play an important role in the inflammatory process of atherosclerosis [ 1 , 2 , 5 , 18 ] . to evaluate the prognostic value of different optical agents for non - invasive detection of the disease , quantitative immunohistochemical analysis of macrophages in aortic arteries using f4/80 monoclonal antibodies
was performed , and the macrophage counts were then correlated with in vivo fmt measurements of the same animals .
quantification of the in vivo fmt signal and ex vivo macrophage count showed a good correlation for both prosense ( r = 0.89 , p < 0.05 ) and cat b fast ( r = .86 , p < 0.05 ) , but not for integrisense ( r = 0.35 , p = 0.44 ) ( figures 6(d)6(f ) ) . to access cathepsin activity or v3 integrin expression as a function of time throughout disease progression , we performed in vivo fmt imaging in both apoe and c57bl/6 mice at time points indicated in figure 7 .
a statistically significant increase in the amount of prosense and integrisense was observed in the chest area of apoe versus c57bl/6 mice starting 25 and 22 weeks on diet , respectively .
we further evaluated the newly developed agents for the detection of reduced inflammation associated with atherosclerotic lesions in apoe mice upon prophylactic treatment with the cholesterol lowering agent , ezetimibe .
ezetimibe is known to lower ldl and inflammation associated with the atherosclerosis in apoe mice . in our experiments
, ezetimibe lowered the ldl and elevated hdl levels about threefold ( table 2 ) .
cat b fast detected a statistically significant effect of the treatment at both 21 and 31 weeks on the diet ( figure 8(c ) ) , while the large , nonselective , polymer - based agent , prosense , identified a statistically selective effect of the treatment only at 31 weeks on diet ( figure 8(a ) ) .
the newly developed nonselective agent , prosense fast , found a statistically significant effect of the treatment only at 12 weeks ( figure 8(b ) ) while integrisense was insensitive to the effect of treatment by ezetimibe ( figure 8(d ) ) .
heart attack and stroke , often fatal clinical outcomes of atherosclerosis , are frequently associated with vulnerable arterial plaques that develop as a product of inflammatory processes initiated by high levels of ldl .
thus , molecular imaging biomarkers of inflammation may play an important role in the monitoring the development and efficacy of new therapeutics as well as in the early detection of the disease . in this paper
, we describe the development and evaluation of new cathepsin activatable agents and their comparison to the new v3 integrin targeted agent in the detection of atherosclerosis associated inflammation in apoe mice using established fmt technology .
we further demonstrate the sensitivity of individual biomarkers to detect inhibition of inflammation in prophylactic studies with the therapeutic ezetimibe .
we have developed new cathepsin agents , prosense fast and cat b fast , based on low molecular weight peptides flanked by fully quenched fluorophores .
once the protease cleaves the low molecular weight peptide , the fluorophores diffuse apart leading to a full fluorescence signal .
these agents reach maximum activation within six to eight hours , while the polymer - based agent prosense [ 27 , 37 ] requires 24 hours for full activation .
activation of prosense fast is nonselective for tested cathepsins similar to the polymer - based prosense , which is only weakly selective for cathepsin b. conversely , the cat b fast agent is highly selective for cathepsin b. thus , parallel measurements using the new agents and polymer - based prosense can address the effect of the protease selectivity and to some extent also the potential effect of the polymer coil on the detection sensitivity of atherosclerosis associated inflammation .
the v3 integrin targeted agent , integrisense , has been used successfully in the detection of tumor growth and rgd - based v3 integrin targeted agents in the detection of inflammation and angiogenesis in mouse and rabbit models of atherosclerosis [ 4042 ] .
we evaluated the sensitivity and specificity of integrisense in the detection of atherosclerotic lesions in the apoe mice model of atherosclerosis and compared it to the cathepsin - activatable agents .
all new agents were developed with the requirements for the design of low molecular weight therapeutics .
fluorophores were selected to align with the parameters needed to stay within the optimal tissue transparency window ( 680950 nm ) [ 27 , 30 ] , with potential for future successful applications in the clinical evaluation of patients .
we employed fmt technology for the quantitative in vivo detection of molecular imaging biomarkers in both apoe and control c57bl/6 mice and confirmed the localization of the measured fluorescence using ct .
we found that the fmt measured concentrations of imaging biomarkers in the thoracic area of apoe mice indicate that these agents accumulated in the aortic arch and the roots of adjacent carotid and subclavian arteries ( sites of histologically confirmed atherosclerosis ) similar to previous reports .
moreover , the in vivo measured agent concentrations of both cathepsin - activated and v3 integrin targeted agents correlated well with the ex vivo fluorescence in the same areas of the dissected arteries of apoe mice .
an early stage of atherosclerotic lesions is characterized by infiltration of monocytes and macrophages and their development into foam cells [ 13 ] controlled by extracellular receptors including v3 integrin .
as the atherosclerotic plaque progresses , endothelial cells , macrophages , and foam cells die , releasing matrix metalloproteinases and other intracellular enzymes such as cathepsins [ 2022 , 24 ] .
the cell proliferation triggers on v3 integrin - dependent angiogenesis , originating from adventitial vasa vasorum and resulting in immature , fragile , and leaky vasculature , triggering intraplaque hemorrhage [ 25 , 26 ] . to understand the cellular profile of the imaging biomarkers , we conducted flow cytometric analysis of cells isolated from dissected aortic tissues of apoe mice and showed that cathepsin agents were activated primarily in myeloid ( monocytes and macrophages ) cells while the v3 integrin agent was found not only in myeloid cells but also in lymphoid and stromal ( endothelial ) cells indicating that integrisense likely detects inflammatory cells , angiogenesis , and cell proliferation .
flow cytometry of blood from animals injected with cathepsin agents detected very low levels ( compared to aortas ) of the agents in both myeloid and lymphoid cells 24 hours after agent injection , while v3 agent was also detected in both cell types , albeit at much higher levels for myeloid cells , indicating again involvement of v3 integrin in the development and proliferation of inflammatory cells .
immunofluorescence detected the activated prosense agent in the center of the atherosclerotic lesions , some of it co - localized with resident macrophages confirming that cathepsins are activated in the inflammatory cells and later released from the dead cells into the core of the plaque .
the v3 agent was found in advanced atherosclerotic lesions densely distributed in the adventitia and some was also co - localized with macrophages within the core of the lesions reflecting expected development of new vasculature and already discussed expression on the inflammatory cells .
fmt measured concentrations of cathepsin agents prosense and cat b fast in apoe mice correlated well with the number of macrophages detected in the six different areas of the aortic arch , supporting the flow cytometric and immunohistochemistry data .
however , the correlation between the macrophage count and fmt measured concentrations of v3 agent was poor , which was expected giving the high levels of v3 on the other cells types within the plaque .
co - localization of cathepsins ( b , l , s and k ) and macrophages in the core of atherosclerotic lesions of apoe mice and human patients has been documented many times [ 2024 , 28 , 29 , 31 , 34 , 35 ] , confirming an important role of these proteases in the progression of atherosclerosis associated inflammation and the development of vulnerable plaques .
activation of newly developed cathepsin agents , prosense fast and cat b fast and previously studied [ 34 , 35 , 37 ] polymer - based , prosense , in atherosclerotic lesions of apoe mice can therefore potentially serve as an imaging biomarker of inflammation .
the co - localization of v3 integrin agent with macrophages and its localization in the adventitia agree well with the previously documented role of v3 integrin in the transformation of monocytes and macrophages in the core of atherosclerotic lesions [ 19 , 3942 ] and with the role of this integrin in the neoangiogenesis of vulnerable plaques [ 26 , 42 , 43 ] .
the v3 integrin agent is therefore , contrary to cathepsin agents , a molecular imaging biomarker of both inflammation and angiogenesis .
the poor correlation between the fmt measured v3 agent concentrations and the macrophage counts may indicate binding of this agent to cells other than inflammatory cells within the atherosclerotic lesions .
elevated levels of ldl in the blood of apoe mice on regular chow and even greater in mice on chow containing elevated levels of fat and/or cholesterol translate into the formation of atherosclerotic lesions in the aortic arch and proximal arteries as early as 3 months after the birth [ 44 , 45 , 47 ] . the onset of the disease and the size of the lesions varied between individual animals .
fmt in vivo measurements of apoe mice fed with a high cholesterol diet found , when compared to c57blk/6 mice fed with regular chow , elevated levels of the cathepsin probe activation as early as 1520 weeks after diet initiation .
the levels of activation started to grow exponentially after 25 weeks with an increasing variability among individual animals .
in a similar set of fmt measurements , the accumulation of v3 agent in atherosclerotic lesion of apoe mice was detected two to five weeks later and then the concentration of the agent was again increasing exponentially .
our data indicate a good correlation between the activation of cathepsin agents and atherosclerosis - associated inflammatory processes in a longitudinal study using these agents which may provide a noninvasive early means of detection of inflammation in developing atherosclerotic lesions of apoe mice
. the somewhat delayed onset of the fmt measured v3 agent accumulation might be the result of the later onset of plaque associated angiogenesis . since both elevated inflammation as well as angiogenesis are characteristics of vulnerable plaques , both cathepsin as well as v3 integrin agents may serve in their in vivo detection .
ezetimibe , a selective inhibitor of intestinal cholesterol absorption , has been shown to reduce plasma ldl , increase hdl , and inhibit atherosclerosis associated inflammation [ 44 , 45 , 48 ] .
we found that all cathepsin agents at some point detect inhibition of inflammation by ezetimibe in prophylactic longitudinal study of apoe mice fed on high cholesterol diet .
to our surprise , the new and nonselective fast acting agent prosense fast showed a statistically significant difference only at the 12 week time point , while the polymer - based prosense detected the statistically different inhibition only at the 31-week time point .
the most sensitive for the detection of inhibition by ezetimibe was cat b fast , finding the statistically significant inhibition at both 21 and 31 weeks .
this may indicate that the selectivity rather than the size of the agent , as might be concluded from previous studies , is critical for an efficient detection of atherosclerosis - associated inflammation using cathepsin probes .
fmt measurements with the v3 integrin selective agent did not detect ezetimibe - induced inhibition of atherosclerosis progression in apoe mice .
this is likely a reflection of a broader spectrum of processes involved in the accumulation of v3 agent in the atherosclerotic lesions .
some of these processes may not be affected by the treatment with ezetimibe or they are affected on different time scales .
hence , a more detailed analysis of the functions of these two types of imaging biomarkers may contribute new details on the development of atherosclerotic lesions .
here we describe the functional in vivo imaging evaluation of new cathepsin activatable and v3 integrin targeted agents , prepared using requirements for the design of new therapeutics . using fmt , we measured quantitative changes in atherosclerosis - associated inflammation in apoe mice and compared these to the previously studied polylysine - based cathepsin agent .
we have demonstrated the importance of cathepsin agent selectivity and capability of all agents to quantitatively detect longitudinal progress in inflammation and angiogenesis of atherosclerotic lesions .
we have further showed that cathepsin agents detect inhibition of inflammation as a result of prophylactic treatment with the anti - inflammatory therapeutic , ezetimibe [ 44 , 45 ] . | inflammation as a core pathological event of atherosclerotic lesions is associated with the secretion of cathepsin proteases and the expression of v3 integrin .
we employed fluorescence molecular tomographic ( fmt ) noninvasive imaging of these molecular activities using cathepsin sensing ( prosense , catb fast ) and v3 integrin ( integrisense ) near - infrared fluorescence ( nirf ) agents . a statistically significant increase in the prosense and integrisense signal
was observed within the chest region of apoe/ mice ( p < 0.05 ) versus c57bl/6 mice starting 25 and 22 weeks on high cholesterol diet , respectively . in a treatment study using ezetimibe ( 7 mg / kg ) , there was a statistically significant reduction in the prosense and catb fast chest signal of treated ( p < 0.05 ) versus untreated apoe/ mice at 31 and 21 weeks on high cholesterol diet , respectively .
the signal of prosense and catb fast correlated with macrophage counts and was found associated with inflammatory cells by fluorescence microscopy and flow cytometry of cells dissociated from aortas .
this report demonstrates that cathepsin and v3 integrin nirf agents can be used as molecular imaging biomarkers for longitudinal detection of atherosclerosis , and cathepsin agents can monitor anti - inflammatory effects of ezetimibe with applications in preclinical testing of therapeutics and potentially for early diagnosis of atherosclerosis in patients . | 1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusions |
parasitological study - laboratory breeding scheme for
snails - the snails used in this study were originally obtained from water
channels running in alexandria governorate ( egypt ) and were maintained for two years in
the parasitology department , faculty of medicine , alexandria university , egypt .
the
snails were housed in glass aquaria containing snail - conditioned water and were fed
lettuce leaves , tetramine fish food and calcium carbonate , under suitable environmental
laboratory conditions , using the method described by eveland and haseeb ( 2011 ) .
source and maintenance of the parasite - s.
mansonicercariae were originally harvested from shedding b.
alexandrinasnails naturally infected with an egyptian strain of s.
mansoni and collected from alexandria water channels .
maintenance of the
s. mansoni life cycle was conducted between our laboratory snails
and swiss strain albino mice ( el naga et al .
groups of 10 snails were placed in 200 ml beakers containing aged
de - chlorinated tap water ( dtw ) under direct sunlight for 2 h. each cercarial suspension
was carefully shaken to obtain an even mixture and the average number of cercariae per
ml was estimated .
mice were infected with 100 cercariae / mouse using the paddling
tail - immersion technique according to the method described by dettman et al .
all work with laboratory animals was
conducted in accordance with the egyptian national animal welfare standards and was
approved by the ethical committee of the faculty of medicine , alexandria university . at
seven weeks post - infection ( p.i . )
, mice were sacrificed and their livers were used as a
source of parasite eggs .
the snails were exposed individually to eight-10 vigorously swimming freshly
hatched miracidia under direct sunlight for 3 - 4 h and then were kept in the dark and
maintained under previously described conditions ( el
naga et al .
2010 , eveland & haseeb
2011 , mostafa & el - dafrawy
2011 ) .
selection of susceptible and resistant snail stocks - separation of
resistant and susceptible isolates was achieved according to zanotti - magalhes ( 1997 ) . for the selection of resistant
individuals , snails that remained uninfected after exposure to infection
susceptible individuals were selected by isolation
of snail progeny that yielded high infection frequencies ( f1 ) and were then
reared singly for self - reproduction ; these progeny were selected as the experimental
susceptible group ( zanotti - magalhes et al .
experimental design - two groups ( f2 ) were used in our
experiments : 400 susceptible snails ( group i ) and 400 resistant snails ( group ii ) .
each
group was further subdivided equally into two subgroups : 200 young susceptible ( subgroup
ia ) and 200 young resistant ( subgroup iia ) snails were infected at the age of two months
( before egg laying was initiated ) , at the size of 3 - 4 mm in diameter .
another 200 adult
susceptible ( subgroup ib ) and 200 adult resistant ( subgroup iib ) snails were infected at
the age of four months ( after egg laying was initiated ) , at the size of 8 - 10 mm in
diameter .
afterwards , the infected snails of each subgroup were returned to separate
plastic aquaria and kept in darkness .
two weeks later , 100 snails from each experimental subgroup were subjected to enzyme
assay testing .
the other 100 snails in each subgroup were used for the determination of
parasitological parameters , starting from the 28th day post - exposure , twice weekly for
three weeks .
1:a scheme showing the followed experimental design with regards to the
parasitological parameters .
2:a scheme showing the followed experimental design with regards to
cytosolic superoxide dismutase enzyme assay .
determination of susceptibility and resistance of individual snails of the four
subgroups - at four weeks post - exposure to the parasite , the snails from
each experimental subgroup were individually checked for cercarial shedding twice
weekly , repeatedly for three weeks .
the susceptible and nonshedding snails of each
subgroup were separated and returned to different containers . on the first day of
detecting cercariae , the susceptible snails in each subgroup
were isolated individually
in small transparent plastic containers , each in 100 ml of aged dtw , and were kept in
darkness throughout the shedding period .
each of the containers contained only one
susceptible snail and was labelled with the experimental subgroup , the snail number , the
pre - patent period ( ppp ) and the number of shed cercariae at each shedding assessment
( zanotti - magalhes et al .
nonshedding individuals from each subgroup were returned together into a separate
aquarium and were tested twice weekly for cercarial shedding until the 49th day p.i . at
the end of the three weeks dedicated to shedding
, nonshedding snails were examined under
the dissecting microscope to make sure there are no arrested developmental stages
present . moreover ,
if any snail died during the ppp , it was squeesed between two slides ,
entirely dissected and inspected under a stereo - dissecting microscope to detect
developing sporocysts , which appear as opaque discrete masses within the snail tissue
( corra & paraense 1971 , niemann & lewis 1990 ) .
each shedding snail in all experimental subgroups was investigated for the following
parasitological parameters .
ppp - ppp is defined as the period from the day of snail exposure to
miracidia to the day immediately before the first shedding of cercariae ( haroun 1996 , yousif
et al .
starting from the 28th day post - exposure , all subgroups were
examined twice weekly until the 49th day post - exposure ; each snail was tested
individually .
infection rate ( ir ) - the snail ir was calculated by dividing the
number of shedding and positive crushed snails by the number of exposed snails .
the
percentage of susceptible and resistant snails in each subgroup was then determined
( frandsen 1979 , yousif et al .
the snail ir was calculated at the end of the
experiment using the following equation :
mean cercarial output ( mco ) - on the first day of cercariae detection ,
positive snails were separated individually and placed in plastic cups .
the cercariae
shed from each susceptible snail were counted under a stereomicroscope twice weekly for
three successive weeks ( chu & dawood 1970 ,
frandsen 1979 ) .
mortality rate ( mr ) - the number of dead snails for each subgroup was
counted starting from the day of exposure to the parasite until the 49th day p.i .
the
number of dead snails in the breeding aquaria was recorded twice weekly . according to
yousif
( 1998 ) , the mr was calculated
using the following equation :
sod1 enzyme assay - one hundred snails from each experimental subgroup
were used for the enzyme assay .
each snail was cleaned gently , dissected and examined under a
stereomicroscope to detect developing sporocysts .
snails that did not show any
sporocysts under the microscope were considered to be resistant .
two control groups were used in the biochemical study : uninfected young snails and
uninfected adult snails .
the soft tissue of the dissected snail was perfused with normal saline , blotted on
tissue paper and weighed .
the tissue was homogenised with phosphate - buffered saline ( ph
7.2 ) by sonication for 2 min in a glass test tube ; to prevent temperature elevation to
protect the enzymes present , the tube was placed in a beaker containing ice .
the
homogenised tissue was centrifuged at 500 g for 4 min and the
supernatant containing the proteins was stored in aliquots at -20c until use .
the collected supernatants were used for an sod1
enzyme assay . the protein concentration for each homogenate supernatant
the sod1 activity was
determined by adding the substrate of the enzyme to the snail tissue homogenate and
measuring the increase in absorbance over time during the reaction with a
spectrophotometer according to todd and gomez
( 2001 ) .
a sod1 enzyme assay kit , purchased from biodiagnostic ( egypt ) , was
used and relies on the ability of sod1 to inhibit the phenazine methosulphate - mediated
reduction of nitroblue tetrazolium .
calculation : where a control = the change in absorbance at 560 nm over 5 min following the addition
of 5-methylphenazinium methyl sulfate ( pms ) ( r4 ) to the reaction mixture in the absence
of the sample , a sample = the change in absorbance at 560 nm over 5 min following the
addition of pms to the reaction mixture in the presence of the sample .
statistical analysis - data were analysed using the ibm spss software
package v.20.0 ( kirkpatrick & feeney 2013 ) .
qualitative data are described using a number and percent and quantitative data are
described using the range ( minimum and maximum ) , mean , standard deviation and median .
comparisons between the different studied subgroups were tested using the chi - square
test .
when more than 20% of the cells showed an expected count of less than 5 ,
correction for chi - square was performed using monte carlo correction .
the distributions
of quantitative variables were tested for normality using the kolmogorov - smirnov test ,
the shapiro - wilk test and the dagstino test .
when a normal data distribution was revealed , parametric tests were
applied ; if the data were abnormally distributed , nonparametric tests were used . for
normally distributed data , comparisons between the different studied subgroups were
analysed using the f - test ( anova ) and the post - hoc test ( scheffe ) for pairwise
comparisons .
the
significance of the obtained results was judged at the 5% level ( kotz et al .
ethics - all work with laboratory animals was conducted in accordance
with the egyptian national animal welfare standards and was approved by the ethica
committee of the faculty of medicine , alexandria university .
parasitological study - ppp - snails of each subgroup were individually
examined for cercarial shedding twice weekly , starting from the 28th day after exposure
to infection until the 49th day ( table i ) .
table inumber of shedding snails in each of the examined pre - patent period ( ppp )
days in the studied subgroupsppp ( n =
100)day
28th32nd36th40th44th49thia372716500iia2
3
5
79
11
ib11
14
16
17
9
7
iib0
0
0
0
0
0
79.451
45.965
23.200
< 0.001
< 0.001
p < 0.001
p < 0.001
a : significance with subgroup ia ( young susceptible ) ;
b : significance with subgroup iia ( young resistant ) ;
c : significance with subgroup ib ( adult susceptible ) ;
d : statistically significant at p 0.05 ; iib : adult
resistant subgroup ; mc : monte carlo test ; n : number of members in each
subgroup ; : value for chi - square test .
a : significance with subgroup ia ( young susceptible ) ;
b : significance with subgroup iia ( young resistant ) ;
c : significance with subgroup ib ( adult susceptible ) ;
d : statistically significant at p 0.05 ; iib : adult
resistant subgroup ; mc : monte carlo test ; n : number of members in each
subgroup ; : value for chi - square test .
the maximum number of shedding snails on the 28th day among the four subgroups was
observed in the young susceptible subgroup ( ia ) .
these differences were demonstrated
among the susceptible young ( ia ) and susceptible adult ( ib ) subgroups at all studied
time points , except on the 36th day . in contrast , there were no significant differences
between the young resistant ( iia ) and adult resistant ( iib ) subgroups at the earlier
time points .
significant differences were found at the 40th , 44th and 49th days . additionally , significant differences between the young subgroups ia and iia at all the
time points , except on the 40th day , were also observed .
furthermore , significant
differences were found between the susceptible and resistant adult subgroups ( ib and
iib ) on all days of shedding .
ir % - the percentage of susceptible and resistant snails in each
subgroup was determined .
irs were 92% , 74% , 37% and 0% in the subgroups ia , ib , iia and
iib , respectively .
statistically significant differences among the susceptible and
resistant groups at different snail ages and among young and adult subgroups with
different compatibility patterns were evident ( table
ii ) .
table iiinfection rate ( ir ) among the studied subgroups iaiiaibiib
pir ( % ) 9237740200.315
< 0.001
sigia - iia , ia - ib , ia - iib , ib - iia , ib - iib , iia - iib
--
a : statistically significant at p 0.05 ; ia : young
susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; sig : significance between subgroups
using chi - square test ( ) .
a : statistically significant at p 0.05 ; ia : young
susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; sig : significance between subgroups
using chi - square test ( ) .
total cercarial production ( tcp ) and mco per susceptible snail - the
total number of cercariae produced by the susceptible snails in subgroups ia , ib and iia
was counted twice weekly over a period of three weeks .
the mean number of cercariae shed
by each susceptible snail in the different studied subgroups was calculated and is
presented in tables iii , iv .
the young susceptible subgroup ( ia ) yielded the highest mco , at
298.1 132.13 ( tables iii , iv ) .
table iiicercarial roduction in the studied subgroupssubgroupsshedding snails
( n)tcpmcoia85151,002298.1 132.13iia379,87744.49 32.35ib7441,73294.13 44.17iib000.0 0.0ia : young ceptible bgroup ; ib : adult ceptible bgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; mco : mean cercarial output ( mean
number of cercariae / snail / shedding time ) ; tcp : total number of
cercariae / subgroup .
ia : young ceptible bgroup ; ib : adult ceptible bgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; mco : mean cercarial output ( mean
number of cercariae / snail / shedding time ) ; tcp : total number of
cercariae / subgroup .
table ivmean cercarial shedding over three successive weeks per susceptible snail
among the studied subgroups iaiiaibiibmin - max97 - 59418 - 17320 - 2140 - 0mean sd298.1 132.144.5 32.3594.13 44.20.0
0.0median271.033.5090.500.0f test234.333
p < 0.001
sigia - iia , ia - ib , ia - iib , iia - ib , ib - iib , iia - iib
a : statistically significant at p
0.05;b : statistically significant at p 0.001 ; ia : young
susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; sig : significant difference between
subgroups using post - hoc test ( scheffe ) .
normally distributed data were
expressed in mean standard deviation ( sd ) and was compared using f test
( anova ) .
a : statistically significant at p
0.05;b : statistically significant at p 0.001 ; ia : young
susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; sig : significant difference between
subgroups using post - hoc test ( scheffe ) .
normally distributed data were
expressed in mean standard deviation ( sd ) and was compared using f test
( anova ) .
mr % - the number of dead snails for each subgroup was counted from the
day of exposure to infection until the 49th day p.i .
the mr for each subgroup was 47% ,
35% , 27% , 14% for subgroups
a positive correlation between both infection and
mrs of each subgroup was demonstrated , with mortality increasing with infection ( table vi ) .
table vmortality rate ( mr ) among the studied subgroups at the different studied
pre - patent periods with compatibility status of the dead snailsmr
( % ) day
28th32nd36th40th44th49thia ( 47)7 ( 7s)02 ( 2s)5 ( 5s)14 ( 14s)19 ( 19s)iia ( 27)2 ( 2r)3 ( 3r)3 ( 1r , 2s)5 ( 2r , 3s)6 ( 2r , 4s)8 ( 4r , 4s)ib ( 35)0
03 ( 3s)6 ( 6s)11 ( 10s , 1r)15 ( 13s , 2r)iib ( 14)1 ( 1r)1 ( 1r)2 ( 2r)3 ( 3r)3(3r)4(4r)
27.085
11.897
0.4101.059.368
13.461
p <
0.001
p = 0.015
p = 1.000
p = 0.8740.025
0.004
a : significance with subgroup ia ( young susceptible ) ;
b : significance with subgroup ib ( adult susceptible ) ;
c : statistically significant at p 0.05 ; iia : young
resistant subgroup ; iib : adult resistant subgroup ; mc : monte carlo test ; r :
resistant ; s : susceptible ; : value for chi - square test .
a : significance with subgroup ia ( young susceptible ) ;
b : significance with subgroup ib ( adult susceptible ) ;
c : statistically significant at p 0.05 ; iia : young
resistant subgroup ; iib : adult resistant subgroup ; mc : monte carlo test ; r :
resistant
table vicorrelation between infection rate ( ir ) and mortality rates ( mr ) in the
studied subgroupscorrelationir ( % ) mr ( % ) pia9247 < 0.001
iia37270.130ib7435 < 0.001
iib014 < 0.001
r
( p)0.983 ( 0.017)-
a : statistically significant at p 0.05 ; ia : young
susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; p : p - value for chi - square test ; r :
pearson coefficient .
a : statistically significant at p 0.05 ; ia : young
susceptible subgroup ; ib : adult susceptible subgroup ; iia : young resistant
subgroup ; iib : adult resistant subgroup ; p : p - value for chi - square test ; r :
pearson coefficient .
enzyme assay - the sod1 enzyme activities in the noninfected ( young and
adult ) subgroups were 0.26 0.04 and 0.30 0.03 u / g tissues , respectively . in the
infected subgroups ,
the highest level of sod1 enzyme activity was obtained in the adult
resistant subgroup ( iib ) , with a mean of 0.90 0.07 u / g tissues , whereas the lowest
enzyme activity was in the susceptible individuals of the young susceptible subgroup
( ia ) , at 0.32 0.03 .
the enzyme activity was higher in the resistant snails , regardless
of their age , in comparison to susceptible and noninfected snails .
furthermore , the sod1
activity was higher in the adult subgroups than in the young subgroups ( table vii ) .
table viithe level of cytosolic superoxide dismutase enzyme ( sod1 ) per snail s gram
tissue in u / g tissues among the studied subgroupssod1
( u / g)cacbia sia riia siia rib sib riibmin - max0.20 - 0.310.25 - 0.350.25 - 0.350.43 - 0.630.34 - 0.450.66 - 0.830.21 - 0.440.61 - 0.730.79 - 0.99mean sd0.26 0.040.30 0.030.32 0.030.55 0.070.40 0.030.76 0.050.34 0.070.68 0.040.90 0.07median0.260.300.320.580.400.770.360.670.91f
-
-
-
-
209.018
-
-
-
-
p
-
-
-
-
< 0.001
-
-
-
-
sig---
b - d
b , c , e
b - f
e , g
b - f , h
b - i
a : statistically significant at p
0.05;b : significance with group uninfected
young;c : significance with group uninfected adult ; d :
significance with subgroup ia ( young susceptible
members);e : significance with subgroup ia ( young resistant
members ) ; f : significance with subgroup iia ( young
susceptible members ) ; g : significance with subgroup iia
( young resistant members ) ; h : significance with subgroup ib
( adult susceptible members);i : significance with subgroup
ib ( adult resistant members ) ; ca : young noninfected ; cb : adult noninfected ;
ia r : young susceptible subgroup ( resistant members ) ; ia s : young
susceptible subgroup ( susceptible members ) ; ib r : adult susceptible subgroup
( resistant members ) ; ib s : adult susceptible subgroup ( susceptible members ) ;
iia r : young resistant subgroup ( resistant members ) ; iia s : young resistant
subgroup ( susceptible members ) ; sig : significance between subgroups was done
using post - hoc test ( scheffe ) .
normally distributed data were expressed in
mean standard deviation ( sd ) and was compared using f test ( anova ) .
a : statistically significant at p
0.05;b : significance with group uninfected
young;c : significance with group uninfected adult ; d :
significance with subgroup ia ( young susceptible
members);e : significance with subgroup ia ( young resistant
members ) ; f : significance with subgroup iia ( young
susceptible members ) ; g : significance with subgroup iia
( young resistant members ) ; h : significance with subgroup ib
( adult susceptible members);i : significance with subgroup
ib ( adult resistant members ) ; ca : young noninfected ; cb : adult noninfected ;
ia r : young susceptible subgroup ( resistant members ) ; ia s : young
susceptible subgroup ( susceptible members ) ; ib r : adult susceptible subgroup
( resistant members ) ; ib s : adult susceptible subgroup ( susceptible members ) ;
iia r : young resistant subgroup ( resistant members ) ; iia s : young resistant
subgroup ( susceptible members ) ; sig : significance between subgroups was done
using post - hoc test ( scheffe ) .
normally distributed data were expressed in
mean standard deviation ( sd ) and was compared using f test ( anova ) .
infection of biomphalaria species by the larval stage of s.
mansoni leads to an intimate , long - term association that largely challenges
the snail s physical integrity and severely compromises its biological fitness ( sandland et al .
although the infective stage
( miracidia ) is small relative to the host , once inside the host s body , miracidia
initiate major developmental changes to support their extensive growth and
transformation to more specialised larval stages ( de jong - brink 1995 ) .
based on a postulation of the short life span of miracidium
in water , which at the same time gives rise to hundreds of cercariae , schistosome
development inside its snail intermediate host can be considered the weakest link in the
parasite s life cycle . additionally ,
although a given parasite strain can only develop
within a compatible snail , no such discrimination occurs in the human host .
therefore ,
snail control is highly effective in eliminating schistosomiasis and is also easier than
changing human habits . for these reasons , searching for snail factors that prevent
parasite development will help in designing tools that can be utilised for controlling
schistosomiasis ( knight et al .
2014 ) . genetic background and the ids are snail - related factors that are known to affect the
compatibility of biomphalaria to s. mansoni ( richards 1975 , negro - corra et al . 2007 ) .
age - dependent variability in susceptibility to
s. mansoni has been studied in b. glabrata(richards 1984 , spada
et al .
2010 )
and it was found that the number of genes controlling resistance differs with the age of
the snail , which in turn affects the compatibility pattern ( richards 1977 , richards et al .
moreover , the number of genes controlling resistance varies
amongbiomphalaria species ( richards
1977 , richards et al .
2005 ) , which highlights the importance
of studying the effect of age on the genetic outcome of b.
alexandrina .
these cells produce the enzyme cytosolic sod1 , an important enzyme that
catalyses the conversion of o2- to h2o2 .
the
h2o2 produced by biomphalaria snails kills
s. mansoni through its potential oxidative damage , thereby affecting
snail susceptibility ( bender et al .
goodall et al . ( 2006 ) found that sod1 enzyme
expression in b. glabrata differs among susceptible and resistant
snails .
however , the effect of the snail s age on the activity of this enzyme has not
been evaluated . within this context ,
the current work examined the effect of the age of b.
alexandrina snails on their genetic outcome and on the activity of sod1 ( as
a representative of ids ) .
the present work was conducted using approaches involving both
parasitological and biochemical techniques and was achieved by including 200 snails in
each of four experimental subgroups : young susceptible ( ia ) , adult susceptible ( ib ) ,
young resistant ( iia ) and adult resistant ( iib ) . in each subgroup ,
100 snails were
dedicated for parasitological examination that began four weeks post - exposure to
miracidia and continued for the following three weeks .
the other 100 snails were used
for the biochemical study ( sod1 enzyme assay ) , which was performed at two weeks
post - exposure to infection .
moreover , the effect of the interaction of snail age , genetics and internal defence on
the outcome of infection was also examined .
our results revealed that younger snails , belonging to either a susceptible or resistant
group , showed higher susceptibility when compared to adult snails .
young susceptible
snails exhibited the shortest range of ppp , with the majority of shedding snails being
recorded by the 28th day , at 37 of 85 shedding snails .
moreover , the remainder of
shedding snails in the same subgroup showed their first shedding before the 40th day
p.i . :
27 snails shed cercariae for the first time on the 32nd day , 16 snails shed on the
36th day and five snails shed on the 40th day . regarding the adult susceptible subgroup ( ib ) , of 74 shedding snails , only 11 shed for
the first time on the 28th day p.i .
the maximum number of shedding snails was 16 and 17
recorded by the 36th and the 40th days , respectively .
( 2011 ) stated that high snail compatibility to
trematode infection is characterised by a short ppp .
the significant differences
observed between subgroups ia and ib with regard to shedding durations indicate that
subgroup ia ( young susceptible ) carries higher susceptibility to s.
mansoni infection , accompanied by rapid development of the parasite inside
the snail . as for subgroup iia ( young resistant ) shedding members , the maximum number of shedding
snails for the first time was 11 that were recorded by the 49th day with only two snails
shed for the first time by the 28th day .
conversely , the snails belonging to the adult
resistant subgroup iib displayed only the resistant phenotype , with no recoded ppp .
subgroup iia ( young resistant ) showed a significant difference compared to subgroup iib
( adult resistant ) , which contained only resistant individuals , indicating higher
susceptibility of the former .
these significant differences between subgroups ia and ib
and between iia and iib can be attributed to the effect of age .
it was not surprising that the compatibility pattern also affected the outcome of
infection , as evidenced by the significant differences between subgroups ia and iia and
between subgroups ib and iib .
moreover , the significant differences recorded between
young resistant , susceptible snails and adult susceptible , susceptible snails ( iia and
ib ) may be attributed to the effect of the interaction between age and genetics on
compatibility patterns .
this was represented by a longer ppp in the susceptible
individuals in subgroup iia than in snails of the aforementioned adult susceptible
subgroup . in the current study
, some individuals of subgroups iia and ib displayed a ppp of 49
days .
the delay in parasite development in these snails indicates their lower
susceptibility to infection . a comparison between the young resistant ( iia ) subgroup and
adult susceptible ( ib ) subgroup revealed that 11 snails shed for the first time on the
49th day in the former group , but only seven in the latter .
however , this difference was
not statistically significant , indicating that both age and compatibility pattern are
important in determining the outcome of infection .
richards ( 1984 ) stated that genetic factors
determining b. glabrata juvenile nonsusceptibility operate throughout
the snail s life , masking the presence of the factors for adult susceptibility and ,
thus , unfortunately complicating studies on the influence of age on snail
susceptibility .
the highest ir in the present study was exhibited by the snails belonging to the young
susceptible subgroup ia . in total , 92% of subgroup
ia ( young susceptible ) , 74% of
subgroup ib ( adult susceptible ) and 37% of subgroup iia ( young resistant ) were
susceptible .
it should be
noted that even the snails that died during the course of the experiment were included
in the statistical analysis ; these snails were examined for developing sporocysts .
although our experiment was carried out on snails resulting from self - reproduction , some
resistant snails were obtained in the progeny of susceptible individuals , indicating the
dominance of the resistance character in b. alexandrina . in their
studies , both shoukry et al .
( 2010 ) observed the appearance of
resistant snails originating from cross of susceptible parents .
a probable explanation
for the resistant members obtained in the susceptible subgroups is that , although
resistance alleles are sometimes not expressed in the snail phenotype , they can
accumulate through generations to produce a resistant phenotype that appears in later
progeny .
that is , the parents of these resistant snails harboured unexpressed resistance
genes , which subsequently accumulated among successive generations ( el naga et al .
similarly , the resistance
character in b. glabrata snails is inherited in a dominant manner
( richards & merritt 1972 , lewis et al .
abdel - hamid et al . ( 2006 ) and el - nassery et
al . (
2013 ) found that both susceptibility and resistance in b.
alexandrina are hereditary characters and are genetically controlled . in contrary to lewis et al .
( 2002 ) , who found
that susceptible b. glabrata parents did not give rise to any resistant
progeny , susceptible subgroups in the current study resulted in 8 - 26% resistant
individuals in the two age groups .
( 2010 ) that a susceptible population
of b. alexandrina may contain more resistance alleles than b.
glabrata , thus accounting for the appearance of resistant progeny
originating from completely susceptible parents .
although the two susceptible subgroups
contained resistant snails , the significant difference noted between them suggests that
the resistant alleles obtained from f1 parents were potentiated by the impact
of age , resulting in the appearance of more resistant individuals in the adult
susceptible subgroup ( ib ) . in the current work ,
this result was also observed in other studies using different species
( lewis et al .
however , 37 susceptible members were recorded in our
young resistant subgroup ( iia ) , which reinforces our assumption on the effect of the age
of b. alexandrina on snail compatibility pattern .
the appearance of
susceptible individuals in the young resistant subgroup could also be explained by its
weaker internal defence when compared to the adult resistant subgroup .
this was proven
by the significant difference noted in sod1 enzyme activity between resistant snails in
the young and adult resistant subgroups , at 0.68 0.04 and 0.90 0.07 u / g tissues ,
respectively .
anderson et al . ( 1982 ) attributed the lower irs of
adult snails compared to younger snails to a number of different factors . first
, older
snails possess physical barriers to penetration imposed by their tough thickened body
covering .
additionally , they produce greater quantities of mucus , which replete with
chemicals that confuse searching miracidia .
older snails also move faster and are thus more difficult to locate by
miracidia .
furthermore , egg - laying capacity could be among the factors causing more
resistance in adult snails , wherein the snail expends all its energy on reproduction , as
stated by richards ( 1977 ) .
our main goal was to ascertain whether resistant alleles transmitted from susceptible
parent snails to their young progeny are more functional at the adult stage and our
results suggest that they are .
we propose that resistance alleles in the susceptible
population may become more functional through the snail aging process , which is
responsible for the lower susceptibility in adult b.
alexandrinasnails .
the highest susceptibility in the young susceptible subgroup was also evidenced by the
highest tcp among the four studied subgroups , at 151,002 .
this was followed by the adult
susceptible subgroup , which produced 41,732 cercariae over the three weeks of shedding .
according
to the frandsen classification , the three subgroups can be categorised in classes 4 , 2
and 1 ( well compatible , poorly compatible and not very compatible , respectively ) .
regarding the adult resistant subgroup ,
no cercariae were produced at all , placing this
subgroup in frandsen class 0 or resistant snails ( frandsen 1979 ) .
the recorded mrs in the present study were 47% , 35% , 27% and 14% in subgroups ia , ib ,
iia and iib , respectively and were directly proportional to the irs in the different
subgroups .
the higher mrs noted in the susceptible compared to the resistant groups
could be explained by the devastating effects of s. mansoni on snail
health .
s. mansoni is considered a parasite that exerts a selective
pressure on the susceptible population . during its development ,
s. mansoni
sporocysts invade biomphalaria s digestive gland and the
adjacent gonads , leading to destruction of these organs ( lemos & andrade 2001 ) .
thus , infection becomes an element of natural
selection , which is highly negative for infected snails ( rosa et al .
moreover , the disturbance in metallic ion concentrations
due to trematode infection is considered another cause of mortality in parasitised
snails ( mostafa & dajem 2010 ) .
furthermore ,
the exposure of susceptible snails to a high dose of infection ( 8 - 10 miracidia ) per
snail at the same time may explain the higher mortality recorded in the laboratory , as
the majority of these miracidia are thought to develop without much restraint . in
contrast , under natural conditions , the snail is not likely to be exposed to such a high
number of miracidia at a given time ( corra &
paraense 1971 ) .
the mrs recorded in the two resistant subgroups were 35% and 14% , subgroups iia ( young
resistant ) and iib ( adult resistant ) , respectively .
the mortality of the resistant
snails can be attributed to a high concentration of the sod1 enzyme , which produces
h2o2 , a highly ros .
although these reactive species are
beneficial for combating the invading parasite , they also seriously damage biological
membranes in the host tissue , the so - called " cost of resistance " ( bender et al . 2007 , bonner et al .
2012 ) ; hence , ros are considered a double - edged sword ( beckman & ames 1998 ) .
( 2010 ) stated that exposure alone of b. alexandrina
snails to s. mansoni miracidia has a negative impact on snail
survival rates . in the present work ,
we functionally investigated the activity of the sod1 enzyme in a
whole - snail soft tissue homogenate at two weeks after exposure to infection .
for the
control noninfected snails , the results were 0.26 0.04 and 0.30 0.03 u / g of sod1
activity in the noninfected young and adult groups , respectively .
cytosolic sod1 is present
during the course of the snail lifetime to protect cells against damage by the free
radicals that are generated due to normal metabolism ( bender et al . 2005 ) .
regarding the results of the experimental subgroups in the current study , the combined
effects of both age and compatibility pattern resulted in the highest sod1 activity in
the adult resistant subgroup , with a mean of 0.90 0.07 u / g tissue .
in contrast , the
lowest enzyme activity was found in the susceptible snails of the young susceptible
subgroup , at 0.32 0.03 u / g tissue .
furthermore , as shown intable vii , the sod1 activities were higher in the resistant
individuals of the studied subgroups than in the corresponding susceptible individuals .
this indicates the importance of this enzyme in determining a snail s compatibility
status .
our results of higher sod1 activity levels in resistant vs. susceptible snails
are in agreement with hahn et al .
( 2004 ) , mahmoud and rizk ( 2004 ) , bender et
al .
( 2005 ) and bayne ( 2009 ) , who
reported an increased number of haemocytes generating superoxide in s.
mansoni resistant snails .
the resulting free radicals were efficient for
encapsulating or eliminating the parasite in resistant snails , but failed in the case of
susceptible snails ( mahmoud & rizk 2004 ) .
it is worth mentioning that studies on the genes of a resistant b.
glabrata strain have revealed that polymorphisms in the gene encoding
cytosolic sod1 are associated with susceptibility / resistance ; such results reinforce the
role of sod1 in schistosome resistance ( goodall et al .
the results of the present work showed that the age of b. alexandrina
snails affects sod1 activity levels .
significant differences in sod1 activity levels
between young and adult snails obtained from the same ( f1 ) parent were
observed , with a higher activity at the adult stage .
goodall et al . ( 2006 ) reported thatbiomphalaria sod1 is
encoded by three alleles and one was found to be significantly associated with
resistance .
furthermore , some alleles controlling compatibility were found to be
affected by snail age ( richards 1977 , richards & shade , richards et al .
, it would be interesting
to assess whether the genes encoding sod1 are among the other genes that are modified by
snail age . in summary ,
the significant differences in all parasitological and biochemical
parameters between young and adult snails in the same group were attributed to the age
effect , which made resistance alleles more functional in adults , with higher sod1 enzyme
activity . moreover , the complex interaction between age , genetic background and ids
between the susceptible and resistant subgroups results in great variability in
compatibility patterns .
this interaction led to the appearance of the highest
significant difference between the young susceptible ( ia ) and adult resistant ( iib )
subgroups .
the results presented herein can have potential epidaemiological implications
inbiomphalaria control . by determining the age
at which a snail is
most susceptible to s. mansoni infection , the optimal timing for the
application of molluscicides could be identified .
this in turn would increase the
efficacy of the applied method , potentiating schistosomiasis control .
moreover ,
identification of the most resistant snails is important for use in later biological
snail control , after studying the compatibility of successive generations .
investigations of genes encoding enzymes involved in the respiratory burst are
recommended . in particular , the genes responsible for the production of
h2o2 should be targeted as potential candidates for genetic
loci causally related to the susceptibility / resistance ofb .
| of the approximately 34 identified biomphalaria
species , biomphalaria alexandrina represents the
intermediate host of schistosoma mansoni in egypt . using
parasitological and sod1 enzyme assay , this study aimed to elucidate the impact of
the age of b. alexandrina snails on their genetic variability and
internal defence against s. mansoni infection .
susceptible and
resistant snails were reared individually for self - reproduction ; four subgroups of
their progeny were used in experiment .
the young susceptible subgroup showed the
highest infection rate , the shortest pre - patent period , the highest total cercarial
production , the highest mortality rate and the lowest sod1 activity . among the young
and adult susceptible subgroups , 8% and 26%
were found to be resistant , indicating
the inheritance of resistance alleles from parents .
the adult resistant subgroup ,
however , contained only resistant snails and showed the highest enzyme activity .
the
complex interaction between snail age , genetic background and internal defence
resulted in great variability in compatibility patterns , with the highest significant
difference between young susceptible and adult resistant snails .
the results
demonstrate that resistance alleles function to a greater degree in adults , with
higher sod1 activity and provide potential implications for biomphalaria
control .
the identification of the most susceptible snail age enables
determination of the best timing for applying molluscicides .
moreover , adult
resistant snails could be beneficial in biological snail control . | MATERIALS AND METHODS
RESULTS
DISCUSSION |
chronic obstructive pulmonary disease ( copd ) is a serious lung disease that severely threatens people s health .
copd has become a global health problem and is expected to be the third leading cause of mortality worldwide by 2020 .
the main characteristics of copd are persistent airflow limitation and acute respiratory symptoms , associated with rapid lung function deterioration , declining health , and increased risk of mortality .
impaired gas exchange leading to hypoxemia is a key feature of copd that contributes to the pathogenesis of copd .
the use of oxygen ( o2 ) to treat respiratory failure in patients with copd and long - term oxygen therapy have been studied since the 1980s .
helium ( he ) is an inert gas with many advantages : it is colorless , odorless , tasteless , nontoxic , and relatively stable . besides , helium does not dissolve in water or interact with other gases in vivo
heliox is a gas mixture with a lower density than oxygen , consisting of oxygen and helium .
recently , the special physical characteristics of heliox have drawn more attention from clinicians because using this gas mixture can reduce the airway resistance by improving respiratory gas exchange and decreasing turbulent flow .
a number of researchers have studied the clinical drug administration effects of heliox - driven aerosol over the recent years , while the findings were strikingly different .
findings from some studies support the benefits of heliox - driven aerosol , whereas other studies have not found clinical benefits from the use of heliox . moreover , the treatment effects were obviously different with different proportions of oxygen and helium . combining the previous findings with our limited knowledge , we surmised that heliox was better than oxygen when used for copd patients , and increasing helium concentration might have a superior efficacy . in order to confirm this conjecture ,
the copd rat model was built up by exposure to smoking and intratracheal instillation of lipopolysaccharide ( lps ) to evaluate the effects of heliox - driven nebulization ( hdn ) .
the pulmonary function testing and blood gas analysis were also conducted for different proportions of hdn .
all animals were handled according to the ethical principles of laboratory animal care , and all experimentations were licensed by animal protection association ( ethics no .
a total of 46 healthy male wistar rats ( weight 20020 g ) were provided from second military medical university experimental animal center ( shanghai , china ) and housed in the animal room with free access to water and food . the animal room was maintained at 252c , 50% to 70% relative humidity , with a 12/12 h light / dark cycle .
a week later , 12 rats were randomly selected as controls and 34 rats were used to establish an experimental copd model by exposure to smoking and intratracheal instillation of lps ( sigma , usa , lot l-2880 ) .
each model rat was exposed to smoked cigarettes ( red card , flue - cured tobacco tar : 12 mg ; nicotine smoke : 1.2 mg ; flue gas of carbon monoxide : 14 mg ) for 30 min / d , for 90 d. on the 61st and 74th days , the rats were intratracheally instilled with 200 g lps using a microinjector . then 6 rats from the experimental copd group and 6 rats from the corresponding unexposed control group were randomly selected to perform clinical characterization , including body weight and lung function factors : airway pressure ( awp ) , inspiratory resistance ( ri ) , expiratory resistance ( re ) , dynamic lung compliance ( cdyn ) , forced vital capacity ( fvc ) , forced expiratory volume at 0.10 s ( fev0.10 ) , forced expiratory volume at 0.20 s ( fev0.20 ) , fev0.10/fvc% , fev0.20/fvc% , peak expiratory flow ( pef ) , and respiratory rate ( rr ) .
the remaining 28 experimental copd rats were randomly divided into 4 groups with 7 rats per group : experimental copd rats without treatment ( group b ) , experimental copd rats with medical oxygen exposure ( group c ) ; experimental copd rats with heliox ( he / o2=63%/37% ) exposure ( group d ) ; and experimental copd rats with heliox ( he / o2=71%/29% ) exposure ( group e ) .
the components of atomized liquid were 5 ml 0.9% saline containing 4000 units chymotrypsin ( shanghai no .
1 biochemical pharmaceutical co. , ltd . ) , inhalation pulmicort ( 1 mg/2 ml ) and atrovent ( 500 g/2 ml ) .
the drugs were atomized by using an atomizer ( parilcd , bonn , germany ) and the inhaled gas flow was adjusted to 6 l / min .
the rats were anesthetized using 3% sodium pentobarbital by intraperitoneal injection as a dose of 0.13 ml/100 g of body weight .
femoral artery was isolated without touching the nerves by using a 1-ml syringe and avoiding the air .
arterial blood samples were immediately placed into gem premier 3000 blood gas analyzer ( instrumentation laboratory , lexington , ma , usa ) to examine partial pressure of oxygen in arterial blood ( pao2 ) , partial pressure of carbon dioxide in arterial blood ( paco2 ) , arterial oxygen saturation ( sao2 ) , and potential of hydrogen ( ph ) .
after anesthetizing the rats , pulmonary function tests were performed using the anires 2005 lung function meter ( peking biolab tech company , beijing , china ) , according to the manufacturer s instructions .
an endotracheal tube was inserted into the rats and connected to the outlet of the ventilator .
after 30 normal respiratory cycles , the lung function parameters , including ri , re , fev0.1/fvc% , cdyn , and pef , were examined and recorded .
all data were expressed as mean plus or minus standard deviation ( sd ) and analyzed using the authorized software of statistical product and service solutions ( spss ) version 19.0 ( spss inc , chicago , il , usa ) .
significance tests among different groups were performed using 1-way analysis of variance ( anova ) followed by post hoc multiple comparisons with least significant different ( lsd ) testing .
meanwhile , anova was conducted for the blood gas indicators , and paired samples t test was also performed . in all cases ,
a total of 46 healthy male wistar rats ( weight 20020 g ) were provided from second military medical university experimental animal center ( shanghai , china ) and housed in the animal room with free access to water and food . the animal room was maintained at 252c , 50% to 70% relative humidity , with a 12/12 h light / dark cycle .
a week later , 12 rats were randomly selected as controls and 34 rats were used to establish an experimental copd model by exposure to smoking and intratracheal instillation of lps ( sigma , usa , lot l-2880 ) .
each model rat was exposed to smoked cigarettes ( red card , flue - cured tobacco tar : 12 mg ; nicotine smoke : 1.2 mg ; flue gas of carbon monoxide : 14 mg ) for 30 min / d , for 90 d. on the 61st and 74th days , the rats were intratracheally instilled with 200 g lps using a microinjector . then 6 rats from the experimental copd group and 6 rats from the corresponding unexposed control group were randomly selected to perform clinical characterization , including body weight and lung function factors : airway pressure ( awp ) , inspiratory resistance ( ri ) , expiratory resistance ( re ) , dynamic lung compliance ( cdyn ) , forced vital capacity ( fvc ) , forced expiratory volume at 0.10 s ( fev0.10 ) , forced expiratory volume at 0.20 s ( fev0.20 ) , fev0.10/fvc% , fev0.20/fvc% , peak expiratory flow ( pef ) , and respiratory rate ( rr ) .
the remaining 28 experimental copd rats were randomly divided into 4 groups with 7 rats per group : experimental copd rats without treatment ( group b ) , experimental copd rats with medical oxygen exposure ( group c ) ; experimental copd rats with heliox ( he / o2=63%/37% ) exposure ( group d ) ; and experimental copd rats with heliox ( he / o2=71%/29% ) exposure ( group e ) .
the components of atomized liquid were 5 ml 0.9% saline containing 4000 units chymotrypsin ( shanghai no .
1 biochemical pharmaceutical co. , ltd . ) , inhalation pulmicort ( 1 mg/2 ml ) and atrovent ( 500 g/2 ml ) .
the drugs were atomized by using an atomizer ( parilcd , bonn , germany ) and the inhaled gas flow was adjusted to 6 l / min .
the rats were anesthetized using 3% sodium pentobarbital by intraperitoneal injection as a dose of 0.13 ml/100 g of body weight .
femoral artery was isolated without touching the nerves by using a 1-ml syringe and avoiding the air .
arterial blood samples were immediately placed into gem premier 3000 blood gas analyzer ( instrumentation laboratory , lexington , ma , usa ) to examine partial pressure of oxygen in arterial blood ( pao2 ) , partial pressure of carbon dioxide in arterial blood ( paco2 ) , arterial oxygen saturation ( sao2 ) , and potential of hydrogen ( ph ) .
after anesthetizing the rats , pulmonary function tests were performed using the anires 2005 lung function meter ( peking biolab tech company , beijing , china ) , according to the manufacturer s instructions .
an endotracheal tube was inserted into the rats and connected to the outlet of the ventilator .
after 30 normal respiratory cycles , the lung function parameters , including ri , re , fev0.1/fvc% , cdyn , and pef , were examined and recorded .
all data were expressed as mean plus or minus standard deviation ( sd ) and analyzed using the authorized software of statistical product and service solutions ( spss ) version 19.0 ( spss inc , chicago , il , usa ) .
significance tests among different groups were performed using 1-way analysis of variance ( anova ) followed by post hoc multiple comparisons with least significant different ( lsd ) testing .
meanwhile , anova was conducted for the blood gas indicators , and paired samples t test was also performed . in all cases ,
the changes of weight and lung function for control and experimental copd rats are shown in table 1 . compared with the control group ,
furthermore , awp , ri , re , and rr were significantly increased , whereas fev0.1/fvc and cdyn were significantly decreased in the model group ( p<0.05 ) .
the obvious differences of main lung function parameters ( ri , re , and cdyn ) between the experimental copd group and the control group showed that changes expected for the copd model were successfully achieved .
results for the pulmonary function tests are list in table 2 and figure 1 . compared with the model group ,
no statistical differences of the ri , re , and cdyn levels were found in the medical oxygen group ( p>0.05 ) . however , ri and re were significantly lower and cdyn was significantly higher in the heliox ( he / o2=63%/37% and he / o2=71%/29% ) groups than in the experimental copd group ( p<0.05 ) .
meanwhile , significant reductions of the fev0.1/fvc values were found in groups c , d , and e. statistical difference of pef was observed between the heliox ( he / o2=71%/29% ) group and the medical oxygen group ( p<0.05 ) .
the blood gas exchange variables were recorded and the differences among these 5 groups were analyzed using anova ( table 3 ) . according to the results , statistical differences were found in the paco2 , ph , and sao2 levels , while not in pao2 levels .
the results of paired samples t test for these 5 groups are shown in table 4 .
the values of paco2 were significantly higher while the ph values were significantly lower after atomization in medical oxygen and heliox ( he / o2=63%/37% ) groups ( p<0.05 ) .
meanwhile , statistical difference of sao2 was only found in heliox ( he / o2=71%/29% ) group after atomization ( p<0.05 ) .
the changes of weight and lung function for control and experimental copd rats are shown in table 1 . compared with the control group ,
furthermore , awp , ri , re , and rr were significantly increased , whereas fev0.1/fvc and cdyn were significantly decreased in the model group ( p<0.05 ) .
the obvious differences of main lung function parameters ( ri , re , and cdyn ) between the experimental copd group and the control group showed that changes expected for the copd model were successfully achieved .
results for the pulmonary function tests are list in table 2 and figure 1 . compared with the model group ,
no statistical differences of the ri , re , and cdyn levels were found in the medical oxygen group ( p>0.05 ) . however , ri and re were significantly lower and cdyn was significantly higher in the heliox ( he / o2=63%/37% and he / o2=71%/29% ) groups than in the experimental copd group ( p<0.05 ) .
meanwhile , significant reductions of the fev0.1/fvc values were found in groups c , d , and e. statistical difference of pef was observed between the heliox ( he / o2=71%/29% ) group and the medical oxygen group ( p<0.05 ) .
the blood gas exchange variables were recorded and the differences among these 5 groups were analyzed using anova ( table 3 ) . according to the results , statistical differences were found in the paco2 , ph , and sao2 levels , while not in pao2 levels .
the results of paired samples t test for these 5 groups are shown in table 4 .
the values of paco2 were significantly higher while the ph values were significantly lower after atomization in medical oxygen and heliox ( he / o2=63%/37% ) groups ( p<0.05 ) .
meanwhile , statistical difference of sao2 was only found in heliox ( he / o2=71%/29% ) group after atomization ( p<0.05 ) .
according to the results of our study , heliox ( he / o2=71%/29% and he / o2=63%/37% ) as the driving gas influenced the lung function parameters ( ri , re , fev0.1/fvc , and cdyn ) and blood gases ( paco2 , ph , and sao2 ) to benefit patients with copd . increasing the inspired oxygen and replacing nitrogen with lower - density helium have been the common and dominant adjuvant treatments for copd patients .
additional oxygen can improve the exercise tolerance of patients but does not improve lung function .
fortunately , a helium - oxygen mixture can be used to reduce airway resistance and lung volume , which is of more benefit to patients with copd .
heliox is also reported to promote drug deposition , especially in the small airways and alveoli by aerosol drug delivery .
thus , there is no doubt that heliox has many effects that would benefit copd treatment .
heliox can improve exercise tolerance by improving oxygen delivery ; however , the clinical effect of heliox on lung functions is controversial .
lung function parameters , such as fev1 and fvc , were increased after hdn , according to the study of chiappa et al . , whereas xiao et al .
demonstrated there were no differences among fev1 , fvc , and maximal voluntary ventilation between the treatment group and the control group . in this paper ,
fev0.1/fvc was significantly lower after oxygen or heliox atomization treatment , especially in the heliox ( 71%/29% ) group ( p<0.05 ) . though the detected parameters were not the same , our results consistently matched those obtained by chiappa et al .
, who demonstrated that hdn had positive effects on lung functions . considering fev0.1/fvc reflects the airflow obstruction , we speculated that heliox could improve lung function by decreasing airflow obstruction .
austen et al . found that decreased cdyn accounted for widespread airway narrowing and increased cdyn could diminish pulmonary resistance as well as improve the elastic recoil and lung ventilation .
in addition , re and ri were significantly lower after hdn , which indicated that the ventilation situation of lung was significantly improved .
thus , we concluded that hdn treatment could improve the lung function by decreasing airflow obstruction , diminishing pulmonary resistance , and improving ventilation ; and he / o2=71%/29% was the superior proportion .
oxygen and heliox therapies are used for the treatment of respiratory failure , which occurs when lungs and other respiratory apparatus become unable to ensure adequate systemic oxygenation .
respiratory failure is further classified as a failure of oxygenation ( a low pao2 ) with a normal or high paco2 level ; the presence of acidosis , a consequence of hypercapnia , is another important variable .
blood gas analysis is helpful for the diagnosis of copd patients with hypoxemia , hypercapnia , or acid - base balance and further provides a reliable basis for determining the presence and type of respiratory failure .
respiratory depression is the main description of oxygen - driven nebulization or heliox with high oxygen concentration by blocking the stimulation of hypoxia on the respiratory center , increasing the difficulty of breathing and causing carbon dioxide retention . according to the results of blood gas analysis , paco2 elevated and/or ph decreased and the similar results were found in oxygen and heliox ( he / o2=63%/37% ) groups in our study .
thus , though the lung function was improved in oxygen and heliox ( he / o2=63%/37% ) groups , the gas exchange might be inhibited .
however , the blood gas situation was obviously different in heliox with low oxygen concentration ( he / o2=71%/29% ) .
pao2 was increased without higher paco2 and the value of paco2 in several rats was even decreased , indicating an improvement in co2 retention by hdn ( he / o2=71%/29% ) treatment .
patients with copd experience oxygen deprivation for prolonged periods , and sao2 is considered an indirect measure for arterial oxygen pressure .
sao2 was significantly associated with qtc interval and electrocardiographic changes were showed in copd patients with low sao2 . in our study , sao2 was significantly increased in heliox ( he / o2=63%/37% ) group ( p<0.05 ) . thus , hdn with the percentage he / o2=71%/29% might significantly improve the respiratory status and reduce the risk of cardiovascular disease in copd patients by increasing arterial oxygen pressure and decreasing co2 retention .
hdn with heliox in the proportion of he / o2=71%/29% was the superior method to improve the lung function and blood gas status in a smoking- and lps - induced copd rat model . | backgroundchronic obstructive pulmonary disease ( copd ) is a serious lung disease that severely threatens people s health .
this study aimed to investigate the effects of heliox - driven nebulization ( hdn ) on lung function and arterial blood gases in a copd rat model.material/methodstwelve healthy male wistar rats were selected as controls and 34 rats were used to establish a copd model induced by lipopolysaccharide .
then 6 rats each from the control and model groups were selected for their symptoms to be observed .
the remaining 6 normal rats were used as control group ( group a ) and the remaining 28 experimental copd rats were randomly assigned to 4 groups : experimental copd group ( group b ) , medical oxygen group ( group c ) , and heliox groups ( group d , he / o2=63%/37% ; group e , he / o2=71%/29% ) . the lung function indicators and arterial blood gases were analyzed to evaluate the effects of different driving gases on copd rats.resultsthe copd model was successfully established with slow growth and severe lung dysfunction .
inspiratory resistance , expiratory resistance , and forced expiratory volume at 0.10 s ( fev0.10)/fvc were significantly decreased , whereas dynamic lung compliance was significantly increased in groups d and e , compared with the experimental copd group ( group b ; p<0.05 ) . meanwhile , compared with the model group , the values of partial pressure of carbon dioxide in arterial blood were significantly higher , whereas the potential of hydrogen values were significantly lower after atomization in groups c and d but not in group e ( p<0.05 ) . the obvious increase in arterial oxygen saturation was found only in group e ( p<0.05).conclusionshdn improved the lung function and arterial blood gas analysis results in experimental copd rats , with an optimal percentage of he / o2=71%/29% . | Background
Material and Methods
Animal model
Grouping and treatment
Blood gas analysis
Pulmonary function testing
Statistical analysis
Results
COPD-model construction
Changes of lung function indicators after atomization
Blood gas analysis results
Discussion
Conclusions |
a fecal specimen was obtained from a piglet experimentally infected with strain ska-1 , which was first isolated from a pig with diarrhea in tottori prefecture , japan .
rna was extracted from a 20% fecal suspension in phosphate - buffered saline by using the isogen - ls kit ( nippon gene ltd . ,
( 13 ) with some modifications ( 14 ) . during single - primer amplification ,
several dna bands were detected that appeared to correspond to rna segments of a rotavirus .
dna was purified by using a wizard sv gel and a pcr clean - up system ( promega , madison , wi , usa ) and cloned into the pcr - topo vector by using a topo ta cloning kit ( invitrogen , carslbad , ca , usa ) .
sequence analysis and comparisons were performed by using genetyx - win software ( genetyx , tokyo , japan ) and mega software ( www.megasoftware.net/ ) .
we selected 4 genes encoding vp4 , vp6 , vp7 , and nsp4 for sequence analysis .
sequence data were obtained for comparative sequence analysis among group a and nongroup a rotaviruses .
there was little identity for any of the 4 genes between ska-1 and group a or c rotaviruses .
in contrast , nucleotide and amino acid sequences of the 4 genes of ska-1 showed relatively high identities with those of a novel group of rotavirus strains ( j19 and b219 ) .
the vp6 gene , which is associated with group specificity , of ska-1 showed highest identities among the 4 genes with those of the novel group human rotaviruses : 72%73% at the nucleotide level and 76%77% at the amino acid level .
vp6 also showed relatively high identities with those of group b rotaviruses : 52%53% at the nucleotide level and 36%40% at the amino acid level ( table ) .
the other 3 genes ( vp4 , vp7 , and nsp4 ) , also showed high identities with those of the novel group of human rotaviruses , although identity values were lower than those for the vp6 gene ( table ) .
* vp , viral protein ; ns , nonstructural protein ; na , not available .
: ska-1 vp4 , ab576625 ; ska-1 vp6 , ab576626 ; ska-1 vp7 ab576627 ; ska-1 nsp4 ; ab576628 .
lengths and nucleotide sequences of the 5 and 3 noncoding regions of the 4 genes of ska-1 were similar to those of the novel group of rotaviruses . using the vp7 gene as a reference , we identified sequences of the 5 noncoding region of vp7 as 5-ggaactttaaagcc-3 for strain ska-1 , 5-ggcaatttgaagcc-3 for the novel group of human rotaviruses , and 5-ggcaataaa-3 for group b rotaviruses .
phylogenetic analysis of the 4 genes also showed that ska-1 is closely related to the novel group of human rotavirus strains ( j19 and b219 ) ( figure ) .
phylogenetic trees for a ) viral protein ( vp ) 4 , b ) vp6 , c ) vp7 , and d ) nonstructural protein 4 genes of group a , b , and c rotaviruses , a novel group of human rotaviruses , and porcine rotavirus strain ska-1 .
although group a rotaviruses have been extensively studied , nongroup a rotaviruses have not been extensively studied . in particular , there is little information on group d , e , f , and g rotaviruses .
a novel group of human rotaviruses ( j19 and b219 ) are not related to any other groups of rotaviruses .
rotavirus strain ska-1 was detected in a fecal specimen from a piglet with diarrhea in japan , and was isolated in ma-104 cells in 1999 .
its group specificity was suggested to be group b on the basis of reverse transcription pcr results with a group b specific primer .
however , our sequence analysis showed that the porcine ska-1 strain is different from group b human and porcine rotaviruses .
nucleotide and amino acid sequences of ska-1 did not show identities with those of group a or c rotaviruses , although they showed some relatedness to those of group b rotaviruses .
however , high identities were observed between ska-1 and a novel group of human rotaviruses . in addition
, there are similarities in the 4 genes analyzed in this study between ska-1 and the novel group of human rotaviruses : 1 ) nucleotide sequences and nucleotide numbers of noncoding regions at the 5 and 3 ends are similar to each other ; 2 ) lengths of nucleotide and deduced amino acid sequences are similar to each other ; and 3 ) phylogenetic analysis showed that these viruses are in the same cluster . except for porcine strain ska-1 , there has been no report of animal rotaviruses being classified into a novel group of human rotaviruses .
a survey of the prevalence of antibodies against ska-1 among humans and animals , including pigs , would be useful .
a classification system for nongroup a rotaviruses has not been established because 1 ) information on nucleotide sequences of group d , e , f , and g rotaviruses is lacking ; 2 ) no expressed reference vp6 proteins are available ; 3 ) reference strains of group d
g rotaviruses have not been adapted to cell culture ; and 4 ) it is not known whether fecal samples or rna or extracted rna of group e
g strains are available in any laboratories . on the basis of serologic characterization and sequence analysis , a classification system for nongroup
a rotaviruses , which includes the novel group of human and porcine rotaviruses such as j19 , b219 , and ska-1 , should be established . | we determined nucleotide sequences and inferred amino acid sequences of viral protein ( vp ) 4 , vp6 , vp7 , and nonstructural protein 4 genes of a porcine rotavirus strain ( ska-1 ) from japan .
the strain was closely related to a novel group of human rotavirus strains ( b219 and j19 ) . | The Study
Conclusions |
the worldwide growth in childhood obesity ( apfelbacher et al . 2008 ; stamatakis et al . 2010 ) in connection with a corresponding decline in physical activity ( pa ) and inappropriate dietary patterns ( edwards et al .
2005 ) has led to the need for open discussion regarding possible effective methods of increasing children s daily pa .
2010 ; sharma 2006 ) and nutrition intervention programs ( evans et al . 2010 ; li and hooker 2010 ; ransley et al . 2007 ; sharma 2006 ) .
as children spend much of their childhood in school ( fox 2004 ) , the school environment can instigate and maintain healthy lifestyle habits ( pate et al .
school - related pa ( guinhouya et al . 2009 ; tudor - locke et al .
2010 ) , physical education ( pe ) lessons ( pate et al . 2006 ; tudor - locke et al .
2009a ) , and morning , lunchtime , and other pa breaks ( ridgers et al . 2009 , 2010
2006 ) have been helpful in effectively increasing the daily pa of school children ( mota et al .
the demonstrable health benefits of pa for children ( e.g. reduction of high blood pressure , obesity and depression ; increases in fitness and bone - mineral density ) are brought about by an accumulation of an average of at least 60 min of pa per day and up to several hours of at least moderate intensity pa ( janssen and leblanc 2010 ; strong et al .
however , some of these health benefits can be achieved through an average of 30 min of moderate - to - vigorous pa ( mvpa ) per day ( janssen and leblanc 2010 ) .
while health - enhancing pa in adults needs to last for 2060 min without a break , it can be carried out by children in shorter , 10- to 15-min intervals adding up to 60 min or more of mvpa per day ( strong et al . 2005 ) .
these shorter pa episodes should be primarily carried out in pe lessons ( cale and harris 2006 ; strong et al . 2005 ) and in break periods ( mota et al .
, medical experts have stated that schools should ensure that all children and youth partake in a minimum of 30 min of mvpa during each school day ( pate et al .
2006 ) and the total health - related minimum of steps should amount to 11,000 steps per day for girls and 13,000 for boys ( president s council on physical fitness and sports 2001 ; vincent and pangrazi 2002 ) .
previous studies demonstrate the appropriateness of the school environment for daily pa for children and the reduction of their sedentary behaviour .
potentially novel approaches for reducing children s sedentary time include activity breaks during class time , delivery of active lessons and homework , realisation of non - elimination games and changes to the classroom environment and furniture ( foster et al .
2010 ) are a proven determinant of increased pa for 9- and 10-year - old children at school .
these involve not only playgrounds with adventure playgrounds , swings , trees , playground markings and sandpits , but also corridors , free classrooms , movement corners and courts ( nielsen et al . 2010 ) .
the present study attempts to uncover the potential of mvpa during school breaks for attaining the health - related amount of overall daily pa in 9- and 10-year - old overweight and non - overweight school children .
this study assessed the contribution of 30 min of mvpa during school breaks to the overall daily pa and to the acquirement of health - related amount of steps in 9- and 10-year - old overweight and non - overweight school children .
the specific objectives were to : describe and compare differences in overall pa in the specific parts of the school day ( before school , pe and another lessons , breaks , after school ) in overweight and non - overweight girls and boys divided based on the duration of mvpa in school breaks.find out and describe the relationship between school breaks and the overall daily pa in the groups of children stratified by gender and obesity levels.describe and compare the intensity of overall pa during school breaks in overweight and non - overweight girls and boys divided based on the duration of mvpa during the school breaks . describe and compare differences in overall pa in the specific parts of the school day ( before school , pe and another lessons , breaks , after school ) in overweight and non - overweight girls and boys divided based on the duration of mvpa in school breaks .
find out and describe the relationship between school breaks and the overall daily pa in the groups of children stratified by gender and obesity levels .
describe and compare the intensity of overall pa during school breaks in overweight and non - overweight girls and boys divided based on the duration of mvpa during the school breaks .
overall daily pa will be assessed according to variables simultaneously measured by the actitrainer monitor ; variables include number of steps , heart rate and duration of pa .
we expect that the children who partake in at least 30 min of mvpa during the school breaks will attain a higher amount of overall daily pa .
however , we do not know to what extent the expected contribution would be in presently overweight children .
elementary schools were selected intentionally based upon uniformly implemented daily school routines , similar size and equipment , number of students and localization in the city s development .
participants included 149 girls and 111 boys aged 9 and 10 in six classes of two elementary schools in katowice ( poland ) who returned signed , informed parental - consent forms enabling them to participate in the study . the final , entire 2-day monitored physical activity ( using an actitrainer activity monitor ) was completed , and 137 girls and 102 boys with a mean age of 9.48 0.40 years ( 20.1% overweight and 19.2% obese ) were included in the analysis of the data .
illnesses and non - participation in the physical education lesson were a reason for the elimination of 15 and 6 children , respectively ( representing 8% of girls and 8% of boys ) .
all of the children assessed wore the actitrainer activity monitor continuously for 2 days ( excluding sleeping , hygiene , and bathing times ) for a minimum of 10 h per day . between the 4th and 5th and
the 11th and 12th of february 2010 , all of the participating children followed their usual daily school routine , including five school lessons , four breaks and one lunchtime break .
school lessons usually started at 8 a.m. , were each 45 min in length and finished between 1 and 2 p.m. each of the children participated in one pe lesson with the same gymnastics content .
one of the four school breaks lasted 30 min , while the others were 5 min long . during the said 30-min break ,
the children played movement games , participated in rhythmic and dance activities as well as used sports and game equipment ( e.g. , a bouncy ball , frisbee , gymball , hopscotch and skipping rope ) in the playground under the supervision of teachers .
the children remained in their classrooms during the shorter breaks or could move around spontaneously in the school corridors under the supervision of teachers .
the actitrainer ( actitrainer , florida , usa ) is small and light ( 8.6 3.3 1.5 cm ; 53 g ) .
it is a multi - functional device composed of a heart - rate monitor , tri - axis solid - state accelerometer , an electronic pedometer , an inclinometer , and an ambient light sensor .
the recording of data can be viewed on the built - in display . upon turning on the display panel ,
the actitrainer can monitor and continually store recorded data over a period of 7 days .
the validity and reliability of the actitrainer - based step counting in non - laboratory conditions was verified with 20 non - obese university students ( neuls 2008 ) .
a right hip - fixed actitrainer activity monitor continuously measured the pa of children in 15-s intervals for the entire body - wearing time .
when monitoring pa , the actitrainer was secured at the waist using a neoprene pouch and an elastic belt . when collecting heart - rate data , the polar chest strap was worn across the sternum .
the overall measured pa was simultaneously represented by number of steps , heart rate and duration of pa variables .
children were classed as being physically active , as opposed to sedentary , when > 25 counts per 15 s were recorded .
intensity zones for overall pa were set based on the percentage of the maximal age - related heart rate , i.e. , 220-age ( edwards 2010 ) .
moderate - to - vigorous physical activity was defined as heart rate above 60% of the maximum age - related heart rate in accordance with published studies ( edwards 2010 ; kirkpatrick and birnbaum 1997 ) .
children s height was measured using an anthropometer a-319 ( trystom corporation , olomouc , czech republic ) while their body weight was measured using calibrated tanita wb 110 s ma ( quick medical corporation , seattle , wa , usa ) .
the first author measured the height and weight to the nearest 0.5 cm and 0.1 kg .
bmi was calculated as body mass in kilograms divided by height in meters squared ( kg / m ) .
children were classified as overweight if their bmi was equal to or greater than the sex- and age - specific 85th percentile from the world health organization growth charts ( world health organization 2007 ) available from the world health organization web site . after completing morning hygiene routines on the first day
, the monitored children s parents fastened the polar chest strap ( wearlink t31 ) around their child s chest and fitted the elastic waist belt with the actitrainer on their right hip . after arriving at school , the first author of the study and the class teachers checked the functioning of the actitrainer and wrote down the time of arrival in the proxy report .
teachers further recorded the beginning times of the school lessons and breaks in the proxy report . in the evening , the parents recorded the time at which both elastic belts were removed . statistical analyses including descriptive , correlations , and analyses of variance were performed using spss 19.0 .
a series of one - way ( at least 30 min or less than 30 min of moderate - to - vigorous pa during school breaks ) anovas were conducted to examine possible differences in overall pa ( number of steps , heart rate , duration of pa ) in each part of the school day , separately for girls and boys . before school time , pe lessons , teaching lessons , breaks , and after - school time were used as the dependent variables . a comparison of the proportion of overweight and obese children with and without 30 min of moderate - to - vigorous pa during school breaks was carried out using mann
three - time and three - way ( gender ; overweight and non - overweight state ; at least 30 min or less than 30 min of mvpa during school breaks ) anovas were used to examine the strength of the tested factors for overall daily pa .
spearman rank correlation rs was used to determine the relationship between school breaks and the overall daily pa .
three - time and three - way ( gender ; overweight and non - overweight state ; at least 30 min or less than 30 min of moderate - to - vigorous pa during school breaks ) anovas were also used to determine the variation in the intensity of overall pa during school break time in the three heart - rate zones . in order to identify the differences in overall pa between overweight and non - overweight girls and boys with or without 30 min of mvpa , a post hoc scheffe test was used .
the estimate of the strength of the relationship between the independent and dependent variables was represented as coefficients effect size
the values of 0.2 , 0.5 , and 0.8 were interpreted as small , medium , and large effects , respectively ( thomas and nelson 2001 ) .
elementary schools were selected intentionally based upon uniformly implemented daily school routines , similar size and equipment , number of students and localization in the city s development .
participants included 149 girls and 111 boys aged 9 and 10 in six classes of two elementary schools in katowice ( poland ) who returned signed , informed parental - consent forms enabling them to participate in the study . the final , entire 2-day monitored physical activity ( using an actitrainer activity monitor ) was completed , and 137 girls and 102 boys with a mean age of 9.48 0.40 years ( 20.1% overweight and 19.2% obese ) were included in the analysis of the data .
illnesses and non - participation in the physical education lesson were a reason for the elimination of 15 and 6 children , respectively ( representing 8% of girls and 8% of boys ) .
all of the children assessed wore the actitrainer activity monitor continuously for 2 days ( excluding sleeping , hygiene , and bathing times ) for a minimum of 10 h per day . between the 4th and 5th and
the 11th and 12th of february 2010 , all of the participating children followed their usual daily school routine , including five school lessons , four breaks and one lunchtime break .
school lessons usually started at 8 a.m. , were each 45 min in length and finished between 1 and 2 p.m. each of the children participated in one pe lesson with the same gymnastics content .
one of the four school breaks lasted 30 min , while the others were 5 min long . during the said 30-min break ,
the children played movement games , participated in rhythmic and dance activities as well as used sports and game equipment ( e.g. , a bouncy ball , frisbee , gymball , hopscotch and skipping rope ) in the playground under the supervision of teachers .
the children remained in their classrooms during the shorter breaks or could move around spontaneously in the school corridors under the supervision of teachers .
the actitrainer ( actitrainer , florida , usa ) is small and light ( 8.6 3.3 1.5 cm ; 53 g ) .
it is a multi - functional device composed of a heart - rate monitor , tri - axis solid - state accelerometer , an electronic pedometer , an inclinometer , and an ambient light sensor .
the recording of data can be viewed on the built - in display . upon turning on the display panel ,
the actitrainer can monitor and continually store recorded data over a period of 7 days .
the validity and reliability of the actitrainer - based step counting in non - laboratory conditions was verified with 20 non - obese university students ( neuls 2008 ) .
a right hip - fixed actitrainer activity monitor continuously measured the pa of children in 15-s intervals for the entire body - wearing time .
when monitoring pa , the actitrainer was secured at the waist using a neoprene pouch and an elastic belt .
when collecting heart - rate data , the polar chest strap was worn across the sternum .
the overall measured pa was simultaneously represented by number of steps , heart rate and duration of pa variables .
children were classed as being physically active , as opposed to sedentary , when > 25 counts per 15 s were recorded .
intensity zones for overall pa were set based on the percentage of the maximal age - related heart rate , i.e. , 220-age ( edwards 2010 ) .
moderate - to - vigorous physical activity was defined as heart rate above 60% of the maximum age - related heart rate in accordance with published studies ( edwards 2010 ; kirkpatrick and birnbaum 1997 ) .
children s height was measured using an anthropometer a-319 ( trystom corporation , olomouc , czech republic ) while their body weight was measured using calibrated tanita wb 110 s ma ( quick medical corporation , seattle , wa , usa ) .
the first author measured the height and weight to the nearest 0.5 cm and 0.1 kg .
bmi was calculated as body mass in kilograms divided by height in meters squared ( kg / m ) .
children were classified as overweight if their bmi was equal to or greater than the sex- and age - specific 85th percentile from the world health organization growth charts ( world health organization 2007 ) available from the world health organization web site . after completing morning hygiene routines on the first day , the monitored children s parents fastened the polar chest strap ( wearlink t31 ) around their child s chest and fitted the elastic waist belt with the actitrainer on their right hip . after arriving at school ,
the first author of the study and the class teachers checked the functioning of the actitrainer and wrote down the time of arrival in the proxy report .
teachers further recorded the beginning times of the school lessons and breaks in the proxy report . in the evening , the parents recorded the time at which both elastic belts were removed .
statistical analyses including descriptive , correlations , and analyses of variance were performed using spss 19.0 .
a series of one - way ( at least 30 min or less than 30 min of moderate - to - vigorous pa during school breaks ) anovas were conducted to examine possible differences in overall pa ( number of steps , heart rate , duration of pa ) in each part of the school day , separately for girls and boys . before school time , pe lessons , teaching lessons , breaks , and after - school time were used as the dependent variables .
a comparison of the proportion of overweight and obese children with and without 30 min of moderate - to - vigorous pa during school breaks was carried out using mann
three - time and three - way ( gender ; overweight and non - overweight state ; at least 30 min or less than 30 min of mvpa during school breaks ) anovas were used to examine the strength of the tested factors for overall daily pa .
spearman rank correlation rs was used to determine the relationship between school breaks and the overall daily pa .
three - time and three - way ( gender ; overweight and non - overweight state ; at least 30 min or less than 30 min of moderate - to - vigorous pa during school breaks ) anovas were also used to determine the variation in the intensity of overall pa during school break time in the three heart - rate zones . in order to identify the differences in overall pa between overweight and non - overweight girls and boys with or without 30 min of mvpa , a post hoc scheffe test was used .
the estimate of the strength of the relationship between the independent and dependent variables was represented as coefficients effect size
the values of 0.2 , 0.5 , and 0.8 were interpreted as small , medium , and large effects , respectively ( thomas and nelson 2001 ) .
the mean ( sd ) values for the children s anthropometric characteristics and physical activity levels during the segmented school day are presented in table 1 . of the participants , 42 girls ( 30.6% ) and 52 boys ( 51% ) were classified as overweight or obese .
children of both gender , with at least 30 min of moderate - to - vigorous school - break pa , show a significantly higher number of steps and duration of pa at school ( p < 0.001 ) and overall daily pa ( p < 0.01 ) than children without 30 min of mvpa during school breaks ( table 1 ) . a higher heart rate at school ( p < 0.001 ) and overall daily pa ( p < 0.01 ) in girls with at least 30 min of mvpa school breaks was also found when compared with girls with less than 30 min of mvpa during school breaks ( table 1).table 1anthropometric characteristics and physical activity levels [ mean ( standard deviation ) ] of 9- and 10-year - old overweight and non - overweight children divided based on the duration of the moderate - to - vigorous physical activity in school breaks ( katowice , poland , 2010)girls ( n = 137)boys ( n = 102)30 min of mvpa breaks ( n = 65)effect size ( d)<30 min of mvpa breaks ( n = 72)30 min of mvpa breaks ( n = 53)effect size ( d)<30 min of mvpa recess ( n = 49)anthropometric data age ( years)9.36 ( 0.38)9.62 ( 0.34)9.35 ( 0.38)9.56 ( 0.44 ) body height ( cm)134.62 ( 6.03)0.29136.64 ( 7.62)136.11 ( 5.84)0.18135.02 ( 6.62 ) body weight ( kg)32.92 ( 8.01)0.3035.39 ( 8.40)35.38 ( 6.83)0.2233.74 ( 8.09 ) bmi ( kg / m)17.99 ( 3.03)0.1018.69 ( 4.21)19.04 ( 3.03)0.2918.32 ( 3.23 ) overweightn = 14 ( 21.5%)0.16n = 11 ( 15.3%)n = 12 ( 22.6%)0.04n = 11 ( 22.5% ) obesityn = 6 ( 9.2%)0.18n = 11 ( 15.3%)n = 17 ( 32.1%)0.21n = 12 ( 24.5%)steps ( number ) before school1,219 ( 880)0.48844 ( 677)**1,315 ( 860)0.141,198 ( 857 ) at school3,049 ( 732)1.811,500 ( 954)***3,474 ( 1,152)1.112,110 ( 1,298 ) * * * pe lesson1,134 ( 674)0.201,244 ( 413)1,218 ( 751)0.491,544 ( 563 ) * * another lessons831 ( 532)0.74492 ( 379)***894 ( 696)0.23742 ( 636 ) breaks1,313 ( 406)2.44456 ( 294)***1,683 ( 628)2.06643 ( 321 ) * * * after school7,089 ( 3,012)0.096,779 ( 3,488)6,644 ( 3,899)0.455,141 ( 2,654 ) * all day11,357 ( 2,888)0.679,124 ( 3,689)***11,433 ( 4,057)0.848,449 ( 2,890)***heart rate ( beats / min ) before school111.96 ( 17.13)0.12110.06 ( 14.22)110.58 ( 18.92)0.42103.93 ( 11.75 ) * at school112.80 ( 9.74)0.69106.15 ( 9.63)***108.59 ( 9.46)0.16107.06 ( 9.73 ) pe lesson128.36 ( 20.42)0.46136.20 ( 13.24)122.34 ( 21.81)0.62134.12 ( 15.52 ) * another lessons107.15 ( 10.13)0.65101.17 ( 8.13)***102.58 ( 10.36)0.17100.97 ( 7.89 ) breaks125.01 ( 11.13)1.00114.26 ( 10.32)***122.77 ( 11.63)0.16120.73 ( 13.23 ) after school108.22 ( 10.45)0.44103.73 ( 9.83)*104.04 ( 13.60)0.24101.17 ( 10.15 ) all day111.00 ( 9.90)0.48106.64 ( 8.14)**107.74 ( 9.44)0.43104.05 ( 7.59)*duration of pa ( min ) before school42.17 ( 32.40)0.5427.21 ( 22.16)**39.68 ( 23.86)0.0942.27 ( 31.84 ) at school135.80 ( 21.33)2.1881.33 ( 27.92)***142.53 ( 27.81)1.7796.95 ( 23.27 ) * * * pe lesson30.49 ( 10.45)0.4434.38 ( 7.29)30.12 ( 11.36)0.7936.77 ( 2.69 ) * * another lessons75.22 ( 17.72)1.3049.20 ( 21.88)***83.15 ( 27.10)0.8960.31 ( 23.95 ) * * * breaks38.52 ( 5.16)3.7716.85 ( 6.23)***40.06 ( 5.58)3.3519.39 ( 6.75 ) * * * after school254.48 ( 66.78)0.21270.10 ( 77.91)233.44 ( 82.27)0.18218.50 ( 87.90 ) all day431.07 ( 65.08)0.68378.64 ( 86.99)***414.80 ( 91.48)0.65357.71 ( 83.68)**n number of participants , mvpa moderate - to - vigorous physical activity , bmi body mass index , pe physical education , pa overall physical activity , d coefficients effect size for one - way analysis of variance ( cohen 1988 ) and the mann
the values of 0.2 , 0.5 , and 0.8 were interpreted as small , medium , and large effects , respectively ( thomas and nelson 2001)statistical significance ( one - way analysis of variance ) of the differences between groups of children 30 min and < 30 min of mvpa during school breaks is expressed as : * p < 0.05 , * * p < 0.01 , * * * p < 0.001overweight or obesity represents bmi from 85th to 97th or greater than 97th of who growth charts ( world health organization 2007 ) anthropometric characteristics and physical activity levels [ mean ( standard deviation ) ] of 9- and 10-year - old overweight and non - overweight children divided based on the duration of the moderate - to - vigorous physical activity in school breaks ( katowice , poland , 2010 ) n number of participants , mvpa moderate - to - vigorous physical activity , bmi body mass index , pe physical education , pa overall physical activity , d coefficients effect size for one - way analysis of variance ( cohen 1988 ) and the mann
the values of 0.2 , 0.5 , and 0.8 were interpreted as small , medium , and large effects , respectively ( thomas and nelson 2001 ) statistical significance ( one - way analysis of variance ) of the differences between groups of children 30 min and < 30 min of mvpa during school breaks is expressed as : * p < 0.05 , * * p < 0.01 , * * * p < 0.001 overweight or obesity represents bmi from 85th to 97th or greater than 97th of who growth charts ( world health organization 2007 ) three - way anovas determined the significant effect of 30 min of mvpa during school breaks on the overall daily pa ( number of steps f = 27.66 , p < 0.001 ; heart rate f = 13.48 , p < 0.001 ; duration of pa f = 19.40 , p < 0.001 ) .
the effect of 30 min of mvpa during school breaks on overall daily pa is more significant than the two other tested factors , gender and overweight . in terms of gender , a significant effect was only found on all - day pa with heart rate ( f = 6.37 , p = 0.012 ) ; with the overweight variable , a significant effect was only found with the number of steps ( f = 4.45 , p = 0.036 ) .
overweight and non - overweight girls and boys with at least 30 min of mvpa during school breaks attained more than 10,000 steps per day .
overweight and obese children with at least 30 min of mvpa during school breaks demonstrated about 2,362 more steps per day than overweight and obese children with less than 30 min of mvpa during school breaks .
taken as a whole , girls and boys with at least 30 min of mvpa during school breaks were more active by more than 2,200 steps per day when compared with girls and boys with less than 30 min of mvpa during school breaks .
pa during pe lessons has a significant effect ( p < 0.001 ) on overall daily pa represented by the number of steps , the heart rate , and the duration of pa .
the effect of the realisation of pa during the pe lesson on overall daily pa was more significant than the other two factors , gender and overweight .
the length of school - break pa is positively associated in a significant fashion with the overall daily pa of girls ( number of steps rs = 0.36 , p < 0.001 ; heart rate rs = 0.55 , p < 0.001 ; duration of pa rs = 0.30 , p < 0.001 ) and boys ( number of steps rs = 0.50 , p < 0.001 ; heart rate rs = 0.49 , p < 0.001 ; duration of pa rs = 0.29 , p = 0.004 ) .
the positive relationship between pa during school breaks and overall daily pa is slightly higher with non - overweight children ( number of steps rs = 0.41 , p < 0.001 ; heart rate rs = 0.46 , p < 0.001 ; duration of pa rs = 0.33 , p < 0.001 ) than in their overweight peers ( number of steps rs = 0.38 , p < 0.001 ; heart rate rs = 0.55 , p < 0.001 ; duration of pa rs = 0.21 , p = 0.05 ) .
the distribution of the intensity of overall pa in school breaks for overweight and non - overweight girls and boys is presented in figs . 1 and 2 . both , overweight and non - overweight children with at least 30 min of mvpa during school breaks demonstrated a significantly longer period of pa in moderate ( f = 93.85 , p < 0.001 , d = 1.33 ) and vigorous ( f = 19.88 , p < 0.001 , d = 0.59 ) heart rate zones than children with less than 30 min of mvpa during school breaks ( figs . 1 , 2).fig . 1comparison of the intensity of overall physical activity in school breaks time for 9- and 10-year - old overweight and non - overweight girls divided based on the duration of moderate - to - vigorous physical activity during school breaks ( katowice , poland , 2010 ) .
p level of statistical significance ( post hoc scheffe test ) , maximum heart - rate values were calculated by a simplified age - related karvonen equation , i.e. , 220-age ( edwards 2010)fig .
2comparison of the intensity of overall physical activity during school breaks for 9- and 10-year - old overweight and non - overweight boys divided based on the duration of moderate - to - vigorous physical activity during school breaks ( katowice , poland , 2010 ) .
p level of statistical significance ( post hoc scheffe test ) , maximum heart - rate values were calculated by a simplified age - related karvonen equation , i.e. , 220-age ( edwards 2010 ) comparison of the intensity of overall physical activity in school breaks time for 9- and 10-year - old overweight and non - overweight girls divided based on the duration of moderate - to - vigorous physical activity during school breaks ( katowice , poland , 2010 ) .
p level of statistical significance ( post hoc scheffe test ) , maximum heart - rate values were calculated by a simplified age - related karvonen equation , i.e. , 220-age ( edwards 2010 ) comparison of the intensity of overall physical activity during school breaks for 9- and 10-year - old overweight and non - overweight boys divided based on the duration of moderate - to - vigorous physical activity during school breaks ( katowice , poland , 2010 ) .
p level of statistical significance ( post hoc scheffe test ) , maximum heart - rate values were calculated by a simplified age - related karvonen equation , i.e. , 220-age ( edwards 2010 )
this study indicated that school - related physical activity ( pe lessons and break - time activities ) represent a substantial part of the daily pa of school children ( loucaides and jago 2008 ; pate et al .
because of similarities in the results of the pa variables ( the number of steps , heart rate , and duration of pa ) of the participants , this discussion will concentrate on the particularly frequent variable , number of steps . in support of tudor - locke et al .
( 2009a ) and loucaides and jago ( 2008 ) , we have confirmed that overweight and non - overweight girls take an average of 24.1 and 22.2% of the daily steps within school , 11.1 and 10.8% during pe lessons , and 9.4 and 8.5% during breaks .
likewise , overweight and non - overweight boys reach 29.8 and 30.4% of the daily steps within school , 10.5 and 15.1% during pe lessons , and 12.2 and 12.5% at breaks .
this study highlighted the considerable contribution of at least 30 min of moderate - to - vigorous school - break pa to daily steps for overweight and non - overweight children ( 15.1 and 13.6% ) .
pa realised in pe lessons represents a similar contribution ( > 13% ) to the total number of daily steps in overweight and non - overweight children . during breaks between school lessons ,
children were able to move about spontaneously in the school corridors or carry out both structured and unstructured pa in the playground under the supervision of teachers .
structured pa consisted of the playing of movement games , rhythmic and dance activity as well as using sports and game equipment .
the children , irrespective of sex or body - weight status , made use of the opportunity to be active .
their involvement in the pa was most clearly marked in activities of a moderate intensity .
children who were not interested in participating in pa during school breaks were offered pa , but were not forced to participate .
2006 ) corroborate the present study , illustrating that pa - friendly school environments ( stratton and mullan 2005 ; ridgers et al .
2007 ) with high levels of equipment ( verstraete et al . 2006 ) and supervision ( sallis et al . 2001 ) stimulated children to be more active , irrespective of sex ( sallis et al . 2001 ; verstraete et al .
participation in at least 30 min of mvpa during school breaks significantly contributes to an increase in overall school- and all - day pa in overweight and non - overweight girls and boys and assists in approaching the health - related minimum of daily amount of steps ( 11,000 and 13,000 steps per day for girls and boys ) for this age category of children ( president s council on physical fitness and sports 2001 ; vincent and pangrazi 2002 ) .
female participants who partook in longer sessions of mvpa during school breaks also demonstrated a higher level of before - school pa .
girls with at least 30 min of mvpa during school breaks had a before - school number of steps equating to 10.7% of their all - day steps , while girls with less than 30 min of mvpa during school breaks had before - school steps of only 9.3% . before - school pa is undoubtedly linked with active school commuting , which is considered another effective way of increasing daily pa for school children ( faulkner et al .
the study s strengths include the triangulation approach to the assessment of pa levels . three different variables ( number of steps , duration of pa , and heart rate ) from one actitrainer monitoring device provide more reliable results than results based on one variable alone . however , this study has three main limitations .
first , it conducted a simplified calculation of the maximum age - related heart rate without knowledge of individuals resting heart rates .
actitrainer wear reactivity effect could influence the results , although two - day instrument monitoring of free - living pa is considered non - reactive ( tudor - locke et al .
for a more accurate measurement of the level of pa in the field using an actitrainer monitor , a longer period of monitoring ( including at least one weekend day ) in different seasons of the year would be needed .
a more comprehensive understanding of the overall daily pa of school - age children also requires valid information as to the possible influence of lifestyle behaviour , parents and peer support , and the environmental characteristics of their residence .
the prevalence of obesity and decreased pa among school - age children underpinned the implementation of effective strategies to increase daily pa and counteract child obesity .
school - related pa , including pe lessons and morning , lunchtime , and other breaks involving pa , has been helpful in terms of increasing the daily pa of school children .
pilot findings imply that 30 min of daily moderate - to - vigorous pa during school breaks significantly contributes to higher school and overall daily pa of overweight and non - overweight 9- and 10-year - old children and assists in the attainment of the health - related minimum of pa .
monitoring children s all - day pa with the use of the actitrainer multifunctional device made possible a comprehensive analysis and assessment of children s behaviour .
to conclude , promotion of an active lifestyle in children and youth is one of the health priorities of developed countries .
involvement of schools in the implementation of such priorities seems to be effective . by facilitating pa during school breaks ,
it is possible to encourage children to have an active lifestyle without regard to their gender , race or obesity level .
this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited . | objectivesthis study examines whether moderate - to - vigorous physical activity ( mvpa ) , in at least 30-min school breaks ( sb ) , helps to achieve the health - related amount of daily physical activity ( pa ) and whether these exercises influence after - school pa.methodsthe actitrainer - based pa was monitored over two school days in 239 children aged from 9 to 10 ( 57.3% female ; 20.1% overweight , and 19.2% obese ) , in katowice , in february 2010 .
pa was assessed based on steps , heart rate , and duration of pa.resultsmvpa , for 30 min during sb , represented an average of 1,258 steps for overweight girls and 1,620 steps for boys , and 1,336 steps for non - overweight girls and 1,758 steps for boys .
children with 30 min of mvpa during sb attained a higher daily amount of steps ( p < 0.001 ) and duration of overall pa ( p < 0.01 ) , in comparison with less physically active children.conclusionthe daily 30 min of mvpa during sb amounts to 12.5% of the overall number of steps for girls and 16.3% for boys , thus contributing to higher school pa and overall pa and leading to the achievement of the health - related minimum of pa . | Introduction
Methods
Participants and settings
Instruments and procedure
Statistical analyses
Results
Discussion
Limitations
Conclusion
Conflict of interest
Open Access |
renal transplantation ( rt ) as an effective mode of treatment of end stage renal disease has gained popularity across the world .
, rt provides a much better overall outcome . with technological advances and availability of effective therapy for immunosuppression the life expectancy after rt has gone up .
that gives us the opportunity to assess the outcome of these patients more holistically , taking morbidity , capacity of work , general life - style into account over and above life expectancy and renal function estimates .
formerly , transplant outcomes were measured mainly from the perspective of the clinicians , focusing on survival , creatinine values , degree of hypertension and so forth .
now , the subjective experiences of the patients are increasingly recognized as an important outcome measure . in this regard quality of life ( qol )
the term qol refers to the physical , psychological and social domains of health seen as distinct areas that are influenced by patients beliefs , experiences , expectations and perception .
qol and psychosocial well - being usually improve after a renal transplant and remain stable for a minimum of several years .
exceptions are often made by non - adherence : the degree to which the patient 's behavior differs from agreed medical recommendations .
moreover , symptom experience has been shown to be related to non - adherence and health related qol in renal transplant patients .
psychological factors , prominent through their presence as comorbidities , are the other sources of concern .
depression , anxiety and alteration of the body image have often been observed as comorbid conditions in patients of rt .
besides that , these conditions also may mar the broader picture of outcome where psychological well - being , satisfaction , capacity to work are considered among others .
we have assessed the patients cross - sectionally and estimated the renal function along with the level of depression , anxiety , adherence and the overall qol .
the outcome was defined as the composite qol including physical and psychological well - being .
the aim of the current study has been to see if and how the psychological factors as well as the parameters relevant to graft function are associated with the outcome of these patients .
this was a cross - sectional observational study carried out at the transplantation clinic in the department of nephrology , institute of post graduate medical education and research , kolkata , india .
it is a renowned hospital in the country with a wide catchment area including the entire eastern india along with other parts of the country .
the study sample consisted of 105 consecutive post - rt patients aged ranges from 18 - 60 years attending the clinic and fulfilling the inclusion criteria . written informed consent
inclusion criteria for the patients were stable clinical condition , absence of any infection or acute complication and an interval of at least 3 months after transplantation .
socio - demographic and clinical data was gathered using a pro - forma specially designed for the study which included all the relevant parameters as described in literature including age , sex , marital status , family type , monthly income , education , residence , current and past employment status , past medical and psychiatric history , family history , current and past intake of psychoactive substance ( s ) among others .
these included duration of renal failure , native kidney disease ( whenever the information is available ) , duration and type of dialysis , complications ( if any ) and general health during dialysis , creatinine level and medications immediately after transplantation ( after discharge from the hospital ) as well as on current assessment , episodes of rejection , infection , hospitalization , new onset diabetes after transplantation ( nodat ) if any , cost of treatment , etc .
patients understanding of the current treatment and course of recovery after transplantation was assessed through a list of questions cited in the structured interview for renal transplantation ( sirt ) which assesses patient 's ability to identify medications , state the purpose of transplantation , the side - effects of medications , the symptoms and risk of rejection , the importance of compliance and the post - operative recovery course . anxiety and depression were assessed by hospital anxiety and depression scale ( hads ) .
so , few modifications were made before using it in the current study , e.g. , the expressions such as blood pressure treatment plan was replaced by
the questions in this scale are relevant to the current research and it has the added advantage of being brief and focused .
the maximum obtainable score in this scale is 8 which denotes high adherence , a score below 6 denotes low adherence and scores between 6 and 8 is considered medium adherence .
qol was assessed through the abbreviated version of world health organization ( who ) qol scale ( whoqol - bref ) .
it is a 26-item questionnaire which is scored on a likert scale from 1 - 5 and generates four domain scores .
all the rating scales were administered on the patients during clinical visit to the transplantation clinic .
the collected data was statistically analyzed using statistical package for social sciences ( spss ) 16.0 for windows . based on hads
subscale score ( 11 or more or < 11 ) , patients were grouped as with or without anxiety and depression .
the groups were compared using mann - whitney u - test and fisher 's exact test for continuous and categorical variables , respectively .
the effect sizes were reported as r ( calculated from z value of mann - whitney u - test ) and cramer 's v. the level of significance was set at p < 0.05 ( two - tailed ) .
this was a cross - sectional observational study carried out at the transplantation clinic in the department of nephrology , institute of post graduate medical education and research , kolkata , india .
it is a renowned hospital in the country with a wide catchment area including the entire eastern india along with other parts of the country .
the study sample consisted of 105 consecutive post - rt patients aged ranges from 18 - 60 years attending the clinic and fulfilling the inclusion criteria . written informed consent
inclusion criteria for the patients were stable clinical condition , absence of any infection or acute complication and an interval of at least 3 months after transplantation .
socio - demographic and clinical data was gathered using a pro - forma specially designed for the study which included all the relevant parameters as described in literature including age , sex , marital status , family type , monthly income , education , residence , current and past employment status , past medical and psychiatric history , family history , current and past intake of psychoactive substance ( s ) among others .
these included duration of renal failure , native kidney disease ( whenever the information is available ) , duration and type of dialysis , complications ( if any ) and general health during dialysis , creatinine level and medications immediately after transplantation ( after discharge from the hospital ) as well as on current assessment , episodes of rejection , infection , hospitalization , new onset diabetes after transplantation ( nodat ) if any , cost of treatment , etc .
patients understanding of the current treatment and course of recovery after transplantation was assessed through a list of questions cited in the structured interview for renal transplantation ( sirt ) which assesses patient 's ability to identify medications , state the purpose of transplantation , the side - effects of medications , the symptoms and risk of rejection , the importance of compliance and the post - operative recovery course .
so , few modifications were made before using it in the current study , e.g. , the expressions such as blood pressure treatment plan was replaced by
the questions in this scale are relevant to the current research and it has the added advantage of being brief and focused .
the maximum obtainable score in this scale is 8 which denotes high adherence , a score below 6 denotes low adherence and scores between 6 and 8 is considered medium adherence .
qol was assessed through the abbreviated version of world health organization ( who ) qol scale ( whoqol - bref ) .
it is a 26-item questionnaire which is scored on a likert scale from 1 - 5 and generates four domain scores .
all the rating scales were administered on the patients during clinical visit to the transplantation clinic .
the collected data was statistically analyzed using statistical package for social sciences ( spss ) 16.0 for windows . based on hads subscale score ( 11 or more or < 11 ) , patients were grouped as with or without anxiety and depression .
the groups were compared using mann - whitney u - test and fisher 's exact test for continuous and categorical variables , respectively .
the effect sizes were reported as r ( calculated from z value of mann - whitney u - test ) and cramer 's v. the level of significance was set at p < 0.05 ( two - tailed ) .
the mean age of the sample was 35.03 [ standard deviations ( sd ) 9.75 ] years and mean years of education was 12.2 ( sd 3.15 ) years . among them
76 ( 72.4% ) were males , 67 ( 63.8% ) were married and 53 ( 50.5% ) were employed .
the median duration of renal failure was 12 [ interquartile range ( iqr ) 15 ] months and median duration of dialysis was 6 ( iqr 5 ) months . the median time elapsed since transplantation was 23 ( iqr 32 ) months . among the patients , 9 ( 8.57% ) had depression and 9 ( 8.57% ) had anxiety as assessed by hads .
the mean hads depression and anxiety score in these patients was 12.78 ( sd 2.22 ) and 10 ( sd 5.77 ) , respectively .
the comparisons of socio - demographic and clinical variables among the groups are summarized in table 1 .
there were significantly higher number of episodes of complication or rejection in post - rt patients with anxiety as compared to those without ( u = 223 , z = 3.23 , p = 0.001 , r = 0.32 ) .
furthermore , the post - rt patients with anxiety had significantly higher number of hospitalizations than those without anxiety ( u = 232 , z = 2.45 , p = 0.014 , r = 0.24 ) .
there was significantly lower creatinine levels in those without anxiety as compared to those with anxiety disorder ( u = 176 , z = 2.93 , p = 0.003 , r = 0.29 ) .
significantly higher number of post - rt patients without depression had a poor general health ( 78% ) during dialysis compared to their counterparts ( 27% ) with depression ( p = 0.004 , cramer 's v = 0.31 ) . in those with anxiety , 70% had any episode of rejection as compared to only 20% without anxiety , which was statistically significant ( p = 0.005 , cramer 's v = 0.31 ) .
for post - rt patients with depression the mean age was higher and mean duration of renal failure was longer than the patients without depression but for both these parameters the differences did not reach statistical significance though a trend could be noticed in both cases .
group differences in demographic and clinical profile the differences between patients with depression or anxiety and those without in various domains of qol , adherence as well as in their awareness of the current treatment and course of recovery are summarized in table 2 . in those with anxiety , qol was significantly lower in four out of five domains , i.e. , general ( u = 60 , z = 4.39 , p < 0.001 , r = 0.43 ) , experience ( u = 175.5 , z = 2.95 , p = 0.003 , r = 0.29 ) , satisfaction
( u = 75.5 , z = 4.13 , p < 0.001 , r = 0.40 ) and depression ( u = 108 , z = 4.03 , p < 0.001 , r = 0.39 ) , whereas it was not significantly different in ability domain . in patients with depression ,
qol was significantly lower in all domains , i.e. , general ( u = 163.5 , z = 3.17 , p = 0.002 , r = 0.31 ) , experience ( u = 109.5 , z = 3.71 , p < 0.001 , r = 0.36 ) , ability ( u = 230.5 , z = 2.33 , p = 0.02 , r = 0.23 ) , satisfaction ( u = 134.5 , z = 3.41 , p = 0.001 , r = 0.33 ) and depression ( u = 87 , z = 4.29 , p < 0.001 , r = 0.42 ) .
the present study examined 105 consecutive rt patients fulfilling the criteria and assessed their comprehensive renal function through reviewing charts and their awareness about treatment and illness along with its course through a validated instrument [ structured interview for renal transplantation ( sirt ) ] made solely for this purpose .
the assessments of their anxiety , depression and qol were made with the help of another set of validated and standardized instruments ( hads and whoqol - bref ) .
a study of this sample size with meticulous assessments of their renal function as well as psychiatric comorbidity has not been carried out so far from this part of the world .
our study reveals a point prevalence of both syndromal anxiety and depression to be 8.57% each ( nine patients out of 105 ) .
the presence of anxiety and depressive symptoms in a sub - syndromal level is more widespread among the post - rt subjects .
alavi et al . in their cross - sectional study of 100 rt and 63 hd patients had shown 65.3% and 51.6% of the rt patients had depression and anxiety respectively using symptom checklist-90 subscales of depression and anxiety .
whereas 24 out of 88 ( 27.27% ) patients had anxiety and 20 out of 88 ( 22.73% ) had clinical depression in another study in iran where , like the current study , the assessments were made using hads and cases were defined when the scores exceeded 11 .
an even higher prevalence ( anxiety in about 50% and depression in 25% of patients ) has also been reported from turkey .
these differences in prevalence across studies might reflect the inherent differences of the study groups as prevalence of depression is known to vary widely across nations . a somewhat unexpected finding was that a significantly higher number of patients without depression had a poor general health during dialysis than their depressed counterparts .
the reports of general health prior to rt while patients were on hd were gathered from the patients themselves based on their recall .
general health was assessed through general health questionnaire-12 and a score of over 3 was considered poor general health .
this is a reflection of health prior to rt and the depression that was assessed in the current study developed after rt with possible psychosocial as well as biological causes underlying it .
therefore , a higher number of non - depressed patients showing poor general health do not produce any serious incongruity to the overall study findings .
another surprising finding was neither anxiety nor depression was seen to be associated with poor adherence .
it is general wisdom among psychiatrists that adherence is a big challenge in treating any of the psychiatric disorders .
depression has specifically been shown to negatively affect immunosuppressive medication adherence in a previous study . however , in the current study neither anxiety nor depression was seen to be associated with poor adherence .
this may be due to the low prevalence of psychiatric comorbidity detected in the current study thereby not adequately reflecting the picture of adherence in rt patients .
another possible explanation is that the patient education delivered by all the members of the transplantation clinic during the attendance of patients in the clinic through individual and group counseling might have improved compliance .
as expected , the various domains of qol were poor in patients with anxiety and depression .
patients with depression had a significantly poorer qol in all domains in comparison with the patients without depression with medium effect sizes for most of the subscales .
for patients with anxiety , however , there was not any significant difference in the subscale of ability in comparison with their non - anxious counterparts .
qol in its various domains as well as the perception of qol have been shown to be poor in rt patients with depression .
the post - rt patients with anxiety had significantly more episodes of complications or rejections with a medium effect size ( r = 0.32 ) and higher number of hospitalizations with a small effect size ( r = 0.24 ) compared to their non - anxious counterparts .
futhermore , among those with anxiety the proportion of subjects with any episode of rejection ( 70% ) was significantly higher than among those without anxiety ( 20% ) and the effect size of this difference was medium ( cramer 's v = 0.31 ) .
another important finding was that the creatinine level was significantly lower in patients without anxiety compared to their anxious counterparts though the effect size was small ( r = 0.29 ) .
these findings in the current study show anxiety being related to parameters directly reflecting post - rt renal function .
such findings have not been reported in any of the previous studies . in a previous study ,
noohi et al . compared the patients with syndromal anxiety against those with sub - syndromal anxiety and could not spot any significant difference in the history of graft rejection
. there has been a concern among the researchers about the presence of psychiatric disorders in rt patients as these conditions might increase the morbidity . since
a process of rt is not just aimed at patient survival , but also at decreasing the morbidity to a minimum , a parallel assessment of these conditions is considered useful . furthermore , the current study showed that a psychiatric comorbidity in the form of anxiety , was not just leading to a poor qol , but also was associated with episodes of graft rejection , complications , higher level of creatinine and more number of hospitalizations , i.e. , the parameters more closely reflecting renal function after rt per se .
the major limitation of our study was the cross - sectional design . a longer period of observation would have given us a better picture of the role of psychiatric morbidity in post - rt renal function and overall outcome .
furthermore , assessment of biological parameters such as cortisol level , could possibly suggest a link between the stress arising out of rt and the development of psychiatric comorbidities .
this also limits the study as a hypothesis on the possible causes of psychiatric comorbidities could not be proposed .
furthermore , medication adherence scale has not been validated in post - rt patients , which limits its use in this population . | the objective of this study was to determine anxiety and depression and its relationship with quality of life ( qol ) in renal transplant ( rt ) recipients .
a total of 105 consecutive patients were assessed cross - sectionally at least 3 months after rt .
hospital anxiety and depression scale was applied to assess anxiety and depression .
qol was assessed through the abbreviated version of world health organization qol scale .
patients awareness of illness and treatment was assessed through structured interview for renal transplantation .
nine ( 8.57% ) patients had syndromal anxiety and 9 ( 8.57% ) had syndromal depression .
both these groups had significantly lower scores in almost all domains of qol compared with their non - anxious and non - depressed counterparts .
there were a higher number of hospitalizations and episodes of complication or rejection in post - rt patients with anxiety as compared to those without ( p = 0.001 ) .
syndromal depression and anxiety are associated with poor qol and syndromal anxiety is associated with significantly higher number of hospitalizations , rejections and complications in post - rt patients . | Introduction
Materials and Methods
Participants
Tools for assessment
Procedure
Statistical analysis
Results
Discussion |
genetic susceptibility and exposure to neurotoxins are contributors to brain tissue malfunction and neuronal cell loss .
environmental toxins influence pd onset ( rajput 2001 ) and are also suggested to be involved in atypical parkinsonian syndromes ( caparros - lefebvre and lees 2005 ) and progressive supranuclear palsy ( psp ) ( davis et al .
furthermore , the age - related neurodegenerative disorders are commonly characterised by the accumulation of insoluble neurotoxic ubiquinated proteins ( johnson 2000 ) .
the neurotoxin hypothesis states that detoxification enzymes can influence susceptibility to pd , but recently another mechanism has also been recognised as important for detoxification of the brain .
proteins that are involved in cellular efflux prevent penetration into the brain by various potentially toxic compounds or their accumulation .
they include p - glycoprotein ( p - gp ) ( cordon - cardo et al .
1989 ) and multidrug resistance associated protein ( mrp ) . at the blood brain barrier ( bbb ) , p - gp is expressed in a high concentration at the luminal side of the brain vascular endothelium ( demeule et al .
2001 ) and functions as an efflux pump by translocating a substrate to the extracellular compartment .
p - gp in the endothelial cells of the choroid plexus has a transport direction towards the csf ( de lange 2004 ) .
the p - gp transporter is associated with the multidrug resistance gene ( mdr1 ) .
interestingly , a significant association was found between parkinsonian patients exposed to pesticides and c3435 t polymorphism of the mdr1 gene , related to lower p - gp function ( drozdzik et al .
thus the mutation of the mdr1 gene with diminished p - gp function increases the vulnerability to the damaging effects of pesticides .
it is assumed that neurotoxic substances such as certain pesticides are substrates of p - gp ( bain and leblanc 1996 ) and cns exposure to such agents may continue to cause neurodegenerative diseases . in a pilot study ( kortekaas et al .
2005 ) , impaired bbb p - gp transport was found in the midbrain of five pd patients compared to five healthy control subjects , measured with [ c]-verapamil pet , pointing to a role of p - gp in the brain to prevent accumulation of toxic substances causing pd neurodegeneration .
this finding , however , could not be replicated in a study that measured p - gp function with [ c]-(r)-verapamil pet in ten early stage pd patients compared to healthy controls ( bartels et al .
decreased expression of p - gp in bbb vasculature has been found in other neurodegenerative diseases , like alzheimer s disease ( ad ) ( vogelgesang et al . 2001 ) and creutzfeldt jakob disease ( cjd ) ( vogelgesang et al
. 2006a ) . in ad , p - gp up - regulation in early pathogenesis
was suggested as a compensatory mechanism to increase the clearance of harmful proteins , while at later disease stages p - gp expression was lost ( vogelgesang et al .
we hypothesize that p - gp - mediated transport out of the brain is involved in clearance of harmful substances and dysfunction of this system also occurs in late stage parkinsonian neurodegeneration . in the present study
, we performed [ c]-verapamil - pet brain imaging of new groups of de novo pd patients , pd patients in more advanced disease stages , progressive supranuclear palsy ( psp ) , and multisystem atrophy ( msa ) patients and a healthy control group , to study regional bbb p - gp function in patients with different parkinsonian pathology .
pet with [ c]-verapamil has been shown to be an appropriate methodology for the in vivo assessment of the functional p - gp status in human bbb ( hendrikse et al .
the distribution volume ( dv ) of the p - gp substrate verapamil can be taken as a measure of p - gp efflux function , higher [ c]-verapamil uptake meaning less p - gp efflux function .
patients were selected from the movement disorders clinic of the university medical centre groningen ; healthy volunteers were asked to participate by advertisement .
the study was approved by the medical ethics committee of the university medical centre groningen and all subjects gave their informed consent .
ten de novo pd patients ( diagnosed in the past year and without pd medication ) , and ten pd patients in more advanced disease stage ( hoehn and yahr stages 23 ) participated in the study ( table 1 ) .
furthermore , five patients with psp and four patients with msa , compliant with the criteria for psp or msa according to the ninds - spsp international workshop ( litvan et al . 1996 ) and the consensus statement for the diagnosis of msa ( gilman et al , 1999 ) were included .
nine healthy age - matched subjects , who did not use medication , participated as controls .
patients did not use known p - gp modulating agents [ ( cardiovascular drugs , antimalarial drugs , cyclosporine a , phenothiazines , hormones ( e.g. , tamoxifen ) ] and certain antibiotics such as cefoperazone , ceftriaxone , and erythromycine [ see for a detailed list ( hendrikse et al . 2001 ) ] .
t1 weighted mri scans were performed on all subjects and did not show abnormalities or significant atrophy of cortical regions .
table 1patient characteristicsdiagnosis ( n)mean agedisease durationupdrs iii medicationpd ( 10)63 sd 85 years sd 221 sd 7 ( onstate)levodopa + dopamine - agonist ( ropinirole or pramipexole or pergolide)pd de novo ( 10)66 sd 9<1 year19 sd 8xpsp ( 5)63 sd 83 years sd 220 sd 8amantadine ( 2 patients)msa ( 4)59 sd 113 years
sd 227 sd 3levodopa ( 2 patients)controls ( 10)59 sd 10none0nonemean values for the groups are given with standard deviations ( sd ) patient characteristics mean values for the groups are given with standard deviations ( sd ) [ c]-verapamil was synthesised as described before ( wegman et al .
pet scans were performed with an ecat exact hr + positron camera ( siemens / cti , knoxville , tn ) .
after cannulation of the radial artery , all subjects underwent 1 h dynamic pet scanning consisting of 21 frames after injection of [ c]-verapamil ( 200400 mbq ) .
serial arterial blood sampling to define the input function for [ c]-verapamil was done using a computerised sampling programme ; six remaining samples were drawn manually . these samples ( collected at intervals of 10 min )
were processed by high liquid chromatography to measure the fraction of unmetabolised parent compound . in this way
pet data were reconstructed to a 128 128 63 matrix with a plane separation of 2.425 and a bin size of 2.250 mm . for anatomical demarcation , the frames 1221 of the dynamic [ c]-verapamil scans were summated using clinical application programming package ( capp ; siemens , erlangen , germany ) . on this summated image ,
capp regions of interest ( rois ) were manually drawn over the midbrain and whole brain to calculate distribution volume ( dv ) in the rois .
using matlab ( the mathworks inc . , natick , usa ) and a linearization according to logan ( 2000 ) , starting at t = 5 min , dv images were constructed from the [ c]-verapamil scans , which was done for the whole brain roi as well as pixelwise to obtain parametric dv images .
logan analysis is a graphical method of analysis , applicable to ligands that bind reversibly to receptors or enzymes , calculating the dv for dynamic pet data before steady state is actually reached . for comparison of regional differences between the groups
1991 ) ( spm99 , wellcome department of cognitive neurology , london , uk ) .
spm99 instead of the latest spm version was used to compare the results with those of the pilot study ( kortekaas et al .
individual dv images were spatially normalised to the [ c]-verapamil dv template that was constructed in the pilot study .
after spatial smoothing by convolution with a gaussian kernel of 20 mm , a two - sample t test was done . to visualize the statistical parametric maps , the threshold was set to p < 0.001 uncorrected . because higher variation in dv between individuals may render smaller differences between the groups statistically insignificant in spm , t tests were repeated after global calculation of the dv images to 100 in spm .
we also used this spm analysis method to compare our results with the previous pilot study ( kortekaas et al .
2005 ) . from each subject 10 ml blood was collected in an edta coated vial for determination of mdr1 c3435 t polymorphism on exon 26 . a polymerase chain reaction ( pcr)-based restriction fragment length polymorphism assay was performed with the primers
mdr1f 5-tgctggtcctgaagttgatctgtaac-3 and mdr1r 5-acattaggcagtgactcgatgaaggca-3. pcr was carried out with an initial denaturation of 12 min at 95c followed by 30 cycles of denaturation at 94c for 1 min , primer annealing for 1 min at 60c , and 1 min extension at 70c .
when the mutation was present ( 3435 t ) , digesting of the 248 bp pcr - product produced the 172- , 60 bp and 16-bp fragments .
the digested pcr products were analysed on a 2% agarose gel with ethidium bromide staining .
whole brain verapamil dv of all subjects was plotted against the c3435 t polymorphism ( cc , ct and tt genotypes ) , and correlation coefficient of genotype with dv was calculated in all subjects and in the separate groups .
[ c]-verapamil uptake was high in the pituitary , ventricles and skin , moderate in grey matter and low in white matter and bone .
the logan analysis provides the dv of a tracer , and is defined by a ratio between the influx of the tracer k1 and the efflux out of the brain k2 , of which the latter reflects the efflux function of p - gp .
the influx k1 of [ c]-verapamil was of the same magnitude in all groups , which confirms that differences in dv reflect differences in p - gp efflux function . mean dv in the more advanced pd group was 0.56 ( sd 0,12 ) , de novo pd showed 0.50 ( sd 0,08 ) , psp 0.66 ( sd 0,05 ) , msa 0.66 ( sd 0,07 ) and controls 0.59 ( sd 0,13 ) .
k1 in the pd group gave 0.037 ( sd 0,013 ) , psp 0.038 ( sd 0,005 ) , msa 0.036 ( sd 0,006 ) and controls 0.036 ( sd 0,006 ) .
pixel by pixel t test ( p < 0.001 uncorrected ) in spm did not show increased midbrain [ c]-verapamil uptake in either of the pd groups as compared to the control group .
the advanced pd group showed significantly increased [ c]-verapamil uptake in frontal white matter regions compared to controls ( spm coordinates 34 , 30 , 26 and 16 , 48 , 12 , near grey matter brodmann areas 9 and 10 , p corrected at cluster level 0.000 , fig .
1 ) ; a cluster with decreased uptake was seen in the cerebellar tonsils ( maximum effect at coordinates 40 , 54 , 46 ) .
the de novo pd group had lower uptake in brainstem and cerebellar tonsils ( brainstem coordinates 2 , 26 , 26 with p corrected at cluster level 0.000 , fig . 2 ) compared to the control group and did not show clusters with higher uptake .
plots of the local spm responses showed a considerable separation of the groups , although there are some overlapping cases , where the pd groups show larger variation in local response values .
comparison of de novo and advanced pd groups revealed a cluster of pixels reflecting higher uptake in the advanced pd group in the anterior cingulate cortex ( coordinates 4 , 28 , 6 ) and in the caudate head ( coordinates 14 , 14 , 2 with p corrected at cluster level 0.01 ) .
increased [ c]-verapamil uptake in the frontal lobe in advanced pd patients compared to healthy controls ; spm coordinates 34 , 30 , 26 and 16 , 48 , 12 ( brodmann areas 9 and 10 ) with p corrected at cluster level 0.000 .
b plot of the frontal effect ( pd patients scan number 110 and controls 1120 ) shows little overlapfig .
2a de novo pd patients showed lower brainstem [ c]-verapamil uptake in the brainstem compared to controls , p < 0.001 ; spm coordinates 2 , 26 , 26 with p corrected at cluster level 0.000 .
b plot of the brainstem effect shows more variation in pd de novo values ( scan number 110 ) with some overlap with the control group ( scan number 1120 ) a spm t test on mri overlay showing p < 0.001 . increased [ c]-verapamil uptake in the frontal lobe in advanced pd patients
compared to healthy controls ; spm coordinates 34 , 30 , 26 and 16 , 48 , 12 ( brodmann areas 9 and 10 ) with p corrected at cluster level 0.000 .
b plot of the frontal effect ( pd patients scan number 110 and controls 1120 ) shows little overlap a de novo pd patients showed lower brainstem [ c]-verapamil uptake in the brainstem compared to controls , p < 0.001 ; spm coordinates 2 , 26 , 26 with p corrected at cluster level 0.000 .
b plot of the brainstem effect shows more variation in pd de novo values ( scan number 110 ) with some overlap with the control group ( scan number 1120 ) in the psp group , significantly increased uptake was seen in basal ganglia regions ( maximum effect in lentiform nucleus , coordinates 26 , 10 , 6 with p corrected at cluster level 0.06 , fig .
when plotting the effect , one psp patient did not show higher uptake compared to the healthy controls ( fig .
notably , this was also the patient with shortest disease duration ( < 1 year ) and only mild parkinsonian symptoms ( updrs motor part score 12 ) .
the psp group had also increased uptake in frontal regions compared both to controls and the advanced pd group , but this did not reach significance and plotting the effect showed high variance between the psp patients .
msa patients did not show significant differences compared to controls , however after lowering the threshold to p < 0.01 , also increased uptake in basal ganglia regions was seen with p corrected at cluster level 0.98 and uncorrected p at 0.056 in putamen ( 28 , 6 , 2 ) .
plot of the spm effect showed more variance between the msa patients with one patient in the control range .
this patient had lower disease severity compared to the other msa patients , with 2 years disease duration and updrs motor score 24 .
3a psp patients showed higher [ 11c]-verapamil uptake in lentiform nucleus and putamen compared to controls , p < 0.001 ; spm coordinates 26 , 10 , 6 with p corrected at cluster level 0.06 .
b plot of the basal ganglia effect shows higher values in four of the five psp patients ( scan number 15 ) .
psp patient number 5 had shortest disease duration ( < 1 year ) a psp patients showed higher [ 11c]-verapamil uptake in lentiform nucleus and putamen compared to controls , p <
0.001 ; spm coordinates 26 , 10 , 6 with p corrected at cluster level 0.06 .
b plot of the basal ganglia effect shows higher values in four of the five psp patients ( scan number 15 ) .
psp patient number 5 had shortest disease duration ( < 1 year ) all results did not remain statistically significant after correction for multiple comparisons in spm ( fdr correction with level of significance set at p < 0 .
t - tests in spm were repeated after global calculation of the dv images to 100 , which showed comparable results and did not reveal additional foci with relative dv differences between the groups .
the frequencies of the determined mdr1 polymorphism in our study group did not differ from the frequencies seen in the general population ( tan et al .
furthermore , c3435 t genotypes did not significantly correlate with the differences in [ c]-verapamil dv in the brain in all groups .
we compared [ c]-verapamil pet data of the present study with data of the previously published pilot study that was performed by our group .
mean injected dose was lower in the pilot study with 111 mbq given as lowest dose ; for a better signal - to - noise ratio , the cut - off dose for the current study was set at 200 mbq .
the previous pilot control group showed relatively lower midbrain uptake compared to the nine control subjects of this study , which , however , only was shown in spm after global calculation of the mean dv to 100 .
neither group used medication nor was there a significant age difference between both the control groups , although the pilot group had one outlier of 86 years .
we could not demonstrate a definite methodological explanation for the midbrain effect in the pilot control subjects .
however in our opinion , global calculation of the mean may have affected the results in the midbrain area , which is a small region with a low uptake signal and is thereby prone to artefacts . finally , because of the lower injected tracer dose , data of the previous pilot study were not further used for the present study .
the results of this study do not support the idea of a primary dysfunctional bbb p - glycoprotein efflux system in the pathogenesis of parkinson s disease , but finds decreased p - gp function at later disease stages .
this is in line with a most recent study that measured bbb p - gp function in mostly early stage pd patients using [ c]-(r)-verapamil , where higher variance in tracer uptake was seen in several regions in the pd group , but no significant decreased p - gp function was found ( bartels et al .
more advanced pd patients showed frontal regions with diminished p - gp function relative to normal controls and de novo pd patients .
regional frontal decreases in both advanced pd and advanced psp possibly reflects the affected frontostriatal neuronal projections with frontal dysfunction in these disease stages ( green et al . 2002 ) .
the increased uptake lies in the white matter regions rather than the cortex . in general ,
involvement of white matter regions and vasculature in neurodegenerative disorders that can be seen as proteinopathies , involving accumulation of misfolded proteins , is a topic of important discussion .
it was shown in vitro that -amyloid is transported by p - gp ( kuhnke et al .
interestingly , the deposition of a140 in cerebral amyloid angiopathy ( caa ) , which is found in around 90% of ad patients , is predominantly found in the penetrating arterioles that supply the periventricular white matter ( lee and markus 2006 ) .
another study associated the failure of interstitial fluid drainage from the white matter in ad with the deposition of a in the perivascular fluid drainage pathways ( roher et al .
thus , decreased p - gp function in white matter regions may influence protein removal and accumulation at sites more distant from the white matter . furthermore
, it is suggested that lewy body - related pathology initially involves the neuronal perikarya , dendrites , and axons , with abundantly present
these authors also find that impairment of axonal transport and synaptic transmission leads to the formation of lewy bodies , a hallmark of functional disturbance long before neuronal cell death .
white matter p - gp dysfunction might also influence removal of toxic substances such as pesticides , which could cause harm to distant sites of the white matter after alterations of axonal transport , leading to dying - back patterns of neurodegeneration ( morfini et al .
greater disease severity and a more rapid neurodegenerative process have been associated with white matter hyperintensities ( piccini et al .
however , although specific methodology or rating scales were not used to assess white matter pathology on the mri s in this study , we could not detect any visually apparent white matter hyperintensities in any subject .
both de novo and more advanced pd patients showed lower [ c]-verapamil uptake in the cerebellum . the cerebellum is closely linked to midbrain nuclei , but is by itself not affected with progression of pd pathology ( braak and braak 2000 ) .
possibly , p - gp function in these regions is increased in pd , but does not deteriorate in later stages , as these regions are not vulnerable to pd pathology . on the other hand , in msa cerebellar degeneration can be part of the disease spectrum .
we did not find changed p - gp function in the cerebellum in the msa patients .
however , the msa patients in this study did not show symptoms of cerebellar dysfunction and mri scans did not show cerebellar atrophy .
psp and msa patients showed basal ganglia regions with increased [ c]-verapamil uptake , pointing at decreased p - gp function in regions with neuropathological features in these disorders .
brain atrophic changes are not likely to play a role in these results , because mri scans did not show overt atrophy in these regions .
psp patients also showed decreased p - gp function in frontal regions . although this result was not significant , plotting this effect showed higher variation between the psp patients with one psp patient in early disease stage who showed values overlapping with the controls ( fig .
these results may possibly point at regionally decreased p - gp function with advancing disease stage . regionally decreased p - gp function could thus occur not only in more advanced pd , but may also play a role in other neurodegenerative diseases with parkinsonism , and likely plays a role in advancing neurodegenerative processes in general .
it has been argued that decreased cerebrovascular p - gp impairs the ability of the brain to expel excess proteins , and that the resulting accumulation of protein in cells could overwhelm cellular ubiquitin - proteasomal degradation ( vogelgesang et al .
p - gp was even found to interact actively with the proteasome complex , and a role for p - gp in the transport of proteasome - derived peptides was suggested ( begley et al .
loss of p - gp function may possibly also lead to increased accumulation of insoluble -synuclein with ongoing neurodegeneration in pd .
p - gp dysfunction in late disease stage may also contribute to neuronal damage due to increased accumulation of toxins .
it is known that environmental factors causing damage , such as pesticides , are closely linked to mechanisms underlying the formation of -synuclein pathology and pd - like neurodegeneration ( norris et al . 2007 ) .
furthermore , it is recognised that the prevalence of dementia is higher in pd patients than in age - matched controls , which often is found to be defined by ad pathology in conjunction to the lewy bodies ( burn 2006 ) .
decreased p - gp function to expel proteins in advanced pd could also play a role in the co - existence of -synuclein and -amyloid in pd with dementia ( pdd ) , a patient group that was not investigated in the present study . in the study presented here , de novo pd patients showed lower [ c]-verapamil uptake compared to normal controls in the brainstem , which may indicate a regional up - regulation of p - gp function in early stage pd .
the brainstem regions lies posterior to the pons , comprising the locus coeruleus and lying slightly superior to the dorsal motor vagal nucleus , a region that is affected early in pd pathology ( del et al .
2002 ) , containing projection neurons of the intermediate reticular zone , coeruleus subcoeruleus complex and caudal raphe nuclei .
a limitation of the methods , using [ c]-verapamil uptake as a measure of p - gp efflux function , is the low uptake signals of this tracer in the normal condition , as it is removed by p - gp .
intersubject variation in tracer uptake can be substantial , whereby small regional effects may be overlooked in the statistical comparison .
on the other hand , global calculation can be applied to investigate regional patterns within disease groups rather than absolute p - gp measures .
this should be applied with care , as small regions with low uptake signals may be prone to artefactual changes . in the case of the present study ,
the brainstem effect was also seen without proportional scaling in spm , and is therefore more likely a significant result . initial up - regulation of p - gp expression or increase of p - gp function in reaction to neuronal damage may be associated with a neuroinflammatory process in parkinson s disease . in pd as well as in other neurodegenerative diseases , extensive microglia activation , the key event in neuroinflammation ,
is seen ( gerhard et al . 2001 ; mcgeer and mcgeer 1998 , 2004 ) .
microglia themselves express p - gp , which increases upon microglia activation ( langford et al .
several factors that are involved in inflammatory processes , like tnf- , il-6 and nitric oxide ( no ) may increase p - gp function ( fernandez et al . 2004 ; mcrae et al .
2002 ) showed that exposure of bbb endothelial cells to activated t - lymphocytes induced endothelial cell death , whereas p - gp expression and function was increased in surviving cells .
the authors conclude that p - gp could play a regulatory role in promoting survival of cerebral endothelial cells and maintenance of bbb function . in conclusion , neuronal damage with a subsequent neuroinflammatory reaction
could , by activating the p - gp system promote the clearance of the brain of potentially toxic compounds . an initially compensatory mechanism with up - regulation of p - gp expression
has also been suggested in the early pathogenesis of alzheimer s disease ( ad ) . at later disease stages ,
p - gp in the brain could thus form a compensatory mechanism to increase clearance of neurotoxic substances that play a role in neurodegenerative diseases .
jakob disease ( cjd ) , where decreased p - gp expression was suggested to facilitate the accumulation of prp prions ( vogelgesang et al .
whether p - gp also transports -synuclein , a major constituent of lewy bodies that causes neurodegeneration in pd when aggregated , has so far not been investigated .
furthermore , it is known that p - gp contributes to the cellular efflux of some pesticides ( bain and leblanc 1996 ) and environmental toxins influence pd onset ( rajput , 2001 ) . however , decreased p - gp function was not seen in de novo pd patients , indicating that impaired bbb p - gp efflux function does not play a primary role in the initiation of pathology in idiopathic pd .
the findings of our study could have implications in the therapeutic options in pd and other proteopathies .
p - gp activity can be modulated by a variety of substances , but most studies focus on p - gp inhibition , e.g. , in the prevention of multidrug resistance in oncological treatments ( tan et al .
. however , p - gp activity can also be up regulated , for example by st johns worth or rifampicin ( fromm 2000 ; hennessy et al . 2002 ) .
selectively increasing p - gp mediated bbb efflux function would provide a possible novel therapeutic means to prevent further accumulation of insoluble toxic compounds or proteins leading to ongoing neurodegeneration .
we conclude that cerebrovascular p - gp function is regionally decreased with advanced stages in several parkinsonian neurodegenerative diseases , implying decreased regulatory and protective function of the bbb with ongoing neurodegeneration . | decreased blood brain barrier ( bbb ) efflux function of the p - glycoprotein ( p - gp ) transport system could facilitate the accumulation of toxic compounds in the brain , increasing the risk of neurodegenerative pathology such as parkinson s disease ( pd ) .
this study investigated in vivo bbb p - gp function in patients with parkinsonian neurodegenerative syndromes , using [ 11c]-verapamil pet in pd , psp and msa patients .
regional differences in distribution volume were studied using spm with higher uptake interpreted as reduced p - gp function .
advanced pd patients and psp patients had increased [ 11c]-verapamil uptake in frontal white matter regions compared to controls ; while de novo pd patients showed lower uptake in midbrain and frontal regions .
psp and msa patients had increased uptake in the basal ganglia . decreased bbb p - gp function seems a late event in neurodegenerative disorders , and could enhance continuous neurodegeneration .
lower [ 11c]-verapamil uptake in midbrain and frontal regions of de novo pd patients could indicate a regional up - regulation of p - gp function . | Introduction
Materials and methods
Results
Discussion |
the demand for an effective fabrication method for a large - area nanostructure array has recently stimulated increased interest and research activities in the fields of optics and optoelectronics , including photovoltaic cells , light - emitting devices , and photo - detectors .
two - dimensional arrays of nanostructures have been reported to enable modulation of both the energy and the path of photons to increase efficiency and sensitivity [ 1 - 3 ] while also providing antireflective properties .
the antireflective property improves the visibility of the transparent window , as well as the light extraction or absorption efficiency , by reducing the reflection of incident light and increasing its transmission accordingly .
in fact , the reflectivity can be greatly suppressed for a wide spectral bandwidth when a nanostructure array with a subwavelength pitch can make a continuous and monotonous change in the effective refractive index from air to the solid surface [ 4 - 6 ] .
stability problems due to the thermal mismatch in the conventional methods applying multilayered thin films can be improved .
the previous fabrication strategy relied on costly e - beam lithography , whereas molding technologies , such as nanoimprinting , have emerged due to high throughput and cost - effective process capabilities [ 5,8 - 16 ] .
master patterns required for molding can be fabricated by e - beam , interference lithography , anodizing aluminum oxide , colloidal nanolithography using polystyrene [ 12 - 14 ] , and electron cyclotron resonance ( ecr ) plasma etching .
although molding technology is favorable for mass production , the high cost of mask preparation and the limited resources for large - area mask patterning frequently restrict its practical applications .
simpler bottom - up fabrication using a molding stamp with a subwavelength nanostructure array can be achieved through thermal dewetting of a metal film .
sputter - coated metal film is transformed into an isolated random array of metal dots when thermally annealed , which can be used as the etch shadow mask for the following substrate etching to make an antireflective nanostructure array .
thermal dewetting of pt / pd alloy film on a si wafer was previously studied for this purpose .
other previous attempts include thermal dewetting of ni film on a sio2 surface , ag sputtering on a heated surface , and ag sputtering followed by thermal dewetting on a curved surface .
the transformation results from the increased surface energy of the metal film and its subsequent move to a minimum energy state , similar to the principle of ostwald ripening .
this approach has more promise for large - area process applications in view of enhanced uniformity and fewer defects compared to the colloidal lithography commonly with polystyrene nanoparticles , which yields more defects and pattern irregularities as the substrate size increases .
however , the vacuum deposition and high process temperatures ( greater than 300 c ) used to increase the surface energy of the metal can often limit its applications and are unfavorable for polymer - coated substrates . in this paper , we report an alternative method for fabricating an etch mask with a subwavelength nanostructure array for antireflective applications using nanosilver colloids and relatively low annealing temperatures . as - received nanosilver colloids with diameters of 2030 nm
were agglomerated into isolated nanosilver islands on a wafer - scale silicon substrate in the range of 5080 nm via a combined mechanism of solvent dewetting and ostwald ripening using spin coating and substrate heating .
several variables were identified , including nanosilver density , spin coating speed , nanosilver colloid size , annealing temperature , and time , to vary the size and coverage rate of the nanosilver islands . due to the sufficiently high etch selectivity of silver to silicon , various configurations and aspect ratios of nanostructures could be easily achieved .
the reflection rate was reduced below 5% , which is much lower than the 40% of bare silicon in the visible zone . in particular , the reduction effect of reflection was maximized in the ultraviolet ( uv ) region of ~300 nm with a rate of over 95% .
korea ) , which include nanosilver particles in the range of 1030 nm dispersed in a non - polar solvent blend ( xylene , ethylene glycol , and others ; the exact information was confidential to inktech inc . ) , was diluted to 110 wt% using the product - customized thinner .
1 ) . the deposition and transformation process into nanosilver islands was presumed to proceed according to diagram shown in fig .
2 . nanosilver colloidal ink was spin - coated onto a full wafer - scale silicon substrate to form a multilayered nanosilver film ( fig .
2a ) and transformed to an isolated random array of nanosilver islands by thermal annealing during the following steps .
the continuous film is broken off and grows with increasing space between neighboring islands ( fig .
this process is affected by several parameters , including the as - received particle size , type of solvent , concentration , spin speed , annealing temperature , and time . in this work
, the average size and coverage rate ( density ) of the nanosilver islands were analyzed using concentrations from 3 to 7 wt% , and spin speeds in the range of 2,0004,000 rpm with other fixed conditions of annealing temperature ( 250 c ) and time ( 15 min ) .
the initial annealing temperature was set at 180 c , lower than nanosilver sintering temperature ( 250 c ) , to prevent instantaneous sintering of nanosilver before sufficient dewetting occurred .
tem image of as - received silver nanoparticles fabrication schematic of nanosilver islands for an antireflective etch mask the antireflective nanostructure array was fabricated via reactive ion etching ( multiplex icp
sts , oxford systems ) with the optimized condition of c4f8 ( 40 sccm ) and sf6 ( 45 sccm ) , following a wet removal process of nanosilver residues to form a pattern array of nanosilver islands as an etch mask .
various structural configurations of the nanopillar array were flexibly achievable due to good etch selectivity of silver to silicon . allowing slight isotropic etching condition ,
the etching condition was optimized to fit into the aspect ratio of one at the present approach , if etching is done for 60 s under the given conditions .
finally , the reflectance from the ultraviolet to the visible region was measured by uv spectroscopy ( model : varian cary 5000 ) at the incidence angle of 7 , located in the oled center of seoul national university in korea .
the antireflective effects for the two most dominant process conditions , concentration and spin speed , were investigated for nanopillar structures with an aspect ratio close to one .
the transformation of multilayered nanosilver film into an isolated random array of nanosilver islands was most likely thermodynamically driven by the combined self - assembly of dewetting and ostwald ripening .
heating the plate made the surface energy of the solvent and nanosilver increase , causing microscopic dewetting of the solvent with tiny nanosilver particles captured inside a dewet droplet . further increasing the plate temperature to 250 c , increased the agglomeration of nanosilver islands due to self - assembly .
finally , the islands were sintered to create a randomly distributed array of nanosilver islands during the extended heating stage . in this process , the surface energy of the applied solvent in the nanosilver ink is the most critical factor controlling the transformation .
3 shows the transformation is completed with the use of a non - polar solvent blend at 3 wt% of nanosilver ( fig .
3a ) , while silver colloids dispersed in isopropyl alcohol ( ipa ) did not complete the transformation ( fig .
3b , c ) since the polar solvent was quite volatile due to the relatively low surface energy compared to non - polar counterparts .
effect of the nanosilver colloidal solvent on the nanosilver island transformation : a 3 wt% in non - polar solvent including xylene , etc .
, b 5 wt% in ipa ( polar solvent ) , c 5 wt% in ipa the effects of nanosilver concentration and spin speed on the formation of nanosilver islands are presented in figs . 4 and 5 , respectively . as the nanosilver concentration increased from 3 to 7 wt% at a spin speed of 4,000 rpm
the nanosilver islands began to form networks with the neighboring nanosilver islands above 7 wt% . increasing
the spin speed reduced the average size of the islands , as shown in fig .
these results imply that the thickness of the as - coated nanosilver multilayer determines the average size of the nanosilver islands proportionally .
nanosilver island size and configuration in terms of ag concentration ( 4,000 rpm applied ) : a 3 wt% , b 5 wt% , c 7 wt% effect of spin speed on the nanosilver deposition for 5 wt% : a 2,000 rpm , b 4,000 rpm a quantitative analysis was performed to more clearly define the effects of the nanosilver islands on the average size and coverage rate in fig . 6 and table 1 .
the average size increased from 58.1 nm for 3 wt% to 62.6 nm for 5 wt% by a rate of 7.7% at a spin speed of 4,000 rpm , whereas the average size increased from 69.5 nm to 79.3 nm by a rate of 14.1% at 2,000 rpm in the central region .
increased rate grows up to be 37.7 , 45.9% at 4,000 and 2,000 rpm , respectively at the outer region as shown in table 1 .
these results suggest the effect of nanosilver concentration gets more critical at lower spin speed , and outer side along with the increased standard deviation , which implies the areal uniformity is limited in the spin - coated substrate . in comparison , on the other hand , the spin speed seems to be more effective to grow up the average size of nanosilver islands .
the increase rates of 19.6 and 26.7% were driven when the spin speed reduces from 4,0002,000 rpm for 3 and 5 wt% , respectively .
for all cases , the standard deviation shows similar tendency to the average size of nanosilver islands with respect to nanosilver concentration and spin speed . it should be noted that in the present investigation , the smaller islands , less than roughly 30 nm , were not counted because they were lifted off during the following substrate etching and did not remain in the final etched nanostructure array . under the given analysis conditions ,
the coverage rate increased in proportion to the nanosilver concentration and spin speed as well .
it was expected that further grown nanosilver islands would expose more open spaces to the substrate as the nanosilver concentration increased .
however , the coverage rate is also directly related to the standard deviation of the island size , which is likely to reduce with increased spin speed because the higher spin speed improves the uniformity of the film .
therefore , the lower spin speed creates a wider spectrum of the island size with an even greater number of smaller islands than were counted in this analysis , even though the lower speed generates a larger average size .
quantitative analysis of nanosilver islands in terms of the concentration and spin speed : a average size , b coverage rate quantitative analysis of nanosilver islands in different zone of the deposited wafer figure 7 shows photographs of both the nanosilver - coated etch mask ( top ) and the etched substrate ( bottom ) . the etch mask colors were nearly the same as the bare wafer , whereas the etched substrate showed a color change to dark with considerably diminished reflection .
figure 8 shows sem images of the etched nanostructure array as a function of etch time ( 40 and 60 s ) for the samples processed at a spin speed of 2,000 rpm and nanosilver concentration of 5 wt% .
the higher nanostructure pillars ( ~110 nm ) were surely obtained as the etch time was extended to 60 s ( fig .
these sem images validate that the smaller islands , less than roughly 30 nm , tends to be lift - off during etching . hence
, the etching under the given condition creates cone - shaped nanopillars for the etch mask of nanosilver islands in the range of roughly 3060 nm , the depth of which is in proportion to the size of nanosilver islands .
this can provide the chances of a continuous and monotonous change in the effective refractive index from air to the solid surface for the reduced light reflections .
full wafer - scale view of the a nanosilver island deposited wafer and b etched wafer antireflective nanostructure array after etching and removal of nanosilver residues for the samples processed at 2,000 rpm and 5 wt% : etch time of a 40 s , b 60 s the reflective properties of the finalized nanostructure arrays on the silicon substrate were measured in the uv visible region ( fig .
9 ) for the etched samples ( 40 and 60 s ) to investigate the effect of etched depth . compared to the bare silicon wafer , the reflections of the array were greatly reduced depending on the process conditions ( spin speed and concentration ) of the nanosilver island fabrication and etched depth .
reflection rates for the nanostructured samples with a bare silicon wafer as a reference : a the etched for 40 s , b the etched for 60 s the highest antireflection occurred at the higher concentration ( 5 wt% ) and for the extended etch time in fig .
9b , which indicates that the larger size and higher nanopillar arrays were more effective at reducing the reflection . the minimum reflection rate in fig .
9b decreased to about 0.7% around a wavelength of 300 nm in the uv region , and 56% in the visible region .
the antireflection rate , compared to the bare silicon wafer , was approximately 98 and 83% in the uv and visible regions , respectively for the given condition .
hence , the antireflection turned out to be more dominant in the uv region , probably because the achieved average size of the nanosilver islands ( 5090 nm in the present experiments ) was more suitable to interact with the uv than in the visible light rays .
this agrees with the result that the reflection reduction rate varied with the nanosilver concentration and spin speed in fig .
9a , where the etched depth is around 50 nm , is about 10% in the visible region for 5 wt% concentration with the doubled antireflection efficiency from in fig . 9b .
furthermore , the reflection data band for the given conditions apparently gets larger along with the etched depth in fig .
9b , which is assumingly due to increasing occurrence of lift - off during etching especially for 3 wt%-4,000 rpm , the coating process condition of the smallest size of nanosilver island array .
an additional minor factor to influence the reflection of silicon substrate may be the coverage rate ( nanopillar density ) . in comparison of ag 5 wt%-4,000 rpm with
9b , the anti - reflection effect is greater for ag 5 wt%-4,000 rpm although its average size of nanosilver islands is smaller than ag 3 wt%-2,000 rpm. this probably results from the higher coverage rate in ag 5 wt%-4,000 rpm than in
ag 3 wt%-2,000 rpm. it represents that the density of nanopillar array is higher for ag 5 wt%-4,000 rpm , which leads to the larger effect of anti - reflection than ag 3 wt%-2,000 rpm. the reversed result in fig .
9a seems to be due to the unexpected data deviation all within the limited range of tolerable inaccuracy , which always comes up for random self - assembly .
we investigated an alternative method to fabricate a wafer - level antireflective nanostructure array using nanosilver colloidal lithography .
the combined action of dewetting and oswald ripening contributed to the transformation of the spin - coated multilayer of nanosilver colloids into randomly distributed nanosilver islands that could be used as an etch mask for antireflective nanostructures .
accordingly , the spin - coated nanosilver colloidal layer began to be dewetted and agglomerate as it gradually solidified from its wet state under various annealing conditions .
identified process parameters , the average size and coverage rate of the nanosilver islands were affected most critically by the concentration and the spin speed .
the as - received colloid size , temperature , and annealing time were less critical parameters .
the average size of the resulting nanosilver islands was in the range of 50100 nm , with a coverage rate of 4060% and the standard deviation of 2050% .
it may not be at the acceptable level in many applications other than nanostructured optics such as antireflection window in which such randomness is even ideal to cover a wide range of wavelength spectrum . compared with sputter - coated metal film ,
reported previously [ 17 - 20 ] , the colloidal form is more readily viscous at relatively lower temperatures .
additionally , the solvent wet state at the beginning of the temperature increase can facilitate dewetting and agglomerating of metal colloids into the isolated islands .
hence , the present approach may provide an improved method for a more effective self - assembled transformation . the wet process and low temperature annealing are advantages of the present process for extended process applications .
etching through silicon substrates produced various structural profiles and nanopillar heights as a function of etch time .
anisotropic etching was performed to generate a nanopillar profile with nearly right - angled edges .
the reflection measurements revealed that the antireflection effect was substantially large in general , and depends on the nanopillar height .
the reflection nearly disappeared in the uv wavelength range and was only 17% of that of a bare silicon wafer in the visible range for the condition of the extended etch time and largest nanosilver concentration that turned out to be ideal process theme for this approach .
the present data level for the antireflection rate is comparable with those reported in previous publications .
the present fabrication method is expected to draw extensive industrial interest for producing large - area nanotemplates in a cost - effective and more accessible manner .
for example , the array could be used as a metal dot layer to derive localized surface plasmon resonance ( lspr)-coupled light emission .
more in - depth understanding and further investigation of the nanosilver island transformation would improve the uniformity , process stability , and throughput .
this research was supported by a grant ( 08k1401 - 00511 ) from the center for nanoscale mechatronics and manufacturing , one of the 21st century frontier research programs , and a platform project grant ( 10033636 - 2009 - 11 ) supported by the ministry of the knowledge economy of korea .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . | an alternative method is presented for fabricating an antireflective nanostructure array using nanosilver colloidal lithography .
spin coating was used to produce the multilayered silver nanoparticles , which grew by self - assembly and were transformed into randomly distributed nanosilver islands through the thermodynamic action of dewetting and oswald ripening .
the average size and coverage rate of the islands increased with concentration in the range of 5090 nm and 4065% , respectively .
the nanosilver islands were critically affected by concentration and spin speed .
the effects of these two parameters were investigated , after etching and wet removal of nanosilver residues .
the reflection nearly disappeared in the ultraviolet wavelength range and was 17% of the reflection of a bare silicon wafer in the visible range . | Introduction
Experimental
Results and Discussion
Conclusion
Acknowledgments
Open Access |
pregnancy - induced hypertension ( pih ) , a leading complication in pregnancy that affects the mother and fetus , is becoming more frequent mainly because of increasing maternal age .
maternal obesity , a basal condition that increases the risk of pih 3-fold , has also increased over the decades . on the other hand ,
increased insulin resistance is observed in pih as well as in successful pregnancies [ 46 ] . in the course of a normal pregnancy , insulin resistance
is correlated with increased maternal adipose tissue deposition and supports placental formation and fetal growth .
although the placenta is a large producer of cytokines during pregnancy , adipose tissue is regarded as the main organ producing insulin resistance and related cytokines [ 5 , 6 , 8 , 9 ] .
recently , free fatty acids ( ffa ; also known as nonesterified fatty acids ( nefa ) ) were shown to be mediators of immune and inflammatory actions in adipose tissue [ 6 , 813 ] .
although increased circulating ffa have been observed in gestational diabetes mellitus , preterm delivery , or other adverse maternal outcomes in pregnant subjects [ 6 , 9 , 14 , 15 ] , increased circulating ffa have also been described in normal pregnancy .
different pathways which do not induce systemic inflammation observed in pih in insulin resistance remain unclear . in this study , we measured peripheral monocyte chemoattractant protein-1 ( mcp-1 ) , a proinflammatory chemokine that induces monocyte action leading to cell adhesion and endothelial dysfunction , ffa , and high - molecular weight ( hmw ) adiponectin , a major adipocytokine that reflects insulin sensitivity , in pih patients and made repeated measurements of these molecules in women with normal pregnancies throughout the course of pregnancy .
we hypothesized that an alteration of the chemokines in the inflammatory pathway protects women with normal pregnancies , but not pih patients , from cardiovascular disorders in a state of physiological insulin resistance .
this study was reviewed and approved by the institutional review board of nara medical university , and informed consent was obtained from each subject . for the preliminary study , we recruited 17 nonpregnant women with body mass index ( bmi ) under 25 kg / m , 25 normal pregnant women at 28 weeks or later of gestation , and 7 severe pih patients .
all women were east asian , and none were taking any medications or showed evidence of any metabolic diseases or complications other than pih .
severe pih was defined as the new onset of 2 consecutive measurements of diastolic blood pressure 110 mmhg and systolic blood pressure 160 mmhg diagnosed after 20 weeks of gestation . after the preliminary study , we recruited 36 normal pregnant women for sample correction by taking measurements 3 times throughout the course of pregnancy ( 1st screening = around 12 weeks of gestation ; 2nd = 28 weeks ; and 3rd = 36 weeks ) for a longitudinal study and paired analysis .
all subjects had serum samples available for analysis and did not have gestational diabetes mellitus , thyroid malfunction , or other complications except hypertension .
serum was separated immediately and stored at 80c for 3 years for the longest and 6 month for the shortest storage .
serum ffa ( mainly palmitic acid ) were measured in duplicate with a commercially available kit ( biovision research products , mountain view , ca ) .
the lower limit of detection was 2 m . to estimate alterations in unbound ffa ,
the data were corrected with serum albumin concentrations ( ffa ( m)/albumin ( g / dl ) ) by using the bcg albumin assay kit ( biochain , hayward , ca ) .
serum hmw adiponectin level and its ratio to total adiponectin were measured on the same 96-well plate in duplicate using a commercially available protease - pretreated elisa kit ( sekisui medical , co. , ltd . ,
japan ) . the lower limit of detection was 0.075 ng / ml . the intraassay coefficient of variation ( cv ) was within 20% , while the inter - assay cv was not more than 15% .
serum leptin and mcp-1 concentrations were measured in duplicate with commercially available elisa kits ( r&d systems , inc . ,
. the lower limit of detection was less than 7.8 pg / ml for leptin and less than 5.0 pg / ml for mcp-1 .
for leptin , the intraassay cv was 3.3% at a concentration of 64.5 pg / ml , 3.0% at 146 pg / ml , and 3.2% at 621
pg / ml , while the inter - assay cv was 5.4% at 65.7 pg / ml , 4.2% at 146 pg / ml , and 3.5% at 581 pg / ml . for mcp-1 ,
the intraassay cv was 7.8% at a concentration of 76.7 pg / ml , 4.7% at 364 pg / ml , and 4.9% at 1121
pg / ml , while the inter - assay cv was 6.7% at 74.2 pg / ml , 5.8% at 352 pg / ml , and 4.6% at 1076 pg / ml . in the longitudinal study , measurement of ffa , albumin , mcp-1 , and hmw adiponectin
, we compared normal pregnant women with nonpregnant women and compared pih patients with normal pregnant women at 28 weeks or later of gestation .
statistical analysis was performed using the mann - whitney u - test ( spss 15.0j ; spss japan inc . ,
results in respective patients were analyzed in pairs using repeated measures of anova with the post hoc test ( bonferroni correction ; spss 15.0j ) .
in the preliminary study , we compared normal pregnant women at 28 weeks or later of gestation with nonpregnant subjects and compared pih patients with normal pregnant women at 28 weeks or later of gestation .
diastolic blood pressure was significantly lower in normal pregnant subjects than in the nonpregnant subjects .
serum concentrations of ffa ( raw data and after correction with albumin ) , mcp-1 , total and hmw adiponectin ( raw data and ratio ) , and leptin are shown in table 1 .
ffa concentrations were significantly higher in pih subjects than in normal pregnant women but no significant difference was observed between normal pregnancy and nonpregnant controls .
however , after the albumin correction ( an estimated value reflecting unbound ffa ) , serum concentrations were significantly higher in normal pregnancy than in nonpregnant controls .
serum concentrations of mcp-1 were significantly lower in normal pregnant subjects than in nonpregnant controls and were significantly higher in pih than in normal pregnant subjects .
hmw adiponectin concentration and its ratio to total adiponectin were significantly lower in normal pregnant subjects than in nonpregnant subjects and were higher in pih subjects than in normal pregnant women .
serum leptin was significantly increased only in pih patients compared to that in normal pregnant subjects .
these trends in hmw adiponectin , leptin , and mcp-1 are similar to those in our former report that included subjects with bmis greater than 25 kg / m ; however , the trends in total adiponectin and hmw - to - total adiponectin ratio differ from those in our previous report .
ffa concentrations were not significantly altered in normal pregnant women over the course of 3 measurements : 1st screening , 97.47 13.05 m ; 2nd , 110.89 12.78 ; 3rd , 120.85 12.24 .
however , after the albumin correction ( 1st screening , 3.15 0.04 g / dl ; 2nd , 2.57 0.03 ; 3rd , 2.52 0.03 ) to estimate the alteration of unbound ffa , the value ( ffa [ m]/albumin [ g / dl ] ) was significantly increased throughout the course of pregnancy : 1st screening , 31.38 4.29 ; 2nd , 42.51 4.82 ; 3rd , 48.45 5.10 ; p = 0.0048 ( figure 1 ) .
in contrast , mcp-1 concentrations decreased significantly during the course of pregnancy : 1st screening , 154.36 20.27 pg / ml ; 2nd , 110.56 33.44 ; 3rd , 108.78 28.17 ; p < 0.0001 ( figure 1 ) .
hmw adiponectin was also significantly decreased during the course of pregnancy : 1st screening , 3.48 0.30 g / ml ; 2nd , 2.88 0.27 ; 3rd , 2.86 0.25 ; p = 0.0001 ( figure 1 ) .
our preliminary study showed increases of ffa ( particularly bioactive unbound ffa ) and mcp-1 in lean severe pih patients .
however , in normal pregnancy , ffa increased but mcp-1 significantly decreased in the serum . in longitudinal study throughout normal pregnancy , increase of ffa and the decrease of mcp-1 during the course have been clarified . a significant decrease in hmw adiponectin , which may be consistent with the physiological increase of insulin resistance in normal pregnancy ,
fatty acids play pivotal roles in the development of several diseases including adult metabolic syndrome [ 8 , 1012 ] and pregnancy complications such as miscarriage or preterm delivery , though fatty acids are also involved in the successful physiological distribution of energy in pregnancy .
it was recently revealed that ffa are mediators of toll - like receptor ( tlr)-4 and the nf - kappab pathway of macrophages within adipose tissue and are regarded as key molecules in systemic inflammation , which plays a role in type 2 diabetes and cardiovascular disease [ 10 , 12 , 13 ] . as we reviewed in this journal , tlrs may contribute to pregnancy pathologies .
several reports described increased ffa in preeclampsia [ 6 , 15 ] but these reports did not estimate unbound ffa . in preeclamptic patients ,
acute inflammation is one of the major features of preeclampsia pathophysiology [ 2 , 4 , 6 , 7 , 15 ] .
a difference between our study and former studies is that we chose only lean subjects , who may not show adipocyte hypertrophy .
we recently reported that increased brain - type natriuretic peptide ( bnp ) correlated with increased adiponectin in pih , similar to the findings in acute coronary syndrome and cardiomyopathy .
we also showed that bnp induced the release of adiponectin from cultured adipocytes in vitro .
in this respect , further research is needed to reveal the conditions of adipose tissue in pih patients without obesity .
mcp-1 is a major chemokine and proinflammatory cytokine that activates monocyte recruitment and strongly contributes systemically to the pathology of inflammation .
it is now well accepted that the insulin - resistant state induces the mitogen - activated protein kinase pathway and increases mcp-1 secretion from adipocytes [ 11 , 12 ] , indicating that mcp-1 potentiates the pathology of insulin resistance .
increased mcp-1 was observed in pregnant women with severe obesity [ 22 , 23 ] and preeclampsia [ 17 , 24 ] .
, a peripheral decrease of mcp-1 in successful human pregnancy has only been described in a single report using multiple cytokine arrays , showing a similar decrease of the serum concentration of this molecule during pregnancy .
this decrease may be a system adaptation in humans to avoid pathologic activation of monocytes in pregnancy - induced insulin resistance . the only evidence to support this hypothesis
mcp-1 expression was increased in the kidney in rat but expression declined significantly after the rats became pregnant , and blood pressure was also decreased .
this paper suggests the existence of an adaptation system during pregnancy via chemokine regulation .
the limitation of this study is that food intake was not equalized between each sampling even though all samples were taken after an overnight fast .
ffa concentrations are altered for several days after different food choices , and albumin may decrease with emesis or anemia
. a larger cohort study or more frequent sampling may reduce the alteration of the results after an unusual dietary event .
although ffa were also increased during the course of normal pregnancy , which may be consistent with physiological insulin resistance , mcp-1 was decreased , which would inhibit mcp-1-mediated pathologic inflammation during the hyperlipidemic state of a successful pregnancy .
ffa and mcp-1 in adipose tissue are regarded as key molecules in homeostatic inflammatory linkage .
this is the first report suggesting a difference between pathological inflammation and reasonable insulin resistance in human pregnancy .
further research in adipocytokines and adipose tissue may lead new statistics for prediction and therapy of pregnancy complications . | increased insulin resistance and inflammatory action are observed in pregnancy - induced hypertension ( pih ) , but similar insulin resistance is observed also in successful pregnancy . to estimate insulin resistance and inflammatory activity in normal pregnancy and pih , serum concentrations of free fatty acids ( ffa ; corrected with albumin to estimate unbound ffa ) ,
monocyte chemoattractant protein ( mcp)-1 , and high - molecular weight ( hmw ) adiponectin were measured in severe pih patients with a bmi less than 25 kg / m2 and were measured 3 times during the course of pregnancy in women with normal pregnancies .
ffa / albumin , mcp-1 , and hmw adiponectin concentrations were significantly higher in pih patients than in women with normal pregnancies .
the 3 measurements of ffa / albumin showed a significant increase through the course of uncomplicated pregnancies .
in contrast , mcp-1 and hmw adiponectin were significantly decreased during the course of pregnancy .
these results suggest that the reduced mcp-1 concentration in normal pregnancy may be a pathway to inhibit the induction of pathological features from physiological insulin resistance and homeostatic inflammation . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
it is essential in child development of language and includes the ability to detect , find , discriminate , memorize , recognize , and comprehend sounds .
all the developmental steps of hearing are important and connected ; therefore , the interruption of one or more of the steps induces important damage in the child.1
more than 800,000 children around the world have bilateral permanent loss hearing , either congenital or acquired.2 the newborn hearing screening ( nhs ) is essential to early detection of hearing loss , a condition with serious vocational , educational , and social consequences.3 the earlier the hearing loss is diagnosed , the soon treatment can be started , reducing the damage on speaking and language development of the child and contributing to better social interaction.2
4
psychoactive drugs are prejudicial to the organism of users , and use of these drugs during pregnancy can harm the fetus.5
6
7 drug use can cause a global impact in the neonate 's development , damaging growth of the central nervous system , and may bring on an alcoholic fetal syndrome,5 in addition to craniofacial malformations , hearing deficits , and prolonged interpeak on evoked hearing potentials.6
7
however , the use of psychoactive drugs during pregnancy is not a risk factor for hearing loss,4 according to the joint committee of infant hearing ( jcih ) , 2007.8 this committee suggested the following risk factors for hearing loss : caregiver concern regarding delays on hearing , speaking , language , or development ; familial history of permanent loss in childhood ; intensive care of the neonate for more than 5 days ; or any of the following occurrences , regardless of the permanence : extracorporeal membrane oxygenation , mechanic ventilation , exposure to ototoxic drugs or loop diuretics , and hyperbilirubinemia that requires exchange transfusion ; in utero infections , craniofacial malformations , physical aspects such as white forelock that are associated with syndromes that include sensorineural hearing loss or permanent conductive hearing loss ; syndromes associated with hearing loss , progressive or late hearing loss manifestation ; neurodegenerative disorders or sensorimotor neuropathies ; postnatal infections with positive culture associated with sensorineural hearing loss ; head trauma ; and chemotherapy .
the fact that psychoactive drugs use is not considered a risk factor for hearing loss by the jcih , 20078 probably justifies the lack of studies about hearing in children exposed to drugs during pregnancy
. however , there is evidence of probable hearing alterations in exposed babies , such as a predisposition to secretory otitis media , with progressive loss of hearing9 ; abnormalities of the hearing process9 ; changes in the formation of outer hair cells9 ; absolute latencies and prolonged interpeak7 ; less blood flow at the cochlea , which can cause a lower amplitude in the evoked potentials of hearing nerves7 ; and delay of hearing system maturation.5
in view of important sequelae of hearing deficits in infants and the possible relation between the use of psychoactive drugs during the gestational period and hearing loss occurrence , this study aims to analyze nhs results , the occurrence of associated risk factors , and the likely incidence of hearing deficit in neonates exposed to psychoactive drugs during pregnancy .
the researched sample comes from the hospital materno infantil presidente vargas ( hmipv ) , a public hospital from the south of brazil , porto alegre 's hospital reference to risk gestation .
14/11 on july 13 , 2011 , and from universidade federal de cincias da sade de porto alegre , no .
the term commitment to the use and disclosure of data ( suited for projects that use clinical data ) was utilized ; the study was ethically and methodologically appropriate according to the guidelines and standards of research involving human ( 196/96 resolution ) from national health board .
all nhs results , performed in outpatient , inpatient , or newborn intensive therapy unit ( itu ) subjects , are registered in the database .
the same is stored in microsoft excel file and updated by local audiologists , according to the actions taken for each baby .
this database was created from worksheets that had these items : date that the nhs was performed , mother 's name , birth weight , gestational age , 1- and 5-minute apgar scores , sex , medical observations , mother 's registered use of drugs , place where nhs was performed , responsible audiologist , newborn situation in nhs protocol , test results and/or retest results , and/or nhs hearing results of each baby .
if data were missing , they were added from physical records if possible . for this database ,
the presence of a medical register about the use of psychoactive drugs by mothers between december 9 , 2008 , and december 29 , 2011 , was considered an inclusion factor ; a total of 146 exposed babies were found ( study group ) .
psychoactive drugs cited on the records were marijuana , crack , alcohol , tobacco , and cocaine , and at least one use was enough to consider a present exposition . then 500 unexposed babies were selected ( control group ) , randomly selected from an initial group of 2,578 unexposed babies .
sample size ( study group and control group ) was statistically calculated to support a 90% power , 5% significance level , and 0.5% effect size of standard deviation between groups or a 20% difference .
it analyzes the answers by an algorithmic statistical technique , allied to an artifact rejection system .
the latter screens by transient otoacoustic emissions , by dispersion product , and also by automated auditory brainstem response ( abr ) testing , which is the screening method chosen for each baby according to the suggested jcih ( 2007)8 protocol , considering the risk factors for hearing loss .
the database was analyzed and organized based on the following variables : sex , presence or absence of risk factors for hearing loss , observations ( when present , documented use of psychoactive drugs ) , first referral after nhs ( retest , monitoring , or discharged ) , monitoring results ( when realized ) , retest results ( when realized ) , high complexity results ( when realized ) , and final results with audiological diagnosis ( when completed ) .
otoacoustic emissions test ( oae ) and/or automated abr test was used in the nhs . in both tests
the possible results are pass ( no alteration ) and fail ( with alteration ) .
discharge occurs when the baby passes through screening in both ears without risk factors for hearing loss .
the retest is a new exam , done 15 days after nhs or according to the schedule availability of the service , after the baby has failed in at least one ear .
hearing monitoring is a new test that is done on newborns with risk factors for hearing loss 6 months after the original screening had been done and after the baby passed with both ears .
babies who failed the nhs retest are sent to high complexity services in the city , where audiological diagnosis can be performed through a complementary audiological evaluation .
sometimes it is not possible to perform the exam ( any step : screening , retest , hearing monitoring , or diagnosis process ) for specific reasons , such as baby agitation . in these cases ,
when the baby did not show for the evaluation , the result was documented as did not attend .
absolute frequency analysis was done , and the prevalence of variables was studied ( failure rate on newborn hearing screening process in children of mothers who were psychoactive drug users , risk factors present for hearing loss by jcih , 2007,8 and necessary referrals for the conclusion of audiological diagnosis ) and compared with the same variables as control babies during pregnancy .
the tests used were chi - square , pearson , or fisher exact test , with the statistical package for social sciences ( spss ) program , 17.0 version .
from the initial sample of 146 babies from the study group and 500 babies from the control group , 72 babies were sent for retest , 20 ( 13.7% ) from the study group and 52 ( 10.4% ) from the control group ; 130 for monitoring , 45 ( 30.8% ) from the study group and 85 ( 17% ) from the control group ; and 444 were discharged , 81 ( 55.5% ) from the study group and 363 ( 72.6% ) from the control group .
1 presents the sample distribution relative to sex , showing a homogeneous sample in the study group and the control group ( p = 0.621 ) .
distribution of the sample in relation to sex ( p = 0.621 ) .
2 shows the study group and the control group comparison relative to the presence of at least one risk factor for hearing loss ( jcih , 20078 ) .
occurrence of hearing loss risk factors was significantly larger in the study group ( p < 0.0001 ) .
presence of risk factors in study and control groups ( * p < 0.0001 ) . the nhs failure rate comparison between groups is shown in fig .
3 ; both groups were similar , although failure in the study group is slightly greater .
exposure to psychoactive drugs during pregnancy was not a statistically significant factor to increased failure rate in the study group ( p = 0.267 ) .
results of the newborn hearing screening in study group and control group ( p = 0.267 ) .
4 shows the actions taken in the study group and the control group after the initial nhs .
a greater prevalence of discharge in the control group and hearing monitoring in the study group can be seen ; these tendencies reached statistical significance ( p < 0.001 ) .
the retest rate was similar between groups ( p > 0.05 ) .
actions taken after initial newborn hearing screening in study and control groups ( * p < 0.001 ) .
fig .
5 presents the actions taken because of hearing monitoring , 6 months after the initial nhs ; the group comparison showed no statistically significant difference ( p = 0.157 ) .
actions taken made from hearing monitoring in study and control groups ( p = 0.157 ) .
the results obtained from retests after a failed nhs in the study and the control groups can be seen in table 1 and are similar between the groups ( p = 0.917 ) .
6 illustrates the comparison between the final results of nhs process and audiological diagnoses in the control group and the study group , showing again the similarity between the groups ( p = 0.168 ) .
final result of newborn hearing screening and audiological diagnosis in study and control groups ( p = 0.168 ) .
two babies in the study group were sent to high complexity services ; one of them died and the other one obtained normal results on both ears . in the control group , there were four cases ; two did not attend for diagnosis and two obtained unclear results , one compatible with otitis media .
this comparison between the study and control groups was not statistically significant ( p = 0.667 ) .
the present study compared nhs results , the occurrence of risk factors for hearing loss in babies exposed to psychoactive drugs during pregnancy , and the hearing loss case incidence , if present .
the results showed that exposure to psychoactive drugs during the gestational period was not a differential factor to failing the nhs ; there was no statistically significant difference between groups in this aspect ( p = 0.267 ) .
however , the study group showed a higher tendency to fail the nhs ( 13% ) relative to the control group ( 9% ) .
no specific studies were found in the literature that associated nhs failure rates and exposure to drugs during the gestational period .
however , research demonstrated neurosensory hearing loss in 18 to 20% of rodents exposed to ethanol since day 7 of pregnancy , through abr.10 in another study , absolute latencies and prolonged interpeaks on abr were evidenced in children exposed to cocaine during the prenatal period.7 the literature also described alterations during auditory system formation , such as changed outer hair cells formation9 and decreased bloody flow on cochlea.7
in relation to the present study , it is believed that the absence of significant nhs failure in newborns exposed to psychoactive drugs during the gestational period can suggest three hypotheses .
the first is an association with type , quantity , and frequency of drug use by the pregnant woman ; this information could not be investigated in this sample . beyond that
, it was also considered that some newborns in the study group had mothers who reported only one episode of drug use , which might explain the absence of alterations on nhs , considering that the quantity of use may have been insufficient to damage the newborns ' hearing system .
another hypothesis is related to the possibility that psychoactive drugs are not directly involved in alterations on hearing structure , and that hearing alterations can occur in this population due to pre- or perinatal risk factors for infant hearing deficit , such as time in itu of more than 5 days .
it is known that these newborns can present prematurity , underweight , low apgar scores , and other complications that require more time in the itu , which could be the real cause of hearing deficits in this babies . in this study , it was not possible in the database to discriminate babies who had different tests on nhs .
the oae points to answers on specific frequencies , generated on frequency bands where outer hair cells are normal or next to normal,11 and detects alterations from the cochlea.12 the abr evaluates the auditory pathway integrity and auditory nerves to the brainstem.13
14 not all babies had automatic abr testing because the protocol at the collection place was followed , and this could be another reason for the nonoccurrence of significant difference on nhs failure rate between groups , as the literature reports neural alteration occurrence.7
10 it is suggested that future studies search nhs results on babies exposed to drugs and standardize the type of test .
it was not viable to identify which psychoactive drugs each pregnant mother used in the present research , as well as the exposure time of each baby and the gestational period of drug abuse , because of the absence of this information n database ( previously structured ) . studies describe the difficulty of quantifying the doses of respective psychoactive drug that are considered prejudicial and potential causers of fetal damage.5
6
9
15 this way , as the database used in this study had a great variety of drugs , without separation of consumed substance , such analysis and interpretation was not possible in this study .
the jcih on infant hearing , 20078 does not include psychoactive drug exposure during pregnancy as a risk for hearing loss .
however , it was confirmed from this study that exposed babies had greater occurrence ( 39.7% ) of associated risk factors compared with unexposed babies ( 22.2% ) , and this difference was statistically significant ( p < 0.0001 ) .
this is an extremely relevant fact due to the evidence about drug exposition sensitizing the population to risk factor onset , as previously described .
this aspect highlights the importance of hearing monitoring in this population ; the incidence of hearing alterations on babies with risk factors can vary from 0.3 to 14.1%,16 which is significantly greater than the hearing loss in eutrophic newborns .
it was not possible to list which risk factors were most prevalent in the study groups due to the sample size .
so we decided to analyze this association in a future study . regarding the statistical difference observed in procedures after nhs , the control group had the greater discharge rate ( 72.60% , p < 0,001 ) and the study group had a large rate of referrals to hearing monitoring ( 31% , p < 0.001 ) .
it should be noted that discharge occurs when a baby goes through nhs and has no associated risk factors for hearing loss , and hearing monitoring occurs when a baby goes through nhs and has associated risk factors .
this information reaffirms the previous premise about the need of continuous monitoring of these babies , emphasizing the importance of hearing monitoring . concerning hearing monitoring ,
a high nonattendance rate was found ( 82.2% in the study group and 77.9% in the control group ) .
another study demonstrated a great escape commonly occurs when the individuals are sent for hearing monitoring.17 this evasion compromises the completion of the evaluation and its respective results.18
19 the disinterest and the difficulty of accessing the place where exams are performed is common,19 and mothers who use drugs tend to be of low socioeconomic status , use multiple drugs , have a family history of drug abuse , have a high occurrence of sexually transmitted diseases , not have prenatal exams , and also have depressive and paranoid characteristics.20 it is believed that the evasion to hearing monitoring in this study can be assigned to multiple factors , such as disinterest and the difficulty in local access where the exams are done , the mothers ' drug use profiles , and association of both . the high nonattendance rate observed also affects the study , because through hearing monitoring , complementary evaluations would be possible , for example , of hearing function development .
further studies should determine the incidence of hearing alterations on babies exposed to psychoactive drugs , as well as analyze the development of hearing maturation on this population .
authors agree that it is an understudied topic5
6
9 and that there are still a lot of questions to be answered , like the safe dose of fetus exposure ; hearing alteration pattern , when present ; and which exams are more appropriate for hearing evaluation of newborns exposed to drugs during pregnancy . despite the small amount of referrals to the high complexity services ,
the difficulty to conclude the process was also evident , because even at the final stage of diagnosis patient abstention was considerable ( in the two occurrences in the study group , one died and the other had normal results ; in the four referrals in the control group , two did not show up and two had altered results ) .
the results obtained in this study demonstrate the need for complementary investigation in babies exposed to psychoactive drugs during gestation , because the literature describes evidence about hearing alterations in these individuals.7
9
10 this study contributed with its findings mainly related to the higher occurrence of risk factors for hearing loss in the study group ( p < 0.0001 ) , the prevalence of hearing monitor referrals in the study group ( p < 0.001 ) , and the high prevalence of discharges in the control group ( p < 0.001 ) .
evidently , social work is needed for the effective accompaniment of these individuals , to decrease evasion rate and provide more answers and more concrete associations about child development .
it is also important to highlight a suggestion for future studies investigating psychoactive drug exposition during pregnancy and the association with risk factors for hearing loss , in addition to the effects of each specific drug related to hearing .
in this study , newborns exposed to psychoactive drugs during the gestational period had a similar nhs failure rate as the control group .
concerning risk factors , it was shown that drug exposure provided a higher occurrence of risk factors for hearing loss .
none of the babies exposed to drugs had hearing loss diagnosis , and in the control group , there was one diagnosed occurrence . |
introduction in pregnancy , the mother and fetus share body structures based on the maternal organism . exposure to psychoactive drugs in this period may have repercussions on the baby 's hearing .
therefore , it is necessary to investigate this association .
aim analyze the results of newborn hearing screening ( nhs ) , the occurrence of associated risk factors , and the incidence of hearing loss in newborn exposed to psychoactive drugs during pregnancy .
methods this is an observational retrospective study done from a database analysis . from this database , records were selected about the use of psychoactive drugs by mothers during pregnancy , then the neonates were divide into two groups : the study group ( 146 babies exposed to drugs ) and the control group ( 500 babies not exposed to drugs ) .
the nhs failure rate , the presence of risk factors for hearing loss , and need for audiological diagnosis were analyzed in both groups . from these variables ,
absolute frequency and prevalence rates were calculated and the results compared between groups .
results there was no statistically significant difference in the comparison of nhs failure rates between the groups ( p = 0.267 ) .
the occurrence of risk factors for hearing loss was greater in babies exposed to drugs ( p < 0.0001 ) .
there was only one diagnosis of hearing loss , which occurred in the control group ( p = 0.667 ) .
conclusion the use of psychoactive drugs by mothers during pregnancy did not affect the nhs failure rate of this sample . however , the occurrence of significant risk factors in the study group showed a possible sensitivity of babies exposed to psychoactive drugs during pregnancy . | Introduction
Methods
Results
Discussion
Conclusion |
crohn 's disease ( cd ) and ulcerative colitis ( uc ) are the major forms of inflammatory bowel diseases ( ibds ) in humans .
the etiology of ibd is still unknown , but evidence has been accumulated to indicate that environmental and genetic factors contribute to promote an immunopathologic process leading to chronic inflammation .
studies of experimental models of ibd and clinical observations also suggest that cd4 + t - cells play a major role in initiating and shaping the pathologic response in ibd .
consistently , t - cell - directed therapies have been employed with some clinical success in ibd patients [ 13 ] .
the ability of cd4 + t - cells to alter the magnitude and outcome of the intestinal tissue - damaging inflammatory responses is mostly dependent on the production of distinct profiles of cytokines .
traditionally , cd lesion has been associated with a predominant activation of t helper type 1 ( th1)-lymphocytes that produce large quantities of interferon ( ifn)- under the stimulus of interleukin ( il)-12 [ 57 ] . by contrast , the uc lesion was believed to be driven by th2-cytokines , such as il-4 and il-13 [ 8 , 9 ] .
more recently , studies from several laboratories had led to the identification of more complex networks of cytokine interactions in ibd tissue , thus shedding light into the role of a distinct subset of th cells , namely , th17 cells , in the pathogenesis of colitis [ 1013 ] . in this paper , we will review the available data supporting the involvement of th17 cells in the modulation of gut inflammation .
th17 cells have derived the name by their ability to produce il-17 , also termed il-17a .
additional cytokines produced by th17 cells include il-17f , il-21 , il-22 , tnf- , and il-6 . while th1 and th2 cell differentiations depend on their respective effector cytokines ( i.e. , ifn- and il-4 , resp . ) , induction of th17 cells does not require il-17a .
by contrast , th17 cell development requires the activity of the transcription factors retinoic acid - related orphan receptor ( ror ) t and ror , and runx1 , and is driven by tgf-1 [ 1416 ] .
th1 and th2-related cytokines inhibit th17 cell differentiation , while il-17 is not able to suppress th1 and th2 cells or does so very weakly .
therefore , suppression of ifn- and il-4 represents a way by which tgf-1 could promote th17 cell differentiation , even though tgf-1-driven th17 cell development can also occur in the absence of ifn- and il-4 [ 18 , 19 ] .
the effects of tgf-1 on th17 cell differentiation are concentration dependent ; low doses induce th17 cells , while high doses inhibit th17 cell development and promote regulatory t - cells ( tregs ) .
importantly , tregs can convert into inflammatory th17 cells in the presence of inflammatory cytokines such as il-6 .
since th17 cells appear to be resistant to the tregs - mediated immunosuppression , it is likely that during chronic inflammatory processes , such as ibd , tregs may augment rather than suppressing th17-mediated immune responses .
these observations and the demonstration that il-21 is made by t - cells in response to il-6 clearly indicate that il-21 acts downstream of il-6 in th17 cell generation .
importantly , il-21 stimulates th17 cells to produce il-21 , thus triggering an autocrine loop that amplifies th17 cell responses .
il-21 enhances the expression of il-23 receptor ( il-23r ) in th17 cells , through a process that is dependent on stat3 and rort , making these cells responsive to il-23 . since il-23 facilitates the expansion and/or stabilization of the th17 phenotype
, these data suggest a mechanism by which il-21 could contribute to amplify th17-cell responses .
th17 cell - derived cytokines are supposed to play an important role in the protection of the host against various bacteria and fungi , particularly at mucosal surfaces , given their ability to enhance the recruitment and facilitate the activation of neutrophils and stimulate the production of defensins by epithelial cells [ 27 , 28 ] . in line with this
it was shown that th17 cells are constitutively present in the human and mouse intestinal mucosa .
studies in mice have also shown that , in the gut , commensal bacteria drive the differentiation of th17 cells through a mechanism which is strictly dependent on the production of adenosine 5-triphosphate ( atp ) .
additionally , it was shown that stimulation of dendritic cells with toll - like receptor ( tlr ) ligands induces synthesis of cytokines that promoted differentiation of il-17 producing cd4 + t - cells .
similarly , stimulation of dendritic cells with bacterial - derived ligands for nod-2 results in enhanced il-17 production by human memory t - cells , through an il-1 and il-23-dependent mechanism .
it has been also demonstrated that th17 cells can be induced independent of tlr activation .
for instance , engagement of the c - type lectin dectin-1 with curdlan stimulates dendritic cells to secrete il-6 , tnf- , and il-23 thus promoting the differentiation of th17 cells .
in fact , in the absence of il-23 , th17 cells may have regulatory functions that correlate , in part , with their ability to produce il-10 [ 33 , 34 ] .
th17-related cytokines can trigger several inflammatory pathways due to their ability to enhance the synthesis of inflammatory cytokines ( e.g. , il-1 , il-6 , tnf- , gm - csf ) , chemokines ( e.g. , il-8 , cxcl1 , cxcl8 , monocyte chemoattractant protein-1 , monocyte - inhibitor protein ( mip)-3 ) , cyclooxygenase-2 , and tissue - degrading matrix metalloproteinases ( mmps ) [ 27 , 35 ] .
in 2003 , fujino et al . showed an increased number of il-17-producing cells in the inflamed gut of patients with cd and patients with uc in comparison to normal and disease controls . by immunohistochemistry
, it was shown that , in active uc , il-17-expressing cells were located mainly within the lamina propria , while in active cd , these cells were scattered throughout the submucosa and muscularis propria .
major sources of il-17 were cd3 + t - cells and cd68 + cells . in line with this , it was shown that rna transcripts for il-17a and il-17f were upregulated in the inflamed mucosa of uc patients and cd patients [ 12 , 36 ] .
more recently annuziato et al . demonstrated that the number of il-17-producing t - cells is higher in cd than in normal gut mucosa , and that some of these cells produce also ifn- .
stimulation of these cells with il-12-enhanced expression of th1-related markers ( i.e. , t - bet and ifn- ) and downregulated rort and il-17 , thus indicating that il-17-secreting t - cells can be induced to differentiate in fullypolarized th1 cells .
there is also evidence that treatment of intestinal lymphocytes with il-23 enhances either il-17a or ifn- in uc or cd , respectively .
indeed we have previously shown that in both cd and uc tissues there is enhanced production of il-21 , a cytokine that is capable of expanding the ongoing th1 cell response in cd , stimulating gut fibroblasts to secrete mmps , and inducing colonic epithelial cells to produce the chemokine mip-3 .
il-22 is also highly expressed in mucosal samples of patients with active cd and to a lesser degree of patients with uc . like other th17 members ,
il-22 stimulates colonic fibroblasts to make inflammatory cytokines ( e.g. , il-6 , il-8 , il-11 , and leukemia inhibitory factor ) , chemokines , and mmps .
moreover , il-22 enhances the expression of tnf- , il-8 , and -defensin .
upregulation of th17-related cytokines does not however represent a specific hallmark of ibd , as increased levels of il-17a and other th17-markers have been seen in patients with rheumatoid arthritis , multiple sclerosis , and psoriasis [ 4446 ] .
moreover , studies in murine models of these pathologies strongly suggest that th17 cells play a pivotal role in propagating tissue - damaging immune responses [ 4749 ] .
by using il-17 receptor a ( il-17ra ) knockout mice , zhang et al . showed that il-17 is necessary for the development of colitis induced by intrarectal administration of trinitrobenzenesulfonic ( tnbs ) acid .
consistently , administration of il-17ra igg1 fusion protein in mice with tnbs - colitis significantly attenuated colonic inflammation and prevented weight loss . in this context
it is however noteworthy that il-17ra mediates the activities of both il-17a and il-17f , making difficult to establish the exact contribution of these cytokines in the pathogenesis of tnbs - colitis .
studies in other models of colitis , such as the dextran sulfate sodium ( dss)-induced colitis , showed that il-17f deficiency results in reduced colitis , whereas il-17a - null mice develop more severe disease .
th17 cells seem to be involved in the pathogenesis of colitis induced by transfer of a cecal bacterial antigen - specific c3h / hejbir ( c3bir ) cd4 ( + ) t - cell line to c3h / hesnj scid mice ( see figure 1 ) . in this model ,
colitis associated with enhanced production of il-17 , and adoptive transfer of il-17-secreting t - cells to scid recipients resulted in a marked gut inflammation , as compared to that caused by transfer of th1 cells .
administration of mice with a monoclonal anti - il-23p19 prevented and treated active colitis , downregulated the synthesis of a broad array of inflammatory cytokines and chemokines in the colon , and promoted apoptosis of colitogenic th17 cells . by using a novel model of cd8 + t - cell - dependent colitis , tajima et al
have recently shown that a single adoptive transfer of nave cd8 + t - cells into syngeneic rag - deficient mice was followed by rapid spontaneous proliferation of these cells in the mesenteric lymph nodes and severe colitis .
analysis of cytokine - secreting cd8 + t - cells in the mesenteric lymph nodes showed the existence of il-17 and ifn--double - positive cells .
notably , adoptive transfer of nave cd8 + t - cells derived from either il-17- or ifn--knockout mice associated with a remarkably less severe colitis , raising the intriguing possibility that il-17 and ifn- can cooperate to cause pathology in this model of colitis .
consistent with these findings is our demonstration that il-21-null mice are largely protected against the development of dss - colitis and tnbs - relapsing colitis .
this protection was associated with a reduced colonic expression of several th17- and th1-related genes .
additionally , blockade of il-21 activity with a specific il-21r - fusion protein reduced intestinal inflammation and th17 response during the course of dss colitis . taken together these findings suggest that th17 cytokines contribute to orchestrate the t - cell response that causes tissue damage in the gut .
il-23 is a heterodimeric cytokine composed by the specific p19 subunit and the p40 chain that is shared with il-12 .
the synthesis of the p40 subunit and the functional heterodimeric il-23 is enhanced in the gut of cd patients [ 5 , 56 ] . in a recent study ,
kamada et al . showed that il-23 is made preferentially by a subset of mucosal cells expressing both macrophage ( i.e. , cd14 , cd33 , cd68 ) and dendritic cell markers ( i.e. , cd205 , cd209 ) and present in large numbers in cd tissue . these cells induce lamina propria mononuclear cells to make ifn- rather than il-17 production through an il-23 and tnf - alpha - dependent mechanism .
this substantiates further the concept that , in cd , il-23 may expand the ongoing th1 cell response in line with these data , a neutralizing p40 monoclonal antibody has shown clinical efficacy in patients with moderate and severe cds . if the therapeutic effect of this novel reagent is due to the neutralization of il-12 and/or il-23 remains to be ascertained .
however , studies conducted in various animal models of colitis would seem to indicate that il-23 is more pathogenic than il-12 in the gut .
for instance , by backcrossing il-10-deficient mice with mice lacking il-12p35 or il-23 p19 , yen et al . showed that il-23 was essential for manifestation of chronic intestinal inflammation , whereas il-12 was not .
cd4 + t - cells from il-10/p19-knockout mice still produced large amounts of ifn- , thus indicating that th1 responses developed normally in the absence of il-23 , but disease manifestations required the presence of il-23 .
moreover , administration of exogenous il-23 in rag mice reconstituted with nave cd4 + t - cells caused a more severe colitis that was associated with enhanced production of il-6 and il-17 and preventable by treatment of mice with a blocking il-6 or il-17 antibody .
although in this model , il-6 and il-17 were made by memory t - cells , there is no doubt that some of the pathogenic functions of il-23 in the gut are mediated by non - t - cell populations .
this was first shown by powrie et al . who analyzed the effect of an agonistic anti - cd40 antibody in rag mice lacking il-23p19 or il-12p35 .
administration of anti - cd40 caused a systemic and local inflammation characterized by wasting disease , splenomegaly , increases in serum proinflammatory cytokines , and colitis .
it was shown that the systemic inflammatory response and the elevated concentrations of proinflammatory cytokines in the serum were driven by il-12 while the local intestinal inflammation and production of il-17 in the intestine were controlled by il-23 .
more recently , using the t - cell transfer model of colitis , the same group has shown that il-17-deficient t - cells are not impaired in their ability to induce colitis in rag mice .
protection of colitis seen in rag mice lacking il-23 was associated with no decrease in the levels of il-17 , as well as lack of il-23 did not significantly affect the relative amounts of the th17-specific factor rort in the colon .
consistent with this study , noguchi et al . showed that transfer of nave cd4 + t - cells prepared from il-17-knockout mice induced severe colitis in rag mice .
together , these data indicate that th17 cell responses are not specifically impaired in the intestine of il-23-deficient mice and that il-23-mediated colitis is not strictly dependent on il-17 production . a more detailed analysis of the mechanisms underling the il-23-dependent pathologies revealed that il-23 can facilitate colitis not only via direct effects on inflammatory mediators but also indirectly by counteracting regulatory mechanisms . indeed ,
protection of colitis seen after transfer of nave t - cells to rag mice lacking il-23 was associated with an increase in the frequency of foxp3-expressing t regulatory cells in the intestine . upon nave
t - cell transfer , administration of mice with either an antagonistic il-10r antibody or a blocking tgf- antibody increased colonic inflammation compared to untreated controls .
moreover nave t - cells isolated from transgenic mice expressing a dominant - negative form of tgf- receptor ii and unable to respond to tgf- induced significant colitis when transferred to il-23-deficient rag mice .
high levels of ifn- , but not il-17 , were seen in colitic mice , thus suggesting that ifn- might drive the chronic intestinal inflammation in this setting . notably , transfer of foxp3-deficient t - cells to il-23-deficient rag mice caused severe colitis , indicating that il-23 is not essential to the pathogenesis of intestinal inflammation , if counterregulatory mechanisms are defective or absent .
these later findings well fit with the notion that the requirement of il-23 for the initiation and progress of gut inflammation varies depending on the model .
in fact , acute colitis induced by tnbs is driven by il-12 and negatively regulated by il-23 .
the recent discovery that il-23 and th17 cells play a major role in the development of chronic intestinal inflammation in mouse models of ibd together with the identification of il-23r gene polymorphisms that influence ibd susceptibility has provided a new picture of the way the local immune response can promote intestinal tissue damage .
these new data suggest that , at least in theory , the il-23/th17 axis could be a promising target for suppressing inflammation in ibd .
this is in line with the demonstration that blockade of cytokines that positively regulate th17 cell polarization can dampen colitis .
for instance , a monoclonal antibody targeting il-6r has been successfully used in patients with active cd .
both in vitro and in vivo data showed that anti - il-6 treatment enhanced mucosal t - cell apoptosis .
however , given the critical role of il-6 in the differentiation of th17 cells , it is tempting to speculate that the beneficial effect of anti - il-6r may in part rely on the control of th17 cell responses . nonetheless , some important issues remain to be resolved .
for example , emerging evidence suggests that th17 cell biological effects are strictly context - dependent , and that th17 cells can promote either protective or pathogenic responses depending on additional factors present within the mucosal milieu .
mechanisms that trigger and regulate th17 cell response in human ibd remain to be elucidated .
similarly it is unknown whether and how these cells interact in vivo with other regulatory and effector mucosal t - cell subsets .
it is also unclear whether the various th17 cytokines make qualititatively and quantitatively different contributions to the initiation and progress of gut inflammation or whether they are redundant in their ability to modulate specific inflammatory pathways .
it remains also to be clarified why mice lacking il-23 or specific th17-related genes are differently susceptible to specific forms of experimental colitis .
as pointed out above , th17-cytokines are constitutively produced in the normal human and mouse gut , raising the possibility that these molecules may be involved in the maintenance of immunological homeostasis and/or in the control of specific inflammatory pathways .
if this is the case , blocking th17-cytokines could have deleterious rather than beneficial effects for the host .
finally , experimental studies are still necessary to ascertain whether prolonged treatment with biological compounds that interfere with th17 cell functions can enhance the risk of infections and cancer , given that th17 cytokines mediate host - defensive mechanisms to bacteria and fungi and exert antitumor activity . | crohn 's disease and ulcerative colitis , the major forms of inflammatory bowel diseases ( ibds ) in humans , have been traditionally associated with exaggerated and poorly controlled t helper ( th ) type 1 or th2 cell response , respectively .
more recent studies have , however , shown that ibds are also characterized by a sustained production of cytokines made by a distinct lineage of th cells , termed th17 cells .
the demonstration that th17-related cytokines cause pathology in many organs , including the gut , and that expansion and maintenance of th17 cell responses require the activity of il-23 , a cytokine made in excess in the gut of ibd patients has contributed to elucidate new pathways of intestinal tissue damage as well as to design new therapeutic strategies . in this review ,
we discuss the available data supporting the role of the il-23/th17 axis in the modulation of intestinal tissue inflammation . | 1. Introduction
2. Th17 Cell Induction and Functions
3. Expression of Th17 Cytokines in Human IBD
4. Role of Th17 Cells in the Pathogenesis of Experimental Colitis
5. IL-23 and Gut Inflammation
6. Conclusions |
each of the options has limitations , such as the need for second site of surgery , limited supply , inadequate size and shape , and the morbidity associated with donor site ; thus , there remains a need for the new option .
synthetic hydroxyapatite is widely used as a bone substitute material because it can bond directly to the living bone and has excellent biocompatibility and bioactivity .
however , hydroxyapatite does not have enough mechanical properties to act as substitute in loading - bearing parts of the human skeleton .
it has been desired to develop a material that has mechanical properties analogous to those of natural bone .
it is known that human bone has a three - dimensional woven apatite - polymer structure made of inorganic apatite crystals and organic collagen fibers .
bone consists of an organic - inorganic hybrid with a characteristic structure that leads to specific mechanical properties such as high fracture toughness and flexibility .
it is a good strategy to mimic bone structure in the design of bone - repairing materials . from this point of view
, the fabrication of hybrid materials consisting of apatite and natural organic polymer can be expected to be a good strategy to obtain bone repairing materials that have both a bone bonding ability and mechanical properties similar to those of natural bone .
previous study reported that apatite - coated silk scaffolds can combine the osteoconductive properties of bioceramics with the mechanical resilience of polymer .
silk scaffolds combined with apatite promoted cellular attachment and bone formation in vitro , providing an appropriate osteogenic environment for tissue engineering . in vivo research on mongrel dogs by zhao
. showed that apatite - silk scaffolds could be successfully used to repair mandibular critical size border defects .
the premineralization of the porous silk protein scaffold provided an increase osteoconductive environment for the cells to regenerate sufficient new bone tissue .
silks are fibrous proteins with remarkable mechanical properties produced in fiber formed by silkworms and spiders .
sericin comprised granular and high - molecular , water soluble glycoproteins and it acted as a protein glue to fix fibroin fibers together in the cocoon . in silk textile processing , sericin is usually removed , resulting in fine silk fibers .
the protein which resists oxidation is antibacterial , uv - resistant , and absorbs and releases moisture easily .
sericin can be cross - linked , copolymerized , and blended with other macromolecular materials , especially artificial polymers , to produce materials with the improved properties .
the protein is also used as an improving reagent or a coating material for natural and artificial fibers , fabrics , and articles .
sericin composites are useful as degradable biomaterials , biomedical materials , polymers for forming articles , functional membranes , fibers , fabrics , and articles [ 8 , 9 ] .
sericin has a potential to facilitate apatite deposition and can be useful as polymer material in the fabrication of hybrid materials analogous to bone through biomimetic processes .
indonesia is well known for its silk textile derived from wild silkworm cocoon of cricula triphenestrata .
cricula triphenestrata is one of the world wild species of silkworms which only habitats in java island .
the use of discarded sericin of cricula triphenestrata cocoon extract from the water waste of silk textile industry as biomaterials will be beneficial for the local silk textile industry and also the development of natural biomaterials as bone substitute .
biomaterials may have low , medium , or high potential risk to human safety depending on the type and the extent of patient contact .
one of the international standards recommends the appropriate steps for the biological assessment of medical devices in vitro assessment of cytotoxicity of new biomaterials . in this primary screening
, we aimed to evaluate the cytotoxicity of cricula triphenestrata cocoon extract on human gingival fibroblasts .
the cocoon shells of the silkworm cricula triphenestrata ( figure 1 ) were obtained from pt yarsilk gora mahottama textile industri at yogyakarta , indonesia .
the cocoon shells were taken from karang tengah forest at kabupaten bantul , yogyakarta , indonesia .
the medium of rpmi 1640 , dulbecco 's modified eagle 's minimum essential medium ( dmem ) , penicillin , streptomycin , amphotericin , and trypsin were obtained from gibco ( carlsbad , ca , usa ) .
all other chemicals were analytical or pharmaceutical grade and obtained from sigma - aldrich chemicals ( bornem , belgium ) .
the cocoon shells of the silkworm cricula triphenestrata , were mixed with an aqueous solution of 0.3% na2co3 ( w / v ) at 98c for 1 hour .
the solution was then dialyzed in cellulose membranes ( mw = 3500 g / mol ) against deionized water for 3 days by changing the water daily to remove the ions and other impurities .
human gingival fibroblasts were obtained from biopsies of the attached gingival of sound permanent molar teeth of healthy persons . informed consent based on an appropriate protocol was obtained from the donors .
the biopsies were stored at 4c for at most 24 hours in collection medium ( rpmi 1640 supplemented with penicillin 100 u / ml , streptomycin 100 mg / ml , and amphotericin 2.5 mg / ml ) prior to amplification .
the gingival tissues were cut into 1 to 2 mm pieces then washed three times by rpmi 1640 .
after that , the cut biopsies were placed into 25 cm tissue culture flasks .
the explants were incubated with culture medium consisting of dmem 90% , 10 mm hepes , glucose ( 4.5
g / l ) , penicillin ( 100 u / ml ) , streptomycin ( 100 mg / ml ) , and amphotericin ( 2.5 mg / ml ) , supplemented with 10% heat - inactivated fetal calf serum ( fcs ) .
the tissue samples were grown at 37c in a humidified atmosphere of 10% carbon dioxide in the air .
when outgrowth of cells was observed , the medium was replaced twice weekly until cells reached confluence .
cells were detached from the monolayer by a brief treatment with trypsin - edta ( 0.25% trypsin , 0.02% edta ) and recultured in 75 cm tissue flasks until confluent monolayer was reobtained .
the mtt cytotoxicity test is based on iso 10993 - 5 of biological evaluation of medical devices , part 5 : tests for in vitro cytotoxicity .
cells ( 1 10 cells / ml ) in dmem of 50 l were seeded into 96-well plates and maintained in culture for 24 hours to form a semiconfluent monolayer .
they were then exposed to the cricula triphenestrata cocoon extracts ( 50 l ) over a range of 0.01100 g / ml concentration .
after 24-hour exposure , the formazan formations were determined for each treatment concentration by elisa reader at a wavelength of 570 nm .
the relative viability of the treated cells as compared to the control cells were expressed as the % cytoviability , using the following formula :
( 1)%cytoviability=100%od570 treatedod570 control ,
where od570 treated is mean value of the measured optical density of the treated cells ; od570 control is mean value of the measured optical density of the control cells .
statistical analysis was performed using analysis of variance ( anova ) to determine the effect of cocoon extract concentration on the fibroblast cells cytoviability .
a standard curve was performed based on the extract concentrations numbers versus the cytoviability percentage value .
the extract concentrations used were 100 , 50 , 25 , 12.5 , 6.25 , 3.125 , 1.563 , 0.781 , and 0.010 g / ml . each concentration was performed in four replicates .
this research used the cytoviability values that only fall in the linear range of the standard curve to reduce error .
a linear regression analysis was performed regarding to the corresponding concentration numbers and cytoviability values .
the regression formula was y = ax + b. the ic50 value was estimated using the fitted line of the linear regression as ic50 = ( 0.5 b)/a .
the result of relative viability of the fibroblast treated cells as compared to the control cells was expressed in table 1 .
table 1 showed that increased concentration of the cricula triphenestrata cocoon extract exposure on the cells resulted in the decreasing of cytoviability percentage of human gingival fibroblast cells .
further statistical analysis by anova was shown in table 2 , which described that there was not any significant influence of the treated extracts concentration on the cytoviability of human gingival fibroblast ( p > 0.05 ) .
the ic50 value of cricula triphenestrata cocoon extract for a battery of fibroblast cells was obtained by regression analysis of the corresponding dose - response curve .
the regression formula for the dose - response curve was y = 67.630 0.390x . by the formula ,
in the field of biomaterials , it is necessary to consider aspects of security , such as elimination of cytotoxicity and other harmful effects of the material to be used . by definition
, the cytotoxicity of an agent means the toxicological risks caused by a material or its extract in a cell culture .
the interactions of the materials and their components with the cells at a molecular level are responsible for tissue reactions , such as inflammation , necrosis , immunological alterations , genotoxicity , and apoptosis . during the last years
, the interest of in vitro systems as an alternative to animal experiments in toxicological research has been steadily increasing .
we used indirect test , in which the rate of cell growth ( cell number ) and the metabolic activity ( mtt ) have indicated the degree of cytotoxicity of cricula triphenestrata cocoon extract .
table 1 shows the effect of cricula triphenestrata cocoon extract on human gingival fibroblast cells viability measured by mtt test .
mtt is a yellow water - soluble tetrazolium dye which is reduced by live cells to a purple formazan product insoluble in aqueous solutions .
as can be seen from table 1 , the cocoon extract exposure of 0.01100 g / ml during 24 hour of incubation induces the cytoviability of the fibroblast to be 71.18464.486% in comparison to the control .
the highest inhibition effect is induced by 100 g / ml extract concentration and the weakest is on 0.01 g / ml one . although further statistical analysis shows no significant difference among the concentrations , but it is proved that there is an increasing cytoviability on the decreasing of the extract concentration exposure .
the half maximal inhibitory concentration ( ic50 ) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function .
this quantitative measure indicates how much a particular substance is needed to inhibit a given biological process by half . in other words ,
it is the half maximal ( 50% ) inhibitory concentration ( ic ) of a substance ( 50% ic , or ic50 ) .
the score of ic50 represents the concentration of a drug that is required for 50% inhibition in vitro . in this study ,
the determination of ic50 by the formula of y = 67.630 0.390x is 172 g / ml . it means that the highest treated concentration of 100 g / ml cocoon extract reveals no significant cytotoxicity against human fibroblasts ( less than 172 g / ml ) .
previous study reported that the ic50 of bombyx mori silkworm cocoon extract on vero cells was 230 g / ml .
comparing to the ic50 of cricula triphenestrata cocoon extract in this study , the cricula triphenestrata cocoon extract is lower than the bombyx mori .
it means that the cricula triphenestrata cocoon extract is more cytotoxic than the bombyx mori .
previous study reported that there was a correlation between environmental condition and the protein production of cocoon .
the difference between the environmental condition and food consumption may influence the cocoon characteristic including the cytotoxicity . in the present study , the cytotoxicity of cricula triphenestrata cocoon extract which is aimed to be used as bone substitute
like other tissues , normal fibroblast function is critical to maintain the periodontal tissue function for optimal healing .
based on the research of the cytotoxicity of indonesian silkworm cocoon extract of cricula triphenestrata on human fibroblasts , it was concluded that cricula triphenestrata cocoon extract up to 100 g / ml was not cytotoxic on human gingival fibroblast cells . by this finding
, further researches are proposed to develop the cricula triphenestrata cocoon extract as an alternative biomaterial for bone substitute . |
objectives . the aim of this paper was to evaluate the cytotoxicity of indonesian silkworm cocoon extract of cricula triphenestrata on human fibroblasts . methods and materials .
the cocoon shells of the silkworm cricula triphenestrata were degumming .
the shells were mixed with an aqueous solution of 0.3% na2co3 at 98c for 1 hour .
the solution was then dialyzed in cellulose membranes against deionized water for 3 days .
the cocoon shells extract powder was collected via rotary evaporation and dried under freeze dryer .
cell culture medium was exposed to cricula triphenestrata cocoon extract ( 0.01100 g / ml ) for 24 hours .
the primary human gingival fibroblasts were exposed to the treated cell culture medium for 24 hours .
cytotoxicity evaluation was done by mtt method .
the data were analyzed by one - way anova .
result .
the result revealed no significant cytotoxicity of cricula triphenestrata cocoon extract against human fibroblasts at a concentration up to 100 g / ml ( p > 0.05 ) .
conclusion .
cricula triphenestrata cocoon extract was not cytotoxic on human gingival fibroblast cells . | 1. Introduction
2. Materials and Method
3. Results
4. Discussion
5. Conclusion |
a triad of high triglycerides , low high - density lipoprotein ( hdl ) cholesterol and elevated small dense low - density lipoprotein ( ldl ) particles occurring in a patient with type 2 diabetes is referred to atherogenic diabetic dyslipidemia ( add ) .
insulin resistance at the level of adipocyte causing increased free fatty acid efflux is thought to be central to the pathogenesis of add .
this results in increased very ldl ( vldl ) cholesterol from the liver facilitated by increased synthesis of coprotein apo b. subsequent actions mediated by cholesterol ester transferase protein in transferring triglycerides from vldl particles to hdl and ldl result in increased apo a1 containing small dense hdl and apo b containing small dense ldl particles .
the triglyceride - enriched hdlis subsequently hydrolyzed by hepatic lipase orlipoprotein lipase resulting in low hdl ; apo a - i dissociates from thereduced - size hdl , which is filtered by the renalglomeruli and degraded in renal tubular cells [ figure 1 ] . high concentration of vldl - transported tg triggers cetp mediated transfer of ldl cholesteryl ester or hdl cholesteryl ester in exchange for tg .
triglyceride - rich hdl cholesterol or ldl cholesterol then undergoes hydrolysis by hepatic lipase or lipoprotein lipase .
abbreviations : apoa-1 , apolipoprotein a-1 ; apob , apolipoprotein b ; ce , cholesteryl ester ; cetp , cholesteryl ester transfer protein ; ffa , free fatty acid ; hl , hepatic lipase ; lpl , lipoprotein lipase ; sd ldl , small dense ldl cholesterol ; tg , triglyceride . nature clinical practice endocrinology and metabolism ( 2009)5 , 150 - 159 reducing ldl cholesterol ( c ) using statins is a proven strategy for primary as well as secondary prevention of cardiovascular events . hence , statin therapy is accepted as a first line in management of dyslipidemia , diabetic , or otherwise .
but , despite statin therapy , a significant residual riskremains potentially attributable to increased triglyceride concentration and low hdl cholesterol , a characteristic hallmark of add . a meta - analysis of 14 trials involving statins that included 18,686 people with diabetes proved that presence of low hdl and high triglyceride limits the efficacy of statin therapy alone in reducing the vascular events despite achieving target ldl - c levels .
similarly , a meta - analysis of 17 prospective studies showed that after adjusting for variables such as hdl - c , total cholesterol , and other risk factors , the relative risk for coronary heart disease with onemmol / l ( 1 mmol / l = 88.4956 mg / dl ) increase in triglyceride was 1.14 [ 95% confidence interval ( ci ) 1.05 - 1.28 ] for men and 1.37 ( 95% ci 1.13 - 1.66 ) for women .
this has led to renewed interest in treatments that could selectively target high triglycerides and low hdl thus , further reducing the cardiovascular risk .
omega 3 fatty acids , nicotinic acid , and fibrates are currently available drugs used to target such dyslipidemia .
a systematic review and meta - analysis of available evidence studying the merits of omega 3 fatty acid supplementation mainly , docosahexaenoic acid ( dha ) and eicosapentaenoic acid ( epa ) have failed to show any cardiovascular benefit .
similarly , recently published randomized placebo controlled origin trial ( outcome reduction with initial glargine intervention ) that included patients with dysglycemia ( n : 12,536 ) showed no cardiovascular benefits in the study population exposed to omega 3 fatty acid compared to placebo during the study period of over 6 years .
this is despite significant reduction ( p < 0.001 ) in triglyceride concentration in study arm compared to placebo ( 23.5+/- 3 mg / dl vs. 9+/-3 mg / dl ) .
it is of interest though to note that baseline triglyceride value in both groups in this study was around 140 mg / dl .
ascend ( astudy of cardiovascular events in diabetes ) a large placebo - controlled randomized prospective trial involving about 15,000 patients with diabetes in currently underway looking at the efficacy of 1 g capsules containing 90% omega 3 fatty acids ( 0.4 g epa , 0.3 g dha ) as a primary preventive measure against cardiovascular events in patients with diabetes mellitus ( clinicaltrails.gov : nct00135226 ) .
nicotinic acid in extended release preparation ( 2 g / day ) has been proven to increase hdl cholesterol by 20% and reduce triglyceride concentration by 25% in addition to lipid lowering by statin therapy .
clinical utility of nicotinic acid is primarily limited by significant side effects such as severe facial flushing and cutaneous rash that are thought to be mediated by prostaglandins .
liropiprant a prostaglandin receptor antagonist was designed as a codrug to minimize these unpleasant side effects .
but , more recently hps2 thrive ( treatment of hdl to reduce the incidence of vascular events ) study done to assess the cardiovascular benefit of nicotinic acid / liropiprant combination in addition to statin + /-
ezetimibe with a prestudy ldl cholesterol concentration of < 130 mg / dl on over 40,000 patients across the globe showed highly significant four fold increased risk of myopathy in the study group compared to placebo and also double ( 0.9 - 0.4% ) the incidence of diabetic complications ( typically hyperglycemia ) needing early termination of the study raising serious concerns about the safety of this approach .
peroxisome proliferator - activated receptor - alpha ( ppar ) , gamma ( ppar ) , and beta / delta ( ppar / ) agonists regulate gene transcription by binding to specific deoxyribonucleic acid response elements upon ligand activation and heterodimerization with 9-cis retinoic acid receptor . depending on the activating ligand ,
different receptor conformations are adopted , leading to different coactivator recruitment and subsequent effects on gene expression . even though all the ppar agonists are from the same pharmaceutical class , their biological activity varies widely based on selective alpha or gamma modulation .
ppar regulates expression of genes encoding enzymes and transport proteins controlling lipid metabolism and is expressed predominantly in tissues with a high capacity for fatty acid oxidation like liver , heart , skeletal muscle , brown fat , and kidney .
ppar not only promotes pre - adipocyte differentiation , but also induces adiponectin expression , which increases fatty acid oxidation by activation of the amp - activated protein kinase pathway and down regulates the expression of genes encoding resistin and tumor necrosis factor together contributing to reduced insulin resistance . while ppar agonism reduces insulin resistance , ppar agonism compliments it by reducing the ffa load on peripheral tissues thereby augmenting glucose uptake .
ppar and ppar receptors have also been found in vascular endothelium , monocytes / macrophages , and smooth muscle cells of vascular lineage exerting specific anti - inflammatory and lipid modulating effects supporting their role in antiatherogenesis figure 2 .
cell research 2010 ; 20:124 - 137 predominant ppar agonists recognized as glitazones proven to improve insulin resistance have been used as antidiabetic agents for a few years now .
pioglitazone , englitazone , troglitazone , rosiglitazone , and darglitazone were synthesized later and among these , troglitazone , pioglitazone , and rosiglitazone were evaluated in clinical studies .
troglitazone was approved for clinical use 1997 but were subsequently withdrawn with in 3 years because of idiosyncratic liver toxicity .
pioglitazone although is still available to be prescribed , but concern regarding increased risk of bladder cancer on cumulative exposure and risk of osteoporosis in women has limited its potential for widespread use . because of predominant ppar agonism , these agents have minimal influence if any on add .
predominant ppar agonists , fibrates as they are known , are proven to influence lipid profile but their role in add is extensively debated .
meta - analysis of several trials involving fibrates by sacks et al . , ( accord , field , bip , hhs , va - hit ) showed that fibrates are very likely to be beneficial as an add on therapy in subgroup of patients characterized by high triglycerides and low hdl cholesterol despite statin usage .
, demonstrated that those patients with a high mean triglyceride concentration obtained greater reduction in cardiovascular outcomes . all fibrates ( except gemfibrozil )
have shown reversible elevation of creatinine ( up to 30% ) of unknown significance . in another meta - analysis , combination therapy with statin and fibrate was shown to be safe with no increased risk of serious myopathy or rhabdomyolysis . despite lack of any significant influence on ppar receptors , fibrates were shown to have some positive influence on glucose metabolism probably secondary to their influence on insulin resistance but clinical outcome study proving their usefulness in improving glycemic control in addition to dyslipidemia is lacking .
a combined ppar/ agonist should ideally be a suitable drug in treatment of type 2 diabetic patients on statin therapy who have residual cardiovascular risk secondary to elevated triglyceride concentration .
this molecule would not only target the dyslipidemia but also contribute to improved glycemic control .
given these benefits of dual ppar/ agonism , several pharmaceutical agents with such action commonly named as
depending on their molecular structure , these molecules exert dual action with varying degrees of ppar and ppar activism .
faglitazar , the first glitazar to be tested , was dropped very early on in development phase secondary to significant edema .
similarly , ragaglitazar was also dropped early on due to its carcinogenic potential on urothelial cells in rodent models .
muraglitazar , although proved successful in improving insulin sensitivity and treating diabetic dyslipidemia , was suspended in 2006 due to significant cardiovascular side effects .
although these tested molecules resulted in adverse events , these have been compound specific and of diverse origin , that is , urothelial , renal , and cardiac rising hopes of a potential drug that could mitigate these side effects and yet have a positive influence on insulin sensitivity and dyslipidaemia dominated by high triglycerides , small dense ldl and low hdl cholesterol figure 3 .
saroglitazar , also a dual ppar/ agonist with predominant ppar activity , is considered novel and unique as it was conceptualized to deliver antidyslipidemic and antihyperglycemic effects without any of the adverse events of its predecessor molecules . in a phase 1 study designed to evaluate pharmacokinetics , safety and tolerability of the drug ,
healthy volunteers were subject to varying doses of saroglitazar ( 0.25 - 128 mg / day ) .
all doses were very well - tolerated with no serious adverse events ( renal / hepatic / cardiac ) reported in the study group . similarly in a 16-week ( 4 week wash out phase ) multicenter , randomized , placebo - controlled , phase 3 trial of diabetic patients on treatment with atorvastatin 10 mg and with residual dyslipidemia consisting of triglycerides of > 200 and < 500 , subjects were put to 12-week therapy with 2 and 4 mg of saroglitazar . at the end of 12 weeks , the results showed statistically significant reduction in triglyceride concentration ( -45.5 mg / dl + /-
3.03% ) with 2mg / d of saroglitazar + atorvastatin and similar reduction ( -46 mg / dl mg / dl + /-
3.02% ) in those on 4 mg / day of saroglitazar + atorvastatin compared with those on placebo + atorvastatin . although non significant in statistical terms , patients in study group reduced glycated hemoglobin levels by 0.3+/- 0.08% with 2 mg / day and 0.2+/- 0.07% with 4 mg / day dosage . at the end of 24 weeks , study group reported no significant renal , hepatic , and cardiac adverse events . for
now as observed in phase 1 and phase 3 trails , saroglitazar appears safe and has not shown any of the adverse effects that are commonly recognized with its class of molecules ( e.g. muraglitazar - cardiovascular and tesaglitazar - renal and bone marrow ) . but given historical concerns , proof of long - term safety remains paramount and is yet to be proven .
although , it has shown positive results in improving glycemic and lipid parameters in patients with add in short term , a longer term clinical trial proving its efficacy in improving cardiovascular outcome is currently nonexistent but much desired . until
such time given recent approval of saroglitazar by indian authorities permitting its clinical use in treatment of add , both the pharmaceutical industry and medical profession should exercise close pharmacovigilance and report any adverse events as soon as observed .
a systematic review and meta - analysis of available evidence studying the merits of omega 3 fatty acid supplementation mainly , docosahexaenoic acid ( dha ) and eicosapentaenoic acid ( epa ) have failed to show any cardiovascular benefit .
similarly , recently published randomized placebo controlled origin trial ( outcome reduction with initial glargine intervention ) that included patients with dysglycemia ( n : 12,536 ) showed no cardiovascular benefits in the study population exposed to omega 3 fatty acid compared to placebo during the study period of over 6 years .
this is despite significant reduction ( p < 0.001 ) in triglyceride concentration in study arm compared to placebo ( 23.5+/- 3 mg / dl vs. 9+/-3 mg / dl ) .
it is of interest though to note that baseline triglyceride value in both groups in this study was around 140 mg / dl .
ascend ( astudy of cardiovascular events in diabetes ) a large placebo - controlled randomized prospective trial involving about 15,000 patients with diabetes in currently underway looking at the efficacy of 1 g capsules containing 90% omega 3 fatty acids ( 0.4 g epa , 0.3 g dha ) as a primary preventive measure against cardiovascular events in patients with diabetes mellitus ( clinicaltrails.gov : nct00135226 ) .
nicotinic acid in extended release preparation ( 2 g / day ) has been proven to increase hdl cholesterol by 20% and reduce triglyceride concentration by 25% in addition to lipid lowering by statin therapy .
clinical utility of nicotinic acid is primarily limited by significant side effects such as severe facial flushing and cutaneous rash that are thought to be mediated by prostaglandins .
liropiprant a prostaglandin receptor antagonist was designed as a codrug to minimize these unpleasant side effects .
but , more recently hps2 thrive ( treatment of hdl to reduce the incidence of vascular events ) study done to assess the cardiovascular benefit of nicotinic acid / liropiprant combination in addition to statin + /-
ezetimibe with a prestudy ldl cholesterol concentration of < 130 mg / dl on over 40,000 patients across the globe showed highly significant four fold increased risk of myopathy in the study group compared to placebo and also double ( 0.9 - 0.4% ) the incidence of diabetic complications ( typically hyperglycemia ) needing early termination of the study raising serious concerns about the safety of this approach .
peroxisome proliferator - activated receptor - alpha ( ppar ) , gamma ( ppar ) , and beta / delta ( ppar / ) agonists regulate gene transcription by binding to specific deoxyribonucleic acid response elements upon ligand activation and heterodimerization with 9-cis retinoic acid receptor . depending on the activating ligand ,
different receptor conformations are adopted , leading to different coactivator recruitment and subsequent effects on gene expression . even though all the ppar agonists are from the same pharmaceutical class , their biological activity varies widely based on selective alpha or gamma modulation .
ppar regulates expression of genes encoding enzymes and transport proteins controlling lipid metabolism and is expressed predominantly in tissues with a high capacity for fatty acid oxidation like liver , heart , skeletal muscle , brown fat , and kidney .
ppar not only promotes pre - adipocyte differentiation , but also induces adiponectin expression , which increases fatty acid oxidation by activation of the amp - activated protein kinase pathway and down regulates the expression of genes encoding resistin and tumor necrosis factor together contributing to reduced insulin resistance . while ppar agonism reduces insulin resistance , ppar agonism compliments it by reducing the ffa load on peripheral tissues thereby augmenting glucose uptake .
ppar and ppar receptors have also been found in vascular endothelium , monocytes / macrophages , and smooth muscle cells of vascular lineage exerting specific anti - inflammatory and lipid modulating effects supporting their role in antiatherogenesis figure 2 .
cell research 2010 ; 20:124 - 137 predominant ppar agonists recognized as glitazones proven to improve insulin resistance have been used as antidiabetic agents for a few years now .
pioglitazone , englitazone , troglitazone , rosiglitazone , and darglitazone were synthesized later and among these , troglitazone , pioglitazone , and rosiglitazone were evaluated in clinical studies .
troglitazone was approved for clinical use 1997 but were subsequently withdrawn with in 3 years because of idiosyncratic liver toxicity .
pioglitazone although is still available to be prescribed , but concern regarding increased risk of bladder cancer on cumulative exposure and risk of osteoporosis in women has limited its potential for widespread use . because of predominant ppar agonism , these agents have minimal influence if any on add .
predominant ppar agonists , fibrates as they are known , are proven to influence lipid profile but their role in add is extensively debated .
, ( accord , field , bip , hhs , va - hit ) showed that fibrates are very likely to be beneficial as an add on therapy in subgroup of patients characterized by high triglycerides and low hdl cholesterol despite statin usage . similar systematic review on usefulness of fibrate therapy by jun et al .
, demonstrated that those patients with a high mean triglyceride concentration obtained greater reduction in cardiovascular outcomes . all fibrates ( except gemfibrozil )
have shown reversible elevation of creatinine ( up to 30% ) of unknown significance . in another meta - analysis , combination therapy with statin and fibrate was shown to be safe with no increased risk of serious myopathy or rhabdomyolysis . despite lack of any significant influence on ppar receptors , fibrates were shown to have some positive influence on glucose metabolism probably secondary to their influence on insulin resistance but clinical outcome study proving their usefulness in improving glycemic control in addition to dyslipidemia is lacking .
a combined ppar/ agonist should ideally be a suitable drug in treatment of type 2 diabetic patients on statin therapy who have residual cardiovascular risk secondary to elevated triglyceride concentration .
this molecule would not only target the dyslipidemia but also contribute to improved glycemic control .
given these benefits of dual ppar/ agonism , several pharmaceutical agents with such action commonly named as
depending on their molecular structure , these molecules exert dual action with varying degrees of ppar and ppar activism .
faglitazar , the first glitazar to be tested , was dropped very early on in development phase secondary to significant edema .
similarly , ragaglitazar was also dropped early on due to its carcinogenic potential on urothelial cells in rodent models .
muraglitazar , although proved successful in improving insulin sensitivity and treating diabetic dyslipidemia , was suspended in 2006 due to significant cardiovascular side effects .
although these tested molecules resulted in adverse events , these have been compound specific and of diverse origin , that is , urothelial , renal , and cardiac rising hopes of a potential drug that could mitigate these side effects and yet have a positive influence on insulin sensitivity and dyslipidaemia dominated by high triglycerides , small dense ldl and low hdl cholesterol figure 3 .
saroglitazar , also a dual ppar/ agonist with predominant ppar activity , is considered novel and unique as it was conceptualized to deliver antidyslipidemic and antihyperglycemic effects without any of the adverse events of its predecessor molecules . in a phase 1 study designed to evaluate pharmacokinetics , safety and tolerability of the drug ,
healthy volunteers were subject to varying doses of saroglitazar ( 0.25 - 128 mg / day ) .
all doses were very well - tolerated with no serious adverse events ( renal / hepatic / cardiac ) reported in the study group . similarly in a 16-week ( 4 week wash out phase ) multicenter , randomized , placebo - controlled , phase 3 trial of diabetic patients on treatment with atorvastatin 10 mg and with residual dyslipidemia consisting of triglycerides of > 200 and < 500 , subjects were put to 12-week therapy with 2 and 4 mg of saroglitazar . at the end of 12 weeks
, the results showed statistically significant reduction in triglyceride concentration ( -45.5 mg / dl + /-
3.03% ) with 2mg / d of saroglitazar + atorvastatin and similar reduction ( -46 mg / dl mg / dl + /-
3.02% ) in those on 4 mg / day of saroglitazar + atorvastatin compared with those on placebo + atorvastatin .
although non significant in statistical terms , patients in study group reduced glycated hemoglobin levels by 0.3+/- 0.08% with 2 mg / day and 0.2+/- 0.07% with 4 mg / day dosage . at the end of 24 weeks , study group reported no significant renal , hepatic , and cardiac adverse events . for
now as observed in phase 1 and phase 3 trails , saroglitazar appears safe and has not shown any of the adverse effects that are commonly recognized with its class of molecules ( e.g. muraglitazar - cardiovascular and tesaglitazar - renal and bone marrow ) . but given historical concerns , proof of long - term safety remains paramount and is yet to be proven .
although , it has shown positive results in improving glycemic and lipid parameters in patients with add in short term , a longer term clinical trial proving its efficacy in improving cardiovascular outcome is currently nonexistent but much desired . until such time given recent approval of saroglitazar by indian authorities permitting its clinical use in treatment of
add , both the pharmaceutical industry and medical profession should exercise close pharmacovigilance and report any adverse events as soon as observed .
predominant ppar agonists recognized as glitazones proven to improve insulin resistance have been used as antidiabetic agents for a few years now .
pioglitazone , englitazone , troglitazone , rosiglitazone , and darglitazone were synthesized later and among these , troglitazone , pioglitazone , and rosiglitazone were evaluated in clinical studies .
troglitazone was approved for clinical use 1997 but were subsequently withdrawn with in 3 years because of idiosyncratic liver toxicity .
pioglitazone although is still available to be prescribed , but concern regarding increased risk of bladder cancer on cumulative exposure and risk of osteoporosis in women has limited its potential for widespread use .
because of predominant ppar agonism , these agents have minimal influence if any on add .
predominant ppar agonists , fibrates as they are known , are proven to influence lipid profile but their role in add is extensively debated .
meta - analysis of several trials involving fibrates by sacks et al . , ( accord , field , bip , hhs , va - hit ) showed that fibrates are very likely to be beneficial as an add on therapy in subgroup of patients characterized by high triglycerides and low hdl cholesterol despite statin usage . similar systematic review on usefulness of fibrate therapy by jun et al .
, demonstrated that those patients with a high mean triglyceride concentration obtained greater reduction in cardiovascular outcomes . all fibrates ( except gemfibrozil )
have shown reversible elevation of creatinine ( up to 30% ) of unknown significance . in another meta - analysis , combination therapy with statin and fibrate was shown to be safe with no increased risk of serious myopathy or rhabdomyolysis . despite lack of any significant influence on ppar receptors , fibrates were shown to have some positive influence on glucose metabolism probably secondary to their influence on insulin resistance but clinical outcome study proving their usefulness in improving glycemic control in addition to dyslipidemia is lacking .
a combined ppar/ agonist should ideally be a suitable drug in treatment of type 2 diabetic patients on statin therapy who have residual cardiovascular risk secondary to elevated triglyceride concentration .
this molecule would not only target the dyslipidemia but also contribute to improved glycemic control .
given these benefits of dual ppar/ agonism , several pharmaceutical agents with such action commonly named as
depending on their molecular structure , these molecules exert dual action with varying degrees of ppar and ppar activism .
faglitazar , the first glitazar to be tested , was dropped very early on in development phase secondary to significant edema .
similarly , ragaglitazar was also dropped early on due to its carcinogenic potential on urothelial cells in rodent models .
muraglitazar , although proved successful in improving insulin sensitivity and treating diabetic dyslipidemia , was suspended in 2006 due to significant cardiovascular side effects .
although these tested molecules resulted in adverse events , these have been compound specific and of diverse origin , that is , urothelial , renal , and cardiac rising hopes of a potential drug that could mitigate these side effects and yet have a positive influence on insulin sensitivity and dyslipidaemia dominated by high triglycerides , small dense ldl and low hdl cholesterol figure 3 .
saroglitazar , also a dual ppar/ agonist with predominant ppar activity , is considered novel and unique as it was conceptualized to deliver antidyslipidemic and antihyperglycemic effects without any of the adverse events of its predecessor molecules . in a phase 1 study designed to evaluate pharmacokinetics , safety and tolerability of the drug ,
healthy volunteers were subject to varying doses of saroglitazar ( 0.25 - 128 mg / day ) .
all doses were very well - tolerated with no serious adverse events ( renal / hepatic / cardiac ) reported in the study group . similarly in a 16-week ( 4 week wash out phase ) multicenter , randomized , placebo - controlled , phase 3 trial of diabetic patients on treatment with atorvastatin 10 mg and with residual dyslipidemia consisting of triglycerides of > 200 and < 500 , subjects were put to 12-week therapy with 2 and 4 mg of saroglitazar . at the end of 12 weeks , the results showed statistically significant reduction in triglyceride concentration ( -45.5 mg / dl + /-
3.03% ) with 2mg / d of saroglitazar + atorvastatin and similar reduction ( -46 mg / dl mg / dl + /-
3.02% ) in those on 4 mg / day of saroglitazar + atorvastatin compared with those on placebo + atorvastatin .
although non significant in statistical terms , patients in study group reduced glycated hemoglobin levels by 0.3+/- 0.08% with 2 mg / day and 0.2+/- 0.07% with 4 mg / day dosage . at the end of 24 weeks , study group reported no significant renal , hepatic , and cardiac adverse events . for now as observed in phase 1 and phase 3 trails , saroglitazar appears safe and has not shown any of the adverse effects that are commonly recognized with its class of molecules ( e.g. muraglitazar - cardiovascular and tesaglitazar - renal and bone marrow ) . but given historical concerns , proof of long - term safety remains paramount and is yet to be proven .
although , it has shown positive results in improving glycemic and lipid parameters in patients with add in short term , a longer term clinical trial proving its efficacy in improving cardiovascular outcome is currently nonexistent but much desired . until such time given recent approval of saroglitazar by indian authorities permitting its clinical use in treatment of
add , both the pharmaceutical industry and medical profession should exercise close pharmacovigilance and report any adverse events as soon as observed . | a triad of high triglycerides , low high - density lipoprotein ( hdl ) cholesterol , and elevated small dense low - density lipoprotein particles occurring in a patient with type 2 diabetes is referred to atherogenic diabetic dyslipidemia ( add ) . despite statin therapy
, a significant residual risk remains potentially attributable to increased triglyceride concentration and low hdl cholesterol , a characteristic hallmark of add .
current therapeutic options in reducing this residual risk include nicotinic acid , omega 3 fatty acids , and selective peroxisome proliferator - activated receptor - alpha ( ppar ) agonists ( fibrates ) .
these drugs are limited in their potential either by lack of evidence to support their role in reducing cardiovascular events or due to their side effects .
this review details their current status and also the role of new glitazar , saroglitazar adual ppar/ agonist with predominant ppar activity in the management of add . | I
A
Omega 3 fatty acids
Nicotinic acid
Peroxisome proliferator-activated receptor agonists
Glitazones
Fibrates
Glitazars |
the gut microbiota , which resides in the gastrointestinal tract ( git ) and is also termed microflora , plays an important role in human health and disease .
the git is comprised of the stomach , the duodenum , the jejunum , the ileum , the colon , the rectum , and the anal canal . the lower digestive tract , specifically the colon , is the primary site of importance for bacterial cell colonization ; however , upper digestive tract microorganisms are also of importance .
the bacterial population of the gut has been studied in diseases such as colon cancer , inflammatory bowel diseases ( ibd ) , hypercholesterolemia , nonalcoholic fatty liver disease ( nafld ) and others .
certain bacterial populations , such as lactic acid bacteria , have been shown to positively influence health .
hence , attempts to modify the microflora , towards those bacteria , for disease treatment and prevention should prove advantageous . for this purpose ,
the delivery of viable probiotic bacteria is impeded by the harsh conditions of the upper git , hence , a vessel for delivering optimum cell viability to the lower git is required .
microcapsules can be used as a vehicle with the capability to protect the viability and activity of orally delivered bacterial cells through the upper git .
this paper will first give an overview of the gut microbiota and its main characteristics , focusing on its role in colon cancer , ibd , and hypercholesterolemia .
modulation of the gut microbiota to promote health will then be described through the use of probiotics , prebiotics , and synbiotics , with probiotics as a main focus .
microencapsulation and types of microcapsules will be described along with their success in the treatment and prevention of diseases .
the gut microbiota contains a broad spectrum of microorganisms , totalling 10 to 10 bacterial cells , but has not been completely explored as of yet .
the importance of the gut microflora is exemplified by the fact that the number of bacterial cells outnumbers human cells by a factor of ten .
the human intestinal habitat contains 300 to 500 different species of bacteria , varying significantly in content between individuals .
most gut bacteria reside in the lower part of the digestive tract , in the large intestine , since the upper tract consists of high levels of acid , bile , and pancreatic secretions which are toxic to most microorganisms , as shown in figure 1 . even though some bacterial species of the gut are potential pathogens , the constant interaction between the host and its microbes usually remains beneficial to the health of the host .
it has been demonstrated that the gut bacterial population plays an important role in their host 's metabolism and energy consumption , especially in the digestion and absorption of nutrients [ 1 , 5 ] .
the upper portion of the git , made up of the stomach and the duodenum , harbours very low numbers of microorganisms , with less than 1000 bacterial cells per gram of contents , with the predominant microorganisms present being lactobacilli and streptococci [ 6 , 7 ] .
the relatively low number of microorganisms found in the upper digestive tract , although some are of great importance in human disease , can be explained by the presence of high levels of acid , bile , and pancreatic secretions , as aforementioned [ 7 , 8 ] .
one important organism found in the stomach , which can withstand these harsh conditions , is helicobacter pylori , a microorganism responsible for ulcers and stomach cancer .
there is also a phasic propulsive motor activity in the upper git which impedes any stable bacterial colonization .
lower in the digestive tract are found the jejunum and the ileum where there is a gradual increase in the bacterial numbers from 10 to approximately 10 cells per gram of contents by the time the distal ileum is reached .
once in the large intestine , the tract is heavily populated by anaerobes with up to 10 cells per gram of luminal contents .
this paper focuses on the lower part of the digestive tract , due to its abundant bacterial population .
bacteria are classified into genera and species based on their individual phenotypic and genotypic characteristics , with a number of different genera found actively residing in the human git .
the dominant anaerobic genera are bacteroides , bifidobacterium , eubacterium , clostridium , peptococcus , peptostreptococcus , and ruminococcus [ 4 , 11 ] .
the main genera of facultative anaerobic bacteria are escherichia , enterobacter , enterococcus , klebsiella , lactobacillus , and proteus [ 4 , 11 ] .
the proportion and numbers of these bacteria can vary , depending on a number of genetic and environmental factors , including disease state and one 's food intake [ 1 , 1113 ] .
the main functions of the microflora were mostly elucidated by investigations with animals bred under germ - free conditions , with the functions broadly qualified as metabolic , trophic , and protective [ 3 , 14 , 15 ] .
the gut microbiota has a significant impact on host metabolism , participating in microbial - mammalian co - metabolism .
the microbiota is considered a multifunctional organ with metabolic capabilities that humans have not yet fully evolved into their own genomes .
it has the ability to break down indigestible plant polysaccharides , termed dietary fibers and also plays an important role in the biotransformation of conjugated bile acids , described in more detail later in this paper [ 1720 ] .
the importance of the gut microbiota in vitamin synthesis was demonstrated many years ago with the use of germ - free animals .
experiments on a chick animal model demonstrated the synthesis of riboflavin , vitamin b , pantothenic acid , vitamin b12 , folic acid , nicotinic acid , thiamine , and biotin by the gut microbiota .
, two resident organisms of the small intestine , were specifically shown to synthesize significant amounts of vitamin b12 .
as described , the gut microbiota has extensive roles to play in normal human metabolism .
the gut microbiota has gained importance in disease aetiology and pathology , with emerging evidence demonstrating its role in disease [ 1 , 24 ] .
a number of diseases have been associated with alterations of the gut microbiota , and if one can elucidate the exact link between the two one can begin to successfully treat and prevent these disorders through the modulation of the number and/or species of microorganisms present .
some disorders associated with the microflora include colon cancer , ibd , hypercholesterolemia and nonalcoholic fatty liver disease , among others [ 13 , 2428 ] .
colorectal cancer is the second most common cause of cancer death in men and women .
although the genetic mechanisms of colorectal cancer are well established , there are several environmental factors that have also been implicated in the development of sporadic colon carcinomas [ 3 , 30 ] .
foods , such as processed meats , which contain high levels of dietary fat , have been associated with an increased risk of colon cancer development when compared to the risk associated with a high intake of fruits , vegetables , grains , and fish [ 30 , 31 ] . it was proposed that the effect of diet could be mediated by changes in the composition of the colonic microflora such that the intestinal bacteria are responsible for the initiation of colon cancer [ 3 , 13 ] .
data shows that bacteria of the bacteroides and clostridium genera were associated with an increase in the incidence and growth rate of colonic tumors in tumor - induced animals , while genera such as lactobacillus and bifidobacterium ( well - characterized bacteria predominantly used in therapeutic probiotic formulations ) appeared to prevent tumorigenesis [ 32 , 33 ] .
the properties of the colonic microflora make it a promising target for the development of a colon cancer therapeutic .
ibd , prominent in western countries , is made up of a group of disorders that are characterized by a chronic and relapsing inflammation of the git .
the two most prominent forms of ibd are crohn 's disease ( cd ) and ulcerative colitis ( uc ) , with the bacterial flora as an important factor and contributor of the inflammation [ 36 , 37 ] .
sufferers of ibd have a higher bacterial attachment to gut epithelial surfaces when compared to that found in healthy individuals .
the link between intestinal mucosal inflammation and the resident bacteria has been further demonstrated , in vivo , using rats and mice treated with broad - spectrum antibiotics .
this treatment mitigates , although only temporarily , mucosal inflammation in animals with ibd , suggesting that the resident bacteria are causing the inflammation .
furthermore , an overpopulation of the bacteroides genera on the gut epithelium leads to an increased occurrence of transmural inflammatory lesions .
early research demonstrated that the presence of escherichia coli is linked to active uc and contributes to the development of inflammation .
e. coli has also been linked to cd , with the presence of specific adherent - invasive species found in the resected ileum of patients [ 41 , 42 ] .
this effect appears to be species specific as only certain phylogenetic groups of e. coli were found to be more frequent in uc and cd patients when compared with healthy controls [ 43 , 44 ] .
it is clear that the gut microflora plays an important role in ibd pathology and an efficient therapy is still required .
hypercholesterolemia is a disorder whereby an individual demonstrates an elevated serum cholesterol level . for many decades now
this disorder has been recognized as a significant risk factor associated with atherosclerosis and coronary heart disease .
current treatment options to lower serum cholesterol levels involve the use of pharmacological agents such as statins which act by inhibiting hmg - coa reductase , the rate - limiting enzyme of cholesterol biosynthesis .
statins make up a group of compounds that are generally well tolerated but remain expensive and have significant side - effects , including gastrointestinal problems , such as diarrhoea , but may also include severe liver and skeletal abnormalities [ 4749 ] .
bearing the potential significant consequences of hypercholesterolemia in mind , the importance of the gut microbiota in cholesterol metabolism and the pathogenesis of hypercholesterolemia , a new paradigm is suggested for the development of a successful treatment . as early as 1959 , research was performed to elucidate the role of the gut microbiota in cholesterol homeostasis with researchers demonstrating that germ - free rats , administered a diet without significant amounts of cholesterol , nonetheless showed higher serum - cholesterol values than control rats administered the same diet .
several mechanisms have been proposed as methods by which the gut microbiota may modulate cholesterol levels within the host .
recent developments have demonstrated that the composition of the microbiota and diet is directly correlated with cholesterol levels in vivo , specifically , the number of bifidobacteria found in the gut is positively correlated with higher levels of high - density lipoprotein ( hdl ) [ 5254 ] .
in contrast , the number of coriobacteriaceae is correlated with higher levels of non - hdl cholesterol .
gut microbial activities influence lipid metabolism , bearing a significant impact on hypercholesterolemia , by the modification of bile acid metabolic patterns , by impacting the emulsification , absorption , and storage properties of bile acids and by influencing the lipoperoxidation through bile acid signalling properties . with these facts in mind
past and current research has demonstrated that the gut microbiota plays an important role in the pathogenesis of a number of diseases .
certain bacteria , considered good , such as bifidobacteria and lactobacilli , are shown to be correlated with a decrease in the occurrence of a number of disorders , suggesting that the targeted increase of these beneficial bacteria could decrease the incidence and severity of prominent diseases .
the colonic delivery of prebiotics and probiotics are methods that have been successfully used to modify the gut microbiota .
antibiotics can prove beneficial in short - term use but their prolonged use may result in significant side - effects .
an important concern is the development of bacterial resistance which reduces the effectiveness of the therapy and further predisposes the patient to life - threatening illnesses caused by potential pathogens with increased resistance to the antibiotic .
current research focuses on prebiotics , probiotics , and a combination of both , termed synbiotics for modulating the gut microbiota .
non - viable food component that confers a health benefit on the host , associated with a modulation of the microbiota .
prebiotic molecules consist of naturally occurring or synthetic sugars used by certain colonic bacteria , especially bifidobacteria , as a carbon source for growth and metabolism .
numerous prebiotics have demonstrated their beneficial effects on disease through modulators of the gut microbiota [ 5761 ] .
prebiotic delivery nonspecifically increases the number of good bacteria not acting at the species level , which may be important in some disease states . on the other hand ,
probiotics are a method by which the gut microbiota can be specifically modulated for an individual to reestablish and maintain a healthy state .
the fao and who define probiotics as live microorganisms which , when administered in adequate amounts , confer a health benefit on the host .
probiotics are inexpensive , safe , free of long - term negative side - effects , and have already demonstrated beneficial effects for treating immunological , digestive , and respiratory diseases .
furthermore , these are naturally occurring organisms found in foods such as milk and yoghurt and , so , are widely accepted by the general public .
the most common types of probiotic microorganisms are the lactic acid bacteria , important components of the healthy gut microbiota and regarded as safe by the american fda .
other microorganisms occasionally used as probiotics are yeasts and filamentous fungi . in this section , we describe the use of probiotics on colorectal cancer , ibd , hypercholesterolemia and nafld .
probiotics have been proposed and investigated as a potential treatment / prevention method for colorectal cancer .
early studies demonstrated that 1,2-dimethylhydrazine-(dmh- ) induced colon cancer in rats showed a decrease in mortality rate if the test animals were fed streptococcus thermophilus - fermented skim milk .
another research group demonstrated that lactobacillus rhamnosus gg , in lyophilized form incorporated in a high - fat diet , was effective at reducing tumor incidence in the rat dmh colon cancer model .
studies using bifidobacterium longum also demonstrated an inhibition of carcinogen - induced colon cancers and precursor lesions [ 96 , 97 ] .
additional studies demonstrate a reduction of colon tumorigenesis markers following the incorporation of lactobacillus acidophilus in a high - fat control diet in dmh colon cancer rats .
the probiotics are suggested to achieve a protective effect by interacting with the carcinogen(s ) in the intestinal lumen ( in the case of the dmh rat model , interaction with the dmh metabolites azoxymethane or methylazoxymethane ) leading to a decrease in the potency / availability of the carcinogenic compound [ 97 , 98 ] .
probiotics have also been investigated as a method of treatment for ibd . a trial in ulcerative colitis ( uc ) patients
was performed to study the effect of the delivery of an oral probiotic capsule on the remission of the disorder .
the probiotic bifidobacteria were administered following treatment with an uc standard therapy . it was demonstrated that 93.3% of the patients in the control group suffered a disease relapse compared to only 20% of the patients administered the probiotic capsule .
a significant reduction in inflammation was also observed in the treatment group when compared to the control group .
another study demonstrated the use of faecalibacterium prausnitzii as a probiotic for treating crohn 's disease ( cd ) .
f. prausnitizii and its supernatant were both found to have anti - inflammatory effects in vitro using peripheral blood mononuclear and colon adenocarcinoma cells and in vivo in a mouse model of induced colitis .
a number of other studies related to the effects of probiotics on the prevention and the treatment of ibd , described in another review , have been done , with varying success .
early studies suggest that probiotic bacteria may have a beneficial effect on hypercholesterolemic patients , by decreasing blood lipid levels .
a study was undertaken with hypercholesterolemic mice administered low levels of the probiotic lactobacillus reuteri for a week .
the mice demonstrated a decrease in cholesterol and triglyceride levels and an increase in the hdl : ldl ratio .
a study was also performed with hyperlipidemic patients who were administered the probiotic lactobacillus sporogenes over a three - month period . following treatment
, these patients showed , on average , a 32% reduction in total cholesterol levels accompanied with a 35% reduction in ldl .
studies have also demonstrated that the delivery of certain strains of lactobacilli can alleviate symptoms associated with ibd [ 103 , 104 ] .
probiotics have also been proposed as a potential treatment option for nafld because of their modulating effect on the gut flora that could influence the gut - liver axis towards a healthy state .
nafld is characterized by the release of inflammatory cytokines and commensal bacteria have been shown to provoke anti - inflammatory responses from the gut epithelia , suggesting a mechanism of action to treat the disease .
probiotics can have an inhibitory impact on the development of nafld by a number of mechanisms : competitive inhibition of pathogenic bacterial strains , alteration of the inflammatory effects of pathogenic strains through changes in cytokine signalling , improvement of the function of the epithelial barrier and direct decreases of proinflammatory cytokines , including tnf- .
vsl#3 is a high - potency medical food probiotic made up of a number of different bacterial strains .
these strains make up 450 billion live lactic acid bacteria per packet : bifidobacterium breve , bifidobacterium longum , bifidobacterium infantis , lactobacillus acidophilus , lactobacillus plantarum , lactobacillus paracasei , lactobacillus bulgaricus , and streptococcus thermophilus .
this combination , in both murine and human trials , demonstrated all of the mechanisms described as potential beneficial targets for the treatment of nafld .
murine models of acute liver injury have also shown a decrease in hepatic injury following the administration of various lactobacillus and bifidobacterium species [ 108110 ] .
there are a number of mechanisms by which probiotics could be exerting their beneficial effects , as shown in figure 2 .
the mechanisms include ( 1 ) by the production of pathogen inhibitory substances ; ( 2 ) by the blocking of pathogenic bacteria adhesion sites ; ( 3 ) by nutrient competition and production ; ( 4 ) by the degradation of toxins and toxin receptors ; ( 5 ) by the modulation of immune responses .
probiotics must be delivered to the target sites in sufficient number and metabolic active phase to be effective .
one of the major limitations is the delivery of probiotics to the lower git , with the presence of acids and bile greatly hindering the viability of the probiotics as they travel through the gut ( specifically the acidic environment of the stomach ) .
another complication is the presence of an immune system which can be induced and potentially attack the delivered cells .
hence , a method is required to protect the probiotic cells while maintaining high levels of probiotic viability and activity when delivered in the git .
this paper introduces microencapsulation and discusses its potentials and limitations in bacterial cell delivery to the git .
microencapsulation is a method defined as the entrapment of a compound or a system inside a dispersed material for its immobilization , protection , controlled release , structuration and functionalization .
there exists a great variety of microcapsules which can differ in size , composition , and function , depending on the final goal of the encapsulated product .
microcapsules can be used to entrap all sorts of substances : solids , liquids , drugs , proteins , bacterial cells , stem cells , and so forth [ 112114 ] .
with such a range of substances that can be entrapped , microcapsules can have an assortment of objectives and applications , whether for drug delivery , enzyme retrieval , artificial cell and artificial organ delivery or , as described in this review , for the delivery of live probiotic bacteria .
there are a number of microcapsule delivery systems that have been proposed for the oral delivery of live bacterial cells , as detailed in table 1 .
sun and griffiths investigated the use of acid - stable beads made of gellan and xanthan gum for the immobilization of bifidobacterium .
the research group demonstrated that immobilized cells survived significantly better than free cells after refrigeration in pasteurized yogurt for a period of 5 weeks .
one common encapsulation method , for viable cell immobilization , utilizes calcium alginate as a polymer . however , one prominent difficulty encountered with the use of alginate beads is that these , alone , are not acid resistant and upon exposure to the low ph conditions encountered in the stomach , display significant shrinkage and a decrease in mechanical strength
. a number of methods utilizing polymer cross - linking have been suggested , including formulations using carrageenan , alginate - poly - l - lysine , starch polyanhydrides , polymethacrylates , and enteric coated polymers .
microencapsulation methods are still being developed and optimized to allow for increased gastrointestinal survival and immunoprotection .
one newly developed type of microcapsule that shows promising results in terms of mechanical stability and ph resistance is the genipin - crosslinked - alginate - chitosan ( gcac ) microcapsule , shown in figure 3 [ 87 , 117 ] .
one of the most commonly utilized and characterized formulations for microencapsulation is the alginate - poly - l - lysine - alginate ( apa ) microcapsule .
this type of microcapsule has been used for many applications including drug , stem cell , and bacterial cell delivery .
this method relies on a polyelectrolyte complexation mechanism for the association of the polymers , alginate and poly - l - lysine ( pll ) .
alginate is a naturally occurring biocompatible polymer , extracted from brown algae , that is increasingly being used in the biotechnology industry for a wide range of applications .
alginate is an unbranched polysaccharide which contains 1,4-linked -d - mannuronic acid and -l - guluronic acid blocks which are interdispersed with regions of the alternating structure , -l - mannuronic acid--l - guluronic acid blocks .
pll is a polypeptide made up of the amino acid l - lysine that is available in a variable number of chain lengths , determined by its molecular weight .
it is a polycationic polymer that can be used during the coating step of microencapsulation .
the addition of this polymer leads to the formation of a capsule membrane that provides selective permeability and immunoprotection .
the alginate bead could not withstand the harsh conditions of the git in the absence of pll , which provides it with an increased mechanical stability .
the microencapsulation technique employed is determined by the type and the size of microcapsules one wants to obtain .
the characteristics of the microcapsule must also take into consideration the function that the microcapsule will ultimately undertake .
the first step is the incorporation of the ingredients into a solution by mixing or dispersion , to make up the core of the microcapsule .
this is then followed by mechanical operations , such as spraying or emulsification , to form the droplets .
the final step of microencapsulation involves product stabilization through coating , followed by a number of physical or chemical processes .
each step of microencapsulation can be optimized according to the desired characteristics of the final formulation .
research has shown that microencapsulated probiotics keep their viability better than free cells under stress in git deliveries .
research into the applications of microencapsulated probiotics is also ongoing , with promising results for the eventual treatment of a number of disorders , described in the following section .
the potential antitumorigenic properties of a microencapsulated formulation of l. acidophilus were studied in min ( multiple intestinal neoplasia ) mice carrying a germline apc mutation which spontaneously develop numerous pretumoric intestinal neoplasms .
the mice were gavaged apa microcapsules of l. acidophilus over a period of 12 weeks followed by the enumeration , the classification and the histopathology of adenomas . unfortunately , no statistically significant difference was observed between the treatment and control group in terms of the number of large intestinal ( colonic ) adenomas . on a more positive note
, there was a statistical difference between the control and treatment groups following analysis of the small intestine number of adenomas and gastrointestinal intraepithelial neoplasias .
these preliminary results suggest that microencapsulated probiotic bacteria could have a role in the development of a successful colon cancer therapeutic .
early research has demonstrated that certain lactobacilli have a bile salt hydrolase ( bsh ) enzyme which can contribute to a significant cholesterol lowering effect in vivo in cardiovascular diseases .
the oral delivery of lactobacillus has , therefore , emerged as a potential mechanism for inducing cholesterol lowering .
demonstrated that microencapsulated bsh - active bacteria are able to survive in a simulated human gastrointestinal model while maintaining cell viability and enzyme activity , which would not be possible with the direct delivery of nonmicroencapsulated bacterial cells . another microencapsulated probiotic lactobacillus demonstrated cholesterol lowering capabilities in hypercholesterolemic animals , albeit with a different mechanism of action involving a feruloyl esterase enzyme .
lactobacillus fermentum , a feruloyl esterase active bacterium , was microencapsulated and delivered to hypercholesterolemic hamsters twice daily by oral gavage , for a period of 18 weeks .
following treatment , hamster serum cholesterol , ldl cholesterol , and the atherogenic index were 21.36% , 31.40% , and 32.59% lower , respectively , in the treatment group when compared to the control group .
histological studies were also performed and demonstrated that the microencapsulated probiotic reduced the progression of atherosclerotic lesions in the test animals .
this probiotic formulation was hence shown to be effective at managing excessive serum cholesterol and triglyceride levels . with these results
, the microencapsulation of probiotics is very promising for the development of a cholesterol - lowering therapeutic in cardiovascular diseases .
it has been shown that , to be effective at reducing colon tumorigenesis , therapeutic probiotic microorganisms must remain viable in vivo .
the same study that demonstrated that the administration of l. acidophilus had an inhibitory effect on colon tumorigenesis showed that the amount of probiotic colonization of the git is directly linked to the rate of inhibition of tumorigenesis . since viability is vital to the mechanism of action of the probiotic , it is crucial to realize that , of the bacteria ingested , only 1% survive the gastric transit , limiting the overall therapeutic effect of any orally delivered bacterial formulation . with unprotected probiotic formulations already demonstrating therapeutic potential ,
although probiotics , prebiotics , and synbiotics have shown great potential for the treatment of a number of disorders , there are still a number of challenges that remain to be addressed before they can be successfully used to treat / prevent disorders .
microencapsulation has provided a significant advancement in the field , allowing for the delivery of a greater number of viable bacteria to the git . however
, a number of issues concerning the formulation of a microencapsulated probiotic still need to be addressed before a successful product can be developed .
there are a number of microencapsulation types being employed , each varying in efficiency and application .
furthermore , the industry scale production of microencapsulated probiotics , at a cost - effective level , and investigation of formulation stability , cell viability and retention of metabolic activity of the encapsulated bacterial cells requires further development for specific bacterial strains and diseases .
most of the probiotics are strain specific , they therefore must be developed and characterised in vitro and evaluated for their suitability and efficacy in proper animal models and human clinical trials .
furthermore , a specific mechanism of action must be developed for each application so that an evidence - based probiotic formulation can be designed that can potentially compete with well - articulated and well - developed drug formulations .
the elucidation of the mechanism of action of the probiotic would allow for a better selection process .
as described before , the administration of bacteria from the same species but of different strains resulted in noncomparable effects , further emphasizing the importance of mechanistic studies as part of the probiotic selection process .
the variability in experimental design poses a great challenge in probiotic research that must be addressed properly .
since the compositions of the gut microflora are not identical , there can be contradicting results as to the beneficial effect of probiotics on the microbiota of the git .
this composition variability also gives rise to potential difficulties in terms of the use of animal models for the investigation of probiotic formulations .
a targeted well - defined formulation should be developed in which microencapsulation will play a critical role .
there are excellent trials available that demonstrate the efficacy of these formulations but safety is an issue that remains to be investigated .
as mentioned earlier , rigorous in vitro and in vivo animal and human clinical studies are needed to demonstrate the efficacy and the long - term safety of microencapsulated and other probiotic formulations .
nevertheless , the literature suggests that probiotics will lead to efficient therapeutic formulations for the treatment and/or prevention of a number of animal and human health disorders . | the gut microbiota plays a crucial role in maintaining health .
alterations of the gut bacterial population have been associated with a number of diseases .
past and recent studies suggest that one can positively modify the contents of the gut microbiota by introducing prebiotics , probiotics , synbiotics , and other therapeutics .
this paper focuses on probiotic modulation of the gut microbiota by their delivery to the lower gastrointestinal tract ( git ) .
there are numerous obstacles to overcome before microorganisms can be utilized as therapeutics .
one important limitation is the delivery of viable cells to the lower git without a significant loss of cell viability and metabolic features through the harsh conditions of the upper git .
microencapsulation has been shown to overcome this , with various types of microcapsules available for resolving this limitation .
this paper discusses the gut microbiota and its role in disease , with a focus on microencapsulated probiotics and their potentials and limitations . | 1. Introduction
2. The Gastrointestinal Bacterial System
3. The Gut Microbiota and Its Role in Human Health and Disease
4. Modulation of the Gut Microbiota for Human Health Benefits
5. Microencapsulation and Delivery of Probiotics
6. Microencapsulated Probiotics
7. Challenges and Future Outlooks |
cancer is a major public health problem all over the world , and has become one of the primary causes of morbidity and mortality .
the epidemiology of cancer is influenced by the aging and growth of the world population and a rise in cancer - causing behaviors ; therefore , the global burden of cancer is rapidly increasing . the etiology of cancer is complex and is still obscure .
recent research suggests that single - nucleotide polymorphisms ( snps ) in genes play critical roles in cancer development and progression [ 36 ] . among these snps , icam-1 polymorphisms have been shown to be particularly important .
icam-1 , a single - chain 76110 kda glycoprotein , is a member of the immunoglobulin superfamily , which is involved in cell adhesion and signalling .
studies indicate that icam-1 plays an important role in tumorigenesis and tumor progression , specifically by facilitating tumor invasion and metastases .
many case - control studies have demonstrated that icam-1 polymorphisms ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) are associated with susceptibility to many cancers , including colorectal cancer , breast cancer , diffuse astrocytoma , prostate cancer , cutaneous malignant melanoma , ovarian cancer , urothelial cell carcinoma , oral cancer , and acute promyelocytic leukanemia ( apl ) . in view of the relevance of icam-1 polymorphisms
( rs5491 , rs3093030 , rs281432 , and rs1799969 ) in susceptibility to cancer , many studies have been conducted [ 918 ] but the results were inconclusive and inconsistent . in 2014 ,
wang et al . performed a meta - analysis of 14 studies and concluded that icam-1 rs5498 polymorphism was associated with cancer susceptibility . in the present study
, we aimed to clarify the relationship between other icam-1 polymorphisms ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) and cancer susceptibility based on all eligible published case - control studies .
all eligible case - control studies on the relationship between polymorphisms of icam-1 ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) and cancer susceptibility up to june 16 , 2015 were identified by a systematic literature search in pubmed , web of science , and google scholar .
neoplasms ) and ( polymorphism or variant or mutation ) .
in addition , for each retrieved publication , a manual search for relevant references was also conducted to find additional case - control studies .
the inclusion criteria were : 1 ) evaluation of the icam-1 polymorphisms and cancer susceptibility , 2 ) case - control studies , and 3 ) presenting available information to assess the odds ratio ( or ) with 95% confidence interval ( ci ) .
major reasons for exclusion of studies were : 1 ) no control population , 2 ) abstracts and reviews , 3 ) no available genotype frequency , 4 ) duplication of the previous publication , and 5 ) non - human studies .
two investigators ( xiaolong zhang and junjie huang ) independently extracted data on icam-1 polymorphisms , first author , year of publication , ethnicity of the case - control studies , genotyping methods , source of controls , type of cancer , and genotype number in cancer cases and controls .
we evaluated the association between icam-1 polymorphisms and cancer susceptibility by or and 95% ci .
the significance of the pooled or was determined by the z - test and p<0.05 was considered statistically significant .
a total of 4 genetic models were selected : allele contrasts , additive genetic model , recessive genetic model , and dominant genetic model separately .
heterogeneity was detected by the -based q statistic test to assess the heterogeneity within the case - control studies .
when there was heterogeneity ( p<0.10 , i>50% ) , the random - effects model was used to calculate the pooled ors ; otherwise , the fixed - effects model was used .
stratification analyses of cancer type , genotyping method , and source of control were conducted .
sensitivity analyses were further performed to calculate the stability of the results by removing each case - control study from the enrolled pooled data to detect the influence of the respective data set on the pooled ors . to examine the potential publication bias ,
the stata 12.0 ( stata corporation , college station , tx ) was used to conduct all statistical analyses .
the study quality was assessed independently by xiaolong zhang and junjie huang by referring to the newcastle - ottawa scale ( nos ) , which examines the quality of non - randomized studies by the selection of participants , comparability of groups , and exposure assessment .
all eligible case - control studies on the relationship between polymorphisms of icam-1 ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) and cancer susceptibility up to june 16 , 2015 were identified by a systematic literature search in pubmed , web of science , and google scholar .
neoplasms ) and ( polymorphism or variant or mutation ) .
in addition , for each retrieved publication , a manual search for relevant references was also conducted to find additional case - control studies .
the inclusion criteria were : 1 ) evaluation of the icam-1 polymorphisms and cancer susceptibility , 2 ) case - control studies , and 3 ) presenting available information to assess the odds ratio ( or ) with 95% confidence interval ( ci ) .
major reasons for exclusion of studies were : 1 ) no control population , 2 ) abstracts and reviews , 3 ) no available genotype frequency , 4 ) duplication of the previous publication , and 5 ) non - human studies .
two investigators ( xiaolong zhang and junjie huang ) independently extracted data on icam-1 polymorphisms , first author , year of publication , ethnicity of the case - control studies , genotyping methods , source of controls , type of cancer , and genotype number in cancer cases and controls .
we evaluated the association between icam-1 polymorphisms and cancer susceptibility by or and 95% ci .
the significance of the pooled or was determined by the z - test and p<0.05 was considered statistically significant .
a total of 4 genetic models were selected : allele contrasts , additive genetic model , recessive genetic model , and dominant genetic model separately .
heterogeneity was detected by the -based q statistic test to assess the heterogeneity within the case - control studies .
when there was heterogeneity ( p<0.10 , i>50% ) , the random - effects model was used to calculate the pooled ors ; otherwise , the fixed - effects model was used .
stratification analyses of cancer type , genotyping method , and source of control were conducted .
sensitivity analyses were further performed to calculate the stability of the results by removing each case - control study from the enrolled pooled data to detect the influence of the respective data set on the pooled ors . to examine the potential publication bias ,
the stata 12.0 ( stata corporation , college station , tx ) was used to conduct all statistical analyses .
the study quality was assessed independently by xiaolong zhang and junjie huang by referring to the newcastle - ottawa scale ( nos ) , which examines the quality of non - randomized studies by the selection of participants , comparability of groups , and exposure assessment .
after a systematic literature search and selection based on the inclusion criteria , 140 publications were considered for eligibility . however , among these eligible articles , 115 are disqualified because they were not about polymorphisms , were not cancer studies , or they were reviews or letters . of the remaining 25 publications ,
7 were based on case - only design , 5 were not polymorphism studies , and 4 were not about susceptibility to cancer . as a result , 9 publications with 14 case - control studies including 4608 cancer cases and 4913 controls were included in the present meta - analysis .
eight studies were conducted in people of asian ethnicity , 6 studies were conducted in people of white ethnicity , and only 1 study was conducted in people of african ethnicity . in addition , there were 10 studies done by pcr , 4 performed by taqman , and only 1 conducted by pcr - rflp .
the control groups consisted of 10 population - based studies and 5 hospital - based studies .
of these included studies , 3 reported urothelial cell carcinoma and 3 reported oral cancer .
two studies were on breast cancer , 2 were on apl , and 2 were on ovarian cancer .
prostate cancer , cutaneous melanoma , and colorectal cancer were also mentioned in other studies .
the results of the meta - analysis for the association between icam-1 polymorphisms ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) and susceptibility to cancer are presented in table 2 . according to the outcomes of heterogeneity analysis ,
obvious heterogeneity was identified in icam-1 rs3093030 polymorphism ( c vs. t : p=0.012 , i=52.6% ; cc vs. tt : p=0.003 , i=62.1% ; cc vs. tc+tt : p=0.003 , i=62.1% ) therefore , the random - effects model was used to estimate the pooled ors in these genetic models . according to the present analysis
, we discovered that icam-1 rs1799969 polymorphism was significantly associated with overall cancer susceptibility ( table 2c , c vs. t : or=1.662 , 95%ci = 1.2882.143 , p=0.141 , figure 2a ; ct vs. tt : or=1.860 , 95%ci=1.3982.474 , p=0.507 , figure 2b ; cc+ct vs. tt : or=1.812 , 95%ci=1.3732.391 , p=0.284 , figure 2c ) .
nevertheless , no relevance was identified between other icam-1 polymorphisms ( table 2a , rs5491 ; table 2b , rs3093030 ; table 2d , rs281432 ) and overall cancer susceptibility . in stratified analysis of the source of controls ,
an increased susceptibility of the population - based group in rs1799969 polymorphism was found in 3 genetic models ( table 2c , c vs. t : or=2.007 , 95%ci=1.4712.737 , p=0.778 ; ct vs. tt : or=2.110 , 95%ci=1.5022.962 , p=0.852 ; cc+ct vs. tt : or=2.151 , 95%ci=1.5393.006 , p=0.907 ) .
interestingly , we also identified an increased susceptibility for asians in rs3093030 polymorphism ( table 2b , cc vs. tc+tt : or=1.728 , 95%ci=1.2342.421 , p=0.787 , figure 3 ) in the stratification analysis by ethnicity .
egger s test and begg s funnel plot were performed to examine the publication bias risk and we found no publication bias ( figure 4a4d ) . additionally , the quality of the enrolled studies is shown in table 3 .
after a systematic literature search and selection based on the inclusion criteria , 140 publications were considered for eligibility . however , among these eligible articles , 115 are disqualified because they were not about polymorphisms , were not cancer studies , or they were reviews or letters . of the remaining 25 publications ,
7 were based on case - only design , 5 were not polymorphism studies , and 4 were not about susceptibility to cancer . as a result , 9 publications with 14 case - control studies including 4608 cancer cases and 4913 controls were included in the present meta - analysis .
eight studies were conducted in people of asian ethnicity , 6 studies were conducted in people of white ethnicity , and only 1 study was conducted in people of african ethnicity . in addition , there were 10 studies done by pcr , 4 performed by taqman , and only 1 conducted by pcr - rflp .
the control groups consisted of 10 population - based studies and 5 hospital - based studies .
of these included studies , 3 reported urothelial cell carcinoma and 3 reported oral cancer .
two studies were on breast cancer , 2 were on apl , and 2 were on ovarian cancer .
prostate cancer , cutaneous melanoma , and colorectal cancer were also mentioned in other studies .
the results of the meta - analysis for the association between icam-1 polymorphisms ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) and susceptibility to cancer are presented in table 2 . according to the outcomes of heterogeneity analysis ,
obvious heterogeneity was identified in icam-1 rs3093030 polymorphism ( c vs. t : p=0.012 , i=52.6% ; cc vs. tt : p=0.003 , i=62.1% ; cc vs. tc+tt : p=0.003 , i=62.1% ) therefore , the random - effects model was used to estimate the pooled ors in these genetic models . according to the present analysis
, we discovered that icam-1 rs1799969 polymorphism was significantly associated with overall cancer susceptibility ( table 2c , c vs. t : or=1.662 , 95%ci = 1.2882.143 , p=0.141 , figure 2a ; ct vs. tt : or=1.860 , 95%ci=1.3982.474 , p=0.507 , figure 2b ; cc+ct vs. tt : or=1.812 , 95%ci=1.3732.391 , p=0.284 , figure 2c ) .
nevertheless , no relevance was identified between other icam-1 polymorphisms ( table 2a , rs5491 ; table 2b , rs3093030 ; table 2d , rs281432 ) and overall cancer susceptibility . in stratified analysis of the source of controls , an increased susceptibility of the population - based group in rs1799969 polymorphism
was found in 3 genetic models ( table 2c , c vs. t : or=2.007 , 95%ci=1.4712.737 , p=0.778 ; ct vs. tt : or=2.110 , 95%ci=1.5022.962 , p=0.852 ; cc+ct vs. tt : or=2.151 , 95%ci=1.5393.006 , p=0.907 ) .
interestingly , we also identified an increased susceptibility for asians in rs3093030 polymorphism ( table 2b , cc vs. tc+tt : or=1.728 , 95%ci=1.2342.421 , p=0.787 , figure 3 ) in the stratification analysis by ethnicity .
egger s test and begg s funnel plot were performed to examine the publication bias risk and we found no publication bias ( figure 4a4d ) . additionally , the quality of the enrolled studies is shown in table 3 .
icam-1 , a cell adhesion molecule with a key role in inflammation and immune surveillance , has been implicated in carcinogenesis by facilitating instability of the tumor environment . in 2014 ,
wang et al . conducted a meta - analysis and concluded that icam-1 rs5498 polymorphism was associated with cancer susceptibility .
however , the association between cancer susceptibility and other polymorphisms of icam-1 ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) remained unclear .
recently , dore et al . demonstrated that no significant association was detected between icam-1 rs1799969 polymorphism and apl in whites .
obtained the opposite results in breast cancer , colorectal cancer , and cutaneous malignant melanoma , respectively .
in addition , several publications ( case - control studies ) indicated that icam-1 polymorphisms ( rs5491 , rs3093030 , and rs281432 ) were also involved in tumorigenesis and tumor progression .
however , the conclusions were inconclusive because of the limited number of relevant published reports .
we aimed to comprehensively define the association between icam-1 polymorphisms ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) and cancer susceptibility in a total of 9 publications , including 14 case - control studies with 4608 cases and 4913 controls .
moreover , in the stratified analysis , significant cancer susceptibility in population - based and asian groups was identified for rs1799969 and rs3093030 , respectively .
it was well - established that hospital - based studies may have selection bias ; the controls may only represent a poorly - defined reference population rather than the general population or the population of interest , especially when the genotypes examined are relevant to disease - related factors that the hospital - based controls may have been exposed to .
the selection of appropriate and representative controls is of great importance in reducing biases in polymorphism association studies .
therefore , we conducted subgroup analysis by source of control and found that the source of control did not influence our conclusions . although we conducted a comprehensive retrieval of all eligible studies , several limitations of this meta - analysis should be acknowledged .
firstly , the number of currently available case - control studies enrolled in our study was small and we could not achieve definitive results . secondly , lack of detailed data on individuals limited the precision of our analysis of adjusted estimates involving other factors such as age and sex .
thirdly , only 1 study discussed the genetic predisposition of every icam-1 polymorphism to each cancer , and we could not evaluate the effects of a single polymorphism of icam-1 on a specific cancer because eligible case - control studies were insufficient for pooled analysis . finally , the effect of icam-1 polymorphisms on cancer susceptibility might be affected by complex factors , such as histological types of cancer and matching criteria .
results of our meta - analysis show that icam-1 polymorphism rs1799969 is significantly associated with increased susceptibility to cancer .
future well - designed studies are warranted to further explore the relationship between icam-1 polymorphisms and cancer susceptibility . | backgroundmany epidemiology studies have indicated that polymorphisms in icam-1 are associated with a variety of cancers , but published data are contradictory and inconclusive .
therefore , we conducted the current meta - analysis to elaborate the effects of icam-1 polymorphisms ( rs5491 , rs3093030 , rs281432 , and rs1799969 ) on cancer susceptibility.material/methodswe conducted a comprehensive literature search in pubmed , web of science , and google scholar .
odds ratios ( ors ) with 95% confidence intervals ( cis ) were estimated to assess the association between icam-1 polymorphisms and cancer susceptibility.resultswe enrolled 14 published case - control studies including 4608 cancer cases and 4913 controls .
we found an increased susceptibility of cancer in polymorphism rs1799969 ( c vs. t : or=1.662 , 95%ci=1.2882.143 , p=0141 ; ct vs. tt : or=1.860 , 95%ci=1.3982.474 , p=0.507 ; cc+ct vs. tt : or=1.812 , 95%ci=1.3732.391 , p=0.284 ) of icam-1 among the overall population .
however , no association between polymorphisms rs5491 , rs3093030 , or rs281432 of icam-1 and cancer susceptibility was identified . in the stratification analysis by ethnicity , we identified an increased susceptibility for asians in rs3093030 polymorphism ( cc vs. tc+tt : or=1.728 , 95% ci=1.2342.421 , p=0.787).conclusionsour results suggest that the icam-1 polymorphism rs1799969 is significantly associated with increased susceptibility to overall cancer .
further studies ( preferably prospective ) are warranted to validate these relationships . | Background
Material and Methods
Literature search strategy
Selection criteria
Data extraction
Statistical analysis
Quality evaluation
Results
Study characteristics
Meta-analysis
Sensitivity analyses and publication bias
Discussion
Conclusions |
the and subunits of pcc were co - expressed in e. coli , with a his - tag on the subunit .
crystals were obtained by the microbatch method under oil , and the structures were determined by the molecular replacement method .
frozen hydrated human pcc particles at 70 g / ml concentration were imaged at 50,000 magnification in a 100 kv cryo - electron microscope .
a featureless gaussian oval was used to obtain a low resolution ( 40 ) model from negative stain electron microscope images .
a 15 resolution 3d reconstruction was obtained from ~10,000 cryo - em particle images , using the structure from the negative stain images as the initial model .
site - specific and deletion mutants were designed based on the structural information , and their effects on the formation of the holoenzyme was assessed by nickel affinity chromatography .
the catalytic activity of pcc was determined by a coupled enzyme assay , monitoring the hydrolysis of atp .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature .
the and subunits of pcc were co - expressed in e. coli , with a his - tag on the subunit .
crystals were obtained by the microbatch method under oil , and the structures were determined by the molecular replacement method .
frozen hydrated human pcc particles at 70 g / ml concentration were imaged at 50,000 magnification in a 100 kv cryo - electron microscope .
a featureless gaussian oval was used to obtain a low resolution ( 40 ) model from negative stain electron microscope images .
a 15 resolution 3d reconstruction was obtained from ~10,000 cryo - em particle images , using the structure from the negative stain images as the initial model .
site - specific and deletion mutants were designed based on the structural information , and their effects on the formation of the holoenzyme was assessed by nickel affinity chromatography .
the catalytic activity of pcc was determined by a coupled enzyme assay , monitoring the hydrolysis of atp .
full methods and any associated references are available in the online version of the paper at www.nature.com/nature . | propionyl - coenzyme a carboxylase ( pcc ) , a mitochondrial biotin - dependent enzyme , is essential for the catabolism of the amino acids thr , val , ile and met , cholesterol , and fatty acids with an odd number of carbon atoms .
deficiencies of pcc activity in humans are linked to the disease propionic acidemia ( pa ) , an autosomal recessive disorder that can be fatal in infants 14 .
the holoenzyme of pcc is an 66 dodecamer , with a molecular weight of 750 kd .
the subunit contains the biotin carboxylase ( bc ) and biotin carboxyl carrier protein ( bccp ) domains , while the subunit supplies the carboxyltransferase ( ct ) activity . here
we report the crystal structure at 3.2 resolution of a bacterial pcc 66 holoenzyme as well as cryo - electron microscopy ( cryo - em ) reconstructionat 15 resolution demonstrating a similar structure for human pcc .
the structure defines the overall architecture of pcc and reveals unexpectedly that the subunits are arranged as monomers in the holoenzyme , decorating a central 6 hexamer .
a hitherto unrecognized domain in the subunit , formed by residues between the bc and bccp domains , is crucial for interactions with the subunit .
we have named it the bt domain .
the structure reveals for the first time the relative positions of the bc and ct active sites in the holoenzyme .
they are separated by approximately 55 , indicating that the entire bccp domain must translocate during catalysis .
the bccp domain is located in the active site of the subunit in the current structure , providing insight for its involvement in the ct reaction .
the structural information establishes a molecular basis for understanding the large collection of disease - causing mutations in pcc , and also has important relevance for the holoenzymes of other biotin - dependent carboxylases , including 3-methylcrotonyl - coa carboxylase ( mcc ) 57 and eukaryotic acetyl - coa carboxylase ( acc ) 8,9 . | Methods summary
Crystallography
Cryo-electron microscopy
Mutagenesis and kinetic studies
Supplementary Material |
46,xx gonadal dysgenesis is a rare genetically heterogeneous disorder characterized by underdeveloped ovaries with consequent , impuberism , primary amenorrhea , and hypergonadotropic hypogonadism and occurs in < 1 in 10,000 women .
the forms of 46,xx gonadal dysgenesis not associated with somatic malformations are more commonly inherited in an autosomal recessive fashion and are more common in consanguineous families .
several pleiotropic genes may be involved in 46,xx gonadal dysgenesis with somatic malformations , and these cases are very rare in occurrence and may be unique to specific families .
several cases of 46,xx gonadal dysgenesis with specific somatic malformations were described as specific syndromes in literature which were acquired either in autosomal dominant or recessive fashion .
mullerian agenesis or mayer - rokitansky - kuster - hauser ( mrkh ) syndrome is characterized by congenital aplasia of the uterus and the upper part ( 2/3 ) of the vagina in a woman with normal development of secondary sexual characteristics and a normal 46,xx karyotype .
it affects 1 in 4500 women , and the usual clinical presentation is primary amenorrhea in a young woman with normal external genitalia and functional ovaries .
the phenotypic manifestations of mrkh syndrome may sometimes overlap with various other syndromes and require accurate delineation .
even though previously this syndrome was considered as a sporadic anomaly , increasing number of familial cases now support the hypothesis of a genetic cause .
a 20-year - old female patient presented to our hospital with complaints of primary amenorrhea , short stature , and absence of secondary sexual characters .
there was no family history of consanguinity , miscarriages , neonatal deaths , or any other family member with primary amenorrhea .
she gives history of delayed milestones and stunted growth from the age of 4 years .
her pulse rate was 86 beats / min and blood pressure was 100/70 mm of mercury . on cardiac auscultation , a systolic murmur was heard in the aortic area .
scoring of pubic , axillary hair growth , and breast development were tanner 's stage 1 [ figure 1 ] .
external genital examination revealed immature labia majora and minora , clitoris with a blind vaginal pouch .
there was gibbus in the lumbar region with short fourth metatarsal in the left foot .
x - ray of the cervical , dorsal , and lumbar spine revealed fusion of l1 and l2 vertebrae and scoliosis of dorsal spine with no abnormalities in cervical spine [ figure 2 ] .
echocardiography revealed dilated aorta and bicuspid aortic valve with no other cardiac abnormalities , and abdominal ultrasound examination revealed single horseshoe - shaped kidney and absence of uterus and ovaries .
the abdominal ultrasound findings were confirmed later by magnetic resonance imaging ( mri ) of the abdomen and pelvis [ figure 3 ] . t2-weighted coronal image of pelvis revealed bladder and rectum without interposition of uterus .
t1-axial fluid attenuation inversion recovery image of the brain at the level of pituitary fossa was normal and karyotyping revealed normal 46,xx complement [ figures 4 and 5 ] .
iu / l ) with low estradiol ( < 5 pg / ml ) levels .
her blood sugar , thyroid function tests , serum cortisol , and prolactin levels were normal . hormonal therapy with ethinyl estradiol 10 g / day was started for the development of secondary sexual characteristics and to prevent osteoporosis .
arrows show the absence of breast development and axillary hair arrow shows fusion of the second and third lumbar vertebrae and scoliosis of dorsal spine t2-weighed coronal image of pelvis shows the absence of uterus and ovaries in the magnetic resonance image of pelvis and the absence of ovaries in bilateral adnexa t1 axial fluid attenuation inversion recovery image at the level of pituitary fossa shows normal pituitary fossa 46,xx karyotype complement
gonadal dysgenesis with female phenotype is defined as the absence or incomplete development of ovaries .
the karyotypes , 45,xo , 45,x/46,xx , 45,x/46 , x , dic(x ) , 46,xx , and 46,xy , have been reported in literature .
46,xx gonadal dysgenesis is relatively rare form of gonadal dysgenesis and wide variations are seen in its clinical presentation .
mrkh syndrome is the second most common cause of primary amenorrhea and more commonly presents with only mullerian agenesis and rarely with many other congenital anomalies .
the association of gonadal dysgenesis and mrkh syndrome is extremely rare . in the present case , both gonadal agenesis and mullerian agenesis existed in the same patient along with multiple congenital malformations .
overlapping association of short stature , cardiac , renal , and bony anomalies is seen in both gonadal dysgenesis with turner 's phenotype and type 2 mrkh syndromes .
the exact genetic mechanisms that underlie the association of 46,xx gonadal dysgenesis with mrkh syndrome is not known as similar congenital malformations were also seen in cases with 45x0 and mosaic forms of gonadal dysgenesis with coexistent mullerian agenesis .
embryologically , ovaries develop from three sources : mesodermal epithelium , underlying mesenchyme , and primordial germ cells .
the mullerian ducts appear lateral to gonads and develop into fallopian tubes uterus and upper part of vagina and broad ligaments .
shah et al . proposed three possibilities for the coexistence of 46,xx gonadal dysgenesis and mrkh syndrome : mutations or deletions in genes commonly involved in the development and migration of germ cells and mullerian ducts , microdeletions or duplications in x chromosomes resulting in abnormal gene transcription factors interrupting the development of both germ cells and mullerian structures , and endocrine disruptors playing a role .
literature search revealed few such cases with 46,xx gonadal dysgenesis and mrkh syndrome , and the salient clinical features were discussed here for comparison .
levinson et al . described the first case of 46,xx gonadal dysgenesis and mrkh syndrome in a 17-year - old female who presented with short stature , absence of vagina , secondary sexual characters , internal genitalia , and gonads .
reported a case of 46,xx gonadal dysgenesis and mrkh syndrome in which prominent features were normal stature , primary amenorrhea , impuberism with ovarian dysgenesis , absent uterus , and fallopian tubes .
bhandari and chaudhary reported a case from dehradun , and the clinical findings of this case were short stature , impuberism , rudimentary vagina , and absent uterus and ovaries .
described similar cases of 46,xx gonadal dysgenesis and mullerian agenesis without any other congenital malformation in women with normal stature .
recently , dutta and taneja et al . reported a case of a 19-year - old female from new delhi with short stature , normal secondary sexual characters , webbed neck , fusion of cervical vertebrae , scoliosis , atrial septal defect , right renal agenesis with malrotated left kidney , and mullerian agenesis .
reported a case of a 17-year - old female with 46,xx gonadal dysgenesis and mrkh syndrome with torticollis , lumbar scoliosis , prominent nevi on face and trunk , poorly developed secondary sexual characters , ectopic pelvic kidney , and absence of gonads and internal genitalia with a blind vagina .
oyer et al . reported a case of neonate with 46,xx gonadal dysgenesis who presented with diaphragmatic hernia , doomed bicuspid aortic valve , and mullerian derivative defects .
reported a 19-week - old fetus with 46,xx karyotype , normal female external genitalia , complete gonadal agenesis , large encephalocele , spina bifida , and omphalocele .
all the studies mentioned above revealed that 46,xx gonadal dysgenesis with mullerian agenesis commonly presents with normal female phenotype with primary amenorrhea , impuberism , and hypergonadotropic hypogonadism with or without somatic malformations , the latter being extremely rare .
the last two case reports suggest that some of the congenital anomalies may not be compatible with normal survival and this might be the reason for the rarity of these cases .
both 46,xx gonadal dysgenesis and mullerian dysgenesis are heterogeneous disorders with rare coexistence and obscure reason .
it is unclear whether the somatic malformations and ovarian failure are caused by ostensibly pleiotropic gene(s ) or closely linked genes . in the present case ,
a clinical diagnosis of turner 's syndrome was made initially as patient presented with short stature , primary amenorrhea , and impuberism , and endocrine evaluation revealed hypergonadotropic hypogonadism .
later , mri scan images confirmed mullerian agenesis and other congenital malformations , and karyotyping has shown 46,xx complement .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . | 46,xx gonadal dysgenesis is a rare genetically heterogeneous disorder characterized by underdeveloped ovaries with consequent , impuberism , primary amenorrhea , and hypergonadotropic hypogonadism .
mullerian agenesis or mayer - rokitansky - kuster - hauser ( mrkh ) syndrome is characterized by congenital aplasia of the uterus and the upper part ( 2/3 ) of the vagina in a woman with normal development of secondary sexual characteristics and a normal 46,xx karyotype .
the phenotypic manifestations of mrkh syndrome may sometimes overlap with various other syndromes and require accurate delineation .
the coexistence of both these disorders is extremely rare . here
, we report a case of 46,xx gonadal dysgenesis and mrkh syndrome with anatomically dispersed congenital anomalies unique among reported cases . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSIONS
Declaration of patient consent
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Conflicts of interest |
clay - shoveler 's fracture is isolated spinous process avulsion fracture classically observed in the lower cervical or upper thoracic spine .
this type of fracture is most common in those who engaged in hard physical labor3,6,7 ) .
recently , several cases of isolated spinous process fractures that are caused by motor vehicle accidents or sports injuries have been reported ( table 1 ) .
isolated spinous process fractures are most common at the level of t1 , followed by c7 , t2 , t3 , and c61,7 ) . among them , multiple spinous process fractures are very rare1 ) .
we present a case of multiple isolated spinous process fractures with twelve contiguous levels of the cervico - thoracic spine , which extended from c4 to t8 . to the best of our knowledge ,
this case is the most multiple spinous process fractures so far reported in the literatures .
a 34-year - old man was transferred our hospital from local clinic after motorcycle accident .
initial spine x - rays showed multiple spinous process fractures in the cervico - thoracic spine . computed tomography
( ct ) scanning of the cervicothoracic spine showed twelve contiguous spinous process fractures ranged from c4 to t8 ( fig .
cervico - thoracic spine magnetic resonance images ( mri ) revealed that spinal cord was normal and there was no additional soft tissue injury ( fig .
after 3 months , we evaluated follow - up cervico - thoracic ct scanning which did n't show union of spinous process fractures yet .
after 6 months follow - up , visual analogue scale score was gradually improved from 6 to 1 .
clay - shoveler 's fracture may occur through direct trauma on the flexed spine or through shear forces2,3,4,5,6,7 ) .
muscle and ligamentous stress is transmitted through the supra - spinous ligaments and results in an avulsion fracture of the spinous processes2 ) .
our case is an unusual example for multiple spinous process fracture that can be produced by several mechanisms , such as a direct blow to the posterior aspect of lower neck and the thorax , upper back hyperextension , and hyperflexion injuries against the supraspinous ligament after motorcycle crash .
a double spinous process sign at the affected level can be detected on simple antero - posterior radiographs6,9 ) .
downward displacement of fractured spinous process fragments ( spinolaminar breach sign ) can be seen on lateral radiograph views3,6,7 ) .
isolated spinous process fracture considered as a warning sign for more severe spinal injury , so it should be evaluated carefully to detect more severe spinal injuries7 ) .
ct and mri of the cervical and thoracic spine may be useful for detecting more serious spine fractures and further injuries to surrounding structures including spinal cord and ligament3,6 ) .
generally , this type of fractures are treated conservatively without the need for surgical intervention3,5,8 ) .
immobilization and restriction of physical activity for 4 to 6 weeks generally result in good outcome3,8 ) .
our patient shows good clinical outcome after 4 weeks immobilization and restriction of physical activity .
we report a case of twelve isolated spinous process fracture of the cervico - thoracic spine after motorcycle accident . | the clay - shoveler 's fracture is an isolated avulsion fracture of the lower cervical or upper thoracic spinous process . among them , multiple spinous process fractures are very rare .
we present 34-year - old male patient who have multiple spinous process fractures with twelve contiguous levels of cervico - thoracic spine ( from c4 to t8 ) after motorcycle accident .
this case is multiple isolated spinous process fractures with good clinical outcome . | INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION |
during june august 2010 , a total of 152 fecal samples were collected from the rectums of piglets ( age 13 months ; weight 624 kg ) , fattening pigs ( age 34 months ; weight 2550 kg ) , and sows ( age 12 years ; weight 180250 kg ) .
the pigs were raised in 6 farrow - to - finish farms , 2 fattening farms , and 1 weaner indoor farm of central italy ( 7 farms in the umbria region and 2 farms in the marche region ) .
fecal samples from 21 pig farmers were collected from 5 of the 9 farms , 17 of which were available for molecular analysis .
microscopic diagnosis of d. fragilis was based on visualization of pleomorphic trophozoites , ranging in size from 4 m to 20 m , with fragmented chromatin and pale gray - blue finely vacuolated cytoplasm after giemsa staining ( figure 1 ) .
dna was extracted directly from 200 mg of feces by using the qiaamp dna stool minikit ( qiagen , hilden , germany ) .
reference d. fragilis dna of genotype 1 ( strains 379 and 1085 ) was used as a positive control .
dientamoeba fragilis trophozoites in a smear of pig feces after giemsa staining , italy , 20102011 .
a taqman real - time pcr that targets the 5.8 s ribosomal locus was performed in a lightcycler 480 apparatus ( roche diagnostics gmbh , mannheim , germany ) as described ( 11 ) . for the 18s rrna gene , a published assay ( 12 )
was used to amplify a 662-bp fragment , followed by amplification of a 366-bp fragment with newly designed primers df322for ( 5-gagaaggcgcctgagagata-3 ) and df687rev ( 5-ttcatactgcgctaaatcatt-3 ) . for the internal transcribed spacer 1 ( its1 ) region ,
the forward primer ssu2 ( 13 ) and the reverse primer df - itsrev ( 5-gcgggtcttcctatataaacaagaacc-3 ) were used , whereas the forward primer df - itsnesfor ( 5-atacgtccctgccctttgta-3 ) and the reverse primer df - itsnesrev ( 5-gcaatgtgcattcaaagatcgaac-3 ) were used in the nested pcr .
the sequences were assembled by using seqman ii ( dnastar , madison , wi , usa ) and compared with those available in public databases by using blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) .
the microscopic examination showed that 52 of the 74 piglets , 11 of the 14 fattening pigs , and 8 of the 64 sows were positive for d. fragilis ( table 1 ) .
more trophozoites were observed in fecal samples from piglets , suggesting a higher susceptibility of young animals to infection ( data not shown ) .
( in 42% of pigs ) , endolimax nana protozoa ( 32% ) , iodoamoeba buetschli protozoa ( 25% ) , and other flagellates ( 4.5% ) . of the 21 samples from pig farmers ,
4 from farmers working on 2 farms were positive for d. fragilis by microscopy ( table 1 ) .
molecular techniques were applied to 38 pig fecal samples , specifically 24 samples positive by microscopy from 6 farms and 14 samples negative by microscopy from 2 farms , and to all 17 human fecal samples .
a comparison of human and pig samples collected from the same farm was possible for farms 2 , 3 , and 5 ( table 2 ) . using real - time pcr ,
all 24 positive pig samples were amplified , with cycle threshold values ranging from 30 to 34 , whereas none of the 14 negative samples were positive to this assay ( table 2 ) . however , because no inhibition controls were run , false - negative results can not be ruled out .
of the 17 human fecal samples , 13 were positive with cycle threshold values of 2940 .
the sequence analysis of 15 amplified products ( 11 from pigs and 4 from humans ) showed 100% homology with d. fragilis genotype 1 ( table 2 ) .
genotype 2 was not found in any of the samples from pigs or humans . *
# 100% identity to dq223442 , dq223450 , dq223452 , dq223454 , dq223456 , and dq167586 .
next , a 366-bp fragment of the 18s rrna gene was analyzed . in this fragment
, genotypes 1 and 2 can be distinguished by 8 substitutions or insertions or deletions ( figure 2 ) , which were further confirmed by sequencing the entire 18s rrna gene from 2 reference isolates and 2 human isolates from this study .
amplification was obtained from 6 of the 24 positive pig samples and from 8 of the 17 human samples .
one human isolate ( h7 ) showed a single nucleotide substitution in the fragment sequenced ( figure 2 ) .
sequences from 3 microscopically negative pig samples ( all from farm 1 ) had a high homology ( 96% ) with trichomitus batrachorum , a flagellate of reptiles , although the sequence could originate from t. rotunda , a flagellate of pigs that has not been described at the molecular level .
multiple alignment of the 366-bp fragment of the 18s rrna gene from dientamoeba fragilis genotypes 1 and 2 .
amplification was obtained from 11 of the 24 pig samples ( table 2 ) , but only 2 sequences could be clearly identified as d. fragilis .
four sequences showed homology ( 80% ) with flagellates from different vertebrate classes whereas the remaining 5 sequences were excluded because of insufficient quality .
the 2 d. fragilis sequences from pigs showed 100% homology with sequences from human isolates from the united kingdom ( table 2 ) , further supporting the presence of genotype 1 in these 2 hosts .
a direct comparison of its1 sequences from humans and pigs from a single farm in italy was not possible because d. fragilis was amplified from only 2 human samples from 2 farms from which no pig samples were available .
the analysis of its1 from the 2 human isolates showed full identity to human isolates from the netherlands and the united kingdom ( table 2 ) .
considering the size of the world s pig population ( > 1 billion ) , the close contact between pigs and humans in many parts of the world , and the difficulties in the proper management of pig fecal waste , the role of these animals as reservoirs of zoonotic pathogens must be carefully evaluated .
we demonstrated that pigs are hosts of d. fragilis , on the basis of molecular analysis of 3 fragments in the ribosomal cluster .
sequence analyses of fragments of the 18s and 5.8s rrna genes showed genotype 1 in isolates collected in the same farm from humans and pigs , suggesting the potential for zoonotic transmission of this parasite .
if a transmissible cyst stage exists , then environmental contamination with pig feces should be considered a key factor in the transmission of this parasite .
pigs also are a fascinating animal model to elucidate the life cycle of this elusive parasite . | the world is home to more than 1 billion pigs , which produce large quantities of feces . we know that some organisms in pig feces can cause human disease , and now we might have another to add to the list .
little is known about where the common intestinal parasite dientamoeba fragilis comes from and how it is spread .
however , recent molecular analysis confirmed that the organism found in pigs is indeed the same as the one found in humans .
therefore , pigs ( or their feces ) might be a source of this parasitic infection in humans . | The Study
Conclusions |
a 3 year old girl initially presented at an outside institution with bruising on her left shoulder at 3 months of age . she was diagnosed with an infantile hemangioma , which did not respond to subsequent standard treatment consisting of six laser therapy treatments and 8 months of propranolol .
continued enlargement of the lesion involving her left shoulder , with worsening pain , prompted presentation to our institution for further evaluation .
magnetic resonance imaging ( mri ) was performed to evaluate for an underlying vascular malformation ( vm ) .
this showed multiple small foci of t2 hyperintensity isolated to the dermis , with no underlying vm present ( figure 1 ) .
the mri results led to a dermatology consultation and subsequent skin biopsy , with pathology showing kaposiform hemangioendothelioma .
follow - up consultation 3 years later noted decreased size of the left shoulder lesion with resolution of pain . a 26 year old male runner with long - standing anterior right knee pain for the preceding 7 years was initially evaluated in a sports medicine clinic and diagnosed with patellofemoral pain syndrome and mild lateral patellar maltracking .
an mri , performed to assess cartilaginous structures of the right knee , showed a partially intra - articular venous vm along the medial aspect of the right knee joint ( figure 2a and b ) with no arterial involvement .
the patient s pain reached a point where he could hardly get out of bed to put clothes on , or walk to the freezer to get ice to put on the knee .
sclero - therapy was offered and subsequently performed during three serial sessions . during the first sclerotherapy ( figure 2c ) , 10 cc of sodium tetradecyl sulfate 3% ( sotradecol ; angiodynamics , inc . ,
latham , ny , usa ) was mixed , using foam technique , and injected into the lesion , avoiding a component involving the weight - bearing articular surface to avoid potential development of secondary osteoarthritis .
foam technique involves mixing the solution back and forth between two syringes attached to a three - way stopcock .
the second sclerotherapy , 5 months later , utilized 10 cc of sotradecol distributed amongst three separate areas of the vm . a follow - up mri
a third sclerotherapy was performed 4 months after the second for residual pain , using 2 cc of sotradecol with immediate stasis of venous flow after injection . at a 3-month follow - up appointment following the third sclerotherapy ,
the patient noted entering a military officer training program and being able to run 3.2 km ( 2 miles ) twice weekly , pain - free .
a 34 year old man was initially diagnosed with a left forearm vm at 14 years of age .
multiple courses of treatment including surgical resection and endovascular sclerotherapy ensued , following a typical complex recurrent and indolent course for a vm .
he presented to our institution with constant pain in his left forearm , requiring scheduled narcotic pain medication .
an mri was performed , which showed a low - flow vm with a clear nidus in the interosseous membrane region of the distal forearm ( figure 3a , and b ) .
a pre - sclerotherapy diagnostic ultrasound of the region depicted suitable percutaneous access of the vm .
subsequent nidus - targeted treatment with endovascular sclerotherapy ( figure 3c ) using ultrasound for access , markedly improved symptoms after three serial treatments . during the first sclerotherapy
finger compression was used to occlude the deep communicating perforator veins to induce stasis in the vm , which was treated with 4 cc of sotradecol . during the second sclerotherapy
5 months later , the existing vm was much smaller than during the prior treatment .
again , the communicating perforator veins were manually occluded and sclerotherapy using 4 cc of sotradecol was performed .
a third sclerotherapy a few months later utilized 4 cc of sotradecol . following the sclerotherapy treatments ,
a follow - up mri depicted substantially decreased size of the vm ( figure 3d ) . also , the patient noted he no longer required scheduled narcotic pain medication . a 27-year - old woman , diagnosed at age 6 with a right hand vm , presented to our institution for further management with increased discomfort , and concern for risk of rebleeding given newly increased distention of her right hand veins . she had four different operations in the past to control a significant spontaneous bleeding episode , with minimal to no improvement in pain symptoms .
a compression garment had been tried only intermittently in the past , with some relief .
a time - resolved magnetic resonance angiography ( mra ) was performed to further characterize the vm , which depicted a high - flow vm ( figure 4 ) with arterial supply from superficial palmar arch branches of both the ulnar and radial artery .
however , given the substantial risk of collateral damage to adjacent nerves , skin , and muscle and potential loss of limb function , the patient elected for more consistent use of a compression garment prior to more invasive management .
a 3 year old girl initially presented at an outside institution with bruising on her left shoulder at 3 months of age . she was diagnosed with an infantile hemangioma , which did not respond to subsequent standard treatment consisting of six laser therapy treatments and 8 months of propranolol .
continued enlargement of the lesion involving her left shoulder , with worsening pain , prompted presentation to our institution for further evaluation .
magnetic resonance imaging ( mri ) was performed to evaluate for an underlying vascular malformation ( vm ) .
this showed multiple small foci of t2 hyperintensity isolated to the dermis , with no underlying vm present ( figure 1 ) .
the mri results led to a dermatology consultation and subsequent skin biopsy , with pathology showing kaposiform hemangioendothelioma .
follow - up consultation 3 years later noted decreased size of the left shoulder lesion with resolution of pain .
a 26 year old male runner with long - standing anterior right knee pain for the preceding 7 years was initially evaluated in a sports medicine clinic and diagnosed with patellofemoral pain syndrome and mild lateral patellar maltracking .
an mri , performed to assess cartilaginous structures of the right knee , showed a partially intra - articular venous vm along the medial aspect of the right knee joint ( figure 2a and b ) with no arterial involvement .
the patient s pain reached a point where he could hardly get out of bed to put clothes on , or walk to the freezer to get ice to put on the knee .
sclero - therapy was offered and subsequently performed during three serial sessions . during the first sclerotherapy ( figure 2c ) , 10 cc of sodium tetradecyl sulfate 3% ( sotradecol ; angiodynamics , inc . ,
latham , ny , usa ) was mixed , using foam technique , and injected into the lesion , avoiding a component involving the weight - bearing articular surface to avoid potential development of secondary osteoarthritis .
foam technique involves mixing the solution back and forth between two syringes attached to a three - way stopcock .
the second sclerotherapy , 5 months later , utilized 10 cc of sotradecol distributed amongst three separate areas of the vm . a follow - up mri after the second sclerotherapy depicted reduced size of the vm ( figure 2d ) .
a third sclerotherapy was performed 4 months after the second for residual pain , using 2 cc of sotradecol with immediate stasis of venous flow after injection . at a 3-month follow - up appointment following the third sclerotherapy ,
the patient noted entering a military officer training program and being able to run 3.2 km ( 2 miles ) twice weekly , pain - free .
a 34 year old man was initially diagnosed with a left forearm vm at 14 years of age .
multiple courses of treatment including surgical resection and endovascular sclerotherapy ensued , following a typical complex recurrent and indolent course for a vm .
he presented to our institution with constant pain in his left forearm , requiring scheduled narcotic pain medication .
an mri was performed , which showed a low - flow vm with a clear nidus in the interosseous membrane region of the distal forearm ( figure 3a , and b ) .
a pre - sclerotherapy diagnostic ultrasound of the region depicted suitable percutaneous access of the vm .
subsequent nidus - targeted treatment with endovascular sclerotherapy ( figure 3c ) using ultrasound for access , markedly improved symptoms after three serial treatments . during the first sclerotherapy
finger compression was used to occlude the deep communicating perforator veins to induce stasis in the vm , which was treated with 4 cc of sotradecol . during the second sclerotherapy 5 months later , the existing vm was much smaller than during the prior treatment .
again , the communicating perforator veins were manually occluded and sclerotherapy using 4 cc of sotradecol was performed .
a third sclerotherapy a few months later utilized 4 cc of sotradecol . following the sclerotherapy treatments ,
a follow - up mri depicted substantially decreased size of the vm ( figure 3d ) . also , the patient noted he no longer required scheduled narcotic pain medication .
a 27-year - old woman , diagnosed at age 6 with a right hand vm , presented to our institution for further management with increased discomfort , and concern for risk of rebleeding given newly increased distention of her right hand veins .
she had four different operations in the past to control a significant spontaneous bleeding episode , with minimal to no improvement in pain symptoms .
a compression garment had been tried only intermittently in the past , with some relief .
a time - resolved magnetic resonance angiography ( mra ) was performed to further characterize the vm , which depicted a high - flow vm ( figure 4 ) with arterial supply from superficial palmar arch branches of both the ulnar and radial artery .
however , given the substantial risk of collateral damage to adjacent nerves , skin , and muscle and potential loss of limb function , the patient elected for more consistent use of a compression garment prior to more invasive management .
vms are present at birth and grow as the patient does , demonstrating a normal rate of endothelial turnover .
they may not become apparent until adulthood , once they enlarge enough to be visualized or produce symptoms .
histologically , they encompass a spectrum of lesions that include arterial , capillary , venous , and lymphatic components in various combinations , with the majority consisting of venous - predominant low - flow malformations .
management of vms requires a multidisciplinary approach due to their prolonged and often progressive clinical course .
this typically involves multiple physicians with expertise in internal medicine , pediatric cardiology , dermatology , surgery , psychiatry , and interventional radiology , in order to correctly classify vms and guide long - term treatment .
treatment is mainly guided by patient symptoms and classification of the vm based on low - flow versus high - flow .
additional considerations include patient age , life expectancy , comorbid conditions and progression of symptoms .
severe symptoms such as disabling pain , swelling , functional impairment , ulceration , or severe bleeding often require more invasive treatment , which is typically endovascular treatment with embolization or sclerotherapy versus surgical resection . both treatment approaches complement each other , and are generally palliative rather than curative , requiring much dedication .
low - flow vms , which represent about 90% of vms , can be successfully treated using endovascular sclerotherapy alone .
this involves percutaneous access to the lesion and direct injection of a sclerosing agent , causing precipitation of endothelium proteins via denaturation and dehydration , and ultimately thrombosis and endothelial destruction . maintaining
therefore , one must not obstruct all major venous pathways of the affected extremity with sclerotherapy .
less common high - flow vms , defined as such by containing an arterial component , often require more aggressive treatment secondary to shunting from high - output failure states , which is associated with increased morbidity and mortality .
high - flow vms are typically treated first with transarterial embolization , which involves occlusion of the vascular inflow and/or outflow for primary treatment alone , or as an adjunctive treatment , prior to subsequent sclerotherapy or surgery .
determining pertinent vm characteristics , including classification of high - flow or low - flow , is important prior to intervention .
noninvasive imaging , particularly mri , plays an important role in the characterization of vms .
classification into low - flow or high - flow dynamics provides a useful way to assist the interventional radiologist in planning treatment .
whether dealing with low - flow or high - flow vms , it is important to identify the nidus , which represents the abnormal focus of tortuous vessels at the center of the vm .
there may be multiple nidi in a single lesion . by targeting the nidus during treatment ,
the most direct pathways of blood flow into the malformation are sclerosed and a longer duration of effect is observed .
occasionally , an intervention can stimulate vascular endothelial cells and paradoxically worsen symptoms . therefore targeting treatment to the nidus and thoughtfully limiting damage to adjacent structures
pretreatment imaging also helps to identify the nidus for targeting treatment ( figure 3 ) .
images can depict vm extent and associated involvement of muscle , bone , joints , or subcutaneous tissue that may predispose to complications ( figure 2 ) .
though clinically apparent , the extent of a vm is often underestimated by physical exam alone .
one study revealed that in greater than 75% of cases there was more extensive involvement demonstrated on mri compared to the physical exam and nearly half of those cases resulted in a change in management .
mri also guides therapy further by characterizing vascular inflow and outflow ; determining the number of feeding vessels avoids potential problems with undertreatment of a single vessel when multiple vessels contribute .
while mri is the mainstay of diagnostic imaging assessment of vms , additional imaging modalities may be helpful .
mri techniques have the benefit of excellent soft tissue characterization and lack of ionizing radiation in patients who are often young and require serial follow - up .
risks of mri include safety concerns related to the strong magnetic field , as well as the risk of nephrogenic systemic fibrosis with the use of intravenous gadolinium in patients with chronic kidney disease .
ultrasound provides real - time visualization during intervention for vascular access and real - time arterial and venous flow velocities .
ultrasound is limited by inability to assess deep lesions or those adjacent to bone or air .
computed tomography ( ct ) and ct angiography provides detail using contrast enhancement and allows assessment of calcification or thrombus , in addition to providing complementary imaging information when mr does not provide a clear answer .
ct is limited by exposure to ionizing radiation , and depicts less blood flow information when compared to mr .
conventional angiography provides the concomitant treatment possibility , while it is limited by its invasive nature , projection format , and exposure to ionizing radiation . at our institution , we begin our vm mri exams with fat suppressed t2-weighted sequences in the axial plane to document vm location for subsequent time - resolved contrast - enhanced images .
then , we inject a gadolinium - based contrast material , usually the blood pool agent gadofosveset trisodium ( ablavar ; lantheus medical imaging , north billerica , ma , usa ) and acquire time - resolved contrast - enhanced mra images . in the extremities , we typically use specialized receiver coils and the cartesian acquisition with projection - like reconstruction ( capr ) technique , which allows creation of both time - resolved three - dimensional ( 3d ) datasets and time - of - arrival maps that depict flow information on a single image ( figures 3b and 4c ) .
capr is a 3d gradient echo sequence that uses elliptical centric sampling , two - dimensional ( 2d ) homodyne reconstruction , view - sharing , and 2d sensitivity - encoding .
clinical advantages of capr include images with both high temporal resolution that allows visualization and clear distinction of arterial and venous phases and high spatial resolution ( 1 mm isotropic ) that allows sharp depiction of small branching vessels .
time - of - arrival maps allow a depiction of the vm on a single image , which patients generally find helpful for understanding subsequent treatment planning and management .
time - resolved mra using capr technique has been shown to demonstrate flow characteristics to guide treatment .
other time - resolved techniques can be used as well , with or without view - sharing .
we typically use non - view - shared , multiphase , breath - hold , 2d accelerated mra when imaging the abdomen or pelvis and view - shared techniques , such as capr , in the extremities .
finally , delayed post - contrast t1-weighted fat - suppressed high - resolution gradient echo images are acquired in the blood pool phase .
these are useful to document the extent of prior ablation or treatment , assess for venous thrombus or outflow obstruction , and further assess soft tissue extent .
our studies were performed on a 3.0-t ge scanner platform system ( ge healthcare , waukesha , wi , usa ) .
this included mri depicting lack of an actual underlying vm leading to skin biopsy , and the correct diagnosis and treatment ( figure 1 ) ; mri depicting the full extent of the underlying vm in relation to sensitive structures , therefore limiting sclerotherapy to safe regions and avoiding possible secondary osteoarthritis ( figure 2 ) ; mri depicting a clear single nidus of the vm , which was targeted for subsequent sclerotherapy ( figure 3 ) ; and mri confirming the high - flow nature of a vm ( figure 4 ) , with endovascular embolization and sclerotherapy offered , but ultimately managed conservatively with compression garment given the clinical setting .
these are only a subset of the advantages pretreatment imaging can provide for these complex vascular lesions .
additional benefits have been described previously , and include determining the focal versus diffuse nature of a vm which may therefore require multiple treatment sessions ; fully characterizing both the superficial and deep venous system drainage of an extremity to ensure adequate residual drainage following treatment , particularly in patients with klippel
trnaunay syndrome with abnormal underlying venous systems ; and defining the vm connection with the deep venous system to determine the risk of deep venous thrombosis following sclerotherapy .
peripheral vms are complex vascular lesions that consist of a variety of combinations of arterial , capillary , venous , and/or lymphatic vessels .
pretreatment imaging , primarily with mri , provides valuable information prior to intervention in order to make the proper diagnosis , determine lesion extent , identify the nidus , and classify flow dynamics into low - flow or high - flow to guide subsequent treatment .
in particular , imaging with mri and time - resolved mra provides both high spatial and temporal resolution allowing for precise lesion characterization and treatment planning . | peripheral vascular malformations ( vms ) are complex and diverse vascular lesions which require individualized pretreatment planning .
pretreatment imaging using various modalities , especially magnetic resonance imaging and time - resolved magnetic resonance angiography , is a valuable tool for classifying peripheral vms to allow proper diagnosis , demonstrate complete extent , identify the nidus , and distinguish between low - flow and high - flow dynamics that determines the treatment approach .
we discuss pretreatment imaging findings in four patients with peripheral vms and how diagnostic imaging helped guide management . | Case reports
Case 1
Case 2
Case 3
Case 4
Discussion
Conclusion |
dama means a rejection of the permission previously given by the patient to allow health services providers to take care of him ( 3 ) .
dama is accounted for one percent of total discharges in canada and varies from 0.8% to 2.2% in the us ( 5 , 7 ) . besides ,
in studies conducted in iran , various rates of dama have been reported ( 810 ) .
dama is regarded as a risk factor for the recurrence of disease , readmission and higher costs for patient ( 1113 ) .
incurred costs due to dama in five years were estimated to be nearly 3 billion dollars ( 14 ) .
patient dissatisfaction with the medical team , personal or family problems , patient s perception of feeling of wellbeing , dissatisfaction with treatment provided , low economic status , improper equipment , the lack of medical and nursing care can be mentioned as the most significant causes for dama ( 1416 ) . through investigating the rate and causes contributing to dama in hospitals ,
their weaknesses can be identified , and accordingly planning should be done to resolve problems and decrease the rate of dama .
such attempts may play an important role not only in promoting the health of population , but also in reducing additional costs and increasing hospital revenue .
this study , therefore , aimed to estimate the rate of and causes contributing to dama in iranian hospitals through conducting a systematic review and meta - analysis of the results of published studies .
a systematic review and meta - analysis study was conducted in 2014 and a search strategy was developed by an experienced librarian .
required data were collected through searching for key words included : dama , leaving against medical advice , causes * , hospital and their persian equivalents using databases including pubmed , ovid , google scholar , embase , scopus , magiran , scientific information database ( sid ) , google search engine , and also gray literature . to increase the confidence in the selection of articles , the reference lists of the articles , certain relevant journals and web sites in this field also were searched .
published articles from january 2000 to december 2014 indicating the rate of dama were included .
the inclusion criteria included : articles in english and persian language ; studies conducted in iranian hospitals , articles reporting the rate of dama .
the following articles were excluded ; letters to editor , presentations in conferences and case reports . to assess the quality of the retrieved articles , two authors evaluated the articles according to the checklist of strengthening the reporting of observational studies in epidemiology ( strobe ) . in the first phase ,
articles non - relevant to the subject of this study were excluded . in the second phase ,
computer software for reference management ( endnote x5 ) was used for organizing and recognizing the duplication articles .
after excluding non - relevant articles and duplicates among different databases as well as matching the exclusion criteria , 17 articles were incorporated into the study ( fig .
the extracted data were summarized in previously designed extraction tables included : author and year , city , sample and sample size , dama prevalence ( % ) , dama - male gender ( % ) , dama cause ( % ) . literature review and retrieval flow diagram to estimate the rate of dama ,
a quantitative meta - analysis method was adopted using computer software ( cma : 2comprehensive meta - analysis ) . in order to report the results , forest plots with 95% confidence interval and
funnel plot were used to report the rate of dama and examine the publication bias , respectively . according to the q statistics indicating heterogeneity of results in different studies , the random effect model was used to perform meta - analyses . to compare the rate of dama between female and male we conducted t - test by using spss version 16 software ( chicago , il , usa ) .
the contributing causes of dama were extracted through the method of manual content - analysis .
this method is used to detect , categorize and report themes from text and is very popular in analyzing qualitative data ( 17 ) .
two researchers coded data by through the following process : data immersion ( getting accustomed to data context ) , identifying and extracting primary codes , themes detection ( assigning the extracted primary codes to the related themes ) , reviewing and completing the detected themes , naming and defining themes , the reliability assurance of extracted codes and themes ( two encoders agreed on removing disputed cases through discussion ) .
a systematic review and meta - analysis study was conducted in 2014 and a search strategy was developed by an experienced librarian .
required data were collected through searching for key words included : dama , leaving against medical advice , causes * , hospital and their persian equivalents using databases including pubmed , ovid , google scholar , embase , scopus , magiran , scientific information database ( sid ) , google search engine , and also gray literature . to increase the confidence in the selection of articles , the reference lists of the articles , certain relevant journals and web sites in this field also were searched .
published articles from january 2000 to december 2014 indicating the rate of dama were included .
the inclusion criteria included : articles in english and persian language ; studies conducted in iranian hospitals , articles reporting the rate of dama .
the following articles were excluded ; letters to editor , presentations in conferences and case reports . to assess the quality of the retrieved articles , two authors evaluated the articles according to the checklist of strengthening the reporting of observational studies in epidemiology ( strobe ) . in the first phase ,
articles non - relevant to the subject of this study were excluded . in the second phase ,
computer software for reference management ( endnote x5 ) was used for organizing and recognizing the duplication articles .
after excluding non - relevant articles and duplicates among different databases as well as matching the exclusion criteria , 17 articles were incorporated into the study ( fig .
the extracted data were summarized in previously designed extraction tables included : author and year , city , sample and sample size , dama prevalence ( % ) , dama - male gender ( % ) , dama cause ( % ) . literature review and retrieval flow diagram
a systematic review and meta - analysis study was conducted in 2014 and a search strategy was developed by an experienced librarian .
required data were collected through searching for key words included : dama , leaving against medical advice , causes * , hospital and their persian equivalents using databases including pubmed , ovid , google scholar , embase , scopus , magiran , scientific information database ( sid ) , google search engine , and also gray literature . to increase the confidence in the selection of articles , the reference lists of the articles , certain relevant journals and web sites in this field also were searched .
published articles from january 2000 to december 2014 indicating the rate of dama were included .
the inclusion criteria included : articles in english and persian language ; studies conducted in iranian hospitals , articles reporting the rate of dama .
the following articles were excluded ; letters to editor , presentations in conferences and case reports .
to assess the quality of the retrieved articles , two authors evaluated the articles according to the checklist of strengthening the reporting of observational studies in epidemiology ( strobe ) .
in the first phase , articles non - relevant to the subject of this study were excluded . in the second phase ,
computer software for reference management ( endnote x5 ) was used for organizing and recognizing the duplication articles .
after excluding non - relevant articles and duplicates among different databases as well as matching the exclusion criteria , 17 articles were incorporated into the study ( fig .
1 ) . the extracted data were summarized in previously designed extraction tables included : author and year , city , sample and sample size , dama prevalence ( % ) , dama - male gender ( % ) , dama cause ( % ) .
to estimate the rate of dama , a quantitative meta - analysis method was adopted using computer software ( cma : 2comprehensive meta - analysis ) . in order to report the results , forest plots with 95% confidence interval and funnel plot
were used to report the rate of dama and examine the publication bias , respectively . according to the q statistics indicating heterogeneity of results in different studies , the random effect model was used to perform meta - analyses . to compare the rate of dama between female and male we conducted t - test by using spss version 16 software ( chicago , il , usa ) .
the contributing causes of dama were extracted through the method of manual content - analysis .
this method is used to detect , categorize and report themes from text and is very popular in analyzing qualitative data ( 17 ) .
two researchers coded data by through the following process : data immersion ( getting accustomed to data context ) , identifying and extracting primary codes , themes detection ( assigning the extracted primary codes to the related themes ) , reviewing and completing the detected themes , naming and defining themes , the reliability assurance of extracted codes and themes ( two encoders agreed on removing disputed cases through discussion ) .
to estimate the rate of dama , a quantitative meta - analysis method was adopted using computer software ( cma : 2comprehensive meta - analysis ) . in order to report the results , forest plots with 95% confidence interval and funnel plot
were used to report the rate of dama and examine the publication bias , respectively . according to the q statistics indicating heterogeneity of results in different studies , the random effect model was used to perform meta - analyses . to compare the rate of dama between female and male we conducted t - test by using spss version 16 software ( chicago , il , usa ) .
the contributing causes of dama were extracted through the method of manual content - analysis .
this method is used to detect , categorize and report themes from text and is very popular in analyzing qualitative data ( 17 ) .
two researchers coded data by through the following process : data immersion ( getting accustomed to data context ) , identifying and extracting primary codes , themes detection ( assigning the extracted primary codes to the related themes ) , reviewing and completing the detected themes , naming and defining themes , the reliability assurance of extracted codes and themes ( two encoders agreed on removing disputed cases through discussion ) .
out of 913 retrieved articles by the initial search , 17 articles were incorporated into the study ( 8 , 1832).the information from the studied articles has been provided in table 1 . in general , there were 244858 individuals studied in the articles .
the articles were published from 2010 to 2014 . as it is observed in table 1 ,
the studies related to the rate measurement of discharge against medical advice have been conducted only in seven cities of iran especially in tehran with the most frequency .
the lowest rate of discharge against medical advice was reported by a study with the participation of 4835 inpatient children in sari ( 2.2% ) .
the highest rate of discharge against medical advice was belonged to a study conducted on 1171 patient form the department of physiotherapy ( 34.4% ) .
the mean score of discharge against medical advice was higher in men compared with women ( 42.36.5 versus 57.66.5 ) .
the statistical test showed that the difference in the rate of discharge against medical advice between two gender is statistically significant ( p<0.05 ) .
out of the 17 reviewed articles , 3 articles have investigated dama among patients in departments of pediatrics , 2 articles in departments of psychiatry , 4 articles in emergency departments , 7 articles in inpatients wards ( whole hospital ) and 1 article in department of cardiology .
patient- related reasons and hospital condition are two main them of dama cause the rates of dama in terms of these groups are provided in table 2 . while the highest rate of dama is associated with patients in departments of psychiatry ,
discharge against medical advice rate in iranian hospital according to study groups in general , using a random effects model , the rate of dama in iranian hospitals was estimated at 7.9% ( fig .
( dama=7.9% [ 6.3% 9.8% ] , df = 16 i2= 99.5 , q= 3613.9 )
. the rate of discharge against medical advice in iranian hospitals using a random effects model in the current study , a funnel plot was used to examine the publication bias in reviewed studies ( fig .
the results of this figure show that there is the possibility of risk of publication bias .
the funnel plot to examine the possibility of the publication bias using content analysis , causes contributing of dama were categorized into two main categories : causes related to patient and his / her family and hospital - related causes .
the most important causes in each category , in terms of their priority , are presented in fig .
shows , among causes related to patient and his / her family , patient s perception of feeling of wellbeing ( 29% ) , financial problems ( 21% ) and family problems ( 16% ) were considered as the most important causes , whereas among hospital related causes .
the most important patient and family related causes contributing to discharge against medical advice the most important hospital related causes contributing to discharge against medical advice as fig . 5 shows , among hospital related causes lack of attention from physicians and nurses ( 31% ) , inappropriate behavior by hospital team ( 19% ) and absences timely physician ( 14% ) were regarded as the most important causes in dama .
the results of the current study showed that the rate of dama in iranian hospitals is 7.9% .
the most important causes contributing to dama included patient s perception of feeling of wellbeing , financial problems , the lack of attention from physicians and nurses as well as inappropriate behavior by hospital team .
the rate of dama in iranian hospitals is too high in comparison with many studies conducted in developed countries ( 11 , 3337 ) .
one of the possible reasons for this issue can be high quality services in hospitals of developed countries . as the results of the review of studies conducted in iran showed , hospital related causes ( such as the lack of attention from physicians and nurses , inappropriate behavior by hospital team , improper equipment and environment ) are the most important reasons for dama .
so , paying more attention to the improvement of the quality of services provided to patients in iranian hospitals and in particular , special focusing on the hospital related causes mentioned in this study , are necessary . according to the results ,
the rate of dama among patients in departments of psychiatry has been higher than those of other patients and such a higher rate has been also reported in previous studies ( 3842 ) . given the nature of the disease of this group of patients which extends average length of stay in hospital , these patients tend more to dama due to the perception of feeling of wellbeing , boring hospital environment and financial problems . another possible reason may stem from cultural issues , because mental illness is considered as a social stigma and an unpleasant phenomenon from social and family perspectives in iran .
most patients and their families , therefore , tend to early discharge from hospital , so that their relatives and others close to them do not become aware of the hospitalization .
it is , therefore , important to pay more attention to and focus on this group of patients . besides , the rate of dama among children was less than other patients .
the results of some studies also indicated the low rate of dama in departments of pediatrics ( 2 , 4 , 43 ) .
main reasons for this issue may be the lack of decision - making authority of children and parents worries for their children s health .
therefore , they try to solve financial problems anyway and the treatment of their children will be continued until full recovery is made and patient can be discharged based on physicians orders . moreover ,
the rate of dama among children is less because they do not work and are less affected by family problems .
the rate of dama in emergency departments in iranian hospitals was estimated at 11.8% . in comparison with results of most studies conducted in emergency departments ,
one of the main reasons for this issue may be overcrowding in emergency departments in iran , because all 4 studies conducted in emergency departments in iran were done in reference hospitals which are usually overcrowded .
thus increasing the space , facilities and human resources working in these departments can be effective in reducing the rate of dama .
the results showed that dama was in men more than women , and this result had been shown in previous studies ( 4648 ) .
the reason of low rate of discharge in women than men could be because of their more cautious and fear .
it may be due to men job and their responsibility to make a living which forces them to go back home sooner , also ; their early recovery feeling in compared with women due to their physiology and masculine nature . in this study , the main reasons for dama were categorized into two main categories : causes related to patient and his / her family and hospital - related causes . in general , patient s perception of feeling of wellbeing , financial problems , the lack of attention from physicians and nurses as well as inappropriate behavior by hospital team were considered as four main causes contributing to dama . in this regard , results of a study conducted by alibis and rami also pointed to dissatisfaction with treatment trends , financial and family problems , patient s perception of feeling of wellbeing , preferring other hospitals , dissatisfaction with hospital environment , inadequate treatment and being a teaching hospital ( 49 ) . in many studies conducted in other countries , the relationships between causes such as insurance status , socioeconomic status , patient history as well as other similar causes and dama were also investigated ( 5053 ) .
however , unfortunately many of these causes have not been paid enough attention by researchers in iran .
different studies throughout the world have addressed impacts of dama such as costs , readmissions , deaths and morbidities ( 6 , 12 , 5462 ) . such issues regarding patients discharged from iranian hospitals have not been addressed in studies which may be primarily due to the poor information system in iranian hospitals .
given the importance of the issue , the investigation of results and impacts of dama in iranian hospitals is considered as an inevitable necessity .
time is another issue related to studies on dama conducted in iran , so that all studies have conducted from 2010 and in our search results , no study was found which had been published before that time .
the introduction of clinical governance in iran can be considered as a possible reason for this issue , because during the indicated time interval which most studies have been conducted , clinical governance model has been implemented in iranian hospitals ( 63 ) . through implementing this model with special emphasis on dama ,
if this assumption is true , then we can argue that implementing models similar to the clinical governance can be effective in this area . fortunately ,
in recent year , iranian hospitals accreditation standards include great attention to the issue of dama which requires careful attention in order to successfully implement it and achieve excellent results in this field . despite the efforts of authors ,
the lack of access to some theses and hospital reports on dama can be mentioned as a limitation of this study .
the rate of dama in iranian hospitals in comparison with those of other countries is relatively high ; thus effective initiatives in this area are required .
in addition , patient s perception of feeling of wellbeing , financial problems , the lack of attention from physicians and nurses as well as inappropriate behavior by hospital team were mentioned as main reasons for dama .
these causes can be given high priority in plans for quality improvement and reducing dama .
furthermore , given the limited studies conducted in iran and the lack of investigation into some variables contributing to dama and also impacts of dama in iranian hospitals , more research is needed in this area .
all ethical issues including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . have been completely observed by the author . | background : discharge against medical advice ( dama ) is a problem for hospitals which may result in increasing readmissions , morbidities , inabilities , deaths and health care costs .
this study , aimed to investigate the rate and causes of dama in iranian hospitals.methods:a systematic review and meta - analysis study was conducted in 2014 .
required data were collected through searching for key words included :
discharge against medical advice , leaving against medical advice
, causes * , hospital and their persian equivalents , over databases including pubmed , ovid , google scholar , embase , scopus , magiran , scientific information database ( sid ) .
the reference lists of the articles , certain relevant journals and web sites in this field were also searched.results:out of 913 articles initially retrieved , finally 17 articles were incorporated into the study .
there were 244858 individuals studied in the articles . using a random effects model , the rate of dama in iranian hospitals
was estimated at 7.9% ( 6.3%9.8% ) .
while the highest rate of dama was associated with patients in departments of psychiatry ( 12% ) , the lowest rate was related to patients in departments of pediatrics ( 3.7 ) .
dama was in men more than women ( p<0.05 ) patient s perception of feeling of wellbeing , financial problems , family problems , the lack of attention from physicians and nurses , inappropriate behavior with patients by hospital team and the lack of timely care were mentioned as main causes for dama.conclusion:the rate of dama in iranian hospitals is relatively high .
thus effective initiatives in this area are required . | Introduction
Methods
None
Search strategy
Including and excluding criteria
Quality Assessment
Data Extraction
Data analysis
Causes
Results
Discussion
Conclusion
Ethical Considerations |
there are controversies surrounding the role of diuretic renal scans when deciding on conservative therapy or surgery in children with ureteropelvic junction obstruction ( upjo ) .
it remains difficult to choose an optimal time for surgery as a result of the high variability in renal function , the degree of obstruction , the extent of damage , and the potential of regeneration in a growing kidney .
in addition , the relative paucity of collagen in the neonatal renal pelvis helps to alleviate the effect of high obstructing pressure .
however , an unrecognized obstruction may result in renal damage and renal failure . to date
however , the value of this investigation in children has been questioned , because of its inherently high false - positive and false - negative rates .
specifically , false - negative results are clinically important because they can result in missed optimal surgical opportunities .
more reliable assessment tools are therefore required to aid in decision making regarding the optimal surgical time .
we retrospectively compared a conventional differential renal function ( drf ) measurement with a new drf measurement that assesses the renal parenchymal areas from imaging studies in children who underwent pyeloplasty due to unilateral upjo .
from september 2001 to january 2008 , 29 children underwent pyeloplasty due to unilateral upjo , and tc - diethylenetriaminepentaacetic acid ( tc - dtpa ) renal scans and other imaging studies , such as magnetic resonance imaging ( mri ) , computed tomography ( ct ) , and renal ultrasonography , were performed .
diuretic renal scans were performed by using the standardized tc - dtpa protocol , as a result of a discussion between the society for fetal urology ( sfu ) and the pediatric nuclear medicine council . on the morning of the study ,
oral fluids were encouraged , followed by intravenous administration of 15 ml / kg of a 0.9% sodium chloride solution 30 min preceding the scan . a renal scan using tc - dtpa
the dosage administered was scaled for body weight and was based on an adult dose of 600 mbq .
intravenous furosemide ( 1 mg / kg ) was given when maximum pelvicaliceal distention was observed .
we proposed a novel method to calculate the renal parenchymal area and make correlations with drf as measured by renal scans .
all imaging studies such as mri , ct , and renal ultrasonography were viewed on the picture archiving & communications system ( pacs ) , and all areas of measurement were conducted with the electronic drawing tool provided by the pacs radiographic software . a dedicated urologist conducted all the measurements of the renal parenchymal areas ( unit areas ) .
the unit areas of both kidneys were measured by manual tracing of the renal system excluding the extrarenal - pelvic area ( region of interest ) on the pacs workstation ( fig .
1 ) . in order to obtain reliable results , we rechecked the images three times in a magnified view ( 2 ) , and an average was taken for each set of results .
we applied the equation , drf per unit area of upjo ( or normal contralateral kidney ; nck)=drf of upjo ( or nck)/ renal parenchymal area of upjo ( or nck ) .
modified drf of upjo was calculated by using the equation , modified drf of upjo ( or nck)=drf per unit area of upjo ( or nck)100/(drf per unit area of upjo+drf per unit area of nck ) ( fig .
the data were further analyzed with calculations using the mcnemar chi - square test and generalized estimation equation for comparison of the modified drf group and the conventional drf group .
the statistical analyses were performed by using spss ( version 12.0 ; spss inc , chicago , il , usa ) computer software .
we reviewed the diuretic renal scan results of 29 pediatric patients ( 26 males and 3 females ) who underwent pyeloplasty due to unilateral upjo ( 23 left kidneys and 6 right kidneys ) .
the mean patient age was 42.652.6 months ( range , 3 - 198 months ) .
indications for pyeloplasty were recurrent urinary tract infection or flank pain ( 11 children ) , drf of less than 40% on the affected side with severe hydronephrosis ( 8 children ) , progressive dilatation on a serial ultrasound ( 7 children ) , and a greater than 10% decrease in drf on a serial renal scan ( 3 children ) ( table 1 ) .
the mean cross - sectional areas of the upjo kidney and of the normal contralateral kidney were 62.129.2 cm and 41.322.5 cm , respectively ( p<0.01 ) .
the conventional and modified drf of the upjo kidney were 45.29.2% and 35.29.5% , respectively ( p<0.01 ) .
children were divided into 2 groups on the basis of the results of the initial drf : group i ( n=8 ) had drf less than 40% , and group ii ( n=21 ) had drf greater than 40% .
thirteen children ( 62% ) who initially belonged to group ii ( n=21 ) were reclassified into group i by the modified drf measurement . in group ii , 7 of 11 children ( 63.6% ) whose modified drf value was less than 40% had recurrent urinary tract infection or flank pain .
a total of 6 of 7 children ( 86% ) showed progressive dilatation on the serial ultrasound ( table 2 ) .
the modified drf measurement demonstrated higher accuracy than the conventional method in drf assessment , with respect to signs and symptoms , reduction in renal function , and hydronephrosis .
the false - negative rates of conventional drf and modified drf were 72.4% and 27.6% , respectively .
in the pediatric population , congenital urinary tract obstruction is the most common fetal anomaly identified in prenatal screening of pregnant women .
koff proposed that ureteral obstruction be defined as a functional or anatomical obstruction of urine flow from the renal pelvis to the ureter that results in renal damage or manifests as clinical symptoms such as recurrent urinary tract infection and flank pain when left untreated .
it is well known that the glomerular filtration rate ( gfr ) is lower in newborns than in older children , and the gfr increases several times during the initial 6 months of life . in this period
, untreated obstruction can lead to early renal atrophy and permanent loss of renal function [ 6 - 8 ] .
diuretic renal scans have become a popular method for differentiating between obstructive and nonobstructive hydronephrosis .
however , the value of this investigation in children has been questioned as a result of the inaccurate results it entails . to obtain maximum benefits from diuretic renal scans ,
factors such as adequate hydration and diuretic use are crucial in overcoming the reservoir or ' mixing chamber ' effect , which may stimulate obstruction in dilated but otherwise unobstructed systems .
adequate hydration must be ensured , and there must be sufficient residual renal function to enable diuretic response in order to define the distensibility and volume of the collecting system .
urinary bladder volume and drainage can also affect the response pattern and the clinician 's ability to interpret lower ureteric drainage , which explains the use of bladder catheter drainage during the study .
nam and lee emphasized that the factors that help to determine true obstruction , such as renogram curves , diuretic half - lives , serial renal imaging scans , and drfs , should be taken into account when determining the optimal surgical time in children with upjo .
it remains unclear whether this supranormal function of the obstructed kidney reflects a true increase or merely a measurement error . the relatively high incidence ( 9% to 21% ) of this paradoxical function
is clinically important because management of hydronephrosis with supranormal function has not been clearly established to date . in our study ,
supranormal function ( 55% or greater ) was present in 4 patients ( 13.8% ) .
ham et al hypothesized that supranormal drf may occur as a result of increased renal blood flow caused by altered renal hemodynamics .
consequently , there are pressing clinical needs for a more reliable test to assess the appropriateness of surgical intervention in children with upjo . to our knowledge ,
the correlation between differential parenchymal areas on imaging studies and drf reported on renal scans has not been reported previously .
feder et al suggested that renal parenchymal areas measured by ct strongly correlate with the results of the renal scans .
the overall averaged difference in calculating differential function by ct versus that of renal scan was only 4.73% . according to these results , measurement of drf in kidneys with a significant size difference
we propose a new methodology : drf on the renal scan is proportional to the renal parenchymal area on imaging studies , and drf per unit area is more accurate .
in addition , kidney dimensions can be easily measured on imaging studies and the treating clinician can rapidly assess the degree and site of obstruction . modified drf was significantly different from conventional drf .
we reviewed 29 children with upjo who underwent pyeloplasty , and as intraoperative findings demonstrate the most reliable diagnostic results , we suggest that there are no methodological problems comparing the false - negative results of conventional drf with that of modified drf : the false - negative rates of conventional drf and of modified drf were 72.4% and 27.6% , respectively . furthermore ,
86% of children with progressive dilatation on the serial ultrasound demonstrated drf of less than 40% on modified drf in group ii .
modified drf measurement according to cross - sectional area showed higher diagnostic accuracy , and it may be considered a valuable method for deciding on pyeloplasty in equivocal circumstances .
early in the debate , a number of authors advocated early intervention to preserve renal function .
there is a risk of deteriorating renal function in the future despite eventual spontaneous improvement or resolution of hydronephrosis .
in addition , there is a possibility of refining our diagnostic armamentarium to detect renal decompensation at a reversible stage before the kidney becomes permanently damaged .
however , until now , evidence that suggests surgery will improve renal function or at least prevent further renal damage is lacking . increasingly , observation has been recommended for most infants , as many appear to do well without aggressive surgical intervention , and the current trend in the treatment of patients with unilateral upjo is nonoperative care .
koff and campbell initially observed and subsequently performed surgery in patients with renal function and drf deterioration .
they reported a study in which 104 neonates with unilateral upjo were managed conservatively and followed up for over 5 years .
only 7% of children required pyeloplasty due to drf deterioration . however , relief of obstruction is more suitable in the following conditions : drf of less than 40% or functional reduction at follow - up , recurrent urinary tract infection despite prophylactic antibiotics treatment , or a strong likelihood of recurrent urinary tract infection regardless of the drf value .
surgery may help to prevent renal parenchymal infection and irreversible renal damage [ 21 - 24 ] .
moreover , the procedure should not be delayed when indicated , because the surgical risks of pyeloplasty in infants are not as high as those of ureteral re - implantation .
our indications for pyeloplasty were recurrent urinary tract infection or flank pain ( 11 children ) , drf of less than 40% on the affected side ( 8 children ) , progressive dilatation of hydronephrosis ( 7 children ) , and a greater than 10% decrease in drf on serial renal scans ( 3 children ) .
this study was limited by the fact that it was performed retrospectively , and the data were analyzed in selected children who underwent pyeloplasty due to unilateral upjo . as a consequence , we were not able to analyze false - positive results and specificity . furthermore , measurements of the unit area were not made with a single imaging tool and therefore measurement error was possible .
finally , our study had a small sample size of 29 children ; therefore , additional confirmatory studies are required in the near future .
currently , drf is one of the most important parameters applied to determine the optimal time for surgical intervention for upjo in children .
however , the value of this investigation in children has been questioned because of its high false - positive and false - negative rates .
our modified drf measurement exhibited a lower false - negative rate and may become a valuable method for deciding on pyeloplasty in children with upjo in equivocal circumstances . | purposediuretic 99mtc - diethylenetriaminepentaacetic acid ( tc - dtpa ) renal scans may show false - negative or false - positive results in children with ureteropelvic junction obstruction ( upjo ) .
we evaluated whether modified differential renal function ( drf ) revised by the renal cross - sectional area on imaging study may be a more valuable predictor than conventional drf on a renal scan for deciding on a proper interventional time.materials and methodsbetween
september 2001 and january 2008 , we reviewed the diuretic renal scan results of 29 pediatric patients who underwent pyeloplasty due to unilateral upjo .
diuretic renal scans using the standard 99mtc - dtpa protocol and imaging studies for renal unit measurement area were done .
conventional drf measurement and modified calculation of drf per unit area were done .
conventional drf was classified into group i ( below 40% ) and group ii ( above 40%).resultsthe mean age of all patients was 42.652.6 months ( range , 3 - 198 months ) .
the mean cross - sectional areas of the upjo kidney and of the normal contralateral kidney were 62.129.2 cm2 and 41.322.5 cm2 , respectively ( p<0.01 ) .
the conventional and modified drf of the upjo kidney were 45.29.2% and 35.29.5% , respectively ( p<0.01 ) .
thirteen children ( 62% ) in group ii ( n=21 ) were classified in group i by the modified drf measurement.conclusionsthe modified drf measurement calculated according to cross - sectional area showed fewer false - negative results and may be a valuable method for deciding on pyeloplasty under equivocal circumstances . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS |
resin composites are the most commonly used aesthetic restorative materials due to their universal usage , resemblance to tooth structure in color and mechanical properties , requiring minimal removal of tooth structure and ease of chair side use . today , the common problem of aesthetic dentistry is discoloration and color mismatch of tooth - colored restorations after consumption of chromogens .
the color change of composite resins depends on intrinsic and extrinsic factors ; intrinsic factors involve changes in the filler , matrix and matrix / filler interface ; extrinsic factors involve absorption or adsorption of dyes from external sources , such as coffee , tea , nicotine and mouthrinses like chlorhexidine . in the recent years , prescription of mouthrinses for the control of caries and periodontal disease has become more common . in mentally retarded cases or after periodontal surgeries , plaque control with mechanical methods is not properly possible ; thus , chemical methods like the use of chlorhexidine mouthrinse as an antiseptic solution are required . in comparison to common antibiotic therapies ,
chlorhexidine is a well - known biguanide compound with effective anti - plaque action and chromogenic potential causing brown staining of teeth , tongue , silicate and resin restorations .
various staining mechanisms have been described for chlorhexidine including degradation of chlorhexidine to release parachloraniline , non - enzymatic browning reactions ( the maillard reactions ) , protein denaturation by chlorhexidine with metal sulfide formation and precipitation of anionic dietary chromogens by cationic antiseptics .
lamba et al , reported that immersion of composite , glass ionomer cement and compomer in mouthrinses can significantly change the color of these materials .
celik et al , investigated the effect of three chlorhexidine mouthrinses on composites and reported that all composites showed acceptable discoloration .
in the recent years , considerable improvements have occurred in formulation of composite materials , mainly by use of nanotechnology .
nanofilled composites are new materials containing inorganic nanofillers scattered in the resin matrix and have improved mechanical strength , optical properties , wear resistance , elasticity modulus and color stability .
microhybrid composites are dominant in dental markets , thus , in this study , we compared the color stability of new efficient composites with each other .
instrumental techniques decrease subjective interpretation as compared with the visual methods . in comparison of these two methods , spectrophotometer is a more accurate method to detect color changes of dental restorations .
a spectrophotometer uses cie system , which is based on human perception and was developed by the commission intrnationale de leclairage in 1978 .
color change is shown with e in this system , which is total color change based on l * , a * and b * values .
the l * value refers to lightness and a * and b * values refer to chromaticity coordinates .
positive a * values show a shift towards red and negative values show a shift towards green ; positive b * values show a shift towards yellow and negative values show a shift towards blue ; when e is less than or equal to 1 unit , it is not visible to the human eye ; when e is between 1 and 3 units , the color change is visually perceptible to the experienced examiner ; when e is greater than or equal to 3.3 units , the color change is clinically unacceptable .
discoloration of aesthetic restorations is problematic , wasting time and cost for replacement of restorations due to dissatisfaction of patients .
but prescription of chlorhexidine mouthrinses for a minimum of two weeks after periodontal surgeries is a common practice , which may cause discoloration of aesthetic composite restorations of patients .
the aim of this study was to evaluate the effects of chlorhexidine mouthrinses available in the iranian market on color stability of microhybrid and nanofilled composites .
in this in - vitro study , two composite resins and seven types of chlorhexidine mouthrinses were used .
artificial saliva ( hypozalix spray , biocodex , france ) was used as the control group . a ph meter ( labtron , tehran ,
the details of materials used in this study are presented in tables 1 and 2 .
mouthrinses used in this study composites used in this study ( shade : a2 ) bisphenol a - glycidyl methacrylate bisphenol a ethoxylated dimethacrylate urethane dimethacrylate triethylene glycol dimethacrylate polyethylene glycol dimethacrylate one - hundred and sixty disc - shaped specimens , 80 from each composite , were made in prefabricated celluloid molds with dimensions of 7x2 mm .
composites were placed in molds and compressed with a 1 mm thick glass slab for 30 seconds to obtain a uniformly smooth surface .
each specimen was light - cured with valo light curing unit ( ultradent , south jordan , usa ) with a light intensity of 800 mw / cm for 20 seconds on both sides . a radiometer ( kerr , demetron , orange , ca , usa )
the bottoms of specimens were coded from one to 10 to facilitate the color measurement of each specimen .
the upper surface of each specimen was ground with 600 , 800 , 1000 , and 2000-grit silicon carbide papers ( matador , cologne , germany ) successively under running water .
all specimens were placed at the bottom of a container in such a way that they were completely dipped in the solution . after polishing ,
the specimens were immersed in artificial saliva at 37c for 24 hours in an incubator in a closed container , allowing post - polymerization .
each specimen was then blotted dry with a filter paper and then subjected to color measurement by placing the probe tip perpendicular to the specimen surface .
the initial color values ( l * , a * , b * ) were measured using vita easy shade advance ( vita zahnfabrik , bad sckingen , germany ) against a white background before insertion of samples in the solutions .
the spectrophotometer was calibrated after color measurement of every three samples by placing the probe tip against the calibration block .
the specimens were immersed in mouthrinses once a day for two minutes ( n=10 ) , which was equivalent to two rinses with mouthwash per day for one minute .
after two minutes of immersion in the mouthrinses , the specimens were immersed in artificial saliva in an incubator at 37c .
this process was repeated for two weeks to simulate the use of chlorhexidine mouthrinses for two weeks , which is commonly prescribed after dental treatments like periodontal surgeries .
after one and two weeks of immersion , color measurements were repeated with the same spectrophotometer , and these measurements were made under the same conditions and in the same manner described for initial color measurement .
the total color change ( e * ) was calculated using the following formula :
e=(l*)2+(a*)2+(b*)22 the collected data were statistically analyzed with spss version 22 using two - way anova to evaluate the interaction effects of the material type and mouthrinse on color change .
one - way anova , tukey 's post hoc test , t - test and paired t - test were used to analyze data at a significance level of 0.05 .
one - hundred and sixty disc - shaped specimens , 80 from each composite , were made in prefabricated celluloid molds with dimensions of 7x2 mm .
composites were placed in molds and compressed with a 1 mm thick glass slab for 30 seconds to obtain a uniformly smooth surface .
each specimen was light - cured with valo light curing unit ( ultradent , south jordan , usa ) with a light intensity of 800 mw / cm for 20 seconds on both sides . a radiometer ( kerr , demetron , orange , ca , usa )
the bottoms of specimens were coded from one to 10 to facilitate the color measurement of each specimen .
the upper surface of each specimen was ground with 600 , 800 , 1000 , and 2000-grit silicon carbide papers ( matador , cologne , germany ) successively under running water .
all specimens were placed at the bottom of a container in such a way that they were completely dipped in the solution . after polishing
, the specimens were immersed in artificial saliva at 37c for 24 hours in an incubator in a closed container , allowing post - polymerization .
each specimen was then blotted dry with a filter paper and then subjected to color measurement by placing the probe tip perpendicular to the specimen surface .
the initial color values ( l * , a * , b * ) were measured using vita easy shade advance ( vita zahnfabrik , bad sckingen , germany ) against a white background before insertion of samples in the solutions . the spectrophotometer was calibrated after color measurement of every three samples by placing the probe tip against the calibration block .
the specimens were immersed in mouthrinses once a day for two minutes ( n=10 ) , which was equivalent to two rinses with mouthwash per day for one minute .
after two minutes of immersion in the mouthrinses , the specimens were immersed in artificial saliva in an incubator at 37c .
this process was repeated for two weeks to simulate the use of chlorhexidine mouthrinses for two weeks , which is commonly prescribed after dental treatments like periodontal surgeries .
after one and two weeks of immersion , color measurements were repeated with the same spectrophotometer , and these measurements were made under the same conditions and in the same manner described for initial color measurement .
the total color change ( e * ) was calculated using the following formula :
e=(l*)2+(a*)2+(b*)22 the collected data were statistically analyzed with spss version 22 using two - way anova to evaluate the interaction effects of the material type and mouthrinse on color change .
one - way anova , tukey 's post hoc test , t - test and paired t - test were used to analyze data at a significance level of 0.05 .
two - way anova revealed that the interaction effects of composite type and mouthrinses on color change were statistically significant ( p=0.049 ) .
the mean values and standard deviations of l , a and b color parameters for each group of composite resin after seven and 14 days of immersion in chlorhexidine mouthrinses are shown in table 3 .
the mean l , a and b values and standard deviation of filtek z250 compared to filtek z350xt in different solutions after seven and 14 days the mean values and standard deviations of color change ( e ) for each group of composite resin after seven days of immersion in chlorhexidine mouthrinses are shown in table 4 .
the mean e values and standard deviation ( sd ) of filtek z250 compared to filtek z350xt in different solutions after seven and 14 days statistically significant difference between the two time points in each group and composite at significance level of 0.05 .
-the similar superscripted uppercase letters in the same column show no significant difference between groups at each time point ( p>0.05 ) -the similar superscripted lowercase letters in the same row show no significant difference between two composites at each time point ( p>0.05 ) column chart showing the mean e17 values and standard deviation of filtek z250 compared to filtek z350xt in different solutions filtek z250 in vi - one mouthrinse showed maximum discoloration ( e=3.051.49 ) after seven days , which had a statistically significant difference with the control group ( p=0.004 ) but it did not show significant differences with other groups .
filtek z350xt in behsa mouthrinse showed maximum discoloration ( e=1.740.7 ) after seven days , which had a statistically significant difference with the control ( p=0.000 ) , najo ( p=0.004 ) and epimax ( p=0.014 ) mouthrinses .
the e values after 14 days revealed that all groups had acceptable color change ( e<3.3 ; fig .
the mean values and standard deviations of color change ( e ) of each group of composite resin after 14 days of immersion in chlorhexidine mouthrinses are shown in table 4 .
column chart showing the mean e114 values and standard deviation of filtek z250 compared to filtek z350xt in different solutions filtek z250 in najo mouthrinse ( e=2.651.46 ) and after that in vi - one ( e=2.432.23 ) showed maximum discoloration ; although it did not have significant differences with other groups ( p>0.05 ) .
filtek z350 xt in behsa showed maximum discoloration ( e=2.340.78 ) , which had significant differences with other groups ( p<0.05 ) .
the e values in filtek z350xt after 14 days in comparison to seven days showed higher discoloration in all groups and the discoloration was significant in all groups except for chlorhexidine and najo .
the e values in filtek z250 after 14 days in comparison to seven days showed higher discoloration in epimax , hexodine , najo and behsa groups and lower discoloration in other groups , and the discoloration was only significant in the control and epimax groups .
the present study evaluated the effects of seven commercially available chlorhexidine mouthrinses in the iranian market on color stability of two types of resin - based composites .
the results revealed that the color change of composites after seven and 14 days of immersion in mouthrinses was clinically acceptable ( e<3.3 ) . with regard to color parameters ( l*a*b * ) , the results showed that l values were negative , which indicated that the specimens became darker in all groups . in a * axis ,
the specimens color shifted towards red in all groups , and in b * axis , the color of microhybrid composite shifted towards yellow and that of nanofilled composite shifted towards blue .
in addition , filtek z250 ( microhybrid ) showed less color change than filtek z350xt ( nanofilled ) .
after the first and second weeks , the color change ( e value ) of filtek z250 with larger filler particles was greater than that of filtek z350xt in all groups .
filtek z250 contains filler particles with an average size of 0.6m ; while filtek z350xt contains filler particles ranging from 5 to 20 nm .
larger particles create a rough surface while polishing and smaller particles create a smooth surface ; thus , composites with larger fillers are more susceptible to discoloration .
in addition , composites with larger filler particles are more susceptible to water sorption by the polymer network and the effect of coloring agents on the quality of the bond between the matrix and filler can cause discoloration .
water sorption may cause micro - cracks by expanding and laminating the resin component and hydrolyzing the silane and decrease the functional life of composite resins ; thus , micro - crack formation and interfacial gaps between the filler and matrix allow soluble dyes of mouthrinses to penetrate and cause discoloration ; these reasons can explain the greater color stability of filtek z350xt .
our findings are in accordance with those of al - qaisi and alrahman and festuccia et al .
after seven days of immersion in mouthrinses , vi - one mouthrinse showed greater discoloration after alcohol - based mouthrinses .
weak acids like citric acid have a softening effect on polymer matrix and decrease the microhardness of composites in addition to alcohol .
kin , epimax , and vi - one mouthrinses have citric acid in their composition but in this study , vi - one showed greater discoloration .
the manufacturers of kin and epimax claim that these mouthrinses have color preservers in their composition that inhibit discoloration of teeth and composites ; therefore , the greater discoloration of vi - one may be due to the absence of color preserver in its formulation . after 14 days of immersion in mouthrinses ,
microhybrid composite in najo with 10% alcohol ( e=2.65 ) and nanofilled composite in behsa with 11.6% alcohol ( e=2.34 ) showed greater discoloration .
alcohol concentration in mouthrinses varies from 0 to 27% , which is comparable to the alcohol percentage of beer ( 4% ) and wine ( 12% ) .
ethanol is a bipolar molecule , which enhances the dissolution of hydrophobic and hydrophilic components .
alcohol in mouthrinses does not have any other therapeutic effect except as a solvent . due to this reason , in the clinical setting ,
alcohol - free mouthrinses have been proven to have the same effect as alcohol - based mouthrinses with fewer side effects .
our findings are similar to those of villalta et al , asmussen , and stober et al , who reported that alcohol had a softening effect on composites , so the chromaticity of composites increases in alcohol - based solutions .
comparison of discoloration between e17 and e114 revealed that filtek z350xt had significantly greater discoloration after 14 days in comparison to seven days in all groups except for two ( chlorhexidine and najo ) ; it means that all specimens showed a significant progressive increase in color change with increasing number of rinses in all chlorhexidine mouthrinses except for two groups , and filtek z250 showed a significant discoloration only in two groups ( control and epimax ) after 14 days in comparison to seven days ; this result may be due to the higher potential of nanofilled composites for discoloration as mentioned above and showed that increasing the frequency and duration of rinses can affect the color stability of nanofilled composites more than microhybrid composites . in this study , despite the low ph of epimax ( ph=4.3 ) , it did not show greater discoloration than other groups .
we did not find a correlation between ph and discoloration of composites , which is in accord with the findings of diab et al , who reported that the ph of test solutions did not have an effect on discoloration of restorative materials . in clinical situations
, different factors may affect the color stability of restorative materials such as the presence of saliva , salivary pellicle , and the effect of different foods and beverages , which are difficult to simulate in an in - vitro setting .
this result may be due to the absence of these chromogenic materials , which could intensify the chromogenic potential of chlorhexidine mouthwashes .
the results of the present study indicated that composites showed an acceptable color change after two weeks of immersion in chlorhexidine mouthrinses .
mouthrinses , which contained alcohol and citric acid showed greater discoloration ; thus , it is better to limit their prescription . | objectives : this study aimed to evaluate the effects of chlorhexidine mouthrinses on color stability of nanofilled and micro - hybrid resin - based composites.materials and methods : in this in - vitro study , 160 disc - shaped specimens ( 7x2 mm ) were fabricated of filtek z250 and filtek z350xt enamel ( a2 shade ) .
the samples of each group were randomly divided into eight subgroups ( n=10 ) .
the specimens were incubated in artificial saliva at 37c for 24 hours .
the baseline color values ( l * , a * , b * ) of each specimen were measured according to cie lab system using a reflection spectrophotometer . after baseline color measurements ,
the control samples were immersed in saliva and the test groups were immersed in kin ( cosmodent ) , vi - one ( rozhin ) , epimax ( emad ) , hexodine ( donyaye behdasht ) , chlorhexidine ( shahrdaru ) , najo ( najo ) and behsa ( behsa ) mouthrinses once a day for two minutes .
the specimens were then immersed again in saliva .
this process was repeated for two weeks .
color measurements were made on days seven and 14 .
two - way and one - way anova and tukey 's post hoc test , t - test and paired t - test were used to analyze data at a significance level of 0.05.results:all specimens displayed color change after immersion in the mouthrinses .
significant interactions were found between the effects of materials and mouthrinses on color change.conclusions:all composite resins tested showed acceptable color change after immersion in different mouthrinses .
filtek z350xt showed less color change than filtek z250 .
mouthrinses containing alcohol ( behsa and najo ) and citric acid ( vi - one ) caused greater discoloration of composites . | INTRODUCTION
MATERIALS AND METHODS
Preparation of samples:
Baseline color measurement:
Immersion of specimens in chlorhexidine mouthrinses:
Second and third color measurements:
RESULTS
DISCUSSION
CONCLUSION |
for many purposes , it is useful to have a list of protein sequences of known structure from entries in the protein data bank ( pdb ) ( 1 ) that fit certain user - defined criteria .
these purposes include statistical analysis of certain structural features , such as side - chain rotamer distributions ( 2 ) , benchmarking sequence alignment and structure prediction methods ( 3 ) , and analysis of a set of proteins , such as those with a certain function ( e.g. dna - binding ) ( 4 ) .
the criteria for limiting the list of sequences can be applied to each chain in a pdb file , to each entry as a whole or to the relationships between protein chains and entries .
chain - specific criteria include function , species , sequence length and whether the chain is presented with only c atoms in the pdb file or all atoms .
entry - specific criteria include the function of a complex , resolution , r - factor , experiment type ( nmr or x - ray ) and other structure quality criteria .
there are a number of servers that provide such lists , including the reprdb ( 5 ) , astral ( 6 ) , uniqueprot ( 7 ) sites and the pdb site itself . each provides a variety of services for specific purposes .
they vary in a number of respects , including how sequence identities are determined and the flexibility and extent of services available to the user . in this paper , we describe extensions to our pisces server that provides some advantages over other such servers , depending on the purposes of the user .
first , we believe in general that local alignments are better than global alignments for the purpose of selecting sequences from the pdb dataset , since many proteins share homology in one domain that may represent only a portion of the complete sequence .
servers that use clustal w ( 8) , which performs global alignments , may underestimate sequence identity for shared domains by aligning over unrelated portions of two sequences .
it is also important that sequence alignments cover the full - length of related regions of each protein pair .
blast [ and to a lesser extent psi - blast ( 9 ) ] may overestimate sequence identity for distantly related proteins by providing incomplete alignments over only the most - conserved regions of the sequence pair . overestimating sequence identities results in lists that are shorter than necessary since
structure - based alignments are likely to provide the most complete and accurate alignments at low - sequence identity than any sequence - based alignment method ( 10 ) . as described below , pisces now uses a combination of structure alignments at low - sequence identity and sequence alignment using psi - blast at high - sequence identity . other criteria than strict sequence identity
second , sequence culling servers may provide services for a variety of purposes . [ the verb to cull can be used in different senses .
the original meaning ( webster 's 1913 ) was to select or gather , as in flowers .
more recently , the word is used in two ways : culling the weakest members of a herd , or culling the herd itself , leaving behind the healthiest animals .
we are generally using it in the second sense , to cull the pdb by removing redundant or low - resolution structures . ] in addition to culling the entire pdb , a user may wish to cull a subset of the pdb .
for instance , a user may have a list of pdb entries that contain antibody structures and may want a subset of these with certain structural quality and mutual sequence identity .
one can also apply the sequence identity by - entry rather than by - chain. we define this as removing a pdb entry ( that may contain more than one chain ) from the list if another entry already in the list has at least one chain that has a sequence identity , higher than the cut - off , to any chain in the proposed entry
. this might be useful if one wants a list of unique complexes in the pdb .
another aspect of such a server is the kind of annotation provided in the results .
each server returns a list of chains and some servers are able to return a list of fasta - formatted sequences . usually , the annotation is simply the chain i d and sometimes what the rcsb provides in its
however , the rcsb does not provide consistent annotation for each chain in each pdb file with multiple chains .
chain b is 2-microglobulin . in the fasta - formatted sequence database from the pdb ,
these two chains are given as :
2hla_a mol : protein length:270 human class i histocompatibility antigen aw2hla_b mol : protein length:99 human class i histocompatibility antigen aw 6
in fact , the lines are unfortunately truncated , but even the annotation from the sequence details resource for the entry 2hla on rcsb 's site has only this for chain b : human class i histocompatibility antigen aw 68.1. there is , however , more information in the full mmcif file , including the name
beta-2-microglobulin as well as the swiss - prot entry accession number from which additional information may be obtained .
2hla_a mol : protein length:270 human class i histocompatibility antigen aw 2hla_b mol : protein length:99 human class i histocompatibility antigen aw 6 our pisces server was developed from a previous version referred to as
culledpdb used blast to determine sequence identities , while pisces has used psi - blast to determine sequence identities ( 11 ) by building a position - specific scoring matrix or profile for each unique sequence in the pdb from a multi - round search of the non - redundant protein sequence database ( 12 ) .
pisces also provides a resource for culling a user - input list of chains or entries from the pdb or from a set of non - pdb sequences provided as either a list of genbank ids or in the fasta format .
we have shown that the use of the psi - blast profiles resulted in much longer lists than while using blast ( 11 ) , while not exhibiting errors associated with using a global alignment algorithm , such as clustal w. we have made several important improvements in pisces that are described in this paper :
we now use the structure alignment program ce ( 13 ) to refine the relationship determination between proteins and recalculate their sequence identities that are found by using psi-blast.pisces now use ce - type sequence identity , i.e. identical pairs divided by all aligned pairs excluding gaps .
psi - blast , in contrast , calculates the sequence identity by the ratio of the number of identical pairs to the full - length of the alignment , including gaps .
this change means that if two closely related proteins are aligned , but one has a large insertion , the sequence identity will remain high .
this may occur for instance if a long disordered loop is engineered out of a protein to facilitate crystallization .
the new definition of sequence identity is a more conservative criterion for sequence culling , since sequence identities are higher than they were previously using the psi - blast values , and more sequences are removed for a target sequence identity.pisces is now able to cull by - entry in addition to by - chain , as described above.pisces now provides more annotation for each chain in the pdb than the pdb itself does .
this annotation includes chain - specific gene names and functional and species information retrieved from the full mmcif files of the pdb , and the swiss - prot and genbank databases.from the re - engineered search engine of the pdb ( now in beta release , ) ( 14 ) , with a single click on the external sitespisces menu on the pdb 's search result page , all the hits or a selected subset of the results of a search can be transmitted to the pisces site for culling using a user 's input criteria for structural quality and sequence identity cut - offs .
we now use the structure alignment program ce ( 13 ) to refine the relationship determination between proteins and recalculate their sequence identities that are found by using psi - blast .
pisces now use ce - type sequence identity , i.e. identical pairs divided by all aligned pairs excluding gaps .
psi - blast , in contrast , calculates the sequence identity by the ratio of the number of identical pairs to the full - length of the alignment , including gaps .
this change means that if two closely related proteins are aligned , but one has a large insertion , the sequence identity will remain high .
this may occur for instance if a long disordered loop is engineered out of a protein to facilitate crystallization .
the new definition of sequence identity is a more conservative criterion for sequence culling , since sequence identities are higher than they were previously using the psi - blast values , and more sequences are removed for a target sequence identity .
pisces is now able to cull by - entry in addition to by - chain , as described above .
pisces now provides more annotation for each chain in the pdb than the pdb itself does .
this annotation includes chain - specific gene names and functional and species information retrieved from the full mmcif files of the pdb , and the swiss - prot and genbank databases . from the re - engineered search engine of the pdb ( now in beta release , ) ( 14 ) , with a single click on the external sitespisces menu on the pdb 's search result page , all the hits or a selected subset of the results of a search can be transmitted to the pisces site for culling using a user 's input criteria for structural quality and sequence identity cut - offs .
pisces uses the mmcif - format files from rcsb to determine sequences , experiment type , resolution , r - factors and other features of pdb entries and chains .
these mmcif files are a result of the uniformity project , an effort by the rcsb to standardize and correct information across all the pdb files ( 15,16 ) .
some missing values for resolution and r - factors are obtained from the pdbfinder database ( 17 ) .
pisces works from a fasta - formatted database of all sequences in the pdb called pdbaa , which is distinct from ncbi 's database of the pdb sequences with the same name ( ) .
our pdbaa database is available to users who want a complete set of sequences in the pdb .
it is also used to provide the sequences and annotations returned by pisces for subsets of the pdb .
pdbaa provides basic information on chain length , experiment type ( x - ray , nmr , etc . ) , resolution , r - factor and free r - factor as appropriate .
these pieces of information are useful if a user wants to use a structure as a template for homology modeling , and wants to choose the best template by searching the whole pdb . in order to integrate as much useful information as possible into the output of pisces
the goal is to have gene names for each sequence , swiss - prot or other database identifiers and species information .
these are obtained from the following sources : the mmcif files themselves , swiss - prot 's index of pdb entries ( pdbtosp.txt , ) , the protein identification resource ( pir ) and ncbi 's non - redundant protein sequence database , in that order to obtain the desired information .
our goal in culling the pdb is to provide the longest list possible of the highest resolution structures that fulfill the sequence identity and structural quality cut - offs . for this purpose , we need to identify potential evolutionary relationships and complete sequence alignment between related regions of each identified pair . historically , 98% of requests to pisces have been for culled lists at 25% identity or higher .
we have determined that psi - blast identifies 99.9% of such relationships with e - value of 1.0 or better ( data not shown ) , and thus we do not use structure alignment to identify evolutionary relationships .
rather , we use the structure alignment program ce ( 13 ) to align structure pairs which psi - blast determines as having a sequence identity of 50% or less or for which the psi - blast alignment covers < 80% of the shorter sequence in each pair .
we have found some cases where structure alignment sequence identities are much lower than those calculated with psi - blast even though the alignment lengths are comparable .
this may occur when one single - domain protein is homologous to each of the two domains in another protein .
ce may align the first protein to either of the two domains of the second protein , but not necessarily the more closely related one . to account for this
, we use the sequence identity obtained by either psi - blast or ce , whichever has the larger number of identically aligned residue pairs . with the sequence identities in hand
, pisces uses the method of hobohm and sander ( 18,19 ) to cull the sequences that pass the chain and entry criteria input by the user .
culling by - chain means treating each chain in each pdb entry as a separate entity .
based on the requests from a number of users of pisces , we have added another functionality to pisces : culling by - entry. for this procedure , the sequence identity between any two entries is defined as the highest sequence identity of any one chain in one entry with any one chain in the other entry . in this way
, no two entries will appear in the same list if they share chains with sequence identity over the cut - off .
pisces further allows the user to choose whether to cull within each entry and allows the user to use another sequence identity cut - off for this culling procedure .
so for instance , if an entry is a homodimer , the user can choose to have both sequences returned ( no culling within entry ) or just one of them ( culling within entry by some value < 100% ) .
pisces first gives the user the option to : ( i ) cull the entire pdb ; ( ii ) cull from a search at the pdb 's re - engineered search engine site this option takes the user to the pdb 's web page ( ) before returning to pisces ; ( iii ) cull from a user - input list of chains or entries ; ( iv ) cull from a list of genbank entries ; and ( v ) cull from a fasta - formatted set of sequences or from blast or psi - blast output . for options ( iv ) and ( v ) , sequence alignments
are obtained by using psi - blast on the list of input sequences to determine sequence identities .
it is assumed that these sequences are not in the pdb and , therefore , structural criteria are not used .
option ( i ) takes a user directly to a page for inputting structural quality criteria ( experiment type , resolution , r - factor , c-status , chain lengths , etc . ) and sequence identity cut - offs . option ( ii )
take a user to the rcsb site or the user may start at the rcsb site directly .
once the rcsb server has returned a list of entries that satisfy search parameters , the user can click on the external sitespisces menu on the rcsb page to return to pisces and to a page displaying the list of hits ( all or selected ) from rcsb .
option ( iii ) takes a user to a page for inputting a list of pdb entries or chains .
after the list of pdb entries or chains to be searched is confirmed by the user for options ( ii ) and ( iii ) , he / she is asked for the structural criteria to be used for culling .
pisces then confirms the input data and asks for user name , institution and email address .
when the results are ready , almost always within minutes , the server sends an email to the user to download the results from a page given in the email .
the results include a list of the input sequences ( if used ) , the input cut - offs , the list of chains or entries that result from culling and a fasta - formatted file of the selected sequences .
in addition to the sequence culling service , pisces also provides databases and programs that may prove useful to the user :
a fully annotated pdbaa database . for each pdb chain ,
the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species.two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries.a standalone package for pisces .
the standalone version of pisces has all the major features of the web - based pisces server . a fully annotated pdbaa database . for each pdb chain ,
the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species .
two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries . a standalone package for pisces .
the standalone version of pisces has all the major features of the web - based pisces server .
pisces uses the mmcif - format files from rcsb to determine sequences , experiment type , resolution , r - factors and other features of pdb entries and chains .
these mmcif files are a result of the uniformity project , an effort by the rcsb to standardize and correct information across all the pdb files ( 15,16 ) .
some missing values for resolution and r - factors are obtained from the pdbfinder database ( 17 ) .
pisces works from a fasta - formatted database of all sequences in the pdb called pdbaa , which is distinct from ncbi 's database of the pdb sequences with the same name ( ) .
our pdbaa database is available to users who want a complete set of sequences in the pdb .
it is also used to provide the sequences and annotations returned by pisces for subsets of the pdb .
pdbaa provides basic information on chain length , experiment type ( x - ray , nmr , etc . ) , resolution , r - factor and free r - factor as appropriate .
these pieces of information are useful if a user wants to use a structure as a template for homology modeling , and wants to choose the best template by searching the whole pdb . in order to integrate as much useful information as possible into the output of pisces
the goal is to have gene names for each sequence , swiss - prot or other database identifiers and species information .
these are obtained from the following sources : the mmcif files themselves , swiss - prot 's index of pdb entries ( pdbtosp.txt , ) , the protein identification resource ( pir ) and ncbi 's non - redundant protein sequence database , in that order to obtain the desired information .
our goal in culling the pdb is to provide the longest list possible of the highest resolution structures that fulfill the sequence identity and structural quality cut - offs . for this purpose
, we need to identify potential evolutionary relationships and complete sequence alignment between related regions of each identified pair .
historically , 98% of requests to pisces have been for culled lists at 25% identity or higher .
we have determined that psi - blast identifies 99.9% of such relationships with e - value of 1.0 or better ( data not shown ) , and thus we do not use structure alignment to identify evolutionary relationships . rather , we use the structure alignment program ce ( 13 ) to align structure pairs which psi - blast determines as having a sequence identity of 50% or less or for which the psi - blast alignment covers < 80% of the shorter sequence in each pair .
we have found some cases where structure alignment sequence identities are much lower than those calculated with psi - blast even though the alignment lengths are comparable .
this may occur when one single - domain protein is homologous to each of the two domains in another protein .
ce may align the first protein to either of the two domains of the second protein , but not necessarily the more closely related one . to account for this
, we use the sequence identity obtained by either psi - blast or ce , whichever has the larger number of identically aligned residue pairs .
with the sequence identities in hand , pisces uses the method of hobohm and sander ( 18,19 ) to cull the sequences that pass the chain and entry criteria input by the user .
culling by - chain means treating each chain in each pdb entry as a separate entity .
based on the requests from a number of users of pisces , we have added another functionality to pisces : culling by - entry. for this procedure , the sequence identity between any two entries is defined as the highest sequence identity of any one chain in one entry with any one chain in the other entry . in this way
, no two entries will appear in the same list if they share chains with sequence identity over the cut - off .
pisces further allows the user to choose whether to cull within each entry and allows the user to use another sequence identity cut - off for this culling procedure .
so for instance , if an entry is a homodimer , the user can choose to have both sequences returned ( no culling within entry ) or just one of them ( culling within entry by some value < 100% ) .
pisces first gives the user the option to : ( i ) cull the entire pdb ; ( ii ) cull from a search at the pdb 's re - engineered search engine site this option takes the user to the pdb 's web page ( ) before returning to pisces ; ( iii ) cull from a user - input list of chains or entries ; ( iv ) cull from a list of genbank entries ; and ( v ) cull from a fasta - formatted set of sequences or from blast or psi - blast output . for options ( iv ) and ( v ) ,
sequence alignments are obtained by using psi - blast on the list of input sequences to determine sequence identities .
it is assumed that these sequences are not in the pdb and , therefore , structural criteria are not used .
option ( i ) takes a user directly to a page for inputting structural quality criteria ( experiment type , resolution , r - factor , c-status , chain lengths , etc . ) and sequence identity cut - offs . option ( ii )
take a user to the rcsb site or the user may start at the rcsb site directly .
once the rcsb server has returned a list of entries that satisfy search parameters , the user can click on the external sitespisces menu on the rcsb page to return to pisces and to a page displaying the list of hits ( all or selected ) from rcsb . option ( iii )
takes a user to a page for inputting a list of pdb entries or chains . after the list of pdb entries or chains to be searched
is confirmed by the user for options ( ii ) and ( iii ) , he / she is asked for the structural criteria to be used for culling .
pisces then confirms the input data and asks for user name , institution and email address .
when the results are ready , almost always within minutes , the server sends an email to the user to download the results from a page given in the email .
the results include a list of the input sequences ( if used ) , the input cut - offs , the list of chains or entries that result from culling and a fasta - formatted file of the selected sequences .
in addition to the sequence culling service , pisces also provides databases and programs that may prove useful to the user :
a fully annotated pdbaa database . for each pdb chain ,
the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species.two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries.a standalone package for pisces .
the standalone version of pisces has all the major features of the web - based pisces server . a fully annotated pdbaa database .
for each pdb chain , the description line includes chain length , resolution and r - value ( for x - ray structures ) , protein descriptions , database name and entry name in the reference database , and species .
two more condensed variants of pdbaa : pdbaa.ent and pdbaa.nr.pdbaa.ent has all redundant chains within entries removed and the removed chain ids are placed at the end of the description line of the representative chains .
pdbaa.nr is similar to pdbaa.ent , but it removes all redundant chains within the whole pdbaa instead of only within entries .
the standalone version of pisces has all the major features of the web - based pisces server .
we investigated the effects of using structure - based alignments on the results of pisces . on the pdb dataset ,
the number of related pairs was : 637 115 according to blast ( e - value better than 1.0 , alignment length > 30 ) and 758 411 according to psi - blast ( e - value better than 1.0 , alignment length > 30 ) . in figure 1 , we have plotted sequence identities and alignment lengths for psi - blast versus blast and ce versus psi - blast .
the bottom two plots show that the psi - blast alignments are generally longer than the blast alignments , and the ce alignments are longer than the psi - blast alignments .
the top two plots show that the sequence identities are lower in psi - blast than in blast and lower in ce than in psi - blast .
as noted earlier , this is due to the fact that incomplete alignments result in artificially higher sequence identities , because the alignment occurs over the most - conserved regions of the two sequences .
we have boxed a region of the top two plots to show pairwise relationships that would be used to cull at 30% sequence identity .
for instance , in the upper right figure these points would be used to cull using psi - blast - determined sequence identities but not ce - determined identities , since the points in the box have psi - blast sequence identity > 30% but ce sequence identity < 30% .
there are far more points in this box than in the corresponding rectangular box for inclusion by psi - blast but not by ce ( to the left and above the green box ) . in table 1
, we show the lengths of lists under various sequence identity cut - offs as determined by using blast , psi - blast and the reprdb server ( 5 ) , which use clustal w and structure alignments .
ce within pisces provides longer lists at low - sequence identity than the sequence - based methods of blast and psi - blast . at very high - sequence identity ,
the results are based primarily on psi - blast calculations and thus are very similar . actually , the difference between ce and psi - blast is not only the list length , but also the content in the lists .
for example , for lists with 25% sequence identity cut - off , the ce list has only 57 more chains than the psi - blast list , but in fact the two lists have 157 different chains that are not in common .
its lists are longer at high - sequence identity , probably because the global alignment algorithm produces alignments over non - homologous regions of sequence pairs , thus lowering the sequence identity compared with a local alignment algorithm .
as shown in introduction , chain - specific information is sometimes missing in the fasta - formatted database available from the rcsb . for 2hla
, our pdbaa provides these annotations :
2hlaa 270 xray 2.60 0.173 na class i histocompatibility antigen ( hla - aw68 ) < sws 1a68_human > [ homo sapiens]2hlab 99 xray 2.60 0.173 na beta 2-microglobulin < sws b2mg_human > [ homo sapiens ]
after the chain identifier is the sequence length , experiment type , resolution , r - factor , free r - factor ( not available in this case ) , the gene name , the database reference in angular brackets ( in this case , swiss - prot entry b2mg_human ) and the species in square brackets .
while this information is available in the mmcif file for 2hla , it is not available in the fasta sequence database provided by the rcsb .
2hlaa 270 xray 2.60 0.173 na class i histocompatibility antigen ( hla - aw68 ) < sws 1a68_human > [ homo sapiens ] 2hlab 99 xray 2.60 0.173 na beta 2-microglobulin < sws b2mg_human
> [ homo sapiens ] finally , we expect the link to pdb 's search capabilities to be very useful . since the establishment of this link , almost 10% of the requests to pisces have come via the pdb search page .
the re - engineered pdb page is only in beta test phase , and so we expect that pisces will find additional utility when rcsb releases the final version of their search site .
scatterplots of sequence identities ( top figures ) and alignment lengths ( bottom figures ) for alignment pairs in common between psi - blast and blast ( left two figures ) and ce and psi - blast ( right two figures ) . on the top two plots , a box is shown that contains points that would be used to eliminate sequences at 30% sequence identity using the method on the x - axis but not by the method on the y - axis .
lengths of lists obtained using different sequence alignment methods criteria for inclusion in the lists : resolution 3.0 ; including c chains ; excluding non - x - ray entries . | pisces is a database server for producing lists of sequences from the protein data bank ( pdb ) using a number of entry- and chain - specific criteria and mutual sequence identity .
our goal in culling the pdb is to provide the longest list possible of the highest resolution structures that fulfill the sequence identity and structural quality cut - offs .
the new pisces server uses a combination of psi - blast and structure - based alignments to determine sequence identities .
structure alignment produces more complete alignments and therefore more accurate sequence identities than psi - blast .
pisces now allows a user to cull the pdb by - entry in addition to the standard culling by individual chains . in this scenario
, a list will contain only entries that do not have a chain that has a sequence identity to any chain in any other entry in the list over the sequence identity cut - off .
pisces also provides fully annotated sequences including gene name and species .
the server allows a user to cull an input list of entries or chains , so that other criteria , such as function , can be used .
results from a search on the re - engineered rcsb 's site for the pdb can be entered into the pisces server by a single click , combining the powerful searching abilities of the pdb with pisces 's utilities for sequence culling .
the server 's data are updated weekly .
the server is available at . | INTRODUCTION
METHODS
Database information and sequence annotation
Sequence identities
Culling by-chain and by-entry
Usage
RESULTS AND DISCUSSION
Figures and Tables |
various surgical techniques have been developed to optimize esthetic outcomes even in operations for malignancy . unlike other parts of the body ,
thus , it is crucial to minimize postoperative scarring in these areas , especially in neck dissection ( nd ) .
robot - assisted and endoscopic - assisted surgeries are typical examples . kang et al.1 first reported robot - assisted modified radical nd in 2010 .
they performed the operation via a transaxillary approach , but difficulties in approach to level i , an important area for oral cavity cancer treatment , were encountered2 . to overcome this limitation , nd via modified face - lift or the retroauricular approach using robotic surgery
robot - assisted nd has many advantages , such as excellent esthetic outcomes and good visualization during surgery , but its high cost is a problem for most patients .
endoscopic - assisted surgery can be an alternative and more cost - effective option compared to robot surgery , as byeon et al.3 recently , we treated two patients who underwent endoscopic - assisted nd via a retroauricular approach and had excellent esthetic outcomes
. herein , we report these cases and discuss the pros and cons of endoscopic nd .
a 63-year - old male patient visited the department of oral and maxillofacial surgery at dental hospital of yonsei university of college of dentistry in korea .
incisional biopsy had been performed by another hospital , which revealed invasive squamous cell carcinoma .
the ulcerative lesion was present on the lingual side of # 36 , # 37 and was 3.02.0 cm in size .
additional diagnostic imaging , including magnetic resonance imaging ( mri ) and positron - emission tomography ( pet ) , were performed to determine extent of surgery .
there was no mandibular or metastatic lymph node invasion , nor were there metastatic lesions on any other organs ( ct2n0m0 , stage ii ) .
thus , wide excision with marginal mandibulectomy , selective neck dissection ( snd ) ( i - iii ) via endoscope , and repair of the soft tissue defect with megaderm ( l&c bio , seongnam , korea ) were performed .
it took almost 5 hours to complete endoscope - assisted snd ( i - iii ) , with 450 cc of blood loss during the operation .
there was mild marginal mandibular branch weakness , which might also have arisen in conventional nd .
a 56-year - old male was referred from a local clinic for evaluation of a tongue mass .
mri and pet demonstrated no regional or distant metastasis ( ct2n0m0 , stage ii ) .
consequently , wide excision with endoscopic nd , reconstruction of the surgical defect with a radial forearm free flap , and repair of the resulting forearm defect with megaderm were performed .
it took almost 3.5 hours to complete endoscope - assisted snd ( i - iii ) , with 200 cc of blood loss during the operation .
the final diagnosis confirmed the clinical staging ( pt2n0m0 , stage ii ) . unlike in case 1 , no postoperative facial nerve weakness was observed .
incision line and anatomical landmarks for endoscpoic neck dissection were outlined on patient 's neck.(fig .
1 ) a retroauricualr incision was used to approach the sternocleidomastoid muscle ( scm ) and a skin flap was created .
the scm muscle was exposed , then we continued subplastysmal dissection inferiorly and anteriorly until we reach the omohyoid muscle , hyoid bone and the midline of the neck . during skin flap elevation ,
a self - retaining retractor ( sejong medical corporation , paju ; sangdosa , seoul , korea ) was used for good visualization of the surgical field .
a nerve stimulator was used to identify the marginal mandibular branch of the facial nerve .
some portions of level ib , iia , and iii were also dissected under direct visualization .
then , a 30-degree endoscope ( 10 mm in diameter , 30 cm in length ; stryker , san jose , ca , usa ) was applied in the level i area followed by fixation with an endoscope holder ( karl storz , tuttlingen , germany).(figs .
2 , 3 ) dissection of the anterior belly area ( level ia ) was performed ; then , level ib , which contains the submandibular gland , was dissected .
harmonic curved shears ( harmonic ace 36p ; johnson & johnson medical , cincinnati , oh , usa ) were used to cauterize minor vessels and dissect tissues .
the distal facial artery and vein were ligated with premium surgiclips ( covidien , dublin , ireland ) ; the submandibular ganglion , wharton 's duct , proximal facial artery and vein were cut after ligation with surgiclips .
we demarcated the level of omohyoid muscle , which is inferior to the border of level iii , and dissected lymphoadipose tissue superiorly from inferior margin using endoscope .
finally , dissection of the carotid artery and internal jugular vein was performed with posterior retraction of the scm .
micro - vascular surgery was performed in case 2 as usual manuals which have been done in conventional neck dissection.(fig .
4 ) donor vessels such as the proximal portion of facial artery , superior thyroid artery , and related veins were observed well under microscope .
. however , an assistant had to retract the skin flap with an army - navy retractor for visualization of the operative field .
a 63-year - old male patient visited the department of oral and maxillofacial surgery at dental hospital of yonsei university of college of dentistry in korea .
incisional biopsy had been performed by another hospital , which revealed invasive squamous cell carcinoma .
the ulcerative lesion was present on the lingual side of # 36 , # 37 and was 3.02.0 cm in size .
additional diagnostic imaging , including magnetic resonance imaging ( mri ) and positron - emission tomography ( pet ) , were performed to determine extent of surgery .
there was no mandibular or metastatic lymph node invasion , nor were there metastatic lesions on any other organs ( ct2n0m0 , stage ii ) .
thus , wide excision with marginal mandibulectomy , selective neck dissection ( snd ) ( i - iii ) via endoscope , and repair of the soft tissue defect with megaderm ( l&c bio , seongnam , korea ) were performed .
it took almost 5 hours to complete endoscope - assisted snd ( i - iii ) , with 450 cc of blood loss during the operation .
there was mild marginal mandibular branch weakness , which might also have arisen in conventional nd .
a 56-year - old male was referred from a local clinic for evaluation of a tongue mass .
mri and pet demonstrated no regional or distant metastasis ( ct2n0m0 , stage ii ) .
consequently , wide excision with endoscopic nd , reconstruction of the surgical defect with a radial forearm free flap , and repair of the resulting forearm defect with megaderm were performed .
it took almost 3.5 hours to complete endoscope - assisted snd ( i - iii ) , with 200 cc of blood loss during the operation .
the final diagnosis confirmed the clinical staging ( pt2n0m0 , stage ii ) . unlike in case 1 ,
incision line and anatomical landmarks for endoscpoic neck dissection were outlined on patient 's neck.(fig .
1 ) a retroauricualr incision was used to approach the sternocleidomastoid muscle ( scm ) and a skin flap was created .
the scm muscle was exposed , then we continued subplastysmal dissection inferiorly and anteriorly until we reach the omohyoid muscle , hyoid bone and the midline of the neck . during skin flap elevation ,
a self - retaining retractor ( sejong medical corporation , paju ; sangdosa , seoul , korea ) was used for good visualization of the surgical field .
a nerve stimulator was used to identify the marginal mandibular branch of the facial nerve .
some portions of level ib , iia , and iii were also dissected under direct visualization .
then , a 30-degree endoscope ( 10 mm in diameter , 30 cm in length ; stryker , san jose , ca , usa ) was applied in the level i area followed by fixation with an endoscope holder ( karl storz , tuttlingen , germany).(figs .
2 , 3 ) dissection of the anterior belly area ( level ia ) was performed ; then , level ib , which contains the submandibular gland , was dissected .
harmonic curved shears ( harmonic ace 36p ; johnson & johnson medical , cincinnati , oh , usa ) were used to cauterize minor vessels and dissect tissues .
the distal facial artery and vein were ligated with premium surgiclips ( covidien , dublin , ireland ) ; the submandibular ganglion , wharton 's duct , proximal facial artery and vein were cut after ligation with surgiclips .
we demarcated the level of omohyoid muscle , which is inferior to the border of level iii , and dissected lymphoadipose tissue superiorly from inferior margin using endoscope .
finally , dissection of the carotid artery and internal jugular vein was performed with posterior retraction of the scm .
micro - vascular surgery was performed in case 2 as usual manuals which have been done in conventional neck dissection.(fig .
4 ) donor vessels such as the proximal portion of facial artery , superior thyroid artery , and related veins were observed well under microscope .
. however , an assistant had to retract the skin flap with an army - navy retractor for visualization of the operative field .
gagner4 introduced endoscopic surgery as an alternative to conventional open neck surgery for parathyroidectomy in 1996 . since then , various trials for endoscopic surgery on the neck , including thyroidectomy5 , submandibular gland excision6 , sentinel node biopsy7 , and nd3 , have been performed .
this technique was developed gradually and is currently used in the treatment of many patients .
although postoperative period is not enough to discuss about results of postoperative scar , it can not be seen easily in the frontal view because the incision line is hidden along the posterior neck.(figs .
5 - 7 ) second , the procedure is minimally invasive and patients typically recover faster .
the incision line is also shorter than that of the conventional method . although more retraction of the skin flap is needed and causes stretching of the flap , this did not lead to any postoperative complications in the present cases .
we found that accurate nd could be obtained by the endoscopic approach . in the conventional method ,
it is difficult to dissect level iib precisely , but the endoscopic approach provides the best field of view on level ii and favorable fields of view on levels ib and iii .
however , this technique may not be appropriate for unskilled operators who have less experience with endoscopy or are unfamiliar with surgical anatomy .
endoscopy provides limited direct visualization compared to the conventional method , so it can be difficult to recognize the exact area where is being operated and control unexpected hemorrhages
first , the overlying skin tends to be thin and punctured easily , since there is no platysma muscle on level ia .
second , it is difficult to dissect the lymphoadipose tissues on level ia precisely compared with the conventional method because of the limited surgical field
. this technique may also not be appropriate for lesions on the floor of the mouth , near the midline , or along the anterior mandible where lymphatic drainage occurs to level ia .
first , they proposed that endoscopic surgery causes inconveniences to the operator and assistant due to limited space .
second , the robotic system has wrist articulated movement , which enables an unlimited operation field in a narrow working space compared to the endoscope .
third , the robotic system provides more assistance with its arm . however , its high cost is a distinct disadvantage of robotic surgery3,8 .
according to our experience , most patients diagnosed with oral cavity cancer are elderly , and are more concerned about cost than neck scarring . prominent postoperative scarring can be a major problem for patients .
endoscopic nd is a little bit more expensive than the conventional approach , but cheaper than robotic surgery , and causes fewer scars compared to the conventional approach .
thus , we consider endoscopic nd as an alternative to robotic surgery in terms of cost and postoperative scarring .
oral and maxillofacial surgeons should make an effort to gain experience in endoscopic surgery and robotic surgery .
unfortunately , there is no long - term follow - up information on patient outcomes involving these surgeries .
thus , it is impossible to compare oncologic outcomes between endoscopic nd and conventional nd . | various surgical techniques , such as endoscopic surgery and robotic surgery , are developed to optimize the esthetic outcome even in operations for malignancy . a modified face - lift or retroauricular approach
are used to minimize postoperative scarring .
recently , robot - assisted surgery is being done in various fields and considered as favorable treatment method by many surgeons .
however its high cost is a nonnegligible fraction for many patients . on the other hand , endoscopic surgery , which is cheaper than robotic surgery , is minimally invasive with contentable neck dissection .
although it is a difficult technique for a beginner surgeon due to its limited operation view , we suppose it as an alternative method for robotic surgery . herein , we report two cases of endoscopic neck dissection via retroauricular incision with a discussion regarding the pros and cons of endoscopic neck dissection . | I. Introduction
II. Cases Report
1. Case 1
2. Case 2
3. Surgical procedure
III. Discussion |
ractopamine hydrochloride ( rachcl , mw 337.85 , ( 1r*,3r*),(1r*,3s*)-4-hydroxy - r-[[[3-(4-hydroxyphenyl)-1-methylpropyl]amino]methyl]-benzenemethanol hydrochloride ( figure 1 ) ) is a phenethanolamine member of the family of -adrenergic agonists ( -agonists ) .
these compounds are mainly used in human and veterinary medicine as tocolytic and bronchodilator agents .
the advantages of feeding animals with -agonists have been reported to include the promotion of repartitioning of fat into muscles , in addition to the ability of increasing average daily weight gain , improving feed efficiency , saving on feed , and decreasing the number of days to market when higher doses are administered [ 25 ] .
furthermore , as a result of better feed utilization efficiency , there are positive environmental benefits for livestock producers in terms of decreased nitrogen and phosphorus excretions , and reduced amount of total animal waste .
however , meat products obtained from illegally treated animals with these compounds may pose potential risks linked to adverse cardiovascular and central nervous system effects .
ractopamine , a veterinary additive drug , was approved by the us food and drug administration for the use in swine production .
recently , it has become more and more popular not only in pig but also in chicken and cattle production , following clenbuterol which had been the most widely used -agonists in livestock production . due to its potential risks for human health
, ractopamine is still used in a restricted manner within a low limit of dosage when used in livestock production , in many countries .
therefore , there has been an increasing number of analytical methods reported to monitor ractopamine in animal urine , feeds , and tissues such as instrument methods like hplc with electrochemical detection and fluorescence , lc - ms [ 10 , 11 ] , and gc - ms .
however , these analytical approaches , which use several clean - up procedures ( liquid - liquid extraction ( lle ) and solid - phase extraction ( spe ) using different sorbents ) , are quite complicated , time - consuming , and expensive .
furthermore , integral production chain systems currently demand faster onsite ( farmhouses ) and/or online ( slaughterhouses ) test systems .
immunoassays as a screening detection method can rapidly detect low amounts of residues in many samples .
shelver et al . generated both polyclonal and monoclonal antibodies to analyze ractopamine by elisa .
wang et al . also reported a monoclonal antibody immunoassay to determine ractopamine in swine feeds with a detection limit of 0.24 g / g sample .
however , all of these elisa methods were mainly used for animal urine or feed samples , and complicated sample clean - up procedures ( lle and spe ) were required when animal tissues were analyzed .
therefore , these tissue- ( except for urine ) extraction methods combined with rac - elisa are too complex as routine test systems . moreover , to the best of our knowledge , there is no report on the determination of ractopamine in chicken muscle . as a result ,
it is necessary to develop a more rapid , sensitive , and effective method for the determination of rac residues in edible animal foods including chicken muscle .
bovine serum albumin ( bsa ) was obtained from merck ( darmstadt , germany ) .
horseradish peroxidase ( hrp ) , ovalbumin ( ova ) , -glucuronidase from escherichia coli , and freund 's complete and incomplete adjutants were purchased from sigma chemical co. ( saint louis , mo , usa ) .
reagent grade 3 , 3 , 5 , 5-tetramethyl - benzidine ( tmb ) , hydrogen peroxide , isobutyl chloroformate , butane-1 , 4-diol diglycidyl ether , and other chemicals were also from sigma .
ractopamine hydrochloride was purchased from pure chemical analysis co. , ltd ( bornew , belgium ) .
protein a - sepharose 4b was purchased from amersham biosciences ( uppsala , sweden ) .
polystyrene 96-well microplates were from nunc ( rockilde , denmark ) , and the microplate washer was from bio - rad ( hercules , calif , usa ) .
immunoassay absorbance was read with a multiskan spectrum purchased from thermo ( labsystems , vantaa , finland ) in the dual - wavelength mode ( 450650 nm ) .
double deionized water ( ddw ) was prepared with a milli - q ( millipore , mass , usa ) water purification system .
phosphate - buffered saline ( 1 pbs ; 38.4 mmol / l na2hpo4h2o , 11.5 mmol / l nah2po4h2o , 154 mmol / l nacl , ph 7.5 ) , phosphate - buffered saline with 0.05% tween 20 ( pbst ) , coating buffer ( cb , 50 mmol / l sodium carbonate buffer , ph 9.6 ) , blocking buffer ( 0.5% skimmed milk powder in 1 pbs ) , and tmb substrate solution ( prepared by adding 3.3 mg tmb in 250 l dmso to 25 ml phosphate - citrate buffer ( 0.1 mol / l citric acid + 0.2 mol / l na2hpo4 ; ph 4.3 ) containing 3.25 l of a 30% h2o2 solution ) , and termination solution ( 2.5 mol / l h2so4 in ddw ) were used . the hapten rac was coupled to bsa as the immunogen , and coupled to hrp as the enzyme tracer .
the coupling agent , butane-1 , 4-diol diglycidyl ether , was used for coupling rac to proteins .
the conjugating procedure of immunogen rac - bsa was adapted from elliott et al . .
in brief , 10 mg bsa was dissolved in 0.5 ml ddw , and the ph was adjusted to 10.8 using 1.0 mol / l sodium hydroxide .
coupling agent solution ( 50 l of 22 l / l butane-1 , 4-diol diglycidyl ether in ddw ) was added to the bsa solution , and the mixture was incubated for 22 hours at room temperature under nitrogen atmosphere .
rachcl ( 17 mg , 50 mol ) was added to 0.5 ml naoh ( 0.5 mol / l ) containing 10% dimethylformamide .
the mixture was then added to the epoxy - activated bsa solution ( precooled at 4c , in the refrigerator ) , slowly in an ice bath , and incubated for 22 hours at room temperature under nitrogen atmosphere .
the conjugating method of rac - hrp was similar to that of the immunogen in the first step except for the amounts of the reagents which were 5 mg enzyme hrp instead of 10 mg , and 8.5 mg rachcl ( 25 mol ) were added to the activated hrp solution .
finally , the mixture was incubated for 40 hours at room temperature under a nitrogen atmosphere .
both the conjugated immunogen and enzyme tracer were dialyzed against pbs for 3 days at 4c .
the conjugated immunogen was stored at 20c , and the enzyme tracer was stored at 4c with 0.01% thiomersal until required .
the hapten rac was coupled to the protein ova using the method of mixed acid anhydride , as follows .
rachcl ( 17 mg , 50 mol ) and 5 mg ( 50 mol ) succinic anhydride were diluted in 1 ml dry pyridine . after slow stirring for 24 hours at room temperature , the mixture was blown dry with a nitrogen flow .
the residue was diluted in a 2 ml mixture of dmf and 1 , 4-dioxane ( 1 : 1 ) , and then stirred for 10 minutes in an ice bath by adding 13 l ( about 0.05 mmol ) tributylamine .
after 7.2 l ( 0.05 mmol ) isobutyl chloroformate were added , the mixture was stirred for 1 hour at room temperature .
subsequently , the activated rac mixture was dripped onto the precooled protein oa solution which was previously prepared with the following method .
oa ( 10 mg ) was diluted in 2.5 ml phosphate buffer ( 0.1 mol / l ) . in order to avoid protein denaturation ,
dripping and stirring should be carried out at the same time at a low temperature , and should be finished within 30 minutes .
the final mixture was stirred gently for 24 hours at room temperature , and purified using extensive dialysis against pbs buffer for 3 days at 4c .
the purified coating conjugate solution was stored at 20c with 0.02% nan3 until required . antibodies were produced in rabbits using an immunization approach similar to that described by wang et al . .
two white rabbits were immunized by intradermal and intramuscular injections of the emulsified rac - bsa immunogen .
blood was collected from the marginal ear vein 10 days after each booster injection for antibody titer assay , and the whole blood was then collected after 6 immunizations .
the anti - rac antiserum was centrifuged at 2000 rpm for 10 minutes ( eppendorf 5804r , hamburg , germany ) and stored in small tubes ( about 2 ml per tube ) at 20c .
the titer for the specific antiserum was monitored by an indirect competitive elisa using the immunized hapten conjugated to ova .
after dialyzing against pbs buffer for 3 days , the purified antibodies were stored at 4c with 0.02% nan3 until required .
( a ) indirect competitive elisaflat - bottom polystyrene microplates were coated with rac - oa conjugates at 1 g per well in 100 l coating buffer , and incubated overnight at room temperature .
plates were washed 3 times with pbst using a 96pw microplate washer , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs per well for 1 hour at room temperature . after the plates were washed 3 times , 50 l of the appropriate antiserum / antibody dilution in pbs ( the other 50 l of rac standards in pbs were added to determine antibody specificity ) were added in each well for titer determination , and incubated for 1 hour at room temperature .
after 4 additional washes , the plates were incubated for 1 hour at room temperature with 100 l peroxidase - labeled goat anti - rabbit immunoglobulins diluted 1 : 10 000 in pbs , in each well .
after washing 5 times with pbst , 150 l tmb substrate solution were added to each well to measure the hrp tracer activity .
the color production of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well .
the absorbance in each well was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) .
flat - bottom polystyrene microplates were coated with rac - oa conjugates at 1 g per well in 100 l coating buffer , and incubated overnight at room temperature .
plates were washed 3 times with pbst using a 96pw microplate washer , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs per well for 1 hour at room temperature . after the plates were washed 3 times , 50 l of the appropriate antiserum / antibody dilution in pbs ( the other 50 l of rac standards in pbs were added to determine antibody specificity ) were added in each well for titer determination , and incubated for 1 hour at room temperature .
after 4 additional washes , the plates were incubated for 1 hour at room temperature with 100 l peroxidase - labeled goat anti - rabbit immunoglobulins diluted 1 : 10 000 in pbs , in each well .
after washing 5 times with pbst , 150 l tmb substrate solution were added to each well to measure the hrp tracer activity .
the color production of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well .
the absorbance in each well was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) .
( b ) direct competitive elisathe microplate well was coated with purified antibodies in 100 l coating buffer , and incubated overnight at room temperature .
the coated plates were washed 3 times with pbst , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs for 1 hour at room temperature . after the plate was washed 4 times , 50 l of the standard solution of rac dissolved in pbs ( or diluted sample extracts ) followed by 50 l of hrp - hapten conjugate solution diluted in pbs were added to each well , and the mixture was incubated in a shaker for 1 hour at room temperature . after washing 5 times with pbst
the color development of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well .
the absorbance was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) .
the microplate well was coated with purified antibodies in 100 l coating buffer , and incubated overnight at room temperature .
the coated plates were washed 3 times with pbst , and unbound active sites were blocked with 200 l 0.5% skimmed milk powder in pbs for 1 hour at room temperature . after the plate was washed 4 times , 50 l of the standard solution of rac dissolved in pbs ( or diluted sample extracts ) followed by 50 l of hrp - hapten conjugate solution diluted in pbs were added to each well , and the mixture was incubated in a shaker for 1 hour at room temperature . after washing 5 times with pbst
the color development of the enzymatic reaction was terminated after 30 minutes at room temperature by adding 50 l h2so4 ( 2.5 mol / l ) per well .
the absorbance was measured with a multiskan spectrum in the dual - wavelength mode ( 450 nm for the test , and the whole plate background is subtracted with the measurement at 650 nm ) .
( c ) optimization of the rac elisa testdirect competitive elisa was used to analyze the parameters of the described method such as the amount of the antibody coating , and the ionic strength and ph of the diluting buffer .
three different amounts of antibodies ( 0.5 g , 1.0 g , and 1.5 g antibody per well ) were tested .
after the optimal antibody coating quantity was decided , the proper ionic strength was investigated by changing the concentration of pbs buffer ranging from 10 to 40 mmol / l .
then , under the optimal conditions , the effects of different phs ( ph = 6.0 , 7.5 , 8.5 , 9.5 ) were tested .
direct competitive elisa was used to analyze the parameters of the described method such as the amount of the antibody coating , and the ionic strength and ph of the diluting buffer .
three different amounts of antibodies ( 0.5 g , 1.0 g , and 1.5 g antibody per well ) were tested .
after the optimal antibody coating quantity was decided , the proper ionic strength was investigated by changing the concentration of pbs buffer ranging from 10 to 40 mmol / l .
then , under the optimal conditions , the effects of different phs ( ph = 6.0 , 7.5 , 8.5 , 9.5 ) were tested .
( d ) sample preparationfour different samples including chicken muscle , pig muscle , pig liver , and pettitoes were chosen to evaluate the performance of elisa .
for the spiking study , each sample was spiked by dropping with the rac standard solution in methanol , making the samples with well - distributed different levels of rac , the samples were thoroughly mixed and then allowed to stand at 4c overnight .
all the samples were prepared for the immunoassay using the same extraction procedure as that for pig muscle as follows . the sample ( 2 g ) was chopped , and mixed with 10 ml pbs for 2 minutes using a waring blender ( omni international , marietta , ga , usa ) .
then , the mixture was centrifuged for 10 minutes at 4,000 rpm ( centrifuge 5804r , eppendorf ag , hamburg , germany ) at room temperature . the fat layer was then discarded , and the upper liquid was transferred into a test tube , and analyzed directly by direct competitive elisa .
four different samples including chicken muscle , pig muscle , pig liver , and pettitoes were chosen to evaluate the performance of elisa .
for the spiking study , each sample was spiked by dropping with the rac standard solution in methanol , making the samples with well - distributed different levels of rac , the samples were thoroughly mixed and then allowed to stand at 4c overnight .
all the samples were prepared for the immunoassay using the same extraction procedure as that for pig muscle as follows . the sample ( 2 g ) was chopped , and mixed with 10 ml pbs for 2 minutes using a waring blender ( omni international , marietta , ga , usa ) .
then , the mixture was centrifuged for 10 minutes at 4,000 rpm ( centrifuge 5804r , eppendorf ag , hamburg , germany ) at room temperature .
the fat layer was then discarded , and the upper liquid was transferred into a test tube , and analyzed directly by direct competitive elisa .
( a ) instrumentation for hplc analysisthe elisa results were verified using an hplc system ( shimadzu , kyoto , japan ) equipped with a lc-20ab pump and a rf-10axl fld ( excitation wavelength , 226 nm ; emission wavelength , 305 nm ) .
a thermo ods-2 hypersil column ( 5 m , 4.6 mm 250 mm ) was used with a mobile phase consisting of 0.087% 1-pentanesulfonic acid sodium salt in 2% glacial acetic acid solution / acetonitrile ( 68 : 32 ) at a flow rate of 1.0 ml / min .
the elisa results were verified using an hplc system ( shimadzu , kyoto , japan ) equipped with a lc-20ab pump and a rf-10axl fld ( excitation wavelength , 226 nm ; emission wavelength , 305 nm ) . a thermo ods-2 hypersil column ( 5 m , 4.6 mm 250 mm )
was used with a mobile phase consisting of 0.087% 1-pentanesulfonic acid sodium salt in 2% glacial acetic acid solution / acetonitrile ( 68 : 32 ) at a flow rate of 1.0 ml / min .
( b ) sample pretreatmenthomogenized tissue sample ( 5 g ) was extracted with acetonitrile ( 2 10 ml ) and stirred for 20 minutes on a shaker ( ika , staufen , germany ) .
after centrifugation ( 1,500 g , 4c , 10 minutes ) , the supernatant was transferred into a separating funnel .
the acetonitrile solution was defatted by washing with 20 ml hexane ( saturated by acetonitrile ) 3 times .
the residue was redissolved in 5 ml 2% acetic acid solution , and was used for the solid - phase extraction . homogenized tissue sample ( 5 g ) was extracted with acetonitrile ( 2 10 ml ) and stirred for 20 minutes on a shaker ( ika , staufen , germany ) .
after centrifugation ( 1,500 g , 4c , 10 minutes ) , the supernatant was transferred into a separating funnel .
the acetonitrile solution was defatted by washing with 20 ml hexane ( saturated by acetonitrile ) 3 times .
the residue was redissolved in 5 ml 2% acetic acid solution , and was used for the solid - phase extraction . ( c )
sample clean - upsolid - phase extraction was carried out using the extraction cartridges packed with multiwalled carbon nanotubes under reduced pressure .
the cartridges were activated with 5 ml methanol , followed by 5 ml ddw , and then equilibrated with 5 ml 2% acetic acid solution .
after the sample extracts were loaded , the cartridges were washed with 5 ml 2% acetic acid solution , and sequentially eluted with 7 ml 5% ammonia in methanol .
the eluted sample was dried , and redissolved in 1 ml 2% acetic acid for hplc analysis , or redissolved in appropriate volume of pbs for elisa analysis .
solid - phase extraction was carried out using the extraction cartridges packed with multiwalled carbon nanotubes under reduced pressure .
the cartridges were activated with 5 ml methanol , followed by 5 ml ddw , and then equilibrated with 5 ml 2% acetic acid solution . after the sample extracts were loaded , the cartridges were washed with 5 ml 2% acetic acid solution , and sequentially eluted with 7 ml 5% ammonia in methanol .
the eluted sample was dried , and redissolved in 1 ml 2% acetic acid for hplc analysis , or redissolved in appropriate volume of pbs for elisa analysis .
the real food sample offered by tianjin entry - exit inspection and quarantine bureau was extracted by pbs according to sample preparation , the extracts were divided into two aliquots , one is analysed by elisa directly and the other is adjusted to ph 6.8 and incubated at 37c for 10 minutes , then dropped with -d - glucuronidase solution , deconjugated by for 60 minutes , the mixture was adjusted to ph 7.5 and analysed by elisa .
after the purification of the antibody and the preparation of the enzyme tracer , direct competitive elisa was optimized .
several parameters including antibody coating quantity , ionic strength , and ph of the diluting buffer were studied in details .
the optimal quantities of the coating antibody and the enzyme conjugate required for the direct competitive elisa were determined by checkerboard titration .
they were chosen according to the lowest ic50 with an absorbance value of 0.71.2 for the control sample during color development .
the results showed that an antibody coating quantity of 1.0 g per well ( 100 l ) , and an enzyme conjugate dilution factor of 30 000 were the best among all testing conditions .
it is reasonable to believe that the increased ionic strength has a detrimental effect on the interaction between the antibody and the analyte or enzyme conjugate where ionic driving forces prevail .
the crystallizability of the buffer at a high salt concentration resulted in the choice of a concentration of 10 mmol / l pbs as the optimal dilution buffer .
we found that the ic50 reached its minimum value at ph 7.5 , that is , the immunoassay for rac was more sensitive at ph 7.5 than at other phs .
this is because acidic and alkaline solutions likely promote the denaturation of the antibody and/or enzyme conjugate , causing changes in their spatial structures with adverse effects on the reactions between the antibody and the analyte or enzyme conjugate .
a 10 mmol / l pbs buffer of ph 7.5 was chosen as the optimal solvent for the rac standard ( or samples ) and enzyme conjugate dilutions , and 1.0 g of antibody per well was the optimal coating amount .
a 6-point ( stepwise dilution of the rac standard solution ) calibration curve was performed in the elisa test , resulting in an average ic50 of 0.6 ng / ml and average ic15 of 0.04 ng / ml . these results showed that the obtained rac antibody was much more sensitive than those reported by previous studies [ 13 , 15 ] .
crossreactions can affect analytical results by either giving false positives or by elevating the predicted concentration of the target compound when both the target and one or more structurally similar compounds are present .
therefore , the specificity of the antibody toward a compound and its most probable crossreactants should be determined .
the crossreactivity profile of the rac antibody was determined by comparing the dose - response curves of rac with those of 7 analogues including ractopamine , clenbuterol , salbutamol , isoproterenol , terbutaline , dobutamine , and isoxsuprine ( figure 1 ) .
all these compounds showed no cross - reactivity with the rac antibody except for dobutamine .
reported that if the antibody is developed against a compound with a very similar structure , crossreactivity will likely occur . in conclusion , it was reasonable that dobutamine which has a very similar structure to rac showed 7.5% crossreactivity with rac .
the assay precision was studied by determining intra - assay and inter - assay reproducibilities .
the variations in percent inhibition in the intra - assay for 20 , 5 , 1.25 , 0.31 , 0.08 , and 0.02 ng / ml rac tested in a microplate were 0.5 , 1.5 , 4.6 , 7.6 , 16.1 , and 29.6% , respectively .
the inter - assay of the same material run over 6 months resulted in deviations from the means of 2.4 , 4.7 , 7.6 , 10.1 , 24.8 , and 32.4% , respectively .
it seemed likely that antibody sensitivity to these low concentrations was poor and so reproducibility was greatly reduced .
appropriate assays in accelerated trials such as the use of half - lives greater than 7 days at 37c are predictive of 612 months stability at 4c .
therefore , stability trials were carried out with rac antibodies stored at 4c , room temperature , and 37c for 30 days .
tables 1 and 2 show the results of the stability assays for the antibodies and enzyme conjugates , respectively .
there was not a striking change in the ic50 value of the rac antibody stored at different temperatures for 30 days , and none for the enzyme conjugates stored for 7 days .
moreover , color loss was not observed for both rapid assays during the experimental period .
this indicated that temperature could not easily affect the activities of the rac antibody and enzyme conjugate .
therefore , it is reasonable to conclude that both the antibody and enzyme tracer are stable enough to be used in subsequent tests , and even to produce a rac - elisa test kit .
immunoassays are a rapid and convenient analysis method for food samples as they usually do not require sample preconcentration and clean - up steps
. however , elisa methods may have high potential risks for nonspecific binding between the nontarget analytes and antibodies , and are consequently prone to matrix interferences .
chemical compounds present in samples or sample extracts such as proteins , fat , and others , might nonspecifically affect the binding of the antibody and analytes , and might also affect other aspects of the assay .
these so - called matrix effects can reduce the sensitivity and reliability of the competitive immunoassay .
matrix effects are more pronounced in direct immunoassays where not only the specific antibodies but also the enzyme conjugate are under the influence of the sample matrix . several methods can be used to determine matrix effects .
typically , interferences are quantified by comparing a standard curve prepared in buffer such as pbs with a calibration curve generated in the sample matrix known to be free of the analyte .
if the 2 curves are superposable , the effect of the matrix is not significant , and the samples can be analyzed using the standard curve prepared in the matrix - free solution . in the present assay , the matrix effects of our samples were analyzed using direct competitive elisa .
rac standard curves were prepared in pbs buffer , and in dilutions of extracts of rac - free samples to determine whether nonspecific interferences could be eliminated . in order to obtain a rapid , simple , and effective sample extraction method ,
several extraction solvents including methanol , ethanol , acetonitrile , and pbs buffer were tested .
significant matrix interference was found when the sample organic solvent extracts were diluted 5 folds in pbs buffer .
further dilution of the sample organic solvent extracts in pbs buffer could not reduce matrix interference .
even the addition of fish skin gelatin ( fg ) , skimmed milk powder , and tween-20 to the organic solvent extracts could not reduce the nonspecific interactions , and lowered the amount of developed color in the assay . when pbs buffer was used as the extraction solvent alone without any organic solvent or enshrouding reagent ,
the standard curves prepared in rac - free sample extract and in pbs buffer alone were superposable , and the matrix interference problem was easily resolved .
the calibration curves were created by direct competitive elisa using blank samples spiked with 6 different concentrations of rac .
figure 5 shows the comparison of the standard curves of rac in extracts prepared in the 4 rac - free samples and in pbs buffer alone . considering the dilutions of the sample extracts ,
the detection limit of the assay in the blank samples was 0.2 g / kg which was much lower than that of the maximum residue levels ( mrls ) for rac in these matrices .
thus , the loss in assay sensitivity was acceptable . to investigate the efficiency of the extraction method , 4 types of edible food samples were fortified with rac at 3 different levels , and were analyzed by the established direct competitive elisa method .
it was found that all the recoveries of the rac residues in these samples were less than 100% , and those in pig liver samples were little lower than those in other samples .
since the presence of water in these samples makes the amount of the whole extract bigger than that of pbs that had been added prior to extraction , the final concentration of rac is lower than that in theory
. therefore , it is reasonable to conclude that pig liver with a higher quantity of water shows a lower recovery than those of other samples . due to its more complicated chemical component structure ,
pig liver samples were spiked with rac at 0 , 1 , 2 , 5 g / kg , respectively .
after sample extraction and clean - up on a solid - phase extraction column , the purified extract was analyzed by hplc and elisa .
the analytical results obtained with the two methods for the same extract were shown in figure 6 .
also , the spiked pig liver was analyzed by the established elisa with a simple extraction procedure .
figure 7 shows the correlations between the results of hplc with more complicated extraction and clean - up procedures and elisa with a simple extraction procedure . despite the fact that in this case the comparison was established with samples subjected to different treatments , the results correlated well ( r = 0.9517 ) .
these results indicate that the developed elisa method can be used as a rapid screening method for the analysis of rac in food samples .
so , it is important to detect the rac metabolites for a newly established elisa . due to the lack of rac glucuronide metabolites
, they were not involved in the cross - reactivity profile of the antibody . before the analysis of the real food samples ,
-glucuronidase was used to deconjugate the rac glucuronide metabolites and prove indirectly if the antibody has the adequate binding of glucuronide metabolites .
real food samples with precise rac concentration ( validated by hplc - ms according to china national analysis standard method , gbt 22147 - 2008 ) were offered by tianjin entry - exit inspection and quarantine bureau ( export and import samples , including pig muscle , pig liver , pettitoes ) .
the results showed that there is no obvious difference in the rac concentration of the sample extracts with and without enzymatic hydrolysis , so we believe that the produced rac antibody has the ability of adequate binding with the rac glucuronide metabolites . theoretically speaking , pbs should be an appropriate extraction solution for both rac and the glucuronide metabolites due to the solubility of them in pbs .
so , rac glucuronide metabolites can be analysed by the established elisa system without deconjugation by glucuronidase .
the results were compared with the validated results ( shown in table 4 ) . in analysis of the 20 food samples using
the direct competitive elisa resulted in 15 samples being negative ( rac concentration was less than lod of the assay ) .
although the results obtained by elisa was lower than the validated results , they correlated well ( r = 0.9605 ) , indicating the precision of the elisa analytical system and the availability of it for the analysis of real food samples .
the developed elisa method with a high sensitivity and specificity is suitable for the routine screening detection of ractopamine residues in chicken muscle , pettitoes , pig muscle , and liver .
the sample extraction method is quite simple and rapid . although the recoveries are not very high , the detection limits ( 0.2 g / kg ) from the direct competitive elisa for these samples are low enough for the levels of mrl ( 10 g / kg for muscle , 90 g / kg for liver in japan ) .
the stabilities of the rac antibody and rac enzyme tracer together with the good correlation ( r = 0.9517 ) between the analytical results of hplc and elisa demonstrate the accuracy of the developed elisa procedure , confirming its reliability for applications in the rapid screening of rac in food samples . | to determine ractopamine residues in animal food products ( chicken muscle , pettitoes , pig muscle , and pig liver ) , we established a rapid direct competitive enzyme - linked immunosorbent assay ( elisa ) using a polyclonal antibody generated from ractopamine - linker - bsa .
the antibody showed high sensitivity and specificity in phosphate buffer , with an ic50 of 0.6 ng / ml , and the limit of detection was 0.04 ng / ml . the matrix effect of the samples was easily eliminated by one - step extraction with pbs , without any organic solution or clean - up procedure such as spe or liquid - liquid extraction , making it a much more simple and rapid method than previously reported ones .
the detection limit in blank samples was 0.2 g / kg . to validate this new rac ( ractopamine hydrochloride ) elisa ,
a rac - free pig liver sample spiked at three different concentrations was prepared and analyzed by hplc and elisa .
the results showed a good correlation between the data of elisa and hplc ( r2 > 0.95 ) , which proves that the established elisa is accurate enough to quantify the residue of rac in the animal derived foods . | 1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions |
about a thousand complete microbial genomes have been sequenced to date [ 961 genomes in the genomes on line database ( gold ) on 1 april 2009 ( 1 ) ] , and many different methods have been used to predict genes , yielding large differences in gene annotation even across closely related species .
furthermore , very few entries have been kept up - to - date in the primary databases such as genbank ( 2 ) .
we therefore felt that it was important to provide a unified interface to the various gene prediction packages to allow biologists to evaluate them in their genomic and evolutionary contexts .
many studies , such as the prediction of gene function , phylogenetic reconstruction and genomics context analyses , depend on accurate predictions of orthology . among genes that share a common ancestor , only genes that are separated by a speciation event are actual orthologs ( 3 ) . to address the need for reliable ortholog sources ,
several initiatives have been created for better ortholog prediction [ see ( 4 ) for a comparison ] . among these resources ,
orthologs matrix project ( oma ) stands out by its efficient and robust computational method allowing continuous updating with novel genomes ( 5 ) and its ability to exclude non - orthologs , conferring a high reliability in the prediction of true orthologous relationships ( 4 ) .
interactive genome browsers have proved invaluable to the community for visualizing genes and experimental data in their genomic context , and as hubs connecting many biomedical databases ( 6,7 ) .
genome browsers also provide comparative genomics information by displaying homologous regions in a single view .
however , most browsers concentrate on eukaryotic genomes , so that biologists working on microbial genomes are restricted to standalone programs such as the artemis comparison tool ( 8) or web sites such as the joint genome institute 's integrated microbial genomes tools ( http://img.jgi.doe.gov/ ) or genedb ( http://www.genedb.org ) that are more complex to use , can only handle a few genomes at a time and do not integrate as much information via a single interface .
derived genomic databases that connect and expand reference databases are important in particular for automated analyses such as dataset comparisons .
the ebi genome reviews database ( 9 ) provides complete genome sequence and annotation data , continuously updated and extended with automated and manual annotation in uniprotkb ( 10 ) .
the ncbi refseq resource ( 11 ) provides a coherent set of sequences , genes and transcripts , some of which have been manually annotated .
frustratingly , the ebi and ncbi resources use distinct sets of identifiers ( uniprotkb accession number and protein_id for ebi ; refseq accession number , geneid and gi number for ncbi ) that make it hard to navigate between databases using different references .
furthermore , uniprotkb curators not only extend and uniformize annotation , but they also modify gene sequences , changing translational start site predictions , correcting frameshifts or adding genes missing from the original submission . this information is propagated to genome reviews but not to the source ddbj / embl / genbank entries , which can only be modified by the original submitter .
this introduces an additional divergence between databases , as it becomes non - trivial to identify the
same gene in two different databases where the gene might have neither the same identifier scheme nor the same coordinates .
the integr8 database ( 9 ) aggregates curated information on completely sequenced genomes , including taxonomy down to the precise strain level , and cross - references to all chromosomes and plasmids comprising the complete genome .
we introduce the microbe browser , a web server that uses the integr8 database to organize and correlate genomic sequences and annotation from the genbank , genome reviews and refseq databases .
we use the powerful ensembl web code ( 7 ) to present the resulting data in a fully interactive , user - friendly and platform - independent manner .
integr8 and genome reviews are the source of genome data , including curated gene sets and annotation and cross - references to uniprotkb , interpro , gene ontology and the protein data bank .
genbank and refseq are the source of ncbi cross - references ( refseq accession , geneid and gi number ) .
pre - computed gene predictions from the glimmer ( 12 ) , genemark , genemarkhmm ( 13 ) and prodigal ( http://compbio.ornl.gov/prodigal ) packages are provided by the ncbi , and predictions by the easygene method ( 14 ) are downloaded from the easygene web site ( http://servers.binf.ku.dk/cgi-bin/easygene/search ) .
the genome reviews data are used as a reference , because it incorporates substantial automatic and manual annotation from the gold standard uniprotkb knowledgebase ( 10 ) .
cross - references from genbank and refseq genes are merged into genome reviews records based on the position of the 3-end of the genes .
this allows to correctly map not only genes for which no cross - references exist between the databases , but also those for which the 5-end ( start site ) has been possibly changed by uniprotkb curators .
the microbe browser home page is used for organism selection and search term input , which can be a gene name or a cross - reference to any of the source databases .
several view pages are available , the three most informative are detailed below . the user can easily navigate across those pages and detailed online help is available .
the gene report page integrates data on gene sequence and annotation , orthologs and cross - references to the major biological databases .
the chromosome view pages ( figure 1 ) display the original genome annotation submitted in the ddbj / embl / genbank source databases , the modified annotation from uniprotkb ( via genome reviews ) and the gene predictions of several popular packages .
genbank source annotation ( black full boxes ) , genome reviews reference annotation from uniprotkb ( coloured full boxes ) and predictions from five software packages ( hollow boxes ) .
uniprotkb curators have altered the most likely translational start site of the mt1457 gene by similarity to other genomes and created a novel conserved gene identical to m. tuberculosis h37rv gene rv1413 .
genbank source annotation ( black full boxes ) , genome reviews reference annotation from uniprotkb ( coloured full boxes ) and predictions from five software packages ( hollow boxes ) .
uniprotkb curators have altered the most likely translational start site of the mt1457 gene by similarity to other genomes and created a novel conserved gene identical to m. tuberculosis h37rv gene rv1413 .
the chromosome comparison pages ( figure 2 ) display regions surrounding orthologous genes in two or more organisms , highlighting orthology relationships between them , and reveal cases of synteny ( co - localized orthologs ) .
this display scales up to comparing a few species with detailed positional information , while specialized software has been proposed to visualize synteny across dozens of species in a summarized display ( 15 ) .
figure 2.chromosome comparison view of regions around the pgl gene in escherichia coli o157:h7 , e. coli k12 and yersinia pestis kim5 .
genes are coloured by interpro families , and orthologous gene pairs are connected . among the adjacent mod , ybh and bio genes present in e. coli k12 ,
chromosome comparison view of regions around the pgl gene in escherichia coli o157:h7 , e. coli k12 and yersinia pestis kim5 .
genes are coloured by interpro families , and orthologous gene pairs are connected . among the adjacent mod , ybh and bio genes present in e. coli k12 ,
programmatic access to the orthology relationships is available via web services through the oma apis at http://microbe.vital-it.ch/api .
designed primarily for biomedical researchers , the microbe browser runs an easy - to - use , interactive view allowing to visualize gene predictions , orthology and synteny relationships and to navigate across databases .
data originates from established bioinformatics databases : ddbj / embl / genbank source genomic data , annotation and cross - references to the major biological databases retrieved from genome reviews and refseq , pairwise gene orthology predictions from oma , and alternative gene predictions from several prediction packages .
swiss institute of bioinformatics . funding for open access charge : swiss institute of bioinformatics and ecole polytechnique fdrale de lausanne . | the microbe browser is a web server providing comparative microbial genomics data .
it offers comprehensive , integrated data from genbank , refseq , uniprot , interpro , gene ontology and the orthologs matrix project ( oma ) database , displayed along with gene predictions from five software packages .
the microbe browser is daily updated from the source databases and includes all completely sequenced bacterial and archaeal genomes .
the data are displayed in an easy - to - use , interactive website based on ensembl software .
the microbe browser is available at http://microbe.vital - it.ch/. programmatic access is available through the oma application programming interface ( api ) at http://microbe.vital-it.ch/api . | INTRODUCTION
METHODS
USAGE
CONCLUSION
FUNDING |
organophosphate ( op ) and paraquat ( pq ) pesticide ingestion is a serious health problem especially in developing countries , since op compounds were involved in 76% of pesticide poisoning [ 1 , 2 ] and pq self - poisoning has the highest case fatality ( 65% ) for any poisoning in sri lanka .
the two important pathologies behind pq toxicity are the generation of reactive oxygen species ( ros ; superoxide anion , hydrogen peroxide , hydroxyl radical ) and the depletion of nadph which is necessary for normal function .
widespread and robust effects of chlorpyrifos on the genes involved in antioxidant activity have been shown by slotkin et al . .
ros are associated with drugs ( cisplatin , aminoglycosides ) [ 7 , 8 ] and noise - induced [ 9 , 10 ] cochlear pathology .
animal studies showed that pq treatment leads to change in hearing threshold , outer and inner hair cell loss . progressively increasing delay in auditory
event - related potential has been reported in vegetable farmers with long - term exposure to op .
in contrast , kimura et al . reported that there was no statistically significant difference in auditory - evoked responses in farmers who were exposed to various pesticides compared to the controls .
the aim of the study was to identify the effects of deliberate ingestion of op or pq on brain stem auditory - evoked potentials ( baeps ) .
consecutive patients with deliberate self - poisoning with op or pq who were admitted to a secondary and a tertiary care hospital in the southern province of sri lanka were recruited .
the study was approved by the ethical review committee , faculty of medicine , university of ruhuna , sri lanka .
baeps were performed at 1 week ( the first assessment ) and 6 weeks ( the second assessment ) after the exposure .
ingestion of op was confirmed with cholinergic features or patients who were treated at peripheral hospitals with atropine .
pq ingestion was confirmed with positive urine to sodium dithionate test [ 14 , 15 ] .
the control group was recruited from persons accompanying the patients to the tertiary care hospital .
age , gender and occupation - matched healthy volunteers who did not have a history of acute pesticide exposure were recruited .
participants with history of diabetes , neuropathies head injury , and ear infections were excluded from the study .
baep is an electrophysiological method that can be used for neurotoxicity testing of auditory function .
a computer - based neuropack s1 electromyography ( emg)/ep measuring system meb-9400 ( nihon kohden ) was used for the baep .
a piece of cotton moistened with alcohol was used to clean the patient s skin where the electrodes were attached and the skin rubbed with dry gauze to remove any moisture and gel from the skin . recording electrodes ( active and reference ) and grounding electrodes were attached on the patient s head with elefix paste ( fig . 1 ) .
the patient was kept on a chair relaxed with eyes closed . to avoid emg of the neck
, patients were asked not to move their head . to avoid emg of the chin , patient was asked to open his / her mouth slightly .
the hi - cut and the low - cut filters were set to 3 khz and 100 hz , respectively .
the skin electrode contact impedance was kept below 5 k. the stimulation intensity was 90 db and 1,000 stimulations were averaged .
2 ) [ 16 , 17 ] . anova and post hoc comparison were used for the analysis.fig .
2probable sites of generation of the human baep electrode placement in baep probable sites of generation of the human baep
there were 70 ( 50 males ) patients recruited following op ingestion and 28 ( 21 males ) patients following pq ingestion .
the mean [ standard deviation ( sd ) ] age of the op and pq patients was 32 ( 12 ) and 29 ( 12 ) years , respectively .
there were 70 controls and their mean ( sd ) age was 33 ( 12 ) years . in op poisoning ,
53/70 came for the second assessment , and in pq poisoning , 18/28 came for the second assessment .
the differences of the mean latency of i iii , iii v , and i v were prolonged in the test group compared to the controls except in right side 1111 difference in the first assessment of the test , right side 1111 and 1v difference in the first and the second assessment in the test group compared to the controls .
none of the differences were statistically significant ( table 1).table 1the mean latency difference of baep in the test and the control groups in op poisoningcontrol group , n = 70test groupmean differences95% ci1st assessment , n = 702nd assessment , n = 53controls vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentcontrols vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentright sidei iii2.2 0.62.1 0.22.1 0.20.070.110.040.04 to 0.10.1 to 0.050.08 to 0.07iii
v1.8 0.21.9 0.21.8 0.50.020.080.050.04 to 0.090.08 to 0.060.09 to 0.05i v4.0 0.34.0 0.23.9 0.20.010.030.040.03 to 0.10.1 to 0.060.1 to 0.07left sidei
iii2.1 0.22.1 0.22.1 0.20.030.030.0050.2 to 0.070.05 to 0.20.2 to 0.1iii v1.8 0.21.9
v3.9 0.23.7 0.23.9 0.20.060.030.030.09 to 0.10.08 to 0.10.15 to 0.07 the mean latency difference of baep in the test and the control groups in op poisoning table 2 shows the difference of mean latency of i iii , iii
there was no significant difference detected in the test group compared to the controls.table 2the mean latency difference of baep in the test and the control groups in pq poisoningcontrol group , n = 70test groupmean differences95% ci1st assessment , n = 282nd assessment , n = 18controls vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentcontrols vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentright sidei
iii2.2 0.62.1 0.32.1 0.30.090.090.010.4 to 0.20.4 to 0.40.4 to 0.4iii
v1.8 0.21.9 0.21.8 0.20.020.010.010.1 to 0.10.1 to 0.10.1 to 0.1i
v4.0 0.33.9 0.33.9 0.30.010.030.010.2 to 0.20.2 to 0.20.2 to 0.2left sidei
iii2.1 0.22.1 0.22.3 1.30.030.30.30.3 to 0.30.6 to 0.030.6 to 0.1iii v1.8 0.21.8 0.21.8 0.10.050.010.050.1 to 0.060.1 to 0.10.9 to 0.1i
v3.9 0.23.9 0.33.9 0.20.010.0020.010.1 to 0.20.2 to 0.10.1 to 0.2 the mean latency difference of baep in the test and the control groups in pq poisoning
the differences of the mean latency of i iii , iii v , and i v were prolonged in the test group compared to the controls except in right side 1111 difference in the first assessment of the test , right side 1111 and 1v difference in the first and the second assessment in the test group compared to the controls .
none of the differences were statistically significant ( table 1).table 1the mean latency difference of baep in the test and the control groups in op poisoningcontrol group , n = 70test groupmean differences95% ci1st assessment , n = 702nd assessment , n = 53controls vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentcontrols vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentright sidei iii2.2 0.62.1 0.22.1 0.20.070.110.040.04 to 0.10.1 to 0.050.08 to 0.07iii
v1.8 0.21.9 0.21.8 0.50.020.080.050.04 to 0.090.08 to 0.060.09 to 0.05i v4.0 0.34.0 0.23.9 0.20.010.030.040.03 to 0.10.1 to 0.060.1 to 0.07left sidei
iii2.1 0.22.1 0.22.1 0.20.030.030.0050.2 to 0.070.05 to 0.20.2 to 0.1iii
v1.8 0.21.9 0.21.8 0.20.030.0070.020.09 to 0.10.2 to 0.050.07 to 0.1i
v3.9 0.23.7 0.23.9 0.20.060.030.030.09 to 0.10.08 to 0.10.15 to 0.07 the mean latency difference of baep in the test and the control groups in op poisoning
table 2 shows the difference of mean latency of i iii , iii v , and i v . there was no significant difference detected in the test group compared to the controls.table 2the mean latency difference of baep in the test and the control groups in pq poisoningcontrol group , n = 70test groupmean differences95% ci1st assessment , n = 282nd assessment , n = 18controls vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentcontrols vs 1st assessmentcontrols vs 2nd assessment1st assessment vs 2nd assessmentright sidei iii2.2 0.62.1 0.32.1 0.30.090.090.010.4 to 0.20.4 to 0.40.4 to 0.4iii
v1.8 0.21.9 0.21.8 0.20.020.010.010.1 to 0.10.1 to 0.10.1 to 0.1i
v4.0 0.33.9 0.33.9 0.30.010.030.010.2 to 0.20.2 to 0.20.2 to 0.2left sidei
iii2.1 0.22.1 0.22.3 1.30.030.30.30.3 to 0.30.6 to 0.030.6 to 0.1iii
v1.8 0.21.8 0.21.8 0.10.050.010.050.1 to 0.060.1 to 0.10.9 to 0.1i
v3.9 0.23.9 0.33.9 0.20.010.0020.010.1 to 0.20.2 to 0.10.1 to 0.2 the mean latency difference of baep in the test and the control groups in pq poisoning
the current study did not show any significant difference in the interpeak latencies of baep in the patients compared to the controls .
the number of the participants in the pq arm was small and this may not be adequate to come to firm conclusions .
the number of participants in the op arm may have not provided adequate power to detect statistical differences .
baep is a sensory - evoked potential used to assess the integrity of the auditory pathway from the eighth nerve to the auditory cortex .
the interpeak latencies ( i iii , iii v , and i v ) of baep provide a straightforward method to roughly localize lesions in the auditory pathway .
animal studies by bielefeld et al . and harris et al . showed shifting of evoked potential threshold and loss of inner and outer hair cells following the application of 10 , 5 , and 3 mm of pq to cochlea through a surgical incision [ 9 , 11 ] .
clerici and yang demonstrated the disruption of generation and transmission of afferent cochlear signals by generation of reactive oxygen species within the perilymphatic space with artificial perilymph and hydrogen peroxide .
even though both op and pq produce reactive oxygen species after ingestion , the concentration of substances reaching the perilymphatic space may not be sufficient to damage the cochlea .
kimura et al . studied the effects of the event - related evoked potentials in tobacco farmers who were exposed to different pesticides including op .
there were no significant lesions in the auditory pathway in op or pq poisoned patients compared to the matched controls .
the generation of reactive oxygen species within the perilymphatic space following the ingestion of op or pq may not be sufficient to cause lesions in the auditory pathway .
further studies with the assessment of auditory threshold in acute op or pq poisoning are needed . | organophosphate ( op ) and paraquat ( pq ) ingestion is a serious health problem .
a common pathology behind op or pq poisoning is the generation of reactive oxygen species ( ros ) which is known to cause ototoxicity .
the aim of the study was to identify the effects of deliberate ingestion of op or pq on brain stem auditory - evoked potentials ( baeps ) .
consecutive patients with deliberate self - poisoning with op or pq who were admitted to a secondary and a tertiary care hospital in the southern province of sri lanka and matched controls were recruited .
baeps were performed at 1 week ( first assessment ) and 6 weeks ( second assessment ) after the exposure .
interpeak latencies of i iii , iii v , and i v were measured .
there were 70 and 28 patients in the op and pq arms with the mean age of 32 12 and 29 12 years , respectively .
there were 70 controls and their mean age was 33 12 years . in op and pq
poisoning , 53/70 and 18/28 came for the second assessment , respectively .
the interpeak latency was not statistically different in the controls vs the first assessment , controls vs the second assessment , and the first vs the second assessment .
there were no significant lesions in the auditory pathway in op or pq poisoned patients .
the generation of ros within the perilymphatic space following the ingestion of op or pq may not be sufficient to cause lesions in the auditory pathway .
further studies with the assessment of auditory threshold are needed . | Introduction
Materials and Methods
Results
BAEP in OP Poisoning
BAEP in PQ Poisoning
Discussion
Conclusion |
there are currently large african immigrant populations living in philadelphia and other united states cities [ 1 , 2 ] . some of these new immigrants arrive hiv - infected and co - infected with tb and other parasitic diseases such as malaria , filariasis and other helminthes [ 3 , 4 ] .
accordingly , the prevalence of hiv in this population is presumed to be high , though exact numbers are not available . due to the relative rarity of parasitic co - infections in the us population ,
health care professionals are less familiar with the symptoms and the appropriate screening tests available to diagnose these ailments . as a result ,
new immigrants who are already infected , remain infected and accordingly experience higher co - morbidities . to effectively address these diseases within the african immigrant community ,
a host of factors must be considered , including but not limited to : the influence of poverty , education , nutrition , gender inequality , marginalization , stigmatization , language barriers , and access to health and social services [ 58 ] .
the targeted west african immigrant population of philadelphia , pennsylvania that this study is focused on , is composed of francophone ( fp ) immigrants from senegal , the ivory coast , and togo ; and anglophone ( ap ) immigrants from nigeria , liberia , and ghana .
these two populations are culturally distinct and are composed of africans from many different countries and tribes .
african immigrants from english - speaking countries formed an earlier pool of west african immigrants to the united states , while the most recent west african immigrants are mainly from french - speaking countries with minimal formal education or any of the other supports which typically aid transition to a new culture . because fp groups are less able to communicate in english , their opportunities for obtaining adequate health insurance are limited .
similar with other non english - speaking immigrant communities , the fp population is underemployed and chronically stressed [ 5 , 811 ] .
a large number of fp immigrants are refugees from their countries of origin who were forced to flee torture , detention , or death due to war [ 1214 ] .
some arrive as from refugee camps in different countries and are prescreened for different diseases before coming to the us .
others arrive on temporary visas and overstay their visa status , while a minority entered illegally .
additionally , the majority of fp africans are muslim , thus they do not have access to the religious support structures that are readily available from the numerous churches that christian ap africans take advantage of . because fps came from these highly stressful situations , or situations which would engender distrust in the authorities ,
these differences in background make access to health care and social services more difficult for these new immigrants in comparison with other immigrants because their integration and roles within american culture is hampered by these linguistic , social and cultural conditions which affect their education and employment two crucial factors in facilitating access to health care .
some of the literature suggests that these immigrants underutilize the health care system ; the direct result being that many conditions that can be easily treated on an outpatient basis , become conditions which require emergency room visits [ 15 , 16 ] . with the influx of africans from fp countries to the philadelphia area ,
new african immigrants face unknown internal obstacles to health and social services in comparison to earlier immigrants from ap countries .
both of the ap and the fp immigrant groups , but especially the fp groups in philadelphia have remained relatively invisible and understudied in terms of obstacles to accessing health and social services .
studies and surveys in philadelphia and other cities tend to lump africans and african - americans together , yet these immigrant groups are distinct and with differing health and social needs .
this current study was initiated out of the observation of these differences , and that these special needs require different methods to improve access to available services .
this descriptive study is comprised of two components : a structured survey and an ethnographic site visit component composed of interviews and observations .
phase 1 consisted of pilot - testing on 9 ap and 9 fp members and leaders of the community , followed by phase 2 ; administering a 90-item questionnaire to 239 participants ( 125 ap and 114 fp ) , and phase 3 ; site visit observations of 15 participant s ( 10ap and 5fp ) interactions with providers during their visits to health centers . in phase 1 , the questionnaires were pilot - tested for clarity with 9 aps in english ( 3 each from nigeria , ghana , and liberia ) and 9 from fps in french ( 3 each from togo , ivory coast , and senegal ) .
they were asked to address the clarity , length , and sensitivity of the questions .
they were asked to indicate if community issues were adequately addressed by the questions . to establish face validity
, professionals familiar with the immigrant populations and with hiv / aids infection in west africa reviewed the instruments .
the questionnaires were reviewed and approved by temple university and philadelphia department of public health institutional review boards . in phase 2 , self - administered questionnaires were given to participants .
the target sample group was composed of 239 adult west african immigrants ( 125 aps and 114 fps ) residing in philadelphia .
given the migratory nature of the target population , a convenience sample of 239 participants was selected .
thirty - five to forty - two participants from each of the three fp countries with the highest immigrant populations and 3750 participants from each of the three ap countries participated in the study .
participants were recruited during health fairs , in churches , mosques , and car washes .
they decided to participate after explanation that the study was on access to health care and social services and having read the consent form in english or french as necessary .
the interviewers were given the option of reading the questionnaire to those who consented but could not read , however all those who consented in the study were able to read english or french . in meetings ,
the questionnaires were administered before other agenda items for that day by the investigator and/or a trained administrator . more specifically , before giving out the survey ,
the administrator read aloud the consent form to the participants in either english or french , depending on the group ; and explained to them that participation was voluntary and that they could withdraw at any point .
no names were used on the surveys , and responses were kept anonymous and under lock and key , accessible only to the investigator .
there were no right or wrong answers , and they were told to answer the questions as honestly and as thoroughly as possible in approximately 3045 min .
the survey administrator was available to answer questions that participants had while filling the questionnaire .
the target population was unique and a literature search revealed that no similar studies had been conducted with similar groups . the investigator designed a culturally sensitive survey instrument that addressed the most pertinent issues faced by the communities .
the questions constructed in english were translated into french by four fps who were fluent in both english and french .
if the translations remained the same no changes were made , but those that differed were corrected with the intended meaning made clear .
the 90 self - administered questionnaire items were broken down into the following areas : general health and social services , knowledge , attitude and belief ( gkab ) , knowledge , attitude , and belief regarding hiv / aids ( skab ) . the items about general health and social services knowledge featured diseases such as tb , diabetes , heart disease , and cancer .
specific hiv / aids knowledge , attitude , and beliefs were addressed by specific questionnaire items such as beliefs about circumcision , wife inheritance , sex with young girls , and the spread of hiv .
social services featured their access and use of legal help , housing , and employment information .
the social support variable , important in normal settlement and adjustment in a new community had two components ( 1 ) instrumental and emotional support ( social support-1 ) composed of items having someone to tell most secret worries and fears , having someone to help resolve problems , and ( 2 ) a more tangible and informational based social support ( social support-2 ) composed of items befriend individuals from own ethnicity , befriend americans , spend most free time with own ethnic group members , and spend most free time with americans .
social network , the web of relationships important in knowing one s community in terms of , for example ; finding jobs , schools , and services was composed of items visited home of a friend of a different race , visited home of a friend of a different religion , and attended religious services .
discrimination and stigma , implicated by most members of the community for the disparity in health and social services delivery , was composed of questionnaire items denied or lost a job due to ethnicity , poor treatment at health center due to ethnicity , and mistreatment of children at school due to ethnicity .
acculturation was considered to be a major factor in successful settlement of the new immigrants in philadelphia .
the items also measured the length of stay in the us and philadelphia , the languages used regularly , the music listened to , and the people with whom they spent their free time .
the instrument s face validity and content validity was evaluated by key members of the community and professionals in the field of hiv / aids familiar with african aids epidemic ( by specifically looking at the instrument s readability , comprehension , clarity and precision of content ) . in phase 3 , the site visit component of the study involved a brief demographic survey of the participants during visits to service delivery centers .
health centers serving the communities where participants resided were identified , and three of the service centers ( scs ) were chosen for the study ( sc1 , sc2 , and sc3 ) .
service center 1 ( sc1 ) and sc2 were part of philadelphia department of health and the third one was a private hospital .
interpreters accompanied the fp participants who could not speak english . for the interview of the participants during the observation , the investigator used a guide encompassing access , hiv infection , hiv stigma , social support , employment , and significance of english language proficiency .
the notes were transcribed at the end of the day , capturing the key themes of the interview with each participant .
the chi - squared statistic was used to analyze the structured survey to identify significant factors contributing to differences between aps and fps in access to health services .
of the total 239 participants , 125 were from ap countries , 50 ( 20.9% ) were from nigeria , 37 ( 15.5% ) from liberia , and 38 ( 15.9% ) from ghana .
one hundred and fourteen participants were from fp countries , 42 ( 17.6% ) were from togo , 35 ( 14.6% ) from ivory coast , and 37 ( 15.5% ) from senegal .
there were more males in both groups , but significantly more females among the ap than fp .
aps had lived in the us longer than fps ( p = 0.015 ) , but had lived in philadelphia about the same length of time as fps ( p = 0.15 ) . more fps reported higher ses ( p = 0.002 ) in philadelphia than in their home countries compared to aps .
there were more employed aps ( p = 0.008 ) , more aps indicating that they were professionals ( p = 0.03 ) , were christian ( p < 0.0001 ) , and fluent in english ( p < 0.0001 ) than fps . more fps ( p < 0.0001 ) reported fluency in french and being muslim ( p < 0.0001 ) .
0.0001 ) health care needs were met by their primary care physicians while fps used traditional care and ordered medicines from their native countries ( table 1).table 1demographic characteristics of anglophone and francophone west africans in philadelphiavariableanglophonefrancophonetotalp value#% row ( % col)#%row ( % col)#% row ( % col)region1255311447239100sex4.70.03 * male7848 ( 62.4)8652 ( 75.4)164100 ( 68.6 ) female4763 ( 37.6)2837 ( 24.6)75100 ( 31.4 ) total12552 ( 100)11448 ( 100)239100 ( 100)age ( years)0.990.8 18301445 ( 17.7)1755 ( 19.1)31100 ( 18.4 ) 31403045 ( 38)3755 ( 41.6)67100 ( 39.9 ) 41502147 ( 26.6)2453 ( 26.9)45100 ( 26.8 ) 51601456 ( 17.7)1144 ( 12.4)25100 ( 14.9 ) total7947 ( 100)8953 ( 100)168100 ( 100)education30.30.0001 * * no education00 ( 0)7100 ( 6.2)7100 ( 3.0 ) primary111 ( 0.8)888 ( 7.0)9100 ( 3.8 ) secondary1042 ( 8.1)1458 ( 12.4)24100 ( 10.2 ) higher1231 ( 9.8)2769 ( 23.9)39100 ( 16.5 ) university10064 ( 81.3)5736 ( 50.5)157100 ( 66.5 ) total12352 ( 100)11348 ( 100)236100 ( 100)length of stay in us12.30.015 * 01 year739 ( 5.7)1161 ( 10.4)18100 ( 7.8 ) 1.13.0 years1950 ( 15.3)1950 ( 17.9)38100 ( 16.5 ) 3.15.0 years1439 ( 11.3)2261 ( 20.8)36100 ( 15.7 ) 5.110.0 years4754(37.9)4046 ( 37.7)87100 ( 37.8 ) 11 + years3773(29.8)1427 ( 13.2)51100 ( 22.2 ) total12454(100)10646 ( 100)230100 ( 100)length of stay in philadelphia6.70.15 01 year1647 ( 12.9)1853 ( 17.0)34100 ( 14.8 ) 1.13.0 years2561 ( 20.2)1639 ( 15.1)41100 ( 17.8 ) 3.15.0 years2235 ( 17.7)2755 ( 25.5)49100 ( 21.3 ) 5.110.0 years3651 ( 29.0)3449 ( 32.1)70100 ( 30.4 ) 11 + years2569 ( 20.2)1131 ( 10.3)36100 ( 15.7 ) total12454 ( 100)10646 ( 100)230100 ( 100)home country ses6.30.09 very well to do1450 ( 11.2)1450 ( 12.3)28100 ( 11.7 ) well to do6047 ( 48)6953 ( 60.5)129100 ( 54.0 ) average3759 ( 29.6)2641 ( 22.8)63100 ( 26.4 ) poor1474 ( 11.2)526 ( 4.4)19100 ( 7.9 ) total12552 ( 100)11448 ( 100)239100 ( 100)philadelphia ses14.40.002 * very well to do930 ( 7.3)2170 ( 18.6)30100 ( 12.7 ) well to do7550 ( 60.5)7550 ( 66.4)150100 ( 63.3 ) average3468 ( 27.4)1632 ( 14.1)50100 ( 21.0 ) poor686 ( 4.8)114 ( 0.9)7100 ( 3.0 ) total12452 ( 100)11348 ( 100)237100 ( 100)employment7.10.008 * yes9757 ( 78.9)7243 ( 63.2)169100 ( 70.7 ) no2638 ( 21.1)4262 ( 36.8)68100 ( 28.3 ) total12352 ( 100)11448 ( 100)237100 ( 100)occupation6.90.03 * student3356 ( 27.3)2644 ( 23.9)59100 ( 26.3 ) professional6758 ( 55.4)4842 ( 44.0)109100 ( 48.7 ) other(self - employed)2138 ( 17.3)3562 ( 32.1)56100 ( 25.0)total12154 ( 100)10946 ( 100)224100 ( 100)religious affiliation51.30.0001 * * christian11168 ( 89.5)5332 ( 46.9)164100 ( 69.2 ) moslem1016 ( 8.1)5184 ( 45.1)61100 ( 25.7 ) native african117 ( 0.8)583 ( 4.4)6100 ( 2.5 ) other150 ( 0.8)150 ( 0.9)2100 ( 0.8 ) none125 ( 0.8)375 ( 2.7)4100 ( 1.7 ) total12455 ( 100)11345 ( 100)226100 ( 100)english proficiency95.50.0001 * * not write or speak113 ( 0.8)787 ( 6.1)8100 ( 3.4 ) speak some not write113 ( 0.8)787 ( 6.1)8100 ( 3.4 ) write and speak moderate35 ( 2.5)5495 ( 47.4)57100 ( 24.1 ) speak not write218 ( 1.6)982 ( 7.9)11100 ( 4.6 ) speak and write11675 ( 94.3)3925 ( 32.5)155100 ( 64.5 ) total12352 ( 100)11648 ( 100)239100 ( 100)french proficiency163.50.0001 * * not write or speak8498 ( 67.7)22 ( 1.8)86100 ( 36.1 ) speak some not write2181 ( 17.0)519 ( 4.4)26100 ( 10.9 ) write and speak moderate1046 ( 8.1)1254 ( 10.5)22100 ( 9.2 ) speak not write533 ( 4.0)1067 ( 8.8)15100 ( 6.4 ) speak and write45 ( 3.2)8595 ( 74.5)89100 ( 37.4 ) total12452 ( 100)11448 ( 100)238100 ( 100)where health need received53.50.0001 * * emergency room2862 ( 24.0)1738 ( 18.0)45100 ( 21.0 ) community health 2248 ( 19.0)2452 ( 25.0)46100 ( 22.0 ) traditional clinic321 ( 3.0)1179 ( 11.0)14100 ( 7.0 ) buy / order traditional medicine321 ( 3.0)1179 ( 11.0)14100 ( 7.0 ) western trained african physician17 ( 1.0)1493 ( 15.0)15100 ( 7.0 ) primary care physician4488 ( 38.0)612 ( 6.0)50100 ( 23.0 ) nowhere945 ( 8.0)1155 ( 11.0)20100 ( 9.0 ) other779 ( 6.0)221 ( 2.0)9100 ( 4.0 ) total117(100)96(100)213(100 ) * statistically significant at p < 0.05 level * * statistically significant at p < 0.001 level demographic characteristics of anglophone and francophone west africans in philadelphia * statistically significant at p < 0.05 level * * statistically significant at p < 0.001 level participant perception of the greatest barriers to access is shown in fig .
it is noted that besides the language barrier , fp immigrants reported immigrant cultural insensitivity to host culture and lack of documents as major barriers to access ; while aps cited host cultural insensitivity to immigrant culture , transportation , and other factors such as a lack of awareness of how the us health care system works , as barriers . during site visit interviews
, fps reported that immigrants were reluctant to adopt the american culture and were attempting to practice their cultures in their host country .
aps felt that in their interactions with mainstream culture , their culture was often ignored and marginalized .
discrimination and stigma were mentioned by fps and aps as a factor that increased the impediment to access .
the analysis of specific items composing the discrimination and stigma set of questions , it was observed that when participants were asked have you ever been denied a job or lost a job because of your ethnic background ? of 63 respondents who stated that they had experienced discrimination , 57% were fps and 43% were aps . from a total of 15 who reported never applying for a job , but perceiving discrimination , 31% were aps and 69% fps ( fig .
2perception of job discrimination by anglophone and francophone africans in philadelphia perceived barriers to access perception of job discrimination by anglophone and francophone africans in philadelphia the stigma of hiv was perceived to be a major contributing factor to the reluctance towards using the health care system by those sick with hiv or contemplating hiv testing . when asked
if a member of your family became ill with hiv , would you want it to remain a secret , aps ( 60% ) and fps ( 40% ) reported they would not ; while among those responding affirmatively , 48% were aps and 52% were fps ( fig . 3 ) . hiv stigma was an issue that both groups complained about in the community , so it was not surprising to find no significant difference between aps and fps.fig .
3hiv stigma among anglophone and francophone africans living in philadelphia hiv stigma among anglophone and francophone africans living in philadelphia significant differences were observed in the languages used . when asked
what language do you use at home ? among a total of 58 who responded to using english alone , 85% were aps , and 15% fps ; out of a total of 48 who reported using english / ethnic , 94% were aps and 6% fps , while out of a total of 21 participants reporting usage of french , 100% were fps , and from 12 participants reporting the use of english / french , 75% were fps and 25% were aps ( table 1 ) .
more aps received their first health care at the emergency room compared to fps . from the pilot and the in - site conversation phases of the study
, we observed that because of their lack of documents , most fps avoided health care centers including emergency room , unless they desperately had to make the visit .
there were more aps going to their primary health care physicians first compared to fps , hence the significance of transportation for aps ( table 1 ) .
aps had greater social support and network than fps through their established churches and other group organizations ( table 2 ) .
fps who were mainly moslem had less social support and a smaller network there was only one french - speaking church in the city compared to over 10 different english - speaking churches .
there were five mosques in which services were conducted separately for men , women , and children.table 2social support and social network among anglophone and francophone africans in philadelphiaregionanglophonefrancophonetotalvalue#variable#% row ( % col)#% row ( % col)#%row ( % col)1someone to share secret worries and fears2.330.510 never2049 ( 16.4)2151 ( 18.9)41100 ( 17.6 ) sometimes6449 ( 52.5)6551 ( 58.6)129100 ( 55.4 ) most of the time2662 ( 21.3)1638 ( 14.4)42100 ( 18.0 ) all of the time1257 ( 9.8)943 ( 8.1)21100 ( 9.0 ) total122(100)111(100)233(100)2someone to help resolve problem2.010.570 never1148 ( 9.0)1252 ( 10.7)23100 ( 9.9 ) sometimes6449 ( 52.5)6651 ( 59.5)130100 ( 55.8 ) most of the time3059 ( 24.6)2141 ( 18.9)51100 ( 21.9 ) all of the time1759 ( 13.9)1241 ( 10.8)29100 ( 12.4 ) total122(100)111(100)233(100)3trust of philadelphia city government14.890.002 * hardly ever1531 ( 13.5)3369 ( 30.8)48100 ( 22.0 ) some of the time6063 ( 54.1)3537 ( 32.7)95100 ( 43.6 ) most of the time2243 ( 19.8)2957 ( 27.1)51100 ( 23.4 ) always1458 ( 12.6)1042 ( 9.3)24100 ( 11.0 ) total111(100)107(100)218(100)4have you been in the home of a friend of different race in the past 12 months14.40.001 * * do nt remember638 ( 5.0)1062 ( 9.0)16100 ( 6.9 ) no3840 ( 31.4)5860 ( 52.3)96100 ( 41.4 ) yes7764 ( 63.6)4336 ( 38.7)120100 ( 51.7 ) total121(100)111(100)232(100)5have you been in the home of a friend of different religion in the past 12 months7.490.020 * do nt remember747 ( 5.7)853 ( 7.4)15100 ( 6.5 ) no4945 ( 40.2)6155 ( 56.5)110100 ( 47.8 ) yes6663 ( 54.1)3937 ( 36.1)105100 ( 45.7 ) total122(100)108(100)230(100)6how often have you attended religious services in the past 12 months7.170.130 do nt know1045 ( 8.3)1255 ( 11.1)22100 ( 9.6 ) few times per year1539 ( 12.4)2461 ( 22.2)39100 ( 17.0 ) once or twice per month2051 ( 16.5)1949 ( 17.6)39100 ( 17.0 ) almost once per week2867 ( 23.1)1433 ( 13.0)42100 ( 18.2 ) once or more per week4855 ( 39.7)3945 ( 36.1)87100 ( 38.0 ) total121(100)108(100)229(100)7what language do you use at home?124.40.000 * * ethnic2251 ( 18.0)2149 ( 19.6)43100 ( 18.8 ) french00 ( 0.0)21100 ( 19.6)21100 ( 9.2 ) english4985 ( 40.1)915 ( 8.4)58100 ( 25.3 ) ethnic / french00 ( 0.0)22100 ( 20.6)22100 ( 9.6 ) ethnic / french / english312 ( 2.5)2288 ( 20.6)25100 ( 10.9 ) ethnic / english4594 ( 36.9)36 ( 2.8)48100 ( 21.0 ) french / english325 ( 2.5)975 ( 8.4)12100 ( 5.2 ) total122(100)107(100)229(100)8what language do you use with friends?154.80.000 * * ethnic1345 ( 10.7)1655 ( 14.7)29100 ( 12.6 ) french00 ( 0.0)28100 ( 25.7)28100 ( 12.1 ) english6791 ( 54.9)79 ( 6.4)74100 ( 32.0 ) ethnic / french00 ( 0.0)21100 ( 19.3)21100 ( 9.1 ) ethnic / french / english29 ( 1.6)2091 ( 18.3)22100 ( 9.5 ) ethnic / english3895 ( 31.2)25 ( 1.8)40100 ( 9.5 ) french / english212 ( 1.6)1588 ( 13.8)17100 ( 7.4 ) total122(100)109(100)231(100)9involvement in ethnic community organizations20.50.000 * * never2447 ( 20.2)2753 ( 24.8)51100 ( 22.4 ) some of the time6370 ( 53.0)2730 ( 24.8)90100 ( 39.5 ) most of the time1440 ( 11.8)2160 ( 19.4)35100 ( 15.5 ) always1835 ( 15.0)3465 ( 31.2)52100 ( 22.8 ) total119(100)109(100)228(100)10involvement in american community organizations22.320.000 * * never5241 ( 43.0)7459 ( 66.7)126100 ( 54.3 ) some of the time5774 ( 47.1)2026 ( 18.0)77100 ( 33.2 ) most of the time939 ( 7.4)1461 ( 12.6)23100 ( 9.9 ) always350 ( 2.5)350 ( 2.7)6100 ( 2.6 ) total121(100)111(100)232(100)11do you befriend individuals from your ethnic group?9.140.030 * never943 ( 7.4)1257 ( 11.0)21100 ( 9.1 ) some of the time5165 ( 41.8)2735 ( 24.5)78100 ( 33.6 ) most of the time3552 ( 28.7)3348 ( 30.0)68100 ( 29.3 ) always2742 ( 22.1)3858 ( 34.5)65100 ( 28.0 ) total122(100)110(100)232(100)12do you befriend americans?5.010.170 never1137 ( 9.2)1963 ( 17.2)30100 ( 13.0 ) some of the time6755 ( 55.8)5545 ( 49.5)122100 ( 52.8 ) most of the time2648 ( 21.7)2852 ( 25.2)54100 ( 23.4 ) always1664 ( 13.3)936 ( 8.1)25100 ( 10.8 ) total120(100)111(100)231(100)13do you spend most of your free time with individuals from your own ethnic group?11.530.009 * never7391161 ( 10.0)18100 ( 7.8 ) some of the time69643936 ( 35.5)108100 ( 46.8 ) most of the time32463854 ( 34.5)70100 ( 30.3 ) always13372263 ( 20.0)35100 ( 15.1 ) total121(100)110(100)231(100)14do you spend most of your free time with americans?23.690.000 * * never1531 ( 12.4)3369 ( 30.3)48100 ( 20.9 ) some of the time8866 ( 72.7)4534 ( 41.3)133100 ( 57.8 ) most of the time1335 ( 10.7)2465 ( 22.0)37100 ( 16.1 ) always542 ( 4 .. 2)758 ( 6.4)12100 ( 5.2 ) total121(100)109(100)230(100)15do you trust people in your neighborhood?9.030.29 not at all1945 ( 15.6)2355 ( 20.9)42100 ( 18.0 ) not very often5862 ( 47.6)3638 ( 32.7)94100 ( 40.5 ) some of the time3056 ( 24.6)2444 ( 21.8)54100 ( 23.3 ) most of the time1536 ( 12.2)2764 ( 24.4)42100 ( 18.1 ) total122(100)110(100)232(100)16do you trust african americans in your neighborhood?32.250.000 * * not at all716 ( 5.7)3684 ( 32.7)43100 ( 18.5 ) not very often4963 ( 40.2)2937 ( 26.4)78100 ( 33.6 ) some of the time5465 ( 44.3)2935 ( 26.4)83100 ( 35.8 ) most of the time1243 ( 9.8)1657 ( 14.5)28100 ( 12.1 ) total122(100)110(100)232(100)17do you trust members of other races in your community?19.550.000 * * not at all840 ( 6.7)1260 ( 10.9)20100 ( 8.6 ) not very often4158 ( 34.2)3042 ( 27.3)71100 ( 30.9 ) some of the time6561 ( 54.1)4239 ( 38.2)107100 ( 46.5 ) most of the time619 ( 5.0)2681 ( 23.6)32100 ( 14.0 ) total120(100)110(100)230(100)18do you trust members of your own country in your community?16.690.001 * * not at all650 ( 5.0)650 ( 5.5)12100 ( 5.2 ) not very often2144 ( 17.4)2756 ( 24.5)48100 ( 20.8 ) some of the time7466 ( 61.2)3934 ( 35.5)113100 ( 48.9 ) most of the time2035 ( 16.5)3865 ( 34.5)58100 ( 25.1 ) total121(100)110(100)231(100 ) * statistically significant at p < 0.05 level * * statistically significant at p < 0.001 level social support and social network among anglophone and francophone africans in philadelphia * statistically significant at p < 0.05 level * * statistically significant at p < 0.001 level sources of information were also a barrier encountered by both aps and fps . the information source that fps used to find out about hiv was mainly the radio and newspapers .
aps obtained their hiv information through the internet , word of mouth , church sponsored health fairs , and professional journals ( table 1 ) .
however , most aps and fps lamented that there was less information on hiv in the us , which led to their perception of low hiv prevalence . according to two participants
a former nurse from an ap region and an engineer from fp region they had more hiv information in their countries on bill boards , buses , trains , schools , the daily papers , radio , on the tv everyday ; than they were witnessing in the us .
the site visit study questionnaire and interviews revealed more information about barriers to access ( table 2 ) .
site visit information from the small sample of aps and fps indicated that language , proximity to health centers , hiv stigma , discrimination and denial , and power balance between men and women were major barriers to the access and utilization of services .
the men made all the decisions including whether to go to health centers when there was sickness in the family .
the interviews also revealed that some people , especially fp men , persistently maintained risky sexual behaviors despite demonstrating high knowledge concerning hiv / aids .
this formative study highlights the major barrier to access faced by ap and fp africans in philadelphia without disparaging the needs and more work required on the african - american community that they are often grouped under .
reported that immigrants living in urban areas encounter more barriers in seeking health care because of poverty , lack of medical insurance , and difficulty understanding english .
our study exhibited similar observations , but with fps being the most affected . whereas aps in philadelphia identified members of the host culture as culturally insensitive , fps felt that the immigrants were not willing to accept the host culture and were not readily integrating themselves into mainstream culture .
yet , fps as a group interacted mostly with people from their own countries and more specifically , with their own ethnic groups in comparison to aps .
while fps were aware of inherent cultural problems in acculturation , they felt powerless to make real changes .
language , and more specifically comprehension , was a critical factor in the utilization of health care .
interpreters were available to address basic language differences ; however , their lack of medical training was a barrier to comprehension .
we observed interactions where patients blank looks were taken as agreement or understanding of the medical issues or implications involved .
we observed that the length of stay in the us was not as critical as language proficiency in terms of access to care ( table 1 ) .
it is significant to note that coming from an english speaking country did not mean one was an english speaker .
there were those from ap countries who spoke very little english and thus had similar language problem as fp immigrants .
one social worker concurred that some of the patients acted as if they understood the directions given , only to do the opposite or do nothing ; then claiming on the next appointment that they did not understand , yet they had not requested clarity . in
most of africa , the medical field is viewed as an elite profession with medical professionals held in high esteem akin to the traditional medicine men from earlier generations ; their actions and requests are beyond reproach or questioning . the patients with these backgrounds
are used to a role as passive users with little personal say in their health care . when encountering a health care system where they are expected to contribute to their own well - being by health care centers that seemed more efficacious compared to what they had been used to ,
the main limitations of the study are the non - randomized sampling method used and the small sample size .
the logistics of this special situation created these issues and therefore the interpretation and generalizability of the results suffer directly .
the same issues restricted models adjusting for demographics or confounding factors to the extent that they could not be entertained .
the findings of this project warrant future investigation utilizing a larger sample and randomized sampling protocol .
other limitations involve the language barrier and the ethics of sensitive questions and issues common in hiv / aids research .
this study highlights the difference in the perceptions of access to health care and social services among aps and fps in philadelphia .
the study showed that though most studies still group africans and african - americans together , the two groups behave differently , and thus should be considered separately .
this study shows that even among immigrants from same continent and country ; variables such as cultural experience , language , and religious differences influence their access to health care when they move to the us . whereas the more recent fp immigrants considered lack of documentation and immigrant cultural insensitivity as major barriers , aps who had lived longer in the us reported host cultural insensitivity and transportation to be the barriers to access to health care . both communities mentioned hiv stigma and discrimination as a real barrier to hiv prevention efforts , which was strong enough to lead some community members to move to other areas in the city .
language was named by both communities to be a more significant barrier to health care access .
our findings suggest that there is discrepancy in access to health care for immigrant africans with anglophone africans having better access than francophone africans .
the poor access was mainly due to fear of visiting the health centers because of lack of documents , transportation , language barrier , inadequate familiarity of the system ; and in the case of the hiv infected , stigma and discrimination .
hiv stigma was strong in both communities and discouraged most infected members from disclosing their sickness , sometimes even to family members .
fps had poorer access than aps and this difference was based on language proficiency , a lack of formal education , and less social support or poor networking . through site visit interviews we observed that some members of the fp community procured medicines from their own countries for a variety of ailments .
an effective prevention control program in the ap and fp community must be culturally sensitive while involving the key stakeholders of the community .
the churches and mosques that remain an integral part of the communities need to be fully engaged in both the development and application of the programs .
involving the church has been shown to be successful by a program started by one ap community that caters to members infected with hiv by finding housing for them , directing them to the health centers , allocating jobs for them , having english classes , and providing social support ( personal communication ) .
providers need a better understanding of the american health care system , along with a vigilance to recognize and address the cultural sensitivities surrounding hiv risk factors and risk perception in these populations .
this cultural awareness can produce the educational tools which can be used to combat stigmatization and discrimination of those infected and affected by hiv , while equilibrating the power balance between men and women within the ap and fp communities . | objectives to describe the social and cultural differences between anglophone and francophone african immigrants which define the impediments that francophone african immigrants face trying to access health and human services in philadelphia , pennsylvania . methods surveys and personal interviews were administered to participants in social events , community meetings , and health centers .
a chi - squared analysis was used to contrast the communities .
results francophone africans demonstrated less acculturation , education , english fluency , and more legal documentation problems , and thus face greater challenges accessing health care .
anglophone africans had a higher level of acculturation , fewer language problems , and perceived fewer barriers in accessing health care than francophone africans .
conclusions educating new immigrants , through a more culturally sensitive infectious disease treatment and prevention program , is integral to achieving a higher access and utilization rates of available services ; especially in recent francophone immigrants . a larger study is needed to extend the findings to other cities where immigrants with similar backgrounds or acculturation issues reside
. | Background
Methods
Statistical Analyses
Results
Site Visit
Discussion
Limitations
Conclusion |
dna origami robots were designed using cadnano 2.0 ( http://cadnano.org ) , and folded using m13mp18 ssdna as scaffold strand in tae buffer containing 10 mm magnesium .
robots were imaged using tem and afm , and their states were measured using flow cytometry .
adult blaberus discoidalis were maintained at constant room temperature in humid plastic cages with dry pet food and fresh fruit ad libitum .
robots ( typically 0.1 - 3 pmol in 10 ul buffer ) were injected into the animal hemocoel between the two last abdominal sternites .
hemolymph was extracted through the arthrodial membrane and mixed with anticoagulant buffer before hemocytes analysis . for detailed methods | biological systems are collections of discrete molecular objects that move around and collide with each other .
cells carry out elaborate processes by precisely controlling these collisions , but developing artificial machines that can interface with and control such interactions remains a significant challenge .
dna is a natural substrate for computing and has been used to implement a diverse set of mathematical problems1 - 3 , logic circuits4 - 6 and robotics7 - 9 .
the molecule also naturally interfaces with living systems , and different forms of dna - based biocomputing have previously been demonstrated10 - 13 . here we show that dna origami14 - 16 can be used to fabricate nanoscale robots that are capable of dynamically interacting with each other17 - 18 in a living animal .
the interactions generate logical outputs , which are relayed to switch molecular payloads on or off . as a proof - of - principle
, we use the system to create architectures that emulate various logic gates ( and , or , xor , nand , not , cnot , and a half adder ) . following an ex vivo prototyping phase
, we successfully employed the dna origami robots in living cockroaches ( blaberus discoidalis ) to control a molecule that targets the cells of the animal . | Methods summary
Supplementary Material |
according to the world health organization ( who ) , depression was ranked as the third leading cause of the global burden of disease in 2004 and will move into the first place by 2030 .
worldwide , the fifth contributor to the burden of disease in 2020 is predicted to be copd.1,2 as defined by american thoracic society ( ats ) and european respiratory society ( ers ) , copd is a preventable and treatable disease state characterized by airflow limitation that is not fully reversible .
the airflow limitation is usually progressive and is associated with an abnormal inflammatory response of the lungs to noxious particles or gases , primarily caused by cigarette smoking .
chronic bronchitis is defined clinically as chronic productive cough for 3 months in each of 2 successive years in a patient in whom other causes of productive chronic cough have been excluded .
emphysema is defined pathologically as the presence of permanent enlargement of the airspaces distal to the terminal bronchioles , accompanied by destruction of their walls and without obvious fibrosis.3 several clinical studies show high rates of anxiety ( 21%96%)4 and affective ( mostly depressive ) disorders ( 27%79% ) in copd,5,6 which can affect prognostic evolution in terms of compliance , disability , and mortality.7 it is not easy to diagnose depression in copd patients because of overlapping symptoms between copd and depression .
severe copd could lead to somatic symptoms that are difficult to distinguish from depressive symptoms : eg , change in appetite / weight versus tearfulness / depressed appearance ; sleep disturbances versus social withdrawal ; fatigue or loss of energy versus brooding / pessimism ; and diminished ability to think or concentrate versus lack of reactivity to environmental events .
the validity of the international classification of diseases ( icd)-10 and diagnostic and statistical manual of mental disorders ( dsm)-5 criteria for depression , in fact , may to a certain degree be questioned , because it is difficult to decide when somatic symptoms are secondary to depression and when they are secondary to copd.8 additional variables that have potential to confound the association between copd and depression regard some common risk factors , including the subject s demographic characteristics ( age , sex , race / ethnicity , and marital status ) , education , body mass index ( bmi ) , alcohol consumption , tobacco use , socioeconomic status ( income ) , respiratory symptoms ( dyspnea and difficulty with walking ) , and comorbid chronic conditions such as heart disease , stroke , diabetes , arthritis , hypertension , congestive heart failure , and cancer.9 the nature of the relationship between copd and depression is not clear . however , it is well documented that depressed patients show a markedly elevated rate of cigarette smoking,10 which is the most important risk factor for copd .
it has been also postulated that ( mild ) hypoxia that is also present in copd patients can induce central neurobiological changes that predispose to depression and suicide.11 furthermore , in a study with a large sample , a relevant percentage of copd patients entering pulmonary rehabilitation experienced clinically relevant symptoms of anxiety and depression.12 however , it could be supposed that both copd and depression are the clinical manifestations of a common underlying pathophysiologic process . indeed , data in the literature show that copd and depression might not be comorbid conditions per se , but rather both are pathophysiologically related with a common substrate.1326 some authors suggest considering copd as a chronic systemic inflammatory syndrome rather than a respiratory disease associated with extrapulmonary symptoms ( including neurocognitive deficits , depression , anxiety , sleep disturbance , and other non - mental chronic disorders , such as coronary and peripheral artery diseases , anemia , osteoporosis , and rheumatoid arthritis).14 some characteristics commonly observed in copd patients , like obesity,27 are indeed associated with inflammation processes related to depression and anxiety,28 suggesting a complex bidirectional association between adiposity and depression.26 in this field , as suggested by some authors , the reduction of adiposity through physical exercise seems related to significant changes in depressive symptoms.29 as a consequence , ats and ers guidelines have emphasized the importance of pulmonary rehabilitation for patients with copd.30 several authors have indeed shown that there is a strong correlation among physical activity , adiposity , and mental health : several data have suggested that aerobic exercise shows significant effects , comparable to pharmacotherapy3133 and to psychotherapy,34 on reducing depressive symptomatology.35 coventry et al specifically demonstrated that structured exercise training significantly reduces both symptoms of depression and anxiety in people with copd ; furthermore , they showed that structured exercise training is more effective than other , complex psychological , behavioral and lifestyle interventions previously thought to improve mental health in people with copd.36 pulmonary rehabilitation shows poor effects on lung physiology ( eg , ventilation parameters ) ; nevertheless , it improves dyspnea , performance , anxiety , depressive symptoms , and quality of life ( qol).3742 it also seems to decrease secretory function of adipocytes , resulting in reduction of their volume measured through fat mass.43 in this regard , although bmi is widely used as an indirect parameter to assess adiposity , it has been demonstrated that bmi would have a poor correlation with fat mass , which probably represents a more accurate measure.43 considering the impact of aerobic exercise on anxious and depressive symptomatology , physical performance , qol , and adiposity ( assessed with bmi and fat mass ) , the primary aim of this study was to evaluate the effects of aerobic exercise , proposed in the context of pulmonary rehabilitation , through the correlation of psychopathological variables ( depressive and anxious symptoms ) and physical / pneumological parameters .
the secondary endpoint regards the evaluation of potential improvement in qol and in cognitive performance .
this prospective cohort study was carried out in pneumology and psychiatry wards in the university hospital policlinico - vittorio emanuele of catania , italy from january to june 2012 .
g. rodolico university hospital , in accordance with the code of ethics of the world medical association , and written informed consent was obtained by all participants .
inclusion criteria were diagnosis of copd and eligibility to perform pulmonary rehabilitation , according to ats and ers guidelines.44 exclusion criteria were indication for restoring treatment of airway patency and current psychopharmacological treatment ( including sleeping pills ) .
fifty - two consecutive subjects ( 46 males and six females ) were enrolled , with a mean age of 71.56.29 years and a mean school education of 8.083.21 years . enrolled
patients started a 6-week rehabilitation protocol , consisting of lower limb / limbs ( treadmill and exercise bike ) and upper art ( arm ergometer davenbike ) training , as well as calisthenic exercises with increasing intensity from a minimum of 80% to a maximum workload for lower arts in a minimum time .
as proposed by the lung specialists in the pneumology ward of our university hospital , training sessions went on for 2 hours over 5 days a week and were supervised by two physiotherapists and a lung specialist . to exclude potential bias ,
no change in diet regime was carried out during the full duration of the trial .
patients were evaluated at first access to pulmonary rehabilitation outpatient wards in order to assess inclusion and exclusion criteria . for eligible patients , psychometric
assessment , completion of the preliminary investigation , and admission in the rehabilitation program were performed .
the assessment , measured both at baseline and at the end of the 3-month rehabilitation program , consisted of spirometry with bronchodilator test , walking test ( 6-minute walk test [ 6mwt ] ) , bmi , medical research council ( mrc ) dyspnea scale , impedentiometry , motor and metabolic evaluation by sensewear armband ( for 3 consecutive days ) , hopkins symptom checklist 90 ( scl-90 ) , hamilton depression rating scale ( ham - d ) , hospital anxiety and depression scale ( subscale hads - a for anxiety ; subscale hads - d for depression ) , and state - trait anxiety inventory ( stai-1 and stai-2 ) .
cognitive functions were also evaluated using raven s colored progressive matrices ( cpm ) , rey auditory verbal learning test ( ravlt ) , clock drawing test ( cdt ) , and trail making test parts a , b , and ba ( tmt - a , tmt - b , and tmt - ba ) .
according to the ham - d score ( cutoff = 8) , the total sample was divided into two subgroups : depression - positive ( d ) and depression - negative ( nd ) subjects .
a two - tailed test was used to assess statistical significance ; a p - value of < 0.05 was considered significant and a p - value of < 0.01 was considered highly significant .
statistical analysis was performed using the intercooled stata 8 for windows software ( statacorp lp , college station , tx , usa ) .
the descriptive analysis of psychometric and pneumological parameters at baseline and at the end of the rehabilitation program ( at the end of month 3 ) are presented in table 1 .
data show that improvement in all pneumological and respiratory parameters was statistically significant , except for tiffeneau index .
the sample was divided into two subgroups consisting of 38 d and 14 nd subjects according to ham - d .
the mean and standard deviation of psychometric and pneumological parameters at baseline and month 3 in both patient subgroups are shown in table 2 .
we did not find a statically significant difference between the two subgroups in age , lung function , and baseline body composition . regarding the mean bmi at baseline , the d subgroup was overweight ( 24/38 patients , = 63% , had a mean bmi 28.376.80 ) and the nd subgroup was normal weight ( 4/14 patients , = 28% , had a mean bmi 24.964.1 ) ( figures 1 and 2 ) , according to who criteria,45 even if the absolute difference in bmi between the two subgroups was not statistically significant .
pneumological and psychological parameters overlapped except for ham - d ( p=0.002 ) , hads - a ( p=0.03 ) , scl-90 ( p=0.02 ) , and tmt - a ( p=0.02 ) : these differences disappeared at 3 months . at baseline 73% of subjects ( 38/52 ) exceeded the cutoff for depression on the ham - d ( d subgroup ) . at month 3
, the rate of d subjects was reduced from 73% to 7.7% ( 4/52 , odds ratio 6.6 , p=0.0002 ) .
the improvement in almost all other psychological and medical / pneumological parameters was also significant both in the d and nd subgroups at the end of the study .
we compared reported differences following the rehabilitation treatment between the d and nd subgroups ( table 3 ) ; the following parameters showed significant improvements :
ham - d ( d subgroup : 92.19 , nd subgroup : 3.146.20 , p=0.02);hads - a ( d subgroup : 3.842.5 , nd subgroup : 1:421.98 , p=0.03 ) ; andscl-90 ( d subgroup : 0.4860.287 , nd subgroup : 0.2470.177 , p=0.05 ) ( table 3 ) .
ham - d ( d subgroup : 92.19 , nd subgroup : 3.146.20 , p=0.02 ) ; hads - a ( d subgroup : 3.842.5 , nd subgroup : 1:421.98 , p=0.03 ) ; and scl-90 ( d subgroup : 0.4860.287 , nd subgroup : 0.2470.177 , p=0.05 ) ( table 3 ) .
a significant correlation between psychometric ( in particular , hads - a , hads - d , stai-2 , scl-90 , tmt - b , tmt - ba , and cpm ) and internistic parameters was found , especially with fat mass and bmi in the total population : the reduction in fat mass and bmi was related to decreased anxiety and depression and improved qol and cognitive performance ( tables 4 and 5 ; figures 36 ) . at baseline in group d , a significant correlation was present between bmi and the following psychometric variables : hads - a ( r=0.47 , p=0.04 ) ; hads - d ( r=0.5 , p=0.02 ) ; scl-90 ( r=0.56 , p=0.01 ) ; stai-2 ( r=0.51 , p=0.02 ) ; tmt - a ( r=0.52 , p=0.02 ) ; tmt - b ( r=0.44 , p=0.05 ) ; ravlt ( r=0.49 , p=0.03 ) ; and cpm ( r=0.63 , p=0.003 ) ( tables 4 and 5 ; figures 5 and 6 ) .
a significant correlation between bmi and fat mass was present at baseline in subgroup d ( r=0.8667 , p=0.0000 ) but not in subgroup nd ( r=0.3929 , p=0.3833 ) . in subgroup
d , the reduction in bmi was significantly related with fat mass ( r=0.6072 , p=0.005 ) , but the same could not be affirmed for patients of the nd subgroup ( tables 4 and 5 ) .
in agreement with other findings,5,6 we found a high prevalence ( 73% ) of patients with depressive symptoms at baseline in our copd subjects that showed a significant decrease at the end of the rehabilitation program ( 7.7% ) .
previous reviews on pulmonary rehabilitation have indicated that 4-week programs can improve fatigue and emotional function , but these reviews included trials that did not specifically address effects on anxiety and depression,36,46 while in our results , the mean ham - d scores ( used in our research to divide the whole population into the two subgroups and for the evaluation of the changes of depressive symptoms ) decreased significantly both in the total sample and in the d and nd subgroups .
another study examined the relationship between depression and aerobic exercise in generic chronic illness patients;47 some others found a correlation between depression and/or anxiety and aerobic exercise in copd patients undergoing pulmonary rehabilitation , but they did not investigate variables such as bmi or obesity.36,4851 moreover , in our trial , all other assessed psychiatric ( depression , anxiety , and cognitive function ) and medical / pneumological parameters demonstrated a significant improvement after the rehabilitation program .
the evaluation of cognitive performances , improved at 3 months , showed some peculiarities : the improvement in visual - spatial attention ( tmt - a ) , although significant , was modest if compared with the tmt - b parameter , which assesses attention shifting ability and visual - spatial attention , as summarized by tmt - ba that represents the difference between the times of part b and those of part a in executive functioning ( table 1 ) .
memory , assessed by ravlt , was significantly enhanced at 3 months both in immediate and in deferred recall ( table 1 ) , such as praxic skills , mental representation , and planning ( assessed by cdt ) and deduction skills ( evaluated through cpm ) ( table 1 ) .
we highlight the interesting correlations among stai-1 , mrc , and bode ( bmi , obstruction , dyspnea , exercise capacity ) index : decrease in anxiety correlated with the improvement of dyspnea ( mrc ) and bode index in the absence of correlation with objective indices such as forced expiratory volume in the 1st second , bmi , and 6mwt ; this fact could be explained by the tendency of anxious depressed patients to amplify dyspnea.52 the improvement in psychological parameters ( anxiety and depressive symptoms , qol , and cognitive performances ) was related to a general decrease of fat mass and bmi in the total sample ( tables 4 and 5 ) . in subgroup d , this correlation was particularly evident between bmi and the following psychometric variables : hads - a , hads - d , scl-90 , stai-2 , tmt - a , tmt - b , ravlt , and cpm ( table 4 ) . despite the general decrease of fat mass and bmi in the total sample , it was not statistically significant in subgroup nd , for which , unlike subgroup d ( table 5 ) , there was no correlation between bmi and fat mass , both at baseline and after the rehabilitation program . in this sense , bmi alone could be insufficient to explain the correlation with psycho - pathological parameters .
the different results observed between the two subgroups could be explained by the fact that d - subgroup patients presented a baseline condition of overweight and a higher fat mass percentage , not recorded in the nd subgroup , which was mainly composed of persons of normal weight .
the only factor that indeed correlated with the majority of psychometric parameters was bmi , but only in subgroup d , where we recorded a correlation between bmi and fat mass . in spite of the interesting findings , at least three limitations must be considered .
first , metabolic and neurobiological analysis ( eg , inflammatory and oxidative markers ) could explain the link between anxiety and depressive symptoms and internistic medical disorders , such as copd , in order to define a new meaning and management of them , but in this study was not performed .
second , the lack of muscle mass evaluation could influence depressive and anxious symptoms.53 third , our sample size was small .
so further studies are needed to evaluate the link between psychopathological symptoms and copd in terms of inflammatory and oxidative markers with specific measures of them and the impact of muscle wasting on depression and anxiety in this specific population and , at the end , to augment the power of statistical analysis .
our results suggest that it might be useful , in the context of multiple chronic diseases , to start a specific rehabilitation program ( eg , of aerobic exercise ) in order to establish a more appropriate and integrated therapeutic approach where there is suspicion , that needs to be confirmed , that a condition of increased adiposity could influence the genesis and evolution of psychiatric disorders . | backgroundseveral clinical studies suggest common underlying pathogenetic mechanisms of copd and depressive / anxiety disorders .
we aim to evaluate psychopathological and physical effects of aerobic exercise , proposed in the context of pulmonary rehabilitation , in a sample of copd patients , through the correlation of some psychopathological variables and physical / pneumological parameters.methodsfifty-two consecutive subjects were enrolled . at baseline , the sample was divided into two subgroups consisting of 38 depression - positive and 14 depression - negative subjects according to the hamilton depression rating scale ( ham - d ) .
after the rehabilitation treatment , we compared psychometric and physical examinations between the two groups.resultsthe differences after the rehabilitation program in all assessed parameters demonstrated a significant improvement in psychiatric and pneumological conditions .
the reduction of bmi was significantly correlated with fat mass but only in the depression - positive patients.conclusionour results suggest that pulmonary rehabilitation improves depressive and anxiety symptoms in copd .
this improvement is significantly related to the reduction of fat mass and bmi only in depressed copd patients , in whom these parameters were related at baseline .
these findings suggest that depressed copd patients could benefit from a rehabilitation program in the context of a multidisciplinary approach . | Introduction
Materials and methods
Results
Discussion
Conclusion |
multiple etiological factors implicated such as exposure to ultraviolet radiation , pregnancy , contraceptive pills , hormone replacement therapy , cosmetics , phototoxic and anti - seizure medications .
in addition to the uv light itself , photo - induced hormones , growth factors , and chemical mediators of inflammation , which influence the function of melanocytes directly or indirectly , might contribute to the uv - induced pigmentation .
different treatment modalities such as topical depigmenting agents , chemical peels , dermabrasion and laser therapies have been utilised in different studies with varying , not so satisfactory outcomes .
the results of recent clinical trials using localised intradermal microinjections of tranexamic acid ( ta ) and transepidermal delivery of ta using microneedling in the treatment of melasma are promising . in this study
, we compare intradermal microinjections to microneedling in transepidermal delivery of ta in melasma patients .
a prospective , randomised , open - label study was conducted with a sample size of 60 , thirty each in each of the treatment arms , for 1 year from march 2010 to march 2011 , after approval from the ethical committee of the institutional review board ( irb ) .
adult males and females between 18 and 50 years of age with moderate - to - severe bilaterally symmetrical distribution of melasma were included in the study after obtaining written informed consent .
pregnant / lactating females , patients on hormone replacement therapy or oral contraceptives , history of bleeding disorders , concomitant use of anticoagulants , any known drug allergy especially to the study drug , associated medical illnesses , and history of any other depigmenting treatment in the past 1 month were excluded from the study . after obtaining detailed personal and medical history ,
a modified masi scoring system was used to assess the severity of melasma [ figure 1 ] . modified masi scoring
about 4 mg of ta is drawn in a 100 u / ml insulin syringe ( about 4 units ) and diluted with normal saline up to 1 ml ( up to 100 u ) to get a concentration of 4 mg / ml of ta .
after gentle cleansing , topical emla cream was applied over the area to be treated for about 45 to 60 min .
the subjects in arm 1 were treated with multiple microinjections of ta ( 4 mg / ml ) intradermally into the melasma lesions at 1 cm intervals , depending on the extent of involvement , to a maximum of 8 mg to the entire affected area , using a 100 u / ml insulin syringe with a 4-mm mesoneedle .
the microneedles used had a width of 2 cm , studded with 192 fine needles of medical - graded stainless steel .
the needle length was 1.5 mm and diameter 0.25 mm . according to the pressure applied , the needles penetrate the skin from 0.1 to 1.3 mm .
for the subjects in arm 2 , the skin was stretched and microneedling was carried out in vertical , horizontal , and both diagonal directions for about four to five times .
tranexamic acid , 0.5 to 1 ml ( 4 mg / ml ) , was applied over this area , and the procedure was repeated four to five times in the above - said directions .
the procedures were done three times at monthly intervals ( 0 , 4 and 8 weeks ) and followed up for further 3 months at monthly intervals . to assess the clinical response ,
masi scoring , physician global assessment ( pga ) , and patient global assessment ( ptga ) were performed at monthly intervals and any adverse events and complications were recorded . the response to treatment in each patient
was graded at the end of the study : no response , no improvement ; mild response , < 25% improvement ; moderate response , 25% to < 50% improvement ; good response , 50% to < 75% improvement ; very good response , > 75% improvement .
each case was followed up for 3 months to look for further improvement / relapse , if any .
the kruskal wallis ( nonparametric anova ) test was used to compare the means of masi scores at each visit and follow up after treatment in each group .
the unpaired t - test with welch correction was used to compare means of masi scores between the microinjections and microneedling group .
out of 60 patients in the study , 41 ( 68.33% ) patients belonged to the age group 3050 years and the number of females ( 54 ) was more compared to males ( 6 ) in both the treatment arms [ table 1 ] .
most patients had a centrofacial or malar pattern of distribution of melasma [ table 2 ] .
forty - six ( 80% ) patients had mixed type of melasma , 5 ( 8.33% ) had epidermal type and 7 ( 11.67% ) had dermal type [ table 2 ] .
twenty - one ( 70% ) patients had a history of melasma in their family .
seven ( 11.67% ) in the microinjection group and 8 ( 13.33% ) in the microneedling group had undertaken treatment for melasma in the form of chemical peels , skin - lightening agents and some native medicines in the past .
two patients ( 3.33% ) in the microinjection group and 3 ( 5% ) in the microneedling group had concomitant dermatological disease such as dermatosis papulosa nigra , freckles and palmoplantar psoriasis .
fifteen patients in the microinjection group and 16 in the microneedling group were using moisturisers .
eight ( 26.67% ) patients dropped out from the study , 7 ( 23.34% ) in the microinjection group and only 1 ( 3.33% ) in the microneedling group , for unknown reasons .
age and sex distribution fitzpatrick skin type and patterns of melasma the total masi score in the microinjection group reduced from 159.3 ( masib ) at the first visit to 113.7 ( masi2 ) at the third visit , to 102.4 ( masi5 ) at the end of third follow - up . in the microneedling group ,
the total masi score was 264.1 ( masib ) at the first visit , which reduced to 156.9 ( masi2 ) at the third visit and 146.8 ( masi5 ) at the end of third follow - up .
the mean masi scores of all the visits and follow - ups in both the treatment arms are shown in table 3 . in the microinjection group ,
the mean masi score at the base line was 6.93 2.16 ( masib ) and at the end of third follow - up ( masi5 ) was 4.45 1.69 , which is 35.72% improvement with p < 0.01 . in the microneedling group ,
the mean masi score at the baseline was 9.11 4.09 ( masib ) and at the end of third follow - up ( masi5 ) was 5.06 2.14 , which is 44.41% improvement with p < 0.001 [ figure 2 ] .
there was no significant difference in the means of the masi scores between the microinjection and microneedling group with the two - tailed p = 0.299 .
mean masi scores , percentage improvement , and p value of both the groups comparative graph depicting the decline in masi score in both groups six ( 26.09% ) patients in the microinjection group showed more than 50% improvement and 12 ( 41.38% ) patients in the microneedling group showed more than 50% improvement .
of these , two ( 6.90% ) patients showed > 75% improvement [ table 4 ] .
percentage improvement of masi scores in both the groups total pga and ptga scores at different visits in the microinjection and microneedling groups clinical improvement is seen in the comparative photographs [ figures 4 and 5 ] treatment with microneedling : before and after treatment with microinjections : before and after no serious side effects apart from mild discomfort , burning sensation and erythema were observed , which lasted for 1 or 2 days in most patients [ table 5 ] .
recent studies have revealed that topical trans-4-(aminomethyl ) cyclohexanecarboxylic acid ( trans - amcha , ta ) , a plasmin inhibitor , prevents uv - induced pigmentation in guinea pigs .
topical trans - amcha inhibits uv - induced plasmin activity in keratinocytes by preventing the binding of plasminogen to the keratinocytes , which ultimately results in decreased free arachidonic acid and a diminished ability to produce prostaglandins , which in turn decrease melanocyte tyrosinase activity .
human keratinocytes secrete the urokinase - type plasminogen activator , which increases the activity of melanocytes in vitro
. the blockade of this effect may be due to the mechanism by which ta reduces hyperpigmentation in melasma patients . by injecting ta intradermally
, it may be possible to treat the dermal - type melasma in addition to the mixed type .
this technology involves the creation of channels in the skin with micron - sized dimensions , thereby enabling the delivery of a broad range of therapeutic molecules including proteins which would not otherwise cross intact skin .
localised microinjections , the so - called mesotherapy , were introduced in france by pistor .
this technique consists of intradermal or subcutaneous microinjections of 0.05 to 0.1 ml of highly diluted drug mixtures or a single drug , at the sites of body having medical or aesthetic problems .
it is aimed at applying an adequate amount of medication directly at the problematic area and avoiding oral medications .
furthermore , direct injection to the involved sites allows lower dosage of drugs to be used .
the microneedling device is a simple , handheld instrument consisting of a handle with a cylinder studded all around with fine , stainless steel needles of 0.5 to 2 mm in length . to achieve therapeutic benefits ,
this needle - studded cylinder is rolled on the skin in multiple directions to create microchannels .
it is currently used in the cosmetic industry to treat several skin conditions such as pigmentation problems , wrinkles , acne and post - burn scars , and also in facial rejuvenation as a part of collagen induction therapy . in this study , we have used localised microinjections ( mesotherapy ) and microneedling to deliver the study drug ta .
we compared both these modes of drug delivery , the safety and efficacy in the treatment of melasma .
the masi scores , ptga , and pga showed a significant decreasing trend from the baseline to the fourth , eighth , and twelfth week of treatment with ta in both the treatment arms .
the scores showed better improvement in patients treated with microneedling than with microinjections though the difference was not statistically significant .
this can be attributed to the fact that microneedling delivers the medication more evenly and deeper into the skin .
all scores remained almost the same during the next 3 months of follow - up .
on the basis of the findings , ta can be used as potentially a safe , effective , and promising therapeutic agent for the treatment of melasma .
it is an office - based procedure with relatively quick results , no significant side effects , and almost no downtime . | background : melasma is a common cause of facial hyperpigmentation with significant cosmetic deformity . although several treatment modalities are available , none is satisfactory.aim:to compare the therapeutic efficacy and safety of tranexamic acid ( ta ) microinjections versus tranexamic acid with microneedling in melasma.materials and methods : this is a prospective , randomised , open - label study with a sample size of 60 ; 30 in each treatment arms .
thirty patients were administered with localised microinjections of ta in one arm , and other 30 with ta with microneedling .
the procedure was done at monthly intervals ( 0 , 4 and 8 weeks ) and followed up for three consecutive months .
clinical images were taken at each visit including modified melasma area severity index masi scoring , patient global assessment and physician global assessment to assess the clinical response.results:in the microinjection group , there was 35.72% improvement in the masi score compared to 44.41% in the microneedling group , at the end of third follow - up visit .
six patients ( 26.09% ) in the microinjections group , as compared to 12 patients ( 41.38% ) in the microneedling group , showed more than 50% improvement .
however , there were no major adverse events observed in both the treatment groups.conclusions:on the basis of these results , ta can be used as potentially a new , effective , safe and promising therapeutic agent in melasma .
the medication is easily available and affordable .
better therapeutic response to treatment in the microneedling group could be attributed to the deeper and uniform delivery of the medication through microchannels created by microneedling . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
due to the changes in work demands of the nursing faculties and clinical setting , nursing teachers are having difficulties fulfilling multiple roles expectations and balancing the various dimensions of their roles ( 1 ) .
nursing teachers inevitably endure a lot of stress ( 2 ) , while complex work - related requirements and role necessities are beyond one s performance .
if they can not conduct those role expectations and obligations , it will result in serious problems in role execution which affects both their personal life and teaching outcomes ( 2 , 3 ) .
goode believed that multiple role obligations and expectations can be a source of role strain ( 4 ) . according to mobily
, role strain is characterized as a subjective status of distress experienced by a role occupant when exposed to role stress
( 5 ) and any person with having multiple roles may be experienced it ( 6 ) .
many studies reported that role stress and strain may lead to physical and emotional problems such as cardiovascular diseases , tension , anxiety , dissatisfaction and depression which are experienced by nursing teachers while facing different work demands and role expectations in a work setting ( 1 - 8 ) . with regard to importance of role strain , role strain scale ( rss )
it is a comprehensive instrument used to detect and predict the role problems and work - related stress situations in nursing teacher s .
mobily s scale could appeal to other researchers and has been implemented in other parts of the u.s and adopted cross - culturally in other countries ( 3 , 4 , 9 , 10 ) .
role strain scale is based on a theoretical framework namely role stress and strain which included three main roots : structure role theory , social exchange theory , and symbolic interaction theory ( 11 ) .
this scale as a self - report questionnaire using the likert scale includes two sections : demographic variables such as age , gender , marital status , work experience and academic degree , and main part consisting of 44 statements of role strain showing stress - causing situations which the respondents usually face .
the structural model of the original scale contains seven factors : inter - sender conflict ( four items ) , intra - sender conflict ( nine items ) , inter - role conflict ( four items ) , role ambiguity ( seven items ) , role overload ( eight items ) , role incongruity ( seven items ) and role incompetence ( five items ) ( 5 - 11 ) .
surprisingly , no research has been conducted in iran because no localized comprehensive scale exists for exploring role strain .
since a scale can be a culture - bound concept , scales from foreign researches could not be used directly due to the fact that cultural bias necessitates assessing psychometric scale ( 12 - 14 ) .
therefore , researchers decided to obtain a comprehensive and psychometrical scale for assessing the role strain in academic population in iran .
the objective of this study was psychometric evaluation of the role strain scale ( rss ) and confirms its structural model in an iranian population .
the ethical approval was achieved from the research ethics committee of shahid beheshti university of medical sciences ( code 1391 - 1 - 86 - 10827 ) .
ethical considerations have been taken into account which contains explanations about the research aims to participants and getting consent notes from participants consciously to participate in the research , and the fact that participation is optional and giving assurance to participants about information confidentiality .
the research design included translation procedure and psychometric evaluation consisting of pilot testing of the face and content validity , assessing the construct validity and reliability .
rode believed that paying careful attention to the process of psychometric methods and cultural adaptation will ensure the validity and reliability of the scale ( 13 ) .
cultural adaptation is aimed at providing compatibility between the original version of the scale and the new one regarding face , content validity ( 15 ) . a common method to prepare scales for cross - cultural adaptation
there are not standard guidelines for the translation of the scale ( 17 ) .
protocol in the process of translation which included : forward translation , reconciliation , back translation , back translation review , harmonization , cognitive debriefing , results and finalization , and final report ( 18 ) .
initially , the role strain scale was translated by two professional and experienced persian translators from english to persian .
the translations were assessed in terms of content and connotation and semantic clarity by the research team and english teachers . using slight changes ,
the final translated persian version of the scale was approved with the most suitable translated phrases .
next , it was once again translated into english by another translator to keep the originality of the main version and its backward translation to english was compared and confirmed by mobily with the original english version so that it was the same as the original scale , both conceptually and linguistically .
first , to evaluate qualitative face validity , translated persian version of rss was piloted with 10 nursing teachers as a target group ( 5 females and 5 males ) .
after the nursing teachers individually completed the questionnaire , they were asked to express their overall perceptions in responding to the statements of questionnaire . moreover , to assess face validity of the translated persian version quantitatively , the impact score calculated for each of the 44 items of the scale .
a 5-point likert scale was considered in which the choice of always standing for the most important strain was scored as 5 and never , which means the least important strain , was scored as 1 . through implementing the formula ,
items impact scores were calculated ( 19 , 20 ) . for qualitative content validity , through interview with 15 experts in the field of nursing and behavioral science , the experts were asked to express their editing ideas ( i.e. grammar , wording and scaling of the scale ) . for quantitative content validity , to assess content validity index ( cvi ) , the experts were also asked to assess relevancy , simplicity and clarity of each item with the content according to waltz and bausell index , values for cvi are as follows : < 0.70 : unacceptable ; 0.7 - 0.78 : revision and correction ; 0.79 : accepted ( 21 ) . to establish construct validity , confirmatory factor analysis ( cfa )
was conducted by lisrel program to ensure that the factor structure of translated persian version of rss is similar to the original version .
lisrel and amos are programs that can conduct confirmatory factor analysis to release model fit indices ( 22 ) .
severinsson reported if fit indices could not fit to possible data model , confirmatory factor analysis changes into exploratory factor analysis ( efa ) ( 17 ) .
fit indices include : the chi - squared goodness - of - fit test ( /df ) 3 , the goodness - of - fit index ( gfi ) 0.9 , the adjusted goodness - of - fit index ( agfi ) 0.9 , the normed fit index ( nfi ) > 0.9 as well as the non - normed fit index ( nnfi ) > 0.9 , root mean square error of approximation ( rmsea ) < 0.1 and comparative fit index ( cfi ) > 0.9 ( 23 ) .
cfa is a pattern of structural equation modeling ( sem ) ( 17 ) .
prerequisite of cfa is determining sample size , there are differences in recommendations of an acceptable sample size for factor analysis , and some authors recommend five person per item and others ten ( 24 , 25 ) . in this study , the recommendent samples for 44 items were among 220 to 440 . to get samples of factor analysis , stratified cluster sampling has been used . to do so
, the country has been divided geographically into north , south , west and center as strata and medical sciences universities ( nursing colleges ) , as cluster , some of the nursing colleges have been selected randomly from among all nursing colleges of the country ( n = 23 ) .
then all nursing teachers of these colleges who qualified the criteria of this study were given the questionnaire .
these criteria include having at least one year of work experience as nursing teacher in clinical and theoretical teaching , in the field of research and being official authorities in the college .
hence , the questionnaire was not given to those who did not meet these criteria .
regarding lisrel program is sensitive to normal assumption ( 23 ) ; kolmogorov - smirnov test has proved data normality .
values of cronbach s 0.70 were explained as satisfactory internal consistency ( 26 , 27 ) . furthermore , to assess the stability reliability of test -re - test , and intraclass correlation coefficient ( icc ) , 30 nursing teachers completed the persian version of rss in two - week intervals .
icc values of 0.40 or above were considered satisfactory ( r 0.81 - 1.0 as excellent , 0.61- 0.80 very good , 0.41 - 0.60 good , 0.21 - 0.40 fair , and 0.0 - 0.20 poor ) ( 28 ) .
, chicago , usa ) and confirmatory factor analysis was performed with lisrel version 8.8 .
the ethical approval was achieved from the research ethics committee of shahid beheshti university of medical sciences ( code 1391 - 1 - 86 - 10827 ) .
ethical considerations have been taken into account which contains explanations about the research aims to participants and getting consent notes from participants consciously to participate in the research , and the fact that participation is optional and giving assurance to participants about information confidentiality .
the research design included translation procedure and psychometric evaluation consisting of pilot testing of the face and content validity , assessing the construct validity and reliability .
rode believed that paying careful attention to the process of psychometric methods and cultural adaptation will ensure the validity and reliability of the scale ( 13 ) .
cultural adaptation is aimed at providing compatibility between the original version of the scale and the new one regarding face , content validity ( 15 ) . a common method to prepare scales for cross - cultural adaptation
severinsson reported that there are not standard guidelines for the translation of the scale ( 17 ) .
protocol in the process of translation which included : forward translation , reconciliation , back translation , back translation review , harmonization , cognitive debriefing , results and finalization , and final report ( 18 ) .
initially , the role strain scale was translated by two professional and experienced persian translators from english to persian .
the translations were assessed in terms of content and connotation and semantic clarity by the research team and english teachers . using slight changes ,
the final translated persian version of the scale was approved with the most suitable translated phrases .
next , it was once again translated into english by another translator to keep the originality of the main version and its backward translation to english was compared and confirmed by mobily with the original english version so that it was the same as the original scale , both conceptually and linguistically .
first , to evaluate qualitative face validity , translated persian version of rss was piloted with 10 nursing teachers as a target group ( 5 females and 5 males ) .
after the nursing teachers individually completed the questionnaire , they were asked to express their overall perceptions in responding to the statements of questionnaire . moreover , to assess face validity of the translated persian version quantitatively , the impact score calculated for each of the 44 items of the scale .
a 5-point likert scale was considered in which the choice of always standing for the most important strain was scored as 5 and never , which means the least important strain , was scored as 1 . through implementing the formula ,
items impact scores were calculated ( 19 , 20 ) . for qualitative content validity , through interview with 15 experts in the field of nursing and behavioral science , the experts were asked to express their editing ideas ( i.e. grammar , wording and scaling of the scale ) . for quantitative content validity , to assess content validity index ( cvi ) , the experts were also asked to assess relevancy , simplicity and clarity of each item with the content according to waltz and bausell index , values for cvi are as follows : < 0.70 : unacceptable ; 0.7 - 0.78 : revision and correction ; 0.79 : accepted ( 21 ) .
to establish construct validity , confirmatory factor analysis ( cfa ) was conducted by lisrel program to ensure that the factor structure of translated persian version of rss is similar to the original version .
lisrel and amos are programs that can conduct confirmatory factor analysis to release model fit indices ( 22 ) .
severinsson reported if fit indices could not fit to possible data model , confirmatory factor analysis changes into exploratory factor analysis ( efa ) ( 17 ) .
fit indices include : the chi - squared goodness - of - fit test ( /df ) 3 , the goodness - of - fit index ( gfi ) 0.9 , the adjusted goodness - of - fit index ( agfi ) 0.9 , the normed fit index ( nfi ) > 0.9 as well as the non - normed fit index ( nnfi ) > 0.9 , root mean square error of approximation ( rmsea ) < 0.1 and comparative fit index ( cfi ) > 0.9 ( 23 ) .
prerequisite of cfa is determining sample size , there are differences in recommendations of an acceptable sample size for factor analysis , and some authors recommend five person per item and others ten ( 24 , 25 ) . in this study ,
the recommendent samples for 44 items were among 220 to 440 . to get samples of factor analysis ,
, the country has been divided geographically into north , south , west and center as strata and medical sciences universities ( nursing colleges ) , as cluster , some of the nursing colleges have been selected randomly from among all nursing colleges of the country ( n = 23 ) .
then all nursing teachers of these colleges who qualified the criteria of this study were given the questionnaire .
these criteria include having at least one year of work experience as nursing teacher in clinical and theoretical teaching , in the field of research and being official authorities in the college .
hence , the questionnaire was not given to those who did not meet these criteria .
regarding lisrel program is sensitive to normal assumption ( 23 ) ; kolmogorov - smirnov test has proved data normality .
values of cronbach s 0.70 were explained as satisfactory internal consistency ( 26 , 27 ) . furthermore , to assess the stability reliability of test -re - test , and intraclass correlation coefficient ( icc ) , 30 nursing teachers completed the persian version of rss in two - week intervals .
icc values of 0.40 or above were considered satisfactory ( r 0.81 - 1.0 as excellent , 0.61- 0.80 very good , 0.41 - 0.60 good , 0.21 - 0.40 fair , and 0.0 - 0.20 poor ) ( 28 ) .
data were analyzed using statistical package ( version 18 , spss inc . , chicago , usa ) and confirmatory factor analysis was performed with lisrel version 8.8 .
the results of the quantitative face validity revealed that all of the items had impact scores 1.5 . besides , in qualitative part , the nursing teachers ideas were satisfactory and approved all items of the scale . according to the expert panel opinions ,
qualitative content validity was approved with some slight changes ; and the cvi scores for all items were 0.80 .
the mean content validity index was 92% for relevancy , 85% for clarity and 88% for simplicity . in construct validity
, findings showed that among 375 nursing teachers , 302 nursing teachers completed the anonymous self - report questionnaire and returned it ( response rate = 80.5% ) .
participants who did not return the questionnaire were equally from all geographical areas which did not hurt the balance of sampling the research .
the mean age and work experience of nursing teachers was respectively 43.5 6.5 and 15.1 7.4 years .
table 1 according to the results of construct validity , cfa revealed that translated persian version of original rss was not good enough to fit .
all indices had poor goodness of fit , they were respectively , /df = 4.5 , cfi = 0.08 , gfi = 0.62 , agfi = 0.58 , nfi = 0.75 , nnfi = 0.79 and rmser = 0.12 .
as no fitness was found at confirmatory factor analysis , the only way was exploratory factor analysis to obtain the better model .
to do so , initially data was examined by the bartlett s test of sphericity to reach sample adequacy , [ bartlett s test of sphericity ; p < 0.001 = 6588.192 and kmo = 0.818 ] .
next , researchers performed several explorative factor analyses among the variables to identify the best model to fit , and finally a five - factor model was selected by item- to factor loading > 0.45 , applying varimax rotation , eigenvalues > 1 .
the number of items was deleted ( 11 items ; 2 , 5 , 6 , 18 , 21 , 22 , 36 , 8 , 20 , 43 , 44 ) and the rest of items were forced into a five factor solution . five - factor model [ modify rss ] as the best modified model was extracted , accounting for 64.4% of the total variance .
five - factor model comprises 33 items , using the likert scale ( 1 - 5 scores ) accepted .
factors are as follows : factor 1 ( conflict ) including 8 items [ 4 , 16 , 17 , 25 , 26 , 27 , 28 and 42 ] .
factor 2 ( incongruity ) including 7 items [ 7 , 11 , 12 , 13 , 14 , 19 and 24 ] .
factor 3 ( incompetence ) including 6 items [ 3 , 15 , 34 , 35 , 37 and 38 ] .
factor 4 ( ambiguity ) 6 items [ 31 , 32 , 33 , 39 , 40 , and 41 ] .
factor 5 ( overload ) 6 items [ 1 , 9 , 10 , 23 , 29 and 30 ] ( table 2 ) . after doing confirmatory factor analysis
finally , this new structural model [ modify rss ] as last persian version of rss ( rss - p ) was good enough to fit in iranian data table 3 .
the results of reliability showed that the cronbach s alpha coefficient for the rss - p was 0.92 vs 0.89 for original rss and for its subscales ranged from 0.71 to 0.84 against 0.57 - 0.86 . in addition , pearson correlation test of the scale approved correlation between the responses in the two times ( r = 0.89 ) , the icc was obtained 0.91 and for the subscales , it was good to excellent ( 0.64 - 0.85 ) . after obtaining persian version of role strain scale as a valid and reliable scale ,
the quantitative analysis conducted and the results revealed that none of nursing teachers had ceiling and floors scores , therefore its effect was not present in this research , moreover , the mean of role strain in nursing teachers has been measured and the role strain scores has been obtained by calculating mean of individual responses to items : very weak ( 1 - 2.59 ) , weak ( 2.60 - 2.99 ) , moderate ( 3 - 3.49 ) and severe ( 3.5 - 5 ) .
the results showed that role strain total score mean is 3.48 ( 0.23 ) table 4 .
abbreviations : agfi , adjusted goodness - of - fit index ; cfi , comparative fit index ; gfi , goodness - of - fit index ; nfi , normed fit index ; nnfi , non - normed fit index ; rmser , rocky mountain service , employment , redevelopment
all in all , the results of study indicated that rss - p is a valid and reliable scale for measuring role strain .
face validity was approved by target groups satisfaction with the quality of the scale items and satisfactory results obtained by calculating impact score .
both quantity and quality of content validity were verified by expert panel ideas with slight differences which were in line with chang and hanna ( 2 - 10 ) .
polit and beck reported that scale response rate of over 50% is satisfactory ( 29 ) .
after analysis by lisrel , in the primary results , fitness indices showed that the structure and relations among concepts of the original scale were not appropriate for iranian data which can be explained by the language and cultural factors as severinsson reported while the translation of questionnaires for cross - cultural research is important , methodological pitfalls problematic for validity may arise ( 17 ) . in these conditions
in other word , confirmatory factor analysis changes into exploratory factor analysis to find a favorite structural model ( 17 ) .
initially , the kaiser - mayer - olkin ( kmo ) measurement showed that the sample size was adequate for exploratory factor analysis , as andersson maintained the kmo value over 0.6 is satisfactory for factor analysis ( 30 ) .
at last , a new model has been developed by doing several exploratory analyses , having a factor loading > 0.45 and deleting 11 items , which was a reason for irrelevance of these items with role strain scale in iranian data ( 31 ) .
garson suggested that factor loadings of < 0.4 are weak and factor loadings 0.6 are very strong ( 32 ) .
this new model ( rss - p ) contains 5 factors which consist of 33 items which were proved by both the statistic evidence ( 64.4% of total variance ) and theoretical framework .
recommended that a newly developed scale should explain 60% of the total variance ( 33 ) .
although , the results of the present research was supported by lin s study , in a sense that it used efa to evaluate scale items and deleted some of items and came up with a five - factor model appropriate with the data ( 2 ) , items array in each factor was completely different from our study which refers to academic structure difference and expectations of multiple roles in that society . in this stage ,
efa followed by cfa is one of the most common approaches to scale development and validation ( 34 ) .
the results revealed an accepted good fitness between data and the model . in other words , the cfa supported the efa results in terms of the dimensions of new scale .
, the construct validity was also examined through efa and cfa , efa was conducted to provide model specification and then cfa was conducted for validation of the model .
as expected , rss - p as a newly developed scale had higher internal consistency reliability than the original scale did , which can be related to , on one hand , both cultural similarity of data and model and doing psychometric stages properly ( 13 ) . on the other hand , modifying model factors which set the ground for increasing internal consistency among the scale items ( 23 ) .
streiner and norman reported that internal consistency related to the homogeneity of the scale ( 31 ) . in well - designed scale ,
in addition , scale stability reliability showed proper correlation among scale items and intraclass coefficient which is supported by other researches ( 9 , 10 ) . as results showed that role strain total score mean , was moderate to high and among sub - scales , the highest mean of role strain belongs to role overload , and the lowest mean belongs to role incompetence .
the researchers believed that stress and felt difficulty and tension in fulfilling role obligations and expectations leading to role strain ( 4 , 5 ) .
likewise , high work pressure , heaviness of work and a lot of working demands with time shortage led to role overload , and its consequences have influence on personal and organizational life ( 37 , 38 ) .
in addition , role incompetence is ranked in the bottom of the table which means nursing teachers benefit from qualification and competence and this sub - scale is no importance in role strain .
as a conclusion , now this reliable and valid scale is academically appropriate to measure role stress and strain .
the rss - p can help detect and predict a multiplicity of role problems and consequently make educational managers aware of nursing teachers difficulties with facing multiple roles and possible future challenges .
moreover , this scale can benefit from needed potential to be used in comparative studies in different academic societies . as , several measurement of role strain by this scale and comparing the results is more likely to lead to change nursing educational policies and strategic programs of nursing faculties .
thus , the researchers offer more and more studies to be conducted in using this scale to detect role strain and consequently to find ways to decrease and remove role stress and strains , and also they suggest comparative studies in this field .
the strength of this study is full accomplishment of psychometric process to provide a valid and reliable scale to determine nursing teachers role strain for the first time in iran .
while , for weakness of this study , it can be said that using this scale for nursing teachers in clinical teaching who did not deal with multiple roles and expectations and necessities of these roles does not help determine role strain for them , and this problem has been left for future researches . finally , with regard to the fact that nursing ( all fields ) is stress - causing in its essence ; therefore , developing and/or psychometric evaluation a proper scale for role stress and strain is suggested in other fields of nursing such as clinical nursing , community nursing , maternity nursing , etc . | background : nursing teachers have difficulties fulfilling multiple roles expectations and balancing the various dimensions of their roles that may lead to role strain . in order to lack of culturally and academically proper scale in iran to measure role strain in nursing teachers
, localizing a foreign scale in this field is necessary.objectives:the objective of this study was psychometric evaluation of the role strain scale ( rss ) and confirming its structural model in an iranian population.materials and methods : the present cross- sectional study was conducted in 2012 , comprising 302 nursing teachers from around the country who were selected using stratified- cluster sampling .
psychometric evaluation process of the rss was carried out by face , content and constructs validity ( confirmatory and exploratory factor analysis ) .
reliability was examined using test - retest and cronbach s alpha for internal consistency reliability.results:in the primary results , in spite of being approved by face and content validity , in construct validity , fitness indices of original role strain scale showed no satisfactory findings in iranian data .
therefore , some items from the structural model of original version were extracted by exploratory factor analysis and a five factor model with 33 items was obtained .
these factors were role conflict , role ambiguity , role overload , role incompetence , and role incongruity .
new model as persian version of rss was confirmed by calculating fitness indices such as gfi = 0.93 , agfi = 0.94 , nfi = 0.91 , rmsea = 0.093 .
internal consistency reliability for the total scale and subscales were respectively 0.92 , and 0.71 - 0.84 .
results from pearson correlation test indicate a high degree of test - retest reliability ( r = 0 .
89 ) .
icc was also 0.91.conclusions:this reliable and valid scale is academically appropriate for nursing teachers to measure role strain and helps detect and predict a multiplicity of role problems and consequently make educational managers aware of nursing teachers difficulties while facing with multiple roles and possible future challenges . | 1. Background
2. Objectives
3. Materials and Methods
3.1. Ethical Views
3.2. Study Design
3.3. Translation
3.4. Face and Content Validity
3.5. Statistical Analysis
3.6. Reliability
4. Results
5. Discussion |
parkinson 's disease ( pd ) is a progressive neurodegenerative disorder associated with various motor and non - motor features that adversely affect function to varying degrees depending on the patient7 ) .
pd typically affects patients in the seventh decade , and the cardinal signs of pd include resting tremor , rigidity , bradykinesia , and postural instability .
in addition , a stooped posture and masked facies are other significant findings in patients with pd .
several spinal deformities including camptocormia or bent spine syndrome , anterocollis , scoliosis , myopathy - induced postural deformity , and pisa syndrome are also associated with pd12 ) .
the frequency of kyphoscoliosis in patients with pd is approximately 7% , which is much higher than that of scoliosis in an age - matched population without pd12 ) .
the therapeutic options for pd range from medical treatment to neurosurgical treatment , including deep brain stimulation of the subthalamic nucleus and intramuscular botox injections .
surgical correction of these deformities should be considered for patients who have reached a severe stage in order to correct the deformity and restore mobility and functions3 ) .
spinal surgery in patients with pd is very challenging , and is associated with a high rate of postoperative complication and surgical revisions due to instrument failure . here
, we report a case of pd with a fixed spinal deformity that was treated with a relatively short segment fixation and corrective fusion surgery .
a 70-year - old female patient presented with complaints of stooping associated with severe back pain radiating to the left lower limb for a period of 3.5 years .
the patient was unable to lie on her back comfortably and reported severe depression due to her condition .
she had previously received a diagnosis of pd , for which she had taken regular oral medications for the past 10 years .
the patient had a history of multiple spinal surgeries ( total of 4 ) due to instrument - related failures for neurogenic intermittent claudication and stooping ; however , none of the surgical interventions had improved her pain or stooping posture .
the patient had significant kyphoscoliosis on examination , but the straight leg raise test , patrick 's sign , and sciatic nerve compression tests were all negative .
detailed motor and sensory examinations were conducted , which showed that her left ankle dorsiflexion was weaker than that of the right ( grade 3/5 and 4/5 respectively ) ; similar results were seen for big toe dorsiflexion ( grade 3/5 and 4/5 respectively ) .
the patient had hypoesthesia in the lt l5 dermatome , and deep tendon reflexes were bilaterally hypoactive .
1 ) showed generalized osteoporotic changes with pulling out of the l5 screws and rods and marked sagittal imbalance .
x - ray also revealed compensatoryreactive thoracic straightening and cervical hyperextension , which was apparent from the patient 's photograph .
pelvic and spinal sagittal parameters were calculated , which clearly revealed the degree of sagittal imbalance present in the patient .
considering the patient 's degree of deformity and pain , we elected to perform revision surgery .
pedicle screws and rods were reinserted from the l1to s1 levels with iliac screws after performing a partial pedicle subtraction osteotomy ( ppso ) at the l4 level with removal of the intervertebral discs at the level of l34 ( fig .
the patient 's intraoperative and postoperative periods were uneventful , and she was able to walk with a straight back without any support a few days postoperatively .
pre and postoperative surgical outcomes were measured with modified scoliosis research society-23 outcome instrument scoring system ( table 1 ) .
a 70-year - old female patient presented with complaints of stooping associated with severe back pain radiating to the left lower limb for a period of 3.5 years .
the patient was unable to lie on her back comfortably and reported severe depression due to her condition .
she had previously received a diagnosis of pd , for which she had taken regular oral medications for the past 10 years .
the patient had a history of multiple spinal surgeries ( total of 4 ) due to instrument - related failures for neurogenic intermittent claudication and stooping ; however , none of the surgical interventions had improved her pain or stooping posture .
the patient had significant kyphoscoliosis on examination , but the straight leg raise test , patrick 's sign , and sciatic nerve compression tests were all negative .
detailed motor and sensory examinations were conducted , which showed that her left ankle dorsiflexion was weaker than that of the right ( grade 3/5 and 4/5 respectively ) ; similar results were seen for big toe dorsiflexion ( grade 3/5 and 4/5 respectively ) .
the patient had hypoesthesia in the lt l5 dermatome , and deep tendon reflexes were bilaterally hypoactive .
1 ) showed generalized osteoporotic changes with pulling out of the l5 screws and rods and marked sagittal imbalance .
x - ray also revealed compensatoryreactive thoracic straightening and cervical hyperextension , which was apparent from the patient 's photograph .
pelvic and spinal sagittal parameters were calculated , which clearly revealed the degree of sagittal imbalance present in the patient .
considering the patient 's degree of deformity and pain , we elected to perform revision surgery .
pedicle screws and rods were reinserted from the l1to s1 levels with iliac screws after performing a partial pedicle subtraction osteotomy ( ppso ) at the l4 level with removal of the intervertebral discs at the level of l34 ( fig .
the patient 's intraoperative and postoperative periods were uneventful , and she was able to walk with a straight back without any support a few days postoperatively .
pre and postoperative surgical outcomes were measured with modified scoliosis research society-23 outcome instrument scoring system ( table 1 ) .
surgical management of fixed spinal deformities in patients with pd is challenging . indeed , due to disease progression itself and ongoing osteoporosis , both the treating surgeon and patient need to be aware of the increased risk of recurrent kyphosis and instrument failure , which can necessitate multiple revision surgeries .
the literature on this topic is relatively sparse , and there is no established consensus regarding standard management of fixed spinal deformities in patients with pd .
however , upadhyaya et al.15 ) suggested a treatment algorithm for patients with pd and spinal deformities , in which they advocated performing deep brain stimulation surgery ( dbs ) first for pd patients followed by a short - segment spinal decompression for myelopathy or radiculopathy .
however , they indicated that if patients do not meet the standard criteria for dbs surgery , then a short - segment spinal decompression and fusion alone can be performed .
they further emphasized the importance of performing long - segment spinal deformity correction and decompression only in those patients with minimal co - morbidities and with high motivation .
unfortunately , long - segment spinal fixation in camptocormic patients is associated with a complication rate of nearly 100%15 ) . in the present case ,
the patient initially underwent an anterior lumbar inter body fusion ( alif ) of the l3 to s1 level followed by posterior decompression and pedicle screw fixation ( psf ) .
unfortunately , the screws and cage pulled out and she underwent an anterior augmented pedicle screw and cage insertion surgery . following augmentation surgery she complained of leg pain due to l4 root irritation .
since all of these procedures were performed outside of our center , no proper surgical records or clinical radiographs could be obtained during the patient 's first visit to our center .
the lumbosacral x - rays taken at our center clearly showed the presence of instrument failure with loosening of the s1 screws and displaced rods , which led to our diagnosis of iatrogenic flat back syndrome .
the previous alif and psf surgeries in our patient had been performed well , and the initial presentation of the patient was attributed to spinal stenosis .
the patient had obvious osteoporosis and was in a progressive state of pd , and thus there was no proper fusion of the inter body cage which led to loosening of the screws . as a result
, the patient had previously undergone bone cement augmentation cage placement with screw fixation in order to resolve instrument failure .
many studies have been performed for the use of bone cement as an augmentation technique during spinal fusion surgeries of osteoporotic bones561017 ) . however , augmentation alone is not sufficient for preventing construct failures . in the patient described in this report ,
we could have corrected both construct failure and sagittal imbalance via a transperitoneal approach ; however , this involves manipulation of several vascular structures and other anatomical structures around the retroperitoneum . as our patient had a history of multiple anterior surgeries , the likelihood of intraoperative and postoperative complications
specifically , the patient had stooping as a result of compensatory thoracic straightening with further cervical hyperextension to maintain the vertical gaze , and through interviews and physical examinations we were able to associate her symptoms to spinal stenosis . based on this diagnosis , we performed a grade iv osteotomy13 ) at the l4 level ( ppso with l34 disc removal ) .
bilateral iliac screws were inserted along with the pedicle screws from l1 to s1 for improved and maximum stability of her spine .
spinal surgery in patients with pd is prone to complications for a number of reasons9 ) .
the factors contributing to these complications include old age and prevalence of severe osteoporosis , which markedly decreases the fusion rate .
surgical outcomes are further worsened by the pulling out of screws and deteriorating motor function .
on the other hand , radiological evidence of good fusion rates among patients treated surgically led moon et al.9 ) to conclude that short - segment fusion confined to the lumbar region generally achieves good results . in our institution
we categorize such patients under one of the two categories including sagittal imbalance disease and sagittal imbalance syndrome . and in this particular case , although the patient had degenerative flat back , we categorized the patient under the sagittal imbalance syndrome group as she had complaints of mild discomfort related to self - image , pain and functions . in such cases
we often perform relatively short level fusion especially in lumbar vertebras even if lumbar lordosis ( ll ) would not have been restored in accordance with pelvic incidence ( pi ) .
had the patient complained of moderate to severe discomfort , we would have categorized under the sagittal imbalance disease category and a relatively long level ( thoracic included ) fusion with resultant ll exceeding the pi value would have been considered . in our case ,
the patient 's major complaints were related to claudication without any complaints related to camptocormia hence we considered relatively short level fusion surgery is justifiable . in a case
series of 14 patients , babat et al.2 ) reported the increased risk of postoperative instrument failures and revision surgeries , and went on to propose a mechanism of ongoing kyphosis adjacent to the levels operated in pd patients .
they suggested the primary causes of the revisions to be adjacent segment disease , material failure or pullout , pseudoarthrosis , and infection .
likewise , a case study of 12 patients with pd undergoing corrective spinal surgeries was published by bourghli et al.3 ) , who reported increased incidence of instrument failure and need for revision surgeries in such patients .
another explanation for surgicalcomplications in pd patients might be the coexistence of osteoporosis and vitamin d deficiency resulting in overall reduction of bone mineral density9 ) .
in addition to progressive degeneration , improper reconstruction of the spino - pelvic balance with a special focus on ll8 ) resulted in progressive kyphosis in our patient , which eventually led to screw failures and revision surgeries .
indeed , radiographic examinations clearly revealed the presence of severe osteoporosis and degenerative changes ; however , after performing osteotomy with pedicle screw insertion from l1 to s1 along with iliac screws insertion , the sagittal balance was maintained , resulting in correction of the deformity .
both bridwell et al.4 ) and wadia et al.16 ) advocated for the use of pso to gain further sagittal plane correction up to 30 to 40. in a case report of camptocormia in pd , peek et al.11 ) emphasized the establishment of substantial forces on the caudal end for stability along with multiple fixation points and anterior column support while performing revision surgeries . with the advent of cutting - edge medical procedures leading to a longer average life span , age - related debilitating diseases are becoming more common .
pd is one such example , and causes not only physical debilitation , but emotional and economic effects as well .
it is estimated that camptocormia is present in 7% of patients with pd14 ) , which indicates the high probability of corrective spinal surgeries and need for multiple revisions in these patients .
although spinal surgeries , especially revision surgeries in patients with pd are regarded as complicated and unfavorable outcomes are common , we were able to achieve a good result in one such patient .
spinal deformitiesare very common in patients with pd , but deformity - correcting surgeries in such patients should be performed cautiously due to the increased incidence of intraoperative and postoperative complications including instrument failure .
thus , we concluded that revision surgery in patients with pd can be undertaken only after proper planning , and should involve restoration of sagittal balance and stabilization .
importantly , even a short segment fixation and corrective fusion surgery can be beneficial in such patients .
nevertheless , the most important aspect of surgical treatment in patients with pd is the realization that unfavorable surgical outcomes are inevitable due to the natural course of the disease itself9 ) . | parkinson 's disease ( pd ) patients frequently have several spinal deformities leading to postural instabilities including camptocormia , myopathy - induced postural deformity , pisa syndrome , and progressive degeneration , all of which adversely affect daily life activities . to improve these postural deformities and relieve the related neurologic symptoms , patients often undergo spinal instrumentation surgery . due to progressive degenerative changes related to pd itself and other complicating factors ,
patients and surgeons are faced with instrument failure - related complications , which can ultimately result in multiple revision surgeries yielding various postoperative complications and morbidities . here , we report a representative case of a 70-year - old pd patient with flat back syndrome who had undergone several revision surgeries , including anterior and posterior decompression and fusion for a lumbosacral spinal deformity . the patient ultimately benefitted from a relatively short segment fixation and corrective fusion surgery . | INTRODUCTION
CASE REPORT
History and examination
Operative procedure & postoperative course
DISCUSSION
CONCLUSION |
intraoperative hemodynamic monitoring used can effectively guide the use of fluid resuscitation with the goal of reducing length of stay- . non- or minimally - invasive methods of determining cardiac output ( co ) may allow guided fluid therapy in a goal - directed fashion and mitigate the risks inherent in insertion of a central line and/or pulmonary arterial catheter . in this case report , we present a comparison of hemodynamic measurements made during a routine surgical case , as measured by three non- and minimally- invasive hemodynamic methods as part of the product evaluation : the vigileo / flotrac system ( edwards lifesciences corporation , irvine , california ) , the cardio - q odm ( deltex medical limited , chichester , west sussex ) , and the nexfin ( bmeye b.v . ,
the purpose of this case report is to examine the measurements obtained from all three devices , with the goal of further understanding their ability to measure co , as well as their ability to trend hemodynamic changes and guide fluid resuscitation therapy .
a : vigileo / flotrac system ( edwards lifesciences corporation , irvine , california ) ; b : the nexfin ( bmeye b.v . ,
amsterdam , the netherlands ) ; c : the cardio - q odm ( deltex medical limited , chichester , west sussex ) ,
a 72 year old , 72 kg man presented with right upper quadrant pain .
he was brought to the operating room for an elective laparoscopic converted to open cholecystectomy with liver resection and portal vein lymphadenectomy .
the patient was placed in the supine position with arms abducted at near ninety degrees from the body .
after an uneventful intravenous induction with 100 mcg of fentanyl , 60 mg of lidocaine , 140 mg of propofol , and 100 mg of rocuronium , a right radial arterial line was placed and connected to a flotrac / vigileo monitor .
the nexfin finger cuff was then placed on the middle phalanx of the right middle finger .
all three monitors were placed , calibrated , and programmed per their respective manufacturer recommendations .
averaging times for the three monitors were programmed as follows : flotrac / vigileo , 20 seconds ; nexfin , 5 beats ; cardio - q odm , 10 beats .
the monitors were run for 30 minutes prior to incision ; during the case , the cardiac index ( ci ) , flotrac / vigileo and nexfinstroke volume variation ( svv ) , left arm non - invasive blood pressure , arterial line blood pressure , and nexfin non - invasive blood pressure were recorded in the anesthesia record in fifteen minute intervals .
additionally , the cardio - q odm transducer was refocused every fifteen minutes or more frequently to maintain an optimized signal .
when allowable , fluid resuscitation was guided by svv increasing beyond 10% , as measured by the flotrac / vigileo monitor . during periods of rapid blood loss , fluid resuscitation was guided by svv in conjunction with visual estimation of blood loss .
total blood loss was 2,200 ml ; total urine output over the case duration was 790 ml ; fluid resuscitation was provided with crystalloid ( 2,800 ml ) , hydroxyethyl starch colloid ( 2,000 ml ) , and packed red blood cells ( 900 ml ) .
this case provides a real world comparison between the nexfin monitor , a completely non - invasive co monitor , with other methods of minimally invasive co measurement , the flotrac / vigileo monitor and cardio - q odm as part of the internal product evaluation . unlike the flotrac / vigileo and cardioq odm , which require an arterial line and an esophageal placed ultrasound transducer respectively , the nexfin monitor is non - invasive by utilizing external pressure to negate any plethysmographic changes in the measured finger , thereby reconstructing the arterial waveform externally .
calculation of the stroke volume ( sv ) and co is based on pulse contour algorithms , . utilizing a proprietary transfer function based on a clinical database that accounts for age , gender , height , and weight
, this finger arterial pressure waveform is used to reconstruct a brachial arterial waveform which is then used as a substitute for aortic pressure . whereas the flotrac / vigileo monitor has found widespread intraoperative use due to its robustness in patients without cardiac rhythm and valvular abnormalities , the use of the cardio - q odm has been more limited due to its susceptibility to electrocautery noise ( fig . 2 ) .
in our experience , the frequency with which electrocautery is used by surgeons is often high enough to prevent measurement , and often negates the usefulness of this monitor as periods of extensive electrocautery use often coincide with significant hemodynamic changes for which these monitors are used .
intraoperative fluid resuscitation in this case was guided by svv when possible ( maintaining svv below 10% ) , and in conjunction with visual estimation during periods of brisk blood loss .
here , we focus our discussion on comparing the performance of the nexfin device , with the vigileo / flotrac and cardio - q odm systems , in regards to measurements of mean arterial pressure , cardiac output , and stroke volume variation .
co : cardiac output ; sv : stroke volume ; ftc : flow time corrected ; pv : peak velocity ; ma : mean acceleration ; hr : heart rate.b : an example of electrocautery interference ( arrow ) with the cardio - q odm signal . throughout the surgery ,
the nexfin estimated a consistently higher mean arterial pressure ( map ) than either the flotrac / vigileo monitor or cardio - q odm .
however , nexfin map trended consistently with both the arterial line and blood pressure cuff , and response time to changes in map were not measurably different between devices , suggesting the likelihood of a measurement bias due to device positioning , or calibration error .
correlations between the nexfin and peripheral arterial blood pressure measurements have been demonstrated in adult and pediatric cardiac surgeries ; however , this correlation may not translate to the adult abdominal case .
although blood pressure correlation has been demonstrated between the nexfin and manual and automatic non - invasive cuff measurements in non - operative environments , bias may be introduced secondary to surgical factors such as positioning .
additionally , the nature of the noninvasive and minimally invasive systems can create questionable results during extreme vasoconstriction , conditions seen with reynaud 's disease or shock as cited by the manufacturer .
the degree to which measurements are sensitive to less severe vaso - constrictive conditions that may be seen in the operating room such as distal hypothermia is unknown .
it has been shown that measured co can differ substantially depending on the method of determining co , with variations as large as 41.3% , 42.1% , and 39% for arterial pulse contour analysis , esophageal doppler , and nexfin respectively , versus thermodilution , .
these figures exceed the 30% criteria suggested by critchley and critchley . for the purposes of intraoperative management , however , the ability to identify trends in co may in itself be of use in guiding fluid resuscitation . in this regard
, the nexfin monitor was able to display ci changes in the same direction as the flotrac / vigileo and cardio - q at several points ( fig .
3 ) , displaying a drop after induction , and increases after each dose of vaso - constrictive medication ( markers a , e , and g ) ; marker j shows a drop in output with all three measurement techniques , due to rapid surgical blood loss .
although a recent study demonstrated that earlier flotrac system did not accurately track changes in co following the administration of phenylephrine , a predominantly 1-adrenergic receptor agonist , newer version has been developed to track the change well .
surgical events are marked by vertical bars as follows : a , phenylepherine administered ; b , oral - gastric tube placed , ephedrine administered ; c , surgical incision ; d , abdominal insufflation , patient positioned in reverse trendelenburg ; e , phenylepherine administered ; f , conversion from laparoscopic to open case , phenylepherine administered , loss of cardio - q monitor signal due to electrocautery interference ; g , phenylepherine administered ; h , end of extensive electrocautery use , cardio - q monitor reinitialized ; i , significant portal vein bleeding noted by surgeon ; j , vaso - active medication given ; k , patient returned to level , supine position ; l , abdominal wall closure . stroke volume variation has been shown to be efficacious in predicting fluid responsiveness , and in this regard may serve as an intraoperative tool to guide fluid resuscitation .
3 compares svv as determined by the flotrac / vigileo and nexfin monitors , demonstrating increase with surgical blood loss , and decrease with fluid resuscitation , and demonstrates correlation between svv and nexfin - determined ci . during this case , increasing trends in svv were associated with decreasing trends in ci , and vice versa .
prior work has questioned the efficacy of minimally invasive cardiac output monitoring versus clinical observation ; but the data was from 2006 - 2007 icu patients who are very different from patients in the operating room .
furthermore , the technology has improved significantly since then . in this case , and intraoperatively in general , we believe that the value of svv can be used safely and effectively to determine fluid responsiveness and guide fluid therapy
. however , when svv / ppv is between 9 - 13% ( gray zone ) , there are about 25% of the cases for whom svv / ppv may not accurately predict the volume responsiveness under general anesthesia . for those instances ,
the averaging times were different in all the three devices that may show the delayed response .
additionally , the value of intraoperative svv must also be interpreted within the surgical context .
this particular case involved surgical manipulation in close proximity to the inferior vena cava , very likely with alternating compression and decompression .
the subsequent effect on cardiac preload can theoretically bias measurements of svv , and any fluctuation must also account for this or any other surgically induced bias . in conclusion , during operative situations where objective determination of co is critical to clinical decision - making , non- or minimally - invasive methods of co may provide measurements that correlate within approximately 40% of thermodilution measurements via a pulmonary arterial catheter .
however , in surgical situations where objective measurement is unnecessary , where the risks of pulmonary artery placement outweigh the utility of thermodilution measurements , or where ci or svv trends alone provide utility in guiding goal - directed fluid management , the nexfin monitor may provide useful hemodynamic information regarding fluid responsiveness for clinical decision making in a completely non - invasive fashion and it is comparable to that obtained by vigileo / flotrac and cardio - q odm monitors .
throughout the surgery , the nexfin estimated a consistently higher mean arterial pressure ( map ) than either the flotrac / vigileo monitor or cardio - q odm .
however , nexfin map trended consistently with both the arterial line and blood pressure cuff , and response time to changes in map were not measurably different between devices , suggesting the likelihood of a measurement bias due to device positioning , or calibration error .
correlations between the nexfin and peripheral arterial blood pressure measurements have been demonstrated in adult and pediatric cardiac surgeries ; however , this correlation may not translate to the adult abdominal case .
although blood pressure correlation has been demonstrated between the nexfin and manual and automatic non - invasive cuff measurements in non - operative environments , bias may be introduced secondary to surgical factors such as positioning .
additionally , the nature of the noninvasive and minimally invasive systems can create questionable results during extreme vasoconstriction , conditions seen with reynaud 's disease or shock as cited by the manufacturer .
the degree to which measurements are sensitive to less severe vaso - constrictive conditions that may be seen in the operating room such as distal hypothermia is unknown .
it has been shown that measured co can differ substantially depending on the method of determining co , with variations as large as 41.3% , 42.1% , and 39% for arterial pulse contour analysis , esophageal doppler , and nexfin respectively , versus thermodilution , .
these figures exceed the 30% criteria suggested by critchley and critchley . for the purposes of intraoperative management , however , the ability to identify trends in co may in itself be of use in guiding fluid resuscitation . in this regard
, the nexfin monitor was able to display ci changes in the same direction as the flotrac / vigileo and cardio - q at several points ( fig .
3 ) , displaying a drop after induction , and increases after each dose of vaso - constrictive medication ( markers a , e , and g ) ; marker j shows a drop in output with all three measurement techniques , due to rapid surgical blood loss .
although a recent study demonstrated that earlier flotrac system did not accurately track changes in co following the administration of phenylephrine , a predominantly 1-adrenergic receptor agonist , newer version has been developed to track the change well .
surgical events are marked by vertical bars as follows : a , phenylepherine administered ; b , oral - gastric tube placed , ephedrine administered ; c , surgical incision ; d , abdominal insufflation , patient positioned in reverse trendelenburg ; e , phenylepherine administered ; f , conversion from laparoscopic to open case , phenylepherine administered , loss of cardio - q monitor signal due to electrocautery interference ; g , phenylepherine administered ; h , end of extensive electrocautery use , cardio - q monitor reinitialized ; i , significant portal vein bleeding noted by surgeon ; j , vaso - active medication given ; k , patient returned to level , supine position ; l , abdominal wall closure .
stroke volume variation has been shown to be efficacious in predicting fluid responsiveness , and in this regard may serve as an intraoperative tool to guide fluid resuscitation .
3 compares svv as determined by the flotrac / vigileo and nexfin monitors , demonstrating increase with surgical blood loss , and decrease with fluid resuscitation , and demonstrates correlation between svv and nexfin - determined ci . during this case , increasing trends in svv were associated with decreasing trends in ci , and vice versa .
prior work has questioned the efficacy of minimally invasive cardiac output monitoring versus clinical observation ; but the data was from 2006 - 2007 icu patients who are very different from patients in the operating room .
furthermore , the technology has improved significantly since then . in this case , and intraoperatively in general , we believe that the value of svv can be used safely and effectively to determine fluid responsiveness and guide fluid therapy
. however , when svv / ppv is between 9 - 13% ( gray zone ) , there are about 25% of the cases for whom svv / ppv may not accurately predict the volume responsiveness under general anesthesia . for those instances ,
the averaging times were different in all the three devices that may show the delayed response .
additionally , the value of intraoperative svv must also be interpreted within the surgical context .
this particular case involved surgical manipulation in close proximity to the inferior vena cava , very likely with alternating compression and decompression .
the subsequent effect on cardiac preload can theoretically bias measurements of svv , and any fluctuation must also account for this or any other surgically induced bias . in conclusion , during operative situations where objective determination of co is critical to clinical decision - making , non- or minimally - invasive methods of co may provide measurements that correlate within approximately 40% of thermodilution measurements via a pulmonary arterial catheter . however , in surgical situations where objective measurement is unnecessary , where the risks of pulmonary artery placement outweigh the utility of thermodilution measurements , or where ci or svv trends alone provide utility in guiding goal - directed fluid management , the nexfin monitor may provide useful hemodynamic information regarding fluid responsiveness for clinical decision making in a completely non - invasive fashion and it is comparable to that obtained by vigileo / flotrac and cardio - q odm monitors . | abstractas part of the enhanced recovery after surgery ( eras ) protocol , the goal - directed fluid management with hemodynamic monitoring can effectively guide perioperative fluid use and significantly improve the outcomes in high - risk patients undergoing major surgeries .
several minimally invasive and non - invasive monitoring devices are commercially available for clinical use . as part of an internal evaluation
, we reported the results from three different hemodynamic monitoring devices used in a patient undergoing a major abdominal surgery . | INTRODUCTION
CASE REPORT
DISCUSSION
Mean arterial pressure
Cardiac output
Stroke volume variation |
superior mesenteric artery syndrome is an uncommon cause of duodenal obstruction , and its manifestations are generally associated with compression on the third part of the duodenum between the abdominal aorta and superior mesenteric artery .
in this report , a patient is described presenting with epigastric pain and weight loss due to superior mesenteric artery syndrome .
the patient has also nutcracker syndrome , which is the compression of the left renal vein between the aorta and the superior mesenteric artery at its origin .
in addition to an appropriate clinical history , ct findings indicating decreased aortomesenteric angle and a shortened aortomesenteric distance can suggest the diagnosis for both the superior mesenteric artery syndrome and accompanying nutcracker syndrome .
superior mesenteric artery ( sma ) syndrome is a rare cause of abdominal pain , nausea , and vomiting that may be undiagnosed in patients presenting to the emergency department .
sma syndrome is arteriomesenteric obstruction of the duodenum , which is characterized by compression of the third portion of the duodenum by sma ( as it passes over this portion of the duodenum ) .
nutcracker syndrome ( ncs ) is characterized by impeded outflow from the left renal vein ( lrv ) into the inferior vena cava ( ivc ) due to extrinsic lrv compression .
we presented this case report in order to discuss the diagnosis of a patient with both sma syndrome and accompanying ncs with computed tomography ( ct ) findings .
a 28-year - old man was admitted to kirikkale university medicine faculty , radiology department , kirikkale , turkey , on 26 april 2013 . he had a history of 1-day vomiting , epigastric pain , and bloating ( table 1 ) .
he also reported weight loss , anorexia and intermittent abdominal pain during the past few years .
blood test results were as follows : mean corpuscular hemoglobin concentration , 36.3 g / dl ; granulocyte ( percentage , count ) , % 77 , 8 10/ul ; creatinine 0.61 mg / dl , with no other abnormalities . urinalysis revealed no hematuria .
abbreviations : mchc , mean corpuscular hemoglobin concentration ; lrv , left renal vein ; ct , computed tomography .
the scan was performed with ge hi - speed , a dual - detector ct scanner , which is regularly calibrated .
ct images showed the reduction of the angle between the abdominal aorta ( aa ) and sma , and decreased aortomesenteric distance .
the angle between the aa and sma was 14.30 ( figure 1a ) , and the aortomesenteric distance was 3.9 mm ( figure 1b ) .
gastric and proximal duodenal dilatation was observed ( figure 1c ) with the compression of the duodenal lumen between the aa and sma ( figures 1d and 1e ) .
the lrv was also compressed by the sma close to its origin , resulting in proximal dilatation ( figure f ) .
a ) sagittal ct scan showing the reduction of the angle ( 14 30 ) between the abdominal aorta and superior mesenteric artery .
b ) axial ct scan showing decreased aortomesenteric distance ( 3 , 9 mm ) .
d ) sagittal ct scan of abdomen showing duodenal compression between the abdominal aorta and superior mesenteric artery ( d : duodenum , lrv : left renal vein ) .
e ) axial ct scan of abdomen demonstrating duodenal compression between the abdominal aorta and superior mesenteric artery ( d , duodenum ; sma , superior mesenteric artery ) .
f ) axial ct scan showing dilated left renal vein owing to superior mesenteric artery compressing ( white arrow ) .
sma syndrome is a rare condition where the third part of the duodenum is compressed between the sma and aa , leading to duodenal outlet obstruction .
the incidence of sma syndrome has been found to be between 0.013% and 0.3% of the general population ( 1 ) .
sma syndrome is more common in females , and usually occurs between 35 and 45 years of age .
clinical diagnosis of sma syndrome is rather challenging owing to nonspecific character of the signs and symptoms in the majority of the patients , requiring a radiological diagnosis or confirmation of diagnosis .
upper gastrointestinal barium x - ray may demonstrate gastric and duodenal dilatation , or blockade of barium flow from duodenum to jejunum .
an endoscopic gastroduodenoscopy or ct scan of the abdomen could be helpful for confirmatory studies .
these modalities may reveal the extrinsic compression of the third portion of the duodenum and the narrow angle between the aa and sma ( 2 ) .
radiographically , sma syndrome is characterized by several findings such as gastric and proximal duodenal dilatation with compression of the duodenum and dilatation of the left renal vein . in normal patients ,
the distance between the aa and sma ( aortomesenteric distance ) is 10 - 34 mm with aortomesenteric angle of 28-65 ( 3 , 4 ) .
angiographic studies have indicated that patients with sma syndrome have an abnormal aortomesenteric angle ( 6-22 ) , and a shortened aortomesenteric distance ( 2 - 8 mm ) compared to normal patients . in a study using multidetector computed tomography ( mdct ) , the average aortomesenteric angle in patients with sma syndrome was 13.5 , with an aortomesenteric distance of 4.4 mm . evidently , any absolute numerical cut - off is arbitrary , but some reports have advocated that an aortomesenteric distance < 8 mm and an angle < 22 are suggestive of the diagnosis providing the right clinical setting ( 5 ) .
the lrv normally passes between the aa and sma , where it may be pressed , resulting in a variable degree of lrv obstruction .
this compression of lrv between aa and sma is entitled nutcracker syndrome ( ncs ) ( 6 ) .
the ncs is the clinical equivalent of nutcracker phenomenon ( ncp ) defined by a set of symptoms with significant alterations ( 7 ) .
while the terms ncp and ncs are sometimes used interchangeably in the literature , shin and lee accentuated that the nutcracker anatomy is not always accompanied with clinical symptoms and that some of the anatomic findings suggestive of nutcracker may stand for a normal variant or be accounted for by other conditions ( 8) .
imaging , such as ultrasonography ( us ) , doppler us , ct , or magnetic resonance imaging ( mri ) , is required to diagnose ncs .
acute narrowing of the lrv at the level of sma ( beak sign ) , lateral ( hilar ) dilatation and the presence of collateral pathways at ct imaging can be helpful for the diagnosis of ncs .
however , the diagnostic procedure ( considering the reference standard for establishing the diagnosis of ncs ) is invasive selective left renal phlebography with the measurement of the pressure gradient between the lrv and ivc ( 2 , 6 ) .
our patient had decreased aortomesenteric distance and aortomesenteric angle , dilated stomach and proximal duodenum and dilated lrv .
english literature search revealed one case in which sma syndrome was found in concomitance with ncp ( 4 ) .
sma syndrome accompanied by ncs , a rare collaboration , is the strong point of our study .
sma syndrome should be considered in the differential diagnosis of duodenal obstruction such as duodenal hypoplasia , duodenal stenosis , duodenal atresia , peptic ulcer disease , congenital bands , intestinal malrotation , annular pancreas , and pre - duodenal portal vein ( 2 ) .
most cases occur in patients with weight loss and reduction of fat stores around superior mesenteric artery because of a variety of reasons ( surgery , malabsorption , trauma , etc . )
( 3 ) . in the differential diagnosis of ncs , pathologies that cause pain , hematuria and proteinuria with an unexplained cause can be considered ( 6 ) .
conservative management based on the correction of electrolyte imbalance , and appropriate enteral or parenteral nutrition could result in symptomatic improvement and weight gain in most patients .
surgery , which may be considered if conservative treatment fails , involves the release of the treitz ligament . during surgery ,
the ligament may be simply released or reconstructive approaches such as the open or close laparoscopic duodenojejunostomy , or roux - en - y procedure are chosen ( 2 ) . in ncs management , supporting treatment is sufficient if the symptoms are minimal .
surgical or radiological treatments are indicated for severe pain , evident hematuria and renal functional deterioration ( 6 ) .
ncs should be considered in the differential diagnosis of patients with unknown cause of hematuria and flank pain . in conjunction with an appropriate clinical history ,
ct findings indicating decreased aortomesenteric angle and a shortened aortomesenteric distance can suggest the diagnosis for both conditions . | introduction : superior mesenteric artery syndrome is an uncommon cause of duodenal obstruction , and its manifestations are generally associated with compression on the third part of the duodenum between the abdominal aorta and superior mesenteric artery.case presentation : in this report , a patient is described presenting with epigastric pain and weight loss due to superior mesenteric artery syndrome .
the patient has also nutcracker syndrome , which is the compression of the left renal vein between the aorta and the superior mesenteric artery at its origin.conclusions:in addition to an appropriate clinical history , ct findings indicating decreased aortomesenteric angle and a shortened aortomesenteric distance can suggest the diagnosis for both the superior mesenteric artery syndrome and accompanying nutcracker syndrome . | Introduction:
Case Presentation:
Conclusions:
1. Introduction
2. Case Presentation
3. Discussion |
recent phase iii trials have demonstrated the benefits of maintenance therapy with pemetrexed for metastatic non - small cell lung cancer ( nsclc ) [ 1 , 2 ] .
pemetrexed maintenance therapy improved progression - free survival and overall survival by both continuation and switch maintenance strategies . with the support of emerging evidence ,
oncologists have become accustomed to prolonging the administration of cytotoxic pemetrexed for advanced nsclc , similar to the use of molecular targeting agents for anaplastic lymphoma kinase ( alk)-positive or epidermal growth factor receptor ( egfr)-mutant nsclc . however , disease progression during maintenance pemetrexed is almost inevitable after a median progression - free survival of 46 months .
local ablative therapies for oligoprogressive disease ( opd ) , including surgery , radiofrequency ablation , and stereotactic ablative radiotherapy ( sabr ) have been reported to extend survival with continued tyrosine kinase inhibitors ( tki ) for alk - positive or egfr - mutant lung cancer [ 4 , 5 ] . a preliminary report from a phase ii trial demonstrated prolonged progression - free survival in oligo - metastatic nsclc patients receiving local consolidative therapy for non - progressive disease , compared to systemic therapy alone .
however , there have been no previous investigations on local ablative therapy for opd in patients receiving maintenance pemetrexed . here ,
we present our preliminary results of sabr eradicating progressive lesions with the continued use of pemetrexed .
patients diagnosed with advanced lung adenocarcinoma were treated with pemetrexed in combination with cisplatin / carboplatin as the first - line treatment , or as the salvage regimen , after systemic progression under tki .
maintenance pemetrexed ( 500 mg / m ) in a 21-day cycle was given after four to six cycles of the initial pemetrexed / platinum if there was no disease progression .
response evaluation criteria in solid tumors ( recist ) version 1.1 for imaging studies was adopted to evaluate treatment responses .
patients with oligoprogression ( 5 discrete progressing lesions ) that continued to derive systemic benets from pemetrexed were prospectively enrolled to receive sabr for locoregional disease .
meanwhile , pemetrexed was continued until further systemic progression not suitable for local therapy developed .
treatment - related toxicities were scored according to common terminology criteria for adverse events ( ctcae ) version 4.0 . here , we present three consecutively enrolled cases receiving sabr for thoracic oligoprogression under pemetrexed with > 18 months of clinical and imaging follow - up .
case 1 involves a 55-year - old woman with stage iv egfr - mutant ( del19/l858r ) nsclc , presenting with multiple lung tumors and pleural nodular seeding .
she initially responded to gefitinib , but 5 months later developed enlarged left upper - lung tumors and increased pleural nodularity .
six cycles of pemetrexed / cisplatin achieved partial remission , and maintenance pemetrexed was given after the induction treatment .
after six cycles of maintenance pemetrexed , slight enlargement of the left lung tumors and pleural lesions were noted by computed tomography ( ct ) of the chest , without other systemic progressions .
she underwent sabr via cyberknife targeting on oligoprogressive tumors ( five lesions in total ) , each with 2022 gy in a single fraction on 5 separate days .
repeat chest imaging 2 and 4 months later demonstrated stationary thoracic disease with grade 1 radiation pneumonitis ( fig .
maintenance pemetrexed was continued for an additional 10 cycles until new nodular lesions in the left lung developed 7 months after sabr .
she later developed t790 m mutations and spine metastases with subsequent failure of erlotinib and gemcitabine , while her chest disease remained relatively stable 2 years after the sabr .
case 2 is a 71-year - old man with metastatic egfr - mutated adenocarcinoma of the lung .
he received first - line afatinib and palliative radiotherapy for a metastatic lesion at t8 of the spine .
the primary tumor in his left upper lung initially regressed but demonstrated apparent enlargement 7 months later .
pemetrexed / cisplatin were given for salvage treatment , and the tumor showed partial regression .
he remained on tri - weekly pemetrexed for 2 years with good locoregional and systemic disease control .
only mild toxicities , such as grade 1 fever , grade 1 anemia / eosinophilia , and grade 1 gastrointestinal discomfort occurred during the maintenance treatment .
follow - up chest ct revealed enlargement of the primary tumor , with no evidence of extra - thoracic progression .
sabr targeted the tumor in his left upper lung with 44 gy in four fractions .
the tumor regressed after sabr treatment and has been unchanged for 2 years with postradiation fibrotic changes ( fig .
currently , the patient has remained on maintenance pemetrexed for more than 48 months with no evidence of disease recurrence .
case 3 demonstrates a 63-year - old man diagnosed with stage iia ( t1bn1m0 ) nsclc in september 2006 .
the patient underwent salvage radiotherapy with 60 gy in 25 fractions to the recurrent mediastinal lymphadenopathies .
, he developed a right hilar mass and a new nodular lesion in the left upper lobe , without other systemic relapse .
salvage pemetrexed / carboplatin were given and maintenance pemetrexed was started after six cycles of induction treatment .
he had good response of thoracic disease in serial follow - up imaging studies , until an oligoprogressive tumor at the left upper lobe showed enlargement after 14 cycles of maintenance pemetrexed .
he received sabr to the left upper lung tumor with 45 gy in 3 fractions in december 2013 .
the patient has remained on maintenance pemetrexed for non - progressive disease at this time with grade 1 fatigue and radiation fibrosis ( fig .
survival benefits for patients with advanced nsclc carried out by maintenance pemetrexed have been clearly demonstrated by a series of clinical trials [ 1 , 2 , 3 ] .
pemetrexed , as a multi - targeted folate antimetabolite , is well tolerated during long - term use . in the paramount trial , more than one third of patients allocated to the maintenance arm received more than six cycles of pemetrexed without worsening quality of life .
though numerous questions were left unanswered regarding optimal patient selection , multi - agent regimens , and administration duration of maintenance pemetrexed . for non - progressive advanced non - squamous nsclc , oncologists have recently tended to prescribe maintenance pemetrexed in a similar way as for tki , which is uninterruptedly until disease progression .
opd arises from subclone expansion resistant to ongoing treatment , and is an early state of disease progression .
before the dissemination of resistant subclones becomes the predominant burden of the patient , opd with a limited number of isolated regions amenable to local ablative therapy offers a chance to delay the start of overwhelming progression . local ablative therapy for opd prevents the early termination of the ongoing treatment , while there is still effective systemic disease suppression by the current regimen .
gomez et al . recently reported local therapy prolonged progression - free survival in advanced nsclc with non - progressive disease after initial systemic treatment , demonstrating the value of aggressive local treatment on consolidating disease control .
by contrast , in this case series , we showed that sabr targeting opd prolonged the treatment with pemetrexed , maintaining clinical benefits ( table 1 ) .
sabr is an effective tool at controlling local disease in opd under tki [ 4 , 5 , 8 ] or oligometastases . in this report ,
all patients had sustained local control of thoracic oligoprogression after the sabr , while there was only mild radiation - related pneumonitis / fibrosis . with only one case having her second progression during follow - up , all of them had longer time - to - further - progression than their initial progression - free time under maintenance pemetrexed , reflecting apparent benefits from sabr . in the paramount trial
, superior survival outcomes were observed in patients receiving longer exposure to pemetrexed . in this series ,
two patients with long progression - free intervals before sabr also continued additional pemetrexed treatment ( 12 cycles in average ) without a second progression . the extended use of maintenance pemetrexed could be attributed to not only early elimination of drug - resistant subclones by sabr , but also the interactions between the pemetrexed response and driver oncogene mutations .
reported that for patients with alk - positive nsclc , the time - to - progression under pemetrexed was significantly longer compared to that of patients with egfr mutations and alk - negative / egfr wild - type control groups .
although the underlying molecular basis behind the different responses to pemetrexed remains unclear , berge et al . have demonstrated that pemetrexed exposure in crizotinib - nave patients did not affect subsequent crizotinib outcomes after pemetrexed failed .
recently , a cost - effectiveness analysis on molecular testing of alk - gene rearrangement and rst - line therapy with crizotinib for patients with advanced nsclc showed that early detection with alk - targeted inhibition in alk - positive cases is not cost - effective . in the present report
, we demonstrated that for opd during maintenance pemetrexed , sabr successfully extended the time of effective systemic disease suppression by pemetrexed , thus reserving the high expenses of tests for alk - driver mutation and possible crizotinib use for definite systemic failure .
this combined - modality strategy should be considered first , especially in low - income or developing countries , where resources for second - line treatment after pemetrexed failure are much more limited . to our knowledge , this is the first report indicating that sabr targeting oligoprogression with continued maintenance pemetrexed is an effective approach for patients with advanced non - squamous nsclc .
although the true benefits from sabr during maintenance pemetrexed warrants a clear demonstration by a randomized trial , this consecutive case series shows that a subset of patients receiving sabr during maintenance pemetrexed for opd can experience long - term disease control without severe treatment - related toxicities , in a similar way to those with driver mutations receiving targeted therapy . | maintenance pemetrexed offers survival benefit with well - tolerated toxicities for advanced non - squamous non - small cell lung cancer ( nsclc ) .
we present 3 consecutively enrolled patients with advanced non - squamous nsclc , receiving stereotactic ablative radiotherapy ( sabr ) for oligoprogressive disease during maintenance pemetrexed .
all of them had sustained local control of thoracic oligoprogression after the sabr , while maintenance pemetrexed were kept for additionally long progression - free interval .
sabr targeting oligoprogression with continued pemetrexed is an effective and safe approach to extend exposure of maintenance pemetrexed , thus maximizing the benefit from it . | Introduction
Patients and Methods
Case Report
Discussion
Statement of Ethics
Disclosure Statement |
hemorrhagic cystitis ( hc ) is a diffuse inflammatory state of the urinary bladder that can occur as a complication of radiotherapy , chemotherapy , or infection .
it may result in hemorrhage of the bladder mucosa , which is observed clinically as hematuria
. the focus of this article will be on radiation - induced hc , a relatively uncommon but serious complication of radiation therapy .
report a 6.5% risk of developing radiation - induced hc in patients treated with radiotherapy due to cervical cancer .
radiation - damage of the bladder does not become clinically evident until six months after initiation of the radiotherapy . irradiated tissue sustains damage that results in microscopic obliterative endarteritis and progressive arteriopathy leaving the affected tissue hypocellular , hypovascular , and ischemic .
collagen and cells are replaced during the healing process , but complete restoration is unlikely to occur [ 3 , 4 ] .
bleeding may also occur as a consequence of the formation of structurally weak and brittle vessels that easily rupture .
the use of hyperbaric oxygen treatment ( hbot ) in the treatment of tissue damaged by radiation was pioneered by marx et al . who suggested that hbot enhances healing of tissues subjected to radiation therapy .
they were the first to describe the therapeutic effects of hbot in humans on patients having undergone protracted radiation therapy of the head and neck . in an animal study predating the human study
, the authors showed that animals subjected 100% o2 in a hyperbaric environment had an 8 - 9 times higher vascular density in irradiated tissue compared to both a normobaric group and an air - breathing control group .
a four - year follow - up study showed that the angiogenic effect was long lasting .
oxygen in hyperbaric conditions leads to an increase of oxygen levels in tissues resulting in angiogenesis , increased collagen formation , and an increased number of fibroblasts .
the angiogenesis is correlated , at least in part , to the macrophages of the affected tissue , which react to the steep oxygen gradient achieved in a hyperbaric environment .
the increase in fibroblasts and collagen creates a connective tissue framework for the new vessels .
the elevated level of oxygen also supports the regeneration of damaged tissue , as well as reducing edema , necrosis , and leukocytic infiltration .
its overall effectiveness in various pathologies has manifested in many clinical studies and trials [ 4 , 5 , 6 ] .
more research is needed to delineate and define the clinical effectiveness and benefits of hbot in each disease entity .
the purpose of this study is to evaluate the efficacy of hbot in radiation - induced hc .
between the years 2006 and 2010 , ten patients ( seven men and three women ) with radiation - induced hc as a consequence of standard methods of treatment were subjected to hbot .
their average age was 68.9 years ; age - range 54 - 81 years . in these patients , four men
were initially treated with radio - hormonal therapy or radiation therapy because of prostate cancer .
patients in this group received 50 gy of teleradiotherapy , with an additional dosage of 20 - 24 gy .
patients in this group received 50 gy of teleradiotherapy with an additional dosage of 16 gy .
two women were treated for cervical cancer and one for endometrial cancer . in the case of cervical and endometrial cancers ,
30 gy of brachytherapy with 45 - 50 gy of teleradiotherapy were utilized ( details in table 1 ) .
patient characteristics abbreviations : m = male , f = female , tuc = transurethral coagulation , c = cyclonamine , e = exacyl , k = vitamin k , b = brachytherapy , t = teleradiotherapy patients in which radiation induced hc was present were initially treated for cancer using radiotherapy and in some cases with additional brachytherapy table 1 contains the details of this treatment and hematuria was recognized with an average onset of 4.3 years ( 0.5 - 8 ) after radiotherapy .
prior to the advent of hbot , treatment of radiation - induced hc was initially performed utilizing cystoscopic electrocoagulation , oral etamsylate , tranexamic acid , and vitamin k. however , a follow - up performed at 7 - 48 ( average 24.5 ) months revealed that this approach only resulted in temporary improvement , as reappearance of hematuria was observed in all patients .
each patient received cystoscopic confirmation of hc , as well as exclusion of urinary tract infection and bladder cancer .
one patient received treatment with palliative intent due to persistent hematuria , seeing as the cancer was advanced and no additional treatment with intention of cure was planned .
the other patients were without detectible presence of cancer . additionally , five of the patients required blood transfusions due to low hemoglobin values .
the transfusions were given both before and during hbot , but were not required after .
the hbot was performed in a delta haux - quadro 2700 hyperbaric chamber , engineered to minimize claustrophobia .
the treatment schedule consisted of five sessions a week , with a duration of 90 minutes per session . during the sessions , patients were provided with 100% o2 at a pressure of 253312.5 pa ( 2.5 atm ) .
sessions were continued until the hematuria disappeared , or until the patient requested to terminate the treatment .
the average number of hbo treatments was 43.4 ( 8 - 60 ) [ 43.5 ( 8 - 120 ) ] .
one patient received a total of 120 treatments in two sets of 60 , interspaced by four months between each series and two other patients completed a series of 60 treatments .
the efficacy of the treatment was assessed every six months by visually evaluating the degree of hematuria . in case of any suspicions of hematuria
complete resolution of hematuria was achieved in six of the ten patients ( 60% ) . the maximum follow up after hyperbarotherapy was 18 months .
none of these patients showed signs of hematuria upon completion of hbot , including the patient that received 120 treatments .
four patients who suffered from bladder , endometrial , or prostate cancers , were withdrawn from the treatment . in one of the patients with primary bladder cancer , treatment was discontinued because of increasing hematuria and suspicion of bladder cancer recurrence .
the other three patients who discontinued their treatments prematurely due to coexisting morbidities had lessened or incomplete cessation of the hematuria .
the four patients who had no complete resolution of symptoms were the same patients who did not complete the treatment .
according to current standards of treatment , electrocoagulation , with or without pharmacotherapy , is the first step in the treatment of hc .
conjugated estrogens have a success rate of 60 - 85% in the treatment of hematuria in hc .
the efficacy of conjugated estrogens applies to different etiology of hc and it should be noticed that these results using post high dose chemotherapy are not directly applicable to radiocystitis .
pentosan polysulfate , tranexemic acid , aminocaproic acid , alprostadil , dinoprost , silver nitrate , and aluminum can also be used [ 1216 ] . when the hematuria can not be controlled using electrocoagulation or pharmacotherapy , selective embolization of hypogastric arteries can be performed .
if this fails , cystectomy and supravesical diversion of urine may have to be performed to control the hematuria .
although many studies have shown the benefit of hbot in the treatment of radiation - induced hc , especially in patients who have failed other forms of management , hbot is currently not included in standard treatment [ 2 , 3 , 1720 ] . in the literature on the subject ,
studies report complete resolution - rates range from 76 - 100% [ 1 , 3 , 2125 ] .
although , our study demonstrated slightly lower rates of complete resolution ( 60% ) , it is clear that the treatment contributed significantly to the healing process and health of the patients .
in addition to its high efficacy , hbot is safe and well tolerated by patients .
side effects are rare and generally well tolerated by the patients [ 9 , 17 ] .
they include pharyngotympanic tube problems , pneumothorax , air emboli , as well as cns and pulmonary oxygen - toxicity .
furthermore , the benefits of hbot are relatively long lasting when compared to standard methods of treatment .
a study by del pizzo involving 11 patients , with a mean follow - up of 5.1 years , showed an asymptomatic period of 2.5 years in eight of the patients , following treatment with hbot .
after five years of observation , five out of the eight patients had a recurrence of hematuria and required supravesical urinary diversion .
many questions regarding patient selection to hbot remain , as factors such as age , total irradiation dose , and duration of hematuria influence the outcome of hbot .
furthermore , there is a lack of data evaluating the effect of hbot in smoking patients or patients with chronic disease .
it should be remembered that hc may be caused by pharmacotherapy , such as cyclophosphamide .
moreover clotting and coagulation profiles in all cases of hc should be performed to rule out hematuria due to clotting and coagulation disorders .
there is no general consensus on the cost - effectiveness of hbot compared to standard methods of treatment , although investigations on the subject seem to suggest that hbot is more cost - effective than standard methods of treatment . in a study similar to this one ,
they concluded that the average cost per patient was $ 10,000 to $ 15,000 , comparing favorably to the cost of multiple conservative treatments to control symptoms . a review article by wang et al .
claims that hbot significantly reduces the length of the patient 's hospital stay , amputation rate , and wound care expenses in musculoskeletal disorders , making it is a cost - effective modality . concerning the treatment of soft tissue radiation necrosis using hbot , boykin et al .
showed that hbot achieved a reduction in patient charges by greater than 30% when compared to traditional management . in a study by hampson et al
. where a hypobaric treatment was used , a positive outcome occurred in 94% of patients with osteoradionecrosis of the jaw ( n = 43 ) , 76% with cutaneous radionecrosis that caused open wounds ( n = 58 ) , 82% with laryngeal radionecrosis ( n = 27 ) , 89% with radiation cystitis ( n = 44 ) , and 63% with gastrointestinal radionecrosis ( n = 73 ) .
a positive outcome of 100% was seen in patients who were treated with oral surgery in a previously irradiated jaw ( n = 166 ) .
cianci et al also reported that a reduction in expenses in the treatment of thermal - burn patients with adjunctive hbot .
, concerning hbot use in diabetic foot ulcers , concludes that hbot is cost - effective compared with standard care .
the authors also mention that additional hbot capacity would be needed if it were to be adopted as the standard care throughout canada .
in conclusion , hbot seems to be a promising treatment alternative in refractory hematuria caused by radiation - induced hc .
many studies have shown that hbot is a cost - effective alternative in various pathologies , including radiation - induced hc .
more cost - benefit analyses should be performed before a definite conclusion can be reached . | introductionwe present the effect of hyperbaric oxygen therapy ( hbot ) after radiotherapy for cancer in the pelvic cavity resulting in hematuria . increasing
the pressure of oxygen ( po2 ) in ischemic tissues favors the formation of new blood vessels and increases the secretion of collagen.material and methodswe evaluated 10 patients who were treated with hbot from october 2006 to december 2010 due to persistent radiation damage to the lining of the bladder leading to recurrent hematuria .
the study group was comprised of seven men and three women . in the case of cervical and endometrial cancers ,
30 gy of brachytherapy with 45 - 50 gy of teleradiotherapy were used . in prostate cancer ( pca ) , we applied 50 gy of teleradiotherapy with an additional dose of 20 - 24 gy , and in the case of bladder cancer ( bca ) , 50 gy of teleradiotherapy was applied with an additional dose of 16 gy .
hbot consisted of 60 hbo2 treatments , in which patients were administered 100% oxygen at a pressure of 2.5 atm.resultsthe group effect of total or partial resolution was observed in six patients . in one case , treatment was discontinued due to an increase in hematuria and the consequent suspicion of bladder tumor recurrence . while in and additional three cases
, the treatment did not produce the desired result.conclusionstreatment of hemorrhagic cystitis is a difficult therapeutic challenge .
one possible method is the implementation of hbot . in very difficult cases
, hbo2 treatment appears to be effective in giving more than half of patients a chance of getting better . | INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS |
one of the goals of pediatrics is children s achievement of maximum growth and development .
periodic surveillance of normal growth and development and screening for abnormalities are the major ways to achieve this goal . to this end
, birth weight is considered the most important index of neonates growth and the most important determinant of infants mortality .
low birth weight along with congenital abnormalities has a considerable role in physical and mental problems of childhood .
in addition , a high percentage of mortality during infancy and neonatal period is associated with low birth weight neonates .
very low birth weight neonates ( vlbw ) include 1.4% of the total births , while 50% of the neonatal mortality and 50% of neonatal disabilities are associated with them .
failure to thrive ( ftt ) risk in preterm infants is higher than term born infants .
it is expected that vlbw neonates reach to growth parameter as full term neonates at the end of their 2 year .
a study in ghana in 2000 on the growth and survival of lbw infants from the age of 0 to 9 years old showed that growth impairment in those children continued during childhood and that a partial compensatory growth had occurred within the first three years of their lives .
attention to growth and development is important in preventing children from mortality , but it is more important to prevent them from physical , mental and social disorders and their complications in the future .
one of the most important measures in this regard is to monitor and follow children s growth .
the prevalence of lbw neonates in 1999 in the urban areas of isfahan was 6.3% while it was reported to be 6.8% 0.6% in 2005 and 2006 .
furthermore , the prevalence of vlbw neonates in 2005 was reported to be 1.3% 0.2% . in recent years
many intensive care units has been developed in iran , so the mortality rate of vlbw infants had decreased but the growth pattern of lbw and vlbw infants are monitored as normal birth weight ( nbw ) infants by nchs growth charts . because of the importance of neonates growth and especially vlbw neonates as the important index of neonates health status , this research aims to compare growth trend in the normal , low and very low birth weight neonates until the age of 18 months .
we are seeking to find answers for the mean of weight , height and head circumference in normal , low and very low birth weight neonates until the age of 18 months as well as any lack of increase in the weight growth curve for these neonates .
the study population in this research consists of all the neonates born with normal weight , low or very low weight that were 18 months old at the time of sampling , which attended health care centers in the city of isfahan and had health care files . in this study
considering = % 5 , 90 children having normal birth weight and 90 children having low birth weight and 34 children with very low birth weight ( d = 0.8 ) ( 214 children in total ) comprised the study population .
the data gathering tool was a researcher - made questionnaire with 3 sections : the first section covered the demographics of the parents including questions about the father s job and education , the mother s job , education , age , number of pregnancies and the kind of delivery .
the second section comprised of questions about the children s demographics like the child s gender , weight , height and head circumference at birth , the birth order , the birth spacing with previous siblings , the duration of breast feeding , manner feeding and child s age at the time of starting the supplementary feeding .
the third section consists of questions about weight , height , head circumference , horizontal trend or decreasing trend of growth curve ( lack of increasing growth curve ) at the end of 2 , 4 , 6 , 9 , 12 , 15 and 18 months of the subjects .
content validity was used to validate data collection tools , and the reliability of the questionnaire was approved through re - test .
inclusion criteria included single pregnancy , being 18 months at the time of completing the questionnaire , and a complete health care profile for the months of 2 , 4 , 6 , 9 , 12 and 15 .
exclusion criteria include suffering from congenital abnormality , non - iranian nationality , being an adopted child , normal birth weight , and low birth weight caused by intra uterine growth retardation ( iugr ) , history of hospitalization in neonatal period for normal birth - weight neonates and after neonatal period for low and very low birth weight babies . from all health care centers in esfahan city , 29 centers were selected using simple random sampling . for determining samples considered for all three groups ( nbw , lbw , vlbw ) childcare notebooks in the departments of family healthcare centers were used .
all the care given to children is registered in these notebooks with dates of admission .
the selection of low birth weight and normal birth weight children in the healthcare centers was made based on the regular random sampling , in such a way that using childcare notebooks , the 18 month neonates with lbw and nbw who were registered , were extracted and in cases of having the inclusion criteria , they were selected using table of random numbers . in order to select the sample for vlbw group , because of inadequate number of such babies , the subjects whom had inclusion criteria were selected by census .
after selecting the samples , one of the researchers called the baby s mother and requested her to bring her baby for routine childcare visit on the arranged date at health care center .
at the time of completing the questionnaire , the researcher attended healthcare center accompanied by the questioner and recorded the information from the date of birth to 15 months old ; however the assessment at the 18 month was made by the researcher . for data analysis , the descriptive and inferential statistical method ( analysis of variance , kruskal - wallis , spearman correlation coefficient and chi - square tests ) were used .
the statistical software applied in the study was spss software version 18 ( spss inc .
from all health care centers in esfahan city , 29 centers were selected using simple random sampling . for determining samples considered for all three groups ( nbw , lbw , vlbw ) childcare notebooks in the departments of family healthcare centers were used .
all the care given to children is registered in these notebooks with dates of admission .
the selection of low birth weight and normal birth weight children in the healthcare centers was made based on the regular random sampling , in such a way that using childcare notebooks , the 18 month neonates with lbw and nbw who were registered , were extracted and in cases of having the inclusion criteria , they were selected using table of random numbers . in order to select the sample for vlbw group , because of inadequate number of such babies , the subjects whom had inclusion criteria were selected by census .
after selecting the samples , one of the researchers called the baby s mother and requested her to bring her baby for routine childcare visit on the arranged date at health care center .
at the time of completing the questionnaire , the researcher attended healthcare center accompanied by the questioner and recorded the information from the date of birth to 15 months old ; however the assessment at the 18 month was made by the researcher . for data analysis ,
the descriptive and inferential statistical method ( analysis of variance , kruskal - wallis , spearman correlation coefficient and chi - square tests ) were used .
the statistical software applied in the study was spss software version 18 ( spss inc .
the mean weight , height and the head circumference is shown in table 1 , 2 and 3 , respectively .
table 1 indicates that the mean weight in children with very low birth weight at 4 , 12 and 18 months old is 3.5 , 6 and 7 times as that at birth respectively ; that for children having low birth weights was 2.5 , 3.5 and 4 times as much , and that for children having a normal birth weight was 2 , 3 and 3.5 times as much , respectively .
mean and standard deviation of the weights of normal birth weight , low birth weight and very low birth weight children at 2 , 4 , 6 , 9 , 12 , 15 and 18 months mean and standard deviation of the heights of normal birth weight , low birth weight and very low birth weight children at 2 , 4 , 6 , 9 , 12 , 15 and 18 months mean and standard deviation of the head circumferences of normal birth weight , low birth weight and very low birth weight children at 2 , 4 , 6 , 9 , 12 , 15 and 18 months the variance analysis was used for comparing means of weight , height and head circumference in the three groups .
the results showed the mean weight of children at the age of 2 months ( p < 0.001 , f = 240.45 ) , 4 months ( p < 0.001 , f = 164.54 ) , 6 months ( p < 0.001 , f = 77.47 ) , 9 months ( p < 0.001 , f = 49.96 ) , 12 months ( p < 0.001 , f = 42.67 ) , 15 months ( p < 0.001 , f = 32.39 ) and 18 months ( p < 0.001 , f = 26.29 ) was significantly different in the three groups .
in addition , the mean height in children at 2 months ( p = 0.00 , f = 134.44 ) , 4 months ( p < 0.001 , f = 113.20 ) , 6 months ( p < 0.001 , f = 93.55 ) , 9 months ( p < 0.001 , f = 50.77 ) , 12 months ( p < 0.001 , f = 47.30 ) , 15 months ( p < 0.001 , f = 33.39 ) and 18 months old ( p <
the mean of head circumference of children at 2 months ( p < 0.001 , f = 112.04 ) , 4 months ( p < 0.001 , f = 62.27 ) , 6 months ( p < 0.001 , f = 40.66 ) , 9 months ( p < 0.001 , f = 31.90 ) , 12 months ( p < 0.001 , f = 23.30 ) , 15 months ( p < 0.001 , f = 21.28 ) and 18 months old ( p <
regarding the frequency distribution of horizontal growth curve , the results showed that there are no horizontal growth curves in the children studied at the age of 2,4 and 9 months . in children with normal birth weights , the relative frequency of horizontal growth curve at 6 , 15 and 18
months were 9.2% , 8.2% and 5.6% , respectively . in children with low birth weight
the relative frequency of horizontal growth curve was 2.2% at 12 months old and for all other ages , there were no horizontal growth curves . also , there were no horizontal growth curves in children having very low birth weights except at 18 months of age and for this age ; the relative frequency was 2.9% ( table 4 ) .
the frequency distribution of horizontal trend in growth curve of children with nbw , lbw and vlbw at 2 , 4 , 6 , 9 , 12 , 15 and 18 months with respect to frequency distribution of descending growth curve , the results showed that no descending trend in the children s growth curve was observed in ages 2 , 4 , 6 and 9 months among children in the three groups ; however , descending trend was noticed in 2.2% , 3.3% and 8.9% of children having normal birth weights at months 12 , 15 and 18 respectively . in low birth weight group
there was a descending trend of growth curve in 1.1% and 3.3% of the samples at post natal months 12 and 15 . in relation with demographic characteristics ,
the chi - square test showed that the father s education ( p = 0.007 ) , mother s education ( p = 0.01 ) , feeding manner ( p = 0.001 ) , and the time for commencement of complementary feeding ( p = 0.001 ) showed a significant difference among three groups , kruskal - wallis test showed that the breast feeding duration ( p < 0.001 ) and mother s age at birth ( p = 0.04 ) have a significant difference among the three groups .
the results showed that there is a statistical difference among three groups in terms of growth indices until 18 months of age .
however , the comparison of growth indices mean within different months demonstrates that the mean weight of children having very low birth weights in ages of 4 , 12 and 18 months has raised to 3.5 , 6 and 7 times , respectively .
the increase of weight in low birth weight group was 2.5 , 3.5 and 4 times and in normal birth weight group were 2.3 and 3.3 times at the same ages . in this
very context , van der mei et al . in 2000 showed that there was lower mean weight in mlbw and vlbw children at 2 , 6 , 18 , 48 and 96 months in comparison with the reference group .
powers et al . in 2008 conducted a study entitled growth and development of vlbw children after being discharged from hospital till 3 years of age .
the results showed that during the first twelve months weight gain pattern of children with the gestational age 27 weeks is not appropriate .
however , it increased from 18 months old faster than first year and became desirable up to 30 months of age , whereas , the growth delay continued till age of 3 in children whose gestational age was 26 . in the present study , we did not review the children s growth on the basis of gestational age and this may be considered as one of the constraints of this study . in light of the table 2 ,
the mean of height in children with very low birth weight raised 30.14 cm from birth to 12 months old . in children with low birth weight ,
the mean height increased till the age of 12 months up to 26.6 cm , whereas , it was 25.6 cm in children having normal weights .
from 1 year old to 18 months old , the height of children with low birth weight and also normal birth weight increased about 6.5 cm and that for children with very low birth weight increased about 7 cm .
ford et al . in 2000 showed that vlbw children at ages of 2 , 5 , 8 and 14 months are significantly shorter than children born with normal weights . in the present study
, we examined the trend of vlbw children s height growth up to the age of 18 months . at birth , the difference between the mean height of vlbw children and nbw group was 10.05 cm , and at month 18 it was 4.94 cm .
however , by considering the little difference in terms of height of these neonates as compared to nbw neonates at the age of 18 months , the results would have been different from that of made by ford if the study of children s growth had been conducted for a longer duration .
thus , it is suggested that more studies should be conducted in long - term .
the findings about the head circumference showed that the mean head circumference within studied durations was lower in lbw neonates as compared to nbw ones and also in vlbw neonates is also lower compared to the other two groups .
nonetheless , taking table 3 into consideration , the increase of head circumference mean from birth till 12 months of age in vlbw children was 16.03 cm and in lbw children was 12.11 cm , while in nbw children it was 11.11 cm . from 12 till 18 months of age the increase of mean head circumference in lbw children and also nbw children was about 1.5 cm and in the vlbw children it was about 1.8 cm .
van der mei et al . demonstrated that the increase of head circumference in moderately low birth weight and vlbw children during the first two months after birth was less than that in lbw children and from 2 to 4 months of age it was more than the reference group and after that , the increase in head circumference was similar for both groups .
the results of the study conducted by constantinou et al . in 2005 showed that in extremely low birth weight group ( elbw ) the mean of head circumference at 4 , 18 and 30 months was significantly lower than the mean in vlbw group . in the present study , due to having some limitations
, we did not deal with the growth of elbw neonates as an independent group and we considered all elbw and vlbw neonates as one group ( vlbw ) .
perhaps , if we had examined this group separately , we would have gained different results . in general
, the results of the present study indicate the desirable growth of lbw and vlbw groups of children compared to the nbw group .
these children have the potential to make up for their low weight after birth and also perhaps the attention and sensitivity of the parents toward these children in terms of nutrition and healthcare regardless of their low weights can be considered as an evidence for their desirable growth rates .
it is worth mentioning that although it is expected that the physical growth of lbw neonates with the absence of congenital abnormalities , injury of the central nervous system and vlbw increase up to a term in infant by the end of the 2 year , these children have a relatively high amount of differences with normal birth weight children in terms of growth indices at 18 months old . therefore , it is necessary to conduct some studies in this regard , prospectively and over a long period of time in order to guess exactly at what time lbw and vlbw iranian children are able to be similar to the nbw children in terms of growth , because this criterion might not be true for the iranian infants .
another necessary point with respect to lbw and vlbw children s growth is having a specific growth chart for them .
this is because currently , the growth status of such infants is examined on the basis of charts for neonates having normal birth weights .
the present study showed that there was horizontal trend in growth curve of normal birth weight children at ages of 6 , 15 and 18 months , in low birth weight children at 12 months and also in children with very low birth weight merely at the age of 18 months .
it may be stated that at the beginning of supplementary feeding and at the start of consuming family food , or starting to walk and its subsequent increased activity , children will experience a period of no increase in growth curve and this can be more noticed in children having normal weight compared to low birth and very low birth weight children .
it seems that all the attention and care given by the patents in relation to growth curve in children with low birth weight , in particular , children with very low birth weight , resulted in the fact that they were immediately taken care of once they suffered from any growth disorder . considering the results obtained by the research , the need for taking more effective measures for preventing birth of low birth weight neonates is more felt and in view of this , the role played by all the employees and authorities of health care system , obstetricians and midwives regarding anticipation and control of the factors affecting on low birth weight in neonates are highly important .
the healthcare authorities are recommended to provide a growth curve suitable for each one of the weight groups so that the children of every group birth weight can be examined and compared based on that curve . | background : birth weight is considered as the most important index of neonates growth as well as the most important determinant of infants mortality .
this study was conducted to investigate the growth pattern in normal , low and very low birth weight neonates for 18 months in the health care centers of isfahan , iran.materials and methods : in a cross - sectional and retrospective study , 214 neonates ( 90 with normal , 90 with low and 34 with very low birth weights ) were enrolled in the health care centers of isfahan and their growth in the 2nd , 4th , 6th , 9th , 12th , 15th and 18th months were investigated .
the data collection tool was a 3-part questionnaire which was validated using content validity .
a part of data was completed based on the health file of the family.findings:the mean of weight , height and head circumference in the months 2 , 4 , 6 , 9 , 12 and 15 in the three groups with normal , low and very low birth weights were significantly different . in the ages of 6 , 15 , 12 and 18
months , there was the highest frequency of horizontal trend in the growth pattern of children in the three groups . in the growth rate of neonates with low birth weight
, there was the highest frequency of horizontal trend at the age of 12 months and there was the highest frequency of descending trend in the 15th months.conclusions:the findings showed that according to national center for health statistics ( nchs ) percentiles , low and very low birth weight infants had lower growth in weight , length and head circumference compared to normal birth weight infants .
therefore , special attention should be given to healthcare and post - birth growth surveillance of these two groups to maintain and improve the health level of them . | I
M
Sampling
F
D |
conventional orthognathic surgery and craniofacial reconstruction have experienced widespread success , but several limitations are associated with these treatment modalities .
distraction osteogenesis is the process of new bone formation at the vascularized margin of bone segments separated by incremental traction .
the most common technique in distraction osteogenesis is mechanical stretching of the reparative bone tissue by a distraction device through an osteotomy or corticotomy site . with this technique ,
new bone is generated in the gap of osteotomy or corticotomy at the approximate rate of 1 mm per day .
this procedure was used as early as 1905 by codivilla and later popularized by the clinical and research studies of ilizarov[35 ] and ilizarov et al . in 1992 ,
distraction osteogenesis was first applied to the human mandible by mccarthy et al . , and since then , it has been applied to all the bones of the craniofacial skeleton , including the mid face and maxilla .
orthodontic tooth movement is a process in which a mechanical force is applied to induce alveolar bone resorption on the pressure side , and alveolar bone deposition on the tension side . on the tension side ,
the periodontal ligament is stretched ( distracted ) and followed by alveolar bone deposition ( osteogenesis ) .
the process of osteogenesis in the periodontal ligament during orthodontic tooth movement is similar to that in the midpalatal suture during rapid palatal expansion or to that in the midface sutures in the growing animal during mid face distraction .
the regular rate of osteogenesis in orthodontic tooth movement during canine retraction is about 1 mm per month , which is much slower than that in distraction osteogenesis .
liou and huang first applied this concept to orthodontic tooth movement and performed rapid canine retraction through distraction , since that there is scarcity of literature about it . in this case report
a 16-year - old post pubertal female with angle 's class i malocclusion with bimaxillary protrusion presented to our department for orthodontic treatment . according to the protocol of our department ,
all the essential orthodontic diagnostic records including facial photographs [ figure 1 ] were taken and analyzed .
treatment plan for this patient was fixed orthodontic mechanotherapy with extraction of all four premolars and retraction of anterior teeth . as with conventional orthodontic mechanotherapy retraction of canine
patient was informed about the treatment procedure , and a written informed consent was obtained .
photographs of patient ( a - d ) before orthodontic treatment the individual canine distractor was a tooth - borne , semi - rigid device .
after the bands were fabricated for the canine and first molar , an impression was obtained , the bands were transferred into the impression material , and the study cast was made . the device consisted of an anterior section , a posterior section , a screw , and a hex wrench to advance the screw [ figure 2 ] .
a 360 activation of the screw produced 0.5 mm of distal movement in the canine tooth .
distraction device and lab procedure ( a - c ) after the first premolar extraction , vertical osteotomies were carried out at the buccal and lingual sites of the interseptal bone adjacent to the canine tooth .
the vertical osteotomies were connected with an oblique osteotomy extending toward the base of the interseptal bone to weaken the resistance [ figures 3 and
4 ] .
slight swelling was observed on the patient 's face after the surgery , and it was resolved after three to five days , postoperatively .
vertical and oblique osteotomies ( dotted line ) for undermining bone distal to canine clinical intraoral photograph ( a - c ) of extracted tooth and surgical technique the distraction was initiated just after the surgery .
an advancement of 0.5 mm was performed per day , until each canine tooth was distracted into the desired position in two weeks .
the patients were closely monitored during the distraction period , and at the time of the activation of the device to retract the canine , patient often felt pain , but pain was subsided after 5 to 10 minutes of the device activation .
intraoral photographs of a patient , before and after rapid canine distalization , are shown on right side [ figures 5 and 6 ] and on left side [ figure 7 ] , in the mandibular arch .
initially , we have planned the distraction of maxillary and mandibular canines simultaneously , but our canine distractor was bulky , as we have used custom made distractor which was inexpensive .
so , we planned to distract the maxillary and mandibular canine separately . intra - oral photograph of patient before distraction on right side in mandibular arch intra - oral photograph of patient at end of distraction on right side in mandibular arch complete canine distraction procedure with retention device on left side in mandibular arch
after the bands were fabricated for the canine and first molar , an impression was obtained , the bands were transferred into the impression material , and the study cast was made . the device consisted of an anterior section , a posterior section , a screw , and a hex wrench to advance the screw [ figure 2 ] .
a 360 activation of the screw produced 0.5 mm of distal movement in the canine tooth .
after the first premolar extraction , vertical osteotomies were carried out at the buccal and lingual sites of the interseptal bone adjacent to the canine tooth .
the vertical osteotomies were connected with an oblique osteotomy extending toward the base of the interseptal bone to weaken the resistance [ figures 3 and
4 ] .
slight swelling was observed on the patient 's face after the surgery , and it was resolved after three to five days , postoperatively .
vertical and oblique osteotomies ( dotted line ) for undermining bone distal to canine clinical intraoral photograph ( a - c ) of extracted tooth and surgical technique
an advancement of 0.5 mm was performed per day , until each canine tooth was distracted into the desired position in two weeks .
the patients were closely monitored during the distraction period , and at the time of the activation of the device to retract the canine , patient often felt pain , but pain was subsided after 5 to 10 minutes of the device activation .
intraoral photographs of a patient , before and after rapid canine distalization , are shown on right side [ figures 5 and 6 ] and on left side [ figure 7 ] , in the mandibular arch .
initially , we have planned the distraction of maxillary and mandibular canines simultaneously , but our canine distractor was bulky , as we have used custom made distractor which was inexpensive .
so , we planned to distract the maxillary and mandibular canine separately . intra - oral photograph of patient before distraction on right side in mandibular arch intra - oral photograph of patient at end of distraction on right side in mandibular arch complete canine distraction procedure with retention device on left side in mandibular arch
reducing the orthodontic treatment time and controlling the anchorage loss are common aims of research in modern orthodontic practice .
rapid canine distalization is a cornerstone for these goals , and was first introduced by liou and huang in 1998 . since then , however , studies of rapid canine distalization through distraction of the periodontal ligament are limited because individual canine retractors are not available on the market and the long - term effects are unknown . we have observed that degree of the patient problem in terms of pain and discomfort vis a vis regular canine retraction and enmasse retraction using microimplants was more with this procedure , especially during the osteotomy , to reduce the bony resistance , and during the activation of the device , to distract the canine .
after the seven days of the surgery , patient was completely comfortable with no report of pain and discomfort .
clinically , after the completion of distraction procedure , periodontium was sound with considerable amount of keratinized gingival and radiographically newly formed bone , which was indistinguisible after three to four months of distraction .
we closely monitored the canine root during distraction period as the canine distraction was completed within two weeks , while the root resorption is initiated two to three weeks , after the orthodontic force is applied , and may continue for the duration of force application .
the best way to minimize the root resorption induced by orthodontic tooth movement is to complete the tooth movement in short duration or even before the initiation of root resorption .
the major advantages of rapid distalization were shortening the treatment time , eliminating the need for additional anchorage , and rapid retraction of incisors using the new bone tissue distal to the lateral incisors . with this technique
by using this concept , canines can be distracted distally in premolar extraction space in three weeks without significant complication .
however , the clinical techniques and procedures in canine distraction still need to be refined .
this new concept and technique is best used on those cases whose anterior teeth are severely crowded or protruded .
the canines can be distracted rapidly , and almost all of the extraction space can be used for anterior dental alignment or retraction . after distraction , the anterior tooth retraction can be rapid as well , while the new bone tissues distal to the lateral incisors are still fibrous .
in addition to orthodontic tooth movement , we might possibly use this new concept and technique to generate new bone and keratinized gingival tissue for treating periodontal disease .
currently , the canine distractors are bulky , unidirectional , and unavailable in the market .
they need to be refined , developed , and oriented with fixed appliances in the future
. moreover , the long - term effects of this method are unknown , and clinical and histological research is needed . | distraction osteogenesis is a method of inducing new bone formation by applying mechanical strains on preexisting bone .
the process of osteogenesis in the periodontal ligament during orthodontic tooth movement is similar to the osteogenesis in the midpalatal suture during rapid palatal expansion .
a new concept of distracting the periodontal ligament is proposed to elicit rapid canine retraction in two weeks . at the time of first premolar extraction , the interseptal bone distal to the canine
was undermined with a bone bur , grooving vertically inside the extraction socket along the buccal and lingual sides and extending obliquely toward the socket base .
then , a tooth - borne , custom - made , intraoral distraction device was placed to distract the canine distally into the extraction space .
it was activated 0.5 mm / day , immediately after the extraction .
canine was distracted 6.5 mm into the extraction space within two weeks . | INTRODUCTION
CASE REPORT
Distraction device
Surgical procedure
Distraction protocol
DISCUSSION
CONCLUSION |
two techniques have been developed for the percutaneous placement of bilateral self - expandable metallic stent ( sems ) for palliative drainage : stent - in - stent and side - by - side deployment techniques , in which two tubular stents were used for bilateral sems deployment , including y and t - configured stent placement [ 2 , 3 ] .
we developed a novel , two - piece , y - shaped sems , with angled main stent and a smaller limb stent ( micro - tech , nanjing , china ) .
this configuration conforms to the hilar biliary tract anatomy and provides strong support forces to the duct ( figure 1 ) . the hilar trifurcated biliary anatomy ( a ) .
the novel y - configured bilateral self - expandable metallic stent ( sems ) consists of two components : the main stent ( angled shape ) and a limb stent .
the intrahepatic branch portion ( star ) of the main stent is 8 mm in diameter and 2.5 cm in length ; the common bile duct portion ( triangle ) is 10 mm in diameter and 3.0 cm in length . at the angle of the main stent
the limb stent ( circle ) is 8 mm in diameter and 2.5 cm in length ( b , c ) .
fluoroscopic images show 4 - 6 radiopaque markers made of gold attached to the outer surface of the stent ( d ) ( short arrow ) high - dose - rate ( hdr)-ir was the most common radioactive source for intraluminal brachytherapy ( ilbt )
. however , an hdr - ir afterloading unit is not available at most hospitals in china , which should be protected by an isolated and well - shielded room .
moreover , because the bile duct tissue is very thin , hdr - ir ilbt can not be completed in some cases due to the computerized remote after loading source where protection is needed .
i seeds are characterized by a long half - life ( 59.6 days ) , which is convenient for their storage .
brachytherapy with i seeds strands has been reported for the treatment of hepatocellular carcinoma with intrahepatic portal vein tumor thrombus .
herein , we used a newly designed y - shaped sems combined with two i seed strands to treat type iv bismuth tumor .
a 62-year - old female was admitted to the hospital complaining of right upper quadrant pain .
initial magnetic resonance ( mr ) showed hepatic hilar obstruction due to cholangiocarcinoma , extending from hilum of extrahepatic bile duct to distal common bile duct ( figure 2a ) .
elevated total bilirubin level ( 204.3 mol / l ) and direct bilirubin level ( 177.3 mol / l ) were found at admission .
percutaneous transhepatic biliary drainage ( ptbd ) through the left intrahepatic bile duct ( figure 3a ) was done .
two temporary 8.5-f drainage catheters ( cook , bloomington , il , united states ) were inserted across the hilar tumor to duodenum for percutaneous ptbd via bilateral approaches .
the patient had severe hemobilia two days after ptbd and it was due to a pseudoaneurysm of the right intrahepatic artery , and was treated successfully with selective transarterial coil embolization .
we developed a newly designed y - configured bilateral sems , which has two components : angled main stent and a smaller limb stent ( figure 1 ) .
the cylindrical titanium capsules of the i seeds ( xingke company , shanghai , china ) were 0.8 mm in diameter and 4.5 mm in length , and seed activity was 1.0 mci .
we placed 15 and 10 seeds in two 4-f sterile catheter to construct i seed strands , respectively .
preoperative magnetic resonance ( mr ) showed intrahepatic bile duct dilation , which was caused by hilar cholangiocarcinoma in a 62-year - old female patient ( a ) .
abdominal computed tomography ( ct ) showed less intrahepatic and extrahepatic bile duct dilatation than before the procedure ( b , c ) .
follow - up ct obtained at second month after stent placement showed adequate biliary drainage , with decompression of the treated biliary ducts in transverse ( b ) and volume - rending imaging ( c )
bismuth type iv cholangiocarcinoma was confirmed by ptcb ( a ) .
the cbd and opening hole portion of stent were deployed and ensure two middle radiopaque markers towards the opening of contralateral bile duct ( b ) .
cholangiography was performed through two 8.5-f drainage catheter two month later ( e ) ; after patency of the expanded stents was confirmed , and the drainage catheter and seed strands were removed ( f )
the procedure was conducted on the tenth day after the initial drainage procedure . super - stiff guide wire ( boston scientific , natick , ma , usa ) were passed through the stenosis simultaneously into the duodenum via bilateral approach .
the main stent was first introduced over the guide wire into the common bile duct ( cbd ) through the right ptbd tract .
the main stent was initially introduced over the guidewire into the cbd through the right ptbd tract .
the proximal end of the main stent was located in the ipsilateral intrahepatic duct ( ihd ) beyond the boundary of the hilar tumor .
the radiopaque markers on the outer surface of the proximal , middle , and distal portions of the stent positioned correctly under fluoroscopy , so that the opening in the side of the stent faced the contralateral bile duct .
firstly , the cbd part of the main stent and the part with the opening in the side were deployed , ensuring that the middle two radiopaque markers were positioned at the opening of the contralateral bile duct .
secondly , a catheter and guidewire were passed via the contralateral bile duct , through the opening in the side of the stent , into the cbd and duodenum .
the catheter was then withdrawn , and the limb stent was introduced over the guidewire and advanced until the distal portion of limb stent ( micro - bell shape , 2 - 3 mm in length ) going into the main piece of stent . after confirming that its proximal end was located in the ihd beyond the boundary of the hilar tumor and its distal end located within the lumen of the main stent , the limb stent was deployed .
thirdly , both 9-f sheath ( cordis , usa ) , was advanced along the stiff guide wire , and two i seed strands were inserted through the 9-f sheath to the trifurcation stenosis , respectively ( figure 3d ) .
finally , fixed seed strands and super stiff guide wire withdraw the 9-f sheath , and two 8.5-f drainage catheters were inserted along super stiff guide wire across the hilar tumor to duodenum .
after 2 months ilbt , two i seed strands and temporary drainage catheter were removed after patency of the expanded stents was confirmed by the cholangiogram ( figure 3e and f ) .
cumulative absorbed dose at the dose reference points , 5 mm , 10 mm , 15 mm from the source axis , were 97.2 , 28.8 , 14.4 gy according to the computerized treatment planning system ( university of beijing aeronautics and astronautics , beijing , china ) ( figure 4 ) , respectively .
cumulative absorbed dose was analyzed according to the computerized treatment planning systems ( tps ) abdominal computed tomography ( ct ) showed less intrahepatic and extrahepatic bile duct dilatation than before the procedure ( figure 2b and c ) . meanwhile ,
total bilirubin and direct bilirubin levels decreased to 29.8 mol / l and 11.7 mol / l , respectively , and remained low throughout the follow - up until patient 's death due to myocardial infarction 381 days after the stenting .
palliation with percutaneous sems placement has been the best option available for the treatment of patients with unresectable malignant hilar biliary obstruction .
stent - in - stent deployment was the most common method , in which a second stent is placed through the mesh of the first stent . in these cases ,
the mesh in the middle portion of the stent is necessarily looser and therefore more likely to allow tumor ingrowth and occlusion .
bile duct stents manufactured in china are constructed using metal wires of thicker gauge ( 0.16 mm ) than that used in south korean stents ( 0.12 mm ) , which results in reduced stent compliance .
the newly designed y - configured bilateral sems was in accordance with hilar biliary trifurcation anatomical structure improving compliance .
moreover , the proximal end with a micro single bell shape ( 2 - 3 mm in length ) were inserted into the lumen of the main piece of stent without excessive overlapping , which was different form traditional stent - in - stent deployment , and another sems could be placed coaxially into the original sems through the ptbd tract without difficulty when second occlusion occurred .
considering that the median stent patency using a t or y configuration was 104 - 250 days in the literatures , total bilirubin levels remained low throughout the follow - up 381 days , which was satisfactory .
another innovation is the treatment of type iv klatskin tumor with placement i seed strands to prolong the patency period .
firstly , the seeds can deliver a high radiation dose to the target tissues that drops rapidly with the distance from the seeds , thereby resulting in low radiation doses to the surrounding tissues . as a result
, the use of i seeds enables the delivery of a high radiation dose to tumor tissues while minimizing injury to normal tissues , which requires less shield .
for example , cumulative absorbed dose at the 5 mm from the source axis was 97.2 gy and it dropped rapidly to 28.8 gy at the 10 mm from the source axis in this patient .
secondly , i seeds are characterized by a long half - life ( 59.6 days ) , and this long half - life resulted in sustained injury to the tumor compared with the use of external irradiation .
thirdly , the ilbt can be stopped immediately when brachytherapy related complications occurred . in conclusion , a newly designed y - shaped sems combined with two i seed strands via bilateral approach represents a feasible treatment for patients with type iv klatskin tumors , providing both brachytherapy and biliary drainage .
thus , this technique may provide a novel method for prolonging survival in these patients . | we report a case in an inoperable patient with type iv klatskin tumor treated by the use of a novel , two piece , y - configured self - expandable metallic stent ( sems ) combined with two 125i seed strands via bilateral approach .
the placement of the y - shaped sems was successful and resulted in adequate biliary drainage .
after 2 months of intraluminal brachytherapy ( ilbt ) , both 125i seed strands and temporary drainage catheter were removed after patency of the expanded stents was confirmed by the cholangiogram .
this technique was feasible and could be considered for the treatment of patients with bismuth type iv klatskin tumors . | Purpose
Case report
Discussion
Disclosure |
paracetamol belongs to the class of medications called analgesic ( pain reliever ) and antipyretic ( fever reducer ) and is widely used as an over - the - counter ( otc ) pain and fever medication to relieve mild to moderate pain from head , muscle , menstrual period , cold , sore throat , tooth , reactions to vaccination ( shot ) , in osteoarthritis ( arthritis caused by the breakdown of the lining of the joints ) and to reduce fever .
paracetamol is known to cause liver and renal injury in humans and experimental animals when administered in higher and repeated doses .
the incidences associated with liver injury include abdominal pain , nausea , vomiting , low blood sugar , low blood ph , easy bleeding , oliguria ( decreased output of urine ) and hepatic encephalopathy .
the excess use of paracetamol leads to liver and renal failure , and even causes death .
the mechanism of paracetamol toxicity in the liver is well described , but in the kidney it is less clearly understood .
paracetamol is primarily metabolized and excreted as paracetamol glucuronide and paracetamol so4 in the hepatocytes .
when paracetamol is taken at a higher dose ( larger amounts of paracetamol are bioactivated ) , it produces a reactive metabolite n - acetyl - p - benzoquinone imine ( napqi ) , generated by several hepatic cytochrome p-450 isoenzymes .
glutathione ( gsh ) can reduce napqi , recycling to paracetamol , or conjugate the napqi forming paracetamol
generation of the reactive oxygen species has appeared as an early event that is preceded by intracellular gsh depletion .
depletion of gsh level permits the protein thiolarylation by napqi that may compromise the protein function and contribute to paracetamol , leading to hepatocellular damage .
paracetamol - induced renal insufficiency depends on the drug , dose , duration of pharmacological effect and the health of the patient .
overdose of paracetamol causes various adverse effects in the kidney , which may partly be due to the vasoconstriction resulting in inhibition of renal prostaglandin - mediated vasodilatation , decreasing renal blood flow and finally decreasing the glomerular filtration rate .
oxidative stress , with decline of gsh and lipid peroxidation , has also been suggested as a mechanism by which paracetamol could induce renal injury .
triphala is the combination ( equal proportion 1:1:1 ) of dried fruits ( phala ) of amalaki ( embelica officinalis ) , bibhitaki ( terminalia bellirica ) and haritaki ( terminalia chebula ) . as per ayurveda , in the indian system of medicine
, it is commonly used to balance the three doshas ( humors ) in the body , viz .
traditionally , the triphala formulation has been used as a laxative in chronic constipation , as a detoxifying agent of the colon , and as a rejuvenator of the body . according to recent researches
, the triphala formulation also shows antidiabetic and hepatoprotective activities and plays an important role in blood pressure control and balances cholesterol .
triphala is an important medicine in rasayana therapy that preserves and promotes health , immunity and longevity .
the individual plants of triphala have been reported to be a rich source of vitamin c , ellagic acid , gallic acid , chebulinic acid , bellericanin , -sitosterol and flavanoids , which possess several other health benefits as well .
these herbs are also reported for their anti - inflammatory , anti - mutagenic , antioxidant , cytoprotective , gastroprotective , anti - cancer activity and anti - bacterial effects .
renal toxicity has not yet been reported till date to the best of our knowledge .
this study was , therefore , undertaken to examine the protective effect of triphala against paracetamol- induced hepatic
potassium dihydrogen phosphate , dibasic monohydrogen phosphate , formaldehyde , paraffin wax , xylene and biochemical kits were purchased from merck chemicals private ltd .
dried fruits of amalaki ( emblica officinalis ) , bibhitaki ( terminalia bellirica ) and haritaki ( terminalia chebula ) were collected from the farm of csir - cimap , lucknow and authenticated by an ayurvedic physician at the hmpd , csir - cimap lucknow , india .
a voucher specimen of amalaki ( emblica officinalis ) ( hmp101 ) , bibhitaki ( terminalia bellirica ) ( hmp102 ) and haritaki ( terminalia chebula ) ( hmp103 ) was deposited in the departmental herbarium of the csir - cimap , lucknow , up , india . dried fruits of amalaki , bibhitaki and haritaki
the dried powder of all the three fruits was mixed in a defined ratio of 1:1:1 and the mixture was extracted with distilled water by using a cold maceration process .
one hundred grams of the above mixture was taken and soaked overnight in 400 ml of distilled water .
the extract was filtered using a filter paper ( whatman filter paper grade 1 ) and then the extract was concentrated in a rotary evaporator ( buchi r-210 , switzerland ) and freeze - dried under vacuum for 50 h using the lobconco freeze drying system ( 42c to 47c temperature with 0.340 mbar pressure ) .
completely dried material was collected and stored in a closed air - tight container at room temperature until use .
swiss albino mice ( weight 2025 g ) were maintained in an air conditioned room ( 22 3c ) with relative humidity of 50 5% and 12-h light/12-h dark cycle and acclimatized for 1 week prior to the study .
they were fed with a certified pellet rodent diet ( dayal industries , lucknow , india ) and water was provided ad libitum .
the institutional animal ethics committee of csir - cimap approved the protocol of the experiment ( ah-2012 - 10 ) .
the guidelines for animal care were followed as recommended by the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) , government of india .
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controlsgroup 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled watergroup 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 daysgroup 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days .
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controls group 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled water group 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 days group 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days . on the 8 day , all the groups except group 1 were given an intraperitoneal injection of paracetamol at 500 mg / kg bw .
after 2.5 h of paracetamol administration , 500 l of blood was collected from the retro - orbital plexus of the mice using non - heparinised hematocrit capillaries . the serum was separated and stored at -20c for experimental analysis .
the collected blood samples were used for the analysis of different biochemical parameters , viz .
aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , alkaline phosphatase ( alp ) , bilirubin , creatinine , urea and uric acid , by using standard diagnostic kits as per the manufacturer 's protocol ( labkit from merck chemicals private ltd . , india ) .
after blood collection , animals were sacrificed and their liver was dissected out and washed with phosphate buffer ( ph 7.4 ) for complete removal of blood stains and clot .
after washing , tissue was fixed in 10% formalin and embedded in molten paraffin wax followed by cutting of sections with microtome .
deparaffinized sections were then stained with hematoxylin and eosin and observed microscopically for histopathological changes .
all the statistical analyses were performed with the help of graph pad instat software ( version 3.0 ) .
difference between means was calculated by one - way analysis of variance ( anova ) followed by the student newman
potassium dihydrogen phosphate , dibasic monohydrogen phosphate , formaldehyde , paraffin wax , xylene and biochemical kits were purchased from merck chemicals private ltd .
dried fruits of amalaki ( emblica officinalis ) , bibhitaki ( terminalia bellirica ) and haritaki ( terminalia chebula ) were collected from the farm of csir - cimap , lucknow and authenticated by an ayurvedic physician at the hmpd , csir - cimap lucknow , india .
a voucher specimen of amalaki ( emblica officinalis ) ( hmp101 ) , bibhitaki ( terminalia bellirica ) ( hmp102 ) and haritaki ( terminalia chebula ) ( hmp103 ) was deposited in the departmental herbarium of the csir - cimap , lucknow , up , india .
dried fruits of amalaki , bibhitaki and haritaki were collected and powdered using a mill .
the dried powder of all the three fruits was mixed in a defined ratio of 1:1:1 and the mixture was extracted with distilled water by using a cold maceration process .
one hundred grams of the above mixture was taken and soaked overnight in 400 ml of distilled water .
the extract was filtered using a filter paper ( whatman filter paper grade 1 ) and then the extract was concentrated in a rotary evaporator ( buchi r-210 , switzerland ) and freeze - dried under vacuum for 50 h using the lobconco freeze drying system ( 42c to 47c temperature with 0.340 mbar pressure ) .
completely dried material was collected and stored in a closed air - tight container at room temperature until use .
swiss albino mice ( weight 2025 g ) were maintained in an air conditioned room ( 22 3c ) with relative humidity of 50 5% and 12-h light/12-h dark cycle and acclimatized for 1 week prior to the study .
they were fed with a certified pellet rodent diet ( dayal industries , lucknow , india ) and water was provided ad libitum .
the institutional animal ethics committee of csir - cimap approved the protocol of the experiment ( ah-2012 - 10 ) .
the guidelines for animal care were followed as recommended by the committee for the purpose of control and supervision of experiments on animals ( cpcsea ) , government of india .
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controlsgroup 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled watergroup 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 daysgroup 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days .
group 1 : ( vehicle control ) animals received distilled water for 7 days and served as normal controls group 2 : ( paracetamol ) animals in this group received ( only the paracetamol challenge ) distilled water group 3 : ( liv-52 ) positive control group , pre - treated with liv-52 at 5.2 ml / kg body weight ( bw ) for 7 days group 4 and group 5 : served as the treatment groups and pre - treated with triphala extract at 100 mg / kg and 300 mg / kg body weight for 7 days . on the 8 day , all the groups except group 1 were given an intraperitoneal injection of paracetamol at 500 mg / kg bw .
after 2.5 h of paracetamol administration , 500 l of blood was collected from the retro - orbital plexus of the mice using non - heparinised hematocrit capillaries . the serum was separated and stored at -20c for experimental analysis .
the collected blood samples were used for the analysis of different biochemical parameters , viz .
aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , alkaline phosphatase ( alp ) , bilirubin , creatinine , urea and uric acid , by using standard diagnostic kits as per the manufacturer 's protocol ( labkit from merck chemicals private ltd . , india ) .
after blood collection , animals were sacrificed and their liver was dissected out and washed with phosphate buffer ( ph 7.4 ) for complete removal of blood stains and clot .
after washing , tissue was fixed in 10% formalin and embedded in molten paraffin wax followed by cutting of sections with microtome .
deparaffinized sections were then stained with hematoxylin and eosin and observed microscopically for histopathological changes .
all the statistical analyses were performed with the help of graph pad instat software ( version 3.0 ) .
difference between means was calculated by one - way analysis of variance ( anova ) followed by the student newman
intraperitonial ( ip ) administration of paracetamol in swissalbino mice ( group 2 ) at the dose of 500 mg / kg bw produced a significant ( p < 0.001 ) increase in the serum levels of ast , alt , alp and bilirubin , while pre - treatment with the liv-52 syrup ( 5.2 ml / kg body weight ) ( group 3 ) and triphala extract at the rate of 100 mg / kg and 300 mg / kg bw ( groups 3 and 4 ) significantly ( p < 0.01 ) prevented the elevation in these liver enzymes [ table 1 , figure 1 ] .
similarly , the serum levels of creatinine , urea and uric acid were increased after paracetamol administration , which were significantly ( p < 0.001 ) different from the vehicle control group .
pre - treatment with the triphala extract at the above - mentioned dose and liv-52 syrup effectively ( p < 0.01 ) prevented the increase in the serum levels of these renal function tests [ table 1 , figure 1 ] .
the effect of triphala extract on liver function tests ( ast , alt , alp and bilirubin ) and renal function tests ( creatinine , urea and uric acid ) shows its effect in a dose - dependent manner .
the triphala extract exhibited more protective effects at 300 mg / kg bw than 100 mg / kg bw [ table 1 and figure 1 ] .
effect of triphala rasayana on liver and kidney bio - markers for assessing hepatic - renal toxicity in swiss albino mice effect of triphala extract on different groups of mice against paracetamol - induced hepatic
the effect of pre - treatment with triphala extract at 100 mg / kg and 300 mg / kg body weight dose on ast , alt , alp , bilirubin , creatinine , urea and uric acid of mice in paracetamol - induced liver and renal toxicity . * p < 0.05 , * * p < 0.01 , * * * p <
0.001 , paracetamol compared with the vehicle control group , p < 0.05 , p < 0.01 , p < 0.001 , liv-52 and treatment groups 4 and 5 compared with the paracetamol group ( n = 6 mice / group ) histopathological observation of liver tissues showed that there were no abnormal morphological changes in the liver specimens of the control group [ figure 2a ] .
the liver samples of the paracetamol group showed necrosis , phagocyte filtration and fat degeneration [ figure 2b ] .
the liver tissues in the groups pre - treated with liv-52 syrup and triphala extract protects the liver cells , central vein and portal vein from paracetamol toxicity and maintains the normal architecture of the liver tissues [ figure 2c e ] .
control group ( a ) mice showing normal histological architecture and chords of hepatocytes are radiating .
toxin ( paracetamol)-induced group ( b ) showing centri - zonal necrotic areas and degenerated liver tissues .
liv-52 ( 5.2 ml / kg body weight ) and triphala extract at 100 mg / kg and 300 mg / kg body weight ( c , d and e ) showing protection of hepatocytes with mild changes and pyknotic nuclei around the central vein indicating necrotic changes
intraperitonial ( ip ) administration of paracetamol in swissalbino mice ( group 2 ) at the dose of 500 mg / kg bw produced a significant ( p < 0.001 ) increase in the serum levels of ast , alt , alp and bilirubin , while pre - treatment with the liv-52 syrup ( 5.2 ml / kg body weight ) ( group 3 ) and triphala extract at the rate of 100 mg / kg and 300 mg / kg bw ( groups 3 and 4 ) significantly ( p < 0.01 ) prevented the elevation in these liver enzymes [ table 1 , figure 1 ] .
similarly , the serum levels of creatinine , urea and uric acid were increased after paracetamol administration , which were significantly ( p < 0.001 ) different from the vehicle control group .
pre - treatment with the triphala extract at the above - mentioned dose and liv-52 syrup effectively ( p < 0.01 ) prevented the increase in the serum levels of these renal function tests [ table 1 , figure 1 ] .
the effect of triphala extract on liver function tests ( ast , alt , alp and bilirubin ) and renal function tests ( creatinine , urea and uric acid ) shows its effect in a dose - dependent manner .
the triphala extract exhibited more protective effects at 300 mg / kg bw than 100 mg / kg bw [ table 1 and figure 1 ] .
effect of triphala rasayana on liver and kidney bio - markers for assessing hepatic - renal toxicity in swiss albino mice effect of triphala extract on different groups of mice against paracetamol - induced hepatic
the effect of pre - treatment with triphala extract at 100 mg / kg and 300 mg / kg body weight dose on ast , alt , alp , bilirubin , creatinine , urea and uric acid of mice in paracetamol - induced liver and renal toxicity . * p < 0.05 , * * p < 0.01 , * * * p < 0.001 , paracetamol compared with the vehicle control group , p
< 0.05 , p < 0.01 , p < 0.001 , liv-52 and treatment groups 4 and 5 compared with the paracetamol group ( n = 6 mice / group )
histopathological observation of liver tissues showed that there were no abnormal morphological changes in the liver specimens of the control group [ figure 2a ] .
the liver samples of the paracetamol group showed necrosis , phagocyte filtration and fat degeneration [ figure 2b ] .
the liver tissues in the groups pre - treated with liv-52 syrup and triphala extract protects the liver cells , central vein and portal vein from paracetamol toxicity and maintains the normal architecture of the liver tissues [ figure 2c e ] .
control group ( a ) mice showing normal histological architecture and chords of hepatocytes are radiating .
toxin ( paracetamol)-induced group ( b ) showing centri - zonal necrotic areas and degenerated liver tissues .
liv-52 ( 5.2 ml / kg body weight ) and triphala extract at 100 mg / kg and 300 mg / kg body weight ( c , d and e ) showing protection of hepatocytes with mild changes and pyknotic nuclei around the central vein indicating necrotic changes
the liver is a multipurpose organ in the body related with regulation of various metabolic pathways .
therefore , damage of the liver tissues caused by a hepatotoxic agent is a serious consequence .
paracetamol is the most commonly used analgesic and antipyretic drug known to possess a hepatotoxic effect in humans as well as in animals at high doses .
it is generally observed that drug- or chemical - induced liver and kidney injuries are associated with the overproduction of free radicals .
parallel to this , renal insufficiency occurs in around 12% of patients with paracetamol overdose .
thus , prevention and treatment of liver injury , together with renal protection , can be a good line of treatment in liver diseases .
herbal medicines have been used since antiquity and have played a pivotal role in the prevention and treatment of various diseases .
a large number of herbs and herbal formulations are used in the indian medicinal systems , viz .
ayurveda , siddha , unani and amchi , etc . , according to the world health organization ( who ) , 80% of the people throughout the world currently use herbal medicines for primary health care , as well as most of the modern plant - derived pharmaceutical medicines are used in ways that correlated directly with their traditional uses .
triphala is considered as one of the most important rasayana formulations in the indian system of medicine ( ism ) . in this current research work ,
triphala extract was screened for its possible hepato renal protection in mice against paracetamol - induced toxicity . in the present study ,
paracetamol - induced hepatotoxicity was evidenced by the biochemical measurements and histopathological changes , while renal damage was indicated by rise in serum levels of renal markers . the increased level of serum ast , alt , alp , bilirubin , creatinine , urea and uric acid in mice treated with paracetamol indicated altered liver and renal functions due to its toxic effects .
pre - treatment of mice with triphala extract for seven consecutive days resulted in a significant protection of paracetamol - induced elevation of serum enzyme markers ( ast , alt , alp , bilirubin , creatinine , urea and uric acid ) given in two doses when compared with the paracetamol group [ figure 1 ] .
the triphala extract significantly reduced the elevated serum enzyme levels in the treatment group , which indicated the ability of triphala to preserve the normal functions of the liver and kidney .
it can be proposed that triphala inhibited the free radical generation and resultant damage to maintain the normal functions of the liver and renal cellular membrane ; [ figure 1 ] although the exact mechanism of action needs further investigation .
the hepatoprotective effect of the triphala extract was further confirmed by histopathological studies of the liver tissues , which basically supported the results obtained from the serum assays . moreover , as revealed by the histopathological study , the triphala extract could provide protection against paracetamol - mediated hepatic alterations by membrane regeneration and , therefore , it may be helpful in limiting the drug - induced membranal damage in the liver and kidney .
furthermore , in our study , the results clearly show that the triphala extract at the dose of 300 mg / kg bw offers more protection to liver tissues that were comparable to liv-52 , but in the case of 100 mg / kg bw , it offers comparatively less protection than liv-52 against paracetamol - induced hepato renal injury .
however , a more elaborative study with special focus on the effects of triphala in maintaining gsh levels and possible measurement of naqpi using n aceyl cysteine as a positive control is still required .
in conclusion , the findings of the current study illustrated that the triphala extract was effective in eliminating the paracetamol - induced hepato
it may be a potential lead for the protection of the liver and kidney against drug - induced toxicity . | background : paracetamol , a widely used analgesic and antipyretic , is known to cause liver and renal injury in humans when administered in higher and repeated doses that cause acute liver injury .
triphala is a well - known ayurvedic rasayana formulation that is prescribed for balancing of vata , pitta and kapha .
traditionally , it is used for the treatment of liver and kidney diseases.objective : the present study was undertaken to examine the protective effect of triphala extract against paracetamol - induced hepato
renal injury in swiss albino mice.materials and methods : swiss albino mice ( weight 2025 g ) were used in this study . the mice were divided into five groups of six animals each .
the aqueous extract of triphala was given orally at two different doses ( 100 and 300 mg / kg body weight ) for seven consecutive days , followed by a single intraperitoneal injection of paracetamol ( 500 mg / kg body weight ) to induce hepato
renal toxicity .
serum levels of liver enzymes , aspartate aminotransferase ( ast ) , alanine aminotransferase ( alt ) , alkaline phosphatase ( alp ) , bilirubin , creatinine , urea and uric acid were measured as indices of liver and renal injury .
all the statistical analyses were performed with the help of one - way analysis of variance ( anova ) followed by student newman
keuls test as post hoc test .
results were considered statistically significant when p < 0.05.results:pre-treatment with triphala extract at 100 mg / kg and 300 mg / kg body weight exhibited a significant ( p < 0.01 ) hepatoprotective activity .
the protective effect of triphala extract at 300 mg / kg body weight appears more effective than 100 mg / kg body weight.conclusion:the present study gives an evidence of the protective role of triphala extract against paracetamol - induced hepato
renal toxicity and validates its traditional claim in the ayurveda system . | INTRODUCTION
MATERIALS AND METHODS
Drugs and chemicals
Plant materials
Preparation of the extract
Experimental animals
Paracetamol-induced hepatorenal toxicity in mice
Biochemical analysis
Histopathological studies
Statistical analysis
RESULTS
Effects of
Effect of
DISCUSSION
CONCLUSION |
mixed gonadal dysgenesis is a condition of unusual and asymmetrical gonadal development leading to an unassigned sex differentiation .
a number of differences have been reported in the karyotype , most commonly a mosaicism 45,x/ 46 , xy .
a 16-year - old person , reared as female , born out of nonconsanguineous marriage , presented to our clinic with complaints of genital ambiguity and primary amenorrhoea along with lack of secondary sexual characters .
there was no h / s / o crisis or cyclical abdominal pain in the past .
however , the patient was taking 5 mg of prednisolone after being labeled as a case of congenital adrenal hyperplasia ( cah ) .
other past history , family history , antenatal history , perinatal history , and developmental history were noncontributory . on earlier evaluations , a small uterus was found on ultrasonogrphy ( usg ) studies but no gonads were visualized . on examination ,
genital examination revealed a single perineal opening , phallic length of 5 cm , with a well - formed glans and a well - formed scrotal sac .
there was no evidence of hypocortisolemia ( cortisol 9.08 g / dl , adrenocorticotropic hormone [ acth ] 82.5 pg / ml ) or elevated level of 17-oh- progesterone ( 0.16 ng / ml ) .
basal testosterone level was 588 ng / dl with basal androstenedione level of 1.83 ng / ml ( male : 0.7 - 3.6 , female : 0.3 - 3.5 ) .
pooled luteinizing hormone ( lh ) was 11.79 miu / ml and follicle - stimulating hormone ( fsh ) was 66.37 miu / ml .
basal and 72 h post beta - human chorionic gonadotropin ( hcg ) levels of androstenedione and testosterone levels were done ( basal testosterone of 652 ng / dl and basal androstenedione of 1.17 ng / ml ; 72 h post hcg testosterone of 896 ng / dl and androstenedione of 1.34 ng / ml ) .
mri pelvis [ figure 1 ] revealed uterus didelphys with obstructed right moiety and bilateral ovarian - like structures . magnetic resonance imaging ( mri ) showing rudimentary uterus and bilateral ovarian - like structures the patient
was managed in collaboration with department of gynecology and obstetrics and discussions with patient party and a decision was taken to remove the gonads and mullerian structures laparoscopically .
however at the time of surgery , only right sided gonads and adjacent tubal structures were visualized and removed .
however , left sided mullerian remnants could not be removed due to adhesion to sigmoid colon .
histopathological examination [ figure 2 ] revealed presence of testicular tissue showing atrophic seminiferous tubules with hyperplasia of leydig cells . epididymal tissue and an epididymal cyst are also present . however , no ovarian tissue was seen .
histopathological examination ( hpe ) of resected gonads showing atrophic seminiferous tubules ( 20 ) the patient is still admitted with us as on the day of writing this manuscript and is being managed with oral estrogens .
there was no evidence of hypocortisolemia ( cortisol 9.08 g / dl , adrenocorticotropic hormone [ acth ] 82.5 pg / ml ) or elevated level of 17-oh- progesterone ( 0.16 ng / ml ) .
basal testosterone level was 588 ng / dl with basal androstenedione level of 1.83 ng / ml ( male : 0.7 - 3.6 , female : 0.3 - 3.5 ) .
pooled luteinizing hormone ( lh ) was 11.79 miu / ml and follicle - stimulating hormone ( fsh ) was 66.37 miu / ml .
basal and 72 h post beta - human chorionic gonadotropin ( hcg ) levels of androstenedione and testosterone levels were done ( basal testosterone of 652 ng / dl and basal androstenedione of 1.17 ng / ml ; 72 h post hcg testosterone of 896 ng / dl and androstenedione of 1.34 ng / ml ) .
mri pelvis [ figure 1 ] revealed uterus didelphys with obstructed right moiety and bilateral ovarian - like structures . magnetic resonance imaging ( mri ) showing rudimentary uterus and bilateral ovarian - like structures
the patient was managed in collaboration with department of gynecology and obstetrics and discussions with patient party and a decision was taken to remove the gonads and mullerian structures laparoscopically .
however at the time of surgery , only right sided gonads and adjacent tubal structures were visualized and removed .
however , left sided mullerian remnants could not be removed due to adhesion to sigmoid colon .
histopathological examination [ figure 2 ] revealed presence of testicular tissue showing atrophic seminiferous tubules with hyperplasia of leydig cells . epididymal tissue and an epididymal cyst are also present . however , no ovarian tissue was seen .
histopathological examination ( hpe ) of resected gonads showing atrophic seminiferous tubules ( 20 ) the patient is still admitted with us as on the day of writing this manuscript and is being managed with oral estrogens .
this 16-year - old normotensive person reared as a girl came with a labeled diagnosis of 46xx disorder of sexual differentiation ( dsd ) ( diagnosed to be 46xx on the basis of buccal smear examination done outside ) , thought to be due to cah and started on prednisolone 5 mg once a day , even without a history suggestive of salt losing crisis . before the karyotype report was available , since the patient could not produce documents or investigation reports favoring the diagnosis of cah and thus justifying the use of prednisolone , we thought of reevaluating the patient completely .
we stopped the corticosteroid for 3 days and then reevaluated the adrenal axis , which was normal .
since the basal testosterone level was high , aromatase deficiency and partial androgen insensitivity syndrome ( pais ) were next in the line of diagnostic possibilities .
pais was ruled out due to presence of mullerian structures visualized on mri pelvis , as mullerian - inhibiting substance ( mis ) secretion is not hampered in pais .
the patient had no clinical or hormonal features suggestive of aromatase deficiency in the form of absence of virilization of mother during pregnancy , presence of significant values of serum estradiol levels , low normal levels of serum androstenedione and an absence of a significant rise of serum androstenedione levels even after beta - hcg stimulation .
lastly , we were left with diagnostic considerations of ovotesticular dsd ( ot - dsd ) and mgd .
the syndromic diagnoses associated with abnormal gonadal development were not suspected as patient did not reveal any of the known features of such defects ( e.g. , wild type [ wt]-1 , steroidogenic factor [ sf]-4 , dax-1 , etc . ) .
since patient was reared as female with history of clitoroplasty and vaginoplasty in the past and functionality of the external genital organs was not expected , a combined decision involving the patient party , gynecologist , and our department was taken to remove the gonads and assign a female sex to the patient .
on histopathological examination ( hpe ) of the gonadal tissue , only testicular tissue was seen , which favored the diagnosis of mgd over ot - dsd .
also a 46xx karyotype is more likely to be found in patients with ot - dsd .
although 46xo/46xy is the most common karyotype in patients with mgd , 46xy karyotype has also found in such patients .
the only method of differentiating 46xy mgd from 46xy ot - dsd is by histopathological demonstration of testicular tissue .
another point favoring mgd over ot - dsd is the presence of mullerian structures and testicular tissue on the same side , which is not seen in ot - dsd , as the testicular tissue is functional and secretes adequate amounts of mis .
however , in mgd , since the testicular tissue is dysgenetic , mullerian structures may be preserved on the side of testicular tissue .
the points that do not favor a diagnosis of mgd in our patient include an absence of short stature and turner 's stigmata .
the most common genotype in these patients is 46xo / xx , however 46xy genotype has also been described , which can be difficult to distinguish from 46xy ot - dsd .
the management of these patients can be difficult as phenotypically females may need multiple staged procedures for clitoroplasty , vaginoplasty , and gonadal biopsies / gonadectomies .
phenotypically male patients may need multiple procedures for hypospadias repair with limited gain in corporal tissue .
these patients are at increased risk of cancer , so it is prudent to remove the nonfunctional gonadal tissues .
these patients usually need a multidisciplinary approach for management , but still long - term data on fertility and functional improvement of each approach of treatment is lacking . | a 16-year - old person , reared as female presented with complaints of genital ambiguity and primary amenorrhoea along with lack of secondary sexual characters , but without short stature and turner 's stigmata .
she was taking steroids after being misdiagnosed as congenital adrenal hyperplasia ( cah ) .
karyotype analysis revealed 46xy karyotype .
there was no evidence of hypocortisolemia ( cortisol 9.08 g / dl , adrenocorticotropic hormone [ acth ] 82.5 pg / ml ) or elevated level of 17-oh - progesterone ( 0.16 ng / ml ) .
pooled luteinizing hormone ( lh ) was 11.79 miu / ml and follicle - stimulating hormone ( fsh ) was 66.37 miu / ml .
serum estradiol level was 25 pg / ml ( 21 - 251 ) .
basal and 72 h post beta - human chorionic gonadotropin ( hcg ) levels of androstenedione and testosterone levels were done ( basal testosterone of 652 ng / dl and basal androstenedione of 1.17 ng / ml ; 72 h post hcg testosterone of 896 ng / dl and androstenedione of 1.34 ng / ml ) . magnetic resonance imaging ( mri )
pelvis ( with ultrasonogrphy [ usg ] correlation ) revealed uterus didelphys with obstructed right moiety and bilateral ovarian - like structures . right sided gonads and adjacent tubal structures were visualized laparoscopically and removed .
left sided gonads were not visualized and mullerian remnants were adhered to sigmoid colon .
histopathological examination revealed presence of testicular tissue showing atrophic seminiferous tubules with hyperplasia of leydig cells . no ovarian tissue was seen .
based on these results a diagnosis of 46xy mixed gonadal dysgenesis ( mgd ) was made , which is rare and is difficult to distinguish from 46xy ovotesticular disorder of sexual differentiation ( ot - dsd ) .
the patient was managed with a multidisciplinary approach and fertility issues discussed with the patient 's caregivers . | I
C
Investigations
Management
D |
the colonic epithelial lining is a highly regenerative tissue , with resident stem cells replenishing the tissue every few days .
this process of regeneration requires the maintenance of a proliferative stem cell compartment . over time , the newly produced daughter cells lose their proliferative potential and migrate toward the luminal surface of the intestine .
coincident with migration , progenitor cells differentiate into mature barrier - forming enterocytes or mucous producing goblet cells .
failure of this differentiation program is thought to contribute to compromised epithelial barrier such as is observed in inflammatory bowel disease and colon cancer .
detailed study of the above processes will require methodologies for isolating crypt - base and surface cell populations .
current methods for the isolation of intestinal epithelial cells include chelating agents and mechanical disassociation , resulting in the isolation of entire crypts , epithelial sheets , or single cells , dependent on experimental conditions .
these are high yield methods , yet changes in intercellular signaling have been reported under these conditions that may not represent the native environment .
laser capture microdissection allows for the collection of spatial distinct material , but achieves low molecular yield . in human colonic tissue , serial horizontal cryosectioning methods have been developed .
however , murine mucosal tissues are prohibitively small for direct appropriation of this technique . in humans , intestinal crypt lengths
( the distant between the crypt - base and surface cells ) in the colon varies between ~1001,000 m . in mice ,
the small size of murine crypts presents a challenge to the isolation of these two cell populations using existing protocols .
we now describe a low cost , high yield method for the isolation of crypt - base and surface epithelial cell population in murine colonic tissue .
tissue harvested in this manner may then be analyzed by a number of standard downstream applications , including immunofluorescence staining , rt pcr ( real time polymerase chain reaction ) or western blotting .
experiments used c57bl/6 mice between 10 and 12 weeks of age . all procedures using animals were reviewed and approved by the emory university institutional animal care and use committee and were performed according to national institutes of health criteria .
set up the cryostat equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade!).fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) . remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min.set the cryostat to 10 m per section and shave the oct block until it is flat .
equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade ! ) .
fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) .
remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min .
set the cryostat to 10 m per section and shave the oct block until it is flat .
prepare the razor blades : prepare 2 razor blades per sample . using a metal file , dull the sharped edge of blades .
place mice in a sealed chamber and euthanize mice by isoflurane inhalation and cervical dislocation , then spray 70% ethanol onto the abdomen and thorax . make a small incision in skin of the abdomen with scissors and then make an incision to expose the peritoneal cavity .
cut the large intestine at the anal verge and tease apart the mesentery until the colon is free .
excise a distal colon segment between 0 and 6 cm from the anus . here , the crypt depth is ~150 m ( see figure 1b ) .
stretch the tissue using dissection pins on a silicon dish in hanks buffer and let rest for 10 min at rt ( figure 1c ) .
slide a razor blade under the desired section of tissue and then pour off the hanks buffer .
pick up the razor / tissue sandwich and allow it to warm slightly by placing a finger on the top blade ( mucosal side up .
this orients the tissue so that the basal muscle layers are sectioned first . ) .
separate the sandwich and cut around the edges of the tissue segment . cut a tissue segment approximately , 0.5 cm x 1 cm . edge areas have a tendency to curl , causing the serial sections to contain many tissue layers .
note that over - cutting is better than under - cutting , so err on the side of caution .
allow the tissue to warm until the ice on top of the tissue begins to melt .
at this point quickly and carefully press the tissue into the oct block in the cryostat . remove the blade by placing a finger over the sample .
analysis of mrna or protein , place the samples in tubes with 5 crypt - base , 5 transitional , and 5 surface sections per tube .
for rna collection add 1 ml commercial isolation reagent and incubate at rt for 10 min . perform rna purification according to manufacturer s instructions .
use 5 - 10 g of total rna for cdna generation . for protein purification ,
add 0.5 ml of cold ripa lysis buffer ( plus protease inhibitors ) per 5 sections .
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c .
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c .
set up the cryostat equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade!).fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) . remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min.set the cryostat to 10 m per section and shave the oct block until it is flat . back
equilibrate cryostat to -20 c , including the microtome blade and chucks ( exercise extreme caution around the blade ! ) .
fill an empty cryomold with oct ( optimal cutting temperature compound ) and equilibrate to -20 c ( ~30 min ) .
remove the frozen oct from the mold and fix it on the chuck with liquid oct .
place the block / chuck in the microtome arm and allow the oct to set for 10 min .
set the cryostat to 10 m per section and shave the oct block until it is flat .
back the microtome arm away from the blade by several centimeters . at this point do not adjust the blade or chuck for the remainder of the experiment .
prepare the razor blades : prepare 2 razor blades per sample . using a metal file , dull the sharped edge of blades .
place mice in a sealed chamber and euthanize mice by isoflurane inhalation and cervical dislocation , then spray 70% ethanol onto the abdomen and thorax .
make a small incision in skin of the abdomen with scissors and then make an incision to expose the peritoneal cavity .
cut the large intestine at the anal verge and tease apart the mesentery until the colon is free .
excise a distal colon segment between 0 and 6 cm from the anus . here , the crypt depth is ~150 m ( see figure 1b )
pin the tissue to the dissection dish with the mucosal surface facing up . stretch the tissue using dissection pins on a silicon dish in hanks buffer and let rest for 10 min at rt ( figure 1c ) .
slide a razor blade under the desired section of tissue and then pour off the hanks buffer .
pick up the razor / tissue sandwich and allow it to warm slightly by placing a finger on the top blade ( mucosal side up .
this orients the tissue so that the basal muscle layers are sectioned first . ) .
separate the sandwich and cut around the edges of the tissue segment . cut a tissue segment approximately , 0.5 cm x 1 cm . edge areas have a tendency to curl , causing the serial sections to contain many tissue layers .
note that over - cutting is better than under - cutting , so err on the side of caution .
allow the tissue to warm until the ice on top of the tissue begins to melt .
at this point quickly and carefully press the tissue into the oct block in the cryostat .
analysis of mrna or protein , place the samples in tubes with 5 crypt - base , 5 transitional , and 5 surface sections per tube . for rna collection
use 5 - 10 g of total rna for cdna generation . for protein purification , add 0.5 ml of cold ripa lysis buffer ( plus protease inhibitors ) per 5 sections .
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c .
add samples to sds page loading buffer ( laemmli buffer ) and incubate at 100 c for 10 min .
subject samples to sds page and transfer , then block in 5% milk / pbs for 2 hr . perform western blot analysis followed by incubation with klf4 antibodies ( 1:500 ) o / n at 4 c .
proceeding toward the lumen , the muscularis mucosa is evident adjacent to the crypt - base epithelial cells , transitional cells are in the middle of the tissue , and finally , surface cell populations face the gut lumen . the serial sectioning method described here
maintains an orientation such that the longitudinal and circular muscularis are encountered first ( figure 2b ) , followed by the muscularis mucosa ( figure 2c ) .
note the appearance of non - muscle interstitial cells in the top left of the image .
the subsequent sections contain epithelial cells and interstitial tissue ( lamina propria ) , starting with the crypt - base cells ( figure 2d ) .
note the absence of a central lumen in most of the crypts , which appear darker due to the closely packed nuclei .
the transitional cells appear as tightly packed glands with prominent central lumens ( figure 2e ) .
lastly , surface cell populations have an irregular structure , with areas of the epithelial monolayer viewed en face ( figure 2f ) . serial sections as described above may also be processed for immunofluorescence labeling and confocal microscopy ( as described here ) . figure 2
g shows isolated crypts fixed and permeabilized in 100% methanol and subsequently stained for nuclei ( blue ) and the cell - cell junction protein zonula occludens 1 ( zo-1 , green ) . in the traditional sectioning orientation , crypt and surface cell can be viewed simultaneously ( figure 2 g ) .
this z0 - 1 lining of the lumen can also be seen in serial sectioned samples of transitional cell populations , in the center of the circular glands ( figure 2 g ) .
enhanced zo-1 stain is seen in surface cell populations ( figure 2i and j ) .
several differentiation factors are known to be expressed in a spatiotemporal fashion along the crypt - to surface axis .
this includes the transcription factor kruppel - like factor 4 ( klf4 ) , which is known to be enriched in surface cell populations . using traditional sectioning methods
, klf4 is found in the nuclei of lumen facing surface cell populations ( figure 2k ) .
we therefore speculated that klf4 mrna would be predominantly expressed in surface cell populations . to test this ,
serial sections were collected and grouped into surface , transitional , and crypt - base samples .
subsequently , rna was harvested using standard trizol extraction , with an additional purification by rneasy column and dnase treatment .
rna was collected from 5 pooled consecutive sections , which yielded between 5 - 10 g of total rna per sample .
these samples were then subjected to semi - quantitative real time pcr for both klf4 and a reference gene , tata box binding protein 1 ( tbp-1 ) ( figure 2l ) . as shown in figure 2l , after normalization to tbp-1 , surface cells were found to express up to 8 times the klf4 mrna that is expressed in crypt cells .
similarly , klf4 protein levels were found to exhibit spatial regulation along the crypt - surface axis .
serial sections were pooled as described above and then incubated in lysis buffer for 20 min at 4 c .
samples were then subjected analysis by sds - page ( figure 2 m ) . as shown in figure 2 m ,
the above findings demonstrate the ability of this protocol to isolate large amounts of surface and crypt epithelial cells from the murine colonic mucosa .
figure 1 : ( a ) schematic showing murine colonic mucosa and external muscle layers .
our method aims to collect discrete surface cell and crypt - base cell populations by serial cryosectioning ( 10 m each ) .
( b ) crypt height varies along the length of the colon ( distance between crypt - base and surface layers ) .
data points indicate individual crypt measurements and symbols indicate biological replicates ( n = 4 ) .
( c ) colon segments collected , bisected , and pinned onto a silicon dissecting dish .
( d ) a tissue segment approximately 3 cm from the anus is pressed between to razor blades and the frozen on dry ice .
( a ) colonic tissue stained with hematoxylin - eosin in cross - section showing the circular muscularis ( c.m . ) , the muscularis mucosa ( m.m . ) , crypt - base cells , transitional cells , and surface cells .
( d ) crypt - base cells . note the absence of a central lumen .
( f ) surface cells . ( g ) immunofluorescence staining of isolated colonic crypts .
zo-1 ( green ) and nuclei ( blue ) are observed in cross - section .
( l ) real - time pcr assessment of klf4 mrna levels between the three compartments .
this includes gaps in our understanding of the cellular signaling environment of the stem cell compartment at the base of the colonic epithelial crypts .
for example , increased understanding of in vivo differentiation is required as a benchmark for studies aimed at producing in vitro primary intestinal systems .
firstly , removing the edges around the frozen tissue segment ( as discussed in section 3.2 ) is required as the tissue edges curl during dissection and freezing .
failure to move these edges results is a mixed population of surface and crypt base cells .
mounting the tissue on a pre - chilled , leveled oct block is also essential .
this protocol can be adapted to other areas of the distal colon by altering the number of sections in each pool .
for example , as shown in figure 1b , mucosal crypt length varies between 150 - 300 m .
therefore , when studying the region between 4 cm-6 cm from the anus , the number of sections should be increased from 15 to 30 .
importantly , this protocol is not suggested for the proximal colon ( 7 cm-9 cm ) due to folds ( plicae obliquae ) which extend into the lumen making it impossible to separate surface and crypt - base cells by serial sectioning .
serial sectioning techniques have been used to study crypt - base and surface epithelial cell populations in humans . however
, our protocol adds additional steps that are required due to the small size of murine tissue .
we propose that the above protocol will allow for the investigation of crypt - base and surface epithelial cell population in genetically tractable mouse systems . indeed ,
however , it should be noted that , like several of the alternative methods described above , the resulting sections contain a mixture of epithelial and interstitial lamina propria cells . in conclusion ,
the protocol described here provides a rapid , high yield method for the analysis of molecular processes in spatially distinct regions of the murine colonic mucosa . | the colonic mucosal tissue provides a vital barrier to luminal antigens . this barrier is composed of a monolayer of simple columnar epithelial cells .
the colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of lieberkhn .
progenitor cells produced in the crypt - bases migrate toward the luminal surface , undergoing a process of cellular differentiation before being shed into the gut lumen . in order to study these processes at the molecular level ,
we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa ; the proliferative crypt zone , and the differentiated surface epithelial cells .
our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of rna and protein . | Introduction
Protocol
1. Experimental Setup
2. Dissection of Distal Colonic Tissue
3. Mount Tissue on the Cryostat for Sectioning
Representative Results
Discussion
Disclosures |
treating cognitive impairment , along with motor recovery and improving performance in daily activities , is one of the key focus areas of stroke rehabilitation .
as cognition is closely related to patient motivation and motor function improvement , implementing cognitive function treatment in a timely and effective manner is crucial for the overall success of poststroke rehabilitation .
previous reports showed 25%-40% of patients suffer from dementia by three months after stroke , and the number increased to 50%-75% when patients with minor symptoms of cognitive impairment are included [ 2 - 4 ] . among patients with brain lesions ,
memory disturbance is commonly a critical challenge in rehabilitating patients to enable them to return to active participation in society and the workforce .
research has suggested that rehabilitation programs designed to maximize neuroplasticity are effective for improving memory functions in these patients .
recently , transcranial direct - current stimulation ( tdcs ) and repetitive transcranial magnetic stimulation ( rtms ) , noninvasive methods for stimulating the cerebral cortex , have been intensively studied as a treatment for brain injury . while both modalities are used to treat brain injury ,
tdcs is the preferred method , as it allows simultaneous application of brain stimulation and conventional rehabilitation .
concluded that applying tdcs to the brain region improved motor functions and increased motor cortex excitability .
recent studies also suggested that tdcs was useful in treating cognitive dysfunction , and improving attention , memory , and executive function [ 7 - 9 ] .
however , there are still insufficient data to confirm its effect on the cognitive functions of stroke patients .
thus , in this study , we determined the effectiveness of tdcs in cognitive recovery in acute and subacute stroke patients .
we selected stroke patients with cognitive impairment and a score of 27 on the korean version of mini mental state exam ( k - mmse ) from patients treated at the department of rehabilitation medicine , asan medical center , from october 1 , 2010 to march 30 , 2012 .
the guardians of the patients indicated that the patients had experienced their first stroke , and had no previous history of abnormal cognitive functions .
patients who were classified as acute or subacute , within 6 months from their stroke , and who showed no damage to their temporal lobe on magnetic resonance imaging , were selected .
subjects were then included when they were willing and available for five treatment sessions per week .
patients with apraxia , aphasia , and neglect were excluded , given that these factors may have an impact on the outcome of cognitive function tests . for the safety of patients , we excluded those who had a history of craniectomy or seizure .
we used the double - blind method to randomly divide the subjects into three test groups : ( 1 ) the left fronto - temporal anode stimulation ( left - ftas ) group , ( 2 ) the right fronto - temporal anode stimulation ( right - ftas ) group , and ( 3 ) the sham group .
patients in each group received tdcs treatment for 30 min , five times a week , for 3 weeks .
the treatments were performed using a phoresor ii auto model pm850 ( iomed , salt lake city , ut , usa ) , an fda - approved device , with 5 5 cm sponge electrodes attached to the patient s scalp .
the intensity of the current was set at 2 ma , which was confirmed as safe by iyer et al .
tdcs anodal electrodes were placed according to the 10 - 20 international eeg system , at t3 for the left - ftas group and at t4 for the right - ftas group . for the sham group ,
sponge electrodes were attached using the same method as for the left - ftas group , but without any current running . the cognitive rehabilitation program used in the study was comcog ( maxmedica inc . ,
seoul , korea ) , with the focus on attention and memory improvement for cognitive disorder patients .
the subjects were evaluated before the treatment and after completing the 3-week program , using the k - mmse and computerized neurocognitive function tests ( cnt , maxmedica , seoul , korea ) to assess cognitive function , and the korean version of the modified barthel index ( k - mbi ) to assess activities of daily living .
the cnt included visual and auditory continuous performance tests ( cpts ) to evaluate continuous concentration on visual and auditory stimuli .
the kruskal - wallis test was used to test the homogeneity among the three groups , and the wilcoxon signed - rank test was used to analyze the preand post - treatment data for each test group .
the kruskal - wallis test was used to compare the three test groups , and the mann - whitney test was used as the follow - up test .
we selected stroke patients with cognitive impairment and a score of 27 on the korean version of mini mental state exam ( k - mmse ) from patients treated at the department of rehabilitation medicine , asan medical center , from october 1 , 2010 to march 30 , 2012 .
the guardians of the patients indicated that the patients had experienced their first stroke , and had no previous history of abnormal cognitive functions .
patients who were classified as acute or subacute , within 6 months from their stroke , and who showed no damage to their temporal lobe on magnetic resonance imaging , were selected .
subjects were then included when they were willing and available for five treatment sessions per week .
patients with apraxia , aphasia , and neglect were excluded , given that these factors may have an impact on the outcome of cognitive function tests . for the safety of patients
we used the double - blind method to randomly divide the subjects into three test groups : ( 1 ) the left fronto - temporal anode stimulation ( left - ftas ) group , ( 2 ) the right fronto - temporal anode stimulation ( right - ftas ) group , and ( 3 ) the sham group .
patients in each group received tdcs treatment for 30 min , five times a week , for 3 weeks .
the treatments were performed using a phoresor ii auto model pm850 ( iomed , salt lake city , ut , usa ) , an fda - approved device , with 5 5 cm sponge electrodes attached to the patient s scalp .
the intensity of the current was set at 2 ma , which was confirmed as safe by iyer et al .
tdcs anodal electrodes were placed according to the 10 - 20 international eeg system , at t3 for the left - ftas group and at t4 for the right - ftas group . for the sham group , sponge electrodes were attached using the same method as for the left - ftas group , but without any current running . the cognitive rehabilitation program used in the study was comcog ( maxmedica inc . ,
seoul , korea ) , with the focus on attention and memory improvement for cognitive disorder patients .
the subjects were evaluated before the treatment and after completing the 3-week program , using the k - mmse and computerized neurocognitive function tests ( cnt , maxmedica , seoul , korea ) to assess cognitive function , and the korean version of the modified barthel index ( k - mbi ) to assess activities of daily living . the cnt included visual and auditory continuous performance tests ( cpts ) to evaluate continuous concentration on visual and auditory stimuli .
the kruskal - wallis test was used to test the homogeneity among the three groups , and the wilcoxon signed - rank test was used to analyze the preand post - treatment data for each test group .
the kruskal - wallis test was used to compare the three test groups , and the mann - whitney test was used as the follow - up test .
the subjects for this study included : ( 1 ) six male and nine female patients in the left - ftas group , ( 2 ) seven male and eight female patients in the right - ftas group , and ( 3 ) seven male and eight female patients in the sham group .
the mean age of patients in each group was as follows : ( 1 ) 60.9 12.9 years for the left - ftas group , ( 2 ) 58.915.0 years for the right - ftas group , and ( 3 ) 68.514.6 years for the control group , with no significant differences between the groups .
the mean time elapsed from the onset of stroke to the start of the treatment program was : ( 1 ) 42.231.9 days for the left - ftas group , ( 2 ) 38.127.0 days for the right - ftas group , and ( 3 ) 39.529.6 days for the sham group , with no significant differences between the groups ( table 1 ) .
the results from post - treatment cnt suggested that the left - ftas group showed significant improvement in the backward digit span test , the verbal learning test - delayed recall , a visual span test , and the k - mmse .
all three groups showed significant improvement in k - mbi scores ( table 2 ) .
comparison of pre- and post - treatment data for each group suggests that the left - ftas group showed significant improvement in auditory memory , compared to the other groups .
however , there were no significant differences between the groups in terms of visual memory and attention .
there were no differences between the groups in terms of k - bmi ( table 3 ) .
the subjects for this study included : ( 1 ) six male and nine female patients in the left - ftas group , ( 2 ) seven male and eight female patients in the right - ftas group , and ( 3 ) seven male and eight female patients in the sham group .
the mean age of patients in each group was as follows : ( 1 ) 60.9 12.9 years for the left - ftas group , ( 2 ) 58.915.0 years for the right - ftas group , and ( 3 ) 68.514.6 years for the control group , with no significant differences between the groups .
the mean time elapsed from the onset of stroke to the start of the treatment program was : ( 1 ) 42.231.9 days for the left - ftas group , ( 2 ) 38.127.0 days for the right - ftas group , and ( 3 ) 39.529.6 days for the sham group , with no significant differences between the groups ( table 1 ) .
the results from post - treatment cnt suggested that the left - ftas group showed significant improvement in the backward digit span test , the verbal learning test - delayed recall , a visual span test , and the k - mmse .
all three groups showed significant improvement in k - mbi scores ( table 2 ) .
comparison of pre- and post - treatment data for each group suggests that the left - ftas group showed significant improvement in auditory memory , compared to the other groups .
however , there were no significant differences between the groups in terms of visual memory and attention .
there were no differences between the groups in terms of k - bmi ( table 3 ) .
the objective of this study was to determine the effect of tdcs treatment on the temporal lobe in terms of cognitive recovery of poststroke patients . to date
, only a small number of studies have been done on tdcs as a treatment method for cognitive impairment . while montie et al .
suggested that cathode stimulation to the left fronto - temporal lobe resulted in improvement in naming in aphasia , other studies concluded that anode stimulation to the left perisylvian region resulted improvement in word memory in healthy patients .
boggio et al . also concluded that anode stimulation to the left fronto - temporal lobe helped to improve memory performance in alzheimer disease .
the results of our study suggested that anodal stimulation to the left fronto - temporal lobe improved auditory memory , confirming the conclusion of earlier studies , although the treatment did not affect visual memory .
cnt test showed that the treatment had been positively effective in all areas , but other three groups , excluding auditory memory , showed no statistical difference .
only few studies have been done on the effect of tdcs on cognitive function in stroke patients . while kang et al .
suggested that anode stimulation at the dorso - lateral prefrontal cortex improved attention , kim et al . concluded that rtms on the same region was only effective in treating depression , and
there have been two reports on patients with stroke who experienced linguistic improvement , but these studies only involved 10 and 13 patients , respectively .
no studies have clearly defined the effects of noninvasive brain stimulation on the cognitive function of stroke patients .
studies have discovered that tdcs affects the brain by activating n - methyl - d - aspartic acid ( nmda ) receptors and suppressing the cerebral cortex via gamma - aminobutyric acid ( gaba ) receptors . the effect of tdcs is known to last for approximately 90 minutes , during which the cerebral cortex remains excited , but there are too few studies to be able to draw conclusions on the accumulated effect .
this report showed that marked improvements are noted after 3 weeks of tdcs treatment , but the accumulated effect could not be determined because serial cognitive function was not assessed .
the findings from this study may have been limited by the small number of patients in each test group .
cerebral infarction and hemorrhage were both included and lesions varied largely , so that spontaneous recovery could not be fully excluded . as well as excluding cathode stimulation from the treatment .
additionally , the subjects were not tested for depression , which may have affected the outcome . as it has been suggested that depression is prominent among stroke patients and as depression may affect attention and memory ,
the possibility remains that the study may have included patients with depression , whose depression symptoms were affected by tdcs , which then subsequently resulted in improved cognitive function . furthermore , poststroke apathy can affect cognitive function , but this could not be addressed in this study . since the subjects were evaluated for cognitive performance at 24 hours after completion of the 3-week treatment program , the long - term effect of tdcs treatment remains undetermined .
moreover , we excluded patients with abnormal cognitive function based on the guardian s statement , but we had not tested the cognitive function of patients before they suffered their stroke , which may have an impact on the study result
. nevertheless , this study confirmed that tdcs is effective in treating poststroke cognitive impairment .
we were also able to conclude that stimulation on the temporal lobe , as well as on the prefrontal lobe , as suggested by earlier studies , is helpful in cognitive recovery .
the results from this study confirmed that applying tdcs to the left temporal lobe effectively improved auditory memory of patients with poststroke cognitive impairment .
we concluded that tdcs can be employed as a treatment sub - protocol in a cognitive treatment program .
however , further studies with a larger subject pool and various stimulation methods should be conducted to confirm these findings . | background and purposeto investigate whether transcranial direct - current stimulation ( tdcs ) can improve cognition in stroke patients.methodsforty-five stroke patients ( 20 males and 25 females , average age : 62.7 years ) with cognitive dysfunction were included in this prospective , double - blinded , randomized case
control study .
all patients were right - handed and the mean elapsed time after stroke was 39.3 days .
three different treatments groups were used : ( 1 ) anodal stimulation of the left anterior temporal lobe , ( 2 ) anodal stimulation of the right anterior temporal lobe , and ( 3 ) sham stimulation .
tdcs was delivered for 30 minutes at 2 ma with 25 cm2 electrodes , five times / week , for a total of 3 weeks , using a phoresor ii auto model pm 850 ( iomed ) .
the evaluation of cognitive impairment was based on a computerized neuropsychological test ( cnt ) , korean mini - mental state examination ( k - mmse ) .
the korean version of the modified barthel index ( k - mbi ) was used to assess activities of daily living functionality .
these evaluations were conducted in all patients before and after treatment.resultseach group included 15 patients .
pre - treatment evaluation showed no significant differences between the three groups for any of the parameters .
there was significant improvement in the verbal learning test on the cnt in the left anodal stimulation group ( p < 0.05 ) .
there were , however , no significant differences in the k - mmse or k - mbi scores among the three groups.conclusionsthese results demonstrated the beneficial effects of anodal tdcs on memory function .
thus , tdcs can successfully be used as a treatment modality for patients with cognitive dysfunction after stroke . | Introduction
Methods
Subjects
Treatments
Statistical analysis
Results
General characteristics of the subjects
Pre- and post-treatment comparison
Comparison between the three groups
Discussion
Conclusions |
cell signaling pathways
are largely organized via a specific recruitment
of signaling effector proteins to their target membranes and a confined
release of calcium ions .
the quintessential example of this is the
action of phospholipase c ( plc ) that binds and hydrolyzes phosphatidylinositol
4,5-bisphosphate ( pi(4,5)p2 ) in the plasma membrane to
diacylglycerol ( dag ) and the water - soluble inositol 1,4,5-trisphosphate
( ip3 ) , the latter inducing the release of ca from the
endoplasmic reticulum ( er ) into the cytosol .
another prominent example is synaptotagmin-1 , the main ca sensor of neuronal exocytosis in the presynaptic axon terminal .
synaptotagmin-1 binding to pi(4,5)p2 directly amplifies
protein cooperativity and thus sensitivity to ca by a
factor of > 40 .
pi(4,5)p2 is
enriched in the inner leaflet of the plasma
membrane and constitutes around 1% of the total anionic
phospholipid content in cellular membranes . in comparison with other phospholipids
, it contains a rather bulky
phosphorylated inositol headgroup with a negative charge ranging from
3 e to 5 e , depending
on the ph and the presence of proteins or ions .
pi(4,5)p2 and other negatively charged lipids
in the cytosolic leaflet are constantly exposed to divalent cations .
in resting cells ,
the cytosolic concentration
of ca upon cell signaling has been reported to span a
wide range from 0.5 m to several hundred m , with a half - life
of 500 s to 26 ms .
ca influx primarily originates from internal stores
within the endoplasmic / sarcoplasmic reticulum or from specialized
channels within the plasma membrane providing an essentially infinite
supply of extracellular calcium . in all
cases
, ca is delivered as brief transients , forming microdomains
at the membrane site of influx , and thus ,
local concentrations of ca can be expected to exceed
cytosolic concentrations by orders of magnitude .
meanwhile , unlike ca , the levels of free ,
cytosolic mg are maintained within a fairly narrow concentration
range of 0.251 mm .
interestingly , calcium
but not magnesium ions have been ascribed a strong propensity to promote
the formation of pi(4,5)p2 clusters as demonstrated in
several studies , primarily by using monolayer techniques .
while the overall effects of divalent cations , including calcium ,
on pi(4,5)p2 lateral organization have been intensely studied ,
the mechanism of ca and pi(4,5)p2 interactions
at the molecular level remain unclear . experiments with
pure pi(4,5)p2 monolayers have suggested partial dehydration of both ca and pi(4,5)p2 upon interaction with each other , triggering an electron density increase in the
pi(4,5)p2 headgroup region as well as acyl chain region
thickening .
these studies , however , have typically focused on single pi(4,5)p2 molecules or used simplified
coarse - grained models that lack sufficient
details to deal with specific chemical features of phosphatidylinositides . herein , we combine protein lipid binding assays and spectroscopic
experiments with atomistic molecular dynamics ( md ) simulations employing
refined state - of - the art force fields to unravel the functional and
structural consequences of the interplay between ca and
pi(4,5)p2 .
our data indicate a hitherto undiscovered role
and mechanism for ca in cellular signaling , namely the
direct organization of the phosphoinositide headgroup conformation
and the selective recognition thereof by the pleckstrin homology ( ph )
domain of plc 1 , the canonical pi(4,5)p2 sensor .
to determine the
equilibrium dissociation constants ( kd ) for divalent cation / pi(4,5)p2 interaction , we employed
a simple fluorescent assay using a supported lipid bilayer platform containing 5 mol % of pi(4,5)p2 ( for details , see the supporting information ) .
significantly , the kd values differed by less than a factor of 2 ,
with a kd of 0.6 0.2 mm for ca compared to 1.2 0.2 mm for mg ( figure s1 ) .
we therefore decided to use a cation
concentration of 1 mm for all follow - up experiments , matching the
free mg concentration in the cytosol . in order to systematically
study the effects of ca on pi(4,5)p2 ,
we produced
100 nm diameter large unilamellar vesicles ( luvs ) , facilitating the
control of membrane lipid composition and properties . for quality
control and physicochemical characterization , all preparations were
first subjected to thin layer chromatography ( tlc ) , dynamic light
scattering ( dls ) , and zeta potential measurements ( figure s2 ) . having the opposite charge of pi(4,5)p2 , it is not surprising that ca and mg equally
reduce the zeta potential of popc liposomes containing 5 mol % of
pi(4,5)p2 , the former being described previously .
in fact , the presence of either cation attenuates
the electrokinetic potential of the membrane down to the level of
popc alone ( figure s2c ) . because
of its extraordinary stereospecificity , the plc 1-ph domain
is widely used as the canonical reporter for cellular pi(4,5)p2 levels at the plasma membrane as well as with in
vitro assays .
size - exclusion chromatography
and dls confirmed that the purified plc 1-ph domain ( figure s3a , b ) was monomeric in solution , even
in the presence of ca and mg ( figure s3c , d ) .
next , we performed liposome flotation
assays to follow plc 1-ph binding efficiency to popc / pi(4,5)p2 vesicles .
interestingly , preincubation with 1 mm ca but not 1 mm mg fully inhibited liposome binding ( figure 1a , b ) . moreover , plc
1-ph did not bind to pure popc liposomes , highlighting its
specificity to pi(4,5)p2 .
( b ) plc 1-ph binding
to luvs with popc / pi(4,5p)2 ( 95/5 mol % ) .
( c ) guvs after
ecfp - plc 1-ph addition ( green ) and dil as membrane marker ( red ) .
( d ) the distribution of
median ecfp - plc 1-ph intensity per pixel of individual guvs
and different sizes of control ( blue ) and after preincubation with
1 mm mg ( green ) or ca ( red ) ( data from two
additional independent experiments are provided in figure s5 ) .
the median intensity
values with mean and standard deviation are depicted in the inset .
the mann
whitney test was used as significance test ( p value < 0.0001 for all cases ) .
circular dichroism ( cd ) spectroscopy excluded a direct effect
for
cations on the secondary structure of the protein ( figure s3e , f ) .
as such , although ca and mg bind to pi(4,5)p2 with comparable kd values and reduce electrokinetic membrane properties
in an equal manner , only ca was capable of inhibiting
plc 1-ph binding .
this indicates that pi(4,5)p2 recognition
by proteins can not be solely based on electrostatic interactions . because a concentration of 1 mm ca corresponds to
twice its kd for pi(4,5p)2 interaction ,
we performed additional flotation assays with lower ca concentrations . here , a significant reduction in protein binding
could be observed already at a concentration of 0.6 mm ca ( figure s4 ) . in this context , recent
data by milovanovic and colleagues show that ca but not
mg promotes syntaxin-1/pi(4,5)p2 domain formation
by an underlying mechanism in which ca clusters pi(4,5)p2 and syntaxin-1 independently from each other
. moreover , ca acts as a charge bridge that merges multiple syntaxin-1/pi(4,5)p2 clusters into larger domains . also here , ca was
found to be effective at a concentration of 0.5 mm while even 1 mm
mg had no effect .
we therefore
additionally followed the binding of monomeric ecfp - plc 1-ph
fusion protein to giant unilamellar vesicles ( guvs ) ( figure 1c ) . despite limited control
over membrane lipid composition at the individual guv level , guvs
provide the most appropriate synthetic
approach for flat and freestanding bilayer systems . in this system ,
the presence of 1 mm ca drastically reduced ecfp - plc
1-ph binding ( figure 1d and figure s5 ) , demonstrating
the robustness of the observed effect , irrespective of membrane curvature .
magnesium , however , also reduced ecfp - plc 1-ph domain binding ,
halfway toward the ca effect . to understand this result ,
it is important to note that liposome flotation experiments with proteins
are nonequilibrium assays because much of the protein stays in the
bottom of the tube . at the same time ,
by contrast ,
protein binding in the guv experiment is at equilibrium and binding
events are quantified at the individual guv level . to analyze the
molecular basis for the cation specificity , vibrational sum frequency
spectroscopy ( vsfs )
was employed to study the effects of ca and mg on pure pi(4,5)p2 monolayers at the
air / water interface .
the spectra were recorded over frequency ranges
corresponding to the headgroup and acyl - chain portions of the lipid
molecules and included the adjacent interfacial water structure .
we present vsfs spectra from the inositol ring and phosphate regions
of pi(4,5)p2 in the absence and presence of 1 mm ca and mg ( figure 2a , detailed peak assignments in figure s6 and table s1 ) . in the absence of cations in the
subphase ,
both the inositol ring vibrations and the phosphate stretches
were rather weak ( black data points ) .
this is because of a relatively
disordered arrangement of the pi(4,5)p2 headgroups adopted
in a pure buffer with a wide range of tilt angles relative to the
surface normal . with 1 mm ca , however , the inositol ring
signal ( 961 cm and 1012 cm peaks from the c c and c o coupled vibrations , respectively ) increased substantially ( red data points ) .
in
fact , the resonances showed 2.7- and 3.6-fold increases , respectively ,
in oscillator strength ( table s1 ) .
these
changes reflect both reorientation of the inositol rings and a narrowing
of their orientational distribution upon cation binding .
significantly ,
the changes were not nearly as strong upon the addition of 1 mm mg ( blue data points ) . in that case ,
the oscillator strength
of the inositol ring vibrations was increased by only a factor of
1.5 and 2.1 , respectively .
such results indicated that ca rigidified the configuration of the pi(4,5)p2 headgroups
much more effectively than mg .
vsfs spectra of ( a ) the
inositol ring and phosphate regions and
( b ) the carbonyl c = o symmetric stretch region of pi(4,5)p2 on a buffer subphase ( black spectra ) containing 1 mm mgcl2 ( blue spectra ) or 1 mm cacl2 ( red spectrum ) at
a surface pressure of 17 mn / m .
the open circles represent vsfs data
points , and the solid lines are fits to the data .
spectra of the same data
offset along the y - axis are provided in figure s7 .
in addition to the inositol ring
modes , the phosphate peaks ( e.g. , sympo3 at 982 cm , sympo2 at 1086 cm , asypo3 at 1115 cm , asypo2 at 1154 cm , detailed assignments
in figure s6 and table s1 ) also showed a substantial intensity increase upon the introduction
of ca to the subphase .
this indicates a strong net orientation
and/or ordering of the headgroup phosphates upon ca binding .
it should be noted that ca binding may help to deprotonate
the second monoesterified phosphate , which
would prompt additional changes in the spectra beyond those related
to ordering and tilt angle .
moreover , upon the addition of ca , the symmetric po3 stretch
exhibited a relatively large 20 cm blue shift ,
while the asymmetric po3 and po2 stretches shifted by 6 cm and 8 cm , respectively ( table s1 ) .
the shifts of both po3 peaks are consistent with phosphate dehydration upon cation binding
and/or a symmetry change of the c3v point group .
the shift of the asymmetric po2 peak also suggests headgroup phosphate
dehydration upon ca binding .
the spectral
change brought about by 1 mm mg in the
phosphate region was much less pronounced overall compared to that
with 1 mm ca .
the difference in the interactions of ca and mg with phosphate could be explained at
least in part by different dehydration penalties for these two cations .
it has been suggested that ca binding to phosphate groups
is favored because ca is more easily dehydrated than
mg .
this difference in the
hydration shell chemistry may , in turn , act to disfavor the bridging
of the inositol rings of pi(4,5)p2 , which would weaken
the ordering effect of mg .
in addition to phosphate
and inositol resonances , vsfs spectra
were also obtained in the carbonyl c = o symmetric stretch ( 1730
cm ) region before
and after addition of 1 mm cacl2 or mgcl2 ( figure 2b ) .
in fact , a 1.6-fold increase in the oscillator
strength of this peak was observed upon binding of ca , while only a 1.3-fold increase was found for mg ( table s2 ) .
this oscillator strength increase
should correspond to a backbone ordering effect , thus helping to reinforce
a more rigid configuration of the headgroup inositol rings . ordering
of the lipid acyl chains was also observed ( figure s8 and table s3 ) .
taken
together , the changes in the vsfs spectra provide strong
experimental evidence for distinct conformational changes within the
lipid headgroup region in the presence of ca , but less
with mg .
such results should be important for the plc
1-ph domain selectivity of pi(4,5)p2 found above
with liposomes and guvs . with the aim
of obtaining mechanistic insights into the effects of ca and
mg on pi(4,5)p2 molecules at a molecular
level , we employed atomistic md simulations . in order to reduce methodological
bias , we used two all - atom force fields ( opls - aa and charmm36 ) as
well as the united - atom force field from berger ( table s4 ) .
importantly , to further account for electronic polarization
effects of charged groups in a mean field manner , for ca interacting with pi(4,5)p2 phosphates we also employed
the recently developed electronic continuum correction with rescaling
( eccr ) method .
this , to a large extent ,
dampens the unrealistically high ion pairing found when employing
nonpolarizable force fields .
it is particularly
useful in the present case where strong electronic polarization can
be expected in the vicinity of multiple - charged moieties .
we
generated multiple sets of 1 s long trajectories for different
initial pi(4,5)p2 distributions prior to and after the
addition of ca or mg .
for all simulations ,
consistently with all force fields used , we find that ca interacts with pi(4,5)p2 and has a pronounced effect
on the lipid headgroup orientation ( figure 3 and figures s9 and s13 ) .
moreover , control simulations with mg showed that
the effects induced by magnesium are much weaker than those induced
by calcium for all simulations ( figure 3c , d and si ) , in full agreement
with experiments .
snapshots from md simulations of the lipid bilayer taken
at 1 s
( a ) without and ( b ) with ca .
( c ) tilt angle distribution
of the pi(4,5)p2 headgroup and ( d ) density profiles of
lipid headgroups without ( blue ) and with mg ( green ) or
ca ( red ) . numbers in ( c ) represent mean tilt angles for
each system . here , only the results of the berger force field simulations
are presented .
additional force field simulations with similar outcomes
can be found in the supporting information .
the addition of ca or mg immediately
leads to a significant reduction of the area per lipid ( figure s10 and table s5 ) .
this macroscopic effect
is in agreement with lateral condensation of the pi(4,5)p2-containing monolayers by ca and our vsfs analysis of the ch stretches ( figure s8 ) . at the microscopic level
, we found
that each pi(4,5)p2 molecule binds on average 1.63.1
ca molecules , depending on the force field that is employed
( table s5 ) .
this is consistent with the
water peak spectral changes , which show that each lipid molecule binds
more than two ca ions ( figure s8 ) .
ca binds mostly to the phosphate groups at positions
4 and 5 , but it also penetrates deeper into the lipid bilayer to interact
with the carbonyl groups ( figure s11 ) .
ca binding to the lipid carbonyl group is consistent
with the vsfs data in the carbonyl stretch region , as documented herein
( figure 2b ) and elsewhere . in agreement with
previously published computational and experimental
results , we observed that ca increases
the order parameters of the pi(4,5)p2 acyl chains ( figure s12 ) .
the acyl chain ordering is also
fully in line with the effects observed in the vsfs spectra ( figure s8 ) .
the most prominent feature
observed by simulations is a pronounced
headgroup reorientation , primarily caused by the ability of ca to bridge two pi(4,5)p2 headgroups ( figure 3a , b ) .
to quantitatively
analyze the headgroup reorientation , we monitored the tilt angle between
the c1c4 atoms of the pi(4,5)p2 inositol ring and
the bilayer normal .
the average tilt angle in the control simulation
without ca was in the range of 3541 , depending
on the employed force field .
this result is in agreement with previously
published md studies . in the presence of ca ions , however ,
the average tilt angle significantly increased for all of the force
fields up to 65 ( figure 3c and figures s9 and s13 ) .
simulations
thus consistently showed bending of the pi(4,5)p2 headgroup
toward the plane of the bilayer and away from bulk water ( table s5 ) .
moreover , consistent with a narrowing
of the inositol ring s distribution as indicated by vsfs results
above ( figure 2a ) ,
ca slowed pi(4,5)p2 headgroup rotational diffusion
as revealed by the rotational correlation function ( figure s9e ) .
the ca effect was also manifested
in the density profiles ( figure 3d ) , where the location of the pi(4,5)p2 headgroups
shifted in the presence of calcium toward the bilayer center . moreover
,
ca significantly decreased the solvent accessible surface
area of pi(4,5)p2 , which correlated with a reduced average
number of hydrogen bonds between the pi(4,5)p2 headgroups
and water molecules ( table s5 ) .
these data
also match the experimentally observed partial dehydration of pi(4,5)p2 in the presence of ca as measured here by vsfs
and elsewhere .
the charge state
of pi(4,5)p2 in lipid membranes is
highly sensitive to the cellular ph and the presence of proteins and
ions . by using
not only the default parametrization
( charmm36 and opls - aa ) but also the eccr corrected charges for the
ions and pi(4,5)p2 phosphate groups ( berger , opls - aa ) ,
we were able to assess the potential effects of the lipid charge state .
namely , the charge used for pi(4,5)p2 varied from 3.75
to 5 , depending on the particular force field ( for more details ,
see the si ) .
reassuringly , we found semiquantitatively
the same effect of ca on the pi(4,5)p2 tilt
angle with ca in all the systems which were tested .
this
indicates that under the conditions of these investigations the protonation
state of pi(4,5)p2 was not particularly critical for the
observed effects .
by means of protein lipid
binding assays and spectroscopic
experiments , together with atomistic md simulations , we have unraveled
and characterized in molecular detail the pronounced effect of ca on pi(4,5)p2 headgroup presentation .
first , we
confirmed the previously observed increase of the pi(4,5)p2 acyl chain order and pi(4,5)p2 cluster formation , as evidenced here by vsfg spectroscopy and md simulations .
second ,
we characterized at the molecular level the interactions of ca with pi(4,5)p2 headgroup phosphates , as well
as the more deeply seated carbonyl groups .
we observed the hitherto
unrecognized consequences of ca binding for pi(4,5)p2 at the molecular level .
namely , we observed a dramatic change
in the pi(4,5)p2 headgroup tilt angle . by means of liposome
flotation and guv binding assays , we show that ca has
a strong propensity to render the pi(4,5)p2 headgroup invisible
to the plc-1 ph domain .
our data lead to the plausible
conjecture that the calcium - induced
switching of phosphoinositide conformational states may serve as a
potential cellular mechanism for lipid recognition and thus play a
decisive role in cell signaling and membrane trafficking
. a systematic
correlation of kinetics and curvature sensitivities at the nanoscale in vitro(58 ) will be key to understanding
the general applicability of our data to other proteins and to different
endomembranes . | the
orchestrated recognition of phosphoinositides and concomitant
intracellular release of ca2 + is pivotal to almost every
aspect of cellular processes , including membrane homeostasis , cell
division and growth , vesicle trafficking , as well as secretion .
although
ca2 + is known to directly impact phosphoinositide clustering ,
little is known about the molecular basis for this or its significance
in cellular signaling . here , we study the direct interaction of ca2 + with phosphatidylinositol 4,5-bisphosphate ( pi(4,5)p2 ) , the main lipid marker of the plasma membrane .
electrokinetic
potential measurements of pi(4,5)p2 containing liposomes
reveal that ca2 + as well as mg2 + reduce the
zeta potential of liposomes to nearly background levels of pure phosphatidylcholine
membranes .
strikingly , lipid recognition by the default pi(4,5)p2 lipid sensor , phospholipase c delta 1 pleckstrin homology
domain ( plc 1-ph ) , is completely inhibited in the presence
of ca2 + , while mg2 + has no effect with 100 nm
liposomes and modest effect with giant unilamellar vesicles .
consistent
with biochemical data , vibrational sum frequency spectroscopy and
atomistic molecular dynamics simulations reveal how ca2 + binding to the pi(4,5)p2 headgroup and carbonyl regions
leads to confined lipid headgroup tilting and conformational rearrangements .
we rationalize these findings by the ability of calcium to block a
highly specific interaction between plc 1-ph and pi(4,5)p2 , encoded within the conformational properties of the lipid
itself .
our studies demonstrate the possibility that switchable phosphoinositide
conformational states can serve as lipid recognition and controlled
cell signaling mechanisms . | Introduction
Results
and Discussion
Conclusion |
stem cells , directed to differentiate into specific cell types , offer the possibility of a renewable source of replacement cells and tissues to treat diseases ( 1 ) .
periodontium has limited capacity for regeneration in early phases of the disease ( 2 ) because of the lack of robust stem cells . in such a scenario exogenous regenerative
tools such as ex vivo expanded / manipulated stem cells can be used to replenish the host cell niche and facilitate tissue regeneration ( 3 ) .
human bone - marrow - derived mesenchymal stem cells ( bm - mscs ) that are extensively used in regenerative approaches require a highly invasive harvest procedure and the frequency , proliferation efficiency and differentiation potential of bm - mscs decline with age .
foetal or neonatal mscs appear to be more primitive and have greater multi - potentiality than their adult counterparts and can thus be considered as an alternative source of mscs .
the umbilical cord , which is discarded at birth , can provide an inexhaustible source of stem cells through a non - invasive , painless and ethically non - controversial collection procedure .
the umbilical cord - derived mscs ( hucmscs ) are more primitive and have faster proliferation rates , wide multi - potency and greater expansion capability compared with adult mscs and do not induce teratomas ( 4 ) .
their intermediate state between adult and embryonic stem cells also makes them an ideal candidate for reprogramming to the pluripotent status .
the arena of tissue engineering for regeneration requires a scaffold , cells and growth factors .
basic fibroblast growth factor ( bfgf ) is a potent mitogen for mesenchymal cells and is also known to induce angiogenesis , chemotaxis and proliferation of periodontal ligament cells .
bfgf has successfully regenerated periodontal tissues , harnessing the ecto - mesenchymal origin of the periodontium and the presence of undifferentiated mesenchymal cells in the periodontal ligament of adults ( 5 ) . a regenerative approach that combines the advantages of both hucmscs and bfgf
can prove beneficial in the management of periodontal defects . to the best of our knowledge
the role of hucmscs in the field of periodontal regeneration has not been extensively researched and no studies have evaluated the proliferative / adhesive property of hucmscs to root surfaces . hence in a first of its kind attempt , the capacity of hucmscs alone and under the influence of bfgf to differentiate and adhere onto periodontally healthy and diseased tooth root surfaces has been evaluated .
the research protocol was approved by the institutional ethical board and review committee of krishnadevaraya college of dental sciences , affiliated to rajiv gandhi university of health sciences , bangalore for human subjects and the study was conducted in accordance with the helsinki declaration of 1975 , as revised in 2000 .
the study protocol was explained to the expectant mothers and a written consent was obtained .
fresh human umbilical cords were obtained from informed healthy mothers in hospitals at the time of delivery , after birth and collected in phosphate buffered saline solution ( 6 ) .
the cords were rinsed twice in phosphate buffered saline in penicillin and streptomycin , and the cord blood was removed during this process .
the washed cords were cut into 1-mm pieces and floated in low - glucose dulbecco s modified eagle s medium containing 10% foetal bovine serum .
the pieces of cord were subsequently incubated at 37c in a humidified atmosphere consisting of 5% co2 .
the medium was replaced every 3 days after the initial plating . when well - developed colonies of cells appeared after 10 days , the culture were trypsinized and passaged into a new flask for further expansion ( fig .
all of the infused umbilical cord mesenchymal stem cells were derived from passages 2~5 , with rigorous purification and quality control ( 7 ) .
cultured ucmscs showed strong positive staining for cd73 , cd44 , cd105 and cd 90 ( fig .
24 periodontally diseased scaffolds were obtained from teeth with proximal attachment loss of 7 mm that were indicated for extraction due to poor prognosis .
24 healthy scaffolds were obtained from teeth indicated for orthodontic extraction / surgical dis - impaction from non - smoker and systemically healthy patients .
teeth with root caries or root restorations , root surface abrasions , fracture , demineralized root surfaces , patients who have undergone scaling and root planning or any other type of regenerative periodontal therapy 6 months prior to the initial examination were not included in the study .
the root surfaces were planed with scalers and curettes * to remove any soft connective tissues .
all the pulpal tissues in root canals were removed with files. test specimen of size 16 mm ( 4 mm4 mm ) and thickness of 2 mm were prepared .
the specimens were then sterilized by autoclaving and kept in saline at 4c until further use ( 6 ) .
the samples were distributed into 4 groups ( 12 specimens per group ) group - i : hucmscs were added to healthy root substrate group - ii : hucmscs were added to periodontally diseased root substrate group - iii : hucmscs and bfgf were added to healthy root substrate group - iv : hucmscs and bfgf were added to periodontally diseased root substrate bfgf-2 was reconstituted by dissolving it in distilled water as per the manufacturer s guidelines .
this solution was then diluted by 100 times to get 1000 l ( 1 ml ) of stock solution with 1 g ( 1,000 ng ) of growth factor .
thus a concentration of 1 ng/1 l of stock solution was prepared , of which 8 l was used for each ml of media ( 8) .
1 ) from cell passages 25 were pipetted out and seeded onto the scaffolds of all groups at a density of 110 per sample ( 6 ) . for groups
iii and iv , bfgf was supplemented to the culture media before seeding the cells on the scaffolds .
following incubation for the selected time period , scaffolds were gently withdrawn from the tissue culture plates .
specimens were fixed in 3% glutaraldehyde in ice cold phosphate buffered saline for 30 minutes .
this was followed by immersion in amyl acetate solution for 2 hours specimens were then critical point dried with liquid co2 ( 6 ) .
once processing was completed , scaffolds were placed in sem * * for viewing and photographing at 500 .
a grid was placed over the images , to facilitate cell counting , repeated cell counts of the number of cells attached to the tooth surface were performed by a blinded observer and each photograph had 5 representative areas , one central and 4 peripheral .
in addition the morphology of individual cells were assessed at a magnification of 500 and were scored using a scale from 1 to 4 , where 1=flat cells , 2=combination of oval or round and flat cells , 3=oval cells and 4=round cells ( 9 ) .
the parameters that were assessed and compared between the groups were number of cells on the scaffold and the morphological score of the adhereing cells .
t test was used to determine whether there was a statistical difference between control and study group in the parameters measured .
one way analyses of variance with tukey s post hoc test were used to determine the difference of group wise comparisons .
the research protocol was approved by the institutional ethical board and review committee of krishnadevaraya college of dental sciences , affiliated to rajiv gandhi university of health sciences , bangalore for human subjects and the study was conducted in accordance with the helsinki declaration of 1975 , as revised in 2000 .
the study protocol was explained to the expectant mothers and a written consent was obtained .
fresh human umbilical cords were obtained from informed healthy mothers in hospitals at the time of delivery , after birth and collected in phosphate buffered saline solution ( 6 ) .
the cords were rinsed twice in phosphate buffered saline in penicillin and streptomycin , and the cord blood was removed during this process .
the washed cords were cut into 1-mm pieces and floated in low - glucose dulbecco s modified eagle s medium containing 10% foetal bovine serum .
the pieces of cord were subsequently incubated at 37c in a humidified atmosphere consisting of 5% co2 .
the medium was replaced every 3 days after the initial plating . when well - developed colonies of cells appeared after 10 days , the culture were trypsinized and passaged into a new flask for further expansion ( fig .
all of the infused umbilical cord mesenchymal stem cells were derived from passages 2~5 , with rigorous purification and quality control ( 7 ) .
cultured ucmscs showed strong positive staining for cd73 , cd44 , cd105 and cd 90 ( fig .
24 periodontally diseased scaffolds were obtained from teeth with proximal attachment loss of 7 mm that were indicated for extraction due to poor prognosis .
24 healthy scaffolds were obtained from teeth indicated for orthodontic extraction / surgical dis - impaction from non - smoker and systemically healthy patients .
teeth with root caries or root restorations , root surface abrasions , fracture , demineralized root surfaces , patients who have undergone scaling and root planning or any other type of regenerative periodontal therapy 6 months prior to the initial examination were not included in the study .
the root surfaces were planed with scalers and curettes * to remove any soft connective tissues .
all the pulpal tissues in root canals were removed with files. test specimen of size 16 mm ( 4 mm4 mm ) and thickness of 2 mm were prepared .
the specimens were then sterilized by autoclaving and kept in saline at 4c until further use ( 6 ) .
the samples were distributed into 4 groups ( 12 specimens per group ) group - i : hucmscs were added to healthy root substrate group - ii : hucmscs were added to periodontally diseased root substrate group - iii : hucmscs and bfgf were added to healthy root substrate group - iv : hucmscs and bfgf were added to periodontally diseased root substrate
bfgf-2 was reconstituted by dissolving it in distilled water as per the manufacturer s guidelines .
this solution was then diluted by 100 times to get 1000 l ( 1 ml ) of stock solution with 1 g ( 1,000 ng ) of growth factor .
thus a concentration of 1 ng/1 l of stock solution was prepared , of which 8 l was used for each ml of media ( 8) .
1 ) from cell passages 25 were pipetted out and seeded onto the scaffolds of all groups at a density of 110 per sample ( 6 ) . for groups
iii and iv , bfgf was supplemented to the culture media before seeding the cells on the scaffolds .
following incubation for the selected time period , scaffolds were gently withdrawn from the tissue culture plates .
specimens were fixed in 3% glutaraldehyde in ice cold phosphate buffered saline for 30 minutes .
ethanol was discarded and specimens were again fixed in 100% fresh ethanol overnight . next day they were kept in fresh solution of 100% ethanol for 1 hour .
this was followed by immersion in amyl acetate solution for 2 hours specimens were then critical point dried with liquid co2 ( 6 ) .
a grid was placed over the images , to facilitate cell counting , repeated cell counts of the number of cells attached to the tooth surface were performed by a blinded observer and each photograph had 5 representative areas , one central and 4 peripheral .
in addition the morphology of individual cells were assessed at a magnification of 500 and were scored using a scale from 1 to 4 , where 1=flat cells , 2=combination of oval or round and flat cells , 3=oval cells and 4=round cells ( 9 ) .
the parameters that were assessed and compared between the groups were number of cells on the scaffold and the morphological score of the adhereing cells .
the students t test was used to determine whether there was a statistical difference between control and study group in the parameters measured .
one way analyses of variance with tukey s post hoc test were used to determine the difference of group wise comparisons .
the purpose of this present study was to determine the ability of hucmscs to differentiate into cells either in the presence or absence of bfgf and its ability to adhere to scaffolds derived from periodontally diseased and healthy root surfaces of human teeth in vitro at two different time periods ( day 14 and day 21 ) .
anova was done to compare the attachment of cells on all the four groups to test the null hypothesis .
p value was less than 0.05 which signifies that the null hypothesis being tested in the study , that umbilical cord stem cells differentiate into fibroblasts or fibroblast like cells and attach to the root surfaces , is accepted and all the groups show attachment of cells .
group i , group iii and group iv showed a significant increase in the attachment of cells from 14th to 21st day ( p value 0.02 , 0.03 and 0.001 respectively ) . in group
ii , there was increase in attachment of cells from 2.2 cells / unit area to 3.4 cells / unit area , from day 14 to day 21 ( graph 1 ) . on day 14 when all the groups were compared by anova
there was a statistically significant increase of cells in group - iii as compared to group - i with a
when group - i was compared with group - ii and group - iv , statistically insignificant results with p value of 0.98 and 0.06 respectively .
comparison of group - ii with group - iii and iv gave statistically insignificant result with
f test , a statistically significant result of p value of 0.003 was achieved which implies that all the groups showed attachment of cells .
a statistically significant increase in attachment of cells was seen in group - iii and group - iv as compared to group - ii with a
p value of 0.98 and this did not show a statistically significant result , when tukeys test was done to compare group - i with group - ii , iii and iv , statistically insignificant result was seen with
p value of 0.014 was seen showing a decrease in morphology score from 14th to 21st day . in group
ii reduction in mean score was seen from 1.1 to 0.8 from day 14 to day 21 suggesting change of morphology from round to flat .
group iv showed reduction in score from 1.06 to 0.9 indicating conversion to a more mature flat morphology of fibroblast ( graph 2 ) . on day 14 the mean score of morphology of the cells in group - i was 1.6 , in group - ii 1.1 , group - iii showed 1.2 and group - iv showed 1.06 ( fig .
the mean score for group - i was 0.9 , group - ii was 0.8 , group - iii had 0.93 , and group - iv had 0.9 .
this implies maturation of cells from 14 day to 21 day ( graph 2 , fig .
anova was done to compare the attachment of cells on all the four groups to test the null hypothesis .
p value was less than 0.05 which signifies that the null hypothesis being tested in the study , that umbilical cord stem cells differentiate into fibroblasts or fibroblast like cells and attach to the root surfaces , is accepted and all the groups show attachment of cells .
group i , group iii and group iv showed a significant increase in the attachment of cells from 14th to 21st day ( p value 0.02 , 0.03 and 0.001 respectively ) . in group
ii , there was increase in attachment of cells from 2.2 cells / unit area to 3.4 cells / unit area , from day 14 to day 21 ( graph 1 ) . on day 14 when all the groups were compared by anova
there was a statistically significant increase of cells in group - iii as compared to group - i with a
when group - i was compared with group - ii and group - iv , statistically insignificant results with
comparison of group - ii with group - iii and iv gave statistically insignificant result with
f test , a statistically significant result of p value of 0.003 was achieved which implies that all the groups showed attachment of cells .
a statistically significant increase in attachment of cells was seen in group - iii and group - iv as compared to group - ii with a
p value of 0.98 and this did not show a statistically significant result , when tukeys test was done to compare group - i with group - ii , iii and iv , statistically insignificant result was seen with
in group i significant difference with a p value of 0.014 was seen showing a decrease in morphology score from 14th to 21st day . in group
ii reduction in mean score was seen from 1.1 to 0.8 from day 14 to day 21 suggesting change of morphology from round to flat .
group iv showed reduction in score from 1.06 to 0.9 indicating conversion to a more mature flat morphology of fibroblast ( graph 2 ) . on day 14 the mean score of morphology of the cells in group -
i was 1.6 , in group - ii 1.1 , group - iii showed 1.2 and group - iv showed 1.06 ( fig .
the mean score for group - i was 0.9 , group - ii was 0.8 , group - iii had 0.93 , and group - iv had 0.9 .
this implies maturation of cells from 14 day to 21 day ( graph 2 , fig .
regeneration requires the formation of new cementum on root surface , new alveolar bone and periodontal ligament fibres making functional connection between the two mineralized tissues adhesion of cells to the root surface is one of the essential processes along with cell migration for an oriented and functional attachment ( 8) .
bm - mscs , and embryonic stem cells ( escs ) have demonstrated the possibility of creating dental tissue replacements from non - dental stem cells by imitating embryonic development mechanisms .
mscs have been procured from human delivery wastes , such as cord blood , umbilical cord tissue and placenta ( 10 ) .
bfgf serves as a potent mitogen and elevates the level of cyclin d protein and cyclin dependent kinase , which drives the cells into s phase ( 10 ) , thus modulating the hucmscs population growth kinetics .
hence the primary objective of the study was to assess the proliferative capacity of hucmscs with and without bfgf and the attachment of these cells to tooth root surface .
there are no previous studies reported in the scientific literature that have evaluated the propensity of hucmscs to differentiate into cells that attach to tooth root surfaces .
the osteogenic , adipogenic potential and immunosuppressive activity of these cells is not altered by bfgf ( 10 ) .
infact bfgf supplementation encourages bm - mscs differentiation to osteogenic and adipogenic phenotype and enhances the immunosuppressive activity ( 11 , 12 ) .
bfgf also preserves the primitive status of hucmscs , reduces cell death , decreases the secretion of vascular endothelial growth factor and mmp-3 ( 10 ) .
one of the primary pre - requisites of optimum periodontal regeneration is to increase the cellular machinery i.e. , the msc directly in sites that requires regeneration ( 13 ) .
hucmscs have great proliferative potential and can differentiate into different cell types depending on the inductive media ( 14 ) .
attachment of cells to the root surface is very important to obtain newly formed periodontal ligament which is a non - mineralised connective tissue between two mineral - ised tissues i.e. bone and cementum ( 15 ) . in a study done by inac et al .
2009 that evaluated the in vitro differentiation of human embryonic stem cells on tooth surface ( 8) a formation of soft - hard tissue relationship in close contact areas was noted .
escs acquired fibroblast like morphology , formed tissue like deposits on cementum surfaces and occasional embryonic body like structures .
2013 successfully isolated cells from wharton s jelly of umbilical cord , principally exhibiting a fibroblast like morphology ( 14 ) .
these cells expanded rapidly reaching 80~90% confluence in 19 days . in groups supplemented with bfgf ,
this further reiterates the important role played by bfgf as an inductive media in propelling hucmscs into a more committed lineage of differentiation .
this role of bfgf in inducing the proliferation of fibroblast or fibroblast like cells has also been noted by ramasamy .
he also mentioned that bfgf preserves the primitive status of ucmsc by increasing expression of nanog , sox2 and rex1 transcription factors ( 10 ) . when paired
t test was done to assess proliferation of cells from day 14 to day 21 , all the groups showed a significant increase in attachment of cells , suggesting the high proliferative capacity of hucmscs .
the proliferated cells also demonstrated the ability to attach to tooth root surface as initially hypothesised in the study . on day 14 ,
when group i was compared with group iii , group iii showed a significant increase in attachment of cell number in accordance with the studies done by hagmann et al and ramasamy et al .
( 10 , 16 ) increase in attachment of cells was also seen in group iv as compared to group ii , showing the mitogenic and proliferative effect of fibroblast growth factor ( fig .
both these groups were under the inductive influence of b fgf . on day 14 , when group iii was compared with group iv there was a decrease in attachment of cells in periodontally diseased root surfaces ( group iv ) .
this could be attributed to the presence of rough surfaces with sharp edges of the periodontally diseased surfaces , due to presence of calculus and debris that could damage the cell membranes leading to diminished cell viability ( 18 ) .
similar observation was seen on day 21 with more number of cells attached in group i than group ii .
the increased number of cells in group i may be explained by the root surface biocompatibility of healthy tooth root surfaces as compared to diseased tooth surfaces .
the decreased cellular adhesion to diseased root surface is similar to a study conducted by belal et al .
2006 where the effectiveness of pdgf - bb application on periodontally diseased root surfaces was assessed through attachment and growth of fibroblast cells .
the diseased group showed the least number of cell attachment as compared to the healthy and scaling and root planing group .
the presence of rough blebs and some debris on the root surfaces of diseased samples may hinder pdl fibroblast cell attachment and growth , and thus explained the presence of incompatible root surfaces in this group ( 6 ) .
when comparison between group i and group ii was done , increased attachment of cells was seen in group ii on day 14 and comparison between group iii and group iv showed profound cell attachment in group iv on day 21 .
procedural effects- such as solutions or sterility of slides maybe responsible for this difference , as also noted by gamal et al .
some samples even showed microbial contamination which might have prevented the attachment of cells and it was decided not to count specimens without cell growth .
it is important to understand the morphological changes that occur during the growth and maturation of cells on a suitable biocompatible substrate .
flat cells with numerous attachment extensions and lamellipodia are generally firmly attached and round cells are poorly attached ( 19 ) . in the current study , all the groups showed presence of round and oval cell morphology on day 14 .
cells isolated from the wharton s jelly displayed a typical fibroblast like morphology which is in accordance with li et al .
. however , group i had the maximum mean score of 1.6 suggesting highest percentage of round and oval cells , followed by group iii with a mean score of 1.2 which suggests the presence of more of flat cells .
similarly , there was a lesser score for group ii , which was 1.1 suggesting more of round or oval cells as compared to a score of 1.06 for group iv showing more number of flat cells , suggesting the positive influence of bfgf on the differential potential of cells ( fig .
studies have demonstrated that human dental pulp cells when cultured with the medium containing bfgf , were highly proliferative and capable of differentiating in vitro into osteoblasts , chondrocytes , and adipocytes ( 20 ) .
stem cells from the apical papilla ( scap ) a type of mesenchymal stem cells found in the developing tissue , apical papilla , of immature permanent teeth when expanded in the presence of bfgf for 1 week in subsequent stimulation of the osteogenic / dentinogenic condition resulted in enhanced differentiation ( 21 ) . on day 21 , most of the cells in all the groups showed flat morphology with similar mean scores , which indicates that the cells proliferated into mature cells in 21 days .
hence it can be concluded that hucmscs can differentiate into cells and attach on tooth surfaces and the supplementation with bfgf enhances the proliferation and attachment of the cells .
the morphology of these cells are similar to fibroblasts and show increase in maturity with addition of bfgf .
previous studies have reported that human umbilical cord tissue demonstrates the advantages of providing an abundant supply of cells from donors via a non - invasive procedure with a low risk of infection and can therefore be considered an attractive alternative source of mscs .
hucmscs can differentiate into cells that possess the fibroblast - like morphology , non - hematopoietic cell surface phenotypes , low immunogenicity , and multipotent differentiation ability .
therefore , mscs obtained from human umbilical cord tissue may serve as a rational candidate cell source for tooth regeneration research .
growth factors when added can modulate many cellular activities , including cell differentiation and proliferation , cell migration and extracellular matrix synthesis and production .
although there are many challenges ahead of us in terms of utilizing stem cells for tissue regeneration , hucmscs are one of the most promising postnatal stem cell populations for tissue repairing and regeneration .
future research can be targeted at characterization of these differentiated cells and better define their role in periodontal regeneration .
the propensity for this promising cellular machinery hucmscs , in periodontal regeneration needs to be further researched . | background and objectivesthe purpose of this first of its kind study was to analyse the growth , development and attachment of cultured human umbilical cord stem cells alone or supplemented with basic fibroblast growth factor ( bfgf ) on both healthy and periodontally diseased tooth surfaces in vitro.methodsfour groups of 12 root surface scaffolds each were classified as group i- healthy root surfaces ; group ii- periodontally diseased ; group iii- healthy with bfgf and group iv- periodontally diseased root with bfgf .
bfgf was applied in the concentration of 8 ng / ml on to the surface followed by incubation of cultured human umbilical cord stem cells ( hucmscs ) on the scaffolds .
scanning electron microscopy observations were made on 14th and 21st days to assess the proliferation and morphology of cells attached on the tooth surface.resultscultured hucmscs demonstrated adhesion to tooth root scaffold .
all the groups showed a significant increase in the number of cell attachment from 14th day to 21st day .
the groups with bfgf showed a significant increase in attachment of cells when compared to the groups without bfgf .
the cells showed an increase in number of flat cells from 14th day to 21st day in all the groups indicating an increased maturity of cells .
periodontally diseased groups had less maturity of cells than healthy groups .
the groups supplemented with bfgf , had more mature cells than the groups without bfgf.conclusionshucmscs have the propensity to differentiate into cells that have the capacity to bind to root surfaces .
hucmscs incubated with bfgf showed better proliferation and attachment to tooth root surfaces . the role of hucmscs can be further explored for periodontal regeneration . | Introduction
Materials and Methods
Isolation and culture of Human Umbilical Cord Stem Cells
Root slice preparation
Specimen preparation, sectioning and sterilization
Preparation of bFGF-2
Cell seeding
Preparation of teeth for SEM Analysis
Statistical Analysis
Results
Cell proliferation assay
Cell morphology assay
Discussion
Conclusion |
early childhood caries ( ecc ) is a common infectious disease involving the maxillary anterior primary teeth more frequently . usually , because of the coronal structure loss
however , the profound psychological impacts of tooth loss , along with its untoward effects on esthetics , mastication , speech and growth , should not be overlooked .
ecc poses a challenge to pediatric dentists , especially when highly mutilated teeth are to be restored by composite resins . in spite of several problems existing naturally in the utility of composite resins ,
these agents are suggested as the materials of choice for restoration of anterior teeth because of esthetic demands . on the other hand , small crowns and relatively large pulp chambers of primary teeth
leave the dentists with insufficient tooth structure to obtain proper bond and retention . the post
, different types of casted or prefabricated post systems have been proposed . in direct restoration of deciduous anterior teeth ,
additionally , it seems that extension of clinical crown to cementum may increase the longevity of the restoration .
this study compared the fracture resistance of conventional and cementum - extended composite restoration with or without intracanal composite posts .
a total of 60 freshly extracted maxillary primary incisors with similar root length and width were used in this study .
the specimens with crack , fracture and root resorption or canal obliteration were discarded . following the tooth extraction ,
they were maintained in normal saline to avoid dehydration and were then disinfected in 0.05% chloramine trihydrate solution for 1 week .
all crowns were cut perpendicular to the long axis using a diamond bur in a high - speed hand piece under a water coolant leaving 1 mm above the cemento enamel junction ( cej ) .
the root canals were prepared to size # 45 by k - files ( mani inc . , tochigi , japan ) 1 mm short of the apex .
then , the canals were obturated with metapex ( meta biomed co. ltd . , republic of korea ) by the same operator ,
mounted 1.5 mm higher than the acrylic resin surface and divided into four groups of 15 each : group 1 : the root canals were sealed and the teeth were restored with composite resin ( cf )
following obturation with metapex , the root canal orifices were covered with a thin layer of light - curing calcium hydroxide ( lime - lite , pulpdent , watertown , ma , usa ) to create a smooth surface for composite placement .
full crown build - up was performed with solid 3 m espe filtek z250 dental composite ( 3 m espe , st .
following obturation with metapex , the root canal orifices were covered with a thin layer of light - curing calcium hydroxide ( lime - lite , pulpdent , watertown , ma , usa ) to create a smooth surface for composite placement .
full crown build - up was performed with solid 3 m espe filtek z250 dental composite ( 3 m espe , st .
group 2 : intracanal composite posts were placed and the teeth were restored with composite resin ( cf + cp )
the first 4 mm of the canal fillings was then removed to leave a 3 mm space for composite post placement . upon etching ( scotchbond etchant , 3 m espe , st .
paul , mn , usa ) and bonding ( single bond , 3 m espe , st .
paul , mn , usa ) with flowable composite ( filtek z350 t flowable , 3 m espe , st .
paul , mn , usa ) , intracanal composites were placed and the crown build - up was performed with solid z250 ( 3 m espe , st .
the first 4 mm of the canal fillings was then removed to leave a 3 mm space for composite post placement . upon etching ( scotchbond etchant , 3 m espe , st .
paul , mn , usa ) and bonding ( single bond , 3 m espe , st .
paul , mn , usa ) with flowable composite ( filtek z350 t flowable , 3 m espe , st .
paul , mn , usa ) , intracanal composites were placed and the crown build - up was performed with solid z250 ( 3 m espe , st .
group 3 : the root canals were sealed and the teeth were restored with composite 0.5 mm extended to the cementum ( cecf )
following pulpectomy , the canals were obturated and covered with a thin layer of light - curing calcium hydroxide to create a smooth surface for composite placement .
full crown build - up was performed with solid 3 m espe filtek z250 composite .
the composite covered 1 mm of the enamel and 0.5 mm of the cementum .
following pulpectomy , the canals were obturated and covered with a thin layer of light - curing calcium hydroxide to create a smooth surface for composite placement .
full crown build - up was performed with solid 3 m espe filtek z250 composite .
group 4 : composite intracanal posts were placed and the teeth were restored with composite resin with 0.5 mm extension to the cementum ( cecf + cp )
similar to group 3 , the first 4 mm of the root canal fillings was removed to leave a 3 mm space for composite post placement . upon etching and bonding with 3 m espe z350 xt flowable composite ,
intracanal composite posts were placed and the crown build - up was performed with solid 3 m espe filtek z250 composite .
the composite covered 1 mm of the enamel and 0.5 mm of the cementum .
similar to group 3 , the first 4 mm of the root canal fillings was removed to leave a 3 mm space for composite post placement . upon etching and bonding with 3 m espe z350 xt flowable composite ,
intracanal composite posts were placed and the crown build - up was performed with solid 3 m espe filtek z250 composite .
the composite restoration height in all groups was equal to 4 mm . to assess the fracture resistance , the teeth were separately mounted into acrylic resin and fixed by a jig .
then , the assembly received a progressively increasing load with a cross - head speed of 0.5 mm / min at 148 to the long axis of the primary incisors on the mid - palatal surface to simulate primary occlusion in a universal testing machine ( zwick , germany ) until the occurrence of the fracture .
the statistical analysis was performed by the one - way analysis of variance ( anova ) test or its non - parametric equivalent , the kruskal
to assess the possible effect of different mesiodistal widths on the fracture resistance , the one - way anova test was applied ; however , no statistically significant difference was found ( p > 0.05 ) .
the average mesiodistal width of each group of the specimens is presented in table 1 .
the highest and the lowest mean fracture resistance ( mfr ) values were recorded for the cecf + cf and the cecf groups , respectively [ table 2 , figure 1 ] .
the mean mesiodistal width of each group in millimeters the mean fracture resistance ( sd ) and the range of fracture resistance values a comparative illustration of the mean fracture resistance ( sd ) value of each group the mfr in group 4 was significantly higher than that in groups 1 and 3 , but not group 2 . on the other hand ,
cf + cp did not show significantly higher mfr compared with cf [ table 3 ] .
statistical differences of the mean fracture resistance between the groups under study ( p value ) * the location of the fracture line against cej was microscopically examined .
a fracture line above the cej was recorded for 12 ( 80% ) specimens in the cf group .
this value equaled 10 ( 66.6% ) for cf + cp and cecf + cp and 9 ( 60% ) for cecf .
no statistically significant difference was found between the study groups according to the location of the fracture line against cej ( p > 0.05 ) .
superior outcomes were achieved in terms of the fracture resistance of primary anterior teeth , especially when intracanal composite resin posts were used .
extension of the composite build - up to the cementum was not likely to significantly enhance the longevity of such restorations .
however , the application of composite posts with or without the extension of the restoration to the cementum might improve the fracture resistance of the composite restorations of severely damaged anterior primary incisors .
based on the present findings , it could be argued that in cases of general anesthesia , instead of extraction , gingivectomy with laser or electrocautering can be used to expose more cementum to receive a larger composite restoration that will increase the longevity of primary teeth when used with proper intracanal retention .
the mechanical behavior of endodontically treated teeth is strongly influenced by the interface between the applied restorative material , remained dental structure and rigidity of the restorative material .
this is even more important when restoring the weakened endodontically treated roots . despite the proper fit of the cast posts and cores ,
their application has been limited due to their solely frictional intracanal retention and their relatively high fracture rate as a factor of their stiffness .
therefore , there has been a shift toward application of resin - based posts , which offers a closer modulus of elasticity to the root and an adhesive along with mechanical retention , leading to superior stress distribution under functional loads .
the flared root in ecc patients lacks sufficient coronal and apical dentin to gain proper fitness for the prefabricated posts .
therefore , these roots are commonly filled with a bulk of luting cements that might jeopardize the longevity of the restoration and the tooth . in order to make amends for the discussed shortcomings , glass ionomer cements , composite resins and accessory glass fiber posts
several tests have been used to assess the effect of masticatory load on the longevity of the restored teeth , including shear bond strength , tensile bond strength and fracture resistance .
fracture resistance testing was used in the present study . because the tooth width is an effective factor in the fracture resistance of the tooth , the groups were matched in this regard .
the least fracture resistance in the present study was slightly above 410 n. in the study of rentes et al .
carried out on 3 - 5.5 year - old children , the bite force was measured to be 213.17 43.97 n. the mean load values resulting in fracture ranged from 410 n to 601 n in the present study , which was well above the reported maximum bite force for the primary incisors .
however , the specimens in the present study did not undergo thermocycling , which might yield lower mfr values , and should be addressed in future studies .
composite resin posts were used in the present study owing to their ease of application , no need for laboratory procedures , lower expenses and superior adaptation .
the successful application of intracanal posts in endodontically treated teeth is not always a factor of post length .
studies have reported 100% success rates for the teeth restored with composites and reinforced with intracanal posts .
it is stated that long posts can adversely affect the underlying development of permanent teeth .
therefore , short posts up to the cervical one - third of the roots were applied in the present study .
studied the effect of application of short posts and their overlying restorations on stress distribution .
they concluded that resin - based restorative materials with higher elastic moduli were not as suitable as short post core materials in endodontically treated maxillary deciduous incisors .
however , it is assumed that the rigidity of post and core systems has no effect on the fracture behavior of the damaged endodontically treated teeth with limited ferrule .
given the large part of coronal breakdown in ecc , other measures have been sought to achieve the effects of ferrule preparation .
for the same reason , to increase the ferrule effect , in the present study a 0.5 mm cement extension of preparation was applied in the cecf and cecf + cp groups .
although this extension resulted in higher fracture resistance values compared with the groups where preparation was ended within enamel , the differences were not statistically significant . on the other hand , where composite posts had been used with a preparation extension to the cementum , the fracture resistance values were significantly higher .
authors have argued that the fracture resistance of parapost prefabricated systems applied with composite cement and core materials is not influenced by ferrule preparation .
crown lengthening to develop a superior ferrule effect has been recommended for the proper restoration of endodontically treated teeth through the literature .
there was no consistency between the present findings and those of the in vitro study of meng et al .
they suggested a significantly increased fracture resistance with increased apical ferrule preparation lengths for simulated forced tooth eruption , but not for simulated crown lengthening .
tang et al . suggested that a 1.5 - 2.0 mm high coronal ferrule would improve the fracture resistance of endodontically restored teeth .
however , an apically extended ferrule might decrease the fracture resistance in narrower roots due to the reduced dentin volume and the increased clinical crown / root length ratio .
restoration of the primary anterior teeth has always posed a challenge to the pediatric clinicians due to the insufficiency of tooth structure , lack of proper bonding of the restorative materials to the primary teeth and the patients non - cooperation . because the amount of remained tooth structure to provide enough bond strength is limited in ecc cases , especially in the anterior teeth where the pulp chamber is relatively larger ,
the results of the present study should be generalized to the clinical situation with caution .
the in vitro literature on testing the post - endodontic restorations suffers from lack of methodological standardization due to the significant heterogeneity in test design and parameters . in line with the present study ,
approximately 60% of the studies performed on the in vitro fracture resistance of post - endodontic restorations have used static loading . only around 15% of similar studies
it has been argued that such studies have applied inconsistent numbers of thermo and load cycles and a wide range of cross - head speed of linear loading ; thus , further studies under thermocyclic and mechanical loading are recommended to better simulate the clinical situation .
this is particularly important because some authors have suggested that the fracture resistance of different posts under thermocycled conditions have not shown any statistical differences .
moreover , the influence of water and naocl storage , host - derived matrix metalloproteinases , ph cycling and food - simulating solutions on the degradation of the adhesive interface may project more of a clinically accurate picture .
the current study showed that the cementum - extended composite fillings with intracanal composite posts could be successful in severely damaged incisors . | objective : the aim of this study was to comparatively assess the fracture resistance of the cementum - extended and conventional composite fillings with or without intracanal composite posts in severely damaged deciduous incisors.materials and methods : this in vitro study was performed on 60 extracted deciduous maxillary incisors that were randomly divided into four groups : group 1 : composite filling ( cf ) ; group 2 : composite filling with composite posts ( cf + cp ) ; group 3 : composite filling extended 0.5 mm to cementum ( cecf ) ; group 4 : composite filling extended 0.5 mm to cementum with composite posts ( cecf + cp ) .
the fracture resistance was assessed by exerting a progressively increasing load with a cross - head speed of 0.5 mm / min in a universal testing machine.statistical analysis : data were analyzed by spss-18 using one - way analysis of variance at < 0.05.results:the mean fracture resistance ( mfr ) values of the experimental groups were 410.57 139.44
n , 564.44 92.63 n , 507.5 76.37 n and 601.08 96.04 n. a significant difference was found between the mfr of groups 1 and 2 , groups 1 and 4 and groups 3 and 4 ( p < 0.05).conclusion : a superior outcome was achieved by intracanal composite posts in both conventional and cementum - extended composite fillings . | INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION |
rotational atherectomy ( ra ) was introduced in the interventional arena in 1988 as a dedicated device for calcified lesions . due to the complexity of the technique , the development of alternative methods such as the cutting balloon procedure , and the high restenosis rate of subsequent bare metal stenting in long lesions , its use had later declined . however , with the increasing use of drug - eluting stents ( des ) and the aggressive treatment of longer lesions , the numbers of procedure performed with ra have increased significantly again in recent years . calcified coronary lesions are a well - known risk factors of short and long - term poor outcomes after both bare metal stent ( bms ) , and des implantation. however , ra can facilitate the delivery of stents in severely calcified coronary lesions by modifying plaque anatomy and smoothing inner vascular lumen .
recent reports have suggested that the use of ra in combination with des implantation to treat severely calcified lesions may achieve high procedural success and an acceptable restenosis rate. however , the long - term efficacy and safety of des combined with ra in patients with calcified coronary lesions is limited .
rotational atherectomy ( rotablator , boston scientific , ma ) usually begins with 1.25 , 1.5 , or 1.75 mm burrs with a speed between 180,000 and 200,000 r / min .
the corresponding anticoagulation therapy consists of weight - adjusted unfractionated heparin ( 100 u / kg ) followed by supplemental boluses to maintain the activated clotting time for more than 300 s. all patients received dual antiplatelet therapy with aspirin and clopidogrel ( 300600 mg per loading dose ) before the procedure .
post - procedural antiplatelet therapy consisted of 100 mg / d of aspirin and 75 mg / d of clopidogrel , in accordance with procedure guidelines .
registry and randomized control trials have failed to show ra can decrease rates of restenosis , or target lesion revascularization ( tlr ) .
although the initial procedural success rate was highest in patients treated with ra ( 89% ( ra ) vs. 77% ( excimer laser ) vs. 80% ( balloon angioplasty ) ) , tlr after one year was significantly increased compared to excimer laser and balloon angioplasty .
similar results were found in the randomized comparison of balloon angioplasty versus rotational atherectomy in complex coronary lesions ( cobra ) .
procedural success rate was originally highest with ra ( 85% vs. 78% ; p < 0.05 ) but no differences were found regarding restenosis , tlr , or symptomatic outcome after six months . the routine use of ra for simple type a / b1 lesions is not recommended .
multiple treatment strategies are available for calcified lesions , including plain old balloon angioplasty ( poba ) , cutting balloons , brachytherapy , drug - eluting balloons and des implantation .
neointimal hyperplasia is the primary mechanism for in - stent restenosis ( isr ) and has been shown in animal models to be more effectively treated with ra than poba .
furthermore , the ra causes less baro- trauma to the artery with a greater acute procedural minimum lumen diameter ( mld ) , which may further decrease the incidence of restenosis .
clinical comparisons of ra to several other methods for the treatment of isr have shown mixed results . in the angioplasty versus rotational atherectomy for treatment of diffuse in - stent restenosis trial ( artist ) ,
furthermore , at 6 months , patients treated with poba had less restenosis ( 51% vs. 65% ; p = 0.039 ) and better event - free survival ( 91.3% vs. 79.6% ; p = 0.0052 ) when compared with ra . in the rotational atherectomy versus balloon angioplasty for diffuse in - stent restenosis trial ( roster ) , 200 patients were randomly selected to receive either ra with adjunctive poba , or poba alone . with an average follow - up of 12 months ,
patients treated with ra had lower tlr rates ( 32% vs. 45% ; p = 0.042 ) , lower repeat stent use , and less residual intimal hyperplasia by intravascular ultrasound ( ivus ) .
the major difference between these two trials is that roster was a single center randomized trial performed in the us , whereas the artist trial was a multicenter randomized trial performed in europe . in addition , patients with underexpanded stents were excluded from the roster trial , whereas they were included in the artist trial . low pressure balloon angioplasty ( < 6 atm ) was applied in both trials , which may not adequately treat under deployed stents .
the somewhat controversial data regarding the efficacy of ra in isr has become less important since the introduction of des .
now , the best way to treat isr is to prevent it in the first place . in multiple studies involving the use of des for the treatment of simple and complex coronary lesions ,
tlr rates have consistently been below 10% with reduced major adverse cardiac events ( mace ) when compared to bms. these results are far superior in comparison to the historical controls for ra as a stand - alone procedure , or with adjunctive poba , even in simple coronary lesions .
a primary concern in the treatment of cto is the ability to completely expand the lesion for the optimal deployment of stents , once the lesion has been crossed with a guide wire .
failure to fully expand stents may increase the risk of both stent thrombosis and isr .
furthermore , restenosis rates of cto lesons after poba and stenting remain extremely high. the modification of vessel compliance with ra has been utilized to address these issues .
similar to its use in isr and native vessels , ra has failed to show a benefit over conventional therapies regarding decreasing long - term restenosis rates , or acute complications .
mechanical debulking prior to the stenting of bifurcated lesions has also been proposed in order to prevent less plaque shifting ( snow plowing ) while increasing the preservation of side branches .
although no randomized trials have yet been performed to see if ra decreases the rate of restenosis or side branch occlusion , clinical studies have shown mixed benefits.
beatriz v , et al . performed a study with a series of 164 calcified coronary lesions in 145 consecutive patients who underwent aggressive plaque modification ( pm ) with either ra and/or the cutting balloon ( cb ) technique before des implantation .
cb was used in moderate calcified lesions and ra alone , or followed by cb in severe calcified lesions .
pm was achieved by using cb in 57% and by ra alone , or followed by cb in 43% .
all patients received their des implantations successfully . at follow - ups at 15 11 months , the overall mace rate was 9.6% ( 3.4% cardiac death , 2.3% myocardial infarction , and 3.4% tlr ) .
the only independent predictor of mace was left ventricular ejection fraction ( lvef ) 50% ( odds ratio ( or ) 3.88 ; 95% confidence interval ( ci ) : 1.1513.1 ; p = 0.03 ) .
there were no significant differences in mace and tlr based on the type of pm used .
clavijo , et al . reported a death rate of 6.8% at a 6-month median follow - up without in - hospital deaths using sirolimus - eluting stents with ra .
the average age in the series was 71.5 9.6 years , 44.4% of patients were diabetic and 83.6% had type c lesions .
reported on a series regarding the use of ra followed by des only in calcified coronary lesions .
the incidence of cumulative mace , defined as death , myocardial infarction ( mi ) and target vessel revascularization , was 15.8% at an average follow - up period of 14.7 months .
death occurred in four patients ( 4.2% ) , non q - wave mi occurred in 3 patients ( 3.2% ) , and q wave mi occurred in 2 patients ( 2.1% ) .
their study also reported on another series that used ra followed by cb plaque modification for des implantation in severely calcified lesions that appeared to be more efficacious than the control group , including significantly larger final stent cross - sectional area by measurement of intravascular ultrasound ( 6.80 1.27 mm
vs. 5.38 1.39 mm ; p = 0.048 ) .
several unique concerns are involved with the use of des in severely tortuous and calcified lesions : ( 1 ) the vigorous manipulation of des through calcified lesions can result in the disruption of the polymer coating and decrease its effectiveness in preventing restenosis ; ( 2 ) suboptimal deployment of des in such complex lesions may further increase the risk of stent thrombosis already posed by the delayed endotheliazation in patients with des ; and ( 3 ) local delivery of drugs through a calcified lesion can be greatly impaired .
, there was a 98% to 100% procedural success rate , no major adverse cardiac events and less than 10% tlr at follow - up examinations .
, , when des was compared directly to bms in patients with heavily calcified lesions that required ra , there was a significant difference in the late lumen loss ( 0.11 0.7 mm in the des group and 1.11 0.9 mm in the bms group , p = 0.001 ) at nine month follow - ups .
this difference was manifest in the clinical event rates at late follow - up ( combined endpoint of all cause mortality , mi , and tlr was 7.4% in the des group and 38.2% in the bms group ; p = 0.004 )
. des continued to show more clinical benefit than bms over two years of follow - ups .
a strategy combining the ra technique and des implantation is a safe and effective treatment option for patients with complex lesions .
we believe that ra with des should be considered as an essential technique in certain lesions , especially the calcified lesions visible by fluoroscopy , more than 180 degree of calcification under ivus study , or the ivus catheter can not pass the lesions , and thus , be available in all catheterization laboratories . | rotational atherectomy ( ra ) was introduced in the interventional arena in 1988 as a dedicated device for calcified lesions . due to the complexity of the technique , the development of alternative methods such as the cutting balloon procedure , and the high restenosis rate of subsequent bare metal stenting in long lesions , its use had later declined . however , with the increasing use of drug - eluting stents ( des ) and the aggressive treatment of longer lesions , the number of procedure performed with ra has increased significantly again in recent years . in this article , we reviewed the application of ra in des era . | Introduction
Basics of rotational atherectomy
Rotational atherectomy for uncomplicated lesions
Rotational atherectomy for in-stent restenosis
Rotational atherectomy for chronic total occlusions (CTO) & bifurcation lesions
Rotational atherectomy for heavily calcified & complicated lesions
Rotational atherectomy and the use of drug-eluting stents
Conclusions |
pancreatic resection is technically challenging due to anatomic factors such as the organ 's retroperitoneal location and close proximity to the duodenum and major vasculature . as a result , the application of laparoscopy to pancreatectomy has been slower than with other abdominal procedures .
the complication rates and oncologic outcomes of this method are similar to those of the open method .
i , n addition tlpd has some advantages , such as comparatively less time to return to daily activities , less scar formation and early initiation of adjuvant treatment .
this method requires experience not only in pancreatic surgery but also in the field of laparoscopic surgery .
therefore , the number of centres where this technique is regularly performed is limited . in particular , using robotic surgery at the time of reconstruction increases the possibility that the procedure can be completed entirely by the laparoscopic method .
we aimed to present the difficulties we experienced in the transition to laparoscopic surgery following 100 classic open whipple operations .
between december 2011 and april 2014 , 100 classic open pancreaticoduodenectomies were performed by the same surgeon .
the difficulties encountered in the transition to laparoscopic surgery are classified as preoperative , intraoperative and postoperative problems .
the most important difficulty encountered during this period was the selection of a suitable patient . in determining the eligibility of patients suitable for laparoscopy , the patients who had tumour size below 2.5 cm or who had intraductal papillary mucinous neoplasm ( ipmn ) and benign conditions were given priority .
advanced tumours and tumours with vascular invasion were excluded from the study . before the procedure ,
all patients were evaluated with triphasic computed tomography ( ct ) and endoscopic ultrasound for appropriate staging .
of 5 patients who underwent surgery , 2 had pancreatic head tumour , 2 had cystic neoplasms of the pancreas and 1 had a diagnosis of chronic pancreatitis .
five trocars of 10 - 12 mm were used , and 5-mm nathanson retractor was used for liver retraction , which was placed in the sub - xiphoid position .
subsequently , the superior mesenteric vein were dissected , a tunnel was opened from the posterior of the pancreas and the pancreas was hanged .
the first portion of the duodenum was transected by means of endostapler and the pancreas was transected by means of ultrasonic dissector .
the treitz ligament was dissected and the specimen was taken to the patient 's right .
the uncinate process of the pancreas and portopancreatic junction was dissected and pancreatic tissue was separated from the portal vein and superior mesenteric artery . during this period , we preferred the second trochar position [ figure 2 ] .
finally , cholecystectomy was performed ; the bile duct was cut and the specimen was put into an endobag .
an end - to - side pancreaticojejunostomy and duct - to - mucosa anastomosis was performed .
subsequently , an end - to side hepaticojejunostomy and end - to - side duodenojejunostomy were performed .
finally , the infraumblical trocar site was extended 5 cm and the specimen was removed [ figure 3 ] .
we can prepare gastrocolic ligament dissection , pancreatic head mobilisation , partial kocher manoeuvre and hilus dissection at this position , we prepare extended kocher manoeuvre and uncinate dissection . additionally , in the final step
the surgeon stand on the right side of the patient final step of our operation postoperative early enteral nutrition was started in a manner similar to classic open pancreaticoduodenectomy .
analgesia was provided with a patient - controlled pump for the early postoperative period of 3 days and with oral narcotic analgesics in the following period .
the patients were discharged on postoperative day 7 if they had an uneventful postoperative period .
the most important difficulty encountered during this period was the selection of a suitable patient . in determining the eligibility of patients suitable for laparoscopy , the patients who had tumour size below 2.5 cm or who had intraductal papillary mucinous neoplasm ( ipmn ) and benign conditions were given priority .
advanced tumours and tumours with vascular invasion were excluded from the study . before the procedure ,
all patients were evaluated with triphasic computed tomography ( ct ) and endoscopic ultrasound for appropriate staging .
of 5 patients who underwent surgery , 2 had pancreatic head tumour , 2 had cystic neoplasms of the pancreas and 1 had a diagnosis of chronic pancreatitis .
five trocars of 10 - 12 mm were used , and 5-mm nathanson retractor was used for liver retraction , which was placed in the sub - xiphoid position .
subsequently , the superior mesenteric vein were dissected , a tunnel was opened from the posterior of the pancreas and the pancreas was hanged .
the first portion of the duodenum was transected by means of endostapler and the pancreas was transected by means of ultrasonic dissector .
the treitz ligament was dissected and the specimen was taken to the patient 's right .
the uncinate process of the pancreas and portopancreatic junction was dissected and pancreatic tissue was separated from the portal vein and superior mesenteric artery . during this period , we preferred the second trochar position [ figure 2 ] .
finally , cholecystectomy was performed ; the bile duct was cut and the specimen was put into an endobag .
an end - to - side pancreaticojejunostomy and duct - to - mucosa anastomosis was performed .
subsequently , an end - to side hepaticojejunostomy and end - to - side duodenojejunostomy were performed .
finally , the infraumblical trocar site was extended 5 cm and the specimen was removed [ figure 3 ] .
we can prepare gastrocolic ligament dissection , pancreatic head mobilisation , partial kocher manoeuvre and hilus dissection at this position , we prepare extended kocher manoeuvre and uncinate dissection . additionally , in the final step
analgesia was provided with a patient - controlled pump for the early postoperative period of 3 days and with oral narcotic analgesics in the following period .
the patients were discharged on postoperative day 7 if they had an uneventful postoperative period .
seventeen cases were completed laparoscopically and 5 cases were converted to open surgical procedure . here , we have given some infomation about cases converted to open surgical procedures .
indications of the other 17 cases were head of pancreas cancer , periampullary tumour , ipmn and cancer doubt of chronic pancreatitis . during surgical procedure of the first five patients , even though the operations were started by laparoscopic method , 3 of them at various stages due to bleeding and 2 of them during the reconstruction of the pancreas , which was of bad quality , underwent conversion to open surgery . on the other hand , the next 17 patients had total laparoscopic pancreatectomy .
the data collected from the first 5 patients operations , which were converted to open surgery , were analysed .
conversion to open surgery was done due to bleeding during dissection of the first branch of the portal vein in 1 patient and due to bleeding during dissection of the uncinate process of the pancreas in 2 patients . in 2 patients , dissection
was completed laparoscopically and conversion to open surgery was done due to the soft quality of the pancreas . according to these early results , bleeding seems to be the most frequent cause of conversation to open surgery .
the bleeding of the first branch of the portal vein due to excessive traction was yet another reason for uncontrollable bleeding . at this stage
the most important preventive method is isolating and separating vessels properly and placing the clips if necessary .
the average length of hospital stay of the patients who had tlpd was 10 days ( 6 - 21 ) . estimated blood loss was 340 ml ( 250 - 440 ) .
we do not prefer to perform jejunostomy on the first day of surgery ; enteral nutrition is started by nasojejunal tube and increased daily . on day 3 or day 4
in the last 25 years , the most significant advance in pancreatic head tumour surgery has been the decrease of the mortality rate to below 1% . in parallel to developments in technical equipment , the use of minimally invasive surgery in pancreatic cancer is another significant advance over this same period .
tlpd , which requires high technical skill levels , is one of the most difficult kinds of laparoscopic surgery . nowadays ,
although there are many centres dealing with this issue , a large case series has not been made . in the initial period of tlpd , besides being a highly skilled pancreatic surgeon , it is necessary to have good skills in laparoscopic surgery . in recent years
, studies comparing open and laparoscopic pancreatectomy have begun to be published which have not revealed any superiority of tlpd to the classic open technique . however , having the same survival advantage compared with open surgery through a smaller incision is likely to be an advantage of this technique .
it is a fact that by performing tlpd in early - stage and cystic pancreatic tumours , better cosmetic results can be expected in this group of patients .
moreover , it is known that tlpd can provide an advantage in terms of earlier onset of patients to chemotherapy .
initially the surgeon has to reach a certain level of experience in pancreatic surgery in order to start performing tlpd because the problems of this technique in the start - up period are similar to the early - stage difficulties of open surgery ( more estimated blood loss , more operation time , more fistula , and the mortality rate ) .
thus , patient selection is very important in the initial phase . for initial cases ,
those with early stage tumours or tumour - like lesions such as ipmn should be selected . in these patients , although dissection can be performed in an easier way , the pancreaticojejunostomy may become problematic .
this is because the quality of the pancreas is softer in early - stage tumours .
according to our experience in the older patients who have early - stage tumours and no serious comorbidities , the pancreatic quality is better and this makes comfortable dissection possible . in tlpd , dissection is different from the classic open technique .
optionally in the conventional method , resection is clockwise : first stomach , then the common bile duct and pancreatic resection is performed whether the retro - pancreatic dissection is done in the last part of the dissection phase . in tlpd
optionally , the posterior pancreas around the superior mesenteric artery is dissected and finally common bile duct and gallbladder dissection is done .
one of the biggest advantages of tlpd including artery- first approach is that it allows the surgeon to evaluate the relationship between the tumour and surrounding arterial structures .
thus , determination of resectability of the tumor can be made at early stage of operation .
however , in our series , there was no patient with major vessel invasion including portal vein or mesenteric vascular structures due to meticulous selection of initial cases .
if there is any doubt about invasion of artery during open procedure , retropancreatic approach is reasonable to determine resectability as it is described in textbooks .
another advantage is that gallbladder can be used for traction of the liver upward . in general ,
in addition , 10-mm trocars are important because their use allows the camera to move , helping the surgeon by working in different angles [ figure 2 ] . in the reconstruction period ,
thus , after doing the back wall , the anterior wall can be complete from the incision which is done for removing the specimen .
the most important disadvantages of tlpd are longer operation time and higher cost compared to open procedures .
in conclusion , it can be suggested that tlpd is a safer procedure , while having comparable technique and oncologic outcomes to open surgery . | background : recently , total laparoscopic pancreatectomy has been performed at many centres as an alternative to open surgery . in this study
, we aimed to present the difficulties that we have encountered in converting from classic open pancreaticoduodenectomy to total laparoscopic pancreatectomy.materials and methods : between december 2012 and january 2014 , we had 100 open pancreaticoduodenectomies .
subsequently , we tried to perform total laparoscopic pancreaticoduodenectomy ( tlpd ) in 22 patients . in 17 of these 22 patients
, we carried out the total laparoscopic procedure .
we analysed the difficulties that we encountered converting to tlpd in three parts : preoperative , operative and postoperative .
preoperative difficulties involved patient selection , preparation of operative instruments , and planning the operation .
operative difficulties involved the position of the trocars , dissection , and reconstruction problems .
the postoperative difficulty involved follow - up of the patient.results:according to our experiences , the most important problem is the proper selection of patients .
contrary to our previous thoughts , older patients who were in better condition were comparatively more appropriate candidates than younger patients .
this is because the younger patients have generally soft pancreatic texture , which complicates the reconstruction .
the main operative problems are trocar positions and maintaining the appropriate position of the camera , which requires continuous changes in its angles during the operation .
however , postoperative follow - up is not very different from the classic procedure.conclusion:tlpd is a suitable procedure under appropriate conditions . | INTRODUCTION
MATERIALS AND METHODS
Preoperative period
Intraoperative period
Postoperative period
RESULTS
DISCUSSION
CONCLUSION
Financial Support and Sponsorship
Conflicts of Interest |
surveillance of viral gastroenteritis outbreaks was initiated just over a decade ago at the national institute for public health and the environment in the netherlands ( rivm ) .
outbreaks were typically reported by the municipal health service or the food inspection services to the rivm , and samples were collected in close collaboration with these agencies . to determine the role of nov and
possible differences between different nov strains in gastroenteritis outbreaks , a minimal set of epidemiologic data ( setting , date of onset , number of persons affected , most probable mode of transmission , and number of hospitalizations ) was collected for reported outbreaks .
these data were supplemented with results of molecular biologic detection and typing techniques to enable more in - depth analysis of surveillance data .
preliminary typing of strains was performed by sequencing region a ( 280 nt in the polymerase gene ) of the virus .
a systematic selection of strains was also typed by sequencing region c ( 277 nt in the capsid gene ) ( 1,2 ) . because nov activity is much higher in winter months
, seasons were analyzed from july through june , rather than per calendar year . from december 18 , 1993
to december 26 , 2005 , a total of 1,032 gastroenteritis outbreaks were reported to the rivm .
, 695 ( 74% ) outbreaks met our inclusion criteria for a nov outbreak ( 25% of samples positive by reverse transcription
overall , we observed an increasing trend in the number of reported outbreaks per year ( figure , panel a ) . in the 199596 ,
200102 , 200203 , and 200405 seasons , more nov outbreaks were reported ( 66 , 90 , 154 , and 161 , respectively ) .
ggii virus strains were predominant in all years and caused 577 ( 91% ) of 631 outbreaks with known genotypes , compared with 36 ( 6% ) of 631 outbreaks caused by ggi or ggiv viruses and 18 ( 3% ) of 631 outbreaks caused by mixed infections with viruses of different genotypes ( table ) .
* mw , mainly waterborne ; f , foodborne ; ptp , person - to - person ; u / o , unknown or other ; t , travel associated ; s / d , school or daycare center ; r / c / c , restaurant , canteen , or caterer ; h , hospital ; ph , private house ; ri , residential institution .
ggii.4 strains have been detected since 1995 , with the highest proportions observed in years with high numbers of outbreaks . in the epidemic seasons of 199596 , 200203 , and 200405 the percentages of outbreaks caused by ggii.4 were 82% , 83% , and 89% , respectively , compared with an overall average of 68% ( figure ) . in seasons after these epidemics
, the percentage caused by ggii.4 decreased to 39% in 199697 , 55% in 200304 , and 32% in the first half of 200506 .
multiple nov genotypes co - circulated throughout the years of the study , but in postepidemic years , outbreaks caused by non - ggii.4 strains were more common ( figure , panel c ) .
the high number of outbreaks in 200102 may be partially explained by emergence of a new variant of ggii.4 in the spring of 2002 ( 4 ) , which caused uncharacteristically high numbers of outbreaks between april and june .
the epidemic increases in the number of outbreaks and seasonality of outbreaks were mainly attributable to ggii.4 .
strains with genotypes other than ggii.4 were found at similar levels throughout the year ( data not shown ) .
of ggii.4 outbreaks by the dotted line ( values on left y - axis ) , bars indicate percentage of ggii.4 outbreaks of the total no . ( values on right y - axis ) , and arrows indicate epidemic seasons .
shading of the bar indicates the percentage of ggii.4 , ranging from white ( 0%20% ) , in steps of 20% , to black ( 80%100% ) .
a total of 548 ( 79% ) outbreaks were reported in healthcare settings ( hospitals and residential institutions ) compared with 102 ( 15% ) in nonhealthcare settings ; for 45 ( 6% ) outbreaks no data were available ( table ) .
a total of 407 ( 81% ) of 502 outbreaks with genotyping information in healthcare settings were caused by ggii.4 nov strains ( table ) . in the nonhealthcare settings ,
ggii.4 was significantly less prevalent ( 39 [ 43% ] of 91 , p<0.0001 ) , resulting in a relative risk at least 2.17 higher for acquiring a ggii.4 infection in a healthcare setting than in other settings . a mode of transmission was reported for 272 ( 39% ) outbreaks .
data confirmed that the main transmission route was person to person ( 60% , 163/272 ) ( table ) ( 5,6 ) .
ggii.4 strains were found in 73% of person - to - person outbreaks compared with 44% of food - related outbreaks ( p<0.0001 ) . when outbreaks for which no genotype was known were counted as non - ggii.4 strains , the relative risk of finding ggii.4 in an outbreak caused by person - to - person transmission was 2.3 greater than finding it in a foodborne outbreak or relative to other genotypes .
multiple ggii nov strains were found in 18 outbreaks ( 3 foodborne , 1 waterborne , 4 person - to - person , and 10 with unknown modes of transmission ) .
detailed molecular epidemiologic data from long - term surveillance on nov outbreaks are rare because nov molecular detection techniques became available only in the mid-1990s . in our 12-year surveillance study , we observed large differences in the magnitude of the annual winter peak of nov infection .
although this finding has been observed in studies covering a shorter period ( 3,57 ) , our data suggest an increase in infections with ggii.4 in recent years , particularly in healthcare settings .
however , the number of outbreaks reported in healthcare settings is likely overrepresented in our study because of mandatory reporting of illness in such settings .
an actual increase in the number of outbreaks can not be proven based on passive surveillance data alone , but an increase is strongly suggested by increased prevalence of ggii.4 in recent years and supported by reported shifts in the predominant ggii.4 variant associated with large numbers of outbreaks ( 8) .
the overall dominance of ggii.4 suggests that this genotype is more transmissible than other genotypes in healthcare settings , where close contact of many persons favors person - to - person transmission .
transmission may also be affected by poorer hygiene or greater susceptibility to infection . increased transmissibility could result from increased levels of shedding of ggii.4 or altered stability of virus particles outside the host compared with other genotypes .
alternatively , changes in circulating viruses may lead to differences in host cell binding or immune recognition , thereby changing the dynamics of infection or size of the population at risk .
our group and other researchers have reported the emergence of distinct ggii.4 lineages in 199596 , 2002 , and 2004 ( 2,4,9,10 ) .
this suggests that the changing phenotype of ggii.4 strains results in increased numbers of outbreaks ( 10 ) . a detailed characterization of ggii.4 strains is ongoing to determine the molecular mechanisms involved in observed epidemiologic patterns .
the marked decrease in the percentage of ggii.4 strains during seasons after epidemic seasons caused by variant strains suggests that populations may acquire immunity against these predominant strains .
the value of this surveillance dataset will increase with its continuation , as well as with its expansion as part of a european surveillance network ( http://www.eufoodborneviruses.co.uk/ ) .
future research will be directed at understanding the molecular basis for observed changes in the epidemiology of nov and control of its spread . | from 1994 through 2005 , gastroenteritis outbreaks caused by norovirus generally increased in the netherlands , with 3 epidemic seasons associated with new ggii.4 strains . increased percentages of ggii.4 strains during these epidemics , followed by a sharp decrease in their absolute and relative numbers , suggest development of immunity . | The Study
Conclusions |
huangqi can tonify qi , and yinyanghuo can support yang ; the two are commonly used in chinese clinical medicine in the treatment of qi deficiency , yang insufficiency , and other diseases .
they can also promote osteoblast function , increase calcified bones , and promote dna synthesis of bone marrow cells ; an in vitro experiment has shown that yinyanghuo can promote osteoblast proliferation and differentiation .
huangqi yinyanghuo group ( hyg ) is the traditional chinese medicine which is used in clinical practice widely [ 2 , 3 ] .
over 90% of tumor cells have telomerase activity , while the majority of somatic cells lack or even do not have telomerase activity .
telomerase synthesizes telomeric dna repetitive sequences by reverse transcription with its own rna as the template and adds them to the telomere ends , in order to compensate for the terminal bases lost due to cell division and ensure the stability and integrity of chromosomes , thus providing the basis for eternal life of tumor cells .
htert is its rate - limiting component , expression of htert genes plays a decisive role in telomerase expression , and bcl-2 gene ( i.e. , b - cell lymphoma / leukemia-2 gene ) is a type of protooncogene which is capable of suppressing apoptosis .
htert and bcl-2 protein are both very good targets for cancer therapy . by studying the effects of hyg on htert and bcl-2 protein in osteosarcoma cells ,
this experiment provides a theoretical basis for clinical application of hyg in treatment of osteosarcoma .
agilent 1100 series hplc system ; kq-300 ultrasonic cleaner , nanjing kunshan ultrasonic instruments co. , ltd .
huangqi and yinyanghuo crude drugs ( proportion of the two herbs was 1 : 1 in this compounded prescription ) were purchased from guangzhou kelaimente medicine company , which were identified by the institute of botany of the guangzhou academy of sciences as the dried root of astragalus membranaceus ( fisch ) bge .
mongholicus ( bge . ) hsiao of the family leguminosae and epimedium brevicornum maxim . of the family berberidaceae .
icariin reference substance ( batch number 130526 - 152369 ) was obtained from guangzhou institute of biological products assay ; the water used was double distilled water , all the detection reagents were of hplc grade , and other reagents were all of chemical grade .
human osteosarcoma cell lines ( hos and cck8 ) were purchased from nanjing dingsheng biotechnology co. , ltd . fetal bovine serum ( hyclone ) was purchased from beijing saiaoruite biochemical products co. , ltd .
htert antibody was purchased from santa cruz biotechnology ; bcl-2 antibody , dab chromogenic reagent kit , and immunohistochemical staining kit ( sp kit ) were all purchased from chengdu hengyuan bioengineering co. , ltd .
investigation was performed on three levels of four factors : amount of water addition ( a ) , soaking time ( b ) , extraction time ( c ) , and extraction times ( d ) by orthogonal design table l9(3 ) using icariin as the indicator .
the chromatographic column used was hypersil ods2 ( 4.6 mm 250 mm , 5 m ) , mobile phase was methanol-0.38% phosphoric acid solution ( 48 : 52 ) , flow rate was 1.0 ml / min , detection wavelength was 270 nm , column temperature was 40c , and sample injection volume was 10 l .
icariin reference substance was accurately weighed and added to 70% methanol solution to prepare 0.01 mg / ml icariin reference solution ; after filtration , 2.00 ml of filtrate was precisely drawn , placed in a 50 ml flask , added to 40 ml of 70% methanol solution , ultrasonicated for 10 min , allowed to cool and stand at room temperature , then diluted to the mark by addition of 70% methanol solution , shaken uniformly , and filtered with 0.45 m membrane , and the subsequent filtrate was collected as the test solution .
reference solution was taken and prepared into solutions with concentrations of 0.010 , 0.020 , 0.050 , 0.100 , 0.200 , and 0.500 g / ml , respectively ; 10 l of the solutions was taken , and their peak areas were measured ; standard curves were plotted with the peak area as ordinate ( y ) and the icariin concentration as abscissa ( x ) , and regression equation was calculated to be y = 2428x 0.6658 , r = 0.9999 .
the results showed that the icariin concentration had a good linear relationship with the peak area within the concentration range of 0.01~0.50 g / ml ; see figure 1
. 10 l of reference solution was precisely drawn , sample was injected 5 times in parallel , peak area value of icariin was measured , and rsd value was calculated to be 1.52% , which indicated good precision of the instrument . test solution was prepared according to the test solution preparation method , sample was injected after standing for 0 , 2 , 4 , 8 , and 12 h , respectively , peak area of icariin was measured , and rsd value resulted to be 2.50% , which indicated that the test solution had good stability within 12 h. test solutions were prepared in quintuplicate according to the test solution preparation method , and peak areas were measured ; rsd of content was calculated to be 1.83% , which indicated good reproducibility of the present method .
approximately 0.20 g of samples with known concentration was accurately weighed in quintuplicate , placed in stoppered erlenmeyer flasks , and added precisely with 2 ml of 0.198 mg / ml icariin solution in dilute ethanol , and peak area was measured according to the method under sample assay , content was determined , and average recovery rate resulted to be 100.5% , with rsd value of 1.72% .
crude drugs were extracted according to the orthogonal design table l9(3 ) , extracts were combined , appropriate volume of extract was taken , and icariin content in the extract was determined according to the method under plotting of standard curves ; the results and analysis are shown in tables 2 and 3 .
it can be judged according to the results in table 2 that the size of influence of various factors on icariin extraction was d > a > b > c in descending order ; optimal extraction process was a3b1c2d3 , that is , addition of a 12-fold volume of water to the quangqi and yinyanghuo crude drugs with a weight ratio of 1 : 1 , soaking for 15 min , and three times of extraction with each time lasting 1.0 h ; the residues were then discarded and the decoctions were combined to give the extract . as can be seen from table 3 , factor d has a significant influence on extraction of icariin , while factors a , b , and c have relatively small influences within the investigation range , which presented no statistical significance .
the above screened optimal extraction process was validated , and the validation results are shown in table 4 . according to the results in table 4 , average content of icariin in the extract is 69.07 g , which is higher than the content in any test number , and does not have relatively large deviation as well .
cells were subcultured in an incubator set at 37c , 5% co2 , and saturated humidity ; subculture medium was 10% fbs and 100 /ml double antibody - containing high glucose dmem ( containing phenol red ) .
cells were seeded in 96-well plates at 5 10 cells per well . after culturing for 24 h ,
each well was filled separately with 200 l of hyg - containing media with final concentrations of 0.125 , 0.25 , 0.5 , 1 , 2 , 4 , and 8
mol / l ; meanwhile , a nondrug treatment group was set up ; after incubation it was continued in the incubator for additional 24 h , 48 h , 72 h , 96 h , and 120 h , and each well was filled with 20 l of cck-8 solution and incubated in the incubator for another 4 h. absorbance of each well was measured at 490 nm wavelength using elisa reader ; cell morphology was observed under a microscope and photographed .
cell growth curve was plotted with time as the horizontal axis and absorbance as the vertical axis .
survival fraction ( sf ) was calculated as follows : sf ( % ) = ( od value of experimental group / od value of control group ) 100% .
the experiment was repeated three times in parallel ; finally , time - survival curves were plotted according to the sf mean standard deviation ( x-s ) of three samples .
cells were seeded in 24-well plates at 4 10 cells per well , two experimental groups were set up , and each group contained three parallel samples ; meanwhile , a control group was set up . after culturing for 24 h ,
each well was filled with 1 ml of medium containing optimal concentration of hyg and then cultured for 3 d and 5 d. streptavidin peroxidase ( sp ) method was used .
cells were fixed conventionally and stained , and the ones with appropriate morphological characteristics were selected under a 10x inverted microscope and photographed .
semiquantitative analysis of immunohistochemical images were performed using image j and image pro plus software . statistical processing : statistical processing was performed using spss software .
data were expressed as ( x-s ) , comparison among groups was performed by t - test , and correlation analysis was performed by paired chi - square test ; p < 0.05 was considered statistically significant .
cell survival curve describes the survival status of osteosarcoma cells at different doses and times ; see figure 1 .
as can be seen from figure 2 , the survival fraction of osteosarcoma hos cells decreases markedly with the passage of time , and no significant late stage cell proliferation is present .
correlation analysis showed that the cell survival fraction was negatively correlated with the drug dose ( p < 0.01 ) .
the morphological changes of osteosarcoma , after hyg treatment , and untreated osteosarcoma cells , under light microscopy , form as shown in figure 3 .
immunohistochemical images were analyzed using image pro plus software ; the results showed a gradual increase in gray scale value of each image , which indicated gradual decrease in protein content of each image .
the data showed that the htert and bcl-2 protein expressions in the experimental groups were apparently lower than the control group and that the expression intensity was negatively correlated with time ( figure 4 and table 5 ) .
positive cell counting was performed on immunohistochemical images using image j software ; the higher the count of positive cells , the larger the number of cells expressing htert and bcl-2 protein , which also means wide distribution of htert and bcl-2 protein - expressing cells , and vice versa .
the difference was significant by paired chi - square test ( p < 0.05 ) ( table 6 ) .
the paired chi - square test found that htert and bcl-2 protein expressions have a high degree of consistency and a significant correlation ( p < 0.05 ) .
icariin is one of the main active ingredients in yinyanghuo ; pharmacological studies have shown that icariin has multiple pharmacological activities such as antitumor , immunity enhancement , cardiovascular function improvement , and endocrine regulation .
many tcm preparations with yinyanghuo as the monarch drug [ 6 , 7 ] used icariin as the investigation index ; therefore , the selection of icariin as the investigation index for screening of extraction process in this study is feasible and reasonable .
referring to the literature , the author also tried to select different mobile phases , acetonitrile - water , methanol - acetic acid , methanol - water - glacial acetic acid , and so forth , which resulted in poor separation effects at the position of icariin peak , with interferences from other small peaks ; when methanol-0.38% phosphoric acid solution ( 48 : 52 ) was used , icariin peak separated well from other impurity peaks , without interfering peaks . through the understanding of many behavioral relationships between htert and bcl-2 protein , this experiment aims to understand the effects of hyg on htert and bcl-2 proteins in osteosarcoma ( hos ) cells and possible correlation ; meanwhile , it provides a theoretical basis for clinical application of hyg .
typical osteosarcoma usually occurs in the 1020 age group ; nevertheless , it may occur in any age group , including infancy , childhood , and old age .
men have a higher incidence than women , with a male - to - female ratio of approximately 2 : 1 .
surgical treatment has greatly improved the prognosis of patients , but disability rate remains very high .
telomerase is expressed at a very high level in 90% of cancer cells , while it is rarely expressed in normal cells ; it is a recognized anticancer target .
htert is the rate - limiting enzyme of telomerase , which regulates the activity of telomerase and has a parallel relationship with telomerase activity [ 14 , 15 ] . at present , many scholars have turned their eyes to htert , hoping to achieve a breakthrough for the treatment of cancer .
bcl-2 gene is the focus of everyone 's attention ; there have been many reports that bcl-2 protein can protect cells from apoptosis and increase the expression of htert [ 1719 ] , but there are also some experiments which demonstrated that bcl-2 protein is not correlated with , or even negatively correlated with , htert [ 20 , 21 ] .
the present study found that untreated osteosarcoma cells were mainly in short spindle shape under light microscope and were arranged tightly , with large nuclei , nuclear division was common , and cells were grown in multilayers when they were dense .
after treatment with hyg , cell morphology changed obviously , cells were mainly in polygonal shape and were arranged in a monolayer , cell size tended to be uniform , and nuclei were small and were deeply stained ; these results indicated the occurrence of morphological differentiation and changes .
osteosarcoma cell apoptosis was significantly increased with the increase of drug concentration ; cell apoptosis was also significantly increased with the extension of drug action time . under the effect of optimal concentration , with time as the axis ,
htert and bcl-2 protein expressions did not change significantly in the control group ; in the experimental group 3 d and experimental group 5 d , htert and bcl-2 protein expressions gradually weakened and were in a negative correlation with the time of drug action , which were consistent with relevant reports [ 22 , 23 ] ; the two were in a time - dependent relationship with the drug and showed a high degree of consistency with respect to expression .
the study results showed the presence of time dependence between hyg and htert but could not confirm that hyg was able to directly influence telomerase activity ; there existed high degree of consistency between bcl-2 protein and htert , but whether there exists mutual assistance between the two needs further research ; due to the discovery of telomerase activity in part of normal tissues , whether hyg has toxic side effects on them also needs further study ; there is also the possibility of false positive and false negative results in nonfull quantitative determination of htert and bcl-2 gene protein expressions ; tumor cells were in a low ph state , but the tumor cells in the present experiment were in a state where ph is 7.27.4 , which can not fully reflect the low ph state of malignant tumor tissues in vivo . | to screen the optimal extraction process and content determination of active component of huangqi yinyanghuo group ( hyg ) and to study the effects of hyg on human telomerase reverse transcriptase ( htert ) and bcl-2 protein in osteosarcoma ( hos ) cells , providing the theoretical basis for clinical application of hyg in treatment of osteosarcoma , orthogonal design table l9(43 ) was used to design the extraction process of hyg , and icariin was taken as the investigation index to optimize the extraction process of hyg .
0.125 , 0.25 , 0.5 , 1 , 2 , 4 , and 8 mol / l hyg were taken to act separately on logarithmic growth phase osteosarcoma hos cells , cck-8 assay was used to determine cell viability , and immunohistochemical sp assay was used to determine the expression of htert and bcl-2 protein .
apoptosis rate was positively correlated with the dose of hyg , and the expressions of htert and bcl-2 protein were significantly decreased with the prolonged duration of action . under the effect of hyg , dose was negatively correlated with osteosarcoma cell survival fraction ; osteosarcoma cell survival fraction was positively correlated with htert and bcl-2 protein ; duration of action was negatively correlated with htert and bcl-2 protein ; and htert and bcl-2 protein were in a synchronous relationship . | 1. Introduction
2. Instruments and Drugs
3. Methods
4. Determination of Icariin Content
5. Results
6. Discussion |
in 2000 , the national research council ( nrc ) , an arm of the national academy of sciences , released a report entitled , " toxicological effects of methylmercury " that nrc committee reviewed the existing literature on the health effects associated with exposure to methylmercury ( mehg ) , including relatively low - level exposure from current levels of fish consumption , and assessed the dose - response relationship based on available epidemiological studies .
the overall conclusion of that report was that , at levels of exposure in some fish- and marine mammal - consuming communities ( including those in the faroe islands and new zealand ) , subtle but significant adverse effects on neuropsychological development were occurring as a result of in utero exposure . based on those findings
, the committee also concluded that " ... the risk to that population [ the women who consume large amounts of fish and seafood during pregnancy ] is likely to be sufficient to result in an increase in the number of children who have to struggle to keep up in school " . since the release of that report
, there has been continuing discussion of the public health relevance of current levels of exposure to mehg .
much of this discussion has been linked to the release of the most recent longitudinal update of the seychelles island study by gary myers et al . , which reports no significant adverse neurological effects of in utero mehg exposure at 9 years of age .
it has been posited by the authors of that study in a letter to the lancet , as well as in a commentary and subsequent letter by constantine g. lyketsos published in the lancet , that these findings supercede those of the nrc committee , and that based on the seychelles findings , there is little or no risk of adverse neurodevelopmental effects at current levels of exposure . as members of the nrc committee that wrote the 2000 report
we , therefore , wish to address some of the key points raised by drs .
in his commentary , lyketsos discounts the nrc report on the grounds that the committee did not have access to the seychelles 9-year follow - up data . however , at the time of its deliberation , the committee knew that this follow - up assessment was in progress and recognized that the consistent negative effects from the earlier stages of the seychelles study made it highly unlikely that adverse effects would emerge in the 9-year follow - up .
the committee 's conclusions , therefore , were based on an the consideration that the consistent adverse effects reported in the faroe islands and new zealand studies would not be confirmed in this seychelles cohort at any point in development .
thus , in our opinion the continued absence of negative effects in the most recent seychelles data does not alter the overall interpretation of the data or the conclusions of the report .
in addition to assertions about how the new data from the seychelles should alter the current view of the public health risk from methylmercury exposure , lyketsos takes the position in that " the seychelles study is methodologically the most advanced " study conducted to date .
the basis for this assertion is unclear . from a research design perspective , the seychelles and faroes studies were very similar , and both were state - of - the - art .
both studies used continuous measures of exposure based on reliable biomarkers , statistical control for a broad range of potential confounders , and measurement of standard , well - respected measures of neuropsychological function .
although the 9-year seychelles follow - up assessed a larger number of developmental end points , adverse effects were seen in the faroes and new zealand studies in multiple domains of cognitive and neuromotor function .
the nrc report noted that there is a strong scientific consensus that blood lead concentrations in excess of 10 g / dl place a child at increased risk for poor developmental outcomes .
nevertheless , not all lead studies have found this association , and substantial variability exists in the magnitudes of the reported effects .
if two studies from this literature were chosen randomly , it is likely that the results would not be entirely concordant .
a similar consensus is emerging regarding the effects of low - level pcb exposure on developmental outcomes despite some studies which failed to detect negative effects .
the uncertainties inherent in conducting human studies , which , for ethical reasons , must rely on statistical rather than experimental control for confounders , stem , in part , from unmeasured confounders and effect modifiers that may be idiosyncratic to the sample being studied , but can interfere with our ability to detect true effects and to replicate those found in other studies .
thus , the failure to detect adverse effects in the seychelles study could well be due to the substantial sample - to - sample variation expected when trying to identify relatively subtle effects on development in an inherently " noisy " system of complex , multi - determined neurobehavioral end points .
the nrc report also emphasized that study - to - study comparisons are best made on the basis of the estimated value of parameters of interest , not simply on whether the studies yield " p < .05 " .
in fact , the nrc analysis noted that comparing the studies with respect to their estimated benchmark doses and associated confidence limits noted much less discrepancy between them .
myers et al . , the authors of the seychelles studies , argue in their letter to the lancet that hair mercury concentration is the only methylmercury exposure marker which has been correlated " against actual brain concentrations " and that cord blood mercury has not .
it should be noted that the correlation that myers refers to is between mercury in maternal hair and infant brains .
the same study also examined the correlation between mercury in infant blood and infant brains , and both sets of correlations were in the same range ( 0.60.8 vs. 0.40.8 , respectively ) . since cord blood is the gestational surrogate of infant blood , the study cited by myers et al .
supports the conclusions reached in the nrc report ; namely , that the use of either cord blood or maternal hair mercury is adequate for estimating the exposure dose . since these metrics
each provide information about different periods of development , use of both metrics will increase the likelihood of uncovering a true dose - response relationship .
both measures were , in fact , employed in the faroes study and gave strikingly similar results although the cord blood measures generally yielded slightly stronger associations ( in terms of p - values ) .
appear to have misconstrued , is that exposure misclassification ( i.e. , errors in matching the exposure - based biomarker of dose to the observed effect ) generally makes it less likely to observe a true relationship and is highly unlikely to result in a spurious relationship .
both cord blood and maternal hair ( and , in fact , all exposure measures ) result in some degree of exposure misclassification .
thus , any exposure metric ( including cord blood ) which yields statistically valid relationships across a range of developmental endpoints provides useful information about the relationship between dose and response . another point which continues to be raised in the discussion of the applicability of the faroes data to exposures in other communities is the notion of " bolus doses " .
it is important to point out that this notion is hypothetical and is supported by few data . because the largest source of methylmercury exposure in the faroese is consumption of whale meat that is relatively high in methylmercury concentration
, it has been suggested that whale meat dinners might lead to isolated large spikes in methylmercury exposure during pregnancy .
grandjean has pointed out however , that in addition to whale dinners , stored ( frozen and dried ) portions of whale meat are also consumed in small amounts as snacks over extended periods of time .
dietary assessments in both the faroes and seychelles studies were limited and the extent of " bolus " doses can not be readily determined in either study . however , grandjean et al .
report that the mercury concentration in the proximal 2 cm of pregnancy - period maternal hair in the faroes study correlated with the concentration in full - duration pregnancy period hair with a coefficient of 0.93 .
this is comparable to the correlation of 0.850.91 seen in a similar analysis in the seychelles using hair segments of about 3 cm and suggests that the influence of " bolus dose , " if any , is comparable in the two studies .
further report that the individual children whose mother 's hair showed the greatest variability in mercury concentration between segment and full length did not influence the outcome of the dose - response assessment , lyketsos appears to misunderstand the intent of such comparisons .
these comparisons do not speak to the effect of variability in exposure per se on developmental outcomes , but speaks directly to the notion of bolus dose .
the larger the bolus dose , the greater the variability that is expected between the mercury concentration in the segment and the full - length hair sample , which reflects average exposure .
thus , " bolus doses " in the faroes cohort do not seem to be responsible for the observed effects of methylmercury on development .
finally , issues have been raised as to the power of the various studies to detect an effect in developmental outcomes .
lyketsos claims that the seychelles study had power of 90% to detect neurodevelopmental effects of mercury toxicity , challenging the nrc report , which estimated that the seychelles study had power of only 50% to detect several of the effects seen in the faroes study .
given that the faroe islands and seychelles studies had similar ranges of exposure , power considerations are driven primarily by sample size , so it stands to reason that the seychelles study ( just under 800 mother / infant pairs ) will have less power than the faroe study ( over 1000 pairs ) . while it may well be true that the seychelles study was designed with 90% power to detect a particular effect , it is also true that the study had only around 50% power to detect five of the eight effects seen in the faroes ( see table 7 - 1 and figure 7 - 2 of the nrc report ) .
in his letter , lyketsos acknowledges that " it is beyond doubt " that mercury is neurotoxic and that there may be a need to warn pregnant women against eating certain seafood .
the reference dose we recommended in the nrc report is derived from a benchmark dose of 58 g / l mehg in blood .
this corresponds to the exposure level that doubles the risk of adverse neurological development from 5% to 10% in the faroes cohort .
this is an important potential public health impact , which is preventable . in the interest of protecting public health ,
we believe it is better to err on the side of caution in the face of three well - designed studies , two of which are positive and one of which is negative .
no evidence that has emerged since the publication of the nrc report changes our view on this issue . once reasonable evidence of adverse effects has been provided , the issue is not whether methylmercury exposure from fish can pose a risk , but rather the dose ( including an appropriate margin of safety ) that is appropriate to provide prudent protection for the most vulnerable individuals in the population . | in 2000 , the national research council ( nrc ) , an arm of the national academy of sciences , released a report entitled , " toxicological effects of methylmercury . "
the overall conclusion of that report was that , at levels of exposure in some fish- and marine mammal - consuming communities ( including those in the faroe islands and new zealand ) , subtle but significant adverse effects on neuropsychological development were occurring as a result of in utero exposure . since the release of that report
, there has been continuing discussion of the public health relevance of current levels of exposure to methylmercury .
much of this discussion has been linked to the release of the most recent longitudinal update of the seychelles island study .
it has recently been posited that these findings supercede those of the nrc committee , and that based on the seychelles findings , there is little or no risk of adverse neurodevelopmental effects at current levels of exposure . in this commentary ,
members of the nrc committee address the conclusions from the nrc report in light of the recent seychelles data .
we conclude that no evidence has emerged since the publication of the nrc report that alters the findings of that report . | Introduction
Discussion
Conclusions
List of abbreviations
Competing interests
Authors' contributions
Pre-publication history |
clinical symptoms in diseased calves : case 1 : in may 2010 , a holstein calf
( female , 84 days old ) showed anorexia , pyrexia and diarrhea in a depositary raising farm in
yamaguchi prefecture . despite treatment with antibiotics ( kanamycin and tylosin ) ,
sulfamonomethoxine and antipyretic , the calf died 20 days after the onset of diarrhea . case
2 : in july 2012 , a japanese black calf ( female , 246 days old ) on another farm in yamaguchi
prefecture repeated diarrhea despite treatment with antibiotics ( kanamycin ) and a probiotic
product after the birth , and then , feces were collected during the acute phase for
diagnosis .
isolation of microorganisms : tissue specimens obtained from major organs
including cerebrum , jejunum , ileum , cecum , colon and ideal contents of the dead calf ( case
1 ) and feces of the second calf ( case 2 ) were minced and homogenized in serum - free eagle s
minimum essential medium with kanamycin ( mem ) ( nissui pharmaceutical co. , tokyo , japan ) .
after centrifugation of the 10% homogenate , the supernatant was filtered through a 450 nm
membrane ( merck millipore ltd . , carrigtwohill , ireland ) , and 0.15 ml of the
supernatants were inoculated onto hamster lung ( hmlu-1 ) cells , mardin - darby bovine kidney
( mdbk ) cells , bovine testicular ( bt ) cells , human rectal adenocarcinoma ( hrt-18 ) cells and
vero cells in 24-well plates .
after adsorption for 60 min at 37c , the cells were washed
with mem , and then , 0.5 ml of mem containing 0.1% bovine serum albumin
( bovogen biologicals , williams avenue , australia ) was added to each well .
the cells were
incubated at 37c , and cytopathic effects ( cpe ) were observed .
after incubation for 10 days ,
cells were frozen and thawed once and then centrifuged .
subsequent passages were carried out
at least twice in the same manner with 0.15 ml of the supernatant .
dhl agar , columbia agar with 5% sheep blood and gam agar
with 5% yolk and 0.1% cysteine were used for bacterial isolation .
the gene of
clostridium perfringens toxin isolated from small - intestinal contents was
identified by pcr .
identification of isolates by pcr , sequence and phylogenetic analysis of c. pecorum
omp1 gene : c. pecorum - infected cells and the supernatants from
organs described above were used for dna extraction .
total dna was extracted using dneasy
blood & tissue kit ( qiagen , hiden , germany ) .
pcr was carried out with takara ex taq hot
start version ( takara bio inc .
primer pairs targeting fragments of
genus - specific and species - specific chlamydia omp1 genes were used for pcr
as described by kaltenbck et al . .
pcr products were electrophoresed on 2.0% agarose gel and visualized using
ethidium bromide staining .
pcr products were purified using minielute pcr purification kit
( qiagen , germantown , md , u.s.a . ) and directly sequenced using a bigdye terminator cycle
sequencing kit v3.1 ( applied biosystems , austin , tx , u.s.a . ) .
phylogenetic trees were
constructed using neighbor - joining methods , and
the reliability of the branch was evaluated by bootstrapping with 1,000 replicates .
antigenic analysis of c. pecorum isolates : the antigenicity of isolates
were compared by immunoblot analysis using rabbit antisera to bo / yokohama , bo / maeda and ov / ipa
strains of c. pecorum .
antisera to bo / maeda and ov / ipa strains were
prepared as described by fukushi and hirai .
hmlu-1
cells were infected with c. pecorum 2258 , bo / yokohama , bo / maeda and ov / ipa
strains and c. psittaci cal10
strain and then incubated at 37c in 5% co2 until cpe was observed .
the cells
were recovered from dishes with 0.02% edta in phosphate - buffered saline ( pbs ) .
after
centrifugation at 200 g for 5 min at 4c , the supernatant was removed ,
and the cells were resuspended in pbs .
cells were then mixed with an equal volume of 2
concentrated sample buffer consisting of 6.25 mm tris - hcl ph 6.8 , 2% sodium dodecyl sulfate
( sds ) , 20% glycerol and 0.001% bromophenol blue .
samples were boiled for 3 min , placed on
ice for 3 min and centrifuged at 13,000 g for 3
min at room temperature .
cell lysates were electrophoresed using 12% sds - page
and transferred to a pvdf membrane ( immobilon - p ; millipore , billerica , ma , u.s.a . ) .
after
blocking with tris - buffered saline ( tbs ) containing 3% gelatin ( eia grade reagent gelatin ;
bio - rad , ca , u.s.a . ) for 45 min at 37c , the membrane was washed three times with tbs
containing 0.05% tween 20 ( t - tbs ) .
after incubation with diluted rabbit antisera for 45 min
at 37c , the membrane was washed 3 times with t - tbs . then , the membrane was reacted with
peroxidase - conjugated purified recombinant protein a / g ( thermo fischer scientific , rockford ,
il , u.s.a . ) for 45 min at 37c .
after washing the membrane with t - tbs and tbs three times
each , specific bands were visualized using 3,3-diaminobenzidine tetrahydrochloride ( dab ;
wako pure chemical industries , ltd . , osaka , japan ) .
each dilution was mixed with an
equal volume of 200 tcid50/0.05 ml of c.
pecorum strains and incubated at 37c for 1
then , 0.1 ml
( approximately 6 10 cells ) of hmlu-1 cells in mem containing 10% fetal bovine
serum was added to each well .
after incubation at 37c for 7 days , antibody titers were
expressed as the reciprocal of the highest dilution of serum which inhibited cpe
completely .
histological and immunohistochemical analyses : tissue specimens obtained
from major organs , intestines and central nervous system of the dead calf ( case 1 ) were
fixed in 10% neutral buffered formalin and embedded in paraffin wax .
paraffin - embedded
tissues were cut at 2 m and stained with hematoxylin - eosin .
after deparaffinization and rehydration of the paraffin
sections , endogenous peroxidase activity was blocked by incubation in 3%
h2o2 for 10 min .
sections were incubated with 10% normal goat serum
for 30 min and then with a rabbit anti - c .
sections were
rinsed three times in pbs containing 0.2% tween 20 for 10 min and then incubated with
histofine simple stain max - po ( r ) ( nichirei biosciences , inc . , tokyo , japan ) for 30 min .
the
sections were rinsed three times in pbs containing 0.2% tween 20 for 10 min .
the sections
were incubated with 3,3-diaminobenzidine tetrahydrochloride under the microscope to control
color development , washed in tap water for 10 min and rinsed with distilled water .
isolation of microorganisms and pcr for virus detection : characteristic
cpe appeared in hmlu-1 cells inoculated with jejunum homogenate from case 1 ( 2258 strain )
at the first passage ( fig .
1.characteristic cpe observed in hmlu-1 cells inoculated with homogenized jejunum
supernatant from case 1 . ) .
cpe was observed seven days after inoculation . on the other hand , at the second
passage , cpe similar to that of 2258 strain appeared in hmlu-1 cells inoculated with calf
feces from case 2 ( 24100 strain ) .
gimenez staining of hmlu-1 cells infected with these
strains revealed many small purplish - red particles . c.
in addition , c. pecorum - specific
genes were detected from jejunum , ileum , cecum and colon of case 1 .
clostridium
perfringens was isolated from jejunal and ileal contents of case 1 at
approximately 10 cfu / g .
the gene of -toxins was shown in these isolates by pcr ,
and isolates were identified as type a ( data not shown ) .
characteristic cpe observed in hmlu-1 cells inoculated with homogenized jejunum
supernatant from case 1 .
no other viruses were isolated from specimens , and pcr was also negative for all specimens
from cases 1 and 2 when tested for bovine viral diarrhea virus , bovine coronavirus ,
rotavirus [ 3 , 10 , 25 ] , bovine torovirus , bovine enterovirus , paramyxoviruses , bovine
adenovirus and bovine herpesvirus 4 .
identification of isolates by pcr , sequence and phylogenetic analysis : two
isolates ( 2258 and 24100 ) were pcr positive when genus - specific and species - specific
omp1 genes of c. pecorum were targeted .
nucleotide
sequences of genus - specific pcr products were determined and compared with those of
published c. pecorum omp1 genes .
omp1 gene partial
sequence of the 2 isolates was 100% identical and showed the highest identity ( 99.8% ) to the
bo / yokohama strain isolated from cattle with enteritis in japan .
nucleotide similarities of
the omp1 gene between the isolates and other published c.
pecorum isolates were 89.2% , 85.2% and 82.0% for bo / maeda strain which was
isolated from cattle with pneumonia in japan , ov / ipa strain which was isolated from sheep
with polyarthritis in united states , and 66p130 strain which was isolated from cattle with
enteritis in united states , respectively ( fig .
2.phylogenetic tree of c. pecorum strains based on the nucleotide
sequence of the omp1 gene ( 534 nucleotides ) .
genbank accession number are 22 - 58
( lc021417 ) , 24 - 100 ( lc021418 ) , bo / yokohama ( lc021422 ) , bo / maeda ( lc021419 ) , ov / ipa
( lc021421 ) , l71 ( af269280 ) , lw613 ( aj440240 ) , sbe ( eu684916 ) , ab10 ( eu684917 ) , m14
( eu684920 ) , 824 ( eu684922 ) , be53 ( eu684923 ) , ib1 ( eu684924 ) , ib2 ( eu684925 ) , ib3
( eu684926 ) , ib4 ( eu684927 ) , ib5 ( eu684928 ) , w73 ( eu684929 ) , r69 ( eu684930 ) , ic3
( eu684932 ) , ic4 ( eu684933 ) , 1710s ( gq228167 ) , 1920brz ( gq228168 ) , 66p130 ( gq228180 ) ,
1708 ( gq228194 ) and mc marsbar ugt ( hq457473 ) .
the nucleotide sequence of c.
psittaci cpx0308 strain ( ab284064 ) was used as an outgroup to root the
tree .
the percentage bootstrap values calculated from 1,000 replications are indicated
above the internal nodes . ,
table 1table 1.nucleotide sequence identities ( % ) of omp1 genes among
c. pecorum strainsstrain225824100bo / yokohamabo / maedaov / ipa66p1302258 - 10099.889.285.282.024100 - 99.889.285.282.0bo / yokohama-89.085.081.8bo / maeda-84.283.0ov / ipa-80.266p130- ) .
phylogenetic tree of c. pecorum strains based on the nucleotide
sequence of the omp1 gene ( 534 nucleotides ) .
genbank accession number are 22 - 58
( lc021417 ) , 24 - 100 ( lc021418 ) , bo / yokohama ( lc021422 ) , bo / maeda ( lc021419 ) , ov / ipa
( lc021421 ) , l71 ( af269280 ) , lw613 ( aj440240 ) , sbe ( eu684916 ) , ab10 ( eu684917 ) , m14
( eu684920 ) , 824 ( eu684922 ) , be53 ( eu684923 ) , ib1 ( eu684924 ) , ib2 ( eu684925 ) , ib3
( eu684926 ) , ib4 ( eu684927 ) , ib5 ( eu684928 ) , w73 ( eu684929 ) , r69 ( eu684930 ) , ic3
( eu684932 ) , ic4 ( eu684933 ) , 1710s ( gq228167 ) , 1920brz ( gq228168 ) , 66p130 ( gq228180 ) ,
1708 ( gq228194 ) and mc marsbar ugt ( hq457473 ) .
the nucleotide sequence of c.
psittaci cpx0308 strain ( ab284064 ) was used as an outgroup to root the
tree .
the percentage bootstrap values calculated from 1,000 replications are indicated
above the internal nodes .
antigenic characterization of c. pecorum isolates : the 38 to 40 kda major
outer membrane protein of 2258 strain strongly
reacted with rabbit antiserum raised against the bo / yokohama strain . on the other hand ,
no
specific reaction was detected by immunoblot analysis with rabbit antisera to bo / maeda or
ov / ipa strains ( fig . 3fig .
antiserum against bo / yokohama ( a ) , bo / maeda ( b ) and ov / ipa ( c )
strains was used for each figure .
lane 1 : mock - infected hmlu-1 cells ; lane 2 , 2258
strain - infected hmlu-1 cells ; lane 3 , bo / maeda strain - infected hmlu-1 cells ; lane 4 ,
bo / yokohama strain - infected hmlu-1 cells ; lane 5 , ov / ipa strain - infected hmlu-1 cells ;
lane 6 , c. psittaci cal10 strain - infected hmlu-1 cells . arrows
indicate a specific band of major outer membrane protein ( 3840 kda ) . ) .
antigenicity between isolates ( 2258 and 24100 ) and the 3 strains ( bo / yokohama ,
bo / maeda and ov / ipa ) was compared using cross - neutralization tests with rabbit antisera
.
isolates 2258 and 24100 were antigenically similar to the bo / yokohama strain , but were a
little different from the bo / maeda and ov / ipa strains ( table 2table 2.cross-reactivity among c. pecorum strains determined by
neutralization teststrainantibody titer to antisera againstbo / yokohamabo / maedaov / ipa22588<2<2241004<2<2bo / yokohama164<2bo / maeda48<2ov / ipa<2<24a ) the reciprocal of the highest dilution of serum which inhibited cpe
completely . ) .
antiserum against bo / yokohama ( a ) , bo / maeda ( b ) and ov / ipa ( c )
strains was used for each figure .
lane 1 : mock - infected hmlu-1 cells ; lane 2 , 2258
strain - infected hmlu-1 cells ; lane 3 , bo / maeda strain - infected hmlu-1 cells ; lane 4 ,
bo / yokohama strain - infected hmlu-1 cells ; lane 5 , ov / ipa strain - infected hmlu-1 cells ;
lane 6 , c. psittaci cal10 strain - infected hmlu-1 cells .
arrows
indicate a specific band of major outer membrane protein ( 3840 kda ) .
a ) the reciprocal of the highest dilution of serum which inhibited cpe
completely .
neutralization test for paired sera of case 2 : a significant increase in
antibody against 24100 strain was confirmed by neutralizing tests using paired sera .
neutralizing titers in sera collected during the acute phase of infection were less than
1:2 , but increased to 1:4 during the convalescent phase .
histopathology and immunohistochemical staining for c. pecorum antigens in tissues
from case 1 : systemic hyperemia and congestion together with necrosis of villi
with short or long rod - shaped bacteria in the upper part of the small intestine were
observed in tissue sections .
hemorrhagic ileitis with edematous and granulomatous changes
was observed in the intestinal crypts of the ileum .
positive reactions were observed in the jejunal villi by immunohistochemical
staining with rabbit anti - c .
in this study , c. pecorum was isolated from specimens obtained from 2
calves with diarrhea . in case 1 ,
the bacterium was isolated from jejunum , and c.
pecorum specific omp1 genes were detected from several locations
in the intestines . additionally , c. pecorum antigens were observed in the
jejunal villi by immunohistochemical staining .
therefore , we
speculated that c. pecorum might exacerbate the disease caused by
clostridium perfringens . in these 2 cases , it is possible that c. pecorum was either the primary
pathogen or an exacerbating factor causing diarrhea . on the other hand
recently , poudel et al . reported that asymptomatic endemic c. pecorum infections
reduce growth rates in calves by up to 48% .
they considered that the mechanism of growth
suppression by subclinical chlamydial infection was malabsorption of nutrients due to a
local inflammatory response to intestinal mucosal infection .
additionally , despite the
absence of clinical signs , chlamydial infection was associated with reduced serum iron
concentrations and lower hematocrit values , and infected calves were leukopenic .
mohamad and rodolakis reported that the persistence of c. pecorum strains
in the intestines and vaginal mucus of ruminants could cause long - term sub - clinical
infection which may affect the animal s health .
this may explain the poor weight gain
observed in case 2 after recovery from diarrhea .
although the standard method for detecting antibodies to chlamydiaceae
spp . in animals is still the complement fixation test , our study showed that a neutralization test was also a useful method for
diagnosis of c. pecorum infections .
in addition , in the dead calf , it was
confirmed that immunohistochemistry for c. pecorum antigens was also
useful .
genetic and antigenic analysis showed that 2258 and 24100 strains were more closely
related to bo / yokohama strain isolated from cattle with enteritis than to bo / maeda strain
isolated from cattle with pneumonia and ov / ipa strain isolated from sheep with
polyarthritis .
bo / yokohama - like c. pecorum strains might cause enteritis
more than other serotypes .
however , the 2 isolates showed higher sequence identities to
bo / maeda and ov / ipa strains than to 66p130 strain isolated from cattle with enteritis in
united states .
thus , sequence identities of c. pecorum omp1 gene might vary
according to their geographical background .
the isolation of similar strains from different locations in separate years suggests that
c. pecorum might be spreading among the cattle population in yamaguchi
prefecture .
an epidemiological study of c. pecorum is being conducted
currently to clarify the seroprevalence and relationship with disease . in conclusion
, this study showed that c. pecorum isolates similar to
bo / yokohama might be endemic in yamaguchi prefecture and cause enteric diseases in
cattle . | chlamydia pecorum ( designated 2258 ) was isolated in 2010 in
hmlu-1 cells from the jejunum of a calf which died of necrotizing enterocolitis in
yamaguchi prefecture , japan .
immunohistochemical staining identified c.
pecorum positive reactions in the jejunal villi .
c. pecorum ,
designated 24100 , was isolated from the feces of a calf with diarrhea in another farm in
yamaguchi prefecture in 2012 . a significant increase in neutralizing antibody titers
against c. pecorum
was confirmed in paired sera .
nucleotide sequence
identities of omp1 genes of the 2 isolates were 100% .
the isolates were
genetically and antigenically more closely related to c. pecorum
bo / yokohama strain isolated from cattle with enteritis in japan than to the other
prototype strains , bo / maeda isolated from cattle with pneumonia and ov / ipa isolated from
sheep with polyarthritis .
these results indicate that c. pecorum strains
similar to 2258 and 24100 might be endemic in yamaguchi prefecture and cause enteric
disease in cattle . | MATERIALS AND METHODS
RESULTS
DISCUSSION |
external - beam radiation therapy ( ebrt ) with intracavitary high - dose - rate ( hdr ) brachytherapy is the standard treatment modality for advanced cervical cancer .
although long - term survival rates in patients with cervical cancer treated with definitive radiotherapy ( rt ) have improved considerably in recent years , the incidence of late gastrointestinal complications has also increased .
the rectum is a major organ at risk during treatment planning because of its close proximity to the pelvic organs and relative immobility ; radiation proctitis has been reported in 5% to 11% of patients treated for gynecological cancer .
sigmoid complications such as obstructive sigmoiditis and perforation are rarely reported , although some previous studies have estimated they occur in 0.6 - 6.4% of cases .
herein we report two uncommon cases of radiation sigmoiditis mimicking sigmoid colon cancer after ebrt with intracavitary hdr brachytherapy for uterine cervical cancer with dosimetric consideration .
we also discuss the importance of radiation dose to the sigmoid colon in three - dimensional image - based brachytherapy .
a 79-year - old woman received definitive rt for international federation of gynecology and obstetrics ( figo ) stage iiib cervical cancer .
ebrt was performed at a total dose of 50.4 gy in 1.8 gy fractions for five days each week using a 15-mv photon .
intracavitary hdr brachytherapy ( microselectron - hdr ) of 24 gy in six fractions was conducted twice each week using a tandem and colpostat . following the completion of rt , the cervical cancer completely disappeared .
after one year , the patient returned to the hospital complaining of intermittent rectal bleeding .
a barium study revealed a 3-cm - long severe narrowing of the sigmoid wall , which was suspicious for sigmoid colon cancer ( fig .
sigmoidoscopy revealed luminal narrowing with a friable hyperemic nodular mucosal change 15 cm from the anal verge ( fig .
a 58-year - old woman received definitive rt with concurrent chemotherapy for figo stage iiia cervical cancer .
ebrt was performed at a total dose of 50.4 gy in 1.8 gy fractions for five days each week using a 15-mv photon .
intracavitary hdr brachytherapy ( microselectron - hdr ) of 24 gy in six fractions was conducted twice each week using a tandem and colpostat .
six cycles of weekly cisplatin ( 30 mg / m ) were given during rt .
after five years , the patient visited our emergency department with complaints of poor oral intake , lower abdominal pain and no stool passage over ten days .
sigmoidoscopy revealed an approximately 5-cm - long section of diffuse luminal narrowing in the sigmoid colon and hyperemic mucosal nodularities with mucosal edema ( fig .
a computed tomography ( ct ) scan also revealed thickening of the wall of the rectosigmoid colon probably caused by radiation or inflammation ; however no evidence of any cancer recurrence was found ( fig .
she has been managed conservatively with laxatives and has been clinically followed with improved symptoms .
we estimated an irradiated dose of brachytherapy of 518.7 to 642.3 cgy at the sigmoid colon point , which was 29.5% to 60.5% higher than prescribed dose at point a per fraction .
a 79-year - old woman received definitive rt for international federation of gynecology and obstetrics ( figo ) stage iiib cervical cancer .
ebrt was performed at a total dose of 50.4 gy in 1.8 gy fractions for five days each week using a 15-mv photon .
intracavitary hdr brachytherapy ( microselectron - hdr ) of 24 gy in six fractions was conducted twice each week using a tandem and colpostat . following the completion of rt , the cervical cancer completely disappeared .
after one year , the patient returned to the hospital complaining of intermittent rectal bleeding .
a barium study revealed a 3-cm - long severe narrowing of the sigmoid wall , which was suspicious for sigmoid colon cancer ( fig .
sigmoidoscopy revealed luminal narrowing with a friable hyperemic nodular mucosal change 15 cm from the anal verge ( fig .
a 58-year - old woman received definitive rt with concurrent chemotherapy for figo stage iiia cervical cancer .
ebrt was performed at a total dose of 50.4 gy in 1.8 gy fractions for five days each week using a 15-mv photon .
intracavitary hdr brachytherapy ( microselectron - hdr ) of 24 gy in six fractions was conducted twice each week using a tandem and colpostat .
six cycles of weekly cisplatin ( 30 mg / m ) were given during rt .
after five years , the patient visited our emergency department with complaints of poor oral intake , lower abdominal pain and no stool passage over ten days .
sigmoidoscopy revealed an approximately 5-cm - long section of diffuse luminal narrowing in the sigmoid colon and hyperemic mucosal nodularities with mucosal edema ( fig .
a computed tomography ( ct ) scan also revealed thickening of the wall of the rectosigmoid colon probably caused by radiation or inflammation ; however no evidence of any cancer recurrence was found ( fig .
she has been managed conservatively with laxatives and has been clinically followed with improved symptoms .
we estimated an irradiated dose of brachytherapy of 518.7 to 642.3 cgy at the sigmoid colon point , which was 29.5% to 60.5% higher than prescribed dose at point a per fraction .
acute and late gi complications are a major concern during and after rt . radiation sigmoiditis is not well - known compared to other gi complications such as proctitis and enteritis because it has been rarely reported .
galland and spencer . reported 23 cases of sigmoid complications among 80 cases of radiation colitis induced by pelvic rt .
ramirez et al . reported that a total of 35 patients developed sigmoid perforation among over 5,000 patients who were treated with rt for stage ib - iiib cervical cancer . in this report
the sigmoiditis occurred one year after rt completion for one patient and five years after rt completion for the other patient .
both cases showed obstructive symptoms , which make clinicians suspicious of sigmoid colon cancer or recurrent cancer . during the follow - up of cancer patients who received rt previously , clinicians need to keep the possibility of rt complications in mind .
holloway et al . reported that the sigmoid - to - tandem distance is significantly related to sigmoid dose using 3d imaging techniques .
concordantly , the simulation films of case 1 showed close proximity between the sigmoid colon and the tandem , which suggests the possibility of a high sigmoid dose during brachytherapy ( fig .
the estimated dose at the sigmoid colon point during brachytherapy was 518.7 to 642.3 cgy , which was 29.5% to 60.5% higher than prescribed dose at point a ( 400 cgy ) per fraction .
the international commission on radiation units ( icru ) 38 did not describe this hot spot in the sigmoid colon .
the rectal and bladder reference dose points are only standard for reporting normal tissue doses with brachytherapy .
indeed , these points are based on plain films and two - dimensional ( 2d ) brachytherapy planning .
as ebrt is moving from conventional to conformal planning , the incorporation of three - dimensional ( 3d ) sectional imaging such as ct and mri into brachytherapy planning is under active investigation .
recently , there has been a proposal to establish image - guided intracavitary brachytherapy guidelines for cervical carcinoma . in the group european
de curietherapies and european society for therapeutic radiology and oncology ( gec - estro ) recommendations , the rectum and bladder , as well as the sigmoid colon , are highlighted as organs at risk .
many studies have reported a sigmoid dose in 3d image - based planning for the treatment of cervical carcinoma with hdr brachytherapy .
kim et al . reported that the sigmoid dose was higher than the rectal dose in 8 of 13 ( 61% ) patients and its volume was discontinuously located as multiple " hot spots " in 9 of 13 ( 69% ) patients on dosimetric analysis of 3d image - based planning of intracavitary brachytherapy for cervical cancer .
currently at our institution , 3d image - based brachytherapy is implemented to decrease the radiation dose to organs at risk , including the sigmoid colon ( fig .
3 ) . 3d image - based brachytherapy planning can be helpful , especially for patients who have risk factors .
patients who have a history of abdominal surgery , pelvic inflammatory disease , or diverticulosis are at a greater risk of developing radiation - induced gi complications because such conditions may create adhesions to the uterus , which prevent the loops of the bowel from moving during rt . in conclusion ,
radiation sigmoiditis is characterized by obstructive symptoms and its radiological findings can mimic sigmoid colon cancer .
radiation oncologists should acknowledge the sigmoid colon as well as rectum and bladder as organs at risk .
3d image - based brachytherapy could be an optimal modality to reduce the radiation - toxicity associated with treatment for uterine cervical cancer . | external - beam radiation therapy with intracavitary high - dose - rate brachytherapy is the standard treatment modality for advanced cervical cancer ; however , late gastrointestinal complications are a major concern after radiotherapy .
while radiation proctitis is a well - known side effect and radiation oncologists make an effort to reduce it , the sigmoid colon is often neglected as an organ at risk .
herein , we report two cases of radiation sigmoiditis mimicking sigmoid colon cancer after external - beam radiation therapy with intracavitary high - dose - rate brachytherapy for uterine cervical cancer with dosimetric consideration . | INTRODUCTION
CASE REPORTS
1. Case 1
2. Case 2
3. Dosimetric analysis
DISCUSSION |
subtle , asymmetric variation of the contralateral structures of the head and face occur commonly in the general population in the absence of any local lesion or condition .
occasionally , a gross asymmetry easily perceptible to the eye may occur which progresses slowly but steadily and exhibits asymmetric development .
one such entity , characterized by marked unilateral overdevelopment of hard and soft tissues of the face is a rare congenital malformation .
this has been termed variously in the literature as facial hemihypertrophy , hemimacrosomia , partial / unilateral gigantism , congenital hemifacial hyperplasia.[13 ] pollock ar ( 1985 ) emphasized the use of the term congenital hemifacial hyperplasia because it is more histologically precise .
congenital hemifacial hyperplasia ( chh ) , first described by meckel j f in 1882 , did not gain widespread recognition until gesell 's classic studies .
gessel ( 1927 ) described chh as essentially a developmental anomaly antedating birth and arising in a same way as a partial deflection of the normal process of birth .
asymmetric enlargement could be manifested in a unilateral / crossed configuration and may involve all the body tissue in the area ( ie , total ) or a single systems such as muscular , vascular , skeletal or nervous ( ie , limited ) .
chh has been classified by rowe(1962 ) to involve :
the entire half of the body : complex hemihypertrophy , one or both limbs : simple hemihypertrophy , the face , head and associated structures : hemifacial hypertrophy .
the entire half of the body : complex hemihypertrophy , one or both limbs : simple hemihypertrophy , the face , head and associated structures : hemifacial hypertrophy .
the purpose of this report is to present the case of a child with congenital hemifacial hyperplasia and to supplement existing clinical knowledge .
a 12-year - old boy was referred to government dental college and hospital , aurangabad ( m.s . ) with request for the treatment of facial asymmetry present since birth [ figure 1 ] .
he was the last of three siblings without any previous family history of facial asymmetry .
diffuse enlargement of the right side of face beginning abruptly at the midline and deviation of nose , mouth and chin toward the left side the physical examination revealed a mentally healthy and active boy .
the patient 's face was asymmetrical with an enlargement of the right side , including the malar , maxillary , and mandibular region .
the pinna of the right ear was slightly enlarged and there was deviation of the nose and the chin to the left side , which appeared to be the normal side [ figure 1 ] .
the skin and hairs appeared normal with the exception of small hyperpigmented nonhairy patches on the right cheek .
there was an obvious enlargement of tongue on the right side , which began abruptly at the midline [ figure 2 ] .
speech was not impaired by this deformity of the tongue and the sense of taste appeared to be equally distributed . the general examination revealed normal symmetrical development of the extremities and the trunk . noticeable enlargement of tongue with the well - demarcated midline erupted teeth in maxilla were 7,6,5,4,3,2,1/1,2,c , d , e,6,7 and in the mandible 7,6,5,4,3,2,1/1,2,3,4,e,6,7 .
the teeth on the affected side had matured earlier , were larger , and had erupted prematurely with early loss of the deciduous teeth followed by replacement of permanent dentition as compared to the normal side .
the dimensions of right first molars were considerably greater than left first molars [ figure 3 ] .
this enlargement extended from the canine region being most prominent in the premolar and molar region [ figures 3 and 4 ] .
malocclusion was evident with an anterior open bite , distortion of dental arches , and spacing between teeth .
obviously enlarged right first molar than left one with spacing between teeth on right side of the arch .
eruption pattern on right side is far advanced than the left side with broad and thick alveolar process wider and thicker mandibular alveolar process on right side , particularly in the premolar - molar region the eadiograph confirmed the clinical findings .
opg view showed that the mandible was unilaterally enlarged with the body of mandible being longer on the right side with an enlarged right condyle resulting in mandibular deviation to the left [ figure 5 ] .
other striking features were increased height of ramus and body of mandible on the right side than on the left .
there was accelerated development of teeth on the right side with early eruption of permanents and closure of root apices than of the left - sided teeth [ figure 5 ] .
unilaterally enlarged ramus , body , and condyle of mandible on the right side . accelerated development of teeth on the right side than on the left side
rowe ( 1962 ) described the criteria for true chh as it is an unusual condition that produces the facial asymmetry by a marked unilateral localized overgrowth of all the tissues in the affected area , that is , facial soft tissues , bone , and teeth . .
the unilateral enlargement of the viscerocranium is bounded by frontal bone superiorly ( not including eye ) , inferiorly by the border of mandible , medially by midline of the face , and laterally by ear , pinna being included within the hypertrophic area .
the disproportionate growth is almost always evident at birth and the enlarged side generally grows at the rate proportional to but slightly faster than the normal side .
the disproportionate growth rate is maintained until the time of skeletal maturation and results in an asymmetry existing throughout life .
the multisystem involvement has resulted in etiological heterogeneity and no single theory explains the etiology adequately .
factors that can be implicated are heredity , chromosomal abnormalities , atypical forms of twinning , altered intrauterine environment , endocrine dysfunctions , anatomical and functional anomalies of vascular / lymphatic systems and disturbances of the central nervous system .
an interesting concept put forth by gesell ( 1927 ) suggests an inequality of regulatory abilities in embryologic development leading to an aberrant twinning mechanism .
pollock and coworkers ( 1985 ) proffer a hypothesis that the neural tube and its precursors are unilaterally hyperplastic
. the enlarged half of the neural tube would give rise to proportionally more numerous neural crest cells on the involved side .
the increased number of crest cells persists throughout much of the prenatal and formative postnatal growth periods of life and lead to unilateral overgrowth of the crest - derived bone , cartilage , teeth , muscles , and soft tissue .
some tube segments may be involved more often than others helping explain why cephalic areas are more often affected clinically than other areas .
involvement of orofacial structures is related to asymmetric morphogenesis of teeth , bone , and soft tissues .
dentition abnormalities are with respect to crown size , root size , and shape and rate of development .
tooth size enlargement is random with the frequency of involvement more in cuspid followed by premolars and first molars and least occurring in incisors , second molars , and third molars .
skeletal findings may be in the form of an asymmetric growth of the frontal bone , maxilla , palate , mandible , or condyles .
abnormal occlusal relationships such as midline deviation , unequal occlusal plane level , open bite , and widely spaced teeth on the involved side have been reported .
soft tissue abnormalities include thickened and enlarged anatomical tissues on involved side with the sharply demarcated midline .
the tongue , which is commonly involved , may show a bizarre picture of enlargement of fungiform papillae with unilateral enlargement and contralateral displacement .
other soft tissues such as lips , buccal mucosa , uvula , and tonsils are also affected . because most of the striking signs of congenital hemihypertrophy are usually manifested in the orofacial region , gorlin and meskin ( 1962 ) suggested them help in differentiation of this condition from other entities that may simulate hypertrophy . among the conditions that closely mimic chh as suggested by various authors
are fibrous dysplasia , dyschondroplasia , congenital lymphedema , arteriovenous aneurysm , hemangioma , lymphangioma , klippel trenaunay syndrome , von recklinghausen 's neurofibromatosis , malignant conditions such as osteosarocma and chondrosarcoma .
all these conditions , however , exhibit sufficient clinical differences with chh and should be distinguished on the basis of specific radiographs , clinical and laboratory findings . to the unfamiliar clinician , hemihypertrophy that is localized to orofacial region can constitute a diagnostic problem .
deformities of the teeth and their related hard tissues in the jaw are key findings for correct diagnosis , particularly in hemihypertrophy limited to the face .
these may include reconstructive procedures such as osteotomies or orthognathic surgical procedure and soft tissue debulking by excision of excess masticatory and subcutaneous tissues , with preservation of neuromuscular functions .
an extensive search of the english language literature revealed no formal reports of malignant transformation . | congenital hemifacial hyperplasia ( chh ) is a rare congenital malformation characterized by marked unilateral overdevelopment of hard and soft tissues of the face .
asymmetry in chh is usually evident at birth and accentuated with age , especially at puberty .
the affected side grows at a rate proportional to the nonaffected side so that the disproportion is maintained thr oughout the life .
multisystem involvement has resulted in etiological heterogeneity including heredity , chromosomal abnormalities , atypical forms of twinning , altered intrauterine environment , and endocrine dysfunctions ; however , no single theory explains the etiology adequately .
deformities of all tissues of face , including teeth and their related tissues in the jaw , are key findings for correct diagnosis of chh . here
an attempt has been made to present a case of chh with its archetypal features and to supplement existing clinical knowledge . | Introduction
Case Report
Discussion |
molecular
dynamics ( md ) has had a long and important history in
computational biology and has made many recent contributions
toward understanding a variety of biological phenomena , including
protein folding , protein ligand binding , and conformational change
of large biomolecules .
though running a large - scale simulation has historically been a
demanding task , recent advancements in both software and hardware
have made simulations of complex systems possible .
nonetheless , because of the high - dimensional nature of the resulting
time series , the generation of many md trajectories is not sufficient
for providing scientific insight .
in fact , data analysis has increasingly
become a major barrier in the application of md simulations .
one analysis technique attempts to estimate
the slowly equilibrating
collective motions , or eigenprocesses , contained in a simulation ( e.g. ,
proteins folding from unfolded conformations , ligands binding to a
target , enzymes shifting from an inactive to active state , etc . ) .
these eigenprocesses are useful as they can be measured experimentally ,
but by constructing them from a simulation , we can provide a level
of detail that is unavailable to experiment alone .
markov state models
( msms ) are one such method that has been useful in a variety of contexts .
briefly , an msm describes
continuous dynamics in phase space as a jump process over a finite
number of states that partition the space .
practically , the construction of
an msm is an active area of research
as there are significant hurdles that must be overcome in order to
build a model that accurately describes the dynamics contained in
a simulation .
for example , an msm requires
the definition of a state decomposition that is , a partition
of phase space into discrete states .
the accuracy of the model relies
heavily on the quality of this state decomposition , but although there
have been a number of improvements to this process , there is no consensus on the best method for defining a state space .
an msm , however , is merely one way to
estimate a system s
eigenprocesses from a simulation .
therefore , we can hope to overcome
the practical challenges faced when constructing an msm by approximating
the dynamics within a different scheme . toward this end
, we introduce
an extension of time - structure based independent components analysis
( tica ) using the kernel trick for estimating the characteristic eigenprocesses
contained in a simulation without using an msm . first ,
we lay the theoretical foundation for the method , then formulate the
problem and derive its solution , and finally , we use the method to
analyze several toy models as well as md simulations of the fip35
ww domain from shaw et al .
in this
section , we briefly discuss markovian dynamics and the transfer operator
approach but leave most details to other works .
we also note that , for clarity , most of our notation is borrowed
from prinz et al .
we are interested
in discrete - time dynamics in some phase space that are markovian ,
ergodic , and time - reversible .
this density is properly normalized such
that dx (x ) = 1 . let x(t ) denote a markov process
in . because of the markov property , there exists a transition
probability density such that p(x , y;)dy is the probability
of being in some volume ( dy ) around y after time given that the system started at x. the dynamical progression of the markov chain can
be described
via a propagator operator , which maps a probability density at time t , pt(x ) , to a new density at time t +
later , pt+(x):1additionally , there exists a corresponding
operator called the transfer operator defined such that2 the functions ut(x ) are related to probability densities by3(while the
propagator provides a more intuitive
operation , the transfer operator s eigenfunctions will be the
eigenprocesses we actually estimate in the following methods . )
the action of the transfer operator can be decomposed into a series
of relaxation processes , which correspond to its eigenfunctions , i(x ) , and their eigenvalues ,
i:4where5 the eigenfunctions of the
transfer operator are orthogonal and
normalized such that6as a result
of this decomposition , we can
model the dynamics of the markov chain directly if we have an estimate
of the eigenfunctions of the transfer operator .
in fact , an msm provides
an estimate of this transfer operator by estimating a finite number
of the eigenfunctions as a sum of indicator functions , where the indicator
functions define the state decomposition in phase space . however ,
the msm is not the only technique that can approximate the transfer
operator .
the eigenvalues of the transfer operator correspond
to a relaxation time scale , ti , which can be measured via the autocorrelation function of
a trajectory { x0,x1, ... ,xt , ... } projected onto
the corresponding eigenfunction:7 in recent work from no and
nske , it was shown that a variational
principle can be derived for the eigenvalues of the transfer operator ,
meaning that given a mean - centered , integrable function , , the autocorrelation of f at time is always
less than the autocorrelation of the first
nonstationary eigenfunction , 1 , of the transfer
operator:8 this is powerful because it implies
that one can find the slowest
dynamical processes in a simulation without building an explicit
estimate of the operator itself , but by directly estimating
its eigenfunctions .
have successfully constructed estimates of the transfer operator s
top eigenfunctions in the span of a prespecified library of basis
functions by leveraging this variational bound . here , we take a different
approach that does not require a predefined basis set . in
what follows , we are working in the space
and will assume that it is a euclidean space with some dimension , d. therefore , if x and y are in
, then xy = xy denotes the inner product
and x y = xy the outer product between the two length - d column vectors .
time - structure based independent
component analysis ( tica ) is an unsupervised learning method for finding
projections that decorrelate slowly . given a markov chain in phase
space : { xt } ,
tica attempts to find a vector v such
that the autocorrelation of xt projected onto v is maximized ( eq 9).9here , and is some lag time greater than
zero .
to compute the expectation values in eq 9 , assume we can sample m pairs of points in phase
space , { ( xt ,yt ) } , where xt is sampled from the equilibrium
distribution , ( ) , and yt from the transition probability density , p(xt, ; ) . then ,
we can estimate the time - lag correlation matrix , covariance matrix ,
and mean vector using a maximum likelihood approach with a multivariate
normal approximation ( see si section
let1011where xt = xt , with
mean given by12 given these matrices , eq 9 becomes13it can be shown that the solution to eq 13 is
also a solution to the generalized eigenvalue
problem:14 furthermore , the remaining
eigenvectors from eq 14 are the slowest projections
constrained to being uncorrelated
with the previously found solutions .
this method has proven
to be very useful for constructing msms
that reproduce the slowest time scales in a simulation .
however , more importantly , since eqs 7 and 9 are the same , the solutions to the tica problem
( tics ) are also solutions to the variational problem and therefore
provide estimates of the slowest eigenfunctions of the transfer operator .
these estimates , however , are crude since the
tics are constrained to be linear functions of the input coordinates ,
but if we can extend the tica problem to include nonlinear degrees
of freedom then we will be able to estimate the true eigenfunctions
more accurately .
the simplest approach
to extending the tica problem beyond linear combinations of coordinates
in is to use a mapping function , : v , which expands the original representation of the data
into a higher - dimensional feature space , v. we can
then calculate the linear tics in the feature space , v , but since is arbitrary , these tics can in fact be nonlinear
functions of the coordinates in . the dimensionality of v , however , could be enormous ( or perhaps infinite ) , and
so , this scheme is impractical except in very simple cases . under
certain conditions ,
however , it is possible to perform analysis in v without ever actually calculating the representations ,
(x ) , by employing what is known as the kernel
trick . for illustration
, we reproduce a simple example
from schlkopf et al . of the kernel
let and define the mapping
function between
phase space , , and feature space , v ,
to be (x ) = ( x12 , , x22 ) .
because of this definition ,
it is easy to show that15equation 15 indicates
that we can calculate the dot product in v in one
of two ways .
we can either compute (x ) and (y ) and explicitly calculate their dot product or we can simply
square the dot product of the vectors in .
either route gives us the same result ,
but the latter has the advantage of being much easier in cases where
the feature space s dimension is very large
imagine
using a polynomial of degree much greater than two in eq 15or infinite .
kernels are used commonly
in machine learning when extending linear
problems to provide nonlinear solutions . any problem that can be rewritten entirely in terms of inner products
can be extended to use the kernel trick , and in the following , we
show how to rewrite the tica problem in this way . remember
that the tica problem searches for a projection that maximizes the
autocorrelation function of a projection onto a vector v. our goal is to rewrite the optimization problem ( eq 13 ) in terms of only inner products and then apply the kernel
trick to solve the tica problem in high - dimensional feature spaces .
this assumption is only violated when one coordinate can be written
as a linear combination of the other coordinates . in these cases ,
we need only project the data into a nonredundant space and continue
the analysis there .
we start with a set of m pairs of points in phase space , { ( xt , yt ) } , where xt is sampled from the equilibrium distribution , ( ) and yt from the transition probability
density , p(xt, ; ) . consider a function : v that maps points in phase space , , into a higher - dimensional
feature space , v. now we want to solve the tica problem
in this new space v. the solution , v v , is a solution to the eigenvalue problem
given in eq 14 .
however , the covariance and
time - lag correlation matrices are calculated in terms of the vectors
in v : here , we have used (x )
(y ) to denote the outer product of the
two vectors (x ) , (y ) v. for now , assume that the (xt ) values and (yt ) values are centered ( see supporting information
( si ) centering data in the feature space )
. it can be shown that the solution , v , is in the span
of the xt values and yt values , which means that we
can write v as a linear combination of the input data
points ( see si
let such that16we will rewrite the tica problem in v in terms of a gram matrix k , where k has 2 m rows and 2 m columns ,
defined as17where
(x)(y ) is the inner
product between the two vectors (x),(y ) v. in
other words , the gram matrix can be broken into four blocks:18such
that each block is an m m matrix and19202122let imm denote the m - dimensional
identity matrix and 0mm denote an m by m matrix
of all zeros . then , let r be a 2 m 2 m matrix given by eq 23.23 in the si derivation of the
ktica solution , we show that the numerator and denominator
of the rayleigh quotient in eq 9 can be rewritten
in terms of and k:2425and so the tica problem in the feature space v can
be written totally in terms of inner products:26which is itself a rayleigh quotient and so
the solutions satisfy the generalized eigenvalue problem:27 however , solving such problems can be challenging because
the matrix kk can have zero eigenvalues , which correspond
to directions
in v that have zero variance associated with them .
these directions are either due to redundant coordinates ( in v ) or under sampling and so are not desirable solutions
to the tica problem . however , these zero variance directions lead
to infinite eigenvalues in eq 27 .
this issue
is not exclusive to the kernel tica problem and is faced in most kernel
learning methods .
several solutions have been reported in the cca ,
lda , and svm literature that introduce a regularization term .
we use the method applied to kernel - cca in mika et al . but realize that another form of regularization
may be even more useful .
the result is a modified objective function
that penalizes the solutions according to the l2 norm on .28 this new
rayleigh quotient can be written as the solution to a
modified generalized eigenvalue problem:29 for large
enough , the result is a positive definite matrix , kk+i2m2 m , which allows us to compute the solutions reliably .
we refer the reader to other kernel methods that use a similar regularization
scheme for a discussion , as well as the results section two - dimensional brownian dynamics . as in the case of linear tica , there are
many solutions to the ktica problem ( herein called ktics ) , where each
is the slowest projection subject to being uncorrelated with all of
the slower ktics .
let v v denote the i ktic and let denote the i eigenvector of eq 29 corresponding to v : because of the variational principle ,
the ktics are direct estimates of the transfer operator s right
eigenfunctions : the first eigenfunction ( 0(x ) =
1 x ) is not estimated by ktica
as it is stationary , and so the second eigenfunction is estimated
by the first ktic .
however , to directly compare to the eigenfunctions
of the transfer operator , we need to ensure that the solutions to
the ktics are normalized correctly .
the eigenfunctions of the
transfer operator are orthonormal with
respect to the inner product defined in eq 5 .
since the first eigenfunction is equal to one for all points x , this means that the eigenfunctions have these properties : 1 .
all dynamic eigenfunctions ( i > 0 ) have
zero
mean ( ) . since the gram matrix is centered in
the feature space ( see si section centering
data in the feature space ) , the ktics have zero mean .
the
eigenfunctions are uncorrelated and are normalized such
that they have unit variance ( cov(i(x ) , j(x ) ) = i , j = ij ) . as shown in the si section derivation of the ktica solution , the variance of the i ktic , is given by so we can normalize each such
that the variance is one .
arbitrary points in can be
projected onto the ktics as
this operation can be written entirely in terms of inner products:31we emphasize here
that it is possible to evaluate
the value of v(x)and therefore i+1(x)without ever specifying or v. in a sense , the ktics behave
like a kernel density estimator , where the value at some point x depends on how close that point is to the training points . to solve the ktica
problem
, we need to calculate the gram matrix , k ,
given in eq 18 , which requires the choice of
a kernel function that specifies inner products in the feature space .
although there are a variety of kernel functions available , for this
work we chose the commonly used gaussian kernel:32here , x and y correspond
to two conformations of a molecule . technically , these vectors are
points in phase space and represent the positions and momenta of all
atoms in the simulation . however , in most bimolecular simulations ,
we do not care to describe the rotation and translation of the molecule .
moreover , because we only have finite sampling ,
if we attempt to describe
rotation and translation in our models , then we will likely do so
only by sacrificing accuracy in describing the conformational change
that we are interested in . for this reason , for each application we
select an internal set of coordinates ( e.g. , all dihedral angles or
contact maps of a protein ) to use in eq 32 .
these coordinates will depend on the particular system being studied ,
and so we leave their specification to the application sections below . with the kernel given in eq 32 ,
the gram
matrix is defined as33 the solutions to eq 29 provide an explicit
form of the estimates of each eigenfunction i+1:34 this expression highlights the analogy to a gaussian kernel
density
estimator , where we can evaluate i+1 at any point x based on the eigenvectors from eq 29 and how close x is to each point in
the training set .
in addition to the kernel , we must choose
the correlation lag time ,
, and the regularization strength , . in previous studies ,
tics were largely unperturbed by the choice of ; hence , for each of our example systems , we fix
a and ask for the best model given this choice . therefore ,
there are two free parameters we must specify before we can solve
eq 29 : and . bayes theorem
demonstrates that the probability of a model given the data is proportional
to the probability of the data given the model , which means that we
can select a model based on maximizing the model s likelihood
of generating new data . here ,
a model is the set of
ktics from eq 29 solved with particular values
for and . therefore , our strategy will be to solve
eq 29 over a range of values for and
and then pick the model that assigns the highest probability
to new data .
as the ktics are estimates of the transfer operator s
eigenfunctions ,
we can define a likelihood based on eq 4 .
let k be a positive integer , then let { ( at ,bt )}t = 1n be pairs of test points
that were not used in computing the ktics , where at is sampled from the true equilibrium distribution ,
( ) , and bt from
the true transition probability density , p(at, ; k ) .
then , the probability of observing all of these transitions can
be written in terms of the infinitely many eigenfunctions of the transfer
operator : however , we do not have access to infinitely
many eigenfunctions , so we instead truncate the expansion at the top m + 1 eigenfunctions to calculate an approximate probability , p.35 this approximation is better when m is large ,
or when { i : i > m } are very small . since the equilibrium probability is
not dependent on either of and , we can ignore the
( ) terms when comparing models .
this is especially clear
when looking at the approximate log - likelihood:36 for all applications ,
we calculate a set of ktics for a variety
of values of and on a subset of the full data set
( the training set ) .
then , eqs 34 and 36 can be used to evaluate the approximate likelihood
on { ( at , bt)}t = 1n the test set that was initially left out of the calculation .
the values of and that are selected are those that
are most likely according to eq 36 .
one
technical issue when calculating these probabilities is that
since we truncate the infinite expansion at relatively few ktics ,
the resulting probability is not guaranteed to be positive . in practice
,
this can be difficult to handle but can be avoided by only testing
pairs of points that are separated by many multiples of . in
this way , the faster components we ignore in eq 36 have equilibrated and so the truncated sum is a better approximation
of the true ( infinite ) expansion .
admittedly , this problem likely
indicates that defining such a probability based solely on the slowest
eigenfunctions of the transfer operator is not possible .
there may
in fact exist a better scheme for evaluating these models ; however ,
in the results below , we show that , at least for simple systems , the
likelihood approach does work .
computationally , solving the
ktica problem requires finding the
solution to eq 29 , which is a generalized eigenvalue
problem .
however , the matrices on either side are 2 m 2 m , where m is the number of data points in the training set .
this is a computational
challenge for all kernel methods , as the solution becomes more difficult
to compute as more data is used . here
. however , there are many sophisticated
solutions that have been developed for other kernel - learning methods ,
and these will certainly be useful in the future .
we simulated
a markov jump process on a one - dimensional , four - well potential as
suggested in prinz et al .
the potential
given by37was discretized into 100 points between 1
and 1 , and a transition matrix was defined such that at each point ,
there was nonzero probability to move left , move right , or stay at
the current point . in each case , the probability to move from point xi to xj was proportional to exp[(v(xj)v(xi ) ) ] .
from the first 1000 pairs of transitions separated by 10 time steps
were used in the ktica method . the euclidean distance between points
was used in conjunction with the gaussian kernel defined in eq 32 to calculate the gram matrix .
the approximate
likelihood scores were calculated for 1000 pairs of points sampled
from the second trajectory separated by 10 timesteps . in this simple
case ,
there were many models that were roughly the same quality as
judged by the likelihood scores ( figure 1 ) .
approximate
log likelihood evaluated on test data provided a similar
score for most models we tested .
models using a value of 0.01 for
or a value of 10 for were given very low
scores , while all other models seemed to be good enough .
the best
model according to the likelihood evaluated on test data correctly
determined the top three eigenfunctions for this system .
the ktics
are shown in blue while the actual eigenfunctions are shown in red .
it is illustrative to remember that the tica solution is trivial in
this one - dimensional case . even in this simple case ,
the nonlinearity
in the ktics prove to be essential for estimating the transfer operator s
eigenfunctions . since the generating
process was itself an msm ,
there are three slow
eigenfunctions , corresponding to transitions across the three barriers
on this system .
the best model correctly determined the slowest three
ktics and are illustrated in figure 1 .
these
results illustrate the value of nonlinearity in estimating the eigenfunctions
of the transfer operator .
in fact , since the dynamics take place in
a one - dimensional space , there is only one solution to the regular
tica problem , which only ( crudely ) estimates the slowest eigenfunction
of the true transfer operator .
we also investigated the mller
potential , and simulated dynamics
that were governed bywhere = 10 , kt = 15 ,
and r(t ) is a
delta - correlated gaussian process with zero mean , and v(x ) was defined aswhere a = ( 1,1,6.5 ,
0.7 ) ; b = ( 0 , 0 , 11 , 0.6 ) ; c = ( 10,10,6.5 ,
0.7 ) ; a = ( 200,100,170 , 15 ) ; x = ( 1 , 0,0.5,1 ) ; y = ( 0 ,
0.5 , 1.5 , 1 ) as suggested by mller and brown . using the euler maruyama method and a time step
of 0.1 , we produced two trajectories of length 10 time
steps .
the initial positions were sampled via a uniform distribution
over the box : [ 1.5,1.2 ] [ 0.2,2.0 ] . the
ktica algorithm was used to analyze the first trajectory by ( regularly )
sampling 2000 pairs of transitions separated by 10 timesteps .
the
cross - validation scores were calculated for 1000 pairs of transitions
separated by 50 timesteps using the top two ktics .
as there are essentially
three local minima in the mller potential , the top two eigenfunctions
should correspond to population transfer between the three wells .
the model selected by the likelihood criterion correctly estimates
these eigenfunctions ( figure 2 ) while numerous
other models do not .
ktica models for the mller potential simulations
are not
as well - behaved as the one - dimensional case .
there were a few models
that assigned a nonpositive value for the likelihood of test data
( white regions above ) .
however , the maximum likelihood model correctly
identified the top two eigenfunctions , which separate the three major
minima in the potential energy surface .
these eigenfunctions are depicted
in the bottom two panels , with the red and blue color corresponding
to the value of the ktic .
the solutions are highly
dependent on the choice of the ,
which indicates the necessity of regularization .
if is too
small , then the ktics focus on only a few points that are not sampled
often in the data set .
when it is too large , the eigenvalues are underestimated
and the eigenfunctions are distorted from the correct functions . the
approximate likelihood evaluated on test data chooses an intermediate
value of whose ktics correctly separate the three wells in
the mller potential ( figure 3 ) .
three models
with = 0.5 but varying values are
depicted above .
these results illustrate the necessity of using regularization .
when is too small ( top ) ,
the solutions focus on only a few
points in the data set that are not sampled heavily , while when it
is too large , the solutions are distorted ( bottom ) .
fortunately , the
likelihood based model selection criterion picks an intermediate value
of ( middle ) whose ktics are better estimates of the actual
eigenfunctions .
a single trajectory of alanine
dipeptide ( ala1 ) was simulated using
the amber99sb - ildn force field in implicit
solvent ( gbsa ) for 500 ns using openmm6.0 with a time step of 2 fs at 300 k using a langevin integrator .
the
trajectory was subsampled every 250 fs for analysis and split in half ,
with the first half serving as the training data and the second serving
as the test data .
the ktica calculation was performed by regularly
sampling 2000 pairs of points separated by 1.25 ps from the training
data .
the gaussian kernel was used in conjunction with a distance
metric defined on intra - atomic distances . for each conformation ,
the
distance between all pairs of heavy atoms were computed , which formed
a vector of length 45 .
then , the distance between two conformations
was the euclidean distance between these vectors . for the likelihood
calculation
, we regularly sampled 5000 pairs of points separated by
6.25 ps to ensure that the approximate likelihoods defined by the
top two ktics were non - negative for most models . we should not expect
this approximate probability to accurately describe motions at 1.25
ps but would hope that as one looks further in time the probability
based on the top two eigenfunctions becomes more accurate .
since alanine
dipeptide has been the subject of many computational studies , it is
known that the slowest two relaxations occur along the axes of the
ramachandran plot of the two dihedral angles , and .
the most likely model recapitulated these two slow processes ( figure 4 ) .
we reanalyzed the two 100 s md trajectories
of the fip35 ww
domain performed by shaw et al .
the ktica
problem was solved by analyzing 10 000 pairs of transitions
separated by 50 ns from the first trajectory .
( note that because of
the size of the data set , the pairs of transitions overlapped one
another .
this is , in a sense , the same as using the sliding window
approach for counting transitions when building an msm , which is common
practice . )
the gaussian kernel defined in eq 32 was used with a distance metric defined on the protein s
contact map .
each conformation was represented as a 528-element vector
of intramolecular residue residue distances . where all pairwise
distances were tracked between residues that were at least three amino
acids away from each other .
the distance between a pair of residues
was computed by taking the distance between the two closest heavy
atoms in the respective residues .
the likelihood defined by the slowest
ktic alone was evaluated on 1000 pairs of transitions separated by
500 ns .
this time was long enough to assign non - negative likelihoods
to a majority of the models we tested .
as in the one - dimensional
case , there are several models that are
very good according to the cross - validation scores , while models with
small values of and perform poorly .
alanine dipeptide
has been studied extensively , and it is known that the slowest two
relaxations occur along the axes of the ramachandran plot .
the projection
of the test data set into these axes shows that the top two ktics
correctly identify the slow motions in the alanine dipeptide simulations .
likelihood defined by only the slowest ktic
was evaluated for the
ww domain test data .
again , small values of and produced
likelihoods that were often negative for test data ; however , there
is a region in which all likelihoods were positive .
the maximum likelihood
model s slowest ktic corresponded to the folding transition ,
as it was correlated with rmsd to the native state .
the time scale
associated with this ktic was 4.4 s , which agrees with previous
analysis of this same data set .
the slowest eigenfunctions of
the transfer operator for the ww
domain are not known , but for the maximum likelihood model , the slowest
ktic is physically reasonable as it separated folded and unfolded
states in the simulation ( figure 5 ) .
additionally ,
the ktic had a corresponding time scale of 4.4 s , which is
consistent with previous analysis of this data set .
though the tica method has proven to be remarkably successful for
building state decompositions for msm analysis , its solutions
are limited because they are constrained to be linear functions of
the input coordinates .
for this reason , the extension to nonlinear
functions by using the kernel trick represents a substantial improvement
that allows ktica to provide estimates of the transfer operator s
eigenfunctions directly , without the use of an msm .
we have shown here that the method can accurately calculate the
slowest eigenprocesses of simple systems but admit that there is still
work to be done before it can be routinely used for analyzing arbitrarily
complex dynamics .
computationally , the matrices in eq 29 scale with the amount of data used , which means that as the
systems become larger and more complicated and more data is needed ,
the eigenvalue problem will become increasingly difficult to solve .
however , this problem is not unique to ktica and , in fact , is ingrained
in all kernel learning methods . for this reason , we expect significant
improvements to be made rapidly as these methods have been extensively
studied in machine learning . | the allure of a molecular dynamics
simulation is that , given a
sufficiently accurate force field , it can provide an atomic - level
view of many interesting phenomena in biology .
however , the result
of a simulation is a large , high - dimensional time series that is difficult
to interpret .
recent work has introduced the time - structure based
independent components analysis ( tica ) method for analyzing md , which
attempts to find the slowest decorrelating linear functions of the
molecular coordinates .
this method has been used in conjunction with
markov state models ( msms ) to provide estimates of the characteristic
eigenprocesses contained in a simulation ( e.g. , protein folding , ligand
binding ) . here ,
we extend the tica method using the kernel trick to
arrive at nonlinear solutions .
this is a substantial improvement as
it allows for kernel - tica ( ktica ) to provide estimates of the characteristic
eigenprocesses directly without building an msm . | Introduction
Theory
Results
Conclusions |
in recent years a lot of effort has been
undertaken in order to
prepare and investigate ordered structures of -conjugated organic
molecules on nanoscopic and mesoscopic scales .
the interest of both
fundamental and applied science was mainly driven by the goal to understand
and improve the properties of materials used for organic electronics
( e.g. , light emitting diodes , field effect transistors and solar cells ) and to establish novel systems
for optical applications ( e.g. , waveguiding and lasing ) . frequently small rodlike molecules like oligothiophenes ,
oligoacenes
and oligophenylenes have been selected as model systems . in the group
of oligothiophenes sexithiophene ( 6 t )
has shown interesting high quality
properties like a charge carrier mobility of up to 0.1 cm/(v s ) .
consequently there has been an interest
in understanding the growth mechanism of such molecules on well - defined
model substrates like single crystalline cu , sheet silicates or patterned sio2 .
the study in this paper
aims at providing a conclusive picture
of the formation of 6 t nanostructures by an in - depth analysis of the
growth on an alkali - halide single crystal , namely potassium chloride
( kcl ) .
kcl exhibits a face centered
cubic crystal structure with a lattice constant of 6.36 .
the unit cell of the ( 100 ) surface can be described
by the 2d - space group p4 mm as shown
in figure 1 .
importantly , this high symmetry
surface exhibits rotation centers of order 2 and 4 as well as mirror
and gliding planes .
it is expected that the symmetry of the substrate
will be reflected in the multiplicity of epitaxial alignments of the
deposited molecules .
( a ) the ( 100 ) surface
of the kcl crystal , with the white rectangle
illustrating the surface unit cell .
( b ) the p4 mm symmetry of the surface unit cell including
the respective symmetry elements .
the hot wall epitaxy ( hwe ) technique
was applied for the deposition of the organic material , which allows
the growth process to be performed close to thermodynamic equilibrium ,
and in further consequence relatively high vapor pressure of the organic
deposit in the substrate region can be achieved .
therefore the requirements
concerning vacuum conditions are reduced as compared to , e.g. , molecular
beam epitaxy .
the source material 6 t
was purified twice by thermal sublimation before filling it into the
quartz tube of the hwe reactor .
kcl substrates were transferred into
the deposition chamber via a load lock and subsequently preheated
at the deposition temperature ( 60135 c ) for 30 min to
clean the surface from adsorbed species and to ensure a stable temperature
during the whole deposition process .
the deposition was performed
thereafter under a base pressure of 9 10 mbar at a nominal deposition rate of 0.4 nm / min .
x - ray diffraction ( xrd ) measurements
were carried out on a philips xpert x - ray diffractometer using
cr k radiation ( = 2.29 ) and a secondary graphite
monochromator .
brentano
configuration by varying the z - component of the scattering
vector q. consequently it is possible to detect lattice
planes which are parallel to the sample surface .
pole figures are acquired by measuring at a
constant length of q and only varying its direction .
based on both
the observed bragg peaks of the specular scan and the direction of
the poles ( net - plane normals ) within the pole figures , the involved
crystallographic phases as well as the azimuthal alignment of the
crystallites with respect to the substrate can be identified .
optical microscope images
have been acquired by a nikon labophot 2a microscope in combination
with a nikon type 115 digital camera .
scanning force microscopy
( sfm ) studies of the deposited organic films were performed using
a digital instruments dimension 3100 in the tapping mode .
the images
have been acquired at scan speeds of 46 m / s using sic
tips exhibiting a cone angle of 40. nominal values for resonance
frequency and tip radius are 325 khz and 10 nm respectively .
in this paper we reported
on epitaxial growth of 6 t on kcl ( 100 ) ,
which was examined by crystallographic and morphological investigations . by means of sfm analysis different morphologies
furthermore , it was
found that the area covered by islands is declining with increasing
substrate temperature , whereas the sample area covered by needles
is constant over the investigated temperature range ( 60135
c ) .
consequently we can draw the conclusion that needle - like
structures are energetically more stable as compared to flat islands
formed of standing 6 t molecules on kcl ( 100 ) .
additionally , different
activation energies for the growth of nanoneedles in height and width
have been deduced from sfm analysis , namely , 0.26 0.02 ev and
0.15 0.04 ev , respectively . by means of xrd specular
scans it was possible to determine the
contact planes of the structures on the surface .
needles with { 010 }
and { 411 } and islands with the { 100 } facet parallel to the
substrate surface have been detected . utilizing xrd pole figures and
fft analysis of optical microscope images we observed that there is
a well - defined azimuthal alignment of the structures on the surface
in addition to the stacking in growth direction . notably structures
with { 100 } and
{ 411 } contact planes are in perfect azimuthal
alignment to each other , which is explained by ledge directed epitaxy
of the islands on the sidewalls of already existing needles .
the orientation of the crystallographic structures which are present
on the substrate surface fulfills the growth model for rodlike molecules
on sheet silicate substrates developed by simbrunner et al .
therein it is explained that the adsorption
of a single molecule dominates in a first step and the nucleation
of organic crystallites which follows thereafter only leads to a slight
reorientation of the lmas .
in particular rotational and mirror symmetries
have to be reflected by the formed needle orientations .
the latter
considerations are substantiated by the results presented in this
paper . the 4-fold rotational symmetry and the mirror symmetry of the
kcl ( 100 ) substrate surface leads to eight distinct growth directions
of each crystal type .
moreover two different crystal contact planes ,
namely , { 010 } and { 411 } , are characterized by an analogous
lma orientation as similarly observed for p - hexaphenyl
on kcl ( 100 ) .
this hints that the adsorption
geometry of single molecules seems to be decisive for the organic
crystal nucleation as expected by the growth model .
the fact that
one adsorption geometry can lead to different crystal contact planes
may be explained by polymorphs of the first monolayer , that are both
based on the same azimuthal orientation of the lma on the substrate
surface .
the argument is supported by the fact that monolayer polymorphs
of 6 t have already been reported on ag ( 001 ) and ag ( 110 ) . concluding the
latter discussion it can be stated that the azimuthal
alignment of the organic crystals and molecules is basically determined
by a two step process .
the initial phase is dominated by the adsorption
of single molecules in their energetically preferred adsorption geometry
on the substrate surface .
this adsorption process is mainly determined
by an interplay between substrate geometry , molecular geometry and
importantly symmetry properties . in the second step , where the organic
crystal nucleation takes place ,
only a slight azimuthal realignment
of the molecules can be caused in order to optimize the lattice match
at the interface . | the morphology and structure of sexithiophene deposited
on kcl
( 100 ) substrates was investigated by scanning force microscopy and
specular x - ray diffraction measurements .
two different needle - like
structures with { 010 } and { 411 } contact planes have been observed
as well as islands of almost upright standing sexithiophene molecules
with a { 100 } contact plane .
furthermore an azimuthal alignment of
all three crystal orientations was observed by x - ray diffraction pole
figure measurements , and the growth directions reflect the 4-fold
rotational symmetry of the substrate surface .
in addition the analysis
of crystals with { 411 } and { 100 } contact planes unveiled that
they share a common crystallographic direction which is explained
by ledge directed epitaxy . | Introduction
Experimental Procedures
Conclusion and Discussion |
null | in humans , absence of fragile x mental retardation protein ( fmrp ) , an rna - binding protein , results in fragile x syndrome ( fxs ) , the most common inherited form of intellectual disability . here
we report through biochemical and electrophysiological studies that fmrp binds the c - terminus of the slack sodium - activated potassium channel to activate the channel .
the findings suggest that slack activity may provide a link between patterns of neuronal firing and changes in protein translation . | Supplementary Material |
mirnas are a class of endogenous and evolutionarily conserved noncoding small rnas ( approximately 22 nucleotides ) that bind to the 3 untranslated regions of target genes . once bound , mirnas repress target gene translation and/or induce the degradation of the target gene mrna .
mirnas are ubiquitously expressed in a wide range of species , including viruses , worms , flies , plants and animals .
it is estimated that 1000 mirnas are encoded in the human genome and expressed in a tissue- and cell type - specific manner .
an individual mirna can target numerous other mrnas and can itself be targeted by multiple mirnas , resulting in a vast regulatory potential .
mirnas have been shown to have fundamental roles in diverse biological and pathological processes , including intracellular signaling pathways , organ morphogenesis and disease processes .
the aberrant expression of mirnas is closely associated with the initiation and progression of pathophysiologic processes .
most circulating mirnas are found in lipid or lipoprotein complexes , such as apoptotic bodies , microvesicles , exosomes and other extracellular vesicles , resulting in a high degree of mirna stability despite the presence of ribonucleases in both plasma and serum .
the existence of mirnas circulating in the blood has raised the possibility that mirnas may possess the capability to serve as novel biomarkers .
recent reports have demonstrated that mirnas represent potential biomarkers with distinct clinical characteristics , such as disease diagnosis or activity .
scleroderma ( systemic sclerosis , ssc ) is a complex heterogeneous autoimmune disease of unknown etiopathogenesis .
ssc is characterized by vascular dysfunction and the excessive accumulation of the extracellular matrix ( ecm ) , resulting in the progressive fibrotic replacement of normal tissue architecture and leading to the failure of affected organs , such as the kidney , heart and lung .
the exact cellular and molecular mechanism of ssc remains unclear , but it involves complex interactions among vascular , immune and fibrotic processes in association with a genetic predisposition . to date , no therapy has been shown to reverse or arrest the progression of fibrosis , representing a major unmet medical need .
the selective targeting of the molecules and pathways that are involved in fibroblast activation , either singly or in combination , may offer new approaches in the treatment of fibrosis .
the elevated expression of profibrotic mirnas and/or reduced expression of antifibrotic mirnas are likely to be important factors in the development of fibrosis in ssc patients .
the regulation of mirna expression could be therapeutically exploited to offer new treatments for ssc , and mirnas circulating in the blood represent potential biomarkers for fibrosis . in this review ,
we summarize recent advances in identifying the specific mirnas involved in ssc pathogenesis , underscored by the potential use of specific mirnas as novel biomarkers and therapeutic targets for the future management of ssc patients .
mirna expression profiles have been successfully utilized in the classification of human cancer , heart disease and autoimmune diseases .
studies of mirna expression profiles in patients with ssc are expected to result in similar findings , revealing a biological and clinical relevance between mirna expression and ssc disease state and severity . in our previous study
the two major subgroups of ssc , diffuse cutaneous scleroderma ( dssc ) and limited cutaneous scleroderma ( lssc ) , displayed different mirna expression signatures .
we identified 42 mirnas that were differentially expressed in dssc and 60 mirnas with altered expression in lssc .
a total of 21 mirnas displaying altered expression in ssc skin samples were common between dssc and lssc . among these mirnas with changed expression , many were predicted to function as inhibitors of numerous mrnas involved in autoimmune , vascular and fibrotic processes .
identified 24 differentially expressed mirnas from patients with ssc , including 9 upregulated mirnas and 15 downregulated mirnas . through bioinformatics analysis and literature retrieval
, they identified the target genes regulated by six of the mirnas whose differential expression was correlated with ssc pathogenesis and hypothesized that the abnormal expression of some mirnas may be important molecular events during the process of ssc genesis , development and evolution .
the overlap between the mirna expression signature identified in our study , which examined patients with ssc , and the signature identified in li 's study is relatively limited . in our study
, we used skin samples from four patients with dssc , two patients with lssc , and three healthy volunteers as normal controls for mirna array analysis . in li 's study , skin samples from three patients with ssc ( the authors did not mention the ssc subgroups ) and three healthy control volunteers were used for mirna array analysis .
the difference between the two studies can be explained by diversity in the disease severity , medical history and ethnicity of ssc patients , mirna profiles of specific cell - types , and even more specifically , the developmental and functional organ status .
both our and li 's study revealed the unique mirna expression signature in ssc patients , demonstrating that many of the dysregulation mirnas exerted pro- or antifibrotic effects and participated in fibrosis .
immune perturbations and vascular injury precede and contribute to the development of fibrosis in ssc , which , in turn , further exacerbate vascular and immune damage .
tissue fibrosis is the hallmark of ssc and is responsible for most ssc clinical manifestations .
microarray and real - time pcr analysis has shown that approximately 40 mirnas are linked to fibrosis in various organs and disease settings .
most of these mirnas are regulated by transforming growth factor ( tgf)- , which has been considered a crucial participant in the pathogenesis of the ssc fibrotic process .
many mirnas have been shown to participate in repairing and remodeling the matrix including collagens , matrix metalloproteinases and integrins .
mir-133 , mir-141 , mir-200a / b , mir-21 and mir-590 have been shown to directly induce or inhibit fibrosis by targeting the tgf-/smad canonical signaling pathway .
an analysis of mir-132 , mir-133 and the mir-1792 cluster ( mir-18a , 19a / b ) revealed that their targets were connective tissue growth factor .
mir-29a / b / c , mir-377 ( clustered with mir-382 ) and mir-449a / b have shown to be involved with ecm structural proteins or enzymes active in ecm remodeling .
let7 , mir-132 , mir-155 , mir-192 and mir-382 have been implicated in indirectly regulating fibrogenesis by affecting the epithelial - to - mesenchymal transition while mir-132 , mir-15b , mir-16 , mir-150 , mir-27a , mir-27b , mir-335 and mir-34a were found to induce myofibroblast proliferation and resistance to apoptosis .
mir-29 has been extensively studied and is the best characterized direct regulator of ecm synthesis .
mir-29 is known to directly repress the expression of collagen i , collagen iii and collagen iv .
platelet - derived growth factor - b and tgf- can downregulate mir-29a expression , leading to the further upregulation of platelet - derived growth factor - b and tgf-. this positive feedback loop can lead to the uncontrolled accumulation of ecm proteins .
evidence has suggested that a strong downregulation of mir-29 expression in tissues and cells can cause a fibrotic response , including cardiac , pulmonary and liver fibrosis .
mir-29 was shown to negatively regulate col1a1 , col1a2 , col3a1 , fbn1 and eln1 gene expression and to be responsible for ecm synthesis in myocardial infarction . in lung fibrosis , mir-29 targeted many fibrosis - associated genes , including both structural ecm genes and enzymes involved in tissue remodeling .
tetrachloride - induced hepatic fibrogenesis mice , the mir-29 family was found to be significantly decreased in livers .
the overexpression of mir-29b in a mouse model resulted in protection against salt - induced hypertensive renal medullary fibrosis after a high - salt diet .
the silencing of mir-29b in the kidney of these animals caused the upregulation of a large number of ecm and ecm - modulating genes .
our previous study demonstrated that mir-29 was decreased in both ssc skin tissues and primary fibroblasts .
identified mir-29 as a key regulator of collagen expression in ssc . in their study , mir-29 was downregulated in cultured dermal fibroblasts derived from ssc skin , resulting in excessive collagen production . in a bleomycin - induced mouse model of skin fibrosis , the tyrosine - kinase inhibitor imatinib mesylate
was shown to inhibit the platelet - derived growth factor - b and tgf- pathways and restore mir-29a levels .
in addition , serum mir-29a levels were found to be significantly decreased in the very early stage of ssc .
all of these studies suggest that mir-29 expression was downregulated by tgf- and that decreased mir-29 expression was involved in ssc tissue fibrosis .
other signaling pathways also involve mir-29 , such as the wnt / b - catenin , nuclear factor - kappab ( nf-b ) and mitogen - activated protein kinases pathways .
in addition to mir-29 , there are many other mirnas known to undergo ssc fibrosis regulation , ( table 1 ) .
our preliminary data showed that mir-21 was increased and that mir-145 was decreased in both ssc skin tissues and fibroblasts .
after stimulation with tgf- , the expression of mir-21 was increased and that of smad7 mrna was decreased .
further , we studied the direct one - to - one relationship between mir-21 and smad7 .
we found that smad7 was a direct target of mir-21 and that mir-21 was induced by tgf- , which in turn promoted the tgf--induced fibrogenic activation of skin fibroblasts through the targeting of smad7 ( data not shown ) .
honda et al . demonstrated decreased mir-150 expression in ssc fibroblasts both in vivo and in vitro .
the transfection of a mir-150 inhibitor into normal fibroblasts induced the expression of integrin 3 , phosphorylated smad3 and col1a1 , whereas the forced overexpression of mir-150 resulted in the downregulation of these genes in ssc fibroblasts .
mir-196a directly contributed to the constitutive upregulation of col1a1 and col1a2 expression in ssc fibroblasts .
the ddr2mir-196a pathway was a negative feedback system and impairment of this pathway may be involved in the pathogenesis of ssc .
the inhibition of mir-196a led to the overexpression of col1a1 and col1a2 in normal fibroblasts , whereas the overexpression of mir-196a resulted in the downregulation of col1a1 and col1a2 in ssc fibroblasts . compared with untreated normal fibroblasts ,
the mirnas described above exert pro- or antifibrotic effects in ssc through different signaling pathways .
they may be common regulators of fibrosis in different organs and disease settings . inhibiting the function of these mirnas through the use of anti - mir oligonucleotides , small - molecule inhibitors , mirna sponges and mirna masks / target protectors and promoting
the activity of antifibrotic mirnas through gene therapy or delivery of mirna mimics can serve as novel therapeutic options in the treatment of fibrosis .
fibrosis is the most important and prominent clinical feature of ssc and occurs in multiple organs . the extent and severity of tissue fibrosis correlates with prognosis and mortality in ssc .
however , there is an unmet need for reliable and accurate biomarkers that reflect the fibrotic process in ssc .
serum mirna levels can be used as valuable biomarkers for the diagnosis , prognosis and therapeutic value of various diseases .
the expression pattern of mirnas is reflective of the underlying pathophysiologic processes specific to various disease states .
disease - associated mirnas can be measured from various sources , such as blood components , biologic fluids and tissue samples .
mirnas are highly stable and appear to be resistant to variations in sample handling ( they can even be detected in formalin - fixed paraffin - embedded samples by microarray profiling or in situ hybridization ) , which increase their appeal as practical biomarkers .
good correlations between serum and tissue mirna profiles have already been shown for several cancers and various other diseases .
reports suggest that mirnas have important roles in systemic rheumatic diseases . various diseases and different stages of the same disease
the elevated expression of profibrotic mirnas and/or reduced expression of antifibrotic mirnas are likely to be important factors in the development of fibrosis in ssc . circulating mirnas are promising biomarker candidates for the diagnosis , prognosis and assessment of disease activity and severity ( table 2 ) .
serum mir-150 levels were found to be decreased in ssc patients , and ssc patients with lower serum mir-150 levels had more severe clinical manifestations .
patients with lower serum mir-196a levels had a significantly higher ratio of dssc : lssc , a higher modified rodnan total skin thickness score , and a higher prevalence of pitting scars than those with higher mir-196a levels .
serum levels of mir-92a were significantly higher in ssc patients than in normal subjects , and patients with increased mir-92a levels tended to have telangiectasia at a lower frequency than those with normal levels .
collected serum samples from 61 patients with ssc , 8 patients with systemic lupus erythematosus , 8 patients with dermatomyositis , 12 patients with scleroderma spectrum disorder and 20 healthy controls .
analysis of the samples by real - time pcr indicated that the mir-142 - 3p levels in patients with ssc were significantly higher than those in patients with systemic lupus erythematosus , dermatomyositis , scleroderma spectrum disorder ( ssd ) and healthy control subjects .
the serum levels of mir-142 - 3p were correlated with the severity of ssc fibrosis and may be useful diagnostic markers for the presence of ssc and differentiation of ssc from scleroderma spectrum disorder .
serum mir-29a levels were not downregulated in ssc , and there was no statistically significant difference between healthy control subjects and ssc patients . however , ssc patients with reduced mir-29a levels had significantly higher right ventricular systolic pressure by doppler echocardiography than those with normal mir-29a levels .
although the cause of pulmonary hypertension in ssc is still uncertain , this result suggested that mir-29a also has a role in the pathogenesis of pulmonary hypertension .
mirnas are important post - transcriptional regulators that may repress more than 60% of all mammalian protein - coding genes . they are strongly associated with the pathogenesis of a wide range of human diseases .
disease - associated mirnas represent a new class of targets for the development of mirna - based therapeutic modalities , which may yield patient benefits that are unobtainable using other therapeutic approaches . as outlined in this review ,
there is increasing evidence that mirnas have a role in the regulation of fibrosis in ssc patients .
similar to antisense molecules that have been used therapeutically for fibrosis , the use of mirna as a therapeutic target provides theoretical advantages over the current drug design strategies in ssc fibrosis , which are focused on single - gene targeting .
the specific inhibition of a mirna or the addition of a single mirna mimetic may produce a phenotype that is derived from a complex set of gene expression changes .
clinical results with mir-122 inhibition in hcv patients validate the concept that mirna modulation in humans is possible .
although the field of mirna therapeutics is in its infancy , promising data in both laboratory animals and in humans already exist . because mirnas exhibit remarkable stability , as well as an ease and reliability of detection , mirnas from blood samples or urine samples of patients hold promise as novel noninvasive clinical biomarkers , especially for early diagnosis .
provocative ideas on mirna and its role in the pathogenesis of diverse autoimmune diseases including ssc have been put forward .
the identification of specific mirnas as biomarkers of disease severity will pave the way for the development of disease- and stage - specific targeted therapies .
a search for original articles published between 2007 and 2013 was performed using the pubmed database .
the search terms used were microrna , ' fibrosis , ' ssc or scleroderma or systemic sclerosis ' and precursor and process , ' alone and in combination .
papers cited in this review were selected based on the authors ' view of their direct relevance to the current topic . | mirnas are important post - transcriptional regulators . the aberrant expression of mirnas is strongly associated with the initiation and progression of pathophysiologic processes in a wide range of human diseases .
scleroderma ( systemic sclerosis ; ssc ) is a highly heterogeneous autoimmune disease that includes the progressive fibrotic replacement of normal tissue architecture in multiple organs .
our previous studies have suggested that ssc skin tissues display a different mirna expression signature than that found in normal controls .
mirnas with pro- or antifibrotic properties are found to be dysregulated in ssc skin fibrosis .
serum mirna levels are associated with ssc activity and severity .
mirnas have the potential to be therapeutic targets and serve as biomarkers for ssc diagnosis and assessment of disease state and severity .
this review summarizes the ssc mirna expression signature and the roles of dysregulation of mirnas in ssc tissues and serum and examines the future therapeutic potential of targeting mirnas in the management of ssc patients . | Introduction
miRNA expression signature in SSc fibrosis
miRNAs dysregulated in SSc fibrosis
miR-29
Other miRNAs
miRNA as biomarkers in SSc
Conclusions
Review criteria |
magnetic resonance imaging studies have used a wide range of techniques to explore different properties of the human connectome . a simplified conceptual overview of how some of the most commonly used imaging techniques relate to these properties is presented in figure 1 . a schematic overview of the different neuroimaging techniques available for measuring structural and functional properties of the human connectome .
dcm , dynamic causal modeling ; dwi , diffusion - weighted imaging ; gca , granger causality analysis ; ica , independent component analysis ; mca , morphometric covariance analysis ; ppi , psychophysiological interactions ; pwc , pair - wise correlations ; tbss , tract - based spatial statistics ; t1 , t1-weighted imaging ; vba , voxel - based analysis ; wm , white matter .
connectome structure refers to the anatomical connections between different brain regions , and is measured in mri studies using either t1- or diffusion - weighted imaging ( dwi ) .
the former indirectly infers connectivity between regions either through voxel - wise mapping of variations in white matter density , or covariance in regional morphometric parameters , such as gray matter volume or cortical thickness .
these morphometric covariance analyses ( mca ) can be performed either between a single seed region and all other brain voxels ( pezawas et al . , 2005 ; meyer - lindenberg et al . , 2007 ) , between ( many ) pairs of a priori defined regions ( he et al .
2008 ) , or following multivariate decomposition of the data using techniques such as independent component analysis ( ica ; xu et al .
dwi provides a more direct measure of the integrity and trajectory of anatomical connections , subject to certain caveats ( mori and zhang , 2006 ; zalesky and fornito , 2009 ; jbabdi and johansen - berg , 2011 ) .
voxel - based analysis ( vba ) techniques applied to estimates of white matter density / volume or dwi - derived measures ( e.g. , axial / radial diffusivity and/or fractional anisotropy ) allow whole - brain mapping of differences in white matter integrity or morphometry .
this includes methods such as tract - based spatial statistics ( tbss ; smith et al . , 2006 ) .
alternatively , connectivity may be indexed with dwi by deterministic or probabilistic tractography of fiber trajectories linking different brain regions , either at the level of specific pairs of brain regions , or between every possible pair of regions defined using some comprehensive cerebral parcelation ( hagmann et al . , 2008 ; zalesky et al . ,
connectivity is estimated either as the number of tracts intersecting each regional pair ( zalesky et al . , 2011 ) , or some index of tissue integrity averaged across the extent of the reconstructed trajectory ( van den heuvel et al . , 2010 ) .
note that all of these anatomical measures only provide undirected estimates of inter - regional connectivity ; i.e. , they may imply that two regions are connected , but they can not resolve whether the connection runs from region a to b or vice - versa .
magnetic resonance imaging studies of connectome function generally use blood - oxygenation - level dependent ( bold ) imaging .
functional interactions between regions can be categorized into one of two broad classes : functional and effective connectivity ( friston , 1994 ) .
effective connectivity refers to the influence that one neuronal system exerts over another and allows inferences concerning causal ( directed ) interactions between regions .
the most rudimentary model of effective connectivity is provided by psychophysiological interaction ( ppi ) analysis ( friston et al . , 1997 ) , which involves analyzing task - related changes in connectivity between a seed region and all other brain voxels .
dynamic causal modeling ( dcm ; friston et al . , 2003 ) and granger causality analysis ( gca ; bressler and seth , 2011 ) provide more elaborate means for inferring causal interactions between brain regions but have seldom been studied in the context of genetically informative designs ( though , see curcic - blake et al .
functional connectivity refers to a statistical dependence between regionally distinct neurophysiological recordings and is undirected in nature .
most frequently , this is measured through simple pearson correlation of regional activity time courses .
this covariance is typically studied between a specific seed region and all other brain voxels ( termed a seed - based correlation analysis ) , or in a pair - wise correlation ( pwc ) analysis where associations between either a single pair of a priori defined regions , or multiple pairs of regions , are computed .
another popular technique for studying functional connectivity involves multivariate spatio - temporal decomposition using techniques such as spatial ica ( beckmann and smith , 2004 ; calhoun et al . , 2004 ) , which offers a data - driven method for identifying spatially independent networks of voxels with temporally coherent activity .
a final important distinction that applies to studies of both connectome function and structure concerns whether inter - regional interactions are studied at the level of connectivity or topology .
studies of connectivity are concerned with measuring variations in the strength and nature ( e.g. , structural or functional , positive or negative ) of connectivity between regions .
studies of topology are concerned with how connections between different brain regions are configured with respect to each other .
these analyses require relatively comprehensive mapping of inter - regional connectivity using a pwc approach combined with graph analysis to characterize such topological variations ( bullmore and sporns , 2009 ; bullmore and bassett , 2011 ) .
briefly , graph analysis involves modeling the brain as a graph of nodes , representing brain regions , connected by edges , representing some measure of inter - regional structural or functional interaction .
the method allows a wide range of topological properties representing diverse aspects of connectome organization to be computed ( bullmore and sporns , 2009 ; rubinov and sporns , 2010 ) . though topology and connectivity are not necessarily independent ( barrat et al .
lynall et al . , 2010 ) , the distinction between them provides a useful heuristic for evaluating findings in imaging connectomics . in the following , we first consider studies examining genetic influences on brain network connectivity before discussing studies of network topology .
most of the connectomic phenotypes studied in imaging genetics studies to date have been relatively specific , focusing on particular neural circuits of theoretical interest .
these have typically been investigated by analyzing connectivity between specific pairs of brain regions , or between one seed region and all other brain voxels .
most of this work has used fmri and therefore focuses on understanding how specific genetic variants influence functional connectivity between regions . here , we focus on studies of three neural circuits that have been widely studied in imaging genetic research
examples of how genetic variants impact other neural systems can be found elsewhere ( e.g. , meyer - lindenberg et al . , 2007 ; tan et al . , 2007 ; kempf et al .
, 2008 ; krugel et al . , 2009 ; rasetti et al . , 2011 ) .
the first mri study to examine genetic influences on a connectomic phenotype examined how variation in the human serotonin ( 5-ht ) transporter gene slc6a4 impacted functional connectivity between the amygdala and medial prefrontal cortex ( mpfc ; heinz et al . , 2005 ) . a variable number of tandem repeats ( vntr ) in the 5 promoter region ( 5-httplr ) of slc6a4 influences mrna and protein expression such that a short ( s ) compared to long ( l ) allele is associated with reduced transcriptional efficiency ( heils et al . , 1996 ) and reduced 5-ht transporter binding in the brain ( heinz et al . , 2000 ) .
accordingly , the s allele has been variably associated with personality traits associated with negative emotionality ( lesch et al .
, 1996 ; munafo et al . , 2009 ) , alcohol dependence ( mchugh et al . , 2010 ) , mood disorders ( clarke et al . , 2010 ) , and suicidality ( li and he , 2007 ) . in some cases ,
these associations may be moderated by environmental factors ( caspi et al . , 2010 ) .
consistent with an association between the s allele and negative emotions , an early fmri study found that human carriers of this allele showed increased amygdala activation during perception of aversive emotional stimuli ( hariri et al . , 2002 ) .
in a separate study using a similar task , an association was reported between the s allele and greater functional connectivity between the amygdala and anterior mpfc ( heinz et al . , 2005 ) .
this finding was noteworthy given the abundant evidence pointing to the amygdala and mpfc as critical nodes within a broader neural network subserving emotional regulation ( phillips et al .
these findings were subsequently replicated by an independent group , who also reported an association between the s allele and reduced functional connectivity between the amygdala and more posterior medial prefrontal regions located in pre- and sub - genual anterior cingulate cortex ( acc ; pezawas et al . , 2005 ) .
an anatomical basis for these functional effects was suggested by a corresponding reduction in gray matter volume covariance between the amygdala and pre - genual acc in s allele carriers compared to l / l homozygotes , a finding replicated in an independent study using dti - derived measures of anatomical connectivity ( pacheco et al . , 2009 ) .
the functional connectivity measures predicted approximately 30% of the variance in temperamental measures of anxiety - related traits such as harm avoidance .
no such association was found with regional indices of activation or volume , indicating that the connectomic measures provided a more direct physiological marker of clinically relevant behavioral phenotypes . collectively , these results suggest that variation in slc6a4 regulates fronto - limbic dynamics in highly specific ways , such that the s allele up - regulates functional connectivity between amygdala and anterior mpfc and down - regulates connectivity between amygdala and perigenual acc ( figure 2 )
cortical surface renderings display the stereotactic peaks of regions in prefrontal cortex where functional connectivity with the amygdala is impacted by common genetic variants .
different colors denote the different genes studied in relation to fronto - limbic functional connectivity ; namely , slc6a4 ( heinz et al . , 2005 ; pezawas et al . , 2005 ; schardt et al . , 2010 ) ,
maoa ( buckholtz et al . , 2008 ) , comt ( rasch et al . , 2010 ) , and drd2 ( blasi et al . , 2009 ) .
arrows indicate whether functional connectivity was increased ( ) or decreased ( ) in carriers of the putative risk allele for each gene .
the distinction between cyan and blue foci for slc6a4 studies differentiates a single study examining functional connectivity during cognitive regulation of emotion ( schardt et al . ,
this distinction illustrates how the risk allele of 5-httplr polymorphism of this gene can be associated with either increased or decreased functional connectivity with the amygdala in adjacent regions of right pre - genual acc , depending on task context . another genetic variant shown to impact
fronto - limbic connectivity is a vntr in the upstream promoter region of the x - linked monoamine oxidase a ( maoa ) gene ( buckholtz et al . , 2008 ) .
maoa is the main enzyme responsible for catabolizing synaptic 5-ht and norepinephrine , and maoa expression is higher when the vntr comprises 3.54 repeats ( maoa - h ) than when 2 , 3 , or 5 repeats are present ( maoa - l ; sabol et al . , 1998 ) .
the low activity allele has been shown to interact with childhood stressors to increase risk for adverse mental health outcomes such as aggressive behavior and mood disturbances ( caspi et al . , 2002 ; kim - cohen et al . , 2006 ;
, functional connectivity between the amygdala and anterior mpfc is higher in maoa - l individuals compared to maoa - h participants performing an emotional perception task ( buckholtz et al . , 2008 ) , a finding that parallels the functional connectivity increases between these two regions found in carriers of the 5-httplr - s allele ( heinz et al . , 2005 ; pezawas et al . , 2005
the common effect of the maoa - l and 5-httplr - s alleles on amygdala mpfc circuitry may result from a shared influence on synaptic serotonin levels , as both are thought to be associated with increased brain 5-ht .
greater functional connectivity between the amygdala and anterior mpfc may therefore represent a final common pathway for genetically mediated increases in synaptic 5-ht concentrations .
contrary to this view however , recent imaging evidence suggests that 5-htpplr genotype has little effect on basal synaptic serotonin levels ( jedema et al . , 2010 ; murthy et al . , 2010
as such , the precise mechanism through which the polymorphism influences mpfc amygdala connectivity remains unclear , but may be related to its emerging role in neurodevelopment ( ansorge et al .
regardless of the precise mechanism , it is noteworthy that the effects seem specific to variants impacting on 5-ht function , as functional polymorphisms in genes that do not directly impact this neurotransmitter show no effect on amygdala mpfc connectivity . for example , two genetic variants that influence synaptic dopamine levels , the val met polymorphism of the catechol - o - methyl transferase ( comt ) gene , and the rs1076560 polymorphism of the d2 receptor gene drd2 , influence functional connectivity between the amygdala and lateral , but not medial , pfc ( buckholtz et al . , 2008 ;
these findings point to highly specific genetic influences on different components of fronto - limbic circuitry .
some of these genetic effects can be expressed in different ways depending on psychological context .
for example , during conditions requiring cognitive regulation of emotion , s allele carriers actually show increased functional connectivity between the amygdala and perigenual acc ( schardt et al . , 2010 ) .
this result indicates that the previously reported reduction in functional coupling between these regions does not reflect a hard - wired deficit , despite evidence for anatomical connectivity reductions between these regions ( pezawas et al . , 2005 ) .
rather , the connectivity reduction may reflect a bias to process aversive stimuli in maladaptive ways , which may be ameliorated given appropriate environmental circumstances . in this regard , the connectivity increase in s allele carriers during cognitive regulation of emotion may reflect the deployment of greater processing resources to overcome this processing bias .
these context - specific effects on connectomic phenotypes are consistent with evidence for environmental factors such as life stress moderating the influence of slc6a4 genotype on functional connectivity of the amygdala ( canli et al . , 2006 ) .
to summarize , studies of the slc6a4 and maoa genes provide examples of how genetic influences on brain connectivity can be expressed in circuit specific ways , and in a manner contingent on environmental context .
the circuit- and context - specificity of these effects is underscored by work on other genes , such as ca1cnac ( erk et al .
, 2010 ) , 5 acetylcholine receptor subunit ( chrna5 ; hong et al . , 2010 ) ,
brain - derived neurotrophic factor ( bdnf ; mukherjee et al . , 2011 ) , and 2b - adrenergic receptor ( adra2b ; rasch et al . , 2009 ) .
variants in each of these genes have been shown to impact fronto - limbic circuitry in a task - dependent manner , and in regions outside the amygdala
understanding how each of these variants impact different components of fronto - limbic circuits , and under what conditions , will no doubt prove to be a challenging task .
fronto - temporal connectivity is of particular interest to schizophrenia researchers , as diverse lines of evidence point to altered structural and functional interactions between frontal and temporal brain regions as a core feature of the disease phenotype ( weinberger et al . , 1992 ; meyer - lindenberg et al . , 2005 ; ellison - wright and bullmore , 2009 ; van den heuvel et al . , 2010 ;
accordingly , variation in several candidate risk genes for schizophrenia has been shown to impact fronto - temporal connectivity , including loci in comt ( bertolino et al . , 2006 ; dennis et al . , 2010b ) ,
rgs4 ( buckholtz et al . , 2007 ) , ppp1r1b encoding daarp-32 ( curcic - blake et al . , 2012 ) , and erbb4 ( konrad et al . , 2009 ) .
one genetic variant receiving increasing attention for its effects on fronto - temporal circuitry is the rs1344706 polymorphism of znf804a .
the gene encodes a zinc finger domain protein with unknown function , although preliminary evidence suggests a role in oligodendrocyte regulation ( riley et al . , 2010 ) and neurodevelopment ( chung et al . , 2010 )
the rs1344706 polymorphism has now been implicated as a genome - wide significant risk variant for both schizophrenia and bipolar disorder in several independent samples ( odonovan et al .
steinberg et al . , 2011 ) , with fine - mapping of nearly all common variants in the gene confirming that rs1344706 is the most strongly associated marker ( williams et al . , 2011 ) .
the first study to investigate the effects of the rs1344706 polymorphism on the brain used fmri to examine activation and functional connectivity during performance of a working memory and emotion perception task ( esslinger et al . ,
the variant was not found to impact measures of regional activation , but did influence inter - regional functional connectivity , suggesting its effects show higher penetrance at the level of distributed neural systems .
specifically , carriers of the risk associated a allele demonstrated reduced functional connectivity of the dorsolateral pfc with other ipsi- and contra - lateral prefrontal regions , as well as increased functional connectivity between dorsolateral pfc and the hippocampal formation , during working memory performance .
increased fronto - hippocampal functional connectivity had previously been reported in a positron emission tomography study of patients with schizophrenia ( meyer - lindenberg et al .
, 2005 ) and was thus interpreted as a possible cip mediating the role of znf804a in risk for the disorder .
the a allele was also associated with enhanced functional connectivity within fronto - limbic circuits during the emotional perception task , which was interpreted as a possible cip for bipolar disorder .
thus , this study pointed to context - specific and pleiotropic effects of the rs1344706 polymorphism with relevance for understanding its role in risk for both schizophrenia and bipolar disorder . a subsequent study in an independent sample using the same working memory task replicated the association between the a allele and both increased fronto - hippocampal functional coupling and decreased fronto - frontal connectivity ( rasetti et al .
, 2011 ) . the lack of a significant effect on regional activation was also replicated .
similar connectivity changes were apparent in schizophrenia patients and their unaffected relatives suggesting that these changes may indeed represent a candidate cip for the disorder ( figure 3 ) .
however , the a allele carriers in this study showed stronger negative fronto - hippocampal functional connectivity , contrasting the original report of higher positive functional connectivity between these two regions ( esslinger et al . , 2011 ) .
these results suggest distinct interpretations : stronger positive connectivity implies greater functional integration or cooperation between regions ; stronger negative connectivity points to antagonistic or competitive dynamics ( clare kelly et al . , 2008 ) .
the reasons for this discrepancy are unclear , although the fact that stronger negative functional connectivity has been found in patients and their unaffected relatives ( meyer - lindenberg et al .
, 2005 ; rasetti et al . , 2011 ) suggests that it may provide a more faithful representation of genetically influenced pathophysiological processes in schizophrenia .
illustration of the hippocampal region where functional connectivity with a seed in right dorsolateral pfc was influenced by znf804a variation and was found to differ between schizophrenia patients , their unaffected relatives and healthy controls .
the two bar charts plot parameter estimates for fronto - hippocampal functional connectivity as a function of znf804a in healthy controls ( middle ) , and in controls , patients , and siblings ( right ) .
carriers of the a risk allele showed an increasing trend toward greater negative functional connectivity , as did patients with schizophrenia .
the effect of the rs1344706 variant on fronto - hippocampal connectivity may be less robust than its effect on fronto - frontal coupling , and may only be expressed in certain contexts .
in one recent study , the variant was found to influence fronto - hippocampal functional connectivity only during working memory performance , whereas it impacted fronto - frontal coupling during working memory , emotion perception , and resting - state paradigms ( esslinger et al . , 2011 ) .
thus , the effect on fronto - frontal connectivity may be generalized , possibly resulting from an influence on anatomical connectivity between these regions ( wei et al . , 2010 ) , whereas the association with fronto - hippocampal coupling may be context - specific .
this result is consistent with research into the effects of slc6a4 variation on fronto - limbic circuitry ( e.g. , schardt et al . , 2010 ) demonstrating the highly context - specific influences of genetic variants of brain functional dynamics .
precisely mapping the circumstances under which these genetic effects are expressed will be an important step in characterizing the functional significance of any putative cips related to znf804a variation , particularly given recent evidence that schizophrenia may also be characterized by a profile of both generalized and context - specific functional connectivity deficits which affect prefrontal regions in particular ( fornito et al . , 2011a ) .
the dmn comprises regions of posterior cingulate cortex ( pcc ) , precuneus , medial pfc and lateral parietal cortices , and functionally couples with the hippocampus under certain task contexts ( buckner et al . , 2008 ) .
the dmn characteristically shows elevated activity during passive rest conditions or tasks requiring introspective processing and deactivates during cognitively demanding tasks ( shulman et al . , 1997 ;
buckner et al . , 2008 ; harrison et al . , 2008 , 2011 ) .
alterations of dmn connectivity have been found in patients suffering a range of neuropsychiatric disorders including schizophrenia ( bluhm et al . , 2007 ) ,
autism ( kennedy and courchesne , 2008 ) , attention - deficit hyperactivity disorder ( castellanos et al . , 2008 ) ,
major depression ( greicius et al . , 2007 ) , and alzheimer s disease ( greicius et al . , 2004 ; see sonuga - barke and castellanos , 2007 ; zhang and raichle , 2010 for reviews ) . in the case of schizophrenia ,
similar alterations have been found in patients unaffected relatives , suggesting an association between dmn connectivity changes and genetic risk for the disorder ( whitfield - gabrieli et al . , 2009 ) .
accordingly , specific variants in genes linked to risk for psychosis , such as the d - amino acid oxidase ( daao ; papagni et al . , 2011 ; prata et al . , 2012 ) and neuregulin-1 ( nrg1 ; winterer et al
confirmation that functional connectivity of the dmn is under genetic control came from a recent resting - state fmri study of a sample of extended pedigrees comprising 333 individuals selected from 29 families ( glahn et al . ,
spontaneous neural dynamics measured during the so - called resting - state , when people lie quietly in the scanner without performing any specific task , are highly organized and correlate in a manner that recapitulates well - known functional networks ( biswal et al .
the topography of these networks is robust over time and individuals ( damoiseaux et al . , 2006 ; shehzad et al . ,
2009 ) and has been linked to neurophysiological measures ( he et al . , 2008 ; shmuel and leopold , 2008 ) .
in addition , spontaneous bold signal fluctuations influence task - evoked activity ( fox et al . , 2006 ; mennes et al . , 2011 ) ,
perception ( hesselmann et al . , 2008 ) , and behavior ( fox et al . , 2007 ) , suggesting that they represent an intrinsic and functionally important component of neural dynamics ( fox and raichle , 2007 ; though , see morcom and fletcher , 2007 ; fornito and bullmore , 2010 for caveats ) .
the extended pedigree design exploited the varying degree of genetic relatedness between participants to estimate genetic and environmental influences on functional connectivity of the dmn , as characterized using ica .
genetic influences on an averaged estimate of functional connectivity of the entire dmn were significant , with heritability ( h ) estimated to be 0.42 .
the degree to which each constituent region of the dmn was functionally connected with the entire network was also heritable , ranging from 0.33 to 0.42 for key nodes such as posterior cingulate / precuneus , mpfc , and lateral parietal cortex . despite the dmn s involvement in a wide range of diseases ,
the most frequently studied genetic locus in relation to dmn connectivity has been the apolipoprotein e ( apoe ) gene .
the gene encodes a lipoprotein that in the central nervous system , plays a role in coordinating mobilization of cholesterol , phospholipids , and fatty acids , and has been implicated in neuronal development , plasticity , and repair ( mahley and rall , 2000 ) .
homozygosity for the last is the most established genetic risk factor for late - onset alzheimer s disease , being associated with a > 10-fold increase in risk ( farrer et al . , 1997 ; bertram et al . , 2007 )
in contrast , average allelic summary odds ratios for non - apoe - related variants are 1.25 ( bertram and tanzi , 2008 ) .
apoe has also been implicated in the pathophysiology of schizophrenia and mood disorders ( gibbons et al . ,
several studies have demonstrated an association between the 4 allele and both increased and decreased functional connectivity of the dmn and medial temporal regions , though reports of increases have been more common in younger samples ( filippini et al . , 2009 ; dennis et al .
one hypothesis proposed to explain this age effect is that 4 carriers inherit a cip characterized by enhanced dmn connectivity early in life .
this enhanced connectivity facilitates the spread of amyloid- plaques , the primary pathological characteristic of alzheimer s disease , throughout the network and exacerbates risk of disease onset and progression ( machulda et al .
these plaques accumulate and result in a deterioration of dmn function and concomitant reduction of network connectivity relative to non - risk allele carriers .
though this postulate requires experimental verification , it is supported by reports that dmn regions are among the first to show aggregation of amyloid- plaques in alzheimer s disease and that these plaques disperse throughout the network with disease progression ( buckner et al . , 2005 , 2009 ) .
it also accords with reports that gray matter volume loss in neurodegenerative disease occurs within functionally connected networks ( seeley et al . , 2009 ) .
the effects of apoe genotype on functional connectivity are often apparent in the absence of any differences in gray matter volume , cognitive impairment , or -amyloid deposition ( dennis et al . , 2010a ; sheline et al .
in addition , one recent study found that resting - state connectivity measures of the dmn were able to differentiate between cognitively unimpaired 4 carriers with a family history of alzheimer s disease and individuals without any apparent genetic risk with an effect size approximately three times greater than was possible using measures of task - related activation during a memory encoding paradigm ( fleisher et al . , 2009 ) .
such findings provide a powerful demonstration of the enhanced sensitivity provided by connectomic measures for indexing the physiological effects of disease risk variants , and support the contention that these variants often show higher penetrance at the level of distributed neural systems .
the first mri study to examine genetic influences on a connectomic phenotype examined how variation in the human serotonin ( 5-ht ) transporter gene slc6a4 impacted functional connectivity between the amygdala and medial prefrontal cortex ( mpfc ; heinz et al . , 2005 ) .
a variable number of tandem repeats ( vntr ) in the 5 promoter region ( 5-httplr ) of slc6a4 influences mrna and protein expression such that a short ( s ) compared to long ( l ) allele is associated with reduced transcriptional efficiency ( heils et al . , 1996 ) and reduced 5-ht transporter binding in the brain ( heinz et al . , 2000 ) .
accordingly , the s allele has been variably associated with personality traits associated with negative emotionality ( lesch et al .
, 1996 ; munafo et al . , 2009 ) , alcohol dependence ( mchugh et al . , 2010 ) , mood disorders ( clarke et al . , 2010 ) , and suicidality ( li and he , 2007 ) . in some cases ,
these associations may be moderated by environmental factors ( caspi et al . , 2010 ) .
consistent with an association between the s allele and negative emotions , an early fmri study found that human carriers of this allele showed increased amygdala activation during perception of aversive emotional stimuli ( hariri et al . , 2002 ) .
in a separate study using a similar task , an association was reported between the s allele and greater functional connectivity between the amygdala and anterior mpfc ( heinz et al . , 2005 ) .
this finding was noteworthy given the abundant evidence pointing to the amygdala and mpfc as critical nodes within a broader neural network subserving emotional regulation ( phillips et al .
these findings were subsequently replicated by an independent group , who also reported an association between the s allele and reduced functional connectivity between the amygdala and more posterior medial prefrontal regions located in pre- and sub - genual anterior cingulate cortex ( acc ; pezawas et al . , 2005 ) .
an anatomical basis for these functional effects was suggested by a corresponding reduction in gray matter volume covariance between the amygdala and pre - genual acc in s allele carriers compared to l / l homozygotes , a finding replicated in an independent study using dti - derived measures of anatomical connectivity ( pacheco et al . , 2009 ) .
the functional connectivity measures predicted approximately 30% of the variance in temperamental measures of anxiety - related traits such as harm avoidance .
no such association was found with regional indices of activation or volume , indicating that the connectomic measures provided a more direct physiological marker of clinically relevant behavioral phenotypes . collectively , these results suggest that variation in slc6a4 regulates fronto - limbic dynamics in highly specific ways , such that the s allele up - regulates functional connectivity between amygdala and anterior mpfc and down - regulates connectivity between amygdala and perigenual acc ( figure 2 )
cortical surface renderings display the stereotactic peaks of regions in prefrontal cortex where functional connectivity with the amygdala is impacted by common genetic variants .
different colors denote the different genes studied in relation to fronto - limbic functional connectivity ; namely , slc6a4 ( heinz et al . , 2005 ; pezawas et al . , 2005 ; schardt et al . , 2010 ) ,
maoa ( buckholtz et al . , 2008 ) , comt ( rasch et al . , 2010 ) , and drd2 ( blasi et al . , 2009 ) .
arrows indicate whether functional connectivity was increased ( ) or decreased ( ) in carriers of the putative risk allele for each gene .
the distinction between cyan and blue foci for slc6a4 studies differentiates a single study examining functional connectivity during cognitive regulation of emotion ( schardt et al . , 2010 ) from others involving passive perception of emotional stimuli
this distinction illustrates how the risk allele of 5-httplr polymorphism of this gene can be associated with either increased or decreased functional connectivity with the amygdala in adjacent regions of right pre - genual acc , depending on task context .
another genetic variant shown to impact fronto - limbic connectivity is a vntr in the upstream promoter region of the x - linked monoamine oxidase a ( maoa ) gene ( buckholtz et al . , 2008 ) .
maoa is the main enzyme responsible for catabolizing synaptic 5-ht and norepinephrine , and maoa expression is higher when the vntr comprises 3.54 repeats ( maoa - h ) than when 2 , 3 , or 5 repeats are present (
the low activity allele has been shown to interact with childhood stressors to increase risk for adverse mental health outcomes such as aggressive behavior and mood disturbances ( caspi et al . , 2002 ; kim - cohen et al .
accordingly , functional connectivity between the amygdala and anterior mpfc is higher in maoa - l individuals compared to maoa - h participants performing an emotional perception task ( buckholtz et al . , 2008 ) , a finding that parallels the functional connectivity increases between these two regions found in carriers of the 5-httplr - s allele ( heinz et al . , 2005 ; pezawas et al . , 2005
; figure 2 ) . the common effect of the maoa - l and 5-httplr - s alleles on amygdala mpfc circuitry may result from a shared influence on synaptic serotonin levels , as both are thought to be associated with increased brain 5-ht .
greater functional connectivity between the amygdala and anterior mpfc may therefore represent a final common pathway for genetically mediated increases in synaptic 5-ht concentrations .
contrary to this view however , recent imaging evidence suggests that 5-htpplr genotype has little effect on basal synaptic serotonin levels ( jedema et al . , 2010 ; murthy et al . ,
2010 ) . as such , the precise mechanism through which the polymorphism influences mpfc
amygdala connectivity remains unclear , but may be related to its emerging role in neurodevelopment ( ansorge et al . , 2004 ) .
regardless of the precise mechanism , it is noteworthy that the effects seem specific to variants impacting on 5-ht function , as functional polymorphisms in genes that do not directly impact this neurotransmitter show no effect on amygdala mpfc connectivity .
for example , two genetic variants that influence synaptic dopamine levels , the val met polymorphism of the catechol - o - methyl transferase ( comt ) gene , and the rs1076560 polymorphism of the d2 receptor gene drd2 , influence functional connectivity between the amygdala and lateral , but not medial , pfc ( buckholtz et al .
these findings point to highly specific genetic influences on different components of fronto - limbic circuitry .
some of these genetic effects can be expressed in different ways depending on psychological context .
for example , during conditions requiring cognitive regulation of emotion , s allele carriers actually show increased functional connectivity between the amygdala and perigenual acc ( schardt et al . , 2010 ) .
this result indicates that the previously reported reduction in functional coupling between these regions does not reflect a hard - wired deficit , despite evidence for anatomical connectivity reductions between these regions ( pezawas et al . , 2005 ) .
rather , the connectivity reduction may reflect a bias to process aversive stimuli in maladaptive ways , which may be ameliorated given appropriate environmental circumstances . in this regard , the connectivity increase in s allele carriers during cognitive regulation of emotion may reflect the deployment of greater processing resources to overcome this processing bias .
these context - specific effects on connectomic phenotypes are consistent with evidence for environmental factors such as life stress moderating the influence of slc6a4 genotype on functional connectivity of the amygdala ( canli et al . , 2006 ) .
to summarize , studies of the slc6a4 and maoa genes provide examples of how genetic influences on brain connectivity can be expressed in circuit specific ways , and in a manner contingent on environmental context .
the circuit- and context - specificity of these effects is underscored by work on other genes , such as ca1cnac ( erk et al . ,
2010 ) , 5 acetylcholine receptor subunit ( chrna5 ; hong et al . , 2010 ) , brain - derived neurotrophic factor ( bdnf ; mukherjee et al . , 2011 ) , and 2b - adrenergic receptor ( adra2b ; rasch et al . , 2009 ) .
variants in each of these genes have been shown to impact fronto - limbic circuitry in a task - dependent manner , and in regions outside the amygdala
understanding how each of these variants impact different components of fronto - limbic circuits , and under what conditions , will no doubt prove to be a challenging task .
fronto - temporal connectivity is of particular interest to schizophrenia researchers , as diverse lines of evidence point to altered structural and functional interactions between frontal and temporal brain regions as a core feature of the disease phenotype ( weinberger et al . , 1992 ; meyer - lindenberg et al . , 2005 ; ellison - wright and bullmore , 2009 ; van den heuvel et al . , 2010 ;
fornito et al . , 2011a , 2012 ; pettersson - yeo et al . ,
accordingly , variation in several candidate risk genes for schizophrenia has been shown to impact fronto - temporal connectivity , including loci in comt ( bertolino et al .
, 2006 ; dennis et al . , 2010b ) , rgs4 ( buckholtz et al . , 2007 ) , ppp1r1b encoding daarp-32 ( curcic - blake et al . , 2012 ) , and erbb4 ( konrad et al . , 2009 ) . one genetic variant receiving increasing attention for its effects on fronto - temporal circuitry is the rs1344706 polymorphism of znf804a .
the gene encodes a zinc finger domain protein with unknown function , although preliminary evidence suggests a role in oligodendrocyte regulation ( riley et al . , 2010 ) and neurodevelopment ( chung et al . , 2010 ) .
the rs1344706 polymorphism has now been implicated as a genome - wide significant risk variant for both schizophrenia and bipolar disorder in several independent samples ( odonovan et al . , 2008 ; stefansson et al . , 2009 ;
riley et al . , 2010 ; steinberg et al . , 2011 ) , with fine - mapping of nearly all common variants in the gene confirming that rs1344706 is the most strongly associated marker ( williams et al . , 2011 ) .
the first study to investigate the effects of the rs1344706 polymorphism on the brain used fmri to examine activation and functional connectivity during performance of a working memory and emotion perception task ( esslinger et al . , 2009 ) .
the variant was not found to impact measures of regional activation , but did influence inter - regional functional connectivity , suggesting its effects show higher penetrance at the level of distributed neural systems .
specifically , carriers of the risk associated a allele demonstrated reduced functional connectivity of the dorsolateral pfc with other ipsi- and contra - lateral prefrontal regions , as well as increased functional connectivity between dorsolateral pfc and the hippocampal formation , during working memory performance . increased fronto - hippocampal functional connectivity
had previously been reported in a positron emission tomography study of patients with schizophrenia ( meyer - lindenberg et al . , 2005 ) and was thus interpreted as a possible cip mediating the role of znf804a in risk for the disorder .
the a allele was also associated with enhanced functional connectivity within fronto - limbic circuits during the emotional perception task , which was interpreted as a possible cip for bipolar disorder .
thus , this study pointed to context - specific and pleiotropic effects of the rs1344706 polymorphism with relevance for understanding its role in risk for both schizophrenia and bipolar disorder . a subsequent study in an independent sample using the same working memory task replicated the association between the a allele and both increased fronto - hippocampal functional coupling and decreased fronto - frontal connectivity ( rasetti et al . , 2011 ) .
similar connectivity changes were apparent in schizophrenia patients and their unaffected relatives suggesting that these changes may indeed represent a candidate cip for the disorder ( figure 3 ) .
however , the a allele carriers in this study showed stronger negative fronto - hippocampal functional connectivity , contrasting the original report of higher positive functional connectivity between these two regions ( esslinger et al .
these results suggest distinct interpretations : stronger positive connectivity implies greater functional integration or cooperation between regions ; stronger negative connectivity points to antagonistic or competitive dynamics ( clare kelly et al . , 2008 ) .
the reasons for this discrepancy are unclear , although the fact that stronger negative functional connectivity has been found in patients and their unaffected relatives ( meyer - lindenberg et al . , 2005 ; rasetti et al . , 2011 )
suggests that it may provide a more faithful representation of genetically influenced pathophysiological processes in schizophrenia .
illustration of the hippocampal region where functional connectivity with a seed in right dorsolateral pfc was influenced by znf804a variation and was found to differ between schizophrenia patients , their unaffected relatives and healthy controls .
the two bar charts plot parameter estimates for fronto - hippocampal functional connectivity as a function of znf804a in healthy controls ( middle ) , and in controls , patients , and siblings ( right ) .
carriers of the a risk allele showed an increasing trend toward greater negative functional connectivity , as did patients with schizophrenia .
the effect of the rs1344706 variant on fronto - hippocampal connectivity may be less robust than its effect on fronto - frontal coupling , and may only be expressed in certain contexts .
in one recent study , the variant was found to influence fronto - hippocampal functional connectivity only during working memory performance , whereas it impacted fronto - frontal coupling during working memory , emotion perception , and resting - state paradigms ( esslinger et al . , 2011 ) .
thus , the effect on fronto - frontal connectivity may be generalized , possibly resulting from an influence on anatomical connectivity between these regions ( wei et al . , 2010 ) , whereas the association with fronto - hippocampal coupling may be context - specific .
this result is consistent with research into the effects of slc6a4 variation on fronto - limbic circuitry ( e.g. , schardt et al . , 2010 ) demonstrating the highly context - specific influences of genetic variants of brain functional dynamics . precisely mapping
the circumstances under which these genetic effects are expressed will be an important step in characterizing the functional significance of any putative cips related to znf804a variation , particularly given recent evidence that schizophrenia may also be characterized by a profile of both generalized and context - specific functional connectivity deficits which affect prefrontal regions in particular ( fornito et al . , 2011a ) .
the dmn comprises regions of posterior cingulate cortex ( pcc ) , precuneus , medial pfc and lateral parietal cortices , and functionally couples with the hippocampus under certain task contexts ( buckner et al . ,
the dmn characteristically shows elevated activity during passive rest conditions or tasks requiring introspective processing and deactivates during cognitively demanding tasks ( shulman et al . , 1997 ;
buckner et al . , 2008 ; harrison et al . , 2008 , 2011 ) .
alterations of dmn connectivity have been found in patients suffering a range of neuropsychiatric disorders including schizophrenia ( bluhm et al . , 2007 ) ,
autism ( kennedy and courchesne , 2008 ) , attention - deficit hyperactivity disorder ( castellanos et al . , 2008 ) ,
major depression ( greicius et al . , 2007 ) , and alzheimer s disease ( greicius et al . , 2004 ; see sonuga - barke and castellanos , 2007 ; zhang and raichle , 2010 for reviews ) . in the case of schizophrenia ,
similar alterations have been found in patients unaffected relatives , suggesting an association between dmn connectivity changes and genetic risk for the disorder ( whitfield - gabrieli et al . , 2009 ) .
accordingly , specific variants in genes linked to risk for psychosis , such as the d - amino acid oxidase ( daao ; papagni et al . , 2011 ; prata et al . , 2012 ) and neuregulin-1 ( nrg1 ; winterer et al . , 2008
confirmation that functional connectivity of the dmn is under genetic control came from a recent resting - state fmri study of a sample of extended pedigrees comprising 333 individuals selected from 29 families ( glahn et al . ,
spontaneous neural dynamics measured during the so - called resting - state , when people lie quietly in the scanner without performing any specific task , are highly organized and correlate in a manner that recapitulates well - known functional networks ( biswal et al .
the topography of these networks is robust over time and individuals ( damoiseaux et al . , 2006 ; shehzad et al . , 2009 ) and
in addition , spontaneous bold signal fluctuations influence task - evoked activity ( fox et al . , 2006 ; mennes et al . , 2011 ) ,
perception ( hesselmann et al . , 2008 ) , and behavior ( fox et al . , 2007 ) , suggesting that they represent an intrinsic and functionally important component of neural dynamics ( fox and raichle , 2007 ; though , see morcom and fletcher , 2007 ; fornito and bullmore , 2010 for caveats ) .
the extended pedigree design exploited the varying degree of genetic relatedness between participants to estimate genetic and environmental influences on functional connectivity of the dmn , as characterized using ica .
genetic influences on an averaged estimate of functional connectivity of the entire dmn were significant , with heritability ( h ) estimated to be 0.42 . the degree to which each constituent region of the dmn was functionally connected with the entire network was also heritable , ranging from 0.33 to 0.42 for key nodes such as posterior cingulate / precuneus , mpfc , and lateral parietal cortex . despite the dmn s involvement in a wide range of diseases ,
the most frequently studied genetic locus in relation to dmn connectivity has been the apolipoprotein e ( apoe ) gene .
the gene encodes a lipoprotein that in the central nervous system , plays a role in coordinating mobilization of cholesterol , phospholipids , and fatty acids , and has been implicated in neuronal development , plasticity , and repair ( mahley and rall , 2000 ) .
homozygosity for the last is the most established genetic risk factor for late - onset alzheimer s disease , being associated with a > 10-fold increase in risk ( farrer et al .
in contrast , average allelic summary odds ratios for non - apoe - related variants are 1.25 ( bertram and tanzi , 2008 ) .
apoe has also been implicated in the pathophysiology of schizophrenia and mood disorders ( gibbons et al . ,
several studies have demonstrated an association between the 4 allele and both increased and decreased functional connectivity of the dmn and medial temporal regions , though reports of increases have been more common in younger samples ( filippini et al . , 2009 ; dennis et al .
, 2010a , b ; machulda et al . , 2011 ; westlye et al . ,
one hypothesis proposed to explain this age effect is that 4 carriers inherit a cip characterized by enhanced dmn connectivity early in life .
this enhanced connectivity facilitates the spread of amyloid- plaques , the primary pathological characteristic of alzheimer s disease , throughout the network and exacerbates risk of disease onset and progression ( machulda et al .
these plaques accumulate and result in a deterioration of dmn function and concomitant reduction of network connectivity relative to non - risk allele carriers .
though this postulate requires experimental verification , it is supported by reports that dmn regions are among the first to show aggregation of amyloid- plaques in alzheimer s disease and that these plaques disperse throughout the network with disease progression ( buckner et al . , 2005 , 2009 ) .
it also accords with reports that gray matter volume loss in neurodegenerative disease occurs within functionally connected networks ( seeley et al . , 2009 ) .
the effects of apoe genotype on functional connectivity are often apparent in the absence of any differences in gray matter volume , cognitive impairment , or -amyloid deposition ( dennis et al . , 2010a ; sheline et al .
in addition , one recent study found that resting - state connectivity measures of the dmn were able to differentiate between cognitively unimpaired 4 carriers with a family history of alzheimer s disease and individuals without any apparent genetic risk with an effect size approximately three times greater than was possible using measures of task - related activation during a memory encoding paradigm ( fleisher et al . , 2009 ) .
such findings provide a powerful demonstration of the enhanced sensitivity provided by connectomic measures for indexing the physiological effects of disease risk variants , and support the contention that these variants often show higher penetrance at the level of distributed neural systems .
the preceding discussion illustrates how genetic variation can influence the nature and strength of connectivity between different brain regions .
we now consider studies of genetic influences on how connections are arranged in the brain ; i.e. , how genes influence the topology of the connectome
the human cerebral cortex comprises an estimated 10 neurons connected by 10 fibers ( braitenberg and schz , 1991 ) .
though numerous , these connections represent only a small fraction of the total 5 10 connections that are possible .
similarly , neuronal connectivity in the nematode worm caenorhabditis elegans , which has been comprehensively mapped at the level of each and every synapse , comprises < 3% of the total number of connections possible while estimates of the degree of inter - regional connectivity ( i.e. , connectivity between distinct cytoarchitectonic regions ) in the macaque monkey macaca mulatta and the feline brain range from 18 to 38% ( latora and marchiori , 2003 ; kaiser and hilgetag , 2006 ) .
this sparsity suggests that connections between brain regions develop according to specific constraints and/or design principles that are likely to be under genetic control .
ramn y cajal ( 1995 ) proposed over a century ago that one critical organizational principle for brain networks involves the minimization of wiring costs .
the adaptive benefit of this principle is clear , as less wiring reduces the total energy required to support neuronal communication ( laughlin and sejnowski , 2003 ) .
accordingly , a substantial body of evidence has accumulated to suggest that pressure to minimize wiring costs can explain numerous aspects of cortical organization , including axonal branching and cortical folding patterns ( van essen , 1997 ; cherniak et al . , 1999 ) , neuronal morphology ( buzsaki et al . , 2004 ; chklovskii , 2004 ) , the spatial location of neurons and cytoarchitectonic fields ( klyachko and stevens , 2003 ; cherniak et al . , 2004 ) , and even the fraction of cortical volume occupied by axons and dendrites ( chklovskii et al . , 2002
however , wiring cost minimization alone can not account for all design features of nervous system networks ( chen et al . , 2006 ; kaiser and hilgetag , 2006 ) , and recent evidence points to a trade - off between cost minimization and the emergence of topological properties such as communication efficiency , which may be behaviorally advantageous but entail a wiring cost premium ( kaiser and hilgetag , 2006 ; bassett et al . , 2010 ) .
the topological efficiency of a network can be readily quantified using graph theoretic techniques . in complex networks ,
communication is more efficient when fewer connections must be traversed to transfer information between any two nodes ; the fewer the connections , the faster the rate of information transmission , and the lower the probability of signal degradation or transmission errors ( latora and marchiori , 2003 ) . in an economically wired network ,
long - range projections act as topological short - cuts that reduce the mean path length between regions and dramatically increase communication efficiency ( buzsaki et al . , 2004 ; kaiser and hilgetag , 2006 ) . however , these short - cuts come at the cost of increased wiring . in principle
it would be possible to maximize network efficiency simply by adding more connections ( latora and marchiori , 2003 ; achard and bullmore , 2007 ) , though the metabolic costs associated with forming and maintaining each connection limit the total volume of wiring that can be supported ( laughlin and sejnowski , 2003 ) .
this balance , between efficiency maximization and connection cost minimization , may be construed as one concerned with the optimization of connection cost - efficiency .
the importance of this balance for the connectome was recently underscored by a study showing that the physical embedding of the brain s connectivity architecture in the three - dimensional space of the skull is optimally cost - efficient , subject to certain higher - dimensional constraints ( bassett et al . , 2010 ) .
we examined the heritability of cost - efficient properties of functional brain network topology using resting - state fmri in healthy twins ( fornito et al . , 2011b ) .
network efficiency was measured using established graph theoretic methods ( latora and marchiori , 2001 ) , whereas connection costs were estimated indirectly using a measure of physical distance between regions .
we found that the balance between these two measures , taken as an index of network cost - efficiency , was highly heritable at a global level , with genetic factors accounting for approximately 60% of the phenotypic variance .
these influences were not uniformly distributed throughout the cortex , with regional estimates of significant genetic influences ranging from 0.30 to 0.80 ( figure 4 ) . some of the largest effects were seen in regions of lateral pfc and core components of the dmn , such as posterior cingulate and medial prefrontal cortices ( figure 4 ) .
heritability estimates in these regions are comparable to those reported for whole - brain and regional gray matter volume ( 0.66 < h < 0.97 ; thompson et al . , 2001 ; peper et al . , 2007 ) , cognitive abilities ( 0.30 < h < 0.80 ; mcclearn et al . , 1997 ; bouchard , 1998 ; boomsma et al . , 2002 ) , and various measures of personality and psychopathology ( 0.50 < h < 0.60 ; boomsma et al . , 2002 ) .
they also compare favorably with heritability estimates for simple measures of functional connectivity within the dmn ( 0.33 < h < 0.42 ; glahn et al . , 2010 ) , as well as regional task - related activation ( 0.40 < h < 0.80 ; koten et al . , 2009 ; blokland et al . ,
2011 ) and dti - derived measures of anatomical connectivity ( 0.55 < h < 0.85 ; chiang et al . , 2009 ) .
cortical regions showing statistically significant ( p < 0.05 , corrected ) heritability for regional cost - efficiency of network functional connectivity .
dmpfc , dorso - medial prefrontal cortex ; pcc , posterior cingulate cortex ; sfg , superior frontal gyrus ; pmfg , posterior middle frontal gyrus ; pcg , post - central gyrus ; spl , superior parietal lobule ; psts , posterior superior temporal sulcus .
the adaptive benefit of a cost - efficient neural architecture is intuitive , and it is plausible that evolution would favor connections that can provide high communication efficiency for low metabolic cost . evidence that greater topological efficiency and/or greater cost - efficiency predicts better performance on tests of intelligence and working memory ( bassett et al .
, 2009 ; li et al . , 2009 ; van den heuvel et al . , 2009 ; zalesky et al . ,
2011 ) , and that alterations of network efficiency and cost - efficiency are apparent in a range of patient groups , including schizophrenia ( bassett et al . , 2009 ; lynall et al . , 2010 ;
2011 ) , adhd ( wang et al . , 2009 ) , and alzheimer s disease ( lo et al . , 2010 ) , indicates that these findings have clear implications for psychiatric disorders . to our knowledge
however , no study to date has examined how variations in specific candidate genes influence brain network cost - efficiency , or any other topological properties of the connectome .
. indeed , graph analytic studies of neuroimaging data have pointed to several other candidate topological properties that may be under genetic control .
for example , both structural and functional human brain networks are characterized by a small - world topology ( achard et al . , 2006 ;
hagmann et al . , 2007 ) , which concurrently supports locally segregated and globally integrated connectivity to provide high dynamical complexity , a property that may have been favored by evolutionary processes ( sporns et al . , 2000
the human connectome also possesses a hierarchical , modular architecture ; i.e. , it can be decomposed into subsets of regions , termed modules , that show relatively high connectivity with each other than with other areas at multiple scales of resolution , allowing the formation of modules within modules and so on ( meunier et al . , 2009 ; bassett et al . ,
this property has been associated with enhanced functional stability and diversity ( kaiser et al . , 2007 ) , the emergence of self - organized , critical dynamics ( rubinov et al . ,
2011 ) , and may be intimately linked to optimization of connection cost - efficiency ( bassett et al . ,
these considerations raise questions as to whether genetic influences are specific to network cost - efficiency , or are related to other topological variations . in our analysis , we found no evidence for genetic influences on a range of other topological properties frequently studied in the graph theoretic literature , including clustering , path length , small - worldness , and global and local efficiency , although we did not examine modularity ( fornito et al . , 2011b ) .
other studies have however reported heritability values as high as 0.80 for some of these measures , computed for functional networks derived from electroencephalographic ( eeg ) recordings ( smit et al . , 2008 ) .
the reasons for these inconsistencies are unclear , but may be related to the imaging techniques employed or the enhanced power provided by the much larger sample analyzed in the eeg study . in general , the high degree of inter - correlation between different topological measures of brain network organization ( alexander - bloch et al . , 2010 ; lynall et al . ,
2010 ) , means that multivariate genetic analyses allowing categorization of different topological measures into distinct groupings defined by common genetic influences will be critical for identifying the primary genetic constraints on connectome development and organization .
this brief overview illustrates the potential power of imaging connectomics in the search for ips for neuropsychiatric disorders .
specifically , the available data indicate that connectomic measures are able to index the physiological effects of disease risk genes when simpler regional measures of activation or volume , or cognition , can not ( esslinger et al . , 2009 ; dennis et al .
, 2010a ; sheline et al . , 2010a ; machulda et al . , 2011 ; rasetti et al
they also indicate that variations in cips correlate with relevant behavioral indices when regional measures do not ( pezawas et al . , 2005 ; buckholtz et al . ,
2008 ) , and that certain connectomic phenotypes are highly heritable ( chiang et al . , 2009 ; glahn et al . , 2010 ;
collectively , these data support the idea that the penetrance of many disease risk genes is higher at the level of distributed brain systems than at the level of isolated brain regions , a conclusion consistent with an emerging consensus that many psychiatric disorders arise from genetically mediated vulnerabilities in discrete neural circuits ( meyer - lindenberg and weinberger , 2006 ; meyer - lindenberg , 2009 , 2010 ; insel , 2011 ) . as such
, cips may offer greater sensitivity for characterizing genetic effects in imaging genetic designs than more traditional , regionally focused neuroimaging measures .
one potential criticism of cips is that , by design , they are more complicated , requiring additional processing steps and assumptions for their derivation . characterizing activation or volume in a single region
is intrinsically univariate , requiring measurement of one property describing the behavior of that region , whereas connectivity measures are intrinsically bi or multivariate as they involve two or more regions by definition .
the problem is worse for graph analytic studies , which often incorporate multiple measures and processing steps when computing topological properties .
each additional measure and step can introduce noise into the analysis , making the resulting phenotypic characterizations less reliable ( habeck and moeller , 2011 ) , and placing an upper limit on power for detecting genetic effects ( kendler and neale , 2010 ; blokland et al . , 2011 ) .
this emphasis on simplicity and reliability must however be balanced with a need to define valid and pathophysiologically relevant ips .
most major psychiatric diseases are disorders of brain connectivity , and the physiological effects of putative disease risk variants are likely to be expressed throughout multiple , interconnected neural circuits .
the findings discussed here support these conclusions , and demonstrate that even highly derived topological properties , such as network cost - efficiency , can show high heritability ( fornito et al .
moreover , recent studies have shown that connectomic measures , despite their increased complexity , represent more robust biomarkers of schizophrenia ( calhoun et al . , 2008 ; erhardt et al . , 2011 ; bassett et al . ,
2012 ) and risk for alzheimer s disease ( fleisher et al . , 2009 ) , than simpler , regional measures .
thus , while parsimony and reliability are always important , candidate ips should be selected for their reliability and their hypothesized role in relevant pathophysiological processes ; not purely for their simplicity .
a more general criticism often leveled at the ip approach concerns whether any candidate ips studied thus far are truly intermediate in the sense that they mediate the effects of candidate genetic variants on disease risk or expression ( walters and owen , 2007 ; kendler and neale , 2010 ) .
under such a mediation model , the risk gene is assumed to influence the ip , which in turn influences illness susceptibility ( i.e. , gene > ip > disorder ) ; thus , the ip is truly interposed between gene and disorder . in this case
the ip is likely to represent a physiological mechanism through which genetic risk is expressed .
an alternative possibility however , is that the gene exerts pleiotropic and possibly independent effects on both the ip and disease risk ( i.e. , ip < gene > disorder ) . under this liability model , the ip is not involved in disease pathophysiology but may represent a biomarker of illness susceptibility . distinguishing between these two possibilities experimentally is challenging ( walters and owen , 2007 ; kendler and neale , 2010 ) and it is thus unclear which category the cips discussed here fall into .
our use of the term ip in reference to the measures discussed here is therefore subject to this caveat . in practice
however , the distinction may be overly simplistic as it assumes near - complete genetic overlap between the ip and psychiatric disorder .
a more likely scenario is that ips and psychiatric disorders posses both common and independent genetic influences which require multivariate analyses in genetically informative samples for complete characterization ( kendler and neale , 2010 ) . to our knowledge ,
though it is as yet unclear whether any of the candidate ips identified to date should be best conceptualized using mediation , liability , or multivariate models , they can certainly provide important clues as to how genetic risk factors influence variation in key phenotypic properties of psychiatric disease .
mapping differences and commonalities in these properties across disorders may prove useful in developing new , biologically informed diagnostic criteria .
in addition , preliminary evidence suggests that ips can augment the search for novel risk genes ( potkin et al . , 2009 ) .
the research reviewed here indicates that this kind work will benefit from a greater focus on identifying novel ips that describe systems - level properties of the brain , and that imaging connectomics provides a powerful methodological and conceptual framework for doing so . | psychiatric disorders are phenotypically heterogeneous entities with a complex genetic basis . to mitigate this complexity , many investigators study so - called intermediate phenotypes ( ips ) that putatively provide a more direct index of the physiological effects of candidate genetic risk variants than overt psychiatric syndromes . magnetic resonance imaging ( mri )
is a particularly popular technique for measuring such phenotypes because it allows interrogation of diverse aspects of brain structure and function in vivo .
much of this work however , has focused on relatively simple measures that quantify variations in the physiology or tissue integrity of specific brain regions in isolation , contradicting an emerging consensus that most major psychiatric disorders do not arise from isolated dysfunction in one or a few brain regions , but rather from disturbed interactions within and between distributed neural circuits ; i.e. , they are disorders of brain connectivity . the recent proliferation of new mri techniques for comprehensively mapping the entire connectivity architecture of the brain , termed the human connectome , has provided a rich repertoire of tools for understanding how genetic variants implicated in mental disorder impact distinct neural circuits . in this article , we review research using these connectomic techniques to understand how genetic variation influences the connectivity and topology of human brain networks .
we highlight recent evidence from twin and imaging genetics studies suggesting that the penetrance of candidate risk variants for mental illness , such as those in slc6a4 , maoa , znf804a , and apoe , may be higher for ips characterized at the level of distributed neural systems than at the level of spatially localized brain regions .
the findings indicate that imaging connectomics provides a powerful framework for understanding how genetic risk for psychiatric disease is expressed through altered structure and function of the human connectome . | A Primer on Imaging Connectomics
The Genetics of Human Brain Network Connectivity
SLC6A4, MAOA, and fronto-limbic connectivity
Fronto-temporal connectivity and ZNF804A
APOE and the default mode network
The Genetics of Human Brain Network Topology
Conclusion
Conflict of Interest Statement |
road traffic accidents constitute the most common cause . among the penetrating injuries , gunshot and
stab injury are the main causes in the western world . in india , proper incidence and causes are not known .
though in western literature it is unheard of the arrow injury , it is a common finding in india , especially in the tribal areas .
delivery of a baby with wound caused by penetrating injury in mother is not commonly encountered . here , we are presenting a case of intra uterine injury of a fetus caused by an arrow striking his mother at term .
a male baby of 2.7 kg weight was brought to outpatient department with the history of delivery with an injury on the right cheek [ figure 1 ] .
the cesarean section was done as the mother had a penetrating arrow injury on her lower abdomen due to an assault 9 h before .
she came to hospital with the arrow still penetrating in her body [ figure 2 ] .
baby with the wound on the face mother with the arrow there was a four cornered star shaped injury of 2.5 cm diameter on the right cheek .
as general anesthesia could not be arranged immediately , the procedure was done under local anesthesia .
trauma during pregnancy is a unique event because two patients , the mother and her fetus , are at risk and need special evaluation and management .
penetrating injury as a cause of maternal - fetal injury during pregnancy is found in one third of such cases .
gunshot , stab wounds , and attempt of illegal abortion are the most common causes of penetrating trauma . in penetrating trauma , probability of maternal organ damage
is about 1540% and fetal injury is about 70% . if compared with blunt trauma fetal injury is more likely in penetrating trauma .
fetal death after gunshot injury is 71% and stabbings 42%.4 muzumdar and his colleague described a bizarre case from canada where a baby had head injury when mother herself fired an air gun through her vagina at term . following a spontaneous precipitous vaginal delivery the baby had convulsion and was found to have intracranial hemorrhage with a metal pellet near the right lateral ventricle .
the newborn had a right temporal swelling and it was diagnosed as a subcutaneous hemangioma . but at 2 years of life , it was symptomatic and diagnosed as growing skull fracture with dural tear due to the prenatal injury
. patient needed neurosurgical procedure . though the injury in the baby and its management was simple in our case , it is presented here because of its fascinating nature and the interesting backdrop .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . | injury of a pregnant lady risks both mother and fetus
. various modes of injuries are possible . but arrow injury is not usually heard of in today 's world .
we have reported a male child delivered with a cut injury on the face .
it was caused by a penetrating arrow hitting his mother in her lower abdomen at term .
the injury of the baby was repaired successfully . | I
C
D
None
D
F
C |
a 37-year - old chinese male presented to the department of ophthalmology and visual sciences , alexandra hospital , singapore , with a history of poor vision in the left eye since childhood .
visual acuity was light perception in the left eye and 20/20 in the right eye .
fundus examination in the left eye revealed mnf extending from the optic disc up to the equator [ fig . 1 ] .
montage fundal photograph of the left eye showing extensive retinal myelination manifest refraction was performed with a best - corrected visual acuity ( bcva ) of 20/20 in the right eye and of light perception in the left eye , with a spherical equivalent of -3.50 diopter sphere ( ds ) and of -12.50 ds , respectively . to further investigate the cause of poor visual acuity , the patient was subjected to electrophysiology testing , which included a pattern perg , pattern visual - evoked potential ( vep ) and a full - field erg , all conducted under the international society for clinical electrophysiology of vision ( iscev ) protocol.[68 ] in the perg findings , p50 and n95 amplitudes were normal in the right eye ( p50 = 6.2 , n95 = 9.5 ) , suggesting normal macula and retinal ganglion cell function , but undetectable in the left eye ( no observable response ) .
pattern reversal vep was also undetected in the left eye ( no observable response ) .
these findings are suggestive of poor visual function in the left eye . in the full - field erg findings ,
the scotopic b - wave ( rod response , a result of the depolarization of the on - bipolar neurons ) was within normal limits in amplitude , although higher in the right eye compared with the left .
the maximal and photopic responses were also within normal limits for both eyes [ table 1 ] .
the perg has been shown to be important in clinical and research applications , as it is useful in providing information about retinal ganglion cell function , including macular function .
therefore , by using a similar stimulus to that for the vep , the perg can aid in its interpretation when the vep is abnormal . in this case ,
an undetectable perg in the left eye is consistent with abnormal ganglion cell function , which correlates with amblyopia in that eye .
however , in addition to studies that suggest that mnf may be considered an oligodendrocytic choristoma , further studies have also shown the disappearance of mnf in patients who had later developed branch retinal artery occlusion due to ischemic atrophy of the ganglion cell and nerve fiber layer
. there has also been an implication of the loss of ganglion cells post - central retinal artery occlusion ( crao ) , where the photopic negative response in cases of crao have been shown to be severely affected despite preservation of the cone b - wave .
this makes it possible , although not easy to establish due to the lack of previous ophthalmic records , that the abnormal retinal ganglion cell function in this patient may be directly associated with the extensive mnf in the retinal nerve fiber layer rather than a result of the secondary amblyopia that had developed as a result of the extensive myelination .
this suggestion may be reinforced from findings in a recent case report where two out of the three patients studied were not only amblyopic but also demonstrated consistent changes in the perg . compared with our patient ,
the p50 and n95 waves in the perg were recordable but decreased amplitude and increased in latency although the mnfs described were only mainly peri - papillary and not extensive . in conclusion
therefore , electrophysiological testing can play an important role in the assessment of visual function in a patient with poor visual acuity associated with extensive myelination , and this diagnostic modality may be able to provide a guide to visual prognosis in such cases . | an observational case report of electrophysiological abnormalities in a patient with anisomyopic amblyopia as a result of unilateral extensive myelinated retinal nerve fibers ( mnfs ) is illustrated .
the electrophysiological readings revealed an abnormal pattern electroretinogram ( perg ) but normal full - field electroretinogram readings in the affected eye .
the visual - evoked potential was also undetectable in that eye .
our findings suggest that extensive mnfs can be associated with electrophysiological abnormalities , in particular the perg , which can aid in diagnosing the cause of impaired vision when associated with amblyopia . | Case Report
Discussion |
the postpartum care ( ppc ) visit is an important opportunity to assess the physical and psychosocial health of the mother .
the american academy of pediatrics ( aap ) and the american college of obstetricians and gynecologists ( acog ) recommend that women , regardless of age , seek postpartum care between 4 and 6 weeks after childbirth .
the postpartum care visit may be utilized to counsel mothers on infant care and family planning , encourage breastfeeding , identify and treat medical conditions common to the postpartum period , and manage preexisting or emerging chronic conditions . despite the known benefits of the ppc visit
, there are many access and utilization barriers to care . as a result , healthy people 2020 aims to increase the proportion of women , across demographic and socioeconomic boundaries , who attend a ppc visit after giving birth , thereby highlighting postpartum care as a national priority to promote the health of women and children .
data on the utilization , content , and quality of the ppc visit are limited and often lack a comprehensive preventive care component . according to the centers for disease control and prevention 's ( cdc ) pregnancy risk assessment monitoring system ( prams ) 2004 , based on data from 11 states and new york city , 89% of us women who deliver attend a ppc visit
. however , the prevalence of women who attend a postpartum care visit is lower among certain subgroups .
approximately 71.2% of women who had received late prenatal care and 71% of women with 8 years of education attend a postpartum care visit .
in addition , only 66% of women who did not receive prenatal care and 59.5% of women whose infants did not have newborn checkups obtain ppc . among the women who do attend the ppc visit ,
many factors influence the decision to seek ppc . a study by kogan et al .
reported that the strongest indicator of whether a woman would obtain ppc was her use of prenatal care .
other studies have reported that women with inadequate or no prenatal care are less likely to adhere to recommendations for continuity of care after birth , which include newborn visits , childhood immunizations , and postpartum visits . among a nationally represented sample of women who had a live birth in the 1988 national maternal and infant health survey ( n = 9953 ) ,
women who had no prenatal care had a 3.39 greater odds of not receiving ppc compared to women who had prenatal care .
a commitment to preventive health during pregnancy ( i.e. , prenatal care ) may be a strong indicator of maternal health care utilization after birth .
new mothers are often sleep deprived , overwhelmed , and have limited time to tend to their personal health care needs .
women with unintended pregnancies may find it difficult to come to terms with their pregnancy .
after birth , postpartum depression and other mental health conditions may result , impacting the woman 's willingness to take initiative and follow through with medical appointments .
alcohol and drug use can further hinder a woman 's ability to attend to her health care needs .
in addition , women who had a baby born preterm or low birth weight may be less likely to seek ppc , as their attention and time are devoted to the care of the newborn [ 5 , 11 ] .
furthermore , enduring traumatic events , such as a still birth or infant death have a major impact on mental health status , which may result in low ppc utilization rates .
some studies report that women with less than a high school education , less than 26 years of age , a household income of less than $ 20,000 , and high parity are associated with a low acceptance rate of the ppc visit [ 2 , 5 , 7 ] .
the disparities in ppc utilization require attention to improve access to quality care for women across socioeconomic and demographic boundaries .
access barriers further decrease the likelihood that a new mother will schedule a ppc visit .
medicaid programs serve a higher proportion of pregnant women of low socioeconomic status with special needs than do private insurers .
although medicaid recipients receive hospital coverage , after birth outpatient visits such as ppc visits , are not always covered expenses . as a result , these women tend to seek acute , reactive care and do not have a long - term health care plan .
recent legal immigrants experience additional barriers to care as they are ineligible for medicaid services and language barriers can be a deterrent to seeking care as well .
lack of comprehensive health insurance coverage is a considerable challenge to meeting maternal healthcare needs .
furthermore , cultural and religious beliefs influence an individual 's regard for the postpartum visit .
social support , including partner involvement , can have a bearing on perceived feasibility of scheduling a ppc visit while caring for a newborn .
logistical barriers such as inaccessible transportation , long waits during appointments , and lack of child care further limit the likelihood of a postpartum visit . in light of the expanding hispanic population in the us ,
our study is focused on a predominantly hispanic multiethnic sample of low - income women . to address gaps in the literature , our study used data from a recent probability sample of mothers from los angeles county to study the determinants of and barriers to ppc utilization . to our knowledge , this is the first study to date to highlight actual and perceived barriers to ppc in this subpopulation .
data from the 2007 los angeles mommy and baby ( lamb ) study ( n = 4 , 075 ) were used to examine the extent to which postpartum care utilization is related to sociodemographic characteristics and to identify factors that reduce the likelihood of postpartum care utilization among women living in los angeles ( la ) , california .
the lamb study was a cross - sectional , population - based study that examined maternal and child health outcomes during the preconception , prenatal , and postpartum periods .
eligible participants were la residents who had a live - birth in la county in 2007 .
this study was a collaborative effort between the university of california , los angeles ( ucla ) and the los angeles county department of maternal , child , and adolescent health .
a total of 10,000 surveys were mailed to eligible women within 6 months after delivery .
the response rate was 56% , after adjusting for faulty addresses , language issues , maternal deaths , and loss of follow - up due to inability to locate the respondent .
the recruitment process consisted of a ( 1 ) notification letter 4 months after child birth , ( 2 ) mailed questionnaires , ( 3 ) mailed reminder letters , ( 4 ) and telephone follow - up to nonresponders .
the questionnaires were translated into spanish and chinese and a telephone translation service provided access in 88 languages .
data from completed surveys were linked to birth certificate data resulting in a final analytic sample of 4,075 .
this study was approved by both the los angeles county department of public health and the ucla institutional review boards ( irbs ) in 2007 .
the current analysis focused on the determinants of , and barriers to , postpartum care .
respondents were asked whether or not they obtained a postpartum care visit ( yes / no ) .
a range of maternal sociodemographic factors including race ( non - hispanic white , hispanic , non - hispanic black , asian / pacific islander ( api ) , and native american ) , age in years ( 016 , 17 - 18 , 1929 , 3039 , 4049 ) , marital status ( married , separated / divorced , widowed , never married but living together , never married but living apart ) , income ( < $ 20,000 , $ 20,00039,000 , $ 40,00059,000 , $ 60,00099,000 , > $ 100,000 ) , and education ( less than high school , high school graduate ,
some college or more , and not stated ) were included in the analyses ( table 1 ) .
when two races / ethnicities were provided , the first listed race / ethnicity was used .
additional variables of interest included insurance type ( no insurance , medi - cal / medicaid , private insurance , other ) , prenatal care utilization ( yes / no ) , care received prior to pregnancy ( reasons for seeking care : to have a healthy pregnancy , chronic medical problem , problem with previous pregnancy , expected to get pregnant , encouraged by doctor or nurse ) , intendedness of pregnancy ( yes , yes but not trying very hard , trying hard to keep from getting pregnant , or neither trying nor preventing pregnancy ) , preterm birth , low birth weight ( yes / no ) , and whether or not the infant attended a newborn visit ( yes / no ) . we analyzed the data using sas 9.3 ( sas institute inc . , cary , nc ) .
weights were added to account for the sampling design and survey nonresponse . to test differences between women who did and did not obtain postpartum care on select socio - demographic factors and other possible determinants , analyses were performed ( table 2 ) .
multivariate logistic regression analyses were used to identify predictors of postpartum care utilization adjusting for selected covariates ( table 3 ) . adjusted odds ratios ( ors ) and 95% confidence intervals ( cis ) were computed using the beta coefficients ( ) and standard errors ( ses ) from the multivariable logistic analyses .
the sample was predominantly hispanic , between 19 and 39 years old , married , had an annual income < $ 20,000 , and obtained a high school - level education or less .
table 2 displays the bivariate analyses of select maternal demographic and socioeconomic characteristics by postpartum visit status . in the bivariate analyses , maternal race / ethnicity , age , income , marital status , education , pregnancy intendedness , prenatal care , care received prior to pregnancy , and preterm
table 3 shows results of the multivariable analysis on the association between predictors and the lack of postpartum care utilization while controlling for confounders . in model 1 ,
hispanic mothers were significantly less likely to lack a ppc visit compared with non - hispanic white mothers ( or = 0.57 , 95% ci 0.38 , 0.87 ) .
mothers aged 30 to 39 ( or = 0.68 , 95% ci 0.51 , 0.91 ) were less likely to lack a ppc visit compared to mothers aged 19 to 29 . mothers from families with income
less than $ 20,000 were almost three times as likely to lack a ppc visit ( or = 2.89 , 95% ci 1.43 , 5.82 ) compared to mothers with households of > $ 100,000 .
separated or divorced and never married mothers were more likely to lack a ppc visit compared to married mothers .
mothers who reported not trying hard to get pregnant or trying to prevent pregnancy were also more likely to lack a ppc visit , compared to those who were trying .
mothers who did not attend prenatal care were more than 3 times as likely to lack a ppc visit ( or = 3.08 , 95% ci 1.68 , 5.63 ) .
when insurance status replaced family income in model 2 , mothers who had medi - cal insurance were twice as likely to lack a ppc visit compared to those on private insurance ( or = 2.19 , 95% ci 1.54 , 3.11 ) .
hispanic ethnicity , maternal age , marital status , pregnancy intendedness , and prenatal care utilization remained significant predictors .
figure 1 shows the most commonly reported reasons for not receiving a postpartum check - up .
women reported that they felt fine , were too busy with the baby , had other things going on , and felt there was no need for ppc .
our paper highlights the many factors influencing a woman 's decision to seek postpartum care .
our study population was multiethnic , predominantly hispanic and low - income , based on the demographics of los angeles county . in the multivariable analyses , postpartum care utilization was lowest among women who were low income , separated
hispanic women were more likely to obtain ppc compared to non - hispanic white women .
consistent with other studies , prenatal care was the strongest predictor of postpartum care utilization [ 5 , 7 , 8 ] .
women trying to get pregnant may be more inclined to seek care prior to , during , and after pregnancy .
one would expect that a continuum of maternal care may reflect a women 's commitment to life - long health .
however , on the contrary to our expectations , care received prior to pregnancy was not a significant determinant of ppc utilization .
the perinatal period represents a window of opportunity to engage and educate women about the importance of postpartum care .
the top 5 reasons for not seeking ppc indicate that women who lacked ppc did not consider the ppc visit a high priority ( figure 1 ) .
interestingly , access to care was not perceived as a top reason for not obtaining ppc .
interventions aimed at changing attitudes and perceptions about the importance of the ppc visit must include individuals , families , and communities .
the hispanic community , where familial support is of high value , had the highest adjusted rates for postpartum care use . a multitiered approach ,
targeting individuals , families , and communities may be necessary to improve ppc utilization rates .
women who do not have an adequate support system in place may find it exceedingly difficult to set aside time to care for their personal healthcare needs .
for the purpose of this analysis , marital status was viewed as a proxy for support .
in addition to a supportive spouse , extended family support can also facilitate a woman 's ability to seek ppc .
encouragement from one 's spouse and/or family member(s ) can provide the necessary impetus for scheduling a postpartum visit .
even gentle nudging from friends , neighbors , and respected individuals in the community can elicit the motivation necessary to seek care .
medi - cal is the medicaid program available to residents in the state of california .
this public health insurance program provides health care services to low - income individuals and families . in california
, certain eligibility requirements apply to receive 60 days of ppc through medi - cal services .
if the eligibility requirements are met , covered services include hospital and scheduled office visits during puerperium , assessment of uterine involution , and contraceptive counseling .
comprehensive postpartum care including nutrition , psychosocial , or health education services is billed fee for service .
furthermore , the postpartum care eligibility period does not cover conditions unrelated to the pregnancy , such as urinary tract infection , respiratory infection , hepatitis , preexisting hypertension , cholecystitis , appendicitis , abnormal pap smear , and cancer .
although some postpartum services are available , eligibility restrictions likely limit the accessibility of postpartum care for select subgroups .
our findings indicate that women enrolled in medi - cal are twice as likely to lack a ppc visit compared to those with private insurance .
these findings further support the need for national and state level policies addressing barriers to postpartum care .
the affordable care act addresses this need through the inclusion of provisions to support pregnant and postpartum women . under the affordable care act ,
furthermore , the affordable care act helps make preventive care affordable and accessible by requiring new health plans to cover and eliminate cost sharing for preventive services .
the lamb study was a cross - sectional study based on a sampling of live birth certificates .
in addition , the mother 's current relationship with the baby 's father may differ from their relationship status during pregnancy and as a result has a bearing on the survey responses . as this study took place in la county
however , this study contributes to the growing body of the literature emphasizing the importance of the postpartum visit and identifying barriers to ppc .
in our study population , despite the many barriers to obtaining postpartum care , hispanic women were more likely to receive postpartum services compared with non - hispanic white mothers .
an interesting finding of our study was that perceptions about the barriers to postpartum care were the result of a perceived low value of the postpartum visit .
this indicates a need for health care facilities and providers to make concerted efforts to increase knowledge about the importance of the postpartum visit , enhance the use or design of medical encounters , identify community resources , and develop targeted interventions .
the prenatal care visit is one early opportunity to educate women about the importance of ppc .
providers can influence perceptions about ppc , increase its acceptability , and contribute to significant improvements in women 's health . | this study aimed to identify actual and perceived barriers to postpartum care among a probability sample of women who gave birth in los angeles county , california in 2007 .
survey data from the 2007 los angeles mommy and baby ( lamb ) study ( n = 4,075 ) were used to identify predictors and barriers to postpartum care use .
the lamb study was a cross - sectional , population - based study that examined maternal and child health outcomes during the preconception , prenatal , and postpartum periods .
multivariable analyses identified low income , being separated / divorced and never married , trying hard to get pregnant or trying to prevent pregnancy , medi - cal insurance holders , and lack of prenatal care to be risk factors of postpartum care nonuse , while hispanic ethnicity was protective .
the most commonly reported barriers to postpartum care use were feeling fine , being too busy with the baby , having other things going on , and a lack of need .
findings from this study can inform the development of interventions targeting subgroups at risk for not obtaining postpartum care .
community education and improved access to care can further increase the acceptability of postpartum visits and contribute to improvements in women 's health .
postpartum care can serve as a gateway to engage underserved populations in the continuum of women 's health care . | 1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions |
goldman and benedek described the structure of shark ( spin dogfish ) , squalus acanthias , and determined the relationship between morphology and nonswelling properties of the cornea .
the authors described the morphology of the corneal stroma which consisted of parallel running lamellae which are interconnected across by sutures .
a comparative study of collagen in the cornea and sclera of young and adult spiny fish showed that corneal sutural fibrils contained collagen type i , whereas the scleral cartilage matrix contained type ii .
the immunofluorescence labelling of collagen type i was observed on the thick fibrils but not on the thin fibrils .
praus and goldman studied the nature of glycosaminoglycans of the shark dogfish and reported that the shark cornea contained 75% galactosaminoglycans of the total glycosaminoglycans present .
recently scott mccall et al . used the shark dogfish as a model to study collagen cross - linking .
the distribution of corneal layers in the fish cornea varies depending on the aquatic environment .
s. p. collin and h. b. collin , in their review , describe the structure of the cornea in various fish .
the florida gar fish ( lepisosteus platyrhincus ) cornea consists of the epithelium , bw , stroma , dm , and endothelium .
the corneas of the sandlance fish ( limnichthyes fasiciatus ) , pipefish ( corythoichthys paxtoni ) , and salamander fish ( lepidogalaxias salamandroides ) are covered by the epithelium which contains goblet cells .
the stroma is composed of a thin dermal stroma which contained aggregates of pigments and sutures , an iridescent layer , a mucous layer , and anterior sclera stroma .
the posterior part of the cornea is covered by the dm and endothelium [ 79 ] . in ratfish ( hydrolagus collie )
( cartilaginous fish ) , the cornea contains the conventional epithelium , bw , stroma with sutures , and dm .
the deep - sea teleost [ coryphaenoides ( nematonurus ) armatus ] contains a very peculiar cornea .
the fish cornea consists of an epithelium followed by a thick dermal stroma and mucous layer .
they have the most primitive vertebrate cornea containing cfs organized in stromal lamellae which are crossed by fibrillar sutures . the primitive morphology of the cornea provides its nonswelling properties and maintains transparency .
there have been no studies carried out on the organization of pgs and cfs of the stingray and shark . in this paper
we investigated the ultrastructure of cfs , pgs , and sutures of the shark and stingray stroma .
we were interested to see whether there would be any ultrastructural differences between these closely related species .
three stingrays ( 6 - 7 year old ) ( species : dasyatis americana ) and three sharks ( 6 - 7 year old ) ( species : squalus acanthias ) were used for the study . the eyes were fixed in 4% paraformaldehyde within 60 minutes of the fish 's death .
the eyes were kept in 4% paraformaldehyde for 12 hours and then washed in phosphate buffer ( 3 15 minutes ) .
the corneas were removed from the eyes and were fixed in 2.5% glutaraldehyde containing 0.05% cuprolinic blue ( bdh ltd . , dorset ) using a critical electrolyte concentration mode sodium acetate + magnesium chloride buffer overnight at room temperature .
the tissue was washed in 2.5% glutaraldehyde in sodium acetate + magnesium chloride ( 3 15 minutes ) followed by another wash in sodium tungstate ( 3 15 minutes ) .
the tissue was then dehydrated through a graded ethanol series ( 50% to 100% ) 15 minutes each and 100% acetone ( 2 30 minutes ) .
they were infiltrated in a mixture of acetone and taab 031 resin ( 1 : 1 ) for 8 hours .
the tissue was further infiltrated in 100% taab 031 resin for 8 hours ( 3 ) .
each quadrate was placed in green mold for embedding into blocks for sections to be cut .
the central part of the cornea in each quadrate was oriented at the cutting surface of the block and polymerized in taab 031 resin for 8 hours at 70c .
all the sections were cut at a 90 degree angle to the surface of the cornea ( cross section ) .
semithin ( 1 m ) sections were collected on glass slides and stained using toluidine blue .
the sections were stained with 2% uranyl acetate ( 10 minutes ) and lead citrate ( 10 minutes ) and observed using transmission electron microscopy jeol 1400 ( jeol ltd . , akishima , japan ) .
all the digital images were taken from the central part of the cornea . to analyze the cfs and pgs , 3 digital images were taken from the 4 lamellae of the anterior stroma , 4 lamellae from the middle stroma , and 4 lamellae from the posterior stroma above descemet 's membrane . to observe the pgs distribution without the structure of cf
the thickness of the lamellae , the minimum cf diameter , center - to - center spacing , and pg area were measured using the soft imaging system ( item , soft imaging system gmbh , mnster , germany ) analysis program .
the electron micrographs of cfs were processed with the item program and color coded according to the size of the cfs and pgs .
the mann whitney u test ( spss ) was used for statistical analysis because the data were not normally distributed . tissue procurement and use
it was ethically approved by the local ethical committee , king saud university , saudi arabia , and king khalid eye specialist hospital , riyadh , saudi arabia .
the corneal stroma of the stingray consisted of the lamellae which were running parallel to the surface of the cornea .
electron microscopic observations showed that the bw consisted of randomly running cfs ( figure 1(b ) ) . in the posterior part of the bw ,
the cfs of the bw ran parallel to the corneal surface and blended ( arrowhead ) into the most anterior stromal lamellae ( figure 1(b ) ) .
the anterior lamellae adjacent to the posterior part of the bw were very thin and some of them consisted of three to four cfs ( figures 1(b ) and 2(a ) ) .
below these thin lamellae , occasionally , orthogonally running cfs were observed ( figures 1(b ) and 2(b ) ) .
the thickness of the most anterior stromal lamellae was from 0.9 m to 2.33 m . the cf diameter in these lamellae was very small ( 20 nm ) ( figure 2(a ) ) .
the whole cornea contained approximately 25 lamellae . under the electron microscope the lamellae thickness varied from the anterior to the posterior stroma .
the thinnest lamellae were in the anterior stroma whereas the thickest lamellae were in the middle stroma .
the mean thickness of the anterior stromal lamellae ( 19 ) , middle stromal lamellae ( 1020 ) , and posterior stromal lamellae ( 2126 ) was 2.62 m ( n = 27 ) , 8.65 m ( n = 30 ) , and 4.88 m ( n = 15 ) , respectively .
the lamellae in the anterior stroma were not interlacing but instead were running parallel to each other ( figure 2(b ) ) .
the cfs within the lamellae in the anterior , middle , and posterior stroma were running parallel to each other .
the cross section of the cfs showed the distribution the cfs of variable diameters within the lamellae ( figure 2(d ) ) .
the posterior part of the cornea was covered by the dm ( figure 2(f ) ) .
the dm consisted of very fine , loosely interwoven fibrils ( figure 2(f ) ) .
the keratocytes were distributed throughout the stroma and contained a large nucleus ( figure 3(a ) ) . throughout the stroma of the stingray , sutures were observed running obliquely to the lamellae ( figure 3(b ) ) . in some places
the longitudinal section of the sutures showed that they consisted of two types of fibrils , thick cfs and thin microfibrils ( figure 3(c ) ) .
the cross section of the suture showed that the thin microfibrils were surrounded by the thick cfs ( figure 3(d ) ) .
the attachment of the pgs was not observed within the microfibrils ( figure 3(f ) ) .
the cfs in the anterior and posterior part of the suture were randomly arranged and blended with the cfs of the lamellae ( figure 4(a ) ) . in some places
the anterior and posterior end of the sutures also contained groups of microfibrils surrounded by the cfs which blended into the lamella ( figures 4(a ) and 4(b ) ) .
at the anterior and posterior end of the sutures , pgs were attached to the cfs but not to the microfibrils ( figure 4(c ) ) .
the longitudinally running microfilaments were occasionally observed in the stroma ( figure 4(a ) ) .
the structure of the shark cornea was very similar to the structure of the stingray cornea except for the distribution of pgs around the cfs . in the shark
, the sutures of the three or four lamellae were connected to each other and ran in straight lines unlike in the stingray .
the pgs were small in size and closely arranged around the cfs ( figure 4(e ) ) .
the cf diameter of the stingray and shark was analyzed by processing with an electron micrograph ( figures 5(a ) and 5(c ) ) using the item program .
after processing , the images were displayed using color coding to demonstrate the distribution of variable diameters of cfs ( figures 5(b ) and 5(d ) ) .
in the stingray , the mean diameters of the cfs in the anterior ( 22.06 3.22 nm , n = 263 ) , middle ( 22.12 4.40 nm , n = 255 ) , and posterior stroma ( 22.21 5.05 , n = 307 ) were very similar to each other ( table 1 ) . the interfibrillar spacing between the cf in the anterior stroma ( 32.74 8.19 nm , n = 263 ) was less than the interfibrillar spacing in the middle stroma ( 36.9 5.12 , n = 255 ) and posterior stroma ( 37.83 7.48 nm , n = 307 ) ( table 1 ) . in the shark , the cf diameters in the anterior ( 23.37 2.92 nm , n = 278 ) , middle ( 24.80 2.51 nm , n = 263 ) , and posterior stroma ( 24.59 2.44 nm , 303 ) were also very similar to each other . in the shark
the interfibrillar spacing in the anterior ( 35.84 4.18 nm , n = 278 ) and posterior stroma ( 35.17 4.37 nm , n = 263 ) was less than in the middle stroma ( 37.86 3.93 nm , n = 303 ) . in the stingray ,
most of the cfs were in the range from 15 to 25 nm ( red and green ) and a few of them were in the range from 25 to 30 nm ( blue ) . in the shark
a very few cfs were in the range from 15 to 20 nm ( red and green ) , and most of the cfs were in the range from 20 to 30 nm ( green and blue ) .
the cf diameter of the stingray in the anterior , middle , and posterior stroma was significantly less ( p < 0.0001 ) than those in the shark ( table 1 ) .
the interfibrillar spacing in the anterior , middle , and posterior stroma of the stingray was also significantly different ( p < 0.0001 ) from those of the shark ( table 1 ) . in the stingray
, the different sizes of pgs were decorated around the cfs of the stroma ( figures 2(d ) , 2(e ) , and 2(f ) ) and around the cfs of the suture ( figures 3(d ) and 3(e ) ) .
the structure of the pgs in the anterior , middle , and posterior stroma was identified in the electron micrographs taken from the corneal sections which were not stained with uranyl acetate and lead citrate ( figures 6(a ) , 6(c ) , and 6(e ) ) .
the electron micrographs ( figures 6(a ) , 6(c ) , and 6(e ) ) were processed using the item program and the variable sizes of the pgs were demonstrated by color coding in the digital images ( figures 6(b ) , 6(d ) , and 6(f ) ) .
the pgs in the anterior and middle stroma were rod shaped and their sizes ranged from 50 to 464 nm ( red and green color ) ( figures 6(b ) and 6(d ) ) .
the pgs in the posterior stroma were very large and ranged from 465 to 1292 nm ( blue , yellow , and pink ) ( figure 6(f ) ) .
the pgs in the posterior stroma were thick in the middle and thin at the edges .
the mean pgs areas in the anterior and middle stroma were similar to each other and not significantly different .
the pgs area in the posterior stroma ( 324.64 182.00 nm , n = 465 ) was significantly ( p < 0.001 ) higher compared to the mean pgs area of the anterior ( 224.54 82.64 nm , n = 384 ) and middle ( 225.52 80.86 nm , n = 460 ) stroma .
the density of the pgs was higher in the middle and posterior stroma compared to the anterior stroma in the stingray ( table 2 ) .
the electron micrographs of the pgs in the anterior , middle , and posterior stroma , shown in figures 7(a ) , 7(c ) , and 7(e ) , were color coded according to their variable sizes and are shown in figures 7(b ) , 7(d ) , and 7(f ) .
the mean area size of the pgs in the anterior stroma ( 194.52 79.26
nm ) was less than those in the middle ( 209.09 92.04 nm ) and posterior ( 201.65 92.67 nm ) stroma ( table 2 ) .
the density of the pgs was also less in the anterior stroma than in the middle and posterior stroma ( table 2 ) .
the pgs analysis also showed that the mean pgs area of the anterior ( p < 0.001 ) , middle ( p < 0.003 ) , and posterior ( p < 0.001 ) stroma of the stingray was significantly higher compared to the mean pgs area of the anterior , middle , and posterior stroma of the shark .
the density of the pgs in the stingray in the anterior , middle , and posterior stroma was less than those in the shark stroma ( table 2 ) .
the spacing between the pgs in the shark was also significantly ( p < 0.001 ) less compared to the spacing between the pgs of the stingray ( table 2 ) .
the normal human cornea is composed of five layers , epithelium , bowman 's layer ( bw ) , stroma , descemet 's membrane ( dm ) , and endothelium .
it is composed of cfs lamellae which contain angular lamellae relative to the transverse lamellae ( fibres ) along the corneal surface .
these lamellae ( fibres ) branch several times and connect to each other and in many cases interleave into the bowman 's layer .
the highest degree of this interconnectivity was observed in the transverse lamellae in the anterior stroma .
it is believed that transverse lamellae and their interconnectivity play an important role in determining corneal biomechanics and stress distribution , resisting the formation of regions with stress , which are more susceptible to deformation .
it was also suggested that interconnectivity of the lamellae in the anterior stroma is likely to serve to stabilize corneal shape and prevent lamellar slippage [ 15 , 16 ] .
the interconnectivity also provides rigidity to the anterior stroma and specifies corneal curvature and shape .
our studies showed that the stroma of the stingray consisted of concentric lamellae lying in parallel to the corneal surface and interconnected across by sutures throughout the stroma .
the structure of the stingray cornea was very similar to the structure of shark cornea described by goldman and benedek .
the interconnectivity of lamellae by sutures in the stingray and shark can be compared with the interconnectivity of lamellae in the anterior human stroma described by winkler et al . .
we believe that interconnectivity of the lamellae by sutures in the stingray and shark plays an important role in determining corneal biomechanics and stabilizing corneal shape , as has been observed in the anterior stroma of the human cornea by winkler et al . .
these sutures were also observed in the stroma of salamander and shark fish [ 1 , 9 ] .
the sutures in the shark run in straight lines continuously across 3 or 4 lamellae and are connected to each other . in the stingray the sutures of different lamellae were not connected to each other and did not run in straight lines .
our observations showed that the sutures in the stingray and shark consisted of thick cfs and thin microfibrils .
the thin filaments were surrounded by thick fibrils as previously reported by goldman and benedek .
thick cfs were composed of collagen type i whereas the thin fibrils were not composed of collagen type i .
the thin fibrils were never twisted whereas the thick fibrils were observed twisting and joining together .
the thick fibrils of the sutures were decorated by pgs but not the thin fibrils .
goldman and benedek reported that thin fibrils never stretched within the sutures and they inhibit the stretching of the sutures .
it has been suggested that the cylindrical core of thin fibrils was the principal bundle of the suture .
we presumed that the cylindrical core of thin fibrils provides mechanical strength and resistance to swelling of the cornea .
if the stretching of sutural fibrils was inhibited then the thickness of the cornea might be limited by the length of the thin fibril bundles .
occasionally orthogonally running cfs were observed suggesting that the fibrils might be entering into the early stages of the formation of a suture and blending with the lamellar cfs .
this phenomenon might be providing mechanical strength to the lamellar cfs and might be restricting the separation of the cfs within the lamellae and the separation of the lamellae from each other . in the cornea , three core proteins ( lumican , keratocan , and mimecan ) bearing glycosaminoglycan ( gag ) chains of keratan sulfate and one protein ( decorin ) bearing gag chains of chondroitin / dermatan sulfate are present . it has been reported that lumican plays an important role in regulating the cf diameter and it was higher in concentration in the posterior part of the stroma compared with that in the anterior stroma .
the cf diameters in the anterior , middle , and posterior stroma were not significantly different .
the density of the cfs was higher in the posterior stroma . in the shark ,
the cf diameter and density were significantly smaller compared with those in the middle and posterior stroma .
this could be due to higher concentrations of lumican in the posterior stroma . comparing the shark and stingray ,
the cf diameters of the stingray were significantly smaller compared to the cf diameters of the shark throughout the stroma .
this could also be due to the reduced amount of lumican present in the stingray compared with that in the shark .
goldman and benedek reported that in the shark the cfs diameters in the anterior stroma ( 32.7 2.1 nm ) were larger than in the posterior stroma ( 28.2 3.8 nm ) . in the present study , the means of minimum cf diameter of shark in the anterior ( 23.37 2.92 nm ) , middle ( 24.80 2.51 nm ) , and posterior ( 24.59 2.44 nm ) stroma were very similar to each other .
the mean of minimum cfs diameters of the shark in our study was smaller than the cfs diameter of the shark reported by goldman and benedek .
this could be due to different processing techniques or methods of calculating the diameter . in our experiments tissue
was processed with glutaraldehyde containing cuprolinic blue in sodium acetate + magnesium buffer and taab 031 resin was used , whereas they processed tissue in glutaraldehyde and osmium tetroxide embedded in epon 812 .
goldman and benedek reported that the density of cfs from anterior to posterior stroma of the shark was approximately in the range from 450/m to 540/m whereas our studies showed that the density of cfs from anterior stroma to posterior stroma was in the range of 547/m to 595/m .
he reported that stained cs / ds filaments are ~70 nm long , whereas ks pgs are ~70 nm long .
lewis et al . reported that pgs composed of cs / ds were very large ( about 300 nm ) whereas pgs composed of ks were small ( about 65 nm ) . in the stingray ,
it is believed that these large pgs in the posterior stroma of stingray are composed of cs / ds pgs .
the presence of the large pgs in the posterior stroma may play an important role in regulating the hydration of the stingray cornea .
the pgs analysis in the shark showed that there was no significant difference in the mean area of the pgs in the anterior , middle , and posterior stroma .
the density of pgs in the posterior and middle stroma was significantly ( p < 0.001 ) higher compared to the density of the pgs in the anterior stroma .
it is surprising that both the stingray and the shark belong to the same class , but the pgs mean areas in the stingray were significantly ( p < 0.001 ) larger compared to the pgs mean area in the shark in the anterior , middle , and posterior stroma .
the density of the pgs however was significantly less in the stingray compared to the pgs density in the shark in all regions ( anterior , middle , and posterior stroma ) .
pgs of stingray and shark , like those in bovine and mouse , show no specific order .
the distance between the pgs in the shark is significantly smaller compared to those in the stingray . it could be possible that this close association of the high density of pgs in the shark may strengthen the stromal cfs which provide strength to their large sized cornea as previous studies showed that large animals such as camels have a large density of small pgs .
it has been suggested that in comparison with the cornea of more advanced vertebrates , the nonswelling shark cornea contains large amounts of highly - sulfated chondroitin sulfate and small amounts of keratan sulfate which regulate hydration in the aquatic environment .
scott mccall et al . suggested that interlamellar bonds increase the mechanical strength of the cornea because they could physically link the entire adjacent lamellae of the corneal stroma .
these physical interactions could occur between separate collagen fibrils both within an individual lamella and in adjacent lamellae .
these interactions between the cfs could arise via the terminal domains of the gag chains that extend from the sides of all collagen fibrils .
in elasmobranch corneas , the sutures provide a physical interlamellar bond supporting the mechanical strength of the cornea and inhibiting the swelling of the cornea in the shark [ 1 , 24 ] .
we suggest that the physical strength of the sutures might be provided by the interaction of the cfs which occurs via the terminal domain of the gag chains that extend from the side of the cfs .
we believe that pgs play an important role in providing the physical strength of sutures that inhibit the swelling of the cornea in the stingray and shark .
the presence of high levels of chondroitin sulfate and low levels of keratan sulfate in the shark and in the stingray may also contribute to the nonswelling properties of their corneas .
further studies are required to investigate the role of keratan sulfate and chondroitin sulfate by using their specific antibodies and employing biochemistry , immunohistochemistry , and immunogold techniques . | we report here the ultrastructural organization of collagen fibrils ( cf ) and proteoglycans ( pgs ) of the corneal stroma of both the stingray and the shark .
three corneas from three stingrays and three corneas from three sharks were processed for electron microscopy .
tissues were embedded in taab 031 resin .
the corneal stroma of both the stingray and shark consisted of parallel running lamellae of cfs which were decorated with pgs . in the stingray ,
the mean area of pgs in the posterior stroma was significantly larger than the pgs of the anterior and middle stroma , whereas , in the shark , the mean area of pgs was similar throughout the stroma .
the mean area of pgs of the stingray was significantly larger compared to the pgs , mean area of the shark corneal stroma .
the cf diameter of the stingray was significantly smaller compared to the cf diameter in the shark .
the ultrastructural features of the corneal stroma of both the stingray and the shark were similar to each other except for the cfs and pgs .
the pgs in the stingray and shark might be composed of chondroitin sulfate ( cs)/dermatan sulfate ( ds ) pgs and these pgs with sutures might contribute to the nonswelling properties of the cornea of the stingray and shark . | 1. Introduction
2. Methods
3. Results
4. Discussion |
successful organ preservation is an important component of transplantationand ensures the maintenance of organ viability until implantationinto the recipient .
currently , heart preservation for transplantationis limited to 4 to 6 hours of cold ischemic storage , and longerperiods of ischemia are known to adversely affect survival .
this is in contrast to preservation of the liver , kidney , andpancreas , which have been successfully preserved for 24 to 36hours although graft function may be transiently compromised .
ischemia initiates a complex injury process that is characterized by the loss of high energy phosphates , the accumulation of intracellular inosine and hypoxanthine , cessation of the na - k pump , followed by cell swelling , cytosolic calcium increase , and enzymatic degradation . in general , lower organ temperatures ( 0c to 4c ) maintain high energy phosphates more effectively .
however , temperatures below 2c significantly increase the risk of cold injury with some proteins denaturing below 0c . at higher temperatures ( 8c to 12c ) , organ function is maintained to a greater extent [ 11 , 12 ] .
however , at temperatures above 12c the higher metabolic demand for oxygen leads to irreversible hypoxic injury and thus significantly impairs organ function .
ideally , there should be a temperature range at which these two tendencies can be balanced .
the instructions for use of organ preservation solutions state a temperature range between 2 - 8c or 4 - 8c .
hence , the balance seemingly forms the cornerstone of cold ischemic storage which is hypothermia at 4c to 8c with an appropriate cold storage solution .
hypothermiadecelerates metabolism and the ionic constituents of the storage solution facilitaterapid cessation of electrical activity .
the formulation of the preservation solution is based on three principles : ( a ) hypothermic arrest of metabolism ; ( b ) provision of a physical and biochemical environment that maintains viability of the structural components of the tissue during hypothermic metabolic arrest ; and ( c ) minimization of the effects of reperfusion injury .
hypothermia does not stop metabolism but it slows down biochemicalreaction rates and decreases the rate at which intracellularenzymes degrade essential cellular components necessary fororgan viability .
most enzymes of hypothermic animals showa 1.5 to 2.0-fold decrease in activity for every 10c decreasein temperature .
hypothermia also retards lysis of organelles like lysosomesthat , in turn , release autolytic enzymes that cause cell death .
the current gold standard for transporting a donor organ to a recipient for transplant involves storing the organ in organ preservation fluid within sequential plastic bags that are placed on ice and transported in a cooler .
some of the potential issues associated with this method of storage include ( a ) variability of packaging materials used ; ( b ) bioincompatibility of the material that touches the organ ; ( c ) tissue injury due to movement / vibration ; and ( d ) potential uneven cooling of the organ tissue . in a multicenter study of 186 organs , recommended packaging guidelines were tested for their ability to maintain organ temperatures at acceptable temperatures .
paragonix technologies , inc . has developed a series of medical devices intended for transport of donor hearts and kidneys to recipients for implant : the sherpa pak cardiac transport system ( cts ) and the sherpa pak kidney transport system ( kts ) .
the sherpa pak system ( figures 1 and 2 ) is intended to provide a safe , consistent method for cold ischemic storage and transport of donor organs .
a , b , c ) future product design of sherpa pak cts and kts .
the systems are designed to overcome many of the potential issues associated with the current systems used for transport .
the sherpa pak s cooling mechanism is based on phase - change material ( pcm ) , which is a substance with a high heat of fusion which is capable of storing and releasing large amounts of energy .
the sherpa pak s pcm panels are designed to hold 5c longer than conventional phase change material cold packs ( which undergo phase change at 0c , and have little heat capacity at 5c ) .
in addition , the pcm can be placed in direct contact with temperature sensitive products because there is no risk of freezing ( please note , however , that the cold panels do not come in direct contact with the donor organ ) .
management of preservation in the sherpa pak system is the same for both kidneys and hearts other than that the heart is connected to a connector in the inner hard shell assembly and the kidney is not .
we have performed multiple temperature profile studies for varying periods of time , from 4 hours up to 30 hours of storage ( tables 1 and 2 ) . preconditioning protocol for components of the sherpa pak systems .
performance data for static hypothermic storage between 4 and 8c for up to 30 hours using the sherpa pak technology at ambient temperature .
all protocols were in conformity with the guide for the care and use of laboratory animals published by the national institutes of health 86 - 23 , revised in 1985 .
anesthesia was induced with 4.4 mg / kg telazol , 2.2 mg / kg xylazine and 0.04 mg / kg atropine intramuscularly and maintained with isoflurane after endotracheal intubation .
median sternotomy was performed and heparin ( 300 units / kg ) was given intravenously .
an aortic root cannula was inserted and , after cross - clamping the ascending aorta , 1 l of celsior ( genzyme , cambridge , ma , usa ) cardioplegia was administered through the cannula .
the heart was vented through the left atrial appendage , and superior and inferior vena cava , and donor cardiectomy was performed .
complete sherpa pak cardiac transport systems ( cts ) and sherpa pak kidney transport systems ( kts ) were utilized for the thermal qualification test . prior to initiating the testing
, the components of the sherpa pak systems were preconditioned according to the instructions for use provided with the device ( table 1 ) .
the sherpa pak system consists of multiple components : 1 ) an outer transport shipper which contains various non - ice - based temperature controlled packaging elements ; 2 ) an inner and outer hard shell assembly ( i.e. sherpa pak/sherpa pak shell ) which provides a double , rigid barrier shipper in which the organ is immersed and suspended in a cold storage fluid cleared for use in storing and transporting donor organs ; 3 ) an off - the - shelf temperature data logger that monitors and displays the temperature of the organ during transport ; and 4 ) an off - the - shelf timer that displays elapsed time during transport .
once conditioned , the systems were assembled in accordance with the instructions for use with the exception that additional thermocouples were placed to monitor temperature at various locations within the system .
these thermocouples were connected to an external display ( the presence of the thermocouples did not alter the closure of the device ) .
temperature readings were monitored and recorded at regular intervals for the period of the experiment .
temperature measurements were taken every 5 min in studies i.a , i.b and iii.a , and every 10 s in studies ii and iii.b . when not otherwise indicated , the storage solution was saline or water . in order to capture any variations in temperature within the device , thermocouples were placed in various locations of the device .
thermocouple locations are designated as follows : center1 = one probe ( n=1 ) located in the center of the fluid filled sherpa pak. wetted probe = one probe ( n=1 ) located at the top of the fluid filled sherpa pak ( within fluid ) .
this assembly was then placed into a third solution - filled bag and placed onto crushed ice in an ice chest . when not otherwise indicated , the storage solution was saline or water . study design .
three different ex - vivo studies have been performed with the paragonix sherpa pak devices : study i : the temperature of the fluid filled device was measured for up to 30 h at an outside temperature set at 22c ( study i.a and study i.b , table 2).study ii : the temperature of the fluid filled device was measured for up to 30 h at extreme outside temperatures set at -8c and 31c ( study ii , table 3).study iii : the temperature of pig hearts attached to the device was measured for up to 12 h ( study iii.a and iii.b ; table 4 ) .
study i : the temperature of the fluid filled device was measured for up to 30 h at an outside temperature set at 22c ( study i.a and study i.b , table 2 ) .
study ii : the temperature of the fluid filled device was measured for up to 30 h at extreme outside temperatures set at -8c and 31c ( study ii , table 3 ) . study iii : the temperature of pig hearts attached to the device was measured for up to 12 h ( study iii.a and iii.b ; table 4 ) .
performance data for static hypothermic storage between 4 and 8c for up to 30 hours using the sherpa pak technology with two 1 hour excursions to extreme hot and cold temperatures .
summary of animal studies of static hypothermic storage between 4 and 8c using the sherpa pak technology .
sherpa pak can keep the temperature of the preservation solution between 4 - 8c for a period of up to 30 hours . the first part of study i ( table 2 ) demonstrated in three separate test runs that all temperatures recorded from the individually placed temperature probes remained below the 8c upper limit of the design specification through four hours .
moreover , all recorded temperatures from all individually placed temperature probes remained below the 8c upper limit through 12 hours of experimentation .
most importantly , the experiment verified that the fluid within the sherpa pak ( in which the donor heart is immersed ) was maintained between 4c and 6c ( as measured by the three probes placed within the fluid ; two at center of the container , one against the side wall ) which is within the accepted 4c - 8c .
the second part of study i ( table 2 ) demonstrated that all temperatures recorded from the individually placed temperature probes remained below the 8c upper limit through 30 hours .
most importantly , the experiment verified that the fluid within the sherpa pak ( in which the donor organ is immersed ) was maintained between 4c and 8c ( as measured by the temperature probes placed within the fluid ) .
sherpa pak consistently maintains fluid temperature at 4 - 8c despite extreme fluctuations in ambient temperature .
results of the four separate test runs ( two test runs each at cold or hot temperatures ) of the experiment ( study ii : table 3 ) demonstrated that the storage solution remained below the 8c upper limit of the design specification through 24 hours .
interestingly , in the second warm temperature test run , the cold storage solution was n't preconditioned according to its instructions for use and the starting temperature of the fluid was 7c ( versus 6c according to manufacturer ifus ) .
even with this slightly elevated temperature , the sherpa pak system was capable of maintaining temperature throughout the 24 hour period .
sherpa pak can maintain the temperature at 4 - 8c when the system is loaded with a pig heart .
both the sherpa pak cts as well as the conventional ice storage demonstrated their ability to cool and maintain temperature throughout the experimental period .
however , the ice system cooled the heart based on the two thermocouples to temperatures as low as 0c at the apex closest to the ice , and < 1c inside the left ventricle of the pig heart .
the experiment verified that the fluid within the sherpa pak ( in which the donor organ is immersed ) was maintained between 4c and 8c ( study iii , table 4 ) .
temperatures were recorded every 5 min , providing up to 145 readings in study iii.a , and every 10 s , providing up to 10801 readings in study iii.b results of 12 temperature test runs during pig heart storage demonstrated that the sherpa pak cts was able to maintain fluid temperatures within a range of 4 - 8c when fully loaded with a pig heart ( table 4 ) .
the purpose of this study was to verify whether the paragonix sherpa pak device was capable of maintaining the desired temperature for organ storage which lies between 4 and 8c .
several studies were performed during which all system components including the cold storage solution were preconditioned according to the instructions for use . in certain studies ,
exterior conditions remained constant as the system will always be under the direct supervision of medical personnel during transport and not anticipated to be exposed to uncontrolled temperatures or environmental conditions .
the purpose of study ii , in which exterior conditions were changed to either high temperatures ( 31c ) or low temperatures ( -8c ) for 1 hour twice during 30 hours of monitoring , was to challenge the system at two extreme temperatures .
the results of the testing verified that the design of the sherpa system was able to maintain the fluid in which the donor organ is contained within the clinically acceptable range of 4c - 8c through 24 hours , even when challenged to high and low temperatures . when ambient temperatures were kept constant ( at 22c ) the system was able to maintain temperatures within a range of 4c - 8c throughout 30 hours of monitoring .
based upon the results of the testing , we conclude that the sherpa pak devices are designed such that they can reliably maintain a clinically acceptable storage temperature for the donor organ .
it is commonly accepted that the 4c - 8c temperature range is the best balance for preservation of high energy phosphate stores , minimization of cold injury , and preservation of post - transplant function .
although temperatures below 4c offer superior maintenance of high energy phosphates , temperatures below 2c significantly increase the risk of cold injury and frostbite [ 8 , 9 ] .
the results of the studies demonstrated that the paragonix sherpa pak device was capable of maintaining a very consistent temperature within the 4c - 8c temperature range through twelve hours .
the conventional ice storage was also very effective at cooling and maintaining temperature of the heart through six hours ; however , the data show that temperatures encountered with this system approached 0c for the last few hours of the experiment , thus increasing the potential for issues with the donor heart .
these data compared well to a multicenter study on transport temperature of organs . studying 186 organs ,
the average organ temperature during transportation was below 2c , and after 6 hours below 0c .
based upon these test results , it appears that the paragonix sherpa pak transport system is effective at maintaining the temperature of the organ in the optimal temperature range . while the study investigated ambient temperatures that might be expected in a clinical situation and temperature excursion that may occur during organ transport
, a limitation of the study is that measurements were taken in a research setting , which may not entirely reflect the clinical setting .
the paragonix sherpa pak device may decrease cold injury of donor organs by maintaining the temperature consistently between 4c and 8c and therefore may decrease primary graft failure after organ transplantation . | introductioncurrently , the gold standard for donor organ preservation in clinical organ transplantation consists of 3 plastic bags and an ice box .
the first plastic bag includes the organ itself immersed in preservation solution ( e.g. celsior ) .
this bag is put in a second bag filled with saline , and then these two are put in a third bag filled with saline which is then put in the ice box .
the disadvantage of this method is that the organ usually gets too cold .
it has been shown that the theoretical perfect temperature for organ preservation is 4c - 8c .
while higher temperatures lead to hypoxic injury of the organ because the metabolism is not decreased efficiently , lower temperatures than 4c increase the risk of cold injury with protein denaturation . in the current study , we investigated a device that keeps the organ temperature consistently in the desired range of 4c - 8c and can potentially decrease cold injury to donor organs.methodsthree different ex vivo studies were performed with the paragonix sherpa pak devices : 1 ) the temperature of the fluid - filled device was measured for up to 30 hours at an outside temperature set at 22c , 2 ) the temperature of the fluid - filled device was measured for up to 30 hours at extreme outside temperatures set at -8c and 31c , 3 ) the temperature of a pig heart attached to the device was measured up to 12 hours.resultsall studies showed that the paragonix sherpa pak can keep the temperature of the heart consistently between 4 and 8c.conclusionsthe paragonix sherpa pak device may decrease cold injury of donor organs by maintaining the temperature consistently between 4c and 8c and therefore may decrease primary graft failure after organ transplantation . | Introduction
Methods
Results
Discussion
Conclusion |
the most important factor in improving the outcome of a patient with out - of - hospital cardiopulmonary arrest ( ohcpa ) is cardiopulmonary resuscitation ( cpr ) performed by lay persons ( bystanders ) on the scene before contact with an organized emergency medical service ( ems ) .
however , insufficient knowledge concerning the confirmation of ohcpa , risks , and complications of cpr may be one of obstructive factors for immediate cpr .
non - trained lay persons tend to be particularly reluctant to perform immediate and aggressive cpr on patients who are not clearly suffering cpa , such as patients with normal body temperature , patients who were active until a few minutes before sudden collapse , or patients who appear to breathe or be able to move . lay persons can not easily recognize these cases as ohcpa , and they may be afraid of complications due to unnecessary cpr if the patient is not ohcpa , which may be harmful to the patient
it is clear that fear of complications of cpr may delay immediate recognition of ohcpa and the initiation of cpr .
however , there have been few reports concerning the risks and complications of bystander cpr for unconscious non - cpa patients outside a hospital and with respiratory arrest or inadequate respiration that have been widely known and accepted .
the object of the present study was to clarify the complications that may arise in such cases and the safety of bystander cpr , and to examine the role of immediate bystander cpr .
in japan , an out - of - hospital ems system has been established by the ambulance service and emergency life - saving technicians ( elsts ) belonging to the fire department and supported by in - hospital emergency departments ( eds ) .
the license of elst was established in 1991 . in yokohama city , pre - hospital and interhospital ems
systems have also been established for critical and severe patients , including those with ohcpa .
yokohama is the second largest city in japan , and our institute is located in its center .
the surface area and population of yokohama are 434 km and 3.37 million , respectively .
we selected 12 hospitals whose eds can receive and treat the most severe patients , including ohcpa patients , and who must accept all patients with ohcpa regardless of their capacity .
the medical doctor who is the ems director belongs to the 12 hospitals and carries out his duties in the central operation center of the city fire department ; the director advises elsts at the scene , decides how patients should be treated by elsts and oversees the transfer of ohcpa patients to the hospital .
any ohcpa patient is transferred to the nearest ed of the 12 hospitals [ figure 1 ] , except those who was diagnosed in some hospital and are expected to die in the near future , who are transferred to the hospital and for whom a death certificate can be written . for all hospitals ,
including ours , all data concerning unexpected ohcpa patients are population - based.[46 ] the number of cases who underwent cpr and cases with ohcpa a working group of the medical control ( mc ) committee of yokohama city , which includes some of these ems directors , has trained fire department members in the protocol of telephone advice on cpr by call takers and dispatchers so that they may offer telephone cpr ( t - cpr ) . until 2006 , following the old protocol for t - cpr , when ems callers and dispatchers received emergency calls about suspected ohcpa , they gave instructions for standard cpr ( mouth - to - mouth ventilation plus chest compression , consisting of 2 ventilations alternating with 15 compressions ) , or compression - only cpr if the caller rejected or was reluctant to perform mouth - to - mouth ventilation ; these instructions were given independent of the condition of the patient , caller or scene , except where there was difficulty in communication . since 2006 , following the new protocol for t - cpr , instructions are given for chest compression - only cpr without mouth - to - mouth ventilation . in our medical dispatching system , one dispatcher or call taker interviews the caller about the condition of the patient and gives instructions for t - cpr , while another sends the ambulance team to the scene .
this study was a population - based observational case series in the single center that examined data from april 2004 to march 2010 .
the subjects were patients who underwent cpr with chest compression by lay persons not belonging to any organized ems , regardless of their training in cpr or whether advised by t - cpr .
the subjects also had to be confirmed as non - ohcpa patients and had to have not undergone any cpr by any organized ems such as an ambulance team .
therefore , the subjects underwent just a short duration of cpr before contact with ems and confirmation as non - cpa by ems .
patients with ohcpa diagnosed after contact with an ems team and patients who received only a precordial thump , tapping of the back or heimlich maneuver without chest compression were excluded .
complications of bystander cpr included rib fracture , lung injuries such as pneumothorax and lung contusion , abdominal organ injuries such as hepatic , splenic and gastric injuries , and chest and/or abdominal pain requiring analgesics . in our ed , one medical doctor gathered information on the patient as in detail as possible from the ems ambulance team , the patient 's family or bystanders , and if necessary , the patient 's family doctor and the ems dispatcher in the central operation center , without directly treating the patient ; these data and information were entered into the patient 's medical record using a special format and in detailed writing .
other medical doctors in the ed checked symptoms and physical findings , and performed x - ray and blood examinations , electrocardiograms , and chest and abdominal ultrasonography in all cases , as well as other advanced examinations on demand such as computed tomography ( ct ) scans .
no clinical but practically unnecessary examinations were carried out solely to identify complications , such as simple x - ray examinations other than chest x - ray , chest ct , consecutive electrocardiograms or troponin i assay .
in japan , an out - of - hospital ems system has been established by the ambulance service and emergency life - saving technicians ( elsts ) belonging to the fire department and supported by in - hospital emergency departments ( eds ) .
the license of elst was established in 1991 . in yokohama city , pre - hospital and interhospital ems
systems have also been established for critical and severe patients , including those with ohcpa .
yokohama is the second largest city in japan , and our institute is located in its center .
the surface area and population of yokohama are 434 km and 3.37 million , respectively .
we selected 12 hospitals whose eds can receive and treat the most severe patients , including ohcpa patients , and who must accept all patients with ohcpa regardless of their capacity .
the medical doctor who is the ems director belongs to the 12 hospitals and carries out his duties in the central operation center of the city fire department ; the director advises elsts at the scene , decides how patients should be treated by elsts and oversees the transfer of ohcpa patients to the hospital .
any ohcpa patient is transferred to the nearest ed of the 12 hospitals [ figure 1 ] , except those who was diagnosed in some hospital and are expected to die in the near future , who are transferred to the hospital and for whom a death certificate can be written . for all hospitals ,
including ours , all data concerning unexpected ohcpa patients are population - based.[46 ] the number of cases who underwent cpr and cases with ohcpa a working group of the medical control ( mc ) committee of yokohama city , which includes some of these ems directors , has trained fire department members in the protocol of telephone advice on cpr by call takers and dispatchers so that they may offer telephone cpr ( t - cpr ) . until 2006 , following the old protocol for t - cpr , when ems callers and dispatchers received emergency calls about suspected ohcpa , they gave instructions for standard cpr ( mouth - to - mouth ventilation plus chest compression , consisting of 2 ventilations alternating with 15 compressions ) , or compression - only cpr if the caller rejected or was reluctant to perform mouth - to - mouth ventilation ; these instructions were given independent of the condition of the patient , caller or scene , except where there was difficulty in communication . since 2006 , following the new protocol for t - cpr , instructions are given for chest compression - only cpr without mouth - to - mouth ventilation . in our medical dispatching system , one dispatcher or call taker interviews the caller about the condition of the patient and gives instructions for t - cpr , while another sends the ambulance team to the scene .
this study was a population - based observational case series in the single center that examined data from april 2004 to march 2010 .
the subjects were patients who underwent cpr with chest compression by lay persons not belonging to any organized ems , regardless of their training in cpr or whether advised by t - cpr .
the subjects also had to be confirmed as non - ohcpa patients and had to have not undergone any cpr by any organized ems such as an ambulance team .
therefore , the subjects underwent just a short duration of cpr before contact with ems and confirmation as non - cpa by ems .
patients with ohcpa diagnosed after contact with an ems team and patients who received only a precordial thump , tapping of the back or heimlich maneuver without chest compression were excluded .
complications of bystander cpr included rib fracture , lung injuries such as pneumothorax and lung contusion , abdominal organ injuries such as hepatic , splenic and gastric injuries , and chest and/or abdominal pain requiring analgesics . in our ed , one medical doctor gathered information on the patient as in detail as possible from the ems ambulance team , the patient 's family or bystanders , and if necessary , the patient 's family doctor and the ems dispatcher in the central operation center , without directly treating the patient ; these data and information were entered into the patient 's medical record using a special format and in detailed writing .
other medical doctors in the ed checked symptoms and physical findings , and performed x - ray and blood examinations , electrocardiograms , and chest and abdominal ultrasonography in all cases , as well as other advanced examinations on demand such as computed tomography ( ct ) scans .
no clinical but practically unnecessary examinations were carried out solely to identify complications , such as simple x - ray examinations other than chest x - ray , chest ct , consecutive electrocardiograms or troponin i assay .
during the study period , 26 cases met the criteria for inclusion in our study .
the patients mean age was 69.0 years old ( ranging from 25 to 97 ) and the male : female ratio was 15:11 . in this period , we experienced 1,844 patients with ohcpa who underwent cpr by organized ems members and hospital staffs , and 884 of them ( 47.9% ) underwent bystander cpr .
we also experienced 910 patients who underwent bystander cpr , 884 of them ( 97.1% ) were confirmed as ohcpa by organized ems and 26 ( 2.9% ) were confirmed as non - ohcpa by organized ems [ figure 1 ] .
chest compression ( cc)-only cpr was performed in 21 cases and standard cpr ( cc and ventilation ) was performed in 5 cases .
the mean duration of bystander cpr was 6.5 minutes ( ranging from 1 to 26 minutes ) with 10 cases ( 38.5% ) over 7 minutes and 3 ( 11.5% ) over 10 minutes [ figure 2 ] .
nine patients died : 7 of intracranial hemorrhage and 1 of acute chronic congestive heart failure ( all of a causative pathological condition of unconsciousness ) , and 1 died of pneumonia secondary to unconsciousness .
the neurological condition at discharge of 8 patients ( 30.8% ) was the same as that just before the event , 5 patients ( 19.2% ) had mild disorder , 2 ( 7.7% ) had severe disorder , and the remaining 4 ( 15.4% ) had vegetative state .
all neurological disorders were due to the causative etiology of the unconsciousness , such as subarachnoid hemorrhage , massive cerebral infarction , etc . , or due to hypoxic brain damage during respiratory arrest .
the duration of bystander cpr and complication ; cc means chest compression and mmv means mouth - to - mouth ventilation , and foot note shows detail of cases with complications three cases ( 11.5% ) suffered from symptoms which can be thought as complications related to bystander cpr .
the rate of complications was evaluated 11.5% at most . in one case , we found tracheal bleeding without preceding hemoptysis immediately following tracheal intubation in the ed without any resistance during the procedure , however the bleeding was minimal and easily controlled with positive pressure mechanical ventilation for insufficient respiration due to neck hanging . in another case , we found minor gastric mucosal laceration during gastrointestinal fiberscopy for examination and treatment of hematemesis , which showed active bleeding from a chronic gastric ulcer in addition to the fresh minor laceration . in the remaining case ,
we found chest pain upon inspiration , indicating a minor rib fracture or muscular damage ; this was easily controlled with analgesics [ figure 2 ] .
although cpr performed by lay persons ( bystanders ) is extremely important to improve the outcome of patients with ohcpa , bystanders are not always able or willing to perform immediate , accurate , and high - quality cpr if they feel they have inadequate knowledge concerning appropriate cpr techniques .
bystanders can be supported to perform appropriate cpr by prior education , such as training courses or by direct advice at the time of the event , such as t - cpr .
call takers or dispatchers can not confirm a patient as cpa , however , nor can they support bystander cpr with full confidence if they lack adequate knowledge of the risks and complications of cpr , particularly cpr for non - ohcpa patients . likewise , cpr educators for lay citizens and for call takers and dispatchers are also hindered in the education and training they offer if they do not have adequate knowledge concerning the risks and complications of cpr , particularly cpr for non - cpa patients .
thus , accurate information concerning the risks and complications of cpr will be helpful to bystanders , call takers , and their educators so that they may be more able to immediately identify ohcpa and perform cpr , resulting in an improvement in the prognosis of ohcpa in the district .
moreover , it is more important to perform immediate cpr on patients who can not be clearly identified as ohcpa than for those with obvious cpa .
however , bystanders tend to be reluctant to perform immediate cpr for unconscious patients with inadequate respiration due to fear of misidentification of ohcpa , and because of the potential risks and complications of cpr . to eliminate hesitation in carrying out immediate and aggressive cpr , to increase the likelihood of bystander cpr for ohcpa patients and to improve the outcome of patients with ohcpa , it is important to clarify these issues and educate the public about the low occurrence of complications in bystander cpr , the easy treatability of these complications , and the safety of bystander cpr , particularly cpr for non - ohcpa patients .
the guarantee of minimal risk of complications of bystander cpr can make bystanders more willing to perform immediate cpr even when they can not confirm the patient as ohcpa . this guarantee can also help call takers or dispatchers of regional ems teams in providing t - cpr with full confidence , and can assist all cpr educators of anyone associated with bystander cpr .
our data indicate a very low occurrence of complications of cpr in non - ohcpa patients .
white et al . also reported that 12% of non - cpa patients who underwent cpr experienced discomfort and only 2% suffered a fracture , and no patients suffered visceral organ injury , in which study they did not specifically mandate chest x - ray or other diagnostic evaluations that might be directed toward complete ascertainment of cpr injury . in our present study , there still remained possibility of overlook of minor rib or sternal fracture , minor liver injuries without intra - abdominal bleeding , minor lung contusion without airway bleeding , minor gastric mucosal injuries without hematemesis , or asymptomatic complications , by chest and abdominal x - ray , chest - abdominal ultrasonography , electrocardiography and blood tests as routine examinations in our ed for all critical patients .
it must be noted , however , that the objective of the present study was not to clarify the occurrence of simply anatomical complications that may cause bystanders to hesitate to perform cpr , but to examine the safety of cpr for non - ohcpa patients .
many authors have shown a higher rate of occurrence of complications of cpr in ohcpa , including rib or sternal fracture , lung contusions , and liver or gastric injuries .
reported that 86% of rib fractures found during autopsy were missed by simple chest x - ray examination .
however , the duration of cpr in our study was short , possibly because in non - ohcpa patients , it must be terminated when the ems team assumes responsibility for the patient .
complications are reported to be more likely with prolonged chest compressions because of increasing fatigue and decreasing attention of the rescuer , deteriorating the quality of the compressions.[1012 ] one of the limitations of this study is that we can not confirm the ohcpa or non - ohcpa status of the patient before ems contact with the patient .
it is possible that the subjects include patients with ohcpa at the time of the event but who achieve return of spontaneous circulation ( rosc ) with bystander cpr before the arrival of the ems team .
another limitation is that this study was very small in size because of the extremely low frequency of t - cpr for non - ohcpa patients in our district , which is due to our protocol of the call takers for identifying ohcpa .
the third limitation is that there is no way to ascertain the direct association of three pathological conditions with bystander cpr , which we presented as complications of cpr
tracheal bleeding just after tracheal intubation without any resistance indicated the high possibility that the injury occurred not during but before the procedure of intubation .
tracheal bleeding or lung injury is commonly seen in patients with prolonged cpa in ed who underwent aggressive cpr for long duration .
fresh minor gastric mucosal laceration indicated the possibility that the injury occurred during cpr but there was also possibility that the injury had occurred before cpr .
however , in this study , we used in - hospital data of single center evaluated and written by medical doctors , which could certify high quality of the data in the view point of accuracy and detail .
additionally , our data do not include the risks and complications of cpr for ohcpa patients or those of cpr in a hospital and performed by an organized medical resuscitation team .
nevertheless , our data do indicate the extremely low frequency of lethal complications of cpr for non - ohcpa patients as well as the ease of treating complications of bystander cpr , both of which can encourage bystanders to perform cpr and call takers to assist bystanders in performing cpr on unconscious patients with inadequate respiration .
the risks and frequency of complications of bystander cpr are thought to be very low .
it is reasonable to perform immediate cpr on unconscious victims with inadequate respiration , and to help bystanders perform cpr using the t - cpr system .
immediate cpr or t - cpr for these victims can thus be performed promptly and confidently . | background : insufficient knowledge of the risks and complications of cardiopulmonary resuscitation ( cpr ) may be an obstructive factor for cpr , however , particularly for patients who are not clearly suffering out of hospital cardiopulmonary arrest ( oh - cpa ) .
the object of this study was to clarify the potential complication , the safety of bystander cpr in such cases.materials and methods : this study was a population - based observational case series . to be enrolled ,
patients had to have undergone cpr with chest compressions performed by lay persons , had to be confirmed not to have suffered ohcpa .
complications of bystander cpr were identified from the patients medical records and included rib fracture , lung injury , abdominal organ injury , and chest and/or abdominal pain requiring analgesics . in our emergency department ,
one doctor gathered information while others performed x - ray and blood examinations , electrocardiograms , and chest and abdominal ultrasonography.results:a total of 26 cases were the subjects .
the mean duration of bystander cpr was 6.5 minutes ( ranging from 1 to 26 ) .
nine patients died of a causative pathological condition and pneumonia , and the remaining 17 survived to discharge .
three patients suffered from complications ( tracheal bleeding , minor gastric mucosal laceration , and chest pain ) , all of which were minimal and easily treated .
no case required special examination or treatment for the complication itself.conclusion:the risk and frequency of complications due to bystander cpr is thought to be very low .
it is reasonable to perform immediate cpr for unconscious victims with inadequate respiration , and to help bystanders perform cpr using the t - cpr system . | INTRODUCTION
MATERIALS AND METHODS
Emergency medical service system in the study area
Study protocol and data collection
RESULTS
DISCUSSION
CONCLUSION |
studies of disease experiences have been conducted to understand the various stories of what patients suffer through from specific diseases .
the purpose of these studies is to evaluate the sickness by analyzing the patient s physical , psychological , and social conditions over time .
furthermore , the studies can gather generalized knowledge that can be used for diagnosing specific diseases by noting the common aspects among the various personal experiences of different patients .
documents such as memoirs of , and interviews with , patients are the usual sources of the current studies .
these sources have been analyzed as stories of their lived experience , but they have a limitation for generalizing the understanding of certain diseases and patients due to the small number of analysis targets .
when patients feel a lack of pertinent information available or a need to share their experience with , be heard by , receive help from , and give help to others , they find internet sites where they can be involved in active dialogue with other patients who share the same problems .
for these reasons , the internet sites related to diseases are greatly expanding and thus presenting an excellent opportunity for quantitative analyses
. an ever - increasing number of memoirs and corresponding comments are being posted online by patients , as well as articles written by professionals . in recent years , this tendency has become more active due to the appearance of blogs , message boards , forums , and snss .
numerous stories of illnesses and medication processes are now elicited by , and archived in , various patient - support platforms , and the amount of information thus generated by patients has formed a huge data set , commonly referred to as big data . to analyze the massive information on the internet , a method called text - mining or text data - mining has been introduced .
text - mining is used for classifying and organizing a large amount of text to extract common themes and lexical , phrasal , or sentential tendencies .
recently , an increasing number of researchers dealing with health - related issues have suggested that the text - mining method was found to be more useful for studying massive amounts of data than the traditional , qualitative , ethnographic methods of participant observation and interviews .
the text - mining tasks such as text classification / clustering , word - frequency / content analysis , and phrase net visualization proved efficient in detecting the emerging issues and needs of the patients and in providing portrayals of collective opinions and concerns .
nevertheless , there has been little research on the potential of text - mining in identifying clinical distinctions and patient concerns .
the present study used a text - mining method to analyze memoirs related to a specific disease posted on the internet because this genre most powerfully captures the patients experience of the disease .
patients stories it differs from the traditional qualitative analyses that are personal and contextual ( at the personal level and within the here and now ) and particularly from a narrative approach to illness experience that usually aims at finding something that is out of average .
in contrast , the text - mining approach transforms the stories into quantitatively estimable data structures and numbers , which generalize the patients distinctive experiences and conditions a - contextually and collectively .
the primary purpose of the present study is to determine whether text - mining the memoires of patients who have had experiences of a specific diseases can form and produce generalized knowledge about a particular disease . as a pilot study for long - term research and an alternative method on massive internet language data , our approach is , by design , focused on a sample of 399 illness stories for the experiment .
the secondary purpose of the present study is to discern what interests and concerns have been reflected in the memoires . in this study
, we used a large sample of on - line memoirs of patients with fibromyalgia ( fm ) syndrome .
the basic reason for focusing on fm is that this disease is often accompanied by a large variety of physical , psychological , and social problems due to the pain of the chronic musculoskeletal disorder [ 1315 ] , so we formulated a hypothesis that these peculiarities could be seen in patient memoirs of the disease .
the main complaint of fm patients is chronic and systemic musculoskeletal pain that yields difficulties in daily life while they are awake , and the pain is hard to relieve and moderate [ 11,1619 ] .
fm patients also suffer from other physical symptoms such as fatigue and sleep disorders , and psychosocial symptoms such as depression , anxiety , and tension [ 11,12,2023 ] .
consequently , these symptoms , as well as the pain , can have a negative effect on the quality of the patients life [ 2428 ] . to confirm whether the data acquired via text - mining represents the previously mentioned characteristics of fm described in the past studies
, we used a method that calculates the frequency of words with the focus on the nouns in a written text . on this basis , we analyzed what types of words were high - frequency nouns , and how these nouns were used in the original text . in many cases ,
whenever necessary we double - checked the result of the text - mining with the text itself to grasp the usage of words more accurately .
experienceproject.com is a popular online social networking community where 3.8 million personal stories are created and more than 1.5 million participants write and read the stories . regarding the demographics of the participants of this site , most of them are from the united states ( 36.8% ) , india ( 17.1% ) , the united kingdom ( 7.5% ) , canada ( 4.4% ) , and philippines ( 2.2% ) ( http://www.alexa.com/siteinfo/experienceproject.com ) . as our data source , we chose the forum under the title of i have fibromyalgia , where various types of patients share their experience of the disease . at
i have fibromyalgia , the participants are fm patients who voluntarily post their stories to share the illness experience , and receive health - related information and emotional support from others .
a total of 399 memoir samples were collected through text - mining tools at http://www.experienceproject.com/groups/have-fibromyalgia/2671?omni . to obtain the language data from the forum , we first collected the unstructured data of memoirs associated with fibromyalgia through a crawling process ( figure 1 ) . to obtain the language data from a webpage that uses web - programing languages such as html and jsp
the data thus retrieved were saved into a text file to make a structured and analyzable dataset .
after this , we generated information such as word frequency lists , keyword lists , and concordance plots through antconc , which is a concordancer available on the internet , often used by corpus linguists and social scientists to sort text data .
we also made a dataset by categorizing the words of the text file into nouns , verbs , and adjectives by using stanford pos tagger , which assigns part of speech tags to individual words .
then we eliminated such unnecessary and functional words as articles ( e.g. , a and the ) , conjunctions ( e.g. , and and but ) , and pronouns ( e.g. , i and you ) to have fm - relevant data . through the process
, we obtained a total of 21,929 nouns ready for analysis . to conduct a lexical analysis and pattern identification
, we used manyeyes , whose distinctive function is to identify phrasal relationships ( figure 1 ) . manyeyes is a web - based visualization tool created by fernanda vigas and martin wattenberg , which offers a graphic representation of patterns and distinctions of a given dataset .
constructed on a public website , it allows users to upload a set of data , visualize them , and gain new insights into patterns occurring in the data .
particularly , the phrase net visualization offers pattern matching and general views of the words in the data and visualizes any 2 words that frequently associate and appear together .
according to the analysis of the frequency of nouns from the memoirs of fm patients ,
this means that pain is the most distinctive issue for fm patients , suggesting it is the most important word characterizing fm .
pain was used in the texts , we were able to extract important information to understand fm . for more detailed information about the use of
we conducted 2 additional analyses : 1 ) keywords that were frequently found in the same sentence as the word
i experience chronic pain , debilitating muscle spasms , joint tenderness and swelling ( see figures 2 and 3 ) , and keywords that were directly preceding or following the word
it s so hard to explain to someone that does nt have chronic pain , and i still am waiting for that elusive pain - free day ( figure 3 ) . by using the concordance and phrase net visualization
specifically , the pain of fm seems to occur mainly in the body ( 9 ) , muscles ( 15 ) , neck ( 46 ) , back ( 60 ) , joints ( 62 ) , and shoulders ( 68 ) ( figures 2 and 3 ) .
fatigue ( 34 ) and depression ( 33 ) also seem to be related with pain ( figure 2 ) .
stiffness , and aching ( figure 2 ) , although these nouns were not in the top 100 .
chronic, and constant ( figure 3 ) . as shown in the analysis of the frequency of nouns ( table 1 ) ,
symptoms ( 12 ) indicates that fm patients are interested in their symptoms in the same way ( table 1 ) .
also frequent are the nouns related to symptoms of fm such as sleep ( 27 ) , depression ( 33 ) , fatigue ( 34 ) , and stress ( 48 ) ( table 1 ) .
these words in particular directly indicate the symptomatic characteristic of fm . there are terms such as body ( 9 ) ,
head ( 42 ) , and neck ( 46 ) that indicate which parts of the physical body are affected by the pain of fm ( table 1 )
overall , the lexically suggested relationship among adjacent words confirms that fm is a disease that causes extensive pain in the musculoskeletal system .
results also support that patients suffer from fm for a long time because it is a chronic disease .
years ( 3 ) that often accompanies the description of fm ( table 1 ) . in most cases
first , fm patients are eager to be free from the pain . as shown in figure 3 , the word
free ( adj . ) and relief ( 65 ) are frequently connected to the word
pain , which reflects the desire of fm patients to have relief from , and be free from , the pain of fm .
also , the word life ( 6 ) has a high frequency ( table 1 ) and is still in the top 20 words when combined with modifying adjectives ( figure 4 ) . in other words , the adjectives such as normal ,
life meaning that fm patients long for changing their present life and/or going back to a normal life ( hope for a normal life back ) . as indicated in figure 4 , the word
it seems that the cravings and painful life of fm patients are connected to the high - frequency words related to help such as support ( 45 ) ,
in many cases , these keywords are used when fm patients are in need of specific help .
furthermore , it has to do with the fm patients interest in the treatment of pain . this phenomenon can be confirmed with the words
medication ( 11 ) and management , which have a connection with the word pain ( figure 3 ) and the high - frequency word
medication ( table 1 ) . however , the specific treatments , methods , and the names of the drugs were not conclusive in the analysis of the frequency of nouns .
this means that from the patients point of view these were not important issues ; rather , they were only a medical point . instead ,
fm patients used everyday language to describe their experiences . along with the treatment of pain and help against fm , words such as work ( 22 ) and job ( 25 ) showed a high frequency ( table 1 ) , indicating that work / occupation is an important part of fm patients concerns ( i.e. , whether they lost a job or are trying to get one ) .
this is because work / occupation is necessary for living life well , and fm can negatively affect these aspects of life .
another observation of this study was that there are many words referring to a particular person or group of people , for example ,
mother ( 18 ) , family ( 20 ) , husband ( 24 ) , friend ( 25 ) , and children ( 38 ) ( table 1 ) .
this supports the notion that fm patients continue to live life under the basis of human relations and have concerns related with them .
according to the analysis of the frequency of nouns from the memoirs of fm patients ,
this means that pain is the most distinctive issue for fm patients , suggesting it is the most important word characterizing fm .
pain was used in the texts , we were able to extract important information to understand fm . for more detailed information about the use of
we conducted 2 additional analyses : 1 ) keywords that were frequently found in the same sentence as the word
i experience chronic pain , debilitating muscle spasms , joint tenderness and swelling ( see figures 2 and 3 ) , and keywords that were directly preceding or following the word
it s so hard to explain to someone that does nt have chronic pain , and i still am waiting for that elusive pain - free day ( figure 3 ) . by using the concordance and phrase net visualization
specifically , the pain of fm seems to occur mainly in the body ( 9 ) , muscles ( 15 ) , neck ( 46 ) , back ( 60 ) , joints ( 62 ) , and shoulders ( 68 ) ( figures 2 and 3 ) .
fatigue ( 34 ) and depression ( 33 ) also seem to be related with pain ( figure 2 ) .
stiffness , and aching ( figure 2 ) , although these nouns were not in the top 100 .
chronic, and constant ( figure 3 ) . as shown in the analysis of the frequency of nouns ( table 1 ) ,
symptoms ( 12 ) indicates that fm patients are interested in their symptoms in the same way ( table 1 ) .
also frequent are the nouns related to symptoms of fm such as sleep ( 27 ) , depression ( 33 ) , fatigue ( 34 ) , and stress ( 48 ) ( table 1 ) .
these words in particular directly indicate the symptomatic characteristic of fm . there are terms such as body ( 9 ) ,
head ( 42 ) , and neck ( 46 ) that indicate which parts of the physical body are affected by the pain of fm ( table 1 )
overall , the lexically suggested relationship among adjacent words confirms that fm is a disease that causes extensive pain in the musculoskeletal system .
results also support that patients suffer from fm for a long time because it is a chronic disease .
years ( 3 ) that often accompanies the description of fm ( table 1 ) . in most cases
first , fm patients are eager to be free from the pain . as shown in figure 3 , the word
free ( adj . ) and relief ( 65 ) are frequently connected to the word
pain , which reflects the desire of fm patients to have relief from , and be free from , the pain of fm . also , the word life ( 6 ) has a high frequency ( table 1 ) and is still in the top 20 words when combined with modifying adjectives ( figure 4 ) . in other words , the adjectives such as normal ,
life meaning that fm patients long for changing their present life and/or going back to a normal life ( hope for a normal life back ) . as indicated in figure 4 , the word
it seems that the cravings and painful life of fm patients are connected to the high - frequency words related to help such as support ( 45 ) , care ( 68 ) , and help ( 78 ) ( table 1 ) . in many cases ,
furthermore , it has to do with the fm patients interest in the treatment of pain .
this phenomenon can be confirmed with the words medication ( 11 ) and management , which have a connection with the word pain ( figure 3 ) and the high - frequency word medication ( table 1 ) .
however , the specific treatments , methods , and the names of the drugs were not conclusive in the analysis of the frequency of nouns .
this means that from the patients point of view these were not important issues ; rather , they were only a medical point . instead ,
fm patients used everyday language to describe their experiences . along with the treatment of pain and help against fm , words such as work ( 22 ) and job ( 25 ) showed a high frequency ( table 1 ) , indicating that work / occupation is an important part of fm patients concerns ( i.e. , whether they lost a job or are trying to get one ) .
this is because work / occupation is necessary for living life well , and fm can negatively affect these aspects of life .
another observation of this study was that there are many words referring to a particular person or group of people , for example ,
doctor ( 7 ) , mother ( 18 ) , family ( 20 ) , husband ( 24 ) , friend ( 25 ) , and children ( 38 ) ( table 1 ) .
this supports the notion that fm patients continue to live life under the basis of human relations and have concerns related with them .
this study used text - mining methods to analyze 399 samples of only memoirs of the specific disease fm that were posted on the internet .
the study confirmed that pain is the biggest issue for the fm patients [ 11,1518,20,21 ] . through the analysis of keywords used in association with the word
pain , we obtained the information related to the pains and accompanying symptoms from which the fm patients suffer .
the pains were affecting body parts , including muscles, leg, neck, back,
joints, and shoulders with accompanying symptoms such as spasms, stiffness, and aching, and were described as being
chronic, and constant. the findings were also consistent with the clinical features of fm revealed from previous research [ 20,2228 ] . besides the fm - related pains , the words with high frequency were found to be the very key words that define the clinical features of fm .
sleep and fatigue were frequently found in the memoirs of the patients , and the high occurrence attests to the significance of sleep deprivation and fatigue severity as issues in their lives the clinical fact being commonly reported in other studies .
in addition , words such as depression and stress that were ranked high in frequency indicate that the fm patients also go through psychological tribulation .
the relationship between the fm - related pains and the clinical features of fatigue and depression were also identified by the phrase net visualization . through the text - mining method , this study quantitatively confirmed the clinical features of fm that were found by qualitative research , and showed the possibility that the memoirs posted online by people with fm may well describe the clinical features of the disease .
furthermore , the same method can be applied to the memoirs of patients suffering from other diseases .
second , the study demonstrated that it is possible to understand the interests and concerns of fm patients through text - mining .
fm patients wanted to escape from the pain and symptoms , so they were interested in medical treatment and help .
also , they seemed to have interest in their work and occupation [ 28,3234 ] .
moreover , it was shown that they hope to continue to live life through relationships with the people around them .
these findings are in accord with the other results from narrative analyses [ 17,19,20,26,3235 ] .
the results of this study show the possibility of recognizing key words from a large database of texts , and even though patient memoirs of illness experiences are personal , the interests of fm patients can be understood by generalizing the patterns of keywords .
consequently , by increasing the understanding of fm patients interests , medical professionals can increase their effectiveness when dealing with them in a psychological and social way .
it should be noted , however , that the keywords obtained through the text - mining process are not concerned with certain previously known aspects of fm patients concerns , such as sexual dysfunction related to fatigue and medication , psychological need for receiving help from health - related professionals ( e.g. , nurse , physical therapist , and clinical psychologist ) , and the struggle to keep up with daily routines or household chores as a coping strategy for pain . regarding these concerns ,
this omission is due to the fact that text - mining represents only dominant patterns and generalizable knowledge . in other words , although the aforementioned concerns are distinctive and important aspects of an individual s experience of fm , those individualized distinctions are averaged and outweighed by the sheer number of more general fm - related verbal phenomena . in that aspect , text - mining fails to focus on qualitative appraisal and assessment of some under - represented concerns , which are otherwise an important aspect of fm patients inner struggles and personal lives .
therefore , the method and analysis in the present study , which is based on occurrence frequency and phrasal relationship , is only useful for identifying dominant patterns and obtaining generalized knowledge .
firstly , textual analysis of keyword extraction can help researchers understand a large amount of information in a short period time , but it can be unclear how the information can be used in specific contexts . in this study , we have tried to examine how certain keywords were used in context by using the phrase net visualization . in spite of this , there were some cases where the keywords did not occur at a high frequency . in such cases
, there was no choice but to find the sentences involving certain keywords to understand the meaning .
secondly , sometimes the quality of the memoirs of disease experience can change depending on the condition of the patient ( e.g. , duration and degree of illness and coexistence of other diseases ) , but this was not addressed in the processing of the data used in this study .
in addition , factors such as age , gender , culture , and the social / occupational status of patients were not specified in the memoirs , so these factors were not included .
the reason for not controlling these patient features is that text - mining utilizes large , unspecified amounts of data , which is one of its inherent limitations .
therefore , the limitations should be considered when interpreting the results based on text - mining .
thirdly , this study was able to grasp the characteristics of fm through a keyword analysis , but extracted keywords may not be the best method for diagnosing diseases . in other words , keywords extracted in the course of medical history can be used by doctors as important information to formulate a hypothesis for the disease of the patients , but this is only a part of the clinical process for making a final diagnosis . to make an accurate diagnosis , clinical check - ups and inferences of doctors are still necessary .
to showcase the text - mining method and its performance , this study analyzed 399 patient memoirs of a specific disease ( fm ) posted on the internet .
this research showed that extracting keywords can confirm the clinical distinction of a certain disease , and it can help objectively understand the concerns of patients by generalizing their large number of various , subjective comments .
however , it is believed that there are limitations to the processes and methods for organizing and classifying large amounts of text , and these limits have to be considered when analyzing the results . though text - mining can greatly contribute to the understanding of symptoms and patients experience of diseases , the development of research methodology to overcome these limitations is greatly needed . | backgroundthe purpose of this study was to evaluate the possibility of using text - mining to identify clinical distinctions and patient concerns in online memoires posted by patients with fibromyalgia ( fm).material / methodsa total of 399 memoirs were collected from an fm group website .
the unstructured data of memoirs associated with fm were collected through a crawling process and converted into structured data with a concordance , parts of speech tagging , and word frequency .
we also conducted a lexical analysis and phrase pattern identification .
after examining the data , a set of fm - related keywords were obtained and phrase net relationships were set through a web - based visualization tool.resultsthe clinical distinction of fm was verified .
pain is the biggest issue to the fm patients .
the pains were affecting body parts including muscles, leg, neck,
back,
joints, and shoulders with accompanying symptoms such as spasms, stiffness, and aching, and were described as
sever, chronic, and
constant. this study also demonstrated that it was possible to understand the interests and concerns of fm patients through text - mining .
fm patients wanted to escape from the pain and symptoms , so they were interested in medical treatment and help .
also , they seemed to have interest in their work and occupation , and hope to continue to live life through the relationships with the people around them.conclusionsthis research shows the potential for extracting keywords to confirm the clinical distinction of a certain disease , and text - mining can help objectively understand the concerns of patients by generalizing their large number of subjective illness experiences .
however , it is believed that there are limitations to the processes and methods for organizing and classifying large amounts of text , so these limits have to be considered when analyzing the results .
the development of research methodology to overcome these limitations is greatly needed . | Background
Material and Methods
Results
Clinical distinction of FM
Concerns of FM patients
Discussion
Conclusions |
nelson syndrome is a rare condition defined by enlargement of a pituitary adenoma following bilateral adrenalectomy for treatment of cushing disease.1 the incidence of nelson syndrome in adults varies between 8 and 29%,2 but it may be seen more frequently in patients who undergo adrenalectomy at a younger age.3 the pathophysiology of the disease is complex and controversial,124 and it is unknown what factors drive corticotroph cells in these patients .
blood tests showing rising adrenocorticotropic hormone ( acth ) levels and imaging showing an enlarging pituitary mass confirm the diagnosis.4 treatment depends on surgery and/or radiotherapy.145 we present the case of a patient with recurrent nelson syndrome whose effective treatment requires novel imaging due to prior sellar reconstruction with a metal plate ; this case suggests that metabolic radionuclide imaging may be a new option for managing patients with nelson syndrome .
in 1968 , the then 8-year - old patient underwent bilateral adrenalectomy for treatment of cushing disease .
four years later , she developed bitemporal hemianopsia , was found to have increased acth , and was diagnosed with nelson syndrome . after external beam radiation and transsphenoidal decompression of her tumor , a right frontal craniotomy for tumor resection and hypophysectomy provided symptomatic relief until 1983 , when her acth again began to rise .
transsphenoidal exploration failed to find tumor ; the surgeon reconstructed the sellar floor with metallic mesh encased in methyl methacrylate .
her acth normalized until 1998 , when another successful endonasal tumor resection was performed . in 2002 , the patient underwent a craniotomy for resection of a radiation - induced left frontal meningioma .
in 2005 , at the age of 45 , she presented with a clinical spectrum characteristic of past acth overabundance : skin pigmentation , retro - orbital headache , and mouth ulcers .
her acth was measured at greater than 3,000 pg / ml ( normal range 0 to 46 pg / ml ) .
pituitary - protocol magnetic resonance imaging ( mri ) demonstrated two potential sites of recurrent tumor : one adjacent to the sella and a second lower on the clivus .
metallic artifact from the previously placed mesh made interpretation of the study difficult ( fig .
1 ) . t1-weighted postgadolinium magnetic resonance imaging ( mri ) . ( a ) midline sagittal .
concern for residual tumor along sellar floor seen in ( a ) and ( c ) , as well as within the sphenoidal body ( b ) .
to better elucidate which region may have been responsible for her elevated acth , the patient was sent for octreotide scintigraphy . despite poor spatial resolution ,
however , in light of her radiation - induced meningioma , the patient refused radiosurgical treatment and insisted on pursing surgical options .
the patient then underwent a fludeoxyglucose positron emission tomography ( fdg pet ) scan with fine - cut computed tomography ( ct ) coregistration to better delineate her tumor and adjacent bony landmarks .
2 , the anatomic relationship between the area of increased glucose uptake and the sellar floor was well defined .
the ct was imported to the brainlab neuronavigation system ( brainlab ag , feldkirchen , germany ) . during surgery ,
the mesh plate was found encased with scar and carefully removed . with the aid of intraoperative navigation ( fig .
3 ) , the left floor of the sella was explored ; a bony crevice was found , and pathologic tissue within it was removed . using gentle suction and various ring curettes , all tissue within the osseous cave
her postoperative course was uneventful , and she was discharged home on postoperative day 4 . at her first postoperative visit 1 week after discharge , her headaches had resolved , her skin tone began lightening , and she had resumed her preoperative activities .
serial acth levels dropped precipitously , eventually reaching a nadir of 90 pg / ml .
she remained asymptomatic for the remainder of her life before succumbing to complications from acute myeloleukemia in 2010 .
( a ) fine - cut axial computed tomography ( ct ) . ( b ) 18-fludeoxyglucose positron emission tomography ( fdg pet ) .
( c ) coregistration overlay demonstrating area of increased glucose uptake along the floor of the sella to the left of midline .
intraoperative screen shot from brainlab neuronavigation system ( brainlab ag , feldkirchen , germany ) displaying a registered probe placed at the spot suggested by coregistration in ( a ) axial , ( b ) coronal , and ( c ) sagittal projections .
rates of recurrence differ according to tumor pathology,6 but it has been suggested that patients with prior target gland destruction , as in patients with nelson syndrome , face higher recurrence rates due to loss of inhibitory feedback.7 in one large series , rates of recurrence for acth tumors was 12% , whereas 25% of those with nelson syndrome faced recurrence within 10 years.6 in the work - up of both de novo and recurrent disease , mri is the imaging modality of choice . unfortunately , reliable direct visualization of microadenomas is limited to tumors greater than 4 mm .
advanced imaging techniques , such as dynamic scanning and delayed postcontrast images , can depict smaller adenomas , but there is a huge potential for misinterpretation.8 thus , acth - secreting adenomas , which are frequently very small , are correctly identified by mri in only about half of cases.9 additionally , for a subset of patients , mri may be contraindicated .
furthermore , imaging of the sella and its contents may be compromised by parasellar metallic artifact from either concomitant previously treated aneurysms11 or as a result of previous sellar surgery . in the latter case ,
metallic artifact may stem from metallic mesh used during buttress repair of the sellar floor1213 or metallic debris from prior drilling.14 if mri is contraindicated or provides insufficient image quality for any of the aforementioned reasons , alternate imaging modalities must be considered .
ct scans , which are routinely ordered to provide surgeons valuable osseous anatomic detail for purposes of operative planning , may be used to visualize sellar - regions tumors , particularly when contrast is administered.15 even on older generation machines , the spatial resolution of ct scanners has allowed for the detection microadenomas as small as 3 to 4 mm,16 but the poor sensitivity of ct for microadenoma detection does not allow this to be done reliably.17 ct scans , like mri , are susceptible to metallic artifact that may degrade image quality18 in patients who have had prior attempts at resection .
scintigraphy , particularly with in - octreotide , may also aid in the diagnosis of pituitary adenomas .
scintigraphy is a nuclear medicine imaging test in which cameras capture emitted radiation from previously administered radioisotopes to form two - dimensional images .
octreotide is a somatostatin analog that binds to several somatostatin receptor subtypes.19 the normal anterior pituitary gland contains these octreotide - binding somatostatin receptor subtypes , but techniques have been developed to detect additional uptake from pituitary adenomas.20 importantly , somatostatin receptor expression , and consequentially scintigraphy visualization , differs by adenoma type .
a high proportion of growth hormone - and thyroid - stimulating hormone - secreting tumors are visualized after in - octreotide administration , whereas prolactin and nonfunctioning adenomas display variable somatostatin receptor expression.2122 acth - secreting tumors present a slightly more complicated picture . in patients with newly diagnosed cushing disease , tumors
do not demonstrate increased uptake,23 but a majority of recurrent acth - secreting tumors are visualized.22 it has been speculated that this difference may be due to decreased somatostatin receptor expression in the setting of long - term cortisol exposure.23 pet scans are another nuclear medicine study in which gamma rays are detected from previously administered radiotracers linked to biologically active molecules . in some cases pet nucleotides
have been used to measure expression of specific receptors , such as dopamine,24 or enzymes , such as monoamine oxidase b.25 much more frequently used , however , are nucleotides targeted at the more general measures of glucose uptake , via the glucose analog fdg , or anabolism , via the amino acid analog c - methionine .
both have found value in the detection of pituitary adenomas,262728293031323334 but c - methionine may have greater utility in detecting microadenomas.29 additional diagnostic utility imaging has been gained by coregistering axial pet images with either ct35 or mri.36 however , although pet coregistration with either mri373839 or ct40 has been used in intraparenchymal tumor surgery , this is the first report of pet coregistration in the setting of pituitary tumor . | recurrent pituitary disease presents unique challenges , including in some cases difficulty localizing a tumor radiographically . here , we present the case of a patient with recurrent nelson syndrome whose radiographic work - up was complicated by a significant parasellar metallic artifact .
positron emission tomography ultimately localized the lesion , and coregistration with computed tomography allowed for accurate intraoperative navigation .
additionally , we review a range of imaging techniques available in the evaluation of pituitary disease . | Introduction
Case Presentation
Discussion |
small cell lung cancer ( sclc ) accounts for 20 - 25% of all bronchogenic carcinomas and is associated with the poorest 5-year survival of all histologic types .
it represents a group of highly malignant tumors , giving rise to early and widespread metastasis at the time of diagnosis at sites such as lymph nodes , adrenal glands , liver , lung , brain and bones .
the pancreas is a relatively infrequent site of metastasis from this neoplasm , and reports on metastatic small cell carcinoma ( scc ) in the pancreas , either of pulmonary or extrapulmonary origin , to be diagnosed by fine needle aspiration ( fna ) cytology are very rare.[46 ] here , we report a case that was diagnosed as metastatic scc by ultrasound ( us)-guided fna cytology and immunocytochemical studies of a pancreatic mass .
a 66-year - old man , a heavy smoker , presented with persistent cough , weight loss and superior vena cava syndrome . computerized tomography ( ct )
ct scan of the abdomen showed a mass in the pancreatic tail , suggestive of metastasis from a primary in the lung .
grnwald giemsa ( mgg ) stain and wet - fixed smears in 95% ethanol were stained by papanicolaou method .
the smears were highly cellular and contained small , round tumor cells with scanty cytoplasm , which showed extensive nuclear molding [ figure 1a and b ] .
the nuclei of the tumor cells were round to oval and had salt and pepper type chromatin .
some singly dispersed neoplastic cells resembled lymphoma cells and were accompanied by a few cytoplasmic fragments [ figure 1c ] .
most of the tumor cells were found to have a positive reaction for pan - cytokeratin [ figure 1d ] , and a variable number were positive for neurone - specific enolase [ figure 1e ] and chromogranin a. occasional tumor cells were positive for synaptophysin , but all were negative for leukocyte common antigen . based on cytomorphology and immunocytochemistry , the diagnosis was metastatic scc .
metastatic small cell carcinoma : ultrasound - guided fna of a pancreatic mass in a 66-year - old man : cytomorphological features and immunocytochemical stainings on fna smears .
( a ) the neoplastic cells show nuclear molding and have round to oval nuclei with a salt and pepper type chromatin pattern ( papanicolaou , 400 ) .
( b ) highly cellular smear show small , round tumor cells with scanty cytoplasm , extensive nuclear molding , and mitotic activity ( mgg , 400 ) .
( c ) dissociated lymphoma - like tumor cells along with a few cytoplasmic fragments resembling lymphoglandular bodies are observed ( mgg , 1000 ) .
( d ) positive reaction for cytokeratin in most of the tumor cells ( ck , 400 ) ( e ) positive reaction for neurone - specific enolase in a few neoplastic cells , including rare large cells ( nse , 1000 ) the patient was subjected to trans - bronchial needle aspiration ( tbna ) and biopsy .
the tbna smears were non - diagnostic but trans - bronchial needle biopsy contained tissue fragments containing bronchial glands , closely associated with aggregates of small , round blue tumor cells .
the tumor cells were positive for chromogranin a , synaptophysin , cd57 and cytokeratin ( ck ) , but staining for cd 20 and cd3 was negative .
metastatic involvement of the pancreas is an infrequent clinical condition . on searching medical records of 850 patients with lung cancer , maeno et al . identified 26 ( 3.1% ) patients with pancreatic metastasis .
yoon et al . reported 53 pathologically proven metastatic tumors of the pancreas ( mtps ) ; the primary malignancies were renal cell carcinoma ( n=14 ) , gastric cancer ( n=11 ) , colorectal cancer ( n=5 ) , lymphoma ( n=4 ) , non - sclc ( n=3 ) , gastrointestinal stromal tumor ( n=2 ) , melanoma ( n=2 ) , sclc ( n=2 ) , gallbladder cancer ( n=2 ) and one case each of hepatocellular carcinoma , thymic carcinoid , liposarcoma , cholangiocarcinoma , osteosarcoma , breast cancer , duodenal cancer and ovarian cancer . among 20 non - hematological metastases in the pancreas evaluated by fna , volmar et al . observed 9 renal - cell carcinomas , 3 melanomas , 2 pulmonary small - cell carcinomas , 2 breast carcinomas and one each case of prostate carcinoma , colon adenocarcinoma , pulmonary squamous cell carcinoma and gastrointestinal stromal tumor .
reported 17 metastatic malignancies to pancreas , which accounted for 0.73% of all pancreatic fnas .
the primary sites included eight renal cell carcinomas , four lymphomas , two squamous cell carcinomas ( one from the lung and the other from the esophagus ) , one medullary thyroid carcinoma , one alveolar rhabdomyosarcoma and an sclc .
the sclc includes three morphologic categories in paraffin sections : ( 1 ) scc , ( 2 ) mixed small cell / large cell and ( 3 ) combined scc . in our case , the smears were made mostly of small cells , interspersed with occasional large cells .
the neoplastic cells had scant cytoplasm and stippled or salt - and - pepper type chromatin .
nuclear molding was conspicuous and scattered cells with paranuclear blue inclusions or apoptotic bodies were present throughout the smear .
extreme nuclear molding by clusters of small tumor cells is considered to be the most characteristic presentation of scc ; moreover , paranuclear blue inclusions , which are 1 - 4 m spherical inclusions demonstrated by romanowsky stain , may act as a diagnostically useful finding . according to arora
, nuclear molding , cell size and scant , basophilic cytoplasm were highly sensitive and specific for distinguishing scc from non - scc ( nscc ) .
other features , such as salt - and - pepper chromatin , crush artifact and apoptotic bodies , had also significantly high specificity ; however , their low sensitivity precluded their usefulness in separating scc from nscc .
difficulties may be observed in differentiating metastatic scc from other small round cell tumors and primary pancreatic scc from metastatic scc . in the present case , we observed dissociation of neoplastic cells and presence of cytoplasmic fragments resembling lymphoglandular bodies , which are common features of non - hodgkin lymphoma , but can be observed in a small percentage in other small round cell tumors . under such situations , a panel of immunohistochemical stains can be useful in differentiating scc from various other small cell neoplasms . in practical work - up of scc , immunohistochemistry is useful to discern scc from high - grade lymphoma ; scc labels with antibodies to high molecular weight cytokeratins , but not with antibodies to leukocyte common antigen ( cd45 ) . in our case ,
the neoplastic cells in fna smears were ck+ and stain for lca showed negative reaction , thus excluding the possibility of non - hodgkin lymphoma .
the most common immuno profile of metastatic ssc from a lung primary is positive staining with cytokeratin 7 and thyroid transcription factor ( ttf)-1 and no reaction to ck20 .
however , immunohistochemical studies are found to have limited value in distinguishing a primary site from metastatic scc . according to lin et al .
, immunohistochemistry has not proven useful in distinguishing primary periampullary scc from metastatic sclc . although , a high percentage of lung primary cancer expresses ttf-1 , this marker has variable positivity in patients with extrapulmonary scc , and the expression of ttf-1 has not been well explored in pancreatic scc , with only one negative reported case in literature to date .
moreover , cytokeratin ( ck7 and ck20 ) switching can occur in the natural history of pulmonary ssc . in our case ,
the pancreatic fna cytology was diagnosed as metastatic scc , which was positive for pan - ck , neurone - specific enolase , chromogranin a and synaptophysin .
however , the immunohistochemical/ immunocytochemical panels did not include ck7 , ck20 and ttf-1 , which could have further confirmed the primary lung cancer . | small cell lung carcinoma represents a group of highly malignant tumors giving rise to early and widespread metastasis at the time of diagnosis .
however , the pancreas is a relatively infrequent site of metastasis by this neoplasm , and there are only occasional reports on its fine needle aspiration ( fna ) cytology diagnosis . a 66-year - old man presented with extensive mediastinal lymphadenopathy and a mass in the pancreatic tail .
ultrasound - guided fna smears from the pancreatic mass contained small , round tumor cells with extensive nuclear molding .
the cytodiagnosis was metastatic small cell carcinoma .
immunocytochemical staining showed that a variable number of neoplastic cell were positive for cytokeratin , chromogranin a , neurone - specific enolase and synaptophysin but negative for leukocyte common antigen .
the trans - bronchial needle aspiration was non - diagnostic , but biopsy was suspicious of a small cell carcinoma .
this case represents a rare metastatic lesion in the pancreas from small cell lung carcinoma , diagnosed by fna cytology . | Introduction
Case Report
Discussion |
in statistical analysis , regression is used to explore the relationship between k input variables x1 , x2 , , xk ( also known as independent variables or explanatory variables ) and the output variable y ( also called dependent variable or response variable ) from n sets of observations . in linear regression , the method of least - squares is applied to find the regression coefficients j , j = 0 , 1 , , k , which describe the contribution of the corresponding independent variable xj in explaining the dependent variable y. the aim of regression analysis is to estimate the parameters on the basis of available / observed empirical data .
traditional studies on regression assume the observations to have crisp values . in the crisp linear regression model ,
the parameters ( regression coefficients are crisp ) appear in a linear form ; that is ,
( 1)y=0+1x1+2x2++kxk+random error .
once the coefficients 0 , 1 , 2 , ,
k are determined from the observed samples , the responses are estimated from any given sets of x1 , x2 , , xk values .
fuzzy set theory , developed by zadeh , has capability to describe the uncertain situations , containing ambiguity and vagueness .
it may be recalled that a fuzzy set a defined on a universe of discourse x is characterized by a membership function a(x ) which takes values in the interval ( i.e. , a : x ) .
the value a(x ) represents the grade of membership of x x in a. this grade corresponds to the degree to which that element or individual is similar or compatible with the concept represented by the fuzzy set .
thus , the elements may belong in the fuzzy set to a greater or lesser degree as indicated by a larger or smaller membership grade .
tanaka et al . [ 2 , 3 ] initiated the research in the area of linear regression analysis in a fuzzy environment , where a fuzzy linear system is used as a regression model .
they consider a regression model in which the relations of the variables are subject to fuzziness , that is , the model with crisp input and fuzzy parameters .
in general , fuzzy regression can be classified into two categories : when the relations of the variables are subject to fuzziness , when the variables themselves are fuzzy . when the relations of the variables are subject to fuzziness , when the variables themselves are fuzzy .
there
exist several conceptual and methodological approaches to fuzzy regression with respect to the characterization mentioned above . tanaka and watada , tanaka et al . , and tanaka and ishibuchi considered more general models in fuzzy regression . in the approaches of tanaka et al . , they considered the l - r fuzzy data and minimized the index of fuzziness of the fuzzy linear regression model .
as described by tanaka and watada , a fuzzy number is a fuzzy subset of the real line whose highest membership values are clustered around a given real number called the mean value ; the membership function is monotonic on both sides of this mean value .
hence , fuzzy number can be decomposed into position and fuzziness , where the position is represented by the element with the highest membership value and the fuzziness of a fuzzy number is represented by the membership function . the comparison among various fuzzy regression models and the difference between the approaches of fuzzy regression analysis and conventional regression analysis
chang and lee and redden and woodall pointed out some weaknesses of the approaches proposed by tanaka et al .
a fuzzy linear regression model based on tanaka 's approach by considering the fuzzy linear programming problem has also been introduced by peters . in fuzzy set theory ,
the entropy is a measure of degree of fuzziness which expresses the amount of average ambiguity / difficulty in making a decision whether an element belongs to a set or not .
the following are the four properties introduced in de luca and termini , which are widely accepted as a criterion for defining any new fuzzy entropy measure h( ) of the fuzzy set a:(i)p1 ( sharpness ) : h(a ) is minimum if and only if a is a crisp set ; that is , a(x ) = 0 or 1 for all x;(ii)p2 ( maximality ) : h(a ) is maximum if and only if a(x ) = 0.5 for all x;(iii)p3 ( resolution ) : h(a ) h(a ) , where a is sharpened version of a;(iv)p4 ( symmetry ) : h(a)=h(a ) , where a is the complement of a ; that is , a(x)=1-a(x ) .
p1 ( sharpness ) : h(a ) is minimum if and only if a is a crisp set ; that is , a(x ) = 0 or 1 for all x ; p2 ( maximality ) : h(a ) is maximum if and only if a(x ) = 0.5 for all x ; p3 ( resolution ) : h(a ) h(a ) , where a is sharpened version of a ; p4 ( symmetry ) : h(a)=h(a ) , where a is the complement of a ; that is , a(x)=1-a(x ) .
dubosis and prade [ 11 , 12 ] interpreted the measure of fuzziness h(a ) as quantity of information which is being lost in going from a crisp number to a fuzzy number .
it may be noted that the entropy of an element with a given membership function a~(x ) is increasing if a(x ) is in [ 0 , 0.5 ] and decreasing if a(x ) is in [ 0.5 , 1 ] .
we accept the definition of fuzzy number given by tanaka and watada , where the mean value is also called apex .
xn ) be a discrete random variable with probability distribution p = ( p1 , p2 , , pn ) in an experiment ; then according to shannon , the information contained in this experiment is given by
( 2)h(p)=i=1npilogpi .
based on this famous shannon 's entropy , de luca and termini indicated the following measure of fuzzy entropy :
( 3)h(a)=kxx[a(x)loga(x ) + ( 1a(x))log(1a(x))]dx .
studied fuzzy linear regression ( flr ) model with some restrictions in the form of prior information and obtained the estimators of regression coefficients with the help of fuzzy entropy for the restricted flr model .
here , we propose an intuitionistic fuzzy regression model and its general form in triangular intuitionistic fuzzy setup is given by
( 4)y~=~0+~1x~1++~kx~k+random error ,
where the value of the output variable y~ defined by ( 4 ) is a triangular intuitionistic fuzzy number ; ~0,~1, ,~k is a vector of intuitionistic fuzzy parameters where ~j=(mj;j,j;j,j ) is a triangular intuitionistic fuzzy number for j = 0,1 , , k and x~1,x~2, ,x~k are triangular intuitionistic fuzzy ( explanatory ) variables .
it may be recalled that a fuzzy set a in x , given by zadeh , is as follows :
( 5)a={(x,a(x)):xx } ,
where a : x is the membership function of the fuzzy set a and a(x ) is the grade of belongingness of x into a. thus in fuzzy set theory the grade of nonbelongingness of an element x into a is equal to 1 a(x ) . however , while expressing the degree of membership of an element in a fuzzy set , the corresponding degree of nonmembership is not always equal to one minus the degree of belongingness .
the fact is that , in real life , the linguistic negation does not always identify with logical negation .
therefore , atanassov [ 1518 ] suggested a generalization of classical fuzzy set , called intuitionistic fuzzy set ( ifs ) .
atanassov 's ifs a~ under the universal set x is defined as
( 6)a~={x,a~(x),a~(x):xx } ,
where a,a~:x are the membership and nonmembership functions such that 0a~+a~1 for all x x. the numbers a~(x ) and a~(x ) denote the degree of membership and nonmembership of an element x x to the set a~x , respectively . for each element
x x , the amount a~(x)=1-a~(x)-a~(x ) is called the degree of indeterminacy ( hesitation part ) .
it is the degree of uncertainty whether x belongs to a~ or not . in literature , burillo and bustince , lee , liu and shi , and
grzegorzewski proposed various research works on intuitionistic fuzzy numbers . in this section , the notion of ifns has been studied and presented by the taking care of these research works .
an intuitionistic fuzzy subset a~={x,a~(x),a~(x):xx } of the real line is called an intuitionistic fuzzy number if the following axioms hold:(i)a~ is normal ; that is , there exist m ( sometimes called the mean value of a~ ) such that a~(m)=1 and a~(m)=0;(ii)the membership function a~ is fuzzy - convex ; that is ,
( 7)a~(x1+(1)x2)min{a~(x1),a~(x2)}x1,x2x , ;(iii)the nonmembership function a~ is fuzzy - concave ; that is
( 8)a~(x1+(1)x2)max{a~(x1),a~(x2)},x1,x2x , ;(iv)the membership and the nonmembership functions of a~ satisfying the conditions 0 f1(x ) + g1(x ) 1 and 0 f2(x ) + g2(x ) 1 have the following form :
( 9)a~(x)={f1(x),for mxm,1,for x = m , f2(x),for mxm+,0,otherwise , where the functions f1(x ) and f2(x ) are strictly increasing and decreasing functions in [ m , m ] and [ m , m + ] , respectively , and
( 10)a~(x)={g1(x),for mxm,0,for x = m , g2(x),for mxm+,1,otherwise , where the functions g1(x ) and g2(x ) are strictly decreasing and increasing functions in [ m , m ] and [ m , m + ] , respectively . here
and are called the left and right spreads of the membership function a~ , respectively . and are called the left and right spreads of the nonmembership function a~(x ) .
an intuitionistic fuzzy subset a~={x,a~(x),a~(x):xx } of the real line is called an intuitionistic fuzzy number if the following axioms hold:(i)a~ is normal ; that is , there exist m ( sometimes called the mean value of a~ ) such that a~(m)=1 and a~(m)=0;(ii)the membership function a~ is fuzzy - convex ; that is ,
( 7)a~(x1+(1)x2)min{a~(x1),a~(x2)}x1,x2x , ;(iii)the nonmembership function a~ is fuzzy - concave ; that is
( 8)a~(x1+(1)x2)max{a~(x1),a~(x2)},x1,x2x , ;(iv)the membership and the nonmembership functions of a~ satisfying the conditions 0 f1(x ) + g1(x ) 1 and 0 f2(x ) + g2(x ) 1 have the following form :
( 9)a~(x)={f1(x),for mxm,1,for x = m , f2(x),for mxm+,0,otherwise , where the functions f1(x ) and f2(x ) are strictly increasing and decreasing functions in [ m , m ] and [ m , m + ] , respectively , and
( 10)a~(x)={g1(x),for mxm,0,for x = m , g2(x),for mxm+,1,otherwise , where the functions g1(x ) and g2(x ) are strictly decreasing and increasing functions in [ m , m ] and [ m , m + ] , respectively . here
and are called the left and right spreads of the membership function a~ , respectively .
and are called the left and right spreads of the nonmembership function a~(x ) .
a~ is normal ; that is , there exist m ( sometimes called the mean value of a~ ) such that a~(m)=1 and a~(m)=0 ; the membership function a~ is fuzzy - convex ; that is ,
( 7)a~(x1+(1)x2)min{a~(x1),a~(x2)}x1,x2x , ; the nonmembership function a~ is fuzzy - concave ; that is
( 8)a~(x1+(1)x2)max{a~(x1),a~(x2)},x1,x2x , ; the membership and the nonmembership functions of a~ satisfying the conditions 0 f1(x ) + g1(x ) 1 and 0 f2(x ) + g2(x ) 1 have the following form :
( 9)a~(x)={f1(x),for mxm,1,for x = m , f2(x),for mxm+,0,otherwise , where the functions f1(x ) and f2(x ) are strictly increasing and decreasing functions in [ m , m ] and [ m , m + ] , respectively , and
( 10)a~(x)={g1(x),for mxm,0,for x = m , g2(x),for mxm+,1,otherwise , where the functions g1(x ) and g2(x ) are strictly decreasing and increasing functions in [ m , m ] and [ m , m + ] , respectively . here
and are called the left and right spreads of the membership function a~ , respectively . and are called the left and right spreads of the nonmembership function a~(x ) . symbolically , an intuitionistic fuzzy number is represented as a~ifn=(m;,;, ) .
an ifn aifn = ( m ; , ; , ) may be defined as a triangular intuitionistic fuzzy number ( tifn ) if and only if its membership and nonmembership functions take the following form :
( 11)a~(x)={1mx,for mxm,1,for x
= m,1xm,for mxm+,0,otherwise,(12)a~(x)={mx,for mxm,0,for x = m , xm,for mxm+,1,otherwise .
an ifn aifn = ( m ; , ; , ) may be defined as a triangular intuitionistic fuzzy number ( tifn ) if and only if its membership and nonmembership functions take the following form :
( 11)a~(x)={1mx,for mxm,1,for x
it may be noted that a tifn a~=(m;,;, ) degenerate to a triangular fuzzy number a = ( m ; , ) if = , = , and a~(x)=1-a~(x ) , x . further , an tifn a~={x,a~(x),a~(x):x } ; that is , a~=(m;,;, ) is a conjunction of two fuzzy numbers a = ( m ; , ) with the membership function a+(x)=a~(x ) and a = ( m ; , ) with the membership function a~(x)=1-a~(x ) .
the entropy calculated using ( 3 ) from the membership function of tifn given by ( 11 ) can be expressed as follows : size
( 13)h(a)=k[x[m,m][a(x)loga(x)+(1a(x ) ) log(1a(x))]dx + x[m , m+][a(x)loga(x)+(1a(x ) ) log(1a(x))]dxx[m,m][a(x)loga(x)+(1a(x))]=hl(a~)+hr(a~ ) ,
where hl(a~)=k/2 and hr(a~)=k/2 .
it follows that h(a~)=k(+)/2 , which does not depend on m. it may be observed that , in the case of symmetrical tifn , the left and the right entropies are identical . on the other hand , in case of nonsymmetric tifn , the left entropy is a function of and the right entropy is a function of . similarly , the left entropy and the right entropy from the nonmembership function ( which we called left to left and right to right entropies ) of the tifn are the functions of and , respectively .
hence , a triangular intuitionistic fuzzy number can be characterized by five attributes : the position parameter m , the left entropy , the right entropy , left to left entropy , and right to right entropy . there is a one - to - one correspondence between a triangular intuitionistic fuzzy number and its entropies . in other words ,
given a triangular intuitionistic fuzzy number , one can determine the unique position and entropies .
conversely , given a position and entropies , one can construct a unique triangular intuitionistic fuzzy number .
sometimes experimenter 's past experiences may be available as prior information about unknown regression coefficients to estimate more efficient estimators . here
, we assume that such prior information is provided in the form of exact linear restrictions on regression coefficients . in the present work ,
we first find the unrestricted estimators of regression coefficients with the help of fuzzy entropy .
further , the restricted estimators of the regression coefficients are obtained by incorporating the prior information in the form of linear restrictions .
,x~ik ) , i = 1 , , n , in the regression analysis are triangular intuitionistic fuzzy numbers .
the notion of regression using fuzzy entropy is to construct five conventional regression equations ( one for apex , one for left entropy of the membership function , one for right entropy of the membership function , one for left entropy of the nonmembership function , and one for right entropy of the nonmembership function ) for the response variable y~ using the corresponding attributes of the k fuzzy explanatory variables x~j . in order to be specific ,
we denote y , x1 , x2 , , xk by the apexes of y~,x~1,x~2, ,x~k , respectively , ey , ex1 , ex2 , , exk by the left entropy of y~,x~1,x~2, ,x~k , respectively , ey , ex1 , ex2 , , exk by the right entropy of y~,x~1,x~2, ,x~k , respectively , ey , ex1 , ex2 , , exk by the left to left entropy of y~,x~1,x~2, ,x~k , respectively , and ey , ex1 , ex2 , , exk by the right to right entropy of y~,x~1,x~2, ,x~k , respectively .
therefore , the five fundamental regression equations in a nonrecursive ( nonadaptive ) setup may be written as
( 14)ya = a0a+i=1k(aiaxia+biaexil+ciaexir+diaexil+eiaexir)+ya;eyl = a0l+i=1k(ailxia+bilexil+cilexir+dilexil+eilexir)+eyl;eyr = a0r+i=1k(airxia+birexil+cirexir+direxil+eirexir)+eyr;eyl=a0l+i=1k(ailxia+bilexil+cilexir+dilexil+eilexir)+eyl;eyr=a0r+i=1k(airxia+birexil+cirexir+direxil+eirexir)+eyr ,
where y , ey , ey , ey , and ey are the error vectors of dimension n 1 .
the compact form of the above mentioned nonrecursive or nonadaptive equations is given by
( 15)ya = x+ya , eyl = x+eyl , eyr = x+eyr , eyl=x+eyl,eyr=x+eyr ,
where
( 16)x=(1 x1a , x2a, ,xka ex1l , ex2l, ,exkl ex1r , ex2r, ,exkr ex1l,ex2l,
,exkl ex1r,ex2r, ,exkr)n(5k+1),=(a0a a1a , a2a, ,aka b1a , b2a, ,bka c1a , c2a , , cka d1a , d2a, ,dka e1a , e2a, ,eka)(5k+1)1t,=(a0l a1l , a2l, ,akl b1l , b2l, ,bkl c1l , c2l , , ckl d1l , d2l, ,dkl e1l , e2l, ,ekl)(5k+1)1t,=(a0r a1r , a2r, ,akr b1r , b2r, ,bkr c1r , c2r , , ckr d1r , d2r, ,dkr e1r , e2r,
,akl b1l,b2l, ,bkl c1l,c2l , , ckl d1l,d2l,
,akr b1r,b2r, ,bkr c1r,c2r , , ckr d1r,d2r,
,ekr)(5k+1)1 t .
in many real life situations , where the measurements are carried out ( for example car speed astronomical distance ) ,
it is natural to think that the spread ( vagueness ) in the measure of a phenomenon is proportional to its intensity .
d'urso and gastaldi have done several simulations and observed that even if we consider an adaptive or recursive regression model along with nonadaptive or nonrecursive regression model , they yield identical solutions when there is only one independent variable .
but if there are more than one independent variable , then the estimated values of the left entropies and right entropies obtained through the recursive fuzzy regression model will have less variance as compared to the nonrecursive fuzzy regression model . with this consideration ,
we rewrite the proposed intuitionistic fuzzy linear regression model ( 15 ) in a recursive / adaptive setup where dynamic of the entropies is dependent on the magnitude of the estimated apexes as follows :
( 17)ya = ya+ya ; where ya=x,eyl = eyl+eyl ; where eyl=xb+1d , eyr = eyr+eyr ; where eyr=xf+1g , eyl=eyl+eyl ; where eyl=xp+1q , eyr=eyr+eyr ; where eyr=xu+1v ,
where x is the n ( 5k + 1)-matrix containing the values of the input variables ( data matrix ) , is a column 5k + 1-vector containing the regression coefficients for the apexes of the first model ( referred to as core regression model ) , y and y are the vector of the observed apexes and the vector of the interpolated apexes , respectively , both having dimension n 1 , ey and ey are the vector of the observed left entropies and the vector of the interpolated left entropies , respectively , both having dimension n 1 , ey and ey are the vector of the observed right entropies and the vector of the interpolated right entropies , respectively , both having dimension n 1 , ey and ey are the vector of the observed left to left entropies and the vector of the interpolated left to left entropies , respectively , both having dimension n 1 , ey and ey are the vector of the observed right to right entropies and the vector of the interpolated right to right entropies , respectively , both having dimension n 1 , and 1 is a ( n 1)-vector of all 1s , b and d are regression parameters for the second regression equation model ( referred to as left entropy regression model ) , f and g are regression parameters for the third regression model ( referred to as right entropy regression model ) , p and q are regression parameters for the fourth regression equation model ( referred to as left to left entropy regression model ) , and u and v are regression parameters for the fifth regression equation model ( referred to as right to right entropy regression model ) . the error term in the regression equation of apexes will remain the same while the error terms in the regression equations of entropies may be different .
the error vectors ey and ey in the left and right entropies are of the dimension ( n 1 ) and the error vectors ey and ey in the left to left and right to right entropies are of the dimension ( n 1 ) .
if some prior information about unknown regression coefficients is available on the basis of past experiences , then it may be used to estimate more efficient estimators .
we assume that such prior information is in the form of exact linear restrictions on regression coefficients . in the present model ,
we associate such restrictions in the equations for the estimation of regression coefficients in the intuitionistic fuzzy linear regression model with fuzzy entropy .
therefore , we make the model capable of taking into account possible linear relations between the size of the entropies and the magnitude of the estimated apexes .
moreover , we assume that the regression coefficients are subjected to the j ( j < 5k + 1 ) exact linear restrictions , which are given by
( 18)h = h ,
where h and h are known and the matrix h is of full row rank .
in many applications , it is possible that the values of the variables are on completely different scales of measurement . also , the possible larger variations in the values will have larger intersample differences , so they will dominate in the calculation of euclidean distances . therefore
consider the euclidean distance between two triangular intuitionistic fuzzy numbers yi = ( yi ; eyi , eyi ; eyi , eyi ) and yi = ( yi next , similar to common linear regression ( based on crisp data ) , the regression parameters are estimated by minimizing the following sum of square errors ( we use a compact matrix notation ) :
( 20)(,b , d , f , g , p , q , u , v ) = i=1nw1(yiayia)2+i=1nw2(eyileyil)2 + i=1nw3(eyireyir)2+i=1nw4(eyileyil)2 + i=1nw5(eyireyir)2 = w1(yaya)t(yaya)+w2(eyleyl)t(eyleyl ) + w3(eyreyr)t(eyreyr ) + w4(eyleyl)t(eyleyl ) + w5(eyreyr)t(eyreyr ) = w1((ya)tya2(ya)tya+(ya)tya ) + w2((eyl)teyl2(eyl)teyl+(eyl)teyl ) + w3((eyr)teyr2(eyr)teyr+(eyr)teyr ) + w4((eyl)teyl2(eyl)teyl+(eyl)teyl ) + w5((eyr)teyr2(eyr)teyr+(eyr)teyr ) = w1((ya)tya2(ya)tx+txtx ) + w2((eyl)teyl2(eyl)t(xb+1d ) ) + w2((xb+1d)t(xb+1d ) ) + w3((eyr)teyr2(eyr)t(xf+1 g ) ) + w3((xf+1g)t(xf+1 g ) ) + w4((eyl)teyl2(eyl)t(xp+1q ) ) + w4((xp+1q)t(xp+1q ) ) + w5((eyr)teyr2(eyr)t(xu+1v ) ) + w5((xu+1v)t(xu+1v ) ) = w1((ya)tya2(ya)tx ) + txtx(w1+w2b2+w3f2+w4p2+w5u2 ) + w2((eyl)teyl2(eyl)txb2(eyl)t1d ) + w3((eyr)teyr2(eyr)txf2(eyr)t1 g ) + w4((eyl)teyl2(eyl)txp2(eyl)t1q ) + w5((eyr)teyr2(eyr)txu2(eyr)t1v ) + 2txt1(w2bd+w3fg+w4pq+w5uv ) + n(w2d2+w3g2+w4q2+w5v2 ) .
differentiating ( , b , d , f , g , p , q , u , v ) , that is , ( 20 ) , partially with respect to and equating it to zero , we get
( 21)(,b , d , f , g , p , q , u , v)=0w1xtya+xtx(w1+w2b2+w3f2+w4p2+w5u2)w2xteylbw3xteyrfw4xteylpw5xteyru+xt1(w2bd+w3fg+w4pq+w5uv)=0=((xtx)1xt[w1ya+w2eylb+w3eyrf + w4eylp+w5eyru 1(w2bd+w3fg+w4pq+w5uv)eyl](xtx)1) (w1+w2b2+w3f2+w4p2+w5u2)1 .
similarly , differentiating ( 20 ) partially with respect to b , d ,
f , g , p , q , u , and v , we get
( 22)b=(txtx)1[(eyl)txtxt1d];(23)d=1n[(eyl)t1txt1b];(24)f=(txtx)1[(eyr)txtxt1g];(25)g=1n[(eyr)t1txt1f];(26)p=(txtx)1[(eyl)txtxt1q];(27)q=1n[(eyl)t1txt1p];(28)u=(txtx)1[(eyr)txtxt1v];(29)v=1n[(eyr)t1txt1u ] ;
respectively . equations ( 21)(29 )
therefore , we rewrite the system of equations explicitly in a recursive way as follows :
( 30)i+1=((xtx)1xt[w1ya+w2eylbi+w3eyrfi+w4eylpi + w5eyrui1(w2bidi+w3figi + w4piqi+w5uivi)w1ya+w2eylbi+w3eyrfi+w4eylpi](xtx)1)(w1+w2bi2+w3fi2+w4pi2+w5ui2)1;bi+1=(i+1txtxi+1)1[(eyl)txi+1i+1txt1di];di+1=1n[(eyl)t1i+1txt1bi];fi+1=(i+1txtxi+1)1[(eyr)txi+1i+1txt1gi];gi+1=1n[(eyr)t1i+1txt1fi];pi+1=(i+1txtxi+1)1[(eyl)txi+1i+1txt1qi];qi+1=1n[(eyl)t1i+1txt1pi];ui+1=(i+1txtxi+1)1[(eyr)txi+1i+1txt1vi];vi+1=1n[(eyr)t1i+1txt1ui ] .
in order to initiate the recursive process of obtaining the estimators , we take some initial values for b , d , f , g , p ,
q , u , v , and . after several numbers of iterations , the values of estimators get corrected to a predefined error of tolerance .
we denote these values by b^ , d^ , f^ , g^ , p^ , q^ , u^ , v^ , and ^ in order to differentiate them from the eventually obtained restricted estimator ~ in the next commutation . in a more general setup , if , in the linear regression model ( 17 ) , we consider k1 crisp and k2 intuitionistic fuzzy input variables , then the dimensions of x and will be n ( k1 + 5k2 + 1 ) and ( k1 + 5k2 + 1 ) 1 , respectively . it may further be noted that the core of the solution 's structure will remain the same and we will have similar kind of estimators .
remark .
if a tifn a~=(m;,;, ) degenerate to a triangular fuzzy number a = ( m ; , ) , then our nonsymmetric intuitionistic fuzzy weighted linear regression model reduces to nonsymmetric fuzzy linear regression model defined by kumar et al . .
next , we assume that the regression coefficients are subjected to the linear restrictions which are given by ( 18 ) .
it may be noted that the unrestricted estimator obtained above in ( 21 ) does not satisfy the given restrictions ( 18 ) .
we aim to obtain the restricted estimator which satisfies the given restrictions under the regression model ( 17 ) . for this
, we propose to minimize the following score function :
( 31)s(,,b , d , f , g , p , q , u , v ) = (,b , d , f , g , p , q , u , v)2(hh ) = w1((ya)tya2(ya)tx ) + txtx(w1+w2b2+w3f2+w4p2+w5u2 ) + w2((eyl)teyl2(eyl)txb2(eyl)t1d ) + w3((eyr)teyr2(eyr)txf2(eyr)t1 g ) + w4((eyl)teyl2(eyl)txp2(eyl)t1q ) + w5((eyr)teyr2(eyr)txu2(eyr)t1v ) + 2txt1(w2bd+w3fg+w4pq+w5uv ) + n(w2d2+w3g2+w4q2+w5v2 ) 2(hh ) , where 2 is the vector of lagrange 's multiplier .
differentiating s( , , b , d , f , g , p , q , u , v ) partially with respect to and equating it to zero , we get
( 32)w1xtya+xtx(w1+w2b2+w3f2+w4p2+w5u2 ) w2xteylbw3xteyrfw4xteylpw5xteyru + xt1(w2bd+w3fg+w4pq+w5uv)h=0 .
here , we again relabel the computed restricted estimator by ~. therefore , in view of ( 21 ) and ( 32 ) , we get size
( 33)~=((xtx)1xt[w1ya+w2eylb+w3eyrf+w4eylp + w5eyru 1(w2bd+w3fg+w4pq+w5uv)eyl](xtx)1 ) (w1+w2b2+w3f2+w4p2+w5u2)1 + ( xtx)1ht(w1+w2b2+w3f2+w4p2+w5u2)~=^+1(w1+w2b2+w3f2+w4p2+w5u2 ) (xtx)1ht. similarly , differentiating s( , , b , d , f , g , p , q , u , v ) partially with respect to and equating it to zero , we get
( 34)h~=hh^+1(w1+w2b2+w3f2+w4p2+w5u2 ) h(xtx)1ht=h^=(w1+w2b2+w3f2+w4p2+w5u2 ) [h(xtx)1ht]1(hh^ ) .
from ( 33 ) and ( 34 ) , we have
( 35)~=^+(xtx)1ht[h(xtx)1ht]1(hh^ ) .
also , differentiating ( 31 ) partially with respect to b , d , f , g , p , q , u , and v and equating all to zero , we get
( 36)b~=b^ , d~=d^ , f~=f^ , g~=g^,p~=p^ , q~=q^ , u~=u^ , v~=v^ ,
respectively . from ( 35 ) we see that
( 37)h~=h^+[h(xtx)1ht][h(xtx)1ht]1 (hh^)h~=h^+(hh^)=h .
we apply our procedure to estimate the intuitionistic fuzzy output value for a data consisting of the crisp input and intuitionistic fuzzy output ( where left entropy and right entropy are equal ) and tabulate the data in table 1.we obtain ^=(-4.4026,3.5733,7.3786,5.6858) , b^=0.2942 , d^=14.7144 , f^=0.2942 , g^=14.7144 , p^=0.2909 , q^=17.4487 , u^=0.2909 , and v^=17.4487 where the number of iterations required is 125 .
we apply our procedure to estimate the intuitionistic fuzzy output value for a data consisting of the crisp input and intuitionistic fuzzy output ( where left entropy and right entropy are equal ) and tabulate the data in table 1 .
we obtain ^=(-4.4026,3.5733,7.3786,5.6858) , b^=0.2942 , d^=14.7144 , f^=0.2942 , g^=14.7144 , p^=0.2909 , q^=17.4487 , u^=0.2909 , and v^=17.4487 where the number of iterations required is 125 .
we apply our procedure to estimate intuitionistic fuzzy output value for a data consisting of crisp input and intuitionistic fuzzy output ( where left and right entropy are not equal ) and tabulate the data in table 2.we obtain ^=(-4.7697,3.5933,7.2030,5.9152) , b^=0.2952 , d^=14.5871 , f^=0.2646 , g^=20.3429 , p^=0.3052 , q^=15.7050 , u^=0.2717 , and v^=23.1201 where the number of iterations required is 113 .
we apply our procedure to estimate intuitionistic fuzzy output value for a data consisting of crisp input and intuitionistic fuzzy output ( where left and right entropy are not equal ) and tabulate the data in table 2 .
we obtain ^=(-4.7697,3.5933,7.2030,5.9152) , b^=0.2952 , d^=14.5871 , f^=0.2646 , g^=20.3429 , p^=0.3052 , q^=15.7050 , u^=0.2717 , and v^=23.1201 where the number of iterations required is 113 .
we apply our procedure to estimate intuitionistic fuzzy output value for a data consisting of crisp input , intuitionistic fuzzy input , and intuitionistic fuzzy output ( where left and right entropy are not equal ) and tabulate the data in table 3.we obtain ^=(-3.2352,0.6811,0.5314,-0.9164,0.0846,-3.1631,2.953) , b^=0.4225 , d^=0.5478 , f^=0.4307 , g^=0.1637 , p^=0.3231 , q^=3.8723 , u^=0.4985 , and v^=1.8659 where the number of iterations required is 51 .
we apply our procedure to estimate intuitionistic fuzzy output value for a data consisting of crisp input , intuitionistic fuzzy input , and intuitionistic fuzzy output ( where left and right entropy are not equal ) and tabulate the data in table 3 .
we obtain ^=(-3.2352,0.6811,0.5314,-0.9164,0.0846,-3.1631,2.953) , b^=0.4225 , d^=0.5478 , f^=0.4307 , g^=0.1637 , p^=0.3231 , q^=3.8723 , u^=0.4985 , and v^=1.8659 where the number of iterations required is 51 .
we apply our procedure to estimate intuitionistic fuzzy output value for a data consisting of intuitionistic fuzzy input and intuitionistic fuzzy output ( where left and right entropy are not equal ) and tabulate the data in table 4.we obtain ^=(11.8141,-0.2161,1.6104,-1.8254,0.5687,-0.1879) , b^=0.3880 , d^=0.3674 , f^=0.3880 , g^=0.3674 , p^=0.3547 , q^=2.2108 , u^=0.3547 , and v^=3.2108 where the number of iterations required is 255 .
we apply our procedure to estimate intuitionistic fuzzy output value for a data consisting of intuitionistic fuzzy input and intuitionistic fuzzy output ( where left and right entropy are not equal ) and tabulate the data in table 4 .
we obtain ^=(11.8141,-0.2161,1.6104,-1.8254,0.5687,-0.1879) , b^=0.3880 , d^=0.3674 , f^=0.3880 , g^=0.3674 , p^=0.3547 , q^=2.2108 , u^=0.3547 , and v^=3.2108 where the number of iterations required is 255 .
an intuitionistic fuzzy weighted linear regression ( ifwlr ) model with and without some linear restrictions in the form of prior information has been studied .
the estimators of regression coefficients have also been obtained with the help of fuzzy entropy for the restricted / unrestricted ifwlr model by assigning some weights in the distance function .
it has been observed that the restricted estimator is better than unrestricted estimator in some sense .
thus , whenever some prior information is available in terms of exact linear restrictions on regression coefficients , it is advised to use restricted estimator ~ in place of unrestricted estimator ^. | in fuzzy set theory , it is well known that a triangular fuzzy number can be uniquely determined through its position and entropies . in the present communication
, we extend this concept on triangular intuitionistic fuzzy number for its one - to - one correspondence with its position and entropies . using the concept of fuzzy entropy
the estimators of the intuitionistic fuzzy regression coefficients have been estimated in the unrestricted regression model .
an intuitionistic fuzzy weighted linear regression ( ifwlr ) model with some restrictions in the form of prior information has been considered .
further , the estimators of regression coefficients have been obtained with the help of fuzzy entropy for the restricted / unrestricted ifwlr model by assigning some weights in the distance function . | 1. Introduction
2. Restricted IFWLR Model with Fuzzy Entropy
3. Estimation of Regression Coefficients
4. Numerical Examples
5. Conclusions |
they are important in numerous processes , such as energy storage , molecular recognition , and mediation in many biological functions [ 13 ] .
carbohydrates form part of the backbone of rna and dna molecules , and they are also linked to proteins and lipids as glycoproteins and glycolipids .
the complex functions of carbohydrate molecules are attributed to their outstanding conformational variety and flexibility .
noncovalent interaction between carbohydrates and neighboring molecules , such as metal ions , water , or other noncovalently bound molecules , can effectively stabilize favored conformations of these complexes and play a vital role in molecular recognition processes .
therefore , exploration and characterization of water carbohydrate interactions as well as noncovalently bound metal ion - carbohydrate systems and their microhydrated complexes is an essential step in understanding the origins of the biochemical functionality of carbohydrates .
sodium and potassium cations are involved in the transport of amino acids and monosaccharides across cell membranes , and play an important role in physiologic and pathologic processes in the extracellular environment .
a number of techniques , such as nmr [ 5 , 6 ] , neutron - scattering , terahertz absorption spectroscopy , and computer simulations [ 811 ] have been used to study the dynamics and hydration properties of different monosaccharide units and short oligosaccharides in water .
water interactions with a variety of carbohydrate molecules have also been studied in the gas phase in the absence of solvent effects to enable the intrinsic behavior of the system to be examined and evaluated in detail .
such gas phase studies eliminate the effects of the surrounding solvent that complicate and may mask behavior of the system in the condensed phase .
hydrogen bonding and the conformational structures of phenyl - substituted mono- and oligosaccharides and their hydrated complexes have been widely investigated by simons and co - workers [ 1217 ] by a combination of mass - selected resonant two - photon ionization ( r2pi ) , uv hole burning , ir - dip , spectroscopy experiments , and computation through molecular mechanics , density functional theory ( dft ) and ab initio calculations .
these studies reveal that the water binding site(s ) to the preferred carbohydrate conformations and structures of their hydrated complexes are dominated by cooperative hydrogen bonding .
a number of studies have been conducted on the interaction of monosaccharides with alkali ions in the gas phase .
bothner et al . characterized monosaccharide / alkali cation ( li , na , k , rb , or cs ) complexes of d - glucose , d - galactose , d - mannose , d - fructose , and a deuterated analog of glucose , 6,6-d - glucose - d2 , using different mass spectrometric techniques and molecular modeling .
cerda and wesdemiotis used the kinetic method and ab initio calculations to investigate the thermochemistry and structures of sodiated mono- and disaccharide stereoisomers .
their results indicate that each saccharide solvates the sodium ion differently , depending on the donor groups available .
more recently , heaton and armentrout investigated the sodium cation complexes of arabinose , xylose , glucose , and galactose using a guided ion beam tandem mass spectrometer equipped with an electrospray ionization source , and quantum chemical calculations .
they found that the experimental na binding affinities follow the trend arabinose < xylose < glucose < galactose . because biomolecules and metal ions are surrounded by water in living systems , interactions of their complexes with water molecules are of significant importance in understanding the molecular mechanisms involved in biological processes .
the structures and energetics of hydrated amino acid / alkali cation complexes have been studied using blackbody dissociation ( bird ) experiments [ 2129 ] , guided ion beam studies [ 3032 ] , high - pressure mass spectrometry [ 33 , 34 ] , infrared multiple photon dissociation ( irmpd ) spectroscopy [ 29 , 35 ] , and calculations [ 2132 , 35 , 36 ] .
very recently , fridgen and co - workers [ 3739 ] used irmpd spectroscopy in combination with computational methods to determine the structures of singly hydrated nucleobase / alkali cation complexes . in this study , we present the first experimental results on the water binding energies to the monosaccharide / cation complexes , arax , xylx , ribx , glcx , and galx , where x = na or k. these results were obtained from high - pressure mass spectrometry equilibria measurements in the gas phase .
the experiments were performed with a home - made 60 magnetic sector high - pressure mass spectrometer ( hpms ) using a pulsed ion - beam esi ion source , which has been previously described in detail .
briefly , cationized monosaccharides were obtained by electrospray from a silica capillary ( 15 m i.d . ; 150 m o.d . ) . a solution containing ~2.0 mm monosaccharide in water / methanol ( 1:1 ) and nacl or ki
was supplied to the capillary by a syringe pump at a rate of 0.8 l / min .
the clustered ions were desolvated by a dry nitrogen gas counter - current and in a heated ( ~80c ) pressure - reducing capillary through which they were introduced into the fore - chamber , and then deflected toward a 3-mm orifice in the interface plate leading to the reaction chamber ( rc ) .
ions drifting across the rc toward the exit slit under the influence of a weak electric field ( 2 v / cm at 10 mbar ) were hydrated and reached equilibrium prior to being sampled to the mass analysis section of the mass spectrometer .
ion detection was provided by a secondary electron scintillation detector of the daly type with an aluminum conversion dynode using a short rise - time photomultiplier ( hamamatsu r-647 - 04 ; ) .
the output pulses of the multiplier were counted using a multichannel scaler with dwell - time per channel of 1 s .
mass spectra were registered with continuous ion sampling , while for equilibrium determination the ion beam was injected into the rc in a pulsed mode by applying short pulses ( + 45 v , 90 s ) to the deflection electrode with repetition of 1 ms .
typically , several thousand injection pulses were sufficient to accumulate a reasonable signal of the ion arrival time distribution ( atd ) for each mass on the multichannel scaler ( figure 1 ) .
figure 1arrival time distributions of the reactant , xylna , and product , xylna(h2o ) , ions .
the inset shows the ratio of ion intensities , [ xylna]/[xylna(h2o ) ] , as a function of ion residence time arrival time distributions of the reactant , xylna , and product , xylna(h2o ) , ions .
the inset shows the ratio of ion intensities , [ xylna]/[xylna(h2o ) ] , as a function of ion residence time the reagent gas mixture consisting of pure n2 as the carrier gas at about 10 mbar and a known partial pressure of water vapor ( 0.020.20 mbar ) was supplied to the rc via the heated reactant gas inlet ( rgi ) at a flow rate of ~100 ml / min .
the pressure was measured with an mks capacitance manometer attached near the inlet of the rgi .
the amount of water introduced into the n2 gas flow was kept constant throughout the temperature - dependent measurements of the equilibrium constants .
water concentrations were controlled continuously with a calibrated temperature and humidity transmitter ( delta ohm , type do 9861 t ; italy ) inserted into the carrier gas flow line .
the rc temperature was monitored by an iron - constantan thermocouple which was embedded close to the ion exit slit ; the temperature could be varied from ambient to 300 c by electrical heaters .
the equilibrium constants kn-1,n equation ( 1 ) for the association reaction equation ( 2 )
1\documentclass[12pt]{minimal }
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\begin{document}$$ { k_{{n - } } } _ { { { 1},n } } = \left ( { { i_n}\cdot { p_o}/{i_{{n - } } } _ { { 1}}\cdot p } \right ) $ $ \end{document}2\documentclass[12pt]{minimal }
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\begin{document}$$ \begin{array}{*{20}{c } } { { \text{sac}}{{\text{x}}^ { + } } \cdot { { \left ( { { { \text{h}}_{{2}}}{\text{o } } } \right)}_{{n - } } } _ { { 1 } } + { { \text{h}}_{{2}}}{\text{o } } \leftrightarrow { \text{sac}}{{\text{x}}^ { + } } \cdot { { \left ( { { { \text{h}}_{{2}}}{\text{o } } } \right)}_n } } \hfill \\ { { \text{sac } } = { \text{ara}},\;{\text{xyl}},\;{\text{rib}},\;{\text{glc}},\;{\text{or}}\;{\text{gal } } } \hfill \\ \end{array } $ $ \end{document}were determined from the measured atd peak areas of the ions sacx(h2o)n and sacx(h2o)n1 , whose ratio , in / in1 , is taken to be equal to the ion concentration ratio , and p is the known partial pressure of h2o ( in mbar ) .
equilibrium attainment was checked by comparing the atds of the reactant and product ions , and testing the ion intensity ratio of [ sacx(h2o)n-1]/[sacx(h2o)n ] as a function of ion residence time .
this is illustrated in figure 1 for the pair of ions xylna and xylna(h2o ) , while the inset shows the ratio of their intensity as a function of time .
clearly , within the error limits and the limits of statistical noise , the ratio of [ xylna]/[xylna(h2o ) ] remains essentially constant , suggesting that the system is at equilibrium under the present experimental conditions .
the equilibrium constants , kn1,n , as functions of temperature for the sacna(h2o)n and sack(h2o ) systems , are shown in the vant hoff plots of figures 2 and 3 .
the standard enthalpy , \documentclass[12pt]{minimal }
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\begin{document}$$ \delta s_n^o $ $ \end{document } , changes for each equilibrium equation ( 2 ) , obtained from the slope and intercept of the vant hoff plot , are shown in tables 1 and 2 . the data for lower case n are given in the tables , since determining the hydration enthalpies for higher hydration steps requires temperatures below room temperature , which are not accessible with the present reaction chamber .
the reported \documentclass[12pt]{minimal }
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\begin{document}$$ \delta s_n^o $ $ \end{document } values are the averages of at least three measurements and the uncertainty corresponds to the standard deviation of the linear least - squares fit .
the free energy , \documentclass[12pt]{minimal }
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\begin{document}$$ \delta g_n^o = \delta h_n^o - t\delta s_n^o $ $ \end{document}.
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\begin{document}$$ { \text{sacn}}{{\text{a}}^ { + } } { \left ( { { { \text{h}}_{{2}}}{\text{o } } } \right)_n } _ { { - { 1 } } } + { } { { \text{h}}_{{2}}}{\text{o } } < = = > { \text{sacn}}{{\text{a}}^ { + } } { \left ( { { { \text{h}}_{{2}}}{\text{o } } } \right)_n } $ $ \end{document } : ( a ) n = 1 , and ( b ) n = 2.figure 3vanhoff plots of equilibrium constants for the gas - phase reactions sack + h2o < = > sack(h2o).table 1experimental enthalpies , entropies , and free energy values for the hydration of the sodiated monosaccharidesionn\documentclass[12pt]{minimal }
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\begin{document}$$ - \delta g_n^o $ $ \end{document } ( kj / mol)d(sac - na ) ( kj / mol)arana+157.3(2)74.9(2)35.0(3)170 ; 173243.1(2)79.5(6)19.4(4)xylna+156.1(2)76.6(5)33.3(4)171 ; 183244.8(3)84.1(7)19.7(5)ribna+157.7(1)87.9(4)31.5(2)176252.3(1)113.0(6)18.6(3)glcna+154.0(1)73.6(6)32.0(3)174 ; 183251.0(2)113.0(4)17.0(3)galna+146.4(1)64.4(2)27.4(2)177 ; 205242.7(2)87.9(4)16.5(3)standard pressure is 1000 mbaruncertainties in parenthesesgn at 298 kexp .
values from ref . at 298 ktable 2experimental enthalpies , entropies , and free energy values for the hydration of the potassiated monosaccharidesionh ( kj / mol)s ( j / mol k)g ( kj / mol)arak49.4(1)92.0(4)22.0(2)xylk52.3(2)108.0(5)20.0(3)ribk52.7(2)113.8(7)18.8(4)glck48.5(2)92.9(8)20.8(4)galk49.8(1)101.3(3)19.6(2)standard pressure is 1000 mbaruncertainties in parenthesesg at 298 k vant hoff plots of equilibrium constants for the gas - phase reactions \documentclass[12pt]{minimal }
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\begin{document}$$ { \text{sacn}}{{\text{a}}^ { + } } { \left ( { { { \text{h}}_{{2}}}{\text{o } } } \right)_n } _ { { - { 1 } } } + { } { { \text{h}}_{{2}}}{\text{o } } < = = > { \text{sacn}}{{\text{a}}^ { + } } { \left ( { { { \text{h}}_{{2}}}{\text{o } } } \right)_n } $ $ \end{document } : ( a ) n = 1 , and ( b ) n = 2 .
vanhoff plots of equilibrium constants for the gas - phase reactions sack + h2o < = > sack(h2o ) .
experimental enthalpies , entropies , and free energy values for the hydration of the sodiated monosaccharides standard pressure is 1000 mbar uncertainties in parentheses exp .
. values from ref . at 298 k experimental enthalpies , entropies , and free energy values for the hydration of the potassiated monosaccharides standard pressure is 1000 mbar uncertainties in parentheses
as mentioned above , in the present experiments ions are generated by electrospray from the aqueous - methanol solution . in solution , monosaccharides exist in an equilibrium between several different tautomeric and anomeric forms . these various forms have different free energies and , in general , the tautomeric equilibrium is dominated by conformers of six - membered ( pyranoid ) ring structures . in an aqueous solution , ara , xyl , glc , and gal exist predominantly ( 94% ) or exclusively in the c4 ( ara ) and c1 ( xyl , glc , and gal ) pyranoid ring conformations [ 41 , 42 ] , ( scheme 1 ) , and their equilibrium occurs between the and anomers at the carbon atom c-1 .
scheme 1ring conformations of the monosaccharides considered in this work , indicating the numbering convention of carbon atoms .
the oxygen bonded to the carbon follows the same number convention ring conformations of the monosaccharides considered in this work , indicating the numbering convention of carbon atoms .
the oxygen bonded to the carbon follows the same number convention the equilibrium mixture of rib contains structures of both pyranoid ( c1 and c4 ) and furanoid ( five - membered ) rings in the approximate ratio 4:1 , with each of these tautomers having and anomers .
therefore , one can expect that the sacx complexes formed in electrospray droplets and then transported into the reaction chamber for each system represent a mixture of their conformational forms .
heaton and armentrout determined the ground state ( gs ) and low energy structures for the and anomeric forms of the arana , xylna , glcna and galna complexes at the b3lyp/6 - 311 + g(d , p ) level with single - point energies calculated at the b3lyp , b3p86 , and mp2(full ) levels of theory using a 6 - 311+g(2d,2p ) bases set .
a schematic representation of the gs structures for these complexes showing sodium cation coordination by the oxygen sites can be seen in scheme 2 .
scheme 2schematic representation of the ground state structures of the and anomeric forms of the sodiated monosaccharide complexes obtained in the theoretical calculations schematic representation of the ground state structures of the and anomeric forms of the sodiated monosaccharide complexes obtained in the theoretical calculations for xylna , glcna , and galna , the calculations at all three levels of theory predict the same gs conformers for each complex shown in scheme 2 . for arana
, these calculations predict that the gss of the bidentate binding conformers , c4 [ o2 , o3 ] , are in the range of 00.3 kj / mol and 01.0 kj / mol for the and forms , respectively .
the gss determined for the tridentate binding orientations , -arana(c4)[o1,o4,o5 ] and -arana(c1)[o1,o2,o5 ] , are in the range of 03.3 kj / mol and 05.3 kj / mol , respectively .
the population analysis by heaton and armentrout indicate that the gs structures obtained for the xylna , -galna , and -glcna systems are predominant in equilibrium at 298 k , and their excited conformers comprise < 1% ( for xylna and -galna ) , and ~3% ( for -glcna ) of the total population .
in contrast , excited conformers of arana comprise 11%47% of the total population and 1%20% for the -glcna and -galna systems ( depending on level of theory ) . for ribna , the most stable and conformers computationally predicted at the hf/6 - 31 g level have c4 ring with [ o1,o2,o4,o5 ] and [ o2,o4,o5 ] binding geometry , respectively .
the numbering of atoms and nomenclature used here are consistent with the literature [ 19 , 20 ] .
it is likely that the gs structures and very low - lying excited conformations of sacna , as discussed above , correspond to the precursor ions for hydration in the present study .
it seems reasonable to assume that in the singly hydrated systems , sacna(h2o ) , the water molecule binds directly to the sodium cation of sacna , as illustrated schematically below for the hydration of the gs tridentate conformations predicted for the and forms of arana .
based on the structural characterization of sacna as the precursors to hydrated structures , one can say that the water binding energies to these complexes measured in the present experiments for each system correspond to a mixture of the precursor ions discussed above .
table 1 shows that with the exception of galna , for all systems studied , the hydration enthalpies ( h1 ) of the first water molecule are very similar ( within ~3 kj /mol ) , and very close to that of the fourth water molecule ( 57.7 4 ; 54.8 6 kj / mol ) in the na(h2o)n system .
this suggests that the solvation effect provided by ara , xyl , rib , and glc on a sodium ion in the sacna systems is comparable to that of three water molecules in the na(h2o)n cluster .
this observation is in line with a multidentate ( 2 ) coordination of na in the arana , xylna , glcna , and galna complexes predicted by heaton and armentrout . for galna(h2o ) , the water binding enthalpy is smaller than that for other sacna(h2o ) systems ( table 1 ) .
this is expected because both the and gss of gal bind the sodium cation more tightly than other monosaccharides studied here .
figure 4 shows the plot of the hydration free energies ( \documentclass[12pt]{minimal }
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\begin{document}$$ - \delta g_1^o $ $ \end{document } ) , table 1 , versus the corresponding na affinities of monosaccharides .
an analogous correlation was also found [ 33 , 34 ] for the binding energies of water to the cationized amino acids ( aas ) , aana , and aak .
this effect can be rationalized by direct interaction of the water molecule with the na ion in the sacna(h2o ) system , and as a consequence of charge delocalization from na to the monosaccharide moiety , resulting in a lower electrostatic interaction between na and h2o . the charge density on na in sacna
figure 4correlation between hydration free energies , g , and corresponding sodium ion affinity of monosaccharides .
t = 298 k. the sodium ion affinity values are taken from ref .
correlation between hydration free energies , g , and corresponding sodium ion affinity of monosaccharides .
t = 298 k. the sodium ion affinity values are taken from ref .
generally , the observed binding energies of the second water molecule in sacna(h2o)2 are smaller than those of the first one ( table 1 ) .
this result can be explained by increasing steric crowding and decreasing effective charge on the sodium ion when the next water molecule is added to the sacna(h2o ) complex . as shown in table 1 ,
the h2 values for all of the systems investigated here vary between 42.7 2 and 52.3 1 kj / mol , such that they are within the range of the binding energies reported in the literature for the binding of the fifth water molecule in the na(h2o)n complex , 44.451.5 kj / mol [ 43 , 45 ] .
this observation could indicate that the second water in the sacna(h2o)2 systems is coordinated directly to the sodium ion , although the slightly higher values of both h2 and s2 for ribna(h2o)2 and glcna(h2o)2 may suggest that the second h2o in these two complexes is bound more tightly than in others , possibly because of the h - bonded interaction with the monosaccharide moiety .
as expected , the enthalpy change values for the association of h2o to the sack complexes are lower than those for sacna ( tables 1 and 2 ) .
these results are fully in line with the predominating direct electrostatic interaction between the water molecule and alkali metal cation in these systems .
because the ionic radius of k is larger than na , its charge density becomes smaller and , therefore , the electrostatic interaction of h2o with sack is weaker compared with sacna .
the h and g values found for sack(h2o ) are identical within the combined error bars ( table 2 ) , and very similar to the corresponding experimental values of 49.4 and 18.4 kj / mol , respectively , reported in the literature for the fourth water in the k(h2o)n system .
this indicates that similarly to the sacna(h2o ) systems discussed above , the solvation effect provided by the monosaccharides in sack(h2o ) is comparable to that of three water molecules in the corresponding k(h2o)n system .
as mentioned above , in the present experiments ions are generated by electrospray from the aqueous - methanol solution . in solution , monosaccharides exist in an equilibrium between several different tautomeric and anomeric forms . these various forms have different free energies and , in general , the tautomeric equilibrium is dominated by conformers of six - membered ( pyranoid ) ring structures . in an aqueous solution , ara , xyl , glc , and gal exist predominantly ( 94% ) or exclusively in the c4 ( ara ) and c1 ( xyl , glc , and gal ) pyranoid ring conformations [ 41 , 42 ] , ( scheme 1 ) , and their equilibrium occurs between the and anomers at the carbon atom c-1 .
scheme 1ring conformations of the monosaccharides considered in this work , indicating the numbering convention of carbon atoms .
the oxygen bonded to the carbon follows the same number convention ring conformations of the monosaccharides considered in this work , indicating the numbering convention of carbon atoms .
the oxygen bonded to the carbon follows the same number convention the equilibrium mixture of rib contains structures of both pyranoid ( c1 and c4 ) and furanoid ( five - membered ) rings in the approximate ratio 4:1 , with each of these tautomers having and anomers .
therefore , one can expect that the sacx complexes formed in electrospray droplets and then transported into the reaction chamber for each system represent a mixture of their conformational forms .
heaton and armentrout determined the ground state ( gs ) and low energy structures for the and anomeric forms of the arana , xylna , glcna and galna complexes at the b3lyp/6 - 311 + g(d , p ) level with single - point energies calculated at the b3lyp , b3p86 , and mp2(full ) levels of theory using a 6 - 311+g(2d,2p ) bases set .
a schematic representation of the gs structures for these complexes showing sodium cation coordination by the oxygen sites can be seen in scheme 2 .
scheme 2schematic representation of the ground state structures of the and anomeric forms of the sodiated monosaccharide complexes obtained in the theoretical calculations schematic representation of the ground state structures of the and anomeric forms of the sodiated monosaccharide complexes obtained in the theoretical calculations for xylna , glcna , and galna , the calculations at all three levels of theory predict the same gs conformers for each complex shown in scheme 2 . for arana
, these calculations predict that the gss of the bidentate binding conformers , c4 [ o2 , o3 ] , are in the range of 00.3 kj / mol and 01.0 kj / mol for the and forms , respectively .
the gss determined for the tridentate binding orientations , -arana(c4)[o1,o4,o5 ] and -arana(c1)[o1,o2,o5 ] , are in the range of 03.3 kj / mol and 05.3 kj / mol , respectively .
the population analysis by heaton and armentrout indicate that the gs structures obtained for the xylna , -galna , and -glcna systems are predominant in equilibrium at 298 k , and their excited conformers comprise < 1% ( for xylna and -galna ) , and ~3% ( for -glcna ) of the total population .
in contrast , excited conformers of arana comprise 11%47% of the total population and 1%20% for the -glcna and -galna systems ( depending on level of theory ) . for ribna , the most stable and conformers computationally predicted at the hf/6 - 31 g level have c4 ring with [ o1,o2,o4,o5 ] and [ o2,o4,o5 ] binding geometry , respectively .
the numbering of atoms and nomenclature used here are consistent with the literature [ 19 , 20 ] .
it is likely that the gs structures and very low - lying excited conformations of sacna , as discussed above , correspond to the precursor ions for hydration in the present study .
it seems reasonable to assume that in the singly hydrated systems , sacna(h2o ) , the water molecule binds directly to the sodium cation of sacna , as illustrated schematically below for the hydration of the gs tridentate conformations predicted for the and forms of arana .
based on the structural characterization of sacna as the precursors to hydrated structures , one can say that the water binding energies to these complexes measured in the present experiments for each system correspond to a mixture of the precursor ions discussed above .
table 1 shows that with the exception of galna , for all systems studied , the hydration enthalpies ( h1 ) of the first water molecule are very similar ( within ~3 kj /mol ) , and very close to that of the fourth water molecule ( 57.7 4 ; 54.8 6 kj / mol ) in the na(h2o)n system .
this suggests that the solvation effect provided by ara , xyl , rib , and glc on a sodium ion in the sacna systems is comparable to that of three water molecules in the na(h2o)n cluster .
this observation is in line with a multidentate ( 2 ) coordination of na in the arana , xylna , glcna , and galna complexes predicted by heaton and armentrout . for galna(h2o ) , the water binding enthalpy is smaller than that for other sacna(h2o ) systems ( table 1 ) .
this is expected because both the and gss of gal bind the sodium cation more tightly than other monosaccharides studied here .
figure 4 shows the plot of the hydration free energies ( \documentclass[12pt]{minimal }
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\begin{document}$$ - \delta g_1^o $ $ \end{document } ) , table 1 , versus the corresponding na affinities of monosaccharides .
an analogous correlation was also found [ 33 , 34 ] for the binding energies of water to the cationized amino acids ( aas ) , aana , and aak .
this effect can be rationalized by direct interaction of the water molecule with the na ion in the sacna(h2o ) system , and as a consequence of charge delocalization from na to the monosaccharide moiety , resulting in a lower electrostatic interaction between na and h2o . the charge density on na in sacna
figure 4correlation between hydration free energies , g , and corresponding sodium ion affinity of monosaccharides .
t = 298 k. the sodium ion affinity values are taken from ref .
correlation between hydration free energies , g , and corresponding sodium ion affinity of monosaccharides .
t = 298 k. the sodium ion affinity values are taken from ref .
generally , the observed binding energies of the second water molecule in sacna(h2o)2 are smaller than those of the first one ( table 1 ) .
this result can be explained by increasing steric crowding and decreasing effective charge on the sodium ion when the next water molecule is added to the sacna(h2o ) complex . as shown in table 1 ,
the h2 values for all of the systems investigated here vary between 42.7 2 and 52.3 1 kj / mol , such that they are within the range of the binding energies reported in the literature for the binding of the fifth water molecule in the na(h2o)n complex , 44.451.5 kj / mol [ 43 , 45 ] .
this observation could indicate that the second water in the sacna(h2o)2 systems is coordinated directly to the sodium ion , although the slightly higher values of both h2 and s2 for ribna(h2o)2 and glcna(h2o)2 may suggest that the second h2o in these two complexes is bound more tightly than in others , possibly because of the h - bonded interaction with the monosaccharide moiety .
as expected , the enthalpy change values for the association of h2o to the sack complexes are lower than those for sacna ( tables 1 and 2 )
. these results are fully in line with the predominating direct electrostatic interaction between the water molecule and alkali metal cation in these systems .
because the ionic radius of k is larger than na , its charge density becomes smaller and , therefore , the electrostatic interaction of h2o with sack is weaker compared with sacna .
the h and g values found for sack(h2o ) are identical within the combined error bars ( table 2 ) , and very similar to the corresponding experimental values of 49.4 and 18.4 kj / mol , respectively , reported in the literature for the fourth water in the k(h2o)n system .
this indicates that similarly to the sacna(h2o ) systems discussed above , the solvation effect provided by the monosaccharides in sack(h2o ) is comparable to that of three water molecules in the corresponding k(h2o)n system .
hydration of sodiated and potassiated monosaccharides ( arabinose , xylose , ribose , glucose , and galactose ) produced by electrospray from an aqueous - methanol solution have been investigated by pulsed high - pressure mass spectrometry .
the thermochemical values measured here for each solvated system correspond to a mixture of the monosaccharide structures involved in the sacx complexes .
thermochemical information indicates that the solvation effects of ara , xyl , rib , glc , and gal on both na and k ions are comparable to that of three water molecules in na(h2o)n and k(h2o)n , respectively , which is consistent with metal cation coordination in the most stable conformations as calculated for the sacna complexes .
a good correlation was found between the free energy changes for the attachment of the first water molecule to the arana , xylna , glcna and galna complexes and the sodium cation binding affinities to the corresponding monosaccharides . | the thermochemical properties hon , son , and gon for the hydration of sodiated and potassiated monosaccharides ( ara = arabinose , xyl = xylose , rib = ribose , glc = glucose , and gal = galactose ) have been experimentally studied in the gas phase at 10 mbar by equilibria measurements using an electrospray high - pressure mass spectrometer equipped with a pulsed ion beam reaction chamber .
the hydration enthalpies for sodiated complexes were found to be between 46.4 and 57.7 kj / mol for the first , and 42.7 and 52.3 kj / mol for the second water molecule . for potassiated complexes ,
the water binding enthalpies were similar for all studied systems and varied between 48.5 and 52.7 kj / mol .
the thermochemical values for each system correspond to a mixture of the and anomeric forms of monosaccharide structures involved in their cationized complexes . | Introduction
Experimental
Results
Discussion
SacNa
SacK
Conclusions |
acute coronary syndromes and acute myocardial infarctions are associated with plaque rupture and the formation of thrombi at the site of the rupture 1,2 .
although the presence of thrombi in the coronaries of patients with acute coronary syndromes has been suggested by coronary angiography 3 and confirmed by autopsy studies 4,5 , there is little information about fresh human coronary thrombi .
newer suction catheters make the percutaneous retrieval of fresh thrombi from the coronaries of patients possible and may contribute to improved perfusion 6,7 .
a recent review by gerd heusch and colleagues 7 provides an overview on the previously published data on the molecular and cellular composition of particulate debris as well as soluble factors retrieved from human coronary arteries , primarily as this pertains to coronary microembolization .
we examined fresh coronary thrombectomy specimens from patients with acute coronary syndromes and assessed their structure and cellularity as well as the potential of thrombi to participate in vascular healing .
the thrombectomy specimens were aspirated from patients presenting with acute coronary syndromes [ st - segment and non - st - segment elevation myocardial infarction ( mi ) ] to the cardiac catheterization laboratory of the yale- new haven hospital .
because of concern about the process of obtaining consent from patients before their acute treatment of myocardial infarction and the fact that this could have delayed life - saving therapy , the study was performed with approval of the yale university human investigation committee under a protocol that specified that consent was not needed if no patient data were collected and that at least one investigator had to have no contact with the patients or with the specimens .
fresh thrombectomy specimens were retrieved with an export ( medtronic , san diego , ca , usa ) aspiration coronary catheter from patients with acute coronary syndromes during clinically indicated coronary angiography and percutaneous coronary intervention procedures .
the decision to use a coronary aspiration catheter was the clinical decision of the interventional cardiologist .
the specimens were placed in the collection filter , which was then immersed in normal saline in a sterile biopsy container .
the specimens were then aspirated with a pipette under a sterile hood and placed in culture bottles with medium .
dmem high glucose medium ( invitrogen , carlsbad , ca , usa ) with penicillin / streptomycin and 20% fbs ( invitrogen ) was used for the culture of some thrombectomy specimens and the cells grew and developed features of mesenchymal cells .
m199 medium ( invitrogen ) with penicillin / streptomycin and 20% fbs ( invitrogen ) , l - glutamine , endothelial cell growth supplement ( bd biosciences , mountain view , ca , usa ) and 100 g / ml porcine intestinal heparin ( sigma , st .
louis , mo , usa ) was used for the culture of other thrombectomy specimens and these yielded cells that differentiated into endothelial cell lineages 8 .
low serum stem cell medium ( hpgm , cambrex , walkersville , md , usa ) was used to select progenitor cells .
when they reached confluence , they were detached with trypsin and passaged onto cell culture dishes and/or onto slides culture chambers and stained as described below .
sections were stained with haematoxylin eosin or were incubated with primary anti - human desmin , pancytokeratin , cluster of differentiation ( cd)31 [ platelet endothelial cell adhesion molecule ( pecam-1 ) ] , tgfb ( transforming growth factor beta ) , von willebrand factor ( vwf ) , cd117 [ mast / stem cell growth factor receptor ( scfr ) ] , cd15 [ stage - specific embryonic antigen 1 or lewis x ( a surface marker for myelomonocytic cells / neutrophils ) ] and cd34 [ a glycoprotein ( l - selectin receptor ) found on bone marrow haematopoietic cells , endothelial progenitor cells and endothelial cells ] antibodies , then with secondary biotinylated antibody and detected with a streptavidin - horseradish peroxidase system after background staining .
cells cultured in cell culture media were also subjected to immunohistochemistry with the above antibodies and also human anti - cd68 [ macrosialin ( a glycoprotein expressed on monocytes / macrophages ) ] and muscle sarcoplasma actin [ ( msa ) a marker for muscle cells ] . to determine the relative contribution of various thrombus components , planimetry using image j software ( http://rsbweb.nih.gov/ij/ ) was used . because the distinction between fibrin material and platelets is difficult , the software was used to detect and express the proportion of red cell material with the rest being non - red cell material ( fibrin , platelets and white cells ) .
cells grown in serum - poor stem cell medium were also plated onto 24-well plates coated with fibronectin and then incubated with dii ldl ( sigma ) to assess their ability to endocytose ldl 9 .
sections were stained with haematoxylin eosin or were incubated with primary anti - human desmin , pancytokeratin , cluster of differentiation ( cd)31 [ platelet endothelial cell adhesion molecule ( pecam-1 ) ] , tgfb ( transforming growth factor beta ) , von willebrand factor ( vwf ) , cd117 [ mast / stem cell growth factor receptor ( scfr ) ] , cd15 [ stage - specific embryonic antigen 1 or lewis x ( a surface marker for myelomonocytic cells / neutrophils ) ] and cd34 [ a glycoprotein ( l - selectin receptor ) found on bone marrow haematopoietic cells , endothelial progenitor cells and endothelial cells ] antibodies , then with secondary biotinylated antibody and detected with a streptavidin - horseradish peroxidase system after background staining .
cells cultured in cell culture media were also subjected to immunohistochemistry with the above antibodies and also human anti - cd68 [ macrosialin ( a glycoprotein expressed on monocytes / macrophages ) ] and muscle sarcoplasma actin [ ( msa ) a marker for muscle cells ] . to determine the relative contribution of various thrombus components , planimetry using image j software ( http://rsbweb.nih.gov/ij/ ) was used . because the distinction between fibrin material and platelets is difficult , the software was used to detect and express the proportion of red cell material with the rest being non - red cell material ( fibrin , platelets and white cells ) .
cells grown in serum - poor stem cell medium were also plated onto 24-well plates coated with fibronectin and then incubated with dii ldl ( sigma ) to assess their ability to endocytose ldl 9 .
thrombectomy specimens were retrieved from 29 patients ; samples from 10 patients were used for histology , but one sample was too small and could not be analysed ; samples from the other 19 patients were cultured , but only 16 grew and could be passaged .
all patients were pretreated with aspirin and clopidogrel and anticoagulated with intravenous heparin to achieve and activated clotting time 250300 sec . as part of a standard protocol . in general , the proportion of patients with acute coronary syndrome treated with glycoprotein iib / iiia inhibitors was less than 20% .
the relative contribution of red cell material in the nine samples used for histology was 51 18% .
mixed thrombi , white thrombi and red thrombi ( a c ) ; platelets , fibrin , red cells and white cells in varying amounts ( d f ) ; cholesterol crystals of various shapes ( g i ) .
immunohistochemistry of thrombi in situ after fixation with paraformaldehyde : cd34 positive , desmin ( a marker for muscle cells ) positive , pancytokeratin ( a marker for epithelial cells ) negative , cd31 ( a marker for endothelial cells ) positive , cd15 ( a marker for neutrophilic polymorphonuclear cells ) positive , tgfb ( a marker for smooth muscle cells and macrophages ) positive , vwf ( a marker for endothelial cells ) positive , cd117 ( a marker for haematopoietic stem cells ) negative ( fig.2 ) .
immunohistochemistry of thrombi in situ after fixation with paraformaldehyde : cd34 , desmin , pancytokeratin ( negative ) , cd31 ( a d ) , cd15 , tgfb , vwf , cd117 ( negative ) ( e h ) .
cells grown in dmem 20% fbs medium became spindle shaped and grew in clusters until they became confluent . there were numerous dividing cells and sporadic multinucleated cells ( fig.3a g ) .
cells grown in m199 20% fbs ( endothelial cells medium ) grew in small clusters and then became confluent in a cobblestone pattern ( fig.3h i ) .
growth pattern of cultured cells grown in dmem 20% fbs ( a c ) ; shapes of cultured cells , dividing cells , multinucleated cells ( d f ) , spindle - shaped cells reminiscent of mesenchymal cells / fibroblasts ( g ) ; cells grown in m199 20% fbs ( ec medium ) grew in small clusters ( h ) and then became confluent in a cobblestone pattern ( i ) .
cells survived in serum - free stem cell medium for 12 months without dividing ( fig.4a ) .
cells grown in serum - poor stem cell medium were then incubated with endothelial cells medium , differentiated to endothelial progenitor cells capable of endocytosing dii - ac ldl ( fig.4b ) .
cells survived in serum - free stem cell medium for 12 months ( a ) ; mononuclear cells from thrombus grown in serum - poor stem cell medium differentiated to endothelial progenitor cells capable of endocytosing dii - ac ldl ( b ) .
cells grown in dmem 20% fbs were cd68 ( a marker for monocytes ) negative , vwf negative , cd34 negative , msa positive , pancytokeratin ( a marker for epithelial cells ) negative , cd15 negative , desmin positive and tgf positive ( fig.5 ) .
immunohistochemistry of cells grown in dmem 20% fbs : cd68 ( negative ) , vwf ( negative ) , cd34 ( negative ) , msa ( a d ) , pancytokeratin ( negative ) , cd15 ( negative ) , desmin , tgf ( e h ) .
cells grown in m199 20% fbs were pancytokeratin negative , cd31 ( a marker for endothelial cells ) positive , cd34 positive , vwf positive ( fig.6 ) .
immunohistochemistry of cells grown in m199 20% fbs : pancytokeratin ( negative ) , cd31 , cd34 , vwf ( a d ) .
coronary thrombi are white , red and mixed thrombi and contain various amounts of platelets , red blood cells and leucocytes 10 .
this is different than the classic teaching that venous thrombi are red and arterial thrombi are white , and is in accordance with previously published data by montalescot et al . who showed that the composition of coronary thrombi can change depending on the timepoint when they are harvested in relation to their formation 11,12 .
leucocyte infiltration of unstable plaques has been described before 14 and may be related to outcome in patients with acute coronary syndromes .
thus , the variability in thrombus composition may be caused by both intrinsic characteristics of the patient ( e.g. peripheral blood white cell count ) and may reflect the lag between plaque rupture / coronary thrombosis and cardiac catheterization and percutaneous coronary intervention and thrombectomy . a recent report that studied the structure of coronary thrombi and its relation to the onset of chest pain , found that in at least 50% of patients with acute stemi , coronary thrombi were days or weeks old .
the authors interpreted this as suggesting that sudden coronary occlusion is often preceded by a variable period of plaque instability and thrombus formation , initiated days or weeks before onset of symptoms 15 .
we also found cholesterol crystals which have been described in plaques before and which may contribute to thrombosis in ruptured plaque and to the so called no - reflow16,17 phenomenon which is seen in the cardiac catheterization laboratory .
blood - derived cells are present in approximately 10% of endothelial cells in the neovasculature formed in response to surgical sponge implantation in mice 18,19 and may also form vascular mural cells 20 . increased epc numbers in the periphrery at the time of mi
on the other hand , monocytosis at the time of mi is associated with an increased left ventricular end - diastolic volume and a reduced ejection fraction 22 .
our findings are in agreement with previously published data about fresh thrombectomy specimens demonstrating the prominent presence of von willebrand factor at the sites of platelet accumulation , presence of tissue factor and platelets at the sites of deposition of fibrin fibrils 23 .
the authors of that study also reported the presence of cd16- , cd45- and cd34-positive cells in the thrombectomy specimens and a weak positive correlation between the number of inflammatory cells involved in the unit area of coronary thrombi specimen and the time of collection of the specimens after the onset of chest pain 23 .
more recently , using dye - staining angioscopy , uchida and colleagues showed in vivo that acute coronary syndrome patients can have predominantly fibrin - rich thrombi that have a low platelet content 24 , underscoring the variability in cellular content of thrombi causing unstable angina and acute mi .
our experiments with dmem culture medium suggest that the cells with progenitor markers were already pre - existing in the thrombectomy specimens .
our experiments with cells in culture using serum - poor stem cell media also suggest that some endothelial progenitor cells are contained in the thrombectomy specimens and persist for prolonged periods of time in culture , but we can not exclude the possibility that these were mature cell lines , which were re - programmed under low serum stem cell medium conditions throughout 12 months . in summary , out data suggest that intracoronary thrombi harvested at the time of primary percutaneous intervention from patients with acute myocardial infarction contain various amounts of platelets , fibrin , red and white blood cells as well as cholesterol crystals
. the cells are mostly of mesenchymal lineage and include cells with features of endothelial and of endothelial progenitor cells .
these findings provide insights into the pathogenesis of plaque rupture , of thrombus lysis and remodelling and of vascular repair . | acute coronary syndromes and acute myocardial infarctions are often related to plaque rupture and the formation of thrombi at the site of the rupture .
we examined fresh coronary thrombectomy specimens from patients with acute coronary syndromes and assessed their structure and cellularity .
the thrombectomy specimens consisted of platelets , erythrocytes and inflammatory cells .
several specimens contained multiple cholesterol crystals .
culture of thrombectomy specimens yielded cells growing in various patterns depending on the culture medium used .
culture in serum - free stem cell enrichment medium yielded cells with features of endothelial progenitor cells which survived in culture for a year .
immunohistochemical analysis of the thrombi revealed cells positive for cd34 , cells positive for cd15 and cells positive for desmin in situ , whereas cultured cell from thrombi was desmin positive but pancytokeratin negative .
cells cultured in endothelial cell medium were von willebrand factor positive .
the content of coronary thrombectomy specimens is heterogeneous and consists of blood cells but also possibly cells from the vascular wall and cholesterol crystals . the culture of cells contained in the specimens yielded multiplying cells , some of which demonstrated features of haematopoietic progenitor cells and which differentiated into various cell - types . | Introduction
Materials and methods
Microscopy
Results
Discussion
Conflict of interest |
breast cancer is the second leading cause of cancer deaths in women today after lung cancer .
metastatic breast cancer ( mbc ) is deemed incurable and median survival for patients with cancer that is estrogen receptor ( er ) negative or no longer hormonally responsive is only 18 to 24 months [ 1 , 2 ] . a variety of molecularly targeted drugs have been developed for mbc , but to date only , trastuzumab ( herceptin , genentech , ca , usa ) has shown an overall survival ( os ) benefit of several months [ 35 ] . clearly , more effective treatment strategies and/or improved patient selection are urgently needed . in the 1990s ,
high dose chemotherapy followed by autologous stem cell transplantation ( hd - asct ) was pioneered to improve survival .
the initial feasibility studies in small cohorts of patients with advanced stage disease showed improved survival when compared to historical controls [ 69 ] .
subsequent prospective randomized clinical trials comparing hd - asct to standard chemotherapy resulted in improved pfs , but failed to demonstrate os benefit [ 1014 ] .
the results of the last trial of these series with 386 patients with metastatic breast cancer employing hd - asct were reported most recently and confirmed the results of earlier studies .
all together , the lack of convincing survival data at the expense of high toxicity resulted in the demise of hd - asct as a treatment modality of mbc .
however , while the vast majority of patients with mbc will succumb to their disease within a relatively short period of time , 510% of patients live longer than 5 years , and observational data suggest that 13% of patients treated with conventional chemotherapy and/or hormonal manipulation may experience long - term survival beyond 1015 years [ 1621 ] .
quite surprisingly , the most impressive long - term survival rates in excess of 10% were reported with hd - asct [ 2224 ] .
response to treatment , site of metastases , hormone receptor status , performance status , and short initial disease - free interval were important prognostic factors for survival after hd - asct and corresponded to prognostic factors known from treatment with conventional , less aggressive therapies [ 2527 ] .
we have studied long - term survival rates as well as the distinct disease- and patient - related characteristics that would be predictive of long - term survival in a cohort of patients with mbc , who were treated at our institution between 1989 and 1999 .
records of all patients in the bone marrow transplant registry at ucsd treated with hd - asct for mbc between 1989 and 1999 were retrospectively reviewed .
patients were followed for a median of 65 months from diagnosis ( range 10.4255.0 months ; quartiles 36.7109.3 months ) .
age , race , stage at diagnosis , histology , estrogen receptor ( er ) and menopausal status , body mass index ( bmi ) in kg / m at the time of hd - asct , time to transplant and death , site of metastasis , and disease status prior to hd - asct were extracted . since all transplants were carried out prior to routine assessment of her-2/neu receptor status , this information was not part of our analysis .
menopause was defined clinically by absence of menstruation for more than 12 months ( http://www.cancer.gov/ ) .
high dose chemotherapy prior to stem cell support was platinum based in combination with thiotepa / etoposide , ifosfamide / etoposide , cyclophosphamide / bcnu , cyclophosphamide / mitoxantrone , or cyclophosphamide / thiotepa .
body surface area was allowed to exceed 2 m. ten patients received hd - asct in first line ; all other patients received at least one line of treatment prior to hd - asct ( median 1 ; range 04 ) .
the other 89 patients received peripherally mobilized stem cells , 23 of which received grafts supplemented with harvested bone marrow cells .
mobilization was achieved with granulocyte- or granulocyte / macrophage - colony stimulating factor with or without cyclophosphamide .
extent of metastatic disease was determined within 12 weeks prior to and within 612 weeks after hd - asct by imaging and physical exam .
thereafter , assessment of disease status was prompted by patient symptoms and carried out at physicians ' discretion .
patients were grouped according to site of metastasis : in visceral or bone metastasis , or local or nodal recurrence .
if multiple sites of metastasis were present , assignment to one metastatic site was prioritized as follows : visceral > bone > nodal > local .
complete response ( cr ) , partial response ( pr ) , stable disease ( sd ) , and progressive disease ( pd ) were determined according to recist criteria .
multiple linear regression models were used to explore the association between a continuous outcome and predictors of interest .
cox models were used to identify independent predictors of a time - to - event variable and schoenfeld tests were used to test for proportional hazard assumptions .
predictors of interest with p < 0.2 in the univariate analysis were included in an initial model and a manual backward selection was made based on the wald test p values .
median age at initial diagnosis was 43 years ( quartiles 3849 ) and 47 years ( quartiles 4143 ) at metastasis .
median time from initial diagnosis to documentation of metastasis was 29 months ( quartiles 16.663.7 ) , and from metastasis to hd - asct 6.6 months ( quartiles 4.516.6 ) , respectively .
twelve patients received hd - asct as their first line of treatment , whereas 84 patients had at least one prior line of chemotherapy for metastatic disease ( 48 patients with 1 line , 24 with 2 lines , 10 patients with 3 lines , and 2 patients with 4 lines of chemotherapy ) .
median bmi at metastasis was 25 ( quartiles 21.429.6 ) and 24% of patients were obese ( bmi > 30 ) .
for all patients ( n = 96 ) , median pfs and os were 3.9 years ( ci 2.95.4 ) and 5.6 years ( ci 4.17.4 ) after initial diagnosis , and 1.6 years ( ci 1.31.9 ) and 2.7 years ( ci 2.33.9 ) after diagnosis of metastatic disease , respectively ( figures 1(a ) and 1(b ) ) .
survival rates at 10 years were 24.5% ( ci 17.234.9% ) from diagnosis and 14.4% ( ci 8.7 , 23.9% ) from when metastatic disease was diagnosed .
as depicted in figure 2 , pfs and os differed significantly for patients with a bmi 30 and > 30 . at diagnosis , median pfs for patients with bmis
30 ( n = 73 ) and > 30 ( n = 23 ) was 4.4 ( ci 3.66.7 ) and 2.5 years ( ci 1.74.9 ) , and median os was 7.1 ( ci 4.48.7 ) and 3.2 years ( 2.416.75 ) , respectively ( figure 2(a ) and 2(c ) ; all p values = 0.001 ; univariate ) .
once metastasis was diagnosed , median pfs for patients with bmis 30 and > 30 was 1.8 ( ci 1.442.77 ) and 1.04 years ( 0.91.96 ) years , and median os was 3.20 ( ci 2.424.30 ) and 2.30 years ( ci 1.564.96 ) , respectively ( figure 2(b ) and 2(d ) ; all p values < 0.02 ; univariate ) .
ethnicity , stage at diagnosis , histology , site of metastasis , remission prior to hd - asct , menopausal status , ages at hd - asct and at diagnosis ( < age 40 versus age 40 ) , bmi ( 30 versus > 30 ) , and er status were evaluated as independent predictors of pfs and os . in a univariate analysis , bmi , stage at initial diagnosis , and histology were predictive of time from initial diagnosis to metastatic disease ( all p values < 0.01 ) .
as depicted in table 2 , only the initial stage at diagnosis and bmi were confirmed as independent predictors in a multivariate multilinear regression analysis .
time to metastasis was significantly shorter for patients with a bmi > 30 ( table 2 ) .
median time to metastasis was 1.14 years for patients with a bmi > 30 compared to 1.85 years for patients with a bmi 30 ( p < 0.04 for time to metastasis ) .
times to metastasis were similar for stage i or ii disease ( approximately 2 years ) , but time to metastasis shortened significantly compared to stage i or ii disease when stage iii was present at diagnosis ( 0.72 years , p < 0.0006 ) . stage at diagnosis , er - status , site of metastasis , remission status prior to hd - asct , and bmi were considered for multivariate analysis since univariate p values were < 0.02
. only bmi , stage at diagnosis and site of metastasis were confirmed to be independent predictors of survival at the time of metastasis ( table 3 ) .
patients with a bmi > 30 had a significantly higher hazard of progression and death compared to patients with a bmi 30 ( hr for progression = 2.23 ; p = 0.005 ; hr for death = 1.82 ; p = 0.04 ) . also ,
once metastasis was diagnosed , hazards of progression and death increased significantly with increasing stages present at diagnosis ( compared to stage i all p values < 0.05 ; table 3 ) or with visceral disease .
in the 1990s , high hd - asct was thought to be a beneficial treatment option in mbc , based on the hypothesis that significant dose escalation would improve the efficacy of chemotherapy .
hd - asct was abandoned . since cure could not be achieved with aggressive treatment , the school of thought changed and sequential single agent chemotherapy with or without biological targeted therapies and /or hormonal manipulation in er+ patients has become the preferred way of symptom control [ 2 , 29 ] .
however , a proportion of patients with mbc can achieve long - term survival which has been reported to exceed 10% with more aggressive chemotherapy such as hd - asct [ 2224 ] .
we were specifically interested if such high percentages of survival could be maintained beyond 10 years , at which point it is possible that at least some of these patients may be cured of their disease .
second , we studied the patient- and disease - specific characteristics predictive of survival . for both questions we analyzed our institutional cohort of 96 patients with mbc who had received hd - asct as part of their treatment algorithm .
since the last patient was transplanted in 1999 , each patient had a minimum follow - up of 10 years .
survival rates at 10 years were 24.5% from initial diagnosis and 14.4% from when metastatic disease was diagnosed .
second , and most surprisingly , in univariate and multivariate analyses , obesity was the only patient - related factor associated with significantly shorter os and pfs from initial diagnosis and from when metastasis was diagnosed .
obesity has previously been reported as a risk factor for survival in patients diagnosed with earlier stages of breast cancer [ 3032 ] and seems associated with higher stages at diagnosis , but , to the best of our knowledge , this is the first study reporting obesity as a risk factor for survival in patients with mbc .
other patient - related factors such as age , ethnicity , and menopausal status were not significant .
weights and obesity status were not available to us at the time of initial diagnosis , but recorded at the time of metastasis prior to first treatment .
the median interval between initial diagnosis and time to metastasis was only approximately 3 years and can probably be considered a time frame where major weight changes are not expected to occur for the majority of patients .
we therefore felt comfortable to use the weight obtained at metastasis as the representative personal weight for each patient .
third , among disease - related characteristics , stage at diagnosis was an independent predictor of time to metastasis .
neu was not part of the analysis since it was not routinely tested prior to 1999 . at the time of metastasis , in addition to stage at diagnosis , visceral metastasis also became an independent predictor of a higher hazard for progression and , suggestively , shortened os .
higher stages at diagnosis are known to be associated with shorter time to recurrence and poorer survival [ 35 , 36 ] , and increased likelihood of occult metastasis in permissive niches such as the bone marrow and/or biologically more aggressive forms of cancer with increased burden of disease were suggested as plausible explanations for poorer survival at higher stages [ 37 , 38 ] . also , visceral metastasis has been reported before as a predictor of poor survival in mbc [ 2527 ] .
the association of poorer outcomes with higher stages at initial diagnosis or visceral metastasis seems uniformly accepted , and our study supports these previous findings . the most important and novel finding was the negative effect of obesity on survival in mbc , and its importance as independent predictor of time to metastasis and progression . to the best of our knowledge ,
these results provide first strong evidence that elevated bmi may contribute significantly to poor survival in mbc and these data were ascertained in the setting of hd - asct . while it has been reported that obesity is associated with higher stages of breast cancer at diagnosis , and that obesity negatively impacts various outcome factors in earlier stages of breast cancer [ 3032 ] , the effects of obesity
have not been directly studied in mbc . specifically in respect to locoregional breast cancer , two prospective studies identified obesity as an independent risk factor for overall mortality , distant and contralateral recurrence , and death from breast cancer [ 31 , 32 ] .
similar findings for all presentations of breast cancer ( some of which had mbc ) were reported in the prospective analysis of case - control studies on environmental factors and health ( pace ) study . in general , it is increasingly recognized that obesity and cancer are linked and that obesity not only confers a higher risk of cancer incidence but also increases cancer mortality as demonstrated in three large population - based cohort studies [ 3941 ] .
the reasons are poorly understood , but tumor growth stimulation by adipokines released from fatty tissue , inflammation [ 4244 ] , and especially in the case of breast cancer increased estrogen levels in obese postmenopausal females fueling tumor growth [ 45 , 46 ] are implicated . also , adjuvant undertreatment of obese patients , if chemotherapy is not strictly weight - based [ 4749 ] , is increasingly recognized as important for early relapse . against physicians ' beliefs , chemotherapy in the obese is well tolerated , and , while dose increases proportional to weight do not confer a higher risk for toxicity [ 49 , 50 ] , under - dosing in the obese breast cancer patient receiving adjuvant chemotherapy demonstrated increased risk for recurrence , shorter dfs or os [ 51 , 52 ] . despite these data , two recent surveys show that only 25% of breast cancer patients receive strictly weight - based dosing during adjuvant chemotherapy and that 19 of 44 cooperative group trials used dose limits for the obese [ 47 , 48 ] . in our study population ,
doses of previous adjuvant regimens were unknown , and suboptimal adjuvant treatment can not be ruled out as a reason for faster time to metastasis in the obese .
however , agents used for treatment in the metastatic setting and for hd - asct were dosed by actual body weight , and body surface areas were allowed to exceed 2 m , such that underdosing does not appear to be the main reason for poorer outcome parameters .
further study limitations are absence of her-2-neu as important prognostic marker , since it was not routinely available in the era of hd - asct , and patient population diversity
. selection bias may be present since not every patient deemed a candidate for hd - asct may have been referred or proceeded to hd - asct .
factors such as , but not limited to , physicians ' or patients ' treatment preferences , patients ' performance status and socioeconomic status may have interfered . until now , obesity and its effect on outcomes in mbc have not been examined in depth , and this is the first report to implicate obesity in poorer outcomes . while this observation was made in the setting of hd - asct , conclusions about the contribution of hd - asct to long - term survivorship
can not be made , given retrospective study design , diverse study patient population , and mixed treatment algorithms .
however , our results demonstrate that ultra - long - term survivorship can be achieved in the setting of more aggressive chemotherapy with hd - asct and that obesity was deleterious in mbc . in contrast to mbc ,
correction of unhealthy life - style behavior associated with obesity is the subject of intense exploration after adjuvant treatment for breast cancer , where patients are officially recognized as breast cancer survivors .
results from the women 's healthy eating and living ( whel ) study suggest that obese patients can experience significant overall survival benefit if compliant with a combination of diet and exercise .
we believe that our findings generate the hypothesis that obesity may be a potentially correctable patient - related adverse survival factor in mbc and , similar to approaches for locoregional stages of breast cancer , can serve as a target for intervention given the prospect of long - term survival in certain settings . | the purpose of the study was to identify predictors of long - term survival in metastatic breast cancer ( mbc ) .
a cohort of 96 patients , who received high - dose chemotherapy with autologous stem cell support ( hd - asct ) as part of their treatment , was analyzed .
percent long - term survival at 10 years was
24.5% ( ci 17.234.9% ) when metastasis was diagnosed and 14.4% ( ci 8.723.9% ) when mbc was diagnosed .
survival was impacted significantly by body mass index ( bmi ) .
median overall survival from initial diagnosis or from time of metastasis for patients with bmis
30 and > 30 ( obese ) was 7.1 ( ci 4.48.7 ) and 3.2 years ( 2.416.75 ) , respectively , or 3.2 or 2.3 years ( all p = 0.02 ) . also , obesity was the only independent patient - related predictor of time to metastasis and of survival .
while obesity is linked with poor outcomes in earlier stages of breast cancer , this has not been previously reported for mbc . | 1. Introduction
2. Methods
3. Results
4. Discussion |
autophagy , ancient system necessary to maintain homeostasis in eukaryotic cells , degrades long - lived cytoplasmic proteins and organelles and provides nutrients in starvation or stress conditions ( 1 )
. there are at least three types of autophagy in eukaryotic cells : macroautophagy , microautophagy , and chaperone - mediated autophagy ( 2 ) .
microautophagy involves the continuous degradation of cytosolic constituents close to the lysosomes via budding of the lysosomal membrane . in chaperone - mediated autophagy ,
proteins containing a kferq motif , which is recognized by hsc70 , are transported into the lysosomal lumen via lamp-2a to be degraded ( 3 - 6 ) .
macroautophagy is the primary route of degradation and has been most widely studied . in this process , cellular components such as protein aggregates and organelles ( e.g. , mitochondria ) are wrapped by an elongated cup - shaped membrane , which forms a double - membrane structure called an autophagosome ( 7 ) .
autophagosomes mature and fuse with lysosomes to facilitate the degradation of the internal contents . recently
autophagic adaptors such as p62 targets polyubiquitylated substrates and are selectively degraded by autophagy ( 8) . the autophagic pathway consists of three distinct stages .
initially , an isolation membrane is formed through complex interaction of the autophagic protein atg6 ( beclin-1 ) and type - iii phosphatidylinositol 3-kinase , along with membrane components .
elongation of the isolation membrane and completion of autophagosome formation are regulated by at least two ubiquitin - like systems : the microtubule - associated protein 1 light chain 3 ( lc3 ; mammalian homolog of the yeast autophagic protein atg8 ) and the atg12 conjugation systems ( 9,10 ) .
atg12 is transferred to the e1- and e2-like enzyme atg7 and atg10 sequentially , and is finally conjugated with a lysine residue of atg5 through its c - terminal glycine residue .
the atg12-atg5 conjugates facilitate the elongation of the isolation membrane and catalyze the lc3 conjugation with the help of atg16 .
the c - terminal amino acids of lc3 are cleaved by atg4 and conjugated with phosphatidylethanolamine ( pe ) by the e1- and e2-like enzymes atg7 and atg3 , respectively .
the lipidated lc3 ( lc3-ii ) is incorporated into the newly formed autophagosomal membrane . upon autophagosome closure , the atg12-atg5-atg16 complex dissociates from the outer autophagosomal membrane , but lc3 remains in the autophagosomal lumen as an autophagosomal marker .
interestingly , lc3 binds to the adaptor protein p62 , which interacts with polyubiquitylated structures to facilitate their autophagic degradation ( 11 ) . finally , the outer membrane of the autophagosome fuses with the lysosomal membrane , followed by degradation of the membrane and its contents
although autophagy is necessary for cellular homeostasis , it is involved in biological processes including development , aging , and degeneration ( 12 ) .
aberrant regulation of autophagy is related to many diseases , such as cancer and neurodegenerative disease ( 13,14 ) .
autophagy can directly eliminate intracellular pathogens or enhance pathogen recognition and elimination ( 15 - 19 ) , and can regulate autoimmunity and inflammatory disorders ( 20 ) .
moreover , autophagy affects the adaptive immune system by contributing to antigen presentation and controlling t- and b - cell homeostasis ( 21 - 26 ) . here , we briefly review the role of autophagy in innate immune control .
because autophagy removes large cytoplasmic proteins and organelles , intracellular pathogens such as bacteria and parasites are cleared by autophagic machinery .
autophagy can target free microbes in the cytosol and pathogens contained in phagosomes ( 27 ) .
( 15 ) showed that group a streptococcus ( gas ) that escapes from endosomes into the cytoplasm becomes enveloped by autophagosome - like compartments and is killed upon fusion of these compartments with lysosomes ( fig .
1 ) . gas survives and multiplies in atg5-deficient cells , which indicates that elimination of gas is autophagy - dependent . in cases of intracellular pathogens that persist within phagosomes such as mycobacterium tuberculosis
, autophagy promotes the maturation of mycobacterial phagosomes into phagolysosomes . using mycobacterium bovis bacille calmette - gurin ( bcg ) , starvation- or rapamycin - induced autophagy leads to mycobacterial phagosome colocalization with the lc3 , resulting in fusion of phagosomes with autophagosomes , which in turn deliver the pathogen - containing vacuoles for lysosomal degradation ( 16 ) . in case of human macrophages ,
it induces promoter activation of the autophagy - related genes atg5 and beclin-1 , and promotes colocalization of bacterial phagosomes and autophagosomes ( 28 ) .
however , the mechanism of autophagy targeting and its role in natural infection without exogenous induction of autophagy remains unclear .
interestingly , a recent study using wild type mycobacterium tuberculosis revealed how m. tuberculosis interfaces with the selective autophagy pathway from within the phagosomes in resting macrophages ( fig .
, m. tuberculosis includes several virulence factors such as the extraembryonic spermatogenic homeobox 1 ( esx-1 ) secretion system ( 30 , 31 ) .
the bacterial esx-1 facilitates the exposure of bacterial dna to the host by permeabilizing the phagosome membrane ( 32 ) .
the exposed bacterial dna is recognized by the cytosolic dna pathway , including stimulator of interferon ( ifn ) genes ( sting ) , and surrounded by a ubiquitin chain .
ubiquitin and lc3-binding autophagy adaptors p62 and nuclear dot protein 52 ( ndp52 ) recruit autophagy components to target the bacilli to the selective autophagy pathway . in this process , atg5 and tank - binding kinase 1 are also required .
consequently , bacilli - containing autophagosomes are fused with lysosomes to facilitate the elimination of mycobacteria .
other intracellular bacteria and parasites such as listeria monocytogenes and salmonella species are also limited by autophagy with various strategies including selective autophagy activation ( 33 - 36 ) .
the innate immune system recognizes conserved microbial molecular structures , so called pathogen - associated molecular patterns ( pamps ) .
pattern recognition receptors ( prrs ) bind to these conserved structures and initiate downstream signaling pathways ( 37 ) . in addition , signaling initiated by prr activation can promote the autophagy induction .
studies have shown that activation of toll - like receptors ( tlrs ) facilitates pathogen elimination by autophagy induction ( 17 , 18 ) .
tlr4 stimulated with lipopolysaccharide ( lps ) induces autophagy in primary human macrophages and the murine macrophage cell line , raw 264.7 .
redistribution of lc3 protein from a diffuse to a punctate pattern and increased levels of the lipidated form of lc3 ( lc3ii ) , both of which are reliable markers of autophagy induction , were observed after stimulation with lps .
this process occurred via the toll / interleukin-1 receptor domain - containing adapter - inducing interferon ( trif)-p38 axis , but not via myd88 , and resulted in formation of the autophagosome colocalized with mycobacteria ( fig .
thus , it was suggested that autophagy induced by tlr activation enhances the elimination of mycobacteria . according to a report showing the effect of tlr agonists on autophagy induction in raw264.7 ( 18 ) , ligands of tlr3 , tlr4 , and
tlr7 signaling activated by two different ligands , single - stranded rna ( ssrna ) and imiquimod , induces the formation of autophagosomes characterized by lc3 puncta formation in murine macrophages ( fig .
tlr7-induced autophagy activation promotes the killing of intracellular mycobacteria , even though mycobacteria are normally not associated with tlr signaling ( 18 ) .
in addition to the formation of autophagosomes fused with pathogen - containing phagosomes , tlr signaling could enhance the maturation of phagosomes into phagolysosomes via autophagic machinery ( 19 ) .
when zymosan ( a component of the fungal cell wall ) is phagocytosed , phagosomes rapidly recruit lc3 and fuse with lysosomes ( fig .
lc3 translocation to phagosomes was dependent on tlr2 but not myd88 , and requires atg 5 and atg7 .
interestingly , lc3 recruitment to phagosomal membranes is not associated with the double - membrane structures characteristic of autophagosomes .
thus , this study identified a new way of using the autophagic machinery to enhance conventional functions of phagocytes after tlr activation ( 38 ) .
nucleotide oligomerization domain ( nod)-like receptors ( nlrs ) are a family of cytoplasmic molecules that recognize bacterial cell wall components , such as peptidoglycan , in the cytosol . upon bacterial invasion ,
nlrs recognize bacterial peptidoglycan by their c - terminal leucine - rich repeat and initiate signal transduction by their n - terminal effector domain .
downstream signals activate nuclear factor kappa - light - chain - enhancer of activated b cells ( nf-b ) and activator protein 1 , and lead to the production of cytokines involved in innate defense ( 39 ) .
studies have shown that nod1 and nod2 are critical for the autophagic response to bacterial invasion ( 40 , 41 ) . in mouse
embryonic fibroblasts ( mefs ) , nod2 recruits atg16l1 to the plasma membrane at the bacterial entry site , which facilitates bacterial trafficking to the autophagosomes .
this process does not require the adaptor receptor interacting protein 2 and the transcription factor nf-b ( 41 ) .
another study showed that nod2 activation by muramyl dipeptide ( mdp ) induces autophagosome formation and subsequently promotes major histocompatibility complex ( mhc ) class ii - associated antigen presentation in human dendritic cells ( dcs ) . in this process ,
atg5 , atg7 , atg16l1 , and receptor - interacting serine - threonine kinase 2 are required ( 40 ) .
thus , autophagy mediates bacterial clearance and bacterial antigen presentation , which are initiated by nod sensing of invasive bacteria ( fig .
interestingly , nod2 and atg16l1 are two of the most important genes associated with crohn 's disease ( 42 - 44 ) , a complicated inflammatory condition .
dcs isolated from patients with crohn 's disease expressing risk alleles for nod2 and atg16l1 are impaired in autophagy induction , bacterial trafficking , and antigen presentation .
overexpression or neuron - specific deletion of atg proteins resulted in increased or decreased survival following intracranial injection with sindbis virus , respectively ( 45,46 ) . in another study , intracranial infection with hsv-1 beclin-1
virulence factor , icp 34.5 , encoded by herpes simplex virus 1 ( hsv-1 ) blocks autophagy induction through binding and inhibiting beclin-1 .
most viral sensing is mediated by viral nucleic acids , which is different from other pathogens containing particular cell wall components triggering tlr activation .
tlr3 , tlr7 , tlr8 , and tlr9 sense endocytosed viral nucleic acids in the endosomal compartment ( 49 ) . in plasmacytoid dcs ( pdcs ) , viral rna or dna recognized by tlr7 or tlr9 , respectively , induces ifn regulatory factor 7 ( irf7 ) activation via myd88 , leading to type - i ifn production . in vesicular stomatitis virus ( vsv ) ,
pdcs recognize replicating virus in cytosol rather than the viral genome via tlr7 in endosomes .
however , how these cytosolic replication intermediates gain entry to the endosomal compartment where tlr7 resides is not completely known .
a study demonstrated that autophagy mediates the delivery of the cytosolic pamp to the lysosomes to activate tlr7 signaling ( fig .
atg5-deficient pdcs are unable to produce ifn- and interleukin ( il)-12p40 in response to vsv .
in addition to vsv , pdcs lacking atg5 failed to produce ifn- in response to hsv-1 , which is recognized by tlr9 , whereas the il-12 response was not impaired in these cells .
autophagy plays an important role in the antiviral response by delivering viral replication intermediates from the cytosol to the endosome .
however , the precise mechanism of the differential control of nf-b versus ifn- induction pathways in pdcs by autophagy remains to be determined ( 50 - 52 ) .
most cell types other than pdcs use cytosolic receptors , retinoic acid - inducible gene ( rig)-i - like receptors ( rlrs ) and melanoma differentiation associated gene 5 ( mda-5 ) , to sense the viral invasion ( 53 - 55 ) . via their caspase - recruiting domains ( cards ) , rlrs recognize double - stranded rna , which is synthesized during active viral replication in the cytosol and signal through ifn- promoter stimulator-1 ( ips-1 ; also known as mavs , visa , or cardif ) , and activate transcription factors irf-3 and nf-b , subsequently leading to the production of type - i ifn and proinflammatory cytokines ( 39 ) .
a study showed that atg5-atg12 conjugates negatively regulate innate viral recognition by rig - i and mda-5 in mefs ( 56 ) .
the atg5-atg12 conjugates , which are directly associated with card domains of rig - i and ips-1 , block rlr signaling and suppress type - i ifn production ( fig .
, atg5- and atg7-deficient mefs , lacking atg5-atg12 conjugates , overproduce type - i ifn in response to vsv .
similarly , atg5-deficient cells show increased ifn production through enhanced rlr signaling in response to vsv infection . in this study ,
in addition , dysfunctional mitochondria and mitochondria - associated ips-1 are accumulated in the absence of autophagy .
collectively , accumulation of ros associated with dysfunctional mitochondria may potentiate rlr signaling in atg5-deficient cells ( fig .
inflammasomes are molecular machinery that promote innate immune defenses by triggering the maturation of proinflammatory cytokines such as il-1 ( 58 ) .
stimulation of tlr4 by lps is unable to induce inflammasome activation in macrophages if lps is not contaminated with other ligands .
however , atg16l1-deficient macrophages exhibit enhanced il-1 and il-18 production in response to lps ( fig .
5a ) , suggesting that autophagy normally suppresses inflammasome activation by lps ( 20 ) .
atg16l1 is an essential component of the autophagosome , forming a complex with atg5-atg12 conjugates and inducing lc3-pe conjugation .
interestingly , atg16l1 is one of the most important genes associated with crohn 's disease .
thus , endotoxin - induced inflammasome activation in atg16l1 deficiency could be involved in the occurrence of crohn 's disease . in a recent study detailing the complex reciprocal regulation of inflammasome and autophagic pathways , blocking autophagy
the activation of absent in melanoma 2 ( aim2 ) or nlr pyrin domain containing 3 ( nlrp3 ) inflammasomes trigger autophagy induction by activating ralb to bind to exo84 , which serves as platform for the formation of isolation membrane ( 60 ) .
autophagy engulfs ubiquitylated assembled inflammasomes through p62 , an autophagic adaptor including both ubiquitin - associated domains and lc3-interacting regions , that recognizes ubiquitinated molecules and assists their degradation by autophagy ( fig .
thus , activation of inflammasomes induces autophagy , which in turn limits the inflammasome activity by physical engulfment .
this may represent a negative regulation of autophagy in maintaining homeostasis by returning to a basal state in some inflammatory conditions .
recent advances in the characterization of autophagic machinery enable us to identify the role of autophagy in immune systems . by virtue of its ability to degrade cytosolic constituents ,
autophagy has been shown to eliminate intracellular pathogens regardless of their location ( cytosol or phagosome ) . moreover ,
autophagic machinery induced by tlr activation facilitates clearance of pathogens through autophagosome formation or maturation of phagosomes .
autophagy also mediates bacterial clearance and adaptive immune responses including antigen presentation to mhc class ii after bacterial sensing via nlrs . in viral infection , autophagy promotes the production of antiviral type - i ifn by delivering the cytosolic replication intermediates to the lysosomes , enabling recognition of virus by endosomal tlrs .
autophagy may also play a critical role in regulating inflammatory conditions including crohn 's disease by controlling inflammasome activity .
autophagy prevents excessive activation of inflammasomes to maintain basal status against endogenous / exogenous irritants .
collectively , autophagy plays multiple roles in immunity , either activating or suppressing immune responses .
comprehensive understanding of autophagy will therefore provide a more integrated picture of how this system controls immune responses . | autophagy is a fundamental cellular process in eukaryotic cells for maintaining homeostasis by degrading cellular proteins and organelles .
recently , the roles of autophagy have been expanded to immune systems , which in turn modulate innate immune responses .
more specifically , autophagy acts as a direct effector for protection against pathogens , as well as a modulator of pathogen recognition and downstream signaling in innate immune responses .
in addition , autophagy controls autoimmunity and inflammatory disorders by negative regulation of immune signaling . in this review ,
we focus on recent advances in the role of autophagy in innate immune systems . | INTRODUCTION
DIRECT ELIMINATION OF PATHOGENS BY AUTOPHAGY
INTERACTION BETWEEN AUTOPHAGY AND TOLL-LIKE RECEPTORS
AUTOPHAGY IN NUCLEOTIDE OLIGOMERIZATION DOMAIN-LIKE RECEPTOR SIGNALING
AUTOPHAGY IN ANTIVIRAL IMMUNITY
AUTOPHAGY IN REGULATION OF INFLAMMASOME
CONCLUSION |
people with hiv are living decades longer than before due to combination art ; however , complications including cvd < osteoporosis and non aids defining malignancies are higher than in the general population .
many of the emerging complications related to chronic hiv infection represent disease processes where vitamin d is known to play an important role .
a single point cross sectional case control study was conducted in a tertiary care hospital to determine the prevalence of vitamin d deficiency and its association with carotid intima media thickness ( cimt ) in hiv patients and to study the association of lipid profile with hiv infection . out of the 45 hiv positive cases ,
vitamin d deficiency was found in 42 cases ( 93.33% ) whereas only 33 out of 45 ( 73.33% ) healthy controls .
a positive association was seen between cd4 levels and 25 hydroxy vitamin d. an inverse co - relation between cd4 count and triglycerides and vldl levels while a positive co - relation with total cholesterol , ldl cholesterol and hdl cholesterol levels was seen .
people infected with hiv-1 are at an increased risk of cardiovascular disease ( cvd ) .
hiv - infected adults have increased levels of proinflammatory cytokines and endothelial activation markers , which have been associated with cvd in the general population .
in addition , the carotid intima - media thickness ( cimt ) , a surrogate marker for atherosclerosis , is associated with levels of proinflammatory cytokines and endothelial activation markers .
dyslipidemia is an important metabolic change that may occur in individuals with hiv infection which may contribute to cardiovascular disease .
the present study was conducted with the aim to determine the prevalence of vitamin d deficiency and its association with carotid intima media thickness in hiv patients and to study the correlation of lipid profile and hiv infection .
the study was conducted in a tertiary care hospital in allahabad from july 2011 to july 2012 .
forty - five hiv positive cases diagnosed by elisa , who were 18 years and above and 45 healthy age and sex matched controls were studied .
thorough clinical and biochemical examination was done for all cases and controls and they were assessed for fasting blood glucose , fasting lipid profile , hiv 1 and 2 , cd4 count , ecg , cimt , 25(oh)vitamin d.
out of the 45 hiv + ve cases , vitamin d deficiency ( 25(oh ) d levels < 30 ng / ml ) was found in 42 cases ( 93.33% ) and 33 out of the 45 ( 73.33% ) age and sex matched healthy controls .
twenty - three cases ( 51.11% ) and only 7 controls ( 15.5% ) has deranged lipid profile ( p = .
cases were grouped into 4 groups according to their cd4 counts : > 500/mm , 201 - 500/mm , 50 - 200/mm , and < 50/mm . an inverse relationship between cd4 count and triglyceride levels ( r value = -0.221 ) and vldl levels ( r value = -0.234 )
was seen while a gradual decline of total cholesterol levels , ldl levels and hdl levels with decreasing cd4 count was observed ( r values = 0.057 , 0.013 and 0.217 respectively ) .
out of the 45 cases , none ( 0% ) had cimt<6 mm , 23(51.11% ) had cimt measuring 6 mm , 7 ( 15.55% ) had cimt measuring 7 mm , 15 ( 13.33% ) had cimt measuring 8 mm and none ( 0% ) measuring > 8 mm compared to controls with 1 ( 2.22% ) who had cimt < 6 mm , 16(35.55% ) with cimt measuring 7 mm , 19 ( 42.22% ) had cimt measuring 7 mm , 9 ( 20% ) had cimt measuring 8 mm and none ( 0% ) measuring > 8 mm . by using statistical analysis , no significant difference in
the cimt of hiv positive cases and hiv negative controls was present ( p = 1.00 ) .
in this study , the prevalence of vitamin d deficiency in hiv positive cases was 93.33% as compared to a prevalence of 66.66% in the healthy population .
lipid profile , in the present study was altered with a prevalence of 51.11% in hiv infected population and 15.55% in the healthy population .
alteration in the lipid profile occurred even during the early stages of hiv infection and , more so , as the disease progressed .
the fasting total cholesterol , hdl and ldl were significantly lower in the hiv positive subjects as compared to the healthy subjects . whereas fasting triglycerides and vldl were significantly higher in hiv positive cases compared to the hiv negative controls .
as the disease progresses with drop in cd4 count , there is a progressive decline in hdl , ldl , and total cholesterol levels and a rise in triglycerides and vldl levels of hiv positive subjects .
all the 45 hiv positive cases in our study were assessed for cimt by ultrasonography .
percentage of cimt > 8 mm , 8 mm , 7 mm , 6 mm and < 6 mm among the hiv positive population was found to be 0% , 33.33% , 15.55% , 51.11% and 0% as compared to 0% , 20% , 42.22% , 35.55% and 2% in the hiv negative population respectively .
there was no significant difference in cimt between the hiv positive cases and the hiv negative controls with a p = 1.00 .
in previous studies , right and left cimt were significantly higher in hiv positive groups as compared to the hiv negative controls . significant predictors of cimt measurements in uninfected controls were body mass index and homocysteine , but only the duration of art therapy was predictive of cimt in the hiv infected group . out of the 45 cases in this study , 42 had vitamin d deficiency and their mean cimt was 0.65 mm compared to the 33 out of the 45 healthy controls who are vitamin d deficient with mean cimt = 0.68 mm .
there was no significant difference between the two groups , which was contrary to findings of previous studies where the relationship between vitamin d insufficiency and increased cimt in hiv positive patients remained significant even after adjustment for traditional risk factors for hardening of the arteries , hiv related factors like viral load and duration of antiretroviral therapy and lipid levels .
vitamin d deficiency was significantly higher in hiv patients as compared to age and sex matched healthy controls and hence such patients would benefit from vitamin d supplementation given at initiation of art as it would offer a safe and effective means of augmenting the immune restoration response to treatment .
it can also be concluded that lipid profile may be a good index of disease progression in hiv patients . | background : people with hiv are living decades longer than before due to combination art ; however , complications including cvd < osteoporosis and non aids defining malignancies are higher than in the general population .
many of the emerging complications related to chronic hiv infection represent disease processes where vitamin d is known to play an important role.objectives:a single point cross sectional case control study was conducted in a tertiary care hospital to determine the prevalence of vitamin d deficiency and its association with carotid intima media thickness ( cimt ) in hiv patients and to study the association of lipid profile with hiv infection . out of the 45 hiv positive cases ,
vitamin d deficiency was found in 42 cases ( 93.33% ) whereas only 33 out of 45 ( 73.33% ) healthy controls .
fifty one point eleven percent of cases had dyslipidemia compared to 15.55% of controls .
a positive association was seen between cd4 levels and 25 hydroxy vitamin d. an inverse co - relation between cd4 count and triglycerides and vldl levels while a positive co - relation with total cholesterol , ldl cholesterol and hdl cholesterol levels was seen .
no significant difference was seen in cimt in cases and controls . | Background:
Objectives:
I
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the palate gets divided into primary and secondary palates . during palatogenesis , fusion of two maxillary outgrowths , named palatal shelves , forms the majority of palate .
these shelves appear as protrusions on the lateral walls of the oronasal cavity , growing vertically and downward around the tongue . after a while ,
the palatal shelves elevate to a horizontal position and fuse on midline above the tongue to form the palate ( figure 1 ) ( 1 ) .
interruption in the palatogenesis process for any reason may lead to cleft palate ( cp ) which is characterized by a defect in oronasal separation .
this malformation is a very frequent congenital defect in human and it may or may not be accompanied by cleft lip ( cl ) ( 2 ) .
reported 1.0 cp along with cl ( cpl ) per 1000 live births in iran , which is lower compared to other countries ( 3 ) .
both genetic and environmental factors could have roles in orofacial clefts , cp and/or cl incidence .
many genes and signaling pathways are involved in palatogenesis , impaired action of each could lead to orofacial clefts ; further information presented in table 1 .
genes can be categorized into four groups : genes expressed in a particular area or period of palate development ; genes with biological activities without direct involvements , like receptors for retinoic acid and folic acid ; homeobox genes which determine the identity of embryonic regions along the anterior - posterior axis ; genes involved in deactivation and excretion of xenobiotics as those in p-450 cytochrome system ( 4 ) .
environmental factors like folic acid deficiency ( 4 ) , toxins in the living area ( 5 ) , teratogens such as phenytoin ( 6 ) , corticosteroids and retinoic acid ( 7 ) , and also consanguinity ( 5 ) may affect the gene expression pattern and cause cp and/or cl . further research is required toward understanding the exact molecular events .
abbreviations : bmp , bone morphogenic protein ; cp , cleft palate ; cl , cleft lip ; pdgf , platelet - derived growth factor ; wnt , wingless type ; tgf3 , transforming growth factor ; foxe1 , forkhead box protein e1 ; vax1 , ventral anterior homeobox 1 ; msx1 , msh homeobox 1 ; shox2 , short stature homeobox 2 ; tbx22 , t - box transcription factor ; osr1 , odd - skipped - related 1 ; osr2 , odd - skipped - related 2 ; fgfr2 , fibroblast growth factor receptor 2 .
palatogenesis happens during weeks 8 - 12 in human intrauterine life ( embryonic days e12-e15 in mice ) .
the palate gets divided into primary and secondary palates . during palatogenesis , fusion of two maxillary outgrowths , named palatal shelves , forms the majority of palate .
these shelves appear as protrusions on the lateral walls of the oronasal cavity , growing vertically and downward around the tongue . after a while ,
the palatal shelves elevate to a horizontal position and fuse on midline above the tongue to form the palate ( figure 1 ) ( 1 ) .
interruption in the palatogenesis process for any reason may lead to cleft palate ( cp ) which is characterized by a defect in oronasal separation .
this malformation is a very frequent congenital defect in human and it may or may not be accompanied by cleft lip ( cl ) ( 2 ) .
reported 1.0 cp along with cl ( cpl ) per 1000 live births in iran , which is lower compared to other countries ( 3 ) .
both genetic and environmental factors could have roles in orofacial clefts , cp and/or cl incidence .
many genes and signaling pathways are involved in palatogenesis , impaired action of each could lead to orofacial clefts ; further information presented in table 1 .
genes can be categorized into four groups : genes expressed in a particular area or period of palate development ; genes with biological activities without direct involvements , like receptors for retinoic acid and folic acid ; homeobox genes which determine the identity of embryonic regions along the anterior - posterior axis ; genes involved in deactivation and excretion of xenobiotics as those in p-450 cytochrome system ( 4 ) .
environmental factors like folic acid deficiency ( 4 ) , toxins in the living area ( 5 ) , teratogens such as phenytoin ( 6 ) , corticosteroids and retinoic acid ( 7 ) , and also consanguinity ( 5 ) may affect the gene expression pattern and cause cp and/or cl . further research is required toward understanding the exact molecular events .
abbreviations : bmp , bone morphogenic protein ; cp , cleft palate ; cl , cleft lip ; pdgf , platelet - derived growth factor ; wnt , wingless type ; tgf3 , transforming growth factor ; foxe1 , forkhead box protein e1 ; vax1 , ventral anterior homeobox 1 ; msx1 , msh homeobox 1 ; shox2 , short stature homeobox 2 ; tbx22 , t - box transcription factor ; osr1 , odd - skipped - related 1 ; osr2 , odd - skipped - related 2 ; fgfr2 , fibroblast growth factor receptor 2 .
pubmed search was undertaken using search terms including ' cleft palate repair ' , ' palatal cleft closure ' , ' cleft palate + stem cells ' , ' cleft palate + plasma - rich platelet ' , ' cleft palate + scaffold ' , ' palatal tissue engineering ' , and ' bone tissue engineering ' .
the found articles were included if they defined a therapeutic strategy and/or assessed a new technique .
the ability to speak and feed normally is the essential goal of cp treatment . in addition
patients with cp , cl or both ( cpl ) , often require long extensive treatments which may cause financial burden , morbidity and other medical complications ( 23 ) .
he incised the cleft edges and placed sutures leading to suppuration and then healing across the defect .
later , vonlangenbeck introduced the use of mucoperiosteal flaps to close clefts involving the hard palate ( 24 ) .
after that , a wide range of surgical techniques has been described to repair clefts , all with relative advantages and disadvantages , and all based on bone grafting ( 25 ) .
one of the major problems in cleft repair is bony reconstruction of alveolar and hard palate defects .
surgical repair by utilizing autogenous bone grafts is the current standard of care ; tibia , mandible , iliac , cranium and ribs are common donor sites ( 26 ) .
bone grafts classified as primary , secondary , and tertiary , are based on the timing of the grafting procedure ( 26 ) .
this procedure has several disadvantages , mostly related to the donor site ; morbidity after the bone graft harvest is the major problem .
complications are seen early and/or late in the donor site which include postoperative pain , wound infection , bleeding , altered sensation , local tissue injury , poor mobility , and possibly of fracture of the donor bone .
late complications include chronic pain , unaesthetic scarring , gait disturbance , and paresthesia ( 26 ) .
this bone can also be absorbed ; thus , full integration into the host site is not achieved ( 27 ) .
meyer et al . reported only 82% successful secondary alveolar bone grafting rate after a long - term follow - up cohort study ( 29 ) .
moreover , grafted bone needs to be very tenacious to support tooth eruption and to tolerate physical stress from muscles of mastication . to solve the problems of donor site morbidity
however , there is still a risk of infection in addition to immune response elicitation and problems with structural integrity ( 27 ) .
midface retrusion makes other therapies necessary to establish a normal occlusion and improves facial appearance ( 31 ) .
in addition , postoperative development of an oronasal fistula in the grafted area still remains a significant problem of the palatal reconstruction surgery .
reports have mentioned an incidence ranging from 11% to 23% , with the most likely site being the junction of the hard and soft palates ( 32 - 35 ) .
depending on size , fistulas may lead to hypernasal speech , nasal regurgitation , and food trapping .
patients with bilateral clefts appeared to have a 2- to 3-fold higher incidence of postoperative fistula development compared with unilateral clefts ( 36 , 37 ) .
another surgical approach for palatal cleft repair is using buccal fat pad applied for treatment of cp less than 20 mm in length , located in the posterior two - thirds of the palate ( 38 ) .
buccal fat pad has been used in both primary palatal cleft repair and treatment of postoperative fistula ( 39 - 42 ) .
use of buccal fat pad may result in a reduction of palatal scarring , which may limit the subsequent maxillary growth disturbance ( 27 ) .
evaluated the utility of combining the pedicled buccal fat covered with lyophilized dermis in adult patients with the successful treatment of oronasal fistula ( 43 ) .
a scaffold provides a solid framework in a desirable site for cell growth , also allowing cell attachment and migration .
surgeons may use an empty scaffold to induce host cell migration , or they may employ it as a carrier for cells in cell therapy ( 44 ) .
they should also be absorbable and the absorption rate must be almost same as bone formation rate
. moreover , a scaffold should be able to allow bone cells to migrate and proliferate . finally , it must be physically stable and easy to manufacture and sterilize and should be handy to use .
it should allow the cell - cell interaction and diffusion of nutrients and metabolites ( 45 ) .
chemical and topographic surface properties of the scaffold can effect cell adhesion , proliferation and migration ( 46 ) .
recently , it was shown that the presence of nano - structures in polymer - based composite scaffolds could be more effective for osteoblasts in comparison with micro - structures ( 47 ) .
therefore , many materials have usually been used as bone tissue engineering scaffolds ; collagen ( 48 , 49 ) , hyaluronic acid ( 50 ) , and hydroxyapatite ( 51 , 52 ) are some common examples .
these materials may be used either alone or in combination with other different substances . recently
, mesoporous bioactive glass has been developed as a scaffold with excellent osteogenic properties for bone regeneration ( 53 ) .
biodegradation and biocompatibility of novel poly ( epsilon - caprolactone)/nano - fluoridated hydroxyapatite ( pcl - fha ) scaffolds were investigated as well .
the osteoblast - like cells were attached to and proliferated on pcl - fha ( 54 ) .
in addition , acellularized dermal matrix ( alloderm ) has been applied for repair of palatal fistulas .
platelet rich fibrin ( prf ) has been also examined as a scaffold for human osteoblast carrier .
metabolic activity and proliferation of human osteoblasts were supported to a significantly high extent using prf membranes , in vitro ( 49 ) .
scaffolds are provided in various forms including porous solid meshes , foams , and injectable gel networks .
injectable gels are desirable because they can be applied for irregular shaped defects ; they can be utilized in a noninvasive procedure , and just by simple mixing they are combined with therapeutic agents or cells ( 58 ) .
tissue engineering is a novel technique which applies stem cells and/or differentiated cells , scaffolds and signaling molecules to achieve clinical therapeutic goals .
characteristics of an optimal cell source include availability in large quantities , no immune rejection , no graft versus host disease , no tumorigenicity , predictable differentiation potential of stem cells , and integration into the surrounding tissues ( 59 ) .
embryonic stem cells ( escs ) are a source that may be used in tissue engineering .
these cells exhibit long - term proliferation in vitro and the ability to differentiate into any cell type in the human body ( 44 ) , but there is a concern about their tumorigenic properties ( 60 ) .
mesenchymal stem cells ( mscs ) are considered as a suitable choice for bone tissue engineering ( 61 ) .
there are different sources for mscs , including umbilical cord blood ( ucb - mscs ) , wharton 's jelly ( uc - mscs ) , bone marrow ( bmmscs ) ( 61 ) , and adipose tissue ( adscs ) ( 62 , 63 ) .
autologous bmmscs possess a high proliferative potential and the ability to differentiate into osteoblastic cells and they can induce bone formation ( 64 , 65 ) .
furthermore , uc - mscs are an available source of cells for bone tissue engineering and could have broad application prospects in tissue repair ( 61 ) .
different kinds of stem cells have been applied for tissue regeneration in cp and alveolar cleft in different studies . among them , there are experimental and clinical studies which suggested that tissue - engineered bone may provide an acceptable alternative for cp repair ( 66 ) .
de kok et al . evaluated msc - based alveolar bone regeneration in a canine alveolar defect model using both autologous and allogeneic bmmscs .
histomorphometrical analysis showed new bone formed within the pores of the matrices loaded with autogenic bmmscs or allogenic bmmscs in equivalent amount .
analysis of circulating antibody levels against bmmscs also supported the hypothesis that mscs did not induce a systemic response by the host .
after nine weeks , the labeled cells were present within the lacunae of newly formed bone , proposing that autologous and allogeneic bmmscs have the capacity to regenerate bone within craniofacial defects ( 67 ) .
used osteogenically differentiated rat fat - derived stem cells , seeded onto poly - l - lactic acid scaffold , to repair rat palatal bone defect models .
they demonstrated the feasibility of using these cells for healing such defects , and also proved bone formation in the treated area ( 63 ) .
kim et al . recruited periodontal ligament stem cells ( pdlscs ) and bone marrow stem cells ( bmscs ) to regenerate alveolar bone in the dog canine peri - implant defect model .
they found these cells effective and bmscs were more impressive than pdlscs in bone formation ( 68 ) .
. investigated restoration of the alveolar cleft with engineered bone , constructed by sponge collagen protein combined with autologous bmmscs , in the dog alveolar cleft model .
twelve weeks after implantation , 3-dimensional ct and histological examination showed acceptable bone formation in the experimental model compared to the control .
it was concluded that these materials can restore the defect of alveolar bone effectively and they can be used clinically to treat alveolar cleft ( 69 )
. mylonas et al . compared the efficiency of bioglass and synthetic hydroxyapatite / tricalcium phosphate granular scaffolds loaded by allogeneic bmmscs for alveolar bone repairmen in a dog model .
no difference was shown in the newly formed bone percent area using these scaffolds , although both exhibited desirable results ( 70 ) .
did a survey to compare bone regeneration of tissue engineered bone from canine adscs and autogenous bone graft in the bilateral maxillary alveolar cleft model in dog .
for osteogenic differentiation of the undifferentiated cells , they were seeded into a hydroxyapatite / beta - tricalcium phosphate scaffold , overwhelmed in specific osteogenic medium for 21 days .
this material was applied on one side and corticocancellous tibial autograft was added on the other side .
there was a significant difference between the tissue - engineered side and autografted side , with bone autograft superiority ( 66 ) .
behnia et al . evaluated the effect of recombinant platelet derived growth factor and in vitro osteogenic differentiated human bone marrow mesenchymal stem cells ( hbmmscs ) in the secondary alveoloplasty .
they reported significant improvement in bone regeneration three months after the operation , suggesting an enhancement effect of recombinant platelet derived growth factor with hmscs on regeneration capacity of the cells ( 71 ) .
gimbel et al . compared donor site pain in both tissue engineering minimally invasive iliac crest bone graft and traditional iliac crest bone graft techniques for alveolar bone defect repair , to determine if a tissue engineering technique with similar bone healing results offered any decreased morbidity .
their results exhibited closure of alveolar cleft defects with an absorbable collagen sponge and bone marrow stem cells eventuated in reduced donor site morbidity and decreased the donor site pain intensity and frequency ( 72 ) .
however , bone marrow aspiration is an undeniable part of bmscs extraction ; so , bmscs application fails to totally obviate the donor site morbidity ( 73 ) . the bmp signaling molecules family has been used to induce osteoblastic differentiation .
the recombination technology now allows production of large , pure quantities of rhbmp-2 which can be used widely in clinics and laboratories . in a proper environment
, rhbmp-2 can cause bone formation by inducing stem cells to differentiate into bone forming cells ( 74 ) .
mayer et al . evaluated rhbmp-2 in maxillary alveolar clefts in 24 adults , skeletally mature foxhound dogs .
there were three treatment groups : ( 1 ) rhbmp-2 combined with the copolymer poly(lactide - co - glycolide ) and autogenous blood , ( 2 ) poly(lactide - co - glycolide ) and autogenous blood , ( 3 ) autograft from the posterior iliac crest ; and an untreated group as control .
the results showed more bone regeneration in the autograft group at two months , but after four months there were no significant differences between this group and the rhbmp-2 group ( 75 ) .
after that , chin et al . demonstrated clinically the feasibility of using rhbmp-2 to repair alveolar clefts without the use of autogenous graft tissue .
in addition , histological assessment proved that it was a normal and vital bone ( 76 ) .
herford et al . examined bony regeneration of premaxillary clefts by using rhbmp-2 within a collagen sponge carrier in clinic .
computed tomographic studies , before and four months after the operation , revealed that such defects can have complete osseous regeneration induced by rhbmp-2 ( 77 ) .
nguyen et al . also compared the therapeutic benefits of absorbable collagen sponge , absorbable collagen sponge plus rhbmp-2 , hydroxyapatite - tricalcium phosphate scaffold , hydroxyapatite - tricalcium phosphate scaffold plus rhbmp-2 , and control group on surgically created defects in rats .
radiomorphometrical and histomorphometrical assessments at four , eight , and 12 weeks showed an increase in bone formation in animals treated with hydroxyapatite - tricalcium phosphate plus rhbmp-2 ( p < 0.05 ; four weeks ) compared with the empty scaffold ( 28 ) .
alonso et al . did a survey to compare the effect of a combination of a resorbable collagen matrix along with rhbmp-2 with traditional iliac crest bone graft for closure of alveolar defects .
the authors concluded that rhbmp-2 therapy had satisfactory bone healing effects and it could reduce tissue morbidity ( 78 ) .
furthermore , a novel technique which encased rhbmp-2 in a demineralized bone matrix scaffold ( bmp / dbm ) was developed as an alternative to autografting for secondary alveolar cleft reconstruction .
reported adverse events after the use of rhbmp-2 as an alternative to autogenous bone graft in oral and maxillofacial surgical procedures .
they found that 66.3% of patients with reconstruction of mandible after fracture or cancer or alveolar cleft repair by means of rhbmp-2 reported local reactions , graft failure , infections and other wound complications and 30.1% required additional surgery to address the reported adverse events ( 80 ) .
such reports make it necessary to use natural materials instead of synthetic ones . in regenerative medicine ,
platelet - rich plasma ( prp ) is a blood product with a high platelet concentration and the rationale beyond using platelets in tissue engineering is the release of bioactive factors which collaborate in tissue regeneration .
platelets contain a number of growth factors such as platelet - derived , vascular endothelial , beta - type transforming , epidermal , fibroblast and insulin - like i , which promote angiogenesis and synthesis of the extracellular matrix ( 81 - 83 ) .
as one of the main components in tissue regeneration , we successfully applied prp to treat nonhealing wounds ( 82 ) , vesicovaginal fistulas ( 83 ) , and stress urinary incontinence ( 81 ) . in one cp case , we injected autologous prp which is described in details in the following section .
there are a few reports about using prp into grafts for alveolar clefts treatment as a source of growth factors for osteogenic differentiation .
osteoregeneration efficacy of autologous bone grafting in prp has been clinically tested by oyama et al .
they reported higher volume ratio of bone regeneration in prp - applied cases than control cases who did not receive prp in their treatments .
it was suggested that prp could enhance bone regeneration and may be beneficial in cp and cl patients treatment procedures ( 84 ) .
ouyang et al . evaluated the effect of prp as an adjunct to bovine porous bone mineral ( bpbm ) graft in treatment of periodontal intra bony defects clinically .
their results showed a significant favorable clinical improvement in treating such defects by prp and bpbm application simultaneously ( 85 ) .
applied autologous mscs combined with prp for an alveolar cleft osteoplasty of a nine - year - old female patient .
results showed promising bone regeneration and bridging of the cleft after six months ( 86 ) .
designed a longitudinal survey to evaluate the outcomes of secondary autogenous bone graft combined with prp in the alveolar cleft .
the study considered two groups including 35 alveolar clefts in 30 patients with grafted autogenous bone and prp and 36 clefts in 30 patients with grafted autogenous bone alone .
the density and resorption of the grafted bone was assessed at one week , and one , three , six , and 12 months postoperatively .
the authors suggested that prp may improve bone remodeling in the early phase ; however , prp seemed to have no effect on bone resorption following secondary bone graft in long term ( 87 ) .
in a clinical study , luaces - rey et al . compared alveolar reconstruction in secondary alveoloplasty with or without prp .
in one group , secondary alveoloplasty was performed using autogenous bone graft and in the second group prp was added to the autogenous bone grafting procedure .
digital orthopantomography was performed immediately after the operation and again three and six months later .
in contrast to the previous study , the data showed no significant differences between the two groups regarding bone regeneration amount ( 89 ) .
there was a significant reduction in postoperative bone resorption , particularly in the prp group ( 90 ) .
the ability to speak and feed normally is the essential goal of cp treatment . in addition
patients with cp , cl or both ( cpl ) , often require long extensive treatments which may cause financial burden , morbidity and other medical complications ( 23 ) .
he incised the cleft edges and placed sutures leading to suppuration and then healing across the defect .
later , vonlangenbeck introduced the use of mucoperiosteal flaps to close clefts involving the hard palate ( 24 ) .
after that , a wide range of surgical techniques has been described to repair clefts , all with relative advantages and disadvantages , and all based on bone grafting ( 25 ) .
one of the major problems in cleft repair is bony reconstruction of alveolar and hard palate defects .
surgical repair by utilizing autogenous bone grafts is the current standard of care ; tibia , mandible , iliac , cranium and ribs are common donor sites ( 26 ) .
bone grafts classified as primary , secondary , and tertiary , are based on the timing of the grafting procedure ( 26 ) .
this procedure has several disadvantages , mostly related to the donor site ; morbidity after the bone graft harvest is the major problem .
complications are seen early and/or late in the donor site which include postoperative pain , wound infection , bleeding , altered sensation , local tissue injury , poor mobility , and possibly of fracture of the donor bone .
late complications include chronic pain , unaesthetic scarring , gait disturbance , and paresthesia ( 26 ) .
this bone can also be absorbed ; thus , full integration into the host site is not achieved ( 27 ) .
meyer et al . reported only 82% successful secondary alveolar bone grafting rate after a long - term follow - up cohort study ( 29 ) .
moreover , grafted bone needs to be very tenacious to support tooth eruption and to tolerate physical stress from muscles of mastication . to solve the problems of donor site morbidity
however , there is still a risk of infection in addition to immune response elicitation and problems with structural integrity ( 27 ) .
midface retrusion makes other therapies necessary to establish a normal occlusion and improves facial appearance ( 31 ) .
in addition , postoperative development of an oronasal fistula in the grafted area still remains a significant problem of the palatal reconstruction surgery .
reports have mentioned an incidence ranging from 11% to 23% , with the most likely site being the junction of the hard and soft palates ( 32 - 35 ) . depending on size
patients with bilateral clefts appeared to have a 2- to 3-fold higher incidence of postoperative fistula development compared with unilateral clefts ( 36 , 37 ) .
another surgical approach for palatal cleft repair is using buccal fat pad applied for treatment of cp less than 20 mm in length , located in the posterior two - thirds of the palate ( 38 ) .
buccal fat pad has been used in both primary palatal cleft repair and treatment of postoperative fistula ( 39 - 42 ) .
use of buccal fat pad may result in a reduction of palatal scarring , which may limit the subsequent maxillary growth disturbance ( 27 ) .
evaluated the utility of combining the pedicled buccal fat covered with lyophilized dermis in adult patients with the successful treatment of oronasal fistula ( 43 ) .
a scaffold provides a solid framework in a desirable site for cell growth , also allowing cell attachment and migration .
surgeons may use an empty scaffold to induce host cell migration , or they may employ it as a carrier for cells in cell therapy ( 44 ) .
they should also be absorbable and the absorption rate must be almost same as bone formation rate
. moreover , a scaffold should be able to allow bone cells to migrate and proliferate . finally , it must be physically stable and easy to manufacture and sterilize and should be handy to use .
it should allow the cell - cell interaction and diffusion of nutrients and metabolites ( 45 ) .
chemical and topographic surface properties of the scaffold can effect cell adhesion , proliferation and migration ( 46 ) .
recently , it was shown that the presence of nano - structures in polymer - based composite scaffolds could be more effective for osteoblasts in comparison with micro - structures ( 47 ) .
therefore , many materials have usually been used as bone tissue engineering scaffolds ; collagen ( 48 , 49 ) , hyaluronic acid ( 50 ) , and hydroxyapatite ( 51 , 52 ) are some common examples .
these materials may be used either alone or in combination with other different substances . recently
, mesoporous bioactive glass has been developed as a scaffold with excellent osteogenic properties for bone regeneration ( 53 ) .
biodegradation and biocompatibility of novel poly ( epsilon - caprolactone)/nano - fluoridated hydroxyapatite ( pcl - fha ) scaffolds were investigated as well .
the osteoblast - like cells were attached to and proliferated on pcl - fha ( 54 ) .
in addition , acellularized dermal matrix ( alloderm ) has been applied for repair of palatal fistulas .
platelet rich fibrin ( prf ) has been also examined as a scaffold for human osteoblast carrier .
metabolic activity and proliferation of human osteoblasts were supported to a significantly high extent using prf membranes , in vitro ( 49 ) .
scaffolds are provided in various forms including porous solid meshes , foams , and injectable gel networks .
injectable gels are desirable because they can be applied for irregular shaped defects ; they can be utilized in a noninvasive procedure , and just by simple mixing they are combined with therapeutic agents or cells ( 58 ) .
tissue engineering is a novel technique which applies stem cells and/or differentiated cells , scaffolds and signaling molecules to achieve clinical therapeutic goals
. characteristics of an optimal cell source include availability in large quantities , no immune rejection , no graft versus host disease , no tumorigenicity , predictable differentiation potential of stem cells , and integration into the surrounding tissues ( 59 ) .
embryonic stem cells ( escs ) are a source that may be used in tissue engineering .
these cells exhibit long - term proliferation in vitro and the ability to differentiate into any cell type in the human body ( 44 ) , but there is a concern about their tumorigenic properties ( 60 ) .
mesenchymal stem cells ( mscs ) are considered as a suitable choice for bone tissue engineering ( 61 ) .
there are different sources for mscs , including umbilical cord blood ( ucb - mscs ) , wharton 's jelly ( uc - mscs ) , bone marrow ( bmmscs ) ( 61 ) , and adipose tissue ( adscs ) ( 62 , 63 ) .
autologous bmmscs possess a high proliferative potential and the ability to differentiate into osteoblastic cells and they can induce bone formation ( 64 , 65 ) . furthermore , uc - mscs are an available source of cells for bone tissue engineering and could have broad application prospects in tissue repair ( 61 ) .
different kinds of stem cells have been applied for tissue regeneration in cp and alveolar cleft in different studies . among them , there are experimental and clinical studies which suggested that tissue - engineered bone may provide an acceptable alternative for cp repair ( 66 ) .
de kok et al . evaluated msc - based alveolar bone regeneration in a canine alveolar defect model using both autologous and allogeneic bmmscs .
histomorphometrical analysis showed new bone formed within the pores of the matrices loaded with autogenic bmmscs or allogenic bmmscs in equivalent amount .
analysis of circulating antibody levels against bmmscs also supported the hypothesis that mscs did not induce a systemic response by the host .
after nine weeks , the labeled cells were present within the lacunae of newly formed bone , proposing that autologous and allogeneic bmmscs have the capacity to regenerate bone within craniofacial defects ( 67 ) .
conejero et al . used osteogenically differentiated rat fat - derived stem cells , seeded onto poly - l - lactic acid scaffold , to repair rat palatal bone defect models .
they demonstrated the feasibility of using these cells for healing such defects , and also proved bone formation in the treated area ( 63 ) .
kim et al . recruited periodontal ligament stem cells ( pdlscs ) and bone marrow stem cells ( bmscs ) to regenerate alveolar bone in the dog canine peri - implant defect model .
they found these cells effective and bmscs were more impressive than pdlscs in bone formation ( 68 ) .
. investigated restoration of the alveolar cleft with engineered bone , constructed by sponge collagen protein combined with autologous bmmscs , in the dog alveolar cleft model .
twelve weeks after implantation , 3-dimensional ct and histological examination showed acceptable bone formation in the experimental model compared to the control .
it was concluded that these materials can restore the defect of alveolar bone effectively and they can be used clinically to treat alveolar cleft ( 69 ) .
mylonas et al . compared the efficiency of bioglass and synthetic hydroxyapatite / tricalcium phosphate granular scaffolds loaded by allogeneic bmmscs for alveolar bone repairmen in a dog model .
no difference was shown in the newly formed bone percent area using these scaffolds , although both exhibited desirable results ( 70 ) .
did a survey to compare bone regeneration of tissue engineered bone from canine adscs and autogenous bone graft in the bilateral maxillary alveolar cleft model in dog .
for osteogenic differentiation of the undifferentiated cells , they were seeded into a hydroxyapatite / beta - tricalcium phosphate scaffold , overwhelmed in specific osteogenic medium for 21 days .
this material was applied on one side and corticocancellous tibial autograft was added on the other side .
there was a significant difference between the tissue - engineered side and autografted side , with bone autograft superiority ( 66 ) .
behnia et al . evaluated the effect of recombinant platelet derived growth factor and in vitro osteogenic differentiated human bone marrow mesenchymal stem cells ( hbmmscs ) in the secondary alveoloplasty .
they reported significant improvement in bone regeneration three months after the operation , suggesting an enhancement effect of recombinant platelet derived growth factor with hmscs on regeneration capacity of the cells ( 71 ) .
gimbel et al . compared donor site pain in both tissue engineering minimally invasive iliac crest bone graft and traditional iliac crest bone graft techniques for alveolar bone defect repair , to determine if a tissue engineering technique with similar bone healing results offered any decreased morbidity .
their results exhibited closure of alveolar cleft defects with an absorbable collagen sponge and bone marrow stem cells eventuated in reduced donor site morbidity and decreased the donor site pain intensity and frequency ( 72 ) .
however , bone marrow aspiration is an undeniable part of bmscs extraction ; so , bmscs application fails to totally obviate the donor site morbidity ( 73 ) .
de kok et al . evaluated msc - based alveolar bone regeneration in a canine alveolar defect model using both autologous and allogeneic bmmscs .
histomorphometrical analysis showed new bone formed within the pores of the matrices loaded with autogenic bmmscs or allogenic bmmscs in equivalent amount .
analysis of circulating antibody levels against bmmscs also supported the hypothesis that mscs did not induce a systemic response by the host .
after nine weeks , the labeled cells were present within the lacunae of newly formed bone , proposing that autologous and allogeneic bmmscs have the capacity to regenerate bone within craniofacial defects ( 67 ) .
conejero et al . used osteogenically differentiated rat fat - derived stem cells , seeded onto poly - l - lactic acid scaffold , to repair rat palatal bone defect models .
they demonstrated the feasibility of using these cells for healing such defects , and also proved bone formation in the treated area ( 63 ) .
kim et al . recruited periodontal ligament stem cells ( pdlscs ) and bone marrow stem cells ( bmscs ) to regenerate alveolar bone in the dog canine peri - implant defect model .
they found these cells effective and bmscs were more impressive than pdlscs in bone formation ( 68 ) .
. investigated restoration of the alveolar cleft with engineered bone , constructed by sponge collagen protein combined with autologous bmmscs , in the dog alveolar cleft model .
twelve weeks after implantation , 3-dimensional ct and histological examination showed acceptable bone formation in the experimental model compared to the control .
it was concluded that these materials can restore the defect of alveolar bone effectively and they can be used clinically to treat alveolar cleft ( 69 )
. mylonas et al . compared the efficiency of bioglass and synthetic hydroxyapatite / tricalcium phosphate granular scaffolds loaded by allogeneic bmmscs for alveolar bone repairmen in a dog model .
no difference was shown in the newly formed bone percent area using these scaffolds , although both exhibited desirable results ( 70 ) .
recently , pourebrahim et al . did a survey to compare bone regeneration of tissue engineered bone from canine adscs and autogenous bone graft in the bilateral maxillary alveolar cleft model in dog .
for osteogenic differentiation of the undifferentiated cells , they were seeded into a hydroxyapatite / beta - tricalcium phosphate scaffold , overwhelmed in specific osteogenic medium for 21 days .
this material was applied on one side and corticocancellous tibial autograft was added on the other side .
there was a significant difference between the tissue - engineered side and autografted side , with bone autograft superiority ( 66 ) .
behnia et al . evaluated the effect of recombinant platelet derived growth factor and in vitro osteogenic differentiated human bone marrow mesenchymal stem cells ( hbmmscs ) in the secondary alveoloplasty .
they reported significant improvement in bone regeneration three months after the operation , suggesting an enhancement effect of recombinant platelet derived growth factor with hmscs on regeneration capacity of the cells ( 71 ) .
gimbel et al . compared donor site pain in both tissue engineering minimally invasive iliac crest bone graft and traditional iliac crest bone graft techniques for alveolar bone defect repair , to determine if a tissue engineering technique with similar bone healing results offered any decreased morbidity .
their results exhibited closure of alveolar cleft defects with an absorbable collagen sponge and bone marrow stem cells eventuated in reduced donor site morbidity and decreased the donor site pain intensity and frequency ( 72 ) .
however , bone marrow aspiration is an undeniable part of bmscs extraction ; so , bmscs application fails to totally obviate the donor site morbidity ( 73 ) .
the recombination technology now allows production of large , pure quantities of rhbmp-2 which can be used widely in clinics and laboratories . in a proper environment ,
rhbmp-2 can cause bone formation by inducing stem cells to differentiate into bone forming cells ( 74 ) .
mayer et al . evaluated rhbmp-2 in maxillary alveolar clefts in 24 adults , skeletally mature foxhound dogs .
there were three treatment groups : ( 1 ) rhbmp-2 combined with the copolymer poly(lactide - co - glycolide ) and autogenous blood , ( 2 ) poly(lactide - co - glycolide ) and autogenous blood , ( 3 ) autograft from the posterior iliac crest ; and an untreated group as control .
the results showed more bone regeneration in the autograft group at two months , but after four months there were no significant differences between this group and the rhbmp-2 group ( 75 ) .
after that , chin et al . demonstrated clinically the feasibility of using rhbmp-2 to repair alveolar clefts without the use of autogenous graft tissue .
in addition , histological assessment proved that it was a normal and vital bone ( 76 ) .
herford et al . examined bony regeneration of premaxillary clefts by using rhbmp-2 within a collagen sponge carrier in clinic .
computed tomographic studies , before and four months after the operation , revealed that such defects can have complete osseous regeneration induced by rhbmp-2 ( 77 ) .
nguyen et al . also compared the therapeutic benefits of absorbable collagen sponge , absorbable collagen sponge plus rhbmp-2 , hydroxyapatite - tricalcium phosphate scaffold , hydroxyapatite - tricalcium phosphate scaffold plus rhbmp-2 , and control group on surgically created defects in rats .
radiomorphometrical and histomorphometrical assessments at four , eight , and 12 weeks showed an increase in bone formation in animals treated with hydroxyapatite - tricalcium phosphate plus rhbmp-2 ( p < 0.05 ; four weeks ) compared with the empty scaffold ( 28 ) .
alonso et al . did a survey to compare the effect of a combination of a resorbable collagen matrix along with rhbmp-2 with traditional iliac crest bone graft for closure of alveolar defects .
the authors concluded that rhbmp-2 therapy had satisfactory bone healing effects and it could reduce tissue morbidity ( 78 ) .
furthermore , a novel technique which encased rhbmp-2 in a demineralized bone matrix scaffold ( bmp / dbm ) was developed as an alternative to autografting for secondary alveolar cleft reconstruction .
reported adverse events after the use of rhbmp-2 as an alternative to autogenous bone graft in oral and maxillofacial surgical procedures .
they found that 66.3% of patients with reconstruction of mandible after fracture or cancer or alveolar cleft repair by means of rhbmp-2 reported local reactions , graft failure , infections and other wound complications and 30.1% required additional surgery to address the reported adverse events ( 80 ) .
in regenerative medicine , tissue repair is strongly dependent on formation of new blood vessels and capillaries .
platelet - rich plasma ( prp ) is a blood product with a high platelet concentration and the rationale beyond using platelets in tissue engineering is the release of bioactive factors which collaborate in tissue regeneration .
platelets contain a number of growth factors such as platelet - derived , vascular endothelial , beta - type transforming , epidermal , fibroblast and insulin - like i , which promote angiogenesis and synthesis of the extracellular matrix ( 81 - 83 ) .
as one of the main components in tissue regeneration , we successfully applied prp to treat nonhealing wounds ( 82 ) , vesicovaginal fistulas ( 83 ) , and stress urinary incontinence ( 81 ) . in one cp case , we injected autologous prp which is described in details in the following section .
there are a few reports about using prp into grafts for alveolar clefts treatment as a source of growth factors for osteogenic differentiation .
osteoregeneration efficacy of autologous bone grafting in prp has been clinically tested by oyama et al .
they reported higher volume ratio of bone regeneration in prp - applied cases than control cases who did not receive prp in their treatments .
it was suggested that prp could enhance bone regeneration and may be beneficial in cp and cl patients treatment procedures ( 84 ) .
ouyang et al . evaluated the effect of prp as an adjunct to bovine porous bone mineral ( bpbm ) graft in treatment of periodontal intra bony defects clinically .
their results showed a significant favorable clinical improvement in treating such defects by prp and bpbm application simultaneously ( 85 ) .
applied autologous mscs combined with prp for an alveolar cleft osteoplasty of a nine - year - old female patient .
results showed promising bone regeneration and bridging of the cleft after six months ( 86 ) .
lee et al . designed a longitudinal survey to evaluate the outcomes of secondary autogenous bone graft combined with prp in the alveolar cleft .
the study considered two groups including 35 alveolar clefts in 30 patients with grafted autogenous bone and prp and 36 clefts in 30 patients with grafted autogenous bone alone .
the density and resorption of the grafted bone was assessed at one week , and one , three , six , and 12 months postoperatively .
the authors suggested that prp may improve bone remodeling in the early phase ; however , prp seemed to have no effect on bone resorption following secondary bone graft in long term ( 87 ) .
they observed satisfactory results both on bone and soft tissue regeneration ( 88 ) . in a clinical study , luaces - rey et al .
, secondary alveoloplasty was performed using autogenous bone graft and in the second group prp was added to the autogenous bone grafting procedure .
digital orthopantomography was performed immediately after the operation and again three and six months later .
in contrast to the previous study , the data showed no significant differences between the two groups regarding bone regeneration amount ( 89 ) .
there was a significant reduction in postoperative bone resorption , particularly in the prp group ( 90 ) .
in this mini - review , we reported a summary of the key points concerning cleft palate development , genes involving this defect , current therapeutic strategies , recently novel aspects , and future advances in treatments for easy and fast understanding of the concepts , rather than a systematic review .
according to the literature review as well as our unpublished experiences , we proposed a novel technique for cp management . on the basis of our hypothesis ,
since we provided a mini - review study , we did not explain all the aspects in details which was the limitation of our study .
the strong point of our study was brief and understandable explanations in each part which helped reader find out the whole subject in a short time . in a one - year - old female case ,
meanwhile , for increasing the success rate , we suggested to inject hprp ( human plasma - rich platelet ) along fistula borders before surgery .
three injections were done at two - month intervals . after the third injection , a reduction was observed in the fistula size .
in addition , free margins turned thick and well vascularized ; tissue grafting was performed successfully . unfortunately
the patient was a candidate for tissue grafting from tongue or buccal area , an invasive procedure , six months later .
surprisingly , sufficient tissue was generated two months after the third injection and the fistula was closed with no more graft needed .
the present experience drove us to the hypothesis that prp can not only reduce the size of defect , but it can also increase angiogenesis as an important factor for tissue grafting ( unfortunately there was no publishable picture available ) . furthermore , the surgical interference is still the gold standard treatment procedure for cp , so we strongly recommend our technique for small defects ; in wider defects we recommend it before surgery to enhance angiogenesis and reduce graft failure rate .
in a one - year - old female case , a fistula was developed after bone autografting for cp treatment .
meanwhile , for increasing the success rate , we suggested to inject hprp ( human plasma - rich platelet ) along fistula borders before surgery .
three injections were done at two - month intervals . after the third injection , a reduction was observed in the fistula size .
in addition , free margins turned thick and well vascularized ; tissue grafting was performed successfully .
the patient was a candidate for tissue grafting from tongue or buccal area , an invasive procedure , six months later .
surprisingly , sufficient tissue was generated two months after the third injection and the fistula was closed with no more graft needed .
the present experience drove us to the hypothesis that prp can not only reduce the size of defect , but it can also increase angiogenesis as an important factor for tissue grafting ( unfortunately there was no publishable picture available ) . furthermore , the surgical interference is still the gold standard treatment procedure for cp , so we strongly recommend our technique for small defects ; in wider defects we recommend it before surgery to enhance angiogenesis and reduce graft failure rate . | context : cleft palate is the second most common birth defect and is considered as a challenge for pediatric plastic surgeons .
there is still a general lack of a standard protocol and patients often require multiple surgical interventions during their lifetime along with disappointing results.evidence acquisition : pubmed search was undertaken using search terms including ' cleft palate repair ' , ' palatal cleft closure ' , ' cleft palate + stem cells ' , ' cleft palate + plasma rich platelet ' , ' cleft palate + scaffold ' , ' palatal tissue engineering ' , and ' bone tissue engineering ' .
the found articles were included if they defined a therapeutic strategy and/or assessed a new technique.results:we reported a summary of the key - points concerning cleft palate development , the genes involving this defect , current therapeutic strategies , recently novel aspects , and future advances in treatments for easy and fast understanding of the concepts , rather than a systematic review .
in addition , the results were integrated with our recent experience.conclusions:tissue engineering may open a new window in cleft palate reconstruction .
stem cells and growth factors play key roles in this field . | 1. Context
1.1. Cleft Palate Development Scenario
2. Evidence Acquisition
2.1. Current Therapeutic Strategies
2.2. Using Scaffolds for Cleft Palate Repair
2.3. Applying Stem Cells to Engineered Palates
2.3.1. Experimental Studies
2.3.2. Clinical Studies
2.4. Synthetic Adjuvant in Engineered Palates
2.5. Platelet-Rich Plasma Usage as a Natural Adjuvant in Engineered Palates
3. Results
4. Conclusions
4.1. Case Report |
infective endocarditis ( ie ) is a disease caused by infection of the inner layer of the heart wall , i.e. , the endocardium .
it develops in the heart valves ( most frequently in the aortic valve ) , ventricles , and atria [ 1 , 2 ] .
infective endocarditis related to an implanted foreign body ( a stimulator electrode or a cardioverter - defibrillator ) should also be mentioned when discussing this matter .
congenital heart defects , previous rheumatic diseases , and mitral valve prolapse syndrome constitute factors predisposing to ie [ 1 , 2 ] . individuals dependent on intravenous drugs or subjected to medical procedures requiring long - term vascular access ( chronic hemodialysis , chronic parenteral nutrition , prolonged intensive care unit ( icu ) stay ) are at risk of developing ie [ 4 , 5 ] .
infective endocarditis simultaneously affecting the left and right side of the heart occurs in approximately 15% of cases .
prosthetic valve endocarditis ( pve ) constitutes 1040% of the total number of ie cases [ 1 , 2 , 7 ] .
this paper presents the case of a patient requiring implantation of aortic , mitral , and tricuspid valve bioprostheses as a result of ie and reimplantation of the aortic valve bioprosthesis on the 46 day after the initial surgery due to ie recurrence .
the aim of this study is to present the case and assess the treatment results of a patient requiring implantation of three valve bioprostheses due to ie ; the procedure was complicated by the necessity to replace the aortic valve prosthesis because of pve .
a 19-year - old patient was admitted to a toxicology ward with significant sleepiness and unclear speech in the middle of a drinking bout which had lasted for the previous 3 months ; he had been addicted to alcohol for the past 5 years as a result of as he claimed mitigating the symptoms of tourette syndrome .
after several days , the patient started exhibiting clinical symptoms of pneumonia with rapid deterioration of his general condition ; he required intubation and continued mechanical ventilation .
the patient was transferred to the intensive care unit of the jzef stru city hospital in poznan .
after admission , he was intubated , mechanically ventilated , and sedated with a continuous infusion of midazolamum and fentanyl .
transthoracic echocardiography ( tte ) revealed grade 4 mitral insufficiency , the presence of 1.2 1.6 cm vegetation on the anterior cusp with rupture of the tendinous cords of this cusp , and vegetation 1.0 cm in length on the posterior cusp .
the cusps of the aortic valve exhibited features of marginal thickening with the presence of vegetation and grade 1 insufficiency .
the tricuspid valve was insufficient with a grade 1 central regurgitation jet and suspected vegetation .
empiric antibiotic therapy was applied ( imipenem , vancomycin , clindamycinum ) , even though bacteriological analysis of venous blood , urine , and bronchial secretions showed them to be sterile .
dental consultation revealed the presence of numerous inflammatory foci in the oral cavity , which resulted in the extraction of 8 carious teeth .
after 7 days of antibiotic therapy , due to the presence of mobile vegetations on the valves and the associated risk of embolization , the patient was transferred to an operating room at the cardiac surgery ward .
the surgical procedure was performed using extracorporeal circulation ( ecc ) via a median sternotomy .
the ascending aorta was cannulated ; venous cannulas were placed in the caval veins via the right atrium .
after aortic cross - clamping and the administration of a crystalloid cardioplegic solution into the coronary ostia , the right ventricle was opened , and the tricuspid valve was removed .
subsequently , the interatrial septum was incised , and the bicuspid valve was removed ; the aortic valve was removed through the incision in the aorta .
all three removed valves exhibited features of dysfunction in the form of insufficiency , cusp perforation , and vegetation . during the next stage of the operation , single sutures on teflon pledgets were used to implant the valve bioprostheses : medtronic hancock ii porcine 29 in the mitral position , edwards lifesciences c - e perimount 21 in the aortic position , and medtronic hancock ii porcine 33 in the tricuspid position . according to the literature
, pve occurs as frequently on biological as on mechanical prostheses [ 3 , 8 ] , but because of the patient 's alcoholism and the resulting high risk of non - adherence to anticoagulation therapy , a decision was made to implant biological valve prostheses .
aortic cross - clamping time was 161 minutes , ecc time was 224 minutes , and total surgery time was 340 minutes .
the patient was transferred to a recovery room in good general condition , hemodynamically stable , requiring moderate doses of catecholamines . on the third postoperative day , after the mediastinal drains were removed , the intubated and mechanically ventilated patient was transferred to the intensive care unit .
the cultures from blood ( drawn twice ) and from all the valves removed during surgery were sterile , but antibiotic therapy was continued regardless . on the 14 day of icu
the patient was transferred to the cardiology ward , where he was diagnosed with sepsis confirmed by numerous bacteriological tests .
pseudomonas aeruginosa ( > 1000 000 colony forming unit ( cfu)/ml ) was cultured from the tip of the central venous catheter , urine , and bronchial secretions .
similar results were obtained in the culture tests of peripheral venous blood ( drawn several times ) . targeted antibiotic therapy
no clinical improvement of the patient 's condition was achieved ; the fever and weakness did not subside .
transesophageal echocardiography ( tee ) revealed the presence of vegetation on the aortic valve bioprosthesis .
computed tomography ( ct ) of the chest , head , and abdomen as well as x - ray of the chest did not show any features of inflammatory lesions or metastatic abscesses .
after echocardiographic examination and cardiac surgical consultations , a decision was made to replace the previously implanted aortic valve bioprosthesis .
on the 46 day after the initial surgery , the patient was once again transferred to an operating room .
after connecting the patient to ecc , aortic cross - clamping , and the administration of cardioplegia to the coronary ostia , the right atrium and the interatrial septum were opened in order to check the condition of the mitral and tricuspid bioprostheses ; no vegetation was found .
after the aorta was opened , a conglomerate of vegetation was revealed on the non - coronary cusp of the subsequently removed aortic valve bioprosthesis .
an edwards lifesciences c - e perimount 21 aortic valve bioprosthesis was reimplanted in its stead .
aortic cross - clamping time was 92 minutes , and ecc time was 140 minutes .
the patient was then transferred to a recovery room in stable condition , where he was extubated after 19 hours .
pseudomonas aeruginosa was cultured from the aortic valve bioprosthesis removed during surgery , while bacteriological tests of blood drawn several times in the postoperative period were sterile .
on the 12 day after reoperation , the patient was transferred in good condition to a cardiac rehabilitation hospital with a recommendation to continue oral anticoagulation for 3 months and antibiotic therapy .
control echocardiographic examination , conducted on the 30 day after leaving the cardiac surgery ward , revealed normal function of all the bioprostheses .
the so - called modified duke criteria are applied for diagnosing ie [ 1 , 2 , 9 ] .
the minor criteria include the existence of a heart condition or other causes predisposing to ie ( drug addiction , chronic hemodialysis , prolonged icu stay ) , fever over 38c , vascular phenomena ( arterial emboli , septic pulmonary infarcts ) , and immunological phenomena ( osler 's nodes , roth 's spots , glomerulonephritis , hemorrhages ) . in order to diagnose ie
, two major , one major and three minor , or five minor criteria need to be met ; however , tee still remains the conclusive examination , with a sensitivity and specificity of approximately 95% .
the patient was in the small group of cases with lesions in three heart valves requiring surgical treatment .
the authors have experience in conducting this type of procedure . there were no complications during or directly after surgery .
unfortunately , so - called early left - sided prosthetic valve endocarditis ( pve within the first postoperative year ) occurred some days after the operation .
patients reoperated due to pve constitute 1040% of all cases of ie [ 1 , 2 , 7 ] .
the risk increases to 1520% if the multiple - valve defect is caused by ie [ 6 , 12 , 13 ] . in 2013 , there were 111 procedures involving three valves in poland , with a hospital mortality of 13.51% .
this is why echocardiographic assessment ( mainly tee ) is so important in cases of ie , as a decision concerning the implantation of more than one prosthetic valve is of great importance for the patient . | the patient was admitted to the department of cardiac surgery of the j. stru city hospital in poznan due to infective endocarditis involving the aortic , mitral , and tricuspid valves .
implantation of three biological valve prostheses proceeded without complications .
starting on day 23 , the patient 's general condition deteriorated , with high fever . despite postoperative antibiotic therapy ,
transesophageal echocardiography revealed the presence of vegetation on the bioprosthetic aortic valve . on the 46th day after the initial surgery ,
the patient required replacement of the aortic bioprosthesis , which exhibited the presence of numerous vegetations .
the bioprosthetic mitral and tricuspid valves were not affected by the degenerative process . on the 12th day after the reimplantation of the bioprosthetic aortic valve ,
the patient was discharged from the hospital in good general condition . | Introduction
Case report
Discussion
Disclosure |
this article investigates the commonly held belief that patients seen in hospital outpatient departments ( opds ) are sicker than those seen in private practice .
this assumed case - mix difference is said to be one of the reasons opd per - visit costs are higher than costs in private practice .
it is generally agreed that stand - by capacity and other costs peculiar to the opd , such as social workers , overhead attributed from the inpatient portion of the hospital , and research and education costs , play a part in the opd 's greatly increased cost structure as well .
these latter variables , however , are beyond the scope of this article . our task here is to examine in detail the case - mix component to see how much sicker if at all opd patients are .
our major finding is that at most only relatively minor case - mix differences exist between the two settings . using a descriptive method we find slight differences and indications that there may actually be no differences . using a newly developed interactive model we find relatively minor case - mix differences , with the opd patients from 5 to 15 percent sicker than their counter - parts in private practice .
it should be emphasized that these differences apply to all opd visits combined , including those in small or non - teaching hospitals .
preliminary data collected from major teaching opds indicate that this subset of patients may be considerably sicker . for both analyses
, we have utilized a large scale data set developed by robert mendenhall at the university of southern california .
this data set has been described in detail elsewhere ( mendenhall , 1978 a and b ) and has been examined for reliability and validity ( perrin et al . , 1978 ) .
previous analyses have been limited mostly to the amount of primary care provided by internal medicine subspecialties ( aiken , 1981 ; girard , 1979 ) .
this paper represents a first attempt to apply this data set to the issue of the relative complexity of patients seen in different settings .
this data set is the only national information currently available which compares patients in private practice with those seen in the hospital outpatient department .
it is not possible to separate opds of different types for separate analysis . a subsequent study which uses the opd as the sampling unit could overcome this limitation .
despite the largely contrary anecdotal evidence in the literature claiming that there are large case - mix differences between patients seen in hospital opds and by physicians in private practice , our previous literature review revealed several data bases which tend to support our findings . using 1970 - 71 social survey data in washington , d.c . , backed up by a second survey of physicians in private practice , prepaid group practices , and hospital opds and emergency rooms , investigators were unable to demonstrate that hospital ambulatory patients were any sicker than private patients ( dutton , 1979 ) .
measures used in the dutton study were a chronic illness index for children and , for adults , the percent reporting a medical problem .
this finding of no major differences in the sickness level of the groups is all the more striking since emergency room visits are included .
dutton found striking socio - economic differences between opd and private practice patients , without using race as a variable . compared to private practice patients ,
these differences were : nearly twice as many patients of the opd came from families headed by women ( 43 percent versus 28 percent ) .
more than twice as many families of the opd patients were below the poverty level ( 33 percent versus 14 percent ) .
nearly three times as many families of the opd patients received public assistance ( 36 percent versus 13 percent ) .
family income in the opd patient group was considerably less than in the private practice patient group ( $ 6,970 versus $ 11,000 ) .
satisfied with most of their patients while 43 percent of the physicians in private practice were .
dutton 's findings of little case - mix difference using different measures provides important corroboration for the usc - mendenhall data while at the same time indicating the importance of socio - economic factors in differentiating patients seen in the outpatient department from those seen in private practice . whether or not these socio - economic differences contribute to increased resource use in the opd
all results reported in this paper are from a data set developed by the university of southern california from 1977 to 1979 . this was a cross - sectional survey of physicians in 24 specialties , using a stratified weighted sample from ama tapes which makes each specialty representative of the u.s .
physicians were asked to rank each patient seen for a three - day period on a five - point scale for complexity , urgency , and severity . they also supplied primary and secondary diagnoses , lab tests , x - rays , and procedures ordered and performed , and minutes spent with patients .
this article examines case - mix difference for four specialties : general practice , family practice , internal medicine and pediatrics .
obstetricians - gynecologists were also analyzed but were found to be diagnostically non - comparable , although no severity differences were found between settings . emergency room patients are omitted from our analysis .
table 1 indicates for the four specialties the number of physicians responding and the number of patient records analyzed .
physicians were mailed a questionnaire and log diary and were asked to fill out data on each patient as that patient was seen .
since three consecutive days were chosen for the patient data collection , it is possible that some patients who had return visits after a brief interval are in the sample more than once . strictly speaking
thus the 834 outpatients seen by the 18 residents in family practice represent groups of about 50 patients seen by the same doctor ; the 18 residents , however , almost certainly represent 18 separate opds .
extensive reliability studies have determined that most of the elements are reliable using a test - retest method ( perrin , et al . , 1978 ) .
some elements dealing with another portion of the questionnaire were deemed unreliable , particularly number of personnel associated with the physician . the only item connected with the analysis in this article which was found unreliable by perrin et al . was the distinction between general practitioner and family practitioner .
physicians had a tendency to report themselves as either a gp or fp to the american medical association , to reverse this designation when filling out the questionnaire , and to reverse it again when replication was requested by perrin et al .
this does not appear to pose a problem for our analysis , since the findings for the gp and fp categories are practically indistinguishable .
while it is difficult to compare the data as a whole to outside sources , validity is indicated by comparison of the most common diagnoses of private practitioners with those obtained by the national ambulatory care survey ( nchs , december , 1981 ) .
internal validity is discussed in subsequent sections of this article , specifically in the issue of severity , urgency , and complexity ratings for diagnoses considered of varying severity .
the major finding to emerge is that patients for all four types of specialties seen in the opd appear to be very little , if at all , sicker than those seen by specialists in private practice .
table 2 shows the comparisons for physician - rated measures for complexity , severity , and urgency . using the complexity measure , family practice physicians and residents in opds did rate their patients as slightly more complex . using the severity coding ,
hospital - based physicians tended to rank their patients as slightly sicker than private practitioners .
for the overwhelming number of patients seen by physicians , the index by specialty between the two settings is the same .
it is consistent with face validity that pediatricians who see a large number of well children for preventive care rate their patients as less severely ill than adult patients .
the supposition that severely ill children predominate in the hospital clinics and outpatient departments is not borne out , however . also consistent with external validity
is the finding that complexity increases somewhat as we move from the true generalists ( general practice and family practice ) to the primary care specialists ( pediatricians and internists ) .
complexity and urgency were scored on a five - point scale and severity on a seven - point scale recoded to four points .
only whole numbers could be used , so that while most scores hover around 2.5 , they are composed of large numbers of patients scored 2 and 3 .
the standard errors for this table are therefore quite large even though the distributions are normal .
that is , physicians may distribute their patient load relatively equally around the same mid - point regardless of how seriously ill their patients are . to test this hypothesis , essential hypertension and respiratory infection were compared for each practice setting for each appropriate specialty .
as can be seen in table 3 there appears to be general agreement that hypertensive patients are about equally sick , regardless of where they are seen or by whom .
this indicates that physicians are indeed calibrating their scales in a similar way regardless of setting .
if anything , patients with hypertension are considered slightly more ill by physicians in opds .
the data for upper respiratory infection are similar to those for hypertension , with all physicians judging upper respiratory infections as about as complex and urgent as hypertension but somewhat less severe .
another measure of case - mix is available comparing the illness level of patients in the opd with those in private practice percent of patients with a secondary diagnosis .
this lack of consistency by site is true for essential hypertension and upper respiratory problems as well . for hypertension , between a third and a half of all patients
have a secondary diagnosis compared with about 10 to 20 percent for upper respiratory infection . taken together , the results indicate that pediatrics is the only specialty with a noticeable difference , and that difference is relatively minor .
this item can be looked at as a measure of the relative efficiency of the various settings if it is conceded that case - mix is indeed similar and that efficiency includes such items as teaching .
table 5 shows consistent differences in time spent with patients even when controlling for diagnosis . in all but one case , the time spent by residents was considerably longer than the time spent by salaried staff physicians in the opd
this may illustrate the importance of a learning function for residents . with few exceptions , salaried staff physicians in opds spent more time than physicians in private practice .
this may illustrate problems in scheduling patient load , either because of no - shows or because physicians may have less incentive to schedule rapidly .
data on x - rays and laboratory tests exist only in a crude state in this data set and will not be reported in detail . in general ,
when diagnosis was controlled for , residents tended to order the most tests and physicians in private practice the fewest .
this difference reaches its peak in pediatrics , where residents order nearly twice as many x - rays and tests as physicians in private practice .
we realize that the findings of this paper are controversial and of great policy importance .
some of the controversy already generated has come from practitioners in the field , especially mds , whose experience has been of a much sicker case mix in their hospital outpatient practice than in their private practice . in going against commonly accepted tenets in any field ,
these caveats and our responses to them are as follows : caveat : emergency rooms , where the sickest opd patients were seen , were not included .
studies in the past have included them , usually by fiat , since with most social survey methodologies it is impossible to omit them .
there is no question that the 25 percent of their patient practice seen by hospital - based physicians in the emergency room as well as the 10 percent seen by private physicians are much sicker .
it should be noted here that while excluding the emergency room weakens a hospital 's claim for additional costs incurred for standby capacity , it by no means eliminates the claim .
long waits , no - shows for appointments , and disrupted treatment schedules , longer hours and unusual staffing requirements are much more common in hospital outpatient departments than in private practice .
caveat : the clinics represented a broad mix of hospital opds , and were not limited to teaching hospitals or to big cities , where the sickest opd patients are seen .
response : all outpatient departments should be included . anecdotal evidence from the larger , more complicated ones does not cancel a case - mix which is not as sick in the smaller , less complicated opds .
data from the american hospital association indicates that , even when emergency room visits are excluded , almost half ( 44.0 percent ) of outpatient department visits take place in hospitals of under 300 beds and 11.4 percent take place in hospitals of less than 100 beds ( aha , 1982 ) .
while some of these visits involve only tests ( the so - called private referred portion ) , visits involving only tests are common to large hospitals as well .
student health services , which make up a large proportion of small , non - teaching hospital visits , are also excluded . in short , while the anecdotal evidence supports case - mix being more serious in large urban hospitals , there is little or no evidence about smaller suburban hospitals and hmos connected with hospitals
. if both our findings from usc - mendenhall and the anecdotal evidence about case - mix in large urban hospitals are correct , then patients in hmos and smaller hospital opds are actually less sick than patients in private practice as a whole .
response : it is a shortcoming of any secondary analysis that variables important to the point being examined are not included in the original study design .
it should be noted , though , that the case - mix differences we attempted to substantiate were physical , rather than socio - economic in nature .
socio - economic variables become important only if a primarily medical explanation of case - mix differences can not be demonstrated and only if we assume that socio - economic problems make a patient more difficult to treat .
dutton 's finding that outpatient department physicians do not like to treat these patients points in this direction , but the particular reasons leading to a more difficult to treat designation have not been isolated .
if socio - economic problems are a factor in case - mix differences , we should find patients with the same severity of diagnosis using more physician time and hospital resources when they have these problems . to this end
we are in the process of designing a small supplementary project in the boston area which will replicate the usc - mendenhall study design and will add to it the following variables : public assistance status occupation of head of household native language ( degree of difficulty speaking english ) psychological problems socio - economic problems environmental problems a specific question about socio - economic and environmental problems was actually asked by the usc - mendenhall study , but only the primary reason for seeking care could be coded .
only 2 percent of hospital outpatients seen by internists sought care for a socio - economic , psychological , or environmental problem as their primary problem .
the percent would have been far higher if a secondary reason for the presenting problem could have been expressed as well .
caveat : the physician may have internally calibrated the mid - point for rating complexity , urgency , and severity in different ways depending on his experience in the setting .
a corollary of this is that , even for specific diagnoses , this internal calibration may have an effect .
for example , a hospital - based physician who sees extremely ill diabetics on an inpatient basis may rate his diabetics seen in the opd as not very ill even though they have clinical signs and symptoms far more severe than those ambulatory diabetics seen in private practice .
response : while the argument that physicians in different settings may have internally calibrated their mid - points for severity , complexity , and urgency around different scales is potentially valid , a series of special analyses we conducted does not seem to support it . specifically : four diagnoses essential hypertension , diabetes , neuroses , and upper respiratory infections were analyzed in detail .
hospital - based physicians scored all four as about as sick as patients with the same diagnosis seen in private practice .
it is possible to argue that the patients with hypertension and diabetes had more serious forms of these illnesses , yet were evaluated as no more sick than private patients because of the hospital - based physician 's greater experience with very sick inpatients with these diagnoses .
this argument can not be made , however , for the upper respiratory infections , and probably not for neuroses .
patients with these four relatively uncomplicated diagnoses account for about the same 25 percent of the practice for patients of hospital - based and private physicians , indicating that the rest of the practice distribution may also be the same .
other measures of severity such as percent of all patients with secondary diagnoses are similar across practice settings . caveat : the data apply only to general practitioners , family practitioners , internists , and pediatricians ;
the very sick and terminally ill patients of these specialists may be treated overwhelmingly in the opd .
response : while this hypothesis is appealing , it should be pointed out that the four tertiary specialties expected to have the sickest patients medical oncology , neurology , nephrology , and hematology account for only 4.4 percent of ail patients seen in the opd , hardly enough to make much overall difference . caveat : the anecdotal evidence is very strong and compelling to mds , particularly to those practitioners who actually work in large inner city opds . response : as indicated , large hospital opds account for only about half of all opd visits .
in addition , these opds , like smaller ones , are prone to several other phenomena which dilute the severity of their case - mix : patients referred to the opd to have stitches removed or other medical conditions checked and patients referred after hospitalization because they do not have a primary physician .
the anecdotal evidence probably applies , then , to a select group of patients seen in a select group of hospital outpatient departments .
these are the highly technically sophisticated opds which are sometimes assumed to be representative of all opds .
the previous findings and caveats have been based upon a descriptive and straightforward analysis of the data . in a follow - up study
, we subjected the same data to an interactional model which is in the process of development at yale ( fetter , 1980 ) .
this system of autogrouping is similar to the more established and better known diagnostic - related group ( drg ) system for inpatient services also developed at yale . using time spent with the physician as the dependent variable
, an automatic interaction detector program divides patient visits into 14 broad groups based on organ system and etiology using the primary diagnosis and then into 154 subgroups based upon nine independent variables which influence patient care time .
the separate groupings were designed to form groups with the greatest possible between - group variance and the least possible within - group variance .
that is , the patients in the final groups very closely approximate each other in terms of time spent with the physician .
the variables used are : presenting problem as defined by chief complaint had the physician seen the patient before ? was the problem acute or chronic ? was the care provided well child or adult care , prenatal care , postnatal care , post - operative care , or none of these ? had the patient been seen on referral ? was psychotherapy provided as part of the visit ?
sex and race were also available as variables , but did not account for any splits and were therefore discarded . the yale system was developed and refined on national ambulatory medical care survey ( namcs ) data . in order to use it for our analysis
the three types of care private practice , residents in the outpatient department , and salaried staff physicians in the outpatient department were run separately and then were summed with weights applied to make the total representative of the u.s .
the four specialties general practice , family practice , internal medicine , and pediatrics were treated separately , as in the previous analysis .
the splits occurring in the namcs data were frozen and assumed to be the ones which would occur naturally in the mendenhall data .
the minimum terminal group size was set at ten , rather than 25 , in order to accommodate the smaller mendenhall data set .
presenting complaint was not available in the mendenhall data set , thus reducing the number of subgroups which could be formed by about 50 .
data were trimmed to eliminate the outliers on both high and low sides of the distribution .
as table 6 indicates , this alternate , more statistically complex method produces results similar to the basic analysis reported previously .
that is to say , the patients of salaried internists are about 5 percent sicker than those of private practitioners , and the patients of residents are about 13 percent sicker .
this more sophisticated analysis thus quantifies the minor differences previously suspected . for the autogrouping analysis producing this table ,
all patients are divided into the final groups developed at yale and then are redistributed into the three practice settings under consideration .
this produces the actual mean length of encounter for each group ( 19.32 minutes for residents , for example ) compared with internists as a whole ( 15.59 ) .
the data for each type of practice are then rerun through the program and are recalculated based on the percent of visits for that type of practice in each of the final groups multiplied by the mean time it takes for all patients in the group to be seen .
the expected length of encounter was calculated by determining for each type of practice what proportion of their patients was in each of the final groups and then multiplying that proportion by the mean encounter time for the group . for residents ,
the calculation indicates that residents should be taking only 17.32 minutes to see their particular mix of patients compared to the 19.32 minutes they are actually taking .
in fact , both types of opd physicians spent significantly more time with their patients than indicated if they were adhering to the mean times established for all patients for each of the groups .
the formal relationship for the time residents take to treat their patients is shown by the ratio of actual time to expected time , which is 1.115 , indicating that residents are taking 11.5 percent longer to treat their patients than indicated by their case - mix .
more important for this paper , the expected length of encounter for residents is longer than the group average length of encounter , giving a ratio of 1.111 .
this indicates that residents in internal medicine actually have a case - mix 11 percent sicker than the average and about 13 percent sicker than internists in private practice , who have an expected length of encounter slightly less than the group average and a case - mix ratio of .983 .
this 13 percent sicker estimation is , probably coincidentally , the same one which would be arrived at if the severity rating were compared for residents ( 2.7 ) and private internists ( 2.3 ) , indicating that the more simplified data , despite its large standard error and descriptive approach , is comparable to this way of examining the data .
table 7 indicates that for seven of the eight comparisons possible , patients seen in the opd were both more complex to treat and took longer to treat , even considering their complexity , than patients in private practice . in the one aberrant case ,
the two specialty primary practices , internal medicine and pediatrics , showed greater differences , again consistent with anecdotal evidence .
the important point of this article is that a 5 to 15 percent case - mix difference overall , even if it can be substantiated for other specialties , is not very large .
it is far smaller than generally supposed and far smaller than needed to explain cost differences which range up to two or three times as high in hospital opds .
obviously , these overall case - mix differences obscure a great range within opds by size and teaching status .
it is quite possible , for example , that the most complicated large , inner city opds have a case - mix twice as medically sick as a private physician 's office while small suburban opds have the same or even a less sick case - mix .
if only 10 percent of opd visits occur in large urban teaching hospitals , then the 5 to 15 percent overall finding might be accurate . recognizing these differences among opds
suggests that we do not treat all opds in a similar manner in any reimbursement reform . further research on case - mix differences in urban teaching and community hospitals would allow us to classify institutions according to case - mix for reimbursement purposes . a pilot study which addresses this subject is now underway as part of our overall ambulatory care project . | the belief that patients seen in hospital outpatient departments are sicker than those patients seen by private practice physicians is examined in this article .
a large scale data set developed by robert mendenhall at the university of southern california and modeled on the national ambulatory medical care survey ( namcs ) is used for secondary analysis .
differences in case - mix complexity were found to be slight , using two separate techniques . | Introduction
Previous Findings on Ambulatory Case-Mix
Methodology
Descriptive Findings
Caveats
Ambulatory Patient Groups Formed by Autogrouping
Conclusions |
diabetes affects almost every tissue in the body and causes significant organ dysfunction that results in diabetes - related morbidity and mortality .
coronary artery disease is the leading cause for the increased cardiovascular morbidity and mortality in diabetes , and atherosclerosis of the coronary vessels is its initial pathogenetic mechanism .
coronary artery disease and hypertension can account for most of the myocardial abnormalities that occur in diabetes .
however , postmortem , experimental , and observational studies also provide evidence for a specific cardiomyopathy in diabetes , which may contribute to myocardial dysfunction in the absence of coronary artery atheroma .
this is also sustained by the fact that patients with diabetes , independently of the severity of coronary artery disease , have increased risk of heart failure in comparison with subjects without diabetes . in general diabetes
affects the heart in 3 ways : ( 1 ) coronary artery disease due to accelerated atherosclerosis ; ( 2 ) cardiac autonomic neuropathy ; and ( 3 ) diabetic cardiomyopathy ( dcp ) .
although there is high awareness among clinicians about the first two disease entities , diabetic cardiomyopathy is poorly recognized by most physicians .
dcp , first defined by rubler in 1972 , is characterized by the myocardial dysfunction in the absence of coronary artery disease , hypertension , or valvular heart disease .
it is associated with both type 1 and type 2 diabetes mellitus ( t1 dm and t2 dm ) . as in the case that we report here , dcp may be subclinical for a long time , before the appearance of clinical symptoms or signs .
we present the case of a 39-year - old man that was admitted in our clinic due to a stroke . according to its past medical history ,
the patient was diagnosed in the primary care setting with elevated blood glucose levels , about 8 months before the stroke .
the rest of his past medical history was free of any other diseases . according to the information we have , at that time the patient was given lifestyle advice on dietary and physical activity , by the primary care physician .
he was also strongly encouraged to lose weight as the patient had body mass index of 34 ( bmi = 34 ) .
unfortunately , there is no record of the specific glucose values identified at that time .
we do nt know also , the time period that this patient had deranged blood sugar levels , since , according to what he reported , he did nt have any medical blood tests done over the past 10 years . during his admission to the hospital , the patient was afebrile , well - oriented in time and space , with paresis of the right upper and lower limbs .
his arterial blood pressure was 140/90 mmhg , electrocardiogram ( ecg ) with sr and his pulse was regular .
his bmi at that time was the same with the bmi originaly calculated about 68 months ago .
his medical history was free of any other diseases , apart from the hyperglycemia mentioned earlier .
the computed tomography ( ct ) brain scan revealed an extensive ischemic infarct of the left parietal lobe in the supply zone of the left middle cerebral artery .
his biochemical tests results were : white blood cell count ( wbc ) = 9000/l , hematocrit value ( hct ) = 44,8% , serum glutamic oxaloacetic transaminase ( sgot ) = 33u / l , serum glutamic - pyruvic transaminase ( sgpt ) = 21u / l , -gt : 53u / l , blood glucose level : 187 mg / dl . during the 2 day of hospitalization we performed an ecg .
we found an enlarged left atrium , an enlarged hypokinetic left ventricle with an ejection fraction of 30% and a large thrombus in it as well as an enlarged right ventricle .
warfarin was added to his drug therapy , that already included antiplatelet agents . during his stay in the hospital ,
the diagnosis of dm for the first time was made and his glycosylated hemoglobin level ( hba1c ) was 10.42% .
the patient followed a stable clinical course with a progressive improvement in his motor functions .
he is now on motor physiotherapy and a regular medical follow - up for his diabetes , his heart failure and the stroke .
dcp is defined as the cardiovascular damage present in diabetes patients , which is characterized by myocardial dilatation and hypertrophy , as well as a decrease in the systolic and diastolic function of the left ventricle , and its presence is independent of the coexistence of ischemic heart disease or hypertension . as in the case of our patient , dcp may be subclinical for a long time , before the appearance of clinical symptoms or signs .
various proposed mechanisms include metabolic disturbances , insulin resistance , microvascular disease , alterations in the renin - angiotensin system ( ras ) , cardiac autonomic dysfunction and myocardial fibrosis .
chronic hyperglycemia is thought to play a central role in the development of dcp , although multiple complex mechanisms and an interplay of many molecular and metabolic events within the myocardium and plasma contribute to the pathogenesis .
the main metabolic abnormalities in diabetes are hyperglycemia , hyperlipidemia and inflammation , all of which stimulate generation of reactive oxygen or nitrogen species that cause most of the diabetic complications , including diabetic nephropathy and dcp .
several adaptive responses caused by these metabolic abnormalities finally result in cardiac dysfunction and heart failure .
however , proper treatment of diabetes and its metabolic abnormalities in the primary care setting reduces the rates of dcp , myocardial infarction , and associated cardiovascular death .
since dcp is now known to have a high prevalence in the asymptomatic diabetic patient , screening for its presence at the earliest stage of development would be appropriate in order to prevent the progression to congestive heart failure and further significant complications . as this high - risk diabetic population is constanly rising , increasing the awareness of physicians for the serious metabolic complications of diabetes , especially in the primary care setting , will help in taking appropriate and early action towards the prevention of full - blown disease and decreasing disability and mortality . | diabetic cardiomyopathy ( dcp ) is defined as the cardiovascular damage present in diabetes patients , which is characterized by myocardial dilatation and hypertrophy , as well as a decrease in the systolic and diastolic function of the left ventricle , and its presence is independent of the coexistence of ischemic heart disease or hypertension . as in the case of the patient that we present here , dcp may be subclinical for a long time , before the appearance of serious clinical symptoms , signs and complications .
dcp is poorly recognized by most physicians .
currently , there is no specific treatment for this pathologic entity .
however , proper treatment of diabetes and its metabolic abnormalities in the primary care setting reduces the rates of this serious metabolic complication of diabetes . as this high - risk diabetic population is constantly rising , increasing the awareness of physicians for the serious metabolic complications of diabetes , especially in the primary care setting , will help in taking appropriate and early action towards the prevention of full -blown disease and decreasing disability and mortality . | Introduction
Case Report
Discussion |
of thyroid nodules examined by the use of an us - fnab at our institution from may 2007 to april 2008 , small thyroid nodules less than 5 mm in the maximum diameter were included in the study .
high - resolution ultrasonography ( us ) was performed with either an hdi 5000 ( phillips medical systems , bothell , wa ) or iu 22 unit ( phillips medical systems ) using a 12 - 15 mhz linear probe for guidance during the us - fnab procedure .
we obtained informed written consent from all patients before performing us - fnabs and our institutional review board approved this study .
malignant sonographic findings included microcalcifications , marked hypoechogenicity , an irregular margin and a taller - than - wide shape . the inclusion criteria for an us - fnab of small thyroid nodules less than 5 mm in the maximum diameter were the following .
the criteria included a thyroid nodule with a likelihood of malignancy as depicted on diagnostic us ( n = 149 ) , a small solid thyroid nodule in the contralateral lobe in a patient with a known thyroid malignancy ( n = 41 ) or the presence of a small solid nodule in patients at a high risk for having a thyroid malignancy ( n = 11 ) .
risk factors for a thyroid malignancy included cervical lymphadenopathy with a positive cytology for a thyroid malignancy ( n = 3 ) , prior head and neck irradiation ( n = 1 ) and a newly developed nodule in the opposite or remaining thyroid lobe after surgery for a known thyroid malignancy ( n = 7 ) .
a repeat us - fnab was performed after the first biopsy whenever the cytology result was ' inadequate ' or when the cytology result did not match the sonographic impression .
a primary malignancy was defined as the largest malignant nodule in the thyroid and a satellite malignancy was defined as a smaller - sized , intraglandular metastatic lesion in the ipsilateral or contralateral lobe .
an experienced radiologist ( seven years experience for performing an us - fnab of thyroid nodules ) performed an us - fnab with the use of a 23-gauge needle attached to a 10-ml plastic syringe .
when the needle tip reached the target nodule , the needle was observed as a small echogenic spot within the nodule on the us monitor ( fig .
after the needle tip was placed in the appropriate area of the target nodule , sampling was commenced using the ' mixed sampling technique . '
this technique is an individual sampling technique where the needle is moved up and down for a few seconds only by movement of the wrist of the operator and without initial aspiration under us guidance . in all cases ,
after a sample was obtained , the specimen was mounted immediately onto a glass slide .
we obtained four to six slides by performing two or three smears from each nodule .
the cytological diagnosis was classified as ' benign , ' ' suspicious for a malignancy , ' ' malignant , ' ' indeterminate ' or ' inadequate . ' ' benign ' included a normal thyroid , nodular hyperplasia , lymphocytic thyroiditis or other benign conditions . '
suspicious for a malignancy ' included specimens showing clusters of atypical follicular cells with some nuclear grooves . ' malignant ' included specimens showing clusters of abnormal follicular cells with discrete nuclear grooves , intranuclear pseudoinclusions or a papillary projection . '
indeterminate ' referred to specimens showing follicular cell proliferative lesions that were indeterminate for a differential diagnosis of nodular hyperplasia , follicular neoplasm or a follicular variant of a ptc . in this study ,
an adequate specimen was defined as having more than six clusters of follicular thyroid cells and showing the presence of an identifiable colloid in each preparation ( 5 ) ; conversely , a specimen with less than six clusters of follicular thyroid cells in each slide was considered as ' inadequate . ' for each primary small malignancy , capsular invasion with tumor invasion into the thyroid capsule , a perithyroidal lymph node metastasis with tumor invasion into lymph nodes in the central neck , multifocality with an intraglandular metastasis in the ipsilateral lobe and bilaterality with an intraglandular metastatic lesion in the contralateral lobe were examined .
of 201 small nodules in 180 patients , 118 small nodules in 110 patients were not removed surgically and these nodules were excluded from the determination of diagnostic indices . of 83 nodules in 75 patients , lesions with ' benign ' cytology were regarded as having a negative cytology classification for a malignancy and lesions with ' suspicious for a malignancy ' or ' malignant ' cytology were regarded as having as a positive cytology classification for a malignancy .
lesions with an ' indeterminate ' ( n = 7 ) and ' inadequate ' ( n = 14 ) cytology as determined after the first us - fnab were excluded in the determination of the sensitivity , specificity , predictive value , accuracy , false positive rate and false negative rate . ultimately , of 62 nodules in 58 patients , the sensitivity , specificity , positive predictive value , negative predictive value , accuracy , false positive rate and false negative rate for the first us - fnab were examined .
of thyroid nodules examined by the use of an us - fnab at our institution from may 2007 to april 2008 , small thyroid nodules less than 5 mm in the maximum diameter were included in the study .
high - resolution ultrasonography ( us ) was performed with either an hdi 5000 ( phillips medical systems , bothell , wa ) or iu 22 unit ( phillips medical systems ) using a 12 - 15 mhz linear probe for guidance during the us - fnab procedure .
we obtained informed written consent from all patients before performing us - fnabs and our institutional review board approved this study .
malignant sonographic findings included microcalcifications , marked hypoechogenicity , an irregular margin and a taller - than - wide shape . the inclusion criteria for an us - fnab of small thyroid nodules less than 5 mm in the maximum diameter were the following .
the criteria included a thyroid nodule with a likelihood of malignancy as depicted on diagnostic us ( n = 149 ) , a small solid thyroid nodule in the contralateral lobe in a patient with a known thyroid malignancy ( n = 41 ) or the presence of a small solid nodule in patients at a high risk for having a thyroid malignancy ( n = 11 ) .
risk factors for a thyroid malignancy included cervical lymphadenopathy with a positive cytology for a thyroid malignancy ( n = 3 ) , prior head and neck irradiation ( n = 1 ) and a newly developed nodule in the opposite or remaining thyroid lobe after surgery for a known thyroid malignancy ( n = 7 ) .
a repeat us - fnab was performed after the first biopsy whenever the cytology result was ' inadequate ' or when the cytology result did not match the sonographic impression .
a primary malignancy was defined as the largest malignant nodule in the thyroid and a satellite malignancy was defined as a smaller - sized , intraglandular metastatic lesion in the ipsilateral or contralateral lobe .
an experienced radiologist ( seven years experience for performing an us - fnab of thyroid nodules ) performed an us - fnab with the use of a 23-gauge needle attached to a 10-ml plastic syringe .
procedures were performed under us guidance using a perpendicular puncture and without local anesthesia . when the needle tip reached the target nodule ,
the needle was observed as a small echogenic spot within the nodule on the us monitor ( fig .
after the needle tip was placed in the appropriate area of the target nodule , sampling was commenced using the ' mixed sampling technique . '
this technique is an individual sampling technique where the needle is moved up and down for a few seconds only by movement of the wrist of the operator and without initial aspiration under us guidance . in all cases ,
after a sample was obtained , the specimen was mounted immediately onto a glass slide .
we obtained four to six slides by performing two or three smears from each nodule .
the cytological diagnosis was classified as ' benign , ' ' suspicious for a malignancy , ' ' malignant , ' ' indeterminate ' or ' inadequate . ' ' benign ' included a normal thyroid , nodular hyperplasia , lymphocytic thyroiditis or other benign conditions . '
suspicious for a malignancy ' included specimens showing clusters of atypical follicular cells with some nuclear grooves . '
malignant ' included specimens showing clusters of abnormal follicular cells with discrete nuclear grooves , intranuclear pseudoinclusions or a papillary projection . '
indeterminate ' referred to specimens showing follicular cell proliferative lesions that were indeterminate for a differential diagnosis of nodular hyperplasia , follicular neoplasm or a follicular variant of a ptc . in this study ,
an adequate specimen was defined as having more than six clusters of follicular thyroid cells and showing the presence of an identifiable colloid in each preparation ( 5 ) ; conversely , a specimen with less than six clusters of follicular thyroid cells in each slide was considered as ' inadequate . ' for each primary small malignancy , capsular invasion with tumor invasion into the thyroid capsule , a perithyroidal lymph node metastasis with tumor invasion into lymph nodes in the central neck , multifocality with an intraglandular metastasis in the ipsilateral lobe and bilaterality with an intraglandular metastatic lesion in the contralateral lobe were examined .
of 201 small nodules in 180 patients , 118 small nodules in 110 patients were not removed surgically and these nodules were excluded from the determination of diagnostic indices . of 83 nodules in 75 patients , lesions with ' benign ' cytology were regarded as having a negative cytology classification for a malignancy and lesions with ' suspicious for a malignancy ' or ' malignant ' cytology were regarded as having as a positive cytology classification for a malignancy .
lesions with an ' indeterminate ' ( n = 7 ) and ' inadequate ' ( n = 14 ) cytology as determined after the first us - fnab were excluded in the determination of the sensitivity , specificity , predictive value , accuracy , false positive rate and false negative rate . ultimately , of 62 nodules in 58 patients , the sensitivity , specificity , positive predictive value , negative predictive value , accuracy , false positive rate and false negative rate for the first us - fnab were examined .
of 201 small thyroid nodules ( maximum diameter range , 1.0 to 5.0 mm ; mean diameter , 3.4 mm ) in 180 patients ( female : male = 152:28 ; mean age , 48.0 years ; age range , 18 to 75 years ) , the incidence of adequate specimens was 162 ( 81% ) after the first us - fnab . of 201 biopsies ,
the cytology was classified as ' benign ' for 107 lesions ( 53% ) , ' suspicious for a malignancy ' for 22 lesions ( 11% ) , ' malignant ' for 16 lesions ( 8% ) , ' indeterminate ' for 17 lesions ( 9% ) and ' inadequate ' for 39 lesions ( 19% ) ( table 1 ) .
of the 75 patients , 41 patients underwent a total thyroidectomy , 21 patients underwent a lobectomy , 12 patients underwent a subtotal thyroidectomy and one patient underwent an isthmectomy .
fifty small malignant thyroid nodules in 49 patients ( female : male = 44:5 ; mean age , 44.2 years ; mean maximum diameter , 3.3 mm ; maximum diameter range , 1.0 to 5.0 mm ) were identified as ptmcs based on a pathological examination and were classified as 23 ( 46% ) primary and 27 ( 54% ) satellite ( smaller - sized ptcs that are distinguished from the largest ptc ) malignancy .
thirty - three small thyroid nodules with ' benign ' cytology in 33 patients showed 28 nodular hyperplasias and five ptmcs on the pathology after thyroid surgery . of the 23 pathologically confirmed primary malignancies , after the first us - fnab two lesions ( 9% ) were classified as ' benign , ' eight lesions ( 35% ) were classified as ' suspicious for a malignancy , ' 10 lesions ( 44% ) were classified as ' malignant , ' two lesions ( 9% ) were classified as ' indeterminate ' and one lesion ( 4% ) was classified as ' inadequate ' based on the cytology .
of five ( two benign , tow indeterminate , and one inadequate ) pathologically confirmed primary malignant nodules with negative cytology results after the first biopsy , four lesions were classified as ' suspicious for a malignancy ' after a second biopsy and one lesion was classified as ' benign ' after a second biopsy .
although one pathologically confirmed malignant nodule demonstrated negative cytology results even after two consecutive us - fnabs , the patient underwent thyroid surgery because of the high likelihood of a malignancy as found on diagnostic us .
of the 27 pathologically confirmed satellite malignancies after the first us - fnab , three lesions ( 11% ) were classified as ' benign , ' 10 lesions ( 37% ) were classified as ' suspicious for a malignancy , ' six lesions ( 22% ) were classified as ' malignant , ' two lesions ( 7% ) were classified as ' indeterminate ' and six lesions ( 22% ) were classified as ' inadequate ' based on the cytology .
eleven satellite malignant nodules that showed a negative cytology after the first biopsy were surgically removed without an additional biopsy as they showed a combined primary thyroid malignancy on simultaneous us - fnabs . for the first us - fnab of 62 nodules
, there were 34 ( 55% ) true positives , 0 ( 0% ) false positives , 23 ( 37% ) true negatives and five ( 8% ) false negatives . the sensitivity ( 87% ) ,
specificity ( 100% ) , positive predictive value ( 100% ) , negative predictive value ( 82% ) , accuracy ( 92% ) , false positive rate ( 0% ) and false negative rate ( 8% ) after an us - fnab for nodules smaller than 5 mm in the maximum diameter were determined ( table 2 ) .
twenty - three patients with a primary ptmc smaller than 5 mm in the maximum diameter underwent thyroid surgery .
capsular extension ( 9% , 2 of 23 ) , a perithyroidal lymph node metastasis ( 30% , 7 of 23 ) , multifocality ( 9% , 2 of 23 ) and bilaterality ( 4% , 1 of 23 ) were identified .
thyroid surgery for these patients included a total thyroidectomy ( n = 7 ) , a subtotal thyroidectomy ( n = 4 ) and a lobectomy ( n = 12 ) .
twenty - one patients ( 12% , 21 of 180 ) experienced mild pain that persisted for several minutes during or after the biopsy , but the pain subsided spontaneously without medication .
us - fnab is the least invasive , most simple and most accurate method to evaluate a thyroid nodular lesion .
however , the size criterion is still controversial for thyroid nodules evaluated on an us - fnab .
the american thyroid association ( ata ) guidelines suggest that , in general , only nodules larger than 10 mm in diameter should be evaluated as the nodules have the potential to represent a clinically significant cancer ( 6 ) . occasionally , there may also be nodules less than 10 mm in the maximum diameter that require evaluation due to suspicious us findings and/or a patient history of thyroid cancer ( 7 ) .
the society of radiologists in ultrasound consensus statement suggests performing us - fnab on thyroid nodules 10 mm or greater in the maximum diameter when the nodules exhibit microcalcifications or other us features associated with thyroid cancer , as an early diagnosis of thyroid cancer does not guarantee a decrease in recurrence or the mortality rate ( 8) .
burman ( 9 ) also mentioned that irrespective of which suggested minimum measurement or guideline is used , the most critical requirement is to monitor these patients with periodic clinical and us examinations .
however , many authorities suggest that it is important to diagnose thyroid cancer at an early stage to reduce the risk of recurrence or mortality ( 10 , 11 ) .
there are a few reports that have evaluated the use of an us - fnab of thyroid nodules less than 5 mm in the maximum diameter .
( 4 ) found a 64% adequate sampling rate for 25 small thyroid nodules less than 5 mm in diameter , but the study did not indicate whether the maximum or minimum diameter of the nodules had been measured .
( 12 ) retrospectively analyzed 243 patients with pathologically confirmed ptmcs , but the investigators did not comment on the adequacy or efficacy of us - fnabs for the thyroid nodules . in this study , the sensitivity and negative predictive value of performing an us - fnab for the diagnosis of small thyroid nodules equal to or less than 5 mm in the maximum diameter were relatively low as compared with values obtained for other diagnostic indices ; however , the specificity and positive predictive value were high . to the best of our knowledge ,
these are the highest rates among previously published reports , although the sensitivity and negative predictive value of an us - fnab for small - diameter thyroid nodules were 87% and 82% , respectively .
however , five ' benign ' nodules identified after the first us - fnab were later confirmed as ptmcs after thyroid surgery and were determined as false negative cytologies ( 8% ) , but false positive cytology was not detected .
this false negative rate for an us - fnab of small thyroid nodules less than 5 mm in the maximum diameter is relatively high ; however , this rate may be subject to change if non - operated nodules are surgically confirmed .
the size of the five ptmcs with false negative cytology as determined after the first us - fnab was less than 3 mm in the maximum diameter as determined on diagnostic us .
therefore , we assumed that the relatively high false negative rate was due to sampling of primarily normal tissue rather than the ptmc during the ' to - and - fro ' cell acquisition . in the present study , the incidence of capsular invasion of a ptmc smaller than 5 mm in the maximum diameter was 9% ( 2 of 23 ) , which was relatively low as compared to the studies of roti et al .
( 12 ) reported that two patients ( 2% , 2 of 86 ) with thyroid cancers less than 5 mm had regional lymph node metastases , but in the present study , 23 primary ptmcs equal to or less than 5 mm in the maximum diameter showed a perithyroidal lymph node metastasis ( 30% , 7 of 23 ) .
we presume that the high rate of a perithyroidal lymph node metastasis in the present study might be associated with the relatively high incidence of the subcapsular location of ptmcs . regarding the multifocality of a ptmc , roti et al .
( 13 ) reported that 32% ( 78 of 243 ) and 31% ( 63 of 203 ) of patients were indentified with multifocal lesions , respectively , but the investigators in both studies did not describe ptmcs less than 5 mm in the maximum diameter .
( 12 ) found that the incidence rate of bilaterality of a ptmc was 19% ( 45 of 243 ) , but the investigators did not separately mention the bilaterality rate of a ptmc less than 5 mm in the maximum diameter . in the present study , multifocality ( 9% , 2 of 23 ) and bilaterality ( 4% , 1 of 23 ) were identified .
to the best of our knowledge , no large - scale study has examined the incidence of regional lymph node metastases , multifocality and bilaterality of a ptmc less than 5 mm in the maximum diameter . in our study , significant complications related to performing an us - fnab were not detected in any of the patients .
several factors have been associated with a low incidence of complications with the use of the us - fnab procedure , including one sampling with one puncture , shortening of the procedure time , accurate targeting and watching the needle tip during the biopsy . in particular ,
continuous watching of the needle tip on the us monitor during the entire procedure is important in order to prevent arterial injury to the carotid or vertebral artery .
second , thyroid surgery for the thyroid malignancies included total , subtotal and a lobectomy and regional lymph node resection was not performed for all of the ptcs .
third , we did not consider if the presence of underlying diffuse thyroid disease influenced the outcome of the us - fnabs .
( 14 ) have suggested that in cases of underlying diffuse thyroid disease , such as thyroiditis or diffuse goiter , the outcome of an us - fnab for small thyroid nodules might have resulted in an negative influence .
finally , our technique of performing an us - fnab of thyroid nodules is not a standard technique .
the importance of early diagnosis of thyroid cancer in patients at low risk remains uncertain as thyroid cancers are typically slow growing and are associated with a low morbidity and mortality ( 6 , 8 , 15 ) .
however , we believe that early diagnosis of thyroid malignancies less than 5 mm in the maximum diameter by the use of diagnostic us and an us - fnab is helpful to improve patient morbidity rates for poorly differentiated thyroid malignancies . in addition , in our study , the diagnosis of 27 satellite small ptmcs after an us - fnab influenced the decision regarding the type of thyroid surgery ( e.g. , a lobectomy or total thyroidectomy ) . in conclusion ,
an us - fnab of thyroid nodules smaller than 5 mm in the maximum diameter is an effective diagnostic method if performed by an experienced radiologist . in the present study ,
primary ptmcs smaller than 5 mm in the maximum diameter had a low incidence of capsular invasion , multifocality and bilaterality , but a relatively high incidence of perithyroidal lymph node metastases . | objectivethe aim of this study was to determine the efficacy of the use of an ultrasound - guided fine - needle aspiration biopsy ( us - fnab ) to diagnose thyroid nodules smaller than 5 mm in the maximum diameter and to evaluate pathological findings of small thyroid malignancies.materials and methodsfrom may 2007 to april 2008 , we evaluated the findings of us - fnabs of small thyroid nodules less than 5 mm in the maximum diameter .
the cytopathological findings were retrospectively reviewed and the diagnostic performance of the use of an us - fnab was examined in all patients.resultsof 201 small thyroid nodules in 180 patients , there were 162 adequate specimens ( 81% ) . among 180 patients ,
75 patients underwent thyroid surgery and 50 malignant and 33 benign nodules were identified based on a pathological examination .
all small malignant thyroid nodules were identified as papillary thyroid microcarcinomas ( ptmcs ) .
there were 34 ( 55% ) true positive , 0 ( 0% ) false positive , 23 ( 37% ) true negative and five ( 8% ) false negative results for malignancy after performing a first us - fnab in 62 surgically confirmed nodules . the sensitivity ( 87% ) ,
specificity ( 100% ) , positive predictive value ( 100% ) , negative predictive value ( 82% ) , accuracy ( 92% ) , false positive rate ( 0% ) and false negative rate ( 8% ) for an us - fnab were determined . in 23 patients with a primary ptmc , capsular invasion ( 9% , 2 of 23 ) , a perithyroidal lymph node metastasis ( 30% , 7 of 23 ) , the rate of multifocality ( 9% , 2 of 23 ) and bilaterality ( 4% , 1 of 23 ) were also determined.conclusionan us
- fnab of thyroid nodules smaller than 5 mm in the maximum diameter is an effective diagnostic procedure . | MATERIALS AND METHODS
Patient Selection
Fine Needle Aspiration Biopsy Technique
Cytopathological Examinations
Diagnostic Performance
RESULTS
DISCUSSION |
there are twenty million blind people in india ; eighty percent of this blindness is due to causes which are preventable .
cataract in india is the most important cause of preventable blindness accounting to 63.7 percent .
lens - induced glaucoma ( lig ) was first described in the year 1900 by gifford and von reuss independent of each other .
while the former described it as a glaucoma associated with hypermature cataract , the latter described it as a glaucoma associated with spontaneous absorption of lens substance through intact lens capsule .
subsequently , various workers [ 46 ] described such types of cases under different names like lig , lens - induced uveitis and glaucoma , phacotoxic glaucoma , phacogenic glaucoma , and finally phacolytic glaucoma .
these terms including the more popular term phacolytic glaucoma have been discarded for various reasons and convenience in favour of the term lig . at present , lig is a clinical condition characterised by ( i ) a violent secondary glaucoma ( resembling acute angle closure glaucoma ) in one eye with senile mature cataract , hyper mature senile cataract ( rarely immature senile cataract ) yet with an open angle , ( ii ) normal intraocular pressure and open angle in other eye , and ( iii ) a prompt relief of symptoms and restoration of vision after cataract extraction in the effected eye .
late reporting for treatment of cataract leading to serious complications like lig remains one of the most important cause of irreversible loss of vision , especially so in the rural population .
this preventable and curable condition , though rare in developed countries , is unfortunately still prevalent in india .
the study is undertaken to study the profile of lens - induced glaucoma leading to blindness in rural population in western maharashtra , india .
the records of cataract cases who were diagnosed as lig and operated at our hospital from 2005 to 2011 were analyzed .
fifty cases included in this study were operated for lens - induced glaucoma between 2005 and 2011 at pravara rural hospital , loni , maharashtra , india .
all of them reported with pain , redness , and watering of acute onset in addition to gradual progressive loss of vision in affected eye as their presenting complaints and were diagnosed as lig based on the clinical findings and raised intraocular pressure .
congenital cataract , secondary cataract , traumatic cataract , complicated cataract , and known cases of glaucoma were excluded from the study .
complete ophthalmic examination with preoperative and postoperative visual acuity , iop measurement , slit lamp examination , and intraoperative and postoperative complications were recorded .
fifty cases recorded over 6 years contributed about 2.4 percent of all cases of senile cataract admitted for cataract extraction during this period .
the youngest patient was a 56-year - old female and the oldest an 81-year - old male .
cases were diagnosed as phacomorphic based on presence of pain and redness of the affected eye associated with the presence of corneal oedema , shallow anterior chamber , dilated pupil , intumescent cataractous lens , and intraocular pressure above 21 mm hg .
symptoms of pain and redness improved in the majority of cases ; however , there was no remarkable improvement in visual acuity after cataract surgery in most cases .
the time gap between onset of acute symptoms of pain , redness , marked reduction of vision , and reporting of patients to hospital is as shown in table 2 .
eight cases ( 16% ) presented to the hospital within 48 hours of onset of the disease .
twenty - two patients ( 44% ) came to the hospital between the 3rd and the 6th day . in twenty patients ( 40% ) , the disease had been present for more than a week before any treatment was sought .
the visual acuity was markedly reduced in all cases due to cataract as well as due to loss of corneal transparency secondary to a sudden rise of intraocular pressure .
thirty - nine patients had only light perception , and in three cases , even perception of light was doubtful .
the mean preoperative intraocular pressure was 44 mm hg ( range 2468 ) . in 25 ( 61% )
patients , the intraocular pressure could be reduced to less than 30 mm hg by hypotensive medications .
it can be seen that 16% of the patients recovered very good vision ( 6/18 or better ) after surgery
. low vision / visual impairment ( < 6/186/60 ) occurred in 12 ( 24% ) cases .
total blindness ( no pl ) occurred in 2 ( 4% ) cases ( table 3 ) .
while performing the surgery , shallow anterior chamber due to posterior vitreous pressure was seen in 12 patients .
posterior capsular tear resulting in loss of the vitreous occurred in five patients , and in three patients , complete removal of the cortical material could not be achieved .
postoperative uveitis was seen in 19 ( 33.3% ) patients and striate keratopathy in 14 ( 24.6% ) cases .
cataract remains the most important cause of blindness in developing countries , affecting mostly the older rural population .
delayed reporting for treatment leads to serious complications like lens - induced glaucoma causing irreversible visual loss .
lig is a condition to reckon with in our ophthalmic patients , especially ours being a rural area . in spite of easy availability of surgical facilities with concerted efforts of the national programme for control of blindness ( npcb ) , ngos , government agencies , and private practitioners , cataract surgery being a very cost effective and rewarding surgery , still many people are becoming blind due to lack of awareness about significance of early management .
illiterate , older , and rural population are the worst affected . in this study , 30 patients got no visual improvement or only marginal improvement in vision after the operation .
this was the group where time lag between development of symptoms of pain / redness and reporting for treatment was the longest .
visual outcome after cataract surgery has been reported normal , that is , 6/18 or better only in 16 percent eyes , and blindness ( <
6/60pl+ ) occurred in 28 ( 56% ) cases . 45.7 percent of eyes with presenting vision of less than 3/60 after cataract surgery could not be improved with correction ( npcb ) .
the most important cause of poor postoperative vision has been attributed to surgical complications ; however , in our study , late reporting for treatment also emerged as one of the most important causes for poor postoperative vision following cataract surgery .
females have been reported to have more adverse outcome ( 36.2 percent ) as compared to males ( 30.5% ) . in our study , 60 percent had postoperative vision less than 6/60 , which is quite less as compared to only 21 percent reported by pradhan et al .
the reason for delayed reporting in spite of services for cataract surgery available so easily appears to be poor health education , acceptance of poor vision as part of aging , fear of operation , lesser expectations and socioeconomic constraints .
also many people especially in rural areas take treatment for redness and pain in eyes from some local practitioners who miss the diagnosis initially .
another factor about late reporting found was that the very elderly visually handicapped persons were left to their own fate as no one bothered to bring them to the hospital . presenting vision of most of the patients was less than 3/60 in our study . presenting vision of less than 6/60
was reported by murthy et al . in a rural population , and poor visual outcome
it is highlighted from our study that lack of awareness in a rural population about early treatment of cataract is a major detrimental factor in the visual outcome after cataract surgery , inspite of the advances in cataract surgery techniques .
the present analysis of 50 cases of lens - induced glaucoma who received intraocular lens implants has shown that visual recovery and control of iop following surgerywas not satisfactory due to late presentation of the majority of cases . |
purpose . to determine the clinical profile of lens - induced glaucoma ( lig ) , reasons for late presentation , and outcome of current management
. methods .
retrospective analysis of 50 eyes with lig over a 6-year period between 2005 and 2011 at a tertiary care centre in rural india . visual acuity and intraocular
pressure ( iop ) were assessed preoperatively and postoperatively along with postoperative complications .
results .
fifty ( 2.4% ) of 12,004 senile cataracts operated at pravara rural hospital , loni , presented with lig .
there were 39 ( 78% ) phacomorphic cases and 11 ( 22% ) phacolytic glaucoma .
following cataract surgery , 21 of 50 operated eyes ( 42% ) had visual acuity 6/60 or worse .
conclusion .
the results highlight the importance of early diagnosis and treatment of visually disabling cataract .
there is a need to educate both the patient and the cataract surgeon about the dangers of lens - induced glaucoma and of about poor outcome if treatment is delayed . | 1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusion |
maintaining the deciduous teeth in the dental arch till it exfoliates is pivotal in maintaining the integrity of the arch , establishing occlusion and function of the permanent dentition .
the main reasons for premature loss of primary teeth are dental trauma or dental caries .
every effort should be taken to preserve the vitality of the pulp as it leads to more favorable treatment outcomes .
indirect pulp treatment ( ipt ) is one of the most preferred vital pulp treatment modality in restorative dentistry today which may be attributed to the introduction of new biocompatible materials and a better understanding of pulp biology .
dorfman et al . in 1943 stated that decalcification of dentin precedes bacterial invasion within the dentin . removing the outer layer of carious dentin , that contain the majority of the microorganisms , reducing the continued demineralization of the deeper dentin layers from bacterial toxins , and sealing the lesion to allow the pulp to generate reparative
ca(oh)2 has been the material of choice for indirect pulp capping for over 200 years .
it served as the gold standard to which newly introduced pulp capping materials were compared .
the introduction of newer materials with bioactive properties such as mineral trioxide aggregate ( mta ) , light cured calcium silicate ( theracal lc ) , biodentin helped surpass the demerits of ca(oh)2 such as nonadherence to dentin , dissolution over time , and tunnel defects and ensured increased success rate for ipt procedure .
the major constituents of this material are dicalcium silicate , tricalcium silicate , tricalcium aluminate , gypsum , and tetracalcium aluminoferrite .
it has good reparative dentin - forming ability as confirmed by many in vivo studies using this material in vital pulp therapies .
the drawbacks of mta include longer setting time , difficult handling characteristics , high cost , and solubility demonstrating 24% loss after 78 days of storage in water .
theracal lc is a new class of materials called resin modified calcium silicates that have been reported to stimulate apatite formation and the formation of reparative dentin .
theracal contains approximately 45% wt mineral material ( type iii portland cement ) , 10% wt radiopaque component , 5% wt hydrophilic thickening agent ( fumed silica ) , and approximately 45% hydrophobic resin .
both mta and theracal have been proved to have the ability to induce reparative dentin formation . in a literature search , no studies were found evaluating ipt using mta and theracal in primary molars .
hence , the present study was done to evaluate the reparative dentin - forming ability of mta and theracal in ipt in primary molars .
the study design was a clinical trial in a sample of 43 primary molars in 21 patients divided into two groups by simple random sampling method .
forty - three healthy primary molars in 21 patients aged between 4 and 7 years with deep carious lesion on either occlusal or proximal surface with no history of spontaneous pain and with a remaining dentin thickness ( rdt ) of more than 0.25 mm in the bitewing radiograph , as measured using coreldraw x3 software ( corel corporation , ottawa , ontario , canada ) ( mean rdt in mta group
periodontally sound teeth with large carious lesion with no history of spontaneous pain , with more than 2/3 of the root present , with no periapical changes and absence of sinus tract , were selected for the study .
teeth with definite pulpal involvement , discoloration , mobility and radiographically signs of internal resorption , pulpal calcification , periapical or furcation involvement were excluded .
the teeth with deep carious lesion with no history of spontaneous pain were subjected to routine standardized periapical and posterior bitewing radiograph to ascertain the inclusion criteria .
after administration of topical anesthetic , the tooth to be treated was isolated using rubber dam .
care was taken to remove caries completely from the lateral walls of the cavity and cavosurface margin .
soft caries at the pulpal floor of the cavity was removed preserving the affected dentin as identified by visual and tactile method .
following caries removal , the cavity was flushed with saline for 10 s and dried with cotton pellets . in patients belonging to group i ,
mta powder was mixed with sterile water for 30 s so as to get a sandy consistency and carried with a ball ended condenser tip and applied by light pressure with moist cotton pellet .
the cotton pellet was placed in the cavity , and temporary restoration with znoe was done .
a base with resin - modified glass ionomer cement ( rmgic ) followed by composite restoration was given post 45 min once the cotton pellet and temporary was removed . in patients belonging to group ii
, theracal lc was applied directly to the dentin surface using the dispensing syringe and light cured for 20 s , followed by rmgic base and composite restoration .
a baseline radiograph was made after the restoration using an extension cone paralleling ( xcp ) film holder .
subsequently , at 3 and 6 months , the teeth were clinically examined and radiographically evaluated .
all the radiographs were standardized using xcp technique and scanned and transferred to the computer for digital analysis .
dentin thickness was measured at the baseline , 3 and 6 months using coreldraw x3 software [ figures 14 ] by an independent examiner who was blinded to the study .
the digitalized images were overlapped using the software keeping cementoenamel junction and the highest point in the floor of pulp chamber as reference points , and increment in dentin thickness was measured .
mineral trioxide aggregate group measurement of reparative dentin formation mineral trioxide aggregate group [ magnified ] measurement of reparative dentin formation
theracal group [ magnified ] intragroup comparison of dentin increment in both the test groups was done between baseline value and baseline to 3 months value ( a ) , baseline value and 36 months value ( b ) , baseline to 3 months value and 36 months value ( c ) , and baseline value and baseline to 6 months value ( d ) .
criteria for clinical evaluation included the absence of spontaneous pain , sensitivity to pressure , fistula , edema , and abnormal mobility .
treatment was considered to be successful when the pulp remained vital with no signs of spontaneous pain and no radiographic evidence of pathologic change at the end of study period .
statistical analysis was performed using spss software version 20 ( ibm corporation , armonk , new york , united states ) .
intragroup and intergroup comparison for an increase in dentin thickness was carried out using independent t - test .
forty - three healthy primary molars in 21 patients aged between 4 and 7 years with deep carious lesion on either occlusal or proximal surface with no history of spontaneous pain and with a remaining dentin thickness ( rdt ) of more than 0.25 mm in the bitewing radiograph , as measured using coreldraw x3 software ( corel corporation , ottawa , ontario , canada ) ( mean rdt in mta group
periodontally sound teeth with large carious lesion with no history of spontaneous pain , with more than 2/3 of the root present , with no periapical changes and absence of sinus tract , were selected for the study .
teeth with definite pulpal involvement , discoloration , mobility and radiographically signs of internal resorption , pulpal calcification , periapical or furcation involvement were excluded .
the teeth with deep carious lesion with no history of spontaneous pain were subjected to routine standardized periapical and posterior bitewing radiograph to ascertain the inclusion criteria .
after administration of topical anesthetic , the tooth to be treated was isolated using rubber dam .
care was taken to remove caries completely from the lateral walls of the cavity and cavosurface margin .
soft caries at the pulpal floor of the cavity was removed preserving the affected dentin as identified by visual and tactile method . following caries removal ,
the cavity was flushed with saline for 10 s and dried with cotton pellets . in patients belonging to group i ,
mta powder was mixed with sterile water for 30 s so as to get a sandy consistency and carried with a ball ended condenser tip and applied by light pressure with moist cotton pellet .
the cotton pellet was placed in the cavity , and temporary restoration with znoe was done .
a base with resin - modified glass ionomer cement ( rmgic ) followed by composite restoration was given post 45 min once the cotton pellet and temporary was removed . in patients belonging to group ii
, theracal lc was applied directly to the dentin surface using the dispensing syringe and light cured for 20 s , followed by rmgic base and composite restoration .
a baseline radiograph was made after the restoration using an extension cone paralleling ( xcp ) film holder .
subsequently , at 3 and 6 months , the teeth were clinically examined and radiographically evaluated .
all the radiographs were standardized using xcp technique and scanned and transferred to the computer for digital analysis .
dentin thickness was measured at the baseline , 3 and 6 months using coreldraw x3 software [ figures 14 ] by an independent examiner who was blinded to the study .
the digitalized images were overlapped using the software keeping cementoenamel junction and the highest point in the floor of pulp chamber as reference points , and increment in dentin thickness was measured .
mineral trioxide aggregate group measurement of reparative dentin formation mineral trioxide aggregate group [ magnified ] measurement of reparative dentin formation
theracal group [ magnified ] intragroup comparison of dentin increment in both the test groups was done between baseline value and baseline to 3 months value ( a ) , baseline value and 36 months value ( b ) , baseline to 3 months value and 36 months value ( c ) , and baseline value and baseline to 6 months value ( d ) .
criteria for clinical evaluation included the absence of spontaneous pain , sensitivity to pressure , fistula , edema , and abnormal mobility .
treatment was considered to be successful when the pulp remained vital with no signs of spontaneous pain and no radiographic evidence of pathologic change at the end of study period .
statistical analysis was performed using spss software version 20 ( ibm corporation , armonk , new york , united states ) .
intragroup and intergroup comparison for an increase in dentin thickness was carried out using independent t - test .
of a total of 22 specimens included in the mta group , four specimens were excluded due to loss of follow - up .
deposition of tertiary dentin in mta group at the end of 3 months varied between 0.073 mm and 0.119 mm . from 36 months , it varied between 0.041 mm and 0.073 mm .
the mean increment in dentin thickness in first 3 months was 0.095 0.014 mm and from 36 months was 0.056 0.009 mm .
the average increase in dentin thickness in a total 6 months was 0.151 0.022 mm .
intragroup comparison of dentin increment in mta group showed a statistically significant increase in dentin thickness when the baseline value was compared with baseline to 3 months value with a p < 0.001 ( a ) [ table 1 ] .
statistically significant increase in dentin thickness was noted when baseline value was compared with that of 36 months ( b ) [ table 1 ] .
comparison of baseline to 3 months value with 36 months value also gave an increase in dentin thickness with a p < 0.001 which was statistically significant ( c ) [ table 1 ] .
similarly when baseline value was compared with total 6 months value , a statistically significant increase in dentin thickness was found with a p < 0.001 ( d ) [ table 1 ] .
intragroup comparison - mineral trioxide aggregate group three of the total 21 specimens included in the theracal group were excluded ; one due to a loss of follow - up and two due to dislodged restoration .
deposition of tertiary dentin in the theracal group at the end of 3 months varied between 0.088 mm and 0.115 mm ; from 3 to 6 months it varied from 0.046 mm to 0.059 mm .
the mean increase in dentin thickness in the first 3 months was 0.101 mm 0.008 mm and from 3 months to 6 months was 0.053 mm 0.004 mm .
the average increase in dentin thickness in total 6 months was 0.154 mm 0.012 mm .
intragroup comparison of dentin increment in theracal group also showed a statistically significant increase in dentin thickness when the baseline value was compared with a baseline to 3 months ( a ) [ table 2 ] and also while comparing baseline value with that of 36 months ( b ) [ table 2 ] .
comparison of baseline to 3 months value with 36 months value gave a statistically significant result with p < 0.001 ( c ) [ table 2 ] .
statistically significant result was obtained when the baseline value was compared with total 6 months value ( d ) [ table 2 ] .
intragroup comparison - theracal group intergroup comparison between mta and theracal using independent t - test was done between baseline to 3 months , 36 months , and total 6 months .
intergroup analysis of dentin increment in the first 3 months and next 3 months between mta and theracal were not significant yielding a p = 0.091 and 0.149 respectively .
increase in dentin increment in a total of 6 months was also compared between both the materials ( p = 0.611 ) [ table 3 ] .
these results showed that even though there is increase in dentin thickness within each group , there is no significant difference in dentin increment when comparing both groups .
intergroup comparison comparing the difference in dentin increment in both groups showed that dentin increment in first 3 months was statistically significant when compared to dentin increment in 36 months ( p < 0.001 ) [ table 4 ] .
intergroup comparison - mineral trioxide aggregate and theracal intergroup comparison ( difference ) between mineral trioxide aggregate and theracal
however , many studies have proved that the success of ipt was more compared to that of pulpotomy . a retrospective study by farooq et al
. showed normal exfoliation pattern for teeth treated with ipt compared to teeth treated by pulpotomy procedure , which showed early exfoliation .
careful diagnosis of the pulpal status is essential for the success of any conservative pulp treatment .
the samples for this study were selected based on the clinical and radiographic criteria for ipt .
correlated clinical and radiographic evaluation of pulp status with histologic findings and found that findings using defined criteria for clinical and histological classification of pulp conditions revealed a good agreement especially for cases with no pulpal disease or reversible disease .
the mean rdt determined from the bitewing radiographs in the mta group was 0.515 0.182 mm and that of the theracal group was 0.522 0.179 mm .
as reported by murray and smith preparation of deep cavities with an rdt of 0.250.004 mm injures underlying odontoblasts , preventing the secretion of reactionary dentin beneath the cavity preparation .
assessment of dentin thickness was done at the baseline , at the end of 3 months and 6 months in the digitalized radiographs keeping cement - enamel junction and highest point on the floor of the pulp chamber as reference points . in an in vitro study in extracted teeth ,
krasner and rankow proposed the presence of identifiable reference points for locating the floor of pulp chamber .
the authors demonstrated that the floor of the pulp chamber is always located in the center of the tooth at the level of the cement - enamel junction and the cement - enamel junction is the most consistent , repeatable landmark for locating the position of the pulp chamber .
large round bur in a slow speed handpiece was used for the caries removal in this study .
falster in an in vivo study comparing the pulp protecting ability of calcium hydroxide and adhesive resin demonstrated that the use of large , round carbide burs allow for better control of the partial caries removal step at the site of potential pulp exposure when compared to the use of spoon excavator . with spoon excavators ,
removal of affected dentin from deeper layers may cause accidental pulp exposure more commonly than with round burs at low speed .
mta is available as grey mta ( gmta ) and white mta ( wmta ) .
the occurrence of discoloration and longer setting time in association with gmta led to the introduction of wmta with the elimination of iron , preventing alumina - ferrite formation which was responsible for the discoloration .
smaller particle size of wmta provides greater specific surface area which increases wetting volume , water - binding capacity and hydration rate .
no significant difference between gmta and wmta in terms of calcified bridge thickness and pulp inflammatory response to the capping materials was observed in the study by eskandarizadeh et al .
owing to its superior properties , wmta was used as one of the test material in this study .
the drawbacks of mta include longer setting time , difficult handling characteristics , high cost , and solubility demonstrating 24% loss after 78 days of storage in water .
theracal has high calcium releasing ability , lower solubility , and comparable shear bond strength when compared with mta .
studied the sealing ability of theracal , biodentin and mta using confocal laser scanning microscopy and found out that theracal exhibits better sealing ability as a pulp capping agent than mta and biodentin . in the histologic study of direct pulp capping with theracal , cannon et al
proved that theracal demonstrated a complete hard tissue formation , adhesiveness to moist substrate and more positive response to the pulp exposure and bacterial contamination .
theracal has higher bond strength than mta in alkaline environment of the dentin as corroborated by the in vitro study comparing the push - out bond strength of biodentin , glass ionomer cement ( gic ) , mta and theracal .
there was no statistical difference in dentin deposition at the end of the first 3 months , second 3 months , and total 6 months between mta and theracal .
this may be attributed to the fact that theracal is chemically similar to mta formulation with the addition of hydrophobic resin . between the groups ,
the dentin increment in first 3 months in theracal group was higher when compared to that of the mta group , though not statistically significant .
in theracal , there is increased calcium ion release at the initial period of 28 days , which decreases later .
the alkaline ph of theracal decreases from 10 - 11 to 8 - 8.5 in 714 days creating a favorable environment for pulp cell viability and metabolic activity with the formation of reparative tertiary dentin
. the average dentin increment in the first 3 months in both groups was significantly higher when compared to second 3 months which may be attributed to decrease in calcium release by both materials over time as explained by in vitro study by gandolfi et al .
present study showed high tertiary dentin formation by both the test materials owing to the fact that both materials release calcium ions in sufficient quantities to promote reparative dentin formation .
the average reparative dentin formation in this study after mta placement in 6 months was found to be 0.151 0.022 mm .
this result is in agreement with the study done by george et al . comparing the reparative dentin formation between mta and ca(oh)2 in which the dentin increment in 6 months in the mta group was found to be 0.143 0.045 mm .
the mean reparative dentin formation in the theracal group in 6 months was found to be 0.154 mm 0.012 mm .
the 83% success rate of ipt restored with stainless steel crowns was not statistically different from the 87% success in teeth restored with intracoronal restoration . in vitro study done by kasraei et al .
shikha et al . in a comparative study of shear bond strength between rmgic and conditioned and unconditioned mta surface concluded that rmgic can be applied as a base over mta when used in direct pulp treatment or furcal repair .
gic placed over 45 min as opposed to 72 h old mta , showed no effect on crazing , setting time or shear bond strength with a noticeable formation of calcium salts at the interface .
rmgic also has a better bonding to composite resin than the conventional gic due to the similar chemistry between rmgic and composite resin .
both are cured by a free radical initiator system which provides a potential for chemical bonding between these two materials . as suggested in the above studies ,
rmgic was placed over both mta and theracal before placing a composite restoration to obtain an optimum marginal seal . in the present study , failure due to dislodged restorations
tooth position in the arch , the restoration size with a large configuration factor , and self - etching primer bonded to enamel , high caries activity challenge may possibly explain the failure rate of occlusal restorations of second primary molars . based on the results of the present study , both mta and theracal showed statistically significant tertiary dentin deposition .
further long - term studies with larger sample size and histologic evaluation are required to confirm these results .
more researches directed toward the development of better economical and biocompatible materials which do not affect the pulp vitality and normal resorption are needed for providing less invasive , quality dental treatment for the pediatric patients .
based on the results of this short - term clinical and radiographic study , the following conclusions were drawn :
clinically and radiographically , both mta and theracal are good ipt medicaments with significant reparative dentin formationradiographically , both mta and theracal showed increased dentin deposition in first 3 months when compared to the second 3 months .
clinically and radiographically , both mta and theracal are good ipt medicaments with significant reparative dentin formation radiographically , both mta and theracal showed increased dentin deposition in first 3 months when compared to the second 3 months . due to the ease of application , comparable tertiary dentin formation , better sealing ability and the convenience to provide the final restoration in a single appointment , theracal may be recommended as an alternative to mta in pediatric restorative dentistry . | aim : to clinically and radiographically evaluate the reparative dentin formation in indirect pulp treatment ( ipt ) using mineral trioxide aggregate ( mta ) and light cured calcium silicate ( theracal ) in primary molars over a period of 6 months.materials and methods : a clinical trial on ipt on 43 primary molars in 21 patients between the age of 47 years , divided into two groups : 22 teeth in mta group and 21 in theracal group .
measurement of the variation in dentin thickness was done on the digitalized radiograph at baseline , 3 months and 6 months using coreldraw x3 software.results:statistical analysis using an independent t - test for intragroup and intergroup comparison showed a significant increase in dentin thickness in both the mta and theracal group ( intragroup comparison [ p < 0.05 ] )
. however , intergroup comparison between mta and theracal showed no statistical difference in reparative dentin formation ( p > 0.05).conclusion : clinically and radiographically , both mta and theracal are good ipt materials . the better handling characteristics and comparable reparative dentin - forming ability of theracal make this material an alternative to mta in pediatric restorative procedures . | Introduction
Materials and Methods
Selection criteria
Sample preparation and experimentation
Results
Discussion
Conclusion
Financial support and sponsorship
Conflicts of interest |
the mission of icrh is to contribute to sexual and reproductive health and to strive for the recognition of sexual and reproductive health rights for every man and woman in every stage of their lives .
icrh has divided this field into four clusters : hiv and sexually transmitted infections ( sti ) including human papilloma virus ( hpv ) with a particular focus on prevention ; maternal health including mother & child health , with specific attention for safe motherhood and family planning ; sexual and gender based violence ( sgbv ) , harmful traditional practices such as female genital mutilation ( fgm ) and forced / child marriage : integration of sexual and reproductive health and rights within health systems . in each of these four clusters
, icrh performs a diversity of projects , most of them involving both scientific research and interventions .
this combination allows for a cross - fertilization between science and action and contributes both to the societal relevance of the scientific research and the other way around- to the scientific underpinning of policy measures .
reproductive health problems most often transcend the field of pure medicine , since they are deeply rooted in the fabric of communities and families , and hugely affected by societal organization , cultural values and economic circumstances . reproductive health research and action are therefore , if they aim to be effective , pre - eminently multidisciplinary ventures , and this is also how icrh s projects are devised .
the research staff of icrh consists of a variety of experts , ranging from physicians , obstetricians and epidemiologists to anthropologists , lawyers and woman s health activists .
the beneficial impact of this interdisciplinary way of working is shown by the multitude of field tools , policy advices and sensitization instruments that have resulted from the projects , as well as by the broad gamut of scientific magazines in which the research findings of icrh have been published .
another key characteristic of icrh is its emphasis on international and cross - sectoral cooperation and networking . in the course of its 15 years of existence
, icrh has built sustainable cooperation platforms with numerous organizations in all parts of the world and in all sectors : universities , ngos , public authorities , thematic networks , international institutions , private sector ?
most projects are conducted in cooperation with several other groups , raising the expertise , know - how and creativity to a higher level than would be the case if only one single organization took forward the project .
explaining how icrh works can best be done by giving some examples of its projects .
we list below just a few examples , but a full list of references can be obtained on the website .
momi ( missed opportunities in maternal and infant health ) : this project , funded by the european commission , started in 2011 , aiming at developing a package of interventions targeting the health of newborns and women in the early postpartum period and throughout the first year after childbirth .
this package will be delivered through a combined facility- and community - based approach designed to integrate services and strengthen health systems .
it is implemented in four african countries ( burkina faso , kenya , malawi and mozambique ) by a consortium consisting of several african and european partners .
example 2 : kesho bora : a better future : this large multicentre study including a nested randomized controlled trial was implemented in five research sites in east , west and south africa .
the overall aim of the study was to optimize the use of antiretroviral drugs during pregnancy , delivery and breastfeeding both to prevent mother - to - child transmission of hiv-1 ( pmtct ) and preserve the health of the hiv-1-infected mother in settings where the majority of women breastfeed .
the findings from the study have been important for informing international and national pmtct guidelines in developing countries where hiv-1 infected women have little or no option other than to breastfeed their babies .
the who guidelines on antiretroviral drugs for treating pregnant women and preventing hiv infection in infants : recommendations for a public health approach were importantly influenced by findings from the kesho bora study .
example 3 : post abortion family planning : an estimated 8 million induced abortions are performed annually in china . however ,
the delivery of post abortion family planning ( pafp ) services is almost inexistent in china . in 2005 , icrh took the initiative , in collaboration with 5 other institutions to conduct the first study in china on the impact of post - abortion family planning services on contraceptive use and abortion rate .
the main purpose of the study was to define optimal ways to introduce pafp services in urban areas in china in order to reduce the rate of unwanted pregnancies and induced abortions .
desafio is an institutional university cooperation programme financed by the university development cooperation of the flemish university council ( vlir - uos ) and coordinated by icrh .
the programme aims to strengthen the university eduardo mondlane ( uem ) as a developmental actor in mozambican society in the area of sexual and reproductive health ( srh ) and hiv / aids .
the innovation of the programme lies primarily in the multi - disciplinary approach , combining medical , social , cultural and legal aspects of reproductive health , in the linkage between research and community outreach , and in the long - term approach gradually developing the local capacity and creating a sustainable environment . in terms of academic output ,
more than 300 peer reviewed scientific articles have been ( co- ) authored by the centre s researchers , more than 20 phd dissertations and more than 100 master dissertations have been promoted .
in terms of organizational structure , icrh has followed a remarkable and quite innovative track .
it has been established as an academic research institute within the structure of the department of gynecology , faculty of medicine and health sciences of ghent university , but through the nature of its activities it developed immediately strong links with communities and organizations in developing countries , which have been consolidated in a multitude of forms of cooperation , but also in the establishment of daughter ngos .
currently , such organizations exist in mozambique and in kenya , and probably more will follow in other parts of the world .
the daughter icrhs are autonomous local organizations established according to the legislation of their countries and governed by representatives of local stakeholder groups . in order to ensure close ties between all icrhs , an additional not - for - profit organization
icrh global serves as a coordination platform between icrhs and in the future it will also develop networking and sensitization activities in the field of sexual and reproductive health . through its unusual structure , icrh as a group has placed itself on the crossroad of four key levels in sustainable development work : academic research , field action , global connectivity and local empowerment . in terms of funding ,
this challenging situation creates a rather insecure financial working environment , but on the other hand it forces icrh not only to apply a prudent and scrupulous financial management , but also to remain creative and innovative in its project development , and to stay in the vanguard of the scientific and societal thinking and acting in the field of reproductive health .
icrh has proven in the last few years that it has been very successful in coping with this situation , as is shown by the fast growing number of projects .
currently , icrh is coordinator of dozens of projects and programmes , some of them with budgets of several millions of dollars . | the international centre for reproductive health ( icrh ) was established by prof .
dr .
marleen temmerman in the aftermath of the un conference on population and development in cairo in 1994 .
this conference called for world - wide action to improve the sexual and reproductive health situation of the global population in general and for vulnerable groups in particular , and this is exactly what icrh is striving for and has been working on for the last 15 years .
icrh is a multidisciplinary centre of excellence , in research , capacity building and fieldwork in sexual and reproductive health and hiv prevention , and an advocate for sexual and reproductive health and rights .
right from the start , icrh has opted for a global approach , which has resulted in a broad geographical spread of activities , with projects in africa , latin america , asia and europe . since its inception ,
icrh has participated in more than 120 projects , often as the coordinator , and through this work it has contributed considerably not only to scientific knowledge , improvement of health systems and increased accessibility of health services for vulnerable groups , but also to the quality of live of numerous individuals . since 2004 , icrh has been recognized as a who collaborating centre for research on sexual and reproductive health . | Objectives and approach
Projects and publications
Structure and funding |
the study sample consisted of 3,210 individuals ( 1,423 men and 1,787 women ) from the study on nutrition and health of aging population in china .
the study population , design , and protocols of this population - based cohort study have been previously described ( 19 ) .
briefly , all participants were unrelated chinese hans , aged 5070 years , with at least 20 years residence in beijing or shanghai . among them , 424 participants had type 2 diabetes ( 267 had previously diagnosed type 2 diabetes and 157 had screen - detected and treatment - naive type 2 diabetes ) , 878 participants had impaired fasting glucose ( ifg ) ( all 878 were screen detected and treatment naive ) , and 1,908 participants had normal fasting glucose ( nfg ) .
type 2 diabetes was defined by either 1999 world health organization criteria ( 20 ) or previously diagnosed type 2 diabetes .
nfg and ifg were defined as fasting glucose < 5.6 mmol / l ( 100 mg / dl ) and 5.6 mmol / l ( 100 mg / dl ) less than or equal to fasting glucose <
the study was conducted simultaneously in both beijing and shanghai from march to june 2005 .
the participants were recruited from two urban districts ( 400 participants for each district ) and one rural district ( 800 participants ) , representing people with high to low socioeconomic status , using a multistage sampling method in each city .
all participants were selected randomly from the eligible candidates listed in the residential registration record .
one person from each household was allowed to participate , and at least 40% of the total participants were men in each district .
individuals with the following conditions were excluded from the study : 1 ) severe psychological disorders , physical disabilities , cancer , cardiovascular disease , alzheimer 's disease , or dementia , within 6 months ; or 2 ) currently diagnosed with tuberculosis , aids , and other communicable diseases .
all participants attended a physical examination , during which standard anthropometric measurements and overnight fasting blood samples were collected .
glucose was measured enzymatically on an automatic analyzer ( hitachi 7080 ; hitachi , tokyo , japan ) with reagents purchased from wako pure chemical industries ( osaka , japan ) .
a1c concentrations were measured by turbidometric immunoassay in red blood cells on the hitachi 7080 analyzer using reagents from roche diagnostics ( indianapolis , in ) . homeostasis model assessment ( homa ) of insulin sensitivity ( homa - s ) and -cell function ( homa - b ) were estimated using levy 's computer model ( 21 ) .
written informed consent was obtained from all participants , and study protocols were approved by the institutional review board of the institute for nutritional sciences .
genomic dna was extracted from peripheral blood leukocytes by the salting - out procedure ( available at http://humgen.wustl.edu/hdk_lab_manual/dna/dna2.html ) .
snp genotyping was performed with the genomelab snpstream genotyping system ( beckman coulter ) , according to the manufacturer 's protocol .
seventeen snps previously reported to be associated with type 2 diabetes by at least one of the gwass ( 712 ) were successfully genotyped in our population .
these include snps near cdkal1 ( rs10946398 , rs7754840 , rs7756992 , and rs9465871 ) , hhex / ide ( rs1111875 , rs5015480 , and rs7923837 ) , ext2 ( rs1113132 , rs11037909 , and rs3740878 ) , cdkn2a / b ( rs10811661 and rs564398 ) , igf2bp2 ( rs4402960 and rs1470579 ) , slc30a8 ( rs13266634 ) ( r325w ) , loc387761 ( rs7480010 ) , and an intergenic snp ( rs9300039 ) in chromosome 11 .
the genotyping success rate was > 97.1% , and the concordance rate was > 99% based on 12% duplicate samples ( n = 384 ) . samples with ambiguous base calling were genotyped again .
genotype frequencies of all 17 snps were consistent with hardy - weinberg equilibrium ( p > 0.01 ) , and most of the minor allele frequencies observed in this study were comparable with those in the hapmap chb ( chinese han in beijing ) sample ( online appendix table 1 [ available at http://dx.doi.org/10.2337/db08-0047 ] ) .
genotypic distributions were similar in beijing and shanghai populations ( p > 0.05 ) , except for the three hhex snps ( p = 0.041 , 0.003 , and 0.005 for rs1111875 , rs5015480 , and rs7923837 , respectively ) .
the association between each snp and the risk of type 2 diabetes and ifg was examined using logistic regression .
generalized linear regression was applied to study the associations between each snp and type 2 diabetes related quantitative traits .
participants with known diabetes or receiving glucose - lowering treatment ( n = 267 ) were excluded from the type 2 diabetes related quantitative trait analyses .
all association analyses assumed an additive effect of the risk allele and were adjusted for sex , age , bmi ( where appropriate ) , and geographical region ( shanghai versus beijing ) .
bmi , insulin , and homa - s were log transformed before analyses , and the data were presented as geometric means .
because of a significant difference in genotype distribution of the three hhex / ide snps ( p < 0.05 ) and in diabetes prevalence between the shanghai and beijing participants ( p < 0.0001 ) , analyses for these snps were performed for shanghai and beijing separately .
gene - gene interactions were assessed by including the respective interaction terms of pairwise snps in logistic regressions using the maximum likelihood estimation .
the combined effect of multiple snps on the risk of type 2 diabetes and/or ifg was determined by logistic regression after categorizing the participants into groups according to the number of the risk alleles they carried .
association analyses were performed with sas version 9.1 ( sas institute , cary , nc ) .
meta - analyses were conducted with stata ( version 9.2 ; stata , college station , tx ) .
power calculations were performed using quanto software ( available at http://hydra.usc.edu/gxe/ ) , and the power shown in online appendix table 1 was calculated for association between each snp and type 2 diabetes using the odds ratios ( 712 ) reported in the original studies and sample size and minor allele frequencies in our own study .
genomic dna was extracted from peripheral blood leukocytes by the salting - out procedure ( available at http://humgen.wustl.edu/hdk_lab_manual/dna/dna2.html ) .
snp genotyping was performed with the genomelab snpstream genotyping system ( beckman coulter ) , according to the manufacturer 's protocol .
seventeen snps previously reported to be associated with type 2 diabetes by at least one of the gwass ( 712 ) were successfully genotyped in our population .
these include snps near cdkal1 ( rs10946398 , rs7754840 , rs7756992 , and rs9465871 ) , hhex / ide ( rs1111875 , rs5015480 , and rs7923837 ) , ext2 ( rs1113132 , rs11037909 , and rs3740878 ) , cdkn2a / b ( rs10811661 and rs564398 ) , igf2bp2 ( rs4402960 and rs1470579 ) , slc30a8 ( rs13266634 ) ( r325w ) , loc387761 ( rs7480010 ) , and an intergenic snp ( rs9300039 ) in chromosome 11 .
the genotyping success rate was > 97.1% , and the concordance rate was > 99% based on 12% duplicate samples ( n = 384 ) . samples with ambiguous base calling were genotyped again .
genotype frequencies of all 17 snps were consistent with hardy - weinberg equilibrium ( p > 0.01 ) , and most of the minor allele frequencies observed in this study were comparable with those in the hapmap chb ( chinese han in beijing ) sample ( online appendix table 1 [ available at http://dx.doi.org/10.2337/db08-0047 ] ) .
genotypic distributions were similar in beijing and shanghai populations ( p > 0.05 ) , except for the three hhex snps ( p = 0.041 , 0.003 , and 0.005 for rs1111875 , rs5015480 , and rs7923837 , respectively ) .
the association between each snp and the risk of type 2 diabetes and ifg was examined using logistic regression .
generalized linear regression was applied to study the associations between each snp and type 2 diabetes related quantitative traits .
participants with known diabetes or receiving glucose - lowering treatment ( n = 267 ) were excluded from the type 2 diabetes related quantitative trait analyses .
all association analyses assumed an additive effect of the risk allele and were adjusted for sex , age , bmi ( where appropriate ) , and geographical region ( shanghai versus beijing ) .
bmi , insulin , and homa - s were log transformed before analyses , and the data were presented as geometric means .
likelihood ratio tests were used to examine genotype distribution in beijing and shanghai . because of a significant difference in genotype distribution of the three hhex / ide snps ( p < 0.05 ) and in diabetes prevalence between the shanghai and beijing participants ( p < 0.0001 ) , analyses for these snps were performed for shanghai and beijing separately .
gene - gene interactions were assessed by including the respective interaction terms of pairwise snps in logistic regressions using the maximum likelihood estimation .
the combined effect of multiple snps on the risk of type 2 diabetes and/or ifg was determined by logistic regression after categorizing the participants into groups according to the number of the risk alleles they carried .
association analyses were performed with sas version 9.1 ( sas institute , cary , nc ) .
meta - analyses were conducted with stata ( version 9.2 ; stata , college station , tx ) .
power calculations were performed using quanto software ( available at http://hydra.usc.edu/gxe/ ) , and the power shown in online appendix table 1 was calculated for association between each snp and type 2 diabetes using the odds ratios ( 712 ) reported in the original studies and sample size and minor allele frequencies in our own study .
we first examined the association with the risk of type 2 diabetes and ifg ( table 2 ) .
the four cdkal1 snps spanned two ld blocks ( r = 1.0 for rs7754840 and rs10946398 and 0.96 for rs7756992 and rs9465871 ) and were each significantly associated with type 2 diabetes ( odds ratios ranged between 1.38 and 1.49 ; p < 1.9 10 ) and with combined ifg / type 2 diabetes ( between 1.20 and 1.22 ; p < 0.0013 ) .
the cdkn2a / b rs10811661 variant was also associated with type 2 diabetes ( odds ratio 1.31 [ 95% ci 1.121.54 ] ; p = 0.001 ) and combined ifg / type 2 diabetes ( 1.26 [ 1.131.41 ] ; p = 2.76 10 ) .
the second cdkn2a / b snp ( rs564398 ) , which was not in ld with rs10811661 ( r = 0 ) , was not associated with type 2 diabetes or combined type 2 diabetes / ifg .
the two snps in igf2bp2 ( r = 0.83 ) and the slc30a8 snp ( rs13266634 ) showed modest association with combined ifg / type 2 diabetes ( odds ratios between 1.12 and 1.17 ; p = 0.0130.033 ) but not with type 2 diabetes alone .
the three ext2 variants were in complete ld ( r = 1.0 ) and occurred less frequently in our population ( 58% ) than in european populations ( 70% ) .
these variants , as well as those in chromosome 11 ( rs7480010 and rs9300039 , r = 0.037 ) , were not associated with type 2 diabetes or ifg .
analyses for the three snps in the hhex / ide ld block were performed separately in shanghai and beijing populations , as the difference in genotype distribution and prevalence of type 2 diabetes and ifg could lead to spurious associations due to population stratification ( table 2 ) .
all three hhex / ide snps were significantly associated with type 2 diabetes in shanghai participants , with rs5015480 and rs7923837 also associated with combined ifg / type 2 diabetes .
meta - analyses suggested that the associations exhibited significant heterogeneity for snps rs1111875 ( p = 0.006 ) and rs5015480 ( p = 0.028 ) between beijing and shanghai populations .
we next examined the association between genetic variants and type 2 diabetes related quantitative traits ( glucose , a1c , insulin , homa - b , homa - s , and bmi ) to investigate whether these variants conferred risk of type 2 diabetes through their effects on any of these intermediate traits ( table 3 ) .
consistent with the case - control analyses , the snps that showed significant evidence for association with diabetes - related phenotypes were those that were also associated with type 2 diabetes or ifg , except for cdkn2a / b rs10811661 and loc387761 rs7480010 .
all four cdkal1 snps were significantly associated with a1c ( p values 0.0360.0096 ) and homa - b ( p values 0.0240.0009 ) .
the snps ( rs7756992 and rs9465871 ) in the second ld block of this locus also showed significant association with fasting glucose levels ( p < 0.04 ) .
interestingly , the allele of slc30a8 snp rs13266634 that increases the risk of combined ifg / type 2 diabetes was significantly associated with lower bmi ( p = 0.0087 ) and marginally associated with decreased homa - b ( p = 0.05 ) . only the associations of cdkal1-rs10946398 , rs7754840 , and igf2bp2-rs4402960 with homa - b remained significant after bonferroni correction for multiple testing ( p = 0.0014 , 0.05/36 tests ) . to examine whether the associations for the cdkal1 variants were independent , we performed additional multiple regression analyses that included all four cdkal1 snps in one model .
results showed that none of the four snps remained significant ( p 0.17 ) .
( rs7754840 and rs7756992 were chosen to represent each of the pairs ) were independent from each other for the associations with type 2 diabetes or related quantitative traits in multiple regression models with both rs7754840 and rs7756992 genotypes in the model , with age , sex , region , and bmi ( where appropriate ) as covariates .
the results revealed that the association seems to be driven by rs7754840 , for the associations with type 2 diabetes , bmi , and homa - b , or by rs7756992 , for the association with a1c , but interestingly , rs7754840 and rs7756992 seem to have independent effects on the associations with homa - s or insulin ( online appendix table 2 ) .
we also performed a meta - analysis with the data from the previously published studies ( 1012 ) , including those from japanese , korean , and hong kong chinese populations ( 2225 ) , to assess the heterogeneity between caucasians and asians for the cdkal1 and cdkn2a / b loci ( rs7754840 and rs10811661 were chosen to represent each of them , respectively ) .
the results showed that for the cdkn2a / b loci ( rs10811661 ) , the heterogeneity between caucasians and asians did not reach significance ( p = 0.059 ) , while a significant heterogeneity was observed between caucasians and asians ( p = 8.872 10 ) for the cdkal1 loci ( rs7754840 ) ( online appendix fig .
1 ) , and this is consistent with the recent finding reported by ng et al . ( 25 ) .
although we did not observe the association among the loc387761 snp rs7480010 and type 2 diabetes or ifg , we found that the allele that increased the diabetes risk in european populations was modestly associated ( p < 0.03 ) with increased insulin sensitivity ( homa - s ) and lower fasting insulin levels . furthermore , despite a strong association between the cdkn2a / b snp rs10811661 and type 2 diabetes , no association was observed with any of the diabetes - related quantitative traits .
the intergenic snp rs9300039 and the three ext2 snps ( rs3740878 , rs11037909 , and rs1113132 ) were not associated with any of the diabetes - related quantitative traits .
we found no evidence of multiplicative gene - gene interactions among the main snps ( rs9465871 , rs10811661 , rs4402960 , and rs13266634 ) in each of the cdkal1 , cdkn2a / b , igf2bp2 , and slc30a8 genes . a significantly higher proportion of participants with type 2 diabetes carry increasing numbers of risk alleles , compared with participants with nfg ( fig .
each additional risk allele increased the risk of type 2 diabetes by 1.24-fold ( p = 2.85 10 ) ( fig .
1b ) and combined ifg / type 2 diabetes by 1.21-fold ( p = 6.31 10 ) ( fig .
participants harboring seven or all eight risk alleles had a 4.44-fold increased risk for type 2 diabetes ( p = 5 10 ) compared with those with one or no risk alleles ( fig .
consistently , participants with increasing numbers of risk alleles tended to have increased fasting levels of plasma glucose ( p = 0.013 ) ( fig .
1e ) , as well as decreased homa - b values ( p = 3.34 10 ) ( fig .
, participants with increasing numbers of risk alleles tended to have significantly lower bmi ( p = 5.3 10 ) ( fig .
1f ) , which is consistent with previous results found for the cdkal1 and slc30a8 polymorphisms ( table 3 ) .
in this study of chinese hans , we replicated associations with several diabetes susceptibility variants recently identified through gwass in white europeans ( 712 ) . variants in cdkal1 , cdkn2a / b , igf2bp2 , slc30a8 , and hhex loci were significantly associated with the risk of type 2 diabetes or combined ifg / type 2 diabetes .
furthermore , variants in cdkal1 and igf2bp2 were strongly associated with -cell function estimated by homa - b .
these associations were stronger than those observed in individuals of european ancestry ( 810,12 ) ( online appendix table 1 ) , and cdkal1 risk allele frequencies are also substantially higher in chinese ( 4355% ) than europeans ( 1531% ) .
moreover , significant heterogeneity between caucasians and asians was found for the cdkal1 loci ( rs7754840 ) in the meta - analysis that combined the data from the previous studies in white europeans , japanese , korean , hong kong chinese , and our study ( p = 8.872 10 ) ( online appendix fig .
1 ) , while no significant heterogeneity was observed among the asians ( p = 0.369 ) .
these observations suggest that these cdkal1 variants might play an even more important role in diabetes susceptibility in chinese .
the risk allele of the first pair of cdkal1 variants was strongly associated with reduced -cell function ( homa - b ) and increased a1c levels , while the second pair of cdkal1 variants showed an association with impaired -cell function ( homa - b ) and higher glucose levels , as well as with increased a1c .
the results from additional multiple regression analyses suggest that the four snps most likely represent the effects of a single cdkal1 locus .
however , none of these four snps stands out as being the variant driving the association .
therefore , we assume that none of them is likely to be the causal variant , but presumably they are in moderate to high ld with the causal snp and are therefore less consistently associated with the traits of interest .
this region would benefit from a detailed fine mapping to identify possible causal variants in future studies .
these results support previous findings ( 9,13,26 ) that the four cdkal1 snps confer the risk of type 2 diabetes through reduced insulin secretion , although the causal snp is yet to be identified .
we also observed significant association between cdkn2a / b rs10811661 and type 2 diabetes and ifg with a slightly higher odds ratio ( 1.3 ) than that observed in europeans ( 1.20 ) ( 1012 ) .
the risk allele is twice as prevalent in chinese hans ( 46% ) as in europeans ( 21% ) .
however , we did not observed significant heterogeneity between caucasians and asians in the meta - analysis with data from the previously published studies ( p = 0.059 ) .
interestingly , none of the diabetes - related traits showed an association with cdkn2a / b rs10811661 .
the second cdkn2a / b variant , rs564398 , which is less frequent in chinese hans ( 13% ) than in europeans ( 38% ) , was not associated with type 2 diabetes or any related traits .
the association between variants in igf2bp2 and type 2 diabetes was not significant , although the odds ratios were similar to those observed in european populations ( 1.15 ) , suggesting that our study may not have been sufficiently powered .
indeed , assuming an additive model and a minor allele frequency of 25% , we had < 50% power to detect previously reported odds ratios at p < 0.05 .
we did , however , find a significant association with combined ifg / type 2 diabetes .
the associations with homa - b suggest that igf2bp2 confer type 2 diabetes risk through a reduced -cell function .
similarly , we found no association between the slc30a8 rs13266634 variant and type 2 diabetes , while an association with combined ifg / type 2 diabetes reached borderline significance .
interestingly , the risk allele that increased diabetes risk in europeans was also associated with a lower bmi in this population .
we also failed to find any evidence for association between type 2 diabetes and the snps in ext2 ( rs3740878 , rs11037909 , and rs1113132 ) and the intergenic snp rs9300039 , despite 80% power to detect previously reported effect estimates ( 7 ) .
although these snps exhibited marginal associations with type 2 diabetes in the original study ( 7 ) , they were largely negative in the subsequent four gwass and other replication studies in samples from u.k .
( 8,12 ) , finnish ( 10,11 ) , swedish ( 10 ) , icelandic ( 9 ) , german ( 27 ) , and japanese ( 23 ) populations .
therefore , the original associations for these snps were either population specific or overestimated due to the winner 's curse
( 28,29 ) , but the consistent lack of replication suggests that these findings were more likely false - positives .
meta - analyses or studies with larger sample sizes will be required to draw definitive conclusions .
although there was no association between rs7480010 ( loc387761 ) and type 2 diabetes or ifg , the allele conferring risk of diabetes in europeans was associated with increased insulin sensitivity and showed a tendency toward a reduced -cell function as well .
for the three snps in hhex / ide gene region , the associations with type 2 diabetes or ifg were observed only in shanghai individuals in whom each risk allele resulted in 1.45- to 1.79-fold increased diabetes risk , suggesting that geographical stratification may exist in our population for these snps and their roles in type 2 diabetes susceptibility .
however , given the relatively small sample size , we can not rule out sampling bias .
we found no evidence of pairwise synergistic gene - gene interactions on type 2 diabetes and the related phenotypes among cdkal1-rs9465871 , cdkn2a / b - rs10811661 , igf2bp2-rs4402960 , and slc30a8-rs13266634 . in joint analyses ,
the risk of type 2 diabetes was increased by 1.24-fold for each additional risk allele , and participants with seven or all eight risk alleles ( 3.8% ) had a 4.44-fold increased risk of type 2 diabetes ( p = 5 10 ) compared with those with one or no risk allele .
( 11 ) , who examined combined effects of 10 risk variants in a gwas of european populations . compared with scott 's study ,
the advantage of our study is that our data are based on the general population .
however , a replication in larger population is required to examine whether combinations of risk alleles from these variants have good predictive and diagnostic potential in chinese hans . in conclusion
, we replicated the association of type 2 diabetes with the snps in cdkal1 and cdkn2a / b genes and confirmed that the snps in slc30a8 and igf2bp2 were associated with the risk of combined ifg / type 2 diabetes .
importantly , the risk variants in cdkal1 , cdkn2a / b , igf2bp2 , and slc30a8 appear to act in an additive manner to increase the risk of type 2 diabetes and related phenotypes .
these results provide solid evidence for the notion that these variants individually or collectively contribute to the risk of type 2 diabetes in the chinese han population , possibly by impairing -cell function or reducing insulin secretion . | objective genome - wide association studies have identified common variants in cdkal1 , cdkn2a / b , igf2bp2 , slc30a8 , hhex / ide , ext2 , and loc387761 loci that significantly increase the risk of type 2 diabetes .
we aimed to replicate these observations in a population - based cohort of chinese hans and examine the associations of these variants with type 2 diabetes and diabetes - related phenotypes.research design and methods we genotyped 17 single nucleotide polymorhisms ( snps ) in 3,210 unrelated chinese hans , including 424 participants with type 2 diabetes , 878 with impaired fasting glucose ( ifg ) , and 1,908 with normal fasting glucose.results we confirmed the associations between type 2 diabetes and variants near cdkal1 ( odds ratio 1.49 [ 95% ci 1.271.75 ] ; p = 8.91 107 ) and cdkn2a / b ( 1.31 [ 1.121.54 ] ; p = 1.0 103 ) .
we observed significant association of snps in igf2bp2 ( 1.17 [ 1.031.32 ] ; p = 0.014 ) and slc30a8 ( 1.12 [ 1.011.25 ] ; p = 0.033 ) with combined ifg / type 2 diabetes .
the snps in cdkal1 , igf2bp2 , and slc30a8 were also associated with impaired -cell function estimated by homeostasis model assessment of -cell function . when combined , each additional risk allele from cdkal1-rs9465871 , cdkn2a / b - rs10811661 , igf2bp2-rs4402960 , and slc30a8-rs13266634 increased the risk for type 2 diabetes by 1.24-fold ( p = 2.85 107 ) or for combined ifg / type 2 diabetes by 1.21-fold ( p = 6.31 1011 ) .
none of the snps in ext2 or loc387761 exhibited significant association with type 2 diabetes or ifg .
significant association was observed between the hhex / ide snps and type 2 diabetes in individuals from shanghai only ( p < 0.013 ) but not in those from beijing ( p > 0.33).conclusions our results indicate that in chinese hans , common variants in cdkal1 , cdkn2a / b , igf2bp2 , and slc30a8 loci independently or additively contribute to type 2 diabetes risk , likely mediated through -cell dysfunction . | RESEARCH DESIGN AND METHODS
Genotyping.
Statistical analyses.
RESULTS
DISCUSSION
Supplementary Material |
cerebral palsy ( cp ) , defined as a nonprogressive injury to the brain , covers numerous
neurological conditions , which result in abnormal development of movement and postural
control .
injury to the brain typically results in impairments in motor function , such as
muscle weakness , spasticity , and decreased selective motor control , which are primary causes
of children with cp limitations in the performance of activities of daily living and
participation1 .
children with cp have a
decreased level of daily physical activity in comparison to their healthy peers .
they do not
use their physical reserves sufficiently during the day to achieve optimal levels of daily
physical activity2 .
children with cp need
to maintain a higher level of physical fitness than healthy children in order to maintain
the natural growth process as they grow older3 .
the peak oxygen uptake ( vo2 peak ) , typically used as a measure of aerobic
fitness , is lower in children with cp , as has been shown through exercise tests using cycle
ergometry or a treadmill4 , 5 .
the maximum aerobic physical fitness , vo2 peak ,
is an internationally acknowledged measure of the fitness of the cardiorespiratory system .
three studies4,5,6 have studied vo2
peak in children and adolescents with cp during a maximal exercise test .
these studies
compared vo2 peak to the values observed in normally developing ( nd ) children and
adolesents5 , 6 . the most functional and appropriate way to assess
vo2
peak in children with cp who are able to walk independently is a progressive walking or
running - based maximal exercise test4 , 7 .
various researchers have developed
treadmill walking maximal exercise tests to assess individuals with cp and to investigate
the effectiveness of rehabilitation programs4,5,6 , 8,9,10,11 .
however , these methods limit the generalization of the results to actual daily walking ,
because a variety of comfortable walking speeds are exhibited by children with different
types of cp5 . therefore , it is important
to test children under general conditions that are comparable to daily living conditions .
for estimating the maximal aerobic capacity , an adapted cycle ergometer test specifically
developed for cp can be used12 , 13 .
this aerobic test has the advantage of reducing errors
that are produced when walking with diverse patterns .
currently , studies of ergometer
aerobic tests14 have provided
insufficient evidence of their effect12 , 15 ; furthermore , no direct cardiorespiratory
results have been considered in the aerobic measurements16 .
although unnitan et al.17 studied the effect of uphill walking on children with diplegic cp ,
and measured the of physical fitness of adult male cp subjects18 , their evaluations were performed using an arm ergometer .
therefore , their score does not reflect changes in fitness resulting from a mainly leg - based
training program . timed
walking tests may offer a less expensive , simpler , and safer alternative to
laboratory testing of children with cp10 , 19 . among such tests ,
the 6-min walk test
( 6mwt ) has been shown to predict cardiorespiratory fitness in both healthy and severely
disabled children , whether used with or without gas collection20 , 21 .
novac et al.22 systematically discussed
the most effective and applicable interventions for children with cp . in their reports of
the literature ,
the most recommended and applicable intervention was green light
intervention , and they emphasized the importance of fitness training for aerobic fitness .
before children with cp perform aerobic fitness tests , their aerobic ability should be
measured .
however , previous studies have reported in lacked consistent results and utilized
various measurement methods and environments .
thus , it is necessary to provide a precise
method and measurement for base data to obtain accurate reference values .
accordingly , this
preliminary study aimed to determine the cardiorespiratory endurance of children with cp
using a case series study in order to provide the reference data required for interventions
appropriate for south korean cp sufferers , since aerobic ability evaluation and
interventions for it for children with cp are not well recognized in south korea .
six children participated in this study . four subjects ( 3 boys , 1 girl ) had a diagnosis of
bilateral or unilateral spastic cp , and 2 subjects ( 1 boy , 1 girl ) were children with normal
development .
four children with cp gmfcs ( gross motor function classification system ) level
i and ii and two normally developing children ( age : 715 years ) were studied ( table 1table 1.general characteristics of subjectssubjectstypegmfcs levelgender ( boys : 0 , girls : 1)age ( years)height ( cm)weight ( kg)nda--01215148b--1711522cpcspastic bilateral101214031dspastic bilateral2015155.446espastic unilateral21711632fspastic unilateral201013838cp : cerebral palsy ; nd ; normally developing children ; gmfcs : gross motor function
classification scale ) .
subjects with cp were recruited from a medical center in gyeonggi - do south .
this study complied with the ethical prinsiples of the declaration of helsinki , and all the
subjects and parents received explanations regarding the purpose and procedure of the study
before voluntarily agreeing to take part .
all parents signed an informed consent statement
before the start of the measurements . before testing , a medical examination was completed
and anthropometric data were collected .
the inclusion criteria were gmfcs levels i to ii ,
uni- or bilateral spastic cp , age of 516 years , ability to walk continuously for 6 min , and
ability to provide informed consent .
potential subjects were excluded if they had received
orthopedic surgery or neurosurgery and/or botulinum toxin injection within the previous 6
months , or had cardiac or respiratory conditions that could have been negatively affected by
the proposed training .
all the selected subjects were also free of intellectual problems
that could have influenced their participation and/or motivation .
cycle ergometer testing
was performed with calf supports ( motomed viva cycle , reek medizinterchnik gm bh ,
betzenweiler , germany ) .
the cycle ergometer test served to familiarize and habituate the
subjects , and it also allowed us to check whether a given subject needed to have his / her
feet attached to the pedals by velcro strapping23 . the concentration and volume of oxygen consumed ( vo2 ) ,
peak respiratory exchange ratio ( rer peak ) , and minute ventilation ( ve ) were measured
simultaneously by portable metabolic measurement systems with prevent
pneumotachs ( medgraphics vo2000-medical graphics , st .
the ergometer
test began with a 5-min warm up followed by 612 min of exercise with increasing intensity
at a pedaling rate of 50 rpm as paced by a metronome .
verbal encouragement was provided
throughout the test to help each child achieve maximal effort .
the subjects were also fitted
with a neoprene face mask that could be used with the vo2000 .
a polar hr strap was also
fitted around the chest of the subjects ( polar electro oy , kempele , finland ) .
gas exchange
and pulmonary ventilation values were recorded using the breathbreath mode with 20-s
average values printed during testing .
cp : cerebral palsy ; nd ; normally developing children ; gmfcs : gross motor function
classification scale the 6-min walk test was performed in the laboratory corridor between two lines set 20 m
apart according to a standard procedure24
under the supervision of the same physiotherapist .
the subjects were instructed to walk as
far as possible in 6 min .
the subjects received standardized and vigorous verbal
encouragement , and they were advised of the distance covered and time remaining every 30 s.
all subjects performed , the cycle - ergometer test first , and the 6-min walking test was
conducted on the next day to avoid fatigue effects .
the ages of the subjects were distributed as follows : two were aged 7 years , two were aged
12 years , one was aged 10 years , and one was aged 15 years ; i.e , the youngest and oldest
subjects were 7 and 15 years of age . regarding the subjects gmfcs assessment , three were
level ii , and one was level i. two normal children of 7 and 12 years age participated in
this experiment to serve as controls for comparison with the children with cp .
the vo2 peak ( ml / kg / min ) of child f ( cp ) was 57.4 , which was the
highest , and that of child d was 22.6 , which was the lowest .
the two normal children , a and
b , had vo2 peaks ( ml / kg / min ) of 22.9 and 56.5 , respectively .
in the 6mwt ,
child c ( cp ) walked 578 m , which was the longest distance , and child e walked 390 m , which
was the shortest distance ( table 2table 2.subjects peak cardiorespiratory responses to ergometrytypesubjectsvo2 peakrer peakve peakhr peak6mwtnda22.90.8218.5105580b56.50.9723.8135450cpc25.91.3625.3145578d22.61.0222.9131527e44.50.8732.5129390f57.40.9759.5147560vo2 peak ( ml / kg / min ) : peak oxygen uptake ; rer peak : peak respiratory
exchange ratio ; ve peak ( l / min ) : maximum ventilation per minute ; hr peak ( beats / min ) :
peak heart rate ; 6mwt ( m ) : 6-min walk test ) .
furthermore , the comparison of vo2 peak between normal child a
and child c ( cp ) showed that the former had a value of 22.9 , which was lower than that of
the latter .
the comparison of vo2 peak between normal child b and child e ( cp )
showed that the latter had a value of 44.5 , which was lower than that of the former .
the
comparison of 6mwt between normal child a and child c ( cp ) and between normal child b and
child e ( cp ) showed that the normal children walked further than the children with cp . vo2 peak ( ml / kg / min ) : peak oxygen uptake ; rer peak : peak respiratory
exchange ratio ; ve peak ( l / min ) : maximum ventilation per minute ; hr peak ( beats / min ) :
peak heart rate ; 6mwt ( m ) : 6-min walk test
this preliminary study aimed to determine the cardiorespiratory endurance of children with
cp using a case series study to provide the reference data required for interventions
appropriate for south korean cp sufferers , since aerobic ability evaluation and
interventions for it for children with cp are not well recognized in south korea .
children with cp have lower vo2 peak values ( ml / kg / min ) as well as shorter
walking distances in set times than normal children .
numerous studies have reported the
lower physical activities of children with cp in terms of aerobic abilities4 , 5 , 1725 ,
and some studies have reported that children and adolescents with cp have lower physical
activity abilities than normal peers26 .
verschuren et al.6 reported that
comparisons with normative data sets suggest that adolescents with cp tend to participate in
less structured and lower intensity physical activities than healthy adolescents .
durstine et al.27 described a vicious
circle in which persons with a chronic disease or disability experience less physical
activity , which then leads to a cycle of deconditioning and further physical deterioration
and reduction in activity .
however , as seen in this study , the child with cp aged 7 ( subject
e ) had a lower vo2 peak value than the normally developing child ( subject b ) , and
the child with cp aged 12 ( subject c ) had a higher vo2 peak value than the
normally developing child ( subject a ) .
subject b ( nd ) had a lower vo2 peak than
subject e ( cp ) , consistent with the results of previous studies6 , 13 .
one study6 reported that a child with cp had a
vo2 peak value of 42 ml / kg / min , which was close to our study result of
44.5 ml / kg / min ; however , the interpretation of the results of this previous are limited ,
because the subjects were aged from 7 to 17 years . with regard to the comparison of the
results of subjects
a and c , it is necessary to determine the reason for the higher
vo2 peak value in children with cp than in normal children , considering of
various possibilities . a child with gmfcs level
i can perform gross motor skills such as
running and jumping and participate in physical and dynamic activities .
in contrast , a child
with cp at gmfcs level ii can not participate in physical and dynamic activities such as
playground activities owing to his / her limited abilities .
despite examining only one case in
this study , our results suggest children with cp who have small body mass and high
functional level have even higher vo2 peak values than normal children .
nsenga et
al.13 reported that children with cp at
gmfcs levels i and ii can have cardiorespiratory endurance similar to that of normal
children of similar ages .
the former showed higher values ( 41.4 ) than
the latter ( 33.6 ) .
this result was used as an explanation for the lower vo2 of
children with cp .
the present study found comparable values for children and adolescents
with cp and normally developing subjects .
the ventilation peak ( vepeak ) value for child c
( cp ) was higher ( 25.3 ) than that ( 18.5 ) of child a ( nd ) .
similarly , the child e ( cp ) showed
a higher value ( 32.5 ) than that ( 23.8 ) of child b ( nd ) .
the distance walked strongly correlated with the 6mwt
vo2 peak ( r = .948 ) and was related more modestly to the
cycle - ergometer vo2 peak ( r = 0.625)19 . in this study
, vo2 was not collected while
performing the 6mwt . however , the results of the present study show that children with cp ( c
and e ) walked shorter distances than normal children ( a and b ) .
nsenga et al.19 reported that the 6mwt distance increased
after aerobic training , showing a correlation between the cardiorespiratory capacity and
6mwt distance .
although a previous study suggested there is no significant correlation
between the ergometer vo2 peak and 6mwt distance , maanum et al.28 reported that the 6mwt distance was a
significant predictor of cardiorespiratory capacity for children with spastic cp .
peak rer is generally used as a secondary criterion for
maximal effort ; however , large individual variations may be present depending on the test
modes and protocols29 .
the average peak
rer values in the present study ( 0.871.36 ) were comparable to the average values reported
in previous studies of children with cp ( 0.91.15 ) , and most studies have reported a peak hr
of 190 bpm4 , 30 .
these results does not support the finding that the children did
not produce their maximal effort because of the lower peak hr values .
however , the present
study conducted ergometer tests and not treadmill tests ; furthermore , there was an age
difference among the subjects . thus far , most studies of children with cp have focused on cycle ergometer or treadmill
walking tests6 , 8,9,10 , seeking to verified the effectiveness of these tests12 ,
13,14,15 , 17 . however , the available literature on the cardio - respiratory capacity of
children with cp focuses on treadmill walking rather than cycle ergometry .
verschuren et al.7 noted that treadmill protocols such as the bruce and balke tests are
not appropriate for children with cp .
furthermore , treadmill testing is difficult for many
children with cp because they suffer from severe balance issues31 .
it is difficult to compare the children from all studies
that have provided information about vo2 peak for children and adolescents with
cp .
although many studies have recognized the importance of fitness for maintaining aerobic
capacity , it is not easy to conduct aerobic tests with children with cp owing to the
limitations described in the studies cited above . among studies with a high evidence level ,
the accuracy of the results may not be that high owing to the use of arm ergometry rather
than a test of the lower extremities .
other studies include a case study15 with a low evidence level and studies with
a small number of subjects12,13,14 .
accordingly ,
more evidence and data that can be used as a reference to provide fitness programs for
school children with cp are needed in the future .
this study aimed to determine the
cardiorespiratory endurance of children with cp using a case series study in order to
provide the reference data required for interventions appropriate for south korean cp
sufferers , since aerobic ability evaluation and interventions for it for children with cp
are not well recognized in south korea . despite success in the study aims , the results
should be interpreted with care , owing to the small number of participating subjects . in conclusion , this case report reports cardiorespiratory parameter values that are similar
to those that have been reported previously .
fitness training for aerobic fitness is an
important intervention method for children with cp .
accordingly , it is necessary to provide
evidence of cardiorespiratory fitness levels for children with cp when such fitness programs
are conducted .
furthermore , it is necessary to provide precise methods and measurements for
base data in every assessment to obtain accurate reference values . in this
regard , our study
will provide information about vo2 peak values for an aerobic - fitness - related
study conducted with children with cp in the future . | [ purpose ] this preliminary study aimed to determine the cardiorespiratory endurance of
children with cerebral palsy ( cp ) using a case series study in order to provide the
reference data required for interventions appropriate for south korean cp sufferers , since
aerobic ability evaluation and interventions for children with cp are not well recognized
in south korea .
[ subjects and methods ] four children and adolescents with cp gmfcs ( gross
motor function classification system ) level i and ii and two normally developing children
( nd ) ( age : 715 years ) were studied .
cycle ergometer testing was performed to determine
their vo2 peak and rer peak concentrations as well as ve peak and 6mwt
distance .
[ results ] the vo2 peak was lower in subject e ( cp ) at 44.5 than in
subject b ( nd ) , and it was lower in subject a ( nd ) at 22.9 than in subject c ( cp ) .
the
6mwt distance was longer in subjects a and b ( nd ) than in age - matched cp subjects .
[ conclusion ] this case report demonstrates that the cardiorespiratory parameters values of
cp children were similar to those reported previously .
further research is required to
evaluate the normative values of cp and the optimal cardiorespiratory parameters . | INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION |