Biomedical subjects

Prem Puri

Publications and source records attributed to Prem Puri.

At least 19 recordsLinked to original sources

PubMed2007

Adriamycin mouse model: a variable but reproducible model of tracheo-oesophageal malformations.

A spectrum of tracheo-oesophageal malformations is seen in humans: oesophageal atresia, tracheal agenesis and laryngotracheo-oesophageal clefts. They are thought to share a common but unknown aetiology. These birth defects are frequently associated with other VACTERL anomalies. The adriamycin rat model (ARM) has proved to be a valuable model of the VACTERL anomalies, illustrating the dysmorphogenesis of oesophageal atresia and tracheal agenesis. As organogenesis relies on temporaspatially co-ordinated signalling systems, the next step would be to study the molecular pathogenesis of tracheo-oesophageal malformations. However, the mouse is the foremost mammal studied by developmental biologists, offering an expanding wealth of knowledge and scientific research techniques with which to investigate these anomalies. A limited dose response analysis of the teratogenicity of adriamycin in the mouse has identified a dose and timing of injections that produced tracheo-oesophageal malformations and other VACTERL anomalies. A clear account of the types and variability of the tracheo-oesophageal malformations produced by this dose is essential in order to be able to plan and interpret any future investigations of early gestation fetuses. CBA/Ca mice were accurately time-mated (n = 10). Nine dams received intraperitoneal injections of adriamycin (6 mg/kg) and one control dam received saline injections, on days 7 and 8. Fetuses were harvested on day 18, near term. Tracheo-oesophageal malformations were examined by dissecting microscope and serial transverse sections. Results are reported in the standard teratological manner as mean percentage per litter (+/-SEM). The resorption rate of the adriamycin treated fetuses was 50.4%. There were 29 adriamycin treated fetuses for inspection. Tracheo-oesophageal malformations were found in 29.2% (+/-10.3), affecting five out of nine litters. Oesophageal atresia occurred in 15.6% (+/-8.1), laryngotracheo-oesophageal cleft in 10.4% (+/-7) and tracheal agenesis in 3.1% (+/-3.1). All of these malformations occurred with a tracheo-oesophageal fistula. Unlike the ARM, the AMM can produce fetuses with complete laryngotracheo-oesophageal cleft as well as oesophageal atresia or tracheal agenesis. Their occurrence was found to be reproducible but variable. These are important considerations when planning and interpreting experiments using this model.

Animals↗

PubMed2007

Altered regulation of retinoic acid synthesis in nitrofen-induced hypoplastic lung.

Retinoids are a group of molecules derived from vitamin A, which play an important role in lung development. Within the cell, retinol can either be oxidized to retinal or esterified to retinyl esters by lecithin : retinol acyltransferase (LRAT) for storage. Retinal is then oxidized to an active metabolite of vitamin A, retinoic acid (RA) by retinal dehydrogenase (RALDH). RA is the active metabolite of vitamin A. Cyp26 (a1,b1, and c1), which is a member of the cytochrome P450 family, acts by reducing the activity of RA. Cyp26 type b1 is the predominant subtype expressed in the murine lung. Several studies have suggested that nitrofen may interfere with the retinoid pathway resulting in congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia. Recently, it was reported that nitrofen may act by inhibiting RALDH2. The aim of this study was to examine the pulmonary expression of Cyp26b1, LRAT, and RALDH2, the key enzymes involved in the synthesis of RA, in order to understand the mechanisms underlying pulmonary hypoplasia in the nitrofen CDH model. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9 of gestation (D9). Fetal lungs were harvested at D15, D17, D19, and D21. D17, D19, and D21 lungs were divided into three groups: control, nitrofen without CDH and nitrofen with CDH, whereas D15 lungs were divided into only two groups; control and nitrofen as the diaphragm is not fully formed yet at this stage. Real- time PCR was performed to evaluate the relative level of Cyp26b1, LRAT, and RALDH2 expression in the lung. Relative levels of Cyp26b1 mRNA were significantly decreased in the lungs of nitrofen with CDH (D17;0.19 +/- 0.09, D19;0.70 +/- 0.20, D21;0.40 +/- 0.36) and nitrofen without CDH (D17;0.14 +/- 0.06, D19;0.54 +/- 0.42, D21;0.51 +/- 0.56) compared to controls (D17;0.35 +/- 0.16, D19;1.15 +/- 0.48, D21;1.28 +/- 0.78) (P < 0.05). LRAT expression was also significantly decreased in nitrofen with CDH (D17; 19.3 +/- 7.8, D19; 4.3 +/- 1.1, D21; 3.3 +/- 1.6) and nitrofen without CDH (D17; 21.2 +/- 11.1, D19; 4.5 +/- 3.6, D21; 4.1 +/- 1.6) compared to controls (D17; 153.7 +/- 29.8, D19; 26.8 +/- 16.8 D21; 10.1 +/- 3.8) (P < 0.05). There was no significant difference in the relative levels of Cyp26b1 and LRAT between nitrofen with CDH and nitrofen without CDH. There were no significant differences in RALDH2 expression among the groups at any stages. Down-regulation of Cryp26b1 and LRAT demonstrates that RA content is decreased in nitrofen induced hypoplastic lungs compared to controls. The finding that RALDH2 expression in the hypoplastic lung is not altered suggests that nitrofen may act by interfering with the retinoid metabolism during the early stage of the retinoid signaling pathway.

Acyltransferases↗

PubMed2007

A new rapid acetylcholinesterase staining kit for diagnosing Hirschsprung's disease.

Conventional acetylcholinesterase (AChE) histochemistry is both time consuming and complicated and requires the mixing of reagents that are toxic to the human body. We developed a rapid technique for performing AChE histochemistry, which has already been published, and now present a kit for performing AChE histochemistry that is a further improvement. Rectal suction biopsy specimens taken from 20 constipated patients and three full thickness biopsy specimens taken from 4 Hirschsprung's disease (HD) patients during pull-through surgery from aganglionic, transitional, and ganglionic bowel segments were tested using our rapid technique and the new kit. Each specimen was incubated for only 6 min. All ganglion cells stained clearly for AchE in just 6 min using both techniques. However, the kit was able to stain AchE positive nerve fibers more clearly and did not detect endogenous peroxidase-containing histiocytes, as did the earlier rapid technique. The kit could also detect AchE positive nerve fibers in the circular and longitudinal muscle layers, unlike the earlier rapid technique. The kit allows AChE histochemistry to be performed rapidly with complete accuracy, without any risk for toxicity. Moreover, the kit provides more focused information on AchE distribution in the bowel itself without any extraneous staining and can be used for diagnosing HD and allied disorders as well as establishing the exact level of innervation for pull-through resection.

Acetylcholinesterase↗

PubMed2007

Lack of association of IL8 gene polymorphisms with familial vesico-ureteral reflux.

Vesico-ureteral reflux (VUR) is the most common inherited disorder of the lower urinary tract. Children with VUR are at risk for ongoing renal damage with subsequent infections. IL8 is an important inflammatory mediator which can be produced by epithelial cells of the renal tract in response to a variety of inflammatory stimuli. High serum concentrations of IL-8 have been reported in patients with chronic renal failure. Elevated IL-8 levels have been reported in the urine of patients with VUR and renal parenchymal scarring (RPS). More recently it was reported that urine IL-8 levels remain elevated in infants with VUR even in the absence of a urinary tract infection (UTI). Increased IL-8 expression has been shown to be associated with polymorphism at position -251 (rs4073) of the IL-8 promoter. The aim of this study was to examine the association of IL-8 gene polymorphism with familial VUR in a cohort of 219 siblings from 109 families affected with VUR, the largest such cohort tested to date. RPS was assessed using dimercaptosuccinic acid scintigraphy. Genotyping was performed in 219 siblings with VUR (157 without RPS, 62 with RPS) and 292 controls for the position -251 of IL-8 gene by polymerase chain reaction with tetra primers and gel analysis. Genotype was compared using the chi square test. Statistical significance was taken as a value of P < 0.05. There were no significant differences in IL-8 -251 genotype frequency between VUR patients and controls. Similarly, gender, severity of VUR and renal parenchymal scarring had no effect on IL-8 -251 genotype frequency. Although IL-8 urinary levels have been reported to be elevated in VUR, our data indicate that IL-8 gene is not involved in the pathogenesis of familial VUR or reflux nephropathy.

Chelating Agents↗

PubMed2007

Kidney development in the nitrofen-induced pulmonary hypoplasia and congenital diaphragmatic hernia in rats.

BACKGROUND/PURPOSE: The relationship of the developing lung and kidney is not completely understood. Renal enlargement has been reported in association with pulmonary hypoplasia in congenital diaphragmatic hernia (CDH). Recent studies suggest that retinoids may be involved in the pathogenesis of CDH. The aims of this study were to investigate the effects of pulmonary hypoplasia on renal development and to evaluate retinoids status of kidneys in the nitrofen model of CDH. METHODS: Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9.5 of gestation. Fetuses were recovered at term and divided into 3 groups: 1, control (n = 69); 2, nitrofen without CDH (n = 25); and 3, nitrofen with CDH (n = 40). Kidneys were dissected, weighed, and processed for biochemical measurements of DNA, proteins, total retinol content, and for immunohistochemical staining of proliferating cells. RESULTS: Kidneys were smaller in nitrofen-exposed animals vs control animals (group 3, 0.65 +/- 0.08; group 2, 0.62 +/- 0.09 vs group 1, 0.73 +/- 0.09% of body weight, P < .001), and there were no differences between right and left kidney weight in all the 3 groups. Regression of total kidney weight on body weight showed a linear direct correlation between them in all the groups. Total amount of DNA was significantly reduced in nitrofen-exposed animals vs controls (group 3, 80.58 +/- 35.65; group 2, 64.71 +/- 20.28 vs group 1, 110.34 +/- 42.15 microg, P < .01), but the DNA concentration remained the same in the 3 groups (group 3, 3.59 +/- 1.26; group 2, 3.06 +/- 1.19; group 1, 3.43 +/- 1.05 microg DNA/mg kidney). Total protein content (group 3, 1145.59 +/- 500.36; group 2, 993.2 +/- 276.62; group 1, 1287.48 +/- 312.52 microg), protein concentration (group 3, 49.76 +/- 11.12; group 2, 43.95 +/- 6.79; group 1, 47.38 +/- 6.93 microg protein/mg kidney), and protein-to-DNA ratio (group 3, 15.12 +/- 5.98; group 2, 16.22 +/- 6.85; group I, 16.16 +/- 7.02 microg/microg) were similar in all groups. Retinol concentration was significantly reduced in both nitrofen-exposed groups compared with the control group (group 3, 1.35 +/- 0.24; group 2, 1.28 +/- 0.11; group 1, 2.53+/-0.61 microg retinol/g kidney). Proliferation index was similar in all 3 groups (group 3, 50.43 +/- 8.81; group 2, 47.96 +/- 6.01; group 1, 47.64 +/- 5.76% of proliferating cells). CONCLUSIONS: Our data clearly show that renal enlargement in association with pulmonary hypoplasia is not seen in the nitrofen-induced CDH. These results rule out any possible relationship between lung and kidney development. Moreover, kidneys are hypoplastic in both nitrofen-exposed groups and have reduced retinol content, suggesting that a retinoid pathway disruption could be the common mechanism in the pathogenesis of lung and kidney hypoplasia in the nitrofen model of CDH.

Animals↗

PubMed2006

Symptomatic anal anomalies in chromosome 22q11 deletion syndrome: a report of three patients.

Deletion of chromosome 22q11 is a common genetic condition with varying clinical presentation ranging from neonatal fatality to patients whose presentation to medical services will be prompted after a few years by speech delay or mild developmental concerns. While most published data relating to patients with 22q11 deletions has focused on the "classical" presentation of the condition with cardiac manifestations, hypocalcaemia and velopharyngeal insufficiency, a much wider range of clinical presentations can characterise this syndrome. Anal anomalies, comprising imperforate anus and symptomatic anal stenosis, are a rarely described presentation of this multisystem disorder. In this report we document three patients presenting to paediatric services with symptoms attributed initially to symptomatic anal stenosis/atresia.

Anal Canal↗

PubMed2006

Long-term outcome of patients with enterocolitis complicating Hirschsprung's disease.

Enterocolitis remains the most serious complication of Hirschsprung's disease (HD). The purpose of this study was to evaluate the risk factors in the development of enterocolitis and the long-term outcome in these patients. The hospital records of 259 consecutive patients with HD during 1975-2003 were examined. The data was analysed for age at presentation, associated anomalies, level of aganglionosis, clinical features, number of episodes of enterocolitis, type of pullthrough, necessity for post-pullthrough sphincterectomy. Follow up was carried out by personal interviews and interviews over the telephone with patients/parents. Enterocolitis was diagnosed on the basis of clinical features of diarrhoea, pyrexia, abdominal distention and vomiting. Of the 259 patients with HD, 74 patients (28.5%) were found to have enterocolitis. Out of 39 patients with Down's syndrome and HD, 19 (48%) had enterocolitis. Fifteen (20%) patients had other associated anomalies. Fifty-six patients (75.6%) were male and 18 (24.3%) were female. In 30 patients enterocolitis was the presenting feature in the neonatal period, 22 of which presented in the first 2 weeks of life. Fifty-six patients (75.6%) had rectosigmoid disease and 18 (24.3%) had long segment disease or total colonic aganglionosis. Eighteen (24.3%) had only preoperative enterocolitis and 31(41.8%) had only postoperative enterocolitis. Twenty-five (33.7%) had both pre- and post-operative enterocolitis. Twenty (27%) patients had more than 2 episodes of enterocolitis. Various pullthrough procedures were performed. Twenty-six patients (35.1%) required internal sphinctermyectomy to treat the enterocolitis. At the time of follow-up, 16 patients were lost to follow-up. Of the remaining 58 patients, 3 patients died, 2 due to enterocolitis and 1 due to sepsis. Six patients remained with a stoma. Twenty-two patients were continent and were stooling normally, but 14 of these were on laxatives for several years after pullthrough procedure. The mean age at achieving full continence was 4.95 years in the 22 patients with normal bowel function. Nineteen patients are still on laxatives and 8 patients are soiling. Eight patients continue to have multiple recurrent episodes of enterocolitis at follow-up. Down's syndrome is an important risk factor in the development of enterocolitis. The majority of patients with enterocolitis complicating HD continue to have disturbances of bowel function many years after surgery for HD.

Cathartics↗

PubMed2006

Retinoic acid rescues lung hypoplasia in nitrofen-induced hypoplastic foetal rat lung explants.

There is increasing evidence to suggest that the retinoid pathway is involved in the pathogenesis of congenital diaphragmatic hernia (CDH). We hypothesised that retinoids are involved in the pathogenesis of associated pulmonary hypoplasia in CDH and therefore designed this study to investigate the effects of retinoid acid on nitrofen-induced hypoplastic lungs. Pregnant rats were exposed to either olive oil or 100 mg nitrofen on day 9.5 of gestation. Foetal lungs were harvested on embryonic day 13.5 and were cultured for 96 h with or without exogenous retinoic acid (RA) (1 muM) added daily to the culture medium. Lungs were divided into four study groups: control (n=31); control + RA (n=19); nitrofen (n=19); and nitrofen + RA (n=12). Lung growth was assessed in each group by measuring branching morphogenesis, total DNA content and the proportion of proliferating cells stained by immunohistochemistry. One-way ANOVA test was used for statistical analysis. Retinoic acid significantly increased the growth of nitrofen-induced hypoplastic lungs, whilst growth of control lungs did not change. The number of lung buds and lung area of nitrofen-exposed hypoplastic lungs after 96 h of culture significantly increased after the addition of RA compared to the non-treated hypoplastic lungs (25.75+/-6.47 vs 15.11+/-3.29 and 0.98+/-0.18 mm(2) vs 0.65+/-0.13 mm(2), respectively; P<0.0001). Lung perimeter was also higher when RA was added to hypoplastic lungs compared to the non-treated ones, although it did not reach significance (12.51+/-2.53 mm vs 11.19+/-2.56 mm; P=0.17). Conversely, the addition of RA to control lungs did not affect the number of lung buds, lung area or lung perimeter after 96 h in culture compared to the non-treated ones (31.28+/-4.66 vs 31.81+/-6.67; 1.29+/-0.18(2) vs 1.29+/-0.23 mm(2) and 18.47+/-3.47 mm vs 17.89+/-2.94 mm, respectively; P=NS). Retinoic acid also increased the total DNA content and the proportion of proliferating cells in hypoplastic lungs compared to the non-treated ones (2.59+/-0.58 mug vs 1.96+/-0.31 mug and 57.89+/-9.46% vs 36.76+/-8.15%, respectively; P<0.001). The addition of RA did not affect either total DNA content or the proportion of proliferating cells in control lungs compared to the non-treated ones (4.04+/-0.64 mug vs 3.79+/-0.85 mug and 58.67+/-11.23% vs 56.03+/-10.36%, respectively; P=NS). This study demonstrates for the first time that RA rescues lung hypoplasia in nitrofen-induced hypoplastic lungs. These results suggest that retinoid pathway may be involved in the pathogenesis of associated pulmonary hypoplasia in CDH.

Analysis of Variance↗

PubMed2006

Altered renin-angiotensin system gene expression causes renal hypoplasia in the rats with nitrofen-induced diaphragmatic hernia.

The association between renal hypoplasia and pulmonary hypoplasia in congenital diaphragmatic hernia (CDH) has become recently appreciated. However, the underlying mechanisms responsible for this association are still unknown. Renin-angiotensin system (RAS) plays an important role in renal and somatic growth, angiogenesis and reproduction. We hypothesized that abnormal expression of RAS components may be responsible for renal hypoplasia in CDH. We therefore designed this study to examine the gene expression of main components of RAS in the kidney of nitrofen-induced CDH in the rat. Pregnant rats were exposed to either olive oil or 100 mg of nitrofen on day 9.5 of gestation. Foetuses were recovered at term and divided into three groups: control (n=8), nitrofen without CDH (n=8) and CDH (n=8). Reverse transcription polymerase chain reaction was performed to evaluate the relative amount of angiotensinogen (AGT), angiotensin II type 1 receptor with 1a and 1b subtypes (AT(1a)R and AT(1b)R), angiotensin II type 2 receptor (AT(2)R), angiotensin-converting enzyme (ACE) and renin expression in the kidney. AT(1a)R, AT(1b)R, AT(2)R, AGT and renin levels were significantly decreased in the kidney of CDH rats compared with controls. We did not find a significant difference in ACE between CDH animals and controls. Our data show that the downregulation of RAS may play an important role in the pathogenesis of renal hypoplasia in the nitrofen-induced CDH.

Abnormalities, Multiple↗

PubMed2006

Enteric nervous system and developmental abnormalities in childhood.

ENS consists of a complex network of neurons, organised in several plexuses, which interact by means of numerous neurotransmitters. It is capable of modulating the intestinal motility, exocrine and endocrine secretions, microcirculation and immune and inflammatory responses within the gastrointestinal tract, independent of the central nervous system. Though the embryological development of various plexuses are completed by mid-way of gestation, the maturation of neurons and nerve plexuses appear to continue well after birth. Therefore, any histological or functional abnormalities related to the gastrointestinal function must be investigated with the ongoing maturational processes in mind.

Animals↗

PubMed2006

Long-term results of bowel function after treatment for Hirschsprung's disease: a 29-year review.

Although various surgical procedures have been described to treat Hirschsprung's disease (HD), few studies have evaluated the long-term results of these children. The purpose of this study was to assess the long-term clinical outcome and bowel function of patients with HD. The hospital records of 259 consecutive patients with a confirmed histological diagnosis of HD during 1975-2003 were examined. Data was assessed for age at presentation, sex, clinical presentation, associated anomalies, level of aganglionosis, surgical procedures, complications and bowel function. Follow up was carried out by personal/telephone interviews with patients or their parents. Of the 259 patients with HD, 200 were males (77.2%) and 59 females (22.8%). Intestinal obstruction was the presenting feature in 147 patients (56.8%), intestinal perforation in 5 (1.9%), enterocolitis in 30 (11.6%) and constipation in 77 (29.7%). Thirty-nine patients (15.1%) had associated Down's syndrome. Two hundred and nine patients (80.7%) had rectosigmoid disease, 31 (12%) had long segment disease and 19 (7.3%) had total colonic aganglionosis. Forty-three patients (16.6%) had preoperative enterocolitis. Primary colostomy was performed in 160 patients and a primary pull through in 90. Seven patients had a sphincteromyectomy for ultrashort HD. Two patients died prior to treatment. Various pull through procedures were performed in these patients. Postoperative complications included: pelvic abcess in 2, rectal stricture in 10, perianal excoriation in 7, anastomotic leak in 8, intestinal obstruction in 3, wound dehiscence in 1, stomal prolapse/stenosis in 5, rectovesical fistula in 2 and enterocolitis in 56. Five patients underwent a redo pull through and 46 required a post pull through sphincterectomy. At the time of follow-up, 27 were lost to follow-up, 9 died, 18 had permanent stomas and 4 were too young to assess bowel function. Of the remaining 194 patients, bowel function was normal in 132 (68%). Twenty patients (10.3%) had soiling and 42 (21.7%) had constipation requiring laxatives or enemas. There was no difference in bowel function in relation to type of pull through operation. Only 34% of patients with Down's syndrome had normal continence. The majority of patients with HD continue to have disturbances of bowel function for many years before attaining normal continence.

Adolescent↗

PubMed2006

Vagal neural crest contribution to the chick embryo cloaca.

Intrinsic innervation of the developing chick cloaca is provided by the enteric nervous system, a network of neurons and glia that lies within its walls. The enteric nervous system originates from neural crest cells that migrate from the vagal and sacral regions of the neural tube during the early stages of development. Abnormal cloacal development can cause a number of anorectal anomalies including persistent cloaca. Our study aimed to investigate the contribution of vagal neural crest cells to the total population of enteric neurons and glia within the chick embryo cloaca, using quail-chick chimeras. Chicken embryos were incubated until the 10-12 somite stage (ss). The vagal neural tube, corresponding to somites 1-7, was then microsurgically ablated in ovo and isochronic and isotopic quail grafts were performed. The eggs were then reincubated until embryos were harvested at E12. Whole embryos were fixed in Bouin's fluid, embedded in paraffin wax and sectioned. Immunohistochemistry was carried out using the HNK-1 antibody to label all neural crest cells, and the quail-specific antibody, QCPN, to label quail cells. QCPN-immunoreactive cells were seen to make up a large proportion of enteric neurons and glia within the walls of the embryonic cloaca. HNK-1 labelled all neural crest cells in the myenteric and submucosal plexuses as well as the sacral crest-derived nerve of Remak, while QCPN-positive cells were evident in both plexuses but mostly in the submucosal plexus, where they appeared to make up the majority of neurons. Results show that the chick embryo cloaca is primarily innervated by vagal neural crest cells. Further studies to investigate the contribution of sacral neural crest cells to the same region will give further insight into the development of the enteric nervous system within the embryonic cloaca.

Animals↗

PubMed2006

Postnatal development of the mucosal plexus in the porcine small and large intestine.

Knowledge regarding the foetal and postnatal development of the enteric nervous system is crucial for the understanding of congenital disorders. While lot of information exists regarding the myenteric and submucosal plexuses, the development of the mucosal plexus has not been previously studied. The mucosal innervation seems to play an important role in the local reflex activity of the gut. In this study, we examined the development of enteric mucosal innervation in the pig at various ages of life. Small and large bowel paraffin-embedded specimens were stained with PGP 9.5 and neurofilament protein in three piglets from six age groups (60 and 90 days gestation, newborn, 4 and 12 weeks old, and adult pigs). Small and large bowel demonstrated identical innervation patterns. Myenteric and submucosal plexuses were stained with PGP 9.5 at 60 days gestation. However, the mucosal staining was first noted clearly at the newborn period. By 4 weeks, PGP 9.5 staining was noted in small amounts within the mucosa. Inner proprial and villous fibres were seen ahead in time to the subepithelial fibres. Both inner proprial and villous staining became quiet prominent by 12 weeks of age and remained unchanged into adulthood. However, the subepithelial fibres appear to increase in adulthood. This study demonstrates for the first time that enteric mucosal innervation first appears only at birth. The immaturity of the mucosa generated reflex activity, and secretory functions may have implication in the management of functional intestinal obstruction in the premature infant.

Age Factors↗

PubMed2006

Elevated expression of IL2 is associated with increased infiltration of CD8+ T cells in biliary atresia.

AIM OF STUDY: Lymphocyte-mediated inflammation within the liver and bile ducts has been proposed as a potential mechanism in the pathogenesis of biliary atresia (BA). Recent reports have suggested that BA is associated with upregulation of TH1-type inflammatory cytokines. Quantitative real-time polymerase chain reaction (qrt-PCR) is a useful tool to assess immune cell density and cytokine gene expression in tissues. The aim of this study was to characterize the inflammation in BA by quantifying lymphocyte density and expression of TH1 cytokines such as IL2 and tumor necrosis factor alpha (TNF-alpha) in liver biopsies using qrt-PCR. METHODS: Total RNA was extracted from archival formalin-fixed paraffin-embedded liver biopsy specimens collected from three age-matched groups of patients: group I, nonsyndromic BA at the time of Kasai portoenterostomy (n = 10); group II, choledochal dilatation at the time of surgery (n = 2); group III, other cholestatic diseases including biliary hypoplasia and parenteral nutrition-associated cholestasis (n = 3). Qrt reverse transcription-PCR was performed to measure the relative levels of messenger RNA (mRNA) expression of CD4, CD8, IL2, and TNF-alpha. Their levels were normalized to the level of beta-actin expression. The numbers of CD4+ and CD8+ lymphocytes infiltrating portal tracts were also measured using immunohistochemistry. Data were expressed as median and ranges, and statistical comparison and correlation was by nonparametric test (Kruskal-Wallis and Spearman rank). RESULTS: CD8 mRNA expression was significantly increased in group I (median 0.26, range 0.11-6.29) compared with groups II and III (0.002, 0-0.004 and 0.02, 0-0.12) (P < .05). Levels of IL2 mRNA expression were also significantly increased in group I (1.22, 0.05-7.54) compared with groups II (0.58, 0-1.17) and III (0, 0-0.03) (P < .05). In contrast, there was no significant increase in CD4 and TNF-alpha expression in group I compared with groups II and III. There was a significant correlation between the number of CD8+ lymphocytes in portal tracts and the levels of CD8 mRNA expression. CONCLUSION: Increased expression of IL2 and increased CD8 T-cell infiltration in liver biopsy specimens from infants with BA suggest the possibility that IL2 plays a role in the pathogenesis of this condition.

Biliary Atresia↗

PubMed2006

Developmental changes in submucosal nitrergic neurons in the porcine distal colon.

BACKGROUND/PURPOSE: As our understanding of the enteric nervous system improves, it becomes clear that it is no longer sufficient to simply determine whether enteric ganglion cells are present but also to determine whether correct number and types of ganglion cells are present. Nitric oxide is recognized as a potent mediator of inhibitory nerves responsible for the relaxation of the smooth muscle of the gastrointestinal tract. The aim of this study was to determine the normal nitrergic neuronal density and morphology in the submucosal plexus of the porcine distal bowel from fetal life to adulthood. METHODS: Distal large bowel specimens were obtained from porcine fetuses of gestational age E60 (n = 5), E90 (n = 5), 1-day-old piglets (n = 5), 4-week-old piglets (n = 5), 12-week-old piglets (n = 5), and adult pigs (n = 5). Whole-mount preparations of the submucosal plexus were made and stained with NADPH diaphorase histochemistry. The ganglia density, the number of ganglion cells per ganglia, and nucleus and cytoplasmic area were measured. RESULTS: Ganglia density decreased progressively and markedly with age until the adulthood (P < .001). On the contrary, ganglion cells increased their size over time predominantly because of increase in cytoplasm (P < .001). The number of ganglion cells per ganglia increased significantly during the fetal life. However, there was a significant reduction in the number of ganglion cells per ganglia during the period from birth to 4 weeks, remaining constant thereafter (P < .001). CONCLUSIONS: The quantitative and qualitative morphometric analysis of the colonic submucous plexus shows that significant developmental changes occur during fetal and postnatal life. These findings indicate that the age of the patient is of utmost importance during histopathologic evaluation of enteric nervous system disorders.

Age Factors↗

PubMed2006

Increased CXCR3 expression associated with CD3-positive lymphocytes in the liver and biliary remnant in biliary atresia.

BACKGROUND: Lymphocyte-mediated inflammatory damage of the bile ducts has been proposed as a potential mechanism in the pathogenesis of biliary atresia (BA). Chemokines regulate leukocyte migration and act as critical organizers of cell distribution in inflammatory responses. The aim of this study was to analyze the infiltration of T lymphocytes and the expression of a chemokine receptor, CXCR3, predominantly expressed on type 1 polarized T cells (T(H)1, T(C)1) in the liver and excised biliary remnants in infants with BA. METHODS: Immunohistochemistry for CD3, CD8, and CXCR3 was performed using liver biopsy specimens collected from the following 3 age-matched groups of patients: group 1, BA (nonsyndromic) at the time of Kasai portoenterostomy (n = 10); group 2, congenital choledochal dilatation (n = 2); and group 3, other cholestatic diseases including paucity of intrahepatic bile ducts and cholestasis (n = 3) related to total parenteral nutrition. Cellular staining on each section was graded from 0 to 4 and compared using nonparametric statistics. RESULTS: Infiltrating CD3+ and CD8+ lymphocytes in the portal tracts were significantly increased in group 1 (3.1 +/- 0.4, 2.8 +/- 0.4), compared with groups 2 (1.0 +/- 0.0, 1.0 +/- 0.0) and 3 (1.7 +/- 0.3, 1.5 +/- 0.5) (P < .01, P < .05). CXCR3+ mononuclear cells were significantly increased in group 1 (2.6 +/- 0.3) compared with groups 2 (0.5 +/- 0.5) and 3 (0.7 +/- 0.3) (P < .05). They were mainly found in the portal tracts with a similar distribution to CD3+ cells. CXCR3+ cells and CD3+ cells also showed a similar distribution in specimens of biliary remnants from just below the portal plate. CONCLUSIONS: Increased expression of CXCR3 associated with a significantly increased CD3 and CD8 T-cell infiltration suggests that CXCR3+ lymphocytes in a type 1 (T(H)1, T(C)1) cytokine milieu may play a role in the pathogenesis of BA.

Bile Ducts↗

PubMed2006

Effect of VEGF on the branching morphogenesis of normal and nitrofen-induced hypoplastic fetal rat lung explants.

PURPOSE: Changes in vascular structures as well as vascular endothelial growth factor (VEGF) downregulation have been reported in hypoplastic lungs associated with congenital diaphragmatic hernia. We hypothesized that VEGF may accelerate branching morphogenesis and thus may modulate lung growth in normal and nitrofen-induced pulmonary hypoplastic lungs. METHODS: A hypoplastic fetal lung model and a normal control lung model were induced by feeding pregnant rats with or without nitrofen, respectively. Fetal lungs harvested on day 13.5 were cultured at ambient oxygen tensions for 72 hours with 0, 25, 50, or 100 ng/mL of exogenous rat VEGF added daily in the serum-free medium. The rates of increase in bud count and airway contour were evaluated. Real-time polymerase chain reaction was carried out to evaluate the expression of surfactant protein C mRNA in the explants at the end of culture. RESULTS: Vascular endothelial growth factor accelerated the increase in bud count and airway contour in normal and hypoplastic lung explants compared to controls. Surfactant protein C mRNA expression was significantly increased at 50 ng/mL VEGF compared to controls in both normal and hypoplastic lung explants. CONCLUSION: These data suggest that VEGF plays an important role in lung morphogenesis and may accelerate lung growth in nitrofen-induced hypoplastic lung.

Animals↗

PubMed2006

Familial vesicoureteral reflux: influence of sex on prevalence and expression.

PURPOSE: Familial clustering of vesicoureteral reflux implies that genetic factors have an important role in the pathogenesis of vesicoureteral reflux. We investigated the impact of sex on familial vesicoureteral reflux. MATERIALS AND METHODS: Between 1998 and 2003 we identified 159 white families with at least 2 children (range 2 to 5) with vesicoureteral reflux confirmed by a voiding cystourethrogram. Families were divided into 3 groups, including group 1 with only boys affected (29 or 18%), group 2 with only girls affected (57 or 36%) and group 3 with boys and girls affected (73 or 46%). Clinical characteristics, reflux grade and associated renal anomalies in these 3 groups were compared using the Mann-Whitney U and chi-square tests for statistical analysis. RESULTS: Sisters of index female patients were at significantly higher risk for vesicoureteral reflux than brothers (p <0.01). Boys in group 1 had a significantly higher grade of vesicoureteral reflux than boys in group 3, and girls in groups 3 and 2 (p = 0.018, <0.001 and <0.0001, respectively). Severe renal scarring was present in 7% of boys in group 1, 6.6% of boys in group 3, 1.3% of girls in group 3 and 1.9% of girls in group 2. Duplex kidneys were present in 15.3% of boys in group 1 but only in 2.3% of boys in group 3 (p <0.01), 5.7% of girls in group 3 and 5.8% of girls in group 2. Age at diagnosis was not significantly different among boys in groups 1 and 3, and girls in group 2 (median 0.9, 0.6 and 1.9 years, respectively). CONCLUSIONS: The risk and severity of vesicoureteral reflux in siblings depends on the sex of the affected child. Brothers of index male patients have higher grade of reflux and higher rate of associated duplex systems. This has implications for genetic counseling and for modeling the inheritance of vesicoureteral reflux.

Child, Preschool↗