Gastroenterology

Comparison Of Platelet-Activating Factor Levels In Infants With Necrotizing Enterocolitis And A Neonatal Pig Model.

S. Piecuch, G. Condemi, P. Dzakpasu, G. Valencia, E. Kornecki, P.M. Gootman, S. Rabinowitz (intr. by K. Bromberg), Department of Pediatrics, State University of New York - Health Sciences Center, Brooklyn, NY.

Objective: Platelet-activating factor (PAF) levels in neonatal pigs subjected to stress factors linked to the development of necrotizing enterocolitis (NEC) were compared with PAF levels observed in infants with clinical NEC. Methods: PAF levels were monitored in 164 infants at risk for NEC, 11 of whom developed Bell stage II or III NEC, in order to determine the relationship between PAF level and NEC. We also determined the effect of acute exposure to stress factors associated with NEC on PAF levels in neonatal pigs. Four pigs, ranging from 2-11 days of age, were fed hyperosmolar formula and subjected to arterial hemorrhage and to hypoxic ventilation with 10% oxygen. The animals were then ventilated with 100% oxygen for a period of 1-10 hours. PAF levels were done following each stressful event, and then intermittently during the subsequent period of observation. We compared the PAF levels in the stressed pigs to those observed in the infants. Results: The mean PAF level was 2.0 ng/ml in the 153 infants without NEC and 13.6 ng/ml in the 11 infants with Bell stage II or III NEC (significantly increased, p<0.05). PAF levels increased from baseline when NEC developed and returned to baseline when it resolved; the degree of PAF elevation correlated positively with the severity of illness. Acute stress did not have a similar effect on PAF levels in the pigs. All 4 animals had PAF level <0.03 ng/ml following instrumentation, prior to stress. Pig #3 became unstable and expired 6 hours post-stress; peak PAF level prior to death was 5.8 ng/ml. PAF levels did not exceed 3.0 ng/ml in the other 3 animals, 2 of which experienced significant periods of physiologic instability following stress. None of the 4 pigs demonstrated an increase in PAF level to the degree seen in human infants with NEC. Conclusions: PAF levels were increased in human infants with NEC, and the degree of increase was positively correlated with the severity of disease. Neonatal pigs subjected to severe acute stress did not develop a comparable degree of elevation in PAF level, suggesting that PAF elevation is not simply a non-specific response to acute stress and may be a relatively specific marker for NEC.

Lipopolysaccharide-Induced Inflammation Results In Loss Of Glycosaminoglycans.

MS Salat, HS Dweck and L Rosenfeld. Neonatal Research Laboratory, Westchester Medical Center, New York Medical College, Valhalla, NY.

Necrotizing enterocolitis (NEC) is an acute inflammatory disease of newboms. In vitro studies by our group have shown that products of nitric oxide degrade extracellular matrix glycosaminoglycans (GAGs), such as heparan sulfate (HS) or chondroitin sulfates (CS). The purpose of this study is to determine whether these GAGs are degraded in intestines of rats treated with lipopolysaccharide (LPS). Methods: WKY rats were given intraperitoneal injections of E. coli LPS (0.1 mglkg/day) or vehicle for 5 consecutive days. On day six the rats were sacrificed and organs were removed. GAGs were extracted from the large intestines, then released from protein and radiolabeledwith NaOH-NaB³H₄ followed by extensive proteolysis. After desalting, the GAGs were analyzed by gel filtration column chromatography (GF). Results: The GAGs extracted from rat large intestine separated as two peaks in GF analysis (Figure). There was a substantial loss of the material in peak 2 in LPS-trealed tissues relative to the control. Preliminary analyses suggest that peak 2 is CS. Conclusion: LPS treatment results in loss of an intestinal GAG. These experiments support our hypothesis that products of nitric oxide, released in excess during inflammation, lead to degradation of extracellular matrix GAGs. This process may be a part of the pathogenesis of NEC.

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Zonula Occludens Toxin (Zot) Inhibits Intracellular Trafficking Of Luciferase Dna In Susceptible Cells.

T. L. Watts, J. Malone, R. Malone, A. Fasano, Center for vaccine Development and Division of Pathology, University of Maryland, Baltimore, MD.

Zonula occludens toxin (Zot) is a protein elaborated by Vibrio Cholerae that modulates the opening of intercellular tight junctions (tj). The mechanism by which Zot modulates tj involves rearrangement of the cytoskeleton with resultant polymerization of actin fibers via a PKC dependent pathway. Thus, Zot promotes an increase in tissue permeability and a reduction in transepithelial resistance by opening tj. Preliminary data suggests that Zot is a microtubule binding protein capable of modulating normal intracellular trafficking patterns. Using a luciferase trasnfection assay, various cell lines were trans-fected with luciferase DNA in the presence or absence of Zot. In addition, cells were exposed to Zot either before or after luciferase transfection.

Three general phenotypes were observed. 410.4 cells showed a reduction in DNA expression either after pre- or post-treatment with Zot. DU145, NIH 3T3, and Cos-7 cells lacked DNA expression only when Zot was administered after DNA transfection. In cells known to be deficient for the Zot receptor (Iva, T84) there was no observable effect on luciferase expression in the presence of Zot. These obser-vations have led to the conclusion that Zot may inhibit DNA expression due to its ability to modulate intracellular trafficking.

An Unusual Presentation Of Extra-Hepatic Biliary Atresia.

J Rosado, A.R. DeFelice, F Zapata, R Ramon, N Sharma, and P.G. Kazlow. Department of Pediatrics, Columbia University, New York, NY.

It is believed that extra-hepatic biliary atresia (EHBA) is typically not found in premature infants. In addition, children with EHBA classically present with light-colored, or acholic, stools. The patient, a 32 week gestation, twin A, was delivered via caesarean section. Birth weight was 1170 g. Apgars were 7 and 8. The patient was fed enterally and never received hyperalimentation. The neonatal course was complicated by sepsis, which responded to antibiotic treatment. The infant was noted to have conjugated hyperbilirubinemia (6.0/2.3mg/dl) on day 2 of life. The direct bilirubin peaked at a level of 9.3/3.7 mg/dl, on day 5 of life, then began to decrease. However, the bilirubin then reached a plateau of 9.3/2.0 mg/dl. Liver function tests were elevated, including alkaline phosphatase 231 u/l, AST 55 u/l, ALT 53 u/l and GGT P 2074 u/l. Work-up for infectious and metabolic etiologies was negative. The patient was then placed on Phenobarbital. An initial DISIDA scan revealed no excretion of isotope into the duodenum. During this entire time, the child continued to produce stools that were greenish-yellow in color. The child was then placed on Actigal, and a repeat DISIDA scan again showed no excretion. A percutaneous liver biopsy, at age 11 weeks, revealed bile ductule proliferation, inflammation, and mild fibrosis. The child was taken to the operating room, where he underwent a Kasai (hepatoportoenterostomy) procedure. This resulted in a fall of the serum bilirubin and an improvement of the liver function tests. EHBA is the result of a progressive, inflammatory process that leads to the obliteration of the biliary tract. Cases in still-births, or in premature infants, are rare. Stools are generally acholic at the time of presentation. The etiology of this condition is unknown. Many theories have been proposed, including infectious, ischemic, immunologic and toxic causes. Our patient was intriguing in that he was premature and maintained pigmented stools throughout the course of his work-up. The Kasai procedure is most successful when performed before 60 days of age. We therefore suggest an aggressive work-up of all infants with neonatal jaundice, even those born prematurely and having pigmented stools, to rule out EHBA as a diagnostic entity.

Epstein-Barr Virus Hepatitis As A Precursor To Scleroderma In An Adolescent.

E Treskova, I Shendrik*, N Galeano, A.R. Depelice, and P.G. Kazlow. Departments of Pediatrics and Pathology*, Columbia University, New York, NY.

Scleroderma has classically been regarded as a dermatological condition. Recent studies have described multi-system involvement, particularly in the gastrointestinal tract. A 14 y.o. female presented to our hospital with complaints of fatigue, dysuria, swollen feet and a 20 lb weight gain. Physical examination was notable for marked bilateral pitting edema of the lower extremities. Laboratory data revealed a normal CBC. There was hypoalbuminemia (2.8 g/dl) and elevation of the liver enzymes (AST 113 u/l; ALT 148 u/l). Alkaline phosphatase and GGT were normal. An autoimmune work-up, including ANA, anti-DNA, anti-cardiolipin antibodies, anti-mitochondrial antibodies, was negative. Smooth muscle antibody titers were positive at 1:80. Serology for hepatitis A, B, and C was negative. Abdominal ultrasound revealed splenomegaly, with a normal-appearing liver. The patient experienced an initial clinical improvement but soon became worse. At reevaluation 3 months later, she had developed hepatomegaly and elevation of all liver function tests (AST 356 u/l; ALT 58 u/l; GGT 202 u/l). EBV-VCA titers, which had initially been negative, were now increased to 1:2500. The patient underwent a percutaneous liver biopsy, which revealed diffuse sinusoidal lymphocytic infiltration consistent with EBV infection. The patient again experienced a clinical remission with the use of vigorous diuretic therapy. Three months later, she presented again, with stiffness of the carpal, metacarpal and tarsal joints. She had also developed areas of patchy hypo- and hyperpigmentation of her skin. There was also the onset of Raynaud's phenomenon and dyspnea upon exertion. At that time, a diagnosis of scleroderma was made. She was treated with Prednisone and 6-MP and experienced significant improvement, both clinically and in all biochemical parameters. Scleroderma is a diagnosis that is only infrequently made in the pediatric age group. There is a great deal of speculation as to the role of infectious agents, particularly the Epstein-Barr Virus, in auto-immune disease. The Epstein-Barr nuclear antigen-1 has a highly antigenic glycine-alanine region which cross reacts with similar epitopes in many human proteins. The clinician should be alerted to look for possible auto-immune disease in the patient who presents with that which appears to be multi-system EBV infection.

Neonatal Cholestasis And Urinary Thiobarbituric Acid Reactive Substance (Utbars) Levels.

Kazimierz Watorek, Richard Strauss, Gisela Wirz, Mark Hiatt, Thomas Hegyi UMDNJ-RWJ Medical School, St. Peter's Medical Center, Div of Neonatology, and Dept of Environmental and Community Medicine, N Brunswick, NJ.

Neonatal cholestasis (Direct bilirubin level, (DBL) >2mg/dl on day >10) was associated with increased levels of uTBARS in a group of preterm infants. Urine samples from 10 cholestaic infants (BW 1034±667g; GA 27.4±4.7wks) and 27 controls (BW 1306±679g; GA 29.4±4.1wks) were collected on 39.4±35.6 days of life and frozen at -70°F for later examination. The pH adjusted sample was combined with TBA, heated, and analyzed by spectrophotometric analysis. The groups were similar with respect to Apgar scores, oxygen requirement, arterial oxygen content, and serum bilirubin levels during the first week of life. uTBARS excretion levels (ng/mg of creatinine) are shown below:

Multiple regression analysis revealed significant relationships between DBL and oxygen requirement, maximal total serum bilirubin concentration, and birth weight. Of note, there were no relationships noted between DBL and intravenous nutrition data. uTBARS levels were not associated with any of these variables. In these infants, cholestasis was associated with abnormal liver function tests, and with a non significant increase in uTBARS, products of lipid peroxidation, suggesting a role for reactive oxygen substances in liver damage.

Linkage Disequilibrium Analysis Of The Gaucher Disease N370S Mutation.

¹GA Diaz, ¹BD Gelb, ²N. Risch, ³T. Nygaard, ⁴I. Maire, ⁵L. Poenaru, ⁵C. Caillaud, ⁶C. Sa Miranda, ⁶O. Amaral, ¹PK. Mistry and ¹RJ. Desnick. ¹Department of Human Genetics, Mount Sinai School of Medicine, New York, NY; ²Department of Genetics, Stanford University, Stanford, CA; ³Department of Neuroscience, UMDNJ-New Jersey Medical School, Newark, NJ; ⁴Hopital Debrousse, Lyon, France; ⁵Laboratoire de Genetique, Hopital Cochin, Paris, France; ⁶Institute de Genetica Medica, Porto, Portugal.

Gaucher disease, a lysosomal storage disease caused by glucocerebrosidase (GBA) deficiency, is far more common in the Ashkenazi Jewish (AJ) population, for reasons which remain controversial. Previous work with GBA polymorphisms suggested that the commonest mutation, N370S, arose from a founder in the AJ population. To investigate the genetic history of this disorder, a 9.2-cM region containing the GBA locus was finely mapped with 10 short tandem repeat markers which were then used for genotyping AJ (n=140) and non-AJ Portuguese (n=27), and French (n=19) chromosomes carrying N370S mutations. Marker allele frequencies were generated for N370S and control chromosomes from each population. Greater than 92% of the AJ N370S haplotypes were conserved at three of the four central markers, indicating the existence of a common founder. The AJ N370S haplotype was observed frequently in the French and Portuguese populations, although about 26% and 33%, respectively, had divergent haplotypes, indicating recurrent N370S mutation events. The linkage disequilibrium parameter, δ, was calculated for the marker loci. The results for the AJ N370S were consistent with a more ancient coalescence for the GBA mutation than for the common AJ idiopathic torsion dystonia mutation which had previously been dated back ~17 generations (~1650). French and Portuguese N370S chromosomes carrying the central AJ N370S haplotype had less overall linkage disequilibrium at loci flanking the conserved core haplotype than did the AJ N370S chromosomes. These findings disprove the hypothesis that the N370S mutation arose in the Ashkenazi population and then was bred into the non-Jewish European populations by admixture. Rather, the AJ N370S was ancient in Europe and rose to high prevalence after its introduction into the AJ population. The finding that AJ and European non-AJ N370S chromosomes share a common origin do not support models positing heterozygote advantage as a mechanism to explain the high frequency of the N370S mutation in the AJ population.

Prevalence Of Celiac Disease Among First And Second Digree Relatives In The U.S.A.

I. Berti, K. Horvat, T. Not, A. Fasano. Division of Pediatric GI and Nutrition, University of Maryland School of Medicine, Baltimore, MD U.S.A.

Introduction: Large epidemiological studies showed a high prevalence (1:300) of Celiac disease (CD) throughout Europe. In contrast, CD is still considered rare in U.S.A, although our blood donor screening study suggested a prevalence similar to that reported in Europe(1). Aims of this study were: 1. To establish the prevalence of CD among first and second degree relatives of celiac patients in U.S.A, and, 2. To compare the sensitivity and specificity of transglutaminase (TG) assay to the antiendomysium antibodies (EmA) test. Methods: Informed consent was obtained from 746 first degree, and 110 second degree relatives of CD subjects recruited through local celiac support groups. A questionnaire was used to collect relationship and medical data from all subjects. Ema IgA antibodies were detected in sera by indirect immunofluorescence (IFA) using both human umbilical cord vein and monkey esophagus cryosections as substrate. IgA and IgG antigliadin (AGA) antibodies were detected using an ELISA method. IgG AGA positive sera were also tested for total IgA level. All Ema-positive and a subgroup of Ema-negative samples were also tested for IgA and IgG anti-TG by ELISA. Intestinal endoscopy and biopsy was recommended to all subjects with positive EmA test and/or elevated AGA, and to subjots AGA IgG positive with total IgA deficiency. Results: The results summarized in the table show that 35/746 (4.7%) first degree relatives were EmA positive. In 8 of them, intestinal biopsy was performed and showed a flat mucosa. Five of the twenty-four first degree relatives that had elevated IgA and IgG AGA underwent to intestinal biopsy. Three of them (60%) showed partial villous atrophy. Four out of 110 (3.6%) second degree relatives were EmA positive. Comparison of EmA-positive sera with anti-TG assay revealed a sensitivity of 91% for IgA and 71% for IgG. EmA-negative samples were also tested for IgA and IgG anti-tTG. 316/323 were IgA anti-tTG negative (specificity 98%) and 312/323 were IgG anti-tTG negative (specificity 96.4%).

Discussion: Our data show a projected high prevalence of CD among first (6.3%) and second (3.6%) degree relatives of U.S. patients. IgA EmA and IgA anti-tTG antibodies have a predictive positive value of 100%. Besides, in 60% of subjects AGA + that underwent intestinal biopsy, we found histologic features of celiac disease, even if the mucosal lesion was moderate. In our study tissue ITG autoantibodies are highly sensitive (IgA: 91%; IgG 76%) and specific (IgA 98%, IgG 96.4%) indicators for untreated CD and represent a valuable objective test to diagnose this condition.

1. Scand J Gastroenterol 1998:33:494-498.