P106

ASSOCIATION FOR ACADEMIC SURGERY AND SOCIETY OF UNIVERSITY SURGEONS—ABSTRACTS P105. SMALL INTESTINAL LYMPHOID AGGREGATES: IMPLICATIONS IN THE PATHOGENESIS OF NECROTIZING ENTERCOLITIS. J. Halkias, B. Podd, K. Goth, V. Camerini; Saban Research Institute and Division of Neonatology, Childrens Hospital Los Angeles and University of Southern California, Los Angeles, CA Defects in the gastrointestinal mucosal immune system may account for the unique susceptibility of premature infants to the development of necrotizing enterocolitis (NEC). Although prematurity and subsequent feeding are risk factors, understanding the development and function of T cells within the mucosa of the gastrointestinal tract may give insight to NEC pathophysiology. Small lymphoid aggregates are found throughout the length of the small intestine of mice, within the lamina propria and below the epithelial crypts. We have previously shown a temporal relationship between the appearance of lymphoid aggregates and the subsequent enrichment in intraepithelial lymphocytes (IEL) coincident with the time of weaning in mice. We hypothesize that defects in T cell or B cell colonization of lymphoid aggregates may correlate with NEC pathogenesis. Consistent with this hypothesis, we observed that T cells were largely absent from lymphoid aggregates in the small intestine of infants with NEC compared to control infants with ileal atresia. In this study, we found that lymphoid aggregates in mice were initially T cell rich and subsequently colonized by B cells. The appearance of B cells was coincident with the time of weaning, suggesting that bacterial flora drive the transition in lymphoid aggregate maturation in the small intestine. Regulatory T cells inhibit the activation and proliferation of effector T cells, leading to antigen-specific suppression of inflammation, immunehomeostasis, and tolerance. The site of induction of tolerance to bacteria flora is currently unknown. Here, we identified T cells bearing a surface phenotype consistent with regulatory T cells within the small lymphoid aggregates and lamina propria of the small intestine. Furthermore, we identified a subset of dendritic cells within these aggregates that have previously been shown to induce tolerance and T regulatory differentiation. We hypothesize that early colonization of the neonatal gut with bacterial flora leads to the generation of tolerance towards commensal organisms, and that the induction of naı¨ve T cells into T regulatory cells occurs in the lymphoid aggregates of the small intestine. We therefore suggest a role for the small lymphoid aggregates as the sites of induction of tolerance to commensal flora, and propose that defects in the development and maturation of lymphoid aggregates contribute to the susceptibility of the premature infant to NEC. Whether mucosal T cells are derived from intestinal lymphoid aggregates, and whether the assembly of lymphoid aggregates in humans mirrors that of mice, is yet unknown.

P106. ENDOTHELIAL CELL INFLUENCE ON HEPATIC PROGENITOR CELL DIFFERENTIATION. M. H. Walkup, N. R. Wright, P. Chandrasekaran, L. L. Samuelson, D. A. Gerber; University of North Carolina, Chapel Hill, NC Introduction: Hepatic stem cell populations have been identified from embryonic stages through adulthood. While this population of cells has a multitude of therapeutic potentials, several challenges exist when working with these cells. One of these challenges involves striking a balance between the proliferative capacity of these cells and their function as hepatocytes or biliary cells. During embryonic development, endothelial cells surround and interact with the liver bud directing its progression towards maturity. While the exact nature of this interaction is unclear, embryos that lack endothelial cells do not develop functional livers. Hypothesis: We propose that creating a co-culture system involving adult hepatic progenitor cells in direct communication with endothelial cells will recapitulate the embryonic milieu and stimulate the cells to either proliferate or differentiate. Methods: Adult hepatic progenitor cells (HPC) were isolated from 6-8 week old mice. The population was enriched for

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those cells expressing the Stem Cell Antigen-1 (Sca-1) surface marker. Miltenyi’s MACS® was used to separate out the Sca-1⫹ population. These cells were placed into co-culture with 3 different endothelial cell lines felt to represent different stages of organ/tissue development. Liver related markers including a-fetoprotein, albumin and CK-19 were analyzed at both the transcriptional and translational levels. Cellular proliferation was studied using colony formation and quantification assays. Therapeutic potential was explored by encapsulating the co-culture system in alginate beads and transplanting the beads into recipient animals. Results: The Sca-1⫹ HPC in co-culture with endothelial cells demonstrated an increase in proliferation compared to control cells that were cultured alone. This result was independent of the endothelial cell line used for co-culture. Culture of the HPC alone demonstrated that the cells are bipotent; during their initial period of proliferation they demonstrate a decrease in expression of liver related markers. Subsequent RT-PCR showed that Sca-1⫹ HPC cultured with Py4.1 cells (hemangioblastoma line) maintained their expression of immature markers. Sca-1⫹ HPC cultured with C-166 (yolk-sac derived) and MEC (myocardial endothelial cells) cell lines expressed mature hepatocyte and biliary markers at later time points. Transplantation experiments confirmed the viability of these cell constructs in an in vivo model. Conclusions: We demonstrated that the HPC population spontaneously loses expression of those markers associated with cellular differentiation. By creating a co-culture model involving Sca-1⫹ HPC and select endothelial cell lines, we have developed a reproducible model that recapitulates the tissue environment from embryogenesis through adulthood. The impact of co-culture leads to an enhanced proliferative rate by the HPC. However, the individual endothelial cell lines have variable effects on HPC differentiation. Future experiments will focus on identifying signaling factors involved in HPC differentiation, as well as further study of the in vivo model.

P107. AN INTERLEUKIN-6 NEUTRALIZING ANTIBODY PREVENTS CYCLOSPORINE INDUCED NEPHROTOXICITY IN MICE. M. Laspina 1, K. G. Maier 1, L. Gatto 2, D. S. Kittur 1; 1Suny Upstate Medical University, Syracuse, NY, 2Suny Cortland, Cortland, NY Background: Chronic use of cyclosporine A (CyA) induces nephrotoxicity primarily due to endothelial dysfunction. In our previous studies, potential mechanisms were identified in vitro and implicated NADPH oxidase and Interleukin-6 (IL-6) as key components in causing endothelial dysfunction. In this study, we tested the hypothesis that NADPH oxidase activity and IL-6 are key components in renal damage in an in vivo model. Methods: Male mice C57B/6 mice from JAX Laboratories at 6-8 weeks were subjected to a low salt diet throughout the trial. After one week on a low salt diet, the mice were injected daily with treatments in 50 ␮L vehicle composed of 75% cremaphor (Sigma) and Ethanol for five weeks. A vehicle alone group was also set aside. Mice were weighed and 25 mg/kg/day cyclosporine (Novartis Pharma) was injected daily. Apocynin (Calbiochem) 20mg/kg were injected either alone or concomitantly with CyA. Another group of mice were administered IL-6 Antibody (R⫹D Systems, Cat # MAB406) at 2 ␮g/day along with CyA. The kidneys were removed en bloc immediately and submitted in formalin for paraffin sections. Trichrome stains were performed. Slides were blinded and ten photographs of cortical areas per treatment group were taken, which covered an estimate of 10% surface area in random fashion. Areas of renal damage, which were determined by tubular necrosis, were identified and quantified by amount of necrosis per photograph. Each photograph was divided into ten blocks, and the number of blocks that contained necrotic tubules per photo were recorded. Results: The two control mice (low salt only) had no damage. The four vehicle mice had trace amounts of tubular necrosis. CyA treatment group demonstrated the highest amount of damage (29/70; 41%). CyA with apocynin, a specific NADPH oxidase inhibitor, was found