Melissa D Halpern

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Chapters

• Halpern, M. D. (2021). Bile Acids in the Pathogenesis of Necrotizing Enterocolitis. In Necrotizing Enterocolitis: Pathogenesis, Diagnosis, and Treatment. CRC Press.

Journals/Publications

• Calton, C. M., Carothers, K., Ramamurthy, S., Jagadish, N., Phanindra, B., Garcia, A., Viswanathan, V. K., & Halpern, M. D. (2024). exacerbates experimental necrotizing enterocolitis via upregulation of the apical sodium-dependent bile acid transporter. American journal of physiology. Gastrointestinal and liver physiology, 326(1), G25-G37.
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature infants. Evidence indicates that bile acid homeostasis is disrupted during NEC: ileal bile acid levels are elevated in animals with experimental NEC, as is expression of the apical sodium-dependent bile acid transporter (Asbt). In addition, bile acids, which are synthesized in the liver, are extensively modified by the gut microbiome, including via the conversion of primary bile acids to more cytotoxic secondary forms. We hypothesized that the addition of bile acid-modifying bacteria would increase susceptibility to NEC in a neonatal rat model of the disease. The secondary bile acid-producing species exacerbated both incidence and severity of NEC. upregulated the bile acid transporter Asbt and increased levels of intraenterocyte bile acids. Treatment with also altered bile acid profiles and increased hydrophobicity of the ileal intracellular bile acid pool. The ability of to enhance NEC requires bile acids, as pharmacological sequestration of ileal bile acids protects animals from developing disease. These findings indicate that bile acid-modifying bacteria can contribute to NEC pathology and provide additional evidence for the role of bile acids in the pathophysiology of experimental NEC. Necrotizing enterocolitis (NEC), a life-threatening gastrointestinal emergency in premature infants, is characterized by dysregulation of bile acid homeostasis. We demonstrate that administering the secondary bile acid-producing bacterium enhances NEC in a neonatal rat model of the disease. -enhanced NEC is dependent on bile acids and driven by upregulation of the ileal bile acid transporter Asbt. This is the first report of bile acid-modifying bacteria exacerbating experimental NEC pathology.
• Calton, C. M., Carothers, K., Jagadish, N., Phanindra, B., Garcia, A., Viswanathan, V., & Halpern, M. D. (2023). Clostridium scindens exacerbates experimental necrotizing enterocolitis via upregulation of the apical sodium-dependent bile acid transporter. . American Journal of Physiology, Liver and GI. doi:doi: 10.1152/ajpgi.00102.2023
• Rothers, J. L., Calton, C., Stepp, J. M., & Halpern, M. D. (2023). Enteral Feeding and Antibiotic Treatment Do Not Influence Increased Coefficient of Variation of Total Fecal Bile Acids in Necrotizing Enterocolitis.. Newborn, 2(2), 128-132. doi:10.5005/jp-journals-11002-0063
• Ramamurthy, S., Viswanathan, V. K., Ramamurthy, S., Phanindra, B., Jagadish, N., Halpern, M. D., & Calton, C. M. (2021). Su143 SECONDARY BILE ACID PRODUCER CLOSTRIDIUM SCINDENS EXACERBATES NECROTIZING ENTEROCOLITIS IN NEONATAL RATS. Gastroenterology, 160(6), S-632. doi:10.1016/s0016-5085(21)02219-8
• Viswanathan, V. K., Ramamurthy, S., Phanindra, B., Jagadish, N., Halpern, M. D., & Calton, C. M. (2021). Su143 SECONDARY BILE ACID PRODUCER CLOSTRIDIUM SCINDENS EXACERBATES NECROTIZING ENTEROCOLITIS IN NEONATAL RATS. Gastroenterology, 160(6). doi:10.1016/s0016-5085(21)02219-8
• Halpern, M. D., & Calton, C. M. (2020). Su1242 A TWO DIMENSIONAL ENTEROID MODEL TO STUDY THE ROLE OF ASBT IN NECROTIZING ENTEROCOLITIS. Gastroenterology, 158(6), S-555. doi:10.1016/s0016-5085(20)32090-4
• Knapp, S., Kehring, A., Stepp, J., Calton, C. M., Gephart, S. M., Bandlamuri, S., Boyle, K. E., Dietz, G. I., Johnson, H., Romo, R. E., Spencer, M., Bedrick, A. D., & Halpern, M. D. (2020). Elevated Coefficient of Variation in Total Fecal Bile Acids Precedes Diagnosis of Necrotizing Enterocolitis. Scientific reports, 10(1), 249.
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  Accumulation of bile acids (BAs) may mediate development of necrotizing enterocolitis (NEC). Serial fecal samples were collected from premature infants with birth weight (BW) ≤ 1800 g, estimated gestational age (EGA) ≤ 32 weeks, and
• Halpern, M. D., & Denning, P. W. (2015). The role of intestinal epithelial barrier function in the development of NEC. Tissue barriers, 3(1-2), e1000707.
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  The intestinal epithelial barrier plays an important role in maintaining host health. Breakdown of intestinal barrier function is known to play a role in many diseases such as infectious enteritis, idiopathic inflammatory bowel disease, and neonatal inflammatory bowel diseases. Recently, increasing research has demonstrated the importance of understanding how intestinal epithelial barrier function develops in the premature neonate in order to develop strategies to promote its maturation. Optimizing intestinal barrier function is thought to be key to preventing neonatal inflammatory bowel diseases such as necrotizing enterocolitis. In this review, we will first summarize the key components of the intestinal epithelial barrier, what is known about its development, and how this may explain NEC pathogenesis. Finally, we will review what therapeutic strategies may be used to promote optimal development of neonatal intestinal barrier function in order to reduce the incidence and severity of NEC.
• Gephart, S. M., Spitzer, A. R., Effken, J. A., Dodd, E., Halpern, M., & McGrath, J. M. (2014). Discrimination of GutCheck(NEC): a clinical risk index for necrotizing enterocolitis. Journal of perinatology : official journal of the California Perinatal Association, 34(6), 468-75.
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  Better measures are needed to identify infants at risk for developing necrotizing enterocolitis (NEC) and facilitate communication about risk across transitions. Although NEC is multi-factorial, quantification of composite risk for NEC in an individual infant is not clearly defined. The objective of this study was to describe the derivation, validation and calibration testing of a novel clinical NEC risk index, GutCheck(NEC). Individual risk factors were weighted to assess composite odds of developing NEC. GutCheck(NEC) is designed to improve communication about NEC risk and coordination of care among clinicians across an infant's clinical course.
• Weitkamp, J. H., Rosen, M. J., Zhao, Z., Koyama, T., Geem, D., Denning, T. L., Rock, M. T., Moore, D. J., Halpern, M. D., Matta, P., & Denning, P. W. (2014). Small intestinal intraepithelial TCRγδ+ T lymphocytes are present in the premature intestine but selectively reduced in surgical necrotizing enterocolitis. PloS one, 9(6), e99042.
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  Gastrointestinal barrier immaturity predisposes preterm infants to necrotizing enterocolitis (NEC). Intraepithelial lymphocytes (IEL) bearing the unconventional T cell receptor (TCR) γδ (γδ IEL) maintain intestinal integrity and prevent bacterial translocation in part through production of interleukin (IL) 17.
• Halpern, M., Cherrington, N. J., Estrada, T. E., Frisk, H. A., Canet, M. J., Hardwick, R. N., Dvorak, B., Lux, K., & Halpern, M. D. (2013). The hepatic bile acid transporters Ntcp and Mrp2 are downregulated in experimental necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 304(1).
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants and is characterized by an extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. We have previously shown that, during the development of experimental NEC, the liver plays an important role in regulating inflammation in the ileum, and accumulation of ileal bile acids (BA) along with dysregulation of ileal BA transporters contributes to ileal damage. Given these findings, we speculated that hepatic BA transporters would also be altered in experimental NEC. Using both rat and mouse models of NEC, levels of Cyp7a1, Cyp27a1, and the hepatic BA transporters Bsep, Ntcp, Oatp2, Oatp4, Mrp2, and Mrp3 were investigated. In addition, levels of hepatic BA transporters were also determined when the proinflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-18, which are both elevated in NEC, are neutralized during disease development. Ntcp and Mrp2 were decreased in NEC, but elevated ileal BA levels were not responsible for these reductions. However, neutralization of TNF-α normalized Ntcp, whereas removal of IL-18 normalized Mrp2 levels. These data show that the hepatic transporters Ntcp and Mrp2 are downregulated, whereas Cyp27a1 is increased in rodent models of NEC. Furthermore, increased levels of TNF-α and IL-18 in experimental NEC may play a role in the regulation of Ntcp and Mrp2, respectively. These data suggest the gut-liver axis should be considered when therapeutic modalities for NEC are developed.
• Halpern, M., Halpern, M. D., Gephart, S. M., McGrath, J. M., & Effken, J. A. (2012). Necrotizing enterocolitis risk: state of the science. Advances in neonatal care : official journal of the National Association of Neonatal Nurses, 12(2).
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  Necrotizing enterocolitis (NEC) is the most common cause of gastrointestinal-related morbidity and mortality in the neonatal intensive care unit (NICU). Its onset is sudden and the smallest, most premature infants are the most vulnerable. Necrotizing enterocolitis is a costly disease, accounting for nearly 20% of NICU costs annually. Necrotizing enterocolitis survivors requiring surgery often stay in the NICU more than 90 days and are among those most likely to stay more than 6 months. Significant variations exist in the incidence across regions and units. Although the only consistent independent predictors for NEC remain prematurity and formula feeding, others exist that could increase risk when combined. Awareness of NEC risk factors and adopting practices to reduce NEC risk, including human milk feeding, the use of feeding guidelines, and probiotics, have been shown to reduce the incidence of NEC. The purpose of this review is to examine the state of the science on NEC risk factors and make recommendations for practice and research.
• Rogan, D. T., Patrick, S. M., Halpern, M. D., Frisk, H. A., & Estrada, T. E. (2012). 411 Developmental Regulation of Sam Pointed Domain Ets Factor (SPDEF) in Rat Ileum Cultured With Bile Acids. Gastroenterology, 142(5), S-91. doi:10.1016/s0016-5085(12)60347-3
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  fibers as a percentage of total tissue area (μm2). The nerve volume densities were significantly decreased in the jejunum (23.7 vs. 31.3; p 0.05) and duodenum (30.1 vs. 28.8; p > 0.05). These findings suggest that endogenous microbiota are necessary for the normal patterning of the ENS in the early postnatal period, particularly in the mid to distal small intestine and colon. Further studies are needed to determine whether the mechanisms by which the endogenous microbiota act on the ENS involve a direct effect on neural cells.
• Bedrick, A. D., Patrick, S. M., Halpern, M. D., & Estrada, T. E. (2011). Bile Acid Level Variability is Increased in Necrotizing Enterocolitis. Gastroenterology, 140(5), S-445. doi:10.1016/s0016-5085(11)61825-8
• Halpern, M., Halpern, M. D., Martin, N. A., Mount Patrick, S. K., Estrada, T. E., Frisk, H. A., Rogan, D. T., & Dvorak, B. (2011). Active transport of bile acids decreases mucin 2 in neonatal ileum: implications for development of necrotizing enterocolitis. PloS one, 6(12).
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants, but its etiology remains unclear. We have previously shown that mucin 2 (Muc2) positive goblet cells are significantly decreased in NEC. We have also shown that ileal bile acids (BAs) are significantly increased during the development of this disease. Because BAs can affect mucins, we hypothesized that elevated ileal BAs contribute to decreased Muc2 in experimental NEC. The role of Muc2 in NEC was evaluated in Winnie +/+ mice, a strain that produces aberrant Muc2. Muc2 and trefoil factor 3 (Tff3) were assessed in neonatal rats subjected to the NEC protocol when bile acids were removed, and in ileal explants from newborn and older rats cultured with and without BAs. Further, the role of active transport of BAs was determined using neonatal rats given the apical sodium dependent bile acid transporter (Asbt) inhibitor SC-435 and in neonatal Asbt knockout mice subjected to the NEC protocol. Mice with aberrant Muc2 had significantly greater incidence and severity of NEC. Using both in vivo and ex vivo techniques, we determined that BAs decrease Muc2 positive cells in neonatal but not older ileum. However, Tff3 positive cells are not decreased by BAs. In addition, active transport of BAs is required for BAs to decrease Muc2 in immature ileum. These data show that functional Muc2 plays a critical role in the prevention of NEC and BAs can potentiate the decreased Muc2 in disease development. Further, BAs have a more profound effect on Muc2 in immature versus older ileum, which may explain at least in part why NEC occurs almost exclusively in premature infants.
• Patrick, S. M., Martin, N., Halpern, M. D., Estrada, T. E., Dvorak, B., & Coursodon, C. F. (2011). IL-1β and TNF-α Do Not Decrease Protein Levels of the Apical Sodium-Dependent Bile Acid Transporter in IEC-6 Cells. Gastroenterology, 140(5), S-654. doi:10.1016/s0016-5085(11)62712-1
• Dvorak, K., Maynard, A., Khailova, L., Halpern, M. D., Dvorak, K., Dvorak, B., & Arganbright, K. M. (2010). M1831 Improvement of Intestinal Mucus Layer After Epidermal Growth Factor Treatment of Necrotizing Enterocolitis in a Rat Model. Gastroenterology, 138(5), S-428. doi:10.1016/s0016-5085(10)61974-9
• Halpern, M. D., Weitkamp, J. H., Mount Patrick, S. K., Dobrenen, H. J., Khailova, L., Correa, H., & Dvorak, B. (2010). Apical sodium-dependent bile acid transporter upregulation is associated with necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 299(3), G623-31.
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. Previously, we showed that luminal bile acids (BAs) are increased and correlated with disease development and that the apical sodium-dependent BA transporter (ASBT), which transports BAs from the ileal lumen into enterocytes, is upregulated in rats with NEC. We hypothesized that intraenterocyte, rather than luminal, BAs are associated with NEC and that upregulation of ASBT may be a mechanism by which this occurs. Neonatal rats with or without the ASBT inhibitor SC-435, mice in which ASBT was knocked out, and mice that overproduce BAs were subjected to the NEC protocol. Disease development, ASBT, and the farnesoid X receptor protein, along with luminal and intraenterocyte BA levels, were assessed. In addition, ileal sections from premature infants with and without NEC were examined for ASBT via immunohistology and real-time PCR. When BAs were not transported into enterocytes (rats given SC-435 and ASBT knockout mice), severity and incidence of NEC were reduced. In contrast, in mice that overproduce BAs, ASBT was elevated, intraenterocyte BAs were increased, and disease development was increased. ASBT staining was more intense on the apical membrane of ileal enterocytes from premature infants with NEC than premature infants with non-NEC diagnoses. In addition, ASBT mRNA levels were significantly higher in infants with NEC. These data show that accumulation of intraenterocyte BAs contributes to disease development, elevated ASBT increases disease severity in experimental models of NEC, and ASBT is elevated in human NEC. These data confirm that BAs and upregulation of ASBT play a crucial role in NEC pathogenesis and suggest that inhibition of ASBT could be utilized as a therapeutic modality against this disease.
• Khailova, L., Mount Patrick, S. K., Arganbright, K. M., Halpern, M. D., Kinouchi, T., & Dvorak, B. (2010). Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 299(5), G1118-27.
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  Necrotizing enterocolitis (NEC) is a devastating intestinal disease of neonates, and clinical studies suggest the beneficial effect of probiotics in NEC prevention. Recently, we have shown that administration of Bifidobacterium bifidum protects against NEC in a rat model. Intestinal apoptosis can be suppressed by activation of cyclooxygenase-2 (COX-2) and increased production of prostaglandin E(2) (PGE(2)). The present study investigates the effect of B. bifidum on intestinal apoptosis in the rat NEC model and in an intestinal epithelial cell line (IEC-6), as a mechanism of protection against mucosal injury. Premature rats were divided into the following three groups: dam fed, hand fed with formula (NEC), or hand fed with formula supplemented with B. bifidum (NEC + B. bifidum). Intestinal Toll-like receptor-2 (TLR-2), COX-2, PGE(2), and apoptotic regulators were measured. The effect of B. bifidum was verified in IEC-6 cells using a model of cytokine-induced apoptosis. Administration of B. bifidum increased expression of TLR-2, COX-2, and PGE(2) and significantly reduced apoptosis in the intestinal epithelium of both in vivo and in vitro models. The Bax-to-Bcl-w ratio was shifted toward cell survival, and the number of cleaved caspase-3 positive cells was markedly decreased in B. bifidum-treated rats. Experiments in IEC-6 cells showed anti-apoptotic effect of B. bifidum. Inhibition of COX-2 signaling blocked the protective effect of B. bifidum treatment in both in vivo and in vitro models. In conclusion, oral administration of B. bifidum activates TLR-2 in the intestinal epithelium. B. bifidum increases expression of COX-2, which leads to higher production of PGE(2) in the ileum and protects against intestinal apoptosis associated with NEC. This study indicates the ability of B. bifidum to downregulate apoptosis in the rat NEC model and in IEC-6 cells by a COX-2-dependent matter and suggests a molecular mechanism by which this probiotic reduces mucosal injury and preserves intestinal integrity.
• Maynard, A. A., Dvorak, K., Khailova, L., Dobrenen, H., Arganbright, K. M., Halpern, M. D., Kurundkar, A. R., Maheshwari, A., & Dvorak, B. (2010). Epidermal growth factor reduces autophagy in intestinal epithelium and in the rat model of necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 299(3), G614-22.
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  Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Epidermal growth factor (EGF) is one of the most promising candidates in NEC prophylaxis. Autophagy regulates cell homeostasis, but uncontrolled activation of autophagy may lead to cellular injury. The aim was to evaluate the effects of EGF on intestinal autophagy in epithelial cells and in the rat NEC model and measure autophagy in NEC patients. Intestinal epithelial cells (IEC-6) and the rat NEC model were used to study the effect of EGF on intestinal autophagy. Protein levels of Beclin 1 and LC3II were measured in the intestinal epithelium in both in vivo and in vitro models. Ultrastructural changes in intestinal epithelium were studied by electron microscopy. Expression of Beclin 1, LC3II, and p62 protein was evaluated in biopsies from NEC patients. Autophagy was induced in IEC-6 cells and inhibited by adding EGF into the culture. In the rat NEC model, EGF treatment of NEC reduced expression of Beclin 1 and LC3II in ileal epithelium. Morphologically, typical signs of autophagy were observed in the epithelium of the NEC group, but not in the EGF group. A strong signal for Beclin 1 and LC3II was detected in the intestine from patients with NEC. Autophagy is activated in the intestinal epithelium of NEC patients and in the ileum of NEC rats. Supplementation of EGF blocks intestinal autophagy in both in vivo and in vitro conditions. Results from this study indicate that EGF-mediated protection against NEC injury is associated with regulation of intestinal autophagy.
• Patrick, S. M., Khailova, L., Halpern, M. D., Dvorak, B., & Dobrenen, H. (2010). T1877 Interleukin-18 Upregulates the Apical Sodium-Dependent Bile Acid Transporter (ASBT) in Neonatal Rat Ileum and IEC-6 Cells. Gastroenterology, 138(5), S-597. doi:10.1016/s0016-5085(10)62755-2
• Patrick, S. M., Martin, N., Kehring, A., Halpern, M. D., & Dobrenen, H. (2010). T1876 Active Transport of Bile Acids is Not Required for Bile Acid-Induced Reduction of Mucin 2. Gastroenterology, 138(5), S-597. doi:10.1016/s0016-5085(10)62754-0
• Weitkamp, J., Patrick, S. M., Halpern, M. D., Dvorak, B., Dobrenen, H., & Correa, H. (2010). 1013 The Apical Sodium-Dependent Bile Acid Transporter is Increased in Human Necrotizing Enterocolitis. Gastroenterology, 138(5), S-146-S-147. doi:10.1016/s0016-5085(10)60672-5
• Yajima, M., Kinouchi, T., Khailova, L., Johnson, A., Halpern, M. D., Dvorak, B., & Arganbright, K. M. (2010). 278 Reduced Apoptosis in In Vivo and In Vitro Models of Necrotizing Enterocolitis After Treatment With Bifidobacterium Bifidum. Gastroenterology, 138(5), S-52. doi:10.1016/s0016-5085(10)60236-3
• Khailova, L., Dvorak, K., Arganbright, K. M., Halpern, M. D., Kinouchi, T., Yajima, M., & Dvorak, B. (2009). Bifidobacterium bifidum improves intestinal integrity in a rat model of necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 297(5), G940-9.
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  Neonatal necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in premature infants. Oral administration of probiotics has been suggested as a promising strategy for prevention of NEC. However, little is known about the mechanism(s) of probiotic-mediated protection against NEC. The aim of this study was to evaluate the effects of Bifidobacterium bifidum treatment on development of NEC, cytokine regulation, and intestinal integrity in a rat model of NEC. Premature rats were divided into three groups: dam fed (DF), hand fed with formula (NEC), or hand fed with formula supplemented with 5 x 10(6) CFU B. bifidum per day (B. bifidum). All groups were exposed to asphyxia and cold stress to develop NEC. Intestinal injury, mucin and trefoil factor 3 (Tff3) production, cytokine levels, and composition of tight junction (TJ) and adherens junction (AJ) proteins were evaluated in the terminal ileum. B. bifidum decreased the incidence of NEC from 57 to 17%. Increased levels of IL-6, mucin-3, and Tff3 in the ileum of NEC rats was normalized in B. bifidum treated rats. Reduced mucin-2 production in the NEC rats was not affected by B. bifidum. Administration of B. bifidum normalized the expression and localization of TJ and AJ proteins in the ileum compared with animals with NEC. In conclusion, administration of B. bifidum protects against NEC in the neonatal rat model. This protective effect is associated with reduction of inflammatory reaction in the ileum, regulation of main components of mucus layer, and improvement of intestinal integrity.
• Khailova, L., Dvorak, K., Arganbright, K. M., Williams, C. S., Halpern, M. D., & Dvorak, B. (2009). Changes in hepatic cell junctions structure during experimental necrotizing enterocolitis: effect of EGF treatment. Pediatric research, 66(2), 140-4.
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  Necrotizing enterocolitis (NEC) is a devastating disease of premature babies. Previously, we have shown that EGF reduces NEC and that overproduction of hepatic TNF-alpha is associated with intestinal damage. Leakage of TNF-alpha may be a consequence of epithelial hepatic cellular junction dysfunction. The aim of this study was to investigate changes in the composition of hepatic tight junctions (TJs) and adherens junctions (AJs). Using an established rat model of NEC, animals were divided into the following groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with EGF (EGF). Serum EGF and histologic localization of major TJ and AJ proteins were evaluated. Distribution patterns of hepatic TJ and AJ proteins were significantly altered in the NEC group compared with those in DF or EGF groups. Cytoplasmic accumulation of occludin, claudin-2, and ZO-1 with reduction of claudin-3 signal was detected in the liver of NEC rats. Localization of beta-catenin was associated with the hepatocyte membrane in EGF and DF groups, but diffused in the NEC group. These data show that hepatic cellular junctions are significantly altered during NEC pathogenesis. EGF-mediated reduction of experimental NEC is associated with protection of hepatic integrity and structure.
• Thomas, M. A., Johnson, C., Halpern, M. D., & Dvorak, B. (2009). T1759 Bile Acids Decrease Mucin 2 and Trefoil Factor 3 in Neonatal Rat Ileal Explants. Gastroenterology, 136(5), A-574. doi:10.1016/s0016-5085(09)62640-8
• Thomas, M. A., Khailova, L., Johnson, C., Halpern, M. D., & Dvorak, B. (2009). T1752 Incidence and Severity of Necrotizing Enterocolitis Is Increased in Mice That Over Express the Hepatic Bile Salt Export Pump. Gastroenterology, 136(5), A-572. doi:10.1016/s0016-5085(09)62633-0
• Thomas, M. A., Khailova, L., Johnson, C., Halpern, M. D., Dvorak, B., & Arganbright, K. M. (2009). 217 Decreased Development of Experimental Necrotizing Enterocolitis in Apical Sodium-Dependent Bile Acid Transporter Knockout Mice. Gastroenterology, 136(5), A-41. doi:10.1016/s0016-5085(09)60188-8
• Yajima, M., Maynard, A., Kinouchi, T., Khailova, L., Johnson, A., Halpern, M. D., Dvorak, B., & Arganbright, K. M. (2009). 1077 Bifidobacterium bifidum in a Rat Model of Necrotizing Enterocolitis: Changes in Toll-Like Receptors and Inflammatory Cytokines. Gastroenterology, 136(5), A-166. doi:10.1016/s0016-5085(09)60750-2
• Dvorak, K., Reynolds, C. A., Khailova, L., Halpern, M. D., Dvorak, K., Dvorak, B., & Arganbright, K. M. (2008). T1823 Regulation of Intestinal Autophagy By Epidermal Growth Factor in An Experimental Model of Necrotizing Enterocolitis. Gastroenterology, 134(4), A-570-A-571. doi:10.1016/s0016-5085(08)62664-5
• Dvorak, K., Yajima, M., Maynard, A., Kinouchi, T., Khailova, L., Halpern, M. D., Dvorak, K., Dvorak, B., & Arganbright, K. M. (2008). W1774 Bifidobacterium Bifidum Improves Intestinal Barrier Function in Experimental Necrotizing Enterocolitis. Gastroenterology, 134(4), A-712-A-713. doi:10.1016/s0016-5085(08)63328-4
• Halpern, M. D., & Dvorak, B. (2008). Does abnormal bile acid metabolism contribute to NEC?. Seminars in perinatology, 32(2), 114-21. doi:10.1053/j.semperi.2008.01.005
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  Bile acids (BAs) facilitate emulsification, absorption, and transport of fats and sterols in the intestine and liver and are essential for normal digestion. However, accumulation of BAs in the intestine can result in damage to the intestinal epithelium. Using the neonatal rat model of necrotizing enterocolitis (NEC), we have recently shown that BAs accumulate in both the ileal lumen and enterocytes of neonatal rats with NEC and the increased BA levels are positively correlated with disease severity. Importantly, when BAs are not allowed to accumulate, neonatal rat pups develop significantly less disease. In addition, BA transporters are altered during disease development. These data indicate that BAs play an important role in the development of experimental NEC, and suggest that the inability of neonatal rats to adequately regulate BA transporters may be a mechanism by which ileal damage occurs.
• Halpern, M. D., Khailova, L., Molla-Hosseini, D., Arganbright, K., Reynolds, C., Yajima, M., Hoshiba, J., & Dvorak, B. (2008). Decreased development of necrotizing enterocolitis in IL-18-deficient mice. American journal of physiology. Gastrointestinal and liver physiology, 294(1), G20-6.
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  Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly of prematurely born infants, characterized in its severest from by extensive hemorrhagic inflammatory necrosis of the distal ileum and proximal colon. Proinflammatory cytokines have been implicated in the development of NEC, and we have previously shown that IL-18 is significantly elevated in the well-established neonatal rat model of NEC. To determine whether IL-18 contributes to intestinal pathology in NEC, we subjected IL-18 knockout mice to the protocol used to develop experimental NEC in newborn rats. Newborn B6.129P2-Il18(tm1Aki)/J (NEC IL-18(-/-)) and wild-type (NEC WT) mice were hand fed every 3 h with cow's milk-based formula and exposed to asphyxia and cold stress twice daily. After 72 h, animals were killed and distal ileum and liver were removed. Disease development was determined via histological changes in the ileum as scored by a blinded evaluator. The number of TNF-alpha-, IL-12-, and IL-1beta-positive cells and macrophages were determined in both ileum and liver via immunohistology. IkappaB-alpha and IkappaB-beta were determined from protein extracts from both ileum and liver using Western blot analysis. The incidence and severity of NEC was significantly reduced in NEC IL-18(-/-) mice compared with NEC WT. Furthermore, mean ileal macrophages and hepatic IL-1beta were significantly reduced in IL-18(-/-) mice subjected to the NEC protocol. There were no statistically significant changes in Kupffer cells, hepatic TNF-alpha, ileal IL-1beta, or IL-12. IkappaB-alpha and IkappaB-beta were significantly increased in NEC IL-18(-/-) mice ileum and liver, respectively. These results confirm that IL-18 plays a crucial role in experimental NEC pathogenesis.
• Halpern, M., Dvorak, B., Khailova, L., Clark, J. A., Hosseini, D. M., Arganbright, K. M., Reynolds, C. A., & Halpern, M. D. (2008). Comparison of epidermal growth factor and heparin-binding epidermal growth factor-like growth factor for prevention of experimental necrotizing enterocolitis. Journal of pediatric gastroenterology and nutrition, 47(1).
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  Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease of prematurely born infants. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) have protective effects against intestinal injury. The aim of this study was to compare the effect of oral administration of HB-EGF, EGF, or both on the incidence of NEC in a neonatal rat model.
• Reynolds, C. A., Khailova, L., Halpern, M. D., Dvorak, B., & Arganbright, K. M. (2008). 127 Bile Acids Decrease Ileal Mucin During Development of Experimental Necrotizing Enterocolitis. Gastroenterology, 134(4), A-22. doi:10.1016/s0016-5085(08)60109-2
• Ito, Y., Doelle, S. M., Clark, J. A., Halpern, M. D., McCuskey, R. S., & Dvorak, B. (2007). Intestinal microcirculatory dysfunction during the development of experimental necrotizing enterocolitis. Pediatric research, 61(2), 180-4.
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  The aim of this study was to evaluate changes in intestinal microcirculation during necrotizing enterocolitis (NEC) and to examine the effect of endothelin (ET)-1 on the intestinal microcirculation. Prematurely born rats were either hand-fed formula (NEC) or dam fed (DF) and were exposed to asphyxia and cold stress twice daily to induce disease. At 0, 2, 3, and 4 d after the birth, the microcirculation in the ileum was examined using in vivo microscopic methods. The nutritive microvascular perfusion in the NEC group was progressively compromised from d 3 to d 4 (35% and 50% decrease, respectively) when compared with DF rats. Concomitantly, intestinal blood flow assessed by laser Doppler flowmetry was significantly reduced at d 2, 3, and 4 (by 31%, 36%, and 73%, respectively). Levels of ET-1 mRNA in the ileum were increased 3.7-fold. Microvascular responses to topically applied ET-1 were significantly increased in the NEC group, which was associated with decreased expression of ETB receptor. These results suggest that microcirculatory dysfunction in the distal ileum of neonatal rats with NEC contributes to disease progression and that enhanced microvascular responsiveness to ET-1 may participate in these microcirculatory disturbances.
• Clark, J. A., Doelle, S. M., Halpern, M. D., Saunders, T. A., Holubec, H., Dvorak, K., Boitano, S. A., & Dvorak, B. (2006). Intestinal barrier failure during experimental necrotizing enterocolitis: protective effect of EGF treatment. American journal of physiology. Gastrointestinal and liver physiology, 291(5), G938-49.
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  Necrotizing enterocolitis (NEC) is the most common intestinal disease of premature infants. Although increased mucosal permeability and altered epithelial structure have been associated with many intestinal disorders, the role of intestinal barrier function in NEC pathogenesis is currently unknown. We investigated the structural and functional changes of the intestinal barrier in a rat model of NEC. In addition, the effect of EGF treatment on intestinal barrier function was evaluated. Premature rats were divided into three groups: dam fed (DF), formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC + EGF); all groups were exposed to asphyxia/cold stress to develop NEC. Intestinal permeability, goblet cell density, mucin production, and composition of tight junction (TJ) proteins were evaluated in the terminal ileum, the site of NEC injury, and compared with the proximal jejunum, which was unaffected by NEC. Animals with NEC had significantly increased intestinal paracellular permeability compared with DF pups. Ileal goblet cell morphology, mucin production, and TJ composition were altered in animals with NEC. EGF treatment significantly decreased intestinal paracellular permeability, increased goblet cell density and mucin production, and normalized expression of two major TJ proteins, occludin and claudin-3, in the ileum. In conclusion, experimental NEC is associated with disruption of the intestinal barrier. EGF treatment maintains intestinal integrity at the site of injury by accelerating goblet cell maturation and mucin production and normalizing expression of TJ proteins, leading to improved intestinal barrier function.
• Dvorak, K., Saunders, T. A., Holubec, H., Halpern, M. D., Dvorak, K., Dvorak, B., Doelle, S. M., & Clark, J. A. (2006). Epidermal growth factor improves intestinal barrier function in a rat model of necrotizing enterocolitis. The FASEB Journal, 20(5).
• Halpern, M. D., Clark, J. A., Saunders, T. A., Doelle, S. M., Hosseini, D. M., Stagner, A. M., & Dvorak, B. (2006). Reduction of experimental necrotizing enterocolitis with anti-TNF-alpha. American journal of physiology. Gastrointestinal and liver physiology, 290(4), G757-64.
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. However, despite significant morbidity and mortality, the etiology and pathogenesis of NEC are poorly understood. Evidence suggests that ileal proinflammatory mediators such as IL-18 contribute to the pathology associated with this disease. In addition, we have previously shown that upregulation of TNF-alpha in the liver is correlated with ileal disease severity in a neonatal rat model of NEC. With the use of a neonatal rat model of NEC, we evaluated the incidence and severity of ileal damage along with the production of both hepatic and ileal proinflammatory cytokines in animals injected with (anti-TNF-alpha; n = 23) or without (NEC; n = 25) a monoclonal anti-TNF-alpha antibody. In addition, we assessed changes in apoptosis and ileal permeability in the NEC and anti-TNF-alpha groups. Ileal damage was significantly decreased, and the incidence of NEC was reduced from 80% to 17% in animals receiving anti-TNF-alpha. Hepatic TNF-alpha and hepatic and ileal IL-18 were significantly decreased in pups given anti-TNF-alpha compared with those sham injected. In addition, ileal luminal levels of both TNF-alpha and IL-18 were significantly decreased in the anti-TNF-alpha-injected group. Ileal paracellular permeability and the proapoptotic markers Bax and cleaved caspase-3 were significantly decreased in the anti-TNF-alpha group. These data show that hepatic TNF-alpha is an important component for the development of NEC in the neonatal rat model and suggest that anti-TNF-alpha could be used as a potential therapy for human NEC.
• Halpern, M. D., Holubec, H., Clark, J. A., Saunders, T. A., Williams, C. S., Dvorak, K., & Dvorak, B. (2006). Epidermal growth factor reduces hepatic sequelae in experimental necrotizing enterocolitis. Biology of the neonate, 89(4), 227-35.
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  Neonatal necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We recently demonstrated that the gut/liver axis plays an important role in the pathophysiology of NEC through the release of inflammatory mediators into the intestinal lumen. We have also shown that supplementation of formula with epidermal growth factor (EGF) dramatically decreases ileal pathology associated with experimental NEC. In this study, we examined the effects of EGF on the liver portion of the gut/liver axis in the neonatal rat model of NEC.
• Halpern, M. D., Holubec, H., Saunders, T. A., Dvorak, K., Clark, J. A., Doelle, S. M., Ballatori, N., & Dvorak, B. (2006). Bile acids induce ileal damage during experimental necrotizing enterocolitis. Gastroenterology, 130(2), 359-72.
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of premature infants. While the effect of bile acids (BAs) on intestinal mucosal injury is known, we investigated the contribution of BAs during the development of NEC in neonatal rats.
• Clark, J. A., Lane, R. H., Maclennan, N. K., Holubec, H., Dvorakova, K., Halpern, M. D., Williams, C. S., Payne, C. M., & Dvorak, B. (2005). Epidermal growth factor reduces intestinal apoptosis in an experimental model of necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 288(4), G755-62.
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  Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Although end-stage NEC is characterized histopathologically as extensive necrosis, apoptosis may account for the initial loss of epithelium before full development of disease. We have previously shown that epidermal growth factor (EGF) reduces the incidence of NEC in a rat model. Although EGF has been shown to protect intestinal enterocytes from apoptosis, the mechanism of EGF-mediated protection against NEC is not known. The aim of this study was to investigate if EGF treatment elicits changes in expression of apoptotic markers in the ileum during the development of NEC. With the use of a well-established neonatal rat model of NEC, rats were divided into the following three experimental groups: dam fed (DF), milk formula fed (NEC), or fed with formula supplemented with 500 ng/ml EGF (NEC+EGF). Changes in ileal morphology, gene and protein expression, and histological localization of apoptotic regulators were evaluated. Anti-apoptotic Bcl-2 mRNA levels were markedly reduced and pro-apoptotic Bax mRNA levels were markedly elevated in the NEC group compared with DF controls. Supplementation of EGF into formula significantly increased anti-apoptotic Bcl-2 mRNA, whereas pro-apoptotic Bax was significantly decreased. The Bax-to-Bcl-2 ratio for mRNA and protein was markedly decreased in NEC+EGF animals compared with the NEC group. The presence of caspase-3-positive epithelial cells was markedly reduced in EGF-treated rats. These data suggest that alteration of the balance between pro-and anti-apoptotic proteins in the site of injury is a possible mechanism by which EGF maintains intestinal integrity and protects intestinal epithelium against NEC injury.
• Dvorak, K., Saunders, T. A., Halpern, M. D., Dvorak, K., Dvorak, B., Doelle, S. M., & Clark, J. A. (2005). 101 Effect of EGF on the Intestinal Tight Junction Barrier in Experimental Necrotizing Enterocolitis. Pediatric Research, 58(2), 372-372. doi:10.1203/00006450-200508000-00130
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  101 Effect of EGF on the Intestinal Tight Junction Barrier in Experimental Necrotizing Enterocolitis
• Saunders, T. A., Halpern, M. D., Dvorak, B., Doelle, S. M., Clark, J. A., & Burger, L. C. (2005). 102 Effect of Anti-TNF-A Treatment on the Incidence of Experimental Necrotizing Enterocolitis. Pediatric Research, 58(2), 372-372. doi:10.1203/00006450-200508000-00131
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  102 Effect of Anti-TNF-A Treatment on the Incidence of Experimental Necrotizing Enterocolitis
• Saunders, T. A., Halpern, M. D., Dvorak, K., Dvorak, B., Doelle, S. M., & Clark, J. A. (2005). TIGHT JUNCTIONS FORMATION IN EXPERIMENTAL MODEL OF NECROTIZING ENTEROCOLITIS - EFFECT OF EGF TREATMENT: OP1-04. Journal of Pediatric Gastroenterology and Nutrition, 40(5), 617. doi:10.1097/00005176-200505000-00023
• Dvorak, B., Halpern, M. D., Holubec, H., Dvorakova, K., Dominguez, J. A., Williams, C. S., & Meza, Y. G. (2004). Rat milk decreases necrotizing enterocolitis in a rat model. Advances in experimental medicine and biology, 554, 471-3.
• Payne, C. M., Saunders, T. A., Payne, C. M., Meza, Y. G., Holubec, H., Halpern, M. D., Dvorakova, K., & Dvorak, B. (2004). P0919 ENTERAL EPIDERMAL GROWTH FACTOR REGULATES ILEAL BILE ACID TRANSPORT IN EXPERIMENTAL NECROTIZING ENTEROCOLITIS. Journal of Pediatric Gastroenterology and Nutrition, 39(Supplement 1), S407. doi:10.1097/00005176-200406001-01043
• Williams, C. S., Saunders, T. A., Meza, Y. G., Halpern, M. D., Dvorak, B., Doelle, S. M., & Clark, J. A. (2004). O0029 ENTERAL ADMINISTRATION OF HB-EGF REDUCES THE INCIDENCE OF NECROTIZING ENTEROCOLITIS IN A RAT MODEL. Journal of Pediatric Gastroenterology and Nutrition, 39(Supplement 1), S18. doi:10.1097/00005176-200406001-00031
• Dvorak, B., Halpern, M. D., Holubec, H., Dvorakova, K., Dominguez, J. A., Williams, C. S., Meza, Y. G., Kozakova, H., & McCuskey, R. S. (2003). Maternal milk reduces severity of necrotizing enterocolitis and increases intestinal IL-10 in a neonatal rat model. Pediatric research, 53(3), 426-33.
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  Necrotizing enterocolitis (NEC) is a devastating intestinal disease of premature infants. Maternal milk has been suggested to be partially protective against NEC; however, the mechanisms of this protection are not defined. The aim of this study was to examine the effect(s) of artificial feeding of rat milk (RM)-versus cow milk-based rat milk substitute (RMS) on the development of NEC in a neonatal rat model and elucidate the role of inflammatory cytokines in NEC pathogenesis. Newborn rats were artificially fed with either collected RM or RMS. Experimental NEC was induced by exposure to asphyxia and cold stress and evaluated by histologic scoring of damage in ileum. Intestinal cytokine mRNA expression was determined by real-time PCR. Cytokine histologic localization was performed by confocal microscopy. Similar to human NEC, artificial feeding of RM reduces the incidence and severity of NEC injury in neonatal rats. Freezing and thawing of collected RM did not eliminate the protective effect of maternal milk. Ileal IL-10 expression was significantly increased in the RM group compared with RMS. Increased IL-10 peptide production was detected in the RM group with signal localized predominantly in the cytoplasm of villus epithelial cells. These results suggest that the protective effect of maternal milk is associated with increased production of anti-inflammatory IL-10 in the site of injury. Better understanding of the mechanisms underlying these protective effects could be beneficial either in the prevention of NEC or in the development of future therapeutic strategies to cure NEC.
• Halpern, M. D., Dominguez, J. A., Dvorakova, K., Holubec, H., Williams, C. S., Meza, Y. G., Ruth, M. C., & Dvorak, B. (2003). Ileal cytokine dysregulation in experimental necrotizing enterocolitis is reduced by epidermal growth factor. Journal of pediatric gastroenterology and nutrition, 36(1), 126-33.
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of premature infants. We have shown in previous studies that proinflammatory interleukin-18 and interleukin-12 are up-regulated in the ileum of rats with experimental NEC and that epidermal growth factor (EGF) reduces the development of disease. Here we investigated whether the protective effects of EGF are a result of changes in ileal interleukin-18, interleukin-12 and/or antiinflammatory interleukin-10.
• Halpern, M. D., Holubec, H., Dominguez, J. A., Meza, Y. G., Williams, C. S., Ruth, M. C., McCuskey, R. S., & Dvorak, B. (2003). Hepatic inflammatory mediators contribute to intestinal damage in necrotizing enterocolitis. American journal of physiology. Gastrointestinal and liver physiology, 284(4), G695-702.
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  Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Along with pathological effects in the ileum, severe NEC is often accompanied by multisystem organ failure, including liver failure. The aim of this study was to determine the changes in hepatic cytokines and inflammatory mediators in experimental NEC. The well-established neonatal rat model of NEC was used in this study, and changes in liver morphology, numbers of Kupffer cells (KC), gene expression, and histological localization of IL-18, TNF-alpha, and inducible nitric oxide synthase were evaluated. Intestinal luminal TNF-alpha levels were also measured. Production of hepatic IL-18 and TNF-alpha and numbers of KC were increased in rats with NEC and correlated with the progression of intestinal damage during NEC development. Furthermore, increased levels of TNF-alpha in the intestinal lumen of rats with NEC was significantly decreased when KC were inhibited with gadolinium chloride. These results suggest an important role of the liver and the gut-liver axis in NEC pathogenesis.
• Williams, C. S., Meza, Y. G., Holubec, H., Halpern, M. D., & Dvorak, B. (2003). Inhibition of kupffer cells decreases the incidence and severity of experimental necrotizing enterocolitis. Gastroenterology, 124(4), A276-A277. doi:10.1016/s0016-5085(03)81386-0
• Williams, C. S., Payne, C. M., Meza, Y. G., Holubec, H., Halpern, M. D., Dvorakova, K., Dvorak, B., & Dominguez, J. A. (2003). Accumulation of bile acids in ileum associated with intestinal damage rats with experimental necrotizing enterocolitis. Gastroenterology, 124(4), A277. doi:10.1016/s0016-5085(03)81387-2
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  Necrotizing enterocolitis (NEC) is associated with the release of a variety of proniflamatory mediators in the small intestine. We have previously shown that through the enterohepatic axis, luminal ileal TNFa was significantly increased and correlated with severity of intestinal damage. Upregulation of IL-113 and TNFet reduces significantly the ileal sodium-dependent bile acid transporter (ASBT) in rats (Gastroenterology 2002; 123) and thus may lead to accumulation of bile in the terminal ileum. Increased concentrations of cytotoxic bile acids (BA) were reported to induce damage to intestinal mucosa. Objective: The aim of this study was to determine if changes in bile acid levels in the terminal ileum and serum are associated with the development of NEC. Design/Methods: Newborn rats were artificially fed cow milkbased rat milk substitute (RMS) and exposed to asphyxia and cold stress for 4 days to induce experimental NEC. Dam fed (DF) littermates were also exposed to asphyxia-cold stresses and served as controls. Total BA levels were determined in ileal flushes using an enzymatic assay (Diazyme). In addition, segments of terminal ileum were processed and evaluated histologicaly and by scanning electron microscopy (SEM). Results: Total BA were significantly increased (p
• Halpern, M. D., Holubec, H., Dominguez, J. A., Williams, C. S., Meza, Y. G., McWilliam, D. L., Payne, C. M., McCuskey, R. S., Besselsen, D. G., & Dvorak, B. (2002). Up-regulation of IL-18 and IL-12 in the ileum of neonatal rats with necrotizing enterocolitis. Pediatric research, 51(6), 733-9.
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  Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Because the proinflammatory cytokines IL-18, IL-12, and interferon (IFN)-gamma have been implicated in other diseases of the small intestine, we hypothesized that these cytokines would play an important role in NEC pathogenesis. NEC was induced in newborn rats via enteral feeding with rat milk substitute and asphyxia and cold stress (RMS). Dam-fed, asphyxia- and cold-stressed littermates were used as controls (DF). After 96 h, the distal ileum was removed from all animals and processed to determine expression and localization of IL-18, IL-12, and IFN-gamma using real-time reverse transcriptase PCR and immunohistology. IL-18 and IL-12 mRNA from the RMS group were increased (p < or = 0.05) compared with DF controls, and there was a correlation between increasing IL-18 and IL-12 mRNA levels and progression of tissue damage (r = 0.629 and 0.588, respectively; p < or = 0.05). Immunohistology revealed IL-18 in the cytoplasm of villi and crypt enterocytes and IL-12-positive monocytes/macrophages were increased with disease progression (r = 0.503, p < or = 0.05). No differences in the number of IFN-gamma-positive cells were observed between groups. These data demonstrate up-regulation of IL-18 and IL-12 in experimental NEC and a correlation between production of these proinflammatory cytokines and progression of tissue damage.
• Philipps, A. F., Pham, T. D., Maclennan, N. K., Lane, R. H., Halpern, M. D., Dvorakova, K., & Dvorak, B. (2002). IGF alters jejunal glucose transporter expression and serum glucose levels in immature rats.. American journal of physiology. Regulatory, integrative and comparative physiology, 283(6), R1450-60. doi:10.1152/ajpregu.00172.2002
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  Milk-borne insulin-like growth factors (IGFs) enhance nutrient absorption in the immature intestine, which is characterized by low levels of glucose oxidation. We therefore hypothesized that feeding a rat milk substitute (RMS) devoid of growth factors to rat pups would lower serum glucose levels relative to dam-fed control rats and that supplementation of RMS with physiological doses of either IGF-I or IGF-II would normalize serum glucose levels via increased jejunal glucose transporter 2 (GLUT2) and high-affinity Na(+)-glucose cotransporter (SGLT1) expression. We found lower serum glucose concentrations in RMS-fed pups; in contrast, serum glucose levels in the IGF-supplemented pups were similar to those of dam-fed controls. RT-PCR and laser scanning confocal microscopy similarly demonstrated that IGF supplementation increased expression of jejunal glucose transporters. Further experiments demonstrated that IGF supplementation altered mRNA levels of key mitochondrial enzymes without altering jejunal lactase activity. We conclude that IGF-I and IGF-II supplementation increases serum glucose levels in the immature rat pup fed artificial formula and alters gene expression of the jejunal glucose transporters.
• Williams, C. S., Stepankova, R., Payne, C. M., Mcwilliam, D. L., Mccuskey, R. S., Holubec, H., Halpern, M. D., Dvorak, B., & Dominguez, J. A. (2002). Epidermal growth factor reduces the development of necrotizing enterocolitis in a neonatal rat model.. American journal of physiology. Gastrointestinal and liver physiology, 282(1), G156-64. doi:10.1152/ajpgi.00196.2001
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  Necrotizing enterocolitis (NEC) is the most common gastrointestinal disease of prematurely born infants. Maternal milk plays an important protective role against NEC development and is the major source of epidermal growth factor (EGF) for neonates. The aim of this study was to examine the effect of orally administered EGF on the incidence of NEC in a neonatal rat model. Newborn rats were artificially fed either with growth factor-free rat milk substitute (RMS) or RMS supplemented with 500 ng/ml of EGF (RMS+EGF). Experimental NEC was induced by exposure to asphyxia and cold stress. Development of NEC was evaluated by gross and histological scoring of damage in the ileum. Ileal EGF receptor (EGF-R), EGF, and transforming growth factor-alpha mRNA expression was assessed by RT competitive-PCR, and the EGF-R was localized by immunohistochemistry. EGF supplementation of formula reduced the incidence and severity of NEC in rats (13/16 RMS vs. 4/13 RMS+EGF). Ileal EGF-R mRNA expression was markedly increased in the RMS group compared with RMS+EGF. Enhanced EGF-R expression in the RMS group was localized predominantly in the epithelial cells of injured ileum. These data suggest a new potential therapeutic approach for the prevention and treatment of NEC.
• Yocum, D. E., & Halpern, M. D. (1991). The paradoxical effects of diethyldithiocarbamate: comparisons between New Zealand black/white F1 hybrid and Balb/c mice.. Clinical immunology and immunopathology, 58(1), 69-79. doi:10.1016/0090-1229(91)90149-5
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  Diethyldithiocarbamate (DTC), an immunomodulative agent suggested to enhance T cells, has been shown to prolong life in autoimmune MRL-lpr/lpr mice. In addition to increased survival, MRL-lpr/lpr mice treated with DTC displayed a number of changes in expression of cell surface antigens as well as decreased serum autoantibody levels. To determine if DTC treatment would have similar positive effects on another murine model of autoimmune disease, we studied the New Zealand Black/White F1 hybrid (NZB/W). In addition, the effects of DTC treatment on cell surface antigen expression were compared between the NZB/W and a normal murine strain, the Balb/c. DTC treatment increased the density of cell surface antigens in the NZB/W, but decreased the density of these antigens in the Balb/c. Treatment with DTC induced distinct changes in the percentage of cells expressing specific surface antigens that differed between the NZB/W and the Balb/c. There was no affect on serum anti-DNA and anti-histone antibody levels or on survival in NZB/W mice treated with DTC. Therefore, while DTC treatment did not successfully influence the disease course in the NZB/W, it did result in specific changes in cell surface antigens. These data demonstrate that DTC is capable of inducing a variety of immunologic changes depending upon the strain treated.
• Yocum, D. E., Hersh, E. M., & Halpern, M. D. (1990). Diethyldithiocarbamate, a novel immunomodulator, prolongs survival in autoimmune MRL-lpr/lpr mice.. Clinical immunology and immunopathology, 55(2), 242-54. doi:10.1016/0090-1229(90)90100-5
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  MRL-lpr/lpr mice die at an early age from a spontaneously developing systemic lupus erythematosus-like disease and are characterized by massive lymphadenopathy, hyperproliferation of Lyt-2/L3T4 (null) T cells, decreased responses to mitogens, thymic atrophy, and very high serum autoantibody levels. Diethyldithiocarbamate (DTC), an immunomodulator suggested to enhance T cells, was used to treat 12-week-old female MRL-lpr/lpr mice (25 mg/kg/week). DTC treatment significantly prolonged survival (50% mortality, 43 weeks vs 20 weeks). Increased survival was associated with decreased lymphadenopathy, decreased proliferation of null cells, restoration of impaired mitogen responses, decreased thymic atrophy, and decreased serum levels of anti-DNA and anti-histone antibodies. Studies of cell surface antigen phenotype demonstrated increased expression of Lyt-2 and macrophage surface antigens. No effect on L3T4 single staining cells was observed. These results show that DTC significantly alters the disease course in these mice and suggest that DTC may be a useful treatment for autoimmune disease.

Presentations

• Calton, C. M., Carothers, K., Ramamurthy, S., Jagadish, N., Phanindra, B., Anett, G., Ridgeway, K., Viswanathan, V., & Halpern, M. D. (2022, May). Secondary Bile Acid Producing Bacteria Enhance Necrotizing Enterocolitis in a Neonatal Rat Model. . Digestive Disease Week. San Diego, CA: American Gastroenterological Association.