Bohuslav Dvorak

New Therapies and Preventive Approaches for Necrotizing Enterocolitis: Report of a Research Planning Workshop

The National Institute of Child Health and Human Development and the Digestive Diseases Interagency Coordinating Committee held a workshop, chaired by Dr. W. Allan Walker, on July 10–11, 2006, to identify promising leads in necrotizing enterocolitis (NEC) research. The goals of the workshop were to identify new approaches to the prevention and treatment of NEC, to define basic and translational mechanisms of potential approaches to NEC, and to develop recommendations for clinical studies to reduce the incidence of NEC. Workshop participants implicated prematurity, introduction of enteral feedings, gastrointestinal bacterial colonization, gut motility, proinflammatory cytokines, impaired gut blood flow, and various neonatal complications in the pathogenesis of NEC. They concluded that a unifying hypothesis encompassing these pathogenetic factors is the uncontrolled exuberant inflammatory response to bacterial colonization that characterizes the intestine of premature infants. The inflammatory cascade appears to offer multiple targets for interventions with a variety of anti-inflammatory agents, including human milk and probiotics. Because of the rapidity with which the inflammatory response gets out of control in infants with NEC, workshop participants agreed that searching for ways to prevent NEC will be more rewarding than trying to identify ways to treat the condition once it has become established.

Up-regulation of IL-18 and IL-12 in the ileum of neonatal rats with necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a common and devastating gastrointestinal disease of premature infants. Because the proinflammatory cytokines IL-18, IL-12, and interferon (IFN)-γ 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-γ using real-time reverse transcriptase PCR and immunohistology. IL-18 and IL-12 mRNA from the RMS group were increased (p ≤ 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 ≤ 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 ≤ 0.05). No differences in the number of IFN-γ–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.

Maternal milk reduces severity of necrotizing enterocolitis and increases intestinal IL-10 in a neonatal rat model

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.

Increased Epidermal Growth Factor Levels in Human Milk of Mothers with Extremely Premature Infants

Maternal milk is the major source of nutrients and growthpromoting substances in the first weeks of life for the majority of neonates. Epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) are trophic peptides present in human milk with significant healing effects on injured gastrointestinal mucosa. Decreasing gestational age of neonates is associated with higher risk of developing gastrointestinal disorders, and human milk provides better protection against these diseases compared with formula. The aim of this study was to evaluate the concentrations of EGF and TGF-α in human milk collected from mothers with infants born: extremely preterm, preterm, and full term. Milk samples were collected at the end of first, second, and fourth week postpartum from each mother of infants born in one of the three gestational age groups: extremely preterm (23-27 wk, n = 16), preterm (32-36 wk, n = 16), and full term (38-42 wk, n = 15). Milk concentrations of EGF and TGF-α were quantified with a homologous RIA in the milk aqueous fraction. Concentrations of EGF in human milk from the extremely preterm group (23-27 wk) were significantly higher compared with values from the preterm and full-term groups throughout the first month of lactation. A similar pattern was observed with human milk TGF-α; however, milk TGF-α levels were lower than EGF. In conclusion, we have found higher concentrations of EGF and TGF-α in human milk of mothers with extremely preterm babies. These data may indicate the potential importance of milk-borne EGF and TGF-α for the development of extremely premature infants.

Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis

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.

Erythropoietin stimulates vasculogenesis in neonatal rat mesenteric microvascular endothelial cells.

Human breast milk is a rich source of growth factors, including erythropoietin (Epo), the endogenous hormonal stimulant of erythropoiesis. Recombinant human Epo (rhEpo) has been shown to stimulate 1) angiogenesis, the process of new blood vessel growth from preexisting vessels;2) vasculogenesis, tubule formation from single-cell suspensions; and 3) endothelial cell proliferation in immortalized endothelial cells and vessel explants. We hypothesized that Epo would induce mitogenesis and stimulate vasculogenesis in primary cultures of microvascular endothelial cells (MVECs) from neonatal rat mesentery. Isolation, purification, characterization, and culture of MVECs were performed. Cell proliferative effects of rhEpo were studied by 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay in cultured MVECs. Vasculogenic effects of rhEpo were examined on cultured MVECs plated on either hormone-rich Matrigel substratum or the extracellular matrix protein, type I collagen. Our findings show that MVECs are isolated and purified, and that rhEpo stimulates MVEC proliferation, with maximal proliferation seen with a concentration of 50 IU/mL rhEpo. Tubule formation assays reveal that an rhEpo concentration of 50 IU/mL produces maximal tubule formation after 12 h on both Matrigel and the simple substratum, type I collagen. Our study is the first to examine the effects of rhEpo on the endothelium of the neonatal gastrointestinal tract. These data suggest that Epo may have a trophic effect on the vasculature of the gastrointestinal tract early in development. Furthermore, as Epo has been measured in breast milk, and its receptor has been shown to exist on the mucosa and gastrointestinal vasculature, Epo may be an endogenous stimulant of vessel growth during neonatal gastrointestinal development.

Increased Expression and Secretion of Interleukin-6 in Patients with Barrett’s Esophagus

Purpose: Barrett’s esophagus (BE) is a common premalignant lesion of the distal part of the esophagus that arises as a consequence of chronic duodenogastroesophageal reflux. Interleukin (IL)-6 is a pleiotropic cytokine that regulates immune defense mechanisms and hematopoiesis. In addition, IL-6 may also be involved in malignant transformation and tumor progression. IL-6 has been shown to inhibit apoptosis. The major aim of this study was to evaluate expression of IL-6 in BE at the protein and mRNA levels. In addition, we tested whether proteins that are associated with IL-6 signaling, phosphorylated signal transducer and activator of transcription 3 and two antiapoptotic proteins, Bcl-xL and Mcl-1, are also expressed in the same tissues. Experimental Design: Biopsies of duodenum, BE, and squamous epithelium were evaluated by using a human cytokine protein array, ELISA, real-time PCR, and immunohistochemistry. Results: Increased IL-6 levels were found to be secreted from BE tissue compared with duodenum or squamous epithelium from sites adjacent or 5 cm away from the BE lesion. IL-6 mRNA was also elevated in BE compared with duodenum or squamous epithelium in five of seven patients. Immunohistochemical studies confirmed IL-6 expression in intestinal glandular epithelium in BE tissue. Activated signal transducer and activator of transcription 3, Mcl-1, and Bcl-xL are present at higher levels in BE glands, with lower levels being found in duodenum or squamous epithelium Conclusions: These data, taken together, suggest that elevated IL-6 levels in BE may contribute to the development of apoptosis resistance, thereby placing this epithelium at higher risk of developing malignancy.

Milk Epidermal Growth Factor and Gut Protection

Maternal milk is a complex fluid, with multifunctional roles within the developing gastrointestinal tract. Epidermal growth factor (EGF) and heparin-binding EGF-like growth factor (HB-EGF) are members of the family of EGF-related peptides. Biological actions of these growth factors are mediated via interaction with the EGF-receptor (EGF-R). In the early postnatal period, breast milk is the major source of EGF for the developing intestinal mucosa. HB-EGF is also detected in breast milk, but in concentrations 2 to 3 times lower than EGF. With normal physiological conditions, the intestinal epithelium undergoes a continuing process of cell proliferation, differentiation, and maturation. EGF plays an important role in these processes. In pathophysiologic situations, EGF contributes to epithelial protection from injury and post-injury mucosal repair. Necrotizing enterocolitis (NEC) is a devastating disease affecting infants born prematurely. The pathogenesis of NEC is not known, and there is no effective treatment for this disease. In an experimental NEC model, oral administration of a physiological dose of EGF significantly reduces the incidence and severity of NEC. HB-EGF provides similar protection against NEC, but only when pharmacological doses are used. Further studies are necessary before EGF can be introduced as an efficient therapeutic approach of intestinal injury.

Activation of the Interleukin-6/STAT3 Antiapoptotic Pathway in Esophageal Cells by Bile Acids and Low pH: Relevance to Barrett's Esophagus

Objectives: The molecular factors contributing to the development of Barretts esophagus (BE) are unclear. Our previous studies showed that BE tissues secrete interleukin-6 (IL-6) and express proteins associated with IL-6 signaling, including IL-6 receptor, activated signal transducer and activators of transcription 3 (STAT3), and antiapoptotic proteins Bcl-xL and Mcl-1. Here, we test the hypothesis that bile acids and gastric acids, two components of refluxate associated with gastresophageal reflux disease, activate the IL-6/STAT3 pathway. Materials and Methods: Immunohistochemistry was used to assess levels of phosphorylated STAT3 in esophageal tissue samples from BE patients with different grades of dysplasia. Seg-1 esophageal adenocarcinoma cells were evaluated for STAT3 activation and IL-6 and Bcl-xL expression by molecular biology techniques, including Western blot, reverse transcription–PCR, and ELISA after exposure to control media (pH 7.4), media supplemented with a 0.1 mmol/L bile acid cocktail with media at pH 4 or media at pH 4 with bile acid cocktail. Results: Immunohistochemical analysis showed that activated, phosphorylated STAT3 is expressed in nuclei of dysplastic BE and cancer tissues. Treatment of Seg-1 cells with media containing bile acid cocktail and acidified to pH 4 resulted in increased activation of STAT3, IL-6 secretion, and increased expression of Bcl-xL. Inhibition of the STAT3 pathway using STAT3 small interfering RNA or Janus-activated kinase inhibitor resulted in increased apoptosis. Conclusions: The IL-6/STAT3 antiapoptotic pathway is induced by short exposure to bile acid cocktail and low pH. This alteration, if persistent in vivo, may underlie the development of dysplastic BE and tumor progression.

Ileal Cytokine Dysregulation in Experimental Necrotizing Enterocolitis Is Reduced by Epidermal Growth Factor

BackgroundNecrotizing 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. MethodsNewborn rats were artificially fed with either growth-factor–free rat milk substitute (RMS) or RMS supplemented with 500 ng/mL EGF (RMS + EGF) and NEC was induced via exposure to asphyxia and cold stress. Cytokine expression and localization were assessed using reverse-transcription real-time polymerase chain reaction and immunohistology/confocal microscopy. ResultsEnteral administration of EGF (RMS + EGF) decreased overproduction of interleukin-18 and increased interleukin-10 production in the ileum. Furthermore, increased interleukin-10 production was associated with up-regulation of the transcription factor Sp1 in RMS + EGF rats. ConclusionsThese data suggest that EGF may reduce NEC via increased interleukin-10 and decreased interleukin-18 and that EGF-mediated up-regulation of Sp1 may account for the increased interleukin-10.