Patricia W. Lin

Necrotizing enterocolitis: recent scientific advances in pathophysiology and prevention.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality among infants in the neonatal intensive care unit. Here we review the epidemiology and pathophysiology of NEC, with an emphasis on the latest research findings and potential areas for future research. NEC continues to be one of the most devastating and unpredictable diseases affecting premature infants. Despite decades of research, the pathogenesis of this disease remains unclear, and prevention and treatment strategies are limited. Hopefully, future studies aimed at understanding premature intestinal defenses, commensal or probiotic bacterial influences, and possible genetic predisposition will lead to the improvement of prevention and treatment strategies.

Probiotic bacteria induce maturation of intestinal claudin 3 expression and barrier function

An immature intestinal epithelial barrier may predispose infants and children to many intestinal inflammatory diseases, such as infectious enteritis, inflammatory bowel disease, and necrotizing enterocolitis. Understanding the factors that regulate gut barrier maturation may yield insight into strategies to prevent these intestinal diseases. The claudin family of tight junction proteins plays an important role in regulating epithelial paracellular permeability. Previous reports demonstrate that rodent intestinal barrier function matures during the first 3 weeks of life. We show that murine paracellular permeability markedly decreases during postnatal maturation, with the most significant change occurring between 2 and 3 weeks. Here we report for the first time that commensal bacterial colonization induces intestinal barrier function maturation by promoting claudin 3 expression. Neonatal mice raised on antibiotics or lacking the toll-like receptor adaptor protein MyD88 exhibit impaired barrier function and decreased claudin 3 expression. Furthermore, enteral administration of either live or heat-killed preparations of the probiotic Lactobacillus rhamnosus GG accelerates intestinal barrier maturation and induces claudin 3 expression. However, live Lactobacillus rhamnosus GG increases mortality. Taken together, these results support a vital role for intestinal flora in the maturation of intestinal barrier function. Probiotics may prevent intestinal inflammatory diseases by regulating intestinal tight junction protein expression and barrier function. The use of heat-killed probiotics may provide therapeutic benefit while minimizing adverse effects.

Lactobacillus rhamnosus blocks inflammatory signaling in vivo via reactive oxygen species generation.

Uncontrolled inflammatory responses in the immature gut may play a role in the pathogenesis of many intestinal inflammatory syndromes that present in newborns or children, such as necrotizing enterocolitis (NEC), idiopathic inflammatory bowel diseases (IBD), or infectious enteritis. Consistent with previous reports that murine intestinal function matures over the first 3 weeks of life, we show that inflammatory signaling in the neonatal mouse gut increases during postnatal maturation, with peak responses occurring at 2-3 weeks. Probiotic bacteria can block inflammatory responses in cultured epithelia by inducing the generation of reactive oxygen species (ROS), which inhibit NF-kappaB activation through oxidative inactivation of the key regulatory enzyme Ubc12. We now report for the first time that the probiotic Lactobacillus rhamnosus GG (LGG) can induce ROS generation in intestinal epithelia in vitro and in vivo. Intestines from immature mice gavage fed LGG exhibited increased GSH oxidation and cullin-1 deneddylation, reflecting local ROS generation and its resultant Ubc12 inactivation, respectively. Furthermore, prefeeding LGG prevented TNF-alpha-induced intestinal NF-kappaB activation. These studies indicate that LGG can reduce inflammatory signaling in immature intestines by inducing local ROS generation and may be a mechanism by which probiotic bacteria can prevent NEC in premature infants or reduce the severity of IBD in children.

The probiotic Lactobacillus GG may augment intestinal host defense by regulating apoptosis and promoting cytoprotective responses in the developing murine gut.

Necrotizing enterocolitis (NEC) remains a leading cause of morbidity and mortality in preterm infants. Although its pathogenesis is poorly understood, inappropriate apoptosis of the mucosal epithelia has been implicated. Recent clinical trials have shown that probiotics may reduce the incidence of NEC, and probiotics have been shown to suppress intestinal epithelial apoptosis in cultured cells. However, little is known about their mechanism of action in the developing intestine in vivo. Here, we confirm that the probiotic Lactobacillus rhamnosus GG (LGG) reduces chemically induced intestinal epithelial apoptosis in vitro. Furthermore, we report for the first time that LGG administered orally to live animals can reduce chemically induced epithelial apoptosis ex vivo, as measured by staining for active caspase 3 and terminal deoxynucleotidyltransferase. Using cDNA microarray analysis from the intestine of live, orally inoculated mice, we show that LGG up-regulates a battery of genes with known and likely cytoprotective effects. These studies indicate that probiotics such as LGG may augment intestinal host defenses in the developing intestine by stimulating antiapoptotic and cytoprotective responses. Because apoptosis may be a precursor to NEC, understanding the mechanism behind probiotic modulation of apoptotic pathways may allow for development of more specifically targeted therapies or preventive strategies in the future.

Developmental biology of gut-probiotic interaction

While our current knowledge of probiotic interaction in the developing gut remains poorly understood, emerging science is providing greater biological insight into their mechanism of action and therapeutic potential for human disease. Given their beneficial effects, probiotics remain promising agents in neonatal gastrointestinal disorders. Probiotics may restore or supply essential bacterial strains needed for gut maturation and homeostasis, particularly in hosts where this process has been disrupted. Here we highlight the unique characteristics of developing intestinal epithelia with a focus on gut development and colonization as well as the inflammatory propensity of immature epithelia. Additionally, we review potential mechanisms of beneficial probiotic interaction with immature intestinal epithelia including immunomodulation, upregulation of cytoprotective genes, prevention and regulation of apoptosis, and maintenance of barrier function. Improved knowledge of gut-probiotic interaction in developing epithelia will allow for a better understanding of how probiotics exert their beneficial effects and help guide their therapeutic use.

Commensal Escherichia coli Reduces Epithelial Apoptosis through IFN-αA–Mediated Induction of Guanylate Binding Protein-1 in Human and Murine Models of Developing Intestine

Appropriate microbial colonization protects the developing intestine by promoting epithelial barrier function and fostering mucosal tolerance to luminal bacteria. Commensal flora mediate their protective effects through TLR9-dependent activation of cytokines, such as type I IFNs (α, β) and IL-10. Although IFN-β promotes apoptosis, IFN-α activates specific antiapoptotic target genes whose actions preserve epithelial barrier integrity. We have recently identified guanylate binding protein-1 (GBP-1) as an antiapoptotic protein, regulated by both type I and type II IFNs, that promotes intestinal epithelial barrier integrity in mature intestine. However, the mechanisms by which commensal bacteria regulate epithelial apoptosis during colonization of immature intestine and the contributions of GBP-1 are unknown. The healthy newborn intestine is initially colonized with bacterial species present in the maternal gastrointestinal tract, including nonpathogenic Escherichia coli. Therefore, we examined the influence of commensal E. coli on cytokine expression and candidate mediators of apoptosis in preweaned mice. Specifically, enteral exposure of 2 wk-old mice to commensal E. coli for 24 h selectively increased both IFN-αA and GBP-1 mRNA expression and prevented staurosporine-induced epithelial apoptosis. Exogenous IFN-αA treatment also induced GBP-1 expression and protected against staurosporine-induced apoptosis in a GBP-1 dependent manner, both in vitro and ex vivo. These findings identify a role for IFN-αA–mediated GBP-1 expression in the prevention of intestinal epithelial apoptosis by commensal bacteria. Thus IFN-αA mediates the beneficial effects of commensal bacteria and may be a promising therapeutic target to promote barrier integrity and prevent the inappropriate inflammatory responses seen in developing intestine as in necrotizing enterocolitis.

Mo1675 Pharmacokinetics and Tolerability of iOWH032, an Inhibitor of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Chloride Channel, in Normal Volunteers and Cholera Patients

Hassan Brim, Edward Lee, Scot Dowd, Adeyinka Laiyemo, Hassan Ashktorab Background: Linkage of specific bacterial markers to colorectal pathogenesis has been hampered by limited knowledge of the colonic microbiota and changing bacterial classification schemes. More than 80% of the colonic microbiota is not cultivatable and can only be assessed through metagenomic analysis. Aim: To perform a metagenomic analysis of 10 colon cancer tumors and their matched normal tissues. Methods: Metagenome sequencing was conducted on the illumina 2x150bp sequencing platform. Briefly DNA was prepared and libraries created using nextera library preparation kits. Sequencing was performed on each sample. For all samples an average of 20 million reads were obtained. Paired Sequencing reads were joined and entered into MG-RAST for metagenome analysis. The data was compared to M5NR using a maximum e-value of 1e-5, a minimum identity of 60 %, and a minimum alignment length of 15 measured in amino acids for protein databases. Results: Metagenomes were compared using best hit classification in two groups (normal and tumor). Based upon this best hit classification at the kingdom level in MG-RAST it was found, using one-way ANOVA, that tumor samples had a significantly higher classification related to Microviridae (bacteriophage) specifically related to Enterobacteria phage with a P= 0.0028. The other notable significant difference was the archaea. It was found at the class level that methanobacteria had higher classification hits in the normal samples (p = 0.03). Based upon functional gene abundance classification it was found that phage (P=0.007) as well as iron acquisition and metabolism genes (p= 0.05) were significantly different between the groups. Further analysis showed that as expected from the taxonomic classification data that tumor samples had higher levels of phage related functional classifications primarily related to phage capsid proteins. Other notable findings consisted of bacterial protein acetylation and deacetylation genes that were found to be significantly higher in normal tissue compared to tumor.

289 TUMOR NECROSIS FACTOR ALPHA LEVELS IN RESPONSE TO PATHOGENIC STIMULI IN THE DEVELOPING GUT OF EMBRYONIC AND NEWBORN MICE.

Background Prematurity is the most important risk factor for necrotizing enterocolitis (NEC), a significant cause of neonatal morbidity and mortality. Premature infants may exhibit a high risk of NEC due to developmental immaturity of the gastrointestinal tract and inability to respond appropriately to pathogenic stimuli, as compared to a more mature intestine. Purpose We used TNF-alpha as a proinflammatory marker to assess the inflammatory response to pathogenic bacteria and their products, during late embryonic and early postnatal gut development. Methods Timed pregnancies in C57BL/6J mice allowed accurate dating of neonatal mice. These mice were sacrificed at embryonic day 18 (sterile gut) and at postnatal days +16 and +21 for development of an ex vivo system of mouse intestinal infection. Intestines were isolated in 2 cm sections and surgically opened lengthwise to expose epithelia. Intestinal sections were washed in warm HBSS+ and then treated with or without wild-type Salmonella typhimurium or LPS in RPMI media at 37C, 5% CO2 for 30, 60, 120, and 180 minutes. Media was collected for further analysis. Proinflammatory response was measured by ELISA for TNF-alpha. Summary of Results Older mice demonstrated a larger proinflammatory response, as measured by increased TNF-alpha secretion, as compared to the embryonic day 18 mice. Peak response occurred 2-3 hours after being stimulated by the bacteria or LPS. Conclusions Our preliminary data indicate that the immature intestine is not appropriately responding to pathogenic stimuli. We further speculate that initial colonization of the neonatal intestine with normal flora may play a key role in regulating this response. Further experiments with gnotobiotic mice will help delineate the exchange of prokaryotic and eukaryotic signals involved.