EP2 Receptor Blockade Attenuates COX-2 Upregulation During Intestinal Inflammation.

High levels of PGE2 have been implicated in the pathogenesis of intestinal inflammatory disorders such as necrotizing enterocolitis (NEC) and peritonitis. However, PGE2 has a paradoxical effect: its low levels promote intestinal homeostasis, whereas high levels may contribute to pathology. These concentration-dependent effects are mediated by four receptors, EP1-EP4. In this study, we evaluate the effect of blockade of the low affinity pro-inflammatory receptors EP1 and EP2 on expression of COX-2, the rate-limiting enzyme in PGE2 biosynthesis, and on gut barrier permeability using cultured enterocytes and three different models of intestinal injury. PGE2 upregulated COX-2 in IEC-6 enterocytes, and this response was blocked by the EP2 antagonist PF-04418948, but not by the EP1 antagonist ONO-8711 or EP4 antagonist E7046. In the neonatal rat model of NEC, EP2 antagonist and low dose of COX-2 inhibitor Celecoxib, but not EP1 antagonist, reduced NEC pathology as well as COX-2 mRNA and protein expression. In the adult mouse endotoxemia and cecal ligation/puncture models, EP2, but not EP1 genetic deficiency decreased COX-2 expression in the intestine. Our results indicate that the EP2 receptor plays a critical role in the positive feedback regulation of intestinal COX-2 by its end-product PGE2 during inflammation and may be a novel therapeutic target in the treatment of NEC.

Effects of artificially introduced Enterococcus faecalis strains in experimental necrotizing enterocolitis.

Enterococcus faecalis is a ubiquitous intestinal symbiont and common early colonizer of the neonatal gut. Although colonization with E. faecalis has been previously associated with decreased pathology of necrotizing enterocolitis (NEC), these bacteria have been also implicated as opportunistic pathogens. Here we characterized 21 strains of E. faecalis, naturally occurring in 4-day-old rats, for potentially pathogenic properties and ability to colonize the neonatal gut. The strains differed in hemolysis, gelatin liquefaction, antibiotic resistance, biofilm formation, and ability to activate the pro-inflammatory transcription factor NF-κB in cultured enterocytes. Only 3 strains, BB70, 224, and BB24 appreciably colonized the neonatal intestine on day 4 after artificial introduction with the first feeding. The best colonizer, strain BB70, effectively displaced E. faecalis of maternal origin. Whereas BB70 and BB24 significantly increased NEC pathology, strain 224 significantly protected from NEC. Our results show that different strains of E. faecalis may be pathogenic or protective in experimental NEC.

Lactobacillus murinus HF12 colonizes neonatal gut and protects rats from necrotizing enterocolitis.

The use of lactobacilli in prevention of necrotizing enterocolitis (NEC) is hampered by insufficient knowledge about optimal species/strains and effects on intestinal bacterial populations. We therefore sought to identify lactobacilli naturally occurring in postnatal rats and examine their ability to colonize the neonatal intestine and protect from NEC. L. murinus, L. acidophilus, and L. johnsonii were found in 42, 20, and 1 out of 51 4-day old rats, respectively. Higher proportion of L. murinus in microbiota correlated with lower NEC scores. Inoculation with each of the three species during first feeding significantly augmented intestinal populations of lactobacilli four days later, indicating successful colonization. L. murinus, but not L. acidophilus or L. johnsonii, significantly protected against NEC. Thus, lactobacilli protect rats from NEC in a species- or strain-specific manner. Our results may help rationalizing probiotic therapy in NEC.

Soybean-derived recombinant human epidermal growth factor protects against experimental necrotizing enterocolitis.

BACKGROUND: Epidermal Growth Factor (EGF) reduces necrotizing enterocolitis (NEC). However, its high cost virtually prohibits clinical use. To reduce cost, soybean expressing human EGF was developed. Here we report effectiveness of soybean-derived EGF in experimental NEC. METHODS: Newborn rats were subjected to the NEC-inducing regimen of formula feeding and hypoxia. Formula was supplemented with extract from EGF-expressing or empty soybeans. NEC pathology was determined microscopically. Localization of tight junction proteins JAM-A and ZO-1 was examined by immunofluorescence and levels of mucosal COX-2 and iNOS mRNAs by real time PCR. RESULTS: Soybean extract amounts corresponding to 150µg/kg/day EGF caused considerable mortality, whereas those corresponding to 75µg/kg/day EGF were well tolerated. There was no significant difference in NEC scores between animals fed plain formula and formula supplemented with empty soybean extract. Soybean-EGF-supplemented formula at 75µg/kg/day EGF significantly decreased NEC, attenuated dissociation of JAM-A and ZO-1 proteins from tight junctions, and reduced intestinal expression of COX-2 and iNOS mRNAs. CONCLUSION: Supplementation with soybean-expressed EGF significantly decreased NEC in the rat model. Soybean-expressed EGF may provide an economical solution for EGF administration and prophylaxis of clinical NEC.

Evolving understanding of neonatal necrotizing enterocolitis.

PURPOSE OF REVIEW: Necrotizing enterocolitis (NEC) is a devastating disease that predominately affects premature neonates. The pathogenesis of NEC is multifactorial and poorly understood. Risk factors include low birth weight, formula-feeding, hypoxic/ischemic insults, and microbial dysbiosis. This review focuses on our current understanding of the diagnosis, management, and pathogenesis of NEC. RECENT FINDINGS: Recent findings identify specific mucosal cell types as potential therapeutic targets in NEC. Despite a broadly accepted view that bacterial colonization plays a key role in NEC, characteristics of bacterial populations associated with this disease remain elusive. The use of probiotics such as lactobacilli and bifidobacteria has been studied in numerous trials, but there is a lack of consensus regarding specific strains and dosing. Although growth factors found in breast milk such as epidermal growth factor and heparin-binding epidermal growth factor may be useful in disease prevention, developing new therapeutic interventions in NEC critically depends on better understanding of its pathogenesis. SUMMARY: NEC is a leading cause of morbidity and mortality in premature neonates. Recent data confirm that growth factors and certain bacteria may offer protection against NEC. Further studies are needed to better understand the complex pathogenesis of NEC.

Colonization with Escherichia coli EC 25 protects neonatal rats from necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a significant cause of morbidity and mortality in premature infants; yet its pathogenesis remains poorly understood. To evaluate the role of intestinal bacteria in protection against NEC, we assessed the ability of naturally occurring intestinal colonizer E. coli EC25 to influence composition of intestinal microbiota and NEC pathology in the neonatal rat model. Experimental NEC was induced in neonatal rats by formula feeding/hypoxia, and graded histologically. Bacterial populations were characterized by plating on blood agar, scoring colony classes, and identifying each class by sequencing 16S rDNA. Binding of bacteria to, and induction of apoptosis in IEC-6 enterocytes were examined by plating on blood agar and fluorescent staining for fragmented DNA. E. coli EC 25, which was originally isolated from healthy rats, efficiently colonized the intestine and protected from NEC following introduction to newborn rats with formula at 106 or 108 cfu. Protection did not depend significantly on EC25 inoculum size or load in the intestine, but positively correlated with the fraction of EC25 in the microbiome. Introduction of EC25 did not prevent colonization with other bacteria and did not significantly alter bacterial diversity. EC25 neither induced cultured enterocyte apoptosis, nor protected from apoptosis induced by an enteropathogenic strain of Cronobacter muytjensii. Our results show that E. coli EC25 is a commensal strain that efficiently colonizes the neonatal intestine and protects from NEC.

Experimental Anti-Inflammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone gp96.

Semapimod, a tetravalent guanylhydrazone, suppresses inflammatory cytokine production and has potential in a variety of inflammatory and autoimmune disorders. The mechanism of action of Semapimod is not well understood. In this study, we demonstrate that in rat IEC-6 intestinal epithelioid cells, Semapimod inhibits activation of p38 MAPK and NF-κB and induction of cyclooxygenase-2 by TLR ligands, but not by IL-1ß or stresses. Semapimod inhibits TLR4 signaling (IC50 ≈0.3 µmol) and acts by desensitizing cells to LPS; it fails to block responses to LPS concentrations of ≥5 µg/ml. Inhibition of TLR signaling by Semapimod is almost instantaneous: the drug is effective when applied simultaneously with LPS. Semapimod blocks cell-surface recruitment of the MyD88 adapter, one of the earliest events in TLR signaling. gp96, the endoplasmic reticulum-localized chaperone of the HSP90 family critically involved in the biogenesis of TLRs, was identified as a target of Semapimod using ATP-desthiobiotin pulldown and mass spectroscopy. Semapimod inhibits ATP-binding and ATPase activities of gp96 in vitro (IC50 ≈0.2-0.4 µmol). On prolonged exposure, Semapimod causes accumulation of TLR4 and TLR9 in perinuclear space, consistent with endoplasmic reticulum retention, an anticipated consequence of impaired gp96 chaperone function. Our data indicate that Semapimod desensitizes TLR signaling via its effect on the TLR chaperone gp96. Fast inhibition by Semapimod is consistent with gp96 participating in high-affinity sensing of TLR ligands in addition to its role as a TLR chaperone.

Toll-like receptor 3 mediates PROMININ-1 expressing cell expansion in biliary atresia via Transforming Growth Factor-Beta.

BACKGROUND: In biliary atresia (BA), epithelial-mesenchymal hepatic progenitor cells (HPC) expressing the stem/progenitor cell marker PROMININ-1 (PROM1) undergo expansion and subsequent transdifferentiation into collagen-producing myofibroblasts within regions of evolving biliary fibrosis under the regulation of Transforming Growth Factor-ß (TGFß) signaling. We hypothesized that pro-inflammatory Toll-like Receptor-3 (TLR3) signal activation promotes the differentiation of PROM1+ HPC via TGFß pathway activation in vitro. METHODS: PROM1+ Mat1a(-/-) HPC were treated with a double-stranded RNA analog, polyionosinic-polycytidylic acid (Poly I:C), ± small molecule inhibitors nafamostat, or SB431542. RESULTS: Poly I:C induced myofibroblastic-like morphologic changes, degradation of IκB-α consistent with TLR3-NFκB activation, a 15-fold increase in the expression of Vimentin, a 9-fold increase in Collagen-1a, a 4.6-fold increase in Snail at 24h (p<0.05), and an 8.2-fold increase in Prom1 at 72h (p<0.0001) by qPCR. Immunofluorescence demonstrated nuclear phosphorylated SMAD3, TLR3, and COLLAGEN-1α staining following Poly I:C treatment. Degradation of IκBα was inhibited by nafamostat. Co-treatment with either nafamostat or SB431542 blocked the morphologic change and abrogated the increased expression of Cd133, Collagen, Vimentin, and Snail1. CONCLUSIONS: TLR3 activation induces myofibroblastic differentiation of PROM1+ HPC in part via TGFß pathway activation to promote BA-associated biliary fibrosis.

Roles of nitric oxide and intestinal microbiota in the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis remains one of the most vexing problems in the neonatal intensive care unit. Risk factors for NEC include prematurity, formula feeding, and inappropriate microbial colonization of the GI tract. The pathogenesis of NEC is believed to involve weakening of the intestinal barrier by perinatal insults, translocation of luminal bacteria across the weakened barrier, an exuberant inflammatory response, and exacerbation of the barrier damage by inflammatory factors, leading to a vicious cycle of inflammation-inflicted epithelial damage. Nitric oxide (NO), produced by inducible NO synthase (iNOS) and reactive NO oxidation intermediates play a prominent role in the intestinal barrier damage by inducing enterocyte apoptosis and inhibiting the epithelial restitution processes, namely enterocyte proliferation and migration. The factors that govern iNOS upregulation in the intestine are not well understood, which hampers efforts in developing NO/iNOS-targeted therapies. Similarly, efforts to identify bacteria or bacterial colonization patterns associated with NEC have met with limited success, because the same bacterial species can be found in NEC and in non-NEC subjects. However, microbiome studies have identified the three important characteristics of early bacterial populations of the GI tract: high diversity, low complexity, and fluidity. Whether NEC is caused by specific bacteria remains a matter of debate, but data from hospital outbreaks of NEC strongly argue in favor of the infectious nature of this disease. Studies in Cronobacter muytjensii have established that the ability to induce NEC is the property of specific strains rather than the species as a whole. Progress in our understanding of the roles of bacteria in NEC will require microbiological experiments and genome-wide analysis of virulence factors.

Low doses of celecoxib attenuate gut barrier failure during experimental peritonitis.

The intestinal barrier becomes compromised during systemic inflammation, leading to the entry of luminal bacteria into the host and gut origin sepsis. Pathogenesis and treatment of inflammatory gut barrier failure is an important problem in critical care. In this study, we examined the role of cyclooxygenase-2 (COX-2), a key enzyme in the production of inflammatory prostanoids, in gut barrier failure during experimental peritonitis in mice. I.p. injection of LPS or cecal ligation and puncture (CLP) increased the levels of COX-2 and its product prostaglandin E2 (PGE2) in the ileal mucosa, caused pathologic sloughing of the intestinal epithelium, increased passage of FITC-dextran and bacterial translocation across the barrier, and increased internalization of the tight junction (TJ)-associated proteins junction-associated molecule-A and zonula occludens-1. Luminal instillation of PGE2 in an isolated ileal loop increased transepithelial passage of FITC-dextran. Low doses (0.5-1 mg/kg), but not a higher dose (5 mg/kg) of the specific COX-2 inhibitor Celecoxib partially ameliorated the inflammatory gut barrier failure. These results demonstrate that high levels of COX-2-derived PGE2 seen in the mucosa during peritonitis contribute to gut barrier failure, presumably by compromising TJs. Low doses of specific COX-2 inhibitors may blunt this effect while preserving the homeostatic function of COX-2-derived prostanoids. Low doses of COX-2 inhibitors may find use as an adjunct barrier-protecting therapy in critically ill patients.

The role of the intestinal microbiota in the pathogenesis of necrotizing enterocolitis.

Development of necrotizing enterocolitis (NEC) requires a susceptible host, typically a premature infant or an infant with congenital heart disease, enteral feedings and bacterial colonization. Although there is little doubt that microbes are critically involved in the pathogenesis of NEC, the identity of specific causative pathogens remains elusive. Unlike established normal adult gut microbiota, which is quite complex, uniform, and stable, early postnatal bacterial populations are simple, diverse, and fluid. These properties complicate studies aimed at elucidating characteristics of the gut microbiome that may play a role in the pathogenesis of NEC. A broad variety of bacterial, viral, and fungal species have been implicated in both clinical and experimental NEC. Frequently, however, the same species have also been found in physiologically matched healthy individuals. Clustered outbreaks of NEC, in which the same strain of a suspected pathogen is detected in several patients suggest, but do not prove, a causative relationship between the specific pathogen and the disease. Studies in Cronobacter sakazakii, the best characterized NEC pathogen, have demonstrated that virulence is not a property of a bacterial species as a whole, but rather a characteristic of certain strains, which may explain why the same species can be pathogenic or non-pathogenic. The fact that a given microbe may be innocuous in a full-term, yet pathogenic in a pre-term infant has led to the idea of opportunistic pathogens in NEC. Progress in understanding the infectious nature of NEC may require identifying specific pathogenic strains and unambiguously establishing their virulence in animal models.

Transactivation of EGFR by LPS induces COX-2 expression in enterocytes.

Necrotizing enterocolitis (NEC) is the leading cause of gastrointestinal morbidity and mortality in preterm infants. NEC is characterized by an exaggerated inflammatory response to bacterial flora leading to bowel necrosis. Bacterial lipopolysaccharide (LPS) mediates inflammation through TLR4 activation and is a key molecule in the pathogenesis of NEC. However, LPS also induces cyclooxygenase-2 (COX-2), which promotes intestinal barrier restitution through stimulation of intestinal cell survival, proliferation, and migration. Epidermal growth factor receptor (EGFR) activation prevents experimental NEC and may play a critical role in LPS-stimulated COX-2 production. We hypothesized that EGFR is required for LPS induction of COX-2 expression. Our data show that inhibiting EGFR kinase activity blocks LPS-induced COX-2 expression in small intestinal epithelial cells. LPS induction of COX-2 requires Src-family kinase signaling while LPS transactivation of EGFR requires matrix metalloprotease (MMP) activity. EGFR tyrosine kinase inhibitors block LPS stimulation of mitogen-activated protein kinase ERK, suggesting an important role of the MAPK/ERK pathway in EGFR-mediated COX-2 expression. LPS stimulates proliferation of IEC-6 cells, but this stimulation is inhibited with either the EGFR kinase inhibitor AG1478, or the selective COX-2 inhibitor Celecoxib. Taken together, these data show that EGFR plays an important role in LPS-induction of COX-2 expression in enterocytes, which may be one mechanism for EGF in inhibition of NEC.

Role of interleukin-10 in the pathogenesis of necrotizing enterocolitis.

BACKGROUND: Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in premature neonates. The pathogenesis of NEC is characterized by an intestinal epithelial injury caused by perinatal insults, leading to the activation of the mucosal innate immune system and exacerbation of the epithelial barrier damage. Cytokines play an important role in mucosal immunity. Interleukin-10 (IL-10) is an anti-inflammatory cytokine that has been shown to play a role in epithelial integrity and modulation of the mucosal immune system. We hypothesized that IL-10 may protect against the development of experimental NEC by blunting the inflammatory response in the intestine. METHODS: Wild-type and IL-10 -/- mice underwent a NEC-inducing regimen of formula feeding in combination with hypoxia and hypothermia (FF+HH). Integrity of the gut barrier was assessed through measurement of epithelial apoptosis, tight junction disruption, and inducible nitric oxide synthase. A total of 5 µg of exogenous IL-10 was administered intraperitoneally to IL-10-/-mouse pups before the initiation of FF+HH to test dependence of gene knockout phenotype on IL-10. RESULTS: IL-10 -/- FF+HH showed more severe morphologic and histologic changes compared with controls as evidenced by increased epithelial apoptosis, decreased junctional adhesion molecule-1 localization, and increased intestinal inducible nitric oxide synthase expression. Administration of exogenous IL-10 alleviated the mucosal injury. CONCLUSIONS: We conclude that IL-10 plays a protective role in the pathogenesis of NEC by attenuating the degree of intestinal inflammation.

The human milk oligosaccharide disialyllacto-N-tetraose prevents necrotising enterocolitis in neonatal rats.

BACKGROUND: Necrotising enterocolitis (NEC) is one of the most common and fatal intestinal disorders in preterm infants. Breast-fed infants are at lower risk for NEC than formula-fed infants, but the protective components in human milk have not been identified. In contrast to formula, human milk contains high amounts of complex glycans. OBJECTIVE: To test the hypothesis that human milk oligosaccharides (HMO) contribute to the protection from NEC. METHODS: Since human intervention studies are unfeasible due to limited availability of HMO, a neonatal rat NEC model was used. Pups were orally gavaged with formula without and with HMO and exposed to hypoxia episodes. Ileum sections were scored blindly for signs of NEC. Two-dimensional chromatography was used to determine the most effective HMO, and sequential exoglycosidase digestions and linkage analysis was used to determine its structure. RESULTS: Compared to formula alone, pooled HMO significantly improved 96-hour survival from 73.1% to 95.0% and reduced pathology scores from 1.98 ± 1.11 to 0.44 ± 0.30 (p<0.001). Within the pooled HMO, a specific isomer of disialyllacto-N-tetraose (DSLNT) was identified to be protective. Galacto-oligosaccharides, currently added to formula to mimic some of the effects of HMO, had no effect. CONCLUSION: HMO reduce NEC in neonatal rats and the effects are highly structure specific. If these results translate to NEC in humans, DSLNT could be used to prevent or treat NEC in formula-fed infants, and its concentration in the mother's milk could serve as a biomarker to identify breast-fed infants at risk of developing this disorder.

P-glycoprotein induction by breast milk attenuates intestinal inflammation in experimental necrotizing enterocolitis.

P-glycoprotein (Pgp), a product of the multi-drug resistance gene MDR1a, is a broad specificity efflux ATP cassette transmembrane transporter that is predominantly expressed in epithelial tissues. Because mdr1a(-/-) mice tend to develop spontaneous colitis in bacteria-dependent manner, Pgp is believed to have a role in protection of the intestinal epithelium from luminal bacteria. Here we demonstrate that levels of Pgp in the small intestine of newborn rodents dramatically increase during breastfeeding, but not during formula feeding (FF). In rats and mice, levels of intestinal Pgp peak on days 3-7 and 1-5 of breastfeeding, respectively. The mdr1a(-/-) neonatal mice subjected to FF, hypoxia, and hypothermia have significantly higher incidence and pathology, as well as significantly earlier onset of necrotizing enterocolitis (NEC) than congenic wild type mice. Breast-fed mdr1a(-/-) neonatal mice are also more susceptible to intestinal damage caused by the opportunistic pathogen Cronobacter sakazakii that has been associated with hospital outbreaks of NEC. Breast milk, but not formula, induces Pgp expression in enterocyte cell lines in a dose- and time-dependent manner. High levels of ectopically expressed Pgp protect epithelial cells in vitro from apoptosis induced by C. sakazakii. Taken together, these results show that breast milk-induced expression of Pgp may have a role in the protection of the neonatal intestinal epithelium from injury associated with nascent bacterial colonization.

Ubiquitin-editing enzyme A20 promotes tolerance to lipopolysaccharide in enterocytes.

Although enterocytes are capable of innate immune responses, the intestinal epithelium is normally tolerant to commensal bacteria. To elucidate the mechanisms of tolerance, we examined the effect of preexposure to LPS on activation of p38, c-Jun, and NF-kappaB in enterocytes by several inflammatory and stress stimuli. Shortly after the initial LPS challenge, enterocytes become tolerant to restimulation with LPS or CpG DNA, but not with IL-17 or UV. The state of tolerance, which lasts 20-26 h, temporally coincides with LPS-induced expression of the anti-inflammatory ubiquitin-editing enzyme A20. Small interfering RNA silencing of A20 prevents tolerance, whereas ectopic expression of A20 blocks responses to LPS and CpG DNA, but not to IL-17 or UV. A20 levels in the epithelium of the small intestine are low at birth and following gut decontamination with antibiotics, but high under conditions of bacterial colonization. In the small intestine of adult rodents, A20 prominently localizes to the luminal interface of villus enterocytes. Lower parts of the crypts display relatively low levels of A20, but relatively high levels of phospho-p38. Gut decontamination with antibiotics reduces the levels of both A20 and phospho-p38. Along with the fact that A20-deficient mice develop severe intestinal inflammation, our results indicate that induction of A20 plays a key role in the tolerance of the intestinal epithelium to TLR ligands and bacteria.

Peroxynitrite-induced p38 MAPK pro-apoptotic signaling in enterocytes.

Enterocyte apoptosis in necrotizing enterocolitis is partly due to the elaboration of toxic intermediates of nitric oxide (NO), such as peroxynitrite (PN). Because p38 mitogen-activated protein kinase (MAPK) and serine-threonine kinase (AKT) are well-characterized pro- and anti-apoptotic mediators, respectively, we hypothesized that PN could induce enterocyte apoptosis via activation of p38 and deactivation of AKT. To test this hypothesis, the rat intestinal cell line, IEC-6, was treated with PN. PN caused phosphorylation of p38, its upstream activator, MKK3/6, and downstream effector, transcription factor ATF-2. PN-induced apoptosis was inhibited by the p38 inhibitor, SB202190, and by p38 siRNA. PN decreased AKT phosphorylation; this effect was abrogated by pre-treatment with SB202190 or p38 siRNA. PN exposure also increased the activity of the protein phosphatase 2A (PP2A). These data demonstrate that PN-mediated apoptosis depends on the p38 pathway and that p38 mediates deactivation of AKT survival pathways possibly by the involvement of PP2A.

Specific and slow inhibition of the kir2.1 K+ channel by gambogic acid.

Although Kir2.1 channels are important in the heart and other excitable cells, there are virtually no specific drugs for this K+ channel. In search of Kir2.1 modulators, we screened a library of 720 naturally occurring compounds using a yeast strain in which mammalian Kir2.1 enables growth at low [K+]. One of the identified compounds, gambogic acid (GA), potently (EC(50) < or = 100 nm) inhibited Kir2.1 channels in mammalian cells when applied chronically for 3 h. This potent and slow inhibition was not seen with Kv2.1, HERG or Kir1.1 channels. However, acutely applied GA acted as a weak (EC(50) = approximately 10 mum) non-selective K+ channel blocker. Intracellular delivery of GA via a patch pipette did not potentiate the acute effect of GA on Kir2.1, showing that slow uptake is not responsible for the delayed, potent effect. Immunoblots showed that total Kir2.1 protein expression was not altered by GA. Similarly, immunostaining of intact cells expressing Kir2.1 with an extracellular epitope tag demonstrated that GA does not affect Kir2.1 surface expression. However, the 3-h treatment with GA caused redistribution of Kir2.1 and Kv2.1 from the Triton X-100-insoluble to the Triton X-100-soluble membrane fraction. Thus, GA changes the K+ channel membrane microenvironment resulting in potent, specific, and slow acting inhibition of Kir2.1 channels.

Current concepts regarding the pathogenesis of necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a devastating disease that predominantly affects premature neonates. The mortality associated with NEC has not changed appreciably over the past several decades. The underlying etiology of NEC remains elusive, although bacterial colonization of the gut, formula feeding, and perinatal stress have been implicated as putative risk factors. The disease is characterized by massive epithelial destruction, which results in gut barrier failure. The exact molecular and cellular mechanisms involved in this complex disease are poorly understood. Recent studies have provided significant insight into our understanding of the pathogenesis of NEC. Endogenous mediators such as prostanoids, cyclooxygenases, and nitric oxide may play a role in the development of gut barrier failure. Understanding the structural architecture of the gut barrier and the cellular mechanisms that are responsible for gut epithelial damage could lead to the development of novel diagnostic, prophylactic and therapeutic strategies in NEC.

Induction of fibroblast growth factor 10 (FGF10) in the ileal crypt epithelium after massive small bowel resection suggests a role for FGF10 in gut adaptation.

We have previously reported that fibroblast growth factor 10 (FGF10) is crucial for the survival and proliferation of progenitor cells during embryonic gastrointestinal development. We sought to characterize the potential role of FGF10 signaling in the adaptive response following small bowel resection. Adult wild-type and Fgf10(LacZ) mice underwent 50% small bowel resection (SBR) or sham operation. Tissues were harvested 24 or 48 hr after surgery for histology, immunohistochemistry, and in situ hybridization. After SBR, Fgf10 expression was demonstrated in the epithelium at the base of the crypts. Moreover, there was a statistically significant increase in proliferating cells and goblet cells after SBR. In vitro studies using rat intestinal epithelial crypt (IEC-6) cells exposed to medium with or without recombinant FGF10 showed increased proliferation and phosphorylation of Raf and AKT with the addition of FGF10. Our results suggest that FGF10 may play a therapeutic role in diseases involving intestinal failure.