Golam Mostafa

Intestinal alkaline phosphatase is a gut mucosal defense factor maintained by enteral nutrition.

Under conditions of starvation and disease, the gut barrier becomes impaired, and trophic feeding to prevent gut mucosal atrophy has become a standard treatment of critically ill patients. However, the mechanisms responsible for the beneficial effects of enteral nutrition have remained a mystery. Using in vitro and in vivo models, we demonstrate that the brush–border enzyme, intestinal alkaline phosphatase (IAP), has the ability to detoxify lipopolysaccharide and prevent bacterial invasion across the gut mucosal barrier. IAP expression and function are lost with starvation and maintained by enteral feeding. It is likely that the IAP silencing that occurs during starvation is a key component of the gut mucosal barrier dysfunction seen in critically ill patients.

Intestinal alkaline phosphatase preserves the normal homeostasis of gut microbiota

Background and aims The intestinal microbiota plays a critical role in maintaining human health; however, the mechanisms governing the normal homeostatic number and composition of these microbes are largely unknown. Previously it was shown that intestinal alkaline phosphatase (IAP), a small intestinal brush border enzyme, functions as a gut mucosal defence factor limiting the translocation of gut bacteria to mesenteric lymph nodes. In this study the role of IAP in the preservation of the normal homeostasis of the gut microbiota was investigated. Methods Bacterial culture was performed in aerobic and anaerobic conditions to quantify the number of bacteria in the stools of wild-type (WT) and IAP knockout (IAP-KO) C57BL/6 mice. Terminal restriction fragment length polymorphism, phylogenetic analyses and quantitative real-time PCR of subphylum-specific bacterial 16S rRNA genes were used to determine the compositional profiles of microbiotas. Oral supplementation of calf IAP (cIAP) was used to determine its effects on the recovery of commensal gut microbiota after antibiotic treatment and also on the colonisation of pathogenic bacteria. Results IAP-KO mice had dramatically fewer and also different types of aerobic and anaerobic microbes in their stools compared with WT mice. Oral supplementation of IAP favoured the growth of commensal bacteria, enhanced restoration of gut microbiota lost due to antibiotic treatment and inhibited the growth of a pathogenic bacterium (Salmonella typhimurium). Conclusions IAP is involved in the maintenance of normal gut microbial homeostasis and may have therapeutic potential against dysbiosis and pathogenic infections.

Intestinal Alkaline Phosphatase Has Beneficial Effects in Mouse Models of Chronic Colitis

Background: The brush border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and is protective against dextran sulfate sodium (DSS)‐induced acute injury in rats. The present study evaluated the potential therapeutic role for orally administered calf IAP (cIAP) in two independent mouse models of chronic colitis: 1) DSS‐induced chronic colitis, and 2) chronic spontaneous colitis in Wiskott‐Aldrich Syndrome protein (WASP)‐deficient (knockout) mice that is accelerated by irradiation. Methods: The wildtype (WT) and IAP knockout (IAP‐KO) mice received four cycles of 2% DSS ad libitum for 7 days. Each cycle was followed by a 7‐day DSS‐free interval during which mice received either cIAP or vehicle in the drinking water. The WASP‐KO mice received either vehicle or cIAP for 6 weeks beginning on the day of irradiation. Results: Microscopic colitis scores of DSS‐treated IAP‐KO mice were higher than DSS‐treated WT mice (52 ± 3.8 versus 28.8 ± 6.6, respectively, P < 0.0001). cIAP treatment attenuated the disease in both groups (KO = 30.7 ± 6.01, WT = 18.7 ± 5.0, P < 0.05). In irradiated WASP‐KO mice cIAP also attenuated colitis compared to control groups (3.3 ± 0.52 versus 6.2 ± 0.34, respectively, P < 0.001). Tissue myeloperoxidase activity and proinflammatory cytokines were significantly decreased by cIAP treatment. Conclusions: Endogenous IAP appears to play a role in protecting the host against chronic colitis. Orally administered cIAP exerts a protective effect in two independent mouse models of chronic colitis and may represent a novel therapy for human IBD. (Inflamm Bowel Dis 2011)

Identification of specific targets for the gut mucosal defense factor intestinal alkaline phosphatase

Intestinal alkaline phosphatase (IAP) is a small intestinal brush border enzyme that has been shown to function as a gut mucosal defense factor, but its precise mechanism of action remains unclear. We investigated the effects of IAP on specific bacteria and bacterial components to determine its molecular targets. Purulent fluid from a cecal ligation and puncture model, specific live and heat-killed bacteria (Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes), and a variety of proinflammatory ligands (LPS, CpG DNA, Pam-3-Cys, flagellin, and TNF) were incubated with or without calf IAP (cIAP). Phosphate release was determined by using a malachite green assay. The various fluids were applied to target cells (THP-1, parent HT-29, and IAP-expressing HT-29 cells) and IL-8 secretion measured by ELISA. cIAP inhibited IL-8 induction by purulent fluid in THP-1 cells by >35% (P < 0.005). HT29-IAP cells had a reduced IL-8 response specifically to gram-negative bacteria; >90% reduction compared with parent cells (P < 0.005). cIAP had no effect on live bacteria but attenuated IL-8 induction by heat-killed bacteria by >40% (P < 0.005). cIAP exposure to LPS and CpG DNA caused phosphate release and reduced IL-8 in cell culture by >50% (P < 0.005). Flagellin exposure to cIAP also resulted in reduced IL-8 secretion by >40% (P < 0.005). In contrast, cIAP had no effect on TNF or Pam-3-Cys. The mechanism of IAP action appears to be through dephosphorylation of specific bacterial components, including LPS, CpG DNA, and flagellin, and not on live bacteria themselves. IAP likely targets these bacterially derived molecules in its role as a gut mucosal defense factor.

W1866 Local Peritoneal Irrigation With Intestinal Alkaline Phosphatase is Protective Against Peritonitis in Mice

Background The brush-border enzyme intestinal alkaline phosphatase (IAP) functions as a gut mucosal defense factor and detoxifies different toll-like receptor ligands. This study aimed to determine the therapeutic effects of locally administered calf IAP (cIAP) in a cecal ligation and puncture (CLP) model of polymicrobial sepsis.

S2064 Intestinal Alkaline Phosphatase Maintains the Normal Homeostasis of Gut Microbiota

Membranous calcium-sensing receptors (CaSR) on intestinal epithelial cells are difficult to study, due to fast loss of function following isolation of the cells. We established a chemical isolation process to maintain receptor function, being able to measure receptor-activity in a fluorescence spectrometry setup. In diarrhea, high intracellular concentrations of cyclic nucleotides in intestine epithelial cells lead to net fluid excretion due to enhanced serosal to mucosal fluid-transport. We showed earlier the reversibility of this effect via activation of CaSR with calcimimetic agents including natural and synthetic small molecules such as the compound R-568. Exposure to these allosteric modifiers in the presence of calcium resulted in a cessation of fluid excretion, and enhanced absorption. In the present study we develop a new isolation method for single cells of all intestinal segments and demonstrate that CaSR remains viable and active upon stimulationwith allostericmodifiers or by increasing the extracellular calcium concentration. These results give an important new tool for intestinal transport screening. METHODS We generated functional individual intestinal epithelial cells from the duodenum, jejunum, ileum and colon of female rats and human colon, using EDTA digestion solution and light mechanical force at 37°C for 20 minutes. Cells were loaded with 2nM Fluo-4 and Fluo-8, calcium-sensing fluorescent dyes. After multiple washout-steps and sedimentation for two hours, the activity of the CaSRwas determined, using a fluorescence spectrometer measuring the fluorescent excitation at 516nm. RESULTS We were able to show the functionality of epithelial CaSR on epithelial cells from four sections of the intestine of the rat: duodenum, jejunum, ileum and colon, and also human colon. The CaSR shows a dose dependent activation over a concentration range of 0.125mM to 2.5mM. The EC50 for calcium for the freshly isolated cells was comparable to that previously obtained for intact native tissue. In a separate study isolated cells were exposed to R-568 prior to the calcium dose curve, resulting in a left phase shift of the activation curve indicative of enhanced calcium binding to the receptor. CONCLUSION Our studies demonstrate that it is now possible to develop viable isolated cells from the entire digestive tract that maintain CaSR density. This technique provides an important new screening tool for use in testing pharmacological agents on intestinal epithelia.

S1670 A Potential Role for Intestinal Alkaline Phosphatase in Colonic Inflammation

Background: Colorectal cancer (CRC) is common and its incidence increases over time with the chronic intestinal inflammation observed in patients with Inflammatory Bowel Disease. Secreted Protein, Acidic and Rich in Cysteine (SPARC) has been show to affect tumour growth, cancer invasion and patient prognosis in numerous human cancers including bowel cancer. SPARC may also affect the colonic inflammatory response. Aim: To investigate SPARCs role on the development of colonic inflammation in the mouse. Methods: SPARC wild-type (WT) and knockout (KO) mice were treated with 3% dextran sodium sulphate (DSS) for 7 days. Intestinal inflammation was assessed on day 6, using the modified murine endoscopic score of colitis severity (MEICS), by high-resolution miniature video endoscopy. Mice were sacrificed and the colons assess histologically. In separate experiments, colons were harvested and total lymphocytes were isolated from the spleen, intra-epithelium (IE) and rest of colon (C) and were analysed for levels of CD4 and FoxP3 +ve cells by flow cytometry. Results: SPARC KO mice demonstrated less endoscopic colonic inflammation than WT mice (MEICS 4 vs 7; p=0.013) and less weight loss (KO 0.48% weight gain vs WT 6.2% weight loss p=0.074). Overall lymphocyte numbers isolated from spleen, IE and C fromKOwasmarkedly less than that isolated fromWTmice (0.77x108/ml vs 1.28x108/ml; 0.82x106/ml vs 16.8x106/ml and 4.4x106/ml vs 8.8x106/ml respectively). The percentage of CD4 +ve cells from spleen, IE and C were between 10-15% from both KO and WT mice. The percentage of FoxP3 +ve cells from spleen, IE and C from both KO and WT mice were 0.05% vs 1.85%, 1.2% vs 2.3%, 1.22% vs 1.75% respectively. Conclusion: SPARC affects the endoscopic colonic inflammatory response. Total lymphocyte counts are lower in inflamed SPARC KO colons and spleen consistent with a lower level of chronic inflammation. The percentage of FoxP3 +ve cells from the spleen and IE were lower in KO mice suggesting an impact of regulation of the immune response. The mechanistic impact of SPARC requires further elucidation.