Membrane prohibitin forms a dynamic complex with p56lck to regulate T cell receptor signaling.

Prohibitin is a highly conserved ubiquitously expressed protein involved in several key cellular functions. Targeting of this protein in the membrane by the virulence polysaccharide, Vi, of human typhoid-causing pathogen, Salmonella enterica serovar Typhi (S. Typhi), results in suppression of IL-2 secretion from T cells activated through the T-cell receptor (TCR). However, the mechanism of this suppression remains unclear. Here, using Vi as a probe, we show that membrane prohibitin associates with the src-tyrosine kinase, p56lck (Lck), and actin in human model T cell line, Jurkat. Activation with anti-CD3 antibody brings about dissociation of this complex, which coincides with downstream ERK activation. The trimolecular complex reappears towards culmination of proximal TCR signaling. Engagement of cells with Vi prevents TCR-triggered activation of Lck and ERK by inhibiting dissociation of the former from prohibitin. These findings suggest a regulatory role for membrane prohibitin in Lck activation and TCR signaling.

Targeting Histone Deacetylases to Modulate Graft-Versus-Host Disease and Graft-Versus-Leukemia.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the main therapeutic strategy for patients with both malignant and nonmalignant disorders. The therapeutic benefits of allo-HSCT in malignant disorders are primarily derived from the graft-versus-leukemia (GvL) effect, in which T cells in the donor graft recognize and eradicate residual malignant cells. However, the same donor T cells can also recognize normal host tissues as foreign, leading to the development of graft-versus-host disease (GvHD), which is difficult to separate from GvL and is the most frequent and serious complication following allo-HSCT. Inhibition of donor T cell toxicity helps in reducing GvHD but also restricts GvL activity. Therefore, developing a novel therapeutic strategy that selectively suppresses GvHD without affecting GvL is essential. Recent studies have shown that inhibition of histone deacetylases (HDACs) not only inhibits the growth of tumor cells but also regulates the cytotoxic activity of T cells. Here, we compile the known therapeutic potential of HDAC inhibitors in preventing several stages of GvHD pathogenesis. Furthermore, we will also review the current clinical features of HDAC inhibitors in preventing and treating GvHD as well as maintaining GvL.

Enterotoxigenic Escherichia coli-blood group A interactions intensify diarrheal severity.

Enterotoxigenic Escherichia coli (ETEC) infections are highly prevalent in developing countries, where clinical presentations range from asymptomatic colonization to severe cholera-like illness. The molecular basis for these varied presentations, which may involve strain-specific virulence features as well as host factors, has not been elucidated. We demonstrate that, when challenged with ETEC strain H10407, originally isolated from a case of cholera-like illness, blood group A human volunteers developed severe diarrhea more frequently than individuals from other blood groups. Interestingly, a diverse population of ETEC strains, including H10407, secrete the EtpA adhesin molecule. As many bacterial adhesins also agglutinate red blood cells, we combined the use of glycan arrays, biolayer inferometry, and noncanonical amino acid labeling with hemagglutination studies to demonstrate that EtpA is a dominant ETEC blood group A-specific lectin/hemagglutinin. Importantly, we have also shown that EtpA interacts specifically with glycans expressed on intestinal epithelial cells from blood group A individuals and that EtpA-mediated bacterial-host interactions accelerate bacterial adhesion and effective delivery of both the heat-labile and heat-stable toxins of ETEC. Collectively, these data provide additional insight into the complex molecular basis of severe ETEC diarrheal illness that may inform rational design of vaccines to protect those at highest risk.

Association of ATG16L1 gene haplotype with inflammatory bowel disease in Indians.

Inflammatory bowel disease (IBD) is characterized by multigenic inheritance. Defects in autophagy related genes are considered to show genetic heterogeneity between populations. We evaluated the association of several single nucleotide polymorphisms (SNPs) in the autophagy related 16 like 1 (ATG16L1) gene with IBD in Indians. The ATG16L1 gene was genotyped for ten different SNPs using DNA extracted from peripheral blood of 234 patients with Crohn's disease (CD), 249 patients with ulcerative colitis (UC) and 393 healthy controls The SNPs rs2241880, rs4663396, rs3792106, rs10210302, rs3792109, rs2241877, rs6737398, rs11682898, rs4663402 and rs4663421 were genotyped using the Sequenom MassArray platform. PLINK was used for the association analysis and pairwise linkage disequilibrium (LD) values. Haplotype analysis was done using Haploview. All SNPs were in Hardy Weinberg equilibrium in cases and controls. The G allele at rs6737398 exhibited a protective association with both CD and UC. The T allele at rs4663402 and C allele at rs4663421 were positively associated with CD and UC. The T allele at rs2241877 exhibited protective association with UC only. The AA genotype at rs4663402 and the GG genotype at rs4663421 were protectively associated with both CD and UC. Haplotype analysis revealed that all the SNPs in tight LD (D' = 0.76-1.0) and organized in a single haplotype block. Haplotype D was positively associated with IBD (P = 5.8 x 10-6 for CD and 0.002 for UC). SNPs in ATG16L1 were associated with IBD in Indian patients. The relevance to management of individual patients requires further study.

Blood Group O-Dependent Cellular Responses to Cholera Toxin: Parallel Clinical and Epidemiological Links to Severe Cholera.

Because O blood group has been associated with more severe cholera infections, it has been hypothesized that cholera toxin (CT) may bind non-O blood group antigens of the intestinal mucosae, thereby preventing efficient interaction with target GM1 gangliosides required for uptake of the toxin and activation of cyclic adenosine monophosphate (cAMP) signaling in target epithelia. Herein, we show that after exposure to CT, human enteroids expressing O blood group exhibited marked increase in cAMP relative to cells derived from blood group A individuals. Likewise, using CRISPR/Cas9 engineering, a functional group O line (HT-29-A(-/-)) was generated from a parent group A HT-29 line. CT stimulated robust cAMP responses in HT-29-A(-/-) cells relative to HT-29 cells. These findings provide a direct molecular link between blood group O expression and differential cellular responses to CT, recapitulating clinical and epidemiologic observations.

Therapeutic targeting of inflammation and tryptophan metabolism in colon and gastrointestinal cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide and the second leading cause of cancer death in the United States. Cytotoxic therapies cause significant adverse effects for most patients and do not offer cure in many advanced cases of CRC. Immunotherapy is a promising new approach to harness the body's own immune system and inflammatory response to attack and clear the cancer. Tryptophan metabolism along the kynurenine pathway (KP) is a particularly promising target for immunotherapy. Indoleamine 2,3-dioxygenase 1 (IDO1) is the most well studied of the enzymes that initiate this pathway and it is commonly overexpressed in CRC. Herein, we provide an in-depth review of how tryptophan metabolism and KP metabolites shape factors important to CRC pathogenesis including the host mucosal immune system, pivotal transcriptional pathways of neoplastic growth, and luminal microbiota. This pathway's role in other gastrointestinal (GI) malignancies such as gastric, pancreatic, esophageal, and GI stromal tumors is also discussed. Finally, we highlight how currently available small molecule inhibitors and emerging methods for therapeutic targeting of IDO1 might be applied to colon, rectal, and colitis-associated cancer.

CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5+ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice.

Hyaluronic acid, a glycosaminoglycan in the extracellular matrix, binds to CD44 and Toll-like receptor 4 (TLR4). We previously addressed the role of hyaluronic acid in small intestinal and colonic growth in mice. We addressed the role of exogenous hyaluronic acid by giving hyaluronic acid intraperitoneally and the role of endogenous hyaluronic acid by giving PEP-1, a peptide that blocks hyaluronic acid binding to its receptors. Exogenous hyaluronic acid increased epithelial proliferation but had no effect on intestinal length. PEP-1 resulted in a shortened small intestine and colon and diminished epithelial proliferation. In the current study, we sought to determine whether the effects of hyaluronic acid on growth were mediated by signaling through CD44 or TLR4 by giving exogenous hyaluronic acid or PEP-1 twice a week from 3-8 wk of age to wild-type, CD44(-/-), and TLR4(-/-) mice. These studies demonstrated that signaling through both CD44 and TLR4 were important in mediating the effects of hyaluronic acid on growth in the small intestine and colon. Extending our studies to early postnatal life, we assessed the effects of exogenous hyaluronic acid and PEP-1 on Lgr5(+) stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5(+) reporter mice from postnatal day 7 to day 14 decreased Lgr5(+) cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5(+) stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4.

The virulence polysaccharide Vi released by Salmonella Typhi targets membrane prohibitin to inhibit T-cell activation.

T cells are critical to immunity against pathogenic Salmonella including Salmonella Typhi which causes systemic infection, typhoid, in humans. The strategies that this pathogen employs to keep T-cell mediated immune responses in check during establishment of systemic infection are not completely understood. Here, we show that the virulence polysaccharide Vi, which distinguishes S. Typhi from localized gastroenteritis-producing nontyphoidal Salmonella serovars, is a potent inhibitor of T-cell activation. Vi released by S. Typhi interacts with the membrane prohibitin complex and inhibits IL-2 secretion from T cells stimulated through the T-cell receptor (TCR) but does not affect PMA-activated interleukin 2 (IL-2) secretion. Treatment with Vi suppresses early activation events including TCR down-regulation, actin polymerization, and phosphorylation of ERK. Coadministration of Vi with anti-CD3 Ab reduces secretion of IL-2 and interferon γ in mice. Our findings reveal a mechanism by which S. Typhi may target T-cell immunity during establishment of typhoid.

NOD2 gene mutations associate weakly with ulcerative colitis but not with Crohn's disease in Indian patients with inflammatory bowel disease.

BACKGROUND: Three mutations (two missense and one frameshift) in the NOD2 gene are associated with Crohn's disease (CD) in a proportion of patients with Crohn's disease in North America, Europe and Australia. These three mutations are not found in Indian patients with CD. We undertook new studies to identify polymorphisms in the NOD2 gene in the Indian population and to detect whether any of these were associated with inflammatory bowel disease (IBD) in this population. METHODS: Individual exons of the NOD2 gene were amplified by PCR and subjected to denaturing high performance liquid chromatography (DHPLC) to detect heteroduplex formation. All 12 exons of the NOD2 gene were amplified and Sanger-sequenced to detect polymorphisms in the NOD2 gene. 310 patients with CD, 318 patients with ulcerative colitis (UC) and 442 healthy controls (HC) were recruited for association studies. DNA from these participants was evaluated for the identified eight polymorphisms by Sequenom analysis. RESULTS: Heteroduplex formation was noted by DHPLC in exons 2 and 4 of the NOD2 gene. Sequencing of the entire NOD2 gene data revealed eight polymorphisms - rs2067085, rs2066842, rs2066843, rs1861759, rs2111235, rs5743266, rs2076753, and rs5743291 - of which the latter four were described for the first time in Indians. None of these polymorphisms was associated with CD. The SNPs rs2066842 and rs2066843 were in significant linkage disequilibrium. Both SNPs showed a significant association with UC (P=0.03 and 0.04 respectively; odds ratio 1.44 and 1.41 respectively). CONCLUSION: Four NOD2 polymorphisms were identified for the first time in the Indian population. Of 8 NOD2 polymorphisms, none were associated with CD but two were weakly associated with UC. NOD2 polymorphisms do not play a major role in CD genesis in India.

Mitochondrial electron transport chain complex dysfunction in the colonic mucosa in ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is characterized by an energy deficiency state of the colonic epithelium. This study evaluated mitochondrial electron transport chain (ETC) complex activity in normal and disease mucosa in patients with UC. Alterations in ETC complexes were also investigated in experimental colitis in mice. METHODS: Biopsies were obtained from macroscopically normal and diseased colonic mucosa of 43 patients with UC and 35 controls undergoing screening colonoscopy and ETC complex activity was assayed biochemically. ETC complex activities were also assayed in colonic epithelial cells isolated from Swiss albino mice with dextran sodium sulfate (DSS)-induced colitis at various stages of induction of colitis. Mucosal nitrite levels and protein carbonyl content were determined. RESULTS: The activity of Complex II was significantly decreased in colonic biopsies from UC patients compared with controls, while activities of other mitochondrial complex were normal. Complex II activity was equally decreased in diseased and normal mucosa in UC; the degree of reduction did not correlate with clinical, endoscopic, or histological grading of disease activity. In DSS-fed mice, a reduction in activity of Complex IV and Complex II was observed. Activity of other complex was not affected. Administration of aminoguanidine, an inducible nitric oxide synthase (iNOS) inhibitor, attenuated all parameters of colitis as well as the reductions in Complex IV and Complex II activity. CONCLUSIONS: Reduction in Complex II activity appears to be a specific change in UC, present in quiescent and active disease. Mitochondrial complex dysfunction occurs in DSS colitis in mice and appears to be mediated by nitric oxide.

Impairment of mitochondrial acetoacetyl CoA thiolase activity in the colonic mucosa of patients with ulcerative colitis.

BACKGROUND AND AIMS: Butyrate oxidation by colonocytes is impaired in ulcerative colitis. This study examined the activity of enzymes involved in butyrate oxidation in ulcerative colitis. METHODS: Activities of mitochondrial acetoacetyl coenzyme A (CoA) thiolase, crotonase and beta-hydroxy butyryl CoA dehydrogenase were estimated spectrophotometrically in rectosigmoid mucosal biopsies from patients with ulcerative colitis and Crohn's colitis, and control subjects undergoing colonoscopy for colon cancer or rectal bleeding. RESULTS: The activity of mitochondrial acetoacetyl CoA thiolase was decreased by 80% in ulcerative colitis (3.4 (0.58) mumol/min/g wet weight, n = 30) compared with control (16.9 (3.5), n = 18) and with Crohn's colitis (17.6 (3.1), n = 12) (p<0.0001). The activity of two other mitochondrial butyrate oxidation enzymes--crotonase and beta-hydroxy butyryl CoA dehydrogenase--as well as of cytoplasmic thiolase was normal in ulcerative colitis. Mitochondrial thiolase activity in ulcerative colitis did not correlate with clinical, endoscopic or histological indices of disease severity. Mitochondrial thiolase activity was reduced in the normal right colon mucosa of patients with left-sided ulcerative colitis. Enzyme kinetic studies revealed a lowered V(max), suggesting inhibition at a site distinct from the catalytic site. Reduced thiolase activity in ulcerative colitis was returned to normal by exposure to 0.3 mM beta-mercaptoethanol, a reductant. Using normal colon mucosal biopsies, redox modulation of thiolase activity by hydrogen peroxide, a mitochondrial oxidant, could be shown. A significant increase in hydrogen peroxide formation was observed in ulcerative colitis biopsies. CONCLUSION: A defect of mitochondrial acetoacetyl CoA thiolase occurs in ulcerative colitis. Increased reactive oxygen species generation in mitochondria of epithelial cells in ulcerative colitis may underlie this defect.