BACKGROUND & AIMS Indoleamine 2,3 dioxygenase-1 (IDO1) catabolizes tryptophan along the kynurenine pathway. Though IDO1 is expressed in inflamed and neoplastic epithelial cells of the colon, its role in colon tumorigenesis is not well understood. We used genetic and pharmacologic approaches to manipulate IDO1 activity in mice with colitis-associated cancer and human colon cancer cell lines. METHODS C57Bl6 wild type (control), IDO1−/−, Rag1−/−, Rag1/IDO1 double knockout mice were exposed to azoxymethane and dextran sodium sulfate (DSS) to induce colitis and tumorigenesis. Colitis severity was assessed by measurements of disease activity, cytokine levels and histologic analysis. In vitro experiments were conducted using HCT116 and HT29 human colon cancer cells. 1-methyl tryptophan and small interfering RNA were used to inhibit IDO1. Kynurenine pathway metabolites were used to simulate IDO1 activity. RESULTS C57Bl6 mice given pharmacologic inhibitors of IDO1 and IDO1−/− mice had lower tumor burdens and reduced proliferation in the neoplastic epithelium following administration of DSS and azoxymethane than control mice. These reductions were also observed in Rag1/IDO1 double knockout mice compared to Rag1−/− mice (which lack mature adaptive immunity). In human colon cancer cells, blockade of IDO1 activity reduced nuclear and activated β-catenin, transcription of its target genes (cyclin D1 and Axin2), and ultimately proliferation. Exogenous administration of IDO1 pathway metabolites kynurenine and quinolinic acid led to activation of β-catenin and proliferation of human colon cancer cells, and increased tumor growth in mice. CONCLUSIONS IDO1, which catabolizes tryptophan, promotes colitis-associated tumorigenesis in mice, independent of its ability to limit T-cell mediated immune surveillance. The epithelial cell-autonomous survival advantage provided by IDO1 to colon epithelial cells indicate its potential as a therapeutic target.

Entamoeba histolytica causes amebic dysentery and amebic liver abscess, major causes of morbidity and mortality worldwide. We have used differential hybridization screening to isolate an E. histolytica-speciflic cDNA clone. The cDNA was found to encode a serine-rich E. histolytica protein (SREHP) containing multiple tandem repeats. The structural motif of SREHP resembles some of the repetitive antigens of malarial species, especially the circumsporozoite proteins. A recombinant trpE fusion protein containing the tandem repeats of SREHP was recognized by immune serum from a patient with amebiasis, demonstrating that SREHP is a naturally immunogenic protein. An antiserum raised against the recombinant fusion protein specifically bound to two distinct bands with apparent molecular masses of 46 and 52 kDa in a crude preparation of E. histolytica trophozoite membranes. This antiserum also inhibited E. histolytica trophozoite adhesion to Chinese hamster ovary cells in vitro. The ability to isolate E. histolytica-specific genes, and to express those genes in Escherichia coli, may be important in studying the molecular basis ofE. histolytica pathogenesis and for the future development of vaccines.The protozoan pathogen Entamoeba histolytica is a major cause of debilitating illness and death worldwide, infecting 500 million people and causing an estimated 50 million cases of diarrhea and 50,000 deaths yearly (1). There is an urgent need for a vaccine that could prevent the establishment of E. histolytica infection or the development of invasive disease. Studies in animal models have demonstrated that immunity to E. histolytica infection can be produced by immunization with E. histolytica lysates (2)(3)(4). However, the difficulty in obtaining large quantities of trophozoites, and the relatively crude nature of the immunizing preparations, have severely limited the scope of these studies. To approach this problem, we isolated genes expressed in a pathogenic strain ofE. histolytica but not in the nonpathogenic E. histolytica-like Laredo strain.

The intestinal protozoan pathogen Entamoeba histolytica lacks mitochondria and derives energy from the fermentation of glucose to ethanol with pyruvate, acetyl enzyme Co-A, and acetaldehyde as intermediates. A key enzyme in this pathway may be the 97-kDa bifunctional E. histolytica alcohol dehydrogenase 2 (EhADH2), which possesses both alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase activity (ALDH). EhADH2 appears to be a fusion protein, with separate N-terminal ALDH and C-terminal ADH domains. Here, we demonstrate that EhADH2 expression is required for E. histolytica growth and survival. We find that a mutant EhADH2 enzyme containing the C-terminal 453 amino acids of EhADH2 has ADH activity but lacks ALDH activity. However, a mutant consisting of the N-terminal half of EhADH2 possessed no ADH or ALDH activity. Alteration of a single histidine to arginine in the putative active site of the ADH domain eliminates both ADH and ALDH activity, and this mutant EhADH2 can serve as a dominant negative, eliminating both ADH and ALDH activity when co-expressed with wild-type EhADH2 in Escherichia coli. These data indicate that EhADH2 enzyme is required for E. histolytica growth and survival and that the C-terminal ADH domain of the enzyme functions as a separate entity. However, ALDH activity requires residues in both the N-and C-terminal halves of the molecule.The anaerobic intestinal protozoan parasite Entamoeba histolytica converts pyruvate to ethanol in its fermentation pathway (1). The last two steps of this pathway are the conversion of acetyl-CoA to acetaldehyde followed by the reduction of acetaldehyde to ethanol (1). E. histolytica possesses at least three enzymes with alcohol dehydrogenase (ADH) 1 activity: a NADP-dependent ADH (EhADH1); a 97-kDa NAD(ϩ)-dependent and Fe 2ϩ -dependent bifunctional enzyme with both ADH and acetaldehyde dehydrogenase (ALDH) activities (EhADH2, also known as EhADHE); and a 43-kDa NADP-dependent ADH with some sequence homology to class III microbial alcohol dehydrogenases (EhADH3) (2-5). There are at least two enzymes with ALDH activity, the EhADH2 enzyme and a NADPdependent ALDH, EhALDH1 (2, 5, 6). Given the presence of multiple ADH and ALDH enzymes in E. histolytica, an important question is whether any of these enzymes are essential for E. histolytica growth and survival and thus potential targets for anti-amebic therapy.The EhADH2 enzyme is part of a newly described family of multifunctional enzymes found in Gram-negative and Grampositive bacteria and the intestinal protozoan parasite Giardia lamblia (2, 7-12). EhADH2 and other members of the family appear to be composed of separate C-terminal ADH and Nterminal ALDH domains linked together to create a fusion enzyme (8). The EhADH2 enzyme utilizes NAD and Fe 2ϩ as co-factors and does not demonstrate homology with the zincdependent ADH enzymes (13). Regions of EhADH2 that could be involved in iron binding and NAD binding have been identified, but a requirement for specific residues in enzymatic activity has not been demo...

Riehl TE, Santhanam S, Foster L, Ciorba M, Stenson WF. CD44 and TLR4 mediate hyaluronic acid regulation of Lgr5 ϩ stem cell proliferation, crypt fission, and intestinal growth in postnatal and adult mice. Am J Physiol Gastrointest Liver Physiol 309: G874 -G887, 2015. First published October 1, 2015; doi:10.1152/ajpgi.00123.2015.-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, CD44Ϫ/Ϫ , and TLR4 Ϫ/Ϫ 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 ϩ stem cell proliferation and crypt fission. Administration of PEP-1 to Lgr5 ϩ reporter mice from postnatal day 7 to day 14 decreased Lgr5 ϩ cell proliferation and decreased crypt fission. These studies indicate that endogenous hyaluronic acid increases Lgr5 ϩ stem cell proliferation, crypt fission, and intestinal lengthening and that these effects are dependent on signaling through CD44 and TLR4. hyaluronic acid; CD44; Toll-like receptor 4; intestinal growth; crypt fission; Lgr5 stem cell proliferation HYALURONIC ACID (HA), a glycosaminoglycan polymer with repeating units of disaccharides composed of D-glucuronic acid and N-acetyl-D-glucosamine, is an important constituent of the extracellular matrix. HA is secreted by many cell types; it is assembled at the plasma membrane by HA synthases (HASs) and extruded into the extracellular space. The HA chain can extend up to 2 ϫ 10 5 disaccharides and up to 25 m long. HA expression is increased in many injury states, including Crohn's disease in humans, dextran sodium sulfate (DSS)-induced colitis in mice, and intestinal radiation injury (8,16,24). The angiogenic, inflammatory, and immunostimulatory effects of HA are mediated by binding to its receptors. The HA receptor CD44 is expressed on the plasma membrane of most cells, including fibroblasts, smooth muscle cells, epithelial cells, and immune cells (19). In addition to binding to CD44, HA also binds to Toll-like receptors 2 and 4 (TLR2 and TLR4), which are components of the innate immune system (7, 20, 24). TLR2 and TLR4 are widel...

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