Published papers

A rare case of chronic GVHD enterocolitis with multiple colon ulcers was reported. A 21-year-old man was admitted on May 2000, because of progressing diarrhea and abdominal pain. He had received allogenic bone marrow transplantation in August 1999. On admission, Colonoscopy showed multiple colon ulcers and mucosal edema. He was treated with antibiotics and virucide, but had no resolution both in clinically and endoscopically. We eventually diagnosed this case as chronic GVHD enterocolitis and treated with immunosuppressant. Endoscopic appearance was remarkably improved and diarrhea was disappeared.

Interaction of OX40 (CD134) on T cells with its ligand (OX40L) on antigen-presenting cells has been implicated in pathogenic T cell activation. This study was performed to explore the involvement of OX40/OX40L in the development of T cell-mediated chronic colitis. We evaluated both the preventive and therapeutic effects of neutralizing anti-OX40L MAb on the development of chronic colitis in SCID mice induced by adoptive transfer of CD4+CD45RBhigh T cells as an animal model of Crohn's disease. We also assessed the combination of anti-OX40L and anti-TNF-α MAbs to improve the therapeutic effect. Administration of anti-OX40L MAb markedly ameliorated the clinical and histopathological disease in preventive and therapeutic protocols. In vivo treatment with anti-OX40L MAb decreased CD4+ T cell infiltration in the colon and suppressed IFN-γ IL-2, and TNF-α production by lamina propria CD4+ T cells. The combination with anti-TNF-α MAb further improved the therapeutic effect by abolishing IFN-γ, IL-2, and TNF-α production by lamina propria CD4+ T cells. Our present results suggested a pivotal role of OX40/OX40L in the pathogenesis of T cell-mediated chronic colitis. The OX40L blockade, especially in combination with the TNF-α blockade, may be a promising strategy for therapeutic intervention of Crohn's disease.

Background & Aims: Inducible costimulator (ICOS)/B7RP-1 represents a newly described receptor/ligand pair involved in costimulation of T cells by antigen-presenting cells. We investigated the involvement of the ICOS/B7RP-1 interaction in the pathogenesis of colitis and the therapeutic potential of anti-ICOS monoclonal antibody (mAb) in experimental colitis. Methods: We administered anti-ICOS or anti-B7RP-1 mAb to mice with experimental colitis induced by transfer of CD4+CD45RBhigh T cells from normal mice into SCID mice. The ability of CD4+CD45RBhigh cells derived from ICOS-/- mice to induce colitis was assessed. Th2 cytokine production and apoptosis in infiltrating T cells was examined after administration of anti-ICOS mAb. Results: ICOS was strongly induced on CD4+ T cells, and B7RP-1 was expressed by macrophages in the inflamed mucosa of colitic mice. Anti-ICOS mAb, but not anti-B7RP-1, ameliorated chronic colitis when administered in prevention or therapeutic protocols. Transfer of CD4+CD45RBhigh T cells from ICOS-/- mice induced colitis. Treatment with anti-ICOS mAb did not enhance the production of Th2 cytokines, but a single dose of anti-ICOS mAb induced massive apoptosis of infiltrating ICOS-expressing T cells. Conclusions: ICOS/B7RP-1 interactions are not required for the development of colitis. However, treatment with anti-ICOS mAb can prevent and reverse intestinal inflammation by inducing apoptosis of ICOS-expressing T lymphocytes.

Crohn's disease (CD) affects more than 400,000 individuals in the United States and more than 18,000 in Japan. Its incidence is CD, one of the chronic inflammatory bowel diseases (IBD), is debilitating disorder of unknown ethiology, characterized by a marked accumulation of activated T cells and monocytes/macrophages in the inflamed mucosa. Various proinflammatory cytokines, such as TNF-α, INF-γ, IL-6, IL-12, and IL-18, might be involved in its pathogenesis. In this review, we describe the role of various cytokines in the development of CD and the new therapeutic trials modulating cytokine networks for the treatment of CD.

OBJECTIVES: Epithelioid granulomas with transmural inflammation are a characteristic histological feature in Crohn's disease (CD). However, these are not frequently detected by histopathological examination in the biopsy specimens. Here, we demonstrated unique structures with T-lymphocyte aggregates (TLAs) that were specifically found in ileal villi of CD. We characterized the histological and phenotypical features of these structures and assessed the diagnostic value of TLAs for CD. METHODS: Tissue samples were obtained from the inflamed and uninflamed areas of ileal and colonic mucosa of 32 patients with CD. For controls, mucosal samples were obtained from unaffected areas of 18 patients with colon cancer and inflamed and uninflamed areas of 12 patients with ulcerative colitis (UC). RESULTS: 1) In 21 of 32 cases of CD (66%), we found unique structures with lymphoid cell aggregates that were localized in villi of ileum. These structures were not detected in any normal or UC intestine. 2) These structures were composed mainly of T cells, and CD4+ CD45RO+ cells dominated. Neither B cells nor c-kit positive immature lymphocytes were found. Moreover, CD68 positive macrophages were demonstrated in these aggregates, and cells positive for interleukin 18, a pivotal cytokine for Th1 differentiation, were expressed. 3) The aggregates were detected in seven of 13 patients with CD in whom granuloma was not detected by precise histopathological examination. Furthermore, we detected TLAs even in the ilea of two of four CD patients (50%) whose affected lesions were limited to the colon. CONCLUSIONS: We demonstrated TLAs in intestinal villi that may contribute to the pathogenesis of CD. Detection of these lymphocyte aggregates is helpful for diagnosis of CD when granuloma is not found. © 2002 by Am. Coll. of Gastroenterology.

OBJECTIVE: The development of T helper type 1 (Th1) CD4+ T cells in the intestinal mucosa is driven by interleukin (IL)-12 produced from activated macrophages and IL-18 produced from activated macrophages and epithelial cells. Each of these two cytokines is important for the mucosal response during intestinal inflammation, but their synergistic effect is not fully understood. To characterize the synergistic effect of IL-12 and IL-18 with respect to human intestinal inflammation, we assessed the effect of IL-12 and IL-18 on lamina propria lymphocytes from normal control subjects (LPL-NL) and patients with Crohn's disease (LPL-CD). METHODS: Expression of IL-12 receptor (IL-12R) β1, β2, and IL-18Rα in LPLs was analyzed by flow cytometry. The functional activity of IL-12 and IL-18 was assessed by the effect of recombinant IL-12 and recombinant IL-18 on interferon-γ production, the proliferative response, and the induction of IL-2R, IL-12R, and IL-18R of LPLs. RESULTS: IL-12Rβ2 expression was significantly greater in LPL-CD compared with LPL-NL. LPL-NL demonstrated a proliferative response and a significant increase in interferon-γ production and IL-2Rα expression when exposed to both IL-12 and IL-18, but neither IL-12 nor IL-18 were able to induce this response on their own. However, IL-12 and IL-18 produced this response in LPL-CD when administered alone. Moreover, a more pronounced synergistic effect of IL-12 and IL-18 was observed in LPL-CD. The response normally observed after administration of IL-12 and IL-18 was significantly inhibited by anti-IL-2 and anti-IL-2Rα monoclonal antibody. Furthermore, IL-12 was observed to upregulate IL-18Rα expression in LPL-CD. CONCLUSIONS: These findings suggest that a combination of IL-12 and IL-18 in the absence of T cell receptor engagement may serve as a potent regulatory factor for LPL and contribute to the maintenance and enhancement of chronic inflammation in CD. © 2002 by Am. Coll. of Gastroenterology.

Based on Pasteur's work on the microbial nature of fermentation, it was widely believed that the presence of bacteria in the intestine was essential for the life of the host. It has also been known for decades that gut commensal microbes effect the activation and development of the systemic immune system through gut-associated lymphoid tissues (GALT). Recent extensive studies have shown that recognition of microbes is mediated by a set of germline-encoded receptors, Toll-like receptors (TLRs), in mammals. This article reviews the role of the innate immunity system in the development of GALT and the pathogenesis of inflammatory bowel diseases (IBD).

For years, medical researchers have striven to develop selective immunotherapies that could specifically ameliorate pathogenic immune responses without immunocompromising the patient. Blockade of many known receptors on T cells can inhibit the initiation of immune responses. However, this approach is problematic in that it is not possible to predict the onset of disease in patients. Current immunotherapies are unsatisfactory for the sporadic exacerbating type of diseases such as multiple sclerosis and inflammatory bowel disease (IBD), because they require either long-term treatment or acute treatment with high-dose immunosuppressants. With regard to this issue, the inducible and inflammatory site-specific molecule, inducible costimulator (ICOS), may be particularly useful as an ideal targeting molecule for the strategy of treatment of human IBD patients.

Studies have shown that bone marrow cells have the potential to differentiate into a variety of cell types. Here we show that bone marrow cells can repopulate the epithelia of the human gastrointestinal tract. Epithelial cells of male donor origin were distributed in every part of the gastrointestinal tract of female bone marrow transplant recipients. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract during epithelial regeneration after graft-versus-host disease or ulcer formation. Regeneration of gastrointestinal epithelia with donor-derived cells in humans shows a potential clinical application of bone marrow-derived cells for repairing severely damaged epithelia, not only in the gastrointestinal tract but also in other tissues.

Background & Aims: Autoimmune responses against colonic epithelium may play a role in the development of colonic inflammation associated with ulcerative colitis (UC). In this study, we established and characterized B-cell lines and clones that produced anticolon antibody from inflamed colonic mucosa of UC subjects. Methods: B-cell lines were generated through Epstein-Barr virus transformation of lamina propria lymphocytes (LPLs) from colonic mucosa and peripheral blood lymphocytes, and these lines were screened for the production of anticolon antibodies. B-cell lines were then cloned by limiting dilution culture, and messenger RNA expression of immunoglobulin heavy-chain variable region (VH) was assessed. Results: VH gene families used in B-cell lines established from LPLs of normal controls were diverse, and B-cell lines from UC LPLs expressed a restricted VH3 family usage. All 15 clones from UC used a restricted VH3 gene family, whereas diverse VH gene families were used by 24 clones from normal controls. The analysis of nucleotide sequences indicated that these clones were derived from various germline gene segments. Conclusions: The restricted VH gene usage in anticolon autoantibodies producing B-cell clones suggests that a particular antigenic stimulus contributes to the pathogenesis of UC.

The etiology and pathogenesis of inflammatory bowel disease remains an area under intense investigation. Crohn's disease (CD) is characterized by a marked accumulation of activated Th1 type CD4+ T cells and macrophages in inflamed intestinal mucosa. IL-18 is a recently described cytokine that mainly exists in activated macrophages and shares biological activities with IL-12 in driving the development of Th1 type CD4+ T cells by inducing interferon-γ. To clarify the role of IL-18 in intestinal inflammation in CD, we assessed the functional role of IL-18 in regulating intestinal mucosal lymphocytes in human CD and murine CD model. Copyright © 2001 S. Karger AG, Basel.

Background & Aims: Crohn's disease (CD) is associated with an increased number of infiltrating macrophages, which release a variety of proinflammatory cytokines. Interleukin (IL)-18 has been implicated in the modulation of mucosal CD4 + T cells towards Th1 responses, which are implicated in the pathogenesis of CD. Here we assess the role of macrophages and of IL-18 in the murine model of intestinal inflammation that mimics the immunologic characteristics of human CD. Methods: Colitis was induced in C57BL/6 mice immunized with 2,4,6-trinitrobenzene sulfonic acid (TNBS) followed by rectal administration of TNBS in ethanol. Mice were treated with either an antibody directed against macrophages conjugated to the ribosome-inactivating protein saporin (anti-Mac-1-saporin) or with a neutralizing antibody against IL-18. In addition, we assessed whether an identical TNBS immunization/challenge protocol could induce colitis in IL-18 -/- mice. Results: The colonic mucosa of TNBS-treated mice was marked by infiltration of Mac-1-positive macrophages and up-regulation of IL-18. The administration of the anti-Mac-1-saporin antibody or the neutralizing anti-IL-18 antibody resulted in a dramatic attenuation of mucosal inflammation in this model. In addition, TNBS was unable to induce significant colitis in the IL-18 -/- mice. Conclusions: Our data underscore the pivotal role of macrophages, and the macrophage-derived IL-18, in the establishment of TNBS-induced colitis in mice. Our results highlight the potential use of therapy directed against IL-18 in the treatment of patients with CD.

To assess the role of interleukin-4 (IL-4) and interleukin-13 (IL-13) in colon cancer cell-cell adhesion, we investigated the effect of both cytokines in human colon cancer cell line, colo205 cell-cell adhesion. IL-4 receptor was expressed on the cell surface of colo205, and recombinant IL-4 inhibited colo205 cell-cell adhesion in a dose-dependent fashion without inhibiting cell proliferation. Flow cytometric analysis revealed that monoclonal antibodies (mAbs) directed against E-cadherin and carcinoembryonic antigen (CEA) inhibited colo205 cell-cell adhesion and IL-4 significantly inhibited the expression of E-cadherin and CEA. IL-13 also inhibited colo205 cell-cell adhesion. These results indicated that IL-4 and IL-13 inhibited colon cancer cell-cell adhesion by down-regulation of E-cadherin and CEA molecules. We then investigated the expression of both cytokines from freshly isolated colon cancer tumour-infiltrating lymphocytes (TILs). With reverse transcription-polymerase chain reaction and flow cytometric analysis. we demonstrated that colon TILs expressed IL-4 and IL-13 mRNA and protein. These results suggest that Th 2 type cytokines IL-4 and IL-13 locally-produced from TILs may regulate colon cancer adhesion by down-regulation of adhesion molecules. (C) 2000 Cancer Research Campaign.

Background & Aims: Crohn's disease (CD) is characterized by a marked accumulation of activated Th1 type CD4+ T cells and macrophages in inflamed intestinal mucosa. Interleukin (IL)-18 is a recently described cytokine that mainly exists in activated macrophages and shares biological activities with IL-12 in driving the development of Th1 type CD4+ T cells by inducing interferon gamma. To clarify the role of IL-18 in intestinal inflammation in CD, we assessed the functional role of IL-18 in regulating intestinal mucosal lymphocytes. Methods: Serum IL-18 concentration was measured by enzyme-linked immunosorbent assay. Expression of IL-18 and IL-18 receptor in human intestinal mucosa was determined using immunohistochemistry and flow cytometry. The functional activity of IL-18 was assessed by the use of recombinant IL-18 to stimulate both the growth of intestinal mucosal lymphocytes and IL-2 receptor induction activity. Results: The serum IL-18 concentration was significantly higher in patients with CD than normal controls. In the inflamed colonic mucosa of CD, many IL-18+CD68+ macrophages had infiltrated the lamina propria. Intestinal mucosal lymphocytes from CD expressed functional IL-18 receptors. Recombinant IL-18 induced significant proliferative responses in freshly isolated mucosal lymphocytes from CD patients, but not from normal controls. 1L-18 up-regulated IL-2 receptor expression in mucosal lymphocytes from patients with CD, but not from normal controls. Conclusions: These findings suggest that infiltrated macrophages in the inflamed intestinal mucosa in CD produce IL-18, and that macrophage-derived IL-18 may serve as a potent regulatory factor for intestinal mucosal lymphocytes, thereby contributing to chronic intestinal inflammation in CD.

To assess a role of microsatellite instability (MSI) in the development of gastric adenocarcinoma or adenoma from chronic gastritis, we analysed mutations of five microsatellite loci in gastritis, adenoma and adenocarcinoma retrospectively. Gastric mucosa was biopsied from the same area in each patient at different periods and examined for MSI. Only one of 55 patients with chronic gastritis revealed MSI-H phenotype and the other 54 patients showed microsatellite stable (MSS) phenotypes. In six of 17 patients with gastric adenoma or well-differentiated adenocarcinoma, MSI-positive phenotypes were demonstrated. Interestingly, all of six patients showing MSI, including three high-level MSI (MSI-H) cases and three low-level (MSH-L) cases, had already revealed MSI at the stage of chronic gastritis. In two of three MSI-H cases, the identical MSI patterns had been observed at the stage of gastritis 1.5-7 years before the final diagnosis of adenocarcinoma. The adjacent gastritis mucosa within 10 mm from the carcinoma demonstrated MSI as well. MSI was not found in any of 35 patients with Helicobacter pylori infection, but found in one of 30 patients without infection. Moreover, two of three cases of gastric adenoma or well-differentiated adenocarcinoma with MSI-H at the stage of chronic gastritis showed no evidence of Helicobacter pylori infection throughout the observation periods. These results indicate that MSI in biopsy specimens at the stage of chronic gastritis may predict the risk of the progression to adenoma and well-differentiated adenocarcinoma, and that Helicobacter pylori infection itself may not induce MSI directly in the gastric mucosa. (C) 2000 Cancer Research Campaign.

OBJECTIVE: Much remains unknown about the pathogenesis of intestinal Behcet's disease. The majority of these patients are treated with surgical intervention, although it has been recently reported that a number of medical treatments are sometimes effective. Only few studies, however, have ever been undertaken to analyze the long-term prognosis of this disease. In this study, we analyzed the clinical course and the recurrences after initial therapy in patients with intestinal Behcet's disease. METHODS: We investigated 20 patients (surgical group, n = 8; nonsurgical group, n = 12) for whom the clinical courses were known for ≥2 yr (2-23 yr). RESULTS: The surgical group tended to have higher rates of complications such as ocular and ileal lesions than the nonsurgical group. In the surgical group, 75% of the patients recurred (and were readmitted) within 2 yr, and 37.5% of the patients required reoperation for intestinal obstruction because of ulcer at the anastomosis. The percentage of peripheral CD8 + DR + lymphocytes in the recurrent group (10.4% ± 2.5%) was significantly higher than that in the nonrecurrent group (4.3% ± 1.2%, p < 0.05). CONCLUSIONS: Our results indicate that more extensive disease involving the ileum and ocular lesions are markers of severity and progression to surgical crisis, and that patients requiring surgery suffer more frequent recurrences. Furthermore, an increased percentage of peripheral CD8 + DR + lymphocytes may be a risk factor for disease recurrence. (C) 2000 by Am. Coll. of Gastroenterology.

To clarify the role of colonic mucin in the autoimmune process of ulcerative colitis, circulating antibodies against human colonic mucin were investigated. Purified colonic mucin, obtained from human colonic mucosa by gel filtration, using a Bio-Gel A-1.5-m column and CsCl equilibrium density gradient, was divided into soluble mucin (S-mucin) secreted extracellularly and membranous mucin (M-mucin) binding to cell membrane. Sodium dodecylsulfate polyacrylamide gel electrophoresis and Western blotting analysis showed that antibodies in the serum samples of some patients with ulcerative colitis recognized purified S- and M-mucin of >180-kD. By enzyme-linked immunosorbent assay (ELISA), anti-mucin antibodies were detected in 11 of 60 patients with ulcerative colitis (18). In contrast, the antibodies were not detected in 22 patients with Crohn's disease. The titers of anti-mucin antibodies against S-mucin and M-mucin were not different in each patient. By ELISA using mucin in which the sugar chains were destroyed by neuraminidase or NaIO4 treatment, it was demonstrated that anti-mucin antibodies recognized the epitopes of either the sugar chain or the core protein exposed through destruction of the sugar chains. We then investigated the relationship between anti-mucin antibodies and the patients' clinical features. Anti-mucin antibodies were detected in 6 of 15 patients with chronic continuous type ulcerative colitis (40) and in 5 of 26 patients with relapsing-remitting type (19), but there was no anti-mucin antibody-positive serum in patients who had had only one attack without any relapse. These results suggest that anti-mucin antibodies could be a disease marker for ulcerative colitis and that immunological abnormalities in colonic mucin contribute to the persistence of colonic mucosal inflammation.

Background and Aim: Although molecular mechanisms underlying ulcerative colitis (UC)-associated neoplasms have been studied for years, understanding of these mechanisms remains incomplete and no good predictable marker for development of colonic neoplasms in patients with UC has been established. The aim of this study was to assess if microsatellite instability (MSI) contributes to the development of colonic neoplasms in patients with UC. Methods: We have examined MSI in chronic inflamed and neoplastic colonic mucosa of UC patients. We have also obtained serial biopsied colonic tissues retrospectively 2-12 years before the final diagnosis from patients with high level MSI (MSI-H+) UC-associated neoplasms, and analyzed MSI using them at different periods. Results: Eight of 12 UC-associated colon cancers (67%), four of six UC-associated high grade dysplasias (67%), and two of six UC-associated low grade dysplasias (33%) revealed MSI-H+ phenotypes. In contrast, 15 of 59 lesions (25%) in inflamed UC mucosa without colonic neoplasm revealed MSI-H+. Interestingly, all four patients with MSI-H+ phenotypes at the final diagnosis of UC-associated colon cancer or dysplasia had already had MSI-H+ at the stage of chronic colitis, 2-12 years before the final diagnosis. Conclusion: These results support the notion that MSI contributes to the carcinogenesis of UC-associated neoplasms, and indicate that this analysis in inflamed colonic mucosa at surveillance colonoscopy is useful for identifying UC patients who have high risk for neoplastic progression. © 2005 Blackwell Publishing Asia Pty Ltd.