Tomoharu Yajima

Damaged epithelia regenerated by bone marrow–derived cells in the human gastrointestinal tract

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.

Interleukin 7 is produced by human intestinal epithelial cells and regulates the proliferation of intestinal mucosal lymphocytes.

The interaction of mucosal lymphocytes and intestinal epithelial cells is thought to be important in regulating immune response in the intestinal mucosa, but conclusive evidence is limited. Here we demonstrate the expression of IL-7 mRNA in human intestinal mucosa by combined reverse transcription PCR and Southern blot hybridization. Immunohistochemistry and in situ hybridization confirm the presence of IL-7 in intestinal epithelial cells, especially in epithelial goblet cells. Moreover, IL-7 receptor expression in mucosal lymphocytes is demonstrated by immunohistochemistry and in situ hybridization, as well as by Southern blot and flow cytometric analysis of freshly isolated lamina propria lymphocytes. In contrast, IL-7 receptor could not be detected in the cell surface of freshly isolated PBLs. The functional activity of IL-7 receptor is demonstrated by the utility of recombinant IL-7 to stimulate the growth of lamina propria lymphocytes, and conversely inhibit CD3-dependent proliferation of these cells. In contrast, IL-7 caused no significant increase in DNA synthesis and cell numbers when added to PBLs. These findings suggest that human intestinal epithelial cells and epithelial goblet cells produce IL-7, and locally produced IL-7 may serve as a potent regulatory factor for intestinal mucosal lymphocytes.

Cytomegalovirus is frequently reactivated and disappears without antiviral agents in ulcerative colitis patients

OBJECTIVE: The clinical significance of cytomegalovirus (CMV) reactivation complicating ulcerative colitis (UC) patients has been uncertain. It has therefore remained undetermined whether or not CMV reactivation should be treated in UC patients under immunosuppression. The aim of the study was to clarify the natural history of CMV reactivation in UC patients.METHODS: Sixty-nine UC patients with moderate to severe activity were enrolled in the study. All of the patients were treated with prednisolone, and/or immunosuppressants such as cyclosporine A. We sequentially monitored CMV reactivation every 2 wk up until 8 wk using the CMV antigenemia (Ag) assay and plasma quantitative real-time polymerase chain reaction (PCR) assay for CMV.RESULTS: Immunoglobulin (Ig) G for CMV was positive in 48 patients (69.6%) and negative in 21 patients (30.4%). CMV was reactivated in 25 patients out of the 48 seropositive patients (52.1%) during the study period. The CMV Ag and PCR values were low and none of the patients showed any evidence of CMV infection on biopsy specimens by hematoxylin and eosin staining. While gancylovir (GCV) was not used except in two patients, clinical outcomes including rates of remission and colectomy were not significantly different among the CMV reactivation-positive, -negative, and CMV IgG negative groups. Furthermore, CMV disappeared without GCV in most of the CMV reactivation-positive patients.CONCLUSIONS: CMV is frequently reactivated in active UC patients; however, it disappears without antiviral agents. Therefore, antiviral therapies should not be necessary for most UC patients with only CMV reactivation as long as CMV Ag values are low.

Risk-adapted pre-emptive therapy for cytomegalovirus disease in patients undergoing allogeneic bone marrow transplantation.

We prospectively evaluated a risk-adapted pre-emptive treatment with ganciclovir for CMV diseases in patients undergoing allogeneic bone marrow transplantation (BMT). High-level CMV antigenemia (10 or more positive cells on two slides) or CMV antigenemia at any level in patients with grade II–IV acute graft-versus-host disease (aGVHD) were chosen as risk factors. We also retrospectively evaluated virus reactivation in plasma using quantitative real-time polymerase chain reaction (PCR). Fifty patients were evaluable. None of the 27 patients with or without grade I aGVHD developed high-level CMV antigenemia or CMV disease. Among the 23 patients with grade II–IV aGVHD, 12 patients (52%) developed CMV antigenemia and were treated pre-emptively, of whom two developed CMV gastroenteritis or retinitis in spite of therapy. Six of the remaining 11 patients developed CMV gastroenteritis before CMV antigenemia was detectable. All of the eight patients with CMV diseases were successfully treated with ganciclovir and no deaths directly related to CMV disease occurred. In four of the seven evaluable patients with CMV gastroenteritis, real-time PCR was able to detect virus reactivation earlier than CMV antigenemia. Although our risk-adapted pre-emptive therapy effectively reduced CMV-related mortality, further refinements of this approach, particularly in the prevention of CMV gastroenteritis, may be achieved by incorporating real-time PCR. Bone Marrow Transplantation (2000) 25, 765–769.

Novel, Objective, Multivariate Biomarkers Composed of Plasma Amino Acid Profiles for the Diagnosis and Assessment of Inflammatory Bowel Disease

Background Inflammatory bowel disease (IBD) is a chronic intestinal disorder that is associated with a limited number of clinical biomarkers. In order to facilitate the diagnosis of IBD and assess its disease activity, we investigated the potential of novel multivariate indexes using statistical modeling of plasma amino acid concentrations (aminogram). Methodology and Principal Findings We measured fasting plasma aminograms in 387 IBD patients (Crohns disease (CD), n = 165; ulcerative colitis (UC), n = 222) and 210 healthy controls. Based on Fisher linear classifiers, multivariate indexes were developed from the aminogram in discovery samples (CD, n = 102; UC, n = 102; age and sex-matched healthy controls, n = 102) and internally validated. The indexes were used to discriminate between CD or UC patients and healthy controls, as well as between patients with active disease and those in remission. We assessed index performances using the area under the curve of the receiver operating characteristic (ROC AUC). We observed significant alterations to the plasma aminogram, including histidine and tryptophan. The multivariate indexes established from plasma aminograms were able to distinguish CD or UC patients from healthy controls with ROC AUCs of 0.940 (95% confidence interval (CI): 0.898–0.983) and 0.894 (95%CI: 0.853–0.935), respectively in validation samples (CD, n = 63; UC, n = 120; healthy controls, n = 108). In addition, other indexes appeared to be a measure of disease activity. These indexes distinguished active CD or UC patients from each remission patients with ROC AUCs of 0.894 (95%CI: 0.853–0.935) and 0.849 (95%CI: 0.770–0.928), and correlated with clinical disease activity indexes for CD (rs = 0.592, 95%CI: 0.385–0.742, p<0.001) or UC (rs = 0.598, 95%CI: 0.452–0.713, p<0.001), respectively. Conclusions and Significance In this study, we demonstrated that established multivariate indexes composed of plasma amino acid profiles can serve as novel, non-invasive, objective biomarkers for the diagnosis and monitoring of IBD, providing us with new insights into the pathophysiology of the disease.

Mucosal T Cells Expressing High Levels of IL-7 Receptor Are Potential Targets for Treatment of Chronic Colitis

The IL-7/IL-7R-dependent signaling pathway plays a crucial role in regulating the immune response in intestinal mucosa. Here we demonstrate the pivotal role of this pathway in the development and treatment of chronic colitis. T cells expressing high levels of IL-7R were substantially infiltrated in the chronic inflamed mucosa of TCR α-chain knockout mice and IL-7 transgenic mice. Transfer of mucosal T cells expressing high levels of IL-7R, but not T cells expressing low levels of IL-7R, from these mice into recombinase-activating gene-2−/− mice induced chronic colitis. Selective elimination of T cells expressing high levels of IL-7R by administrating small amounts of toxin-conjugated anti-IL-7R Ab completely ameliorated established, ongoing colitis. These findings provide evidence that therapeutic approaches targeting mucosal T cells expressing high levels of IL-7R are effective in the treatment of chronic intestinal inflammation and may be feasible for use in the therapy of human inflammatory bowel disease.

Functional expression of costimulatory molecule CD86 on epithelial cells in the inflamed colonic mucosa.

BACKGROUND & AIMS Costimulatory signals are essential for T-cell activation. The aim of this study was to demonstrate expression of costimulatory molecules CD86 and CD80 in human colonic epithelial cells and assess their functional roles in the activation of T cells in inflamed colonic mucosa. METHODS CD86 and CD80 expression was assessed by immunohistochemistry of colonic mucosa, reverse-transcription polymerase chain reaction, and flow cytometric analysis of isolated colonic epithelial cells and cell lines. Costimulatory effect of CD86-expressing colonic epithelial cells on purified CD4(+) T-cell proliferation was also assessed at suboptimal phytohemagglutinin stimulation. RESULTS CD86 and CD80 messenger RNA was detected in isolated colonic epithelial cells from normal and inflamed mucosa of patients with ulcerative colitis. Immunohistochemistry and flow cytometric analysis of colonic epithelial cells confirmed cell surface expression of CD86 protein in inflamed colonic mucosa. Cell surface expression of CD86 protein was increased in the colonic epithelial cell line HT29-18-N2 after interferon gamma stimulation. Purified CD4(+) T cells proliferated in response to suboptimal phytohemagglutinin costimulated with interferon gamma-stimulated HT29-18-N2, and these proliferative responses were efficiently inhibited by the addition of anti-CD86 monoclonal antibody. Costimulatory effect of CD86-expressing colonic epithelial cells isolated from inflamed mucosa of ulcerative colitis was also demonstrated at suboptimal phytohemagglutinin stimulation in CD4(+) T cells. CONCLUSIONS Colonic epithelial cells may act as antigen-presenting cells, and contribute to the activation of T cells through costimulatory molecule CD86 expression in inflamed colonic mucosa.

A synthetic mimetic of CD4 is able to suppress disease in a rodent model of immune colitis

CD4+ mucosal T cells mediate the intestinal inflammation in Crohns disease and may serve as an important target for immune intervention. Here we assessed the therapeutic effect of a synthetic mimetic of CD4 designed to mimic both the sequence and conformation of the complementarity‐determining region 3 of murine CD4 V1 domain (rD‐mPGPtide) in a mouse colitis model using immunization with 2,4,6‐trinitrobenzene sulfonic acid (TNB). i.  v. administration of the rD‐mPGPtide but not control scrambled peptide could suppress severe inflammation in the chronic colitis mouse model. After treatment with the rD‐mPGPtide, a striking improvement of diarrhea and acute wasting disease was observed with decreased mortality. Serum anti‐TNB antibody titers, CD45RBlowCD4+ T cells in the lamina propria and IFN‐γ mRNA expression in the mucosa were significantly decreased with the rD‐mPGPtide treatment. Anti‐CD4 antibody also suppressed disease by depletion of CD45RBhighCD4+ T cells in the colonic mucosa. The observation that the synthetically engineered analogue of murine CD4 inhibits inflammation in a rodent disease model by different mechanisms than anti‐CD4 antibody suggests that a human version of this peptide has potential therapeutic utility in CD4+ mucosal T cell‐mediated intestinal inflammation in Crohns disease.

Dose-adjusted preemptive therapy for cytomegalovirus disease based on real-time polymerase chain reaction after allogeneic hematopoietic stem cell transplantation

We have prospectively evaluated the efficacy of real-time PCR-guided preemptive therapy for CMV diseases in allogeneic hematopoietic stem cell transplant recipients with grades II–IV acute GVHD. The dose of ganciclovir was adjusted according to the viral load determined by real-time polymerase chain reaction (PCR). On detecting CMV reactivation in the plasma, ganciclovir was initiated at a dose of 5 mg/kg body weight once daily, and the dose was increased to twice daily if viral load continued to increase after initiating ganciclovir. In 39 evaluable patients, CMV reactivation assessed by real-time PCR became positive in 30 (77%). One developed CMV gastroenteritis before PCR became positive. Thus the remaining 29 patients were treated preemptively with ganciclovir. The dose of ganciclovir was increased in 12 patients (41%) of preemptively treated patients for increasing viral load. CMV diseases were diagnosed in two patients (one gastroenteritis and one retinitis), and late CMV disease was diagnosed in one patient (gastritis). The treatment was generally well-tolerated, but three patients (10%) developed neutropenia (neutrophil count less than 1.0 × 109/l). In conclusion, real-time PCR-guided preemptive therapy with decreased dose of ganciclovir is feasible and does not increase the frequency of CMV diseases if the dose is adjusted according to the viral load.

Regulatory effect of interleukin-4 and interleukin-13 on colon cancer cell adhesion

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.