Minoru Matsuura

Usefulness of quantitative real-time PCR assay for early detection of cytomegalovirus infection in patients with ulcerative colitis refractory to immunosuppressive therapies

Background: Studies suggest that cytomegalovirus (CMV) infection exacerbates ulcerative colitis (UC) refractory to immunosuppressive therapies. Early and accurate diagnosis of CMV infection is important for the treatment of UC. We evaluated the usefulness of quantitative real‐time polymerase chain reaction (PCR) for detecting CMV infection in inflamed colonic mucosa of patients with UC refractory to immunosuppressive therapies. Methods: From 2003 to 2006, 30 patients (mean age: 41 ± 18 years; 14 men, 16 women) with UC refractory to immunosuppressive therapies were enrolled in the study. We evaluated CMV infection by CMV antigenemia, histologic examination, and quantitative real‐time PCR for CMV using colonic mucosa and investigated the clinical outcomes of antiviral therapy. Results: CMV‐DNA was detected only in the inflamed colonic mucosa in 17 (56.7%) of 30 patients. Of the 17 CMV‐DNA‐positive patients, 4 were positive for CMV antigenemia or inclusion bodies on histologic examination; of the 13 CMV‐DNA‐negative patients none was positive for CMV antigenemia or inclusion bodies. Of the 17 CMV‐DNA‐positive patients, 12 (70.6%) were treated with ganciclovir for 2 weeks and 10 patients went into remission. Two other patients required colectomy after antiviral therapy. In contrast, of the 13 CMV‐DNA‐negative patients 12 (92.3%) achieved remission after intensifying their immunosuppressive therapies. Conclusions: Quantitative real‐time PCR assay for detecting CMV‐DNA is useful for early, accurate diagnosis of CMV infection in patients with UC refractory to immunosuppressive therapies, enabling prompt and appropriate treatment. (Inflamm Bowel Dis 2007)

Elimination of local macrophages in intestine prevents chronic colitis in interleukin-10-deficient mice.

Uncontrolled activation of T cells and macrophages is involved in the development of inflammatory bowel disease (IBD). However, the precise role of intestinal macrophages for development of IBD is till unclear. To investigate the role of local macrophages for development of IBD, we developed poly-D, L-lactic acid microspheres containing dichloromethylene diphosphonate, which was specifically taken up by macrophages and depleted them, and then the animal model for human IBD was treated with this reagent. We have shown that rectal administration of the microspheres reduced the numbers of resident macrophages in the intestinal lymphoid follicles of interleukin-10-deficient mice. Importantly, depletion of intestinal macrophages was associated with suppression of development of chronic colitis. These results suggest that local macrophages in the intestine play a critical role in the development of chronic colitis in an animal model for IBD. Our study implies that elimination of resident macrophages in the intestine may become a therapeutic approach to IBD.

Long-term effect of tacrolimus therapy in patients with refractory ulcerative colitis

Background  Little is known about long‐term outcome of tacrolimus therapy for ulcerative colitis.

A Case of Autoimmune Pancreatitis Associated with Sclerosing Cholangitis, Retroperitoneal Fibrosis and Sjögren’s Syndrome

We report a very rare case of autoimmune pancreatitis (AIP) associated with sclerosing cholangitis, retroperitoneal fibrosis and Sjögren’s syndrome. The patient had an enlarged pancreas, and autoantibodies were detected in the serum. Serum IgG and IgG4 concentrations were also elevated. Endoscopic retrograde cholangiopancreatography revealed an irregular narrowing of the main pancreatic duct from the head to the body and sclerotic change in the intrapancreatic common bile duct, which later extended to the intrahepatic bile ducts. In addition, histological examination of the liver revealed lymphocytic sclerosis around the bile ducts, similar to the histology in the pancreas of AIP. Retroperitoneal tumors were diagnosed as retroperitoneal fibrosis by histological examination. Serological and functional abnormalities suggestive of Sjögren’s syndrome were detected, and histological findings of the lip were compatible with Sjögren’s syndrome. Immunohistochemistry of each lesion disclosed that most of the infiltrating lymphocytes were T cells with similar levels of both CD4+ and CD8+ cells. Moreover, some of the infiltrating plasma cells were positive for anti-IgG4 monoclonal antibody. These diseases were dramatically improved by steroid therapy. Although the pathophysiology of AIP is still unclear, the present case suggests a common pathophysiological mechanism for AIP, sclerosing cholangitis, retroperitoneal fibrosis and Sjögren’s syndrome.

Involvement of myeloid dendritic cells in the development of gastric secondary lymphoid follicles in Helicobacter pylori-infected neonatally thymectomized BALB/c mice

ABSTRACT We previously described an animal model of Helicobacter pylori-induced follicular gastritis in neonatally thymectomized (nTx) mice. However, it is still not clear whether antigen-presenting dendritic cells (DCs) in the stomach have a role in the development of secondary follicles in H. pylori-infected nTx mice. We investigated the distribution of DC subsets using this model and examined their roles. To identify lymphoid and myeloid DCs, sections were stained with anti-CD11c (pan-DC marker) in combination with anti-CD8α (lymphoid DC marker) or anti-CD11b (myeloid DC marker) and were examined with a confocal microscope. Expression of macrophage inflammatory protein 3α (MIP-3α), which chemoattracts immature DCs, was analyzed by real-time PCR and immunohistochemistry. Follicular dendritic cells (FDCs) were stained with anti-SKY28 antibodies. In noninfected nTx mice, a few myeloid and lymphoid DCs were observed in the bottom portion of the lamina propria, whereas in H. pylori-infected nTx mice, there was an increased influx of myeloid DCs throughout the lamina propria. FDC staining was also observed in the stomachs of members of the infected group. MIP-3α gene expression was upregulated in the infected nTx group, and the immunohistochemistry analysis revealed MIP-3α-positive epithelial cells. These data suggest that H. pylori infection upregulates MIP-3α gene expression in gastric epithelial cells and induces an influx of myeloid DCs in the lamina propria of the gastric mucosa in nTx mice. Myeloid DCs and FDCs might contribute to the development of gastric secondary lymphoid follicles in H. pylori-infected nTx mice.

Reduced numbers and proapoptotic features of mucosal-associated invariant T cells as a characteristic finding in patients with inflammatory bowel disease

Background:Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in the homeostasis of mucosal immunity; however, their role in inflammatory bowel disease (IBD) is unclear. Methods:Flow cytometry was used to enumerate peripheral blood MAIT cells in 88 patients with ulcerative colitis (UC), 68 with Crohns disease (CD), and in 57 healthy controls. Immunohistochemistry identified MAIT cells in intestinal tissue samples from patients with UC (n = 5) and CD (n = 10), and in control colon (n = 5) and small intestine (n = 9) samples. In addition, expression of activated caspases by MAIT cells in the peripheral blood of 14 patients with UC and 15 patients with CD, and 16 healthy controls was examined. Results:Peripheral blood analysis revealed that patients with IBD had significantly fewer MAIT cells than healthy controls (P < 0.0001). The number of MAIT cells in the inflamed intestinal mucosae of patients with UC and CD was also lower than that in control mucosae (P = 0.0079 and 0.041, respectively). The number of activated caspase-expressing MAIT cells in the peripheral blood of patients with UC and CD was higher than that in healthy controls (P = 0.0061 and 0.0075, respectively), suggesting that the reduced MAIT cell numbers in IBD are associated with an increased level of apoptosis among these cells. Conclusions:The number of MAIT cells in the peripheral blood and inflamed mucosae of patients with UC and CD was lower than that in non-IBD controls. Also, MAIT cells from patients with IBD exhibited proapoptotic features. These data suggest the pathological involvement and the potential for therapeutic manipulation of these cells in patients with IBD.

The effect of proteasome inhibitor MG132 on experimental inflammatory bowel disease

Immunoproteasome up‐regulation enhances the processing of nuclear factor‐κB (NF‐κB) and degradation of IκBα, which correlates with increased amounts of NF‐κB in the various cells. Aberrant activation of NF‐κB is involved in the pathogenesis of inflammatory bowel disease (IBD). The aim of this study was to elucidate the effect of proteasome inhibitor MG132 on experimental IBD. We investigated the effects of MG132 on intestinal inflammation and epithelial regeneration in both interleukin‐10‐deficient (IL‐10−/−) mice and mice with dextran sulphate sodium (DSS)‐induced colitis. Body weight, histological findings and tumour necrosis factor (TNF)‐α mRNA expression, epithelial cell proliferation and NF‐κB p65 activity in colonic tissues were examined. The effects of MG132 on cell proliferation, migration and multiple drug resistance 1 (MDR1) gene expression were determined in vitro. MG132 ameliorated intestinal inflammation of IL‐10−/− mice by decreasing TNF‐α mRNA expression in the colonic tissues, which was associated with suppression of NF‐κB activation, and reduced significantly the number of Ki‐67‐positive intestinal epithelial cells. On the other hand, MG132 did not reduce intestinal inflammation in mice with DSS‐induced colitis, and delayed significantly the recovery of body weight and epithelial regeneration. MG132 also suppressed significantly epithelial cell proliferation, cell migration and MDR1 gene expression in vitro. Proteasome inhibition reduces T cell‐mediated intestinal inflammation, but may interrupt both epithelial regeneration and barrier function of colonic mucosa. Optimal use of proteasome inhibitor should be kept in mind when we consider its clinical application for patients with IBD.

Tacrolimus therapy as an alternative to thiopurines for maintaining remission in patients with refractory ulcerative colitis.

Background Although the efficacy of tacrolimus for inducing remission of refractory ulcerative colitis (UC) is established, its efficacy for maintaining remission of UC has not been evaluated. Aim The aim of this study was to evaluate the efficacy of tacrolimus compared with thiopurines for maintaining remission in patients with refractory UC. Methods Twenty-four UC patients treated with tacrolimus and 34 treated with thiopurines to maintain remission were enrolled as the tacrolimus group and the thiopurine group, respectively. In the tacrolimus group, 82.8% of the patients were treated with tacrolimus for induction of the remission, whereas 70% of the patients in the thiopurine group were induced remission with either corticosteroid or cytapheresis. Proportions of patients who kept steroid-free remission between the tacrolimus and the thiopurine groups were compared. Maintenance of remission using tacrolimus or thiopurines was defined as no need for other therapies other than aminosalicylates without relapse for at least 3 months. Secondarily, to determine whether the response to thiopurines affects the long-term efficacy of tacrolimus maintenance therapy, the overall cumulative relapse-free survival based on the Kaplan-Meier method was estimated in thiopurine-naive or thiopurine-intolerant patients and thiopurine-refractory ones in the tacrolimus group. Results Remission was successfully maintained in 17 patients (70.8%) of the tacrolimus group, and 28 patients (82.4%) of the thiopurine group. The overall cumulative relapse-free survival of thiopurine-naive or thiopurine-intolerant patients in the tacrolimus group was similar to that in the thiopurine group, and significantly higher than that of thiopurine-refractory patients in the tacrolimus group. Conclusion Maintenance therapy with tacrolimus for patients with UC could be considered an alternative to thiopurine therapy.

Specific antibodies against recombinant protein of insertion element 900 of Mycobacterium avium subspecies paratuberculosis in Japanese patients with Crohn's disease

Background: Mycobacterial avium subspecies paratuberculosis (MAP) infection has been hypothesized as an etiological factor of Crohns disease (CD). However, the involvement of MAP in the pathophysiology of CD is controversial. The aim of this study is to investigate whether MAP is involved in the pathogenesis of CD with the glutathione S‐transferase fusion recombinant protein encoding a portion of insertion element (IS) 900 (IS900‐GST), which is specific for MAP. Methods: Serum samples from the patients with CD (n = 50), ulcerative colitis (n = 40), colonic tuberculosis (n = 20), and non‐IBD controls (n = 44), were applied for solid‐phase enzyme‐linked immunosorbent assay (ELISA) to detect antibodies against MAP and Saccharomyces cerevisiae. IS900‐GST, which was made by the pGST‐4T‐2 vector inserted with polymerase chain reaction‐amplified IS900DNA, was used as an antigen of MAP. Moreover, we studied the relationship between antibodies against IS900‐GST and clinical characteristics. Results: ELISA showed that the serum level of immunoglobulin G and immunoglobulin A antibodies against IS900‐GST (anti‐IS900) in patients with CD were significantly higher than those with ulcerative colitis, colonic tuberculosis, and control subjects. The levels of anti‐IS900 tended to be higher in CD patients with small intestinal involvement than with colonic involvement alone. Anti‐IS900 in patients with penetrating‐ and stricture‐type CD was significantly higher than with inflammatory‐type CD. Furthermore, a negative correlation was found between the titer of anti‐IS900 and disease duration. Anti‐IS900 was not associated with surgical treatment nor was it associated with the use of immunosuppressants. No significant correlation was observed between the serum levels of anti‐IS900 and anti‐S cerevisiae antibody. Conclusions: This is the first demonstration of the ELISA system of detecting antibodies against IS900 in IBD patients. MAP could be involved in the pathophysiology of Japanese patients with CD.

Helicobacter felis-induced gastritis was suppressed in mice overexpressing thioredoxin-1

Thioredoxin-1 (TRX-1) is a redox-active protein involved in scavenging reactive oxygen species and regulating redox-sensitive transcription factors. TRX-1 is induced in various inflammatory conditions and shows cytoprotective action. We investigated the roles of TRX-1 in the host defense mechanism against Helicobacter felis (H. felis) infection. Transgenic (TG) mice overexpressing human TRX-1 and wild-type (WT) mice were orally inoculated with H. felis. After 2 months, histology, oxidative damage, and gene expression of several cytokines, including macrophage inflammatory protein-2 (MIP-2), a murine equivalent to interleukin (IL)-8, in the gastric mucosa were investigated. Furthermore, the effects of TRX-1 on oxidative stress and neutrophil migration were studied both in vivo and in vitro. The gastric mucosa was thickened in H. felis-infected WT mice, but not in infected TRX-1-TG mice. Histologically, all H. felis-infected WT mice developed moderate-to-severe gastritis, whereas the development of gastritis was significantly suppressed in infected TRX-1-TG mice. Oxidative damage markers, 8-hydroxy-2′-deoxyguanosine and malondialdehyde, increased in the stomach of infected WT mice, but not TRX-1-TG mice. Upregulation of IL-1β and tumor necrosis factor-α gene expression in H. felis-infected TRX-1-TG mice was significantly lower than in WT mice. However, upregulation of MIP-2 and IL-7 was not different between the two groups. TRX-1 suppressed oxidative cytotoxicity and DNA damage, and inhibited neutrophil migration both in vivo and in vitro. The present study suggests that overexpression of TRX-1 suppresses H. felis-induced gastritis by inhibiting chemotaxis of neutrophils and reducing oxidative stress.