Kayoko Matsumura

Immunosuppressive effects of tacrolimus on macrophages ameliorate experimental colitis

Background: Tacrolimus is a novel immunomodulator for inflammatory bowel diseases. Immunosuppressive effects of tacrolimus on T cells are well known; however, the effects of tacrolimus on macrophages remain unclear. The aim of this study was to investigate the effects of tacrolimus on activated macrophages and to examine its efficacy in murine colitis models. Methods: Proinflammatory cytokine production from lipopolysaccharide (LPS)–stimulated peritoneal macrophages of IL‐10‐knockout (KO) mice with and without tacrolimus was measured. We investigated the effects of tacrolimus on nuclear factor‐&kgr;B (NF‐&kgr;B), mitogen‐activated protein kinase (MAPK), and caspase activation in macrophages and the induction of apoptosis in macrophages in vitro and examined the in vivo apoptotic effect of tacrolimus on colonic macrophages in IL‐10‐KO mice. We evaluated the effect of the rectal administration of tacrolimus on colonic inflammation in IL‐10‐KO mice and dextran sulfate sodium (DSS)–induced colitis in CB.17/SCID mice. Results: Proinflammatory cytokine production from tacrolimus‐treated macrophages was significantly lower than that from untreated cells. Tacrolimus suppressed LPS‐induced activation of both NF‐&kgr;B and MAPK in macrophages and induced apoptosis of macrophages via activation of caspases 3 and 9. Rectal administration of tacrolimus evoked apoptosis of colonic macrophages in IL‐10‐KO mice. Moreover, the rectal administration of tacrolimus ameliorated colitis in IL‐10‐KO mice and DSS‐induced colitis in CB.17/SCID mice. Gene expression of inflammatory cytokines in colonic mucosa was significantly lower in tacrolimus‐treated mice than in untreated mice. Conclusions: Tacrolimus suppresses the function of activated macrophages and promotes their apoptosis, which may lead to the amelioration of colonic inflammation. Inflamm Bowel Dis 2010

Systematic review: cytomegalovirus infection in inflammatory bowel disease.

Human cytomegalovirus (HCMV) is a member of the herpesvirus family. HCMV infection is a common viral infection in humans, occurring in 50%–80% of adults, depending on the population studied. Acute infections are frequently asymptomatic, but once the infection is acquired, similar to other herpesvirus infections, HCMV infection persists lifelong in a latent state. The ability of HCMV to escape from control by the host immune system, resulting in its reactivation, suggests the importance of ongoing immune surveillance to prevent HCMV reactivation. HCMV is a common cause of opportunistic infection and causes severe and fatal disease in immune-compromised individuals. Gastrointestinal cytomegalovirus (CMV) disease can occur in persons with normal immune function, but it most frequently occurs in adults with immune defi ciency, such as those with acquired immunodefi ciency syndrome (AIDS) or who have undergone organ transplantation, cancer chemotherapy, or steroid therapy. In gastrointestinal CMV disease, the gross appearance and location of the lesions are similar regardless of the cause of the host’s immunodefi ciency. Ulceration, erosions, and mucosal hemorrhage are the primary macroscopic fi ndings. However, the pathogenesis of intestinal lesions associated with CMV infection is very complicated and still unclear. Many recent studies have reported the prevalence of CMV infection in patients with infl ammatory bowel disease (IBD). In IBD patients, particularly those with ulcerative colitis (UC), HCMV is often reactivated, because such patients are frequently treated with immunosuppressive agents. This reactivation is known to cause exacerbation of colitis. Moreover, CMV infection can induce severe colitis, even in patient with UC who have never been treated with immunosuppressive agents. However, the signifi cance of CMV in colonic infl ammation of patients with IBD remains unclear. Indeed, some investigators have suggested that CMV behaves in the intestine as a nonpathogenic bystander, and others have reported that CMV has a crucial role in triggering the onset of infl ammation, which causes the complications. Therefore, when CMV is detected either by blood tests or in mucosal biopsy specimens in patients with severe IBD, clinicians are faced with a therapeutic dilemma; should antiviral therapy be initiated and the level of immunosuppression be reduced, with the risk of further worsening the IBD, or should the dose of immunosuppressive agents be increased to suppress the IBD activity? This article reviews diagnostic and therapeutic strategies for CMV infection in patients with IBD. Sources for this review were located by using multiple search strategies in MEDLINE and by examination of relevant reference lists, with a particular focus on Englishlanguage articles concentrating on the diagnosis and treatment of CMV infection and IBD.

Effect and Safety of Granulocyte-Monocyte Adsorption Apheresis for Patients with Ulcerative Colitis Positive for Cytomegalovirus in Comparison with Immunosuppressants

Background: Cytomegalovirus (CMV) infection exacerbates ulcerative colitis (UC) refractory to immunosuppressive therapies (IMT). However, the underlying UC remained active in some UC patients, despite the fact that CMV-DNA in colonic mucosa became negative after antiviral therapy. Therefore, new therapeutic strategies for UC patients concomitant with CMV infection in mucosa are required. Aims: The aim of this study was to evaluate the effect and safety of granulocyte-monocyte adsorption apheresis (GMA) in UC patients positive for CMV infection after antiviral therapy. Methods: From October 2003 to December 2008, 64 patients with UC refractory to IMT, including steroids and immunomodulators, were enrolled in this retrospective, observational, multicenter study, which was reviewed and approved by the Institutional Review Board of Kyoto University. CMV infection was investigated by 3 methods (histologic examination, CMV antigenemia, and polymerase chain reaction). We investigated the clinical outcomes of GMA and IMT after 2 weeks of treatment with ganciclovir. Results: Thirty-one (48.4%) of 64 patients with UC refractory to IMT were positive for CMV. Of the 31 patients, 4 (12.9%) underwent colectomy. Twenty-seven patients (87.1%) underwent antiviral therapy. Of those 27 patients, 7 achieved remission following antiviral therapy alone. Of the remaining 20 patients who did not achieve remission despite the disappearance of CMV-DNA, 11 and 9 patients were treated with additional GMA (GMA group) and IMT (IMT group), respectively. Of 11 patients (GMA group), 9 achieved remission and 2 underwent colectomy. Out of the remaining 9 patients (IMT group), 4 achieved remission and 5 underwent colectomy. CMV-DNA was not detected in 11 patients after GMA, but it was detected again in all 5 patients of the IMT group who underwent colectomy. The total colectomy rate in UC patients positive for CMV was 35.5% (11/31). In addition, colectomy-free survival in the CMV relapse (+) group was estimated to be 12.9% at 65 months, while that in the CMV relapse (–) group was estimated to be 100% at 60 months. Conclusion: The colectomy ratio tends to be high in refractory UC patients with recurrent CMV reactivation or infection. Therefore, GMA might be a safe and effective treatment for UC patients positive for CMV because it does not induce CMV reactivation.

Efficacy of oral tacrolimus on intestinal Behcet's disease

To the Editor: Behcet’s disease (BD) is a chronic, relapsing, multisystem inflammatory disorder with mucocutaneous, ocular, joint, vascular, central nervous system, and gastrointestinal involvement. Although the deposition of immune complexes in the walls of small blood vessels has been proposed as the underlying pathological mechanism, the etiology still remains unknown. About 3%–26% of patients with BD have gastrointestinal tract involvement. Intestinal BD is intractable and medical treatment for intestinal BD has not been fully established. In this regard, various kinds of therapeutic agents such as 5-aminosalicilic acid (5-ASA), corticosteroids, immunomodulators, and infliximab have been tried to treat intestinal BD, although some patients with intestinal BD are still refractory to these immunosuppressive drugs. Therefore, more effective therapies will be required for patients with intestinal BD. Tacrolimus is a macrolide antibiotic with potent immunosuppressive activity, isolated from Sterptomyces tsukubaenesis. Tacrolimus is widely used for the prevention of allograft rejection in patients undergoing liver transplantation. Recently, much attention has been paid to tacrolimus for patients with inflammatory bowel disease (IBD) refractory to conventional therapy because of its more reliable intestinal absorption and immunosuppressive effects compared to those of cyclosporine A (CyA). Several reports showed that tacrolimus had therapeutic effects on uveitis with BD. However, the efficacy of tacrolimus on intestinal BD is still unknown. We herein report the first case of ulceration at the ascending colon in a patient with BD, which was successfully treated with oral tacrolimus. This case suggests that tacrolimus can be a promising therapy for BD patients with intestinal involvement. A 27-year-old woman with BD and myelodysplastic syndrome (trisomy of chromosome 8) complained of repeated right lower abdominal pain. The colonoscopic finding revealed a punched-out ulcer at the ascending colon near the Bauhin valve. She was diagnosed with intestinal involvement of BD. Administration of 5-aminosalicilic acid (mesalazine) (3 g/day), prednisolone (PSL) (45 mg/day), and CyA (200 mg/day) was started for treatment of her intestinal BD. Despite the use of these drugs, she relapsed repeatedly and needed an increased dose of PSL to achieve clinical remission. In July 2006 she was admitted to our hospital because of severe abdominal pain despite oral administration of 30 mg of PSL. Laboratory data revealed white cell counts 4000/lL; hemoglobin 11.2g/dL; platelets 17.8 10/lL; c-reactive protein (CRP) 1.4 mg/dL; total protein 6.2 g/dL; and albumin 3.9 g/dL. Colonoscopic findings revealed round and oval ulceration at the ascending colon near the Bauhin valve (Figure 1A). After informed consent was obtained from the patient and her family, oral tacrolimus was initiated to adjust the serum trough levels of tacrolimus from 10–15 ng/mL. Three weeks after starting oral tacrolimus therapy her abdominal pain disappeared and CRP became negative. Five months after initiating oral tacrolimus administration of PSL was completely tapered off without relapse of abdominal symptoms. Then we tapered the dose of tacrolimus to achieve trough levels of 5–10 ng/mL. In February 2009 she was in clinical remission. Colonoscopic examination 33 months after starting tacrolimus revealed complete disappearance of the ulcerative lesion at the ascending colon near the Bauhin valve (Figure 1B). Established guidelines for the treatment for intestinal BD remains unclear, although several therapies including 5-ASA, steroid, immunomodulators, thalidomide, and infliximab have been tried. Several reports showed that tacrolimus played an important role in the treatment of uveitis in BD patients who did not respond to cyclosporin. Our case suggested that tacrolimus could be an alternative option for patients with intestinal BD refractory to conventional therapy. In general, tacrolimus has immunosuppressive properties similar to CyA, but is 100 times more potent than CyA in vitro. It interacts with calcineurin and inhibits the translocation process of the nuclear factor of activated T-cells. This leads to a decrease in interleukin-2 levels, which in turn reduces the activation and proliferation of T-cells. In this case she repeated the complaint of abdominal pain irrespective of therapy with the combination of CyA and PSL. Therefore, we tried to administer tacrolimus, expecting a strong immunosuppressive effect. As a result she was successfully treated with tacrolimus. We carefully monitored the whole blood levels of tacrolimus to adjust from 10–15 ng/mL for induction of remission. Tacrolimus was tapered to achieve trough levels of 5–10 ng/ mL for maintenance according to the therapeutic design for patients with IBD. She did not have any serious side effects related to tacrolimus, although further studies will be necessary to decide its optimal trough level for patients with intestinal BD. This is the first report showing the therapeutic effect of tacrolimus on intestinal lesion of BD. We think that tacrolimus can be a new therapeutic CopyrightVC 2009 Crohn’s & Colitis Foundation of America, Inc. DOI 10.1002/ibd.20970 Published online 5 June 2009 in Wiley InterScience (www.interscience.wiley.com).

Prior use of immunomodulatory drugs improves the clinical outcome of endoscopic balloon dilation for intestinal stricture in patients with Crohn's disease

Endoscopic balloon dilation is a promising procedure to improve symptoms of intestinal stricture in patients with Crohns disease (CD). However, the long‐term efficacy of endoscopic balloon dilation combined with immunomodulatory drugs remains unclear. The aim of the present study is to investigate whether prior use of immunomodulatory drugs affects the clinical outcome of endoscopic balloon dilation for intestinal stricture in CD.

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis.

BACKGROUND AND AIMS Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis. METHODS HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo. RESULTS Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice. CONCLUSIONS Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.

Modulation of the Th1/Th2 balance by infliximab improves hyperthyroidism associated with a flare-up of ulcerative colitis

To the Editor: Several reports have suggested an association between inflammatory bowel disease and autoimmune thyroid disease.1,2 Hyperthyroidism in patients with ulcerative colitis (UC) is very rare, with an incidence reported to be 0.82%3 to 3.7%.2 However, chronic thyroiditis is well known as an extramanifestation of UC. The cause of the association between UC and Graves’ disease remains unclear because the colon and thyroid do not have a common trigger antigen for embryological origins. Alteration in T-cell function might be a possible mechanism, accounting for their association. We report a case of a patient with both UC and hyperthyroidism successfully treated with infliximab. Intracellular cytokine assay demonstrated that infliximab modulated the Th1/Th2 balance and finally led to the improvement of both diseases.

Heparan sulfate on intestinal epithelial cells plays a critical role in intestinal crypt homeostasis via Wnt/β-catenin signaling

Heparan sulfate (HS), a constituent of HS proteoglycans (HSPGs), is a linear polysaccharide present on the cell surface. HSPGs modulate functions of several growth factors and signaling molecules. We examined whether small intestinal epithelial HS plays some roles in crypt homeostasis using intestinal epithelium cell (IEC)-specific HS-deficient C57Bl/6 mice. Survival rate after total body irradiation was significantly reduced in HS-deficient mice due to profound intestinal injury. HS-deficient IECs exhibited Wnt/β-catenin pathway disruption, decreased levels of β-catenin nuclear localization, and reduced expression of Wnt target genes, including Lgr5 during crypt regeneration. Moreover, epithelial HS increased Wnt binding affinity of IECs, promoted phosphorylation of Wnt coreceptor LRP6, and enhanced Wnt/β-catenin signaling following ex vivo stimulation with Wnt3a, whereas activation of canonical Wnt signaling following direct inhibition of glycogen synthase kinase-3β by lithium chloride was similar between HS-deficient and wild-type mice. Thus HS influences the binding affinity of IECs to Wnt, thereby promoting activation of canonical Wnt signaling and facilitating regeneration of small intestinal crypts after epithelial injury.

IL-17 Promotes HSP47 Expression and Intestinal Fibrosis in Crohn's Disease

investigate the role of IL-17 in HSP47 expression and the potential for IL-17 as a new therapeutic target for intestinal fibrosis in CD. Methods: In Vivo study, serum levels of HSP47 in CD, UC and normal subjects were measured by enzyme-linked immunosorbent assay (ELISA). Localization of HSP47 positive cells in the intestine were evaluated by immunohistochemistry (IHC) using intestinal surgical specimens of patients with CD and UC. Gene expressions of HSP47, TGF-β-1, IL-13, HSP70 and IL-17 in the intestinal tissues were examined by quantitative real-time PCR (qRT-PCR). In Vitro study, gene expression of HSP47 in NIH3T3 cells after stimulation with TGF-β-1 or IL-17 was determined by qRT-PCR. Results: (1) Serum levels of HSP47 in patients with CD were significantly higher than in patients with UC and normal subjects. (2) IHC study revealed that HSP47 positive cells were mainly observed in mesenchymal site, submucosa and fibrostenotic site, and that the number of HSP47 positive cells were greater in the intestinal tissues of patients with CD than those with UC. (3) Gene expressions of HSP47, TGF-β-1 and IL-17 were significantly higher in the intestinal tissues of patients with CD than those with UC, while there were no significant differences of HSP70 and IL-13 gene expressions in the intestinal tissues between patients with CD and UC. (4) Gene expression of HSP47 in NIH3T3 cells were up-regulated by IL-17 stimulation as well as TGF-β-1. In addition, p38MAPK inhibitor suppressed HSP47 gene expression in NIH3T3 cells stimulated with IL-17. Conclusion: Our data suggest that IL-17 might be involved in intestinal fibrosis of CD by up-regulation of HSP47 expression in intestinal fibroblasts through p38MAPK pathway. IL-17 could be a new therapeutic target for not only intestinal inflammation but also intestinal fibrosis in CD.

205 Role of Heat Shock Protein 47 in the Pathogenesis of Patients With Crohn's Disease

The definitive diagnosis of inflammatory bowel disease (IBD) is currently based on a combination of clinical, radiographic, endoscopic, and histological criteria. Patients often present with varying complaints of abdominal pain, rectal bleeding, weight loss, anemia and diarrhea that guide the physician toward IBD. Some of these symptoms overlap with other more common gastrointestinal disorders, including irritable bowel syndrome and celiac disease. In cases where IBD is strongly suspected, physicians recommend invasive and costly procedures such as endoscopy and radiologic evaluation to determine the diagnosis. Given the promise of GM-CSF as a potential therapy for IBD, we screened levels of surface CD116 on circulating leukocytes in 43 IBD patients and 43 healthy controls. The median age of IBD patients was 40 wherein 57% were male. 28 patients had Crohns disease (CD) and 15 patients had ulcerative colitis (UC). The median age of normal patients was 36 wherein 55% were males. The physician caring for the patient made the diagnosis of IBD. Initial studies revealed that granulocyte surface CD116 levels in IBD (MFI 843±27)were significantly lower than healthy controls (MFI 1468±75, p 0.05) and were independent of any medications (p > 0.05). Within IBD populations, UC patients have significantly lower granulocyte (729±33 vs 916±32, p 0.05). Based on these findings, we conclude that CD116 expression is a sensitive and selective biomarker for IBD. Accordingly, we propose that development of a surface CD116 diagnostic test could prove beneficial to the diagnosis of IBD and may help differentiate from other gastrointestinal disorders.