Yusuke Honzawa

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

Low prevalence of CMV infection in patients with Crohn's disease in comparison with ulcerative colitis: Effect of different immune response on prevalence of CMV infection

We read with great interest the original article of Kim et al. [1] on cytomegalovirus (CMV) infection in patients with active inflammatory bowel disease. In this article, the authors focused on incidence of CMV infection in patients with UC. However, we were very interested in the lower incidence of CMV infection in CD patients in comparison with that in UC patients. The authors demonstrated that 12 of the 120 patients with UC and none of the 20 patients with CD had CMV infection, although there were a limited number of patients with CD enrolled in this study. Why was the incidence of CMV infection lower in patients with CD than in those with UC? We have reported the data of quantitative real-time polymerase chain reaction using colonic mucosa (mucosal PCR) for diagnosing reactivation of CMV in patients with UC refractory to immunosuppressive therapies [2, 3]. In addition, we also evaluated the involvement of CMV infection in patients with CD refractory to immunosuppressive therapies. Ten patients with CD refractory to immunosuppressive therapies including corticosteroids, immunomodulators and infliximab (IFX) were evaluated by using CMV antigenemia, histologic examination, and mucosal PCR. The mean age of the ten patients was 34.4 ± 9.6 years. The involvement site of disease was small bowel type (40%), small and large bowel type (40%), and large bowel type (20%). The mean Crohn’s disease activity index score was 228.1 ± 67.5. Of the ten patients, two (20%) had been treated with corticosteroids alone, two (20%) with a combination of corticosteroids and immunomodulators (IM), one (10%) with the combination of IM and IFX, and five (50%) with IM alone. As a result, CMV infection and reactivation was not detected in all of these CD patients despite treatment with immunosuppressive drugs (unpublished data). This data was consistent with the report of Kim et al. Kim et al. discussed that TNF-a and IFN-c may activate viral latency of CMV in the setting of IBD exacerbation. However, does IFN-c really induce reactivation of CMV? Indeed, it is well known that TNF-a plays an important role in reactivation of CMV in both monocytes and dendritic cells [4]. In addition, a recent in vitro study showed CMV could force monocytes to acquire an M1 proinflammatory phenotype and differentiate into long-lived macrophages. CMV infected monocytes with M1 phenotype induce cytokine expression of IL-6, TNF-a, and IL-1b, which can contribute to increasing activated macrophages and permissive cells supporting active replication of CMV. Thus, TNF-a would be significantly associated with CMV reactivation and infection in patients with IBD. On the contrary, it was reported that IFN-c, which is produced from CD4? T cells, could suppress CMV reactivation [5]. Moreover, CD is considered a Th1-type inflammatory process with greater expression of IFN-c [6]. These findings suggested that CMV reactivation might not easily occur in patients with CD, supporting lower incidence of CMV infection in patients with CD. In conclusion, we consider that different immune response between UC and CD might reflect different incidence of CMV infection. However, further investigation will be necessary for elucidating the mechanism of CMV infection in IBD. H. Nakase (&) T. Yoshino Y. Honzawa T. Chiba Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 54, Shogoin-Kawaracho, Sakyo-ku, Kyoto 606-8507, Japan e-mail: hiropy_n@kuhp.kyoto-u.ac.jp

Efficacy and safety of infliximab as rescue therapy for ulcerative colitis refractory to tacrolimus.

Background and Aim:  Little is known about the efficacy and safety of infliximab for ulcerative colitis refractory to tacrolimus. The aim of this study was to evaluate the efficacy and safety of infliximab in the induction of remission in ulcerative colitis patients with persistent symptoms despite tacrolimus therapy.

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.

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.

Diagnosis and Treatment of Ulcerative Colitis with Cytomegalovirus Infection: Importance of Controlling Mucosal Inflammation to Prevent Cytomegalovirus Reactivation

Human cytomegalovirus (HCMV) is a member of the herpesvirus family. HCMV infection persists throughout the host lifespan in a latent state following primary infection. The ability of HCMV to escape control by the host immune system and its resulting reactivation suggests the importance of ongoing immune surveillance in the prevention of HCMV reactivation. HCMV is a common cause of opportunistic infection that causes severe and fatal disease in immune-compromised individuals. In inflammatory bowel disease patients, particularly those with ulcerative colitis (UC), HCMV is often reactivated because these patients are frequently treated with immunosuppressive agents. This reactivation exacerbates colitis. Additionally, HCMV infection can induce severe colitis, even in patients with UC who have never been treated with immunosuppressive agents. However, the role of HCMV in colonic inflammation in patients with UC remains unclear. Here, we present previous and current clinical data on the diagnosis and treatment of HCMV infection in UC. Additionally, our experimental data from a newly established mouse model mimicking UC with concomitant CMV infection clearly demonstrate that inflammation could result in the exacerbation of UC disease activity with induction of HCMV reactivation. In summary, optimal control of colonic inflammation should be achieved in UC patients who are refractory to conventional immunosuppressive therapies and are positive for HCMV.

Clinical significance of serum diamine oxidase activity in inflammatory bowel disease: Importance of evaluation of small intestinal permeability

The intestinal mucosa serves as a major anatomic and functional barrier to potentially harmful intraluminal components such as bacteria and several antigens. The intestinal barrier is formed by epithelial cells and the junctional complex, including the tight junction (TJ) complex. Alterations of the composition of TJs was reported for both Crohn’s disease (CD) and ulcerative colitis (UC) and consisted of upregulated expression of the pore-forming TJ protein claudin-2. In addition, the impaired permeability may represent the early onset of inflammatory bowel disease (IBD) because increased intestinal epithelial permeability precedes clinical relapse in patients with asymptomatic CD and in some healthy close relatives of the patients. These data suggest that small intestinal permeability plays a critical role in the disease onset of IBD and an examination of small intestinal permeability would be useful for diagnosing IBD and predicting disease relapse. We note with great interest the ‘‘Importance of disrupted intestinal barrier in inflammatory bowel disease’’ by Salim et al. In that review the authors highlighted the importance of intestinal barrier function in the pathogenesis of IBD and techniques for evaluating intestinal permeability in vivo and in vitro. However, it is not easy to evaluate small intestinal permeability in vivo, despite several modalities, such as the cellobiose/mannitol permeability test or CrEDTA test. Diamine oxidase (DAO) is an enzyme that catalyzes the oxidation of diamines such as histamine, putrescine, and cadaverine. In humans and rodents, DAO is specifically located at the apical end of mature villous cells with high activity and its activity reflects the integrity and maturity of the small intestinal mucosa. Several studies of humans and animals revealed that DAO activity in serum inversely correlates with intestinal permeability of small intestine. Ayuso et al reported that disease activity in patients with UC was related to the nonsynonymous single nucleotide polymorphism (SNP) in the DAO. On the other hand, a recent study demonstrated that a nonsynonymous SNP of the histaminedegrading enzyme DAO is not useful for assessing susceptibility to CD. Therefore, the significance of measuring DAO for IBD patients remains unclear. We measured serum DAO activity levels in patients with IBD and evaluated the clinical significance of DAO in IBD.

Refractoriness of Intestinal Behçet's Disease with Myelodysplastic Syndrome Involving Trisomy 8 to Medical Therapies - Our Case Experience and Review of the Literature

Background/Aims: Gastrointestinal lesions of Behçets disease (BD) are often refractory to medical therapy and sometimes result in serious comorbidities such as gastrointestinal perforation and massive bleeding. There are several reports of patients with BD comorbid with myelodysplastic syndrome (MDS) involving trisomy 8 that frequently have intestinal lesions refractory to conventional medical therapy. Little is known about the efficacy of infliximab (IFX) for these intestinal lesions. Methods: We present 2 cases of intestinal BD with MDS involving trisomy 8 who did not respond to IFX, and review previous reports of BD with MDS involving trisomy 8 concerning their responsiveness to conventional medical therapy. Results: Among 31 previously reported cases that received medical treatment for BD, 19 (61.3%) showed temporary improvement of the BD symptoms, 9 (29.0%) deteriorated, and 3 (9.7%) showed no change. All of the 9 cases that showed deterioration had intestinal lesions. Our 2 cases failed to respond to IFX, resulting in a poor prognosis. Conclusions: IFX might not be effective for improving intestinal BD comorbid with MDS involving trisomy 8. Trisomy 8 is associated with the BD prognosis and refractoriness to conventional medical therapy.

Tacrolimus or infliximab for severe ulcerative colitis: short-term and long-term data from a retrospective observational study

Objective Treatment of severe ulcerative colitis (UC) is challenging. Although the efficacy of tacrolimus (TAC) and infliximab (IFX) have been evaluated in patients with severe UC, the safety and efficacy levels of sequential therapies (TAC→IFX/IFX→TAC) in these patients remain unclear. The aim of this study was to assess short-term and long-term outcomes in patients with severe UC treated with TAC and IFX. Methods From October 2001 to February 2014, 29 patients with consecutive severe UC treated with TAC or IFX were retrospectively evaluated. Median follow-up duration was 27 months (range 0.5–118 months). The primary end point was short-term outcomes at 8 weeks after induction of TAC (TAC group, n=22) or IFX (IFX group, n=7). The secondary end point included long-term outcomes and colectomy-free survival. The clinical response was evaluated based on a partial Mayo score. Results The clinical remission (CR) rate at 8 weeks in the TAC and IFX groups was 63.6% and 71.4%, respectively. In 13 of the 29 patients (10 in the TAC group, 3 in the IFX group), sequential therapies were used in their clinical courses. In 9 of these 13 patients (6 in the TAC group, 3 in the IFX group), CR was achieved and maintained by sequential therapies. Overall cumulative colectomy-free survival was 79.3% at 118 months. Conclusions TAC and IFX had similar effects on remission induction in patients with severely active UC. Sequential therapies could rescue patients with UC who failed initial treatment with TAC or IFX. In clinical practice, sequential therapies might be deliberately performed.

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.