Takehiko Katsurada

Rikkunshito Ameliorates the Aging-Associated Decrease in Ghrelin Receptor Reactivity via Phosphodiesterase III Inhibition

Aging is associated with decreased food intake, a phenomenon termed the anorexia of aging. In this study, we sought to clarify changes in peripheral and central appetite-related factors in aged mice. Furthermore, we investigated the effects of rikkunshito, a traditional Japanese medicine, on age-related anorexia. C57BL/6J mice that were 6 or 75 wk old were studied. We investigated changes in food intake, ghrelin and leptin levels, and the expression of appetite-related genes with age. In addition, we verified the effects of ghrelin, rikkunshito, phosphodiesterase 3 (PDE3), and phosphoinositide 3-kinase inhibitors on appetite. Food intake was significantly decreased in 75-wk-old mice compared with the 6-wk-old mice. In 75-wk-old mice, plasma acylated ghrelin levels under fasting conditions were lower than in 6-wk-old mice, whereas leptin levels under feeding conditions were substantially higher. The expression levels of hypothalamic preproghrelin under feeding conditions and the expression levels of neuropeptide Y and agouti-related protein under fasting conditions were lower compared with those of the 6-wk-old mice. Ghrelin supplementation (33 microg/kg) failed to increase food intake in 75-wk-old mice. Conversely, oral administration of LY294002, a phosphoinositide 3-kinase inhibitor, and cilostamide, a PDE3 inhibitor, increased food intake in 75-wk-old mice. Moreover, rikkunshito increased food intake in aged mice. The components of rikkunshito (nobiletin, isoliquiritigenin, and heptamethoxyflavone) had inhibitory effects on PDE3. These results suggest that dysregulation of ghrelin secretion and ghrelin resistance in the appetite control system occurred in aged mice and that rikkunshito ameliorated aging-associated anorexia via inhibition of PDE3.

A novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates inflammatory colonic injury in mice

BACKGROUND In inflammatory bowel disease (IBD), gut inflammation is associated with the activation of nuclear factor kappa B (NF-κB), a key pro-inflammatory transcription factor. AIM To investigate the therapeutic potential of a novel, specific NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), we examined its effect on IBD using murine experimental colitis models. METHODS The in vitro effect of DHMEQ was evaluated by inflammatory cytokine production and p65 immunostaining using HT-29 and RAW264.7 cells. The in vivo therapeutic effect of DHMEQ was studied in colitis induced by dextran sulphate sodium (DSS) and trinitrobenzenesulphonic acid (TNBS). In these, progression and severity of colitis was mainly assessed by the disease activity index (DAI), histopathology, cellular infiltration, and mRNA expression levels of pro-inflammatory cytokines in the colonic tissues. RESULTS In RAW264.7 cells, DHMEQ significantly inhibited tumour necrosis factor (TNF)-α and interleukin (IL)-6 production induced by LPS in a dose-dependent manner by blocking the nuclear translocation of NF-κB. In addition, DHMEQ inhibited IL-8 production induced by LPS in HT-29 cells. DHMEQ significantly ameliorated DSS colitis as assessed by DAI scores, colonic oedema, and histological scores. Immunohistochemistry revealed that DHMEQ inhibited colonic infiltration of nuclear p65(+) cells, CD4(+) lymphocytes, and F4/80(+) macrophages. mRNA expression levels of the pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-12p40, IL-17, and MCP-1 were also suppressed by DHMEQ administration. Furthermore, DHMEQ significantly ameliorated TNBS colitis as assessed by body-weight changes and histological scores. CONCLUSION DHMEQ ameliorated experimental colitis in mice. These results indicate that DHMEQ appears to be an attractive therapeutic agent for IBD.

Investigation of gastric and duodenal mucosal defects caused by low-dose aspirin in patients with ischemic heart disease.

Background Low-dose aspirin is used for secondary prevention of ischemic heart disease and ischemic cerebrovascular disease. Currently, the frequency of gastrointestinal disorder among usersof low-dose aspirin is unknown. Aims To investigate through endoscopic examination the frequency of gastroduodenal disorder associated with buffered and enteric-coated aspirin (ECA). Methods Screening upper endoscopic examinations were prospectively performed on 236 patients with ischemic heart disease. Endoscopic findings including ulcers and flat erosions were assessed as mucosal defects. Results Mucosal defects were found in 92 of 190 (48.4%) users of low-dose aspirin and 6 of 46 (13.0%) nonusers. There were significantly more mucosal defects among users of low-dose aspirin than among those using no aspirin (P<0.0001). Mucosal defects were found in 54 of 98 (60.7%) users of buffered aspirin (BA), whereas 38 of 101 (37.6%) users of ECA had mucosal defects. Users of ECA had significantly fewer erosions than did those of BA (P=0.0015). The frequency of ulcer is similar between BA users and ECA users. Conclusions As endoscopy frequently reveals gastroduodenal disorder among low-dose aspirin users, both administration of BA and of enteric-coated aspirin warrant concern for gastroduodenal ulcer.

Endoscopic survey of low-dose-aspirin-induced gastroduodenal mucosal injuries in patients with ischemic heart disease.

Background and Aim:  Low‐dose aspirin is effective for the prevention of cardiovascular and cerebrovascular events, but the frequency of gastrointestinal injuries among users of low‐dose aspirin in Japan is currently unknown. In the present study endoscopic examination was performed to investigate the frequency of gastroduodenal injuries associated with low‐dose aspirin in patients with ischemic heart disease (IHD).

Human Amnion-Derived Mesenchymal Stem Cell Transplantation Ameliorates Dextran Sulfate Sodium-Induced Severe Colitis in Rats

Mesenchymal stem cells (MSCs) are a valuable cell source in regenerative medicine. Recently, several studies have shown that MSCs can be easily isolated from human amnion. In this study, we investigated the therapeutic effect of human amnion-derived MSCs (AMSCs) in rats with severe colitis. Colitis was induced by the administration of 8% dextran sulfate sodium (DSS) from day 0 to day 5, and AMSCs (1 × 106 cells) were transplanted intravenously on day 1. Rats were sacrificed on day 5, and the colon length and histological colitis score were evaluated. The extent of inflammation was evaluated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and immunohistochemistry. The effect of AMSCs on the inflammatory signals was investigated in vitro. AMSC transplantation significantly ameliorated the disease activity index score, weight loss, colon shortening, and the histological colitis score. mRNA expression levels of proinflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and migration inhibitory factor (MIF) were significantly decreased in the rectums of AMSC-treated rats. In addition, the infiltration of monocytes/macrophages was significantly decreased in AMSC-treated rats. In vitro experiments demonstrated that activation of proinflammatory signals induced by TNF-α or lipopolysaccharide (LPS) in immortalized murine macrophage cells (RAW264.7) was significantly attenuated by coculturing with AMSCs or by culturing with a conditioned medium obtained from AMSCs. Although the phosphorylation of IκB induced by TNF-α or LPS was not inhibited by the conditioned medium, nuclear translocation of NF-κB was significantly inhibited by the conditioned medium. Taken together, AMSC transplantation provided significant improvement in rats with severe colitis, possibly through the inhibition of monocyte/macrophage activity and through inhibition of NF-κB activation. AMSCs could be considered as a new cell source for the treatment of severe colitis.

Clinical and Pharmacokinetic Factors Associated With Adalimumab-Induced Mucosal Healing in Patients With Crohn’s Disease

Background & Aims We previously reported results from a prospective randomized controlled trial comparing the efficacy of adalimumab monotherapy versus combination with azathioprine for patients with Crohn’s disease (CD) who were naive to biologics and thiopurines. We performed a subanalysis of data from this study to evaluate factors associated with endoscopic response and mucosal healing in study participants. Methods We compared simple endoscopic scores for CD between patients with moderate to severe active CD randomly assigned groups that received adalimumab monotherapy (n = 85) or adalimumab in combination with azathioprine (n = 91), from June 2011 to June 2014 in Japan. We evaluated associations of simple endoscopic scores for CD with clinical factors and trough levels of adalimumab. Ultimately, 135 patients at Week 26 and 139 patients at Week 52 from 44 referral sites were analyzed for the present investigation. Results The odds for endoscopic response were significantly higher in the combination group than in the monotherapy group at Week 26 (odds ratio [OR], 2.12; 95% confidence interval [CI], 1.04–4.32) but not at Week 52 (OR, 1.50; 95% CI, 0.77–2.94). The odds of mucosal healing did not differ significantly between groups at Weeks 26 or 52. Simple endoscopic scores for CD at Week 0 was significantly associated with mucosal healing at Week 26 (OR, 0.80; 95% CI, 0.72–0.90) and at Week 52 (OR, 0.91; 95% CI, 0.84–0.99). Higher adalimumab trough level at Week 26 associated with mucosal healing at Week 52 (OR, 1.34; 95% CI, 1.14–1.58; P for trend = .001) and was significantly higher in patients with endoscopic response than in patients without endoscopic response at Weeks 26 and 52 (P < .001). Conclusions In a post hoc analysis of data from a randomized controlled trial of patients with moderate to severe CD, we found that adalimumab in combination with azathioprine increased trough levels of adalimumab. Higher trough levels of adalimumab associated with endoscopic response and mucosal healing at Weeks 26 and 52. UMIN registration No: 000005146.

M1114 Evaluation of Gastric Mucosal Blood Flow Using Contrast-Enhanced Ultrasonography During Low-Dose Aspirin Therapy and During NSAID and Low-Dose Aspirin Combined Therapy

Background: Low-dose aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) are now used extensively for prevention of vascular disorders or treatment of chronic pain. The frequency of gastric mucosal lesions is expected to increase with combined use of these medicines. A histamine-2 receptor antagonist (H2RA) suppresses development of acid secretion and is expected to prevent development of gastric mucosal lesions associated with NSAIDs and low-dose aspirin. Evaluation of gastrointestinal blood flow is important for understanding the pathogenesis of various gastrointestinal diseases. Aim: we used a noninvasive examination method, transabdominal contrast-enhanced ultrasonography (low mechanical harmonic index imaging), and assessed gastric mucosal blood flow during low-dose aspirin therapy and combined therapy with low-dose aspirin and an NSAID (loxoprofen) and then investigated the effect of an H2RA (lafutidine) on gastric mucosal blood flow. Subjects and methods: The subjects were sixteen healthy volunteers without H. pylori infection. Their mean age was 24.4 years. Subjects in the placebe took low-dose aspirin (81mg) once a day for 14 days (days 1-14), 60 mg of loxoprofen three times a day for 7 days (days 8-14), and a placebo twice a day for 14 days (days 1-14). Subjects in the lafutidine followed the same protocol for low-dose aspirin and loxoprofen but took 10 mg of lafutidine twice a day for 14 days (days 1-14). We carried out contrast-enhanced ultrasonography with perflubutane on day 1, day 8, day 15 and measured gastric mucosal blood flow of the anterior wall of the gastric antrum. A time-intensity curve (TIC) for gastric mucosal blood flow was plotted from recorded ultrasonographic images. Three values were calculated from the TIC: AUC (area under the curve), TIC peak value and TIC peak time. We used these three values to measure gastric mucosal blood flow. Results: The AUC decreased significantly after taking low-dose aspirin (day 8) in both the placebo and lafutidine groups. However, no statistically significant decrease in the AUC was found between day 8 and day 15. The results for TIC peak value were the same as those for AUC. No significant extension of TIC peak time was shown between day 1, day 8 and day 15. No significant difference in AUC, TIC peak value and TIC peak time was found between the placebo and laftidine groups. Conclusion: Low-mechanical index imaging with perflubutane enables noninvasive assessment of gastric mucosal blood flow in real time. The use of this method in a daily primary care setting is expected to lead to prevention and early detection of gastric mucosal injury caused by aspirin and NSAIDs.

Evaluation of amnion-derived mesenchymal stem cells for treatment-resistant moderate Crohn’s disease: study protocol for a phase I/II, dual-centre, open-label, uncontrolled, dose–response trial

Introduction The medical treatment options for patients with Crohn’s disease (CD) are limited and patients resistant to those therapies are left requiring surgical operations that usually only achieve some symptomatic relief. Mesenchymal stem cells (MSC) have been shown to be effective for the treatment of CD, and we have demonstrated in animal experiments that human amnion-derived MSCs (AMSC) are a potential new therapeutic strategy. Therefore, we designed this study to investigate the safety and efficacy of AMSCs in patients with treatment-resistant CD. Methods and analysis This is the protocol for an ongoing phase I/II, dual-centre, open-label, uncontrolled, dose–response study. The estimated enrolment is 6–12 patients with treatment-resistant, moderate CD. A dose of 1.0×106 cells/kg will be administered intravenously in the low-dose group at days 0 and 7. After confirming the safety of low-dose administration, a dose of 4.0×106 cells/kg will be administered intravenously in the high-dose group on days 0 and 7. The primary endpoint will measure the occurrence of adverse events related to acute infusion toxicity, and secondary endpoints will include long-term adverse events and efficacy of AMSC administration. Ethics and dissemination The Institutional Review Board of Hokkaido University Hospital approved this study protocol (approval number H29-6). A report releasing study results will be submitted to an appropriate journal. Discussion This study is the first to investigate the safety and efficacy of AMSC use for CD treatment. Our results will advance studies on more efficient and convenient methods to overcome the limits of available CD treatments. Trial registration number UMIN000029841.

PWE-119 Decreased Frequency Of Peripheral And Intestinal Nkg2a-positive T Cells In Ulcerative Colitis Patients

Introduction The intestinal tract is home to a numerous immune cellular components that continuously encounter abundant exogenous stimuli, where remain in a state of controlled inflammation normally. Inflammatory bowel disease (IBD) is characterised by chronic and refractory intestinal inflammation complising ulcerative colitis (UC) and Crohn’s disease (CD) in main. Inhibitory natural killer receptors (iNKRs) are expressed on NK cells, involved in NK-cell tolerance to self. There is increasing evidence that iNKRs like NKG2A expressed on T cells are importantly involved in the regulation of immune responses though no studies have addressed the potential role in the pathogenesis of IBD. We analysed the expression of NKG2A on T cells in dextran sulfate sodium (DSS)-induced colitis model mouse and IBD patients. Methods Male BALB/c mice were administrated 5% DSS in distilled water for 7 days ad libitum for induction of experimental colitis. For controls, age-matched BALB/c mice were given distilled water. NKG2A+ T cells in peripheral blood mononuclear cells (PBMCs) and lamina propria mononuclear cells (LPMCs) were analysed by flow cytometry. For histological analysis of DSS-induced colitis mice, hematoxylin and eosin (HandE) staining and immunohistochemistry were performed. For blocking experiments using antibody (Ab), mice were intraperitoneally injected with 300 µg of anti-NKG2A monoclonal Ab or control IgG after oral administration of 5% DSS. PBMCs samples from 23 healthy controls, 20 UC and 16 CD patients were analysed by flow cytometry. Tissue sections from 7 controls, 6 UC and 5 CD patients were subjected to histological analysis. Data were evaluated using the Student’s t-test or the Mann-Whitney U-test. Results In the active phase of DSS-induced colitis mouse, the frequency of NKG2A+ T cells was significantly decreased in the peripheral blood and increased in the intestine, suggesting the mobilisation of this T cell subset to the sites of inflammation. Administration of anti-NKG2A Ab increased the number of inflammatory foci in DSS-induced colitis, suggesting the involvement of NKG2A+ T cells in this colitis model. In UC patients, the frequency of NKG2A+ T cells in PBMCs was significantly decreased, compared with CD patients and healthy controls, regardless of clinical conditions. Notably, in sharp contrast to the DSS-induced colitis mouse, the frequency of NKG2A+ cells in LPMCs was also decreased in UC patients. Conclusion These results suggest that inadequate local infiltration of NKG2A+ T cells may be involved in the pathogenesis of UC. Our study demonstrates the frequency of NKG2A+ T cells is decreased in both PBMCs and LPMCs in UC patients, implicating this T cell subset as a potential therapeutic target for UC. Disclosure of Interest None Declared.

A case of toxic epidermal necrolysis with extensive intestinal involvement.

Toxic epidermal necrolysis (TEN) is life-threatening cutaneous adverse drug reaction characterized by extensive keratinocyte death. Gastrointestinal (GI) involvement in TEN is rare, however it contributes to poor prognosis. Here, we report a case of TEN with extensive intestinal involvement. A 56-year-old female was treated by zonisamide for fibromyalgia. Ten days later, generalized erythema appeared with blister, superficial punctate keratitis, and severe diarrhea. Skin biopsy specimen revealed that marked keratinocyte death. She was diagnosed with Stevens-Johnson syndrome (SJS). In spite of methylprednisolone pulse therapy for three days and plasma exchange three times, the bullous lesions spread to cover 60% of the body surface area. She was finally diagnosed with TEN, and treated with high-dose intravenous immunoglobulins for three days and another methylprednisolone pulse therapy. After these therapies, skin lesions were gradually improved. Despite skin manifestation improvement, the patients diarrhea persisted (2000ml/day), resulting in hypoalbuminemia (0.8g/dL). Upper endoscopy revealed extensive erosions in esophagus and duodenum. Colonoscopy revealed multiple erosions and edematous structures in distal ileum and colon. Biopsy specimens of ileum and colon showed extensive mucosal detachment and granulation tissues. Examinations of Clostridium difficile toxin and cytomegalovirus antigen were negative. According to these findings, we considered her diarrhea to be consistent with GI lesions related in TEN. We treated her with mucoprotective agent, total parenteral nutrition management, albumin supplementation, and tapered systemic corticosteroid slowly. GI symptoms were gradually improved. Approximately 15 cases of SJS/TEN with GI manifestations have been reported in English literature. The mortality rate of SJS/TEN with GI manifestations is about 45%. There is no specific treatment, and treatment has largely been supportive. GI symptoms are often delayed, as in our case. GI mucosal damage may result in enteral nutrition difficulty, hypoalbuminemia, poor wound healing and bacterial translocation. The prolonged intestinal lesions may be attributable to difficulties with re-epithelization of intestinal mucosa by digestive fluid and formation of granulation tissue. Intestinal involvement of SJS/TEN is refractory and life-threatening complication. Regardless of the severity of skin manifestations, we should carefully monitor the GI symptoms.