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Background The mortality and risk factors of severe disease and death due to arterial and venous thromboembolism (ATE and VTE, respectively) in patients with inflammatory bowel disease (IBD) remain unclear, especially in Asia. Aims This study aimed to reveal the mortality and risk factors of TE in IBD patients in Japan. Methods In the primary surveillance, responses to questionnaires regarding the number of cases of severe TE and TE-associated death in IBD patients in a span of over the past 10 years were obtained from 32 institutions in Japan. In the secondary surveillance, detailed data about IBD patients with TE were collected. The characteristics, laboratory data, therapy status, and situation at the time of TE development were retrospectively collected, and the data were compared between the patients with and without severe TE and TE-associated death. Results The incidence of TE was 1.89% among 31,940 IBD patients. The frequencies of severe TE and TE-associated mortality were 10.7% and 1.0% among the total IBD and TE with IBD patients, respectively. The only risk factor for severe ATE and ATE-associated death was ischemic heart disease. The independent risk factors for severe VTE and VTE-associated death were age (≤ 45 years old), the site of VTE, and disease severity, with anti-TNF therapy as a potential negative risk factor. Patients with severe VTE had a high risk of developing persistent VTE and sequelae. Conclusion Unlike ATE, the incidence of VTE was comparable in Asian and Western countries. Therapeutic and prophylactic strategies for managing IBD-associated TE in Asia are urgently needed.

Background Given the increasing health concerns for patients with inflammatory bowel disease (IBD), amidst the COVID-19 pandemic, we investigated the impact of the pandemic on the anxiety and behavioral changes in Japanese patients with IBD. Methods We analyzed 3032 questionnaires from patients with IBD, aged 16 years or older visiting 30 hospitals and 1 clinic between March 2020 and June 2021. The primary outcome was the score of the anxiety experienced by patients with IBD during the pandemic. Results Participants reported a median age of 44 years; 43.3% of the patients were women. Moreover, 60.6% and 39.4% were diagnosed with ulcerative colitis and Crohn’s disease, respectively, with a median disease duration of 10 years. Participants indicated an average of disease-related anxiety score of 5.1 ± 2.5 on a ten-point scale, with a tendency to increase, 1 month after the number of infected persons per population increased. The top three causes for anxiety were the risk of contracting COVID-19 during hospital visits, SARS-CoV-2 infection due to IBD, and infection by IBD medication. Factors associated with anxiety were gender (women), being a homemaker, hospital visit timings, mode of transportation (train), use of immunosuppressive drugs, and nutritional therapy. Most patients continued attending their scheduled hospital visits, taking their medications, experienced the need for a family doctor, and sought guidance and information regarding COVID-19 from primary doctors, television, and Internet news. Conclusions Patients with IBD experienced moderate disease-related anxiety due to the pandemic and should be proactively informed about infectious diseases to relieve their anxiety.

Background This multicenter prospective study (UMIN000019958) aimed to evaluate the usefulness of serum leucin-rich alpha-2 glycoprotein (LRG) levels in monitoring disease activity in inflammatory bowel disease (IBD). Methods Patients with moderate-to-severe IBD initiated on adalimumab therapy were enrolled herein. Serum LRG, C-reactive protein (CRP), and fecal calprotectin (fCal) levels were measured at week 0, 12, 24, and 52. Colonoscopy was performed at week 0, 12, and 52 for ulcerative colitis (UC), and at week 0, 24, and 52 for Crohn’s disease (CD). Endoscopic activity was assessed using the Simple Endoscopic Score for Crohn’s Disease (SES-CD) for CD and the Mayo endoscopic subscore (MES) for UC. Results A total of 81 patients was enrolled. Serum LRG levels decreased along with improvements in clinical and endoscopic outcomes upon adalimumab treatment (27.4 ± 12.6 μg/ml at week 0, 15.5 ± 7.7 μg/ml at week 12, 15.7 ± 9.6 μg/ml at week 24, and 14.5 ± 6.8 μg/ml at week 52), being correlated with endoscopic activity at each time point (SES-CD: r = 0.391 at week 0, r = 0.563 at week 24, r = 0.697 at week 52; MES: r = 0.534 at week 0, r = 0.429 at week 12, r = 0.335 at week 52). Endoscopic activity better correlated with LRG compared to CRP and fCal on pooled analysis at all time points (SES-CD: LRG: r = 0.636, CRP: r = 0.402, fCal: r = 0.435; MES: LRG: r = 0.568, CRP: 0.389, fCal: r = 0.426). Conclusions Serum LRG is a useful biomarker of endoscopic activity both in CD and UC during the adalimumab treatment.

Although indigo naturalis (IN) is effective for patients with active ulcerative colitis (UC), IN was associated with adverse events (AEs), including pulmonary arterial hypertension (PAH). Our aim was to evaluate the occurrence of IN-associated AEs and to evaluate any IN dose–effect on AEs. A nationwide survey, using questionnaires, was conducted by conducted by the research group funded by the Ministry of Health, Labour and Welfare of Japan, between June 2017 and September 2018. A first questionnaire determined the occurrence of AEs associated with the therapeutic use of IN or herbal medicines containing IN in patients with UC. A second survey identified the clinical characteristics of patients who developed IN-associated critical AEs, namely, liver dysfunction, PAH, and intussusception. Across 337 participating institutions, 49,320 patients with UC were identified, with IN used in 877 (1.8%). AEs were reported in 91 patients (107 events), including liver dysfunction (n = 40), gastrointestinal symptoms (n = 21), headache (n = 13), and PAH (n = 11). No dose–effect relationship between IN and AEs was identified. Liver dysfunction tended to be mild and reversible. Ten cases of intussusception were reported, with 40% of these patients requiring surgical resection. IN-induced PAH was recovered in patients who discontinued to use IN. No IN-associated deaths were reported. IN-associated AEs were identified among patients with UC, with liver dysfunction often being reversible, while surgical resection was required in a high proportion of patients who developed intussusception. Both healthcare workers and patients should adequately recognize the potential for AEs with the use of IN.

Environmental factors are suggested to affect the pathogenesis of several diseases, including inflammatory bowel disease (IBD). The seasonality of disease onset and exacerbation in IBD, however, are not well established. We herein aimed to clarify the disease seasonality and to investigate the underlying characteristics in IBD patients exhibiting seasonality of the disease course. This was a multicenter observational study comprising 20 institutions (Osaka Gut Forum) in Japan. Data were collected from November 2013 to August 2014 using survey forms for physicians and questionnaires for patients. Multivariate analysis was performed to clarify the independent factors affecting disease seasonality. A total of 1055 patients, including 298 patients with Crohn’s disease (CD) and 757 patients with ulcerative colitis (UC), were enrolled. The proportion of CD patients with disease onset in the summer was significantly larger than that in the other seasons, while UC patients exhibited no seasonality of disease onset. More than half of the IBD patients (51.1%) experienced seasonal exacerbation of IBD, and winter was the most common season for disease exacerbation in both CD and UC patients. Seasonality of disease onset and exacerbation was observed in young-onset patients (≤40 years old), but not in elderly-onset patients. Age at onset was independently associated with the seasonality of both disease onset and exacerbation. Seasonality of disease onset and exacerbation was observed especially in young-onset IBD patients. Underlying pathophysiologic triggers for disease initiation and exacerbation may be influenced by age at disease onset.

Indigo Naturalis (IN) is used as a traditional herbal medicine for ulcerative colitis (UC). However, the mechanisms of action of IN have not been clarified. We aimed to evaluate the efficacy of IN for ameliorating colonic inflammation. We further investigated the mechanisms of action of IN. Colitis severity was assessed in dextran sodium sulfate-induced colitis and trinitrobenzene sulfonic acid-induced colitis models with or without the oral administration of IN or indigo, which is a known major component of IN. Colonic lamina propria (LP) mononuclear cells isolated from IN-treated mice were analyzed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and flow cytometry. LP and splenic mononuclear cells cultured in vitro with IN or indigo were also analyzed. The role of the candidate receptor for indigo, the aryl hydrocarbon receptor (AhR), was analyzed using Ahr-deficient mice. Colitis severity was significantly ameliorated in the IN and indigo treatment groups compared with the control group. The mRNA expression levels of interleukin (Il)-10 and Il-22 in the LP lymphocytes were increased by IN treatment. The treatment of splenocytes with IN or indigo increased the expression of anti-inflammatory cytokines and resulted in the expansion of IL-10-producing CD4+ T cells and IL-22-producing CD3−RORγt+ cells, but not CD4+Foxp3+ regulatory T cells. The amelioration of colitis by IN or indigo was abrogated in Ahr-deficient mice, in association with diminished regulatory cytokine production. IN and indigo ameliorated murine colitis through AhR signaling activation, suggesting that AhR could be a promising therapeutic target for UC.

Oligosaccharide structures and their alterations have important roles in modulating intestinal inflammation. N-Acetylglucosaminyltransferase V (GnT-V) is involved in the biosynthesis of N-acetylglucosamine (GlcNAc) by β1,6-branching on N-glycans and is induced in various pathologic processes, such as inflammation and regeneration. GnT-V alters host immune responses by inhibiting the functions of CD4+ T cells and macrophages. The present study aimed to clarify the role of GnT-V in intestinal inflammation using GnT-V transgenic mice. Colitis severity was compared between GnT-V transgenic mice and wild-type mice. β1,6-GlcNAc levels were investigated by phytohemagglutinin-L4 lectin blotting and flow cytometry. We investigated phagocytosis of macrophages by measuring the number of peritoneal-macrophage-ingested fluorescent latex beads by flow cytometry. Cytokine production in the culture supernatant of mononuclear cells from the spleen, mesenteric lymph nodes, and bone-marrow-derived macrophages was determined by enzyme-linked immunosorbent assay. Clodronate liposomes were intravenously injected to deplete macrophages in vivo. Chronic-colitis-associated tumorigenesis was assessed after 9 months of repeated administration of dextran sodium sulfate (DSS). DSS-induced colitis and colitis induced by trinitrobenzene sulfonic acid were markedly exacerbated in GnT-V transgenic mice compared with wild-type mice. Production of interleukin-10 and phagocytosis of macrophages were significantly impaired in GnT-V transgenic mice compared with wild-type mice. Clodronate liposome treatment to deplete macrophages blocked the exacerbation of DSS-induced colitis and impairment of interleukin-10 production in GnT-V transgenic mice. Chronic-colitis-associated tumorigenesis was significantly increased in GnT-V transgenic mice. Overexpression of GnT-V exacerbated murine experimental colitis by inducing macrophage dysfunction, thereby enhancing colorectal tumorigenesis.

Treatment with endoscopic submucosal dissection (ESD) for gastric noninvasive neoplasia (NIN) diagnosed by endoscopic forceps biopsy specimen, whether as a follow-up or "total incisional biopsy", is controversial. To validate the use of ESD for total incisional biopsy in NIN, we examined the underdiagnosis rate of NIN and the rates of complication associated with ESD.This is a cross-sectional multicenter retrospective study from 10 hospitals. Subjects diagnosed with NIN (equivalent to category 3 or 4.1 of the Vienna classification) by endoscopic forceps biopsy and treated with ESD were included. From March 2003 to December 2009, a total of 468 subjects were included and analyzed. The underdiagnosis rate was defined as the proportion of lesions diagnosed with adenocarcinoma after ESD. We assessed the complete en-bloc resection rate and the complication rate of ESD.Among the 468 subjects with NIN, 205 were diagnosed with adenocarcinoma after ESD, with an underdiagnosis rate of 44% (95% confidence interval: 39-49%). Two submucosal cancer lesions had invaded beyond 500 μm and one had lymphatic involvement. The complete en-bloc resection rate was 97%. The incidences of post-ESD bleeding, perforation, and serious complications were 5.5, 4.7, and 0.43%, respectively. There were no procedure-related deaths.In this large-scale, multicenter cross-sectional study, over 40% of the noninvasive gastric neoplasia specimens were determined to have adenocarcinoma, and the ESD-related complication rate was relatively low. Therefore, ESD was useful and may be a therapeutic option for gastric NIN.

Double-balloon endoscopy (DBE) examinations are not yet widely accepted as routine procedures for examining the small bowel of patients with Crohn's disease (CD).To evaluate the feasibility and usefulness of DBE for CD in tertiary-care hospitals.Between July 2004 and September 2008, 1444 DBE procedures were performed for 704 patients in 6 tertiary-care hospitals. Patient profile, indication, diagnosis and treatment of DBE were evaluated using a multicenter database.DBE examinations were most frequently performed in 75 patients with CD, corresponding to 10.5% of all the patients examined by DBE. Fifty patients were diagnosed with CD before DBE, while DBE was performed for the diagnosis of 25 new CD patients. Small bowel lesions were often detected even when the terminal ileum was not involved. In the 75 patients, 21 patients were asymptomatic at the time of DBE examinations. Active inflammatory lesions were detected in 51.2% of the CD patients, and were even detected in 33.3% of the asymptomatic CD patients. The treatment was altered in 53.3% of the CD patients after the DBE evaluation. No severe complications were experienced.DBE procedures can be safely performed in patients with CD and should be considered for the precise evaluation of and to determine the treatment strategy for CD.

Interstitial cells of Cajal (ICC) play important roles in autonomic gut motility as electrical pacemakers and mediators of neural regulation of smooth muscle functions. Insufficiency of ICC has been reported in a wide range of gut dysmotilities. Thus, restoration of ICC may be a therapeutic modality in these diseases. Here we provide evidence that transplanted bone marrow (BM) cells can restore gut dysmotility in part via transdifferentiation to ICC. Bone marrow cells obtained from Kit insufficient W/W v mice or syngeneic GFP-transgenic mice with wild-type Kit were transferred to W/W v recipients. Whole gut transit time and gastric emptying were examined 5 and 6 weeks after BM transplantation, respectively, and ICCs were identified in whole mounts, frozen sections and transmission electron immunomicroscopy of the gut smooth muscle layers using specific antibodies. Transplantation of wild-type BM into W/W v mice significantly improved whole gut transit time and gastric emptying. Fluorescent immunohistochemistry revealed GFP+Kit+ cells in the myenteric plexus, deep muscular plexus, and submucosal plexus smooth muscle layers of the stomach, small intestine, and colon, respectively. In the whole mounts, GFP+Kit+ cells were bipolar and spindle shaped, and transmission electron immunomicroscopy showed GFP+ cells rich in mitochondria and endoplasmic reticulum between gut smooth muscle layers, suggesting the presence of GFP+ cells with morphological characteristics of ICC. These results suggest that BM contains cells that may incorporate into ICC networks and improve dysmotility in W/W v mice. Thus, BM transplantation may become to a new therapeutic modality for gut dysmotilities due to ICC insufficiency.

Bone marrow contains hematopoietic stem cells, nonhematopoietic mesenchymal stem cells, and several precursor cells for osteoblasts, chondrocytes, adipocytes, myocytes, hepatocytes, and even neural cells. Research findings indicate that multipotent stem cells in the adult body may be used to recover the lost functions of damaged tissues. This study examined the involvement of bone marrow-derived cells in the regeneration of the stomach after experimental gastric ulcers were produced in rats. We transplanted the bone marrow of transgenic rats that expressed green fluorescence protein (GFP) throughout the body. Twenty-one days after the bone marrow transplantation (BMT), gastric ulceration was induced, using absolute ethanol. Control animals received saline. After various observation periods, rats harboring GFP-positive bone marrow-derived cells were killed, and the tissues were removed and processed to prepare paraffin-embedded sections. Cells expressing GFP were identified by conventional immunohistochemistry, using anti-GFP antibody. To identify whether cells expressing GFP were epithelial cells or interstitial cells such as fibroblasts, serial sections were examined with anti-cytokeratin antibody or anti-vimentin antibody, respectively. Furthermore, to confirm that cells expressing GFP were epithelial cells or interstitial cells, we used double-staining analysis with anti-GFP antibody or anti-cytokeratin antibody, respectively. GFP-positive, bone marrow-derived cells were found in the cytokeratin-positive gastrointestinal epithelium, as well as among vimentin-positive interstitial cells. Interestingly, the proportions of GFP-positive, cytokeratin-positive epithelial cells and vimentin-positive interstitial cells were significantly greater in the ethanol-treated damaged stomachs than in the saline-treated controls. The present study clearly demonstrates that bone marrow-derived cells are involved in the regeneration of the stomach after ethanol-induced ulcers in rats.

Patients with inflammatory bowel disease (IBD) often exhibit vitamin K deficiency. Vitamin K has been shown to inhibit inflammation via interleukin (IL)-6 suppression. This study aimed to evaluate the effect of vitamin K in a murine model of colitis.Colitis was induced using dextran sulfate sodium (DSS) in mice fed either a vitamin K-deficient (K-def) or a vitamin K-supplemented (K-sup) diet. The clinical and histological severity of colitis was assessed, and levels of cytokine production from the spleen and colonic lamina propria were measured by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction. Cytokine expression levels in CD4(+), CD11b(+), and CD19(+) cells in the presence and absence of vitamin K [menatetrenone (MK-4)] were measured in vitro and apoptosis was determined by caspase 3/7 activity and Annexin V staining.DSS administration resulted in significantly more severe body weight loss, shorter colon length, and higher histological scores in mice fed a K-def diet than those fed a K-sup diet. IL-6 expression in lamina propria mononuclear cells was significantly higher in the K-def group than in the K-sup group. IL-6 expression was significantly decreased in the presence of MK-4 in CD19(+) cells, but not in the CD4(+) and CD11b(+) subpopulations. Apoptotic cell population in CD19(+) cells was increased in the presence of MK-4 in vitro and in vivo.Vitamin K exerts a protective effect against DSS colitis; this effect is associated with IL-6 downregulation. Vitamin K could be a potential treatment target for IBD.

Abrogating tolerance is a critical step in the pathogenesis of Crohn’s disease (CD). T cell-anergy is one of the main mechanisms of tolerance and is regulated by the gene related to anergy in lymphocytes (GRAIL). This study investigated the expressions and regulation of GRAIL in CD and murine colitis models. Expressions of GRAIL mRNA and protein in CD4+ T cells were investigated in the peripheral blood and mucosal tissues of patients with CD, mice with dextran sodium salt (DSS)-induced colitis, and Il-10-deficient mice. MicroRNAs responsible for the regulation of GRAIL were examined by miRNA microarray. GRAIL-overexpressing T cells were intravenously injected in mice with DSS-induced colitis. The GRAIL expression was higher in the lamina propria (LP) CD4+ T cells of CD patients than of the control subjects, while it was lower in the peripheral blood CD4+ T cells of the CD patients than of the control subjects. The GRAIL mRNA expression was lower, but the GRAIL protein expression was higher in the LP of colitic mice than that of non-colitic mice. The miRNA microarray identified miR-290-5p as an miRNA that inhibits expression of the GRAIL protein and that is highly expressed in the LP of non-colitic mice. GRAIL-expressing T cells expressed regulatory T cell markers and showed suppressive effects in murine DSS-induced colitis. Our results show that expression of GRAIL is uniquely regulated by the specific miRNA in the intestinal mucosa, and suggest that GRAIL may associate with the pathophysiology of CD.

The therapeutic potential of bone marrow (BM)-derived cells in ulcers is not known. This study aimed to clarify (1) cell types that are derived from the BM which infiltrate ulcers; (2) whether BM-derived cells or gastric myofibroblasts can be used for cell transplantation to treat ulcers; and (3) the phenotypes of such transplantable cells.(1) Wild-type mice were transplanted with BM cells of green fluorescent protein (GFP)-transgenic mice. Acetic acid-induced gastric ulcers were produced in mice after BM transplantation. (2) BM cells and gastric myofibroblasts were isolated from GFP-transgenic mice. Bone marrow cells attached to plastic dishes were selected for expansion. Gastric ulcers were induced, and BM-derived cells, myofibroblasts, or phosphate-buffered saline were injected around ulcers. The ulcer healing process was examined macroscopically and histologically. (3) Expression of growth factors and cytokines in transplantable cells was examined by reverse transcriptase-polymerase chain reaction.(1) GFP-positive cells with interstitial phenotypes were observed at the ulcerated area. (2) Ulcer healing was significantly promoted by the injection of BM-derived cells compared to controls on day 7, but not on day 3. The BM-derived cells were observed in the tissue surrounding the ulcer. However, myofibroblasts were not found. (3) The BM-derived cells expressed hepatocyte growth factor, transforming growth factor-beta(1), and other stromal factors before transplantation, and had mesenchymal phenotypes after transplantation.BM-derived cells are involved in the ulcer healing. BM-derived cells, but not myofibroblasts, are locally implantable to ulcers. Thus, BM-derived cells can be transplanted to accelerate ulcer healing.