Tomoki Aomatsu

Comparison of the fecal microbiota profiles between ulcerative colitis and Crohn’s disease using terminal restriction fragment length polymorphism analysis

BackgroundTerminal restriction fragment length polymorphism (T-RFLP) analysis is a powerful tool to assess the diversity of a microbial community. In this study, we performed T-RFLP analysis of the fecal microbiota from patients with ulcerative colitis (UC) and those with Crohn’s disease (CD).MethodsThirty-one patients with UC, 31 patients with CD, and 30 healthy individuals were enrolled. The polymerase chain reaction (PCR) products obtained from the 16S rRNA genes of fecal samples were digested with BslI, and T-RF lengths were determined.ResultsThe fecal microbial communities were classified into 5 clusters. Twenty-eight of the 30 healthy individuals and 17 of the 18 patients with inactive UC were classified into clusters I, II, and III, but these clusters included a small number of patients with active UC and inactive/active CD. In contrast, 8 of the 13 patients with active UC and the majority of CD patients (12 of the 16 patients with inactive CD, and 11 of the 15 patients with active CD) were included in clusters IV and V. Based on the BslI-digested T-RFLP database, the bacteria showed a significant decrease in the Clostridium family in patients with active UC and inactive/active CD. In contrast, Bacteroides were significantly increased in CD patients. No significant differences were observed between patients with active UC and those with active CD.ConclusionThe fecal microbial communities of IBD patients were different from those of healthy individuals. The gut microbiota of patients with inactive UC tended to be closer to that of healthy individuals, suggesting different roles for the fecal microbiota in the pathophysiology of UC and CD.

Interleukin-33 suppresses Notch ligand expression and prevents goblet cell depletion in dextran sulfate sodium-induced colitis

Interleukin (IL)-33 is a cytokine belonging to the IL-1 family. IL-33 plays an important role in Th2 immune responses, and induces goblet cell hyperplasia in the intestinal mucosa. In this study, to elucidate the molecular mechanisms underlying IL-33-induced goblet cell hyperplasia, we investigated how IL-33 modulates the Notch signaling pathway in dextran sulfate sodium (DSS)-induced experimental colitis. DSS colitis was induced in BALB/c mice with intraperitoneal administrations of IL-33 (1 µg/body) every 48 h. Tissue samples were evaluated by standard immunohistochemical procedures. The mucosal mRNA expression of the Notch ligands was analyzed by a real-time polymerase chain reaction. The mucosal mRNA expression of Notch ligands [Jagged1 (Jag1) and Delta-like (Dll) 1 and 4] was significantly increased in DSS-colitis mice. IL-33-induced goblet cell hyperplasia in the control mice. In the DSS-colitis mice, the goblet cells were depleted in the colon, but IL-33 completely prevented goblet cell depletion in the DSS-colitis mice. IL-33 induced a significant decrease in Jag1 and Dll4 mRNA expression in the mucosa of the control mice. Mucosal mRNA expression for Jag1, Dll1 and 4 was significantly elevated in the DSS-colitis mice, but this elevation was significantly blocked by the administration of IL-33. IL-33 dose-dependently decreased Jag1 mRNA expression in mouse colonic subepithelial myofibroblasts. In contrast to its preventive effects on goblet cell depletion, IL-33 aggravated DSS colitis. IL-33 prevented goblet cell depletion via its inhibitory actions against Notch ligand expression in DSS colitis, but exacerbated the disease activity. IL-33 plays two counter actions in mucosal inflammation; the first is a protective action via goblet cell induction, and the second is a pro-inflammatory action as a Th2 cytokine.

Febrile seizures associated with influenza A

To clarify the clinical impact of influenza A on the development of febrile seizures (FS), consecutive FS patients brought to our hospital between October 2003 and September 2004 were prospectively surveyed. Patients infected with influenza A (influenza A patients) and those uninfected with influenza (non-influenza patients) were compared with regard to clinical characteristics of FS. Influenza infection was determined by rapid antigen test and/or serologically. Associations of influenza A with atypical findings of FS, including partial seizures, prolonged seizures, multiple seizures during the same illness, and 30-min or longer prolonged postictal impairment of consciousness (PPIC), were analyzed by multiple logistic regression. A total of 215 patients (47 influenza A and 168 non-influenza patients) were enrolled in the study. Age was significantly higher in the influenza A group (39.85+/-22.16 months vs. 27.51+/-17.14 months, P<0.001). Of 42 patients aged 48 months or older, which corresponded to the 80th percentile for age, 15 (35.7%) were influenza A patients, with a significantly higher incidence of such patients than in the subgroup of patients aged 47 months or younger (32/173, 18.5%) (P=0.015). On multiple logistic regression analysis, influenza A was independently associated with PPIC (odds ratio: 4.44, 95% confidence interval: 1.52-12.95, P=0.006), but not with other atypical findings. The positive association of influenza A with PPIC suggests that influenza may affect state of consciousness at the same time that it induces seizures with fever.

Faecal chitinase 3-like-1: a novel biomarker of disease activity in paediatric inflammatory bowel disease

Aliment Pharmacol Ther 2011; 34: 941–948

Terminal Restriction Fragment Length Polymorphism Analysis of the Gut Microbiota Profiles of Pediatric Patients with Inflammatory Bowel Disease

Background/Aim: We analyzed the fecal microbiota profiles of pediatric patients with inflammatory bowel disease. Method: Terminal restriction fragment length polymorphism analysis was performed in 10 fecal samples from Crohn’s disease (CD), 14 samples from ulcerative colitis (UC) and 27 samples from healthy individuals. The bacterial diversity was evaluated by the Shannon diversity index. Result: In CD patients, a setting of similarity generated three major clusters. The majority of CD patients were classified into CD clusters I and II (9 out of 10), but the majority of healthy individuals (21 of 27) were classified into CD cluster III. In UC patients, a setting of similarity also generated three major UC clusters, but each cluster was not characteristic for UC patients or healthy individuals. The changes in simulated bacterial composition indicated that the class Clostridia, including the genus Faecalibacterium, was significantly decreased in CD patients as compared to UC patients and/or healthy individuals. The genus Bacteroides was also decreased as compared to healthy individuals. The bacterial diversity measured by the Shannon diversity index was significantly reduced in CD patients as compared to healthy individuals. Conclusion: The gut microbiota profile of pediatric CD patients was different from that of healthy children.

Tacrolimus (FK506) suppresses TNF-α-induced CCL2 (MCP-1) and CXCL10 (IP-10) expression via the inhibition of p38 MAP kinase activation in human colonic myofibroblasts.

In order to investigate the molecular mechanisms underlying the immunosuppressive effects of tacrolimus (FK506) on intestinal inflammation, we examined whether FK506 effects cytokine/chemokine secretion in human colonic myofibroblasts. Human colonic myofibroblasts were isolated from normal human colonic tissue. The mRNA and protein expression for human CCL2 and CXCL10 were analyzed by real-time PCR and ELISA, respectively. p38 MAP kinase activation was evaluated by western blotting. Tacrolimus (1 µM) suppressed tumor necrosis factor (TNF)-α-induced CCL2 and CXCL10 mRNA expression, but did not modulate TNF-α-induced interleukin (IL)-6 or CXCL8 mRNA expression. Dose-dependent, inhibitory effects of tacrolimus on CCL2 and CXCL10 expression were observed at the mRNA and protein levels. Significant inhibitory effects of tacrolimus were observed at concentrations as low as 0.5 µM for CCL2 and 0.1 µM for CXCL10, respectively. TNF-α-induced CCL2 and CXCL10 expression depended on p38 MAP kinase activation, and tacrolimus strongly inhibited the TNF-α-induced phosphorylation of p38 MAP kinase. Tacrolimus did not affect interferon (IFN)-γ-induced signaling transducer and activator of transcription (STAT)-1 phosphorylation, nor did it modulate CXCL10 mRNA and protein expression. In conclusion, tacrolimus suppressed CCL2 and CXCL10 expression in human colonic myofibroblasts. These inhibitory effects of tacrolimus may play key roles in the therapeutic effects of colonic inflammation in inflammatory bowel disease (IBD) patients.

Fulminant hepatitis B and acute hepatitis B due to intrafamilial transmission of HBV after chemotherapy for non-Hodgkin’s lymphoma in an HBV carrier

Hepatitis B virus (HBV) reactivation after chemotherapy has been investigated, but little is known about the risk of horizontal transmission from an immunocompromised host with HBV reactivation. We treated two children with fulminant hepatitis B and acute hepatitis B, respectively, whose grandmother, an HBV carrier, had been undergoing rituximab plus cyclophosphamide, doxorubicin, vincristine, and prednisone (R+CHOP) therapy for lymphoma. The grandmother was also suffering from fulminant hepatitis when both children became ill. The complete HBV DNA sequences of the three family members were identical. The full genome sequence analysis of HBV provided strong evidence of intrafamilial transmission of HBV. Treatments that cause immunosuppression, such as R+CHOP therapy for lymphoma, can increase the levels of serum HBV DNA and the risk of intrafamilial HBV infection when given to HBV carriers. In conclusion, specific antiviral prophylaxis is indispensable for preventing horizontal transmission as well as reactivation of HBV in chemotherapy-treated HBV carriers.

Fasting and bed rest, even for a relatively short period, are risk factors for ceftriaxone-associated pseudolitiasis.

Cholelithiasis is one of the side‐effects of ceftriaxone (CTRX). Reportedly, the cholelithiasis resolves relatively soon after cessation of CTRX, hence, it is called pseudolithiasis. Previous reports have suggested that biliary pseudolithiasis can cause not only gallstone attacks, but also severe adverse events, such as cholecystitis and pancreatitis. The purpose of this study was to prospectively elucidate the risk factors and clinical features of CTRX‐associated pseudolithiasis in pediatric patients.

Chitinase 3-like 1 induces survival and proliferation of intestinal epithelial cells during chronic inflammation and colitis- associated cancer by regulating S100A9

Many host-factors are inducibly expressed during the development of inflammatory bowel disease (IBD), each having their unique properties, such as immune activation, bacterial clearance, and tissue repair/remodeling. Dysregulation/imbalance of these factors may have pathogenic effects that can contribute to colitis-associated cancer (CAC). Previous reports showed that IBD patients inducibly express colonic chitinase 3-like 1 (CHI3L1) that is further upregulated during CAC development. However, little is known about the direct pathogenic involvement of CHI3L1 in vivo. Here we demonstrate that CHI3L1 (aka Brp39) knockout (KO) mice treated with azoxymethane (AOM)/dextran sulphate sodium (DSS) developed severe colitis but lesser incidence of CAC as compared to that in wild-type (WT) mice. Highest CHI3L1 expression was found during the chronic phase of colitis, rather than the acute phase, and is essential to promote intestinal epithelial cell (IEC) proliferation in vivo. This CHI3L1-mediated cell proliferation/survival involves partial downregulation of the pro-apoptotic S100A9 protein that is highly expressed during the acute phase of colitis, by binding to the S100A9 receptor, RAGE (Receptor for Advanced Glycation End products). This interaction disrupts the S100A9-associated expression positive feedback loop during early immune activation, creating a CHI3L1hi S100A9low colonic environment, especially in the later phase of colitis, which promotes cell proliferation/survival of both normal IECs and tumor cells.

A new isoform of interleukin-32 suppresses IL-8 mrna expression in the intestinal epithelial cell line ht-29

Interleukin (IL)-32 plays a role in the pathophysiology of inflammatory bowel disease (IBD). We isolated a new isoform of the IL-32 transcript in the process of cloning the full-length IL-32 gene from human colonic subepithelial myofibroblasts (SEMFs). The expression of mRNA in the samples was assessed by RT-PCR and real-time PCR analyses. The PCR products from the IL-32 genes were ligated into the expression vector pIRESneo2. The new isoform of the IL-32 transcript (336 nucleotides) completely lacked exon 4 of the IL-32γ gene, and was 60 bp shorter than IL-32α. TNF-α induced the mRNA expression of the new IL-32 isoform in a dose- and time-dependent manner. Stable transfection of this new isoform significantly decreased TNF-α-induced IL-8 mRNA expression in HT-29 cells, but the expression of the IL-32α gene had no effect. The mRNA expression of this new isoform was significantly elevated in the inflamed mucosa of IBD patients. A new isoform of the IL-32 transcript may play an anti-inflammatory role in the inflamed mucosa of IBD.