Tokiyoshi Ayabe

Graft-versus-host disease disrupts intestinal microbial ecology by inhibiting Paneth cell production of α-defensins

Allogeneic hematopoietic stem cell transplantation (SCT) is a curative therapy for various hematologic disorders. Graft-versus-host disease (GVHD) and infections are the major complications of SCT, and their close relationship has been suggested. In this study, we evaluated a link between 2 complications in mouse models. The intestinal microbial communities are actively regulated by Paneth cells through their secretion of antimicrobial peptides, α-defensins. We discovered that Paneth cells are targeted by GVHD, resulting in marked reduction in the expression of α-defensins, which selectively kill noncommensals, while preserving commensals. Molecular profiling of intestinal microbial communities showed loss of physiologic diversity among the microflora and the overwhelming expansion of otherwise rare bacteria Escherichia coli, which caused septicemia. These changes occurred only in mice with GVHD, independently on conditioning-induced intestinal injury, and there was a significant correlation between alteration in the intestinal microbiota and GVHD severity. Oral administration of polymyxin B inhibited outgrowth of E coli and ameliorated GVHD. These results reveal the novel mechanism responsible for shift in the gut flora from commensals toward the widespread prevalence of pathogens and the previously unrecognized association between GVHD and infection after allogeneic SCT.

Efficacy of high-frequency ultrasound probes for the preoperative staging of invasion depth in flat and depressed colorectal tumors ☆ ☆☆ ★

BACKGROUND Flat and depressed nonpolypoid types of colorectal tumors have drawn much attention. Since endoscopic mucosal resection technique is available, it is of great importance to distinguish intramucosal carcinoma from invasive carcinoma because determination of the invasion depth is essential for choosing this therapy. The usefulness of high-frequency (20 MHz) ultrasound probes for preoperative staging of invasion depth in this type of colorectal tumor was evaluated. METHODS Forty-nine cases of flat and depressed tumors were examined with the ultrasound probe and diagnostic accuracy was confirmed by comparing ultrasonic images with the pathologic findings of the specimens resected either by endoscopic mucosal resection or surgical operation. RESULTS The normal colonic wall was visualized as a nine-layered structure and the muscularis mucosae was depicted in 37 (76%) of 49 cases. Flat and depressed tumors were visualized as hypoechoic lesions and the invasion depth was accurately diagnosed in 43 (88%) of 49 lesions. CONCLUSIONS High-frequency ultrasound probes proved to be useful in determining the invasion depth and therapeutic strategy in flat and depressed colorectal tumors.

Invasion depth diagnosis of depressed type early colorectal cancers by combined use of videoendoscopy and chromoendoscopy

BACKGROUND Depressed type early colorectal cancers are found less frequently than other polypoid cancers although they have a higher submucosal invasion rate. Recently videocolonoscopy and chromoendoscopy have become available and precise descriptions of these lesions are now routine. Because endoscopic mucosal resection is designated for intramucosal and focally extended submucosal (m-sm1) cancers, an evaluation of the characteristic findings indicating invasion depth with these modalities is important. METHODS Between January 1991 and March 1996, 64 depressed type early colorectal cancers were detected and treated. When a faint abnormality of the mucosa was suspected by routine videocolonoscopy, 0.1% of indigo carmine solution was sprayed on the mucosal surface (chromoendoscopy). Colonoscopic findings of m-sm1 cancers and moderately and massively extended submucosal (sm2-3) cancers were retrospectively reviewed and compared with confirmed histologic findings. RESULTS Characteristic colonoscopic findings needed for surgical operation were as follows: (1) expansion appearance, (2) deep depression surface, (3) irregular bottom of depression surface, and (4) folds converging toward the tumor. By using these findings, the invasion depth of depressed type early colorectal cancers could be correctly determined in 58 of 64 lesions (91%). CONCLUSIONS Characteristic colonoscopic findings obtained by a combination of videocolonoscopy and chromoendoscopy are useful for determination of the invasion depth of depressed type colorectal cancers, an essential factor in choosing a treatment modality.

Quantitative analysis for human glucocorticoid receptor α/β mRNA in IBD ☆

We have previously reported that in peripheral blood mononuclear cells (PBMC), the augmented expression of the β isoform of the human glucocorticoid receptor (hGRβ), as a putative dominant negative regulator of glucocorticoid action, is associated with glucocorticoid (GC) unresponsiveness of UC patients. In this study, we quantified the levels and serial changes of hGR transcripts in PBMC of IBD patients by a real-time fluorescence monitoring of PCR. As results, relative hGRβ mRNA expression was significantly higher in the active stage of UC than in inactive periods of UC or CD patients. Longitudinal analysis revealed that hGRβ mRNA expression in UC was increased after the relapse of inflammation, suggesting that the overproduction of cytokines during inflammation may be responsible. In in vitro culture experiments of human lymphoid cell (CEM) and human PBMC, IL-7, and IL-18 increased hGRβ mRNA expression in these cells but GC itself did not. Through these analyses, it is indicated that the inflammatory cytokines altered the splicing condition of the primary transcript of hGR gene in IBD patients.

Minute findings by magnifying colonoscopy are useful for the evaluation of ulcerative colitis

BACKGROUND Colonoscopy has an important role in the diagnosis of ulcerative colitis. However, colonoscopic findings are inadequate for the prediction of relapse without histologic examination. In this study, the role of magnifying colonoscopy in ulcerative colitis was evaluated. METHODS One hundred sixteen magnifying colonoscopy observations were made in 61 patients with ulcerative colitis between January 1994 and October 1998. A simple classification of magnifying colonoscopic findings into 5 categories was devised as follows: regularly arranged crypt openings, villous-like, minute defects of epithelium, small yellowish spots, and coral reef-like appearance. The colonoscopic findings by classification were compared with histopathologic findings, and the usefulness of the classification for predicting relapse was prospectively analyzed in 18 patients. RESULTS Compared with grade as determined by conventional colonoscopy, there was a better correlation between the classification of findings by magnifying colonoscopy and histopathologic findings (r(2) = 0.665, 0.807, respectively). Of 18 patients studied prospectively, 7 of 9 with minute defects of epithelium relapsed within 6 months, and the cumulative nonrelapsing rate was significantly lower in patients with minute defects of epithelium compared with those without minute defects of epithelium (p = 0.0059). Moreover, minute defects of epithelium was found to be a significant independent predictive factor for relapse (multivariate analysis, Cox proportional hazards model; p = 0.0203). CONCLUSIONS Our proposed classification of magnifying colonoscopic findings in patients with ulcerative colitis is useful for the evaluation of disease activity and for the prediction of periods of remission.

Mouse Paneth Cell Secretory Responses to Cell Surface Glycolipids of Virulent and Attenuated Pathogenic Bacteria

ABSTRACT Mouse Paneth cells respond to bacteria and bacterial cell surface antigens by discharging secretory granules into the lumen of small intestinal crypts (T. Ayabe et al., Nat. Immunol. 1:113-118, 2000). To investigate mechanisms regulating these responses, purified surface glycolipid molecules with known acyl chain modifications and attenuated properties were tested for the ability to stimulate Paneth cell secretion. The antigens included lipopolysaccharide (LPS) from wild-type and msbB-null Escherichia coli and phoP-null and phoP-constitutive Salmonella enterica serovar Typhimurium strains, as well as LPS, lipid A, and lipoteichoic acid from Pseudomonas aeruginosa and Listeria monocytogenes grown in Mg2+-limited media. Measurements of total secreted protein, secreted lysozyme, and the bactericidal peptide activities of collected secretions showed that the purified antigens elicited similar secretory responses from Paneth cells in mouse crypts ex vivo, regardless of glycolipid acyl chain modification. Despite their impaired Tlr4 pathway, Paneth cells in ex vivo C3H/HeJ mouse crypts released equivalent amounts of bactericidal peptide activity in response to purified bacterial antigens, including lipid A. Thus, mouse Paneth cells respond equivalently to purified bacterial cell envelope glycolipids, regardless of functional Tlr4, the structural properties of glycolipid acyl chains, or their association with virulence in humans.

Autoantibodies against the specific epitope of human tropomyosin(s) detected by a peptide based enzyme immunoassay in sera of patients with ulcerative colitis show antibody dependent cell mediated cytotoxicity against HLA-DPw9 transfected L cells

BACKGROUND AND AIMS Recent studies suggest that tropomyosin (TM) may act as a putative autoantigen in ulcerative colitis (UC). Recently, we identified, by computer homology analysis, a specific peptide (HIAEDADRK) in human TM that can bind to HLA-DPw9. The aim of this study was to investigate the presence of autoantibodies against this peptide in UC. METHODS Antibodies were measured by ELISA with a synthetic peptide in 20 healthy volunteers, 48 patients with UC, 26 with Crohns disease (CD), eight with primary sclerosing cholangitis (PSC), and six with primary biliary cirrhosis (PBC). The functional significance of antibodies was investigated by antibody dependent cell mediated cytotoxicity (ADCC) against DPw9 transfected L cells using a standard 51Cr release assay. RESULTS Optical density values (mean (SD)) of sera from patients with UC (1.40 (0.52)) and PSC (1.65 (0.12)) were significantly higher than those from healthy volunteers (0.32 (0.28)) (p<0.05), CD (0.50 (0.34)) (p<0.05) and PBC (0.14 (0.09)) (p<0.05). Values in UC decreased with clinical improvement. The ADCC activity of UC sera correlated well with antibody titre against this synthetic peptide. CONCLUSIONS Anti-TM antibody was detected in UC sera by a specific peptide based ELISA with high reproducibility. This peptide may be an antigenic epitope of TM involved in the immunopathogenesis of UC and, perhaps, PSC.

Bactericidal Activity of Mouse α-Defensin, Cryptdin-4 Predominantly Affects Noncommensal Bacteria

Mouse Paneth cell α-defensins, termed cryptdins, are secreted into the intestinal lumen, have microbicidal activity, and contribute to intestinal innate immunity. Among them, cryptdin-4 (Crp4) has the most potent microbicidal activity. In the intestinal lumen, commensal bacteria colonize and elicit beneficial effects in the host. However, the effects of Crp4 against commensal bacteria are poorly understood. Thus, we investigated the bactericidal activities of Crp4 against commensal bacteria compared to noncommensal bacteria. Oxidized Crp4 showed only minimal or no bactericidal activity against 8 out of 12 commensal bacterial species, including Bifidobacterium bifidum and Lactobacillus casei. We further addressed a role of the conserved disulfide bonds of Crp4 by analyzing reduced Crp4 (r-Crp4). r-Crp4 demonstrated significantly greater bactericidal activities against 7 of 12 commensal bacteria than did oxidized Crp4. Oxidized Crp4 and r-Crp4 elicited equivalently potent bactericidal activities against 11 of the 11 noncommensal bacteria tested, such as Salmonella enterica serovar Typhimurium,and against 5 of 12 commensal bacteria. Furthermore, when r-Crp4 was exposed to a processing enzyme of cryptdins, i.e. MMP-7, r-Crp4 was degraded and the bactericidal activities disappeared. These findings suggest that Crp4 has selective bactericidal activities against intestinal microbiota and that the activities are dependent on the disulfide bonds.

Molecular characteristics and physiological functions of major royal jelly protein 1 oligomer.

Royal jelly contains numerous components, including proteins. Major royal jelly protein (MRJP) 1 is the most abundant protein among the soluble royal jelly proteins. In its physiological state, MRJP 1 exists as a monomer and/or oligomer. This study focuses the molecular characteristics and functions of MRJP 1 oligomer. MRJP 1 oligomer purified using HPLC techniques was subjected to the following analyses. The molecular weight of MRJP 1 oligomer was found to be 290 kDa using blue native‐PAGE. MRJP 1 oligomer was separated into 55 and 5 kDa spots on 2‐D blue native/SDS‐PAGE. The 55 kDa protein was identified as MRJP 1 monomer by proteome analysis, whereas the 5 kDa protein was identified as Apisimin by N‐terminal amino acid sequencing, and this protein may function as a subunit‐joining protein within MRJP 1 oligomer. We also found that the oligomeric form included noncovalent bonds and was stable under heat treatment at 56°C. Furthermore, MRJP 1 oligomer dose dependently enhanced and sustained cell proliferation in the human lymphoid cell line Jurkat. In conclusion, MRJP 1 oligomer is a heat‐resistant protein comprising MRJP 1 monomer and Apisimin, and has cell proliferation activity. These findings will contribute to further studies analyzing the effects of MRJP 1 in humans.

Expression of the antimicrobial peptide α‐defensin/cryptdins in intestinal crypts decreases at the initial phase of intestinal inflammation in a model of inflammatory bowel disease, IL‐10‐deficient mice

Background: The etiology of inflammatory bowel disease (IBD) is associated with an altered microflora due to a failure of the immune system. This study investigated the expression of the intestinal antimicrobial peptide &agr;‐defensin, which plays a pivotal role in the regulation of the intestinal microflora in a representative model of IBD, interleukin (IL)‐10‐deficient mice. Methods: The expression of &agr;‐defensin/cryptdins in IL‐10‐deficient mice was assessed by real‐time polymerase chain reaction (PCR) and acid/urea polyacrylamide gel (AU‐PAGE). The alteration of &agr;‐defensin/cryptdins expression was compared with the inflammatory grade of mice intestine at various weeks from birth. Results: The weight, length, and inflammation grade of the mouse intestines were assessed at 5, 7, 9, 11, 13, and 15 weeks from birth. While the weight of the large intestine was heavier at 15 weeks after birth in the IL‐10‐deficient mice than in the control mice, histological inflammation began from 7 weeks after birth. Real‐time PCR and AU‐PAGE identified a significant decrease in the expression of &agr;‐defensin/cryptdins at 7 weeks after birth in the IL‐10 knockout mice, thus illustrating the involvement of &agr;‐defensin/cryptdins in the etiology of the intestinal inflammation in IBD. This study also identified the expression of &agr;‐defensin/cryptdins to be inversely proportional to age until 11 weeks, suggesting a relationship between the formation of the intestinal microflora and a reduction in the expression of &agr;‐defensin/cryptdins. Conclusions: The altered expression of antimicrobial peptide &agr;‐defensin may cause the onset of intestinal inflammation due to a failure to regulate intestinal microflora. (Inflamm Bowel Dis 2010)