Mariko Negi

Localization of Propionibacterium acnes in granulomas supports a possible etiologic link between sarcoidosis and the bacterium

Sarcoidosis likely results from the exposure of a genetically susceptible subject to an environmental agent, possibly an infectious one. Mycobacterial and propionibacterial organisms are the most commonly implicated potential etiologic agents. Propionibacterium acnes is the only microorganism, however, found in sarcoid lesions by bacterial culture. To evaluate the pathogenic role of this indigenous bacterium, we screened for the bacterium in sarcoid and non-sarcoid tissues using immunohistochemical methods with novel P. acnes-specific monoclonal antibodies that react with cell-membrane-bound lipoteichoic acid (PAB antibody) and ribosome-bound trigger-factor protein (TIG antibody). We examined formalin-fixed and paraffin-embedded samples of lungs and lymph nodes from 196 patients with sarcoidosis, and corresponding control samples from 275 patients with non-sarcoidosis diseases. The samples were mostly from Japanese patients, with 64 lymph node samples from German patients. Immunohistochemistry with PAB antibody revealed small round bodies within sarcoid granulomas in 20/27 (74%) video-assisted thoracic surgery lung samples, 24/50 (48%) transbronchial lung biopsy samples, 71/81 (88%) Japanese lymph node samples, and 34/38 (89%) German lymph node samples. PAB antibody did not react with non-sarcoid granulomas in any of the 45 tuberculosis samples or the 34 samples with sarcoid reaction. In nongranulomatous areas, small round bodies detected by PAB antibody were found in alveolar macrophages of lungs and paracortical macrophages of lymph nodes from many sarcoid and some non-sarcoid patients. Large-spheroidal acid-fast bodies, Hamazaki–Wesenberg bodies, which were found in 50% of sarcoid and 15% of non-sarcoid lymph node samples, reacted with both PAB and TIG antibodies. Electron microscopy revealed that these Hamazaki–Wesenberg bodies had a single bacterial structure and lacked a cell wall with occasional protrusions from the body. The high frequency and specificity of P. acnes, detected by PAB antibody within sarcoid granulomas, indicates that this indigenous bacterium might be the cause of granuloma formation in many sarcoid patients.

γ-Glutamylcyclotransferase as a novel immunohistochemical biomarker for the malignancy of esophageal squamous tumors

Recently, overexpression of γ-glutamylcyclotransferase (GGCT) has been reported in various cancer tissues suggesting that it has significant potential as a diagnostic marker. The aim of this study was to examine the suitability of GGCT for the detection of high-risk lesions at an early stage of esophageal squamous cell carcinoma (ESCC). A total of 200 lesions, including 120 invasive ESCC, 80 esophageal squamous intraepithelial neoplasia consisting of 40 low-grade intraepithelial neoplasia (LGIEN) and 40 high-grade intraepithelial neoplasia (HGIEN), as well as 20 confounding lesions, were examined by immunohistochemical (IHC) staining for GGCT. IHC staining for Ki-67 and p53 was also performed in esophageal squamous intraepithelial neoplasia to compare the diagnostic power of GGCT. Increased expression of GGCT was common in invasive ESCC and HGIEN (87.5% and 85.0%, respectively), but was much less frequently observed in LGIEN (17.5%). GGCT expression significantly correlated with the presence of lymph node metastasis and the degree of differentiation. In the differential diagnosis of LGIEN and HGIEN, GGCT possessed both high sensitivity and high specificity, while Ki-67 and p53 only possessed either high sensitivity or high specificity. Additionally, GGCT expression was higher in 7 out of 8 ESCC cell lines (KYSE series) than in a normal esophageal squamous cell line (Het-1A). The expression levels strongly correlated with enzymatic activity (r=.92; P<.001). These results indicate that overexpressed GGCT retains its enzymatic activity and can become a valuable biomarker for the diagnosis of HGIEN that is likely to progress to subepithelial invasion.

Atonal homolog 1 protein stabilized by tumor necrosis factor α induces high malignant potential in colon cancer cell line

Patients with inflammatory bowel disease (IBD) have an increased risk of developing colitis‐associated colorectal cancer (CAC). CAC cells often develop chemoresistance, resulting in a poorer prognosis than that of sporadic colorectal cancer (CRC). The mechanism by which CAC enhances malignant potential remains unknown. We have previously reported that the proteasomal degradation of the transcription factor Atonal homolog 1 (Atoh1) protein results in the non‐mucinous form of CRC. It also remains unknown whether Atoh1 protein is expressed in CAC. Therefore, in the present study, we investigated whether Atoh1 protein stabilizes in CAC. Consequently, the treatment with TNF‐α stabilized Atoh1 protein through the inactivation of GSK‐3β via Akt, resulting in the mucinous form of CRC cell lines. Atoh1 protein also enriched cancer stem cells with upregulated Lgr5 expression and cells in G0/G1 cell cycle phase, resulting in both the chemoresistance to 5‐fluorouracil and oxaliplatin and the promotion of cell migration. Immunofluorescence of the human mucinous CAC specimens showed the accumulation of NF‐κB p65 at nuclei with the expression of Atoh1 in mucinous cancer. In conclusion, the inflammation associated with carcinogenesis may preserve the differentiation system of intestinal epithelial cell (IEC), resulting in the acquisition of both the mucinous phenotype and high malignant potential associated with the enrichment of cancer stem cell.

First autopsy analysis of a neovascularized arterial network induced by indirect bypass surgery for moyamoya disease: case report.

The object of this study was to analyze the pathology of collateral vessels newly induced by indirect bypass surgery for moyamoya disease (MMD). An autopsy analysis was conducted on a 39-year-old woman with MMD who had died of a brainstem infarction. The patient had undergone bilateral indirect bypass surgeries 22 years earlier. Sufficient revascularization via bilateral external carotid arterial systems was confirmed by cerebral angiography before her death. Macroscopic observation of the operative areas revealed countless meandering vessels on the internal surface of the dura mater connected with small vessels on the brain surface and in the subpial brain tissue. Notably, microscopic analysis of these vessels revealed the characteristic 3-layer structure of an arterial wall. This autopsy analysis was the first to confirm that indirect bypass surgery had induced the formation of a new arterial network (arteriogenesis) and that this network had been maintained for more than 20 years to compensate for the chronic cerebral ischemia caused by the MMD.

Diagnosis and treatment of microscopic colitis.

Microscopic colitis (MC) designates two types of chronic diarrhea diseases, which are lymphocytic colitis and collagenous colitis. The prevalence of microscopic colitis is increasing in both Western and Eastern countries, possibly due to the high incidence of colonoscopic survey in chronic diarrhea patients. Although the overall prognosis of MC patients is mostly good, it should be noted that appropriate diagnosis and choice of treatment is required to assure a good clinical outcome for MC patients. Also, a certain population of MC patients may take a severe and refractory clinical course, and thus require advanced clinical care using medications supported by less evidence. In this review, we would like to feature the essential points regarding the diagnosis of MC, and also describe the current standard of treatments for MC patients. In addition, we would like to add some findings from the national survey and research carried out in Japan, to compare those data with the western countries.

Reduced Human α-defensin 6 in Noninflamed Jejunal Tissue of Patients with Crohn’s Disease:

Background:Mucosal barrier dysfunction is considered a critical component of Crohn’s disease (CD) pathogenesis after the identification of susceptibility genes. However, the precise mechanism underlying mucosal barrier dysfunction has not yet been elucidated. We therefore aimed to elucidate the molecular mechanism underlying the expression of human &agr;-defensin 6 (HD6) in patients with CD. Methods:HD6 expression was induced by the transfection of an atonal homolog 1 (Atoh1) transgene and was assessed by reverse transcription polymerase chain reaction. The HD6 promoter region targeted by Atoh1 and &bgr;-catenin was determined by reporter analysis and chromatin immunoprecipitation assay. HD5/HD6/Atoh1/&bgr;-catenin expression in noninflamed jejunal samples collected by balloon endoscopy from 15 patients with CD and 9 non-inflammatory bowel disease patients were assessed by immunofluorescence. Results:Both promoter activity and gene expression of HD6 was significantly upregulated by the Atoh1 transgene in human colonic cancer cell line. We identified a TCF4 binding site and an E-box site, critical for the regulation of HD6 transcriptional activity by directly binding of Atoh1 in the 200-bp HD6 promoter region. The treatment with &bgr;-catenin inhibitor also decreases HD6 promoter activity and gene expression. Moreover, HD6 expression, but not HD5 expression, was found to be decreased in noninflamed jejunal regions from patients with CD. In HD6-negative crypts, nuclear accumulation of &bgr;-catenin was impaired. Conclusions:HD6 expression was found to be regulated by cooperation between Atoh1 and &bgr;-catenin within the HD6 promoter region. Downregulation of HD6 in noninflamed mucosa may contribute to mucosal barrier dysfunction of patients with CD.

Endoscopic features and genetic background of inflammatory bowel disease complicated with Takayasu arteritis

Takayasu arteritis (TA) is occasionally complicated with inflammatory bowel disease (IBD). This study assessed the endoscopic and genetic features of IBD complicated with TA (IBD‐TA).

Histologically Confirmed IgG4-Related Small Intestinal Lesions Diagnosed via Double Balloon Enteroscopy

Immunoglobulin G4 (IgG4)-related disease is a systemic disease characterized by chronic inflammation with abundant IgG4-positive cells and elevated serum IgG4. Previous reports describe infiltration of IgG4-positive cells in multiple organs [1]. In the gastrointestinal (GI) tract, previous reports indicate that inflammation was observed in the esophagus, stomach, and colon [1–6]. However, there have been few reports regarding small intestinal lesions. We report a case of IgG4-related disease with small and large intestinal lesions. A 55-year-old man presented to our hospital with 4 months of general fatigue and lower abdominal pain. He had the past history of cholecystectomy and did not take any non-steroidal anti-inflammatory drugs. He was admitted because his abdominal pain was worsening. The findings of abdominal computed tomography (CT) scan indicated that his pancreas was swollen; however, he did not have any ocular, salivary gland, and retroperitoneal lesions. Blood tests revealed high levels of C-reactive protein (4.1 mg/dL), elevated IgG (5,636 mg/dL), and IgG4 (2,480 mg/dl) levels. Nephritis was suspected because urinary tests revealed elevated beta-2 microglobulin and N-acetyl-b-D-glucosaminidase although renal biopsy was not done. Histological findings from cervical lymph node indicated infiltration of IgG4-positive cells while malignant lymphoma and Castleman’s disease were excluded. Esophagogastroduodenoscopy (EGD) and colonoscopic findings revealed multiple erosions and ulcerations in the stomach and colon (Fig. 1a,b). The abdominal CT also indicated increased bowel wall thickness of the proximal small intestine, therefore we performed double balloon enteroscopy (DBE). Small erosions and irregular ulcerations were observed at the jejunum (Fig. 1c,d) while inflammation was not observed in the ileum. These lesions included neither longitudinal nor circular ulcerations. Membranous strictures were not observed. Histological findings from biopsy specimens revealed infiltration of inflammatory cells, including plasma cells (Fig. 2a,b) in the jejunum. The percentage of IgG4-positive cells among IgG-positive plasma cells was approximately 70 % (Fig. 2c,d). IgG4-positive cells were detected not only from specimens containing erosions but also from endoscopically normal mucosa in the ileum (Fig. 2e,f). A total of 40 mg daily of oral prednisolone was given and his symptoms rapidly improved. At 3 months after administration of corticosteroids, the colonoscopy indicated colonic ulceration was improved (Fig. 3). However, IgG4positive cells remained even after clinical remission was obtained by the use of corticosteroids. Thus, dose of steroid has been gradually decreased to avoid relapsing remission. On the basis of the elevated serum IgG4 level and histological analysis (ratio of IgG4/IgG [ 50 % in the cervical lymph node and GI tracts), this patient was diagnosed K. Fujita M. Naganuma (&) E. Saito S. Suzuki A. Araki M. Watanabe Departments of Gastroenterology and Hepatology, School of Medicine, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo 113-8519, Japan e-mail: mnaganuma.gast@tmd.ac.jp

Propionibacterium acnes-derived insoluble immune complexes in sinus macrophages of lymph nodes affected by sarcoidosis

Background Propionibacterium acnes is thought to be a causative agent of sarcoidosis. Patients with sarcoidosis have circulating immune complexes. We attempted to detect P. acnes-derived immune complexes in sarcoid lesions. Methods We evaluated formalin-fixed and paraffin-embedded lymph node samples from 38 sarcoidosis patients and 90 non-sarcoidosis patients (27 patients with necrotizing lymphadenitis, 28 patients with reactive lymphadenitis, 16 patients with colon cancer, 19 patients with gastric cancer) by immunohistochemistry using anti-human immunoglobulins (IgG, IgA, and IgM) and complement (C1q and C3c) antibodies, and a P. acnes-specific monoclonal antibody (PAB antibody) that reacts with the membrane-bound lipoteichoic acid of P. acnes. Results Small round bodies (SRBs) bound to IgA, IgM, or IgG were detected in sinus macrophages, in 32 (84%), 32 (84%), or 11 (29%) sarcoid samples, respectively, and in 19 (21%), 26 (29%), or no (0%) control samples, respectively. Some of these insoluble immune complexes (IICs) also bound to C1q and C3c. We developed a microwave treatment followed by brief trypsin digestion (MT treatment) to detect PAB-reactive SRBs bound to immunoglobulins (IIC-forming P. acnes). MT treatment revealed abundant IIC-forming P. acnes in most (89%) of the sarcoid samples and sparse distribution in some (20%) of the control samples with lymphadenitis, but no IIC-forming P. acnes was detected in control samples without inflammation. IIC-forming P. acnes were mostly bound to both IgA and IgM. The PAB-reactive antigen and immunoglobulins were both located at the peripheral rim of the IIC-forming P. acnes. Conventional electron microscopy identified many SRBs (0.5–2.0 μm diameter) in sinus macrophages of sarcoid lymph nodes with many IIC-forming P. acnes, some of which were in phagolysosomes with a degraded and lamellar appearance. Conclusions P. acnes-derived IICs in sinus macrophages were frequent and abundant in sarcoid lymph nodes, suggesting a potential etiologic link between sarcoidosis and this commensal bacterium.

Caudal type homeobox 2 expression induced by leukocytapheresis might be associated with mucosal healing in ulcerative colitis: CDX2 by LCAP for mucosal healing in UC

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with an intractable, recurrent course. Although the goal of UC therapy has recently been to target mucosal healing, the molecular mechanism of mucosal healing remains unknown. In this study, we aimed to elucidate the molecular dynamics related to the proliferation and differentiation of intestinal epithelial cells during cytapheresis therapy in a short duration.