Yoshinobu Eishi

A new clinicopathological entity of IgG4-related autoimmune disease.

BackgroundAutoimmune pancreatitis (AIP) is occasionally associated with other autoimmune diseases.MethodsTo investigate the pathophysiology of AIP, we immunohistochemically examined the pancreas and other organs in eight patients with AIP, and in controls, using anti-CD4-T and CD8-T cell subsets, as well as IgG4 antibodies.ResultsIn AIP patients, severe or moderate infiltration of IgG4-positive plasma cells associated with CD4- or CD8-positive T lymphocytes was detected in the peripancreatic tissue (6/6), bile duct (8/8), gallbladder (8/8), portal area of the liver (3/3), gastric mucosa (5/7), colonic mucosa (2/2), salivary glands (1/2), lymph nodes (6/6), and bone marrow (2/2), as well as in the pancreas (8/8). There were few IgG4-positive plasma cells at the same sites in controls.ConclusionsThese results suggest that AIP is not simply pancreatitis but that it is a pancreatic lesion involved in IgG4-related systemic disease with extensive organ involvement. We propose a new clinicopathological entity, of a systemic IgG4-related autoimmune disease in which AIP and its associated diseases might be involved.

Autophagy-deficient mice develop multiple liver tumors

Autophagy is a major pathway for degradation of cytoplasmic proteins and organelles, and has been implicated in tumor suppression. Here, we report that mice with systemic mosaic deletion of Atg5 and liver-specific Atg7⁻/⁻ mice develop benign liver adenomas. These tumor cells originate autophagy-deficient hepatocytes and show mitochondrial swelling, p62 accumulation, and oxidative stress and genomic damage responses. The size of the Atg7⁻/⁻ liver tumors is reduced by simultaneous deletion of p62. These results suggest that autophagy is important for the suppression of spontaneous tumorigenesis through a cell-intrinsic mechanism, particularly in the liver, and that p62 accumulation contributes to tumor progression.

Quantitative Analysis of Mycobacterial and Propionibacterial DNA in Lymph Nodes of Japanese and European Patients with Sarcoidosis

ABSTRACT The cause(s) of sarcoidosis is unknown. Mycobacterium spp. are suspected in Europe and Propionibacterium spp. are suspected in Japan. The present international collaboration evaluated the possible etiological links between sarcoidosis and the suspected bacterial species. Formalin-fixed and paraffin-embedded sections of biopsy samples of lymph nodes, one from each of 108 patients with sarcoidosis and 65 patients with tuberculosis, together with 86 control samples, were collected from two institutes in Japan and three institutes in Italy, Germany, and England. Genomes of Propionibacterium acnes, Propionibacterium granulosum, Mycobacterium tuberculosis, Mycobacterium avium subsp. paratuberculosis, and Escherichia coli (as the control) were counted by quantitative real-time PCR. Either P. acnes or P. granulosum was found in all but two of the sarcoid samples. M. avium subsp. paratuberculosis was found in no sarcoid sample. M. tuberculosis was found in 0 to 9% of the sarcoid samples but in 65 to 100% of the tuberculosis samples. In sarcoid lymph nodes, the total numbers of genomes of P. acnes or P. granulosum were far more than those of M. tuberculosis. P. acnes or P. granulosum was found in 0 to 60% of the tuberculosis and control samples, but the total numbers of genomes of P. acnes or P. granulosum in such samples were less than those in sarcoid samples. Propionibacterium spp. are more likely than Mycobacteria spp. to be involved in the etiology of sarcoidosis, not only in Japanese but also in European patients with sarcoidosis.

Quantitative PCR of mycobacterial and propionibacterial DNA in lymph nodes of Japanese patients with sarcoidosis

BACKGROUND The causes of sarcoidosis are not known. The DNA of Mycobacterium tuberculosis has been detected in some sarcoid lesions. In Japan, Propionibacterium acnes has been isolated from such lesions, but whether this indigenous bacterium is related to the disease is unclear. We used PCR to estimate the number of genomes of these bacteria in sarcoid lesions, to identify any link between sarcoidosis and these two bacterial species. METHODS We examined formalin-fixed and paraffin-embedded sections of biopsy and surgical samples from lymph nodes of 15 patients with sarcoidosis, 15 patients with tuberculosis, and 15 patients with gastric cancer (controls). Quantitative PCR was done to amplify segments of 16 S ribosomal RNA of P. acnes and P. granulosum and of insertion sequence 6110 of M. tuberculosis. PCR products were identified and the quantities of the products were estimated in terms of the fluorescence of oligonucleotide reporter probes. The numbers of bacterial genomes in samples were estimated from standard curves of serially diluted bacterial DNA. FINDINGS Genomes of M. tuberculosis were found in samples from all 15 patients with tuberculosis, from three patients with sarcoidosis, and in one control sample. Genomes of P. acnes were found in 12 of the 15 patients with sarcoidosis, in two tuberculosis patients, and three controls. The difference in the estimated number of P. acnes genomes between individuals with and without sarcoidosis was similar to that in the number of M. tuberculosis between people with and without tuberculosis. There were 5x10(5) P. acnes genomes in sarcoidosis and 3x10(6) M. tuberculosis genomes in tuberculosis, respectively, on average per microg of total DNA. The three patients with sarcoidosis but without P. acnes all had P. granulosum DNA in their biopsy samples; the number of genomes of the bacterium was 5x10(5). INTERPRETATION These findings suggest that propionibacteria had resided or proliferated ectopically in the sarcoid lesions, whether there was a connection with the disease or not. Propionibacteria are a more likely cause than mycobacteria of sarcoidosis.

Improvement in Atrophic Gastritis and Intestinal Metaplasia in Patients in Whom Helicobacter pylori Was Eradicated

Few long-term studies of chronic gastritis associated with Helicobacter pylori have been published. Kuipers (1) and Valle (2) and their colleagues showed that in about one third of infected patients, nonatrophic gastritis progressed to glandular atrophy and intestinal metaplasia before final follow-up (mean duration of follow-up, 11.5 years and 32 years, respectively). Two studies of 10 patients (3) and 35 patients (4) infected with H. pylori found long-term improvement in intestinal metaplasia after H. pylori eradication. However, in one study of more than 100 patients with H. pylori infection, glandular atrophy and intestinal metaplasia did not improve during long-term follow-up (5). We evaluated histologic changes 12 to 15 months after attempted eradication of H. pylori to explore whether glandular atrophy and intestinal metaplasia improve after eradication. Methods Patients We enrolled all consecutive patients with dyspepsia and H. pylori infection who were seen at our gastroenterology clinic in Tokyo, Japan, for diagnostic upper gastrointestinal endoscopy. The proportions of patients who were self-referred, were referred from within our hospital, and were referred from other hospitals were roughly equal. Patients with possible allergy to penicillin, amoxicillin, or proton-pump inhibitors and those with previous gastrectomy were excluded. Written informed consent was obtained from participants in accordance with the Declaration of Helsinki and its later revision. The protocol was approved by the institutional review board of Tokyo Medical and Dental University, Tokyo, Japan. Study Design At entry, patients underwent upper gastrointestinal endoscopy; gastric mucosa was histologically evaluated by performing biopsy. Helicobacter pylori status was assessed by using bacteriologic culture, histologic results, the rapid urease test, and the urea breath test. Eligible patients were treated for 1 week with a proton-pump inhibitor (omeprazole or lansoprazole), amoxicillin, and clarithromycin with or without a mucoprotective agent (ecabet sodium, rebamipide, or sofalcone). At 1 to 3 months (short-term follow-up) and at 12 to 15 months (long-term follow-up) after eradication therapy, patients again underwent upper gastrointestinal endoscopy and gastric biopsy specimens were examined histopathologically. H. pylori Assessment At each endoscopy, three biopsy specimens each were taken from the greater curvature of the antrum and the greater curvature of the corpus of the stomach. One sample from each site was cultured, one was used for the rapid urease test, and one was used for histologic examination. Cultures on modified Skirrow agar with 10% horse blood were incubated for 5 to 7 days at 37 C in a microaerobic atmosphere. We identified H. pylori by observing colony structure and performing biochemical tests for urease, catalase, and oxidase activities. Biopsy specimens for histologic examination were stained with Giemsa stain or hematoxylineosin. After an overnight fast, a [13C]urea breath test was done with 100 mg of [13C]urea per 100 mL of distilled H2O. The results were considered negative if the ratio of 13CO2 to 12CO2 in 15 minutes increased by less than 5 parts per million. The presence of H. pylori was investigated at each endoscopic examination. The bacterium was considered to be present if results of at least two of four tests (bacteriologic culture, histologic results, rapid urease test, and urea breath test) were positive. Helicobacter pylori was considered eradicated if results were negative for all four tests at both short-term and long-term follow-up. No patient had only one positive test result. Gastritis Scores The endoscopists were blinded to the results of treatment. For each patient, two pathologists who were blinded to the results of treatment made independent histologic diagnoses by examining one biopsy specimen each from the antrum and corpus. The pathologists disagreed on the diagnosis in 99 of the 978 specimens examined (49 specimens from the antrum and 50 specimens from the corpus; the final diagnoses were chronic inflammation in 24 specimens, neutrophil activity in 39, glandular atrophy in 27, and metaplasia in 9). Consensus was reached by re-examination and discussion. Severity of chronic inflammation, neutrophil activity, glandular atrophy, and intestinal metaplasia was graded from 0 (normal) to 3 (markedly abnormal) according to the visual analogue scales of the updated Sydney System (6). Specimens stained with Giemsa were evaluated for H. pylori by another investigator. Statistical Analysis We divided patients into two groups on the basis of the success or failure of H. pylori eradication. We examined background factors and histologic findings at baseline to identify pretreatment differences between the two groups. We used the unpaired t-test for age, the chi-square test for sex ratios in the two groups, both the chi-square test and the Fisher exact test for comorbid conditions, and the Wilcoxon rank-sum test for histologic findings. For the antrum and corpus, we performed the Wilcoxon signed-rank test to compare histologic findings at baseline with those at follow-up (1 to 3 months and 12 to 15 months after treatment). To evaluate differences in changes in the histologic findings during follow-up, we performed mixed-effects ordinal logistic regression by using the computer program MIXOR (7). This method prevents problems caused by regression to the mean. We used SAS software, version 6.12 (SAS Institute, Inc., Cary, North Carolina), to perform all other analyses. A P value less than 0.05 was considered statistically significant. Results We studied 207 consecutive patients with H. pylori infection and dyspepsia. Patients underwent endoscopy before treatment and at least once after treatment, at 1 to 3 months. In addition, 163 patients agreed to undergo another endoscopy at 12 to 15 months after treatment (mean, 14 months). Thirty-three patients declined to undergo a second follow-up examination, and 11 patients were untraceable because they had moved out of the area. Helicobacter pylori was eradicated without serious side effects in 115 of the 163 patients who had long-term follow-up. The 115 patients in whom H. pylori was eradicated were similar to the 48 who remained infected in terms of mean (SD) age (54 12 years vs. 59 10 years), sex distribution (84 men and 31 women vs. 30 men and 18 women), and diagnosis (47 and 18 patients with chronic gastritis, 30 and 15 with duodenal ulcers, 25 and 15 with gastric ulcer, and 13 and 0 with gastroduodenal ulcers). The Table shows histologic findings for the gastric mucosa before treatment and again at the two follow-up points. The two groups did not differ significantly in histologic findings before treatment. In patients with successful eradication, scores for chronic inflammation and neutrophil activity at both sites were lower at both follow-up points than before treatment, and scores for glandular atrophy in the corpus and intestinal metaplasia in the antrum were lower at 12 to 15 months than before treatment. Glandular atrophy in the corpus improved in 34 (89%) of the 38 patients with atrophy before treatment, and intestinal metaplasia in the antrum improved in 28 (61%) of the 46 patients with metaplasia before treatment. Table. Histologic Results before Treatment To Eradicate Helicobacter pylori and at Short- and Long-Term Follow-up In patients with unsuccessful eradication of H. pylori, no significant histologic changes were observed at follow-up, except for progression of chronic inflammation. Mixed-effects ordinal logistic regression showed statistically significant differences between groups in changes at follow-up for chronic inflammation and neutrophil activity at both sites and for intestinal metaplasia in the antrum (Table, Figure). Individual histologic changes were as follows (some percentages do not total 100 because of rounding). Among patients with successful eradication of H. pylori, glandular atrophy in the antrum decreased in 29% and 27% at short- and long-term follow-up, respectively, was unchanged in 40% and 30%, and increased in 31% and 43%; in the corpus, glandular atrophy decreased in 24% and 30%, was unchanged in 70% and 64%, and increased in 6% and 6%. Intestinal metaplasia in the antrum decreased in 16% and 25% of patients of these patients, was unchanged in 78% and 71%, and increased in 5% and 4%; intestinal metaplasia in the corpus decreased in 3% and 4%, was unchanged in 88% and 91%, and increased in 10% and 5%. Among patients without eradication of H. pylori, glandular atrophy in the antrum decreased in 23% and 38%, at short- and long-term follow-up, respectively, was unchanged in 44% and 29%, and increased in 33% and 33%; glandular atrophy in the corpus decreased in 25% and 19%, was unchanged in 46% and 65%, and increased in 29% and 17%. Intestinal metaplasia in the antrum decreased in 0% and 0%, was unchanged in 98% and 96%, and increased in 2% and 4%; intestinal metaplasia in the corpus decreased in 12% and 8%, was unchanged in 81% and 83%, and increased in 6% and 8%. Figure. Predicted curves obtained by mixed-effects ordinal logistic regression models for histologic changes in the gastric mucosa in patients in whom Helicobacter pylori infection was or was not successfully eradicated . H. pylori H. pylori P Discussion Tucci and colleagues (8) reported that atrophic gastritis of the corpus of the stomach had regressed by 12 months after discontinuation of treatment in 10 of 20 patients with fundic atrophic gastritis in whom H. pylori was successfully eradicated. In contrast, Witteman and colleagues (10) found no changes at 57 weeks in glandular atrophy of the antrum and corpus of patients in whom the bacterium was eradicated. In a prospective study with a mean follow-up of 1 year (range, 6 to 18 months), van der Hulst and colleagues (5) found that the degree of atrophy did not change; however, their grading of a

In situ localization of Propionibacterium acnes DNA in lymph nodes from sarcoidosis patients by signal amplification with catalysed reporter deposition

Sarcoidosis is a systemic granulomatous disease of unknown aetiology. Many genomes of Propionibacterium acnes and P. granulosum have been detected in lymph nodes from patients with sarcoidosis. In situ localization of propionibacterial genomes in sarcoid lymph nodes may help to establish an aetiological link between sarcoidosis and these indigenous bacteria. Formalin‐fixed and paraffin‐embedded biopsy samples of lymph nodes from nine patients with sarcoidosis, nine patients with tuberculosis, and nine patients with non‐specific lymphadenitis as controls were examined by quantitative real‐time PCR (QPCR) for P. acnes and by in situ hybridization (ISH) that used catalysed reporter deposition (CARD) for signal amplification with digoxigenin‐labelled oligonucleotide probes that complemented 16S rRNA of P. acnes. The signals per 250 µm2 of tissue sections were counted from inside and outside the granulomas of sarcoidosis and tuberculosis and from control lymph nodes. The number of genomes by QPCR was examined for correlation with the mean signal count by ISH with CARD. In sarcoid samples, one or several signals were detected in the cytoplasm of some epithelioid cells in granulomas and of many mononuclear cells around granulomas. The mean signal counts were higher (p < 0.001) in granulomatous areas than in other areas of sarcoid lymph nodes. Even in their non‐granulomatous areas, counts were higher than in granulomatous areas (p = 0.0023) and non‐granulomatous areas (p < 0.001) of tuberculous lymph nodes and control lymph nodes (p = 0.0071). Correlation between the results by QPCR and ISH with CARD was significant (r = 0.86, p < 0.001). The accumulation of P. acnes genomes in and around sarcoid granulomas suggests that this indigenous bacterium may be related to the cause of granulomatous inflammation in sarcoidosis. Copyright

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.

Gastric mucosal density of Helicobacter pylori estimated by real-time PCR compared with results of urea breath test and histological grading.

The accuracy of the urea breath test (UBT) and histological grading for estimation of the density of Helicobacter pylori in gastric mucosa is not known. Real-time (TaqMan) PCR was used to estimate the total number of H. pylori genomes in biopsy samples. These values were compared with those obtained by the UBT and the histological grade obtained by the Sydney system. The UBT and endoscopy with antral and corporal biopsies were performed in 88 consecutive untreated patients with dyspepsia. Bacterial culture and the rapid urease test were done with fresh biopsy materials. TaqMan PCR and histological examination were done on serial paraffin sections of the biopsy samples. Of the five methods tested, TaqMan PCR had the highest sensitivity and specificity (both 100%) in the diagnosis of H. pylori infection. The mean density of H. pylori genomes for pairs of biopsy samples from individual patients was compared with the individual values obtained by the UBT; correlation between the results was significant. The density of H. pylori genomes was higher in histological grades 1, 2 and 3 than in grade 0, without significant differences between adjacent grades from 1 to 3. These results suggest that the severity of H. pylori infection of the stomach can be estimated by the UBT and that histopathologists might state whether the organism is present or absent, rather than making a quantitative statement as recommended in the Sydney system.

Basophils preferentially express mouse mast cell protease 11 among the mast cell tryptase family in contrast to mast cells

Tryptases and chymases are the major proteins stored and secreted by mast cells, and they have various biological functions. However, the nature of proteases produced by basophils has been poorly characterized, particularly in mice. mMCP‐11 is the most recently discovered mast cell tryptase in mice and was originally identified as Prss34, which is transcribed in some mast cell‐like cell lines and at the early stage in the culture of BMMC with IL‐3. Curiously, Prss34 is preferentially expressed in the BM and spleen among normal tissues in contrast to other mast cell tryptases. Therefore, it remains elusive what types of cells express mMCP‐11 in vivo. Here, we show that mMCP‐11 is highly expressed by primary basophils and to a much lesser extent, by some mast cells. Prss34 transcripts were detected abundantly in primary and cultured basophils and very weakly in peritoneal mast cells or cultured BMMC. Conversely, transcripts for mMCP‐6 and mMCP‐7 tryptases were preferentially expressed by cultured and peritoneal mast cells but not basophils. We established a mMCP‐11‐specific mAb and showed that mMCP‐11 proteins are indeed expressed by primary basophils and those infiltrating the affected tissues during allergic inflammation and parasitic infections. Some primary mast cells also expressed mMCP‐11 proteins, albeit at a much lower level. Thus, basophils rather than mast cells are the major source of mMCP‐11. This is the first study to demonstrate that mouse basophils produce a trypsin‐like protease.

Aurora kinase B is a predictive factor for the aggressive recurrence of hepatocellular carcinoma after curative hepatectomy

Patterns of cancer recurrence hold the key to prognosis after curative resection. This retrospective study aimed to identify a predictor and therapeutic candidate for aggressive recurrence of hepatocellular carcinoma (HCC).