CHAC1 overexpression in human gastric parietal cells with Helicobacter pylori infection in the secretory canaliculi.

BACKGROUND: Cation transport regulator 1 (CHAC1), a newly discovered enzyme that degrades glutathione, is induced in Helicobacter pylori (H. pylori)-infected gastric epithelial cells in culture. The CHAC1-induced decrease in glutathione leads to an accumulation of reactive oxygen species and somatic mutations in TP53. We evaluated the possible correlation between H. pylori infection and CHAC1 expression in human gastric mucosa. MATERIALS AND METHODS: Both fresh-frozen and formalin-fixed paraffin-embedded tissue samples of gastric mucosa with or without H. pylori infection were obtained from 41 esophageal cancer patients that underwent esophago-gastrectomy. Fresh samples were used for real-time polymerase chain reaction for H. pylori DNA and CHAC1 mRNA, and formalin-fixed samples were used for immunohistochemistry with anti-CHAC1 and anti-H. pylori monoclonal antibodies. Double-enzyme or fluorescence immunohistochemistry and immuno-electron microscopy were used for further analysis. RESULTS: Significant CHAC1 overexpression was detected in H. pylori-infected parietal cells that expressed the human proton pump/H,K-ATPase α subunit, whereas a constitutively low level of CHAC1 mRNA expression was observed in the other samples regardless of the H. pylori infection status, reflecting the weak CHAC1 expression detected by immunohistochemistry in the fundic-gland areas. Immuno-electron microscopy revealed intact H. pylori cells in the secretory canaliculi of infected parietal cells. Some parietal cells exhibited positive nuclear signals for Ki67 in the neck zone of the gastric fundic-gland mucosa with H. pylori infection. CONCLUSION: Cation transport regulator 1 overexpression in H. pylori-infected parietal cells may cause the H. pylori-induced somatic mutations that contribute to the development of gastric cancer.

Helicobacter pylori invades the gastric mucosa and translocates to the gastric lymph nodes.

Helicobacter pylori has been considered to be non-invasive and to rarely infiltrate the gastric mucosa, even though there is an active Th1 immune response in the lamina propria of the H. pylori-infected stomach. To elucidate whether H. pylori invades the lamina propria and translocates to the gastric lymph nodes, we examined H. pylori in formalin-fixed and paraffin-embedded tissue sections of stomach and gastric lymph nodes obtained from 51 cancer patients using real-time PCR and immunohistochemistry (IHC) with a novel anti-H. pylori monoclonal antibody that recognizes lipopolysaccharides. Fresh gastric lymph nodes were used to culture for H. pylori. In 46 patients with H. pylori in the stomach, the bacterium was found in the lymph nodes from 21 patients by culture, 37 patients by PCR, and 29 patients by IHC. H. pylori captured by macrophages was found in the lamina propria of 39 patients. In the lymph nodes, the bacterium was found in many macrophages and a few interdigitating dendritic cells at the paracortical areas. H. pylori was also found in the intracellular canaliculi of parietal cells in 21 patients, but intracytoplasmic invasion into gastric epithelial cells was not identified. When compared to the commercially available anti-H. pylori antibodies, the novel antibody showed the highest sensitivity to detect H. pylori-positive macrophages, whereas no difference was found for H. pylori in the mucous layer. The H. pylori-positive macrophages in the lamina propria correlated with chronic gastritis as well as translocation of such cells to the lymph nodes. These results suggest that H. pylori-induced gastric epithelial damage allows the bacteria to invade the lamina propria and translocate to the gastric lymph nodes, which may chronically stimulate the immune system. The bacteria captured by macrophages, whether remaining alive or not, may contribute to the induction and development of H. pylori-induced chronic gastritis.

Helicobacter pylori induces somatic mutations in TP53 via overexpression of CHAC1 in infected gastric epithelial cells.

Infection with Helicobacter pylori is known to decrease the level of glutathione in gastric epithelial cells and increase the production of reactive oxygen species (ROS), which can lead to DNA damage and the development of gastric cancer. Cation transport regulator 1 (CHAC1) has γ-glutamylcyclotransferase activity that degrades glutathione. We found that cagA-positive H. pylori infection triggered CHAC1 overexpression in human gastric epithelial (AGS) cells leading to glutathione degradation and the accumulation of ROS. Nucleotide alterations in the TP53 tumour suppressor gene were induced in AGS cells overexpressing CHAC1, whereas no mutations were detected in cells overexpressing a catalytically inactive mutant of CHAC1. A high frequency of TP53 mutations occurred in H. pylori-infected AGS cells, but this was prevented in cells transfected with CHAC1 siRNA. These findings indicate that H. pylori-mediated CHAC1 overexpression degrades intracellular glutathione, allowing the accumulation of ROS which subsequently causes mutations that could contribute to the development of gastric cancer.

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.

Sarcoidosis and NOD1 variation with impaired recognition of intracellular Propionibacterium acnes.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. NOD2 mutations have been shown to predispose to granulomatous diseases, including Crohn's disease, Blau syndrome, and early-onset sarcoidosis, but not to adult sarcoidosis. We found that intracellular Propionibacterium acnes, a possible causative agent of sarcoidosis, activated NF-kappaB in both NOD1- and NOD2-dependent manners. Systematic search for NOD1 gene polymorphisms in Japanese sarcoidosis patients identified two alleles, 796G-haplotype (156C, 483C, 796G, 1722G) and 796A-haplotype (156G, 483T, 796A, 1722A). Allelic discrimination of 73 sarcoidosis patients and 215 healthy individuals showed that the frequency of 796A-type allele was significantly higher in sarcoidosis patients and the ORs were significantly elevated in NOD1-796G/A and 796A/A genotypes (OR [95% CI]=2.250 [1.084, 4.670] and 3.243 [1.402, 7.502], respectively) as compared to G/G genotype, showing an increasing trend across the 3 genotypes (P=0.006 for trend). A similar association was found when 52 interstitial pneumonia patients were used as disease controls. Functional studies showed that the NOD1 796A-allele was associated with reduced expression leading to diminished NF-kappaB activation in response to intracellular P. acnes. The results indicate that impaired recognition of intracellular P. acnes through NOD1 affects the susceptibility to sarcoidosis in the Japanese population.

Widespread expression of γ-glutamyl cyclotransferase suggests it is not a general tumor marker.

γ-Glutamyl cyclotransferase (GGCT) contributes to the γ-glutamyl cycle that regulates glutathione metabolism. Although GGCT has been implicated in several studies as a possible cancer marker, little is known about its distribution in cells and tissues. The authors investigated GGCT expression in normal tissues and tumors using Western blots and immunohistochemistry with a novel anti-GGCT monoclonal antibody. GGCT was detected in most organs and was mainly found in epithelial cells. Although the intracellular distribution was mainly cytoplasmic, in some situations, nuclear staining was strong. A significant increase in the expression of GGCT was found in tumors of the lung, esophagus, stomach, bile duct, and uterine cervix. In contrast, there was a significant decrease in expression in renal and urothelial tumors. These results suggest that GGCT may be a biomarker of tumors in a limited range of organs.

Characterization of Propionibacterium acnes isolates from sarcoid and non-sarcoid tissues with special reference to cell invasiveness, serotype, and trigger factor gene polymorphism.

Sarcoidosis is a systemic granulomatous disease of unknown etiology. Propionibacterium acnes is the only microorganism so far isolated from sarcoid lesions. To examine whether P. acnes isolates from sarcoid tissues differ from those obtained from non-sarcoid tissues, we studied cell invasiveness, serotype, and polymorphisms of the P. acnes trigger factor protein and the two invasion-associated proteins (named PAmce and PAp60) in 35 P. acnes isolates from sarcoid lymph nodes and 127 isolates from non-sarcoid tissues. Most of the serotype I isolates (79/112; 71%), but none of the serotype II isolates (0/50) were cell-invasive. Two prominent types of trigger factors, one with and one without a 15 amino acid-residue deletion, corresponded to serotype II and serotype I, respectively. Non-invasive isolates had genomic mutations that caused more than one amino acid change in either the PAmce or PAp60 gene, with four exceptional isolates. P. acnes was finally classified into nine isotypes, and isolates obtained from sarcoid and non-sarcoid tissue did not differ. Although the finding did not link P. acnes to sarcoidosis, the present study clarified the cell invasiveness of P. acnes and the close correlation of cell invasiveness to the serotype and genotype of the two invasion-associated P. acnes genes.

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 micro m(2) 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.