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DyP-type peroxidases are a family of heme peroxidases named for their ability to degrade persistent anthraquinone dyes. DyP-type peroxidases are subclassified into three classes: classes P, I and V. Based on its genome sequence, Streptomyces avermitilis, eubacteria, has two genes presumed to encode class V DyP-type peroxidases and two class I genes. We have previously shown that ectopically expressed SaDyP2, a member of class V, indeed has the characteristics of a DyP-type peroxidase. In this study, we analyzed SaDyP1, a member of the same class V as SaDyP2. SaDyP1 showed high amino acid sequence identity to SaDyP2, retaining a conserved GXXDG motif and catalytic aspartate. SaDyP1 degraded anthraquinone dyes, which are specific substrates of DyP-type peroxidases but not azo dyes. In addition to such substrate specificity, SaDyP1 showed other features of DyP-type peroxidases, such as low optimal pH. Furthermore, immunoblotting using an anti-SaDyP2 polyclonal antibody revealed that SaDyP1 and/or SaDyP2 is expressed in mycelia of wild-type S. avermitilis.

Bacterial catalase is important for intracellular survival of the bacteria. This protein of Propionibacterium acnes, one of possible causes of sarcoidosis, induces hypersensitive Th1 immune responses in sarcoidosis patients. We examined catalase expression in cultured P. acnes isolated from 19 sarcoid and 18 control lymph nodes and immunohistochemical localization of the protein in lymph nodes from 43 sarcoidosis and 102 control patients using a novel P. acnes-specific antibody (PAC) that reacts with the catalase protein, together with the previously reported P. acnes-specific PAB and TIG antibodies. High catalase expression of P. acnes cells was found during stationary phase in more isolates from sarcoid than from non-sarcoid lymph nodes and was associated with bacterial survival under H2O2-induced oxidative stress. In many sarcoid and some control lymph nodes, catalase expression was detected at the outer margins of PAB-reactive Hamazaki-Wesenberg (HW) bodies in sinus macrophages, the same location as catalase expression on the surface of cultured P. acnes and the same distribution as bacterial cell membrane-bound lipoteichoic acid in HW bodies. Some or no catalase expression was detected in sarcoid granulomas with PAB reactivity or in clustered paracortical macrophages packed with many PAB-reactive small-round bodies. HW bodies expressing catalase may be persistent P. acnes in sinus macrophages whereas PAB-reactive small-round bodies with undetectable catalase may be activated P. acnes proliferating in paracortical macrophages. Intracellular proliferation of P. acnes in paracortical macrophages may lead to granuloma formation by this commensal bacterium in sarcoidosis patients with Th1 hypersensitivity to certain P. acnes antigens, including catalase.

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.

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.

Objective: Proliferation of fibroblasts (desmoplastic reaction) in the lung adenocarcinomas is an important phenomenon that correlates with metastases and poor prognosis. Because basement membranes are often involved in the desmoplastic areas and many cytokines have binding capacity to basement membrane molecules, we hypothesized that basement membrane modify the paracrine effects between cancer cells and fibroblasts via the fibrogenic cytokines and this hypothesis was experimentally investigated. Methods: The effects of conditioned media derived from ten lung carcinoma cell lines and normal airway epithelial cells on DNA synthesis of fetal lung fibroblasts were determined. We focused on fibroblast growth factor 2 (FGF-2) as the candidate paracrine cytokines and examined their diffusion through an experimental basement membrane matrix model, Matrigel™. Results: All the conditioned media promoted DNA synthesis of fetal lung fibroblasts. Detection by ELISA methods and the neutralizing antibodies suggested that FGF-2 was one of the responsible factors for the growth promotion. Diffusion of FGF-2 across the polycarbonate membrane was suppressed by coating with Matrigel. When FGF-2-secreting A549 cells were covered with Matrigel, FGF-2 was stored in Matrigel and its diffusion into the culture media was significantly reduced. Binding of FGF-2 to Matrigel was completely blocked by a basic protein, protamine sulfate. In the presence of protamine sulfate in Matrigel overlaid on A549 cells, diffusion of FGF-2 increased 7-fold as much as that without overlaid Matrigel. Conclusion: These results suggest that the basement membrane acts as a barrier to the diffusion and a reservoir of cytokines secreted by cancer cells, and that the subsequent degradation of the basement membrane by cancer cells could release the stored cytokines and promote growth of fibroblasts.

Sarcoidosis, of unknown etiology, may result from exposure of a genetically susceptible subject to a specific environmental agent(s), possibly an infectious one, although none has been identified. Propionibacterium acnes is so far the only bacterium to be isolated from sarcoid lesions. Genomes of P. acnes have been detected in large numbers in sarcoid lymph nodes by the quantitative polymerase chain reaction. By in situ hybridization, P. acnes genomes were found in sarcoid lymph nodes in and around sarcoid granulomas. These results point to an etiological link between P. acnes and some cases of sarcoidosis. Host factors may be more critical than agent factors in the etiology of sarcoidosis, as already suggested from the phenomenon of the Kveim test, in which a suspension of sarcoid tissues injected intracutaneously causes sarcoid granulomas in patients with sarcoidosis but not in healthy people or patients with other diseases. A recombinant trigger-factor protein, RP35, from P. acnes causes a cellular immune response in some patients with sarcoidosis, but not in subjects without sarcoidosis. RP35 caused pulmonary granulomas in mice sensitized with the protein and adjuvant. Sarcoid granulomas may form during hypersensitivity to antigens of P. acnes indigenous to or proliferating in the affected organ.

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-κB 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-κB 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.

Etiology of sarcoidosis remains unknown. A trigger factor from Propionibacterium acnes causes a cellular immune response in some sarcoid patients but not in nonsarcoid subjects. We examined whether experimentally induced hypersensitivity to the trigger factor gives rise to granulomas. Female C57BL/6 mice primed intravenously with P. acnes or not were sensitized with recombinant-protein RP35, a fragment of P. acnes trigger factor, and complete Freund's adjuvant. In controls, RP35 was replaced with P. acnes or one of two control proteins. In primed and unprimed mice, pulmonary granulomas were found in some of the mice sensitized with RP35 or P. acnes but in no control-protein-sensitized mice. Detection of pulmonary granulomas (25-57%) did not differ significantly between mice sensitized with RP35 or P. acnes, primed or not. No difference in popliteal lymph-node-cell reactivity and serum antibodies to these two antigens was found between mice with and without pulmonary granulomas. P. acnes was cultured from the lungs of 8 (33%) of 24 untreated mice. The recombinant trigger-factor protein of P. acnes caused pulmonary granulomas in primed and unprimed mice sensitized with the protein and adjuvant. Sarcoid granulomas may form during hypersensitivity to antigens of P. acnes indigenous to the affected organ.