HSP70 and HSP90 Differentially Regulate Translocation of Extracellular Antigen to the Cytosol for Cross-Presentation.

Antigens (Ag) from cancer or virus-infected cells must be internalized by dendritic cells (DCs) to be presented to CD8(+) T cells, which eventually differentiate into Ag-specific cytotoxic T lymphocytes (CTLs) that destroy cancer cells and infected cells. This pathway is termed cross-presentation and is also implicated as an essential step in triggering autoimmune diseases such as Type I diabetes. Internalized Ag locates within endosomes, followed by translocation through a putative pore structure spanning endosomal membranes into the cytosol, where it is degraded by the proteasome to generate antigen peptides. During translocation, Ag is believed to be unfolded since the pore size is too narrow to accept native Ag structure. Here, we show that paraformaldehyde-fixed, structurally inflexible Ag is less efficient in cross-presentation because of diminished translocation into the cytosol, supporting the "unfolded Ag" theory. We also show that HSP70 inhibitors block both endogenous and cross-presentation. ImageStream analysis revealed that the inhibition in cross-presentation is not due to blocking of Ag translocation because a HSP70 inhibitor rather facilitates the translocation, which is in marked contrast to the effect of an HSP90 inhibitor that blocks Ag translocation. Our results indicate that Ag translocation to the cytosol in cross-presentation is differentially regulated by HSP70 and HSP90.

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.

Heat shock protein 90 (HSP90) contributes to cytosolic translocation of extracellular antigen for cross-presentation by dendritic cells.

In antigen (Ag) cross-presentation, dendritic cells (DCs) take up extracellular Ag and translocate them from the endosome to the cytosol for proteasomal degradation. The processed peptides can enter the conventional MHC I pathway. The molecules responsible for the translocation of Ag across the endosomal membrane into the cytosol are unknown. Here we demonstrate that heat shock protein 90 (HSP90) is critical for this step. Cross-presentation and -priming were decreased in both HSP90α-null DCs and mice. CD8α(+) DC apoptosis mediated by translocation of exogenous cytochrome c to the cytosol was also eliminated in HSP90α-null mice. Ag translocation into the cytosol was diminished in HSP90α-null DCs and in DCs treated with an HSP90 inhibitor. Internalized Ag was associated with HSP90 and translocated to the cytosol, a process abrogated by the HSP90 inhibitor. Ag within purified phagosomes was released in an HSP90-dependent manner. These results demonstrate the important role of HSP90 in cross-presentation by pulling endosomal Ag out into the cytosol.

Quantitative PCR of Propionibacterium acnes DNA in samples aspirated from sebaceous follicles on the normal skin of subjects with or without acne.

To elucidate whether people with hair follicles containing many Propionibacterium acnes cells are prone to acne, we developed a novel method to count the number of P. acnes in hair follicles. We sampled sebaceous material in hair follicles by aspiration at a constant negative pressure from the nose, forehead, and upper arm of 86 patients with acne vulgaris and 209 control subjects with healthy skin, including 84 subjects age-matched to the patients. Genome-equivalents of P. acnes in samples were estimated by real-time quantitative PCR (TaqMan). Numbers of P. acnes genome-equivalents were extremely low in control subjects less than 10 years of age and generally higher at greater ages, with much variation in subjects in the same decade of life. In men, the median count was highest in controls aged 15-19 years; in women, it peaked twice, in controls aged 15-19 years and again in those aged 40 years or older. P. acnes counts on the forehead and nose were higher in the acne patients aged 10-14 years than in the age-matched controls in both sexes. The counts at three sites were similar in acne patients and controls aged 15 to 29 years in both sexes. The results suggest that people with hair follicles containing many P. acnes cells are not particularly prone to acne, except for younger teenagers. Our aspiration method with estimation by real-time PCR can be used to examine the cutaneous microflora of P. acnes.

Quantitative cellular level analysis of mitochondrial DNA 3243A > G mutations in individual tissues from the archival temporal bones of a MELAS patient.

CONCLUSION: We could represent the first quantitative analysis of the mutation rate at the cellular level in human inner ear of a patient with MELAS (mitochondrial encephalopathy, lactic acidosis and stroke-like episodes) by combining laser capture microdissection (LCM) and quantitative real time PCR. OBJECTIVES: We previously reported combining LCM and PCR to isolate mtDNA from the cells of specific tissues within a human archival celloidin-embedded temporal bone section without known otological history. Using this method, we quantitatively analyzed the rate of mtDNA 3243A > G mutation in the inner ear of a MELAS patient, and examined the correlation of the mutation rate at the cellular level and their histopathological condition. METHODS: We extracted each inner ear organs using LCM from temporal bone sections of a MELAS patient, and studied the mutation rate, which was calculated as the ratio of the amount of mutant mtDNA to the total mtDNA. RESULTS: We found that the mtDNA mutation rate was high in spiral ganglion cells and the saccular macula, but was comparatively low in hair cells of the organ of Corti, the stria vascularis and the facial nerve. With the exception of the stria vascularis, there was a good correlation between the mutation rate and the histological findings.

Comparison of mesenchymal stem cells derived from arterial, venous, and Wharton's jelly explants of human umbilical cord.

We isolated mesenchymal stem cells (MSC) from arteries (UCA), veins (UCV), and Wharton's jelly (UCWJ) of human umbilical cords (UC) and determined their relative capacities for sustained proliferation and multilineage differentiation. Individual UC components were dissected, diced into 1-2 mm(3) fragments, and aligned in explant cultures from which migrating cells were isolated using trypsinization. Preparations from 13 UCs produced 13 UCWJ, 11 UCV, and 10 UCA cultures of fibroblast-like, spindle-shaped cells negative for CD31, CD34, CD45, CD271, and HLA-class II, but positive for CD13, CD29, CD44, CD73, CD90, CD105, and HLA-class I. UCV cells exhibited a significantly higher frequency of colony-forming units fibroblasts than did UCWJ and UCA cells. Individual MSCs could be selectively differentiated into osteoblasts, chondrocytes, and adipocytes. When compared for osteogenic potential, UCWJ cells were the least effective precursors, whereas UCA-derived cells developed alkaline phosphatase activity with or without an osteogenic stimulus. UC components, especially blood vessels, could provide a promising source of MSCs with important clinical applications.

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.

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.

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.

Clonality analysis for normal and cancerous colon tissues with human androgen receptor gene polymerase chain reaction.

We assessed the feasibility of clonality analysis with human androgen receptor gene polymerase chain reaction in terms of the sensitivity and specificity for normal and cancerous colonic tissues taken from fourteen informative cases selected from 22 women with colonic adenocarcinoma. Ten crypts microdissected from each 10-microm-thick cryostat sections and whole tissues were used as samples. DNA was extracted from the samples and amplified with and without prior enzyme digestion. These products were analyzed by capillary electrophoresis for clonality. Of the whole-tissue DNA, none of the normal tissues and seven (50.0%) of the cancerous tissues showed monoclonality. Of the microdissected samples, monoclonality was found in 88.4% (107/121) of normal crypts and 95.9% (117/122) of cancerous crypts. Samples composed of crypts with short and long alleles were found in eight of the 14 normal colonic mucosae, but in none of the cancerous tissues. We concluded that the sensitivity of this method is limited for both whole-tissue DNA and microdissected-tissue DNA, because monoclonality from small samples does not always indicate monoclonality of the entire lesion. The high specificity of this method, however, allows polyclonal results in whole tissues to be confirmed by additional analysis of microdissected tissues.

Detection of mitochondrial DNA from human inner ear using real-time polymerase chain reaction and laser microdissection.

CONCLUSIONS: In this study we were able to amplify and analyze extremely small amounts of template DNA from only a few individually dissected cells. We anticipate that this approach will facilitate the detection and analysis of mitochondrial (mt) DNA mutations in specific cell types in the inner ear, which should shed new light on genetic disorders leading to hearing loss. OBJECTIVE: To isolate mtDNA from selected tissues in the inner ear. Although several methods for extracting DNA from formalin-fixed, celloidin-embedded, archival human temporal bones have been reported, the isolation of DNA from the inner ear by means of laser microdissection has not been previously demonstrated. MATERIAL AND METHODS: This was a retrospective study. Temporal bones were obtained from subjects with no known otological history at autopsy. The combined method of laser microdissection and real-time polymerase chain reaction was used to isolate mtDNA from selected tissues in the inner ear. RESULTS: mtDNA could be isolated from the stria vascularis, spiral ligament, spiral ganglion cells and organ of Corti.

Propionibacterium acnes is the most common bacterium commensal in peripheral lung tissue and mediastinal lymph nodes from subjects without sarcoidosis.

BACKGROUND: In studies of the unknown etiology of sarcoidosis, Propionibacterium acnes (a possible agent) was found in the lungs and lymph nodes of many sarcoidosis patients and some control subjects. P. acnes might be commensal not only to the skin, conjunctivae, and intestine, but also to the lungs and lymph nodes of individuals without sarcoidosis. METHODS: We cultured peripheral lung tissue and various lymph nodes obtained from patients with diseases other than sarcoidosis. DNA of 45 isolates of P. acnes from these patients, 67 isolates from normal skin, conjunctiva, and intestine, and 39 isolates from sarcoid lymph nodes were compared by random amplified polymorphic DNA analysis. RESULTS: P. acnes was isolated from half of 43 lungs and 8 of 11 mediastinal lymph nodes, mostly in pure culture. P. acnes was isolated from half of 20 gastric and 3 of 12 intestinal lymph nodes; intestinal bacteria were also numerous. In general, fewer than 500 colony-forming units of P. acnes per gram tissue were isolated, but 4 lung tissue specimens, 2 of which had a few granulomas, had many more. P. acnes strains from a particular site (lung, lymph node, skin or conjunctivae, and intestine) were genetically similar, more than isolates obtained from different sites. Lymph-node isolates from subjects with and without sarcoidosis differed little. CONCLUSION: These results suggest that P. acnes normally resides in peripheral lung tissue and mediastinal lymph nodes and that the strains of P. acnes isolated from sarcoid lymph nodes were not specific to sarcoidosis.

Granulocyte-macrophage colony-stimulating factor and lung immunity in pulmonary alveolar proteinosis.

The anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibody is inferred to cause idiopathic pulmonary alveolar proteinosis (iPAP): the antibody neutralizes GM-CSF and thereby impairs differentiation of alveolar macrophages. Administration of GM-CSF improves respiratory function of patients with iPAP, as confirmed in this study using aerosolized GM-CSF. To elucidate its mechanism, we characterized bronchoalveolar lavage fluid and alveolar macrophages obtained from three patients with iPAP who were treated successfully with aerosolized GM-CSF. Cell number, expressions of surface mannose receptor and the transcription factor PU.1, and phagocytic ability of alveolar macrophages were all restored to control levels. With treatment, the neutralizing capacity of GM-CSF activity was reduced markedly, concomitant with the decreasing autoantibody levels. Interestingly, the amount of GM-CSF autoantibody complex also decreased. In one case in which the complex was analyzed, the majority of GM-CSF binding the complex was endogenous protein, suggesting that the complex is removed immediately from the lung after treatment. Our study shows that GM-CSF administration engenders a decrease in the neutralizing capacity against the protein in the lungs. Thereby, it facilitates restoration of the normal function of alveolar macrophages.

High-affinity autoantibodies specifically eliminate granulocyte-macrophage colony-stimulating factor activity in the lungs of patients with idiopathic pulmonary alveolar proteinosis.

Deficiency of granulocyte-macrophage colony-stimulating factor (GM-CSF) in mice results in pulmonary alveolar proteinosis (PAP) from impaired surfactant catabolism by alveolar macrophages (AMs). Recently, we have shown that neutralizing anti-GM-CSF autoantibodies develop specifically in patients with idiopathic pulmonary alveolar proteinosis (iPAP). Analogous to murine PAP models, it is plausible that the autoantibodies reduce GM-CSF activity, resulting in AM dysfunction and surfactant accumulation. To examine this hypothesis, we estimated the neutralizing activity of the autoantibodies in the lungs of patients and characterized their biologic properties. GM-CSF bioactivity was completely abrogated in the bronchoalveolar lavage fluid (BALF) of patients with iPAP but not in healthy subjects. Autoantibodies were present in the alveoli in high concentrations and colocalized with GM-CSF. They recognized human GM-CSF with high avidity (K(AV) = 20.0 +/- 7.5 pM) and high specificity, reacting with its superstructure and neutralizing GM-CSF activity to a level 4000 to 58 000 times the levels of GM-CSF normally present in the lung. Although target epitopes varied among patients, GM-CSF amino acids 78 to 94 were consistently recognized. Thus, autoantibodies bind GM-CSF with high specificity and high affinity, exist abundantly in the lung, and effectively block GM-CSF binding to its receptor, inhibiting AM differentiation and function. Our data strengthen the evidence associating anti-GM-CSF autoantibodies with the pathogenesis of this disease.

Pulmonary granulomas caused experimentally in mice by a recombinant trigger-factor protein of Propionibacterium acnes.

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.

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.

Quantitative analysis of bacterial DNA from Mycobacteria spp., Bacteroides vulgatus, and Escherichia coli in tissue samples from patients with inflammatory bowel diseases.

BACKGROUND: The etiology of inflammatory bowel diseases is unknown. Mycobacteria spp., Bacteroides vulgatus, and Escherichia coli have been suspected to be involved. The aim of the present study was to examine the possible relationship between inflammatory bowel diseases and these microbes. METHODS: We studied 45 patients; 16 with Crohn's disease, 11 with ulcerative colitis, and 18 with colon cancer as controls. We used a real-time quantitative polymerase chain reaction to detect and estimate numbers of bacterial genomes in formalin-fixed, paraffin-embedded tissue samples from the subjects. The bacteria studied were Mycobacterium tuberculosis, M. avium, M. paratuberculosis, B. vulgatus, and E. coli. Immunohistochemical staining was done to locate B. vulgatus and E. coli in tissue samples. RESULTS: The three Mycobacterium species were not detected. B. vulgatus and E. coli were detected more frequently and in greater numbers in samples from patients with inflammatory bowel diseases than in samples from control patients with colon cancer. The frequency and numbers were not related to the severity of the disease. Many bacteria of these species were found within the mucous layer, underneath erosions, in necrotic ulcer bed tissues, and in abscesses. E. coli cells were found in perivascular areas in the proper muscle layer and in germinal centers of lymph follicles. CONCLUSIONS: Our results suggest that Mycobacteria spp. are not involved in the etiology of Crohn's disease and that mucosa-associated B. vulgatus and E. coli are not a direct cause of inflammatory bowel diseases, although they may contribute to the diseases by preventing or delaying remission.

Quantitative analysis of mycobacterial and propionibacterial DNA in lymph nodes of Japanese and European patients with sarcoidosis.

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.