Tamiko Takemura

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.

Pathological studies on sarcoidosis autopsy. I. Epidemiological features of 320 cases in Japan

Three hundred and twenty autopsy cases of sarcoidosis during a 32 year period were collected from the Annuals of the Pathological Autopsy Cases in Japan, published yearly since 1958, and from a literature survey. A statistical analysis of these reviewed autopsy cases was carried out on the epidemiological features of the disease and on the causes of death.

Pulmonary vascular involvement in sarcoidosis : a report of 40 autopsy cases

We examined pulmonary vascular involvement in 40 autopsy cases of sarcoidosis. In these cases granulomatous involvement was observed at all levels from large elastic pulmonary arteries to venules, and venous involvement was more prominent than arterial involvement. The extent of granulomatous vascular involvement was related to that of parenchymal granuloma. No significant difference was found between upper and lower lobes in the incidence of granulomatous vascular involvement. The distribution of granulomata in the blood vessels was segmental and adventitial, and medial involvement was prominent in the larger vessels. Healed lesions of granulomatous vascular involvement also were observed at various levels in blood vessels. Prominent granulomatous involvement was found in the lymphatic capillaries and collecting lymphatic vessels in lungs with sarcoidosis. Serial sections of the lungs demonstrated interstitial granuloma directly connecting the lymphatic capillaries around small blood vessels. Granulomatous involvement in vasa vasorum and lymphatic capillaries is likely to be an important factor in the pathogenesis of granulomatous vascular involvement in lungs with sarcoidosis. The present study suggests that granulomatous vascular involvement and its sequelae may contribute to the development of pulmonary sarcoidosis.

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

Frequent loss of imprinting of IGF2 and MEST in lung adenocarcinoma

Genomic imprinting is a parental origin–specific chromosomal modification that causes differential expression of maternal and paternal alleles of a gene. Accumulating evidence suggests that deregulation of imprinted genes, including loss of imprinting (LOI), plays a role in oncogenesis. In the present study, we investigated allelic expression of six imprinted genes in human lung adenocarcinomas as well as in matched normal lung tissue. Informative cases showing heterozygosity for the gene of interest were selected from 35 patients. LOI of the insulin‐like growth factor 2 gene (IGF2) and mesoderm‐specific transcript (MEST, also known as paternally expressed gene 1) was noted in 47% (seven of 15) and 85% (11 of 13) of informative cases, respectively. Monoallelic expression was maintained in all the matched normal tissues examined. LOI of IGF2 was seen more frequently in moderately to poorly differentiated adenocarcinomas. In contrast, H19, small nuclear ribonucleoprotein–associated polypeptide N gene (SNRPN), necdin gene (NDN), and long QT intronic transcript 1 (LIT1) exhibited consistent monoallelic expression in all the informative samples. These findings indicated that independent deregulation took place in imprinted genes and suggested that aberrant imprinting of IGF2 and MEST was involved in the development of lung adenocarcinoma.

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.

Pathological studies on sarcoidosis autopsy. II. Early change, mode of progression and death pattern.

Three hundred and twenty autopsy cases of sarcoidosis in Japan were analyzed to determine the pathological changes in the early stage, the mode of progression in each organ and the changes in the final stage of the disease.

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.

Pathology of hypersensitivity pneumonitis.

Purpose of review Hypersensitity pneumonitis, caused by inhalation of various antigens, is characterized by interstitial mononuclear cell infiltration, nonnecrotizing granulomas, cellular bronchiolitis, and fibrosis. The pathological picture of chronic hypersensitivity pneumonitis is, however, complicated; it is sometimes difficult to differentiate chronic hypersensitivity pneumonitis from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, and connective-tissue-related lung disease. The clinical, radiological, and pathological features of chronic hypersensitivity pneumonitis have recently been described. This study reviews the previously reported information and provides new insights into the pathological features of chronic hypersensitivity pneumonitis. Recent findings The pathological features of chronic hypersensitivity pneumonitis comprise overlapping usual interstitial pneumonia-like pattern with subpleural patchy fibrosis, alternating normal alveoli and fibroblastic foci, a nonspecific interstitial pneumonia-like pattern, and centrilobular fibrosis. In contrast to pathological features of acute and subacute hypersensitivity pneumonitis, epithelioid cell granulomas are sparse or absent, but giant cells are seen in the interstitium. Bridging fibrosis between peribronchiolar area and perilobular areas is an outstanding feature of chronic hypersensitivity pneumonitis. Autopsy cases of chronic hypersensitivity pneumonitis have demonstrated not only upper lobe contraction but also lower lobe contraction, mimicking usual interstitial pneumonia pattern and diffuse alveolar damage. Summary The present review focuses on the pathological features of chronic hypersensitivity pneumonitis and presents that centrilobular fibrosis and bridging fibrosis are the important hallmarks of chronic hypersensitivity pneumonitis, even with a usual interstitial pneumonia-like pattern.