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.

Proliferative response of peripheral blood mononuclear cells and levels of antibody to recombinant protein from Propionibacterium acnes DNA expression library in Japanese patients with sarcoidosis.

BACKGROUND AND AIM OF THE WORK: The causes of sarcoidosis are unknown. Propionibacterium acnes has been isolated from sarcoid lesions, and many genomes of P. acnes or P. granulosum have been detected in all biopsy samples tested from Japanese patients with sarcoidosis. We searched for protein antigens from propionibacteria that caused immune responses in patients with sarcoidosis but not in subjects without sarcoidosis. METHODS: A lambda gt11 genomic DNA expression library of P. acnes was screened with sera from patients with sarcoidosis. Antibodies to a recombinant protein from the insert recovered by the screening were measured in serum and bronchoalveolar lavage (BAL) fluid from patients with or without sarcoidosis by an immunofluorescence-based method. Peripheral blood mononuclear cells from patients with and without sarcoidosis were used to examine the lymphoproliferative response to the protein. RESULTS: Of 180,000 plaques screened, two clones coded for an identical recombinant protein, termed RP35, were recognized by sera. RP35 was the C-terminal region of P. acnes trigger factor. RP35 caused sarcoidosis specific proliferation of the mononuclear cells from 9 (18%) of the 50 patients with sarcoidosis; in a similar way, purified protein derived from Mycobacterium tuberculosis evoked specific responses in 8 (38%) of 21 patients with tuberculosis. Serum levels of IgG and IgA antibodies to RP35 were high in patients with sarcoidosis and other lung diseases. In BAL fluid levels IgG or IgA antibodies were high in 7 (18%) and 15 (39%), respectively, of 38 patients with sarcoidosis, and in 2 (3%) and 2 (3%), respectively, of 63 patients with other lung diseases. CONCLUSIONS: The RP35 protein from P. acnes causes a cellular immune response in some patients with sarcoidosis but not in subjects without 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.

Immunolocalization of platelet-derived growth factor, transforming growth factor-beta, and fibronectin in acute megakaryoblastic leukemia manifesting tumor formation.

Acute megakaryoblastic leukemia (AMKL) manifesting myelofibrosis and tumor formation in the liver with marked increase of reticulin is described. The megakaryoblastic nature of the leukemic cells of the bone marrow and the hepatic tumor nodule was established by positive immunohistochemical stains for CD41a and CD41b on frozen tissue sections. Immunolocalization of the platelet-derived growth factor (PDGF) protein and transforming growth factor (TGF)-beta protein also was demonstrated in the leukemic cells of the bone marrow and the hepatic tumor. Further, the deposition of fibronectin that has been known as the ligand of CD41a molecule and collagen types I and IV were recognized in the extracellular matrix of the bone marrow and the hepatic tumor. These results suggest that specific expression of growth factor proteins by the leukemic cells may selectively regulate the fibrosis of the bone marrow as well as the tumor formation of AMKL. The expression of adhesion molecules and growth factor proteins by the leukemic cells and the deposition of extracellular matrix are discussed in relation to the myelofibrosis as well as the tumor-forming nature of AMKL.