Staphylococcus aureus Cowan I was incubated with monolavers of cells derived from several portions of mouse kidney, and found to be ingested by all types of the renal cells. Intracellular localization of S. aureus was determined by resistance of intracellular cocci against lysostaphin digestion and confirmed by electron microscopy. From renal medulla, three morphological variants of the hyperosmolarity-tolerant (HOT) cells were obtained. The rate of cocci-ingesting cells varied from 16.9% to 93.4% among those of the HOT cells at the end of 3-hr incubation. From renal cortex, three morphological variants of epithelial cells grew in medium RK-1. Among them, only the cells on the edge of colony ingested Cowan I, while the epithelial cells on the center of colony ingested few cocci. Transferred from medium RK-1 to MEM supplemented with 10% FBS, part of the cortical cells changed into fibroblast-like appearance and obtained the capacity to ingest Cowan I. This result may indicate the correlation between ingesting capacity and cellular morphology. From a glomerulus, epithelial (GE) cells and fibroblast-like (GF) cells were obtained. The GE cells ingested not only S. aureus Cowan I but Staphylococcus epidermidis and Staphylococcus saprophyticus after 30-min incubation, while the GF cells, like both of the HOT cells and the cortical cells, ingested only S. aureus. These results suggest a possibility that S. aureus is located within nonprofessional phagocytes during its infection and intracellular coccus plays an important role in its pathogenicity.

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Identification of methicillin-resistant Staphylococcus aureus by drug-susceptibility tests alone poses a serious problem, because a considerable number of clinical S. aureus isolates are borderline resistant to methicillin. To circumvent this problem, we have developed a quick and sensitive method of PCR amplification for the detection of mecA gene, which, coding for PBP2', is the specific genetic element of methicillin-resistant Staphylococcus aureus. This method made it possible to identify MRSA strains in a short time using as few as 30 cells as a starting material for template DNA. Using this method, we found that the strains of borderline methicillin-resistance could be accurately identified. We also found one S. aureus clinical strain, T3, which lacked mecA gene in spite of its resistance to methicillin.

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In the immunoblot analysis, sera from patients infected with Mycoplasma pneumoniae reacted with the 168kDa (P1) and the 85kDa proteins of virulent strain FH-P24 and P24-S1 mutant strain but not with the 85kDa protein of P24-S11. Sera of hamsters and BALB/c mice, which had been immunized with live vaccines, were tested. In FH-P24 immunized animals, 100% or 80%, and in P24-S1, 40% of hamsters and 60% of BALB/c mice, developed antibodies against the 85kDa protein, but antibodies were not detected in sera of P24-S11 immunized animals. The correlation between the development of antibodies to 85kDa protein in the sera of vaccinated animals and the effects of protection by living vaccines were suggested.

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A histopathological scoring system was developed to assess the pathology of acute Mycoplasma pneumoniae pulmonary infection in a hamster model. A final score per animal (ranging 0-26) is obtained by averaging scores from each lung which have been accumulated by the addition of subscores from the assessments of quantity and quality of peribronchiolar and peribronchial infiltrates, luminal exudates, perivascular infiltrates, and parenchymal pneumonia. The scoring scheme was then applied to test the ability of a heat-killed inoculum to induce pulmonary pathology and to the trial of a 43kDa protein-associated antigen as a vaccine immunogen. A heat-killed inoculum delivered by both intratracheal and intranasal routes did not induce pulmonary pathology compared to a live inoculum (respective mean scores 0.1, 6.7; P<0.01). Animals prevaccinated with the 43kDa antigen developed an accentuated pathological response after live challenge compared to those unvaccinated (respective mean scores 16.8, 5.8; P=0.00007). Hypersensitization to growth medium components may, however, have contributed to the accentuated disease since the lungs of vaccinated animals challenged with culture-negative media also were affected (mean score 5.4). Reproducibility of the scoring system was measured by duplicate reading of histology slides which were randomized to the observer upon the second reading (r=0.93; P=0.000009). The scoring system has the ability to differentiate disease severity in small groups of animals.

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Two EBV-negative human B-lymphoma cell lines, BJAB and DG75, were transfected with an Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA-2) gene, which plays a critical role in the EBV-induced immortalization of primary B lymphocytes. Furthermore, DG75 cells were co-transfected with the EBNA-2 gene and a latent membrane protein (LMP) gene. Expression of eight surface antigens on the resultant EBNA-2-expressing cell clones was analyzed by flowcytometry. None of the EBNA-2-expressing cell clones derived from BJAB and DG75 showed a significant increase in the expression of cell surface marker CD23, of which enhancement by EBNA-2 in a different EBV-negative human B cell line, Louckes, was previously reported. Expression of CD25 (IL-2R/Tac) on cell surface, however, was induced in two of six DG75-derived cell clones. One of the two CD25-induced cell clones was expressing EBNA-2 only, and the other was co-expressing EBNA-2 and LMP. The results suggest that EBNA-2 has a potential to up-regulate CD25 independently of CD23 on human B cells.

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The assay for detecting IgM neutralizing (NT) antibody activity to human herpesvirus-6 (HHV-6) was developed by using pretreatment of blood sample with staphylococcal protein A. The activity was mostly present in IgM fractions of serum but not in IgA fractions separated by ultracentrifugation. The assay was used for seroepidemiological studies for HHV-6 infection. In primary HHV-6 infection, IgM NT antibodies appeared 5 to 7 days after onset of exanthem subitum, reached maximum titers at 2 to 3 weeks, and tended to decline to undetectable levels after 2 months. In contrast, reactivation of HHV-6 observed in organ transplants showed somewhat greater degree of IgM NT antibody responses that persisted for 2 to 3 months and became undetectable 5 to 6 months after transplantation. The level and persistence of NT antibody titers measured by the conventional method was generally greater than those of the IgM titers. The prevalence of the IgM NT antibodies was examined in healthy individuals. The antibody was first detected at 4 to 7 months of age (5%), reached maximum level at 8 to 11 months (40%), and was detectable by 4 to 6 years (17%). A few (4 to 5%) of adolescents and adults were positive for the antibody.

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Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48hr, and the activities of interleukin-1β (IL-1β), IL-2, IL-4, IL-6, tumor necrosis factor-α (TNF-α) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1β, IL-6 and TNF-α, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-α. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-β and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro.

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We found that the number of peripheral blood polymorphonuclear leukocytes (PMN) dramatically increased in both sarcoma 180 (S-180) and MM-46 mammary carcinoma (MM-46) ascites tumor-bearing mice, and mice required a remarkable resistance to Candida albicans infection via intravenous route. When the resistance was determined by number of cells of C. albicans in the kidney, a significant decrease in the number of fungal cells was observed in the kidneys of infected ascites tumor-bearing mice. An increase of active oxygen levels of PMN from ascites tumor-bearing mice was observed, suggesting that this factor is important in developing of resistance in ascites tumor-bearing mice. Additionally, a culture supernatant of tumor cells co-cultivated with bone-marrow cells in vitro increased the number of granulocytes and macrophages differentiated from the bone-marrow cells.

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Group a mutants of Clostridium perfringens are deficient in theta toxin but release a dialyzable substance (“substance A”), which restores theta toxin activity to group b mutants, into a culture supernatant; group b mutants are defective in “substance A” release. “Substance A” activity appeared in the exponentially growing phase of group a mutants and disappeared in the stationary phase. “Substance A” activity was most stable at pH 5.0 and 0C and even increased threefold in the first 5hr, but gradually decreased during the following 15hr. It was quickly inactivated at neutral and higher pHs at 0C.

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Mycobacterium avium and Mycobacterium intracellulare are differentiated from each other by thin-layer chromatography of lipid fraction extracted after incubation with [³⁵S]methionine. The former contained a petroleum ether-soluble sulfolipid and the latter did not.

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The presence or absence of the phosphoenolpyruvate: carbohydrate phosphotransferase system (PTS) in obligately heterofermentative group III lactobacilli including Lactobacillus brevis (3 strains), L. buchneri (2 strains) and L. fermentum (3 strains) was surveyed systematically for a series of sugars utilizable by these organisms. Contrary to common expectation, PTSs were found in two strains of L. fermentum: sucrose-PTS in one strain; sucrose- and mannose-PTSs in the other. All these activities were found to be constitutive.

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Genetic variation among Malaysian isolates of Salmonella typhi was determined by analysis of ribosomal RNA gene restriction patterns. Of the 20 isolates analyzed, eight different pattern combinations were detected. The amount of variation observed was also dependent upon the restriction endonuclease used; PstI produced more different patterns than did SmaI. The results suggested that disease activity was due to a number of different clones circulating simultaneously rather than a single strain. Further implications of the data are discussed.

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A conspicuous adhesion of Staphylococcus aureus organisms to murine cutaneous fibroblasts and NIH/3T3 cells cultured in vitro and subsequent ingestion of S. aureus organisms by these fibroblasts are described. In the present experimental system, only fibroblasts-adhering S. aureus organisms were efficiently ingested by fibroblasts unlike S. epidermidis and S. saprophyticus. These findings might suggest a correlation between the pathogenesis of S. aureus and its intracellular localization in non-professional phagocytes such as fibroblasts in a special reference to its higher pathogenicity than those of coagulase negative counterparts.

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