Yozo Miyakawa

Characterization of Pathogenic Constituents of Cryptococcus neoformans Strains

We examined seven strains, comprising five serotypes, of Cryptococcus neoformans to determine what constituents of the organisms are responsible for pathogenicity and virulence in BALB/c mice. C. neoformans strains were divided into three virulence classes by survival rates after intravenous inoculation of 1 × 105 or 1 × 107 viable cells, and virulence was found not to be correlated with serotype or capsular size. C. neoformans cells resisted phagocytosis in different degrees in the presence of normal serum. Sensitivity of the C. neoformans strains to singlet oxygen ranged from resistance to susceptibility. Histological examination revealed that a weakly encapsulated virulent strain induced inflammatory responses with granuloma formation in the liver, lung, and kidney in addition to formation of cystic foci in the brain. In contrast, although the heavily encapsulated virulent strain produced granulomatous lesions in the liver, this strain preferably produced mucinous cystic foci in the lung, kidney, and brain. Correlation between virulence, and biological, histopathological and physiological evidence suggests that C. neoformans strains are endowed with the implicated multiple pathogenic constituents in various degrees and proportions. The following are suggested as the most important pathogenic constituents: (i) a polysaccharide capsule responsible for resistance to phagocytosis and formation of cystic foci; (ii) a cell surface structure for responsible for resistance to intra‐ or extracellular killing and induction of the granulomatous lesion; (iii) a growth rate suitable for interacting with phagocytic elimination.

Mercuric reductase enzymes from Streptomyces species and group B Streptococcus

Mercury volatilization (Hg2+ reductase) activity has been found with Hg2+-resistant isolates of three Streptomyces species and with three Hg2+-resistant strains of group B Streptococcus from clinical sources in Japan. Hg2+ reductase activities in crude cell extracts showed the temperature sensitivity, the requirement for an added thiol compound and the characteristic dependence on NAD(P)H cofactors of similar enzymes isolated from other bacteria.

Suppression of HIV-1 Reverse Transcriptase Activity by Culture Supernatants of Mycoplasmas

Coinfection with mycoplasmas has been shown to enhance cytopathic changes in T lymphocytes in culture brought about by human immunodeficiency virus type‐1 (HIV‐1), accelerate disease progression, and suppress reverse transcriptase (RT) activity simultaneously. We attempted to identify the components in culture supernatants of mycoplasmas which suppress RT activity. The marked inhibitory effect on RT by culture supernatants was dependent upon Mg2+. The culture supernatants exhibited the activities of DNase and RNase, which degraded the products and substrates in RT assay, respectively. Gel filtration studies revealed that two major protein peaks, peak 1 (MW 67‐100 kDa) and peak 2 (MW 10‐25 kDa), exhibited DNase and/or RNase activities, and that both peaks contained a significant degree of inhibitory activity on RT. These results indicate that suppression of RT activity by the culture supernatants of mycoplasmas is due to DNase and RNase activities in the culture supernatants. The results of the present investigation suggest that RT assay of certain biological materials that are contaminated with mycoplasmas must be conducted carefully.

Electrophoretic Patterns of Extracellular Deoxyribonuclease (DNase) and Their Correlation with T-Type in Group A Streptococci

Molecular heterogeneity of the extracellular deoxyribonuclease (DNase) in group A streptococci was demonstrated in 42 clinical isolates. Although Polyacrylamide gel electrophoretic patterns of the extracellular DNase of all the isolates were heterogeneous, they could be divided into five main patterns with respect to the presence or absence of three DNase components including DNase B. By comparing the electrophoretic patterns of DNase in all the isolates with their T‐types, we found that the patterns were quite characteristic for their T‐types, especially in the prevalent T‐types 12 and 1, and that the isolates of T‐types 12 and 1 produced DNase B as their major extracellular DNase. Relative DNase B activity in the total extracellular DNase activity of group A, B, and G isolates was determined by the rapid method of neutralization with anti‐DNase B antibody. The results showed neutralization of DNase activity in all the isolates of group A streptococci, largely corresponding to their T‐types, but not of the isolates of groups B and G. These results indicate that the electrophoretic patterns of the extracellular DNase of group A streptococci are closely correlated with their T‐types, suggesting the physicochemical taxonomic value of these properties.

Antigen-specific suppression of antibody responses by T lymphocytes cytotoxic for antigen-presenting cells

In this study we demonstrated an alternative model of the antigen (Ag)‐specific suppression of antibody response in mice. Splenocytes that were taken from BALB/c mice immunized by i.v. injection of soluble human serum albumin (HSA) or ovalbumin exhibited MHC‐restricted Ag‐specific cytotoxicity for the respective antigen‐presenting cells (APC). When HSA‐primed splenocytes cultured with Ag and interleukin‐2 (IL‐2) were treated with anti‐CD4 or anti‐CD8 monoclonal antibody (mAb) plus complement, CD8+ and CD4+ T cells exhibited nearly the same level of cytotoxicity against APC. Furthermore, HSA‐primed CD4+ and CD8+ T cells released the same amount of interferon‐γ (IFN‐γ) when stimulated with Ag and IL‐2. Recombinant IFN‐γ was shown to suppress the in vitro plaque‐forming cell (PFC) response to sheep red blood cells (SRBC) only when it was added within 24 h after addition of Ag. The supernatants from both HSA‐primed CD4+ and CD8+ T cells suppressed the PFC response to SRBC in vitro, and the suppressive activity was abrogated by anti‐IFN‐γ mAb, but increased by anti‐IL 4 mAb. These results suggest that in our system the effector cells for Ag‐specific suppression of the antibody response in mice are both the cytotoxic type 1 clones (IFN‐γ‐producing) of CD4+ and CD8+ T cells for APC, and that IFN‐γ is a major extracellular effector molecule for such suppression.

Characteristics of Macrophage Activation by Gamma Interferon for Tumor Cytotoxicity in Peritoneal Macrophages and Macrophage Cell Line J774.1

We investigated the characteristics of macrophage‐mediated tumor cytotoxicity (MTC) against Meth A target, H2O2 generation and release of effector molecule(s) for MTC, by comparing with those of peritoneal macrophages (PMP) and macrophage cell line J774.1 during stimulation with recombinant gamma interferon (IFN‐γ). In PMP, MTC was demonstrated when they were stimulated with IFN‐γ for 12 hr (short‐term stimulation) and was abrogated when they were stimulated for 48 hr (long‐term stimulation). Enhanced H2O2 generation was observed in PMP activated by long‐term stimulation followed by triggering with PMA, but not observed by triggering with Meth A cells. By contrast, whereas non‐treated J774.1 cells have already attained a definite level of MTC, a higher MTC level was demonstrated both by short‐ and long‐term stimulations. Conversely, J774.1 cells were unable to generate H2O2 at any stage of IFN‐γ stimulation followed by triggering both with PMA or Meth A cells. The time course for stimulation of PMP by IFN‐γ for release of cytotoxic factor (CF) corresponded to that for MTC by PMP, and activities of the CF released from both activated PMP and J774.1 cells also closely corresponded to those of MTC by both cells. The serological and physicochemical characteristics of CF released from both activated PMP and J774.1 cells were determined to be closely related to those of tumor necrosis factor (TNF). These results indicate that in contrast to PMP, the J774.1 cell line is free from suppression stage for MTC and CF release during stimulation with IFN‐γ. The results suggest that TNF‐like CF plays a crucial role for MTC against Meth A target, and that H2O2 is irrelevant for MTC against Meth A.