Search

Our previous reports have shown that animals inoculated for 8 weeks with 1 X 10(5) pseudohyphal, avirulent C. neoformans exhibited prolonged survival upon challenge with virulent cryptococci. This paper described a transient phase of immunosuppression which occurs during the initial weeks of the immunization protocol. Animals injected with pseudohyphal organisms had depressed responses to T and B-cell mitogens. In addition, they had lowered responses to immunization with sheep erythrocytes. Both humoral and cell mediated responses were affected.

Three antigens were compared for their ability to detect immune responses in C57Bl/6 mice sensitized to Cryptococcus neoformans. Elicitation of responses in vitro was greatest with a urea extract antigen, followed in efficiency by an alkali extract and a soluble capsular polysaccharide preparation. The reactivity paralleled the protein content of the preparation.

Subpopulations of spleen cells responsible for responsiveness and unresponsiveness to cryptococcal antigen in vitro were identified. Lymphocytes which responded in lymphocyte transformation (LT) assays were nylon wool nonadherent and theta antigen positive. These lymphocytes required the presence of an accessory cell which could be supplied by normal peritoneal exudate cells. Spleen cells taken from mice which had been infected for 3 to 15 days were tested to determine their ability to respond to cryptococcal antigen in LT assays. A minimal response was detected at the ninth day of infection. The response of infected spleen cells was attributed to a nonadherent lymphocyte. Nonadherent spleen cells of infected animals had enhanced responses after removal of adherent cells and addition of normal peritoneal exudate cells. Suppressor cells were detected in the spleens of infected mice by the 12th day of infection and thereafter. A nonadherent suppressor cell was identified, but indirect evidence suggested that an adherent cell could also be present in infected spleens.

Soluble extracts of Cryptococcus neoformans were examined for their ability to induce a macrophage-regulatory T-suppressor cell known to appear in the spleens of mice infected with cryptococci. Suppressor cells were induced by injection of extracts of encapsulated or thinly encapsulated strains of cryptococci. Dose-response analysis showed that as little as 25 micrograms of soluble capsular polysaccharide antigen could induce significant suppressor cell activity, with maximum suppression occurring at a dose of 100 micrograms. The suppressor cells appeared within 1 week of injection of antigen and persisted for at least 2 months. Suppressor cells were induced in animals given tolerogenic doses of levan, human gamma globulin, and soluble capsular polysaccharide antigen. When these same antigens were administered in immunogenic form, no suppressor cell activity was detected. Therefore, the suppressive mechanism was common to models of immunologic tolerance and was not unique to cryptococcal disease or cryptococcal capsular polysaccharide antigen. The phagocytosis-inhibiting lymphokine produced by the suppressor cell population completely inhibited the phagocytic activity of only a portion of peritoneal exudate cells. Other macrophages in the population were not totally inhibited but exhibited a reduction in the number of yeast cells engulfed.

The action of Merthiolate on the pathogenic yeasts Blastomyces, dermatitidis, Histoplasma capsulatum, and Sporothrix schenckii was compared to the effect of treatment with formaldehyde. Concentrations of 1:10,000 and 1:5,000 Merthiolate for three exposure times (24, 48, and 72 h) at 4 and 25 degrees C were tested on three media (brain heart infusion with and without blood, and modified Sabouraud agar). The effect of Merthiolate on these three yeasts was primarily fungistatic, with maximum effect using 1:5,000 Merthiolate at 25 degrees C for at least 48 h. Mycelial suspensions of B. dermatitidis, H. capsulatum, S. shenckii, and the yeast phase of Cryptococcus neoformans were susceptible to the 1:5,000 Merthiolate concentration after 24 h of treatment. The antifungal effect of Methiolate varies with species and growth phase of the fungus. Concentration, time of exposure, and temperature of incubation are important variables.

Mice were immunized with a viable, avirulent strain of Cryptococcus neoformans. Lymphocyte blastogenic assays showed a 10-fold increase in reactivity of sensitized spleen cells, and histopathologic examination revealed marked splenic hyperplasia. Thirty-two days after intravenous inoculation with a virulent strain of C. neoformans, none of the control animals survived whereas 60 percent of the immunized mice were alive with no clinical evidence of disease. This animal model shows that protective immunity can be established, and once developed, provide a better model for the study of important aspects of immunity in fungal disease.

A murine model was developed to study the cell-mediated immune response of mice immunized with one of two live, avirulent forms of Cryptococcus neoformans: a nonencapsulated mutant and a thinly encapsulated pseudohyphal variant. A lymphocyte transformation assay was used to evaluate the cellular response of control and sensitized spleen cells after in vitro incubation with three merthiolate-killed whole-cell antigens of C. neoformans. An antigen-to-spleen cell ratio of 10:1 and 5 days of incubation of antigen-spleen cell mixtures were established as optimal conditions for maximum lymphocyte transformation. Maximum responses occurred from 2 to 3 weeks after the last of eight weekly intraperitoneal inoculations of C. neoformans. This assay provided an accurate, reproducible method of studying cell-mediated immunity to C. neoformans, and applications to the study of cryptococcal pathogenesis are proposed.