Nancy K. Hall

Immunization of mice with an avirulent pseudohyphal form of Cryptococcus neoformans

Mice were immunized with a viable, avirulent strain ofCryptococcus 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 ofC. 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.

Role of macrophages in immunity and pathogenesis of experimental cryptococcosis induced by the airborne route--Part II: Phagocytosis and intracellular fate of Cryptococcus neoformans.

Alveolar and peritoneal macrophages isolated from mice during different stages of cryptococcal infection were able to phagocytize Cryptococcus neoformans. Alveolar macrophages exhibited a fungistatic effect on Cr. neoformans at day 14 post infection, whereas peritoneal macrophages displayed a fungicidal effect on the organism at days 21 and 28 post respiratory exposure to Cr. neoformans. However, these effects were transient and both macrophage populations provided a favorable environment for the intracellular growth of Cr. neoformans as a result of “an immunosuppressive state” which occurred during progressive stages of cryptococcal infection. Therefore, rather than being the main effector arm of acquired immunity against progressive cryptococcosis, the macrophages may contribute to the spread of Cr. neoformans by acting as a mediator for the dissemination of the organism to the central nervous system.

Non-specific immunosuppression by Cryptococcus neoformans infection

Cryptococcus neoformans-infected animals were found to be immunosuppressed when tested by a variety of assays for immune competence. Primary humoral immune responses and delayed-type hypersensitivity reactions to sheep erythrocytes were suppressed in animals which had been infected for two weeks. Lymphocyte proliferation (LP) assays to sRBC stroma were also significantly diminished at two weeks of infection. Spleen cells of infected mice suppressed the LP response of sRBC immunized, normal mice in vitro. At least a part of the suppression could be attributed to a nylon wool non-adherent cell. Suppressor cells continued to be present in spleen cell suspensions following treatment with anti-T cell serum or anti-immunoglobulin and complement. When infected spleen cells were separated by adherence to plastic, both the adherent and non-adherent fractions exhibited suppressive activity. Incubation of infected spleen cells in tissue culture for 48 hr resulted in the elaboration of soluble immunosuppreessive factors into the tissue culture medium. These data indicated that immune suppression in cryptococcosis can occur as a result of infection with Cryptococcus neoformans, and that at least one mechanism involved is the induction of adherent and non-adherent suppressor cells in the spleens of infected mice.

Suppression of responses to cryptococcal antigen in murine cryptococcosis.

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.

Immunosuppression by avirulent, pseudohyphal forms ofCryptococcus neoformans

Our previous reports have shown that animals inoculated for 8 weeks with 1 × 105 pseudohyphal, avirulentC. 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.

Production of specific antibody to Cryptococcus neoformans by hybridomas in vitro

Cells of spleen homogenates from sensitized BALB/c or C57BL/6J mice were fused with cells of a mouse myeloma line (P3X63Ag8 or SP2/0 Ag 14) using polyethylene glycol. Nine of 40 hybridomas from a BALB/c, P3X63Ag8 cross produced anti-cryptococcal antibody. Antibodies of at least four different specificities were identified.

Functional testing and chemical composition of cryptococcal extracts.

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.

Chemical and Serological Comparison of Two Antigen Extracts of Thermoactinomyces candidus

Summary: Immunologically reactive fractions were isolated from two antigen extracts of Thermoactinomyces candidus (Thermoactinomyces pyridine extract‐TPE and double dialysis antigen‐DDA). Activity was demonstrated using immunoelectrophoresis and crossed immunoelectrophoresis against rabbit antisera to T. candidus spores. Chemical analysis of DDA revealed 11 to 16 bands on polyacrylamide gel electrophoresis including five glycoprotein bands. TPE contained 8 to 11 protein bands with 3 glycoprotein components. Both antigens showed variation in sequential preparations.