M. 20), infecting approximately 500, 000 individuals each year. The spectrum of diseases due to this fungus is usually broad. The majority of individuals develop a moderate, subclinical contamination, but symptomatic diseases range from an influenza-like illness to a chronic cavitary pulmonary disease or a highly lethal disseminated contamination, particularly in immunosuppressed individuals, such as individuals with AIDS (21, 24) or patients receiving potent immunomodulators, such as tumor necrosis factor alpha (TNF-) inhibitors (10, 11). Host protective immunity can Chloroxylenol be induced via conversation of var. var. (14, 19, 36). In particular, a heat shock protein with a molecular mass of 62 kDa (Hsp60) is an immunodominant antigen that can induce protective cellular responses, which are strictly dependent on the presence and activation of a specific V8.1/8.2+ subset of CD4+ T lymphocytes (14, 19). Antibody responses in histoplasmosis have been characterized previously (23-25, 35, 40), but the role of the antibodies in pathogenesis is usually unclear. Depletion of CD4+ and CD8+ T cells in B-lymphocyte knockout mice results in fungal burdens in organs after contamination with var. that are markedly higher than those in wild-type animals (3), suggesting that an antibody may collaborate with T cells in combating histoplasmosis. Consistent with the conflicting results of previous experiments with polyclonal sera for fungal infections such as cryptococcosis (8, 9, 29), administration of polyclonal serum from mice immune to var. did not protect na?ve mice from histoplasmosis (39). Interestingly, in vivo experiments with specific monoclonal Chloroxylenol antibodies (MAbs) to the capsular polysaccharide of the fungus have revealed the presence of protective, nonprotective, and disease-enhancing MAbs, suggesting that this divergent results obtained with polyclonal preparations may be a result of the relative proportions of protective and nonprotective antibodies in immune sera (9, 16). We recently described MAbs that change the pathogenesis of experimental histoplasmosis. Administration of immunoglobulin M (IgM) isotype MAbs that bind a histone 2B-like protein (H2B) on the surface of var. yeast cells reduces the fungal burden, decreases pulmonary inflammation, and prolongs survival in a murine model of contamination (32, 33). These protective MAbs have been associated with enhanced levels of IL-4, IL-6, and IFN- in the lungs of infected mice. Additionally, the IgM MAbs increase phagocytosis of the yeast by J774.16 cells through a CR3-dependent process and inhibit yeast cell growth in macrophages, altering the intracellular fate of the fungus (32, 33, 37). However, the penetration of IgM MAbs into the lung is usually problematic, and the protective results obtained with the MAbs to H2B were improved when the fungus was opsonized with the MAbs prior to contamination. Our current work sought to ascertain whether the administration of IgG isotype MAbs was more effective in modifying histoplasmosis. Therefore, we generated a panel of IgG MAbs against Hsp60 from var. and found that the IgG1 and IgG2a MAbs dramatically altered var. pathogenesis. Interestingly, an IgG2b MAb was not protective, indicating that isotype may impact efficacy. (The data in this paper are from a thesis to be submitted by A. J. Guimar?es in partial fulfillment of the requirements for a Ph.D. degree from the Sue Golding Graduate Division of Medical Science, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY.) MATERIALS AND METHODS Fungal strains and growth conditions. The reference strain var. G217B was obtained from the American Type Culture Collection (Rockville, MD). var. yeast cells were produced in Ham’s F-12 medium at 37C with rotary shaking as described previously (4). Generation of MAbs against rHsp60. Animal experiments were performed according to the guidelines of the Institute for Animal Studies of the Albert Einstein College of Medicine. made up of the pET21 plasmid harboring the var. Hsp60 gene was a gift from G. Deepe (University of Cincinnati, Cincinnati, OH). Recombinant Hsp60 (rHsp60) was purified as described previously (14). Two female BALB/c mice (6 to 8 8 weeks old; Jackson Mouse monoclonal to CER1 Immunoresearch Labs, West Grove, PA) were immunized intraperitoneally with 50 g of rHsp60 suspended in a 1:1 (vol/vol) emulsion of complete Freund’s adjuvant (Sigma-Aldrich) and phosphate-buffered saline (PBS). Additional doses were administered 2 and 4 weeks after the first immunization in 1:1 (vol/vol) emulsions of incomplete Freund’s adjuvant (Sigma-Aldrich). Sera were obtained before each immunization and 2 weeks Chloroxylenol after the last immunization and analyzed for the presence of antibodies to rHsp60 from var. using an indirect enzyme-linked immunosorbent assay (ELISA) that.
