ABSTRACT
Paracoccidioidomycosis (PCM) is caused by the dimorphic fungus Paracoccidioides brasiliensis. Immunostimulatory effects of P. brasiliensis DNA and CpG-oligodeoxyribonucleotides (CpG-ODN) have shown a Th2-Th1 immunomodulation of the isogenic murine model of susceptibility, which develops a progressive and disseminating disease. In this study, we investigated the optimum time interval and doses of CpG-ODN which are able to induce Th2-Th1 immunomodulation. The optimum concentrations for the induction of a decrease in antibody production were 0.5 and 1 microg. Mice immunized twice with CpG-ODN and gp43 (5 and 7 days before the challenge) showed a 60% higher chance of survival compared with the control group (nonimmunized), and an increase in Th1 isotype (IgG2a) was also observed. In vitro assays of naive and preimmunized mice showed discrete cellular proliferation when stimulated by suitable concentrations of CpG-ODN. Type 1 cytokines interleukin-12 (IL-12) and interferon-gamma were increased in cell culture supernatants, but no significant difference was found in Th2 IL-4 cytokines in stimulated or nonstimulated cell cultures. Concerning the Th2-Th1 kinetics in experimental PCM models by adjuvant effect of CpG-ODN, there are still many questions to be answered and clarified. However, the gathering of data obtained in this investigation has led us to suggest that the modulation of Th2-Th1 in experimental PCM depends on time and CpG-ODN concentration.
Subject(s)
Adjuvants, Immunologic/pharmacology , Antigens, Fungal/genetics , Fungal Proteins/genetics , Glycoproteins/genetics , Oligodeoxyribonucleotides/pharmacology , Paracoccidioides/genetics , Paracoccidioidomycosis/therapy , Th1 Cells/immunology , Th2 Cells/immunology , Animals , Antibodies, Fungal/biosynthesis , Antibodies, Fungal/blood , Cell Proliferation , Cells, Cultured , Cytokines/metabolism , Dose-Response Relationship, Immunologic , Fungal Proteins/chemical synthesis , Genes, Fungal/immunology , Glycoproteins/chemical synthesis , Lymph Nodes/cytology , Lymph Nodes/immunology , Lymph Nodes/metabolism , Mice , Oligodeoxyribonucleotides/chemical synthesis , Paracoccidioides/growth & development , Paracoccidioides/immunology , Paracoccidioidomycosis/immunology , Paracoccidioidomycosis/pathology , Spleen/microbiology , Spleen/pathology , Th1 Cells/drug effects , Th2 Cells/drug effectsABSTRACT
Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature-sensitive phenotype. These mutant cells arrest growth at 37 degrees C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23 degrees C and 37 degrees C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.