RESUMEN
Memory T cell populations in patients with paracoccidioidomycosis (PCM) were analyzed before and after chemotherapy treatment. Peripheral blood mononuclear cells (PBMC) collected from patients infected by Paracoccidioides brasiliensis or from non-infected individuals were stimulated in vitro with either membrane and extra-cellular antigens (MEXO) or yeast cell antigen preparation (PbAg) of P. brasiliensis. An increase in the level of CD4(+) memory T cells was determined in PBMC from PCM patients before (NT) and after treatment (TR) and in those with PCM relapsed (RE) compared to that from non-infected controls (NINF). The CD8(+) memory T cells were increased in PBMC from RE patients stimulated with MEXO, but not in NT or TR. The distribution of memory B cells did not differ between NT and TR patients, while a significant elevation was determined in RE patients and higher antibody levels were also detected. The cytokine analysis showed low production of IFN-gamma by cells from RE patients compared with NT or TR patients. In contrast, high production of IL-4 was detected in NT and RE patients, and moderate levels were produced by RE patients. These results suggest that IFN-gamma production may participate in the maintenance of immunological memory in the acquired protection against P. brasiliensis infection and this data can contribute to future development of successful treatment of PCM to avoid relapsing.
Asunto(s)
Antifúngicos/uso terapéutico , Memoria Inmunológica , Paracoccidioides/inmunología , Paracoccidioidomicosis/inmunología , Subgrupos de Linfocitos T/inmunología , Adolescente , Adulto , Anciano , Anticuerpos Antifúngicos/sangre , Anticuerpos Antifúngicos/inmunología , Linfocitos B/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Interferón gamma/sangre , Interferón gamma/inmunología , Interleucina-4/sangre , Interleucina-4/inmunología , Leucocitos Mononucleares/inmunología , Persona de Mediana Edad , Paracoccidioidomicosis/tratamiento farmacológico , Paracoccidioidomicosis/microbiologíaRESUMEN
Allelic variants of cytokine genes seem to be involved in mechanisms of resistance or susceptibility to several diseases. The aim of this study was to investigate the frequency of genotypes with the tumor necrosis factor-alpha TNF-alpha gene polymorphism G/A at position -308 and the IL-10 gene polymorphism G/A at position -1082, and to verify a possible association of these polymorphisms with paracoccidioidomycosis (PCM) caused by Paracoccidioides brasiliensis. Genotyping was performed by allele-specific polymerase chain reaction (ASPCR) and restriction fragment length polymorphism (RFLP) on genomic DNA isolated of granulocytes from 54 PCM patients and 31 noninfected individuals. The analysis of SNP at position -1082 IL-10 showed a high frequency of GA genotype in both patients and controls (51% and 55%, respectively), while the allelic frequency showed 54% of G allele in the patients and 66% of A allele in the controls. The GG genotype was more frequent in patients (85%) and controls (68%) when we analyze the SNP at position -308 of TNF-alpha gene. Otherwise, 91% of PCM patients and 84% of noninfected individuals carried the G allele in -308 TNF-alpha SNP. Stimulation of cells from individuals with PCM phenotyped as A+ (GA or AA genotypes) presented elevation of TNF-alpha producing cells when compared with IL-10-producer cells. These findings reinforce the critical role of IL-10 and TNF-alpha in the paracoccidioidomycosis and can strongly suggest that the genetic screening of the -308G/A and -1082G/A polymorphisms may be a valid tool for identification of subjects needing a more appropriate therapy.
Asunto(s)
Interleucina-10/genética , Paracoccidioides , Paracoccidioidomicosis/genética , Polimorfismo de Nucleótido Simple/genética , Factor de Necrosis Tumoral alfa/genética , Adolescente , Adulto , Anciano , Frecuencia de los Genes , Genotipo , Granulocitos/inmunología , Humanos , Persona de Mediana Edad , Paracoccidioidomicosis/inmunologíaRESUMEN
In this work, we analyzed serological responses of paracoccidioidomycosis (PCM) patients to membrane and extracellular antigens (Mexo) of Paracoccidioides brasiliensis by ELISA, immunoblot technique and immunofluorescence assays to identify a specific antigen profile. Among 140 PCM serum samples analyzed, a homogeneous IgG response to Mexo was observed. The specificity of this antigen was 96.6% in relation to control sera and 81.2% to sera from patients with diverse infections. Patients undergoing treatment for more than 1 year showed a reduced antibody response against Mexo. These results suggest that the presence of anti-Mexo antibodies might be an indicator of active disease. A protein from Mexo with a molecular weight of 28 kDa (Pb28) was the most specific antigen in humoral immune responses to PCM, since it reacted with 100% of patient sera and did not react with heterologous serum samples tested. This protein was purified by molecular filtration chromatography in FPLC system and, when tested by immunoblotting, it maintained its reactivity and specificity of 100% with PCM sera. The Pb28 N-terminal amino acid sequence comparison analysis in the non-redundant GenBank database at NCBI revealed no significant homology to known PCM proteins or to other fungal proteins of known function. Since the 28-kDa protein of P. brasiliensis seems to be specific for PCM, it can be used as an alternative antigen in immunoblotting diagnostic methods.