RESUMEN
Adult Swiss (susceptible) and BALB/c (non-susceptible) mice were inoculated by the intravenous route with 1 x 10(6) yeast cells of Paracoccidioides brasiliensis, strain 18. Immunologic parameters, histopathology and features of the bronchoalveolar lavage (BAL) were evaluated at week 2, 4, 8 and 16 post-infection. The pulmonary infection was progressive in Swiss mice and regressive in Balb/c mice. The numbers of total cells, lymphocytes and polymorphonuclear neutrophils increased in BAL, as well as the percentages of giant cells, and CD4 and CD8 positive cells. The ultrastructural study of BAL cells revealed a predominance of macrophages and a frequency of 13.2% of type II pneumocytes. As the infection progressed, the number of fungal cells and spreading macrophages, as well as the stimulated release of H2O2 by macrophages, increased. The animals exhibited an exacerbation of the humoral immune response and a depression of cellular immunity during the infection. There was a good correlation between the intensity and the pattern of the pulmonary histopathology and the cellular findings in the BAL. The present model reproduces some anatomoclinical patterns of the human disease and shows that BAL may be a useful tool in monitoring the pulmonary infection caused by P. brasiliensis.
Asunto(s)
Líquido del Lavado Bronquioalveolar/citología , Enfermedades Pulmonares Fúngicas/patología , Pulmón/patología , Paracoccidioidomicosis/patología , Animales , Anticuerpos Antifúngicos/sangre , Líquido del Lavado Bronquioalveolar/inmunología , Líquido del Lavado Bronquioalveolar/microbiología , Recuento de Células , Ensayo de Inmunoadsorción Enzimática , Peróxido de Hidrógeno/metabolismo , Hipersensibilidad Tardía , Inmunidad Innata , Inmunodifusión , Pulmón/inmunología , Pulmón/microbiología , Enfermedades Pulmonares Fúngicas/inmunología , Enfermedades Pulmonares Fúngicas/microbiología , Macrófagos Alveolares/fisiología , Macrófagos Alveolares/ultraestructura , Masculino , Ratones , Ratones Endogámicos BALB C , Paracoccidioides/inmunología , Paracoccidioides/aislamiento & purificación , Paracoccidioidomicosis/inmunología , Paracoccidioidomicosis/microbiología , Subgrupos de Linfocitos TRESUMEN
The effect of macrophage blockade on the natural resistance and on the adaptative immune response of susceptible (B10.D2/oSn) and resistant (A/Sn) mice to Paracoccidioides brasiliensis infection was investigated. B10.D2/oSn and A/Sn mice previously injected with colloidal carbon were infected ip with yeast cells to determine the 50% lethal dose, and to evaluate the anatomy and histopathology, macrophage activation, antibody production and DTH reactions. Macrophage blockade rendered both resistant and susceptible mice considerably more susceptible to infection, as evidenced by increased mortality and many disseminated lesions. P. brasiliensis infection and/or carbon treatment increased the ability of macrophages from resistant mice to spread up to 25 days after treatment. In susceptible mice the enhanced spreading capacity induced by carbon treatment was impaired at all assayed periods except at 1 week after infection. Macrophage blockade enhanced DTH reactions in resistant mice, but did not alter these reactions in susceptible mice, which remained anergic. To the contrary, macrophage blockade enhanced specific antibody production by susceptible mice, but did not affect the low levels produced by resistant mice. The effect of macrophage blockade confirms the natural tendency of resistant animals to mount DTH reactions in the course of the disease and the preferential antibody response developed by susceptible mice after P. brasiliensis infection. On the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms to P. brasiliensis infection.
Asunto(s)
Macrófagos Peritoneales/inmunología , Paracoccidioidomicosis/inmunología , Animales , Anticuerpos Antifúngicos/sangre , Carbono/farmacología , Movimiento Celular/efectos de los fármacos , Coloides/farmacología , Susceptibilidad a Enfermedades , Femenino , Hipersensibilidad Tardía , Inmunidad Innata , Inmunoglobulina G/sangre , Dosificación Letal Mediana , Activación de Macrófagos , Ratones , Ratones Endogámicos , Paracoccidioides/inmunología , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/patologíaRESUMEN
The specific delayed-type hypersensitivity (DTH) response was evaluated in resistant (A/SN) and susceptible (B10.A) mice intraperitoneally infected with yeasts from a virulent (Pb18) or from a non-virulent (Pb265) Paracoccidioides brasiliensis isolates. Both strains of mice were footpad challenged with homologous antigens. Pb18 infected A/SN mice developed an evident and persistent DTH response late in the course of the disease (90th day on) whereas B10.A animals mounted a discrete and ephemeral DTH response at the 14th day post-infection. A/SN mice infected with Pb265 developed cellular immune responses whereas B10.A mice were almost always anergic. Histological analysis of the footpads of infected mice at 48 hours after challenge showed a mixed infiltrate consisting of predominantly mononuclear cells. Previous infection of resistant and susceptible mice with Pb18 did not alter their DTH responses against heterologous unrelated antigens (sheep red blood cells and dinitrofluorobenzene) indicating that the observed cellular anergy was antigen-specific. When fungal related antigens (candidin and histoplasmin) were tested in resistant mice, absence of cross-reactivity was noted. Thus, specific DTH responses against P. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.
Asunto(s)
Hipersensibilidad Tardía , Paracoccidioidomicosis/inmunología , Animales , Antígenos Fúngicos , Reacciones Cruzadas , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos A , Paracoccidioides/inmunología , Paracoccidioides/patogenicidad , Paracoccidioidomicosis/patología , Virulencia/inmunologíaRESUMEN
The interaction of human monocytes or monocyte-derived macrophages and yeast-form Paracoccidioides brasiliensis was studied in vitro. Yeast cells were readily ingested by adherent monocytes or macrophages. Multiplication of P. brasiliensis, measured by growth as colony forming units (cfu) on a supplemented medium with good plating efficiency, was greater in monocyte co-cultures compared to the number of cfu obtained from complete tissue-culture medium (CTCM). Multiplication increased with time in macrophage co-cultures, e.g., from two-six-fold in 24 h to nine-fold in 72 h. Microscopic observations indicated that ingested yeast cells multiplied inside macrophages. When monocytes were treated with supernate cytokines (CK) from concanavalin-A-stimulated mononuclear cells, then co-cultured with P. brasiliensis, multiplication was significantly inhibited compared with control monocyte co-cultures. Treatment of macrophages--derived from monocytes by culture in vitro for 3 days--for a further 3 days with CK resulted in maximal inhibition of multiplication over the subsequent 72 h. Similarly, when monocyte-derived macrophages (after culture for 7 days) were treated for 3 days with recombinant human gamma-interferon (IFN; 300 U/ml) or CK they restricted multiplication of P. brasiliensis by 65% and 95%, respectively, compared with control macrophages. Antibody to IFN abrogated the effect of IFN or CK treatment. These findings show that ingested P. brasiliensis can multiply in human monocytes or macrophages and that this multiplication can be restricted by activated monocytes or macrophages.
Asunto(s)
Citocinas/inmunología , Macrófagos/microbiología , Monocitos/microbiología , Paracoccidioides/crecimiento & desarrollo , Fagocitos/microbiología , Adulto , Adhesión Celular , Células Cultivadas , Recuento de Colonia Microbiana , Medios de Cultivo , Humanos , Interferón gamma/inmunología , Linfocitos/microbiología , Activación de Macrófagos , Macrófagos/inmunología , Monocitos/inmunología , Paracoccidioides/inmunología , Fagocitos/inmunología , Fagocitosis , Proteínas RecombinantesRESUMEN
The density and distribution of T cells, T helper cells, macrophages and B cells at the site of skin tests with a cytoplasmic Paracoccidioides brasiliensis antigen (paracoccidioidin) was studied at 24 and 48 h post-challenge in 10 patients with the chronic form of paracoccidioidomycosis and in 5 non-infected individuals. The in situ study was carried out using immunoperoxidase techniques and monoclonal antibodies. The controls showed negative skin test. In the patients, the great majority of the cells in the perivascular foci were T cells (CD43-positive cells) making up 47% and 48.6% of the total number of cells at 24 and 48 h respectively. Most of the T cells showed a T helper phenotype (CD45RO-positive cells). Approximately 25% of the cells were macrophages (CD68-positive cells) and there were very few B lymphocytes (CD20-positive cells). The present data on the microanatomy of paracoccidioidin skin test sites were consistent with a delayed type hypersensitivity pattern. Our results were comparable to those reported on skin tests for other granulomatous chronic diseases.
Asunto(s)
Proteínas Fúngicas/inmunología , Hipersensibilidad Tardía/inmunología , Leucocitos Mononucleares/inmunología , Paracoccidioides , Paracoccidioidomicosis/inmunología , Adulto , Enfermedad Crónica , Humanos , Técnicas para Inmunoenzimas , Inmunofenotipificación , Pruebas Intradérmicas , MasculinoRESUMEN
Immunohistochemical techniques using monoclonal antibodies to T lymphocyte subsets were used to characterize granulomas caused by Paracoccidioides brasiliensis. Punch biopsies of skin or mucosa from eight patients and of lymph nodes from two patients with disseminated paracoccidioidomycosis were studied. The T lymphocytes were distributed either in a localized pattern related to epithelioid granulomas or in a diffuse arrangement, predominantly around the vessels. In the granulomas, T cells formed a peripheral mantle surrounding central aggregates of macrophages. The majority of lymphocytes were T-helper cells with few suppressor cells. In contrast, patients presented with a decreased number of peripheral T-helper lymphocytes and a corresponding decrease in helper-suppressor cell ratios. There was no clear-cut relationship between tissue helper-suppressor cell ratios and the level of cellular immunodepression of the patients. The lowest P. brasiliensis antibody titers were detected in patients with the highest tissue helper-suppressor cell ratios. The distribution pattern of T lymphocytes in P. brasiliensis granulomas, with a predominance of helper phenotype, suggests that these cells are actively involved in the disease process.
Asunto(s)
Granuloma/inmunología , Paracoccidioidomicosis/inmunología , Linfocitos T , Adolescente , Adulto , Anticuerpos Antifúngicos/biosíntesis , Biopsia con Aguja , Niño , Femenino , Humanos , Inmunidad Celular , Inmunohistoquímica , Ganglios Linfáticos/patología , Masculino , Persona de Mediana Edad , Membrana Mucosa/patología , Paracoccidioides/inmunología , Piel/patología , Linfocitos T Colaboradores-Inductores , Linfocitos T ReguladoresRESUMEN
We carried out a comparative study of the histopathology (lung, liver, spleen, kidney and adrenals) and the anti-P. brasiliensis humoral (immunodiffusion test) and cellular (footpad test) immune response of mice intravenously inoculated with yeast forms of three P. brasiliensis isolates (Pb 18, Pb 192, Pb 265). Pb 265 (avirulent strain) did not evoke specific lesions or antibody production; the levels of cellular immunity were significantly lower than those of the two other isolates. Lung granulomas induced by strain Pb 18 were richer in fungi and neutrophils and poorer in mononuclear cells when compared to those induced by strain Pb 192. Extrapulmonary lesions were more frequent in mice infected with strain Pb 18. Strains Pb 18 and Pb 192 raised similar humoral and cellular anti-P. brasiliensis responses. Cell wall analysis did not suggest striking differences among the strains. Slightly higher levels of the water soluble fraction 3 (which contains the immunogenic galactomannan and protein) were detected in strain Pb 265.