L. M. Singer-Vermes

The source of the growth-promoting factor(s) affects the plating efficiency of Paracoccidioides brasiliensis

We studied the influence of the growth factor (GF) source, concentration and production time on the plating efficiency of Paracoccidioides brasiliensis yeast cells. The highest plating efficiencies were achieved when the GF was derived from a fast growing P. brasiliensis isolate which was not homologous to the plated samples.

Experimental murine paracoccidioidomycosis: relationship among the dissemination of the infection, humoral and cellular immune responses

The dissemination of Paracoccidioides brasiliensis cells to the heart, omentum/pancreas, spleen, liver and lungs, assessed by colony forming unit (CFU) counts, the levels of specific antibodies to this fungal agent (by ELISA), and the specific DTH reaction were studied in susceptible (B10.A) and resistant (A/Sn) mice. The animals were infected intraperitoneally with P. brasiliensis yeast cells and were evaluated 2, 4, 12 and 16 weeks later. The mosl remarkable differences between the two mouse strains were observed 16 weeks after infection, when B10.A mice displayed high numbers ofCFU in all examined organs, except the heart, high antibody titres, and depressed DTH response. At this point. A/Sn mice presented low or absent CFU in all organs, low antibody litres and expressive DTH response. The CFU counts were shown to be a reliable parameter to discriminate susceptible from resistant animals. The fungal load in the most affected organs correlated wilh the antibody litres and was inversely correlated with the intensity of the DTH reaction. The patterns of immune response in this model mimic human paracoccidioidomycosis, in which high specific antibody levels and depressed DTH reactions are found in multifocal and severe forms of the disease.

In vivo and in vitro characteristics of six Paracoccidioides brasiliensis strains.

The yeast-like forms of six P. brasiliensis strains were characterized and compared using in vitro (growth curve determination) and in vivo (pathogenicity to sensitive inbred mice) criteria. Strains Pb 18 and Pb 265 which behaved similarly in vitro, showing low counts of fungi and long mean generation times, were respectively the most and the least pathogenic strains. Strains Pb 2052 and IVIC Pb 267, which grow abundantly in vitro were, respectively virulent and avirulent. Strains Pb SN and IVIC Pb 9 behaved similarly both in vitro and in vivo displaying an intermediate pattern of virulence and growing conditions.

Alterations in the pathogenicity of oneParacoccidioides brasiliensis isolate do not correlative with itsin vitro growth

Thein vitro subcultivation of some microorganisms for long periods causes measurable loss of their pathogenicity, which can be reverted by reisolation from infected hosts. We compared the pathogenicity and thein vitro growth pattern of oneP. brasiliensis isolate (Pb 18) in its yeast phase, using the following samples: 1) The original pathogenic Pb 18 (OP). 2) Pb 18 attenuated by continuousin vitro subcultivation (AT). 3) Pb 18 (AT) reisolated from susceptible B 10.A mice (RS). 4) Pb 18 (AT) reisolated from resistant A/SN mice (RR). Pathogenicity was evaluated by anatomopathology and mortality of mice infected i.p. with 5×106 fungi. Median survival times of mice infected with OP ranged from 74 to 117 days during the first 51 months of subculturing; with more cycles of subculturing the median survival time increased, reaching 250 days at the 64th month. This indicated decreasing virulence of OP during this period of subculturing. Survival of mice infected with RS and RR was respectively 112 and 123 days, which is similar to the behavior of the OP variant. Thein vitro growth curve profile of RR showed significantly higher numbers of total and viable yeasts than the other studied variant. These results show that: 1) Pb 18 isolate loses its pathogenicity by continuous subcultivation. This phenomenon is reverted by reisolation from mice, independently from their susceptibility to the fungus; 2) thein vitro growth patterns of Pb 18 do not correlate with alterations in pathogenicity but are influenced by the hosts environment.

Depletion of CD8+ T Cells In Vivo Impairs Host Defense of Mice Resistant and Susceptible to Pulmonary Paracoccidioidomycosis

ABSTRACT Using a pulmonary model of infection, we demonstrated previously that A/Sn and B10.A mice are, respectively, resistant and susceptible to Paracoccidioides brasiliensis infection. Employing the same experimental model, we examined herein the role of CD8+ T cells in the course of paracoccidioidomycosis. Treatment with anti-CD8 monoclonal antibodies caused a selective depletion of pulmonary and splenic CD8+ T cells in both mouse strains. The number of pulmonary CD4+ T cells and immunoglobulin-positive cells was independent of the number of CD8+ T cells. In susceptible mice, the loss of CD8+ T cells by in vivo treatment with anti-CD8 monoclonal antibodies impaired the clearance of yeasts from the lungs and increased the fungal dissemination to the liver and spleen. The same treatment in resistant mice increased fungal dissemination to extrapulmonary tissues but did not alter the pulmonary fungal load. Furthermore, CD8+ T-cell depletion did not modify delayed-type hypersensitivity reactions of A/Sn mice but increased these reactions in B10.A mice. The production of P. brasiliensis-specific antibodies by resistant and susceptible mice depleted of CD8+ T cells was similar to that of mice given control antibody. Histopathologically, depletion of CD8+ T cells did not disorganize the focal granulomatous lesions developed by both mouse strains. These results indicate that CD8+ T cells are necessary for optimal clearance of the fungus from tissues of mice infected with P. brasiliensisand demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.

DELAYED-TYPE HYPERSENSITIVITY RESPONSE IN AN ISOGENIC MURINE MODEL OF PARACOCCIDIOIDOMYCOSIS

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 againstP. brasiliensis depend on both the hosts genetically determined resistance and the virulence of the fungal isolate.

Growth curves, morphology and ultrastructure of ten Paracoccidioides brasiliensis isolates

The yeast phase of ten P. brasiliensis isolates were studied to characterize their growth pattern, morphology and ultrastructure. Growth curves were determined after counts of total and viable fungi units (FU) during 20 days. Three growth patterns were observed: slow, reaching approximately 10–30× 106 FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60–150×106 FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180–370×106 FU/tube (Pb 2052 and Pb 192). The highest percentage of viable cells occurred on the 6th day of culture for Pb 192, Pb Campo Grande, Pb 2052 and IVIC Pb 9; on the 8th day for Pb Vitor, Pb SN, Pb 18 and IVIC Pb 267; on the 10th day for Pb 265 and on the 12th day of culture for Pb 2. Mean generation times varied from approximately 21.2 (Pb 2052) to 102.6 hours (Pb 265). The isolates showed similar morphology, except IVIC Pb 267 which did not present a typical yeast-phase at 35°C and the two fast-growing isolates (Pb 2052 and Pb 192) that presented smaller cell sizes and less tendency to clump. The ultrastructure of the isolates was similar: the cell walls presented a width of 0.1 to 0.2 °; the mitochondria presented few cristae and had equivalent patterns of distribution and morphology; the endoplasmic reticulum was scanty, presenting narrow cisternae; the vacuoles, empty or filled with electrondense material, were numerous and two to five nuclei with pores were constantly observed.

Effect of macrophage blockade on the resistance of inbred mice to Paracoccidioides brasiliensis infection.

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 toParacoccidioides 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 afterP. brasiliensis infection. On the whole, macrophage functions appear to play a fundamental role in the natural and acquired resistance mechanisms toP. brasiliensis infection.

Pathogenicity and immunogenicity of Paracoccidioides brasiliensis isolates in the human disease and in an experimental murine model.

The pathogenicity and immunogenicity of six recently isolated Paracoccidioides brasiliensis samples derived from patients presenting distinct and well defined clinical forms of paracoccidioidomycosis (PCM) were compared as to their virulence, tropism to different organs and ability to induce specific cellular and humoral immune response in susceptible (B10.A) inbred mice. Isolates Pb44 and Pb47 were obtained from acute cases, Pb50 from a chronic severe form, Pb45 from a chronic moderate ease and both Pb56 and Pb57 from chronic mild forms of PCM. Pathogenicity and tropism of each fungal sample were evaluated by LD50% estimation, examination of gross lesions on various organs at 2, 4, 12 and 16 weeks post‐in feet ion, and by colony‐forming unit (CFU) counts in the lungs at week 16 post‐infection of mice. Fungal tropism in human PCM and in B10. A mice was always dissociated. A well defined relationship between virulence of the fungal sample and the clinical findings of the correspondent patient was not evident, although a tendency to higher LD50% and less intense paracoccidioidic lesions was observed in mice infected with Pb56 and Pb57. The specific DTH response patterns varied according to the infectant sample, but positive DTH reactions at the beginning of the infection and a tendency to anergy or low DTH responses at week 12 and/or week 16 post‐infection were always observed. A correspondence between the DTH response in humans and in mice was noticeable only when the isolates from the most benign cases (Pb56 and Pb57) were considered. The specific antibody patterns in mice and in the correspondent patients were also not analogous. Collectively, these results indicate that an association between the fungal pathogenicity and immunogenicity in the human disease and in susceptible mice was discernible only when isolates obtained from very mild eases (Pb56 and Pb57) were considered.

Influence of the genetic pattern and sex of mice in experimental paracoccidioidomycosis

Eight genetically different strains of mice were compared regarding the dissemination of Paracoccidioides brasiliensis to the lungs, liver and omentum/pancreas, DTH responses and specific antibody production at 16 weeks after intraperitoneal infection with Pb18, a virulent P. brasiliensis isolate. The degree of dissemination of the infection varied: B10.A and C57Bl/6, the most susceptible mouse strains, had positive cultures and high colony‐forming unit (CFU) counts in all analysed organs. DBA/2 and A/Sn mice had negative cultures, being thus classified as the most resistant strains. CBA/J, C3H/HeJ, F1(A/SnxB10.A) and BALB/c mice were regarded as relatively resistant, since discrete fungal growth was observed only in one or two of the studied organs. All mouse strains, except B10.A mice, produced specific DTH responses which did not seem to be associated with the severity of disease. Production of high levels of specific antibodies was found in all strains except in the DBA/2 and C57B1/6 mice. The influence of the host sex on the outcome of paracoccidioidomycosis was evident only in susceptible animals: female B10.A mice displayed lower CFU counts in the three examined organs, whereas no differences were found between male and female A Sn animals. The higher resistance of female B10.A mice was not accompanied by differences in their capacity to maintain a DTH reaction, nor in their production of antibody. This fact argues against the widely believed association of susceptibility to P. brasiliensis infection with both impaired DTH reactivity and increased humoral response.