Depletion of CD8(+) T cells in vivo impairs host defense of mice resistant and susceptible to pulmonary paracoccidioidomycosis.

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. brasiliensis and demonstrate more prominent protective activity by those cells in the immune responses mounted by susceptible animals.

Protective role of gamma interferon in experimental pulmonary paracoccidioidomycosis.

We have developed a murine model of pulmonary infection by Paracoccidioides brasiliensis in which resistance was associated with immunological activities governed by gamma interferon (IFN-gamma). To better characterize this model, we measured type 1 and type 2 cytokines in the lungs and investigated the effect of endogenous IFN-gamma depletion by monoclonal antibodies in the course of infection of susceptible (B10.A) and resistant (A/Sn) mice. At weeks 4 and 8 after infection, lungs from susceptible animals presented levels of IFN-gamma, interleukin-4 (IL-4), IL-5, and IL-10 higher than those in resistant mice. In both mouse strains, neutralization of endogenous IFN-gamma induced exacerbation of the pulmonary infection, earlier fungal dissemination to the liver and spleen, impairment of the specific cellular immune response resulting in significantly lower delayed-type hypersensitivity reactions, and increased levels of immunoglobulin G1 (IgG1)- and IgG2b-specific antibodies. Histopathological analysis demonstrated that depletion of IFN-gamma changes the focal granulomatous lesions found in the lungs of B10.A and A/Sn mice into coalescent granulomata which destroy the pulmonary architecture. These results suggest that irrespective of the mouse strain, IFN-gamma plays a protective role and that this cytokine is one major mediator of resistance against P. brasiliensis infection in mice.

Comparative studies on the antibody repertoire produced by susceptible and resistant mice to virulent and nonvirulent Paracoccidioides brasiliensis isolates.

The specific recognition pattern of antibodies produced by susceptible and resistant mice infected with the low virulence Paracoccidioides brasiliensis isolate (Pb265) was examined by an immunoblotting procedure and compared with that of antibodies produced by highly virulent isolate (Pb18) in infected mice. Both mouse strains produced IgG antibodies to 13 of the 16 major antigen bands, and showed a recognition pattern similar to sera from mice infected with the virulent isolate. Nevertheless, the reaction to components interposed among major bands (intermediate antigen bands of 75, 73, 68, 64, 33, 23, 22, and 12.5 kD) were detected exclusively with antibodies raised in response to the virulent P. brasiliensis isolate independent of the resistance pattern of the host. It was also demonstrated here that the most diversified repertoire of specific IgA was produced when the susceptible host and virulent fungus were associated.

Paracoccidioides brasiliensis infection in nude mice: studies with isolates differing in virulence and definition of their T cell-dependent and T cell-independent components.

Athymic and euthymic BALB/c mice infected with highly (Pb18) or slightly (Pb265) virulent Paracoccidioides brasiliensis isolates were compared regarding mortality, presence of viable yeasts, specific immunoglobulin M (IgM) and IgG titers, and the antigen recognition patterns of these antibodies. Isolate Pb18 caused a more severe disease in athymic mice, as supported by higher number of infected organs and shorter survival times. These animals, however, were resistant to Pb265 infection. High titers of antibodies were found only in euthymic mice, seven weeks after Pb18 infection. At this time, euthymic animals presented IgG antibodies to numerous protein bands that were not detected at four weeks postinfection or after Pb265 inoculation. In contrast, antibodies from athymic mice always reacted with few antigen bands. Although the majority of P. brasiliensis antigens are T cell-dependent, the immunodominant gp43 and also the 41.5- and 27.5-kD antigens are here, for the first time, characterized as T cell-independent antigens of P. brasiliensis.

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 C57B1/6, 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.

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 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.

Pulmonary paracoccidioidomycosis in resistant and susceptible mice: relationship among progression of infection, bronchoalveolar cell activation, cellular immune response, and specific isotype patterns.

Using the intraperitoneal route of infection, we demonstrated previously that A/Sn mice are resistant and B10.A mice are susceptible to Paracoccidioides brasiliensis infection. Since paracoccidioidomycosis is a deep systemic granulomatous disorder that involves primarily the lungs and then disseminates to other organs and systems, we herein investigated the course of the infection and the resulting immune responses developed by A/Sn and B10.A mice after intratracheal infection with P. brasiliensis yeast cells. It was observed that A/Sn mice develop a chronic benign pulmonary-restricted infection, whereas B10.A mice present a chronic progressive disseminated disease. A/Sn animals were able to restrict fungal infection to the lungs despite the increased fungal load at the beginning of the infection. This behavior was associated with low mortality rates, the presence of adequate and persistent delayed-type hypersensitivity reactions, oxidative burst by bronchoalveolar cells, and production of high levels of specific antibodies in which immunoglobulin G2a (IgG2a) and IgG3 isotype titers were significantly higher than those observed in the susceptible mice. In contrast, B10.A animals showed a constant pulmonary fungal load and dissemination to the liver and spleen. This infection pattern resulted in high mortality rates, discrete delayed-type hypersensitivity reactivity, poorly activated or nonactivated bronchoalveolar cells, and production of specific IgG2b isotype titers significantly higher than those observed in the resistant mice at week 4 of infection. Thus, A/Sn and B10.A mice maintain the same resistance patterns as those observed previously with the intraperitoneal route of infection. Furthermore, the obtained results suggest that resistance to paracoccidioidomycosis is associated with T-cell, macrophage, and B-cell activities that are known to be mediated by gamma interferon.

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 case 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-infection, 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 cases (Pb56 and Pb57) were considered.

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 against P. brasiliensis depend on both the host's genetically determined resistance and the virulence of the fungal isolate.

Plasma amylase levels as a marker of disease severity in an isogenic murine model of paracoccidioidomycosis.

Survival patterns after peritoneal infection with Paracoccidioides brasiliensis vary according to the mouse strain and to the virulence of the fungal isolate. It has previously been observed that a significant increase in plasma amylase levels occurs only when susceptible mice (B10.A) were infected with a virulent isolate (Pb18). In order to verify if increased amylase levels correlate with susceptibility to P. brasiliensis infection, 12 mouse strains with different susceptibility patterns to this fungus were investigated after infection with Pb18. When compared with their respective controls, C57BI/6, B10D2/oSn, B10D2/nSn, C3H/HeJ, B10.A and BALB/c mice showed a conspicuous amylase increase and AKR, (NZB x NZW)F1, CBA/J, (A/Sn x B10.A)F1, A/Sn and DBA/2 absence of alteration. The influence of the infecting fungal isolate on this enzymatic parameter was investigated using B10.A mice and fungal isolates with diverse degrees of virulence. When compared with their non-infected controls, mice infected with Pb45 or Pb47 showed a very high amylase increase, with Pb44 or Pb18 a high one and with Pb50 or Pb265 a discrete increase. On the whole, there is an inverse correlation between survival times after infection and the increase in amylase levels. Thus, measurement of plasma amylase is a satisfactory parameter to evaluate the severity of paracoccidioidomycosis in mice.

Gelatinase activity of exoantigens from virulent and non-virulent isolates of Paracoccidioides brasiliensis.

Differences in the occurrence of components with gelatinase activity were detected among four isolates of Paracoccidioides brasiliensis: Pb339 and Pb18 (highly virulent), and Pb265 and Pb18AV (very low virulence). Culture filtrates from these isolates were electrophoresed in substrate gels and tested for gelatinase activity. Pb339 showed three enzyme bands of apparent molecular masses: 43, 53 and 78 kDa; Pb18 had two bands, one at 59 kDa and another with molecular mass higher than 78 kDa. Isolate Pb18AV showed only one band at 78 kDa and Pb265 exhibited a component of molecular mass which failed to enter the separating gel.

Cytokines in the host response to mycotic agents.

In summary, different approaches have been taken to understand cytokine responses to different fungal infections. Singer-Vermes and co-investigators indirectly examined cytokine responses to paracoccidioidomycosis by studying the types of cellular and humoral immune responses that were induced in resistant and susceptible mouse strains. Their results implicated Th1 cell responses in the resistant mouse strain and Th2 cell responses in the mouse strain susceptible to paracoccidioidomycosis. By measuring cytokine production and through cytokine depletion experiments, Wu-Hsieh showed that besides IFN gamma, TNF alpha was important in host defences against the intracellular pathogen, H. capsulatum. Both cytokines play important roles in the regulation of other cytokines. In histoplasmosis, the dynamics of the complex interactions amongst cytokines govern the efficiency of host clearance of the fungus from tissues. Ferrante and collaborators, examining TNF alpha and TNF alpha receptors on neutrophils presented data showing that TNF alpha plays an important role in the activation of neutrophils for anti-Candida activity. Through the detection of cytokine mRNAs with RT-PCR, Moser and co-workers found that cytokine mRNAs of macrophage origin were produced preferentially in the lungs of mice infected with Histoplasma or Blastomyces. A great challenge still lies ahead of us. It is well understood that the interactions of cytokines are extremely complex at the levels of the induction and expression of the immune responses as well as on effects on natural cellular defences. Work accomplished thus far has laid the ground work for future studies in the effort to dissect host cytokine responses and to understand the roles of cytokines in protection against fungal infections.

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 most remarkable differences between the two mouse strains were observed 16 weeks after infection, when B10.A mice displayed high numbers of CFU 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 titres 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 with the antibody titres 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.

Advances in experimental paracoccidioidomycosis using an isogenic murine model.

A genetically controlled murine model of paracoccidioidomycosis which allowed us to investigate several parameters of the host-parasite interactions was established in our laboratory. Natural resistance and acquired immune responses to P. brasiliensis infection were investigated employing resistant and susceptible mice infected with highly virulent or slightly virulent P. brasiliensis isolates. Resistant mice inoculated with a highly virulent P. brasiliensis isolate present efficient macrophage activation, presence of DTH response, low levels of specific antibody and a tendency to resolution of the infectious process, suggesting that a T helper-1 mode of immune response is mounted. Susceptible mice, on the contrary, seem to mount a predominantly T helper-2 type of immune response activation in which an inefficient macrophage activation, depressed DTH reactions and high levels of antibodies result in progressive disease. The crucial role of the fungal virulence on the outcome of the infection of susceptible and resistant mice is also demonstrated, thus reinforcing the idea that both the innate resistance of the host and the pathogenicity of the fungal cells are determinant on the outcome of the disease. This model is proposed as a framework of our current knowledge of the host-parasite interactions in paracoccidioidomycosis and as a basis for future challenge in continuing analyses.

Specific recognition pattern of IgM and IgG antibodies produced in the course of experimental paracoccidioidomycosis.

Specific IgM and IgG responses to Paracoccidioides brasiliensis produced in resistant and susceptible mice during experimental paracoccidioidomycosis were examined by the immunoblotting procedure. Sera from infected mice recognized 51 antigen bands with apparent molecular masses from 8 to 86 kD. Sixteen of these were defined as major antigen bands because of almost universal presence of antibodies to them, and their intense staining. All sera, including those from normal control mice, tested for both IgM and IgG antibody reacted with the major E antigen which appeared as a large diffuse band from 43 to 47 kD. Comparisons between resistant and susceptible mice showed some significant differences in IgM responses to many antigen bands. While IgG responses were quite similar for both strains, differences were apparent in the response to the antigens at 62 and 68 kD.

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.

Alterations in the pathogenicity of one Paracoccidioides brasiliensis isolate do not correlative with its in vitro growth.

The in 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 the in vitro growth pattern of one P. brasiliensis isolate (Pb 18) in its yeast phase, using the following samples: 1) The original pathogenic Pb 18 (OP). 2) Pb 18 attenuated by continuous in vitro subcultivation (AT). 3) Pb 18 (AT) reisolated from susceptible B10.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 x 10(6) 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. The in 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) the in vitro growth patterns of Pb 18 do not correlate with alterations in pathogenicity but are influenced by the host's environment.

Evaluation of the pathogenicity and immunogenicity of seven Paracoccidioides brasiliensis isolates in susceptible inbred mice.

In this investigation the pathogenicity and immunogenicity of seven isolates of Paracoccidioides brasiliensis were compared. The pathogenicity of each isolate was determined by 50% lethal dose estimations and histopathology analysed during a 24-week-period. Four basic patterns of virulence could be defined after intraperitoneal infection of susceptible, genetically homogenous, B10.A mice, namely, slightly virulent (isolates Pb 265 and IVIC Pb 267), intermediate (isolates Pb 192, IVIC Pb 9 and Pb SN), virulent (isolate Pb 2052) and highly virulent (isolate Pb 18). The granulomas induced by the individual isolates were similar although the evolution of inflammation and the organs affected varied according to the isolate used. The immunogenicity of each P. brasiliensis isolate was evaluated by measuring IgG titres with an ELISA method. The intermediate and the slightly virulent isolates induced weak antibody production whereas isolates Pb 18 and Pb 2052 induced stronger specific humoral responses. Differences in the kinetics of antibody production elicited by the different fungal isolates were also observed.

Suppression of IgE antibody production against an unrelated antigen in experimental murine paracoccidioidomycosis.

A suppression of the IgE antibody response to ovalbumin was obtained in susceptible mice infected with Paracoccidioides brasiliensis yeast cells a few days prior to immunization with the former antigen plus adjuvant. A direct relationship between the number of injected fungi and the suppressive effect was established. When infection with a pathogenic isolate of P. brasiliensis (Pb 18) was compared to a non-pathogenic isolate (IVIC Pb267), the IgE anti-ovalbumin response was reduced by both. A similar effect was observed if mice were injected with dead yeast cells prior to immunization. Two strains of mice with completely opposite susceptibilities to infection with Pb18 cells (B10.A--susceptible and A/SN--resistant) both showed suppressed IgE anti-ovalbumin antibody production when infected 3 days prior to immunization. Injection of both strains of mice with P. brasiliensis antigen on the same day as immunization also had the same suppressive effect. These results suggest that the suppression of IgE response to an unrelated antigen in experimental murine paracoccidioidomycosis could be due to antigenic competition or to a suppressive component present in P. brasiliensis cells.

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 X 10(6) FU/tube (Pb 18, Pb 265 and PB 2); intermediate, reaching 60-150 X 10(6) FU/tube (IVIC Pb 9, IVIC Pb 267, Pb SN, Pb Vitor and Pb Campo Grande) and fast, reaching 180-370 X 10(6) 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 degrees 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 mu; 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 electron-dense material, were numerous and two to five nuclei with pores were constantly observed.