Differential correlation between interleukin patterns in disseminated and chronic human paracoccidioidomycosis.

In an attempt to understand better the immunoregulatory disorders in paracoccidioidomycosis (PCM), the possible correlation between interleukin pattern, lymphoproliferation, C-reactive protein (CRP) and specific antibody levels was investigated in the polarized clinical forms of this disease. We studied 16 PCM patients, eight with the disseminated disease (four under treatment and four non-treated) and eight with the chronic disease. The patients with disseminated disease exhibited high antibody titres specific to Paracoccidioides brasiliensis antigen compared with patients with the chronic form of disease. Tumour necrosis factor (TNF), IL-1, IL-6 and CRP in the serum of non-treated disseminated PCM patients were increased, which correlated positively with the low mitogenic response of peripheral blood mononuclear cells (PBMC) to phytohaemagglutinin (PHA) (P < 0.01) and with the high antibody titres (P < 0.001) of these patients. Moreover, we found in the disseminated PCM patients positive correlations between IL-1 and IL-6 (P = 0.0007); IL-1 and TNF (P = 0.0045); IL-1 and IL-6 with the high antibody titres (P = 0.0834 and P = 0.0631, respectively); IL-1, IL-6 and TNF with CRP levels. By contrast, no correlations were found with those interleukins in the treated disseminated and chronic patients or in controls. It was interesting to find an inverse correlation between IL-4 and antibody production in non-treated disseminated PCM (r = -0.4770); moreover, a significant correlation (P = 0.0820) was found in chronic PCM patients with respect to the low level of either IL-4 and antibody titres against fungus antigen. Chronic PCM patients also had IL-2 levels inversely correlated with antibody production (r = -0.6313; P = 0.0628). Inverse correlations were also observed between IL-2 and IL-6 levels in non-treated disseminated patients (P = 0.0501) and between IL-2 and IL-4 in chronic patients (P = 0.0131). The inflammatory cytokines might have a pivotal role in the genesis and in control of some aspects of the disease, such as granulomatous reaction, hypergammaglobulinaemia and depression of T cell-mediated immunity in PCM.     

Paracoccidioidomycosis infection in domestic and wild mammals by Paracoccidioides lutzii

Paracoccidioidomycosis (PCM) is a systemic mycosis that occurs in several Latin American countries, especially in Brazil. It is caused by the thermo‐dimorphic fungus Paracoccidioides spp. Serological studies to detect animal infection represent an excellent strategy for data on the agent's ecology. Although the state of Rio Grande do Sul (RS) is an endemic area for PCM in humans, there is scarce information available on the ecology of the agent in the region. This study aimed to investigate the infection by Paracoccidioides lutzii in animals living in RS, Brazil. A total of 85 wild mammals, 200 horses and 196 domestic dogs, previously tested for infection by P. brasiliensis, were included in this study. Serum samples from the animals were tested by ELISA to detect anti‐ P. lutzii antibodies. From the 481 animals tested, 105 (21.8%) were seropositive for IgG anti‐P. lutzii. Of these, 54 were also positive for P. brasiliensis. A total of 11 horses (10.5%), 30 dogs (28.8%) and 10 wild mammals (9.5%) were positive only for P. lutzii (n=51). The detection of anti‐P. lutzii antibodies in animals of RS suggests that the fungus can be found in southern Brazil, despite being described mainly in the midwest and southeast of the country.     

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 ceja‐1 sequence as a potential new molecular marker for Paracoccidioides brasiliensis infection

We have developed a two‐step PCR assay that amplifies a region of the ceja‐1 sequence that is specific for virulent strains of Paracoccidioides brasiliensis. An internal region of the ceja‐1 sequence was chosen for designing primers that were utilised in a single tube heminested PCR protocol to amplify DNA from six virulent strains. PCR specificity was determined by the absence of amplified products with genomic DNA from four non‐virulent strains of P. brasiliensis and from eight fungal pathogens, one bacterium, two protozoa, one worm and mouse and human genomic DNA (leucocytes). The fact that the PCR product was only obtained with the genetic material from virulent isolates of P. brasiliensis suggested that this partial amplified sequence might be a marker of virulence for this fungus. The diagnostic potential of this PCR was confirmed by the successful amplification of this fragment with genomic DNA obtained in lymph node aspirate from a patient with paracoccidioidomycosis.     

Human {alpha}{beta} and {gamma}{delta} T cells from unexposed individuals respond to protein antigens of the yeast form of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis, a dimorphic fungus, causes chronicgranuiomatous mycosis in susceptible individuals. Differentreports have shown that cell-mediated immunity is essentialfor protection against systemic mycosis, including paracoccldloidomycosis.We analyzed the reactivity of ß and T cells fromunexposed Caucasian donors to P. brasiliensis yeast form components.Our results indicate: (I) ß and T cells proliferateafter in vitro stimulation with lysates of P. brasiliensis;(II) similar numbers of ß T cells (f = 1/21,000) andof T cells (f = 1/8000) respond to P. braslllensls; (III) P.braslllensls-reactive T cells express the V9V2 TCR; (Iv) thestimulatory activity of P. brasilensis for both ßand T cells primarily resides in a high molecular weight (100kDa) and in a low molecular weight (<<1 kDa) fraction;(v) the ligands responsible for stimulation of both ßand T cells are sensitive to proteinase treatment We concludethat both ß and T cells from healthy individualsrespond to ubiquitous protein antigens of P. brasiliensis.     

Localization of glycoconjugates and glycogen in yeast-like cells and protoplasts of Paracoccidioides brasiliensis

Summary. The presence and localization of storage polysaccharides and of polysaccharides as cell structure constituents of Paracoccidioides brasiliensis yeast-like cells and protoplasts were studied using the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) technique. Yeast-like cells presented glycogen particles in the form of rosettes, which were mostly concentrated in regions of the cytoplasm. Cells in the budding process presented small amounts of glycogen in their matrix. The intracellular membranes and the attachment of the mother cell to the bud were clearly labelled by silver, demonstrating a large amount of glycoconjugates. The protoplasts presented a small amount of glycogen in their cytoplasm, a reduction probably due to the enzymatic treatment to which the cells were submitted.
Zusammenfassung. Vorkommen und Lokalisation von Speicherpolysacchariden und Struktur-polysacchariden wurden mittels PATAg-Technik an der Hefephase und an Protoplasten von Paracoccidioides brasiliensis untersucht. Zellen der Hefephase zeigten Glykogen-Rosetten meist in zytoplasmatischen Regionen konzentriert. Knospende Hefezellen hatten geringe Glykogen-Mengen in ihrer Matrix. Die intrazellulären Membranen und der Grenzbereich zwischen Mutterzelle und Knospe waren deutlich silbermarkiert, was auf eine hohe Konzentration von Glykokonjugaten hinweist. Protoplasten zeigten geringe Glykogen-Konzentrationen im Zytoplasma, wahrscheinlich bedingt durch die Enzymbehandlung zur Protoplastentransformation.     

Tumour necrosis factor production in vivo and in vitro in response to Paracoccidioides brasiliensis and the cell wall fractions thereof.

Tumour necrosis factor (TNF) was detected in serum from mice challenged with Paracoccidioides brasiliensis. The serum TNF levels of mice challenged with an avirulent strain were significantly higher than those of mice challenged with a virulent strain, and the same was observed for the TNF levels of mice challenged with a cell wall fraction (F1) from the two fungal strains. Fraction F1 consisted of chitin and beta-glucan; but although the chitin contents were similar for the two strains, the avirulent strain allowed a greater content of beta-glucan. The beta-glucan, purified from both strains, increased serum TNF levels in an identical dose-dependent manner, whereas purified chitin did not induce serum TNF levels. P. brasiliensis, the F1 fractions and beta-glucan induced macrophages to secrete TNF in vitro. The differences in TNF levels, induced by the different fungal strains, were correlated with the beta-glucan concentrations in the cell walls of both the avirulent and virulent strains of P. brasiliensis. These findings support a role for TNF in the pathogenicity of P. brasiliensis.     

Paracoccidioides brasiliensis conidia recognize fibronectin and fibrinogen which subsequently participate in adherence to human type II alveolar cells: involvement of a specific adhesin

We examined the ability of Paracoccidioides brasiliensis conidia to interact with fibronectin, fibrinogen and with A549 cells, in order to establish the nature of the molecules involved. Conidia bound to immobilized proteins in a concentration-dependent manner. Antibodies against fibronectin and fibrinogen inhibited the fungal adherence to the corresponding proteins; as did laminin and fibronectin, but not fibrinogen when added in soluble form; however, the fibrinogen fragment D interfered with adhesion in a significant manner. Various monosaccharides and RGD/RGDS peptides had no effect on adherence to fibronectin or fibrinogen, while N-acetylneuraminic acid (NANA) abolished adherence to both proteins. Additionally, these proteins were detected on the surface of A549 cells. Inhibition assays showed a significant decrease in fungal adherence when A549 cells were treated with anti-fibrinogen, anti-fibronectin antibodies and a purified adhesin of P. brasiliensis (32-kDa protein); or when conidia were treated with these soluble proteins, mAb anti-32-kDa protein, RGD peptides and NANA. These results suggest that fibrinogen and fibronectin facilitate the adherence of conidia to A549 cells probably through the interaction with adhesin-type molecules or a sialic acid based recognition system. These interactions appear to play a role in the initial fungal attachment to the lung, and consequently, also in the pathogenesis of paracoccidioidomycosis.