Differential production of interleukin-1 family cytokines (IL-1ß, IL-18, IL-33 and IL-37) in patients with paracoccidioidomycosis: correlation with clinical form and antifungal therapy.

Besides interleukin (IL)-1ß and IL-18, the newly described cytokines of IL-1 family IL-33 and IL-37 can contribute to the differentiation and maintenance of different population of T cells. IL-33 acts as an allarmin and promotes a predominant Th2 inflammatory response, whereas IL-37 plays an important role as an antagonist of inflammation. In paracoccidioidomycosis (PCM), caused by the dimorphic fungi Paracoccidioides brasiliensis and P. lutzii, it has been shown that the acquired immune responses are associated with the diverse clinical manifestations. The severe and disseminated forms (acute form [AF] and multifocal chronic form [CF-MF]) are characterized by high Th2 cytokines and antibody production, impaired cellular immune response, and eosinophilia. In contrast, in the localized form (unifocal chronic form [CF-UF]), the cellular immune response is preserved, with high production of Th1 and Th17 cytokines, and low antibody titers. This study aimed to quantify interleukin-1 family cytokines (IL-1ß, IL-18, IL-37, IL-33, and the soluble IL-33 receptor sST2) in sera of patients presenting different clinical forms of PCM before, during, and after antifungal treatment, as well as to analyze the expression of these cytokines in lesions of PCM patients. We found that AF patients presented high serum levels of IL-1ß, IL-18, IL-33, sST2, and IL-37, and that these cytokines are strongly expressed in lymph nodes lesions. Furthermore, antifungal therapy resulted in the diminution of circulating cytokines and sST2 levels in all groups of patients. These results indicate that, besides IL-1ß and IL-18, IL-33, IL-37, and sST2 can be associated with the disease activity and severity.

Subtractive phage display selection for screening and identification of peptide sequences with potential use in serodiagnosis of paracoccidioidomycosis caused by Paracoccidioides brasiliensis.

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease endemic in Latin America whose aetiologic agents are the thermodimorphic fungi Paracoccidioides brasiliensis and Paracoccidioides lutzii. Despite technological advances, some problems have been reported for the fungal antigens used for serological diagnosis, and inconsistencies among laboratories have been reported. The use of synthetic peptides in the serological diagnosis of infectious diseases has proved to be a valuable strategy because in some cases, the reactions are more specific and sensitive. In this study, we used a subtractive selection with a phage display library against purified polyclonal antibodies for negative and positive PCM sera caused by P. brasiliensis. The binding phages were sequenced and tested in a binding assay to evaluate its interaction with sera from normal individuals and PCM patients. Synthetic peptides derived from these phage clones were tested in a serological assay, and we observed a significant recognition of LP15 by sera from PCM patients infected with P. brasiliensis. Our results demonstrated that subtractive phage display selection may be useful for identifying new epitopes that can be applied to the serodiagnosis of PCM caused by P. brasiliensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Currently, there is no standardized method for the preparation of paracoccidioidomycosis (PCM) antigens, which has resulted in differences in the antigens used for serological diagnosis. Here, we report a procedure that uses subtractive phage display selection to select and identify new epitopes for the serodiagnosis of PCM caused by Paracoccidioides brasiliensis. A synthetic peptide obtained using this methodology was successfully recognized by sera from PCM patients, thus demonstrating its potential use for improving the serodiagnosis of this mycosis. The development of synthetic peptides for the serodiagnosis of PCM could be a promising alternative for the better standardization of diagnoses among laboratories.

Increased peripheral blood TCD4+ counts and serum SP-D levels in patients with chronic paracoccidioidomycosis, during and after antifungal therapy.

BACKGROUND: The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE: To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS: Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS: In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION: Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.

Phenotypic and functional evaluations of peripheral blood monocytes from chronic-form paracoccidioidomycosis patients before and after treatment.

BACKGROUND: Paracoccidioidomycosis (PCM) is systemic mycosis caused by the thermal dimorphic fungus of genus Paracoccidioides, leading to either acute/subacute (AF) or chronic (CF) clinical forms. Numerous CF patients after treatment exhibit sequels, such as pulmonary and adrenal fibrosis. Monocytes are cells that are involved in the inflammatory response during active infection as well as in the fibrogenesis. These cells comprise a heterogeneous population with distinct phenotypic and functional activities. The scope of this study was to identify changes regarding functional and phenotypical aspects in monocytes comparing CF PCM patients on antifungal treatment versus non-treated patients (PMC-p). METHODS: Twenty-three CF PCM composed of 11 non-treated patients (NTG) and 12 patients in apparent cure (ACG) were studied. Sixteen healthy individuals were used as control group (CG). Monocyte subsets were determined by immunophenotyping based on CD14 and CD16 expression. Cellular function was measured in vitro with and without stimulation with lipopolysaccharide (LPS) and P. brasiliensis exoantigen (PbAg) for 24 hours. Independent samples were compared using unpaired t tests, dependent samples were analyzed by paired t-test. Groups of more than two independent samples were analyzed using an ANOVA, with Tukey's post-test. Significance was set up at p <0.05. RESULTS: Our results showed high counts of peripheral blood CD14+CD16+ and CD14+CD16++ monocytes in untreated PCM-p accompanied by intense production of pro-inflammatory cytokines (IL-1ß and TNF-α) and profibrotic growth factors (TGF-ß1 and bFGF) by monocytes challenged with P. brasiliensis antigens. After the introduction of antifungal therapy, the counts of CD14+CD16+ cells returned to baseline while CD14+CD16++ counts remained high. Interestingly, counts of CD14+CD16++ monocytes remained elevated even 52 ± 7 months after successful antifungal treatment. Furthermore, the ACG-patients showed preserved pro-inflammatory activity in the presence of specific antigen stimuli and high spontaneous production of TNF-α by monocytes. CONCLUSIONS: Infection with Paracoccidioides leads to initiation of a specific proinflammatory response by monocytes of PCM-p during active disease and in the apparent cure. A profibrotic profile by monocytes was observed only at admission. Furthermore, PCM-p with apparent cure showed high spontaneous production of TNF-α and high counts of CD14+CD16++ monocytes, probably induced by hypoxia duo to fibrotic sequelae.

Detection of antibodies against Paracoccidioides brasiliensis in free-range domestic pigs (Sus scrofa).

Paracoccidioidomycosis, caused by the thermodimorphic fungus Paracoccidioides brasiliensis, is a human systemic mycosis prevalent in Latin America. Paracoccidioidomycosis affects mainly male rural workers, causing granulomatous lesions in several organs such as the lungs, liver and spleen. The participation of other animal species in the fungus epidemiology is not well understood. The objective of this study was to evaluate the infection of free-range domestic pigs by P. brasiliensis. Serum samples from 106 pigs were analyzed by ELISA and the immunodiffusion test, using P. brasiliensis gp43 and exoantigen as antigens, respectively. The overall positivity to gp43 in ELISA was 37.7 %, although no reactivity was observed in the immunodiffusion test and nor was P. brasiliensis detected in tissue samples (spleen, lung, liver and lymph nodes) from slaughtered animals submitted to culture, histopathological examination and PCR analysis. Five pigs seronegative to gp43 were exposed to natural infection by P. brasiliensis, and all animals seroconverted 3 months after exposure. The results suggest that free-range pigs are frequently infected with P. brasiliensis but are resistant to disease development. This is the first report of paracoccidioidomycosis in pigs.

Wild animals as sentinels of Paracoccidioides brasiliensis in the state of Rio Grande do Sul, Brazil.

Paracoccidioides brasiliensis, a dimorphic pathogenic fungus, causes the principal form of systemic mycosis in Brazil. The literature furnishes only limited data on the ecology of this fungus in the state of Rio Grande do Sul, the southernmost state of Brazil. The purpose of this study was to evaluate the prevalence of fungal infection in wild animals, using serological tests and using the animals as sentinels of the presence of P. brasiliensis in three specified mesoregions of Rio Grande do Sul. A total of 128 wild animals from the three mesoregions were included in the study. The serum samples were evaluated by immunodiffusion and the enzyme-linked immunosorbent assay (ELISA) technique to detect anti-gp43 antibodies from P. brasiliensis. Two conjugates were tested and compared with the ELISA technique. Although no positive samples were detected by immunodiffusion, 26 animals (20%), belonging to 13 distinct species, were found to be seropositive by the ELISA technique. The seropositive animals were from two mesoregions of the state. The results were similar according to the gender, age, and family of the animals, but differed significantly according to the conjugate used (p < 0.001), showing more sensitivity to protein A-peroxidase than to protein G-peroxidase. The finding that wild animals from the state of Rio Grande do Sul are exposed to P. brasiliensis suggests that the fungus can be found in this region despite the often-rigorous winters, which frequently include below-freezing temperatures.

Interrelationship of clinical, histomorphometric and immunohistochemical features of oral lesions in chronic paracoccidioidomycosis.

BACKGROUND: This study aimed to analyze the oral lesions of chronic paracoccidioidomycosis concerning their histomorphometric, immunohistochemical, and clinical features in a standardized sample. METHODS: Fifty biopsy specimens of oral lesions of chronic paracoccidioidomycosis were submitted to hematoxylin and eosin (H&E), Grocott-Gomori and immunohistochemical staining. Data regarding disease duration and size and number of oral lesions, as well as erythrocytes, leukocytes, lymphocytes, hematocrit, hemoglobin, and erythrocyte sedimentation rate, were collected from medical charts. Granuloma density and number and diameter of buds and fungal cells, and IL-2, TNF-alpha and IFN-gamma expression, as well as clinical and hematological features, were quantified and correlated. RESULTS: Bud diameter was significantly greater in intermediate density granulomas compared to higher density granulomas. The other variables (number of buds, number and diameter of fungi, expression of IL-2, TNF-alpha and IFN-gamma, and clinical and hematological features) did not significantly change with the density of granulomas. There was a positive correlation between bud number and fungal cell number (r = 0.834), bud diameter and fungal cell diameter (r = 0.496), erythrocytes and number of fungi (r = 0.420), erythrocytes and bud number (r = 0.408), and leukocytes and bud number (r = 0.396). Negative correlation occurred between number and diameter of fungi (r = -0.419), bud diameter and granuloma density (r = -0.367), TNF-alpha expression and number of fungi (r = -0.372), and TNF-alpha expression and bud number (r = -0.300). CONCLUSION: The histological, immunological, and clinical features of oral lesions evaluated did not differ significantly between patients in our sample of chronic paracoccidioidomycosis. TNF-alpha levels were inversely correlated with intensity of infection.

Proteomic analysis of serum samples of paracoccidioidomycosis patients with severe pulmonary sequel.

BACKGROUND: Pulmonary sequelae (PS) in patients with chronic paracoccidioidomycosis (PCM) typically include pulmonary fibrosis and emphysema. Knowledge of the molecular pathways involved in PS of PCM is required for treatment and biomarker identification. METHODOLOGY/PRINCIPAL FINDINGS: This non-concurrent cohort study included 29 patients with pulmonary PCM that were followed before and after treatment. From this group, 17 patients evolved to mild/ moderate PS and 12 evolved severe PS. Sera from patients were evaluated before treatment and at clinical cure, serological cure, and apparent cure. A nanoACQUITY UPLC-Xevo QT MS system and PLGS software were used to identify serum differentially expressed proteins, data are available via ProteomeXchange with identifier PXD026906. Serum differentially expressed proteins were then categorized using Cytoscape software and the Reactome pathway database. Seventy-two differentially expressed serum proteins were identified in patients with severe PS compared with patients with mild/moderate PS. Most proteins altered in severe PS were involved in wound healing, inflammatory response, and oxygen transport pathways. Before treatment and at clinical cure, signaling proteins participating in wound healing, complement cascade, cholesterol transport and retinoid metabolism pathways were downregulated in patients with severe PS, whereas signaling proteins in gluconeogenesis and gas exchange pathways were upregulated. At serological cure, the pattern of protein expression reversed. At apparent cure pathways related with tissue repair (fibrosis) became downregulated, and pathway related oxygen transport became upregulated. Additionally, we identified 15 proteins as candidate biomarkers for severe PS. CONCLUSIONS/SIGNIFICANCE: Development of severe PS is related to increased expression of proteins involved in glycolytic pathway and oxygen exchange), indicative of the greater cellular activity and replication associated with early dysregulation of wound healing and aberrant tissue repair. Our findings provide new targets to study mechanisms of PS in PCM, as well as potential biomarkers.

IgG reactivity profile to Paracoccidioides spp. antigens in people with asymptomatic Paracoccidioidomycosis.

Introduction. Paracoccidioidomycosis (PCM) is a systemic mycosis caused by Paracoccidioides spp. As the disease is known to affect mostly men over 40 years old who previously worked handling soil, some cities of agricultural economy in endemic regions may have more cases of paracoccidioidal infection.Gap statement. The true frequency of PCM cannot be established in Brazil because it is not a disease of mandatory reporting. The detection of paracoccidioidal infection may assist in the planning of health services, in order to provide early detection of the disease and to prevent its worsening or even progression to death. In addition, little is described about sera reactivity with antigens from different species of Paracoccidiodes, especially P. lutzii.Aim. Current research was conducted in an inland municipality of southern Brazil, in order to assess infection rate within this endemic region of PCM disease.Methodology. ELISA was employed to evaluate 359 sera from random volunteers from Guarapuava, Paraná, Brazil, to detect IgG against cell-free antigens (CFA) from P. restrepiensis B339, P. americana LDR3 and P. lutzii LDR2. Confirmatory ELISA employed gp43 from B339. Reduction of cross-reactions was sought by treatment with sodium metaperiodate (SMP-CFA, SMP-gp43). Immunoblot was performed with 37 selected sera among those reactive in ELISA. Epidemiological profile was assessed by questionnaire.Results. ELISA reactivity was: CFA/SMP-CFA in general 37.3/17.8 %, B339 25.3/14.5 %, LDR3 24.5/1.4 %, LDR2 8.3/5.8 %; gp43/SMP-gp43 7.2/4.7 %. There were sera reactive with multiple CFAs. In immunoblot, five sera showed the same reaction profile with P. lutzii's antigens as PCM disease sera. Rural residence and soil-related professions were risk factors for paracoccidioidal infection.Conclusion. The low prevalence is in accordance with previous reports of lower PCM disease endemicity in Guarapuava than in other areas of Paraná. Although P. brasiliensis seems to be the prevalent strain of the region, 21 sera from people who only lived in Guarapuava reacted with P. lutzii LDR2. CFA-ELISA with whole antigens seems a good option for serological screening in epidemiological surveys.

Characteristics of environmental Paracoccidioides brasiliensis isolates.

The ecological niche or exact habitat of the fungus Paracoccidioides brasiliensis is not known, and few isolates have been obtained from the environment. In this study, ten isolates were analyzed with respect to antigenic composition, serology, pathogenicity, and molecular aspects. Gp43 is considered to be the molecular basis for the serodiagnosis of paracoccidioidomycosis; however, in this study only six of the environmental isolates secreted this molecule (four in great amounts and two in small amounts). Other molecules were also produced. When exoantigens from these isolates were tested using immunodiffusion, only four preparations were positive by ID tests. However, when these exoantigens were tested by ELISA, all of them except one were able to detect anti-P. brasiliensis antibodies. In Western blot assays, these exoantigens showed different reactivities. Isolates that secreted gp43 presented positive reactions for this molecule, and isolates that did not secrete gp43 gave positive reactions for other minor molecules. RAPD analysis revealed that there is great genetic variation between these environmental isolates. These isolates were non-pathogenic: no mortality was observed among the inoculated mice during an 18-month follow-up period.

Dynamics of plasma micronutrient concentrations and their correlation with serum proteins and thyroid hormones in patients with paracoccidioidomycosis.

Minerals, such as zinc, copper, and iron are reported to play roles in chronic infectious diseases; however, their role in paracoccidioidomycosis (PCM) remains unknown. This study aimed to examine the micronutrient dynamics and their correlation with serum proteins and thyroid hormones in patients with PCM. In 14 patients with PCM and 10 healthy subjects, we evaluated the body mass index (BMI) along with serum levels of hemoglobin, iron, ferritin, zinc, copper, magnesium, albumin, globulin, thyroid stimulating hormone (TSH), thyroxine (free T4), and triiodothyronine (T3). Evaluations were conducted at the first appointment, before treatment, and at the end of the first, second, fourth, and sixth month of PCM treatment. The control group was only evaluated once. We observed that before treatment, patients with PCM, had higher levels of copper and lower level of iron than those of the control group. After one month of treatment, the iron levels increased, whereas the levels of copper after six months of treatment. Reduction in inflammatory activity, indicated by the normalization of C-reactive protein, ferritin, albumin, and globulin levels, was observed during treatment. However, no correlation was observed between the serum levels of minerals and inflammatory activity or thyroid function in this study. In conclusion, our results showed higher serum copper levels in control group compared to those in pretreatment patients; the clinical importance of this observation should be investigated in further studies. After treatment, serum copper levels showed a tendency to decrease. In addition, serum iron levels were decreased at the stage of active disease, and were increased after treatment. Thus, serum iron levels can be used as a better biomarker for treatment control.

Ceruloplasmin, transferrin and apolipoprotein A-II play important role in treatment's follow-up of paracoccidioidomycosis patients.

Paracoccidioidomycosis (PCM) is a systemic disease caused by thermodymorphic fungi of the Paracoccidioides brasiliensis complex, (Paracoccidioides spp.). Patients with PCM reveal specific cellular immune impairment. Despite the effective treatment, quiescent fungi can lead to relapse, usually late, the serological diagnosis of which has been deficient. The present study was carried out with the objective of investigating a biomarker for the identification of PCM relapse and another molecule behaving as an immunological recovery biomarker; therefore, it may be used as a cure criterion. In the evolutionary analysis of the proteins identified in PCM patients, comparing those that presented with those that did not reveal relapse, 29 proteins were identified. The interactions observed between the proteins, using transferrin and haptoglobin, as the main binding protein, were strong with all the others. Patient follow-up suggests that cerulosplamin may be a marker of relapse and that transferrin and apolipoprotein A-II may contribute to the evaluation of the treatment efficacy and avoiding a premature decision.

Aseptic cure of pulmonary paracoccidioidomycosis can be achieved after a previous subcutaneous immunization of susceptible but not resistant mice.

The murine model of paracoccidioidomycosis, the most important South American endemic mycosis, mimics the human disease: resistance is associated with preserved cellular immunity while T-cell anergy is related with susceptibility. In the present study we asked whether a previous s.c. infection which induces strong cellular immunity would protect mice against a lethal pulmonary challenge. It was found that susceptible but not resistant mice developed immunoprotection and aseptic cure of infection. Immunoprotection led to reversal of DTH anergy, increased levels of antibodies and pulmonary IL-12, IL-2 and IL-4 indicating a balanced type 1/type 2 response. On the contrary, no marked differences in A/Sn infection and immunity were observed. Depletion experiments showed that immunoprotection required the cooperative action of CD4(+) and CD8(+) T cells in association with IFN-gamma and IL-12. Altogether, these observations demonstrated that susceptible hosts can develop sterilizing immunity and defined the main immunological requirements to control secondary paracoccidioidomycosis.

Monoclonal antibodies against complement 3 neoantigens for detection of immune complexes and complement activation. Relationship between immune complex levels, state of C3, and numbers of receptors for C3b.

C3-bearing immune complexes and C3 activation products were detected by using two monoclonal antibodies, one specific for a neoantigenic determinant on C3c and the other for C3d. To quantitate immune complexes, the anti-C3c or anti-C3d antibodies were fixed to microtiter plates and reacted with test plasma. The binding of C3-bearing immune complexes in this plasma was then measured with radioisotope- or enzyme-labeled anti-human IgG. To test for C3 breakdown products, solid-phase monoclonal antibody to the C3d neoantigen was reacted with EDTA-plasma samples, and fixed iC3b or C3d was measured with a polyclonal anti-C3 antibody. Patients with autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, and Sjogren's syndrome, and paracoccidioidomycosis were found to contain immune complexes bearing C3b/iC3b or C3d. In most conditions, there were more C3d-containing immune complexes than C3b/iC3b. Although CR1 (C3b receptors) rapidly converted immune complex-bound iC3b to C3dg/C3d and lupus patients had reduced CR1, no correlation between the state of C3 on circulating immune complexes or levels of immune complexes and CR1 numbers was seen. However, levels of C3-fixing ICs correlated with levels of C3 activation products. This assay system with monoclonal antibodies to neoantigens expressed on activated, but not native, C3 provides sensitive and specific means for detecting and classifying C3-fixing immune complexes and for assessing C3 activation.

Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

Adrenal function in 23 children with paracoccidioidomycosis.

Adrenal involvement by Paracoccidioides brasiliensis was described at necropsies and in many clinical studies, but only in adults. Therefore, the aim of this study was to evaluate adrenal function in children with paracoccidioidomycosis. Twenty-three children with the systemic form of paracoccidioidomycosis were evaluated and divided in two Groups: Group A (n = 8) included children before treatment and Group B (n = 15) children after the end of treatment. Plasma cortisol (basal and after ACTH test), ACTH, renin activity, aldosterone, sodium and potassium were measured. They were within normal range in all cases, except for renin activity and aldosterone, which were elevated in some cases. Group A patients showed basal and post-ACTH cortisol levels significantly greater than Group B patients. The results showed that adrenal function was not compromised in these children with paracoccidioidomycosis.

[Laboratory evolutive aspects of children under paracoccidioidomycosis treatment]. / Aspectos laboratoriais evolutivos de crianças em tratamento da paracoccidioidomicose.

The study of 38 children with paracoccidioidomycosis, aged up to 14, treated for 24 to 30 months with either a sulfonamide derivative or ketoconazole either alone or, after the use of amphotericin B. Laboratory data at admission were analyzed and compared with those of sequential tests after up to 30 months follow-up. Anemia, eosinophilia, increased bilirubin and aminotransferases normalized, in most patients, after three months treatment and hypoalbuminemia normalized after six months, suggesting that these laboratory findings are useful for monitoring early therapeutic response. Peripheral leucocytes, erythrocyte sedimentation rate, IgG, and serological titers for Paracoccidioides brasiliensis were increased and frequently normalized after nine to 12 months of treatment. They may be useful for monitoring the entire treatment and emphasize the need for long term treatment of paracoccidioidomycosis in children.

Membrane and extracellular antigens of Paracoccidioides brasiliensis (Mexo): identification of a 28-kDa protein suitable for immunodiagnosis of paracoccidioidomycosis.

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.

Identification of immunogenic proteins from Paracoccidioides brasiliensis antigenic fractions F0, FII and FIII.

Paracoccidioides brasiliensis causes a chronic granulomatous mycosis prevalent in South America, and cell-mediated immunity is the principal mode of protection against this fungal infection. In this context, one of the strategies to discover proteins that are target of an effective immune response against P. brasiliensis is the partial sequencing of cDNA from an expression library previously screened with immunoglobulins (Ig) to generate antigen sequence tags (AST). In the present work, a P. brasiliensis yeast cDNA expression library was screened with affinity chromatography-purified IgG from rabbit sera immunized with P. brasiliensis antigenic fractions (F0, FII or FIII) or from paracoccidioidomycosis (PCM) patient sera by indirect ELISA. From 119 clones selected by the immunoscreening procedure, 40% were recognized by IgG from PCM patients, 25% were recognized by anti-F0, 8% were selected by anti-FII and 11% recognized by FIII specific antibodies. The remaining clones presented cross-reaction to all anti-sera tested. The AST homologies with previously reported sequences in the nonredundant GenBank at NCBI revealed high significant homology to fungal proteins of known function. One of them matched calcineurin B of Neurospora crassa with 35% identity and 55% similarity in amino acid sequence. We also identified an AST homologous to a Kinesin like protein from Ustilagus maydis and other fungi with 86% identity and 91% similarity. On the other hand, the vast majority of selected cDNA clones are new genes and represent 60% of the total. Prediction of transmembrane regions with the prediction transmembrane protein topology with a hidden markov model (TMHMM) revealed consensus sequences representing structural membrane segments in 28 encoded proteins.

Leukocyte immunophenotypes in bronchoalveolar lavage fluid and peripheral blood of paracoccidioidomycosis, sarcoidosis and silicosis.

Leukocyte subsets in bronchoalveolar lavage (BAL) fluid and peripheral blood of patients with paraccoccidioidomycosis, sarcoidosis and silicosis were characterized using monoclonal antibodies and an immunoperoxidase technique. In paraccocidioidomycosis, the number of T-helper/inducer CD4-positive lymphocytes was lower in peripheral blood than in BAL fluid. Additional analysis showed that the expression of HLA-DR was very similar in alveolar macrophages, lung and blood T-cells. In sarcoidosis and silicosis there were higher proportions of T-helper/inducer cells in peripheral blood than in BAL fluid. The alterations in the T-helper/inducer/T-suppressor/cytoxic CD4/CD8 ratio in sarcoidosis and silicosis were more appreciable in peripheral blood than in BAL fluid, contrasting with the results in paracoccidioidomycosis. The expression of HLA-DR by alveolar macrophages in sarcoidosis was the highest of all the disease studied. No statistically significant differences were observed between chronic multifocal and chronic unifocal paracoccidioidomycosis disease, stage II and stage III sarcoidosis, and chronic and accelerated silicosis. The three granulomatous diseases analyzed had a few alveolar macrophages expressing the CD4 molecule on their surface. These findings and the technique of analyzing both peripheral blood and BAL leukocyte subsets may help to understand the pathogenesis of interstitial lung diseases.