Negative immunodiffusion test results obtained with sera of paracoccidioidomycosis patients may be related to low-avidity immunoglobulin G2 antibodies directed against carbohydrate epitopes

Immunodiffusion (ID) is the serologic test most frequently used for the diagnosis and posttherapy follow-up of patients with paracoccidioidomycosis (PCM). The ID test is highly specific (100%), but its sensitivity is relatively low (90%), leading to false-negative results. The aim of this study was to determine the profiles of antibodies in sera from patients with proven PCM and with negative results in the ID test (IDneg) versus positive results in the ID test (IDpos). We analyzed 46 sera from patients with active PCM for total immunoglobulin G (IgG) and IgG subclass responses to Paracoccidioides brasiliensis gp43 antigen (treated or not treated with sodium metaperiodate) by enzyme-linked immunosorbent assay and immunoblotting. Immunoblotting showed that both IDneg and IDpos sera recognized predominantly the gp43 fraction of the P. brasiliensis antigen used in the ID test. IDneg sera contain low-avidity antibodies, low levels of specific IgG (total) and IgGI, and high levels of IgG2 compared with IDpos sera. The antibodies present in IDpos sera were predominantly directed against carbohydrate epitopes, since treatment with sodium metaperiodate resulted in a significant decrease in antibody reactivity. These data suggest that the lack of reactivity of sera from PCM patients in the ID test may be related to the production of low-avidity IgG2 antibodies directed against carbohydrate epitopes.10580280

Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients’ sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost

Flow-cytometric analysis of cytokine production in human paracoccidioidomycosis

Human infection with Paracoccidioides brasiliensis may result in three major outcomes: paracoccidioidomycosis-infection (PI), which is observed in healthy carriers living in endemic areas and the adult form (AF) and juvenile form (JF) of the disease. In this study we proposed to examine the intracellular expression of IFN-gamma, TNF-alpha, IL-2, IL-10, IL-12, CXCL8, CXCL9 and CXCL10 by peripheral blood mononuclear cells (PBMC) of patients with the JF and AF of the disease, as well as of PI individuals stimulated with PMA plus ionomycin.. LPS or anti-CD3 plus anti-CD28, by flow cytometry. The results showed that PI individuals present a higher percentage of cells producing IFN-gamma, TNF-alpha, IL-2, CXCL9 and CXCL10 when compared to AF and JF patients. IFN-gamma was predominantly detected in CD3(+)CD8(+) T cells, whereas IL-2 and TNF-alpha were mainly expressed in CD3(+)CD4(+) cells. Monocytes of PI individuals also presented higher expression of CD80 and lower expression of CD86 when compared to JF and AF patients, and higher expression of HLA-DR, only when compared to JF patients. These results indicate that the differential production of cytokines and chemokines, as well as the expression of co-stimulatory molecules involved in antigen presentation, may influence the outcome of PCM infection. (c) 2006 Elsevier Ltd. All rights reserved.354173220721

TLR-2, TLR-4 and dectin-1 expression in human monocytes and neutrophils stimulated by Paracoccidioides brasiliensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Paracoccidioidomycosis (PCM) is an endemic mycosis in Latin America caused by the dimorphic fungus Paracoccidioides brasiliensis. The pattern of the immune responses to P. brasiliensis determines the disease progression and clinical outcome. Innate immune response is mediated by phagocytic cells, such as macrophage and neutrophils, which ingest and kill invading pathogens and then trigger the adaptive immune system through the secretion of cytokines and chemokines. The C-type like lectin receptors (CLR) and Toll-like receptors (TLRs) are the two main pattern recognition receptors in phagocytic cells that recognize fungal components. Therefore, the purpose of the present study was to evaluate the expression of TLR-1, TLR-2, TLR-4 and dectin-1 (CLR) in monocytes and neutrophils from healthy individuals after stimulation with Pb18 (high virulence) and Pb265 (low virulence) yeasts of P. brasiliensis. As positive controls we used specific ligands to TLR-4 (LPS), TLR-2 and dectin-1 (zymosan). Our results demonstrated a decreased of TLRs and dectin-1 expression mainly on monocytes as opposes on neutrophils, as soon as 30 minutes after yeast cells stimulation. This decrease was similar to the one caused by zymosan stimulation and indicates that up binding the complexes are rapidly internalized. There was a tendency towards an increased TLR2 and dectin-1 mRNA expression in response to fungal cells, mainly to Pb265. P. brasiliensis yeast cells induced the production of pro-inflammatory and anti-inflammatory cytokines, but the low ratio between TNF-alpha and IL-10 in response to zymosan and Pb265 indicates a balanced production of IL-10 and TNF-alpha, while Pb18 predominantly induced TNF-alpha secretion. Fungal cells also induced an elevated production of PGE(2) by monocytes and neutrophils showing their potential to provoke an intense inflammatory response. Altogether our results suggest the participation of TLR2, TLR-4 and dectin-1 in P. brasiliensis recognition, internalization and consequent activation of the immune response against the fungus. Moreover, the preferential recognition of zymosan and Pb265 by TLR-2 and dectin-1 would result in the production of adequate concentration of IL-10, which would be able to counterbalance the excessive inflammatory response mediated by TNF-alpha and PGE2. With these attributes the low virulence strain of P. brasiliensis would induce a controlled immune response beneficial to the host.477722733Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Capture enzyme-linked immunosorbent assay to detect specific immunoglobulin E in sera of patients with paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is the most frequent systemic mycosis in South America. The disease is characterized by a polyclonal activation of B cells, resulting in hyperimmunoglobulinemia. The production of immunoglobulin (Ig) E in deep mycosis has been related to the severity of the disease. However, the detection of specific IgE in sera of patients is difficult because of the competition with the IgG. We compared a capture and an indirect enzyme-linked immunosorbent assay (ELISA) technique to detect Paracoccidioides brasiliensis IgE. We found that the capture ELISA presented higher performance and lower background values than the indirect assay, resulting in a significant quantitative discrimination between sera from patients with the 2 major clinical forms of PCM. Patients with the juvenile form presented significantly higher levels of P. brasiliensis IgE, as compared with patients with the adult form. The capture ELISA was used in the follow-up of patients receiving treatment, showing that the levels of specific IgE decreased as the patient's clinical conditions improved.o TEXTO COMPLETO DESTE ARTIGO, ESTARÁ DISPONÍVEL À PARTIR DE AGOSTO DE 2015.65323724