Carlos P. Taborda

Mortality due to systemic mycoses as a primary cause of death or in association with AIDS in Brazil: a review from 1996 to 2006

Deaths caused by systemic mycoses such as paracoccidioidomycosis, cryptococcosis, histoplasmosis, candidiasis, aspergillosis, coccidioidomycosis and zygomycosis amounted to 3,583 between 1996-2006 in Brazil. When analysed as the underlying cause of death, paracoccidioidomycosis represented the most important cause of deaths among systemic mycoses (approximately 51.2%). When considering AIDS as the underlying cause of death and the systemic mycoses as associated conditions, cryptococcosis (50.9%) appeared at the top of the list, followed by candidiasis (30.2%), histoplasmosis (10.1%) and others. This mortality analysis is useful in understanding the real situation of systemic mycoses in Brazil, since there is no mandatory notification of patients diagnosed with systemic mycoses in the official health system.

More Is Not Necessarily Better: Prozone-Like Effects in Passive Immunization with IgG

Despite a century of study, the relationship between Ag-specific Ig concentration and protection remains poorly understood for the majority of pathogens. In certain conditions, administration of high Ab doses before challenge with an infectious agent can be less effective than smaller Ab doses, a phenomenon which is consistent with a prozone-like effect. In this study, the relationship between IgG1, IgG2a, IgG2b, and IgG3 dose, infective inocula, and protection was investigated in a mouse model of Cryptococcus neoformans infection. The activity of each IgG subclass ranged from protective to disease-enhancing depending on both the Ab dose and infective inocula used. Enhanced dissemination to the brain was observed in mice given a high IgG2a dose and a relatively low inoculum. Ab administration had immunomodulatory effects, with cytokine expression in lung, brain, and spleen varying as a function of the infective inoculum Ab dose and IgG subclass. In vitro studies did not predict or explain the mechanism of in vivo prozone-like effects, because all isotypes were opsonic and elicited NO release from macrophages. IgG2a was most efficient in inducing a macrophage oxidative burst. These results reveal that an individual Ab can be protective, nonprotective, or disease-enhancing depending on its concentration relative to a challenge inoculum. Our findings have implications for the potential contribution of Ab responses to defense against microbial diseases because Ab-mediated immunity may be protective, nonprotective, or even deleterious to the host.

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (“ghosts”) can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.

The use of cell-free antigens of Paracoccidioides brasiliensis in serological tests

A simple and rapid method for extracting specific cell-free antigens (CFA) from the yeast form of Paracoccidioides brasiliensis grown on agar slants was developed. Extracts were analysed mainly by immunodiffusion (ID) tests, for the immunodiagnosis of paracoccidioidomycosis. Extracts were obtained from 2 to 7-day-old cultures. The extract obtained after the third day, which contained 200-300 micrograms of protein/ml, gave the best ID reactions, with 100% sensitivity and specificity. The main precipitation band was intense and showed total identity with that formed by the specific 43 kDa glycoprotein. Other tests such as counterimmunoelectrophoresis and immunoelectrophoresis were also used successfully with CFA. The 3-day CFA extract showed a pattern of polypeptides in the 10-110 kDa range after SDS-polyacrylamide gel electrophoresis. Five components of 20, 43, 45, 55 and 70 kDa reacted on immunoblots with sera from patients with active chronic paracoccidioidomycosis. The specific diagnostic antigen of 43 kDa always predominated in all CFA preparations. The present method is thus very useful for the rapid production of an antigenic extract which can be readily characterized and used in the serodiagnosis of paracoccidioidomycosis.

Treatment options for paracoccidioidomycosis and new strategies investigated

Paracoccidioidomycosis is the most prevalent systemic endemic mycosis in South America with most reported cases in Brazil. It is a major cause of disability and death among young adult rural workers during their most productive years of life. Sequels are frequent and the evolution of the disease and mortality burden are strongly influenced by the socio–economic status of the patients. Although long periods of antifungal therapy (sulfamethoxazole/trimethoprim, itraconazole and amphotericin B) are used in clinical practice, relapses remain a significant unresolved problem. Early diagnosis is hampered by structural factors, ranging from the high costs of reagents, the lack of trained personnel and limited access to the healthcare system by rural workers. A peptide vaccine aimed at immunotherapy of paracoccidioidomycosis, as an adjuvant to chemotherapy, is being studied. The protective effects obtained in mice intratracheally infected with Paracoccidioides brasiliensis, and the promiscuous binding of the peptide P10 to HLA-DR molecules, suggest that it could be used as a vaccine to reduce the duration of chemotherapy and the risk of relapse.

The Monoclonal Antibody against the Major Diagnostic Antigen of Paracoccidioides brasiliensis Mediates Immune Protection in Infected BALB/c Mice Challenged Intratracheally with the Fungus

ABSTRACT The protective role of specific antibodies against Paracoccidioides brasiliensis is controversial. In the present study, we analyzed the effects of monoclonal antibodies on the major diagnostic antigen (gp43) using in vitro and in vivo P. brasiliensis infection models. The passive administration of some monoclonal antibodies (MAbs) before and after intratracheal or intravenous infections led to a reduced fungal burden and decreased pulmonary inflammation. The protection mediated by MAb 3E, the most efficient MAb in the reduction of fungal burden, was associated with the enhanced phagocytosis of P. brasiliensis yeast cells by J774.16, MH-S, or primary macrophages. The ingestion of opsonized yeast cells led to an increase in NO production by macrophages. Passive immunization with MAb 3E induced enhanced levels of gamma interferon in the lungs of infected mice. The reactivity of MAb 3E against a panel of gp43-derived peptides suggested that the sequence NHVRIPIGWAV contains the binding epitope. The present work shows that some but not all MAbs against gp43 can reduce the fungal burden and identifies a new peptide candidate for vaccine development.

In Vitro Activity of the Antifungal Plant Defensin RsAFP2 against Candida Isolates and Its In Vivo Efficacy in Prophylactic Murine Models of Candidiasis

ABSTRACT We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Additive effect of P10 immunization and chemotherapy in anergic mice challenged intratracheally with virulent yeasts of Paracoccidioides brasiliensis

Paracoccidioidomycosis is a systemic granulomatous disease manifested in the acute/subacute or chronic forms. The anergic cases of the acute/subacute form are most severe, leading to death threatening conditions. Drug treatment is required to control the disease but the response in anergic patients is generally poor. A 15-mer peptide from the major diagnostic antigen gp43, named P10, induces a T-CD4+ helper-1 immune response in mice of different haplotypes and protects against intratracheal challenge with virulent P. brasiliensis. Presently, P10 immunization and chemotherapy were associated in an attempt to improve antifungal treatment in Balb/c mice made anergic by adding dexamethasone to the drinking water. The combined drug/peptide treatment significantly reduced the lung CFUs in infected anergic mice, largely preserved lung alveolar structure and prevented fungal dissemination to liver and spleen. Results recommend that a P10-based vaccine should be associated to chemotherapy for improved treatment of paracoccidioidomycosis aiming especially at anergic cases.

Paracoccidioides brasiliensis vaccine formulations based on the gp43-derived P10 sequence and the Salmonella enterica FliC flagellin.

ABSTRACT Paracoccidioidomycosis (PCM) is a systemic granulomatous disease caused by the dimorphic fungus Paracoccidioides brasiliensis. Anti-PCM vaccine formulations based on the secreted fungal cell wall protein (gp43) or the derived P10 sequence containing a CD4+ T-cell-specific epitope have shown promising results. In the present study, we evaluated new anti-PCM vaccine formulations based on the intranasal administration of P. brasiliensis gp43 or the P10 peptide in combination with the Salmonella enterica FliC flagellin, an innate immunity agonist binding specifically to the Toll-like receptor 5, in a murine model. BALB/c mice immunized with gp43 developed high-specific-serum immunoglobulin G1 responses and enhanced interleukin-4 (IL-4) and IL-10 levels. On the other hand, mice immunized with recombinant purified flagellins genetically fused with P10 at the central hypervariable domain, either flanked or not by two lysine residues, or the synthetic P10 peptide admixed with purified FliC elicited a prevailing Th1-type immune response based on lung cell-secreted type 1 cytokines. Mice immunized with gp43 and FliC and intratracheally challenged with P. brasiliensis yeast cells had increased fungal proliferation and lung tissue damage. In contrast, mice immunized with the chimeric flagellins and particularly those immunized with P10 admixed with FliC reduced P. brasiliensis growth and lung damage. Altogether, these results indicate that S. enterica FliC flagellin modulates the immune response to P. brasiliensis P10 antigen and represents a promising alternative for the generation of anti-PCM vaccines.

Synthesis and immunological activity of a branched peptide carrying the T-cell epitope of gp43, the major exocellular antigen of Paracoccidioides brasiliensis

The 43 kDa glycoprotein (gp43) of Paracoccidioides brasiliensis is the major diagnostic antigen of paracoccidioidomycosis (PCM), a prevalent fungal infection in South America. A 15‐mer sequence from gp43, denominated P10, induced T‐CD4+ T helper 1 cellular immune responses in mice of three different haplotypes and protected against intratracheal challenge by a virulent isolate of P. brasiliensis. In an attempt to improve delivery of P10, a promiscuous antigen also presented by human leucocyte antigen‐DR alleles, aiming at immunotherapy, we synthesized a multiple antigen peptide with the protective T‐cell epitope expressed in a tetravalent 13‐mer analog of P10 (M10). M10 induced specific lymph node cell proliferation in mice preimmunized with peptides in complete Freunds adjuvant (CFA). In addition, M10 immunization without CFA significantly protected intratracheally infected mice. We conclude that M10 is a candidate for an anti‐PCM vaccine. In this report we describe: (1) the synthesis of M10; (2) the induction of M10‐elicited T‐cell response and (3) in vivo protection of mice immunized with M10 and challenged by a virulent strain of P. brasiliensis.