Cytokine production in lungs and adrenal glands of high and low antibody producing mice infected with Paracoccidioides brasiliensis

Mice genetically selected for high (H) and low (L) antibody production (HIV-A and L-IV-A) were used in an experimental model of paracoccidioidomycosis. In a previous work, it was observed that male HIV-A animals were more susceptible to the infection due to adrenal gland damage. Male HIV-A and LIV-A animals were intravenously inoculated with Paracoccidioides brasiliensis (strain 18) and sacrificed 2, 4, 6, 8 and 10 weeks after inoculation. At each time interval, lungs and adrenals were removed to estimate recoverability of the fungus, as well as to determine Th1 (IFN-gamma, TNF-alpha) and Th2 (IL-4 and IL-10) cytokine profiles. While viable fungi recoverability from the lungs of HIV-A mice was higher after 4 and 8 weeks, there was less fungal recovery from the adrenals of LIV-A animals after the 2nd week, with total fungal elimination after the 8th week. With regard to Th2 cytokines, there was an inhibition in IL-4 production in the organs from infected animals, the extent of which varied according to the organ and the time period after initiation of infection. IL-10 production was found to be lower in both organs. Determination of Th1 cytokines revealed that IFN-gamma production increased in both organs, mainly in the adrenal of LIV-A after 8 and 10 weeks, when these animals showed a total fungal elimination. A significant difference was observed between HIV-A and LIV-A concerning TNF-alpha production in both organs and at all recovery times, in that LIV-A produced a higher level of this cytokine, mainly in the adrenal. These results may explain the high susceptibility of HIV-A to P. brasiliensis infection, is due, at least in part, to adrenal involvement. The higher production of Th1 cytokines by LIV-A in comparison to HIV-A mice may account for LIV-A resistance to P. brasiliensis infection. Our data reveal the importance of this experimental model in the study of the adrenal involvement in paracoccidioidomycosis, since this gland may be highly compromised in the patients, leading to the development of Addison's Disease.

Production of reactive oxygen (H2O2) and nitrogen (NO) intermediates and TNF-α in mice genetically selected for high (H) and low (L) antibody response and experimentally infected with Leptospira serovar pomona

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The effects of propolis on antibody production by laying hens

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Plasmodium falciparum: sequence diversity and antibody recognition of the Merozoite surface protein-2 (MSP-2) in Brazilian Amazonia

The merozoite surface protein-2 (MSP-2) of Plasmodium falciparum comprises repeats flanked by dimorphic domains defining the allelic families FC27 and IC1. Here, we examined sequence diversity at the msp-2 locus in Brazil and its impact on MSP-2 antibody recognition by local patients. Only 25 unique partial sequences of msp-2 were found in 61 isolates examined. The finding of identical msp-2 sequences in unrelated parasites, collected 6-13 years apart, suggests that no major directional selection is exerted by variant-specific immunity in this malaria-endemic area. To examine antibody cross-reactivity, recombinant polypeptides derived from locally prevalent and foreign MSP-2 variants were used in ELISA. Foreign IC1-type variants, such as 3D7 (currently tested for human vaccination), were less frequently recognized than FC27-type and local IC1-type variants. Antibodies discriminated between local and foreign IC1-type variants, but cross-recognized structurally different local IC1-type variants. The use of evolutionary models of MSP-2 is suggested to design vaccines that minimize differences between local parasites and vaccine antigens. (C) 2004 Elsevier B.V. All rights reserved.

Detection of anti-Giardia lamblia serum antibody among children of day care centers

OBJETIVOS: Detectar anticorpos séricos anti-Giardia lamblia entre crianças atendidas em creches e estimar a freqüência de infecção por Giardia lamblia em área endêmica. MÉTODOS: Foram coletadas três amostras de fezes de cada uma das 147 crianças de três creches da rede municipal de Botucatu, SP, com idade variando de 0 a 6 anos, e as amostras foram processadas pelos métodos de sedimentação espontânea e flutuação pelo sulfato de zinco. Amostras de sangue foram obtidas da polpa digital, coletadas em papel de filtro e testadas pelos métodos de imunofluorescência indireta (IFI) e de reação imunoenzimática (Elisa) para pesquisa de IgG anti-Giardia. RESULTADOS E CONCLUSÕES: de um total de 147 crianças, 93 (63,3%) apresentaram cistos de Giardia nas fezes. Dos 147 eluatos testados, 93 (63,3%) e 100 (68%) foram positivos para Giardia em IFI e em Elisa, respectivamente. A sensibilidade de IFI foi de 82% e de Elisa, 72%. Contudo, Elisa foi menos específica (39%) do que IFI (70%). A imunofluorescência indireta apresentou maior concordância com o exame de fezes do que Elisa.

Determining the structural basis for specificity of ligands using crystallographic screening

Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.

ZapA, a possible virulence factor from Proteus mirabilis exhibits broad protease substrate specificity

The opportunistic bacterium Proteus mirabilis secretes a metalloprotease, ZapA, considered to be one of its virulence factors due to its IgA-degrading activity. However, the substrate specificity of this enzyme has not yet been fully characterized. In the present study we used fluorescent peptides derived from bioactive peptides and the oxidized ß-chain of insulin to determine the enzyme specificity. The bradykinin- and dynorphin-derived peptides were cleaved at the single bonds Phe-Ser and Phe-Leu, with catalytic efficiencies of 291 and 13 mM/s, respectively. Besides confirming already published cleavage sites, a novel cleavage site was determined for the ß-chain of insulin (Val-Asn). Both the natural and the recombinant enzyme displayed the same broad specificity, demonstrated by the presence of hydrophobic, hydrophilic, charged and uncharged amino acid residues at the scissile bonds. Native IgA, however, was resistant to hydrolysis by ZapA.

Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach

Background: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma.Objective: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides.Methods: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb IA6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program.Results: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb IA6.Conclusion: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.

Thrombomodulin-independent activation of protein C and specificity of hemostatically active snake venom serine proteinases - Crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator

Protein C activation initiated by the thrombin-thrombomodulin complex forms the major physiological anticoagulant pathway. Agkistrodon contortrix contortrix protein C activator, a glycosylated single-chain serine proteinase, activates protein C without relying on thrombomodulin. The crystal structures of native and inhibited Agkistrodon contortrix contortrix protein C activator determined at 1.65 and 1.54 angstrom resolutions, respectively, indicate the pivotal roles played by the positively charged belt and the strategic positioning of the three carbohydrate moieties surrounding the catalytic site in protein C recognition, binding, and activation. Structural changes in the benzamidine-inhibited enzyme suggest a probable function in allosteric regulation for the anion-binding site located in the C-terminal extension, which is fully conserved in snake venom serine proteinases, that preferentially binds Cl1- instead of SO42-.

Venom peptide analysis of Vipera ammodytes meridionalis (Viperinae) and Bothrops jararacussu (Crotalinae) demonstrates subfamily-specificity of the peptidome in the family Viperidae

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Imunossensor amperométrico

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development of an enzyme-linked immunosorbent assay for detection of IgM antibodies to Babesia bigemina in cattle

A crude antigenic preparation of Babesia bigemina was used to develop an ELISA for the detection of IgM antibodies. Optimal dilutions of the antigen, using positive and negative reference sera, were determined by checkerboard titrations. Negative sera from cattle imported from tick-free areas, serum samples collected from infected B. bigemina cattle were used to validate the test. The specificity was 94% and sensitivity of the Elisa 87.5%. Sera from 385 cattle deriving from areas free from tick-borne diseases, which were submitted to a preimmunization process, were screened by this technique. The Elisa detected seroconversion on the 14th day post-inoculation in animals either infested with Boophilus microplus ticks (infected with B. bigemina), or inoculated with B. bigemina infected blood. Antibody titers decreased after day 33; however, all animals remained positive until the end of the experiment (124 days). The ELISA described may prove to be an appropriate serological test for the detection of IgM antibodies against B. bigemina.

Demonstration of antibody and cellular immune response to brain extract in West and Lennox-Gastaut syndromes

Investigamos a resposta imunológica celular e humoral frente a extrato salino de tecido cerebral em 9 pacientes com síndrome de Lennox-Gastaut, 15 pacientes com síndrome de West e 20 crianças normais. A técnica de imunodifusão dupla em gel de agar (Ouchterlony) evidenciou em todos os pacientes, altos níveis de um anticorpo precipitante contra o extrato salino de tecido cerebral. O teste de inibição de migração de leucócitos com o mesmo antígeno mostrou-se positivo na maioria dos pacientes. O possível papel destas respostas autoimmunes na patogenia das sindromes de West e Lennox-Gastaut é discutido.