Differences in reactivity of paracoccidioidomycosis sera with gp43 isoforms

The glycoprotein gp43 from Paracoccidioides brasiliensis is the main antigenic component in paracoccidioidomycosis (PCM) because it is recognized by 100% of PCM patients. It has also been shown that different fungal strains produce gp43 with at least four isoform profiles. In this study, different isoform profiles from gp43, with pIs ranging from 5.8 to 8.5, were affinity purified from various P. brasiliensis (B-339, S.S., 1925 and I-9) exoantigens. Because of the isoform heterogeneity, we questioned whether those isoform profiles could be similarly recognized by acute or chronic PCM patients. By using a specific and sensitive method for detection of human IgG anti-gp43 antibodies, the monoclonal antibody capture immunoassay, we report that not all gp43 isoform profiles are equally recognized in PCM sera when anti-gp43 MAb 17c was employed as capturing antibody. Our result showed that recognition of pI 8.5 gp43 isoform was significantly lower for both acute (56%) and chronic patients (71%), compared with gp43 isoforms from the standard strain B-339. on the other hand, when anti-gp43 MAb 8a, which recognizes a different antigenic epitope was used to capture the different gp43 isoform profiles, all patient's sera reacted similarly. The results described suggest that not all the antigenic epitopes expressed by gp43 are equally present in all P. brasiliensis strains.

Survey for Tick-Borne Zoonoses in the State of Espirito Santo, Southeastern Brazil

Blood samples collected from 201 humans, 92 dogs, and 27 horses in the state of Espirito Santo, Brazil, were tested by polymerase chain reaction, indirect immunofluorescence assays, and indirect enzyme-linked immunosorbent assay for tick-borne diseases (rickettsiosis, ehrlichiosis, anaplasmosis, borreliosis, babesiosis). Our results indicated that the surveyed counties are endemic for spotted fever group rickettsiosis because sera from 70 (34.8%) humans, 7 (7.6%) dogs, and 7 (25.9%) horses were reactive to at least one of the six Rickettsia species tested. Although there was evidence of ehrlichiosis (Ehrlichia canis) and babesiosis (Babesia cams vogeli, Theileria equi) in domestic animals, no human was positive for babesiosis and only four individuals were serologically positive for E. canis. Borrelia burgdorferi-serologic reactive sera were rare among humans and horses, but encompassed 51% of the canine samples, suggesting that dogs and their ticks can be part of the epidemiological cycle of the causative agent of the Brazilian zoonosis, named Baggio-Yoshinari Syndrome.

Use of crude, FML and rK39 antigens in ELISA to detect anti-Leishmania spp. antibodies in Felis catus

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

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.

Detection of Leptospira spp. in the semen and urine of bulls serologically reactive to Leptospira interrogans serovar hardjo

O presente trabalho avaliou a PCR na detecção de leptospiras em sêmen e urina de dez touros sorologicamente reagentes, comparando seus resultados com aqueles obtidos por outras técnicas de diagnóstico. Foram realizadas duas colheitas de materiais em dias alternados. As amostras de sêmen e de urina foram separadas em alíquotas para visualização direta em microscopia de campo escuro, inoculação em hamsters (apenas para o sêmen), isolamento em meio de cultura e PCR. Nenhum hamster apresentou positividade na prova de soroaglutinação microscópica (SAM); fragmentos de rins e fígado desses animais foram utilizados para a tentativa de isolamento em meio de cultura, sendo positivo o cultivo a partir do rim de hamster inoculado com semen de um touro, e do fígado de hamsters inoculados com o semen de três touros. O isolamento em meio de cultura foi negativo para todas as amostras de sêmen, mas foi positivo para cinco amostras de urina. Na PCR não houve resultado positivo para as amostras de sêmen, e apenas uma amostra de urina apresentou resultado positivo, sendo coincidente com uma das culturas positivas. Não foi possível visualizar leptospiras em nenhuma das amostras por exame direto em microscopia de campo escuro.

Pesquisa de anticorpos anti-Neospora caninum em bovinos leiteiros, cães e trabalhadores rurais da região Sudoeste do Estado de Mato Grosso

Considerando a importância da neosporose interferindo na produtividade animal, avaliou-se a frequência de anticorpos anti-Neospora caninum em amostras de soros de bovinos leiteiros da Região Sudoeste do Estado de Mato Grosso, Brasil, complementando-se com amostras sorológicas colhidas de cães e de humanos que conviviam nas mesmas propriedades rurais amostradas. Um total de 1.036 amostras de soros foram analisadas, sendo 932 de fêmeas bovinas leiteiras, 37 de cães e 67 de humanos, provenientes de 24 propriedades e examinados por meio da reação de imunofluorescência indireta (RIFI). As amostras de soros humanos reagentes foram testadas novamente por Western-blotting para confirmação dos resultados. Anticorpos anti-N. caninum foram encontrados em 499 bovinos (53,5%), em pelo menos um animal positivo por propriedade, em 25 caninos (67,6%) e em sete humanos (10,5%). Não houve diferença significativa no número de bovinos positivos por faixa etária. Os resultados obtidos indicam uma ampla disseminação de N. caninum na região estudada.

Antigenic characterization of Trypanosoma evansi using sera from experimentally and naturally infected bovines, equines, dogs, and coatis

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

Effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs on the production of reactive oxygen species by activated rat neutrophils

The release of reactive oxygen specie (ROS) by activated neutrophil is involved in both the antimicrobial and deleterious effects in chronic inflammation. The objective of the present investigation was to determine the effect of therapeutic plasma concentrations of non-steroidal anti-inflammatory drugs (NSAIDs) on the production of ROS by stimulated rat neutrophils. Diclofenac (3.6 µM), indomethacin (12 µM), naproxen (160 µM), piroxicam (13 µM), and tenoxicam (30 µM) were incubated at 37ºC in PBS (10 mM), pH 7.4, for 30 min with rat neutrophils (1 x 10(6) cells/ml) stimulated by phorbol-12-myristate-13-acetate (100 nM). The ROS production was measured by luminol and lucigenin-dependent chemiluminescence. Except for naproxen, NSAIDs reduced ROS production: 58 ± 2% diclofenac, 90 ± 2% indomethacin, 33 ± 3% piroxicam, and 45 ± 6% tenoxicam (N = 6). For the lucigenin assay, naproxen, piroxicam and tenoxicam were ineffective. For indomethacin the inhibition was 52 ± 5% and diclofenac showed amplification in the light emission of 181 ± 60% (N = 6). Using the myeloperoxidase (MPO)/H2O2/luminol system, the effects of NSAIDs on MPO activity were also screened. We found that NSAIDs inhibited both the peroxidation and chlorinating activity of MPO as follows: diclofenac (36 ± 10, 45 ± 3%), indomethacin (97 ± 2, 100 ± 1%), naproxen (56 ± 8, 76 ± 3%), piroxicam (77 ± 5, 99 ± 1%), and tenoxicam (90 ± 2, 100 ± 1%), respectively (N = 3). These results show that therapeutic levels of NSAIDs are able to suppress the oxygen-dependent antimicrobial or oxidative functions of neutrophils by inhibiting the generation of hypochlorous acid.

Inhibition of peroxidase activity and scavenging of reactive oxygen species by astilbin isolated from Dimorphandra mollis (Fabaceae, Caesalpinioideae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Spliceosomal factors are recognized by Chagasic sera

We investigated the possibility that Chagas' patients develop an autoimmune response to human UsnRNPs or Sm epitopes. Using purified human UsnRNPs we detected anti-human UsnRNPs antibodies in sera from patients with Chagas' disease. The antibodies were also detected using peptide-ELISA containing the Sm-motif 1 domain, showing that 61% (31/51) of the Chagas sera contained antibodies against the Sm-motif 1. Our preliminary results obtained by immunoprecipitation using Chagas chronically infected Balb/C sera and HeLa nuclear extracts showed that some autoantibodies could also be found during the course of the disease. Groups of 20 mice tone-month-old) were infected i.p. with Y strain 30 bloodstream trypomastigotes and killed at 60 days post-infection and sera were used for immunoprecipitation analysis as mentioned, These antibodies cross-reacted with U2 snRNP in HeLa nuclear extract and revealed many different bands in T. cruzi nuclear extract. These results suggest that the autoantibodies may have a role in the pathogenesis of the disease,

Intermediate reactive oxygen and nitrogen from macrophages induced by Brazilian lichens

The activity of ten compounds isolated from Brazilian lichen over the release of hydrogen peroxide and nitric oxide was evaluated in the culture of peritoneal macrophage cells from mice. Salazinic, secalonic A and fumarprotocetraric acids were the compounds that induced the greatest release of H2O2, whereas 12R-usnic and diffractaic acids induced the release of NO. These results indicate that lichen products have potential immunological modulating activities. (C) 2004 Elsevier B.V. All rights reserved.

Presence of autoantibodies against small nuclear ribonucleoprotein epitopes in Chagas' patients' sera

We investigated the possibility that Chagas' patients develop an autoimmune response to human UsnRNPs (small nuclear ribonucleoprotein) or Sm epitopes. Using purified human UsnRNPs, we detected anti-human UsnRNPs antibodies in sera from patients suffering from Chagas' disease. The antibodies it-ere also detected using peptide enzyme-linked immunosorbent assays containing the Sm-motif 1 domain. The latter technique showed that 61% (31/51) of the Chagas' patients' sera contained antibodies against Sm-motif I. The detection of anti-UsnRNPs autoantibodies in Chagas patients' sera strongly encourages further studies using animal models to determine how these autoantibodies appear.

Yersinia enterocolitica O : 3 isolated from patients with or without reactive arthritis induces polyclonal activation of B cells and autoantibody production in vivo

The mechanisms by which arthritis-provoking pathogens such as Yersinia enterocolitica interact with the human immune system to produce inflammatory synovitis are not well known. One of the immunomodulating mechanisms used against these pathogens is the polyclonal activation of lymphocytes. In this study, we investigated the extent of the B-lymphocyte activation induced in mice by a strain of Y. enterocolitica O:3 (FCF 526) isolated from a patient with arthritis, and compared it with two other strains, a virulent one (FCF 397[+]) isolated from a patient without arthritis and its plasmidless isogenic pair (FCF397[-]). Also we investigated the production of autoantibodies in mice infected with these different strains. SPF Swiss mice were infected intravenously with a suspension of Y. enterocolitica . Spleen cells were taken on days 7, 14, 21 and 28 after infection and the number of cells secreting nonspecific and specific antibodies of IgG 1 , IgG 2a , IgG 2b , IgG 3 , IgM and IgA isotypes were determined by the ELISPOT technique. The presence of autoantibodies in mouse serum was investigated by the dot-blot assay. The pattern of infection of the three bacterial strains were almost the same. We observed a general increase in the number of nonspecific Ig-secreting cells with all three strains, and the greatest increases observed were in the IgG 2a and IgG 3 isotypes. Only a small fraction of the immunoglobulins detected were antibacterial, suggesting that the rest resulted from polyclonal B cell activation. The strain isolated from the patient with arthritis (FCF526) induced the greatest production of autoantibodies, coinciding with the period in which the greatest activation of nonspecific B lymphocytes was seen. There were no signs of arthritis or inflammation in the joints of the infected animals. Based on our results, we were unable to determine whether there is an association between the arthritogenic capability of Y. enterocolitica and polyclonal activation of B cells.

Columnar microstructure of nanocrystalline Ga1-xMnxN films deposited by reactive sputtering

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