RESUMEN

BACKGROUND: Shiga toxin (Stx)-producing Escherichia coli (STEC) infection is associated with hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. Stx is essential in the pathogenesis of HUS, which has been mostly related to Stx2-producing isolates. Very limited data exist on the immune response to STEC in the Brazilian population. In this study, the prevalence of immunoglobulin G (IgG) antibodies to Stx2 was investigated in sera of children diagnosed with HUS and of healthy children in the city of São Paulo, Brazil. METHODS: IgG-antibody reactivity to Stx2 was determined by immunoblotting (WB) and enzyme-linked immunosorbent assay (ELISA) in sera from 13 children with HUS aged 8 months to 6 years and 54 healthy urban children aged 5 months to 7 years. RESULTS: A positive immune response to the A and B subunits of Stx2 was observed in 46.1% HUS patients and in 16.6% healthy individuals by WB. All HUS patients and 62.9% healthy children showed IgG antibodies to the Stx2 A subunit. The frequency of antibodies to both subunits or only to the A subunit of Stx2 was significantly higher in HUS patients than controls (p<0.05). Also, the mean OD value obtained by ELISA was higher in that group. Considering children's age, the frequency of reactivity to either the A subunit or both subunits of Stx2 was considerably higher in HUS children up to three years old compared to controls in the same age range. Moreover, in almost 37% of healthy children, no immune response to Stx2 was detected independently of the child's age. CONCLUSIONS: The seroepidemiolgy of anti-Stx2 antibodies was described for the first time in healthy children and children with HUS in Brazil. The percentage of individuals showing antibodies against Stx2 was higher among HUS patients than controls, and in spite of the low number of notified HUS cases, STEC strains are circulating in our settings. In addition, the results obtained also corroborated previous data on the increased sensitivity and specificity of WB compared to toxin-based enzyme immunoassays.

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RESUMEN

Enterohemorrhagic Escherichia coli (EHEC) strains can cause hemolytic uremic syndrome (HUS), a leading cause of childhood renal failure. The adhesin intimin and the secreted proteins A (EspA) and B (EspB) contribute to the occurrence of EHEC attaching and effacing lesions. In this study, immunoblot assays were performed to determine immunoglobulin G (IgG) antibodies reactive with these proteins in sera from 13 children diagnosed with HUS and in sera from 54 healthy Brazilian children. In general, high frequencies of serum IgG antibodies reactive with EspA, EspB and the conserved region of intimin were observed in both HUS patients and controls with no statistically significant differences. However, a marked difference in immune response to these proteins was observed in HUS patients compared to controls in infants less than two years of age. In addition, IgG against the variable region of intimin γ was more frequently detected in HUS patients than in children with no signs of infection (p<0.05) regardless of age, suggesting that the detection of antibodies directed to the variable region of intimin γ can be useful in serodiagnostic tests of EHEC-infected patients. The immune response against intimin and structural proteins encoded by the locus of enterocyte effacement pathogenicity island in patients with HUS has previously not been described in Brazil. The results presented here may contribute to the development of diagnostic tools and complement information concerning EHEC epidemiology in our setting.

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The presence of subAB was investigated for 3,453 Escherichia coli strains of various pathogenic categories. The occurrence of other virulence genes in subAB-positive strains was investigated. The subAB operon was detected among some Shiga toxin-producing E. coli (STEC) serotypes devoid of eae and carrying ehxA. Most subAB-positive strains also harbored stx2, iha, saa, and lpfA(O113).

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Pouco se conhece sobre os epítopos derivados de promastigotas metacíclicos de Leishmania que são importantes para a regulação ou destruição do parasita, como alvos de ação imunológica no hospedeiro vertebrado. Neste estudo, nós investigamos um método alternativo para obter promastigotas metacíclicos de Leishmania major, pela avaliação do curso da infecção e reação de hipersensibilidade do tipo retardado (HTR) em hospedeiros resistentes e susceptíveis. Promastigotas não-infectantes (procíclicos) de L. major, recentemente isolados de amastigotas, foram selecionados pela adesão a colunas de lã de vidro negativamente carregadas, enquanto que promastigotas metacíclicos não se aderem à coluna e podem ser recuperados com facilidade. Condições ótimas de cromatografia foram validadas por análise estatística. O rendimento médio de parasitas obtidos após separação em colunas de lã de vidro e a viabilidade dos promastigotas foram estimados por microscopia óptica. Os promastigotas metacíclicos tiveram um rendimento médio de 43,5% a 57,5%. Camundongos BALB/c (susceptíveis) e camundongos C57BL/6 (resistentes) apresentaram padrões distintos de lesões cutâneas, os primeiros com lesões mais agressivas, induzidas por promastigotas metacíclicos. As respostas à reação de HTR foram maiores nos grupos de camundongos C57BL/6, submetidos à infecção com promastigotas metacíclicos. Estes resultados indicam que o novo método poderia ser integrado aos protocolos existentes para estudar a metaciclogênese de parasitas do gênero Leishmania in vivo.

RESUMEN

Little is known on the epitopes derived from metacyclic promastigotes of Leishmania that are important on the regulation or destruction of the parasite, as targets of immune attack in the vertebrate host. In this study we investigated an alternative method to obtain metacyclic promasigotes of Leishmania major, as evaluated by the course of infection and delayed-type hipersensitivity (DTH) in resistant versus susceptible inbred mice. Non-infective (procyclic) promastigotes of L. major recently transformed from tissue amastigotes were attached to a negatively charged glass-wool column, whereas metacyclic promastigotes were not bound to columns and could be easily recovered. Optimal chromatography conditions were validated through statistical analyses. Parasite average yield from glass wool columns and promastigote viability were estimated by light microscopy. Metacyclic promastigotes yielded 43.5% to 57.5%. Different patterns of cutaneous lesions were obtained in BALB/c (susceptible) and C57BL/6 (resistant) mice, the former with highly infective lesions induced by metacyclic promastigotes. DTH responses proved to be higher in groups of C57BL/6 mice which were infected with metacyclic promastigotes. These results indicate that the new method could be integrated with the investigation of metacyclogenesis of Leishmania in vivo.

Pouco se conhece sobre os epítopos derivados de promastigotas metacíclicos de Leishmania que são importantes para a regulação ou destruição do parasita, como alvos de ação imunológica no hospedeiro vertebrado. Neste estudo, nós investigamos um método alternativo para obter promastigotas metacíclicos de Leishmania major, pela avaliação do curso da infecção e reação de hipersensibilidade do tipo retardado (HTR) em hospedeiros resistentes e susceptíveis. Promastigotas não-infectantes (procíclicos) de L. major, recentemente isolados de amastigotas, foram selecionados pela adesão a colunas de lã de vidro negativamente carregadas, enquanto que promastigotas metacíclicos não se aderem à coluna e podem ser recuperados com facilidade. Condições ótimas de cromatografia foram validadas por análise estatística. O rendimento médio de parasitas obtidos após separação em colunas de lã de vidro e a viabilidade dos promastigotas foram estimados por microscopia óptica. Os promastigotas metacíclicos tiveram um rendimento médio de 43,5% a 57,5%. Camundongos BALB/c (susceptíveis) e camundongos C57BL/6 (resistentes) apresentaram padrões distintos de lesões cutâneas, os primeiros com lesões mais agressivas, induzidas por promastigotas metacíclicos. As respostas à reação de HTR foram maiores nos grupos de camundongos C57BL/6, submetidos à infecção com promastigotas metacíclicos. Estes resultados indicam que o novo método poderia ser integrado aos protocolos existentes para estudar a metaciclogênese de parasitas do gênero Leishmania in vivo.

RESUMEN

Little is known on the epitopes derived from metacyclic promastigotes of Leishmania that are important on the regulation or destruction of the parasite, as targets of immune attack in the vertebrate host. In this study we investigated an alternative method to obtain metacyclic promasigotes of Leishmania major, as evaluated by the course of infection and delayed-type hipersensitivity (DTH) in resistant versus susceptible inbred mice. Non-infective (procyclic) promastigotes of L. major recently transformed from tissue amastigotes were attached to a negatively charged glass-wool column, whereas metacyclic promastigotes were not bound to columns and could be easily recovered. Optimal chromatography conditions were validated through statistical analyses. Parasite average yield from glass wool columns and promastigote viability were estimated by light microscopy. Metacyclic promastigotes yielded 43.5% to 57.5%. Different patterns of cutaneous lesions were obtained in BALB/c (susceptible) and C57BL/6 (resistant) mice, the former with highly infective lesions induced by metacyclic promastigotes. DTH responses proved to be higher in groups of C57BL/6 mice which were infected with metacyclic promastigotes. These results indicate that the new method could be integrated with the investigation of metacyclogenesis of Leishmania in vivo.

Pouco se conhece sobre os epítopos derivados de promastigotas metacíclicos de Leishmania que são importantes para a regulação ou destruição do parasita, como alvos de ação imunológica no hospedeiro vertebrado. Neste estudo, nós investigamos um método alternativo para obter promastigotas metacíclicos de Leishmania major, pela avaliação do curso da infecção e reação de hipersensibilidade do tipo retardado (HTR) em hospedeiros resistentes e susceptíveis. Promastigotas não-infectantes (procíclicos) de L. major, recentemente isolados de amastigotas, foram selecionados pela adesão a colunas de lã de vidro negativamente carregadas, enquanto que promastigotas metacíclicos não se aderem à coluna e podem ser recuperados com facilidade. Condições ótimas de cromatografia foram validadas por análise estatística. O rendimento médio de parasitas obtidos após separação em colunas de lã de vidro e a viabilidade dos promastigotas foram estimados por microscopia óptica. Os promastigotas metacíclicos tiveram um rendimento médio de 43,5% a 57,5%. Camundongos BALB/c (susceptíveis) e camundongos C57BL/6 (resistentes) apresentaram padrões distintos de lesões cutâneas, os primeiros com lesões mais agressivas, induzidas por promastigotas metacíclicos. As respostas à reação de HTR foram maiores nos grupos de camundongos C57BL/6, submetidos à infecção com promastigotas metacíclicos. Estes resultados indicam que o novo método poderia ser integrado aos protocolos existentes para estudar a metaciclogênese de parasitas do gênero Leishmania in vivo.