A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques

The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi.Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage) it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50 percent similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.

A técnica de Western blot foi utilizada para demonstrar a presença de anticorpos do soro de cães, que apresentavam leishmaniose visceral canina, contra algumas moléculas específicas no lisado da forma promastigota de Leshmania chagasi.Através da associação da técnica de Western blot com as alterações morfológicas observadas como resultado da técnica de soro-neutralização em células McCoy (que mimetizam o macrófago) foi possível observar o papel de algumas moléculas de maior relevância para a determinação da doença em cães sintomáticos bem como o papel de outras moléculas na predição de cães infectados assintomáticos com o potencial de serem transmissores e ainda diferenciá-los como cães assintomáticos resistentes. Na análise dos soros durante a reação de immunoblotting observou-se uma variação de 9 a 27 bandas imunorreagentes, que foram comparadas utilizando-se o coeficiente de similaridade de Dice. No dendrograma construído com base no coeficiente, observou-se 50 por cento de similaridade entre as bandas totais reagentes com o lisado de promastigota formando cinco agrupamentos. A principal diferença foi observada com respeito à condição clínica, ou seja, cães sintomáticos e assintomáticos ficaram em grupos separados. Os soros dos cães assintomáticos distribuídos em dois grupos diferentes do dendrograma apresentaram padrões de comportamento diferentes, quanto à morfologia celular na reação de soro-neutralização, ou seja, a presença ou ausência de lise celular. De acordo com esta análise foi possível avaliar o status imunitário e associá-lo com determinados marcadores específicos observados na reação encontrada nas fitas de Western blot.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Cinéticas de crescimento de Leishmania realizadas in vitro após a internalização da forma promastigota na célula e a ocorrência da transformação do parasito na forma amastigota foram descritas por vários autores, seja com a utilização de explantes de macrófagos em células de baço de hamster ou atualmente da célula de linhagem de macrófago humano U937. Aliando a microscopia à descrição das inter-relações morfológicas e à síntese de moléculas específicas foi possível esclarecer pontos sobre a biologia do parasito. O presente estudo mostra o acompanhamento do ciclo de crescimento da Leishmania chagasi em uma cinética realizada com células de linhagem McCoy, no período de 144 horas. Durante o processo, as transformações morfológicas foram reveladas pela reação de imunofluorescência indireta (RIFI) e as moléculas liberadas no meio extracelular foram observadas pelo método de SDS-PAGE, em intervalos de 24 horas no período de 144 horas. Observou-se que nas primeiras 72 horas, a forma promastigota da L. chagasi fica aderida à membrana das células com aspecto arredondado (amastigota-like). Em 96 horas as células infectadas apresentaram alterações morfológicas; em 120 horas, as células liberaram, para o meio extracelular, antígenos fluorescentes solúveis; e em 144 horas foram observadas novas formas alongadas dos parasitos como se fossem promastigotas. No SDS-PAGE, proteínas com pesos moleculares específicos são observadas em cada ponto da cinética, mostrando que a célula McCoy parece mimetizar o macrófago e que pode ser um modelo útil para o estudo da infecção do binômio leishmânia/célula.

McCoy cell line as a possible model containing CD4+ receptors for the study of HIV-1 replication

Several studies have recently shown the use of recombinant rabies virus as potential vector-viral vaccine for HIV-1. The sequence homology between gp 120 and rabies virus glycoprotein has been reported. The McCoy cell line has therefore been used to show CD4+ or CD4+ like receptors. Samples of HIV-1 were isolated, when plasma of HIV-1 positive patients was inoculated in the McCoy cell line. The virus infection was then studied during successive virus passages. The proteins released in the extra cellular medium were checked for protein activity, by exposure to SDS Electrophoresis and blotting to nitro-cellulose filter, then reacting with sera of HIV positive and negative patients. Successive passages were performed, and showed viral replication, membrane permeabilization, the syncytium formation, and the cellular lysis (cytopathic effect). Flow cytometry analysis shows clear evidence that CD4+ receptors are present in this cell line, which enhances the likelihood of easy isolation and replication of HIV. The results observed allow the use of this cell line as a possible model for isolating HIV, as well as for carrying out studies of the dynamics of viral infection in several situations, including exposure to drugs in pharmacological studies, and possibly studies and analyses of the immune response in vaccine therapies

Frequency of serum anti-cysticercus antibodies in the population of a rural Brazilian community (Cassia dos Coqueiros, SP) determined by ELISA and immunoblotting using Taenia crassiceps antigens

Considering the impact of cysticercosis on public health, especially the neurologic form of the disease, neurocysticercosis (NC), we studied the frequency of positivity of anti-Taenia solium cysticercus antibodies in serum samples from 1,863 inhabitants of Cássia dos Coqueiros, SP, a municipal district located 80 km from Ribeiräo Preto, an area considered endemic for cysticercosis. The 1,863 samples were tested by enzyme linked immunosorbent assay (ELISA) using an antigenic extract from Taenia crassiceps vesicular fluid (Tcra). The reactive and inconclusive ELISA samples were tested by immunoblotting. Of the 459 samples submitted to immunoblotting, 40 were strongly immunoreactive to the immunodominant 18 and 14 kD peptides. Considering the use of immunoblotting as confirmatory due to its high specificity, the anti-cysticercus serum prevalence in this population was 2.1 percent

Production of monoclonal antibodies anti-Taenia crassiceps cysticerci with cross-reactivity with Taenia solium antigens

We describe the production of the potential monoclonal antibodies (MoAbs) using BALB/c mice immunized with vesicular fluid (VF)-Tcra (T. crassiceps) antigen. Immune sera presented anti-VF-Tcra (<20kD) IgG and IgM antibodies with cross-reactivity with T. solium (Tso) antigen (8-12, 14, and 18 kD). After cell fusion, we selected 33 anti-Tcra and anti-Tso reactive IgM-clones and 53 anti-Tcra specific IgG-clones, 5 of them also recognizing Tso antigens. Two clones identified the 8-14 and 18kD peptides of VF-Tcra.

A comparative study on IgG-ELISA, IgM-IFT and Kato-Katz methods for epidemiological purposes in a low endemic area for schistosomiasis

The high sensitivity and the possibility of automation of the enzyme-linked-immunosorbent-assay (ELISA) has indicated this technique as one of the most useful serological test for epidemiological studies. In the present study, an ELISA for detection of IgG antibodies against adult worm antigens (IgG-ELISA) was investigated for epidemiological purposes, in a rural area of the municipality of Itariri (Säo Paulo, Brazil). Blood on filter paper (1,180 samples) from about 650 school children were submitted to ELISA and the data compared to the results of the parasitogical method of Kato-Katz and also to the IgM-IFT (immunofluorescence test for IgM antibodies to gut associated antigens). The prevalence rates respectively of 8.5 per cent, 43.0 per cent and 56.2 per cent by the Kato-Katz, IgG-ELISA, and IgM-IFT methods suggest the poor sensitivity of the parasitological method for detection of Schistosoma mansoni eggs in individuals with low worm burden, situation commonly observed in low endemic areas. These results can partially explain the poor degree of agreement between the IgG-ELISA and the Kato-Katz, as suggested by the Kappa index of 0.170. Otherwise, the Kappa index of 0.675 showed substantial agreement between the two serological tests. Some discrepancy of results between the two serological techniques must be better investigated.

Teste de hemaglutinação para o diagnóstico da neurocisticercose humana: desenvolvimento de reagente estável utilizando antígeno heterólogo / Hemmagglutination test for the diagnosis of human neurocysticercosis: development of a stable reagent using heterologous antigens

Foi padronizado o teste de hemaglutinação (HA) empregando hemácias de ganso formolizadas, taninizadas e sensibilizadas com extrato antigênico de líquido vesicular de C. longicollis (HA-CI) e extrato salino total de C. cellulosae (HA-Cc). Foram ensaiados 61LCR de dois grupos: 41 de pacientes com neurocisticercose e 20 de grupo controle, respectivamente, reativos e não-reativos no teste ELISA empregando C. cellulosae. Nos LCR do grupo controle não foi observada reatividade e 34 (82,9%) e 35 (85,4%) LCR de doentes foram reativos, respectivamente, nos testes HA-CI e HA-Cc. O estudo da estabilidade dos reagentes pronto para uso mostrou vantagens para o armazenamento a 4°C, em glicerol a 50%, por até 6 meses. Os resultados obtidos indicam que o reagente utilizando Cysticercus longicollis e estabilizado com glicerol pode ser empregado como alternativa no diagnóstico imunológico da neutocisticercose

Hemagglutination test for the diagnosis of human neurocysticercosis: development of a stable reagent using homologous and heterologous antigens

Foi padronizado o teste de hemaglutinacao (HA) utilizando as hemacias formolizadas e taninizadas de ganso sensibilizadas com extrato salino total de C. cellulosae (HA-Cc) e liquido vesicular de Cysticercus longicollis (HA-Cl). Foram ensaiadas 61 amostras de liquido cefalorraquiano (LCR), 41 de pacientes com neurocisticercose e 20 de um grupo de controle, respectivamente, regentes e nao-regentes no teste ELISA utilizando antigenos de C. cellulosae...

Frequência de anticorpos anti-Cysticercus cellulosae em população de risco para cisticercose e em segmento de população considerado supostamente normal, em regiões do Estado de São Paulo, Brasil / Frequency of anti-cysticercus cellulosae antibodies in high risk population for cysticercosis and in a segment considered as normal population, in areas of Säo Paulo state, Brazil

Examinaram-se, através de reação de fixação de complemento, os soros de 234 pacientes internados em hospital psiquiátrico localizado no município de Presidente Prudente, considerados de risco para infecção cisticercótica, além de 454 soros de gestantes procedentes da Região Administrativa de Santos e 397 soros de indivíduos considerados supostamente normais, procedentes da Região Administrativa de Presidente Prudente. O antígeno utilizado na reação de fixação de complemento foi obtido através de extração metílica, à temperatura ambiente, dos císticercos tratados com acetona. Consideraram-se positivas as reações em que ocorreu fixação de complemento a partir da diluição 1 :2. Dos 1.085 soros testados, 27 apresentaram atividade anticomplementar e 17 (1,6%) mostraram-se reagentes. Todavia, quando se consideraram, separadamente, os grupos procedentes de Santos, Presidente Prudente e os doentes mentais, percebe-se diferença significativa nos resultados: assim, os índices de freqüência foram, respectivamente, 0,88% e 1,00% para os indivíduos procedentes de Santos e Presidente Prudente e considerados supostamente normais e 3,8% para os doentes mentais. Os resultados indicam que não é desprezível a ocorrência de anticorpos anti-Cysticercus cellulosae em nosso meio, especialmente entre pacientes de hospitais psiquiátricos (AU).