Extract of Ascaris suum induces TGF-ß and early production of IgG1 in experimental autoimmune hepatitis / Extrato de Ascaris suum induz TGF-ß e produção precoce de IgG1 na hepatite autoimune experimental

Abstract In experimental autoimmune hepatitis (EAH) of Th1 profile, an extract of adult Ascaris suum worms (ASC) was previously found to deviate the immune response to a Th2/IL-10 pattern. Here, the effects of treatment with ASC on production of TGF-β and the anti-Ascaris isotypes IgG1 and IgG2a in EAH were evaluated. EAH was induced in BALB/c mice, intravenously with concanavalin A. Two hours later, these animals received ASC (EAH+ASC group) or PBS vehicle (EAH group). IgG1 and IgG2a were evaluated 8 h, 24 h and 7 d after induction. TGF-β was measured in a splenocyte culture at this last time. The isotype levels in the EAH group were low throughout the kinetics. In the EAH+ASC group, there was significant production of IgG1 at 24 h and 7 d, but of IgG2a only at 7 d. There was statistically greater production of TGF-β in the EAH+ASC group. The higher levels of IgG1 and TGF-β in this group suggest that an additional Th1 response control route exists in EAH, which needs to be investigated.

Resumo Na hepatite autoimune experimental (HAE) de perfil Th1, o extrato de vermes adultos Ascaris suum (ASC) desviou a resposta imune para um padrão Th2/IL-10. Neste trabalho, foram avaliados os efeitos do tratamento com ASC na produção TGF-β e dos isótipos de IgG1 e IgG2a anti-Ascaris na HAE. Esta foi induzida em camundongos BALB/c intravenosamente com Concanavalina A. Após duas horas, os animais receberam ASC (grupo HAE+ASC) ou veículo PBS (grupo HAE). IgG1 e IgG2a foram avaliados em 8 horas, 24 horas e 7 dias após indução. TGF-β foi mensurado em cultura de esplenócitos nesse último tempo. Os níveis dos isótipos no grupo HAE foram baixos durante toda a cinética. No grupo HAE+ASC, houve produção significativa de IgG1 em 24 horas e 7 dias, mas somente em 7 dias para IgG2a. A produção de TGF-β foi estatisticamente maior no grupo HAE+ASC. Níveis mais altos de IgG1 e TGF-β nesse grupo sugerem uma via adicional de controle da resposta Th1 na HAE que precisa ser investigada.

Seropositivity for ascariosis and toxocariosis nd cytokine expression among the indigenous people in the Venezuelan delta region / Seropositividad para ascariosis y toxocariosis y expresión de citocinas entre la población indígena de la región del delta Venezolano

The present study aimed at measuring seropositivities for infection by Ascaris suum and Toxocara canis using the excretory/secretory (E/S) antigens from Ascaris suum (AES) and Toxocara canis (TES) within an indigenous population. In addition, quantification of cytokine expressions in peripheral blood cells was determined. A total of 50 Warao indigenous were included; of which 43 were adults and seven children. In adults, 44.1% were seropositive for both parasites; whereas children had only seropositivity to one or the other helminth. For ascariosis, the percentage of AES seropositivity in adults and children was high; 23.3% and 57.1%, respectively. While that for toxocariosis, the percentage of TES seropositivity in adults and children was low; 9.3% and 14.3%, respectively. The percentage of seronegativity was comparable for AES and TES antigens in adults (27.9%) and children (28.6%). When positive sera were analyzed by Western blotting technique using AES antigens; three bands of 97.2, 193.6 and 200.2 kDas were mostly recognized. When the TES antigens were used, nine major bands were mostly identified; 47.4, 52.2, 84.9, 98.2, 119.1, 131.3, 175.6, 184.4 and 193.6 kDas. Stool examinations showed that Blastocystis hominis, Hymenolepis nana and Entamoeba coli were the most commonly observed intestinal parasites. Quantification of cytokines IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 and IL-4 expressions showed that there was only a significant increased expression of IL-4 in indigenous with TES seropositivity (p < 0.002). Ascaris and Toxocara seropositivity was prevalent among Warao indigenous.

El objetivo del presente estudio fue determinar la seropositividad de infección por Ascaris suum y Toxocara canis, utilizando antígenos de excreción/secreción (E/S) de Ascaris suum (AES) y Toxocara canis (TES) en una población indígena. Adicionalmente, se cuantificó la expresión de citocinas a partir de células de sangre periférica. Un total de 50 indígenas Warao se incluyeron en el estudio; 43 fueron adultos y 7 niños. Entre los adultos, 44,1% fueron seropositivos para ambos parásitos; mientras que los niños sólo mostraron seropositividad a uno u otro de los helmintos. Para ascariosis, el porcentaje de seropositividad para los antígenos AES fue alto tanto en adultos como en niños; 23,3% y 57,1%, respectivamente. Para toxocariosis, el porcentaje de seropositividad para los antígenos TES fue bajo en adultos así como en niños; 9,3% y 14,3%, respectivamente. El porcentaje de seronegatividad fue similar tanto para los antígenos AES como para TES en adultos (27,9%) y niños (28,6%). Cuando la seropositividad fue analizada a través de la técnica de Western blotting utilizando los antígenos AES; 3 bandas de 97,2, 193,6 y 200,2 kDas fueron principalmente reconocidas. Para los antígenos TES, 9 bandas fueron mayormente identificadas; 47,4, 52,2, 84,9, 98,2, 119,1, 131,3, 175,6, 184,4 y 193,6 kDas. Los análisis coproparasitológicos mostraron que los parásitos Blastocystis hominis, Hymenolepis nana y Entamoeba coli fueron los parásitos intestinales más comúnmente observados. La cuantificación de la expresión de las citocinas IFN-γ, IL-2, IL-6, TGF-β, TNF-α, IL-10 e IL-4 mostró que hubo un significante incremento de la expresión de IL-4 entre los indígenas con seropositividad para los antígenos TES (p < 0.002). La seropositividad para Ascaris y Toxocara fue prevalente entre los indígenas Warao.

Boosting the suppressive effects of Ascaris suum components in IFN-γ-deficient mice / Potencialização do efeito supressor de componentes do Ascaris suum em camundongos deficientes em IFN-γ

High molecular weight components from Ascaris suum extract suppress ovalbumin-specific immunity in mice. In IFN-γ-deficient mice, ovalbumin-specific delayed-type hypersensitivity reactions are more strongly downregulated by these suppressive components. Here, the cellularity of the delayed-type hypersensitivity reaction in IFN-γ-deficient mice and the increased downregulation induced by Ascaris suum components were analyzed. IL-12p40-dependent neutrophilic influx was predominant. Suboptimal doses of the suppressive fraction from this nematode completely inhibited the hypersensitivity reaction, thus indicating intensification of the immunosuppression under conditions of intense recruitment of IFN-γ-independent neutrophils.

Componentes de alto peso molecular do extrato de Ascaris suum suprimem a imunidade específica à ovalbumina em camundongos. Em camundongos geneticamente deficientes de IFN-γ a reação de hipersensibilidade tardia específica para ovalbumina foi mais fortemente prejudicada por estes componentes supressivos. Aqui, a celularidade da reação de hipersensibilidade tardia em camundongos deficientes de IFN-γ e o incremento na supressão induzida por componentes do Ascaris suum foram analisados. Influxo neutrofílico, dependente de IL-12p40, foi predominante. Dose sub-ótima da fração supressiva do nematódeo inibiu completamente a reação de hipersensibilidade, indicando uma intensificação da imunossupressão em condições de recrutamento intenso de neutrófilos independente de IFN-γ.

Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component

Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 10(9) M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography

Effect of the injection of an extract of Ascaris suum on macrophage activation during the early phase of Mycobacterium bovis BCG infection in C57Bl/6 mice

Injection of an Ascaris suum extract (Asc) affects both the humoral and cellular immune responses to unrelated antigens when it is co-administered with these antigens. In the present study we evaluated the effect of Asc on macrophage activation in the early phase of Mycobacterium bovis BCG (Pasteur strain TMCC 1173) infection in C57Bl/6 mice. C57Bl/6 mice were injected intraperitoneally (ip) with 0.1 mg BCG (BCG group) or BCG plus 1 mg Asc (BCG + Asc group). The peritoneal exudates were obtained at 2, 7 and 14 days after infection. The numbers of IFN-g-secreting cells were assessed by the ELISPOT assay. Nitric oxide (NO) production was measured by the Griess method and by the evaluation of NADPH diaphorase activity in the peritoneal exudates. The administration of Asc extract increased NADPH diaphorase activity (2 days: control = 0, BCG = 7 per cent, BCG + Asc = 13 per cent, and Asc = 4 per cent; 7 days: control = 4, BCG = 13 per cent, BCG + Asc = 21 per cent, and Asc = 4.5 per cent) and TNF-a levels (mean + or - SD; 2 days: control = 0, BCG = 169 + or - 13, BCG + Asc = 202 + or - 37, and Asc = 0; 7 days: control = 0, BCG = 545 + or - 15.5, BCG + Asc = 2206 + or - 160.6, and Asc = 126 + or - 26; 14 days: control = 10 + or - 1.45, BCG = 9 + or - 1.15, BCG + Asc = 126 + or - 18, and Asc = 880 + or - 47.67 pg/ml) in the early phase of BCG infection. Low levels of NO production were detected at 2 and 7 days after BCG infection, increasing at 14 days (mean + or - SD; 2 days: control = 0, BCG = 3.7 + or - 1.59, BCG + Asc = 0.82 + or - 0.005, Asc = 0.48 + or - 0.33; 7 days: control = 0, BCG = 2.78 + or - 1.54, BCG + Asc = 3.07 + or - 1.05, Asc = 0; 14 days: control = 0, BCG = 9.05 + or - 0.53, BCG + Asc = 9.61 + or - 0.81, Asc = 10.5 + or - 0.2 (2 x 106) cells/ml). Furthermore, we also observed that Asc co-injection induced a decrease of BCG-colony-forming units (CFU) in the spleens of BCG-infected mice during the first week of infection (mean + or - SD; 2 days: BCG = 1.13 + or - 0.07 and BCG + Asc = 0.798 + or - 0.305; 7 days: BCG = 1.375 + or - 0.194 and BCG + Asc = 0.548 + or - 0.0226; 14 days: BCG = 0.473 + or - 0.184 and BCG + Asc = 0.675 + or - 0.065 (x 102) CFU). The present data suggest that Asc induces the enhancement of the immune response in the early phase of BCG infection.

Histological changes in hamster liver by ES antigen of Ascaris suum.

Polyacrylamide gel electrophoresis with SDS (PAGE-SDS) of the ES antigens of A. suum revealed several protein molecules which differed from those obtained in ES antigens of A. lumbricoides. Nature of liver damage caused by ES antigens of A. suum was studied in hamsters to find out the nature of damage and to compare with those caused by ES antigens of A. lumbricoides. Feeding of ES antigens of A. suum was carried out in 7 hamsters for 75 days. After such feeding gross hepatic damage was noticed. This was characterized by pericentrivenular degeneration and necrosis of liver parenchyma, the lesions being different and much more severe than those observed in hamster challenged by ES products of A. lumbricoides. The lesions appear to be immune mediated.

Effect of purified components of Ascaris suum extracts on IgE antibody response in guinea pigs

The effect of components P530 and P29, isolated from Ascaris suum adult worm extract (ASC), on the heterologous IgE antibody response was studied in guinea pigs. Groups of 7 guinea pigs were immunized ip with 50 micrograms of ovalbumin (OA) alone or mixed with 200 micrograms of each component precipitated in an alum gel. The primary and secondary IgE antibody response was evaluated by passive cutaneous anaphylaxis reaction (PCA). Immunization of guinea pigs with P29 plus ovalbumin (OA) resulted in a significant increase in the level of serum IgE anti-OA antibodies, especially in the secondary response (almost 8-fold higher when compared with control group). This potentiated response was not observed when the animals received OA plus P530 or the crude extract. Indeed, the P530 component, as well as the crude extract, induced a depression of the anti-OA IgE antibody response (2-3 fold decrease when compared with OA-immunized animals). It was also shown that P29, but not P530 or ASC, was capable of eliciting a strong anti-ASC IgE antibody response. These results demonstrate that in guinea pigs these two Ascaris suum components have antagonistic biological effects, one inducing potentiation and the other suppression of the heterologous IgE antibody response