A new procedure has been developed to concentrate the phosphofructokinase from muscle of Ascaris suum with minimum loss of activity. By utilizing this method, 50 ml fraction was concentrated to a final volume of 3 ml in about 1.5 h without loss in enzyme activity. The concentrated enzyme had a specific activity of 64 units per mg. Ascaris muscle-cuticle was incubated in 50 1M solutions of either acetylcholine, serotonin, y-aminobutyric acid, levamisole, or saline alone. Phosphate analysis of the isolated phosphofructokinase from each incubation revealed that the enzyme contained the following moles of phosphate per subunit: 2.9 (acetylcholine), 2.2 (serotonin), 2.0 (y-aminobutyric acid), 1.5 (levamisole), and 3.4 (salne alone). The present study did not establish a direct correlation between degree of phosphorylation and phosphofructokinase activity. Phosphofructokinase from muscle of Ascaris suum appears to contain several phosphorylation sites, and one of these sites is required to be phosphorylated in order for the enzyme to exhibit maximum activity under physiological conditions.

O extrato de Ascaris suum (Asc total), preparado a partir de uma mistura de vermes machos e fêmeas (albergando ovos), suprime a resposta imune específica a ovalbumina (OA). A partir do fracionamento deste extrato por gel filtração demonstrou-se que componentes protéicos de alto peso molecular (PM), eluídos no primeiro pico (PI), eram supressores da resposta a OA e os eluídos no terceiro pico (PIII) estimularam uma resposta maior de anticorpos IgE anti-Asc. Uma resposta do tipo Th2 foi preferencialmente estimulada por este extrato, sendo as citocinas IL-4 e IL-10 atuantes na supressão dos parâmetros da resposta anti-OA mediados por células Th1. Em algumas espécies de helmintos a resposta Th2 é estimulada de forma estágio-específica. Assim, analisamos neste trabalho quais dos componentes do Asc total seriam responsáveis pelo efeito supressor. Verificamos que os extratos dos vermes adultos apresentaram perfis cromatográficos semelhantes ao do extrato total. O perfil cromatográfico do Asc O foi distinto, com um segundo pico (PII) mais evidente e apresentou um quarto pico de menor PM. O fracionamento eletroforético confirmou uma concentração de proteínas com PM entre 107 e 52 kDa e a presença de proteínas adicionais entre 27,2 e 19 kDa neste extrato. Os extratos de vermes e dos ovos suprimiram a resposta imune celular específica a OA, avaliada por reações de hipersensibilidade tardia, resposta proliferativa de células de linfonodos drenantes e secreção de citocinas por estas células. A produção de anticorpos IgG1 e IgG2a anti-OA foi também diminuída acentuadamente pelos mesmos extratos. Observamos, ainda, que as citocinas predominantes na resposta aos extratos dos vermes e dos ovos foram diferentes, com maior secreção de IL-4 e IL-10 nos primeiros e IFN-g no segundo. Com relação às diferentes frações do Asc O, a produção de anticorpos IgE anti-OA foi suprimida por componentes de PI e PIII, enquanto que os de PII estimularam a síntese maior de IgE anti-Asc O. Os componentes de PIV não tiveram nenhum efeito. Nossos resultados indicam que o efeito supressivo do Asc total é uma propriedade que pode ser atribuída aos vermes machos e fêmeas. No entanto, o extrato preparado a partir dos ovos também apresenta este efeito, o qual parece ser mediado por mecanismos diferentes. / The extract of Ascaris suum (whole Asc), prepared from male and female worms (with stored eggs) suppress the specific immune response to ovalbumin (OA). This extract was fractionated by gel filtration and high and low molecular weight (MW) proteins were obtained in the first (PI) and third peak (PIII), respectively. PI suppressed the OA-specific cellular and humoral responses and PIII stimulated more anti-Asc IgE antibodies. The whole Asc stimulated a Th2 response predominantly, with high levels of IL-4 and IL-10. These cytokines had an important role in the down-regulation of the Th1 response to OA. In some helminthic infections the Th2 response is stimulated by specific stages of the life cycle. Thus, in this work we investigated which components of whole Asc were responsible for immunosuppression. The worm extracts presented similar chromatographic profiles. The profile of Asc O was distinct, with the second peak being more evident and showing a fourth peak with much lower MW components. By polyacrylamide gel electrophoresis we observed a high concentration of proteins between 107 and 52 kDa. In addition, some bands between 27,2 and 19 kDa were only present in this extract. The extracts from worms and eggs suppressed the cell-mediated immune response to OA, measured by delayed-type hypersensitivity, proliferative response of draining lymph node cells and cytokine secretion. The anti-OA IgG1 and IgG2a antibody production were as drastically reduced by these extracts. The cytokines IL-4 and IL-10 were mainly secreted in response to worm extracts, whereas the egg extract stimulated more IFN-g. Regarding the different fraction of Asc O, PI and PIII components diminished the anti-OA IgE antibody production, whereas PII components stimulated higher anti-Asc O IgE synthesis. PIV components did not display any effect. Our results indicate that the immunosuppressive effect of whole Asc is due to male and female body components. However, extracts prepared from eggs do also display this effect, that seems to be mediated by different mechanisms.

LEITE, Ana Karine Rocha de Melo. Efeito protetor dos extratos de Ascaris suum e Coccidioides posadasii e da lectina da semente de Dioclea violacea na artrite por zymosan em ratos e camundongos. 2009. 70 f. Tese (Doutorado) - Universidade Federal do Ceará. Faculdade de Medicina. Programa de Pós-Graduação em Ciências Médicas, Fortaleza, 2009. / Submitted by denise santos (denise.santos@ufc.br) on 2011-10-05T13:37:05Z No. of bitstreams: 1 2009_tese_akrmleite.pdf: 1567980 bytes, checksum: 9f13d2575ffdfa87c6cfdfc7a9ccfc75 (MD5) / Approved for entry into archive by Eliene Nascimento(elienegvn@hotmail.com) on 2011-10-07T12:10:24Z (GMT) No. of bitstreams: 1 2009_tese_akrmleite.pdf: 1567980 bytes, checksum: 9f13d2575ffdfa87c6cfdfc7a9ccfc75 (MD5) / Made available in DSpace on 2011-10-07T12:10:24Z (GMT). No. of bitstreams: 1 2009_tese_akrmleite.pdf: 1567980 bytes, checksum: 9f13d2575ffdfa87c6cfdfc7a9ccfc75 (MD5) Previous issue date: 2009 / The interactions between innate and acquired immune responses participate in the pathophysiology of the autoimmune diseases. Though infections are associate with the development of the chronic arthritis it is possible that exposure to some germs as helminthes and fungi influences potentially the prevalence and/or gravity of the immune diseases. Lectins derivate of the plants can modulate the inflammation by action in receptors of the innate response. We investigated the effect of extracts from Ascaris suum (AS), Coccidioides posadasii (CS) and a lectin isolated from Dioclea violacea (Dviol) in zymosan-induced arthritis (ZyA). Wistar rats and Swiss mice received 1 mg or 0.1 mg zymosan intra-articular (i.art.), respectively. Groups were pretreated (30 min) with AS (0.25 - 2.5 mg/animal; i.p. or p.o.) CP (1 - 100 µg/animal; i.art. i.p. or p.o) or Dviol (0.3 - 30 µg; i.art. or 1 - 6 mg/kg; i.v.). Non-treated group (NT) received Zy (i.art.) and the vehicle. Naive animals received just saline (i.art.) and the vehicle. The hypernociception was evaluated through articular incapacitation test in s/1min. The joint exudate was used for evaluation of cell influx (CI), nitrite and cytokine levels. The synovium was used for histopatology. The glycosaminoglycan (GAG) content of the cartilage was quantificated for the measured of the structural damage. The AS extract both i.p. and p.o. significantly and dose-dependently inhibited CI and hypernociception in ZyA as compared to NT (P<0.01) as well as reverted articular damage assessed by quantification of the GAG and by synovitis observed in the histology. The administration of the AS extract reduced significantly levels of nitrite, interleukin-1β (IL-1β) and IL-10, but not tumor necrosis factor alpha (TNF-α) as compared to NT. In mice, it reduced IL-10 but not IL-1β and TNF- α. The treatment with CP extract both i.p. and p.o. inhibited hypernociception and CI in ZyA as compared to NT, but not reverted articular injury measured by GAG and histology. The administration of the Dviol in naïve animals promoted CI significant, though just the highest dose (30 g) promoted hypernociception. In ZyA, Dviol (i.art.) reduced the CI and hypernociception dose-dependently (P<0.01). The administration of Dviol (i.v.) significantly reduced both the hyperalgesia and CI in ZyA as compared to NT (P<0.01). The effect of the Dviol was reverted when it was pre-incubated with mannose (1M). The date show that AS extract promote functional improve and protect of the articular damage in ZyA that are associate with reduction of the NO and cytokine (i.art.) liberation. This effect is species independent and functions orally. An extract of the fungi CP has anti-inflammatory activity in ZyA. A lectin isolated of the Dviol reduces CI and hypernociception in ZyA probably by coupling the mannose receptor. Together the results show that substances that act in receptors of the innate response modulate the immunomediate articular inflammation. / Interações entre a resposta imune inata e adquirida participam na fisiopatologia de doenças auto-imunes. Embora infecções estejam associadas ao desenvolvimento de artrites crônicas, é possível que exposição a alguns germes, como helmintos e fungos, potencialmente influencie a prevalência e/ou gravidade de doenças imunomediadas. Lectinas derivadas de plantas, por ação em receptores de resposta inata, podem modular inflamação. Nós investigamos o efeito dos extratos de Ascaris suum (AS) e de Coccidioides posadasii (CP) e de uma lectina isolada da Dioclea violacea (Dviol) na artrite induzida por zymosan (AZy). Ratos Wistar e camundongos Swiss receberam 1 mg ou 0,1 mg de zymosan intra-articular (i.art.), respectivamente. Grupos foram pré-tratados (30 min) com os extratos de AS (0,25 - 2,5 mg/animal; i.p ou p.o.), CP (1 - 100 µg/animal; i.art., i.p. ou p.o.) ou Dviol (0,3 - 30 µg i.art. ou 1 - 6 mg/Kg e.v.). Grupo não-tratado (NT) recebeu Zy (i.art.) e veículo. Animais naive receberam apenas salina (i.art.) e veículo. A hipernocicepção foi avaliada através do teste de incapacitação articular em s / 1min. O lavado articular foi usado para análise do influxo celular (IC), níveis de nitrito e citocinas. A sinóvia foi utilizada para histopatologia. O conteúdo de glicosaminoglicanos (GAG) da cartilagem foi quantificado para medir dano estrutural. O extrato de AS, seja i.p. ou p.o., inibiu de forma dose-dependente a hipernocicepção e o IC na AZy em relação ao grupo NT (P<0,01), bem como reverteu o dano articular avaliado pela quantificação de GAG e a sinovite vista à histologia. A administração do extrato de AS, reduziu significantemente os níveis de nitrito, inteleucina-1β (IL-1β) e IL-10, mas não de fator de necrose tumoral alfa (TNF-α), em relação ao NT. Em camundongos, o extrato de AS reduziu os níveis de IL-10, mas não de IL-1β ou TNF-α. O tratamento com o extrato de CP, seja i.p. ou p.o., inibiu significantemente a hipernocicepção e o IC na AZy, em relação ao NT, no entanto, não reverteu a lesão articular medida pela quantidade de GAG e histologia. A administração da Dviol, em animais naive promoveu IC significante, embora apenas a maior dose (30µg) promoveu hipernocicepção. Na AZy, a injeção i.art. da Dviol reduziu o IC e hipernocicepção de forma dose-dependente, em relação ao NT (P<0,01). A administração da Dviol (i.v.) reduziu ambos hipernocicepção e IC na AZy, em relação ao NT (P<0,01). O efeito da Dviol foi revertido quando essa lectina foi pré-incubada com manose 1 M. Os dados mostram que um extrato de AS promove melhora funcional e protege do dano estrutural na AZy, que são associados com redução na liberação de NO e citocinas i.art. Esse efeito independe da espécie e ocorre por via oral. Um extrato do fungo CP tem ação anti-inflamatória na AZy. Uma lectina isolada da Dviol reduz IC e hipernocicepção na AZy, provavelmente por acoplamento a um receptor de manose. Em conjunto, os resultados mostram que substâncias que agem em receptores de resposta inata modulam a inflamação articular imunomediada.

O extrato de Ascaris suum (Asc total), preparado a partir de uma mistura de vermes machos e fêmeas (albergando ovos), suprime a resposta imune específica a ovalbumina (OA). A partir do fracionamento deste extrato por gel filtração demonstrou-se que componentes protéicos de alto peso molecular (PM), eluídos no primeiro pico (PI), eram supressores da resposta a OA e os eluídos no terceiro pico (PIII) estimularam uma resposta maior de anticorpos IgE anti-Asc. Uma resposta do tipo Th2 foi preferencialmente estimulada por este extrato, sendo as citocinas IL-4 e IL-10 atuantes na supressão dos parâmetros da resposta anti-OA mediados por células Th1. Em algumas espécies de helmintos a resposta Th2 é estimulada de forma estágio-específica. Assim, analisamos neste trabalho quais dos componentes do Asc total seriam responsáveis pelo efeito supressor. Verificamos que os extratos dos vermes adultos apresentaram perfis cromatográficos semelhantes ao do extrato total. O perfil cromatográfico do Asc O foi distinto, com um segundo pico (PII) mais evidente e apresentou um quarto pico de menor PM. O fracionamento eletroforético confirmou uma concentração de proteínas com PM entre 107 e 52 kDa e a presença de proteínas adicionais entre 27,2 e 19 kDa neste extrato. Os extratos de vermes e dos ovos suprimiram a resposta imune celular específica a OA, avaliada por reações de hipersensibilidade tardia, resposta proliferativa de células de linfonodos drenantes e secreção de citocinas por estas células. A produção de anticorpos IgG1 e IgG2a anti-OA foi também diminuída acentuadamente pelos mesmos extratos. Observamos, ainda, que as citocinas predominantes na resposta aos extratos dos vermes e dos ovos foram diferentes, com maior secreção de IL-4 e IL-10 nos primeiros e IFN-g no segundo. Com relação às diferentes frações do Asc O, a produção de anticorpos IgE anti-OA foi suprimida por componentes de PI e PIII, enquanto que os de PII estimularam a síntese maior de IgE anti-Asc O. Os componentes de PIV não tiveram nenhum efeito. Nossos resultados indicam que o efeito supressivo do Asc total é uma propriedade que pode ser atribuída aos vermes machos e fêmeas. No entanto, o extrato preparado a partir dos ovos também apresenta este efeito, o qual parece ser mediado por mecanismos diferentes. / The extract of Ascaris suum (whole Asc), prepared from male and female worms (with stored eggs) suppress the specific immune response to ovalbumin (OA). This extract was fractionated by gel filtration and high and low molecular weight (MW) proteins were obtained in the first (PI) and third peak (PIII), respectively. PI suppressed the OA-specific cellular and humoral responses and PIII stimulated more anti-Asc IgE antibodies. The whole Asc stimulated a Th2 response predominantly, with high levels of IL-4 and IL-10. These cytokines had an important role in the down-regulation of the Th1 response to OA. In some helminthic infections the Th2 response is stimulated by specific stages of the life cycle. Thus, in this work we investigated which components of whole Asc were responsible for immunosuppression. The worm extracts presented similar chromatographic profiles. The profile of Asc O was distinct, with the second peak being more evident and showing a fourth peak with much lower MW components. By polyacrylamide gel electrophoresis we observed a high concentration of proteins between 107 and 52 kDa. In addition, some bands between 27,2 and 19 kDa were only present in this extract. The extracts from worms and eggs suppressed the cell-mediated immune response to OA, measured by delayed-type hypersensitivity, proliferative response of draining lymph node cells and cytokine secretion. The anti-OA IgG1 and IgG2a antibody production were as drastically reduced by these extracts. The cytokines IL-4 and IL-10 were mainly secreted in response to worm extracts, whereas the egg extract stimulated more IFN-g. Regarding the different fraction of Asc O, PI and PIII components diminished the anti-OA IgE antibody production, whereas PII components stimulated higher anti-Asc O IgE synthesis. PIV components did not display any effect. Our results indicate that the immunosuppressive effect of whole Asc is due to male and female body components. However, extracts prepared from eggs do also display this effect, that seems to be mediated by different mechanisms.

The NAD+-linked malic enzyme from Ascaris suum has been studied with regard to its kinetic and catalytic properties. Possible relationships between these properties and the physiological functioning of the malic enzyme were examined.

Glycogen synthase, the enzyme that catalyzes the rate-limiting reaction of glycogen syntheses has been purified and characterized from Ascaris suum muscle. Glycogen in the crude extract was digested to release the enzyme, eluted from a DE52 cellulose column and then applied to a Sepharose affinity column. The purified Ascaris enzyme was found to be homologous to the mammalian enzyme with regard to subunit and holoenzyme Mr^3 allosteric activation, substrate affinity and covalent modification. However, the association between Ascaris glycogen synthase and endogenous glycogen differed from that in mammalian systems.

One molecular form of fumarase from Ascaris suum was demonstrated by cellulose acetate electroporesis and isoelectric focusing. The enzyme was partially purified by ammonium sulfate fractionation and ion-exchange chromatography to a specific activity of 49 units per mg protein. Enzymatic assay of the partially purified by ammonium sulfate fractionation amd ion-exchange chromatography to a specific activity of 49 units per mg protein. Enzymatic assay of the partially purified preparation showed glyceraldehyde-3-phosphate dehydrongenase to be the major preparative contaminant.

A complex which synthesized glycogen de novo has been purifiedfrom Ascaris suum. This complex (GS-2) consists of a 66 KDa protein, a 140 KDa protein, and a>330 KDa glycoprotein.

Neste trabalho, investigamos os mecanismos da resposta imune estimulados por PAS-1 para o desencadeamento do seu efeito modulatório na inflamação alérgica pulmonar. Para tanto, camundongos C57BL/6 selvagens, IL-12-/-, IFN-<font face=\"Symbol\">g-/- e IL-10-/- foram imunizados e desafiados com OVA ou PAS-1 ou OVA + PAS-1. Ainda, camundongos C57BL/6 imunizados e desafiados com OVA receberam transferência adotiva de células CD19+, B220+, CD3+, CD4+, CD8+, CD4+CD25-, CD4+CD25+ PAS-1-primadas. Nossos resultados demonstraram que o efeito imunomodulatório de PAS-1 é mediado por IFN-<font face=\"Symbol\">g e IL-10, mas não por IL-12, e que somente camundongos que receberam células CD8+ ou CD4+CD25+ não desenvolveram inflamação pulmonar e produziram, respectivamente, IFN-<font face=\"Symbol\">g e IL-10/TGF-ß. Em conjunto, estes resultados demonstraram que o efeito imunomodulatório de PAS-1 é devido à estimulação de células T CD8+ produtoras de IFN-<font face=\"Symbol\">g e por CD4+CD25+ secretoras de IL-10 e TGF-ß. / In this work, we investigate the immune response mechanisms triggered by PAS-1 to promote its immunomodulatory effect in the lung allergic inflammation. For that, wild type, IL-12-/-, IFN-<font face=\"Symbol\">g-/- and IL-10-/- C57BL/6 mice were immunized and challenged with OVA or PAS-1 or OVA + PAS-1. Moreover, wild type C57BL/6 mice were adoptively transferred with PAS-1-primed CD19+, B220+, CD3+, CD4+, CD8+, CD4+CD25-, and CD4+CD25+ lymphocytes. Our results demonstrated that the immunomodulatory effect induced by PAS-1 is mediated by IFN-<font face=\"Symbol\">g and IL-10, but not by IL-12, and only mice which received CD8+ or CD4+CD25+ T cells did not present lung allergic inflammation and produced IFN-<font face=\"Symbol\">g and IL-10/TGF-ß, respectively. Taken together, these results demonstrated that the imunomodulatory effect induced by PAS-1 is due to stimulating CD8+ T cells, which secrete IFN-<font face=\"Symbol\">g, and CD4+CD25+ T cells, which secrete IL-10 and TGF-ß.

Actuellement, les infections des humains, des plantes et des animaux par les nématodes parasites ont un impact économique majeur. En l’absence de vaccin efficace, les anthelminthiques sont les principaux agents chimiothérapeutiques utilisés pour le traitement et la prophylaxie des infections à nématodes. Cependant, la résistance est apparue pour la plupart des anthelminthiques. Par conséquent il est urgent de comprendre la génétique des récepteurs ciblés par ces anthelminthiques et de trouver des cibles alternatives afin de développer de nouveaux anthelminthiques. Nous avons démontré les effets du nouvel anthelminthique cyclooctadepsipeptide, emodepside, sur le potentiel de membrane et les courants voltagedépendants chez le parasite du porc Ascaris suum. Enfin, nous avons cloné quatre gènes codant des sous-unités du récepteur de l'acétylcholine d'un autre parasite du porc, Oesophagostomum dentatum et caractérisé dans des ovocytes de Xenopus laevis quatre sous-types de récepteurs au lévamisole. / Parasitic nematode infections of humans, plants and animals are of major economic impact. Anthelmintics are the main chemotherapeutic agents used for treatment and prophylaxis of nematode infections because there is presently no effective vaccine on the market. However, resistance has been reported to the mainstay anthelmintics. There is therefore the urgent need to understand the genetics of the receptors targeted by these anthelmintics and to find alternative targets for developing new anthelmintics. We have demonstrated the effects of the new novelacting cyclooctadepsipeptide anthelmintic, emodepside, on the membrane potential and voltageactivated currents in the pig parasite Ascaris suum. Finally, we show the cloning of four acetylcholine receptor subunit genes from another pig parasite, Oesophagostomum dentatum and the expression and characterization of four levamisole receptor subtypes in Xenopus laevis oocytes.