A toxoplasmose é uma protozoose de alta prevalência no Brasil, causada pelo Toxoplasma gondii, sendo transmitida pela ingestão de alimentos contaminados com oocistos, excretados em fezes de felinos, ou cistos, em carnes cruas ou mal cozidas. A doença é usualmente assintomática, mas em fetos ou pacientes com imunodepressão, pode ser devastadora. Neste trabalho, estudamos a prevalência sorológica da infecção em animais de diferentes regiões do estado de São Paulo, tanto de vida livre, cães (200/ABC) como indicadores ambientais, e gatos (100/São Paulo) como hospedeiros definitivos, e em animais de produção, bovinos (200/Taquarituba), suínos (200/Osasco), caprinos( 200/Botucatu), ovinos (200/São Manuel) e frangos de corte(185/Botucatu), além de estudos parasitológicos em gatos e águas sob suspeita. Foram padronizados ELISA para cada espécie animal, utilizando índices adequados e reprodutíveis, com confirmação por Western Blotting e determinação da avidez em amostras positivas. A prevalência da toxoplasmose animal foi determinada sendo crescente em suínos (8.5%), bovinos (11%), caprinos (17%), ovinos (31%), felinos (40%) e cães (50,5%), não sendo encontrada em frangos de corte. Em suínos, caprinos, cães e gatos, a freqüência de anticorpos de baixa avidez sugere que a transmissão da infecção é constante durante a vida do animal, mas em bovinos e ovinos não foram encontrados anticorpos de baixa avidez, sugerindo infecção precoce ou sazonal na vida do animal. Pela alta taxa de infecção recente em felinos, é possível prever uma fração significativa de animais excretando oocistos, embora sem comprovação parasitológica. A avaliação da presença de anticorpos anti-T.gondii deve ser criteriosa, sendo que os reagentes de hemaglutinação para uso humano fornecem resultados erráticos nesta medida. A pesquisa de oocistos na água é de baixa sensibilidade, devendo ser feita em materiais colhidos no período de suspeita da transmissão. Em São Paulo, o risco de transmissão da toxoplasmose está relacionado a quase todas as fontes de infecção pesquisadas, tornando necessários estudos para o melhor manejo dos animais de consumo humano e tratamento de água, com eliminação de gatos errantes. / Toxoplasmosis, caused by Toxoplasma gondii and highly prevalent protozoan disease in Brazil, is mainly transmitted by ingestion of contaminated food and water, both by oocysts, excreted in cat feces, or cysts from undercooked meat from warm-blooded animals. Usually asymptomatic, it is extremely severe in the fetus or immunosuppressed patients. In this work, we studied the serological prevalence of toxoplasmosis in animals from several regions of the São Paulo State, both free living, as dogs (ABC) as environmental contamination index, and cats (São Paulo, as definitive hosts, or livestock as cattle (Taquarituba), swine (Osasco), goats (Botucatu), sheep (São Manuel) and fowls (São Paulo), with parasitological studies in cats and suspicious drinking water. We standardized ELISA for each species, using reproducible and adequate indexes, with Western blot confirmation and avidity assays in positive samples. Toxoplasmosis prevalence was increasing in swine (8.5%), cattle (11%), goats (17%), sheep (31%), cats (40%) and dogs (50.5%), without positive sample in fowls. Goats, pigs, dogs and cats presented 5-20% low avidity antibody samples, suggesting sustained transmission during animal life, but cattle and sheep presented only high avidity samples, suggesting an seasonal or early in life infection. Due to the high recent infection rate in cats, it is possible to preview a significant oocyst excreting cat frequency, despite parasitological evidence. Antibody determination must be carefully evaluated, as human hemagglutination reagents give erratic information. Oocyst detection in drinking water presented very low sensitivity and must be performed only in water collected at the period of the infection. In São Paulo, almost all of tested sources are able of toxoplasmosis transmission, reinforcing the need of better management of livestock, adequate water treatment and elimination of free living cats.

A toxoplasmose é uma protozoose de alta prevalência no Brasil, causada pelo Toxoplasma gondii, sendo transmitida pela ingestão de alimentos contaminados com oocistos, excretados em fezes de felinos, ou cistos, em carnes cruas ou mal cozidas. A doença é usualmente assintomática, mas em fetos ou pacientes com imunodepressão, pode ser devastadora. Neste trabalho, estudamos a prevalência sorológica da infecção em animais de diferentes regiões do estado de São Paulo, tanto de vida livre, cães (200/ABC) como indicadores ambientais, e gatos (100/São Paulo) como hospedeiros definitivos, e em animais de produção, bovinos (200/Taquarituba), suínos (200/Osasco), caprinos( 200/Botucatu), ovinos (200/São Manuel) e frangos de corte(185/Botucatu), além de estudos parasitológicos em gatos e águas sob suspeita. Foram padronizados ELISA para cada espécie animal, utilizando índices adequados e reprodutíveis, com confirmação por Western Blotting e determinação da avidez em amostras positivas. A prevalência da toxoplasmose animal foi determinada sendo crescente em suínos (8.5%), bovinos (11%), caprinos (17%), ovinos (31%), felinos (40%) e cães (50,5%), não sendo encontrada em frangos de corte. Em suínos, caprinos, cães e gatos, a freqüência de anticorpos de baixa avidez sugere que a transmissão da infecção é constante durante a vida do animal, mas em bovinos e ovinos não foram encontrados anticorpos de baixa avidez, sugerindo infecção precoce ou sazonal na vida do animal. Pela alta taxa de infecção recente em felinos, é possível prever uma fração significativa de animais excretando oocistos, embora sem comprovação parasitológica. A avaliação da presença de anticorpos anti-T.gondii deve ser criteriosa, sendo que os reagentes de hemaglutinação para uso humano fornecem resultados erráticos nesta medida. A pesquisa de oocistos na água é de baixa sensibilidade, devendo ser feita em materiais colhidos no período de suspeita da transmissão. Em São Paulo, o risco de transmissão da toxoplasmose está relacionado a quase todas as fontes de infecção pesquisadas, tornando necessários estudos para o melhor manejo dos animais de consumo humano e tratamento de água, com eliminação de gatos errantes. / Toxoplasmosis, caused by Toxoplasma gondii and highly prevalent protozoan disease in Brazil, is mainly transmitted by ingestion of contaminated food and water, both by oocysts, excreted in cat feces, or cysts from undercooked meat from warm-blooded animals. Usually asymptomatic, it is extremely severe in the fetus or immunosuppressed patients. In this work, we studied the serological prevalence of toxoplasmosis in animals from several regions of the São Paulo State, both free living, as dogs (ABC) as environmental contamination index, and cats (São Paulo, as definitive hosts, or livestock as cattle (Taquarituba), swine (Osasco), goats (Botucatu), sheep (São Manuel) and fowls (São Paulo), with parasitological studies in cats and suspicious drinking water. We standardized ELISA for each species, using reproducible and adequate indexes, with Western blot confirmation and avidity assays in positive samples. Toxoplasmosis prevalence was increasing in swine (8.5%), cattle (11%), goats (17%), sheep (31%), cats (40%) and dogs (50.5%), without positive sample in fowls. Goats, pigs, dogs and cats presented 5-20% low avidity antibody samples, suggesting sustained transmission during animal life, but cattle and sheep presented only high avidity samples, suggesting an seasonal or early in life infection. Due to the high recent infection rate in cats, it is possible to preview a significant oocyst excreting cat frequency, despite parasitological evidence. Antibody determination must be carefully evaluated, as human hemagglutination reagents give erratic information. Oocyst detection in drinking water presented very low sensitivity and must be performed only in water collected at the period of the infection. In São Paulo, almost all of tested sources are able of toxoplasmosis transmission, reinforcing the need of better management of livestock, adequate water treatment and elimination of free living cats.

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc??? receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.

Immune heparin-induced thrombocytopenia (HIT) is a potentially serious complication of heparin therapy and is associated with antibodies directed against a complex of platelet factor 4 (PF4) and heparin. Early diagnosis of HIT is important to reduce morbidity and mortality. I developed an enzyme immunoassay that detects the binding of HIT IgG to PF4-heparin in the fluid phase. This required techniques to purify and biotinylate PF4. The fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. I was able to detect anti-PF4-heparin IgG in 93% of HIT patients. I also investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 88% of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration. While HIT patients possess antibodies to PF4-heparin, I observed that HIT antibodies will also bind to PF4 alone adsorbed on polystyrene ELISA wells but not to soluble PF4 in the absence of heparin. Having developed a technique to affinity-purify anti-PF4-heparin HIT IgG, I provide the first estimates of the avidity of HIT IgG. HIT IgG displayed relatively high functional affinity for both PF4-heparin (Kd=7-30nM) and polystyrene adsorbed PF4 alone (Kd=20-70nM). Furthermore, agarose beads coated with PF4 alone were almost as effective as beads coated with PF4 plus heparin in depleting HIT plasmas of anti-PF4-heparin antibodies. I conclude that the HIT antibodies which bind to polystyrene adsorbed PF4 without heparin are largely the same IgG molecules that bind PF4-heparin and thus most HIT antibodies bind epitope(s) on PF4 and not epitope(s) formed by part of a PF4 molecule and part of a heparin molecule. Binding of PF4 to heparin (optimal) or polystyrene/agarose (sub-optimal) promotes recognition of this epitope. Under conditions that are more physiological and sensitive than previous studies, I observed that affinity-purified HIT IgG will cause platelet aggregation upon the addition of heparin. Platelets activated with HIT IgG increased their release and surface expression of PF4. I quantitated the binding of affinity-purified HIT 125I-IgG to platelets as they activate in a plasma milieu. Binding of the HIT IgG was dependent upon heparin and some degree of platelet activation. Blocking the platelet Fc??? receptor-II with the monoclonal antibody IV.3 did not prevent HIT IgG binding to activated platelets. I conclude that anti-PF4-heparin IgG is the only component specific to HIT plasma that is required to induce platelet aggregation. The Fab region of HIT IgG binds to PF4-heparin that is on the surface of activated platelets. I propose that only then does the Fc portion of the bound IgG activate other platelets via the Fc receptor. My data support a dynamic model of platelet activation where released PF4 enhances further antibody binding and more release.

Fundação de Amparo a Pesquisa do Estado de Minas Gerais / Recombinant proteins from Toxoplasma gondii have been used in several experimental models, as well as for serodiagnosis of human toxoplasmosis, particularly to differentiate acute from chronic phases of the infection. In the present study, we evaluated the kinetics of IgM, IgA, and IgG isotypes, in addition to IgG1 and IgG3 subclasses, by testing sequential serum samples from patients with acute toxoplasmosis. It was carried out immunoassays by using SAG2A recombinant antigen and soluble antigen of Toxoplasma (STAg). The avidity of IgG1 antibody was assessed using slot blot assay. Additionally, a ratio between IgG1 and IgG3 subclasses (IgG3:IgG1) was determined and evaluated its degree of association with levels of IgM and IgA specific for STAg and the avidity index of IgG1 specific for SAG2A. The results showed a decreasing kinetic profile for SAG2A and STAg for IgM and IgA. The kinetic profile for the IgG antibody was increasing for both antigens. Compared to the avidity for IgG1, it was observed that sera from an early stage showed a low average avidity of IgG1 for SAG2A while the same samples showed intermediate mean avidity of IgG1 when STAg was used as antigen. In a later phase, the average avidity observed was high for STAg and intermediate for SAG2A in the same tested sera suggesting that SAG2A may be a promising tool for the detection of avidity. Associations between IgG3/IgG1 and specific IgM and IgA levels for STAg and the avidity index of specific IgG1 for SAG2A were found, and together these parameters could be used as valuable tools in the diagnosis of human toxoplasmosis, especially in situations when the determination of different phases is critical. / Proteínas recombinantes de Toxoplasma gondii têm sido utilizadas em diversos modelos experimentais, assim como para o diagnóstico sorológico da infecção humana por este parasito, principalmente com o intuito de diferenciar as fases aguda e crônica da toxoplasmose. Neste estudo, foi avaliada a cinética dos anticorpos IgM, IgA ,IgG e subclasses (IgG1 e IgG3) através de imunoensaios realizados em amostras seqüenciais de soros humanos, provenientes de pacientes com toxoplasmose aguda. Estas amostras foram testadas frennte ao antígeno recombinante SAG2A, utilizando-se como paradigma de comparação o antígeno solúvel total de Toxoplasma (STAg). A avidez do anticorpo IgG1 foi avaliada utilizando a metodologia slot-blot. Adicionalmente, a razão entre as subclasses IgG3 e IgG1 (IgG3:IgG1) foi determinada e avaliada quanto ao grau de associação com os níveis de IgM e IgA específicos para STAg e aos índices avidez de IgG1 específicos para SAG2A. Os resultados demonstraram a presença de níveis decrescentes de IgM e IgA para ambos os antígenos utilizados, enquanto que para o isotipo IgG o perfil cinético demonstrou níveis crescentes para ambas preparações antígênicas. Em relação aos índices de avidez para IgG1, foi observado que amostras de soros de uma fase inicial apresentaram baixa avidez média de anticorpos IgG1 dirigidos para SAG2A, enquanto que as mesmas amostras demonstraram avidez média intermediária de IgG1 quando STAg foi utilizado como antígeno. Já em uma fase mais tardia, a avidez média observada foi alta para STAg e intermediária com SAG2A. A razão entre IgG3:IgG1 obtida no primeiro bimestre foi significantemente maior para SAG2A em comparação com STAg. Tomados em conjunto, os resultados obtidos no presente estudo indicam que a proteína recombinantes SAG2A pode se constituir em uma ferramenta efetiva na diferenciação das fases da infecção humana por T. gondii. / Mestre em Imunologia e Parasitologia Aplicadas