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.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2014-11-03T20:17:54Z No. of bitstreams: 2 Dissertação - Fernanda Rassi Alvarenga - 2009.pdf: 1178426 bytes, checksum: 23c15006d0ef89ee6336d71ed66e974e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Erika Demachki (erikademachki@gmail.com) on 2014-11-03T20:18:53Z (GMT) No. of bitstreams: 2 Dissertação - Fernanda Rassi Alvarenga - 2009.pdf: 1178426 bytes, checksum: 23c15006d0ef89ee6336d71ed66e974e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2014-11-03T20:18:53Z (GMT). No. of bitstreams: 2 Dissertação - Fernanda Rassi Alvarenga - 2009.pdf: 1178426 bytes, checksum: 23c15006d0ef89ee6336d71ed66e974e (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2009-06-26 / Toxoplasmosis is a parasitic disease widespread around the world caused by Toxoplasma gondii. Infection acquired during pregnancy may cause intrauterine damage and sequelae in the newborn. Serological testing for IgG/IgM anti-Toxoplasma antibodies may fail to differentiate between recent and past infection. Despite that, rapid diagnosis of acute infection during pregnancy allows rapid treatment and prevents or attenuates congenital toxoplasmosis. PURPOSE: to establish the frequency of acute toxoplasmosis in pregnant women, the vertical transmission rate and the value of specific IgG-avidity test to date infection in pregnancy; to evaluate the relationship between IgG-avidity and congenital toxoplasmosis. MATERIAL AND METHODS: this report summarizes a retrospective study performed on 235,993 pregnant women attended by “The Pregnancy Protection Program” – public health system (SUS) of the State of Goiás – Brazil, from January 2004 to December 2007. ELISA (IgG / IgM) and IgG-avidity test were performed for maternal screening of toxoplasmosis. Fetal and newborn investigation of the infection was performed by “The Toxoplasmosis Vertical Transmission Control Program” protocols. The association between data was statistically analyzed by the x2 test (p < 0,05 was considered statistically significant). RESULTS: the frequency of IgM-positive among pregnant women studied population was 0,7%. Among IgM-positive women, only 207 (12,5%) performed the screening test in first 3-month period of pregnancy and 91% of pregnant women presented high avidity ( > 40%). The vertical transmission rate was 62%. There was no statistically significant relationship between higher (> 40%) or lower (≤ 25%) IgG-avidity test and presence of congenital infection (p= 0,08 e p= 0,57, respectively). There was no statistically significant association between maternal diagnosis in first trimester, low avidity and vertical transmission rate. CONCLUSIONS: the frequency of IgM-positive in pregnant women was lower than Brazilian rates founded in other studies. The study showed high persistent vertical transmission rate besides prenatal management and treatment. The IgG-avidity was not useful to predict vertical transmission. These results indicate that the IgG-avidity test must be not carried out in all IgM-positive pregnant women in the State of Goiás-Brazil, as a confirmatory test for the diagnosis of maternal toxoplasmosis. / A toxoplasmose, parasitose prevalente em todo o mundo, quando adquirida na gestação pode ser transmitida para o feto e ocasionar agravos que limitarão o desenvolvimento da criança para o resto da vida. O diagnóstico laboratorial da toxoplasmose com os testes imunoenzimáticos disponíveis ainda tem limitada capacidade para determinar se a mulher grávida adquiriu infecção aguda durante a gestação, ou anteriormente. Por outro lado, o diagnóstico precoce de infecção aguda na gestante, associado à medicação específica adequada, pode mudar o prognóstico da infecção congênita, diminuindo as sequelas nas crianças. OBJETIVO: estabelecer a frequência de toxoplasmose aguda gestacional, a taxa de transmissão vertical e o valor do teste de avidez da IgG como marcador de doença aguda ou crônica, bem como sua associação com comprometimento do concepto. MATERIAL E MÉTODOS: estudo retrospectivo de análise dos casos de toxoplasmose aguda identificados em gestantes atendidas pelo serviço público de saúde (SUS), no Programa de Proteção à Gestante do Estado de Goiás (APAE/SES/SMS), no período de janeiro de 2004 a dezembro de 2007. O rastreamento de infecção aguda na gravidez foi realizado através da pesquisa de anticorpos IgM específicos em gota de sangue digital coletada em papel filtro, e em soro, bem como determinação da avidez da IgG nesta mesma amostra, todos pela técnica ELISA. O diagnóstico de infecção fetal e/ou neonatal foi realizado segundo protocolo utilizado no centro de referência do Programa de Controle Vertical da Toxoplasmose (HC/FM/IPTSP/UFG). A correlação entre as variáveis foi avaliada pelo teste do x2. Foi considerado estatisticamente significante valores de p < 0,05. RESULTADOS: foram realizados 235.993 exames no período de janeiro de 2004 a dezembro de 2007, no Estado de Goiás. A frequência de soropositividade para IgM no rastreamento foi de 0,7%. Somente 207 mulheres (12,5%) realizaram o teste no primeiro trimestre, sendo que 91% das gestantes apresentaram alta avidez (> 40%). A taxa de transmissão vertical foi de 62% no grupo de gestantes acompanhadas no HC/FM/UFG. Não houve relação estatisticamente significante entre baixa (≤ 25%) ou alta (> 40%) avidez com xii comprometimento do concepto (p=0,08 e p=0,57, respectivamente). Não houve associação significativa entre diagnóstico gestacional no primeiro trimestre com baixa avidez e transmissão vertical. CONCLUSÕES: a frequência de soropositividade na triagem pré-natal (provável toxoplasmose aguda) ficou abaixo da média nacional, mas a taxa de transmissão vertical permaneceu alta apesar do acompanhamento e do tratamento pré-natal. O teste de avidez da IgG teve pouco valor, em nosso meio, para datar a infecção adquirida, pois a maioria das gestantes iniciou o pré-natal após o primeiro trimestre; também não mostrou associação com transmissão vertical, não sendo oportuna sua realização sistemática ou utilização como segundo teste na triagem pré-natal em Goiás.

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.

Die vorliegende Arbeit hatte das Ziel, die Korrelation zwischen der Avidität der Anti-Masern-IgG-Antikörper und deren In-Vitro-Neutralisationsfähigkeit zu untersuchen, sowie mittels Datenbankanalyse die Seroprävalenz von schützenden Antikörpern gegen Masern und den Impfstatus der Kinder- und Jugendlichen festzustellen. Die lineare Korrelation zwischen Neutralisationsfähigkeit und Avidität war in dieser Stichprobe schwach (ρ=0,240, p=0,006). Für hohe IgG Konzentrationen über 1000 mIU/ml fand sich eine mittlere Korrelation zwischen Avidität und Neutralisationstiter (ρ=0,612; p<0,001). Bei den untersuchten Jahren von 1997 bis 2013 zur Seroprävalenz (n=8611) wiesen im Durchschnitt 93,4 % der Patienten IgG-Konzentrationen im positiven Bereich (>200 mIU/ml) auf. In allen Jahrgängen lag der Anteil über 90 %. Zur Ermittlung des Impfstatus wurde eine Stichprobe 2- bis 18-Jähriger aus dem Jahr 2012 untersucht. Insgesamt hatten 81,1 % die erste Masernimpfung erhalten. Die zweite Masernimpfung erhielten noch 59,7 % der Kinder und Jugendlichen.

In dieser Arbeit wurden zwei serologische Teste zur Unterscheidung inzidenter und prävalenter Proben etabliert und validiert, welche auf der Reifung des Immunsystems in der frühen HIV-Infektion basieren. Im BED-ELISA werden anhand von anti-HIV-gp41-spezifischen IgG-Antikörperspiegeln die Proben klassifiziert. Die Aviditäts-Methode (AI) unterscheidet die Bindungsfähigkeit der Antikörper an spezifische Antigene. Das Probenpanel wurde in einem weiteren Inzidenztest, dem IDE-V3-ELISA (Kooperation F. Barin), gemessen welcher den Anstieg der Antikörperreaktivität gegen zwei verschiedene immundominante Epitope nutzt. Wirtsspezifische Marker und virale Eigenschaften wurden untersucht, um Merkmale zu identifizieren, welche die Sensitivität und Spezifität der Teste verbessern könnten. Mit dem BED-ELISA wurde eine Pilotstudie unter Berliner HIV-Patienten durchgeführt. Zur Vereinfachung des Probentransports in Studien wurde der Einfluss einer Filtertrocknung der Plasmaproben auf die Infektiosität von HIV, Stabilität der HIV-RNA und die Antikörperreaktivität im BED-ELISA untersucht. In den Testen wurden inzidente Plasmaproben mit folgenden Sensitivitäten und Spezifitäten richtig klassifiziert: 80% und 86% (BED-ELISA); 74% und 82% (AI); 73% und 84% (IDE-V3). Von allen untersuchten wirtsspezifischen Faktoren korrelierte nur der Gehalt an Antikörpern der IgG3-Subklasse mit der Fehlklassifikation der Proben. Für die Pilotstudie wurden zwischen Feb. 2005 und Nov. 2007 von 132 erstmalig HIV-1 positiv diagnostizierten Patienten Proben genommen und im BED-ELISA analysiert (51% Anteil inzidente Infektionen). Die Antikörperreaktivitäten blieben nach der Filtertrocknung erhalten, so das auf der Grundlage der Ergebnisse eine deutschlandweite Inzidenzstudie mit Filter-getrockneten Plasmaproben geplant wurde, die seit Januar 2008 im Auftrag des BMG zur Verbesserung der Datenlage zur HIV-Inzidenz in Deutschland durchgeführt wird. / In this PhD thesis two methods that can differentiate between incident and prevalent infections were established and validated. Both tests are based on the maturation of the immune system during early HIV-infection. The BED-ELISA uses anti-HIV-gp41 specific IgG-antibody levels for differentiation. The avidity method (AI) is based on the binding-capacity of the antibodies to specific antigens in the presence of a chaotropic agent. The sample panel was also evaluated using an additional incidence ELISA, the IDE-V3-ELISA (cooperation with F. Barin). This test is also based on the antibody''s reactivity to two different immune dominant epitopes, allowing incident and prevalent infections to be differentiated. Host specific factors and viral determinants were analysed to provide information that could lead to improvements in the sensitivity and specificity of the incidence tests. With the BED-ELISA a pilot study with HIV-infected patients in Berlin was carried out. The inactivation of HIV-1 after filter-drying of samples, the stability of viral RNA and reactivity of antibodies in the BED-ELISA were analysed to simplify the transport of samples in future studies. Incident plasma samples were identified correctly with following sensitivities and specificity’s: 80% and 86% (BED-ELISA); 74% and 82% (AI); 73% and 84% (IDE-V3). Of all host factors analysed, only the titre of IgG3-antibodies correlated with the incorrect classification of samples. In the pilot study samples from 132 newly diagnosed HIV-positive patients were obtained between February 2005 and November 2007 and analysed in the BED-ELISA (51% proportion of incident infections). It could be shown that filter-drying of plasma samples rendered HIV non-infectious but did not influence antibody reactivity. Based on these results, a German HIV incidence study using filter-dried plasma samples, designed to improve knowledge of HIV-incidence in Germany, was sponsored by the BMG and has been ongoing since January 2008.

Neisseria meningitidis é um diplococo Gramnegativo, aeróbio e encapsulado, causador mais comum de meningite e septicemia. Este agente é o principal causador de infecções bacterianas invasivas no mundo. Apesar de existirem 13 sorogrupos de N. meningitidis, apenas 6 são capazes de causar infecção: A, B, C, W135, X e Y. O sorogrupo B difere dos outros sorogrupos patogênicos por sua cápsula polissacáride ter composição idêntica ao ácido policiálico, presente em muitas glicoproteínas humanas, particularmente encontrados no tecido cerebral fetal, e bioquimicamente homóloga com a estrutura molecular de adesão do neurônio. Sendo assim, a cápsula polissacáride não pode ser usada em vacinas conjugadas, pois pode causar autoimunidade, sendo pouco imunogênica. Doenças meningocócicas causadas pelos sorogrupos A, C, Y e W135 podem ser prevenidas pelas vacinas que contêm polissacarídeos capsulares específicos conjugados. Para que uma vacina seja eficaz contra o sorogrupo B, é importante que esta abranja todos os sorotipos e seja capaz de promover imunidade duradoura, principalmente em crianças abaixo de dois anos, as mais acometidas. Vacinas baseadas em vesículas de membrana externa (OMVs, do inglês Outer Membrane Vesicles) de N. meningitidis B são amplamente estudadas. No presente estudo, OMVs de meningococo B (B:4:P1.9) foram associadas a um lipídio catiônico, o dioctadecildimetilamônio (DDA-BF) em preparação antigênica testada em camundongos fêmeas não isogênicos, e comparamos os títulos de anticorpos IgG, IgG1, IgG2a e IgG2b com os anticorpos produzidos por camundongos imunizados com a mesma OMVS associada ao hidróxido de alumínio, por ELISA. As análises foram realizadas com soros de cada animal colhidos individualmente, após 60 dias de imunização. A avidez dos anticorpos também foi analisada por ELISA. Immunoblot e Dot-ELISA avaliaram a reação específica entre a cepa homóloga usada na imunização e a reação a antígenos cruzados com outras cepas de meningococo. A hipersensibilidade tardia (HTT) foi comparada entre os dois grupos experimentais, após o desafio com cepa homóloga em uma das patas, depois de 24 horas da injeção, após 14 dias da primeira dose de imunização. / Neisseria meningitidis is an encapsulated Gram-negative aerobic diplococcus, the most commom meningitidis and sepsis agent , and the major bacterial invasive disease agent worldwide. Infections are caused by only 6 of 13 pathogenic serogroups: A,B,C, W135 and Y. Meningococcal serogroup B differs from the other pathogenic serogroups because it has a capsular polysaccharide identical to the polysialic acid present in many human glycoproteins, in particular, it is similar to carbohydrates found in fetal brain tissue. This is the reason that it does not allow the use of polysaccharide protein in conjugate vaccine, and for its low immunogenic. An effective meningococcal B vaccine development should cover all serotypes and be able to promote long term immunity, mainly in children under 2 years, the most affected age. Meningococcal outer membrane vesicles (OMVs) vaccines are widely studied. In this present study, meningococcal serogroup B OMVs (B:4:P1.9) was associated with a cationic lipid, dioctadecyldimetylammonium (DDA-BF) in an antigenic preparation tested in female outbred mice. Individual serum was collected, and antibodies titles IgG, IgG1, IgG2a were compared with animals immunized with OMVs and aluminium hydroxide, analyzed by ELISA. Analyses were carried out 60 days after first immunization. Antibodies avidity index were also analyzed by ELISA. Immunoblot and Dot-ELISA were carried out to evaluate specific reaction for homologous stranis and cross-reactive antigens present in other meningococcal strains. Delayed type hypersensitivity (DTH) was compared between two experimental groups, 24 hours before injection of homologous strain challenge.

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.

Les composantes innée et acquise de l'immunité travaillent ensemble pour assurer une protection efficace de l'organisme. Les cellules dendritiques, cellules sentinelles de l’immunité capturent via des récepteurs de surface les agents pathogènes et les présentent aux lymphocytes T pour stimuler les réponses immunitaires adaptatives spécifiques. Une famille de ces récepteurs, nommée Récepteurs Lectines de type C (CLRs) ont un rôle important dans la reconnaissance de motifs oligosaccharides des pathogènes. Leurs fonctions sont parfois détournées par certains pathogènes à leur avantage et notamment le VIH. La reconnaissance du virus par DC-SIGN, une des CLRs, favorise la dissémination du virus. A l’inverse, la langerine, autre CLR, est considérée comme une barrière naturelle au VIH. Ainsi, DC-SIGN est devenue une cible thérapeutique prometteuse mais sa reconnaissance des ligands osidiques est largement partagée par la langerine.Ce travail vise à développer des antagonistes de DC-SIGN spécifiques et de hautes affinités permettant de rivaliser avec la présentation multivalente des glycosylations de gp120 du VIH avec DC-SIGN. Une approche rationnelle a été employée dans le développement de ligands glycomimétiques hautement sélectifs pour DC-SIGN à partir de l’étude du site de liaison des deux CLRs. Puis, des plates-formes de présentations de ces glycomimétiques, de valences et de géométries différentes, sont comparées par SPR. Les améliorations spectaculaires d'affinités parfois observées sont liées à différents mécanismes d’interactions multivalentes responsables d’un phénomène d’avidité.Sur une des architecture de présentation sélectionnée (RODs), un travail de caractérisation fine des mécanismes responsables de ce gain d’affinité et/ou d’avidité a été conduit par la combinaison de plusieurs techniques biophysiques (SPR, ITC, polarisation de fluorescence et AUC). L’influence de la topologie de cette structure sur les mécanismes d’interactions est ainsi mise en évidence. Par les travaux menés, plusieurs ligands multivalents ont montré des affinités sans précédent pour DC SIGN atteignant des affinités du nanomolaire et représentant les meilleurs inhibiteurs connus à ce jour.Associé au développement d’antagonistes multivalents, une CLR (DCIR) a été identifiée récemment comme impliquée dans la dissémination du VIH, comme DC¬SIGN. Dans une perspective future de développement de glycomimétique, des travaux ont été menés sur la caractérisation structurale et fonctionnelle de ce nouvel acteur dans la problématique VIH. / The innate and acquired immunity components work together to provide efficient protection of organisms. Dendritic cells, sentinel cells of the immunity, are able to capture pathogens through their receptors on the surface and they can present the antigens to lymphocytes T in order to stimulate specific adaptive immune responses. Among these receptors, there is a family named C-type lectin receptors (CLRs), which has an important role in the recognition of pathogenic oligosaccharide motifs. CLRs can be hijacked by many pathogens including HIV. DC-SIGN, one of the CLRs, interacts with the virus and promotes its dissemination. Unlike DC-SIGN, langerin, another CLR, has a protective role against the HIV infection. In this context, DC-SIGN became a promising therapeutic target but it shares ligand specificities with langerin.This work aims to develop highly specific antagonists against DC-SIGN in order to compete with the multivalent glycosylated gp120 protein of HIV. Using the study of the two lectins binding sites as starting point, a rational approach has been exploited to develop highly selective glycomimetics against DC SIGN. The SPR technique was used to investigate multivalent platforms with different valencies as well as ligand presentation in space. The amazing improvement of the affinity observed in some cases can be linked to different mechanisms of multivalent interactions, leading to an avidity phenomenon. On a selected scaffold (RODs), we characterized the different mechanisms responsible for the affinity and/or avidity gains, using a combination of different biophysical techniques (SPR, ITC, fluorescence polarization, AUC). In this work, we highlighted that the topology of this structure can influence the mechanisms of interactions. Overall, different multivalent ligands showed unique affinities for DC-SIGN, reaching the nanomolar affinity range, and they represent the best inhibitors to date.Finally, another CLR has been recently identified as one of the protein involved in the HIV infection as well as DC-SIGN. In a future perspective of glycomimetic development, structural and functional characterization has been done on this new actor involved in the HIV issue.

Neisseria meningitidis é um diplococo Gramnegativo, aeróbio e encapsulado, causador mais comum de meningite e septicemia. Este agente é o principal causador de infecções bacterianas invasivas no mundo. Apesar de existirem 13 sorogrupos de N. meningitidis, apenas 6 são capazes de causar infecção: A, B, C, W135, X e Y. O sorogrupo B difere dos outros sorogrupos patogênicos por sua cápsula polissacáride ter composição idêntica ao ácido policiálico, presente em muitas glicoproteínas humanas, particularmente encontrados no tecido cerebral fetal, e bioquimicamente homóloga com a estrutura molecular de adesão do neurônio. Sendo assim, a cápsula polissacáride não pode ser usada em vacinas conjugadas, pois pode causar autoimunidade, sendo pouco imunogênica. Doenças meningocócicas causadas pelos sorogrupos A, C, Y e W135 podem ser prevenidas pelas vacinas que contêm polissacarídeos capsulares específicos conjugados. Para que uma vacina seja eficaz contra o sorogrupo B, é importante que esta abranja todos os sorotipos e seja capaz de promover imunidade duradoura, principalmente em crianças abaixo de dois anos, as mais acometidas. Vacinas baseadas em vesículas de membrana externa (OMVs, do inglês Outer Membrane Vesicles) de N. meningitidis B são amplamente estudadas. No presente estudo, OMVs de meningococo B (B:4:P1.9) foram associadas a um lipídio catiônico, o dioctadecildimetilamônio (DDA-BF) em preparação antigênica testada em camundongos fêmeas não isogênicos, e comparamos os títulos de anticorpos IgG, IgG1, IgG2a e IgG2b com os anticorpos produzidos por camundongos imunizados com a mesma OMVS associada ao hidróxido de alumínio, por ELISA. As análises foram realizadas com soros de cada animal colhidos individualmente, após 60 dias de imunização. A avidez dos anticorpos também foi analisada por ELISA. Immunoblot e Dot-ELISA avaliaram a reação específica entre a cepa homóloga usada na imunização e a reação a antígenos cruzados com outras cepas de meningococo. A hipersensibilidade tardia (HTT) foi comparada entre os dois grupos experimentais, após o desafio com cepa homóloga em uma das patas, depois de 24 horas da injeção, após 14 dias da primeira dose de imunização. / Neisseria meningitidis is an encapsulated Gram-negative aerobic diplococcus, the most commom meningitidis and sepsis agent , and the major bacterial invasive disease agent worldwide. Infections are caused by only 6 of 13 pathogenic serogroups: A,B,C, W135 and Y. Meningococcal serogroup B differs from the other pathogenic serogroups because it has a capsular polysaccharide identical to the polysialic acid present in many human glycoproteins, in particular, it is similar to carbohydrates found in fetal brain tissue. This is the reason that it does not allow the use of polysaccharide protein in conjugate vaccine, and for its low immunogenic. An effective meningococcal B vaccine development should cover all serotypes and be able to promote long term immunity, mainly in children under 2 years, the most affected age. Meningococcal outer membrane vesicles (OMVs) vaccines are widely studied. In this present study, meningococcal serogroup B OMVs (B:4:P1.9) was associated with a cationic lipid, dioctadecyldimetylammonium (DDA-BF) in an antigenic preparation tested in female outbred mice. Individual serum was collected, and antibodies titles IgG, IgG1, IgG2a were compared with animals immunized with OMVs and aluminium hydroxide, analyzed by ELISA. Analyses were carried out 60 days after first immunization. Antibodies avidity index were also analyzed by ELISA. Immunoblot and Dot-ELISA were carried out to evaluate specific reaction for homologous stranis and cross-reactive antigens present in other meningococcal strains. Delayed type hypersensitivity (DTH) was compared between two experimental groups, 24 hours before injection of homologous strain challenge.