Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Corynebacterium diphtheriae pode ser isolado tanto de quadros de difteria clássica, quanto de infecções sistêmicas, como endocardite. O fibrinogênio (Fbn) e a fibronectina (Fn) são glicoproteínas presentes na matriz extracelular de tecidos conjuntivos. A influência destas proteínas na patogênese das infecções locais e invasivas causadas por C. diphtheriae é objeto de estudo devido ao fato do bacilo diftérico poder ser encontrado em lesões nas quais o Fbn e a Fn são predominantes, incluindo a pseudomembrana diftérica e vegetações cardíacas presentes na endocardite infecciosa. São crescentes as evidências de que o C. diphtheriae pode, além de aderir, ser internalizado por células em cultura. No presente estudo, investigou-se a participação de C. diphtheriae e das proteínas de superfície 67-72p na aderência à Fn e ao Fbn de plasma humano e a eritrócitos. A aderência às células HEp-2 e internalização também foram analisadas. A participação de 67-72p nos mecanismos de morte celular foi avaliada através das colorações por Azul de Tripan e 46-diamidino-2-fenil indol (DAPI), pelo ensaio de redução utilizando dimetil-tiazol-difenil tetrazólio (MTT) e por citometria de fluxo. As 67-72p foram extraídas da superfície da amostra toxigênica C. diphtheriae subsp. mitis CDC-E8392 através de processos mecânicos e precipitação com sulfato de amônio saturado. Análises por SDS-PAGE e immunoblotting detectaram a presença das bandas protéicas de 67 e 72kDa nas amostras toxinogênicas e atoxinogênicas analisadas, as quais pertenciam aos biotipos fermentador e não fermentador de sacarose. C. diphtheriae foi capazes não só de formar agregados na presença de plasma de coelho, mas também de converter Fbn em fibrina independentemente da presença do gene tox. No entanto, a amostra atoxinogênica ATCC 27010 (tox-) foi menos aderente ao Fbn do que a homóloga ATCC 27012 (tox+). A interação bacteriana com eritrócitos foi inibida somente pela Fn. Ligações entre Fn e/ou Fbn com 67-72p foram demonstradas por dot blotting, ELISA e/ou ensaios utilizando fluorescência. As 67-72p foram capazes de inibir as interações bacterianas com o Fbn, indicando que 67-72p podem participar do processo de aderência do patógeno aos tecidos do hospedeiro. Através da microscopia óptica, demonstrou-se a ligação de 67-72p adsorvidas em microesferas de látex com células HEp-2. Anticorpos de coelho do tipo IgG anti 67-72p interferiram somente com a expressão do padrão de aderência do tipo difuso, normalmente apresentado pela amostra CDC-E8392. A Microscopia Eletrônica de Transmissão (MET) e a inibição da internalização bacteriana pela IgG anti 67-72p ou por 67-72p indicaram o papel de 67-72p como invasina. Alterações do citoesqueleto de células HEp-2 com acumulação de actina polimerizada, induzida por microesferas sensibilizadas com 67-72p, foi observada pelo fluorescent actin staining (FAS) test. Foi visualizado um aumento no número de bactérias viáveis no compartimento intracelular após tratamento de células HEp-2 ou dos microrganismos com Fn. A presença de partículas de látex adsorvidas com 67-72p no interior de vacúolos frouxos em células HEp-2 sugeriu que estas proteínas podem causar efeito citotóxico. A avaliação através das colorações com Azul de Tripan, DAPI e os ensaios de redução utilizando MTT demonstraram um decréscimo na viabilidade de células tratadas com 67-72p. As mudanças morfológicas observadas 3 horas após o início do tratamento com 67-72p incluíram vacuolização, fragmentação nuclear e formação de corpúsculos apoptóticos. A citometria de fluxo revelou um decréscimo de 15,13% no volume/tamanho de células tratadas com 67-72p. Além disso, o ensaio utilizando Iodeto de Propídio (IP) e Anexina V (AV)-FITIC demonstrou que havia 66,1% de células vivas (IP-/AV-), 16,6% de células em apoptose inicial (IP-/AV+) e 13,8% de células em apoptose tardia ou necrose secundária. Em conclusão, as 67-72p estão diretamente envolvidas na interação com Fn e Fbn. As proteínas não fimbriais 67-72p são hemaglutininas implicadas na aderência a células respiratórias e na internalização. Além disso, estas proteínas podem atuar como fatores de virulência em potencial para induzir apoptose de células epiteliais nos estágios iniciais da difteria e nas infecções invasivas causadas pelo C. diphtheriae / Corynebacterium diphtheriae have been isolated from classical diphtheria and systemic infections such as endocarditis. Fibrinogen (Fbn) and fibronectin (Fn) are high molecular-weight glycoproteins that may be found in extracellular matrix of connective tissues. Their influence in the pathogenesis of local and in invasive C. diphtheriae infection is object of interest due to the fact that diphtheria bacilli is recovered from lesions where such proteins are predominant, including pharyngeal pseudomembrane and valve heart vegetations in infectious endocarditis. There is growing evidence that C. diphtheriae may adhere to and be internalized by cells in culture. The present study investigated the participation of C. diphtheriae strains and 67-72p, a surface protein, in adherence to human plasma Fn, Fbn, erythrocytes, adherence to and internalization by HEp-2 cells. The participation of 67-72p in promoting cell death was evaluated by the Trypan blue, DAPI staining methods, methylthiazole tetrazolium (MTT) reduction assay and flow cytometry. The 67-72p was extracted from C. diphtheriae subsp. mitis CDC-E8392 toxigenic strain, by mechanical process and ammonium sulfate fractionation. SDS-PAGE and immunoblotting analysis detected the polypeptide bands of 67 and 72 kDa in all toxigenic and nontoxigenic strains from both sucrose-fermenting and non-fermenting biotypes. Diphtheria bacilli were capable to both form bacterial aggregates in rabbit plasma and to convert Fbn to fibrin independently to the presence of tox gene, albeit the ATCC 27010 (tox-) strain was less adherent to Fbn than the parental strain ATCC 27012 (tox+). Bacteria-erythrocytes interaction was inhibited only by Fn. Interactions of Fn and/or Fbn with 67-72p were demonstrated by dot blotting, ELISA and/or fluorescence assays. Bacteria-Fbn interaction was inhibited by 67-72p, indicating that 67-72p may participate in the adhesion of the pathogen to host tissues. The interaction of HEp-2 cells with 67-72p-adsorbed latex microspheres was demonstrated by light microscopy. Rabbit IgG anti 67-72p was shown to interfere with diffuse adherence phenotype to HEp-2 cells displayed by CDC-E8392, but not by other phenotypes. Transmission electron microscopy (TEM) and the inhibition of bacterial internalization by anti 67-72p IgG and 67-72p indicated the role of 67-72p as invasin. Cytoskeletal accumulation of polymerized actin in HEp-2 cells induced by 67-72p-microspheres was observed by the fluorescent actin staining (FAS) test. Fn enhanced the intracellular viability of all strains tested. The presence of 67-72p-latex particles inside spacious vacuoles (SV) in HEp-2 cells, suggested a citotoxic effect of these proteins. Evaluation by the Trypan blue staining method, 4,6-Diamidine-2-phenylindole dihydrochloride DAPI and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay showed a significant decrease in viability of HEp-2 cells treated with 0.2 mg/ml 67-72p. Morphological changes in HEp-2 cells (vacuolization, nuclear fragmentation, and the formation of apoptotic bodies) was observed after 3 h post-treatment with 67-72p. Flow cytometry revealed a 15.13% reduction apoptotic volume of HEp-2 cells treated with 0.2 mg/ml 67-72p. Moreover, a double-staining assay using Propidium Iodide (PI)/Annexin V (AV) demonstrated the numbers of vital (PI-/AV-) (66.1%) vs. early apoptotic (PI-/AV+) (16.6%) cells and late apoptotic or secondary necrotic cells (PI+/AV+) (13.8%). In conclusion, the 67-72p are directly implicated in C. diphtheriae interaction with Fn and Fbn. The non-fimbrial Fn/Fbn binding 67-72p are hemagglutinins directly implicated in adherence to and internalization by respiratory epithelial cells. Moreover, 67-72p may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stages of diphtheria and C. diphtheriae invasive infection

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Corynebacterium diphtheriae pode ser isolado tanto de quadros de difteria clássica, quanto de infecções sistêmicas, como endocardite. O fibrinogênio (Fbn) e a fibronectina (Fn) são glicoproteínas presentes na matriz extracelular de tecidos conjuntivos. A influência destas proteínas na patogênese das infecções locais e invasivas causadas por C. diphtheriae é objeto de estudo devido ao fato do bacilo diftérico poder ser encontrado em lesões nas quais o Fbn e a Fn são predominantes, incluindo a pseudomembrana diftérica e vegetações cardíacas presentes na endocardite infecciosa. São crescentes as evidências de que o C. diphtheriae pode, além de aderir, ser internalizado por células em cultura. No presente estudo, investigou-se a participação de C. diphtheriae e das proteínas de superfície 67-72p na aderência à Fn e ao Fbn de plasma humano e a eritrócitos. A aderência às células HEp-2 e internalização também foram analisadas. A participação de 67-72p nos mecanismos de morte celular foi avaliada através das colorações por Azul de Tripan e 46-diamidino-2-fenil indol (DAPI), pelo ensaio de redução utilizando dimetil-tiazol-difenil tetrazólio (MTT) e por citometria de fluxo. As 67-72p foram extraídas da superfície da amostra toxigênica C. diphtheriae subsp. mitis CDC-E8392 através de processos mecânicos e precipitação com sulfato de amônio saturado. Análises por SDS-PAGE e immunoblotting detectaram a presença das bandas protéicas de 67 e 72kDa nas amostras toxinogênicas e atoxinogênicas analisadas, as quais pertenciam aos biotipos fermentador e não fermentador de sacarose. C. diphtheriae foi capazes não só de formar agregados na presença de plasma de coelho, mas também de converter Fbn em fibrina independentemente da presença do gene tox. No entanto, a amostra atoxinogênica ATCC 27010 (tox-) foi menos aderente ao Fbn do que a homóloga ATCC 27012 (tox+). A interação bacteriana com eritrócitos foi inibida somente pela Fn. Ligações entre Fn e/ou Fbn com 67-72p foram demonstradas por dot blotting, ELISA e/ou ensaios utilizando fluorescência. As 67-72p foram capazes de inibir as interações bacterianas com o Fbn, indicando que 67-72p podem participar do processo de aderência do patógeno aos tecidos do hospedeiro. Através da microscopia óptica, demonstrou-se a ligação de 67-72p adsorvidas em microesferas de látex com células HEp-2. Anticorpos de coelho do tipo IgG anti 67-72p interferiram somente com a expressão do padrão de aderência do tipo difuso, normalmente apresentado pela amostra CDC-E8392. A Microscopia Eletrônica de Transmissão (MET) e a inibição da internalização bacteriana pela IgG anti 67-72p ou por 67-72p indicaram o papel de 67-72p como invasina. Alterações do citoesqueleto de células HEp-2 com acumulação de actina polimerizada, induzida por microesferas sensibilizadas com 67-72p, foi observada pelo fluorescent actin staining (FAS) test. Foi visualizado um aumento no número de bactérias viáveis no compartimento intracelular após tratamento de células HEp-2 ou dos microrganismos com Fn. A presença de partículas de látex adsorvidas com 67-72p no interior de vacúolos frouxos em células HEp-2 sugeriu que estas proteínas podem causar efeito citotóxico. A avaliação através das colorações com Azul de Tripan, DAPI e os ensaios de redução utilizando MTT demonstraram um decréscimo na viabilidade de células tratadas com 67-72p. As mudanças morfológicas observadas 3 horas após o início do tratamento com 67-72p incluíram vacuolização, fragmentação nuclear e formação de corpúsculos apoptóticos. A citometria de fluxo revelou um decréscimo de 15,13% no volume/tamanho de células tratadas com 67-72p. Além disso, o ensaio utilizando Iodeto de Propídio (IP) e Anexina V (AV)-FITIC demonstrou que havia 66,1% de células vivas (IP-/AV-), 16,6% de células em apoptose inicial (IP-/AV+) e 13,8% de células em apoptose tardia ou necrose secundária. Em conclusão, as 67-72p estão diretamente envolvidas na interação com Fn e Fbn. As proteínas não fimbriais 67-72p são hemaglutininas implicadas na aderência a células respiratórias e na internalização. Além disso, estas proteínas podem atuar como fatores de virulência em potencial para induzir apoptose de células epiteliais nos estágios iniciais da difteria e nas infecções invasivas causadas pelo C. diphtheriae / Corynebacterium diphtheriae have been isolated from classical diphtheria and systemic infections such as endocarditis. Fibrinogen (Fbn) and fibronectin (Fn) are high molecular-weight glycoproteins that may be found in extracellular matrix of connective tissues. Their influence in the pathogenesis of local and in invasive C. diphtheriae infection is object of interest due to the fact that diphtheria bacilli is recovered from lesions where such proteins are predominant, including pharyngeal pseudomembrane and valve heart vegetations in infectious endocarditis. There is growing evidence that C. diphtheriae may adhere to and be internalized by cells in culture. The present study investigated the participation of C. diphtheriae strains and 67-72p, a surface protein, in adherence to human plasma Fn, Fbn, erythrocytes, adherence to and internalization by HEp-2 cells. The participation of 67-72p in promoting cell death was evaluated by the Trypan blue, DAPI staining methods, methylthiazole tetrazolium (MTT) reduction assay and flow cytometry. The 67-72p was extracted from C. diphtheriae subsp. mitis CDC-E8392 toxigenic strain, by mechanical process and ammonium sulfate fractionation. SDS-PAGE and immunoblotting analysis detected the polypeptide bands of 67 and 72 kDa in all toxigenic and nontoxigenic strains from both sucrose-fermenting and non-fermenting biotypes. Diphtheria bacilli were capable to both form bacterial aggregates in rabbit plasma and to convert Fbn to fibrin independently to the presence of tox gene, albeit the ATCC 27010 (tox-) strain was less adherent to Fbn than the parental strain ATCC 27012 (tox+). Bacteria-erythrocytes interaction was inhibited only by Fn. Interactions of Fn and/or Fbn with 67-72p were demonstrated by dot blotting, ELISA and/or fluorescence assays. Bacteria-Fbn interaction was inhibited by 67-72p, indicating that 67-72p may participate in the adhesion of the pathogen to host tissues. The interaction of HEp-2 cells with 67-72p-adsorbed latex microspheres was demonstrated by light microscopy. Rabbit IgG anti 67-72p was shown to interfere with diffuse adherence phenotype to HEp-2 cells displayed by CDC-E8392, but not by other phenotypes. Transmission electron microscopy (TEM) and the inhibition of bacterial internalization by anti 67-72p IgG and 67-72p indicated the role of 67-72p as invasin. Cytoskeletal accumulation of polymerized actin in HEp-2 cells induced by 67-72p-microspheres was observed by the fluorescent actin staining (FAS) test. Fn enhanced the intracellular viability of all strains tested. The presence of 67-72p-latex particles inside spacious vacuoles (SV) in HEp-2 cells, suggested a citotoxic effect of these proteins. Evaluation by the Trypan blue staining method, 4,6-Diamidine-2-phenylindole dihydrochloride DAPI and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay showed a significant decrease in viability of HEp-2 cells treated with 0.2 mg/ml 67-72p. Morphological changes in HEp-2 cells (vacuolization, nuclear fragmentation, and the formation of apoptotic bodies) was observed after 3 h post-treatment with 67-72p. Flow cytometry revealed a 15.13% reduction apoptotic volume of HEp-2 cells treated with 0.2 mg/ml 67-72p. Moreover, a double-staining assay using Propidium Iodide (PI)/Annexin V (AV) demonstrated the numbers of vital (PI-/AV-) (66.1%) vs. early apoptotic (PI-/AV+) (16.6%) cells and late apoptotic or secondary necrotic cells (PI+/AV+) (13.8%). In conclusion, the 67-72p are directly implicated in C. diphtheriae interaction with Fn and Fbn. The non-fimbrial Fn/Fbn binding 67-72p are hemagglutinins directly implicated in adherence to and internalization by respiratory epithelial cells. Moreover, 67-72p may act as a potential virulence factor to induce apoptosis of epithelial cells in the early stages of diphtheria and C. diphtheriae invasive infection

Evidências recentes sugerem que as doenças periodontais podem desempenhar um papel relevante na etiologia e patogênese de doenças cardiovasculares e hipertensão arterial. A resposta inflamatória, com conseqüente elevação de marcadores sistêmicos como proteína C-reativa, fibrinogênio e interleucina-6, e a disfunção endotelial, podem ser os responsáveis por essa associação. Alguns estudos têm relatado maiores níveis pressóricos, maior massa ventricular esquerda e disfunção endotelial em pacientes com doenças periodontais. Ao mesmo tempo, estudos clínicos vêm mostrando que a terapia periodontal pode levar à redução dos níveis plasmáticos dos marcadores de inflamação e redução do risco cardiovascular. O presente estudo teve como objetivo avaliar os efeitos da terapia periodontal não-cirúrgica em 26 pacientes (idade média de 53.68.0 anos) hipertensos refratários. Foram avaliados marcadores plasmáticos de inflamação (proteína C-reativa, fibrinogênio e interleucina-6), pressão arterial sistólica e diastólica, massa ventricular esquerda e rigidez arterial. A terapia periodontal foi eficaz na redução da média de todos os marcadores de risco cardiovascular avaliados. Os níveis de proteína C-reativa baixaram 0.7mg/dl 6 meses após a terapia periodontal, os de IL-6, 1.6pg/dl e os de fibrinogênio 55.3mg/dl (p<0.01). A pressão arterial sistólica apresentou redução média de 16.7mmHg e a diastólica de 9.6mmHg. A massa ventricular esquerda diminuiu em média 12.9g e a velocidade da onda de pulso, um marcador de rigidez arterial, e consequentemente de disfunção endotelial, apresentou redução de seus valores médios de 0.9m/s (p<0.01). Dessa forma, conclui-se que a terapia periodontal foi eficaz na redução dos níveis de proteína C-reativa, interleucina-6, fibrinogênio, pressão arterial, massa ventricular esquerda e rigidez arterial. / Recent evidences suggest that periodontal diseases may play a relevant role in the etiology and pathogenesis of cardiovascular diseases and hypertension. The inflammatory response, and the consequent elevation of systemic markers such as C-reactive protein, fibrinogen and interleukin-6, and endothelial dysfunction, may be responsible for this association. Some studies have reported higher blood pressure levels, left ventricle mass and endothelial dysfunction in patients presenting periodontal diseases. At the same time, clinical trials have been showing that periodontal therapy can lead to the reduction of plasmatic levels of inflammatory markers and reduction of the cardiovascular risk. The present study aims to evaluate the effects of non-surgical periodontal therapy in 26 patients (mean age: 53.68.0 years old) diagnosed as having refractory hypertension. The study measured plasmatic markers of inflammation (C-reactive protein, fibrinogen and interleukin-6), systolic and diastolic blood pressure, left ventricle mass and arterial stiffness. Periodontal therapy was effective in reducing all cardiovascular risk markers evaluated. The levels of C-reactive protein lowered 0.7mg/dl 6 months after periodontal therapy, the IL-6 levels, 1.6pg/dl and fibrinogen levels 55.3mg/dl (p<0.01). Systolic blood pressure lowered 16.7mmHg and diastolic 9.6mmHg (means). Left ventricle mass lowered 12.9g (means) and pulse wave velocity, a marker of arterial stiffness, and consequently endothelial dysfunction, presented reduction of 0.9m/s (means) (p<0.01). So, the study conclusion is that periodontal therapy was effective in reducing levels of C-reactive protein, interleukin-6, fibrinogen, blood pressure, left ventricle mass and arterial stiffness.

Streptococcus agalactiae est la première cause d’infections invasives chez le nouveau né et, malgré la mise en place de stratégies de prévention, cette bactérie reste le principal agent étiologique des infections néonatales. Les souches de séquence type 17, dites hyper-virulentes, sont particulièrement associées avec les méningites, type d’infection ayant des conséquences lourdes en terme de mortalité et morbidité. Ce clone possède des caractéristiques uniques, telle que la fixation au fibrinogène, ainsi qu’un répertoire de protéines de surface qui lui sont spécifiques. Parmi ces protéines, Srr2 appartient à une famille de larges glycoprotéines streptococcales et staphylococcales impliquées dans la pathogénicité. Un domaine central de Srr2, le domaine BR, est responsable de la fixation spécifique du fibrinogène par le clone ST-17, ainsi qu’au plasminogène et à divers composants de la matrice extracellulaire. Cette protéine promeut ainsi l’adhésion et le franchissement des barrières cellulaires. L’interaction de Srr2 avec les systèmes fibrinolytique et de coagulation de l’hôte favorise la dissémination bactérienne par l’activation de la fibrinolyse, et la persistance de la bactérie dans l’organisme par la formation d’agrégats bactériens. La liaison de Srr2 avec le fibrinogène semble également promouvoir la persistance bactérienne en favorisant l’internalisation et la survie dans les macrophages. Ainsi, la protéine Srr2 confère un avantage pour le processus infectieux du clone ST-17 dans l’hôte, et constitue une cible vaccinale intéressante pour la prévention des infections à S. agalactiae. / Streptococcus agalactiae is the leading cause of invasive infections in neonates. Despite the implementation of prevention strategies, this bacterium remains the main etiological agent of neonatal infections. Hyper-virulent sequence-type 17 strains are particularly associated with meningitis, a type of infection with serious consequences in terms of mortality and morbidity. This clone has unique characteristics, such as fibrinogen binding, and a panel of specific surface proteins. Among these proteins, Srr2 belongs to a family of large streptococcal and staphylococcal glycoproteins involved in pathogenicity. A central domain of Srr2, BR domain, is responsible for the specific binding of fibrinogen by the ST -17 clone and also binds plasminogen and various components of the extracellular matrix. Thereby, it promotes adhesion and crossing of cellular barriers. The interaction of Srr2 with fibrinolytic and coagulation systems of the host could promote bacterial spread through the activation of fibrinolysis and the persistence of the bacteria in the host by the formation of bacterial aggregates. The interaction of Srr2 with fibrinogen also seems to promote bacterial persistence in promoting the internalization and survival of the bacteria in macrophages. Thus, Srr2 confers an advantage to the infectious process of the ST- 17 clone in the host and is an attractive vaccine candidate for the prevention of S. agalactiae infections.

L'étude systématique de l'hémostase post-thrombolytique a été peu étudiée. Chez 80 malades thrombolysés consécutifs, une étude prospective a comporté l'étude – aux heures 0, 2 et 24 – des facteurs de l'hémostase suivants: fibrinogène, plasminogène, PDF (produits de dégradation de la fibrine et du fibrinogène), D-dimères, alpha2-antiplasmine et facteur XIII, ainsi que l'hématocrite et la numération plaquettaire. Des calculs statistiques approfondis ont exploré les corrélations des variations des facteurs hémostatiques entre eux et avec 37 paramètres cliniques et étiologiques. Processus moléculaires post-thrombolytiques. Le rt-PA induit deux processus, indépendants statistiquement à la 2ème heure: d'une part une élévation des PDF et des D-dimères; d'autre part, une baisse du fibrinogène, corrélée à une baisse du plasminogène (r=0,48, p=0.01), de l'alpha2-antiplasmine (r=0.48, p =0.004) et du facteur XIII (r=0.44, p=0.01). La baisse du plasminogène est corrélée significativement avec celle de l'alpha2-antiplasmine (r=0.77, p<0.001), et du facteur XIII (r=0.47, p=0.02). La mise en jeu de facteurs anti-fibrinolytiques, qui n'avait jamais été décrite précédemment, peut jouer un rôle dans une limitation de la fibrinolyse et dans la rethrombose. Des corrélations sont notées entre la baisse précoce du plasminogène et l'étiologie cardioembolique (p=0.04), et un mauvais pronostic final (p=0.03), possiblement en rapport la thrombolyse intense de gros caillots. Les hématomes intra-cérébraux parenchymateux (HP) sont liés significativement à la baisse du fibrinogène (p=0.01) et à l'augmentation des PDF (p=0.01). Une baisse du fibrinogène au-dessous de 2g/L multiplie la probabilité de HP précoce par un facteur 12,82. Ainsi est confirmé le modèle d'une “coagulopathie précoce avec dégradation du fibrinogène”», prédictive de l'hématome, proposé par l'équipe lyonnaise de thrombolyse en 2004 / A systematic study of post-thrombolytic haemostasis has rarely been performed. In 80 consecutive patients, we have prospectively studied at hours 0, 2 and 24 the following parameters: fibrinogen, plasminogen, alpha2-antiplasmin, factor XIII, fibrin(ogen) Degradation Products (FDP), D-dimers, haematocrit and platelet count. Comprehensive statistical studies calculated correlations of the haemostatic values betwen themselves and with 38 etiological and clinical parameters. Molecular dynamics. Two changes between h0 and h2 were statistically independent: an increase in FDP and D-Dimers; a decrease in fibrinogen, plasminogen, alpha2-antiplasmin and factor XIII. At h2, the decrease in fibrinogen was significantly correlated with that of plasminogen (0.48, p = 0.01), alpha2-antiplasmin (0.48, p = 0.004), and factor XIII (0.44, p = 0.01). The decrease in plasminogen was significantly correlated with those of antifibrinolytic components, alpha2-antiplasmin (r=0.77, p<0.001) and factor XIII (0.47, p=0.02). To our knowledge, such an activation of antifibrinolytic components had not hitherto been mentioned. The h2 decrease of plasminogen was correlated with cardioembolic etiology (p=0.04) and final poor oucome (p=0.03), a fact possibly due to intense thrombolysis of large clots. Patients having early parenchymal hematomas (PH) showed h2 haemostasis disturbances: high FDP (p=0.01), and low fibrinogen (p=0.01). The decrease in fibrinogen less than 2g/L multiplies the odds of early PH by a factor 12.82. Thus, we confirm the model of an “early fibrinogen degradation coagulopathy” predictive of hematomas, which had been coined by the Lyon thrombolysis team in 2004

<div>Large protein molecules are abundant in biological cells but are very difficult to study in physiological conditions due to molecular disorder. For large proteins, most structural information is obtained in crystalline states which can be achieved in certain conditions at very low temperature. X-ray and neutron crystallography methods can then be used for determination of crystalline structures at atomic level. However, in solution at room or physiological temperatures such highly resolved descriptions cannot be obtained except in very few cases. Scattering methods that can be used to study this type of structures at room temperature include small-angle x-ray and neutron scattering. These methods are used here to study two distinct proteins that are both classified as glycoproteins, which are a large class of proteins with diverse biological functions. In this study, two specific plasma glycoproteins were used: Fibrinogen (340 kDa) and Alpha 1-Antitrypsin or A1AT (52 kDa). These proteins have been chosen based on the fact that they have a propensity to form very large molecular aggregates due to their tendency to polymerize. One goal of this project is to show that for such complex structures, a combination of scattering methods that include SAXS, SANS, and DLS can address important structural and interaction questions despite the fact that atomic resolution cannot be obtained as in crystallography. A1AT protein has been shown to have protective roles of lung cells against emphysema, while fibrinogen is a major factor in the blood clotting process. A systematic approach to study these proteins interactions with lipid membranes and other proteins, using contrast-matching small-angle neutron scattering (SANS), small angle x-ray scattering (SAXS) and dynamic light scattering (DLS), is presented here. A series of structural reference points for each protein in solution were determined by performing measurements under osmotic stress controlled by the addition of polyethylene glycol-1,500 MW (PEG 1500) in the samples. Osmotic pressure changes the free energy of the molecular mixture and has consequences on the structure and the interaction of molecular aggregates. In particular, the measured radius of gyration (Rg) for A1AT shows a sharp structural transition when the concentration of PEG 1500 is between 33 wt\% and 36 wt\%. Similarly, a significant structural change was observed for fibrinogen when the concentration of PEG 1500 was above 40 wt\%. This analysis is applied to a study of A1AT interacting with lipid membranes and to a study of fibrinogen polymerization in the presence of the enzyme thrombin, which catalyzes the formation of blood clots. The experimental approach presented here and the applications to specific questions show that an appropriate combination of scattering methods can produce useful information on the behavior and the interactions of large protein systems in physiological conditions despite the lower resolution compared to crystallography.</div>

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No description available.

Que o envenenamento pela serpente Bothrops jararaca causa distúrbios hemorrágicos sistêmicos, com alteração da coagulação e fibrinólise sangüíneas, é notório. Contudo, pouco se sabe sobre a ação in vivo desse veneno sobre as plaquetas. Em estudos recentes, demonstrou-se que esse veneno causa trombocitopenia, distúrbios da agregação e diminuição do número de corpos densos plaquetários, que, dessarte, sugeriam a ativação das plaquetas circulantes. Com o escopo de comprovar esta hipótese e melhor caracterizar as ações in vivo desse veneno sobre as plaquetas, serviu-se de um modelo experimental que empregava coelhos para o envenenamento pela B. jararaca. No grupo experimental, os animais foram injetados i.v. com o veneno da B. jararaca (60 µg/kg) e no grupo controle com salina. Previamente à administração de salina ou veneno, os coelhos tiveram suas plaquetas marcadas ex vivo com NHS-biotina. Para a avaliação das alterações plaquetárias, amostras de sangue foram coletadas seqüencialmente, em intervalos de tempo que variaram de 1 a 144 horas após a administração do veneno ou salina. Durante o envenenamento, houve trombocitopenia, hipofibrinogenemia, elevação dos níveis plasmáticos do fator de von Willebrand, diminuição da função plaquetária no sangue total induzida pela botrocetina e pelo colágeno e diminuição da secreção de ATP. Não obstante, os níveis plasmáticos de fator plaquetário 4, um marcador específico da ativação plaquetária in vivo, e os níveis intraplaquetários de serotonina se mantiveram constantes. Pela citometria de fluxo, observou-se um decréscimo significativo da expressão do epítopo da GPIIb-IIIa reconhecido pelo anticorpo monoclonal P2, porém isso não foi observado ao utilizar-se anticorpos policlonais. A expressão de fibrinogênio ou dos produtos de degradação do fibrinogênio/fibrina (PDF) na membrana plaquetária também não sofreu alteração significativa ao longo do tempo. Houve, todavia, elevações significativas da P-selectina plaquetária, um receptor cuja expressão é indicativa de ativação plaquetária, e do epítopo induzido por ligantes (LIBS1) da GPIIIa. A porcentagem de plaquetas reticuladas na circulação, assim como os tempos de sobrevivência plaquetária, não foram estatisticamente diferentes entre os dois grupos. As análises histológicas e imuno-histoquímicas dos órgãos dos coelhos mostraram que as plaquetas circulantes são retidas entre redes de fibrina nos capilares pulmonares. Os resultados obtidos sugerem que a trombina engendrada pelos componentes pró-coagulantes deste veneno desempenha uma função essencial na patogenia dos distúrbios da coagulação e plaquetários observados neste modelo de envenenamento. O aumento da expressão de P-selectina no grupo experimental comprovou a hipótese inicial de que as plaquetas dos coelhos envenenados são verdadeiramente ativadas na circulação. Os dados ora apresentados demonstram definitivamente que a diminuição do fibrinogênio ou o aumento dos PDF não são a causa fundamental da disfunção plaquetária observada no envenenamento botrópico e que outro(s) composto(s) parece(m) estar envolvido(s) com estes distúrbios plaquetários. / In spite of being well established that Bothrops jararaca snake venom causes blood coagulation and fibrinolysis disturbances in patients, scant information about blood platelet disorders during envenomation is available. In recent investigations, thrombocytopenia, platelet aggregation disturbances and decreased numbers of platelet dense bodies were observed following venom administration, suggesting that circulating platelets had been activated. In order to prove this hypothesis and to gain a better characterization of the in vivo role of this venom on platelets, an experimental model of B. jararaca envenomation was utilized. Rabbits were injected i.v. either with B. jararaca venom (60 µg/kg) (experimental group) or saline (control group). Previously to saline or venom administration, rabbit platelets were labeled ex vivo with NHS-biotin. To evaluate platelet disturbances, blood samples were collected consecutively, at time intervals that varied from 1 to 144 hours after venom or saline administration. During envenomation, there were thrombocytopenia, hypofibrinogenemia, elevation of von Willebrand factor plasma levels, reduced botrocetin- and collagen-induced platelet aggregation in whole blood, and decreased ATP secretion. However, plasma levels of platelet factor 4, a specific marker of in vivo platelet activation, and intraplatelet serotonin levels remained constant. By flow cytometry, a significant decrease on the expression of GPIIb-IIIa epitope recognized by P2 monoclonal antibody was observed; however, this was not observed when polyclonal antibodies were employed. Fibrinogen or fibrin(ogen) degradation product (FDP) expression on platelet surface showed no significant alteration. Nonetheless, significant elevations of platelet P-selectin, a receptor whose expression is indicative of platelet activation, and of ligand-induced binding sites (LIBS1) of GPIIIa were noted. The percentage of circulating reticulated platelets, as well as platelet survival times, were not statistically different between the two groups. Histopathological and immunohistochemical analyses of rabbit organs demonstrated that circulating platelets were sequestered among fibrin deposits in pulmonary capillaries. These results suggest that thrombin generated by procoagulating components of B. jararaca venom has an essential role in the pathogenesis of platelet and coagulation disorders in this experimental model. Increased expression of P-selectin in the experimental group proves the initial hypothesis that platelets of envenomed rabbits are indeed activated in the circulation. The data presented herein demonstrate definitively that decreased fibrinogen or increased FDP levels are not the primary cause of the platelet dysfunction observed in bothropic envenomation, but other substances seem to be responsible for it.

Que o envenenamento pela serpente Bothrops jararaca causa distúrbios hemorrágicos sistêmicos, com alteração da coagulação e fibrinólise sangüíneas, é notório. Contudo, pouco se sabe sobre a ação in vivo desse veneno sobre as plaquetas. Em estudos recentes, demonstrou-se que esse veneno causa trombocitopenia, distúrbios da agregação e diminuição do número de corpos densos plaquetários, que, dessarte, sugeriam a ativação das plaquetas circulantes. Com o escopo de comprovar esta hipótese e melhor caracterizar as ações in vivo desse veneno sobre as plaquetas, serviu-se de um modelo experimental que empregava coelhos para o envenenamento pela B. jararaca. No grupo experimental, os animais foram injetados i.v. com o veneno da B. jararaca (60 µg/kg) e no grupo controle com salina. Previamente à administração de salina ou veneno, os coelhos tiveram suas plaquetas marcadas ex vivo com NHS-biotina. Para a avaliação das alterações plaquetárias, amostras de sangue foram coletadas seqüencialmente, em intervalos de tempo que variaram de 1 a 144 horas após a administração do veneno ou salina. Durante o envenenamento, houve trombocitopenia, hipofibrinogenemia, elevação dos níveis plasmáticos do fator de von Willebrand, diminuição da função plaquetária no sangue total induzida pela botrocetina e pelo colágeno e diminuição da secreção de ATP. Não obstante, os níveis plasmáticos de fator plaquetário 4, um marcador específico da ativação plaquetária in vivo, e os níveis intraplaquetários de serotonina se mantiveram constantes. Pela citometria de fluxo, observou-se um decréscimo significativo da expressão do epítopo da GPIIb-IIIa reconhecido pelo anticorpo monoclonal P2, porém isso não foi observado ao utilizar-se anticorpos policlonais. A expressão de fibrinogênio ou dos produtos de degradação do fibrinogênio/fibrina (PDF) na membrana plaquetária também não sofreu alteração significativa ao longo do tempo. Houve, todavia, elevações significativas da P-selectina plaquetária, um receptor cuja expressão é indicativa de ativação plaquetária, e do epítopo induzido por ligantes (LIBS1) da GPIIIa. A porcentagem de plaquetas reticuladas na circulação, assim como os tempos de sobrevivência plaquetária, não foram estatisticamente diferentes entre os dois grupos. As análises histológicas e imuno-histoquímicas dos órgãos dos coelhos mostraram que as plaquetas circulantes são retidas entre redes de fibrina nos capilares pulmonares. Os resultados obtidos sugerem que a trombina engendrada pelos componentes pró-coagulantes deste veneno desempenha uma função essencial na patogenia dos distúrbios da coagulação e plaquetários observados neste modelo de envenenamento. O aumento da expressão de P-selectina no grupo experimental comprovou a hipótese inicial de que as plaquetas dos coelhos envenenados são verdadeiramente ativadas na circulação. Os dados ora apresentados demonstram definitivamente que a diminuição do fibrinogênio ou o aumento dos PDF não são a causa fundamental da disfunção plaquetária observada no envenenamento botrópico e que outro(s) composto(s) parece(m) estar envolvido(s) com estes distúrbios plaquetários. / In spite of being well established that Bothrops jararaca snake venom causes blood coagulation and fibrinolysis disturbances in patients, scant information about blood platelet disorders during envenomation is available. In recent investigations, thrombocytopenia, platelet aggregation disturbances and decreased numbers of platelet dense bodies were observed following venom administration, suggesting that circulating platelets had been activated. In order to prove this hypothesis and to gain a better characterization of the in vivo role of this venom on platelets, an experimental model of B. jararaca envenomation was utilized. Rabbits were injected i.v. either with B. jararaca venom (60 µg/kg) (experimental group) or saline (control group). Previously to saline or venom administration, rabbit platelets were labeled ex vivo with NHS-biotin. To evaluate platelet disturbances, blood samples were collected consecutively, at time intervals that varied from 1 to 144 hours after venom or saline administration. During envenomation, there were thrombocytopenia, hypofibrinogenemia, elevation of von Willebrand factor plasma levels, reduced botrocetin- and collagen-induced platelet aggregation in whole blood, and decreased ATP secretion. However, plasma levels of platelet factor 4, a specific marker of in vivo platelet activation, and intraplatelet serotonin levels remained constant. By flow cytometry, a significant decrease on the expression of GPIIb-IIIa epitope recognized by P2 monoclonal antibody was observed; however, this was not observed when polyclonal antibodies were employed. Fibrinogen or fibrin(ogen) degradation product (FDP) expression on platelet surface showed no significant alteration. Nonetheless, significant elevations of platelet P-selectin, a receptor whose expression is indicative of platelet activation, and of ligand-induced binding sites (LIBS1) of GPIIIa were noted. The percentage of circulating reticulated platelets, as well as platelet survival times, were not statistically different between the two groups. Histopathological and immunohistochemical analyses of rabbit organs demonstrated that circulating platelets were sequestered among fibrin deposits in pulmonary capillaries. These results suggest that thrombin generated by procoagulating components of B. jararaca venom has an essential role in the pathogenesis of platelet and coagulation disorders in this experimental model. Increased expression of P-selectin in the experimental group proves the initial hypothesis that platelets of envenomed rabbits are indeed activated in the circulation. The data presented herein demonstrate definitively that decreased fibrinogen or increased FDP levels are not the primary cause of the platelet dysfunction observed in bothropic envenomation, but other substances seem to be responsible for it.