Os vírus da Influenza Equina (EIV) (H3N8 e H7N7) pertencem à família Orthomyxoviridae, gênero Influenza A. Apesar de existirem poucos relatos de infecção humana pelo EIV, é conhecido o risco zoonótico e infecção interespécies. Serviços de vigilância epidemiológica da OIE e WHO informam que o subtipo H3N8 é isolado de surtos que ocorrem mundialmente, enquanto o subtipo H7N7, menos patogênico, não é isolado desde 1980, sendo então considerado um vírus extinto. Embora o EIV seja endêmico em nosso meio, há poucos trabalhos nacionais que tenham versado sobre a avaliação atual de anticorpos (Ac) anti-EIV presentes nos equídeos do Estado de São Paulo, o que motivou a realização do presente estudo. Os objetivos do presente trabalho foram: 1) avaliar a ação de diferentes tratamentos de soro descritos pela OIE e WHO para a remoção de inibidores inespecíficos da hemaglutinação em soros de 10 equinos vacinados (H3N8 A/Equi/Kentucky/1/1997), sendo eles: a) TPH: tripsina, metaperiodato de potássio seguido de adsorção de hemácias; b) KH: kaolin 20% seguido de adsorção de hemácias; e c) RDEH: RDE seguido de adsorção de hemácias; 2) avaliar a presença de Ac contra os vírus H3N8 e H7N7, em 84 equídeos não vacinados do Estado de São Paulo; 3) comparar a frequência de Ac contra H3N8 entre equídeos amostrados do estado de SP e de um painel de soros de equídeos do município de Mossoró - RN, região onde não há estudos sobre a circulação do EIV. Constatou-se que não houve diferença estatística entre os tratamentos de soro para a remoção de inibidores inespecíficos da hemaglutinação (p>0,05; confiança de 95%), todavia o tratamento RDEH apresentou resultados mais consistentes, corroborando a recomendação da OIE e da WHO de utilizar preferencialmente este tratamento. O perfil sorológico dos animais amostrados de SP sugere que circule o subtipo H3N8 e que o subtipo H7N7 circule de forma subclínica nos equídeos, o que é sustentado por outros trabalhos realizados no Brasil. Há evidências no Brasil sobre a detecção de anticorpos em equinos contra o subtipo H7N7, mesmo não havendo o isolamento deste no mundo desde 1980. No painel de soros do RN, onde a espécie Equus asinus era maioria, verificou-se a igualdade estatística entre as frequências de Equus caballus e Equus asinus positivos no teste de HI para o subtipo H3N8 (p>0,05; confiança de 95%), dado inédito em nosso meio. A frequência dos equídeos positivos no teste de HI para o subtipo H3N8 foi estatisticamente maior (p<0,05; confiança de 95%) em SP do que em RN. / The Equine influenza Virus (EIV) (H3N8 and H7N7) belong to Orthomyxoviridae family and Influenza A genus. Although there are few reports of human infection with EIV zoonotic and interspecies infection risk is known. OIE and WHO services on epidemiological surveillance report that H3N8 subtypes are isolated and characterized from worldwide outbreaks while H7N7 subtype less pathogenic has not being isolated since 1980 considered an extinct virus. Although the EIV is endemic in our country there are few national studies that had versed on the current evaluation of horses antibodies (Ab) from São Paulo State which motivated the present study. The study objectives were: 1) to evaluate the effects of different serum treatments described by OIE and WHO for the removal of nonspecific inhibitors of hemagglutination in 10 vaccinated (H3N8 - A/Equi/Kentucky/1/1997) horses sera being: a) TPH: trypsin, potassium metaperiodate followed by adsorption of erythrocytes b) KH: kaolin 20% followed by adsorption of erythrocytes and c) RDEH: RDE followed by adsorption of erythrocytes; 2) investigate the presence of antibodies against H3N8 and H7N7 viruses in 84 unvaccinated equines in São Paulo State; 3) compare the frequency of antibodies against H3N8 sampled between São Paulo State and a panel of equines sera from Mossoró - RN where there are no studies on EIV circulation. There was no statistical difference between the treatments for serum nonspecific inhibitors of hemagglutination removal (p>0.05; 95% confidence) however RDEH treatment showed results more consistent confirming OIE and WHO recommendation to use this treatment with priority. The serological profile of SP samples suggests H3N8 subtype circulates in those animals and H7N7 subtype might circulate in a subclinical form in equines, which is supported by other studies conducted in Brazil. There is evidence of antibodies detection against equine H7N7 subtype in Brazil, even without since 1980 isolation in the world. In animals from RN State which had Equus asinus representing a major fraction there was statistical equal frequencies of Equus caballus and Equus asinus positivity in HI test against H3N8 subtype (p>0.05; 95% of confidence), as unprecedented in the world. The frequency of positive equine against H3N8 subtype on HI test in SP was statistically higher (p<0.05; 95% of confidence) than in RN.

Os vírus da Influenza Equina (EIV) (H3N8 e H7N7) pertencem à família Orthomyxoviridae, gênero Influenza A. Apesar de existirem poucos relatos de infecção humana pelo EIV, é conhecido o risco zoonótico e infecção interespécies. Serviços de vigilância epidemiológica da OIE e WHO informam que o subtipo H3N8 é isolado de surtos que ocorrem mundialmente, enquanto o subtipo H7N7, menos patogênico, não é isolado desde 1980, sendo então considerado um vírus extinto. Embora o EIV seja endêmico em nosso meio, há poucos trabalhos nacionais que tenham versado sobre a avaliação atual de anticorpos (Ac) anti-EIV presentes nos equídeos do Estado de São Paulo, o que motivou a realização do presente estudo. Os objetivos do presente trabalho foram: 1) avaliar a ação de diferentes tratamentos de soro descritos pela OIE e WHO para a remoção de inibidores inespecíficos da hemaglutinação em soros de 10 equinos vacinados (H3N8 A/Equi/Kentucky/1/1997), sendo eles: a) TPH: tripsina, metaperiodato de potássio seguido de adsorção de hemácias; b) KH: kaolin 20% seguido de adsorção de hemácias; e c) RDEH: RDE seguido de adsorção de hemácias; 2) avaliar a presença de Ac contra os vírus H3N8 e H7N7, em 84 equídeos não vacinados do Estado de São Paulo; 3) comparar a frequência de Ac contra H3N8 entre equídeos amostrados do estado de SP e de um painel de soros de equídeos do município de Mossoró - RN, região onde não há estudos sobre a circulação do EIV. Constatou-se que não houve diferença estatística entre os tratamentos de soro para a remoção de inibidores inespecíficos da hemaglutinação (p>0,05; confiança de 95%), todavia o tratamento RDEH apresentou resultados mais consistentes, corroborando a recomendação da OIE e da WHO de utilizar preferencialmente este tratamento. O perfil sorológico dos animais amostrados de SP sugere que circule o subtipo H3N8 e que o subtipo H7N7 circule de forma subclínica nos equídeos, o que é sustentado por outros trabalhos realizados no Brasil. Há evidências no Brasil sobre a detecção de anticorpos em equinos contra o subtipo H7N7, mesmo não havendo o isolamento deste no mundo desde 1980. No painel de soros do RN, onde a espécie Equus asinus era maioria, verificou-se a igualdade estatística entre as frequências de Equus caballus e Equus asinus positivos no teste de HI para o subtipo H3N8 (p>0,05; confiança de 95%), dado inédito em nosso meio. A frequência dos equídeos positivos no teste de HI para o subtipo H3N8 foi estatisticamente maior (p<0,05; confiança de 95%) em SP do que em RN. / The Equine influenza Virus (EIV) (H3N8 and H7N7) belong to Orthomyxoviridae family and Influenza A genus. Although there are few reports of human infection with EIV zoonotic and interspecies infection risk is known. OIE and WHO services on epidemiological surveillance report that H3N8 subtypes are isolated and characterized from worldwide outbreaks while H7N7 subtype less pathogenic has not being isolated since 1980 considered an extinct virus. Although the EIV is endemic in our country there are few national studies that had versed on the current evaluation of horses antibodies (Ab) from São Paulo State which motivated the present study. The study objectives were: 1) to evaluate the effects of different serum treatments described by OIE and WHO for the removal of nonspecific inhibitors of hemagglutination in 10 vaccinated (H3N8 - A/Equi/Kentucky/1/1997) horses sera being: a) TPH: trypsin, potassium metaperiodate followed by adsorption of erythrocytes b) KH: kaolin 20% followed by adsorption of erythrocytes and c) RDEH: RDE followed by adsorption of erythrocytes; 2) investigate the presence of antibodies against H3N8 and H7N7 viruses in 84 unvaccinated equines in São Paulo State; 3) compare the frequency of antibodies against H3N8 sampled between São Paulo State and a panel of equines sera from Mossoró - RN where there are no studies on EIV circulation. There was no statistical difference between the treatments for serum nonspecific inhibitors of hemagglutination removal (p>0.05; 95% confidence) however RDEH treatment showed results more consistent confirming OIE and WHO recommendation to use this treatment with priority. The serological profile of SP samples suggests H3N8 subtype circulates in those animals and H7N7 subtype might circulate in a subclinical form in equines, which is supported by other studies conducted in Brazil. There is evidence of antibodies detection against equine H7N7 subtype in Brazil, even without since 1980 isolation in the world. In animals from RN State which had Equus asinus representing a major fraction there was statistical equal frequencies of Equus caballus and Equus asinus positivity in HI test against H3N8 subtype (p>0.05; 95% of confidence), as unprecedented in the world. The frequency of positive equine against H3N8 subtype on HI test in SP was statistically higher (p<0.05; 95% of confidence) than in RN.

A finalidade desse trabalho foi a análise de aspectos estruturais e biológicos de uma lectina do veneno de B. atrox, denominada galatrox. A purificação da galatrox envolveu dois passos cromatográficos, sendo o primeiro por afinidade em coluna de agarose-lactose e o segundo referente a aplicação do material retido no gel de agarose-lactose (LacR), em coluna de Sephadex G-25. As etapas de purificação foram monitoradas por leitura de absorbância em 280nm e SDS-PAGE. Preparações de galatrox foram submetidas à digestão in situ com tripsina e a massa molecular e o sequenciamento dos peptídeos obtidos foram determinados por espectrometria de massa (MALDI-TOF-MS e ESI-CID-MS/MS). A seqüência N-terminal de aminoácidos da galatrox foi obtida pela reação automatizada de Edman (PROCISE-419®). A atividade lectínica dessa proteína foi caracterizada por meio de testes de aglutinação de eritrócitos humanos, na presença ou ausência de diferentes carboidratos como lactose (4mM) e galactose (20mM), EDTA (5mM) e aquecimento (100ºC/10min). A desgranulação mastocitária foi determinada pela liberação de -Hexosaminidase por células RBL-2H3 sensibilizadas com IgE anti-DNP(dinitrofenil) e estimuladas com galatrox, veneno bruto, fração não retida na coluna de agarose-lactose (Lac-nR), HSA-DNP (controle positivo) ou PBS (controle negativo). O nível de indução de apoptpse e/ou necrose de células RBL-2H3 tratadas com galatrox foi avaliado pela marcação com anexina V-FITC e/ou iodeto de propídeo e analisado por citometria de fluxo (FACScanto®) com o auxílio do software (CBA-DIVA®). A camptotecina foi utilizada como referência de apoptose/necrose. A atividade edematogênica foi testada em camundongos pela injeção intraplantar de galatrox; fração Lac-nR, veneno bruto e PBS. Análise por SDS-PAGE indicou que as preparações de galatrox eram homogêneas e continham bandas de 14.000 e 28.000 de massa molecular relativa em condições redutoras ou não, respectivamente. A seqüência N-terminal foi correspondente aos seguintes aminoácidos: NNXPQDWLPMNGLXYKIFD, e a seqüência de alguns aminoácidos internos corresponderam a KDFSWEWTDR e GHSEVWLGLWDK. A atividade hemaglutinante dessa lectina foi dose-dependente e inibida por EDTA (5mM), aquecimento (100ºC/10min) e lactose (32mM, 16mM, 8mM, 4mM, 2mM, 1mM, 0,5mM). Ao contrário do veneno bruto e da fração Lac-nR, esta lectina não foi edematogênica e nem promoveu apoptose e necrose de células RBL- 2H3. A galatrox induziu uma discreta desgranulação mastocitária em comparação com o veneno bruto e a fração Lac-nR. Com base nos dados obtidos sugere-se que a galatrox é uma lectina homodímérica com 28.000 de massa molecular relativa, ligante de -galactosídeos e que apresenta similaridade estrutural com outras lectinas tipo-C do veneno de Bothrops ssp. Além disso, a galatrox comportou-se como um fraco agente pró-inflamatório e não induziu efeitos apoptótico e necrótico significantes. Finalmente, esse trabalho poderá contribuir para o melhor entendimento do impacto biológico da presença de lectinas no veneno e nos envenenamentos, bem como na geração de novos produtos biotecnológicos. / The aim of this work was the analysis of structural and biological aspects of a B. atrox venom lectin, named galatrox. The galatrox purification involved two chromatographic steps, starting with an affinity column of Lactosyl-Sepharose followed by application of the retained material on a Lactosyl-Sepharose gel (LacR) in Sephadex G-25 column. The purification steps were monitored by absorbance at 280nm and SDSPAGE. Galatrox samples were submitted to digestion in situ with trypsin and the sequencing mass of the obtained peptides were determined by mass spectrometry (MALDI-TOF-MS and ESI-CID-MS/MS). The N-terminal sequence of amino acids from galatrox was obtained by automatized Edman degradation (PROCISE-491®). The lectin activity of this protein was characterized by human erytrocytes agglutination test in presence or absence of different carbohydrates as lactose (4mM) and galactose (20mM), EDTA (5mM) and heat. The mast cells degranulation was determined by -hexosaminidase release in RBL-2H3 cells sensibilized with anti-DNP IgE and challenged with galatrox, crude venom in a non retained fraction in Lactosyl-Sepharose (Lac-nR) , HAS-DNP (positive control) or PBS (negative control). The apoptosis and/or necrosi induced level in treated RBL- 2H3 cells with galatrox was evaluated using AnnexinV and/or Propidium iodide and analysed by flow citometry (FACSCanto®) with the help software (CBA-DIVA®). The camptotecin was used with apoptosi/necrosi reference. The edematogenic was tested in mice by the intraplantar injection of galatrox, Lac-nR fraction, crude venom and PBS. SDS-PAGE analyse indicated that the galatrox preparations were homogenous and contained a single band of 14,000 or 28,000 relative molecular weight showed in reducting or non-reducting conditions, respectively. N-terminal amino acid sequence was determined as following: NNXPQDWLPMNGLXYKIFD, and some internal amino acid sequence was obtained which correspond to KDFSWEWTDR and GHSEVWLGLWDK. The hemagglutination activity of this lectin was dose dependent and inhibited by EDTA (5mM), heating (100ºC/10min) and lactose (32mM, 16mM, 8mM, 4mM, 2mM, 1mM, 0,5mM). In the other hand, the crude venom and Lac-nR fraction, this lectin was not edematogenic nor promoted apoptosis and necrosi of RBL-2H3 cells. The galatrox induced a discret mast cells degranulation compared with crude venom and Lac-nR fraction. Based in obtained datas is suggested that galatrox is a homodimeric lectin with relative molecular mass of 28,000, -galactoside bindings and that shows structural similarity with other Ctype lectins from Bothrops ssp. venom. Even more the galatrox showed to be a weak proinflamatory agent and did not induced significant apoptotic and necrotic effect. Finally this work would contribute to better understanding of the biological impact that the lectin presence in venom and in poisonings as well in the generation of a new biotechnological products.

In this thesis I develop and implement “antibody landscapes”, a method to profile immunity against a pathogen as a function of antigenic differences between a range of strains. Theoretically applicable to any antigenically variable pathogen and measurement of immunity, the work here focusses on antibody-mediated immunity against the A/H3N2 influenza subtype. Applying the methodology to study annual serum samples from individuals monitored for influenza infection over a period of six years, patterns of influenza immunity were found to be remarkably distinct and maintained almost unchanged over time in the absence of influenza exposure. Upon infection, the initial response is strikingly antigenically broad, including responses against viruses far beyond the extent of cross-reactivity observed after a primary infection. Analysis of two vaccination cohorts, one receiving an antigenically advanced vaccine strain and one a more typical vaccine strain choice, revealed many of the same patterns of response as seen with infection. Antigenically advanced vaccination generated greater responses against later strains but surprisingly, due to equivalent boosting of prior immunity, this came at no cost to responses generated against contemporary or older strains. Exploring in more detail the development of immunity over time, analysis of a cohort of children demonstrated that - in contrast to adults with diverse exposure histories - antibody responses to a first infections were remarkably similar in pattern and magnitude. Interestingly, for second infections, although post-infection antibody titres against circulating strains were comparable to those after first infections, overall cross-reactivity of the response against future antigenic variants appeared to be diminished. The findings here underline the significant role prior-immunity plays in affecting the response to new exposures and the importance of understanding it. An important conclusion is that by failing to account for it, current approaches to influenza vaccine strain selection may be suboptimal and pre-emptive vaccine strain updates may improve overall vaccine efficacy where immunity to current strains already exists in the population. Building on the work presented here should help to optimise strain choice and vaccine efficacy even further.

Being among the most prevalent of persistent infectious agents in humans worldwide, Helicobacter pylori induces chronic inflammation (gastritis), which may progress to peptic ulceration and stomach cancer. The ability to adhere to the gastric mucosa is considered to be both a colonization and virulence property of H. pylori. For adherence, H. pylori expresses surface-located attachment proteins (adhesins) that bind to specific receptors in the gastric mucosa. The best characterized H. pylori adhesin-receptor interaction is that between the blood group antigen binding adhesin (BabA) and the fucosylated blood group antigens, which are glycans highly expressed in the gastric mucosa. Our recent results have changed the view of the blood group antigen-specific binding mode of H. pylori. We have tested clinical isolates of H. pylori from human populations worldwide for their ability to bind to ABO blood group antigens. The results revealed that more than 95% of isolates from Sweden, Germany, Spain, Japan and Alaska that bind fucosylated blood group antigens, bind both the Lewis b antigen (Leb) (of blood group O) and the blood group A-related antigen A-Lewis b, i.e. they exhibit a generalist type of binding mode. In contrast, the majority of strains (62%) from South American Amerindians bound best to Leb, i.e. they exhibit a specialist blood group “O antigen” binding mode. This specialization in binding coincides with the unique predominance of blood group O in the South American Amerindian populations. Furthermore, we also showed that H. pylori could switch from specialist to generalist binding modes by chromosomal integration of foreign babA gene fragments. A mutant strain lacking the babA gene turned out to adhere to inflamed gastric epithelium, despite the fact that it did not bind Leb. We identified the receptor to which the mutant binds to as the sialyl-dimeric-Lewis x antigen (sdiLex) and found its expression to be associated with persistent H. pylori infection and chronic inflammation, both in humans and Rhesus monkeys. The cognate sialic acid binding adhesin (SabA) was identified by our ReTagging technique. Deletion of sabA caused loss of H. pylori binding to sialylated glycans, and screening of single colony isolates revealed a high frequency of spontaneous on⇒off phase variation in sLex binding. Using erythrocytes as a model for sialyl dependent cell adhesion, we could show that SabA is the sought-after H. pylori sialyl-dependent hemagglutinin. Swedish clinical H. pylori isolates were analyzed for sialyl-dependent hemagglutination (sia-HA), and the sia-HA titers were found to be highly correlated to the levels of sLex binding. Clinical isolates were shown to exhibit several distinct binding modes for sialylated glycans, which suggest that SabA exhibit polymorphism in binding. We also found that SabA binds to sialylated glycans on neutrophil surfaces by mechanisms involving “selectin mimicry”, and that SabA plays an important role in nonopsonic activation of neutrophils. In the human stomach, H. pylori is exposed to selective pressures such as immune and inflammatory responses, and this is reflected by changes in mucosal glycosylation patterns. The high mutation and recombination rates of H. pylori in combination with bio selection will continuously generate clones that are adapted to changes in individual gastric mucosa. Such adaptive selection contributes to the remarkable diversity in binding modes and to the extraordinary chronicity of H. pylori infections worldwide.

Darbo tikslas: Kokybinė - kiekybinė baltymų frakcijų, praturtintų lektinais, iš Urtica dioica L. žolės, sausojo ekstrakto analizė ir antimikrobinio poveikio įvertinimas. Darbo uždaviniai: 1. Išskirti baltymų frakcijas, praturtintas lektinais, iš Urtica dioica L. šviežios ir džiovintos žolės bei sausojo ekstrakto; 2. Sausos Urtica dioica L. žolės frakcijose nustatyti baltymų dalelių dydį; 3. Įvertinti baltymų frakcijų, išskirtų iš Urtica dioica L. šviežios ir džiovintos žolės bei sausojo ekstrakto, hemagliutinacinį aktyvumą; 4. Kiekybiškai įvertinti baltymų kiekį gautose frakcijose; 5. Atlikti kiekybinę lektinų analizę pagal hemagliutinacijos titrą; 6. Įvertinti lektinais praturtintų frakcijų, išskirtų iš Urtica dioica L. džiovintos žolės bei sausojo ekstrakto, antimikrobinį aktyvumą. Darbo metodai: 1. Lektinais praturtintas baltymų frakcijas išskyrėme, taikydami tirpalų prisotinimą amonio sulfatu iki skirtingos procentinės koncentracijos; 2. Lektinai identifikuoti, pritaikius triušio eritrocitų hemagliutinacijos reakciją; 3. SDS – PAGE elektroforezės metodu nustatytas frakcijų dalelių dydis; 4. Bradfordo metodu kiekybiškai įvertinta baltymų sudėtis išskirtose frakcijose; 5. Lektinai kiekybiškai įvertinti pagal hemagliutinacinį aktyvumą; 6. Antibakterinis aktyvumas įvertintas standžiosiose Miulerio – Chintono agaro mitybos terpėse cilindriukų metodu. Tyrimo rezultatai: 1. Hemagliutinacinis aktyvumas nustatytas visose išskirtose baltymų frakcijose. 2. Didžiausiu... [toliau žr. visą tekstą] / Aim of experiment: The qualitative – quantitaive analysis and antibacterial activity evaluation of lectin enriched protein fractions from Urtica dioica L. fresh and dry herb and dry extract; Experiment tasks: 1. To extract the lectin enriched protein fractions from Urtica dioica L. fresh and dry herb and dry extract; 2. To assess the size of protein particles from Urtica dioica L. dry herb by SDS-PAGE assay; 3. To determine the hemagglutinating activity of lectin enriched fractions from Urtica dioica L. fresh and dry herb and dry extract; 4. To evaluate the protein amount in prepared fractions by Bradford assay; 5. To analyse the lectin amount by hemagglutination titre; 6. To evaluate the antibacterial activity of lectin enriched fractions from Urtica dioica L. fresh and dry herb and dry extract; Methods: 1. We used the precipitation with ammonium sulphate for the extraction of lectin enriched protein fractions; 2. Lectins were identified by hemagglutination assay; 3. We determined the size of protein particles using SDS-PAGE method. 4. We evaluated protein amount in fractions by Bradford assay; 5. Lectin quantity was evaluated by hemagglutination titer; 6. We evaluated the antibacterial activity on solid medium with Peni cylinders. Results: 1. The hemagglutinating activity was found in all protein fractions. 2. Maximum of hemagglutinating activity (2,95) was noticed in the first lectin enriched protein fraction from fresh Urtica dioica L. herb. 3. The particle size was... [to full text]

Thesis (M.Sc. (Clinical Pathology))--Mahidol University, 1978. / Supported by the National Research Council.

A finalidade desse trabalho foi a análise de aspectos estruturais e biológicos de uma lectina do veneno de B. atrox, denominada galatrox. A purificação da galatrox envolveu dois passos cromatográficos, sendo o primeiro por afinidade em coluna de agarose-lactose e o segundo referente a aplicação do material retido no gel de agarose-lactose (LacR), em coluna de Sephadex G-25. As etapas de purificação foram monitoradas por leitura de absorbância em 280nm e SDS-PAGE. Preparações de galatrox foram submetidas à digestão in situ com tripsina e a massa molecular e o sequenciamento dos peptídeos obtidos foram determinados por espectrometria de massa (MALDI-TOF-MS e ESI-CID-MS/MS). A seqüência N-terminal de aminoácidos da galatrox foi obtida pela reação automatizada de Edman (PROCISE-419®). A atividade lectínica dessa proteína foi caracterizada por meio de testes de aglutinação de eritrócitos humanos, na presença ou ausência de diferentes carboidratos como lactose (4mM) e galactose (20mM), EDTA (5mM) e aquecimento (100ºC/10min). A desgranulação mastocitária foi determinada pela liberação de -Hexosaminidase por células RBL-2H3 sensibilizadas com IgE anti-DNP(dinitrofenil) e estimuladas com galatrox, veneno bruto, fração não retida na coluna de agarose-lactose (Lac-nR), HSA-DNP (controle positivo) ou PBS (controle negativo). O nível de indução de apoptpse e/ou necrose de células RBL-2H3 tratadas com galatrox foi avaliado pela marcação com anexina V-FITC e/ou iodeto de propídeo e analisado por citometria de fluxo (FACScanto®) com o auxílio do software (CBA-DIVA®). A camptotecina foi utilizada como referência de apoptose/necrose. A atividade edematogênica foi testada em camundongos pela injeção intraplantar de galatrox; fração Lac-nR, veneno bruto e PBS. Análise por SDS-PAGE indicou que as preparações de galatrox eram homogêneas e continham bandas de 14.000 e 28.000 de massa molecular relativa em condições redutoras ou não, respectivamente. A seqüência N-terminal foi correspondente aos seguintes aminoácidos: NNXPQDWLPMNGLXYKIFD, e a seqüência de alguns aminoácidos internos corresponderam a KDFSWEWTDR e GHSEVWLGLWDK. A atividade hemaglutinante dessa lectina foi dose-dependente e inibida por EDTA (5mM), aquecimento (100ºC/10min) e lactose (32mM, 16mM, 8mM, 4mM, 2mM, 1mM, 0,5mM). Ao contrário do veneno bruto e da fração Lac-nR, esta lectina não foi edematogênica e nem promoveu apoptose e necrose de células RBL- 2H3. A galatrox induziu uma discreta desgranulação mastocitária em comparação com o veneno bruto e a fração Lac-nR. Com base nos dados obtidos sugere-se que a galatrox é uma lectina homodímérica com 28.000 de massa molecular relativa, ligante de -galactosídeos e que apresenta similaridade estrutural com outras lectinas tipo-C do veneno de Bothrops ssp. Além disso, a galatrox comportou-se como um fraco agente pró-inflamatório e não induziu efeitos apoptótico e necrótico significantes. Finalmente, esse trabalho poderá contribuir para o melhor entendimento do impacto biológico da presença de lectinas no veneno e nos envenenamentos, bem como na geração de novos produtos biotecnológicos. / The aim of this work was the analysis of structural and biological aspects of a B. atrox venom lectin, named galatrox. The galatrox purification involved two chromatographic steps, starting with an affinity column of Lactosyl-Sepharose followed by application of the retained material on a Lactosyl-Sepharose gel (LacR) in Sephadex G-25 column. The purification steps were monitored by absorbance at 280nm and SDSPAGE. Galatrox samples were submitted to digestion in situ with trypsin and the sequencing mass of the obtained peptides were determined by mass spectrometry (MALDI-TOF-MS and ESI-CID-MS/MS). The N-terminal sequence of amino acids from galatrox was obtained by automatized Edman degradation (PROCISE-491®). The lectin activity of this protein was characterized by human erytrocytes agglutination test in presence or absence of different carbohydrates as lactose (4mM) and galactose (20mM), EDTA (5mM) and heat. The mast cells degranulation was determined by -hexosaminidase release in RBL-2H3 cells sensibilized with anti-DNP IgE and challenged with galatrox, crude venom in a non retained fraction in Lactosyl-Sepharose (Lac-nR) , HAS-DNP (positive control) or PBS (negative control). The apoptosis and/or necrosi induced level in treated RBL- 2H3 cells with galatrox was evaluated using AnnexinV and/or Propidium iodide and analysed by flow citometry (FACSCanto®) with the help software (CBA-DIVA®). The camptotecin was used with apoptosi/necrosi reference. The edematogenic was tested in mice by the intraplantar injection of galatrox, Lac-nR fraction, crude venom and PBS. SDS-PAGE analyse indicated that the galatrox preparations were homogenous and contained a single band of 14,000 or 28,000 relative molecular weight showed in reducting or non-reducting conditions, respectively. N-terminal amino acid sequence was determined as following: NNXPQDWLPMNGLXYKIFD, and some internal amino acid sequence was obtained which correspond to KDFSWEWTDR and GHSEVWLGLWDK. The hemagglutination activity of this lectin was dose dependent and inhibited by EDTA (5mM), heating (100ºC/10min) and lactose (32mM, 16mM, 8mM, 4mM, 2mM, 1mM, 0,5mM). In the other hand, the crude venom and Lac-nR fraction, this lectin was not edematogenic nor promoted apoptosis and necrosi of RBL-2H3 cells. The galatrox induced a discret mast cells degranulation compared with crude venom and Lac-nR fraction. Based in obtained datas is suggested that galatrox is a homodimeric lectin with relative molecular mass of 28,000, -galactoside bindings and that shows structural similarity with other Ctype lectins from Bothrops ssp. venom. Even more the galatrox showed to be a weak proinflamatory agent and did not induced significant apoptotic and necrotic effect. Finally this work would contribute to better understanding of the biological impact that the lectin presence in venom and in poisonings as well in the generation of a new biotechnological products.

A galectina-1 (Gal-1) é uma lectina homodimérica multifuncional capaz de reconhecer e se ligar a beta-galactosídeos por meio de um domínio denominado carbohydrate recognition domain (CRD). A Gal-1 humana (Gal-1h) e a Gal-1 de camundongo (Gal-1c) mantêm 88,15% de homologia e, apesar de não existirem mutações em aminoácidos-chave do CRD, há substituições próximas a esses resíduos. Considerando as implicações dessas diferenças em estrutura e função, e que é comum a utilização de modelos murinos para estudar a função Gal-1, o presente trabalho objetiva analisar comparativamente a Gal-1c e a Gal-1h por meio de ensaios de cristalização e determinação estrutural da Gal-1c, além da avaliação comparativa da atividade lectínica da Gal-1h e da Gal-1c por glycan array e hemaglutinação. Também foi avaliada a capacidade de ambas as Gal-1 em induzir a exposição de fosfatidilserina (FS) em neutrófilos ativados provenientes de medula de camundongos normais ou deficientes de ?-2 integrina (Mac-1), de modo a investigar se a interação Gal-1/Mac-1 estaria envolvida nesse processo. Preparações homogêneas e ativas de Gal-1c e Gal-1h foram utilizadas nos ensaios. Os cristais de Gal-1c foram obtidos em 20% de polietilenoglicol 3350 e 0,2 M de fluoreto de amônio. Os dados de difração de raios X foram coletados e processados, obtendo-se uma estrutura com resolução de 2,4 Å. Observou-se que substituições de aminoácidos entre a Gal-1c e a Gal-1h estão localizadas em regiões expostas ao solvente, próximas do CRD e distantes da interface de dimerização. A análise comparativa entre Gal-1c e Gal-1h mostrou que estas substituições conferem a Gal-1c um caráter mais polar, com consequente aumento da distribuição de volume molecular. Nos ensaios de hemaglutinação, pode-se observar que é necessária uma concentração 2 vezes maior de Gal-1c para aglutinar eritrócitos humanos, de carneiro e de coelho na mesma proporção que a Gal-1h. Por meio do glycan array, pode-se determinar o perfil de ligação a glicanas de ambas as Gal-1. As duas Gal-1 apresentam afinidade por glicanas ramificadas contendo galactose terminal, e a Gal-1h apresentou maior intensidade de ligação às glicanas quando comparada à Gal-1c. Preparações de Gal-1c e Gal-1h induzem níveis semelhantes de exposição de FS na superfície de neutrófilos deficientes ou não de Mac-1, sugerindo que a interação Gal-1/Mac-1 não esteja envolvida no processo de exposição de FS na superfície de neutrófilos ativados. Assim, a diferença sequencial entre a Gal-1c e a Gal-1h é capaz de gerar diferenças estruturais consideráveis que implicam no reconhecimento diferencial de glicanas, o que, entretanto, não se reflete na capacidade de indução de FS na superfície de neutrófilos ativados. Além disso, a interação Gal-1/Mac-1 parece não participar desse processo, o que pode indicar que o papel da Gal-1 no turnover de neutrófilos, via reconhecimento fagocítico, seja um processo complexo e independente dessa interação. / Galectin-1 (Gal-1) is a homodimeric and multifunctional lectin that recognizes and binds to beta-galactoside by a carbohydrate recognition domain (CRD). Human Gal-1 (hGal-1) and mouse Gal-1 (mGal-1) are 88.15% identical, and although there are no mutations in key amino acids within the CRD, there are differences in the amino acids sequence near the CRD. Given the potential of these differences to alter overall structure and function, and the common utilization of murine models to study Gal-1 function, we sought to directly compare key biochemical features of hGal and mGal-1. Thus, we performed crystallization and structure determination assays of mGal-1, and determined the carbohydrate binding specificy of mGal-1 and hGal-1 using a glycan array and using hemagglutination assay. We also evaluated the ability of both Gal-1 to induce exposure of phosphatidylserine (PS) in activated neutrophils from the bone marrow of normal or ?-2 integrin (Mac-1) deficient mice, in order to investigate the involvement of Gal-1/Mac-1 interaction in this process. To accomplish this, homogeneous and active preparations of hGal-1 and mGal-1 were used in the study. mGal-1 crystals were obtained in 20% polyethylene glycol 3350 and 0.2 M ammonium fluoride. Data from X-ray diffraction were collected and processed, yielding a structure with a final resolution of 2.4 Å. The amino acid substitutions found between mGal-1 and hGaI-1 are detected on the solvent-exposed surfaces where the CRDs are located and not on the proteins dimerization surfaces. A comparative structural analysis between mGal-1 and hGal-1 shows that these amino acid substitutions confer to mGal-1 a greater number of ionizable residues, polar character, appearance of the acid regions clustered, and a slight increase of volume distribution. In hemagglutination assays, twice the concentration of mGal-1 was required to cause equivalent agglutination of human, sheep or rabbit erythrocytes as hGal-1. Glycan array analysis demonstrated that both galectins have affinity for branched glycans containing terminal galactose residues. However, hGal-1 appeared to display higher levels of binding that mGal-1. Preparations of mGal-1 and hGal-1 induced similar levels of PS exposure on normal or Mac-1 deficient neutrophils, suggesting that the interaction Gal-1/Mac-1 is not involved in this process. Thus, hGal-1 and mGal-1 appear to possess considerable differences in glycan recognition that likely reflects subtle difference in amino acid sequence. Furthermore, the interaction Gal-1/Mac-1 do not appear to participate in this PS exposure process, which suggest that other Gal-1 receptors are likely important in this process.

<p>U radu je ispitana zastupljenost influenca A virusa, njihova antigenska i genetička svojstva i osetljivost na antivirotik oseltamivir.</p><p>Ispitivanje je sprovedeno u toku četiri uzastopne sezone, od 2010/2011 do 2013/2014 &nbsp;i obuhvatilo je 887 briseva nosa i grla pacijenata sa simptomima gripa, sa teritorije Južnobačkog okruga. Svi uzorci su&nbsp;testirani na prisustvo influenca A(H1N1)pdm09, A(H3N2), A(H1N1), A(H5) i A(H7) i influenca B virusa, real-time RT PCR testom. Pozitivni uzorci iz sezona 2012/2013 i 2013/2014, podvrgnuti su izolaciji na MDCK ćelijskim kulturama, a zatim je izvr&scaron;eno ispitivanje sposobnosti dobijenih izolata da aglutiniraju eritrocite koko&scaron;ke, čoveka i zamorca u reakciji virusne hemaglutinacije. Antigenska svojstva izolata sa hemaglutinacionim titrom &ge;40, ispitana su reakcijom inhibicije hemaglutinacije. Genetičkoj karakterizaciji, sekvenciranjem hemaglutinin i neuraminidaza gena, podvrgnuti su reprezentativni izolati iz sezona 2012/2013 i 2013/2014. Za ispitivanje osetljivosti odabranih izolata virusa na oseltamivir upotrebljen je hemiluminiscentni test inhibicije aktivnosti neuraminidaze.</p><p>Ukupno 46,3% (411/887) uzoraka bilo je influenca pozitivno, od čega je 73% (300/411) bilo influenca A pozitivno, a 27% (111/411)influenca B pozitivno (p&lt;0,0001). &nbsp;Influenca A(H1N1)pdm09 podtip je detektovan u 48% (144/300), a A(H3N2) podtip u&nbsp;52% (156/300) influenca Apozitivnih uzoraka. Najveći procenat influencaA pozitivnih zabeležen je u uzrastnoj grupi 5-14 godina (48,2%, 77/160) i kod pacijenata sa lak&scaron;im kliničkim manifestacijama gripa (43,7%, 153/350).</p><p>Influenca A(H1N1)pdm09 podtip preovladavao je u uzrastnoj grupi 15-29 godina (66%, 31/47, p=0,0400) i 30-64 godina (55,9%,71/127, p=0,0215), kao i kod pacijenata sa te&scaron;kom akutnom respiratornom bole&scaron;ću (63,5%, 80/126, p&lt;0,0001), fatalnih slučajeva (100%,9/9, p=0,0039) i pacijenata sa hroničnim bolestima i stanjima (68,8%, 84/122, p&lt;0,0001).&nbsp;</p><p>Influenca A(H3N2) podtip dominirao je kod dece uzrasta do 4 godine (72,2%,13/18, p=0,0381) i 5-14 godina (75,3%, 58/77, p&lt;0,0001), kod pacijenata sa lak&scaron;im oblikom bolesti (69,3%,106/153, p&lt;0,0001) i bez hroničnih bolesti ili stanja (66,3%, 118/178,&nbsp;p&lt;0,0001).</p><p>Najznačajniji predikcioni faktori komplikacija influence bili su: prisustvo hroničnih bolesti ili stanja i uzrast &ge;15 godina. Prisustvo hroničnih bolesti ili stanja nosilo je 34 puta, a uzrast &ge;15 godina 10 puta veći rizik od nastanka te&scaron;kih oblika bolesti.</p><p>Izolacija influenca virusa na MDCK ćelijskim kulturama, bila je uspe&scaron;na u 34,3% (70/204) slučajeva, pri čemu je u grupi uzoraka sa real-time RT-PCR Ct vrednostima &lt;30 ona iznosila 80,5% (62/77), kod uzoraka sa Ct vrednostima 30-34 svega 8,7% (8/92), a izolacija iz uzoraka sa Ct vrednostima &gt;34 nije bila moguća. U reakciji hemaglutinacije, najbolji rezultati su postignuti sa eritrocitima zamorca, koje je u titru &ge;40 aglutiniralo 56% (14/25) A(H1N1)pdm09 virusa i 62,5% (15/24) A(H3N2) virusa. Sa humanim eritrocitima dobar titar dalo je 16% (4/25) influenca A(H1N1)pdm09 i 8,3% (2/24) A(H3N2) virusa, a sa koko&scaron;ijim eritrocitima 8% (2/25) A(H1N1)pdm09 virusa i nijedan virus A(H3N2) podtipa.</p><p>Rezultati antigenske karakterizacije pokazali su da je svih 23 influenca virusa A(H1N1)pdm09 podtipa, iz sezona 2012/2013 i 2013/2014, antigenski bilo slično referentnom, vakcinalnom virusu A/California/7/2009. Nasuprot tome, samo 1 od 7 ispitanih A(H3N2) virusa iz sezone 2012/2013, antigenski je bio sličan vakcinalnom virusu A/Victoria/361/2011, a samo 2 od 20 iz sezone 2013/2014 antigenski je bilo slično vakcinalnom A/Texas /50/2012 virusu.</p><p>Filogenetska analiza hemaglutinin gena influenca A(H1N1)pdm09 virusa iz sezone 2012/2013, pokazala je da su u na&scaron;oj sredini, bili prisutni virusi iz dve različite genogrupe, 6C i 7, dok su naredne sezone svi analizirani virusi pripadali genogrupi 6B. Virusi iz na&scaron;e sredine bili su filogenetski srodni A(H1N1)pdm09 virusima iz drugih evropskih zemalja. Svi ispitani A(H3N2) virusi iz sezone 2012/2013 i2013/2014, pripadali su genetičkoj grupi&nbsp; 3C.3.Filogenetski su bili srodni sa virusima iz drugih gografskih regiona Evrope.</p><p>Svih 20 izolata influenca A(H1N1)pdm09 podtipa i 23 A(H3N2) podtipa pokazali su normalnu inhibiciju aktivnosti neuraminidaze pod dejstvom oseltamivira.ekvenciranje neuraminidaza gena jednog A(H3N2) virusa, koji je imao 8 puta redukovanu inhibiciju aktivnosti neuraminidaze oseltamivirom, ukazalo jena prisustvo retke mutacije Q391H, povezane sa rezistencijom na inhibitore neuraminidaze.</p><p>Rezultati ovog rada ukazali su na značaj influenca A virusa kao etiolo&scaron;kih uzročnika akutnih respiratornih obolenja u na&scaron;oj sredini, naročito za osobe sa hroničnim bolestima koje su pod povećanim rizikom od razvoja te&scaron;kih oblika gripa. U ovom istraživanju stečena su i saznanja koja imaju praktičnu primenu u postupku antigenske karakterizacije influenca A virusa, koja je jedna od ključnih faza u procesu pripreme vakcine protiv gripa. Značajna antigenska razlika A(H3N2) virusa koji su cirkulisali u sezonama 2012/2013 i 2013/2014 u odnosu na viruse koji su bili u sastavu vakcina u datim sezonama, ukazala je na neophodnost unapređenja proizvodnje vakcine protiv gripa. Dobijeni su i prvipodaci orezistenciji na antivirotik oseltamivir, kao i o filogenetskim odnosima i genetičkim grupama virusa koji su&nbsp; cirkulisali u na&scaron;oj sredini.</p> / <p>In this study we investigated the representation, antigenic and genetic properties, and sensitivity to antiviral drug oseltamivir of influenza A viruses. The study was conducted&nbsp; during 4 consecutiveseasons 2010/2011 - 2013/2014, and included 887 nasal and throat swabs taken from patients with influenza-like symptoms from South&nbsp; Backa district. All samples were tested for influenza A(H1N1)pdm09, A(H3N2), A(H1N1), A(H5), A(H7) and influenza B viruses, by real-time RT-PCR. Isolation on MDCK cell culture was performed with positive samples from seasons 2012/2013 and 2013/2014, and virus isolates were tested for ability&nbsp; to agglutinate guinea pig, chicken and human red blood cells in reaction of virus hemagglutination. Antigenic properties of isolates with hemagglutination titre &ge;40, were investigated using reaction&nbsp; of hemagglutination inhibition. Genetic characterization was performed by sequencing of neuraminidase and hemagglutination genes of representative isolates from seasons 2012/2013 and 2013/2014. Testing for sensitivity to oseltamivir was done with chemiluminescent neuraminidase inhibition assay.</p><p>Total of 46,3% (411/887) of samples were influenza positive, out of which 73% (300/411) were influenza A positive and 27% (111/4111, p&lt;0,0001) were influenza&nbsp; B positive. Influenza A(H1N1)pdm09 subtype was detected in 48% (144/300), and A(H3N2) subtype in 52% (156/300) of influenza A positive samples. The highest proportion of influenza A positive samples wasfound in age group 5-14&nbsp; years (48,2%,&nbsp; 77/160) and among patients with uncomplicated influenza (43,7%, 153/350).</p><p>Influenza A(H1N1)pdm09 subtype predominated in age group 15-29 years (66%, 31/47, p=0,0400) and 30-64 years (55,9%,71/127, p=0,0215), in patients with severe acute respiratory illness (63,5%, 80/126, p&lt;0,0001), in fatal cases (100%, 9/9, p=0,0039), and among patients with underlying chronic diseases and conditions (68,8%,84/122, p&lt;0,0001).</p><p>Influenza A(H3N2) subtype predominated in age group &le;4 years (72,2%, 13/18, p=0,0381) and 5-14 years (75,3%,58/77, p&lt;0,0001), in patients with mild form of influenza (69,3%,106/153, p&lt;0,0001), and in group of patients without chronic diseases and conditions (66,3%,60/478, p&lt;0,0001).</p><p>The most significant risk factors for severe influenza were: the presence of underlying diseases and conditions and age &ge;15 years. Patients with chronic illnesses and conditions had 34 times higher and patients &ge;15 years of age 10 times higher risk from severe influenza.</p><p>Isolation rate of influenza A viruses in MDCK cell cultures was 34,3% (70/204). For samples with real time RT-PCR Ct values &lt;30 isolation rate was 80,5% (62/77), for samples with Ct values 30-34 it was 8,7% (8/92), while isolation of viruses from samples with Ct values &gt;34 was not successful. In the reaction of virus hemagglutination, the best results were achieved with guinea pig red blood cells which agglutinated in titre &ge;40, 56% (14/25) of influenza A(H1N1)pdm09 viruses and&nbsp; 62,5% (15/24) of A(H3N2) viruses. With human erythrocytes, good titre gave 16% (4/25) of influenza A(H1N1)pdm09 and 8,3% (2/24)of A(H3N2) viruses and with chicken erythrocytes 8% (2/25) A(H1N1)pdm09 viruses and none of the A(H3N2) viruses.</p><p>Results of the antigenic characterization of 23 influenza A(H1N1)pdm09 viruses, showed that they were antigenically similarto referent, vaccine virus A/California/7/2009. On the contrary, only 1 out of 7 influenza A(H3N2) viruses from season 2012/2013,was antigenically similar to A/Victoria/361/2011 vaccine virus, and only 2 out of 20 from season 2013/2014 were antigenically similar to A/Texas/50/2012&nbsp; vaccine virus.</p><p>Filogenetic analysis of hemagglutinin genes indicated co-circulation of 2 distinct genetic groups, 6C and 7, of A(H1N1)pdm09 viruses during the season 2012/2013, while during the season 2013/2014 all tested viruses were from genetic group 6B. Influenza A(H1N1)pdm09 viruses from our region, were closely related to viruses from other European countries. All influenza A(H3N2) viruses from season 2012/2013 and 2013/2014 belonged to genetic clade 3C.3 and were closely related to viruses from different European countries.</p><p>Total of 20 A(H1N1)pdm09 isolates and 23 A(H3N2) isolates were tested for sensitivity to oseltamivir, and all of them showed normal inhibition of neuraminidase activity with oseltamivir. Sequencing of&nbsp; neuraminidase gene of one A(H3N2) virus with 8-fold reduced inhibition by oseltamivir, revealed rare mutation Q391H associated with antiviral resistance.</p><p>Results of this study indicate the significance of influenza A viruses as etiological factors of acute respiratory diseases in our area, especially for persons with chronic medical conditions who are at higher risk for severe influenza. Data gathered during&nbsp;the process of virus isolation and investigation of hemagglutination abilities of&nbsp; isolated viruses, have practical application in antigenic testing of influenza A viruses which is one of the key points of process of anti-flu vaccine production. Significant &nbsp;antigenic difference between influenza A(H3N2) viruses from seasons 2012/2013 and&nbsp; 2013/2014 and vaccine viruses, emphasis the importance of vaccine production improvement. During this study, the first data about antiviral resistance, filogenetic relationships and genetic groups of influenza viruses from our region, were obtained.</p>