Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

A doença hemolítica perinatal (DHPN) é caracterizada pela destruição das hemácias fetais por anticorpos da classe IgG presentes na circulação materna. Esses anticorpos, dirigidos contra antígenos eritrocitários presentes nas hemácias do feto, atravessam a barreira placentária e promovem a hemólise prematura dos eritrócitos, podendo levar à anemia fetal. O presente trabalho teve como objetivo principal determinar a prevalência dos anticorpos irregulares em gestantes atendidas em maternidades públicas da hemorrede de Santa Catarina por meio de um estudo observacional, com coorte retrospectiva, entre janeiro de 2012 a dezembro de 2015, englobando a análise de dados arquivados no laboratório de Imunohematologia do Centro de Hematologia e Hemoterapia de Santa Catarina, situado na cidade de Florianópolis. As variáveis investigadas nas amostras de cordão e de recém nascido (RN) foram: Anticorpo(s) materno(s) identificado(s) no teste de eluato de amostras com teste de antiglobulina direto positivo; fenotipagem ABO/RhD da mãe do RN. No caso das amostras de gestantes com suspeita de aloimunização e mães dos RNs, avaliaram-se as seguintes variáveis: anticorpo(s) antieritrocitário(s) identificado (s); fenotipagem ABO/RhD da gestante. O número total de casos analisados ao longo dos 4 anos foi equivalente a 585, sendo que o número de casos de anticorpos irregulares maternos na população estudada foi equivalente a 45. A distribuição do total de anticorpos irregulares maternos (TAIM) para cada ano demonstrou forte tendência de crescimento linear (coeficiente de correlação linear igual a 0,9311). Os dados coletados apontaram as seguintes especificidades de anticorpos, citados de acordo com a ordem decrescente de distribuição de frequência : anti-D (57,8%); anti-D,-C (11,1%); anti-c (6,7%); anti-c,-E (4,4%); anti-D,-E (4,4%); anti-M (4,4%); anti-C (2,2%); anti-E (2,2%); anti-K (2,2%); anti-Fy(b) (2,2%); anti-Jk(a) (2,2%) com prevalência de 41% do anti-D sob as outras classes de anticorpos. A associação entre o fenótipo RhD da gestante e a realização do teste de Pesquisa de Anticorpos Irregulares (PAI) demonstrou que o fato do fenótipo RhD ser positivo ou negativo não influencia na execução do teste (p=0,1672; Coeficiente de Cramer equivalente a 0,1783), entretanto, foi possível observar uma predominância do número de casos de testes de PAI não realizados sob os realizados, em especial em gestantes RhD positivo. Os resultados encontrados contribuíram para a elaboração do material clínico na forma de cartilha intitulado: DHPN - DOENÇA HEMOLÍTICA PERINATAL E TESTES IMUNOHEMATOLÓGICOS PARA DIAGNÓSTICO LABORATORIAL. A cartilha foi elaborada na forma de perguntas e respostas englobando principalmente esclarecimentos sobre os testes imunohematológicos que auxiliem no diagnóstico da DHPN. / Perinatal hemolytic disease is characterized by the destruction of fetal red blood cells by IgG antibodies present in the maternal circulation. These antibodies directed against erythrocyte antigens present in red blood cells of the fetus, cross the placental barrier and promote the premature haemolysis of red blood cells and can lead to fetal anemia. This study aimed to determine the prevalence of irregular antibodies in pregnant women in public hospitals in hemorrede of Santa Catarina through an observational study with retrospective cohort from January 2012 to December 2015, involving the analysis of archived data in Immunohematology laboratory of Center of Hematology and Hemoterapy of Santa Catarina, located in Florianópolis. The variables to be investigated in the cord samples and newborn (NB) were: maternal antibody identified in eluate test of samples with a positive direct antiglobulin test; phenotyping ABO / RhD of newborn`s mother. For samples of pregnant women with suspected alloimmunization and mothers of newborns, the following variables will be evaluated: Antierythrocyte(ies) antibody(ies) identified; phenotyping ABO / RhD of the pregnant woman. The total number of cases analyzed over the four years was equivalent to 585, and the number of cases of maternal irregular antibodies in the population studied was equivalent to 45. The distribution of total maternal irregular antibodies (TMIA) for each year showed strong linear growth trend (linear correlation coefficient of 0.9311). The data collected showed the following specific antibodies, cited according to the descending order of frequency distribution: Anti-D (57.8%); anti-D, -C (11,1%); anti-c (6.7%); anti-c E (4.4%); anti-D, E (4.4%); Anti-M (4.4%); Anti-C (2.2%); Anti-E (2.2%); anti-K (2.2%); anti-Fy (b) (2.2%); antiJk(a) (2.2%) with a prevalence of 41% of anti-D in relation to other antibody specificities. The association between RhD phenotype of pregnant women and the realization of Irregular antibody screening test (PAI) showed that the fact of the RhD phenotype be positive or negative does not influence the test execution (p = 0.1672; Cramer coefficient equivalent to 0.1783), however, it was possible to observe a predominance of the number of cases of PAI tests not performed under performed, especially in RhD positive pregnant women. The results contributed to the development of the clinical material in the form of spelling book entitled: DHPN - PERINATAL HEMOLYTIC DISEASE AND IMMUNOHEMATOLOGICAL TESTS FOR LABORATORY DIAGNOSTICS. The spelling book was prepared in the form of questions and answers covering mainly clarifications on immunohematological tests to assist in the diagnosis of DHPN.

A piometra é uma condição mórbida caracterizada pela inflamação do útero com acúmulo de exsudatos, resultante de ações hormonais e geralmente associada à presença de bactérias no lúmen uterino. A anemia é a alteração hematológica mais frequentemente observada em cadelas com piometra e está associada à cronicidade da doença, diminuição da eritropoiese, devido ao efeito toxêmico na medula óssea, diminuição da disponibilidade de ferro ou perda de sangue para o útero. Adicionalmente, o efeito das toxinas bacterianas e os radicais livres gerados pelo metabolismo oxidativo dos neutrófilos podem resultar na modificação da estrutura antigênica da membrana do eritrócito, permitindo a ligação de imunoglobulinas em sua superfície e acelerando a destruição eritrocitária. Essa hipótese pode ser comprovada pela detecção de imunocomplexos na superfície eritrocitária de cadelas com piometra. O diagnóstico de piometra foi estabelecido em 33 cadelas atendidas no Serviço de Obstetrícia/Ginecologia do Hospital Veterinário da Universidade de São Paulo com base na anamnese, exame físico e exames subsidiários (ultrassonografia, hemograma e concentrações séricas de ureia e creatinina). As amostras sanguíneas foram coletadas em dois momentos. A primeira anterior a ovariosalpingohisterectomia (OSH) e a segunda, sete a dez dias após a OSH. A quantificação de hemácias com deposição de imunocomplexos IgG e IgM foi realizada utilizando-se anticorpos anti-IgG e anti-IgM (Bethyl®Laboratories) conjugadas a fluoresceína de isotiocianato (FITC), e a leitura realizada com citômetro de fluxo (FACS Calibur; Becton, Dickinson and Company© 2007 BD), sendo os resultados expressos em percentual de hemácias marcadas. Foram utilizados o Teste de Shapiro-Wilk para a avaliação da distribuição de dados e a comparação entre os grupos controle, pré e pós-OSH foi realizada valendo-se do Teste t ou Teste t pareado e Correlação de Pearson, e do Teste U de Mann-Whitney e Correlação de Spearman, para as variáveis com distribuição normal e não-normal, respectivamente. O valor de alfa estipulado foi de 0,05. Analisando os valores hematológicos de cada um dos cães incluídos no estudo, observa-se que 19 (57,6%) apresentavam anemia normocítica normocrômica não regenerativa no momento pré-OSH e cinco (15,2%) no momento pós-OSH. Em cães do grupo controle foram observadas 0,14 - 0,77% (0,43±0,18%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (0,68±0,29%) para anticorpos anti-IgM FITC. Já nos cães com piometra, foram encontradas 0,14 - 4,19% (0,96±0,86%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (1,37±1,71%) com anticorpos anti-IgM FITC, antecedendo a OSH. No momento pós-OSH observou-se 0,18 - 16,2% (2,77±3,67%) de hemácias marcadas para anticorpos anti-IgG FITC e 0,15 - 19,8% (4,01±4,46%) para anticorpos anti-IgM FITC. O percentual de hemácias marcadas com anticorpos anti-IgG FITC diferiu entre os grupos controle e piometra, pré-OSH (p<0,001) e pós-OSH (p<0,001). Em relação a anticorpos anti-IgM FITC, não foram observadas diferenças entre os grupos controle e pré-OSH (p=0,09), porém, após a OSH houve aumento na marcação de hemácias, quando comparado ao grupo controle (p<0,001). Apenas alguns animais apresentaram mais de 5% de hemácias marcadas, e isto ocorreu, principalmente, no momento pós-OSH. Entretanto, não resultou no agravamento da anemia, indicando que a piometra em cadelas está associada à deposição de imunoglobulinas G ou M na superfície das hemácias, sem, no entanto, promover hemólise ou agravamento da anemia / The pyometra is a morbid condition characterized by inflammation of the uterus with exudates accumulation resulting from hormonal action and usually associated with the presence of bacteria in the uterine lumen. Anemia is the hematologic changes most frequently observed in bitches with pyometra and is associated with chronic disease, diminished erythropoiesis due to toxemic effect on the bone marrow, decreased iron availability or loss of blood to the uterus. Additionally, the effect of bacterial toxins and free radicals generated by neutrophil oxidative metabolism may result in the modification of antigenic structure of the erythrocyte membrane, allowing the binding of immunoglobulins on their surface and accelerating erythrocyte destruction. This hypothesis can be confirmed by detection of immune complexes in the erythrocyte surface dogs with pyometra. The diagnosis of pyometra was established in 33 dogs attended by the Obstetrics Service/Gynecology of Veterinary Hospital of the University of São Paulo based on history, physical examination and additional tests (ultrasound, blood count and serum concentrations of urea and creatinine). Blood samples were collected in two stages. The first prior to ovariohysterectomy (OSH) and the second, seven to ten days after OSH. Quantification of erythrocyte with deposition of IgG and IgM immune complexes was performed using antibodies anti-IgG and anti-IgM (Bethyl® Laboratories) conjugated to fluorescein isothiocyanate (FITC), and the reading performed with flow cytometry (FACS Calibur; Becton, Dickinson and Company© 2007 BD), and the results expressed as a percentage of red blood cells marked. The Shapiro-Wilk test was used to assess the distribution of data and the comparison between the control group, pre- and post-OSH was carried out making use of the t test or paired t test and Pearson correlation, and test U Mann-Whitney and Spearman correlation for the variables with normal and non-normal distribution, respectively. The stipulated alpha value was 0.05. The analysis of the hematological values of each of the dogs enrolled in the study, it was observed that 19 (57.6%) with anemia normocytic normochromic non-regenerative in the pre-OSH moment and 5 (15.2%) in the post-OSH moment. In control group of dogs were observed from 0.14 to 0.77% (0.43± 0.18%) of red blood cells marked with antibodies anti-IgG FITC and 0.29 to 9.58% (0.68± 0.29%) for antibodies anti-IgM FITC. Already in dogs with pyometra were found 0.14 to 4.19% (0.96 ± 0.86%) of red blood cells marked with antibodies anti-IgG FITC and 0.29 to 9.58% (1.37 ± 1.71%) with antibodies anti-IgM FITC, prior to OSH. In the post-OSH time it was observed 0.18 to 16.2% (2.77 ± 3.67%) of red blood cells marked for antibodies anti-IgG FITC and 0.15 to 19.8% (4.01 ± 4.46%) for antibodies anti-IgM FITC. The percentage of red blood cells marked with antibodies anti-IgG FITC differ between groups control and pyometra, pre-OSH (p<0.001) and post-OSH (p<0.001). Regarding the antibodies anti-IgM FITC, no differences were observed between the control and pre-OSH (p=0.09), however, after the OSH was no increase in labeling of red blood cells compared to the control group (p<0.001). Only a few animals have more than 5% of marked red blood cells, and this was mainly in the post-OSH time. However, not result in the worsening of anemia, indicating that pyometra in dogs is related to the deposition of immunoglobulin G or M on the surface of erythrocytes, without, however, promote hemolysis or worsening of anemia

A Síndrome Hemolítica Urêmica (SHU) é a principal doença associada à infecção com linhagens de Escherichia coli produtoras de toxina de Shiga (Stx), doença para qual não há uma vacina ou tratamento específico. A toxina Stx é formada por uma subunidade A enzimaticamente ativa e uma B pentamérica responsável pela ligação da toxina na célula hospedeira. Neste trabalho propomos o uso de Bacillus subtilis, uma bactéria não patogênica e formadora de esporos, como veículo vacinal para a expressão de formas atóxicas da Stx2, sob o controle de um promotor induzível por estresse (PgsiB). Camundongos BALB/c imunizados com células vegetativas ou esporos das linhagens vacinais de B. subtilis, por diferentes vias, induziram baixos níveis de anticorpos anti-Stx em soro (IgG) e fezes (IgA). Avaliamos também o potencial imunogênico da Stx gerada em linhagens recombinates de E. coli, mas os anticorpos gerados não foram capazes de neutralizar a toxina nativa. Os resultados indicam que formas alternativas de expressão e/ou o uso de adjuvantes são necessárias para gerar formulações vacinais eficazes contra a SHU. / The Hemolytic Uremic Syndrome (HUS) is the main disease associated with infections with Shiga toxin (Stx) - producing Escherichia coli strain and no effective vaccine or treatment exist. The Stx toxin consist of an enzymatically active A subunit and a pentameric B subunit responsible toxin binding to host cells. In this work we propose the use of Bacillus subtilis, a harmless spore form bacteria as a vaccine vehicle for the expression atoxic forms of Stx2, under the control of stress inducible (PgsiB) promoter. BALB/c mice immunized with vegetative cells and spores of the B. subtilis vaccine strain using different immunization routes elicited low specific antibody levels at serum (IgG) or fecal extracts (IgA). We also investigated the immunogenic potencial of StxB purified from recombinant E. coli strain, but the induced anti-StxB antibodies did not neutralize the native toxin. The results indicate that alternative expression system or the incorporation of the adjuvants are required for the generation of vaccine formulation active against HUS.

Membros termofílicos do gênero Campylobacter são reconhecidos como importantes enteropatógenos para o ser humano e animais. A grande diversidade ecológica destes microorganismos em diferentes habitats tais como: água, animais e alimentos predispõem ao aparecimento de novos fatores de virulência. Este trabalho teve por objetivo detectar os genes codificantes da Toxina Distensiva Citoletal (CDT) por meio da técnica de PCR, pesquisar a atividade de hemolisinas e a influência de soluções quelantes e de íons nesta atividade. Foram utilizadas 45 amostras de C. jejuni de origem avícolas para pesquisa de atividade hemolítica, cultivadas em Caldo Triptona de Soja (TSB). Após o crescimento bacteriano, as amostras foram semeadas em Ágar tríptico de soja (TSA) contendo 5% de sangue de ovino, equino e bovino, sendo cada sangue testado isoladamente. Para verificar a influência de agentes quelantes e solução de íons na atividade hemolítica, as amostras de C. jejuni foram cultivadas em TSB contendo separadamente os quelantes EDTA, ácido acético, soluções de íons CaCl2 , MgCl2 e FeCl3, em atmosfera de microaerofilia. Quanto à atividade de hemolisina de Campylobacter jejuni em placas de TSA – sangue, foi possível observar que houve hemólises em 48,89% das amostras quando utilizado sangue eqüino, em 40% em sangue de bovino e em 31,11% quando de ovino. Quanto à influência de agentes quelantes e íons em caldo TSB na atividade de hemolisinas em amostras de Campylobacter jejuni semeadas em placas de TSA – sangue ovino, foi observada atividade hemolítica em 26,67% quando utilizado CaCl2, 15,55% (FeCl3), 22,22% (EDTA), 11,11% (MgCl2) e apenas 2,22% (ácido acético). No tocante à atividade hemolítica, o TSA - sangue bovino apresentou 15,55% (CaCl2), 24,44% (FeCl3), 26,26% (EDTA), 20% (MgCl2) e 11,11% (ácido acético). A atividade hemolítica para o sangue equino foi de 24,44% (CaCl2), 22,22% (FeCl3), 28,89% (EDTA), 28,89% (MgCl2) e 8,89% (ácido acético). Para detecção dos genes cdtA, cdtB e cdtC através da técnica da Reação da Polimerase em Cadeia (PCR), foram utilizadas 119 amostras de C. jejuni de origem avícolas. Foi possível observar que 38% possuíam os três genes, e foram identificados somente os genes cdtA e cdtC em 19% do total de amostras, sendo que o gene cdtB foi encontrado em 14%, o gene cdtC foi observado em 12%, os genes cdtA e cdtB em somente 1%, os genes cdtB e cdtC em 1% e para cdtA em 1%. Observou-se que os resultados são dignos de atenção, pois demonstraram em amostras avícolas a presença de estirpes de C. jejuni com potencial virulento. A atividade hemolítica apresentou significativo aumento quando utilizado sangue de origem equina. A mesma foi diminuída quando utilizados agentes quelantes ou íons, nos três tipos de sangue. / Thermophilic members of the Campylobacter genus are recognized as important enteropathogenics for humans and also for other animals. The great diversity of ecological habitats in different organisms such as water, food, and animals may promote new virulence factors. This study aimed at detecting the distending cytolethal toxin (CDT) encoding genes by PCR, studying the activity of hemolysin and also the influence of chelation solutions and ions. A total of 45 samples of C. jejuni from poultry origin, grown in Tryptone Soy Broth (TSB) were used for investigating hemolytic activity. After bacterial growth, samples were plated on Tryptic Soy Agar (TSA) containing 5% sheep, equine or bovine blood, being each blood tested individually. In order to check the influence of chelation agents and ions solution on the hemolytic activity, samples of C. jejuni strains were grown in TSB containing chelation agents individually: EDTA, acetic acid, CaCl2 ion, MgCl2 and FeCl3 solutions, all in microaerophilic atmosphere. Regarding the detection of Campylobacter jejuni hemolysin activity on TSA plates, blood hemolysis were observed in 48.89 % of samples when equine blood was used; in 40% of samples when bovine blood was used and in 31.11 % when the blood used was of sheep origin. The influence of ions and chelation agents in hemolysin activity in TSB when Campylobacter jejuni was plated on TSA with sheep blood can be described as: hemolytic activity was observed at 26.67% of samples when CaCl2 was used, at 15.55 % for FeCl3, 22 22 % for EDTA, 11.11 % for MgCl2 and only 2.22% when acetic acid was used. The hemolytic activity detected when bovine blood - TSA was used indicated 15.55% for CaCl2, 24.44% for FeCl3, 26.26 % for EDTA, 20 % for MgCl2 and 11.11% for acetic acid. In terms of the hemolytic activity when equine blood was used, the results indicated 24.44% for CaCl2, 22.22 % for FeCl3, 28.89 % for EDTA, 28.89 % for MgCl2 and 8.89% for acetic acid. Finally, regarding the detection of cdtA, cdtB and cdtC through PCR, 119 samples of C. jejuni from poultry origin were used. The results indicated that all three genes were present in 38 % of the samples, whereas only two genes were identified in 19 % of samples, while the cdtB gene was singly found in 14%, the cdtC gene was independently observed in 12%, cdtA and cdtB genes together were found in 1% of the samples; the cdtB and cdtC genes associated were detected in 1%, while cdtA alone answered for 1% of detections. The results also showed the presence of C. jejuni strains with virulence potential. The hemolytic activity increased significantly when blood of equine origin was used, and that this activity was reduced when ions or chelating agents were used in combination with the three types of blood cells.

Since Shiga-toxigenic Escherichia coli (STEC) strains are not considered to be enteroinvasive, the mechanism(s) by which Shiga toxin (Stx) gains access to the circulation and to target tissues expressing its target receptor Gb3 is crucial to the disease process. There is increasing evidence that by facilitating translocation of Stx across the intestinal epithelium and by transporting bound toxin to remote sites such as the renal endothelium, polymorphonuclear leucocytes (PMNs) play a key role in the pathogenesis of serious STEC disease. Plasma levels of PMN-attracting CXC chemokines such as IL-8 also appear to correlate in humans with the severity of disease. Thus, the capacity of STEC strains to elicit CXC chemokine responses in intestinal epithelial cells may be a crucial step in pathogenesis. In order to determine which STEC factor(s) are responsible for the induction of CXC chemokine responses by intestinal epithelial (HCT-8) cells, a real-time reverse transcription PCR assay was developed to quantitatively measure relative expression of chemokine mRNA for IL-8, ENA-78, GCP-2, MGSA, MIP-2α and MIP-2β. Similarly, a commercially available sandwich ELISA was used to measure levels of IL-8 protein secreted by HCT-8 cells in response to infection with STEC. When HCT-8 cells were infected with the wellcharacterised locus of enterocyte effacement (LEE)-negative O113:H21 strain 98NK2 or the LEE-positive STEC strain EDL933, there were significant differences in the levels of chemokine mRNA and IL-8 protein expression. In particular, the LEE-negative strain 98NK2 induced significantly higher and earlier levels of chemokine mRNAs, including IL-8, MIP-2α and MIP-2β at 1 and 4 h, and ENA-78 at 4 h. However, EDL933 elicited no significant upregulation of any of the chemokine mRNAs at 1 h, and only modest increases in IL-8, MIP-2α and MIP-2β by 4 h, post-infection. These results were confirmed by IL-8 ELISA which showed that 98NK2 elicited significant levels of IL-8 protein by 2 h post-infection, and remained high until 4 h post-infection. In comparison, EDL933 did not elicit significant IL-8 induction over that of control cells, even at 4 h post-infection. When a range of STEC isolates from clinical samples were tested for their capacity to induce chemokine production in HCT-8 cells, highly significant differences were observed between the strains. Infection of HCT-8 cells with a range of LEE-negative STEC strains isolated from patients with severe STEC disease resulted in significantly higher and earlier upregulation of IL-8 and MIP-2α mRNA than that elicited by several LEE-positive STEC strains. Similarly, levels of IL-8 protein in LEE-negative STEC-infected HCT-8 culture supernatants were significantly higher than in LEE-positive STEC-infected culture supernatants. Only one LEE-positive strain, an O26 strain 95ZG1, was capable of inducing chemokine responses comparable to that induced by infection with the LEE-negative STEC strains. These results were also shown not to be attributable to differences in the adherence, initial doses or growth of the strains during the assay, or to a loss of viability of the HCT-8 cells. These results, therefore, suggest that there may be interesting differences in the ability of STEC strains to induce chemokine production in intestinal epithelial cells. The factor(s) that contribute to chemokine induction by epithelial cells in response to STEC were then examined. The difference in responses could not be attributed to the expression or non-expression of LEE genes, the presence or absence of an STEC megaplasmid or to differences in O serogroup. Although purified Stx1 and Stx2 were able to induce IL-8 and MIP-2α mRNA, and IL-8 protein, the levels of chemokine induction in response to wild-type STEC did not correlate with the type or amount of Stx produced by these strains in vitro. Similarly, deletion of the single stx2 gene from 98NK2 had no significant effect on chemokine induction compared to wild-type 98NK2-infected HCT-8 cells. Interestingly, several of the LEE-negative STEC strains eliciting the strongest chemokine responses belonged to flagellar serotype H21. Incubation of HCT-8 cells with purified H21 flagella elicited IL-8 and MIP-2α mRNA responses similar to those seen in the presence of the most potent LEE-negative STEC strains. Deletion of the fliC gene largely abolished the capacity of 98NK2 to elicit IL-8 and MIP-2α mRNA and IL-8 protein responses in HCT-8 cells. Similarly, deletion of both stx2 and fliC from 98NK2 elicited a response similar to that observed with deletion of fliC alone. Flagella were then purified from the high chemokine-inducing STEC strains 95HE4 (O91:H7) and 95ZG1 (O26:H11). Purified H7 and H11 flagella were similarly able to induce both IL-8 and MIP-2α mRNA, and IL-8 protein, in HCT-8 cells at levels similar to their respective wild-type strains. Deletion of fliC from two other STEC strains, 97MW1 (O113:H21) and 86-24 (O157:H7), confirmed that flagellin was responsible for the majority of chemokine responses in these wild-type strains. However, an inability of EDL933 to induce these responses was unexpected and later found to be due to a lack of expression of H7 flagella by this strain. Purified H21 FliC (His6-FliC) alone was able to induce chemokine production (including IL-8, MIP-2α and MIP-2β at 1 and 4 h, and ENA-78 at 4 h) by HCT-8 cells at similar levels to that observed for 98NK2. Taken together, these data suggest that although Stx is capable of inducing CXC chemokine responses, the elevated responses observed in cells infected with certain STEC strains are largely attributable to the production of flagellin. Purified His6-H21 flagellin was also able to induce p38 MAPK activation in vitro and IL-8 and MIP-2α mRNA were superinduced in the presence of both Stx2 and H21 flagellin. Blockade of the p38 pathway with SB203580 resulted in a down-regulation of IL-8 protein levels (by up to 61%) in response to H21 flagellin, but not IL-8 mRNA, suggesting that this inhibition may occur post-transcriptionally. Blocking the ERK and JNK pathways similarly decreased IL-8 secretion in response to H21 flagellin, suggesting that all three MAPK pathways are involved in this response. Indeed, concurrent inhibition of all three pathways resulted in virtually complete inhibition of IL-8 protein production (98%). Transfected HeLa and MDCK cells stably expressing TLR5 activated p38 in the presence of purified H21 flagellin, whereas dominant-negative (DN) TLR5-expressing cells did not, supporting previous studies that show that flagellin acts via TLR5. These data suggest that TLR5 and the p38, ERK and JNK MAPK pathways all play an important role in the response of intestinal epithelial cells to H21 flagellin from STEC, and that the combined effects of Stx and flagellin on host intestinal epithelial cells may result in an augmented inflammatory response. A role for flagellin in virulence was then investigated. BALB/c mice were orally inoculated with wild-type 98NK2 or 98NK2ΔfliC. Of the 16 mice challenged with the wildtype strain 98NK2, 9 (56%) died during the experiment (median survival time 7.6 days). However, only 3 of 16 mice (19%) challenged with 98NK2ΔfliC died (median survival time > 14 days). The difference in survival rate was statistically significant. No significant differences in the level of intestinal colonisation of 98NK2 or 98NK2ΔfliC were observed. Thus, flagellin directly contributes to the virulence of STEC in streptomycin-treated mice. Since the streptomycin-treated mouse is a model for systemic Stx-mediated pathology, these results suggest that the pro-inflammatory effects of flagellin play an important role in the pathogenesis of Stx-mediated STEC disease in vivo. / Thesis (Ph.D.)--School of Molecular and Biomedical Science, 2004.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Normal endothelial cell function is controlled in part by a tightly regulated balance between angiopoietin-1 and -2 (Ang-1 and Ang-2). Angiopoietin dysregulation (decreased Ang-1 and increased Ang-2) leads to an activated endothelium that is contractile, adhesive, and prothrombotic. Since an activated endothelial phenotype is seen in invasive group A streptococcal infection, E. coli O157:H7-induced hemolytic-uremic syndrome (HUS), and sepsis, we hypothesized that angiopoietin dysregulation might also be present in these syndromes, and to that end, measured angiopoietin levels in several well-characterized patient cohorts. Decreased Ang-1 and/or increased Ang-2 were found in all three syndromes, and were predictive of clinical outcome in HUS and sepsis. The prognostic utility of Ang-2 in sepsis was further enhanced by combination with biomarkers of inflammation. Angiopoietin dysregulation may therefore represent a shared final common pathway to endothelial activation as well as a clinically useful prognostic biomarker in streptococcal toxic shock, HUS, and sepsis.

Shiga toxin-producing E. coli (STEC) cause diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome (HUS). All STEC produce one or both of two Shiga toxins, Stx1 and Stx2. STEC strains that produce Stx2 are more strongly associated with HUS than strains that produce Stx1 or both Stx1 and Stx2. Epidemiologic evidence indicates a recent increase in the rate of HUS among STEC outbreaks. The increasing rate of HUS could be explained by a shift in the toxin profiles of STEC strains. The purpose of this study was to examine trends in toxin profiles of human STEC O157 isolates from 1996 to 2008 and to assess whether an increase in the number of Stx2-only-producing strains could be correlated with a recent increase in HUS cases. Data from three independent datasets, collected from PulseNet, eFORS and NARMS, were used. Additionally, trends such as seasonal variations, geographical variations, gender differences, and age differences were examined for each toxin profile. Results from this study show a shift in the toxin profile of human STEC O157 strains in the United States, in that the proportion of Stx2-only producing strains has increased dramatically since 1996.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal