One of the most effective immunological interventions for human disease prevention is the administration of anti-red blood cell (RBC) IgG, more specifically, anti-D IgG, for prevention of hemolytic disease of the fetus and newborn (HDN), a serious and potentially fatal condition caused by the maternal immune response against the Rhesus (Rh) blood group system D antigen on fetal RBC. Despite its widespread clinical use, the mechanism of the suppressive anti-RBC IgG effect is not fully understood. In a murine model of immunity to foreign RBCs, transfusion of mice with IgG-opsonized RBCs strongly attenuated the antibody response compared to transfusion of untreated RBCs. This model was used to study the anti-RBC IgG effect on the host immune response. Contrary to the predominant theories of the anti-D effect, here it is shown that IgG-mediated RBC clearance is not sufficient for the attenuation of antibody responses. IgG-opsonized RBCs internalized by the mononuclear phagocytic cells could stimulate T and B cell responses against RBC antigens. This thesis also shows that the adaptive tolerance at the T or B cell level is not the reason for the attenuation of the antibody response. Instead, IgG selectively prevented the appearance of antigen-primed RBC-specific B cells and, surprisingly, induced the host B cell response against the IgG in complex with RBCs. These results suggest that the inability of RBC-specific B cells to recognize and present RBC-specific epitopes may explain the inhibitory IgG effect.

[Truncated abstract] Iron is a trace mineral used by the body in many physiological processes that are essential to athletic performance. Commonly, the body's iron stores are compromised by exercise via several well established mechanisms. One such mechanism is the destruction of red blood cells (hemolysis), in response to the mechanical stress and circulatory strain of exercise. Although it appears that a force-dependent relationship between the heel-strike of the running gait and ground contact exists, the effects of the intensity trained at and the ground surface type trained upon have not been documented. Similarly, the effects of a cumulative training stress (i.e. multiple daily sessions) has not been examined. In addition to hemolysis, exercise also invokes an inflammatory response that results in an up-regulation of the cytokine interleukin-6 (IL-6). This cytokine is the primary mediator of hepcidin expression, a liver-produced hormone that regulates iron metabolism in the gut and in macrophages. The influence of exercise on hepcidin expression is relatively unknown, and as such it is possible that this hormone may be a mitigating factor implicated in athletic-induced iron deficiency. Therefore, the purpose of this thesis was to investigate the effect of different training frequencies, intensities and ground surfaces on the hemolytic response. In addition, the impact of exercise-induced inflammation on hepcidin expression in the 24 h post-exercise was investigated, with the aim of determining whether this hormone may be a potential new mechanism associated with athletic-induced iron deficiency. Finally, an interaction between hemolysis and hepcidin activity was examined to investigate their potential combined effect on iron status in the 24 h post-exercise. ... Venous blood and urine samples were collected pre- and immediately post-exercise, and at 3 and 24 h of recovery. Samples were analysed for circulating levels of IL-6, free Hb, Hp, serum iron, ferritin and urinary hepcidin activity. At the conclusion of both the T1 and T2 interval runs, the free Hb and serum Hp were significantly increased (p<0.05) from pre-exercise levels. Furthermore, a cumulative effect of two running sessions was shown in the T2 trial, via a further significant fall in serum Hp. The IL-6 and hepcidin activity were significantly increased after each running session (p<0.05) with no cumulative effect seen. Serum iron and ferritin were significantly increased post-exercise after each interval run (p<0.05), but were not influenced by the addition of a prior LSD run 12 h earlier. As a result, this investigation showed a cumulative effect of consecutive training sessions on RBC destruction in male athletes. Furthermore, post-exercise increases to serum iron and hepcidin, and their interaction was suggested to have potential implications for an athlete's iron status. Overall, the findings of this thesis show that hemolysis is evident at the conclusion of endurance running, and is influenced by training intensity and frequency. The results enabled a time-line for hepcidin expression post-exercise to be established, and the implications of increases to the activity of this hormone, in association with the hemolytic changes seen with endurance exercise are discussed.

Thesis (Ph.D.)--University of Adelaide, School of Molecular and Biomedical Science, 2003. / "February 2003." Addendum and corrigenda inserted at back. Includes bibliographical references (leaves 249-272).

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / Includes bibliographical references. Online version available on the World Wide Web.

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&reg;Laboratories) conjugadas a fluoresceína de isotiocianato (FITC), e a leitura realizada com citômetro de fluxo (FACS Calibur; Becton, Dickinson and Company&copy; 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&#177;0,18%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (0,68&#177;0,29%) para anticorpos anti-IgM FITC. Já nos cães com piometra, foram encontradas 0,14 - 4,19% (0,96&#177;0,86%) de hemácias marcadas com anticorpos anti-IgG FITC e 0,29 - 9,58% (1,37&#177;1,71%) com anticorpos anti-IgM FITC, antecedendo a OSH. No momento pós-OSH observou-se 0,18 - 16,2% (2,77&#177;3,67%) de hemácias marcadas para anticorpos anti-IgG FITC e 0,15 - 19,8% (4,01&#177;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&lt;0,001) e pós-OSH (p&lt;0,001). Em relação a anticorpos anti-IgM FITC, não foram observadas diferenças entre os grupos controle e pré-OSH (p&#61;0,09), porém, após a OSH houve aumento na marcação de hemácias, quando comparado ao grupo controle (p&lt;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&reg; Laboratories) conjugated to fluorescein isothiocyanate (FITC), and the reading performed with flow cytometry (FACS Calibur; Becton, Dickinson and Company&copy; 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&plusmn; 0.18%) of red blood cells marked with antibodies anti-IgG FITC and 0.29 to 9.58% (0.68&#177; 0.29%) for antibodies anti-IgM FITC. Already in dogs with pyometra were found 0.14 to 4.19% (0.96 &#177; 0.86%) of red blood cells marked with antibodies anti-IgG FITC and 0.29 to 9.58% (1.37 &#177; 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 &#177; 3.67%) of red blood cells marked for antibodies anti-IgG FITC and 0.15 to 19.8% (4.01 &#177; 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&lt;0.001) and post-OSH (p&lt;0.001). Regarding the antibodies anti-IgM FITC, no differences were observed between the control and pre-OSH (p&#61;0.09), however, after the OSH was no increase in labeling of red blood cells compared to the control group (p&lt;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

Made available in DSpace on 2014-06-11T19:27:15Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-07-11Bitstream added on 2014-06-13T19:14:30Z : No. of bitstreams: 1 moraes_lf_me_botfmvz.pdf: 428197 bytes, checksum: bc0cb7abb11b1fca9cd1b81b73ec75e5 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A anemia hemolítica imuno-mediada (AHIM) é a causa mais comum das anemias hemolíticas e tem sido associada a estados de hipercoagulabilidade. Neste trabalho objetivou-se: avaliar a ocorrência de alterações hematológicas, hemostáticas e renais e a prevalência das classes de imunoglobulinas (Ig) envolvidas nas AHIM. 87 cães anêmicos foram submetidos ao teste de Coomb’s, citometria de fluxo (CF) e auto-aglutinação, além de hemograma e contagem de reticulócitos, perfil hemostático e renal, pesquisa de hematozoários e testados para Ehrlichia sp e leptospirose. Os resultados foram analisados pelo teste t, Mann-Whitney ou Kruskal-Wallis ao nível de 5% de significância. 61 cães (70,11%) foram positivos para AHIM na CF, 31 (35,63%) para o Coomb’s e 24 (27,58%) para auto-aglutinação. Não houve predomínio de uma classe de Ig envolvida. As alterações clínico-laboratoriais para cães com AHIM incluíram anemia regenerativa, macrocítica, hipocrômica e reticulocitose, além de icterícia, febre, hiperglobulinemia e bilirrubinúria. 9,8% dos cães positivos apresentaram esferocitose, 29,5% leucocitose, 39,6% neutrofilia e 72,1% trombocitopenia. A proteinúria e o aumento na relação proteína: creatinina urinárias revelam indícios de lesão renal. Os cães anêmicos positivos e negativos para AHIM apresentaram diferença estatística em relação ao grupo controle para contagem de plaquetas, TP, TTPA, AT, PDF e Dímeros D. 25 cães positivos e sete cães negativos para AHIM apresentaram um estado tromboembólico. Estas alterações hemostáticas podem ser atribuídas à causa base predominante para a anemia em questão, uma vez que 74,6% dos casos de AHIM foram atribuídos às doenças infecciosas, a maioria associados à Ehrlichia sp / Immune-mediated hemolytic anemia (IMHA) is the most common cause of hemolytic anemia and has been associated with hypercoagulable states. The objectives of this study were: to evaluate the incidence of hemostatic, renal and hematological abnormalities, and the prevalence immunoglobulin’s classes involved in IMHA. 87 anemic dogs were selected and submitted to Coomb’s test, flow cytometry (FC) and auto-agglutination, along with CBC, reticulocyte count, hemostatic and renal profile, hemoparasite search in peripheral blood smears, and Ehrlichia sp and leptospirosis tests. The results were analyzed by t test, Mann-Whitney or Kruskal-Wallis at 5% of significance. 61 dogs (70.11%) were positive to IMHA by CF, 31 (35.63%) by Coomb's test and 24 (27.58%) by auto-agglutination. There wasn’t a predominance of IgG or IgM involvement. The hematological and clinical changes in dogs with IMHA included macrocytic, hypochromic regenerative anemia, and reticulocytosis, as well as icterus, fever, auto-agglutination, hyperglobulinemia and bilirubinuria. 9.8% of dogs with IMHA had spherocytosis, 29.5% leukocytosis, 39.6% neutrophilia and 72.1% thrombocytopenia. Proteinuria and the increase in urine protein creatinine ratio reveal evidences of renal injury. There were statistical differences between the control group and anemic dogs positive or negative for IHMA to the following tests: platelet count, PT, APTT, AT, FDP and D-dimer. 25 positive dogs and seven negative dogs for IMHA were in a thromboembolic state. These hemostatic changes observed in both groups of anemic dogs may be attributed to the predominant underlying cause of anemia, since 74.6% of IHMA cases were attributed to infectious diseases; mostly Ehrlichia sp related

Orientador: Regina Kiomi Takahira / Banca: Raimundo Souza Lopes / Banca: Áureo Evangelista Santana / Resumo: A anemia hemolítica imuno-mediada (AHIM) é a causa mais comum das anemias hemolíticas e tem sido associada a estados de hipercoagulabilidade. Neste trabalho objetivou-se: avaliar a ocorrência de alterações hematológicas, hemostáticas e renais e a prevalência das classes de imunoglobulinas (Ig) envolvidas nas AHIM. 87 cães anêmicos foram submetidos ao teste de Coomb's, citometria de fluxo (CF) e auto-aglutinação, além de hemograma e contagem de reticulócitos, perfil hemostático e renal, pesquisa de hematozoários e testados para Ehrlichia sp e leptospirose. Os resultados foram analisados pelo teste t, Mann-Whitney ou Kruskal-Wallis ao nível de 5% de significância. 61 cães (70,11%) foram positivos para AHIM na CF, 31 (35,63%) para o Coomb's e 24 (27,58%) para auto-aglutinação. Não houve predomínio de uma classe de Ig envolvida. As alterações clínico-laboratoriais para cães com AHIM incluíram anemia regenerativa, macrocítica, hipocrômica e reticulocitose, além de icterícia, febre, hiperglobulinemia e bilirrubinúria. 9,8% dos cães positivos apresentaram esferocitose, 29,5% leucocitose, 39,6% neutrofilia e 72,1% trombocitopenia. A proteinúria e o aumento na relação proteína: creatinina urinárias revelam indícios de lesão renal. Os cães anêmicos positivos e negativos para AHIM apresentaram diferença estatística em relação ao grupo controle para contagem de plaquetas, TP, TTPA, AT, PDF e Dímeros D. 25 cães positivos e sete cães negativos para AHIM apresentaram um estado tromboembólico. Estas alterações hemostáticas podem ser atribuídas à causa base predominante para a anemia em questão, uma vez que 74,6% dos casos de AHIM foram atribuídos às doenças infecciosas, a maioria associados à Ehrlichia sp / Abstract: Immune-mediated hemolytic anemia (IMHA) is the most common cause of hemolytic anemia and has been associated with hypercoagulable states. The objectives of this study were: to evaluate the incidence of hemostatic, renal and hematological abnormalities, and the prevalence immunoglobulin's classes involved in IMHA. 87 anemic dogs were selected and submitted to Coomb's test, flow cytometry (FC) and auto-agglutination, along with CBC, reticulocyte count, hemostatic and renal profile, hemoparasite search in peripheral blood smears, and Ehrlichia sp and leptospirosis tests. The results were analyzed by t test, Mann-Whitney or Kruskal-Wallis at 5% of significance. 61 dogs (70.11%) were positive to IMHA by CF, 31 (35.63%) by Coomb's test and 24 (27.58%) by auto-agglutination. There wasn't a predominance of IgG or IgM involvement. The hematological and clinical changes in dogs with IMHA included macrocytic, hypochromic regenerative anemia, and reticulocytosis, as well as icterus, fever, auto-agglutination, hyperglobulinemia and bilirubinuria. 9.8% of dogs with IMHA had spherocytosis, 29.5% leukocytosis, 39.6% neutrophilia and 72.1% thrombocytopenia. Proteinuria and the increase in urine protein creatinine ratio reveal evidences of renal injury. There were statistical differences between the control group and anemic dogs positive or negative for IHMA to the following tests: platelet count, PT, APTT, AT, FDP and D-dimer. 25 positive dogs and seven negative dogs for IMHA were in a thromboembolic state. These hemostatic changes observed in both groups of anemic dogs may be attributed to the predominant underlying cause of anemia, since 74.6% of IHMA cases were attributed to infectious diseases; mostly Ehrlichia sp related / Mestre

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.

Orientadores: Antonio Condino Neto, Sara Teresinha Olalla Saad / Tese (doutorado)- Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T18:06:47Z (GMT). No. of bitstreams: 1 Almeida_AnaCarolinade_D.pdf: 1227023 bytes, checksum: 25438a379e180eeb0d1972b464b909b0 (MD5) Previous issue date: 2009 / Resumo: SIRP 'alfa'(Signal Regulatory Protein 'alfa') é um receptor que medeia funções inibidoras em fagócitos. Sua ativação e conseqüente fosforilação dos ITIMs ocorre pela ligação ao CD47 presente na membrana dos eritrócitos, e permite o recrutamento e a ativação de SHP-1, a qual desfosforila substratos específicos envolvidos na mediação de diversos efeitos fisiológicos. O objetivo deste trabalho foi avaliar o papel da dexametasona (dexa) e de IFN?/TNFa sobre a expressão de SIRPa e SHP-1; a consequência desta regulação sobre a eritrofagocitose; e o nível de expressão gênica de SIRPa e SHP-1 em monócitos de pacientes com anemia hemolítica autoimune (AHAI) antes e depois de corticoterapia. Monócitos de doadores sadios e células mielomonocíticas U937 foram cultivados por 48 horas com dexa (1µM) ou IFN? (100U/ml) e TNFa (1000U/ml), por 6 horas com Hemina® (30uM), ou por 72 horas com prednisolona (0,15 e 1mg/l). Monócitos foram isolados de pacientes com AHAI antes e depois da corticoterapia. A expressão gênica de SIRPa e SHP-1 foi determinada por PCR em Tempo Real, a expressão protéica de SIRPa e SHP-1 foi determinada por Western Blotting, e a capacidade de eritrofagocitose foi determinada por microscopia. IFN? e TNFa, in vitro, promoveram o aumento da expressão gênica e protéica de SIRPa e a expressão gênica de SHP-1, em paralelo com a redução da capacidade de eritrofagocitose em monócitos normais. Em contrapartida, embora tenha aumentado a expressão gênica de SIRPa e SHP-1, dexa in vitro não alterou a expressão destas proteínas, assim como não alterou a capacidade de eritrofagocitose de monócitos normais. A expressão gênica de SIRPa e SHP-1 foi maior em monócitos de pacientes com AHAI em comparação a doadores sadios. Após corticoterapia, a expressão gênica de SIRPa e SHP-1 em monócitos de pacientes com AHAI se mostrou similar a doadores sadios. Pacientes com AHAI estudados antes da corticoterapia apresentaram baixos níveis de hemoglobina e após corticoterapia esse índice de mostrou normal. A expressão gênica de SIRPa foi aumentada pela cultura de monócitos com hemina, mas a expressão de proteína permaneceu a mesma. Nossos resultados confirmam o papel fundamental da SIRPa na regulação da eritrofagocitose e sugere que a expressão de mRNA de SIRPa em monócitos de pacientes com AHAI antes de corticoterapia é aumentada pela liberação de heme, e que a redução da expressão gênica de SIRPa após corticoterapia se deve a um efeito indireto desta droga pela redução da eritrofagocitose e diminuição da disponibilidade de heme. / Abstract: SIRPa (Signal Regulatory Protein a) is an inhibitory receptor in phagocytes. Its activation and consequent phosphorylation of ITIMs occurs by the binding to CD47 on erythrocyte membrane, what allows SHP-1 recruitment, which dephosphorylates specific substrates involved in the mediation of several physiologic effects. The aim of this work was to determine the role of dexamethasone and IFN?/TNFa upon SIRPa and SHP-1 expression, and the consequence of this regulation over erythrophagocytosis; and to evaluate the regulation of SIRPa and SHP-1 in peripheral blood monocytes (PBM) of autoimmune hemolytic anemia (AIHA) patients before and after glucocorticoid (GC) therapy. PBM from healthy donors and U937 myelomonocytic cells were cultured for 48 hours with dexamethasone (1µM) or IFN? (100U/ml) and TNFa (1000U/ml), for 6 hours with Hemin (30uM), or for 72 hours with prednisolone (0.15 and 1mg/l). PBM were isolated from AIHA patients under GC therapy or not. SIRPa and SHP-1 gene expression was determined by Real Time PCR, SIRPa and SHP-1 protein level was determined by Western Blotting, and erythrophagocytosis was determined by microscopy. IFN? and TNFa increased SIRPa gene and protein expression and SHP-1 gene expression, in parallel with a decrease in erythrophagocytosis ability in PBM. On the other hand, although SIRPa and SHP-1 gene expression was significantly increased, dexamethasone did not alter SIRPa and SHP-1 protein expression, and did not alter erythrophagocytosis ability in monocytes. SIRPa and SHP-1 expression was significantly higher in PBM from AIHA patients compared to normal. After GC therapy, SIRPa and SHP-1 expression was similar in PBM of AIHA patients compared to healthy donors. AIHA patients studied before glucocorticoid therapy showed low hemoglobin and after glucocorticoid therapy the level of hemoglobin was normal. SIRPa gene expression was increased by culture with hemin, but protein expression remained the same. Our results confirm the key role of SIRPa in erythrophagocytosis regulation and suggest that SIRPa mRNA expression in AIHA patients before glucocorticoid therapy is increased by heme release, and the decrease of SIRPa gene expression after glucocorticoid therapy is due to an indirect effect of this drug by the reduction of erythophagocytosis and free heme availability. / Doutorado / Doutor em Farmacologia

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.