Abstract not available

Un contexte réglementaire de plus en plus strict dans le domaine de l’évaluation environnementale de l’impact des composés chimiques d’origine anthropique et plus précisément du risque écotoxicologique dans l’environnement aquatique, exige de renforcer les outils d’évaluation et d’affiner la connaissance de leur valeur prédictive. A ce titre, l’étude des biomarqueurs de génotoxicité doit être privilégiée, compte tenu du rôle central de l’ADN dans le fonctionnement du vivant et des effets trans-générationnels susceptibles de conduire à un fardeau génétique affectant les populations exposées à un environnement contaminé. Dans cette perspective, et après avoir défini la problématique à l’issue d’une analyse bibliographique, ce travail de thèse a permis de développer un biomarqueur de génotoxicité chez l’épinoche (Gasterosteus aculeatus), espèce modèle en écotoxicologie, en utilisant la mesure des dommages primaires à l’ADN par le test des comètes en conditions alcalines. Il visait à : i) apporter une information complémentaire au sein d’une batterie de biomarqueurs utilisée en biomonitoring chez cette espèce, 2) apporter des éléments de compréhension sur la valeur prédictive d’un effet génotoxique sur le tissu germinal des organismes aquatiques exposés au stress chimique. Concernant le premier point, les résultats soulignent l’intérêt de ce biomarqueur de génotoxicité développé sur le tissu sanguin au laboratoire, sa grande sensibilité amenant à un pouvoir discriminant significatif dans le cadre d’approches multiparamétriques menées dans un second temps sur le terrain. Pour ce qui est de l’étude de la signification écotoxicologique de ce biomarqueur de génotoxicité, le but était d’explorer le lien entre l’intégrité de l’ADN des cellules germinales de ce poisson et la qualité de la descendance. Les résultats de ce travail démontrent de manière originale la contribution majoritaire du génome paternel à la transmission d’une information génétique délétère ayant pour conséquence des anomalies de survie et de développement mesurées sur la descendance. Ils permettent de disposer d’un élément d’évaluation prédictive pertinent de l’effet des contaminants à potentiel génotoxique dans les écosystèmes aquatiques, dans un contexte d’intérêt grandissant de la compréhension du déclin de populations de poisson universellement recensé. / A rule context in the field of environmental impact assessment of anthropogenic chemicals and specifically ecotoxicological risk to the aquatic environment, requires strengthening the assessment tools and refine knowledge of their predictive value. As such, the study of biomarkers of genotoxicity should be choose, given the central role of DNA in living organisms function and trans-generational effects could lead to a genetic load affecting populations exposed to a contaminated environment . In this context, and after defining the problem at the end of a literature review, this thesis has developed a biomarker of genotoxicity in the stickleback (Gasterosteus aculeatus), model species in ecotoxicology, using the measurement primary DNA damage by the comet assay under alkaline conditions. It aimed to: i) provide additional information in a battery of biomarkers used in biomonitoring in this species, 2) provide some understanding of the predictive value of a genotoxic effect on the germinal tissue of aquatic organisms exposed to chemical stress. On the first point, the results underline the importance of this biomarker of genotoxicity developed on blood tissue in the laboratory, its high sensitivity leading to a significant discriminating power under multiparameter approaches taken in a second time on the ground. Regarding the study of the ecotoxicological significance of this biomarker of genotoxicity, the aim was to explore the relationship between DNA integrity in germ cells of the fish and the quality of the offspring. The results of this work show an original way the majority of the paternal genome contribution to the transmission of a deleterious genetic information that result in abnormal survival and development measured on the offspring. They allow you to have a predictive element relevant evaluation of the effect of genotoxic potential contaminants in aquatic ecosystems, in a context of growing interest in understanding the decline of fish populations identified universally.

Red blood cells (RBCs) are vital for oxygen delivery to tissues and constitute the vast majority of all cells in blood. After leaving the red bone marrow as mature cells, RBCs have a lifespan of approximately 120 days before they are removed from the circulation by macrophages, mainly in the spleen and liver. RBC transfusion is a common therapy in modern healthcare. Major surgery, numerous cancer treatments and other, often lifesaving, interventions would be unthinkable without available blood supply. For this reason, hospitals store donated RBCs in blood banks. The metabolic and structural changes that occur during prolonged storage of RBCs (the storage lesion) have been studied in detail in vitro and include oxidative stress, a reduction in glycolysis, increased membrane rigidity and shedding of microparticles from the RBC membrane. Stored RBCs share several features of senescent RBCs, but also with RBCs undergoing an apoptotic-like process called eryptosis. A consequence of the storage lesion is the fact that as much as 25% of stored RBCs could be rapidly removed from the circulation within 24 hours after transfusion. The mechanisms behind this rapid macrophage-mediated recognition and removal of stored RBCs, and its immunological consequences, remain largely unknown. Therefore, the aims of this thesis were to investigate if cryopreserved human RBCs induced an inflammatory response following autologous transfusion into healthy volunteers, and to further understand the mechanisms behind macrophage recognition of stored RBCs in vitro and in vivo. Autologous transfusion of two units of cryopreserved RBCs into healthy human recipients was found to be associated with an increased extravascular RBC elimination already at 2 hours after transfusion. However, there were no signs of an increased production of any of the investigated pro-inflammatory cytokines, indicating that an increase in the destruction of RBCs per se did not induce an inflammatory response. Eryptosis is a form of induced RBC death associated with an increased cytoplasmic Ca2+ uptake. We found that a subset of human RBCs increased their Ca2+ permeability during prolonged storage at +4°C. Using a murine model, to further understand how RBCs with an increased Ca2+ permeability were eliminated by phagocytic cells in the spleen, it was found that such RBCs were taken up by marginal zone macrophages and dendritic cells (DCs) in a manner distinct from that of naturally senescent RBCs. The DC population particularly efficient in this process expressed CD207 and are known for their ability to promote immunological tolerance. Eryptotic cell uptake was not regulated by the phagocytosis-inhibitory protein CD47 on the RBCs. To investigate how RBCs damaged during liquid storage are recognized and taken up by macrophages, a model to store and transfuse murine RBCs was developed. This storage model generated murine RBCs with several characteristics similar to that of stored human RBCs (i.e. loss of ATP, formation of RBC microparticles and rapid clearance of up to 35% of the RBCs during the first 24 h after transfusion). In vitro phagocytosis of human as well as murine stored RBCs was serum dependent and could be inhibited by blocking class A scavenger receptors using fucoidan or dextran sulphate. In conclusion, the findings of this thesis contribute to further understanding how changes inflicted to RBCs during storage direct the fate of these cells in their interaction with cells of the immune system after transfusion. The observation of an increased Ca2+ permeability of stored RBCs, and the possible recognition of such cells by tolerance-promoting DCs, in combination with the findings that class A scavenger receptors and serum factors may mediate recognition of stored RBCs, may result in novel new directions of research within the field of transfusion medicine.

Objetivo: Comparar a evolução clínica de pacientes críticos idosos (>= 60 anos) com pacientes mais jovens (< 60 anos) após a implementação de uma estratégia liberal ou restritiva de transfusão de hemácias. Métodos: Trata-se de uma análise dos seguintes ensaios clínicos randomizados: Transfusion Requirements After Cardiac Surgery (TRACS), Transfusion Requirements in Surgical Oncologic Patients (TRISOP) e Transfusion Requirements In Critically ill Oncologic Patients (TRICOP). Nós estratificamos os pacientes em idade abaixo de 60 anos ou maior ou igual a 60 anos que foram randomizados para uma estratégia liberal ou restritiva de transfusão de hemácias. O desfecho composto foi um combinado de mortalidade em 30 dias e complicações graves. Resultados: Dos 1000 pacientes incluídos, 567 (57%) apresentavam 60 anos ou mais e 433 (43%) apresentavam menos de 60 anos e foram incluídos no estudo. O desfecho primário nos pacientes mais jovens (< 60 anos) ocorreu em 57% dos pacientes do grupo liberal e em 48% dos pacientes do grupo restritivo (p=0,060). O desfecho primário nos pacientes idosos foi alcançado em 63,3% dos pacientes no grupo liberal e em 69,4% dos pacientes no grupo restritivo (p=0,123). Entretanto, nos pacientes mais idosos, o choque cardiogênico foi mais frequente nos pacientes no grupo submetido a estratégia restritiva (14,8% vs 6%, p=0,001). Conclusões: Embora não tenha sido demonstrada nenhuma diferença no desfecho primário entre os grupos, a estratégia restritiva de transfusão foi associada a aumento da ocorrência de choque cardiogênico nos pacientes idosos críticos quando comparada a estratégia liberal. O risco cardiovascular da anemia pode ser mais deletério do que o risco da transfusão de sangue em pacientes idosos / Objective: The aim of this study was to compare outcomes in critically ill patients who are aged 60 years or more or less than 60 years after implementation of a restrictive or a liberal transfusion strategy. Methods: This is an analysis of the Transfusion Requirements After Cardiac Surgery (TRACS) randomized clinical trial of the Transfusion Requirements in Surgical Oncologic Patients (TRISOP) randomized clinical trial and of the Transfusion Requirements In Critically ill Oncologic Patients (TRICOP) randomized clinical trial. In this analysis, we separated patients into those aged 60 years or more (elderly) and those aged less than 60 years randomized to a restrictive or a liberal strategy of red blood cell transfusion. The primary outcome was a composite defined as a combination of 30-day all-cause mortality and severe morbidity. Results: Of the 1000 patients included in the studies, 567 (57%) were aged 60 years or more and 433 (43%) were aged less than 60 years and were included in this study. The primary outcome in younger patients ( < 60 years-old) occurred in 56.9% of patients in the liberal strategy group and in 47.9% of patients in the restrictive strategy group (p=0.060). The primary outcome in elderly patients (>= 60 years-old) occurred in 63.3% of patients in the liberal strategy group and in 69.4% of patients in the restrictive strategy group (p=0.123). However, in the older patients, cardiogenic shock was more frequent in patients in the restrictive transfusion group (14.8% vs 6%, p=0.001). Conclusions: Although there was no difference between groups regarding the primary outcome, a restrictive transfusion strategy was associated with an increased rate of cardiogenic shock in elderly critical patients compared with a more liberal strategy. Cardiovascular risk of anemia may be more harmful than the risk of blood transfusion in older patients

A demanda por transfusões sanguíneas tem aumentado no Brasil e o número de doações de sangue permanecem insuficientes. Há escassez de componentes de sangue para transfusão, principalmente de concentrados de células vermelhas do sangue. As células-tronco pluripotentes induzidas humanas (hiPSCs) possuem um grande potencial para se tornar uma fonte de CÉLULAS VERMELHAS DO SANGUE, pois podem se diferenciar em qualquer tipo celular, incluindo CÉLULAS VERMELHAS DO SANGUE de fenótipo específico. O objetivo deste trabalho é a geração de hiPSCs para partir de células mononucleares de sangue periférico (PBMCs) de candidatos a doação de sangue que possuem fenótipo eritrocitário de baixa imunogenicidade, bem como a diferenciação eritroide das hiPSCs geradas. As amostras de sangue periférico (PB) de 11 indivíduos foram coletadas e caracterizadas quanto ao genótipo para os seguintes antígenos eritrocitários: Sistema Rh (RHCE*01/RHCE*02/RHCE*03/RHCE*04/RHCE*05), Kell (KEL*01/KEL*02), Duffy (FY*01/FY*02 and FY*02N.01), Kidd (JK*01/JK*02) e MNS (GYPB*03/GYPB*04). Outros antígenos de grupos sanguíneos distintos foram determinados por meio de fenotipagem. Duas amostras (PBMCs PB02 e PB12) foram selecionadas para a reprogramação devido ausência de múltiplos antígenos eritrocitários e, portanto, considerados de baixa imunogenicidade. Os PBMCs foram enriquecidos em eritroblastos e em seguida, as células foram transfectadas com os vetores episomais pEB-C5 e pEB-Tg e então, co-cultivados sobre fibroblastos de embriões murinos (MEFs) até o surgimento de colônias semelhantes a hiPSCs (hiPSC PB02 e hiPSC PB12). Estas colônias foram transferidas para condições de cultivo próprias e posteriormente caracterizadas quanto à sua pluripotência. A expressão dos genes de pluripotência OCT4, SOX2 e NANOG demonstrou níveis de expressão maior em comparação às linhagens não pluripotentes. As análises de imunofenotipagem por citometria de fluxo revelaram que em torno de 86% das células expressaram Nanog, 88% Oct4 e 88% Sox2. Os níveis de expressão de genes de pluripotência e marcadores foram consistentes com o estado indiferenciado encontrado em células pluripotentes conhecidas. A análise funcional para avaliação da pluripotência foi realizado pela injeção das hiPScs em camundongos imunodeficientes, demonstrando a formação de teratoma nas linhagens geradas. A metodologia para diferenciação hematopoética das hiPSCs geradas a partir dos corpos embrioides estão em progresso. O potencial de diferenciação foi confirmado durante a padronização deste processo, utilizando ensaio de formação de colônias em metilcelulose. Uma média de 10,5 colônias de precursores eritroide foram obtidas a partir de 50x103 hiPSC PB02 em diferenciação e uma colônia mista (mieloide e linfoide) a partir de 15x103 hiPSC PB12 foram obtidas. Neste trabalho foi possível gerar duas linhagens de hiPSCs com fenótipos de antígenos eritrocitários de interesse que podem ser mantidas em cultura por um longo período (26 passagens) e demonstram um potencial de diferenciação hematopoética. / The demand for blood transfusion has increased in Brazil and the number of blood donations remains insufficient. Therefore, there is a shortage of blood components for transfusion, mainly concentrates of red blood cells (RBCs). Human induced pluripotent stem cells (hiPSCs) have great potential to become a source of RBCs, because they can differentiate into every cellular type, including RBCs of a particular phenotype. The objective of this work was to generate hiPSC from mononuclear cells of peripheral blood (PBMCs) from blood donors who presented low immunogenic phenotype for transfusion, and erythroid differentiation of the generated hiPSCs. Peripheral blood samples from 11 individuals were collected and characterized for the following erythrocyte antigens: Rh system (RHCE*01/RHCE*02/RHCE*03/RHCE*04/RHCE*05), Kell (KEL*01/KEL*02), Duffy (FY*01/FY*02 and FY*02N.01), Kidd (JK*01/JK*02), MNS (GYPB*03/GYPB*04). Additionally, other antigens of different blood groups were determined by phenotyping. The samples PBMC PB02 and PBMC PB12 were chosen for iPS generation due to their multiple negative erythrocyte antigens. They were isolated, expanded into erythroblasts, and transfected using the reprogramming episomal vectors PEB-C5 and PEB-Tg. This population was co-cultured on mouse embryonic fibroblasts (MEFs) until the appearance of hiPSC like colonies (hiPSC PB02 and hiPSC PB12). These colonies were transferred to human embryonic stem cells (hESCs) culture conditions and characterized regarding their pluripotency. The expression of OCT4, SOX2 and NANOG pluripotency genes demonstrated that the expression of both lineages was higher in comparison with non-pluripotent lineages. Immunophenotyping performed by flow cytometry revealed that 86% of cells expressed Nanog, 88% Oct4 and 88% Sox2. Expression levels of pluripotency genes and markers were consistent with undifferentiated state found in known pluripotent cells. Functional analysis for pluripotency was achieved by the hiPSC injection in immunodeficient mice showing that both hiPSC cell lines were able to induce teratoma tumor. The hematopoietic differentiation potential was confirmed using methylcellulose assay, with an average of 10.5 erythroid colonies from 50x103 single cells and a mixed colonies of myeloid and lymphoid cells) and finally a colony composed of white cells from 15x103 PB12 hiPSC. In conclusion, it was possible to generate a hiPSC from a red blood cell phenotype that are negative for multiple antigens, and this cell line can be maintained for a long period in culture (26 passages) and show potential for hematopoietic differentiation.

A aloimunização contra antígenos eritrocitários decorre normalmente de gestações ou transfusões prévias e torna-se um problema mais frequente entre pacientes submetidos à transfusão, até mesmo entre pacientes transfundidos de forma esporádica, como nos casos de pacientes com câncer de mama. A detecção de anticorpos irregulares deve ser realizada com técnica sensível, capaz de detectar os anticorpos de maior relevância clínica. A falha na detecção de um aloanticorpo pode levar a reação transfusional hemolítica aguda ou tardia de intensidade variável que podem agravar ainda mais a condição clínica do receptor. Atualmente no Hospital do Câncer III, a detecção de anticorpo irregular é realizada na técnica em gel-teste na fase de antiglobulina humana. O presente trabalho teve como objetivos avaliar o impacto da implantação da técnica enzimática na pesquisa de anticorpos irregulares (P.A.I) na rotina pré-transfusional em associação à técnica utilizada na rotina, e estudar o perfil de aloimunização em portadores do câncer de mama atendidos nesse serviço. Entre junho de 2015 e maio de 2016, 429 amostras de sangue de pacientes com câncer de mama coletadas para testes pré-transfusionais foram submetidas à P.A.I pelas metodologias em Liss\\AGH e Nacl\\Enzima. Quando a P.A.I resultava positiva, a identificação do anticorpo era realizada utilizando a técnica correspondente. A frequência de aloimunização encontrada pela técnica de Liss/AGH foi de 1,86% (8/429), enquanto a técnica enzimática revelou uma taxa de aloimunização de 7,6% (32/421) e com associação dos resultados de ambas técnicas obtivemos 9,32% (40/429). . Assim como na literatura, os anticorpos dos sistemas Rh foram os mais frequentes. A rotina institucional apresentou o Anti-D como predominante em 5 amostras (41,6%), seguido por 2 Anti-E (16,6%), 2 Anti-C (16,6%), 1 Anti-Lea (8,4%) , 1 Anti-Jka (8,4%) e 1 Anti-S (8,40). Enquanto que em enzima, o Anti-E foi o mais predominante em 13 amostras (35%), seguido por 9 (24%) autoanticorpos públicos quentes, 7 Anti-Lea (19%), 4 Anti-D (11%), 1 Anti-C (2,75%), 1 Anti-Cw (2,75%), 1 Anti-K (2,75%) e 1 Anti- Dia (2,75%). Para comparar proporções do perfil de aloimunização desses pacientes, foram utilizados os testes Qui-quadrado e Teste G, com valores de p<0,10 considerados significantes. Foram observadas diferenças significantes entre aloimunizados e não aloimunizados quanto à cor da pele, classificação RhD, histórico transfusional e tempo de incidência de aloanticorpo. Observamos que a aloimunização não está correlacionada ao número de transfusões de concentrados de hemácias na Instituição e sim pelo histórico transfusional. Antecedentes transfusionais foram identificados em 27,5% dos aloimunizados e 14% dos não aloimunizados (p) = 0.0398. O predomínio de RhD 7 positivo foi verificado tanto nos aloimunizados quanto nos grupos dos não aloimunizados com percentuais de 75% e 90%, respectivamente (p) = 0.0147. De acordo com a estimativa do tempo para geração de aloanticorpos, todas as aloimunizadas (100%) apresentaram resultado considerado longo, sendo superior a 72 horas. Dentre as não aloimunizadas o mesmo tempo apresentouse em 41 (82%) das pacientes (p) = 0.0583, demonstrando que a técnica enzimática pode ser utilizada com o intuito de se detectar aloanticorpos em reduzido intervalo de tempo pós-exposição a antígenos eritrocitários. Diante desses fatos, propomos a aplicação da fenotipagem eritrocitária para os antígenos dos sistemas Kell e Rh, para os indivíduos portadores do câncer de mama. Também propomos a ampliação deste projeto na rotina pré- transfusional sobre os demais grupos de pacientes oncológicos tratados pelo INCA. Tal medida certamente contribuirá para reduzir o risco de transfusões fenótipo imcompatíveis que poderiam acarretar reações transfusionais hemolíticas ou transfusões ineficazes. / The alloimmunization from erythrocyte antigens usually happens from previous pregnancies or transfusions and becomes a frequent problem among patients undergoing transfusion, even among those transfused sporadically, as in the cases of patients with breast cancer. The detection of irregular antibodies should be performed with sensitive technique capable of detecting the most clinically relevant antibodies. The failure of detecting an alloantibody can provoke acute or delayed hemolytic transfusion reaction of varying intensity that can further worsen the clinical condition of the recipient. Currently in the Cancer Hospital III, irregular antibody detection is performed in the gel-test technique at the stage of human antiglobulin. This study aimed to evaluate the impact of enzymatic technique implementation in irregular antibody screening (PAI) in the pre-transfusion routine in association with the technique used in routine, and study the alloimmunization profile in patients with breast cancer treated in this service. Between June 2015 and May 2016 XXX blood samples (serum? Plasma?) Of patients with breast cancer collected for pre-transfusion tests were submitted to the P.A.I methodologies Liss \\ AGH and NaCl \\ enzyme. When P.A.I resulted positive, identification of the antibody was carried out using the corresponding technique. The frequency of alloimmunization found by the technique Liss / AGH was 1.86% (8/429), whereas the enzymatic technique revealed an alloimmunization rate of 7.6% (32/421) and association of the results of both techniques was 9.32% (40/429). As found in literature, the antibodies of the Rh systems were the most frequent. The institutional routine presented anti-D as prevalent in 5 samples (41.6%), followed by anti-E 2 (16.6%) 2 Anti-C (16.6%) 1 Anti-Lea (8, 4%), anti-Jka 1 (8.4%) and anti-S 1 (8.40). While in enzyme technique, Anti-E was the most predominant in 13 samples (35%), followed by 9 (24%) hot public autoantibodies 7 Anti-Lea (19%) 4 Anti-D (11%), 1 Anti-C (2.75%), Anti-Cw 1 (2.75%) 1 Anti-K (2.75%) and Anti Day 1 (2.75%). To compare alloimmunization profile proportions of these patients, the Chi-square test and test G were used, with p <0.10 considered significant. Significant differences were observed between alloimmunized and not alloimmunized as ethnicity, RhD classification, transfusional historic and time of incidence of alloantibodies. We note that alloimmunization is not correlated to the number of red cell concentrate transfusions in the institution but by transfusional historic. Past transfusions have been identified in 27.5% of alloimmunized and 14% of non-alloimmunized (p) = 0.0398 patients. The prevalence of RhD positive was observed in both groups alloimmunized and non-alloimmunized with 75% and 90%, respectively (p) = 0.0147. According to the estimated time for the generation of alloantibodies, all isoimmunized (100%) had 9 results considered long, and more than 72 hours. Among the non-alloimmunized, 41 (82%) patients (p) = 0.0583 presented the same time, indicating that the enzymatic technique can be used in order to detect alloantibodies in a reduced post-exposure interval to erythrocyte antigens. Given these facts, we propose the application of phenotyping erythrocyte to the antigens of the Kell and Rh systems, for all individuals of breast cancer. We also propose the extension of this project in the pre-transfusion routine on other groups of cancer patients treated by INCA. This measure will certainly help to reduce the risk of transfusion incompatible phenotype that could cause hemolytic transfusion reactions or ineffective transfusions.

by Yu Chung Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 85-102). / Abstract also in Chinese. / Chapter Section 1 --- Literature review / Chapter 1.1 --- Physiology of anemia in the prematurity --- p.1 / Chapter 1.11 --- Erythropoiesis and Erythropoietin --- p.2 / Chapter 1.12 --- Postnatal changes in oxygen transport and delivery --- p.3-6 / Chapter 1.2 --- The concept of oxygen supply and demand --- p.6-7 / Chapter 1.3 --- Compensatory response and adverse effects of anemia --- p.8 -9 / Chapter 1.4 --- Treatments of anemia --- p.9 / Chapter 1.41 --- Red blood cell transfusion --- p.9-13 / Chapter 1.42 --- Recombinant human erythropoietin (rHuEpo) --- p.13 -14 / Chapter 1.5 --- Methods of cardiac output measurements --- p.15 / Chapter 1.51 --- Determination of cardiac output by invasive method --- p.15 -19 / Chapter 1.52 --- Determination of cardiac output by non-invasive methods --- p.20 -30 / Chapter 1.6 --- Methods of oxygen consumption measurements --- p.31 -37 / Chapter 1.7 --- Haemodynamic effects of red blood cell transfusion in preterm infants . --- p.38 -39 / Chapter Section 2 --- Introduction --- p.40 -42 / Chapter Section 3 --- Methodology --- p.43-47 / Chapter Section 4 --- Results --- p.48-51 / Chapter Section 5 --- Discussion --- p.52 -56 / Chapter Section 6 --- Conclusion --- p.57 / Chapter Section 7 --- Future Direction --- p.58 / Chapter Section 7 --- Tables and Figures --- p.59-84 / Chapter Section 8 --- References --- p.85 -102

Introdução: Desde a década de 1970, quando se observou que a transfusão pré-transplante renal diminuía a rejeição ao enxerto, o efeito transfusion-related immunomodulation (TRIM) vem sendo estudado, não havendo, ainda, um entendimento completo de seu mecanismo. Recentemente, a descrição das células T reguladoras (TREG) trouxe novas possibilidades de compreensão desse fenômeno, com grande implicação terapêutica. Objetivos: Avaliar a proporção de células TREG antes e após a transfusão de concentrado de hemácias em uma amostra de pacientes clínicos não imunossuprimidos e correlacionar os resultados com o volume transfundido e o tempo de estocagem do sangue. Métodos: Pacientes com diversas patologias clínicas e com indicação de transfusão de concentrado de hemácias foram recrutados consecutivamente. Após assinatura do consentimento informado, o número de células TREG pré e pós-transfusão foi avaliado, bem como sua correlação com o número de unidades transfundidas e o tempo de estocagem. Resultados: Observou-se um aumento estatisticamente significativo na proporção de células TREG (CD4+, CD25+, FOXP3+) 72 horas após a transfusão (p=0,003). Tal aumento mostrou correlação com o número de unidades transfundidas, mas não com o tempo de estocagem (p=0,017 e 0,49, respectivamente). Conclusão: O aumento do numero de células TREG parece estar presente após transfusão de hemácias não leucodepletadas mesmo em uma amostra heterogênea, o que reforça sua importância clínica. A influência do número de unidades mas não do tempo de estocagem corrobora estudos anteriores. O mecanismo molecular do efeito TRIM, bem como seu impacto clínico em um cenário real, ainda precisam ser melhor avaliados. / Background: Transfusion-related immunomodulation (TRIM) has been the subject of research since the 1970s, when transfusion before kidney transplantation was found to decrease the rates of allograft rejection, but the mechanisms underlying this effect have yet to be fully elucidated. The recent description of regulatory T cells (TREG) has paved the way for a new understanding of this phenomenon, with major therapeutic implications. Objectives: To assess the levels of TREG cells before and after transfusion of packed red blood cells in a clinical sample of non-immunosuppressed patients and to correlate findings with the volume of blood transfused and length of storage. Methods: Patients with a variety of clinical conditions and with an indication for transfusion of packed red blood cells were recruited consecutively. After providing informed consent, pre- and post-transfusion TREG levels were measured, and their correlation with number of units transfused and length of storage was assessed. Results: There was a significant increase in the levels of TREG cells (CD4+/CD25+/FOXP3+) within 72 hours of transfusion (p=0.003). This increase correlated with the number of units transfused, but not with length of storage (p=0.017 and 0.49, respectively). Conclusion: An increase in TREG cell levels appears to follow transfusion of non-leukoreduced packed red blood cells even in a heterogeneous sample, which highlights the clinical relevance of this phenomenon. The influence of number of transfused units, but not of length of storage, on the results corroborates the findings of previous non-clinical studies. Further research is warranted to better investigate the molecular mechanism of the TRIM effect, as well as its clinical impact in real-world settings.

Šiame darbe analizavome DEA 1.1 kraujo tipo pasiskirstymą šunų populiacijoje, kraujo morfologinių rodiklių dinamiką prieš ir po kraujo donacijos praėjus 7, 14 ir 21 dienai. Tyrimai atlikti Kauno smulkių gyvūnų veterinarijos klinikose – ,,Kauno veterinarijos praktika“ ir ,,Santakos veterinarija“, 2012 – 2013 metais. Tyrimo metu nustatyta, kad didžiąją dalį donorų sudaro patelės (67,35 proc.), tarp šunų donorų plačiau paplitęs DEA 1.1-teigiamas kraujo tipas (53,06%). Išanalizavus kraujo morfologinius rodiklius, praėjus 7, 14 ir 21 dienai po kraujo donacijos nustatėme, kad šunų kraujas į normos ribas grįžta per 14 dienų. / In this work was analyze DEA 1.1 blood type distribution in the canine population, dynamics of morphological parameters of blood before and after blood donation at 7, 14 and 21 days. The study was carried out in a small animal clinics – ,,Kauno veterinarijos praktika” and ,,Santakos veterinarija” in Kaunas, 2012 – 2013. The investigation revealed, that most of the donors is a female (67,35 proc.), between the canine donors most prevalence DEA 1.1-positive blood type (53,06%). Analysis of morphological parameters after 7, 14 and 21 days, blood donation revealed, that dog blood returns to the normal range within 14 days.

Severe anemia is the most prevalent life-threatening complication of malaria infection. In addition to destruction of red blood cells (RBC), decreased RBC production or erythropoietic suppression has been shown to contribute to malarial anemia. The mechanism of this suppression is unknown, but it is considered to be multifactorial since erythropoietic suppression can be observed in the presence of both inflammatory mediators and parasite-derived factors. Experiments presented in this thesis aimed at determining the role of host cytokines released in response to blood-stage malaria infection and parasite-derived factors in erythropoietic suppression underlying the development of malarial anemia. Pro-inflammatory cytokines released during malaria infection have been proposed to play a central role in erythroid suppression. To dissect the discrete roles of these cytokines in the processes leading to anemia, mice were treated with CpG-oligodeoxynucleotides (CpG-ODN) which, like malaria infection in humans and experimental mouse models, induces an acute type 1 pro-inflammatory response. CpG-ODN treatment induced anemia, which was associated with suppressed erythropoiesis and reduced RBC survival. Importantly, CpG-ODN-induced IFN-gamma was found to be the major factor mediating erythropoietic suppression but not decreased RBC survival. We also studied the roles of Th1, Th2 and anti-inflammatory cytokines produced in response to Plasmodium chabaudi AS infection in the development of erythropoietic suppression during blood-stage malaria. Signal transducer and activator of transcription (STAT)6, required for signaling of the Th2 cytokines IL-4 and IL-13, was shown to play a critical role in malarial anemia by inhibiting the proliferation and differentiation of erythroid cells. We also observed that suppressed erythropoiesis is a general feature in mice infected with various rodent Plasmodium species that differ in their clinical manifestations and immune responses. Since parasite-derived factors have been shown to contribute to malarial pathogenesis including anemia, the contribution of P. falciparum - and P. yoelii-derived products to erythropoietic suppression was investigated. Both Plasmodium-derived and synthetic hemozoin (Hz) suppressed the proliferation but not the maturation of erythroid progenitor cells in vitro. However, P. yoelii-derived Hz but not synthetic Hz induced transient anemia in mice. These findings provide novel insights into the complex interactions between the parasite and host immune system and the regulation of erythropoiesis during severe malarial anemia.