15 resultados para Chronic lymphoproliferative disorders. Immunophenotyping. Immune system lymphoma
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Dissertation presented to obtain the Ph.D degree in Biology
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Abstract The investigation of the web of relationships between the different elements of the immune system has proven instrumental to better understand this complex biological system. This is particularly true in the case of the interactions between B and T lymphocytes, both during cellular development and at the stage of cellular effectors functions. The understanding of the BT cells interdependency and the possibility to manipulate this relationship may be directly applicable to situations where immunity is deficient, as is the case of cancer or immune suppression after radio and chemotherapy. The work presented here started with the development of a novel and accurate tool to directly assess the diversity of the cellular repertoire (Chapter III). Contractions of T cell receptor diversity have been related with a deficient immune status. This method uses gene chips platforms where nucleic acids coding for lymphocyte receptors are hybridized and is based on the fact that the frequency of hybridization of nucleic acids to the oligonucleotides on a gene chip varies in direct proportion to diversity. Subsequently, and using this new method and other techniques of cell quantification I examined, in an animal model, the role that polyclonal B cells and immunoglobulin exert upon T cell development in the thymus, specifically on the acquisition of a broader repertoire diversity by the T cell receptors (Chapter IV and V). The hypothesis tested was if the presence of more diverse peptides in the thymus, namely polyclonal immunoglobulin, would induce the generation of more diverse T cells precursors. The results obtained demonstrated that the diversity of the T cell compartment is increased by the presence of polyclonal immunoglobulin. Polyclonal immunoglobulin, and particularly the Fab fragments of the molecule, represent the most diverse self-molecules in the body and its peptides are presented by antigen presenting cells to precursor T cells in the thymus during its development. This probably contributes significantly to the generation of receptor diversity. Furthermore, we also demonstrated that a more diverse repertoire of T lymphocytes is associated with a more effective and robust T cell immune function in vivo, as mice with a more diverse T cell receptors reject minor histocompatiblility discordant skin grafts faster than mice with a shrunken T cell receptor repertoire (Chapter V). We believe that a broader T cell receptor diversity allows a more efficient recognition and rejection of a higher range of external and internal aggressions. In this work it is demonstrated that a reduction of TCR diversity by thymectomy in wild type mice significantly increased survival of H-Y incompatible skin grafts, indicating decrease on T cell function. In addiction reconstitution of T-cell diversity in mice with a decreased T cell repertoire diversity with immunoglobulin Fab fragments, lead to a increase on TCR diversity and to a significantly decreased survival of the skin grafts (Chapter V). These results strongly suggest that increases on T cell repertoire diversity contribute to improvement of T cell function. Our results may have important implications on therapy and immune reconstitution in the context of AIDS, cancer, autoimmunity and post myeloablative treatments. Based on the previous results, we tested the clinical hypothesis that patients with haematological malignancies subjected to stem cell transplantation who recovered a robust immune system would have a better survival compared to patients who did not recover such a robust immune system. This study was undertaken by the examination of the progression and overall survival of 42 patients with mantle cell non-Hodgkin lymphoma receiving autologous hematopoietic stem cell transplantation (Chapter VI). The results obtained show that patients who recovered higher numbers of lymphocytes soon after autologous transplantation had a statistically significantly longer progression free and overall survivals. These results demonstrate the positive impact that a more robust immune system reconstitution after stem cell transplantation may have upon the survival of patients with haematological malignancies. In a similar clinical research framework, this dissertation also includes the study of the impact of recovering normal serum levels of polyclonal immunoglobulin on the survival of patients with another B cell haematological malignancy, multiple myeloma, after autologous stem cell transplantation (Chapter VII). The relapse free survival of the 110 patients with multiple myeloma analysed was associated with their ability to recover normal serum levels of the polyclonal compartment of immunoglobulin. These results suggest again the important effect of polyclonal immunoglobulin for the (re)generation of the immune competence. We also studied the impact of a robust immunity for the response to treatment with the antibody anti CD20, rituximab, in patients with non- Hodgkins lymphoma (NHL) (Chapter VIII). Patients with higher absolute counts of CD4+ T lymphocytes respond better (in terms of longer progression free survival) to rituximab compared to patients with lower number of CD4+ T lymphocytes. These observations highlight again the fact that a competent immune system is required for the clinical benefit of rituximab therapy in NHL patients. In conclusion, the work presented in this dissertation demonstrates, for the first time, that diverse B cells and polyclonal immunoglobulin promote T cell diversification in the thymus and improve T lymphocyte function. Also, it shows that in the setting of immune reconstitution, as after autologous stem cell transplantation for mantle cell lymphoma and in the setting of immune therapy for NHL, the absolute lymphocyte counts are an independent factor predicting progression free and overall survival. These results can have an important application in the clinical practice since the majority of the current treatments for cancer are immunosuppressive and implicate a subsequent immune recovery. Also, the effects of a number of antineoplastic treatments, including biological agents, depend on the immune system activity. In this way, studies similar to the ones presented here, where methods to improve the immune reconstitution are examined, may prove to be instrumental for a better understanding of the immune system and to guide more efficient treatment options and the design of future clinical trials. Resumo O estudo da rede de inter-relaes entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreenso deste complexo sistema biolgico. Tal particularmente verdade no caso das interaces entre os linfcitos B e T, quer durante o desenvolvimento celular, quer ao nvel das funes celulares efectoras. A compreenso da interdependncia entre linfcitos B e T e a possibilidade de manipular esta relao pode ser directamente aplicvel a situaes em que a imunidade est deficiente, como o caso das doenas neoplsicas ou da imunossupresso aps radio ou quimioterapia. O trabalho apresentado nesta dissertao iniciou-se com o desenvolvimento de um novo mtodo laboratorial para medir directamente a diversidade do reportrio celular (Captulo III). Redues da diversidade do reportrio dos receptores de clulas T tm sido relacionadas com um estado de imunodeficincia. O mtodo desenvolvido utiliza gene chips, aos quais hibridizam os cidos nucleicos codificantes das cadeias proteicas dos receptores linfocitrios. A diversidade calculada com base na frequncia de hibridizao do cido nucleico da amostra aos oligonucletidos presentes no gene chip. De seguida, e utilizando este novo mtodo e outras tcnicas de quantificao celular examinei, num modelo animal, o papel que as clulas policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocitrio T no timo, especificamente na aquisio de um reportrio diverso de receptores T (Captulos IV e V). Testei, ento, a hiptese de que a presena no timo de pptidos mais diversos, como a imunoglobulna policlonal, induzisse a gnese de precursores T mais diversos. Demonstrmos que a diversidade do compartimento T aumentado pela presena de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta molcula, representam as molculas autlogas mais diversas presentes nos organismos vertebrados. Estes pptidos so apresentados por clulas apresentadoras de antignio s clulas precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a gnese da diversidade dos receptores. Tambm demonstrmos que a presena de um reportrio mais diverso de linfcitos T se associa a um incremento da funo imunolgica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejeio mais eficientes de um maior nmero de agressores internos e externos. Demonstrmos que ratinhos com receptores de clulas T (RCT) com maior diversidade rejeitam transplantes cutneos discordantes para antignios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor reportrio T (Captulo V). Por outro lado, uma reduo da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobrevivncia de transplantes cutneos incompatveis para o antignio H-Y (antignio minor de histocompatibilidade), indicando uma diminuio da funo linfocitria T. Alm disso, a reconstituio da diversidade dos linfcitos T em ratinhos com uma diversidade de reportrio T diminuda, induzida pela administrao de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminuio significativa da sobrevivncia dos enxertos cutneos (Captulo V). Estes resultados sugerem que o aumento do reportrio de clulas T contribui para uma melhoria das funes celulares T e podero ter implicaes importantes na teraputica e reconstitutio imunolgica em contexto de SIDA, neoplasias, autoimunidade e aps tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hiptese clnica de que doentes com neoplasias hematolgicas sujeitos a transplantao de precursores hematopoiticos e com recuperao imunolgica precoce aps transplante teriam uma sobrevivncia mais longa do que doentes que no recuperassem to bem a sua imunidade. Analismos a sobrevivncia global e sobrevivncia sem doena de 42 doentes com linfoma no Hodgkin de clulas do manto sujeitos a transplante autlogo de precursores hematopoiticos (Captulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linfcitos imediatamente aps o transplante autlogo, apresentaram uma sobrevivncia global e sem progresso mais longa do que doentes que no recuperaram contagens linfocitrias to precocemente. Estes resultados demonstram o efeito positivo de uma reconstitutio imunolgica robusta aps transplante de presursores hematopoiticos, sobre a sobrevivncia de doentes com neoplasias hematolgicas. Do mesmo modo, estudmos o efeito que a recuperao de nveis sricos normais de imunoglobulina policlonal tem na sobrevivncia de doentes com outras neoplasias hematolgicas de linfcitos B, como o mieloma mltiplo,aps transplante autlogo de precursos hematopoiticos (Captulo VII). A sobrevivncia livre de doena dos 110 doentes com mieloma mltiplo analizados est associada com a sua capacidade de recuperar nveis sricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a importncia da imunoglobulina policlonal para a gnese de competncia imunolgica. Tambm estudmos o impacto de um sistema imunitrio eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma no Hodgkin (LNH) (Captulo VIII). Os resultados mostram que doentes com valores mais elevados de linfcitos T CD4+ respondem melhor (em termos de maior sobrevida livre de doena) ao rituximab, do que doentes com valores mais baixos. Estas observaes ilustram a necessidade de um sistema imunitrio competente para o benefcio clnico da teraputica com rituximab em doentes com LNH. Em concluso, o trabalho apresentado nesta dissertao demonstra que as clulas B e a imunoglobulina policlonal promovem a diversidade das clulas T no timo e melhoram a funo linfocitria T perifrica. Concomitantemente, tambm demonstrmos que, no contexto de reconstituio imune, por exemplo, aps transplante autlogo de precursores hematopoiticos em doentes com linfomas de clulas do manto, o nmero absoluto de linfcitos uma factor independente da sobrevivncia. Os resultados demonstram, tambm, a importncia dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princpio se prova pelo facto de que doentes com mieloma mltiplo sujeitos a transplante autlogo de precursores hematopoiticos que recuperam valores normais sricos de imunoglobulinas policlonais, terem melhores taxas de resposta em comparao com doentes que no recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplicaes na prtica clnica dado que a maioria dos tratamentos de doenas neoplsicas implica imunossupresso e, subsequente, recuperao imunolgica. Estes estudos podem ser um instrumento fundamental para uma melhor compreenso do sistema imune e guiar uma escolha mais eficiente de opes teraputicas bem como contribuir para a concepo de futuros estudos clnicos.
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Dissertation presented to obtain the Doctorate degree (Ph.D.) in Biology at Instituto de Tecnologia Qumica e Biolgica da Universidade Nova de Lisboa
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Dissertation presented to obtain the Ph.D degree in Biochemistry
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ABSTRACT: Background: Sleep is integral to biological function and sleep disruption can result in both physiological and psychological dysfunction. The acute cognitive consequences of sleep loss has been an active field of recent investigation, evidence suggests that sleep disruption in critically ill older adults can result in acute decrements in cognitive functioning. Surgery activates the innate immune system, inducing neuroinflammatory changes that interfere with cognition. The fact that patients with sleep disorders have an increased likelihood of exhibiting postoperative delirium encourages us to investigate the contribution of perioperative SF to the neuroinflammatory and cognitive responses of surgery. Methods: The effects of 24h sleep fragmentation (SF) and surgery were explored on adult C57BL/6J male mice. SF procedure started at 7 am with the home-cages being placed on a large platform orbital shaker cycled every 120 seconds (30 sec on/90 sec off). This procedure lasted for 24h. Stabilized tibia fracture was performed either before or after the 24h SF procedure. Separate cohorts of mice were tested for systemic and hippocampal inflammation and cognition. Results: Twenty-four hours of SF induced non-hippocampal memory dysfunction and increase in systemic IL-6. SF and surgery caused hippocampal-dependent memory impairment, although memory impairment was not exacerbated by combining SF with surgery. One day after either SF or surgery there was a significant increase in IL6 mRNA and TNF-alpha mRNA. These increments were more pronounced when either pre or post operative SF was combined with surgery. Conclusions: We show that while SF and surgery can independently produce significant memory impairment, perioperative SF significantly increased hippocampal inflammation without further cognitive impairment. The dissociation between neuroinflammation and cognitive decline may relate to our use of a sole memory paradigm that does not capture other aspects of cognition, especially learning.
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Dissertation presented to obtain the Ph.D. degree in Biochemistry at the Instituto de Tecnologia Qumica e Biolgica, Universidade Nova de Lisboa
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Dissertation presented to obtain the Ph.D. degree in Biochemistry
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Dissertation presented to obtain the PhD degree in Biology
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RESUMO:Aterosclerose uma das principais causas de morbilidade e mortalidade no mundo ocidental. responsvel, direta ou indiretamente, pela maior percentagem de gastos com a sade na maioria dos pases europeus. A teoria lipdica da aterosclerose, que se baseia na dislipidemia como causa primria para a doena vascular tem algumas implicaes prticas importantes: permite a definio de linhas de orientao e protocolos simples e ainda estabelece alvos teraputicos que podem ser atingidos na maior parte dos casos com a atual interveno farmacolgica. A associao da aterosclerose com o sistema imunolgico (a teoria imunolgica), forneceu por sua vez novas formas de explorar os mecanismos envolvidos e abriu novas perspetivas para um conhecimento mais completo da doena. No entanto, levanta dificuldades evidentes no que diz respeito s possibilidades teraputicas. De todos os intervenientes no processo aterosclertico (bioqumicos, imunolgicos e anatmicos), as lipoprotenas de elevada densidade (HDL) so atualmente reconhecidas como um dos fatores mais importantes na aterognese. Isto baseado no reconhecimento das mltiplas propriedades anti-aterognicas das HDL como por exemplo: a anti-oxidante, a anti-inflamatria e a antitrombtica, bem como o seu importante papel na melhoraria da funo endotelial. Atualmente, consensual que as funes anti-aterognicas das HDL vo alm do seu papel no transporte reverso do colesterol (RCT) e a importncia das HDL no processo aterosclertico baseia-se no apenas no seu papel protetor impedindo a formao da placa de ateroma, mas tambm na estabilizao destas, prevenindo a sua ruptura e, consequentemente o evento trombtico. Como fundamentais no processo aterosclertico esto reconhecidos dois principais conjuntos de eventos: um caracterizado por alteraes no metabolismo das lipoprotenas que resultam em lipoprotenas pr-inflamatrias e pr-oxidantes que interagem com os componentes celulares da parede arterial e que conduzem formao da placa de ateroma; o outro evento a resposta imunolgica desencadeada contra um novo conjunto de antignios que por sua vez leva produo de citoquinas pr-inflamatrias. Dada a complexidade da HDL e das suas mltiplas funes estas lipoprotenas tornaram-se um potencial alvo para a resposta auto-imune, e cujas consequncias podem explicar algumas das associaes identificados em estudos clnicos e epidemiolgicos. Contudo esta interao entre o sistema imunolgico e HDL nunca foi exaustivamente estudada. Portanto, pomos a hiptese de que em condies oxidativas e pr-inflamatrias, um aumento do antignio (HDL) conduz a um consequente acrscimo na produo de anticorpos anti-HDL (aHDL) responsveis pela alterao quantitativa e / ou qualitativa das HDL. O conceito de que estes anticorpos podem contribuir tanto para a evoluo a longo prazo do processo aterosclertico, como para o desencadeamento de eventos clnicos pode tambm explicar a heterogeneidade encontrada em cada doente e nos grandes estudos clnicos, no que diz respeito aos fatores de risco e outcomes clnicos. Para alm disso, a confirmao desta hiptese pode permitir explicar porque que as intervenes teraputicas atualmente em desenvolvimento para aumentar os nveis de HDL, no conseguem mostrar a to esperada reduo do risco vascular. O objetivo geral desta tese foi identificar e caracterizar a resposta humoral contra os componentes da HDL, e avaliar possveis mecanismos que possam contribuir para a modificao das propriedades anti-aterognicas das HDL. Para alcanar este objetivo investigou-se: 1) A presena de anticorpos aHDL em doentes com lpus eritematoso sistmico (SLE) e em doentes com manifestaes clnicas de aterosclerose, como os doentes com doena arterial coronria (CAD), acidente vascular cerebral isqumico (IS) e diabetes tipo 2; 2) Os principais alvos antignicos dentro do complexo das HDL e a associao entre os ttulos de anticorpos aHDL e diferentes caractersticas clnicas destas doenas; 3) As modificaes das funes normais associadas s HDL, em particular da funo anti-oxidante e anti-inflamatria; 4) A atividade biolgica dos anticorpos aHDL isolados do soro de doentes atravs de um conjunto de experincias in vitro de inibio da atividade da paraoxonase 1 (PON1) e da expresso de molculas de adeso em culturas de clulas endoteliais. Para tal foi necessrio estabelecer um mtodo de isolamento dos anticorpos. Os anticorpos aHDL isolados do soro de doentes foram utilizados de forma a identificar as potenciais alteraes dos sistemas celulares utilizados; 5) O efeito de frmacos usados no tratamento das dislipidemias, em particular o cido nicotnico e as estatinas, na variao dos ttulos de anticorpos aHDL atravs de ensaios clnicos randomizados, controlados com placebo e em dupla ocultao. Os mtodos utilizados neste trabalho incluram: tcnicas imunolgicas (como por exemplo, enzyme-linked immunoabsorbent assay - ELISA, ensaio imunoturbidimetrico e cromatografia de imuno-afinidade) tcnicas bioqumicas (tais como a quantificao de atividade enzimtica por espectrofotometria e por luminescncia), experincias com cultura de clulas e citometria de fluxo. Os nossos resultados mostram que: 1) A presena de anticorpos aHDL, e mais especificamente anticorpos contra alguns do seus principais componentes como a apolipoprotena A-I (ApoA-I, principal apolipoprotena presente nas HDL) e a PON1 (o enzima que mais contribui para a propriedade anti-oxidante das HDL), quer em doentes com doenas auto-imunes, como o SLE, quer em doentes com manifestaes clnicas de aterosclerose, como CAD, IS e diabetes tipo 2. Os doentes apresentaram ttulos de anticorpos IgG aHDL, aApoA-I e aPON1 significativamente mais elevados do que controlos saudveis com a mesma idade e sexo. 2) A correlao positiva estatisticamente significativa entre os ttulos de aHDL e aApoA-I e aPON1 sugere que estes sejam dois dos principais alvos antignicos dentro do complexo das HDL. Os anticorpos encontrados nestes doentes esto associados com a diminuio da atividade da PON1 e a uma reduo da capacidade anti-oxidante total (TAC) do soro, um aumento dos biomarcadores de disfuno endotelial (como por exemplo dos metabolitos do xido ntrico - NO2- e NO3-, as molculas de adeso vascular e intracelular - VCAM-1 e ICAM-1 e os nveis de 3-nitrotirosina). Nos doentes com SLE os ttulos destes esto associados a um aumento do dano cardiovascular e atividade global da doena avaliados pelas escalas SLICC/ACR DI e BILAG score, respetivamente. Enquanto que nos doentes com diabetes tipo 2 estes anticorpos esto associados com um aumento dos nveis de glicemia em jejum (FGP) e hemoglobina glicada (HbA1c). 3) Aps se ter estabelecido um mtodo de isolamento dos anticorpos que permite isolar quantidades significativas de anticorpos do soro de doentes sem perder a sua especificidade, foi identificada a capacidade dos anticorpos isolados do soro de doentes inibirem de uma forma dependente da concentrao a atividade da PON1 at um mximo de 70% no caso dos doentes com SLE e ente 7-52% no caso dos anticorpos isolados de doentes com CAD e IS. 4) O efeito anti-inflamatrio das HDL na inibio da produo de VCAM-1 induzida por citoquinas (como o TNF-) foi revertido em mais de 80% pelos anticorpos aHDL isolados do soro de doentes. 5) A angiogenesis induzida por HDL atravs do aumento do fator de crescimento do endotlio vascular (VEGF) foi anulada em 65% pelos anticorpos aHDL isolados do soro de doentes. 6) Os atuais agentes farmacolgicos disponveis para aumentar as concentraes de HDL-C esto associados a um aumento dos ttulos de anticorpos.-------- ABSTRACTAtherosclerosis is the major cause of morbidity and mortality in the western world. It is also responsible, directly or indirectly, for the highest percentage of health costs in most European countries. Despite the use of new technologies for the diagnosis of vascular disease and regardless of the major advances in treatment, the atherosclerosis-related clinical burden is still raising. The lipid theory of atherogenesis, which identifies dyslipidemia as the primary cause of this vascular disease has some important practical implications: it allows the definition of simple guidelines and establishes therapeutic targets which can be generally met with current pharmacologic intervention. The association between atherosclerosis an the immune system (the immune concept) has in turn provided new ways of exploring the mechanisms involved in this condition and has opened new perspectives in the understanding of the disease. However, it raises obvious difficulties when it comes to treatment options. Of all the players (biochemical, immunological and anatomical) involved in this matter, high-density lipoproteins (HDL) are currently recognised as one of the most important factors in atherogenesis. This is based on the recognition of HDL's multiple anti-atherogenic properties: anti-oxidant, anti-inflammatory and antithrombotic, as well as its capacity to improve endothelial function. Nowadays, it is widely recognized that the anti-atherogenic functions of HDL go beyond reverse cholesterol transport (RCT), and the importance of HDL is based not just on its ability to reduce atheroma formation but also on its ability to stabilise plaques, therefore preventing their rupture and ultimately thrombosis. Two main set of events have been recognised as fundamental in atherogenesis: one, characterized by lipoprotein metabolism alterations, resulting in pro-inflammatory and pro-oxidative lipoproteins, which interact with the normal cellular elements of the arterial wall leading to atheroma formation; the other, the immune cellular response towards new sets of antigens which lead to the production of pro-inflammatory cytokines. Given to HDL complexity and multiple functions this lipoprotein has became a potential target for an auto-immune response, the consequences of which may explain some of the association identified in epidemiological and clinical studies, though the interaction between the immune system and HDL has never been thoroughly addressed. Therefore, we hypothesized that under oxidative and pro-inflammatory conditions, the increase in the antigen (HDL) would lead to a consequent increase in the production of anti-HDL (aHDL) antibodies be responsible for quantitative and/or qualitative changes of HDL. The concept that these antibodies may contribute either to the long-term evolution of atherosclerosis or to the triggering of clinical events may also explain the heterogeneity found in individual patients and in large cohorts regarding risk factors and clinical outcomes. Moreover this may be a major breakthrough in understanding why therapeutic interventions that increase HDL levels, failed to show the anticipated reduction in vascular risk. The overall aims of this thesis were to identified and characterize the humoral response towards HDL components and to evaluate the possible mechanisms that may contribute to the modifications of the anti-atherogenic properties of HDL. To achieve this objective we investigated: 1) the presence of aHDL antibodies in patients with systemic lupus erythematosus (SLE) and in patients with atherosclerosis-related clinical events, such as coronary artery disease (CAD), ischemic stroke (IS) and type 2 diabetes; 2) the association between the titres of aHDL antibodies and different clinical features of these diseases; 3) the modifications of the anti-atherogenic properties of HDL; 4) the biologic effect of aHDL antibodies isolated from serum of patients on the anti-oxidant and anti-inflammatory properties of HDL; 5) the effect of different pharmacologic treatments for dyslipidemia on the prevalence and activity of aHDL antibodies. The methodologies used in this work included immunologic-related techniques (e.g. enzyme-linked immunoabsorbent assay ELISA, immunoturbidimetric immunoassay and immunoaffinity chromatography), biochemical techniques (enzymatic assays with quantification by spectrophotometry and luminescence methods), cell culture experiments and flow cytometry. Our results indicate that: 1) The titres of IgG aHDL, anti-apolipoprotein A-I (aApoA-I) and anti-paraoxonase 1 (aPON1) antibodies were higher in patients with SLE, CAD, IS and type 2 diabetes when compared with age and sex matched healthy controls. 2) The antibodies found in these patients were associated with decreased PON1 activity, (the enzyme responsible for most of the anti-oxidant effect of HDL), reduced total anti-oxidant capacity (TAC) of serum and increased biomarkers of endothelial dysfunction (nitric oxide metabolites, adhesion molecules, nitrotyrosine). In patients with SLE the antibody titres were associated with an increase in disease-related cardiovascular damage and activity whereas in patients with type 2 diabetes they were directly related with the fasting glucose plasma (FGP) levels and the glycosylated haemoglobin (HbA1c). 3) The antibodies isolated from serum of our patients, directly inhibited HDL-associated PON1 activity in a dose dependent way ranging from 7 to 52%. 4) The anti-inflammatory effect of HDL, measured by the percentage of inhibition of the cytokine-induced production of vascular adhesion molecules (VCAM-1), was reduced in more than 80% by aHDL antibodies isolated from our patients. 5) The HDL-induced angiogenesis by increasing vascular endothelial growth factor (VEGF) levels was abrogated in 65% by the antibodies isolated from serum of patients. 6) The current available pharmacologic agents for increasing HDL-C concentrations were associated with an increase in the titres of IgG aApoA-I antibodies. This increase was higher in the extended release niacin when compared to statins probably due to their dampening effect on oxidative stress. In conclusion, aHDL antibodies are present in different pathologic conditions. aHDL antibodies represent a family of self-reacting immunoglobulins, of which ApoA-I and PON1 might be the most relevant targets. These antibodies are biologically active, interfering with the HDL anti-oxidant and anti-inflammatory properties and, consequently, with the atherosclerotic process. The pathogenic potential of these antibodies may lead to the identification of a new biomarker for vascular disease, whilst presenting itself as a novel target for a different treatment approach which may redefine the treatment strategies and clinical trials design for HDL interventions in the future.
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RESUMO - O gnero Listeria contm oito espcies (L. monocytogenes, L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, L. grayi, L. marthii, e a L. rocoutiae), das quais duas so patognicas. L. monocytogenes patognica para humanos e animais; L. ivanovii primeiramente infecta animais e raramente causa doenas em humanos. A Listeria monocytogenes uma bactria patognica Gram-positiva facultativa intracelular, ubqua na natureza. Nos ltimos anos o nmero de casos de listeriose tem vindo a aumentar. Pode causar uma doena rara e grave chamada listeriose, especialmente nas mulheres grvidas, nos idosos ou em indivduos com o sistema imunitrio debilitado, de maneira espordica ou em forma surtos. Realizou-se um estudo observacional, descritivo com o objectivo de se fazer a descrio e a caracterizao do surto de listeriose ocorrido na regio de Lisboa e Vale do Tejo entre 2009-2011. O perodo de maior nmero de casos diagnosticados de listeriose ocorreu entre o ms de Abril e Agosto de 2010. Mas a janela temporal em que ocorreu o surto estendeu-se de Maro de 2009 a Janeiro de 2012. Ocorreram 51 casos de internamento com diagnstico de listeriose, dos quais 25 casos foram confirmados, pela tcnica de PFGE, pertencer mesma estirpe I, sorotipo 4b e pulsotipo 070 e 0101. Na maioria dos casos eram do sexo feminino, com uma mdia de 57,14 anos de idade e com residncia na regio de Lisboa e Vale do Tejo. Em 96,08% dos doentes internados com listeriose apresentavam factores de predisposio, comorbilidade e /ou imunossupresso. A bacteriemia foi a manifestao clnica mais frequente, seguida da meningite. A letalidade da listeriose foi de 15,69%.
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Dissertation presented to obtain the Ph.D degree in Biology.
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RESUMO: A prevalncia das doenas atpicas tem vindo a aumentar, em especial ao nvel dos pases ocidentalizados. Vrios fatores tm sido apontados para justificar este aumento de prevalncia,destacando-se o reduzido tamanho das famlias, o elevado uso de antibiticos, a melhoria das condies sanitrias, bem como a diminuio quer das infees de helmintas, quer da contaminao orofecal. Alguns estudos tm tambm avaliado a influncia do ambiente pr-natal no desenvolvimento de atopia e asma. Da anlise da literatura, parece inegvel a importncia deste perodo para o desenvolvimento do sistema imunitrio. Neste mbito, a transmisso de atopia descendncia em mulheres atpicas, e concretamente com asma alrgica, poder ser moldada desde este perodo. A possibilidade de identificar marcadores de risco precoces para o desenvolvimento de atopia poder ser o primeiro passo para o desenvolvimento de estratgias de preveno para os indivduos em risco. Este trabalho pretendeu abordar o sistema imunitrio materno de forma a enriquecer a sua caraterizao desde o terceiro trimestre da gravidez at ao fim do puerprio. Para alm da explorao de perfis celulares e citocnicos maternos (nos quais se incluiu sobretudo a avaliao de diferentes populaes de clulas T e B, com funes efetoras e reguladoras), foi tambm considerada a sua eventual relao com o desenvolvimento de atopia nas crianas. Foram recrutadas 135 mulheres com critrios para serem includas num dos 4 grupos do estudo: grvidas atpicas GA (n=24), no grvidas atpicas NGA (n=32), grvidas saudveis GS (n=44) e no grvidas saudveis NGS (n=35). Foram caraterizadas por Citometria de Fluxo populaes de leuccitos e linfcitos, com particular interesse nos perfis maturativos de linfcitos T e B, bem como nas subpopulaes de clulas T e B reguladoras. Foi ainda efetuada uma anlise funcional, para avaliar a capacidade de produo de citocinas pelos linfcitos T e B. Foram igualmente avaliadas as concentraes de citocinas sricas por ensaios imunoenzimticos. Estes parmetros imunolgicos maternos foram acompanhados desde o terceiro trimestre de gestao, at depois do puerprio (primeiras 6 semanas ps parto), e aos seis meses de idade, foi efetuada uma avaliao clnica das crianas. As mulheres no grvidas atpicas apresentaram contagens celulares mais elevadas para a generalidade das populaes leucocitrias e linfocitrias (em relao a mulheres no grvidas saudveis). Destaca-se ainda uma maior presena de eosinfilos nas mulheres NGA (p=0,0009; teste de Mann-Whitney U), que tinham igualmente os seus compartimentos linfocitrios T e B mais ricos em clulas de memria, em relao s mulheres NGS. Para os perfis de regulao, verificou-se que as clulas T reguladoras se encontravam percentualmente aumentadas (p0,003; teste de Mann-Whitney U), tal omo as clulas T produtoras de IL10 aps estimulao (p0,03; teste de Mann-Whitney U) em mulheres NGA. Tambm se observou uma maior expresso de Foxp3 (p=0,0002; teste de Mann-Whitney U), e ainda a diminuio dos nveis sricos de IFN- nas mulheres NGA (p=0,0019; teste de Mann-Whitney U), em relao a mulheres NGS. De um modo geral, as alteraes verificadas nos parmetros imunolgicos de mulheres grvidas atpicas no terceiro trimestre da gravidez foram semelhantes s observadas em mulheres grvidas saudveis. Comparadas com mulheres NGA, nas mulheres grvidas atpicas ocorreu uma alterao substancial da frmula leucocitria, com um importante incremento de neutrfilos (p<0,0001; teste de Mann-Whitney U) e diminuio dos valores das restantes populaes leucocitrias. A diminuio nas contagens de linfcitos totais estendeu-se a grande parte das subpopulaes linfocitrias caraterizadas. Nos compartimentos linfocitrios T e B foi possvel observar uma diminuio das subpopulaes de clulas de memria. Verificou-se igualmente na gravidez uma menor expresso de Foxp3 em mulheres GA (p<0,0001; teste de Mann-Whitney U) e ainda menos clulas B CD24HiCD38Hi circulantes (p=0,0012; teste de Mann-Whitney U). Ocrreu ainda uma diminuio relativa das clulas T CD4 produtoras de IFN- em mulheres GA (p0,024; teste de Mann-Whitney U), e uma maior presena de clulas T CD8 produtoras de IL17 (p=0,0172; teste de Mann-Whitney U), em relao ao observado em mulheres NGA. Depois do puerprio, no compartimento T de mulheres do grupo GA, verificou-se um aumento das populaes de clulas de memria. Em comparao com a gravidez, aps o puerprio o compartimento B, apresentou nas mulheres GA um aumento significativo da subpopulao de clulas B de transio (p<0,0001; teste de Wilcoxon). Verificou-se, igualmente em mulheres GA aps o puerprio, uma maior expresso de Foxp3 nas clulas T reguladoras (p<0,0001; teste de Wilcoxon) e o aumento das populaes de clulas T circulantes produtoras de IFN- (p0,0234; teste de Wilcoxon). As modulaes das populaes T e B desde a gravidez at depois do puerprio ocorreram de forma semelhante nas mulheres dos grupos GA e GS. Apesar de as mulheres GA manterem um perfil imunolgico prximo do das mulheres GS depois do puerprio, aconteceu tambm neste perodo um processo de reaproximao ao perfil observado nas mulheres NGA. As mulheres GA com manifestaes de risco para atopia na descendncia (comparadas com mulheres GA sem manifestaes de risco para atopia na descendncia at aos 6 meses de vida) apresentaram uma maior proporo de clulas T e menor proporo de clulas B, percentagens mais elevadas de clulas T CD8 de memria efetoras, de clulas B de transio e de clulas B CD24HiCD38Hi, e contagens mais baixas de clulas B de memria. Na avaliao destes parmetros como marcadores de risco para o desenvolvimento de atopia verificou-se que o parmetro com melhor desempenho foi a percentagem de clulas B de transio, com uma Odds-Ratio de 54,0 [IC 95%: 4,2-692,9; (p=0,0005)], sensibilidade de 90,0% [IC 95%: 55,5 99,8] e especificidade de 85,7% [IC 95%: 57,2 98,2]. Este estudo foi pioneiro em Portugal, e no mundo, no que se refere ao acompanhamento do compartimento linfocitrio B circulante, abordando o seu perfil de maturao, e em particular as clulas B com funes reguladoras, desde a gravidez at ao fim do puerprio, em mulheres atpicas e no atpicas. A este nvel, encontram-se estudos na literatura a documentar a alterao do compartimento B durante a gravidez. O presente trabalho reporta agora que alteraes, como a diminuio do nmero de clulas B em circulao, so impostas tambm na mulher atpica. Em suma, demonstrou-se a existncia de um perfil imunolgico caraterstico em mulheres atpicas, que sofre alteraes significativas durante a gravidez, tendendo os parmetros imunolgicos a normalizar aps o puerprio. O compartimento T, para o qual a literatura mais rica em estudos e abordagens, demonstrou tambm neste trabalho oscilaes caratersticas entre o perodo pr e ps-natal. Verificaram-se sobretudo variaes nos compartimentos de clulas T de memria, sem grandes alteraes ao nvel das clulas Treg no que se refere sua presena em circulao. Apenas a registar a menor expresso de Foxp3 nas clulas Treg durante a gestao observada em mulheres atpicas, tal como em mulheres saudveis (como tambm j foi relatado em estudos anteriores). Apesar de muitos dos dados se encontrarem em concordncia com a literatura, quer no que se refere s subpopulaes de clulas de memria, quer no que se refere s clulas Treg, tambm se encontram resultados discordantes, por exemplo documentando variaes numricas nas clulas Treg em circulao em mulheres atpicas e mulheres atpicas grvidas. A importncia de harmonizar protocolos e fentipos, parece crucial na abordagem de estudos futuros. Ao nvel do risco para a atopia na descendncia de mulheres atpicas, acrescentou-se ainda a possibilidade de definir marcadores no invasivos para a criana, em particular as clulas B de transio. Estas clulas, cuja maior presena em circulao no recm-nascido foi recentemente associada com manifestaes alrgicas subsequentes, so agora apontadas j na mulher atpica, grvida do terceiro trimestre, como um elemento de risco para o desenvolvimento de atopia. Os marcadores de risco descritos, para alm de facilmente poderem vir a ser englobados no mbito dos normais rastreios maternos durante a gravidez, apresentam ainda a vantagem da precocidade do diagnstico, permitindo no s a possibilidade de preveno ps-natal, mas estendendo esta possibilidade ao perodo gestacional.----------------------------ABSTRACT: The prevalence of atopic diseases has been increasing, especially in Westernized countries. Several factors have been suggested to justify this increase in prevalence, as the small size of families, the high use of antibiotics, the improvement in sanitation conditions, as well as the reduction of both helminth infections, and orofecal contamination. A few studies have adressed the influence of prenatal environment on the development of atopy and asthma. From literature, it seems undeniable the importance of the prenatal period for the development of the immune system. In this context, the transmission of atopy to the progeny in atopic women, and specifically in women with allergic asthma, can be modulated from this period on. The ability to detect early risk markers for the development of atopic diseases may be the first step in the development of prevention strategies for individuals at risk. This study aimed to approach the maternal immune system in order to enrich its characterization from the third trimester of pregnancy until the end of the puerperium period. In addition to the evaluation of the maternal cellular profiles (in which, mostly, diferente populations of T and B cells with effector and regulatory functions were included) and citokines, the relation between these profiles and the development of atopy in the progeny was also assessed. 135 women were recruited for this study, and fullfiled the inclusion criteria necessary to be included in one of the four groups preset: atopic pregnant women - GA (n = 24), atopic nonpregnant women - NGA (n = 32), healthy pregnant women - GS (n = 44) and healthy nonpregnant women - NGS (n = 35). Populations of leukocytes and lymphocytes, and particularty maturation profiles of T and B lymphocytes, as well as subpopulations of T and B cells with regulatory functions, were characterized by flow cytometry. Functional assays were also performed, to assess the ability of cytokine production by T and B lymphocytes. Serum cytokine concentrations were assessed as well by enzymatic immunoassays. These maternal imune parameters were monitored since the third trimester of pregnancy until the end of the puerperium period (first six weeks after delivery). A clinical evaluation of all the newborn children was performed at the age of six months. Non-atopic pregnant women presented higher cell counts for most leukocyte and lymphocyte populations (compared to healthy non-pregnant women). We should also highlight the increased presence of eosinophils in NGA women (p = 0,0009; Mann-Whitney U test). Again compared to NGS women, NGA women showed increased memory cells within the circulating T and B lymphocyte compartments. Considering the regulatory profiles, NGA women presented higher percentages of regulatory T cells (p0,003; Mann-Whitney U test) and IL10 producing T cells after stimulation (p0,03; Mann Whitney U), as well as increased expression of Foxp3 (p = 0,0002; Mann-Whitney U test), and also decreased serum levels of IFN- (p = 0,0019; test Mann-Whitney U test) compared to NGS women. In general, the changes observed in immune parameters of atopic pregnant women in the third trimester of gestation were similar to those observed in healthy pregnant women. Comparing pregnant and non-pregnant atopic women, an important change in leukocyte subsets was observed, with a significant increase of neutrophils (p <0,0001; Mann-Whitney U test) and the consequent diminution of the remaining leukocyte populations in the GA group. The decrease in total lymphocyte counts was extended to most of the lymphocyte subsets characterized. It was possible to detect a decrease in memory cell subsets within the T and B lymphocyte compartments, also. During pregnancy, a lower expression of Foxp3 was reported in GA women (p <0,0001; Mann-Whitney U test) and, besides, lesser CD24HiCD38Hi B cells were present in circulation in these women, compared to NGA women (p = 0,0012; Mann-Whitney U test). There was still a decrease in the percentages of IFN--producing CD4 T cells in GA women (p0,024; Mann-Whitney U test) and a greater presence of IL17-producing CD8 T cells (p = 0,0172; Mann-Whitney U test), compared to the levels observed in NGA women. At the end of the puerperium, there was an increase in memory cell subpopulations within the T cell compartment of GA women. Compared with the pregnancy evaluation, after puerperium, the B cell compartment showed a significant increase in the transitional subpopulation (p<0,0001; Wilcoxon test), in GA women. Moreover, after puerperium, GA women exhibited a greater expression of Foxp3 in Treg cells (p <0,0001; Wilcoxon test) and there was an increase in circulating IFN--producing T cells (p0,0234; Test Wilcoxon). The modulations of T and B cell subpopulations from pregnancy until the end of puerperium were similar in women of GA and GS groups. Although at the end of puerperium, GA women still kept an immune profile close the one observed in GS women, at this time point, there were also signs of rapprochement between the immune profiles observed in women of GA and NGA groups. GA women with atopic manifestations in the offspring (compared to GA women without atopic manifestations in the offspring at the age of 6 months) presented higher proportions of T cells and lower proportions of B cells, higher percentages of effector memory CD8 T cells, transitional B cells and CD24HiCD38Hi B cells, and, finally, lower absolute counts of memory B cells. In the evaluation of these parameters as risk markers for the development of atopy, the parameter which presented the best performance was the percentage of transitional B cells, with an Oddsratio of 54,0 [95% CI: 4,2 to 692,9; (p = 0,0005)], sensitivity of 90,0% [95% CI: 55,5 to 99,8] and a specificity of 85,7% [95% CI: 57,2 to 98,2]. This study was a pioneer in Portugal, and in the world, in what concerns the monitoring of the circulating B cell compartment, addressing not only the maturation profile, but, in particular, B cells with regulatory functions, from pregnancy untill after puerperium, in atopic and non-atopic women. Literature presents evidence of a typical change in circulating B cells during pregnancy. This study now reports that changes, such as the decrease in the number of circulating B cells,/ are also imposed by pregnancy in atopic woman. In brief, it demonstrated the existence of a characteristic immune profile in atopic women, which undergoes significant alterations during pregnancy, tending to normalize after the puerperium. As for the T cell compartment, for which the literature is richer in studies and approaches, this study also showed characteristic fluctuations between the pre- and postnatal periods. There were variations mostly in the memory subsets within the T cell compartment, without major changes in regulatory T cells regarding their presence in circulation. Only the expression of Foxp3 in Treg cells presented lower levels during pregnancy, in both atopic and healthy women (as previously reported in other studies). Although much of the data now reported are in agreement with literature, regarding either memory cell subsets or regulatory T cells, there are also conflicting results, for example documenting changes in the numbers of regulatory T cells circulating in atopic pregnant and atopic non-pregnant women. The importance of harmonizing protocols and phenotypes seems crucial for the establishement of future studies. Considering the risk for atopy in the offspring of atopic women, this study added the possibility to define non-invasive markers for the child, in particular transitional B cells. These cells, whose greater presence in circulation in newborns has recently been associated with subsequent allergy development, are here identified in atopic pregnant women in the third trimester of gestation as a risk factor in the development of atopy in their progeny. The risk factors described, besides having the capacity to easily become integrated within the normal maternal screening protocols during pregnancy, also have the advantage of an early diagnosis, allowing not only the possibility of postnatal prevention but extending this possibility to the prenatal period.
Resumo:
Many viruses have developed numerous strategies to recruit and take advantage of cellular protein degradation pathways to evade the cellular viral immune system. One such virus is the Kaposis Sarcoma associated herpesvirus (KSHV), first discovered in Kaposis Sarcoma lesions found in AIDS patients. Latency-Associated Nuclear Antigen (LANA) is a KSHV multifunctional protein responsible for tethering viral DNA to the chromosome ensuring maintenance and segregation of the viral genome during cell division. Besides its main role of viral maintenance, LANA also physically interacts with several host proteins to modulate cell functions. One such function is to recruit the EC5S ubiquitin-ligase complex by interacting with Elongin BC complex and Cullin 5 protein, which in turn ubiquitinate substrates such as NF-B and p53 to allow persistent viral infection. Like any other post-translation modifications, ubiquitination is reversible through deubiquitination enzymes (DUBs). LANA also interacts with ubiquitin specific protease 7 (USP7), a deubiquitination enzyme involved in regulation of several proteins including p53. Interaction with USP7 is made through a conserved peptide motif, which is also present in LANA. This work addresses the role of LANA in the recruitment and modulation of the ubiquitination and deubiquitination pathways. Despite the continued efforts in uncovering new LANA interacting partners to form a functional EC5S ubiquitin-ligase complex, only MHV-68 LANA interacted directly with Elongin BC, other interactions were not direct and may require a linker protein. On the other hand, LANA interaction with USP7 was able to be analysed by X-ray structure determination. In addition to a conserved P/AxxS motif, a novel Glutamine (Gln) residue from KSHV LANA was shown to make a specific interaction with USP7. This Gln residue is also present in other herpesvirus protein and hence it might be a conserved motif within herpesviruses.
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Cancer remains as one of the top killing diseases in first world countries. Its not a single, but a set of various diseases for which different treatment approaches have been taken over the years. Cancer immunotherapy comes as a new breath on cancer treatment, taking use of the patients immune system to induce anti-cancer responses. Dendritic Cell (DC) vaccines use the extraordinary capacity of DCs antigen presentation so that specific T cell responses may be generated against cancer. In this work, we report the ex vivo generation of DCs from precursors isolated from clinical-grade cryopreserved umbilical cord blood (UCB) samples. After the thawing protocol for cryopreserved samples was optimized, the generation of DCs from CD14+ monocytes, i.e., moDCs, or CD34+ hematopoietic stem cells (HSCs), i.e, CD34-derived DCs, was followed and their phenotype and function evaluated. Functional testing included the ability to respond to maturation stimuli (including enzymatic removal of surface sialic acids), Ovalbumin-FITC endocytic capacity, cytokine secretion and T cell priming ability. In order to evaluate the feasibility of using DCs derived from UCB precursors to induce immune responses, they were compared to peripheral blood (PB) moDCs. We observed an increased endocytosis capacity after moDCs were differentiated from monocyte precursors, but almost 10-fold lower than that of PB moDCs. Maturation markers were absent, low levels of inflammatory cytokines were seen and T cell stimulatory capacity was reduced. Sialidase enzymatic treatment was able to mature these cells, diminishing endocytosis and promoting higher T cell stimulation. CD34-derived DCs showed higher capacity for both maturation and endocytic capacity than moDCs. Although much more information was acquired from moDCs than from CD34-derived DCs, we conclude the last as probably the best suited for generating an immune response against cancer, but of course much more research has to be performed.
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The immune system comprises of different cell types whose role is to protect us against pathogens. This thesis investigates a very important mechanism for our organism protection in a specific disorder: cross-presentation in Wiskott-Aldrich Syndrome (WAS). WAS is caused by loss-of-function mutations in the cytoskeletal regulator WASp and WAS patients suffer from eczema, thrombocytopenia, and immunodeficiency. X-linked neutropenia (XLN) is caused by gain-of-function mutations in WASp and XLN patients suffer from severe congenital neutropenia and immunodeficiency. This thesis was focused on the role of B and T lymphocytes and dendritic cells (DCs). This work will be divided into two main topics: 1) In the first part I studied the capacity of B cells to take up, degrade and present antigen. Moreover I studied the capacity of B cells to induce T cell proliferation. 2) In the second part, I studied T cell proliferation induced by dendritic cells. To increase our understanding about this mechanism, additional experiments were performed, including acidification capacity of CD8+ and CD8- DCs, reactive oxygen species (ROS) production since it is directly connected to acidification. These assays were measured by flow cytometry. Localization of Rac1 and Rac2 GTPases was assessed by confocal microscopy. Proliferation, acidification and ROS production assays were performed also with cells from X-linked neutropenia (XLN) mice. From this study we concluded that B cells cannot induce CD8+ T cell proliferation however they take up and present antigen. Moreover I have shown that increased cross-presentation by WASp KO DCs with ovalbumin is associated with decreased capacity to acidify endosomal compartment; and WASp KO CD8- DCs have increased Rac2 localization to the phagosome. XLN dendritic cells have similar acidification and ROS production capacity than wildtype cells. In conclusion, our data suggests that WASp regulates antigen processing and presentation in DCs.
