Immune reconstitution after autologous hematopoietic stem cell transplantation��

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 B���T 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- Hodgkin���s 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-rela����es entre os diversos elementos do sistema immune tem-se mostrado um instrumento essencial para uma melhor compreens��o deste complexo sistema biol��gico. Tal �� particularmente verdade no caso das interac����es entre os linf��citos B e T, quer durante o desenvolvimento celular, quer ao n��vel das fun����es celulares efectoras. A compreens��o da interdepend��ncia entre linf��citos B e T e a possibilidade de manipular esta rela����o pode ser directamente aplic��vel a situa����es em que a imunidade est�� deficiente, como �� o caso das doen��as neopl��sicas ou da imunossupress��o ap��s radio ou quimioterapia. O trabalho apresentado nesta disserta����o iniciou-se com o desenvolvimento de um novo m��todo laboratorial para medir directamente a diversidade do report��rio celular (Cap��tulo III). Redu����es da diversidade do report��rio dos receptores de c��lulas T t��m sido relacionadas com um estado de imunodefici��ncia. O m��todo desenvolvido utiliza ���gene chips���, aos quais hibridizam os ��cidos nucleicos codificantes das cadeias proteicas dos receptores linfocit��rios. A diversidade �� calculada com base na frequ��ncia de hibridiza����o do ��cido nucleico da amostra aos oligonucle��tidos presentes no ���gene chip���. De seguida, e utilizando este novo m��todo e outras t��cnicas de quantifica����o celular examinei, num modelo animal, o papel que as c��lulas policlonais B e a imunoglobulina exercem sobre o desenvolvimento linfocit��rio T no timo, especificamente na aquisi����o de um report��rio diverso de receptores T (Cap��tulos IV e V). Testei, ent��o, a hip��tese de que a presen��a no timo de p��ptidos mais diversos, como a imunoglobulna policlonal, induzisse a g��nese de precursores T mais diversos. Demonstr��mos que a diversidade do compartimento T �� aumentado pela presen��a de imunoglobulina policlonal. A imunoglobulina policlonal, e particularmente os fragmentos Fab desta mol��cula, representam as mol��culas aut��logas mais diversas presentes nos organismos vertebrados. Estes p��ptidos s��o apresentados por c��lulas apresentadoras de antig��nio ��s c��lulas precursoras T no timo, durante o desenvolvimento celular T. Tal, provavelmente, contribui para a g��nese da diversidade dos receptores. Tamb��m demonstr��mos que a presen��a de um report��rio mais diverso de linf��citos T se associa a um incremento da fun����o imunol��gica T in vivo. Uma diversidade de receptores T mais extensa parece permitir um reconhecimento e rejei����o mais eficientes de um maior n��mero de agressores internos e externos. Demonstr��mos que ratinhos com receptores de c��lulas T (RCT) com maior diversidade rejeitam transplantes cut��neos discordantes para antig��nios minor de histocompatibilidade mais rapidamente do que ratinhos com um menor report��rio T (Cap��tulo V). Por outro lado, uma redu����o da diversidade do RCT, causada por timectomia de ratinhos de estirpes selvagens, mostrou aumentar significativamente a sobreviv��ncia de transplantes cut��neos incompat��veis para o antig��nio H-Y (antig��nio minor de histocompatibilidade), indicando uma diminui����o da fun����o linfocit��ria T. Al��m disso, a reconstitui����o da diversidade dos linf��citos T em ratinhos com uma diversidade de report��rio T diminu��da, induzida pela administra����o de fragmentos Fab de imunoglobulina, conduz a um aumento da diversidade dos RCT e a uma diminui����o significativa da sobreviv��ncia dos enxertos cut��neos (Cap��tulo V). Estes resultados sugerem que o aumento do report��rio de c��lulas T contribui para uma melhoria das fun����es celulares T e poder��o ter implica����es importantes na terap��utica e reconstituti����o imunol��gica em contexto de SIDA, neoplasias, autoimunidade e ap��s tratamentos mieloablativos. Baseado nos resultados anteriores, decidimos testar a hip��tese cl��nica de que doentes com neoplasias hematol��gicas sujeitos a transplanta����o de precursores hematopoi��ticos e com recupera����o imunol��gica precoce ap��s transplante teriam uma sobreviv��ncia mais longa do que doentes que n��o recuperassem t��o bem a sua imunidade. Analis��mos a sobreviv��ncia global e sobreviv��ncia sem doen��a de 42 doentes com linfoma n��o Hodgkin de c��lulas do manto sujeitos a transplante aut��logo de precursores hematopoi��ticos (Cap��tulo VI). Os resultados obtidos mostraram que os doentes que recuperaram contagens mais elevadas de linf��citos imediatamente ap��s o transplante aut��logo, apresentaram uma sobreviv��ncia global e sem progress��o mais longa do que doentes que n��o recuperaram contagens linfocit��rias t��o precocemente. Estes resultados demonstram o efeito positivo de uma reconstituti����o imunol��gica robusta ap��s transplante de presursores hematopoi��ticos, sobre a sobreviv��ncia de doentes com neoplasias hematol��gicas. Do mesmo modo, estud��mos o efeito que a recupera����o de n��veis s��ricos normais de imunoglobulina policlonal tem na sobreviv��ncia de doentes com outras neoplasias hematol��gicas de linf��citos B, como o mieloma m��ltiplo,ap��s transplante aut��logo de precursos hematopoi��ticos (Cap��tulo VII). A sobreviv��ncia livre de doen��a dos 110 doentes com mieloma m��ltiplo analizados est�� associada com a sua capacidade de recuperar n��veis s��ricos normais do compartmento policlonal de imunoglobulina. Estes resultados pioneiros indicam a import��ncia da imunoglobulina policlonal para a g��nese de compet��ncia imunol��gica. Tamb��m estud��mos o impacto de um sistema imunit��rio eficiente sobre a resposta ao tratamento com o anticorpo anti CD20, ituximab, em doentes com linfoma n��o Hodgkin (LNH) (Cap��tulo VIII). Os resultados mostram que doentes com valores mais elevados de linf��citos T CD4+ respondem melhor (em termos de maior sobrevida livre de doen��a) ao rituximab, do que doentes com valores mais baixos. Estas observa����es ilustram a necessidade de um sistema imunit��rio competente para o benef��cio cl��nico da terap��utica com rituximab em doentes com LNH. Em conclus��o, o trabalho apresentado nesta disserta����o demonstra que as c��lulas B e a imunoglobulina policlonal promovem a diversidade das c��lulas T no timo e melhoram a fun����o linfocit��ria T perif��rica. Concomitantemente, tamb��m demonstr��mos que, no contexto de reconstitui����o imune, por exemplo, ap��s transplante aut��logo de precursores hematopoi��ticos em doentes com linfomas de c��lulas do manto, o n��mero absoluto de linf��citos �� uma factor independente da sobreviv��ncia. Os resultados demonstram, tamb��m, a import��ncia dos valores de linfocitos T na resposta ao tratamento com rituximab no caso de doentes com LNH. O mesmo princ��pio se prova pelo facto de que doentes com mieloma m��ltiplo sujeitos a transplante aut��logo de precursores hematopoi��ticos que recuperam valores normais s��ricos de imunoglobulinas policlonais, terem melhores taxas de resposta em compara����o com doentes que n��o recuperam valores normais de imunoglobulinas policlonais. Estes resultados podem ter importantes aplica����es na pr��tica cl��nica dado que a maioria dos tratamentos de doen��as neopl��sicas implica imunossupress��o e, subsequente, recupera����o imunol��gica. Estes estudos podem ser um instrumento fundamental para uma melhor compreens��o do sistema imune e guiar uma escolha mais eficiente de op����es terap��uticas bem como contribuir para a concep����o de futuros estudos cl��nicos.

Massive shift in the pneumococcal nasopharyngeal flora after the 7-valent conjugate vaccine: epidemiological studies and testing pathogenic potential in animal models

Dissertation presented to obtain the PhD degree in Biology/Molecular Biology by Universidade Nova de Lisboa, Instituto de Tecnologia Qu��mica e Biol��gica

The materials, technique, conservation treatment and after-care of interior de um convento

Disserta����o apresentada na Faculdade de Ci��ncias e Tecnologia da Universidade Nova de Lisboa para a obten����o do grau de Mestre em Conserva����o e Restauro, especializa����o em pintura sobre tela

What is the value of value-at-risk after all?: A conditional approach using quantile regressions

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA ��� School of Business and Economics

Proxy statement proposals after the 2008 financial crash

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Finance from the NOVA ��� School of Business and Economics

Facing unemployment after being at the top: impact, job search strategies and crucial competences

A Work Project, presented as part of the requirements for the Award of a Masters Degree in Management from the NOVA ��� School of Business and Economics

Electoral opportunism and fiscal policy before and after the EMU

A Masters Thesis, presented as part of the requirements for the award of a Research Masters Degree in Economics from NOVA ��� School of Business and Economics

Osteoclast activity in ankylosing spondylitis patients before and after TNF-blocking therapy

Ankylosing spondylitis (AS) is a chronic inflammatory rheumatic disease of the axial skeleton. The major outcome of this disease is defined by new bone formation, commonly observed in the ligaments of the intervertebral joints, that can lead to the formation of bony spurs, known as syndesmophytes. Previous studies have shown that serum levels of TNF, IL-6 and IL-17 are increased in AS patients and may be implicated in the development of secondary osteoporosis, since these cytokines are able to induce osteoclast (OC) differentiation and, therefore, bone resorption. In this work we aimed to assess the effects of TNF-blocking therapy in the systemic inflammatory environment of AS patients with active disease as well as in OC differentiation and activity. To accomplish this objective, we cultured circulating monocytes from AS patients, before and after therapy, under osteoclastogenic conditions and we performed two functional assays (TRAP staining and resorption pit assay) and analyzed the expression of osteoclast specific genes. We have shown that AS patients with active disease have increased levels of pro-inflammatory cytokines when compared with healthy subjects. We also found that IL-17, TGF-�� and osteoprotegerin are decreased after TNF-blocking therapy. Interestingly, we also observed that after TNF-blocking therapy the expression of some genes is favoring osteoclastogenesis and that differentiated OCs have increased resorption activity. These results suggest that in active AS there may be an uncoupling between inflammation and OC activity that is reset by TNF-blocking therapy.

Health systems efficiency after the crisis in the OECD

This work evaluates the efficiency position of the health system of each OECD country. It identifies whether, or not, health systems changed in terms of quality and performance after the financial crisis. The health systems performance was calculated by fixed-effects estimator and by stochastic frontier analysis. The results suggest that many of those countries that the crisis affected the most are more efficient than the OECD average. In addition, some of those countries even managed to reach the top decile in the efficiency ranking. Finally, we analyze the stochastic frontier efficiency scores together with other health indicators to evaluate the health systems��� overall adjustments derived from the crisis.

How to promote and enhance communication between a big retailer's after-sales department and its suppliers

W is the biggest electronic goods retailer in Portugal accounting with almost fifty percent of market share in its area. During the last years, many small W suppliers had to close their doors, and many others are in huge troubles. Among the reason for this situation, the huge bargaining power of W in the relationship seems crucial. The focus of the directed research will be in the after sales department where I did an internship from September 2014 to January 2015.

Changes in prescribing patterns of benzodiazepines after training of general practitioners

RESUMO: Introdu����o: As benzodiazepinas s��o os f��rmacos ansiol��ticos e hipn��ticos mais utilizados. O elevado consumo destes f��rmacos tem representado uma preocupa����o devido aos efeitos secund��rios do seu uso prolongado e depend��ncia. Portugal tem a maior utiliza����o de benzodiazepinas na Europa. Este estudo pretende analisar a altera����o do padr��o de prescri����o de benzodiazepinas ap��s uma interven����o com cl��nicos gerais. M��todos: A interven����o consistiu numa sess��o educacional a um grupo de cl��nicos gerais. Foi comparado o padr��o de prescri����o de benzodiazepinas dos m��dicos intervencionados com o de um grupo de m��dicos n��o intervencionado da mesma regi��o e com o de um grupo de m��dicos n��o intervencionados de outra regi��o. Analisaram-�����se as prescri����es de 12 meses antes e depois da interven����o. A an��lise do padr��o de prescri����o utilizou como metodologia a Dose Di��ria Definida (DDD) e a Dose Di��ria Definida por 1000 pacientes por dia (DHD). A an��lise estat��stica recorreu a m��todos de regress��o segmentada. Resultados: Houve uma diminui����o no padr��o de prescri����o de benzodiazepinas no grupo intervencionado ap��s a interven����o (p=0.005). Houve tamb��m uma redu����o no padr��o de prescri����o no grupo n��o intervencionada da mesma regi��o (p=0.037) e no grupo n��o-intervencionado da regi��o diferente (p=0.010). Analisando por g��nero, prescritores do g��nero feminino prescrevem uma quantidade maior de benzodiazepinas. Os cl��nicos gerais do g��nero feminino intervencionados tiveram a maior redu����o na prescri����o ap��s a interven����o (p=0.008). Discuss��o: Os dados demonstraram que a interven����o reduziu a prescri����o de benzodiazepinas ap��s a interven����o. A diminui����o geral do padr��o de prescri����o poder�� ser explicada pelo efeito de Hawthorne ou pela contamina����o entre os tr��s grupos de cl��nicos gerais. Os dados dispon��veis n��o explicam as diferen��as nos padr��es de prescri����o por g��nero. Conclus��o: Este estudo demonstra como uma ��nica interven����o tem um impacto positivo na melhoria dos padr��es de prescri����o. A replica����o desta interven����o poder�� representar uma oportunidade para alterar a prescri����o de benzodiazepinas em Portugal. -----------------------------ABSTRACT: Introduction: Benzodiazepines are the most utilized anxiolytic and hypnotic drugs. The high consumption of benzodiazepines has been a concern due to the reported side effects of long-�����term use and dependence. Portugal has the highest benzodiazepine utilisation in Europe. This study aims to analyse the change in General Practitioners��� (GPs) benzodiazepine prescription pattern after na intervention period. Methods: An educational session was delivered to a group of intervened GPs. The benzodiazepine prescription pattern of the intervened group was compared to the pattern of a non-�����intervened matched group from the same region, and to the pattern of another non-�����intervened matched group from a diferente region. The research time frame was 12 month before and after intervention. The analysis of the prescription trends used the Defined Daily Dose (DDD) and Defined Daily Dose per 1000 patients per day (DHD) methodology. The statistical methods consisted of segmented regression analysis. Results: There was a decrease in benzodiazepine prescription pattern of intervened GPs after intervention (p=0.005). There was also a decrease in benzodiazepine prescription pattern for the non-�����intervened group from the same region (p=0.037) and for the non-����� intervened group from a diferente region (p=0.010). Concerningthe analysis by gender, female gender prescribed a higher amount of benzodiazepines. The intervened female gender prescribers presented the highest decrease in prescription trend after intervention (p=0.008). Discussion: The data demonstrated that the intervention was effective in reducing benzodiazepine prescription after intervention. The general decrease in prescription trend might be explained by a Hawthorne effect or a contamination effect between the three groups of GPs. The available data couldn��t explain the diferences in prescription patterns by gender. Conclusion: This study demonstrates how a single intervention has a positive impact on improving prescription trends. The replication of this intervention might be an opportunity to changing the worrying benzodiazepine utilisation in Portugal.

Proxy statement proposals after the 2008 financial crash

In traumatic financial times, both shareholders and the media promptly blame companies for lack of decent corporate governance mechanisms. Proxy statement proposals have increasingly been used by the more active shareholders as to vindicate managers to correct anomalies and restore financial markets��� confidence. I examine the proposals of the largest companies in the S&P 500 index after the Lehmann Brothers crash and their effect on stock prices. Proposals initiated by shareholders negatively impact the company���s stock price, particularly if the proposers are unions, pension funds and institutional investors. Also, I find corporate governance proposals to harm firm���s market performance, unlike compensation and social policy proposals whose effects are intangible. The exception to these disappointing attempts to improve companies��� conduct relies on proposals shared by several investors.