Les vecteurs lentiviraux (LV) sont des outils efficaces de transfert de gène, largement utilisés en thérapie génique, en particulier pour la transduction ex vivo de cellules souches et progénitrices hématopoïétiques (CSPH). Afin d’étudier simultanément la fusion et la transduction dans les CSPH avec les LV, nous avons adapté une méthode basée sur latechnologie du transfert d’énergie entre deux molécules fluorescentes (FRET). Pour mettre en place cette technique, des LV capables d’incorporer spécifiquement une enzyme, la bétalactamase (BLAM-LV) et de coder une forme tronquée du récepteur au facteur de croissance nerveuse (DELTA-NGFR), sont produits. Nos résultats montrent que les LV sont soumis à une restriction post-entrée forte dans les cellules hématopoïétiques, que ce soit dans des lymphocytes T immortalisés ou bien des CSH CD34+. Nous montrons également que cette inhibition post-entrée peut être partiellement saturée après une forte augmentation de la multiplicité d’infection ou en présence d’additifs de culture, comme la Vectofusin-1® ou laRetronectin®. De plus, nous avons montré lors de la transduction de CSPH avec des vecteurs BLAM-LV que la Vectofusin-1® agit sur l’étape d’entrée en augmentant l’adhésion et la fusion entre les membranes virale et cellulaire. Cette technique représente donc un nouvel outil sensible et efficace pour étudier de façon concomitante l’étape de fusion et le niveau de transduction dans les cellules cibles. A terme, ce travail permettra une meilleure compréhension de la biologie des LV mais pourra également conduire à l’élaboration de protocoles de transduction lentivirale plus efficaces. / Lentiviral vectors (LV) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking to precisely evaluate parameters that control the efficiency of transduction in relation with the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer (FRET)-based HIV-1 fusion assay to measure the entry of non-replicative recombinant LV in various cell types, including primary human hematopoietic stem and progenitor cells, and to quantify the level of transduction of he same initially-infected cells. The assay utilizes recombinant LV containing betalactamase (BLAM)-Vpr chimeric proteins (BLAM-LV) and encoding a truncated form of thelow affinity nerve growth factor receptor (DELTA-NGFR). This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing for instance the extent of lentiviral post-entry restrictions occuring in cells of hematopoietic origin. The assay also shows that transduction enhancers like Vectofusin®-1 or Retronectin® can partially relieve this post-entry block but their effects differ in the way to promote LV entry. Furthermore, our results show that Vectofusin®-1 acts at the entry step by promoting the adhesion and the fusion between lentiviral and cellular membranes. In conclusion, one such assay should be useful to study hematopoietic post-entry restrictions directed against LV and should allow improvements in various LV-based gene therapy protocols.

Thesis (Ph. D.)--University of Nevada, Reno, 2005. / Includes bibliographical references. Online version available on the World Wide Web.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20992号 / 医博第4338号 / 新制||医||1027(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小柳 義夫, 教授 髙折 晃史, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

O pacu, Piaractus mesopotamicus, é um teleósteo da Família Characidae, intensivamente cultivado no Brasil por causa de sua rusticidade, crescimento rápido e fácil adaptação, além de seu excelente sabor. Para uma melhor produção de peixes, informações sobre seu sistema imunológico incluindo a histologia dos órgãos linfóides se faz necessária. Sendo assim, o objetivo deste estudo foi descrever histomorfologicamente os órgãos linfóides: rim cefálico, fígado, baço e timo do Piaractus mesopotamicus, analisando os perfis celulares de cada órgão. Foram utilizados 30 animais juvenis, com idade que variaram entre 5 meses a um ano, com peso médio de 588.1 g e comprimento total médio de 27,51 cm. Os órgãos foram pós-fixados em solução de paraformaldeído 4% e Karnovsky, depois desidratados, diafanizados em xilol e incluídos em parafina. Os cortes foram obtidos com uma espessura de 4µm e corados em hematoxilina-eosina. Os esfregaços sanguíneos foram corados em Giemsa-May Grünwald para microscopia de luz. Para microscopia eletrônica de transmissão as amostras fixadas em Karnovsky foram lavadas em sacarose e pósfixadas em tetróxido de ósmio 1%, desidratadas e emblocadas em resina Spurr. O resultado da análise macroscópica mostra que a distribuição do rim, rim cefálico, timo, fígado e baço são as mesmas encontradas na maioria dos teleósteos. Quanto à forma desses órgãos, o fígado apresentou uma variação anatômica na forma e números de lobos, sendo constituído por três lobos. O rim apresentou uma forma em \"H\", onde a região central deste se expandia sobre a bexiga natatória. O rim cefálico, em animais com idade mais avançada, se apresentou como uma dilatação na região cranial do rim, se mostrando bem visível. O timo e o baço apresentaram localização e forma similares aos de outros teleósteos. A microscopia mostrou similaridades entre os órgãos do Piaractus mesopotamicus e outros teleósteos. Na microscopia eletrônica de transmissão foram observadas similaridades ultraestruturais das células encontradas no rim cefálico, fígado, timo e baço e as já descritas em peixes teleósteos. Hemácias, trombócitos, linfócitos, eosinófilos e células granulocíticas especiais encontrados no sangue periférico, tanto de animais jovens quanto dos animais com idade mais avançada, foram os mesmos tipos celulares descritos na literatura de teleósteos. Observando nossos resultados concluímos que histologicamente os órgão linfóides de Piaractus mesopotamicus são similares aos de outros teleósteos e comparando os resultados de microscopia de transmissão concluímos que as estruturas encontradas nas nossas análises são as mesmas das descritas em outras espécies. Os resultados das nossas observações macroscópicas mostraram que o pacu é uma espécie que apresenta algumas características morfológicas singulares e que podem estar ligadas ao tipo de alimentação e a adaptações evolutivas. Esses achados demonstram que a anatomia dos órgãos de peixes e suas variações precisam ser analisados e correlacionados com suas funções e forma de vida de cada espécie. / Piaractus mesopotamicus (Pacu) is a fish from the Characidae Family, it is intensively culture in Brazil because of its rusticity, easy raising and adaptation, besides its excellent flavour. In order to produce a healthy fish, information on its immunological system, including the histology of the lymphoid organs is needed. Therefore, the objective of this study is to describe histomorphologicaly the lymphoid organs: head kidney, liver, spleen and thymus of the Piaractus mesopotamicus, analyzing the cellular profiles of each organ. Thirty young animals, with age varying between 5 months to a year, with average weight and total length of 588.1 g and a 27.51 cm, respectively, were used. The organs were fixed in 4% paraformaldehyde solution, and Karnovsky, afterwards dehydrated, diafanized in xilol and included in paraffin. Four µm sections were obtained and stained with hematoxilin-eosin. Blood smears were stained with Giemsa-May Grünwald for light microscopy. Samples for transmission electric microscopy were fixed in Karnovsky, washed in sacarose and post-fixed with osmium tetroxide 1%, dehydrated and embedded in resin Spurr. The macroscopic analysis show that the localization of the kidney, head kidney, thymus, liver and spleen are very similar to most of the teleosts. Some particularities were observed in the liver, which presented an anatomical variation in the shape and number of lobes, being constituted by three lobes. The kidney presented existe \"H\" shape, where the central region overlaps the swimming bladder. The head-kidney, in adult animals, presented an evident dilation in the cranial region of the kidney. The thymus and spleen presented a similar location and shape to that of the teleosts. The light microscopy studies showed similarities between the organs of the Piaractus mesopotamicus and other teleost fishes. The transmission electron microscopy studies showed ultrastructural similarities, of the cells from the head kidney, liver, thymus and spleen with the literature. Eritrocytes, trombocytes, lymphocytes, eosinophils and special granulocytic cells were found in peripheral blood of both juvenile and adult animals and were similar to the cellular profiles described in the literature to the other teleost fishes. Our studies were successful in describing the macro, micro and ultrastructure of Piaractus mesopotamicus organs and may be used as reference for further research aiming the improvement of fish health status in aquaculture.

The transplantation of bone marrow - more generally said of hematopoietic cells - is in the present time one of the modern therapeutical methods used in cases of malfunction of haematogenesis inside of the bone. The ususal reason for going through with transplantation is leukemia, lymphomas, also anemia, congenital metabolic disorders, autoimmune disorders or some of the solid tumors. Thanks to the transplantation of hematopoietic cells, it is possible to highten the chance of restoration in more than 50 % of patients suffering from oncological disease. The submitted thesis is processed in theoretical form, it is goal is to present the important points leading to progress of this presently very much used treatment and also thorough descripton of present trends in the area of transplantation of hematopoietic cells, that are almost identical in local transplatation centers as they are in the world. Another goal is to describe the role of a general nurse during providing of the care for a patient before and after transplantation of hematopoietic cells. The thesis is focused on the key areas of the issues of transplatation of hematopoietic cells and it is divided into several parts. The first part deals with the general description of the topic, it presents short introduction into the transplantation of hematopoietic cells, the basic division, historical and current indications and short preamble into todays trends. It describes closely the area of the registry of the bone marrow donors, which is the basics for the choosing of the suitable unrelated donor. The second part of the thesis deals with the desciption of historical progress since the very beginnings, through the progress during postwar period, the development of 70s and 80s of the previous century and also the 90s of the 20th century up until the present day. The third part is focused on current methods of collection of hematopoietic cells and following transplatation, it also describes closely the ways of donating hematopoietic cells, the preparation of the patient for transplantation, the course of transplantation and what should the patient prepare for during the early posttransplantation period. The fourth part is focused on nursing, it states thoroughly the role of a nurse in the nursing care before and after transplantation of hematopoietic cells, the principles a nurse has to abide by, and it also closely describes the role of a nurse during transplant application. It also describes dietary and important regimen measures the patient is set to follow after transplantation. The last part is focused especially on complications that are possible after hemopoetic cells transplantation. The necessary data have been obtained through thorough study of professional Czech and foreign monoghraphs and scientific periodicals. The acquisition of the data was significantly contributed to by the attended internsip in Internal hematological and oncological clinic of the Faculty hospital Brno Bohunice. The findings regarding nursing were obtained especially by the study of nursing standards. This thesis points out the important role of transplatation of hemopoetic cells as a method of treatment predominantly of hemooncological illnesses in current medicine and the most important roles of a nurse during the nursing of transplantation patients. The thesis provides a comperehensive overview on the issue of transplants of the hematopoietic cells and describes key areas that are considered as important. It focuses on the historical progress and the desciption of present trends thanks to which the transplantology can be considered as a highly professional and top field. It also highlights the main roles of a nurse during nursing that must be proffesional, qualified, meticulous, considerate and individualized. The results of the survey are supposed to provide the professional and amateur public enough information about the issue of transplantation of the hematopoietic cells.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Maintenance of hematopoiesis depends upon rare hematopoietic stem cells (HSCs), which can persist over an organism’s lifetime. It is conceivable that they must maintain a high degree of genetic stability; otherwise recurring exposure to genotoxins and accumulation of genetic changes could result in genomic instability and malignancy or cell death. We have focused on the response of HSCs and primitive hematopoietic cells to highly toxic DNA double-strand breaks (DSBs). Using assays to detect break rejoining and kinetics of early DSB response foci, we determined that non-cycling human HSC-containing cells display delayed break rejoining kinetics and persistent γH2AX and 53BP1 foci compared to cycling counterparts, more differentiated hematopoietic cells and human primary fibroblasts. In contrast, when stimulated to cycle, these HSC-containing cells are quite efficient at repairing breaks and resolving foci. These data suggest that the DNA damage response may be unusually prolonged in non-cycling primitive hematopoietic cells.

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2009. / Title from title page screen (viewed on February 4, 2010). Research advisor: John M. Cunningham, M.D. Document formatted into pages (xiv, 115 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 89-103).

Chronic myelogenous leukemia (CML) is a clonal hematopoetic stem cell disorder characterized by proliferation of cells expressing BCR-ABL fusion protein. In the BCR-ABL+ leukemia murine model, 12B1, we explored the therapeutic applicability of chaperone-rich cell lysate (CRCL) in the context of syngeneic hematopoietic cell transplantation (HCT) to treat pre-existing leukemia. Our results demonstrate that tumor growth is significantly delayed in mice receiving syngeneic HCT from 12B1 tumor CRCL immunized donors compared to animals receiving HCT from non-immunized donors. CRCL immunization post-immune HCT further hindered tumor growth when compared to immune HCT without post-transplant vaccination. The magnitude of the immune response was consistent with the anti-tumor effects observed in vivo. We also demonstrated that cured mice had developed long-term tumor specific immunity against 12B1 tumor cells. In addition, we documented that both T cells and NK cells contributed to the anti-tumor effect of CRCL vaccination as depletion of either subset hampered tumor growth delay. Thus, our results suggest that CRCL represents a promising vaccine capable of generating specific immune responses. This anti-tumor immunity can be effectively transferred to a host via HCT and further enhanced post-HCT with additional tumor CRCL immunizations.CD4+CD25+ regulatory T cells (Treg) play a central role in the prevention of autoimmunity and in the control of immune responses by down-regulating the function of effector CD4+ or CD8+ T cells. The role of Treg in Mycobacterium tuberculosis infection and persistence is inadequately documented. Therefore, the current study was designed to determine whether CD4+CD25+ FoxP3+ regulatory T cells may modulate immunity against human tuberculosis (TB). Ourresults indicate that the number of CD4+CD25+FoxP3+ Treg increases in the blood or at the site of infection in active TB patients. The frequency of CD4+CD25+FoxP3+ Treg in pleural fluid inversely correlates with local MTB-specific immunity(p<0.002). These CD4+CD25+FoxP3+ T lymphocytes isolated from the blood and pleural fluid are capable of suppressing MTB-specific IFN-γ and IL-10 production in TB patients. Therefore, CD4+CD25+FoxP3+ Treg expanded in TB patients suppress Mycobacterium tuberculosis immunity and may therefore contribute to the pathogenesis of human TB.

Thesis (Ph. D.)--University of Nevada, Reno, 1998. / Includes bibliographical references. Online version available on the World Wide Web.