Dentre as enfermidades que causam alterações hematológicas em bovinos podemos citar a leucose enzoótica bovina (LEB). Podendo em alguns animais, determinar formações tumorais por infiltração de células mononucleares (linfócitos, pró-linfócitos e linfócitos atípicos), em diferentes tecidos. A doença possui quadro sintomático pleomórfico e alterações hematológicas, evidenciadas por leucocitose e linfocitose persistente, com aumento de formas linfocitárias atípicas. Desta forma o presente estudo tem como objetivo avaliar a atipia linfocitária em bovinos da raça Holandesa Preto e Branco em animais soropositivos com ou sem a presença de linfocitose persistente e animais soronegativos para LEB e correlacioná-la com a apoptose de células CD5+ e a proliferação linfocitária. Assim, foram selecionados 56 animais, divididos de acordo com o leucograma e o sorodiagnóstico da LEB pela imunodifusão em ágar gel e pelo ensaio imunoenzimático em: negativos (n = 25), alinfocitóticos (AL, n= 12) e com linfocitose persistente (LP, n=19). Destes, 15 animais tiveram suas amostras sanguíneas avaliadas quanto a proliferação de linfócitos e apoptose. Os resultados deste estudo mostraram que, a contagem de leucócitos totais e os valores absolutos e relativos de linfócitos atípicos foram maiores no grupo LP. Em relação aos linfócitos atípicos, os linfócitos sombra de Gümprecht, linfócito monocitóide e linfócito com núcleo duplo, foram encontrados nos três grupos de estudo, porém mais comumente em animais infectados pelo VLEB, predominantemente no grupo manifestando linfocitose persistente. Este trabalho demonstrou uma menor proliferação de linfócitos nos animais infectados pelo VLEB manifestando LP, associado a uma menor apoptose de células CD5+. Na análise da correlação entre a apoptose de células CD5+ e a porcentagem das populações linfocitárias entre os 15 animais, observou-se que a apoptose tem uma correlação positiva com os linfócitos com núcleo duplo, o que não ocorreu nas demais populações celulares. Na correlação entre a proliferação, foi observado nos 15 animais, que quanto maior a porcentagem de linfócitos menor é a proliferação e quanto maior a porcentagem de linfócitos monocitóides menor a proliferação, o que não ocorreu nas demais populações celulares. Portanto, a manifestação da LP em animais naturalmente infectados pelo VLEB, pode ser associada ao aumento de linfócitos atípicos do tipo sombra de Gümprecht, linfócito monocitóide e linfócito núcleo duplo e da menor apoptose das células CD5 + e da menor proliferação de linfócitos / Among the diseases that cause hematological changes in cattle can cite the enzootic bovine leukemia (EBL). And in some animals, to determine tumor formations by infiltration of mononuclear cells (lymphocytes, pro-lymphocytes and atypical lymphocytes) in different tissues. The disease has symptoms very pleomorphic and hematological changes, evidenced by leukocytosis, and persistent lymphocytosis, an increase of atypical lymphocyte forms. Thus this study aims to evaluate the atypical lymphocyte in Holstein Black and White in seropositive animals with or without the presence of persistent lymphocytosis and animals seronegative LEB and correlate them with apoptosis of cells CD5 + and lymphocyte proliferation. Thus, we selected 56 animals, divided according to the WBC and serodiagnosis of LEB by agar gel immunodiffusion and by enzyme immunoassay in: negative (n = 25), non- lymphocytic (AL, n = 12) and with persistent lymphocytosis (PL, n = 19). Of these, 15 animals had their blood samples evaluated for lymphocyte proliferation and apoptosis. The results of this study showed that in total leukocytes and absolute and relative values of atypical lymphocytes were higher in group LP. Regarding atypical lymphocytes, lymphocytes Gumprecht shadow, lymphocyte and monocytoid lymphocytes with dual core, were found in the three study groups, but more commonly in animals infected VLEB, predominantly in the group with persistent lymphocytosis. This study demonstrated a reduced proliferation of lymphocytes in animals infected VLEB expressing LP, associated with lower apoptosis of CD5 + cells. In analyzing the correlation between apoptosis of cells and the percentage of CD5 + lymphocyte populations among the 15 animals, we observed that apoptosis has a positive correlation with lymphocytes with dual core, which did not occur in other cell populations. Correlation between proliferation, was observed in 15 animals, the higher the percentage of lymphocyte proliferation and lower the higher the percentage of monocytoid lymphocytes less proliferation, which did not occur in other cell populations. Therefore, the expression of LP in animals naturally infected VLEB may be associated with increased atypical lymphocytes of the type of shadow Gumprecht, lymphocyte and lymphocyte monocytoid dual-core and reduced apoptosis of CD5 + cells and reduced lymphocyte proliferation

La neuropiline-1 (Nrp-1) est une protéine transmembranaire agissant comme récepteur des sémaphorines de classe 3 (Sema3) et du facteur de croissance de l'endothélium vasculaire (VEGF). En plus de son rôle crucial dans le développement des systèmes nerveux et cardiovasculaires, Nrp-1 est impliquée dans des processus physiopathologiques impliquant certains de ses ligands classiques (Sema3 et VEGF) ou récemment caractérisés (TGF-β1 et PDGF) dans les tissus adultes. Dans le système immunitaire, Nrp-1 participe aux interactions entre les lymphocytes T et les cellules dendritiques, transmet les effets immunorégulateurs de Sema3A sur les lymphocytes T, et est impliqué dans le mécanisme suppresseur des lymphocytes T régulateurs (Treg) Foxp3+. Cependant, l'expression de Nrp-1 dans les lymphocytes T non-Treg humains et murins n'a été que peu étudiée. L'objectif de ce projet était de caractériser les populations de lymphocytes T Nrp-1+ non-Treg chez la souris, d'analyser leurs fonctions, d'identifier les mécanismes conduisant à l'expression de Nrp-1 dans ces cellules, et de comprendre le rôle joué par Nrp-1 dans les réponses immunitaires T. Mon travail s'est d'une part intéressé à une population de lymphocytes T non-conventionnels appelés lymphocytes « invariant natural killer T » (iNKT). Les lymphocytes iNKT sont des lymphocytes Tαβ dérivés du thymus aux propriétés immunomodulatrices reposant sur la sécrétion rapide de cytokines TH1 et TH2 après engagement de leur TCR semi-invariant par des complexes CD1d/glycolipide. Un sous-type distinct de cellules iNKT, dont l'origine et l'homéostasie sont encore mal connues, produit la cytokine pro-inflammatoire IL-17. Dans ce travail, j'ai montré que les lymphocytes iNKT émigrés thymiques récents sont identifiés spécifiquement par l'expression de Nrp-1. Les lymphocytes iNKT producteurs d'IL-17 expriment Nrp-1 et dépendent de l'export thymique. D'autre part, Nrp-1 est exprimé par les thymocytes immatures en division et certains lymphocytes Tαβ conventionnels mémoires en prolifération homéostatique rapide. In vitro, l'activation des lymphocytes T par le TCR induit l'expression de Nrp-1 de manière dépendante de la voie de signalisation Ca2+/calcineurine/NFAT. L'expression de Nrp-1 dans les lymphocytes T activés les sensibilise aux effets régulateurs de Sema3A et TGF-β1. En conclusion, ces résultats apportent de nouvelles données concernant l'expression et la fonction de Nrp-1 dans le système immunitaire. Plus généralement, cette étude permet d'envisager des stratégies thérapeutiques ciblant les processus dépendants de Nrp-1 dans les pathologies du système immunitaire et du système nerveux ou les cancers.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Thesis (Ph.D. in Immunology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 146-169). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Introdução: O transplante de células tronco hematopoiéticas é capaz de curar as doenças hematológicas. O papel da repopulação linfocitária precoce no período pós transplan te visa combater a células neoplásicas que resistiram ao regime de condicionamento e prevenir as infecções oportunistas graves. Sendo assim, uma contagem elevada de linfócitos no período pós transplante é capaz de reduzir a mortalidade relacionada ao transplante (TRM), melhorar a sobrevida livre de doença e reduzir a taxa de recidiva. Objetivos: Avaliar a recuperação linfocitária precoce no D+21 e D+30 pós transplante correlacionado com a taxa de recidiva da doença de base, mortalidade, sobrevida global e livre de doença. Analisar a freqüência das complicações infecciosas neste período. Métodos: Analisado o número absoluto de linfócitos no D+21 e D+30 pós transplante de células tronco hematopoiéticas. Conforme dados da literatura definiu-se no D+21 e no D+30 aqueles com número absoluto de linfócitos abaixo e acima de 300 e se correlacionou os dados obtidos com a taxa de óbito, taxa de recidiva, sobrevida global em 5 anos, sobrevida livre de doença, em 5 anos, TRM em 100 dias. e mortalidade não relacionada a recaída (NRM). Resultados: Neste estudo foram incluídos 100 pacientes portadores das seguintes neoplasias hematológicas: leucemia mielóide aguda, leucemia linfocítica aguda, leucemias secundárias e síndrome mielodisplásica. Destes, 55 pacientes eram do sexo masculino e 45 do sexo feminino. A média de idade foi de 27,9 anos (mínima 9 meses e máxima 55 anos). A mediana do tempo de seguimento foi de 601 dias (IC 95% 106-1845). A mediana de CD 34 infundidos foi de 4,0 (IC 95% 2,4-5,7) e quanto a origem destas células CD 34 infundidas 85% foram de medula óssea (MO), 12% periférica (PBSC) e 3% sangue de cordão umbilical (SCU). Quanto ao tipo de condicionamento realizado 22% foram não mieloablativos e 78% mieloablativos.A mediana de linfócitos no D+21 foi de 460 (IC 95% 0 - 6250) e no D+30 foi de 760 (IC 95% 40-6370). Com relação a taxa de infecções observou-se que 19% das infecções foram de etiologia viral, 65 % bacteriana e 17% fúngicas. A sobrevida global (OS) em 5 anos foi de 44 % , sobrevida livre de doença (DFS) foi de 37,7% , a mortalidade relacionada ao transplante (TRM) em 100 dias foi de 32,5%. E a mortalidade não relacionada a recidiva (NRM) em 5 anos foi de 40,2%. No desfecho óbito observamos que 69% dos pacientes que foram a óbito no D+21 tinham linfócitos abaixo de 300, e 43,9% tinham linfócitos acima de 300 (p<0,05). Pacientes com valores menores que 300 no dia 30 tem 2,20 vezes o risco de irem a óbito quando comparados com aqueles com valores acima de 300 (IC 95% 1,03-4,69) ajustado para DECH e CD34. Pacientes com valores menores que 300 no dia 30 tem 3,76 vezes o risco de irem a óbito em menos de 100 dias quando comparados com aqueles com valores acima de 300 (IC 95% 1,23-11,46) Conclusões: A reconstituição linfocitária precoce (> 300) no D+21 e no D+30 melhora a sobrevida global e livre de doença, bem como reduz a taxa de recidiva da doença de base e reduz a mortalidade. / Background: The role of repopulating lymphocyte after allogenic stem cell transplantation (SCT) includes the prevention of serious infections and attacking residual tumor cells in the early post transplant phase. Therefore, the current study analysed the role of the absolute lymphocyte count (ALC) on day 21 and 30 after SCT in predicting transplant outcomes of patients in terms of the risk of transplant related mortality (TRM) recurrence of original disease and risk of opportunistic infections. Objective: Evaluate early lymphocyte recovery on D +21 and D +30 posttransplant correlated with the rate of recurrence of the underlying disease, mortality, overall survival and disease free survival. Analyzed the frequency of infectious complications in this period. Methods: Analyzed the absolute lymphocyte count in the D +21 and D +30 after hematopoietic stem cell transplantation. According to literature data set the we correlate the absolute lymphocyte count in the D +21D +30 below and above 300 these data with the rate of death, relapse rate, overall survival in 5 years, disease-free survival in 5 years , TRM in 100 days and mortality unrelated to relapse (NRM). Results : Included in the study 100 patients with the following hematologic malignancies: acute myeloid leukemia, acute lymphocytic leukemia, secondary leukemia and myelodysplastic syndrome. Of these, 55 patients were male and 45 female. The average age was 27.9 years (minimum 9 months and maximum 55 years). The median follow-up was 601 days (95% CI 106-1845). The CD 34 median that was infused was 4.0 (95% CI 2.4 to 5.7).The source of stem cells infused was 85% of bone marrow (BM), peripheral 12% (PBSC) and 3 % of umbilical cord blood (UCB). Regarding the type of conditioning performed 22% were non myeloablative and 78% of lymphocytes were mieloablativos. The median of absolute lymphocyte count in the D +21 was 460 (95% CI 0 to 6250) and D +30 was 760 (95% CI 40- 6370 ). Regarding the rate of infections were observed 19% viral infections , bacterial in 65% and fungal in 17%. Overall survival (OS) at 5 years was 44%, disease-free survival (DFS) was 37.7%, transplant related mortality (TRM) in 100 days was 32.5%. Non relapsed mortality (NRM) at 5 years was 40.2%. The death rate found that 69% of patients who died at the D +21 had presented lymphocytes count below 300, and 43.9% were above 300 lymphocytes (p <0.05). Patients with counts less than 300 in D+30 presented 2.20 times risk of death when compared with those who presented values above 300 (95% CI 1.03 to 4.69) adjusted for GVHD and CD34. Patients presenting values less than 300 in 30 days have 3.76 times more risk of death in less than 100 days compared with those with values above 300 (95% CI 1.23 to 11.46). Conclusions: The early lymphocyte reconstitution (> 300) in D +21 D +30 improves overall survival and disease-free and reduces the relapse rate of the underlying disease and reduces mortality.

Introdução: O transplante de células tronco hematopoiéticas é capaz de curar as doenças hematológicas. O papel da repopulação linfocitária precoce no período pós transplan te visa combater a células neoplásicas que resistiram ao regime de condicionamento e prevenir as infecções oportunistas graves. Sendo assim, uma contagem elevada de linfócitos no período pós transplante é capaz de reduzir a mortalidade relacionada ao transplante (TRM), melhorar a sobrevida livre de doença e reduzir a taxa de recidiva. Objetivos: Avaliar a recuperação linfocitária precoce no D+21 e D+30 pós transplante correlacionado com a taxa de recidiva da doença de base, mortalidade, sobrevida global e livre de doença. Analisar a freqüência das complicações infecciosas neste período. Métodos: Analisado o número absoluto de linfócitos no D+21 e D+30 pós transplante de células tronco hematopoiéticas. Conforme dados da literatura definiu-se no D+21 e no D+30 aqueles com número absoluto de linfócitos abaixo e acima de 300 e se correlacionou os dados obtidos com a taxa de óbito, taxa de recidiva, sobrevida global em 5 anos, sobrevida livre de doença, em 5 anos, TRM em 100 dias. e mortalidade não relacionada a recaída (NRM). Resultados: Neste estudo foram incluídos 100 pacientes portadores das seguintes neoplasias hematológicas: leucemia mielóide aguda, leucemia linfocítica aguda, leucemias secundárias e síndrome mielodisplásica. Destes, 55 pacientes eram do sexo masculino e 45 do sexo feminino. A média de idade foi de 27,9 anos (mínima 9 meses e máxima 55 anos). A mediana do tempo de seguimento foi de 601 dias (IC 95% 106-1845). A mediana de CD 34 infundidos foi de 4,0 (IC 95% 2,4-5,7) e quanto a origem destas células CD 34 infundidas 85% foram de medula óssea (MO), 12% periférica (PBSC) e 3% sangue de cordão umbilical (SCU). Quanto ao tipo de condicionamento realizado 22% foram não mieloablativos e 78% mieloablativos.A mediana de linfócitos no D+21 foi de 460 (IC 95% 0 - 6250) e no D+30 foi de 760 (IC 95% 40-6370). Com relação a taxa de infecções observou-se que 19% das infecções foram de etiologia viral, 65 % bacteriana e 17% fúngicas. A sobrevida global (OS) em 5 anos foi de 44 % , sobrevida livre de doença (DFS) foi de 37,7% , a mortalidade relacionada ao transplante (TRM) em 100 dias foi de 32,5%. E a mortalidade não relacionada a recidiva (NRM) em 5 anos foi de 40,2%. No desfecho óbito observamos que 69% dos pacientes que foram a óbito no D+21 tinham linfócitos abaixo de 300, e 43,9% tinham linfócitos acima de 300 (p<0,05). Pacientes com valores menores que 300 no dia 30 tem 2,20 vezes o risco de irem a óbito quando comparados com aqueles com valores acima de 300 (IC 95% 1,03-4,69) ajustado para DECH e CD34. Pacientes com valores menores que 300 no dia 30 tem 3,76 vezes o risco de irem a óbito em menos de 100 dias quando comparados com aqueles com valores acima de 300 (IC 95% 1,23-11,46) Conclusões: A reconstituição linfocitária precoce (> 300) no D+21 e no D+30 melhora a sobrevida global e livre de doença, bem como reduz a taxa de recidiva da doença de base e reduz a mortalidade. / Background: The role of repopulating lymphocyte after allogenic stem cell transplantation (SCT) includes the prevention of serious infections and attacking residual tumor cells in the early post transplant phase. Therefore, the current study analysed the role of the absolute lymphocyte count (ALC) on day 21 and 30 after SCT in predicting transplant outcomes of patients in terms of the risk of transplant related mortality (TRM) recurrence of original disease and risk of opportunistic infections. Objective: Evaluate early lymphocyte recovery on D +21 and D +30 posttransplant correlated with the rate of recurrence of the underlying disease, mortality, overall survival and disease free survival. Analyzed the frequency of infectious complications in this period. Methods: Analyzed the absolute lymphocyte count in the D +21 and D +30 after hematopoietic stem cell transplantation. According to literature data set the we correlate the absolute lymphocyte count in the D +21D +30 below and above 300 these data with the rate of death, relapse rate, overall survival in 5 years, disease-free survival in 5 years , TRM in 100 days and mortality unrelated to relapse (NRM). Results : Included in the study 100 patients with the following hematologic malignancies: acute myeloid leukemia, acute lymphocytic leukemia, secondary leukemia and myelodysplastic syndrome. Of these, 55 patients were male and 45 female. The average age was 27.9 years (minimum 9 months and maximum 55 years). The median follow-up was 601 days (95% CI 106-1845). The CD 34 median that was infused was 4.0 (95% CI 2.4 to 5.7).The source of stem cells infused was 85% of bone marrow (BM), peripheral 12% (PBSC) and 3 % of umbilical cord blood (UCB). Regarding the type of conditioning performed 22% were non myeloablative and 78% of lymphocytes were mieloablativos. The median of absolute lymphocyte count in the D +21 was 460 (95% CI 0 to 6250) and D +30 was 760 (95% CI 40- 6370 ). Regarding the rate of infections were observed 19% viral infections , bacterial in 65% and fungal in 17%. Overall survival (OS) at 5 years was 44%, disease-free survival (DFS) was 37.7%, transplant related mortality (TRM) in 100 days was 32.5%. Non relapsed mortality (NRM) at 5 years was 40.2%. The death rate found that 69% of patients who died at the D +21 had presented lymphocytes count below 300, and 43.9% were above 300 lymphocytes (p <0.05). Patients with counts less than 300 in D+30 presented 2.20 times risk of death when compared with those who presented values above 300 (95% CI 1.03 to 4.69) adjusted for GVHD and CD34. Patients presenting values less than 300 in 30 days have 3.76 times more risk of death in less than 100 days compared with those with values above 300 (95% CI 1.23 to 11.46). Conclusions: The early lymphocyte reconstitution (> 300) in D +21 D +30 improves overall survival and disease-free and reduces the relapse rate of the underlying disease and reduces mortality.

Introdução: O transplante de células tronco hematopoiéticas é capaz de curar as doenças hematológicas. O papel da repopulação linfocitária precoce no período pós transplan te visa combater a células neoplásicas que resistiram ao regime de condicionamento e prevenir as infecções oportunistas graves. Sendo assim, uma contagem elevada de linfócitos no período pós transplante é capaz de reduzir a mortalidade relacionada ao transplante (TRM), melhorar a sobrevida livre de doença e reduzir a taxa de recidiva. Objetivos: Avaliar a recuperação linfocitária precoce no D+21 e D+30 pós transplante correlacionado com a taxa de recidiva da doença de base, mortalidade, sobrevida global e livre de doença. Analisar a freqüência das complicações infecciosas neste período. Métodos: Analisado o número absoluto de linfócitos no D+21 e D+30 pós transplante de células tronco hematopoiéticas. Conforme dados da literatura definiu-se no D+21 e no D+30 aqueles com número absoluto de linfócitos abaixo e acima de 300 e se correlacionou os dados obtidos com a taxa de óbito, taxa de recidiva, sobrevida global em 5 anos, sobrevida livre de doença, em 5 anos, TRM em 100 dias. e mortalidade não relacionada a recaída (NRM). Resultados: Neste estudo foram incluídos 100 pacientes portadores das seguintes neoplasias hematológicas: leucemia mielóide aguda, leucemia linfocítica aguda, leucemias secundárias e síndrome mielodisplásica. Destes, 55 pacientes eram do sexo masculino e 45 do sexo feminino. A média de idade foi de 27,9 anos (mínima 9 meses e máxima 55 anos). A mediana do tempo de seguimento foi de 601 dias (IC 95% 106-1845). A mediana de CD 34 infundidos foi de 4,0 (IC 95% 2,4-5,7) e quanto a origem destas células CD 34 infundidas 85% foram de medula óssea (MO), 12% periférica (PBSC) e 3% sangue de cordão umbilical (SCU). Quanto ao tipo de condicionamento realizado 22% foram não mieloablativos e 78% mieloablativos.A mediana de linfócitos no D+21 foi de 460 (IC 95% 0 - 6250) e no D+30 foi de 760 (IC 95% 40-6370). Com relação a taxa de infecções observou-se que 19% das infecções foram de etiologia viral, 65 % bacteriana e 17% fúngicas. A sobrevida global (OS) em 5 anos foi de 44 % , sobrevida livre de doença (DFS) foi de 37,7% , a mortalidade relacionada ao transplante (TRM) em 100 dias foi de 32,5%. E a mortalidade não relacionada a recidiva (NRM) em 5 anos foi de 40,2%. No desfecho óbito observamos que 69% dos pacientes que foram a óbito no D+21 tinham linfócitos abaixo de 300, e 43,9% tinham linfócitos acima de 300 (p<0,05). Pacientes com valores menores que 300 no dia 30 tem 2,20 vezes o risco de irem a óbito quando comparados com aqueles com valores acima de 300 (IC 95% 1,03-4,69) ajustado para DECH e CD34. Pacientes com valores menores que 300 no dia 30 tem 3,76 vezes o risco de irem a óbito em menos de 100 dias quando comparados com aqueles com valores acima de 300 (IC 95% 1,23-11,46) Conclusões: A reconstituição linfocitária precoce (> 300) no D+21 e no D+30 melhora a sobrevida global e livre de doença, bem como reduz a taxa de recidiva da doença de base e reduz a mortalidade. / Background: The role of repopulating lymphocyte after allogenic stem cell transplantation (SCT) includes the prevention of serious infections and attacking residual tumor cells in the early post transplant phase. Therefore, the current study analysed the role of the absolute lymphocyte count (ALC) on day 21 and 30 after SCT in predicting transplant outcomes of patients in terms of the risk of transplant related mortality (TRM) recurrence of original disease and risk of opportunistic infections. Objective: Evaluate early lymphocyte recovery on D +21 and D +30 posttransplant correlated with the rate of recurrence of the underlying disease, mortality, overall survival and disease free survival. Analyzed the frequency of infectious complications in this period. Methods: Analyzed the absolute lymphocyte count in the D +21 and D +30 after hematopoietic stem cell transplantation. According to literature data set the we correlate the absolute lymphocyte count in the D +21D +30 below and above 300 these data with the rate of death, relapse rate, overall survival in 5 years, disease-free survival in 5 years , TRM in 100 days and mortality unrelated to relapse (NRM). Results : Included in the study 100 patients with the following hematologic malignancies: acute myeloid leukemia, acute lymphocytic leukemia, secondary leukemia and myelodysplastic syndrome. Of these, 55 patients were male and 45 female. The average age was 27.9 years (minimum 9 months and maximum 55 years). The median follow-up was 601 days (95% CI 106-1845). The CD 34 median that was infused was 4.0 (95% CI 2.4 to 5.7).The source of stem cells infused was 85% of bone marrow (BM), peripheral 12% (PBSC) and 3 % of umbilical cord blood (UCB). Regarding the type of conditioning performed 22% were non myeloablative and 78% of lymphocytes were mieloablativos. The median of absolute lymphocyte count in the D +21 was 460 (95% CI 0 to 6250) and D +30 was 760 (95% CI 40- 6370 ). Regarding the rate of infections were observed 19% viral infections , bacterial in 65% and fungal in 17%. Overall survival (OS) at 5 years was 44%, disease-free survival (DFS) was 37.7%, transplant related mortality (TRM) in 100 days was 32.5%. Non relapsed mortality (NRM) at 5 years was 40.2%. The death rate found that 69% of patients who died at the D +21 had presented lymphocytes count below 300, and 43.9% were above 300 lymphocytes (p <0.05). Patients with counts less than 300 in D+30 presented 2.20 times risk of death when compared with those who presented values above 300 (95% CI 1.03 to 4.69) adjusted for GVHD and CD34. Patients presenting values less than 300 in 30 days have 3.76 times more risk of death in less than 100 days compared with those with values above 300 (95% CI 1.23 to 11.46). Conclusions: The early lymphocyte reconstitution (> 300) in D +21 D +30 improves overall survival and disease-free and reduces the relapse rate of the underlying disease and reduces mortality.

Issues d'un gène ancestral commun, les molécules CD5 et CD6 sont présentes à la surface de tous les lymphocytes T (LT) matures ainsi qu'à la surface de certains lymphocytes B (LB). Ces deux protéines font partie de la famille des « Scavenger Receptor Cystein Rich » (SRCR) protéines mais la régulation, l'expression et les fonctions de ces deux molécules ne sont pas totalement résolues. Ainsi, CD5 est impliquée dans la régulation du récepteur à l’antigène des LB et des LT, dans la tolérance des LB et elle est présente à la surface des LB régulateurs. A l’opposé, CD6 possède un rôle dans la prolifération des lymphocytes, la survie, la migration et l’adhésion cellulaire. Les expressions de CD5 et CD6 diffèrent au sein des LB normaux ainsi qu'en pathologie. Dans le cadre du lupus érythémateux systémique (LES), le nombre de molécules CD5 à la surface des LB CD5+ est réduit. Dans une autre maladie auto-immune (MAI), le Syndrome de Gougerot Sjögren (SGS), l'expression de CD6 à la membrane des LB n'est pas affectée mais la distribution des LB mémoires CD6+, mais pas des LB naïfs, est modifiée par rapport aux témoins sains. En effet, les LB CD6+ sont sous représentés dans le sang périphérique et ce, en raison de leur délocalisation dans les glandes salivaires (GS) des patients. Cette délocalisation est liée à la surexpression d’ALCAM, le ligand naturel de CD6, par les cellules épithéliales au cours du SGS. L'étude de la diminution de l'expression de CD5 à la surface des LB de patients atteints de LES a permis de montrer qu'il existait un défaut dans le processus de la méthylation de l'ADN chez ces patients. Ce même défaut a été retrouvé dans les GS de patients atteints de SGS. Enfin, les LB de patients atteints de leucémie lymphoïde chronique (LLC) sont porteurs des deux molécules à leur membrane. Nous avons testé l'effet in vitro et in vivo d'un l'anticorps monoclonal (Acm) humanisé anti-CD6, T1H, dans la LLC. De façon intéressante, il s’avère que cet Acm favorise la lyse des LB de LLC et ceci, de façon équivalente au rituximab dans un modèle murin in vivo. T1H est internalisé par les LT et est donc inefficace sur ces cellules. Ces résultats nous permettent de conclure que même si les molécules CD5 et CD6 sont proches phylogénétiquement, elles possèdent des fonctions et des modes de régulation différents entre les LB et les LT mais aussi au sein des différentes populations de LB. Une meilleure compréhension des fonctions et des modes d'actions de ces deux protéines ouvre des perspectives thérapeutiques dans le traitement des MAI et de la LLC. / Derived from a common ancestral gene, CD5 and CD6 molecules are present on the surface of all mature T lymphocytes (LT) and some B lymphocytes (LB). These two proteins are members of the "Scavenger Receptor Cystein Rich" family proteins (SRCR) but the regulation, the expression and the function of these molecules is not totally resolved. CD5 is involved in the regulation of antigen receptor in LB and in LT, in the LB tolerance and is present on the surface of regulator B cells. In contrast, CD6 plays a role in lymphocyte proliferation, survival, migration, and cell adhesion. Expressions of CD5 and CD6 differ within normal LB and and LB present in different in pathologies. In the context of systemic lupus erythematosus (LES), the number of CD5 molecules on the surface of CD5+ LB is reduced. In another autoimmune disease (MAI), the Sjogren Syndrome (SS), the expression of CD6 at the membrane of B cells is not affected but the distribution of CD6 + memory LB but not naive LB is changed compared to healthy controls. Indeed, LB CD6 + are underrepresented in the peripheral blood and, it’s due to their relocation in the salivary glands (GS) of patients. This relocation is related to overexpression of ALCAM, the natural ligand of CD6, by epithelial cells in SS. The study of the decreased expression of CD5 on the surface of LB SLE patients has shown that there was a defect in the process of DNA methylation in these patients. The same defect was found in the GS of SS patients. Finally, B cells from chronic lymphocytic leukemia patients (CLL) are holders of the two molecules on their membrane. We tested the effect in vitro and in vivo of a humanized monoclonal antibody (mAb) anti-CD6, T1H, in the CLL. Interestingly, it appears that this mAb promotes lysis of CLL LB and this effect is equivalent to the one that rituximab was shown to have in an in vivo mouse model. T1H is internalized by LT and is therefore ineffective on these cells. These results allow us to conclude that even if the CD5 and CD6 molecules are phylogenetically close, they have different functions and modes of regulation between LB and LT but also within different populations of LB. A better understanding of their functions and action pathway of these two proteins opens up therapeutic perspectives for the treatment of MAI and CLL.

La différenciation terminale des lymphocytes B qui se déroule dans les centres germinatifs des organes lymphoïdes secondaires est l’étape ultime de la réponse T dépendante et aboutit à la production de plasmocytes (PC) à longue durée de vie qui sécrètent des anticorps hautement affins spécifiques de l’antigène et caractéristiques de la réponse immune adaptative. La transition d’une cellule B naïve vers un PC est gouvernée par un réseau de régulation génique bien décrit et est largement influencée par l’intégration de stimuli externes qui contrôlent le devenir des cellules B tels que l’interaction BCR-antigène et les cytokines produites par les cellules T. La stimulation précoce des lymphocytes B humains activés par IL-2, induit la différenciation en PC via une signalisation ERK prolongée entraînant la baisse d’expression de BACH2, un facteur de transcription clef des cellules B. La répression transitoire de BACH2 est suffisante pour déclencher la différenciation en plasmablastes en l’absence d’IL-2, suggérant ainsi qu’il joue un rôle de « verrou moléculaire » de la différenciation en PC. Il est à noter que cette répression forcée de BACH2 aboutit à la production de plasmablastes caractérisés par un phénotype lymphoplasmocytaire. Ce travail de recherche s’est focalisé sur la caractérisation des mécanismes moléculaires régulant l’expression de BACH2 via la voie de signalisation ERK induite par IL-2. Nous avons identifié ELK-1 comme un médiateur de la répression de BACH2 par la voie IL-2/ERK, comme l’atteste sa capacité à se lier avec un élément de régulation d’un enhancer localisé dans l’intron 1 de BACH2, induisant ainsi la répression de l’enhancer et déverrouillant la différenciation en PC. La caractérisation de cet enhancer de BACH2 a confirmé qu’il est régulé de manière dynamique au cours de la différenciation terminale B et qu’il est localisé dans une région sujette aux mutations suggérant qu’il pourrait être impliqué dans la lymphomagenèse. / The terminal differentiation of B cells, which takes places within germinal centres of secondary lymphoid organs, is the ultimate step of a T cell dependent response and results in the generation of long-lived plasma cells (PCs) that secrete protective, antigen-specific, high-affinity antibodies as part of adaptive immunity. The transition of a naive B cell into a PC is governed by a well-characterised gene regulatory network and is heavily influenced by the integration of externally received signals, including BCR-antigen binding and T cell help, such as cytokines which guide B cell fate. The early IL-2 priming of human primary activated B cells triggers PC differentiation through sustained ERK signalling resulting in the down regulation of B cell transcription factor BACH2. Transient BACH2 repression is sufficient to trigger plasmablast differentiation in the absence of IL-2 suggesting that it acts as a key lock of PC differentiation. Importantly, this enforced BACH2 repression results in the generation of plasmablasts with a lymphoplasmacytic phenotype. The focus of this thesis was to characterise the molecular mechanisms regulating BACH2 expression via the IL-2 ERK transduction pathway. We identify ELK-1 as the mediator of IL-2 ERK induced BACH2 downregulation as it binds to a regulatory enhancer element located within intron 1 of BACH2 instigating its repression and unlocking the PC programme triggering differentiation. The characterisation of this BACH2 enhancer confirms that it is dynamically regulated during PC differentiation and is located within a region targeted for mutation suggesting that it may have a potential role in lymphomagenesis.

Les pénicillines font partie des molécules chimiques les plus fréquemment impliquées dans l’allergie médicamenteuse. Selon l’hypothèse de l’haptène, les molécules chimiques de petite taille doivent se lier aux protéines pour êtres immunogènes. Cependant, très peu est connu sur le processus d’immunisation des patients aux bioconjugués pénicilline-protéine. Notre groupe a récemment synthétisé des bioconjugués albumine sérique humaine-benzylpénicilline (HSA-BP) et a démontré l'existence de lymphocytes T CD4+ naïfs spécifiques du bioconjugué HSA-BP chez des donneurs sains. L'objectif de ce travail de thèse est d'identifier des séquences peptidiques issus de la HSA hapténisées avec la BP, impliquées dans l’activation des cellules T naïves ainsi que l’immunisation des patients allergiques et par conséquent les manifestations cliniques. Notre stratégie combine la spectrométrie de masse, la modélisation moléculaire et le criblage virtuel, la synthèse chimique orientée et la validation biologique sur des lignées de cellules T de longues durées chez les donneurs sains, et à l’aide du test de transformation lymphocytaire ainsi que les lignées de cellules T de courte durée chez les patients allergiques. Cette étude a permis: (1) l’identification des résidus lysine présents sur la HSA hapténisés par la BP par spectrométrie de masse, (2) la sélection par une approche in silico des peptides de 15-mer potentiellement immunogènes, (3) la synthèse orientée de ces peptides-BP à l’aide d’un monomère lysine-BP, (4) l’identification des épitopes reconnus par les cellules T naïves de donneurs sains, (5) la validation de deux épitopes situés sur les lysines 159 et 525 chez les patients allergiques aux pénicillines et (6) confirmation de la HSA comme un bon modèle pour l’hapténisation de la BP. / Penicillins are among the most prevalent drug-inducing allergy. According to the hapten hypothesis small chemical molecules needs to bind to proteins to be immunogenic. However, little is known on the process of patients immunization to penicillin-protein conjugates. Our group has recently synthesized benzylpenicillin-human serum albumin (BP-HSA) bioconjugate and demonstrated the existence of naïve CD4+ T lymphocytes specific to BP-HSA in healthy donors. The objective of this work was to identify peptides sequences from HSA haptenized with BP involved in naïve T-cells activation, immunization of patients and consequently the clinical manifestations. Our strategy combines mass spectrometry, molecular modeling and virtual screening, chemical oriented synthesis and biological validation using long-term T-cell lines in healthy donors and the lymphocyte transformation test as well as short-term T-cell lines in allergic patients. This study allowed: (1) the identification of lysine residues involved in the BP binding to HSA using mass spectrometry, (2) the selection of BP-peptides containing the lysine residues likely to induce immune response using an in silico approach, (3) the synthesis of the selected BP-15 mer peptide bioconjugates using a lysine-BP monomer, (4) the identification of epitopes recognized by naïve T cells from healthy donors, (5) the validation of two epitopes located on lysines 159 and 525 in allergic patients to penicillins and (6) the confirmation of HSA as a good model for BP haptenation.