CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O linfoma difuso de grandes cÃlulas B (LDGCB) corresponde a 50 % dos casos de linfoma nÃo-Hodgkin (LNH). Seu tratamento de escolha à a quimioterapia de associaÃÃo, em especial o esquema CHOP (ciclofosfamida, doxorrubicina, vincristina e prednisona), considerado o tratamento inicial padrÃo dos LDGCB. VariaÃÃes deste esquema, como o protocolo CHOP-Bleo (ciclofosfamida, doxorrubicina, vincristina, prednisona e bleomicina) tem sido utilizadas com a intenÃÃo de se obter maiores taxas de remissÃo completa pelos pacientes. No Brasil, pouco se conhece a respeito da incidÃncia, do comportamento clÃnico, da resposta Ãs terapÃuticas utilizadas e da sobrevida de pacientes com LDGCB. Este estudo teve como objetivos traÃar o perfil epidemiolÃgico dos pacientes portadores de linfoma difuso de grandes cÃlulas B, atendidos no Hospital UniversitÃrio Walter CantÃdio (HUWC), com data de primeiro atendimento de janeiro de 1989 a dezembro de 2003, e que fizeram uso dos esquemas quimioterÃpicos CHOP e/ou CHOP-Bleo; avaliar a eficÃcia e seguranÃa terapÃutica dos esquemas propostos atravÃs da anÃlise do tipo de resposta terapÃutica, achados clÃnicos e laboratoriais destes pacientes. A coleta dos dados foi realizada a partir dos prontuÃrios mÃdicos dos 31 pacientes analisados. Destes, 21 (67,74%) eram do sexo masculino e 10 (32,26%) do feminino, com idade mÃdia de 45,81  16,3 anos. A ocupaÃÃo trabalhador agrÃcola representou 25,82% (8/31). O estÃdio III da classificaÃÃo de Ann Arbor foi o mais freqÃente (32,26%), mas apenas 45% dos pacientes apresentaram sintomas B. A lactato desidrogenase (LDH) sÃrica de 49% dos pacientes encontrava-se elevada à Ãpoca do diagnÃstico, sendo que outros 16% dos pacientes nÃo apresentavam resultado desta enzima em seus prontuÃrios. Quanto ao IPI, 71% foram classificados como de risco baixo e intermediÃrio, 13% de alto risco intermediÃrio, nenhum dos pacientes do estudo apresentou IPI compatÃvel com de alto risco e em 16% dos pacientes nÃo foi possÃvel estabelecer a classificaÃÃo devido à ausÃncia de dados nos prontuÃrios. Quanto à utilizaÃÃo dos protocolos quimioterÃpicos, 58% (18/31) dos pacientes fizeram uso do esquema CHOP, 36% (11/31) utilizaram CHOP-Bleo e 6% (2/31) utilizaram os dois esquemas quimioterÃpicos. Entre os pacientes que utilizaram o esquema CHOP, 78% atingiram a remissÃo completa (RC), 17% apresentaram recidiva da doenÃa e apenas 5 % foram a Ãbito. No grupo que utilizou o esquema CHOP-Bleo, 63% atingiram a RC, 18% apresentaram recidiva da doenÃa e 19% foram a Ãbito. Os 2 pacientes que utilizaram os dois esquemas como tratamento apresentaram recidiva da doenÃa. Os valores de LDH dos pacientes apÃs a quimioterapia apresentam-se reduzidos tanto em pacientes que atingiram a remissÃo completa como naqueles que tiveram recidiva. Verificamos que a sobrevida global (SG) e a sobrevida livre de doenÃa (SLD) nÃo foram influenciadas pelo estÃdio clÃnico e LDH inicial dos pacientes. A regressÃo logÃstica nÃo mostrou significÃncia estatÃstica quando analisou a remissÃo completa dos pacientes a partir dos resultados das variÃveis em estudo pÃs QT, com exceÃÃo da proporÃÃo de reduÃÃo da LDH e a resposta ao tratamento. Os resultados mostraram a eficÃcia e seguranÃa dos esquemas terapÃuticos CHOP e CHOP-Bleo em nossa populaÃÃo de estudo. Os resultados demonstram ainda que se faz necessÃrio o estudo epidemiolÃgico de diferentes populaÃÃes com LDGCB para que haja seguranÃa na escolha de esquemas quimioterÃpicos, bem como a uniformidade em descrever e classificar os linfomas e os seus fatores prognÃstico por parte dos patologistas e oncologistas. / Diffuse Large B-Cell Lymphomas (DLBCL) corresponds to 50% of non-Hodgkinâs lymphomas (LNH). Their treatment of choice is the association chemotherapy, in special the CHOP therapy (cyclophosphamide, doxorubicin, vincristine and prednisone) considered the standard treatment initial of the DLBCL. Variations of this therapy, with the CHOP-Bleo protocol (cyclophosphamide, doxorubicin, vincristine, prednisone and bleomycin) have been used with the intention of obtaining complete response rates for the patients. In Brazil, little is known about the incidence, clinical behavior, response to therapy and survival of the patients with DLBCL. This study aimed to set out the epidemiological profile of patients with diffuse large B-Cell lymphomas, who received medical care at Hospital UniversitÃrio Walter CantÃdio (HUWC), outline in Cearà state, with the first attendment from January 1989 to December 2003, who the used the CHOP and/or CHOP-Bleo therapy; Evaluating the security and efficiency of the protocols proposed by analysis of the kind of therapeutical response, clinical and laboratorial outcomes of these patients. The data collection was performed from medical recording of the 31 patients analyzed. These, 21 (67,74%) were the men and 10 (32,26%) women. The average age was 45,81  16,3 anos. Agriculturists represented 25,82% (8/31) of all patients. The stage III the Ann Arbor classification were the most frequent (32,26%), but only 45% of the patients had B symptoms. The values of lactate dehydrogenises (LDH) enzyme were elevated in 49% of the patients at diagnosis, but in 16% of the patients these values at diagnosis were unknown. As much as the IPI, 71% were classified as an IPI low and intermediate risk, 13% as an IPI intermediate-high risk, none of the study patients showed as an IPI high risk and 16% there is not possible the classification to establish due to the data is unknown. As much as the chemotherapy protocols used, 58% (18/31) of the patients was received CHOP chemotherapy, 36% (11/31) CHOP-Bleo chemotherapy and 6% (2/31) received CHOP associated with CHOP-Bleo chemotherapy. Among the patients who used CHOP chemotherapy, 78% was achieving complete response (CR), 17% was achieving relapse of the disease and only 5% were the death. In the group who used CHOP-Bleo chemotherapy, 63% was achieving RC, 18% was achieving relapse of the disease and 19% died. The 2 patients who used CHOP and CHOP-Bleo chemotherapy were achieving relapse of the disease. The values of the LDH after chemotherapy showed decreased in patients with RC as much as the relapsed patients. We verified that the overall survival (OS) and disease-free survival (DFS) were not influenced by the clinic stage and initial values of the LDH patients. The logistic regression did not show statistical differences when the complete response was analyzed comparing to outcomes the studied variables after QT, except for the proportion of reduction of LDH levels and response to the treatment. The results stress the security and efficiency of the protocols CHOP e CHOP-Bleo in our study population. The data obtained also the need epidemiological studies in different DLBCL populations for the security in the choice chemotherapy, well as standardized the classification and description of the DLBCL and prognoses factures by pathologists and oncologists.

Orientador: Oslei Paes de Almeida / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-23T09:15:14Z (GMT). No. of bitstreams: 1 ToralRizo_VictorHugo_D.pdf: 3216317 bytes, checksum: f10b9daa311d7ae2e304dc1ea8925abf (MD5) Previous issue date: 2013 / Resumo: O linfoma difuso de grandes células B (LDGCB) é o linfoma da cavidade bucal mais comum. Alguns dos LDGCB podem apresentar células grandes morfologicamente similares às células Hodgkin e Reed/Sternberg (HRS) dos linfomas de Hodgkin clássico (LHC). O objetivo deste estudo foi comparar os LDGCB bucal que apresentem células HRS-like (LDGCB-HRS) com o linfoma de Hodgkin primário nodal, considerando os aspectos histológicos e imunoistoquímicos (IQs), angiogênese, índice de mastócitos e células dendríticas (CD), por meio de um amplo painel IQ. Quize casos foram estudados, nos quais sete eram LDGCB-HRS like e oito eram LHC nodal. Para a análise dos aspectos histológicos e IQs foram utilizados os seguintes anticorpos: CD3, CD15, CD20, CD30, CD43, LCA, CD45RO, CD79a, CD83, EMA, MUM-1, PAX-5, perforina, granzyme B, FASN, Ki-67, LMP-1; e EBER1/2. Já para a análise da angiogênese foram utilizados os anticorpos CD34, CD31, D2-40, CD105, vWF e VEGF; e para o índice de mastócitos utilizou-se o mast cell triptase. Finalmente, para avaliar a expressão IQ das CD os anticorpos CD1a, CD83, CD123, CD207, S-100 e FXIIIa foram utilizados. Todas as lâminas foram escaneadas e as células HRS-like, mastócitos e CD imunopositivas foram analisados, assim como os parâmetros morfométricos da angiogênese. Os resultados mostraram que a imunoexpressão foi postiva em 100% de casos de LHC e em 57% dos casos de LDGCB de boca, enquanto que LCA, CD20 e CD79a foram exclusivos para todos os LDGCB, e apenas CD15 foi exclusivo para os LHC. Angiogênese e o índice de mastócitos estavam aumentados em ambas as lesões, e entre elas, o LHC obteve maiores valores que o LDGCB da cavidade bucal em todos os anticorpos analisados. Por fim, o índice de CDs foram estatisticamente significante entre os grupos, exceto para CD83, que não mostrou nenhuma diferença estatística. A distribuição de CD foi reconhecida principalmente na área tumoral e ao redor das células neoplásicas em ambas as entidades. Foi possível concluir que os LDGCB com células HRS-like da cavidade bucal devem ser incluídos no diagnóstico diferencial de LHC da cavidade bucal. Quando da avaliação destes casos, a analise morfológica detalhada assim como o uso de um amplo painel de IQ são recomendados para realizar o diagnóstico correto. A angiogênese é essencial para o desenvolvimento de LDGCB da cavidade bucal, e quaisquer dos anticorpos CD34, CD31 e vWF podem ser utilizados para avaliar os parâmetros morfométricos. A presença significativa de CD nestes linfomas provavelmente desempenha um papel patologicamente relevante nos linfomas. Nossos resultados sugerem que o aumento no número de CD parece ser um fator contribuinte para a resposta imune estimulada pelo crescimento tumoral / Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most common oral lymphoma. Some DLBCLs can present large cells morphologically similar to Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). The objective of this study was to compare oral DLBCL presenting HRS-like cells (DLBCL-HRS like) with primary nodal cHL, considering the following aspects: histological and immunohistochemical (IHC), angiogenesis, index of mast cells and dendritic cells (DCs); through a broad immunohistochemical panel. Fifteen cases were studied, of which, seven were DLBCL-HRS like and eight were nodal cHL. For histological and IHC aspects, immunoexpression of CD3, CD15, CD20, CD30, CD43, LCA, CD45RO, CD79a, CD83, EMA, MUM-1, PAX-5, perforin, granzyme B, FASN, Ki-67, LMP-1; and EBER1/2, were assessed. As for angiogenesis analysis, the antibodies used were CD34, CD31, D2-40, CD105, vWF and VEGF; and for the index of mast cell were used the mast cell tryptase. Finally, for IHC expression of DCs, the antibodies used were CD1a, CD83, CD123, CD207, S-100, and FXIIIa. All slides were scanned and positive immunoreactive cells HRS-like, mast cell and DCs were analyzed, as well as morphometric parameters of angiogenesis. The results showed that the immunoexpression of CD30 was 100% positive in cHL and 57% in oral DLBCL HRS-like, while LCA, CD20 and CD79a were exclusive for all oral DLBCL, and only CD15 was exclusive for cHL. Angiogenesis and mast cell index values were increased in both lesions and between them, cHL was greater than oral DLBCL with all antibodies studied. Finally, DC subsets were statistically significant between groups, except CD83, which did not show statistical significance. The distribution of DCs was mainly in the tumor area, around neoplastic cells in both entities. It was possible to conclude that DLBCL-HRS should be included in the differential diagnosis of oral cHL. When evaluating these cases, a detailed morphologic and a broad IHC analyses for the correct diagnosis are recommended. Angiogenesis is essential to the development of DLBCL of the oral cavity and any of the antibodies CD34, CD31 and vWF could be used to evaluate morphometric parameters. The presence of significantly higher numbers of DCs in these lymphomas could suggest that these cells are likely to play a pathological relevant role in lymphomas. Our findings suggest that increased number of DCs in lymphomas appears to be a factor contributing to the immune response against tumor growth / Doutorado / Patologia / Doutor em Estomatopatologia

L'infection par le cytomégalovirus humain (HCMV) est une cause majeure de mortalité chez les patients immunodéprimés et représente la première cause d'infection congénitale. HCMV est un virus complexe qui s'est adapté au système immunitaire humain en développant de multiples mécanismes d'évasion. L'infection primaire à HCMV est associée à une réplication virale prolongée avant l'établissement de la latence. Il a été montré que cette intense réplication lors de la phase initiale de l'infection était associée à une épuisement fonctionnel des lymphocytes T CD4 spécifiques du virus. Alors que les anticorps jouent un rôle dans la limitation de la dissémination virale et la prévention de l'infection à HCMV, les réponses des lymphocytes B sont peu caractérisées. Dans le présent travail, nous avons étudié l'impact de l'infection à HCMV sur le phénotype et la fonctionnalité des sous-populations de LB du sang circulant chez une cohorte de femme enceintes avec une primo-infection par HCMV en utilisant comme contrôles des sujets sains séropositifs et séronégatifs pour HCMV ainsi que des femmes enceintes séronégatives. Nous montrons que l'infection primaire par HCMV induit une expansion significative et prolongée de deux sous-populations de LB :les LB mémoires activés (CD27+CD21low) et mémoires atypiques (CD27-CD21low), précédemment décrites lors d'infection chroniques. Les LB mémoires atypiques démontrent des signes d'épuisement fonctionnel comme en témoigne une expression élevée de récepteurs inhibant le BCR et une moindre réponse à la stimulation in vitro mesurée par la production de TNF-α. Les expansions de ces deux sous-populations sont corrélées entre elles et liées à la virémie. Ces résultats contribuent à la compréhension de la régulation des réponses des LB lors d'infections virales, en montrant que l'épuisement fonctionnel de LB, précédemment décrit lors d'infections chroniques, peut également survenir lors d'infections primaires.<p>Dans un deuxième temps, nous avons étudié l'acquisition des réponses B mémoires spécifiques de HCMV dirigées contre la principale glycoprotéine de surface, la glycoprotéine B (gB), et deux polypeptides du tégument. Lors de l'infection primaire par HCMV, la production d'anticorps neutralisant le virus, dirigés contre les glycoprotéines d'enveloppe, est retardée par rapport aux anticorps dirigés contre le tégument qui sont non neutralisant. Nous montrons que le phénotype des LB mémoires spécifiques de gB est différent de celui des LB mémoires spécifiques du tégument. La majorité des LB mémoires spécifiques de gB exprime un phénotype CD27+CD21+ alors que la majorité de ceux du tégument exprime le phénotype CD27+CD21low. Nous montrons par la suite chez des sujets sains que ces deux sous-populations de LB mémoires présentent des différences phénotypiques, au niveau de l'expression de récepteurs liés au "trafficking" cellulaire ainsi qu'au niveau de la fonctionnalité. Les LB mémoires CD21low, contrairement au LB mémoires CD21high, expriment des taux bas des récepteurs CXCR5 et CCR7, qui permettent la migration vers les centres germinatifs, mais des taux élevés de CD11c promouvant la migration vers les tissus périphériques. Après stimulation in vitro, les LB mémoires CD21low vont avoir une capacité de production d'immunoglobulines immédiate mais une réponse proliférative plus faible comparée aux LB mémoires CD21+. Nous démontrons la relevance de cette division des LB mémoires sur base de l'expression du CD21 dans un modèle de vaccination de rappel contre la toxoïde tétanique (TT). Après rappel, nous observons une expansion significative de LB mémoires spécifiques de la TT exprimant un phénotype CD27+CD21lowCXCR5lowCD11chigh. Nous proposons ainsi un nouveau mécanisme de manipulation des réponses humorales par des pathogènes qui se traduit par une limitation de l'induction de réponses B effectrices. Nos travaux permettraient également une meilleure approche des réponses B mémoires physiologiques chez l'homme en proposant une classification des LB mémoires basées sur leur fonctionnalité et leur phénotype.<p><p>Human cytomegalovirus (HCMV) infection is a major cause of mortality in immunocompromised patients and is the first cause of congenital infection worldwide. HCMV is a complex virus that has developed multiples immune evasions mechanisms during its co-evolution with mankind. Although often asymptomatic, primary HCMV infection is associated with an intense and prolonged viral replication. It has been previously shown that this intense viral replication is associated with functional exhaustion of virus-specific CD4+ T cells. Although neutralizing antibodies limits viral dissemination and play a role in the prevention of HCMV infection, B cell responses during HCMV infection have been poorly studied so far.<p>In this work, we have studied the impact of HCMV infection on the phenotype and functionality of peripheral-blood B cell subsets in a cohort of pregnant women with a primary HCMV infection. Controls were healthy seronegative and seropositive HCMV donors and HCMV seronegative pregnant women. We show that primary HCMV infection induces a significant and prolonged expansion of two B-cell subsets, previously described in chronic infections :activated memory B cells (MBC) (CD27+CD21low) and atypical MBC (CD27-CD21low). Atypical MBC display signs of functional exhaustion with increased expression of inhibitory receptors and a lower response to in vitro stimulation as assessed by TNF-α production. Expansion of these two subsets are correlated and higher in subjects with detectable viremia. These results contribute to the understanding of the regulation of B cell responses during viral infections and indicate that B cell exhaustion, previously described during chronic infections, can be observed in primary infection.<p>Next, we have characterized the acquisition of HCMV-specific B cell responses directed against envelope glycoprotein B (gB) and two tegument polypeptides (pp150 and pp52). During primary HCMV infection, the production of neutralizing antibodies targeting envelope glycoproteins is delayed when compared to non-neutralizing anti-tegument antibodies. We show that gB and tegument-specific MBC have distinct phenotype during primary HCMV infection. The majority of gB-specific MBC have a CD27+CD21+ phenotype while the majority of tegument-specific MBC have a CD27+CD21low phenotype. We show that CD27+CD21+ and CD27+CD21low MBC express different pattern of chemokine receptors pattern but also have distinct functionality. CD27+CD21low MBC, on the contrary to CD27+CD21+ MBC, express low levels of CXCR5 and CCR7 that favor migration to lymph nodes and germinal centers but express high levels of CD11c that promotes migration to inflammatory tissues.<p>In vitro stimulation of sorted subsets of healthy individuals indicates that CD27+CD21low MBC have higher capacity of immediate immunoglobulin production but a lower proliferative potential as compared to CD27+CD21+ MBC. We further show the relevance of a division of MBC subsets based on CD21 expression in a model of TT booster immunization. Following booster immunization, a significant expansion of TT-specific MBC expressing the phenotype CD27+CD21lowCXCR5lowCD11chigh is observed. <p>We propose that HCMV manipulates the host humoral response by limiting the induction of gB-specific CD27+CD21low "effector" MBC. Our work also indicates that human MBC physiological responses should be studied according to their respective phenotype and functions.<p> / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

Le Lupus Erythémateux Systémique (LES) est une maladie autoimmune sévère dont laquelle les lymphocytes B (LB) jouent un rôle central. Afin de rechercher des anomalies génétiques propre à ces cellules, notre laboratoire a étudié l’expression des gènes dans les LB de patients atteints d’un LES, par rapport à des sujets témoins. Ce projet a pour but d’étudier un de ces gènes (FKBP11), surexprimé dans les LB au cours du LES. Pour cela nous avons créé, à l’aide de lentivirus, des souris transgéniques reproduisant de manière ubiquitaire la surexpression de ce gène. Ces souris développent une hyperplasie des organes lymphoïdes, une hyper gamma globulinémie de type IgG3, et présentent une réponse humorale augmentée après immunisation avec un antigène T-indépendant. Notre étude met en évidence que la surexpression de FKBP11 favorise le développement des plasmocytes dans leur phase initiale dépendante de l’expression de Pax5, après induction de la différenciation plasmocytaire in vitro. Enfin, les souris développent des autoanticorps variés. De plus, le rôle d’une surexpression de FKBP11 dans la rupture de tolérance B a été montré chez des souris transgéniques anti-ADN 56R, croisées avec notre modèle lentigénique, qui voient leur production d’autoanticorps augmentée. La surexpression de FKBP11 dans le modèle C57BL/6lpr/lpr accentue le caractère lymphoprolifératif et l’hyper gamma globulinémie présents dans ces souris. Au cours de ce projet de thèse, j’ai également initié l’étude de deux autres gènes surexprimés dans les LB de patients lupiques (PRDX4, TRIB1), par le développement de modèles transgéniques murins. / Systemic Lupus Erythematosus (SLE) is a severe autoimmune disease where B cells play a central role and carry intrinsic genetic abnormalities.Today, only a few SLE genes have been validated in humans. Looking for these intrinsic B cell abnormalities in SLE, we have performed a microarray analysis of the human transcriptoma in B cells from quiescent SLE patients, in comparison to normal subjects. The project proposes to explore the consequences of overexpression of one of these genes: FKBP11. For this purpose, a lentiviral construct allowing the ubiquitous overexpression of FKBP11 was produced, and has been used to generate lentigenic mice. These mice develop a hyperplasia of lymphoid organs, an IgG3 hypergammaglobulinemia and an increase of humoral immune response after immunisation with a T-independent antigen. Our study points out that the overexpression of FKBP11 promotes the plasmocyte development, at the initial stage which is dependent on Pax5 expression, after in vitro induction of the plasmacytic differentiation. Finally, these mice produce various autoantibodies. The role of FKBP11 overexpression in B cell central tolerance breakdown has been demonstrated in anti-DNA 56R transgenic mice crossed with our lentigenic model. These mice have an increase of autoantibody production. The FKBP11 overexpression in C57BL/6lpr/lpr model increases the lymphoproliferative syndrome and the hypergammaglobulinemia in this model. During this thesis, I have also initiated the study of two others genes which were found overexpressed in B cells of lupus patients (PRDX4, TRIB1), by the development of transgenic murine models.

Le développement actuel très rapide des différentes approches « omiques » génère un grand nombre de biomarqueurs potentiels, en particulier dans le domaine de la cancérologie.L’analyse protéomique par spectrométrie de masse a particulièrement bénéficié des progrès technologiques récents qui ont permis la mise au point de nouvelles approches quantitatives globales ou ciblées. Néanmoins, peu de biomarqueurs potentiels finissent par être concrètement utilisés en pratique clinique, nécessitant l’optimisation des différentes étapes de leur développement.Dans la continuité des travaux ayant abouti à l’identification de biomarqueurs diagnostiques dans les hémopathies lymphoïdes B, ce travail de thèse a permis le développement d’une méthode d’analyse protéomique ciblée pour la vérification et la validation de nouveaux biomarqueurs potentiels. La possibilité d’appliquer des stratégies quantitatives globales à un très grand nombre d’échantillons a pu être démontrée. L’application de ces stratégies quantitatives globales à du tissu ganglionnaire provenant de lymphomes agressifs a permis d’identifier des biomarqueurs potentiellement associés à une résistance au traitement. Enfin,le mode de préparation tube-gel, facilitant l’étude d’un grand nombre d’échantillons, a été validé pour la réalisation d’analyses protéomiques différentielles. / The current development of new « omics » technologies has led to the discovery of a large number of potential biomarkers, particularly in the field of oncology. Proteomics analysis bymass spectrometry have particularly benefited from these technological advances with the development of new global or targeted quantitative approaches. Nevertheless, only a few numbers of potential biomarkers are finally used in clinical practice, requiring further optimization of the development process. Following the initial identification of biomarkers in the diagnosis of lymphoid malignancies performed previously, this thesis has allowed the development of a targeted proteomics method that can be used for the validation of new potential biomarkers. The ability of analysing a large number of samples with a global quantitative approach has also been demonstrated. The application of these global quantitative strategies on lymph node tissue of aggressive lymphoma has permitted the identification of potential new biomarkers associated with chemorefractoriness. Lastly, a tube-gel protocol facilitating the analysis of a large number of samples has been validated for differential proteomics studies.

B-lymphocytes are lymphoid cells, which are a part of the adaptive/innate immune system and generate antibodies. Recently, many studies have supported hypothesis that different rather minor B-lymphocyte subpopulations may play a direct and indirect role in immunopathogenesis in human pathologies such as Crohn's disease (CD). The aim of current study was therefore to investigate distribution of frequencies of B lymphocyte subpopulations (from transient to mature effector B cell stages) in peripheral blood of healthy subjects (CO), patients with Crohn's disease (CD) and ulcerative colitis (UC). Thus, using 11-colour flow cytometry we have analysed 30 blood samples of individuals, including 14 healthy controls, 11 patients with Crohn's disease and 5 with UC. In 6 patients with CD we have had an opportunity to analyze blood samples collected 2 hours after an administration of anti-TNF therapy. Higher frequencies of memory B-lymphocytes (CD19+ CD27+ , CD19+ CD20+ CD27+ and CD19+ CD20+ CD27+ IgM+) were found in patients with CD as compared to COs. (20.06±13.58%; 17.61±13.48%; 88.60±20.56% vs. 11.75±26.47%; 11.25±26.50%; and 66.82±22.60%), in case of CD19+CD20-CD27-IgM+ B-lymphocytes the difference was statistically significant (57.15±17.21% in CD vs. 19.59±31.79% in CO; p=0.0341), which is in accordance...

Abstract In the present study, some of the difficulties in the treatment of the most common malignant lymphoma, diffuse large B-cell lymphoma (DLBCL), were evaluated. Some patients develop local or central nervous system (CNS) relapse after first-line treatment. The treatment of relapsed disease is challenging and despite all efforts, some patients die of the disease. Chemoresistant disease also remains challenging because some patients suffer from refractory disease of a progressive nature. The antioxidant enzymes peroxiredoxins (Prxs) and thioredoxin-1 (Trx) were evaluated as prognostic and predictive markers of DLBCL. High cytoplasmic expression of Prx VI was found to correlate with poor prognosis in patients with DLBCL. Trx knockdown in lymphoma cell culture revealed a possible predictive role of Trx. Trx knockdown sensitized cells to doxorubicin, a widely used chemotherapeutic agent in treatment schemas of DLBCL. Etoposide, another widely used chemotherapeutic agent, on the other hand, killed more native DLBCL cells than did doxorubicin. Patients with high Trx expression at the diagnostic stage of the disease benefitted from etoposide-containing high-dose chemotherapy and autologous stem cell transplantation and did not develop post-transplantation relapses which Trx-negative patients did. Antithrombin III (AT III) in cerebrospinal fluid has been suggested to be a biomarker in previous studies of CNS lymphoma. In the present study, AT III was evaluated in patients with CNS lymphoma and with neurological diseases. High concentrations of AT III in cerebrospinal fluid reflected the magnitude of blood-brain barrier leakage and because of this, AT III should not be used as a biomarker in clinical practice. / Tiivistelmä Tutkimuksessa arvioitiin osaa yleisimmän pahanlaatuisen imukudossyövän eli lymfooman, diffuusin suurisoluisen B-solulymfooman, hoidon haasteista. Osa potilaista saa ensilinjan hoidon jälkeen joko paikallisen tai aivoston alueen taudin uusiutuman. Uusiutuneen taudin hoito on haasteellista, ja hoitoyrityksistä huolimatta osa potilaista kuolee tautiinsa. Solunsalpaajille resistentti tauti on myös yksi haastavista hoitotilanteista, ja osa potilaista kärsiikin hoitojen läpi etenevästä taudista. Antioksidatiivisia entsyymejä, kuten peroksiredoksiineja ja tioredoksiinia, arvioitiin ennusteellisina ja ennakoivina merkkiaineina diffuusissa suurisoluisessa B-solulymfoomassa. Peroksiredoksiini VI:n korkea sytosolinen ilmaantuvuus korreloi tavallista huonompaan diffuusin suurisoluisen B-solulymfooman ennusteeseen. Tioredoksiinin hiljentäminen lymfoomasoluviljelyssä paljasti sen mahdollisen ennakoivan merkityksen hoitoon liittyvässä päätöksenteossa. Solut herkistyivät tiodredoksiinin hiljentämisen vuoksi doksorubisiinille, jota käytetään laajalti diffuusin suurisoluisen B-solulymfooman solunsalpaajahoidoissa. Etoposidi, joka on huomattavasti myrkyllisempi solunsalpaaja, päinvastoin tappoi enemmän tavallisia diffuusia suurisoluisia B-solulymfoomaa edustavia soluja kuin doksorubisiini. Potilaat, joilla oli korkea tioredoksiinin määrä taudin diagnostisessa vaiheessa, hyötyivät etoposidia sisältävästä korkea-annoshoidosta sekä autologisesta kantasolusiirrosta. Näille potilaille ei kehittynyt kantasolusiirron jälkeisiä taudin uusiutumia kuin taas niitä kehittyi potilaille, joilla oli tioredoksiini negatiivinen. Antitrombiini III:a on ehdotettu soveltuvaksi aikaisempien tutkimusten perusteella aivoston lymfooman merkkiaineeksi aivo-selkäydinnesteestä. Tässä tutkimuksessa antitrombiini III:n määrää mitattiin potilailta, joilla oli aivoston lymfooma tai neurologinen sairaus. Korkeat konsentraatiot antitrombiini III:a aivo-selkäydinnesteessä kuitenkin vain heijastivat veri-aivoesteen vuotamisen määrää, ja näin ollen antitrombiini III:a ei tulisi käyttää kliinisessä käytössä.

Les lymphomes B sont des cancers du système lymphatique se développant à partir des cellules B. Il devient évident que le développement des cellules B malignes dépend d’interactions avec les cellules immunes dans leur microenvironnement. Nous avons étudié la caractérisation des lymphocytes T intra tumoraux afin de comprendre leur contribution dans la lymphomagenèse et leur potentiel thérapeutique dans les lymphomes B comme le lymphome diffus à grandes cellules B (DLBCL), le lymphome folliculaire (FL) et le lymphome Hodgkinien classique (cHL)Nous avons mis en évidence une différence importante, quantitative et qualitative, entre la composition immunitaire de différents lymphomes B, notamment au niveau des lymphocytes T intra tumoraux. Le FL se caractérise par une accumulation des lymphocytes T régulateurs (Tregs) exprimant ICOS, pouvant supprimer les cellules B lymphomateuses. La génération des Tregs ICOS+ est favorisée par le contact avec les cellules B lymphomateuses exprimant ICOSL. Quant à lui, le DLBCL a beaucoup de lymphocytes TCD8 coexprimant PD1 et TIM3 possédant un état de dysfonctionnement dit « épuisement », lymphocytes dont la proportion est corrélée à leur niveau de dysfonctionnement et à leur capacité de réponse au blocage des récepteurs inhibiteurs. Enfin, dans certains lymphomes B, en particulier le cHL, nous avons découvert une sous population de TCD8, dite « TFH-like » pour leur similarité phénotypique et fonctionnelle avec les lymphocytes T auxiliaires folliculaires (TFH). Ces données indiquent l’hétérogénéité des composants immunitaires entre différents lymphomes B et sont une piste pour une future thérapie ciblée dans le traitement du lymphome. / B-cell lymphomas represent a heterogeneous group of cancers that affect B cells in the lymphatic system. It has become evidence that malignant B cells depend on various interactions with microenvironmental immune cells for their development. Our study focuses on characterization of intra-tumoral T cells in order to understand their contribution in pathogenesis and their therapeutic potentials in the most frequent B cell-lymphoma such as Diffuse large B-cell lymphoma (DLBCL), Follicular lymphoma (FL) and classical Hodgkin lymphoma (cHL).During this work, we have demonstrated a significant quantitative and qualitative difference between different B-cell lymphoma immune composition, especially between their intra-tumoral T cells. FL is characterized by the accumulation of regulatory T cells (Tregs) expressing ICOS, with ability to suppress lymphoma B cells. Generation of Tregs ICOS+ is prompted by cell contact with the lymphoma B cells expressing ICOSL. On the other hand, DLBCL have high level of TCD8 coexpressing PD1 and TIM3 displaying an exhaustion state, which proportion is correlated with their dysfunction level and with their responsiveness to inhibitor receptors blockade. Finally, in some B-cell lymphoma cases, especially cHL, we found the existence of a TCD8 subset, called TFH-like due to their phenotypic and functional similarity with follicular helper T cells (TFH).These data show heterogeneity of immune components between the different B lymphomas, and give opportunity for targeted therapy in lymphoma treatment

Durant l’ontogénie B, le locus des chaines lourdes d’immunoglobulines (IgH) subit trois processus de réarrangements géniques. Lors des phases précoces du développement B, indépendamment de la rencontre avec un antigène, les réarrangements VDJ permettent l’obtention d’un répertoire d’Ig fonctionnelles. Lors des phases tardives, l’hypermutationsomatique (SHM) permet l’augmentation de l’affinité de l’Ig pour son antigène tandis que larecombinaison isotypique (CSR) modifie ses fonctions effectrices. Ces évènements impliquent l’induction de lésions de l’ADN potentiellement oncogéniques, ce qui impose unerégulation très stricte. Cette régulation est assurée par divers éléments cis-régulateurs répartis tout au long du locus IgH, dont la région régulatrice en 3’ (3’RR). La 3’RR s’étend sur 30 kb et contient quatre activateurs transcriptionnels, les trois premiers formant une structure palindromique. Lors de ma thèse, j’ai utilisé plusieurs modèles murins porteurs de délétions de tout ou partie de la 3’RR pour étudier son rôle, ainsi que celui des différents éléments qui la compose lors des diverses étapes de l’ontogénie B. Nous avons pu déterminer comment la 3’RR régule précisément la CSR en ciblant spécifiquement la région switch acceptrice et caractériser le phénomène encore peu connu de CSR vers IgD. D’autre part, nous avons démontré l’importance de la 3’RR lors de la SHM et dans le développement des différentes sous populations lymphocytaires B. Enfin, la comparaison des résultats obtenus lors de l’analyse des différents modèles nous a permis de déterminer que la structure palindromique de la 3’RR est importante pour une SHM efficace, mais relativement dispensable lors de la CSR. / During B-cell development, the heavy chains locus (IgH) undergoes three genic rearrangement events. During the early stages, before encountering the antigen, VDJ rearrangements allow the generation of a functional Ig repertoire. During the late stages, somatic hypermutation (SHM) increases the affinity of the Ig for its antigen, while class switch recombination (CSR) modifies its effector functions. These events imply thegeneration of potentially oncogenic DNA lesions, and thus require a strict regulation. This regulation is assured by several cis-regulatory elements spread along the IgH locus, including the 3’ regulatory region (3’RR). The 3’RR extends on more than 30kb and contains four transcriptional enhancers, the first three displaying a palindromic conformation. During my PhD, I investigated several mouse models carrying deletion of part or totality of the 3’RR to investigate its role during B cell development. We demonstrated how she precisely regulates CSR by specifically targeting the acceptor switch region, and described the poorly known mechanism of CSR toward IgD. Otherwise, we have demonstrated its importance during SHM and in the correct development of the different B cell subpopulations. Finally, by comparing the results obtained during the analysis of the various mouse models, we have demonstrated that the palindromic structure of the 3’RR is required for optimal SHM, but not for CSR.

Heavy chain isotype switch recombination is preceded by the appearance of RNA initiating 5' of the specific switch region which will undergo recombination. In an effort to understand the potential function of germline transcripts in switch recombination and the degree to which the regulation of germline transcripts correlates with the regulation of switching, we studied this process in the murine B-lymphoma cell line I.29μ, which in the presence of bacterial lipopolysaccharide (LPS) switches primarily to IgA and less frequently to IgE. Levels of α-germline transcripts initiating upstream of α switch (Sα) sequences are elevated in clones of this line which switch well as compared to clones which switch less frequently. TGFβ1 has been shown to increase α-germline transcripts and switching to IgA expression in LPS-stimulated murine splenic B-cells. We now demonstrate in I.29μ cells that TGFβ also increases switching to IgA and increases the level of α-germline transcripts 5 to 9 fold. Nuclear run-on analysis shows that this increase is at the level of transcription. Thus, TGFβ appears to direct switching to IgA by inducing transcription from the unrearranged Sα- CαDNA segment. Germline α RNA is quite stable in I.29μ cells, having a half life of about 3 to 5 hours, and we find only slight stabilization in the presence of TGFβ. Levels of ε-germline transcripts are not increased by TGFβ . IL-4, which modestly increases switching to IgA in I.29μ cells, slightly increases trancription of α-germline RNA. However, we present evidence suggesting that endogenously produced IL-4 may also act at additional levels to increase switching to IgA. IFNγ, which reduces IgA expression in these cells, also reduces the level of α-germline transcripts. IFNγ also reduces the level of ε-germline transcripts induced by IL-4. Our results support the hypothesis that the regulation of transcription of particular switch sequences by cytokines in turn regulates the specificity of recombination. In studies aimed at identifying other signalling pathways that promote class switching, we discovered that inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase (PARP) increase lipopolysaccharide (LPS)-induced switching to IgA in the B cell lymphoma I.29μ and to IgG1 in LPS + IL-4-treated splenic B cells. PARP, which binds to and is activated by DNA strand breaks, catalyzes the removal of ADP-ribose from NAD+ and poly(ADP-ribosylation) of chromatin-associated acceptor proteins. This enzyme is believed to function in cellular processes involving DNA strand breaks as well as in modulating chromatin structure. In I.29μ cells, PARP inhibitors increase IgA switching by day 2 and cause a 5-fold average increase in switching on day 3 as assayed by immunofluorescence microscopy. The PARP inhibitor, nicotinamide, also causes a reduced intensity of hybridization of Cμ and Cα specific probes to genomic DNA fragments containing the expressed VDJ-Cμ and the unrearranged Sα - Cα segments, respectively, indicating that PARP inhibition increases rearrangment of these fragments. Induction of switching by PARP inhibitors is not mimicked by treatment with cAMP analogs or reduced by inhibitors of protein kinase A (PKA). Induction of switching by PARP inhibitors does not appear to involve increased levels of transcription of the unrearranged Cα gene, although TGFβ is required for optimal induction by PARP inhibitors, consistent with a requirement for transcription of the unrearranged CH gene. PARP inhibitors do not overcome the requirement for endogenously produced IL-4.