Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Hematogones are B-lineage lymphoid precursors cells normally found in bone marrow. The B-cell Acute Lymphocytic Leukemia (B-ALL) is a lymphocytic precursors neoplasia with consequent accumulation of immature cells called lymphoblasts in bone marrow. The increase of hematogones after treatment for B-ALL or marrow transplantation may cause doubt to the prognosis due to morphological and immunophenotypic similarities between these normal and malignant precursors cells. The aim of this study was to differentiate subpopulations of hematogones and B lymphoblasts by immunophenotyping using flow cytometry, attempting to identify the hematological laboratory profile at diagnosis and the environmental factors that characterize patients with B-ALL. Group 1 consisted of healthy controls free of hematologic malignancy and group 2 consisted of pediatric patients recently diagnosed with B-ALL before the start of chemotherapy, both in bone marrow samples. Antibodies directed against CD45, CD10, CD19, CD34 and mIgM (membrane) were used, which enabled us to distinguish the degree of cell maturation. Despite of the morphological and immunophenotypic similarities, hematogones and B lymphoblasts were differentiated by flow cytometry through distribution and continuous expression of antibodies on the association of CD45 x CD10 and mIgM x CD34 in B population. This technique allowed a better characterization of hematogones in healthy patients, with a progressive maturational pattern within the same cell population, with immature, intermediate and mature cells. The B lymphoblasts exhibited an incomplete maturation spectrum represented by a single population with immaturity characteristics. Among leukemia patients evaluated, 100% were white, with a predominance of children under 10 years (73%) and female sex (64%). These patients had lower hemoglobin levels (91% of cases), platelets (82%) and leukocytes (45.45%), with elevated leukocyte count in 27.3% of subjects. Predominant signs and symptoms were pallor, weakness, petechiae or bruising presence and fever. The same proportion of leukemic patients resided in both rural and urban zones. Patients living in rural area had features that can justify the etiology of ALL, as the professional connection of their parents to contact with toxic products. Morphological revision combined with immunophenotyping has allowed the correct cell identification and distinction between phenotypic subpopulations of hematogones and B lymphoblasts, contributing to an accurate laboratorial prognostic. Due to number of patients with B-ALL of this study, further researches are suggested to correlate the exposure of parents and leukemia patients to potentially oncogenic environmental factors, with the objective of preventing this neoplasia. / As hematogônias são células jovens linfoides de linhagem B normalmente encontradas na medula óssea. A leucemia linfocítica aguda de células B (LLA-B) é uma neoplasia de precursores linfocíticos com consequente acúmulo de células imaturas denominadas linfoblastos na medula. O aumento das hematogônias após o tratamento para LLA-B ou um transplante medular pode causar dúvidas quanto ao prognóstico, devido às semelhanças morfológicas e imunofenotípicas entre essas células jovens normais e malignas. O objetivo deste trabalho foi diferenciar subpopulações de hematogônias e linfoblastos B por imunofenotipagem através da citometria de fluxo, buscando identificar o perfil laboratorial hematológico ao diagnóstico e os fatores ambientais que caracterizam os pacientes de LLA-B. O grupo 1 foi constituído de controles sadios isentos de neoplasia hematológica e o grupo 2 constou de pacientes pediátricos recém-diagnosticados com LLA-B antes do início do tratamento quimioterápico, ambos em amostras de medula óssea. Foi utilizado um painel de anticorpos monoclonais composto por: CD45, CD10, CD19, CD34 e mIgM (membrana), que possibilitaram a distinção do grau de maturação das células. Apesar das semelhanças morfológicas e imunofenotípicas, hematogônias e linfoblastos B foram diferenciados por citometria de fluxo através da distribuição e expressão contínua dos anticorpos diante da associação de CD45 x CD10 e mIgM x CD34 na população B. Esta técnica permitiu uma melhor caracterização das hematogônias nos pacientes sadios, apresentando um padrão maturacional progressivo dentro da mesma população celular, com células imaturas, intermediárias e maduras. Os linfoblastos B exibiram um espectro maturacional incompleto representado por única população com características de imaturidade. Entre os pacientes com leucemia avaliados, 100% eram de raça branca, com predomínio de crianças menores de 10 anos (73%) e do sexo feminino (64%). Estes pacientes apresentaram valores reduzidos de hemoglobina (91% dos casos), plaquetas (82%) e leucócitos (45,45%), com leucometria elevada em 27,3% dos indivíduos. Os sinais e sintomas predominantes foram: palidez, astenia, presença de petéquias ou equimoses e febre. A mesma proporção de pacientes leucêmicos residia nas zonas rural e urbana. Os pacientes que moram na área rural apresentaram características que podem justificar a etiologia da LLA, como a ligação profissional dos pais ao contato com produtos tóxicos. A revisão morfológica combinada com a imunofenotipagem permitiu a correta identificação celular e a distinção entre as subpopulações fenotípicas das hematogônias e dos linfoblastos B, contribuindo para um preciso prognóstico laboratorial. Devido ao número de portadores de LLA-B deste estudo, pesquisas adicionais são sugeridas para correlacionar a exposição de pais e pacientes com leucemia aos fatores ambientais potencialmente oncogênicos, com o objetivo de prevenir essa neoplasia.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated for the first time in 1991 and since then it has been associated with significant economic losses to the pig industry worldwide. Although vaccination against PRRSV is widely used, an important advance would be the development of marker vaccines allowing serologic discrimination between vaccinated and naturally infected animals. The present study aimed to identify immunogenic and conserved regions dispensable to viral replication in different PRRSV proteins, which could be used as negative serologic markers in a new generation of liveattenuated vaccines. A fine mapping of B-cell linear epitopes in different PRRSV proteins by Pepscan is presented in the first part of this thesis. The results indicated the presence of several B-cell linear epitopes in the non-structural protein 2 (Nsp2) and in all structural proteins encoded by PRRSV, which were consistently recognized by antibodies raised in pigs experimentally infected with a North American strain of the virus (NVSL97-7895). The Nsp2 was found to harbor the highest frequency of immunodominant epitopes (n=18) when compared to structural proteins. In the structural proteins, epitopes consistently recognized by immune sera were located in all studied proteins. Overall, the highest degree of immunogenicity and conservation was exhibited by two epitopes identified in the C-terminal end of the M protein (ORF6). The antibodies recognizing the immunodominant epitopes of each protein were detected as early as days 7 to 15 post-infection (p.i.) and remained detectable until the end of the experiment (day 90 p.i). Based on their immunodominance and level of amino acid (aa) conservation, two target epitopes were selected to serve as serological marker candidates in each of the following PRRSV proteins: Nsp2, GP3 and M. These epitopes were deleted in the wild-type cDNA infectious clone (FL-12) by site-directed mutagenesis. The results of this study are presented in the second part of this thesis. A Nsp2 mutant virus (FLdNsp2/44) was successfully rescued following RNA transfection in MARC 145 cells. This epitope deletion mutant fulfilled the requirements for a differential vaccine virus such as efficient growth in vitro and in vivo and induction of active seroconversion as measured by a commercial ELISA kit associated with the absence of a marker-specific peptide-ELISA response in 100% (n=15) of the vaccinated animals. In vitro and in vivo characterization of the mutant virus clearly showed that removal of a 15-mer Nsp2 epitope had no effect on the immunogenicity, growth properties or virulence when compared to the wild type virus. On the other hand, deletions of previously identified peptide marker candidates within GP3 and M genes were shown to be lethal for virus viability in vitro. Alternatively, by substitution of 5aa at a time within a M peptide marker candidate, a viable mutant virus could be recovered although it still resulted in a positive marker virus. In summary, our results provide proof of concept that PRRSV marker vaccines can be developed using such methodology. Taken together, these data indicate that the combination of a mutant virus carrying a deletion of an immunodominant epitope and the corresponding peptide ELISA represents an attractive approach for the development of PRRSV differential modified-live vaccines. / O vírus da síndrome respiratória e reprodutiva dos suínos (PRRSV) foi isolado pela primeira vez em 1991 e, desde então, tem sido associado a perdas significativas para a suinocultura mundial. Apesar da vacinação contra o PRRSV ser amplamente utilizada, um grande avanço seria alcançado com a elaboração de vacinas diferenciais que permitam a discriminação sorológica entre animais vacinados e naturalmente infectados. O presente estudo teve como objetivo a identificação de regiões imunogênicas, conservadas e dispensáveis a replicação viral, em diferentes proteínas do PRRSV, que pudessem ser utilizadas como marcadores sorológicos negativos em uma nova geração de vacinas atenuadas. Na primeira parte desta tese estão apresentados os resultados de um mapeamento de epítopos lineares de linfócitos B em diferentes proteínas do PRRSV, pelo uso da tecnologia de Pepscan. Os resultados indicam a presença de diversas regiões imunodominantes na proteína não estrutural 2 (Nsp2) e em todas as proteínas estruturais do vírus. Essas regiões foram consistentemente reconhecidas pelo soro de suínos experimentalmente infectados com uma cepa norte-americana do PRRSV (NVSL97-7895). A maior freqüência de epítopos imunodominantes foi identificada na Nsp2 (n=18) e o mais alto grau de imunogenicidade e nível de conservação de aminoácidos foi observado em dois epítopos identificados na extremidade carboxi-terminal da proteína M (ORF6). Anticorpos reagentes com epítopos imunodominantes de cada proteína foram detectados inicialmente entre os dias 7-15 pós-infecção (pi), permanecendo em altos títulos até o final do experimento (dia 90 pi). Com base na imunodominância e nível de conservação de amino ácidos (aa) das seqüências mapeadas, dois epítopos alvos foram selecionados como candidatos a marcadores sorológicos negativos em cada uma das proteínas Nsp2, Gp3 e M. Esses epítopos foram então deletados em um clone infeccioso de cDNA (FL12) por mutagênese sítio-direcionada. Os resultados desses experimentos encontram-se descritos na segunda parte da tese. Um vírus mutante carreando a deleção de um epítopo imunodominante da Nsp2 (FLdNsp2/44) foi obtido após transfeccção de RNA viral em células MARC145. A caracterização in vitro e in vivo do vírus mutante demonstrou que a remoção dos 15 aa da Nsp2 não produziu efeito sobre a imunogenicidade, replicação ou virulência quando comparado ao vírus parental. Além disso, observou-se indução de soroconversão contra o PRRSV em animais infectados, detectada pelo uso de um teste ELISA comercial. Por outro lado, não foi detectada resposta humoral específica contra a região deletada nos animais imunizados com o FLdNsp2/44, conforme resultados de um teste ELISA contendo como antígeno um peptídeo sintético correspondente a seqüência removida. Por outro lado, deleções dos epítopos previamente identificados na Gp3 e proteína M foram letais à viabilidade viral in vitro. Alternativamente, um outro vírus mutante foi gerado pela substituição de 5 aa do epítopo identificado na proteína M, embora a alteração de resíduos não tenha sido suficiente para eliminar a imunogenicidade da região. Em resumo, os resultados do presente estudo se constituem em uma prova de conceito no sentido do desenvolvimento de vacinas diferenciais contra o PRRSV. A utilização de um vírus mutante carreando a deleção de um epítopo imunodominante, associado com um teste de ELISA baseado no peptídeo sintético correspondente a região deletada, representam uma alternativa para o desenvolvimento de vacinas diferenciais atenuadas contra o PRRSV.

Abstract Diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL) are two of the most common lymphomas in the Western world. DLBCL is an aggressive disease with a good response to treatment; about 75% of patients achieve permanent remission after first-line treatment. In patients with relapses or primary refractory disease, prognosis is dismal; only 10–20% of them can be cured, even with aggressive treatments. FL is an indolent lymphoma with a very good response to treatment and slow progression. Median survival with modern treatments is over 15 years. Nevertheless, some patients have short remissions and succumb to disease. Oxidative stress, TP53 mutations, and translocations of MYC, Bcl-2 and Bcl-6 have been linked in many neoplasms to aetiology and poor prognosis. This thesis concerns oxidative stress and redox-state-regulating enzymes in DLBCL and FL, and TP53 mutations and translocations of MYC, Bcl-2 and Bcl-6 in DLBCL. High expression levels of the antioxidant enzyme thioredoxin and a marker of oxidative stress, nitrotyrosine, were related to poor prognosis in DLBCL. In FL, high-level expression of peroxiredoxin was associated with good prognosis. TP53 mutations in specific regions LSH and L3 and concurrent translocation of Bcl-2 were associated with poor prognosis in DLBCL. Not all TP53 mutations predicted survival. High expression levels of Bcl-2 and MYC were associated with poor prognosis in DLBCL. Based on the results presented here, antioxidant function may have protective roles, but also may cause resistance to treatment. TP53 mutations have prognostic roles in DLBCL, but should be further defined. Novel therapies could be developed in connection with these mechanisms. / Tiivistelmä Diffuusi suurisoluinen B-solulymfooma (DLBCL) ja follikulaarinen lymfooma (FL) ovat kaksi yleisintä lymfoomaa länsimaissa. DLBCL on aggressiivinen syöpä, joka reagoi hyvin hoitoihin, jopa 75 % paranee. Kuitenkin potilailla, joilla syöpä uusiutuu hoitojen jälkeen tai etenee hoidon aikana, on erittäin huono ennuste, noin 10-20 % näistä potilaista voidaan parantaa. FL on hyväennusteinen lymfooma, joka yleensä reagoi hyvin hoitoihin. Mediaani elossaoloaika kaikilla FL potilailla on yli 15 vuotta taudin toteamisesta. Osalla potilaista FL kuitenkin on aggressiivisempo. Oksidatiivinen stressin, TP53- mutaatioiden, MYC, Bcl-2 ja Bcl-6 -translokaatioiden on todettu olevan huonoon ennusteeseen yhteydessä olevia tekijöitä monissa syövissä, kuten lymfoomissa. Tämä väitöskirja tutki oksidatiivisen stressin ja hapetus-pelkistys reaktioon liittyvien entsyymien osuutta R-CHOP-hoidetuissa DLBCL:ssa ja FL:ssa immunohistokemian (IHC) avulla. DLBCL:ssa tutkittiin lisäksi TP53 mutaatioita, MYC, Bcl-2 ja Bcl-6 translokaatioiden roolia taudin kulussa. Korkea ekspressio oksidatiivisen stressin merkkiainetta nitrotyrosiinia ja antioksidantti thioredoksiinia olivat yhteydessä huonoon ennusteeseen DLBCL:ssa. FL:ssa runsas ilmentyminen antioksidativiisiin entsyymeihin kuuluvia peroksiredoksiineja olivat yhteydessä hyvään ennusteeseen. TP53 mutaatiot LSH ja L3 alueella ja Bcl-2 -translokaatiot yhdessä olivat yhteydessä huonoon ennusteeseen DLBCL:ssa. Kaikki TP53-mutaatiot eivät olleet assosioituneet huonoon ennusteeseen. DLBCL:ssa Bcl-2 ja MYC –proteiinien runsas ilmentyminen IHC:llä arvioituna liittyi huonoon ennusteeseen. Tulosten perusteella solujen hapetus-pelkistystilaa säätelevillä entsyymeillä voi olla dualistinen rooli, osittain suojeleva ja osittain vahingoittava lymfoomissa. TP53 -mutaatioilla voi olla ennusteellista merkitystä, mutta tämä vaatii lisää tutkimuksia.

Estudo dos processos linfoproliferativos cutâneos de células B objetivando demonstrar a difícil distinção diagnóstica entre os linfomas cutâneos e pseudolinfomas. Foram avaliados 38 casos de processos linfoproliferativos cutâneos de células B. Foi realizada revisão de prontuários médicos e exames anátomo-patológicos. Foram estudados 25 casos de linfomas cutâneos, 7 de pseudolinfomas e 6 casos onde não foi possível a distinção diagnóstica entre as entidades em estudo. As características clínicas, histológicas e imunoistoquímicas foram descritas para cada grupo. A análise estatística foi realizada demonstrando a similaridade entre os linfomas cutâneos e os pseudolinfomas / Study of cutaneous B-cell lymphoproliferative process with the purpose of demonstrating the difficulty of distinguishing between cutaneous lymphomas and pseudolymphomas. 38 cases of cutaneous B-cell lymphoproliferative processes were evaluated. A review of medical records and histophatologic material was performed. The study comprised 25 cases of cutaneous lymphomas, 7 cases of pseudolymphomas and 6 cases where a diagnosis distinguishing between the entities in study was not possible. It described clinical, histophatologic and immunohistochemical characteristics of each group. A statistical analysis showing the similarity between lymphomas and pseudolymphomas was performed

Introdução: A Imunodeficiência Comum Variável (ICV) faz parte de um grupo de imunodeficiências primárias na qual os pacientes apresentam defeitos na maturação e diferenciação dos linfócitos B (LB), resultando em distúrbios funcionais além de alterações na distribuição de seus subtipos. Consequentemente, estes pacientes apresentam hipogamaglobulinemia, susceptibilidade a infecções e ausência de produção de anticorpos a antígenos específicos. Na tentativa de reduzir os episódios de infecções recorrentes, alguns trabalhos têm recomendado a vacinação com patógenos mortos ou subunidades e em trabalho anterior demonstramos a eficácia clínica da vacinação de pacientes com ICV, porém, a experiência com a administração de vacinas em imunocomprometidos é limitada. Objetivos: Avaliar a cinética da distribuição das subpopulações de linfócitos B antes e após a vacinação com antígenos proteicos e polissacarídicos em pacientes com ICV acompanhados no Ambulatório de Imunodeficiências Primárias do Hospital das Clínicas, FMUSP, além da produção de anticorpos específicos aos antígenos vacinais. Pacientes e Métodos: Um grupo de 35 pacientes com ICV e 16 controles foram vacinados contra Influenza, H1N1 e S. pneumoniae. Após as coletas nos tempos pré e pós 1, 3 e 6 meses foram realizados a separação de PBMC e cultura de linfócitos com lisado viral e hemaglutinina de Influenza, além da citometria de fluxo para identificação das subpopulações de LB naive, zona marginal (MZB), memória com troca de isotipo (SMB) e plasmoblastos (PBL). Foram dosados os anticorpos específicos e no grupo dos pacientes foi aplicado um score de sintomas antes e após a imunização. Resultados: Apesar da redução significativa na pontuação do score de sintomas, a maioria dos pacientes não produziu anticorpos específicos para Influenza, H1N1 e S. pneumoniae. A análise da cinética das subpopulações de LB revelou que em indivíduos saudáveis, a resposta contra Influenza apresentou duração de 6 meses, observada por meio da redução da subpopulação naive e aumento gradual da frequência de SMB a partir do primeiro mês. Observamos também redução da população de memória por volta do 3º mês, com aumento da população de PBL que permaneceu elevada até o 6º mês. Por outro lado, a despeito de os pacientes apresentarem aumento de SMB no primeiro mês após a vacinação, sua frequência foi inferior ao observado nos controles, decaindo ao terceiro mês. A população de PBL apresentou aumento precoce no primeiro mês após a vacinação, também muito menor do que observado nos controles, não sendo mantido no terceiro mês. Ainda, observamos uma correlação entre o aumento da expressão destas duas subpopulações no primeiro mês. Apenas a população de MZB apresentou aumento significativo no terceiro mês nos pacientes quando comparados aos controles. Ao dividirmos os pacientes de acordo com a expressão de SMB e PBL após 1 mês da administração das vacinas, observamos que os pacientes que apresentaram aumento na expressão de células B de memória foram os que exibiram uma melhora clínica mais expressiva, soroconverteram e desenvolveram soroproteção para H1N1.Conclusões: Apesar de não apresentarem eficaz diferenciação em células de memória e efetoras, resultando na resposta precoce e de curta duração, observamos que os pacientes foram capazes de reconhecer e responder às vacinas. Além disso, a elevada expressão de MZB no terceiro mês após a vacinação pode sugerir a atuação desta subpopulação na apresentação para os LT. Estes achados reforçam a necessidade de uma melhor compreensão da ativação do sistema imune em pacientes com ICV, para uma adequada subdivisão de acordo com o perfil de resposta após a vacinação / Introduction: Common Variable Immunodeficiency (CVID) is a primary antibody deficiency characterized by defects in B lymphocyte maturation, resulting in disturbed differentiation, distribution and functional variations on its subtypes. As a result , CVID patients have hypogammaglobulinemia and poor antibody response to specific antigens with increased susceptibility to infections. In an effort to minimize the recurrent episodes of infections, some studies have recommended immunization with inactivated pathogens or subunits and in a former study we have shown the clinical improvement determined by immunization in CVID patients, but the experience with vaccines\' administration to immunodeficient patients is limited. Objectives: To evaluate the changes in distribution of B cell subtypes before and after vaccination of CVID patients followed at the Division of Clinical Immunology and Allergy of University of São Paulo Medical School with protein and polysaccharide antigens, as well as specific antibody production . Methods: A group of 35 CVID patients and 16 controls were vaccinated against Influenza, H1N1 and S. pneumoniae vaccines. Blood samples were collected before and 1, 3 and 6 months post vaccination. PBMCs were stimulated with Influenza viral lysate and hemagglutinin peptide. Flow cytometry was performed to identify naïve B cells, marginal zone (MZB), switched memory B cells (SMB) and plasmablasts (PBL). Specific antibody production was measured and a symptoms score was applied for clinical evaluation before and after immunization. Results: In spite of the significant reduction in symptoms score after vaccination, most patients didn\'t produce specific antibodies to Influenza, H1N1 and S. pneumoniae. The analyzes of B cell subtypes changes in healthy individuals upon in vitro Influenza stimulation showed that the response endured up to 6 months post immunization. We observed a reduction in naïve B cell frequency while gradual increase in SMB frequency occurred already at 1 month after vaccination. Moreover, as the memory cell population declined, PBL population increased at the third month post vaccination until the sixth month. Although patients had an increase of SMB on the first month after vaccination, it was lower than that observed in controls, decreasing by the third month post vaccination. Plasmablast frequency had an early increase on the first month, also much lower than the observed in controls decreasing by the third month. In addition, we observed a correlation between the increased expression of SMB and PBL on the first month post vaccination. In patients, only MZB subtype presented a significant increase on the third month when compared to controls. We divided the patients according SMB and PBL expression after 1 month post vaccination and we observed that patients who were able to produce memory B cells showed a better clinical improvement, developed H1N1 seroconversion and seroprotection. Conclusion: Despite the defect on differentiation into memory and effector B cells resulting in early response with lowduration, we observed that patients were able to recognize and respond to vaccines. In addition, the over expression of MZB on the third month after vaccination may suggest the role of this subpopulation as an antigen presenting cell for T cells. These findings reinforce the need of a better understanding of immune system activation and response in CVID patients to propose a division according to vaccine (antigen) responders and non responders

A exposição limitada ao Plasmodium chabaudi induz proeminente imunidade celular, associada à proteção de células T da apoptose. Este estudo tem como objetivo verificar a influência da carga parasitária na geração e manutenção dos linfócitos T e B de memória ao P. chabaudi. Assim, camundongos C57BL/6 foram submetidos à infecção tratada (subpatente) ou não (patente) com cloroquina após a inoculação de 106 eritrócitos parasitados (EP) e analisados nos dias 0, 20, 60, 120 e 200. Com relação à memória de linfócitos T, no dia 20, as freqüências de células CD4+ memória/ativadas e respondedoras aos EP foram significativamente maiores nos animais do grupo subpatente. Os níveis máximos de IgG2a específica foram encontrados no dia 120 em ambos os grupos. O desafio dos animais com 108 EP mostrou que a imunidade protetora declina progressivamente, mas os grupos ainda são capazes de estabelecer resposta secundária eficiente que elimine o parasita. Assim, podemos concluir que a carga parasitária influencia a fase aguda, mas não impede a geração e manutenção das células T e B de memória. / One of the main characteristics of malaria is the intense policlonal activation of splenic T and B lymphocytes induced by the parasite and the consequent elimination, through apoptosis, of part of these cells. However, the limited exposure to the bloodstage malaria seems to induce a prominent cellular immunity, associated with the protection of T lymphocytes from apoptosis. With this in mind, this study aimed to verify the influence of the parasite load in the generation and maintenance of memory T and B cells specific for Plasmodium chabaudi chabaudi AS. In order to evaluate this idea, C57BL/6 mice were infected with 106 parasitized red blood cells (pRBC) and submitted to a patent (untreated) or subpatent infection (controlled with sub-curative doses of chloroquine every time parasitemia reached 1%). Splenocytes from these mice were analyzed at 20, 60, 120 and 200 days after infection, regarding the pRBC-specific T cell proliferation and the expression of surface molecules, as CD4, CD8, CD62L, CD45RB, CD44, CD45R-B220 and IgG. The parasitemia and the splenocyte phenotype were also monitored after the challenge with 108 pRBC. Regarding T cell memory, at day 20 of infection, the frequencies of effector/activated CD4+ T cells (CD62LLOW CD45RBLOW/HIGH) were significantly increased in animals from the patent group, which was strict linked with the highest cellular activation observed in these animals. On the other hand, the total numbers of pRBCproliferating T (CD4+ and CD8+) cells per spleen were approximately 3-fold increased in subpatent animals, indicating that these cells were protected from apoptosis as a result of the limited exposure to the parasite. However, in both groups, these parameters decreased to values similar to those in controls at day 200. The splenocytes from both groups produced Th1 cytokines in response to pRBC in all times of analysis, but at the early phase of infection, Th2 cytokines were also observed, but without differences between the infected groups. Regarding memory B cells, the frequency of sIgG+ cells was increased at day 20 of infection, when 11% and 9% of CD45R+ cells from patent and subpatent animals were positive, respectively. For both groups, specific IgG2a antibodies attained maximum serum levels at day 120, but at day 200, it is possible to observe a significant decrease of these levels only in the serum of patent mice. Moreover, at day 200 of infection, mice of subpatent group showed significantly higher amounts of IgG2a that recognized the intra-erythrocytic forms of the parasite and the surface of infected erythrocytes. Challenge of mice with 108 pRBC showed that protective immunity progressively decline with time and despite the higher levels of specific antibody in subpatent mice, both groups showed similar protection. In experiments of adoptive transference to CD28-/- mice, cells from 200-day infected mice were able to produce specific IgG2a antibodies, in a T CD4+ cell dependent way. In addition, we verified that CD45R+ cells of subpatent mice, when transferred to CD28-/- mice, secreted higher amounts of specific IgG2a and IgG1 antibodies, comparing to cells of patent mice. So, from this work, we can conclude that the parasite load has a great influence in the early immune response to P. chabaudi malaria and it also affects the generation and/or maintenance of memory B cells. Furthermore, according to our data, at least during the analyzed period, the loss of protective immunity against this parasite does not seem to be influenced by the acute-phase parasite load, but it can be a consequence of the progressive decline of T-cell memory response that occurs in patent and subpatent groups with time of infection.

Para avaliar a influência dos polimorfismos de nucleotídeo único (SNPs) do CYP2B6, CYP3A5, GSTM1, GSTP1, GSTT1, PON1, NQO1 e MDR1 na resposta ao tratamento com R-CHOP e CHOP, 82 pacientes com Linfoma Difuso de Grandes Células B, sem evidências de infecção por HIV, foram selecionados nesse estudo. Amostras de sangue periférico foram coletadas para extração de DNA. Os SNPs foram analisados por PCR-RFLP. Em relação aos pacientes que apresentaram resposta completa (RC) ao tratamento (70%), 51% foram tratados com R-CHOP. Sobre o tratamento, 50% dos pacientes com RC apresentaram classificação de ECOG 0-1 (p=0,0193) e a maioria desses pacientes (41%) não apresentaram envolvimento extranodal (p=0,0377). Não houve associação entre os SNPs do CYP2B6, CYP3A5, GSTT1, NQO1 e MDR1 (C3435T) e as variáveis estudadas. Apenas CYP3A5 (sexo p=0,0519), GSTM1 (idade p=0,016; tratamento p=0,0372), GSTP1 (envolvimento extranodal p=0,0307), PON1 (sintomas B p=0,0201; Bulky p=0,0148) e MDR1 C1236T (sexo p=0,0316) mostraram associação. Em relação à sobrevida global, apenas tratamento (p=0,0129), IPI (p=0,000342), idade (p=0,0155), estadiamento (p=0,00281) e ECOG (p=0,00869) apresentaram resultados significantes. Quanto à sobrevida livre de doença (SLD), apenas idade (p=0,0292), estadiamento (p=0,0402) e ECOG (p=0,0142) apresentaram resultados significantes / To evaluated the influence of single nucleotide polymorphisms (SNPs) of CYP2B6, CYP3A5, GSTM1, GSTP1, GSTT1, PON1, NQO1 and MDR1 in the treatment response with R-CHOP and CHOP, 82 patients with Diffuse Large B-cell Lymphoma, without evidence of HIV infection, were enrolled in this study. Peripheral blood samples were collected for DNA extraction. The SNPs were analyzed by PCR-RFLP. In relation the patients that showed complete response (CR) to the treatment (70%), 51% were treated with R-CHOP. About the treatment, 50% of the patients with CR showed ECOG classification of 0-1 and the most of these patients (41%) did not showed extranodal involvement (p=0,0377). There was no association between CYP2B6, CYP3A5, GSTT1, NQO1 and MDR1 (C3435T) SNPs and the variables studied. Only CYP3A5 (gender p=0,0519), GSTM1 (age p=0,016; treatment p=0,0372), GSTP1 (extranodal involvement p=0,0307), PON1 (B symptoms p=0,0201; Bulky p=0,0148) e MDR1 C1236T (gender p=0,0316) showed association. In relation to overall survival, only treatment (p=0,0129), IPI (p=0,000342), age (p=0,0155), stadiament (p=0,00281) and ECOG (p=0,00869) showed significant results. To disease-free survival, only age (p=0,0292), stadiament (p=0,0402) e ECOG (p=0,0142) showed significant results

Abstract Lymphomas are a group of more than 70 different malignancies arising from lymphoid tissues and diffuse large B-cell lymphoma (DLBCL) is the most common subtype of lymphoma. More than 70% of DLBCL patients can be cured with modern therapy, but some patients still die of the disease. The recognition of patients with adverse prognosis, justifying deviation from standard treatment and risking severe side effects, is problematic. The aim of this study was to identify potential biological factors for the prediction of poor treatment response and central nervous system (CNS) relapse in DLBCL patients. The study included 263 lymphoma patients. 205 patients had a DLBCL, and 37 of these represented primary CNS lymphoma (PCNSL). Immunohistochemistry was used to determine the expression of oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine, as well as antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin (Trx) and gamma cysteine ligase (GCL) from samples representing reactive lymphoid tissue and B-cell derived lymphomas. From DLBCL samples staining was also conducted for cell cycle regulating proteins p16, p53, p21 and p27 and chemokine receptors CXCR4, CXCR5 and CCR7. Immunoelectron microscopy (IEM) for CXCR4 and CXCR5, and their ligands CXCL12 and CXCL13 was performed on additional samples from reactive lymphoid tissue, nodal DLBCL, secondary CNS lymphoma and PCNSL. Factors associated with adverse prognosis included expression of nitrotyrosine, Trx and GCL. A prognostic score reflecting the degree of cell cycle dysregulation within each patient’s tumour identified 3 distinct prognostic groups among DLBCL patients. High cytoplasmic CXCR5 expression was associated with CNS involvement, whereas nuclear CXCR4 expression correlated with nodal disease. These results demonstrate the considerable biological heterogeneity seen within DLBCL, but further research is needed to confirm them. High antioxidant activity and the accumulation of damage to cell cycle regulating pathways separated patient groups with a poor prognosis that might benefit from new types of treatment. Chemokine receptor expression seems to play a role in the CNS tropism of DLBCL, and, if confirmed, could in the future contribute to more effective targeting of CNS prophylactic therapies. / Tiivistelmä Lymfoomat ovat yli 70 erilaisen maligniteetin muodostama ryhmä imukudoksen syöpiä, ja diffuusi suurisoluinen B-solulymfooma (engl. DLBCL) on yleisin lymfoomatyyppi. Yli 70 prosenttia DLBCL-potilaista pystytään parantamaan nykyaikaisilla hoidoilla, mutta osa potilaista kuolee edelleen tautiin. Nämä potilaat tarvitsisivat tehokkaampia hoitoja vakavien haittavaikutusten riskistä huolimatta, mutta huonon ennusteen potilaiden tunnistaminen etukäteen on vaikeaa. Tutkimuksen tavoitteena oli löytää biologisia tekijöitä DLBCL-potilaiden hoitovasteen ja taudin keskushermostossa (engl. CNS) uusiutumisen ennustamiseen. Aineisto sisältää 263 lymfoomapotilasta. 205 potilaalla on DLBCL, ja 37:llä näistä primaari aivolymfooma (PCNSL). Immunohistokemiallisilla värjäyksillä määritettiin oksidatiivisen stressin markkereiden 8-hydroksideoksiguanosiinin (8-OHdG) ja nitrotyrosiinin, sekä antioksidanttientsyymien mangaanisuperoksidi-dismutaasin (MnSOD), tioredoksiinin (Trx) ja gammakysteiiniligaasin (GCL) ilmentyminen reaktiivista imukudosta sekä B-soluperäisiä lymfoomia edustavissa näytteissä. DLBCL-näytteistä määritettiin lisäksi solusykliä säätelevien proteiinien p16, p53, p21 ja p27 sekä kemokiinireseptorien CXCR4, CXCR5 ja CCR7 ilmentyminen. Lisäksi reaktiivista imukudosta, imusolmuke-DLBCL:aa, sekundaarista CNS-lymfoomaa ja PCNSL:aa edustavista näytteistä määritettiin immunoelektronimikroskooppisesti reseptorien CXCR4 ja CXCR5 sekä ligandien CXCL12 ja CXCL13 ilmentyminen. Tulosten mukaan voimakas nitrotyrosiini-, Trx- ja GCL-positiivisuus ovat yhteydessä huonoon ennusteeseen. Solusyklin säätelyhäiriön vaikeusastetta kuvaava ennusteellinen pisteytys jaotteli DLBCL-potilaat kolmeen ennusteelliseen ryhmään. Runsas sytoplasminen CXCR5-positiivisuus oli yhteydessä CNS-tautiin, kun taas tumapositiivisuus CXCR4:lle korreloi imusolmuketautiin. Tutkimustulokset kuvaavat DLBCL:n merkittävää biologista heterogeenisyyttä, mutta tulosten varmistamiseksi tarvitaan lisää tutkimuksia. Korkea antioksidanttiaktiivisuus ja solusyklin säätelyhäiriöiden kasautuminen erottivat huonoennusteisia potilasryhmiä, jotka voisivat hyötyä uudenlaisista hoidoista. Kemokiinireseptorien ilmentyminen vaikuttaisi olevan yhteydessä DLBCL:n CNS-hakuisuuteen, ja tulosten varmistuessa ekspressioprofiilien analysointia voitaisiin tulevaisuudessa hyödyntää ennaltaehkäisevien hoitojen tehokkaammassa kohdentamisessa.

Abstract Hematopoietic cells, i.e., erythrocytes, platelets and white blood cells, differentiate from hematopoietic stem cells in a process that is similar in vertebrates. Hematopoiesis is regulated by molecules expressed by both the hematopoietic stem and progenitor cells and the surrounding microenvironments. Knowledge of these molecules is important since many of the genes involved in normal hematopoiesis are mutated in leukemia. Furthermore, this information can be utilized in more efficient isolation and expansion of hematopoietic cells in vitro. However, these molecules are not yet sufficiently characterized. Transmembrane immunoglobulin and mucin domain (TIM) genes form a known family of immunoregulators. In mammals, TIM-4 is expressed by antigen presenting cells, while TIM-1, TIM-2 and TIM-3 are expressed by T cells, in which they regulate differentiation of TH cells. The role of TIM molecules in hematopoiesis has not yet been investigated. The aim of this thesis work was to identify and analyze novel molecules involved in embryonic hematopoiesis using chicken and mouse as model organisms. This was carried out by generating a cDNA library of hematopoietic stem and progenitor cells from embryonic chicken para-aortic region. Both previously known and novel candidate genes were identified from the library. Among them, we found homologs to tim genes. Their expression and role in hematopoiesis was studied further. TIM-2 expression was shown to be tightly governed during B cell development. It is expressed by common lymphoid progenitors and highly proliferative large-pro and large pre-B cells during both fetal liver and adult bone marrow hematopoiesis. In mouse, tim-4 expression was restricted to fetal liver CD45+F4/80+ cells. Furthermore, two distinct populations were identified: F4/80hiTIM-4hi and F4/80loTIM-4lo. The results suggest that the F4/80hiTIM-4hi cells are yolk sac-derived macrophages and the F4/80loTIM-4lo cells myeloid progenitors. This work shows for the first time that TIM family molecules are expressed during hematopoiesis. TIM-2- and TIM-4 are expressed by specific cell types during hematopoietic cell development, and in the future they may be utilized as markers in isolation of hematopoietic progenitor cells. / Tiivistelmä Verisolut eli punasolut, verihiutaleet ja immuunipuolustuksessa tärkeät valkosolut kehittyvät alkion veren kantasoluista prosessissa, joka on kaikissa selkärankaisissa samankaltainen. Veren kanta- ja esisolujen sekä ympäröivän mikroympäristön tuottamat molekyylit säätelevät hematopoieesia eli verisolujen kehitystä. Näiden molekyylien tunteminen on tärkeää, sillä useat normaalia verisolujen kehitystä säätelevät geenit ovat osallisena myös verisyöpien synnyssä. Lisäksi tätä tietoa on mahdollista hyödyntää verisolujen tehokkaammassa eristämisessä ja kasvattamisessa hoitoja varten. Immuunipuolustuksen solut, kuten syöjäsolut eli makrofagit ja T-solut, ilmentävät TIM-molekyylejä (Transmembrane Immunoglobulin and Mucin). Ne toimivat immunologisen vasteen säätelyssä sekä solusyönnissä, mutta niiden roolia verisolujen kehittymisessä ei ole selvitetty aikaisemmin. Tässä väitöstutkimuksessa etsittiin uusia hematopoieesiin vaikuttavia geenejä käyttäen mallieläiminä sekä kanaa että hiirtä. Tutkimuksessa luotiin geenikirjasto kanan alkion para-aortaalisen alueen veren kanta- ja esisoluista. Kirjastosta tunnistettiin useita ennalta tiedettyjä sekä uusia verisolujen kehitykseen vaikuttavia geenejä. Tutkimuksessa analysoitiin tarkemmin kirjastosta löytyneiden TIM-geeniperheen jäsenten ilmentymistä ja roolia verisolujen kehityksessä. Tutkimuksessa osoitettiin, että TIM-2 proteiinin ilmentymistä säädellään tarkasti B-solujen kehityksen aikana. Lymfosyyttien yhteiset esisolut sekä suuret pro-B- ja pre-B-solut ilmentävät TIM-2 proteiinia B-solukehityksen aikana sekä alkion maksassa että aikuisen luuytimessä. Hiiren alkiossa tim-4 geenin ilmentyminen oli rajoittunut maksaan, jossa erottui kaksi erillistä solupopulaatiota: F4/80hiTIM-4hi ja F4/80loTIM-4lo. Tutkimuksen tulokset viittaavat siihen, että maksan F4/80hiTIM-4hi solut ovat ruskuaispussista lähtöisin olevia syöjäsoluja ja F4/80loTIM-4lo solut myeloidisen linjan esisoluja. Tämä tutkimus on ensimmäinen osoitus TIM-molekyylien ilmentymisestä kehittyvissä verisoluissa. Havaitsimme, että TIM-2 ja TIM-4-molekyylejä ekspressoidaan tietyissä soluissa verisolujen erilaistumisen aikana, joten tulevaisuudessa niitä on mahdollista käyttää merkkiproteiineina hematopoieettisten solujen esiasteita eristettäessä.

Abstract Lymphomas are a heterogeneous group of malignancies that arise from lymphatic tissues. Diffuse large B-cell lymphoma (DLBCL) is the most common lymphoma sub-type. It is an aggressive malignancy with an increasing incidence. The prognosis of DLBCL has improved significantly, but problems also remain. The clinical significance of central nervous system (CNS) relapses has become increasingly important. As secondary CNSL (sCNSL) and primary CNS lymphoma (PCNSL) are known to have poor prognoses; the prevention of sCNSL is of crucial importance. Peripheral T-cell lymphomas (PTCL) are rare neoplasms and include several lymphoma subtypes that possess complex and also overlapping morphological and immunophenotypic characteristics. The identification of different entities has improved, but the biological knowledge remains scarce when compared to DLBCL. The optimal treatment schemas for PTCLs are still lacking and they have long been treated with the same therapies as B-cell lymphomas, mainly with suboptimal treatment results. The aim of this study was to identify poor prognostic markers in DLBCL and PTCLs and potential biological markers for the prediction of DLBCL CNS relapse. The study material included patients with systemic DLBCL without CNS affision (sDLBCL), sCNSL, PCNSL and PTCLs. The expression of epithelial-mesenchymal transition (EMT) transcription factors (TFs), chemokines and their receptors and adhesion-, migration- and inflammatory responses-associated molecules were studied by means of immunohistochemistry. IEM was used to verify the specific subcellular location of the studied molecules. GEP was performed on 12 PTCL samples in order to compare the poor prognosis group with the good prognosis group and on one sDLBCL and one sCNSL sample from the time of primary diagnosis. The EMT TFs were found to be expressed in both DLBCL and PTCLs, where they ultimately proved to have prognostic relevance as well. In PTCLs, these TFs were able to delineate a disease group with a specific gene-expression profile. CXCR4, CXCR5, ITGA10, PTEN and CD44 were found to be differently expressed between DLBCL cases with CNS affision when compared to those without CNS disease. These molecules seem to play a role in the development of CNS relapse and hopefully, if further verified, will lead towards the identification of biological markers for CNS relapse prediction. / Tiivistelmä Lymfoomat ovat heterogeeninen ryhmä imukudossyöpiä, joista diffuusi suurisoluinen B-solulymfooma (DLBCL) on yleisin alatyyppi. Se on aggressiivinen maligniteetti, jonka insidenssi on noussut viime vuosina. DLBCL potilaiden ennuste on parantunut merkittävästi, mutta yhä osa potilaista menehtyy tautiinsa. DLBCL:n keskushermostorelapsin kliininen merkitys on tänä päivänä aiempaa suurempi. Sekundaarisen keskushermostolymfooman (sCNSL) ja primaarin aivolymfooman (PCNSL) ennusteet ovat nykyhoidoilla huonoja, joten keskushermostorelapsin ennaltaehkäiseminen on tärkeää. Perifeeriset T-solulymfoomat (PTCLs) ovat ryhmä harvinaisia neoplasioita, joka sisältää useita eri alatyyppejä, joiden morfologiset ja immunofenotyyppiset ominaisuudet ovat monimuotoisia ja osin päällekkäisiä. Eri entiteettien indentifiointi on parantunut, mutta PTCL:ien biologinen tietämys on yhä DLBCL:aa heikompaa. PTCL:ien optimaalinen hoito ei ole selvillä ja tätä tautiryhmää on pitkään hoidettu samoilla hoidoilla kuin DLBCL:aa, mutta huonommilla hoitotuloksilla. Tutkimuksen tavoitteena oli löytää huonon ennusteen markkereita, joilla myös pystyttäisiin ennustamaan DLBCL:n keskushermostorelapsia. Aineisto koostui DLBCL, sCNSL, PCNSL ja PTCL näytteistä. Immunohistokemiallisilla värjäyksillä tutkittiin epiteliaalis-mesenkymaalisen transition (EMT) transkriptiotekijöitä (TF), kemokiinireseptoreita sekä adheesioon-, migraatioon ja inflammaatioon assosioituja molekyylejä. Immunoelektronimikroskopialla varmennettiin molekyylien lokalisaatio soluissa. Geeniekspressioprofiloinnilla (GEP) verrattiin kahdentoista hyvän ja huonon ennusteen ryhmään kuuluvan PTCL näytteen välisiä geeniekspressioeroja sekä kahden DLBCL potilaan näytteitä, joista toiselle kehittyi keskushermostorelapsi. EMT TF:ien ekspressiota nähtiin DLBCL ja PTCL näytteissä, joissa niillä myös todettiin olevan ennusteellista merkitystä. PTCL:ssa TF:t pystyivät erottelemaan tautiryhmän, jolla oli oma spesifinen geeniekspressioprofiilinsa. CXCR4, CXCR5, ITGA10, PTEN ja CD44 ekspressio oli erilaista systeemisissä DLBCL tapauksissa verrattuna sCNSL tapauksiin. Edellä mainituilla molekyyleillä näyttää olevan oma roolinsa keskushermostotaudin kehittymisessä ja jos nämä tulokset pystytään vahvistamaan tulevissa tutkimuksissa, johtavat ne toivottavasti kohti keskushermostorelapsiriskin tarkempaa tunnistamista.