CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / A Leucemia LinfocÃtica Aguda (LLA) e o Linfoma nÃo-Hodgkin (LNH) estÃo entre os mais frequentes tipos de neoplasias em crianÃas. A prevenÃÃo e controle do cÃncer devem ser priorizados, tendo em vista sua alta prevalÃncia e crescente relevÃncia como causa de morte em muitos paÃses, alÃm do grande volume de recursos financeiros consumidos. O nordeste brasileiro à pobre em estudos epidemiolÃgicos sobre o cÃncer infantil. O objetivo deste estudo foi traÃar o perfil dos pacientes portadores de LLA e LNH admitidos no perÃodo de 2001 a 2005 no Hospital Infantil Albert Sabin. Estudo observacional, descritivo e retrospectivo. 325 prontuÃrios (254 casos de LLA e 71 de LNH) de pacientes entre 0 e 18 anos e 11 meses foram revisados. Os dados foram inseridos em bancos de dados dos programas SPSS 14.0, Epi Info 3.3.2 e Microsoft Excel 2007. O ponto de corte para desnutriÃÃo foi o escore Z igual a -2 desvios-padrÃo. Na anÃlise estatÃstica foram utilizados o teste exato de Fisher, Qui-quadrado, Student (t), Mann-Whitney, Shapiro-Wilk, Levene, Log-rank, modelo de regressÃo de Cox e mÃtodo de Kaplan Meier para anÃlise de sobrevida. O nÃvel de significÃncia foi p<0,05. A populaÃÃo deste estudo constituiu-se predominantemente por pacientes do sexo masculino (63,4%), faixa etÃria de 02 a 06 anos (49,8%), cor nÃo-branca (62,5%); provenientes da capital e regiÃo metropolitana (56,9%) e com prognÃstico de alto risco (59,1%). 38,3% evoluÃram a Ãbito. As principais manifestaÃÃes clÃnicas iniciais foram febre, anemia, emagrecimento e cansaÃo nos casos de LLA; e febre, massa tumoral palpÃvel, anemia e dor abdominal nos casos de LNH. O tempo mÃdio de duraÃÃo das queixas foi de 3,9 meses. CrianÃas de 0-1 e de 13-18 anos apresentaram pior prognÃstico. A cor da pele, o prognÃstico, o protocolo de tratamento e os sintomas/sinais iniciais febre, cansaÃo e vÃmito mostraram associaÃÃo significativa em relaÃÃo aos Ãbitos. Os protocolos terapÃuticos mais utilizados foram adaptados do LLA 93 e LNH 95. 31% dos pacientes em uso do LLA 93 e 49,2% em uso do LNH 95 evoluÃram a Ãbito. Maior percentual de Ãbito ocorreu no grupo de alto risco (56,94%) e durante a fase de induÃÃo (36,11%) do protocolo LLA 93. Entre os pacientes de baixo risco, 39,28% faleceram durante a fase de manutenÃÃo e 17,85% apÃs o fim do protocolo. 48,78% dos pacientes de alto risco faleceram durante a fase de induÃÃo. Entre os 25 casos que utilizavam o protocolo LNH 95 e faleceram, 4% correspondiam a Linfoma de alto risco oriundos de cÃlulas T e 96% de cÃlulas B. 53,31% (n=15) dos pacientes com Linfoma de cÃlulas B e risco intermediÃrio para recaÃda faleceram durante o Ciclo A do tratamento. Nove pacientes apresentavam alto risco para recaÃda e 33,34% faleceram na fase de CitorreduÃÃo. O Ãndice de desnutriÃÃo para os pacientes com LLA foi de 8,3%, 6,0% e 5,6% e para LNH foi 12,3%, 14,1% e 15,9% em relaÃÃo a peso/estatura, peso/idade e estatura/idade, respectivamente. Pacientes com LLA apresentaram dÃficit maior no Ãndice peso/estatura, indicativo de um processo de desnutriÃÃo aguda. Maior dÃficit no Ãndice estatura/idade entre os pacientes com LNH indica um processo de desnutriÃÃo crÃnica. Os resultados acerca da frequÃncia dessas patologias, faixa etÃria e sexo foram equivalentes aos encontrados na maioria dos estudos. O prognÃstico inicial e o protocolo terapÃutico indicam uma possÃvel influÃncia sobre o desfecho do tratamento. Estudos adicionais sÃo necessÃrios para avaliar a influÃncia da quimioterapia, cor da pele, estado nutricional e outros fatores sobre o tempo de sobrevida do paciente com cÃncer. Os profissionais de saÃde e a populaÃÃo leiga precisam conhecer melhor e estar atentos Ãs manifestaÃÃes clÃnicas iniciais das neoplasias a fim de facilitar o diagnÃstico precoce. / The Acute Lymphocytic Leukemia (ALL) and the Non-Hodgkin Lymphoma (NHL) are among the most frequent types of cancer in children. The prevention and control of the cancer must be prioritized, in view of its high prevalence and increasing relevance as cause of death in many countries, beyond the great sum of consumed financial resources. The Brazilian northeast is poor in epidemiological studies about cancer in the children. The objective of this paper was to set the profile of the patients with LLA and LNH admitted in the Hospital Infantil Albert Sabin between 2001 and 2005. Observational descriptive and retrospective study. 325 medical registers (254 cases of LLA and 71 of LNH) of patients among 0 and 18 years and 11 months had been revised. The data had been inserted in data bases of the programs SPSS 14.0, Epi 3.3.2 Info and Microsoft Excel 2007. A Z-score cut-off point of <-2 SD was used to classify the malnutrition. The Fisherâs Exact Test, Qui-square, Student (t), Mann-Whitney, Shapiro-Wilk, Levene, Log-rank, Cox regression and Kaplan Meier Survival Probability Estimates were used in the statistical analyses. The level of significance was p<0,05. The population of this study was predominantly male (63.4%), 02 to 06 years age-group (49.8%), non-white (62.5%), from the capital and metropolitan region (56.9%) and with prognostic of high risk (59.1%). 38.3% died. The main clinical manifestations had been fever, anaemia, loss of weight and fatigue in the LLA cases; and fever, anaemia, palpable tumor mass and abdominal pain in the LNH cases. The mean duration time of the complaints was 3.9 months. Children with 0 to 1 and 13 to 18 years had presented worse prognosis. The color of the skin, the prognosis, the treatment protocol and the initial clinical manifestations (fever, fatigue and vomit) had shown significant association in relation to the deaths. The therapeutical protocols more used were adapted of the LLA 93 and LNH 95. 31% of the patients in use of LLA 93 and 49.2% in use of LNH 95 died. The largest percent of deaths were in the group of high risk (56.94%) and in the induction phase (36.11%) of protocol LLA 93. Between the patients of low risk, 39.28% died during the maintenance phase and 17.85% after the end of the protocol. 48.78% of the patients of high risk died during the induction phase. Among the 25 cases that used protocol LNH 95 and died, 4% corresponded to lymphoma of high risk deriving of the cells T and 96% of the cells B. 53.31% (n=15) of the patients with lymphoma of the cells B and intermediate risk to fallen died during the Cycle A of the treatment. Nine patients presented high risk for fallen and 33.34% died in the cytoreduction phase. The malnutrition indices to the LLA patients were of 8.3%, 6.0% and 5.6% and to LNH were 12.3%, 14.1% and 15.9% in relation the weight/height, weight/age and height/age, respectively. Patients with LLA had presented larger deficit in the index weight/height, indicative of a process of acute malnutrition. Larger deficit in the height/age index between the patients with LNH indicates a process of chronic malnutrition. The results about the frequency of these disease, age-group and gender were equivalents to those encountered in the majority of studies. The initial prognosis and the therapeutical protocol indicate an influence on the outcome of the treatment. Other studies are necessary to evaluate the influence of the chemotherapy, color of the skin, nutritional status and other factors on the survival time of the patient with cancer. The professionals of health and the laypeople need to know better and to be intent to the initial clinical manifestations of the neoplasm disease in order to facilitate the precocious diagnosis.

Marginal zone (MZ) lymphomas (MZLs) represent a group of lymphomas originating from B lymphocytes of the “marginal zone” which is the external part of the secondary lymphoid follicles. The WHO classifies MZL into 3 entities; extranodal MZL, splenic MZL and nodal MZL. Extranodal marginal zone lymphoma (EMZL) can arise in different tissues, including the stomach, salivary gland, lung, small bowel, thyroid, ocular adnexa and skin. We present a 25 years old female with a history of angioedema and chronic cutaneous eczema who developed an unusual EMZL. Patient presented with a history of rapidly enlarging skin nodule on her left elbow that had been present for almost one year. Over a period of 2-3 weeks she felt the nodule rapidly changed in size and shape. Excisional biopsy of the mass revealed a lymphoid infiltrate based in the reticular dermis and focally extending into the subcutaneous adipose tissue with formation of disrupted lymphoid follicles positive for CD20, CD23 and BCL2 but negative for CD10, Cyclin D1 and SOX11. Diagnosis was consistent with extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT lymphoma). Patient on presentation did not have any B symptoms other cutaneous lesions, lymphadenopathy or hepatosplenomegaly. PET scan revealed no evidence of abnormal uptake leading to a final Stage IE definition. Patient initiated definitive radiation therapy. EMZL accounts for 5 -10 % of non-Hodgkin lymphoma. It has been described often in organs that are normally devoid of germinal centers. It may arise in reactive lymphoid tissue induced by chronic inflammation in extranodal sites. Primary cutaneous marginal zone lymphoma (PCMZL) is associated with infectious etiologies such as Borrelia burgdorferi and less commonly with viral infections or in relation to autoimmune disorders. Autoimmune disorders, specifically Sjögren's syndrome is associated with a 30-fold increased risk of marginal zone lymphoma. Localized disease can be treated by local radiotherapy, intralesional injections or excision. Widespread skin disease is usually treated with a CD20 directed monoclonal antibody-Rituximab. Patients with PCMZL usually have an indolent clinical course. Extracutaneous dissemination of MALT Lymphoma is uncommon and happens in 6-8 % of patients. The 5 years overall survival is between 98-100%. Family physicians and dermatologists should have a high index of suspicion for this rare lymphoma subtype especially in patients with inflammatory chronic skin conditions and atopy.

Mantle cell lymphoma (MCL) represents a heterogenous subtype of non-Hodgkin lymphoma (NHL), which can present in three distinct clinicopathologic variants: indolent type MCL, classic type MCL and blastoid type MCL. Despite the different variations, MCL, in general, is almost always associated with advanced-stage disease at diagnosis, with a strong predilection for significant extranodal involvement, usually to the bone marrow, CNS, peripheral blood and the gastrointestinal tract. However, the literature review reveals ocular involvement is a more rarely described extranodal site of involvement by MCL. Among the reported cases, the orbit was most commonly involved, followed by the eyelid and the lacrimal gland. We report a 63-year-old male who presented with a nine-month history of progressive symptoms of periorbital swelling and eyelid apraxia, causing bilateral visual disturbances. The patient was initially treated for presumed blepharospasm by his ophthalmologist with botulinum toxin injections; however, his periorbital edema continued to worsen, and he developed a discrete nodule in his right lower eyelid. Biopsy of the right eyelid nodule revealed classic type MCL with immunohistochemical testing positive for CD20, CD5, cyclin D1, SOX11 and Ki67 proliferative index of 40%. Fluorescence in situ hybridization (FISH) analysis detected (11;14) translocation. Mantle Cell Lymphoma International Prognostic Index Combined Biologic Index (MIPIb) score was calculated to be 6.5 points based on his age, ECOG performance status of 0-1, normal serum LDH, normal white blood cell count and elevated Ki67 proliferative index, stratifying patient into the high-risk group, with an estimated median overall survival of 37 months. Due to the bulky MCL involvement in the palpebral conjunctiva affecting his vision and eyelid function, he was immediately treated with radiation therapy to the bilateral orbits. PET-CT revealed adenopathy above and below the diaphragm. Bone marrow biopsy revealed focal involvement (5-10%) by MCL. Brain MRI revealed MCL infiltration in the bilateral orbits and lacrimal glands. Upper and lower endoscopy revealed multiple polyps positive for MCL. Given the advanced stage of the disease and his high-risk stratification, he was started on intensive induction chemotherapy with rituximab, dexamethasone, cytarabine, and carboplatin and received prophylactic intrathecal methotrexate. Systemic imaging after completion of four cycles of treatment revealed near resolution of the majority of the lymphadenopathy and all of the lymph nodes no longer demonstrated any significant metabolic activity. He completed two additional cycles of systemic chemotherapy and is currently being evaluated for autologous hematopoietic stem cell transplantation in complete remission-1 given his excellent response to treatment, his young age, high-risk disease at diagnosis, and good performance status. Despite the diffuse and extensive systemic disease, interestingly, our patient did not exhibit any constitutional or metastasis-associated symptoms and only presented with isolated periorbital swelling. Our case emphasizes the rare extranodal site of involvement by MCL and encourages all medical providers to remain cognizant of the varying ways in which MCL can present clinically.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24496号 / 医博第4938号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 滝田 順子, 教授 上野 英樹, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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<b><u>Introduction:</b></u> Some cancer survivors are known to have an elevated risk of morbidity and mortality from cardiovascular disease. An important cause of this elevated risk is recognised to be irradiation of normal tissues during radiotherapy received as part of cancer therapy. There are substantial difficulties in studying radiation-related cardiovascular disease (RRCD). The reasons for this include the complexities of measuring radiation normal tissue doses retrospectively and the prolonged latencies of many of the cardiovascular endpoints. A variety of complimentary research methodologies can help provide additional knowledge to guide the appropriate management of patients treated in the past and of new patients in the future. <b><u>Methods:</b></u> 1) A cohort study of mortality from circulatory disease in the nationwide British Childhood Cancer Survivor Study (BCCSS). 2) A case-control study of valvular heart disease (VHD) in Dutch Hodgkin lymphoma (HL) survivors, including retrospective radiation dosimetry to estimate the radiation dose to heart valves. 3) A dosimetric study of cardiovascular radiation doses in patients entered into the UK NCRI Lymphoma Study Group RAPID trial, including predictions of 15-year cardiac mortality using innovative methods. 4) A modelling study to predict mean whole heart dose (MWHD) from involved field radiotherapy (IFRT) for HL using anatomical measures. 5) A prospective study using cardiovascular magnetic resonance (CMR) imaging to characterise the heart in women receiving radiotherapy for breast cancer. <b><u>Results:</b></u> 1) The risks of all types of circulatory mortality are elevated in survivors of childhood cancer. The absolute excess risks continue to increase 40+ years following diagnosis. The risk of death from cardiomyopathy and heart failure increased substantially with the introduction of anthracycline chemotherapy. There is no evidence of a reduction in risk of circulatory mortality in more recent eras of diagnosis. 2) There is a strong relationship between estimated radiation dose to the affected heart valve and the risk of subsequent VHD (p<0.001). This effect was modelled to allow prediction of the risk of VHD. 3) A proportion of patients treated with IFRT received a substantial cardiac radiation dose (MWHD = 8.8 Gy, SD = 5.6) but, on average, the predicted 15-year cardiac mortality following treatment is low (absolute risk 0.2%, range 0.0 to 2.7%). 4) It is possible to estimate the mean whole heart dose from IFRT prior to detailed radiotherapy planning based on pre-treatment diagnostic imaging to an accuracy of 5-6% of the prescribed dose. 5) Although women received low cardiac doses (MWHD = 1.5 Gy, SD = 0.8) and have a low predicted risk of cardiac radiation-related morbidity and mortality, there is some evidence of subclinical effects on strain and strain rate imaging of the anterior portions of the left ventricle that receive the highest radiation dose. <b><u>Conclusions:</b></u> Using a variety of methods these studies have all succeeded in adding to knowledge about the nature, magnitude and timing of RRCD. This knowledge can be used to help the future management of cancer patients. In addition, each of the studies has natural and planned extensions and will continue to contribute further knowledge into the future.

Les récepteurs à dépendance présentent la particularité d’induire deux voies de signalisation différentes selon la disponibilité de leur ligand. En présence de leur ligand, ils transduisent une voie de signalisation favorisant notamment la survie cellulaire (signalisation positive), tandis qu’en son absence, ils induisent activement l’apoptose des cellules (signalisation négative). La dualité fonctionnelle de ces récepteurs pourrait leur conférer un rôle central au cours du développement embryonnaire, mais aussi dans le maintien de l’homéostasie cellulaire chez l’adulte. En effet, du fait de leur signalisation pro-apoptotique, les récepteurs à dépendance pourraient limiter le nombre de cellules (en fonction de la disponibilité en ligand), et réguler leur migration (dans des zones dépourvues de ligand), au cours des processus développementaux mais aussi en cas de prolifération tumorale et de dissémination métastatique. Au cours de ma thèse, j’ai donc développé différentes thématiques de recherche visant à préciser le rôle du contrôle apoptotique exercé par les couples nétrine-1/récepteurs à dépendance (DCC et UNC5B) au cours de la tumorigenèse et du développement embryonnaire. Dans ce cadre, nous avons montré que les souris exprimant une forme mutée du récepteur DCC, ayant perdu la capacité d’induire l’apoptose des cellules en absence de son ligand la nétrine-1, étaient prédisposées à la survenue de (1) cancers colorectaux dans un contexte prédisposant, et de (2) lymphomes non hodgkiniens (LNH) dans un modèle de vieillissement. Ces résultats suggèrent que DCC puisse agir comme un suppresseur de tumeur conditionnel, via sa voie de signalisation négative, en absence de son ligand nétrine-1. De plus, nous avons montré qu’il existe un gain de nétrine-1 dans une fraction significative de LNH de type B (LNH-B) chez l’Homme, gain qui leur confère un avantage sélectif en bloquant constitutivement l’apoptose induite par le récepteur DCC. Réciproquement, nous avons montré que l’utilisation d’un anticorps ciblant la nétrine-1 était capable de restaurer l’apoptose induite par le récepteur DCC non lié, dans des lignées tumorales humaines de LNH-B in vitro et ex-vivo dans un modèle de xénogreffe. Ainsi, nos résultats suggèrent que cibler l’interaction nétrine-1/DCC dans les LNH-B pourrait être une stratégie thérapeutique prometteuse. La partie « développement » de mon travail de thèse est encore préliminaire. Elle se focalise sur le rôle du contrôle apoptotique exercé par les couples nétrine-1/récepteurs à dépendance (DCC et UNC5B) au cours de la mise en place des réseaux vasculaire et nerveux chez l’embryon de souris / Dependence receptors share the common property of inducing two types of signaling cascades according to the availability of their ligand. In presence of their ligand, they induce a positive signal allowing cell survival whereas in its absence, they induce cell death by apoptosis. Dependence receptors dual functionality could play a major role both during the embryonic development and in the regulation of tissue homeostasis in adult. Indeed, apoptosis induced by dependence receptors would be a safeguard mechanism which regulates the number of cells and their migration during developmental or tumorigenic process. Thus, during my PhD, I have investigated the involvement of netrin- 1/dependence receptors (DCC and UNC5B) apoptotic control during tumorigenesis and embryonic development. We observed that DCC-mutant mice in which DCC pro-apoptotic signaling is genetically silenced show a higher propensity to develop colorectal cancers in a predisposing context and B-cell type non-Hodgkin lymphoma (B-NHL) with age, suggesting than DCC could act as a conditional tumor suppressor gene through its pro-apoptotic signaling, in absence of netrin-1. Moreover, we show that netrin-1 is overexpressed in a significant fraction of Human B-NHL, which confers tumor cells a selective advantage by blocking DCC-induced apoptosis. Reciprocally, we showed that inhibiting the interaction between netrin-1 and its DCC receptor restore apoptosis in lymphoma cells in vitro and in vivo in xenografts experiments, suggesting that targeting netrin-1 and DCC interaction in lymphoma could represent a promising anti-tumor therapeutic strategy. The "developmental" part of my PhD work is in progress and we obtained only few preliminary data. It focuses on the role of apoptotic control mediated by netrin1-/dependence receptor (DCC/UNC5B) during the development of vascular and neural networks in the mouse embryo

Rituximab (IDEC C2B8) ist ein chimärer monoklonaler Antikörper, der gegen das CD20-Antigen auf B-Lymphozyten gerichtet ist. In klinischen Studien konnten Ansprechraten von 50 Prozent bei Patienten mit niedrigmalignen NHL erzielt werden [61, 62]. Bei den hochmalignen NHL waren die Ansprechraten geringer, doch auch hier konnte eine therapeutische Wirksamkeit von Rituximab nachgewiesen werden [108]. Der genaue Wirkungsmechanismus, durch welchen Rituximab seinen therapeutischen Effekt erzielt, ist weiterhin nicht vollständig geklärt. Hauptsächlich werden dabei Apoptose, Komplement-vermittelte zelluläre Zytotoxizität (CDC) und die Antikörper-vermittelte Zytotoxizität (ADCC) diskutiert. In der vorliegenden Arbeit wurde in vitro die Proliferationsinhibition und die Komplement-vermittelte zelluläre Zytotoxizität durch Rituximab an verschiedenen humanen B-Zellinien geprüft. Anschließend wurde die in vivo-Komplementaktivierung während einer Rituximab-Infusion untersucht. Es konnte gezeigt werden, dass Rituximab in vitro eine unterschiedliche Wirksamkeit bei verschiedenen Lymphomzellinien aufweist. Durch Zugabe von humanem Komplement konnte bei zwei Zellinien eine Rituximab-induzierte CDC beobachtet werden. Bei sechs von zehn Patienten mit unterschiedlichen NHL wurde in vivo ein Anstieg der C3a-desArg-Konzentration im Plasma beobachtet. Die Ergebnisse dieser Arbeit zeigen, dass das Komplementsystem ein wichtiger Mechanismus für die Wirkung von Rituximab zu sein scheint. Die klinischen Erfolge sind viel versprechend, zeigen jedoch auch, dass der therapeutische Nutzen von Rituximab als Monotherapie begrenzt ist. Derzeit laufende prospektive Studien untersuchen die Wirksamkeit von Rituximab in Kombination mit verschiedenen Chemotherapeutika [106, 107]. Erste Ergebnisse sind viel versprechend, doch es bleibt abzuwarten, ob sich diese Ergebnisse auch langfristig in einer Verbesserung der Überlebensrate widerspiegeln. / The chimeric anti-CD20 monoclonal antibody rituximab is a new therapeutic tool for treatment of relapsed B-cell lymphomas. Because rituximab mediates complement-dependent cellular cytotoxicity (CDCC) and antibody-dependent cellular cytotoxicity (ADCC) in the lymphoblastoid cell line SB, it is suggested, that these two mechanisms are responsible for the in vivo antilymphoma effect. We tested the antiproliferative effect of rituximab in 6 CD20 positive human B cell lymphoma cell lines. In the follicular lymphoma cell line, DOHH-2, 1 µg/ml rituximab induces an inhibition of proliferation of 75.5 % (n=5), and in the diffuse large cell lymphoma cell line, Daudi, an inhibition of proliferation of 27.3 % (n=5). No effect was seen in the prelymphocytic leukemia cell line, JVM-2, the hairy cell leukemia cell line, Bonna-12, or the two high-grade lymphoma cell lines, Granta-519 and Raji. To test, whether complement increases the effect of rituximab, we studied the combination of rituximab plus complement. Guinea pig complement (dilution 1:10) increases the inhibitory effect of rituximab on the proliferation of Daudi cells. The degradation product C3a(desArg) is produced during complement cascade activation and could be used as a sensitive and specific marker of complement activation in vivo. So, we measured plasma C3a(desArg) concentration in two CLL patients receiving rituximab monotherapy. In one patient, no increase of plasma C3a(desArg) concentration could be measured during infusion of rituximab. In the other patient, in two treatment cycles C3a(desArg) increases drastically after 2 h of rituximab infusion. We conclude differential in vitro and in vivo effects of rituximab on CD20 positive lymphoma cells. Rituximab inhibits in vitro the proliferation of follicular and diffuse large cell lymphoma cells, which could be amplified by complement in Daudi cells. Furthermore, rituximab mediates in vivo complement cascade activation, highly suggestive of in vivo CDCC.

Linfomas são cânceres que se iniciam a partir da transformação maligna de um linfócito no sistema linfático. Os linfomas são divididos em duas categorias principais: os linfomas de Hodgkin e todos os outros linfomas, denominados linfomas não-Hodgkin (LNH). Os pacientes com LNH são comumente tratados com radioterapia apenas ou combinada com quimioterapia utilizando-se de anticorpo monoclonal anti-CD20, principalmente o rituximab (MabThera&reg). O uso de anticorpos monoclonais (Acm) conjugados à quelantes bifuncionais radiomarcados com radionuclídeos metálicos ou lantanídeos é uma realidade de tratamento para portadores de LNH pelo princípio de radioimunoterapia (RIT). Este estudo concentrou-se nas condições de conjugação do anticorpo monoclonal rituximab (MabThera&reg) com grupamentos quelantes bifuncionais DOTA e DTPA. Na marcação dos Acm conjugados com lutécio-177, foram estudadas as condições de pré-purificação do Acm, condições de conjugação, determinação de número de quelantes acoplados à molécula do anticorpo, purificação do anticorpo conjugado, radiomarcação do anticorpo conjugado, com lutécio-177, purificação do anticorpo marcado, a ligação específica in vitro dos compostos marcados às células Raji, e distribuição biológica em camundongos BALB/c sadios. As três metodologias empregadas na pré-purificação do anticorpo (diálise, cromatografia de exclusão molecular com coluna Sephadex G-50 e ultrafiltração) demonstram-se eficientes e proporcionaram recuperação da amostra superior a 90%. A metodologia de ultrafiltração foi considerada a mais simples e prática, podendo ser aplicada a procedimentos rotineiros de produção de radiofármacos. Além disso, proporcionou a recuperação final de amostra de 97% em microlitros. Nas conjugações do anticorpo com os quelantes DOTA e DTPA em razões molares diferentes do Acm:quelante, observou-se número de grupamentos quelantes acoplados à molécula do Acm proporcional à razão molar estudada. Quando foi avaliada a influência de condições diferentes de conjugação no número de quelantes acoplados à molécula do Acm, não foram observadas diferenças significativas, com resultados de pureza radioquímica (PR) inferior a 80% em todas as condições estudadas. Na comparação de métodos de purificação do Acm conjugado, a abordagem inédita apresentada neste estudo, na qual a cromatografia de exclusão molecular foi combinada com a ultrafiltração resultou em maior eficiência na purificação e preservação da estrutura do anticorpo. Nos estudos de radiomarcação do anticorpo conjugado com DOTA e DTPA, os imunoconjugados de DTPA apresentaram, de forma geral, maior eficiência de marcação com resultados reprodutíveis quando comparados com os imunoconjugados de DOTA, considerando-se as diferentes razões molares utilizadas. As metodologias cromatográficas empregadas no controle de pureza radioquímica do composto radiomarcado proporcionaram a discriminação das diferentes espécies radioquímicas no meio de marcação. A metodologia de purificação do composto conjugado e radiomarcado utilizada proporcionou a obtenção de compostos com alta pureza radioquímica, 97,4&plusmn;1,3% (DOTA 1:50) e 98,7&plusmn;0,2% (DTPA 1:50). Nos estudos de ligação específica às células tumorais Raji, o anticorpo conjugado com quelante DTPA nas razões molares de 1:50 e 1:20 apresentaram perfil semelhante de ligação, com aumento da porcentagem de ligação específica proporcional à concentração celular, enquanto que o imunoconjugado na razão molar de 1:10 apresentou alta porcentagem de ligação não específica. Os resultados obtidos nos estudos de biodistribuição in vivo do anticorpo conjugado e radiomarcado nem sempre se mostraram compatíveis com a biodistribuição de anticorpos radiomarcados íntegros. No caso do quelante DOTA, o imunoconjugado obtido a partir da razão molar 1:20, apresentou melhores características de biodistribuição. No caso do quelante DTPA, a razão molar utilizada pareceu refletir diretamente no clareamento sanguíneo do anticorpo e todas as razões molares utilizadas apresentaram instabilidade in vivo. / Lymphomas are malignancies or cancers that start from the malign transformation of a lymphocyte in the lymphatic system. Lymphomas are divided in two major categories: Hodgkin lymphoma and non-Hodgkin lymphoma (NHL). Patient with NHL are generally treated with radiotherapy alone or combined with immunotherapy using monoclonal antibody rituximab (MabThera&reg). Currently, monoclonal antibodies (Mab) conjugated with bifunctional chelate agents and radiolabeled with metallic or lanthanides radionuclides are a treatment reality for patients with NHL by the principle of radioimmunotherapy (RIT). This study focused on the conditions of conjugation of Acm rituximab (MabThera&reg) with bifunctional chelating agents DOTA and DTPA and labeling with 177-luthetium. Various parameters were studied: method of Acm purification, conditions of Acm conjugation and the determination of the number of chelate coupled to the Acm, the purification of the conjugated Mab, labeling conditions with lutetium-177, purification of the radiolabeled immunoconjugate, radiochemical purity (RP), in vitro specific binding determination to Raji cells (Human Burkitt) and biological distribution performed in normal BALB/c mouse. The three methodologies employed in pre-purification of Acm (dialysis, size exclusion chromatograph and ultrafiltration) demonstrated to be efficient; they provided sample recovery exceeding 90%. However, the methodology of ultrafiltration resulted in greater sample recovery and in microliters. The number of chelate attached to the Mab molecule was proportional to the molar ratio studied. When the influence of different conditions of conjugation in the number of chelate bounded to the Mab was studied, no notable differences were observed. The RP < 80% was observed in all the methods applied. Purification of the conjugated antibody by different methods showed that the innovative combination of Sephadex and ultrafiltration methods resulted in higher efficiency of purification. The optimized conditions for purification of the conjugated antibody preserved the protein integrity. Radiolabelling studies of DOTA and DTPA immunoconjugated showed that DTPA derivatives presented, in general, radiochemical yield superior than DOTA conjugated Mab, considering the different molar ratios studied. The chromatographic methods employed in the RP determination were efficient to separate the different radiochemical species presented in the reaction medium. The methodology used in the purification of the labeled Mab resulted in labeled compounds with high radiochemical purity, 97.4&plusmn;1.3% (DOTA 1:50) and 98.7&plusmn;0.2% (DTPA 1:50). Considering specific cell binding assays (Raji cells), the Mab conjugated to DTPA at 1:50 and 1:20 molar ratios presented similar results, and the percent of cell binding were proportional to the cell concentration, whereas the cell binding for 1:10 molar ratio showed high percent of nonspecific cell binding. The results of in vivo biodistribution studies of labeled Mab not always were compatible with the biodistribution of intact radiolabelled antibody. The DOTA immunoconjugated produced at 1:20 molar ratio, showed better performance in biodistribution studies. In the case of DTPA immunoconjugated, the blood clearance seems to be influenced by the molar ratio applied and the immunoconjugated produced with DTPA chelate at different molar ratio resulted in high in vivo instability compounds.

<p>The eosinophil cationic protein (ECP) is usually associated with the eosinophil granulocyte. In this thesis the presence and production of this protein has been studied in two other cells. The circulating monocyte was found to contain ECP mRNA and small amounts of ECP, one thousand times less than that found in the eosinophil. The production decreased by differentiation of the myelomonoblastic cell line U937 into a macrophage phenotype. Submucosal lung macrophages did not stain for ECP and alveolar macrophages did not contain ECP mRNA. The circulating neutrophil contains ECP at a level hundred fold less than the eosinophil. We found that the protein is located to the primary granules of the neutrophil but could detect no ECP mRNA in the cell. It was shown in vitro that the protein was taken up by the cell and partly transported to the primary granules. The uptake did not seem to be receptor mediated. Upon stimulation of the neutrophils, ECP previously taken up, was re-secreted. </p><p>The ECP protein is heterogeneous both to molecular characteristics and to function. To evaluate if a genetic component is involved, the ECP gene was analysed in 70 individuals. Three single nucleotide polymorphisms (SNP´s) were found, denoted 277(C>T), 434(G>C) and 562(G>C). The two first were located to the mature peptide-coding region and would change the amino acids, arg45cys and arg97thr. The prevalence of the most common SNP, 434, was evaluated in two eosinophil-related diseases, allergy/asthma and Hodgkin Lymphoma (HL). Forty-three HL patients were evaluated and it was found that the 434GG was significantly more prevalent in patients having nodular sclerosis (NS) as compared to other histologies (p=0.03). Erythrocyte sedimentation rate was also related to the 434GG genotype (p=0.009). In 209 medical students 434GG was more common (p=0.002) in those who indicated allergy. The genotype was unrelated to the production of IgE antibodies to allergens. In analysis of 76 subjects with asthma it was found that the 434GG genotype was significantly more common among allergic asthmatics (p=0.04). Asthma and HL-NS are characterized by fibrosis and eosinophils and ECP has been suggested in fibrosis development. </p>