Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 5 uppsatser.

The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs.

No description available.

Background: Heart damage from cancer therapy is a significant problem for survivors. Some of the most effective treatments, such as anthracyclines, cause heart toxicity that can lead to significant morbidity and mortality. Cardiotoxicity also contributes to the loss of promising cancer drugs in early development and is notoriously difficult to predict. This translational project employs parallel pre-clinical and clinical studies to explore circulating biomarkers and cardiac magnetic resonance imaging (CMR) during development of anthracycline associated cardiotoxicity with the aim of finding biomarkers to aid clinical decision making and enable forward/back translation. Methods: Pre-clinical work: A rat model of chronic anthracycline-induced cardiomyopathy was developed involving 8 weekly intravenous boluses of doxorubicin followed by a 4 week ‘washout’ period. A time course assessment of cardiac function using multiple MRI parameters was performed alongside a panel of circulating biomarkers measured prior to dosing. Clinical work: In parallel following ethical approval, 30 cancer patients receiving standard anthracycline chemotherapy were recruited. Serial CMR scans were performed using standard and new exploratory techniques before, during and after treatment and blood was taken to evaluate a similar panel of cardiotoxicity biomarkers using multiplex ELISA at corresponding time points. Results: Pre-clinical results: Systolic and diastolic function declined progressively, culminating in left ventricular dysfunction (LVEF < 50%) by 12 weeks. Myocardial electron microscopy revealed myofibrillar and mitochondrial damage after one dose and gross histopathological damage after 5 doses. Myocardial contrast enhancement and troponin I increased significantly after eight doses and preceded LV dysfunction. Extensive fibrosis was seen 1 month after drug cessation. Clinical results: LVEF declined progressively in all patients and 7 patients (23%) had persistent LV dysfunction 12 months after therapy. Troponin I elevations were seen towards the end of therapy and peak troponin I corresponded with LVEF decline. None of the other circulating biomarkers correlated strongly with outcome. Lower baseline extracellular volume (ECV) was associated with greater LVEF decline but little change in ECV was seen over time. Baseline dyssynchrony was associated with worse outcome and deteriorated with time alongside LVEF decline. Conclusions: Results suggest that troponin I and cardiac MRI are sensitive translational tools in drug induced cardiotoxicity. However, troponin I is a relatively late marker, peaking after substantial myocardial damage, too late to halt or change reatment. The imaging suggests that fibrosis and inflammation cannot be detected within a year of chemotherapy but baseline ECV and strain analysis may have a role in risk stratification.

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.

Made available in DSpace on 2014-12-17T14:14:03Z (GMT). No. of bitstreams: 1 AldairSP_DISSERT.pdf: 3401140 bytes, checksum: 29fad07626595e61c30bdf3a90539133 (MD5) Previous issue date: 2014-02-26 / Chronic lymphoproliferative disorders (DLPC) are lymphoid system diseases characterized by the abnormal proliferation of mature lymphocytes that affect B cells, T lymphocytes and NK cells. The aim of the study was to demonstrate the relevance of immunophenotyping by flow cytometry in patients with prolonged lymphocytosis and / or cytomorphological changes compatible with lymphoproliferative diseases. In this study 460 patients (244 men and 216 women) with DLPC were evaluated. Were analyzed by flow cytometry with a panel of monoclonal antibodies consisting of CD3, CD4, CD5, CD8, CD10, CD19, CD22, CD23, CD25, CD38, CD45, CD16/CD56, and HLADR heavy and light chains of immunoglobulins. It also examines information regarding age, gender of patients and laboratory data as leucocytes, cytomorphological analysis, platelet count and hemoglobin determination. The results showed 398 cases of chronic lymphoproliferative disorders and 62 of DLPC B cell lymphoproliferative diseases T. B showed the following distribution : 253 cases of chronic lymphocytic leukemia (CLL), 42 cases of multiple myeloma ( MM ), 37 cases of lymphoma non - Hodgkin lymphoma in leukemic phase (NHL) , 17 cases of pro- B lymphocytic leukemia ( B -PLL), 15 cases of mantle cell lymphoma (MCL ), 12 cases of plasma cell leukemia ( PCL), 9 cases of lymphoma Burkitt (Linf B), 8 cases of leukemia villous cells ( LCV), 3 cases of splenic lymphoma with villous cells (LECV), a case of follicular lymphoma (LF) and a Waldenstr?n macroglobulinemia ( MW). The diseases source NK / T were 23 cases of peripheral T cell lymphoma (LCTP), 14 cases of T prolymphocytic leukemia (T -PLL), 10 cases of leukemia T of large granular lymphocytes (LGL -T) 9 cases of leukemia cells of adult T (LCTA), 5 cases of Sezary syndrome (SS) and a case of large granular NK leukemia (LGL -NK) lymphocytes. In conclusion, the combined use of the monoclonal antibody panel careful cytomorphological analysis was shown to be essential in immune diagnosis and classification of chronic lymphoproliferative disorders. This study was approved by the IRB - HUOL under number 356 / 09 / Os transtornos linfoproliferativos cr?nicos (DLPC) s?o doen?as do sistema linfoide caracterizadas pela prolifera??o anormal de linf?citos maduros que acometem c?lulas B, linf?citos T e c?lulas NK. O objetivo do estudo foi demonstrar a relev?ncia da imunofenotipagem por citometria de fluxo em pacientes portadores de linfocitose prolongada e/ou altera??es citomorfol?gicas compat?veis com doen?as linfoproliferativas. Neste estudo foram avaliados 460 pacientes (244 homens e 216 mulheres) com DLPC. Foram analisados por citometria de fluxo com um painel de anticorpos monoclonais constitu?do por CD3, CD4, CD5, CD8, CD10, CD19, CD22, CD23, CD25, CD38, CD45, CD16/CD56, HLADR e cadeias leves e pesadas de imunoglobulinas. Foram tamb?m investigadas informa??es referentes ? idade, ao g?nero dos pacientes e aos dados laboratoriais como: leucometria, an?lise citomorfol?gica, contagem de plaquetas e determina??o da hemoglobina. Os resultados demonstraram 398 casos de doen?as linfoproliferativas cr?nicas B e 62 de DLPC de c?lula T. As doen?as linfoproliferativas B apresentaram a seguinte distribui??o: 253 casos de leucemia linfoc?tica cr?nica (LLC), 42 casos de mieloma m?ltiplo (MM), 37 casos de linfoma n?o Hodgkin em fase leuc?mica (LNH), 17 casos de leucemia pr?-linfoc?tica B (LPL-B), 15 casos de linfoma de c?lulas do manto (LCM), 12 casos de leucemia de c?lulas plasm?ticas (LCP), 9 casos de linfoma de Burkitt (Linf B), 8 casos de leucemia de c?lulas vilosas (LCV), 3 casos de linfoma espl?nico de c?lulas vilosas (LECV), um caso de linfoma folicular (LF) e um de macroglobulinemia de Waldenstr?n (MW). As doen?as de origem T/NK foram: 23 casos de linfoma de c?lulas T perif?ricas (LCTP), 14 casos de leucemia pr?linfoc?tica T (LPL-T), 10 casos de leucemia de grandes linf?citos T granulares (LGLT), 9 casos de leucemia de c?lulas T do adulto (LCTA), 5 casos de S?ndrome de Sezary (SS) e um caso de leucemia de grandes linf?citos NK granulares (LGL-NK). Em conclus?o, o uso do painel de anticorpos monoclonais combinado ? an?lise citomorfol?gica cuidadosa mostrou-se essencial no diagn?stico e classifica??o imune das doen?as linfoproliferativas cr?nicas. O presente estudo foi aprovado pelo CEP HUOL sob n?mero 356/09

Abstract Hodgkin lymphoma (HL) is among a heterogeneous group of lymphomas. Over 80% of all patients can be cured with chemo- and radiotherapy. HL has become a model to study long-term effects of radio- and chemotherapy, because of the excellent prognosis. There are a significant number of patients who suffer or die because of the treatment-related long-term toxicity. The aim of this work was to discover new possible biological factors to predict poor prognosis and offer new aspects to individualize patient treatment in a convenient manner in HL. The retrospective study involved HL patients uniformly treated in 1997–2015. Immunohistochemistry was used to determine the expression of various biological markers, including oxidative stress markers 8-hydroxydeoxyguanosine (8-OHdG) and nitrotyrosine and the antioxidant enzymes manganese superoxide dismutase (MnSOD) as well as peroxiredoxins (Prx II, Prx III, Prx V, Prx VI) in HL patient samples. Using immunohistochemistry, we also evaluated expression of hypoxia-inducible factors (HIF-1&#945;, HIF-2&#945;), prolyl hydroxylase domain enzymes (PHD1, PHD2, PHD3), the epigenetic regulator lysine (K)-specific demethylase 4 (KDM4A, KDM4B, KDM4D) as well as sirtuins (SIRT1, SIRT4, SIRT6), the DNA-repair proteins Human Rap1 interacting factor 1 (Rif1) and O6-alkylguanine DNA alkyltransferase (MGMT) from representative classical Hodgkin lymphoma (cHL) patient samples. Low-level expression of 8-OHdG was associated with poorer relapse-free survival (RFS) in advanced-stage HL and a high extent of MnSOD predicted early relapse in the whole HL cohort. Strong expression of PHD1, KDM4B and KDM4D predicted dismal RFS in radiotherapy-treated cHL patients. The results also showed that strong expression of HIF-1&#945;, SIRT6 and Rif1, and SIRT6 together with Rif1, were associated with prolonged RFS, especially in advanced-stage radiotherapy-treated cHL patients. In multivariate analysis, PHD1, MnSOD, 8-OHdG and Rif1 separately and together with SIRT6 were statistically significant predictors of RFS. The results reflect the significance of the studied biomarkers in HL, especially in radiotherapy-treated patients. This might be beneficial when individualizing treatment strategies, avoiding overtreatment and controlling long-term treatment-related toxicity. Further research, however, is needed to confirm these preliminary findings. / Tiivistelmä Hodgkinin lymfooma (engl. HL) kuuluu heterogeeniseen imukudossyöpien eli lymfoomen ryhmään. Yli 80 % lymfoomapotilaista voidaan parantaa solunsalpaaja- ja sädehoidon avulla. Hyvän ennusteen takia HL- tutkimuksen tärkeä painopiste on säde- ja solunsalpaajahoidon pitkän ajan haittavaikutukset. Huomattava määrä potilaista kärsii tai jopa kuolee hoitoon liittyvistä pitkäaikaishaitoista johtuen. Tämän tutkimuksen tarkoituksena oli löytää uusia mahdollisia biologisia tekijöitä, jotka ennakoisivat taudin huonoa ennustetta ja samalla antaa uusia näkökulmia HL potilaiden hoidon yksilöllistämiseen. Tämä retrospektiivinen tutkimus käsitti vuosina 1997-2015 samanlaisesti hoidettuja Hodgkinin lymfooma -potilaita. Immunohistokemiallisilla värjäyksillä määritettiin biologisten merkkiaineiden, mukaan lukien oksidatiivisen stressin markkereiden 8- hydroksideoksiguanosiinin (8-OHdG) ja nitrotyrosiinin, sekä antioksidanttientsyymien mangaanisuperoksidi-dismutaasin (MnSOD) sekä peroksiredoksiinien (Prx II, Prx III, Prx V, Prx VI) ilmentymistä HL -potilasnäytteissä. Määrittelimme myös immunohistokemiallisilla värjäyksillä epigeneettisten säätelijöiden lysiinin spesifisen demetylaasientsyymin 4 (KDM4A, KDM4B, KDM4D) sekä sirtuiinien (SIRT1, SIRT4, SIRT6), hypoksiaa indusoivien tekijöiden (HIF-1&#945;, HIF-2&#945;), prolyylihydroksylaasientsyymien (PHD1, PHD2, PHD3) ja DNA:ta korjaavien proteiinien Rap1 vaikuttuvan tekijä 1 (Rif1) ja O6-metyyliguaniini-DNA metyylitransferaasin (MGMT) ilmentymistä edustavissa klassista Hodgkinin lymfoomaa sairastavien potilaiden (engl. cHL) näytteissä. Heikko 8-OHdG värjäytyminen ennusti ennenaikaista taudin uusiutumaa levinneessä HL:ssa ja korkea MnSOD ilmaantuvuus ennusti ennenaikaista taudin uusiutumaa koko HL -ryhmässä. Sädehoidetuilla cHL potilailla voimakas PHD1, KDM4B ja KDM4D värjäytyminen ennusti ennenaikaista taudin uusiutumaa. Tulokset osoittivat myös, että erityisesti sädehoidetuilla levinneen taudin cHL potilailla voimakas HIF-1&#945;, SIRT6, Rif1 ja SIRT6 yhdessä Rif1:n kanssa oli yhteydessä pidentyneeseen uusiutumavapaaseen aikaan. Monimuuttuja-analyysissä PHD1, MnSOD, 8-OHdG ja Rif1 itsenäisenä ja yhdessä SIRT6 kanssa ennustivat tilastollisesti merkitsevästi taudin ennenaikaista uusiutumaa. Tulokset osoittavat näiden eri biomarkkereiden merkittävyyden HL:ssä, erityisesti sädehoitoa saaneilla potilailla. Tuloksista voi olla hyötyä, kun hoitokäytäntöjä yksilöidään, mikä voisi helpottaa välttämään liiallista hoitoa ja hallitsemaan pitkäaikaisiin hoitoihin liittyviä haittoja. Näiden alustavien havaintojen vahvistamiseksi tarvitaan kuitenkin lisätutkimuksia.

Orientadores: Eliane Maria Ingrid Amstalden, Jose Vasallo / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T14:26:55Z (GMT). No. of bitstreams: 1 Lima_FranciscoPignataro_D.pdf: 4114475 bytes, checksum: 852f416deccb941fe4dc3e63c2553510 (MD5) Previous issue date: 2008 / Resumo: Cerca de 20% a 40% dos linfomas não-Hodgkin (LNH) B são extranodais e caracterizam-se por comportamento biológico distinto, de acordo com o seu sítio de origem e variante histológica. Linfoma ósseo primário (LOP), uma forma extranodal rara, apresenta-se na maior parte dos casos como LNH B de alto grau e evolui com bom prognóstico. Os estudos sobre o LOP são escassos e suas características imunofenotípicas e citogenéticas ainda não estão bem definidas. Neste trabalho, foram analisados três grupos de linfomas difusos de grandes células B (LDGCB): a) ósseos primários, B) nodais e c) metastáticos (com envolvimento ósseo secundário). Foram investigados, nesses grupos, dados clínicos e de sobrevida, aspectos morfológicos, imunofenotípicos e citogenéticos. Na análise imunoistoquímica, foi verificado o estudo das moléculas envolvidas na apoptose e regulação do ciclo celular (Bcl-2, Bax, p53, Bcl-6, p21, pRB) moléculas envolvidas na adesão celular (CD29, CD62L, CD44, CD51) índice de proliferação celular (Ki-67) e expressão das subpopulações de células infiltrantes do sistema imune (CD3, CD4, CD5, CD8, CD57, CD68, Foxp3) com objetivo de melhor caracterizar o LOP. Foram selecionados no total 138 casos de LDGCB, provenientes dos departamentos de Anatomia Patológica FCM-UNICAMP, Hospital AC.-Camargo - Fundação Antônio Prudente, Instituto de Traumatologia e Ortopedia da USP e Centre Hospitalier Universitarie (CHU Purpan). Do total de 138, 76 casos eram linfomas ósseos primários, 46 casos linfomas nodais e 16 casos linfomas com envolvimento ósseo secundário. Nossos resultados demonstraram que o LOP possui características evolutivas que diferem dos demais LDGCB extranodais. Observamos que a maioria dos LDGCB ósseos primários (66%) pertenceram ao imunofenótipo centro germinativo (CG) e não exibiram diferenciação plasmacítica. Os três grupos de LDGCB analisados exibiram forte imunoexpressão das proteínas relacionadas à apoptose e ao ciclo celular enquanto que a imunoexpressão de moléculas de adesão foi fraca ou ausente, na maioria dos casos. O estudo da sobrevida, envolvendo a ausência da imunoexpressão das moléculas de adesão CD29, CD62L e CD51, nos três grupos, revelou-se significativamente associada com melhor sobrevida. O significado biológico deste achado ainda está para ser esclarecido. O CD44 foi o mais fortemente expresso nas células tumorais. As moléculas envolvidas na apoptose Bax e p53 foram relacionadas à pior evolução e o pRB à evolução favorável nos LOP. As proteínas Bcl-2 e Bcl-6 foram expressas nos três grupos de linfomas. Com a análise em 32 casos de LOP das principais translocações em LNH (MYC, BCL2/IGH, BCL6, ALK, IGH/CCND1 e PAX5), conseguimos caracterizar melhor esta entidade. Rearranjos, envolvendo o BCL2 e MYC foram encontrados, entretanto não ocorreram rearranjos do BCL6. Os demais genes não demonstraram translocações. Estes achados indicam que o LOP deve ser considerado uma categoria distinta de LDGCB extranodal, com aspectos genéticos e moleculares mais próximos dos nodais / Abstract: The incidence of extranodal lymphomas is increasing in the last decades. Around 20 to 40% of B-cell non-Hodgkin lymphomas (NHL) arise outside the lymph nodes and may present distinct biological behavior, as compared to the corresponding nodal counterpart of similar histological grade. The heterogeneity of large B-cell lymphomas (LBCL) has been matter of various studies, although morphology alone may be insufficient to separate groups of clinical relevance, immunophenotyping and molecular techniques have demonstrated that different variants of LBCL may present diverse clinical and prognostic features. Primary bone lymphoma (PBL) is rare form of extra nodal NHL, which generally presents a more favorable prognosis than the nodal counterpart. The reason for this is unclear. We selected a large series (n=138) of nodal NHL (N=46), primary (n=76) and metastatic (n=16) bone diffuse large cell lymphomas collected in tissue micro-arrays from several centers in France and Brazil. We investigated to be comparing clinical, morphological and immunophenotypic features. Immunohistochemical detection of proliferation markers, apoptosis related proteins and adhesion molecules was be marked. Our results demonstrated that primary bone LDGCB possesses peculiar clinical characteristics that you differ of the other extranodal LDGCB as the longest evolution and smaller recurrence tax. The three groups of LDGCB exhibited strong immunoexpression of the related proteins the apoptosis and cellular cycle while the immunoexpression of adhesion molecules was weak or absentee in most of the cases. These cases were classified according to the expression of antigens with germinal center (GC) (n=37/62) or non germinal center (non-GC) (n=26/62) stages of B-cell differentiation. The study of the survival involving the absence of the imunoexpressão of the adhesion molecules CD29, CD62L and CD51 in the three groups was revealed significantly associated with better outcome. The biological meaning of this discovery is still to be explained. CD44 was it more strongly expressed in the tumor cells and it demonstrated relationship away with inferior survival without statistical significance. The molecules involved in the apoptosis Bax and p53 were related the worst evolution and the pRB the favorable evolution in PBL. The proteins Bcl-2 and Bcl-6 were expressed in the three lymphomas groups and associated the worst evolution, however without significance. By fluorescence in situ hybridization, we found a substantial number of cases with a rearrangement of BCL2 (n=9/32) and MYC (n= 3/32) whereas PAX5, BCL6, BCL-1/Cyclin D1 and ALK genes were in germ line configuration. Interestingly, one case, with GC phenotype, showed a dual BCL2 and MYC rearrangement. We observed that the majority of the cases with rearrangements were of GC phenotype. These results, associated with the lack of BCL6 rearrangement, suggest that bone DLBCL represents a specific group within extranodal B-cell lymphomas. Keywords: diffuse large cell lymphoma, adhesion molecules, apoptosis, fluorescent in situ hybridization / Doutorado / Anatomia Patologica / Doutor em Ciências Médicas

Linfomas pertencem a um grupo de neoplasias que tem em comum a origem em células linforreticulares, sendo a forma anatômica gastrointestinal a mais prevalente na espécie felina. A maioria dos animais responde inicialmente ao tratamento, contudo, sabe-se que o desequilíbrio no sistema imune, de modo geral, pode facilitar a ocorrência e a disseminação das neoplasias. Para equilibrar a atividade do sistema imune, as células com função regulatória (Tregs) são fundamentais e por isso, alvo de diversas pesquisas isoladamente ou associadas a outros marcadores, como a IL-17A e o CD8, já que tanto as Tregs quanto os linfócitos Th17 originam-se de uma mesma célula T progenitora. Assim, alterações na relação Treg/Th17 podem levar à supressão na produção de linfócitos T CD8, invertendo a relação Tregs/CD8. Também há indícios de que as Tregs alterem a função efetora de células T contra neoplasias em seres humanos e em cães. Foram objetivos deste estudo avaliar as características do linfoma gastrointestinal em felinos, a proliferação celular por meio do índice mitótico e do marcador Ki-67, a expressão imunoistoquímica de CD4, CD8, CD25 (IL-2R), FOXP3 e IL-17A e correlacionar a expressão desses marcadores com o tipo celular, imunofenótipo, resposta ao tratamento e sobrevida global. Para análise estatística foram utilizados os testes de Mann-Whitney, Kruskal-Wallis, Quiquadrado de Pearson, de correlação de Spearman e a curva Kaplan-Meyer. Dos 47 gatos, 85% apresentaram a forma linfocítica de células T, com mediana de sobrevida de 24 meses; 15% apresentaram a forma linfoblástica de células B ou T, com mediana de sobrevida de 5 meses. A expressão de Ki-67 foi mais evidente naqueles pacientes com linfoma linfoblástico de células B ou T se comparado ao linfocítico de células T, com mediana de 40,5% e 8,2%, respectivamente e p=0,002. Na análise dos marcadores do sistema imune, não foi observada marcação de IL-2R. Não houve diferença significante na expressão de IL-17 e FOXP3 entre os tipos celulares e imunofenótipo, bem como não houve correlação entre FOXP3 e IL-17 e entre FOXP3 e CD8. Considerando somente os casos de linfoma linfocítico de células T, na análise do padrão de remissão parcial (n=12) versus completa (n=27), constatou-se diferença significante quanto à sobrevida (p&lt;0,0001) e CD8 (p&#61;0,015). Na análise de sobrevida, o tratamento com L-asparaginase, clorambucil e prednisolona, os níveis de albumina sérica iguais ou maiores que 2,5g/dL e o ganho de peso no início do tratamento foram preditores de maior tempo de sobrevida. Pode-se concluir que o linfoma linfocítico de células T foi o mais prevalente na espécie felina, envolvendo principalmente o intestino delgado e com um tempo mediano de sobrevivência de 24 meses; a expressão de ki-67 foi melhor marcador para determinar a proliferação celular se comparada ao índice mitótico; a expressão de FOXP3 foi baixa e não se relacionou à resposta terapêutica e ao tempo de sobrevida. Na análise específica dos casos de linfoma linfocítico de células T, não houve correlação entre FOXP3/IL-17 e FOXP3/CD8, enquanto que a baixa expressão de CD8 correlacionou-se a maior sobrevida naqueles pacientes em remissão completa / Lymphomas belong to a group of neoplasia that have in common the origin in lymphoreticular cells, and gastrointestinal is the most prevalent anatomical form in feline species. Most animals initially respond to treatment, however, it is known that the imbalance in the immune system generally may facilitate the occurrence and spread of tumors. To balance the activity of immune system, cells with regulatory function (Treg) are essential and therefore the target of many researches, alone or in combination with other markers such as IL-17A and CD8, as well Tregs as Th17 lymphocytes originate from a same progenitor T-cell. Thus, the relation between Treg / Th17 is important since changes in this relation can lead to suppressed production of CD8&#43; T lymphocytes, inverting the relation Treg/CD8. There is also evidence that Tregs alter the effector function of T cells against tumors in human beings and dogs. The objectives of this study was to evaluate the characteristics of gastrointestinal (GI) lymphoma in cats, cell proliferation by mitotic index and Ki-67 marker, immunohistochemical expression of CD4, CD8, CD25 (IL-2R), FOXP3 and IL-17A and correlate the expression of these markers on the cell-type, immunophenotype, response to treatment and overall survival. Statistical analysis was performed by Mann-Whitney test, Kruskal-Wallis test, Pearson\'s Chi-squared test, Spearman correlation and Kaplan-Meier curve. Of the 47 cats, 85% showed lymphocytic T cells, with a median survival of 24 months; and 15% had lymphoblastic B or T cells, with a median survival of 5 months. Ki-67 expression was most evident in patients with lymphoblastic B- or T-cell lymphoma compared to lymphocytic T cell lymphoma, with a median of 40.5% and 8.2%, respectively and p &#61 0.002. In the analysis of immune system markers, immunostaing was not observed IL-2R. There was no significant difference in IL-17A and FOXP3 expression between cell-type and immunophenotypes, as well as no correlation between FOXP3 and IL-17A and between FOXP3 and CD8. Considering only the cases of lymphocytic T-cell lymphoma, there was a significant difference in survival time (p <0.0001) and CD8 expression (p &#61; 0.015) considering partial (n &#61; 12) versus complete remission (n &#61; 27). In survival analysis, treatment A (L-asparaginase, chlorambucil and prednisolone), as well as serum albumin levels equal or higher than 2.5g/dL and weight gain after 30 days of treatment were predictive of increased survival time. As conclusion, lymphocytic T-cell lymphoma was more prevalent in feline species, mainly involving the small intestine and with a median survival of 24 months. Ki-67 expression was better marker to determine cell proliferation than mitotic index. FOXP3 expression was low and did not correlate to therapeutic response and survival time in GI lymphoma. In the specific analysis of lymphocytic T-cell lymphoma, there was no correlation between FOXP3/IL-17A and FOXP3/CD8, while low CD8 expression was correlated with increased survival in patients in complete remission

O linfoma é uma doença neoplásica comum em cães, requerendo quimioterapia para aumentar a sobrevida dos pacientes. Durante o tratamento, são freqüentes as recidivas, que motivam alteração do protocolo medicamentoso. Proteína C-reativa e amilóide A sérica são mediadores de fase aguda produzidos no fígado que apresentam elevações de concentrações séricas em condições inflamatórias, infecciosas e neoplásicas de maneira geral. O objetivo do trabalho foi avaliar o papel dessas proteínas na monitorização da remissão e recidiva do linfoma em cães, utilizando 2 protocolos de tratamento. O protocolo COP (ciclofosfamida, vincristina e prednisona) caracterizou-se por fase de indução de 1 mês e ciclos de manutenção a cada 21 dias e o protocolo VCM (vincristina, ciclofosfamida, metotrexato e L- asparaginase) foi empregado em um regime semanal contínuo. Constituíram-se 5 grupos de estudo: Normal (20 cães hígidos), Controle COP (4 cães hígidos submetidos a quimioterapia com o protocolo COP), Controle VCM (4 cães hígidos submetidos a quimioterapia com o protocolo VCM), Linfoma COP (10 cães com linfoma multicêntrico tratados com o protocolo COP) e Linfoma VCM (10 cães com linfoma multicêntrico tratados com o protocolo VCM). Proteína C-reativa e amilóide A sérica foram determinadas pela técnica de Elisa e a eletroforese foi feita em tiras de acetato de celulose. Nos cães do grupo Normal, o estabelecimento das concentrações de proteína C-reativa, amilóide A sérica e as rações eletroforéticas ocorreu uma única vez; nos cães dos grupos Controle COP e Controle VCM na 1ª, 2ª, 3ª, 4ª, 7ª, 10ª, 13ª e 16ª semanas de quimioterapia e, nos cães dos grupos Linfoma COP e Linfoma VCM, na 1ª, 2ª, 3ª e 4ª semanas, bem como na recidiva e num momento de estabilidade da doença imediatamente antes da recidiva. Os resultados foram interpretados por análise de variância com medidas repetidas, tendo como fatores de controle os grupos e as semanas de observação, seguida de comparações múltiplas de Tukey, ao nível de significância de 5 %. Concluiu-se que: o linfoma induz a resposta de fase aguda em cães, sendo a intensidade da resposta amenizada durante o tratamento bem-sucedido dos pacientes; incrementos de proteína C-reativa e amilóide A sérica não estão relacionados à recidiva do linfoma; a quimioterapia do linfoma com os protocolos COP e VCM não altera a resposta de fase aguda, avaliada por meio dessas proteínas, nem existe diferença na resposta de fase aguda entre tais protocolos; existe relação direta entre os níveis de proteína C-reativa e amilóide A sérica no curso do linfoma e da quimioterapia; aumentos das concentrações séricas de proteína C-reativa são acompanhados de elevações da fração de &beta;2-globulinas e aumentos de amilóide A sérica são acompanhados de elevações de &beta;1-globulinas. / Lymphoma is a common neoplasm in dogs and chemotherapy is indicated to achieve long-term survivals. During the treatment, frequent relapses require drug regimen modifications. C-reactive protein (CRP) and serum amyloid A (SAA) are hepatic acute-phase mediators and usually are increased in inflammatory, infectious and neoplastic conditions. The aim of this study was to evaluate the role of these proteins in remission and relapse monitoring of dogs with lymphoma, under 2 chemotherapy protocols. COP protocol (cyclophosphamide, vincristine and prednisone) included an one-month induction period and maintenance cycles each 21 days and VCM protocol (vincristine, cyclophosphamide, methotrexate and L-asparaginase) was administered in a continuous weekly schedule. Five groups were composed: Normal (20 healthy dogs), COP Control (4 healthy dogs submitted to chemotherapy with COP protocol), VCM Control (4 healthy dogs submitted to chemotherapy with VCM protocol), COP Lymphoma (10 dogs with multicentric lymphoma treated with COP protocol) and VCM Lymphoma (10 dogs with multicentric lymphoma treated with VCM protocol). CRP and SAA were determined by Elisa tests and the electrophoresis was performed in cellulose acetate strips. In Normal dogs, CRP and SAA levels, as well the electrophoretic fractions, were measured only one time; in COP Control and VCM Control groups of dogs at the chemotherapy weeks 1, 2, 3, 4, 7, 10, 13 and 16; in dogs from groups COP Lymphoma and VCM Lymphoma, at the weeks 1, 2, 3 and 4, beside the relapse and a called stability moment immediately before the relapse. Results were compared by means of repeated measures variancy analyses, considering the groups and the observation weeks as the control factors, followed by Tukey´s multiple comparisons, at 5 % of significance level. It was concluded that: lymphoma induces an acute phase response in dogs and the intensity of response declines along the disease remission; increases of CRP and SAA are not related to lymphoma relapse; neither COP nor VCM chemotherapy changes the acute-phase response, when CRP and SAA are taken in account, and there is not difference on acute-phase response between both regimens; there is a positive correlation between CRP and SAA levels during lymphoma assessment and chemotherapy; increases of CRP levels are followed by &beta;2-globulin elevations and increases of SAA levels are related to &beta;1-globulin elevations.