O desenvolvimento da imunoterapia do câncer baseada em células dendríticas (DCs) é alvo de vários estudos. Para tumores sólidos, a abordagem baseada no uso de DCs alogenêicas fundidas com células tumorais tem se mostrado relativamente eficaz. Por outro lado, esta estratégia necessita uma massa tumoral considerável de cada paciente para a geração das células híbridas. Para contornar este problema o uso de DCs transfectadas com mRNA tumoral, o qual pode ser amplificado in vitro a partir de uma pequena amostra inicial do tumor, tem sido investigado. Para isto, uma transfecção eficiente e tradução correta do mRNA tumoral nas DCs são etapas críticas. Sabendo-se que as DCs de pacientes com câncer possuem atividade aloestimuladora defeituosa, DCs derivadas de doadores saudáveis poderiam ser uma alternativa para induzir uma resposta imune mais eficiente. Assim, este trabalho pretendeu aprimorar a metodologia de transfecção de DCs alogenêicas, derivadas de monócitos de doadores saudáveis, com mRNA de antígenos tumorais (survivina e RPSA) super-expressos na leucemia linfóide crônica (LLC) e avaliar sua capacidade em estimular a resposta linfocitária. Ao mesmo tempo, foram estabelecidas as metodologias para amplificação e síntese do RNA destes antígenos tumorais específicos, assim como do RNA mensageiro total, contidos nas células tumorais de pacientes com LLC. Os resultados mostraram ser possível a amplificação do mRNA total extraído das células leucêmicas com manutenção da expressão dos antígenos tumorais. Ainda, várias condições de transfecção com mRNA da survivina, transcrito in vitro, foram testadas, encontrando-se na lipofecção, a melhor maneira de transfectar as DCs. A lipofecção mostrou-se com baixa toxicidade quando comparada à técnica de eletroporação. Observou-se uma eficiência em torno de 40% de células transfectadas num intervalo de tempo entre 12 e 48 horas. Estas células foram usadas como estimuladoras em ensaios de proliferação usando-se linfócitos T alogenêicos como células respondedoras. As células transfectadas com mRNA da survivina foram capazes de estimular resposta linfoproliferativa com maior produção de IFN-gama, avaliado por ELISA. Além disso, a transfecção não alterou o padrão de expressão dos marcadores de superfície característicos das DCs. Estes dados mostram que a transfecção das DCs com mRNA pode afetar a resposta imune induzida por estas APCs. Nossos resultados suportam o uso de DCs transfectadas com mRNA para produção de vacinas anti-tumorais e mostram a survivina como um potente antígeno indutor da resposta linfocitária. / The development of dendritic cell (DC)-based cancer immunotherapy has been the target of many studies. For solid tumors, a promising approach based in allogeneic DCs fused to tumor cells has been relatively effective. On the other hand, this approach needs large tumor samples to generate enough DC-tumor cell hybrids. To overcome this problem, tumor mRNA-transfected DCs can be used, since mRNA can be amplified in vitro and allow unlimited vaccine production. For this, efficient transfection and optimal translation of tumor mRNA in DCs are critical. Moreover, DC derived from cancer patients has defective alostimulatory activity. In this case, DC derived from healthy donors may be an alternative to induce immune response more efficiently. Here, we established the methodology of allogeneic DC transfection with mRNA for tumor antigens (survivin and RPSA) overexpressed in chronic lymphocytic leukemia (CLL), and evaluated their ability for T cell stimulation. At the same time, we established mRNA amplification and mRNA in vitro transcription methodology for specific tumor antigens and total messenger RNA, present in CLL tumor cells. Our results showed it to be possible to amplify total mRNA derived from leukemic cells maintaining tumor antigen expression. Moreover, several transfection conditions using survivin mRNA obtained from in vitro transcript reactions were evaluated, defining lipofection as the better way to transfect DC. We obtained nearly 40% of transfected DCs between 12 and 48 hours. Transfected DCs were used as stimulator cells in proliferation assays using allogeneic T cells as responder cells. Survivin mRNA transfected DCs were able to stimulate T cell proliferation with increased IFN-gama production, measured by ELISA. Furthermore, the transfection did not change the pattern of surface molecules expression characteristic of DC. These data show that mRNA DC transfection can affect immune responses induced by these APCs. These findings support the use of tumor mRNA transfected DCs for anti-cancer vaccine production and show survivin as a potent antigen to induce T cell responses.

by Keung Ka Man. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 163-184). / Abstracts in English and Chinese. / Abstract --- p.ii / 摘要 --- p.iv / Acknowledgements --- p.vi / Table of Contents --- p.vii / List of Abbreviations --- p.xiv / List of Tables --- p.xvi / List of Figures --- p.xvi / Chapter CHAPTER 1. --- INTRODUCTION / Chapter 1.1. --- Events happened after brain injury --- p.1 / Chapter 1.2. --- Glial cells --- p.3 / Chapter 1.2.1. --- Microglia --- p.4 / Chapter 1.2.2. --- Oligodendrocytes --- p.5 / Chapter 1.2.3. --- Astrocytes --- p.5 / Chapter 1.2.3.1. --- Uptake of neurotransmitters --- p.7 / Chapter 1.2.3.2. --- Maintenance of extracellular homeostasis --- p.8 / Chapter 1.2.3.3. --- Induction of blood-brain-barrier --- p.8 / Chapter 1.2.3.4. --- Guidance of migrating neurons during development --- p.9 / Chapter 1.2.3.5. --- Immunocompetent cells of the brain --- p.9 / Chapter 1.2.3.6. --- Contribution to astrogliosis --- p.10 / Chapter 1.3. --- Cytokines and astrogliosis --- p.11 / Chapter 1.3.1. --- IL-6 and astrogliosis --- p.12 / Chapter 1.3.2. --- IL-1 and astrogliosis --- p.13 / Chapter 1.3.3. --- IFN-γ and astrogliosis --- p.14 / Chapter 1.3.4. --- TNF-α and astrogliosis --- p.14 / Chapter 1.3.4.1. --- General properties of TNF-α --- p.15 / Chapter 1.3.4.2. --- TNF receptors (TNFRs) --- p.17 / Chapter 1.3.4.3. --- NFkB induction --- p.18 / Chapter 1.3.4.4. --- Intermediate early genes --- p.19 / Chapter 1.3.4.5. --- iNOS is the target of NFkB and AP-1 --- p.20 / Chapter 1.4. --- β-Adrenergic receptors (P-ARs) --- p.21 / Chapter 1.4.1. --- β-ARs and astrogliosis --- p.22 / Chapter 1.4.2. --- General properties of β-ARs --- p.23 / Chapter 1.4.3. --- Interactions between β-adrenergic mechanism and TNF-α --- p.24 / Chapter 1.5. --- Aims and scopes of the project --- p.25 / Chapter CHAPTER 2. --- MATERIALS & METHODS / Chapter 2.1. --- Materials --- p.29 / Chapter 2.1.1. --- Rats for astrocyte culture --- p.29 / Chapter 2.1.2. --- Cell culture materials --- p.29 / Chapter 2.1.2.1. --- Complete Dulbecco's Modified Eagle Medium:F12 (DF12) --- p.29 / Chapter 2.1.2.2. --- Phosphate buffered saline (PBS) --- p.30 / Chapter 2.1.3. --- Drugs preparation --- p.30 / Chapter 2.1.3.1. --- Recombinant cytokines --- p.30 / Chapter 2.1.3.2. --- Modulators of protein kinase A (PKA) --- p.30 / Chapter 2.1.3.3. --- Modulators of protein kinase C (PKC) --- p.31 / Chapter 2.1.3.4. --- β-Agonists and -antagonists --- p.31 / Chapter 2.1.3.5. --- Antibodies used in western blot analysis --- p.31 / Chapter 2.1.4. --- Reagents for cell proliferation determination --- p.32 / Chapter 2.1.5. --- Reagents for RNA isolation --- p.32 / Chapter 2.1.6. --- Reagents for reverse transcription-polymerase chain reaction (RT-PCR) --- p.32 / Chapter 2.1.7. --- Reagents for Electrophoresis --- p.33 / Chapter 2.1.8. --- Reagents and buffers for western blotting --- p.35 / Chapter 2.2. --- Methods --- p.36 / Chapter 2.2.1. --- Preparation of primary astrocytes --- p.36 / Chapter 2.2.2. --- Preparation of cells for assays --- p.36 / Chapter 2.2.3. --- Determination of cell proliferation --- p.36 / Chapter 2.2.3.1. --- [3H]-Thymidine incorporation assay --- p.37 / Chapter 2.2.3.2. --- MTT assay --- p.37 / Chapter 2.2.3.3. --- Data analysis --- p.38 / Chapter 2.2.4. --- Determination of RNA expression by RT-PCR analysis --- p.38 / Chapter 2.2.4.1. --- RNA extraction --- p.38 / Chapter 2.2.4.2. --- Spectrophotometric Quantitation of DNA and RNA --- p.38 / Chapter 2.2.4.3. --- RNA gel electrophoresis --- p.39 / Chapter 2.2.4.4. --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.39 / Chapter 2.2.4.5. --- Separation of PCR products by agarose gel electrophoresis --- p.40 / Chapter 2.2.4.6. --- Quantification of band density --- p.41 / Chapter 2.2.5. --- Determination of protein expression by Western blotting --- p.41 / Chapter 2.2.5.1. --- Total protein extraction --- p.41 / Chapter 2.2.5.2. --- Western blotting analysis --- p.42 / Chapter CHAPTER 3. --- RESULTS / Chapter 3.1. --- Effects of pro-inflammatory cytokines on astrocyte proliferation --- p.43 / Chapter 3.1.1. --- Effects of TNF-α on astrocyte proliferation --- p.44 / Chapter 3.1.2. --- Effects of TNF-R1 and -R2 antibodies on astrocyte proliferation --- p.47 / Chapter 3.1.3. --- Effects of other cytokines on astrocyte proliferation --- p.50 / Chapter 3.1.4. --- Comparisons of the effects of cytokines on astrocyte proliferation --- p.53 / Chapter 3.2. --- Effects of β-agonist and -antagonist on astrocyte proliferation --- p.55 / Chapter 3.3. --- Effects of TNF-α on the expression of TNFR and endogenous TNF-α in astrocytes --- p.60 / Chapter 3.3.1. --- Effects of TNF-α on the expression of TNF-R1 and -R2 in astrocytes --- p.60 / Chapter 3.3.1.1. --- Effects of TNF-α on the expression of TNF-R1 and -R2 mRNA --- p.60 / Chapter 3.3.1.2. --- TNFR subtypes involved in the TNF-α-induced TNF-R2 mRNA expression --- p.62 / Chapter 3.3.1.3. --- Signaling pathways of the TNF-α-induced TNF-R2 mRNA expression --- p.67 / Chapter 3.3.1.4. --- Effects of TNF-α on the expression of TNF-R1 and -R2 --- p.68 / Chapter 3.3.2. --- Effects of TNF-α on the expression of endogenous TNF-α in astrocytes --- p.73 / Chapter 3.3.2.1. --- Effects of TNF-α on the expression of TNF-α mRNA --- p.73 / Chapter 3.3.2.2. --- TNFR subtypes involved in the TNF-α-induced TNF-α mRNA expression --- p.73 / Chapter 3.3.2.3. --- Signaling pathways of the TNF-α-induced TNF-α mRNA expression --- p.74 / Chapter 3.3.2.4. --- Effects of other cytokines on the expression of TNF-α mRNA --- p.75 / Chapter 3.4. --- Effects of TNF-α on the expression of β1- and β2-AR in astrocytes --- p.85 / Chapter 3.4.1. --- Effects of TNF-α on the expression of β1- and β2-AR mRNA --- p.85 / Chapter 3.4.2. --- TNFR subtypes involved in the TNF-a-induced β1 and β2-AR mRNA expressions --- p.88 / Chapter 3.4.3. --- Signaling pathways of the TNF-α -induced β1- and β2-AR mRNA expressions --- p.88 / Chapter 3.4.4. --- Effects of TNF-α on the expression of β1- and β2-AR protein --- p.100 / Chapter 3.4.5. --- Effects of other cytokines on the expression of β1- and β2-AR mRNA --- p.100 / Chapter 3.5. --- Interactions between TNF-α and β-adrenergic mechanism in astrocytes --- p.107 / Chapter 3.5.1. --- Effects of β-agonists and -antagonists on the TNF-α-induced endogenous TNF-α expression in astrocytes --- p.107 / Chapter 3.5.1.1. --- Effects of ISO and PROP on the expression of TNF-α mRNA --- p.107 / Chapter 3.5.1.2. --- β-AR subtypes involved in the TNF-α-induced TNF-α mRNA expression --- p.108 / Chapter 3.5.2. --- Effects of β-agonists and -antagonists on the TNF-α-induced TNFRs expression in astrocytes --- p.112 / Chapter 3.5.2.1. --- Effects of ISO and PROP on the expression of TNFRs mRNA --- p.112 / Chapter 3.5.2.2. --- β-AR subtypes involved in the TNF-α-induced TNF-R2 mRNA expression --- p.115 / Chapter 3.6. --- Effects of TNF-α on the expression of transcription factors in astrocytes --- p.117 / Chapter 3.6.1. --- "Effects of TNF-α on c-fos, c-jun and NFKB/p50 expression" --- p.118 / Chapter 3.6.2. --- Effects of other cytokines on the expression of NFKB/p50 mRNA --- p.119 / Chapter 3.6.3. --- "TNFR subtypes involved in the TNF-α-induced c-fos, c-jun and NFKB/p50 mRNA expression" --- p.125 / Chapter 3.7. --- Effects of TNF-α on the expression of iNOS in astrocytes --- p.130 / Chapter 3.7.1. --- Effects ofTNF-α the expression of iNOS mRNA --- p.130 / Chapter 3.7.2. --- TNFR subtypes involved in the TNF-α-induced iNOS mRNA expression --- p.131 / Chapter 3.7.3. --- Signaling pathways of the TNF-α-induced iNOS mRNA expression --- p.136 / Chapter 3.7.4. --- Effects of other cytokines on the expression of iNOS mRNA --- p.139 / Chapter 3.7.5. --- Effects of β-agonists and -antagonists on the TNF-α-induced iNOS expression --- p.142 / Chapter 3.7.5.1. --- Effects of ISO and PROP on the expression of iNOS mRNA --- p.142 / Chapter 3.7.5.2. --- β-AR subtypes involved in the TNF-α-induced iNOS mRNA expression --- p.143 / Chapter CHAPTER 4. --- DISCUSSIONS & CONCLUSIONS / Chapter 4.1. --- Effects of TNF-α on astrocyte proliferation --- p.148 / Chapter 4.2. --- Roles of endogenous TNF-α and TNFR in astrocyte proliferation --- p.150 / Chapter 4.3. --- Interactions between TNF-α and β-adrenergic mechanism in astrocytes --- p.154 / Chapter 4.4. --- Induction of transcription factors by TNF-α in astrocytes --- p.157 / Chapter 4.5. --- Possible source of β-agonists --- p.159 / Chapter 4.6. --- Conclusions --- p.160 / REFERENCE --- p.163

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os mÃtodos tradicionais de tratamento do cÃncer, como a quimioterapia e a radioterapia, embora sejam eficazes em vÃrios tipos de tumores, encontram freqÃentemente populaÃÃes de cÃlulas neoplÃsicas resistentes, alÃm de apresentarem uma baixa margem de seguranÃa para os pacientes. A utilizaÃÃo da hipertermia associada à quimioterapia e/ou radioterapia jà se encontra fartamente relatada como vantajosa na literatura especializada, principalmente em pacientes portadores de cÃncer em estÃdio avanÃado, submetidos previamente aos mÃtodos clÃssicos de tratamento. A aplicaÃÃo de calor nos tecidos atravÃs de ultrassom contÃnuo torna mais Ãgil Ãste procedimento e com eficÃcia comprovada. O uso dos antiangiogÃnicos tambÃm vem sendo relatado como eficaz na literatura especializada e atualmente, a lista destas substÃncias vem aumentando consideravelmente. O estudo da aÃÃo da hipertermia associada a alguns agentes antiangiogÃnicos tem sido sugerida uma vez que os vasos tumorais ao encontrarem-se dilatados, nÃo promoverÃo a diminuiÃÃo da temperatura no tecido tumoral e, consequentemente, os efeitos desta associaÃÃo serÃo mais intensos do que no tecido normal devendo contribuir para a morte celular. Objetiva-se neste trabalho, avaliar o efeito antitumoral e antiangiogÃnico da hipertermia induzida por US isolada e combinada com etoricoxibe e pegaptanibe,no carcinossarcoma de Walker 256 implantado na tela subcutÃnea do dorso de ratos por meio de utilizaÃÃo do modelo experimental de hipertermoterapia por US assim como o estudo dos efeitos da hipertermia por US isolada e em combinaÃÃo com etoricoxibe e pegaptanibe,na angiogÃnese tumoral.O mÃtodo utilizado para o estudo teve inÃcio com o implante de cÃlulas de tumor de Walker 256 no dorso de ratos Wistar machos. Os animais foram tratados com hipertermia aplicada atravÃs de aparelho de ultrassom, mantida a nÃvel de 45o C durante cinco minutos no terceiro dia apÃs a inoculaÃÃo e tambÃm tratados com etoricoxibe e pegaptanibe por via oral e intraperitoneal respectivamente a partir do dia da inoculaÃÃo. Cada grupo de animais foi submetido a medidas do crescimento tumoral durante o perÃodo de 30 dias, assim como tambÃm à avaliaÃÃo da microdensidade vascular atravÃs de estudo mesoscÃpico fotogrÃfico, validado pelo estudo microscÃpico. A aplicaÃÃo do calor atravÃs de aparelho de ultrasom demonstrou eficiÃncia e agilidade A hipertermia, o etoricoxibe e o pegaptanibe, apresentaram capacidade antiangiogÃnica, expressada tanto pela curva de sobrevida, como pela avaliaÃÃo da microdensidade vascular. Particularmente, a hipertermia isoladamente apresentou um efeito antiangiogÃnico significativo tanto na curva de crescimento tumoral como na diminuiÃÃo da densidade microvascular. A associaÃÃo da hipertermia com o pegaptanibe, demonstrou uma eficiÃncia na diminuiÃÃo da densidade microvascular significativamente maior do que os demais grupos. O modelo de aplicaÃÃo de hipertermia gerada por um aparelho de ultrassom na modalidade contÃnua foi satisfatÃria, demonstrando ter sido efetiva tanto na diminuiÃÃo do crescimento tumoral, como na diminuiÃÃo da densidade microvascular. / The traditional methods of cancer treatment, like the chemotherapy and radiotherapy, even though they are effective in many types of tumours, they find frequently resistant neoplasic cellsâ population, beyond presenting a low security margin to the patients. The hyperthermia use associated to the chemotherapy and radiotherapy are plenty mentioned as profitable in the specialist literature, especially in patients with cancer in advanced stage, submitted previously to the classic methods of the treatment. The tissuesâ heat insertion through the continuous ultrasound becomes the procedure faster and with proved efficacy. The antiangiogenic use also is being related like effective in the specialist literature and, at this moment, the substancesâ list has grown vastly. The study of the hyperthermia action associated to antiangiogenic agents has been suggested, once the tumour vessels are dilated, would not promote the temperature reduction in the tumour vessels and, therefore, the effects of this association would be more intense than in the normal tissue, what might contribute to the death cell. The study objective is to evaluate the antitumor and antiangiogenic effect of the hyperthermia induced by isolated ultrasound and combined with etoricoxibe and pegaptanibe in the 256 Walker carcinossarcoma implanted in the subcutaneous back screen of mouse by using the hyperthermia experimental model by ultrasound, like the hyperthermia effects study by isolated ultrasound and linked with etoricoxibe and pegaptanibe, in the tumour angiogenesis. The method used describes the insertion of 256 Walker Tumour cells in the back of male Wistar mouse. The animals were treated with hyperthermia applied through the ultrasound equipment, kept to a 45 C level during five minutes in the third day after the inoculation and also treated with etoricoxibe and pegaptanibe by oral and intraperitoneal respectively since the inoculation day. Each animal group was submitted to a tumour growth measurement during a period of 30 days, as well as also a vascular microdensity evaluation through a photographic mesoscopic study, validated by the microscopic study. The heat application through the ultrasound equipment demonstrated efficiency and agility. The hyperthermia, the etoricoxibe and the pegaptanibe showed antiangiogenic capacity, expressed by the over life curve, and also by the vascular microdensity evaluation. Particularly, the isolated hyperthermia showed a significative antiangiogenic effect in the tumour growth curve and also in the decreasing of the micro vascular density. The association between the hyperthermia and the pegaptanibe demonstrated a greater efficiency in the reducing of the micro vascular density than the other groups. The hyperthermia application model generated by the ultrasound equipment in the continuous modality was satisfactory, demonstrating by being effective as much in the reducing of the tumour growth as in the decreasing of the micro vascular density.

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No. of bitstreams: 2 Dissertação - Flávio Silva de Carvalho - 2011.pdf: 2542668 bytes, checksum: 8b150260143e835293c132099b3cbf68 (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2011-03-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / As part of a line of research aimed at the pharmacological and toxicological evaluation of new candidates for antitumor drug prototypes, this work was conducted to evaluate pharmacological and toxicological compounds LQFM030, and LQFM031 LQFL032, drawn from nutlins prototypes. Additionally, we also evaluated whether the compounds LQFM004, LQFM005, and LQFM028 LQFM029 initially envisioned as candidates for prototypes of antipsychotic drugs, drawn from LASSBio579. Initially there was a cytotoxic screening all candidates, using the cytotoxicity test of exclusion of Trypan Blue in K-562 cells. Given this, it was observed that the best IC50 values were obtained with the candidates and LQFM030 LQFM029 (0.55 and 0.56 mM, respectively) and from then on had as starting point for these two molecules to a more detailed and thorough cytotoxicity, where it was observed that the molecule had the best LQFM030 cytotoxic profile in the test of exclusion of Trypan blue and the MTT reduction test, at intervals of 24, 48 and 72, compared with LQFM029 the molecule, using the cell line K-562. Given this, the candidate LQFM030 was the target of other studies conducted to study the survival, safety tests (Capture the Neutral Red dye, Acute Toxicity Oral), cell cycle, cell death mechanism of apoptosis and antioxidant activity. The data showed that the molecule LQFM030 increased life expectancy of the animals, and an IC50 value commensurate with the value of Nutlins found in literature, on the evaluation of acute toxicity, according to OECD 423, the molecule was rated 5 or non- classified according to the classification GSH, the cell cycle was observed an increase in G2 / M phase (73%) and sub-G1 phase (73.6%), and a decrease in S phase (40.9%) ; to assess the mechanism of cell death was characterized increased expression of Bax (8 times compared to untreated group), and a decrease of Bcl-2 (84%) as well as an increase in membrane potential and a decrease of ROS (reactive oxygen species), where there was not a change in protein expression of TNF and NFκβ; antioxidant activity did not show a significant effect, and before the candidate cyclic voltammetry showed only the presence of peak oxidation, not being characterized as a good redox system. When tested for safety in basal cell 3T3, the candidate presented a cytotoxicity with IC50 value of approximately 0.14 mM Thus, the candidate prototype antitumor drug LQFM030 presented as a good candidate for antitumor prototype with important data for such purpose, and the need for further detailed studies to better conceptualization. / No âmbito de uma linha de pesquisa que visa a avaliação farmacológica e toxicológica de novos candidatos a protótipos de fármacos antitumorais, foi realizado neste trabalho a avaliação farmacológica e toxicológica dos compostos LQFM030, LQFM031 e LQFL032, desenhados a partir dos protótipos nutlins. Adicionalmente, também avaliou-se os compostos LQFM004, LQFM005, LQFM028 e LQFM029, inicialmente idealizados como candidatos a protótipos de fármacos antipsicóticos, desenhados a partir do LASSBio579. Inicialmente realizou-se uma triagem citotóxica com todos os candidatos, utilizando o teste de citotoxicidade de Exclusão do Azul de Tripano, em células K-562. Diante disto, observou-se que os melhores valores de IC50 foram obtidos com os candidatos LQFM030 e LQFM029 (0,55 e 0,56 mM, respectivamente) e a partir de então teve-se como ponto de partida essas duas moléculas para um estudo mais detalhado e aprofundado da citotoxicidade, onde observou-se que a molécula LQFM030 apresentou o melhor perfil citotóxico no teste de Exclusão do Azul de Tripano e para o teste de Redução do MTT, no intervalos de tempo de 24, 48 e 72h, quando comparado com a molécula LQFM029, utilizando a linhagem celular K-562. Frente a isso, o candidato LQFM030 foi alvo dos demais estudos realizados visando estudo de sobrevida, ensaios de segurança (Captura do Corante Vermelho Neutro, Toxicidade Aguda Oral), ciclo celular, mecanismo de morte celular por apoptose e atividade antioxidante. Os dados mostraram que a molécula LQFM030 aumentou a expectativa de vida dos animais, e um valor de IC50 compatível com o valor dos Nutlins encontrado na literatura; diante da avaliação da toxicidade aguda, segundo a OECD 423, a molécula teve classificação 5 ou não-classificada de acordo com a classificação GSH; no ciclo celular observou-se um aumento da fase G2/M (73%) e da fase sub-G1 (73,6%), e uma diminuição da fase S (40,9%); para a avaliação do mecanismo de morte celular foi caracterizado um aumento da expressão da proteína Bax (8 vezes em relação ao grupo não-tratado), e uma diminuição de Bcl-2 (84%), assim como um aumento do potencial de membrana e uma diminuição de ROS (espécie reativa de oxigênio), onde não observou-se uma variação na expressão das proteínas TNF e NFκβ; porém um aumento da modulação da proteína citocromo-c de 85%; a atividade antioxidante não apresentou um efeito significativo, e perante a voltametria ciclica o candidato mostrou apenas a presença do pico de oxidação, não sendo caracterizado como um bom sistema de oxirredução. Para o ensaio de segurança, em célula basal 3T3, o candidato apresentou uma citotoxicidade com o valor de IC50 de 0,14 mM aproximadamente. Assim, o candidato a protótipo a fármaco antitumoral LQFM030 apresentou como um bom candidato a protótipo antitumoral com dados importantes para tal finalidade, e a necessidade de estudos ainda mais aprofundados para uma melhor conceituação.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 4 uppsatser.

Thesis (Ph.D. in Immunology) -- University of Colorado, 2005. / Typescript. Includes bibliographical references (leaves 190-212). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

O desenvolvimento da imunoterapia do câncer baseada em células dendríticas (DCs) é alvo de vários estudos. Para tumores sólidos, a abordagem baseada no uso de DCs alogenêicas fundidas com células tumorais tem se mostrado relativamente eficaz. Por outro lado, esta estratégia necessita uma massa tumoral considerável de cada paciente para a geração das células híbridas. Para contornar este problema o uso de DCs transfectadas com mRNA tumoral, o qual pode ser amplificado in vitro a partir de uma pequena amostra inicial do tumor, tem sido investigado. Para isto, uma transfecção eficiente e tradução correta do mRNA tumoral nas DCs são etapas críticas. Sabendo-se que as DCs de pacientes com câncer possuem atividade aloestimuladora defeituosa, DCs derivadas de doadores saudáveis poderiam ser uma alternativa para induzir uma resposta imune mais eficiente. Assim, este trabalho pretendeu aprimorar a metodologia de transfecção de DCs alogenêicas, derivadas de monócitos de doadores saudáveis, com mRNA de antígenos tumorais (survivina e RPSA) super-expressos na leucemia linfóide crônica (LLC) e avaliar sua capacidade em estimular a resposta linfocitária. Ao mesmo tempo, foram estabelecidas as metodologias para amplificação e síntese do RNA destes antígenos tumorais específicos, assim como do RNA mensageiro total, contidos nas células tumorais de pacientes com LLC. Os resultados mostraram ser possível a amplificação do mRNA total extraído das células leucêmicas com manutenção da expressão dos antígenos tumorais. Ainda, várias condições de transfecção com mRNA da survivina, transcrito in vitro, foram testadas, encontrando-se na lipofecção, a melhor maneira de transfectar as DCs. A lipofecção mostrou-se com baixa toxicidade quando comparada à técnica de eletroporação. Observou-se uma eficiência em torno de 40% de células transfectadas num intervalo de tempo entre 12 e 48 horas. Estas células foram usadas como estimuladoras em ensaios de proliferação usando-se linfócitos T alogenêicos como células respondedoras. As células transfectadas com mRNA da survivina foram capazes de estimular resposta linfoproliferativa com maior produção de IFN-gama, avaliado por ELISA. Além disso, a transfecção não alterou o padrão de expressão dos marcadores de superfície característicos das DCs. Estes dados mostram que a transfecção das DCs com mRNA pode afetar a resposta imune induzida por estas APCs. Nossos resultados suportam o uso de DCs transfectadas com mRNA para produção de vacinas anti-tumorais e mostram a survivina como um potente antígeno indutor da resposta linfocitária. / The development of dendritic cell (DC)-based cancer immunotherapy has been the target of many studies. For solid tumors, a promising approach based in allogeneic DCs fused to tumor cells has been relatively effective. On the other hand, this approach needs large tumor samples to generate enough DC-tumor cell hybrids. To overcome this problem, tumor mRNA-transfected DCs can be used, since mRNA can be amplified in vitro and allow unlimited vaccine production. For this, efficient transfection and optimal translation of tumor mRNA in DCs are critical. Moreover, DC derived from cancer patients has defective alostimulatory activity. In this case, DC derived from healthy donors may be an alternative to induce immune response more efficiently. Here, we established the methodology of allogeneic DC transfection with mRNA for tumor antigens (survivin and RPSA) overexpressed in chronic lymphocytic leukemia (CLL), and evaluated their ability for T cell stimulation. At the same time, we established mRNA amplification and mRNA in vitro transcription methodology for specific tumor antigens and total messenger RNA, present in CLL tumor cells. Our results showed it to be possible to amplify total mRNA derived from leukemic cells maintaining tumor antigen expression. Moreover, several transfection conditions using survivin mRNA obtained from in vitro transcript reactions were evaluated, defining lipofection as the better way to transfect DC. We obtained nearly 40% of transfected DCs between 12 and 48 hours. Transfected DCs were used as stimulator cells in proliferation assays using allogeneic T cells as responder cells. Survivin mRNA transfected DCs were able to stimulate T cell proliferation with increased IFN-gama production, measured by ELISA. Furthermore, the transfection did not change the pattern of surface molecules expression characteristic of DC. These data show that mRNA DC transfection can affect immune responses induced by these APCs. These findings support the use of tumor mRNA transfected DCs for anti-cancer vaccine production and show survivin as a potent antigen to induce T cell responses.

No description available.

Glioblastomas are the most common malignant brain tumors in adults. Despite resection of the tumor, combined radio- and chemotherapy and new-targeted therapy approaches, the average life expectancy is only 15 months with a 3-year survival rate of less than 5%. Invasive growth of tumor cells into the surrounding brain tissue complicates treatment and causes recurrence. Imaging techniques such as magnetic resonance imaging (MRI) are conventionally used for diagnosis and for monitoring after surgery in order to detect tumor lesions. However, next to tumor progression, contrast enhancement could also result from pseudoprogression or radiation necrosis. Similarly, next to therapy response, a reduction of contrast enhancement could also mimic a success in therapy (pseudoresponse). Furthermore, the use of targeted therapies requires the molecular detection of biomarkers, which is often performed on tissue biopsies. During therapy, resistance mechanisms to therapy may develop, which have a significant impact on the success of the therapy. Repeated biopsies are needed to distinguish tumor progress from therapy-associated tissue changes and simultaneously detect therapy resistance. Often they are not feasible due to the high risk for the patient. Currently, there are methods to improve monitoring in glioblastoma patients. Besides improved imaging techniques, liquid biopsies are a promising method to detect tumor progression. At the same time, liquid biopsies also allow molecular characterization of the lesions. A key advantage over tissue biopsies is that they are minimally invasive, making them well suited for longitudinal tumor monitoring. For clinical application of liquid biopsies in the form of blood and CSF, components such as circulating tumor cells, circulating tumor DNA, extracellular vesicles, or tumor-educated platelets are used, with the former two probably being the most common tumor markers at present. In this study, a highly sensitive next generation sequencing-based method was established to detect tumor mutations in plasma and CSF of glioblastoma patients. To this end, crucial sample preparation steps were initially optimized. Therefore, four isolation kits extracting cell-free DNA from plasma were compared and the best-performing kit was chosen for the analysis of patient material. Furthermore, a multi-gene next generation sequencing panel (cfDNA-GBM panel) was designed specifically for use in liquid biopsies from glioblastoma patients and tested on a reference standard for cell-free DNA with mutations of different allele frequencies. The newly designed cfDNA-GBM panel, which uses hybrid-capture based enrichment, was compared to a primer-extension based panel. Due to the more consistent coverage of target regions, superior sensitivity, and better clinical applicability due to its smaller size, the cfDNA-GBM panel was used for subsequent clinical investigations. In both enrichments, unique molecular barcodes were used to label all original fragments and thus identify and eliminate errors in subsequent data analysis that occur during amplification in library preparation This increases the sensitivity of the method. During the establishment of the methodology, the use of molecular barcodes clarified the limitation of sequence information due to the small input amount of cell-free DNA. For tumor tissue analysis, an already established larger panel was adapted and extended based on the results of a proof of concept study. After the establishment of the sensitive method, clinical samples of glioblastoma patients were analyzed. Isolation and analysis of cell-free DNA from plasma and CSF resulted in highly variable amounts of cell-free DNA with characteristic oligonucleosomal fragment lengths. Furthermore, no difference in the amount or fragmentation of cell-free DNA from pre- or post-surgery plasma was determined. To increase the amount of cell-free DNA for sequencing, the amount of blood samples was increased from 10 ml (5 patients) to 50 ml (9 patients). Detection of circulating tumor DNA in plasma and CSF from glioblastoma patients was performed using two strategies. In a first approach, somatic tumor mutations were detected in genomic DNA from tumor tissue, which were searched for in the sequence data of liquid biopsies in a second step. In this approach, an increased detection rate (in 56% of patients compared to 25% in 10 ml blood samples) of circulating tumor DNA was detected in blood samples with a volume of 50 ml. Almost all tumor mutations lying in the target region of the smaller cfDNA-GBM panel could be detected in CSF (92%) with similar allele frequencies to those detected in the tumor. In a second strategy, somatic variants were detected in plasma and CSF without prior knowledge of variants in tumor tissue. Interestingly, three variants were detected in the CSF of one patient, which occurred with an allele frequency over 5% in the CSF, while they were not detectable in the analyzed tumor tissue. These variants may be indicators for tumor heterogeneity that was not accessible by analyzing sections of tumor tissue. Finally, mutation-specific probes were designed for three tumor-somatic variants detected in the patients' plasma to validate them by digital PCR. A pair of probes for a mutation in PTEN was successfully established to robustly detect minimal allele frequencies down to 0.1% but could not validate the mutation in cell-free DNA from plasma of the respective patient. Overall, it was shown that analysis of tumor somatic mutations using the cfDNA-GBM sequencing panel designed and established in this study is limited in the analysis of cell-free DNA from plasma of patients with glioblastomas, whereas the detection of circulating tumor DNA from CSF is promising.

Die Intra-Tumor-Heterogenität spiegelt eine kontinuierliche Entwicklung zwischen den Zellen eines einzelnen Tumors wider. Sie ist eine der Hauptursachen für Arzneimittelresistenz bei der Krebsbehandlung. Um dieses Problem anzugehen, ist es daher wichtig, die Tumorevolution innerhalb eines einzelnen Patienten zu verstehen und erfolgreich zu modellieren. Bisherige Arbeiten haben sich nicht erfolgreich mit der Evolution von Tumoren befasst, deren Treiber strukturelle Veränderungen im Genom sind, wie z. B. somatische Kopienzahlveränderungen (SCNAs). Diese Arbeit befasst sich mit der Herausforderung, die Tumorevolution als Folge solcher Veränderungen zu charakterisieren. Wir verwenden einen phylogenetischen Ansatz zur Analyse von multiregionalen Datensätzen in einer großen Pan-Krebs-Kohorte. Wir untersuchen häufige SCNAs in verschiedenen Stadien der Tumorentwicklung und führen eine neue Methode, MEDICC2, ein, die die Tumorevolution innerhalb eines einzelnen Patienten rekonstruiert. In dieser Arbeit haben wir häufige SCNAs charakterisiert, die früh in der Tumorentwicklung auftreten. Aufgrund der Struktur der Kohorte ist die Charakterisierung der subklonalen SCNAs nicht eindeutig. Unsere neue Methode, MEDICC2, akzeptiert höhere Kopienzahlzustände und berücksichtigt die Verdopplung des gesamten Genoms, ein häufiges Ereignis in Tumoren, was eine genauere Modellierung der Tumorevolution ermöglicht. / Intra-tumor heterogeneity reflects an ongoing evolution among cells of a single tumor. It is one of the leading causes of drug resistance in cancer treatments. Therefore, to address this issue, it is important to understand and successfully model tumor evolution within a single patient. Previous work has failed to successfully address the evolution of tumors whose drivers are structural changes in the genome, such as somatic copy number alterations (SCNAs). This work addresses the challenge of characterizing tumor evolution as a result of such changes. We use a phylogenetic approach to analyze multi-region datasets in a large pan-cancer cohort. We investigate frequent SCNAs at different stages of tumor development, and introduce a new method, MEDICC2, which reconstructs tumor evolution within a single patient. In this work, we characterized frequent SCNAs that occur early in tumor development. Due to the structure of the cohort, the characterization of subclonal SCNAs remains inconclusive. Our new method, MEDICC2, accepts higher copy number states and takes into account whole-genome doubling, a frequent event in tumors, which allows for a more precise modeling of tumor evolution.