A foundation for the control of tumors is presented, based upon the formulation of a realistic, knowledge-based mathematical model of the interaction between tumor cells and the immune system. The parametric control variables relevant to the latest experimental data, e.g., the sigmoidal dose-response relationship and Michaelis-Menten dynamics, are also considered. The model consists of 12 states, each composed of first-order, nonlinear differential equations based on cellular kinetics and each of which can be modeled bilinearly. In recent years a great deal of clinical progress has been achieved in the use of optimal controls to improve cancer therapy patient care. For this study, a cancer immunotherapy problem is investigated in which the aim is to minimize the tumor burden at the end of the treatment period, while penalizing excessive administration of interleukin-2 as a limit of toxicity. The optimal solution developed for this investigation is a mixture of an initially large dose of interleukin-2, followed by a gradually decreased dosage and a continuing infusion to maintain the tumor cell population at its allowable limit. Sensitivity analysis is applied to an investigation of the influences of system parameters. It has been found that the immune system is influenced greatly by several parameters such as macrophage level, tumor killing rate, tumor growth rate, and IL-2 level. The simulation results suggest that parametric control variables are important in the destruction of tumors and that the application of exacerbation theory is a good method of tumor control. / Graduation date: 1991

Les maladies allergiques sont devenues un enjeu de santé publique majeur en raison d’une prévalence en constante augmentation ces dernières années, d’un risque accru de choc anaphylactique et de l’absence de traitement curatif existant. Les allergies alimentaires sont caractérisées par une évolution fréquente vers le choc anaphylactique. Les lymphocytes T régulateurs (Treg), cellules permettant le maintien de l’homéostasie immunitaire et le contrôle des réponses, sont au centre du processus de régulation des réponses allergiques. Il a été démontré qu’il existait un déficit quantitatif et qualitatif de ces cellules chez les patients allergiques et que les manifestations allergiques étaient exacerbées en l’absence de Treg. L’induction, in vivo, de ces cellules semble donc être une alternative thérapeutique prometteuse afin de prévenir et traiter les maladies allergiques en particulier, l’allergie alimentaire. L’interleukine-2 (IL-2), cytokine du système immunitaire permettant la survie, l’expansion et la différenciation des lymphocytes, notamment les Tregs lorsqu’elle est utilisée à faible dose, constitue une avancée thérapeutique majeure. L’objectif de cette thèse a été d’évaluer le potentiel thérapeutique de l’IL-2 faible dose dans l’allergie alimentaire. L'IL-2 faible dose induit l’expansion et l’activation de Treg permettant la mise en place d’une protection contre les manifestations cliniques d'allergie alimentaire dans deux modèles de souris avec l'ovalbumine et l'arachide. L’abolition de cet effet clinique chez les souris dont les Treg ont été éliminés démontre la contribution majeure des Treg dans l'efficacité de la thérapie IL-2. Les mécanismes associés à la protection peuvent être corrélés à une modification locale de la balance Th1 / Th2 et une inhibition du recrutement et de l'activation des mastocytes. Nous avons démontré que l’IL-2 induit une protection clinique durable, supérieure à 7 mois, aussi bien lorsqu’elle est administrée de façon préventive (avant la phase de sensibilisation), que curative (après la sensibilisation ou le déclenchement de l’allergie). Cette protection à long terme est également dépendante des Treg dans la mesure où leur déplétion entraine la perte de protection de souris. Ces résultats, combinés à l’absence visible d’augmentation des Treg en fin de protocole, nous ont conduits à caractériser les mécanismes immunorégulateurs inhérents à ce contrôle effectif à distance et sur le long terme après traitement, en analysant notamment les sous populations de cellules régulatrices particulièrement efficace pour contrôler les réponses allergiques. Les résultats préliminaires obtenus, qu’il conviendra de reproduire, ne nous permettent pas de conclure clairement quant à la sélection des Treg spécifiques de l’allergène, ou de sous population de Treg, réputées protectrices dans l’allergie. Toutefois, nos résultats constituent la preuve de concept de l’utilisation de l’IL-2 faible dose dans l’allergie alimentaire. De même l’IL-2 a également montré son efficacité dans un modèle murin d’allergie respiratoire (ovalbumine). Nous avons aussi démontré que des voies d’administration alternatives étaient possibles, notamment la voie orale. De ce fait, un essai clinique de phase II testant l’administration d’IL-2 faible dose dans la rhinoconjonctivite va prochainement être mise en place par notre centre d’investigation clinique. Ainsi, nous espérons développer une nouvelle biothérapie, sûre, efficace et peu coûteuse, dans la prise en charge thérapeutique des allergies. / Regulatory T cells (Treg) are pivotal for maintenance of immune self-tolerance, and also regulate immune responses to exogenous antigens, including allergens. Both decreased Treg number and function have been reported in allergic patients, offering new therapeutic perspectives. The in vivo induction of these cells therefore, appears to be a promising therapeutic option in order to prevent and treat allergic diseases in particular, food allergy. Interleukin-2 (IL-2), allowing the survival, expansion and differentiation of lymphocytes, especially Treg when used at low doses, is a major therapeutic advance. Here, we evaluated the ability of low dose IL-2 (intraperitoneal route to control) allergy in an experimental model of food allergy. Low dose IL-2 (ld-IL-2) induced Treg expansion and activation that elicited protection against clinical manifestations of food allergy in two mouse models with ovalbumin and peanut. This clinical effect was lost in Treg-depleted mice demonstrating the major contribution of Treg in ld-IL-2 efficacy. Also, protection from allergy could be explained by a Treg-dependent local modification of the Th1/Th2 balance and an inhibition of mast cell recruitment and activation. Then, preventive and therapeutic effects of ld-IL-2 were observed over a 7-month period, highlighting its long-term efficacy. This long-term protection is also dependent on Treg insofar as their depletion results in the loss of protection of mice. These results, with the loss of increase of Treg at the end of the protocol, led us to better characterize the immunoregulatory mechanisms involved in the ld-IL2 long term efficacy, in particular by analyzing subpopulations of Treg particularly effective in controlling allergic responses. The preliminary results obtained, which should be reproduced, do not allow us to conclude clearly to the selection of allergen-specific Treg, or Treg subpopulation involved in the regulation of allergic responses. However, our results demonstrate the proof of concept of the use of low-dose IL-2 in food allergy. Similarly IL-2 has also shown its efficacy in a mouse model of asthma (ovalbumin). Alternative routes of administration were also demonstrated, including oral route. As a result, a clinical trial testing low-dose IL-2 in rhinoconjunctivitis will soon be initiated by our center. The present data show for the first time the therapeutic potential of ld-IL-2 for the treatment of food allergy, and should prompt hope, in the therapeutic management of allergies. Patients will be offered the first curative treatment developed in food allergies to improve their quality of life. Keywords: Allergy, Interleukin 2, biotherapy, therapeutic option, allergen-specific Treg, peripheral Treg.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-09-04T16:57:00Z No. of bitstreams: 1 Tiago Landim d'Avila Administração de plasmídeos....pdf: 1447180 bytes, checksum: b65ad3c9ece2b6f29cc032b2fa22c49a (MD5) / Made available in DSpace on 2012-09-04T16:57:00Z (GMT). No. of bitstreams: 1 Tiago Landim d'Avila Administração de plasmídeos....pdf: 1447180 bytes, checksum: b65ad3c9ece2b6f29cc032b2fa22c49a (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A interleucina-12 (IL-12) é uma glicoproteína heterodímerica, codificada por dois genes distintos co-expressados na mesma célula. IL-12 precisa ser glicosilada para exibir atividade funcional. Essa glicoproteína promove a diferenciação de células T e é capaz de estimular a proliferação de células T ativadas e células NK. Além disso, IL-12 promove a produção IFN-γ por células NK e o aumento da atividade lítica de células NK e linfócitos T CD+ citotóxicos. Várias das atividades de IL-12 são desempenhadas ou são amplificadas pela associação com IL-2. Em nosso laboratório, foi gerada uma construção plasmideal capaz de expressar IL-12 canina, na forma de proteína de fusão de cadeia única (pcDNA3.1-scca-IL-12), e outra construção plasmideal capaz de expressar IL-2 canina (pcDNA3.1-ca-IL-2) em células de mamífero. Experimentos preliminares, a combinação de IL-12 e IL-2 expressas em sobrenadantes de células COS-7 mostrou-se capaz de promover produção de IFN-γ em células mononucleares de sangue periférico (CMNSP) de cães sadios. Visando a avaliação do potencial do uso de IL-12 e/ou IL-2 para o desenvolvimento de vacina ou método imunoterapico, no presente trabalho, grupos de cães sadios foram injetados três vezes, com intervalo de 2 dias entre cada duas doses consecutivas, com plasmídeo pcDNA3.1-scca-IL-12 e/ou pcDNA3.1-ca-IL-2 por via muscular por eletroporação. Em seguida, os cães foram submetidos à avaliação clinica, clinico-laboratorial (hemograma, determinação da concentração de transaminases, proteínas, ureia e creatinina no soro) e ensaios foram realizados para a detecção da expressão das proteínas recombinantes (sensibilização de CMNSP para produção de IFN-γ e determinação da concentração de IL-2 no soro). Não foram observadas diferenças nos parâmetros avaliados entre os grupos de animais. Visando a produção de IL-12, células BTI-Tn-5B1-4 foram infectadas com baculovírus recombinante contendo cDNA scca-IL-12 e capazes de adicionar uma cauda de 6 histidinas na extremidade carboxila. Para isso, duas construções diferentes em baculovírus foram elaboradas nosso laboratório, sendo uma delas no presente trabalho. Células BTI-Tn-5B1-4 infectadas com as construções em baculovírus apresentaram a proteína recombinante: a) parcialmente degradada e em grande quantidade no sedimento celular e b) integra e em baixa quantidade no sobrenadante da cultura; de acordo com os resultados de obtidos por SDS-PAGE e Western blot. / Interleukin-12 (IL-12) is a heterodimeric glycoprotein, encoded by two distinct genes co-expressed in the same cell. IL-12 needs to be glycosylated to display functional activity. This glycoprotein promotes differentiation of T cells and is capable of stimulating proliferation of activated T cells and NK cells. In addition, IL-12 promotes IFN-γ production by NK cells and increased lytic activity of NK cells and cytotoxic Tlymphocyte CD +. Several of the activities of IL-12 are held or amplified by the association with IL-2. In our laboratory, a plasmideal construction was generated to express canine IL-12 like single-chain fusion protein (pcDNA3.1-scca-IL-12), and other construction to express canine IL-2 (pcDNA3.1-CA-IL-2) in mammalian cells. Preliminary experiments, the combination of IL-12 and IL-2 supernatants expressed in COS-7 cells was able to promote IFN-γ in peripheral blood mononuclear cells (PBMC) of healthy dogs. To assess the potential of using IL-12 and/or IL-2 for the development of vaccine or immunotherapy method, in this study, groups of healthy dogs were injected three times with an interval of 2 days between each two consecutive doses with plasmid pcDNA3.1-scca-IL-12 and/or pcDNA3.1-ca-IL-2 intramuscularly by electroporation. Then the dogs were submitted to clinical evaluation, clinical and laboratory (blood count, determination of the concentration of transaminases, proteins, urea and serum creatinine) and tests were performed to detect the expression of recombinant proteins (PBMC sensitization to produce IFN-γ concentration and determination of serum IL-2). No differences were observed in all evaluated parameters between the groups of animals. Aiming the production of IL-12 cells, BTI-Tn-5B1-4 were infected with recombinant baculovirus containing cDNA scca-IL-12 and capable of adding a tail of 6 histidines at the carboxyl end. For this, two different constructs were prepared in our laboratory baculovirus, one of them in this work. Cells BTI-Tn-5B1-4 infected with the constructs presented in the recombinant baculovirus: a) partially degraded in large quantities in the sediment cell and b) incorporates and in low quantities in the culture supernatant, according to the results obtained by SDS-PAGE and Western blo

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-11-27T17:22:07Z No. of bitstreams: 1 Andréa Mendes Pereira Ativação de celulas... 2006.pdf: 40276494 bytes, checksum: 9f4656b98e4c74bae9c81e7797e85988 (MD5) / Made available in DSpace on 2012-11-27T17:22:07Z (GMT). No. of bitstreams: 1 Andréa Mendes Pereira Ativação de celulas... 2006.pdf: 40276494 bytes, checksum: 9f4656b98e4c74bae9c81e7797e85988 (MD5) Previous issue date: 2006 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Visando avaliar futuramente o potencial de citocinas na indução de resposta imune celular específica do tipo ThI quando associadas a antígeno(s) recombinante(s) de Leishmania chagasi/infantum no cão, a combinação de IL-2 e IL-12 caninas recombinantes é analisada no presente trabalho. Aqui, descrevemos a clonagem do DNA complementar (cDNA) e, pela primeira vez, a expressão de IL-2 canina recombinante biologicamente ativa em Escherichia coli e por células de mamífero. Para expressão em E. coli, utilizou-se a construção pRSET-calL- 2, anteriormente gerada por nosso grupo. Para expressão em células de mamíferos, foi realizada a clonagem do cDNA de IL-2, sintetizado por reação de transcrição reversa seguida de reação da polimerase em cadeia (RT-PCR) a partir do RNA total de células mononucleares do sangue periférico (CMSP) de cão estimuladas com concanavalina A (Con-A), no vetor pcDNAS.l, gerando a construção pcDNA3.1-caIL-2. O sucesso da clonagem em ambos os vetores de expressão foi confirmado a partir do sequenciamento de DNA e comparação dos resíduos de nucleotídeos com a seqüência de IL-2 canina previamente descrita por outro grupo de investigadores. A IL-2 canina recombinante (rcaIL-2) foi obtida como proteína de fiisão contendo cauda de histidina a partir da transformação de E. coli BL21(DE3)pLysS com pRSET- caIL-2, purificada por cromatografia de afinidade e renaturada por diálise. Além disso, a forma nativa de rcaIL-2 foi secretada no sobrenadante de cultura de células COS-7 transfectadas com a construção pcDNA3.1-caIL-2. A atividade proliferativa de rcaIL-2 sobre células CTLL-2 foi demonstrada em concentrações de até 220 pg/mL da citocina purificada a partir da expressão em E. coli e até a diluição de 1:256 do sobrenadante de COS-7 contendo rcaIL-2. A proteína biologicamente ativa foi capaz de manter a proliferação de CMSP de cães sadios por até 12 dias de cultivo quando as células foram tratadas com 50 ng/mL de IL-2 canina obtida de E. coli e por 10 dias com diluições de até 1:200 do sobrenadante de COS-7 contendo a citocina, na ausência de estímulo prévio ou concomitante. A proliferação foi dose-dependente, com ponto máximo ocorrendo no 8° dia de cultivo. A produção de interferon gama (IFN-y) por CMSP de cães sadios estimuladas com sobrenadante de COS-7 contendo IL-2 ou contendo IL-12 não foi significantemente maior que a produção basal. No entanto, um efeito sinérgico sobre a produção in vitro de IFN-y ocorreu quando concentrações subótimas de ambas as citocinas foram associadas. Diante dos resultados obtidos, a construção pcDNA3.1-caIL-2 e ambas as formas de IL-2 canina recombinante obtidas, assim como as condições experimentais aqui descritas, poderão ser utilizadas no futuro para o estudo do potencial de IL-2, associada ou não a IL-12, como modulador da resposta imune in vitro e in vivo de cães, durante o desenvolvimento de uma vacina ou imunoterapia para leishmaniose visceral canina. / Aiming to study in the fiiture the role of cytokines in inducing specific ThI cellular immune response when associated to Leishmania chagasi/infantum recombinant antigen(s) in dogs, the combination of recombinant canine IL-2 and IL-12 is analysed in the present study. Herein, we describe complementary DNA (cDNA) cloning and, for the first time, the expression of biologically active recombinant canine IL-2 in Escherichia coli and mammal cells. The construction pRSET-caIL-2, previously generated by our group, was used for E. coli expression. For mammalian expression, canine IL-2 cDNA was synthesized by reverse transcription followed by polymerase chain reaction (RT-PCR), using cDNA from a healthy dog’s peripheral blood mononuclear cells (PBMC) stimulated with concanavalin A (Con-A) and cloned into pcDNA3.1, generating the construction pcDNA3.1-caIL-2. Success in canine IL-2 cDNA cloning was accessed in both expression vectors by DNA sequencing and was confirmed by comparing nucleotides residues with canine IL-2 sequence previously described by other investigators. Recombinant canine IL-2 (rcaIL-2) was expressed as His-tag fiision protein after E. coli BL21(DE3)pLysS transformation with pRSET-caIL-2, purified by affinity chromatography and renatured through dialysis. In addition, the native form was secreted in culture supematants of pcDNA3.1-caIL-2 transfected COS-7 cells. A proliferative activity was demonstrated in CTLL-2 cells when the recombinant protein was diluted at 220 pg/mL of purified cytokine from E. coli expression or when COS-7 supernatant was diluted at 1:256. The biologically active protein was able to induce proliferation of PBMC of six healthy dogs until 12 days of culture when cells were treated with 50 ng/mL of E. coli expressed-IL-2 or until 10 days when treated with 1:200 COS-7 supernatant dilution containing the cytokine, without previous or concomitant stimulus. The proliferative effect was dose-dependent and maximum at 8th day of culture. Interferon-gamma (IFN-y) production by PBMC of eight healthy dogs induced by COS-7 IL-2 or IL-12-containing supematants was not significantly higher than the baseline production. However, the association of suboptimal concentrations of both cytokines induced synergistic effect upon in vitro IFN-y production by PBMC. Based on the presented results, the construction pcDNA3.1-caIL-2 or both recombinant protein forms obtained, as well as the experimental conditions described here, can be used in the future to evaluate the potential role of IL-2, associated or not to canine IL-12, as a modulator of in vitro and in vivo immune response of dogs during the development of a vaccine or immunotherapy for canine visceral leishmaniasis.