Techn. Hochsch., Diss., 2003--Aachen.

Universiẗat, Diss., 2004--Kiel.

Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 177-212).

Despite improvements in chemotherapy and radical surgical debulking, peritoneal carcinomatosis (PC) remains among the most common causes of death for abdominal cancers. Immunotherapies have demonstrated efficacy in selected solid malignancies but their potential in PC is poorly explored. Here I report that intraperitoneal injection of an infected cell vaccine (ICV), consisting of autologous tumor cells infected ex-vivo with an oncolytic Maraba MG1 virus expressing interleukin-12 (IL-12), promotes the migration of activated natural killer (NK) cells to the peritoneal cavity in response to the secretion of interferon gamma-induced protein-10 (IP-10) from dendritic cells. This recruitment of cytotoxic, IFNγ-secreting NK cells is associated with a dramatic reduction in tumor burden and improved survival in a colon cancer model of PC. Even in mice with bulky PC (tumors >8 mm), a complete radiological response was demonstrated within 8-14 weeks, associated with 100% long-term survival. Importantly, these results were recapitulated in human lymphocytes exposed to human tumor cell lines infected with MG1-IL12. Finally, I demonstrate that MG1-IL12-ICV generates an effective CD4 and CD8 T cell response in mice following prophylactic immunization associated with the maturation of peritoneal dendritic cells and enrichment of tumor-specific peritoneal T cells. The research presented in this thesis suggests that an MG1-IL12-ICV is a promising therapy that could provide benefit to the thousands of patients diagnosed with PC each year.

Made available in DSpace on 2014-06-11T19:25:35Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-08-03Bitstream added on 2014-06-13T20:14:13Z : No. of bitstreams: 1 000739847.pdf: 1497375 bytes, checksum: 323121923378634f36af730ba11e938a (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Proposição: As células epiteliais das vias respiratórias desempenham uma importante função na patogênese da asma. Elas são responsáveis pela liberação de mediadores químicos ativadores do sistema imunológico na resposta inflamatória das vias aéreas. Citocinas e quimiocinas produzidas por leucócitos ativam as células estruturais dos brônquios e incitam a síntese de outros mediadores químicos que potencializam a inflamação no local. A interleucina-9 (IL-9) é uma importante citocina ativadora das células epiteliais, regula a produção de muco e induz a expressão de quimiocinas e citocinas. Os objetivos deste estudo foram investigar se células epiteliais de pulmão murino (LA-4) estimuladas com a citocina IL-9 produzem a quimiocina do timo regulada por ativação (CCL17/TARC) e o mediador lipídico leucotrieno-C4 (LTC4), e quais vias de sinalização intracelular estariam envolvidas nesse processo. Julga-se que as proteínas quinases desempenhem uma função primordial na expressão e ativação de citocinas e quimiocinas nas vias aéreas, portanto, foi investigada a função da p38 proteína quinase ativada por mitógeno (MAPK), MAPK p42/44, e fosfatidilinositol 3-quinase (PI3K) sobre o efeito da IL-9 na expressão da quimiocina CCL17/TARC em células LA-4. Abordagem experimental: a expressão de CCL17/TARC por LA-4 foi avaliada utilizando Reação em Cadeia da Polimerase Transcriptase Reversa (RT-PCR). A produção de leucotrieno C4 (LTC4) e CCL17/TARC foi avaliada por ELISA. As células LA-4 foram pré-tratados com o antagonista do receptor de cisteinil leucotrieno (CysLT) (MK 571) (10 μM), com o inibidor da p42/44 MAPK (PD98059) (30 μM), inibidor... / Background and propose: The airway epithelial cells play an important role in the pathogenesis of asthma, are responsible for the release of chemical mediators of the immune system triggers inflammation in the airways. Cytokines and chemokines produced by leukocytes activate structural bronchial cells and induce the synthesis of other chemical mediators which enhance the site inflammation. Interleukin-9 (IL-9) is an important cytokine activating epithelial cells, regulating mucus production and induces the expression of chemokines and cytokines. The objectives of this study were to investigate whether murine lung epithelial cells (LA-4) IL-9-stimulated produce the thymus and activation-regulated chemokines (CCL17/TARC) and lipid mediator leukotriene-C4 (LTC4) and what intracellular signaling pathways would be involved in this process. Kinases are believed to play a crucial role in the expression and activation of cytokines and chemokines in the airways. Therefore the role of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and phosphatidylinositol 3-kinase (PI3K) upon the effect of IL-9 on the CCL17/TARC expression in murine lung alveolar epithelial cells (LA-4) was investigated. Experimental approach: CCL17/TARC expression in LA-4 was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Leukotriene C4 (LTC4) and CCL17/TARC production were assessed by ELISA. LA-4 had been pre-treated with cysteinyl leukotriene receptor antagonist (CysLT) (MK 571) (10 μM), p42/44 MAPK inhibitor (PD98059) (30 M), p38 MAPK inhibitor (SB203580) (30 M), and PI3K inhibitor (Wortmannin) (0.1 M) 30 min prior to IL-9 (40 ng.mL-1) stimulation. Key results: IL-9-stimulated LA-4 induced CCL17/TARC mRNA expression after 24 hours. PD98059 and...

Orientador: Sandra Helena Penha de Oliveira / Banca: Lucia Helena Faccioli / Banca: Ana Paula Campanelli / Resumo: Proposição: As células epiteliais das vias respiratórias desempenham uma importante função na patogênese da asma. Elas são responsáveis pela liberação de mediadores químicos ativadores do sistema imunológico na resposta inflamatória das vias aéreas. Citocinas e quimiocinas produzidas por leucócitos ativam as células estruturais dos brônquios e incitam a síntese de outros mediadores químicos que potencializam a inflamação no local. A interleucina-9 (IL-9) é uma importante citocina ativadora das células epiteliais, regula a produção de muco e induz a expressão de quimiocinas e citocinas. Os objetivos deste estudo foram investigar se células epiteliais de pulmão murino (LA-4) estimuladas com a citocina IL-9 produzem a quimiocina do timo regulada por ativação (CCL17/TARC) e o mediador lipídico leucotrieno-C4 (LTC4), e quais vias de sinalização intracelular estariam envolvidas nesse processo. Julga-se que as proteínas quinases desempenhem uma função primordial na expressão e ativação de citocinas e quimiocinas nas vias aéreas, portanto, foi investigada a função da p38 proteína quinase ativada por mitógeno (MAPK), MAPK p42/44, e fosfatidilinositol 3-quinase (PI3K) sobre o efeito da IL-9 na expressão da quimiocina CCL17/TARC em células LA-4. Abordagem experimental: a expressão de CCL17/TARC por LA-4 foi avaliada utilizando Reação em Cadeia da Polimerase Transcriptase Reversa (RT-PCR). A produção de leucotrieno C4 (LTC4) e CCL17/TARC foi avaliada por ELISA. As células LA-4 foram pré-tratados com o antagonista do receptor de cisteinil leucotrieno (CysLT) (MK 571) (10 μM), com o inibidor da p42/44 MAPK (PD98059) (30 μM), inibidor... / Abstract: Background and propose: The airway epithelial cells play an important role in the pathogenesis of asthma, are responsible for the release of chemical mediators of the immune system triggers inflammation in the airways. Cytokines and chemokines produced by leukocytes activate structural bronchial cells and induce the synthesis of other chemical mediators which enhance the site inflammation. Interleukin-9 (IL-9) is an important cytokine activating epithelial cells, regulating mucus production and induces the expression of chemokines and cytokines. The objectives of this study were to investigate whether murine lung epithelial cells (LA-4) IL-9-stimulated produce the thymus and activation-regulated chemokines (CCL17/TARC) and lipid mediator leukotriene-C4 (LTC4) and what intracellular signaling pathways would be involved in this process. Kinases are believed to play a crucial role in the expression and activation of cytokines and chemokines in the airways. Therefore the role of p38 mitogen-activated protein kinase (MAPK), p42/44 MAPK, and phosphatidylinositol 3-kinase (PI3K) upon the effect of IL-9 on the CCL17/TARC expression in murine lung alveolar epithelial cells (LA-4) was investigated. Experimental approach: CCL17/TARC expression in LA-4 was evaluated using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Leukotriene C4 (LTC4) and CCL17/TARC production were assessed by ELISA. LA-4 had been pre-treated with cysteinyl leukotriene receptor antagonist (CysLT) (MK 571) (10 μM), p42/44 MAPK inhibitor (PD98059) (30 M), p38 MAPK inhibitor (SB203580) (30 M), and PI3K inhibitor (Wortmannin) (0.1 M) 30 min prior to IL-9 (40 ng.mL-1) stimulation. Key results: IL-9-stimulated LA-4 induced CCL17/TARC mRNA expression after 24 hours. PD98059 and... / Mestre

Made available in DSpace on 2014-06-11T19:23:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-15Bitstream added on 2014-06-13T18:50:11Z : No. of bitstreams: 1 bannwart_cf_me_botfm.pdf: 462065 bytes, checksum: 2e3988993f9db6f1ef7a8c8058e75e6a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Universidade Estadual Paulista (UNESP) / A interleucina-15 (IL-15) é uma citocina pró-inflamatória produzida principalmente por monócitos e macrófagos em resposta a agentes infecciosos, desempenhando importante papel modulador na imunidade inata e adaptativa. O objetivo do presente trabalho foi avaliar o efeito da IL-15 sobre a atividade fungicida, metabolismo oxidativo e a produção de citocinas por monócitos humanos, infectados in vitro com cepa virulenta de Paracoccidioides brasiliensis (Pb18). Monócitos de sangue periférico, obtidos de indivíduos saudáveis, foram préincubados na ausência ou presença de IL-15 (12,5, 25 e 50 ng/mL) por 24 h a 37oC e infectados com Pb18 na proporção de 50 monócitos para uma célula fúngica durante 4 h e 18 h. A atividade fungicida de monócitos foi determinada após 4 h pela recuperação de fungos viáveis por plaqueamento das co-culturas em meio BHI-ágar. O metabolismo oxidativo foi avaliado pela liberação de peróxido de hidrogênio (H2O2) e de ânion superóxido (O2 -) nas culturas desafiadas com Pb18 e estimuladas com phorbol myristate acetate (PMA) durante 60 mim. A produção de fator de necrose tumoral-alfa (TNF-a), IL-6, IL-10 e IL-15 foi determinada por ensaio imunoenzimático (ELISA) nos sobrenadantes das coculturas obtidos após 4 e 18 h de incubação. Os resultados mostraram que a préincubação de monócitos com IL-15 induziu aumento significativo na atividade fungicida contra Pb18 de maneira dose-dependente, sendo esse efeito neutralizado pela adição de anticorpo monoclonal anti-IL-15. O tratamento com IL- 15 não interferiu na capacidade de liberação de H2O2 e O2 - por monócitos desafiados com Pb18 sugerindo que a atividade fungicida estimulada por IL-15 ocorre por mecanismos independentes do metabolismo oxidativo. Monócitos infectados com o fungo, na ausência de IL-15, produziram níveis de TNF-a, IL-6 e IL-10... / Interleukin-15 (IL-15) is a pro-inflammatory cytokine especially produced by monocytes and macrophages against infectious agents and that play a pivotal role in teh innatte and adaptive immune response. The aim of this study was to analyze the effects of IL-15 on fungicidal activity, oxidative metabolism and cytokine production by human monocytes challenged in vitro with a virulente strain of Paracoccidioides brasiliensis (Pb18). Peripheral blood monocytes obtained from healthy individuals were pre-incubated for 24h with or without human recombinant IL-15 (12.5,25 and 50 ng/mL), and then challenged with Pb18 in a ratio of 50:1 monocytes:fungi. Fungicidal activity of monocytes against Pb18 was assessed by viable fungi recovery from 4 h co-cultures after plating in BHI-agar. Oxidative metabolism was evaluated by hydrogen peroxide (H2O2) and superoxide anion (O2 -) release in the monocyte cultures challenged by Pb18 and stimulated with phorbol myristate acetate (PMA) for 60 min. Tumor necrosis factor-alpha (TNF-a), IL-6, IL-10 and IL-15 production by monocytes were determined in culture supernatants by enzyme immunoassay (ELISA). The results showed that IL-15 ehanced fungicidal activity against Pb18 in a dose-dependent pattern. This effect was abrogated by additon of anti-IL-15 monoclonal antibody to the co-cultures. No significant effect of IL-15 on H2O2 and O2 - release by monocytes was observed suggesting that the fungicidal activity was independent of oxidative metabolism activation. Monocytes infected with P. brasiliensis in the absence of IL-15, produced significantly higher levels of TNF-a, IL-6 and IL-10 after 18 h of coculture in comparison to experiments of 4h-incubation with the fungus. The pretreatment of monocytes with IL-15 induced significant higher levels of TNF-a, IL-10 and IL-15 production by these cells challenged with the fungus... (Complete abstract click eletronic address below)

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / IntroduÃÃo: MGI à o termo que descreve os efeitos da quimioterapia antineoplÃsica nas mucosas, podendo acometer o trato alimentar de maneira global ou localizada. 15 a 40% dos pacientes em quimioterapia apresentam algum grau de mucosite. Muito da fisiopatologia da MGI permanece desconhecido. A IL-18 à uma citocina pleiotrÃpica, com funÃÃo regulatÃria sobre o sistema imune e envolvimento nas fases iniciais da inflamaÃÃo. Objetivo: Avaliar o envolvimento de IL-18 na patogÃnese da MGI induzida por CPT-11. Materiais e MÃtodos: camundongos Balb/C IL-18Wt ou IL-18KO, machos, foram tratados durante quatro dias consecutivos com CPT-11 (60mg/Kg, i.p.) ou veÃculo (0,5 mL, i.p.). Um grupo de animais IL-18Wt recebeu, alÃm do CPT-11, a proteÃna ligante de IL-18 (IL-18Bp; 200 Âg, i.p.). Os seguintes parÃmetros foram avaliados: diarrÃia, leucograma, sobrevida, anÃlise histopatolÃgica, atividade de mieloperoxidade (MPO), dosagem de citocinas (TNF-α, IL-1β ) por ELISA e imunoistoquÃmica para TNF-α e IL-1β nas mucosas ileais. Resultados: CPT-11 induziu diarrÃia significante e promoveu alteraÃÃes intestinais exuberantes (encurtamento de vilos, achatamento e vacuolizaÃÃo de enterÃcitos, necrose em criptas e infiltrado inflamatÃrio de leucÃcitos polimorfonucleares e cÃlulas mononucleares) Observou-se aumento da atividade de MPO e dos nÃveis tissulares de TNF-α e IL-1β dosados por ELISA e intensa imunomarcaÃÃo com anticorpos anti-TNFα e anti-IL-1β nesses animais. AlÃm disso, tais animais apresentaram resposta contrÃtil intestinal exacerbada ao estÃmulo com colinÃrgicos (acetilcolina e betanecol). Os animais IL-18KO tratados com CPT-11 apresentaram diarrÃia estatisticamente menos severa e menor intensidade de alteraÃÃes morfomÃtricas e histolÃgicas. NÃo houve aumento de TNF-α, mas houve de IL-1β , a atividade de MPO nÃo diferiu da observada nos animais que receberam veÃculo e nÃo houve aumento de resposta contrÃtil ao estÃmulo colinÃrgico. Os animais que receberam CPT-11 e IL-18Bp apresentaram diarrÃia estatisticamente menos intensa que aqueles que receberam apenas CPT-11, resposta contrÃtil estatisticamente menos acentuada e menor atividade de MPO. Entretanto, as alteraÃÃes morfomÃtricas e histolÃgicas nÃo diferiram das encontradas nos animais tratados sà com CPT-11. ConclusÃo: IL-18 està envolvida na patogÃnese da MGI induzida por CPT-11. IL-18Bp atenua os eventos envolvidos na MGI induzida por CPT-11 e à um possÃvel candidato a modulador farmacolÃgico desse processo. As alteraÃÃes contrÃteis no intestino promovidas pelo CPT-11 parecem ter um componente inflamatÃrio / Introduction: The term GIM refers to the adverse effects of cancer chemotherapy on mucosal surfaces, affecting different portions of the alimentary tract. 15% to 40% of patients in chemotherapy present some degree of mucositis. By now, much of GIM pathophysiology remains unknown. IL-18 is a pleiotropic cytokine that exerts regulatory functions over the immune system and is also involved in inflammation. Purpose: The aim of the present study was to elucidate the involvement of IL-18 in the pathogenesis of CPT-11-induced mucositis. Materials and methods: Male IL-18 wild type (Wt) or IL-18 knockout (KO) Balb/C mice received CPT-11 (60mg/kg/day, i.p.) or vehicle (0.5ml, i.p.) in a four day schedule. A group of IL-18Wt also received IL-18 binding protein (IL-18Bp, 200 Âg, i.p., 1h before CPT-11). Animals were sacrificed on the fifth day. The following parameters were assessed: histologycal analysis, diarrhea, survival curve, leucogram, myeloperoxidase (MPO) activity assay, ileum levels of TNF-α and IL-1β by ELISA, immunohistochemistry for TNF-α and IL-1β and contractility assay. Results: IL-18Wt animals receiving CPT-11 presented diarrhea and intense histological alterations in the ileum (shortening of villi, flattening and vacuolization of enterocytes, cript necrosis and the presence of inflammatory infiltrate). There was also increased MPO activity, increased levels of TNF-α and IL-1β and strong immunostaining for TNF-α and IL-1β in the ileum. In addition, the CPT-11 treated mice presented increased intestinal contractility when stimulated with cholinergic drugs (bethanecol, BCh and acetylcholine, ACh). In contrast, IL-18KO animals treated with the same dose of CPT-11 presented less diarrhea and less intestinal histological alterations. There was no increase in MPO activity nor in TNF levels, but in IL-1 levels. In addition, TNFα and IL-1β immunostaining was weaker IL-18KO animals. Also, the intestinal contractility in IL-18KO animals was not exarcerbated after a cholinergic challenge. IL-18Wt animals receiving CPT-11 and IL-18Bp did not present significant diarrhea and there was no significant alterations on intestinal contractility after Ach administration. Although MPO activity was not increased in IL-18Bp treated animals, the ileal mucosa presented moderate to severe histological alterations. Conclusion: IL-18 participates in the CPT-11-induced GIM. IL-18Bp attenuates some inflammatory (cell infiltrate) and functional (diarrhea and contractility) events of CPT-11-induced GIM. The contractility alterations in CPT-11 treated animals seem to have an inflammatory related component

Background: COPD is associated with an increased lung macrophage burden. Whilst lung macrophages may self-renew, recruitment of peripheral blood monocytes from the systemic circulation is considered to represent their principal means of replenishment. Through modulating expression of monocytic chemokines CCL2/CCL3 and their respective receptors (CCR2/CCR1+CCR5), IL-6 could play a key role in facilitating the recruitment of monocytes to the lungs of COPD patients. COPD is associated with enhanced pulmonary and systemic IL-6 levels; concentrations of the soluble IL-6 receptor sIL-6R may be an important determinant of IL-6 signalling in COPD. Trans-signalling through sIL-6R, IL-6 may facilitate recruitment of monocytes in COPD by influencing chemokine and chemokine receptor expression. Aims: 1) To compare levels of IL-6, sIL-6R, CCL2 and CCL3 in the plasma and sputum of COPD and controls. 2) To examine of the effects of IL-6 stimulation on monocyte chemokine receptor gene expression (CCR1, CCR2 and CCR5). 3) To compare subtypes (CD14++CD16-, CD14+CD16+, CD14-CD16++) and chemokine receptor expression (CCR1, CCR2, CCR5) of monocytes in COPD (paired stable & exacerbating) and controls. 4) To compare the migratory ability of monocytes from COPD and controls. 5) To compare numbers of marginated CX3CR1+ monocytes in the pulmonary microvasculature and proliferation status (Ki67 positivity) of alveolar macrophages in COPD and controls. Methods: 1) MSD soluble marker analysis was performed on plasma and sputum supernatant. 2) Monocytes underwent stimulation with IL-6 and sIL-6R; chemokine receptor expression was determined by quantitative PCR. 3) Flow cytometry was performed on whole blood to determine monocyte subtype and chemokine receptor expression. 4) Monocyte migration towards sputum supernatant was assessed using a transwell system incorporating fluorescence based detection of DNA from migrated cells. 5) Immunofluorescence and immunohistochemistry was performed on lung tissue (obtained from patients undergoing surgical resection of lung carcinoma) to identify marginated (CX3CR1+CD14+, CX3CR1+CD16+) monocytes and proliferating alveolar macrophages (Ki67) respectively. Results and Conclusion: Levels of sIL-6R were increased in the lungs and systemic circulation of COPD patients implying potential for enhanced IL-6 trans-signalling: monocytes cultured in the presence of IL-6+sIL-6R upregulated expression of the CCR5 gene. A greater proportion of circulating COPD CD14++CD16- and CD14+CD16+ monocytes were demonstrated to express CCR5 compared to controls indicating that CCR5 ligands may have an important influence over monocyte migration in COPD. Levels of CCR5 ligand CCL3 were significantly elevated in COPD sputum supernatant; IL-6 levels were positively associated with CCL3 indicating that IL-6 trans-signalling may mediate lung chemokine expression. Nevertheless, COPD monocytes demonstrated impaired migration towards sputum supernatant and reduced margination to pulmonary microvessels. Despite this, the number of alveolar macrophages in COPD was increased; however this was not likely to be related to self-replication owing to low alveolar macrophage Ki67 expression.

The pro-inflammatory cytokine interleukin 1 (IL-1) is involved in numerous physiological and pathological processes. It contributes to thermoregulation, sleep, feeding behaviour and notably to the exacerbation of non-communicable disorders such as cancer, heart disease, stroke and epilepsy, which are the greatest cause of mortality worldwide. Given this important role, IL-1 is tightly regulated, with regulation mechanisms present at the level of its synthesis, activation and receptor engagement. However, when studying IL-1 in vitro, little notice is taken of the disease microenvironment in which it acts. Acidosis is a hallmark of disease, often due to poor perfusion resulting in a shift to anaerobic respiration, a build-up of lactic acid and poor clearance of CO2. Additionally, highly active infiltrating immune cells favour anaerobic respiration and can contribute to this local acidosis. This thesis utilised primary cell cultures, cell lines and reporter cells to explore the mechanisms of IL-1 signalling under disease-relevant acidic conditions. Subsequently, a murine seizure model was developed to further explore IL-1 signalling in disease conditions in vivo. This work demonstrated that acidic pH itself did not induce IL-1β release, however, it did promote release of minimally active 20 kDa IL-1β in response to damage associated molecular patterns (DAMPs) such as ATP, monosodium urate crystals or calcium pyrophosphate dihydrate crystals. The cleavage of pro-IL-1β into 20 kDa IL-1β was mediated by cathepsin D and was also induced on addition of lactic acid to the culture media. This 20 kDa IL-1β was not further cleaved to the active mature 17 kDa IL-1β thus its production limits the spread of inflammation. The intranasal administration of kainic acid induces seizures in C57Bl/6J mice, however, IL-1β was not observed acutely in this model thus the presence of 20 kDa IL-1β in vivo was not confirmed. In recent years, the contribution of IL-1 to disease has become well established. However, despite successes in the development of novel therapeutics targeted at blocking IL-1 activity, such as anakinra, canakinumab or rilonacept to treat cryopyrin associated periodic syndromes, a number of studies have demonstrated poor efficacy and only minor improvements in patients when targeting IL-1. Thus further knowledge of the mechanisms of IL-1 signalling in disease is required to understand this system and develop improved novel therapeutics.