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

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2013-10-15T16:35:40Z No. of bitstreams: 1 Bianca_Petitinga_de_Paiva. Produção e avaliação...2011.pdf: 1434436 bytes, checksum: f5c3b8722c06f5784b6ec44bc9937084 (MD5) / Made available in DSpace on 2013-10-15T16:35:40Z (GMT). No. of bitstreams: 1 Bianca_Petitinga_de_Paiva. Produção e avaliação...2011.pdf: 1434436 bytes, checksum: f5c3b8722c06f5784b6ec44bc9937084 (MD5) Previous issue date: 2011 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, Brasil / A maioria dos cães com leishmaniose visceral submetida à quimioterapia convencional apresenta recaída após a interrupção do tratamento, talvez, pelo fato desses animais desenvolverem resposta imune celular específica apenas transitoriamente. Uma vez que as citocinas IL-7 e IL-15 são descritas na literatura como sendo capazes de promover resposta imune celular de longa duração (memória), o presente trabalho teve como objetivo a clonagem, a expressão e a avaliação da atividade biológica de IL7 e IL-15 recombinantes caninas (rca-IL-7 e rcaIL-15). Diversos estudos têm demonstrado o papel essencial da IL-7 e IL-15 na homeostase de células-T. A Interleucina-7 é um fator de crescimento e anti-apoptótico de linfócitos T, sendo essencial para a sobrevida de células T maduras, naïve e células de memória, especialmente CD4+. IL-15 apresenta um amplo espectro de atividades biológicas. É crucial para o desenvolvimento, proliferação, sobrevivência e diferenciação de múltiplas células tanto da imunidade inata, quanto da imunidade adaptativa, e apresenta papel essencial na manutenção de células T CD8+ de memória. Nesse trabalho descreveu-se a clonagem do DNA complementar (cDNA) e a expressão de IL-7 e IL-15 recombinantes caninas biologicamente ativas em Escherichia coli. Para expressão em E. coli foram realizadas as clonagens do cDNA de IL-7 e de IL-15 no vetor pRSET, gerando as construções pRSET-caIL-7 e pRSET-caIL-15. O sucesso da clonagem em ambos os vetores de expressão foi confirmado a partir do sequenciamento. As proteínas foram expressas como proteína de fusão com seis moléculas de histidina na extremidade amina da cadeia polipeptídica e após serem solubilizadas com uréia, as proteínas foram purificadas por cromatografia de afinidade e renaturadas por diálise. A avaliação da atividade biológica de IL-15 canina purificada foi realizada em células CTLL-2 e foi demonstrada uma relação dose dependente na faixa de concentração proteica de 0,9 ng/mL até 1 µg/mL. O mesmo efeito foi demonstrado quando essas células foram cultivadas na presença de diferentes diluições de T-STIM (controle positivo do ensaio). Visando determinar as condições em que rca-IL-7 e rca-IL-15 são capazes de induzir efeitos biológicos in vitro, foram realizados experimentos com diferentes doses de ambas as citocinas e demonstrado pela primeira vez que tanto IL-7 quanto IL-15 são capazes de induzir a proliferação de CMNSP de cães pré-ativadas com PHA. Adicionalmente, as citocinas foram avaliadas quanto à capacidade de estimulação da proliferação de CMNSP de cães sem estímulo prévio ou concomitante e foi possível observar que rca-IL-7 foi capaz de manter a proliferação dessas células por até 12 dias de cultivo, enquanto rca-IL-15 foi capaz de fazê-lo por até 14 dias. Diante dos resultados obtidos a rca-IL-7 e rca-IL-15 poderão ser utilizadas no futuro em estudos que visam o estabelecimento de uma resposta de longa duração em cães com LVC submetidos à quimioterapia convencional. / Most dogs with LV subjected to conventional chemotherapy has relapsed after discontinuation of treatment, possibly due these animals develop specific cellular immune response transiently. Since IL-7 and IL-15 are described in the literature asbeing capable of promoting cellular immune response of long-term (memory), this study aimed at cloning, expression and biological activity evaluation of rca-IL7 and rca IL-15. Many studies have demonstrated the essential role of IL-7 and IL- 15 in the homeostasis of T-cells. The Interleukin-7 is a growth and anti-apoptotic factor of T lymphocytes, essential for the occurrence of mature, naive, and memory T cells, especially CD4+. IL-15 presents a wide spectrum of biological activities. It is crucial for the development, proliferation, survival, and differentiation of multiple cells of innate immunity as well as of adaptive immunity, and presents an essential role in the maintenance of CD8+ memory T cells. This work describes the cloning of complementary DNA (cDNA) and the expression of IL-7 and IL-15 canine recombinants biologically active in Escherichia coli. For expression in E. coli, we cloned cDNA of IL-7 and IL- 15 in the vector pRSET, generating the constructions pRSET-caIL7 and pRSETcaIL- 15. The success of the cloning in both the expression vectors was confirmed from the sequencing. The proteins were expressed as fusion protein with six molecules of histidine in the amine extremity of the polypeptide chain and afterwards were solubilized with urea, the proteins were purified by affinity chromatography and renatured by dialysis. The evaluation of the biological activity of purified canine IL-15 was achieved in CTLL-2 cells and a dose-dependent relation in the range of protein concentration of 0.9 ng/mL to 1 ƒÊg/mL was demonstrated. The same effect was demonstrated when these cells were cultivated in the presence of different dilutions of T-STIM (positive control of the test). With the goal of determining the conditions in which rca-IL-7 and rca-IL-15 are capable of inducing biological effects in vitro, we carried out experiments with different doses with both the cytokines and demonstrated for the first time that IL-7 as well as IL- 15 are capable of inducing proliferation of PBMC of dogs pre-activated with PHA. Also, the cytokines were evaluated as to the capacity of stimulation of the proliferation of PBMC of dogs without previous or concomitant stimulation and it was possible to observe that rca-IL-7 was capable of maintaining the proliferation of these cells for up to 12 days of cultivation, while rca-IL-15 was capable of doing it for 14 days. Considering the results obtained rca-rca-7 and IL-IL-15 may be used in future studies that aim to establish a long-termresponse in dogs with CVL undergoing conventional chemotherapy

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

Resumo: O objetivo deste estudo foi o de avaliar possíveis interações clínicas, microbiológicas, imunológicas e genéticas que possam influenciar o sucesso de implantes osseointegrados. Foram avaliados 47 implantes, em 47 pacientes, sendo 31 em condições de saúde (G I) e 16 com perimplantite (G II) e 47 dentes, dos quais 31 estavam sadios e eram dos pacientes com implantes sadios (G III) e 16 dentes sadios dos pacientes com perimplantite (G IV). Foi realizado exame clínico completo em todos os implantes e dentes selecionados. Amostras de fluido crevicular perimplantar/gengival do sítio com maior profundidade de sondagem foram coletadas. A avaliação das bactérias A. actinomycetemcomitans, P. gingivalis, P. intermedia, P. nigrescens e T. forsythia foi realizada pela técnica de PCR e, a quantificação das citocinas IL-1β e IL-6 foi realizada pelo teste ELISA. Células da mucosa bucal foram coletadas para avaliação dos polimorfismos IL1B+3954, IL1B-511 e IL6-174. A avaliação estatística dos parâmetros clínicos SS, SUP, PS e NI revelaram que os implantes do grupo G II apresentaram piores condições clínicas em comparação ao grupo G I. O grupo G II também apresentou piores condições clínicas que o grupo G IV para PS e NI. A análise microbiológica revelou que a bactéria A. actinomcetemycomitans não estava presente em nenhum sítio avaliado. P. intermedia também não foi encontrada no grupo G II. As bactérias estudadas apresentaram proporções semelhantes em todos os grupos avaliados, não havendo diferença entre os grupos. Na análise da concentração de IL-1β e IL-6, não houve diferenças significativas entre os grupos. A população estudada está em Equilíbrio de Hardy-Weinberg. Os polimorfismos estudados não demonstraram predominância dos alelos e dos genótipos. Nenhum polimorfismo foi associado à condição de doença. / Abstract: The aim of the present study was to evaluate clinical, microbiological, immunological and genetics parameters in patients with implant loaded at least for one year. It was examined 47 implants and teeth in 47 patients. Thirty one of those implants were healthy implants (G I), sixteen had peri-implantits (G II) and, 31 healthy teeth was from patients with healthy implants (G III) and 16 healthy teeth from patients with peri-implantits (G IV). Clinical parameters were recorded from all implants and teeth. Gingival crevicular fluid from the highest pocket depth was collected to evaluate the presence of A. actinomycetemcomitans, P. gingivalis, P. intermedia, P. nigrescens e T. ForsythiaI and to evaluate the concentration of interleukin-1β and interleukin-6. Cells from buccal mucosa were collected for genomic DNA extraction and analyze the polymorphism IL-1B +3954, IL-1B -511 e IL-6 -174. G II demonstrated worst results for PD, BPD, Sup and NI when compared to G I and, when compared with G IV it was worst for PD and NI. Microbiological did not detect A. actinomycetemcomitans in any of the sites analysed and, P. intermedia was not detected in G II. Bacteria analyzed was present in same proportion in all analyzed sites showing, no differences between groups. There was no difference in the concentration of IL-1β an IL-6 detected between groups. The population studied was in Hardy-Weinberg Equilibrium. There was no differences in the alleles and polymorphism distribution on the studied population. / Orientador: Glória Maria de Azevedo Thompson Galli / Coorientador: Rosemary Adriana Chierici Marcantonio / Coorientador: Elcio Marcantonio Junior / Banca: Débora Pallos / Banca: Eduardo Saba-Chujfi / Banca: José Eduardo César Sampaio / Banca: Joni Augusto Cirelli / Doutor

La maladie d'Alzheimer est devenue le désordre neurodégénérarif le plus important chez les personnes âgées chez qui elle se manifeste par un déclin cognitif. Cette maladie se caractérise par l'accumulation dans le cerveau de peptides amyloïdes, particulièrement le peptide Aβ42, qui s'avère être le plus toxique. Ce peptide doit être éliminé du cerveau et le transporteur P-glycoprotéine présent dans la Barrière Hémato Encéphalique (BHE) a été proposé comme intervenant dans ce processus. L'objectif essentiel de cette thèse, était de savoir si le peptide Aβ42 est pris en charge par la P-gp. Nos résultats suggèrent que ce transport semble ne pas être possible. Dans le but d'explorer quels sont les changements conduisant à la mort neuronale lors de la maladie d'Alzheimer, une étude sur la modification de deux paramètres contribuant à la vie cellulaire a été aussi réalisée, a savoir : le potentiel membranaire et la concentration intercellulaire des ions chlorure. Des altérations dans ces deux paramètres ont été observées lors de l'incubation des cellules neuronales avec Aβ42. Finalement afin de mieux comprendre l'interaction des molécules avec la P-gp pouvant aboutir à la conception de nouveaux traitements pour la maladie d'Alzheimer, une étude sur l'effet inhibiteur de différentes molécules sur la P-gp parachève cette thèse. Les résultats obtenus lors de ces trois études portent une contribution à la compréhension dans le domaine du développement de cette maladie. / Alzheimer's disease has become the most important degenerative disorder in elderly people, being cognitive decline its main result for patients who exhibit such medical condition. A hallmark of this disease is the brain accumulation of amyloïd peptides, particulary Aβ42 peptide, wich is revealed to be the most toxic. This peptide must be eliminated of brain, and the transporter P-glycoprotein at the blood brain barrier (BBB) has been proposed for this process. The principal aim of this thesis was to know if Aβ42 is transported by P-gp. Our results suggested that such transport mechanism di not seem to be possible.In order to explore the underlying changes that drive neuronal death in Alzheimer's disease, a study of variations in two parameters importants for cell survival (membrane potential and intracellular concentration of chloride) has been made. Alterations in theses parameters were observed in neuronal cells incubated in the presence of Aβ42 . Finally, a study on inhibitor effect of different molecules on P-gp activity completes this thesis, to accomplish the aim understanding molecules interactions with P-gp, so as to establish some possible guidelines related with new treatments for Alzheimer's disease. The overall results obtained in the three studies herein contribute to understand the development of this disease.

O quelóide é um tumor fibroso benigno que ocorre durante a cicatrização da pele em indivíduos geneticamente predispostos. A cicatrização é um processo biológico complexo e depende da interação de diferentes estruturas teciduais e de um grande número de tipos celulares residentes e infiltrativos, que produzem citocinas. A interleucina 8 (IL-8), citocina pró-inflamatória, é super-expressa pelos fibroblastos durante o desenvolvimento do tecido de granulação, acelerando o processo de cicatrização. Como o quelóide resulta de uma reparação tecidual anormal após lesão da pele, o presente estudo teve por objetivo determinar a expressão dos receptores da IL-8, CXCR1 e CXCR2, e a capacidade proliferativa, pelo ciclo celular, dos fibroblastos queloideanos cultivados e extraídos ex vivo, por citometria de fluxo. Fibroblastos de cicatriz queloideana e de pele normal foram obtidos de 21 pacientes da raça negra, com idade variando entre 10 e 40 anos, de lesões com até 2 anos de evolução. Em nosso estudo constatamos expressão reduzida dos receptores para a IL-8, CXCR1(35,7%±11,2) e CXCR2 (27,8%±11,3), em fibroblastos de cicatriz queloideana cultivados, comparando com a pele normal (44,1±16,2 e 46,3±27,1 respectivamente). Entretanto, essa diferença só foi significante para o receptor CXCR2. A baixa expressão desses receptores poderia ser decorrente da atividade de metaloproteinases, que regulam a expressão de proteínas da superfície celular, através de clivagem enzimática, ou a capacidade reduzida de internalização e a reciclagem de receptores, mantida por filamentos de actina do citoesqueleto, que nos fibroblastos do quelóide estão diminuídos. Em relação ao ciclo celular de fibroblastos cultivados do quelóide e da pele normal, verificamos diferenças não significantes da capacidade de replicação (fase S do ciclo celular) e de apoptose. No quelóide observamos significante aumento de células na fase G2/M, indicando aumento da velocidade de divisão celular. Para confirmar esses achados estudamos o ciclo celular de fibroblastos extraídos ex vivo, da porção periférica e central do quelóide e da pele normal. Os fibroblastos da porção periférica apresentaram porcentagem significantemente maior de células com capacidade replicativa, fase S do ciclo (22,9% ± 11,6), em relação à porção central (4,7% ± 2,9) e à pele normal (6,8% ± 4,9), e maior velocidade de divisão celular, fase G2/M (18,6 ± 12,0), em relação à porção central (35,6 ± 7,0) e pele normal (32,3 ± 6,9). Verificamos que a porção central apresentou maior porcentagem de células em apoptose (7,0% ± 2,1), comparado à porção periférica (4,9% ± 1,9) e pele normal (2,0% ± 0,86). Esses dados indicam que as células da porção periférica do quelóide parecem ser responsáveis pela elevada taxa de proliferação, justificando o crescimento expansivo a partir das margens da cicatriz queloideana, com desenvolvimento de lesão semelhante a tumor, bem como a porção central ser responsável pela fibrose, contendo células quiescentes e apoptóticas. Esses resultados sugerem modulação diferencial das reações celulares através das vias de sinalização para proliferação ou morte celular programada. Neste sentido, a baixa expressão dos receptores da IL-8, CXCR1 e principalmente de CXCR2, nos fibroblastos do quelóide sugere capacidade reduzida da IL-8 em promover cicatrização acelerada. A baixa atividade da IL-8 sobre os fibroblastos queloideanos estaria promovendo desregulação da resposta inflamatória e com isso atraindo novas células inflamatórias para o local e produzindo sinais alterados, como grande produção da citocina TGFβ. Essa desregulação do processo de cicatrização, com alteração de citocinas e da matriz extracelular, poderia ser responsável pelas duas populações de fibroblastos, uma proliferativa na periferia e outra quiescente e apoptótica na porção central. Finalizando, podemos concluir que nossos resultados correspondem às alterações histológicas e clínicas do quelóide que se expande nos limites da lesão. / A keloid is a benign fibrous tumor that occurs during wound healing in genetically predisposed individuals. Healing is a complex biological process and depends on the interaction of different tissue structures and a great number of resident and infiltrative cell types. The interleukin-8 (IL-8), a proinflammatory chemokine, showed higher expression in fibroblasts during the development of the granulation tissue, promoting more rapid tissue maturation. Since keloids result from abnormal wound healing, the objective of the present study was to determine the expression of CXCR1 and CXCR2, IL-8 receptors, and the proliferation capacity, throughout the cell cycle, of the keloid fibroblasts extracted ex vivo and those submitted to in vitro cultivation. Normal skin and keloid scar fibroblasts were obtained from 21 African-Brazilian patients, aged from 10 to 40 years, whose lesions had evolved for no longer than 2 years. Expression of receptors and the cell cycle was assessed by flow cytometry. We showed lower expression of the CXCR1 (35,7% ± 11,2) and CXCR2 (27,8%±11,3) in keloid fibroblasts, when compared with normal skin (44,1 ± 16,2 e 46,3 ± 27,1 respectively), but the difference was not significant for the CXCR1 receptor. This lower expression of IL-8 receptors in keloid fibroblasts could be due to the action of metalloproteinases, which regulate the surface protein enzymatically, or fibroblastic cytoskeleton conditions, which influence receptor internalization and recycling. The distribution assessment of cell cycle phases of fibroblasts cultivated from keloid scars and normal skin did not show significant difference in replication capacity and apoptosis. The keloid fibroblasts presented a significantly higher proportion of cells in the G2/M phase, suggesting higher rate of cell division. To confirm these results we studied the cell cycle of fibroblasts extracted ex vivo, now separated by central and peripheral portions of keloid and normal skin. The peripheral fibroblasts showed significant high cell proportions in phase S (22,9% ± 11,6), compared with the central portion (4,7% ± 2,9) and normal skin (6,8% ± 4,9), and higher cells in division phase G2/M (18,6% ± 12,0), compared with the central portion (35,6% ± 7,0) and normal skin (32,3% ± 6,9). The central portion showed higher proportion of apoptosis (7,0% ± 2,1), compared with the peripheral portion (4,9% ± 1,9) and normal skin (2,0% ± 0,86). These results suggest that the keloid peripheral cells could be responsible for the proliferation rate, justifying the expansive keloid growth at the borders of the keloid scar, in a similar fashion to tumor development and the central portion being responsible for fibrosis, with quiescent and apoptotic cells. These results suggest a differentiated modulation of cell reactions by signal pathways for programmed cellular proliferation or death. In this sense, the low expression of the IL-8 receptors CXCR1 and CXCR2 in keloid fibroblasts suggests a diminished capacity of IL-8 to promote accelerated healing. This low expression of IL-8 receptors in keloid fibroblasts could promote the dysregulation of the inflammatory response and thus attract more inflammatory cells to the site, producing different signals, such as a high production of the TGFβ cytokine. This dysregulation of the healing process, with changed cytokine and extracellular matrix expression, could be responsible for two different cell populations of fibroblasts, one proliferation at the periphery and the other fibrotic at the center of the lesion, with apoptotic and quiescent cells. Finally, we conclude that our results correspond to the histological and clinical changes of keloids that grow beyond the wound boundaries.

Vitamin A and the T helper 2 cytokines IL-4 and IL-13 play important roles in the induction of mucin gene expression and mucus hypersecretion. However, the effects of these agents on enzymes responsible for mucin glycosylation have received little attention. Here, we report the upregulation of core 2 β1,6 N-acetylglucosaminyltransferase (C2GnT) activity both by all-trans retinoic acid (RA) and by IL-4 and IL-13 in the H292 airway epithelial cell line. Northern blotting analysis showed that the M isoform of C2GnT, which is expressed in mucus-secreting tissues and can form all mucin glycan β1,6-branched structures, including core 2, core 4, and blood group I antigen, was upregulated by both RA and IL-4/13. The L isoform, which forms only the core 2 structure, was moderately upregulated by IL-4/13 but not by RA. Enhancement of the M isoform of C2GnT by RA was abolished by an inhibitor, of RA receptor α, implicating RA receptor α in the effect of RA. Likewise, an inhibitor of the Janus kinase 3 pathway blocked the enhancing effects of IL-4/13 on the L and M isoforms of C2GnT, suggesting a role of this pathway in the upregulation of these two C2GnTs by these cytokines. Taken together, the results suggest that IL-4/13 T helper 2 cytokines and RA can alter the activity of enzymes that synthesize branching mucin carbohydrate structure in airway epithelial cells, potentially leading to altered mucin carbohydrate structure and properties.

Exposure to traumatic events can lead to many varied psychological and physiological difficulties, including an increased risk for chronic physical health problems and chronic pain disorders, which are thought to be mediated through the three major biological systems involved in the human stress response. The objective of the present study was to examine the relationships between psychological symptoms and proinflammatory immune markers, Interleukin-1β (IL-1β) and Interleukin-6 (IL-6), which are thought to be related to many of the physical health problems associated with posttraumatic psychopathology. Female participants (N=12) were recruited from a trauma specialty clinic and participated in approximately one research session per month for up to one year of psychotherapy. Five participants had at least three data points and were further examined for longitudinal correlations. Baseline measurements of urinary IL-1β were associated with self-report measures of trait anxiety and dissociative symptoms. One participant, who completed nine research sessions over nearly 12 months, showed improvements in depressive symptoms, state and trait anxiety, and dissociative symptoms that seemed to correspond with decreases in IL-6. IL-1β did not seem to be related to any of her symptom measures. A second participant, with five data points over almost four months, showed less marked change in symptomatology, but her IL-6 levels seemed to correspond with depressive and dissociative symptoms, and her IL-1β levels seemed to be associated with trends in state anxiety and dissociative symptoms. Three other participants had between three and four data points, and the trends obtained were inadequate to determine whether any true relationship existed among the longitudinal variables. These results provide preliminary evidence that it may be possible to reduce chronic pro-inflammatory dysregulation through psychotherapy-facilitated symptom reduction.

Indiana University-Purdue University Indianapolis (IUPUI) / More than 200,000 American women are diagnosed with breast cancer each year. Although therapies effective in treating metastatic breast cancer currently exist, each year approximately 40,000 women die from this disease. Current evidence indicates that anti-cancer immune responses can be induced by vaccination in situ to the growth of metastasis and protect patients from the tumor recurrence. However, induction of anticancer immune responses may be limited in their efficacy due to immune suppression mechanisms induced by the developing cancer. Myeloid-derived suppressor cells are one population of immune regulators comprised of immature cells of myeloid origin with important roles in blocking immune activation and promoting tumor progression. Elimination or maturation of these cells has been found to promote enhanced anti-tumor effects and improve overall survival. This thesis identifies a new role for interleukin-12 as a modulator of myeloid-derived suppressor cell activity. Interleukin-12 was found to promote up-regulation of cell maturation markers on the surface of myeloid-derived suppressor cells with an accompanying decrease in factors responsible for conferring suppressive activity such as nitric oxide synthase 2 and arginase I. The alterations in myeloid-derived suppressor cells were observed following both in vitro and in vivo treatment with interleukin-12. Further analysis of the anti-tumor efficacy of interleukin-12 revealed that at least part of its suppression of tumor growth can be linked to reductions in myeloid-derived suppressor cell populations in the tumor microenvironment and an influx of active CD8+ T cells into the tumor microenvironment. The findings outlined in this thesis show that interleukin-12 alters the suppressive function of myeloid-derived suppressor cells leading to significant immune infiltration and activation resulting in increased overall survival and a reduction in metastasis.

Background: Knee osteoarthritis (OA) is the most common form of arthritis in older adults. Knee OA is associated with limitations in physical function. Functional limitations are also associated with another geriatric condition, frailty. Frailty is characterized by reduced strength, endurance and physiological function. Purpose: The primary purpose of this study is to determine if there is a difference in radiographic or symptomatic knee OA severity between non-frail and pre-frail women with knee OA. Secondary objectives include: a) the relationship between radiographic and symptomatic OA severity with serum inflammatory cytokines, and b) if there is a difference in inflammatory cytokines between non-frail and pre-frail women with knee OA. Methods: We included 21 community-dwelling women with knee OA. Frailty was assessed using the Fried Frailty Phenotype. Knee OA severity was characterized by the Kellgren and Lawrence (KL) score and the Knee Injury and Osteoarthritis Outcome Questionnaire (KOOS). Inflammatory cytokines included serum interleukin-6 (IL-6), interleukin-10 (IL-10), tumor necrosis alpha (TNF α) and C reactive protein (CRP). Results: Data from 20 participants (66.1 [9.6] years, BMI 29.7 [4.9] kg/m2, non-frail=55%; pre-frail=45%) were analyzed. Radiographic severity was not different between frailty groups (p= 0.11). There was no difference in symptomatic knee OA severity, measured using the KOOS subscales, between frailty groups (p>0.17). Radiographic OA severity and inflammatory markers were not associated (p>0.30). There was a negative relationship between TNF α and self-reported pain (r=0.26), no relationships between inflammatory cytokines with any other KOOS sub-scales. Lastly, there was no difference in any inflammatory cytokines between non-frail and pre-frail groups. Conclusion: Despite the relatively young age, nearly 50% of our participants were pre-frail. Pre-frailty was unrelated to the severity of the knee OA, or inflammatory cytokines. TNF α may be involved in the experience of pain in these women. While it appears women with knee OA frequently demonstrate pre-frail status, more work is necessary to examine the link between these diseases. / Thesis / Master of Science (MSc) / Arthritis is a chronic disease that has a debilitating effect on the lives of more than 4.6 million Canadians. In 2015, the cumulative economic burden of osteoarthritis was 195.2 billion dollars and is expected to increase significantly in the next two years. Knee osteoarthritis is the most common form of arthritis in older adults. Knee osteoarthritis is associated with increased pain, decreased physical function and decreased quality of life (QOL). In vulnerable older adults increased exhaustion, decreased physical function and muscle loss can increase the risk of developing frailty. Frail older adults are at higher risk of adverse health outcomes such as falls, hospitalization and death. Previous research has shown that older adults with knee OA are at higher risk of developing frailty however, it is not understood what underlying mechanisms increase this risk. This thesis provides fundamental information aimed at understanding potential mechanisms associated with knee osteoarthritis and frailty in women. Our study found that despite their relatively young age, nearly half of the women with knee OA are pre-frail. This data shows that inflammatory cytokines in particular, tumor necrosis factor alpha is related to symptomatic knee osteoarthritis severity in particular, self-reported pain. Overall, early detection of frailty is important when managing this condition. These data suggest that chronic knee pain associated with OA may be a useful trigger for early assessments of frailty in women.