Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2016-08-22T12:13:39Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO FINAL (com ficha catalografica).pdf: 1190266 bytes, checksum: 5fb8d7ae60e8775661fb41f33bcb771e (MD5) / Made available in DSpace on 2016-08-22T12:13:39Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) DISSERTAÇÃO FINAL (com ficha catalografica).pdf: 1190266 bytes, checksum: 5fb8d7ae60e8775661fb41f33bcb771e (MD5) Previous issue date: 2016-02-29 / CAPEs / A sepse, apesar do melhor entendimento de sua fisiopatologia e da aplicação de novos recursos terapêuticos, diferentes parâmetros clínicos e laboratoriais podem ser associados à mortalidade de pacientes criticamente enfermos, dentre eles as citocinas, como por exemplo, as interleucinas (ILs). Concentrações basais mais elevadas de IL-6 e IL-10, além de polimorfismos genéticos nesses genes estão associadas à evolução desfavorável de disfunção orgânica. O objetivo deste estudo foi avaliar os polimorfismos genéticos das ILs 6 (-634C>G) e 10 (-1082G>A), correlacionando-os aos níveis de biomarcadores inflamatórios e escores prognósticos (APACHE II e SOFA) em pacientes diagnosticado com sepse na UTI do Pronto Socorro Cardiológico de Pernambuco, Recife. Um total de 120 pacientes (incluindo 70 pacientes com sepse e 50 controles) foram analisados. Os níveis de citocinas no plasma foram determinados por citometria de fluxo com kit CBA Th1/Th2. Os Polimorfismos IL10-1082G>A e IL6-634C> G foram estudados por meio de análise PCR-RFLP. Os escores de prognóstico e os níveis de interleucinas foram comparados entre os grupos de pacientes com diferentes genótipos IL6 e IL10. Entre os pacientes incluídos no estudo, o grupo com sepse mostrou pontuações mais altas no APACHE II e SOFA e maiores níveis séricos de IL-6 e IL-10. Os polimorfismos avaliados nas regiões promotoras IL6 e IL10 não tiveram efeito sobre os níveis séricos destas citocinas e não estavam associados à sepse ou com os escores, exceto o alelo G de IL10(-1082) que foi associado com valor de SOFA superior a 7. Portanto, o APACHE II e SOFA foram bons indicadores de mortalidade. Níveis mais elevados de IL-6 e IL-10 foram observados nos pacientes com sepse. Neste estudo, embora o polimorfismo IL6 (-634C> G) não demonstrou estar envolvido no processo de agravamento da sepse, o alelo G do gene IL10 (-1082G>A) agiu como um fator de risco quando relacionado com o escore SOFA. No entanto, como esses genes são muito polimórficos, é necessário estudos mais amplos de associação de escores prognósticos incluindo outros polimorfismos. / Sepsis, despite the better understanding of its pathophysiology and application of new therapeutic resources, different clinical and laboratory parameters can be associated with mortality in critically ill patients, including cytokines, such as interleukins (ILs). Higher baseline levels of IL-6 and IL-10, besides genetic polymorphisms in these genes are associated with unfavorable outcomes organ dysfunction. The aim of this study was to evaluate the genetic polymorphisms of ILs 6 (-634C>G) and 10 (-1082G>A), correlating the levels of inflammatory biomarkers and prognostic scores (APACHE II and SOFA) in patients diagnosed with sepsis in Ready ICU Cardiac Emergency of Pernambuco, Recife. A total of 120 patients (including 70 patients with sepsis and 50 controls) were analyzed. Levels of cytokine in plasma were determined by flow cytometry with the CBA Th1 / Th2 kit. The polymorphisms IL10-1082G>A and IL6-634C>G were studied by PCR-RFLP analysis. The prognostic scores and interleukins levels were compared between groups of patients with different genotypes IL6 and IL10. Among the patients included in the study, the group with sepsis showed higher scores on the APACHE II and SOFA and higher serum levels of IL-6 and IL-10. Polymorphisms evaluated in the promoter regions of IL-6 and IL-10 had no effect on serum levels of these cytokines and were not associated with sepsis or with the scores except the G allele of IL-10 (-1082) who was associated with SOFA value than 7. Therefore, the APACHE II and SOFA scores were good indicators of mortality. Higher levels of IL-6 and IL-10 were observed in patients with sepsis. In this study, although the IL6 polymorphism (-634C>G) has not demonstrated to be involved in the process of worsening sepsis, the G allele of the IL10 gene (-1082G>A) acted as a risk factor when related to the SOFA score. However, as these genes are very polymorphic, we need larger studies of association of prognostic scores including other polymorphisms.

Orientadores: Sandra Cecilia Botelho Costa, Maria Elena Guariento / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-15T05:01:43Z (GMT). No. of bitstreams: 1 Caldeira_MarceloHenriqueReis_M.pdf: 3599956 bytes, checksum: 3303daa2040a8322424c8bb12dedc3a9 (MD5) Previous issue date: 2009 / Resumo: Há crescente interesse no entendimento da evolução e da eventual prevenção da Fragilidade, bem como do processo fisiopatológico das doenças desencadeante desta síndrome de importante repercussão funcional. Pesquisas têm demonstrado que a infecção crônica latente por CMVH na população geronte apresenta alta prevalência em idosos frágeis sendo um importante desencadeante de limitações, mas poucos artigos são encontrados ao buscar associação entre esta síndrome e infecção ativa por CMVH. Objetivos: Detectar e comparar a incidência de infecção ativa por CMVH em idosos que apresentem critérios de Fragilidade, utilizando Real-Time -PCR para CMVH, e verificar a correlação entre estes critérios de Fragilidade e a positividade para CMVH. Metodologia: trata-se de um estudo transversal com 55 idosos, acompanhados no Ambulatório de Geriatria e em outros ambulatórios do Hospital de Clínicas da Unicamp, sendo utilizados 04 dos 05 critérios de Fragilidade proposto por Fried e colaboradores (2001); avaliou-se a presença de infecção ativa por CMVH através do Real-Time PCR e posteriormente fez-se à análise dos prontuários desses pacientes. Resultados: o número de pacientes por cada critério de Fragilidade e as médias de medicamentos em uso foram concordantes com a literatura. Relacionando critérios para Fragilidade com o número de medicamentos em uso e número de enfermidades por pacientes, observou-se associação positiva entre esses fatores. Quanto ao número de medicamentos em uso, houve prevalência do sexo feminino; as quatro principais categorias de enfermidades classificadas pelo CID-10, apresentadas pelos pacientes incluídos no estudo foram: doenças do aparelho circulatório (76,47%), doenças endócrinas (47,05%), doenças do sistema osteomuscular e do tecido conjuntivo (45,09%) e doenças metabólicas (33,33%). A reação de amplificação em tempo real das 55 amostras para detecção do CMVH foi negativa em todas amostras, caracterizando a ausência de atividade do CMVH nesta população estudada. Conclusão: mesmo havendo importante associação entre citomegalovirose latente e Síndrome da Fragilidade, neste estudo não foi possível verificar a presença de infecção ativa do CMVH e sua relação com os critérios de Fragilidade estudados; achamos necessária a realização de novos estudos para elucidação da provável associação entre infecção ativa por CMVH e critérios de Fragilidade / Abstract: There is growing interest in understanding the evolution and possible prevention of Frailty, as well as the pathophysiological process of diseases that trigger this syndrome with important functional consequences. Research has shown that chronic latent HCMV in the elderly population is highly prevalent in the frail elderly and is an important trigger of limitations, but few articles are found to seek an association between this syndrome and active infection by HCMV. Objectives: To determine and compare the incidence of HCMV active infection in the elderly presenting Frailty's criteria, using Real-Time PCR for HCMV, and verify the correlation between these criteria of Frailty and positivity HCMV. Methodology: This is a cross-sectional study with 55 individuals, followed at the Department of Geriatrics and other outpatient clinics of the Hospital de Clinicas da Unicamp, being used 04 of the 05 Frailty's criteria proposed by Fried and colleagues (2001), we evaluated the presence of active infection through Real-Time PCR and subsequently became the analysis of records of these patients. Results: The number of patients for each criterion of Frailty and the averages of medications were consistent with the literature. Frailty's criteria relating to the number of medications and number of illness for patients, we observed a positive association between these factors. The number of medications in use were females prevalence, and the four main categories of diseases classified by ICD-10, presented by the patients included in the study were: cardiovascular diseases (76.47%), endocrine disorders (47, 05%), diseases of the musculoskeletal system and connective tissue (45.09%) and metabolic diseases (33.33%). The amplification reaction in real time from 55 samples for detection of HCMV was negative in all samples, characterizing the absence of HCMV activity in this population. Conclusion: Even with significant association between latent cytomegalovirus infection and Frailty syndrome, in this study was not possible to verify the presence of HCMV active infection and its relation to the criteria of Frailty studied, we find necessary to carry out further studies to elucidate the possible association between active infection by HCMV and Frailty criteria / Mestrado / Gerontologia / Mestre em Gerontologia

O osso alógeno fresco-congelado (FFBA, do inglês fresh-frozen bone allograft) é uma alternativa para os procedimentos cirúrgicos de enxerto ósseo, principalmente na preparação do rebordo alveolar para a instalação de implantes osseointegráveis. No entanto, existem alguns paradigmas que envolvem a relação entre resposta do sistema imunológico à aloantígenos presentes no enxerto e o seu comportamento clínico. Procurando entender essa relação, o FFBA foi avaliado como enxerto para preservar o rebordo alveolar pós-extração. Os resultados mostraram que embora tenha ocorrido uma redução estatisticamente significante na altura, espessura e volume do rebordo entre a avaliação inicial e final, essa redução não foi clinicamente significante, permitindo a instalação de implantes osseointegráveis. Em adição, as análises histológicas sugerem um bom comportamento do enxerto, com ausência de reação do tipo corpo estranho e formação de novo osso em todos os sítios analisados. Ao analisar o comportamento da resposta imune, os resultados mostraram que a injeção intradérmica de aloantígenos presentes no FFBA, não induziu uma reação de hipersensibilidade tardia nos pacientes após 4 meses do enxerto. Além disso, os monócitos do sangue periférico (PBMCs) dos pacientes não proliferaram frente aos aloantígenos in vitro. No entanto, os dados também demonstraram que os aloantígenos aumentam a produção de IL-2 e IFN-, mas não alteram a produção de IL-4 e IL-10, por PBMCs dos pacientes. Ao avaliar a relação entre a produção dessas citocinas e o comportamento clínico do enxerto, os dados mostram que existe uma correlação estatisticamente significante entre a produção de IL-2 in vitro e a redução (em %) da altura do rebordo alveolar, embora essa redução não tenha sido clinicamente significante. De fato, a presença de aloantígenos no FFBA não é suficiente para sua contraindicação como material de enxertia. / The fresh-frozen bone allograft (FFBA) is an alternative to surgical procedures of bone grafts, mainly in the preservation of alveolar ridge prior the installation of osseointegrated implants. However there are paradigms that surround the relation between immune response to alloantigens present inside the graft and the clinical response of the graft. An attempt to understand this relationship, the FFBA was evaluated as a graft to preserve the alveolar ridge post-extraction. The results show a statistically significant reduction in height, thickness and volume of the ridge between the initial and final examination, however this reduction was not clinically significant. The ridge preservation allowed implant installation and osseointegration. In addition, histologic analysis suggests a good performance of the graft with no foreign body reaction and formation of new bone at all sites. In analyzing the behavior immune response, the results showed that stimulation with alloantigens present in bone allograft induced no delayed hypersensitivity reaction in vivo. Additionally, periphery blood mononuclear cells (PBMC) from patients no proliferate in response to alloantigens in vitro. However, the data also demonstrated that the alloantigens increase IL-2 and IFN- production, but no IL-4 and IL-10 production, by PBMCs from patients. When evaluate the relation between the cytokines production and clinical parameters, the results demonstrate that there statistically significant correlation between IL-2 production in vitro and ridge height changes (%), although this clinical parameter is not clinically significant. In fact, the alloantigens in FFBA are not sufficient for its contraindications as grafting material.

Orientador: Egle Cristina Couto de Carvalho / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T11:06:18Z (GMT). No. of bitstreams: 1 Guimaraes_JoaoAgripino_M.pdf: 1627287 bytes, checksum: b5aedfb0c0a3b2d807c0d3324ae57317 (MD5) Previous issue date: 2012 / Resumo: Contexto e objetivo: Alterações imunológicas podem estar associadas à infertilidade e endometriose. O objetivo deste estudo foi avaliar se, na infertilidade inexplicada e na infertilidade associada à endometriose, ocorre polarização da resposta imunológica Th1ou Th2. Tipo de estudo e local: Foi realizado um estudo de corte transversal no Centro de Atenção Integral a Saúde da Mulher (CAISM), Universidade de Campinas (Unicamp), Campinas, São Paulo, Brasil, de janeiro a junho de 2010. Métodos: 142 mulheres foram alocadas em três grupos (52 com infertilidade inexplicada, 38 inférteis com endometriose e 52 férteis) para dosagem de citocinas Th1 (interleucina 2, fator de necrose tumoral-ALFA' e do interferon-y ) e citocinas Th2 (interleucinas 4 e 10). Os dados foram descritos através de média, desvio-padrão e mediana, e através de frequências absolutas e relativas. Os grupos foram comparados em relação às variáveis através dos testes de qui-quadrado ou exato de Fisher. As citocinas foram comparadas entre os grupos através do teste de Kruskal-Wallis; em caso de diferença significativa, foram feitas comparações dois a dois através do teste de Mann-Whitney e aceitou-se nível de significância 5%. Resultados: As mulheres com infertilidade inexplicada apresentaram menores níveis de interferon-y (p = 0,0012) e maiores níveis de interleucina-4 (p < 0,0001) do que as mulheres férteis, e maiores níveis de interferon-y (p = 0,0001) e de interleucina-4 (p = 0,0005) do que as mulheres inférteis com endometriose. Aquelas com infertilidade primária apresentaram menores níveis de interferon-y e maiores de interleucina-4 que as mulheres férteis. Conclusão: Não foi detectado qualquer tipo de polarização da resposta imunológica Th1 ou Th2 nas mulheres com infertilidade inexplicada ou com endometriose, quando comparadas com mulheres férteis / Abstract: Context and objective: Immunological alterations may be associated with infertility and endometriosis. The aim of this study was to evaluate the polarization of Th1 and Th2 immune response in women with unexplained infertility and infertility associated with endometriosis. Design and setting: A cross-sectional study was performed at the Center for Integral Attention to Women's Health (CAISM) from University of Campinas (UNICAMP), SP, Brazil, from January to June 2010. Methods: 142 women were allocated into three groups (52 with unexplained infertility, 38 infertile women with endometriosis and 52 fertile women) for measurement of Th1 (interleukin 2, tumor necrosis factor-? and interferon-?) and Th2 cytokines (interleukins 4 and 10). Data were reported as mean, standard deviation, median, and by absolute and relative frequencies. The groups were compared with respect to variables using the chi-square or Fisher exact test. Cytokines were compared between groups using the Kruskall-Wallis test and, if significant differences were noted, comparisons were made in pairs by Mann-Whitney test, with 5% significance level. Results: Women with unexplained infertility showed lower levels of interferon-? (p = 0.0012) and increased levels of interleukin-4 (p <0.0001) than fertile women, and higher levels of interferon-? (p = 0.0001) and interleukin-4 (p = 0.0005) than infertile women with endometriosis. Those with primary infertility had lower levels of interferon-? and interleukin-4 over than fertile women. Conclusion: We could not detect any polarization of Th1 or Th2 immune response in women with unexplained infertility or infertility with endometriosis, when compared with fertile women / Mestrado / Saúde Materna e Perinatal / Mestre em Ciências da Saúde

Orientador: Marco Antônio Alvarenga / Resumo: A endometrite persistente pós cobertura (EPPC) é uma das principais causas de infertilidade na égua devido uma inflamação exacerbada uterina pós cobertura/Inseminação Artificial (IA) diminuindo os índices de fertilidade. O uso do meio condicionado por células tronco mesenquimais demonstra grande efetividade na modulação inflamatória, podendo ser uma alternativa no tratamento e EPPC. O objetivo desse trabalho foi avaliar pela primeira vez diferentes concentrações de meio condicionado (MC) por células tronco mesenquimais equinas sobre a cinética espermática equina e capacidade imunomodulatória do MC na inflamação uterina pós IA em éguas resistentes e susceptíveis assim como sua fertilidade. Foram realizados 2 experimentos. No experimento 1 foi avaliado o efeito da adição de diferentes concentrações de MC sobre a cinética e integridade espermática equina. No Experimento 2 foi avaliado in vivo a capacidade moduladora do MC sobre o processo inflamatório endometrial de éguas resistentes e susceptíveis após inseminação artificial. O MC alterou o VCL (P<0,05) porém não alterou o restante dos parâmetros da cinética espermática (P>0,05). A integridade de membrana plasmática do sêmen equino não foi alterada (P<0,05). O MC reduziu a porcentagem de células polimorfonucleares (PMN) em éguas resistentes 6 horas após a inseminação artificial (P<0,05). Nas éguas susceptíveis o MC diminuiu as porcentagens de PMN e fluido intrauterino 6 e 24 horas após IA (p<0,05), com incremento na fertilidade... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Persistent mating-induce endometritis (PMIE) is a major cause of subfertility in the mare due to exacerbated uterine inflammation post breeding, so therapies have been studied to immunomodulate the inflammatory response. The objective of this work was to evaluate different concentrations of conditioned medium (MC) by equine mesenchymal stem cells on equine sperm kinetics and MC immunomodulatory capacity in uterine inflammation after breeding in resistant and susceptible mares as well as their fertility. Two experiments were performed. In experiment 1 the effect of the addition of different MC concentrations on the equine sperm kinetics and integrity was evaluated. In Experiment 2, the modulatory capacity of MC on the endometrial inflammatory process of resistant and susceptible mares after artificial insemination was evaluated, as well as its effect on conception rates. The MC did not significantly alter sperm kinetics and plasma membrane integrity of equine semen. Even in resistant mares MC reduced the percentage of neutrophils 6 hours after artificial insemination (p <0.05), not negatively interfering with fertility. In susceptible mares, MC decreased the percentages of neutrophils and intrauterine fluid 6 and 24 hours after AI (p <0.05). It was concluded that equine MC does not negatively affect the quality of equine semen or its fertility, modulating the inflammatory response after coverage in resistant and susceptible mares. / Mestre

The gut microbiota and its metabolites – namely short chain fatty acids (SCFAs) – interact with the digestive, immune, and nervous systems. Microbiota with disrupted composition are highly associated with obesity, gastrointestinal symptoms, and chronic inflammation. Levels of SCFAs in the feces can represent dynamics of the microbiota, and they represent one mechanism by which the microbiota interacts with its host. This study aimed to further our understanding of associations between microbiota bacterial diversity and SCFAs, immune markers, BMI, and GI symptoms and to identify bacteria that are differentially abundant in different BMI groups and with synbiotic supplementation. Data (SCFAs, immunoglobulins, body mass index, fecal fiber, fecal protein, measures of GI symptoms, and 16s RNA sequences, n=11) was extracted from a randomized control trial investigating the effects of synbiotic supplementation in non-celiac gluten-sensitive participants. QIIME2 was used to process 16s RNA data, analyze quantitative, qualitative, phylogenetic quantitative, and phylogenetic qualitative measures of alpha and beta diversity and to perform an analysis of composition of microbiomes (ANCOM) for identification of differential abundances. Multiple metrics of alpha diversity were found to significantly correlate with IgG4, IgM, IL-2, acetate, propionate, isobutyrate, valerate, isovalerate, caproate, heartburn, urgent need to defecate, and feelings of incomplete evacuation. Multiple metrics of beta diversity were significantly different between normal and overweight, normal and obese, and overweight and obese BMI classification groups. Beta diversity was also found to significantly correlate with IgG1, IgG3, IgG4, IgA, IL-6, IL-8, fecal fiber, propionate, butyrate, heartburn, acid regurgitation, nausea and vomiting, bloating, abdominal distension, increased gas, and eructation. The synbiotic intervention did not significantly alter alpha or beta diversity. An ANCOM identified bacterial taxa differentially abundant with BMI shifts and synbiotic supplementation, though these taxa were not those included in the synbiotic. Findings demonstrate alpha and beta diversity associations with various SCFAs, GI symptoms, immune markers, and BMI, and the results of the placebo-controlled intervention suggest careful consideration of placebo contents moving forward. This research supports plans to apply analysis to larger sample sizes to elucidate changes microbial profiles that are associated with clinically relevant biomarkers and symptoms.

Previous reports indicate that anti-tumor activity arising from systemically injected P. acnes is macrophage-mediated, whereas anti-tumor activity arising from locally injected P. acnes is T cell-mediated. It is possible these P. acnes-induced cytotoxic T cells arise via the Interleukin cascade. Therefore, this study investigated the involvement of Interleukin 2 (IL 2) and Interleukin 3 (IL 3), known components of the Interleukin cascade, in local P. acnes-mediated anti-tumor action. A 500 ug dose of heat-killed stationary phase P. acnes given simultaneously with 10⁴ tumor cells was found to inhibit tumor formation completely, therefore this amount was used as a standard dose throughout the study. Unvaccinated counterparts developed palpable tumors two weeks after tumor cell administration. Lower doses of vaccine protected animals from tumor growth to a lesser degree. A vaccine prepared from logarithmic phase P. acnes exerted a moderate anti-tumor effect in some cases. IL 2 and IL 3 levels were measured in vitro in normal BALB/c mice (N), tumor-bearing mice (TBH), normal vaccinated mice (N+V), and mice receiving both tumor cell and vaccine injection (T+V). IL 2 and IL 3 production was maintained in both N and N+V host splenocyte cultures throughout the study. In a similar fashion, levels of IL 2 and IL 3 in T+V host splenocyte cultures were comparable to those of N+V hosts. However, TBH splenocyte production of IL 2 and IL 3 began to decline when tumors became palpable, at Day 14 after tumor cell inoculation. By Day 28, TBH IL 2 and IL 3 levels were <15% of normal control levels. Causes for this suppression of IL 2 and IL 3 production in TBH were examined. From reports of others it appeared that suppression may be mediated through prostaglandin(s). Addition of the prostaglandin inhibitor indomethacin to splenocyte cultures greatly enhanced IL 2 production by N, N+V and T+V splenocytes, but failed to restore IL 2 production in TBH splenocyte cultures to normal levels. Thus, it appeared prostaglandins were not directly responsible for the majority of suppression seen in TBH. In the non-tumor-burdened host, prostaglandin appeared to play a homeostatic role regarding IL 2 production. Indomethacin-treatment had little effect on IL 3 production. Nylon wool fractionation of N, TBH, N+V and T+V splenocytes suggested a cell removed by nylon wool treatment was largely responsible for the suppression of IL 2 and IL 3 production in TBH. No obvious presence of functional suppressor cells was noted in N, N+V or T+V splenocytes. From these results, it appeared that P. acnes administration maintains and/or restores IL 2 and IL 3 production, thus favoring the production of CTL. In addition, the suppression of IL 2 and IL 3 production seen in TBH may be due to a nylon wool adherent suppressor cell. A model describing the effect of P. acnes administration on local anti-tumor activity was presented. / Master of Science

Besides the molecular mechanisms regulating activation of FLS mentioned above, we also investigated anti-inflammatory activities of Chinese herbal medicine sinomenine and Liang Miao San on activated human FLS in RA. Sinomenine, an alkaloid isolated from the root of Sinomenium acutum, has been used to alleviate the symptoms of rheumatic diseases. Liang Miao San (LMS), composed of the herbs Rhizoma Atractylodis (Cangzhu) and Cotex Phellodendri (Huangbai), is another traditional Chinese medicine formula for RA treatment. Since the potential anti-inflammatory activities of sinomenine and LMS have been demonstrated, we investigated the in vitro anti-inflammatory effects of sinomenine and LMS on inflammatory cytokine TNF-alpha activation of human normal and RA-FLS and the underlying intracellular mechanisms. In the present study, sinomenine was found to significantly inhibit TNF-alpha induced cell surface expression of VCAM-1 and release of inflammatory cytokine and chemokine IL-6, CCL2 and CXCL8 from both normal and RA-FLS (all p &lt; 0.05). Our results provide a new insight into the differential anti-inflammatory activities of sinomenine and LMS through the suppression of TNF-alpha activated FLS by modulating distinct intracellular signaling pathways in RA, and help to provide a biochemical basis for the development of a cost-effective human synoviocyte model for the future screening of traditional Chinese medicine (TCM) possessing potential anti-rheumatic activities. (Abstract shortened by UMI.) / IL-27, a novel member of the IL-12 family that is produced early by antigen-presenting cells (APCs), can promote T cell proliferation as well as the production of interferon-gamma by naive T lymphocytes. Recent studies have found that elevated expression of IL-27 has been detected in the synovial membranes and fluid of RA. Herein we investigated the in vitro effects of IL-27, alone or in combination with inflammatory cytokine TNF-alpha or IL-Ibeta on the pro-inflammatory activation of human primary FLS isolated from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also studied. We found that the plasma concentration of IL-27 in RA patients (n=112) was significantly higher than that in control subjects (n=46). Both normal and RA-FLS constitutively express functional IL-27 receptor heterodimer, gp130 and WSX-1, with more potent IL-27-mediated activation of signal transducers and activators of transcription (STAT)1 in RA-FLS. IL-27 was found to induce significantly higher cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 and release of inflammatory cytokine IL-6, chemokine CCL2, CXCL9, CXCL10 and matrix metalloproteinase (MMP)-1 of RA-FLS than that of normal FLS (all p &lt; 0.05). The above findings therefore provide a new insight into the IL-27-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation of RA and may have important therapeutic implications. / In the present study, we have investigated the mechanisms of the activation of human fibroblast-like synoviocytes (FLS) induced by various stimuli including interleukin (IL)-27, tumor necrosis factor (TNF)-alpha and IL-beta. The activation of human FLS was studied in terms of the release of cytokines and chemokines and the expression of adhesion molecules. / We investigated the in vitro effects of uric acid crystals, alone or in combination with inflammatory cytokine TNF-alpha or IL-beta on the pro-inflammatory activation of human FLS from RA patients and normal control subjects, and the underlying intracellular signaling molecules were also determined. In the present study, uric acid crystals were found to result in a significant increase of inflammatory cytokine IL-6, chemokine CXCL8 and MMP-1 from both normal and RA-FLS (all p &lt; 0.05). Moreover, additive or synergistic effect was observed in the combined treatment of uric acid crystals and TNF-alpha or IL-1beta on the release of IL-6, CXCL8 and MMP-1 from both normal and RA-FLS. Further investigations showed that the release of inflammatory cytokine, chemokine and matrix metalloproteinase stimulated by uric acid crystals was differentially regulated by intracellular activation of extracellular signal-regulated kinase (ERK) and JNK pathways. Our results therefore provide a new insight into the endogenous danger signal uric acid crystals-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation, and also provide biochemical basis for the development of new modality for inflammatory rheumatic diseases. / Chen, Dapeng. / Adviser: Wong Chun Kwok. / Source: Dissertation Abstracts International, Volume: 73-04, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 203-240). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

by Li Pok Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 94-114). / Abstracts in English and Chinese. / Acknowledgements --- p.I / Abbreviations --- p.II / Abstract --- p.V / 摘要 --- p.VIII / Publications --- p.XI / Table of contents --- p.XII / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Human Th lymphocytes and their immunopathogenic roles --- p.1 / Chapter 1.1.1 --- Characteristics of Th lymphocytes --- p.1 / Chapter 1.1.2 --- Migration and activation --- p.1 / Chapter 1.1.3 --- Th cell differentiation --- p.2 / Chapter 1.1.4 --- Pathological roles --- p.4 / Chapter 1.2 --- Cytokines as modulator in Th lymphocyte activation --- p.6 / Chapter 1.2.1 --- IL-18 --- p.6 / Chapter 1.2.2 --- IL-25 --- p.7 / Chapter 1.3 --- Surface marker expression in Th lymphocytes --- p.8 / Chapter 1.3.1 --- Adhesion molecules --- p.8 / Chapter 1.3.2 --- Cytokine and chemokine receptors --- p.9 / Chapter 1.3.3 --- Costimulatory molecules --- p.11 / Chapter 1.4 --- Cytokine and chemokine release from Th lymphocytes / Chapter 1.4.1 --- Thl cytokines --- p.13 / Chapter 1.4.2 --- Th2 cytokines --- p.14 / Chapter 1.4.3 --- Chemokines --- p.15 / Chapter 1.5 --- Intracellular signaling pathways in Th lymphocytes --- p.19 / Chapter 1.5.1 --- p38 MAPK pathway --- p.19 / Chapter 1.5.2 --- ERK pathway --- p.20 / Chapter 1.5.3 --- JNK pathway --- p.20 / Chapter 1.5.4 --- NF- k B pathway --- p.21 / Chapter 1.6 --- Pharmacological intervention of signaling pathways --- p.22 / Chapter 1.7 --- Aims and scope of the study --- p.24 / Chapter Chapter 2 --- Materials and Methods / Chapter 2.1 --- Materials --- p.26 / Chapter 2.1.1 --- Blood samples --- p.26 / Chapter 2.1.2 --- Media and reagents for cell culture --- p.26 / Chapter 2.1.3 --- Antibodies for costimulation of Th cells --- p.28 / Chapter 2.1.4 --- Recombinant human cytokines --- p.28 / Chapter 2.1.5 --- "Signaling pathway inhibitors: SB203580, PD98035, SP600125 and BAY117082" --- p.28 / Chapter 2.1.6 --- Monoclonal antibodies and reagents for immunofluorescent staining --- p.29 / Chapter 2.1.7 --- Reagents and buffers for the purification of human Th lymphocytes --- p.31 / Chapter 2.1.8 --- Reagents and buffers for protein array --- p.32 / Chapter 2.1.9 --- Reagents and buffers for Thl/2 cytokine and chemokine detection --- p.32 / Chapter 2.1.10 --- Reagents and buffers for protein extraction --- p.32 / Chapter 2.1.11 --- Reagents and buffers for SDS-polyacrylamide gel electrophoresis --- p.33 / Chapter 2.1.12 --- Reagents and buffers for Western blot analysis --- p.35 / Chapter 2.1.13 --- Reagents and buffers for non-radioactive electromobility shift assay (EMSA) --- p.37 / Chapter 2.1.14 --- Reagents and buffers for cell viability and proliferation assay --- p.39 / Chapter 2.1.15 --- Reagent kit for endotoxin level assay --- p.39 / Chapter 2.1.16 --- Other reagent kits --- p.40 / Chapter 2.2 --- Methods --- p.41 / Chapter 2.2.1 --- Purification of human Th lymphocytes and cell culture --- p.41 / Chapter 2.2.2 --- Measurement of total and allergen-specific IgE concentrations --- p.41 / Chapter 2.2.3 --- Immunophenotyping of cells by flow cytometry --- p.42 / Chapter 2.2.4 --- Protein array --- p.42 / Chapter 2.2.5 --- Quantitative analysis of cytokines and chemokines by flow cytometry --- p.43 / Chapter 2.2.6 --- Quantitative analysis of IFN-γ by ELISA --- p.43 / Chapter 2.2.7 --- SDS-PAGE --- p.44 / Chapter 2.2.8 --- Western blot analysis --- p.44 / Chapter 2.2.9 --- EMSA / gel shift assay --- p.45 / Chapter 2.2.10 --- MTT assay --- p.46 / Chapter 2.2.11 --- Cell proliferation assay --- p.46 / Chapter 2.2.12 --- Endotoxin level assay --- p.47 / Chapter 2.2.13 --- Statistical analysis --- p.47 / Chapter Chapter 3 --- Results / Chapter 3.1 --- Effects of IL-18 and IL-25 on the induction of Thl/2 cytokine and chemokine release from costimulated Th lymphocytes --- p.48 / Chapter 3.1.1 --- IL-18 and IL-25 could up-regulate the protein expression of cytokines and chemokines --- p.48 / Chapter 3.1.2 --- IL-18 but not IL-25 induced the release of IFN-γ and TNF-α --- p.48 / Chapter 3.1.3 --- "IL-18 and IL-25 induced the release of IL-5, IL-6 and IL-10" --- p.49 / Chapter 3.1.4 --- "IL-18 induced the release of IP-10, MIG, RANTES, MlP-lα and IL-8" --- p.49 / Chapter 3.1.5 --- "IL-25 induced the release of IP-10, MIG and RANTES" --- p.49 / Chapter 3.1.6 --- IL-18 and IL-25 did not enhance the proliferation of costimulated Th cells --- p.49 / Chapter 3.2 --- "Effects of IL-18 and IL-25 on the activation of p38 MAPK, ERK, JNK and NF- k B" --- p.58 / Chapter 3.2.1 --- "Costimulation with or without IL-18 and IL-25 could activate p38 MAPK, ERK and JNK" --- p.58 / Chapter 3.2.2 --- Costimulation with or without IL-18 and IL-25 could induce NF- k B activity --- p.58 / Chapter 3.3 --- Effects of inhibitors on the IL-18 and IL-25-induced release of Thl/2 cytokines and chemokines --- p.63 / Chapter 3.3.1 --- "Optimal dosage of SB203580, PD98035, SP600125 and BAY117082" --- p.63 / Chapter 3.3.2 --- "SB203580, PD98035 and BAY 117082 but not SP600125 suppressed the IL-18 and IL-25-induced release of Thl/2 cytokines" --- p.63 / Chapter 3.3.3 --- SP600125 suppressed the IL-18 and IL-25-induced release of chemokines --- p.64 / Chapter 3.4 --- Effects of inhibitors on the cell surface expression of IL-18 and IL-25 receptors --- p.72 / Chapter 3.4.1 --- "SB203580, PD98035, BAY 117082 but not SP600125 could suppress IL-18 receptor on costimulated Th cells" --- p.72 / Chapter 3.4.2 --- "SB203580, SP600125, PD98035 and BAY 117082 could not suppress IL-25 receptor on costimulated Th cells" --- p.72 / Chapter 3.5 --- Effects of costimulation on the expression of cell surface markers on Th lymphocytes --- p.75 / Chapter Chapter 4 --- Discussion / Chapter 4.1 --- Effects of IL-18 and IL-25 on the release of Th1/2 cytokines and chemokines --- p.80 / Chapter 4.2 --- "Regulation of Thl/2 cytokines and chemokines through intracellular p38 MAPK, ERK, JNKand NF-kB" --- p.83 / Chapter 4.3 --- Effects of costimulation on different surface markers in Th cells --- p.87 / Chapter 4.4 --- Concluding remarks and future perspectives --- p.90 / References --- p.94

INTRODUÇÃO: A Pitiríase liquenoide (PL) é doença cutânea de etiologia desconhecida. Foi recentemente classificada no grupo das discrasias linfóides de células T. Excetuando-se os estudos sobre as características fenotípicas e moleculares dos linfócitos T na PL, trabalhos relativos aos demais componentes da resposta tecidual cutânea nesta doença são escassos. MÉTODOS: Biopsias de 34 pacientes com diagnóstico clínico e histopatológico de PL foram classificadas de acordo com características histopatológicas nos grupos de pitiríase liquenóide aguda (PLA) (n = 15) e crônica (PLC) (n = 19), e submetidas a técnica imunoistoquímica para demonstração de células de Langerhans, dendrócitos dérmicos fator XIIIIa+, expressão da forma induzida da óxido nítrico sintase (iNOS), fator de necrose tumoral alfa (TNFalfa), interferon gama (IFNy) e interleucinas (IL) 12 e 10. Fez-se a comparação dos resultados obtidos entre os grupos de PLA e PLC. A expressão de iNOS foi também comparada com grupo controle de pele normal (n = 10). RESULTADOS: A população de células de Langerhans epidérmicas foi menor no grupo de PLA. O número de dendrócitos dérmicos fator XIIIa+ não diferiu entre os grupos. Foi observada expressão epidérmica e dérmica de iNOS em ambos os grupos de PL. Três espécimes do grupo controle de pele normal apresentaram fraca expressão de iNOS epidérmica e dérmica. O grupo de lesões de PLA mostrou maior expressão dérmica de TNFalfa e IFNy. A depleção de células de Langerhans epidérmicas foi acompanhada de maior expressão epidérmica de TNFalfa e IL-10. Houve correlação entre a expressão de iNOS e a população de dendrócitos dérmicos fator XIIIa+. CONCLUSÕES: Na PLA a população de células de Langerhans é menor que na PLC e se correlacionou com maior expressão epidérmica de TNFalfa e IL-10. Não houve diferenças na população de dendrócitos dérmicos fator XIIIa+ nos dois grupos de lesão. Demonstrou-se, pela primeira vez, expressão epidérmica e dérmica de iNOS nas lesões de PL. A expressão de iNOS dérmica correlacionou-se com a população dendrocítica Fator XIIIa+. Houve correlação entre a expressão de TNFalfa e de IFNy com as alterações inflamatórias da PLA. Houve correlação negativa entre a expressão dérmica de IL-12 e IL-10 nas lesões da PL. No espectro da resposta tecidual da PL participam as células dendríticas da pele, em ambiente de padrão imunológico Th1 predominante, com conseqüente indução da expressão de iNOS nos sítios de lesão. / BACKGROUND: Pityriasis lichenoides (PL) is a cutaneous disease of unknown etiology which has been regarded as an immunologically mediated reaction. Recently, it was reclassified in the group of Cutaneous Lymphoid T cell Dyscrasia. There are few reports addressing mainly T cell subsets in PL tissue reaction. METHODS: Skin biopsies taken from 34 patients with confirmed diagnosis of PL where classified as pityriasis lichenoides et varioloformis acuta (PLEVA) (n = 15) and pityriasis lichenoides chronica (PLC) (n = 19) according to histopathological features. The skin biopsies where subjected to immunohistochemical technique to demonstrate Langerhans cells, Factor XIIIa+ dermal dendrocytes, inducible nitric oxide synthase (iNOS) expression, tumor necrosis factor alfa (TNFalfa), interferon gama (IFNy) and interleukins (IL) 12 and 10. The ensuing results were compared among the two PL groups. The iNOS results in PL group were also compared to a normal skin control group (n = 10). RESULTS: In PLEVA lesions, there was a decrease in Langerhans cells population when compared to PLC lesions. The factor XIIIa+ dermal dendrocytes number did not differ among PL groups. There was a strong epidermal and dermal iNOS expression in both PL groups. A faint iNOS expression was observed in three specimens of the control group. A higher TNFalfa and INFy expression was observed in PLEVA lesions. The Langerhans cells decrease observed in those lesions was accompanied by higher TNFalfa and IL-10 expression. There was a significant correlation between factor XIIIa+ dermal dendrocytes population and dermal iNOS expression. CONCLUSIONS: PLEVA lesions displayed a decrease in Langerhans cells number, accompanied by higher TNFalfa and IL-10 expression. There was no difference in the amount of factor XIIIa+ dermal dendrocytes in PLEVA and PLC lesions. The study demonstrated, for the first time, the iNOS expression in PL lesions. The factor XIIIa+ dermal dendrocytes population correlated to dermal iNOS expression. TNFalfa and INFy expression correlated to inflammatory alterations observed in PLEVA lesions. There was a negative correlation between IL-12 and IL-10 expression in PL lesions. Dendritic cells participate in the PL spectrum of tissue reaction which is characterized by predominant TH1 cytokines milieu that favors iNOS expression.