Many inflammatory diseases are growing increasing common in the aging society of Taiwan. Inflammation cascades can cause diseases such as rheumatoid arthritis, osteoarthritis, chronic asthma, multiple sclerosis, and so on. The clinically used anti-inflammatory drugs have many side effects and are expensive. Therefore, it is imperative that we find alternatives to these drugs. Marine natural compounds offer great hope in the development of drugs for treating inflammatory diseases. In the present study, we found that Chao-10, which is a marine-derived compound isolated from Formosan soft coral, significantly inhibited the expression of the pro-inflammatory protein, inducible nitric oxide synthase (iNOS), in the lipopolysaccharides (LPS)-stimulated RAW 264.7 macrophage cell line. We suggest that Chao-10 may serve as a potential new anti-inflammatory agent. However, the mechanism by which the anti-inflammatory effects of Chao-10 are mediated is yet unclear. Therefore, we performed two-dimensional electrophoresis (2-DE) to investigate the regulatory mechanism for the anti-inflammatory effect of Chao-10. We isolated some proteins that may be involved in the anti-inflammatory mechanism of Chao-10. In addition, we used immunoprecipitation to find that nucleophosmin (NPM) could interact with nuclear factor kappa B (NF-£eB). Therefore, we hypothesize that nucleophosminmay be involved in the regulation of NF-£eB to enhance the down-regulation of iNOS proteins. In summary, the anti-inflammatory effects of Chao-10 are probably mediated through the some other signaling pathway. Importantly, Chao-10 not only offers some new biomarkers of inflammation but also provides an encouraging outlook on therapeutic approaches.

Le virus de l'hépatite B (HBV) infecte chroniquement plus de 250 millions de personnes. Des traitements existent permettant de contrôler la production de particules infectieuses. Cependant, aucun des traitements actuels ne permet d'éradiquer complètement l'infection. Il est donc nécessaire de développer de nouvelles stratégies thérapeutiques, incluant des approches immunothérapeutiques pour permettre un meilleur contrôle immunologique des infections HBV. Dans une étude récente menée au sein du laboratoire, il a été montré que l'IL-1ß est la cytokine ayant le plus fort effet antiviral contre la réplication d'HBV dans les hépatocytes. Dans le foie, la cytokine IL-1ß est principalement produite par les macrophages résidents (les cellules de Kupffer) ou infiltrant (monocytes inflammatoires différenciés en macrophages). De nombreuses études récentes ont montrés qu'HBV était capable de bloquer partiellement l'induction des réponses immunitaires innées. Il est donc important de déterminer si HBV est capable d'empêcher la production d'IL-1ß par les différents types de macrophages. L'objectif de cette thèse était d'étudier l'effet du virus sur le phénotype des macrophages et les implications de ces modifications phénotypiques sur l'établissement de l'infection dans les hépatocytes. Des monocytes du sang ou des macrophages du foie ont été purifiés, respectivement, du sang périphérique ou de résections hépatiques, et ont été exposés au virus pendant leur différentiation et/ou leur activation pour les monocytes, ou seulement pendant leur activation pour les macrophages hépatiques déjà différenciés. Il a été démontré que le virus de l'hépatite B est capable d'inhiber la sécrétion d'IL-6 et d'IL-1ß par les macrophages pro-inflammatoires. De plus, HBV est capable d'inhiber la sécrétion d'IL-1ß par les macrophages hépatiques stimulés par différents ligands. Finalement, les surnageants de macrophages pro-inflammatoires sont capables de bloquer l'établissement de l'infection, ce qui n'est pas le cas quand les macrophages ont été exposés au virus. Il apparait donc qu'HBV est capable de modifier le phénotype des macrophages pour favoriser l'établissement et la persistance de l'infection. La compréhension des mécanismes de subversion du phénotype des macrophages par le virus de l'hépatite B serait un premier pas vers le développement de nouvelles stratégies thérapeutiques / Hepatitis B virus (HBV) chronically infects over 250 million people worldwide. Several treatments can be used to prevent the formation of de novo particles. However, they do not allow the total eradication of the infection. Therefore, it is necessary to develop new therapeutic strategies, including immune-therapeutic ones, which would be more likely to lead to an immunological control of HBV infections. We have recently shown that IL-1ß is the most effective antiviral cytokine against the replication of HBV in vitro. In the liver, IL-1ß is mainly produced by resident macrophages (also called Kupffer cells) or infiltrating cells (inflammatory monocytes differentiated into macrophages). Recent studies have shown that HBV is able to partially inhibit the induction of innate immune responses. Hence, it was necessary to determine if HBV was also able to block the production of IL-1ß by the different types of macrophages.The objective of this thesis was to study the effect of HBV on macrophage phenotypes and the impact of those modifications on the establishment of HBV infection in hepatocytes.Blood monocytes and liver macrophages were purified, respectively, from peripheral blood or hepatic resections, and were exposed to HBV during their differentiation and/or activation for monocytes, or only during activation for liver macrophages which are already in a differentiated state. HBV was able to partially inhibit the secretion of IL-6 and IL-1ß by pro-inflammatory macrophages. Moreover, HBV was able to inhibit IL-1ß secretion by liver macrophages stimulated by different ligands and, conditioned medium of pro-inflammatory macrophages could inhibit the establishment of infection in hepatocytes. This effect was reverted when macrophages were exposed to HBV, concomitantly with a lower production of IL-6 and IL-1ß.In summary, HBV is able to modify macrophage phenotypes to favour the establishment and persistence of HBV infection. The full understanding of the mechanistic basis of how HBV phenotypically modifies macrophages will be a first step towards the development of new therapeutic strategies

Made available in DSpace on 2014-06-11T19:27:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T20:57:18Z : No. of bitstreams: 1 ramos_bra_me_botfm.pdf: 447116 bytes, checksum: 446e118d63a97d1a69658b7e651b69aa (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Microbial invasion of the amniotic cavity has been described in term deliveries and its role on the immune modulation is of interest to the better understanding of the underlying labor processes. The aim of this study was to determine the prevalence of Mycoplasma hominis and Ureaplasma urealyticum in the amniotic fluid of term pregnancies and to evaluate its influence on cytokines production at the end of pregnancy. A cross sectional study was conducted with fifty five pregnant women out of labor with intact membranes and gestational age between 37 and 41 weeks seen at the Bom Jesus hospital in Ariquemes, Rondônia, between June 2009 and May 2010. Amniotic fluid samples and fragments of chorioamniotic membranes were collected at cesarean section. M. hominis and U. urealyticum detection was performed by PCR and Interleukin (IL)-1β, IL-6, IL-8, IL-10 and Tumor Necrosis Factor (TNF)- levels were determined by ELISA. Chorioamniotic membranes were submitted to histopatological analyses. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not shown detectable concentrations of TNF- and IL-1β. The median concentration of IL-6 and IL-8 were 107.9 pg/mL (0-517.1) and 208.1 pg/mL (0-1897.4), respectively. Interleukin-1, IL-6, IL-8 and TNF- concentrations were not associated with the presence of M. hominis in amniotic fluid, regardless the gestational age. No sample had detectable IL-10 levels. The histopatological analyses have shown no chorioamnionitis in any of the membranes, only a discreet mononuclear infiltration in the decidua could be observed in 40.4% of the samples. Presence of M. hominis was detected in 36.4% of amniotic fluid samples and any of them was positive for U. urealyticum. Regarding cytokines levels, 63.6% and 90.9% of samples have not... (Complete abstact click electronic access below)

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Lectinas de plantas podem ser definidas como proteÃnas de origem nÃo imune que possuem pelo menos um domÃnio nÃo catalÃtico que se liga reversivelmente de maneira especÃfica a um mono- ou oligossacarÃdeo. A famÃlia das lectinas de leguminosas representa o grupo destas proteÃnas mais bem estudadas, em especial destaque a subtribo Diocleinae, que possui o representante mais bem caracterizado, a ConA. As lectinas de Diocleinae apresentam um alto grau de similaridade estrutural, porÃm o mesmo nÃo ocorre quanto Ãs atividades biolÃgicas e especificidade a carboidratos variados, o que torna importante a investigaÃÃo em nÃveis estruturais e biolÃgicos dos seus diversos membros, dentre eles a Canavalia grandiflora Benth. A lectina de sementes de Canavalia grandiflora (ConGr) foi purificada de acordo com Ceccato (2001) e foi cristalizada pelo mÃtodo de difusÃo de vapor a 293 K. Cristais foram obtidos em uma condiÃÃo contendo 0,5 M de sulfato de cÃdmio hidratado, 0,1 M de HEPES pH 7.5 e 1,0 M de acetato de sÃdio triidratado. Os cristais apresentam o grupo espacial ortorrÃmbico I222, a cela unitÃria tem como dimensÃes a=67,70 Ã, b=55,90à e c=107,46 à e Ãngulos ?=?=?=90Â, sendo observado um monÃmero na unidade assimÃtrica e um conteÃdo de 42,53% de solvente no cristal. A estrutura foi resolvida a 2,19à e o problema de fase foi solucionado pelo mÃtodo de substituiÃÃo molecular utilizando as coordenadas da Canavalia gladiata como modelo (PDB: 2D7F). O refinamento satisfatÃrio apresentou âRfactorâ e âRfreeâ com 22,6 e 27,4 respectivamente e apenas um resÃduo de aminoÃcido em regiÃo nÃo permitida do Ramachandran. Apesar da alta similaridade estrutural, pequenas mudanÃas na orientaÃÃo de aminoÃcidos chaves, podem ser responsÃveis pela diversidade nos resultado de aplicaÃÃes biolÃgicas como os resÃduos do sÃtio de ligaÃÃo a carboidrato, que apesar de conservados possuem modificaÃÃes na orientaÃÃo espacial. A seqÃÃncia primÃria da lectina de C. grandiflora apresenta grande similaridade com lectinas do mesmo gÃnero, porÃm ela concentra o maior nÃmero de mutaÃÃes representativas do gÃnero Dioclea, caracterizando-o como o subgÃnero de Canavalia mais prÃximo de Dioclea, e dentre as canavalias à a mais primitiva. A ConGr apresentou atividade edamatogÃnica em modelo de edema de pata (s.c.) e efeito relaxante em mÃsculo liso de aortas de ratos endotelizadas, porÃm os efeitos mostram-se fracos frente a outras lectinas de Diocleinae. / Plant Lectins can be defined as non-immune origin proteins having at least one non-catalytic domain that binds reversibly in a specific fashion to a mono-or oligosaccharide. The legume lectin family group represents the best studied of these proteins, in particular highlighted the subtribe Diocleinae, which has the best characterized representative, ConA. Lectins Diocleinae show a high degree of structural similarity, but the same does not regarding biological activities and specificity to different carbohydrates, which makes it important to research in structural and biological levels of its various members, among them the Canavalia grandiflora Benth. The lectin from Canavalia grandiflora (Congr) was purified according to Ceccato (2001) and was crystallized by vapor diffusion method at 293 K. Crystals were obtained in a state containing 0.5 M of cadmium sulfate hydrate, 0.1 M HEPES pH 7.5 and 1.0 M sodium acetate trihydrate. The crystals have the orthorhombic space group I222, the unit cell has the dimensions a = 67.70 Ã, b = 55.90 à and c = 107.46 à and angles? =? =? = 90 Â, giving a monomer in the asymmetric unit and a content of 42.53% solvent in the crystal. The structure was solved to 2.19 à and phase problem was solved by the method of molecular replacement using the coordinates of Canavalia gladiata as a template (PDB: 2D7F). The refinement showed satisfactory "rfactor" and "Rfree" with 22.6 and 27.4 respectively and only one amino acid residue in a region of the Ramachandran disallowed. Despite the high structural similarity, small changes in the direction of key amino acids may be responsible for the diversity in the biological applications as a result of the residues of the carbohydrate binding site which are retained despite changes in spatial orientation. The primary sequence of the lectin from C. grandiflora has great similarity with lectins of the same genus, but it has the largest number of mutations representative of the genus Dioclea, characterizing it as the subgenus Canavalia nearest Dioclea and canavalias is among the most primitive. The Congregation edamatogÃnica showed activity in a model of paw edema (sc) and relaxing effect on smooth muscle of rat aorta endothelial, but the effects appear to be weak compared to other lectins Diocleinae.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / FundaÃÃo de Amparo à Pesquisa do Estado do Cearà / As sementes de Bauhinia bauhinioides Mart, uma espÃcie pertencente à FamÃlia Leguminosae, SubfamÃlia Caesalpinoideae, Tribo Cercideae, possuem uma lectina Galactose/Lactose especÃfica, que aglutina eritrÃcitos de coelho e humanos nativos ou tratados com enzimas proteolÃticas. A lectina de sementes de B. bauhinioides foi purificada por precipitaÃÃo com sulfato de amÃnio seguida por cromatografias de trocas iÃnica em DEAE-Sephacel e em HiTrap SP, essa Ãltima acoplada a HPLC. Esse procedimento resultou na lectina purificada, nomeada BBL. O processo de purificaÃÃo da BBL foi monitorado por SDS-PAGE e observou-se que a lectina purificada à caracterizada por um perfil eletroforÃtico composto por uma banda Ãnica, com massa molecular aparente de aproximadamente 31 kDa, tanto na presenÃa quanto na ausÃncia de um agente redutor. A anÃlise por espectrometria de massas indicou que BBL possui massa molecular de 28310 Da e, por cromatografia de exclusÃo molecular, observou-se que a lectina, na sua forma nativa, parece assumir uma estrutura tetramÃrica. A lectina de sementes de B. bauhinioides nÃo à uma glicoproteÃna e demonstra elevada estabilidade, sendo capaz de manter sua atividade hemaglutinante em uma ampla faixa de pH, apÃs exposiÃÃo a temperaturas de atà 60 C por 1 hora e apÃs diÃlise contra EDTA. BBL foi testada quanto a atividade prÃ-inflamatÃria utilizando o modelo de edema de pata em ratos, tendo sido observada uma induÃÃo significativa de edema nos animais tratados com BBL na dose de 1,0 mg/kg sendo esta atividade prÃ-inflamatÃria inibida pela incubaÃÃo prÃvia da lectina com 0,1 M de D-galactose. Foi avaliado tambÃm o efeito prÃ-inflamatÃrio da BBL em presenÃa de bloqueadores farmacolÃgicos de dois importantes mediadores inflamatÃrios e observou-se que o L-NAME, um inibidor nÃo-seletivo da enzima Ãxido NÃtrico Sintase, inibiu significativamente o edema causado pela lectina, sugerindo que a atividade prÃ-inflamatÃria causada por BBL envolve a participaÃÃo do oxido nÃtrico. Esses resultados reforÃam a idÃia da utilizaÃÃo de lectinas vegetais como ferramentas biotecnolÃgicas em estudos sobre os mecanismos implicados na resposta inflamatÃria. / A new galactose-specific lectin, named BBL, was purified from seeds of the Caesalpinoideae plant, Bauhinia bauhinioides, by precipitation with solid ammonium sulfate followed by two steps of ion exchange chromatography, and its pro-inflammatory activity was evaluated. The lectin haemagglutinated rabbit and humans erythrocytes (native and treated with proteolytic enzymes), which occurred independent on presence of divalent cations, was stable at 60 ÂC for 60 min, had an optimum pH between pH 8.0 and 9.0 and was inhibited after incubation with D-galactose and its derivatives, especially α-methyl-D-galactopyranoside. The pure protein was found to have a molecular mass of 31 kDa by SDSâPAGE and 28,305 Da by electrospray ionization mass spectrometry. The s.c. injection of BBL induced dose-dependent paw edema at all doses tested, an effect that occurred via carbohydrate site interaction and that was significantly reduced by L-NAME, indicating the important participation of nitric oxide in the late phase of the edema induced by BBL. These findings indicate that this lectin can be used as a tool to better understand the mechanisms involved in inflammatory responses.

The aim of the study was to investigate the association between depression and low bone mineral density (BMD) in premenopausal females. The rationale for the study was that depression is often characterized by cortisol hypersecretion. The role of cortisol includes effects on bone metabolism and the immune system: cortisol is a bone resorption agonist through its support of osteoclastogenesis. The release of pro-inflammatory cytokines, (especially IL-1, IL-6 and TNF-alpha) which induce cortisol secretion, also pushes the balance of bone remodelling in favour of resorption, consequently causing loss of bone mineral density. Significant results have been reported in studies of various groups across the USA, Europe and Asia, indicating a causal role for depression in osteoporosis. However, some studies could not support this association. With both osteoporosis and depression representing growing public health concerns in South Africa, the aim of this study was to examine the association between depression and loss of BMD in a South African sample with varying levels of depression. The study was approached from two starting points: the first used low BMD as the departure point and the second was undertaken from the diagnosis of depression. This was achieved by first investigating women where the primary concern was possible low BMD (referred to as Study 1) and secondly by assessing women whose primary diagnosis was clinically confirmed major depression (Study 2). Study 1 involved investigation of BMD in a volunteer-based sample of 40 premenopausal women drawn from three different sources. All volunteers underwent a DEXA scan, were assessed for depression and supplied saliva for cortisol analysis. Study 2 examined the BMD of five psychiatric patients diagnosed with severe, recurrent major depression and four healthy controls. These volunteers were required to undergo the same testing as subjects in Study 1. In addition, blood and urine samples were taken to examine bone turnover markers (bone specific alkaline phosphate, osteocalcin, urine pyridinoline cross-linked C-telopeptide and deoxypyridinoline). The pro-inflammatory status of the psychiatric patients was compared to reference ranges. The latter served as a small exploratory study and an introduction to further avenues of research. Study 1 revealed no clear general association between depression and bone density on DEXA scores. However, a correlation was found between left femoral neck BMD and depression in those women with low BMD only. Significant differences were found though between subjects with normal and low BMD in terms of body mass index (BMI) and contraception use. Study 2 on the other hand, indicated a trend of association between depression and low BMD: subjects suffering with severe major depression were noted to have lower bone density (on DEXA) and higher bone turnover (as measured by markers of bone turnover) as well as higher cortisol levels than healthy controls. In addition, depressed subjects exhibited elevated IL-1-alpha levels but normal TNF-alpha levels when compared to normative data. In conclusion, the study indicated that the effect of depression on bone density is dependent on the intensity and duration of depression. IL-1-alpha and cortisol may be instrumental in this loss of BMD. / Dissertation (MSc)--University of Pretoria, 2008. / Physiology / unrestricted

Autism spectrum disorder (ASD) is associated with functional abnormalities of the anterior cingulate cortex (ACC), a brain area that mediates social behavior. Given evidence of a role of inflammation in ASD, markers of pro-inflammatory and anti-inflammatory microglia were studied using postmortem ACC tissues from ASD and age-matched typically developed control donors. Gene expression levels of pro-inflammatory (CD68, HLA-DRA, IL1B, NOS2, PTGS2) and anti-inflammatory (ARG1, IGF1, MRC1, PPARG) microglial genes were measured using quantitative real-time PCR. Additionally, brain sections were immunohistochemically stained for a microglial marker. Expression levels of IGF1 were modestly higher, while the expression of MRC1 was modestly lower in ASD donors when compared to control donors. No other differences in gene expression levels between the two groups of donors were observed. Statistical significance for changes in expression levels IGF1 and MRC1 did not survive correction for multiple comparisons. Further research on anti-inflammatory microglial involvement in ASD is warranted.

<p>  </p> <p>Osteoarthritis (OA) is prevalent in equine and can be career-ending for performance horses due to lameness limitations and decreased quality of life. OA is a progressive, multifactorial disease that compromises the synovial joints' normal function, resulting in subchondral bone and articular cartilage deterioration over time. OA is a complex disease that impacts the entire joint, wherein activation of the innate immune system has an essential role in the disease progression and the development of pain. The synovial membrane, or the synovium, is a crucial contributor to the inflammation of diseased joints, regardless of the intra-articular tissue type initially affected. Synoviocytes are a predominant cell type of the synovium and contribute to inflammation by releasing key mediators and degradative enzymes, such as interleukin (IL)-6, IL-1β, a disintegrin, and metalloproteinase (ADAM) domains, and matrix metalloproteinases (MMPs). The production of pro-inflammatory molecules sequentially influences the expression of degradative enzymes and cartilage destruction. Therefore, the pathophysiological processes within synovial joints afflicted by OA can be further understood by studying the characteristics of synoviocytes.</p> <p>We aimed to investigate the inflammatory component of OA in an <em>in vitro</em> model using a primary cell line of equine fibroblast-like synoviocytes (eqFLS) stimulated with tumor necrosis factor-alpha (TNF-α) to represent an initial inflammatory stimulus. Our studies have shown that stimulating eqFLS with TNF-α for 24 hours significantly increased the gene expression of pro-inflammatory biomarkers. Among several pro-inflammatory candidate genes assayed, only pro-inflammatory cytokine IL-6 gene expression could be detected reproducibly following stimulation with the TNF-α gene in eqFLS. We characterized the pro-inflammatory response of eqFLS and utilized this system to examine the impact of novel therapeutic compounds designed <em>in-silico</em> with the goal of reducing the inflammatory response of eqFLS. A piperazine-based compound (C3) and its derivative (02-09) were primarily designed to mimic the interactions of the growth factor pigment epithelium-derived factor (PEDF) with its receptor, the non-integrin laminin receptor 1 (LAMR1). Based on previous <em>in vitro</em> studies in the laboratory, C3 and 02-09 had been proposed to have a strong potential for inhibiting inflammation while reducing angiogenesis and chondrocyte hypertrophy. The efficacy of these two novel compounds on eqFLS was examined in the present work by assessing the gene expression levels of inflammatory biomarkers, including IL-6, IL-1β, IL-8, ADAMs, and MMPs relative to a control housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in various study designs. An <em>in-vitro</em> screen with the IL-1β promoter driving a reporter green fluorescent protein (GFP) was also designed to detect and track the inflammatory response of eqFLS by imaging following stimulation with or without (+/-) TNF-α relative to controls. This screen will be utilized in future studies to potentially identify more effective compounds in the LAMR1-interacting series. The current findings suggest that the novel compounds, especially 02-09, might exhibit an anti-inflammatory effect on eqFLS; therefore, it is a potential therapeutic agent in modulating inflammation during OA development. </p> <p><br></p>

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第20690号 / 工博第4387号 / 新制||工||1682(附属図書館) / 京都大学大学院工学研究科都市環境工学専攻 / (主査)教授 高野 裕久, 教授 米田 稔, 准教授 上田 佳代 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

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