Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-10-10T10:45:14Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-10-16T13:33:10Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-10-16T13:33:10Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-29 / A Artrite Reumatóide (AR) é uma doença inflamatória crônica, caracterizada por inflamação das articulações e associada à incapacidade motora e laboral dos pacientes. Tendo em vista que as estratégias atuais para o tratamento da AR podem apresentar sérios efeitos colaterais e nem sempre resultam em melhora clínica, a terapia com laser de baixa potência (LLLT) surgiu como alternativa para o tratamento da artrite, devido às suas propriedades anti-inflamatórias e de regeneração tecidual. Entretanto, os mecanismos anti-inflamatórios da LLLT nos tecidos biológicos ainda não são completamente conhecidos. Assim, o presente estudo teve por objetivo avaliar os mecanismos imunológicos e a participação das metaloproteinases de matriz (MMP) e seu inibidor (TIMP2), na resolução do processo inflamatório articular após o tratamento com LLLT. Para isso, um processo inflamatório foi induzido nas articulações talocrural e subtalar dos membros posteriores de camundongos C57BL/6, através da administração de zymosan na região periarticular. Os animais foram divididos em 4 grupos (n = 8): (ZY) artrite induzida por zymosan e não tratado, (ZY + 3Jcm-2) artrite induzida por zymosan + LLLT em 3Jcm-2, (ZY + 30Jcm-2) artrite induzida por zymosan + LLLT em 30Jcm-2 e (ZY + DEXA) artrite induzida por zymosan e tratado com dexametasona. As condições da LLLT foram: 830nm, 10mW e densidades energéticas de 3Jcm-2 e 30Jcm-2, no modo contínuo de emissão. A irradiação foi realizada durante 4 dias consecutivos, iniciando 5 horas após a indução da inflamação. Os animais foram eutanasiados 24h após a última aplicação da LLLT e as seguintes análises foram realizadas no tornozelo: morfológica, níveis relativos de mRNA de quimiocinas, citocinas, MMPs e TIMP2 por RT-qPCR, quantificação de citocinas por ELISA e quantificação da expressão tecidual de MMP13 e TIMP2 por imunohistoquímica. No linfonodo poplíteo foi realizada análise de citometria de fluxo para identificação e quantificação das populações de leucócitos. As análises morfológicas revelaram a presença de infiltrado celular no tecido conjuntivo adjacente à região periarticular, após a administração de zymosan. Foi observada diminuição dos níveis teciduais de citocinas após LLLT e alteração nos níveis de mRNA de citocinas e quimiocinas com importância na fisiopatologia da AR, diferentemente, dependendo da densidade de energia utilizada. O grupo ZY + 3Jcm-2 demonstrou aumento na maioria das populações de leucócitos analisadas, bem como na expressão de proteínas co-estimulatórias em macrófagos e células dendríticas do linfonodo proximal à inflamação. Adicionalmente, foi observado maior população de células Treg nos grupos LLLT, e no grupo ZY + 30Jcm-2, mais células Treg CD8+ expressando níveis elevados de CD25. Embora LLLT tenha diminuído os níveis de mRNA da maioria das MMPs analisadas e aumentado de TIMP2, foi observado aumento da expressão tecidual de MMP13 e TIMP2 na região inflamatória do grupo ZY + 3Jcm-2. Tendo em vista a totalidade dos resultados, o LLLT em ambas as densidades de energia, apresentou efeitos anti-inflamatórios positivos para o tratamento da AR, uma vez que diminuiu os níveis da maioria das citocinas, quimiocinas e MMPs nos tecidos inflamados, além de modificar o quantitativo e o fenótipo dos leucócitos linfonodais, especialmente de células Treg. / Rheumatoid Arthritis (AR) is a chronic inflammatory disease, characterized by joint inflammation and associated with motor and labor incapacity. Since treatment strategies could present a serious side effects and not always achieved clinic improvement, the Low Level Laser Therapy (LLLT) have being considered as an alternative to arthritis treatment, due to its anti-inflammatory and healing abilities. However, LLLT anti-inflammatory mechanisms in biological tissues is not completely understood. Then, this study aimed to evaluate immunological mechanisms and contribution of matrix metaloproteinases (MMPs) and its inhibitor (TIMP) on inflammation resolution process induced by LLLT. Arthritis was induced in C57BL/6 mice by zymosan injection into periarticular region. Experimental animals were divided in 4 groups (n=8): (ZY) arthritis induced by zymosan and untreated; (ZY + 3Jcm-2) arthritis induced by zymosan and treated with LLLT at 3Jcm-2; (ZY + 30Jcm-2) arthritis induced by zymosan and treated with LLLT at 30Jcm-2; (ZY + DEXA) arthritis induced by zymosan and treated with dexamethasone. LLLT parameters were: 830nm, 10mW energy densities at 3 Jcm-2 e 30 Jcm-2, in continuous wave emission mode. Irradiation sections and dexamethasone treatment were carried out 4 days consecutively, starting 5 hours after arthritis induction. Animals were euthanized 24h after the last treatment section and the following analysis were procedure at ankle: morphological, cytokine mRNA relative levels, chemokines, MMPs and TIMP2 by RT-qPCR, quantification of cytokines tissue expression by ELISA and MMP13 and TIMP2 by imunohistochemistry. In popliteal lymph node, leukocytes populations were identificated and quantificated by flow cytometry. Morphological analysis showed inflammatory infiltration at adjacent connective tissue of joints, after zymosan injection. After LLLT, it was observed cytokines tissue expression decrease and mRNA levels alteration of important cytokines and chemokines in pathophisiology of AR, depending of energy density used. The ZY + 3Jcm-2 group showed an increase leukocytes populations, as well as, higher expression of costimulatory proteins in macrophages and dendritic cells of popliteal lymph node. Additionally, it was observed higher Treg population in LLLT groups than ZY group. More, ZY + 30Jcm-2 showed higher Treg CD8+ population presenting CD25high. LLLT decreased mRNA levels of almost MMPs analyzed and increased of TIMP2, except for ZY + 3Jcm-2 group in which was observed MMP13 and TIMP2 tissue expression increase. Taken togheter, all results showed that LLLT, at both energy densities used, presente positive anti-inflammatory effects for RA treatment, since it decreases the levels of cytokines, chemokines and MMPs at inflammed tissues. LLLT could also alter number and phenotype leukocytes in lymph node, especially Treg cells.

Orientador: Carlos Manuel Giles Antunez de Mayolo / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-08-19T11:26:29Z (GMT). No. of bitstreams: 1 Souza_ThaisDiniz_M.pdf: 31446941 bytes, checksum: 75f1c0838febdfa063257bf308ddba57 (MD5) Previous issue date: 2011 / Resumo: Esta dissertação de mestrado teve como objetivo realizar imagens por contraste de fase de amostras in vitro de articulações e cartilagens utilizando duas técnicas: a Imagem Realçada por Difração (IRD) e o Método da Propagação. O sistema para a IRD foi montado no Laboratório Nacional de Luz Síncrotron (XRD2-LNLS), utilizando uma fonte de radiação síncrotron. O sistema para o Método da Propagação foi montado no Laboratório de Cristalografia Aplicada e Raios X (LCARX-UNICAMP), utilizando uma fonte de raios X microfoco, instalada dentro de uma cabana experimental blindada com chumbo. As amostras utilizadas foram corpos de provas e amostras de cartilagens e articulações fornecidos pelo grupo do Prof. Dr. William Dias Belangero da Faculdade de Ciências Médicas (FCM-UNICAMP). No Laboratório de Cristalografia foi realizado todo o processo de montagem e instalação da estação experimental com a fonte de raios X microfoco. Nessa estação foram realizadas imagens pelo método convencional e pelo método da propagação. No mesmo laboratório foi também realizado um estudo de caso em colaboração ao grupo da FCM, usando casos clínicos de implantes de próteses metálicas em articulações, estudou-se a regeneração do tecido ósseo após a lesão para fixação da prótese, utilizando amostras in vitro de patas lesionadas de ratos. Por último foram realizadas aquisições tomográficas no método convencional e de propagação de um joelho de rato e de um joelho de coelho. As imagens obtidas pelo método da propagação tanto dos corpos de prova quanto das amostras biológicas mostram que há uma melhor qualidade na imagem, com maior contraste nas bordas e visualização de estruturas que não são visíveis nas radiografias convencionais. O mesmo acontece com as reconstruções tomográficas, que mostram detalhes da anatomia da amostra, facilitando a visualização de danos em tecidos moles.Essa técnica é vantajosa por não precisar de nenhuma instrumentação ótica, sendo explorado somente o tamanho focal da fonte de raios X e a distância entre a amostra e o detector. No LNLS foi usada a linha XRD2 com 10,34 keV de energia para realizar imagens realçadas por difração usando o detector Pilatus. As imagens realçadas por difração obtidas no LNLS têm qualidade superior devido à utilização de um cristal analisador colocado após a amostra. Este cristal funciona como uma estreita fenda angular resultando em imagens com ganho de contraste em relação às imagens obtidas pelos métodos convencionais / Abstract: In this dissertation we have produced in vitro joint and cartilages images by two especific methods of phase contrast imaging: Diffraction enhanced imaging (DEI) method and Propagation method. The DEI setup was implemented at the XRD2 beamline of the Brazilian National Synchrotron Light Laboratory (LNLS) in Campinas. The propagation method setup was implemented at the X-ray applied Crystallography Laboratory (LCARX) using a microfocus source (5 µm), that was installed inside of an experimental hut shielded with lead. The samples used were test objects and specimens of joints and cartilages provided by the group of Dr. William Dias Belangero of School of Medical Sciences (FCM-UNICAMP). In the Crystallography Laboratory was built an experimental station to perform tomography with a microfocus source. At this station pictures were realized by the conventional method and the propagation method. At the same laboratory was realized a study in order to colaborate the FCM group, using clinical cases of metal implants in prosthetic joints, we studied the regeneration of bone tissue after the injury to fixation of the prosthesis, using in vitro samples of injured legs of rats. Finally, tomographic acquisitions of rat and rabbit knees were performed in the conventional method and propagation method. The images obtained by the propagation method of both test objects and biological samples show that there is a better image quality, with high contrast in the edges and visualization of structures that are not visible on conventional radiographs. The same happens with the tomographic reconstructions, showing details of the anatomy of the sample, facilitating the visualization of soft tissue damage. This technique has the advantage of not requiring any optical instrumentation, only exploring the focal size of the X-ray source and the distance between sample and detector. In the LNLS was used the XRD2 beamline with 10,34 keV of energy to perform diffraction enhanced images using the detector Pilatus. The diffraction enhanced images had higher quality because this system uses an analyzer crystal placed after the sample. This crystal serves as a narrow angular slit which resulted in images with gain in contrast as compared to the conventional images methods / Mestrado / Física / Mestra em Física

Class of 2012 Abstract / Specific Aims: To evaluate the presence of statistical information from clinical studies in official product labeling specific for disease-modifying anti-rheumatic drugs (DMARDs) used in the treatment of rheumatoid arthritis. Methods: Data were abstracted from official product labeling DMARDs with FDA approval for treatment of rheumatoid arthritis. Each document was examined for the presence of statement regarding a priori type 1 error rate, p-values, and measures of variance. Medications were classified as either biologic or non-biologic. Main Results: A total of 14 DMARDs, 7 biologics (50%) and 7 non-biologics (50%), were found to be FDA approved for the treatment of rheumatoid arthritis. Primary outcomes consisted of American College of Rheumatology (ACR) response rates, radiographic changes, and health assessment questionnaire score (HAQ). Any measure of variance and the presence of a p-value were both found in six (43%) of the drug labels. Inclusion of p-values was found to be significantly greater in biologics compared to non-biologics for both ACR and radiographic results. Inclusion of variance was found to be significantly greater in biologics compared to non-biologics for radiographic changes only. No package inserts contained statements regarding the a priori type I error rate. Conclusions: Measures of variance are not frequently included in product labeling for either biologic or non-biologic DMARDs. However, inclusion of variance and p-values for ACR response rates and radiographic changes were more likely to be reported for biologics therapies as compared to non-biologics. A statement regarding Type 1 error rates were absent from labels regardless of outcome assessed.

M.A. (Psychology) / Numerous amounts ofliterature has confirmed the positive correlation that exists between exercise and psychological well-being. With the increased interest in the associated psychological factors ofpain, the present study was undertaken to investigate the effects of chronic pain on rheumatoid arthritic and osteoarthritic patients, with the overall aim of reducing, and alleviating these factors. The psychological factors studied were depression, effects ofthe impact ofarthritis, for example, on the independent, physical and psychosocial aspects oftheir lives, as well as coping. It was hypothesized that by engaging in an aquatic exercise program there would be a reduction/alleviation ofpain, which in tum would demonstrate a reduction in the psychological components ofpain. Evidence was found to support the hypothesis, due to the fact that, after exposure to the aquatic therapy program, subjects demonstrated a reduction in pain followed by a reduction in depression, slight improvement in coping with their arthritis, and the impact of arthritis was partially alleviated. The need for a larger sample group, as well a longer period of investigation will be needed for future research.

Rheumatoid arthritis (RA) is a chronic, multisystem inflammatory disease characterized by symmetric and erosive synovitis. The purinergic signaling system plays an important role in the modulation of inflammatory and immune responses through extracellular biomolecules as nucleotides adenine and its adenosine nucleoside derivative, whose extracellular concentrations are controlled by ectoenzymes action as ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase (E-ADA) present on the surface of various cells. The deficiency of vitamin D, a hormone known to play an important role in calcium homeostasis, has been related the several autoimmune diseases, including RA. This fact is due to its immunomodulatory role, where it inhibits the proliferation of T lymphocytes, especially Th1 lymphocytes as well as in the production and action of cytokines. Due to high doses of vitamin D can lead to hypercalcemia and intoxication it was tested the use of a nanoencapsulation system able to release in a controlled and sustainable manner, a reduced dose on site of action. Based on these principles, the effect of vitamin D3 free and nanoencapsulated was evaluated in the score for arthritis, thermal hyperalgesia and paw edema as well as histological analyzes and in the activity of the E-NTPDase and E-ADA enzyme lymphocytes from animals with arthritis induced by complete Freud‟s adjuvant (CFA). Hematological and biochemical parameters, including serum concentrations of vitamin D, were determined. Adult Wistar rats were divided into ten groups: I: control (CN); II: adjuvant-induced arthritis (AR); III: control vehicle (CV); IV: AR+V; V: vitamin D3 free at a dose of 120 IU/day (VD3); VI: AR+VD3 (120 IU/day); VII: control of white formulation (CF); VIII: AR+F; IX: vitamin D3 lipid-core nanocapsules formulation at a dose of 15.84 IU/day (LNC-D3) and X: AR+LNC-D3 (15.84 IU/day). The initiation of treatment was 15 days after the induction of arthritis in a period of 15 days. The results of this study demonstrated that VD3 was able to reduce arthritis score, thermal hyperalgesia and paw edema in rats with AR. At the same time, treatment with LNC-D3 only reduced thermal hyperalgesia and paw edema. The histological analyzes showed that both formulations were able to reduce inflammatory changes induced by CFA. The levels AST showed an increased in the CN group compared to the others. In the group treated with VD3 it was observed an increase in the levels of 25 (OH)D. The activity of the E-NTPDase in lymphocytes from rats that developed AR was higher in comparison with the control group, whereas the activity of E-ADA was lower. This effect was reversed after 15 days of treatment with VD3 and LNC-D3. In addition, both vitamin D3 formulations did not alter the activity of E-NTPDase and E-ADA enzymes in healthy animals. Data from this study indicate that vitamin D3, either in free or nanoencapsulated forms, seems to contribute mitigating the inflammatory process induced by CFA, possibly by modulating the activities of ectonucleotidases, can be used, after further studies, as a complementary therapeutic agent for the treatment of rheumatoid arthritis. / A artrite reumatoide (AR) é uma doença inflamatória crônica, multissistêmica, caracterizada por sinovite simétrica e erosiva. O sistema de sinalização purinérgica desempenha um papel importante na modulação das respostas inflamatórias e imunes, através de biomoléculas extracelulares, como os nucleotídeos de adenina e seu derivado nucleosídeo adenosina, cujas concentrações extracelulares são controladas por ação de ectoenzimas, como ecto-nucleosídeo trifosfato difosfoidrolase (E-NTPDase) e ecto-adenosina desaminase (E-ADA) presentes na superfície de diversas células. A deficiência da vitamina D, um hormônio conhecido por desempenhar importante papel na homeostase do cálcio, vem sendo relacionada a diversas doenças autoimunes, entre elas a AR. Este fato deve-se a seu papel imunomodulador, onde atua na inibição da proliferação de linfócitos T, especialmente linfócitos Th1, bem como na produção e na ação de citocinas. Como altas doses podem levar à hipercalcemia e à intoxicação, foi testada a utilização de um sistema de nanoencapsulação capaz de liberar, de forma controlada e sustentada, uma dose reduzida no local de ação. Com base nestes princípios, foi avaliado o efeito da vitamina D3 livre e nanoencapsulada no escore de artrite, na hiperalgesia termal e no edema de pata, bem como em análises histológicas e na atividade das enzimas E-NTPDases e E-ADA de linfócitos de animais com artrite induzida por adjuvante Completo de Freund (CFA). Parâmetros hematológicos e bioquímicos, entre eles a concentração sérica de vitamina D, foram determinados. Ratas adultas Wistar foram divididas em dez grupos: I: controle (CN); II: artrite (AR); III: controle veículo (CV); IV: AR+V; V: vitamina D3 livre na dose de 120 UI/dia (VD3); VI: AR+VD3 (120 UI/dia); VII: controle da formulação branca (CF); VIII: AR+F; IX: vitamina D3 nanoencapsulada na dose de 15,84 UI/dia (LNC-D3) e X: AR+LNC-D3 (15,84 UI/dia). O início do tratamento foi após 15 dias da indução da artrite, em um período de 15 dias. Os resultados do presente estudo demonstraram que VD3 foi capaz de reduzir escore de artrite, hiperalgesia térmica e edema da pata em ratos com AR. Ao mesmo tempo, o tratamento com LNC-D3 reduziu apenas a hiperalgesia térmica e o edema da pata. As análises histológicas mostraram que ambas as formulações foram capazes de reduzir as alterações inflamatórias induzidas por CFA. Os níveis de AST apresentaram um aumento no grupo AR em comparação com os demais. No grupo tratado com a VD3 observou-se um aumento nos níveis de 25(OH)D. A atividade da E-NTPDase em linfócitos de ratos que desenvolveram AR foi maior em comparação ao grupo controle, ao passo que a atividade da E-ADA foi menor. Este efeito foi revertido após 15 dias de tratamento com VD3 e LNC-D3. Além disso, ambas as formulações de vitamina D3 não alteraram a atividade das enzimas E-NTPDase e E-ADA em animais saudáveis. Os dados do presente estudo apontam que a vitamina D3, tanto na forma livre quanto nanoencapsulada, parece contribuir amenizando o processo inflamatório induzido pelo CFA, possivelmente por modular as atividades das ectonucleotidases, podendo após estudos adicionais ser utilizada como um agente terapêutico complementar para o tratamento da artrite reumatoide.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Gout, or gouty arthritis, has been associated of a range of comorbidities as hypertension and kidney diseases. Some studies have indicated that certain antihypertensive drugs, such as angiotensin I-converting enzyme inhibitors (ACEi), increase the risk of gout. Notably, ACEi block the metabolism of several peptides, in particular bradykinin hydrolysis. Bradykinin is also a substrate for kininase I, which forms des-Arg-kinin B1R agonists. Therefore, ACE inhibition can activate the B1R pathway. Moreover, ACEi are allosteric enhancers of kinin receptors and B1R are linked to inflammatory cardiovascular diseases. However, despite the body of evidence demonstrating a clear contribution of the kinin system to the pathogenesis of some arthritic conditions, the role of the kinin B1 receptor in monosodium urate (MSU) crystals crystal-induced gout is currently unknown, especially in respect to ACEi. Thus, the aim of the present study was to verify the role of the bradykinin B1 receptors and the effect of ACEi on acute gout induced by MSU crystals in rodents. Painful (overt pain and allodynia) and inflammatory parameters (joint edema, leukocyte trafficking, interleukin-1β levels) of acute gout attacks were assessed several hours after intra-articular (IA) injection of MSU (1.25 or 0.5 mg/articulation) in the ankle of rats or mice, respectively. The role of B1R was investigated by pharmacological antagonism or gene deletion. In addition, B1R immunoreactivity on ankle tissue and sensory neurons, kininase I activity and des-Arg9-bradykinin synovial levels were also measured. Similar tools were used to investigate the effects of ACEi on low dose of MSU (0.0125 mg/articulation)-induced inflammation. Bouth, bradykinin B1R antagonism or gene deletion largely reduced all painful and inflammatory signs of gout. Furthermore, MSU increased not only the B1R expression in articular tissues, but also the content of the B1 agonist des-Arg9-bradykinin and the activity of the B1 agonist-forming enzyme kininase I. Finally, low dose of MSU crystals (which did not induced inflammation in control animals) was capable of causing signs of acute gout attacks in ACEi-treated animals, which was B1R-dependent. Concluding, bradykinin B1R activation contributes to acute gouty attacks, including the ones facilitated by ACEi. Thus, B1R is a potential therapeutic target for treatment and prophylaxis of gout, especially in patients taking ACEi. / A gota, ou artrite gotosa, está associada à ocorrência de diversas comorbidades como doenças renais e cardiovasculares. Alguns dos fármacos utilizados para o tratamento destas comorbidades podem ainda precipitar ataques agudos de gota. Um exemplo são os inibidores da enzima cininase II ou enzima conversora de angiotensina (iECA). Muitos dos efeitos cardioprotetores dos iECA são mediados pelos receptores B1 para cininas. Além disso, a inibição da ECA é capaz de bloquear a hidrólise da bradicinina, que por sua vez é também um substrato para a formação do agonista do receptor B1, des-Arg9-bradicinina, pela ação da enzima cininase I. Apesar dos estudos demonstrando o envolvimento do sistema das cininas na gota e do papel do receptor B1 na iniciação e manutenção da inflamação, não se conhece a relação do receptor B1 com a gota, especialmente durante a inibição da ECA. Assim, o presente estudo tem por objetivo verificar o papel do receptor B1 para cininas durante o ataque agudo de gota induzido por cristais de urato monossódico (MSU) em roedores, incluindo aqueles tratados com iECA. Para isso, a dor (alodínia ao toque e comportamento doloroso espontâneo) e alguns parâmetros inflamatórios (edema articular, concentração de proteínas, infiltração leucocitária, avaliação da cinética de leucócitos e níveis de interleucina-1β) causados pelo ataque agudo de gota foram avaliados em diferentes tempos após a injeção intra-articular (IA) de uma alta dose de MSU no tornozelo de roedores. O papel do receptor B1 foi investigado através de seu antagonismo farmacológico, pela utilização de roedores com deleção gênica do receptor e pela inibição da enzima cininase I. Ainda, a expressão do receptor B1 no tecido do tornozelo e neurônios sensoriais, atividade da enzima cininase I e os níveis de des-Arg9-bradicinina foram analisados no fluido sinovial dos roedores submetidos ao modelo de gota. Ferramentas semelhantes foram utilizadas para a investigação do envolvimento dos receptores B1 na inflamação causadas pela inibição da ECA em animais submetidos a injeção IA de uma baixa dose de MSU, que não apresentava efeito per se. Tanto o uso de antagonistas do receptor B1, como sua deleção gênica, foram capazes de reduzir a inflamação neste modelo de gota. Além disso, o MSU causou um aumento na expressão de receptores B1 na articulação, e também um aumento dos níveis do agonista B1, des-Arg9-bradicinina e da atividade da enzima cininase I. Por último, a baixa dose de MSU foi capaz de causar um ataque agudo de gota em animais pré-tratados com iECA, por um mecanismo que envolve a ativação de receptores B1. Concluímos que a ativação do receptor B1 contribui para o desenvolvimento do ataque agudo de gota, incluindo aqueles que são precipitados pelo tratamento com iECA. Assim, o B1 apresenta um potencial terapêutico para o tratamento e profilaxia da gota, especialmente em indivíduos que fazem uso de iECA.

Bone is a dynamic tissue that is continuously remodelled, a process that requires equal amounts of osteoclastic bone resorption and osteoblastic bone formation. Inflammation may disturb the equilibrium and result in local and/or systemic bone loss. Negative bone mass balance occurs in several chronic inflammatory diseases, e.g. periodontitis and rheumatoid arthritis (RA). The aetiology of periodontitis is infectious, while RA is an autoimmune disease. Despite aetiological differences, an association between the two diseases has been established but it is not known if they are causally related. Periodontitis may develop when the inflammatory process, initially restricted to the gingiva (gingivitis), further invades the periodontium and causes bone resorption. The cellular and molecular mechanisms underlying the transition from gingivitis to periodontitis are not fully elucidated. Osteoclast formation is dependent on receptor activator of nuclear factor kappa B ligand (RANKL), but how osteoclast precursors are recruited to the jawbone is poorly understood. A family of cytokines named chemokines has been reported to possess such properties and increasing evidence points towards their involvement in the pathogenesis of chronic inflammatory diseases. The overall aim of this thesis was to gain extended knowledge about the role of chemokines and a newly discovered family of leukocytes named innate lymphoid cells (ILCs) in periodontitis and concomitant inflammatory disturbed bone remodelling. Furthermore, the aim was also to study the association between periodontitis and RA. We identified increased serum levels of monocyte chemoattractant protein (MCP)-1 and CCL11 in individuals with periodontitis. Moreover, a robust correlation between the two chemokines and periodontitis was detected in a weighted analysis of inflammatory markers, subject characteristics and periodontitis parameters. We detected higher MCP-1 levels in periodontitis tissue compared to non-inflamed. Furthermore we demonstrated that human gingival fibroblasts express MCP-1 and CCL11 in response to pro-inflammatory cytokines through NF-κB signalling. Using an inflammatory bone lesion model and primary cell cultures, we discovered that osteoblasts express CCL11 in vivo and in vitro and that the expression increased under inflammatory conditions. Osteoclasts did not express CCL11, but its high affinity receptor CCR3 was upregulated during osteoclast differentiation and found to co-localise with CCL11 on the surface of osteoclasts. Exogenous CCL11 was internalised in osteoclasts, stimulated the migration of osteoclast precursors and increased bone resorption in vitro. To analyse if periodontitis precedes RA we analysed marginal jawbone loss in dental radiographs taken in pre-symptomatic RA cases and matched controls. The prevalence of jawbone loss was higher among cases, and the amount of jawbone loss correlated with plasma levels of RANKL. In the search of the newly discovered ILCs, we performed flow cytometry analyses on gingivitis and periodontitis tissue samples. We detected twice as many ILCs in periodontitis as in gingivitis. In addition we found RANKL expression on ILC1s (an ILC subset). In conclusion, we demonstrated that CCL11 is systemically and locally increased in periodontitis and that the CCL11/CCR3 axis may be activated in inflammatory disturbed bone remodelling. We also found that marginal jawbone loss correlated with plasma levels of RANKL and preceded clinical onset of symptoms of RA. Furthermore, we demonstrated that ILCs are present in periodontitis and represent a previously unknown source of RANKL. / Skelettet har flera viktiga funktioner i kroppen såsom att möjliggöra en upprätt hållning, utgöra fäste för muskler och mediera rörelse, skydda benmärgen och de inre organen samt reglera mängden av lösligt mineral i blodet. Med tiden uppstår mikroskador i skelettet vilket innebär att benvävnaden måste byggas om för att vara fortsatt funktionell. Ombyggnaden kallas remodellering och är en kontinuerlig process som huvudsakligen utförs av benbildande celler kallade osteoblaster och bennedbrytande celler kallade osteoklaster. Remodelleringen är strikt reglerad av olika signalmolekyler och under friska förhållanden råder jämvikt mellan mängden ben som bryts ner och mängden ben som bildas, vilket innebär att benmassan hålls konstant. Vid sjukdomar som medför långvariga inflammationsprocesser i benvävnad eller i närheten av benvävnad, exempelvis parodontit (tandlossningssjukdom) och ledssjukdomen reumatoid artrit (RA), kan den rådande jämvikten rubbas, vilket oftast resulterar i minskad benmängd. Vid parodontit är den bakomliggande orsaken till inflammationen bakterier som finns i placket på tänderna, men vid RA tros anledningen vara att immunförsvaret attackerar kroppsegna celler. Trots olikheterna delar de två sjukdomarna flera gemensamma drag med avseende på riskfaktorer, vilka signalmolekyler som återfinns i blodet samt hur inflammationsprocessen fortskrider. Parodontit föregås av gingivit (tandköttsinflammation). Hos vissa individer övergår gingivit till parodontit, en process som inkluderar nedbrytning av tandens stödjevävnader inklusive käkben. Det är inte helt klarlagt vilka celler och molekyler som finns närvarande vid gingivit respektive parodontit eller vilka mekanismer som ligger bakom skiftet mellan de två tillstånden. Det är sedan tidigare känt att molekylen RANKL är viktig för osteoklastbildning, men det är delvis okänt hur osteoklastförstadieceller rekryteras från blodcirkulationen till käkbenet. En grupp av molekyler kallade kemokiner, som även finns i förhöjda nivåer i blod vid parodontit och RA, har visat sig ha sådana egenskaper. För att finna läkemedel som kan förhindra bennedbrytning till följd av den inflammatoriskt störda benremodellering som sker vid både parodontit och RA är det viktigt att studera sambandet mellan sjukdomarna och få en tydlig bild av vilka celler som är närvarande vid inflammationsprocessen. Det är även av betydelse att kartlägga vilka celler och molekyler som främjar rekrytering av osteoklastförstadieceller och bidrar till bennedbrytning. Syftet med den här avhandlingen var att undersöka betydelsen av kemokiner vid inflammatoriskt störd benremodellering och vid parodontit samt att undersöka sambandet mellan parodontit och RA. För att skapa en tydligare bild av vilka cellertyper som är närvarande vid inflammationsprocessen vid parodontit undersöktes även förekomsten av en nyligen upptäckt celltyp vid namn ILCimmunceller (ILCs) samt om dessa celler uttrycker RANKL. Först analyserades förekomsten av olika inflammatoriska signalmolekyler i blod från individer med parodontit samt från friska kontroller. Individer med parodontit hade förhöjda nivåer av kemokinerna MCP-1 och CCL11. Genom att använda en statistisk analysmetod som utöver inflammatoriska signalmolekyler även inkluderade kliniska variabler kunde ett samband mellan de två kemokinerna och parodontit påvisas. Vidare undersöktes möjliga ursprung till de i blodet förhöjda kemokinnivåerna genom att analysera tandkött från tänder med parodontit samt friskt tandkött. Vid parodontit uppmättes högre nivåer av MCP-1. Gingivala fibroblaster (en celltyp som producerar bindväv och ansvarar för tandköttets uppbyggnad) från människa bildade MCP-1 och CCL11 när de stimulerats med inflammationsfrämjande substanser, vilket krävde aktivering en intracellulär signaleringsväg kallad NF-κB. För att utreda betydelsen av CCL11 vid inflammatoriskt störd benremodellering analyserades bencellers bildning av CCL11 in vivo i skalltak från möss samt in vitro i cellodlingar. Osteoblaster bildade CCL11 in vivo och in vitro och bildningen ökade under inflammatoriska förhållanden. Osteoklaster bildade inte CCL11, men däremot fanns ett uttryck av receptorn CCR3, vilket är en mottagarmolekyl till CCL11. I vävnadssnitt från skalltak visades att CCL11 och CCR3 ser ut att binda till varandra på osteoklasternas yta. Dessutom hade CCL11 en positiv effekt på rekrytering av osteoklastförstadieceller och CCL11 som tillsattes till cellodlingar togs upp av osteoklaster och stimulerade benresorption. För att studera sambandet mellan parodontit och RA analyserades käkbensförlust vid tänder med hjälp av röntgenbilder tagna på individer som senare utvecklade RA (pre-symptomatiska) samt matchade kontroller. De presymptomatiska individerna hade en högre grad av käkbenförlust och det fanns också ett samband mellan käkbensförlust och nivåer av RANKL i blodet. Förekomsten av ILCs i tandkött från tänder med gingivit respektive parodontit analyserades med flödescytometri. Dubbelt så många ILCs återfanns vid parodontit än vid gingivit, varav majoriteten bestod av ILC1 (en undergrupp till ILCs). Vidare analyser visade att ILC1 cellerna bildar RANKL. Sammanfattningsvis, vid parodontit finns förhöjda nivåer av CCL11 i vävnaden och i blodet, och interaktionen mellan CCL11 CCR3 kan vara av betydelse vid inflammatoriskt störd benremodellering. Käkbensförlust föregår RA och korrelerar med nivåer av den osteoklaststimulerande molekylen RANKL i blodet, vilket stödjer teorin om att det finns ett samband mellan de två sjukdomarna. De nyligen upptäckta ILCs återfinns vid både gingivit och parodontit och utgör dessutom en tidigare okänd källa till RANKL.

Patients with rheumatoid arthritis (RA) have a shorter life span than the general population. An increased death due to cardiovascular disease (CVD) has been reported. RA is characterized by synovitis and joint destruction accompanied by an acute phase reaction and systemic features. The present work investigates the epidemiology of CVD in patients with RA in the county of Västerbotten and the influence of inflammation on lipid metabolism and haemostasis. In a retrospective cohort study on 606 RA patients, the overall mortality was significantly higher than in the general population, with an excess death rate for CVD and for ishemic heart diseae (IHD) in both sexes. Multiple Cox regression, showed that male sex, higher age at disease onset and cardiovascular event increased the risk for death. Male sex, high age at disease onset and hypertension increased the risk for cardiovascular event. Diabetes mellitus, treatment with corticosteroids, disease modifying antirheumatic drugs and postmenopausal estrogen neither influenced survival nor the risk of cardiovascular event. In 93 patients with active RA, the levels of cholesterol, high density- (HDL) and low density (LDL) lipoprotein cholesterol were significantly lower, and Lipoprotein(a) was significantly higher compared to controls. In a follow-up on 53 patients, a relation between the change of Lp(a) and acute phase proteins was found only in patients with high levels of Lp(a). Preheparin lipoprotein lipase (LPL) activity and mass were significantly decreased in 17 postmenopausal women with active RA. Preheparin LPL mass correlated inversely to several acute phase proteins and interleukin-6. Low levels of LPL mass may implicate increased hepatic clearence but also increased macrophage ingestion of lipoproteins via the LDL receptor-related protein (LRP). Haemostasis of the circulation was investigated in 74 of the 93 patients with active RA. In patients with extraarticular disease, the release of tissue plasminogen activator (tPA) was significantly decreased, and its inhibitor (PAI-1) was significantly increased compared to patients with nonsystemic disease, implicating hypofibrinolysis. In a two year follow-up, patients with thromboembolic events had significantly elevated levels of von Willebrand factor, PAI-1, triglycerides and haptoglobin compared to event-free patients. In 29 RA patients and 18 spondylarthropathy patients with gonarthritis, radiological joint destruction correlated to PAI-1 antigen in synovial fluid and, inversely, to plasminogen. A relationship between activation of fibrin degrading proteolytic enzymes and joint destruction was implicated. In conclusion, several processes involved in lipid metabolism and haemostasis are influenced in active RA. In view of the increased death rate due to CVD, an efficient control of inflammation should be important, not only for reducing joint destruction, but also for reducing systemical atherogenic and thrombogenic effects. / <p>s. 1-54: sammanfattning, s. 55-133: 6 uppsatser</p> / digitalisering@umu.se

Abstract Rheumatoid arthritis (RA) is an autoimmune disease causing inflammation of synovial joints, which may lead to permanent changes in cartilage and bone tissues. RA patients have antibodies binding to citrullinated and also to carbamylated proteins. Antibodies binding to citrulline are associated with more severe disease progression and may already appear years before clinical disease onset. Citrulline and the lysine carbamylation product, homocitrulline, are similar in structure. Citrullinated proteins have been studied in RA and in neurological diseases, but researchers have been unaware of the effect of homocitrulline in citrulline detection methods. The purpose of the present study was to clarify the features of protein-bound citrulline and homocitrulline in relation to research done on citrullination and in immunological reactions related to rheumatoid arthritis. In the first study of this thesis the confounding role of homocitrulline in citrulline detection was shown. In the first and second study the features of experimentally induced antibodies were assessed. The antibodies induced with citrulline- and homocitrulline-containing protein structures were shown to react both with the ureido groups and the protein structures. The antibodies were able to distinguish between citrulline and homocitrulline in the same sequence even though binding to both. In the third study the simultaneous presence of citrulline and homocitrulline in RA synovial tissue was shown. In conclusion, considering the simultaneous presence of citrulline and homocitrulline and the binding features of the experimentally induced antibodies, homocitrulline could have a yet unsolved role in RA. Secondly, the existence of homocitrulline should be borne in mind in studies on citrullination. / Tiivistelmä Nivelreuma on niveltulehduksen aiheuttava autoimmuunitauti, joka voi johtaa pysyviin muutoksiin nivelen rusto- ja luukudoksessa. Nivelreumaa sairastavilla esiintyy vasta-aineita sitrullinoituneita ja karbamyloituneita proteiineja vastaan. Sitrulliiniin sitoutuvia vasta-aineita voi esiintyä elimistössä jo vuosia ennen taudin puhkeamista, ja niiden esiintyminen on yhdistetty vaikeampaan taudinkuvaan. Sitrulliini ja lysiinin karbamylaatiotuote homositrulliini ovat rakenteellisesti samankaltaisia. Proteiinien sitrullinaatiota on tutkittu nivelreumassa ja neurologisissa taudeissa, mutta homositrulliinin olemassaoloa tai sen vaikutusta tutkimusmenetelmiin ei ole huomioitu. Tämän tutkimuksen tarkoituksena oli selvittää proteiineihin sitoutuneiden sitrulliinin ja homositrulliinin ominaisuuksia aikaisempiin tutkimuksiin ja nivelreuman immunologisiin reaktioihin liittyen. Tässä tutkimuksessa homositrulliinin osoitettiin häiritsevän sitrulliinin tunnistamista. Ensimmäisessä ja toisessa osatyössä aiheutettiin kokeellisesti vasta-aineita sitrulliinia ja homositrulliinia sisältävillä proteiinirakenteilla. Vasta-aineiden havaittiin reagoivan sekä ureidoryhmän että proteiinirakenteen kanssa. Vasta-aineet pystyivät erottamaan sitrulliinin ja homositrulliinin toisistaan samassa rakenteessa, vaikka sitoutuivat kumpaankin. Kolmannessa osatyössä osoitettiin, että sitrulliinia ja homositrulliinia esiintyy samanaikaisesti nivelreumapotilaan tulehtuneessa nivelkalvossa. Tutkimus osoitti, että sitrulliinin ja homositrulliinin samanaikainen esiintyminen ja kokeellisesti aiheutettujen vasta-aineiden ominaisuudet huomioiden homositrulliinilla voi olla jokin toistaiseksi selvittämätön rooli nivelreumassa. Homositrulliinin olemassaolo on syytä huomioida sitrullinaatiota tutkittaessa.

Abstract Reduced physical function and persistent pain are serious consequences of rheumatoid arthritis (RA). Clinical trials have shown that patients with RA suffer from a poor health-related quality of life (HR-QoL). However, limited information is available on the HR-QoL of patients treated in normal clinical practice. The purpose of the present study was to obtain information of how RA can influence on the patients´ HR-QoL and functional ability in a clinical setting and to investigate the impact of disease-related and demographic factors on the HR-QoL. The theoretical framework of the study was the biopsychosocial concept of health, i.e. the ICF model (International Classification of Functioning, Disability and Health) endorsed by WHO. HR-QoL was measured by the Nottingham Health Profile (NHP) questionnaire and functional ability by the Health Assessment Questionnaire (HAQ). The contents of these instruments and the NHP results were evaluated also within the ICF categories. The study consisted of a cross-sectional series and a longitudinal cohort carried out in two central hospitals in Finland in the late 1990s. The cross-sectional group of 122 consecutive out-patients had a mean disease duration of 11 years. The HR-QoL values of the patients were compared with those of an age and gender matched ”healthy” control group living in the same area. The HR-QoL values of the patients were examined also at different functional ability levels. The longitudinal group of 62 consecutive patients had symptom duration ≤ 24 months and no prior use of antirheumatic drugs or glucocorticosteroids at inclusion. First, the impact of the treatment response assessed by the EULAR (DAS28) criteria on HR-QoL was examined at six months from disease onset. Secondly, the HR-QoL changes and their associations with age and gender and with the changes in disease activity, radiographic assessments of hands and feet and functional ability were examined for ten years after the onset of RA. RA patients had poorer scores than the controls in the NHP in the dimensions measuring mobility, pain and energy. These dimensions, along with sleep, displayed also a significant linear association with poorer HAQ levels (p&#160;&#60;&#160;0.001). A better treatment response during the first six months was linearly associated with better HR-QoL with respect to the pain, energy and mobility dimensions (p&#160;&#60;&#160;0.001). Those patients exhibiting no response to treatment had already at baseline the poorest HR-QoL in the dimension for pain and emotional reaction compared with those in the moderate and good responders. During the ten years´ follow-up, all NHP dimensions except social isolation displayed significant improvements, these being most marked during the first six months. Changes in disease activity correlated with changes in pain, energy and emotional reaction (p&#160;&#60;&#160;0.001). The mean level of the HAQ was well preserved over the ten years of this study and its changes were correlated with changes in pain, mobility and energy (p&#160;&#60;&#160;0.001). Women had somewhat poorer NHP improvements in the dimensions assessing energy, emotional reaction and social isolation than males. Disease duration associated strongly with poorer mobility and pain dimensions (p&#160;&#60;&#160;0.01). Within the ICF framework, pain, mobility, energy and sleep were identified as being the most important categories from the patient's perspective. The results of the present study demonstrate that RA has a major influence on the patients´ HR-QoL but early and active treatment can improve the situation. In the ICF framework, the NHP covers a broader spectrum of the ICF categories than can be assessed by the HAQ. / Tiivistelmä Alentunut fyysinen toimintakyky ja jatkuva kipu ovat nivelreumaan liittyviä vakavia seurannaisvaikutuksia. Kliiniset tutkimukset ovat osoittaneet, että nivelreumaa sairastavat potilaat kärsivät huonontuneesta terveyteen liittyvästä elämänlaadusta. Normaalista kliinisestä käytännöstä saatava tieto potilaiden terveyteen liittyvästä elämänlaadusta on kuitenkin niukkaa. Tämän tutkimuksen tarkoituksena oli saada tietoa nivelreuman vaikutuksista potilaiden terveyteen liittyvään elämänlaatuun ja toimintakykyyn kliinisessä asetelmassa ja tutkia sekä tautiin liittyvien että demografisten tekijöiden vaikutusta terveyteen liittyvään elämänlaatuun. Tutkimuksen teoreettisena viitekehyksenä käytettiin WHO:n hyväksymää toimintakyvyn, toimintarajoitteiden ja terveyden kansainvälistä luokitusta (ICF-malli). Terveyteen liittyvää elämänlaatua arvioitiin mittarilla Nottingham Health Profile (NHP) ja toimintakykyä mittarilla Health Assessment Questionnaire (HAQ). Kyseisten mittareiden sisältö samoin kuin NHP- tuloksia arvioitiin käyttämällä myös ICF- luokitusta. Tutkimus käsitti poikkileikkaus- ja pitkittäistutkimuksen, jotka toteutettiin 1990-luvun lopulla kahdessa suomalaisessa keskussairaalassa. Poikkileikkaustutkimukseen osallistui 122 perättäistä polikliinistä potilasta, joilla taudin kesto oli ollut keskimäärin 11 vuotta. Potilaiden terveyteen liittyvää elämänlaatua verrattiin samalla seudulla elävään iän ja sukupuolen suhteen kaltaistettuun verrokkiryhmään. Potilaiden terveyteen liittyvää elämänlaatua arvioitiin myös eri toimintakykytasoilla. Pitkittäistutkimus käsitti 62 perättäistä potilasta, joilla oireet olivat kestäneet ≤&#160;24 kuukautta ja jotka tutkimuksen alkaessa eivät olleet käyttäneet edeltävästi antireumaatteja tai kortikosteroideja. Näillä potilailla hoitovasteen vaikutusta terveyteen liittyvään elämänlaatuun arvioitiin EULAR DAS28- kriteerein kuuden kuukauden kohdalla taudin alusta. Lisäksi tutkittiin terveyteen liittyvän elämänlaadun muutoksia kymmenen vuoden ajalta nivelreuman alusta ja näiden muutosten yhteyttä ikään ja sukupuoleen, taudin aktiviteetissa ja käsissä ja jalkaterissä todettuihin röntgenmuutoksiin samoin kuin toimintakyvyn muutoksiin. Hoitovasteetta jääneillä potilailla oli jo lähtötilanteessa huonoin terveyteen liittyvä elämänlaatu kipu- ja tunnereaktiot- ulottuvuuksissa verrattuna kohtalaisen ja hyvän hoitovasteen saaneisiin. Kymmenen vuoden seurannassa kaikki NHP- ulottuvuudet sosiaalista eristyneisyyttä lukuun ottamatta osoittivat merkittävää paranemista. Selvintä se oli ensimmäisen kuuden kuukauden aikana. Taudin aktiviteetin muutokset korreloivat kipu-, tarmokkuus- ja tunnereaktiot- ulottuvuuksien muutoksiin (p&#160;&#60; &#160;0.001). Keskimääräinen HAQ- taso säilyi hyvänä kymmenen vuoden seurannassa ja HAQ- muutokset korreloivat kipu-, liikkuminen- ja tarmokkuus- ulottuvuuksien muutosten kanssa (p&#160;&#60; &#160;0.001). Naisilla oli miehiä jonkin verran huonompi NHP-paraneminen tarmokkuus-, tunnereaktiot- ja sosiaalinen eristyneisyys- ulottuvuuksissa. Taudin kesto oli selvästi yhteydessä huonompiin liikkuminen- ja kipu- ulottuvuuksiin (p&#160;&#60; &#160;0.01). ICF- luokitusta käytettäessä potilaiden näkökulmasta kipu, liikkuminen, tarmokkuus ja uni nousivat tärkeimmiksi kategorioiksi. Tämän tutkimuksen tulokset osoittavat, että nivelreumalla on huomattava vaikutus potilaiden terveyteen liittyvään elämänlaatuun, jota varhainen ja aktiivinen hoito voi kuitenkin parantaa. ICF- viitekehyksessä NHP kattaa laajemman spektrin ICF- luokista kuin HAQ.