No description available.

Disease progression in Multiple Sclerosis (MS), an autoimmune disease of the CNS, is widely accepted to be due to persistent myelin loss (demyelination) coinciding with lost nerve cells and nerve fibres (neuroaxonal loss). Current treatments are immunomodulatory and do not address the neuroaxonal or demyelinating pathology of the disease. It is hypothesised that a lack of growth factors within the CNS may result in the failure of remyelination. Therefore, biologics such as recombinant therapeutic proteins used for gene therapy offer a promising therapeutic intervention to the progressive stages of the disease. However, due to the short half-lives of these therapeutics and their pleiotropic effects, there is cause for concern over their safety and efficacy. Using LAP technology (the fusion of the therapeutic protein with the latent associated peptide [LAP] of TGFβ), the half-life of the therapeutic protein can be increased and can be targeted to sites of inflammation and disease. This study aimed to investigate the potential neuroprotective, remyelinating and anti-inflammatory effects of latent versions of the growth factors erythropoietin (EPO), insulin-like growth factor 1 (IGF1) and transforming growth factor beta (TGF) respectively. Firstly, using molecular cloning techniques, these growth factors were individually fused and linked to the LAP of TGF via a matrix metalloproteinase (MMP) cleavage site resulting in three latent growth factors. Secondly, these latent growth factors were shown to be expressed, and to be biologically active in vitro when released by MMP cleavage. Finally, syngeneic fibroblasts were engineered to express the latent growth factors. It was found that, in CREAE, the fibroblasts engineered to produce latent TGF significantly reduced the disease clinical score as compared to controls whilst latent EPO produced by transduced fibroblasts failed to exert a statistically significant effect on disease progression. Nonetheless, this study demonstrates the feasibility of the latency platform technology to generate latent therapeutics with the ability to act as an intervention to disease progression in MS.

Osteoarthritis (OA) is the leading cause of musculoskeletal disability in people and horses, and is characterized by progressive joint degeneration. There is a critical need for a better understanding of disease processes leading to OA in order to develop more efficient therapies. A shared feature among different arthritic conditions is chronic synovitis. Macrophages are the main drivers of synovitis and can display pro-inflammatory (M1) or pro-resolving responses (M2). Macrophages promote joint health through phagocytic and secretory activities; however, when these functions are overwhelmed, macrophages upregulate inflammation, recruiting more cells to counteract damage. Once cell recruitment is efficiently accomplished, macrophages coordinate tissue repair and further resolution of inflammation. Bone marrow mononuclear cells (BMNC) are a source of macrophages used to treat inflammation and produce essential molecules for cartilage metabolism; however, little information exists regarding their use in joints. The studies presented in this dissertation focus on understanding the dual role of macrophages in driving and resolving synovitis and how to harness their therapeutic potential. In the first study, patterns of macrophage phenotypes (M1:M2) in healthy and osteoarthritic equine synovium were compared and correlated with gross pathology, histology, and synovial fluid cytokines. M1 and M2 markers were co-expressed in normal and osteoarthritic joints, varying in intensity of expression according to degree of inflammation. Concentrations of synovial fluid IL-10, a macrophage-produced cytokine that is vital for chondrocyte recovery from injury, was lower in OA joints. The combined findings of this study suggest homeostatic mechanisms from synovial macrophages in OA may be overwhelmed, preventing inflammation resolution. In the second study we investigated the response of BMNC to normal (SF) and inflamed synovial fluid (ISF). BMNC cultured in autologous SF or ISF developed into macrophage cultures that were more confluent in ISF (~100%) than SF (~25%), and exhibited phenotypes that were ultimately similar to cells native to normal joints. BMNC cultured in SF or ISF were neither M1 nor M2, but exhibited aspects of both phenotypes and a regulatory response, characterized by increasing counts of IL-10+ macrophages, decreasing concentrations of IL-1β, and progressively increasing concentrations of IL-10 and IGF-1, all more marked in ISF. These findings suggest that homeostatic mechanisms were preserved over time, and potentially favored by macrophage proliferation. Our data suggest that BMNC therapy could potentiate the macrophage- and IL-10-associated mechanisms of joint homeostasis lost in OA. Finally, using an equine model of synovitis, the last study investigated the response of normal and inflamed joints to autologous BMNC injection. Inflamed joints treated with BMNC showed gross and analytical improvements in synovial fluid and synovial membrane, with increasing numbers of regulatory macrophages and synovial fluid concentrations of IL-10, not observed in saline-treated controls. Autologous BMNC are readily available, downregulate synovitis through macrophage-associated effects, and can benefit thousands of patients with OA. Combined, the results of these studies support the role of macrophage-driven synovial homeostasis and identified a therapeutic way to recover homeostatic mechanisms of synovial macrophages lost during chronic inflammation. Our findings also uncover new research directions and methods for future studies targeting modulation of joint inflammation. / Doctor of Philosophy / Osteoarthritis (OA) is a common cause of joint deterioration in people and horses. Current treatments provide limited recovery of joint function, creating an urgent need for more efficient therapies; however, development of new treatments requires better understanding of the mechanism causing OA. A shared characteristic among many arthritic conditions is long-standing inflammation. Cells called macrophages are the main drivers of joint inflammation and can exert pro- and anti-inflammatory effects. Macrophages promote joint health by clearing aggressor agents and secreting molecules required for optimal joint function. However, when these housekeeping functions are overwhelmed by damage, macrophages drive inflammation recruiting more cells to cope with increased demands for repair. If this process is efficiently accomplished, macrophages then resolve inflammation, recovering joint health. Macrophages in the bone marrow (BMNC - bone marrow mononuclear cells) are used to treat inflammation in several tissues and are known to produce molecules essential for joint health. Although little information exists regarding their use in joints, studies treating different organs suggest it can provide high rewards. The studies presented in this dissertation focused on understanding the dual function of macrophages in driving and controlling joint inflammation, and harnessed their therapeutic potential. In the first study, macrophages were investigated in normal and OA-affected joints, and curiously exhibited a hybrid pro- and anti-inflammatory identity in both groups. The indicators of this mixed identity were more markedly expressed in arthritic joints showing gross inflammation. Low levels of a macrophage-derived anti-inflammatory protein called IL-10 were detected in OA joints. The results of this study suggest that anti-inflammatory mechanisms from macrophages may be overwhelmed in OA-affected joints, preventing inflammation to be resolved, and that recovering this anti-inflammatory function may aid in the treatment of OA. In the second study we investigated how the incubation of BMNC in fluid from normal and inflamed joints affects the response of macrophages. Similar to what we observed in the first study, BMNC incubated in both normal and inflamed joint fluid induced macrophages to develop a hybrid identity that was ultimately similar to native cells from normal joints. Macrophages proliferated more when incubated in fluid from inflamed joints. Macrophages in both groups produced anti-inflammatory effects with high levels of IL-10 that were highest in ISF cultures. These observations suggest that higher proliferation of macrophages in inflamed joint fluid helped preserve anti-inflammatory mechanisms. Therefore, our study suggests that joint injection with BMNC could maximize macrophage- and IL-10-associated mechanisms required to resolve joint inflammation. The third and final study investigated the response of normal and inflamed joints to BMNC injection using a model of joint inflammation in horses. Inflamed joints treated with BMNC showed visual and laboratorial markers of improvement, with increasing numbers of macrophages and concentrations of IL-10 in the joint fluid, which remained lower in joints treated with placebo. BMNC provide means to recover macrophage-associated effects required to control joint inflammation and can benefit thousands of patients with OA. Together, the results of these studies show that macrophages are biased promoters of joint health, leading to inflammation when their anti-inflammatory mechanisms are overwhelmed. Replenishing inflamed joints with healthy macrophages maximizes their anti-inflammatory effects, favoring the recovery of a healthy articular environment.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Background: Rheumatoid arthritis (RA) is a common chronic autoimmune disorder characterised by persistent synovitis, typically manifested as symmetric polyarthritis of small hand joints with various extra-articular manifestations. Accurate disease activity measurement is a key component of RA management that facilitates therapeutic optimalization in order to slow down the disease progression and to prevent an irreversible joint damage. The aim of this work was to study the role of candidate serum inflammatory markers and their associations with the disease activity in patients with RA presented by traditional variables of disease activity as well as by musculoskeletal ultrasonography. Results: The first part of our work pointed out relationship between serum calprotectin and clinical as well as ultrasound activity in RA. We have revealed that serum calprotectin is an independent predictor of ultrasound synovitis. Moreover, we have demonstrated the potential of calprotectin to identify patients with residual activity in spite of achieving clinical remission. In the second part, we have provided a detailed analysis of 20 candidate serum markers and found out a tight associations between IL-6, IL-7, IL-22, IL-34, YKL-40, CXCL-13, MMP-3, resistin and visfatin with clinical and ultrasound activity....

Made available in DSpace on 2016-06-02T20:18:15Z (GMT). No. of bitstreams: 1 3957.pdf: 2895420 bytes, checksum: 6582876d42cf86a1b96fcbcd3db2a9c4 (MD5) Previous issue date: 2011-11-29 / Universidade Federal de Minas Gerais / The joint inflammation is a frequent cause of activity limitation in daily life of the population. Usually, this condition also affects the muscles that are functionally related to the inflamed joint. The possible relationship between joint inflammation and the expression of genes related to muscle atrophy, differentiation and growth and muscle mass control in muscles functionally related to the inflamed joint, has not been studied. The aim of this thesis was to evaluate the effect of the tibiotarsical joint inflammation, induced by carrageenan, on the expression of genes related to muscle atrophy (atrogin-1 and MuRF-1), growth and differentiation (MyoD), muscle mass regulation (myostatin), and proinflammatory factors (p38MAPK, NFkB and TNF-alpha); and the expression of TNF-alpha protein in the tibialis anterior (TA) and soleus rat muscle. Changes in the muscle fiber cross-sectional area (CSA) were also evaluated. Wistar rats were randomly divided into four periods (2 days, 7 days and 15 days) and were assigned into four groups within each experimental period: Control, Sham, Inflammation and Immobilization. Real-time polymerase chain reaction, Western blot, immunofluorescence and muscle fiber CSA analyses were performed. The joint inflammation altered the mRNA levels of genes related to muscle atrophy, growth and differentiation, muscle mass regulation and proinflammatory factors in TA and soleus rat muscle after 2, 7, and 15 day. The joint inflammation increased the TNF-alpha protein expression only in the TA muscle at 7 days. The muscle fiber CSA was reduced in the TA at 7 days and, in the soleus muscle at 7 and 15 days. In both muscles TA and soleus, acute joint inflammation was able to stimulate the molecular pathway related to muscle atrophy with no reduction in AST fibers. Conversely, the chronic joint inflammation led to a differential response according to the muscle studied. In the TA muscle, the muscle fiber CSA reduction was related to the proteolytic pathway, while in the soleus, the muscle atrophy occurred without overexpression of genes related to the classical proteolysis pathway. These results suggest that, in soleus muscle, the atrophy could be mediated by other pathway than the ubiquitin-proteasome; suggesting the importance of the decreased protein synthesis in the reduction of protein content in the skeletal muscle. In addition, joint effusion also induced changes in gene expression, although without changes in the muscle fiber CSA. The results of this thesis have clinical relevance and indicate the importance of therapeutic interventions with the attempt to reduce the deleterious effects on muscles related to an inflamed joint. / A inflamação articular é uma causa frequente de limitação nas atividades de vida diária da população. Geralmente essa afecção afeta também os músculos que estão funcionalmente relacionados à articulação inflamada. A possível relação entre a inflamação articular e a expressão de genes relacionados à atrofia muscular, diferenciação e crescimento muscular e com o controle da massa em músculos funcionalmente relacionados à articulação inflamada, ainda não foram estudadas. O objetivo desta tese foi avaliar o efeito da inflamação aguda e crônica da articulação tíbio-társica induzida por carragenina, sobre a expressão de genes musculares relacionados à atrofia (atrogina-1 e MuRF-1), diferenciação e crescimento (MyoD), regulação da massa muscular (miostatina), e fatores próinflamatórios (TNF-alfa, p38MAPK e NFkB) nos músculos TA e sóleo de rato. Foram também avaliadas a expressão da proteína TNF-alpha e a área de secção transversa (AST) das fibras musculares. Ratos Wistar foram distribuídos em três períodos experimentais 2, 7 e 15 dias. Em cada período experimental os animais foram distribuídos em 4 grupos: Controle, inflamação, sham, imobilização. Foram realizadas análises da AST nos músculos TA e sóleo, da reação em cadeia de polimerase (PCR) em tempo real para todos os genes, e análise da expressão da proteína TNF-alpha com as técnicas Western blot e immunofluorescência. A inflamação articular gerou mudanças nos níveis de RNAm dos genes relacionados com a atrofia muscular, diferenciação e crescimento, regulação da massa muscular e de fatores pró-inflamatórios no músculo TA e sóleo de ratos, nos dias 2, 7 e 15. A inflamação articular também aumentou a expressão da proteína TNF-alfa apenas no músculo TA no dia 7. A AST das fibras musculares foi reduzida no TA nos dia 7, e no músculo sóleo nos dias 7 e 15. Em conclusão, em ambos os músculos TA e sóleo, a inflamação articular aguda estimulou a via molecular envolvida na atrofia muscular, sem redução na AST das fibras. Contrariamente, a inflamação articular crônica levou a um padrão de resposta diferente segundo o músculo analisado. No músculo TA, a redução na AST das fibras esteve relacionando com a atividade da via proteolítica, enquanto no sóleo, a atrofia muscular acorreu sem aumento na expressão de genes relacionados com a clássica via de degradação de proteína. Estes resultados sugerem que no músculo sóleo a atrofia pode ter sido regulada por outra via diferente da ubiquitina-proteosoma; indicando a importância que possui a redução da síntese protéica no controle do conteúdo total de proteína no músculo esquelético. Os resultados desta tese têm relevância clinica e orientam sobre a importância de utilizar recursos terapêuticos visando diminuir o efeito deletério que se sucedem nos músculos relacionados com uma articulação inflamada.

Made available in DSpace on 2016-06-02T20:19:10Z (GMT). No. of bitstreams: 1 2169.pdf: 1164220 bytes, checksum: 1a022742edbedbb87cd4f0eb001d00a4 (MD5) Previous issue date: 2008-12-15 / Universidade Federal de Minas Gerais / Clinic evidences show that joint injuries affect muscles related to that joint making them more sensitive to atrophy and loss of force. However, muscle molecular responses in those conditions has not been investigated yet. Objective: To evaluate the effects of tibiotarsal acute joint inflammation on the expression of genes related to muscle atrophy (atrogin-1 and MuRF-1), differentiation and growing (MyoD) and mass regulation (myostatin) in soleus and tibialis anterior (TA) rat muscles. Changes in muscular crosssectional area (CSA) will also be identified. Methods: Fifteen Wistar rats were randomized and divided into three groups: 1) Control, 2) Inflammation, 3) Sham. A 0.03 ml of τ-carragenan or saline solution was injected into right tibiotarsal joint, in both Inflammation and Sham groups, respectively. The muscle fiber CSA analysis was performed in soleus and TA muscles. Real time polymerase chain reaction (PCR) was used to investigate gene expression in soleus and TA muscles, after 48 h of inflammation or effusion joint induction. Results: No significant changes were observed either in muscle and animal weights or in muscle fiber CSA among the groups (p>0.05). The Inflammation group enhanced atrogin-1, MuRF-1 and myostatin gene expressions; however, it decreased MyoD expression in TA muscle. On the other hand, the soleus muscle increased MuRF-1 and MyoD expressions. The Sham group presented an increase of atrogin-1 and myostatin in the TA, while soleus muscle decreased myostatin and enhanced MyoD expression. Conclusion: Both the tibiotarsal acute joint inflammation and the effusion regulate the expression of the genes related to atrophy, differentiation and mass control in rat muscles differently. In addition, this regulation between fast (TA) and slow (soleus) twitch muscles is done differently. / Evidências clínicas indicam que afecções articulares acometem músculos relacionados à articulação tornando-os mais suscetíveis à atrofia e perda de força. Entretanto, as respostas moleculares dos músculos esqueléticos nessas condições não foram ainda estudadas. Objetivo: Avaliar o efeito da inflamação aguda da articulação tíbio-társica sobre a expressão de genes musculares relacionados à atrofia (atrogina-1 e MuRF-1), diferenciação e crescimento (MyoD), e regulação da massa (miostatina) nos músculos Sóleo e Tibial anterior (TA) do rato. Possíveis alterações na área de secção transversa (AST) das fibras musculares foram também identificadas. Métodos: Ratos Wistar foram distribuídos em três grupos (n=5/cada): 1) Controle, 2) Inflamação, 3) Sham. Uma solução de 0,03 ml de carragenina ou salina foi injetada na articulação tíbio-társica direita dos animais nos grupos Inflamação e Sham, respectivamente. Quarenta e oito horas após foram realizadas análises da AST em ambos os músculos sóleo e TA, análise da reação em cadeia de polimerase (PCR) em tempo real para todos os genes. Resultados: Não houve diferença na AST das fibras musculares entre os diferentes grupos experimentais, tanto para os músculos sóleo como os TA (p>0,05). O TA do grupo Inflamação apresentou um aumento na expressão de atrogina-1, MuRF-1 e miostatina com diminuição de MyoD. No sóleo houve aumento na expressão de MuRF-1 e MyoD. O TA do grupo Sham apresentou aumento na expressão de atrogina-1 e miostatina, enquanto que o músculo sóleo apresentou diminuição na expressão de miostatina e aumento de MyoD. Conclusão: Tanto a inflamação como a efusão da articulação tíbio-tarsica regulam diferentemente a expressão de genes relacionados à atrofia, diferenciação e controle de massa muscular. Essa regulação é diferente entre músculos de contração lenta (sóleo) e rápida (TA).