Osteoarthritis (OA) is a degenerative disease of diarthrodial joints causing pain and loss of joint function. Etiology is heterogeneous, but commonly involves inflammation arising from impairment of normal tissue homeostasis and/or function. A cycle of low-grade inflammation and global tissue degradation causes alteration of tissue morphology and function via primary mechanisms or inability to withstand physiological forces. Current therapies variably ameliorate symptoms but do not modify progression. Mesenchymal stem cells (MSCs) have multi-modal properties but are ineffective in ameliorating equine OA. However, anti-inflammatory activities of bone marrow derived MSCs (BMSCs) are enhanced by three-dimensional spheroid culture so equine BMSC (eBMSC) spheroids could inhibit intra-articular inflammation. The overarching hypothesis is that eBMSCs can be enhanced to produce an allogeneic eBMSC therapy that inhibits intra-articular inflammation. In vitro experiments compared differences in anti-inflammatory phenotype between spheroid and traditionally cultured monolayer eBMSCs, the viability and health of eBMSC spheroids administered through needles, and the effects of allogeneic donor on the anti-inflammatory potential of eBMSC spheroids. A model of equine LPS induced synovitis was used to investigate anti-inflammatory efficacy of spheroid eBMSCs compared to placebo or monolayer eBMSCs in vivo. eBMSCs aggregate into spheroids that have stable stem cell marker expression with increased secretion and gene expression of IL-6 and PGE2, and gene expression of SDF-1 and TSG-6. IFN𝛾 and TNFα were not produced by eBMSC spheroids and IL-10 production varied between individuals. Spheroids maintain higher viability and lower senescence than monolayer eBMSCs after injection through a needle and form in high-throughput culture without detrimental effects on expression of TSG-6, IL-6 and PGE synthases that denote an anti-inflammatory phenotype. Additionally, there is significant variation in this phenotype depending on the eBMSC donor. eBMSC spheroids reduced total nucleated cell counts and objective lameness measurements at peak levels of intra-articular inflammation compared to monolayer cultured eBMSCs in vivo. In summary, spheroids increase anti-inflammatory potential of eBMSCs and are practical for clinical use. Increased anti-inflammatory efficacy was demonstrated in a model of in vivo inflammation. This dissertation provides an understanding of the anti-inflammatory activities of eBMSC spheroids that can be used to develop an OA therapy. / Ph. D.

Osteoarthritis (OA) can be debilitating and career-ending for horses. Current treatments offer temporary and symptomatic relief, but potentially deleterious side effects. Bone marrow mononuclear cells (BMNC) are a rich source of macrophage progenitors that are anti-inflammatory and promote inflammation resolution. The objective of this study was to evaluate the ability of intra-articular BMNC therapy to improve clinical signs of naturally occurring equine OA. Horses presenting with clinical and radiographic evidence of moderate OA in a single joint were randomly assigned to 1 of 3 treatments: saline (negative control), triamcinolone (positive control), or BMNC (treatment group). Horses were subjectively and objectively evaluated for lameness and synovial fluid collected (cytology and cytokine/growth factor quantification) at 0, 7, and 21 days post-injection. Data were analyzed using General Estimating Equations with significance set at P<0.05. There were no adverse effects noted in any treatment group. No significant differences in synovial fluid cytology parameters, objective/subjective lameness scores, nor joint circumference were found between treatment groups at any time point. Within treatment groups, joint circumference did not change over time for saline- and triamcinolone-treated horses. However, joint circumference and objective lameness decreased significantly within BMNC-treated horses between Days 0 and 21 and Days 7 and 21. Lameness improved in saline-treated horses from 0 to 21 days, but did not improve in triamcinolone-treated horses. The decreased lameness and lack of adverse effects in the BMNC-treated horses in our study support a larger clinical trial using BMNC. / Master of Science / Osteoarthritis (OA) is a common source of joint pain in people and horses. Current treatments provide only partial and/or temporary relief. As a result, there is an urgent need for more effective and long-lasting treatment options. Arthritis is characterized by uncontrolled joint inflammation and progressive cartilage and bone destruction. Macrophages are cells within normal joints that function to resolve mild inflammation, maintaining joint health. However, when physiologic functions are overwhelmed, macrophages perpetuate inflammation through the recruitment of additional cell types to cope with the increased demands for repair. If this process is appropriately accomplished, macrophages resolve the inflammation, thereby enabling recovery and repair within the joint. Bone marrow aspirate is an excellent source of bone marrow-derived macrophage precursors (bone marrow mononuclear cells or BMNC) that have been shown to reduce joint inflammation and lameness in people and horses. The objective of our clinical trial was to evaluate the ability of intra-articular BMNC to improve clinical signs of naturally occurring OA in horses. BMNC treatment was compared to a placebo injection of saline and a standard-of-care in horses, corticosteroids. There were no adverse effects of BMNC treatment and BMNC-treated horses had significantly reduced joint circumference and lameness after 21 days. Synovial fluid cytology parameters did not differ significantly between treatment groups at any time point. In summary, BMNC are exciting because a horse can be treated with its own cells without the need for specialized equipment, and have the potential to naturally benefit thousands of people and horses suffering from arthritis.

Made available in DSpace on 2014-07-29T15:28:59Z (GMT). No. of bitstreams: 1 TESE final vander fernandes.pdf: 1050911 bytes, checksum: ccd739107090ffd6787f00090aa72602 (MD5) Previous issue date: 2012-08-09 / RA is debilitating autoimmune disease with serious physical, emotional and economic consequences, which affects about 0.5 to 1% of the adult population worldwide. Significant advances in the recognition of the pathophysiology of disease and injury mechanisms allowed the development of new treatment options with drugs that block inflammatory mediators involved in the mechanism of the disease, known as biologics. These drugs are expensive and of continuous use. They are complex to use due care and management of storage and the risk of immediate and delayed adverse reactions. These therapies are available at the Health System for 12 years, is necessary to evaluate the effectiveness of this drug group in the Brazilian public health system in the treatment of rheumatoid arthritis. OBJECTIVES: a) analyze the effectiveness of biological therapies in patients with rheumatoid arthritis of the State of Mato Grosso, through the assessment of disease activity index DAS28-ESR b) Review the laboratory characteristics of these patients; c) examine the safe use these therapies and to describe adverse events. d) analyze the efficiency of the program dispensing of biological therapies in the State of Mato Grosso, Brazil. METHODS: Two groups of patients with rheumatoid arthritis users Pharmacy High Cost of the State Secretariat of Health of Mato Grosso (FAC / SES) or the University General Hospital. Group I included 68 patients who were already in use of biological therapies. These were assessed for disease activity at the time of inclusion in the study and asked about the historical use of biological drugs, satisfaction with access to treatment and adverse events. Laboratory tests prior to treatment were obtained retrospectively. In group II were enrolled and followed prospectively for 24 weeks, 30 patients with early use of biologic therapies. They were evaluated at baseline and at weeks 4, 8, 12 and 24, the disease activity by DAS28-ESR, laboratory tests, satisfaction with access to treatment and adverse events. RESULTS: The groups were similar in demographic characteristics and laboratory related to rheumatoid arthritis. In group I, 80.6% of patients expressed satisfaction with the care of the FAC / SES. The average use of biological was 12.3 months, and 20.9% (14/68) reported an adverse event, all non-serious. The first medication was discontinued in 26.5% (18/68) of patients, 55.6% (10/18) for loss of efficacy and 22.2% (4/18) adverse events. The mean value of DAS28-ESR was 3.8, and 41.2% were in remission or low disease activity, 39,7% in moderate activity and 19,1% in intense activity. In group II, 91.7% of patients expressed satisfaction with the care provided by FAC / SES. Laboratory evaluation was significant change in the conversion of ANA from 13.3% to 23.3% and anti-DNA (ds), 0 to 6.7%, but no clinical significance. The anti-CCP (2) remained positive and stable at 63.3%. There was an adverse reaction in 23% (7/30) of patients. The only suspension of the first plants were by no severe adverse event (hypersensitivity). Following the DAS28-ESR was a significant reduction in the initial mean value of 4.3 to 3.3 (p = 0.002) after 24 weeks of treatment, 60% of patients were in remission or low disease activity, 23, 3% in moderate activity and 16.7% in intense activity. The average value of ESR-DAS28 after use of the biologic group II was significantly lower than in group I (p= 0.042). CONCLUSIONS: 1) Treatment with biologic therapies in the State of Mato Grosso was effective in prospective group. The response in DAS28-ESR was significantly better in the group where the monitoring and control has been programmed with a routine test and calculate the index of disease activity were used. 2) Among the laboratory characteristics were significant conversion in the profile of autoantibodies, confirming the potential immunogenicity of these drugs inducing the formation of autoantibodies, but no clinical significance in the study group. The anti-CCP remained stable in 63% of patients (group II). 3) Biological therapies were safe. There were no serious adverse events reported. There were no reports of infection or death. The most common reason for discontinuation of the first was the loss of biological effectiveness. 4) Patients with rheumatoid arthritis in the use of biologic therapies in the State of Mato Grosso showed satisfaction with the services offered by SES / MT and the majority of users in the metropolitan area of Cuiabá. The program dispensing biological therapies showed higher efficiency when linked to a reference center for this particular type of treatment. / A artrite reumatoide é doença autoimune, debilitante, com graves consequências físicas, econômicas e emocionais, que afeta cerca de 0,5 a 1% da população adulta mundial. Avanços significativos no reconhecimento da fisiopatologia da doença e mecanismos de lesão permitiram desenvolvimento de novas opções de tratamento através de medicamentos que bloqueiam mediadores inflamatórios envolvidos no mecanismo da doença, denominados biológicos. Esses medicamentos são de alto custo e de uso contínuo. São de uso complexo devido aos cuidados de armazenagem e administração além do risco de reações adversas imediatas e tardias. Essas terapias estão disponíveis no Sistema Único de Saúde há 12 anos, sendo necessário avaliar a efetividade deste grupo de medicamentos no sistema de saúde público brasileiro no tratamento da artrite reumatoide. OBJETIVOS: a) analisar a eficácia das terapias biológicas em pacientes com artrite reumatoide do Estado de Mato Grosso, através da avaliação do índice de atividade da doença DAS28-VHS; b) analisar as características laboratoriais destes pacientes; c) analisar a segurança no uso destas terapias e descrever os eventos adversos. d) analisar a eficiência do programa de dispensação das terapias biológicas no Estado de Mato Grosso-Brasil. MÉTODOS: Utilizou-se dois grupos de pacientes com artrite reumatoide usuários da Farmácia de Alto Custo da Secretaria de Estado de Saúde de Mato Grosso (FAC/SES) ou do Hospital Geral Universitário. No grupo I foram incluídos 68 pacientes já em uso de terapias biológicas. Esses foram avaliados quanto à atividade da doenca no momento de inclusão no estudo e questionados sobre o histórico de uso das medicações biológicas, satisfação com o acesso ao tratamento e eventos adversos. Os exames laboratoriais prévios ao tratamento foram obtidos de forma retrospectiva. No grupo II foram admitidos e acompanhados de forma prospectiva por 24 semanas, 30 pacientes em início de uso de terapias biológicas. Esses foram avaliados no início do tratamento e nas semanas 4, 8, 12 e 24, quanto à atividade da doença através do DAS28-VHS, exames laboratoriais, satisfação com o acesso ao tratamento e eventos adversos. RESULTADOS: O grupos estudados foram semelhantes em características demográficas e laboratoriais relacionados a doença reumatoide. No grupo I 80,6% dos pacientes demonstraram satisfação com o atendimento da FAC/SES. O tempo médio de uso de biológicos foi de 12,3 meses, sendo que 20,9% (14/68) relataram algum evento adverso, todos não graves. A primeira medicação foi suspensa em 26,5% (18/68) dos pacientes, sendo 55,6% (10/18) destes por perda de eficácia e 22,2% (4/18) por eventos adversos. O valor médio do DAS28-VHS foi 3,8, sendo que 41,2% estavam em remissão ou baixa atividade da doença, 39,7% em atividade moderada e 19,1% em atividade intensa. No grupo II, 91,7% dos pacientes demonstraram satisfação com o atendimento prestado pela FAC/SES. Na avaliação laboratorial houve alteração significativa na conversão do FAN de 13,3% para 23,3 % e do anti-DNA(ds), de 0 para 6,7%, porém sem repercussão clínica. O anti-CCP(2) manteve-se positivo e estável em 63,3%. Houve reação adversa em 23% (7/30) dos pacientes. A única suspensão do primeiro biológico foi por evento adverso não grave (hipersensibilidade cutânea). No seguimento do DAS28-VHS houve uma redução significativa do valor médio inicial de 4,3 para 3,3 (p:0,002) após 24 semanas de tratamento, sendo que 60% dos pacientes estavam em remissão ou baixa atividade da doença, 23,3% em atividade moderada e 16,7% em atividade intensa. O valor médio do DAS28-VHS após uso de biológicos no grupo II foi significativamente menor do que no grupo I (p:0,042). CONCLUSÕES: 1) O tratamento com terapias biológicas no Estado de Mato Grosso foi eficaz no grupo prospectivo. A resposta no DAS28-VHS foi significativamente melhor no grupo onde o seguimento foi programado e o controle com exames de rotina e cálculo do índice de atividade da doença foram utilizados. 2) Dentre as características laboratoriais houve conversão significativa no perfil de auto-anticorpos, confirmando o potencial de imunogenicidade destas drogas induzindo a formação de auto-anticorpos, porém sem repercussão clínica no grupo estudado. O anti-CCP manteve-se estável em 63% dos pacientes. 3) As terapias biológicas foram seguras. Foram relatados eventos adversos não sérios. Não houve relato de infecção ou óbito. O motivo mais frequente de suspensão do primeiro biológico foi a perda de eficácia. 4) Os pacientes com artrite reumatoide em uso de terapias biológicas no Estado de Mato Grosso demonstraram satisfação com a prestação de serviços oferecida pela SES/MT sendo a maioria dos usuários da região metropolitana de Cuiabá. O programa de dispensação de terapias biológicas mostrou maior eficiência quando vinculado à um centro de referência específico para esse tipo de tratamento.