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dc.contributor.authorAnsari, Umer Saeed-
dc.description.abstractRheumatoid arthritis (RA) is a crippling, chronic inflammatory disease caused by an impaired immune response and mediated by a complex interplay of cytokines and a variety of inflammatory mediators. In Pakistan, rheumatoid arthritis affects a significant number of elderly population, leading to multiple extra-articular complications and eventual mortality. Unfortunately, few studies have been conducted which demonstrated the complex interactions of different inflammatory mediators implicated in the pathogenesis of RA in adult population in Pakistan. Limited data is, however, available on small population groups demonstrating the significance of few selected biomarkers of oxidative stress and cytokines implicated in the pathogenesis of RA. The current study is based on measuring the levels of a spectrum of cytokines, inflammatory mediators and anti-oxidants within the affected joints of 288 patients diagnosed with RA in the adult male population in province Punjab of Pakistan, and comparing the findings with 100 healthy controls. The 288 diagnosed patients of RA were selected and synovial fluid was aspirated from the affected joints by needle aspiration under aseptic conditions. The levels of many cytokines, antioxidants, biomarkers of oxidative stress and inflammatory mediators were measured in the synovial fluid samples, includingmalondialdehyde (MDA), isoprostanes, 8-hydroxy-deoxy guanosine (8-OHdG), 4-hydroxy 2-nonenal (HNE), antioxidants glutathione (GSH), glutathione peroxidase (GPx), glutathione reductase (GRx), catalase (CAT), superoxide dismutase (SOD), Interleukins (IL-2, IL-4, IL-6, IL-8, IL-13, IL-17, IL- 21 & TNF-α ), inducible nitric oxide synthase enzyme (iNOS), Nitric oxide (NO), advanced oxidation protein products (AOPPs), advanced glycation end products (AGEs), basic calcium phosphate (BCP), matrix metalloproteinases (MMPs 1, 2, 3, 8, 9 & 10), lipopolysaccharides (LPS), calcium, pyrophosphates, neutrophils and cyclo-oxygenase-1 (COX-1). The characteristic autoimmunity in RA was triggered by activation of resident dendritic cells in the affected joints, due to altered or implanted antigens, which presented these antigens to T-helper cells, notably, CD4+ cells. These released a cascade of cytokines, thus contributing to the chronicity of RA. Subsequent release of IL-17 by Th-17 lymphocytes recruited and activated macrophages within the joint synovium, which were pivotal in the pathogenesis of RA. These activated macrophages release IL-1, which not only activated the joint fibroblasts but CHAPTER SIX SUMMARY UMER, 2017 88 recruited neutrophils in the joints of RA patients, which causedjoint destruction in RA. Release of IL-6 by macrophages led to enhanced B-cells differentiation into plasma cells, which synthesized and secreted large number of gamma-globulins, initiating a vicious auto-immune response. In the present study, however, IL-21 was more strongly related to joint damage by increasing the activity of TNF-α and MMP-3 released early in the disease. In addition, MMP-3 activity was most significant in the present study, as compared to other MMPs involved in the pathogenesis of RA. Macrophages generated NO in excess, causing production of reactive nitrogen species (RNS), such as nitroxyl ions, nitrosyl ions and peroxynitrites, which accentuated the oxidative damage to synovial membrane in joints of RA patients. IL-21 secreted by macrophages, potentially acted on distant sites, induced osteoclastogenesis by highly expressing the RANKL genes, and these activated osteoclasts were primarily responsible for bone resorption and subsequent pannus formation in the inflamed joints. The activated fibroblasts of synovial tissue also led to enhanced expression of RANKL, thus promoting osteoclasts activity within the inflamed joints. Angiogenesis was typically promoted in affected joints, mediated by TNF-α released by activated macrophages via enhanced expression of VEGF within the narrow joint vicinity. The newly recruited neutrophils dominated in the inflammatory and oxidative destructive processes in the RA joints by generating large amounts of ROS, MMPs and simultaneously releasing cytokines and chemokines which activated the underlying chondrocytes and further promote macrophage activation, thus forming a vicious, positive feed-back loop in RA joints. The destructive proteases released by neutrophils eroded the articulating bones and underlying joint cartilages and degraded the matrix glycoasaminoglycans such as hyaluronic acid. The antioxidant systems in RA were significantly compromised in RA contributing to enhanced lipid peroxidation and progressive joint destruction. Activity of CAT and GRx were observed to be significantly reduced in the current study on synovial fluids of affected joints, compared with healthy controls. It was observed that NO was more strongly correlated with oxidative damage as compared singlet oxygen. Measurement of important biomarkers of oxidative stress showed significant increase in the levels of AOPPs, AGEs and notably MDA. High levels of MDA were observed in the joint fluids of patients with RA, suggesting a role of reactive radicals in the pathogenesis of RA. 8-OHdG was another critical biomarker of oxidative stress and DNA damage, and its levels in joint fluid were CHAPTER SIX SUMMARY UMER, 2017 89 observed to be more strongly correlated to MMP-3 activity, compared with other oxidative biomarkers. Similarly, high AOPPs levels suggested impaired activation of joint synoviocytes, as well as enhanced apoptosis of joint chondrocytes, which, in turn, generated excessive amounts of reactive oxygen radicals (ROS) perpetuating the oxidative insult. Excessive, non-enzymic, glycation of joint proteins forms AGES, which activated the pro-inflammatory Nf-kB pathway, leading to high tendency of bone damage in RA due to enhanced proteolysis. Deposition of excessive amounts of basic calcium phosphate (BCP) was observed in the current study, suggesting its pathogenic role in RA. BCP deposition activated joint fibroblasts, which, in turn, released MMPs 1, 8 and 9, which were most critical in synovial remodeling and cartilage erosion. BCP deposition activated macrophages, which released a cascade of cytokines and chemokines, such as TNF-α which has multi-dimensional roles in the pathological damage to joints of patients with RA. The present study demonstrated high BCP levels in the joints of RA patients, suggesting its critical role in aggravation of inflammatory destruction of RA joints, in particular, its‟ strong correlationship with MMP-3 activity. Lipopolysaccharides (LPS) are bacterial cell wall components, known to induce immune responses in sensitized individuals, notably activation of B-cell induced humoral immunity, contributing to exaggeration of autoimmune response characteristically seen in RA. Presence of LPS in joints vicinity increased the release of Gamma interferons (γ-IFN), which further exacerbated the immune response in RA. No clear relationship, however, was observed between the pathogenesis of RA and synovial fluid levels of calcium and pyrophosphates (PPi) in the inflamed joints. It was noted that the activity of COX-1 was significantly increased in the joints of RA patients, suggesting the possible role of prostaglandins in accentuating the local inflammatory damage. PGE2 in particular enhanced the apoptosis of joint chondrocytes leading to progressive cartilage destruction in the joints of patients with RA. In the present study, COX-1 activity was observed to be strongly correlated with BCP deposition in the joints of patients with RA. The current study, therefore, elucidated the activity and synovial fluid concentrations of a variety of cytokines, chemokines and inflammatory mediators, oxidative biomarkers and pro inflammatory pathways involved in the pathogenesis of RA in the selected adult Pakistani population, which might provide better understanding of the disease progression and interplay of cytokines in the joint destruction characteristically seen in the patients of rheumatoid arthritis.en_US
dc.description.sponsorshipHigher Education Commission Pakistanen_US
dc.publisherThe University of Lahore, Lahore.en_US
dc.subjectBiological & Medical Sciencesen_US
dc.titleAssessment of Circulating Biochemical Markers of Diagnostic Importance and Role of Antioxidative Status in Rheumatoid Arthritis: Study from Local Population of Punjab-Pakistanen_US
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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