A Artrite Reumatóide (AR) é uma doença autoimune associada a poliartropatia inflamatória que acomete principalmente as articulações periféricas. Cerca de 1% da população mundial é afetada, sendo duas a três vezes mais prevalente em mulheres. Apresenta uma patogênese complexa e multifatorial. A sinóvia das articulações afetadas é infiltrada por linfócitos T e B, macrófagos e granulócitos. A sinóvia reumatóide adquire características proliferativas, formando o pannus, e invade a cartilagem articular e o osso, levando à destruição da arquitetura normal da articulação e perda de função. Em vários modelos de doenças autoimunes, a ausência ou diminuição da expressão de proteínas reguladoras do complemento tem sido observada, associada com o agravamento dos sintomas clínicos, sendo que, muitos destes casos, a superativação do sistema complemento pode ser a causa da exacerbação da doença. O presente artigo tem por objetivo revisar os principais aspectos relacionados à regulação do sistema complemento na artrite reumatóide, a fim de propiciar uma melhor compreensão do potencial papel desse sistema na fisiopatologia da doença. / Rheumatoid arthritis (RA) is an autoimmune disease associated with polyarticular inflammatory synovitis that affects mainly the peripheral joints. About 1% of the world population is affected, and it is two to three times more prevalent in women. RA has a complex and multifactorial pathogenesis. The rheumatoid synovium acquires proliferative characteristics, forming the pannus, and invades cartilage and bone, leading to the destruction of normal architecture and loss of function. In several models of autoimmune diseases, the absence or decreased expression of complement regulatory proteins has been observed, associated with worsening of the clinical symptoms, and many of these cases the over-activation of the complement system is the cause of disease exacerbation. This article aims to review the main aspects related to regulation of the complement system in rheumatoid arthritis in order to provide a better understanding of the potential role of this system in the pathophysiology of the disease.

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Myasthenia gravis (MG) is an autoimmune disease whose name means "grave muscular weakness". MG is a rare disease affecting only 200-400 persons per million and the characteristic symptoms include muscle weakness, particularly in highly active voluntary muscles. MG affects the neuromuscular junction in an antibody-mediated manner, resulting in impaired nerve-muscle cell communication in affected individuals. Specifically, two main proteins are targeted: nicotinic acetylcholine receptors (ACh receptors) and a muscle-specific tyrosine kinase (MuSK). Previous studies have discovered the mechanism of MG pathogenesis but the exact mechanisms which cause the failure to maintain self-tolerance have not been discovered. Based on current knowledge of MG, this paper will explore potential causes of the disease and provide numerous hypotheses directed at future research opportunities.

C1QTNF5 is a 25kDa short chain collagen of unknown function which is mutated in late-onset retinal macular degeneration (L-ORMD). L-ORMD is an autosomal dominant disease characterised by sub-retinal pigment epithelial deposits leading to photoreceptor death and visual loss and shows several similarities to age-related macular degeneration (AMD). A Tyr402His polymorphism in complement factor H (CFH), a regulatory protein in the innate immune system, has been associated with increased risk of AMD. C1QTNF5 and CFH are both expressed and secreted by the retinal pigment epithelium (RPE) which supports photoreceptors and is responsible for phagocytosis of shed rod photoreceptor outer segments (ROS). The properties of the normal C1QTNF5 and disease-associated Ser163Arg mutation were examined in detail, including protein characterisation, cellular processing and function. Recombinant wild type and mutant C1QTNF5 were produced and their multimerisation and solubility functions compared. Both proteins were found to be soluble and to form similar multimeric species which were resistant to reducing conditions, as seen in other short chain collagens. Due to the similarities between LORMD and AMD, a proposed interaction between C1QTNF5 and CFH was investigated. CFH is composed of 20 short consensus repeats (SCR) and interactions were confirmed between C1QTNF5 and both CFH and SCR modules 7-8 and 19-20. CFH showed a greater affinity for mutant C1QTNF5 compared with wild type on the basis of surface plasmon resonance assays. Stably transfected RPE-derived cell lines were created which expressed either wild type or mutant C1QTNF5. Both proteins were found to be secreted and showed similar cellular processing with no evidence of aggregation or retention of the mutant protein within the endoplasmic reticulum. In order to investigate C1QTNF5 function, phagocytosis of ROS by the stably transfected cell lines was carried out. Cells expressing wild type C1QTNF5 showed greater ROS phagocytosis compared with mutant C1QTNF5-expressing or untransfected cells. Addition of anti-C1QTNF5 antibody increased ROS phagocytosis further. In summary, it is proposed that wild type and mutant C1QTNF5 are secreted by the RPE where they interact with CFH. C1QTNF5 is also shown to have a role in ROS phagocytosis, with mutation in C1QTNF5 affecting phagocytosis efficiency, which may contribute to sub-RPE deposit formation. The results suggest that CFH may also be involved in this process, suggesting a common pathogenic pathway between L-ORMD and AMD.

The complement system is an important immune system mechanism involved in both the recognition and elimination of invading pathogens. It is activated by three different pathways: The classical pathway, which relies on binding of C1, and results in the cleavage of C4 and C2 through activation of C1r and C1s; the alternative pathway that relies on the spontaneous hydrolysis of C3 and the lectin pathway. The lectin pathway is activated by binding of Mannan-binding lectin (MBL) or the ficolins (L-ficolin, H-ficolin and M-ficolin) to microbial binding motifs, and the subsequent activation of the MBL-associated serine proteases (MASP) 1/ 2/ 3. Of these MASP2 has been identified as the enzyme responsible for the activation of complement by C4 and C2 cleavage. The work presented here will focus on four different aspects of the lectin pathway: specificity and stoichiometry of the L-ficolin protein complex, expression of H-ficolin, substrate characterization for MASP1 and investigation of the prothrombin activation potential of MASP2. L-ficolin binding specificity was investigated using glycan array technology, and it was found that L-ficolin, instead of recognizing single monosaccharides like MBL, instead binds to extended oligosaccharide structures. The binding to these was dependent not only on the presence of acetyl groups, but also on their orientation in space. It was also found that L-ficolin in serum is found as a multimeric protein complex composed of 18 polypeptide chains and associated with one MASP dimer. The expression of H-ficolin resulted in the generation of a stable mammalian cell line producing oligomerized and biologically functional H-ficolin. MASP1 substrate specificity was investigated by two different procedures. Firstly fractionated plasma was subjected to MASP1 treatment in an attempt to identify a plasma protein substrate. This did not yield any substrate candidates, since only cleavage of the protease inhibitor α-2-macroglobulin could be detected. Additionally the thrombin-like activity of MASP1 was investigated through cleavage experiments done with factor XIII and fibrinogen. These experiments showed that the factor XIII cleavage site for MASP1 and thrombin is identical. This was also found for the fibrinogen β-chain but not for the α-chain showing that MASP1 interaction with fibrinogen is distinct from that of thrombin. An earlier observation that MASP2 was capable of activating prothrombin and generating thrombin was further characterized. Here it was shown that the activation of prothrombin by MASP2 is identical to that by factor Xa, which is the enzyme undertaking this role in the coagulation system, and that the activation can result in deposition of fibrin on the surface upon which MASP2 is bound. The prothrombin activation potential of MASP2 was also utilized to develop a new MASP2 activity assay, which was shown to be capable of measuring MASP2 activity, when MASP2 is bound, via MBL (or L-ficolin) to appropriate surfaces.

<p>Complement factor H (CFH) is a major susceptibility gene for age-related macular degeneration (AMD); however, its impact on AMD pathobiology is unresolved. Here, the role of CFH in the development of AMD pathology in vivo was interrogated by analyzing aged Cfh+/- and Cfh-/- mice fed a high fat, cholesterol-enriched diet. Strikingly, decreased levels of CFH led to increased sub-retinal pigmented epithelium (RPE) deposit formation, specifically basal laminar deposits, following high fat diet. Mechanistically, our data show that deposits are due to CFH competition for lipoprotein binding sites in Bruch’s membrane. Interestingly and despite sub-RPE deposit formation occurring in both Cfh+/- and Cfh-/- mice, RPE damage accompanied by loss of vision occurred only in old Cfh+/- mice. We demonstrate that such pathology is a function of excess complement activation and C5a production, associated with monocyte recruitment, in Cfh+/- mice versus complement deficiency in Cfh-/- animals. Due to the CFH dependent increase in sub-RPE deposit height we interrogated the potential of CFH as a novel regulator of Bruch’s membrane lipoprotein binding and show, using human Bruch’s membrane explants, that CFH removes endogenous human lipoproteins in aged donors. Interestingly, although the CFH H402 variant shows altered binding to BrM, this does not affect its ability to remove endogenous lipoproteins. This new understanding of the complicated interactions of CFH in AMD-like pathology provides an improved foundation for the development of targeted therapies for AMD.</p> / Dissertation

Master of Science / Division of Biology / Sherry D. Fleming / Professional phagocytes play a critical role in maintaining homeostasis within a host through phagocytic, microbicidal, and inflammatory activity. Complement receptors (CR) and toll-like receptors (TLRs) aid in phagocytosis and stimulate these cells to enhance the immune response. Environmental factors such as hypoxia, prevalent at sites of tissue damage or infection, induce a similar effect. Systemic components such as opsonins may further enhance phagocyte activity. Similar to professional phagocytes, B1 B cells exhibit a broad range of immunological activity as well as expression of CRs and TLRs. Despite extensive studies with other phagocytes, the effects of CRs and TLRs expression, hypoxic stimulation, or opsonization on B1 B cell function remain unclear. We tested the hypothesis that TLR2 stimulation, hypoxia, CR2 expression, or opsonins would enhance B1 B cell phagocytic and inflammatory activity. Negatively selected peritoneal cavity B1 B cells from the (PerC) of wild type, Tlr2[superscript]-[superscript]/[superscript]-, and Cr2[superscript]-[superscript]/[superscript]- mice, or a B1 B-like cell line, Wehi 231, were subjected to normoxia or hypoxia with or without particles for phagocytosis, TLR2 agonists, or CR2 ligands. The PerC of Tlr2[superscript]-[superscript]/[superscript]- mice contained an altered B1 B cell subset distribution while Cr2[superscript]-[superscript]/[superscript]- mice exhibited a normal repertoire. We demonstrated that hypoxia significantly downregulated inflammatory cytokine production by B1 B cells, while upregulating phagocytic activity in a TLR2 or CR2 dependent manner. TLR2 or CR2 deficiency altered constitutive production of B1 B cell associated cytokines. The CR2 ligand C3d, an opsonin, significantly enhanced the phagocytic activity of B1 B cells but failed to stimulate cytokine production. However, Cr2[superscript]-[superscript]/[superscript]- B1 B cells phagocytosed C3d-coated particles suggesting multiple CR may play a role in B1 B cell phagocytosis. Overall, the data suggest TLRs, CRs, hypoxia, and opsonization all contribute to B1 B cell effector function similar to professional phagocytes.