Although the role of B-cells in autoimmunity is not completely understood, their importance in the pathogenesis of autoimmune diseases has been more appreciated in the past few years. It is now well known that they have roles in addition to (auto) antibody production and are involved by different mechanisms in the regulation of T-cell mediated autoimmune disorders. The evolution of an autoimmune disease is a dynamic process, which takes a course of years during which complex immunoregulatory mechanisms shape the immune repertoire until the development of clinical disease. During this course, the B-cell repertoire itself is influenced and a change in the distribution of immunoglobulin heavy and light chain genes can be observed. B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis (RA), highlighting also the central role of B-cells in the pathogenesis of this disease. Nevertheless, the mechanism of action is unclear. It has been hypothesised that B-cell depletion is able to reset deviated humoral immunity. Therefore we wanted to investigate if transient B-cell depletion results in changes of the peripheral B-cell receptor repertoire. To address this issue, expressed immunoglobulin genes of two patients suffering from RA were analysed; one patient for the heavy chain repertoire (patient H), one patient for the light chain repertoire (patient L). Both patients were treated with rituximab, an anti-CD20 monoclonal antibody that selectively depletes peripheral CD20+ B-cells for several months. The B-cell repertoire was studied before therapy and at the earliest time point after B-cell regeneration in both patients. A longer follow-up (up to 27 months) was performed in patient H who was treated a second time with rituximab after 17 months. Heavy chain gene analysis was carried out by nested-PCR on bulk DNA from peripheral B-cells using family-specific primers, followed by subcloning and sequencing. During the study, patient H received two courses of antibody treatment. B-cell depletion lasted 7 and 10 months, respectively and each time was accompanied by a clinical improvement. Anti-CD20 therapy induced two types of changes in this patient. During the early phase of B-cell regeneration, we noticed the presence of an expanded and recirculating population of highly mutated B-cells. These cells expressed very different immunoglobulin VH genes compared before therapy. They were class-switched and could be detected for a short period only. The long-term changes were more subtle. Nevertheless, characteristic changes in the VH2 family, as well as in specific mini-genes like VH3-23, 4-34 or 1-69 were noticed. Some of these genes have already been reported to be biased in autoimmune diseases. Also in autoimmune diseases, in particular in RA, clonal B-cells have been frequently found in the repertoire. B-cell depletion with anti-CD20 antibody resulted in a long term loss of clonal B-cells in patient H. Thus, temporary B-cell depletion induced significant changes in the heavy chain repertoire. For the light chain gene analysis, the repertoire changes were analysed separately for naive (CD27-) and memory (CD27+) B-cells. Individual CD19+ B-cells were sorted into CD27- and CD27+ cells and single cell RT-PCR was performed, followed by direct sequencing. During the study, patient L received one course of antibody treatment. B-cell depletion lasted 10 months and the light chain repertoire was studied before and after therapy. Before therapy, some differences in the distribution of VL and JL genes were observed between naive and memory B-cells. In particular, the predominant usage of Jk-proximal Vk genes by the CD27- naive B-cells indicated that the receptor editing was less frequent in this population compared to memory cells. In VlJl rearrangements also, some evidence for decreased receptor editing was noticed, with the overrepresentation of the Jl2/3 gene segments. The CDR3 regions of naive and memory cells showed different characteristics: the activity of the terminal deoxynucleotidyl transferase and exonuclease in Vl(5’) side was greater in memory cells. Also in the light chain repertoire, we observed some changes induced by the B-cell depletive therapy. There was a tendency of a less frequent usage of Jk-proximal Vk genes in the naive population. Some Vl genes, previously described in autoimmune diseases and connected to rheumatoid factor activity, such as 3p, 3r, 1g, were not found after therapy. The different characteristics of the CDR3 regions of VlJl rearrangements were not observed anymore. Very significantly, the ratio Vk to Vl was shifted toward a greater usage of Vk genes in the naive population after therapy. Taken together, these results indicate that therapeutic transient B-cell depletion by anti-CD20 antibody therapy modulates the immunoglobulin gene repertoire in the two RA patients studied. Measurable changes were observed in the heavy chain as well as in the light chain repertoire, which may be relevant to the course of the disease. This also supports the notion that the composition of the B-cell repertoire is influenced by the disease and that B-cell depletion can reset biases that are typically found in autoimmune diseases. / Obwohl die Rolle der B-Zellen für den Verlauf einer Autoimmunerkrankung noch nicht vollständig klar ist, so hat ihre Bedeutung für die Autoimmunpathogenese in den letzten Jahren zugenommen. Man weiß mittlerweile sehr wohl, dass ihre Rolle über die reine Produktion von (Auto-)antikörpern hinausgeht und sie z.B. an der Regulation T-Zellvermittelter Autoimmunstörungen beteiligt sind. Das B-Zellrepertoire selbst wird im Laufe einer autoimmunen Erkrankung durch solche immunregulatorische Prozesse beeinflusst. B-Zelldepletive Therapien zeigen vorteilhafte Effekte bei Patienten mit Rheumatoider Arthritis, was wiederum die zentrale Rolle von B-Zellen in der Pathogenese unterstreicht. Nichtsdestotrotz bleibt der genaue Mechanismus unklar. So wird z.B. spekuliert, dass mittels B-Zell-Depletion eine Neugenerierung der humoralen Immunität erreicht wird. Vor diesem Hintergrund untersuchten wir, ob eine vorübergehende B-Zell-Depletion zu Veränderungen im peripheren B-Zellrepertoire führt. Hierfür wurde das B-Zell-Rezeptorrepertoire von zwei Patienten mit Rheumatoider Arthritis im einen Fall bezüglich der schweren Kette (Patient H) und im anderen Fall bezüglich der leichten Kette (Patient L) analysiert. Beide Patienten wurden mit Rituximab, einem monoklonalen anti-CD20 Antikörper, der zu einer selektiven und über mehrere Monate anhaltenden Depletion peripherer CD20+-B-Zellen führt, behandelt. Das B-Zellrepertoire wurde für beide Patienten unmittelbar vor Therapie sowie in der frühen B-Zellregeneration untersucht. Bei Patient H erfolgte eine zweite Behandlung mit Rituximab nach 17 Monaten. Das Follow-up erfolgte in diesem Fall bis Monat 27. Die Analyse der Schwerketten erfolgte mittels nested-PCR mit DNA aus peripheren B-Zellen unter Verwendung familienspezifischer Primer und anschließendem Subklonieren und Sequenzieren. Während der Studie erhielt Patient H zwei Zyklen der Antikörperbehandlung. Die B-Zell-Depletion hielt 7 bzw. 10 Monate an und war zu jedem Zeitpunkt mit einer klinischen Besserung des Patienten verbunden. Die Anti-CD20 Therapie bewirkte zwei Arten von Veränderungen in diesem Patienten. Während der frühen Phase der B-Zellregeneration konnten wir eine ausgedehnte und rezirkulierende Population hochmutierter B-Zellen nachweisen. Diese Zellen exprimierten verglichen mit dem Zeitpunkt vor Therapie sehr unterschiedliche VH Gene. Es handelte sich hierbei um „class-switched“ Zellen, welche nur für kurze Zeit nachweisbar waren. Die Langzeit Veränderungen dagegen waren eher diskret. Nichtsdestotrotz konnten Veränderungen sowohl in der Familie VH2 als auch in spezifischen Mini-Genen wie VH3-23, 4-34 oder 1-69 festgestellt werden. Eine Häufung einiger dieser Gene wurde bereits im Zusammenhang mit Autoimmunerkrankungen berichtet. Zusätzlich bewirkte die Rituximabtherapie ein Verschwinden klonaler B-Zellen für die gesamte Periode. Folglich bewirkte die vorübergehende B-Zell-Depletion signifikante Veränderungen im Schwerkettenrepertoire. Für die Genanalyse der leichten Kette wurden die Veränderungen getrennt für naive (CD27-) und Gedächtnis- (CD27+) B-Zellen untersucht. Einzelne CD19+ B-Zellen wurden in CD27- und CD27+-Zellen sortiert und anschließend eine Single-cell-RT-PCR mit direkter Sequenzierung der Produkte durchgeführt. Während der Studie erhielt Patient L eine Behandlung. Die B-Zell-Depletion dauerte 10 Monate und das Leichtkettenrepertoire wurde vor und nach Therapie analysiert. Gewisse Unterschiede bezüglich der Verteilung von VL und JL Genen zwischen den Populationen naiver und Gedächtnis-B-Zellen konnten vor Therapie nachgewiesen werden. Insbesondere die bevorzugte Nutzung von Vκ Genen Jκ-proximal in CD27- naive B-Zellen spricht für ein vermindertes „receptor editing“ in dieser Population. Ferner fanden sich in VλJλ-Rearrangements Hinweise für ein vermindertes „receptor editing“ mit einer Überrepräsentation der Jλ2/3 Gensegmente. Die CDR3-Regionen der naiven und Gedächtnis-B-Lymphozyten zeigten unterschiedliche Eigenschaften: die Aktivität der Terminalen-Deoxynucleotidyl-Transferase und Exonuklease im Vλ-Abschnitt (5´) war höher als in Gedächtniszellen. Die B-Zelldepletive Therapie führtete zu signifikanten Veränderungen. So fand sich die Tendenz einer weniger häufigen Nutzung von Jκ-proximal gelegenen Vκ Gensegmenten in der naiven B-Zellpopulation. Einige Vλ Gene, wie z.B. 3p, 3r oder 1g, welche kürzlich in Verbindung mit Rheumafaktor bei Autoimmunerkränkungen beschrieben wurden, waren nach der Therapie nicht mehr nachweisbar. Ebenso verschwanden die unterschiedlichen Eigenschaften der CDR3-Regionen von VλJλ-Rearrangements. Besonders auffällig war das Verhältnis Vκ zu Vλ zugunsten einer häufigeren Nutzung von Vκ Genen in der naiven Population nach Therapie verschoben. Zusammenfassend zeigen diese Ergebnisse, dass mittels therapeutischer und vorüber- gehender B-Zell-Depletion durch anti-CD20-Antikörper das Immunglobulingenrepertoire in den beiden untersuchten Patienten moduliert wurde. Messbare Unterschiede fanden sich sowohl im Schwerketten- als auch im Leichtkettenrepertoire. Es erscheint wahrscheinlich, dass diese Veränderungen mit dem beobachteten therapeutischen Ansprechen in Verbindung stehen. Die Ergebnisse stützen die Annahme, dass die Zusammensetzung des B-Zellrepertoires durch die Krankheit beeinflusst wird und für Autoimmunerkrankungen charakteristische Veränderungen mittels B-Zell-Depletion aufgehoben werden können.

ABSTRACT All members registered on the managed care database for the chronic condition Rheumatoid Arthritis (RA), for the period 01 January 2003 to 30 June 2003, were evaluated to determine the prescribing pattern of the cyclo-oxygenase (COX) II inhibitors and non-selective non-steroidal anti-inflammatory (NSAIDs). A total of 2818 members were registered on the managed care database of the chronic condition RA and 1372 members were identified as using COX II inhibitors and 827 members were using nonsteroidal anti-inflammatory (NSAIDs). The prescribing frequency determined for the COX II inhibitors were 48.60% and 29.35% for the NSAIDs. The members identified as either using a COX II inhibitor or a NSAIDs were divided into two groups. The prescribing patterns of each group such as age, gender, co-morbid conditions, concomitant medication use and frequency were analysed and compared to the national institute of clinical excellence (NICE) and the South African Rheumatism and Arthritis Association (SARAA) guidelines for the appropriate prescribing of the COX II inhibitors. Celecoxib was the most frequently prescribed COX II inhibitor accounting for 46% of all the COX II inhibitors identified and diclofenac was the most frequently prescribed NSAID accounting for 34% of all the NSAIDs prescriptions. COX II inhibitors were prescribed more frequently to females with a mean age of 55 years than males. A similar prescribing trend was found with the NSAIDs. The COX II inhibitors were frequently prescribed to patients over the age of 56 with co morbid gastro-oesophageal disease and concomitant warfarin and steroid use. The prescribing patterns found in the managed care environment were similar to those recommended by the NICE and SARAA guidelines. The managed care data showed that the COX II inhibitors, which are supposed to have less gastric adverse side effects, were frequently used in combination with gastroprotective agents (GPA’s). This study indicates that even though COX II inhibitors were prescribed more frequently than NSAIDs in the managed care environment the recommended clinical guidelines and protocols employed by the managed care environment were adhered to. However, there v is a need to closely monitor patients on concomitant GPA’s treatment and COX II inhibitors. This study helped to evaluate the current prescribing patterns of COX II inhibitors in the managed health care environment. This study confirmed that guidelines and protocols were adhered to. These are excellent tools to be used in the managed health care environment to ensure effective and appropriate prescribing.

A dissertation submitted to the Faculty of Health Science, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Master of Science in medicine. Johannesburg 2014 / Introduction: The presence of pain during sleep causes patients with chronic daily pain, such as in Rheumatoid and Osteoarthritis, to experience insomnia, fragmented sleep and an increased number of night-time awakenings. This poor sleep results in an increased sensitivity to pain during the day. The effect of improving sleep on pain, sleep and mood after taking Zolpidem MR was the aim of this study. Methods: 11 patients from Chris Hani Baragwanath Hospital in Soweto South Africa who reported insomnia and daily pain spent 4 weeks in this crossover design, double blinded, placebo controlled study. Week 1- baseline, week 2 and 4 were Intervention weeks – either placebo or Zolpidem MR, week 3 was a Washout week. Data collected included actigraphy, McGill Pain Questionnaire, PSQI, BDI, physical activity questionnaire and daily sleep and pain diaries containing VAS scales for sleep and pain. Results: No significant changes were found in the pain or physical activity levels in any of the patients. Sleep quality, as measured by an isolated PSQI question, was improved by Zolpidem MR (p=0.0075). PSQI was decreased in the final week of the study compared to baseline (8.7 vs. 11.3, p=0.0106) and BDI was lower in week 4 than baseline (7.7 vs. 15.85, p=0.0003), BDI was also lower in week 4 compared to week 2 (7.7 vs. 12.8, p<0.05). However the changes in PSQI and BDI were a result of the order of the weeks, with patients interacting with the researcher and were not due to either Zolpidem MR or placebo. Anecdotal reports include feeling more energised and capable of living life. Conclusion: This study has shown that human interaction is an important component of treatment for insomnia and chronic pain as there is a positive effect on sleep disruption and depression.

Ultrasound (US) imaging has made significant progress over the past 20 years in relation to its role in inflammatory arthritis, and in particular, Rheumatoid Arthritis. Modern US machines provide crisp, detailed images of superficial anatomical structures which has facilitated the uptake of US imaging as an important assessment tool within the Rheumatology department. Diagnostic and prognostic information can now assist clinicians decisions with the goal of improving patient treatment and subsequent outcome. In addition, 3D US imaging has recently been suggested as an additional imaging modality with potential benefits in the assessment of in?ammatory arthritis. Recent work has focused on providing a reliable, responsive US joint count which can be assimilated into routine care as well as providing a platform for clinical research. Thus, my first aim was to show that a defined limited US data set, including 2D and 3D imaging, shows acceptable reliability. I demonstrate that both imaging modalities are reliable in terms of reading and image acquisition when restricted to a limited US data set. My second aim, was to demonstrate that a limited US data set is responsive. Using both a physiological and pharmacological trigger, I demonstrate that both 2D and 3D imaging are responsive and that combining US endpoints with DAS28 (Disease Activity Score - 28) increased the effect size and identifies treatment effects early. Despite notable advances in musculoskeletal US research, there is still need for better understanding of the pathophysiological correlates of ultrasound imaging. Therefore my final aim was to examine the relationship of Power Doppler Signal (PDS) and gray-scale synovial thickening with histological features of synovitis at a single joint level and with an extended joint US data set. I Firstly show that the harvesting of synovial tissue, using a minimally invasive US-guided biopsy technique, is safe and well tolerated by patients and that the quality of tissue and RNA extracted is good. Using this tissue collection method, I demonstrate a good correlation of US and histological parameters of synovitis (specifically CD68+ sub-lining macrophages) at a single joint level, in both an early and established RA cohort. This relationship is maintained if the US assessment is extended to a discrete US joint data set. Furthermore, within the knee joint I demonstrated that PDS correlates well with synovial tissue expression of inflammatory mediators of neoangiogenesis and histological assessment of synovial vascular area.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease which predominantly affects the synovial joints. Local gene therapy represents an approach to produce therapeuticmolecules (i.e. soluble TNF receptor (sTNFR)-Fc and interleukin-1 receptor antagonist (IL-1Ra)) directly in arthritic joints. Gene therapy could be designed to link the level of therapeutic gene expression directly to disease activity, through the use of transcriptional and posttranscriptional regulatory elements. The experiments in this thesis describe the construction of multi-responsive, composite synthetic promoters, comprised of the binding sites for an array of transcription factors activated in arthritic joints. Optimal spatial arrangements of binding sites in relation to each other and to the TATA box were determined by Assembly PCR cloning and the functionality of the resulting synthetic promoters revealed additive or synergistic induction of luciferase reporter gene expression in response to combined stimulation. Candidate synthetic promoters were cloned into a lentiviral vector between insulator elements and displayed significantly enhanced induction, in excess of 1,500 fold in response to combined stimulation. Inflammation-specific activation of lentiviral synthetic promoters was confirmed in a carrageenan-induced paw inflammation mouse model, which demonstrated the strong correlation between local luciferase gene expression and paw inflammation. Post-transcriptional gene regulation was also investigated by exploiting the differential expression of endogenous miR-23b during inflammation. Insertion of miR-23b target sites into the 3’UTR of the luciferase gene subjected luciferase mRNA to regulation by miR-23b. Experiments demonstrated that high basal gene expression driven by constitutive and inducible promoters was significantly downregulated by miR-23b without significantly impairing high gene expression upon stimulation. Overall, the experiments in this thesis have confirmed the induction of inflammation-specific gene expression, regulated by inflammationresponsive endogenous transcriptional and post-transcriptional elements.

Bone makes up a framework that provides protection for internal body organs. The homeostasis of bone is maintained by a balanced process involving old bone degradation and new bone formation. However, this balance can be altered in pathophysiological conditions such as in postmenopausal osteoporosis and in patients with rheumatoid arthritis (RA). In recent years, new therapies have been developed to reduce bone resorption. However, there is disparity in patients’ response to these therapies. The reasons are unclear although age, gender, ethnic background and lifestyle have all been suggested to play a part. For patients with chronic inflammatory conditions, treatment was revolutionised by the discovery and application of biologic therapies that target pro-inflammatory proteins and/or pathways. However, whilst the anti-inflammatory effect of these biologic agents is well-established, their effect on bone loss is just emerging. In RA, it is not clear whether the beneficial anti-inflammatory effects of biologic anti-tumour necrosis factor alpha (TNFα) agents are accompanied by parallel improvements in bone erosion/density, whether there are differences between patient groups and what factors influence the response. In order to address these issues, a database on the factors that influence responsiveness of patients with osteoporosis to bisphosphonates, a treatment that suppresses bone resorption, was established. Based on the outcome of this study, the influence of the key factor(s) that affect bone response to treatment in combination with excess pro-inflammatory cytokine production on bone response in RA patients was determined. Significant improvement in bone mineral density (BMD) and plasma levels of bone biomarkers has been shown in this study with biologic anti-TNFα agents. The improvement in BMD was not always consistent with improvement the clinical response to treatment as assessed by changes in disease activity score 28(DAS28). The study also provides a mechanistic explanation for how blockade of TNFα in patients can reverse the balance of bone loss in patients with RA. Thus, the data show that treatment of patients with biologic anti-TNFα agents reduces the number of osteoclast precursors (OCs) in the blood.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of autoimmune origin affecting approximately 1% of adult population worldwide. The clinical course of RA is highly variable, ranging from self-limiting to severe disease, with considerable individual and socio-economic implications. It is now well acknowledged that early diagnosis and treatment equates to better long-term outcomes. However, despite major therapeutic advances in recent decades, the management of RA remains challenging as a significant proportion of patients presents with active disease despite maximization of therapy. It is also difficult to predict which patients will respond adequately to various treatment regimens. The identification of biomarkers of clinical outcome capable of stratifying patients into accurate prognostic categories and guide pharmacological intervention is therefore urgently needed. Notably, along with clinical variability, RA is characterised by high biological heterogeneity at the tissue level. The cellular infiltrate of the RA synovium can be distinguished into at least three main patterns according to the degree and organisation of the immune cells: the 'Lymphoid' pattern characterised by predominant B and T lymphocytes which tend to cluster in discrete aggregates resembling ectopic lymphoid structures; the 'Myeloid' pattern characterised by absence of lymphocytic aggregates but significant expression of sublining macrophages; the 'Pauci-immune' pattern, that hardly shows any infiltrating immune cells. The hypothesis of this thesis was to determine whether these distinct synovial pathotypes may define specific disease subsets and predict response to therapy in patients with RA. Specifically, this work aims at: 1. evaluating whether the synovial pathotype associates with the presence of specific clinical, serological, radiological and ultrasonographic findings in an early RA cohort (< 1 year onset); 2. exploring the potential role of the synovial pathotype as a predictor of response to conventional synthetic disease-modifying antirheumatic drugs (csDMARD) after 6 months in an early RA cohort; 3. exploring the potential role of the synovial pathotype as a predictor of response to anti-TNFα treatment after 3 months in a csDMARD-failure established RA cohort.

Rheumatoid arthritis (RA) is a chronic, heterogeneous, autoimmune disease that causes inflammation of synovial joints leading to pain, stiffness and swelling. If left untreated, RA results in irreversible joint destruction and long term disability. Initial treatment with glucocorticoids and other immunosuppressive agents suppresses inflammation. However, many of these drugs are not well-tolerated due to extensive side effects or are simply ineffective. The discovery of tumour necrosis factor-α (TNF) as a key mediator of inflammation in RA led to the development of monoclonal anti-TNF antibody therapy. Since then, other biologic drugs targeting immune pathways have been developed for RA, including interleukin-6 (IL-6) blockade, B cell depletion, and T cell co-stimulation blockade. Not all patients will respond to their first biologic drug and currently there is no way to predict which patient will respond to each different class of drug. Generally, 3 – 6 months are required to determine clinical efficacy, during which time joint inflammation proceeds. Therefore, discovering biomarkers to predict treatment response is a research priority. Biologic drugs target immune pathways. As single cell technology advances and has increasing capacity to identify subtle changes in many cell subsets, I hypothesise that studying the blood immune cell landscape will define cellular biomarker profiles relevant to each individual patient’s disease.

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