A dissertation submitted in partial compliance with the requirements for the Masters Degree in Technology: Chiropractic, Technikon Natal, 2001. / The purpose of this study was to determine the efficacy of Genesen Acutouch pointers in the treatment of osteoarthritis of the knee. This was a prospective, randomized clinical trial consisting of sixty patients who volunteered from the general population of Durban. The patients, diagnosed as having osteoarthritis of the knee, were randomly divided into two different treatment groups. Each group consisted of thirty patients between the ages of eighteen and eighty five years. One group received active Genesen Acutouch Therapy, while the second group received placebo Genesen Acutouch Therapy. Data capturing took place for both groups on the first, fifth and ninth consultations. Subjective data was collected using the Numerical Pain Rating Scale-l Ol, the Visual Analogue Scale and the Patient Specific Functional Scale. Objective data was gathered from goniometer and algometer readings. For statistical analysis, only parametric tests were used in all hypothesis tests due to the large sample size. All readings were considered to be continuous variables. The twosample (unpaired) two-tailed t-test was used to compare two independent samples. The two-sample paired t-test was used to compare results from related samples. All tests were conducted at a = 0.05 level of significance / M

Die zentrale Rolle von B- Zellen in der Pathogenese und Therapie von Autoimmunerkrankungen hat in den letzten Jahren zu unterschiedlichen therapeutischen Ansätzen geführt, B- Zellen direkt oder indirekt zu targetieren. Der bisher effektivste Ansatz stellt der monoklonale anti- CD20 Antikörper Rituximab dar. Nach Gabe von Rituximab kommt es zu einer passageren, in der Regel sechs bis neun Monate anhaltenden peripheren B- Zelldepletion. Die anti- CD20 vermittelte B- Zelldepletion stellt zwar ein vielversprechendes Therapieverfahren in der Behandlung der Rheumatoiden Arthritis dar, derzeit ist noch wenig über das Regenerationsverhalten von B- Zellen nach Therapie mit Rituximab bekannt. Daher wurde in dieser Arbeit die B-Zellrepopulation insbesondere hinsichtlich der Modulation des Mutationsmusters des B- Zellrezeptors untersucht. Dazu wurde die VH4- Familie des Immunglobulinrezeptors- prospektiv vor und nach anti- CD20 vermittelter B- Zelldepletion analysiert. Bei drei Patienten (A-C) wurden die Ig-VH4 Gene aus genomischer DNA peripherer B- Zellen amplifiziert, subkloniert und sequenziert. Die Analyse erfolgte zu drei verschiedenen Zeitpunkten: Vor Therapie, in der frühen Regenerationsphase mit einem B- Zellanteil von 1% bis 1,3% im peripheren Blut und in der späten Regenerationsphase, zwei bis drei Monate nach der frühen Regenerationsphase Mini Gene wie VH4-34 und VH4-39, die in Verbindung mit Autoimmunität stehen, waren vor Einleitung der Therapie relativ überexprimiert. Die Behandlung mit Rituximab führte bei allen drei Patienten zu einer Veränderung des Repertoires der regenerierenden B- Zellen mit einer reduzierten Benutzung der VH4-39 Gene, außerdem bei Patient A zusätzlich der VH4-34 Gene. Tief greifende Veränderungen fanden sich während der frühen Regenerationsphase durch den Nachweis einer rezirkulierenden Population hochmutierter B- Zellen, die in einer durchgeführten Immunophänotypisierung mit spezifischen Oberflächenmarkdern als Plasmazellen identifiziert wurden. Da Plasmazellen kein CD20 Molekül exprimieren, werden sie durch eine anti-CD20 vermittelte Therapie nicht direkt eliminiert. Sie zirkulieren während der Phase der B- Zelldepletion aber auch nicht im peripheren Blut. Interessanterweise sind sie in der frühen Regenerationsphase in der Peripherie als erste mit hohem relativem Anteil nachweisbar. Daher wurde untersucht, ob auch Plasmazellen durch die Therapie moduliert werden, obwohl sie durch Rituximab nicht direkt targetiert werden. Hierfür wurden die Sequenzen mit hochmutiertem Ig- Rezeptor (>9 Mutationen/Sequenz) im Verlauf einer detaillierten Analyse zugeführt. Dabei wurde insbesondere das Mutationsmuster in RGYW/WRCY Hotspot Motiven und in den CDR- Regionen untersucht. Die Analyse der Mutationshäufigkeit in RGYW/WRCY Hotspot Motiven erlaubt eine Abschätzung, in wieweit die somatische Hypermutation der B- Zellen durch T- Zell abhängige Differenzierung erfolgte. Die als Plasmazellen identifizierten hochmutierten Sequenzen zeigten vor der Therapie Charakteristika einer aktiven Erkrankung mit einem verminderten Targeting der RGYW/WRCY Motive. Dagegen zeigte sich in den rezirkulierenden Plasmazellen während der frühen Regenerationsphase ein zunehmendes Targeting der RGYW/WRCY Motive. Dies spricht für einen Repertoire Shift zu mehr T- Zell abhängigen B- Zell Mutationen. Ein Zustand, wie er bei Gesunden beobachtet wird. Um die Hypothese einer Rituximab- induzierten Modulation des Plasmazellkompartimentes weiter zu untermauern, wurde der R/S Quotient, d.h. das Verhältnis von Silent zu Replacement Mutationen in den hypervariablen Regionen (CDRs) der hochmutierten Plasmazell-Ig Sequenzen bestimmt. Interessanterweise fanden sich in der Regenerationsphase signifikant erhöhte R/S Ratios in den rezirkulierenden Plasmazellen.. Die signifikante Zunahme an Replacement Mutationen in den CDR- Regionen, welche sich in einer Zunahme des R/S Verhältnisses wiederspiegelt, kann als Entwicklung des Ig- Repertoires durch positive Antigenselektion interpretiert werden und weist somit eine Rituximab- induzierte Veränderung auf, wie man sie sonst bei gesunden Individuen findet. Zusammenfassend zeigt unsere Studie, dass die transiente anti- CD20 vermittelte B- Zelldepletion auch zu einer indirekten Modulation des Plasmazellkompartimentes führt. Insbesondere werden postrekombinatorische Imprints des B- Zell Rezeptors, wie somatische Hypermutation und Antigen Selektion, verändert, die mit hoher Wahrscheinlichkeit für die Entstehung von Autoimmunität bei der Rheumatoiden Arthritis eine Rolle spielen. Zusätzlich kann die Modulation des genetischen Imprints der Ig Rezeptoren bei der Rheumatoiden Arthritis eventuell als möglicher Biomarker entwickelt werden, um ein Ansprechen auf die Therapie vorherzusagen. Dies bedarf weiterer Untersuchungen, um tiefer greifende Einblicke in Prozesse zu erlangen, die durch zukünftige Therapien beeinflussbar werden. / Given that B cells play a central role in the pathogenesis of autoimmune diseases, different therapeutic attempts that target B cells directly or indirectly have been developed in the recent years. Anti CD20 monoclonal antibody, Rituximab poses to be an effective therapeutic agent, to date, in such treatment. Administration of Rituximab leads to transient depletion of B cells, with persistent deletion of peripheral B cells for a period of six to nine months. Although anti CD20 mediated B cell depletion seems to be promising in the treatment of rheumatoid arthritis, little is known about the regeneration profile of B cells following therapy. Therefore, B cell recapitulation, in particular, modulation of mutations in the regenerating B cell receptor was examined in the current study. In this context the immunoglobulin VH4 family receptors were analyzed. In 3 patients A-C, the peripheral B cell Ig-VH4 rearrangements were amplified from genomic DNA, sub cloned and sequenced from three different time points. The first time point was before B cell depletive therapy, the second one refers to the early regeneration phase, the earliest time point after therapy when significant amount of B cells were found in periphery. This corresponds to approximately 1-1.5% B cells in the peripheral circulation and was achieved in five to seven months following rituximab therapy. The third time point namely the late regeneration phase commences three months after the early regeneration phase and the corresponding B cell proportion in the peripheral blood is 1.5-2%. B cell depletion caused prominent changes in the regenerating repertoire in context to the usage of the single variable gene segments and also mutational aspects and distribution of mutations in the RGYW/WRCY hotspot motifs. Mini genes like VH4-34 and 4-39, previously linked to autoimmunity, were predominantly expressed in the repertoire before therapy. Rituximab introduced significant modulation in the recapitulating repertoire which was evident by reduced usage on VH4-39 genes in all three patients and additionally also VH4-34 in patient A. Striking changes were observed in the early regeneration phase with a wave of highly mutated B cells in the peripheral circulation. Immunophenotyping revealed that these were plasma cells. As plasma cells do not express CD20 molecule on their surface they are not directly targeted by anti CD20 therapy. However, these cells were not found in peripheral circulation following rituximab therapy. Interestingly they were found to recirculate in higher levels in the early regeneration phase. Therefore it was intriguing to investigate if plasma cells were also modulated by anti CD20 therapy although they were not directly targeted by the therapy. In this connection, the highly mutated Ig sequences (corresponding to plasma cell sequences with >9 mutations per sequence) were analyzed in detail. In particular, the mutational targeting to the RGYW/WRCY hotspot motifs and mutations in the complementary determining regions (CDRs) were examined. Analysis of mutational confinement to the RGYW/WRCY motifs reveals if the somatic hypermutation in the B cells were T cell driven or not. In the current study the plasma cells during active rheumatoid arthritis showed reduced levels of RGYW/WRCY mutations. In contrast the plasma cells in the regeneration phase represented increased mutations in the RGYW/WRCY motifs suggesting a T cell induced pathway of B cell mutations as expected in healthy state. To further substantiate the hypothesis of plasma cell modulation by rituximab therapy, the ratio of replacement to silent (R/S) mutations in the CDRs of highly mutated plasma cell sequences were analyzed. The plasma cells in the early regeneration phase showed elevated levels of R/S mutations in the CDRs compared to that before therapy. The significantly higher number of replacement mutations in the CDRs leading to increased R/S mutations could be interpreted as a rituximab induced shift of Ig repertoire towards a positive antigen selected B cells which is a characteristic of healthy individuals. To summarize, our study shows that anti CD20 mediated transient B cell depletion leads to an indirect modulation of the plasma cell compartment. In particular post recombinatorial processes such as somatic hypermutaion and antigenic selection which were supposed to play a role in the autoimmune mechanisms of rheumatoid arthritis. Moreover, modulation of the genetic imprints of the Ig receptor could be developed as a potential biomarker candidate in the study of efficacy of such treatment in rheumatoid arthritis. Additional investigations to gain further insights into the processes are required to design potential therapies for future.

B-Lymphozyten leisten unterschiedliche Beiträge zur Pathophysiologie der Rheumatoiden Arthritis. Sie produzieren Autoantikörper, präsentieren Autoantigene und schütten verschiedene Zytokine, die am proinflammatorischen Prozess beteiligt sind, aus. Aufbauend auf diesen Ergebnissen wurden in den letzten Jahren Therapien entwickelt, die gezielt B-Lymphozyten ansteuern um direkt oder indirekt in den autoimmunen Krankheitsverlauf einzugreifen. Die zeitlich begrenzte B-Zell-Depletion mit Rituximab (anti CD20-Antikörper) hat dabei in den letzten Jahren einen hohen Stellenwert erlangt und wird im klinischen Alltag insbesondere bei der Behandlung von Patienten mit rheumatoider Arthritis angewandt. Rituximab induziert im peripheren Blut bemerkenswerte Veränderungen in der Homöostase der B-Zell-Subpopulationen. Nach Therapie mit dem anti-CD20 Antikörper Rituximab beginnt die Repletionsphase mit der peripheren Aussaat von transitionalen unreifen B-Zellen. Im weiteren Verlauf kommt es zu einer Normalisierung des naiven B-Zell-Pools. Das B-Zell Gedächtnis und in besonderem Maße die IgD+CD27+ Gedächtniszellen erholen sich nach Therapie nur langsam. In einer prospektiven klinischen Studie hat unsere Arbeitsgruppe gezeigt, dass die Gesamtzahl der Gedächtniszellen gut mit der Dauer der klinischen Antwort auf Rituximab korreliert. Es ist wenig über die speziellen molekularen Veränderungen innerhalb der Gedächtnis B-Zellen nach Rituximab Therapie bekannt. Um die Veränderungen im peripheren Blut zu verstehen untersuchten wir die somatische Mutationsfrequenz und das Muster der Ig-VH3 Gen Rearrangements, indem wir prä- und posttherapeutisch bei 18 Patienten einzelne B-Zellen isolierten und den individuellen B-Zellrezeptor durch eine Einzelzell RT-PCR amplifizierten und sequenzierten. Wir verglichen das Mutationsmuster nach erfolgreicher B-Zelldepletion in den neu rezirkulierenden Gedächtnis B-Zellen mit dem Mutationsmuster von vier Gesunden Blutspendern und sechs nicht-RA Patienten, die eine Hochdosis Chemotherapie mit anschließender autologer oder allogener Stammzelltransplantation erhalten hatten. Zunächst haben wir die Zusammensetzung der Gedächtniszellen im peripheren Blut analysiert. Der Phänotyp der peripheren prä-switch (IgD+CD27+) und post-switch (IgD-CD27+) Gedächtniszellen zeigte keine quantitativen Unterschiede in RA-Patienten im Vergleich zu Gesunden. Bei der direkten Analyse des B-Zell Immunglobulin Rezeptors fanden sich jedoch zwischen klassengeswitchten und ungeswitchten Gedächtnis B-Zellen signifikante Unterschiede in der Anzahl der Mutationen in der variablen Region der Ig Rezeptors. Die Population der IgD+CD27+ Gedächtniszellen beinhaltete sowohl nicht mutierte, wenig mutierte und stark mutierte (Median= 9 Mutationen pro Sequenz) rearrangierte Ig- Rezeptoren, wohingegen die IgD-CD27+ Gedächtniszellen einen durchgehend hoch mutierten (Median = 18 Mutationen pro Sequenz) Rezeptor aufwiesen. Der Unterschied zwischen beiden Gruppen war signifikant (Mutationsfrequenzen 3.83±0.19% vs. 7.1±0.53%; P=0.0001). Grundlegende Veränderungen wurden bei den rezirkulierenden ungeswitchten Gedächtniszellen (IgD+CD27+) nach vorübergehender B-Zell Depletion mit Rituximab festgestellt. Diese Zellen wurden bis 6 Jahre nach Rituximab beobachtet und zeigten eine stark verzögerte Zunahme an Mutationen im Ig-Rezeptor. Ein Jahr nach einmaliger Gabe von Rituximab waren 84% der einzelnen zirkulierenden IgD+/CD27+ B-Zellen unmutiert. Zu diesem Zeitpunkt fanden sich keine stark mutierten Ig-VH3 Gen Rearrangements (P=0.0001). Mit zunehmendem Abstand zur B-Zell depletierenden Therapie konnten in der Repopulationsphase zunehmende Zahlen an Mutationen in den B-Zell Ig Rezeptoren festgestellt werden. Beispielsweise waren während des 2. Jahres der Regeneration (P=0.0001) 7.8%, sowie nach 4 Jahren nur 14% der Ig Rezeptoren mutiert. Sogar 6 Jahre nach Behandlung, waren VH Mutationen in IgD+ Gedächtniszellen noch deutlich vermindert. Selbst nach dieser Zeit fanden sich in der prä-switch Gedächtnispopulation nur 27% hochmutierte Sequenzen während vor der passageren B-Zelldepletion 52% ein hohe Zahl an Mutationen trugen (P=0.0001). Die posttherapeutische Analyse der CDR3 Länge der regenerierten IgD+ Gedächtniszellen ergab eine erhöhte CDR3 Länge, die signifikant mit der Anzahl der nicht mutierten VH Genrearrangements während der Repletionsphase korreliert. Interessanterweise regenerierten Patienten nach Hochdosis Chemotherapie und allogener Stammzelltransplantation ihre IgD+ Gedächtniszellen mit einer deutlich höheren Anzahl an Mutationen. Ein Jahr nach Transplantation zeigten die Ig Rezeptoren schon 22% hoch mutierte und 42% unmutierte VH Rearrangements. Das zeigt, dass eine gegen CD20 gerichtete Behandlung nicht nur eine Verzögerung der Produktion der ungeswitchten Gedächtniszellen zur Folge hat, sondern darüber hinaus einen signifikanten Effekt auf die Mutationsrate im präswitch Gedächtnis B-Zellpool besitzt. Im Gegensatz zum Mutationsmuster der IgD+ Gedächtniszellen regenerierten die klassengeswitchten Gedächtniszellen nach anti-CD20 Depletion im peripheren Blut mit quantitativ normalen Mutationen im Ig Rezeptor. Interessanterweise fand sich allerdings eine Änderung der exprimierten Isotypen mit deutlicher Dominanz IgA exprimierender B Zellen. Weitere Analysen der klassengeswitchten Gedächtnis B-Zellen zeigen außerdem eine Therapie induzierte qualitative Veränderung dieses B-Zellpools. So waren posttherapeutisch die Mutationen in bestimmten T-Zell abhängigen Mutationshotspots, dem RGYW/WRCY Motiv, signifikant vermehrt (Mutationstargeting vor Therapie 27% vs. 43% nach Rituximab, P=0.0003). Dies weist darauf hin, dass die Mechanismen der Affinitätsreifung im klassengeswitchten B-Zellgedächtnis vor und nach B-Zelldepletion unterschiedlich funktionieren. Der Mutationsmechanismus selbst ist allerdings in diesen Zellen quantitativ nicht eingeschränkt. Zusammenfassend zeigt unsere Arbeit zum erstem mal, dass es nach einer passageren B-Zelldepletion mit anti-CD20 Antikörpern zu einer über Jahre hinweg nachweisbaren ausgeprägten Verzögerung in der Aquisition von somatischen Mutationen in rearrangierten VH Genen der IgD+ Gedächtniszellen kommt. Demgegenüber erholt sich das klassengeswitchte B-Zellgedächtnis mit uneingeschränkter Zahl von Mutationen im Ig Rezeptor. Diese Resultate zeigen, dass anti-CD20 gerichtete Therapien in besonderem Maße IgD+ Gedächtniszellen beeinflussen. Der Selektionsdruck durch Antigene und/oder die Selektion der Ig Rezeptoren erscheint unter diesen Bedingungen speziell bei IgD-Gedächtnis B-Zellen reduziert. Die Daten unterstützen die Hypothese, dass prä-switch Gedächtnis B-Zellen im Vergleich zu post-switch Gedächtnis B-Zellen andere Bedingungen für die Aktivierung der Mutationsmaschinerie benötigen. Die Resultate eröffnen neue Wege für das Verständnis der Pathophysiologie der B-Zell Gedächtnisentwicklung und können helfen neue zielgerichtete Therapien zur Behandlung von Autoimmunerkrankungen zu konzipieren. / Diverse roles of B cells in the pathophysiology of rheumatoid arthritis are now well established. B cells contribute to autoimmunity by producing autoantibodies, processing autoantigen and the production of different cytokines which are involved in the inflammatory cascade. Therefore approaches to target B lymphocytes directly or indirectly are developed for clinical practice to treat autoimmune diseases including rheumatoid arthritis. Transient B cell depletion by rituximab (anti-CD20 antibody) has gained prime importance in recent years. Meanwhile anti-CD20 mediated transient B cell depletion therapy is now used with clinical efficiency in the treatment of patients with rheumatoid arthritis. Rituximab induces noteworthy changes in the homeostasis of peripheral B cell subpopulations during the repletion phase with emerging immature B cells in peripheral blood followed by normalization of the naïve B cell pool and a longterm delay in memory B cell subsets in patients with rheumatoid arthritis. Particularly IgD+CD27+ memory B cells repopulate very slowly during B cell regeneration. In a prospective clinical study, our laboratory has shown that the overall number of memory B cells correlates well to the duration of clinical response to rituximab. Little is known about the particular molecular changes in the memory B cell repertoire after rituximab therapy. To better understand peripheral memory B cell subsets, we explored in detail the somatic mutational frequency and pattern of Ig-VH3 gene rearrangements by using a single B cell sorting technique followed by nested PCR before and up to 6 years after rituximab therapy in 18 RA patients. We compared rituximab inflicted dynamics of mutational acquisition to memory B cell repopulation in 4 healthy donors and 6 non RA patients undergoing high dose chemotherapy followed by autologous or allogeneic stem cell transplantation (SCT). Firstly we analyzed the peripheral composition of memory B cell subsets. The phenotypic analysis of peripheral pre-switch (IgD+CD27+) and post-switch (IgD-CD27+) memory B cells did not reveal any quantitative differences in RA patients prior to B cell depletion therapy compared to healthy donors. However extending those studies in directly analysing the B cell immunoglobulin receptor from individual B cells of RA patients and healthy controls brought interesting results. Pre-switched and post-switched memory B cells showed a highly significant difference in the amount of mutations/sequence. The population of IgD+CD27+ memory B cells is comprised of non-mutated, low and highly mutated (median= 9 mutations/ sequence) rearranged Ig receptors whereas the IgD-CD27+ memory B cell compartment shows quite uniformly highly mutated (median 18 mutations/ sequence) sequences indicating a significant difference between these two groups (mutational frequencies 3.83±0.19% vs. 7.1±0.53%; P=0.0001). Profound changes were noted in the re-emerging pre-switch memory B cells (IgD+/ CD27+) after transient B cell depletion with rituximab. These cells showed over a time period of 6 years after treatment with rituximab significantly delayed acquisition of mutations in Ig receptors on the single B cell level. One year after a single course of rituximab 84% of single repopulating IgD+/CD27+ B cells were unmutated and no highly mutated Ig-VH gene rearrangements were found(P=0.0001). Over time increasing numbers of mutations could be detected i-e 7.8% during 2nd year of regeneration (P=0.0001), 14% after 4 years (n=2). Nevertheless even 6 years after rituximab, VH mutations in IgD+ memory B cells were still reduced with 27% highly mutated sequences compared to 52% pre therapy(P=0.0001). Post-therapy analysis of CDR3 length of regenerated IgD+ memory B cells revealed increased CDR3 length which also correlates well with elevated number of non-mutated VH gene rearrangements observed during repletion phase. In comparison patients undergoing high dose chemotherapy followed by allogeneic stem cell transplantation repopulated IgD+ memory cells earlier with higher numbers of mutations in IgD+ memory B cells. One year after transplantation Ig receptors showed already 22% highly mutated and 42 % unmutated VH rearrangements. These findings indicated that anti-CD20 mediated B cell depletion seems not only to delay the production of pre-switch memory B cells but also significantly affects the acquisition of mutations in the IgD+ memory B cell pool. In contrary to the mutational pattern of IgD+ memory B cells after rituximab class switched memory B cells repopulate in the periphery with quantitatively normal mutations in their Ig receptors. Although the numeric replenishment of these recirculating class-switched memory B cells was also reduced after rituximab, we found no delay in quantitative acquisition of mutations also an increased proportion of IgA expressing B cells in this memory B cell subset was detected. Our data showed that post-therapy mutational targeting in RGYW/WRCY motifs were significantly increased as compared with that of pre-treatment (27% before rituximab vs. 43% after therapy, P=0.0003) indicating that affinity maturation may operate differently in class-switched memory B cells before and after B cell depletion. These results indicate a normal development process with an unimpaired mechanism of mutational acquisition in class-switched memory B cells. These data argue for different requirements to undergo somatic hypermutations in IgD+ memory B cells in comparison to class switched memory B cells. To conclude, our work has demonstrated for the first time a delayed acquisition of somatic hypermutations at single Ig receptor VH gene rearrangements of IgD+ memory B cells in comparison to class-switched memory B cells. These results demonstrate that IgD+ memory B cells are particularly susceptible to anti-CD20 treatment in patients with rheumatoid arthritis. In addition antigenic pressure and/or selection are substantially reduced by rituximab therapy which is basically not seen in the class-switched memory compartment. These data are in line with the hypothesis that IgD+ memory B cells have distinct requirements for activating their mutational machinery compared to class-switched memory B cells which recover normal mutations during regeneration phase. The results have implications in understanding the pathophysiology of memory B cell in rheumatoid arthritis and may be helpful in designing new targeted therapies.

M.Sc.(Med.), Faculty of Health Sciences, University of the Witwatersrand, 2009 / Introduction Foot involvement is common in rheumatoid arthritis (RA). Foot pain, instability and deformity affect ambulation and impacts on health-related quality of life. The aim of this study was to determine the nature, extent and functional impact of rheumatoid foot problems in established RA. Patients and Methods One hundred RA patients were studied. Functional status was evaluated using the modified Health Assessment Questionnaire (mHAQ) and Foot Health Status Questionnaire (FHSQ). Foot deformity and footwear suitability was assessed using the Foot Problems Survey (FP Survey) and Footwear Suitability Scale (FWS Scale). Results In this predominantly female group of 95%, with a mean (± SD) disease duration of 12.2 (7.9) and moderate functional disability [mHAQ: 1.3 (0.6)], the FP Survey showed all patients had one or more foot deformity. Foot function was impaired with a mean (± SD) FHSQ score of 41.3 (12.4) and the FWS Scale showed that 93% wore unsuitable footwear. A strong correlation was observed of the global FHSQ (r=-0.5489, p<0.0001), its pain domain (r=-0.472, p<0.0001) and foot function domain (r=-0.599, p<0.0001), with the global mHAQ score. Despite the high frequency of foot problems observed only 27% had visited a podiatrist. Conclusion In conclusion foot problems and foot function disability is common in Black South African patients with established RA. Furthermore the strong correlation between mHAQ and FHSQ showed that foot functional disability was a major driver of overall functional disability in RA.

Objectives: To determine the impact of human immunodeficiency (HIV) infection on rheumatoid arthritis (RA) disease activity. Patients & Methods: Retrospective record review of RA patients who HIV sero-converted, compared to a HIV negative RA control group. DAS28-ESR and -CRP scores were collected at the initial presentation (T0), time when HIV diagnosis made (TH) and the last clinic visit (TL). Results: Forty three HIV positive RA patients were included. At TL disease activity was similar between the groups, despite methotrexate (MTX) being continued in only 11.6% of the HIV group (vs. 83.7% in the control group, p=0.0002). In the HIV group, all clinical parameters improved except the ESR, which accounted for the significantly higher DAS28-ESR compared to the DAS28-CRP at TL (p=0.004). At TL only 13.9% HIV patients had ongoing moderate to high disease activity. Conclusion: Overall disease activity improved with HIV seroconversion in spite of stopping MTX in the majority of patients. The DAS28-ESR overestimated disease activity compared to DAS28-CRP following HIV seroconversion.

Li, Martin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 187-208). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Microparticles are small subcellular vesicles, which function in inter-cellular communication by transferring RNA, bioactive lipids, proteins and receptors to target cells. Neutrophil microparticles are abundant in rheumatoid arthritis synovial fluids. Current dogma dictates that cartilage is an impenetrable avascular matrix through which metabolites from the synovial fluid must diffuse; we present the first evidence that microparticles can access chondrocytes through the cartilage. Addition of neutrophil microparticles to chondrocytes in vitro and in vivo afforded protection from arthritogenesis, evidenced by reduced extracellular matrix degradation, increased expression of genes involved in cartilage matrix synthesis and reduced inflammatory mediator production. Adoptive transfer of fluorescently labelled neutrophils into mice with inflammatory arthritis migrated to the inflamed joints and released microparticles, which could be found abundantly within the cartilage. We propose a mechanism whereby microparticles deliver the pro-resolving protein Annexin A1, which engages the receptor FPR2/ALX on the chondrocyte, eliciting tissue protection. This protection could be blocked in part by TGF-β neutralising antibodies and by preventing microparticle phosphatidylserine interaction with chondrocytes by coating with Annexin V. Intriguingly, microparticle treatment directly inhibited the phosphorylation of Hsp27. Hsp27 exists as large oligomers within the resting cell, and upon phosphorylation are released as monomers which function to stabilise the mRNA of certain proinflammatory genes such as IL-8, IL-6 and COX-2; which were effects seen during microparticle/chondrocyte coculture. Thus, microparticles may directly reprogram chondrocytes to prevent the expression of pro-inflammatory cytokines and mediators, but also they exert anti-inflammatory effects via the exposure of phosphatidylserine and induce the production of protective TGF-β by chondrocytes. As cartilage has limited capacity for self-repair, there is an unmet need for therapies that actively repair or protect cartilage, especially in Rheumatoid Arthritis. We envisage these microparticles could offer therapeutic possibilities in the protection of cartilage in situ in these difficult-to-treat patients.

Despite the success of current biological agents, achievement of broader efficacy and improved safety profile remains an unmet need in rheumatoid arthritis therapy. Neovasculogenesis plays a vital role in the progression and perpetuation of rheumatoid arthritis and significant evidence has demonstrated molecular heterogeneity within the endothelium (MVE) of different tissues. The heterogeneity of the synovial MVE can be exploited for the development of organ-specific therapeutic and diagnostic reagents. A novel recombinant antibody fragment, scFv A7, with specificity for human arthritic synovium, was isolated in our laboratory following in vivo phage display. The aim of the project described in this thesis is to characterise the antibody reactivity and develop a novel tissue specific therapeutic. The scFv A7 antibody proved to specifically target the microvasculature of human arthritic synovium with no detectable reactivity in a comprehensive range of normal tissues. Furthermore, the detected reactivity was not a common feature of chronic inflammatory conditions. Hence, the A7 antibody represents a unique and versatile tool with great potential for the development of diagnostic and/or therapeutic agents. The unique properties of A7 were combined with the anti-TNF Adalimumab, forming a bispecific antibody with neutralising activity and synovial homing properties. The new construct was able to retain the synovial specificity and showed comparable TNF binding kinetics and biological activity to the parent Adalimumab antibody in vitro. In conclusion, these results demonstrate that scFv A7 reactivity is specific to the microvasculature of human arthritic synovium, suggesting that the target molecule may have potential as a biomarker in arthritis and applications as an immunotherapeutic target. The bispecific antibody format developed showed unaltered TNF blocking capacity and synovial specificity that may allow reduction in the dosage and/or administration frequency, with the ultimate goal to reduce the systemic exposure, achieve a better therapeutic index and decreasing health care costs.

Psoriatic arthritis (PsA) is an inflammatory arthritis that is associated with psoriasis. PsA is a complex disease that is influenced by both genetic and environmental factors. Genetic studies have been successful in identifying risk loci for PsA, the majority of which also confer risk for psoriasis. As PsA has been estimated to have a stronger genetic component compared to psoriasis, this suggests that there should be genetic loci that are associated with PsA and not with psoriasis, which I have defined to be PsA-specific. Therefore, the overall aim of my PhD project is to apply genetic experimental techniques to identify a novel risk locus that is associated with PsA and not psoriasis. Firstly, genotyping of 1,962 PsA cases was performed with the Immunochip, where I contributed to the identification of a novel PsA-specific association on chromosome 5q31, mapping to rs715285 (P=4.38x10-13). Bioinformatic analysis within the association region identified 3 potential causal genes and 4 potential causal variants. Statistical analysis identified CD8+ memory T cells to be the most critical cell type for PsA, and also gave evidence to suggest that rs10065787 is the causal variant within the 5q31 region. A cell-specific eQTL study was performed within this cell type which identified SLC22A5 to be a potential causal gene for PsA. The findings may suggest a potential mechanism by which the 5q31 region contributes towards PsA susceptibility. Variants that reached nominal association during the Immunochip study (P &lt;1.0x10-4) were selected to follow-up in a candidate SNP study, in addition to other SNPs of interest which had been investigated for psoriasis risk but not PsA alone. This was performed within an independent cohort (914 PsA cases, 6,945 controls). Analysis of the independent samples replicated a PsA-specific association with rs12044149 (P = 4.03x10-6) at the IL23R locus. This association reached genome-wide significance during meta-analysis with the discovery Immunochip genotype data (P = 1.28x10-20). The PsA association with IL23R was found to be independent of the psoriasis association at the same locus when performing conditional analysis (Pcond = 4.86x10-06). The effect estimates for rs12044149 in PsA and psoriasis were found to be significantly different (P = 2.0x10-3). Furthermore, direct comparison of the genotypes for rs12044149 showed a significantly increased risk for PsA compared to psoriasis. Functional annotation was performed with credible SNPs sets defined for the PsA and psoriasis association signals at IL23R. This also revealed potential differences in the mechanisms of the two diseases, supporting the independence of their association with IL23R.A rare variant study was also performed as part of this PhD in order to identify a casual gene for PsA. 1,980 PsA cases and 5,913 controls were genotyped with the Illumina HumanCoreExome and HumanExome chips. Single-variant analysis was performed using the Fisher's Exact test. Analysis of common variants (MAF &gt; 0.05) uncovered associations at known PsA and psoriasis loci that reached genome-wide significance, including TRAF3IP2 (P = 3.88x10-18). Analysis of rare (MAF &lt;0.01) and low-frequency (MAF &lt;0.05) variants identified an association with a coding-variant at IFIH1 (rs35667974, P = 2.39x10-6). This association was replicated within an independent cohort, and was found to be significantly associated during multiple-variant testing (P = 2.30x10-6, Pcorr = 5.60x10-6). The minor allele of the rare variant was found to exist in the same haplotype as the minor allele of the PsA-associated common variant at the IFIH1 locus. The rs36557974 SNP found to be associated with PsA independently of the common variant during conditional logistic regression and conditional haplotype analysis. Although the rare-coding variant has been previously reported for psoriasis, these findings demonstrate the use of rare variant analysis to find evidence of a casual gene for PsA.In summary, genetic and statistical approaches have been used to find evidence of causal genes and variants within PsA-specific association regions, as well as to identify a PsA association mapping to a coding region.

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