La polyarthrite rhumatoïde (PR) est une maladie inflammatoire chronique caractérisée par des atteintes articulaires. Les synoviocytes, cellules qui tapissent la membrane synoviale, deviennent réfractaires à l‘apoptose et, en produisant la cytokine inflammatoire Interleukin 6 (IL-6), participent à la chronicité de l‘inflammation qui est à l‘origine de la perte osseuse dans la PR.Le zinc (Zn) et le cadmium (Cd) partagent leurs propriétés physico-chimiques et leurs mécanismes de transport cellulaire. Le Zn régule des fonctions du système immunitaire, contrairement au Cd avec des propriétés sur le système immunitaire peu décrites dans la littérature.Notre travail vise à identifier les mécanismes impliqués dans la modification de l‘homéostasie des métaux dans les synoviocytes par l‘inflammation et les conséquences de cette altération.L‘inflammation induit l‘accumulation des métaux dans les synoviocytes en augmentant l‘expression de l‘importeur ZIP-8. En revanche, l‘expression de l‘exporteur ZnT1 et des metallothionéines (MTs, régulateurs de l‘homéostasie des métaux) est dépendante de la présence des métaux. L‘affinité Cd-MTs permet une accumulation irréversible du Cd dans les cellules qui réduit leur prolifération et la production d‘IL-6.Les effets antiprolifératif et anti-inflammatoire du Cd ont été testés dans le modèle d‘arthrite chez le rat où une seule injection intra-articulaire de Cd à faible dose prévient la perte osseuse et réduit les scores cliniques d‘arthrite. Cela pourrait représenter une nouvelle approche thérapeutique pour le traitement de la PR et d'autres pathologies caractérisées par une hyperplasie et une inflammation localisées / Rheumatoid arthritis (RA) is a chronic inflammatory disease. Synoviocytes, cells forming the inner layer the synovium, become refractory to apoptosis and participate in the chronicity of inflammation through the production of IL-6. The perpetuation of inflammation causes an important induction of bone loss in joints.Zinc (Zn) and Cadmium (Cd) share many physico-chemical properties and cell transport mechanism. Zn is known as a regulator of the normal function of the immune system, while Cd properties on the immune system are not well defined.Our aim was to provide information on the metal homeostasis mechanisms in synoviocytes during chronic inflammation and on the consequences of metal homeostasis changes. After studying the differential effect of Zn and Cd at the cellular level, we could provide an innovative tool to control synoviocyte contribution to rheumatoid arthritis, which was tested on an in vivo model of arthritis.Results show that IL-17/TNF-a combination drives the accumulation of metals inside synoviocytes through the enhancing of ZIP-8 importer expression and regardless of the concentration of metals in the culture medium. In contrast, the expression of the metal exporter ZnT1 and of the homeostasis regulators metallothioneins (MTs) was primarily dependent on metal levels.Addition of Zn stimulated the inflammatory response, while addition of Cd can reduce both viability and inflammation.The anti-proliferative and anti-inflammatory properties of Cd were used in the rat model of arthritis as intra-articular treatment to reduce local inflammation and joint destruction and it may represent a new local therapeutic approach for RA treatment

Introduction: Rheumatoid arthritis (RA) is a chronic disease and one of the more common auto-immune diseases. Patients with RA rely almost solely on pharmaceutical intervention to manage the disease. Autonomic impairment has been proven in previous studies on patients with RA. The positive effect of exercise on autonomic impairment has also previously been demonstrated, but not in the RA population. The purpose of this study was firstly to confirm autonomic impairment in a South African based female population with RA and secondly to evaluate the effect of exercise on the autonomic cardiac function (as measured by short-term heart rate variability), disease activity and functional capacity. Methods: The study was conducted at the University of Pretoria during 2009 and 2010. In the first phase of the study female RA patients were recruited from all rheumatology practices in Pretoria and healthy controls were recruited from family and friends of the research team and of the RA group. Cardiac autonomic function was compared between the two groups by means of short-term heart rate variability. Three techniques were used: time domain, frequency domain and Poincare plot analysis. In the second phase of the study, females with confirmed RA were randomly assigned to an exercise group and a control group. The exercise group was requested to train under supervision two to three times per week for a period of twelve weeks, while the control group continued with their sedentary lifestyle. At study completion the two groups were compared for the effect of exercise intervention on the following three aspects: <ul><li> Autonomic function (as measured by heart rate variability) </li><li> Disease activity (as measured by Disease Activity Score, Visual Analogue Scale and Health Activity Questionnaire) </li><li> Functional capacity (as measured by strength, flexibility and aerobic capacity) </li></ul> Results: In the first phase of the study comparing females with RA (n=45) to healthy females (n=39), the basal heart rate was significantly higher in the RA group. In the supine position significant differences existed between the RA group and the control group (p ≤ 0.01). Indicators of parasympathetic activity showed significantly lower variation in the RA group [RMSSD=14.70, pNN50=0.50, SD1=10.50, HF(ms2)=31] compared to the control group [RMSSD=29.40, pNN50=7.8, SD1=20.9, HF(ms2)=141.00]. Indicators of sympathetic variation were also significantly lower in the RA Group [SD2=36.70, LF(ms2)=65) compared to the Control group (SD2=49.50, LF(ms2)=175]. In the standing position 8 variables indicated autonomic impairment by significant differences (p≤0.01) between the 2 groups. The response of the RA Group to an orthostatic stressor showed less vagal withdrawal, [p-values for RMSSD=0.038, pNN50=0.022, SD1=0.043 and HF(ms2)=0.008 respectively]; and lower sympathetic response [p-values for SD2=0.001 and LF(ms2)<0.001] when compared to the Control group. In the second phase of the study, comparing an RA exercise group to a RA sedentary group, three aspects were evaluated: 1. Heart rate variability At baseline the control group (n=18) had significantly higher variability compared to the exercise group (n=19) for most heart rate variability (HRV) indicators. At study completion the variables showing significant changes (p=0.01 to 0.05) favoured the exercise group in all instances. Wilcoxon signed rank tests were performed to assess changes within groups from start to end. The exercise group showed significant improvement for most of the standing variables, including measurements of combined autonomic influence e.g. SDRR (p=0.002) and variables indicating only vagal influence e.g. pNN50 (p=0.014). The control group mostly deteriorated with emphasis on variables measuring vagal influence [RMSSD, pNN50, SD1 and HF(ms2)]. 2. Disease activity At baseline the two groups were comparable. At the end of the intervention, the exercise group had significant improvement for the tender joint count (p=0.015), swollen joint count (p=<0.001), physician global assessment (p=0.003) and DAS score (p=0.003) compared to the control group. To assess changes that happened within each group from start to end, Wilcoxon signed rank tests were performed. The exercise group improved significantly with regards to tender joint count (p=0.002), swollen joint count (p=0.001), physician global assessment (p=0.001), DAS score (0.001) and the visual analogue scale (p=0.032). The sedentary group improved significantly only in the health assessment questionnaire (p=0.032). 3. Functional capacity Comparing the groups at baseline the exercise group had better knee- and hip flexion on the left hand side but it took them longer to complete the arm curl test. At study completion the exercise group was mostly favoured with regards to flexibility (significant p-values ranging between 0.001 – 0.049), strength (handgrip right p<0.001, leg strength p=0.035, arm curl test p=0.010, sit to stand test p=0.025) and aerobic fitness (1 mile walk test p<0.001 and VO2 max p=0.007). Changes within each group were assessed by Wilcoxon signed rank tests. The exercise groups showed significant changes for many parameters in the three categories, i.e. flexibility (8 of 18), strength (5 of 5), and aerobic fitness (4 of 8). The control group mostly deteriorated in flexibility, while their strength also improved, but not to the same extent as for the exercise group. Their aerobic fitness did not change. Discussion: In the first phase of this study, using standardised methods to measure short-term HRV, females with RA showed less variability compared to a healthy age- and sex matched control group. An inability of the autonomic nervous system to efficiently compensate to internal and external environmental changes may predispose RA patients to arrhythmias thereby increasing cardiovascular mortality. All 3 methods used showed the same outcome, implying decreased HRV and thus an increased risk for arrhythmias in RA patients. Evaluating the autonomic nervous system might be critical in planning management of RA. In the second phase study results indicated that twelve weeks of exercise intervention, had a positive effect on cardiac autonomic function as measured by short-term HRV, in females with RA. Several of the standing variables indicated improved vagal influence on the heart rate. Exercise can thus potentially be used as an instrument to improve cardiac health in a patient group known for increased cardiac morbidity. The exercise programme was also effective in decreasing perception of pain as well as disease activity in female RA patients. Given our findings it seems warranted to include physical exercise as part of the treatment prescription of patients with class I and II RA. Lastly this research has shown that regular, controlled exercise for RA patients with controlled disease can decrease joint stiffness and improve joint mobility, strength and aerobic capacity without exacerbating pain or disease activity. Also, if one observes the decline in the sedentary group for many parameters, it is important to note that this happened over a relative short time period and that even a small change may have a detrimental impact on the RA patient. The current report supports previous literature on autonomic impairment in patients suffering from RA as well as the meaningful positive effect of exercise on disease activity and functional capacity. It is the only study on the effect of an exercise intervention on the cardiac autonomic function of RA patients. Future research in this field should aim for larger study samples, longer intervention periods and perhaps add analysis of blood pressure variability to support results obtained by HRV analysis. / Thesis (MD)--University of Pretoria, 2012. / Internal Medicine / unrestricted

The inflammatory response caused by the deposition of crystals of monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD) in the synovial fluid of joints, results from the interaction of the crystals with neutrophils. Neutrophils (whose function in the body is to remove hazardous microorganisms and inflammatory debris) are activated by the binding of the crystals to the neutrophil cellular membrane, which leads to respiratory burst activity, engulfment of the crystals and release of proteolytic enzymes. Furthermore, we have found that crystals delay the normal “cell death program” or apoptosis, thus allowing for the accumulation of these cells, and extended inflammatory responses. Very little is known about the mechanisms of activation and delay of apoptosis, however, bcl-2 family proteins have been implicated in the control of neutrophil apoptosis. This study helps to define the role of several bcl-2 family proteins (both pro- and anti-apoptotic) by examining the differential expression of these proteins upon stimulation with crystals. Subsequent identification of signaling targets that function to regulate this process in response to crystals could lead to potential therapeutics for crystal-induced inflammatory diseases. / Medicine, Faculty of / Medicine, Department of / Experimental Medicine, Division of / Graduate

Les monocytes sont des leucocytes dérivés de précurseurs de la moelle osseuse pouvant se différencier en macrophages, cellules dendritiques (CD), ou ostéoclastes (OC). Ils jouent un rôle critique dans la persistance de l'inflammation et la destruction des articulations par le biais de mécanismes encore mal connus. Les monocytes sont composés de deux grandes sous-populations chez la souris, discriminées sur la base du marqueur de surface Ly6C. Il a été suggéré que les OC pouvait être préférentiellement différenciés à partir de la sous-population monocytaire Ly6Chigh, dont l'activation excessive et prolongée est une signature clé de nombreuses pathologies inflammatoires. Parmi les acteurs moléculaires responsables de la régulation de l'expression des gènes, les miARNs jouent un rôle majeur dans de nombreux processus physiologiques, dont la réponse inflammatoire, en régulant finement les programmes génétiques au niveau post-transcriptionnel. Leur implication dans l'ostéoclastogénèse est encore mal connue. Par ailleurs, leur expression est perturbée dans de nombreuses pathologies, dont la polyarthrite rhumatoïde qui implique à la fois un dysfonctionnement de la réponse inflammatoire et de l'homéostasie osseuse. Mon projet de thèse vise à mieux comprendre l'implication des sous-populations monocytaires dans la persistance de l'inflammation et de l'activité des OC au travers de l'étude du rôle de miR-146a dans des conditions physiologiques et inflammatoires. J'ai montré que miR-146a est le miARN le plus différemment exprimé entre monocytes classiques Ly6Chigh et non-classiques Ly6Clow, et que son expression est diminuée dans les Ly6Chigh en conditions arthritiques. J'ai également montré que la perte de miR-146a augmente l'ostéoclastogénèse in vitro et l'érosion osseuse in vivo chez les souris arthritiques. Enfin, la surexpression artificielle de miR-146a dans les monocytes Ly6Chigh inhibe la différenciation osteoclastique et la perte osseuse dans l'arthrite expérimentale chez la souris. Mes résultats suggèrent que miR-146a contrôle l'hétérogénéité fonctionnelle des monocytes et qu'une diminution de son expression dans la sous-population Ly6Chigh serait responsable de l'augmentation de l'osteoclastogénèse et de l'érosion osseuse observées en conditions arthritiques. Pour finir, mes résultats montrent également qu'augmenter l'expression de miR-146a dans les monocytes Ly6Chigh présente un intérêt thérapeutique pour contrecarrer la perte osseuse associée à l'arthrite. / Introduction : Monocytes represent a prototypic cell type when investigating the interplay between immune and skeletal systems as they can give rise to different cell types including dendritic cells, macrophages and osteoclasts (OC), which play key roles in immunity and bone homeostasis. Circulating monocytes consist of at least two main functional subsets, Ly6Chigh and Ly6Clow monocytes. It has been suggested that OC might develop preferentially from the Ly6Chigh monocyte subset, which excessive and prolonged activation is a hallmark of many inflammatory diseases. Among key molecular rheostats of cell fate, micro(mi)RNAs are a class of regulatory RNAs that control basic biological functions and orchestrate inflammatory responses. Few miRNAs have been involved in osteoclastogenesis. The present study aimed at investigating the role of miRNAs in osteoclastogenesis in the context of monocyte subsets, under steady state and inflammatory conditions. Methods & Results : Using genome-wide miRNA expression study we have identified miRNAs and putative targeted pathways that are differentially expressed between Ly6Chigh and LyC6low FACS sorted mouse monocytes, and common to their human counter parts CD14+CD16- and CD14dimCD16+ monocytes, respectively. Among these, miR-146a showed higher expression in Ly6Clow monocytes when compared to Ly6Chigh monocytes. Under inflammatory arthritis conditions, expression of miR-146a in Ly6Chigh monocytes was down regulated as compared to healthy controls. Using mouse deficient for miR-146a, we showed that knockdown of miR-146a increased OC differentiation in vitro. While no bone phenotype was evidenced in miR-146a deficient mice, nor under steady state or ovariectomized conditions, arthritis-induced bone resorption and bone loss were increased in miR-146a knockout mice. Finally, using a liposomal formulation able to delivermiR-146a mimics to Ly6Chigh monocytes upon intravenous injection, we showed that enforced expression of miR-146a led to decreased number of TRAP positive cells within the synovium of arthritic mice, and efficiently reduced bone erosion in inflammatory arthritis. This effect was associated with decreased RelB expression in miR-146a-overexpressing Ly6Chigh osteoclast progenitors. Conclusion : Overall, our results show that specific over-expression of miR-146a in Ly6Chigh monocytes altered OC differentiation and decreased bone erosion in inflammatory arthritis. These data suggest a novel role for miR-146a in controlling osteoclast fate of Ly6Chigh monocyte progenitors and that reduced miR-146a expression in Ly6Chigh monocytes under arthritic conditions contributes to pathogenic bone loss. Finally, delivery of miR-146a mimics to Ly6Chigh monocytes may offer valuable therapeutic potential to interfere with pathological bone loss.

Ce mémoire porte sur l’étude de pyrophosphates de calcium hydratés (CPP : Ca2P2O7•nH2O), composés rencontrés dans des micro-calcifications pathologiques associées à l’arthrose et, dans certains cas, responsables d’arthropathie destructive. Ces cristaux, présents dans les articulations de patients arthritiques, possèdent un fort potentiel inflammatoire susceptible d’engendrer une dégradation aigüe du cartilage. Cependant, les mécanismes de formation des phases de CPP et d’activation de leur potentiel inflammatoire n’ont pas encore été entièrement décrits. Nous nous sommes intéressés à l’étude des conditions de formation des différentes phases de pyrophosphates de calcium hydratés in vitro ainsi qu’à la caractérisation fine de chacune des phases d’intérêt biologique avec des outils de laboratoire et de grands instruments afin de mieux comprendre leurs propriétés physico-chimiques et d’améliorer leur identification in vitro et in vivo. Dans un premier temps, un protocole de synthèse a été établi permettant la synthèse de quantités importantes de chacune des phases pures de CPP (CPP amorphe, CPP dihydratés monoclinique et triclinique et CPP tétrahydraté). Les conditions de synthèse associées à la formation de chacune de ces phases, pH et température notamment, ont été explorées. Des échantillons purs ont été utilisés comme références pour les différentes études physico-chimiques et structurales qui ont ensuite été menées. Les échantillons de référence ont été caractérisés finement, d’un point de vue structural avec notamment la résolution de plusieurs structures cristallines (diffraction des rayons X et des neutrons, sur poudre et monocristal) mais aussi au travers de différentes analyses spectroscopiques (spectroscopies FTIR et Raman, RMN du solide) et d’analyses de la morphologie des cristaux (microscopies électroniques à balayage et en transmission, diffraction électronique). Chacune de ces analyses complémentaires, couplées à des modélisations ab initio, a permis de préciser les hypothèses suggérant un rôle de la surface des cristaux dans le potentiel inflammatoire de ces phases. Une troisième partie est consacrée à l’exploration de différentes techniques de synthèse mettant en œuvre différents milieux (cristallisation en solution et en gel). Ces expériences ont permis d’établir des comparaisons avec les processus de formation observés in vivo et d’évolution in vitro à haute température des phases de CPP. Finalement des études ex vivo de cartilages calcifiés seront présentées, mettant en évidence les avantages de ces techniques de caractérisation de laboratoire comme outils de diagnostic. Ce travail permet ainsi de préciser les mécanismes physico-chimiques liés aux différentes phases de CPP in vitro et in vivo afin de mieux comprendre la formation de ces phases et leur potentiel inflammatoire associé, tout en améliorant les possibilités de diagnostic des arthropathies microcristallines. / The present work concerns the study of hydrated calcium pyrophosphates (CPP: Ca2P2O7•nH2O), a group of phases detected in pathological microcalcifications and associated with arthritis. These crystals are frequently observed in the synovial fluid of arthritic patients and they were described as having a high inflammatory potential which could induce a severe degradation of cartilage. However, the mechanisms involved in the formation of the CPP crystals and the activation of their inflammatory potential are not fully understood. This work is focused on the study of the synthesis conditions of CPP in vitro and on the fine characterization of CPP phases of biological interest using laboratory equipments and large-scale facilities. The aim of this work was to describe the physico-chemical properties of these materials, including inflammatory potential, and to improve their identification in vivo and in vitro. First, a synthesis protocol was designed for the production of significant amounts of pure samples for each of the CPP phases (amorphous CPP, monoclinic and triclinic dihydrated CPP and tetrahydrated CPP). Different conditions, including pH and temperature, were studied to achieve the synthesis of reference materials. These samples were precisely characterized using complementary techniques to determine their crystalline structures (powder and single crystal X-ray diffraction and neutron diffraction) as well as using spectroscopic (FTIR and Raman spectroscopies, MAS-NMR) and morphologic analyses (SEM, TEM and electron diffraction). These analyses, combined with ab initio modeling, clarified the hypotheses concerning the role of the crystal surface on the adsorption properties of CPP crystals and their inflammatory potential. The third part of this thesis is focused on the study of CPP synthesis conditions, by using different experimental setups to study crystallization in solution and in gel. A comparison with in vivo formation processes and in vitro high temperature evolution phenomena of these phases was established based on the results of these experiments. Finally, ex vivo analyses of pathological cartilage are presented, highlighting the advantages of different laboratory characterizations as medical diagnostic tools. This work contributes to clarify the physico-chemical characteristics of CPP phases in vitro and in vivo, to improve the knowledge on the formation and the evolution of these phases, their properties including inflammatory potential, and to facilitate their identification in vivo.

Background: Rheumatoid arthritis (RA) is a common complex autoimmune disorder which is influenced by both genetic and environmental factors. While multiple factors that influence susceptibility to and outcome of disease have been identified there is still a large proportion of missing heritability and limited understanding of disease pathogenesis. In recent years, biologic drug therapies have advanced treatment of RA; however good disease control is achieved in just 30% of patients, making identification of predictors of treatment response important. One area of research which is yet to be explored in relation to treatment response, and requires further evaluation in RA susceptibility, is epigenetics. Epigenetics is the study of modifications of the DNA which can influence gene expression but do not alter genetic sequence, and the most commonly studied epigenetic phenomenon, to date, is DNA methylation. Objectives: To identify DNA methylation signatures predictive of treatment response to anti-TNF biologics, to explore the role of DNA methylation in RA susceptibility using disease discordant monozygotic (MZ) twins, and to assess the effect of cryopreservation of cells on DNA methylation. Methods: Genome-wide DNA methylation levels were measured using the HumanMethylation450 BeadChip in pre-treatment whole blood DNA samples from individuals who had extremely good or extremely poor response to the anti-TNF therapies, etanercept and adalimumab, and in MZ twins discordant for RA (n=79 pairs). I also compared genome-wide methylation in cells which had been cryopreserved with fresh cells, to investigate if this technique is suitable for epigenetic investigations. Results: I identified four methylation sites which were significantly related to response to etanercept at a false discovery rate of 5%, the most significantly differentially methylated of which maps to the LRPAP1 gene (p=1.46E-8). Indeed, four other sites at the same locus also showed evidence for differential methylation indicating that this represents a differentially methylated region. No sites were significantly associated with response to adalimumab after correction for multiple testing. I identified subtle differences in DNA methylation between RA discordant twins. Although these were not statistically significant following adjustment for cell composition, one of the most differentially methylated positions mapped to the ZNF74 gene (p=4.97E-6), and replicated a methylation difference identified in the largest previous epigenome-wide association study of RA cases and unrelated healthy controls. I found that cryopreservation of cells does not significantly alter the methylome, an important observation that will impact upon design of future studies. Conclusions: In the largest studies of DNA methylation in RA treatment response and RA discordant MZ twins to date, I identified significant differential methylation in etanercept response, but not adalimumab response, and found small differences in methylation in RA discordant MZ twins. I also concluded that cryopreservation does not significantly alter the methylome.

A problems/needs inventory enquired into special social-psychological-emotional problems experienced by seniors in care facilities, due to arthritis. The inventory was based on three questions: are there any such problems? If so, what are they? And, what could be done by facility staff to help relieve such problems? This was the first stage in a proposed larger project: to develop a Social Work component dealing with such special problems, in order to augment an existing staff training program, and to test, evaluate, revise and implement the complete program package. A community-based approach was used. The Department of Social Work Services at the Arthritis Society produced a listing of key areas of concern at a brainstorming session; health-care and social-service professionals were surveyed by interview or by the Delphi Technique; a seniors' arthritis support group participated in a Nominal Group Technique session; care-facility staff met to discuss relevant issues; and, data was gleaned from current literature by a content analysis method. There was general agreement that seniors in care with arthritis do have special social-psychological-emotional problems because of the disease, and also there was consistency regarding the nature of these problems. Analysis of the data led to findings that included the following propositions: A sense of self-reliance and of social integration are essential to well-being. Limitations on movement, and the effects of chronic pain and of pain medication are associated with significant social-psychological emotional problems among seniors in care with arthritis. Two principles of care were identified: 1) support and encourage independence; and, 2) support and encourage social integration. The propositions will be applied to the development of the content of the Social Work component of the training program. Also, many respondents made important recommendations regarding methods and format for the training program. These are included in this paper. / Arts, Faculty of / Social Work, School of / Graduate

Very few good empirical investigations of pain and coping in children and adolescents currently appear in the published literature. In contrast to the adult literature, for ethical reasons, a foundation of basic research using experimentally-induced pain does not exist in the pediatric literature (McGrath, in press). This remaining deficiency in knowledge about children's spontaneous abilities to cope with pain is particularly harmful because it means that clinicians must base their assessment and treatment of pain in children on their knowledge of adults (Jeans, 1983). The need to consider cognitive-developmental issues has been emphasized in several recent papers (Lavigne, Schulein, & Hahn, 1986; Maddux, Roberts, Sledden, & Wright, 1986; Thompson & Varni, 1986). The present investigation evaluated the pain experienced and spontaneous coping strategies used by 39 children and adolescents with various forms of arthritis, during a painful joint-measuring task which is typically part of physiotherapy treatments for this illness. The two purposes of the study were: 1) to assess age/cognitive-developmental differences and 2) to compare "effective copers" versus children who were having some difficulties coping with pain (i.e. pain was interfering with their activities of daily living). Three age groups (5-7 years, 8-10 years, and 11-18 years), corresponding to the Piagetian stages of preoperational, concrete operational and formal operational thought, were compared. Subjects were videotaped while the range of motion in their joints was measured by the physiotherapist. Videotapes were subsequently coded for behavioral coping strategy use. Immediately following the joint measurement task, subjects were interviewed regarding thoughts they recalled experiencing. Transcribed interviews were subsequently coded for cognitive coping strategies reportedly used and catastrophizing cognitions reportedly experienced. In addition, parents completed two questionnaires rating the degree to which pain interferes with their child's activities of daily living, and the physiotherapist made a global rating of each child's functional capacity. The overall MANOVA using age group as a between groups factor, with self-reported pain variables entered as dependent measures was nonsignificant. A significant multivariate effect did emerge, however, when the coping variables were entered as dependent measures in a second overall MANOVA. Follow up univariate analyses revealed an age/cognitive-developmental trend in behavioral and cognitive coping strategy use. Children in the youngest group (preoperational) used primarily behavioral strategies to cope with pain elicited by the physiotherapy joint-measuring task, whereas slightly older children (concrete operational) began to supplement their repertoire of behavioral coping strategies with some cognitive coping strategies. A significant rise in reported cognitive coping strategy use was observed in the oldest group (formal operational). In addition, a discriminant function revealed that the two most important discriminators between "effective copers" versus children having some difficulties coping with pain were the amount of pain expression (vocal or nonvocal) coded and the amount of catastrophizing thoughts reportedly experienced during the physiotherapy task. Implications of these results for the treatment of children having difficulties coping with arthritic pain are discussed. / Arts, Faculty of / Psychology, Department of / Graduate

The immune system is composed of innate and adaptive mechanisms. Innate immune responses are significantly modulated by immunomodulatory factors that act through the induction of specific patterns of cytokine production in responding cells. Human leukocytes have been shown to respond to substance(s) present in acid extracts of commercial shark cartilage (SC). Shark cartilage is a food supplement taken by consumers as a prophylaxis and for the treatment of conditions ranging from arthritis to cancer. No reliable scientific evidence in the literature supports the alleged usefulness of shark cartilage supplements, but their use remains popular. Cartilage extracts exhibit immunomodulatory properties by inducing various inflammatory, Th1-type cytokines and potent chemokines in human peripheral blood leukocytes (HPBL) in vitro. The objectives of the study were to (1) to determine the nature of the active component(s), (2) to define the scope of cellular response to SC extract, and (3) to elucidate the molecular mechanisms underlying bioactivity. Results showed that there are at least two cytokine-inducing components which are acid stable. One anionic component has been identified as a small (14-21 kDa) glycoprotein with at least 40% carbohydrate content. Shark cartilage stimulated HPBL to produce cytokines resembling an inflammatory, Th1 polarized response. Leukocyte-specific responses consist of both initial cytokine responses to SC directly (i.e., TNF-a) and secondary responses such as the IFN-γ response by lymphocytes following initial SC stimulation. Response of RAW cells, a murine macrophage cell line, indicated that TNF-α could be induced in macrophages of another mammalian species in the absence of other cell types. The results suggest that the human monocyte/macrophage is most likely to be the initial responding cell to SC stimulation. Stimulation of cells appears to engage at least one ligand-receptor interaction with TLR 4, although the role of TLR 2 cannot be ruled out. Initial activation is likely followed by the activation of the JNK and p38 MAPK signal transduction pathways resulting in activation, release, and translocation of transcription factor nuclear factor κB (Nf-kB). This dissertation research study represents the first in-depth study into characterizing the bioactive component(s) of commercial shark cartilage responsible for its immunomodulating properties and defining cellular responses at the molecular level.

Artrite reumatóide é uma doença auto-imune que apresenta acentuado quadro inflamatório e proliferação celular o que, provavelmente, determina a alta prevalência de doenças cardiovasculares quando comparados a população mundial. A mortalidade e a morbidade conseqüentes das doenças cardiovasculares estão 2 vezes aumentadas em pacientes com artrite reumatóide e um dos principais fatores de risco relacionados ao desenvolvimento da aterosclerose é a dislipidemia. Esse importante fator de risco vem sendo associado à artrite reumatóide e as concentrações plasmáticas de lípides são constantemente avaliadas, já que se encontra bem estabelecido a relação entre dislipidemia e alta incidência de doença cardiovascular. No entanto, o verdadeiro impacto das alterações lipídicas na artrite reumatóide não é bem conhecido, já que os resultados de perfil lipídico são contraditórios. Alterações nas concentrações plasmáticas de lípides não necessariamente acompanham distúrbios no metabolismo das lipoproteínas plasmáticas. O objetivo do presente estudo foi avaliar aspectos do metabolismo da LDL e da HDL, em pacientes com artrite reumatóide. Nesse sentido, foi avaliada a cinética plasmática de uma nanoemulsão lipídica artificial com comportamento metabólico semelhante ao da LDL em 30 pacientes com artrite reumatóide divididos em 2 grupos de acordo com a atividade da doença, alta atividade (n=14) e remissão (n=16) e 30 indivíduos controle. A nanoemulsão marcada com éster de colesterol 14EC (EC-14C) e colesterol livre 3H (CL-3H) foi injetada endovenosamente após 12 horas de jejum. As amostras de sangue foram coletadas em tempos pré-determinados (5 min, 1, 2, 4, 6, 8 e 24 horas) após a injeção, para determinação das curvas de decaimento plasmático e da taxa fracional de remoção (TFR) dos lípides marcados, por análise compartimental. As TFR-EC-14C e TFR-CL-3H foram maiores no grupo AR quando comparado ao grupo controle (49%, p<0,05 e 44%, p<0,05, respectivamente), não havendo diferença entre os subgrupos de artrite reumatóide. No grupo artrite reumatóide e em seus subgrupos, as concentrações de HDL-C e apo E foram maiores quando comparados ao grupo controle (33%, p<0,0001 e 20%, p<0,01, respectivamente), enquanto os níveis de apo B foram menores na artrite reumatóide quando comparados ao grupo controle (16%, p<0,05). A transferência de colesterol esterificado radioativo da nanoemulsão para a HDL foi menor na artrite reumatóide, comparando-se com o grupo controle. A transferência dos outros lípides foi similar nos dois grupos. A HDL dos pacientes com artrite reumatóide foi menor do que a dos controles. Esses resultados podem contribuir com a melhor compreensão de possíveis mecanismos relacionados a uma maior incidência de doenças cardiovasculares em pacientes com artrite reumatóide / Mortality and morbidity, as a consequence of cardiovascular diseases, is twice as high in patients with rheumatoid arthritis than in the general worldwide population. This autoimmune disease has predominant inflammatory and cell proliferation background probably explains the high prevalence of cardiovascular disease. Dyslipidemias are important risk factors for cardiovascular disease. This study investigated the link between RA and plasma lipids as a predisposition to this high cardiovascular disease incidence. However, the impact of lipids on cardiovascular risk in rheumatoid arthritis is unclear. So much so, that lipid profiles in patients with rheumatoid arthritis in published studies is contradictory. The events of intravascular lipoprotein metabolism do not necessarily produce altered levels of plasma lipids. In an attempt to unravel novel dysfunctional mechanisms that could trigger pro-atherogenic processes beyond the concentration of the plasma lipids, plasma clearance of a lipidic nanoemulsion that resembles the LDL metabolic behavior were investigated in rheumatoid arthritis patients and compared to control subjects without the disease. 30 patients with rheumatoid arthritis divided into 2 groups according to disease activity, high activity (n=14) and remission (n=16), and 30 controls were studied. A nanoemulsion labeled with 14C-cholesteryl esther (14C-CE) and 3H-free cholesterol (3H-FC) were endovenously injected after which blood samples were collected at pre-determined periods (5 min, 1, 2, 4, 6, 8 and 24 hours), in order to determine the radioactivity of the plasma decay curves and calculate the fractional clearance rate (FCR) of the labeled lipids for compartmental analysis. In the rheumatoid arthritis group and subgroups the HDL-C and apo E concentration were higher when compared to control group (33%, p<0,0001 e 20%, p<0,01, respectively) while apo B concentration was lower (16%, p<0,05). The 14-CE-FCR and 3H-FC-FCR were greater in rheumatoid arthritis group and subgroups when compared to controls (49%, p<0,05 e 44%, p<0,05, respectively). There were no differences between the rheumatoid arthritis subgroups. Therefore, rheumatoid arthritis accelerates the LDL plasma removal, as indicated by a higher 14-CE-FCR and 3H-FC-FCR. The transfer of other lipids was also similar in both groups. The HDL of the rheumatoid arthritis patients was lower than that of the control group. These results could clarify possible mechanisms that can be related to a higher cardiovascular incidence in patients with rheumatoid arthritis