Nous avons développé des nanoparticules d’une prodrogue de glucocorticoïde, la dexaméthasone palmitate (DXP) à visée thérapeutique dans le traitement de la polyarthrite rhumatoïde (PR). Cette maladie auto-immune est caractérisée par une inflammation articulaire, une érosion osseuse et cartilagineuse et une dérégulation du système immunitaire. Parmi les traitements indiqués dans la PR, l'utilisation des glucocorticoïdes est limitée par leurs effets secondaires importants induits par leur pharmacocinétique défavorable. Afin de traiter la PR par voie intraveineuse, la formulation de nanoparticules PEGylées semble indispensable afin d’échapper au phénomène d’opsonisation et d’espérer obtenir une accumulation spécifique au niveau des articulations inflammées. Pour cela, nous avons développé des nanoparticules de DXP (DXP-NP) stabilisées par le DSPE-PEG2000.Les caractéristiques physico-chimiques des nanoparticules obtenues ont été évaluées ainsi que leur stabilité au cours du temps. La structure interne des nanoparticules définie comme amorphe ainsi que leur très fort taux de charge prouvent ainsi l’impact du DSPE-PEG2000 dans l’organisation moléculaire des DXP-NP. In vivo, l’étude de la pharmacocinétique et de la biodistribution des DXP-NP suite à leur administration intraveineuse a démontré une circulation prolongée du système. Dans un modèle murin de polyarthrite rhumatoïde, les DXP-NP ont démontré leur accumulation spécifique dans les articulations inflammées en corrélation avec une supériorité thérapeutique significative en comparaison avec la molécule libre hydrosoluble. Des études histologiques ainsi que l’évaluation du traitement sur l’apparition d’effets indésirables complètent l’étude in vivo. / We developed nanoparticles of a glucocorticoid prodrug, dexamethasone palmitate (DXP) for the treatment of rheumatoid arthritis (RA). Joint inflammation, bone and cartilage erosion and dysregulation of the immune system characterize this autoimmune disease. Among the treatments indicated in RA, the use of glucocorticoids is hampered by their side effects induced by their unfavorable pharmacokinetics. To treat RA intravenously, the formulation of PEGylated nanoparticles seems essential in order to escape from opsonization and to obtain a specific accumulation in the joints inflamed. Therefore, we developed DXP nanoparticles (DXP-NPs) stabilized by the DSPE-PEG2000.The physicochemical characteristics of the nanoparticles obtained were evaluated as well as their stability over time. The amorphous internal structure of the nanoparticles and their very high drug loading thus prove the impact of DSPE-PEG2000 in the molecular organization of DXP-NPs. In vivo, the study of the pharmacokinetics and biodistribution of DXP-NPs following intravenous administration demonstrated prolonged circulation of the system. In a mouse model of rheumatoid arthritis, DXP-NPs their demonstrated specific accumulation in inflamed joints in correlation with significant therapeutic superiority in comparison with the water-soluble free molecule. Histological studies as well as adverse events evaluation supplemented the in vivo study.

The pain relieving efficacy of music listening combined with vibrotactile cutaneous stimulation was determined. Music with mechanical vibration (30min. session; average amplitude of 26μm; frequency range of 60-600Hz.) was applied to subjects with rheumatoid arthritis using the Music Vibration Table (MVT). Scores from pain relief visual analogue scales (VAS) and McGill Pain Questionnaires (MPQ) were compared to groups with music alone and placebo. ANOVA and post hoc analysis indicated that VAS scores from music with vibration were significantly greater than music alone or placebo. MPQ scores also indicated larger percentages of change in pain perception for the music with vibration condition. However, subjects receiving music alone showed a large percentage of change on the affective dimension of the MPQ. This investigation supports the application of music with a controlled, measurable music vibration for the relief of pain. The results of this study warrant further evaluation and development of treatment protocols using music and music vibration.

Ample studies have shown that perturbation of metabolic phenotype is correlated with gene mutation and pathogenesis of colorectal cancer (CRC) and rheumatoid arthritis (RA). Mass spectrometry (MS)-based metabolomics as a powerful and stable approach is widely applied to bridge the gap from genotype/metabolites to phenotype. In CRC suffers, KRAS mutation accounts for 35%-45%. In previous study, SLC25A22 that encodes the mitochondrial glutamate transporter was found to be overexpressed in CRC tumor and thus to be essential for the proliferation of CRC cells harboring KRAS mutations. However, the role of SLC25A22 on metabolic regulation in KRAS-mutant CRC cells has not been comprehensively characterized. We performed non-targeted metabolomics, targeted metabolomics and isotope kinetic analysis of KRAS-mutant DLD1 cells with or without SLC25A22 knockdown using ultra-high performance liquid chromatography (UHPLC) coupled to Orbitrap MS and tandem MS (MS/MS). In global metabolomics analysis, 35 differentially regulated metabolites were identified, which were primarily involved in alanine, aspartate and glutamate metabolism, urea cycle and polyamine metabolism. Then targeted metabolomics analysis on intracellular metabolites, including tricarboxylic acid (TCA) cycle intermediates, amino acids and polyamines, was established by using LC-MS/MS coupled with an Amide BEH column. Targeted metabolomics analysis revealed that most TCA cycle intermediates, aspartate (Asp)-derived asparagine, alanine and ornithine (Orn)-derived polyamines were strongly down-regulated in SLC25A22 knockdown cells. Moreover, the targeted kinetic isotope analysis using [U-13C5]-glutamine as isotope tracer showed that most of the 13C-labeled TCA cycle intermediates were down-regulated in SLC25A22-silencing cells. Orn-derived polyamines were significantly decreased in SLC25A22 knockdown cells and culture medium. Meanwhile, accumulation of Asp in knockdown of GOT1 cells indicated that oxaloacetate (OAA) was majorly converted from Asp through GOT1. Exogenous addition of polyamines could significantly promote cell proliferation in DLD1 cells, highlighting their potential role as oncogenic metabolites that function downstream of SLC25A22-mediated glutamine metabolism. SLC25A22 acts as an essential metabolic regulator during CRC progression as promotes the synthesis of TCA cycle intermediates, Asp-derived amino acids and polyamines in KRAS-mutant CRC cells. Moreover, OAA and polyamine could promote KRAS-mutant CRC cell growth and survival. Rheumatoid arthritis (RA) is a chronic, inflammatory and symmetric autoimmune disease and a major cause of disability. However, there is insufficient pathological evidence in term of metabolic signatures of rheumatoid arthritis, especially the metabolic perturbation associated with gut microbiota (GM). Based on consistent criteria without special diet and therapeutic intervention to GM, we enrolled 50 RA patients and 50 healthy controls. On basis of the platform of UHPLC-MS and GC-MS, were performed for the non-targeted metabolomics to investigate alterations of endogenous metabolites in response to RA inflammation and interaction with GM. 32 and 34 significantly changed metabolites were identified in urine and serum of patients with RA, respectively. The altered metabolites were identified by HMDB, METLIN database or authentic standards, and mostly metabolites were attributed into tryptophan and phenylalanine metabolism, valine, leucine and isoleucine biosynthesis, aminoacyl-tRNA biosynthesis and citrate cycle. To obtain alterations of more components in tryptophan and phenylalanine metabolism, we developed and validated a targeted metabolomics method of 19 metabolites by using LC-QqQ MS. Combining the results of targeted metabolomics with global metabolomics, significantly up-regulated kynurenine (KYN), anthranilic acid (AA) and 5-hydroxylindoleacetic acid (HIAA) simultaneously in urine and serum was found to implicate the activation of tryptophan metabolism under the condition of RA, which acted pro-inflammatory roles in inflammation and was closely correlated with GM. IDO/TDO functioned as a pro-inflammation mediator was overexpressed in RA patients. Urinary kynurenic acid and serum serotonin that have impacts on anti-inflammation in immune system were down-regulated in RA patients. The levels of phenylacetic acid and phenyllactic acid serving as a pro-inflammatory and an anti-inflammatory agent, respectively, increased in serum of patients with RA. Moreover, certain essential amino acids (EAAs), and mostly conditional EAAs were decreased in RA patients, which have been reported to inhibit cell proliferation of immune cells. In particular, deficiency of branched chain amino acids (BCAAs, valine and isoleucine) was observed in serum of patients with RA, which may lead to muscle loss and cartilage damage. The specificity of all altered metabolites resulted from RA was considerably contributed through the GM-derived metabolites. The findings revealed that GM-modulated RA inflammation was mainly resulted from tryptophan and phenylalanine metabolism, and amino acid biosynthesis, which may provide more information for better understanding the RA mechanism.

Die Republik Moldova, ein Anrainerstaat der EU, gilt als ärmstes Land Europas. Trotz vieler gesellschaftlicher Umbrüche und wirtschaftlicher Errungenschaften steht vor allem der Gesundheitssektor immer noch vor großen strukturellen Problemen. Um die Entwicklung im Bereich Rheumatologie zu unterstützen wurde 2011 das Projekt „Curriculare Modernisierung und bessere Versorgung von Rheumapatienten in der Republik Moldau“ ins Leben gerufen. Ein zentraler Punkt ist dabei die Implementierung einer einheitlichen Kerndokumentation für rheumatische Erkrankungen, wie sie als Vorbild schon seit vielen Jahren in Deutschland besteht. Bisher war noch wenig über die genaue Situation der Rheumaerkrankten in Moldova bekannt. Die Auswertung der Datenerhebungen aus dem Jahr 2012 und 2013 soll daher erste Erkenntnisse hinsichtlich der Versorgungssituation der Patienten liefern. Es wurden die Daten von insgesamt 842 Patienten erhoben. Dabei wurden sowohl von den Patienten selbst, als auch von den betreuenden Ärzten vor Ort Fragebögen ausgefüllt. Die Formulare lehnen sich an die deutschen Exemplare der Kerndokumentation an. Neben der körperlichen Untersuchung und Anamneseerhebung wurden auch laborchemische Parameter bestimmt. Es nahmen zwei großen Kliniken sowie neun niedergelassene Rheumatologen am Programm teil. Ausgewertet wurden neben Krankheitshäufigkeiten und demografischen Daten aller Erfassten, vor allem die Krankheitsaktivität, Therapielatenz, medikamentöse Therapie und Lebensqualität der an folgenden Erkrankungen leidenden Patienten: Rheumatoider Arthritis Spondylitis ankylosans Arthritis psoriatica Gicht Systemische Sklerose Systemischer Lupus erythematodes In der Auswertung fallen im Gegensatz zu Deutschland vor allem sehr hohe Krankheitsaktivitäten bei allen betrachten Gruppen auf. Das Therapieziel der Remission oder niedrigen Krankheitsaktivität wird kaum erreicht. Dabei werden die Patienten oftmals nicht leitliniengerecht behandelt. Eine wesentliche Ursache ist dabei die fehlende Möglichkeit der Therapieeskalation mittels Biologika, welche in den Jahren 2012 und 2013 in Moldova gar nicht verfügbar waren. Ein weiteres Problem ist der eingeschränkte Zugang zu Rheumatologen. Zum einen mangelt es im Land an Fachärzten, zum anderen bestehen infrastrukturelle Schwierigkeiten, die eine Vorstellung beim Arzt erschweren. Damit verzögert sich die Therapie oder sie kann nicht rechtzeitig angepasst werden. Die hohen Krankheitsaktivitäten, wie auch die meist lange Therapielatenz führen zu deutlichen Einschränkungen der Lebensqualität. Ursächlich sind dabei vor allem chronische Schmerzen und funktionelle Einschränkungen durch Gelenkdestruktionen. Für ältere Patienten führt dies vor allem zu einem Versorgungsproblem, unter jüngeren Betroffenen sinkt die Arbeitskraft und die Teilhabe am Berufsleben. Mithilfe der Untersuchung konnten erhebliche Probleme und Defizite bei der Versorgung der Erkrankten identifiziert werden. Auf Grund der erhobenen Daten können zukünftig Lösungsstrategien zur Verbesserung der Versorgungssituation erarbeitet werden.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(人間健康科学) / 甲第23127号 / 人健博第89号 / 新制||人健||6(附属図書館) / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 杉本 直三, 教授 黒木 裕士, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

Rheumatoid arthritis (RA) is a chronic, inflammatory autoimmune disease that targets joints, resulting in in permanent disability. Synovial macrophages have been implicated in the pathogenesis of RA; however, their exact origins and functions remains unclear. In this study, we show evidence that synovial macrophages are mostly derived from embryonic origin during normal development. Macrophages are derived from either hematopoietic stem cells (HSC) or erythro-myeloid progenitors (EMP), and it is postulated that different subpopulations of synovial macrophages may have distinct functions contributing to either homeostasis or inflammation. To investigate the phenotypes of synovial macrophage populations and characterize their lineage-specific functions in arthritic joints, we utilized both cell lineage-tracing and K/BxN serum-transfer arthritis mouse models. Utilizing Flt3Cre;Rosa26LSL-YFP mice to label HSC-derived cells, we demonstrated that there is minimal HSC contribution to synovial macrophage populations during homeostasis. Use of RankCre;Rosa26LSL-YFP and Cx3cr1CreERT2;Rosa26LSL-tdTomato mice to label EMP-derived cells corroborated the finding that the EMP compartment maintains the largest contribution to synovial macrophage populations during normal development. Analysis of macrophages in Csf1rMericreMer;Rosa26-LSLtdTomato mice provided definitive prove that synovial macrophages derived from yolk-sac EMP precursors in adult mice. Use of serum transfer arthritis (STA) mice demonstrated that while most macrophages in the inflamed synovium were EMP-derived, there was a marked increase in HSC-derived cells compared to those present in homeostasis. Although this study has contributed to eluding that the heterogeneity of synovial macrophages in both homeostasis and inflammatory arthritis (IA) is complex and lineage-specific, further studies are needed to clearly define lineage-specific functions of macrophages in synovial tissues and in IA.

The aim of the Diploma Thesis is to propose the methodology of a 3D model creation of a femoral part of a knee joint replacement. As the knee joint is the most loaded joint of the human body, the function, description and biomechanics relation is described in first part of the Thesis. The second part is focused on degenerative damage of knee joints that leads to the implantation of standard knee joint replacements. The third part deals with the construction of standard knee joint replacements and their surgery implantation. The last part of the Thesis describes the creation of an individual knee joint replacement, especially the femoral part.

La polyarthrite rhumatoïde (PR) est le rhumatisme chronique le plus fréquent caractérisé par une augmentation du risque de mortalité cardiovasculaire (CV). Ce sur-risque est la conséquence d’une athérogenèse accélérée, mais aussi d’atteintes microvasculaires, secondaires à une dysfonction endothéliale (DE). La réversibilité de la DE en fait une cible thérapeutique pertinente pour réduire la surmortalité en cas de PR. Ces dernières années, la physiopathologie de la DE au niveau des artères de conductance a été comprise grâce aux modèles animaux d’arthrite, principalement par le modèle d’arthrite induite à l’adjuvant (AIA). Toutefois, les effets du méthotrexate (MTX), traitement de première intention de la PR, sur la DE sont toujours inconnus. De même, les études sur la DE des vaisseaux coronariens et cérébraux font défaut.L’objectif de ce travail de thèse a été d’étudier les effets du MTX sur la DE aortique et de comprendre les mécanismes impliqués, mais aussi d’explorer et caractériser la DE au niveau des artères cérébrales moyennes et des artères coronaires dans le modèle AIA chez le rat.Dans une première étude, nos résultats ont montré qu’un traitement de 21 jours par MTX (1 mg/kg/semaine) n’améliore pas la DE aortique en dépit d’effets positifs surla sévérité clinique et l’inflammation systémique.Dans une seconde étude, nos données révèlent que le modèle AIA est associé à une DE au niveau des artères cérébrales moyennes, avec un rôle déterminant de la suractivité de l’arginase, comme en témoigne la capacité d’un traitement par nor-NOHA (40 mg/kg/jour), un inhibiteur d’arginase, à complétement inverser la DE cérébrovasculaire.Dans une troisième étude, nous avons démontré que le modèle AIA est caractérisé par une DE coronarienne positivement corrélée avec les taux plasmatiques d’endothéline-1 et d’angiotensine II, par une inflammation et une hypertrophie cardiaque, et une augmentation de la susceptibilité à l’ischémie myocardique.En conclusion, nos données montrent que les effets positifs du MTX sur le risque CV ne sont pas dus à une amélioration de la DE aortique, soulignant la nécessité de trouver des traitements ciblant spécifiquement le système CV en tant que traitements adjuvants des médicaments antirhumatismaux actuels. De plus, nos données montrent que le modèle AIA est un bon modèle pour mimer les atteintes cardiaques et cérébrovasculaires et pour étudier l’impact des nouveaux traitements visant à réduire le risque CV dans la PR. / Rheumatoid arthritis (RA) is the more frequent chronic rheumatism characterized by an increased risk of cardiovascular (CV) mortality. This over-risk is the consequence of accelerated atherosclerosis, but also of microvascular damages, secondary to an endothelial dysfunction (ED). The reversibility of the ED makes of it a relevant therapeutic target to reducing excessive mortality in RA. In recent years, pathophysiology of ED in conductance arteries has been understood thank to animal models of arthritis, mainly the adjuvant-induced arthritis (AIA) model in rats. However, the effects of methotrexate (MTX), first-line treatment in RA, on ED are still unknown. Likewise, studies on ED of the coronary and cerebral vasculature are lacking.The aim of this thesis work was to study the effect of MTX on aortic ED and to dissect the mechanisms involved, but also to explore and characterize ED on MCA and coronary arteries in the widely-used model of adjuvant-induced arthritis (AIA) in rats.In a first study, our results showed that a 21-days treatment with MTX (1 mg/kg/week) did not improveaortic ED despite positive effects on clinical severity and systemic inflammation.In a second study, our data reported that the AIA model was associated with ED in middle cerebral arteries, involving a seminal role of arginase upregulation, as attested by the capacity of a treatment with nor-NOHA (40 mg/kg/day), an arginase inhibitor, to fully reverse cerebrovascular ED.In a third study, we demonstrated that the AIA model was characterized by a coronary ED positively correlated to plasma levels of endothelin-1 and angiotensin II, by a cardiac inflammation and hypertrophy, and an increased susceptibility to myocardial ischemia.In conclusion, our data indicate that positive effects on CV risk of MTX do not involve improving aortic ED, underlying the necessity to find accurate therapies targeting specifically the CV system as adjuvant treatments to the current anti-rheumatic drugs. Moreover, our data showed that the AIA model is relevant for mimicking cardiac and cerebrovascular impairments, and to study the impact of new treatments for reducing CV risk in RA.

Rheumatoid arthritis (RA) is a chronic inflammatory condition of unknown etiology for which there is no cure. It is one of the most disabling diseases and affects about 1% of the world‟s population. Recent developments in the field of molecular biology have resulted in the production of new drugs used in the treatment RA. Despite these advancements, achieving optimal disease control and prevention of disease progression is still difficult in many patients, leading to a continued search for treatment methods that will improve patient outcomes. Non-biologic forms of treatment that are still being investigated include the use of statins as an adjunct therapy due to their reported antiinflammatory effects. Some studies have shown that the use of statins in patients with RA help in reducing disease activity and swollen joint count in addition to lowering cardiovascular risk. As evidence continue to increase on the anti-inflammatory effect of statins, researchers have started investigating possible benefits that may result from the use of statins in treatment of RA, a chronic disease marked by high levels of systemic and local inflammation. This study investigated the anti-inflammatory effect of statins in rheumatoid arthritis patients with associated risks for cardiovascular disease, using simvastatin as the investigational product. Patients with moderately active RA despite being on maximum disease-modifying antirheumatic drugs (DMARDs) therapy and having associated risks for cardiovascular disease were screened for the study. Eligible patients were randomized into two groups, 1 and 2. Patients in group 1 received simvastatin treatment (20mg/day) for a period of 3 months in addition to their usual DMARDs after which they stopped simvastatin treatment and were followed up for a further 3 months off simvastatin treatment. Those in group 2 were allowed to continue on their usual DMARDs without simvastatin treatment for the first 3 months of the study after which they received 20mg/day simvastatin for a period of 3 months in addition to their usual DMARDs. The anti-inflammatory effect of simvastatin was assessed by monitoring the inflammatory variables; erythrocyte sedimentation rate (ESR) and c-reactive protein (CRP) and disease activity in the two groups at screening, at the crossover point and at end of the study. Disease activity was significantly reduced with simvastatin treatment in the two groups. The mean change in disease activity score with simvastatin treatment was 1.30 (p = 0.01) in group 1 and 1.74 (p = 0.01) in group 2. ESR was significantly reduced with simvastatin treatment in group 1 with a mean change of 19.0 (p = 0.005) and marginally reduced in group 2 with a mean change 26.0 (p = 0.09). There was no significant change in CRP with simvastatin treatment in the two groups. The mean change in CRP with simvastatin treatment was 7.0 (p = 0.24) in group 1 and 14.7 (p =0.20) in group 2. All the patients benefited from the lipid-lowering effect of simvastatin. These findings suggest that statins may have mild anti-inflammatory properties and will be good adjuvant in RA patients with associated risks for cardiovascular disease. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Pharmacology / unrestricted

Rheumatic diseases are common, usually chronic, painful and to some extent invalidating medical conditions. Understanding of the disease pathogenesis is still very fragmentary. Hyperreactivity of the immune system and defect of autotolerance are probably contributed by local factors, which helps to explain, why some joints/muscles are more affected than others. All this results from a complex net of interactions between immune cells, synovial fibroblasts, chondrocytes, osteocytes, myocytes and other cells. In the submitted PhD thesis I have focused on three groups of molecules: regulatory RNAs, S100 proteins and autoantibodies. In the theoretical part, I sum up the current knowledge on their biogenesis, function and the role in rheumatology. In the investigative part, I present six original publications and one review on the role of those molecules in development of rheumatoid arthritis (RA) and idiopathic inflammatory myositis (IIM). One of the main studies was focused on expression of PIWI-interacting RNAs (piRNAs) in RA synovial fibroblasts (SF). piRNAs are small regulatory RNAs which in complex with PIWIL proteins regulate gene expression and silence transpozoms. piRNA expression was considered to be limited to germline and cancer cells. We have found 267 PIWI-interacting RNAs to be expressed...