Background: Rheumatoid arthritis (RA) is characterised by excessive production of tumour necrosis factor alpha (TNFα). This leads to rheumatoid cachexia, a condition characterised by increased resting energy expenditure (REE) and loss of fat-free mass (FFM) leading to functional disability, decreased strength and balance. The aims of this research work was to: a) to develop a new REE equation in order to continuously monitor abnormal changes in REE in the RA population, b) to investigate if smoking further enhances hypermetabolism and c) to examine if the new anti-TNFα medication reverses this metabolic abnormality. Methods: 68 patients with RA were assessed for demographic and anthropometrical characteristics, REE (indirect calorimetry), body composition (bioelectrical impedance), and disease activity [C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), disease activity score 28 (DAS28) and health assessment questionnaire (HAQ)]. 20 of the total 68 patients, about to start anti-TNFα therapy, underwent the exact same aforementioned procedures but on three separate occasions (Baseline: two weeks prior to anti-TNFα treatment, Time-1 and Time-2: two weeks and three months, respectively, after the drug had been introduced. Results: Study 1: Based on FFM and CRP, a new equation was developed which had a prediction power of R2=0.76. The new equation revealed an almost identical mean with measured REE (1645.2±315.2 and 1645.5±363.1 kcal/day, p>0.05), and a correlation coefficient of r=0.87 (p=0.001). Study 2: Smokers with RA demonstrated significantly higher REE (1513.9±263.3 vs. 1718.1±209.2 kcal/day; p=0.000) and worse HAQ (1.0±0.8 vs. 1.7±0.8; p=0.01) compared to age and FFM matched RA non-smokers. The REE difference was significantly predicted by the interaction smoking/gender (p=0.04). Study 3: Significant increases were observed in REE (p=0.002), physical activity (p=0.001) and protein intake (p=0.001) between the three times of assessment. Moreover, disease activity significantly reduced [ESR (p=0.002), DAS28 (p=0.000), HAQ (p=0.000) and TNFα (p=0.024)] while FFM and total body fat did not change (both at p>0.05). Physical activity and protein intake were found to be significant within-subject factors for the observed REE elevation after 12-weeks on anti-TNFα treatment (p=0.001 and p=0.024, respectively). Conclusions: Findings from the first study revealed that the newly developed REE equation provides an accurate prediction of REE in RA patients. Moreover, the results from the second study showed that cigarette smoking further increases REE in patients with RA and has a negative impact on patients’ self-reported functional status. Finally, our data from the third study suggest that REE remains elevated not because of the maintenance of the RA-related hypermetabolism but due to the concomitant significant increases in physical activity and protein intake.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

No description available.

Base teórica: Artrite reumatoide (AR) é uma doença autoimune que afeta as articulações e progride de maneira simétrica e erosiva. Além dos achados articulares, pode ocorrer de perda muscular e síndrome da caquexia. Atualmente, não existe um marcador que sirva de preditor da síndrome de caquexia reumatoide. Estudos metabolômicos em pacientes com AR demonstram uma complexidade em encontrar um biomarcador para caquexia. Ademais, não há modelo experimental de caquexia descrito na literatura, mas o modelo de artrite induzida por colágeno (CIA) possui potencial de ser modelo de caquexia reumatoide. A partir deste modelo, pode-se fazer a busca por biomarcadores de caquexia reumatoide via metabolômica. Objetivo: Avaliar o modelo de CIA como modelo experimental de caquexia reumatoide. Avaliar o perfil metabólico da urina no modelo de CIA e correlacionar com parâmetros clínicos de caquexia reumatoide em busca de possíveis biomarcadores. Métodos: Camundongos machos DBA/1J foram induzidos (CIA; n=13) no dia zero e receberam reforço 18 dias após, e grupo mantidos saudáveis sem indução (CO; n=11). Nos dias 0, 18, 25, 35, 45, 55 e 65 após a indução, foram realizados: coleta de urinas; teste de desempenho físico; teste de locomoção espontânea; teste de força; medida do volume do edema da pata traseira; avaliação do escore clínico; pesagem; e avaliação da ingestão alimentar. Após os 65 dias, os animais foram eutanasiados e tecidos musculares (gastrocnêmio – GA; e tibial anterior – TA) foram dissecados para pesagem e realização da razão sarcoplasmática. Os dados foram analisados por ANOVA de duas vias, seguido de Bonferroni, ou teste t de Pearson, com significância a partir de um p<0,05. A urina coletada foi submetida à ressonância nuclear magnética (1D e 2D J-res). Os metabolitos foram identificados via Chenomx (1D) e pelo Birmingham Metabolite Library (BML; 2D J-res). Utilizou-se a o modelo estatístico de PCA, PLSDA e PLSR para criar ranqueamento de metabolitos (significância a partir de um p<0,05). Analizou-se as rotas metabólicas via Metaboanalyst a partir do ranqueamento de metabólitos obtidos. Os metabólitos obtidos foram filtrados para rotas metabólicas que ocorrem no músculo para identificação de potenciais biomarcadores de perda muscular. Resultados: O grupo CIA apresentou redução de até 24% na locomoção espontânea, de até 66% na força e de até 24% no teste de desempenho físico após 35 dias da indução, bem como redução no peso do GA (24%) e TA (25%), e relação sarcoplasmática (22 e 23%, respectivamente) em relação ao grupo CO. Os modelos estatísticos de PCA, PLSDA e PLSR, e o filtro pelas rotas metabólicas relacionadas com o músculo geraram uma lista de 28 metabólitos e relacionados com o desenvolvimento da doença, sendo eles: 3-metilhistidina, 4-aminobutirato, acetilcolina, arginina, aspartato, carnosina, creatina, creatinina, glutamina, histamina, histidina, isoleucina, leucina, metionina, lisina, mio-inositol, dimetilglicina, acetilalanina, acetilmetionina, pantotenato, fenilalanina, fosfocolina, fosfocreatina, piridoxina, sarcosina, succinilacetona, tiamina, e urocanato. Conclusão: Em concordância com os resultados de redução nos parâmetros de: massa muscular, locomoção espontânea, força e desempenho físico, somando-se a ausência de anorexia bem como mudança no peso, o modelo animal de CIA representa um modelo experimental próprio para caquexia reumatoide. A análise do perfil metabólico deste modelo permite sugerir 28 metabólitos relacionados ao processo de perda muscular, que podem vir a ser biomarcadores de caquexia reumatoide, objetivando prognóstico, diagnóstico e acompanhamento da síndrome. Destes metabólitos, os principais são pertencentes ao metabolismo de: histidina; arginina e prolina; glicina, serina e treonina; fosfocreatina, bem como outros aminoácidos e vitaminas do complexo B. / Background: Rheumatoid Arthritis (RA) is an autoimmune disease that affects the joints and has a symmetric development and it is erosive. Besides joint damage, it can develop muscle loss progress into cachexia syndrome. Currently, there is no marker that can predict it development in rheumatoid patients. Metabolomics in RA have shown to be complex to find out a biomarker for this syndrome. Also, there is no experimental model of cachexia described in literature yet; however the collageninduced arthritis (CIA) animal model seems to be a feasible model for rheumatoid cachexia. With this model, the research for a biomarker of rheumatoid cachexia can be done by metabolomics. Objectives: It will be evaluated if the CIA animal model can be also an animal model of rheumatoid cachexia. Afterwards, it will be evaluated a metabolic profile from urine of this animal model and correlate with clinical signs of rheumatoid cachexia to find out plausible biomarkers of it. Methods: Male DBA/1J mice were submitted to CIA (n=13), immunization occurred at day zero and a booster was performed 18 days after, and a healthy group with no induction (CO; n=11). At the 0,18, 25, 35, 45, 55 and 65 days after the first injection, it was done: urine collection; physical performance test; free exploratory locomotion test; strength test; hindpaw edema volume measurement; follow up disease development; weighted; and food intake. After the 65 days, animals were euthanized and muscle (gastrocnemious – GA; and tibial anterior – TA) were dissected, and weighted for sarcoplasmic ratio. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc, and t-test of Pearson, and statistical critical limit was set for p<0.05. The collected urine was used for nuclear magnetic resonance (1D and 2D J-res). Metabolites were identified by Chenomx (1D) and by the Birmingham Metabolite Library (BML; 2D J-res). Statistical model were performed using PCA, PLSDA and PLSR to create a ranking list of the metabolites (statistical critical limit was set for p<0.05). It was analyzed the metabolic pathway by Metaboanalyst from the data of metabolite ranking list. Then, the metabolite list was filtered by the metabolic pathways that take place in muscle tissue, in order to identify plausible biomarkers of muscle loss. Results: CIA group has shown reduction in up to 24% of free locomotion fatigue, up to 66% of strength and up to 24% of endurance physical performance after 35 days of the induction, as well as a decrease in GA (24%) and TA (25%) weight, and sarcoplasmic ratio also reduced (22 and 23%, respectivamente) related to CO group. The PCA, PLSDA and PLSR statistical models, and the filter by metabolic pathways related to muscle provided a list of 28 metabolites related to disease development, as can be listed: 3-methylhistidine, 4-aminobutyrate, acetylcholine, arginine, aspartate, carnosine, creatine, creatinine, glutamine, histamine, histidine, isoleucine, leucine, methionine, lysine, myo-inositol, dimethylglycine, acetylalanine, acetylmethionine, pantothenate, phenylalanine, phosphocholine, phosphocreatine, pyridoxine, sarcosine, succinylacetone, thiamine, and urocanate. Conclusions: Accordingly with the data with reduction of: muscle mass, spontaneous locomotion, strength and physical performance, added with absence of anorexia as well as weight change, CIA animal model is a feasible experimental model for rheumatoid cachexia. Concerning the metabolic profile from this model, it can be suggested 28 metabolites related to muscle loss in which can be tested for biomarker of rheumatoid cachexia, targeting prognosis, diagnosis, and syndrome follow up. From those metabolites, the main ones are engaged to metabolism of: histidine; arginine and proline; glycine, serine and threionine; phosphorcreatine, as well as other amino acids and vitamins from B complex.

Base teórica: Artrite reumatoide (AR) é uma doença autoimune que afeta as articulações e progride de maneira simétrica e erosiva. Além dos achados articulares, pode ocorrer de perda muscular e síndrome da caquexia. Atualmente, não existe um marcador que sirva de preditor da síndrome de caquexia reumatoide. Estudos metabolômicos em pacientes com AR demonstram uma complexidade em encontrar um biomarcador para caquexia. Ademais, não há modelo experimental de caquexia descrito na literatura, mas o modelo de artrite induzida por colágeno (CIA) possui potencial de ser modelo de caquexia reumatoide. A partir deste modelo, pode-se fazer a busca por biomarcadores de caquexia reumatoide via metabolômica. Objetivo: Avaliar o modelo de CIA como modelo experimental de caquexia reumatoide. Avaliar o perfil metabólico da urina no modelo de CIA e correlacionar com parâmetros clínicos de caquexia reumatoide em busca de possíveis biomarcadores. Métodos: Camundongos machos DBA/1J foram induzidos (CIA; n=13) no dia zero e receberam reforço 18 dias após, e grupo mantidos saudáveis sem indução (CO; n=11). Nos dias 0, 18, 25, 35, 45, 55 e 65 após a indução, foram realizados: coleta de urinas; teste de desempenho físico; teste de locomoção espontânea; teste de força; medida do volume do edema da pata traseira; avaliação do escore clínico; pesagem; e avaliação da ingestão alimentar. Após os 65 dias, os animais foram eutanasiados e tecidos musculares (gastrocnêmio – GA; e tibial anterior – TA) foram dissecados para pesagem e realização da razão sarcoplasmática. Os dados foram analisados por ANOVA de duas vias, seguido de Bonferroni, ou teste t de Pearson, com significância a partir de um p<0,05. A urina coletada foi submetida à ressonância nuclear magnética (1D e 2D J-res). Os metabolitos foram identificados via Chenomx (1D) e pelo Birmingham Metabolite Library (BML; 2D J-res). Utilizou-se a o modelo estatístico de PCA, PLSDA e PLSR para criar ranqueamento de metabolitos (significância a partir de um p<0,05). Analizou-se as rotas metabólicas via Metaboanalyst a partir do ranqueamento de metabólitos obtidos. Os metabólitos obtidos foram filtrados para rotas metabólicas que ocorrem no músculo para identificação de potenciais biomarcadores de perda muscular. Resultados: O grupo CIA apresentou redução de até 24% na locomoção espontânea, de até 66% na força e de até 24% no teste de desempenho físico após 35 dias da indução, bem como redução no peso do GA (24%) e TA (25%), e relação sarcoplasmática (22 e 23%, respectivamente) em relação ao grupo CO. Os modelos estatísticos de PCA, PLSDA e PLSR, e o filtro pelas rotas metabólicas relacionadas com o músculo geraram uma lista de 28 metabólitos e relacionados com o desenvolvimento da doença, sendo eles: 3-metilhistidina, 4-aminobutirato, acetilcolina, arginina, aspartato, carnosina, creatina, creatinina, glutamina, histamina, histidina, isoleucina, leucina, metionina, lisina, mio-inositol, dimetilglicina, acetilalanina, acetilmetionina, pantotenato, fenilalanina, fosfocolina, fosfocreatina, piridoxina, sarcosina, succinilacetona, tiamina, e urocanato. Conclusão: Em concordância com os resultados de redução nos parâmetros de: massa muscular, locomoção espontânea, força e desempenho físico, somando-se a ausência de anorexia bem como mudança no peso, o modelo animal de CIA representa um modelo experimental próprio para caquexia reumatoide. A análise do perfil metabólico deste modelo permite sugerir 28 metabólitos relacionados ao processo de perda muscular, que podem vir a ser biomarcadores de caquexia reumatoide, objetivando prognóstico, diagnóstico e acompanhamento da síndrome. Destes metabólitos, os principais são pertencentes ao metabolismo de: histidina; arginina e prolina; glicina, serina e treonina; fosfocreatina, bem como outros aminoácidos e vitaminas do complexo B. / Background: Rheumatoid Arthritis (RA) is an autoimmune disease that affects the joints and has a symmetric development and it is erosive. Besides joint damage, it can develop muscle loss progress into cachexia syndrome. Currently, there is no marker that can predict it development in rheumatoid patients. Metabolomics in RA have shown to be complex to find out a biomarker for this syndrome. Also, there is no experimental model of cachexia described in literature yet; however the collageninduced arthritis (CIA) animal model seems to be a feasible model for rheumatoid cachexia. With this model, the research for a biomarker of rheumatoid cachexia can be done by metabolomics. Objectives: It will be evaluated if the CIA animal model can be also an animal model of rheumatoid cachexia. Afterwards, it will be evaluated a metabolic profile from urine of this animal model and correlate with clinical signs of rheumatoid cachexia to find out plausible biomarkers of it. Methods: Male DBA/1J mice were submitted to CIA (n=13), immunization occurred at day zero and a booster was performed 18 days after, and a healthy group with no induction (CO; n=11). At the 0,18, 25, 35, 45, 55 and 65 days after the first injection, it was done: urine collection; physical performance test; free exploratory locomotion test; strength test; hindpaw edema volume measurement; follow up disease development; weighted; and food intake. After the 65 days, animals were euthanized and muscle (gastrocnemious – GA; and tibial anterior – TA) were dissected, and weighted for sarcoplasmic ratio. Data were analyzed by two-way ANOVA followed by Bonferroni post hoc, and t-test of Pearson, and statistical critical limit was set for p<0.05. The collected urine was used for nuclear magnetic resonance (1D and 2D J-res). Metabolites were identified by Chenomx (1D) and by the Birmingham Metabolite Library (BML; 2D J-res). Statistical model were performed using PCA, PLSDA and PLSR to create a ranking list of the metabolites (statistical critical limit was set for p<0.05). It was analyzed the metabolic pathway by Metaboanalyst from the data of metabolite ranking list. Then, the metabolite list was filtered by the metabolic pathways that take place in muscle tissue, in order to identify plausible biomarkers of muscle loss. Results: CIA group has shown reduction in up to 24% of free locomotion fatigue, up to 66% of strength and up to 24% of endurance physical performance after 35 days of the induction, as well as a decrease in GA (24%) and TA (25%) weight, and sarcoplasmic ratio also reduced (22 and 23%, respectivamente) related to CO group. The PCA, PLSDA and PLSR statistical models, and the filter by metabolic pathways related to muscle provided a list of 28 metabolites related to disease development, as can be listed: 3-methylhistidine, 4-aminobutyrate, acetylcholine, arginine, aspartate, carnosine, creatine, creatinine, glutamine, histamine, histidine, isoleucine, leucine, methionine, lysine, myo-inositol, dimethylglycine, acetylalanine, acetylmethionine, pantothenate, phenylalanine, phosphocholine, phosphocreatine, pyridoxine, sarcosine, succinylacetone, thiamine, and urocanate. Conclusions: Accordingly with the data with reduction of: muscle mass, spontaneous locomotion, strength and physical performance, added with absence of anorexia as well as weight change, CIA animal model is a feasible experimental model for rheumatoid cachexia. Concerning the metabolic profile from this model, it can be suggested 28 metabolites related to muscle loss in which can be tested for biomarker of rheumatoid cachexia, targeting prognosis, diagnosis, and syndrome follow up. From those metabolites, the main ones are engaged to metabolism of: histidine; arginine and proline; glycine, serine and threionine; phosphorcreatine, as well as other amino acids and vitamins from B complex.

Orientador: Manoel Barros Bertolo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T03:46:59Z (GMT). No. of bitstreams: 1 Pina_FabianaPompeode_M.pdf: 947949 bytes, checksum: 7e9310579d25142d2d37758ff60f7796 (MD5) Previous issue date: 2009 / Resumo: A associação de antígenos de histocompatibilidade com a Artrite Reumatóide (AR) vem sendo demonstrada em inúmeros estudos. No entanto, a avaliação em populações afro-descendentes ainda foi pouco estudada. Os propósitos deste estudo foram os de determinar a freqüência dos alelos HLA-DRB1 e as contribuições do polimorfismo desses alelos na susceptibilidade da AR na população afrobrasileira. Este estudo avaliou também, se a teoria do epitopo semelhante (SE) e o modelo de proteção da Artrite Reumatóide (RAP Model) se aplicam aos pacientes afro-brasileiros com AR. Os alelos HLA-DRB1 de 72 pacientes afro-brasileiros com AR, diagnosticados pelos critérios do American College of Rheumatology (ACR), e de 75 indivíduos saudáveis foram tipados e subtipados utilizando-se a técnica de reação em cadeia de polimerase de DNA amplificado hibridizado, com seqüência de primers específicos de alta e baixa resolução, e depois comparados. Os alelos HLADRB1 *0404 e *0405 apresentaram freqüência maior nos pacientes do que no grupo controle. Já, alelo HLA-DRB1*0102 apresentou uma freqüência aumentada no grupo controle (9,3%), quando comparada com a freqüência nos pacientes. Os alelos DRB1 considerados como pertencentes ao grupo do epitopo semelhante estavam presentes em 39 pacientes (54,2%) e em 20 controles (26,7%), indicando que teoria do epitopo semelhante se aplica à população afro-brasileira com Artrite Reumatóide. Os alelos HLA-DRB1 que apresentavam a sequência DERAA (alelos protetores) reduziram, de forma independente, o risco de desenvolver AR. Os dados obtidos apontam para uma intensa miscigenação racial presente no Brasil. Assim, como nos faz concluir que a susceptibilidade da Artrite Reumatóide em afro-brasileiros é, provavelmente, mediada pela interação de fatores genéticos e étnicos / Abstract: The association of histocompatibility antigens with Rheumatoid Arthritis (RA) comes being demonstrated in innumerable studies. However, the evaluation in afrodescendents populations still little was studied. The aim of this study has been to determine the frequency of HLA-DRB1 alleles, and the contributions of the polymorphism of these alleles in the susceptibility of RA in the Afro-Brazilian population as well. This study, also evaluated, if the theory of the shared epitope (SE) and the model of protection of the Rheumatoid Arthritis (RAP Model) can also be applied to the Afro-Brazilian patients suffering RA. The HLA-DRB1 alleles in 72 Afro-Brazilian patients suffering RA, diagnosed in accordance to the criteria of the American College of Rheumatology (ACR), and of 75 healthful volunteers had been typed and sub-typed using the technique of the polymerase chain reaction of the amplified hybridized DNA, with specific sequence of primers of high and low resolution, were then compared. The HLA-DRB1 *0404 and *0405 alleles had presented higher frequency in the patients group than in the control group. The HLADRB1 *0102 alleles presented a frequency increased (9,3%) in the control group, when compared with the patients group. The DRB1 alleles considered as pertaining to the group of shared epitope were present in 39 patients (54.2%) and in 20 members of the control group(26.7%), indicating that the theory of the shared epitope it is also applied to the Afro-Brazilian population with Rheumatoid Arthritis. The HLADRB1 alleles that presented DERAA sequence (protectors alleles), had reduced, of independent form, the risk to develop RA. The gotten data point to an intense racial miscegenation in Brazil. Thus, as in it makes them to conclude that the susceptibility of the Rheumatic Arthritis in Afro-Brazilian is, probably, determined by the interaction of genetic and ethnic factors / Mestrado / Clinica Medica / Mestre em Clinica Medica

Les patients atteints de polyarthrite rhumatoide (PR) ont une diminution de l'espérance de vie de 10 à 15 ans. Cette augmentation de la mortalité semble liée à un processus athéromateux accéléré. La dysfonction endothéliale (DE) joue un rôle clé dans ce processus. L'arginase est une enzyme qui régule le niveau de NO par compétition avec la NO Synthase (NOS) pour leur substrat commun, la L-arginine.Nous avons montré que la vasodilatation Acétylcholine (ACh) dépendante était altérée dans le modèle d'Arthrite Induite à Adjuvant (AIA) chez le rat Lewis, témoignant d'une DE. L'incubation des anneaux aortiques avec la nor-NOHA un inhibiteur sélectif d'arginase a amélioré la réponse vasculaire à l'ACh chez les rats AIA. L'activité et l'expression vasculaire de l'arginase se sont révélées augmentées chez les rats AIA et corrélées positivement à la sévérité de l'arthrite.Nous avons caractérisé les mécanismes impliqués dans la DE du rat AIA. Nos résultats ont montré que la DE mettait enjeu une diminution de l'activité de la NO synthase, un déficit en EDHF, une suractivité de la COX-2, ainsi qu'une production excessive des anions superoxydes. Le traitement curatif des rats AIA par la nor-NOHA pendant 3 semaines, a permis de restaurer la fonction endothéliale. L'inhibition de l'arginase n'a pas modifié l'atteinte articulaire des rats AIA.Nos travaux ont permis de mieux comprendre la physiopathologie de la DE associée à la PR et ont déterminé pour la première fois le rôle délétère de l'arginase dans cette anomalie. Ils ouvrent des perspectives quant à l'utilisation des inhibiteurs d'arginase comme futurs traitements pharmacologiques de l'atteinte vasculaire du patient PR / Patients with RA are characterized by a decrease in life expectancy of 10 to 15 years. This increase in mortality seems to be related to an accelerated atheroma process. Endothelial dysfunction (ED) has a key role in these processes. The arginase is an enzyme which regulates the level of NO by competing with the NO synthase (NOS) to their common substrate, L-arginine.We showed that acetylcholine (ACh) dependent vasodilation was altered in the model of Adjuvant Induced Arthritis (AIA) in Lewis rats, indicating a endothelial dysfunction. The incubation of aortic rings with nor-NOHA has improved the vascular response to ACh in AIA rats. The activity and expression of vascular arginase are increased in AIA rats and positively correlated with the severity of arthritis.We characterized the mechanisms involved in DE in AIA rats. Our results showed that ED involved a decrease of activity of NO synthase, a decrease of EDHF, overactiviry of COX-2, thromboxane synthase and prostacyclin synthase, as well as excessive superoxide anions. The cure of AIA rats by a selective inhibitor of arginase, nor-NOHA for 3 weeks, has restored endothelial function. In contrast, inhibition of arginase activity did not change the weight, the diameter of ankles, radiological or histological articular damage in AIA rats.Our work has led to a better understanding of pathophysiology of ED associated with rheumatoid arthritis and determined for the first time the deleterious role of arginase in this vascular anomaly. These results open prospects for the use of arginase inhibitors as future pharmacological treatment of vascular patient PR.

Die Rheumatoide Arthritis (RA) ist eine der häufigsten Autoimmunerkrankungen des Menschen. Sie ist durch einen chronischen Verlauf mit Allgemeinsymptomen und erosiven Gelenkentzündungen gekennzeichnet. Die klinischen Beschwerden reichen von Morgensteifigkeit der Gelenke bis zu deren Funktionsverlust mit Deformationen. Extraartikuläre Manifestationen, vor allem das kardiovaskuläre System betreffend, erschweren den Krankheitsverlauf und verkürzen die Lebenserwartung. In der Pathogenese der RA steht die Synovitis, die Entzündung der Gelenkinnenhaut, im Mittelpunkt. Die Ansammlung von Lymphozyten und Monozyten in der Synovialmembran und sezernierte proinflammatorische Zytokine bewirken eine Aufrechterhaltung des Entzündungsgeschehens. Das hauptsächlich von Monozyten produzierte Zytokin Tumornekrosefaktor (TNF) spielt eine entscheidende Rolle in diesem Immunprozess. TNF stimuliert Fibroblasten zur Sezernierung destruktiver Enzyme und regt zur Produktion weiterer, proinflammatorischer Botenstoffe an. Klassischerweise wirkt TNF in seiner löslichen Form und bindet an TNF-Rezeptoren auf nahezu allen Körperzellen. Ein weiterer Wirkmechanismus ist die retrograde Signaltransduktion über membranständiges TNF (tmTNF). Die Funktion von tmTNF, als Rezeptor Signale in die tmTNF-tragende Zelle zu vermitteln, wird als Reverse Signaling bezeichnet. Es ist bekannt, dass Reverse Signaling via tmTNF in Monozyten von RA-Patienten Apoptose auslöst und in weiteren komplexen Immunprozessen involviert ist. Die Bedeutung von TNF für die Rheumatoide Arthritis wird nicht zuletzt dadurch unterstrichen, dass die anti-TNF-Therapie einen sehr wirksamen Therapieansatz darstellt. Die Behandlung führt bei ca. zwei Drittel der Patienten zu einer Reduktion der entzündlichen und schmerzhaften Gelenkschwellung und zu einem Sistieren der Gelenkdestruktion. Es ist jedoch bekannt, dass ein Teil der Patienten schlecht auf die Therapie anspricht. Das dadurch verzögerte Erreichen eines guten Therapieerfolgs führt zu einer Verlängerung des Leidens und zu unnötigen, zum Teil schwerwiegenden Nebenwirkungen. Gesundheitsökonomisch ist dies ebenfalls kritisch zu sehen, da mit hohen Behandlungskosten zu rechnen ist. Eine Vorhersage des Therapieansprechens ist jedoch aktuell nicht möglich. Ziel dieser Studie war es, einen prädiktiven Marker für das anti-TNF-Therapieansprechen bei Patienten mit RA zu finden. Dabei galt der Bedeutung des Reverse Signaling via tmTNF großes Interesse. In der Studie wurden 20 Patienten mit Rheumatoider Arthritis vor und während einer Therapie mit dem TNF-Antagonist Etanercept insgesamt 24 Wochen betreut. Die Erhebung klinischer Daten, wie die Anzahl der druckschmerzhaften und geschwollenen Gelenke, die Einschätzung der Krankheitsaktivität durch den Patienten auf einer visuellen Analogskala (VAS) und die Untersuchung der Entzündungsaktivität mit CRP und BSG, erfolgte einmalig vor und alle vier Wochen unter Therapie. Zur Einschätzung der Krankheitsaktivität wurde der Disease Activity Score (DAS) genutzt. Das Therapieansprechen wurde entsprechend einer Klassifikation und der Veränderung der einzelnen klinischen Parameter im Therapieverlauf gewertet. Auf der Suche nach einem prädiktiven Faktor wurden ebenfalls einmalig vor und alle vier Wochen während der Therapie mit Etanercept laborchemische Experimente durchgeführt. Dazu wurden aus dem Blut der RA-Patienten die Monozyten mit Hilfe einer Dichtegradientenzentrifugation und einer Magnetseparation isoliert. Diese wurden hinsichtlich ihrer Expression von tmTNF sowie TNFR1 und TNFR2 nach Inkubation mit entsprechenden Antikörpern durchflusszytometrisch untersucht. Weiterhin wurden die RA-Monozyten mit Etanercept bzw. einer Negativkontrolle inkubiert. Im Anschluss wurde die Apoptose der Monozyten mittels Durchflusszytometrie und Färbung mit Propidiumiodid bzw. Annexin V quantifiziert. Entsprechend den EULAR-Kriterien kam es bei 10 Patienten (53%) zu einem guten, bei 7 Patienten (37%) zu einem mittleren und bei 2 Patienten (10%) zu einem schlechten Ansprechen. Aufgrund der klinischer Beurteilung erfolgte eine Einteilung in zwei Gruppen, sodass unter den Patienten 10 Responder (53%) und 9 (47%) Non-Responder waren. Die Monozyten der RA-Patienten exprimierten tmTNF mit einer mittleren Fluoreszenzintensität (MFI) von 14,23 ± 3,04. Es wurde eine MFI für TNFR1 mit 31,84 ± 12,99 und für TNFR2 mit 30,02 ± 8,9 gemessen. Die Inkubation der RA-Monozyten mit Etanercept bzw. mit der Negativkontrolle ergab unterschiedliche Resultate innerhalb der Patientengruppe. Dabei wurde die Apoptose der Monozyten in Spontanapoptose (Negativkontrolle) und in Reverse Signaling induzierte Apoptose eingeteilt. Vor Beginn der anti-TNF-Therapie zeigte ein Teil der Patienten eine niedrige Spontanapoptose und eine erhöhte Reverse Signaling induzierte Apoptose. Die Monozyten der anderen RA-Patienten zeigten umgekehrt eine hohe Spontanapoptose und eine niedrige Reverse Signaling induzierte Apoptose. Im Folgenden wurde der Einfluss der Etanercept-Therapie auf die Apoptose der Monozyten untersucht. Es wurde ein Anstieg der niedrigen Spontanapoptose bzw. Reverse Signaling induzierte Apoptose und ein Abfall der hohen Spontanapoptose bzw. Reverse Signaling induzierte Apoptose gesehen. Bezieht man in die Analyse das Therapieansprechen mit ein, so ergibt sich für Responder eine initial hohe Spontanapoptose, die unter Therapie signifikant sinkt. Non-Responder hingegen haben vor Therapie eine niedrige Spontanapoptose, die unter einer Therapie mit Etanercept ansteigt. Für den Verlauf der Reverse Signaling induzierte Apoptose unter der anti-TNF-Therapie gab es keine signifikanten Unterschiede hinsichtlich des Therapieansprechens. Im Fisher-Exact-Test zeigte sich eine deutliche Tendenz (p=0,07), dass Patienten mit niedriger Spontanapoptose bzw. hoher Reverse Signaling induzierter Apoptose zu Studienbeginn schlecht auf die Therapie ansprechen. Dieses Ergebnis konnte mit Einzelparametern verifiziert werden. Die Studie kam zu dem Schluss, dass RA-Patienten, deren Monozyten eine niedrige Reverse Signaling induzierte Apoptose bzw. eine hohe Spontanapoptose aufwiesen, besser auf eine anti-TNF-Therapie mit Etanercept ansprachen. Dieses Ergebnis kann hilfreich für die Entwicklung einer entscheidenden Diagnostik vor Therapieeinstellung sein und leistet einen Beitrag für die Vorhersage des Therapieansprechens.

Es stellt sich die Frage, wie diese Zellen im entzündeten Gewebe überleben können und wie sie ihren Stoffwechsel an die dortigen Bedingungen anpassen. Diese Arbeit beschreibt am Beispiel der Juvenilen Idiopathischen Arthritis (JIA) die Analyse des Stoffwechsels von CD4+ T Lymphozyten, die chronische Entzündungen antreiben undim entzündeten Gewebe lange Zeit persistieren. Pathogene CD4+ CD45RO+ PD1+ CXCR5-T Zellen wurden hierfür aus Synovialflüssigkeit von Patienten mit JIA isoliert und gezeigt, dass auch bei diesen Zellen der Stoffwechsel auf Fettsäureoxidation beruht. Wurde die Fettsäureoxidation durch Etomoxir blockiert, starben die Zellen. Die Störung des Stoffwechsels dieser Zellen könnte somit eine Option für einen neuen Therapieansatzdarstellen. Zusätzlich war die Expression des Transkriptionsfaktors Twist 1 in diesen CD4+CD45RO+ PD1+ CXCR5- T Zellen hochreguliert. Twist 1 ist ein Marker für T Lymphozyten, die in entzündetem Gewebe von Patienten mit chronisch-entzündlichen Erkrankungen der Gelenke oder des Darmes persistieren. Untersuchung in vitro ergaben außerdem, dass Twist 1 spezifisch von Th1 Lymphozyten exprimiert wird, die mehrfach restimuliert wurden. Dieser Transkriptionsfaktor wirkt einerseits der Gewebszerstörung, die von T Zellen verursacht wird, entgegen, und unterstützt anderseits die Persistenz der Zellen im Gewebe durch die Induktion der microRNA148a, die die Expression des pro-apoptotischen Proteins Bim reguliert. In dieser Arbeit zeigen wir dessen Einfluss auf die Regulation des Metabolismus von CD4+ T Lymphozyten bei chronischen Entzündungen. Dabei wird die Glycolyse verringert und vermehrt Fettsäuren synthetisiert, um die Zellen vor reaktiven oxidierenden Spezies (ROS) zu schützen. Zusätzlich konnten wir nachweisen, dass mehrfach re-stimulierte, Twist-defizienteTh1 Zellen unfähig sind, durch Fettsäureoxydation zu überleben, sondern den Stoffwechsel auf Lipid- Peroxidierung umstellen / How do CD4+ T cells adapt their metabolism for survival in the inflamed tissue? This thesis describes analysis of the metabolism of CD4 T lymphocytes driving chronic inflammation and persisting at the site of inflammation, exemplified by cells that reside in the inflamed tissue of patients with the rheumatic disease juvenile idiopathic arthritis. To specifically take aim at the CD4+ T lymphocytes persisting at the site of inflammation, it is important to determine how these cells adapt their metabolism. We show that pathogenic CD4+ CD45RO+ PD1+ CXCR5- T cells that were isolated from the synovial fluid of patients with juvenile idiopathic arthritis are dependent on a fatty acid oxidation for survival ex vivo. Their survival can be blocked by blocking FAO with Etomoxir, pointing to the option of targeting such cells by metabolic interference. Furthermore, CD4+ CD45RO+ PD1+ CXCR5- T cells had upregulated expression of Twist1, a hallmark transcription factor of T lymphocytes persisting in the inflamed tissues of patients with chronic-inflammatory diseases of joints or the gut. Expression of Twist1 is specific for Th1 lymphocytes which have repeatedly been re-stimulated in vitro, or isolated from inflamed. This transcription factor dampens immunopathology caused by the T cells and supports their persistence, by inducing microRNA148a, which regulates expression of the proapoptotic protein Bim. This thesis shows, through conditional genetic inactivation of Twist1 in CD4+ T lymphocytes, that Twist1 regulates the metabolism ofCD4 T lymphocytes of chronic inflammation, by downregulating glycolysis, promoting fatty acid synthesis and protecting the cells from ROS. Additionally, we show that Twist1 deficient repeatedly reactivated murine Th1 cells are unable to survive on fatty acid oxidation and have increased levels of lipid peroxidation.