O hidrogênio molecular (H2) exerce efeito antioxidante, anti-apoptótico e antiinflamatório. Nesse estudo testamos a hipótese que o H2 modula as mudanças cardiovasculares, inflamatórias e termorregulatórias na inflamação sistêmica (IS) induzida por lipopolissacarídeo (LPS) em diferentes doses (0,1 ou 1,5 mg/kg, intravenoso, induzindo IS moderada ou severa) em ratos machos Wistar (250-300 g). LPS ou salina foi injetada imediatamente antes do início dos 360 minutos de inalação do H2 (2% H2, 21% O2, balanceado com nitrogênio) ou ar ambiente (21% O2, balanceado com nitrogênio). A temperatura corporal (Tc) foi mensurada por datalogger pré-implantados na cavidade peritoneal. O H2 não causou mudança nos parâmetros cardiovasculares, inflamatórios e na Tc dos ratos controle (tratados com salina). Durante a IS moderada o H2 reduziu o surgimento das citocinas pró-inflamatórias no plasma (TNF-? e IL-6) enquanto causou um aumento da IL-10 plasmática (citocina anti-inflamatória) e preveniu a febre. Durante a IS severa o H2 potencializou a hipotermia e preveniu a febre e a hipotensão. Além disso, o H2 causou uma redução no surgimento das citocinas pró-inflamatórias (TNF-? e IL-1? do plasma) e prostaglandina E2 [(PGE2), no plasma e no hipotálamo], e um aumento da IL-10 plasmática. Esses dados são consistentes com o entendimento que o H2 atenua a febre na IS moderada e durante a IS severa potencializa a hipotermia, previne a hipotensão e exerce um efeito antiinflamatório forte o suficiente para prevenir a febre alterando a sinalização febrigênica e alterando a produção hipotalâmica de PGE2 / Molecular hydrogen (H2) exerts anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Here we tested the hypothesis that H2 modulates cardiovascular, inflammatory, and thermoregulatory changes in systemic inflammation (SI) induced by lipopolysaccharide (LPS) at different doses (0.1 or 1.5 mg/kg, intravenously, to induce mild or severe SI) in male Wistar rats (250-300 g). LPS or saline was injected immediately before the beginning of 360- minute inhalation of H2 (2% H2, 21% O2, balanced with nitrogen) or room air (21% O2, balanced with nitrogen). Deep body temperature (Tb) was measured by dataloggers preimplanted in the peritoneal cavity. H2 caused no change in cardiovascular, inflammatory parameters, and Tb of control rats (treated with saline). During mild SI, H2 reduced plasma surges of proinflammatory cytokines (TNF-? and IL-6) while caused an increase in plasma IL-10 (anti-inflammatory cytokine) and prevented fever. During severe SI, H2 potentiated hypothermia, and prevented fever and hypotension. Moreover, H2 caused a reduction in surges of proinflammatory cytokines (plasma TNF-? and IL-1?) and prostaglandin E2 [(PGE2), in plasma and hypothalamus], and an increase in plasma IL-10. These data are consistent with the notion that H2 blunts fever in mild SI, and during severe SI potentiates hypothermia, prevents hypotension and exerts anti-inflammatory effects strong enough to prevent fever by altering febrigenic signaling and ultimately down-modulating hypothalamic PGE2 production

O hidrogênio molecular (H2) exerce efeito antioxidante, anti-apoptótico e antiinflamatório. Nesse estudo testamos a hipótese que o H2 modula as mudanças cardiovasculares, inflamatórias e termorregulatórias na inflamação sistêmica (IS) induzida por lipopolissacarídeo (LPS) em diferentes doses (0,1 ou 1,5 mg/kg, intravenoso, induzindo IS moderada ou severa) em ratos machos Wistar (250-300 g). LPS ou salina foi injetada imediatamente antes do início dos 360 minutos de inalação do H2 (2% H2, 21% O2, balanceado com nitrogênio) ou ar ambiente (21% O2, balanceado com nitrogênio). A temperatura corporal (Tc) foi mensurada por datalogger pré-implantados na cavidade peritoneal. O H2 não causou mudança nos parâmetros cardiovasculares, inflamatórios e na Tc dos ratos controle (tratados com salina). Durante a IS moderada o H2 reduziu o surgimento das citocinas pró-inflamatórias no plasma (TNF-? e IL-6) enquanto causou um aumento da IL-10 plasmática (citocina anti-inflamatória) e preveniu a febre. Durante a IS severa o H2 potencializou a hipotermia e preveniu a febre e a hipotensão. Além disso, o H2 causou uma redução no surgimento das citocinas pró-inflamatórias (TNF-? e IL-1? do plasma) e prostaglandina E2 [(PGE2), no plasma e no hipotálamo], e um aumento da IL-10 plasmática. Esses dados são consistentes com o entendimento que o H2 atenua a febre na IS moderada e durante a IS severa potencializa a hipotermia, previne a hipotensão e exerce um efeito antiinflamatório forte o suficiente para prevenir a febre alterando a sinalização febrigênica e alterando a produção hipotalâmica de PGE2 / Molecular hydrogen (H2) exerts anti-oxidative, anti-apoptotic, and anti-inflammatory effects. Here we tested the hypothesis that H2 modulates cardiovascular, inflammatory, and thermoregulatory changes in systemic inflammation (SI) induced by lipopolysaccharide (LPS) at different doses (0.1 or 1.5 mg/kg, intravenously, to induce mild or severe SI) in male Wistar rats (250-300 g). LPS or saline was injected immediately before the beginning of 360- minute inhalation of H2 (2% H2, 21% O2, balanced with nitrogen) or room air (21% O2, balanced with nitrogen). Deep body temperature (Tb) was measured by dataloggers preimplanted in the peritoneal cavity. H2 caused no change in cardiovascular, inflammatory parameters, and Tb of control rats (treated with saline). During mild SI, H2 reduced plasma surges of proinflammatory cytokines (TNF-? and IL-6) while caused an increase in plasma IL-10 (anti-inflammatory cytokine) and prevented fever. During severe SI, H2 potentiated hypothermia, and prevented fever and hypotension. Moreover, H2 caused a reduction in surges of proinflammatory cytokines (plasma TNF-? and IL-1?) and prostaglandin E2 [(PGE2), in plasma and hypothalamus], and an increase in plasma IL-10. These data are consistent with the notion that H2 blunts fever in mild SI, and during severe SI potentiates hypothermia, prevents hypotension and exerts anti-inflammatory effects strong enough to prevent fever by altering febrigenic signaling and ultimately down-modulating hypothalamic PGE2 production

Introduction : Le TNF-a, impliqué dans l’inflammation et la défense de l’hôte, aaussi des propriétés anti-tumorales et est utilisé dans le traitement local des sarcomesdes membres. P53 est un anti-oncogène dont la mutation est associée audéveloppement de tumeurs. Des données expérimentales ont démontré une relationentre activité anti-tumorale de TNF-a et le statut de p53. Matériel et méthodes : Notre objectif a été d’étudier en immunohistochimie lestatut de p53 chez 110 patients atteints de sarcomes et traités par TNF-a. Ensuite,dans 8 sarcomes cultivés ex-vivo, nous avons étudié la localisation de l’apoptoseinduite par le TNF-a par microscopie confocale. Puis, dans 9 lignées de sarcomeshumains, nous avons testé la relation p53/TNF-a en abrogeant p53 grâce à un sh-RNA, ou en utilisant des petites molécules telles que CP-31398 ou Nutlin-3a aptes àrestaurer p53. Enfin, pour mieux comprendre les mécanismes de résistance au TNF-a,nous avons mesuré par méthode EMSA la liaison de NF-kB à l’ADN et recherché parRT-PCR quels gènes de l’apoptose étaient différentiellement régulés.Résultats : Le statut muté de p53 corrèle avec la réponse histologique autraitement par TNF-a chez l’homme. L’apoptose induite par le TNF-a est trouvée aussibien au niveau de la cellule endothéliale que de la cellule tumorale. De plus, laréponse au TNF-a est modulée par le statut de p53 puisque dans les lignées étudiées,l’abrogation de p53 supprime celle-ci alors que la réparation d’une activité p53 permetde l’augmenter. Enfin, une potentialisation de l’apoptose induite par TNF-a estobservée lorsqu’il est associé avec CP-31398 ou Nutlin-3a et elle corrèle avec ladiminution de la liaison du NF-kB à l’ADN. Une augmentation de l’expression desgènes RIPK2, TP53BP2 et GADD45 et une diminution de l’expression de TGF-b1 etFAIM est observée lorsque l’association de Nutlin-3a et TNF-a est synergique sur lamort cellulaire.Conclusions : Ces résultats suggèrent que l’utilisation de molécules capablesde restaurer l’activité de p53 peut inverser la résistance des sarcomes des tissusmous au traitement par TNF-a. Dans ce contexte, des études cliniques pourraientexploiter cette approche pour l’utiliser dans le cadre des sarcomes particulièrementrésistants aux traitements conventionnels ainsi que dans d’autres tumeurs. / Introduction: Although named for its antitumor properties, TNF-a is implicatedin a wide spectrum of diseases including chronic inflammation, autoimmunity andcancer. It is used for the loco regional treatment of limb’s sarcoma. P53 is an antioncogenewhose mutation is associated with tumour development. Experimental datademonstrated that TNF-a cytotoxic activity and p53 status are related.Material and methods: Our objective was to study by immunohistochemistrythe p53 status in 110 sarcoma patients treated by isolated limb perfusion with TNF-a. Then, we studied by confocal microscopy in 8 freshly obtained sarcoma tumours,the localization of apoptosis. Finally, in 9 sarcoma cell lines with different p53 status,we tested the p53/TNF-a relationship by abrogating p53 with a sh-RNA and by usingsmall molecules known to restore p53 functions. To better understand the mechanismsof resistance to TNF-a, we measured by EMSA the NF-kB-binding to DNA and withRT-PCR, we explored the regulation of some apoptosis related genes.Results: We demonstrated a relationship between p53 status and thehistological response to TNF-a use in humans. TNF-a induced apoptosis was presentin endothelial cells as well as in the tumour cells. TNF-a cytotoxicity was dependent onthe p53 status since in the cell lines studied, p53 abrogation reduced it and p53restoration allowed it to increase. Moreover, a potentiation of TNF-a cytotoxic effectwas observed when it was combined to CP-31398 or Nutlin-3a. The killing magnitudewas therefore related to the decrease in the NF-kB-binding to DNA when Nutlin-3awas added. A gene expression increase for RIPK2, TP53BP2 and GADD45 and adecrease for TGF-b1 and FAIM was observed if the combined treatment wassynergistic on tumour death cells.Conclusions: These results suggest that the use of compounds able to restorep53 activity could reverse the soft tissue sarcoma’s resistance to TNF-a treatment.Clinical studies should be performed in order to utilize this approach and to use it inthe context of sarcomas that are particularly resistant to conventional treatments.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / nÃo hà / Paclitaxel (PCX) foi o 1 antineoplÃsico efetivo no tratamento de cÃnceres refratÃrios a quimioterapia convencional. Clinicamente, induz artralgias e mialgias de carÃter incapacitante que comprometem a qualidade de vida e limitam o tempo de tratamento antitumoral, acometendo atà 57% dos doentes. Apesar destas repercussÃes clÃnicas nada foi descrito atà o momento, visando elucidar o envolvimento de citocinas prÃ-inflamatÃrias, na gÃnese da atividade hipernociceptiva do PCX, jà que a droga compartilha com o LPS uma via de sinalizaÃÃo desencadeada por receptores Toll (TLR-4 e TLR-2) para geraÃÃo de genes que codificam TNF-&#945;. Dados da literatura sugerem que ocorra um âcross-talkâ entre esses dois membros da famÃlia Toll e, que agonistas diferentes de TLR-2 e TLR-4 sÃo capazes de induzir a ativaÃÃo de NF-&#945;B, AP1 e MAP kinase e a geraÃÃo de TNF-&#945;, citocina chave na cascata de liberaÃÃo de mediadores inflamatÃrios finais que atuam diretamente no nociceptor. Dados do laboratÃrio registraram que o zymosan (ZY) intrarticular em joelhos de ratos produz uma periartrite caracterÃstica da hipernocicepÃÃo no teste de incapacitaÃÃo articular (IA), e que PCX (8mg/kg) amplificou essa resposta quando se injetou  da dose do ZY. Tal amplificaÃÃo foi inibida com o prÃ-tratamento com inibidores de citocina e de prostanÃides. Objetivo. Investigar a participaÃÃo do TLR-4 e TNF-&#945; na gÃnese do efeito potencializador do PCX na artralgia experimental induzida por ZY. Material e MÃtodos. Ratos foram prÃ-tratados Sc com talidomida (TLD), pentoxifilina, dexametasona, indometacina ou celecoxib e estimulados com subdose de ZY (250&#956;g/animal; i-art). ApÃs a 1 medida do tempo de suspensÃo de pata (TSP) no teste de IA, os animais receberam PCX (8mg/kg; ip). Numa segunda etapa os ratos receberam durante trÃs dias consecutivos o prÃ-tratamento com atorvastatina (3, 10, 30mg/kg/dia; VO). Os seguintes parÃmetros foram avaliados: modulaÃÃo da hipernocicepÃÃo no teste de incapacitaÃÃo articular, dosagem de citocinas em lavado de joelho de ratos (TNF-&#945;, IL-1&#945;, Il-6, KC e CINC) e imunohistoquÃmica para TNF-&#945;, IL-1&#945; e TLR-4 no tecido sinovial. Resultados. Ficou demonstrado que PCX (8mg/kg) potencializa a artralgia experimental induzida por ZY em ratos avaliada pelo aumento significativo do TSP (p<0,001) na 4Âh de artrite em relaÃÃo ao controle no teste de IA. Tal efeito foi inibido de maneira significativa pelo prÃ-tratamento com TLD (45mg/kg) e essa inibiÃÃo foi associada à reduÃÃo dos nÃveis de TNF-&#945; produzido pelas cÃlulas do tecido sinovial no lavado articular e da marcaÃÃo imunohistoquÃmica para TNF-&#61537;. Da mesma forma a inibiÃÃo dessa resposta amplificadora do PCX foi ratificado pelo prÃ-tratamento com atorvastatina nas trÃs doses utilizadas no modelo, tambÃm sendo associado à diminuiÃÃo significativa dos nÃveis de TNF-&#61537; no lavado articular e visÃvel reduÃÃo na marcaÃÃo imunohistoquÃmica para TNF-&#61537;, IL-1&#61538; e TLR-4, nas trÃs doses utilizadas. ConclusÃes. PCX potencializa a hipernocicepÃÃo induzida por ZY por um mecanismo indireto sobre cÃlulas residentes da membrana sinovial que liberam TNF-&#61537; provavelmente pela ativaÃÃo da NF-&#61547;B via TLR-4/MD2, pois esse efeito potencializador foi inibido pela atorvastatina, um provÃvel antagonista de TLR-4. O TNF-&#61537; liberado age iniciando a cascata de mediadores envolvidos com a dor inflamatÃria, o que justifica em parte as artralgias dos pacientes em tratamento com PCX. / Paclitaxel (PCX) was the first effective antineoplastic medicine in the treatment of tumors that do not respond to conventional chemotherapy. Clinically, it induces incapacitating arthralgias and myalgias that interfere with the patient quality of life and limit the duration of the treatment. This is observed in up to 57% of the patients using the drug. Despite these clinical manifestations, nothing has been published that could explain the involvement of pro-inflammatory cytokines in the triggering of the hypernociceptive effect of PCX, even though it is known that the drug shares with LPS a signaling pathway started by Toll-like receptors (TLR-2 and TLR-4) that activates genes coding for TNF-&#945;. The literature suggests that there is a crosstalk between these two members of the Toll family and that different agonists of TLR-2 and TLR-4 are able to induce the activation of NF-kB, AP1 and MAP kinase in the generation of TNF-&#945;, a key cytokines in the cascade liberating the final inflammatory mediators that act directly on the nociceptor. Data obtained in laboratory show that the injection of zymozan into rat knee-joints produces a periarthritis characteristic of the hypernociception seen in the knee joint incapacitation test and that PCX (8mg/kg) amplified the response when  of the zymozan (ZY) doses was injected. The amplification was inhibited when animals were pre-treated with inhibitors of cytokines and prostanoids. Objective: To study the role of TNF-&#945; and TLR-4 on the initiation of the potentiating effect of PCX on the experimental arthralgia induced by ZY. Material and Methods: Rats were pre-treated Sc with thalidomide, pentoxifiline, dexametazone, indometacin and celecoxib and then stimulated with an intra-articular subdoses of ZY (250&#956;g/animal). After the first measurement of the paw elevation time in the knee joint incapacitation test, the animals were treated with PCX (8mg/kg ip). On a second trial, rats were treated for three consecutive days with atorvastatin (3, 10, 30mg/kg/day; VO). The following parameters were evaluated: modulation of the effect on the knee joint incapacitation test (JIT), amount of cytokines in the ratâs knee lavage (TNF-&#945;, IL-1 &#946;, IL-6, KC and CINC) and immunohistochemistry for TNF-&#945;, IL-1&#946; and TLR-4 on synovial tissue. Results: It was shown that PCX (8mg/kg) potentiates the experimental arthralgia induced by ZY in the rats as evaluated by the significant increase in paw elevation time (p<0.001) at the 4th h of arthritis in relations to controls. Such effect was significantly inhibited by pre-treatment with thalidomide (45mg/kg) and the inhibition was associated with a decrease in the amount of TNF-&#945; produced by synovial tissue cells and detected in the joint lavage and in the immunohistochemistry for TNF-&#945;. Likewise the inhibition of the amplifying response to PCX was seen with pre-treatment with atorvastatin at the three doses used in the experiment, which was also associated with a lower TNF-&#945; in the joint lavage and perceptible decrease in the immunohistochemistry for TNF-&#945;, IL-1&#946; and TLR-4. Conclusions: PCX potentiates the hypernociception induced by ZY through an indirect effect on synovial membrane resident cells that release TNF-&#945; probably through activation of the NF-kB pathway by TLR-4/MD2, since the potentiating effect was inhibited by atorvastatin, a TLR-4 antagonist. Released TNF-&#945; act starting the cascade of mediators involved in the inflammatory pain and this partially explains the arthralgia in patients treated with PCX.

In the studies this thesis is based upon, microarrays were applied to profilemRNA populations in biological samples to gain insights into transcriptionalpatterns and their relation to inflammatory disease.Rheumatoid arthritis (RA) is a chronic inflammatory disease, which leads todegradation of cartilage and bone. RA is characterized by synovial inflammationwith varying levels of tissue heterogeneity. This was confirmed by microarrayanalyses of multiple biopsies from the joints of 13 patients, which showed interindividualvariation in transcript populations to be higher than intra‐individualvariationTherapeutic antibodies targeting TNF‐α have revolutionized treatment of RA,although some patients do not respond well. Identification of non‐responders isimportant, not only because anti‐TNF treatment elevates the risk of infections,but also because of the cost of treatment. A proof‐of‐concept study to investigatetranscriptional effects of anti‐TNF treatment demonstrated that differencesbetween response groups could be identified and that these differences revealedbiological themes related to inflammatory disease.A subsequent study was therefore initiated with a larger cohort of 62 patients toinvestigate gene expression patterns in the synovium prior to anti‐TNFtreatment. Here, the heterogeneity was even more pronounced, thetranscriptional patterns were confounded by the presence of synovial aggregatesand only a weak therapy‐correlated signature was detected. The presence oflymphocyte aggregates was found to correlate to response to therapy, which isconsistent with previous findings indicating a higher level of inflammation ingood responding patients.Periodontitis is an inflammatory disease with many similarities to RA. Both areincurable chronic auto‐immune diseases, characterized by tissue destructionwith common genetic associations. Individuals with RA are at higher risk ofaccumulating significant periodontal problems than the general population. PGE2(prostaglandin E2) is known to stimulate inflammation and bone resorption inperiodontitis. In further studies, microarrays were applied in a time seriesdesign on human gingival fibroblats to explore the signal transduction pathwayscontrolling TNF‐α induced PGE2 synthesis in order to identify novel therapeutictargets. The JNK and NF‐kb pathways were identified as being differentiallyaffected by TNF‐a treatment. The transcriptional patterns were further verifiedusing antibodies against phosphorylated JNK/NF‐kb molecules and specificinhibitors of the JNK and NF‐kb signaling cascades. / QC 20100820

In den letzten Jahrzehnten haben sich die so genannten Biologika auch zur Therapie der Psoriasis etabliert. Zu diesen Medikamenten gehört auch Adalimumab, welches als vollständig humaner Antikörper eines der Schlüsselzytokine in der Pathogenese der Psoriasis, TNFα, neutralisiert. Allerdings führt die Therapie nicht bei allen Patienten zu ausreichendem Wirkerfolg. Da bisher vor Beginn der Therapie nicht zwischen den Patienten, die von der Therapie profitieren, und denen, die keine ausreichende Wirkung erfahren, unterschieden werden kann, werden die letzteren unnötigerweise den Risiken und Nebenwirkungen dieser Therapie ausgesetzt. In dieser Arbeit wurden die Interaktionen kultivierter Endothelzellen und Lymphozyten ex vivo unter dem Einfluss von Adalimumab untersucht. Insbesondere auf mögliche Unterschiede zwischen „Respondern“ und „Non-Respondern“ wurde im Hinblick auf die mögliche Entwicklung eines prädiktiven Tests für das Ansprechen auf Adalimumab ein Schwerpunkt gelegt. Lymphozyten gesunder Probanden und von Psoriasis-Patienten wurden ex vivo hinsichtlich ihrer Interaktion mit kultivierten Endothelzellen (HUVEC), mit und ohne TNFα-Stimulation, untersucht. Hierbei wurden sowohl frisch isolierte als auch kryokonservierte Lymphozyten verwendet, da sich zwischen diesen keine Unterschiede in den funktionellen Flusskammer-Versuchen zeigten. Nach Stimulation der Endothelzellen mit TNFα kam es zu einem deutlichen Anstieg des Rollens und der festen Adhäsion aller Lymphozyten an den Endothelzellen. Allerdings zeigten sich im Ausmaß dieser Interaktion deutliche inter-individuelle Unterschiede. Obwohl diese auch bei Psoriasis-Patienten auftraten, konnten keine signifikanten Unterschiede zwischen „Respondern“ und „Non-Respondern“ beobachtet werden. In den Untersuchungen zum Einfluss von Adalimumab auf TNFα-stimulierte Endothelzellen sowie die Interaktion dieser mit Lymphozyten ex vivo zeigte sich eine deutliche Abhängigkeit der Auswirkung vom Zeitpunkt der Behandlung. Erfolgte die Adalimumab-Behandlung vor oder gleichzeitig mit der TNFα-Stimulation der Endothelzellen, kam es zur Aufhebung der TNFα-induzierten Effekte sowohl in der Transkription der Adhäsionsmoleküle (PCR), der Expression dieser (Immunfluoreszenz-Mikroskopie) sowie der dynamischen Interaktionen mit Lymphozyten (Flusskammer). Eine dem TNFα nachfolgende Behandlung blieb ohne Wirkung, sodass davon auszugehen ist, dass sich die bereits induzierten Prozesse nicht mehr rückgängig machen lassen. Hier könnte eine Erklärung für das späte Eintreten der Adalimumab-Wirkung in vivo liegen. Somit konnten in dieser Arbeit vier zentrale Ergebnisse erzielt werden: Erstens, es wurde erstmals gezeigt, dass wichtige funktionelle Eigenschaften humaner Lymphozyten während der Kryokonservierung erhalten bleiben. Zweitens, es wurden erstmals deutliche inter-individuelle Unterschiede im Ausmaß der Interaktion zwischen Lymphozyten ex vivo mit TNFα-stimulierten Endothelzellen nachgewiesen. Adalimumab unterdrückte diese dynamischen Interaktionen, sofern seine Zugabe vor oder gleichzeitig mit der TNF-Exposition erfolgte. Drittens, diese inter-individuellen Unterschiede bestanden gleichermaßen bei gesunden Probanden und Psoriasis-Patienten. Viertens, die funktionellen Unterschiede erlaubten keine Unterscheidung zwischen Psoriasis-Patienten, deren Erkrankung sich durch Adalimumab besserte („Responder“), und denen, deren Erkrankung nicht auf diese Therapie ansprach („Non-Responder“).

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.

Made available in DSpace on 2016-08-10T10:38:36Z (GMT). No. of bitstreams: 1 Rodrigo Bernini de Brito.pdf: 1595621 bytes, checksum: eef53307cc2b9e3d872ec9478aeef786 (MD5) Previous issue date: 2012-12-20 / Olanzapine is a second generation antipsychotic that show low incidence of extrapyramidal side effects and has been recommended as the first line drug for the treatment of schizophrenia and is also used in the treatment of bipolar disorder. But it has the weight gain as a side effect which is common in the chronic use of this medicine. A comprehensive review of the literature revealed that olanzapine induces more weight gain than most other antipsychotics, except clozapine. The incidence of weight gain induced by olanzapine and associated diseases such as diabetes and cardiovascular diseases is higher in this group of patients than in the general population. These unwanted side effects have decreased patients' adherence to treatment. Many clinical observations and studies have attempted to elucidate the possible mechanism involved. However, to date, the mechanism underlying the weight gain induced by olanzapine remains unclear. This present study retrospective evaluates 21 patients using olanzapine for a period of 20 to 119 months, compared within the sample, patients who lost weight or remained stable (<7% gain in relation to BMI) group which gained weight at a moderate or severe way during the use of olanzapine (> 7% gain in relation to BMI). We also evaluated the levels of glucose and plasma lipids of all patients. For the group of patients were also analyzed genetic polymorphisms of TaqIA DRD2 gene and G-308A TNF-&#945; gene by PCR-RFL and ARMS-PCR, respectively. TaqIA of genetic polymorphism (C32806T) in the DRD2 gene was correlated with prolonged use of olanzapine with relevant statistical significance in relation to weight gain and biochemical changes observed in plasma of patients. Regarding the genetic variants of the SNP G-308A TNF-&#945; gene, the findings of this study failed to corroborate or refute the findings of other studies. / A olanzapina é um antipsicótico de segunda geração que exibe uma baixa incidência de efeitos colaterais extrapiramidais e tem sido recomendada como fármaco de primeira linha para o tratamento da esquizofrenia e também é utilizada no tratamento do transtorno bipolar, mas tem o ganho de peso como efeito colateral comum no uso crônico deste medicamento. Uma análise abrangente da literatura revelou que a olanzapina induz maior ganho de peso do que a maioria dos outros antipsicóticos, com exceção da clozapina. A incidência de ganho de peso induzido pela olanzapina e doenças associadas, como diabetes e doenças cardiovasculares, é maior entre o grupo de pacientes do que a da população em geral. Estes efeitos secundários indesejados têm diminuído a adesão dos pacientes ao tratamento. Muitas observações clínicas e estudos têm tentado elucidar o possível mecanismo envolvido. No entanto, até o momento, o mecanismo subjacente ao ganho de peso induzido pela olanzapina permanece obscuro. No presente estudo foi realizada uma investigação retrospectiva, que avaliou 21 pacientes em uso de olanzapina por um período de 20 a 119 meses, comparando dentro da amostra, pacientes que perderam peso ou ficaram estáveis (< 7% ganho em relação ao IMC) ao grupo que ganhou peso de forma moderada ou grave durante o uso da olanzapina (>7% ganho em relação ao IMC). Também foram avaliados os níveis de glicose e lipídeos plasmáticos de todos os pacientes. Para o grupo de pacientes ainda foram analisados os polimorfismos genéticos de TaqIAno gene DRD2 e G-308A do gene TNF-&#945; por PCR-RFL e ARMS-PCR, respectivamente. O polimorfismo genéticos da TaqIA (C32806T) no gene DRD2 apresentou relação com o uso prolongado de olanzapina com relevante significância estatística em relação ao ganho de peso e às alterações bioquímicas observadas no plasma dos pacientes. Em relação as variantes genética do SNP G-308A no gene TNF-&#945;, os achados do presente estudo não permitiram corroborar ou refutar as conclusões de outros estudos.

La sévérité du paludisme est influencée par des interactions complexes entre de nombreux facteurs dont la génétique de l’hôte. Plusieurs études de liaison génétique menées dans différentes ethnies africaines ont montré une liaison entre le locus 6p21 et le paludisme simple. De plus, différents variants au sein des gènes TNF et NCR3, retrouvés dans ce locus, ont été indépendamment associés à ce phénotype au Burkina Faso.Ainsi, nous nous sommes tout d’abord intéressés aux polymorphismes du TNF. Nos résultats montrent que les variants TNF-308, TNF-244, et TNF-238 sont associés à la parasitémie maximale ou aux accès simples au Congo. Les approches moléculaires indiquent que le TNF-244 a un effet cis-régulateur avec une activité promotrice réduite en présence du variant A ainsi qu’une fixation altérée de protéines nucléaires en présence de ce même variant. Enfin, nos analyses bio-informatiques suggèrent que le TNF-244 et le TNF-238 agissent en synergie pour modifier le site de fixation d’au moins un facteur de transcription.Nous avons ensuite confirmé l’association du NCR3-412 avec le paludisme simple et le nombre d’accès fébrile au Congo. Les analyses fonctionnelles montrent que ce SNP a aussi un effet cis-régulateur avec une activité promotrice accrue en présence de l’allèle G et une liaison altérée de deux complexes protéiques en présence de l’allèle C. Les approches in silico et in vitro indiquent que les facteurs STAT4 et RUNX3 sont ceux dont la fixation est altérée.NCR3-412 altérant la résistance à la forme simple du paludisme, nous avons souhaité déterminer s’il est aussi impliqué dans la résistance au paludisme sévère mais nous n’avons détecté aucune association. / The severity of malaria is influenced by complex interactions between many factors including host genetics. Numerous genetic studies conducted in different African ethnic groups have shown a significant linkage between the 6p21 locus and mild malaria attack. In addition to their linkage, several polymorphisms found under the linkage peak, and more precisely within TNF and NCR3, were also independently associated with different sub-phenotypes of mild malaria in Burkina Faso.Thus, we first focused on TNF polymorphisms. Among the 4 polymorphisms analyzed, we found associations between TNF-238, TNF-244, TNF-308 and either mild malaria attack or maximum parasitemia. Molecular approaches showed that TNF-244 has a cis-regulatory effect. Indeed, we observe a decreased promoter activity and an altered binding of nuclear proteins in the presence of the A variant. In addition, our bioinformatics analyses suggested a cooperative effect of TNF-244 and TNF-238 in modifying the binding of at least one transcription factor.We then confirmed the association of NCR3-412 with both mild malaria and the number of febrile episodes in Congo. Functional analyses have shown that this SNP has also a cis-regulatory effect with a decreased promoter activity and an altered binding of two nuclear protein complexes in the presence of the C allele. Finally, in silico and in vitro approaches indicated that STAT4 and RUNX3 are the two transcription factors affected.As NCR3-412 is associated with resistance to mild malaria, we therefore investigated whether this SNP is also involved in severe malaria resistance, but we did not detect any association neither with severe anemia nor with cerebral malaria.

Submitted by Ana Maria Fiscina Sampaio (fiscina@bahia.fiocruz.br) on 2012-07-19T19:54:59Z No. of bitstreams: 1 Gisele Batista Carvalho. Análise da expressão dos Genes e IGF-1, HGF, VEGF - CPqGM - Dissertação de Mestrado - 2008.pdf: 634399 bytes, checksum: c4a7c8d54e5c114e87ff5e2c2cd711b0 (MD5) / Made available in DSpace on 2012-07-19T19:54:59Z (GMT). No. of bitstreams: 1 Gisele Batista Carvalho. Análise da expressão dos Genes e IGF-1, HGF, VEGF - CPqGM - Dissertação de Mestrado - 2008.pdf: 634399 bytes, checksum: c4a7c8d54e5c114e87ff5e2c2cd711b0 (MD5) Previous issue date: 2008 / Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / Nos últimos anos estudos têm demonstrado a participação de citocinas e fatores de proliferação e diferenciação na regeneração do miocárdio em condições de agressão. Os fatores que influenciam a regeneração do miocárdio na fase aguda da infecção por T. cruzi na qual ocorre um processo massivo de destruição do miocárdio, ainda são desconhecidos. Neste trabalho foi investigada a expressão dos genes de HGF, IGF-1, VEGF e TNF-α no coração de camundongos durante o curso da infecção aguda por T. cruzi. Fragmentos de corações de camundongos C57Bl/6 infectados com cepa Colombiana de T. cruzi, sacrificados com 15, 25 30, 40 e 60 dias pós-infecção, foram analisados pela técnica de PCR em tempo real. A expressão dos genes de HGF, IGF-1, VEGF e TNF-α no coração foi avaliada e correlacionada ao grau de inflamação, fibrose e parasitemia nos tempos de infecção analisados. Foi observado um aumento significativo na expressão dos genes de TNF-α e IGF-1 durante toda a fase aguda da infecção, enquanto que a expressão de HGF apresentou-se diminuída. Já a expressão do gene de VEGF não foi significativamente alterada no decorrer da infecção. As expressões dos genes de HGF, IGF-1 e TNF-α apresentaram uma correlação negativa com a parasitemia e a inflamação. Estes resultados indicam que o miocárdio produz vários mediadores solúveis em resposta à infecção por T. cruzi, que podem ter uma participação tanto nos mecanismos de lesão, como o TNF-α, como na recuperação da lesão tecidual promovida pela infecção por T. cruzi, como é o caso do IGF-1. Outros estudos devem ser realizados no sentido de melhor entender o papel destes mediadores produzidos localmente ou trazidos pela circulação no processo de reparo do miocárdio pela infecção por T. cruzi. / Studies carried out in the past few years have demonstrated the participation of cytokines and growth factors related to cell proliferation and differentiation in the regeneration of the myocardium after injury. The factors involved in the regeneration of the myocardium during the acute phase of T. cruzi infection, when a process of massive destruction of the myocardium occurs, are not known. In this work we investigated the gene expression of HGF, IGF-1, VEGF e TNF-α in the hearts of mice during the acute infection by T. cruzi. Heart fragments of C57Bl/6 mice infected by Colombian strain T. cruzi were obtained 15, 25 30, 40 e 60 days after infection and analyzed by real time PCR. The gene expression of HGF, IGF-1, VEGF, and TNF-α in the heart was evaluated and correlated to the degree of inflammation, fibrosis and parasitemia at the time points analyzed. A significant increase in TNF-α and IGF-1 gene expression was observed during the acute phase of infection, whereas the expression of HGF was decreased. The expression of VEGF gene was not significantly altered during infection. The expression of HGF, IGF-1, and TNF-α genes showed a negative correlation with parasitemia and inflammation. The results indicate the miocardium as a source of soluble mediators, in response to T. cruzi infection, which may participate both in the mechanisms of lesion induction, in the case of TNF-α, as well as in the repair of tissue lesions promoted by T. cruzi infection, such as IGF-1. Additional studies should be carried out in order to understand the role of these mediators produced locally or brought by the circulation in the process of lesion repair in the miocardium during T. cruzi infection.