The skeleton is a dynamic organ that undergoes a continual process known as bone remodeling. Bone remodeling is necessary to maintain structural integrity, heal micro-fractures caused by from daily wear and tear, and to store and release essential ions and minerals. Remodeling is a highly regulated process, with bone resorption precisely balanced by bone formation under homeostatic conditions. In the setting of rheumatoid arthritis (RA), an inflammatory condition affecting joints, this balance is lost and bone around inflamed joints is eroded. These so-called "bone erosions" compromise joint function, causing disability. Osteoclasts, multinucleated cells of hematopoietic origin, are the only cells known to resorb bone. Osteoclasts are found at erosion sites in human joints, and data from mouse models of inflammatory arthritis suggest that osteoclasts are required for erosions to form in bone. The canonical pathway of osteoclast differentiation requires stimulation of myeloid precursors by the cytokine Receptor Activator of NF-Kappa B ligand (RANKL) through its receptor, RANK. In the inflamed joint, RANKL expression can be induced on mesenchymal lineage cells by inflammatory cytokines such as tumor necrosis factor alpha (TNF alpha). Surprisingly, our lab observed bone erosions and osteoclast formation in a mouse model of RA in the absence of RANK. Thus we hypothesized that in addition to RANKL expression, the cytokine milieu in RA may directly stimulate osteoclast formation. It was recently reported that the inflammatory cytokines TNF alpha and interleukin-6 (IL-6) in combination stimulate osteoclast differentiation, independent of exogenous RANKL. We have reproduced these results and shown that these osteoclast-like cells form entirely independently of RANK signaling. However, TNF alpha/IL-6 induced osteoclast formation still requires the transcription factor Nuclear Factor of Activated T cells (NFATc1), a master regulator of RANK-mediated osteoclast differentiation, as well as co-stimulatory signaling provided by the immunoreceptor tyrosine based activation motif (ITAM)-containing DNAX-activating protein (DAP12) molecules. We also showed that TNF alpha/IL-6 induced osteoclast formation requires activity of IL-6 receptor (IL-6R), as osteoclast formation can be inhibited through co-culture with an IL-6R blocking antibody (MR16-1). Finally, using an in vivo mouse model of RA in RANK-deficient mice, we tested whether blocking IL-6R with MR16-1 antibody protects against the formation of periarticular bone erosions. Our results suggest that a RANK-independent pathway of osteoclast formation contributes to inflammatory bone erosions. Targeting this pathway may improve outcomes for RA patients.

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Agradecemos a compreensão. on 2017-05-03T14:13:29Z (GMT) / Submitted by SARAH MARIA BARNEZE COSTA null (sarahbarneze@hotmail.com) on 2017-05-03T16:11:16Z No. of bitstreams: 1 Dissertação Final para o Repositório da Unesp - Aluna Sarah Barneze.pdf: 2656758 bytes, checksum: 41757de66dd6c78c3165310b774a5fcb (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-05-03T16:16:11Z (GMT) No. of bitstreams: 1 costa_smb_me_bot.pdf: 2656758 bytes, checksum: 41757de66dd6c78c3165310b774a5fcb (MD5) / Made available in DSpace on 2017-05-03T16:16:11Z (GMT). No. of bitstreams: 1 costa_smb_me_bot.pdf: 2656758 bytes, checksum: 41757de66dd6c78c3165310b774a5fcb (MD5) Previous issue date: 2017-02-16 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A regulação da remodelação óssea ocorre por meio de fatores locais e sistêmicos. Entre os fatores locais estão as citocinas: Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), presente nos osteoblastos, e Receptor Activator of Nuclear Factor Kappa B (RANK), presente nos osteoclastos, além de outras citocinas como o Tumor Necrosis Factor Alpha (TNF-α) que podem agir no processo de deposição e/ou reabsorção óssea in vivo. Entre os fatores sistêmicos que participam da remodelação óssea estão os hormônios estrógeno (E2) e triiodotironina (T3). O objetivo do trabalho foi verificar a ação do E2, infrafisiológico (simulando a menopausa) e T3, suprafisiológico (simulando hipertireoidismo) em osteoblastos humanos derivados de células troncos mesenquimais (CTMs) na síntese proteica de RANKL e TNF-α. Os osteoblastos foram incubados por 72 horas na presença de E2 em dose fisiológica (E2F/10-8M) e infrafisiológica (E2I/10-9M), e T3 em dose fisiológica (T3F/10-9M) e suprafisiológica (T3S/10-8M), e quantificada a matriz mineralizada óssea e a síntese proteica de RANKL e TNF-α por Western Blot. A análise estatística dos dados foi realizada pelo teste de variância ANOVA complementada pelo teste de Tukey, sendo o nível de significância considerado p<0,05. O tratamento de E2 em dose E2F (1,96± 0,48; p<0,05) e E2I (3,18±0,31; p<0,001) elevou a síntese proteica de RANKL comparado ao controle (C) (1,00±0,32), e de TNF-α em dose de E2F (3,61±0,45; p<0,05) e E2I (2,45±0,07; p<0,05) também aumentou em relação ao C (1,13±0,19). Assim como o E2, o T3 elevou os níveis proteicos de RANKL em T3F (1,18±0,10; p<0,05) e T3S (1,14±0,004; p<0,05) comparado ao controle (1,00±0,02), porém, TNF-α diminui em dose de T3S (0,82±0,09; p<0,05) relacionado ao T3F (1,00±0,09) e C (0,99±0,04). Com isso, E2I diminuiu a matriz mineralizada óssea e aumentou a síntese de ambas as proteínas estudadas, o que nos leva a especulação de que a reabsorção óssea observada na menopausa seja via RANKL e TNF-α. Por outro lado, na mimetização de hipertireoidismo com o T3S, observamos diminuição da matriz mineralizada óssea, supressão da síntese proteica de TNF-α e aumento de RANKL, o que nos leva a inferir que o mecanismo de reabsorção óssea no hipertireoidismo seja via RANKL. / The regulation of bone remodeling is intermediated by local and systemic factors. The cytokines are among the local factors: Receptor Activator of Nuclear Factor Kappa B Ligand (RANKL), present in osteoblasts, and Receptor Activator of Nuclear Factor Kappa B (RANK), present in osteoclasts, in addition to other cytokines such as Tumor Necrosis Factor Alpha (TNF-α) that can act in the process of bone deposition and/or resorption in vivo. Hormones are among the systemic factors involved in bone remodeling, estrogen (E2) and triiodothyronine (T3). The aim of the study was to verify the action of E2, infraphysiological (simulating menopause) and T3, supraphysiological (simulating hyperthyroidism) in human osteoblasts derived from mesenchymal stem cells (CTMs) in the protein synthesis of RANKL and TNF-α. Osteoblasts were incubated during 72 hours in the presence of E2 at physiological dose (E2F/10 -8 M) and infraphysiological (E2I/10 -9 M), and T3 at physiological (T3F/10 -9 M) and supraphysiological (T3S/10-8 M) and quantified the bone mineralized matrix and the protein synthesis of RANKL and TNF-α by Western Blot. Statistical analysis of the data was performed by the ANOVA variance test complemented by the Tukey test, and the significance level was considered p<0.05. The treatment of E2F in E2F dose (1.96 ± 0.48, p<0.05) and E2I (3.18 ± 0.31; p <0.001) increased RANKL protein synthesis compared to control (C) (1.00 ± 0.32), and TNF-α in the dose of E2F (3.61 ± 0.45; p<0.05) and E2I (2.45 ± 0.07; p <0.05) also increased in relation to C (1.13 ± 0.19). As with E2, T3 increased RANKL protein levels in T3F (1.18 ± 0.10, p <0.05) and T3S (1.14 ± 0.004; p <0.05) compared to control (1 , 00 ± 0.02), but TNF-α decreased in dose T3S (0.82 ± 0.09; p <0.05) related to T3F (1.00 ± 0.09) and C (0, 99 ± 0.04). Thus, E2I decreased the bone mineralized matrix and increased the synthesis of both proteins studied which leads to speculation that bone resorption observed at menopause by RANKL and TNF-α pathway. On the other hand, in the mimicry of hyperthyroidism with T3S, we observed a reduction of the bone mineralized matrix, suppression of TNF-α protein synthesis and increase of RANKL, which leads us to infer that the mechanism of action for bone resorption in hyperthyroidism is by RANKL. / FAPESP: 2014/15529-0

In Deutschland unterliegt die Reproduktionsmedizin umfassenden gesetzlichen Regelungen. Fertilisierte Oozyten müssen im Pronukleus-Stadium selektiert werden, hierbei darf maximal eine Anzahl von drei Embryonen kultiviert werden. Studien der vergangenen Jahre zielten vornehmlich auf die Entwicklung eines detaillierten Scoring-Systemes (Zygoten Screening im Pronukleus Stadium), um jeweils die Embryonen mit dem größten Entwicklungspotenzial zu selektieren. 99 Patientinnen wurden inkludiert und durchliefen entweder eine IVF oder ICSI Prozedur. Die fertilisierten Oozyten wurden im Pronukleus-Stadium beurteilt. TNF alpha und LIF, beides in der Reproduktionsmedizin bekannte Zytokine, wurden in gepoolten Kulturmedien an den Tagen 3 und 5 gemessen. 865 Oozyten wurden hierbei gewonnen, 438 zeigten positive Fertilisations-Zeichen, es fand sich eine Fertilisationsrate von 62,6%. Die Schwangerschaftsrate betrug 24,7%. Die mittlere PN Scores zeigten sich signifikant niedriger bei nicht konzipierenden Frauen (15.8 versus 17.2). Die mittlere TNF alpha Konzentration zeigte sich sowohl an Tag 3 als auch an Tag 5 signifikant erniedrigt in schwangeren Frauen gegenüber denen, welche nicht konzipierten (0.43pg/ml versus 0.59pg/ml). Die mittlere LIF Konzentration hingegen war signifikant erhöht bei schwangeren Frauen (56.2pg/ml versus 22.2pg/ml an Tag 3). Zusammenfassung: Das PN-Scoring bleibt eine gute Methode zur prognostischen Einschätzung des weiteren Entwicklungspotenziales von Präimplantationsembryonen. Höhere Konzentrationen von LIF und niedrigere Konzentrationen von TNF alpha in Kulturmedien scheinen eine favorable Rolle in der Embryogenese zu spielen. / German law comprehensively regulates IVF procedures and the selection of embryos for further cultivation. By law, fertilized oocytes have to be selected at the pronucleus-stadium, and a maximum of three embryos are allowed to be cultivated. Studies of the past years therefore aimed to elaborate a detailed scoring system (zygote scoring system at the pronucleus stage) in order to select those embryos with the highest potential for further favorable development. METHODS: 99 patients were included in our prospective trial and underwent either IVF or ICSI procedure. Fertilized oocytes were assessed at the pronucleus stage (day 1). TNF alpha and LIF, both cytokines known to be involved in embryonic development, were measured in pooled culture media on day 3 and 5. RESULTS: A total of 865 oocytes were collected, 438 showed positive signs of fertilisation, a fertilisation-rate of 62.6 % was achieved. Pregnancy rate per embryo transfer was 24.7 %. Mean PN-scores were significantly lower in women conceiving (15.8) than in those not conceiving (17.2). Mean TNF alpha concentration in culture media on day 3 was 0,54pg/ml and 0.37pg/ml on day 5 and was significantly lower in women conceiving (0.43pg/ml versus 0.59pg/ml on day 3). Mean LIF concentration on day 3 was 31.5pg/ml and 35.5pg/ml on day 5 and was significantly higher in women conceiving (56.2pg/ml versus 22.2pg/ml on day 3). CONCLUSION: PN-scoring remains a valuable tool for assessing fertilized oocytes for their further developmental potential at the pronucleus stage. Higher concentrations of LIF and lower concentrations of TNF alpha in culture media seem to play a putative favorable role in embryogenesis.

Reconstructive flaps are a mainstay form of treatment for anatomical defects in plastic surgery, and despite extensive progress in the areas of flap anatomy and design, the mechanism of flap healing and the factors that regulate this process are poorly understood. This thesis investigates the regulation of flap healing, and tests the hypothesis that the introduction of genes for angiogenic growth factors can be used to augment the vascularisation and wound healing of ischaemic flaps. Using a modified McFarlane ischaemic skin flap model in Sprague Dawley rats, endogenous angiogenic regulatory factors that included the VEGF and angiopoietin families and their receptors were investigated. Twelve specific quantitative real-time PCR assays documented a general up-regulation of angiogenic genes and their receptors with time following flap elevation. There was not a readily identifiable “master regulator”. Angiogenic protein levels were more variable with a decrease VEGF-A and TNF-α levels along the flap. Debridement studies of the necrotic distal flaps demonstrated for the first time that VEGF-A164 and TNF-α protein levels stabilised, while angiogenic genes of VEGF-A164, VEGF-A120, angiopoietins and their receptors were down-regulated and VEGF-B186 and HIF-1α mRNA increased, compared to non-debrided flaps. Leucocyte proteolysis in devitalised tissue is discussed as a possible mechanism for reduced angiogenic proteins levels in ischaemic flaps. The impact of angiogenic gene therapy using adenoviral vectors in the flap model revealed for the first time that recombinant adenoviruses containing the VEGF-B186 transgene could significantly augment neovascularisation and improve flap survival. This neovascularisation correlated with up-regulation of the expression of multiple endogenous angiogenic genes that included VEGF-A164, the angiopoietins and their receptors. Erythematous plaques were documented as a side effect of Ad VEGF-A165 and Ad VEGF-B186 treatment of rat skin, although in the latter treatment they were very mild. Weals induced by the presence of VEGF-A165 transgene were associated with a marked acute inflammatory cell infiltrate and oedema consistent with the increased vascular permeability effects of VEGF-A165. Ad VEGF-A165 plus Ad ANG-1* induced weals were less prominent with reduced oedema highlighting the stabilising effect of Ad ANG-1* on vascular permeability.

The tumor necrosis factor (TNF) receptor-associated factors (TRAFs) were initially discovered as proteins that inducibly interact with the intracellular region of TNF receptors (TNFRs). Because the TNFRs lack intrinsic catalytic activity, the TRAFs are hypothesized to orchestrate signaling activation downstream of the TNFR superfamily, however their mechanism of activation remains unclear (Inoue et al., 2000; Bishop, 2004). Originally, the TRAFs were compared to the signal transducers and activators of transcription (STAT) protein family, due to their sequence homology, and the presence of multiple RING- and zinc-finger domains, suggesting that their function may be to regulate transcriptional activity (Rothe et al., 1994; Hu et al., 1994; Sato et al. 1995; Cheng et al., 1995). However, subsequent research focused predominantly on their cytoplasmic functions, and more recently on their function as E3 ubiquitin ligases (Pineda et al., 2007). In my research, I analyzed the subcellular localizations of the TRAFs following CD40 ligand (CD40L)-stimulation, and found that TRAF2 and 3 rapidly translocate into the nucleus of primary neurons and Neuro2a cells. Interestingly, similar analysis conducted in pre-B lymphocytes (Daudi cells) revealed a different response to CD40L-stimulation, with TRAF2 and 3 being rapidly degraded within 5-minutes of stimulation. These findings are significant because they demonstrate for the first time that the TRAFs translocate into the nucleus and suggest that they may function within the nucleus in a cell-specific manner. I next analyzed the ability of TRAF2 and 3 to bind to DNA, and found that they both bind to chromatin and the NF-kappaB consensus element in Neuro2a cells, following CD40L-stimulation. Similar analyses of the chromatin binding of TRAF2 and 3 in Daudi cells revealed that they were rapidly degraded, similar to the results from my analysis of their subcellular localization. These findings show for the first time that the TRAFs interact with DNA, and therefore support the hypothesis that the TRAFs may function within the nucleus as transcriptional regulators. Finally, I analyzed the ability of the TRAFs to regulate transcriptional activity by luciferase assay. Previous studies showed that overexpression of TRAF2 and 6 could induce NF-kappaB transcriptional activity; however researchers have not been able to determine the mechanism by which they do so. In my studies, I found that every TRAF can directly regulate transcriptional activity either as co-activators or co-repressors of transcription, in a cell- and target protein-specific manner. Additionally, I found that TRAF2 can act as a transcriptional activator, and that its ability to regulate transcription is largely dependent upon the presence of its RING-finger domain. In conclusion, these studies have revealed an entirely novel function for the TRAFs as immediate-early transcriptional regulators. Future research into the genes that are regulated by the specific TRAF complexes will further elucidate how the TRAFs regulate TNFR signaling, as well as whether dysfunctions in TRAF signaling may be associated with known disorders. If specific TRAF complexes are found to regulate specific genes, then pharmacological targeting of the individual TRAF complexes may allow for the highly specific inhibition of signaling events downstream of the TNFRs, without compromising overall receptor signaling, transcription factor pathways, or cellular systems.

<p>Det har skett en utveckling av flera nya biologiska läkemedel sedan slutet av 1990-talet för patienter med en reumatisk sjukdom. Några av dessa läkemedel administreras som intravenösa infusioner, vilket innebär att patienterna är tvungna att komma till en poliklinik för att erhålla sitt läkemedel. Detta kan påverka det dagliga livet samt patienternas oberoende och att undersöka hur patienterna uppfattar denna situation är av betydelse. Inom reumatologin är det ingen studie gjord som undersökt detta. </p><p>Syftet med studien var att beskriva hur patienter med reumatiska sjukdomar uppfattar beroendet vid intravenös anti TNF-alfa behandling administrerad av en sjuksköterska.</p><p>Studien genomfördes med en deskriptiv kvalitativ design och fenomenografisk ansats. Tjugo patienter som får intravenös anti TNF-alfa behandling har intervjuats.</p><p>I resultatet framkom tre beskrivningskategorier: det trygga beroendet (möta kontinuitet, möta kompetens, erhålla tillgänglighet och få information), det delaktiga beroendet (kunna påverka och få frihet), det energigivande beroendet (få avkoppling, bli bemött, bli omhändertagen och möta miljön). Patienterna uppfattade en trygghet och delaktighet i sin behandling och uppgav att det var energigivande att regelbundet träffa en sjuksköterska. Vid utformning av sjuksköterskans roll i polikliniskt omvårdnadsarbetet vid intravenös anti TNF-alfa behandling bör patienternas uppfattning utgöra grunden. Vidare skulle det vara intressant att undersöka hur patienter som sköter sin anti TNF-alfa behandling själva genom subkutana injektioner och som aldrig haft en regelbunden kontakt med en sjuksköterska beskriver sin situation.</p>

Les recherches menées au sein de notre laboratoire ont montré que la Nicotinamide Phosphoribosyltransférase (Nampt) est surexprimée lors de l’activation de plusieurs cellules immunes. Cette surexpression est surprenante si l’on considère que cette enzyme intervient dans le métabolisme de base de toute cellule (immune et non immune) en participant à la synthèse du cofacteur NAD. Au cours de ce travail, nous avons essayé de comprendre quelle est la contribution de cette enzyme aux fonctions immunes. Puisque les souris génétiquement invalidées pour le gène de la Nampt meurent à un stade embryonnaire précoce nous avons invalidé ce gène de façon conditionnelle uniquement dans les lymphocytes T et B. Nos résultats suggèrent que la délétion de la Nampt dans les cellules T et B compromet leur survie. De plus, nous avons testé l’effet d’un inhibiteur spécifique de la Nampt sur la sécrétion de TNF par des macrophages et des cellules dendritiques. Nos résultats montrent que l’inhibition de la Nampt s’accompagne d’une diminution du taux de NAD intracellulaire et, parallèlement, d’une réduction de la quantité de TNF synthétisé. De même, nous avons montré qu’en augmentant le taux de NAD cellulaire il est possible d’augmenter la quantité de TNF produit, ce qui laisse supposer que la capacité de la cellule à synthétiser du TNF serait directement liée à son contenu en NAD. Cette régulation semble impliquer une étape post-transcriptionnelle puisque l’ARNm du TNF ne paraît pas être affecté par ces traitements. Puisque le taux de NAD peut influencer directement l’activité d’enzymes NAD-dépendantes, et en particulier l’activité des enzymes de la famille des sirtuines, nous nous sommes demandés si une sirtuine était impliquée dans la synthèse du TNF. Une approche pharmacologique et une approche génétique nous ont permis de montrer que SIRT6 serait capable d’augmenter la production de TNF à une étape traductionnelle. Cette conclusion semble être confirmée par le fait que des cellules dendritiques dérivées de souris invalidées pour le gène SIRT6 produisent moins de TNF en réponse à un stimulus microbien par rapport à des cellules sauvages. En conclusion nos observations suggèrent la Nampt, en affectant le niveau intracellulaire de NAD, joue un rôle important dans le contrôle de la production du TNF par les cellules de système immunitaire

Det har skett en utveckling av flera nya biologiska läkemedel sedan slutet av 1990-talet för patienter med en reumatisk sjukdom. Några av dessa läkemedel administreras som intravenösa infusioner, vilket innebär att patienterna är tvungna att komma till en poliklinik för att erhålla sitt läkemedel. Detta kan påverka det dagliga livet samt patienternas oberoende och att undersöka hur patienterna uppfattar denna situation är av betydelse. Inom reumatologin är det ingen studie gjord som undersökt detta. Syftet med studien var att beskriva hur patienter med reumatiska sjukdomar uppfattar beroendet vid intravenös anti TNF-alfa behandling administrerad av en sjuksköterska. Studien genomfördes med en deskriptiv kvalitativ design och fenomenografisk ansats. Tjugo patienter som får intravenös anti TNF-alfa behandling har intervjuats. I resultatet framkom tre beskrivningskategorier: det trygga beroendet (möta kontinuitet, möta kompetens, erhålla tillgänglighet och få information), det delaktiga beroendet (kunna påverka och få frihet), det energigivande beroendet (få avkoppling, bli bemött, bli omhändertagen och möta miljön). Patienterna uppfattade en trygghet och delaktighet i sin behandling och uppgav att det var energigivande att regelbundet träffa en sjuksköterska. Vid utformning av sjuksköterskans roll i polikliniskt omvårdnadsarbetet vid intravenös anti TNF-alfa behandling bör patienternas uppfattning utgöra grunden. Vidare skulle det vara intressant att undersöka hur patienter som sköter sin anti TNF-alfa behandling själva genom subkutana injektioner och som aldrig haft en regelbunden kontakt med en sjuksköterska beskriver sin situation.

Ankylosing spondylitis (AS) is a chronic, inflammatory rheumatic disease that has a highly variable disease course. Three biologic agents, adalimumab, etanercept, and infliximab, have been developed for the treatment of AS. We conducted three studies: 1) an exploratory analysis of a year-long longitudinal dataset to gain insight into the variability of disease activity, physical function, and well-being and to explore the relationship between these outcome measures; 2) a systematic review of the available evidence for the efficacy of biologic treatment; 3) a systematic review of potential adverse effects of this treatment. We found that repeated measures of disease activity, function and well-being fluctuate considerably between patients, with complex patterns occurring over time within patients. There was mostly high quality evidence that these biologics are efficacious against placebo. We did not find evidence of an increase in serious adverse events or serious infections from short-term randomized controlled trials.

The inter-relation between inflammation, the immune system and leukocytes is multifaceted, with communication between stroma and immune cells mediated by cytokines, growth factors, chemokines, integrins and other molecules. Proteolysis plays an important role in regulating these molecules. Proteolytic cleavage can not only destroy some molecules but can activate or shed others, converting local juxtacrine signalling proteins into effectors that act at a distance. Shedding can also convert membrane-bound receptors into soluble ligand-binding inhibitors. Finally, cleavage can convert agonist molecules into antagonists. As a wide-ranging inhibitor of metalloproteinases, tissue inhibitor of metalloproteinase 3 (TIMP3) has the potential to down-regulate many of these activities. We explore the role of TIMP3 in the regulation of inflammation, revealing that loss of TIMP3 leads to a more rapid increase of soluble TNF, higher levels of soluble TNF receptors and ultimately to increased TNF signalling in systemic inflammation. We also demonstrate TIMP3 loss impacts local inflammation. In addition we investigate the importance of TIMP3 in the expansion of hematopoietic cells.