Die rheumatoide Arthritis (RA) ist eine chronische, progressive und systemische Autoimmunerkrankung, in deren Zentrum das dauerhaft entzündete Synovialgewebe der Gelenke steht. Aufgrund vielfältiger Knochen- und Knorpel-destruierender Prozesse kommt es zu irreversiblen Funktionalitätsverlusten der betroffenen Gelenke. Eine tragende Rolle bei der Ausprägung der klinischen Manifestationen wird dabei der exzessiven Synthese des proinflammatorischen Cytokins IL-1 zugesprochen. Dessen Aktivität kann durch kompetitive Blockade des IL-1 Rezeptors Typ I mit dem natürlich vorkommenden, antiinflammatorischen IL-1 Rezeptorantagonisten (IL-1Ra) inhibiert werden. Der Cytokin-blockierende Therapieansatz mit Anakinra, einem rekombinant hergestellten IL-1Ra, konnte die pharmakologischen Behandlungsmöglichkeiten der RA seit 2001 wesentlich erweitern. Gleichwohl erfordern die geringen Halbwertszeiten von IL-1Ra regelmäßige subkutane Injektionen, um hinreichende therapeutische Wirkstoffspiegel im Patienten aufrecht zu erhalten. Vor diesem Hintergrund bieten somatische Gentherapiekonzepte eine vielversprechende Alternative zu den konventionellen Behandlungsstrategien bei der RA-Therapie. Ein IL-1Ra-Gentransfer ins Gelenk soll die persistierende, lokale, endogene Synthese des therapeutischen IL-1Ra-Proteins ermöglichen und lässt in dieser Hinsicht eine nachhaltige Verbesserung der klinischen Symptomatik erwarten. In dieser Arbeit wurden dafür gentherapeutische Foamyvirus-Adenovirus-Hybridvektoren (FAD) zur Expression des IL-1Ra entwickelt und die Funktionalität der Konstrukte evaluiert. Die Vektoren sollten die effizienten adenoviralen Transduktionsmechanismen mit dem Potential der foamyviralen somatischen Integration für einen direkten in vivo Gentransfer kombinieren. Das System besteht aus einem adenoviralen Hochkapazitätsvektor vom Serotyp 5, der eine selbstinaktivierende PFV-Vektorkassette unter Kontrolle des Reversen Tetracyclin Transaktivator Systems (Tet-On) enthält. In FAD-transduzierten Zellen wurde die funktionelle Induzierbarkeit der PFV-Vektorexpression nachgewiesen und die Kinetik der PFV-Partikelfreisetzung charakterisiert. Nach Induktion der PFV-Vektorkassette konnte in FAD-transduzierten Zellen ein langfristig-stabiler IL-1Ra-Gentransfer gezeigt werden. Ferner konnten protektive Effekte eines FAD-vermittelten IL-1Ra-Gentransfers im Zellkulturmodell nachgewiesen werden. Tierexperimentelle Untersuchungen zeigten eine erfolgreiche Transduktion von Synovialzellen nach intraartikulärer Applikation von FAD-Vektoren. Das Tetracyclin-regulierbare Hybridvektorsystem zur Expression des IL-1Ra, das in der vorliegenden Arbeit geschaffen wurde, könnte zukünftig die Basis für ein effektives Werkzeug zum intraartikulären Gentransfer in der klinischen Praxis bieten. / Rheumatoid arthritis (RA) is a chronic, progressive and systemic autoimmune disease, characterized by invasive synovial hyperplasia. Several inflammatory cartilage- and bone- destroying processes lead to an irreversible loss of joint functionality. The excessive synthesis of the pro-inflammatory cytokine IL-1 has been implicated as a primary mediator of pathology in RA. The activity of IL-1 is initiated upon binding to the IL-1 receptor type I and can be inhibited by the naturally occurring anti-inflammatory IL-1 receptor antagonist (IL1-Ra) protein. The cytokine-blocking therapeutic approach with anakinra, a recombinant form of IL-1Ra, has significantly improved the pharmacological treatment of RA since 2001. Nevertheless, due to the short half-life of IL-1Ra, repeated subcutaneous injections are required to maintain therapeutic concentrations in the patient. Thus, somatic gene therapy may offer a promising alternative to conventional therapeutic strategies for treating RA. Following gene delivery of IL-1Ra, it may be expected that a sustained improvement of clinical symptoms is achievable due to the endogenous cellular synthesis and local secretion of the therapeutic IL-1Ra protein. In this work, foamy virus-adenovirus hybrid vectors (FAD) were developed for the expression of IL-1Ra and the functionality of the constructs was evaluated. The hybrids combine the high transduction efficiency of adenovirus vectors with the integrative potential provided by prototype foamy virus (PFV) vectors, for direct in vivo gene transfer. In the system, a complete expression cassette for self-inactivating PFV vectors, which is under the control of the tetracycline-dependent regulatory system (Tet-On), was inserted into the backbone of a serotype 5-based high-capacity adenoviral vector. In FAD-transduced cells, the induction of the PFV vector cassette was demonstrated and the release of secondary infectious PFV vectors was characterized. After the induction of the PFV vector cassette in FAD-transduced cells, a stable long-term IL1-Ra expression was shown. Furthermore, the anti-inflammatory potential of the FAD-mediated IL-1Ra gene transfer was successfully evaluated in a cell culture model. Animal studies indicated successful transduction of cells in the synovium after intra-articular application of FAD-vectors. The tetracycline-inducible hybrid vector system for the expression of IL-1Ra, which was created in the present work, may provide the future basis for an effective tool for intra-articular gene transfer in clinical settings.

Orientador: Cláudia Vianna Maurer-Morelli / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T13:52:07Z (GMT). No. of bitstreams: 1 Barbalho_PatriciaGoncalves_M.pdf: 8177547 bytes, checksum: 9a3923a19cb47c5609f933774ee68213 (MD5) Previous issue date: 2013 / Resumo: O Danio rerio é um peixe teleósteo popularmente conhecido como zebrafish que têm se destacado como modelo animal favorável para investigações genéticas devido à facilidade de manipulação in vivo, por sua transparência nas fases embrionária e larval e por seu desenvolvimento externo. Recentemente, foi demonstrado que o zebrafish é capaz de exibir padrão comportamental e alteração da atividade eletrográfica durante crise epiléptica como visto em roedores, tornando-o um modelo promissor para as investigações moleculares das epilepsias. Os estudos sobre os diferentes eventos já conhecidos das epilepsias no zebrafish estão apenas começando e, portanto, há ainda muito que ser investigado para uma melhor caracterização deste modelo para estudos em epilepsia. Estudos clínicos e em modelos animais mostraram que a crise epiléptica eleva os níveis da interleucina-1 beta e induz morte neuronal. Nesse sentido, o presente trabalho se propôs a investigar (i) o perfil temporal de expressão do transcrito do gene da interleucina-1 beta (il1b) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol por transcriptase reversa-PCR quantitativa e também, sua relação da expressão com a idade em que é realizada a indução da crise no cérebro imaturo e (ii) a morte neuronal pela histoquímica do Fluoro-Jade B (FJB) no cérebro imaturo e adulto do zebrafish após a indução de crise epiléptica pelo agente químico Pentilenotetrazol. Neste trabalho conseguimos estabelecer com sucesso as condições ideais para o acasalamento desta espécie e obtenção de embriões e, para a criação de larvas. Além disso, padronizamos o processamento histológico para a criosecção do cérebro do zebrafish. A análise temporal do transcrito do gene il1b revelou um padrão de expressão similar ao observado em roedores. A marcação por FJB não identificou inequivocadamente a presença de morte neuronal após crise epiléptica / Abstract: Danio rerio is a teleost fish popular named as zebrafish that has emerged as a suitable animal model for genetic investigations due to its features such as: facility for in vivo manipulation, transparency during embryonic and larval stages and external development. Recently, It was demonstrated that larvae and adult zebrafish (Danio rerio) seizure model mimics the behavior, eletrographic and c-fos features that are well established in rodent models of epilepsy, making the zebrafish as a promising model for studying the molecular mechanisms underlying epilepsies. However, the potential of the zebrafish model for epilepsy studies has been partly described since the molecular changes and neuronal loss after seizure has not yet been investigated. Proinflammatory cytokines, such as interleukin-1beta (il1b), has shown to be upregulated in surgical specimens of pharmacoresistent patients and in experimental rodent models of seizure, however the profile of this cytokine in zebrafish model for epilepsy is unknown. For this reason, and in view of the importance of inflammatory response in the pathophysiology of epilepsy, we sought to investigate the temporal transcript profile of interleukin-1b (il1b), a proinflammatory cytokine, in adult and immature (larvae) zebrafish brain after Pentylenetetrazole-evoked seizure, as well its aged-related expression in the developing brain by reverse transcriptase-quantitative PCR and neuronal death by Fluoro-Jade B (FJB) staining in adult zebrafish brain. Zebrafish breeding was successfully established during this work as well as the protocol for histological procedures using the zebrafish brain. Our results showed a short up-regulation of il1b mRNA levels after seizure in immature and adult zebrafish brain similar as with those patterns observed in rodent models. FJB did not show a reliable neuronal death staining in the zebrafish brain after seizure / Mestrado / Ciencias Biomedicas / Mestra em Ciências Médicas

Depuis les dernières décennies, plusieurs études épidémiologiques montrent des associations entre l’infection/inflammation durant la grossesse, les accouchements prématurés, les lésions cérébrales périnatales et les troubles neuro-développementaux ultérieurs tels que l’autisme. Le pathogène le plus fréquemment rencontré durant la grossesse est le streptocoque de groupe B (SGB). Le SGB colonise le tractus gastro-intestinal et/ou vaginal de 10 à 30% des femmes enceintes et provoque une combinaison d’infection et d’inflammation dont la cible la plus fréquente est le placenta (chorioamnionite). Nous avions précédemment montré, à l’aide d’un modèle animal pré-clinique (rat), que l’exposition systémique au SGB inactivé en fin de gestation induit une chorioamnionite, des lésions cérébrales ainsi que des traits comportementaux de type autistique dans la progéniture mâle. Dans le cadre de ce travail, nous avons précisé la nature de la réaction inflammatoire sous-jacente à l’exposition maternelle au SGB inactivé en fin de gestation. Cette réaction inflammatoire est caractérisée par une surexpression d’IL-1β à la fois dans le plasma maternel, le placenta et le plasma foetal. Les placentas présentent une chorioamnionite sévère, principalement caractérisée par des infiltrations de cellules inflammatoires (majoritairement des cellules polymorphonucléaires neutrophiles (PMN), s’étendant jusqu’au versant foetal placentaire. De manière générale, les tissus associés aux foetus mâles présentent des niveaux d’inflammation supérieurs, autant par le profil d’expression des cytokines pro-inflammatoires (IL-1β, IL-6 et TNF-α) que par les infiltrations de PMN. Puisque l’IL-1 est une cytokine pro-inflammatoire associée au travail préterme, aux lésions cérébrales ainsi qu’à l’autisme, nous avons tenté de valider son rôle dans la genèse des troubles neuro-développementaux SGB-induits dans notre modèle. Toutefois, le rôle de l’IL-1 n’a pu être élucidé. Ces résultats supportent les évidences croissantes que le sexe foetal impacte la susceptibilité du foetus face aux agressions inflammatoires. Ces résultats ouvrent plusieurs nouvelles avenues de recherche, notamment sur l’identification de joueurs clés dans la réaction inflammatoire materno-foetale suivant l’exposition au SGB et ainsi développer de nouvelles mesures pour protéger le placenta et le cerveau du foetus en développement. / Abstract : For the last decades, epidemiological studies have associated preterm birth, cerebral lesions and neurodevelopmental disorders to infection and/or inflammation during pregnancy. One of the most common pathogen encountered during gestation is Group B Streptococcus (GBS), which colonizes 10 to 30% of pregnant women’s gastro-intestinal and/or vaginal tracts. We have previously shown - with a preclinical rat model - that exposure to killed GBS at the end of gestation leads to placental and cerebral lesions. Moreover, only male offspring from mothers who experienced GBS-induced gestational inflammation displayed autistic-like behavior. In this work, we analyzed the inflammatory response to maternal inactivated GBS exposure at the end of gestation. This inflammatory response involves IL-1β, which is over expressed in maternal plasma, placenta and fetal plasma. Placentas displayed acute signs of histological chorioamnionitis, with polymorphonuclear cells (PMN) infiltrations even on the fetal side of placenta. Following GBS-induced inflammation, tissues associated to male fetuses generally showed increased inflammatory response as compared to females (IL-1β, IL-6 and TNF-α) and higher PMN placental infiltrations. Because IL-1β is associated to preterm birth, cerebral damage and autism, we wanted to validate the role of IL-1 in the onset of GBS-induced autism-like behavior in our animal model. However, at this stage, we have not been able to demonstrate this role. These results support the evidences that fetal sex matters for fetal susceptibility to inflammatory aggressions during pregnancy. These results pave the way toward the identification of key molecules in chorioamnionitis, brain damage and subsequent neurobehavioral disorders. This will help to find new strategies to protect the placenta and the fetal brain.

Adult neurogenesis, is a lifelong process by which relatively few cells are added into two restricted regions of the brain. Integration of the cells into the existing neuronal circulatory, with the unique properties involved in the maturation of these cells, is possibly critical to the acquisition and retrieval of new memories. With the chronological aging of the organism a process of cellular senescence occurs throughout the body; a portion of which is independent of primary alterations to the stem cells; instead, it appears to be dependent on the environment where the cells reside, and is in part regulated by inflammation. Microglia, the resident immune cells in the brain, are neuroprotective but chronic activation of the microglia, such as the chronic activation that occurs with advanced age, can promote neurotoxic inflammation. However, it not clear if the aged-related increase in neuroinflammation is at least partly responsible for the aged related decrease in neurogenesis. To address the involvement in neuroinflammation in regulating neurogenesis we used 3 different potential therapeutically relevant manipulations. The first was a targeted approach directed at disrupting the synthesis of Interleukin-1beta (IL-1B), which is a proinflammatory cytokine that is consistently found elevated in the aged brain. The second was a cell therapy approach in which human umbilical cord blood cells were injected into the systemic circulation. The final approach was directed at a chemokine system, fractalkine/CX3CR1, which has been shown as an important paracrine signal, from neurons that regulates the activation state of microglia. While the three approaches used to manipulate, aging-rodent model system were different, a consistent finding was reached in all three studies. In the aged brain, microglia which are the predominate produces of IL-1B, negatively regulate neurogenesis. When IL-1B is decreased or microglia activation is decreased, neurogenesis can be partially restored in the aged brain. The results of these studies, demonstrate a key role for microglia in regulating the neurogenic neiche, which are amendable to therapeutic manipulations.

No description available.

Elevated levels of hyaluronan are associated with numerous inflammatory diseases including ulcerative colitis, Crohn's disease and wound healing. Various proinflammatory cytokines have shown to influence hyaluronan expression in cells originating from connective tissue. The overall purposes of this study included: 1. To determine the effects of IL-1β stimulation on HA and HAS2 steady state transcript levels and the signaling pathways involved in its effects. The signaling pathways utilized by proinflammatory mediators to modulate hyaluronan expression have only begun to be elucidated. In this aim, the effects of IL-1β on hyaluronan and HAS expressions in jejunum-derived mesenchymal cells were determined. Inhibition studies were utilized to determine the signaling pathways involved. The overall hypothesis of this aim was that stimulation of jejunum-derived mesenchymal cells with IL-1β activates the mitogen activated protein kinase pathways resulting in increased HAS2 steady state transcript and hyaluronan levels.Results: The results suggest that IL-1β induction of HAS2 expression involves, in part, the mitogen activated protein kinase signaling pathways that act in concert thus leading to an increase in expression of hyaluronan by jejunum-derived mesenchymal cells.2. To determine the effects of dexamethasone on IL-1β mediated increase in hyaluronan and HAS2 expressions and the mechanisms utilized by this glucocorticoid. Glucocorticoids are a mainstay treatment for the inflammatory component of inflammatory bowel disease. Given the recent evidence demonstrating increased hyaluronan in inflamed tissue from patients affected with inflammatory bowel disease, the objective of this aim was to determine the effect of dexamethasone on IL-1β-mediated induction of hyaluronan. The hypothesis of this aim was that pre-treatment with dexamethasone suppressed the ability of IL-1β to increase HAS2 transcript and hyaluronan levels via inhibition of the p38 MAP kinase pathway. Results: Pre-treatment with dexamethasone inhibited IL-1β-mediated hyaluronan and HAS2 induction by blocking the activation of the p38 MAP kinase pathways. 3. To identify the transcriptional and post-transcriptional mechanisms utilized by IL-1β to upregulate HAS2 steady state transcript levels. Very little is known about transcriptional and post-transcriptional regulation of the hyaluronan synthase 2 gene. In this aim, 5' and 3' mapping, luciferase analyses and actinomycin D studies were used to determine the transcriptional and post-transcriptional mechanisms utilized by IL-1β to regulate HAS2 steady state transcript levels. The hypothesis of this aim was that IL-1β used post-transcriptional mechanisms to regulate the HAS2 gene.Results: Dermal fibroblasts were used to find the 5'- and 3'-termini of the HAS2 message. Promoter constructs extending approximately 1 kb upstream from the transcription start site demonstrated no IL-1β response. Blocking protein synthesis prior to the addition of IL-1β dramatically increased HAS2 steady state transcript levels, while inhibition of transcription suppressed the effect of IL-1β on HAS2. Northern blot analysis revealed that cycloheximide and IL-1β exerted differential effects on the two HAS2 transcripts.

To date, the cellular mechanisms involved in the progression of diseases characterized by chronic inflammation, such as rheumatoid arthritis (RA), remain largely unknown. However, cell-to-cell contact interactions between CD4+ helper T (Th) cells and monocytes have been implicated in the induction and maintenance of pro-inflammatory cytokine synthesis that is characteristic to the pathogenesis of RA. One such cytokine produced during monocyte-Th cell contact is interleukin (IL)-1 β, a mediator directly involved in the characteristic tissue destruction that occurs in the synovia of individuals with RA. Previous studies in our laboratories have shown that ligation of CD40 on monocytes with CD40 ligand (CD40L) present on activated Th cells induces monocyte IL-1β synthesis and rescues monocytes from apoptosis. These findings suggest a role for CD40 signaling of monocyte activation in the exacerbation and maintenance of chronic inflammatory responses. This dissertation represents efforts to elucidate components of the CD40 signaling pathway critical to monocyte activation and how CD40-mediated signaling events are modulated by the anti-inflammatory cytokines IL-4 and IL-10. Using either monocytes isolated from human peripheral blood or a monocytic cell line (THP-1), cellular kinases and transcription factors activated upon CD40 ligation were examined by western blot analyses and electrophoretic mobility shift assays (EMSA), respectively. CD40-dependent interleukin-1β synthesis in monocytes was abrogated by inhibitors of protein tyrosine kinase (PTK) activity but not by inhibitors of protein kinase C (PKC). The extracellular signal-regulated kinases 1 and 2 (Erk1/Erk2) mitogen-activated protein kinases (MAPK's) were specifically activated upon CD40 ligation, and specific inhibition of Erk1/Erk2 activation diminished IL-10 production in a dose-dependent manner. Both IL-4 and IL-10 reduced Erk1/Erk2 activation and synergized in this effect. Finally, STAT3, a member of the family of transcription factors involved in cytokine signaling, was highly phosphorylated in monocytes treated with IL-10 or with IL-10 and IL-4 in combination but not with IL-4 alone. Together these results suggest that in monocytes (1) CD40-mediated IL-1β synthesis and NF-κB activation require PTK activity, (2) CD40-mediated IL-1β production is critically dependent upon Erk1/Erk2 activity, (3) both IL-4 and IL-10 target the Erk1/Erk2 signaling cascade in the downregulation of IL-1β synthesis, (4) IL-4 and IL-10 have divergent effects on the CD40 signaling pathway in that these cytokines are synergistic with respect to their ability to inhibit CD40-mediated Erk1/Erk2 activation and IL-1β synthesis, and differ in their ability to block CD40-mediated rescue from apoptosis, and (5) STAT3 activation may be directly involved in the downregulatory effects of IL-10 on CD40 signaling. (Abstract shortened by UMI.)

No description available.

The molecular mechanisms underlying susceptibility to severe respiratory syncytial virus (RSV) infection remain poorly understood. Herein, we report on the role of osteopontin (OPN) in regulation of RSV infection in human epithelial cells and how interleukin-1 beta (IL-1β), a cytokine secreted soon after RSV infection, when persistently expressed can induce OPN expression leading to increased viral infection. We first compared OPN expression in two human epithelial cell lines: HEK-293 and HEp-2. In contrast to HEp-2, HEK-293 expresses low levels of pro-caspase-1 resulting in decreased IL-1β expression in response to RSV infection. We found a correlation between low IL-1β levels and a delay in induction of OPN expression in RSV-infected HEK-293 cells compared to HEp-2. This phenomenon could partially explain the high susceptibility of HEp-2 cells to RSV infection versus the moderate susceptibility of HEK-293 cells. Also, HEK-293 cells expressing low levels of pro-caspase-1 exhibit decreased IL-1β expression and delayed OPN expression in response to RSV infection. HEK-293 cells incubated with human rIL-1β showed a dose-dependent increase in OPN expression upon RSV infection. Also, incubation with rOPN increased RSV viral load. Moreover, HEp-2 cells or mice infected with a mucogenic RSV strain RSV-L19F showed elevated levels of OPN in contrast to mice infected with the laboratory RSV strain rA2. This correlated with elevated levels of OPN following infection with RSV-L19F compared to rA2. Together, these results demonstrate that increased OPN expression is regulated in part by IL-1β, and the interplay between IL-1β and OPN signaling has a pivotal role in the spread of RSV infection.

Inflammation is a biological defence mechanism applied by living organisms against foreign invaders. In the response to DAMPs and PAMPs, organisms use inflammatory multi-protein complexes to fight the attackers. The most studied inflammasome proteins are NLRP3, ASC and Caspase-1. This study is aimed at understanding the role of NEK7 protein in the NLRP3 inflammasome’s activation, using CRISPR/Cas9 system. To determine the effect of CRISPR/Cas9 and transfection, mRNA expression was analyzed. The results obtained suggest that neither the transfection nor the NEK7 protein knockout have sufficiently worked. This study could not experimentally establish that NEK7 triggers NLRP3 inflammasome activation because ELISA was not conducted to verify the levels of cytokines emitted, due to there being no statistical differences between the samples. Above all, the research question in this thesis project was not answered because the instability of the ACTB reference gene negatively influenced the results. However, previous related studies conclude that NEK7 plays a crucial role in the activation of the NLRP3 inflammasome.