Orientador: Sisi Marcondes Paschoal / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-26T18:31:06Z (GMT). No. of bitstreams: 1 Bonfitto_PedroHenriqueLeite_M.pdf: 791921 bytes, checksum: 967c29bbcdf8e6df7bd3a2d919b84111 (MD5) Previous issue date: 2015 / Resumo: As plaquetas são importantes células na inflamação, entretanto, os trabalhos que estudam as citocinas na reatividade plaquetária são raros. O objetivo do presente trabalho foi estudar os efeitos do fator de necrose tumoral-alfa (TNF-?) em plaquetas. Ensaios de agregação foram realizados incubando-se plaquetas com crescentes concentrações de TNF-? (1 - 3000 pg/ml) por diferentes intervalos de tempo (5 - 60 min), na ausência ou presença do antagonista não seletivo dos receptores TNFR1 e TNFR2, o R7050. Também foi estudado o efeito do TNF-? na viabilidade plaquetária utilizando-se o MTT. O efeito do TNF-? na mobilização de Ca2+ em plaquetas foi investigado através de ensaios de fluorescência utilizando-se o fluo-3-AM; os ensaios de western blotting foram realizados para o estudo da ativação da enzima c-Src e do receptor de fibrinogênio. Finalmente, foram determinados os níveis intraplaquetários de AMPc e GMPc por ELISA. O TNF-? inibiu a agregação plaquetária induzida por ADP ou trombina de forma dependente da concentração da citocina e do tempo de incubação. O efeito inibitório máximo do TNF-? na agregação induzida por ADP (5 ?M) foi obtido com a concentração de 300 pg/ml por um tempo de incubação de 30 min (90 ± 7% de inibição), o qual foi significativamente prevenido pela pré-incubação das plaquetas com o R7050. A viabilidade plaquetária não foi modificada pela incubação por 60 min com o TNF-? (30 e 3000 pg/ml). A incubação de plaquetas com TNF-? (300 pg/ml, 30 min) reduziu em 53% o aumento da concentração de Ca2+ total causado pela adição de trombina (200 mU/ml). A queda da concentração de Ca2+ citosólica plaquetária causada pelo TNF-? foi em decorrência da diminuição em 1,8 e 3,4 vezes da mobilização interna do íon e do influxo do mesmo, respectivamente. O TNF-? reduziu em 60% os níveis de AMPc em plaquetas ativadas com ADP. Por outro lado, o TNF-? aumentou significativamente os níveis de GMPc em plaquetas ativadas por ADP (aumento de 51%). A pré-incubação de plaquetas com o inibidor da guanilil ciclase ODQ não reduziu o efeito inibitório do TNF-? na agregação induzida por ADP. Os ensaios de western blotting mostraram que o TNF-? reduziu significativamente a fosforilação do resíduo de Tyr416 da c-Src em plaquetas ativadas. Da mesma forma, o TNF-? reduziu em 37% a fosforilação do resíduo de Tyr773 da subunidade ?3 da integrina ?IIb?3 (receptor de fibrinogênio) em plaquetas ativadas por ADP. Portanto concluímos que o TNF-? inibe a agregação plaquetária via receptores TNFR1 e/ou TNFR2, sem reduzir a viabilidade das plaquetas. O efeito inibitório do TNF-? na agregação é acompanhado pela redução de Ca2+ citosólico e inibição de c-Src e do receptor de fibrinogênio em plaquetas, sendo estes independentes de AMPc ou GMPc / Abstract: Platelets have been described as important cells in inflammation; however, the effects of cytokines on platelet reactivity are rarely studied. The objective of the present work was to investigate the effects of the tumor necrosis factor-alpha (TNF-?) in platelets. Aggregation assays were carried out incubating platelets with increasing TNF-? concentrations (1 - 3000 pg/ml) for different intervals of times (5 - 60 min), in the absence or in presence of the non-selective antagonist of TNFR1 and TNFR2, R7050. Effect of TNF-? on platelet viability was determined using MTT. The effect of TNF-? on the Ca2+ mobilization in platelets was investigated through fluorescence assays using fluo-3AM and Western blotting assays were carried out to determine the activation of c-Src and the fibrinogen receptor. Finally, the cAMP and cGMP levels in platelets were determined by ELISA. TNF-? dose- and time-dependently inhibited ADP or thrombin-induced platelet aggregation. The inhibitory effect of TNF-? on ADP(5 ?M)-induced platelet aggregation was maximum in a concentration of 300 pg/ml incubated with platelets for 30 min (90 ± 7% of inhibition), which was significantly prevented by the incubation of platelets with R7050. Platelet viability was not modified by TNF-? (30 and 3000 pg/ml) incubated for 5 to 60 min. Incubation of platelets with TNF-? (300 pg/ml, 30 min) reduced the increased total Ca2+ concentration induced by thrombin (200 mU/ml) by 53%. Decreasing Ca2+ internal mobilization (1,8 fold) and decreasing in external Ca2+ influx (3,4 fold) led to a reduction of total cytosolic Ca2+ in TNF-? activated platelets. TNF-? reduced the cAMP levels in ADP-activated platelets by 60%. On the other hand, TNF-? significantly increased cGMP levels in ADP-activated platelets (51% increase). Pre-incubation of platelets with the guanylyl cyclase inhibitor ODQ did not modify the inhibitory effect of TNF-? on ADP-induced platelet aggregation. Western blotting analysis showed that TNF-? significantly reduced phosphorylation on Tyr416 of c-Src in activated platelets. Similarly, TNF-? reduced by 37% the Tyr773 phosphorylation of ?3 subunit of ?IIb?3 integrin (fibrinogen receptor) in ADP-activated platelets. Therefore, our results show that TNF-? inhibits platelet aggregation via TNFR1 and/or TNFR2 receptors, without affecting platelet viability. The inhibitory effect of TNF-? on aggregation is accompanied by a reduction in cytosolic Ca2+ and the inhibition of c-Src and fibrinogen receptor activation, which are cAMP and cGMP-independent effects / Mestrado / Farmacologia / Mestre em Farmacologia

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Celiaki (glutenintolerans) är en kronisk, autoimmun sjukdom med diffusa symtom. Vid förtäring av glutenhaltig mat uppstår en allergisk reaktion hos glutenintoleranta individer. Gluten kan inte fullständigt brytas ned av kroppens enzymer, vilket betyder att icke nedbrutna peptidfragment (såsom glutamin) absorberas i tarmslemhinnan. Enzymet transglutaminas katalyserar omvandlingen av glutamin till glutamat. Glutenkänsliga T-celler aktiveras av glutamat att utsöndra proinflammatoriska cytokiner såsom interferon-gamma (IFN-γ) och tumörnekrosfaktor-alfa (TNF-⍺). Syftet med studien var att undersöka om gluten- och gliadinstimulering av celler i helsaliv in vitro kan inducera produktion av IFN-γ och TNF-⍺. Pilotstudier med 2 försökspersoner utfördes, där celler i salivprover stimulerades med enzymatiskt nedbrutet gliadin (<40 mg gliadin), samt PHA (5 µg/mL), PMA (50 ng/mL) och LPS (1 µg/mL) 20 timmar vid 37 ˚C. Cytokinproduktionen i salivproverna kvantifierades med ELISA och uppreglering av IFN-γ och TNF-⍺ undersöktes med RT-qPCR. Efter metodutveckling upprepades stimulering och ELISA med salivprov från 12 försökspersoner (6 individer med och utan celiaki). Immunreaktionen som uppstår hos glutenintoleranta individer in vivo kunde inte återskapas i saliv in vitro med den framtagna metoden. Hos övervägande delen av salivproverna var cytokinproduktionen under detektionsgränsen, 4 pg/mL för IFN-γ och 15,6 pg/mL för TNF-⍺. Det finns risk för att outforskade detaljer eller agens saknades från reaktionskedjan och därmed kunde den förväntade immunreaktionen inte återskapas. En annan felkälla kan vara för låg koncentration av immunceller i saliven. / Celiac disease is a chronic, autoimmune disease that has diffuse symptoms. Upon consuming gluten containing food, an allergic reaction occurs in gluten-sensitive individuals. Gluten cannot be fully digested by human enzymes, which leads to non-digested peptide fragments (such as glutamine) to be absorbed in the gastrointestinal wall. The transglutaminase enzyme catalyzes the conversion of glutamine to glutamate. Glutamate activates gluten-specific T-lymphocytes to produce proinflammatory cytokines e.g., interferon-gamma (IFN-γ) and tumor necrosis factor alpha (TNF-⍺). The aim of this study was to investigate whether stimulation of cells in whole saliva in vitro with gluten and gliadin can induce production of IFN-γ and TNF-⍺. Pilot studies were conducted, where cells in saliva from 2 subjects was stimulated with enzymatically digested gliadin (<40 mg gliadin) together with PHA (5 µg/mL), PMA (50 ng/mL) and LPS (1 µg/mL) for 20 hours at 37 ˚C. The production of cytokines was quantified by ELISA, and the upregulation of IFN-γ and TNF-⍺ was analyzed by RT-qPCR. After method development, the stimulations and ELISA quantifications of the proinflammatory cytokines were repeated in saliva samples from 12 subjects (6 individuals with and without celiac disease). The immune reaction that occurs in people with celiac disease could not be recreated in saliva in vitro with the developed method. In most of the samples the production of cytokines was under the detection range, 4 pg/mL for IFN-γ and 15,6 pg/mL for TNF-⍺. There is risk of unstudied details or agents missing from the reaction chain, and therefore the expected immune reaction could not be recreated. Another source of error could be low concentration of immune cells in saliva.

TNFα-induced programmed necrosis is a caspase-independent cell death program that is contingent upon the formation of a multiprotein complex termed the necrosome. The association of two of the components of the necrosome, receptor interacting protein 1 (RIP1) and RIP3, is a critical and signature molecular event during necrosis. Within this complex, both RIP1 and RIP3 are phosphorylated which are consequential for transmission of the pro-necrotic signal. Namely, it has been demonstrated that RIP3 phosphorylation is required for binding to downstream substrates. Nevertheless, the regulatory mechanisms governing necrosome activation remain unclear. Since necrosis is implicated in a variety of different diseases, understanding the biochemical signaling pathway can potentially yield future drug targets. I was interested in identifying other regulators of necrosis in hope of gaining a better understanding of the necrosis signaling pathway and regulators of the necrosome. To address this, I screened a cancer gene siRNA library in a cell line sensitive to necrosis. From this, I independently identified CYLD as a positive regulator of necrosis. Previous studies suggest that deubiquitination of RIP1 in the TNF receptor (TNFR)-1 signaling complex is a prerequisite for transition of RIP1 into the cytosol and assembly of the RIP1-RIP3 necrosome. The deubiquitinase cylindromatosis (CYLD) is presumed to promote programmed necrosis by facilitating RIP1 deubiquitination in this membrane receptor complex. Surprisingly, I found that TNFα could induce RIP1-dependent necrosis in CYLD-/- cells. I show that CYLD does not regulate RIP1 ubiquitination at the receptor complex. Strikingly, assembly of the RIP1-RIP3 necrosome was delayed, but not abolished in the absence of CYLD. In addition to the TNFR-1 complex, I found that RIP1 within the necrosome was also ubiquitinated. In the absence of CYLD, RIP1 ubiquitination in the NP-40 insoluble necrosome was greatly increased. Increased RIP1 ubiquitination correlated with impaired RIP1 and RIP3 phosphorylation, a signature of kinase activation. My results show that CYLD regulates RIP1 ubiquitination in the NP-40 insoluble necrosome, but not in the TNFR-1 signaling complex. Contrary to the current model, CYLD is not essential for necrosome assembly. Rather, it facilitates RIP1 and RIP3 activation within the necrosome and the corollary is enhancement of necrosome functionality and subsequent necrosis. My results therefore indicate that CYLD exerts its pro-necrotic function in the NP-40 insoluble necrosome, and illuminates the mechanism of necrosome activation.

Studien utvärderar egenskaper hos mesenkymala stromaceller (MSC) i passage 2 och 3. I ett samarbete mellan Cellcolabs och Karolinska Institutet (KI) genomfördes projektet med Katarina Le Blancs forskargrupp. Genom att studera membranmarkörer från tre friska MSC-donatorer, tillsammans med deras förmåga att differentiera till osteoblaster, adipocyter och kondrocyter, samt deras förmåga att inhibera profileringen av CD8+ och CD4+ T-lymfocyter, och slutligen deras möjlighet att öka uttrycket av Indoleamine 2,3-dioxygenase 1 (IDO1) och interleukin 6 (IL6), kunde en jämförelse mellan passage 2 och 3 göras. I korta drag kunde enbart en tydlig skillnad göras mellan de två passagerna. Skillnaden sågs i förmågan att differentiera till osteoblaster, där passage 3 inte kunde prestera på samma nivå som passage 2. Utöver detta var resultaten för de två typerna jämförbara och antydde inte till några större förändringar mellan passage 2 och 3. För att stärka tillförlitligheten av resultatet bör dock fler MSC-donatorer jämföras. / The study evaluates the characteristics and consistency of mesenchymal stromal cells (MSCs) in two different passages, specifically 2 and 3. In a collaboration between Cellcolabs and Karolinska Institutet (KI), the project was conducted with Katarina Le Blanc’s research group. Through studying the surface expression on cells from three distinct MSC donors, along with their differentiation ability into osteoblasts, adipocytes and chondrocytes, their capability to suppress the proliferation of CD8+ and CD4+ T lymphocytes, and finally their possibility to increase the expression of indoleamine 2,3-dioxygenase 1 (IDO1) and interleukin 6 (IL6), a comparison between passage 2 and 3 could be done. It was seen that a clear distinction could be made between the two passages while looking at their ability to differentiate into osteoblasts. The remaining results showed comparable outcomes between passage 2 and 3, suggesting minor changes with the increased passage number. However, to strengthen the reliability of the outcome, more MSC donors should be compared.

Weaning piglets are susceptible to the invasive Streptococcus (S.) suis infection, which can result in septicemia. The aim of this study was to investigate the cytokine profile induced upon S. suis infection of blood, to determine the cellular sources of those cytokines, and to study the potential effects of the induced cytokines on bacterial killing. We measured TNF-α, IL-6, IFN-γ, IL-17A and IL-10 after an experimental intravenous infection with S. suis serotype 2 in vivo, and analyzed whole blood, peripheral blood mononuclear cells (PBMC) and separated leukocytes to identify the cytokine-producing cell type(s). In addition, we used a reconstituted whole blood assay to investigate the effect of TNF-α on bacterial killing in the presence of different S. suis-specific IgG levels. An increase in IL-6 and IL-10, but not in IFN-γ or IL-17A, was observed in two of three piglets with pronounced bacteremia 16 to 20 h after infection, but not in piglets with controlled bacteremia. Our results confirmed previous findings that S. suis induces TNF-α and IL-6 and could demonstrate that TNF-α is produced by monocytes in vitro. We further found that IL-10 induction resulted in reduced secretion of TNF-α and IL-6. Rapid induction of TNF-α was, however, not crucial for in vitro bacterial killing, not even in the absence of specific IgG.

Indiana University-Purdue University Indianapolis (IUPUI) / External Apical Root Resorption (EARR) can be an undesirable side effect of orthodontic treatment. Several studies have already recognized a genetic predisposition to EARR, and some have suggested possible candidate genes that may be involved. The objective of this prospective study was to explore one possible candidate gene that may predispose individuals to EARR during orthodontic treatment. The TNFRSF11A gene encodes the receptor activator of nuclear factor-kappa β (RANK). Together with the RANK ligand, RANK mediates cell signaling that leads to osteoclastogenesis. A diallelic marker was used to investigate the possible relationship between a nonsynonymous TNFRSF11A (RANK) polymorphism and the individuals' development of EARR concurrent with orthodontic treatment. Buccal swab cells of 112 patients who had completed orthodontic treatment were collected for DNA isolation and analysis. EARR of the maxillary central incisors was calculated based on measurements from pre and post treatment occlusal radiographs. Linear regression analysis indicated that length of treatment, overjet, and molar classification are significant predictors of EARR (p=0.05). Other factors, including age, gender, and overbite, were not found to be significantly associated with EARR. An ANOVA was performed to examine the relationship of the genotyped TNFRSF11A marker with the dependent variable EARR. When individuals having at least one copy of allele 2 (1,2 and 2,3 genotypes) were pooled together, a marginally significant association was found between EARR and the marker. Further analysis using logistic regression revealed that individuals with a (1,1) genotype are 4.3 times more likely to be affected by EARR than a person with a (1,2) or (2,2) genotype. From these findings it was concluded that EARR is a complex condition influenced by several treatment variables with the TNFRSF11A gene and its product (RANK) contributing to the individuals' predisposition.

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Background & Aims: Oxidative stress and apoptosis are proposed mechanisms of cellular injury in studies of xenobiotic hepatotoxicity. The aim of this work is to find early signal markers of drug-induced injury of the liver by focusing on select antioxidant/oxidant and apoptotic genes. As well, to address the relationship between conventional liver dysfunction markers and the measured mRNA and protein expressions in the D-galactosamine/lipopolysaccharide and tert-butylhydroperoxide hepatotoxicity models. Furthermore, potential hepatoprotective capabilities of antioxidant polyphenols quercetin and curcumin were evaluated in relation to its modulation of the oxidative stress and apoptotic parameters in the given xenobiotic hepatotoxicity models. Methods: Biochemical markers testing the hepatic function included aminotransferases (ALT, AST) and bilirubin. Measurements of TBARS and conjugated dienes were used to assess lipoperoxidation. Plasma levels of catalase and reduced glutathione were used as indicators of the oxidative status of the cell. Real time PCR was used to analyse the mRNA expressions of the inducible nitric oxide synthase (NOS-2), heme oxygenase-1 (HO-1), superoxide dismutase (SOD-1), glutathione peroxidase (Gpx-1), caspase 3 (Casp3), BH3 interacting domain death agonist (Bid) and Bcl-2...