Functional interferences in host inflammatory immune response by airway allergic inflammation restrain experimental periodontitis development in mice

Aims Periodontal disease (PD) and airway allergic inflammation (AL) present opposing inflammatory immunological features and clinically present an inverse correlation. However, the putative mechanisms underlying such opposite association are unknown. Material and Methods Balb/C mice were submitted to the co-induction of experimental PD (induced by Actinobacillus actinomycetemcomitans oral inoculation) and AL [induced by sensitization with ovalbumin (OVA) and the subsequent OVA challenges], and evaluated regarding PD and AL severity, immune response [cytokine production at periodontal tissues, and T-helper transcription factors in submandibular lymph nodes (LNs)] and infection parameters. Results PD/AL co-induction decreased PD alveolar bone loss and periodontal inflammation while experimental AL parameters were unaltered. An active functional interference was verified, because independent OVA sensitization and challenge not modulate PD outcome. PD+AL group presented decreased tumour necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta, -gamma, IL-17A, receptor activator of nuclear factor kappa-light-chain-enhancer of activated B cells ligand and matrix metalloproteinase (MMP)-13 levels in periodontal tissues, while IL-4 and IL-10 levels were unaltered by AL co-induction. AL co-induction also resulted in upregulated T-bet and related orphan receptor gamma and downregulated GATA3 levels expression in submandibular LNs when compared with PD group. Conclusion Our results demonstrate that the interaction between experimental periodontitis and allergy involves functional immunological interferences, which restrains experimental periodontitis development by means of a skewed immune response.

Involvement of proteinase-activated receptors 1 and 2 in spreading and phagocytosis by murine adherent peritoneal cells: Modulation by the C-terminal of S100A9 protein

Proteinase-activated receptors (PAR) are widely recognized for their modulatory properties in inflammatory and immune responses; however, their direct role on phagocyte effector functions remains unknown. S100A9, a protein secreted during inflammatory responses, deactivates activated peritoneal macrophages, and its C-terminal portion inhibits spreading and phagocytosis of adherent peritoneal cells. Herein, the effect of PAR1 and PAR2 agonists was investigated on spreading and phagocytosis by adherent peritoneal cells, as well as the ability of murine C-terminal of S100A9 peptide (mS100A9p) to modulate this effect. Adherent peritoneal cells obtained from mouse abdominal cavity were incubated with PAR1 and PAR2 agonists and spreading and phagocytosis of Candida albicans particles were evaluated. PAR1 agonists increased both the spreading and the phagocytic activity, but PAR2 agonists only increased the spreading index. mS100A9p reverted both the increased spreading and phagocytosis induced by PAR1 agonists, but no interference in the increased spreading induced by PAR2 agonists was noticed. The shorter homologue peptide to the C-terminal of mS100A9p, corresponding to the H(92)-E(97) region, also reverted the increased spreading and phagocytosis induced by PAR1 agonists. These findings show that proteinase-activated receptors have an important role for spreading and phagocytosis of adherent peritoneal cells, and that the pepticle corresponding to the C-terminal of S100A9 protein is a remarkable candidate for use as a novel compound to modulate PAR1 function. (C) 2009 Elsevier B.V. All rights reserved.

Photoluminescence behavior of Eu(3+) ion doped into gamma- and alpha-alumina systems prepared by combustion, ceramic and Pechini methods

Al(2)O(3):Eu(3+)(1%) samples were prepared by combustion, ceramic, and Pechini methods annealed from 400 to 1400 degrees C. XRD patterns indicate that samples heated up to 1000 degrees C present disordered character of activated alumina (gamma-Al(2)O(3)). However, alpha-Al(2)O(3) phase showed high crystallinity and thermostability at 1200-1400 degrees C. The sample characterizations were also carried out by means of infrared spectroscopy (IR), scanning electron microscopy (SEM) and specific surface areas analysis (BET method). Excitation spectra of Al(2)O(3):Eu(3+) samples present broaden bands attributed to defects of Al(2)O(3) matrices and to LMCT state of O -> Eu(3+), however, the narrow bands are assigned to (7)F(0) -> (5)D(J),(5)H(J) and (5)L(J) transitions of Eu(3+) ion. Emission spectra of samples calcined up to 1000 degrees C show broaden bands for (5)D(0) -> (7)F(J) transitions of Eu(3+) ion suggesting that the rare earth ion is in different symmetry sites showed by inhomogeneous line broadening of bands, confirming the predominance of the gamma-alumina phase. For all samples heated from 1200 to 1400 degrees C the spectra exhibit narrow (5)D(0) -> (7)F(J) transitions of Eu(3+) ion indicating the conversion of gamma to alpha-Al(2)O(3) phases, a high intensity narrow peak around 695 nm assigned to R lines of Cr(3+) ion is shown. Al(2)O(3):Eu(3+) heated up to 1100 degrees C presents an increase in the Omega(2) intensity parameter with the increase of temperatures enhancing the covalent character of metal-donor interaction. The disordered structural systems present the highest values of emission quantum efficiencies (eta). CIE coordinates of Al(2)O(3):Eu(3+) are also discussed. (C) 2007 Elsevier Inc. All rights reserved.

Papel do receptor tipo-2 ativado por protease (PAR-2) na doença periodontal

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito Diabetes mellitus gestacional na modulação de genes relacionados ao metabolismo mitocondrial e a implicação na predisposiçao do recém-nascido à obesidade

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Thrombin-Receptor Antagonist Vorapaxar in Acute Coronary Syndromes

BACKGROUND Vorapaxar is a new oral protease-activated receptor 1 (PAR-1) antagonist that inhibits thrombin-induced platelet activation. METHODS In this multinational, double-blind, randomized trial, we compared vorapaxar with placebo in 12,944 patients who had acute coronary syndromes without ST-segment elevation. The primary end point was a composite of death from cardiovascular causes, myocardial infarction, stroke, recurrent ischemia with rehospitalization, or urgent coronary revascularization. RESULTS Follow-up in the trial was terminated early after a safety review. After a median follow-up of 502 days (interquartile range, 349 to 667), the primary end point occurred in 1031 of 6473 patients receiving vorapaxar versus 1102 of 6471 patients receiving placebo (Kaplan-Meier 2-year rate, 18.5010 vs. 19.9%; hazard ratio, 0.92; 95% confidence interval [CI], 0.85 to 1.01; P=0.07). A composite of death from cardiovascular causes, myocardial infarction, or stroke occurred in 822 patients in the vorapaxar group versus 910 in the placebo group (14.7% and 16.4%, respectively; hazard ratio, 0.89; 95% CI, 0.81 to 0.98; P=0.02). Rates of moderate and severe bleeding were 7.2% in the vorapaxar group and 5.2% in the placebo group (hazard ratio, 1.35; 95% CI, 1.16 to 1.58; P<0.001). Intracranial hemorrhage rates were 1.1% and 0.2%, respectively (hazard ratio, 3.39; 95% CI, 1.78 to 6.45; P<0.001). Rates of nonhemorrhagic adverse events were similar in the two groups. CONCLUSIONS In patients with acute coronary syndromes, the addition of vorapaxar to standard therapy did not significantly reduce the primary composite end point but significantly increased the risk of major bleeding, including intracranial hemorrhage. (Funded by Merck; TRACER ClinicalTrials.gov number, NCT00527943.)

Role of transient receptor potential vanilloid 4 in rat joint inflammation

Objective To determine whether activation of transient receptor potential vanilloid 4 (TRPV-4) induces inflammation in the rat temporomandibular joint (TMJ), and to assess the effects of TRPV-4 agonists and proinflammatory mediators, such as a protease-activated receptor 2 (PAR-2) agonist, on TRPV-4 responses. Methods Four hours after intraarticular injection of carrageenan into the rat joints, expression of TRPV-4 and PAR-2 in trigeminal ganglion (TG) neurons and in the TMJs were evaluated by real-time reverse transcriptionpolymerase chain reaction and immunofluorescence, followed by confocal microscopy. The functionality of TRPV-4 and its sensitization by a PAR-2activating peptide (PAR-2AP) were analyzed by measuring the intracellular Ca2+ concentration in TMJ fibroblast-like synovial cells or TG neurons. Plasma extravasation, myeloperoxidase activity, and the head-withdrawal threshold (index of mechanical allodynia) were evaluated after intraarticular injection of selective TRPV-4 agonists, either injected alone or coinjected with PAR-2AP. Results In the rat TMJs, TRPV-4 and PAR-2 expression levels were up-regulated after the induction of inflammation. Two TRPV-4 agonists specifically activated calcium influx in TMJ fibroblast-like synovial cells or TG neurons. In vivo, the agonists triggered dose-dependent increases in plasma extravasation, myeloperoxidase activity, and mechanical allodynia. In synovial cells or TG neurons, pretreatment with PAR-2AP potentiated a TRPV-4 agonistinduced increase in [Ca2+]i. In addition, TRPV-4 agonistinduced inflammation was potentiated by PAR-2AP in vivo. Conclusion In this rat model, TRPV-4 is expressed and functional in TG neurons and synovial cells, and activation of TRPV-4 in vivo causes inflammation in the TMJ. Proinflammatory mediators, such as PAR-2 agonists, sensitize the activity of TRPV-4. These results identify TRPV-4 as an important signal of inflammation in the joint.

PPAR gamma activation attenuates cold-induced upregulation of thyroid status and brown adipose tissue PGC-1 alpha and D2

Festuccia WT, Blanchard PG, Oliveira TB, Magdalon J, Paschoal VA, Richard D, Deshaies Y. PPAR gamma activation attenuates cold-induced upregulation of thyroid status and brown adipose tissue PGC-1 alpha and D2. Am J Physiol Regul Integr Comp Physiol 303: R1277-R1285, 2012. First published October 24, 2012; doi:10.1152/ajpregu.00299.2012.-Here, we investigated whether pharmacological PPAR gamma activation modulates key early events in brown adipose tissue (BAT) recruitment induced by acute cold exposure with the aim of unraveling the interrelationships between sympathetic and PPAR gamma signaling. Sprague-Dawley rats treated or not with the PPAR gamma ligand rosiglitazone (15 mg.kg(-1).day(-1), 7 days) were kept at 23 degrees C or exposed to cold (5 degrees C) for 24 h and evaluated for BAT gene expression, sympathetic activity, thyroid status, and adrenergic signaling. Rosiglitazone did not affect the reduction in body weight gain and the increase in feed efficiency, VO2, and BAT sympathetic activity induced by 24-h cold exposure. Rosiglitazone strongly attenuated the increase in serum total and free T4 and T3 levels and BAT iodothyronine deiodinase type 2 (D2) and PGC-1 alpha mRNA levels and potentiated the reduction in BAT thyroid hormone receptor (THR) beta mRNA levels induced by cold. Administration of T3 to rosiglitazone-treated rats exacerbated the cold-induced increase in energy expenditure but did not restore a proper activation of D2 and PGC-1 alpha, nor further increased uncoupling protein 1 expression. Regarding adrenergic signaling, rosiglitazone did not affect the changes in BAT cAMP content and PKA activity induced by cold. Rosiglitazone alone or in combination with cold increased CREB binding to DNA, but it markedly reduced the expression of one of its major coactivators, CREB binding protein. In conclusion, pharmacological PPAR gamma activation impairs short-term cold elicitation of BAT adrenergic and thyroid signaling, which may result in abnormal tissue recruitment and thermogenic activity.

Synthesis of high-surface-area gamma-Al2O3 from aluminum scrap and its use for the adsorption of metals: Pb(II), Cd(II) and Zn(II)

Several types of alumina were synthesized from sodium aluminate (NaAlO2) by precipitation with sulfuric acid (H2SO4) and subsequently calcination at 500 degrees C to obtain gamma-Al2O3. The precursor aluminate was derived from aluminum scrap. The various gamma-Al2O3 synthesized were characterized by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), adsorption-desorption of N-2 (S-BET) and scanning electron microscopy (SEM). XRD revealed that distinct phases of Al2O3 were formed during thermal treatment. Moreover, it was observed that conditions of synthesis (pH, aging time and temperature) strongly affect the physicochemical properties of the alumina. A high-surface-area alumina (371 m(2) g(-1)) was synthesized under mild conditions, from inexpensive raw materials. These aluminas were tested for the adsorption of Cd(II), Zn(II) and Pb(II) from aqueous solution at toxic metal concentrations, and isotherms were determined. (C) 2012 Elsevier B.V. All rights reserved.

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor

Convulxin, a powerful platelet activator, was isolated from Crotalus durissus terrificus venom, and 20 amino acid N-terminal sequences of both subunits were determined. These indicated that convulxin belongs to the heterodimeric C-type lectin family. Neither antibodies against GPIb nor echicetin had any effect on convulxin-induced platelet aggregation showing that, in contrast to other venom C-type lectins acting on platelets, GPIb is not involved in convulxin-induced platelet activation. In addition, partially reduced/denatured convulxin only affects collagen-induced platelet aggregation. The mechanism of convulxin-induced platelet activation was examined by platelet aggregation, detection of time-dependent tyrosine phosphorylation of platelet proteins, and binding studies with 125I-convulxin. Convulxin induces signal transduction in part like collagen, involving the time-dependent tyrosine phosphorylation of Fc receptor gamma chain, phospholipase Cgamma2, p72(SYK), c-Cbl, and p36-38. However, unlike collagen, pp125(FAK) and some other bands are not tyrosine-phosphorylated. Convulxin binds to a glycosylated 62-kDa membrane component in platelet lysate and to p62/GPVI immunoprecipitated by human anti-p62/GPVI antibodies. Convulxin subunits inhibit both aggregation and tyrosine phosphorylation in response to collagen. Piceatannol, a tyrosine kinase inhibitor with some specificity for p72(SYK), showed differential effects on collagen and convulxin-stimulated signaling. These results suggest that convulxin uses the p62/GPVI but not the alpha2beta1 part of the collagen signaling pathways to activate platelets. Occupation and clustering of p62/GPVI may activate Src family kinases phosphorylating Fc receptor gamma chain and, by a mechanism previously described in T- and B-cells, activate p72(SYK) that is critical for downstream activation of platelets.

L-amino acid oxidase from Naja atra venom activates and binds to human platelets

An L-amino acid oxidase (LAAO), NA-LAAO, was purified from the venom of Naja atra. Its N-terminal sequence shows great similarity with LAAOs from other snake venoms. NA-LAAO dose-dependently induced aggregation of washed human platelets. However, it had no activity on platelets in platelet-rich plasma. A low concentration of NA-LAAO greatly promoted the effect of hydrogen peroxide, whereas hydrogen peroxide itself had little activation effect on platelets. NA-LAAO induced tyrosine phosphorylation of a number of platelet proteins including Src kinase, spleen tyrosine kinase, and phospholipase Cgamma2. Unlike convulxin, Fc receptor gamma chain and T lymphocyte adapter protein are not phosphorylated in NA-LAAO-activated platelets, suggesting an activation mechanism different from the glycoprotein VI pathway. Catalase inhibited the platelet aggregation and platelet protein phosphorylation induced by NA-LAAO. NA-LAAO bound to fixed platelets as well as to platelet lysates of Western blots. Furthermore, affinity chromatography of platelet proteins on an NA-LAAO-Sepharose 4B column isolated a few platelet membrane proteins, suggesting that binding of NA-LAAO to the platelet membrane might play a role in its action on platelets.

Loss of activator protein-2alpha results in overexpression of the thrombin receptor and correlates with the malignant phenotype of human melanoma

Increasing evidence demonstrates that the thrombin receptor (protease activated receptor-1, PAR-1) plays a major role in tumor invasion and contributes to the metastatic phenotype of human melanoma. We demonstrate that the metastatic potential of human melanoma cells correlates with overexpression of PAR-1. The promoter of the PAR-1 gene contains multiple putative AP-2 and Sp1 consensus elements. We provide evidence that an inverse correlation exists between the expression of AP-2 and the expression of PAR-1 in human melanoma cells. Re-expression of AP-2 in WM266-4 melanoma cells (AP-2 negative) resulted in decreased mRNA and protein expression of PAR-1 and significantly reduced the tumor potential in nude mice. ChIP analysis of the PAR-1 promoter regions bp −365 to −329 (complex 1) and bp −206 to −180 (complex 2) demonstrates that in metastatic cells Sp1 is predominantly binding to the PAR-1 promoter, while in nonmetastatic cells AP-2 is bound. In vitro analysis of complex 1 demonstrates that AP-2 and Sp1 bind to this region in a mutually exclusive manner. Transfection experiments with full-length and progressive deletions of the PAR-1 promoter luciferase constructs demonstrated that metastatic cells had increased promoter activity compared to low and nonmetastatic melanoma cells. Our data shows that exogenous AP-2 expression decreased promoter activity, while transient expression of Sp1 further activated expression of the reporter gene. Mutational analysis of complex 1 within PAR-1 luciferase constructs further demonstrates that the regulation of PAR-1 is mediated through interactions with AP-2 and Sp1. Moreover, loss of AP-2 in metastatic cells alters the AP-2 to Sp1 ratio and DNA-binding activity resulting in overexpression of PAR-1. In addition, we evaluated the expression of AP-2 and PAR-1 utilizing a tissue microarray of 93 melanocytic lesions spanning from benign nevi to melanoma metastasis. We report loss of AP-2 expression in malignant tumors compared to benign tissue while PAR-1 was expressed more often in metastatic melanoma cells than in benign melanocytes. We propose that loss of AP-2 results in increased expression of PAR-1, which in turn results in upregulation of gene products that contribute to the metastatic phenotype of melanoma. ^

STAT3 activation by the ErbB2 receptor and development of peptide-based STAT3 inhibitors

Stats (s&barbelow;ignal t&barbelow;ransducer and a&barbelow;ctivator of t&barbelow;ranscription) are latent transcription factors that translocate from the cytoplasm to nucleus. Constitutive activation of Stat3α by upstream oncoproteins and receptor tyrosine kinases has been found in many human tumors and tumor-derived cell lines and it is often correlated with the activation of ErbB-2. In order to explore the involvement of ErbB-2 in the activation of Stat3 and the mechanisms underlying this event, an erbB-2 point mutant was used as a model of a constitutively activated receptor. Phenylalanine mutations (Y-F) were made in the receptor's autophosphorylation sites and their ability to activate Stat3α was evaluated. Our results suggest that Stat3α and Janus tyrosine kinase 2 associates with ErbB-2 prior to tyrosine phosphorylation of the receptor and that full activation of Stat3α by ErbB-2 requires the participation of other non-receptor tyrosine kinases. Both Src and Jak2 kinases contribute to the activation of Stat3α while only Src binds to ErbB-2 only when the receptor is tyrosine phosphorylated. Our results also suggest that tyrosine 1139 may be important for Src SH2 domain association since a mutant lacking this tyrosine reduces the ability of the Src SH2 domain to bind to ErbB-2 and significantly decreases its ability to activate Stat3α. ^ In order to disrupt aberrant STAT3α activation which contributes to tumorigenesis, we sought small molecules which can specifically bind to the STAT3 SH2 domain, thereby abolishing its ability of being recruited into receptors, and also blocking the dimer formation required for STAT3α activation. A phosphopeptide derived from gp130 was found to have a high affinity to STAT3 SH2 domain, and we decided to use this peptide as the base for further modifications. A series of peptide based compounds were designed and tested using electrophoretic mobility shift assay and fluorescence polarization assay to evaluate their affinity to the STAT3 SH2 domain. Two promising compounds, DRIV-73C and BisPOM, were used for blocking STAT3α activity in cell culture. Either can successfully impair STAT3α activation induced by IL-6 stimulation in HepG2 cells. BisPOM proved to be the more effective in blocking STAT3α tyrosine phosphorylation in induced cells and tumor cell lines, and was the more potent in inhibiting STAT3 dependent cell growth. ^

The role of coactivator associated arginine methyltransferase 1 (CARM1) in nuclear receptor mediated transcription

Arginine methylation has been implicated in the regulation of gene expression. The coactivator-associated arginine methyltransferase 1 (CARMI/PRMT4) binds the p160 family of steroid receptor coactivators (SRCs). This association enhances transcriptional activation by nuclear receptors. Here, we generated and characterized CARM1 knockout mice. Embryos with a targeted disruption of CARM1 are 35% smaller in size than the wild-type littermates and die perinatally. We also generated Carm1-/- and Carm1+/+ mouse embryonic fibroblasts and tested gene expression in response to estrogen. Estrogenresponsive gene expression was aberrant in Carm1-/- fibroblasts and embryos, thus emphasizing the role of arginine methylation as a transcription activation tag. We subsequently studied the role of CARM1 in estrogen signaling in viva in the mammary gland. Conditional knockout of CARM1 in mammary gland and Carml-1-embryonic mammary anlagen transplant experiments did not show any defects in growth and development of the glands. To further dissect the role of CARM1 in estrogen receptor mediated transactivation, we performed cDNA microarray and serial analysis of gene expression on Carm1-/- and Carm1+/+ embryos treated with the estrogen analog, DES. Our results indicate global changes in estrogen regulated genes as well as genes involved in lipid homeostasis. Marker genes for Peroxisome Proliferator Activated Receptor γ (PPARγ) activity, adipsin and aP2, are downregulated in the Carm1-/- embryos. Furthermore, OCT frozen sections of 18.5dpc embryos, processed simultaneously for oil red O staining to look for neutral fat, reveals greatly reduced brown fat accumulation in the Carm1-/- embryos in contrast to wild-type and gain-of-function Carm1 transgenic (ubiquitous) embryo. We used a well-established 3T3-L1 preadipocyte cell line to knockdown CARM1 by short hairpin RNA. 3T3-L1 cells with CARM1 knockdown showed greatly reduced potential to differentiate into mature lipid accumulating adipocytes upon administration of adipogenic stimuli. Ligand-dependent activation of reporter genes by the PPARγ receptor showed that PPRE-luciferase reporter activity was enhanced in the presence of CARM1, additionally, luciferase activity was reduced to background levels when enzyme dead CARM1 (CARM1-VLD) was used. Thus, in this study, we have identified novel pathways that use CARM1 as coactivator and showed that CARM1 functions as a key component of PPARγ receptor mediated gene expression. ^