Broadband NIR Emission in Sol-Gel Er(3+)-Activated SiO(2)-Ta(2)O(5) Glass Ceramic Planar and Channel Waveguides for Optical Application

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Investigação de polimorfismos no gene do receptor 2 da interleucina 8 em indivíduos com periodontite

Pós-graduação em Odontologia - FOAR

Graves ophthalmopathy: low-dose glucocorticoid increases peroxisome proliferator-activated receptor-gamma gene expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of Toll-Like Receptor 2 and 4 RNA Expression and the Cytokine Profile in Postmenopausal Women with Metabolic Syndrome

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Porphyromonas gingivalis stimulates bone resorption by enhancing RANKL (Receptor Activator of NF-κB Ligand) through activation of toll-like receptor 2 in osteoblasts

Periodontitis has been associated with rheumatoid arthritis. In experimental arthritis, concomitant periodontitis caused by oral infection with Porphyromonas gingivalis enhances articular bone loss. The aim of this study was to investigate how lipopolysaccharide (LPS) from P. gingivalis stimulates bone resorption. The effects by LPS P. gingivalis and four other TLR2 ligands on bone resorption, osteoclast formation, and gene expression in wild type and Tlr2-deficient mice were assessed in ex vivo cultures of mouse parietal bones and in an in vivo model in which TLR2 agonists were injected subcutaneously over the skull bones. LPS P. gingivalis stimulated mineral release and matrix degradation in the parietal bone organ cultures by increasing differentiation and formation of mature osteoclasts, a response dependent on increased RANKL (receptor activator of NF-κB ligand). LPS P. gingivalis stimulated RANKL in parietal osteoblasts dependent on the presence of TLR2 and through a MyD88 and NF-κB-mediated mechanism. Similarly, the TLR2 agonists HKLM, FSL1, Pam2, and Pam3 stimulated RANKL in osteoblasts and parietal bone resorption. LPS P. gingivalis and Pam2 robustly enhanced osteoclast formation in periosteal/endosteal cell cultures by increasing RANKL. LPS P. gingivalis and Pam2 also up-regulated RANKL and osteoclastic genes in vivo, resulting in an increased number of periosteal osteoclasts and immense bone loss in wild type mice but not in Tlr2-deficient mice. These data demonstrate that LPS P. gingivalis stimulates periosteal osteoclast formation and bone resorption by stimulating RANKL in osteoblasts via TLR2. This effect might be important for periodontal bone loss and for the enhanced bone loss seen in rheumatoid arthritis patients with concomitant periodontal disease.

Evidence of the Importance of the First Intracellular Loop of Prokineticin Receptor 2 in Receptor Function

Prokineticin receptors (PROKR) are G protein-coupled receptors (GPCR) that regulate diverse biological processes, including olfactory bulb neurogenesis and GnRH neuronal migration. Mutations in PROKR2 have been described in patients with varying degrees of GnRH deficiency and are located in diverse functional domains of the receptor. Our goal was to determine whether variants in the first intracellular loop (ICL1) of PROKR2 (R80C, R85C, and R85H) identified in patients with hypogonadotropic hypogonadism interfere with receptor function and to elucidate the mechanisms of these effects. Because of structural homology among GPCR, clarification of the role of ICL1 in PROKR2 activity may contribute to a better understanding of this domain across other GPCR. The effects of the ICL1 PROKR2 mutations on activation of signal transduction pathways, ligand binding, and receptor expression were evaluated. Our results indicated that the R85C and R85H PROKR2 mutations interfere only modestly with receptor function, whereas the R80C PROKR2 mutation leads to a marked reduction in receptor activity. Cotransfection of wild-type (WT) and R80C PROKR2 showed that the R80C mutant could exert a dominant negative effect on WT PROKR2 in vitro by interfering with WT receptor expression. In summary, we have shown the importance of Arg80 in ICL1 for PROKR2 expression and demonstrate that R80C PROKR2 exerts a dominant negative effect on WT PROKR2. (Molecular Endocrinology 26: 1417-1427, 2012)

Toll-like Receptor 2 Knockout Mice Showed Increased Periapical Lesion Size and Osteoclast Number

Introduction: The aim of this study was to characterize the formation and progression of experimentally induced periapical lesions in TLR2 knockout (TLR2 KO) mice. Methods: Periapical lesions were induced in molars of 28 wild type (WT) and 27 TLR2 KO mice. After 7, 21, and 42 days, the animals were euthanized, and the mandibles were subjected to histotechnical processing. Hematoxylin-eosin-stained sections were examined under conventional light microscopy for the description of pulpal, apical, and periapical features and under fluorescence microscopy for the determination of the periapical lesion size. The subsequent sections were evaluated by tartrate resistant acid phosphatase histoenzymology (osteoclasts), Brown and Brenn staining (bacteria), and immunohistochemistry (RANK, RANKL, and OPG). Data were analyzed by the Mann-Whitney U and Kruskal-Wallis tests (alpha = 0.05), Results: The WT group showed significant differences (P < .05) in the periapical lesion size and the osteoclast number between 7 and 42 days and between 21 and 42 days. In the TLR2 KO group, significant differences (P < .05) in the periapical lesion size and the osteoclast number were found between 7 days and the other periods. There was a significant difference (P < .05) between the 2 types of animal regarding the periapical lesion size, which was larger in the TLR2 KO animals. No significant differences (P > .05) were found between WT and TLR2 KO mice related to the pulpal, apical, and periapical features; bacteria localization; and immunohistochemical results (except for RANK expression). Conclusions: TLR2 KO animals developed larger periapical lesions with a greater number of osteoclasts, indicating the important role of this receptor in the host's immune and inflammatory response to root canal and periradicular infection. (J Endod 2012;38:803-813)

Toll-like receptor 2/MyD88 signaling mediates zymosan-induced joint hypernociception in mice: Participation of TNF-alpha, IL-1 beta and CXCL1/KC

Arthritic pain is a serious health problem that affects a large number of patients. Toll-like receptors (TLRs) activation within the joints has been implicated in pathophysiology of arthritis. However, their role in the genesis of arthritic pain needs to be demonstrated. In the present study, it was addressed the participation of TLR2 and TLR4 and their adaptor molecule MyD88 in the genesis of joint hypernociception (a decrease in the nociceptive threshold) during zymosan-induced arthritis. Zymosan injected in the tibio-tarsal joint induced mechanical hypernociception in C57BL/6 wild type mice that was reduced in TLR2 and MyD88 null mice. On the other hand, zymosan-induced hypernociception was similar in C3H/HePas and C3H/Hej mice (TLR4 mutant mice). Zymosan-induced joint hypernociception was also reduced in TNFR1 null mice and in mice treated with IL-1 receptor antagonist or with an antagonist of CXCR1/2. Moreover, the joint production of TNF-alpha, IL-1 beta and CXCL1/KC by zymosan was dependent on TLR2/MyD88 signaling. Investigating the mechanisms by which TNF-alpha, IL-1 beta and CXCL1/KC mediate joint hypernociception, joint administration of these cytokines produced mechanical hypernociception, and they act in an interdependent manner. In last instance, their hypernociceptive effects were dependent on the production of hypernociceptive mediators, prostaglandins and sympathetic amines. These results indicate that in zymosan-induced experimental arthritis, TLR2/MyD88 is involved in the cascade of events of joint hypernociception through a mechanism dependent on cytokines and chemokines production. Thus, TLR2/MyD88 signaling might be a target for the development of novel drugs to control pain in arthritis. (C) 2011 Elsevier B.V. All rights reserved.

Toll-like receptor 2 knockdown modulates Interleukin (IL)-6 and IL-8 but not Stromal Derived Factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Control of Steroid 21-oic Acid Synthesis by Peroxisome Proliferator-activated Receptor and Role of the Hypothalamic-Pituitary-Adrenal Axis

A previous study identified the peroxisome proliferator-activated receptor alpha (PPARalpha) activation biomarkers 21-steroid carboxylic acids 11beta-hydroxy-3,20-dioxopregn-4-en-21-oic acid (HDOPA) and 11beta,20-dihydroxy-3-oxo-pregn-4-en-21-oic acid (DHOPA). In the present study, the molecular mechanism and the metabolic pathway of their production were determined. The PPARalpha-specific time-dependent increases in HDOPA and 20alpha-DHOPA paralleled the development of adrenal cortex hyperplasia, hypercortisolism, and spleen atrophy, which was attenuated in adrenalectomized mice. Wy-14,643 activation of PPARalpha induced hepatic FGF21, which caused increased neuropeptide Y and agouti-related protein mRNAs in the hypothalamus, stimulation of the agouti-related protein/neuropeptide Y neurons, and activation of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in increased adrenal cortex hyperplasia and corticosterone production, revealing a link between PPARalpha and the HPA axis in controlling energy homeostasis and immune regulation. Corticosterone was demonstrated as the precursor of 21-carboxylic acids both in vivo and in vitro. Under PPARalpha activation, the classic reductive metabolic pathway of corticosterone was suppressed, whereas an alternative oxidative pathway was uncovered that leads to the sequential oxidation on carbon 21 resulting in HDOPA. The latter was then reduced to the end product 20alpha-DHOPA. Hepatic cytochromes P450, aldehyde dehydrogenase (ALDH3A2), and 21-hydroxysteroid dehydrogenase (AKR1C18) were found to be involved in this pathway. Activation of PPARalpha resulted in the induction of Aldh3a2 and Akr1c18, both of which were confirmed as target genes through introduction of promoter luciferase reporter constructs into mouse livers in vivo. This study underscores the power of mass spectrometry-based metabolomics combined with genomic and physiologic analyses in identifying downstream metabolic biomarkers and the corresponding upstream molecular mechanisms.

Peroxisome proliferator-activated receptor {gamma} coactivator 1{alpha} (PGC-1{alpha}) promotes skeletal muscle lipid refueling in vivo by activating de novo lipogenesis and the pentose phosphate pathway

Exercise induces a pleiotropic adaptive response in skeletal muscle, largely through peroxisome proliferator-activated receptor coactivator 1 (PGC-1 ). PGC-1 enhances lipid oxidation and thereby provides energy for sustained muscle contraction. Its potential implication in promoting muscle refueling remains unresolved, however. Here, we investigated a possible role of elevated PGC-1 levels in skeletal muscle lipogenesis in vivo and the molecular mechanisms that underlie PGC-1 -mediated de novo lipogenesis. To this end, we studied transgenic mice with physiological overexpression of PGC-1 and human muscle biopsies pre- and post-exercise. We demonstrate that PGC-1 enhances lipogenesis in skeletal muscle through liver X receptor -dependent activation of the fatty acid synthase (FAS) promoter and by increasing FAS activity. Using chromatin immunoprecipitation, we establish a direct interaction between PGC-1 and the liver X receptor-responsive element in the FAS promoter. Moreover, we show for the first time that increased glucose uptake and activation of the pentose phosphate pathway provide substrates for RNA synthesis and cofactors for de novo lipogenesis. Similarly, we observed increased lipogenesis and lipid levels in human muscle biopsies that were obtained post-exercise. Our findings suggest that PGC-1 coordinates lipogenesis, intramyocellular lipid accumulation, and substrate oxidation in exercised skeletal muscle in vivo.

Loss of the C terminus of melanocortin receptor 2 (MC2R) results in impaired cell surface expression and ACTH insensitivity

Mutations in melanocortin receptor 2 (MC2R) and its related melanocortin receptor accessory protein (MRAP) cause familial glucocorticoid deficiency. We identified a novel MC2R mutation, K289fs. This unique mutation in the C terminus of MC2R is located in the intracellular part of the protein for which the exact function is unknown.

Elevated level of metabotropic glutamate receptor 2/3 in the prefrontal cortex in major depression

Clinical, postmortem and preclinical research strongly implicates dysregulation of glutamatergic neurotransmission in major depressive disorder (MDD). Recently, metabotropic glutamate receptors (mGluRs) have been proposed as attractive targets for the discovery of novel therapeutic approaches against depression. The aim of this study was to examine mGluR2/3 protein levels in the prefrontal cortex (PFC) from depressed subjects. In addition, to test whether antidepressants influence mGluR2/3 expression we also studied levels of mGluR2/3 in fluoxetine-treated monkeys. Postmortem human prefrontal samples containing Brodmann's area 10 (BA10) were obtained from 11 depressed and 11 psychiatrically healthy controls. Male rhesus monkeys were treated chronically with fluoxetine (dose escalated to 3mg/kg, p.o.; n=7) or placebo (n=6) for 39 weeks. The mGluR2/3 immunoreactivity was investigated using Western blot method. There was a robust (+67%) increase in the expression of the mGlu2/3 protein in the PFC of depressed subjects relative to healthy controls. The expression of mGlu2/3 was unchanged in the PFC of monkeys treated with fluoxetine. Our findings provide the first evidence that mGluR2/3 is elevated in the PFC in MDD. This observation is consistent with reports showing that mGluR2/3 antagonists exhibit antidepressant-like activity in animal models and demonstrates that these receptors are promising targets for the discovery of novel antidepressants.