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 1 N248S single-nucleotide polymorphism is associated with leprosy risk and regulates immune activation during mycobacterial infection

Conflicting findings about the association between leprosy and TLR1 variants N248S and I602S have been reported. Here, we performed case-control and family based studies, followed by replication in 2 case-control populations from Brazil, involving 3162 individuals. Results indicated an association between TLR1 248S and leprosy in the case-control study (SS genotype odds ratio [OR], 1.81; P = .004) and the family based study (z = 2.02; P = .05). This association was consistently replicated in other populations (combined OR, 1.51; P < .001), corroborating the finding that 248S is a susceptibility factor for leprosy. Additionally, we demonstrated that peripheral blood mononuclear cells (PBMCs) carrying 248S produce a lower tumor necrosis factor/interleukin-10 ratio when stimulated with Mycobacterium leprae but not with lipopolysaccharide or PAM3cysK4. The same effect was observed after infection of PBMCs with the Moreau strain of bacillus Calmette-Guerin but not after infection with other strains. Finally, molecular dynamics simulations indicated that the Toll-like receptor 1 structure containing 248S amino acid is different from the structure containing 248N. Our results suggest that TLR1 248S is associated with an increased risk for leprosy, consistent with its hypoimmune regulatory function.

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.

Effect of neoadjuvant chemotherapy on low-density lipoprotein (LDL) receptor and LDL receptor-related protein 1 (LRP-1) receptor in locally advanced breast cancer

Low-density lipoprotein (LDL) receptors are overexpressed in most neoplastic cell lines and provide a mechanism for the internalization and concentration of drug-laden nanoemulsions that bind to these receptors. The aim of the present study was to determine whether the administration of standard chemotherapeutic schemes can alter the expression of LDL and LDL receptor-related protein 1 (LRP-1) receptors in breast carcinoma. Fragments of tumoral and normal breast tissue from 16 consecutive volunteer women with breast cancer in stage II or III were obtained from biopsies before the beginning of neoadjuvant chemotherapy and after chemotherapy, from fragments excised during mastectomy. Tissues were analyzed by immunohistochemistry for both receptors. Because complete response to treatment was achieved in 4 patients, only the tumors from 12 were analyzed. Before chemotherapy, there was overexpression of LDL receptor in the tumoral tissue compared to normal breast tissue in 8 of these patients. LRP-1 receptor overexpression was observed in tumors of 4 patients. After chemotherapy, expression of both receptors decreased in the tumors of 6 patients, increased in 4 and was unchanged in 2. Nonetheless, even when chemotherapy reduced receptors expression, the expression was still above normal. The fact that chemotherapy does not impair LDL receptors expression supports the use of drug carrier systems that target neoplastic cells by the LDL receptor endocytic pathway in patients on conventional chemotherapy.

Role of soluble triggering receptor expressed on myeloid cells-1 for diagnosing ventilator-associated pneumonia after cardiac surgery: an observational study

Abstract Background The diagnosis of ventilator-associated pneumonia (VAP) is a challenge, particularly after cardiac surgery. The use of biological markers of infection has been suggested to improve the accuracy of VAP diagnosis. We aimed to evaluate the usefulness of soluble triggering receptor expressed on myeloid cells (sTREM)-1 in the diagnosis of VAP following cardiac surgery. Methods This was a prospective observational cohort study of children with congenital heart disease admitted to the pediatric intensive care unit (PICU) after surgery and who remained intubated and mechanically ventilated for at least 24 hours postoperatively. VAP was defined by the 2007 Centers for Disease Control and Prevention criteria. Blood, modified bronchoalveolar lavage (mBAL) fluid and exhaled ventilator condensate (EVC) were collected daily, starting immediately after surgery until the fifth postoperative day or until extubation for measurement of sTREM-1. Results Thirty patients were included, 16 with VAP. Demographic variables, Pediatric Risk of Mortality (PRISM) and Risk Adjustment for Congenital Heart Surgery (RACHS)-1 scores, duration of surgery and length of cardiopulmonary bypass were not significantly diferent in patients with and without VAP. However, time on mechanical ventilation and length of stay in the PICU and in the hospital were significantly longer in the VAP group. Serum and mBAL fluid sTREM-1 concentrations were similar in both groups. In the VAP group, 12 of 16 patients had sTREM-1 detected in EVC, whereas it was undetectable in all but two patients in the non-VAP group over the study period (p = 0.0013) (sensitivity 0.75, specificity 0.86, positive predictive value 0.86, negative predictive value 0.75, positive likelihood ratio (LR) 5.25, negative LR 0.29). Conclusion Measurement of sTREM-1 in EVC may be useful for the diagnosis of VAP after cardiac surgery.

MiRNA-208a and miRNA-208b are triggered in thyroid hormone-induced cardiac hypertrophy - role of type 1 Angiotensin II receptor (AT1R) on miRNA-208a/α-MHC modulation

Hyperthyroidism promotes cardiac hypertrophy and the Angiotensin type 1 receptor (AT1R) has been demonstrated to mediate part of this response. Recent studies have uncovered a potentially important role for the microRNAs (miRNAs) in the control of diverse aspects of cardiac function. Then, the objective of the present study was to investigate the action promoted by hyperthyroidism on β-MHC/miR-208b expression and on α-MHC/miR-208a expression, as well as the possible contribution of the AT1R in this event. The findings of this study confirmed that AT1R is a key mediator of the cardiac hypertrophy induced by hyperthyroidism. Additionally, we demonstrated that like β-MHC, miR-208b was down-regulated in the hyperthyroid group. Similarly, like the expression of its host gene, α-MHC, miR-208a expression was up-regulated in response to hyperthyroidism. Finally, our data suggest for the first time that AT1R mediates the hyperthyroidism-induced increase on cardiac miRNA-208a/α-MHC levels, while does not influence on the reduction of miRNA-208b/β-MHC levels.

Regulación de la proliferación tumoral y la apoptosis en tumores epidermoides de cavidad oral: papel del receptor para el factor de crecimiento insulínico tipo 1 (IGF-1R) y major vault proteína (MVP)

Programa de doctorado: Cáncer: Biología y Clínica

Molecular analysis of Sigma-1 receptor modulation of the dopamine transporter

Sigma (σ) receptors are well established as a non-opioid, non-phencyclidine, and haloperidol-sensitive receptor family with its own binding profile and a characteristic distribution in the central nervous system (CNS) as well as in endocrine, immune, and some peripheral tissues. Two σ receptors subtypes, termed σ1 and σ2, have been pharmacologically characterized, but, to date, only the σ1 has also been cloned. Activation of σ1 receptors alter several neurotransmitter systems and dopamine (DA) neurotrasmission has been often shown to constitute an important target of σ receptors in different experimental models; however the exact role of σ1 receptor in dopaminergic neurotransmission remains unclear. The DA transporter (DAT) modulates the spatial and temporal aspects of dopaminergic synaptic transmission and interprer the primary mechanism by wich dopaminergic neurons terminate the signal transmission. For this reason present studies have been focused in understanding whether, in cell models, the human subtype of σ1 (hσ1) receptor is able to directly modulate the human DA transporter (hDAT). In the first part of this thesis, HEK-293 and SH-SY5Y cells were permanently transfected with the hσ1 receptor. Subsequently, they were transfected with another plasmid for transiently expressing the hDAT. The hDAT activity was estimated using the described [3H]DA uptake assay and the effects of σ ligands were evaluated by measuring the uptaken [3H]DA after treating the cells with known σ agonists and antagonists. Results illustrated in this thesis demonstrate that activation of overexpressed hσ1 receptors by (+)-pentazocine, the σ1 agonist prototype, determines an increase of 40% of the extracellular [3H]DA uptake, in comparison to non-treated controls and the σ1 antagonists BD-1047 and NE-100 prevent the positive effect of (+)-pentazocine on DA reuptake DA is likely to be considered a neurotoxic molecule. In fact, when levels of intracellular DA abnormally invrease, vescicles can’t sequester the DA which is metabolized by MAO (A and B) and COMT with consequent overproduction of oxygen reactive species and toxic catabolites. Stress induced by these molecules leads cells to death. Thus, for the second part of this thesis, experiments have been performed in order to investigate functional alterations caused by the (+)-pentazocine-mediated increase of DA uptake; particularly it has been investigated if the increase of intracellular [DA] could affect cells viability. Results obtained from this study demonstrate that (+)-pentazocine alone increases DA cell toxicity in a concentration-dependent manner only in cells co-expressing hσ1 and hDAT and σ1 antagonists are able to revert the (+)-pentazocine-induced increase of cell susceptibility to DA toxicity. In the last part of this thesis, the functional cross-talking between hσ1 receptor and hDAT has been further investigated using confocal microscopy. From the acquired data it could be suggested that, following exposure to (+)-pentazocine, the hσ1 receptors massively translocate towards the plasma membrane and colocalize with the hDATs. However, any physical interaction between the two proteins remains to be proved. In conclusion, the presented study shows for the first time that, in cell models, hσ1 receptors directly modulate the hDAT activity. Facilitation of DA uptake induced by (+)-pentazocine is reflected on the increased cell susceptibility to DA toxicity; these effects are prevented by σ1 selective antagonists. Since numerous compounds, including several drugs of abuse, bind to σ1 receptors and activating them could facilitate the damage of dopaminergic neurons, the reported protective effect showed by σ1 antagonists would represent the pharmacological basis to test these compounds in experimental models of dopaminergic neurodegenerative diseases (i.e. Parkinson’s Disease).

La molecola IRTA1 (immunoglobulin superfamily receptor trans location associated 1) risulta selettivamente espressa nei linfomi non Hodgkin B della zona marginale

La diagnosi di linfoma non Hodgkin B della zona marginale si basa su criteri morfologici e sulla sostanziale negatività per marcatori immunoistochimici espressi in altri sottotipi di linfoma B. L’ obiettivo di questo lavoro è stato, quindi, quello di ricercare una molecola specifica associata ai linfomi della zona marginale. Materiali e Metodi. Sono stati esaminati 2.104 linfomi periferici di entità nosologia eterogenea mediante un anticorpo monoclonale, diretto contro la molecola IRTA1, che riconosce la zona marginale nei tessuti linfoidi umani. Risultati. Si è riscontrata espressione di IRTA1 nel 93% dei linfomi della zona marginale ad insorgenza extranodale e nel 74% di quelli primitivi linfonodali suggerendo la possibilità che questi linfomi possano originare dalle cellule perifollicolari o monocitoidi IRTA1+ riscontrabili nei linfonodi reattivi. La valutazione immunoistochimica mediante doppia colorazione (IRTA1/bcl6), ha inoltre dimostrato come vi sia una modulazione fenotipica nelle cellule marginali neoplastiche nel momento in cui esse colonizzano i follicoli linfoidi e durante la loro circolazione nei centri germinativi. Le cellule marginali neoplastiche che differenziano in senso plasmacellulare perdono l’ espressione di IRTA1 Discussione. In conclusione, tali evidenze hanno permesso di ampliare la conoscenza sulla biologia dei linfomi marginali e sottolineano come IRTA1 sia il primo marcatore diagnostico positivo per queste neoplasie.

MicroRNAs may mediate the down-regulation of neurokinin-1 receptor in chronic bladder pain syndrome

Bladder pain syndrome (BPS) is a clinical syndrome of pelvic pain and urinary urgency-frequency in the absence of a specific cause. Investigating the expression levels of genes involved in the regulation of epithelial permeability, bladder contractility, and inflammation, we show that neurokinin (NK)1 and NK2 tachykinin receptors were significantly down-regulated in BPS patients. Tight junction proteins zona occludens-1, junctional adherins molecule -1, and occludin were similarly down-regulated, implicating increased urothelial permeability, whereas bradykinin B(1) receptor, cannabinoid receptor CB1 and muscarinic receptors M3-M5 were up-regulated. Using cell-based models, we show that prolonged exposure of NK1R to substance P caused a decrease of NK1R mRNA levels and a concomitant increase of regulatory micro(mi)RNAs miR-449b and miR-500. In the biopsies of BPS patients, the same miRNAs were significantly increased, suggesting that BPS promotes an attenuation of NK1R synthesis via activation of specific miRNAs. We confirm this hypothesis by identifying 31 differentially expressed miRNAs in BPS patients and demonstrate a direct correlation between miR-449b, miR-500, miR-328, and miR-320 and a down-regulation of NK1R mRNA and/or protein levels. Our findings further the knowledge of the molecular mechanisms of BPS, and have relevance for other clinical conditions involving the NK1 receptor.

Glucagonlike peptide-1 receptor: an example of translational research in insulinomas: a review

Glucagonlike peptide-1 receptors (GLP-1R) play an increasingly important role in endocrine gastrointestinal tumor management. In particular, virtually all benign insulinomas express GLP-1R in high density. Exendin-4 is a GLP-1 analog that has a longer half-life than GLP-1. Targeting GLP-1R by (111)In-DOTA-exendin-4 or (111)In-DPTA-exendin-4 offers a new approach that permits the successful localization of small benign insulinomas. It is likely that this new noninvasive technique has the potential to replace the invasive localization by selective arterial stimulation and venous sampling.

Cannabinoid receptor 1 and 2 agonists increase lipid accumulation in hepatocytes

Cannabinoid receptors CB1 and CB2 are expressed in the liver, but their regulation in fatty hepatocytes is poorly documented. The aim of this study was to investigate the effects of selective CB1 or CB2 agonists on the expression of key regulators of lipid metabolism.

Glucagon-like peptide-1 versus somatostatin receptor targeting reveals 2 distinct forms of malignant insulinomas

Glucagon-like peptide-1 (GLP-1) receptor imaging is superior to somatostatin receptor subtype 2 (sst(2)) imaging in localizing benign insulinomas. Here, the role of GLP-1 and sst(2) receptor imaging in the management of malignant insulinoma patients was investigated.

The Ras inhibitors caveolin-1 and docking protein 1 activate peroxisome proliferator-activated receptor ? through spatial relocalization at helix 7 of its ligand-binding domain

Peroxisome proliferator-activated receptor ? (PPAR?) is a transcription factor that promotes differentiation and cell survival in the stomach. PPAR? upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPAR? is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPAR? signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPAR? and enhanced nuclear translocation and ligand-independent transcription of PPAR? target genes. In contrast, Cav1 overexpression sequestered PPAR? in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPAR?'s ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPAR? and to inhibit cell proliferation. Ligand-activated PPAR? also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPAR? regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPAR? to ligands, limiting proliferation of gastric epithelial cells.