The Role of Skin Opioid Receptor System in Itch

Opioid receptors are key players in induction of chronic itch. This could be confirmed using opiate receptor knockout mice experiments and clinical studies on patients with chronic itch. We have induced a dry skin dermatitis as a model for chronic itching on -(MOR) and -(KOR) opioid receptor knockout (KO) mice. MOR KO mice scratched significantly less than wild type (WT). Additionally the epidermal hypertrophy caused by chronic dermatitis and the amount of epidermal nerve endings in MOR KO mice were significantly decreased than in WT mice. KOR KO mice showed similar scratching behavior as MOR KO mice; however the changes were less significant. In addition, we performed a double blind, placebo controlled, cross over study using topically applied opioid receptor antagonist, Naltrexone, on patients with pruritus in atopic dermatitis. The results revealed significant effects of the topical application of Naltrexone in patients with chronic pruritus (45% improvement of pruritus by VAS compared to placebo, n=24), but not in patients with acute pruritus (7%, n=15). These studies establish the clinical relevance of MOR system and the peripheral, epidermal nerve endings in chronic pruritus and warrant further research and therapeutic potential for such research.

Protein-protein interaction investigated by steered molecular dynamics: the TCR-pMHC complex.

We present a novel steered molecular dynamics scheme to induce the dissociation of large protein-protein complexes. We apply this scheme to study the interaction of a T cell receptor (TCR) with a major histocompatibility complex (MHC) presenting a peptide (p). Two TCR-pMHC complexes are considered, which only differ by the mutation of a single amino acid on the peptide; one is a strong agonist that produces T cell activation in vivo, while the other is an antagonist. We investigate the interaction mechanism from a large number of unbinding trajectories by analyzing van der Waals and electrostatic interactions and by computing energy changes in proteins and solvent. In addition, dissociation potentials of mean force are calculated with the Jarzynski identity, using an averaging method developed for our steering scheme. We analyze the convergence of the Jarzynski exponential average, which is hampered by the large amount of dissipative work involved and the complexity of the system. The resulting dissociation free energies largely underestimate experimental values, but the simulations are able to clearly differentiate between wild-type and mutated TCR-pMHC and give insights into the dissociation mechanism.

Antigen-induced pleural eosinophilia is suppressed in diabetic rats: role of corticosteroid hormones

Previous studies have evidenced for the existence of interactive regulatory mechanisms between insulin and steroid hormones in different systems. In this study, we have investigated whether endogenous corticosteroids could be implicated in the hyporeactivity to antigen challenge observed in sensitized diabetic rats. Alloxinated rats showed a long-lasting increase in the blood glucose levels and a reduction in the number of pleural mast cells at 48 and 72 hr, but not at 24 hr after alloxan administration. In parallel, they also showed a significant elevation in the plasma levels of corticosterone together with an increase in the adrenal/body weight ratio. Antigen-evoked eosinophil accumulation appeared significantly reduced in rats pretreated with dexamethasone as well as in those rendered diabetic 72 hr after alloxan. In the same way, naive animals treated with dexamethasone also responded with a significant decrease in the number of pleural mast cells. Interestingly, when sensitized diabetic rats were pretreated with the steroid antagonist RU 38486 a reversion of the reduction in the allergen-induced eosinophil accumulation was noted. We conclude that the down-regulation of the allergic inflammatory response in diabetic rats is close-related to reduction in mast cell numbers and over expression of endogenous corticosteroids.

Noninvasive imaging of 5-HT3 receptor trafficking in live cells: from biosynthesis to endocytosis.

Sequential stages in the life cycle of the ionotropic 5-HT(3) receptor (5-HT(3)R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT(3)R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real-time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT(3)R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT(3) R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT(3) receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT(3) receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.

Effets hémodynamiques d'un inhibiteur de l'effet vasculaire de la vasopressine chez des patients avec insuffisance cardiaque congestive [Hemodynamic effects of an inhibitor of the vascular effects of vasopressin in patients with congestive heart failure]

To assess the role of vasopressin (AVP) in congestive heart failure (CHF), we investigated 10 patients with CHF refractory to conventional treatment, before and 60 minutes after intravenous administration of 5 micrograms/kg of d(CH2)5Tyr(Me)AVP, a specific antagonist of AVP at the vascular receptor level. Heart rate, systemic arterial pressure, pulmonary arterial pressure, pulmonary capillary wedge pressure, cardiac index by thermodilution, and cutaneous blood flow by laser-Doppler technique were measured. In 9 patients there was no significant hemodynamic and cutaneous blood flow response to the AVP antagonist. Plasma AVP was 2.3 +/- 0.8 pg/ml and plasma osmolality 284 +/- 14 mosm/kg H2O. The tenth patient had the most severe CHF. His plasma AVP was 55 pg/ml and plasma osmolality 290 mosm/kg. He responded to the AVP antagonist with a marked decrease in systemic arterial pressure from 115/61 to 79/41 mm Hg, in pulmonary arterial pressure from 58/31 to 33/13 mm Hg and in pulmonary capillary wedge pressure from 28 to 15 mm Hg. Simultaneously cardiac index increased from 1.1 to 2.21 X min-1 X m-2 and cutaneous blood flow rose 5-fold. Thus, most patients with CHF have only moderately elevated plasma AVP and its role in determining peripheral vascular resistance appears to be limited. AVP may become important in rare patients presenting with marked hemodynamic instability and very high plasma AVP.

Octacalcium Phosphate (OCP) Crystals Induce Synovial Inflammation and Cartilage

Introduction: The presence of intra-articular basic calcium phosphate (BCP) crystals, including OCP, carbonated-apatite, hydroxyapatite and tricalcium phosphate crystals, is associated with severe osteoarthritis and destructive arthropathies such as Milwaukee shoulder. Although BCP crystals displayed, in vitro, mitogenic, anabolic and catabolic responses, their intra-articular effect was never assessed.Objective: To determine the effects of OCP crystals in joints in vivo.Methods: OCP crystals (200 ug in 20 ml PBS) were injected into the right knee joint (the contra-lateral knee joint injected with 20 ul of PBS serving as a control) of wild-type mice treated or not by the IL1R antagonist Anakinra or mice deficient for the inflammasome proteins ASC and NALP3. 4 days and 17 days after crystal injection, mice were sacrificed and knee joints dissected. Histological scoring for synovial inflammation and characterisation of macrophages, neutrophils and T cells were performed. Technetium (Tc) uptake was measured at 6h, 1 and 4 days after OCP injection. Cartilage degradation was evaluated by Safranin O staining and VDIPEN immunohistochemistry. Intra-articular localisation of injected OCP crystals was evidenced by Von Kossa staining.Results: The intra-articular localisation of injected OCP crystals was evidenced by Von Kossa staining performed on non-decalcified samples embedded in methyl-metacrylate. Injection of OCP crystals into knee joints led at day 4 to an inflammatory response with intense macrophage staining and also some neutrophil recruitment in the synovial membrane. This synovitis was not accompanied by increased Tc uptake into the knee joint, Tc uptake being similar in OCP crystal injected knee or control knee at all time points investigated (6h, 1 day, 4 days). The histological modifications persisted over 17 days, with an additional fibrosis evidenced at this later time-point. The OCP crystal-induced synovitis was totally IL-1a and IL-1 independent as shown by the absence of inhibitory effects of anakinra injected into wild-type mice. Accordingly, OCP crystal-induced synovitis was similar in ASC-/- and NALP3-/- mice as no alterations of inflammation were demonstrated between these mice groups. Concerning cartilage matrix degradation, OCP crystals induced a strong breakdown of proteoglycans 4 and 17 days after injection, as measured by loss of red staining from Safranin O-stained sections of cartilage surfaces. In addition, we also measured advanced cartilage matrix destruction mediated by MMPs, as evidenced by VDIPEN staining of cartilage. OCP-mediated cartilage degradation was similar in all experimental conditions tested (WT+Anakinra, or ASC or NALP3 deficient mice).Conclusion: These data indicate in vivo that the intra-articular presence of OCP crystals is associated with cartilage destruction along with synovial inflammation. This is an interesting and new model of destructive arthropathy related to BCP crystals which will allow to assess new therapies in this disease.

Hypertension. L'etude ACCOMPLISH: des résultats vraiment inattendus [The ACCOMPLISH trial: are results really unexpected?]

The ACCOMPLISH trial consists of a randomized morbidity-mortality study involving 11506 hypertensive patients at high cardiovascular risk, randomly allocated to a fixed dose combination containing an angiotensin converting enzyme inhibitor (B, benazepril) and either a calcium antagonist (A, amlodipine) or a diuretic (HCTZ, hydrochlorothiazide). The target blood pressure (< 140/90 mmHg) was achieved after a 6 month titration period in 75.4% of patients receiving B+A, versus 72.4% in those on B + HCTZ. Over a mean follow-up of 3 years, the B + A drug regimen was found to reduce significantly more effectively the relative risk cardiovascular mortality (-20%), fatal and non fatal myocardial infarction (-22%) and coronary revascularization (-14%), appearing therefore particularly effective to prevent complications due to myocardial ischemia.

Increased Pro-inflammatory Cytokines (TNF-a and IL-6) and Anti-inflammatory Compounds (sTNFRp55 and sTNFRp75) in Brazilian Patients during Exanthematic Dengue Fever

Pro-inflammatory cytokines, tumor necrosis factor (TNF-a), interleukin-6 (IL-6) and interleukin-1b (IL-1b) as well as anti-inflammatory compounds, soluble TNF-Receptor p55 (sTNFRp55), sTNFRp75 and IL-1 receptor antagonist (sIL-1Ra), were investigated in 34 Brazilian cases of dengue fever (DF) originated from a study of exanthematic virosis. The presence of pro-inflammatory cytokines was detected in sera from these patients by ELISA. TNF-a and IL-6 levels were significantly higher than control subjects in 32% and 52% patients, respectively. To our knowledge this was the first time a receptor antagonist and soluble receptors for cytokines were detected in sera obtained during exanthematic DF without hemorrhagic manifestations. Both sTNFRp55 and sTNFRp75 were consistently elevated in 42% and 84% patients, respectively. Most patients had IL-1b levels not different from those of normal subjects, except for one case. Only 16% patients had altered levels of IL-1Ra. Previous studies in dengue hemorrhagic fever patients demonstrated production of these soluble factors; here we observed that they are found in absence of hemorrhagic manifestations. The possible role of these anti-inflammatory compounds in immune cell activation and in regulating cytokine-mediated pathogenesis during dengue infection is discussed.

5-HT2A and alpha1b-adrenergic receptors entirely mediate dopamine release, locomotor response and behavioural sensitization to opiates and psychostimulants.

Addictive properties of drugs of misuse are generally considered to be mediated by an increased release of dopamine (DA) in the ventral striatum. However, recent experiments indicated an implication of alpha1b-adrenergic receptors in behavioural responses to psychostimulants and opiates. We show now that DA release induced in the ventral striatum by morphine (20 mg/kg) is completely blocked by prazosin (1 mg/kg), an alpha1-adrenergic antagonist. However, morphine-induced increases in DA release in the ventral striatum were found to be similar in mice deleted for the alpha1b-adrenergic receptor (alpha1b-AR KO) and in wild-type (WT) mice, suggesting the presence of a compensatory mechanism. This acute morphine-evoked DA release was completely blocked in alpha1b-AR KO mice by SR46349B (1 mg/kg), a 5-HT2A antagonist. SR46349B also completely blocked, in alpha1b-AR KO mice, the locomotor response and the development of behavioural sensitization to morphine (20 mg/kg) and D-amphetamine (2 mg/kg). Accordingly, the concomitant blockade of 5-HT2A and alpha1b-adrenergic receptors in WT mice entirely blocked acute locomotor responses but also the development of behavioural sensitization to morphine, D-amphetamine or cocaine (10 mg/kg). We observed, nevertheless, that inhibitory effects of each antagonist on locomotor responses to morphine or D-amphetamine were more than additive (160%) in naïve WT mice but not in those sensitized to either drug. Because of these latter data and the possible compensation by 5-HT2A receptors for the genetic deletion of alpha1b-adrenergic receptors, we postulate the existence of a functional link between these receptors, which vanishes during the development of behavioural sensitization.

Delayed mortality and attenuated thrombocytopenia associated with severe malaria in urokinase- and urokinase receptor-deficient mice.

We explored the role of urokinase and tissue-type plasminogen activators (uPA and tPA), as well as the uPA receptor (uPAR; CD87) in mouse severe malaria (SM), using genetically deficient (-/-) mice. The mortality resulting from Plasmodium berghei ANKA infection was delayed in uPA(-/-) and uPAR(-/-) mice but was similar to that of the wild type (+/+) in tPA(-/-) mice. Parasitemia levels were similar in uPA(-/-), uPAR(-/-), and +/+ mice. Production of tumor necrosis factor, as judged from the plasma level and the mRNA levels in brain and lung, was markedly increased by infection in both +/+ and uPAR(-/-) mice. Breakdown of the blood-brain barrier, as evidenced by the leakage of Evans Blue, was similar in +/+ and uPAR(-/-) mice. SM was associated with a profound thrombocytopenia, which was attenuated in uPA(-/-) and uPAR(-/-) mice. Administration of aprotinin, a plasmin antagonist, also delayed mortality and attenuated thrombocytopenia. Platelet trapping in cerebral venules or alveolar capillaries was evident in +/+ mice but absent in uPAR(-/-) mice. In contrast, macrophage sequestration in cerebral venules or alveolar capillaries was evident in both +/+ and uPAR(-/-) mice. Polymorphonuclear leukocyte sequestration in alveolar capillaries was similar in +/+ and uPAR(-/-) mice. These results demonstrate that the uPAR deficiency attenuates the severity of SM, probably by its important role in platelet kinetics and trapping. These results therefore suggest that platelet sequestration contributes to the pathogenesis of SM.

Resolution of foveal detachment in dome-shaped macula after treatment by spironolactone: report of two cases and mini-review of the literature.

Dome-shaped macula (DSM) was recently described in myopic patients as a convex protrusion of the macula within a posterior pole staphyloma. The pathogenesis of DSM and the development of associated serous foveal detachment (SFD) remain unclear. The obstruction of choroidal outflow and compressive changes of choroidal capillaries have been proposed as causative factors. In this paper, we report two cases of patients with chronic SFD associated with DSM treated with oral spironolactone. After treatment, there was a complete resolution of SFD in both patients. To the best of our knowledge, this is the first report of successful treatment of SFD in DSM by a mineralocorticoid receptor antagonist.

Regulation of gastric and pancreatic lipase secretion by CCK and cholinergic mechanisms in humans.

Gastric lipase (HGL) contributes significantly to fat digestion. However, little is known about its neurohormonal regulation in humans. We studied the role of CCK and cholinergic mechanisms in the postprandial regulation of HGL and pancreatic lipase (HPL) secretion in six healthy subjects. Gastric emptying of a mixed meal and outputs of HGL, pepsin, acid, and HPL were determined with a double-indicator technique. Three experiments were performed in random order: intravenous infusion of 1) placebo, 2) low-dose atropine (5 micrograms.kg-.h-1), and 3) the CCK-A receptor antagonist loxiglumide (22 mumol.kg-.h-1). Atropine decreased postprandial outputs of HGL, pepsin, gastric acid, and HPL (P < 0.03) while slowing gastric emptying (P < 0.05). Loxiglumide markedly increased the secretion of HGL, pepsin, and acid while distinctly reducing HPL outputs and accelerating gastric emptying (P < 0.03). Plasma CCK and gastrin levels increased during loxiglumide infusion (P < 0.03). Atropine enhanced gastrin but not CCK release. Postprandial HGL, pepsin, and acid secretion are under positive cholinergic but negative CCK control, whereas HPL is stimulated by cholinergic and CCK mechanisms. We conclude that CCK and cholinergic mechanisms have an important role in the coordination of HGL and HPL secretion to optimize digestion of dietary lipids in humans.

Impaired expression of the inducible cAMP early repressor accounts for sustained adipose CREB activity in obesity.

OBJECTIVEIncrease in adipose cAMP response binding protein (CREB) activity promotes adipocyte dysfunction and systemic insulin resistance in obese mice. This is achieved by increasing the expression of activating transcription factor 3 (ATF3). In this study we investigated whether impaired expression of the inducible cAMP early repressor (ICER), a transcriptional antagonist of CREB, is responsible for the increased CREB activity in adipocytes of obese mice and humans.RESEARCH DESIGN AND METHODSTotal RNA and nuclear proteins were prepared from visceral adipose tissue (VAT) of human nonobese or obese subjects, and white adipose tissue (WAT) of C57Bl6-Rj mice that were fed with normal or high-fat diet for 16 weeks. The expression of genes was monitored by real-time PCR, Western blotting, and electromobility shift assays. RNA interference was used to silence the expression of Icer.RESULTSThe expression of Icer/ICER was reduced in VAT and WAT of obese humans and mice, respectively. Diminution of Icer/ICER was restricted to adipocytes and was accompanied by a rise of Atf3/ATF3 and diminution of Adipoq/ADIPOQ and Glut4/GLUT4. Silencing the expression of Icer in 3T3-L1 adipocytes mimicked the results observed in human and mice cells and hampered glucose uptake, thus confirming the requirement of Icer for appropriate adipocyte function.CONCLUSIONSImpaired expression of ICER contributes to elevation in CREB target genes and, therefore, to the development of insulin resistance in obesity.

A monoclonal antibody which blocks the function of factor D of human complement.

Factor D is an essential enzyme for activation of complement by the alternative pathway (AP). It has been difficult to obtain mouse monoclonal antibodies (Mabs) which block the function of factor D. We have developed a strategy to obtain such Mabs using a double screening procedure of the initial clones. We selected the clone whose supernatant had the lowest level of anti-factor D Ab by ELISA and abolished factor D haemolytic activity. Addition of this Mab to human serum was shown to abolish conversion of C3 by cobra venom factor, haemolysis of rabbit erythrocytes, and activation of C3 and C5 by cuprophane dialysis membranes.

Ampakine CX546 bolsters energetic response of astrocytes: a novel target for cognitive-enhancing drugs acting as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators.

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.