A template-assembled synthetic protein surface mimetic of the von Willebrand factor A1 domain inhibits botrocetin-induced platelet aggregation.

Platelet adhesion, the initial step of platelet activation, is mediated by the interaction of von Willebrand factor (VWF) with its platelet receptor, the GPIb-IX complex. The binding of VWF to GPIb-IX is induced either by increased shear stress or by exogenous modulators, such as botrocetin. At a molecular level, this interaction takes place between the A1 domain of VWF and the GPIb alpha chain of the GPIb-IX complex. We report here the design and functional characteristics of a VWF template-assembled synthetic protein (TASP), a chimeric four-helix-bundle TASP scaffold mimicking the surface of the A1 domain. Twelve residues located on helices alpha 3 and alpha 4 in the native A1 domain were grafted onto a surface formed by two neighboring helices of the TASP. VWF TASP was found to inhibit specifically botrocetin-induced platelet aggregation and to bind both botrocetin and GPIb alpha. However, in contrast to the native A1 domain, VWF TASP did not bind simultaneously to both ligands. Modeling studies revealed that the relative orientation of the alpha helices in VWF TASP led to a clash of bound botrocetin and GPIb alpha. These results demonstrate that a chimeric four-helix-bundle TASP as a scaffold offers a suitable surface for presenting crucial residues of the VWF A1 domain; the potential of the TASP approach for de novo protein design and mimicry is thereby illustrated.

Tumor necrosis factor-alpha and alphaB-crystallin up-regulation during antibody-mediated demyelination in vitro: a putative protective mechanism in oligodendrocytes.

By using an in vitro model of antibody-mediated demyelination, we investigated the relationship between tumor necrosis factor-alpha (TNF-alpha) and heat shock protein (HSP) induction with respect to oligodendrocyte survival. Differentiated aggregate cultures of rat telencephalon were subjected to demyelination by exposure to antibodies against myelin oligodendrocyte glycoprotein (MOG) and complement. Cultures were analyzed 48 hr after exposure. Myelin basic protein (MBP) expression was greatly decreased, but no evidence was found for either necrosis or apoptosis. TNF-alpha was significantly up-regulated. It was localized predominantly in neurons and to a lesser extent in astrocytes and oligodendrocytes, and it was not detectable in microglial cells. Among the different HSPs examined, HSP32 and alphaB-crystallin were up-regulated; they may confer protection from oxidative stress and from apoptotic death, respectively. These results suggest that TNF-alpha, often regarded as a promoter of oligodendroglial death, could alternatively mediate a protective pathway through alphaB-crystallin up-regulation.

Angiotensin-converting enzyme inhibition improves vascular function in rheumatoid arthritis.

BACKGROUND: The excess in cardiovascular risk in patients with rheumatoid arthritis provides a strong rationale for early therapeutical interventions. In view of the similarities between atherosclerosis and rheumatoid arthritis and the proven benefit of angiotensin-converting enzyme inhibitors in atherosclerotic vascular disease, it was the aim of the present study to delineate the impact of ramipril on endothelial function as well as on markers of inflammation and oxidative stress in patients with rheumatoid arthritis. METHODS AND RESULTS: Eleven patients with rheumatoid arthritis were included in this randomized, double-blind, crossover study to receive ramipril in an uptitration design (2.5 to 10 mg) for 8 weeks followed by placebo, or vice versa, on top of standard antiinflammatory therapy. Endothelial function assessed by flow-mediated dilation of the brachial artery, markers of inflammation and oxidative stress, and disease activity were investigated at baseline and after each treatment period. Endothelial function assessed by flow-mediated dilation increased from 2.85+/-1.49% to 4.00+/-1.81% (P=0.017) after 8 weeks of therapy with ramipril but did not change with placebo (from 2.85+/-1.49% to 2.84+/-2.47%; P=0.88). Although systolic blood pressure and heart rate remained unaltered, diastolic blood pressure decreased slightly from 78+/-7 to 74+/-6 mm Hg (P=0.03). Tumor necrosis factor-alpha showed a significant inverse correlation with flow-mediated dilation (r=-0.408, P=0.02), and CD40 significantly decreased after ramipril therapy (P=0.049). CONCLUSIONS: Angiotensin-converting enzyme inhibition with 10 mg/d ramipril for 8 weeks on top of current antiinflammatory treatment markedly improved endothelial function in patients with rheumatoid arthritis. This finding suggests that angiotensin-converting enzyme inhibition may provide a novel strategy to prevent cardiovascular events in these patients.

Diurnal variation in the release of alpha-MSH from rat hypothalamus and pituitary

Release of alpha-MSH from rat hypothalamic slices was characterized with respect to ionic requirements and possible diurnal variations using a sensitive radioimmunoassay. Addition of 47 mM KCl to the superfusion medium resulted in a twofold increase in alpha-MSH functions as a neurotransmitter or neuromodulator in the hypothalamus. Both spontaneous and potassium-induced alpha-MSH release compared to spontaneous release. Removal of calcium from the superfusion medium abolished the potassium-evoked release of alpha-MSH. This supports the concept that alpha-MSH release were related to diurnal variation. Marked release from the slices was observed at 10.10 h, corresponding to a peak in the alpha-MSH concentration in the hypothalamus [18] and to a lower levels of alpha-MSH in the blood. Contrarily, no significant release from the hypothalamus was obtained at 17.00 h when hypothalamic alpha-MSH content was low, but blood levels exhibited a peak. These findings suggest that there are differences in the regulation of the alpha-MSH from the pituitary and that in the hypothalamus.

NSAIDs inhibit alpha V beta 3 integrin-mediated and Cdc42/Rac-dependent endothelial-cell spreading, migration and angiogenesis.

Cyclooxygenase-2 (COX-2), a key enzyme in arachidonic acid metabolism, is overexpressed in many cancers. Inhibition of COX-2 by nonsteroidal anti-inflammatory drugs (NSAIDs) reduces the risk of cancer development in humans and suppresses tumor growth in animal models. The anti-cancer effect of NSAIDs seems to involve suppression of tumor angiogenesis, but the underlying mechanism is not completely understood. Integrin alpha V beta 3 is an adhesion receptor critically involved in mediating tumor angiogenesis. Here we show that inhibition of endothelial-cell COX-2 by NSAIDs suppresses alpha V beta 3-dependent activation of the small GTPases Cdc42 and Rac, resulting in inhibition of endothelial-cell spreading and migration in vitro and suppression of fibroblast growth factor-2-induced angiogenesis in vivo. These results establish a novel functional link between COX-2, integrin alpha V beta 3 and Cdc42-/Rac-dependent endothelial-cell migration. Moreover, they provide a rationale to the understanding of the anti-angiogenic activity of NSAIDs.

Evidence that extrapancreatic GLUT2-dependent glucose sensors control glucagon secretion.

GLUT2-/- mice reexpressing GLUT1 or GLUT2 in their beta-cells (RIPGLUT1 x GLUT2-/- or RIPGLUT2 x GLUT2-/- mice) have nearly normal glucose-stimulated insulin secretion but show high glucagonemia in the fed state. Because this suggested impaired control of glucagon secretion, we set out to directly evaluate the control of glucagonemia by variations in blood glucose concentrations. Using fasted RIPGLUT1 x GLUT2-/- mice, we showed that glucagonemia was no longer increased by hypoglycemic (2.5 mmol/l glucose) clamps or suppressed by hyperglycemic (10 and 20 mmol/l glucose) clamps. However, an increase in plasma glucagon levels was detected when glycemia was decreased to < or =1 mmol/l, indicating preserved glucagon secretory ability, but of reduced sensitivity to glucopenia. To evaluate whether the high-fed glucagonemia could be due to an abnormally increased tone of the autonomic nervous system, fed mutant mice were injected with the ganglionic blockers hexamethonium and chlorisondamine. Both drugs lead to a rapid return of glucagonemia to the levels found in control fed mice. We conclude that 1) in the absence of GLUT2, there is an impaired control of glucagon secretion by low or high glucose; 2) this impaired glucagon secretory activity cannot be due to absence of GLUT2 from alpha-cells because these cells do not normally express this transporter; 3) this dysregulation may be due to inactivation of GLUT2-dependent glucose sensors located outside the endocrine pancreas and controlling glucagon secretion; and 4) because fed hyperglucagonemia is rapidly reversed by ganglionic blockers, this suggests that in the absence of GLUT2, there is an increased activity of the autonomic nervous system stimulating glucagon secretion during the fed state.

Constitutively active mutants of the alpha 2-adrenergic receptor.

We have mutated a single residue, Thr373 [corrected], in the C-terminal portion of the third intracellular loop of the alpha 2C10-adrenergic receptor into five different amino acids. In analogy with the effect of similar mutations in the alpha 1B- and beta 2-adrenergic receptors, these substitutions resulted in two major biochemical modifications: 1) increased constitutive activity of the alpha 2-adrenergic receptor leading to agonist-independent inhibition of adenylyl cyclase and 2) increased affinity of the receptor for binding agonist but not antagonists. The increased constitutive activity of the mutated alpha 2-adrenergic receptors could be inhibited by pertussis toxin, clearly indicating that it results from spontaneous ligand-independent receptor coupling to Gi. In contrast, the increased affinity of the mutant receptors for binding agonists was unaffected by pertussis toxin treatment, indicating that this is an inherent property of the receptors not dependent on interaction with Gi. Coexpression of the receptor mutants with the receptor-specific kinase, beta ARK1, indicated that the constitutively active alpha 2-adrenergic receptors are substrates for beta-adrenergic receptor kinase (beta ARK)-mediated phosphorylation even in the absence of agonist. These findings strengthen the idea that constitutively active adrenergic receptors mimic the "active" state of a G protein-coupled receptor adopting conformations similar to those induced by agonist when it binds to wild type receptors. In addition, these results extend the notion that in the adrenergic receptor family the C-terminal portion of the third intracellular loop plays a general role in the processes involved in receptor activation.

An index of 5-HT synthesis changes during early antidepressant treatment: α-[11C]methyl-l-tryptophan PET study

The antidepressant selective serotonin transporter inhibitors (SSRIs) are clinically active after a delay of several weeks. Indeed, the rapid increase of serotonin (5-HT) caused by SSRIs, stimulates the 5-HT1A autoreceptors, which exert a negative feedback on the 5-HT neurotransmission. Only when autoreceptors are desensitized, can SSRIs exert their therapeutic activity. The 5-HT1A receptor antagonist pindolol has been used to accelerate the clinical effects of antidepressant by preventing the negative feedback. Using the a-[11C]methyl-L-tryptophan/positron emission tomography (PET), the goal of the present double-blind, randomized study was to compare the changes in a-[11C]methyl-L-tryptophan trapping, an index of serotonin synthesis, in patients suffering from unipolar depression treated with the SSRI citalopram (20 mg/day) plus placebo versus patients treated with citalopram plus pindol (7.5 mg/day). PET and Hamilton depression rating scale (HDRS-17) were performed at baseline, and after 10 and 24 days of antidepressant treatment. Results show that the combination citalopram plus pindol, compared to citalopram alone shows a more rapid and greater increase of an index of 5-HT synthesis in prefrontal cortex (BA 9). This research is the first human PET study demonstrating that, after 24 days, the combination SSRIs plus pindolol produces a greater increase of the metabolism of serotonin in the prefrontal cortex, an area associated to depressive symptoms.

Interaction between neuropeptide Y (NPY) and brain-derived neurotrophic factor in NPY-mediated neuroprotection against excitotoxicity : a role for microglia

The neuroprotective effect of neuropeptide Y (NPY) receptor activation was investigated in organotypic mouse hippocampal slice cultures exposed to the glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Exposure of 2-week-old slice cultures, derived from 7-day-old C57BL/6 mice, to 8 microm AMPA, for 24 h, induced degeneration of CA1 and CA3 pyramidal cells, as measured by cellular uptake of propidium iodide (PI). A significant neuroprotection, with a reduction of PI uptake in CA1 and CA3 pyramidal cell layers, was observed after incubation with a Y(2) receptor agonist [NPY(13-36), 300 nm]. This effect was sensitive to the presence of the selective Y(2) receptor antagonist (BIIE0246, 1 microm), but was not affected by addition of TrkB-Fc or by a neutralizing antibody against brain-derived neurotrophic factor (BDNF). Moreover, addition of a Y(1) receptor antagonist (BIBP3226, 1 microm) or a NPY-neutralizing antibody helped to disclose a neuroprotective role of endogenous NPY in CA1 region. Cultures exposed to 8 microm AMPA for 24 h, displayed, as measured by an enzyme-linked immunosorbent assay, a significant increase in BDNF. In such cultures there was an up-regulation of neuronal TrkB immunoreactivity, as well as the presence of BDNF-immunoreactive microglial cells at sites of injury. Thus, an increase of AMPA-receptor mediated neurodegeneration, in the mouse hippocampus, was prevented by neuroprotective pathways activated by NPY receptors (Y(1) and Y(2)), which can be affected by BDNF released by microglia and neurons.

Nitrate reductase activity and spad readings in leaf tissues of guinea grass submitted to nitrogen and potassium rates

Nitrogen and K deficiency are among the most yield limiting factors in Brazilian pastures. The lack of these nutrients can hamper the chlorophyll biosynthesis and N content in plant tissues. A greenhouse experiment was carried out to evaluate the relationship among N and K concentrations, the indirect determination of chlorophyll content (SPAD readings), nitrate reductase activity (RNO3-) in newly expanded leaf lamina (NL) and the dry matter yield for plant tops of Mombaça grass (Panicum maximum Jacq.). A fractionated 5² factorial design was used, with 13 combinations of N and K rates in the nutrient solution. The experimental units were arranged in a randomized block design, with four replications. Plants were harvested twice. The first harvest occurred 36 days after seedling transplanting and the second 29 days after the first. Significance occurred for the interaction between the N and K rates to SPAD readings and to RNO3- assessment taken on the NL during the first growth. Besides, RNO3- and SPAD readings increased only with the NL N concentration, reaching the highest values of both variables up to about 25 g kg-1, but were ratively constant at higher leaf N. Significant relationships either between SPAD readings or RNO3- activity and shoot dry mass weight were also observed. The critical levels of N concentration in the NL were, respectively, 22 and 17g kg-1 in the first and second harvest. Thus, SPAD instrument and RNO3- assessment can be used as complementary tools to evaluate the N status in forage grass.

Anti-TNF-alpha therapy in patients with chronic non-infectious uveitis: the experience of Jules Gonin Eye Hospital.

BACKGROUND: The purpose of this study is to describe the experience of Jules Gonin Eye Hospital on the long-term outcome of anti-TNF-alpha therapy in chronic non-infectious uveitis. PATIENTS AND METHODS: We identified and followed those patients with chronic non-infectious uveitis who received systemic anti-TNF-alpha therapy. Anti-TNF-alpha therapy was administered when no response had been obtained with classical immunosuppressive therapies or in the presence of severe rheumatoid disease. RESULTS: Fifteen patients (28 eyes), 7 male and 8 female (mean age, 43 years; range: 7 to 70 years) were identified. Diagnoses included HLA-B27-associated anterior uveitis (n = 4), sarcoidosis (n = 2), juvenile idiopathic arthritis (n = 2), idiopathic retinal vasculitis with uveitis (n = 2), pars planitis (n = 2), Adamantiades-Behçet disease (n = 1), birdshot retinochoroidopathy (n = 1), and Crohn's disease (n = 1). Mean duration of ocular disease was 8 years (range: 1 to 29 years). Treatment with infliximab (n = 11), etanercept (n = 2), or adalimumab (n = 2) was initiated. One patient with etanercept was switched to infliximab due to lack of clinical response. Clinical and angiographic regression of uveitis was observed within the first two months of therapy in all patients, and was maintained throughout the entire follow-up period (mean 18 months; range: 3 - 72 months). Recurrence was observed in 3 patients, and resolved after adjustment of therapy. Adverse events were recorded in only one patient (arterial hypotension). CONCLUSIONS: In this series of patients with chronic non-infectious uveitis, anti-TNF-alpha therapy was effective and safe. Further clinical studies are needed to determine an adequate duration of therapy.

The cooperative induction of hypoxia-inducible factor-1 alpha and STAT3 during hypoxia induced an impairment of tumor susceptibility to CTL-mediated cell lysis.

Hypoxia is an essential component of tumor microenvironment. In this study, we investigated the influence of hypoxia (1% PO(2)) on CTL-mediated tumor cell lysis. We demonstrate that exposure of target tumor cells to hypoxia has an inhibitory effect on the CTL clone (Heu171)-induced autologous target cell lysis. Such inhibition correlates with hypoxia-inducible factor-1alpha (HIF-1alpha) induction but is not associated with an alteration of CTL reactivity as revealed by granzyme B polarization or morphological change. Western blot analysis indicates that although hypoxia had no effect on p53 accumulation, it induced the phosphorylation of STAT3 in tumor cells by a mechanism at least in part involving vascular endothelial growth factor secretion. We additionally show that a simultaneous nuclear translocation of HIF-1alpha and phospho-STAT3 was observed. Interestingly, gene silencing of STAT3 by small interfering RNA resulted in HIF-1alpha inhibition and a significant restoration of target cell susceptibility to CTL-induced killing under hypoxic conditions by a mechanism involving at least in part down-regulation of AKT phosphorylation. Moreover, knockdown of HIF-1alpha resulted in the restoration of target cell lysis under hypoxic conditions. This was further supported by DNA microarray analysis where STAT3 inhibition resulted in a partly reversal of the hypoxia-induced gene expression profile. The present study demonstrates that the concomitant hypoxic induction of phospho-STAT3 and HIF-1alpha are functionally linked to the alteration of non-small cell lung carcinoma target susceptibility to CTL-mediated killing. Considering the eminent functions of STAT3 and HIF-1alpha in the tumor microenvironment, their targeting may represent novel strategies for immunotherapeutic intervention.

Induction of tolerogenic lung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitor ameliorated experimental allergic asthma.

Signal transducer and activator of transcription (STAT)-3 inhibitors play an important role in regulating immune responses. Galiellalactone (GL) is a fungal secondary metabolite known to interfere with the binding of phosphorylated signal transducer and activator of transcription (pSTAT)-3 as well of pSTAT-6 dimers to their target DNA in vitro. Intra nasal delivery of 50 μg GL into the lung of naive Balb/c mice induced FoxP3 expression locally and IL-10 production and IL-12p40 in RNA expression in the airways in vivo. In a murine model of allergic asthma, GL significantly suppressed the cardinal features of asthma, such as airway hyperresponsiveness, eosinophilia and mucus production, after sensitization and subsequent challenge with ovalbumin (OVA). These changes resulted in induction of IL-12p70 and IL-10 production by lung CD11c(+) dendritic cells (DCs) accompanied by an increase of IL-3 receptor α chain and indoleamine-2,3-dioxygenase expression in these cells. Furthermore, GL inhibited IL-4 production in T-bet-deficient CD4(+) T cells and down-regulated the suppressor of cytokine signaling-3 (SOCS-3), also in the absence of STAT-3 in T cells, in the lung in a murine model of asthma. In addition, we found reduced amounts of pSTAT-5 in the lung of GL-treated mice that correlated with decreased release of IL-2 by lung OVA-specific CD4(+) T cells after treatment with GL in vitro also in the absence of T-bet. Thus, GL treatment in vivo and in vitro emerges as a novel therapeutic approach for allergic asthma by modulating lung DC phenotype and function resulting in a protective response via CD4(+)FoxP3(+) regulatory T cells locally.

Simultaneous quantification of selective serotonin reuptake inhibitors and metabolites in human plasma by liquid chromatography-electrospray mass spectrometry for therapeutic drug monitoring.

A simple and sensitive liquid chromatography-electrospray ionization mass spectrometry method was developed for the simultaneous quantification in human plasma of all selective serotonin reuptake inhibitors (citalopram, fluoxetine, fluvoxamine, paroxetine and sertraline) and their main active metabolites (desmethyl-citalopram and norfluoxetine). A stable isotope-labeled internal standard was used for each analyte to compensate for the global method variability, including extraction and ionization variations. After sample (250μl) pre-treatment with acetonitrile (500μl) to precipitate proteins, a fast solid-phase extraction procedure was performed using mixed mode Oasis MCX 96-well plate. Chromatographic separation was achieved in less than 9.0min on a XBridge C18 column (2.1×100mm; 3.5μm) using a gradient of ammonium acetate (pH 8.1; 50mM) and acetonitrile as mobile phase at a flow rate of 0.3ml/min. The method was fully validated according to Société Française des Sciences et Techniques Pharmaceutiques protocols and the latest Food and Drug Administration guidelines. Six point calibration curves were used to cover a large concentration range of 1-500ng/ml for citalopram, desmethyl-citalopram, paroxetine and sertraline, 1-1000ng/ml for fluoxetine and fluvoxamine, and 2-1000ng/ml for norfluoxetine. Good quantitative performances were achieved in terms of trueness (84.2-109.6%), repeatability (0.9-14.6%) and intermediate precision (1.8-18.0%) in the entire assay range including the lower limit of quantification. Internal standard-normalized matrix effects were lower than 13%. The accuracy profiles (total error) were mainly included in the acceptance limits of ±30% for biological samples. The method was successfully applied for routine therapeutic drug monitoring of more than 1600 patient plasma samples over 9 months. The β-expectation tolerance intervals determined during the validation phase were coherent with the results of quality control samples analyzed during routine use. This method is therefore precise and suitable both for therapeutic drug monitoring and pharmacokinetic studies in most clinical laboratories.