Xanthine oxidase inhibition by febuxostat attenuates experimental atherosclerosis in mice.

Atherosclerosis is a chronic inflammatory disease due to lipid deposition in the arterial wall. Multiple mechanisms participate in the inflammatory process, including oxidative stress. Xanthine oxidase (XO) is a major source of reactive oxygen species (ROS) and has been linked to the pathogenesis of atherosclerosis, but the underlying mechanisms remain unclear. Here, we show enhanced XO expression in macrophages in the atherosclerotic plaque and in aortic endothelial cells in ApoE(-/-) mice, and that febuxostat, a highly potent XO inhibitor, suppressed plaque formation, reduced arterial ROS levels and improved endothelial dysfunction in ApoE(-/-) mice without affecting plasma cholesterol levels. In vitro, febuxostat inhibited cholesterol crystal-induced ROS formation and inflammatory cytokine release in murine macrophages. These results demonstrate that in the atherosclerotic plaque, XO-mediated ROS formation is pro-inflammatory and XO-inhibition by febuxostat is a potential therapy for atherosclerosis.

Detection of the common acute lymphoblastic leukemia antigen in the serum of leukemia patients.

The common acute lymphoblastic leukemia antigen (CALLA) has been detected in biological fluids using a radioimmunoassay based on the inhibition of binding of 125I-labeled monoclonal anti-CALLA antibody to glutaraldehyde-fixed NALM-1 cells. With this assay, we showed first that CALLA was released in culture fluids from NALM-1 and Daudi cell lines but was absent from culture fluids from CALLA negative cell lines. Then, we found that the sera of 34 out of 42 patients (81%) with untreated common acute lymphoblastic leukemia (c-ALL) contained higher CALLA levels than any of the 42 serum samples from healthy controls. The specificity of these results was further demonstrated by testing in parallel the sera from 48 patients with CALLA negative leukemias, including 26 acute myeloid leukemia (AML), 12 T-cell acute lymphoblastic leukemia (T-ALL), and 10 acute undifferentiated leukemia (AUL). All of these sera gave negative results, except for one patient with AUL, who had a significantly elevated circulating CALLA level, and one patient with AML, who had a borderline CALLA level, 3 SD over the mean of the normal sera. Preliminary results suggest that circulating CALLA is associated with membrane fragments or vesicles, since the total CALLA antigenic activity was recovered in the pellet of the serum samples centrifuged at 100,000 g. In addition, the CALLA-positive pellets contained an enzyme considered as a membrane marker, 5'-nucleotidase. Evaluation of the clinical importance of repeated serum CALLA determinations for the monitoring of c-ALL patients deserves further investigation.

Electro-cortical correlates of motor inhibition : a comparison between selective and non-selective stop tasks

In this study, we compared a selective stop task (transition from a bimanual in-phase to a unimanual index fingers' tapping), with a non-selective stop task (stopping a bimanual in-phase tapping at all), and with a switching task (transition from in-phase to anti-phase bimanual tapping). The aim was twofold: 1) to identify the electro-cortical correlates of selective and non-selective inhibition processes and 2) to investigate which type of inhibition - selective or not - is required when switching between two bimanual motor patterns. The results revealed that all tasks led to enhanced activation (alpha power) of the left sensorimotor and posterior regions which seems to reflect an overall effort to stop the preferred bimanual in-phase tendency. Each task implied specific functional connectivity reorganizations (beta coherence) between cerebral motor areas, probably reflecting engagement in a new unimanual or bimanual movement.

Inhibition of antigen-induced T-cell clone proliferation by antigen-specific antibodies.

Attempts to inhibit the recognition of soluble antigens by T lymphocytes using antibodies specific for the antigen in question have been uniformally unsuccessful, in contrast to the observed specific inhibition of antibody generation by B cells. One exception is the unique situation whereby anti-hapten antisera inhibit the T-cell proliferative responses observed when hapten-specific T lymphocytes or clones are cultured with hapten-derivatized cells or proteins. The inability to inhibit T-cell functions by antigen-specific antibodies has been interpreted in several ways: (1) T cells possess a different repertoire from B cells; (2) the antibodies tested recognize epitopes present on the native antigen, whereas T cells recognize non-native (processed) structures; (3) the antigenic determinant(s) recognized by T cells on the surface of antigen presenting cells are either not accessible to antibodies, or are present in low amounts. The development of antigen-specific T-cell clones and monoclonal antibodies both specific for the same antigenic determinants now allows this question to be investigated definitively. Here, we report for the first time the specific inhibition of antigen-induced T-cell clone proliferation by a monoclonal antibody directed against the relevant soluble protein antigen.

Interleukin-4 and interleukin-5 as targets for the inhibition of eosinophilic inflammation and allergic airways hyperreactivity

Clinical and experimental investigations suggest that allergen-specific CD4+ T-cells, IgE and the cytokines IL-4 and IL-5 play central roles in initiating and sustaining an asthmatic response by regulating the recruitment and/or activation of airways mast cells and eosinophils. IL-5 plays a unique role in eosinophil development and activation and has been strongly implicated in the aetiology of asthma. The present paper summarizes our recent investigations on the role of these cytokines using cytokine knockout mice and a mouse aeroallergen model. Investigations in IL-5-/- mice indicate that this cytokine is critical for regulating aeroallergen-induced eosinophilia, the onset of lung damage and airways hyperreactivity during allergic airways inflammation. While IL-4 and allergen-specific IgE play important roles in the regulation of allergic disease, recent investigations in IL4-/- mice suggest that allergic airways inflammation can occur via pathways which operate independently of these molecules. Activation of these IL-4 independent pathways are also intimately associated with CD4+ T-cells, IL-5 signal transduction and eosinophilic inflammation. Such IL-5 regulated pathways may also play a substantive role in the aetiology of asthma. Thus, evidence is now emerging that allergic airways disease is regulated by humoral and cell mediated processes. The central role of IL-5 in both components of allergic disease highlights the requirements for highly specific therapeutic agents which inhibit the production or action of this cytokine.

Genetic and Antigenic Analysis of Adenovirus Type 3 Strains Showing Intermediate Behavior in Standard Seroneutralization Test

During an epidemiological survey of acute respiratory infection in Rio de Janeiro, among 208 adenovirus isolates, we found two strains that we were not able, by a standard neutralization procedure, to distinguish between type 3 or 7. However, DNA restriction pattern for the two strains with different enzymes were analyzed and showed a typical Ad3h profile. Using a cross-neutralization test in which both Ad3p and Ad7p antisera were used in different concentration against 100 TCID50 of each adenovirus standard and both isolates, we were able to confirm that the two isolates belong to serotype 3. An hemagglutination inhibition test also corroborated the identification of both strains as adenovirus type 3. Comparing Ad3h and Ad3p genome, we observed 16 different restriction enzyme sites, three of which were located in genomic regions encoding polypeptides involved in neutralization sites

Properties of ß-lactamase from Neisseria gonorrhoeae

ß-lactamase activity was studied in Neisseria gonorrhoeae strains. Optimum temperature was found to be 37°C. The enzyme was inactivated at temperatures higher than 60°C, but remained active during storage at low temperatures (4°C, -30°C and -70°C) for two months. Enzyme activity was observed within a pH range of 5.8-8.0, while the optimum pH was 7.0-7.2. Addition of Ni2+, Fe2+, Fe3+, Mn2+ and p-chloromercurybenzoate to the reaction buffer exerted a negative effect upon the activity, whereas Hg2+ and ethylene diamine tetra-acetic acid produced complete inhibition. These results would indicate the presence of -SH groups at the catalytic site of the enzyme.

Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo.

OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.

Cathepsin B-like and cell death in the unicellular human pathogen Leishmania.

In several studies reporting cell death (CD) in lower eukaryotes and in the human protozoan parasite Leishmania, proteolytic activity was revealed using pan-caspase substrates or inhibitors such as carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (Z-VAD-FMK). However, most of the lower eukaryotes do not encode caspase(s) but MCA, which differs from caspase(s) in its substrate specificity and cannot be accountable for the recognition of Z-VAD-FMK. In the present study, we were interested in identifying which enzyme was capturing the Z-VAD substrate. We show that heat shock (HS) induces Leishmania CD and leads to the intracellular binding of Z-VAD-FMK. We excluded binding and inhibition of Z-VAD-FMK to Leishmania major metacaspase (LmjMCA), and identified cysteine proteinase C (LmjCPC), a cathepsin B-like (CPC) enzyme, as the Z-VAD-FMK binding enzyme. We confirmed the specific interaction of Z-VAD-FMK with CPC by showing that Z-VAD binding is absent in a Leishmania mexicana strain in which the cpc gene was deleted. We also show that parasites exposed to various stress conditions release CPC into a soluble fraction. Finally, we confirmed the role of CPC in Leishmania CD by showing that, when exposed to the oxidizing agent hydrogen peroxide (H(2)O(2)), cpc knockout parasites survived better than wild-type parasites (WT). In conclusion, this study identified CPC as the substrate of Z-VAD-FMK in Leishmania and as a potential additional executioner protease in the CD cascade of Leishmania and possibly in other lower eukaryotes.

Inhibition of glial cell proinflammatory activities by peroxisome proliferator-activated receptor gamma agonist confers partial protection during antimyelin oligodendrocyte glycoprotein demyelination in vitro.

Peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a member of the nuclear hormone superfamily originally characterized as a regulator of adipocyte differentiation and lipid metabolism. In addition, PPAR-gamma has important immunomodulatory functions. If the effect of PPAR-gamma's activation in T-cell-mediated demyelination has been recently demonstrated, nothing is known about the role of PPAR-gamma in antibody-induced demyelination in the absence of T-cell interactions and monocyte/macrophage activation. Therefore, we investigated PPAR-gamma's involvement by using an in vitro model of inflammatory demyelination in three-dimensional aggregating rat brain cell cultures. We found that PPAR-gamma was not constitutively expressed in these cultures but was strongly up-regulated following demyelination mediated by antibodies directed against myelin oligodendrocyte glycoprotein (MOG) in the presence of complement. Pioglitazone, a selective PPAR-gamma agonist, partially protected aggregates from anti-MOG demyelination. Heat shock responses and the expression of the proinflammatory cytokine tumor necrosis factor-alpha were diminished by pioglitazone treatment. Therefore, pioglitazone protection seems to be linked to an inhibition of glial cell proinflammatory activities following anti-MOG induced demyelination. We show that PPAR-gamma agonists act not only on T cells but also on antibody-mediated demyelination. This may represent a significant benefit in treating multiple sclerosis patients.

A single mutation in enzyme I of the sugar phosphotransferase system confers penicillin tolerance to Streptococcus gordonii.

Tolerance is a poorly understood phenomenon that allows bacteria exposed to a bactericidal antibiotic to stop their growth and withstand drug-induced killing. This survival ability has been implicated in antibiotic treatment failures. Here, we describe a single nucleotide mutation (tol1) in a tolerant Streptococcus gordonii strain (Tol1) that is sufficient to provide tolerance in vitro and in vivo. It induces a proline-to-arginine substitution (P483R) in the homodimerization interface of enzyme I of the sugar phosphotransferase system, resulting in diminished sugar uptake. In vitro, the susceptible wild-type (WT) and Tol1 cultures lost 4.5 and 0.6 log(10) CFU/ml, respectively, after 24 h of penicillin exposure. The introduction of tol1 into the WT (WT P483R) conferred tolerance (a loss of 0.7 log(10) CFU/ml/24 h), whereas restitution of the parent sequence in Tol1 (Tol1 R483P) restored antibiotic susceptibility. Moreover, penicillin treatment of rats in an experimental model of endocarditis showed a complete inversion in the outcome, with a failure of therapy in rats infected with WT P483R and the complete disappearance of bacteria in animals infected with Tol1 R483P.

c-Jun N-terminal kinase pathway inhibition in intracerebral hemorrhage.

Background: Inhibition of the c-Jun N-terminal kinase (JNK) pathway by the TAT-coupled peptide XG-102 (formerly D-JNKI1) induces strong neuroprotection in ischemic stroke in rodents. We investigated the effect of JNK inhibition in intracerebral hemorrhage (ICH). Methods: Three hours after induction of ICH by intrastriatal collagenase injection in mice, the animals received an intravenous injection of 100 mu g/kg of XG-102. The neurological outcome was assessed daily and the mice were sacrificed at 6 h, 1, 2 or 5 days after ICH. Results: XG-102 administration significantly improved the neurological outcome at 1 day (p < 0.01). The lesion volume was significantly decreased after 2 days (29 +/- 11 vs. 39 +/- 5 mm(3) in vehicle-treated animals, p < 0.05). There was also a decreased hemispheric swelling (14 +/- 13 vs. 26 +/- 9% in vehicle-treated animals, p < 0.05) correlating with increased aquaporin 4 expression. Conclusions: XG-102 attenuates cerebral edema in ICH and functional impairment at early time points. The beneficial effects observed with XG-102 in ICH, as well as in ischemic stroke, open the possibility to rapidly treat stroke patients before imaging, thereby saving precious time.

Effects of a dual inhibitor of angiotensin converting enzyme and neutral endopeptidase, MDL 100,240, on endocrine and renal functions in healthy volunteers.

OBJECTIVE: To investigate the endocrine and renal effects of the dual inhibitor of angiotensin converting enzyme and neutral endopeptidase, MDL 100,240. DESIGN: A randomized, placebo-controlled, crossover study was performed in 12 healthy volunteers. METHODS: MDL 100,240 was administered intravenously over 20 min at single doses of 6.25 and 25 mg in subjects with a sodium intake of 280 (n = 6) or 80 (n = 6) mmol/day. Measurements were taken of supine and standing blood pressure, plasma angiotensin converting enzyme activity, angiotensin II, atrial natriuretic peptide, urinary atrial natriuretic peptide and cyclic GMP excretion, effective renal plasma flow and the glomerular filtration rate as p-aminohippurate and inulin clearances, electrolytes and segmental tubular function by endogenous lithium clearance. RESULTS: Supine systolic blood pressure was consistently decreased by MDL 100,240, particularly after the high dose and during the low-salt intake. Diastolic blood pressure and heart rate did not change. Plasma angiotensin converting enzyme activity decreased rapidly and dose-dependently. In both the high- and the low-salt treatment groups, plasma angiotensin II levels fell and renin activity rose accordingly, while plasma atrial natriuretic peptide levels remained unchanged. In contrast, urinary atrial natriuretic peptide excretion increased dose-dependently under both diets, as did urinary cyclic GMP excretion. Effective renal plasma flow and the glomerular filtration rate did not change. The urinary flow rate increased markedly during the first 2 h following administration of either dose of MDL 100,240 (P < 0.001) and, similarly, sodium excretion tended to increase from 0 to 4 h after the dose (P = 0.07). Potassium excretion remained stable. Proximal and distal fractional sodium reabsorption were not significantly altered by the treatment. Uric acid excretion was increased. The safety and clinical tolerance of MDL 100,240 were good. CONCLUSIONS: The increased fall in blood pressure in normal volunteers together with the preservation of renal hemodynamics and the increased urinary volume, atrial natriuretic peptide and cyclic GMP excretion distinguish MDL 100,240 as a double-enzyme inhibitor from inhibitors of the angiotensin converting enzyme alone. The differences appear to be due, at least in part, to increased renal exposure to atrial natriuretic peptide following neutral endopeptidase blockade.

Oncogenic Ras inhibits Fas ligand-mediated apoptosis by downregulating the expression of Fas.

Tumor growth is the result of deregulated tissue homeostasis which is maintained through the delicate balance of cell growth and apoptosis. One of the most efficient inducers of apoptosis is the death receptor Fas. We report here that oncogenic Ras (H-Ras) downregulates Fas expression and renders cells of fibroblastic and epitheloid origin resistant to Fas ligand-induced apoptosis. In Ras-transformed cells, Fas mRNA is absent. Inhibition of DNA methylation restores Fas expression. H-Ras signals via the PI 3-kinase pathway to downregulate Fas, suggesting that the known anti-apoptotic effect of the downstream PKB/Akt kinase may be mediated, at least in part, by the repression of Fas expression. Thus, the oncogenic potential of H-ras may reside on its capacity not only to promote cellular proliferation, but also to simultaneously inhibit Fas-triggered apoptosis.