Inhibition of natural killer cells by a cytomegalovirus MHC class I homologue in vivo

Herpesviruses, such as murine and human cytomegalovirus (MCMV and HCMV), can establish a persistent infection within the host and have diverse mechanisms as protection from host immune defences'. Several herpesvirus genes that are homologous to host immune modulators have been identified, and are implicated in viral evasion of the host immune response(2,3). The discovery of a viral major histocompatibility complex (MHC) class I homologue, encoded by HCMV(4), led to speculation that it might function as an immune modulator and disrupt presentation of peptides by MHC class I to cytotoxic T cells(5). However, there is no evidence concerning the biological significance of this gene during viral infection. Recent analysis of the MCMV genome has also demonstrated the presence of a MHC class I homologue(6). Here we show that a recombinant MCMV,in which. the gene encoding the class I homologue has been disrupted, has severely restricted replication during the acute stage of infection compared with wild-type MCMV, We demonstrate by in vivo depletion studies that natural killer (NK) cells are responsible for the attenuated phenotype of the mutant. Thus the viral MHC dass I homologue contributes to immune evasion through interference with NK cell-mediated clearance.

Platelet protease-activated receptor 1 and membrane expression of P-selectin in pulmonary arterial hypertension

Introduction: Pulmonary arterial hypertension (PAH) is frequently associated with thrombotic events, particularly involving the pulmonary microcirculation at sites of vascular injury. We therefore decided to analyse protease-activated receptor 1 (PAR1), a key element in the activation of human platelets by thrombin, in PAH patients in stable clinical condition. Methods: Using flow cytometry, we analyzed platelet PAR1 density, PAR1-mediated exposure of P-selectin and the formation of platelet-leukocyte aggregates in 30 PAH patients aged 11 to 78 years (median 50.5 years). The control group consisted of 25 healthy subjects with the same age range as patients. Results: In patients, total platelet PAR1 density and uncleaved PAR1 density correlated negatively with platelet count (r(2) = 0.33 and r(2) = 0.34 respectively, p < 0.0015). In patients with a low platelet count (<150 x 10(9) platelets/L), both densities were increased relative to controls (82% and 33% respectively, p < 0.05). Thrombin peptide-induced platelet exposure of P-selectin was directly related to total and uncleaved PAR1 density (respectively, r(2) = 0.33 and r(2) = 0.29, p < 0.0025) and increased in subjects with low platelet count (46% versus those with normal platelet count, p < 0.05). Patients with low platelet count had decreased in vitro thrombin-induced formation of platelet-leukocyte aggregates (57% decrease versus controls, p < 0.05). Conclusions: There seems to be a subpopulation of PAH patients with increased propensity to thrombotic events as suggested by increased platelet PAR1 expression and PAR-mediated surface exposure of P-selectin associated with decreased platelet count. (C) 2009 Elsevier Ltd. All rights reserved.

Ticagrelor vs. clopidogrel in patients with acute coronary syndromes and diabetes: a substudy from the PLATelet inhibition and patient Outcomes (PLATO) trial

Patients with diabetes mellitus (DM) have high platelet reactivity and are at increased risk of ischaemic events and bleeding post-acute coronary syndromes (ACS). In the PLATelet inhibition and patient Outcomes (PLATO) trial, ticagrelor reduced the primary composite endpoint of cardiovascular death, myocardial infarction, or stroke, but with similar rates of major bleeding compared with clopidogrel. We aimed to investigate the outcome with ticagrelor vs. clopidogrel in patients with DM or poor glycaemic control. We analysed patients with pre-existing DM (n = 4662), including 1036 patients on insulin, those without DM (n = 13 951), and subgroups based on admission levels of haemoglobin A1c (HbA1c; n = 15 150). In patients with DM, the reduction in the primary composite endpoint (HR: 0.88, 95% CI: 0.76-1.03), all-cause mortality (HR: 0.82, 95% CI: 0.66-1.01), and stent thrombosis (HR: 0.65, 95% CI: 0.36-1.17) with no increase in major bleeding (HR: 0.95, 95% CI: 0.81-1.12) with ticagrelor was consistent with the overall cohort and without significant diabetes status-by-treatment interactions. There was no heterogeneity between patients with or without ongoing insulin treatment. Ticagrelor reduced the primary endpoint, all-cause mortality, and stent thrombosis in patients with HbA1c above the median (HR: 0.80, 95% CI: 0.70-0.91; HR: 0.78, 95% CI: 0.65-0.93; and HR: 0.62, 95% CI: 0.39-1.00, respectively) with similar bleeding rates (HR: 0.98, 95% CI: 0.86-1.12). Ticagrelor, when compared with clopidogrel, reduced ischaemic events in ACS patients irrespective of diabetic status and glycaemic control, without an increase in major bleeding events.

The effect of the aged garlic extract, 'Kyolic', on the development of experimental atherosclerosis

The aged garlic extract 'Kyolic' lowers serum cholesterol levels in humans and experimental animals and thus is presumed to have a protective effect against atherosclerosis. However, to date no studies have examined the effect of this substance on the actual development of the disease. In the present study, the right carotid artery of 24 rabbits was de-endothelialized by balloon catheterisation in order to produce a myointimal thickening. After 2 weeks the rabbits were randomly assigned to four groups: Group I received a standard diet; Group II received the standard diet supplemented with 800 mu 1/kg body weight/day 'Kyolic'; Group III received a 1% cholesterol supplemented standard diet; and Group IV received a 1% cholesterol supplemented standard diet plus 'Kyolic'. After 6 weeks, the cholesterol diet caused a 6-fold increase in serum cholesterol level (Group III; 6.4 +/- 0.6 mmol/1) compared to normal diet (Group I; 1.2 +/- 0.4 mmol/1) (P < 0.05) with only a minor, non-significant reduction seen by the addition of 'Kyolic' (Group IV; 6.2 +/- 0.7 mmol/l). Group III rabbits developed fatty streak lesions covering approximately 70 +/- 8% of the surface area of the thoracic aorta, which was significantly reduced to 25 +/- 3% in the 'Kyolic'-treated Group IV. No lesions were present in Groups I and II. The hypercholesterolaemic diet caused an increase in aortic arch cholesterol (2.1 +/- 0.1 mg cholesterol/g tissue) which was significantly reduced by 'Kyolic' supplementation (1.7 +/- 0.2 mg cholesterol/g tissue) (P < 0.05). 'Kyolic' significantly inhibited the development of thickened, lipid-filled lesions in the pre-formed neointimas produced by balloon-catheter injury of the right carotid artery in cholesterol-fed rabbits (intima as percent of artery wall, Group III 42.6 +/- 6.5% versus Group IV 23.8 +/- 2.3%, P < 0.01), but had little effect in rabbits on a standard diet (Group II 18.4 +/- 5.0% versus Group I 16.7 +/- 2.0%). In vitro studies showed that 'Kyolic' has a direct effect on inhibition of smooth muscle proliferation. In conclusion,'Kyolic' treatment reduces fatty streak development, vessel wall cholesterol accumulation and the development of fibro fatty plaques in neointimas of cholesterol-fed rabbits, thus providing protection against the onset of atherosclerosis. (C) 1997 Elsevier Science Ireland Ltd.

Inhibition of angiotensin II receptor 1 limits tumor-associated angiogenesis and attenuates growth of murine melanoma

Purpose We evaluated the involvement of angiotensin II (AngII)-dependent pathways in melanoma growth, through the pharmacological blockage of AT1 receptor by the antihypertensive drug losartan (LOS). Results We showed immunolabeling for both AngII and the AT1 receptor within the human melanoma microenvironment. Like human melanomas, we showed that murine melanomas also express the AT1 receptor. Growth of murine melanoma, both locally and at distant sites, was limited in mice treated with LOS. The reduction in tumor growth was accompanied by a twofold decrease in tumorassociated microvessel density and by a decrease in CD31 mRNA levels. While no differences were found in the VEGF expression levels in tumors from treated animals, reduction in the expression of the VEGFR1 (Flt-1) at the mRNA and protein levels was observed. We also showed downregulation of mRNA levels of both Flt-4 and its ligand, VEGF-C. Conclusions Together, these results show that blockage of AT1 receptor signaling may be a promising anti-tumor strategy, interfering with angiogenesis by decreasing the expression of angiogenic factor receptors.

Prion protein ablation increases cellular aggregation and embolization contributing to mechanisms of metastasis

Cellular Prion Protein (PrP(C)) is a cell surface protein highly expressed in the nervous system, and to a lesser extent in other tissues. PrP(C) binds to the extracellular matrix laminin and vitronectin, to mediate cell adhesion and differentiation. Herein, we investigate how PrP(C) expression modulates the aggressiveness of transformed cells. Mesenchymal embryonic cells (MEC) from wildtype (Prnp(+/+)) and PrP(C)-null (Prnp(0/0)) mice were immortalized and transformed by co-expression of ras and myc. These cells presented similar growth rates and tumor formation in vivo. When injected in the tail vein, PrnP(0/0)raS/myc cells exhibited increased lung colonization compared with Prnp(+/+)ras/myc cells. Additionally, Prnp(0/0)ras/myc cells form more aggregates with blood components than Prnp(+/+)ras/myc cells, facilitating the arrest of Prnp(0/0)ras/myc cells in the lung vasculature. Integrin alpha(v)beta(3) is more expressed and activated in MEC and in transformed Prnp(0/0) cells than in the respective Prnp(+/+) cells. The blocking of integrin alpha(v)beta(3) by RGD peptide reduces lung colonization in transformed Prnp(0/0) cells to similar levels of those presented by transformed Prnp(+/+) cells. Our data indicate that PrP(C) negatively modulates the expression and activation of integrin alpha(v)beta(3) resulting in a more aggressive phenotype. These results indicate that PrP(C) may have main implications in modulating metastasis formation. (C) 2009 UICC

Conditioned inhibition of autonomic Pavlovian conditioning in humans

The present study aimed to demonstrate conditioned inhibition of Pavlovian conditioning of autonomic responses in humans. Subjects (N = 21) were presented initially with four geometric shapes (A, B, C and D). An electric shock served as the unconditioned stimulus (US) during acquisition. Conditional stimuli lasted for 8 s and US onset coincided with CS offset. Subjects were trained with A-US, C-US, and AC-US pairings and AB alone and B alone presentations. The subsequent summation test consisted of C-US pairings and CB alone and CD alone presentations. Conditioning was evident in self-reported US expectancy and first and second interval electrodermal responses. Evidence for conditioned inhibition during the summation test was found in US expectancy and second interval electrodermal responses. (C) 1997 Elsevier Science B.V.

Latent inhibition and autonomic responses: a psychophysiological approach

Latent inhibition, retarded learning after preexposure to the to-be-conditioned stimulus, has been implied as a tool for the investigation of attentional deficits in schizophrenia and related disorders. The present paper reviews research that used Pavlovian conditioning as indexed by autonomic responses (electrodermal, vasomotor, cardiac) to investigate latent inhibition in adult humans. Latent inhibition has been demonstrated repeatedly in healthy subjects in absence of a masking task that is required in other latent inhibition paradigms. Moreover, latent inhibition of Pavlovian conditioning is stimulus-specific and increases with an increased number of preexposure trials which mirrors results from research in animals. A reduction of latent inhibition has been shown in healthy subjects who score high on questionnaire measures of psychosis proneness and in unmedicated schizophrenic patients. The latter result was obtained in a within-subject paradigm that holds promise for research with patient samples. (C) 1997 Elsevier Science B.V.

Inhibition of phospholipase A(2) increases Tau phosphorylation at Ser214 in embryonic rat hippocampal neurons

Background: Arachidonic acid is released from cellular membranes by the action of phospholipase A(2) (PLA(2)) and is implicated in microtubule-associated protein Tau phosphorylation. Tau hyperphosphorylation affects its ability to stabilize microtubules. Objective: To determine the effect of PLA(2) inhibition on the phosphorylation state of Tau phosphoepitopes in primary cultures of hippocampal neurons. Methods: 4 DIC neurons were incubated at different concentrations of methyl-arachidonylfluorophosphonate (MAFP), an irreversible inhibitor of cPLA(2) and iPLA(2). Changes on Tau phosphorylation were determined by Western blotting with a panel of anti-Tau antibodies (C-terminal, Ser199/202, Ser202/205, Ser396 and Ser214). Results: The Ser214 site was hyperphosphorylated upon MAFP treatment. Significant differences were observed with 10 mu M (p = 0.01), 50 mu M (p = 0.01) and 100 mu M (p = 0.05) of MAFP. Less-intense changes were found in other phosphoepitopes. Conclusion: The present findings indicate that the phosphorylation of Ser214 is regulated by c- and/or iPLA(2), whereas other phosphoepitopes primarily regulated by GKS3b were not affected. (C) 2009 Elsevier Ltd. All rights reserved.

Inducible nitric oxide synthase inhibition attenuates lung tissue responsiveness and remodeling in a model of chronic pulmonary inflammation in guinea pigs

We evaluated the influence of iNOS-derived NO on the mechanics, inflammatory, and remodeling process in peripheral lung parenchyma of guinea pigs with chronic pulmonary allergic inflammation. Animals treated or not with 1400W were submitted to seven exposures of ovalbumin in increasing doses. Seventy-two hours after the 7th inhalation, lung strips were suspended in a Krebs organ bath, and tissue resistance and elastance measured at baseline and after ovalbumin challenge. The strips were submitted to histopathological measurements. The ovalbumin-exposed animals showed increased maximal responses of resistance and elastance (p < 0.05), eosinophils counting (p < 0.001), iNOS-positive cells (p < 0.001), collagen and elastic fiber deposition (p < 0.05), actin density (p < 0.05) and 8-iso-PGF2 alpha expression (p < 0.001) in alveolar septa compared to saline-exposed ones. Ovalbumin-exposed animals treated with 1400 W had a significant reduction in lung functional and histopathological findings (p < 0.05). We showed that iNOS-specific inhibition attenuates lung parenchyma constriction, inflammation, and remodeling, suggesting NO-participation in the modulation of the oxidative stress pathway. (C) 2008 Elsevier B.V. All rights reserved.

Effects of inducible nitric oxide synthase inhibition in bronchial vascular remodeling-induced by chronic allergic pulmonary inflammation

Vascular remodeling is an important feature in asthma pathophysiology. Although investigations suggested that nitric oxide (NO) is involved in lung remodeling, little evidence established the role of inducible NO synthase (iNOS) isoform in bronchial vascular remodeling. The authors investigated if iNOS contribute to bronchial vascular remodeling induced by chronic allergic pulmonary inflammation. Guinea pigs were submitted to ovalbumin exposures with increasing doses (1 similar to 5 mg/mL) for 4 weeks. Animals received 1400W (iNOS-specific inhibitor) treatment for 4 days beginning at 7th inhalation. Seventy-two hours after the 7th inhalation, animals were anesthetized, mechanical ventilated, exhaled NO was collected, and lungs were removed and submitted to picrosirius and resorcin-fuchsin stains and to immunohistochemistry for matrix metalloproteinase-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), and transforming growth factor-beta (TGF-beta). Collagen and elastic fiber deposition as well as MMP-9, TIMP-1, and TGF-beta expression were increase in bronchial vascular wall in ovalbumin-exposed animals. The iNOS inhibition reduced all parameters studied. In this model, iNOS inhibition reduced the bronchial vascular extracellular remodeling, particularly controlling the collagen and elastic fibers deposition in pulmonary vessels. This effect can be associated to a reduction on TGF-beta and on metalloproteinase-9/TIMP-1 vascular expression. It reveals new therapeutic strategies and some possible mechanism related to specific iNOS inhibition to control vascular remodeling.

CpG-Induced Th1-Type Response in the Downmodulation of Early Development of Allergy and Inhibition of B7 Expression on T Cells of Newborn Mice

Several differences have been described between neonatal and adult immune responses. The predisposition in early life to Th2-type response or tolerance makes it a susceptible period for infections and allergic sensitization. The aim of this work was to evaluate the effects of CpG-containing oligodeoxynucleotides on neonatal and adult immunization with ovalbumin and Blomia tropicalis extract and compare the CpG effects on B and T cells of neonatal and adult mice. Mice that received CpG showed reduced immunoglobulin E (IgE) antibody production in both neonatal and adult periods, in parallel to increased IgG2a antibody levels. We observed that spleen cells of mice that received CpG in early life produced increased amounts of interferon-gamma upon anti-CD3 stimulation. Negative regulation of IgE response was more pronounced in adult than neonate mice; further, CpG decreased anaphylactic antiovalbumin IgG1 only in adults. Also, an upregulation of toll-like receptor 9 expression was detected in adult B cells, but not in neonatal, upon CpG stimuli. Neonatal B cells showed enhanced interleukin (IL)-10 expression and decreased IL-6 levels than adult B cells in response to CpG. When we analyzed in vitro activation of CD4+ T cells, an increased expression of B7 molecules on T cells in neonates was suppressed by CpG. Altogether, we verified qualitative and quantitative evidences regarding CpG effect on neonatal and adult allergens immunizations, which points to the importance of understanding neonatal immune system to establish immunomodulatory strategies for prevention of allergic diseases.

Anisotropic Reversible Aggregation of Latex Nanoparticles Suspended in a Lyotropic Nematic Liquid Crystal: Effect of Gradients of Biaxial Order

We studied the anisotropic aggregation of spherical latex particles dispersed in a lyotropic liquid crystal presenting three nematic phases; calamitic, biaxial, and discotic. We observed that in the nematic calamitic phase aggregates of latex particles are formed, which become larger and anisotropic in the vicinity of the transition to the discotic phase, due to a coalescence process. Such aggregates are weakly anisotropic and up to 50 mu m long and tend to align parallel to the director field. At the transition to the discotic phase, the aggregates dissociated and re-formed when the system was brought back to the calamitic phase. This shows that the aggregation is due to attractive and repulsive forces generated by the particular structure of the nematic phase. The surface-induced positional order was investigated by surface force apparatus experiments with the lyotropic system confined between mica surfaces, revealing the existence of a presmectic wetting layer around the surfaces and oscillating forces of increasing amplitude as the confinement thickness was decreased. We discuss the possible mechanisms responsible for the reversible aggregation of latex particles, and we propose that capillary condensation of the N(C) phase, induced by the confinement between the particles, could reduce or remove the gradient of order parameter, driving the transition of aggregates from solidlike to liquidlike and gaslike.

Inhibition of phospholipase A(2) in rat brain decreases the levels of total Tau protein

The microtubule-associated protein Tau promotes the assembly and stability of microtubules in neuronal cells. Six Tau isoforms are expressed in adult human brain. All six isoforms become abnormally hyperphosphorylated and form neurofibrillary tangles in Alzheimer disease (AD) brains. In AD, reduced activity of phospholipase A(2) (PLA(2)), specifically of calcium-dependent cytosolic PLA(2) (cPLA(2)) and calcium-independent intracellular PLA(2) (iPLA(2)), was reported in the cerebral cortex and hippocampus, which positively correlated with the density of neurofibrillary tangles. We previously demonstrated that treatment of cultured neurons with a dual cPLA(2) and iPLA(2) inhibitor, methyl arachidonyl fluorophosphonate (MAFP), decreased total Tau levels and increased Tau phosphorylation at Ser(214) site. The aim of this study was to conduct a preliminary investigation into the effects of in vivo infusion of MAFP into rat brain on PLA(2) activity and total Tau levels in the postmortem frontal cortex and dorsal hippocampus. PLA(2) activity was measured by radioenzymatic assay and Tau levels were determined by Western blotting using the anti-Tau 6 isoforms antibody. MAFP significantly inhibited PLA(2) activity in the frontal cortex and hippocampus. The reactivity to the antibody revealed three Tau protein bands with apparent molecular weight of close to 40, 43 and 46 kDa in both brain areas. MAFP decreased the 46 kDa band intensity in the frontal cortex, and the 43 and 46 kDa band intensities in the hippocampus. The results indicate that in vivo PLA(2) inhibition in rat brain decreases the levels of total (nonphosphorylated plus phosphorylated) Tau protein and corroborate our previous in vitro findings.

Inhibition of motor cortex excitability with 15 Hz transcranial alternating current stimulation (tACS)

There remains a lack of solid evidence showing whether transcranial stimulation with weak alternating current (transcranial alternating current stimulation, tACS) can in fact induce significant neurophysiological effects. Previously, a study in which tACS was applied for 2 and 5 min with current density = 0.16-0.25 A/m(2) was unable to show robust effects on cortical excitability. Here we applied tACS at a significantly higher current density (0.80 A/m(2)) for a considerably longer duration (20 min) and were indeed able to demonstrate measurable changes to cortical excitability. Our results show that active 15 Hz tACS of the motor cortex (electrodes placed at C3 and C4) significantly diminished the amplitude of motor evoked potentials and decreased intracortical facilitation (ICF) as compared to baseline and sham stimulation. In addition, we show that our method of sham tACS is a reliable control condition. These results support the notion that AC stimulation with weak currents can induce significant changes in brain excitability; in this case, 15 Hz tACS led to a pattern of inhibition of cortical excitability. We propose that tACS may have a dampening effect on cortical networks and perhaps interfere with the temporal and spatial summation of weak subthreshold electric potentials. (C) 2010 Elsevier Ireland Ltd. All rights reserved.