CD27 mediates interleukin-2-independent clonal expansion of the CD8+ T cell without effector differentiation

The clonal expansion of antigen-specific CD8+ T cells in response to microbial infections is essential for adaptive immunity. Although IL-2 has been considered to be primarily responsible for this process, quantitatively normal expansion occurs in the absence of IL-2 receptor signaling. Here, we show that ligating CD27 on CD8+ T cells that have been stimulated through the T cell receptor causes their expansion in the absence of IL-2 by mediating two distinct cellular processes: enhancing cell cycling and promoting cell survival by maintaining the expression of IL-7 receptor alpha. This pathway for clonal expansion of the CD8+ T cell is not associated with the development of a capacity either for production of IFN-gamma or for cytotoxic T lymphocyte function and, therefore, is uncoupled from differentiation. Furthermore, ligating CD27 increases the threshold concentration at which IL-2 induces IFN-gamma-producing capability by the CD8+ T cell, suggesting that CD27 signaling may suppress effector differentiation. Finally, CD8+ T cells that have been stimulated by the TCR/CD27 pathway maintain their capacity for subsequent expansion and effector differentiation in response to a viral challenge in vivo. Thus, the TCR/CD27 pathway enables the CD8+ T cell to replicate by a process of self-renewal, which may contribute to the continuous generation of new effector CD8+ T cells in persistent viral infections.

Comparing correlations of continuous observations from two independent populations using a sequential approach

A sequential study design generally makes more efficient use of available information than a fixed sample counterpart of equal power. This feature is gradually being exploited by researchers in genetic and epidemiological investigations that utilize banked biological resources and in studies where time, cost and ethics are prominent considerations. Recent work in this area has focussed on the sequential analysis of matched case-control studies with a dichotomous trait. In this paper, we extend the sequential approach to a comparison of the associations within two independent groups of paired continuous observations. Such a comparison is particularly relevant in familial studies of phenotypic correlation using twins. We develop a sequential twin method based on the intraclass correlation and show that use of sequential methodology can lead to a substantial reduction in the number of observations without compromising the study error rates. Additionally, our approach permits straightforward allowance for other explanatory factors in the analysis. We illustrate our method in a sequential heritability study of dysplasia that allows for the effect of body mass index and compares monozygotes with pairs of singleton sisters. Copyright (c) 2006 John Wiley & Sons, Ltd.

A middleware independent grid workflow builder for scientific applications

Grid workflow authoring tools are typically specific to particular workflow engines built into Grid middleware, or are application specific and are designed to interact with specific software implementations. g-Eclipse is a middleware independent Grid workbench that aims to provide a unified abstraction of the Grid and includes a Grid workflow builder to allow users to author and deploy workflows to the Grid. This paper describes the g-Eclipse Workflow Builder and its implementations for two Grid middlewares, gLite and GRIA, and a case study utilizing the Workflow Builder in a Grid user's scientific workflow deployment.

Environmental variation at the onset of independent foraging affects full-grown body mass in the red fox

The period following the withdrawal of parental care has been highlighted as a key developmental period for juveniles. One reason for this is that juveniles cannot forage as competently as adults, potentially placing them at greater risk from environmentally-induced changes in food availability. However, no study has examined this topic. Using a long-term dataset on red foxes (Vulpes vulpes), we examined (i) dietary changes that occurred in the one-month period following the attainment of nutritional independence, (ii) diet composition in relation to climatic variation, and (iii) the effect of climatic variation on subsequent full-grown mass. Diet at nutritional independence contained increased quantities of easy-to-catch food items (earthworms and insects) when compared with pre-independence. Interannual variation in the volume of rainfall at nutritional independence was positively correlated to the proportion of earthworms in cub diet. Pre-independence cub mass and rainfall immediately following nutritional independence explained a significant proportion of variance in full-grown mass, with environmental variation affecting full-grown mass of the entire cohorts. Thus, weather-mediated availability of easy-to-catch food items at a key developmental stage has lifelong implications for the development of juvenile foxes by affecting full-grown mass, which in turn appears to be an important component of individual reproductive potential.

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.

CCL3, acting via the chemokine receptor CCR5, leads to independent activation of Janus kinase 2 (JAK2) and G(i) proteins

The interaction of the chemokine receptor, CCR5, expressed in recombinant cells, with different G proteins was investigated and CCR5 was found to interact with G(i), G(o) and G(q) species. Interaction with Gi leads to G protein activation, whereas G. does not seem to be activated. Additionally, CCR5 activation also leads to phosphorylation of Janus kinase 2 (JAK2). Activation of JAK2 is independent of Gi or Gq activation. Gi protein activation was not prevented by inhibition of JAK, showing that heterotrimeric G protein activation and activation of the JAK/signal transducer and activator of transcription (STAT) pathway are independent of each other. (C) 2004 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Moderate Champagne consumption promotes an acute improvement in acute endothelial-independent vascular function in healthy human volunteers

Epidemiological studies have suggested an inverse correlation between red wine consumption and the incidence of CVD. However, Champagne wine has not been fully investigated for its cardioprotective potential. In order to assess whether acute and moderate Champagne wine consumption is capable of modulating vascular function, we performed a randomised, placebo-controlled, cross-over intervention trial. We show that consumption of Champagne wine, but not a control matched for alcohol, carbohydrate and fruit-derived acid content, induced an acute change in endothelium-independent vasodilatation at 4 and 8 h post-consumption. Although both Champagne wine and the control also induced an increase in endothelium-dependent vascular reactivity at 4 h, there was no significant difference between the vascular effects induced by Champagne or the control at any time point. These effects were accompanied by an acute decrease in the concentration of matrix metalloproteinase (MMP-9), a significant decrease in plasma levels of oxidising species and an increase in urinary excretion of a number of phenolic metabolites. In particular, the mean total excretion of hippuric acid, protocatechuic acid and isoferulic acid were all significantly greater following the Champagne wine intervention compared with the control intervention. Our data suggest that a daily moderate consumption of Champagne wine may improve vascular performance via the delivery of phenolic constituents capable of improving NO bioavailability and reducing matrix metalloproteinase activity.

Age-related increases in circulating inflammatory markers in men are independent of BMI, blood pressure and blood lipid concentrations

Objective: To examine whether age-related increase in concentrations of circulating inflammatory mediators is due to concurrent increases in cardiovascular risk factors or is independent of these. Methods and results: Cytokines (IL-6, IL-18), chemokines (6Ckine, MCP-1, IP-10), soluble adhesion molecules (sICAM-1, sVCAM-1, sE-selectin) and adipokines (adiponectin) were measured in the plasma of healthy male subjects aged 18-84 years (n = 162). These were related to known cardiovascular risk factors (age, BMI, systolic and diastolic blood pressure, plasma total cholesterol, LDL cholesterol, HDL cholesterol and triacylglycerol concentrations) in order to identify significant associations. Plasma concentrations of sVCAM-1, sE-selectin, IL-6, IL-18, MCP-1, 6Ckine, IP-10 and adiponectin, but not sICAM-1, were significantly positively correlated with age, as well as with several other cardiovascular risk factors. The correlations with other risk factors disappeared when age was controlled for. In contrast, the correlations with age remained significant for sVCAM-1, IL-6, MCP-1, 6Ckine and IP-10 when other cardiovascular risk factors were controlled for. Conclusions: Plasma concentrations of some inflammatory markers (sVCAM-1, IL-6, MCP-L 6Ckine, IP-10) are positively correlated with age, independent of other cardiovascular risk factors. This suggests that age-related inflammation may not be driven by recognised risk factors. (C) 2006 Elsevier Ireland Ltd. All rights reserved.

The effect of free Ca2+ on the heat stability and other characteristics of low-heat skim milk powder

Low-heat skim milk powder (SMP), reconstituted to 25% total solids, was found to have poor heat stability. This could be improved by reducing the free Ca2+ concentration to 1.14 mm, or lower, by the addition of either Amberlite IR-120 ion-exchange resin in its sodium form or tri-sodium citrate in skim milk prior to evaporation and spray drying. Reduction in Ca2+ concentration was accompanied by increases in pH, particle size, and kinematic viscosity, and by a reduction in zeta-potential and changes in colour. In-container sterilisation of the reconstituted powder increased particle size, zeta-potential, kinematic viscosity and a* and b* values. However. Ca2+ concentration, pH and whiteness decreased. This study elucidated the importance of Ca2+ concentration and pH on heat stability of low-heat SMP, suggesting that Ca2+ concentration and pH in bulk milk are useful indicators for ensuring that spray dried milk powder has good heat stability. (C) 2009 Elsevier Ltd. All rights reserved.

G protein ss gamma subunits mediate presynaptic inhibition of transmitter release from rat superior cervical ganglion neurones in culture

The activation of presynaptic G protein-coupled receptors (GPCRs) is widely reported to inhibit transmitter release; however, the lack of accessibility of many presynaptic terminals has limited direct analysis of signalling mediators. We studied GPCR-mediated inhibition of fast cholinergic transmission between superior cervical ganglion neurones (SCGNs) in culture. The adrenoceptor agonist noradrenaline (NA) caused a dose-related reduction in evoked excitatory postsynaptic potentials (EPSPs). NA-induced EPSP decrease was accompanied by effects on the presynaptic action potential (AP), reducing AP duration and amplitude of the after-hyperpolarization (AHP), without affecting the pre- and postsynaptic membrane potential. All effects of NA were blocked by yohimbine and synaptic transmission was reduced by clonidine, consistent with an action at presynaptic alpha 2-adrenoceptors. NA-induced inhibition of transmission was sensitive to pre-incubation of SCGNs with pertussis toxin (PTX), implicating the involvement of G alpha(i)/(o)beta y subunits. Expression of G alpha transducin, an agent which sequesters G protein beta gamma (G beta y) subunits, in the presynaptic neurone caused a time-dependent attenuation of NA-induced inhibition. Injection of purified G beta gamma subunits into the presynaptic neurone inhibited transmission, and also reduced the AHP amplitude. Furthermore, NA-induced inhibition was occluded by pre-injection of G beta gamma subunits. The Ca2+ channel blocker Cd2+ mimicked NA effects on transmitter release. Cd2+, NA and G beta gamma subunits also inhibited somatic Ca2+ current. In contrast to effects on AP-evoked transmitter release, NA had no clear action on AP-independent EPSPs induced by hypertonic solutions. These results demonstrate that G beta gamma subunits functionally mediate inhibition of transmitter release by alpha 2-adrenoceptors and represent important regulators of synaptic transmission at mammalian presynaptic terminals.

Neuroprotective effects of hesperetin in mouse primary neurones are independent of CREB activation

Dietary flavonoids, including the citrus flavanone hesperetin, may have stimulatory, effects on cytoprotective intracellular signalling pathways. In primary mouse cortical neurone cultures, but not SH-SY5Y human neuroblastoma cells or human primary dermal fibroblasts (Promocells), hesperetin (100-300 nM, 15 min) caused significant increases in the level of ERK1/2 phosphorylation, but did not increase CREB phosphorylation. Administration of hesperetin for 18 h did not alter gene expression driven by the cyclic AMP response element (CRE), assessed using a luciferase reporter system, but 300 nM hesperetin partially reversed staurosporine-induced cell death in primary neurones. Our data show that hesperetin is a neuroprotective compound at concentrations where antioxidant effects are unlikely to predominate. The effects of hesperetin are cell-type dependent and, unlike the flavanol (-)epicatechin, neuroprotection in vitro is not associated with enhanced CREB phosphorylation or CRE-mediated gene expression. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Analysis of second messenger pathways stimulated by different chemokines acting at the chemokine receptor

The chemokine receptor, CCR5, responds to several chemokines leading to changes in activity in several signalling pathways. Here, we investigated the ability of different chemokines to provide differential activation of pathways. The effects of five CC chemokines acting at CCR5 were investigated for their ability to inhibit forskolin- stimulated 3'-5'-cyclic adenosine monophosphate (cAMP) accumulation and to stimulate Ca2+ mobilisation. in Chinese hamster ovary (CHO) cells expressing CCR5. Macrophage inflammatory protein 1 alpha (D26A) (MIP-1 alpha (D26A), CCL3 (D26A)), regulated on activation, normal T-cell expressed and secreted (RANTES, CCLS), MIP-1 beta (CCL4) and monocyte chemoattractant protein 2 (MCP-2, CCL8) were able to inhibit forskolin -stimulated CAMP accumulation, whilst MCP-4 (CCL13) could not elicit a response. CCL3 (D26A), CCL4, CCLS, CCL8 and CCL13 were able to stimulate Ca2+ mobilisation. through CCRS, although CCL3 (D26A) and CCL5 exhibited biphasic concentration-response curves. The Ca2+ responses induced by CCL4, CCL5, CCL8 and CCL13 were abolished by pertussis toxin, whereas the response to CCL3 (D26A) was only partially inhibited by pertussis toxin, indicating G(i/o)-independent signalling induced by this chemokine. Although the rank order of potency of chemokines was similar between the two assays, certain chemokines displayed different pharmacological profiles in cAMP inhibition and Ca2+ mobilisation assays. For instance, whilst CCL13 could not inhibit forskolin-stimulated cAMP accumulation, this chemokine was able to induce Ca2+ mobilisation via CCR5. It is concluded that different chemokines acting at CCR5 can induce different pharmacological responses, which may account for the broad spectrum of chemokines that can act at CCRS. (C) 2007 Elsevier Inc. All rights reserved.