Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen

Induction of humoral responses to HIV at mucosal compartments without inflammation is important for vaccine design. We developed charged wax nanoparticles that efficiently adsorb protein antigens and are internalized by DC in the absence of inflammation. HIV-gp140-adsorbed nanoparticles induced stronger in vitro T-cell proliferation responses than antigen alone. Such responses were greatly enhanced when antigen was co-adsorbed with TLR ligands. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG. Importantly, intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhanced serum and vaginal IgG and IgA responses. Our results show that HIV-gp140-carrying wax nanoparticles can induce strong cellular/humoral immune responses without inflammation and may be of potential use as effective mucosal adjuvants for HIV vaccine candidates.

Nuclear Dbf2-related protein kinases (NDRs) in isolated cardiac myocytes and the myocardium: activation by cellular stresses and by phosphoprotein serine-/threonine-phosphatase inhibitors

The nuclear Dbf2-related protein kinases 1 and 2 (NDR1/2) are closely-related AGC family kinases that are strongly conserved through evolution. In mammals, they are activated inter alia by phosphorylation of an hydrophobic domain threonine-residue [NDR1(Thr-444)/NDR2(Thr-442)] by an extrinsic protein kinase followed by autophosphorylation of a catalytic domain serine-residue [NDR1(Ser-281)/NDR2(Ser-282)]. We examined NDR1/2 expression and regulation in primary cultures of neonatal rat cardiac myocytes and in perfused adult rat hearts. In myocytes, transcripts for NDR2, but not NDR1, were induced by the hypertrophic agonist, endothelin-1. NDR1(Thr-444) and NDR2(Thr-442) were rapidly phosphorylated (maximal in 15-30 min) in myocytes exposed to some phosphoprotein Ser-/Thr-phosphatase 1/2 inhibitors (calyculin A, okadaic acid) and, to a lesser extent, by hyperosmotic shock, low concentrations of H(2)O(2), or chelerythrine. In myocytes adenovirally-transduced to express FLAG-NDR2 (which exhibited a mainly-cytoplasmic localisation), the same agents increased FLAG-NDR2 activity as assessed by in vitro protein kinase assays, indicative of FLAG-NDR2(Ser-282/Thr-442) phosphorylation. Calyculin A-induced phosphorylation of NDR1(Thr-444)/NDR2(Thr-442) and activation of FLAG-NDR2 were inhibited by staurosporine, but not by other protein kinase inhibitors tested. In ex vivo rat hearts, NDR1(Thr-444)/NDR2(Thr-442) were phosphorylated in response to ischaemia-reperfusion or calyculin A. From a pathological viewpoint, we conclude that activities of NDR1 and NDR2 are responsive to cytotoxic stresses in heart preparations and this may represent a previously-unidentified response to myocardial ischaemia in vivo.

Beyond antioxidants: the cellular and molecular interactions of flavonoids and how these underpin their actions on the brain

The consumption of flavonoid-rich foods and beverages has been suggested to limit the neurodegeneration associated with a variety of neurological disorders and to prevent or reverse normal or abnormal deteriorations in cognitive performance. Flavonoids mediate these effects via a number of routes, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation and a potential to promote memory, learning and cognitive function. Originally, it was thought that such actions were mediated by the antioxidant capacity of flavonoids. However, their limited absorption and their low bioavailability in the brain suggest that this explanation is unlikely. Instead, this multiplicity of effects appears to be underpinned by three separate processes: first, through their interactions with important neuronal and glial signalling cascades in the brain, most notably the phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase pathways that regulate pro-survival transcription factors and gene expression; second, through an ability to improve peripheral and cerebral blood flow and to trigger angiogenesis and neurogenesis in the hippocampus; third, by their capacity to directly react with and scavenge neurotoxic species and pro-inflammatory agents produced in the brain as a result of both normal and abnormal brain ageing. The present review explores the potential inhibitory or stimulatory actions of flavonoids within these three systems and describes how such interactions are likely to underlie neurological effects.

Cellular and molecular investigations into the development of the pectoral girdle

The forelimbs of higher vertebrates are composed of two portions: the appendicular region (stylopod, zeugopod and autopod) and the less prominent proximal girdle elements (scapula and clavicle) that brace the limb to the main trunk axis. We show that the formation of the muscles of the proximal limb occurs through two distinct mechanisms. The more superficial girdle muscles (pectoral and latissimus dorsi) develop by the “In–Out” mechanism whereby migration of myogenic cells from the somites into the limb bud is followed by their extension from the proximal limb bud out onto the thorax. In contrast, the deeper girdle muscles (e.g. rhomboideus profundus and serratus anterior) are induced by the forelimb field which promotes myotomal extension directly from the somites. Tbx5 inactivation demonstrated its requirement for the development of all forelimb elements which include the skeletal elements, proximal and distal muscles as well as the sternum in mammals and the cleithrum of fish. Intriguingly, the formation of the diaphragm musculature is also dependent on the Tbx5 programme. These observations challenge our classical views of the boundary between limb and trunk tissues. We suggest that significant structures located in the body should be considered as components of the forelimb.

Identification of potential mechanisms of toxicity after di-(2-ethylhexyl)-phthalate (DEHP) adult exposure in the liver using a systems biology approach

Phthalates are industrial additives widely used as plasticizers. In addition to deleterious effects on male genital development, population studies have documented correlations between phthalates exposure and impacts on reproductive tract development and on the metabolic syndrome in male adults. In this work we investigated potential mechanisms underlying the impact of DEHP on adult mouse liver in vivo. A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHP doses was performed. Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways, including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profiles revealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesis and to the Rev-erbalpha pathway that senses endogenous heme level. We further confirmed the combined impact of DEHP on the hepatic expression of Alas1, a critical enzyme in heme synthesis and on the expression of Rev-erbalpha target genes involved in the cellular clock and in energy metabolism. This work shows that DEHP interferes with hepatic CAR and Rev-erbalpha pathways which are both involved in the control of metabolism. The identification of these new hepatic pathways targeted by DEHP could contribute to metabolic and endocrine disruption associated with phthalate exposure. Gene expression profiles performed on microdissected testis territories displayed a differential responsiveness to DEHP. Altogether, this suggests that impacts of DEHP on adult organs, including testis, could be documented and deserve further investigations.

A 96-well plate fluorescence assay for assessment of cellular permeability and active efflux in Salmonella enterica serovar Typhimurium and Escherichia coli

Multiply antibiotic-resistant (MAR) mutants of Escherichia coli and Salmonella enterica are characterized by reduced susceptibility to several unrelated antibiotics, biocides and other xenobiotics. Porin loss and/or active efflux have been identified as a key mechanisms of MAR. A single rapid test was developed for MAR. The intracellular accumulation of the fluorescent probe Hoechst (H) 33342 (bisbenzimide) by MAR mutants and those with defined disruptions in efflux pump and porin genes was determined in 96-well plate format. The accumulation of H33342 was significantly (P < 0.0001) reduced in MAR mutants of S. enterica serovar Typhimurium (n = 4) and E. coli (n = 3) by 41 +/- 8% and 17.3 +/- 7.2%, respectively, compared with their parental strains, which was reversed by the transmembrane proton gradient-collapsing agent carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) and the efflux pump inhibitor phenylalanine-arginine-beta-naphthylamide (PA beta N). The accumulation of H33342 was significantly reduced in mutants of Salmonella Typhimurium with defined disruptions in genes encoding the porins OmpC, OmpF, OmpX and OmpW, but increased in those with disruptions in efflux pump components TolC, AcrB and AcrF. Reduced accumulation of H33342 in three other MAR mutants of Salmonella Typhimurium correlated with the expression of porin and efflux pump proteins. The intracellular accumulation of H33342 provided a sensitive and specific test for MAR that is cheap and relatively rapid. Differential sensitivity to CCCP and PA beta N provided a further means to phenotypically identify MAR mutants and the role of active efflux in each strain.

Peroxynitrite mediates disruption of Ca2+ homeostasis by carbon monoxide via Ca2+ ATPase degradation

CO stimulates formation of NO and reactive oxygen species which, via peroxynitrite formation, inhibit Ca(2+) extrusion via PMCA, leading to disruption of Ca(2+) signaling. We propose this contributes to the neurological damage associated with CO toxicity.

What have dendritic cells ever done for adjuvant design? Cellular and molecular methods for the rational development of vaccine adjuvants

Our new molecular understanding of immune priming states that dendritic cell activation is absolutely pivotal for expansion and differentiation of naïve T lymphocytes, and it follows that understanding DC activation is essential to understand and design vaccine adjuvants. This chapter describes how dendritic cells can be used as a core tool to provide detailed quantitative and predictive immunomics information about how adjuvants function. The role of distinct antigen, costimulation, and differentiation signals from activated DC in priming is explained. Four categories of input signals which control DC activation – direct pathogen detection, sensing of injury or cell death, indirect activation via endogenous proinflammatory mediators, and feedback from activated T cells – are compared and contrasted. Practical methods for studying adjuvants using DC are summarised and the importance of DC subset choice, simulating T cell feedback, and use of knockout cells is highlighted. Finally, five case studies are examined that illustrate the benefit of DC activation analysis for understanding vaccine adjuvant function.

A mesoscopic modelling approach to anaesthetic action on brain electrical activity

Relating the measurable, large scale, effects of anaesthetic agents to their molecular and cellular targets of action is necessary to better understand the principles by which they affect behavior, as well as enabling the design and evaluation of more effective agents and the better clinical monitoring of existing and future drugs. Volatile and intravenous general anaesthetic agents (GAs) are now known to exert their effects on a variety of protein targets, the most important of which seem to be the neuronal ion channels. It is hence unlikely that anaesthetic effect is the result of a unitary mechanism at the single cell level. However, by altering the behavior of ion channels GAs are believed to change the overall dynamics of distributed networks of neurons. This disruption of regular network activity can be hypothesized to cause the hypnotic and analgesic effects of GAs and may well present more stereotypical characteristics than its underlying microscopic causes. Nevertheless, there have been surprisingly few theories that have attempted to integrate, in a quantitative manner, the empirically well documented alterations in neuronal ion channel behavior with the corresponding macroscopic effects. Here we outline one such approach, and show that a range of well documented effects of anaesthetics on the electroencephalogram (EEG) may be putatively accounted for. In particular we parameterize, on the basis of detailed empirical data, the effects of halogenated volatile ethers (a clinically widely used class of general anaesthetic agent). The resulting model is able to provisionally account for a range of anaesthetically induced EEG phenomena that include EEG slowing, biphasic changes in EEG power, and the dose dependent appearance of anomalous ictal activity, as well as providing a basis for novel approaches to monitoring brain function in both health and disease.

Rescue of gene expression by modified REST decoy oligonucleotides in a cellular model of Huntington's disease

Transcriptional dysfunction is a prominent hallmark of Huntington's disease (HD). Several transcription factors have been implicated in the aetiology of HD progression and one of the most prominent is repressor element 1 (RE1) silencing transcription factor (REST). REST is a global repressor of neuronal gene expression and in the presence of mutant Huntingtin increased nuclear REST levels lead to elevated RE1 occupancy and a concomitant increase in target gene repression, including brain-derived neurotrophic factor. It is of great interest to devise strategies to reverse transcriptional dysregulation caused by increased nuclear REST and determine the consequences in HD. Thus far, such strategies have involved RNAi or mutant REST constructs. Decoys are double-stranded oligodeoxynucleotides corresponding to the DNA-binding element of a transcription factor and act to sequester it, thereby abrogating its transcriptional activity. Here, we report the use of a novel decoy strategy to rescue REST target gene expression in a cellular model of HD. We show that delivery of the decoy in cells expressing mutant Huntingtin leads to its specific interaction with REST, a reduction in REST occupancy of RE1s and rescue of target gene expression, including Bdnf. These data point to an alternative strategy for rebalancing the transcriptional dysregulation in HD.

Aluminium and breast cancer: sources of exposure, tissue measurements and mechanisms of toxicological actions on breast biology

This review examines recent evidence linking exposure to aluminium with the aetiology of breast cancer. The human population is exposed to aluminium throughout daily life including through diet, application of antiperspirants, use of antacids and vaccination. Aluminium has now been measured in a range of human breast structures at higher levels than in blood serum and experimental evidence suggests that the tissue concentrations measured have the potential to adversely influence breast epithelial cells including generation of genomic instability, induction of anchorage-independent proliferation and interference in oestrogen action. The presence of aluminium in the human breast may also alter the breast microenvironment causing disruption to iron metabolism, oxidative damage to cellular components, inflammatory responses and alterations to the motility of cells. The main research need is now to investigate whether the concentrations of aluminium measured in the human breast can lead in vivo to any of the effects observed in cells in vitro and this would be aided by the identification of biomarkers specific for aluminium action.

Development of substorm cross-tail current disruption as seen from the ground

We discuss substorm observations made near 2100 magnetic local time (MLT) on March 7, 1991, in a collaborative study involving data from the European Incoherent Scatter radar, all-sky camera data, and magnetometer data from the Tromsø Auroral Observatory, the U.K. Sub-Auroral Magnetometer Network (SAMNET) and the IMAGE magnetometer chain. We conclude that for the substorm studied a plasmoid was not pinched off until at least 10 min after onset at the local time of the observations (2100 MLT) and that the main substorm electrojet expanded westward over this local time 14 min after onset. In the late growth phase/early expansion phase, we observed southward drifting arcs probably moving faster than the background plasma. Similar southward moving arcs in the recovery phase moved at a speed which does not appear to be significantly different from the measured plasma flow speed. We discuss these data in terms of the “Kiruna conjecture” and classical “near-Earth neutral line” paradigms, since the data show features of both models of substorm development. We suggest that longitudinal variation in behavior may reconcile the differences between the two models in the case of this substorm.

Simple method for sub-diffraction resolution imaging of cellular structures on standard confocal microscopes by three-photon absorption of quantum dots

This study describes a simple technique that improves a recently developed 3D sub-diffraction imaging method based on three-photon absorption of commercially available quantum dots. The method combines imaging of biological samples via tri-exciton generation in quantum dots with deconvolution and spectral multiplexing, resulting in a novel approach for multi-color imaging of even thick biological samples at a 1.4 to 1.9-fold better spatial resolution. This approach is realized on a conventional confocal microscope equipped with standard continuous-wave lasers. We demonstrate the potential of multi-color tri-exciton imaging of quantum dots combined with deconvolution on viral vesicles in lentivirally transduced cells as well as intermediate filaments in three-dimensional clusters of mouse-derived neural stem cells (neurospheres) and dense microtubuli arrays in myotubes formed by stacks of differentiated C2C12 myoblasts.