Cold Exposure Promotes Atherosclerotic Plaque Growth and Instability via UCP1-Dependent Lipolysis

Molecular mechanisms underlying the cold-associated high cardiovascular risk remain unknown. Here, we show that the cold-triggered food-intake-independent lipolysis significantly increased plasma levels of small low-density lipoprotein (LDL) remnants, leading to accelerated development of atherosclerotic lesions in mice. In two genetic mouse knockout models (apolipoprotein E(-/-) [ApoE(-/-)] and LDL receptor(-/-) [Ldlr(-/-)] mice), persistent cold exposure stimulated atherosclerotic plaque growth by increasing lipid deposition. Furthermore, marked increase of inflammatory cells and plaque-associated microvessels were detected in the cold-acclimated ApoE(-/-) and Ldlr(-/-) mice, leading to plaque instability. Deletion of uncoupling protein 1 (UCP1), a key mitochondrial protein involved in thermogenesis in brown adipose tissue (BAT), in the ApoE(-/-) strain completely protected mice from the cold-induced atherosclerotic lesions. Cold acclimation markedly reduced plasma levels of adiponectin, and systemic delivery of adiponectin protected ApoE(-/-) mice from plaque development. These findings provide mechanistic insights on low-temperature-associated cardiovascular risks.

Hypothermic preservation up-regulates calpain expression and increases ubiquitination in cultured vascular endothelial cells: influence of dopamine pretreatment.

BACKGROUND Prolonged hypothermia, as occurs during solid organ transplantation, negatively influences transplantation outcome. Proteolysis is one of the deleterious events implicated in preservation injury of organ allografts. This strongly affects graft quality and hence immediate organ function. Since donor catecholamine treatment improves transplantation outcome after renal transplantation, the present study was conducted to examine the influence of dopamine (DA) pretreatment on hypothermia induced proteolysis in endothelial cells subjected to prolonged cold storage. MATERIALS AND METHODS Lactate dehydrogenase (LDH) assay, two-dimensional electrophoresis, ubiquitination analysis, intracellular calcium measurement, and Western blot analysis were performed on human umbilical vein endothelial cells (HUVEC) subjected to hypothermic preservation or not. RESULTS HUVEC were highly susceptible to cold storage, which was reflected by morphological changes, loss of viability, and by significant changes in cellular proteome. DA pretreatment prevented cell death during cold storage. Western blot analysis demonstrated a time dependent up-regulation of calpain 1 and 2 during cold storage, which could be prevented by addition of EDTA. DA pretreatment abolished autoproteolysis of calpain 1. Analysis of ubiquitination revealed a significant increase in ubiquitinated conjugates after cold storage. This was not prevented by DA pretreatment. Neither proteasome nor calpain inhibitors prevented cell death during cold storage. CONCLUSION In endothelial cells subjected to cold preservation, activation of the calpain pathway and the ubiquitin proteasome system occur. Although DA pretreatment inhibits the former, calpain inhibition did not protect endothelial cells during cold storage. DA pretreatment might influence proteolysis, but proteolysis is not the major cause of endothelial cell death.

The role of free radicals in cold injuries.

Cold injury is a tissue trauma produced by exposure to freezing temperatures and even brief exposure to a severely cold and windy environment. Rewarming of frozen tissue is associated with blood reperfusion and the simultaneous generation of free oxygen radicals. In this review is discussed the current understanding of the mechanism of action of free oxygen radicals as related to cold injury during rewarming. Decreased energy stores during ischaemia lead to the accumulation of adenine nucleotides and liberation of free fatty acids due to the breakdown of lipid membranes. On rewarming, free fatty acids are metabolized via cyclo-oxygenase and adenine nucleotides are metabolized via the xanthine oxidase pathway. These may be the source of free oxygen radicals. Leukocytes may also play a major role in the pathogenesis of cold injury. Oxygen radical scavengers, such as superoxide dismutase and catalase, may help to reduce the cold induced injury but their action is limited due to the inability readily to cross the plasma membrane. Lipid soluble antioxidants are likely to be more effective scavengers because of their presence in membranes where peroxidative reactions can be arrested.

Alternatively activated macrophages produce catecholamines to sustain adaptive thermogenesis

All homeotherms use thermogenesis to maintain their core body temperature, ensuring that cellular functions and physiological processes can continue in cold environments. In the prevailing model of thermogenesis, when the hypothalamus senses cold temperatures it triggers sympathetic discharge, resulting in the release of noradrenaline in brown adipose tissue and white adipose tissue. Acting via the β(3)-adrenergic receptors, noradrenaline induces lipolysis in white adipocytes, whereas it stimulates the expression of thermogenic genes, such as PPAR-γ coactivator 1a (Ppargc1a), uncoupling protein 1 (Ucp1) and acyl-CoA synthetase long-chain family member 1 (Acsl1), in brown adipocytes. However, the precise nature of all the cell types involved in this efferent loop is not well established. Here we report in mice an unexpected requirement for the interleukin-4 (IL-4)-stimulated program of alternative macrophage activation in adaptive thermogenesis. Exposure to cold temperature rapidly promoted alternative activation of adipose tissue macrophages, which secrete catecholamines to induce thermogenic gene expression in brown adipose tissue and lipolysis in white adipose tissue. Absence of alternatively activated macrophages impaired metabolic adaptations to cold, whereas administration of IL-4 increased thermogenic gene expression, fatty acid mobilization and energy expenditure, all in a macrophage-dependent manner. Thus, we have discovered a role for alternatively activated macrophages in the orchestration of an important mammalian stress response, the response to cold.

The Effect of Submaximal Exercise Preceded by Single Whole-Body Cryotherapy on the Markers of Oxidative Stress and Inflammation in Blood of Volleyball Players

The aim of the study was to determine the effect of single whole-body cryotherapy (WBC) session applied prior to submaximal exercise on the activity of antioxidant enzymes, the concentration of lipid peroxidation products, total oxidative status, and the level of cytokines in blood of volleyball players. The study group consisted of 18 male professional volleyball players, who were subjected to extremely cold air (-130°C) prior to exercise performed on cycloergometer. Blood samples were taken five times: before WBC, after WBC procedure, after exercise preceded by cryotherapy (WBC exercise), and before and after exercise without WBC (control exercise). The activity of catalase statistically significantly increased after control exercise. Moreover, the activity of catalase and superoxide dismutase was lower after WBC exercise than after control exercise (P < 0.001). After WBC exercise, the level of IL-6 and IL-1β was also lower (P < 0.001) than after control exercise. The obtained results may suggest that cryotherapy prior to exercise may have some antioxidant and anti-inflammatory properties. The relations between the level of studied oxidative stress and inflammatory markers may testify to the contribution of reactive oxygen species in cytokines release into the blood system in response to exercise and WBC.

Chronic stress, glucocorticoid receptor resistance, inflammation, and disease risk.

We propose a model wherein chronic stress results in glucocorticoid receptor resistance (GCR) that, in turn, results in failure to down-regulate inflammatory response. Here we test the model in two viral-challenge studies. In study 1, we assessed stressful life events, GCR, and control variables including baseline antibody to the challenge virus, age, body mass index (BMI), season, race, sex, education, and virus type in 276 healthy adult volunteers. The volunteers were subsequently quarantined, exposed to one of two rhinoviruses, and followed for 5 d with nasal washes for viral isolation and assessment of signs/symptoms of a common cold. In study 2, we assessed the same control variables and GCR in 79 subjects who were subsequently exposed to a rhinovirus and monitored at baseline and for 5 d after viral challenge for the production of local (in nasal secretions) proinflammatory cytokines (IL-1β, TNF-α, and IL-6). Study 1: After covarying the control variables, those with recent exposure to a long-term threatening stressful experience demonstrated GCR; and those with GCR were at higher risk of subsequently developing a cold. Study 2: With the same controls used in study 1, greater GCR predicted the production of more local proinflammatory cytokines among infected subjects. These data provide support for a model suggesting that prolonged stressors result in GCR, which, in turn, interferes with appropriate regulation of inflammation. Because inflammation plays an important role in the onset and progression of a wide range of diseases, this model may have broad implications for understanding the role of stress in health.

Brown and white adipose tissues: intrinsic differences in gene expression and response to cold exposure in mice

Brown adipocytes dissipate energy, whereas white adipocytes are an energy storage site. We explored the plasticity of different white adipose tissue depots in acquiring a brown phenotype by cold exposure. By comparing cold-induced genes in white fat to those enriched in brown compared with white fat, at thermoneutrality we defined a "brite" transcription signature. We identified the genes, pathways, and promoter regulatory motifs associated with "browning," as these represent novel targets for understanding this process. For example, neuregulin 4 was more highly expressed in brown adipose tissue and upregulated in white fat upon cold exposure, and cell studies showed that it is a neurite outgrowth-promoting adipokine, indicative of a role in increasing adipose tissue innervation in response to cold. A cell culture system that allows us to reproduce the differential properties of the discrete adipose depots was developed to study depot-specific differences at an in vitro level. The key transcriptional events underpinning white adipose tissue to brown transition are important, as they represent an attractive proposition to overcome the detrimental effects associated with metabolic disorders, including obesity and type 2 diabetes.

Interleukin-2 and inflammation induce distinct transcriptional programs that promote the differentiation of effector cytolytic T cells.

Interleukin(IL)-2 and inflammation regulate effector and memory cytolytic T-lymphocyte (CTL) generation during infection. We demonstrate a complex interplay between IL-2 and inflammatory signals during CTL differentiation. IL-2 stimulation induced the transcription factor eomesodermin (Eomes), upregulated perforin (Prf1) transcription, and repressed re-expression of memory CTL markers Bcl6 and IL-7Ralpha. Binding of Eomes and STAT5 to Prf1 cis-regulatory regions correlated with transcriptional initiation (increased recruitment of RNA polymerase II to the Prf1 promoter). Inflammation (CpG, IL-12) enhanced expression of IL-2Ralpha and the transcription factor T-bet, but countered late Eomes and perforin induction while preventing IL-7Ralpha repression by IL-2. After infection of mice with lymphocytic choriomeningitis virus, IL-2Ralpha-deficient effector CD8(+) T cells expressed more Bcl6 but less perforin and granzyme B, formed fewer KLRG-1(+) and T-bet-expressing CTL, and killed poorly. Thus, inflammation influences both effector and memory CTL differentiation, whereas persistent IL-2 stimulation promotes effector at the expense of memory CTL development.

Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species.

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2(-/-) mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.

Triglyceride depletion of brown adipose tissue enables analysis of mitochondrial respiratory function in permeabilized biopsies.

The research on mitochondrial functions in adipocytes has increasingly evidenced that mitochondria plays an important role in the onset and/or progression of obesity and related pathologies. Mitochondrial function in brown adipose tissue (BAT) has been classically assessed by measuring either the levels/activity of mitochondrial enzymes, or the respiration in isolated mitochondria. Isolation of mitochondria is not advantageous because it demands significant time and amount of tissue and, as tissue homogenates, disrupts biochemical and physical connections of mitochondria within the cell. Here, we described a new and efficient protocol to analyze the mitochondrial respiratory states in BAT biopsies that relies on intracellular triglyceride depletion followed by tissue permeabilization. In addition to minimizing tissue requirements to ∼17 mg wet weight, the proposed protocol enabled analysis of all mitochondrial respiratory states, including phosphorylation (OXPHOS), no-phosphorylation (LEAK), and uncoupled (ETS) states, as well as the use of substrates for complex I, complex II, and cytochrome c; together, these features demonstrated mitochondrial integrity and validated the preparation efficacy. Therefore, the protocol described here increases the possibilities of answering physiological questions related to small BAT regions of human and animal models, which shall help to unravel the mechanisms that regulate mitochondrial function in health and disease.

Increased gene expression of brown fat uncoupling protein (UCP)1 and skeletal muscle UCP2 and UCP3 in MAC16-induced cancer cachexia.

Weight loss in cancer cachexia is attributable to decreased food intake and/or enhanced energy expenditure. We investigated the roles of the uncoupling proteins (UCPs) UCPI, -2, and -3 in a murine model of cachexia, the MAC16 adenocarcinoma. Weight fell to 24% below that of non-tumor-bearing controls (P < 0.01) 18 days after MAC16 inoculation, with significant reductions in fat-pad mass (-67%; P < 0.01) and muscle mass (-20%; P < 0.01). Food intake was 26-60% lower (P < 0.01) than in controls on days 17-18. Non-tumor-bearing mice, pair-fed to match MAC16-induced hypophagia, showed less weight loss (10% below controls, P < 0.01; 16% above MAC-16, P < 0.01) and smaller decreases in fat-pad mass (21% below controls, P < 0.01). Core temperature in MAC16 mice was significantly lower (-2.4 degrees C, P < 0.01) than in controls, and pair-feeding had no effect. MAC16 mice showed significantly higher UCP1 mRNA levels in brown adipose tissue (BAT) than in controls (+63%, P < 0.01), and pair-feeding had no effect. UCP2 and -3 expression in BAT did not differ significantly between groups. By contrast, UCP2 mRNA levels in skeletal muscle were comparably increased in both MAC16 and pair-fed groups (respectively, 183 and 163% above controls; both, P < 0.05), with no significant difference between these two groups. Similarly, UCP3 mRNA was significantly higher than controls in both MAC16 (+163%, P < 0.05) and pair-fed (+253%, P < 0.01) groups, with no significant difference between the two experimental groups. Overexpression of UCP1 in BAT in MAC16-bearing mice may be an adaptive response to hypothermia, which is apparently induced by tumor products; increased thermogenesis in BAT could increase total energy expenditure and, thus, contribute to tissue wasting. Increased UCP2 and -3 expression in muscle are both attributable to reduced food intake and may be involved in lipid utilization during lipolysis in MAC16-induced cachexia.

Relationship between reduced forced expiratory volume in one second and the risk of lung cancer: a systematic review and meta-analysis.

BACKGROUND Individuals with severely impaired lung function have an increased risk of lung cancer. Whether milder reductions in forced expiratory volume in 1 second (FEV(1)) also increase the risk of lung cancer is controversial. Moreover, there is little consensus on whether men and women have similar risks for lung cancer for similar decreases in FEV(1). METHODS A search was conducted of PubMed and EMBASE from January 1966 to January 2005 and studies that examined the relationship between FEV1 and lung cancer were identified. The search was limited to studies that were population based, employed a prospective design, were large in size (> or = 5000 participants), and adjusted for cigarette smoking status. RESULTS Twenty eight abstracts were identified, six of which did not report FEV1 and eight did not adjust for smoking. Included in this report are four studies that reported FEV1 in quintiles. The risk of lung cancer increased with decreasing FEV1. Compared with the highest quintile of FEV1 (> 100% of predicted), the lowest quintile of FEV1 (< approximately 70% of predicted) was associated with a 2.23 fold (95% confidence interval (CI) 1.73 to 2.86) increase in the risk for lung cancer in men and a 3.97 fold increase in women (95% CI 1.93 to 8.25). Even relatively small decrements in FEV1 ( approximately 90% of predicted) increased the risk for lung cancer by 30% in men (95% CI 1.05 to 1.62) and 2.64 fold in women (95% CI 1.30 to 5.31). CONCLUSION Reduced FEV1 is strongly associated with lung cancer. Even a relatively modest reduction in FEV1 is a significant predictor of lung cancer, especially among women.

Environmental factors that influence the cutaneous production of vitamin D

All vertebrates, including humans, obtain most of their daily vitamin D requirement from casual exposure to sunlight. During exposure to sunlight, the solar ultraviolet B photons (290-315 nm) penetrate into the skin where they cause the photolysis of 7-dehydrocholesterol to precholecalciferol. Once formed, precholecalciferol undergoes a thermally induced rearrangement of its double bonds to form cholecalciferol. An increase in skin pigmentation, aging, and the topical application of a sunscreen diminishes the cutaneous production of cholecalciferol. Latitude, season, and time of day as well as ozone pollution in the atmosphere influence the number of solar ultraviolet B photons that reach the earth's surface, and thereby, alter the cutaneous production of cholecalciferol. In Boston, exposure to sunlight during the months of November through February will not produce any significant amounts of cholecalciferol in the skin. Because windowpane glass absorbs ultraviolet B radiation, exposure of sunlight through glass windows will not result in any production of cholecalciferol. It is now recognized that vitamin D insufficiency and vitamin D deficiency are common in elderly people, especially in those who are infirm and not exposed to sunlight or who live at latitudes that do not provide them with sunlight-mediated cholecalciferol during the winter months. Vitamin D insufficiency and deficiency exacerbate osteoporosis, cause osteomalacia, and increase the risk of skeletal fractures. Vitamin D insufficiency and deficiency can be prevented by encouraging responsible exposure to sunlight and/or consumption of a multivitamin tablet that contains 10 micrograms (400 IU) vitamin D.

Experimental Evolution of an RNA Virus in Wild Birds: Evidence for Host-Dependent Impacts on Population Structure and Competitive Fitness

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Cold-related respiratory symptoms in the general population.

INTRODUCTION Cold-related respiratory symptoms are common among northern populations, especially among people suffering from respiratory diseases. However, the prevalence of such symptoms in the general population and the threshold temperatures at which the symptoms start to emerge are poorly known. OBJECTIVES The present study determined the prevalence and threshold temperatures of self-reported respiratory symptoms related to cold, separately for healthy people and those with respiratory disease. MATERIALS AND METHODS Six thousand five hundred ninety-one men and women aged 25 years-74 years from the national FINRISK study were queried about cold-related respiratory symptoms. The results were expressed as age-adjusted prevalence figures and coefficients from multivariate regressions. RESULTS Cold-related respiratory symptoms were more often reported by people with asthma (men 69%/women 78%) and by subjects with chronic bronchitis (65%/76%) than the healthy subjects (18%/21%). A binomial regression showed an increase of symptom prevalence by age and excesses of 4%, 50% and 21% units because of female sex, asthma and chronic bronchitis, respectively. The reported threshold temperature for cold-related symptoms was -14 degrees C for males and -15 degrees C for females, and it showed some increase by age (0 degrees C-5 degrees C), asthma (2 degrees C) and chronic bronchitis (3 degrees C). The threshold temperature for mucus production was exceptional as it decreased by age (2 degrees C-5 degrees C) and asthma (2 degrees C). The effects of smoking and education were marginal. CONCLUSION Cold-related respiratory symptoms are common in patients with chronic respiratory diseases, but they start to emerge at relatively low temperatures. In a cold climate, the cold-related symptoms may have an impact on the health-related quality of life.

Mortality risk attributable to high and low ambient temperature: a multicountry observational study

BACKGROUND Although studies have provided estimates of premature deaths attributable to either heat or cold in selected countries, none has so far offered a systematic assessment across the whole temperature range in populations exposed to different climates. We aimed to quantify the total mortality burden attributable to non-optimum ambient temperature, and the relative contributions from heat and cold and from moderate and extreme temperatures. METHODS We collected data for 384 locations in Australia, Brazil, Canada, China, Italy, Japan, South Korea, Spain, Sweden, Taiwan, Thailand, UK, and USA. We fitted a standard time-series Poisson model for each location, controlling for trends and day of the week. We estimated temperature-mortality associations with a distributed lag non-linear model with 21 days of lag, and then pooled them in a multivariate metaregression that included country indicators and temperature average and range. We calculated attributable deaths for heat and cold, defined as temperatures above and below the optimum temperature, which corresponded to the point of minimum mortality, and for moderate and extreme temperatures, defined using cutoffs at the 2·5th and 97·5th temperature percentiles. FINDINGS We analysed 74,225,200 deaths in various periods between 1985 and 2012. In total, 7·71% (95% empirical CI 7·43-7·91) of mortality was attributable to non-optimum temperature in the selected countries within the study period, with substantial differences between countries, ranging from 3·37% (3·06 to 3·63) in Thailand to 11·00% (9·29 to 12·47) in China. The temperature percentile of minimum mortality varied from roughly the 60th percentile in tropical areas to about the 80-90th percentile in temperate regions. More temperature-attributable deaths were caused by cold (7·29%, 7·02-7·49) than by heat (0·42%, 0·39-0·44). Extreme cold and hot temperatures were responsible for 0·86% (0·84-0·87) of total mortality. INTERPRETATION Most of the temperature-related mortality burden was attributable to the contribution of cold. The effect of days of extreme temperature was substantially less than that attributable to milder but non-optimum weather. This evidence has important implications for the planning of public-health interventions to minimise the health consequences of adverse temperatures, and for predictions of future effect in climate-change scenarios. FUNDING UK Medical Research Council.

Induction of the heat-shock response by carbon dioxide in Chironomus thummi.

The effects of a set of stress treatments on gene expression of Chironomus thummi salivary gland cells have been analyzed. Among the treatments assayed, only during recovery from carbon dioxide have we observed a response similar to that previously described after heat-shock treatment: induction of the heat-shock puffs and synthesis of the heat-shock polypeptides. In these conditions, puffing and transcription of telomeric regions were observed, which led to the appearance of the temperature-inducible telomeric Balbiani ring T-BR-III. Other treatments failed to induce the heat-shock response, despite promoting real stress conditions to C. thummi larvae or salivary gland cells.

A subset of 26S proteasomes is activated at critically low ATP concentrations and contributes to myocardial injury during cold ischemia.

Molecular mechanisms leading to myocardial injury during warm or cold ischemia are insufficiently understood. Although proteasomes are thought to contribute to myocardial ischemia-reperfusion injury, their roles during the ischemic period remain elusive. Because donor hearts are commonly exposed to prolonged global cold ischemia prior to cardiac transplantation, we evaluated the role and regulation of the proteasome during cold ischemic storage of rat hearts in context of the myocardial ATP content. When measured at the actual tissue ATP concentration, cardiac proteasome peptidase activity increased by 225% as ATP declined during cold ischemic storage of hearts in University of Wisconsin (UW) solution for up to 48h. Addition of the specific proteasome inhibitor epoxomicin to the UW solution inhibited proteasome activity in the cardiac extracts, significantly reduced edema formation and preserved the ultrastructural integrity of the cardiomyocyte. Utilizing purified 20S/26S proteasome enzyme preparations, we demonstrate that this activation can be attributed to a subset of 26S proteasomes which are stable at ATP concentrations far below physiological levels, that ATP negatively regulates its activity and that maximal activation occurs at ATP concentrations in the low mumol/L range. These data suggest that proteasome activation is a pathophysiologically relevant mechanism of cold ischemic myocardial injury. A subset of 26S proteasomes appears to be a cell-destructive protease that is activated as ATP levels decline. Proteasome inhibition during cold ischemia preserves the ultrastructural integrity of the cardiomyocyte.

Insecticide impregnated curtains to control domestic transmission of cutaneous leishmaniasis in Venezuela: cluster randomised trial.

OBJECTIVE To measure the impact on transmission of leishmaniasis of curtains impregnated with insecticide. DESIGN Cluster randomised controlled trial: household interview survey, observational study of people's behaviour, entomological study with light trap captures of sandflies inside houses. SETTING 14 urban sectors in Trujillo, Venezuela. PARTICIPANTS 2913 inhabitants of 569 houses. INTERVENTION Sectors were paired according to their 12 month cumulative incidence of cutaneous leishmaniasis, one sector in each pair was randomly allocated to receive polyester curtains impregnated with lambdacyhalothrin (intervention group) while the other sector received curtains without insecticide or no curtains (control groups). After 12 months a follow up household survey was conducted. MAIN OUTCOME MEASURES Reduction in abundance of sandflies indoors and 12 month incidence of clinical cases of cutaneous leishmaniasis. RESULTS Transmission of cutaneous leishmaniasis occurred mainly in the domestic setting, with the incidence over 12 months of 4%. The mean number of sandflies per trap per night was 16. After follow up the 12 month incidence of cutaneous leishmaniasis was 0% in the intervention group and 8% in the six pairs in the control group that received unimpregnated curtains (mean difference 8, 95% confidence interval 4.22 to 11.78; P=0.001). There were significantly fewer sandflies in the intervention group (2 v 15, mean difference 13 sandflies per trap; 9 to 17; P<0.001). CONCLUSION Curtains impregnated with insecticide provide a high degree of protection against indoor transmission of cutaneous leishmaniasis.

Involvement of sympathetic nervous system and brown fat in endotoxin-induced fever in rats.

The object of this study was to assess the role of brown adipose tissue (BAT) and the sympathetic nervous system in the rise in heat production associated with endotoxin-induced fever. Oxygen consumption (VO2) was found to be significantly increased (28%) over a 4-h period after two doses of endotoxin (Escherichia coli lipopolysaccharide, 0.3 mg/100 g body wt) given 24 h apart. Injection of a mixed beta-adrenoceptor antagonist (propranolol) reduced VO2 by 14% in endotoxin-treated rats, whereas the selective beta 1- (atenolol) or beta 2- (ICI 118551) antagonists suppressed VO2 by 10%. These drugs did not affect VO2 in control animals. BAT thermogenic activity assessed from measurements of in vitro mitochondrial guanosine 5'-diphosphate (GDP) binding was elevated by 54% in interscapular BAT and by 171% in other BAT depots. Surgical denervation of one lobe of the interscapular depot prevented these responses. Endotoxin failed to stimulate GDP binding in rats fed protein-deficient diets. This may have been because BAT thermogenic activity was already elevated in control rats fed these diets or because endotoxin caused a marked suppression of food intake in the protein-deficient animals. The results indicate that sympathetic activation of BAT is involved in the thermogenic responses to endotoxin and that these can be modified by dietary manipulation.

Beta interferon controls West Nile virus infection and pathogenesis in mice.

Studies with mice lacking the common plasma membrane receptor for type I interferon (IFN-αβR(-)(/)(-)) have revealed that IFN signaling restricts tropism, dissemination, and lethality after infection with West Nile virus (WNV) or several other pathogenic viruses. However, the specific functions of individual IFN subtypes remain uncertain. Here, using IFN-β(-)(/)(-) mice, we defined the antiviral and immunomodulatory function of this IFN subtype in restricting viral infection. IFN-β(-)(/)(-) mice were more vulnerable to WNV infection than wild-type mice, succumbing more quickly and with greater overall mortality, although the phenotype was less severe than that of IFN-αβR(-)(/)(-) mice. The increased susceptibility of IFN-β(-)(/)(-) mice was accompanied by enhanced viral replication in different tissues. Consistent with a direct role for IFN-β in control of WNV replication, viral titers in ex vivo cultures of macrophages, dendritic cells, fibroblasts, and cerebellar granule cell neurons, but not cortical neurons, from IFN-β(-)(/)(-) mice were greater than in wild-type cells. Although detailed immunological analysis revealed no major deficits in the quality or quantity of WNV-specific antibodies or CD8(+) T cells, we observed an altered CD4(+) CD25(+) FoxP3(+) regulatory T cell response, with greater numbers after infection. Collectively, these results suggest that IFN-β controls WNV pathogenesis by restricting infection in key cell types and by modulating T cell regulatory networks.

A Conserved Histidine in the RNA Sensor RIG-I Controls Immune Tolerance to N1-2'O-Methylated Self RNA.

The cytosolic helicase retinoic acid-inducible gene-I (RIG-I) initiates immune responses to most RNA viruses by detecting viral 5'-triphosphorylated RNA (pppRNA). Although endogenous mRNA is also 5'-triphosphorylated, backbone modifications and the 5'-ppp-linked methylguanosine ((m7)G) cap prevent immunorecognition. Here we show that the methylation status of endogenous capped mRNA at the 5'-terminal nucleotide (N1) was crucial to prevent RIG-I activation. Moreover, we identified a single conserved amino acid (H830) in the RIG-I RNA binding pocket as the mediator of steric exclusion of N1-2'O-methylated RNA. H830A alteration (RIG-I(H830A)) restored binding of N1-2'O-methylated pppRNA. Consequently, endogenous mRNA activated the RIG-I(H830A) mutant but not wild-type RIG-I. Similarly, knockdown of the endogenous N1-2'O-methyltransferase led to considerable RIG-I stimulation in the absence of exogenous stimuli. Studies involving yellow-fever-virus-encoded 2'O-methyltransferase and RIG-I(H830A) revealed that viruses exploit this mechanism to escape RIG-I. Our data reveal a new role for cap N1-2'O-methylation in RIG-I tolerance of self-RNA.

Rational design of a flavivirus vaccine by abolishing viral RNA 2'-O methylation.

Viruses that replicate in the cytoplasm cannot access the host nuclear capping machinery. These viruses have evolved viral methyltransferase(s) to methylate N-7 and 2'-O cap of their RNA; alternatively, they "snatch" host mRNA cap to form the 5' end of viral RNA. The function of 2'-O methylation of viral RNA cap is to mimic cellular mRNA and to evade host innate immune restriction. A cytoplasmic virus defective in 2'-O methylation is replicative, but its viral RNA lacks 2'-O methylation and is recognized and eliminated by the host immune response. Such a mutant virus could be rationally designed as a live attenuated vaccine. Here, we use Japanese encephalitis virus (JEV), an important mosquito-borne flavivirus, to prove this novel vaccine concept. We show that JEV methyltransferase is responsible for both N-7 and 2'-O cap methylations as well as evasion of host innate immune response. Recombinant virus completely defective in 2'-O methylation was stable in cell culture after being passaged for >30 days. The mutant virus was attenuated in mice, elicited robust humoral and cellular immune responses, and retained the engineered mutation in vivo. A single dose of immunization induced full protection against lethal challenge with JEV strains in mice. Mechanistically, the attenuation phenotype was attributed to the enhanced sensitivity of the mutant virus to the antiviral effects of interferon and IFIT proteins. Collectively, the results demonstrate the feasibility of using 2'-O methylation-defective virus as a vaccine approach; this vaccine approach should be applicable to other flaviviruses and nonflaviviruses that encode their own viral 2'-O methyltransferases.

Preexisting helminth infection induces inhibition of innate pulmonary anti-tuberculosis defense by engaging the IL-4 receptor pathway

Tuberculosis and helminthic infections coexist in many parts of the world, yet the impact of helminth-elicited Th2 responses on the ability of the host to control Mycobacterium tuberculosis (Mtb) infection has not been fully explored. We show that mice infected with the intestinal helminth Nippostrongylus brasiliensis (Nb) exhibit a transitory impairment of resistance to airborne Mtb infection. Furthermore, a second dose of Nb infection substantially increases the bacterial burden in the lungs of co-infected mice. Interestingly, the Th2 response in the co-infected animals did not impair the onset and development of the protective Mtb-specific Th1 cellular immune responses. However, the helminth-induced Th2 environment resulted in the accumulation of alternatively activated macrophages (AAMs) in the lung. Co-infected mice lacking interleukin (IL) 4Rα exhibited improved ability to control Mtb infection, which was accompanied by significantly reduced accumulation of AAMs. Moreover, IL-4Rα(-/-) mice adoptively transferred with wild-type macrophages had a significantly higher Mtb load in their lungs compared with those that received IL-4Rα(-/-) macrophages, suggesting a direct contribution for the IL-4R pathway to the heightened susceptibility of co-infected animals. The Th2 response can thus enhance the intracellular persistence of Mtb, in part by mediating the alternative activation of macrophages via the IL-4Rα signaling pathway.

Autocrine WNT signaling contributes to breast cancer cell proliferation via the canonical WNT pathway and EGFR transactivation

De-regulation of the wingless and integration site growth factor (WNT) signaling pathway via mutations in APC and Axin, proteins that target β-catenin for destruction, have been linked to various types of human cancer. These genetic alterations rarely, if ever, are observed in breast tumors. However, various lines of evidence suggest that WNT signaling may also be de-regulated in breast cancer. Most breast tumors show hypermethylation of the promoter region of secreted Frizzled-related protein 1 (sFRP1), a negative WNT pathway regulator, leading to downregulation of its expression. As a consequence, WNT signaling is enhanced and may contribute to proliferation of human breast tumor cells. We previously demonstrated that, in addition to the canonical WNT/β-catenin pathway, WNT signaling activates the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in mouse mammary epithelial cells via epidermal growth factor receptor (EGFR) transactivation. Using the WNT modulator sFRP1 and short interfering RNA-mediated Dishevelled (DVL) knockdown, we interfered with autocrine WNT signaling at the ligand-receptor level. The impact on proliferation was measured by cell counting, YOPRO, and the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay; β-catenin, EGFR, ERK1/2 activation, and PARP (poly [ADP-ribose]polymerase) cleavages were assessed by Western blotting after treatment of human breast cancer cell lines with conditioned media, purified proteins, small-molecule inhibitors, or blocking antibodies. Phospho-DVL and stabilized β-catenin are present in many breast tumor cell lines, indicating autocrine WNT signaling activity. Interfering with this loop decreases active β-catenin levels, lowers ERK1/2 activity, blocks proliferation, and induces apoptosis in MDA-MB-231, BT474, SkBr3, JIMT-1, and MCF-7 cells. The effects of WNT signaling are mediated partly by EGFR transactivation in human breast cancer cells in a metalloprotease- and Src-dependent manner. Furthermore, Wnt1 rescues estrogen receptor-positive (ER+) breast cancer cells from the anti-proliferative effects of 4-hydroxytamoxifen (4-HT) and this activity can be blocked by an EGFR tyrosine kinase inhibitor. Our data show that interference with autocrine WNT signaling in human breast cancer reduces proliferation and survival of human breast cancer cells and rescues ER+ tumor cells from 4-HT by activation of the canonical WNT pathway and EGFR transactivation. These findings suggest that interference with WNT signaling at the ligand-receptor level in combination with other targeted therapies may improve the efficiency of breast cancer treatments.

Diminished sympathetic nervous system activity in genetically obese (ob/ob) mouse.

The genetically obese (ob/ob) mouse exhibits defective thermoregulatory responses to cold exposure. Pathophysiological explanations for this phenomenon have focused on abnormalities in intracellular metabolism or insensitivity of peripheral tissues to the thermogenic effects of catecholamines. Because the sympathetic nervous system (SNS) is subject to feedback regulation, a peripheral impairment in thermogenesis should be associated with a compensatory increase in SNS activity. To examine SNS activity in the ob/ob mouse, norepinephrine (NE) turnover was measured in heart and interscapular brown adipose tissue (IBAT) of ob/ob and lean mice. The results from studies utilizing radiolabeled NE or inhibition of NE biosynthesis with alpha-methyl-p-tyrosine to measure NE turnover demonstrated reductions in SNS activity of 33-56% in heart and of 45-73% in IBAT in ob/ob mice at ambient temperature (22 degrees C) compared with measurements in lean controls. During cold exposure (4 degrees C) NE turnover increased in heart and IBAT to a similar extent in both ob/ob and lean mice, but NE turnover rates in heart, and probably in IBAT as well, remained lower in the obese mice than in the lean despite the gradual development of hypothermia in the ob/ob mice during this period. Administration of naltrexone, a long-acting opiate antagonist, failed to reverse the suppression of SNS activity observed in the ob/ob mice. These data indicate that diminished SNS activity in ob/ob mice may be an additional factor contributing to the defective thermogenesis characteristic of these animals.

The helicase DDX41 senses intracellular DNA mediated by the adaptor STING in dendritic cells

The recognition of pathogenic DNA is important to the initiation of antiviral responses. Here we report the identification of DDX41, a member of the DEXDc family of helicases, as an intracellular DNA sensor in myeloid dendritic cells (mDCs). Knockdown of DDX41 expression by short hairpin RNA blocked the ability of mDCs to mount type I interferon and cytokine responses to DNA and DNA viruses. Overexpression of both DDX41 and the membrane-associated adaptor STING together had a synergistic effect in promoting Ifnb promoter activity. DDX41 bound both DNA and STING and localized together with STING in the cytosol. Knockdown of DDX41 expression blocked activation of the mitogen-activated protein kinase TBK1 and the transcription factors NF-κB and IRF3 by B-form DNA. Our results suggest that DDX41 is an additional DNA sensor that depends on STING to sense pathogenic DNA.

Genetic Tests for Ecological and Allopatric Speciation in Anoles on an Island Archipelago

From Darwin's study of the Galapagos and Wallace's study of Indonesia, islands have played an important role in evolutionary investigations, and radiations within archipelagos are readily interpreted as supporting the conventional view of allopatric speciation. Even during the ongoing paradigm shift towards other modes of speciation, island radiations, such as the Lesser Antillean anoles, are thought to exemplify this process. Geological and molecular phylogenetic evidence show that, in this archipelago, Martinique anoles provide several examples of secondary contact of island species. Four precursor island species, with up to 8 mybp divergence, met when their islands coalesced to form the current island of Martinique. Moreover, adjacent anole populations also show marked adaptation to distinct habitat zonation, allowing both allopatric and ecological speciation to be tested in this system. We take advantage of this opportunity of replicated island coalescence and independent ecological adaptation to carry out an extensive population genetic study of hypervariable neutral nuclear markers to show that even after these very substantial periods of spatial isolation these putative allospecies show less reproductive isolation than conspecific populations in adjacent habitats in all three cases of subsequent island coalescence. The degree of genetic interchange shows that while there is always a significant genetic signature of past allopatry, and this may be quite strong if the selection regime allows, there is no case of complete allopatric speciation, in spite of the strong primae facie case for it. Importantly there is greater genetic isolation across the xeric/rainforest ecotone than is associated with any secondary contact. This rejects the development of reproductive isolation in allopatric divergence, but supports the potential for ecological speciation, even though full speciation has not been achieved in this case. It also explains the paucity of anole species in the Lesser Antilles compared to the Greater Antilles.

A chikungunya fever vaccine utilizing an insect-specific virus platform

Traditionally, vaccine development involves tradeoffs between immunogenicity and safety. Live-attenuated vaccines typically offer rapid and durable immunity but have reduced safety when compared to inactivated vaccines. In contrast, the inability of inactivated vaccines to replicate enhances safety at the expense of immunogenicity, often necessitating multiple doses and boosters. To overcome these tradeoffs, we developed the insect-specific alphavirus, Eilat virus (EILV), as a vaccine platform. To address the chikungunya fever (CHIKF) pandemic, we used an EILV cDNA clone to design a chimeric virus containing the chikungunya virus (CHIKV) structural proteins. The recombinant EILV/CHIKV was structurally identical at 10 Å to wild-type CHIKV, as determined by single-particle cryo-electron microscopy, and it mimicked the early stages of CHIKV replication in vertebrate cells from attachment and entry to viral RNA delivery. Yet the recombinant virus remained completely defective for productive replication, providing a high degree of safety. A single dose of EILV/CHIKV produced in mosquito cells elicited rapid (within 4 d) and long-lasting (>290 d) neutralizing antibodies that provided complete protection in two different mouse models. In nonhuman primates, EILV/CHIKV elicited rapid and robust immunity that protected against viremia and telemetrically monitored fever. Our EILV platform represents the first structurally native application of an insect-specific virus in preclinical vaccine development and highlights the potential application of such viruses in vaccinology.

RBM3 regulates temperature sensitive miR-142–5p and miR-143 (thermomiRs), which target immune genes and control fever

Fever is commonly used to diagnose disease and is consistently associated with increased mortality in critically ill patients. However, the molecular controls of elevated body temperature are poorly understood. We discovered that the expression of RNA-binding motif protein 3 (RBM3), known to respond to cold stress and to modulate microRNA (miRNA) expression, was reduced in 30 patients with fever, and in THP-1-derived macrophages maintained at a fever-like temperature (40 °C). Notably, RBM3 expression is reduced during fever whether or not infection is demonstrable. Reduced RBM3 expression resulted in increased expression of RBM3-targeted temperature-sensitive miRNAs, we termed thermomiRs. ThermomiRs such as miR-142-5p and miR-143 in turn target endogenous pyrogens including IL-6, IL6ST, TLR2, PGE2 and TNF to complete a negative feedback mechanism, which may be crucial to prevent pathological hyperthermia. Using normal PBMCs that were exogenously exposed to fever-like temperature (40 °C), we further demonstrate the trend by which decreased levels of RBM3 were associated with increased levels of miR-142-5p and miR-143 and vice versa over a 24 h time course. Collectively, our results indicate the existence of a negative feedback loop that regulates fever via reduced RBM3 levels and increased expression of miR-142-5p and miR-143.

Cryptochrome Mediates Circadian Regulation of cAMP Signaling and Hepatic Gluconeogenesis

During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)--two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)α. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.

Inflammatory cytokines in depression: neurobiological mechanisms and therapeutic implications.

Mounting evidence indicates that inflammatory cytokines contribute to the development of depression in both medically ill and medically healthy individuals. Cytokines are important for development and normal brain function, and have the ability to influence neurocircuitry and neurotransmitter systems to produce behavioral alterations. Acutely, inflammatory cytokine administration or activation of the innate immune system produces adaptive behavioral responses that promote conservation of energy to combat infection or recovery from injury. However, chronic exposure to elevated inflammatory cytokines and persistent alterations in neurotransmitter systems can lead to neuropsychiatric disorders and depression. Mechanisms of cytokine behavioral effects involve activation of inflammatory signaling pathways in the brain that results in changes in monoamine, glutamate, and neuropeptide systems, and decreases in growth factors, such as brain-derived neurotrophic factor. Furthermore, inflammatory cytokines may serve as mediators of both environmental (e.g. childhood trauma, obesity, stress, and poor sleep) and genetic (functional gene polymorphisms) factors that contribute to depression's development. This review explores the idea that specific gene polymorphisms and neurotransmitter systems can confer protection from or vulnerability to specific symptom dimensions of cytokine-related depression. Additionally, potential therapeutic strategies that target inflammatory cytokine signaling or the consequences of cytokines on neurotransmitter systems in the brain to prevent or reverse cytokine effects on behavior are discussed.

Chronic Hepatitis B Infection: A Review

Importance More than 240 million individuals worldwide are infected with chronic hepatitis B virus (HBV). Among individuals with chronic HBV infection who are untreated, 15% to 40% progress to cirrhosis, which may lead to liver failure and liver cancer. Observations Pegylated interferon and nucleos(t)ide analogues (lamivudine, adefovir, entecavir, tenofovir disoproxil, and tenofovir alafenamide) suppress HBV DNA replication and improve liver inflammation and fibrosis. Long-term viral suppression is associated with regression of liver fibrosis and reduced risk of hepatocellular carcinoma in cohort studies. The cure (defined as hepatitis B surface antigen loss with undetectable HBV DNA) rates after treatment remain low (3%-7% with pegylated interferon and 1%-12% with nucleos[t]ide analogue therapy). Pegylated interferon therapy can be completed in 48 weeks and is not associated with the development of resistance; however, its use is limited by poor tolerability and adverse effects such as bone marrow suppression and exacerbation of existing neuropsychiatric symptoms such as depression. Newer agents (entecavir, tenofovir disoproxil, and tenofovir alafenamide) may be associated with a significantly reduced risk of drug resistance compared with older agents (lamivudine and adefovir) and should be considered as the first-line treatment. Conclusions and Relevance Antiviral treatment with either pegylated interferon or a nucleos(t)ide analogue (lamivudine, adefovir, entecavir, tenofovir disoproxil, or tenofovir alafenamide) should be offered to patients with chronic HBV infection and liver inflammation in an effort to reduce progression of liver disease. Nucleos(t)ide analogues should be considered as first-line therapy. Because cure rates are low, most patients will require therapy indefinitely.

ANKTM1, a TRP-like Channel Expressed in Nociceptive Neurons, Is Activated by Cold Temperatures

Mammals detect temperature with specialized neurons in the peripheral nervous system. Four TRPV-class channels have been implicated in sensing heat, and one TRPM-class channel in sensing cold. The combined range of temperatures that activate these channels covers a majority of the relevant physiological spectrum sensed by most mammals, with a significant gap in the noxious cold range. Here, we describe the characterization of ANKTM1, a cold-activated channel with a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 is a distant family member of TRP channels with very little amino acid similarity to TRPM8. It is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1/VR1 (the capsaicin/heat receptor) but not TRPM8. Consistent with the expression of ANKTM1, we identify noxious cold-sensitive sensory neurons that also respond to capsaicin but not to menthol.

Screening for Lung Cancer: U.S. Preventive Services Task Force Recommendation Statement

The U.S. Preventive Services Task Force (USPSTF) makes recommendations about the effectiveness of specific preventive care services for patients without related signs or symptoms. It bases its recommendations on the evidence of both the benefits and harms of the service and an assessment of the balance. The USPSTF does not consider the costs of providing a service in this assessment. The USPSTF recognizes that clinical decisions involve more considerations than evidence alone. Clinicians should understand the evidence but individualize decision making to the specific patient or situation. Similarly, the USPSTF notes that policy and coverage decisions involve considerations in addition to the evidence of clinical benefits and harms. Summary of Recommendation and Evidence The USPSTF recommends annual screening for lung cancer with low-dose computed tomography (LDCT) in adults aged 55 to 80 years who have a 30 pack-year smoking history and currently smoke or have quit within the past 15 years. Screening should be discontinued once a person has not smoked for 15 years or develops a health problem that substantially limits life expectancy or the ability or willingness to have curative lung surgery. (B recommendation) See the Clinical Considerations section for suggestions for implementation in practice. See the Figure for a summary of the recommendation and suggestions for clinical practice. Figure. Screening for lung cancer: clinical summary of U.S. Preventive Services Task Force recommendation. Appendix Table 1 describes the USPSTF grades, and Appendix Table 2 describes the USPSTF classification of levels of certainty about net benefit. Appendix Table 1. What the USPSTF Grades Mean and Suggestions for Practice Appendix Table 2. USPSTF Levels of Certainty Regarding Net Benefit Supplement. Consumer Fact Sheet. Rationale Importance Lung cancer is the third most common cancer and the leading cause of cancer-related death in the United States (1). The most important risk factor for lung cancer is smoking, which results in approximately 85% of all U.S. lung cancer cases (2). Although the prevalence of smoking has decreased, approximately 37% of U.S. adults are current or former smokers (2). The incidence of lung cancer increases with age and occurs most commonly in persons aged 55 years or older. Increasing age and cumulative exposure to tobacco smoke are the 2 most common risk factors for lung cancer. Lung cancer has a poor prognosis, and nearly 90% of persons with lung cancer die of the disease. However, early-stage nonsmall cell lung cancer (NSCLC) has a better prognosis and can be treated with surgical resection. Detection Most lung cancer cases are NSCLC, and most screening programs focus on the detection and treatment of early-stage NSCLC. Although chest radiography and sputum cytologic evaluation have been used to screen for lung cancer, LDCT has greater sensitivity for detecting early-stage cancer (3). Benefits of Detection and Early Treatment Although lung cancer screening is not an alternative to smoking cessation, the USPSTF found adequate evidence that annual screening for lung cancer with LDCT in a defined population of high-risk persons can prevent a substantial number of lung cancerrelated deaths. Direct evidence from a large, well-conducted, randomized, controlled trial (RCT) provides moderate certainty of the benefit of lung cancer screening with LDCT in this population (4). The magnitude of benefit to the person depends on that person's risk for lung cancer because those who are at highest risk are most likely to benefit. Screening cannot prevent most lung cancerrelated deaths, and smoking cessation remains essential. Harms of Detection and Early Intervention and Treatment The harms associated with LDCT screening include false-negative and false-positive results, incidental findings, overdiagnosis, and radiation exposure. False-positive LDCT results occur in a substantial proportion of screened persons; 95% of all positive results do not lead to a diagnosis of cancer. In a high-quality screening program, further imaging can resolve most false-positive results; however, some patients may require invasive procedures. The USPSTF found insufficient evidence on the harms associated with incidental findings. Overdiagnosis of lung cancer occurs, but its precise magnitude is uncertain. A modeling study performed for the USPSTF estimated that 10% to 12% of screen-detected cancer cases are overdiagnosedthat is, they would not have been detected in the patient's lifetime without screening. Radiation harms, including cancer resulting from cumulative exposure to radiation, vary depending on the age at the start of screening; the number of scans received; and the person's exposure to other sources of radiation, particularly other medical imaging. USPSTF Assessment The USPSTF concludes with moderate certainty that annual screening for lung cancer with LDCT is of moderate net benefit in asymptomatic persons who are at high risk for lung cancer based on age, total cumulative exposure to tobacco smoke, and years since quitting smoking. The moderate net benefit of screening depends on limiting screening to persons who are at high risk, the accuracy of image interpretation being similar to that found in the NLST (National Lung Screening Trial), and the resolution of most false-positive results without invasive procedures (4). Clinical Considerations Patient Population Under Consideration The risk for lung cancer increases with age and cumulative exposure to tobacco smoke and decreases with time since quitting smoking. The best evidence for the benefit of screening comes from the NLST, which enrolled adults aged 55 to 74 years who had at least a 30 pack-year smoking history and were current smokers or had quit within the past 15 years. As with all screening trials, the NLST tested a specific intervention over a finite period. Because initial eligibility extended through age 74 years and participants received 3 annual screening computed tomographic scans, the oldest participants in the trial were aged 77 years. The USPSTF used modeling studies to predict the benefits and harms of screening programs that use different screening intervals, age ranges, smoking histories, and times since quitting. A program that annually screens adults aged 55 to 80 years who have a 30 pack-year smoking history and currently smoke or have quit within the past 15 years is projected to have a reasonable balance of benefits and harms. The model assumes that persons who achieve 15 years of smoking cessation during the screening program discontinue screening. This model predicts the outcomes of continuing the screening program used in the NLST through age 80 years. Screening may not be appropriate for patients with substantial comorbid conditions, particularly those at the upper end of the screening age range. The NLST excluded persons who were unlikely to complete curative lung cancer surgery and those with medical conditions that posed a substantial risk for death during the 8-year trial. The baseline characteristics of the NLST showed a relatively healthy sample, and fewer than 10% of enrolled participants were older than 70 years (5). Persons with serious comorbid conditions may experience net harm, no net benefit, or at least substantially less net benefit. Similarly, persons who are unwilling to have curative lung surgery are unlikely to benefit from a screening program. Assessment of Risk Age, total exposure to tobacco smoke, and years since quitting smoking are important risk factors for lung cancer and were used to determine eligibility in the NLST. Other risk factors include specific occupational exposures, radon exposure, family history, and history of pulmonary fibrosis or chronic obstructive lung disease. The incidence of lung cancer is relatively low in persons younger than 50 years but increases with age, especially after age 60 years. In current and former smokers, age-specific incidence rates increase with age and cumulative exposure to tobacco smoke. Smoking cessation substantially reduces a person's risk for developing and dying of lung cancer. Among persons enrolled in the NLST, those who were at highest risk because of additional risk factors or a greater cumulative exposure to tobacco smoke experienced most of the benefit (6). A validated multivariate model showed that persons in the highest 60% of risk accounted for 88% of all deaths preventable by screening. Screening Tests Low-dose computed tomography has shown high sensitivity and acceptable specificity for the detection of lung cancer in high-risk persons. Chest radiography and sputum cytologic evaluation have not shown adequate sensitivity or specificity as screening tests. Therefore, LDCT is currently the only recommended screening test for lung cancer. Treatment Surgical resection is the current standard of care for localized NSCLC. This type of cancer is treated with surgical resection when possible and also with radiation and chemotherapy. Annual LDCT screening may not be useful for patients with life-limiting comorbid conditions or poor functional status who may not be candidates for surgery. Other Approaches to Prevention Smoking cessation is the most important intervention to prevent NSCLC. Advising smokers to stop smoking and preventing nonsmokers from being exposed to tobacco smoke are the most effective ways to decrease the morbidity and mortality associated with lung cancer. Current smokers should be informed of their continuing risk for lung cancer and offered cessation treatments. Screening with LDCT should be viewed as an adjunct to tobacco cessation interventions. Useful Resources Clinicians have many resources to help patients stop smoking. The Centers for Disease Control and Prevention has developed a Web site with many such resources, including information on tobacco quit lines, available in several languages (www.cdc.gov/tobacco/campaign/tips). Quit l

(99m)Tc-HisoDGR as a Potential SPECT Probe for Orthotopic Glioma Detection via Targeting of Integrin α5β1.

Integrins, a large family of cell adhesion receptors, have been shown to play an important role for glioma proliferation and invasion. Several integrin receptors, including αvβ3, αvβ5, and α5β1, have generated clinical interest for glioma diagnosis and antitumor therapy. Integrin α5β1 has been highlighted as a prognostic and diagnostic marker in glioma, and its expression is correlated with a worse prognosis in high-grade glioma. However, unlike extensively studied integrins αvβ3 and αvβ5, very few integrin α5β1-specific radiotracers have been reported. Developing α5β1-specific radiotracers may provide alternative diagnosis and evaluation options in addition to well-studied αvβ3/αvβ5-specific tracers, and they may add new documents for profiling tumor progression. Here, a novel integrin α5β1-specific probe (99m)Tc-HisoDGR was fabricated for SPECT (single-photon emission computed tomography) imaging of glioma. To confirm its selective targeting of integrin α5β1 in vivo, the mouse models of α5β1-positive U87MG human glioma were subjected to SPECT/CT scans, and biodistribution experiments and blocking studies were performed. Small-animal SPECT/CT imaging experiments demonstrated that the tumors were clearly visualized in both subcutaneous and orthotopic glioma tumor models with clear background at 0.5, 1, and 2 h p.i. The tumor accumulation of (99m)Tc-HisoDGR showed significant reduction when excess cold isoDGR peptide was coinjected, suggesting that the tumor uptake was specifically mediated. Our work revealed that (99m)Tc-HisoDGR represented a powerful molecular probe for integrin α5β1-positive cancer imaging; moreover, it might be a promising tool for evaluating malignancy, predicting prognosis, selecting subpopulations of patients who might be sensitive to integrin α5β1-targeted drugs, and assessing and monitoring the response to integrin α5β1-targeted drugs in clinical trials.

Membrane association of the CD3ε signaling domain is required for optimal T cell development and function.

The TCR:CD3 complex transduces signals that are critical for optimal T cell development and adaptive immunity. In resting T cells, the CD3ε cytoplasmic tail associates with the plasma membrane via a proximal basic-rich stretch (BRS). In this study, we show that mice lacking a functional CD3ε-BRS exhibited substantial reductions in thymic cellularity and limited CD4- CD8- double-negative (DN) 3 to DN4 thymocyte transition, because of enhanced DN4 TCR signaling resulting in increased cell death and TCR downregulation in all subsequent populations. Furthermore, positive, but not negative, T cell selection was affected in mice lacking a functional CD3ε-BRS, which led to limited peripheral T cell function and substantially reduced responsiveness to influenza infection. Collectively, these results indicate that membrane association of the CD3ε signaling domain is required for optimal thymocyte development and peripheral T cell function.

Caveolin-1 deletion exacerbates cardiac interstitial fibrosis by promoting M2 macrophage activation in mice after myocardial infarction.

Adverse remodeling following myocardial infarction (MI) leading to heart failure is driven by an imbalanced resolution of inflammation. The macrophage cell is an important control of post-MI inflammation, as macrophage subtypes secrete mediators to either promote inflammation and extend injury (M1 phenotype) or suppress inflammation and promote scar formation (M2 phenotype). We have previously shown that the absence of caveolin-1 (Cav1), a membrane scaffolding protein, is associated with adverse cardiac remodeling in mice, but the mechanisms responsible remain to be elucidated. We explore here the role of Cav1 in the activation of macrophages using wild type C57BL6/J (WT) and Cav1(tm1Mls/J) (Cav1(-/-)) mice. By echocardiography, cardiac function was comparable between WT and Cav1(-/-) mice at 3days post-MI. In the absence of Cav1, there were a surprisingly higher percentage of M2 macrophages (arginase-1 positive) detected in the infarcted zone. Conversely, restoring Cav1 function after MI in WT mice by adding back the Cav1 scaffolding domain reduced the M2 activation profile. Further, adoptive transfer of Cav1 null macrophages into WT mice on d3 post-MI exacerbated adverse cardiac remodeling at d14 post-MI. In vitro studies revealed that Cav1 null macrophages had a more pronounced M2 profile activation in response to IL-4 stimulation. In conclusion, Cav1 deletion promotes an array of maladaptive repair processes after MI, including increased TGF-β signaling, increased M2 macrophage infiltration and dysregulation of the M1/M2 balance. Our data also suggest that cardiac remodeling can be improved by therapeutic intervention regulating Cav1 function during the inflammatory response phase.

The inflammasome component NLRP3 impairs antitumor vaccine by enhancing the accumulation of tumor-associated myeloid-derived suppressor cells.

The inflammasome is a proteolysis complex that generates the active forms of the proinflammatory cytokines interleukin (IL)-1β and IL-18. Inflammasome activation is mediated by NLR proteins that respond to microbial and nonmicrobial stimuli. Among NLRs, NLRP3 senses the widest array of stimuli and enhances adaptive immunity. However, its role in antitumor immunity is unknown. Therefore, we evaluated the function of the NLRP3 inflammasome in the immune response using dendritic cell vaccination against the poorly immunogenic melanoma cell line B16-F10. Vaccination of Nlrp3(-/-) mice led to a relative 4-fold improvement in survival relative to control animals. Immunity depended on CD8(+) T cells and exhibited immune specificity and memory. Increased vaccine efficacy in Nlrp3(-/-) hosts did not reflect differences in dendritic cells but rather differences in myeloid-derived suppressor cells (MDSC). Although Nlrp3 was expressed in MDSCs, the absence of Nlrp3 did not alter either their functional capacity to inhibit T cells or their presence in peripheral lymphoid tissues. Instead, the absence of Nlrp3 caused a 5-fold reduction in the number of tumor-associated MDSCs found in host mice. Adoptive transfer experiments also showed that Nlrp3(-/-) MDSCs were less efficient in reaching the tumor site. Depleting MDSCs with an anti-Gr-1 antibody increased the survival of tumor-bearing wild-type mice but not Nlrp3(-/-) mice. We concluded that Nlrp3 was critical for accumulation of MDSCs in tumors and for inhibition of antitumor T-cell immunity after dendritic cell vaccination. Our findings establish an unexpected role for Nlrp3 in impeding antitumor immune responses, suggesting novel approaches to improve the response to antitumor vaccines by limiting Nlrp3 signaling.

Sputum inflammation predicts exacerbations after cessation of inhaled corticosteroids in COPD.

INTRODUCTION The GOLD guidelines advocate not to institute inhaled corticosteroids (ICS) in patients with mild-to-moderate COPD. However, many patients do use ICS and in some patients, withdrawal is associated with deteriorating lung function and increased exacerbation rates. Unfortunately, physicians do not know in which patients they can stop ICS treatment safely. AIM To identify predictors of COPD exacerbations after ICS withdrawal. METHODS During ICS treatment, post-bronchodilator spirometry, body plethysmography, and health status assessment were performed in 68 COPD patients using ICS. Additionally, sputum cell differentials, supernatant leukotriene B(4), eosinophilic cationic protein, and myeloperoxidase, serum C-reactive protein and soluble intracellular adhesion molecule, and urinary desmosine were assessed. Sputum was also analysed for mRNA levels of haemoxygenase-1, tumour necrosis factor-α, RANTES, interleukin 5(IL-5), IL-10, IL-12, IL-13, transforming growth factor-β, and interferon-γ. STATISTICS Cox regression analyses were performed using time to exacerbation as outcome variable to identify significant hazards for a COPD exacerbation after ICS withdrawal. RESULTS Higher sputum % eosinophils, higher sputum MPO/neutrophil level, longer duration of COPD symptoms, <40 packyears smoking, and ICS withdrawal in November, December or January were significant hazards (all p<0.05) for experiencing a COPD exacerbation after ICS withdrawal in a monovariate model. In a multivariate model, all factors proved independent predictors except for sputum MPO/neutrophil level. CONCLUSIONS Decisions on whether or not inhaled corticosteroids can be safely withdrawn in mild-to-moderate COPD can be facilitated by assessment of sputum inflammation, particularly eosinophil numbers, next to packyears smoking, season, and duration of COPD symptoms.

Human Cytomegalovirus-Encoded Interleukin-10 Homolog Inhibits Maturation of Dendritic Cells and Alters Their Functionality

Interleukin-10 (IL-10) suppresses the maturation and cytokine production of dendritic cells (DCs), key regulators of adaptive immunity, and prevents the activation and polarization of naïve T cells towards protective gamma interferon-producing effectors. We hypothesized that human cytomegalovirus (HCMV) utilizes its viral IL-10 homolog (cmvIL-10) to attenuate DC functionality, thereby subverting the efficient induction of antiviral immune responses. RNA and protein analyses demonstrated that the cmvIL-10 gene was expressed with late gene kinetics. Treatment of immature DCs (iDCs) with supernatant from HCMV-infected cultures inhibited both the lipopolysaccharide-induced DC maturation and proinflammatory cytokine production. These inhibitory effects were specifically mediated through the IL-10 receptor and were not observed when DCs were treated with supernatant of cells infected with a cmvIL-10-knockout mutant. Incubation of iDCs with recombinant cmvIL-10 recapitulated the inhibition of maturation. Furthermore, cmvIL-10 had pronounced long-term effects on those DCs that could overcome this inhibition of maturation. It enhanced the migration of mature DCs (mDCs) towards the lymph node homing chemokine but greatly reduced their cytokine production. The inability of mDCs to secrete IL-12 was maintained, even when they were restimulated by the activated T-cell signal CD40 ligand in the absence of cmvIL-10. Importantly, cmvIL-10 potentiates these anti-inflammatory effects, at least partially, by inducing endogenous cellular IL-10 expression in DCs. Collectively, we show that cmvIL-10 causes long-term functional alterations at all stages of DC activation.

Endothelial-to-mesenchymal transition contributes to cardiac fibrosis

Cardiac fibrosis, associated with a decreased extent of microvasculature and with disruption of normal myocardial structures, results from excessive deposition of extracellular matrix, which is mediated by the recruitment of fibroblasts. The source of these fibroblasts is unclear and specific anti-fibrotic therapies are not currently available. Here we show that cardiac fibrosis is associated with the emergence of fibroblasts originating from endothelial cells, suggesting an endothelial-mesenchymal transition (EndMT) similar to events that occur during formation of the atrioventricular cushion in the embryonic heart. Transforming growth factor-β1 (TGF-β1) induced endothelial cells to undergo EndMT, whereas bone morphogenic protein 7 (BMP-7) preserved the endothelial phenotype. The systemic administration of recombinant human BMP-7 (rhBMP-7) significantly inhibited EndMT and the progression of cardiac fibrosis in mouse models of pressure overload and chronic allograft rejection. Our findings show that EndMT contributes to the progression of cardiac fibrosis and that rhBMP-7 can be used to inhibit EndMT and to intervene in the progression of chronic heart disease associated with fibrosis.

Histone acetyltransferase PCAF regulates inflammatory molecules in the development of renal injury.

Kidney diseases, including chronic kidney disease (CKD) and acute kidney injury (AKI), are associated with inflammation. The mechanism that regulates inflammation in these renal injuries remains unclear. Here, we demonstrated that p300/CBP-associated factor (PCAF), a histone acetyltransferase, was overexpressed in the kidneys of db/db mice and lipopolysaccharide (LPS)-injected mice. Moreover, elevated histone acetylation, such as H3K18ac, and up-regulation of some inflammatory genes, such as ICAM-1, VCAM-1, and MCP-1, were found upon these renal injuries. Furthermore, increased H3K18ac was recruited to the promoters of ICAM-1, VCAM-1, and MCP-1 in the kidneys of LPS-injected mice. In vitro studies demonstrated that PCAF knockdown in human renal proximal tubule epithelial cells (HK-2) led to downregulation of inflammatory molecules, including VCAM-1, ICAM-1, p50 subunit of NF-κB (p50), and MCP-1 mRNA and protein levels, together with significantly decreased H3K18ac level. Consistent with these, overexpression of PCAF enhanced the expression of inflammatory molecules. Furthermore, PCAF deficiency reduced palmitate-induced recruitment of H3K18ac on the promoters of ICAM-1 and MCP-1, as well as inhibited palmitate-induced upregulation of these inflammatory molecules. In summary, the present work demonstrates that PCAF plays an essential role in the regulation of inflammatory molecules through H3K18ac, which provides a potential therapeutic target for inflammation-related renal diseases.

Biofeedback and relaxation in the treatment of migraine headaches: comparative effectiveness and physiological correlates.

Twenty-seven migraine headache patients were divided into three equal groups which received thermal biofeedback, frontalis EMG biofeedback, or relaxation training. Training was given under "massed" practice conditions (nine sessions per week) and consisted of 18 training sessions and six test-generalisation sessions. While improvements in headaches were observed in all groups, the best improvements took place in the thermal biofeedback group, which had almost complete elimination of migraine attacks by the end of training, and maintained that performance as long as six months after training. Examination of the patterns of skin temperature and EMG changes in the three groups over the course of training also points to a relationship between skin temperature control and reduction in migraine headache symptomatology, and suggests that this relationship is worthy of further investigation.

Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses.

The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.

Vegan proteins may reduce risk of cancer, obesity, and cardiovascular disease by promoting increased glucagon activity.

Amino acids modulate the secretion of both insulin and glucagon; the composition of dietary protein therefore has the potential to influence the balance of glucagon and insulin activity. Soy protein, as well as many other vegan proteins, are higher in non-essential amino acids than most animal-derived food proteins, and as a result should preferentially favor glucagon production. Acting on hepatocytes, glucagon promotes (and insulin inhibits) cAMP-dependent mechanisms that down-regulate lipogenic enzymes and cholesterol synthesis, while up-regulating hepatic LDL receptors and production of the IGF-I antagonist IGFBP-1. The insulin-sensitizing properties of many vegan diets--high in fiber, low in saturated fat--should amplify these effects by down-regulating insulin secretion. Additionally, the relatively low essential amino acid content of some vegan diets may decrease hepatic IGF-I synthesis. Thus, diets featuring vegan proteins can be expected to lower elevated serum lipid levels, promote weight loss, and decrease circulating IGF-I activity. The latter effect should impede cancer induction (as is seen in animal studies with soy protein), lessen neutrophil-mediated inflammatory damage, and slow growth and maturation in children. In fact, vegans tend to have low serum lipids, lean physiques, shorter stature, later puberty, and decreased risk for certain prominent 'Western' cancers; a vegan diet has documented clinical efficacy in rheumatoid arthritis. Low-fat vegan diets may be especially protective in regard to cancers linked to insulin resistance--namely, breast and colon cancer--as well as prostate cancer; conversely, the high IGF-I activity associated with heavy ingestion of animal products may be largely responsible for the epidemic of 'Western' cancers in wealthy societies. Increased phytochemical intake is also likely to contribute to the reduction of cancer risk in vegans. Regression of coronary stenoses has been documented during low-fat vegan diets coupled with exercise training; such regimens also tend to markedly improve diabetic control and lower elevated blood pressure. Risk of many other degenerative disorders may be decreased in vegans, although reduced growth factor activity may be responsible for an increased risk of hemorrhagic stroke. By altering the glucagon/insulin balance, it is conceivable that supplemental intakes of key non-essential amino acids could enable omnivores to enjoy some of the health advantages of a vegan diet. An unnecessarily high intake of essential amino acids--either in the absolute sense or relative to total dietary protein--may prove to be as grave a risk factor for 'Western' degenerative diseases as is excessive fat intake.

Batf is important for IL-4 expression in T follicular helper cells

Apart from T helper (Th)-2 cells, T follicular helper (Tfh) cells are a major class of IL-4-producing T cells, required for regulation of type 2 humoral immunity; however, transcriptional control of IL-4 production in Tfh cells remains mainly unknown. Here, we show that the basic leucine zipper transcription factor ATF-like, Batf is important for IL-4 expression in Tfh cells rather than in canonical Th2 cells. Functionally, Batf in cooperation with interferon regulatory factor (IRF) 4 along with Stat3 and Stat6 trigger IL-4 production in Tfh cells by directly binding to and activation of the CNS2 region in the IL-4 locus. In addition, Batf-to-c-Maf signalling is an important determinant of IL-4 expression in Tfh cells. Batf deficiency impairs the generation of IL-4-producing Tfh cells that results in protection against allergic asthma. Our results thus indicate a positive role of Batf in promoting the generation of pro-allergic IL-4-producing Tfh cells.

The Bile Acid Chenodeoxycholic Acid Increases Human Brown Adipose Tissue Activity.

The interest in brown adipose tissue (BAT) as a target to combat metabolic disease has recently been renewed with the discovery of functional BAT in humans. In rodents, BAT can be activated by bile acids, which activate type 2 iodothyronine deiodinase (D2) in BAT via the G-coupled protein receptor TGR5, resulting in increased oxygen consumption and energy expenditure. Here we examined the effects of oral supplementation of the bile acid chenodeoxycholic acid (CDCA) on human BAT activity. Treatment of 12 healthy female subjects with CDCA for 2 days resulted in increased BAT activity. Whole-body energy expenditure was also increased upon CDCA treatment. In vitro treatment of primary human brown adipocytes derived with CDCA or specific TGR5 agonists increased mitochondrial uncoupling and D2 expression, an effect that was absent in human primary white adipocytes. These findings identify bile acids as a target to activate BAT in humans.

EMSY overexpression disrupts the BRCA2/RAD51 pathway in the DNA-damage response: implications for chromosomal instability/recombination syndromes as checkpoint diseases

EMSY links the BRCA2 pathway to sporadic breast/ovarian cancer. It encodes a nuclear protein that binds to the BRCA2 N-terminal domain implicated in chromatin/transcription regulation, but when sporadically amplified/overexpressed, increased EMSY level represses BRCA2 transactivation potential and induces chromosomal instability, mimicking the activity of BRCA2 mutations in the development of hereditary breast/ovarian cancer. In addition to chromatin/transcription regulation, EMSY may also play a role in the DNA-damage response, suggested by its ability to localize at chromatin sites of DNA damage/repair. This implies that EMSY overexpression may also repress BRCA2 in DNA-damage replication/checkpoint and recombination/repair, coordinated processes that also require its interacting proteins: PALB2, the partner and localizer of BRCA2; RPA, replication/checkpoint protein A; and RAD51, the inseparable recombination/repair enzyme. Here, using a well-characterized recombination/repair assay system, we demonstrate that a slight increase in EMSY level can indeed repress these two processes independently of transcriptional interference/repression. Since EMSY, RPA and PALB2 all bind to the same BRCA2 region, these findings further support a scenario wherein: (a) EMSY amplification may mimic BRCA2 deficiency, at least by overriding RPA and PALB2, crippling the BRCA2/RAD51 complex at DNA-damage and replication/transcription sites; and (b) BRCA2/RAD51 may coordinate these processes by employing at least EMSY, PALB2 and RPA. We extensively discuss the molecular details of how this can happen to ascertain its implications for a novel recombination mechanism apparently conceived as checkpoint rather than a DNA repair system for cell division, survival, death, and human diseases, including the tissue specificity of cancer predisposition, which may renew our thinking about targeted therapy and prevention.

Sudden infant death syndrome.

Despite declines in prevalence during the past two decades, sudden infant death syndrome (SIDS) continues to be the leading cause of death for infants aged between 1 month and 1 year in developed countries. Behavioural risk factors identified in epidemiological studies include prone and side positions for infant sleep, smoke exposure, soft bedding and sleep surfaces, and overheating. Evidence also suggests that pacifier use at sleep time and room sharing without bed sharing are associated with decreased risk of SIDS. Although the cause of SIDS is unknown, immature cardiorespiratory autonomic control and failure of arousal responsiveness from sleep are important factors. Gene polymorphisms relating to serotonin transport and autonomic nervous system development might make affected infants more vulnerable to SIDS. Campaigns for risk reduction have helped to reduce SIDS incidence by 50-90%. However, to reduce the incidence even further, greater strides must be made in reducing prenatal smoke exposure and implementing other recommended infant care practices. Continued research is needed to identify the pathophysiological basis of SIDS.

Dietary factors in chronic inflammation: food tolerances and intolerances of a New Zealand Caucasian Crohn's disease population.

Diet is known to play a major role in the symptoms of the inflammatory bowel disease, Crohn's disease (CD). Although no single diet is appropriate to all individuals, most CD patients are aware of foods that provide adverse or beneficial effects. This study seeks to categorise foods in relation to their effects on symptoms of CD, in a New Zealand Caucasian population. Four hundred and forty-six subjects from two different centres in New Zealand were recruited into the study. An extensive dietary questionnaire (257 food items in 15 groups) recorded self-reported dietary tolerances and intolerances. Across each of the food groups, there were statistically significant differences among responses to foods. A two-dimensional graphical summary enabled stratification of foods according to the probability that they will be either beneficial or detrimental. A small number of foods are frequently considered to be beneficial, including white fish, salmon and tuna, gluten-free products, oatmeal, bananas, boiled potatoes, sweet potatoes (kumara), pumpkin, soya milk, goat's milk and yoghurt. Foods that are typically considered detrimental include grapefruit, chilli or chilli sauce, corn and corn products, peanuts, cream, salami, curried foods, cola drinks, high energy drinks, beer, and red wine. For a number of the food items, the same item that was beneficial for one group of subjects was detrimental to others; in particular soya milk, goat's milk, yoghurt, oatmeal, kiwifruit, prunes, apple, broccoli, cauliflower, linseed, pumpkin seed, sunflower seed, ginger and ginger products, beef, lamb, liver, and oily fish. It was not possible to identify a specific group of food items that should be avoided by all CD patients. The wide range of detrimental items suggests that dietary maintenance of remission is likely to be difficult, and to exclude a substantial number of foods. Personalised diets may be especially important to these individuals.

Deregulated DNA polymerase β strengthens ionizing radiation-induced nucleotidic and chromosomal instabilities

DNA polymerase β (Pol β) is an error-prone enzyme which has been found to be overexpressed in several human tumors. By using a couple of recombinant CHO cells differing only from the exogenous expression of Pol β, we showed here that cells overexpressing Pol β are much more sensitive to IR treatments by increasing apoptosis. We also found that the surviving cells displayed an hypermutator phenotype which could be explained by different pathways involving Pol β, such as (i) an increased capacity to incorporate into DNA the mutagenic dGTP analog, 8-oxo-dGTP, one of the most abundant purine-derived nucleotides exposed to γ-irradiation, (ii) the induction of IR-induced DNA breaks and (iii) accumulation of chromosome aberrations induced by radiation. Alteration of Pol β expression in irradiated cells thus appears to strengthen both cell death and genetic changes associated with a malignant phenotype. These data provide new insights into the cellular response to radiations and the associated carcinogenic consequences.

Improving population representation through geographic health information systems: mapping the MURDOCK study.

This paper highlights methods for using geospatial analysis to assess, enhance, and improve recruitment efforts to ensure representativeness in study populations. We apply these methods to the Measurement to Understand Reclassification of Disease of Cabarrus/Kannapolis (MURDOCK) study, a longitudinal population health study focused on the city of Kannapolis and Cabarrus County, NC. Although efforts have been made to recruit a participant registry that is representative of the 18 ZIP code catchment region inclusive of Cabarrus County and Kannapolis, bias in such recruitment is inevitable. Participants in the MURDOCK study are geospatially referenced at entry, providing information that can be used to monitor and guide recruitment efforts. MURDOCK participant population representativeness was assessed using chi-squared tests to compare the MURDOCK population with 2010 Census data, relative to both the entire 18 ZIP code catchment area and for individual Census tracts. A logistic regression model was fit to characterize Census tracts with low recruitment, defined by fewer than 56 participants from that tract. The distance to the site at which participants enrolled was calculated, and median distance to enrollment site was used in the logistic regression. Tracts with low recruitment rates contained higher minority and younger populations, suggesting specific strategies for improving recruitment in these areas. Areal units farther away from enrollment sites were also not well-sampled, despite being in the specified study area, indicating that distance traveled to enrollment may be a barrier. These results have implications for targeting recruitment efforts and representative samples more generally, including in other population-based studies.

Phenotypic deficits in the HIV-1 envelope are associated with the maturation of a V2-directed broadly neutralizing antibody lineage

Broadly neutralizing antibodies (bnAbs) to HIV-1 can evolve after years of an iterative process of virus escape and antibody adaptation that HIV-1 vaccine design seeks to mimic. To enable this, properties that render HIV-1 envelopes (Env) capable of eliciting bnAb responses need to be defined. Here, we followed the evolution of the V2 apex directed bnAb lineage VRC26 in the HIV-1 subtype C superinfected donor CAP256 to investigate the phenotypic changes of the virus populations circulating before and during the early phases of bnAb induction. Longitudinal viruses that evolved from the VRC26-resistant primary infecting (PI) virus, the VRC26-sensitive superinfecting (SU) virus and ensuing PI-SU recombinants revealed substantial phenotypic changes in Env, with a switch in Env properties coinciding with early resistance to VRC26. Decreased sensitivity of SU-like viruses to VRC26 was linked with reduced infectivity, altered entry kinetics and lower sensitivity to neutralization after CD4 attachment. VRC26 maintained neutralization activity against cell-associated CAP256 virus, indicating that escape through the cell-cell transmission route is not a dominant escape pathway. Reduced fitness of the early escape variants and sustained sensitivity in cell-cell transmission are both features that limit virus replication, thereby impeding rapid escape. This supports a scenario where VRC26 allowed only partial viral escape for a prolonged period, possibly increasing the time window for bnAb maturation. Collectively, our data highlight the phenotypic plasticity of the HIV-1 Env in evading bnAb pressure and the need to consider phenotypic traits when selecting and designing Env immunogens. Combinations of Env variants with differential phenotypic patterns and bnAb sensitivity, as we describe here for CAP256, may maximize the potential for inducing bnAb responses by vaccination.

Naringenin inhibits allergen-induced airway inflammation and airway responsiveness and inhibits NF-kappaB activity in a murine model of asthma.

Naringenin, a flavonoid, has antiinflammatory and immunomodulatory properties. We investigated whether naringenin could attenuate allergen-induced airway inflammation and its possible mechanism in a murine model of asthma. Mice were sensitized and challenged with ovalbumin. Some mice were administered with naringenin before ovalbumin challenge. We evaluated the development of airway inflammation and airway reactivity. Interleukin (IL)4, IL13, chemokine (C-C motif) ligand (CCL)5, and CCL11 in bronchoalveolar lavage fluid and serum total IgE were detected by ELISA. IkappaBalpha degradation and inducible nitric oxide synthase (iNOS) in lungs were measured by Western blot. We also tested NF-kappaB binding activity by electrophoretic mobility shift assay. The mRNA levels of iNOS, CCL5, and CCL11 were detected by real-time PCR. Naringenin attenuated ovalbumin-induced airway inflammation and airway reactivity in experimental mice. The naringenin-treated mice had lower levels of IL4 and IL13 in the bronchoalveolar lavage fluid and lower serum total IgE. Furthermore, naringenin inhibited pulmonary IkappaBalpha degradation and NF-kappaB DNA-binding activity. The levels of CCL5, CCL11, and iNOS were also significantly reduced. The results indicated that naringenin may play protective roles in the asthma process. The inhibition of NF-kappaB and the decreased expression of its target genes may account for this phenomenon.

Disruption of the immune-checkpoint VISTA gene imparts a proinflammatory phenotype with predisposition to the development of autoimmunity.

V domain-containing Ig suppressor of T-cell activation (VISTA) is a negative checkpoint regulator that suppresses T cell-mediated immune responses. Previous studies using a VISTA-neutralizing monoclonal antibody show that VISTA blockade enhances T-cell activation. The current study describes a comprehensive characterization of mice in which the gene for VISTA has been deleted. Despite the apparent normal hematopoietic development in young mice, VISTA genetic deficiency leads to a gradual accumulation of spontaneously activated T cells, accompanied by the production of a spectrum of inflammatory cytokines and chemokines. Enhanced T-cell responsiveness was also observed upon immunization with neoantigen. Despite the presence of multiorgan chronic inflammation, aged VISTA-deficient mice did not develop systemic or organ-specific autoimmune disease. Interbreeding of the VISTA-deficient mice with 2D2 T-cell receptor transgenic mice, which are predisposed to the development of experimental autoimmune encephalomyelitis, drastically enhanced disease incidence and intensity. Disease development is correlated with the increase in the activation of encephalitogenic T cells in the periphery and enhanced infiltration into the CNS. Taken together, our data suggest that VISTA is a negative checkpoint regulator whose loss of function lowers the threshold for T-cell activation, allowing for an enhanced proinflammatory phenotype and an increase in the frequency and intensity of autoimmunity under susceptible conditions.

Brahma-related gene 1-dependent STAT3 recruitment at IL-6-inducible genes.

IL-6 is an immunoregulatory cytokine with multiple functions in hemopoiesis, proliferation, and tumorigenesis. IL-6 triggers phosphorylation, dimerization, and nuclear translocation of STAT3, which binds to target promoters and activates transcription. Brahma-related gene 1 (BRG1), the enzymatic engine of the yeast-mating type-switching and sucrose-nonfermenting chromatin-remodeling complex, is essential for recruitment of STAT1 or STAT1/STAT2-containing complexes to IFN targets. We hypothesized that BRG1 might also be required for STAT3 recruitment. In this study, we show that induction of a subset of human IL-6-responsive genes is BRG1 dependent. BRG1 is constitutively present at these targets and is required for STAT3 recruitment, downstream histone modifications, and IL-6-induced chromatin remodeling. IL-6-induced recruitment of STAT3 to the IFN regulatory factor 1 promoter and subsequent mRNA synthesis is BRG1 dependent, even though IFN-gamma-mediated STAT1 recruitment to this locus is BRG1 independent. BRG1 also increased basal expression of IFN-induced transmembrane protein 3 and IFN-gamma-induced protein 16, and the basal chromatin accessibility at the promoter of IFN regulatory factor 1. The effect on basal expression was STAT3 independent, as revealed by small interfering RNA knockdown. Together with prior observations, these data reveal that BRG1 has a broad role in mediating STAT accessibility at multiple cytokine-responsive promoters and exposes promoter specific differences in both the effect of BRG1 on basal chromatin accessibility and on access of different STAT proteins to the same target.

Exercise-induced asthma screening of elite athletes: field versus laboratory exercise challenge.

PURPOSE The purpose of this study was to compare a laboratory based exercise challenge (LBC) to a field based exercise challenge (FBC) for pulmonary function test (PFT) exercise-induced asthma (EIA) screening of elite athletes. METHODS Twenty-three elite cold weather athletes (14 men, 9 women) PFT positive for EIA (FBC screened) served as subjects. Twenty-three gender and sport matched controls (nonasthmatics) were randomly selected to establish PFT reference values for normal elite athletes. Before FBC, athletes completed a medical history questionnaire for EIA symptoms. FBC evaluations consisted of baseline spirometry, actual or simulated competition, and 5, 10, and 15 min postexercise spirometry. PFT positive athletes were evaluated in the laboratory using an exercise challenge simulating race intensity (ambient conditions: 21 degrees C, 60% relative humidity). PFT procedures were identical to FBC. RESULTS 91% of PFT positive and 48% of PFT normal athletes reported at least one symptom of EIA, with postrace cough most frequent. Baseline spirometry was the same for PFT positives and normal controls. Lower limit reference range (MN - 2 SD) of FEV1 for controls suggests that postexercise decrements of greater than approximately -7% indicate abnormal airway response in this population. Exercise time duration did not effect bronchial reactivity; 78% of FBC PFT positives were PFT normal post-LBC. CONCLUSION Self-reported symptoms by elite athletes are not reliable in identifying EIA. Reference range criterion for FEV1 decrement in the elite athlete postexercise contrasts current recommended guidelines. Moreover, a large number of false negatives may occur in this population if EIA screening is performed with inadequate exercise and environmental stress.

Loss of immune escape mutations during persistent HCV infection in pregnancy enhances replication of vertically transmitted viruses

Globally, about 1% of pregnant women are persistently infected with the hepatitis C virus (HCV). Mother-to-child transmission of HCV occurs in 3-5% of pregnancies and accounts for most new childhood infections. HCV-specific CD8(+) cytotoxic T lymphocytes (CTLs) are vital in the clearance of acute HCV infections, but in the 60-80% of infections that persist, these cells become functionally exhausted or select for mutant viruses that escape T cell recognition. Increased HCV replication during pregnancy suggests that maternofetal immune tolerance mechanisms may further impair HCV-specific CTLs, limiting their selective pressure on persistent viruses. To assess this possibility, we characterized circulating viral quasispecies during and after consecutive pregnancies in two women. This revealed a loss of some escape mutations in HLA class I epitopes during pregnancy that was associated with emergence of more fit viruses. CTL selective pressure was reimposed after childbirth, at which point escape mutations in these epitopes again predominated in the quasispecies and viral load dropped sharply. Importantly, the viruses transmitted perinatally were those with enhanced fitness due to reversion of escape mutations. Our findings indicate that the immunoregulatory changes of pregnancy reduce CTL selective pressure on HCV class I epitopes, thereby facilitating vertical transmission of viruses with optimized replicative fitness.

Oxidative damage of DNA confers resistance to cytosolic nuclease TREX1 degradation and potentiates STING-dependent immune sensing.

Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3' repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity.

The processing of Holliday junctions by BLM and WRN helicases is regulated by p53.

BLM, WRN, and p53 are involved in the homologous DNA recombination pathway. The DNA structure-specific helicases, BLM and WRN, unwind Holliday junctions (HJ), an activity that could suppress inappropriate homologous recombination during DNA replication. Here, we show that purified, recombinant p53 binds to BLM and WRN helicases and attenuates their ability to unwind synthetic HJ in vitro. The p53 248W mutant reduces abilities of both to bind HJ and inhibit helicase activities, whereas the p53 273H mutant loses these abilities. Moreover, full-length p53 and a C-terminal polypeptide (residues 373-383) inhibit the BLM and WRN helicase activities, but phosphorylation at Ser(376) or Ser(378) completely abolishes this inhibition. Following blockage of DNA replication, Ser(15) phospho-p53, BLM, and RAD51 colocalize in nuclear foci at sites likely to contain DNA replication intermediates in cells. Our results are consistent with a novel mechanism for p53-mediated regulation of DNA recombinational repair that involves p53 post-translational modifications and functional protein-protein interactions with BLM and WRN DNA helicases.

Suppression of inflammatory and neuropathic pain by uncoupling CRMP-2 from the presynaptic Ca2+ channel complex

The use of N-type voltage-gated calcium channel (CaV2.2) blockers to treat pain is limited by many physiological side effects. Here we report that inflammatory and neuropathic hypersensitivity can be suppressed by inhibiting the binding of collapsin response mediator protein 2 (CRMP-2) to CaV2.2 and thereby reducing channel function. A peptide of CRMP-2 fused to the HIV transactivator of transcription (TAT) protein (TAT-CBD3) decreased neuropeptide release from sensory neurons and excitatory synaptic transmission in dorsal horn neurons, reduced meningeal blood flow, reduced nocifensive behavior induced by formalin injection or corneal capsaicin application and reversed neuropathic hypersensitivity produced by an antiretroviral drug. TAT-CBD3 was mildly anxiolytic without affecting memory retrieval, sensorimotor function or depression. At doses tenfold higher than that required to reduce hypersensitivity in vivo, TAT-CBD3 caused a transient episode of tail kinking and body contortion. By preventing CRMP-2–mediated enhancement of CaV2.2 function, TAT-CBD3 alleviated inflammatory and neuropathic hypersensitivity, an approach that may prove useful in managing chronic pain.

Preferential occupancy of histone variant H2AZ at inactive promoters influences local histone modifications and chromatin remodeling.

The yeast histone variant H2AZ (Htz1) is implicated in transcription activation, prevention of the ectopic spread of heterochromatin, and genome integrity. Our genome-wide localization analysis revealed that Htz1 is widely, but nonrandomly, distributed throughout the genome in an SWR1-dependent manner. We found that Htz1 is enriched in intergenic regions compared with coding regions. Its occupancy is inversely proportional to transcription rates and the enrichment of the RNA polymerase II under different growth conditions. However, Htz1 does not seem to directly regulate transcription repression genome-wide; instead, the presence of Htz1 under the inactivated condition is essential for optimal activation of a subset of genes. In addition, Htz1 is not generally responsible for nucleosome positioning, even at those promoters where Htz1 is highly enriched. Finally, using a biochemical approach, we demonstrate that incorporation of Htz1 into nucleosomes inhibits activities of histone modifiers associated with transcription, Dot1, Set2, and NuA4 and reduces the nucleosome mobilization driven by chromatin remodeling complexes. These lines of evidence collectively suggest that Htz1 may serve to mark quiescent promoters for proper activation.

Tissue exit: a novel control point in the accumulation of antigen-specific CD8 T cells in the influenza a virus-infected lung.

Memory/effector T cells efficiently migrate into extralymphoid tissues and sites of infection, providing immunosurveillance and a first line of defense against invading pathogens. Even though it is a potential means to regulate the size, quality, and duration of a tissue infiltrate, T cell egress from infected tissues is poorly understood. Using a mouse model of influenza A virus infection, we found that CD8 effector T cells egressed from the infected lung in a CCR7-dependent manner. In contrast, following antigen recognition, effector CD8 T cell egress decreased and CCR7 function was reduced in vivo and in vitro, indicating that the exit of CD8 T cells from infected tissues is tightly regulated. Our data suggest that the regulation of T cell egress is a mechanism to retain antigen-specific effectors at the site of infection to promote viral clearance, while decreasing the numbers of bystander T cells and preventing overt inflammation.

Katanin p60-like1 promotes microtubule growth and terminal dendrite stability in the larval class IV sensory neurons of Drosophila.

Dendrite shape is considered a defining component of neuronal function. Yet, the mechanisms specifying diverse dendritic morphologies, and the extent to which their function depends on these morphologies, remain unclear. Here, we demonstrate a requirement for the microtubule-severing protein katanin p60-like 1 (Kat-60L1) in regulating the elaborate dendrite morphology and nocifensive functions of Drosophila larval class IV dendritic arborization neurons. Kat-60L1 mutants exhibit diminished responsiveness to noxious mechanical and thermal stimuli. Class IV dendrite branch number and length are also reduced, supporting a correspondence between neuronal function and the full extent of the dendritic arbor. These arborization defects occur particularly in late larval development, and live imaging reveals that Kat-60L1 is required for dynamic, filopodia-like nascent branches to stabilize during this stage. Mutant dendrites exhibit fewer EB1-GFP-labeled microtubules, suggesting that Kat-60L1 increases polymerizing microtubules to establish terminal branch stability and full arbor complexity. Although loss of the related microtubule-severing protein Spastin also reduces the class IV dendrite arbor, microtubule polymerization within dendrites is unaffected. Conversely, Spastin overexpression destroys stable microtubules within these neurons, while Kat-60L1 has no effect. Kat-60L1 thus sculpts the class IV dendritic arbor through microtubule regulatory mechanisms distinct from Spastin. Our data support differential roles of microtubule-severing proteins in regulating neuronal morphology and function, and provide evidence that dendritic arbor development is the product of multiple pathways functioning at distinct developmental stages.

The mammalian Scribble polarity protein regulates epithelial cell adhesion and migration through E-cadherin

Scribble (Scrib) is a conserved polarity protein required in Drosophila melanogaster for synaptic function, neuroblast differentiation, and epithelial polarization. It is also a tumor suppressor. In rodents, Scrib has been implicated in receptor recycling and planar polarity but not in apical/basal polarity. We now show that knockdown of Scrib disrupts adhesion between Madin–Darby canine kidney epithelial cells. As a consequence, the cells acquire a mesenchymal appearance, migrate more rapidly, and lose directionality. Although tight junction assembly is delayed, confluent monolayers remain polarized. These effects are independent of Rac activation or Scrib binding to βPIX. Rather, Scrib depletion disrupts E-cadherin–mediated cell–cell adhesion. The changes in morphology and migration are phenocopied by E-cadherin knockdown. Adhesion is partially rescued by expression of an E-cadherin–α-catenin fusion protein but not by E-cadherin–green fluorescent protein. These results suggest that Scrib stabilizes the coupling between E-cadherin and the catenins and are consistent with the idea that mammalian Scrib could behave as a tumor suppressor by regulating epithelial cell adhesion and migration.

Fingolimod provides long-term protection in rodent models of cerebral ischemia.

OBJECTIVE The sphingosine-1-phosphate (S1P) receptor agonist fingolimod (FTY720), that has shown efficacy in advanced multiple sclerosis clinical trials, decreases reperfusion injury in heart, liver, and kidney. We therefore tested the therapeutic effects of fingolimod in several rodent models of focal cerebral ischemia. To assess the translational significance of these findings, we asked whether fingolimod improved long-term behavioral outcomes, whether delayed treatment was still effective, and whether neuroprotection can be obtained in a second species. METHODS We used rodent models of middle cerebral artery occlusion and cell-culture models of neurotoxicity and inflammation to examine the therapeutic potential and mechanisms of neuroprotection by fingolimod. RESULTS In a transient mouse model, fingolimod reduced infarct size, neurological deficit, edema, and the number of dying cells in the core and periinfarct area. Neuroprotection was accompanied by decreased inflammation, as fingolimod-treated mice had fewer activated neutrophils, microglia/macrophages, and intercellular adhesion molecule-1 (ICAM-1)-positive blood vessels. Fingolimod-treated mice showed a smaller infarct and performed better in behavioral tests up to 15 days after ischemia. Reduced infarct was observed in a permanent model even when mice were treated 4 hours after ischemic onset. Fingolimod also decreased infarct size in a rat model of focal ischemia. Fingolimod did not protect primary neurons against glutamate excitotoxicity or hydrogen peroxide, but decreased ICAM-1 expression in brain endothelial cells stimulated by tumor necrosis factor alpha. INTERPRETATION These findings suggest that anti-inflammatory mechanisms, and possibly vasculoprotection, rather than direct effects on neurons, underlie the beneficial effects of fingolimod after stroke. S1P receptors are a highly promising target in stroke treatment.

Varenicline, an alpha4beta2 nicotinic acetylcholine receptor partial agonist, selectively decreases ethanol consumption and seeking.

Alcohol dependence is a disease that impacts millions of individuals worldwide. There has been some progress with pharmacotherapy for alcohol-dependent individuals; however, there remains a critical need for the development of novel and additional therapeutic approaches. Alcohol and nicotine are commonly abused together, and there is evidence that neuronal nicotinic acetylcholine receptors (nAChRs) play a role in both alcohol and nicotine dependence. Varenicline, a partial agonist at the alpha4beta2 nAChRs, reduces nicotine intake and was recently approved as a smoking cessation aid. We have investigated the role of varenicline in the modulation of ethanol consumption and seeking using three different animal models of drinking. We show that acute administration of varenicline, in doses reported to reduce nicotine reward, selectively reduced ethanol but not sucrose seeking using an operant self-administration drinking paradigm and also decreased voluntary ethanol but not water consumption in animals chronically exposed to ethanol for 2 months before varenicline treatment. Furthermore, chronic varenicline administration decreased ethanol consumption, which did not result in a rebound increase in ethanol intake when the varenicline was no longer administered. The data suggest that the alpha4beta2 nAChRs may play a role in ethanol-seeking behaviors in animals chronically exposed to ethanol. The selectivity of varenicline in decreasing ethanol consumption combined with its reported safety profile and mild side effects in humans suggest that varenicline may prove to be a treatment for alcohol dependence.

Interaction of TNF with TNF receptor type 2 promotes expansion and function of mouse CD4+CD25+ T regulatory cells.

Although TNF is a major proinflammatory cytokine, increasing evidence indicates that TNF also has immunosuppressive feedback effects. We have demonstrated in this study that, in both resting and activated states, mouse peripheral CD4(+)CD25(+) T regulatory cells (Tregs) expressed remarkably higher surface levels of TNFR2 than CD4(+)CD25(-) T effector cells (Teffs). In cocultures of Tregs and Teffs, inhibition of proliferation of Teffs by Tregs was initially transiently abrogated by exposure to TNF, but longer exposure to TNF restored suppressive effects. Cytokine production by Teffs remained continually suppressed by Tregs. The profound anergy of Tregs in response to TCR stimulation was overcome by TNF, which expanded the Treg population. Furthermore, in synergy with IL-2, TNF expanded Tregs even more markedly up-regulated expression of CD25 and FoxP3 and phosphorylation of STAT5, and enhanced the suppressive activity of Tregs. Unlike TNF, IL-1beta and IL-6 did not up-regulate FoxP3-expressing Tregs. Furthermore, the number of Tregs increased in wild-type mice, but not in TNFR2(-/-) mice following sublethal cecal ligation and puncture. Depletion of Tregs significantly decreased mortality following cecal ligation and puncture. Thus, the stimulatory effect of TNF on Tregs resembles the reported costimulatory effects of TNF on Teffs, but is even more pronounced because of the higher expression of TNFR2 by Tregs. Moreover, our study suggests that the slower response of Tregs than Teffs to TNF results in delayed immunosuppressive feedback effects.

Chimeric Antigen Receptor-Redirected Regulatory T Cells Suppress Experimental Allergic Airway Inflammation, a Model of Asthma

Cellular therapy with chimeric antigen receptor (CAR)-redirected cytotoxic T cells has shown impressive efficacy in the treatment of hematologic malignancies. We explored a regulatory T cell (Treg)-based therapy in the treatment of allergic airway inflammation, a model for asthma, which is characterized by an airway hyper-reactivity (AHR) and a chronic, T helper-2 (Th2) cell-dominated immune response to allergen. To restore the immune balance in the lung, we redirected Tregs by a CAR toward lung epithelia in mice upon experimentally induced allergic asthma, closely mimicking the clinical situation. Adoptively transferred CAR Tregs accumulated in the lung and in tracheobronchial lymph nodes, reduced AHR and diminished eosinophilic airway inflammation, indicated by lower cell numbers in the bronchoalveolar lavage fluid and decreased cell infiltrates in the lung. CAR Treg cells furthermore prevented excessive pulmonary mucus production as well as increase in allergen-specific IgE and Th2 cytokine levels in exposed animals. CAR Tregs were more efficient in controlling asthma than non-modified Tregs, indicating the pivotal role of specific Treg cell activation in the affected organ. Data demonstrate that lung targeting CAR Treg cells ameliorate key features of experimental airway inflammation, paving the way for cell therapy of severe allergic asthma.

Differential role of CbpA and PspA in modulation of in vitro CXC chemokine responses of respiratory epithelial cells to infection with Streptococcus pneumoniae.

Respiratory epithelial cells play an active part in the host response to respiratory pathogens, such as Streptococcus pneumoniae, by releasing chemokines responsible for neutrophil recruitment. In order to investigate the role of specific pneumococcal virulence factors in eliciting CXC chemokine responses, type II pneumocytes (A549) and nasopharyngeal cells (Detroit-562) were infected with S. pneumoniae D39 or mutants lacking choline-binding protein A (CbpA), pneumococcal surface protein A (PspA), or specific domains thereof. In response to wild-type D39, both A549 and Detroit-562 cells showed a significant increase in CXC chemokine mRNA and interleukin-8 protein. This response was increased twofold when a cbpA deletion mutant (DeltaCbpA) was used, suggesting that CbpA inhibits CXC chemokine induction. All three N-terminal domains of CbpA are required for this effect, as in-frame deletion of the respective region of cbpA had the same effect on the CXC chemokine response as deletion of cbpA altogether. Infection with a pspA deletion mutant (DeltaPspA) led to a twofold decrease in the CXC chemokine response of A549 but not Detroit-562 cells, compared to infection with D39 at 2 h. Thus, PspA appears to have the ability to stimulate early CXC chemokine release from A549 cells. Deletion of the region of pspA encoding the first N-terminal alpha-helical domain reduced the ability of S. pneumoniae to elicit a chemokine response to the same degree as deletion of pspA altogether. Thus, the N termini of CbpA and PspA exert differential effects on CXC chemokine induction in epithelial cells infected with S. pneumoniae.

Effects of Bifidobacterium animalis administered during lactation on allergic and autoimmune responses in rodents.

Probiotics are promoted as being beneficial to health and positive effects on the immune system have been reported. Beneficial immune effects have been attributed to several mechanisms, including stimulating T helper 1 (Th1) immunity. To explore the effects of the probiotic Bifidobacterium animalis on Th1- and Th2-mediated immune responses, two different animal models representing either Th1- or Th2-mediated immune responses were used: a rat model for experimental autoimmune encephalomyelitis (EAE) (Th1) and a mouse model for respiratory allergy induced by ovalbumin (OVA) (Th2). B. animalis administration started when the mice or rats were 2 weeks old. Respiratory allergy or EAE were induced when the animals were 6-7 weeks old. In the allergy model, B. animalis modestly reduced the number of infiltrating eosinophils and lymphocytes in the lungs, but no effects on allergen-specific serum immunoglobulin E levels were found. Cytokine profiles assessed after culturing spleen cells with the mitogen concanvalin A (ConA) showed that B. animalis skewed the Th1/Th2 balance towards Th1 in females. However, allergen-induced cytokine production in females was not affected by B. animalis. In males, B. animalis significantly decreased ConA-induced interleukin-13 and a trend towards lower levels of OVA-induced Th2 cytokines. In the EAE model, B. animalis significantly reduced the duration of clinical symptoms by almost 2 days in males and improved the body weight gain during the experimental period compared with the control group. Our data show that B. animalis reduced several immune parameters in the allergy as well as in the autoimmunity model.

tBid induces alterations of mitochondrial fatty acid oxidation flux by malonyl-CoA-independent inhibition of carnitine palmitoyltransferase-1

Recent studies suggest a close relationship between cell metabolism and apoptosis. We have evaluated changes in lipid metabolism on permeabilized hepatocytes treated with truncated Bid (tBid) in the presence of caspase inhibitors and exogenous cytochrome c. The measurement of β-oxidation flux by labeled palmitate demonstrates that tBid inhibits β-oxidation, thereby resulting in the accumulation of palmitoyl-coenzyme A (CoA) and depletion of acetyl-carnitine and acylcarnitines, which is pathognomonic for inhibition of carnitine palmitoyltransferase-1 (CPT-1). We also show that tBid decreases CPT-1 activity by a mechanism independent of both malonyl-CoA, the key inhibitory molecule of CPT-1, and Bak and/or Bax, but dependent on cardiolipin decrease. Overexpression of Bcl-2, which is able to interact with CPT-1, counteracts the effects exerted by tBid on β-oxidation. The unexpected role of tBid in the regulation of lipid β-oxidation suggests a model in which tBid-induced metabolic decline leads to the accumulation of toxic lipid metabolites such as palmitoyl-CoA, which might become participants in the apoptotic pathway.

Estimation of the dietary requirement for vitamin D in healthy adults.

BACKGROUND Knowledge gaps have contributed to considerable variation among international dietary recommendations for vitamin D.   OBJECTIVE We aimed to establish the distribution of dietary vitamin D required to maintain serum 25-hydroxyvitamin D [25(OH)D] concentrations above several proposed cutoffs (ie, 25, 37.5, 50, and 80 nmol/L) during wintertime after adjustment for the effect of summer sunshine exposure and diet. DESIGN A randomized, placebo-controlled, double-blind 22-wk intervention study was conducted in men and women aged 20-40 y (n = 238) by using different supplemental doses (0, 5, 10, and 15 microg/d) of vitamin D(3) throughout the winter. Serum 25(OH)D concentrations were measured by using enzyme-linked immunoassay at baseline (October 2006) and endpoint (March 2007). RESULTS There were clear dose-related increments (P < 0.0001) in serum 25(OH)D with increasing supplemental vitamin D(3). The slope of the relation between vitamin D intake and serum 25(OH)D was 1.96 nmol x L(-1) x microg(-1) intake. The vitamin D intake that maintained serum 25(OH)D concentrations of >25 nmol/L in 97.5% of the sample was 8.7 microg/d. This intake ranged from 7.2 microg/d in those who enjoyed sunshine exposure, 8.8 microg/d in those who sometimes had sun exposure, and 12.3 microg/d in those who avoided sunshine. Vitamin D intakes required to maintain serum 25(OH)D concentrations of >37.5, >50, and >80 nmol/L in 97.5% of the sample were 19.9, 28.0, and 41.1 microg/d, respectively. CONCLUSION The range of vitamin D intakes required to ensure maintenance of wintertime vitamin D status [as defined by incremental cutoffs of serum 25(OH)D] in the vast majority (>97.5%) of 20-40-y-old adults, considering a variety of sun exposure preferences, is between 7.2 and 41.1 microg/d.

Combining indoor residual spraying and insecticide-treated nets for malaria control in Africa: a review of possible outcomes and an outline of suggestions for the future

Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) are currently the preferred methods of malaria vector control. In many cases, these methods are used together in the same households, especially to suppress transmission in holoendemic and hyperendemic scenarios. Though widespread, there has been limited evidence suggesting that such co-application confers greater protective benefits than either ITNs or IRS when used alone. Since both methods are insecticide-based and intradomicilliary, this article hypothesises that outcomes of their combination would depend on effects of the candidate active ingredients on mosquitoes that enter or those that attempt to enter houses. It is suggested here that enhanced household level protection can be achieved if the ITNs and IRS have divergent yet complementary properties, e.g. highly deterrent IRS compounds coupled with highly toxic ITNs. To ensure that the problem of insecticide resistance is avoided, the ITNs and IRS products should preferably be of different insecticide classes, e.g. pyrethroid-based nets combined with organophosphate or carbamate based IRS. The overall community benefits would however depend also on other factors such as proportion of people covered by the interventions and the behaviour of vector species. This article concludes by emphasizing the need for basic and operational research, including mathematical modelling to evaluate IRS/ITN combinations in comparison to IRS alone or ITNs alone.

N348I in HIV-1 reverse transcriptase can counteract the nevirapine-mediated bias toward RNase H cleavage during plus-strand initiation.

Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (+)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.

Plasmid-encoded proinsulin preserves C-peptide while specifically reducing proinsulin-specific CD8⁺ T cells in type 1 diabetes.

In type 1 diabetes (T1D), there is an intense inflammatory response that destroys the β cells in the pancreatic islets of Langerhans, the site where insulin is produced and released. A therapy for T1D that targets the specific autoimmune response in this disease while leaving the remainder of the immune system intact, has long been sought. Proinsulin is a major target of the adaptive immune response in T1D. We hypothesized that an engineered DNA plasmid encoding proinsulin (BHT-3021) would preserve β cell function in T1D patients through reduction of insulin-specific CD8⁺ T cells. We studied 80 subjects over 18 years of age who were diagnosed with T1D within the past 5 years. Subjects were randomized 2:1 to receive intramuscular injections of BHT-3021 or BHT-placebo, weekly for 12 weeks, and then monitored for safety and immune responses in a blinded fashion. Four dose levels of BHT-3021 were evaluated: 0.3, 1.0, 3.0, and 6.0 mg. C-peptide was used both as an exploratory efficacy measure and as a safety measure. Islet-specific CD8⁺ T cell frequencies were assessed with multimers of monomeric human leukocyte antigen class I molecules loaded with peptides from pancreatic and unrelated antigens. No serious adverse events related to BHT-3021 were observed. C-peptide levels improved relative to placebo at all doses, at 1 mg at the 15-week time point (+19.5% BHT-3021 versus -8.8% BHT-placebo, P < 0.026). Proinsulin-reactive CD8⁺ T cells, but not T cells against unrelated islet or foreign molecules, declined in the BHT-3021 arm (P < 0.006). No significant changes were noted in interferon-γ, interleukin-4 (IL-4), or IL-10 production in CD4 T cells. Thus, we demonstrate that a plasmid encoding proinsulin reduces the frequency of CD8⁺ T cells reactive to proinsulin while preserving C-peptide over the course of dosing.

[Neutrophils and macrophages related to the pathogenesis and disease development of chronic obstructive pulmonary disease by the inflammatory reaction].

Chronic obstructive pulmonary disease (COPD) is a chronic airway disorder characterized by obstructive airflow limitation which is not completely reversible with treatment. Inflammatory changes in the peripheral airways, especially those with the diameter less than 2mm (so-called small airway disease) have been speculated to be initial steps of COPD. And so it must be quite clear that neutrophils and macrophages play an essential role in the pathogenesis of these lesions. Studies with bronchoalveolar lavage demonstrated an increase in neutrophil numbers and the neutrophil chemoattractant interleukin-8. Recent studies demonstrated that neutrophils and macrophages are increased and contain a variety of proteases, which are involved in cell infiltration and activation. Studies with gene-engineered animals and anti-cytokine treatment will facilitate better understanding the role of neutrophils and macrophages, and eventual novel therapy.

Lung dendritic cells imprint T cell lung homing and promote lung immunity through the chemokine receptor CCR4

T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC-activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC-imprinted T cells protect against influenza more effectively than do gut and skin DC-imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC-activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC-activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4.

Extensive diversity of Ig-superfamily proteins in the immune system of insects.

The extensive somatic diversification of immune receptors is a hallmark of higher vertebrates. However, whether molecular diversity contributes to immune protection in invertebrates is unknown. We present evidence that Drosophila immune-competent cells have the potential to express more than 18,000 isoforms of the immunoglobulin (Ig)-superfamily receptor Down syndrome cell adhesion molecule (Dscam). Secreted protein isoforms of Dscam were detected in the hemolymph, and hemocyte-specific loss of Dscam impaired the efficiency of phagocytic uptake of bacteria, possibly due to reduced bacterial binding. Importantly, the molecular diversity of Dscam transcripts generated through a mechanism of alternative splicing is highly conserved across major insect orders, suggesting an unsuspected molecular complexity of the innate immune system of insects.

A dynamic population model to investigate effects of climate on geographic range and seasonality of the tick Ixodes scapularis.

A dynamic population model of Ixodes scapularis, the vector of a number of tick-borne zoonoses in North America, was developed to simulate effects of temperature on tick survival and seasonality. Tick development rates were modelled as temperature-dependent time delays, calculated using mean monthly normal temperature data from specific meteorological stations. Temperature also influenced host-finding success in the model. Using data from stations near endemic populations of I. scapularis, the model reached repeatable, stable, cyclical equilibria with seasonal activity of different instars being very close to that observed in the field. In simulations run using data from meteorological stations in central and eastern Canada, the maximum equilibrium numbers of ticks declined the further north was the station location, and simulated populations died out at more northerly stations. Tick die-out at northern latitudes was due to a steady increase in mortality of all life stages with decreasing temperature rather than a specific threshold event in phenology of one life stage. By linear regression we investigated mean annual numbers of degree-days >0 degrees C (DD>0 degrees C) as a readily mapped index of the temperature conditions at the meteorological stations providing temperature data for the model. Maximum numbers of ticks at equilibrium were strongly associated with the mean DD>0 degrees C (r2>0.96, P<0.001), when the Province of origin of the meteorological station was accounted for (Quebec>Ontario, beta=103, P<0.001). The intercepts of the regression models provided theoretical limits for the establishment of I. scapularis in Canada. Maps of these limits suggested that the range of southeast Canada where temperature conditions are currently suitable for the tick, is much wider than the existing distribution of I. scapularis, implying that there is potential for spread. Future applications of the model in investigating climate change effects on I. scapularis are discussed.

Metabolic modifiers as performance-enhancing technologies for livestock production

With the current and predicted increase in world population, growing global demand and consumption of food will result in increasing competition for land, water, and energy. In turn, this will severely affect our ability to produce food as will the urgent requirement to reduce the impact of food systems on the environment (Godfray et al., 2010). Globalization has boosted trade in livestock inputs and products and resulted in industry growth, and concurrently livestock production has undergone a complex process of technical and geographical change (Gerber et al., 2007). This has resulted in a challenge to livestock producers; growing demand for their produce with a dwindling supply of resources, with the only solution being a significant increase in efficiency. Also, as incomes increase in the burgeoning economies, so does the demand for high-quality animal proteins such as meat, milk, and eggs; thus the Food and Agriculture Organization of the United Nations suggests that food requirements will increase by 70% by 2050 (Anonymous, 2009). The recent success in developed and emerging technologies suggests that the animal industries are well placed to prosper through these new challenges. To ensure these technologies—such as metabolic modifiers including somatotropin, immunization against gonadotropin-releasing factor, and orally active dietary additives like ractopamine, zilpaterol, cysteamine, chromium, betaine, and dietary neuroleptics—can be effectively utilized throughout the animal industries, further emphasis is required on their acceptance and development. As a result of these technological advancements, producers have benefited because of improved production efficiencies while meat packers have improved processing efficiencies because of increased lean meat yield. Ostensibly, the consumer has also benefited because meat is leaner and less expensive to purchase. However, there have been some concerns that the focus on increasing production efficiency and lean meat yield has been to the detriment of meat quality (Dunshea et al., 2005). It is also interesting that at a time of apparently greater need for these technologies, there are external influences such as market differentiation and trade barriers as well as consumer resistance that challenge the use of technologies. A tenet of this article is that if we are to meet the increased global demand for animal protein, then we must continue to develop and adopt technologies to improve livestock efficiency, but we must be cognizant of the potential barriers affecting acceptance.

High prevalence of asthma in cross country skiers.

OBJECTIVES To study the prevalence of asthma (asthma symptoms and bronchial hyperresponsiveness) in Swedish cross country skiers compared with non-skiers and monitor changes in symptoms and bronchial hyperresponsiveness during the year. DESIGN Cross sectional study during the winter ski season and in the summer. SETTING Six ski clubs for élite skiers (total 47) in two different areas of Sweden. SUBJECTS 42 élite cross country skiers and 29 non-skiing referents. MAIN OUTCOME MEASURES Bronchial responsiveness, asthma symptoms, and lung function. RESULTS Bronchial responsiveness was significantly greater and asthma symptoms more prevalent in the skiers than in the referents. There was no difference in bronchial responsiveness within either group between winter and summer. 15 of the 42 skiers used antiasthmatic drugs regularly and 23 had a combination of asthma symptoms and hyperresponsive airways or physician diagnosed asthma, or both. Altogether 33 skiers had symptoms of asthma or bronchial hyperresponsiveness. One of the referents had symptoms of asthma and bronchial hyperresponsiveness, and none used antiasthmatic drugs regularly. CONCLUSIONS Asthma, asthma-like symptoms, and bronchial hyperresponsiveness are much more common in cross country skiers than in the general population and non-skiers. Strenuous exercise at low temperatures entailing breathing large volumes of cold air is the most probable explanation of persistent asthma in skiers.

painDETECT: a new screening questionnaire to identify neuropathic components in patients with back pain.

OBJECTIVE Nociceptive and neuropathic components both contribute to pain. Since these components require different pain management strategies, correct pain diagnosis before and during treatment is highly desirable. As low back pain (LBP) patients constitute an important subgroup of chronic pain patients, we addressed the following issues: (i) to establish a simple, validated screening tool to detect neuropathic pain (NeP) components in chronic LBP patients, (ii) to determine the prevalence of neuropathic pain components in LBP in a large-scale survey, and (iii) to determine whether LBP patients with an NeP component suffer from worse, or different, co-morbidities. METHODS In co-operation with the German Research Network on Neuropathic Pain we developed and validated the painDETECT questionnaire (PD-Q) in a prospective, multicentre study and subsequently applied it to approximately 8000 LBP patients. RESULTS The PD-Q is a reliable screening tool with high sensitivity, specificity and positive predictive accuracy; these were 84% in a palm-top computerised version and 85%, 80% and 83%, respectively, in a corresponding pencil-and-paper questionnaire. In an unselected cohort of chronic LBP patients, 37% were found to have predominantly neuropathic pain. Patients with NeP showed higher ratings of pain intensity, with more (and more severe) co-morbidities such as depression, panic/anxiety and sleep disorders. This also affected functionality and use of health-care resources. On the basis of given prevalence of LBP in the general population, we calculated that 14.5% of all female and 11.4% of all male Germans suffer from LBP with a predominant neuropathic pain component. CONCLUSION Simple, patient-based, easy-to-use screening questionnaires can determine the prevalence of neuropathic pain components both in individual LBP patients and in heterogeneous cohorts of such patients. Since NeP correlates with more intense pain, more severe co-morbidity and poorer quality of life, accurate diagnosis is a milestone in choosing appropriate therapy.

Role of the Parasite-Derived Prostaglandin D2 in the Inhibition of Epidermal Langerhans Cell Migration during Schistosomiasis Infection

Epidermal Langerhans cells (LCs) play a key role in immune defense mechanisms and in numerous immunological disorders. In this report, we show that percutaneous infection of C57BL/6 mice with the helminth parasite Schistosoma mansoni leads to the activation of LCs but, surprisingly, to their retention in the epidermis. Moreover, using an experimental model of LC migration induced by tumor necrosis factor (TNF)-α, we show that parasites transiently impair the departure of LCs from the epidermis and their subsequent accumulation as dendritic cells in the draining lymph nodes. The inhibitory effect is mediated by soluble lipophilic factors released by the parasites and not by host-derived antiinflammatory cytokines, such as interleukin-10. We find that prostaglandin (PG)D2, but not the other major eicosanoids produced by the parasites, specifically impedes the TNF-α–triggered migration of LCs through the adenylate cyclase–coupled PGD2 receptor (DP receptor). Moreover, the potent DP receptor antagonist BW A868C restores LC migration in infected mice. Finally, in a model of contact allergen-induced LC migration, we show that activation of the DP receptor not only inhibits LC emigration but also dramatically reduces the contact hypersensitivity responses after challenge. Taken together, we propose that the inhibition of LC migration could represent an additional stratagem for the schistosomes to escape the host immune system and that PGD2 may play a key role in the control of cutaneous immune responses.

A Novel Adaptor Protein Orchestrates Receptor Patterning and Cytoskeletal Polarity in T-Cell Contacts

Recognition of antigen by T cells requires the formation of a specialized junction between the T cell and the antigen-presenting cell. This junction is generated by the recruitment and the exclusion of specific proteins from the contact area. The mechanisms that regulate these events are unknown. Here we demonstrate that ligand engagement of the adhesion molecule, CD2, initiates a process of protein segregation, CD2 clustering, and cytoskeletal polarization. Although protein segregation was not dependent on the cytoplasmic domain of CD2, CD2 clustering and cytoskeletal polarization required an interaction of the CD2 cytoplasmic domain with a novel SH3-containing protein. This novel protein, called CD2AP, is likely to facilitate receptor patterning in the contact area by linking specific adhesion receptors to the cytoskeleton.

Estrogen receptor-alpha promotes alternative macrophage activation during cutaneous repair.

Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERβ, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.

Secondary aerosolization of viable Bacillus anthracis spores in a contaminated US Senate Office.

CONTEXT Bioterrorist attacks involving letters and mail-handling systems in Washington, DC, resulted in Bacillus anthracis (anthrax) spore contamination in the Hart Senate Office Building and other facilities in the US Capitol's vicinity. OBJECTIVE To provide information about the nature and extent of indoor secondary aerosolization of B anthracis spores. DESIGN Stationary and personal air samples, surface dust, and swab samples were collected under semiquiescent (minimal activities) and then simulated active office conditions to estimate secondary aerosolization of B anthracis spores. Nominal size characteristics, airborne concentrations, and surface contamination of B anthracis particles (colony-forming units) were evaluated. RESULTS Viable B anthracis spores reaerosolized under semiquiescent conditions, with a marked increase in reaerosolization during simulated active office conditions. Increases were observed for B anthracis collected on open sheep blood agar plates (P<.001) and personal air monitors (P =.01) during active office conditions. More than 80% of the B anthracis particles collected on stationary monitors were within an alveolar respirable size range of 0.95 to 3.5 micro m.   CONCLUSIONS Bacillus anthracis spores used in a recent terrorist incident reaerosolized under common office activities. These findings have important implications for appropriate respiratory protection, remediation, and reoccupancy of contaminated office environments.

Activin–like kinase–3 activity is important for kidney regeneration and reversal of fibrosis

Molecules associated with the transforming growth factor β (TGF-β) superfamily, such as bone morphogenic proteins (BMPs) and TGF-β, are key regulators of inflammation, apoptosis and cellular transitions. Here we show that the BMP receptor activin-like kinase 3 (Alk3) is elevated early in diseased kidneys after injury. We also found that its deletion in the tubular epithelium leads to enhanced TGF-β1-Smad family member 3 (Smad3) signaling, epithelial damage and fibrosis, suggesting a protective role for Alk3-mediated signaling in the kidney. A structure-function analysis of the BMP-Alk3-BMP receptor, type 2 (BMPR2) ligand-receptor complex, along with synthetic organic chemistry, led us to construct a library of small peptide agonists of BMP signaling that function through the Alk3 receptor. One such peptide agonist, THR-123, suppressed inflammation, apoptosis and the epithelial-to-mesenchymal transition program and reversed established fibrosis in five mouse models of acute and chronic renal injury. THR-123 acts specifically through Alk3 signaling, as mice with a targeted deletion for Alk3 in their tubular epithelium did not respond to therapy with THR-123. Combining THR-123 and the angiotensin-converting enzyme inhibitor captopril had an additive therapeutic benefit in controlling renal fibrosis. Our studies show that BMP signaling agonists constitute a new line of therapeutic agents with potential utility in the clinic to induce regeneration, repair and reverse established fibrosis.

Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model.

Pertussis is a highly contagious respiratory illness caused by the bacterial pathogen Bordetella pertussis. Pertussis rates in the United States have been rising and reached a 50-y high of 42,000 cases in 2012. Although pertussis resurgence is not completely understood, we hypothesize that current acellular pertussis (aP) vaccines fail to prevent colonization and transmission. To test our hypothesis, infant baboons were vaccinated at 2, 4, and 6 mo of age with aP or whole-cell pertussis (wP) vaccines and challenged with B. pertussis at 7 mo. Infection was followed by quantifying colonization in nasopharyngeal washes and monitoring leukocytosis and symptoms. Baboons vaccinated with aP were protected from severe pertussis-associated symptoms but not from colonization, did not clear the infection faster than naïve animals, and readily transmitted B. pertussis to unvaccinated contacts. Vaccination with wP induced a more rapid clearance compared with naïve and aP-vaccinated animals. By comparison, previously infected animals were not colonized upon secondary infection. Although all vaccinated and previously infected animals had robust serum antibody responses, we found key differences in T-cell immunity. Previously infected animals and wP-vaccinated animals possess strong B. pertussis-specific T helper 17 (Th17) memory and Th1 memory, whereas aP vaccination induced a Th1/Th2 response instead. The observation that aP, which induces an immune response mismatched to that induced by natural infection, fails to prevent colonization or transmission provides a plausible explanation for the resurgence of pertussis and suggests that optimal control of pertussis will require the development of improved vaccines.

Morin Attenuates Ovalbumin-Induced Airway Inflammation by Modulating Oxidative Stress-Responsive MAPK Signaling.

Asthma is one of the most common inflammatory diseases characterized by airway hyperresponsiveness, inflammation, and remodeling. Morin, an active ingredient obtained from Moraceae plants, has been demonstrated to have promising anti-inflammatory activities in a range of disorders. However, its impacts on pulmonary diseases, particularly on asthma, have not been clarified. This study was designed to investigate whether morin alleviates airway inflammation in chronic asthma with an emphasis on oxidative stress modulation. In vivo, ovalbumin- (OVA-) sensitized mice were administered with morin or dexamethasone before challenge. Bronchoalveolar lavage fluid (BALF) and lung tissues were obtained to perform cell counts, histological analysis, and enzyme-linked immunosorbent assay. In vitro, human bronchial epithelial cells (BECs) were challenged by tumor necrosis factor alpha (TNF-α). The supernatant was collected for the detection of the proinflammatory proteins, and the cells were collected for reactive oxygen species (ROS)/mitogen-activated protein kinase (MAPK) evaluations. Severe inflammatory responses and remodeling were observed in the airways of the OVA-sensitized mice. Treatment with morin dramatically attenuated the extensive trafficking of inflammatory cells into the BALF and inhibited their infiltration around the respiratory tracts and vessels. Morin administration also significantly suppressed goblet cell hyperplasia and collagen deposition/fibrosis and dose-dependently inhibited the OVA-induced increases in IgE, TNF-α, interleukin- (IL-) 4, IL-13, matrix metalloproteinase-9, and malondialdehyde. In human BECs challenged by TNF-α, the levels of proteins such as eotaxin-1, monocyte chemoattractant protein-1, IL-8 and intercellular adhesion molecule-1, were consistently significantly decreased by morin. Western blotting and the 2',7'-dichlorofluorescein assay revealed that the increases in intracellular ROS and MAPK phosphorylation were abolished by morin, implying that ROS/MAPK signaling contributes to the relief of airway inflammation. Our findings indicate for the first time that morin alleviates airway inflammation in chronic asthma, which probably occurs via the oxidative stress-responsive MAPK pathway, highlighting a novel profile of morin as a potent agent for asthma management.

Costs and financial feasibility of malaria elimination

The marginal costs and benefits of converting malaria programmes from a control to an elimination goal are central to strategic decisions, but empirical evidence is scarce. We present a conceptual framework to assess the economics of elimination and analyse a central component of that framework-potential short-term to medium-term financial savings. After a review that showed a dearth of existing evidence, the net present value of elimination in five sites was calculated and compared with effective control. The probability that elimination would be cost-saving over 50 years ranged from 0% to 42%, with only one site achieving cost-savings in the base case. These findings show that financial savings should not be a primary rationale for elimination, but that elimination might still be a worthy investment if total benefits are sufficient to outweigh marginal costs. Robust research into these elimination benefits is urgently needed.

Ketamine reduces LPS-induced HMGB1 via activation of the Nrf2/HO-1 pathway and NF-κB suppression.

BACKGROUND Ketamine, as an anesthetic agent, has an anti-inflammatory effect. In the present study, we investigated whether ketamine inhibits release of high mobility group box 1 (HMGB1), a late-phase cytokine of sepsis, in lipopolysaccharide (LPS)-stimulated macrophages through heme oxygenase-1 (HO-1) induction. METHODS Macrophages were preincubated with various concentrations of ketamine and then treated with LPS (1 μg/mL). The cell culture supernatants were collected to measure inflammatory mediators (HMGB1, nitric oxide, tumor necrosis factor-α, and interleukin 1β) by enzyme-linked immunosorbent assay. Moreover, HO-1 protein expression, the phosphorylation and degradation of IκB-α, and the nuclear translocation of nuclear factor E2-related factor 2 and nuclear factor κB (NF-κB) p65 were tested by Western blot analysis. In addition, to further identify the role of HO-1 in this process, tin protoporphyrin (SnPP), an HO-1 inhibitor, was used. RESULTS Ketamine treatment dose-dependently attenuated the increased levels of proinflammatory mediators (HMGB1, nitric oxide, tumor necrosis factor α, and interleukin 1β) and increased the HO-1 protein expression in LPS-activated macrophages. Furthermore, ketamine suppressed the phosphorylation and degradation of IκB-α as well as the LPS-stimulated nuclear translocation of NF-κB p65 in macrophages. In addition, the present study also demonstrated that ketamine induced HO-1 expression through the nuclear translocation of nuclear factor E2-related factor 2 in macrophages. The effects of ketamine on LPS-induced proinflammatory cytokines production were partially reversed by the HO inhibitor tin protoporphyrin (SnPP). CONCLUSION Ketamine inhibits the release of HMGB1 in LPS-stimulated macrophages, and this effect is at least partly mediated by the activation of the Nrf2/HO-1 pathway and NF-κB suppression.

Cytosolic viral sensor RIG-I is a 5'-triphosphate-dependent translocase on double-stranded RNA.

Retinoic acid inducible-gene I (RIG-I) is a cytosolic multidomain protein that detects viral RNA and elicits an antiviral immune response. Two N-terminal caspase activation and recruitment domains (CARDs) transmit the signal, and the regulatory domain prevents signaling in the absence of viral RNA. 5'-triphosphate and double-stranded RNA (dsRNA) are two molecular patterns that enable RIG-I to discriminate pathogenic from self-RNA. However, the function of the DExH box helicase domain that is also required for activity is less clear. Using single-molecule protein-induced fluorescence enhancement, we discovered a robust adenosine 5'-triphosphate-powered dsRNA translocation activity of RIG-I. The CARDs dramatically suppress translocation in the absence of 5'-triphosphate, and the activation by 5'-triphosphate triggers RIG-I to translocate preferentially on dsRNA in cis. This functional integration of two RNA molecular patterns may provide a means to specifically sense and counteract replicating viruses.

Beta adrenergic blockade decreases the immunomodulatory effects of social disruption stress

During physiological or psychological stress, catecholamines produced by the sympathetic nervous system (SNS) regulate the immune system. Previous studies report that the activation of β-adrenergic receptors (βARs) mediates the actions of catecholamines and increases pro-inflammatory cytokine production in a number of different cell types. The impact of the SNS on the immune modulation of social defeat has not been examined. The following studies were designed to determine whether SNS activation during social disruption stress (SDR) influences anxiety-like behavior as well as the activation, priming, and glucocorticoid resistance of splenocytes after social stress. CD-1 mice were exposed to one, three, or six cycles of SDR and HPLC analysis of the plasma and spleen revealed an increase in catecholamines. After six cycles of SDR the open field test was used to measure behaviors characteristic of anxiety and indicated that the social defeat induced increase in anxiety-like behavior was blocked by pre-treatment with the β-adrenergic antagonist propranolol. Pre-treatment with the β-adrenergic antagonist propranolol did not significantly alter corticosterone levels indicating no difference in activation of the hypothalamic-pituitary-adrenal axis. In addition to anxiety-like behavior the SDR induced splenomegaly and increase in plasma IL-6, TNFα, and MCP-1 were each reversed by pre-treatment with propranolol. Furthermore, flow cytometric analysis of cells from propranolol pretreated mice reduced the SDR-induced increase in the percentage of CD11b(+) splenic macrophages and significantly decreased the expression of TLR2, TLR4, and CD86 on the surface of these cells. In addition, supernatants from 18h LPS-stimulated ex vivo cultures of splenocytes from propranolol-treated SDR mice contained less IL-6. Likewise propranolol pre-treatment abrogated the glucocorticoid insensitivity of CD11b(+) cells ex vivo when compared to splenocytes from SDR vehicle-treated mice. Together, this study demonstrates that the immune activation and priming effects of SDR result, in part, as a consequence of SNS activation.

Loss of allergen 1 confers a hypervirulent phenotype that resembles mucoid switch variants of Cryptococcus neoformans.

Microbial survival in a host is usually dependent on the ability of a pathogen to undergo changes that promote escape from host defense mechanisms. The human-pathogenic fungus Cryptococcus neoformans undergoes phenotypic switching in vivo that promotes persistence in tissue. By microarray and real-time PCR analyses, the allergen 1 gene (ALL1) was found to be downregulated in the hypervirulent mucoid switch variant, both during logarithmic growth and during intracellular growth in macrophages. The ALL1 gene encodes a small cytoplasmic protein that is involved in capsule formation. Growth of an all1Delta gene deletion mutant was normal. Similar to cells of the mucoid switch variant, all1Delta cells produced a larger polysaccharide capsule than cells of the smooth parent and the complemented strain produced, and the enlarged capsule inhibited macrophage phagocytosis. The mutant exhibited a modest defect in capsule induction compared to all of the other variants. In animal models the phenotype of the all1Delta mutant mimicked the hypervirulent phenotype of the mucoid switch variant, which is characterized by decreased host survival and elevated intracranial pressure. Decreased survival is likely the result of both an ineffective cell-mediated immune response and impaired phagocytosis by macrophages. Consequently, we concluded that, unlike loss of most virulence-associated genes, where loss of gene function results in attenuated virulence, loss of the ALL1 gene enhances virulence by altering the host-pathogen interaction and thereby impairing clearance. Our data identified the first cryptococcal gene associated with elevated intracranial pressure and support the hypothesis that an environmental opportunistic pathogen has modified its virulence in vivo by epigenetic downregulation of gene function.

Immune dysregulation in major depression: a critical review of existing evidence.

It is now well established that depression is associated with immune dysregulation. It is not, however, known whether this immune dysregulation plays a role in the pathophysiology of major depression or whether it increases the susceptibility of the depressed patient to immune-related disorders. This article presents a critical review of existing evidence for immune dysregulation in major depression, including changes in leucocyte trafficking, lymphocyte function, and markers of immune activation. Possible mediators of immune dysregulation in major depression are briefly discussed. Finally, the relationship between major depression and several medical conditions such as infection, allergy and autoimmune disorders, cardiovascular diseases, cancer and AIDS is critically reviewed.

Schistosoma mansoni worms induce anergy of T cells via selective up-regulation of programmed death ligand 1 on macrophages.

Infectious pathogens can selectively stimulate activation or suppression of T cells to facilitate their survival within humans. In this study we demonstrate that the trematode parasite Schistosoma mansoni has evolved with two distinct mechanisms to suppress T cell activation. During the initial 4- to 12-wk acute stages of a worm infection both CD4(+) and CD8(+) T cells are anergized. In contrast, infection with male and female worms induced T cell anergy at 4 wk, which was replaced after egg laying by T cell suppression via a known NO-dependent mechanism, that was detected for up to 40 wk after infection. Worm-induced anergy was mediated by splenic F4/80(+) macrophages (Mphi) via an IL-4-, IL-13-, IL-10-, TGF-beta-, and NO-independent, but cell contact-dependent, mechanism. F4/80(+) Mphi isolated from worm-infected mice were shown to induce anergy of naive T cells in vitro. Furthermore, naive Mphi exposed to live worms in vitro also induced anergy in naive T cells. Flow cytometry on in vivo and in vitro worm-modulated Mphi revealed that of the family of B7 costimulatory molecules, only programmed death ligand 1 (PD-L1) was selectively up-regulated. The addition of inhibitory mAb against PD-L1, but not PD-L2, to worm-modulated Mphi completely blocked the ability of these cells to anergize T cells. These data highlight a novel mechanism through which S. mansoni worms have usurped the natural function of PD-L1 to reduce T cell activation during early acute stages of infection before the subsequent emergence of egg-induced T cell suppression in the chronic stages of infection.

β-Adrenergic receptor antagonism prevents anxiety-like behavior and microglial reactivity induced by repeated social defeat.

Psychosocial stress is associated with altered immune function and development of psychological disorders including anxiety and depression. Here we show that repeated social defeat in mice increased c-Fos staining in brain regions associated with fear and threat appraisal and promoted anxiety-like behavior in a β-adrenergic receptor-dependent manner. Repeated social defeat also significantly increased the number of CD11b(+)/CD45(high)/Ly6C(high) macrophages that trafficked to the brain. In addition, several inflammatory markers were increased on the surface of microglia (CD14, CD86, and TLR4) and macrophages (CD14 and CD86) after social defeat. Repeated social defeat also increased the presence of deramified microglia in the medial amygdala, prefrontal cortex, and hippocampus. Moreover, mRNA analysis of microglia indicated that repeated social defeat increased levels of interleukin (IL)-1β and reduced levels of glucocorticoid responsive genes [glucocorticoid-induced leucine zipper (GILZ) and FK506 binding protein-51 (FKBP51)]. The stress-dependent changes in microglia and macrophages were prevented by propranolol, a β-adrenergic receptor antagonist. Microglia isolated from socially defeated mice and cultured ex vivo produced markedly higher levels of IL-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 after stimulation with lipopolysaccharide compared with microglia from control mice. Last, repeated social defeat increased c-Fos activation in IL-1 receptor type-1-deficient mice, but did not promote anxiety-like behavior or microglia activation in the absence of functional IL-1 receptor type-1. These findings indicate that repeated social defeat-induced anxiety-like behavior and enhanced reactivity of microglia was dependent on activation of β-adrenergic and IL-1 receptors.

microRNA-31/factor-inhibiting hypoxia-inducible factor 1 nexus regulates keratinocyte differentiation.

Notch plays a critical role in the transition from proliferation to differentiation in the epidermis and corneal epithelium. Furthermore, aberrant Notch signaling is a feature of diseases like psoriasis, eczema, nonmelanoma skin cancer, and melanoma where differentiation and proliferation are impaired. Whereas much is known about the downstream events following Notch signaling, factors responsible for negatively regulating Notch receptor signaling after ligand activation are incompletely understood. Notch can undergo hydroxylation by factor-inhibiting hypoxia-inducible factor 1 (FIH-1); however, the biological significance of this phenomenon is unclear. Here we show that FIH-1 expression is up-regulated in diseased epidermis and corneal epithelium. Elevating FIH-1 levels in primary human epidermal keratinocytes (HEKs) and human corneal epithelial keratinocytes (HCEKs) impairs differentiation in submerged cultures and in a "three-dimensional" organotypic raft model of human epidermis, in part, via a coordinate decrease in Notch signaling. Knockdown of FIH-1 enhances keratinocyte differentiation. Loss of FIH-1 in vivo increased Notch activity in the limbal epithelium, resulting in a more differentiated phenotype. microRNA-31 (miR-31) is an endogenous negative regulator of FIH-1 expression that results in keratinocyte differentiation, mediated by Notch activation. Ectopically expressing miR-31 in an undifferentiated corneal epithelial cell line promotes differentiation and recapitulates a corneal epithelium in a three-dimensional raft culture model. Our results define a previously unknown mechanism for keratinocyte fate decisions where Notch signaling potential is, in part, controlled through a miR-31/FIH-1 nexus.