Differential effects of simvastatin on IL-13-induced cytokine gene expression in primary mouse tracheal epithelial cells

Background

Asthma causes significant morbidity worldwide in adults and children alike, and incurs large healthcare costs. The statin drugs, which treat hyperlipidemia and cardiovascular diseases, have pleiotropic effects beyond lowering cholesterol, including immunomodulatory, anti-inflammatory, and anti-fibrotic properties which may benefit lung health. Using an allergic mouse model of asthma, we previously demonstrated a benefit of statins in reducing peribronchiolar eosinophilic inflammation, airway hyperreactivity, goblet cell hyperplasia, and lung IL-4 and IL-13 production.

Objectives

In this study, we evaluated whether simvastatin inhibits IL-13-induced pro-inflammatory gene expression of asthma-related cytokines in well-differentiated primary mouse tracheal epithelial (MTE) cell cultures. We hypothesized that simvastatin reduces the expression of IL-13-inducible genes in MTE cells.

Methods

We harvested tracheal epithelial cells from naïve BALB/c mice, grew them under air-liquid interface (ALI) cell culture conditions, then assessed IL-13-induced gene expression in MTE cells using a quantitative real-time PCR mouse gene array kit.

Results

We found that simvastatin had differential effects on IL-13-mediated gene expression (inhibited eotaxin-1; MCP-1,-2,-3; and osteopontin (SPP1), while it induced caspase-1 and CCL20 (MIP-3α)) in MTE cells. For other asthma-relevant genes such as TNF, IL-4, IL-10, CCL12 (MCP-5), CCL5 (RANTES), and CCR3, there were no significant IL-13-inducible or statin effects on gene expression.

Conclusions

Simvastatin modulates the gene expression of selected IL-13-inducible pro-inflammatory cytokines and chemokines in primary mouse tracheal epithelial cells. The airway epithelium may be a viable target tissue for the statin drugs. Further research is needed to assess the mechanisms of how statins modulate epithelial gene expression.     

Effectiveness of community-based comprehensive healthy lifestyle promotion on cardiovascular disease risk factors in a rural Vietnamese population: a quasi-experimental study

ABSTRACT: BACKGROUND: Health promotion is a key component for primary prevention of cardiovascular disease (CVD). This study evaluated the impact of healthy lifestyle promotion campaigns on CVD risk factors (CVDRF) in the general population in the context of a community-based programme on hypertension management. METHODS: A quasi-experimental intervention study was carried out in two rural communes of Vietnam from 2006 to 2009. In the intervention commune, a hypertensive-targeted management programme integrated with a community-targeted health promotion was initiated, while no new programme, apart from conventional healthcare services, was provided in the reference commune. Health promotion campaigns focused on smoking cessation, reducing alcohol consumption, encouraging physical activity and reducing salty diets. Repeated cross-sectional surveys in local adult population aged 25 years and over were undertaken to assess changes in blood pressure (BP) and behavioural CVDRFs (smoking, alcohol consumption, physical inactivity and salty diet) in both communes before and after the 3-year intervention. RESULTS: Overall 4,650 adults above 25 years old were surveyed, in four randomly independent samples covering both communes at baseline and after the 3-year intervention. Although physical inactivity and obesity increased over time in the intervention commune, there was a significant reduction in systolic and diastolic BP (3.3 and 4.7 mmHg in women versus 3.0 and 4.6 mmHg in men respectively) in the general population at the intervention commune. Health promotion reduced levels of salty diets but had insignificant impact on the prevalence of daily smoking or heavy alcohol consumption. CONCLUSION: Community-targeted healthy lifestyle promotion can significantly improve some CVDRFs in the general population in a rural area over a relatively short time span. Limited effects on a context-bound CVDRF like smoking suggested that higher intensity of intervention, a supportive environment or a gender approach are required to maximize the effectiveness and maintain the sustainability of the health intervention.     

Divergent effects of compounds on the hydrolysis and transpeptidation reactions of γ-glutamyl transpeptidase

A novel class of inhibitors of the enzyme γ-glutamyl transpeptidase (GGT) were evaluated. The analog OU749 was shown previously to be an uncompetitive inhibitor of the GGT transpeptidation reaction. The data in this study show that it is an equally potent uncompetitive inhibitor of the hydrolysis reaction, the primary reaction catalyzed by GGT in vivo. A series of structural analogs of OU749 were evaluated. For many of the analogs, the potency of the inhibition differed between the hydrolysis and transpeptidation reactions, providing insight into the malleability of the active site of the enzyme. Analogs with electron withdrawing groups on the benzosulfonamide ring, accelerated the hydrolysis reaction, but inhibited the transpeptidation reaction by competing with a dipeptide acceptor. Several of the OU749 analogs inhibited the transpeptidation reaction by slow onset kinetics, similar to acivicin. Further development of inhibitors of the GGT hydrolysis reaction is necessary to provide new therapeutic compounds.     

Mitochondria-derived superoxide links to tourniquet-induced apoptosis in mouse skeletal muscle

Our previous study has reported that superoxide mediates ischemia-reperfusion (IR)-induced necrosis in mouse skeletal muscle. However, it remains poorly understood whether IR induces apoptosis and what factors are involved in IR-induced apoptosis in skeletal muscle. Using a murine model of tourniquet-induced hindlimb IR, we investigated the relationship between mitochondrial dysfunction and apoptosis in skeletal muscle. Hindlimbs of C57/BL6 mice were subjected to 3 h ischemia and 4 h reperfusion via placement and release of a rubber tourniquet at the greater trochanter. Compared to sham treatment, tourniquet-induced IR significantly elevated mitochondria-derived superoxide production, activated opening of mitochondrial permeability transition pore (mPTP), and caused apoptosis in the gastrocnemius muscles. Pretreatment with a superoxide dismutase mimetic (tempol, 50 mg/kg) or a mitochondrial antioxidant (co-enzyme Q(10), 50 mg/kg) not only decreased mitochondria-derived superoxide production, but also inhibited mPTP opening and apoptosis in the IR gastrocnemius muscles. Additionally, an inhibitor of mPTP (cyclosporine A, 50 mg/kg) also inhibited both mPTP opening and apoptosis in the IR gastrocnemius muscles. These results suggest that mitochondria-derived superoxide overproduction triggers the mPTP opening and subsequently causes apoptosis in tourniquet-induced hindlimb IR.     

Time trends in blood pressure, body mass index and smoking in the vietnamese population: a meta-analysis from multiple cross-sectional surveys

Introduction

Data for trends in cardiovascular disease (CVD) risk factors are needed to set priorities and evaluate intervention programmes in the community. We estimated time trends in blood pressure (BP), anthropometric variables and smoking in the Vietnamese population and highlighted the differences between men and women or between rural and urban areas.

Methods

A dataset of 23,563 adults aged 25–74 from 5 cross-sectional surveys undertaken within Vietnam from 2001 to 2009 by the Vietnam National Heart Institute was used to estimate mean BP, weight, waist circumference (WC), body mass index (BMI), the prevalence of hypertension, adiposity or smoking, which were standardised to the national age structure of 2009. Multilevel mixed linear models were used to estimate annual changes in the variables of interest, adjusted by age, sex, residential area, with random variations for age and surveyed provinces.

Findings

Among the adult population, the age-standardised mean systolic and diastolic BP increased by 0.8 and 0.3 mmHg in women, 1.1 and 0.4 mmHg in men, while the mean BMI increased by 0.1 kgm⁻² in women, 0.2 kgm⁻² in men per year. Consequently, the prevalence of hypertension and adiposity increased by 0.9 and 0.3% in women, 1.1 and 0.9% in men with similar time trends in both rural and urban areas, while smoking prevalence only increased in women by 0.3% per year. A U-shaped association was found between age-adjusted BP and BMI in both sexes and in both areas.

Conclusions

From 2001 to 2009, mean BP, weight and WC significantly increased in the Vietnamese population, leading to an increased prevalence of hypertension and adiposity, suggesting the need for the development of multi-sectoral cost-effective population-based interventions to improve CVD management and prevention. The U-shaped relationship between BP and BMI highlighted the hypertension burden in the underweight population, which is usually neglected in CVD interventions.     

Characterization of a high-affinity human antibody with a disulfide bridge in the third complementarity-determining region of the heavy chain

Disulfide bridges are common in the antigen-binding site from sharks (new antigen receptor) and camels (single variable heavy-chain domain, VHH), in which they confer both structural diversity and domain stability. In human antibodies, cysteine residues in the third complementarity-determining region of the heavy chain (CDR-H3) are rare but naturally encoded in the IGHD germline genes. Here, by panning a phage display library designed based on human germline genes and synthetic CDR-H3 regions against a human cytokine, we identified an antibody (M3) containing two cysteine residues in the CDR-H3. It binds the cytokine with high affinity (0.4?nM), recognizes a unique epitope on the antigen, and has a distinct neutralization profile as compared with all other antibodies selected from the library. The two cysteine residues form a disulfide bridge as determined by mass spectrometric peptide mapping. Replacing the cysteines with alanines did not change the solubility and stability of the monoclonal antibody, but binding to the antigen was significantly impaired. Three-dimensional modeling and dynamic simulations were employed to explore how the disulfide bridge influences the conformation of CDR-H3 and binding to the antigen. On the basis of these results, we envision that designing human combinatorial antibody libraries to contain intra-CDR or inter-CDR disulfide bridges could lead to identification of human antibodies with unique binding profiles.     

The Thai phase III trial (RV144) vaccine regimen induces T cell responses that preferentially target epitopes within the V2 region of HIV-1 envelope

The Thai HIV phase III prime/boost vaccine trial (RV144) using ALVAC-HIV (vCP1521) and AIDSVAX B/E was, to our knowledge, the first to demonstrate acquisition efficacy. Vaccine-induced, cell-mediated immune responses were assessed. T cell epitope mapping studies using IFN-γ ELISPOT was performed on PBMCs from HIV-1-uninfected vaccine (n = 61) and placebo (n = 10) recipients using HIV-1 Env peptides. Positive responses were measured in 25 (41%) vaccinees and were predominantly CD4(+) T cell-mediated. Responses were targeted within the HIV Env region, with 15 of 25 (60%) of vaccinees recognizing peptides derived from the V2 region of HIV-1 Env, which includes the α(4)β(7) integrin binding site. Intracellular cytokine staining confirmed that Env responses predominated (19 of 30; 63% of vaccine recipients) and were mediated by polyfunctional effector memory CD4(+) T cells, with the majority of responders producing both IL-2 and IFN-γ (12 of 19; 63%). HIV Env Ab titers were higher in subjects with IL-2 compared with those without IL-2-secreting HIV Env-specific effector memory T cells. Proliferation assays revealed that HIV Ag-specific T cells were CD4(+), with the majority (80%) expressing CD107a. HIV-specific T cell lines obtained from vaccine recipients confirmed V2 specificity, polyfunctionality, and functional cytolytic capacity. Although the RV144 T cell responses were modest in frequency compared with humoral immune responses, the CD4(+) T cell response was directed to HIV-1 Env and more particularly the V2 region.     

Antidotes to anthrax lethal factor intoxication. Part 3: Evaluation of core structures and further modifications to the C2-side chain

Four core structures capable of providing sub-nanomolar inhibitors of anthrax lethal factor (LF) were evaluated by comparing the potential for toxicity, physicochemical properties, in vitro ADME profiles, and relative efficacy in a rat lethal toxin (LT) model of LF intoxication. Poor efficacy in the rat LT model exhibited by the phenoxyacetic acid series (3) correlated with low rat microsome and plasma stability. Specific molecular interactions contributing to the high affinity of inhibitors with a secondary amine in the C2-side chain were revealed by X-ray crystallography.