Christopher McCrae

Budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses

BackgroundCombination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells.MethodsTo test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 μM), formoterol (0.1 μM), both, or neither, and then inoculated with RV-16 (5×106 plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFα, IFN-β, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL28A&B) were analyzed.ResultsRV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFα under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma.ConclusionsBudesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.

Euodenine A: A Small-Molecule Agonist of Human TLR4

A small-molecule natural product, euodenine A (1), was identified as an agonist of the human TLR4 receptor. Euodenine A was isolated from the leaves of Euodia asteridula (Rutaceae) found in Papua New Guinea and has an unusual U-shaped structure. It was synthesized along with a series of analogues that exhibit potent and selective agonism of the TLR4 receptor. SAR development around the cyclobutane ring resulted in a 10-fold increase in potency. The natural product demonstrated an extracellular site of action, which requires the extracellular domain of TLR4 to stimulate a NF-κB reporter response. 1 is a human-selective agonist that is CD14-independent, and it requires both TLR4 and MD-2 for full efficacy. Testing for immunomodulation in PBMC cells shows the induction of the cytokines IL-8, IL-10, TNF-α, and IL-12p40 as well as suppression of IL-5 from activated PBMCs, indicating that compounds like 1 could modulate the Th2 immune response without causing lung damage.

Development of a high-throughput human rhinovirus infectivity cell-based assay for identifying antiviral compounds

Asthma and chronic obstructive pulmonary disease exacerbations are associated with human rhinovirus (HRV) lung infections for which there are no current effective antiviral therapies. To date, HRV infectivity of cells in vitro has been measured by a variety of biochemical and immunological methods. This paper describes the development of a high-throughput HRV infectivity assay using HeLa OHIO cells and a chemiluminescent-based ATP cell viability system, CellTiter-Glo from Promega, to measure HRV-induced cytopathic effect (CPE). This CellTiter-Glo assay was validated with standard antiviral agents and employed to screen AstraZeneca compounds for potential antiviral activity. Compound potency values in this assay correlated well with the quantitative RT-PCR assay measuring HRV infectivity and replication in human primary airway epithelial cells. In order to improve pan-HRV screening capability, compound potency was also measured in the CellTiter-Glo assay with a combination of 3 different HRV serotypes. This HRV serotype combination assay could be used to identify quickly compounds with desirable broad spectrum antiviral activity.

Seasonal and geographic variations in the incidence of asthma exacerbations in the United States

ABSTRACT Background: Exacerbations drive the burden of asthma and lead to significant morbidity and consumption of health care resources. Many prior studies of the epidemiology of asthma exacerbations have relied upon data from hospital care. Objective: The objective of this study was to determine US patterns of geographic and seasonal variations of asthma exacerbations being defined as asthma episodes requiring hospital care and/or a prescription for oral steroid. Methods: The study was a retrospective observational cohort study using administrative claims data for insured individuals from the HealthCore Integrated Research Database, including around 43 million members in the United States. Analyses examined 3 age groups, 6–17, 18–64, and ≥65 years and four US regions, Northeast, Southeast, Central, and Western. Results: Monthly rates of asthma exacerbations showed the greatest variation over the year in children, less so in adults and in the elderly. Clinically important differences in rates of asthma exacerbation were observed between regions with the Western Region having the lowest in all three age groups followed by the Northeast, Central, and Southeast regions. Peaks in children occurred in the early fall following troughs in the summer months, and peaks at year-end occurred in adults, particularly in those over 65 years. Conclusions: There is a striking seasonal variation in asthma exacerbations in the United States. Substantial differences between regions of the United States in asthma exacerbation rates cannot readily be explained and invite further investigation.

A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

Colds as predictors of the onset and severity of COPD exacerbations

Rationale Common colds are associated with acute respiratory symptom exacerbations in COPD patients. Objective To determine exacerbation risk and severity in COPD patients with/without coincident self-reported colds. Methods Global initiative for chronic Obstructive Lung Disease stage I–IV COPD patients electronically transmitted respiratory symptom diaries to research staff daily between December 2006 and April 2009. Respiratory symptom worsening prompted contact by a study nurse and patient assessment to determine if a cold was present or an exacerbation underway. A composite daily symptom score was derived for each subject from diarized symptom data. The exacerbation/cold/virus relation was examined using a Poisson regression model, the relation of colds to respiratory symptom severity using generalized estimating equation models. Results Daily diary transmission compliance of >97% enabled detection of all possible exacerbations. Among 262 exacerbations meeting Anthonisen criteria, 218 (83%) had cold-like symptoms present at their inception, but respiratory viruses were detected in only 106 (40%). Within-subject exacerbation risk was 30 times (95% confidence interval [CI]: 20, 47; P<0.001) greater with colds present. Compared to cold- and virus-negative exacerbations (n=57), the mean increase in composite symptom score in those cold and virus positive (n=79) was 0.93 (95% CI: 0.61, 1.25; P<0.001), cold-positive and virus-negative exacerbations (n=100) 0.51 (95% CI: 0.21, 0.81; P<0.001), cold-negative and virus-positive exacerbations (n=26) 0.58 (95% CI: 0.23, 0.94; P<0.001). Conclusion This study emphasizes the importance of colds in COPD exacerbation risk and severity, even in the absence of virus detection. COPD patients should act promptly when cold symptoms appear to facilitate early intervention for exacerbation prevention or management.

Study on risk factors and phenotypes of acute exacerbations of chronic obstructive pulmonary disease in Guangzhou, China— design and baseline characteristics

BACKGROUND To describe a study design that focuses on risk factors and patterns of chronic obstructive pulmonary disease (COPD) exacerbations. METHODS A 2-year, single centre, observational study was conducted in Guangzhou in China. The study enrolled 318 subjects with COPD aged 40-79 years, stratified into different but equally sized groups according to global initiative for chronic obstructive lung disease (GOLD) stage (including Stage 0) and 86 lung healthy controls. An assessment each year was scheduled including questionnaires, lung function testing, Chest X-ray and blood collection. A sub-group, called sub-group X, consisting of 203 subjects with COPD and 51 lung healthy controls, was selected to answer a symptom questionnaire daily (EXACT-PRO) via a BlackBerry Personal Digital Assistant (PDA) device. Upon an alert that indicated a change in daily symptom pattern, the patients were contacted by the clinic to decide whether they had experienced an exacerbation and should have an extra visit within 24-48 hours. At an extra visit, nasal and throat swabs, induced sputum and blood were collected. Air pollution, temperature and humidity were also monitored daily. A subset of sub-group X, called sub-group M that consisted of 52 COPD patients and 15 healthy controls was dedicated to measure muscle strength and a dexa scan. RESULTS More than 78% of the enrolled patients completed the study successfully. There appeared a difference between the patient groups and the controls in gender, age, body mass index (BMI), forced expiratory volume in 1 second (FEV1), FEV1/FVC and smoking at baseline. In sub-group X 90 out of 203 (44.4%) selected COPD patients developed one or more exacerbations in the 2-year observation period. They were more severe COPD patients according to GOLD stage at study start. On average most exacerbations occurred in the month March and the least number of exacerbations occurred in October. CONCLUSIONS This study with the obtained patient dataset will allow a better insight in many aspects of exacerbations in COPD (e.g., the identification, the risk factors, phenotypes and the biomarkers).

Selection of a screening panel of rhinoviral serotypes

A novel methodology for the selection of a representative primary and secondary screening panel of rhinoviral serotypes for the purposes of identifying potential antiviral agents is presented. This methodology focuses on the active-sites of the rhinoviral proteins but does not invoke historical SAR data, thereby avoiding compound bias.

Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs

The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.

Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells

&NA; Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration‐dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24‐hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN‐&bgr;. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV‐induced exacerbations.