Immunogenetic responses in calves to intranasal delivery of bovine respiratory syncytial virus (BRSV) epitopes encapsulated in poly (DL-lactide-co-glycolide) microparticles

Bovine respiratory syncytial virus (BRSV) is the principal aetiological agent of the bovine respiratory disease complex. A BRSV subunit vaccine candidate consisting of two synthetic peptides representing putative protective epitopes on BRSV surface glycoproteins in soluble form or encapsulated in poly(lactide-co-glycolide) (PLG) microparticles were prepared. Calves (10 weeks old) with diminishing levels of BRSV-specific maternal antibody were intranasally administered a single dose of the different peptide formulations. Peptide-specific local immune responses (nasal secretion IgA), but not systemic humoral (serum IgG) or cellular responses (serum IFN-γ), were generated by all forms of peptide. There was a significant reduction in occurrence of respiratory disease in the animals inoculated with all peptide formulations compared to animals given PBS alone. Furthermore no adverse effects were observed in any of the animals post vaccination. These results suggest that intranasal immunisation with the peptide subunit vaccine does induce an as yet unidentified protective immune response.

Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

Ataluren for the treatment of nonsense-mutation cystic fibrosis: a randomised, double-blind, placebo-controlled phase 3 trial

Background: Ataluren was developed to restore functional protein production in genetic disorders caused by nonsense mutations, which are the cause of cystic fibrosis in 10% of patients. This trial was designed to assess the efficacy and safety of ataluren in patients with nonsense-mutation cystic fibrosis. 

Methods: This randomised, double-blind, placebo-controlled, phase 3 study enrolled patients from 36 sites in 11 countries in North America and Europe. Eligible patients with nonsense-mutation cystic fibrosis (aged ≥6 years; abnormal nasal potential difference; sweat chloride >40 mmol/L; forced expiratory volume in 1 s [FEV₁] ≥40% and ≤90%) were randomly assigned by interactive response technology to receive oral ataluren (10 mg/kg in morning, 10 mg/kg midday, and 20 mg/kg in evening) or matching placebo for 48 weeks. Randomisation used a block size of four, stratified by age, chronic inhaled antibiotic use, and percent-predicted FEV₁. The primary endpoint was relative change in percent-predicted FEV₁ from baseline to week 48, analysed in all patients with a post-baseline spirometry measurement. This study is registered with ClinicalTrials.gov, number NCT00803205. 

Findings: Between Sept 8, 2009, and Nov 30, 2010, 238 patients were randomly assigned, of whom 116 in each treatment group had a valid post-baseline spirometry measurement. Relative change from baseline in percent-predicted FEV₁ did not differ significantly between ataluren and placebo at week 48 (-2·5% vs -5·5%; difference 3·0% [95% CI -0·8 to 6·3]; p=0·12). The number of pulmonary exacerbations did not differ significantly between treatment groups (rate ratio 0·77 [95% CI 0·57-1·05]; p=0·0992). However, post-hoc analysis of the subgroup of patients not using chronic inhaled tobramycin showed a 5·7% difference (95% CI 1·5-10·1) in relative change from baseline in percent-predicted FEV₁ between the ataluren and placebo groups at week 48 (-0·7% [-4·0 to 2·1] vs -6·4% [-9·8 to -3·7]; nominal p=0·0082), and fewer pulmonary exacerbations in the ataluern group (1·42 events [0·9-1·9] vs 2·18 events [1·6-2·7]; rate ratio 0·60 [0·42-0·86]; nominal p=0·0061). Safety profiles were generally similar for ataluren and placebo, except for the occurrence of increased creatinine concentrations (ie, acute kidney injury), which occurred in 18 (15%) of 118 patients in the ataluren group compared with one (<1%) of 120 patients in the placebo group. No life-threatening adverse events or deaths were reported in either group. I

nterpretation: Although ataluren did not improve lung function in the overall population of nonsense-mutation cystic fibrosis patients who received this treatment, it might be beneficial for patients not taking chronic inhaled tobramycin. 

Funding: PTC Therapeutics, Cystic Fibrosis Foundation, US Food and Drug Administration's Office of Orphan Products Development, and the National Institutes of Health. 

Relative Contribution of P5 and Hap Surface Proteins to Nontypable Haemophilus influenzae Interplay with the Host Upper and Lower Airways

Nontypable Haemophilus influenzae (NTHi) is a major cause of opportunistic respiratory tract disease, and initiates infection by colonizing the nasopharynx. Bacterial surface proteins play determining roles in the NTHi-airways interplay, but their specific and relative contribution to colonization and infection of the respiratory tract has not been addressed comprehensively. In this study, we focused on the ompP5 and hap genes, present in all H. influenzae genome sequenced isolates, and encoding the P5 and Hap surface proteins, respectively. We employed isogenic single and double mutants of the ompP5 and hap genes generated in the pathogenic strain NTHi375 to evaluate P5 and Hap contribution to biofilm growth under continuous flow, to NTHi adhesion, and invasion/phagocytosis on nasal, pharyngeal, bronchial, alveolar cultured epithelial cells and alveolar macrophages, and to NTHi murine pulmonary infection. We show that P5 is not required for bacterial biofilm growth, but it is involved in NTHi interplay with respiratory cells and in mouse lung infection. Mechanistically, P5NTHi375 is not a ligand for CEACAM1 or α5 integrin receptors. Hap involvement in NTHi375-host interaction was shown to be limited, despite promoting bacterial cell adhesion when expressed in H. influenzae RdKW20. We also show that Hap does not contribute to bacterial biofilm growth, and that its absence partially restores the deficiency in lung infection observed for the ΔompP5 mutant. Altogether, this work frames the relative importance of the P5 and Hap surface proteins in NTHi virulence.

Primary airway epithelial cell culture and asthma in children-lessons learnt and yet to come

Until recently the airway epithelial cell (AEC) was considered a simple barrier that prevented entry of inhaled matter into the lung parenchyma. The AEC is now recognized as having an important role in the inflammatory response of the respiratory system to inhaled exposures, and abnormalities of these responses are thought to be important to asthma pathogenesis. This review first explores how the challenges of studying nasal and bronchial AECs in children have been addressed and then summarizes the results of studies of primary AEC function in children with and without asthma. There is good evidence that nasal AECs may be a suitable surrogate for the study of certain aspects of bronchial AEC function, although bronchial AECs remain the gold standard for asthma research. There are consistent differences between children with and without asthma for nasal and bronchial AEC mediator release following exposure to a range of pro-inflammatory stimulants including interleukins (IL)-1β, IL-4, and IL-13. However, there are inconsistencies between studies, e.g., release of IL-6, an important pro-inflammatory cytokine, is not increased in children with asthma relative to controls in all studies. Future work should expand current understanding of the "upstream" signalling pathways in AEC, study AEC from children before the onset of asthma symptoms and in vitro models should be developed that replicate the in vivo status more completely, e.g., co-culture with dendritic cells. AECs are difficult to obtain from children and collaboration between centers is expected to yield meaningful advances in asthma understanding and ultimately help deliver novel therapies. 

Induction of the inflammatory regulator A20 by gibberellic acid in airway epithelial cells

BACKGROUND AND PURPOSE: NF-κB-driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA3 ) is a plant-derived diterpenoid with documented anti-inflammatory activity, which is reported to induce A20-like zinc finger proteins in plants. Here, we sought to investigate the anti-inflammatory effect of GA3 in airway epithelial cells and determine if the anti-inflammatory action relates to A20 induction.

EXPERIMENTAL APPROACH: Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o-) were used. Cells were pre-incubated with GA3 , stimulated with Pseudomonas aeruginosa LPS; IL-6 and IL-8 release, A20, NF-κB and IκBα expression were then evaluated. To determine if any observed anti-inflammatory effect occurred via an A20-dependent mechanism, A20 was silenced using siRNA.

KEY RESULTS: Cells pre-incubated with GA3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL-6 and IL-8 release. This effect was mediated via reduced IκBα degradation and reduced NF-κB (p65) expression. Furthermore, the anti-inflammatory action of GA3 was abolished in A20-silenced cells.

CONCLUSIONS AND IMPLICATIONS: We showed that A20 induction by GA3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF-κB and IκBα. GA3 or gibberellin-derived derivatives could potentially be developed into anti-inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction.

Cigarette Smoke, Airway Epithelial Cells and Host Defence

In COPD inflammation driven by exposure to tobacco smoke results in impaired innate immunity in the airway and ultimately to lung injury and remodeling. To understand the biological processes involved in host interactions with cigarette derived toxins submerged epithelial cell culture is widely accepted as a model for primary human airway epithelial cell culture research. Primary nasal and bronchial epithelial cells can also be cultured in air-liquid interface (ALI) models. ALI and submerged culture models have their individual merits, and the decision to use either technique should primarily be determined primarily by the research hypothesis.

Cigarette smoke has gaseous and particulate matter, the latter constituent primarily represented in cigarette smoke extract (CSE). Although not ideal in order to facilitate our understanding of the responses of epithelial cells to cigarette smoke, CSE still has scientific merit in airway cell biology research. Using this model, it has been possible to demonstrate differences in levels of tight junction disruption after CSE exposure along with varied vulnerability to the toxic effects of CSE in cell cultures derived from COPD and control study groups.

Primary nasal epithelial cells (PNECs) have been used as an alternative to bronchial epithelial cells (PBECs). However, at least in subjects with COPD, PNECs cannot consistently substitute for PBECs. Although airway epithelial cells from patients with COPD exhibit a constitutional pro-inflammatory phenotype, these cells have a diminished inflammatory response to CSE exposure. COPD epithelial cells have an increased susceptibility to undergo apoptosis, and have reduced levels of Toll-like receptor-4 expression after CSE exposure, both of which may account for the reduced inflammatory response observed in this group.

The use of CSE in both submerged and ALI epithelial cultures has extended our understanding of the cellular mechanisms that are important in COPD, and helped to unravel important pathways which may be of relevance in its pathogenesis.

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection

Respiratory Syncytial Virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanised monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F-protein with subnanomolar affinity. ALX-0171 demonstrated superior in vitro neutralisation compared to palivizumab against prototypic RSV A and B strains. Moreover, ALX-0171 completely blocked replication below limit of detection in 87% of the viruses tested versus 18% for palivizumab at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.

Eight International London Cough Symposium 2014: Cough hypersensitivity syndrome as the basis for chronic cough

At the Eighth International London Cough Conference held in London in July 2014, the focus was on the relatively novel concept of cough hypersensitivity syndrome (CHS) as forming the basis of chronic cough. This concept has been formulated following understanding of the neuronal pathways for cough and a realisation that not all chronic cough is usually associated with a cause. The CHS is defined by troublesome coughing triggered by low level of thermal, mechanical or chemical exposure. It also encompasses other symptoms or sensations such as laryngeal hypersensitivity, nasal hypersensitivity and possibly also symptoms related to gastrooesopahgeal reflux. The pathophysiologic basis of the CHS is now being increasingly linked to an enhancement of the afferent pathways of the cough reflex both at the peripheral and central levels. Mechanisms involved include the interactions of inflammatory mechanisms with cough sensors in the upper airways and with neuronal pathways of cough, associated with a central component. Tools for assessing CHS in the clinic need to be developed. New drugs may be developed to control CHS. A roadmap is suggested from the inception of the CHS concept towards the development of newer antitussives at the Symposium.

Facial cleanliness and risk of trachoma in families

Trachoma is the leading infectious cause of blindness worldwide, and epidemiologic studies of factors that may increase the transmission of ocular Chlamydia trachomatis are needed. In two villages in a hyperendemic area of Central Tanzania, 472 (90%) of 527 preschool-aged children were examined for specific signs of unclean faces and presence of trachoma. The odds of trachoma were 70% higher in children with flies and nasal discharge on their faces. Other facial signs were not important. In large families, the odds of trachoma increased 4.8-fold if a sibling had trachoma and 6.8-fold if a sibling had trachoma and an unclean face. Health education strategies aimed at improving face washing need to target cleaning nasal discharge and keeping flies off children's faces.

Exposure to children and risk of active trachoma in Tanzanian women

The authors surveyed the trachoma status of 515 women aged 18-60 years and 527 children aged 1-7 years in the trachoma hyperendemic region of Kongwa, Tanzania, in 1989 to further describe the importance of exposure to young children as a risk factor for active trachoma in women. The women were identified as caretakers, who currently cared for children aged 1-7 years; noncaretakers, who lived with, but did not care for, children aged 1-7; or those without children aged 1-7 in the household. The age-adjusted odds ratios for active trachoma seemed to rise with greater exposure to young children, from 1.00 for women without such children, to 1.63 for noncaretakers and 2.43 for caretakers (trend test, p = 0.08). Among those who lived in households with young children, the prevalence of active trachoma in women increased with the total number of young children cared for and with the number of infected children cared for. The prevalence of active trachoma was 40% (6 of 15) for caretakers of three or more infected children, compared with 0 (0 of 88) for caretakers with no infected children (p < 0.0001). Caring for infected children also appeared to be associated with signs of chronic trachoma in caretakers. Noncaretakers who lived with infected children were not at a significantly increased risk for trachoma compared with noncaretakers who were not exposed to such children (5.4% (three of 56) vs. 5.6% (one of 18); p > 0.4). None of the facial signs observed in the children (flies on the face, nasal discharge, etc.) appeared to increase the odds ratio of active trachoma in caretakers beyond the increase associated with trachoma alone in the child. These data support the hypothesis that active disease in women is associated with direct caretaking of young children with active disease. Strategies that interrupt household transmission may affect the binding sequelae of trachoma in women.

Heritability of peripheral refraction in Chinese children and adolescents: the Guangzhou Twin Eye study.

PURPOSE:

To estimate the heritability of peripheral refraction in Chinese children and adolescents.

METHODS:

The authors examined 72 monozygotic (MZ) twins and 48 dizygotic (DZ) twins aged 8 to 20 years from a population-based twin registry. Temporal and nasal peripheral refraction, each 40° from the visual axis, and axial refraction were measured using an autorefractor. Relative peripheral refractive error (RPRE) was defined as the peripheral refraction minus the axial refraction. Heritability was assessed by structural equation modeling after adjustment for age and sex.

RESULTS:

The mean and SD of temporal refraction (T(40)), nasal refraction (N(40)), RPRE-T(40), RPRE-N(40), and T(40)-N(40) asymmetry were -0.27 ± 2.0 D, 0.36 ± 2.19 D, 1.18 ± 1.39 D, 1.80 ± 1.69 D, and -0.62 ± 1.58 D, respectively. The intraclass correlations for T(40) refraction, N(40) refraction, RPRE-T(40), RPRE-N(40), and T(40)-N(40) asymmetry were 0.87, 0.83, 0.65, 0.74, and 0.58 for MZ pairs and 0.49, 0.42, 0.30, 0.41, and 0.32 for DZ pairs, respectively. A model with additive genetic and unique environmental effects was the most parsimonious, with heritability values estimated as 0.84, 0.76, 0.63, 0.70, and 0.55, respectively, for the peripheral refractive parameters.

CONCLUSIONS:

Additive genetic effects appear to explain most of the variance in peripheral refraction and relative peripheral refraction when adjusting for the effects of axial refraction.

A high-value, low-cost bubble continuous positive airway pressure system for low-resource settings:technical assessment and initial case reports

Acute respiratory infections are the leading cause of global child mortality. In the developing world, nasal oxygen therapy is often the only treatment option for babies who are suffering from respiratory distress. Without the added pressure of bubble Continuous Positive Airway Pressure (bCPAP) which helps maintain alveoli open, babies struggle to breathe and can suffer serious complications, and frequently death. A stand-alone bCPAP device can cost $6,000, too expensive for most developing world hospitals. Here, we describe the design and technical evaluation of a new, rugged bCPAP system that can be made in small volume for a cost-of-goods of approximately $350. Moreover, because of its simple design--consumer-grade pumps, medical tubing, and regulators--it requires only the simple replacement of a <$1 diaphragm approximately every 2 years for maintenance. The low-cost bCPAP device delivers pressure and flow equivalent to those of a reference bCPAP system used in the developed world. We describe the initial clinical cases of a child with bronchiolitis and a neonate with respiratory distress who were treated successfully with the new bCPAP device.

Novel methods of drug administration for the treatment and care of older patients

The number of older people globally is increasing, contributing to a growing burden of morbidity and mortality. With this shift in population demographic, comes a new challenge in terms of appropriate healthcare for the over 65 years age group. As medication is the principal therapeutic intervention, it is essential that it be fully optimised, to meet the needs of this heterogeneous population. The most common routes of drug administration are oral and injectable, which may display some limitations for older people, in cases of dysphagia or frailty for example. This review considers alternative methods of drug delivery to the norm, specifically discussing the nasal, pulmonary and transdermal routes, as well as novel orally disintegrating tablets. The changing physiology as ageing occurs must be considered in the development of novel drug delivery devices. This review considers the various aspects of ageing that will influence future drug formulation design and development.