Terry L. Noah

A Controlled Study of Adenoviral-Vector–Mediated Gene Transfer in the Nasal Epithelium of Patients with Cystic Fibrosis

BACKGROUND Cystic fibrosis is a monogenic disease that deranges multiple systems of ion transport in the airways, culminating in chronic infection and destruction of the lung. The introduction of a normal copy of the cystic fibrosis transmembrane conductance regulator (CFTR) gene into the airway epithelium through gene transfer is an attractive approach to correcting the underlying defects in patients with cystic fibrosis. We tested the feasibility of gene therapy using adenoviral vectors in the nasal epithelium of such patients. METHODS An adenoviral vector containing the normal CFTR complementary DNA in four logarithmically increasing doses (estimated multiplicity of infection, 1, 10, 100, and 1000), or vehicle alone, was administered in a randomized, blinded fashion to the nasal epithelium of 12 patients with cystic fibrosis. Gene transfer was quantitated by molecular techniques that detected the expression of CFTR messenger RNA and by functional measurements of transepithelial potential differences (PDs) to assess abnormalities of ion transport specific to cystic fibrosis. The safety of this treatment was monitored by nasal lavage and biopsy to assess inflammation and vector replication. RESULTS The adenoviral vector was detected in nasal-lavage fluid by culture, the polymerase chain reaction (PCR), or both in a dose-dependent fashion for up to eight days after vector administration. There was molecular evidence of gene transfer by reverse-transcriptase PCR assays or in situ hybridization in five of six patients treated at the two highest doses. However, the percentage of epithelial cells transfected by the vector was very low (< 1 percent), and measurement of PD across the epithelium revealed no significant restoration of chloride transport or normalization of sodium transport. At the lower doses of vector, there were no toxic effects. However, at the highest dose there was mucosal inflammation in two of three patients. CONCLUSIONS In patients with cystic fibrosis, adenoviral-vector-mediated transfer of the CFTR gene did not correct functional defects in nasal epithelium, and local inflammatory responses limited the dose of adenovirus that could be administered to overcome the inefficiency of gene transfer.

Pseudomonas aeruginosa Anaerobic Respiration in Biofilms: Relationships to Cystic Fibrosis Pathogenesis

Recent data indicate that cystic fibrosis (CF) airway mucus is anaerobic. This suggests that Pseudomonas aeruginosa infection in CF reflects biofilm formation and persistence in an anaerobic environment. P. aeruginosa formed robust anaerobic biofilms, the viability of which requires rhl quorum sensing and nitric oxide (NO) reductase to modulate or prevent accumulation of toxic NO, a byproduct of anaerobic respiration. Proteomic analyses identified an outer membrane protein, OprF, that was upregulated approximately 40-fold under anaerobic versus aerobic conditions. Further, OprF exists in CF mucus, and CF patients raise antisera to OprF. An oprF mutant formed poor anaerobic biofilms, due, in part, to defects in anaerobic respiration. Thus, future investigations of CF pathogenesis and therapy should include a better understanding of anaerobic metabolism and biofilm development by P. aeruginosa.

Obesity is associated with impaired immune response to influenza vaccination in humans

Background:Obesity is an independent risk factor for morbidity and mortality from pandemic influenza H1N1. Influenza is a significant public health threat, killing an estimated 250 000–500 000 people worldwide each year. More than one in ten of the worlds adult population is obese and more than two-thirds of the US adult population is overweight or obese. No studies have compared humoral or cellular immune responses to influenza vaccination in healthy weight, overweight and obese populations despite clear public health importance.Objective:The study employed a convenience sample to determine the antibody response to the 2009–2010 inactivated trivalent influenza vaccine (TIV) in healthy weight, overweight and obese participants at 1 and 12 months post vaccination. In addition, activation of CD8+ T cells and expression of interferon-γ and granzyme B were measured in influenza-stimulated peripheral blood mononuclear cell (PBMC) cultures.Results:Body mass index (BMI) correlated positively with higher initial fold increase in IgG antibodies detected by enzyme-linked immunosorbent assay to TIV, confirmed by HAI antibody in a subset study. However, 12 months post vaccination, higher BMI was associated with a greater decline in influenza antibody titers. PBMCs challenged ex vivo with vaccine strain virus, demonstrated that obese individuals had decreased CD8+ T-cell activation and decreased expression of functional proteins compared with healthy weight individuals.Conclusion:These results suggest obesity may impair the ability to mount a protective immune response to influenza virus.

Lipid-laden macrophage index and inflammation in bronchoalveolar lavage fluids in children

The presence of lipids in alveolar macrophages has been used clinically as an indicator of aspiration, a process associated with increased lung inflammation in animal models. The hypothesis is that the quantity of lipids in alveolar macrophages, measured as lipid-laden index (LLI), would correlate with lung inflammation in paediatric patients. Children with chronic respiratory symptoms (21 cystic fibrosis (CF), 24 non-CF) underwent flexible bronchoscopy with bronchoalveolar lavage (BAL) and 24-h intraoesophageal pH monitoring for clinical indications. Total cell counts, number and per cent of neutrophils and macrophages, and LLI were determined in the bronchoalveolar lavage fluids (BALF) from all children. BALF were also obtained from eight healthy, young nonsmoking adults for comparison. LLI in non-CF children were 6.9 +/- 3.5 (mean +/- SEM) which were higher than LLI in healthy adults (1.0 +/- 0.4), (p=0.045). Children with CF had very high LLIs (19.2 +/- 4.5) compared with both healthy adults (p=0.014) and children without CF (p=0.045). LLI did not correlate with airway inflammation in any group. LLI in children with abnormal pH probes had a tendency to be higher than in children with normal pH probes, but the difference was not significant (p=0.098). It is concluded that the lipid-laden index was significantly elevated in children with chronic respiratory symptoms compared with healthy adults, and in children with cystic fibrosis compared with those who have other chronic respiratory conditions. However, the lipid-laden index did not correlate with the quantity of bronchoalveolar lavage fluid inflammation. The lipid-laden index in children may, in part, reflect processes other than aspiration, such as airways obstruction.

Association of Lower Airway Inflammation With Physiologic Findings in Young Children With Cystic Fibrosis

The relationship between lower airway markers of inflammation and infection with physiologic findings is poorly understood in young children with cystic fibrosis (CF). The goal of this study was to evaluate the association of bronchoalveolar lavage fluid (BALF) markers of infection and inflammation, including mediators linked to airway remodeling, to infant lung function values in young children with CF undergoing clinically indicated bronchoscopy.

The effect of respiratory synctial virus on chemokine release by differentiated airway epithelium

Respiratory synctial virus (RSV) infection of undifferentiated airway epithelial cells has been shown to induce the production of chemokines. The purpose of this study was to investigate the vectorial release of interleukin (IL-8) and Released on Activation, Normal T-cell Expressed and Secreted (RANTES) by polarized, well-differentiated respiratory epithelial cells after RSV infection. Human bronchial epithelial cultures were differentiated under air-liquid interface conditions and infected with RSV by the apical or basolateral route. RSV infection was specific to the apical surface. Supernatants were collected at 6 and 48 hours after RSV inoculation, and IL-8 and RANTES were measured by enzyme-linked immunosorbent assay (ELISA). Both IL-8 and RANTES were significantly released by 48 hours following inoculation with RSV. The secretion of each chemokine was greatest after apical inoculation, and secretion was polarized to the basolateral supernatant. Immunohistochemical staining confirmed that RSV infection was specific to ciliated cells, and immunohistochemical staining for chemokines was localized to RSV-infected ciliated cells. The authors conclude that, in differentiated human airway epithelium in vitro, RSV-induced increases in IL-8 and RANTES release are predominantly in the basolateral direction. In epithelial layers, virus-containing cells are the predominant source of the increased chemokine release. The authors speculate that similar processes in vivo influence recruitment of leukocytes to sites of RSV infection.

Overweight and obese adult humans have a defective cellular immune response to pandemic H1N1 influenza A virus

Obese adults have a greater risk of morbidity and mortality from infection with pandemic H1N1 influenza A virus (pH1N1). The objective of the present study was to elucidate the specific mechanisms by which obesity and overweight impact the cellular immune response to pH1N1.

Reduced Expression of IRF7 in Nasal Epithelial Cells from Smokers after Infection with Influenza

Smokers are more susceptible to respiratory viral infections, including influenza virus, but the mechanisms mediating this effect are unknown. To determine how epithelial cells contribute to the enhanced susceptibility seen in smokers, we established an in vitro model of differentiated nasal epithelial cells (NECs) from smokers, which showed enhanced mucin expression. The NECs from smokers responded to influenza infection with greater cytotoxicity, release of interleukin-6, and viral shedding than NECs from nonsmokers. Focusing on type I interferon (IFN) expression, we observed that influenza-infected NECs from smokers produced significantly less IFN-alpha than NECs from nonsmokers. Similarly, the expression of IRF7, a key transcription factor controlling the expression of IFN-alpha, was significantly decreased in influenza-infected and IFN-beta-stimulated NECs from smokers. Furthermore, our data indicate that the DNA methylation of the IRF7 gene and expression of the DNA (cytosine-5-)-methyltransferase 1 was enhanced in NECs from smokers. To confirm these findings in vivo, we initiated a study in which smoking and nonsmoking healthy volunteers were inoculated nasally with the live-attenuated influenza virus (LAIV) vaccine, and nasal biopsies were obtained before and after the administration of LAIV. The LAIV-induced expression of IRF7 was lower in the nasal epithelium from smokers, supporting our in vitro observations. These data demonstrate that infection with influenza results in the reduced expression of transcription factor IRF7 in NECs from smokers, and that these effects may be mediated by an epigenetic modification of the IRF7 gene, thus providing a potential mechanism rendering smokers more susceptible to respiratory virus infections.

Tobacco smoke exposure and altered nasal responses to live attenuated influenza virus.

Background Epidemiologic evidence links tobacco smoke and increased risk for influenza in humans, but the specific host defense pathways involved are unclear. Objective We developed a model to examine influenza-induced innate immune responses in humans and test the hypothesis that exposure to cigarette smoke alters nasal inflammatory and antiviral responses to live attenuated influenza virus (LAIV). Methods This was an observational cohort study comparing nasal mucosal responses to LAIV among young adult active smokers (n = 17), nonsmokers exposed to secondhand smoke (SHS; n = 20), and unexposed controls (n = 23). Virus RNA and inflammatory factors were measured in nasal lavage fluids (NLF) serially after LAIV inoculation. For key end points, peak and total (area under curve) responses were compared among groups. Results Compared with controls, NLF interleukin-6 (IL-6) responses to LAIV (peak and total) were suppressed in smokers. Virus RNA in NLF cells was significantly increased in smokers, as were interferon-inducible protein 10:virus ratios. Responses in SHS-exposed subjects were generally intermediate between controls and smokers. We observed significant associations between urine cotinine and NLF IL-6 responses (negative correlation) or virus RNA in NLF cells (positive correlation) for all subjects combined. Conclusions Nasal inoculation with LAIV results in measurable inflammatory and antiviral responses in human volunteers, thus providing a model for investigating environmental effects on influenza infections in humans. Exposure to cigarette smoke was associated with suppression of specific nasal inflammatory and antiviral responses, as well as increased virus quantity, after nasal inoculation with LAIV. These data suggest mechanisms for increased susceptibility to influenza infection among persons exposed to tobacco smoke.

Plasma TGF-β1 in pediatric cystic fibrosis: Potential biomarker of lung disease and response to therapy

Transforming growth factor beta‐1 (TGF‐β1) is an important genetic modifier of lung disease severity in cystic fibrosis (CF), yet the mechanism behind this disease association remains unknown. Initial steps in the investigation of the relationship between TGF‐β1 and CF lung disease include determining the most appropriate available biospecimen for TGF‐β1 protein measurement.