Neurotrophic and neuroimmune responses to early-life Pseudomonas aeruginosa infection in rat lungs.

Early-life respiratory infection with Pseudomonas aeruginosa is common in children with cystic fibrosis or immune deficits. Although many of its clinical manifestations involve neural reflexes, little information is available on the peripheral nervous system of infected airways. This study sought to determine whether early-life infection triggers a neurogenic-mediated immunoinflammatory response, the mechanisms of this response, and its relationship with other immunoinflammatory pathways. Weanling and adult rats were inoculated with suspensions containing P. aeruginosa (PAO1) coated on alginate microspheres suspended in Tris-CaCl(2) buffer. Five days after infection, rats were injected with capsaicin to stimulate nociceptive nerves in the airway mucosa, and microvascular permeability was measured using Evans blue as a tracer. PAO1 increased neurogenic inflammation in the extra- and intrapulmonary compartments of weanlings but not in adults. The mechanism involves selective overexpression of NGF, which is critical for the local increase in microvascular permeability and for the infiltration of polymorphonuclear leukocytes into infected lung parenchyma. These effects are mediated in part by induction of downstream inflammatory cytokines and chemokines, especially IL-1beta, IL-18, and leptin. Our data suggest that neurogenic-mediated immunoinflammatory mechanisms play important roles in airway inflammation and hyperreactivity associated with P. aeruginosa when infection occurs early in life.

Respiratory syncytial virus infection in Fischer 344 rats is attenuated by short interfering RNA against the RSV-NS1 gene.

BACKGROUND: Respiratory syncytial virus (RSV) causes severe bronchiolitis and is a risk factor for asthma. Since there is no commercially available vaccine against RSV, a short interfering RNA against the RSV-NS1gene (siNS1) was developed and its potential for decreasing RSV infection and infection-associated inflammation in rats was tested. METHODS: Plasmids encoding siNS1 or an unrelated siRNA were complexed with a chitosan nanoparticle delivery agent and administered intranasally. Control animals received a plasmid for a non-specific siRNA. After expression of the plasmid in lung cells for 24 hours, the rats were intranasally infected with RSV. RESULTS: Prophylaxis with siNS1 significantly reduced lung RSV titers and airway hyperreactivity to methacholine challenge compared to the control group. Lung sections from siNS1-treated rats showed a sizable reduction in goblet cell hyperplasia and in lung infiltration by inflammatory cells, both characteristics of asthma. Also, bronchoalveolar lavage samples from siNS1-treated animals had fewer eosinophils. Treatment of rats with siNS1 prior to RSV exposure was effective in reducing virus titers in the lung and in preventing the inflammation and airway hyperresponsiveness associated with the infection that has been linked to development of asthma. CONCLUSION: The use of siNS1 prophylaxis may be an effective method for preventing RSV bronchiolitis and potentially reducing the later development of asthma associated with severe respiratory infections.

Nerve growth factor mediates steroid-resistant inflammation in respiratory syncytial virus infection.

Neurotrophic factors and receptors are upregulated in the respiratory tract of humans and rodents infected by the respiratory syncytial virus, leading to airway inflammation and hyperreactivity. The contribution of neurotrophic pathways to the recruitment of immuno-inflammatory cells and their response to anti-inflammatory therapy remains unclear. We sought to determine whether selective nerve growth factor inhibition prevents the immuno-inflammatory response against infection, and explored the effect of inhaled corticosteroids on virus-induced neurotrophic upregulation and the consequent recruitment of immuno-inflammatory cells into the airways. We tried to inhibit the recruitment of lymphocytes and monocytes into the airways of infected weanling rats using immunologic inhibition of nerve growth factor with a specific blocking antibody, or chemical inhibition of receptor tyrosine kinase with K252a. The anti-inflammatory activity of inhaled corticosteroids was studied in infected rats treated with budesonide, fluticasone, or vehicle. Immunological or chemical inhibition of nerve growth factor or its high-affinity receptor tyrosine kinase pathway inhibited the recruitment of inflammatory cells triggered by nociceptive irritation of infected rat airways, thereby reducing local and systemic immuno-inflammatory responses against the virus. Neurotrophic upregulation in infected airways was not affected by inhaled corticosteroids. As a logical consequence, these commonly used drugs were also unable to stop the recruitment of immune and inflammatory effector cells into infected airways. Overexpression of neurotrophic factors and receptors in airways infected by respiratory syncytial virus is critical for the development of airway inflammation and hyperreactivity, which is resistant to the anti-inflammatory effect of inhaled corticosteroids.

Safety of Pirfenidone in Patients with Idiopathic Pulmonary Fibrosis: Experience from 92 Sites in an Open-Label US Expanded Access Program.

INTRODUCTION: In phase 3 clinical trials, pirfenidone significantly slowed disease progression with a well-defined and medically manageable safety profile in patients with idiopathic pulmonary fibrosis (IPF). This study examined safety events related to pirfenidone in patients with IPF in an expanded access program in the US. METHODS: The Expanded Access Program allowed patients with IPF access to pirfenidone prior to US Food and Drug Administration approval. Patients had an IPF diagnosis including a definite or possible usual interstitial pneumonia (UIP) pattern, predicted forced vital capacity ≥50%, and predicted diffusing capacity for carbon monoxide ≥30%. Clinical laboratory data and adverse drug reactions (ADRs) deemed causally related to pirfenidone were analyzed using descriptive summary statistics. RESULTS: Of the 1620 patients treated, 1221 (75.4%) completed the program: 66.5% had definite UIP, and 33.2% had possible UIP. Mean (SD) pirfenidone exposure was 22.8 (9.6) weeks, and mean (SD) daily dose during the course of treatment was 2058.7 (399.2) mg. ADRs occurred in 64.9% of patients: 3.3% were severe and 0.2% life threatening. The most common ADRs were nausea (22.6%) and fatigue (19.6%); 13.0% of patients discontinued due to ADRs. Serious ADRs occurred in 24 patients (1.5%), which were primarily related to elevated liver function enzymes (ten patients, 0.6%). No ADRs led to death. CONCLUSIONS: In this open-label study of 1620 patients with IPF, including those with possible UIP, the safety profile of pirfenidone was consistent with that of earlier clinical trials, and no new safety signals were identified. NCT02141087. FUNDING: Genentech, Inc., a member of the Roche group.

Anti-inflammatory effect of albuterol enantiomers during respiratory syncytial virus infection in rats.

Every year in the United States, respiratory syncytial virus (RSV) infections in infants and young children cause more than 120,000 hospitalizations, often complicated by the need for mechanical ventilation; yet no effective therapy is currently available for this disease. We showed previously that RSV infection is associated with neurogenic inflammation in the lower respiratory tract. In the present study, we sought to determine whether aerosolized beta(2)-receptor agonists inhibit neurogenic-mediated albumin extravasation in the airways of RSV-infected, mechanically ventilated rats, and to compare the anti-inflammatory effects of racemic albuterol ((RS)-albuterol) to its individual enantiomeric components (R)-albuterol and (S)-albuterol. Albumin extravasation evoked by sensorineural stimulation with capsaicin was inhibited only partially by 0.63 mg (RS)-albuterol, and higher doses had minimal incremental effects. In contrast, the anti-inflammatory effect of (R)-albuterol was already larger at the lowest dose of 0.31 mg, and complete inhibition of neurogenic exudation was observed at higher doses. (S)-albuterol had no significant inhibitory effect up to 1.25 mg, and had only partial inhibitory effect at higher doses. The anti-inflammatory effect of (R)-albuterol was independent from the expression of substance P neurokinin 1 receptors, suggesting a direct vascular effect. Our data show that (R)-albuterol has a much stronger inhibitory effect on neurogenic inflammation in RSV-infected airways when aerosolized in enantiomerically pure form, rather than in a racemic mixture with (S)-albuterol. Based on these data, we speculate that (R)-albuterol may be more effective than other adrenergic agents in the management of bronchiolitis.

Leukotriene synthesis during respiratory syncytial virus bronchiolitis: influence of age and atopy.

Respiratory syncytial virus (RSV) infection is the most common cause of bronchiolitis in infants and an important risk factor for the development of recurrent wheezing and asthma. Cysteinyl leukotrienes were implicated in the pathophysiology of these diseases, and are being targeted for their diagnosis and therapy. We measured urinary leukotriene E4 (LTE4) in infants with RSV bronchiolitis in comparison with controls without respiratory infection, and investigated whether medical and family history, age, and passive exposure to tobacco smoke are related to urinary leukotriene excretion. We studied 33 infants with bronchiolitis and 25 controls, 1-12 months of age. Demographic and historical data were obtained from informed-consent forms and questionnaires completed by the parents. RSV was detected in nasal secretions by enzyme-linked immunoassay. Urine samples were collected on day of admission and were analyzed for LTE4 with an enzyme-linked immunoassay. Urinary LTE4 was 8-fold higher in infants with bronchiolitis than in controls. Leukotriene excretion was significantly higher in infected infants <6 months of age with a medical history of eczema or dry cough and/or family history of asthma. Multivariate analysis revealed that eczema and dry cough are independently associated with high LTE4 excretion during bronchiolitis. Exposure to tobacco smoke did not affect urinary LTE4. Our study shows that leukotriene synthesis during bronchiolitis is particularly elevated in younger infants with an atopic/asthmatic background. Urinary LTE4 may become a valuable, noninvasive marker for the identification of patients who will benefit most from therapy with leukotriene modifiers for management of bronchiolitis.

Advances in viral respiratory infections: new experimental models.

The wide array of models available for the study of respiratory viral infections is extremely valuable for the development of novel therapeutic and prophylactic options against these highly prevalent diseases. In addition, through these models we have gathered considerable insight into the cellular and molecular mechanisms involved in the pathogenesis of these infections and the inflammatory and immune responses they elicit in the host. This article reviews new promising models introduced recently in this field.

Combined effects of chronic nicotine and acute virus exposure on neurotrophin expression in rat lung.

Strong epidemiologic evidence indicates that tobacco smoke influences frequency and severity of respiratory infections. Previously, we have shown that infection with respiratory syncytial virus upregulates expression of neurotrophic factors and receptors in the lungs, but the effect of tobacco exposure on neurotrophins is unknown. Therefore, we first sought to determine the expression of neurotrophic pathways in lungs of rats chronically exposed to nicotine, and then we studied the interactions between pollution and infection by inoculating virus after nicotine exposure. Expression of the neurotrophins nerve growth factor (NGF) and brain-derived neurotrophic factor, of their high-affinity tyrosine kinase receptors (trkA and trkB, respectively), and of the low-affinity receptor p75(NTR) was measured in the lungs of nicotine-exposed rats both at the mRNA level by reverse-transcription polymerase chain reaction and at the protein level by enzyme-linked immunoassay. Nicotine increased NGF expression both at the mRNA and protein level and also created a receptor imbalance deriving from increased expression of the pro-inflammatory p75(NTR) receptor without any concomitant change in the high-affinity trkA receptor. Viral infection after chronic nicotine exposure exerted an additive effect on NGF expression, and resulted in exaggerated neurogenic airway inflammation that was abolished by selective inhibition. In conclusion, nicotine levels comparable to those found in smokers are per se able to upregulate the expression of critical neurotrophic molecules in the respiratory tract, and combination of an acute infection following chronic nicotine exposure produces more severe neurotrophic dysregulation and neurogenic-mediated inflammation compared to either infection or nicotine alone.

Less air pollution leads to rapid reduction of airway inflammation and improved airway function in asthmatic children.

OBJECTIVE: Air pollution can promote airway inflammation, posing significant health risks for children with chronic respiratory problems. However, it is unknown whether this process is reversible, so that limiting pollution will benefit these children. We measured the short-term response of allergic asthmatic children exposed to a real-life reduction in outdoor air pollution by using noninvasive biomarkers of airway inflammation and function. PATIENTS AND METHODS: Thirty-seven untreated allergic children with mild persistent asthma were recruited from a highly polluted urban environment and relocated to a less polluted rural environment. Air pollution, pollen counts, and meteorological conditions were carefully monitored at both sites. Nasal eosinophils, fractional exhaled nitric oxide, peak expiratory flow, and urinary leukotriene E(4) were measured first in the urban environment and then again 7 days after relocation to the rural environment. RESULTS: One week after relocation to the rural environment, we measured, on average, a fourfold decrease in nasal eosinophils and significant decrease in fractional exhaled nitric oxide. We also noted an improvement in lower airway function, reflected by highly significant increase in peak expiratory flow. In contrast, mean urinary leukotriene E(4) concentration remained unchanged after 1 week of exposure to the rural environment. CONCLUSIONS: Better air quality is associated with a rapid reduction of airway inflammation in allergic asthmatic children. Nasal eosinophils and fractional exhaled nitric oxide are sensitive indicators of this effect, and their rapid decline is paralleled by improved airway function measured by peak expiratory flow. Leukotriene synthesis has a more variable response to environmental modifications.

Growth factors gene expression in the developing lung.

AIM: This is the first systematic study using quantitative real-time PCR to analyze and compare the expression profiles for critical members of the epidermal growth factor (EGF), transforming growth factor beta (TGFbeta), and vascular endothelial growth factor (VEGF) families in developing rat lungs. METHODS: mRNA expression was quantified at embryonic (E) day 15, 17, 19, 21, and postnatal age 1 day, 2 weeks, 12 weeks. RESULTS: EGF and EGFR increased during gestation and development, then decreased in adulthood, whereas TGFalpha was highest at birth and remained unchanged afterwards. All TGFbeta isoforms increased slightly during pregnancy, reached highest expression during development, and returned to neonatal levels in adulthood. TGFbetaRI and TGFbetaRII patterns were similar to TGFbeta2 and TGFbeta1 respectively, whereas TGFbetaRIII expression was lowest at the postnatal time points. VEGF(164) and VEGF(120) showed a steady increase up to 2 weeks and declined at 12 weeks, whereas highest VEGF(188) expression occurred at 12 weeks. VEGF-A receptors expression paralleled the summation of all three isoforms, increasing steadily with age. CONCLUSION: Expression of growth factors in the developing lung is characterized by highly regulated distinctive patterns that may be critical to understand the early origin and progression of pulmonary diseases in childhood as well as in adulthood. Quantitative real-time PCR analysis revealed several differences compared to previously reported expression patterns defined with older methodologies.

Palivizumab in the prophylaxis of respiratory syncytial virus infection.

Respiratory syncytial virus infection continues to be one of the most important health problems in infancy. Active prophylaxis against this infection (i.e., vaccination) is not available. Therefore, protection of high-risk infants is possible only by passive prophylaxis with specific antibodies. Palivizumab (Synagis) and respiratory syncytial virus intravenous immune globulin are licensed by the US Food and Drug Administration for the prevention of severe lower respiratory tract infections caused by respiratory syncytial virus in infants with bronchopulmonary dysplasia, infants with a history of premature birth (< or =35 weeks gestational age) and children with hemodynamically significant congenital heart disease. Palivizumab is a humanized monoclonal antibody produced by recombinant DNA technology, directed to an epitope in the A antigenic side of the F-protein of the respiratory syncytial virus. This review discusses the characteristics of this drug in detail.

Effect of respiratory syncytial virus on apnea in weanling rats.

Apnea is a common complication of respiratory syncytial virus (RSV) infection in young infants. The purpose of this study was to determine whether this infection affects apnea triggered by sensorineural stimulation in weanling rats. We also studied which neurotransmitters are involved in this response and whether passive prophylaxis with a specific neutralizing antibody (palivizumab) confers protection against it. Weanling rats were inoculated intranasally with RSV, adenovirus, or virus-free medium. Changes in respiratory rate and apnea in response to nerve stimulation with increasing doses of capsaicin were measured by plethysmography. Capsaicin-induced apnea was significantly longer in RSV-infected rats at postinoculation days 2 (upper airways infection) and 5 (lower airways infection), and apnea-related mortality occurred only in the RSV-infected group. Reduction in the duration of apnea was observed after selective inhibition of central gamma-aminobutyric acid (GABA) type A receptors and neurokinin type 1 receptors for substance P. Prophylactic palivizumab protected against apnea and apnea-related mortality. These results suggest that sensorineural stimulation during RSV infection is associated with the development of apnea and apnea-related death in early life, whose mechanism involves the release of GABA acting on central GABA type A receptors and substance P acting on neurokinin type 1 receptors.

Persistent airway inflammation after resolution of respiratory syncytial virus infection in rats.

Neurogenic inflammation is markedly potentiated in airways that are infected with respiratory syncytial virus (RSV). Aims of this study were to determine whether this potentiation persists after the virus is cleared, investigate the mechanism of postviral potentiation, and define whether prophylaxis with a MAb against the RSV fusion protein (palivizumab) prevents this effect. Thirty days after inoculation, no evidence of active RSV infection was found in the airway epithelium by plaque assay or immunostaining and no viral nucleic sequences were detected by PCR, yet capsaicin-induced plasma extravasation in the airways that were infected 30 d earlier with RSV was still significantly larger compared with pathogen-free controls. Substance P content in lung tissues and capsaicin-induced release of this peptide from sensory nerves were significantly increased at 30 d. The administration of palivizumab 24 h before virus inoculation prevented the development of abnormal neurogenic inflammatory responses. Our data suggest that the airways remain abnormally susceptible to the proinflammatory effects of sensory nerves after RSV infection is cleared, as a result of changes in sensory innervation, and that this abnormality can be prevented by passive prophylaxis against RSV.

Immunomodulatory effects of sensory nerves during respiratory syncytial virus infection in rats.

Respiratory syncytial virus (RSV) infection is associated with exaggerated neurogenic inflammation in the airways. This study sought to determine whether irritation of the mucosal sensory fibers affects the recruitment of lymphocytes and monocytes to RSV-infected airways. Pathogen-free rats were inoculated with RSV or with virus-free medium and were injected 5 days later with capsaicin to stimulate airway sensory nerves. Bronchoalveolar lavage was performed 1, 5, or 10 days after nerve stimulation, and samples were analyzed by differential cell count and flow cytometry. Without nerve stimulation, RSV caused a minimal increase in the number of lymphocytes and monocytes above pathogen-free control levels. After nerve stimulation, numerous lymphocytes, predominantly CD4+ T cells, and monocytes were recruited in the airways of infected rats, whereas no difference was found in pathogen-free controls. RSV induced overexpression of the neurokinin 1 (NK1) receptor for substance P on discrete lymphocyte subpopulations within the bronchial-associated lymphoid tissue (BALT), and treatment with a specific NK1 receptor antagonist abolished the recruitment of both lymphocytes and monocytes to infected airways. Our data suggest that airborne irritants stimulating mucosal sensory fibers during RSV infection exert important immunomodulatory effects by attracting to the infected airways selected lymphocyte subpopulations from the local BALT as well as monocytes.

Nerve growth factor and nerve growth factor receptors in respiratory syncytial virus-infected lungs.

Nerve growth factor (NGF) controls sensorineural development and responsiveness and modulates immunoinflammatory reactions. Respiratory syncytial virus (RSV) potentiates the proinflammatory effects of sensory nerves in rat airways by upregulating the substance P receptor, neurokinin 1 (NK(1)). We investigated whether the expression of NGF and its trkA and p75 receptors in the lungs is age dependent, whether it is upregulated during RSV infection, and whether it affects neurogenic inflammation. Pathogen-free rats were killed at 2 (weanling) to 12 (adult) wk of age; in addition, subgroups of rats were inoculated with RSV or virus-free medium. In pathogen-free rats, expression of NGF and its receptors in the lungs declined with age, but RSV doubled expression of NGF, trkA, and p75 in weanling and adult rats. Exogenous NGF upregulated NK(1) receptor expression in the lungs. Anti-NGF antibody inhibited NK(1) receptor upregulation and neurogenic inflammation in RSV-infected lungs. These data indicate that expression of NGF and its receptors in the lungs declines physiologically with age but is upregulated by RSV and is a major determinant of neurogenic inflammation.