Total RNA was extracted using RNEasy Midi-Kits (Qiagen GmbH, Hilden, Germany) according to the manufacturers specifications. 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 p75NTRreceptor Quinine without any concomitant change in the high-affinity trkA receptor. Viral contamination 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 crucial neurotrophic molecules in the respiratory tract, and combination of an acute contamination following chronic nicotine exposure produces more severe neurotrophic dysregulation and neurogenic-mediated inflammation compared to either contamination or nicotine alone. Keywords:asthma, airway inflammation, bronchiolitis, nerve growth factor, tobacco smoke == INTRODUCTION == Strong epidemiologic evidence indicates that exposure to tobacco smoke increases frequency and severity of respiratory tract infections. In particular, several studies15have found a significant association between parental smoking and the rate of hospitalization for respiratory syncytial computer virus (RSV) bronchiolitis in infants and children. Vice versa, viral infections may amplify and prolong the adverse effects of pollution, and recent data suggest that although tobacco smoking remains the major cause of chronic obstructive pulmonary disease, RSV contamination may sustain airway inflammation in these patients leading Rabbit Polyclonal to PIAS3 to accelerated and progressive loss of lung function. 6 Despite the large body of evidence underscoring the importance of interactions between tobacco smoke and viral respiratory infections, the pathophysiologic mechanisms involved remain largely unclear and controversial. Surprisingly, previous studies have reported a decrease in lung inflammation and mucus production when rodents exposed to side-stream cigarette smoke or to nicotine were infected with RSV7or influenza computer virus.8Although this finding is consistent with the hypothesis that certain components of tobacco smoke (specifically nicotine) might have anti-inflammatory properties,9,10it does not fit well with the epidemiologic link between tobacco smoke exposure and virus-induced airway inflammation and hyperreactivity in humans. Previous studies in animal models have shown that RSV contamination upregulates the expression of neurotrophic factors and receptors in the lungs.11,12The increased neurotrophic activity leads to pre-synaptic and post-synaptic modifications responsible for the inflammation and hyperreactivity that develop in virus-infected airways when airborne irritants stimulate intra- and subepithelial nociceptive nerves.1315The same pathophysiologic mechanisms appear to be implicated in human disease, as in recent clinical studies we have shown increased expression of neurotrophic factors and receptors in the airways of RSV-infected infants.16Another environmental agent capable of modulating the activity of sensory neurons and contribute to the process of neurogenic inflammation is usually nicotine, the major tobacco alkaloid present in both the gaseous and particulate phase of cigarette smoke,1719but its effect on neurotrophins expression has not been investigated. Therefore, in the present study, we first analyzed the expression of neurotrophic pathways in lungs chronically exposed to nicotine, Quinine which was infused into pathogen-free rats using micro-osmotic pumps implanted subcutaneously. Expression of nerve growth factor (NGF) and related neurotrophic factors and receptors was measured in the lungs of uncovered rats both at the mRNA level by reverse-transcription polymerase chain reaction (RT-PCR) and at the protein level by enzyme-linked immunoassay. In the second part of this study, we explored the interactions between pollution and contamination by inoculating RSV in rats previously exposed to nicotine for 28 days. In these rats we also measured neurogenic-mediated airway inflammation and assessed the pathophysiologic role played Quinine by the NGF pathway using a specific blocking antibody. == MATERIALS AND METHODS == == Animals == We used pathogen-free Fischer 344 (F-344) rats purchased from Charles River Breeding Laboratories (Raleigh, NC). All rats were housed in polycarbonate cages isolated by polyester filter covers and placed on racks that provided positive individual ventilation with class-100 air to each cage at the rate of 1 1 cage change of air/min (Maxi-Miser, Thoren Caging System, Hazleton, PA). We used individual rooms for housing infected and pathogen-free rats, both of which were serviced by specifically trained husbandry professionals. All manipulations were conducted inside class-100 laminar flow hoods. Bedding, water, and food were autoclaved before use and unpacked only under laminar flow. Cages and water bottles were run through a tunnel washer after every use and disinfected with.