Nasal Lavage Samples Research Articles

Antiviral IFN-γ responses of monocytes at birth predict respiratory tract illness in the first year of life

BackgroundViral respiratory tract infections are the leading cause of acute illness during infancy and are closely linked to chronic inflammatory airway diseases later in life. However, the determinants of susceptibility to acute respiratory tract infections still need to be defined.ObjectiveWe investigated whether the individual variation in antiviral response at birth determines the risk for acute respiratory tract illness in the first year of life.MethodsWe studied 82 children who were enrolled in a birth cohort study of inner-city children with at least 1 parent with allergy or asthma. We cultured cord blood monocytes and assessed IFNG and CCL5 mRNA production at 24 hours after inoculation with respiratory syncytial virus. We also monitored the frequency of acute respiratory tract illness at 3-month intervals and analyzed nasal lavage samples for respiratory tract viruses at the time of illness during the first year.ResultsRespiratory tract infection was reported for 88% of subjects, and respiratory tract viruses were recovered in 74% of symptomatic children. We observed a wide range of antiviral responses in cord blood monocytes across the population. Furthermore, a decrease in production of IFNG (but not CCL5) mRNA in response to respiratory syncytial virus infection of monocytes was associated with a significant increase in the frequency of upper respiratory tract infections (r = −0.42, P < .001) and the prevalence of ear and sinus infections, pneumonias, and respiratory-related hospitalizations.ConclusionIndividual variations in the innate immune response to respiratory tract viruses are detectable even at birth, and these differences predict the susceptibility to acute respiratory tract illness during the first year of life.

Age-related differences in the pathogenesis of chronic rhinosinusitis

Age-related differences in the pathogenesis of chronic rhinosinusitis

S50 Nitrative stress is increased in COPD exacerbations following experimental rhinovirus infection

Introduction and ObjectivesThe majority of acute exacerbations of COPD are associated with viral infection and rhinoviruses are the most frequently detected species. Exacerbations represent a major unmet health need and...

S15 Detection of bacteria in sputum following experimental rhinovirus infection is more common in COPD than controls subjects

Introduction and ObjectivesThere is increasing evidence that the majority of acute exacerbations of COPD (AECOPD) are caused by virus infection, and rhinoviruses are the most frequently identified species. Bacteria are...

S14 Histone deacetylase activity is reduced in COPD subjects during rhinovirus induced exacerbations

Introduction and ObjectivesHistone deacetylase (HDAC) enzymes have a role in suppressing inflammatory gene transcription. There is evidence for reduced HDAC activity in COPD which correlates with the severity of airflow...

Accelerated Shedding of Prions following Damage to the Olfactory Epithelium

In this study, we investigated the role of damage to the nasal mucosa in the shedding of prions into nasal samples as a pathway for prion transmission. Here, we demonstrate that prions can replicate to high levels in the olfactory sensory epithelium (OSE) in hamsters and that induction of apoptosis in olfactory receptor neurons (ORNs) in the OSE resulted in sloughing off of the OSE from nasal turbinates into the lumen of the nasal airway. In the absence of nasotoxic treatment, olfactory marker protein (OMP), which is specific for ORNs, was not detected in nasal lavage samples. However, after nasotoxic treatment that leads to apoptosis of ORNs, both OMP and prion proteins were present in nasal lavage samples. The cellular debris that was released from the OSE into the lumen of the nasal airway was positive for both OMP and the disease-specific isoform of the prion protein, PrP(Sc). By using the real-time quaking-induced conversion assay to quantify prions, a 100- to 1,000-fold increase in prion seeding activity was observed in nasal lavage samples following nasotoxic treatment. Since neurons replicate prions to higher levels than other cell types and ORNs are the most environmentally exposed neurons, we propose that an increase in ORN apoptosis or damage to the nasal mucosa in a host with a preexisting prion infection of the OSE could lead to a substantial increase in the release of prion infectivity into nasal samples. This mechanism of prion shedding from the olfactory mucosa could contribute to prion transmission.

Interferon regulatory factor 7 is a major hub connecting interferon-mediated responses in virus-induced asthma exacerbations in vivo

Exacerbations are responsible for a substantial burden of morbidity and health care use in children with asthma. Most asthma exacerbations are triggered by viral infections; however, the underlying mechanisms have not been systematically investigated. The objective of this study was to elucidate the molecular networks that underpin virus-induced exacerbations in asthmatic children in vivo. We followed exacerbation-prone asthmatic children prospectively and profiled global patterns of gene expression in nasal lavage samples obtained during an acute, moderate, picornavirus-induced exacerbation and 7 to 14 days later. Coexpression network analysis and prior knowledge was used to reconstruct the underlying gene networks. The data showed that an intricate modular program consisting of more than 1000 genes was upregulated during acute exacerbations in comparison with 7 to 14 days later. The modules were enriched for coherent cellular processes, including interferon-induced antiviral responses, innate pathogen sensing, response to wounding, small nucleolar RNAs, and the ubiquitin-proteosome and lysosome degradation pathways. Reconstruction of the wiring diagram of the modules revealed the presence of hyperconnected hub nodes, most notably interferon regulatory factor 7, which was identified as a major hub linking interferon-mediated antiviral responses. This study provides an integrated view of the inflammatory networks that are upregulated during virus-induced asthma exacerbations in vivo. A series of innate signaling hubs were identified that could be novel therapeutic targets for asthma attacks.

Reproducibility of neutrophil percentages in pooled and non-pooled nasal lavage samples

Results The mean (±SEM) neutrophil percentage was not significantly different between the two methods (80 ±12 for SSL, 86 ±8 for MSL, p=0.2). At a minimum total cell count (TCC) cutoff of ≥20, the neutrophil percentage intraclass correlation (ICC) was similar but not sufficiently reproducible for SSL at 0.588 compared to MSL at 0.641. For samples with TCC≥100, the neutrophil percentage ICC of SSL was 0.93 (excellent correlation) and that of MSL was 0.676 (satisfactory). The evaluable samples for TCC≥100 were 60/84 for SSL and 67/84 for MSL. We previously demonstrated in the same experiment that a minimal TCC cutoff of ≥100 cells gave excellent eosinophil percentage ICC for both methods (>0.8). Conclusion SSL ICC was superior to MSL in measuring nasal lavage neutrophil percentage. This method could be used to assess the effects of inhalant stimuli on the nasal cavity.

An exercise in molecular epidemiology: Human rhinovirus prevalence and genetics

Human rhinovirus (HRV) is one of the most common human respiratory pathogens and is responsible for the majority of upper respiratory illnesses. Recently, a phylogeny was constructed from all known American Type Culture Collection (ATCC) HRV sequences. From this study, three HRV classifications (HRVA, HRVB, and HRVC) were determined and techniques for classifying new isolates of HRV were reported. The genetic change of this virus in specific populations over time is of great interest to understand the evolution and epidemiology of viruses. To facilitate the collections of HRV sequences over a number of years, a virology experiment was designed in which students test nasal lavage samples to look for HRV infection. Students will learn a variety of techniques including RNA isolation, cDNA synthesis, qPCR, and agarose gel electrophoresis as well as bioinformatic skills though examination of sequences from the HRV‐field isolates. Furthermore, students can look at symptom data from subjects to investigate correlations between symptom severity and factors such as stress and sleep patterns. Such information can be used to examine hypotheses regarding HRV mutation, symptom severity and epidemiology. BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 39, No. 6, pp. 457–458, 2011

Effect of vitamin supplementation on lung injury and running performance in a hot, humid, and ozone‐polluted environment

In this study, the effect of vitamin C and E supplementation on lung injury and performance of runners were analyzed. Using a randomized, double-blinded, crossover design, nine runners participated in two experimental trials: a 2-week Vitamin trial (vitamin C = 500 mg/day + vitamin E = 100 IU/day) and a 2-week Placebo trial. At the end of each supplementation period the runners performed an 8-km time-trial run in a hot (31°C), humid (70% rh), and ozone-polluted (0.10 ppm O(3)) environmental chamber. Nasal lavage and blood samples were collected pre-, post-, and 6-h post-exercise to assess antioxidant status and CC16 as lung injury marker. Higher plasma (pre- and post-exercise) and nasal lavage (post-exercise) antioxidant concentration were found for the Vitamin trial. Nevertheless, this did not result in performance differences (Vitamin trial: 31:05 min; Placebo trial: 31:54 min; P = 0.075) even though significant positive correlations were found between antioxidant concentration and improvement in time to complete the run. CC16 was higher post-exercise in the Placebo trial (P < 0.01) in both plasma and nasal lavage. These findings suggest that antioxidant supplementation might help to decrease the lung injury response of runners when exercising in adverse conditions, but has little effect on performance.

Viral Profile in Chronic Adenotonsillar Diseases

Objective: Although there is a known role of virus in acute upper respiratory infections, few studies have explored the relationship between chronic diseases of Waldeyer’s ring and viral infections. This study aimed to analyze the presence of the main respiratory viruses in patients with chronic adenotonsillar diseases. Method: Nasal lavage and peripheral blood from 60 children with chronic adenotonsillitis or adenotonsillar hypertrophy were analyzed by RT-PCR for bocavirus, adenovirus, rhinovirus 1 and 2, enterovirus, respiratory syncytial virus, parainfluenza virus type 1 and 3, influenza virus A and B, and human coronavirus. Results: Virus infection was found in 66% of nasal lavage samples (40/60). The most prevalent virus was adenovirus (33.3%), followed by rhinovirus (30%), bocavirus (10%), enterovirus (8.3%), respiratory syncitial virus (5%), and influenza virus (5%). Parainfluenza virus and coronavirus were not detected in any sample. Co-infection was present in 24% of patients (14/60), especially adenovirus (9/14) and bocavirus (8/14). In peripheral blood, none of the patients had virus genome detected. Conclusion: The high prevalence of local genomic viruses in chronic adenotonsillar diseases may reflect a tissue persistence or virus latency, and may be an important etiology that could trigger inflammatory mechanisms involved in chronic diseases of the Waldeyer’s ring.

Quantitative PCR method for detection of mycoplasma spp. DNA in nasal lavage samples from the desert tortoise (Gopherus agassizii)

Mycoplasma agassizii and M. testudineum have been associated with upper respiratory tract disease (URTD) in the threatened desert tortoise (Gopherus agassizii). Because microbiological culture methods have proven difficult to employ in wild desert tortoises, our goal was to develop a sensitive and specific qPCR method for detecting and quantifying mycoplasma DNA in nasal lavage fluid collected in the field. Primers for 16S ribosomal RNA gene sequences specific for M. agassizii and M. testudineum were designed, together with primers that recognize conserved sequences of both microorganisms. Standard curves generated with DNA extracted from known numbers of mycoplasma cells revealed a lower detection limit of approximately 5fg. The qPCR method did not recognize normal flora DNA, and nasal lavage fluid contained no interfering substances. Nasal lavage samples collected from 20 captive desert tortoises housed at the Desert Tortoise Conservation Center (Clark County, Nevada, USA) revealed the presence of M. agassizii DNA in 100% of the tortoises. Concentrations ranged from a low of 6pg ml−1 to a high of 72,962pg ml−1. Only one of the tortoises was positive for M. testudineum. Interestingly, not all of the qPCR positive tortoises showed evidence of seroconversion, suggesting that they were colonized but not infected. This new quantitative method will provide a critical tool for managing threatened populations of the desert tortoise.

Comparison of ozone-specific (OZAC) and oxygen radical (ORAC) antioxidant capacity assays for use with nasal lavage fluid

Antioxidants in respiratory mucus protect the underlying airway epithelium from damage by ozone (O3), a common outdoor air pollutant. To understand O3–antioxidant interactions and the variation of these interactions among individuals, in vitro assays are needed to measure the total antioxidant capacity of airway lavage fluid, a convenient source of (diluted) mucous samples. Here, we compare the oxygen radical absorbance capacity (ORAC), a general method that uses peroxyl radicals as a reactive substance, to the recently developed ozone specific antioxidant capacity (OZAC), a procedure that directly employs O3. For prepared model mucous antioxidant solutions containing uric acid, ascorbic acid or glutathione, the ORAC and OZAC methods yielded comparable antioxidant capacities. The addition of EDTA or DETAPAC, necessary to prevent auto-oxidation of test solutions during the ORAC assay, unpredictably altered ORAC measurements. EDTA did not have a significant effect on OZAC measurements in either prepared uric acid or ascorbic acid solutions. When assessing antioxidant capacities of nasal lavage samples, the ORAC and OZAC assays were no longer comparable. Because the OZAC of nasal lavage samples was positively related to measured uric acid concentrations whereas the ORAC data were not, the OZAC method appears to provide more realistic mucous antioxidant capacities than the ORAC method.

Reproducibility of cell counts in nasal lavage: a comparison of pooled versus non-pooled nasal lavage samples

Results A higher mean cell count was obtained using pooled lavage (means of 342 and 304 vs. 243 and 246, p=0000.4). The mean eosinophil percentage was comparable for both methods (for >100 cell count samples, 7% and 4% for single compared to 5% and 5% for pooled lavage, p=0.2). Single sample lavage produced a higher intraclass correlation (ICC) for eosinophil percentage (0.695 vs. 0.583). A cutoff of 100 total cells gave the most reproducible eosinophil % with ICC of >0.8. The ICC was 0.87 for single sample lavage and 0.81 for pooled lavage with >100 and 0.671 for SSL and 0.535 for MSL with >20 cells. Neutrophil (p=0.2), lymphocyte (p=0.2), monocyte (p=0.3), or basophil (p=0.3) percentages were not significantly different.

Proinflammatory mediators in nasal lavage of subjects with occupational rhinitis

We sought to investigate the type and kinetics of late-phase nasal inflammatory response after nasal challenge with occupational allergens. Participants were 10 subjects experiencing work-related rhinitis symptoms who underwent specific inhalation challenge and tested positive for occupational rhinitis. During challenge, we monitored changes in inflammatory cells, eosinophil cationic protein, myeloperoxidase, and interleukin-8 in nasal lavage samples. The challenge with the active agent induced a significant increase in the percentage of eosinophils at 30 minutes as compared with prechallenge values ( P = 0.04). A significant increase in eosinophil cationic protein levels after challenge with the control ( P = 0.01) and active agent ( P = 0.02) was observed in the late phase after challenge. No significant changes in nasal levels of neutrophils, myeloperoxidase, and interleukin-8 were observed on both control and active challenge days. Our results suggest a predominant nasal eosinophilic inflammatory response after occupational allergen challenge.

Limited airway effects in mild asthmatics after exposure to air pollution in a road tunnel

Ambient air pollution is a contributing factor to respiratory morbidity and mortality and asthmatics are a particularly vulnerable population. The aim of the study was to investigate whether acute exposure to traffic related air pollution in a road tunnel would increase bronchial responsiveness in mild asthmatics, and if this would be accompanied by increased measures of inflammatory markers in the airways assessed by nasal lavage (NAL) and induced sputum. Fourteen mild asthmatics (7 treated with inhaled corticosteroids) were exposed for 2h in a road tunnel and a control environment, respectively, separated by at least 3 weeks. Symptoms and peak expiratory flow (PEF) were recorded. Seven hours following exposure sessions, subjects underwent measurements of fraction of exhaled nitric oxide (FENO), spirometry, and a bronchial provocation test. NAL, induced sputum and blood samples were collected. The median PM(2.5) and PM(10) levels during the exposure occasions in the road tunnel were 80 (range 41-93) μg/m(3) and 183 (72-213) μg/m(3) respectively. Irritative symptoms from the airways increased and PEF decreased after road tunnel exposure. Increased levels of IL-10, IL-12 and TNF-α were observed in NAL fluid from subjects without ongoing inhaled corticosteroid treatment. Forced expiratory volume in 1s (FEV(1)) and the degree of bronchial responsiveness in asthmatics did not change significantly after tunnel exposure. We conclude that asthmatics exhibit increased symptoms, decreased PEF and signs of inflammatory response in the upper airways, after a 2h road tunnel exposure. Our findings may further emphasize asthmatics as a vulnerable group to common air pollutants.

Chemosensory Loss: Functional Consequences of the World Trade Center Disaster

BackgroundIndividuals involved in rescue, recovery, demolition, and cleanup at the World Trade Center (WTC) site were exposed to a complex mixture of airborne smoke, dust, combustion gases, acid mists, and metal fumes. Such exposures have the potential to impair nasal chemosensory (olfactory and trigeminal) function.ObjectiveThe goal of this study was to evaluate the prevalence of chemosensory dysfunction and nasal inflammation among these individuals.MethodsWe studied 102 individuals who worked or volunteered at the WTC site in the days and weeks during and after 11 September 2001 (9/11) and a comparison group with no WTC exposure matched to each participant on age, sex, and job title. Participants were comprehensively evaluated for chemosensory function and nasal inflammation in a single session. Individual exposure history was obtained from self-reported questionnaires.ResultsThe prevalence of olfactory and trigeminal nerve sensitivity loss was significantly greater in the WTC-exposed group relative to the comparison group [prevalence ratios (95% confidence intervals) = 1.96 (1.2–3.3) and 3.28 (2.7–3.9) for odor and irritation thresholds, respectively]. Among the WTC responders, however, individuals caught in the dust cloud from the collapse on 9/11 exhibited the most profound trigeminal loss. Analysis of the nasal lavage samples supported the clinical findings of chronic nasal inflammation among the WTC-exposed cohort.ConclusionsThe prevalence of significant chemosensory impairment in the WTC-exposed group more than 2 years after their exposure raises concerns for these individuals when the ability to detect airborne odors or irritants is a critical safety factor.Relevance to clinical practiceThis outcome highlights the need for chemosensory evaluations among individuals with exposure to acute high or chronic levels of airborne pollutants.

Weekly monitoring of children with asthma for infections and illness during common cold seasons

BackgroundExacerbations of childhood asthma and rhinovirus infections both peak during the spring and fall, suggesting that viral infections are major contributors to seasonal asthma morbidity.ObjectivesWe sought to evaluate rhinovirus infections during peak seasons in children with asthma and to analyze relationships between viral infection and illness severity.MethodsFifty-eight children aged 6 to 8 years with asthma provided 5 consecutive weekly nasal lavage samples during September and April; symptoms, medication use, and peak flow were recorded. Rhinoviruses were identified by using multiplex PCR and partial sequencing of viral genomes.ResultsViruses were detected in 36% to 50% of the specimens, and 72% to 99% of the viruses were rhinoviruses. There were 52 different strains (including 16 human rhinovirus C) among the 169 rhinovirus isolates; no strains were found in more than 2 collection periods, and all but 2 children had a respiratory tract infection. Virus-positive weeks were associated with greater cold and asthma symptom severity (P < .0001 and P = .0002, respectively). Furthermore, virus-positive illnesses had increased duration and severity of cold and asthma symptoms and more frequent loss of asthma control (47% vs 22%, P = .008). Although allergen-sensitized versus nonsensitized children had the same number of viral infections, the former had 47% more symptomatic viral illnesses (1.19 vs 0.81 per month, P = .03).ConclusionsRhinovirus infections are nearly universal in children with asthma during common cold seasons, likely because of a plethora of new strains appearing each season. Illnesses associated with viruses have greater duration and severity. Finally, atopic asthmatic children experienced more frequent and severe virus-induced illnesses.

Human Rhinovirus Infection Up-Regulates MMP-9 Production in Airway Epithelial Cells via NF-κB

Human rhinovirus (HRV) infections up-regulate proinflammatory mediators and growth factors that are associated with exacerbations of inflammatory airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Matrix metalloproteinase (MMP)-9 was shown to be increased in the airways of patients with asthma and COPD. We sought to determine whether HRV infection modulated the expression of MMP-9 and its highest-affinity inhibitor, the tissue inhibitor of metalloproteinase (TIMP)-1, and we explored the mechanism by which this modulation occurs. In vitro studies, using RT-PCR, ELISA, zymography, and a fluorescent activity assay, demonstrated that MMP-9 mRNA, protein, and activity were increased upon infection with HRV, whereas TIMP-1 mRNA and protein remained unchanged. These results were verified in vivo, using nasal lavage samples obtained from subjects with confirmed rhinovirus infections. Human rhinovirus infections were shown to up-regulate NF-kappaB, and NF-kappaB has also been reported to play a role in the expression of MMP-9. We therefore investigated the role of NF-kappaB in HRV-induced MMP-9 expression. Using two inhibitors of IkappaBalpha kinase beta, we observed a concentration-dependent decrease in HRV-induced MMP-9 expression. The role of NF-kappaB in HRV-induced MMP-9 expression was further confirmed using MMP-9 promoter luciferase constructs, which demonstrated that an NF-kappaB site at -620/-607 base pairs was necessary for HRV-induced MMP-9 expression. Electrophoretic mobility shift assays and supershift assays confirmed the nuclear translocation and binding of p50/p65 NF-kappaB subunits to an MMP-9-specific NF-kappaB oligonucleotide. This increase in MMP-9 may be a mechanism by which rhinovirus infections contribute to airway inflammation and, potentially, to airway remodeling.

No evidence for WU polyomavirus infection in chronic obstructive pulmonary disease

Human polyomaviruses are known to cause persistent or latent infections, which are reactivated under immunosuppression. Polyomaviruses have been found to immortalize cell lines and to possess oncogenic properties. Moreover, the recently discovered Merkel cell polyomavirus shows a strong association with human Merkel cell carcinomas. Another novel human polyomavirus, WU polyomavirus (WUPyV), has been identified in respiratory specimens from patients with acute respiratory tract infections (ARTI). WUPyV has been proposed to be a pathogen in ARTI in early life and immunocompromised individuals, but so far its role as a causative agent of respiratory disease remains controversial.The objective of our study was to determine the prevalence of WUPyV infections in adult hospitalized patients with acute exacerbation of chronic obstructive pulmonary disease (COPD) and to establish its potential clinical relevance by comparison to patients with stable COPD hospitalized for other reasons than acute exacerbation of COPD (AE-COPD).A total of 378 respiratory specimens, each 189 induced sputum and nasal lavage samples from 189 patients, who had been recruited in a prospective 2:1 ratio case-control set-up between 1999 and 2003, were evaluated for the presence of WUPyV DNA by real-time PCR.In the present study we could not detect WUPyV DNA in 378 respiratory specimens from 189 adult hospitalized patients with AE-COPD and stable COPD in four consecutive years.Persistence of viral replication or reactivation of latent WUPyV infection did not occur. WUPyV may not play a major role in adult immunocompetent patients with AE-COPD and stable COPD.