Development of a Rhinovirus Inoculum Using a Reverse Genetics Approach.

BACKGROUND: Experimental inoculation is an important tool for common cold and asthma research. Producing rhinovirus (RV) inocula from nasal secretions has required prolonged observation of the virus donor to exclude extraneous pathogens. We produced a RV-A16 inoculum using reverse genetics and determined the dose necessary to cause moderate colds in seronegative volunteers. METHODS: The consensus sequence of RV-A16 from a previous inoculum was cloned, and inoculum virus was produced using reverse genetics techniques. After safety testing, volunteers were inoculated with either RV-A16 (n = 26) or placebo (n = 10), Jackson cold scores were recorded, and nasal secretions were tested for shedding of RV-A16 ribonucleic acid. RESULTS: The reverse genetics process produced infectious virus that was neutralized by specific antisera and had a mutation rate similar to conventional virus growth techniques. The 1000 median tissue culture infectious dose (TCID50) dose produced moderate colds in most individuals with effects similar to that of a previously tested conventional RV-A16 inoculum. CONCLUSIONS: Reverse genetics techniques produced a RV-A16 inoculum that can cause clinical colds in seronegative volunteers, and they also serve as a stable source of virus for laboratory use. The recombinant production procedures eliminate the need to derive seed virus from nasal secretions, thus precluding introduction of extraneous pathogens through this route.

Characterisation of asthma subgroups associated with circulating YKL-40 levels.

The chitinase-like protein YKL-40 mediates airway inflammation and serum levels are associated with asthma severity. However, asthma phenotypes associated with YKL-40 levels have not been precisely defined.We conducted an unsupervised cluster analysis of asthma patients treated at the Yale Center for Asthma and Airways Disease (n=156) to identify subgroups according to YKL-40 level. The resulting YKL-40 clusters were cross-validated in cohorts from the Severe Asthma Research Programme (n=167) and the New York University/Bellevue Asthma Repository (n=341). A sputum transcriptome analysis revealed molecular pathways associated with YKL-40 subgroups.Four YKL-40 clusters (C1-C4) were identified. C3 and C4 had high serum YKL-40 levels compared with C1 and C2. C3 was associated with earlier onset and longer duration of disease, severe airflow obstruction, and near-fatal asthma exacerbations. C4 had the highest serum YKL-40 levels, adult onset and less airflow obstruction, but frequent exacerbations. An airway transcriptome analysis in C3 and C4 showed activation of non-type 2 inflammatory pathways.Elevated serum YKL-40 levels were associated with two distinct clinical asthma phenotypes: one with irreversible airway obstruction and another with severe exacerbations. The YKL-40 clusters are potentially useful for identification of individuals with severe or exacerbation-prone asthma.

Mind-body interactions in the regulation of airway inflammation in asthma: A PET study of acute and chronic stress.

BACKGROUND: Psychological stress has long been recognized as a contributing factor to asthma symptom expression and disease progression. Yet, the neural mechanisms that underlie this relationship have been largely unexplored in research addressing the pathophysiology and management of asthma. Studies that have examined the mechanisms of this relationship in the periphery suggest that it is the superimposition of acute stress on top of chronic stress that is of greatest concern for airway inflammation. METHODS: We compared asthmatic individuals with high and low levels of chronic life stress in their neural and peripheral physiological responses to the Trier Social Stress Test and a matched control task. We used FDG-PET to measure neural activity during performance of the two tasks. We used both circulating and airway-specific markers of asthma-related inflammation to assess the impact of acute stress in these two groups. RESULTS: Asthmatics under chronic stress had a larger HPA-axis response to an acute stressor, which failed to show the suppressive effects on inflammatory markers observed in those with low chronic stress. Moreover, our PET data suggest that greater activity in the anterior insula during acute stress may reflect regulation of the effect of stress on inflammation. In contrast, greater activity in the mid-insula and perigenual anterior cingulate seems to reflect greater reactivity and was associated with greater airway inflammation, a more robust alpha amylase response, and a greater stress-induced increase in proinflammatory cytokine mRNA expression in airway cells. CONCLUSIONS: Acute stress is associated with increases in markers of airway inflammation in asthmatics under chronic stress. This relationship may be mediated by interactions between the insula and anterior cingulate cortex, that determine the salience of environmental cues, as well as descending regulatory influence of inflammatory pathways in the periphery.

Genetic variation in chitinase 3-like 1 (CHI3L1) contributes to asthma severity and airway expression of YKL-40.

BACKGROUND: Single nucleotide polymorphisms (SNPs) in the chitinase 3-like 1 (CHI3L1) promoter, the gene encoding YKL-40, are associated with circulating YKL-40 levels and asthma prevalence. However, the effects of gene polymorphisms on asthma severity and airway expression of YKL-40 have not been examined. OBJECTIVE: We sought to determine the effect of genetic variation in CHI3L1 on asthma severity and YKL-40 expression in subjects from the Yale Center for Asthma and Airways Disease and the Severe Asthma Research Program. METHODS: SNPs spanning the CHI3L1 gene were genotyped in 259 Yale Center for Asthma and Airways Disease and 919 Severe Asthma Research Program subjects. Association and haplotype analyses were conducted to identify effects on airflow obstruction, YKL-40 levels, and asthma severity. RESULTS: Fifteen SNPs in CHI3L1 were associated with FEV1, serum YKL-40 levels, or both. rs12141494 (intron 6) was the only SNP in subjects of European ancestry in both cohorts that was associated with serum YKL-40 levels and postbronchodilator FEV1. Conditional analysis demonstrated that the effect on lung function was independent of the promoter SNP rs4950928, and haplotype analysis demonstrated that G alleles at rs12141494 and rs4950928 are associated with lower YKL-40 expression and higher FEV1 percent predicted values. In asthmatic subjects the risk allele A at rs12141494 was associated with severe asthma and higher YKL-40 expression in the airway (P ≤ .05). CONCLUSION: In contrast to the promoter SNP rs4950928, the intronic SNP rs12141494 in CHI3L1 is associated with asthma severity, lung function, and YKL-40 expression in the blood and airway. These data suggest that SNP rs12141494 modulates YKL-40 expression in the airway and contributes to airway remodeling and asthma severity.

Lower airway rhinovirus burden and the seasonal risk of asthma exacerbation.

RATIONALE: Most asthma exacerbations are initiated by viral upper respiratory illnesses. It is unclear whether human rhinovirus (HRV)­induced exacerbations are associated with greater viral replication and neutrophilic inflammation compared with HRV colds. OBJECTIVES: To evaluate viral strain and load in a prospective asthma cohort during a natural cold. METHODS: Adults were enrolled at the first sign of a cold, with daily monitoring of symptoms, medication use, and peak expiratory flow rate until resolution. Serial nasal lavage and induced sputum samples were assessed for viral copy number and inflammatory cell counts. MEASUREMENTS AND MAIN RESULTS: A total of 52 persons with asthma and 14 control subjects without atopy or asthma were studied for over 10 weeks per subject on average; 25 participants developed an asthma exacerbation. Detection of HRVs in the preceding 5 days was the most common attributable exposure related to exacerbation. Compared with other infections, those by a minor group A HRV were 4.4- fold more likely to cause exacerbation (P = 0.038). Overall, sputum neutrophils and the burden of rhinovirus in the lower airway were similar in control subjects without atopy and the asthma group. However, among HRV-infected participants with asthma, exacerbations were associated with greater sputum neutrophil counts (P = 0.005). CONCLUSIONS: HRV infection is a frequent cause of exacerbations in adults with asthma and a cold, and there may be group-specific differences in severity of these events. The absence of large differences in viral burden among groups suggests differential lower airway sensitization to the effects of neutrophilic inflammation in the patients having exacerbations.

Obstructive Sleep Apnea Risk, Asthma Burden, and Lower Airway Inflammation in Adults in the Severe Asthma Research Program (SARP) II.

BACKGROUND: Obstructive sleep apnea (OSA) may worsen asthma, but large studies are lacking and the underlying mechanisms are unknown. OBJECTIVE: The objective of this study was to determine the prevalence of OSA risk among patients with asthma of different severity compared with normal controls (NC), and among asthmatics, to test the relationship of OSA risk with asthma burden and airway inflammation. METHODS: Subjects with severe (SA, n = 94) and nonsevere asthma (NSA, n = 161), and NC (n = 146) were recruited in an add-on substudy, to the observational Severe Asthma Research Program (SARP) II; subjects completed sleep quality, sleepiness and OSA risk (Sleep Apnea scale of the Sleep Disorders Questionnaire [SA-SDQ]) questionnaires, and clinical assessments. Sputum was induced in a subset of asthmatics. RESULTS: Relative to NC, despite similar sleep duration, the subjects with SA and NSA had worse sleep quality, were sleepier, and had higher SA-SDQ scores. Among asthmatics, higher SA-SDQ was associated with increased asthma symptoms, ß-agonist use, health care utilization, and worse asthma quality of life. A significant association of SA-SDQ with sputum polymorphonuclear cells% was noted: each increase in SA-SDQ by its standard deviation (6.85 units) was associated with a rise in % sputum neutrophils of 7.78 (95% CI 2.33-13.22, P = .0006), independent of obesity and other confounders. CONCLUSIONS: OSA symptoms are more prevalent among asthmatics, in whom they are associated with higher disease burden. OSA risk is associated with a neutrophilic airway inflammation in asthma, which suggests that OSA may be an important contributor to the neutrophilic asthma. Further studies are necessary to confirm these findings and better understand the mechanistic underpinnings of this relationship.

Are there neurophenotypes for asthma? Functional brain imaging of the interaction between emotion and inflammation in asthma.

BACKGROUND: Asthma is a chronic inflammatory disease noteworthy for its vulnerability to stress and emotion-induced symptom intensification. The fact that psychological stress and mood and anxiety disorders appear to increase expression of asthma symptoms suggests that neural signaling between the brain and lung at least partially modulates the inflammatory response and lung function. However, the precise nature of the neural pathways implicated in modulating asthma symptoms is unknown. Moreover, the extent to which variations in neural signaling predict different phenotypes of disease expression has not been studied. METHODS AND RESULTS: We used functional magnetic resonance imaging to measure neural signals in response to asthma-specific emotional cues, following allergen exposure, in asthmatics with a dual response to allergen challenge (significant inflammation), asthmatics with only an immediate response (minimal inflammation), and healthy controls. The anterior insular cortex was differentially activated by asthma-relevant cues, compared to general negative cues, during the development of the late phase of the dual response in asthmatics. Moreover, the degree of this differential activation predicted changes in airway inflammation. CONCLUSIONS: These findings indicate that neurophenotypes for asthma may be identifiable by neural reactivity of brain circuits known to be involved in processing emotional information. Those with greater activation in the anterior insula, in response to asthma-relevant psychological stimuli, exhibit greater inflammatory signals in the lung and increased severity of disease and may reflect a subset of asthmatics most vulnerable to the development of psychopathology. This approach offers an entirely new target for potential therapeutic intervention in asthma.

Neural circuitry underlying the interaction between emotion and asthma symptom exacerbation.

Asthma, like many inflammatory disorders, is affected by psychological stress, suggesting that reciprocal modulation may occur between peripheral factors regulating inflammation and central neural circuitry underlying emotion and stress reactivity. Despite suggestions that emotional factors may modulate processes of inflammation in asthma and, conversely, that peripheral inflammatory signals influence the brain, the neural circuitry involved remains elusive. Here we show, using functional magnetic resonance imaging, that activity in the anterior cingulate cortex and insula to asthma-relevant emotional, compared with valence-neutral stimuli, is associated with markers of inflammation and airway obstruction in asthmatic subjects exposed to antigen. This activation accounts for > or =40% of the variance in the peripheral markers and suggests a neural basis for emotion-induced modulation of airway disease in asthma. The anterior cingulate cortex and insula have been implicated in the affective evaluation of sensory stimulation, regulation of homeostatic responses, and visceral perception. In individuals with asthma and other stress-related conditions, these brain regions may be hyperresponsive to disease-specific emotional and afferent physiological signals, which may contribute to the dysregulation of peripheral processes, such as inflammation.

The avian chB6 alloantigen induces apoptosis in DT40 B cells.

In avian species, B-lymphocytes develop in the bursa of Fabricius. Cells developing in the bursa are subject to signals regulating their survival, with the majority of cells dying by apoptosis within the bursa. However, the molecules delivering the signals influencing this life and death decision remain enigmatic. We have previously shown that antibodies against the chB6 alloantigen present on avian B-lymphocytes can induce a rapid form of cell death. Here we extend this finding by showing that anti-chB6 antibodies induce true apoptosis in DT40 cells without visible membrane damage. This apoptosis results in DNA degradation and morphologic changes characteristic of apoptosis. Furthermore, this apoptosis is coincident with a loss of mitochondrial membrane potential and is inhibited by either overexpression of bcl-x(L) or the presence of inhibitors of caspase 8, 9, or 3 activity. Collectively these data argue that chB6 may function as a novel death receptor on avian B-lymphocytes and support the use of DT40 as an amenable model to study the signaling involved in chB6-induced apoptosis.