IL-4 receptor polymorphisms predict reduction in asthma exacerbations during response to an anti-IL-4 receptor α antagonist.

BACKGROUND: This is the first large pharmacogenetic investigation of the inflammatory IL-4/IL-13 pathway in patients with moderate-to-severe asthma. We analyzed genomic DNA from participants in a 12-week placebo-controlled efficacy trial of pitrakinra (1, 3, or 10 mg twice daily), a novel IL-4/IL-13 pathway antagonist (Clinicaltrials.govNCT00801853). OBJECTIVES: The primary hypothesis for this analysis is that amino acid changes in the 3' end of the IL-4 receptor α gene (IL4RA) or closely proximal variants would predict reductions in asthma exacerbations for subjects randomized to pitrakinra therapy. METHODS: Nineteen IL4RA single nucleotide polymorphisms (SNPs) were tested in 407 non-Hispanic white subjects for association with the primary clinical end point of asthma exacerbations and changes in secondary end points for asthma symptom scores. RESULTS: The most consistent pharmacogenetic associations were observed for the correlated tagging SNPs rs8832 and rs1029489 in the IL4RA 3' untranslated and proximal regions, respectively. Subjects homozygous for the rs8832 common G allele randomized to pitrakinra (placebo group nonsignificant) had decreased asthma exacerbations and decreased nocturnal awakenings and activities limited by asthma. There was also a significant pitrakinra dose-response relationship (placebo/1 mg/3 mg/10 mg) for exacerbations in subjects homozygous for the common allele in rs1029489 (P = .005) and rs8832 (P= .009) and the intronic SNPs rs3024585, rs3024622, and rs4787956 (P = .03). CONCLUSION: This study demonstrates a significant pharmacogenetic interaction between anti-IL-4 receptor α therapy and IL4RA gene variation, identifying an asthma subgroup that is more responsive to therapy with this antagonist.

Mutations in RAI1 associated with Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a mental retardation syndrome associated with deletions involving chromosome 17p11.2. Persons with SMS have characteristic behavioral abnormalities, including self-injurious behaviors and sleep disturbance, and distinct craniofacial and skeletal anomalies. We identified dominant frameshift mutations leading to protein truncation in RAI1 in three individuals who have phenotypic features consistent with SMS but do not have 17p11.2 deletions detectable by standard fluorescence in situ hybridization techniques.

Sequential activation of protein kinase C isoforms by organic dust is mediated by tumor necrosis factor.

Dust samples collected from Nebraska swine confinement facilities (hog dust extract [HDE]) are known to elicit proinflammatory cytokine release from human bronchial epithelial (HBE) cells in vitro. This response involves the activation of two protein kinase C (PKC) isoforms: PKCalpha and PKCepsilon. Experiments were designed to investigate the relationship between the two isoenzymes and the degree to which each is responsible for cytokine release in HBE. Experiments also examined the contribution of TNF-alpha to IL-6 and IL-8 release. PKCalpha and PKCepsilon activities were inhibited using isoform-specific pharmacologic inhibitors and genetically modified dominant-negative (DN) expressing cell lines. Release of the proinflammatory cytokines IL-6, IL-8, and TNF-alpha was measured and PKC isoform activities assessed. We found that HDE stimulates PKCalpha activity by 1 hour, and within 6 hours the activity returns to baseline. PKCalpha-specific inhibitor or PKCalphaDN cells abolish this HDE-mediated effect. Both IL-6 and IL-8 release are likewise diminished under these conditions compared with normal HBE, and treatment with TNF-alpha-neutralizing antibody does not further inhibit cytokine release. In contrast, PKCepsilon activity was enhanced by 6 hours after HDE treatment. TNF-alpha blockade abrogated this effect. HDE-stimulated IL-6, but not IL-8 release in PKCepsilonDN cells. The concentration of TNF-alpha released by HDE-stimulated HBE is sufficient to have a potent cytokine-eliciting effect. A time course of TNF-alpha release suggests that TNF-alpha is produced after PKCalpha activation, but before PKCepsilon. These results suggest a temporal ordering of events responsible for the release of cytokines, which initiate and exacerbate inflammatory events in the airways of people exposed to agricultural dust.

Rhinitis associated with pesticide use among private pesticide applicators in the agricultural health study.

Farmers commonly experience rhinitis but the risk factors are not well characterized. The aim of this study was to analyze cross-sectional data on rhinitis in the past year and pesticide use from 21,958 Iowa and North Carolina farmers in the Agricultural Health Study, enrolled 1993-1997, to evaluate pesticide predictors of rhinitis. Polytomous and logistic regression models were used to assess association between pesticide use and rhinitis while controlling for demographics and farm-related exposures. Sixty-seven percent of farmers reported current rhinitis and 39% reported 3 or more rhinitis episodes. The herbicides glyphosate [odds ratio (OR) = 1.09, 95% confidence interval (95% CI) = 1.05-1.13] and petroleum oil (OR = 1.12, 95% CI = 1.05-1.19) were associated with current rhinitis and increased rhinitis episodes. Of the insecticides, four organophosphates (chlorpyrifos, diazinon, dichlorvos, and malathion), carbaryl, and use of permethrin on animals were predictors of current rhinitis. Diazinon was significant in the overall polytomous model and was associated with an elevated OR of 13+ rhinitis episodes (13+ episodes OR = 1.23, 95% CI = 1.09-1.38). The fungicide captan was also a significant predictor of rhinitis. Use of petroleum oil, use of malathion, use of permethrin, and use of the herbicide metolachlor were significant in exposure-response polytomous models. Specific pesticides may contribute to rhinitis in farmers; agricultural activities did not explain these findings.

How much is too much? Phenotypic consequences of Rai1 overexpression in mice.

The retinoic acid induced 1 (RAI1) gene when deleted or mutated results in Smith-Magenis syndrome (SMS), while duplication of 17p11.2, including RAI1, results in the dup(17)(p11.2) syndrome characterized by mental retardation, growth and developmental delays, and hyperactivity. Mouse models for these human syndromes may help define critical roles for RAI1 in mammalian development and homeostasis that otherwise cannot be deduced from patient studies. A mouse model for duplication, Dp(11)17+, involving Rai1 has been reported. However, this mutant was engineered on a mixed genetic background confounding phenotypic effects due to possible modifier genes. We have therefore created and evaluated mice with a graded series of four (hemizygous) and six (homozygous) copies of Rai1, and overexpressing Rai1>1.5-fold and >2-fold, respectively. Data show that Rai1-transgenic mice have growth retardation, increased locomotor activity, and abnormal anxiety-related behavior compared to wild-type littermates. Rai1-transgenic mice also have an altered gait with short strides and long sways, impaired ability on a cage-top hang test, decreased forelimb grip strength, and a dominant social behavior. Further, analyses of homozygous transgenic mice revealed a dosage-dependent exacerbation of the phenotype, including extreme growth retardation, severe neurological deficits, and increased hyperactivity. Our results show that Rai1 dosage has major consequences on molecular processes involved in growth, development, and neurological and behavioral functions, thus providing evidence for several dosage-thresholds for phenotypic manifestations causing dup(17)(p11.2) syndrome or SMS in humans.

RACK1, a PKC targeting protein, is exclusively localized to basal airway epithelial cells.

The novel isoform of protein kinase C (PKC), PKCepsilon, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCepsilon signaling in the airways and the potential role of the PKCepsilon-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, beta-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCepsilon signaling in the basal airway cell may involve RACK1; however, PKCepsilon regulation in ciliated cells uses RACK1-independent pathways.

Genetics of asthma susceptibility and severity.

This article summarizes major findings in genome-wide studies of asthma susceptibility and severity. Two large meta-analyses identified four chromosomal regions which were consistently associated with development of asthma. Genes that are associated with asthma subphenotypes such as lung function, biomarker levels, and asthma therapeutic responses can provide insight into mechanisms of asthma severity and disease progression. Future genetic studies will incorporate sequencing in comprehensively phenotyped asthmatics to lead to the development of personalized therapy.

Hog barn dust slows airway epithelial cell migration in vitro through a PKCalpha-dependent mechanism.

Agricultural work and other occupational exposures are responsible for approximately 15% of chronic obstructive pulmonary disease (COPD). COPD involves airway remodeling in response to chronic lung inflammatory events and altered airway repair mechanisms. However, the effect of agricultural dust exposure on signaling pathways that regulate airway injury and repair has not been well characterized. A key step in this process is migration of airway cells to restore epithelial integrity. We have previously shown that agents that activate the critical regulatory enzyme protein kinase C (PKC) slow cell migration during wound repair. Based on this observation and direct kinase measurements that demonstrate that dust extract from hog confinement barns (HDE) specifically activates the PKC isoforms PKCalpha and PKCepsilon, we hypothesized that HDE would slow wound closure time in airway epithelial cells. We utilized the human bronchial epithelial cell line BEAS-2B and transfected BEAS-2B cell lines that express dominant negative (DN) forms of PKC isoforms to demonstrate that HDE slows wound closure in BEAS-2B and PKCepsilon DN cell lines. However, in PKCalpha DN cells, wound closure following HDE treatment is not significantly different than media-treated cells. These results suggest that the PKCalpha isoform is an important regulator of cell migration in response to agricultural dust exposure.

Feedlot dust stimulation of interleukin-6 and -8 requires protein kinase Cepsilon in human bronchial epithelial cells.

Individuals exposed to dusts from concentrated animal feeding operations report increased numbers of respiratory tract symptoms, and bronchoalveolar lavage samples from such individuals demonstrate elevated lung inflammatory mediators, including interleukin (IL)-8 and IL-6. We previously found that exposure of bronchial epithelial cells to hog barn dusts resulted in a protein kinase C (PKC)-dependent increase in IL-6 and IL-8 release. We hypothesized that cattle feedlot dusts would also generate bronchial epithelial interleukin release in vitro. To test this, we used interleukin ELISAs and direct PKC isoform assays. We found that a dust extract from cattle feedlots [feedlot dust extract (FLDE)] augments PKC activity of human bronchial epithelial cells in vitro. A 5-10% dilution of FLDE stimulated a significant release of IL-6 and IL-8 at 6-24 h in a PKC-dependent manner vs. control medium-treated cells. An increase in PKCalpha activity was observed with 1 h of FLDE treatment, and PKCepsilon activity was elevated at 6 h of FLDE exposure. The PKCalpha inhibitor, Gö-6976, did not inhibit FLDE-stimulated IL-8 and IL-6 release. However, the PKCepsilon inhibitor, Ro 31-8220, effectively inhibited FLDE-stimulated IL-8 and IL-6 release. Inhibition of FLDE-stimulated IL-6 and IL-8 was confirmed in a dominant-negative PKCepsilon-expressing BEAS-2B cell line but not observed in a PKCalpha dominant negative BEAS-2B cell line. These data support the hypothesis that FLDE exposure stimulates bronchial epithelial IL-8 and IL-6 release via a PKCepsilon-dependent pathway.

Bronchodilator responsiveness in swine veterinarians.

OBJECTIVE: Swine veterinarians are known to be at risk for respiratory symptoms and airflow obstruction. The present study reassessed the prevalence of respiratory complaints and pulmonary function abnormalities in swine veterinarians and sought to characterize their response to bronchodilators. METHODS: A cross-sectional study was conducted during the American Association of Swine Veterinarians annual meeting. Subjects completed a respiratory symptom and workplace exposure history questionnaire and spirometry. Subjects with airflow obstruction were assessed for a post-bronchodilator response with beta2 agonist administration. RESULTS: Participants included 58 veterinarians (mean age, 45.5 years). Work-related symptoms assessed by questionnaire included rhinitis symptoms (60.3%), cough and chest tightness (55.2%), and wheezing (35.1%). Airflow obstruction was detected in 11/58 (19%) of subjects by spirometry. Only 2/9 (22.2%) met American Thoracic Society criteria for reversibility with bronchodilator administration. CONCLUSIONS: Respiratory symptoms and airway obstruction remain common findings in swine veterinarians. Airflow obstruction was not consistently reversible with beta agonists, suggesting that swine barn exposure may be a risk factor for irreversible airflow obstruction.

Inhibition of protein kinase C epsilon causes ciliated bovine bronchial cell detachment.

This study defines the in vitro phenomenon of ciliated bovine bronchial epithelial cell (BBEC) detachment from the basal epithelium and regulation of cilia motility mediated through protein kinase C epsilon (PKCepsilon). The authors determined the time course of activation and downregulation of PKCepsilon by the known PKC activator phorbol 12-myristate 13-acetate (PMA) and demonstrate that chemical inhibition of PKC by calphostin C or the novel PKC isoform inhibitor Ro 31-8220 induced striking detachment of ciliated BBECs from the basal cell monolayer within 1 hour, independent of apoptosis or necrotic cell death. The results of this study support a possible novel PKCepsilon-mediated signaling pathway through which ciliated cell attachment is maintained.