A kinase inhibitor screen identifies signaling pathways regulating mucosal growth during otitis media.

Otitis media, the most common disease of childhood, is characterized by extensive changes in the morphology of the middle ear cavity. This includes hyperplasia of the mucosa that lines the tympanic cavity, from a simple monolayer of squamous epithelium into a greatly thickened, respiratory-type mucosa. The processes that control this response, which is critical to otitis media pathogenesis and recovery, are incompletely understood. Given the central role of protein phosphorylation in most intracellular processes, including cell proliferation and differentiation, we screened a library of kinase inhibitors targeting members of all the major families in the kinome for their ability to influence the growth of middle ear mucosal explants in vitro. Of the 160 inhibitors, 30 were found to inhibit mucosal growth, while two inhibitors enhanced tissue proliferation. The results suggest that the regulation of infection-mediated tissue growth in the ME mucosa involves multiple cellular processes that span the kinome. While some of the pathways and processes identified have been previously implicated in mucosa hyperplasia others are novel. The results were used to generate a global model of growth regulation by kinase pathways. The potential for therapeutic applications of the results are discussed.

Role of the PI3K/AKT pathway and PTEN in otitis media.

Mucosal hyperplasia is common sequela of otitis media (OM), leading to the secretion of mucus and the recruitment of leukocytes. However, the pathogenic mechanisms underlying hyperplasia are not well defined. Here, we investigated the role of the AKT pathway in the development of middle mucosal hyperplasia using in vitro mucosal explants cultures and an in vivo rat model. The Akt inhibitor MK2206 treatment inhibited the growth of middle ear mucosal explants in a dose-dependent manner. In vivo, MK2206 also reduced mucosal hyperplasia. Unexpectedly, while PTEN is generally thought to act in opposition to AKT, the PTEN inhibitor BPV reduced mucosal explant growth in vitro. The results indicate that both AKT and PTEN are mediators of mucosal growth during OM, and could be potential therapeutic targets.

Obesity's effect on asthma extends to diagnostic criteria.

BACKGROUND: The use of inflammatory biomarkers to delineate the type of lung inflammation present in asthmatic subjects is increasingly common. However, the effect of obesity on these markers is unknown. OBJECTIVES: We aimed to determine the effect of obesity on conventional markers of inflammation in asthmatic subjects. METHODS: We performed secondary analysis of data from 652 subjects previously enrolled in 2 Asthma Clinical Research Network trials. We performed linear correlations between biomarkers and logistic regression analysis to determine the predictive value of IgE levels, blood eosinophil counts, and fraction of exhaled nitric oxide values in relationship to sputum eosinophil counts (>2%), as well as to determine whether cut points existed that would maximize the sensitivity and specificity for predicting sputum eosinophilia in the 3 weight groups. RESULTS: Overall, statistically significant but relatively weak correlations were observed among all 4 markers of inflammation. Within obese subjects, the only significant correlation found was between IgE levels and blood eosinophil counts (r = 0.33, P < .001); furthermore, all other correlations between inflammatory markers were approximately 0, including correlations with sputum eosinophil counts. In addition, the predictive value of each biomarker alone or in combination was poor in obese subjects. In fact, in obese subjects none of the biomarkers of inflammation significantly predicted the presence of high sputum eosinophil counts. Obese asthmatic subjects have lower cut points for IgE levels (268 IU), fraction of exhaled nitric oxide values (14.5 ppb), and blood eosinophil counts (96 cells/µL) than all other groups. CONCLUSIONS: In obese asthmatic subjects conventional biomarkers of inflammation are poorly predictive of eosinophilic airway inflammation. As such, biomarkers currently used to delineate eosinophilic inflammation in asthmatic subjects should be approached with caution in these subjects.

Otitis Media and Nasopharyngeal Colonization in ccl3-/- Mice.

We previously found CC chemokine ligand 3 (CCL3) to be a potent effector of inflammation during otitis media (OM): exogenous CCL3 rescues the OM phenotype of tumor necrosis factor-deficient mice and the function of macrophages deficient in several innate immune molecules. To further delineate the role of CCL3 in OM, we evaluated middle ear (ME) responses of ccl3-/-mice to nontypeable Haemophilus influenzae (NTHi). CCL chemokine gene expression was evaluated in wild-type (WT) mice during the complete course of acute OM. OM was induced in ccl3-/- and WT mice, and infection and inflammation were monitored for 21 days. Phagocytosis and killing of NTHi by macrophages were evaluated by an in vitro assay. The nasopharyngeal bacterial load was assessed in naive animals of both strains. Many CCL genes showed increased expression levels during acute OM, with CCL3 being the most upregulated, at levels 600-fold higher than the baseline. ccl3-/- deletion compromised ME bacterial clearance and prolonged mucosal hyperplasia. ME recruitment of leukocytes was delayed but persisted far longer than in WT mice. These events were linked to a decrease in the macrophage capacity for NTHi phagocytosis and increased nasopharyngeal bacterial loads in ccl3-/- mice. The generalized impairment in inflammatory cell recruitment was associated with compensatory changes in the expression profiles of CCL2, CCL7, and CCL12. CCL3 plays a significant role in the clearance of infection and resolution of inflammation and contributes to mucosal host defense of the nasopharyngeal niche, a reservoir for ME and upper respiratory infections. Therapies based on CCL3 could prove useful in treating or preventing persistent disease.

ABAI's MOC Assessment of Knowledge Program Matures: Adding Value with Continuous Learning and Assessment.

Rapid changes in modern medicine along with advances in the science of learning and memory have necessitated a shift in the way physician knowledge is assessed. Physician recertification beyond initial certification has historically consisted of retaining large amounts of knowledge over a long time span. The adult learning theory has shown that the maintenance and improvement of our knowledge base is more effective by being exposed to new concepts at regular intervals throughout one's career and reinforcing these concepts on an ongoing basis. These philosophies have spurred several American Board of Medical Specialties member boards to embark on a variety of continuous assessment models that are designed to keep physicians up to date with the use of new technologies and innovative and flexible question formats. This article describes the new American Board of Allergy and Immunology (ABAI) Continuous Assessment Program. As the ABAI departs from the traditional secure examination/test center model and embarks on its new pilot, the focus remains firmly rooted in the core competencies that patients and the public demand and deserve. Through surveys, the ABAI has laid the groundwork for initial program design by asking its diplomates to rank the most relevant aspects of a sound clinical assessment. Periodic surveys to follow will enable the ABAI to adjust program design to provide the most pertinent content to practicing physicians to improve patient care, promote professionalism, and ensure public trust.

Innate Immunity: Orchestrating Inflammation and Resolution of Otitis Media.

Otitis media (OM) is a common disease in young children, accounting for more office visits and surgeries than any other pediatric condition. It is associated with an estimated cost of five billion dollars annually in the USA. Moreover, chronic and recurrent middle ear (ME) disease leads to hearing loss during critical periods of language acquisition and learning leading to delays in reaching developmental milestones and risking permanent damage to the ME and inner ear in severe cases. Therefore, research to understand the disease pathogenesis and identify new therapeutics is important. Although OM is a multifactorial disease, targeting the molecular mechanisms that drive inflammation and OM resolution is critical. In this review, we discuss the current evidence suggesting that innate immune receptors and effectors play key roles in OM by mediating both the ME inflammatory responses and recovery.

The transcriptome of a complete episode of acute otitis media.

BACKGROUND: Otitis media is the most common disease of childhood, and represents an important health challenge to the 10-15% of children who experience chronic/recurrent middle ear infections. The middle ear undergoes extensive modifications during otitis media, potentially involving changes in the expression of many genes. Expression profiling offers an opportunity to discover novel genes and pathways involved in this common childhood disease. The middle ears of 320 WBxB6 F1 hybrid mice were inoculated with non-typeable Haemophilus influenzae (NTHi) or PBS (sham control). Two independent samples were generated for each time point and condition, from initiation of infection to resolution. RNA was profiled on Affymetrix mouse 430 2.0 whole-genome microarrays. RESULTS: Approximately 8% of the sampled transcripts defined the signature of acute NTHi-induced otitis media across time. Hierarchical clustering of signal intensities revealed several temporal gene clusters. Network and pathway enrichment analysis of these clusters identified sets of genes involved in activation of the innate immune response, negative regulation of immune response, changes in epithelial and stromal cell markers, and the recruitment/function of neutrophils and macrophages. We also identified key transcriptional regulators related to events in otitis media, which likely determine the expression of these gene clusters. A list of otitis media susceptibility genes, derived from genome-wide association and candidate gene studies, was significantly enriched during the early induction phase and the middle re-modeling phase of otitis but not in the resolution phase. Our results further indicate that positive versus negative regulation of inflammatory processes occur with highly similar kinetics during otitis media, underscoring the importance of anti-inflammatory responses in controlling pathogenesis. CONCLUSIONS: The results characterize the global gene response during otitis media and identify key signaling and transcription factor networks that control the defense of the middle ear against infection. These networks deserve further attention, as dysregulated immune defense and inflammatory responses may contribute to recurrent or chronic otitis in children.

The inflammasome adaptor ASC contributes to multiple innate immune processes in the resolution of otitis media.

This study was designed to understand the contribution of the inflammasome and IL-1ß activation in otitis media (OM). We examined the middle ear (ME) response to non-typeable Haemophilus influenzae (NTHi) in wild type (WT) mice using gene microarrays and a murine model of acute OM. Expression of members of the NOD domain-like receptor family of inflammasome genes was significantly up-regulated early in NTHi infection of the ME, potentially activating specific downstream regulatory cascades that contribute to the proliferative inflammatory response observed during OM. Expression of the pro-forms of the inflammasome targets IL-1ß and IL-18 were also up-regulated. To evaluate the role of inflammasome-mediated cytokine maturation, NTHi-induced OM was examined in Asc(-/-)-deficient mice and compared with that seen in WT mice. Mice lacking the Asc gene showed near absence of IL-1ß maturation in the ME and a reduction in leukocyte recruitment and infiltration to the cavity, and their macrophages exhibited reduced phagocytosis of NTHi. These inflammatory defects were linked to an increase in the degree and duration of mucosal epithelial hyperplasia in the ME of Asc(-/-) mice, as well as a delay in bacterial clearance from their MEs. These data demonstrate an important role for the inflammasome and cytokine processing in the course and resolution of OM.

C-Jun N-terminal kinase (JNK) isoforms play differing roles in otitis media.

BACKGROUND: Innate immunity and tissue proliferation play important roles in otitis media (OM), the most common disease of childhood. CJUN terminal kinase (JNK) is potentially involved in both processes. RESULTS: Genes involved in both innate immune and growth factor activation of JNK are upregulated during OM, while expression of both positive and negative JNK regulatory genes is altered. When compared to wildtypes (WTs), C57BL/6 mice deficient in JNK1 exhibit enhanced mucosal thickening, with delayed recovery, enhanced neutrophil recruitment early in OM, and delayed bacterial clearance. In contrast, JNK2-/- mice exhibit delayed mucosal hyperplasia that eventually exceeds that of WTs and is slow to recover, delayed recruitment of neutrophils, and failure of bacterial clearance. CONCLUSIONS: The results suggest that JNK1 and JNK2 play primarily opposing roles in mucosal hyperplasia and neutrophil recruitment early in OM. However, both isoforms are required for the normal resolution of middle ear infection.

A mouse model of otitis media identifies HB-EGF as a mediator of inflammation-induced mucosal proliferation.

OBJECTIVE: Otitis media is one of the most common pediatric infections. While it is usually treated without difficulty, up to 20% of children may progress to long-term complications that include hearing loss, impaired speech and language development, academic underachievement, and irreversible disease. Hyperplasia of middle ear mucosa contributes to the sequelae of acute otitis media and is of important clinical significance. Understanding the role of growth factors in the mediation of mucosal hyperplasia could lead to the development of new therapeutic interventions for this disease and its sequelae. METHODS: From a whole genome gene array analysis of mRNA expression during acute otitis media, we identified growth factors with expression kinetics temporally related to hyperplasia. We then tested these factors for their ability to stimulate mucosal epithelial growth in vitro, and determined protein levels and histological distribution in vivo for active factors. RESULTS: From the gene array, we identified seven candidate growth factors with upregulation of mRNA expression kinetics related to mucosal hyperplasia. Of the seven, only HB-EGF (heparin-binding-epidermal growth factor) induced significant mucosal epithelial hyperplasia in vitro. Subsequent quantification of HB-EGF protein expression in vivo via Western blot analysis confirmed that the protein is highly expressed from 6 hours to 24 hours after bacterial inoculation, while immunohistochemistry revealed production by middle ear epithelial cells and infiltrating lymphocytes. CONCLUSION: Our data suggest an active role for HB-EGF in the hyperplasia of the middle ear mucosal epithelium during otitis media. These results imply that therapies targeting HB-EGF could ameliorate mucosal growth during otitis media, and thereby reduce detrimental sequelae of this childhood disease.

Predictors of response to tiotropium versus salmeterol in asthmatic adults.

BACKGROUND: Tiotropium has activity as an asthma controller. However, predictors of a positive response to tiotropium have not been described. OBJECTIVE: We sought to describe individual and differential responses of asthmatic patients to salmeterol and tiotropium when added to an inhaled corticosteroid, as well as predictors of a positive clinical response. METHODS: Data from the double-blind, 3-way, crossover National Heart, Lung, and Blood Institute's Asthma Clinical Research Network's Tiotropium Bromide as an Alternative to Increased Inhaled Glucocorticoid in Patients Inadequately Controlled on a Lower Dose of Inhaled Corticosteroid (ClinicalTrials.gov number, NCT00565266) trial were analyzed for individual and differential treatment responses to salmeterol and tiotropium and predictors of a positive response to the end points FEV1, morning peak expiratory flow (PEF), and asthma control days (ACDs). RESULTS: Although approximately equal numbers of patients showed a differential response to salmeterol and tiotropium in terms of morning PEF (n = 90 and 78, respectively) and ACDs (n = 49 and 53, respectively), more showed a differential response to tiotropium for FEV1 (n = 104) than salmeterol (n = 62). An acute response to a short-acting bronchodilator, especially albuterol, predicted a positive clinical response to tiotropium for FEV1 (odds ratio, 4.08; 95% CI, 2.00-8.31; P < .001) and morning PEF (odds ratio, 2.12; 95% CI, 1.12-4.01; P = 0.021), as did a decreased FEV1/forced vital capacity ratio (FEV1 response increased 0.39% of baseline for every 1% decrease in FEV1/forced vital capacity ratio). Higher cholinergic tone was also a predictor, whereas ethnicity, sex, atopy, IgE level, sputum eosinophil count, fraction of exhaled nitric oxide, asthma duration, and body mass index were not. CONCLUSION: Although these results require confirmation, predictors of a positive clinical response to tiotropium include a positive response to albuterol and airway obstruction, factors that could help identify appropriate patients for this therapy.

Genome-wide association study identifies TH1 pathway genes associated with lung function in asthmatic patients.

BACKGROUND: Recent meta-analyses of genome-wide association studies in general populations of European descent have identified 28 loci for lung function. OBJECTIVE: We sought to identify novel lung function loci specifically for asthma and to confirm lung function loci identified in general populations. METHODS: Genome-wide association studies of lung function (percent predicted FEV1 [ppFEV1], percent predicted forced vital capacity, and FEV1/forced vital capacity ratio) were performed in 4 white populations of European descent (n = 1544), followed by meta-analyses. RESULTS: Seven of 28 previously identified lung function loci (HHIP, FAM13A, THSD4, GSTCD, NOTCH4-AGER, RARB, and ZNF323) identified in general populations were confirmed at single nucleotide polymorphism (SNP) levels (P < .05). Four of 32 loci (IL12A, IL12RB1, STAT4, and IRF2) associated with ppFEV1 (P < 10(-4)) belong to the TH1 or IL-12 cytokine family pathway. By using a linear additive model, these 4 TH1 pathway SNPs cumulatively explained 2.9% to 7.8% of the variance in ppFEV1 values in 4 populations (P = 3 × 10(-11)). Genetic scores of these 4 SNPs were associated with ppFEV1 values (P = 2 × 10(-7)) and the American Thoracic Society severe asthma classification (P = .005) in the Severe Asthma Research Program population. TH2 pathway genes (IL13, TSLP, IL33, and IL1RL1) conferring asthma susceptibility were not associated with lung function. CONCLUSION: Genes involved in airway structure/remodeling are associated with lung function in both general populations and asthmatic subjects. TH1 pathway genes involved in anti-virus/bacterial infection and inflammation modify lung function in asthmatic subjects. Genes associated with lung function that might affect asthma severity are distinct from those genes associated with asthma susceptibility.

Comparison of physician-, biomarker-, and symptom-based strategies for adjustment of inhaled corticosteroid therapy in adults with asthma: the BASALT randomized controlled trial.

CONTEXT: No consensus exists for adjusting inhaled corticosteroid therapy in patients with asthma. Approaches include adjustment at outpatient visits guided by physician assessment of asthma control (symptoms, rescue therapy, pulmonary function), based on exhaled nitric oxide, or on a day-to-day basis guided by symptoms. OBJECTIVE: To determine if adjustment of inhaled corticosteroid therapy based on exhaled nitric oxide or day-to-day symptoms is superior to guideline-informed, physician assessment-based adjustment in preventing treatment failure in adults with mild to moderate asthma. DESIGN, SETTING, AND PARTICIPANTS: A randomized, parallel, 3-group, placebo-controlled, multiply-blinded trial of 342 adults with mild to moderate asthma controlled by low-dose inhaled corticosteroid therapy (n = 114 assigned to physician assessment-based adjustment [101 completed], n = 115 to biomarker-based [exhaled nitric oxide] adjustment [92 completed], and n = 113 to symptom-based adjustment [97 completed]), the Best Adjustment Strategy for Asthma in the Long Term (BASALT) trial was conducted by the Asthma Clinical Research Network at 10 academic medical centers in the United States for 9 months between June 2007 and July 2010. INTERVENTIONS: For physician assessment-based adjustment and biomarker-based (exhaled nitric oxide) adjustment, the dose of inhaled corticosteroids was adjusted every 6 weeks; for symptom-based adjustment, inhaled corticosteroids were taken with each albuterol rescue use. MAIN OUTCOME MEASURE: The primary outcome was time to treatment failure. RESULTS: There were no significant differences in time to treatment failure. The 9-month Kaplan-Meier failure rates were 22% (97.5% CI, 14%-33%; 24 events) for physician assessment-based adjustment, 20% (97.5% CI, 13%-30%; 21 events) for biomarker-based adjustment, and 15% (97.5% CI, 9%-25%; 16 events) for symptom-based adjustment. The hazard ratio for physician assessment-based adjustment vs biomarker-based adjustment was 1.2 (97.5% CI, 0.6-2.3). The hazard ratio for physician assessment-based adjustment vs symptom-based adjustment was 1.6 (97.5% CI, 0.8-3.3). CONCLUSION: Among adults with mild to moderate persistent asthma controlled with low-dose inhaled corticosteroid therapy, the use of either biomarker-based or symptom-based adjustment of inhaled corticosteroids was not superior to physician assessment-based adjustment of inhaled corticosteroids in time to treatment failure. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00495157.

The role of DNA sensing and innate immune receptor TLR9 in otitis media.

Otitis media (OM), a common infectious disease in children, is associated with bacterial middle ear (ME) infection. Toll-like receptors (TLRs) are important mediators of innate immune responses, and TLR9 specifically recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motifs in bacterial DNA. Additional sensors of foreign DNA have recently been identified. The role of DNA sensing and TLR9 was investigated in a murine model of OM induced by non-typeable Haemophilus influenzae (NTHi). Expression of genes related to DNA-sensing pathways involved in innate immunity was assessed via DNA microarray, qPCR and immunohistochemistry. Middle ear responses to NTHi were examined in wild-type and TLR9(-/-) mice by histopathology and bacterial culture. Expression of TLR9 signaling genes was modestly up-regulated during OM, as was TLR9 protein in both ME mucosal cells and infiltrating leukocytes. However, genes known to be regulated by CpG DNA were dramatically up-regulated, as were genes involved in DNA sensing by DIA, Pol-III and AIM2. Toll-like receptor 9 deletion significantly prolonged the inflammatory response induced by NTHi in the ME and delayed bacterial clearance. The results suggest that DNA sensing via TLR9 plays a role in OM pathogenesis and recovery. Alternative forms of DNA sensing may also contribute to OM.

Asthma in the elderly: Current understanding and future research needs--a report of a National Institute on Aging (NIA) workshop.

Asthma in the elderly is underdiagnosed and undertreated, and there is a paucity of knowledge on the subject. The National Institute on Aging convened this workshop to identify what is known and what gaps in knowledge remain and suggest research directions needed to improve the understanding and care of asthma in the elderly. Asthma presenting at an advanced age often has similar clinical and physiologic consequences as seen with younger patients, but comorbid illnesses and the psychosocial effects of aging might affect the diagnosis, clinical presentation, and care of asthma in this population. At least 2 phenotypes exist among elderly patients with asthma; those with longstanding asthma have more severe airflow limitation and less complete reversibility than those with late-onset asthma. Many challenges exist in the recognition and treatment of asthma in the elderly. Furthermore, the pathophysiologic mechanisms of asthma in the elderly are likely to be different from those seen in young asthmatic patients, and these differences might influence the clinical course and outcomes of asthma in this population.

Biologic modulators in allergic and autoinflammatory diseases.

PURPOSE OF REVIEW: The advent of molecular techniques has resulted in the ability to tailor medications to specific protein targets. This review will emphasize several biological therapies, specifically directed toward cytokine receptors and inhibitors, and their role in the treatment of atopic and autoinflammatory diseases. RECENT FINDINGS: Translational research and the identification of the molecular pathophysiology of diseases have led to more targeted treatment approaches. The biologic modulators encompassing monoclonal antibodies as cytokine inhibitors, receptor blocking antibodies, and new fusion receptors are now being applied to diseases beyond their original application. SUMMARY: The expanded use of biological therapies has experienced success in the treatment of numerous disorders, especially in subsets of patients with disease that has been refractory to conventional therapies.

The role of monoclonal antibodies in the treatment of severe asthma.

A number of therapeutic agents are available for the treatment of asthma, including inhaled corticosteroids, long- and short-acting beta-agonists, leukotriene-modifying agents, long- and short-acting anticholinergic agents, chromones, theophylline, allergen immunotherapy, and oral corticosteroid therapy. All available therapies, despite their proven efficacy, are purely symptomatic including the topical steroids. This issue has led to the development of several biologic agents to aid in asthma management and to potentially alter the course of the disease by interfering with specific aspects of inflammation which may modify remodeling in the airways. Monoclonal antibodies have offered a class of therapeutic agents that enhance treatment options for patients with moderate-to-severe persistent asthma. As such, this article provides an overview of present and future monoclonal antibody therapies for the treatment of patients with severe asthma.