Longitudinal stability of asthma characteristics and biomarkers from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) study.

BACKGROUND: Asthma is a biologically heterogeneous disease and development of novel therapeutics requires understanding of pathophysiologic phenotypes. There is uncertainty regarding the stability of clinical characteristics and biomarkers in asthma over time. This report presents the longitudinal stability over 12 months of clinical characteristics and clinically accessible biomarkers from ADEPT. METHODS: Mild, moderate, and severe asthma subjects were assessed at 5 visits over 12 months. Assessments included patient questionnaires, spirometry, bronchodilator reversibility, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum. RESULTS: Mild (n = 52), moderate (n = 55), and severe (n = 51) asthma cohorts were enrolled from North America and Western Europe. For all clinical characteristics and biomarkers, group mean data showed no significant change from visit to visit. However, individual data showed considerable variability. FEV1/FVC ratio showed excellent reproducibility while pre-bronchodilator FEV1 and FVC were only moderately reproducible. Of note bronchodilator FEV1 reversibility showed low reproducibility, with the nonreversible phenotype much more reproducible than the reversible phenotype. The 7-item asthma control questionnaire (ACQ7) demonstrated moderate reproducibility for the combined asthma cohorts, but the uncontrolled asthma phenotype (ACQ7 > 1.5) was inconstant in mild and moderate asthma but stable in severe asthma. FENO demonstrated good reproducibility, with the FENO-low phenotype (FENO < 35 ppb) more stable than the FENO-high phenotype (FENO ≥ 35 ppb). Induced sputum inflammatory phenotypes showed marked variability across the 3 sputum samples taken over 6 months. CONCLUSIONS: The ADEPT cohort showed group stability, individual stability in some parameters e.g. low FEV1/FVC ratio, and low FENO, but marked individual variability in other clinical characteristics and biomarkers e.g. type-2 biomarkers over 12 months. This variability is possibly related to seasonal variations in climate and allergen exposure, medication changes and acute exacerbations. The implications for patient selection strategies based on clinical biomarkers may be considerable.

Asthma characteristics and biomarkers from the Airways Disease Endotyping for Personalized Therapeutics (ADEPT) longitudinal profiling study.

BACKGROUND: Asthma is a heterogeneous disease and development of novel therapeutics requires an understanding of pathophysiologic phenotypes. The purpose of the ADEPT study was to correlate clinical features and biomarkers with molecular characteristics, by profiling asthma (NCT01274507). This report presents for the first time the study design, and characteristics of the recruited subjects. METHODS: Patients with a range of asthma severity and healthy non-atopic controls were enrolled. The asthmatic subjects were followed for 12 months. Assessments included history, patient questionnaires, spirometry, airway hyper-responsiveness to methacholine, fractional exhaled nitric oxide (FENO), and biomarkers measured in induced sputum, blood, and bronchoscopy samples. All subjects underwent sputum induction and 30 subjects/cohort had bronchoscopy. RESULTS: Mild (n = 52), moderate (n = 55), severe (n = 51) asthma cohorts and 30 healthy controls were enrolled from North America and Western Europe. Airflow obstruction, bronchodilator response and airways hyperresponsiveness increased with asthma severity, and severe asthma subjects had reduced forced vital capacity. Asthma control questionnaire-7 (ACQ7) scores worsened with asthma severity. In the asthmatics, mean values for all clinical and biomarker characteristics were stable over 12 months although individual variability was evident. FENO and blood eosinophils did not differ by asthma severity. Induced sputum eosinophils but not neutrophils were lower in mild compared to the moderate and severe asthma cohorts. CONCLUSIONS: The ADEPT study successfully enrolled asthmatics across a spectrum of severity and non-atopic controls. Clinical characteristics were related to asthma severity and in general asthma characteristics e.g. lung function, were stable over 12 months. Use of the ADEPT data should prove useful in defining biological phenotypes to facilitate personalized therapeutic approaches.

TLR4 mutations are associated with endotoxin hyporesponsiveness in humans.

There is much variability between individuals in the response to inhaled toxins, but it is not known why certain people develop disease when challenged with environmental agents and others remain healthy. To address this, we investigated whether TLR4 (encoding the toll-like receptor-4), which has been shown to affect lipopolysaccharide (LPS) responsiveness in mice, underlies the variability in airway responsiveness to inhaled LPS in humans. Here we show that common, co-segregating missense mutations (Asp299Gly and Thr399Ile) affecting the extracellular domain of the TLR4 receptor are associated with a blunted response to inhaled LPS in humans. Transfection of THP-1 cells demonstrates that the Asp299Gly mutation (but not the Thr399Ile mutation) interrupts TLR4-mediated LPS signalling. Moreover, the wild-type allele of TLR4 rescues the LPS hyporesponsive phenotype in either primary airway epithelial cells or alveolar macrophages obtained from individuals with the TLR4 mutations. Our findings provide the first genetic evidence that common mutations in TLR4 are associated with differences in LPS responsiveness in humans, and demonstrate that gene-sequence changes can alter the ability of the host to respond to environmental stress.

Early-life co-administration of cockroach allergen and endotoxin augments pulmonary and systemic responses.

BACKGROUND: Environmental exposures to cockroach allergen and endotoxin are recognized epidemiological risk factors for the early development of allergies and asthma in children. Because of this, it is important to examine the role of early-life concurrent inhalation exposures to cockroach allergen and endotoxin in the pathogenesis of allergic airways disease. OBJECTIVE: We examined the effects of repeated concomitant endotoxin and cockroach allergen inhalation on the pulmonary and systemic immune responses of newborn and juvenile mice. METHODS: C3H/HeBFeJ mice were exposed to inhaled endotoxin and cockroach allergen via intranasal instillation from day 2 to 21 after birth, and systemic and pulmonary responses were examined in serum, bronchoalveolar lavage fluid, and lung tissue. RESULTS: Cockroach allergen exposures induced pulmonary eosinophilic inflammation, total and allergen-specific IgE, IgG(1), and IgG(2a) production, and alveolar remodelling. Co-exposures with endotoxin and cockroach allergen significantly increased serum IgE and IgG(1), lung inflammation, and alveolar wall thickness, and decreased airspace volume density. Importantly, compared with exposures with individual substances, the responses to co-exposures were more than additive. CONCLUSIONS: Repeated inhalation exposures of neonatal and juvenile mice to endotoxin and cockroach allergen increased the pulmonary inflammatory and systemic immune responses in a synergistic manner and enhanced alveolar remodelling in the developing lung. These data underscore the importance of evaluating the effect of multiple, concurrent environmental exposures, and of using an experimental model that incorporates clinically relevant timing and route of exposures.

Effects of CpG DNA on Th1/Th2 balance in asthma.

Thus, in our studies, we demonstrated that CpG ODN are effective in preventing the development of eosinophilic airway inflammation and bronchial hyper-reactivity in a murine model of asthma. Antigen-associated elevation of serum IgE levels is also suppressed. CpG ODN, administered in conjunction with antigen, is also effective in down-regulation of established Th2 responses. This protection is neither murine strain-dependent nor model-dependent. Although these effects of CpG ODN are associated with the induction of the Th1 cytokines IFN-gamma and IL-12, neither cytokine is absolutely required for the protection. These results suggest that CpG ODN may be an effective immunomodulatory agent in the treatment, and possibly prevention, of asthma.

Alveolar macrophages inhibit retrovirus-mediated gene transfer to airway epithelia.

Gene transfer with integrating vectors such as recombinant retrovirus has the potential to correct inherited lung diseases permanently. As a gene therapy target, the pulmonary epithelium presents several challenges to vector delivery in vivo. Many of the host defenses that have evolved to prevent infection from inhaled bacteria or viruses represent potential barriers to gene transfer to the lung. We performed in vitro studies to determine whether two components of the innate immune system of the lung, airway surface fluid and alveolar macrophages, inhibit retroviral gene transfer to airway epithelia. Human alveolar macrophages obtained by bronchoalveolar lavage from normal subjects were left untreated or activated with lipopolysaccharide (LPS) for 3 hr in the presence of subconfluent human bronchial epithelial cells (HBE); than 4 x 10(5) cfu DA-luciferase retrovirus was added. Three days after infection, luciferase activity was measured in cell lysates. When the epithelial cells were co-cultured with LPS-activated macrophages, retroviral gene transfer to HBE cells was reduced by approximately 60%. Nonactivated macrophages decreased the transfection to approximately 55% of control values. In control experiments with either activated or inactivated macrophages but without epithelia, no luciferase activity was detected, suggesting that terminally differentiated alveolar macrophages are not infected by the recombinant retrovirus. Pretreatment of alveolar macrophages with dexamethasone restored gene transfer to approximately 60% of control values. In contrast, incubation of retrovirus with airway surface fluid had no inhibitory effect on gene transfer. These experiments document that AM inhibit retrovirus-mediated gene transfer to airway epithelia in vitro, and may represent a barrier to retroviral gene transfer in vivo. These barriers may be overcome, at least partially, with pharmacological agents.

CpG oligodeoxynucleotides in asthma.

Asthma is a major health problem, of which the prevalence and severity are increasing, particularly in industrialized nations. One hypothesis for this is that diminished exposure to childhood infections in modern society has led to decreased Th1-type inflammation. Reduced Th1 responses may lead to enhanced Th2-type inflammation, important in promoting asthma and allergic disease. The most common current treatment for asthma is corticosteroids; while these agents inhibit the function of inflammatory cells, they are ineffective in altering the initial Th2-type response to allergen in a sensitized individual. A novel therapeutic approach, recently reported in the preclinical setting, is the use of oligodeoxynucleotides (ODNs), which contain unmethylated motifs centered on CG dinucleotides. These CpG ODNs potently induce Th1 cytokines and suppress Th2 cytokines, and can prevent manifestations of asthma in animal models. These agents have the potential to reverse Th2-type responses to allergens and thus restore balance to the immune system. Clinical trials are ongoing.

Pulmonary edema after freebase cocaine smoking--not due to an adulterant.

A case of pulmonary edema following smoking freebase cocaine is described. We did not clearly establish the mechanism, but this case is unique since adulterants and contaminants were excluded unlike all previously reported patients.

Pulmonary embolization and infarction. An iatrogenic complication of transcatheter embolization of a cerebral arteriovenous malformation with polyvinyl alcohol sponge.

Transcatheter embolization therapy of vascular abnormalities is a well-accepted technique. It is particularly useful in the preoperative management of cerebral arteriovenous malformations, in which the risk of significant hemorrhage at surgery would otherwise be unacceptably high. A patient developed symptomatic pulmonary emboli and infarction following an uneventful embolization using polyvinyl alcohol particles. The authors believe this to be the first reported case of this complication of embolic therapy using polyvinyl alcohol.

Relative release of interleukin-1 beta and interleukin-1 receptor antagonist by alveolar macrophages. A study in asbestos-induced lung disease, sarcoidosis, and idiopathic pulmonary fibrosis.

We examined the influence of untreated interstitial lung disease (ILD) on the in vitro release of interleukin-1 beta (IL-1 beta) and interleukin-1 receptor antagonist (IL-Ira) from alveolar macrophages (AM); AM were harvested from normal volunteers, ILD patients, and patients with asbestos-related pleural disease but no ILD. AM were cultured for 24 h and assays for IL-1 beta and IL-1ra were done using sensitive and specific enzyme-linked immunosorbent assay. A greater amount of IL-1 beta was detected in AM supernatants from asbestosis, sarcoidosis, and IPF patients than in those from normal subjects. The IL-1 beta:IL-1ra ratio (IL-1 beta activity index [IL-1AI]) was significantly lower in supernatants of normal macrophages compared with macrophage supernatants from individuals with ILD. The IL-1AI correlated with bronchoalveolar lavage cellularity, a marker of disease activity. Current smoking was associated with lower IL-1 beta and IL-1ra release in ILD. The IL-1AI is a convenient method for comparison of IL-1 beta activity between patient populations.

IL-1 receptor antagonist release is regulated differently in human alveolar macrophages than in monocytes.

These studies compared the release of interleukin-1 receptor antagonist (IL-1 RA) from alveolar macrophages and peripheral blood monocytes. The cells were cultured in medium containing various amounts of heat-inactivated fetal calf serum (FCS), granulocyte-macrophage colony-stimulating factor (GM-CSF), lipopolysaccharide (LPS), and immunoglobulin G (IgG). In serum-free medium alone, IL-1 RA release was similar from macrophages and monocytes. Increasing FCS concentration caused a significant upregulation of IL-1 RA release in macrophages but not in monocytes. GM-CSF caused a small increase in both cell types. LPS caused downregulation of IL-1 RA release from monocytes but not from macrophages. IgG did not affect IL-1 RA release in either cell group. These studies demonstrate that regulation of IL-1 RA release is different in monocytes and macrophages.

Bronchial hyperreactivity is associated with enhanced grain dust-induced airflow obstruction.

Bronchial hyperreactivity (BHR) is associated with the presence of airway inflammation in asthma and is seen in individuals occupationally exposed to grain dust. To better understand the relationship between BHR and pulmonary inflammation after grain dust exposure, we carried out an inhalation challenge to corn dust extract (CDE) on seven subjects with BHR [a 20% or greater decrease in forced expiratory volume in 1 s (FEV(1)) compared with diluent FEV(1) with a cumulative dose of histamine

Exploration of the volumetric composition of human lung cancer nodules in correlated histopathology and computed tomography.

Gaining a complete and comprehensive understanding of lung cancer nodule histological compositions and how these tissues are represented in radiological data is important not only for expanding the current knowledge base of cancer growth and development but also has potential implications for classification standards, radiological diagnosis methods and for the evaluation of treatment response. In this study we generate large scale histological segmentations of the cancerous and non-cancerous tissues within resected lung nodules. We have implemented a processing pipeline which allows for the direct correlation between histological data and spatially corresponding computed tomography data. Utilizing these correlated datasets we evaluated the statistical separation between Hounsfield Unit (HU) histogram values for each tissue type. The findings of this study revealed that lung cancer nodules contain a complex intermixing of cellular tissue types and that trends exist in the relationship between these tissue types. It was found that the mean Hounsfield Unit values for isolated lung cancer nodules imaged with computed tomography, had statistically significantly different values for non-solid bronchoalveolar carcinoma, solid cancerous tumor, blood, and inactive fibrotic stromal tissue.

Oral administration of CpG-ODNs suppresses antigen-induced asthma in mice.

Oligodeoxynucleotides containing CpG motifs (CpG-ODNs) can protect against eosinophilic airway inflammation in asthma. Previously we have found that parenteral or mucosal administration of CpG-ODNs is effective in preventing (as well as reversing established) disease. In this study, we examined the effect of oral CpG-ODNs on the development of immune tolerance. Using an ovalbumin (OVA)-induced murine model of asthma, we found that CpG-ODNs, administered orally around the time of sensitization, prevented eosinophilic airway inflammation in a dose-dependent manner. Although oral co-administration of CpG-ODNs with OVA (known to induce tolerance) did not significantly change the inhibition of OVA-induced airway eosinophilia, it did modulate OVA-specific immunoglobulin responses: oral administration of OVA alone suppressed OVA-specific IgG1 production, but only mice that received CpG-ODNs demonstrated enhanced levels of OVA-specific IgG2c. Finally, we examined whether oral administration of CpG-ODNs, alone or with OVA, could reverse established eosinophilic airway inflammation. Again, neither OVA nor CpG-ODNs alone modulated established eosinophilic airway inflammation, but a combination of the OVA and CpG-ODNs successfully desensitized the mice. This desensitization was associated with suppression of OVA-specific IgE and enhancement of OVA-specific IgG2c production. These findings provide the first indication that oral administration of CpG-ODNs is effective in preventing and reversing antigen-induced eosinophilic airway inflammation. CpG-ODNs may be useful as a component of oral immunotherapy to promote tolerance in established asthma.

T-lymphocyte dysregulation in asthma.

In this paper, we have reviewed the evidence suggesting that T-cell dysregulation is important in the pathogenesis of asthma. The history, clinical presentation, and an overview of the appropriate management of asthma have been briefly reviewed. T cells obtained from the airways of asthmatics display signs of activation; these changes mirror the intramural inflammation found at biopsy. There is debate about the importance of T helper/suppressor ratios in this population of cells, but alterations in these ratios have been noted with experimental allergen exposure, as well as during acute asthma attacks; some of these changes revert toward normal with steroid therapy. The division of T helper lymphocytes into Th1 and Th2 cells, first described in studies of murine immunology, appear to be relevant in humans, particularly in allergic disease. Although IL-4 and IL-5, prototypical Th2 cytokines, have been most clearly implicated in asthma, there is some evidence supporting a role for Th1 cells/cytokines as well.

CpG DNA and immunotherapy of allergic airway diseases.

Atopic asthma is a highly prevalent and serious health problem for which no therapy currently offers the hope of a cure. Preindustrialized and rural populations appear relatively protected from the asthma epidemic; the hygiene hypothesis ascribes this protection to the effects of microbes and microbial products. An important immunostimulant component of microbes is DNA; bacterial DNA contains sequence motifs centred on the CpG dinucleotide, which are suppressed in mammalian DNA. Oligonucleotides containing these motifs (CpG ODN), like bacterial DNA, promote Th1 and regulatory-type immune responses. Using CpG ODN, we and others have demonstrated in murine studies that CpG ODN are effective in preventing the development of atopic airways disease. Moreover, when administered in conjunction with experimental allergen, they promote the reversal of established eosinophilic inflammation. These data suggest that CpG ODN may be a novel therapeutic tool for the treatment of atopic asthma.

Regulation of interleukin-1 receptor antagonist by Th1 and Th2 cytokines.

Airway inflammation is an important aspect of asthma. Recent studies of airway inflammation in asthma have focused attention on cytokines released by T helper lymphocyte type 1 (Th1)- and Th2-like T cells. Interleukin (IL)-1 is also increased in the airways of asthmatics, and it is most likely derived from airway and alveolar macrophages. The effects of Th1 or Th2 cytokines on the release of IL-1 or its specific antagonist, IL-1ra, have not been well studied. We examined the response of THP-1 cells, a myelomonocytic cell line, to stimulation with various Th1 and Th2 cytokines and found that IL-4, IL-10, and IFN-gamma increased IL-1ra mRNA and protein release. The increase in mRNA was not due to an increase in IL-1ra mRNA stability. IL-4 (10 ng/ml) increased IL-1ra release from 9,641 +/- 322 [from cells stimulated with lipopolysaccharide (LPS) alone] to 50,796 +/- 1,917 pg/ml (from cells stimulated with LPS and IL-4). IL-10 (10 ng/ml) caused a similar upregulation of IL-1ra from LPS-stimulated cells: 87,478 +/- 7,808 compared with 8,004 +/- 1,166 pg/ml released from the cells stimulated with LPS alone. Cells stimulated with IFN-gamma (100 U/ml) and LPS released 27,854 +/- 3,626 pg/ml of IL-1ra, compared with 9,069 +/- 236 pg/ml in the presence of LPS alone. In addition, the Th1 cytokine, IFN-gamma, but not the Th2 cytokines, IL-4 and IL-10, upregulated IL-1 beta mRNA and increased the release of IL-1 beta protein. Similar studies were performed using freshly isolated monocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Mycobacterium tuberculosis in lung transplant recipients.

The diagnosis and treatment of Mycobacterium tuberculosis, although difficult in normal hosts, are even more complex in transplant recipients. As a result of the use of immunosuppressive agents, transplant recipients are not only predisposed to primary tuberculous infections, but they are also uniquely at risk for reactivation of latent infection acquired prior to transplantation or transmitted via the donor organ. The diagnosis of pulmonary tuberculosis can be even more elusive in the setting of lung transplantation where other pulmonary complications can make diagnosis difficult. Here we report a case of a patient who died of disseminated M. tuberculosis 12 wk after lung transplantation, and we review tuberculous infections in lung transplant recipients.

Synergistic activation of the human cytomegalovirus major immediate early promoter by prostaglandin E2 and cytokines.

Human cytomegalovirus (HCMV) is a common cause of morbidity and mortality in immunosuppressed patients, especially transplant recipients. In this population, infection is frequently due to reactivation of latent virus. The major immediate early promoter of HCMV controls production of immediate early gene products, which are both trans- and cis-active and are responsible for reactivation. Activation of this promoter is therefore a crucial step in regulation of reactivation infection. It is known that there are cAMP-response elements in the HCMV major immediate early promoter. We hypothesized that prostaglandins (PG), like PGE2, which are known to increase cAMP, as well as cytokines known to be released during acute inflammation, may be important in the regulation of this promoter and thus in reactivation of HCMV. To examine this, we transfected pCAT760, a plasmid containing the major immediate early promoter of HCMV upstream of a chloramphenicol acetyltransferase (CAT) gene, into THP-1 cells. These cells were subsequently stimulated with PGE2 and/or one of a variety of cytokines. We found that PGE2, tumor necrosis factor (TNF)-alpha, and interleukin (IL)-1 beta each upregulated the HCMV major immediate early promoter. TNF-alpha, IL-1 beta, IL-6, and IL-10 were each synergistic or additive with PGE2 in upregulating the promoter. Since PGE2 and the cytokines are all products of activated macrophages, we suggest that acute inflammation and macrophage activation may predispose to reactivation of latent HCMV.

Asthma guidelines: an assessment of physician understanding and practice.

In 1997 the NHLBI updated guidelines for the diagnosis and management of asthma. We hypothesized that not all components of the updated guidelines are well understood by the physicians who care for asthmatics. To develop appropriate educational interventions that address areas of physician misunderstanding, it is important to identify these components. Based upon NHLBI guidelines, we developed a multiple-choice test of asthma knowledge that was distributed to physicians at the University of Iowa; 108 physicians completed the test, including 20 asthma specialists, 11 asthma specialty fellows, 11 General Medicine faculty, five Family Medicine faculty, 51 Internal Medicine residents, and five Family Medicine residents. The mean correct total score for all physicians was 60 +/- 2% (mean +/- SEM). Asthma specialists scored higher in total score and in pharmacology and prevention. However, no group performed well on estimating disease severity. We further identified deficits in the use of spirometry and anti-inflammatory agents in caring for asthmatic patients. Thus, deficits exist in physician understanding and implementation of the NHLBI guidelines for the diagnosis and management of asthma. By identifying specific areas of misunderstanding, we can design better educational interventions. Clearly, educational programs should emphasize new models for estimating chronic disease severity.