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International audience; Eosinophils play a homeostatic role in the body's immune responses. These cells are involved in combating some parasitic, bacterial, and viral infections and certain cancers and have pathologic roles in diseases including asthma, chronic rhinosinusitis with nasal polyps, eosinophilic gastrointestinal disorders, and hypereosinophilic syndromes. Treatment of eosinophilic diseases has traditionally been through nonspecific eosinophil attenuation by use of glucocorticoids. However, several novel biologic therapies targeting eosinophil maturation factors, such as interleukin (IL)-5 and the IL-5 receptor or IL-4/IL-13, have recently been approved for clinical use. Despite the success of biologic therapies, some patients with eosinophilic inflammatory disease may not achieve adequate symptom control, underlining the need to further investigate the contribution of patient characteristics, such as comorbidities and other processes, in driving ongoing disease activity. New research has shown that eosinophils are also involved in several homeostatic processes, including metabolism, tissue remodeling and development, neuronal regulation, epithelial and microbiome regulation, and immunoregulation, indicating that these cells may play a crucial role in metabolic regulation and organ function in healthy humans. Consequently, further investigation is needed into the homeostatic roles of eosinophils and eosinophil-mediated processes across different tissues and their varied microenvironments. Such work may provide important insights into the role of eosinophils not only under disease conditions but also in health. This narrative review synthesizes relevant publications retrieved from PubMed informed by author expertise to provide new insights into the diverse roles of eosinophils in health and disease, with particular emphasis on the implications for current and future development of eosinophil-targeted therapies.

Background: Asthma is a complex disease with multiple phenotypes that may differ in disease pathobiology and treatment response. IL33 single nucleotide polymorphisms (SNPs) have been reproducibly associated with asthma. IL33 levels are elevated in sputum and bronchial biopsies of patients with asthma. The functional consequences of IL33 asthma SNPs remain unknown. Objective: This study sought to determine whether IL33 SNPs associate with asthma-related phenotypes and with IL33 expression in lung or bronchial epithelium. This study investigated the effect of increased IL33 expression on human bronchial epithelial cell (HBEC) function. Methods: Association between IL33 SNPs (Chr9: 5,815,786-6,657,983) and asthma phenotypes (Lifelines/DAG [Dutch Asthma GWAS]/GASP [Genetics of Asthma Severity & Phenotypes] cohorts) and between SNPs and expression (lung tissue, bronchial brushes, HBECs) was done using regression modeling. Lentiviral overexpression was used to study IL33 effects on HBECs. Results: We found that 161 SNPs spanning the IL33 region associated with 1 or more asthma phenotypes after correction for multiple testing. We report a main independent signal tagged by rs992969 associating with blood eosinophil levels, asthma, and eosinophilic asthma. A second, independent signal tagged by rs4008366 presented modest association with eosinophilic asthma. Neither signal associated with FEV 1, FEV 1/forced vital capacity, atopy, and age of asthma onset. The 2 IL33 signals are expression quantitative loci in bronchial brushes and cultured HBECs, but not in lung tissue. IL33 overexpression in vitro resulted in reduced viability and reactive oxygen species–capturing of HBECs, without influencing epithelial cell count, metabolic activity, or barrier function. Conclusions: We identify IL33 as an epithelial susceptibility gene for eosinophilia and asthma, provide mechanistic insight, and implicate targeting of the IL33 pathway specifically in eosinophilic asthma.

To-date, there has not been a clear signal suggesting that asthma or treatment with inhaled steroids are a risk factor for severe COVID-19 disease. We have therefore explored ACE2 receptor mRNA expression, and co-factors for Sars-CoV-2 infectivity (TMPRSS2 and furin) in bronchial brushes and biopsies from people with asthma and healthy controls, and looked for relationships between asthma severity, Th2- and IL-17 dependent gene signatures, and clinical demographics (age, sex). We have looked at a cohort of 356 research participants from previously described studies. The only significant association was a positive correlation between ACE2 and IL-17-dependent gene expression, and an inverse correlation between ACE2 and Th2-cytokine-dependent gene expression. These data suggest that differences in ACE2, TMPRSS2 and furin epithelial and airway gene expression are unlikely to confer enhanced COVID-19 pneumonia risk in patients with asthma across all treatment intensities and severity., Expression of mRNA for ACE2, the Sars-CoV-2 receptor, is similar in the lower airways of healthy controls and people with mild-severe asthma. Altered ACE2 expression is unlikely to confer enhanced COVID-19 pneumonia risk in asthma.

Introduction Ameliorer le controle de l’asthme est un objectif essentiel de la prise en charge de l’asthme. L’effet du mepolizumab sur le controle de l’asthme evalue par l’Asthma Control Questionnaire-5(ACQ-5) a ete rapporte dans les essais cliniques, mais les donnees en vraie vie sont limitees. Methodes REALITI-A, etude prospective en vraie vie, multicentrique, internationale, a enrole des patients avec un asthme severe a eosinophiles (ASE), ayant initie un traitement par mepolizumab sur 51 centres dans 7 pays. Une periode de 12 mois de donnees precedant l’inclusion etait necessaire pour decrire les donnees demographiques des participants, le fardeau de l’ASE et les traitements. Le controle de l’asthme a ete evalue par l’ACQ-5. Les scores a l’initiation ont ete compares aux scores a 3,6,9 et 12 mois. Les changements du controle depuis l’initiation et le taux de participants ayant une difference minimale importante [DMI] depuis l’initiation de 0,5 unites ont ete evalues. Des modeles mixtes de mesures repetees ont ete utilises pour l’analyse. Resultats Les donnees initiales etaient disponibles pour 368 patients qui ont complete un suivi de 12 mois ( Fig. 1 ). Dix-neuf pour cent (n = 70/368) des patients ont arrete le mepolizumab dans l’annee suivant l’initiation ; les raisons les plus frequentes etaient : la decision du patient (7 % ; n = 27/368), la discretion de l’investigateur (4 % ; n = 14/368), le manque d’efficacite (4 % ; n = 13/368). A l’inclusion, les patients avaient un score moyen ACQ-5 en moindres carres (IC95 %) de 2,99 (2,85 ; 3,13). L’initiation du mepolizumab a ete associee a une amelioration du controle de l’asthme, avec des moyennes MC (IC95 %) du score ACQ-5 a 3,6,9 et 12 mois de 1,81 (1,66 ; 1,96), 1,73 (1,56 ; 1,89), 1,84 (1,62 ; 2,05), et 1,84 (1,63 ; 2,05). Dans les 3 mois, 65 % (152/234) des patients ont obtenu une DMI, passant a 71 % (84/119) apres 12 mois. Les ameliorations ont ete observees dans chaque tranche de taux d’eosinophiles sanguins a l’inclusion, avec 56 % (10/18), 60 % (9/15), et 75 % (62/83) des patients avec des taux d’eosinophiles sanguins Conclusion Cette etude prospective en vie reelle confirme l’efficacite du mepolizumab chez les patients avec un ASE en termes d’amelioration du controle de l’asthme a 3 mois, avec des ameliorations significatives du score ACQ-5. Ces resultats montrent un benefice rapide et significatif du controle de l’asthme avec le mepolizumab en vraie vie, coherent avec les resultats des essais cliniques controles.

Allergen-specific responses in atopic dermatitis (AD) are skewed toward a Th2 profile. However, individuals with AD have been shown to make effective virus-specific Th1 responses, raising the possibility that the skin itself contributes to driving the AD Th2 immunophenotype. Therefore, to explore the programming of immunological sensitization by the skin, we examined the outcome of sensitization through non-lesional skin of individuals with AD and healthy controls. Volunteers (controls, AD individuals with filaggrin gene (FLG) mutations (ADFM), and AD individuals without FLG mutations (ADWT)) were sensitized by cutaneous application of 2,4-dinitrochlorobenzene (DNCB), a small, highly lipophilic chemical sensitizer. At the doses tested, DNCB showed equal penetration into skin of all groups. Clinical reactions to DNCB were significantly reduced in AD. Although both controls and AD made systemic DNCB-specific Th1 responses, these were reduced in AD and associated with significantly Th2-skewed DNCB-specific T-cell responses. Th2 skewing was seen in both ADFM and ADWT, with no difference between these groups. After 3 months, DNCB-specific Th2 responses were persistent in individuals with AD, and Th1 responses persisted in controls. These data provide evidence that when antigen penetration is not limiting, AD skin has a specific propensity to Th2 programming, suggesting the existence of altered skin immune signaling that is AD-specific and independent of FLG status.

Background Tissue factor (TF), a primary initiator of blood coagulation, also plays a pivotal role in angiogenesis. TF expression in the airways is associated with asthma, a disease characterized in part by subepithelial angiogenesis. Objectives To determine potential sources of TF and the mechanisms of its availability in the lung microenvironment. Methods Normal human bronchial epithelial cells grown in air-liquid interface culture were subjected to a compressive stress of 30 cm H 2 O; this is comparable to that generated in the airway epithelium during bronchoconstriction in asthma. Conditioned media and cells were harvested to measure TF mRNA and TF protein. We also tested bronchoalveolar lavage fluid and airway biopsies from asthmatic patients and healthy controls for TF. Results TF mRNA was upregulated 2.2-fold after 3 hours of stress compared with unstressed cells. Intracellular and secreted TF proteins were enhanced 1.6-fold and more than 50-fold, respectively, compared with those of control cells after the onset of compression. The amount of TF in the bronchoalveolar lavage fluid from patients with asthma was found at mean concentrations that were 5 times greater than those of healthy controls. Immunohistochemical staining of endobronchial biopsies identified epithelial localization of TF with increased expression in asthma. Exosomes isolated from the conditioned media of normal human bronchial epithelial cells and the bronchoalveolar lavage fluid of asthmatic subjects by ultracentrifugation contained TF. Conclusions Our in vitro and in vivo studies show that mechanically stressed bronchial epithelial cells are a source of secreted TF and that exosomes are potentially a key carrier of the TF signal.

BACKGROUND: Some patients with severe asthma have recurrent asthma exacerbations associated with eosinophilic airway inflammation. Early studies suggest that inhibition of eosinophilic airway inflammation with mepolizumab-a monoclonal antibody against interleukin 5-is associated with a reduced risk of exacerbations. We aimed to establish efficacy, safety, and patient characteristics associated with the response to mepolizumab. METHODS: We undertook a multicentre, double-blind, placebo-controlled trial at 81 centres in 13 countries between Nov 9, 2009, and Dec 5, 2011. Eligible patients were aged 12-74 years, had a history of recurrent severe asthma exacerbations, and had signs of eosinophilic inflammation. They were randomly assigned (in a 1:1:1:1 ratio) to receive one of three doses of intravenous mepolizumab (75 mg, 250 mg, or 750 mg) or matched placebo (100 mL 0·9% NaCl) with a central telephone-based system and computer-generated randomly permuted block schedule stratified by whether treatment with oral corticosteroids was required. Patients received 13 infusions at 4-week intervals. The primary outcome was the rate of clinically significant asthma exacerbations, which were defined as validated episodes of acute asthma requiring treatment with oral corticosteroids, admission, or a visit to an emergency department. Patients, clinicians, and data analysts were masked to treatment assignment. Analyses were by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT01000506. FINDINGS: 621 patients were randomised: 159 were assigned to placebo, 154 to 75 mg mepolizumab, 152 to 250 mg mepolizumab, and 156 to 750 mg mepolizumab. 776 exacerbations were deemed to be clinically significant. The rate of clinically significant exacerbations was 2·40 per patient per year in the placebo group, 1·24 in the 75 mg mepolizumab group (48% reduction, 95% CI 31-61%; p

Background: Limited data describe the association between the frequency of asthma exacerbations and the decline in lung function in severe asthma. Objective: To determine whether asthma exacerbations are associated with enhanced decline in lung function. Methods: Changes in lung function were analyzed retrospectively using data from the DREAM and MENSA studies of mepolizumab intervention in patients with severe asthma. Patients were either nonsmokers or former smokers. A linear regression model was used to analyze the relationship between the number of exacerbations and decline in FEV1 across treatment groups. Results: In a combined post hoc analysis, 57% (n = 572) of patients had no exacerbations and experienced an improvement in postbronchodilator FEV1 of 143 mL. In contrast, in patients who experienced 3 or more exacerbations, there was a decrease in postbronchodilator FEV1 of 77 mL in the combined analysis. The linear modeling analysis estimated that for each exacerbation seen during the observational period, there was a decrease of 50 mL in FEV1 (P < .001). Conclusions: A direct relationship between the number of exacerbations in patients with severe eosinophilic asthma and decline in lung function was observed. Repeated exacerbations may be associated with accelerated loss of lung function.

Although asthma is defined in terms of reversibility of airflow obstruction, as the disease becomes more severe and chronic, it adopts different characteristics, including a degree of fixed airflow obstruction and corticosteroid refractoriness. Underlying these phenotypes is evidence of airway wall remodeling, which should be distinguished from the increase in smooth muscle linked to airways hyperresponsiveness. Aberrant epithelial-mesenchymal communication leads to a chronic wound scenario, which is characterized by activation of the epithelial-mesenchymal trophic unit, epithelial damage, the laying down of new matrix, and greater involvement of neutrophils in the inflammatory response. In allergic asthmatic patients who remain symptomatic despite high-dose corticosteroid therapy, blockade of IgE with omalizumab confers appreciable clinical benefit. Chronic severe asthma is also accompanied by a marked increase in TNF-α production that might contribute to corticosteroid refractoriness. Based on this, TNF blockade with the soluble fusion protein entanercept produces improvement in asthma symptoms, lung function, and quality of life paralleled by a marked reduction in airways hyperresponsiveness. Identification of novel susceptibility genes, such as a disintegrin and metalloprotease 33 (ADAM33), will provide further targets against which to direct novel therapies for asthma, especially at the more severe end of the disease spectrum.

Nasal obstruction can be monitored objectively by measurement of nasal airflow, as evaluated by nasal peak flow, or as airways resistance/conductance as evaluated by rhinomanometry. Peak flow can be measured during inspiration or expiration. Of these measurements, nasal inspiratory peak flow is the best validated technique for home monitoring in clinical trials. The equipment is portable, relatively inexpensive, and simple to use. One disadvantage, however, is that nasal inspiratory peak flow is influenced by lower airway as well as upper airway function. Rhinomanometry is a more sensitive technique that is specific for nasal measurements. The equipment, however, requires an operator, is more expensive, and is not portable. Thus, it is applicable only for clinic visit measures in clinical trials. Measurements require patient cooperation and coordination, and not all can achieve repeatable results. Thus, this objective measure is best suited to laboratory challenge studies involving smaller numbers of selected volunteers. A nonphysiological measure of nasal patency is acoustic rhinometry. This sonic echo technique measures internal nasal luminal volume and the minimum cross-sectional area. The derivation of these measures from the reflected sound waves requires complex mathematical transformation and makes several theoretical assumptions. Despite this, however, such measures correlate well with the nasal physiological measures, and the nasal volume measures have been shown to relate well to results obtained by imaging techniques such as computed tomography scanning or magnetic resonance imaging. Like rhinomanometry, acoustic rhinometry is not suitable for home monitoring and can be applied only to clinic visit measures or for laboratory nasal challenge monitoring. It has advantages in being easy to use, in requiring little patient cooperation, and in providing repeatable results. In addition to nasal obstruction, allergic rhinitis is recognized to be associated with impaired mucociliary clearance and altered nasal responsiveness. Measures exist for the monitoring of these aspects of nasal dysfunction. Although measures of mucociliary clearance are simple to perform, they have a poor record of reproducibility. Their incorporation into clinical trials is thus questionable, although positive outcomes from therapeutic intervention have been reported. Measures of nasal responsiveness are at present largely confined to research studies investigating disease mechanisms in allergic and nonallergic rhinitis. The techniques are insufficiently standardized to be applied to multicenter clinical trials but could be used in limited-center studies to gain insight into the regulatory effects of different therapeutic modalities.

The subjective recording in diary cards of symptoms of itch, sneeze, nose running, and blockage, with the use of a rating scale to indicate the level of severity, is usual for clinical trials in allergic rhinitis. The primary outcome measure is usually a composite score that enables a single total symptoms score endpoint. It is appreciated, however, that rhinitis has a greater effect on the individual than is reflected purely by the recording of anterior nasal symptoms. Nasal obstruction is troublesome and may lead to sleep disturbance in addition to impaired daytime concentration and daytime sleepiness. These impairments affect school and work performance. Individuals with rhinitis find it socially embarrassing to be seen sneezing, sniffing, or blowing their nose. To capture these and other aspects of the disease-specific health-related quality of life, questionnaires such as the Rhinoconjunctivitis Quality of Life Questionnaire have been developed and validated for clinical trial use. The adoption of health-related quality of life questionnaires into clinical trials broadens the information obtained regarding the effect of the therapeutic intervention and helps focus on issues relevant to the individual patient. It must be appreciated that it is not only the disease that may adversely affect health-related quality of life; administered therapy, although intended to be beneficial, may also cause health impairment. Adverse-event monitoring is thus essential in clinical trials. The first-generation H 1 -histamines, because of their effect on central H 1 -receptors, are classically associated with central nervous system (CNS) effects such as sedation. Although this is not always perceived by the patient, it is clearly evident with objective performance testing, and positron emission tomography scanning has directly demonstrated the central H 1 -receptor occupancy. The second-generation H 1 -antihistamines have reduced central H 1 -receptor occupancy and considerably reduced or absent CNS sedative effects. Therefore, the CNS effects are entirely avoidable, and the first-generation H 1 -antihistamines should no longer be used in the management of allergic rhinitis. The considerably rarer but potentially very serious cardiac arrhythmogenic effects of H 1 -antihistamines are appreciated to be molecule-specific rather than class-specific. The in vitro screening of new compounds to eliminate the further development of those with cardiotoxicity ideally will lead to this adverse effect being historic. The incorporation of electrocardiogram recording in clinical trials provides direct information relating to prolongation of QT interval corrected for heart rate. Although administered at low doses, intranasal steroids still have the potential for systemic absorption and adverse consequences. However, it is appreciated that meaningful differences exist in the bioavailability of different steroid molecules, and although a small but statistically significant effect on growth in children has been identified with the long-term use of intranasal beclomethasone when administered twice daily for 1 year, this is not evident with all intranasal steroids. In addition, twice-daily intranasal steroid administration may have more effect—from the endocrinologic perspective—than once-daily administration in the morning, which coincides better with the natural diurnal variation in cortisol. Thus, once-daily intranasal steroid administration is preferable, and when used in studies in children, measurement of height change during the study period is an important outcome variable together with other indices of systemic steroid bioavailability (eg, tests of hypothalamic-pituitary-adrenal axis function). These considerations have even greater relevance if children are concurrently also receiving inhaled steroids for asthma, because the total steroid load will be greater.

Allergic rhinitis is an inflammatory nasal disorder in which a range of different cells participates. A variety of approaches has been used to monitor nasal inflammation objectively to investigate disease processes and to evaluate the effect of therapeutic intervention. These approaches include nasal lavage, nasal cytology, and nasal biopsy, together with the more recently established measurement of nasal nitric oxide (NO) concentration. Although all provide information about nasal mucosal inflammation, the extent of information that can be obtained by each approach, the ease of sampling, and the complexity of sample handling differ. Such considerations influence the choice of approach when measurement of nasal inflammation is to be an objective outcome parameter in a clinical trial. In addition, the choice of approach is also determined by the questions or hypotheses that are to be addressed. Nasal lavage is simple and rapid to perform, is well tolerated, and provides a sample that can provide information about luminal cell recruitment, cell activation, and plasma protein extravasation. Nasal cytology involves sampling and recovering mucosal surface cells. It is also easy to perform and is well tolerated in general, although some find that the procedure causes a transient unpleasant sensation. A differential cell count from the sample provides information about relative cell populations. Both nasal lavage and nasal cytology are readily applicable to clinical trials. Nasal cytology sample handling is easier, but nasal lavage offers the advantage of providing considerably greater information from the sample. Nasal biopsy is a considerably more invasive procedure and requires expertise not only in tissue sampling but also in biopsy processing. Therefore, it is applicable only in specialist centers. However, nasal biopsy is the only sampling technique that directly informs about tissue cellular events, although these may be implied, in part from the other sampling approaches. Tissue specimens can be used to evaluate both protein and gene expression. Measurement of nasal NO involves expensive equipment but provides an instantaneous result, unlike the other approaches, all of which require sample processing and analysis. Recommendations for standardization of measurement have been made, and measures are considered in part to reflect allergic inflammation within the nasal mucosa. The limitations of nasal NO are that it reflects only a certain aspect of allergic mucosal inflammation, and that because a proportion of nasally measured NO is derived from the sinuses under normal circumstances, nasal NO is not specific for nasal disease. The high contribution from the sinus mucosa limits the discriminatory ability of nasal NO to reflect nasal tissue-specific alterations. The incorporation of measures of nasal inflammation in clinical trials has distinguished anti-inflammatory therapy from symptomatic therapy and has the potential to provide information about the efficacy of novel therapies for allergic rhinitis.

Background Allergic rhinitis is characterized by the epithelial accumulation of cells, particularly mast cells and eosinophils. There is little information relating to the chemotaxins responsible for mast cell epithelial accumulation in this disease. Objective Expression of the mast cell chemoattractants TGF-β, eotaxin, and stem cell factor and their receptors was investigated in tissue sections from biopsy specimens obtained from patients with naturally occurring allergic rhinitis. Methods Specific immunohistochemical staining was performed on thin sections of inferior turbinate biopsy specimens from patients with perennial and seasonal allergic rhinitis and, for comparison, from nonatopic and, where relevant, atopic healthy volunteers without rhinitis. Sequential staining of adjacent 2-μm sections was undertaken to colocalize TGF-β receptors to mast cells. Results Evidence was found of significantly increased epithelial immunoreactivity for TGF-β1, TGF-β2, TGF-β3, TGF-β receptor I, TGF-β receptor II, and TGF-β receptor III in patients with perennial and seasonal allergic rhinitis compared with that seen in healthy control subjects. TGF-β receptors I and II were found to colocalize to mast cells. Eotaxin epithelial immunoreactivity was significantly increased in the perennial group, although there were no corresponding disease-related differences found in relation to CCR-3 immunoreactivity at this site. There was no increase in stem cell factor immunoreactivity within the epithelium in naturally occurring disease. Significant correlations were found between epithelial immunoreactivity for TGF-β1, TGF-β2, TGF-β receptor I, TGF-β receptor II, and the number of epithelial mast cells. Conclusion These findings of enhanced epithelial TGF-β immunoreactivity in patients with rhinitis, the correlation with intraepithelial mast cell numbers, and the colocalization of TGF-β receptors to mast cells suggest that the epithelial expression of TGF-β might represent an important biologic process involved in either the recruitment or retention of mast cells within the epithelium in naturally occurring allergic rhinitis.

Human airway smooth muscle (ASM) has several properties and functions that contribute to asthma pathogenesis, and increasing attention is being paid to its synthetic capabilities. ASM can promote the formation of the interstitial extracellular matrix, and in this respect, ASM from asthmatic subjects compared with normal subjects responds differently, both qualitatively and quantitatively. Thus, ASM cells are important regulating cells that potentially contribute to the known alterations within the extracellular matrix in asthma. In addition, through integrin-directed signaling, extracellular matrix components can alter the proliferative, survival, and cytoskeletal synthetic function of ASM cells. ASM also functions as a rich source of biologically active chemokines and cytokines that are capable of perpetuating airway inflammation in asthma and chronic obstructive pulmonary disease by promoting recruitment, activation, and trafficking of inflammatory cells in the airway milieu. Emerging evidence shows that airway remodeling may also be a result of the autocrine action of secreted inflammatory mediators, including T H 2 cytokines, growth factors, and COX-2–dependent prostanoids. Finally, ASM cells contain both β 2 -adrenergic receptors and glucocorticoid receptors and may represent a key target for β 2 -adrenergic receptor agonist/corticosteroid interactions. Combinations of long-acting β 2 -agonists and corticosteroids appear to have additive and/or synergistic effects in inhibiting inflammatory mediator release and the migration and proliferation of ASM cells.

Asthma is regarded as an inflammatory disorder of the conducting airways characterized by a mast cell, eosinophil and T lymphocyte inflammatory response that is responsive to anti-inflammatory therapy, such as corticosteroids. In more severe and chronic disease, corticosteroids become less effective. As in other chronic inflammatory diseases, the tissue in which the cellular and mediator processes occur plays a major role in maintaining the response and creating a basis for disease persistence. Herein, we describe evidence that the airway epithelium interacting with the underlying mesenchymal cells recapitulates branching morphogenesis, as observed in the developing lung, to create airway wall remodeling. The reciprocal signaling between the susceptible epithelium and responsive mesenchyme (epithelial mesenchymal trophic unit) offers a new paradigm for asthma and creates new opportunities for developing therapeutics based on reversing the 'chronic wound' phenotype of asthmatic airways.

Airway remodeling in asthma refers to a collection of chronic structural changes including subepithelial fibrosis, airway smooth muscle hypertrophy/hyperplasia, and possibly angiogenesis. The mechanisms leading to remodeling are not well defined. One molecule of possible relevance is basic fibroblast growth factor (bFGF), which is a potent mitogen for fibro-blasts, airway smooth muscle cells, and endothelial cells. To test the hypothesis that bFGF expression is increased in asthma, we measured levels of the growth factor in bronchoalveolar lavage (BAL) fluid. Basally, BAL fluid bFGF concentrations were significantly higher in subjects with atopic asthma than in control subjects without asthma (median 0.22 vs 0.06 pg/mL, P = .003). The effect of acute allergen exposure was examined with a segmental bronchoprovocation model in a separate group of subjects with atopic asthma. Ten minutes after segmental bronchoprovocation there was a 5-fold increase in bFGF levels in BAL fluid recovered from allergen-challenged sites compared with control saline-challenged sites (1.52 vs 0.30 pg/mL, P < .002). We conclude that basal levels of BAL fluid bFGF are increased in atopic asthma and that a further increase occurs in response to acute allergen exposure. These findings lend support to the hypothesis that bFGF is implicated in airway remodeling in asthma.

Background: The epidermal growth factor (EGF) family of growth factors plays an important role in maintenance and repair in a variety of epithelial tissues. However, very little is known about coexpression of these factors and their receptors, the c-erbB family of receptor tyrosine kinases, in human nasal epithelium. Objective: We sought to investigate the expression of these molecules in cultured nasal epithelial cells and nasal mucosa from healthy individuals. Methods: Identification of c-erbB receptors and their ligands was sought by using reverse transcription PCR, Western blotting, and immunohistochemistry. Results: Messenger RNA encoding the EGF receptors (EGFR) c-erbB2 and c-erbB3, but not c-erbB4, was detected in primary cultures of human nasal epithelial cells. Transcripts encoding EGF, heparin-binding EGF, transforming growth factor (TGF) α, and amphiregulin were also detected. Receptor and ligand expression was confirmed by using immunocytochemical staining of the cells and Western blotting of the cell lysates. Immunohistochemical analysis of tissue sections obtained from biopsy specimens of nasal mucosa revealed intense membrane staining for the EGFR within the respiratory nasal epithelium, which was predominantly localized at the level of the columnar epithelial layers. Similar staining patterns were observed for c-erbB2 and c-erbB3 in the respiratory nasal epithelium. Evidence for EGF, transforming growth factor α, heparin-binding EGF, amphiregulin, and betacellulin immunostaining in the nasal epithelium was also obtained; their staining patterns paralleled that of EGFR immunostaining. Conclusion: Colocalization of c-erbB receptors and ligands establishes a basis on which to investigate c-erbB receptor– mediated effects in human nasal epithelium. (J Allergy Clin Immunol 2000;106:1124-31.)

Early observations suggested that the inhibition by long-acting β2-agonists (LABAs) of non-specific bronchial hyperresponsiveness following allergen challenge was unrelated to bronchodilation or functional antagonism and might be a reflection of anti-inflammatory activity.Investigation of the effect of LABAs on airway inflammatory responses has demonstrated an inhibition of eosinophil recruitment in allergen challenge studies. Nevertheless, results from biopsy and other studies suggest that the chronic inflammatory process in asthma patients is unaffected by these drugs. There is no evidence from biopsy studies that LABAs are pro-inflammatory or that they mask existing inflammation.The beneficial effects of LABAs in allergen challenge are probably mediated through stabilization of mast cells. Recent evidence suggests that LABAs may reduce numbers of neutrophils and their associated markers; this observation needs to be confirmed in future studies and its relevance to the treatment of asthma determined.

Background: Fexofenadine hydrochloride (HCl) is a new H 1 antihistamine used twice daily in some countries. Objective: A multicenter, double-blind, parallel-group, placebo-controlled trial compared the efficacy and safety of fexofenadine HCl (120 and 180 mg administered once daily) and cetirizine (10 mg once daily) in the treatment of seasonal allergic rhinitis. Methods: After a 3- to 5-day run-in period, patients meeting entrance criteria were randomized to receive placebo, fexofenadine HCl 120 mg once daily, fexofenadine HCl 180 mg once daily, or cetirizine 10 mg once daily (active control) for 2 weeks. Eight hundred twenty-one patients comprised the intention-to-treat population and 722 patients completed the study. Symptom assessments were conducted 12 hours after the dose for the previous 12 hours and again at 24 hours after the dose for the previous 12 hours. In addition, assessment was made immediately before dosing in the morning for the previous 30 minutes. Total symptom score was calculated as the sum of scores for the 4 individual symptoms: (1) sneezing, (2) rhinorrhea, (3) itchy nose, palate, or throat, and (4) itchy, watery, or red eyes; the nasal congestion score was also recorded. Results: Both doses of fexofenadine HCl were superior to placebo in reducing the total symptom score. Efficacy was maintained for the entire dosing interval (ie, for 24 hours). There were no differences in efficacy between the 2 doses of fexofenadine HCl or between either dose of fexofenadine HCl and cetirizine. There was no major side effect, but the combined incidence of drowsiness or fatigue was greater with ce-tirizine (9%) than with placebo (4%) ( P = .07) or fexofenadine (4%) ( P = .02). Conclusions: Once-daily fexofenadine is thus a valuable addition to the nonsedating group of H 1 receptor antagonists currently available for the treatment of seasonal allergic rhinitis. (J Allergy Clin Immunol 1999;104:927-33.)

The characterisation of chronic asthma as an inflammatory condition of the human airway, associated with heightened airway responsiveness to a variety of bronchial stimuli, has lead to the clarification of therapeutic strategies. These strategies have focused on bronchodilation and attenuation of airway inflammation. Inhaled corticosteroids effectively reduce chronic inflammation and produce substantial symptomatic relief in most patients. This article examines the pathophysiology of asthma and discusses the interpretation of current methods of assessment, and the targets and actions of inhaled antiasthmatic drugs in relationship to central and peripheral airway events.

Background: Repeated inhalation of bradykinin and hypertonic saline leads to refractoriness of the bronchoconstrictor response in asthma. It is not known whether cross-refractoriness exists between these stimuli. Objective: We postulated that repeated bradykinin and hypertonic saline bronchial challenges might reduce the airway response to subsequent hypertonic saline and bradykinin challenges, respectively. Methods: Eleven atopic asthmatic subjects underwent two concentration-response studies, separated by 1 hour, with either inhaled histamine or bradykinin. After recovery, a hypertonic saline challenge was performed. During the next phase, nine subjects underwent two concentration-response studies, separated by 1 hour, with hypertonic saline. After recovery, a bradykinin challenge was performed. Results: On the histamine study day, the mean provocative volume of agonist required to produce 20% drop in forced expiratory volume in 1 second (PD 20 ) hypertonic saline was 220.7 L (±42.7 L) and this was not significantly different from that measured at baseline. On the bradykinin study day, the geometric mean provocative concentration of agonist required to produce a 20% drop in forced expiratory volume in 1 second (PC 20 ) was 0.39 mg/ml (0.01 to 11.73 mg/ml) for the first test and significantly higher at 1.38 mg/ml (0.01 to >16.0 mg/ml) for the second test ( p = 0.006). The hypertonic saline PD 20 increased significantly from a baseline of 159.2 L (±27.3 L) to 377.6 (±64.7 L) ( p = 0.003). On the hypertonic saline study day, the mean PD 20 was 152.8 L for the first test, and 337.7 L for the second test ( p = 0.01). PC 20 bradykinin increased significantly from a baseline of 0.57 to 2.56 mg/ml ( p = 0.02). A significant correlation was found between loss of response to bradykinin and to hypertonic saline (r s , 0.63 and 0.76). Conclusion: Refractoriness produced by repeated exposure of the airways to bradykinin and hypertonic saline results in loss of responsiveness to hypertonic saline and bradykinin respectively, suggesting a shared mechanism for refractoriness produced by these stimuli. (J Allergy Clin Immunol 1995;96:502-9.)

Local hyperthermia reduces mast cell degranulation, the severity of acute lung injury, and exercise-induced asthma and decreases symptoms of rhinitis. We have investigated the effect of local hyperthermia on mast cell degranulation and symptom generation in allergic rhinitis to assess its effect and mechanism of action within the nose.In a randomized, double-blind, placebo-controlled, crossover study, 10 subjects with rhinitis were treated for 30 minutes with local hyperthermia or placebo, which was followed 30 minutes later by nasal allergen challenge. During the first two visits nasal lavages were performed to assess vascular leakage and mediator release. During the last two visits nasal airway resistance, the number of sneezes, and mucus secretion were monitored.Local hyperthermia significantly reduced both nasal airway resistance (p0.05) and vascular leakage (p0.02) but had no significant effect on the number of sneezes, on mucus secretion, or on tryptase release.Local hyperthermia reduces allergen-provoked nasal blockage and vascular leakage but has no effect on sneezing, rhinorrhea, or tryptase release. Nasal blockage occurs predominantly via newly formed lipid mediators and kinins, whereas sneezing and rhinorrhea occur predominantly via preformed mediators. We propose that local hyperthermia inhibits newly formed mediator production or release or reduces the sensitivity of the vasculature to inflammatory mediators in general. Further investigation into the mechanisms and potential uses of local hyperthermia is warranted.

Background: Nasal lavage and challenge studies in allergic rhinitis implicate prostaglandin (PG) D 2 in the genesis of nasal blockage. PG D 2 is known to act via at least two receptors, the thromboxane prostanoid receptor and the PG D 2 prostanoid (DP) receptor; the lower airway effects are mediated chiefly by the TP receptor. The receptor involved in the genesis of PG D 2 -induced nasal blockage is unknown. BAY u 3405 is a potent selective competitive TP receptor antagonist, which inhibits the lower airway response to PG D 2 , and shifts the dose-response curve to the right by up to 16-fold. Methods: The efficacy of a single oral dose of 20 mg of BAY u 3405 was examined in comparison with PG D 2 nasal insufflation in a randomized, double-blind, placebo-controlled crossover study, with objective measurement of nasal resistance by active posterior rhinomanometry. Results: BAY u 3405 afforded no protection against PG D 2 -induced nasal blockage. Conclusions: This suggests that PG D 2 -induced nasal blockage may be mediated by the DP receptor rather than the TP receptor and that TP receptor antagonists are unlikely to be of benefit in the treatment of allergic rhinitis. In vivo investigation with specific potent DP receptor antagonists is awaited.

There is increasing evidence for the role of basophils in the allergen-induced late asthmatic response (LAR). To study the effect of inhaled allergen on basophil function in subjects with asthma, ex vivo basophil spontaneous histamine release (SHR) in peripheral blood and plasma histamine was measured before and 2, 5, 10, and 15 minutes, and 2, 4, 6, and 8 hours after allergen bronchial challenge (allergen study day) in six subjects with atopic asthma. Allergen inhalation induced an early response and LAR consisting of a mean (+/- SD) 32.5% (+/- 7.9%) and 28.8% (+/- 7.7%) fall in FEV1, respectively. As a control for the effects of bronchoconstriction, on another occasion, methacholine challenge was performed to produce a mean 33.4% (+/- 3.4%) fall in FEV1 during the early response and no LAR, and blood was obtained to measure basophil histamine release (HR) and plasma histamine. There was a small, but significant (p less than 0.05), rise in median SHR from 4.6% to 6.1% of total basophil histamine after allergen but not after methacholine inhalation. HR remained high after allergen inhalation during the 8 hours of study, whereas it demonstrated a steady, significant, decrease between 4 to 8 hours after methacholine inhalation. No significant changes in plasma histamine were recorded on either allergen or methacholine study days. On a third occasion, SHR was measured after challenge with physiologic saline to control for any effects of methacholine on SHR, and a decrease in HR was recorded during the day similar to HR observed after methacholine challenge. These studies suggest an enhancing effect of inhaled allergen on SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

There is extensive pharmacological and physiological evidence that endothelin-1 influences airway calibre. In mammals, endothelin receptor occur on airway smooth muscle, local storage and release of the peptide have been demonstrated, and inhalation of endothelin-1 induces bronchoconstriction. To investigate the relation between endothelins and asthma the expression of this peptide in endobronchial biopsy specimens was examined immunohistochemically with an antiserum against endothelin-1. Biopsy specimens from 17 asthmatic patients and 11 atopic and non-atopic healthy controls revealed striking differences, with endothelin expression being evident in airways epithelium and vascular endothelium in 11 of the 17 asthmatic patients but in only 1 of 11 controls. These results suggest that endothelins may play a part in the exaggerated bronchomotor tone of asthma.

Incomplete understanding of mechanisms and clinicopathobiological heterogeneity in asthma hinders research progress. Pathogenic roles for T-helper-type 17 (Th17) cells and invariant T cells implied by murine data have yet to be assessed in man. We aimed to investigate the role of Th17 and mucosal associated invariant T (MAIT) cells in airway inflammation; to characterise associations between diverse clinical and immunological features of asthma; and to identify novel multidimensional asthma endotypes.In this single-centre, cross-sectional observational study in the UK, we assessed volunteers with mild-to-severe asthma and healthy non-atopic controls using clinical and physiological assessment and immunological sampling of blood, induced sputum, endobronchial biopsy, and bronchoalveolar lavage for flow cytometry and multiplex-electrochemiluminescence assays. Primary outcomes were changes in frequencies of Th17 and MAIT cells between health and asthma using Mann-Whitney U tests and the Jonckheere-Terpstra test (linear trend across ranked groups). The study had 80% power to detect 60% differences in T-cell frequencies at p0·05. Bayesian Network Analysis (BNA) was used to explore associations between parameters. Topological Data Analysis (TDA) was used to identify multidimensional endotypes. The study had local research ethics approval. All participants provided informed consent.Participants were 84 male and female volunteers (60 with mild-to-severe asthma and 24 healthy, non-atopic controls) aged 18-70 years recruited from clinics and research cohorts. Th17 cells and γδ17 cells were not associated with asthma, even in severe neutrophilic forms. MAIT-cell frequencies were strikingly reduced in asthma compared with health (median frequency in blood 0·9% of CD3+ cells [IQR 0·3-1·8] in asthma vs 1·6 [1·2-2·6] in health, p=0·005; in sputum 1·1 [0·7-2·0] vs 1·8 [1·6-2·3], p=0·002; and in biopsy samples 1·3 [0·7-2·3] vs 3·9% [1·3-5·3%], p=0·02), especially in severe asthma where BAL regulatory T cells were also reduced compared with those in health (4·4, 3·1-6·1, vs 8·1, 5·6-10; p=0·02). BNA and TDA identified six novel clinicopathobiological clusters of underlying disease mechanisms, with elevated mast cell mediators tryptase (p0·0001), chymase (p=0·02), and carboxypeptidase A3 (p=0·02) in severe asthma.This study suggests that Th17 cells do not have a major pathogenic role in human asthma. We describe a novel deficiency of MAIT cells in severe asthma. We also provide proof of concept for application of TDA to identification of multidimensional clinicopathobiological endotypes. Endotypes will require validation in further cohorts.Wellcome Trust.

FEBRUARY 2015 AB40 Abstracts S A T U R D A Y 123 Adult Eosinophilic Oesophagitis: A UK Based Case Series Efrem Eren, MBBS, MRCP, FRCPath, PhD, Tak Chin, William Rae, BSc, BM, MRCP, Syed H. Arshad, DM, FRCP, Peter H. Howarth, MD, Anthony Williams, Elena Salagean, SpR, Bryan N. Fernandes, MD, Ramesh Kurukulaaratchy, Carina Venter, PhD, RD; Southampton General Hospital, UK, Southampton, United Kingdom, Southampton, University Hospital Southampton, Southampton, United Kingdom, The David Hide Asthma and Allergy Research Centre, United Kingdom, University of Southampton, United Kingdom, Southampton General Hospital, Southampton, United Kingdom, University of Southampton, University Hospital of NHS Foundation Trust, United Kingdom, University of Southampton, Southampton, United Kingdom, University of Portsmouth, United Kingdom. RATIONALE: Adult onset Eosinophilic Oesophagitis (EoE) presents frequently to the allergy clinics in the UK. However, no study to date has attempted to describe these patients. . This case series set out to clinically characterise adults patients with EoE seen over the past four years in a tertiary care center in the UK. METHODS: We screened Allergy/Immunology clinic lists and the edocument systems at Southampton General Hospital (SGH) for patients given a diagnosis of EoE since 2010. RESULTS: Thirty six patients were identified with a diagnostic label of EoE. They were predominantly male (77.8%; 28/36) with an age range of 19 – 71 years. The majority (61%, 22/36) suffered from other atopic conditions/or showed a positive total IgE level and suspected a wide range of potential foods triggers. Interestingly, patients often considered food that are difficult to swallow such as meat, chicken and starchy food as the cause of their problems. SPT and specific IgE tests were not helpful in identifying offending foods. As expected the majority (58.3%; 21/36) were sensitised to aero-allergens with 14 being sensitised to tree/birch pollen. Dietary interventions varied widely between these patients, ranging from elemental diets, 6 food elimination diets to test directed diets. CONCLUSIONS: Male sex and atopy were found to be primary characteristics, but age did not influence the diagnosis. A wide range of foods are suspected to trigger EoE pathology in adults, and dietarymeasure seems to vary greatly. Studies are needed to identify which foods are implicated in adult EoE in the UK.

Background Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted. Objectives We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma. Methods Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors. Results TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1β, IL-8, thymic stromal lymphoprotein, and pro–matrix metalloprotease 9 release. SU6656 reduced TNF-α–mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways. Conclusions Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.

Background Rhinoviruses are the major cause of asthma exacerbations. Previous studies suggest that primary bronchial epithelial cells (PBECs) from asthmatic subjects are more susceptible to rhinovirus infection because of deficient IFN-β production. Although augmenting the innate immune response might provide a novel approach for treatment of virus-induced asthma exacerbations, the potential of IFN-β to modulate antiviral and proinflammatory responses in asthmatic epithelium is poorly characterized. Objectives We sought to compare responses of PBECs from nonasthmatic and asthmatic subjects to exogenous IFN-β and test the inflammatory effects of IFN-β in response to rhinovirus infection. Methods PBECs were treated with IFN-β and infected with a low inoculum of human rhinovirus serotype 1B to simulate a natural viral infection. Expression of interferon-responsive genes and inflammatory responses were analyzed by using reverse transcription–quantitative real-time PCR, cytometric bead arrays, or both; viral titers were assessed by using the 50% tissue culture infection dose. Results Expression of IFN-β–stimulated antiviral genes was comparable in PBECs from nonasthmatic or asthmatic donors. Exogenous IFN-β significantly protected PBECs from asthmatic donors against rhinovirus infection by suppressing viral replication. Interferon-inducible protein 10 (IP-10), RANTES, and IL-6 release in response to rhinovirus infection was triggered only in PBECs from asthmatic donors. Although exogenous IFN-β alone stimulated some release of IP-10 (but not IL-6 or RANTES), it significantly reduced rhinovirus-induced IP-10, RANTES, and IL-6 expression when tested in combination with rhinovirus. Conclusions PBECs from asthmatic donors have a normal antiviral response to exogenous IFN-β. The ability of IFN-β to suppress viral replication suggests that it might limit virus-induced exacerbations by shortening the duration of the inflammatory response.

BACKGROUND: Wild plants harbour a variety of viruses and these have the potential to alter the composition of pollen. The potential consequences of virus infection of grasses on pollen-induced allergic disease are not known. METHODS: We have collected pollen from Dactylis glomerata (cocksfoot; a grass species implicated as a trigger of allergic rhino-conjunctivitis) from Wytham Wood, Oxfordshire UK. Extracts were prepared from pollen from uninfected grass, and from grass naturally infected by the Cocksfoot streak potyvirus (CSV). Preparations of pollen from virus-infected and non-infected grasses were employed in skin testing 15 grass pollen-allergic subjects with hayfever. Allergen profiles of extracts were investigated by Western blotting for IgE with sera from allergic subjects. RESULTS: The prevalence of CSV infection in cocksfoot grasses sampled from the study site varied significantly over an eight-year period, but infection rates of up to 70% were detected. Virus infection was associated with small alterations in the quantities of pollen proteins detected by polyacrylamide gel electrophoresis, and in the patterns of allergens identified by Western blotting with IgE from grass pollen allergic subjects. For individual subjects there were differences in potencies of standardised extracts of pollen from virus-free and virus-infected plants as assessed by skin testing, though a consistent pattern was not established for the group of 15 subjects. CONCLUSION: Infection rates for CSV in cocksfoot grass can be high, though variable. Virus-induced alterations in components of grass pollen have the potential to alter the allergenic potency.

Rationale After exposure to ozone, humans develop neutrophilic infiltration of the nasal and bronchial mucosa. Methods To investigate the events contributing to inflammatory cell recruitment in the bronchial mucosa we exposed 10 healthy nonsmoking volunteers to 400 ppb ozone or filtered air for 2h at rest on two separate occasions. Bronchial biopsies were performed 6h after ozone/filtered air exposure. The biopsies were embedded in glycol mathacrylate and immunostained for inflammatory cells, including neutrophils, mast cells, total T-cells (CD3), T-cell subsets CD8 and CD4, macrophages, eosinophils, adhesion molecules (P-selectin, E-selectin, ICAM-1, VCAM-1), cytokines (TNF-α, IL-1β, GM-CSF, IL-6), chemokines (IL-8 and RANTES), and nuclear factor NF-κB. Results No significant changes were seen in the number of T-cells and T-cell subsets, macrophages, eosinophils or percentages of vessels expressing P-selectin, VCAM-1, GM-CSF, IL-6 and RANTES in the biopsies. The number of neutrophils and mast cells in the submucosa was significantly higher after ozone exposure (p=0.009 and p=0.005 respectively). The percentage of vessels expressing E-selectin (p=0.01), ICAM-1 (p=0.005), IL-8 (p=0.02), TNF-α (p=0.02), IL-1β (p=0.009), and NF-κB (p=0.05) increased significantly after ozone exposure versus filtered air exposure. Conclusions Exposure of normal subjects to ozone increases the expression of proinflammatory cytokines resulting in upregulation of IL-8 and adhesion molecules via activation of NF-κB, leading to neutrophilic infiltration in the bronchial mucosa.

Rationale TGF-β exists in an inactive complex form bound to tissue proteoglycans, such as decorin, and requires release to exert its biological effects. Plasmin, a product of plasminogen activation, has been implicated in activating latent TGF-β and will be generated in association with tissue inflammation. We investigated the effects of plasmin on cytokine and TGF-β1 release and gene expression in primary fibroblast cultures from asthmatics and healthy controls. Methods Fibroblasts were grown from endobronchial biopsies of volunteers and used at passages 3-5. They were serum deprived for 24 hours, then challenged with plasmin for a further 24 hours. Cell supernatants were assayed for growth factors and cytokines by ELISA. Fibroblast mRNA was reverse transcribed and subjected to real-time PCR using Taqman probes. Results Plasmin induced a concentration-dependent (P Conclusions Plasmin promoted the liberation of TGF-β1, a cytokine associated with remodeling, but did not induce pro-inflammatory cytokines. The plasmin-induced release of immunomodulatory TGF-β1 may serve to regulate the inflammatory response and its effects on structural cells will promote tissue remodeling.