The role of ACT score in mepolizumab discontinuation.

BACKGROUND: Mepolizumab is a therapy for severe asthma. We have little knowledge of the characteristics of people in the US that discontinue mepolizumab in clinical care. OBJECTIVE: To investigate the real-world efficacy and time to clinical discontinuation of mepolizumab, we evaluated individuals with asthma started on mepolizumab at the Cleveland Clinic. We hypothesized that individuals that discontinue mepolizumab have more severe and uncontrolled asthma at baseline. METHODS: Between 2016 and 2022, patients who started on mepolizumab consented to be assessed over 18 months. At baseline, a questionnaire including demographic and medical history was collected. Laboratory findings such as ACT score, FENO (Fractional Excretion of Nitric Oxide), and spirometry were recorded. At the conclusion of the observation period, the participants were divided into two categories: Group A and Group B. RESULTS: Group B [N = 28] discontinued mepolizumab (p < 0.05) at an average of 5.8 months (SD 4.2 months). Group A [N = 129] stayed on the therapy for at least 1 year. A participant with an ACT score less than 13 has an odds ratio of 6.64 (95% CI, 2.1 - 26.0) of discontinuing mepolizumab therapy. For a male, the odds of discontinuing mepolizumab therapy is 3.39 (95% CI, 1.1-11.2). CONCLUSION: In this real-world study, we find that high eosinophil count may not be adequate in screening which individuals will benefit from mepolizumab. Up to 17% of patients fail therapy within 6 months, with male sex and low ACT score increasing risk of mepolizumab discontinuation at Cleveland Clinic.

The Precision Interventions for Severe and/or Exacerbation-Prone (PrecISE) Asthma Network: An overview of Network organization, procedures, and interventions.

Asthma is a heterogeneous disease, with multiple underlying inflammatory pathways and structural airway abnormalities that impact disease persistence and severity. Recent progress has been made in developing targeted asthma therapeutics, especially for subjects with eosinophilic asthma. However, there is an unmet need for new approaches to treat patients with severe and exacerbation-prone asthma, who contribute disproportionately to disease burden. Extensive deep phenotyping has revealed the heterogeneous nature of severe asthma and identified distinct disease subtypes. A current challenge in the field is to translate new and emerging knowledge about different pathobiologic mechanisms in asthma into patient-specific therapies, with the ultimate goal of modifying the natural history of disease. Here, we describe the Precision Interventions for Severe and/or Exacerbation-Prone Asthma (PrecISE) Network, a groundbreaking collaborative effort of asthma researchers and biostatisticians from around the United States. The PrecISE Network was designed to conduct phase II/proof-of-concept clinical trials of precision interventions in the population with severe asthma, and is supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health. Using an innovative adaptive platform trial design, the PrecISE Network will evaluate up to 6 interventions simultaneously in biomarker-defined subgroups of subjects. We review the development and organizational structure of the PrecISE Network, and choice of interventions being studied. We hope that the PrecISE Network will enhance our understanding of asthma subtypes and accelerate the development of therapeutics for severe asthma.

Resting Energy Expenditure Is Elevated in Asthma.

Background: Asthma physiology affects respiratory function and inflammation, factors that may contribute to elevated resting energy expenditure (REE) and altered body composition. Objective: We hypothesized that asthma would present with elevated REE compared to weight-matched healthy controls. Methods: Adults with asthma (n = 41) and healthy controls (n = 20) underwent indirect calorimetry to measure REE, dual-energy X-ray absorptiometry (DEXA) to measure body composition, and 3-day diet records. Clinical assessments included spirometry, fractional exhaled nitric oxide (FENO), and a complete blood count. Results: Asthmatics had greater REE than controls amounting to an increase of ~100 kcals/day, even though body mass index (BMI) and body composition were similar between groups. Inclusion of asthma status and FENO in validated REE prediction equations led to improved estimates. Further, asthmatics had higher white blood cell (control vs. asthma (mean ± SD): 4.7 ± 1.1 vs. 5.9 ± 1.6, p < 0.01) and neutrophil (2.8 ± 0.9 vs. 3.6 ± 1.4, p = 0.02) counts that correlated with REE (both p < 0.01). Interestingly, despite higher REE, asthmatics reported consuming fewer calories (25.1 ± 7.5 vs. 20.3 ± 6.0 kcals/kg/day, p < 0.01) and carbohydrates than controls. Conclusion: REE is elevated in adults with mild asthma, suggesting there is an association between REE and the pathophysiology of asthma.

Proinflammatory Pathways in the Pathogenesis of Asthma.

There are multiple proinflammatory pathways in the pathogenesis of asthma. These include both innate and adaptive inflammation, in addition to inflammatory and physiologic responses mediated by eicosanoids. An important component of the innate allergic immune response is ILC2 activated by interleukin (IL)-33, thymic stromal lymphopoietin, and IL-25 to produce IL-5 and IL-13. In terms of the adaptive T-lymphocyte immunity, CD4+ Th2 and IL-17-producing cells are critical in the inflammatory responses in asthma. Last, eicosanoids involved in asthma pathogenesis include prostaglandin D2 and the cysteinyl leukotrienes that promote smooth muscle constriction and inflammation that propagate allergic responses.

IL-27 targets Foxp3+ Tregs to mediate antiinflammatory functions during experimental allergic airway inflammation.

Foxp3+ CD4 Tregs are central regulators of inflammation, including allergic inflammation in the lung. There is increasing evidence that inflammatory factors undermine adequate Treg functions and homeostasis, resulting in prolonged and exacerbated inflammation. Therefore, identifying the factors is of the utmost important. IL-27 is an antiinflammatory cytokine implicated in immune regulation and tolerance. However, the cellular mechanisms underlying IL-27-mediated immune regulation in vivo remain largely unknown. Utilizing a cockroach antigen-induced allergic inflammation model in mice, we sought to test the roles of Tregs during IL-27-mediated regulation of allergic inflammation. Intranasally delivered IL-27 significantly reduced the development of airway inflammation. Unexpectedly, the IL-27-induced reduction occurred only in the presence of Tregs. Il27ra-/- and Treg-specific Il27ra-/- mice developed severe airway inflammation, and IL-27 treatment had little impact on diminishing the inflammatory responses. IL-27-induced treatment was restored following transfer of WT Tregs but not of Tregs deficient in Lag3, a molecule induced by IL-27 in Tregs. Finally, Tregs from asthmatic patients exhibited blunted STAT1 phosphorylation following IL-27 stimulation. Taken together, our results uncover that Tregs are the primary target cells of IL-27 in vivo to mediate its antiinflammatory functions, suggesting that altered IL-27 responsiveness in Tregs may underlie inadequate Treg functions and perpetuation of inflammation.

IL-17A Recruits Rab35 to IL-17R to Mediate PKCα-Dependent Stress Fiber Formation and Airway Smooth Muscle Contractility.

IL-17A is a critical proinflammatory cytokine for the pathogenesis of asthma including neutrophilic pulmonary inflammation and airway hyperresponsiveness. In this study, by cell type-specific deletion of IL-17R and adaptor Act1, we demonstrated that IL-17R/Act1 exerts a direct impact on the contraction of airway smooth muscle cells (ASMCs). Mechanistically, IL-17A induced the recruitment of Rab35 (a small monomeric GTPase) and DennD1C (guanine nucleotide exchange factor [GEF]) to the IL-17R/Act1 complex in ASMCs, resulting in activation of Rab35. Rab35 knockdown showed that IL-17A-induced Rab35 activation was essential for protein kinase Cα (PKCα) activation and phosphorylation of fascin at Ser39 in ASMCs, allowing F-actin to interact with myosin to form stress fibers and enhance the contraction induced by methacholine. PKCα inhibitor or Rab35 knockdown indeed substantially reduced IL-17A-induced stress fiber formation in ASMCs and attenuated IL-17A-enhanced, methacholine-induced contraction of airway smooth muscle. Taken together, these data indicate that IL-17A promotes airway smooth muscle contraction via direct recruitment of Rab35 to IL-17R, followed by PKCα activation and stress fiber formation.

High molecular weight hyaluronan ameliorates allergic inflammation and airway hyperresponsiveness in the mouse.

Allergic asthma is a major cause of morbidity in both pediatric and adult patients. Recent research has highlighted the role of hyaluronan (HA), an extracellular matrix glycosaminoglycan, in asthma pathogenesis. Experimental allergic airway inflammation and clinical asthma are associated with an increase of shorter fragments of HA (sHA), which complex with inter-α-inhibitor heavy chains (HCs) and induce inflammation and airway hyperresponsiveness (AHR). Importantly, the effects of sHA can be antagonized by the physiological counterpart high molecular weight HA (HMWHA). We used a mouse model of house dust mite-induced allergic airway inflammation and demonstrated that instilled HMWHA ameliorated allergic airway inflammation and AHR, even when given after the establishment of allergic sensitization and after challenge exposures. Furthermore, instilled HMWHA reduced the development of HA-HC complexes and the activation of Rho-associated, coiled-coil containing protein kinase 2. We conclude that airway application of HMWHA is a potential treatment for allergic airway inflammation.

Nitric oxide alters hyaluronan deposition by airway smooth muscle cells.

Asthma is a chronic inflammatory disease that is known to cause changes in the extracellular matrix, including changes in hyaluronan (HA) deposition. However, little is known about the factors that modulate its deposition or the potential consequences. Asthmatics with high levels of exhaled nitric oxide (NO) are characterized by greater airway reactivity and greater evidence of airway inflammation. Based on these data and our previous work we hypothesized that excessive NO promotes the pathologic production of HA by airway smooth muscle cells (SMCs). Exposure of cultured SMCs to various NO donors results in the accumulation of HA in the form of unique, cable-like structures. HA accumulates rapidly after exposure to NO and can be seen as early as one hour after NO treatment. The cable-like HA in NO-treated SMC cultures supports the binding of leukocytes. In addition, NO produced by murine macrophages (RAW cells) and airway epithelial cells also induces SMCs to produce HA cables when grown in co-culture. The modulation of HA by NO appears to be independent of soluble guanylate cyclase. Taken together, NO-induced production of leukocyte-binding HA by SMCs provides a new potential mechanism for the non-resolving airway inflammation in asthma and suggests a key role of non-immune cells in driving the chronic inflammation of the submucosa. Modulation of NO, HA and the consequent immune cell interactions may serve as potential therapeutic targets in asthma.

Endothelial cells in the innate response to allergens and initiation of atopic asthma.

Protease-activated receptor 2 (PAR-2), an airway epithelial pattern recognition receptor (PRR), participates in the genesis of house dust mite-induced (HDM-induced) asthma. Here, we hypothesized that lung endothelial cells and proangiogenic hematopoietic progenitor cells (PACs) that express high levels of PAR-2 contribute to the initiation of atopic asthma. HDM extract (HDME) protease allergens were found deep in the airway mucosa and breaching the endothelial barrier. Lung endothelial cells and PACs released the Th2-promoting cytokines IL-1α and GM-CSF in response to HDME, and the endothelium had PAC-derived VEGF-C-dependent blood vessel sprouting. Blockade of the angiogenic response by inhibition of VEGF-C signaling lessened the development of inflammation and airway remodeling in the HDM model. Reconstitution of the bone marrow in WT mice with PAR-2-deficient bone marrow also reduced airway inflammation and remodeling. Adoptive transfer of PACs that had been exposed to HDME induced angiogenesis and Th2 inflammation with remodeling similar to that induced by allergen challenge. Our findings identify that lung endothelium and PACs in the airway sense allergen and elicit an angiogenic response that is central to the innate nonimmune origins of Th2 inflammation.

Omalizumab can inhibit respiratory reaction during aspirin desensitization.

BACKGROUND: Aspirin desensitization has been associated with benefit in management of aspirin-exacerbated respiratory disease (AERD). An intervention that would encourage aspirin desensitization to be performed more frequently has substantial potential for improving outcomes and quality of life in patients with AERD. OBJECTIVE: We investigated whether omalizumab administration would be associated with attenuation of aspirin-provoked bronchospasm in patients with AERD undergoing aspirin desensitization. METHODS: We carried out a randomized, double-blind, placebo-controlled study in which subjects with AERD who fulfilled label criteria for omalizumab received omalizumab or placebo for 16 weeks, and then underwent aspirin desensitization. RESULTS: Eleven subjects completed aspirin desensitization. Of the 7 who were randomized to omalizumab, 5 had no respiratory reaction during aspirin desensitization. Compared with placebo, omalizumab was associated with a significantly greater likelihood for subjects with AERD to have no respiratory reaction during desensitization (P = .04, Fisher exact test). There was an overall difference in urinary leukotriene E4 (LTE4) levels in subjects who received omalizumab and did not have a respiratory reaction during desensitization compared with subjects randomized to placebo (P = .035, mixed model with interaction). Urinary LTE4 levels were significantly higher with respiratory reaction in placebo subjects compared with levels obtained after the 100-mg dose in AERD subjects who had no respiratory reaction (P < .001, mixed model with interaction). CONCLUSION: In atopic AERD subjects, omalizumab administration for 16 weeks was associated with "clinically silent" desensitization. Further studies to investigate the therapeutic utility of omalizumab in patients with AERD who are candidates for aspirin desensitization are warranted based on these findings. TRIAL REGISTRATION: ClinicalTrials.gov Identifier NCT00555971.

IL-17-receptor-associated adaptor Act1 directly stabilizes mRNAs to mediate IL-17 inflammatory signaling.

Mechanisms that degrade inflammatory mRNAs are well known; however, stabilizing mechanisms are poorly understood. Here, we show that Act1, an interleukin-17 (IL-17)-receptor-complex adaptor, binds and stabilizes mRNAs encoding key inflammatory proteins. The Act1 SEFIR domain binds a stem-loop structure, the SEFIR-binding element (SBE), in the 3' untranslated region (UTR) of Cxcl1 mRNA, encoding an inflammatory chemokine. mRNA-bound Act1 directs formation of three compartmentally distinct RNA-protein complexes (RNPs) that regulate three disparate events in inflammatory-mRNA metabolism: preventing mRNA decay in the nucleus, inhibiting mRNA decapping in P bodies and promoting translation. SBE RNA aptamers decreased IL-17-mediated mRNA stabilization in vitro, IL-17-induced skin inflammation and airway inflammation in a mouse asthma model, thus providing a therapeutic strategy for autoimmune diseases. These results reveal a network in which Act1 assembles RNPs on the 3' UTRs of select mRNAs and consequently controls receptor-mediated mRNA stabilization and translation during inflammation.

Lung-Infiltrating Foxp3+ Regulatory T Cells Are Quantitatively and Qualitatively Different during Eosinophilic and Neutrophilic Allergic Airway Inflammation but Essential To Control the Inflammation.

Understanding functions of Foxp3+ regulatory T cells (Tregs) during allergic airway inflammation remains incomplete. In this study, we report that, during cockroach Ag-induced allergic airway inflammation, Foxp3+ Tregs are rapidly mobilized into the inflamed lung tissues. However, the level of Treg accumulation in the lung was different depending on the type of inflammation. During eosinophilic airway inflammation, ∼30% of lung-infiltrating CD4 T cells express Foxp3, indicative of Tregs. On the contrary, only ∼10% of infiltrating CD4 T cells express Foxp3 during neutrophilic airway inflammation. Despite the different accumulation, the lung inflammation and inflammatory T cell responses were aggravated following Treg depletion, regardless of the type of inflammation, suggesting regulatory roles for Tregs. Interestingly, however, the extent to which inflammatory responses are aggravated by Treg depletion was significantly greater during eosinophilic airway inflammation. Indeed, lung-infiltrating Tregs exhibit phenotypic and functional features associated with potent suppression. Our results demonstrate that Tregs are essential regulators of inflammation, regardless of the type of inflammation, although the mechanisms used by Tregs to control inflammation may be shaped by environmental cues available to them.

TNF-stimulated gene 6 promotes formation of hyaluronan-inter-α-inhibitor heavy chain complexes necessary for ozone-induced airway hyperresponsiveness.

Exposure to pollutants, such as ozone, exacerbates airway inflammation and hyperresponsiveness (AHR). TNF-stimulated gene 6 (TSG-6) is required to transfer inter-α-inhibitor heavy chains (HC) to hyaluronan (HA), facilitating HA receptor binding. TSG-6 is necessary for AHR in allergic asthma, because it facilitates the development of a pathological HA-HC matrix. However, the role of TSG-6 in acute airway inflammation is not well understood. Here, we hypothesized that TSG-6 is essential for the development of HA- and ozone-induced AHR. TSG-6-/- and TSG-6+/+ mice were exposed to ozone or short-fragment HA (sHA), and AHR was assayed via flexiVent. The AHR response to sHA was evaluated in the isolated tracheal ring assay in tracheal rings from TSG-6-/- or TSG-6+/+, with or without the addition of exogenous TSG-6, and with or without inhibitors of Rho-associated, coiled-coil-containing protein kinase (ROCK), ERK, or PI3K. Smooth-muscle cells from mouse tracheas were assayed in vitro for signaling pathways. We found that TSG-6 deficiency protects against AHR after ozone (in vivo) or sHA (in vitro and in vivo) exposure. Moreover, TSG-6-/- tracheal ring non-responsiveness to sHA was reversed by exogenous TSG-6 addition. sHA rapidly activated RhoA, ERK, and Akt in airway smooth-muscle cells, but only in the presence of TSG-6. Inhibition of ROCK, ERK, or PI3K/Akt blocked sHA/TSG-6-mediated AHR. In conclusion, TSG-6 is necessary for AHR in response to ozone or sHA, in part because it facilitates rapid formation of HA-HC complexes. The sHA/TSG-6 effect is mediated by RhoA, ERK, and PI3K/Akt signaling.

The flavonoid cyanidin blocks binding of the cytokine interleukin-17A to the IL-17RA subunit to alleviate inflammation in vivo.

Cyanidin, a key flavonoid that is present in red berries and other fruits, attenuates the development of several diseases, including asthma, diabetes, atherosclerosis, and cancer, through its anti-inflammatory effects. We investigated the molecular basis of cyanidin action. Through a structure-based search for small molecules that inhibit signaling by the proinflammatory cytokine interleukin-17A (IL-17A), we found that cyanidin specifically recognizes an IL-17A binding site in the IL-17A receptor subunit (IL-17RA) and inhibits the IL-17A/IL-17RA interaction. Experiments with mice demonstrated that cyanidin inhibited IL-17A-induced skin hyperplasia, attenuated inflammation induced by IL-17-producing T helper 17 (TH17) cells (but not that induced by TH1 or TH2 cells), and alleviated airway hyperreactivity in models of steroid-resistant and severe asthma. Our findings uncover a previously uncharacterized molecular mechanism of action of cyanidin, which may inform its further development into an effective small-molecule drug for the treatment of IL-17A-dependent inflammatory diseases and cancer.

Eosinophil Peroxidase Catalyzed Protein Carbamylation Participates in Asthma.

The biochemical mechanisms through which eosinophils contribute to asthma pathogenesis are unclear. Here we show eosinophil peroxidase (EPO), an abundant granule protein released by activated eosinophils, contributes to characteristic asthma-related phenotypes through oxidative posttranslational modification (PTM) of proteins in asthmatic airways through a process called carbamylation. Using a combination of studies we now show EPO uses plasma levels of the pseudohalide thiocyanate (SCN-) as substrate to catalyze protein carbamylation, as monitored by PTM of protein lysine residues into Nϵ-carbamyllysine (homocitrulline), and contributes to the pathophysiological sequelae of eosinophil activation. Studies using EPO-deficient mice confirm EPO serves as a major enzymatic source for protein carbamylation during eosinophilic inflammatory models, including aeroallergen challenge. Clinical studies similarly revealed significant enrichment in carbamylation of airway proteins recovered from atopic asthmatics versus healthy controls in response to segmental allergen challenge. Protein-bound homocitrulline is shown to be co-localized with EPO within human asthmatic airways. Moreover, pathophysiologically relevant levels of carbamylated protein either incubated with cultured human airway epithelial cells in vitro, or provided as an aerosolized exposure in non-sensitized mice, induced multiple asthma-associated phenotypes including induction of mucin, Th2 cytokines, IFNγ, TGFß, and epithelial cell apoptosis. Studies with scavenger receptor-A1 null mice reveal reduced IL-13 generation following exposure to aerosolized carbamylated protein, but no changes in other asthma-related phenotypes. In summary, EPO-mediated protein carbamylation is promoted during allergen-induced asthma exacerbation, and can both modulate immune responses and trigger a cascade of many of the inflammatory signals present in asthma.

Soluble guanylate cyclase as an alternative target for bronchodilator therapy in asthma.

Asthma is defined by airway inflammation and hyperresponsiveness, and contributes to morbidity and mortality worldwide. Although bronchodilation is a cornerstone of treatment, current bronchodilators become ineffective with worsening asthma severity. We investigated an alternative pathway that involves activating the airway smooth muscle enzyme, soluble guanylate cyclase (sGC). Activating sGC by its natural stimulant nitric oxide (NO), or by pharmacologic sGC agonists BAY 41-2272 and BAY 60-2770, triggered bronchodilation in normal human lung slices and in mouse airways. Both BAY 41-2272 and BAY 60-2770 reversed airway hyperresponsiveness in mice with allergic asthma and restored normal lung function. The sGC from mouse asthmatic lungs displayed three hallmarks of oxidative damage that render it NO-insensitive, and identical changes to sGC occurred in human lung slices or in human airway smooth muscle cells when given chronic NO exposure to mimic the high NO in asthmatic lung. Our findings show how allergic inflammation in asthma may impede NO-based bronchodilation, and reveal that pharmacologic sGC agonists can achieve bronchodilation despite this loss.

Subepithelial Accumulation of Versican in a Cockroach Antigen-Induced Murine Model of Allergic Asthma.

The extracellular matrix (ECM) is an important contributor to the asthmatic phenotype. Recent studies investigating airway inflammation have demonstrated an association between hyaluronan (HA) accumulation and inflammatory cell infiltration of the airways. The ECM proteoglycan versican interacts with HA and is important in the recruitment and activation of leukocytes during inflammation. We investigated the role of versican in the pathogenesis of asthmatic airway inflammation. Using cockroach antigen (CRA)-sensitized murine models of allergic asthma, we demonstrate increased subepithelial versican in the airways of CRA-treated mice that parallels subepithelial increases in HA and leukocyte infiltration. During the acute phase, CRA-treated mice displayed increased gene expression of the four major versican isoforms, as well as increased expression of HA synthases. Furthermore, in a murine model that examines both acute and chronic CRA exposure, versican staining peaked 8 days following CRA challenge and preceded subepithelial leukocyte infiltration. We also assessed versican and HA expression in differentiated primary human airway epithelial cells from asthmatic and healthy children. Increases in the expression of versican isoforms and HA synthases in these epithelial cells were similar to those of the murine model. These data indicate an important role for versican in the establishment of airway inflammation in asthma.

The Rise and Fall of Hyaluronan in Respiratory Diseases.

In normal airways, hyaluronan (HA) matrices are primarily located within the airway submucosa, pulmonary vasculature walls, and, to a lesser extent, the alveoli. Following pulmonary injury, elevated levels of HA matrices accumulate in these regions, and in respiratory secretions, correlating with the extent of injury. Animal models have provided important insight into the role of HA in the onset of pulmonary injury and repair, generally indicating that the induction of HA synthesis is an early event typically preceding fibrosis. The HA that accumulates in inflamed airways is of a high molecular weight (>1600 kDa) but can be broken down into smaller fragments (<150 kDa) by inflammatory and disease-related mechanisms that have profound effects on HA pathobiology. During inflammation in the airways, HA is often covalently modified with heavy chains from inter-alpha-inhibitor via the enzyme tumor-necrosis-factor-stimulated-gene-6 (TSG-6) and this modification promotes the interaction of leukocytes with HA matrices at sites of inflammation. The clearance of HA and its return to normal levels is essential for the proper resolution of inflammation. These data portray HA matrices as an important component of normal airway physiology and illustrate its integral roles during tissue injury and repair among a variety of respiratory diseases.

Hyaluronan and Its Heavy Chain Modification in Asthma Severity and Experimental Asthma Exacerbation.

Hyaluronan (HA) is a large (>1500 kDa) polysaccharide of the extracellular matrix that has been linked to severity and inflammation in asthma. During inflammation, HA becomes covalently modified with heavy chains (HC-HA) from inter-α-inhibitor (IαI), which functions to increase its avidity for leukocytes. Our murine model of allergic pulmonary inflammation suggested that HC-HA may contribute to inflammation, adversely effecting lower airway remodeling and asthma severity. Our objective was to characterize the levels of HA and HC-HA in asthmatic subjects and to correlate these levels with asthma severity. We determined the levels and distribution of HA and HC-HA (i) from asthmatic and control lung tissue, (ii) in bronchoalveolar lavage fluid obtained from non-severe and severe asthmatics and controls, and (iii) in serum and urine from atopic asthmatics after an experimental asthma exacerbation. HC-HA distribution was observed (i) in the thickened basement membrane of asthmatic lower airways, (ii) around smooth muscle cells of the asthmatic submucosa, and (iii) around reserve cells of the asthmatic epithelium. Patients with severe asthma had increased HA levels in bronchoalveolar lavage fluid that correlated with pulmonary function and nitric oxide levels, whereas HC-HA was only observed in a patient with non-severe asthma. After an experimental asthma exacerbation, serum HA was increased within 4 h after challenge and remained elevated through 5 days after challenge. Urine HA and HC-HA were not significantly different. These data implicate HA and HC-HA in the pathogenesis of asthma severity that may occur in part due to repetitive asthma exacerbations over the course of the disease.