An Investigation of Organizational Correlates of Distress in Non-Clinician Biomedical Researchers in the United States.

Purpose: Challenges ushered by the COVID-19 pandemic led to an increased focus on the mental well-being of the healthcare workforce. Despite the important contribution non-clinician biomedical researchers make to the mission of academic medical centers, the well-being of this unique population remains understudied in the United States. The purpose of this study was to examine the individual and organizational correlates of distress among non-clinician biomedical researchers. Methods: A survey was delivered to employees of a large academic medical center in the southeastern United States, including non-clinician biomedical researchers. Participants were asked to assess their own well-being using the validated Well-Being Index (WBI) tool, resilience, work and nonwork-related stressors and demographic descriptors. Descriptive statistics and bivariate analyses were conducted, and binary logistic regression was used to examine predictors of increased odds of overall distress. Results: Nearly 44% of surveyed non-clinician biomedical researchers met the threshold for high distress which indicates an increased risk of suicidal ideation, turnover intention, and burnout. The major correlates of distress were at the organizational level, including perceived organizational support (OR 0.79, 95% CI 0.70-0.90), heavy workload and long hours (OR 3.25, 95% CI 1.53-6.88), inability or lack of support to take time off (OR 2.80, 95% CI 1.03-7.66) and conflict with supervisor (OR 5.03, 95% CI 1.13-22.1). While lower individual resilience (OR 0.69, 95% CI 0.54-0.88) was statistically significantly associated with greater distress, it accounted for less than 10% of the overall variance when controlling for other work-related factors. Conclusion: These findings suggest that developing organizational interventions that address institutional support for non-clinician biomedical researchers within academic medical centers represents an important opportunity to reduce distress within this population. While emphasizing individual resiliency as an important in the pursuit of well-being, it is also the responsibility of the organization to create and foster an environment in which employees can access their own resilience.

Therapeutic potential of an orally effective small molecule inhibitor of plasminogen activator inhibitor for asthma.

Asthma is one of the most common respiratory diseases. Although progress has been made in our understanding of airway pathology and many drugs are available to relieve asthma symptoms, there is no cure for chronic asthma. Plasminogen activator inhibitor 1 (PAI-1), a primary inhibitor of tissue-type and urokinase-type plasminogen activators, has pleiotropic functions besides suppression of fibrinolysis. In this study, we show that administration of TM5275, an orally effective small-molecule PAI-1 inhibitor, 25 days after ovalbumin (OVA) sensitization-challenge, significantly ameliorated airway hyperresponsiveness in an OVA-induced chronic asthma model. Furthermore, we show that TM5275 administration significantly attenuated OVA-induced infiltration of inflammatory cells (neutrophils, eosinophils, and monocytes), the increase in the levels of OVA-specific IgE and Th2 cytokines (IL-4 and IL-5), the production of mucin in the airways, and airway subepithelial fibrosis. Together, the results suggest that the PAI-1 inhibitor TM5275 may have therapeutic potential for asthma through suppressing eosinophilic allergic response and ameliorating airway remodeling.

Exposure to cigarette smoke impacts myeloid-derived regulatory cell function and exacerbates airway hyper-responsiveness.

Cigarette smoking enhances oxidative stress and airway inflammation in asthma, the mechanisms of which are largely unknown. Myeloid-derived regulatory cells (MDRC) are free radical producing immature myeloid cells with immunoregulatory properties that have recently been demonstrated as critical regulators of allergic airway inflammation. NO (nitric oxide)-producing immunosuppressive MDRC suppress T-cell proliferation and airway-hyper responsiveness (AHR), while the O2(•-) (superoxide)-producing MDRC are proinflammatory. We hypothesized that cigarette smoke (CS) exposure may impact MDRC function and contribute to exacerbations in asthma. Exposure of bone marrow (BM)-derived NO-producing MDRC to CS reduced the production of NO and its metabolites and inhibited their potential to suppress T-cell proliferation. Production of immunoregulatory cytokine IL-10 was significantly inhibited, while proinflammatory cytokines IL-6, IL-1ß, TNF-α and IL-33 were enhanced in CS-exposed BM-MDRC. Additionally, CS exposure increased NF-κB activation and induced BM-MDRC-mediated production of O2(•-), via NF-κB-dependent pathway. Intratracheal transfer of smoke-exposed MDRC-producing proinflammatory cytokines increased NF-κB activation, reactive oxygen species and mucin production in vivo and exacerbated AHR in C57BL/6 mice, mice deficient in Type I IFNR and MyD88, both with reduced numbers of endogenous MDRC. Thus CS exposure modulates MDRC function and contributes to asthma exacerbation and identifies MDRC as potential targets for asthma therapy.

Development and maintenance of a biospecimen repository for clinical samples derived from pulmonary patients.

The Pulmonary Biospecimen Repository (PBR) at the University of Alabama at Birmingham (UAB) was launched in 2009. The purpose of the UAB PBR is to provide investigators within the pulmonary community at UAB and elsewhere with clinical samples derived from multiple lung diseases, including transplant recipients, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, cystic fibrosis, and asthma. Cell and fluid samples isolated from bronchoalveolar lavage (BAL), plasma, and serum are collected and stored; samples are assessed routinely for viability. Each sample is linked directly with the respective patient information via the Pulmonary Translational Research and Clinical Database, a Health Insurance Portability and Accountability Act compliant database that includes detailed information allowing for the study of specific patient cohorts. To access samples, investigators must complete a request form, which is reviewed by the UAB PBR Steering Committee. To date, more than 800 patients have provided approximately 7,000 BAL, serum and plasma fluid, and cell samples. Over the past 4 years, nearly 800 of these samples have been distributed to investigators at UAB and elsewhere. Future plans for the UAB PBR include expanding sample collection to additional pulmonary diseases, such as mycobacterial infections, increasing the number of sample users and obtaining external funding to ensure its continued sustainability.

Deletion of airway cilia results in noninflammatory bronchiectasis and hyperreactive airways.

The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.

Use of the cockroach antigen model of acute asthma to determine the immunomodulatory role of early exposure to gastrointestinal infection.

The increased incidence of asthma over the last 50 years in developed countries has been associated with a decrease in infections acquired early in childhood. These early infections are thought to shape subsequent immune responses. Although there have been multiple clinical associations between gastrointestinal infections and decreased asthma incidence, it has been difficult to move beyond a simple correlation when studying human patients. This section describes an acute asthma model in C57BL/6 mice designed to specifically evaluate the effect of prior gastric Helicobacter colonization and inflammation in a murine model of cockroach allergen-induced asthma.

Moderate aerobic exercise alters migration patterns of antigen specific T helper cells within an asthmatic lung.

Studies have indicated increased incidence and severity of allergic asthma due to western lifestyle and increased sedentary activity. Investigations also indicate that exercise reduces the severity of asthma; however, a mechanism of action has not been elucidated. Additional work implicates re-distribution of T helper (Th) cells in mediating alterations of the immune system as a result of moderate aerobic exercise in vivo. We have previously reported that exercise decreases T helper 2 (Th2) responses within the lungs of an ovalbumin (OVA)-sensitized murine allergic asthma model. Therefore, we hypothesized that exercise alters the migration of OVA-specific Th cells in an OVA-challenged lung. To test this hypothesis, wildtype mice received OVA-specific Th cells expressing a luciferase-reporter construct and were OVA-sensitized and exercised. OVA-specific Th cell migration was decreased in OVA-challenged lungs of exercised mice when compared to their sedentary controls. Surface expression levels of lung-homing chemokine receptors, CCR4 and CCR8, on Th cells and their cognate lung-homing chemokine gradients revealed no difference between exercised and sedentary OVA-sensitized mice. However, transwell migration experiments demonstrated that lung-derived Th cells from exercised OVA-sensitized mice exhibited decreased migratory function versus controls. These data suggest that Th cells from exercised mice are less responsive to lung-homing chemokine. Together, these studies demonstrate that moderate aerobic exercise training can reduce the accumulation of antigen-specific Th cell migration into an asthmatic lung by decreasing chemokine receptor function.

Site-specific dynamics of CD11b+ and CD103+ dendritic cell accumulations following ozone exposure.

Pulmonary dendritic cells (DCs) are among the first responders to inhaled environmental stimuli such as ozone (O(3)), which has been shown to activate these cells. O(3) reacts with epithelial lining fluid (ELF) components in an anatomically site-specific manner dictated by O(3) concentration, airway flow patterns, and ELF substrate concentration. Accordingly, the anatomical distribution of ELF reaction products and airway injury are hypothesized to produce selective DC maturation differentially within the airways. To investigate how O(3) affects regional airway DC populations, we utilized a model of O(3)-induced pulmonary inflammation, wherein C57BL/6 mice were exposed to 0.8 ppm O(3) 8 h/day for 1, 3, and 5 days. This model induced mild inflammation and no remarkable epithelial injury. Tracheal, but not more distant airway sites, and mediastinal lymph node (MLN) DC numbers were increased significantly after the third exposure day. The largest increase in each tissue was of the CD103(+) DC phenotype. After 3 days of exposure, fewer DCs expressed CD80, CD40, and CCR7, and, at this same time point, total MLN T cell numbers increased. Together, these data demonstrate that O(3) exposure induced site-specific and phenotype changes in the pulmonary and regional lymph node DC populations. Possibly contributing to ozone-mediated asthma perturbation, the phenotypic changes to DCs within pulmonary regions may alter responses to antigenic stimuli. Decreased costimulatory molecule expression within the MLN suggests induction of tolerance mechanisms; increased tracheal DC number may raise the potential for allergic sensitization and asthmatic exacerbation, thus overcoming O(3)-induced decrements in costimulatory molecule expression.

The ΔF508-CFTR mutation inhibits wild-type CFTR processing and function when co-expressed in human airway epithelia and in mouse nasal mucosa.

BACKGROUND: Rescue or correction of CFTR function in native epithelia is the ultimate goal of CF therapeutics development. Wild-type (WT) CFTR introduction and replacement is also of particular interest. Such therapies may be complicated by possible CFTR self-assembly into an oligomer or multimer. RESULTS: Surprisingly, functional CFTR assays in native airway epithelia showed that the most common CFTR mutant, ΔF508-CFTR (ΔF-CFTR), inhibits WT-CFTR when both forms are co-expressed. To examine more mechanistically, both forms of CFTR were transfected transiently in varying amounts into IB3-1 CF human airway epithelial cells and HEK-293 human embryonic kidney cells null for endogenous CFTR protein expression. Increasing amounts of ΔF-CFTR inhibited WT-CFTR protein processing and function in CF human airway epithelial cells but not in heterologous HEK-293 cells. Stably expressed ΔF-CFTR in clones of the non-CF human airway epithelial cell line, CALU-3, also showed reduction in cAMP-stimulated anion secretion and in WT-CFTR processing. An ultimate test of this dominant negative-like effect of ΔF-CFTR on WT-CFTR was the parallel study of two different CF mouse models: the ΔF-CFTR mouse and the bitransgenic CFTR mouse corrected in the gut but null in the lung and airways. WT/ΔF heterozygotes had an intermediate phenotype with regard to CFTR agonist responses in in vivo nasal potential difference (NPD) recordings and in Ussing chamber recordings of short-circuit current (ISC) in vitro on primary tracheal epithelial cells isolated from the same mice. In contrast, CFTR bitransgenic +/- heterozygotes had no difference in their responses versus +/+ wild-type mice. CONCLUSIONS: Taken altogether, these data suggest that ΔF-CFTR and WT-CFTR co-assemble into an oligomeric macromolecular complex in native epithelia and share protein processing machinery and regulation at the level of the endoplasmic reticulum (ER). As a consequence, ΔF-CFTR slows WT-CFTR protein processing and limits its expression and function in the apical membrane of native airway epithelia. Implications of these data for the relative health of CF heterozygous carriers, for CFTR protein processing in native airway epithelia, and for the relative efficacy of different CF therapeutic approaches is significant and is discussed.

Feasibility of exercising adults with asthma: a randomized pilot study.

BACKGROUND: Aerobic exercise appears to have clinical benefits for many asthmatics, yet a complete understanding of the mechanisms underlying these benefits has not been elucidated at this time. PURPOSE: The objective of this study was to determine feasibility for a larger, future study that will define the effect of aerobic exercise on cellular, molecular, and functional measures in adults with mild-moderate asthma. DESIGN: Recruited subjects were randomized into usual care (sedentary) or usual care with moderate intensity aerobic exercise treatment groups. SETTING / PARTICIPANTS: Nineteen adults with mild-moderate asthma but without a recent history of exercise were recruited at the UAB Lung Health Center, Birmingham, AL. INTERVENTION: The exercise group underwent a 12 week walking program exercising at 60 - 75% of maximum heart rate (HRmax). Subjects self-monitored HRmax levels using heart rate monitors; exercise diaries and recreation center sign-in logs were also used. MAIN OUTCOME MEASURES: Functional measures, including lung function and asthma control scores, were evaluated for all subjects at pre- and post-study time-points; fitness measures were also assessed for subjects in the exercise group. Peripheral blood and nasal lavage fluid were collected from all subjects at pre- and post-study visits in order to evaluate cellular and molecular measures, including cell differentials and eosinophilic cationic protein (ECP). RESULTS: Sixteen subjects completed the prescribed protocol. Results show that subjects randomized to the exercise group adhered well (80%) to the exercise prescription and exhibited a trend toward improved fitness levels upon study completion. Both groups exhibited improvements in ACQ scores. No changes were observed in lung function (FEV1, FEV1/FVC), cell differentials, or ECP between groups. CONCLUSIONS: Results indicate that a moderate intensity aerobic exercise training program may improve asthma control and fitness levels without causing asthma deterioration in adult asthmatics. As such, these findings demonstrate the feasibility of the study protocol in preparation for a larger, clinical trial that will elucidate the functional consequences of aerobic exercise on asthmatic cellular and molecular responses.

The ß-glucan receptor dectin-1 promotes lung immunopathology during fungal allergy via IL-22.

Sensitization to fungi, such as the mold Aspergillus fumigatus, is increasingly becoming linked with asthma severity. We have previously shown that lung responses generated via the ß-glucan receptor Dectin-1 are required for lung defense during acute, invasive A. fumigatus infection. Unexpectedly, in an allergic model of chronic lung exposure to live A. fumigatus conidia, ß-glucan recognition via Dectin-1 led to the induction of multiple proallergic (Muc5ac, Clca3, CCL17, CCL22, and IL-33) and proinflammatory (IL-1ß and CXCL1) mediators that compromised lung function. Attenuated proallergic and proinflammatory responses in the absence of Dectin-1 were not associated with changes in Ido (IDO), Il12p35/Ebi3 (IL-35), IL-10, or TGF-ß levels. Assessment of Th responses demonstrated that purified lung CD4(+) T cells produced IL-4, IL-13, IFN-γ, and IL-17A, but not IL-22, in a Dectin-1-dependent manner. In contrast, we observed robust, Dectin-1-dependent IL-22 production by unfractionated lung digest cells. Intriguingly, the absence of IL-22 alone mimicked the attenuated proallergic and proinflammatory responses observed in the absence of Dectin-1, suggesting that Dectin-1-mediated IL-22 production potentiated responses that led to decrements in lung function. To this end, neutralization of IL-22 improved lung function in normal mice. Collectively, these results indicate that the ß-glucan receptor Dectin-1 contributes to lung inflammation and immunopathology associated with persistent fungal exposure via the production of IL-22.

Postexposure administration of a {beta}2-agonist decreases chlorine-induced airway hyperreactivity in mice.

Exposure to chlorine (Cl(2)) damages airway and alveolar epithelia, resulting in acute lung injury and reactive airway dysfunction syndrome. We evaluated the efficacy and mechanisms by which arformoterol, a long-term ß(2)-agonist, administered after exposure, mitigated the extent of this injury. Exposure of C57BL/6 mice to 400 ppm Cl(2) for 30 minutes increased respiratory system resistance and airway responsiveness to aerosolized methacholine (assessed by FlexiVent) up to 6 days after exposure, and decreased Na(+)-dependent alveolar fluid clearance (AFC). Inducible Nitric Oxide Synthase (iNOS) knockout mice developed similar degrees of airway hyperreactivity as wild-type controls after Cl(2) exposure, indicating that reactive intermediates from iNOS do not contribute to Cl(2)-induced airway dysfunction in our model. Intranasal administration of arformoterol mitigated the Cl(2) effects on airway reactivity and AFC, presumably by increasing lung cyclic AMP level. Arformoterol did not modify the inflammatory responses, as evidenced by the number of inflammatory cells and concentrations of IL-6 and TNF-α in the bronchoalveolar lavage. NF-κB activity (assessed by p65 Western blots and electrophoretic mobility shift assay) remained at control levels up to 24 hours after Cl(2) exposure. Our results provide mechanistic insight into the effectiveness of long-term ß(2)-agonists in reversing Cl(2)-induced reactive airway dysfunction syndrome and injury to distal lung epithelial cells.

Repeated bouts of aerobic exercise enhance regulatory T cell responses in a murine asthma model.

We have reported previously that moderate intensity aerobic exercise training attenuates airway inflammation in a murine asthma model. Recent studies implicate regulatory T (Treg) cells in decreasing asthma-related airway inflammation; as such, the current study examined the effect of exercise on Treg cell function in a murine asthma model. Mice were sensitized with ovalbumin (OVA) prior to the start of exercise training at a moderate intensity 3x/week for 4weeks; exercise was performed as treadmill running (13.5m/min, 0% grade). Mice were OVA challenged repeatedly throughout the exercise protocol. At protocol completion, mice were analyzed for changes in the number and suppressive function of CD4(+)CD25(+)Foxp3(+) cells isolated from lungs, mediastinal lymph nodes, and spleens. Results show that exercise increased significantly the number of Foxp3(+) cells within the lungs and mediastinal lymph nodes, but not the spleens, of OVA-treated mice as compared with sedentary controls. Exercise also enhanced the suppression function of CD4(+)CD25(+)Foxp3(+) Treg cells derived from OVA-treated mice as compared with sedentary controls. Specifically, Treg cells from exercised, OVA-treated mice more effectively suppressed CD4(+)CD25(-) cell proliferation and Th2 cytokine production in vitro. Enhanced suppression was associated with increased protein levels of TGF-beta and lesser amounts of IL-10 and IL-17; however, blocking TGF-beta had no effect on suppressive functions. These data demonstrate that exercise-mediated increases in Treg cell function may play a role in the attenuation of airway inflammation. Further, these results indicate that moderate intensity aerobic exercise training may alter the Treg cell function within the asthmatic airway.

Repeated bouts of moderate-intensity aerobic exercise reduce airway reactivity in a murine asthma model.

We have reported that moderate-intensity aerobic exercise training attenuates airway inflammation in mice sensitized/challenged with ovalbumin (OVA). The current study determined the effects of repeated bouts of aerobic exercise at a moderate intensity on airway hyperresponsiveness (AHR) in these mice. Mice were sensitized/challenged with OVA or saline and exercised at a moderate intensity 3 times/week for 4 weeks. At protocol completion, mice were analyzed for changes in AHR via mechanical ventilation. Results show that exercise decreased total lung resistance 60% in OVA-treated mice as compared with controls; exercise also decreased airway smooth muscle (ASM) thickness. In contrast, exercise increased circulating epinephrine levels 3-fold in saline- and OVA-treated mice. Because epinephrine binds beta(2)-adrenergic receptors (AR), which facilitate bronchodilatation, the role of beta(2)-AR in exercise-mediated improvements in AHR was examined. Application of the beta(2)-AR antagonist butoxamine HCl blocked the effects of exercise on lung resistance in OVA-treated mice. In parallel, ASM cells were examined for changes in the protein expression of beta(2)-AR and G-protein receptor kinase-2 (GRK-2); GRK-2 promotes beta(2)-AR desensitization. Exercise had no effect on beta(2)-AR expression in ASM cells of OVA-treated mice; however, exercise decreased GRK-2 expression by 50% as compared with controls. Exercise also decreased prostaglandin E(2) (PGE(2)) production 5-fold, but had no effect on E prostanoid-1 (EP1) receptor expression within the lungs of OVA-treated mice; both PGE(2) and the EP1 receptor have been implicated in beta(2)-AR desensitization. Together, these data indicate that moderate-intensity aerobic exercise training attenuates AHR via a mechanism that involves beta(2)-AR.

Epithelial cells as immune effector cells: the role of CD40.

Through the expression of inflammatory mediators and immune-related molecules, epithelial cells function as immune effector cells in a wide variety of tissues; the expression of the CD40 receptor on these cells contributes this role. Engagement of CD40 activates epithelial cells and results in their release of pro- and anti-inflammatory mediators as well as pro-fibrotic molecules. As such, epithelial CD40 has been implicated in the pathogenesis of inflammatory disorders, generation of self-tolerance, and rejection of allografts.

Requisite role for the dectin-1 beta-glucan receptor in pulmonary defense against Aspergillus fumigatus.

Immune suppression increases the incidence of invasive fungal infections, particularly those caused by the opportunistic mold Aspergillus fumigatus. Previous investigations revealed that members of the TLR family are not absolutely required for host defense against A. fumigatus in nonimmunosuppressed hosts, suggesting that other pattern recognition receptors are involved. We show in this study that naive mice (i.e., not pharmacologically immunosuppressed) lacking the beta-glucan receptor Dectin-1 (Dectin-1(-/-)) are more sensitive to intratracheal challenge with A. fumigatus than control mice, exhibiting >80% mortality within 5 days, ultimately attributed to a compromise in respiratory mechanics. In response to A. fumigatus challenge, Dectin-1(-/-) mice demonstrated impaired IL-1alpha, IL-1beta, TNF-alpha, CCL3/MIP-1alpha, CCL4/MIP-1beta, and CXCL1/KC production, which resulted in insufficient lung neutrophil recruitment and uncontrolled A. fumigatus lung growth. Alveolar macrophages from Dectin-1(-/-) mice failed to produce proinflammatory mediators in response to A. fumigatus, whereas neutrophils from Dectin-1(-/-) mice had impaired reactive oxygen species production and impaired killing of A. fumigatus. We further show that IL-17 production in the lung after A. fumigatus challenge was Dectin-1 dependent, and that neutralization of IL-17 significantly impaired A. fumigatus clearance. Collectively, these results support a requisite role for Dectin-1 in in vivo defense against A. fumigatus.

Loss of Thy-1 inhibits alveolar development in the newborn mouse lung.

Transforming growth factor (TGF)-beta mediates hypoxia-induced inhibition of alveolar development in the newborn lung. TGF-beta is regulated primarily at the level of activation of latent TGF-beta. Fibroblasts expressing Thy-1 (CD90) inhibit TGF-beta activation. We hypothesized that loss of Thy-1 due to hypoxia may be a mechanism by which hypoxia increases TGF-beta activation and that animals deficient in Thy-1 will simulate the effects of hypoxia on lung development. To determine if loss of Thy-1 occurred during hypoxia, non-transgenic (C57BL/6) wild-type (WT) mice exposed to hypoxia were evaluated for Thy-1 mRNA and protein. To determine if Thy-1 deficiency simulated hypoxia, WT and Thy-1 null (Thy-1(-/-)) mice were exposed to air or hypoxia from birth to 2 wk, the critical period of lung development, and lung histology, function, parameters related to TGF-beta signaling, and extracellular matrix protein content were measured. To test if the phenotype in Thy-1(-/-) mice was due to excessive TGF-beta signaling, measurements were also performed in Thy-1(-/-) mice administered TGF-beta neutralizing antibody (1D11). We observed that hypoxia reduced Thy-1 mRNA and Thy-1 staining in WT mice. Thy-1(-/-) mice had impaired alveolarization, increased TGF-beta signaling, reduced lung epithelial and endothelial cell proliferation but increased fibroblast proliferation, and increased collagen and elastin. Lung compliance was lower, and tissue but not airway resistance was higher in Thy-1(-/-) mice at 2 wk. Thy-1(-/-) mice given 1D11 had improved alveolar development and lung function. These data support the hypothesis that hypoxia, by reducing Thy-1, increases TGF-beta activation, and thereby inhibits normal alveolar development.

Acute exercise decreases airway inflammation, but not responsiveness, in an allergic asthma model.

Previous studies have suggested that the asthmatic responses of airway inflammation, remodeling, and hyperresponsiveness (AHR) are interrelated; in this study, we used exercise to examine the nature of this interrelationship. Mice were sensitized and challenged with ovalbumin (OVA); mice were then exercised via running on a motorized treadmill at a moderate intensity. Data indicate that, within the lungs of OVA-treated mice, exercise attenuated the production of inflammatory mediators, including chemokines KC, RANTES, and MCP-1 and IL-12p40/p80. Coordinately, OVA-treated and exercised mice displayed decreases in leukocyte infiltration, including eosinophils, as compared with sedentary controls. Results also show that a single bout of exercise significantly decreased phosphorylation of the NFkappaB p65 subunit, which regulates the gene expression of a wide variety of inflammatory mediators. In addition, OVA-treated and exercised mice exhibited decreases in the levels of Th2-derived cytokines IL-5 and IL-13 and the prostaglandin PGE(2), as compared with sedentary controls. In contrast, results show that a single bout of exercise had no effect on AHR in OVA-treated mice challenged with increasing doses of aerosolized methacholine (0-50 mg/ml) as compared with sedentary mice. Exercise also had no effect on epithelial cell hypertrophy, mucus production, or airway wall thickening in OVA-treated mice as compared with sedentary controls. These findings suggest that a single bout of aerobic exercise at a moderate intensity attenuates airway inflammation but not AHR or airway remodeling in OVA-treated mice. The implication of these findings for the interrelationship between airway inflammation, airway remodeling, and AHR is discussed.

SNO-hemoglobin is not essential for red blood cell-dependent hypoxic vasodilation.

The coupling of hemoglobin sensing of physiological oxygen gradients to stimulation of nitric oxide (NO) bioactivity is an established principle of hypoxic blood flow. One mechanism proposed to explain this oxygen-sensing-NO bioactivity linkage postulates an essential role for the conserved Cys93 residue of the hemoglobin beta-chain (betaCys93) and, specifically, for S-nitrosation of betaCys93 to form S-nitrosohemoglobin (SNO-Hb). The SNO-Hb hypothesis, which conceptually links hemoglobin and NO biology, has been debated intensely in recent years. This debate has precluded a consensus on physiological mechanisms and on assessment of the potential role of SNO-Hb in pathology. Here we describe new mouse models that exclusively express either human wild-type hemoglobin or human hemoglobin in which the betaCys93 residue is replaced with alanine to assess the role of SNO-Hb in red blood cell-mediated hypoxic vasodilation. Substitution of this residue, precluding hemoglobin S-nitrosation, did not change total red blood cell S-nitrosothiol abundance but did shift S-nitrosothiol distribution to lower molecular weight species, consistent with the loss of SNO-Hb. Loss of betaCys93 resulted in no deficits in systemic or pulmonary hemodynamics under basal conditions and, notably, did not affect isolated red blood cell-dependent hypoxic vasodilation. These results demonstrate that SNO-Hb is not essential for the physiologic coupling of erythrocyte deoxygenation with increased NO bioactivity in vivo.

CD40 ligation decreases its protein half-life at the cell surface.

CD40 is expressed on a variety of tumors; anti-CD40 agonists promote tumor cell apoptosis and subsequent tumor regression. Because the effectiveness of anti-CD40- agonists is dependent upon CD40 surface expression, the current study examined ligation-mediated changes in CD40 protein half-life (t(1/2))( )at the cell surface. This study utilized a CD40(+) epithelial cell line (9HTEo-), a CD40 null epithelial cell line (HT-29) engineered to express either wild-type (WT) or mutant (T254A, Q263A, E235A, Delta201) CD40, and the anti-CD40 antibody G28.5. Ligation of endogenous CD40 expressed on 9HTEo- cells decreased CD40 surface protein t(1/2 )from 13 to 4 h (p <0.05). Ligation of WT-, Q263A-, or T254A-CD40 expressed on engineered HT-29 cells decreased CD40 surface protein t(1/2) from an average of 8 to 4 h (p <0.05); T254A and Q263A contain mutated TNF receptor-associated factor (TRAF)2/3-binding sites. In contrast, ligation of E235A and Delta201-CD40 had no affect on its surface protein t(1/2) (p <0.05); E235A contains a mutated TRAF6-binding site while Delta201 lacks an intact cytoplasmic tail. These results suggest that anti-CD40 agonists decrease CD40 surface protein t(1/2) via a mechanism that involves TRAF6 but not TRAF2/3. The therapeutic implications for CD40-mediated tumor regression are discussed.