Cell-free DNA levels associate with COPD exacerbations and mortality.

THE QUESTION ADDRESSED BY THE STUDY: Good biological indicators capable of predicting chronic obstructive pulmonary disease (COPD) phenotypes and clinical trajectories are lacking. Because nuclear and mitochondrial genomes are damaged and released by cigarette smoke exposure, plasma cell-free mitochondrial and nuclear DNA (cf-mtDNA and cf-nDNA) levels could potentially integrate disease physiology and clinical phenotypes in COPD. This study aimed to determine whether plasma cf-mtDNA and cf-nDNA levels are associated with COPD disease severity, exacerbations, and mortality risk. MATERIALS AND METHODS: We quantified mtDNA and nDNA copy numbers in plasma from participants enrolled in the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE, n = 2,702) study and determined associations with relevant clinical parameters. RESULTS: Of the 2,128 participants with COPD, 65% were male and the median age was 64 (interquartile range, 59-69) years. During the baseline visit, cf-mtDNA levels positively correlated with future exacerbation rates in subjects with mild/moderate and severe disease (Global Initiative for Obstructive Lung Disease [GOLD] I/II and III, respectively) or with high eosinophil count (≥ 300). cf-nDNA positively associated with an increased mortality risk (hazard ratio, 1.33 [95% confidence interval, 1.01-1.74] per each natural log of cf-nDNA copy number). Additional analysis revealed that individuals with low cf-mtDNA and high cf-nDNA abundance further increased the mortality risk (hazard ratio, 1.62 [95% confidence interval, 1.16-2.25] per each natural log of cf-nDNA copy number). ANSWER TO THE QUESTION: Plasma cf-mtDNA and cf-nDNA, when integrated into quantitative clinical measurements, may aid in improving COPD severity and progression assessment.

Loss of ANT1 Increases Fibrosis and Epithelial Cell Senescence in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease characterized by progressive lung scarring and remodeling. Although treatments exist that slow disease progression, IPF is irreversible, and there is no cure. Cellular senescence, a major hallmark of aging, has been implicated in IPF pathogenesis, and mitochondrial dysfunction is increasingly recognized as a driver of senescence. Adenine nucleotide translocases (ANTs) are abundant mitochondrial ATP-ADP transporters critical for regulating cell fate and maintaining mitochondrial function. We sought to determine how alterations in ANTs influence cellular senescence in pulmonary fibrosis. We found that SLC25A4 (solute carrier family 25 member 4) (ANT1) and SLC25A5 (ANT2) expression is reduced in the lungs of patients with IPF, particularly within alveolar type II (AT2) cells, by single-cell RNA sequencing and tissue staining. Loss of ANT1 by siRNA in lung epithelial cells resulted in increased senescence markers such as ß-galactosidase and p21, with a reduction in the ratio of nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide. Bleomycin-treated ANT1 knockdown cells also had increased senescence markers compared with bleomycin-treated control cells. Loss of ANT1 in AT2 cells resulted in a reduction in alveolar organoid growth, with an increase in p21 by staining. Global loss of ANT1 resulted in worse lung fibrosis and increased senescence in the bleomycin- and asbestos-induced mouse models of pulmonary fibrosis. In summary, loss of ANT1 contributes to IPF pathogenesis through mitochondrial dysfunction, increased senescence, and decreased regenerative capacity of AT2 cells, resulting in enhanced lung fibrosis. Modulation of ANTs presents a new therapeutic avenue that may alter cellular senescence pathways and limit pulmonary fibrosis.

Adenine nucleotide translocase regulates airway epithelial metabolism, surface hydration and ciliary function.

Airway hydration and ciliary function are critical to airway homeostasis and dysregulated in chronic obstructive pulmonary disease (COPD), which is impacted by cigarette smoking and has no therapeutic options. We utilized a high-copy cDNA library genetic selection approach in the amoeba Dictyostelium discoideum to identify genetic protectors to cigarette smoke. Members of the mitochondrial ADP/ATP transporter family adenine nucleotide translocase (ANT) are protective against cigarette smoke in Dictyostelium and human bronchial epithelial cells. Gene expression of ANT2 is reduced in lung tissue from COPD patients and in a mouse smoking model, and overexpression of ANT1 and ANT2 resulted in enhanced oxidative respiration and ATP flux. In addition to the presence of ANT proteins in the mitochondria, they reside at the plasma membrane in airway epithelial cells and regulate airway homeostasis. ANT2 overexpression stimulates airway surface hydration by ATP and maintains ciliary beating after exposure to cigarette smoke, both of which are key functions of the airway. Our study highlights a potential for upregulation of ANT proteins and/or of their agonists in the protection from dysfunctional mitochondrial metabolism, airway hydration and ciliary motility in COPD.This article has an associated First Person interview with the first author of the paper.

Regulation of mitochondrial fragmentation in microvascular endothelial cells isolated from the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a morbid disease characterized by progressive right ventricle (RV) failure due to elevated pulmonary artery pressures (PAP). In PAH, histologically complex vaso-occlusive lesions in the pulmonary vasculature contribute to elevated PAP. However, the mechanisms underlying dysfunction of the microvascular endothelial cells (MVECs) that comprise a significant portion of these lesions are not well understood. We recently showed that MVECs isolated from the Sugen/hypoxia (SuHx) rat experimental model of PAH (SuHx-MVECs) exhibit increases in migration/proliferation, mitochondrial reactive oxygen species (ROS; mtROS) production, intracellular calcium levels ([Ca2+]i), and mitochondrial fragmentation. Furthermore, quenching mtROS with the targeted antioxidant MitoQ attenuated basal [Ca2+]i, migration and proliferation; however, whether increased mtROS-induced [Ca2+]i entry affected mitochondrial morphology was not clear. In this study, we sought to better understand the relationship between increased ROS, [Ca2+]i, and mitochondrial morphology in SuHx-MVECs. We measured changes in mitochondrial morphology at baseline and following inhibition of mtROS, with the targeted antioxidant MitoQ, or transient receptor potential vanilloid-4 (TRPV4) channels, which we previously showed were responsible for mtROS-induced increases in [Ca2+]i in SuHx-MVECs. Quenching mtROS or inhibiting TRPV4 attenuated fragmentation in SuHx-MVECs. Conversely, inducing mtROS production in MVECs from normoxic rats (N-MVECs) increased fragmentation. Ca2+ entry induced by the TRPV4 agonist GSK1017920A was significantly increased in SuHx-MVECs and was attenuated with MitoQ treatment, indicating that mtROS contributes to increased TRPV4 activity in SuHx-MVECs. Basal and maximal respiration were depressed in SuHx-MVECs, and inhibiting mtROS, but not TRPV4, improved respiration in these cells. Collectively, our data show that, in SuHx-MVECs, mtROS production promotes TRPV4-mediated increases in [Ca2+]i, mitochondrial fission, and decreased mitochondrial respiration. These results suggest an important role for mtROS in driving MVEC dysfunction in PAH.

Reactive oxygen species induced Ca2+ influx via TRPV4 and microvascular endothelial dysfunction in the SU5416/hypoxia model of pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by elevations in pulmonary arterial pressure, in part due to formation of occlusive lesions in the distal arterioles of the lung. These complex lesions may comprise multiple cell types, including endothelial cells (ECs). To better understand the molecular mechanisms underlying EC dysfunction in PAH, lung microvascular endothelial cells (MVECs) were isolated from normoxic rats (N-MVECs) and rats subjected to SU5416 plus hypoxia (SuHx), an experimental model of PAH. Compared with N-MVECs, MVECs isolated from SuHx rats (SuHx-MVECs) appeared larger and more spindle shaped morphologically and expressed canonical smooth muscle cell markers smooth muscle-specific α-actin and myosin heavy chain in addition to endothelial markers such as Griffonia simplicifolia and von Willebrand factor. SuHx-MVEC mitochondria were dysfunctional, as evidenced by increased fragmentation/fission, decreased oxidative phosphorylation, and increased reactive oxygen species (ROS) production. Functionally, SuHx-MVECs exhibited increased basal levels of intracellular calcium concentration ([Ca2+]i) and enhanced migratory and proliferative capacity. Treatment with global (TEMPOL) or mitochondria-specific (MitoQ) antioxidants decreased ROS levels and basal [Ca2]i in SuHx-MVECs. TEMPOL and MitoQ also decreased migration and proliferation in SuHx-MVECs. Additionally, inhibition of ROS-induced Ca2+ entry via pharmacologic blockade of transient receptor potential vanilloid-4 (TRPV4) attenuated [Ca2]i, migration, and proliferation. These findings suggest a role for mitochondrial ROS-induced Ca2+ influx via TRPV4 in promoting abnormal migration and proliferation in MVECs in this PAH model.

A Prediction Model to Help with the Assessment of Adenopathy in Lung Cancer: HAL.

RATIONALE: Estimating the probability of finding N2 or N3 (prN2/3) malignant nodal disease on endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in patients with non-small cell lung cancer (NSCLC) can facilitate the selection of subsequent management strategies. OBJECTIVES: To develop a clinical prediction model for estimating the prN2/3. METHODS: We used the AQuIRE (American College of Chest Physicians Quality Improvement Registry, Evaluation, and Education) registry to identify patients with NSCLC with clinical radiographic stage T1-3, N0-3, M0 disease that had EBUS-TBNA for staging. The dependent variable was the presence of N2 or N3 disease (vs. N0 or N1) as assessed by EBUS-TBNA. Univariate followed by multivariable logistic regression analysis was used to develop a parsimonious clinical prediction model to estimate prN2/3. External validation was performed using data from three other hospitals. MEASUREMENTS AND MAIN RESULTS: The model derivation cohort (n = 633) had a 25% prevalence of malignant N2 or N3 disease. Younger age, central location, adenocarcinoma histology, and higher positron emission tomography-computed tomography N stage were associated with a higher prN2/3. Area under the receiver operating characteristic curve was 0.85 (95% confidence interval, 0.82-0.89), model fit was acceptable (Hosmer-Lemeshow, P = 0.62; Brier score, 0.125). We externally validated the model in 722 patients. Area under the receiver operating characteristic curve was 0.88 (95% confidence interval, 0.85-0.90). Calibration using the general calibration model method resulted in acceptable goodness of fit (Hosmer-Lemeshow test, P = 0.54; Brier score, 0.132). CONCLUSIONS: Our prediction rule can be used to estimate prN2/3 in patients with NSCLC. The model has the potential to facilitate clinical decision making in the staging of NSCLC.

A comparison of visual and quantitative methods to identify interstitial lung abnormalities.

BACKGROUND: Evidence suggests that individuals with interstitial lung abnormalities (ILA) on a chest computed tomogram (CT) may have an increased risk to develop a clinically significant interstitial lung disease (ILD). Although methods used to identify individuals with ILA on chest CT have included both automated quantitative and qualitative visual inspection methods, there has been not direct comparison between these two methods. To investigate this relationship, we created lung density metrics and compared these to visual assessments of ILA. METHODS: To provide a comparison between ILA detection methods based on visual assessment we generated measures of high attenuation areas (HAAs, defined by attenuation values between -600 and -250 Hounsfield Units) in >4500 participants from both the COPDGene and Framingham Heart studies (FHS). Linear and logistic regressions were used for analyses. RESULTS: Increased measures of HAAs (in ≥ 10 % of the lung) were significantly associated with ILA defined by visual inspection in both cohorts (P < 0.0001); however, the positive predictive values were not very high (19 % in COPDGene and 13 % in the FHS). In COPDGene, the association between HAAs and ILA defined by visual assessment were modified by the percentage of emphysema and body mass index. Although increased HAAs were associated with reductions in total lung capacity in both cohorts, there was no evidence for an association between measurement of HAAs and MUC5B promoter genotype in the FHS. CONCLUSION: Our findings demonstrate that increased measures of lung density may be helpful in determining the severity of lung volume reduction, but alone, are not strongly predictive of ILA defined by visual assessment. Moreover, HAAs were not associated with MUC5B promoter genotype.

Excessive hepatomegaly of mice with hepatocyte-targeted elimination of integrin linked kinase following treatment with 1,4-bis [2-(3,5-dichaloropyridyloxy)] benzene.

UNLABELLED: TCBOPOP (1,4-bis [2-(3,5-dichaloropyridyloxy)] benzene) an agonist of the constitutive androstane receptor (CAR), produces rapid hepatocyte hyperplasia and hepatomegaly in the absence of hepatic injury. In this study we demonstrate that integrin-linked kinase (ILK), which is involved in transmission of the extracellular matrix (ECM) signaling by way of integrin receptors, plays an important role in regulating TCPOBOP-induced proliferation of hepatocytes and hepatomegaly. Hepatocyte-specific ILK knockout mice (ILK/liver-/- mice) and wildtype mice (WT) were given a single dose of TCPOBOP (3 mg/kg) by oral gavage. Mice were sacrificed at days 1, 2, 5, and 7 after TCPOBOP administration. WT mice showed maximum proliferation on days 1 and 2, which came back to baseline levels by days 5 and 7 after TCPOBOP administration. The ILK/liver-/- mice, on the other hand, showed a prolonged and a sustained proliferative response as evident by an increased number of proliferative cell nuclear antigen assay (PCNA)-positive cells even at days 5 and 7 after TCPOBOP administration. At day 7 the WT mice showed close to a 2.5-fold increase in liver weight, whereas the ILK/liver-/- mice showed a 3.7-fold increase in liver weight. The prolonged proliferative response in the ILK/liver-/- mice seems to be due to sustained induction of CAR leading to sustained induction of c-Myc, which is known to be a key mediator of TCPOPOP-CAR induced direct liver hyperplasia. CONCLUSION: The data indicate that ECM-mediated signaling by way of ILK is essential for adjustment of final liver size and proper termination of TCPOBOP-induced proliferation of hepatocytes.

Strategies for Achieving Gender Equity and Work-Life Integration in Physician-Scientist Training.

Substantial gender inequities persist across academic medicine. These issues are not new: Recent evidence still points to a chilly climate for women in academic medicine, including those in physician-scientist training. The discussion for how to address gender equity and issues of work-life integration typically centers around faculty and rarely includes trainees. The authors delineate specific strategies to address gender inequity in physician-scientist training by identifying key stakeholders for implementation and proposing areas to integrate these strategies with current training timelines. Strategies discussed include multiple-role mentoring, allyship training for trainees and faculty, early implementation of professional development sessions, incorporation of childcare and family-friendly policies, and additional policies for funding bodies to prioritize gender equity practices. The goal of this article is to equip trainees and the academic community with proactive strategies to create a more equitable environment for future generations of trainees in academic medicine.

Predoctoral MD-PhD grants as indicators of future NIH funding success.

MD-PhD trainees constitute an important source of physician-scientists. Persistence on this challenging path is facilitated by success in garnering independent (R grant) support from the NIH. Published research tracks academic appointments and global R01 success for MD-PhD trainees but has not included information on future funding success of individual MD-PhD predoctoral grant holders. Here, we used data from the NIH RePORTER database to identify and track the funding trajectory of physician-scientists who received predoctoral grant support through the F30 mechanism, which is specific for dual-degree candidates. Male and female F30 awardees did not differ in their success in garnering K (postdoctoral training) grants, but, among F30 grant awardees, men were 2.6 times more likely than women to receive R funding. These results underscore the need for analysis of factors that contribute to the disproportionate loss of NIH-supported female physician-scientists between the predoctoral F30 and the independent R grant-supported stages.

VEGF Receptor 1 Promotes Hypoxia-Induced Hematopoietic Progenitor Proliferation and Differentiation.

Although it is well known that hypoxia incites unleashed cellular inflammation, the mechanisms of exaggerated cellular inflammation in hypoxic conditions are not known. We observed augmented proliferation of hematopoietic stem and progenitor cells (HSPC), precursors of inflammatory leukocytes, in mice under hypoxia. Consistently, a transcriptomic analysis of human HSPC exposed to hypoxic conditions revealed elevated expression of genes involved in progenitor proliferation and differentiation. Additionally, bone marrow cells in mice expressed high amount of vascular endothelial growth factor (VEGF), and HSPC elevated VEGF receptor 1 (VEGFr1) and its target genes in hypoxic conditions. In line with this, VEGFr1 blockade in vivo and in vitro decreased HSPC proliferation and attenuated inflammation. In silico and ChIP experiments demonstrated that HIF-1α binds to the promoter region of VEGFR1. Correspondingly, HIF1a silencing decreased VEGFr1 expression in HSPC and diminished their proliferation. These results indicate that VEGF signaling in HSPC is an important mediator of their proliferation and differentiation in hypoxia-induced inflammation and represents a potential therapeutic target to prevent aberrant inflammation in hypoxia-associated diseases.

COVID-19 and the Early-Career Physician-Scientist. Fostering Resilience beyond the Pandemic.

The coronavirus disease (COVID-19) pandemic has created significant stressors for the academic and scientific community, with unique challenges for early-career physician-scientists. The pandemic-related disruptions have significantly affected research productivity, access to mentoring, professional development and networking opportunities, funding, and personal wellness. This is especially true for pulmonary and critical care medicine faculty because of the burden of specialized clinical care responsibilities that the COVID-19 pandemic has demanded. Departmental, institutional, and national leadership should foster open dialogue to identify and mitigate these challenges to promote ongoing career development of early-career physician-scientists. Implementation of thoughtful interventions to address these challenges will provide essential support for junior faculty and help retain a generation of physician-scientists.

Extracellular superoxide dismutase regulates cardiac function and fibrosis.

Extracellular superoxide dismutase (EC-SOD) is an antioxidant that protects the heart from ischemia and the lung from inflammation and fibrosis. The role of cardiac EC-SOD under normal conditions and injury remains unclear. Cardiac toxicity, a common side effect of doxorubicin, involves oxidative stress. We hypothesize that EC-SOD is critical for normal cardiac function and protects the heart from oxidant-induced fibrosis and loss of function. C57BL/6 and EC-SOD-null mice were treated with doxorubicin, 15 mg/kg (i.p.). After 15 days, echocardiography was used to assess cardiac function. Left ventricle (LV) tissue was used to assess fibrosis and inflammation by staining, Western blot, and hydroxyproline analysis. At baseline, EC-SOD-null mice have LV wall thinning and increases in LV end diastolic dimensions compared to wild-type mice but have normal cardiac function. After doxorubicin, EC-SOD-null mice have decreases in fractional shortening not apparent in WT mice. Lack of EC-SOD also leads to increases in myocardial apoptosis and significantly more LV fibrosis and inflammatory cell infiltration. Administration of the metalloporphyrin AEOL 10150 abrogates the loss of cardiac function, and potentially fibrosis, associated with doxorubicin treatment in both wild-type and EC-SOD KO mice. EC-SOD is critical for normal cardiac morphology and protects the heart from oxidant-induced fibrosis, apoptosis, and loss of function. The antioxidant metalloporphyrin AEOL 10150 effectively protects cardiac function from doxorubicin-induced oxidative stress in vivo. These findings identify targets for the use of antioxidant agents in oxidant-induced cardiac fibrosis.

Gremlin-mediated decrease in bone morphogenetic protein signaling promotes pulmonary fibrosis.

RATIONALE: Members of the transforming growth factor (TGF)-beta superfamily, including TGF-betas and bone morphogenetic proteins (BMPs), are essential for the maintenance of tissue homeostasis and regeneration after injury. We have observed that the BMP antagonist, gremlin, is highly up-regulated in idiopathic pulmonary fibrosis (IPF). OBJECTIVES: To investigate the role of gremlin in the regulation of BMP signaling in pulmonary fibrosis. METHODS: Progressive asbestos-induced fibrosis in the mouse was used as a model of human IPF. TGF-beta and BMP expression and signaling activities were measured from murine and human fibrotic lungs. The mechanism of gremlin induction was analyzed in cultured lung epithelial cells. In addition, the possible therapeutic role of gremlin inhibition was tested by administration of BMP-7 to mice after asbestos exposure. MEASUREMENTS AND MAIN RESULTS: Gremlin mRNA levels were up-regulated in the asbestos-exposed mouse lungs, which is in agreement with the human IPF biopsy data. Down-regulation of BMP signaling was demonstrated by reduced levels of Smad1/5/8 and enhanced Smad2 phosphorylation in asbestos-treated lungs. Accordingly, analyses of cultured human bronchial epithelial cells indicated that asbestos-induced gremlin expression could be prevented by inhibitors of the TGF-beta receptor and also by inhibitors of the mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase pathways. BMP-7 treatment significantly reduced hydroxyproline contents in the asbestos-treated mice. CONCLUSIONS: The TGF-beta and BMP signaling balance is important for lung regenerative events and is significantly perturbed in pulmonary fibrosis. Rescue of BMP signaling activity may represent a potential beneficial strategy for treating human pulmonary fibrosis.

Mitochondrial biogenesis in the pulmonary vasculature during inhalational lung injury and fibrosis.

Cell survival and injury repair is facilitated by mitochondrial biogenesis; however, the role of this process in lung repair is unknown. We evaluated mitochondrial biogenesis in the mouse lung in two injuries that cause acute inflammation and in two that cause chronic inflammation and pulmonary fibrosis. By using reporter mice that express green fluorescent protein (GFP) exclusively in mitochondria, we tracked mitochondrial biogenesis and correlated it with histologic lung injury, proliferation, and fibrosis. At 72 hours after acute LPS or continuous exposure to hyperoxia (Fio2, 1.0), the lungs showed diffuse infiltration by inflammatory cells in the alveolar region. In reporter mice, patchy new mitochondrial fluorescence was found in the alveolar region but was most prominent and unexpected in perivascular regions. At 14 days after instillation of asbestos or bleomycin, diffuse chronic inflammation had developed, and green fluorescence appeared in inflammatory cells in the expanded interstitium and was most intense in smooth muscle cells of pulmonary vessels. In all four lung injuries, mitochondrial fluorescence colocalized with mitochondrial superoxide dismutase, but not with proliferating cell nuclear antigen. These data indicate that vascular mitochondrial biogenesis is activated in diverse inhalational lung injuries along with oxidative stress. This finding indicates a unique and unexpected mechanism of metabolic adaptation to pulmonary fibrotic injuries.

Extracellular superoxide dismutase protects against matrix degradation of heparan sulfate in the lung.

Asbestosis is a form of interstitial lung disease caused by the inhalation of asbestos fibers, leading to inflammation and pulmonary fibrosis. Inflammation and oxidant/antioxidant imbalances are known to contribute to the disease pathogenesis. Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that has been shown to protect the lung from oxidant-mediated damage, inflammation, and interstitial fibrosis. Extracellular matrix (ECM) components, such as collagen and glycosaminoglycans, are known to be sensitive to oxidative fragmentation. Heparan sulfate, a glycosaminoglycan, is highly abundant in the ECM and tightly binds EC-SOD. We investigated the protective role of EC-SOD by evaluating the interaction of EC-SOD with heparan sulfate in the presence of reactive oxygen species (ROS). We found that ROS-induced heparin and heparan sulfate fragments induced neutrophil chemotaxis across a modified Boyden chamber, which was inhibited by the presence of EC-SOD by scavenging oxygen radicals. Chemotaxis in response to oxidatively fragmented heparin was mediated by Toll-like receptor-4. In vivo, bronchoalveolar lavage fluid from EC-SOD knockout mice at 1, 14, and 28 days after asbestos exposure showed increased heparan sulfate shedding from the lung parenchyma. We demonstrate that one mechanism through which EC-SOD inhibits lung inflammation and fibrosis in asbestosis is by protecting heparin/heparan sulfate from oxidative fragmentation.

CD4+ T cell-independent DNA vaccination against opportunistic infections.

Depletion or dysfunction of CD4+ T lymphocytes profoundly perturbs host defenses and impairs immunogenicity of vaccines. Here, we show that plasmid DNA vaccination with a cassette encoding antigen (OVA) and a second cassette encoding full-length CD40 ligand (CD40L), a molecule expressed on activated CD4+ T lymphocytes and critical for T cell helper function, can elicit significant titers of antigen-specific immunoglobulins in serum and Tc1 CD8+ T cell responses in CD4-deficient mice. To investigate whether this approach leads to CD4+ T cell-independent vaccine protection against a prototypic AIDS-defining infection, Pneumocystis (PC) pneumonia, we used serum from mice vaccinated with PC-pulsed, CD40L-modified DCs to immunoprecipitate PC antigens. Kexin, a PC antigen identified by this approach, was used in a similar DNA vaccine strategy with or without CD40L. CD4-deficient mice receiving DNA vaccines encoding Kexin and CD40L showed significantly higher anti-PC IgG titers as well as opsonic killing of PC compared with those vaccinated with Kexin alone. Moreover, CD4-depleted, Kexin-vaccinated mice showed a 3-log greater protection in a PC challenge model. Adoptive transfer of CD19+ cells or IgG to SCID mice conferred protection against PC challenge, indicating a role of humoral immunity in the protection. The results of these studies show promise for CD4-independent vaccination against HIV-related or other opportunistic pathogens.

Neutrophil elastase promotes myofibroblast differentiation in lung fibrosis.

IPF is a progressive lung disorder characterized by fibroblast proliferation and myofibroblast differentiation. Although neutrophil accumulation within IPF lungs has been negatively correlated with outcomes, the role played by neutrophils in lung fibrosis remains poorly understood. We have demonstrated previously that NE promotes lung cancer cell proliferation and hypothesized that it may have a similar effect on fibroblasts. In the current study, we show that NE(-/-) mice are protected from asbestos-induced lung fibrosis. NE(-/-) mice displayed reduced fibroblast and myofibroblast content when compared with controls. NE directly both lung fibroblast proliferation and myofibroblast differentiation in vitro, as evidenced by proliferation assays, collagen gel contractility assays, and αSMA induction. Furthermore, αSMA induction occurs in a TGF-ß-independent fashion. Treatment of asbestos-recipient mice with ONO-5046, a synthetic NE antagonist, reduced hydroxyproline content. Thus, the current study points to a key role for neutrophils and NE in the progression of lung fibrosis. Lastly, the study lends rationale to use of NE-inhibitory approaches as a novel therapeutic strategy for patients with lung fibrosis.