Mitochondrial DNA copy number variation in asthma risk, severity, and exacerbations.

BACKGROUND: Asthma pathophysiology is associated with mitochondrial dysfunction. Mitochondrial DNA copy number (mtDNA-CN) has been used as a proxy of mitochondrial function, with lower levels indicating mitochondrial dysfunction in population studies of cardiovascular diseases and cancers. OBJECTIVES: We investigated whether lower levels of mtDNA-CN are associated with asthma diagnosis, severity, and exacerbations. METHODS: mtDNA-CN is evaluated in blood from 2 cohorts: UK Biobank (UKB) (asthma, n = 39,147; no asthma, n = 302,302) and Severe Asthma Research Program (SARP) (asthma, n = 1283; nonsevere asthma, n = 703). RESULTS: Individuals with asthma have lower mtDNA-CN compared to individuals without asthma in UKB (beta, -0.006 [95% confidence interval, -0.008 to -0.003], P = 6.23 × 10-6). Lower mtDNA-CN is associated with asthma prevalence, but not severity in UKB or SARP. mtDNA-CN declines with age but is lower in individuals with asthma than in individuals without asthma at all ages. In a 1-year longitudinal study in SARP, mtDNA-CN was associated with risk of exacerbation; those with highest mtDNA-CN had the lowest risk of exacerbation (odds ratio 0.333 [95% confidence interval, 0.173 to 0.542], P = .001). Biomarkers of inflammation and oxidative stress are higher in individuals with asthma than without asthma, but the lower mtDNA-CN in asthma is independent of general inflammation or oxidative stress. Mendelian randomization studies suggest a potential causal relationship between asthma-associated genetic variants and mtDNA-CN. CONCLUSION: mtDNA-CN is lower in asthma than in no asthma and is associated with exacerbations. Low mtDNA-CN in asthma is not mediated through inflammation but is associated with a genetic predisposition to asthma.

Studying the Pulmonary Endothelium in Health and Disease: An Official American Thoracic Society Workshop Report.

Lung endothelium resides at the interface between the circulation and the underlying tissue, where it senses biochemical and mechanical properties of both the blood as it flows through the vascular circuit and the vessel wall. The endothelium performs the bidirectional signaling between the blood and tissue compartments that is necessary to maintain homeostasis while physically separating both, facilitating a tightly regulated exchange of water, solutes, cells, and signals. Disruption in endothelial function contributes to vascular disease, which can manifest in discrete vascular locations along the artery-to-capillary-to-vein axis. Although our understanding of mechanisms that contribute to endothelial cell injury and repair in acute and chronic vascular disease have advanced, pathophysiological mechanisms that underlie site-specific vascular disease remain incompletely understood. In an effort to improve the translatability of mechanistic studies of the endothelium, the American Thoracic Society convened a workshop to optimize rigor, reproducibility, and translation of discovery to advance our understanding of endothelial cell function in health and disease.

Genetic and epigenetic regulation of cortactin (CTTN) by inflammatory factors and mechanical stress in human lung endothelial cells.

RATIONALE: Cortactin, an actin-binding cytoskeletal protein, plays a crucial role in maintaining endothelial cell (EC) barrier integrity and regulating vascular permeability. The gene encoding cortactin, CTTN, is implicated in various lung inflammatory disorders. Despite this, the transcriptional regulation of CTTN by inflammatory stimuli and promoter SNPs remains unexplored. METHODS: We transfected human lung ECs with a full-length CTTN promoters linked to a luciferase reporter to measure promoter activity. SNP-containing CTTN promoter was created via site-directed mutagenesis. Transfected ECs were exposed to LPS (PAMP), TNF-α (cytokine), cyclic stretch (CS), FG-4592 (HIF-inducer), NRF2 (anti-oxidant modulator), FTY-(S)-phosphate (endothelial barrier enhancer), and 5'-Aza (demethylation inducer). Immunohistochemistry was used to assess cortactin expression in mouse lungs exposed to LPS. RESULTS: LPS, TNF-α, and 18%CS significantly increased CTTN promoter activities in a time-dependent manner (P<0.05). The variant rs34612166 (-212T/C) markedly enhanced LPS- and 18%CS- induced CTTN promoter activities (P<0.05). FG-4592 significantly boosted CTTN promoter activities (P<0.01), which were partially inhibited by HIF1α (KC7F2) and HIF2α (PT2385) inhibitors (P<0.05). NRF2 activator Bixin increased CTTN promoter activities, whereas NRF2 inhibitor Brusatol reduced them (P<0.05). 5'-Aza increased CTTN promoter activities by 2.9-fold (P<0.05). NF-κB response element mutations significantly reduced CTTN promoter activities response to LPS and TNFα. FTY-(S)-phosphate significantly increased CTTN promoter activities in 24 h. In vivo, cortactin levels were significantly elevated in inflammatory mouse lungs exposed to LPS for 18 h. CONCLUSION: CTTN transcriptional is significantly influenced by inflammatory factors and promoter variants. Cortactin, essential in mitigating inflammatory edema, presents a promising therapeutic target to alleviate severe inflammatory disorders.

Pro- and anti-inflammatory cytokines: the hidden keys to autoimmune gastritis therapy.

Autoimmune gastritis (AIG) is an autoimmune disorder characterized by the destruction of gastric parietal cells and atrophy of the oxyntic mucosa which induces intrinsic factor deficiency and hypo-achlorhydria. AIG predominantly affects the antral mucosa with AIG patients experiencing increased inflammation and a predisposition toward the development of gastric adenocarcinoma and type I neuroendocrine tumors. The exact pathogenesis of this autoimmune disorder is incompletely understood although dysregulated immunological mechanisms appear to major contributors. This review of autoimmune gastritis, an unmet medical need, summarizes current knowledge on pro- and anti-inflammatory cytokines and strategies for the discovery of novel biomarkers and potential pharmacological targets.

Origami-inspired soft fluidic actuation for minimally invasive large-area electrocorticography.

Electrocorticography is an established neural interfacing technique wherein an array of electrodes enables large-area recording from the cortical surface. Electrocorticography is commonly used for seizure mapping however the implantation of large-area electrocorticography arrays is a highly invasive procedure, requiring a craniotomy larger than the implant area to place the device. In this work, flexible thin-film electrode arrays are combined with concepts from soft robotics, to realize a large-area electrocorticography device that can change shape via integrated fluidic actuators. We show that the 32-electrode device can be packaged using origami-inspired folding into a compressed state and implanted through a small burr-hole craniotomy, then expanded on the surface of the brain for large-area cortical coverage. The implantation, expansion, and recording functionality of the device is confirmed in-vitro and in porcine in-vivo models. The integration of shape actuation into neural implants provides a clinically viable pathway to realize large-area neural interfaces via minimally invasive surgical techniques.

eNAMPT is a novel therapeutic target for mitigation of coronary microvascular disease in type 2 diabetes.

AIMS/HYPOTHESIS: Individuals with diabetes are at high risk of cardiovascular complications, which significantly increase morbidity/mortality. Coronary microvascular disease (CMD) is recognised as a critical contributor to the increased cardiac mortality observed in people with diabetes. Therefore, there is an urgent need for treatments that are specific to CMD. eNAMPT (extracellular nicotinamide phosphoribosyltransferase) is a damage-associated molecular pattern and TLR4 ligand, whose plasma levels are elevated in people with diabetes. This study was thus designed to investigate the pathogenic role of intracellular nicotinamide phosphoribosyltransferase (iNAMPT) and eNAMPT in promoting the development of CMD in a preclinical murine model of type 2 diabetes. METHODS: An inducible type 2 diabetic mouse model was generated by a single injection of low-dose streptozocin (75 mg/kg, i.p.) combined with a high-fat diet for 16 weeks. The in vivo effects of i/eNAMPT inhibition on cardiac endothelial cell (CEC) function were evaluated by using Nampt+/- heterozygous mice, chronic administration of eNAMPT-neutralising monoclonal antibody (mAb) or use of an NAMPT enzymatic inhibitor (FK866). RESULTS: As expected, diabetic wild-type mice exhibited significantly lower coronary flow velocity reserve (CFVR), a determinant of coronary microvascular function, compared with control wild-type mice. eNAMPT plasma levels or expression in CECs were significantly greater in diabetic mice than in control mice. Furthermore, in comparison with diabetic wild-type mice, diabetic Nampt+/- heterozygous mice showed markedly improved CFVR, accompanied by increased left ventricular capillary density and augmented endothelium-dependent relaxation (EDR) in the coronary artery. NAMPT inhibition by FK866 or an eNAMPT-neutralising mAb significantly increased CFVR in diabetic mice. Furthermore, administration of the eNAMPT mAb upregulated expression of angiogenesis- and EDR-related genes in CECs from diabetic mice. Treatment with either eNAMPT or NAD+ significantly decreased CEC migration and reduced EDR in coronary arteries, partly linked to increased production of mitochondrial reactive oxygen species. CONCLUSIONS/INTERPRETATION: These data indicate that increased i/eNAMPT expression contributes to the development of diabetic coronary microvascular dysfunction, and provide compelling support for eNAMPT inhibition as a novel and effective therapeutic strategy for CMD in diabetes.

Nocturnal Hypoxemia Is Associated with Sarcopenia in Patients with Chronic Obstructive Pulmonary Disease.

Rationale: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. Our previous studies have identified that nocturnal hypoxemia causes skeletal muscle loss (i.e., sarcopenia) in in vitro models of COPD. Objectives: We aimed to extend our preclinical mechanistic findings by analyzing a large sleep registry to determine whether nocturnal hypoxemia is associated with sarcopenia in patients with COPD. Methods: Sleep studies from patients with COPD (n = 479) and control subjects without COPD (n = 275) were analyzed. Patients with obstructive sleep apnea, as defined by apnea-hypopnea index ⩾ 5, were excluded. Pectoralis muscle cross-sectional area (PMcsa) was quantified using computed tomography scans performed within 1 year of the sleep study. We defined sarcopenia as less than the lowest 20% residuals for PMcsa of control subjects, which was adjusted for age and body mass index (BMI) and stratified by sex. Youden's optimal cut-point criteria were used to predict sarcopenia based on mean oxygen saturation during sleep. Additional measures of nocturnal hypoxemia were analyzed. The pectoralis muscle index (PMI) was defined as PMcsa normalized to BMI. Results: On average, males with COPD had a 16.6% lower PMI than control males (1.41 ± 0.44 vs. 1.69 ± 0.56 cm2/BMI; P < 0.001), whereas females with COPD had a 9.4% lower PMI than control females (0.96 ± 0.27 vs. 1.06 ± 0.33 cm2/BMI; P < 0.001). Males with COPD with nocturnal hypoxemia had a 9.5% decrease in PMI versus COPD with normal O2 (1.33 ± 0.39 vs. 1.47 ± 0.46 cm2/BMI; P < 0.05) and a 23.6% decrease compared with control subjects (1.33 ± 0.39 vs. 1.74 ± 0.56 cm2/BMI; P < 0.001). Females with COPD with nocturnal hypoxemia had an 11.2% decrease versus COPD with normal O2 (0.87 ± 0.26 vs. 0.98 ± 0.28 cm2/BMI; P < 0.05) and a 17.9% decrease compared with control subjects (0.87 ± 0.26 vs. 1.06 ± 0.33 cm2/BMI; P < 0.001). These findings were largely replicated using multiple measures of nocturnal hypoxemia. Conclusions: We defined sarcopenia in the pectoralis muscle using residuals that take into account age, BMI, and sex. We found that patients with COPD have a lower PMI than patients without COPD and that nocturnal hypoxemia was associated with an additional decrease in the PMI of patients with COPD. Additional prospective analyses are needed to determine a protective threshold of oxygen saturation to prevent or reverse sarcopenia due to nocturnal hypoxemia in COPD.

Deficiency of the Deubiquitinase UCHL1 Attenuates Pulmonary Arterial Hypertension.

BACKGROUND: The ubiquitin-proteasome system regulates protein degradation and the development of pulmonary arterial hypertension (PAH), but knowledge about the role of deubiquitinating enzymes in this process is limited. UCHL1 (ubiquitin carboxyl-terminal hydrolase 1), a deubiquitinase, has been shown to reduce AKT1 (AKT serine/threonine kinase 1) degradation, resulting in higher levels. Given that AKT1 is pathological in pulmonary hypertension, we hypothesized that UCHL1 deficiency attenuates PAH development by means of reductions in AKT1. METHODS: Tissues from animal pulmonary hypertension models as well as human pulmonary artery endothelial cells from patients with PAH exhibited increased vascular UCHL1 staining and protein expression. Exposure to LDN57444, a UCHL1-specific inhibitor, reduced human pulmonary artery endothelial cell and smooth muscle cell proliferation. Across 3 preclinical PAH models, LDN57444-exposed animals, Uchl1 knockout rats (Uchl1-/-), and conditional Uchl1 knockout mice (Tie2Cre-Uchl1fl/fl) demonstrated reduced right ventricular hypertrophy, right ventricular systolic pressures, and obliterative vascular remodeling. Lungs and pulmonary artery endothelial cells isolated from Uchl1-/- animals exhibited reduced total and activated Akt with increased ubiquitinated Akt levels. UCHL1-silenced human pulmonary artery endothelial cells displayed reduced lysine(K)63-linked and increased K48-linked AKT1 levels. RESULTS: Supporting experimental data, we found that rs9321, a variant in a GC-enriched region of the UCHL1 gene, is associated with reduced methylation (n=5133), increased UCHL1 gene expression in lungs (n=815), and reduced cardiac index in patients (n=796). In addition, Gadd45α (an established demethylating gene) knockout mice (Gadd45α-/-) exhibited reduced lung vascular UCHL1 and AKT1 expression along with attenuated hypoxic pulmonary hypertension. CONCLUSIONS: Our findings suggest that UCHL1 deficiency results in PAH attenuation by means of reduced AKT1, highlighting a novel therapeutic pathway in PAH.

Basic psychological need satisfaction of collegiate athletes: the unique and interactive effects of team identification and LMX quality.

Purpose: The present study sought to understand the relationships between team identification, leader-member exchange (LMX) quality, and the basic psychological need satisfaction of collegiate athletes, as well as the moderating role of coach-athlete LMX quality. Methods: Self-reported data from 319 collegiate athletes were analyzed using SPSS version 29. The relationships between the study variables were tested by moderation analysis using PROCESS macro model 1. Results: Regression analyses showed team identification to be positively related to the satisfaction of the needs for competence and relatedness, while LMX quality was positively related to the satisfaction of the needs for competence and autonomy. Furthermore, moderation analyses showed that LMX quality positively moderated the relationship between team identification and the satisfaction of the needs for competence and relatedness. Conclusion: The results of this study highlight the important role that team identification and LMX quality play in the satisfaction of the basic psychological needs of collegiate athletes. The implications of these results for the optimal functioning of collegiate athletes are discussed.

Development of a cloud-based flow rate tool for eNAMPT biomarker detection.

Increased levels of extracellular nicotinamide phosphoribosyltransferase (eNAMPT) are increasingly recognized as a highly useful biomarker of inflammatory disease and disease severity. In preclinical animal studies, a monoclonal antibody that neutralizes eNAMPT has been generated to successfully reduce the extent of inflammatory cascade activation. Thus, the rapid detection of eNAMPT concentration in plasma samples at the point of care (POC) would be of great utility in assessing the benefit of administering an anti-eNAMPT therapeutic. To determine the feasibility of this POC test, we conducted a particle immunoagglutination assay on a paper microfluidic platform and quantified its extent with a flow rate measurement in less than 1 min. A smartphone and cloud-based Google Colab were used to analyze the flow rates automatically. A horizontal flow model and an immunoagglutination binding model were evaluated to optimize the detection time, sample dilution, and particle concentration. This assay successfully detected eNAMPT in both human whole blood and plasma samples (diluted to 10 and 1%), with the limit of detection of 1-20 pg/mL (equivalent to 0.1-0.2 ng/mL in undiluted blood and plasma) and a linear range of 5-40 pg/mL. Furthermore, the smartphone POC assay distinguished clinical samples with low, mid, and high eNAMPT concentrations. Together, these results indicate this POC assay, which utilizes low-cost materials, time-effective methods, and a straightforward immunoassay (without surface immobilization), may reliably allow rapid determination of eNAMPT blood/plasma levels to advantage patient stratification in clinical trials and guide ALT-100 mAb therapeutic decision-making.

Extracellular Nicotinamide Phosphoribosyltransferase Is a Therapeutic Target in Experimental Necrotizing Enterocolitis.

Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency of prematurity. Postulated mechanisms leading to inflammatory necrosis of the ileum and colon include activation of the pathogen recognition receptor Toll-like receptor 4 (TLR4) and decreased levels of transforming growth factor beta (TGFß). Extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a novel damage-associated molecular pattern (DAMP), is a TLR4 ligand and plays a role in a number of inflammatory disease processes. To test the hypothesis that eNAMPT is involved in NEC, an eNAMPT-neutralizing monoclonal antibody, ALT-100, was used in a well-established animal model of NEC. Preterm Sprague-Dawley pups delivered prematurely from timed-pregnant dams were exposed to hypoxia/hypothermia and randomized to control-foster mother dam-fed rats, injected IP with saline (vehicle) 48 h after delivery; control + mAB-foster dam-fed rats, injected IP with 10 µg of ALT-100 at 48 h post-delivery; NEC-orally gavaged, formula-fed rats injected with saline; and NEC + mAb-formula-fed rats, injected IP with 10 µg of ALT-100 at 48 h. The distal ileum was processed 96 h after C-section delivery for histological, biochemical, molecular, and RNA sequencing studies. Saline-treated NEC pups exhibited markedly increased fecal blood and histologic ileal damage compared to controls (q < 0.0001), and findings significantly reduced in ALT-100 mAb-treated NEC pups (q < 0.01). Real-time PCR in ileal tissues revealed increased NAMPT in NEC pups compared to pups that received the ALT-100 mAb (p < 0.01). Elevated serum levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), interleukin-8 (IL-8), and NAMPT were observed in NEC pups compared to NEC + mAb pups (p < 0.01). Finally, RNA-Seq confirmed dysregulated TGFß and TLR4 signaling pathways in NEC pups that were attenuated by ALT-100 mAb treatment. These data strongly support the involvement of eNAMPT in NEC pathobiology and eNAMPT neutralization as a strategy to address the unmet need for NEC therapeutics.

Novel Machine Learning Identifies 5 Asthma Phenotypes Using Cluster Analysis of Real-World Data.

BACKGROUND: Asthma classification into different subphenotypes is important to guide personalized therapy and improve outcomes. OBJECTIVES: To further explore asthma heterogeneity through determination of multiple patient groups by using novel machine learning (ML) approaches and large-scale real-world data. METHODS: We used electronic health records of patients with asthma followed at the Cleveland Clinic between 2010 and 2021. We used k-prototype unsupervised ML to develop a clustering model where predictors were age, sex, race, body mass index, prebronchodilator and postbronchodilator spirometry measurements, and the usage of inhaled/systemic steroids. We applied elbow and silhouette plots to select the optimal number of clusters. These clusters were then evaluated through LightGBM's supervised ML approach on their cross-validated F1 score to support their distinctiveness. RESULTS: Data from 13,498 patients with asthma with available postbronchodilator spirometry measurements were extracted to identify 5 stable clusters. Cluster 1 included a young nonsevere asthma population with normal lung function and higher frequency of acute exacerbation (0.8 /patient-year). Cluster 2 had the highest body mass index (mean ± SD, 44.44 ± 7.83 kg/m2), and the highest proportion of females (77.5%) and Blacks (28.9%). Cluster 3 comprised patients with normal lung function. Cluster 4 included patients with lower percent of predicted FEV1 of 77.03 (12.79) and poor response to bronchodilators. Cluster 5 had the lowest percent of predicted FEV1 of 68.08 (15.02), the highest postbronchodilator reversibility, and the highest proportion of severe asthma (44.9%) and blood eosinophilia (>300 cells/µL) (34.8%). CONCLUSIONS: Using real-world data and unsupervised ML, we classified asthma into 5 clinically important subphenotypes where group-specific asthma treatment and management strategies can be designed and deployed.

Circadian disruption dysregulates lung gene expression associated with inflammatory lung injury.

Rationale: Circadian systems drive the expression of multiple genes in nearly all cells and coordinate cellular-, tissue-, and system-level processes that are critical to innate immunity regulation. Objective: We examined the effects of circadian rhythm disorganization, produced by light shift exposure, on innate immunity-mediated inflammatory lung responses including vascular permeability and gene expression in a C57BL/6J murine model of inflammatory lung injury. Methods: A total of 32 C57BL/6J mice were assigned to circadian phase shifting (CPS) with intratracheal phosphate-buffered saline (PBS), CPS with intratracheal lipopolysaccharide (LPS), control (normal lighting) condition with intratracheal PBS, and control condition with intratracheal LPS. Bronchoalveolar lavage (BAL) protein, cell counts, tissue immunostaining, and differentially expressed genes (DEGs) were measured in lung tissues at 2 and 10 weeks. Measurements and results: In mice exposed to both CPS and intratracheal LPS, both BAL protein and cell counts were increased at both 2 and 10 weeks compared to mice exposed to LPS alone. Multiple DEGs were identified in CPS-LPS-exposed lung tissues compared to LPS alone and were involved in transcriptional pathways associated with circadian rhythm disruption, regulation of lung permeability, inflammation with Rap1 signaling, and regulation of actin cytoskeleton. The most dysregulated pathways included myosin light chain kinase, MAP kinase, profilin 2, fibroblast growth factor receptor, integrin b4, and p21-activated kinase. Conclusion: Circadian rhythm disruption results in exacerbated immune response and dysregulated expression of cytoskeletal genes involved in the regulation of epithelial and vascular barrier integrity-the mechanistic underpinnings of acute lung injury. Further studies need to explore circadian disorganization as a druggable target.

Differentiable simulation to develop molecular dynamics force fields for disordered proteins.

Implicit solvent force fields are computationally efficient but can be unsuitable for running molecular dynamics on disordered proteins. Here I improve the a99SB-disp force field and the GBNeck2 implicit solvent model to better describe disordered proteins. Differentiable molecular simulations with 5 ns trajectories are used to jointly optimise 108 parameters to better match explicit solvent trajectories. Simulations with the improved force field better reproduce the radius of gyration and secondary structure content seen in experiments, whilst showing slightly degraded performance on folded proteins and protein complexes. The force field, called GB99dms, reproduces the results of a small molecule binding study and improves agreement with experiment for the aggregation of amyloid peptides. GB99dms, which can be used in OpenMM, is available at https://github.com/greener-group/GB99dms. This work is the first to show that gradients can be obtained directly from nanosecond-length differentiable simulations of biomolecules and highlights the effectiveness of this approach to training whole force fields to match desired properties.

A Razor's Edge: Vascular Responses to Acute Inflammatory Lung Injury/Acute Respiratory Distress Syndrome.

Historically considered a metabolically inert cellular layer separating the blood from the underlying tissue, the endothelium is now recognized as a highly dynamic, metabolically active tissue that is critical to organ homeostasis. Under homeostatic conditions, lung endothelial cells (ECs) in healthy subjects are quiescent, promoting vasodilation, platelet disaggregation, and anti-inflammatory mechanisms. In contrast, lung ECs are essential contributors to the pathobiology of acute respiratory distress syndrome (ARDS), as the quiescent endothelium is rapidly and radically altered upon exposure to environmental stressors, infectious pathogens, or endogenous danger signals into an effective and formidable regulator of innate and adaptive immunity. These dramatic perturbations, produced in a tsunami of inflammatory cascade activation, result in paracellular gap formation between lung ECs, sustained lung edema, and multi-organ dysfunction that drives ARDS mortality. The astonishing plasticity of the lung endothelium in negotiating this inflammatory environment and efforts to therapeutically target the aberrant ARDS endothelium are examined in further detail in this review.

TLR4 Ligation by eNAMPT, a Novel DAMP, is Essential to Sulfur Mustard- Induced Inflammatory Lung Injury and Fibrosis.

Objective: Human and preclinical studies of sulfur mustard (SM)-induced acute and chronic lung injuries highlight the role of unremitting inflammation. We assessed the utility of targeting the novel DAMP and TLR4 ligand, eNAMPT (extracellular nicotinamide phosphoribosyltransferase), utilizing a humanized mAb (ALT-100) in rat models of SM exposure. Methods: Acute (SM 4.2 mg/kg, 24 hrs), subacute (SM 0.8 mg/kg, day 7), subacute (SM 2.1 mg/kg, day 14), and chronic (SM 1.2 mg/kg, day 29) SM models were utilized. Results: Each SM model exhibited significant increases in eNAMPT expression (lung homogenates) and increased levels of phosphorylated NFkB and NOX4. Lung fibrosis (Trichrome staining) was observed in both sub-acute and chronic SM models in conjunction with elevated smooth muscle actin (SMA), TGFß, and IL-1ß expression. SM-exposed rats receiving ALT-100 (1 or 4 mg/kg, weekly) exhibited increased survival, highly significant reductions in histologic/biochemical evidence of lung inflammation and fibrosis (Trichrome staining, decreased pNFkB, SMA, TGFß, NOX4), decreased airways strictures, and decreased plasma cytokine levels (eNAMPT, IL-6, IL-1ß. TNFα). Conclusion: The highly druggable, eNAMPT/TLR4 signaling pathway is a key contributor to SM-induced ROS production, inflammatory lung injury and fibrosis. The ALT-100 mAb is a potential medical countermeasure to address the unmet need to reduce SM-associated lung pathobiology/mortality.

An eNAMPT-neutralizing mAb reduces post-infarct myocardial fibrosis and left ventricular dysfunction.

Myocardial infarction (MI) triggers adverse ventricular remodeling (VR), cardiac fibrosis, and subsequent heart failure. Extracellular nicotinamide phosphoribosyltransferase (eNAMPT) is postulated to play a significant role in VR processing via activation of the TLR4 inflammatory pathway. We hypothesized that an eNAMPT specific monoclonal antibody (mAb) could target and neutralize overexpressed eNAMPT post-MI and attenuate chronic cardiac inflammation and fibrosis. We investigated humanized ALT-100 and ALT-300 mAb with high eNAMPT-neutralizing capacity in an infarct rat model to test our hypothesis. ALT-300 was 99mTc-labeled to generate 99mTc-ALT-300 for imaging myocardial eNAMPT expression at 2 hours, 1 week, and 4 weeks post-IRI. The eNAMPT-neutralizing ALT-100 mAb (0.4 mg/kg) or saline was administered intraperitoneally at 1 hour and 24 hours post-reperfusion and twice a week for 4 weeks. Cardiac function changes were determined by echocardiography at 3 days and 4 weeks post-IRI. 99mTc-ALT-300 uptake was initially localized to the ischemic area at risk (IAR) of the left ventricle (LV) and subsequently extended to adjacent non-ischemic areas 2 hours to 4 weeks post-IRI. Radioactive uptake (%ID/g) of 99mTc-ALT-300 in the IAR increased from 1 week to 4 weeks (0.54 ± 0.16 vs. 0.78 ± 0.13, P < 0.01). Rats receiving ALT-100 mAb exhibited significantly improved myocardial histopathology and cardiac function at 4 weeks, with a significant reduction in the collagen volume fraction (%LV) compared to controls (21.5 ± 6.1% vs. 29.5 ± 9.9%, P < 0.05). Neutralization of the eNAMPT/TLR4 inflammatory cascade is a promising therapeutic strategy for MI by reducing chronic inflammation, fibrosis, and preserving cardiac function.