Idiopathic pulmonary fibrosis (IPF) is characterized by progressive deposition of extracellular matrix, worsening dyspnea, and eventual mortality. Pathogenesis of IPF is poorly understood and the role inflammation and activated leukocytes play in the disease process is controversial. Previous studies demonstrated that activated leukocyte subsets characterize IPF patients. We sought to validate this observation in a well-defined cohort of 35 IPF patients and to correlate the observed leukocyte phenotypes with robust parameters of disease progression. We demonstrate that in univariate and multivariate analyses, increases in the CD14hi, CD16hi subset of monocytes measured at baseline correlated with disease progression, with a threshold value >0.5% of the total peripheral blood mononuclear cells being a significant predictor for worse outcome. In addition, several T cell subsets, including CD25 expressing CD4 cells, and CXCR3 expressing CD4 and CD8 subsets correlated with disease progression when found in increased percentages in the peripheral blood of IPF patients when sampled at baseline. Somewhat surprising in comparison to previous literature, the CD4 T cells did not appear to have lost expression of the co-stimulatory molecule, CD28, but the CD8 T cells did. Taken together, these results are consistent with the presence of an inflammatory process in IPF patients who eventually progress. However, when longitudinal measurements of these same markers were examined, there was significant heterogeneity of expression and these biomarkers did not necessarily remain elevated in IPF patients with progressive disease. We interpret this heterogeneity to suggest that IPF patients experience episodic inflammatory events that once triggered, may lead to disease progression. This longitudinal heterogeneity in biomarker analyses may explain why such markers are not consistently measured in all IPF cohorts.

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with few treatment options. N-acetylcysteine (NAC) is a well-tolerated, inexpensive treatment with antioxidant and anti-fibrotic properties. The National Heart, Lung, and Blood Institute (NHLBI)-sponsored PANTHER (Prednisone Azathioprine and NAC therapy in IPF) trial confirmed the harmful effects of immunosuppression in IPF, and did not show a benefit to treatment with NAC. However, a post hoc analysis revealed a potential beneficial effect of NAC in a subgroup of individuals carrying a specific genetic variant, TOLLIP rs3750920 TT genotype, present in about 25% of patients with IPF. Here, we present the design and rationale for the Phase III, multi-center, randomized, double-blind, placebo-controlled Prospective Treatment Efficacy in IPF Using Genotype for NAC Selection (PRECISIONS) clinical trial.

Methods

The PRECISIONS trial will randomize 200 patients with IPF and the TOLLIP rs3750920 TT genotype 1:1 to oral N-acetylcysteine (600 mg tablets taken three times a day) or placebo for a 24-month duration. The primary endpoint is the composite of time to 10% relative decline in forced vital capacity (FVC), first respiratory hospitalization, lung transplantation, or death from any cause. Secondary endpoints include change in patient-reported outcome scores and proportion of participants with treatment-emergent adverse events. Biospecimens, including blood, buccal, and fecal will be collected longitudinally for future research purposes. Study participants will be offered enrollment in a home spirometry substudy, which explores time to 10% relative FVC decline measured at home, and its comparison with study visit FVC.

Discussion

The sentinel observation of a potential pharmacogenetic interaction between NAC and TOLLIP polymorphism highlights the urgent, unmet need for better, molecularly focused, and precise therapeutic strategies in IPF. The PRECISIONS clinical trial is the first study to use molecularly-focused techniques to identify patients with IPF most likely to benefit from treatment. PRECISIONS has the potential to shift the paradigm in how trials in this condition are designed and executed, and is the first step toward personalized medicine for patients with IPF. Trial Registration ClinicalTrials.gov identifier: NCT04300920. Registered March 9, 2020. https://clinicaltrials.gov/ct2/show/NCT04300920.

Purpose

To preserve photoreceptor cell structure and function in a rodent model of retinitis pigmentosa with P23H rhodopsin by selective inhibition of the mutant rhodopsin allele using a second generation antisense oligonucleotide (ASO).

Methods

Wild-type mice and rats were treated with ASO by intravitreal (IVT) injection and rhodopsin mRNA and protein expression were measured. Transgenic rats expressing the murine P23H rhodopsin gene (P23H transgenic rat Line 1) were administered either a mouse-specific P23H ASO or a control ASO. The contralateral eye was injected with PBS and used as a comparator control. Electroretinography (ERG) measurements and analyses of the retinal outer nuclear layer were conducted and correlated with rhodopsin mRNA levels.

Results

Rhodopsin mRNA and protein expression was reduced after a single ASO injection in wild-type mice with a rhodopsin-specific ASO. Transgenic rat eyes that express a murine P23H rhodopsin gene injected with a murine P23H ASO had a 181 ± 39% better maximum amplitude response (scotopic a-wave) as compared with contralateral PBS-injected eyes; the response in control ASO eyes was not significantly different from comparator contralateral eyes. Morphometric analysis of the outer nuclear layer showed a significantly thicker nuclear layer in eyes injected with murine P23H ASO (18%) versus contralateral PBS-injected eyes.

Conclusions

Allele-specific ASO-mediated knockdown of mutant P23H rhodopsin expression slowed the rate of photoreceptor degeneration and preserved the function of photoreceptor cells in eyes of the P23H rhodopsin transgenic rat. Our data indicate that ASO treatment is a potentially effective therapy for the treatment of retinitis pigmentosa.

Protein tyrosine phosphatase (PTP)-1B antagonizes insulin signaling and is a potential therapeutic target for insulin resistance associated with obesity and type 2 diabetes. To date, studies of PTP-1B have been limited by the availability of specific antagonists; however, treatment of rodents with antisense oligonucleotides (ASOs) directed against PTP-1B improves insulin sensitivity, inhibits lipogenic gene expression, and reduces triglyceride accumulation in liver and adipose tissue. Here we investigated ASO-mediated PTP-1B inhibition in primates. First, PTP-1B ASO (ISIS 113715) dose-dependently inhibited PTP-1B mRNA and protein expression in cultured monkey hepatocytes. Subcutaneous administration of ISIS 113715 reduced PTP-1B mRNA expression in liver and adipose tissue of normal-weight monkeys by 40-50% and improved insulin sensitivity during an iv glucose tolerance test (IVGTT). In obese, insulin-resistant rhesus monkeys, treatment with 20 mg/kg ISIS 113715 for 4 wk reduced fasting concentrations of insulin and glucose and reduced insulin responses during an IVGTT. In these animals, adiponectin concentrations were also increased by 70%, most of which was an increase of high-molecular-weight oligomers. These effects were not observed in monkeys on a lower, dose-escalation regimen (1-10 mg/kg over 9 wk). Overall, the increase of adiponectin concentrations during ISIS 113715 treatment was correlated with the lowering of insulin responses during IVGTT (r = -0.47, P = 0.042). These results indicate that inhibition of PTP-1B with ASOs such as ISIS 113715 may be a viable approach for the treatment and prevention of obesity-associated insulin resistance and type 2 diabetes because they potently increase adiponectin concentrations in addition to improving insulin sensitivity.

Rationale

Daily azithromycin decreases acute exacerbations of chronic obstructive pulmonary disease (AECOPD), but long-term side effects are unknown.

Objectives

To identify the types of exacerbations most likely to be reduced and clinical subgroups most likely to benefit from azithromycin, 250 mg daily, added to usual care.

Methods

Enrollment criteria included irreversible airflow limitation and AECOPD requiring corticosteroids, emergency department visit, or hospitalization in the prior year or use of supplemental oxygen. Recurrent events and cumulative incidence analyses compared treatment received for AECOPD by randomization group, stratified by subgroups of interest. Cox proportional hazards models estimated treatment effects in subgroups adjusted for age, sex, smoking status, FEV1% predicted, concomitant COPD medications, and oxygen use.

Measurements and main results

Azithromycin was most effective in reducing AECOPD requiring both antibiotic and steroid treatment (n = 1,113; cumulative incidence analysis, P = 0.0002; recurrent events analysis, P = 0.002). No difference in treatment response by sex (P = 0.75), presence of chronic bronchitis (P = 0.19), concomitant inhaled therapy (P = 0.29), or supplemental oxygen use (P = 0.23) was observed. Older age and milder Global Initiative for Chronic Obstructive Lung Disease stage were associated with better treatment response (P = 0.02 and 0.04, respectively). A significant interaction between treatment and current smoking was seen (P = 0.03) and azithromycin did not reduce exacerbations in current smokers (hazard ratio, 0.99; 95% confidence interval, 0.71-1.38; P = 0.95).

Conclusions

Azithromycin is most effective in preventing AECOPD requiring both antibiotic and steroid treatment. Adjusting for confounders, we saw no difference in efficacy by sex, history of chronic bronchitis, oxygen use, or concomitant COPD therapy. Greater efficacy was seen in older patients and milder Global Initiative for Chronic Obstructive Lung Disease stages. We found little evidence of treatment effect among current smokers. Clinical trial registered with www.clinicaltrials.gov (NCT0011986 and NCT00325897).

Background

The identification of smoking-related lung disease in current and former smokers with normal FEV₁ is complex, leading to debate regarding using a ratio of forced expiratory volume in 1 s to forced vital capacity (FEV₁/FVC) of less than 0.70 versus the predicted lower limit of normal (LLN) for diagnosis of airflow obstruction. We hypothesized that the discordant group of ever-smokers with FEV₁/FVC between the LLN and 0.70 is heterogeneous, and aimed to characterize the burden of smoking-related lung disease in this group.

Methods

We compared spirometry, chest CT characteristics, and symptoms between 161 ever-smokers in the discordant group and 940 ever-smokers and 190 never-smokers with normal FEV₁ and FEV₁/FVC > 0.70 in the SPIROMICS cohort. We also estimated sensitivity and specificity for diagnosing objective radiographic evidence of chronic obstructive pulmonary disease (COPD) using different FEV₁/FVC criteria thresholds.

Results

The discordant group had more CT defined emphysema and non-emphysematous gas trapping, lower post-bronchodilator FEV₁ and FEF_25-75, and higher respiratory medication use compared with the other two groups. Within the discordant group, 44% had radiographic CT evidence of either emphysema or non-emphysematous gas trapping; an FEV₁/FVC threshold of 0.70 has greater sensitivity but lower specificity compared with LLN for identifying individuals with CT abnormality.

Conclusions

Ever-smokers with normal FEV₁ and FEV₁/FVC <  0.70 but > LLN are a heterogeneous group that includes significant numbers of individuals with and without radiographic evidence of smoking-related lung disease. These findings emphasize the limitations of diagnosing COPD based on spirometric criteria alone.

Background

Growth differentiation factor-15 (GDF-15), a cytokine associated with cardiovascular mortality, increases during chronic obstructive pulmonary disease (COPD) exacerbations, but any role in stable COPD is unknown. We tested associations between GDF-15 and subclinical coronary atherosclerosis, assessed by coronary artery calcium (CAC) score, in COPD subjects free of clinical cardiovascular disease (CVD).

Methods

Cross-sectional analysis of COPD participants (GOLD stages 2-4) in the COPDGene cohort without CVD at enrollment, using baseline CAC (from non-EKG-gated chest computed tomography) and plasma GDF-15 (by custom ELISA). We used multinomial logistic modeling of GDF-15 associations with CAC, adjusting for demographics, baseline risk (calculated using the HEART: Personal Heart Early Assessment Risk Tool (Budoff et al. 114:1761-1791, 2006) score), smoking history, measures of airflow obstruction, emphysema and airway disease severity.

Results

Among 694 participants with COPD (47% women, mean age 63.6 years) mean GDF-15 was 1,304 pg/mL, and mean CAC score was 198. Relative to the lower GDF-15 tertile, higher tertiles showed bivariate association with increasing CAC score (mid tertile odds ratio [OR] 1.80, 95% confidence interval [CI] 1.29, 2.51; higher tertile OR 2.86, CI 2.04, 4.02). This association was maintained after additionally adjusting for baseline CVD risk, for co-morbidities and descriptors of COPD severity and impact, markers of cardiac stress (N-terminal pro-B-type natriuretic peptide, troponin T) and of inflammation (Interleukin-6), and in subgroup analysis excluding men, diabetics, current smokers or those with limited ambulation.

Conclusions

In ever-smokers with COPD free of clinical CVD, GDF-15 contributes independently to subclinical coronary atherosclerosis.

Trial registration

ClinicalTrials.gov, NCT00608764 . Registered 28 January 2008.

Metabolomics is an emerging science that can inform pathogenic mechanisms behind clinical phenotypes in COPD. We aimed to understand disturbances in the serum metabolome associated with respiratory outcomes in ever-smokers from the SPIROMICS cohort. We measured 27 serum metabolites, mostly amino acids, by 1H-nuclear magnetic resonance spectroscopy in 157 white ever-smokers with and without COPD. We tested the association between log-transformed metabolite concentrations and one-year incidence of respiratory exacerbations after adjusting for age, sex, current smoking, body mass index, diabetes, inhaled or oral corticosteroid use, study site and clinical predictors of exacerbations, including FEV1% predicted and history of exacerbations. The mean age of participants was 53.7 years and 58% had COPD. Lower concentrations of serum amino acids were independently associated with 1-year incidence of respiratory exacerbations, including tryptophan (β = -4.1, 95% CI [-7.0; -1.1], p = 0.007) and the branched-chain amino acids (leucine: β = -6.0, 95% CI [-9.5; -2.4], p = 0.001; isoleucine: β = -5.2, 95% CI [-8.6; -1.8], p = 0.003; valine: β = -4.1, 95% CI [-6.9; -1.4], p = 0.003). Tryptophan concentration was inversely associated with the blood neutrophil-to-lymphocyte ratio (p = 0.03) and the BODE index (p = 0.03). Reduced serum amino acid concentrations in ever-smokers with and without COPD are associated with an increased incidence of respiratory exacerbations.

Nonhuman primates living in proximity to humans increase risks for sylvatic arbovirus transmission. We collected serum samples from nonhuman primates in Hlawga National Park near Yangon, Myanmar, and detected antibodies against chikungunya (33%) and Japanese encephalitis (4%) viruses. Buffer zones between primate and human communities might reduce cross-species arbovirus transmission.

Rationale

Ever-smokers without airflow obstruction scores greater than or equal to 10 on the COPD Assessment Test (CAT) still have frequent acute respiratory disease events (exacerbation-like), impaired exercise capacity, and imaging abnormalities. Identification of these subjects could provide new opportunities for targeted interventions.

Objectives

We hypothesized that the four respiratory-related items of the CAT might be useful for identifying such individuals, with discriminative ability similar to CAT, which is an eight-item questionnaire used to assess chronic obstructive pulmonary disease impact, including nonrespiratory questions, with scores ranging from 0 to 40.

Methods

We evaluated ever-smoker participants in the Subpopulations and Intermediate Outcomes in COPD Study without airflow obstruction (FEV₁/FVC ≥0.70; FVC above the lower limit of normal). Using the area under the receiver operating characteristic curve, we compared responses to both CAT and the respiratory symptom-related CAT items (cough, phlegm, chest tightness, and breathlessness) and their associations with longitudinal exacerbations. We tested agreement between the two strategies (κ statistic), and we compared demographics, lung function, and symptoms among subjects identified as having high symptoms by each strategy.

Results

Among 880 ever-smokers with normal lung function (mean age, 61 yr; 52% women) and using a CAT cutpoint greater than or equal to 10, we classified 51.8% of individuals as having high symptoms, 15.3% of whom experienced at least one exacerbation during 1-year follow-up. After testing sensitivity and specificity of different scores for the first four questions to predict any 1-year follow-up exacerbation, we selected cutpoints of 0-6 as representing a low burden of symptoms versus scores of 7 or higher as representing a high burden of symptoms for all subsequent comparisons. The four respiratory-related items with cutpoint greater than or equal to 7 selected 45.8% participants, 15.6% of whom experienced at least one exacerbation during follow-up. The two strategies largely identified the same individuals (agreement, 88.5%; κ = 0.77; P < 0.001), and the proportions of high-symptoms subjects who had severe dyspnea were similar between CAT and the first four CAT questions (25.9% and 26.8%, respectively), as were the proportions reporting impaired quality of life (66.9% and 70.5%, respectively) and short walking distance (22.4% and 23.1%, respectively). There was no difference in area under the receiver operating characteristic curve to predict 1-year follow-up exacerbations (CAT score ≥10, 0.66; vs. four respiratory items from CAT ≥7 score, 0.65; P = 0.69). Subjects identified by either method also had more depression/anxiety symptoms, poor sleep quality, and greater fatigue.

Conclusions

Four CAT items on respiratory symptoms identified high-risk symptomatic ever-smokers with preserved spirometry as well as the CAT did. These data suggest that simpler strategies can be developed to identify these high-risk individuals in primary care.