Hypoxia Inducible Factor 1A Supports a Pro-Fibrotic Phenotype Loop in Idiopathic Pulmonary Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The IPF-conditioned matrix (IPF-CM) system enables the study of matrix-fibroblast interplay. While effective at slowing fibrosis, nintedanib has limitations and the mechanism is not fully elucidated. In the current work, we explored the underlying signaling pathways and characterized nintedanib involvement in the IPF-CM fibrotic process. Results were validated using IPF patient samples and bleomycin-treated animals with/without oral and inhaled nintedanib. IPF-derived primary human lung fibroblasts (HLFs) were cultured on Matrigel and then cleared using NH4OH, creating the IPF-CM. Normal HLF-CM served as control. RNA-sequencing, PCR and western-blots were performed. HIF1α targets were evaluated by immunohistochemistry in bleomycin-treated rats with/without nintedanib and in patient samples with IPF. HLFs cultured on IPF-CM showed over-expression of 'HIF1α signaling pathway' (KEGG, p < 0.0001), with emphasis on SERPINE1 (PAI-1), VEGFA and TIMP1. IPF patient samples showed high HIF1α staining, especially in established fibrous tissue. PAI-1 was overexpressed, mainly in alveolar macrophages. Nintedanib completely reduced HIF1α upregulation in the IPF-CM and rat-bleomycin models. IPF-HLFs alter the extracellular matrix, thus creating a matrix that further propagates an IPF-like phenotype in normal HLFs. This pro-fibrotic loop includes the HIF1α pathway, which can be blocked by nintedanib.

Inhaled nintedanib is well-tolerated and delivers key pharmacokinetic parameters required to treat bleomycin-induced pulmonary fibrosis.

Oral nintedanib is marketed for the treatment of idiopathic pulmonary fibrosis (IPF), Systemic Sclerosis-Associated Interstitial Lung Disease and Chronic Fibrosing Interstitial Lung Diseases with a Progressive Phenotype. While effective at slowing fibrosis progression, as an oral medicine nintedanib has limitations. To reduce side effects and maximize efficacy, nintedanib was reformulated as a solution for nebulization and inhaled administration. To predict effectiveness treating IPF, inhalation was used as a tool to dissect the pharmacokinetic components required for nintedanib pulmonary anti-fibrotic activity. Following oral administration, nintedanib extensively partitioned into tissue and exhibited flip-flop pharmacokinetics, whereby resulting lung Cmax and AUC were substantially higher than plasma. By comparison, inhaled nintedanib was capable of delivering an oral-equivalent lung Cmax with lower local and systemic AUC. Using a multi-challenge bleomycin rat model, this distinct inhaled pharmacokinetic profile was dose responsive (0.05, 0.25 and 0.375 mg/kg), delivering oral-superior pulmonary anti-fibrotic activity with an equivalent delivered lung Cmax (QD inhaled 0.375 mg/kg versus BID oral 60 mg/kg). Possibly assisting this improvement, the infrequent high inhaled dose also improved bleomycin-challenged animal weight gain to levels equivalent to sham. By comparison, BID oral weight gain was substantially less than controls, suggesting a negative health impact on oral administered animals combating fibrosis. Both oral and inhaled administration exhibited anti-inflammatory activity, with oral achieving significance. In summary, inhalation (short-duration nintedanib lung Cmax without high local or systemic AUC) was well-tolerated and was effective reducing bleomycin-induced pulmonary fibrosis.

A 3-month Safety Assessment of Human Bone Marrow Derived Mesenchymal Stromal Cells Administered Once by the Intramuscular Route to Immunodeficient Mice.

Critical limb ischemia (CLI) represents the severest manifestation of peripheral arterial disease and is a major unmet medical need. This disease occurs when the arterial blood supply within the limb fails to meet the metabolic demands of the resting muscle or tissue, resulting in chronic ischemic rest pain and/or tissue necrosis. Human mesenchymal stromal cells, termed hMSCs, represent an exciting therapeutic modality for the treatment of this disease due to their immunomodulatory and tissue reparative functions. The aim of the study was to assess the preclinical toxicity profile of human bone marrow-derived MSCs in support of their use as a treatment for CLI. A 3-month toxicity study was carried out under good laboratory practices in immunodeficient mice who received, intramuscularly, a single dose of 3 × 105 (approximately 15 × 106 cells/kg) hMSCs manufactured under good manufacturing practices. No significant changes in body weight, food consumption, clinical signs, or histopathological changes were observed in the hMSC-treated mice in comparison to the controls. These results highlight that the administration of hMSCs during the 3-month study period was well tolerated and not associated with any test item-related tumors. This data set supported the initiation of a phase 1b first in human study in "no option" for revascularization patients with CLI.

Inhaled biopharmaceutical drug development: nonclinical considerations and case studies.

Biopharmaceuticals are complex molecules often manufactured from living systems and their specificity and novelty holds great promise for the treatment of chronic diseases for which there are currently no cures. The inhalation route of biopharmaceutical drug delivery is attractive because the large surface area of the lung, and close proximity of the alveolar and vascular systems, maximizes the potential for drug delivery to the lung and/or systemic circulation. In addition, costs of delivery to the patient are potentially much reduced, in comparison with parental administration, since inhalation is non-invasive and likely to promote patient compliance. However, in comparison with small molecule drug development, developing an inhaled biopharmaceutical that is effective and safe for human use is associated with many challenges. This review considers some general principles of drug delivery to lung and issues associated with the translation of proof of concept studies to toxicology safety studies (e.g. aerosol generation, species selection, exaggerated pharmacology, and immunogenicity). This review also presents a summary of nonclinical and clinical data from inhaled biopharmaceuticals which are either marketed for human use or in Phase II clinical trials (e.g. DNase, insulin, human growth hormone, vaccines, therapeutic plasmid DNA complexes).

Differential alveolar epithelial injury and protein expression in pneumococcal pneumonia.

The integrity of the alveolar epithelium is a key factor in the outcome of acute lung injury. Here, we investigate alveolar epithelial injury and the expression of epithelial-selective markers in Streptococcus pneumoniae-induced acute lung injury. S. pneumoniae was instilled into rat lungs and alveolar type I (RTI(40)/podoplanin, MMC6 antigen) and alveolar type II (MMC4 antigen, surfactant protein D, pro-surfactant protein C, RTII(70)) cell markers were quantified in lavage fluid and lung tissue at 24 and 72 hours. The alveolar epithelium was also examined using electron, confocal, and light microscopy. S. pneumoniae induced an acute inflammatory response as assessed by increased total protein, SP-D, and neutrophils in lavage fluid. Biochemical and morphological studies demonstrated morphologic injury to type II cells but not type I cells. In particular, the expression of RTI(40)/podoplanin was dramatically reduced, on the surface of type I cells, in the absence of morphologic injury. These data demonstrate that type II cell damage can occur in the absence of type I cell injury without affecting the ability of the lung to return to a normal morphology. These data also demonstrate that RTI(40)/podoplanin is not a type I cell phenotypic marker in experimental acute lung injury caused by S. pneumoniae. Given that RTI(40)/podoplanin is an endogenous ligand for the C-type lectin receptor and this receptor plays a role in platelet aggregation and neutrophil activation, we hypothesize that the reduction of RTI(40)/podoplanin on type I cells might be important for the regulation of platelet and/or neutrophil function in experimental acute lung injury.

General and arthritis-specific barriers to moderate physical activity in women with arthritis.

BACKGROUND: most women with arthritis are insufficiently active, despite the health benefits derived from participation in moderate physical activity (MPA). Understanding perceived barriers that make it difficult for women with arthritis to be active is needed to inform interventions. Barriers are often assessed through investigator-provided lists, containing mainly general, personal, and situational barriers, common across populations (e.g., lack of time). However, identifying an encompassing range of problematic barriers that challenge women's activity participation is needed. Such barriers may be general and arthritis specific (e.g., pain). Problematic barriers may be best identified through assessment of whether individuals actually experience these barriers (i.e., are present) and, for present barriers, their extent of limitation on activity. Thus, the primary study purpose was to examine whether the presence of general and arthritis-specific barrier categories and the limitation of these overall categories were significant predictors of participation in MPA among women with arthritis (n = 248). METHODS: on-line measures of barriers and MPA were completed. FINDINGS: a multiple regression model predicting activity was significant (r(2)(adjusted) = .22; p < .01). Both arthritis-specific and general barrier limitation were the strongest predictors of activity. Arthritis-specific personal barriers were reported as being present most often (e.g., pain). CONCLUSION: interventions should identify problematic barriers, taking into account the extent to which both general and disease-specific barriers limit activity, and then target their alleviation through the use of coping strategies as a way to improve activity adherence and health among women with arthritis.

Validity of recall of tobacco use in two prospective cohorts.

This project studied the convergent validity of current recall of tobacco-related health behaviors, compared with prospective self-report collected earlier at two sites. Cohorts were from the Oregon Research Institute at Eugene (N = 346, collected 19.5 years earlier) and the University of Pittsburgh, Pennsylvania (N = 294, collected 3.9 years earlier). Current recall was examined through computer-assisted interviews with the Lifetime Tobacco Use Questionnaire from 2005 through 2008. Convergent validity estimates demonstrated variability. Validity estimates of some tobacco use measures were significant for Oregon subjects (age at first cigarette, number of cigarettes/day, quit attempts yes/no and number of attempts, and abstinence symptoms at quitting; all P < 0.03). Validity estimates of Pittsburgh subjects' self-reports of tobacco use and abstinence symptoms were significant (P < 0.001) for all tobacco use and abstinence symptoms and for responses to initial use of tobacco. These findings support the utility of collecting recalled self-report information for reconstructing salient lifetime health behaviors and underscore the need for careful interpretation.

Clinical trial participant characteristics and saliva and DNA metrics.

BACKGROUND: Clinical trial and epidemiological studies need high quality biospecimens from a representative sample of participants to investigate genetic influences on treatment response and disease. Obtaining blood biospecimens presents logistical and financial challenges. As a result, saliva biospecimen collection is becoming more frequent because of the ease of collection and lower cost. This article describes an assessment of saliva biospecimen samples collected through the mail, trial participant demographic and behavioral characteristics, and their association with saliva and DNA quantity and quality. METHODS: Saliva biospecimens were collected using the Oragene(R) DNA Self-Collection Kits from participants in a National Cancer Institute funded smoking cessation trial. Saliva biospecimens from 565 individuals were visually inspected for clarity prior to and after DNA extraction. DNA samples were then quantified by UV absorbance, PicoGreen(R), and qPCR. Genotyping was performed on 11 SNPs using TaqMan(R) SNP assays and two VNTR assays. Univariate, correlation, and analysis of variance analyses were conducted to observe the relationship between saliva sample and participant characteristics. RESULTS: The biospecimen kit return rate was 58.5% among those invited to participate (n = 967) and 47.1% among all possible COMPASS participants (n = 1202). Significant gender differences were observed with males providing larger saliva volume (4.7 vs. 4.5 ml, p = 0.019), samples that were more likely to be judged as cloudy (39.5% vs. 24.9%, p < 0.001), and samples with greater DNA yield as measured by UV (190.0 vs. 138.5, p = 0.002), but reduced % human DNA content (73.2 vs. 77.6 p = 0.005) than females. Other participant characteristics (age, self-identified ethnicity, baseline cigarettes per day) were associated with saliva clarity. Saliva volume and saliva and DNA clarity were positively correlated with total DNA yield by all three quantification measurements (all r > 0.21, P < 0.001), but negatively correlated with % human DNA content (saliva volume r = -0.148 and all P < 0.010). Genotyping completion rate was not influenced by saliva or DNA clarity. CONCLUSION: Findings from this study show that demographic and behavioral characteristics of smoking cessation trial participants have significant associations with saliva and DNA metrics, but not with the performance of TaqMan(R) SNP or VNTR genotyping assays. TRIAL REGISTRATION: COMPASS; registered as NCT00301145 at clinicaltrials.gov.

Test-retest reliability of web-based retrospective self-report of tobacco exposure and risk.

BACKGROUND: Retrospectively collected data about the development and maintenance of behaviors that impact health are a valuable source of information. Establishing the reliability of retrospective measures is a necessary step in determining the utility of that methodology and in studying behaviors in the context of risk and protective factors. OBJECTIVE: The goal of this study was to examine the reliability of self-report of a specific health-affecting behavior, tobacco use, and its associated risk and protective factors as examined with a Web-based questionnaire. METHODS: Core tobacco use and risk behavior questions in the Lifetime Tobacco Use Questionnaire-a closed, invitation-only, password-controlled, Web-based instrument-were administered at a 2-month test-retest interval to a convenience sample of 1229 respondents aged 18 to 78 years. Tobacco use items, which covered cigarettes, cigars, smokeless tobacco, and pipe tobacco, included frequency of use, amount used, first use, and a pack-years calculation. Risk-related questions included family history of tobacco use, secondhand smoke exposure, alcohol use, and religiosity. RESULTS: Analyses of test-retest reliability indicated modest (.30 to .49), moderate (.50 to .69), or high (.70 to 1.00) reliability across nearly all questions, with minimal reliability differences in analyses by sex, age, and income grouping. Most measures of tobacco use history showed moderate to high reliability, particularly for age of first use, age of first weekly and first daily smoking, and age at first or only quit attempt. Some measures of family tobacco use history, secondhand smoke exposure, alcohol use, and religiosity also had high test-retest reliability. Reliability was modest for subjective response to first use. CONCLUSIONS: The findings reflect the stability of retrospective recall of tobacco use and risk factor self-report responses in a Web-questionnaire context. Questions that are designed and tested with psychometric scrutiny can yield reliable results in a Web setting.

Reliability of adult retrospective recall of lifetime tobacco use.

Retrospective assessment of tobacco use underlies much of the data collected in epidemiological and genetic epidemiological research. Although individuals are asked to report lifetime tobacco use for periods spanning months to decades, the test-retest reliability intervals of the instruments often span only a few weeks to several months. The present analyses examined the test-retest reliability of retrospective tobacco use measures, including details of first use, circumstances of first use, and initial subjective reactions. The questions were part of the Lifetime Tobacco Use Questionnaire (LTUQ), a Web-based questionnaire designed to assess use of most forms of tobacco or nicotine retrospectively across the lifespan. A convenience sample of 236 men and women with history of tobacco use (Time 1 mean age, 44.9 years; 74.2% females; 75.1% regular monthly tobacco use) responded verifiably to invitations to self-administer the LTUQ two times, 2 years apart. Test-retest reliability analyses reflected high reliability for salient tobacco-use questions. Acceptable levels of reliability were observed for initial subjective reactions to smoking, if the scaled response options were dichotomized. Few differences in the reliability of recall were apparent between sexes and between age groups. These results indicate that recall of important tobacco use information can form a reliable basis for research.

Barriers to moderate physical activity in adult lesbians.

Adult lesbians are not sufficiently physically active to achieve physical and psychological health benefits. Lesbians are one of the least understood minority groups. Therefore, the purpose of this study was to use an ecological framework to identify factors internal to individuals and present in their social environments that may impede participation in regular physical activity. Twenty-one self-identified lesbians aged 22 to 61 years participated in one of four focus groups. The lesbian participants reported many general barriers (i.e., obstacles to participation regardless of sexual orientation) similar to previous research with other populations of women, not stratified by sexual orientation, such as being too tired and the lack of a physical activity partner. A number of lesbian-specific barriers (i.e., obstacles unique to being a lesbian) were also identified, such as the lack of lesbian-focused physical activity groups and the lack of same-sex family memberships to fitness facilities. In conclusion, for many of the general barriers, some of the proven and effective traditional intervention strategies are likely to be effective in increasing physical activity participation rates in the lesbian population. However, barriers related to sexual orientation are likely deeply entrenched in the socio-cultural system of American society and require a societal rethinking of attitudes towards lesbians, a cultural change that is not as easily amenable to traditional health promotion interventions.

Coexpression of RTI40 with alveolar epithelial type II cell proteins in lungs following injury: identification of alveolar intermediate cell types.

Injured alveolar epithelial type (AT) I cells are replaced following the proliferation and transformation of ATII cells to new ATI cells. RTI(40) is an ATI cell-specific protein required for normal lung development. We hypothesized that intermediate cell types in the ATII-to-ATI cell transformation would coexpress RTI(40) and ATII cell-selective proteins. To test this hypothesis, we used a rat model of Staphylococcus aureus-induced acute lung injury and a panel of ATI and ATII cell-specific and -selective antibodies. S. aureus induced an acute inflammatory reaction that was resolving by day 3 postinoculation. At day 3 postinoculation, the alveolar wall was thickened secondary to ATII cell hyperplasia. With the use of confocal microscopy, there was a fivefold increase in the fractional surface area of alveolar walls stained with ATII cell membrane proteins (RTII(70) and MMC4) and a decrease in the fractional surface area associated with RTI(40)-expressing cells. S. aureus-treated lungs also contained unique cell types that coexpressed the RTI(40) and ATII markers RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells. These cells were not observed in control lungs. RTI(40)/MMC4-positive cells were also found in cultured ATII cells before they transformed to an ATI-like phenotype. Our data suggest that RTI(40)/MMC4/RTII(70)- and RTI(40)/MMC4-positive cells are intermediates in the ATII-to-ATI cell transformation. These data also suggest that the coexpression of RTI(40) with ATII cell proteins may be used to identify and investigate ATII cell transdifferentiation to ATI cells following injury.

Pharmacogenetics of nicotine metabolism in twins: methods and procedures.

This article describes a pharmacogenetic investigation of nicotine metabolism in twins. One hundred and thirty-nine twin pairs (110 monozygotic and 29 dizygotic) were recruited and assessed for smoking status, zygosity, and health conditions known or suspected to affect drug metabolism. Participants underwent a 30-minute infusion of stable isotope-labeled nicotine and its major metabolite, cotinine, followed by an 8-hour in-hospital stay. Blood and urine samples were taken at regular intervals for analysis of nicotine, cotinine, and metabolites by gas chromatography-mass spectrometry or liquid chromatography-mass spectrometry and subsequent characterization of pharmacokinetic phenotypes. DNA was genotyped to confirm zygosity and for variation in the primary gene involved in nicotine metabolism, CYP2A6. Univariate and multivariate biometric analyses planned for the future will determine genetic and environmental influences on each pharmacokinetic measure individually and in combination with each other, and in the presence and absence of covariates, including measured genotype. When the analyses are completed, this study will result in a more complete characterization of the impact of genetic and environmental influences on nicotine and cotinine metabolic pathways than has heretofore been reported. The approach taken, with its use of a quantitative model of nicotine metabolism, highly refined metabolic phenotypes, measured genotype, and advanced tools for biometric genetic analysis, provides a model for the use of twins in next-generation studies of complex drug-metabolism phenotypes.

Increased virulence of a fibronectin-binding protein mutant of Staphylococcus aureus in a rat model of pneumonia.

Fibronectin-binding proteins mediate Staphylococcus aureus internalization into nonphagocytic cells in vitro. We have investigated whether fibronectin-binding proteins are virulence factors in the pathogenesis of pneumonia by using S. aureus strain 8325-4 and isogenic mutants in which fibronectin-binding proteins were either deleted (DU5883) or overexpressed [DU5883(pFnBPA4)]. We first demonstrated that fibronectin-binding proteins mediate S. aureus internalization into alveolar epithelial cells in vitro and that S. aureus internalization into alveolar epithelial cells requires actin rearrangement and protein kinase activity. Second, we established a rat model of S. aureus-induced pneumonia and measured lung injury and bacterial survival at 24 and 96 h postinoculation. S. aureus growth and the extent of lung injury were both increased in rats inoculated with the deletion mutant (DU5883) in comparison with rats inoculated with the wild-type (8325-4) and the fibronectin-binding protein-overexpressing strain DU5883(pFnBPA4) at 24 h postinfection. Morphological evaluation of infected lungs at the light and electron microscopic levels demonstrated that S. aureus was present within neutrophils from both 8325-4- and DU5883-inoculated lungs. Our data suggest that fibronectin-binding protein-mediated internalization into alveolar epithelial cells is not a virulence mechanism in a rat model of pneumonia. Instead, our data suggest that fibronectin-binding proteins decrease the virulence of S. aureus in pneumonia.