CD151 Maintains Endolysosomal Protein Quality to Inhibit Vascular Inflammation.

BACKGROUND: Tetraspanin CD151 is highly expressed in endothelia and reinforces cell adhesion, but its role in vascular inflammation remains largely unknown. METHODS: In vitro molecular and cellular biological analyses on genetically modified endothelial cells, in vivo vascular biological analyses on genetically engineered mouse models, and in silico systems biology and bioinformatics analyses on CD151-related events. RESULTS: Endothelial ablation of Cd151 leads to pulmonary and cardiac inflammation, severe sepsis, and perilous COVID-19, and endothelial CD151 becomes downregulated in inflammation. Mechanistically, CD151 restrains endothelial release of proinflammatory molecules for less leukocyte infiltration. At the subcellular level, CD151 determines the integrity of multivesicular bodies/lysosomes and confines the production of exosomes that carry cytokines such as ANGPT2 (angiopoietin-2) and proteases such as cathepsin-D. At the molecular level, CD151 docks VCP (valosin-containing protein)/p97, which controls protein quality via mediating deubiquitination for proteolytic degradation, onto endolysosomes to facilitate VCP/p97 function. At the endolysosome membrane, CD151 links VCP/p97 to (1) IFITM3 (interferon-induced transmembrane protein 3), which regulates multivesicular body functions, to restrain IFITM3-mediated exosomal sorting, and (2) V-ATPase, which dictates endolysosome pH, to support functional assembly of V-ATPase. CONCLUSIONS: Distinct from its canonical function in strengthening cell adhesion at cell surface, CD151 maintains endolysosome function by sustaining VCP/p97-mediated protein unfolding and turnover. By supporting protein quality control and protein degradation, CD151 prevents proteins from (1) buildup in endolysosomes and (2) discharge through exosomes, to limit vascular inflammation. Also, our study conceptualizes that balance between degradation and discharge of proteins in endothelial cells determines vascular information. Thus, the IFITM3/V-ATPase-tetraspanin-VCP/p97 complexes on endolysosome, as a protein quality control and inflammation-inhibitory machinery, could be beneficial for therapeutic intervention against vascular inflammation.

Harmful and Potentially Harmful Constituents in E-Liquids and Aerosols from Electronic Nicotine Delivery Systems (ENDS).

In 2012, the U.S. Food & Drug Administration (FDA) published an established list of 93 harmful and potentially harmful constituents (HPHCs) targeting four tobacco product types (cigarettes, cigarette tobacco, roll-your-own tobacco, smokeless tobacco). In 2016, the FDA finalized the deeming rule to regulate electronic nicotine delivery systems (ENDS). However, knowledge gaps exist regarding whether certain HPHCs are present in ENDS e-liquids and aerosols. We identified and addressed these gaps by conducting literature searches and then experimentally quantifying HPHCs in the e-liquid and aerosol of 37 ENDS brands based on gaps in the literature. The literature searches identified 66 e-liquid HPHCs and 68 aerosol HPHCs that have limited to no information regarding the quantifiability of these constituents. A contracted ISO 17025 accredited laboratory performed the HPHC quantifications. The availability of validated analytical methods in the contracted laboratory determined the HPHCs included in the study scope (63/66 for e-liquids, 64/68 for aerosols). Combining the results from the quantifications and literature searches, 36 (39%) and 34 (37%) HPHCs were found quantifiable (≥limit of quantification [LOQ]) in ENDS e-liquids and aerosols, respectively, with 25 HPHCs being quantifiable in both matrices. Quantifiability results imply potential HPHC transfers between matrices, leaching from components, or formations from aerosol generation. The study results can inform the scientific basis for manufacturers and regulators regarding regulatory requirements for HPHC reporting. The HPHC quantities can also inform evaluations of the public health impact of ENDS and public communications regarding ENDS health risks.

Distributed cognition: Theoretical insights and practical applications to health professions education: AMEE Guide No. 159.

Distributed cognition (DCog) is a member of the family of situativity theories that widens the lens of cognition from occurring solely inside the head to being socially, materially and temporally distributed within a dynamic system. The concept of extending the view of cognition to outside the head of a single health professional is relatively new in the healthcare system. DCog has been increasingly used by researchers to describe many ways in which health professionals perform in teams within structured clinical environments to deliver healthcare for patients. In this Guide, we expound ten central tenets of the macro (grand) theory of DCog (1. Cognition is decentralized in a system; 2. The unit of analysis is the system; 3. Cognitive processes are distributed; 4. Cognitive processes emerge from interactions; 5. Cognitive processes are interdependent; 6. Social organization is a cognitive architecture; 7. Division of labour; 8. Social organization is a system of communication; 9. Buffering and filtering; 10. Cognitive processes are encultured) to provide theoretical insights as well as practical applications to the field of health professions education.

The emerging role of NOTCH3 receptor signalling in human lung diseases.

The mammalian respiratory system or lung is a tree-like branching structure, and the main site of gas exchange with the external environment. Structurally, the lung is broadly classified into the proximal (or conducting) airways and the distal alveolar region, where the gas exchange occurs. In parallel with the respiratory tree, the pulmonary vasculature starts with large pulmonary arteries that subdivide rapidly ending in capillaries adjacent to alveolar structures to enable gas exchange. The NOTCH signalling pathway plays an important role in lung development, differentiation and regeneration post-injury. Signalling via the NOTCH pathway is mediated through activation of four NOTCH receptors (NOTCH1-4), with each receptor capable of regulating unique biological processes. Dysregulation of the NOTCH pathway has been associated with development and pathophysiology of multiple adult acute and chronic lung diseases. This includes accumulating evidence that alteration of NOTCH3 signalling plays an important role in the development and pathogenesis of chronic obstructive pulmonary disease, lung cancer, asthma, idiopathic pulmonary fibrosis and pulmonary arterial hypertension. Herein, we provide a comprehensive summary of the role of NOTCH3 signalling in regulating repair/regeneration of the adult lung, its association with development of lung disease and potential therapeutic strategies to target its signalling activity.

TEARS: a longitudinal investigation of the prevalence, psychological associations and trajectory of poststroke emotionalism.

OBJECTIVE: There are few longitudinal studies of poststroke emotionalism (PSE) and our understanding of the psychological associations of PSE is limited, constraining assessment of existing interventions and the development of new therapies. This study aimed to assess the prevalence and course of PSE over the first year poststroke, and its psychological associations. METHODS: Consenting stroke survivors who were physically and cognitively able to participate were assessed within 2 weeks, 6 and 12 months of stroke to determine PSE point prevalence using a diagnostic, semistructured PSE interview (Testing Emotionalism After Recent Stroke-Diagnostic Interview). At the same assessments, neuropsychological and disability status were determined using Hospital Anxiety and Depression Scale, Abbreviated Mental Test, National Institute of Health Stroke Scale, Barthel Index and Euro-Qol. RESULTS: Two hundred and seventy seven stroke survivors were recruited between 1 October 2015 and 30 September 2018. Diagnostic data were available at baseline for 228 of 277 cohort participants. Point prevalence for PSE was 27.2% at 2 weeks; estimated prevalence at 6 months adjusted for baseline was 19.9% and at 12 months 22.3%. PSE was associated with symptoms of anxiety and event-related distress. INTERPRETATION: PSE affects at least one in five stroke patients acutely following their stroke, and continues to affect one in eight longer term. PSE is associated with anxiety and event-related distress but is not simply a manifestation of mood disorder over time. Such psychological correlates may have implications for longer term social rehabilitation.

Sharing the Bandwidth in Cognitively Overloaded Teams and Systems: Mechanistic Insights from a Walk on the Wild Side of Clinical Reasoning.

IssuePrevious work from the diagnostic error literature has provided indirect evidence that faulty clinical reasoning may be the most frequent cause of error when attaching a diagnostic label. The precise mechanisms underlying diagnostic error are unclear and continue to be subject to considerable theory informed debate in the clinical reasoning literature. Evidence: We take a theoretical approach to merging these two worlds of literature by first zooming out using distributed cognition as a social cognitive lens (macro theory) to develop a view of the process and outcome of clinical reasoning occurring in the wild - defined as the integrated clinical workplace - the natural habitat of clinicians working within teams. We then zoom in using the novel combination of cognitive load theory and distributed cognition to provide additional theoretical insights into the potential mechanisms of error. Implications: Through the lenses of distributed cognition and cognitive load theory, we can begin to prospectively investigate how cognitive overload is represented and shared within interprofessional teams over time and space and how this influences clinical reasoning performance and leads to error. We believe that this work will help teams manage cognitive load and prevent error.

Cigarette Smoke Activates NOTCH3 to Promote Goblet Cell Differentiation in Human Airway Epithelial Cells.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the United States and is primarily caused by cigarette smoking. Increased numbers of mucus-producing secretory ("goblet") cells, defined as goblet cell metaplasia or hyperplasia (GCMH), contributes significantly to COPD pathophysiology. The objective of this study was to determine whether NOTCH signaling regulates goblet cell differentiation in response to cigarette smoke. Primary human bronchial epithelial cells (HBECs) from nonsmokers and smokers with COPD were differentiated in vitro on air-liquid interface and exposed to cigarette smoke extract (CSE) for 7 days. NOTCH signaling activity was modulated using 1) the NOTCH/γ-secretase inhibitor dibenzazepine (DBZ), 2) lentiviral overexpression of the NICD3 (NOTCH3-intracellular domain), or 3) NOTCH3-specific siRNA. Cell differentiation and response to CSE were evaluated by quantitative PCR, Western blotting, immunostaining, and RNA sequencing. We found that CSE exposure of nonsmoker airway epithelium induced goblet cell differentiation characteristic of GCMH. Treatment with DBZ suppressed CSE-dependent induction of goblet cell differentiation. Furthermore, CSE induced NOTCH3 activation, as revealed by increased NOTCH3 nuclear localization and elevated NICD3 protein levels. Overexpression of NICD3 increased the expression of goblet cell-associated genes SPDEF and MUC5AC, whereas NOTCH3 knockdown suppressed CSE-mediated induction of SPDEF and MUC5AC. Finally, CSE exposure of COPD airway epithelium induced goblet cell differentiation in a NOTCH3-dependent manner. These results identify NOTCH3 activation as one of the important mechanisms by which cigarette smoke induces goblet cell differentiation, thus providing a novel potential strategy to control GCMH-related pathologies in smokers and patients with COPD.

Carotid Intima-Media Thickness Progression as Surrogate Marker for Cardiovascular Risk: Meta-Analysis of 119 Clinical Trials Involving 100 667 Patients.

BACKGROUND: To quantify the association between effects of interventions on carotid intima-media thickness (cIMT) progression and their effects on cardiovascular disease (CVD) risk. METHODS: We systematically collated data from randomized, controlled trials. cIMT was assessed as the mean value at the common-carotid-artery; if unavailable, the maximum value at the common-carotid-artery or other cIMT measures were used. The primary outcome was a combined CVD end point defined as myocardial infarction, stroke, revascularization procedures, or fatal CVD. We estimated intervention effects on cIMT progression and incident CVD for each trial, before relating the 2 using a Bayesian meta-regression approach. RESULTS: We analyzed data of 119 randomized, controlled trials involving 100 667 patients (mean age 62 years, 42% female). Over an average follow-up of 3.7 years, 12 038 patients developed the combined CVD end point. Across all interventions, each 10 µm/y reduction of cIMT progression resulted in a relative risk for CVD of 0.91 (95% Credible Interval, 0.87-0.94), with an additional relative risk for CVD of 0.92 (0.87-0.97) being achieved independent of cIMT progression. Taken together, we estimated that interventions reducing cIMT progression by 10, 20, 30, or 40 µm/y would yield relative risks of 0.84 (0.75-0.93), 0.76 (0.67-0.85), 0.69 (0.59-0.79), or 0.63 (0.52-0.74), respectively. Results were similar when grouping trials by type of intervention, time of conduct, time to ultrasound follow-up, availability of individual-participant data, primary versus secondary prevention trials, type of cIMT measurement, and proportion of female patients. CONCLUSIONS: The extent of intervention effects on cIMT progression predicted the degree of CVD risk reduction. This provides a missing link supporting the usefulness of cIMT progression as a surrogate marker for CVD risk in clinical trials.

Long-term cardiovascular safety of febuxostat compared with allopurinol in patients with gout (FAST): a multicentre, prospective, randomised, open-label, non-inferiority trial.

BACKGROUND: Febuxostat and allopurinol are urate-lowering therapies used to treat patients with gout. Following concerns about the cardiovascular safety of febuxostat, the European Medicines Agency recommended a post-licensing study assessing the cardiovascular safety of febuxostat compared with allopurinol. METHODS: We did a prospective, randomised, open-label, blinded-endpoint, non-inferiority trial of febuxostat versus allopurinol in patients with gout in the UK, Denmark, and Sweden. Eligible patients were 60 years or older, already receiving allopurinol, and had at least one additional cardiovascular risk factor. Those who had myocardial infarction or stroke in the previous 6 months or who had severe congestive heart failure or severe renal impairment were excluded. After a lead-in phase in which allopurinol dose was optimised towards achieving a serum urate concentration of less than 0·357 mmol/L (<6 mg/dL), patients were randomly assigned (1:1, with stratification according to previous cardiovascular events) to continue allopurinol (at the optimised dose) or start febuxostat at 80 mg/day, increasing to 120 mg/day if necessary to achieve the target serum urate concentration. The primary outcome was a composite of hospitalisation for non-fatal myocardial infarction or biomarker-positive acute coronary syndrome; non-fatal stroke; or cardiovascular death. The hazard ratio (HR) for febuxostat versus allopurinol in a Cox proportional hazards model (adjusted for the stratification variable and country) was assessed for non-inferiority (HR limit 1·3) in an on-treatment analysis. This study is registered with the EU Clinical Trials Register (EudraCT 2011-001883-23) and ISRCTN (ISRCTN72443728) and is now closed. FINDINGS: From Dec 20, 2011, to Jan 26, 2018, 6128 patients (mean age 71·0 years [SD 6·4], 5225 [85·3%] men, 903 [14·7%] women, 2046 [33·4%] with previous cardiovascular disease) were enrolled and randomly allocated to receive allopurinol (n=3065) or febuxostat (n=3063). By the study end date (Dec 31, 2019), 189 (6·2%) patients in the febuxostat group and 169 (5·5%) in the allopurinol group withdrew from all follow-up. Median follow-up time was 1467 days (IQR 1029-2052) and median on-treatment follow-up was 1324 days (IQR 870-1919). For incidence of the primary endpoint, on-treatment, febuxostat (172 patients [1·72 events per 100 patient-years]) was non-inferior to allopurinol (241 patients [2·05 events per 100 patient-years]; adjusted HR 0·85 [95% CI 0·70-1·03], p<0·0001). In the febuxostat group, 222 (7·2%) of 3063 patients died and 1720 (57·3%) of 3001 in the safety analysis set had at least one serious adverse event (with 23 events in 19 [0·6%] patients related to treatment). In the allopurinol group, 263 (8·6%) of 3065 patients died and 1812 (59·4%) of 3050 had one or more serious adverse events (with five events in five [0·2%] patients related to treatment). Randomised therapy was discontinued in 973 (32·4%) patients in the febuxostat group and 503 (16·5%) patients in the allopurinol group. INTERPRETATION: Febuxostat is non-inferior to allopurinol therapy with respect to the primary cardiovascular endpoint, and its long-term use is not associated with an increased risk of death or serious adverse events compared with allopurinol. FUNDING: Menarini, Ipsen, and Teijin Pharma Ltd.

Practical guidance on use of TEARS-Q to diagnose post-stroke emotionalism.

OBJECTIVE: To evaluate, using a classification tree methodology, the ability of the Testing Emotionalism After Recent Stroke - Questionnaire (TEARS-Q) to determine the need for further assessment of post-stroke emotionalism and to identify those whose emotionalism is sufficiently clear that they need assessment for potential intervention. SETTING: Acute stroke units of nine Scottish hospitals in the context of a longitudinal cohort study of post-stroke emotionalism. SUBJECTS: A total of 228 stroke survivors recruited between October 1st 2015 and September 30th 2018, within two weeks of stroke. MEASURES: The measure was the self-report questionnaire TEARS-Q, constructed based on recognised diagnostic features of post-stroke tearful emotionalism. The reference standard was presence/absence of emotionalism on a diagnostic, semi-structured post-stroke emotionalism interview, administered at the same assessment point. RESULTS: Nine of 159 subjects scoring 0 or 1 on TEARS-Q were diagnosed with post-stroke emotionalism on the reference standard, compared to 11 of 21 subjects scoring 2 to 5 on TEARS-Q and 42 of 48 participants scoring 6 and above. Adding age, sex, deprivation, stroke type, stroke severity, mood, cognition, daily functioning and education did not improve the prediction accuracy sufficiently to change the classification tree. CONCLUSION: TEARS-Q reliably identifies those who need no further post-stroke emotionalism assessment, those who need further assessment to clarify diagnosis, and those who almost certainly have post-stroke emotionalism and may benefit from intervention.

Psychometric evaluation of a newly developed measure of emotionalism after stroke (TEARS-Q).

OBJECTIVE: To evaluate, psychometrically, a new measure of tearful emotionalism following stroke: Testing Emotionalism After Recent Stroke - Questionnaire (TEARS-Q). SETTING: Acute stroke units based in nine Scottish hospitals, in the context of a longitudinal cohort study of post-stroke emotionalism. SUBJECTS: A total of 224 clinically diagnosed stroke survivors recruited between October 1st 2015 and September 30th 2018, within 2 weeks of their stroke. MEASURES: The measure was the self-report questionnaire TEARS-Q, constructed based on post-stroke tearful emotionalism diagnostic criteria: (i) increased tearfulness, (ii) crying comes on suddenly, with no warning (iii) crying not under usual social control and (iv) crying episodes occur at least once weekly. The reference standard was presence/absence of emotionalism on a diagnostic, semi-structured post-stroke emotionalism interview, administered at the same assessment point. Stroke, mood, cognition and functional outcome measures were also completed by the subjects. RESULTS: A total of 97 subjects were female, with a mean age 65.1 years. 205 subjects had sustained ischaemic stroke. 61 subjects were classified as mild stroke. TEARS-Q was internally consistent (Cronbach's alpha 0.87). TEARS-Q scores readily discriminated the two groups, with a mean difference of -7.18, 95% CI (-8.07 to -6.29). A cut off score of 2 on TEARS-Q correctly identified 53 of the 61 stroke survivors with tearful emotionalism and 140 of the 156 stroke survivors without tearful emotionalism. One factor accounted for 57% of the item response variance, and all eight TEARS-Q items acceptably discriminated underlying emotionalism. CONCLUSION: TEARS-Q accurately diagnoses tearful emotionalism after stroke.

Non-invasive brain stimulation in Stroke patients (NIBS): A prospective randomized open blinded end-point (PROBE) feasibility trial using transcranial direct current stimulation (tDCS) in post-stroke hemispatial neglect.

Up to 80% of people who experience a right-hemisphere stroke suffer from hemispatial neglect. This syndrome is debilitating and impedes rehabilitation. We carried out a clinical feasibility trial of transcranial direct current stimulation (tDCS) and a behavioural rehabilitation programme, alone or in combination, in patients with neglect. Patients >4 weeks post right hemisphere stroke were randomized to 10 sessions of tDCS, 10 sessions of a behavioural intervention, combined intervention, or a control task. Primary outcomes were recruitment and retention rates, with secondary outcomes effect sizes on measures of neglect and quality of life, assessed directly after the interventions, and at 6 months follow up. Of 288 confirmed stroke cases referred (representing 7% of confirmed strokes), we randomized 8% (0.6% of stroke cases overall). The largest number of exclusions (91/288 (34%)) were due to medical comorbidities that prevented patients from undergoing 10 intervention sessions. We recruited 24 patients over 29 months, with 87% completing immediate post-intervention and 67% 6 month evaluations. We established poor feasibility of a clinical trial requiring repeated hospital-based tDCS within a UK hospital healthcare setting, either with or without behavioural training, over a sustained time period. Future trials should consider intensity, duration and location of tDCS neglect interventions.Trial registration: ClinicalTrials.gov identifier: NCT02401724.

Characteristics and treatment outcomes of newly diagnosed epilepsy in older people: A 30-year longitudinal cohort study.

OBJECTIVES: To describe the clinical characteristics and evaluate the long-term treatment outcomes in older people with newly diagnosed epilepsy over the past 30 years. METHODS: We included patients newly diagnosed with epilepsy and commenced on antiseizure medications (ASMs) at age 65 years or older between July 1982 and October 2012 at the Western infirmary in Glasgow, Scotland. They were followed up until April 2016 or death. Seizure freedom was defined as no seizure for at least 1 year on unchanged medication at the last follow-up. RESULTS: A total of 201 patients (median age 73 years, 59% male) were included. The median duration from initial seizure to starting treatment was 8 months (interquartile range: 3.0-24.0 months); 42.2% (85/201) patients had more than five seizures before commencing treatment. Brain imaging showed potentially epileptogenic lesions in 19.7% (38/193) of patients and other abnormalities in 56.5% (109/193); 78.6% patients (158/201) were seizure-free at the last follow-up, of whom 94.9% were taking monotherapy. Concomitant aspirin use (n = 80) was associated with a lower probability of being seizure-free (relative risk 0.82, 95% confidence interval 0.70-0.97; P = .02). The use of second-generation ASMs as the initial monotherapy increased from 31.5% (23/73) before 2000 to 70.3% (90/128, P < .001) from 2000 onward. However, the seizure freedom rates (67.1% vs 55.5%; P = .35) and intolerable adverse-effect rates (16.4% vs 19.5%; P = .45) did not show any significant difference. SIGNIFICANCE: There was often a long interval between seizure onset and the initiation of treatment in older people with new-onset epilepsy, although the majority responded well to ASM treatment. Brain imaging showed a high rate of abnormalities. Despite the increased use of second-generation ASMs, treatment outcomes in later-onset epilepsy have not improved over time. The possible effect of aspirin on treatment response warrants further investigation.

The Prospective Studies of Atherosclerosis (Proof-ATHERO) Consortium: Design and Rationale.

Atherosclerosis - the pathophysiological mechanism shared by most cardiovascular diseases - can be directly or indirectly assessed by a variety of clinical tests including measurement of carotid intima-media thickness, carotid plaque, -ankle-brachial index, pulse wave velocity, and coronary -artery calcium. The Prospective Studies of Atherosclerosis -(Proof-ATHERO) consortium (https://clinicalepi.i-med.ac.at/research/proof-athero/) collates de-identified individual-participant data of studies with information on atherosclerosis measures, risk factors for cardiovascular disease, and incidence of cardiovascular diseases. It currently comprises 74 studies that involve 106,846 participants from 25 countries and over 40 cities. In summary, 21 studies recruited participants from the general population (n = 67,784), 16 from high-risk populations (n = 22,677), and 37 as part of clinical trials (n = 16,385). Baseline years of contributing studies range from April 1980 to July 2014; the latest follow-up was until June 2019. Mean age at baseline was 59 years (standard deviation: 10) and 50% were female. Over a total of 830,619 person-years of follow-up, 17,270 incident cardiovascular events (including coronary heart disease and stroke) and 13,270 deaths were recorded, corresponding to cumulative incidences of 2.1% and 1.6% per annum, respectively. The consortium is coordinated by the Clinical Epidemiology Team at the Medical University of Innsbruck, Austria. Contributing studies undergo a detailed data cleaning and harmonisation procedure before being incorporated in the Proof-ATHERO central database. Statistical analyses are being conducted according to pre-defined analysis plans and use established methods for individual-participant data meta-analysis. Capitalising on its large sample size, the multi-institutional collaborative Proof-ATHERO consortium aims to better characterise, understand, and predict the development of atherosclerosis and its clinical consequences.

Safety, tolerability, and pharmacology of AB928, a novel dual adenosine receptor antagonist, in a randomized, phase 1 study in healthy volunteers.

Adenosine suppresses antitumor immune responses via A2a and A2b receptors expressed on intratumoral immune cells. This effect is mediated by increased cyclic adenosine 5'-monophosphate (AMP) levels and phosphorylation of cyclic AMP response element binding protein (CREB). We conducted a phase 1, placebo-controlled, single-ascending-dose (SAD) and multiple-ascending-dose (MAD) study to assess the safety, tolerability, pharmacokinetics (PK), including food effect (FE), and pharmacodynamics (PD) of oral AB928, a novel dual A2aR/A2bR antagonist, in healthy volunteers. AB928 doses between 10 and 200 mg once daily and 100 mg twice daily were evaluated. The study enrolled 85 subjects (randomized 3:1, AB928:placebo), 40 each in the SAD and MAD cohorts, and 5 in the FE cohort. AB928 was well tolerated up to the highest dose tested and did not affect any physiologic parameters potentially sensitive to adenosine inhibition. No safety concern was identified. The PK profile of AB928 was linear and dose-proportional, and a clear PK/PD correlation was demonstrated. Significant inhibition of adenosine receptor-mediated phosphorylated CREB was observed at peak plasma concentrations in all dose cohorts and at trough plasma concentrations in the higher-dose cohorts. AB928 plasma levels ≥1 µM were associated with ≥90% adenosine receptor inhibition. In the postprandial state, the rate of AB928 absorption decreased but the extent of absorption was unchanged. Together, these data support further clinical development of oral AB928 in cancer patients.

Role of KRAS in regulating normal human airway basal cell differentiation.

BACKGROUND: KRAS is a GTPase that activates pathways involved in cell growth, differentiation and survival. In normal cells, KRAS-activity is tightly controlled, but with specific mutations, the KRAS protein is persistently activated, giving cells a growth advantage resulting in cancer. While a great deal of attention has been focused on the role of mutated KRAS as a common driver mutation for lung adenocarcinoma, little is known about the role of KRAS in regulating normal human airway differentiation. METHODS: To assess the role of KRAS signaling in regulating differentiation of the human airway epithelium, primary human airway basal stem/progenitor cells (BC) from nonsmokers were cultured on air-liquid interface (ALI) cultures to mimic the airway epithelium in vitro. Modulation of KRAS signaling was achieved using siRNA-mediated knockdown of KRAS or lentivirus-mediated over-expression of wild-type KRAS or the constitutively active G12 V mutant. The impact on differentiation was quantified using TaqMan quantitative PCR, immunofluorescent and immunohistochemical staining analysis for cell type specific markers. Finally, the impact of cigarette smoke exposure on KRAS and RAS protein family activity in the airway epithelium was assessed in vitro and in vivo. RESULTS: siRNA-mediated knockdown of KRAS decreased differentiation of BC into secretory and ciliated cells with a corresponding shift toward squamous cell differentiation. Conversely, activation of KRAS signaling via lentivirus mediated over-expression of the constitutively active G12 V KRAS mutant had the opposite effect, resulting in increased secretory and ciliated cell differentiation and decreased squamous cell differentiation. Exposure of BC to cigarette smoke extract increased KRAS and RAS protein family activation in vitro. Consistent with these observations, airway epithelium brushed from healthy smokers had elevated RAS activation compared to nonsmokers. CONCLUSIONS: Together, these data suggest that KRAS-dependent signaling plays an important role in regulating the balance of secretory, ciliated and squamous cell differentiation of the human airway epithelium and that cigarette smoking-induced airway epithelial remodeling is mediated in part by abnormal activation of KRAS-dependent signaling mechanisms.

Ontogeny and Biology of Human Small Airway Epithelial Club Cells.

RATIONALE: Little is known about human club cells, dome-shaped cells with dense cytoplasmic granules and microvilli that represent the major secretory cells of the human small airways (at least sixth-generation bronchi). OBJECTIVES: To define the ontogeny and biology of the human small airway epithelium club cell. METHODS: The small airway epithelium was sampled from the normal human lung by bronchoscopy and brushing. Single-cell transcriptome analysis and air-liquid interface culture were used to assess club cell ontogeny and biology. MEASUREMENTS AND MAIN RESULTS: We identified the club cell population by unbiased clustering using single-cell transcriptome sequencing. Principal component gradient analysis uncovered an ontologic link between KRT5 (keratin 5)+ basal cells and SCGB1A1 (secretoglobin family 1A member 1)+ club cells, a hypothesis verified by demonstrating in vitro that a pure population of human KRT5+ SCGB1A1- small airway epithelial basal cells differentiate into SCGB1A1+KRT5- club cells on air-liquid interface culture. Using SCGB1A1 as the marker of club cells, the single-cell analysis identified novel roles for these cells in host defense, xenobiotic metabolism, antiprotease, physical barrier function, monogenic lung disorders, and receptors for human viruses. CONCLUSIONS: These observations provide novel insights into the molecular phenotype and biologic functions of the human club cell population and identify basal cells as the human progenitor cells for club cells.

Cilia dysfunction in lung disease.

A characteristic feature of the human airway epithelium is the presence of ciliated cells bearing motile cilia, specialized cell surface projections containing axonemes composed of microtubules and dynein arms, which provide ATP-driven motility. In the airways, cilia function in concert with airway mucus to mediate the critical function of mucociliary clearance, cleansing the airways of inhaled particles and pathogens. The prototypical disorder of respiratory cilia is primary ciliary dyskinesia, an inherited disorder that leads to impaired mucociliary clearance, to repeated chest infections, and to the progressive destruction of lung architecture. Numerous acquired lung diseases are also marked by abnormalities in both cilia structure and function. In this review we summarize current knowledge regarding airway ciliated cells and cilia, how they function to maintain a healthy epithelium, and how disorders of cilia structure and function contribute to inherited and acquired lung disease.

CC chemokine receptor 2 promotes recruitment of myeloid cells associated with insulin resistance in nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) is a common disease, closely associated with obesity and insulin resistance. We investigated the presence of a subset of myeloid cells associated with metabolic disturbance in the liver of patients with NAFLD and a murine model of obesity-induced liver disease. Gene and protein expression in liver and serum was investigated with RT-PCR or ELISA and correlated to clinical disease. Liver-infiltrating immune cells were isolated from normal or diseased human liver for flow cytometric analysis. In animal experiments, mice were fed a high-fat diet (60% of calories from fat) for 16 wk, or high-fat diet with 30% fructose for 32 wk to induce steatohepatitis and fibrosis. A small molecule inhibitor of CC chemokine receptor 2 (CCR2), CCX872, was administered to some mice. A subset of CD11c+CD206+ immune cells was enriched in human liver tissue, and greater infiltration was observed in NAFLD. The presence of CD11c+CD206+ myeloid cells correlated with systemic insulin resistance. CD11c+CD206+ cells expressed high levels of CCR2, and liver CC chemokine ligand 2 (CCL2) expression was increased in nonalcoholic steatohepatitis and correlated with disease activity. In mice, CCR2 inhibition reduced infiltration of liver CD11b+CD11c+F4/80+ monocytes, which are functional homologs of human CD11c+CD206+ cells, and improved liver injury and glycemic control. A role for CCR2/CCL2 in human NAFLD has long been postulated. These data confirm a role for this chemokine/receptor axis, through mediating adipose and hepatic infiltration of myeloid cells. Inhibition of CCR2 improved hepatic inflammation and fibrosis in murine models of NAFLD. These data confirm the rationale for targeting CCR2 to treat NAFLD. NEW & NOTEWORTHY These data show for the first time that CD11c+CD206+ myeloid cells, previously associated with human adipose tissue inflammation, infiltrate into liver tissue in nonalcoholic fatty liver disease. These cells express CCR2. Inhibition of CCR2 in mice inhibits hepatic inflammation caused by a murine homolog of these myeloid cells and improves experimental liver disease.