Adult expression of the cell adhesion protein Fasciclin 3 is required for the maintenance of adult olfactory interneurons.

The proper functioning of the nervous system is dependent on the establishment and maintenance of intricate networks of neurons that form functional neural circuits. Once neural circuits are assembled during development, a distinct set of molecular programs is likely required to maintain their connectivity throughout the lifetime of the organism. Here, we demonstrate that Fasciclin 3 (Fas3), an axon guidance cell adhesion protein, is necessary for the maintenance of the olfactory circuit in adult Drosophila. We utilized the TARGET system to spatiotemporally knockdown Fas3 in selected populations of adult neurons. Our findings show that Fas3 knockdown results in the death of olfactory circuit neurons and reduced survival of adults. We also demonstrated that Fas3 knockdown activates caspase-3-mediated cell death in olfactory local interneurons, which can be rescued by overexpressing baculovirus p35, an anti-apoptotic protein. This work adds to the growing set of evidence indicating a crucial role for axon guidance proteins in the maintenance of neuronal circuits in adults.

A scalable Drosophila assay for clinical interpretation of human PTEN variants in suppression of PI3K/AKT induced cellular proliferation.

Gene variant discovery is becoming routine, but it remains difficult to usefully interpret the functional consequence or disease relevance of most variants. To fill this interpretation gap, experimental assays of variant function are becoming common place. Yet, it remains challenging to make these assays reproducible, scalable to high numbers of variants, and capable of assessing defined gene-disease mechanism for clinical interpretation aligned to the ClinGen Sequence Variant Interpretation (SVI) Working Group guidelines for 'well-established assays'. Drosophila melanogaster offers great potential as an assay platform, but was untested for high numbers of human variants adherent to these guidelines. Here, we wished to test the utility of Drosophila as a platform for scalable well-established assays. We took a genetic interaction approach to test the function of ~100 human PTEN variants in cancer-relevant suppression of PI3K/AKT signaling in cellular growth and proliferation. We validated the assay using biochemically characterized PTEN mutants as well as 23 total known pathogenic and benign PTEN variants, all of which the assay correctly assigned into predicted functional categories. Additionally, function calls for these variants correlated very well with our recent published data from a human cell line. Finally, using these pathogenic and benign variants to calibrate the assay, we could set readout thresholds for clinical interpretation of the pathogenicity of 70 other PTEN variants. Overall, we demonstrate that Drosophila offers a powerful assay platform for clinical variant interpretation, that can be used in conjunction with other well-established assays, to increase confidence in the accurate assessment of variant function and pathogenicity.

A randomized trial of vonapanitase (PATENCY-1) to promote radiocephalic fistula patency and use for hemodialysis.

OBJECTIVE: Arteriovenous fistulas created in patients with chronic kidney disease often lose patency and fail to become usable. This prospective trial evaluated the efficacy of vonapanitase, a recombinant human elastase, in promoting radiocephalic fistula patency and use for hemodialysis. METHODS: PATENCY-1 was a double-blind, placebo-controlled trial that enrolled 349 patients on or approaching hemodialysis and being evaluated for radiocephalic arteriovenous fistula creation. Of these, 313 were randomized and 311 treated. Patients were assigned to vonapanitase (n = 210) or placebo (n = 103). The study drug solution was applied topically to the artery and vein for 10 minutes immediately after fistula creation. The primary and secondary end points were primary patency (time to first thrombosis or corrective procedure) and secondary patency (time to abandonment). Tertiary end points included use of the fistula for hemodialysis, fistula maturation by ultrasound, and procedure rates. RESULTS: The Kaplan-Meier estimates of 12-month primary patency were 42% (95% confidence interval [CI], 35-49) and 31% (95% CI, 21-42) for vonapanitase and placebo (P = .25). The Kaplan-Meier estimates of 12-month secondary patency were 74% (95% CI, 68-80) and 61% (95% CI, 51-71) for vonapanitase and placebo (P = .048). The proportions of vonapanitase and placebo patients were 39% and 25% (P = .035) with unassisted use for hemodialysis and 64% and 44% (P = .006) with unassisted plus assisted use. CONCLUSIONS: Vonapanitase treatment did not significantly improve primary patency but was associated with increased secondary patency and use for hemodialysis. Further research is needed to evaluate these end points.

Semaphorin 5B is a repellent cue for sensory afferents projecting into the developing spinal cord.

During vertebrate development, centrally projecting sensory axons of the dorsal root ganglia neurons first reach the embryonic spinal cord at the dorsolateral margin. Instead of immediately projecting into the grey matter, they bifurcate and extend rostrally and caudally to establish the longitudinal dorsal funiculus during a stereotyped waiting period of approximately 48 h. Collateral fibres then extend concurrently across multiple spinal segments and project to their appropriate targets within the grey matter. This rostrocaudal extension of sensory afferents is crucial for the intersegmental processing of information throughout the spinal cord. However, the precise cues that prevent premature entry during the waiting period remain to be identified. Here, we show that semaphorin 5B (Sema5B), a member of the semaphorin family of guidance molecules, is expressed in the chick spinal cord during this waiting period and dorsal funiculus formation. Sema5B expression is dynamic, with a reduction of expression apparent in the spinal cord concomitant with collateral extension. We show that Sema5B inhibits the growth of NGF-dependent sensory axons and that this effect is mediated in part through the cell adhesion molecule TAG-1. Knockdown of Sema5B in the spinal cord using RNA interference leads to the premature extension of cutaneous nociceptive axons into the dorsal horn grey matter. These premature projections predominantly occur at the site of dorsal root entry. Our results suggest that Sema5B contributes to a repulsive barrier for centrally projecting primary sensory axons, forcing them to turn and establish the dorsal funiculus.

Arteriovenous fistula patency in the 3 years following vonapanitase and placebo treatment.

OBJECTIVE: This study explored the long-term outcomes of arteriovenous fistulas treated with vonapanitase (recombinant human elastase) at the time of surgical creation. METHODS: This was a randomized, double-blind, placebo-controlled trial of 151 patients undergoing radiocephalic or brachiocephalic arteriovenous fistula creation who were randomized equally to placebo, vonapanitase 10 µg, or vonapanitase 30 µg. The results after 1 year of follow-up were previously reported. The current analysis occurred when the last patient treated was observed for 3 years. For the current analysis, the primary end point was primary patency; the secondary end points included secondary patency, use of the fistula for hemodialysis, and rate of procedures to restore or to maintain patency. RESULTS: There was no significant difference in the risk of primary patency loss with vonapanitase 10 µg or 30 µg vs placebo. When seven initial patency loss events related to cephalic arch and central vein balloon angioplasty were excluded, the risk of patency loss was reduced with vonapanitase overall (hazard ratio [HR], 0.63; P = .049) and 30 µg (HR, 0.51; P = .03). In patients with radiocephalic fistulas (n = 67), the risks of primary and secondary patency loss were reduced with 30 µg (HR, 0.37 [P = .02] and 0.24 [P = .046], respectively). The rate of procedures to restore or to maintain fistula patency was reduced with 30 µg vs placebo (0.23 vs 0.72 procedure days/patient/year; P = .03) and also reduced in patients with radiocephalic fistulas with 30 µg vs placebo (0.17 vs 0.85 procedure days/patient/year; P = .048). CONCLUSIONS: In this study, vonapanitase did not significantly improve primary patency in the primary analysis but did significantly improve primary patency in an analysis that excluded patency loss due to cephalic arch and central vein balloon angioplasty. In patients with radiocephalic fistulas, 30 µg significantly improved primary and secondary patency. Vonapanitase 30 µg decreased the rate of procedures to restore or to maintain patency in the analysis that included all patients and in the subset with radiocephalic fistulas.

Human type I pancreatic elastase treatment of arteriovenous fistulas in patients with chronic kidney disease.

OBJECTIVE: This study explored the safety and efficacy of recombinant type I pancreatic elastase (PRT-201) topically applied once to the external surface of an arteriovenous fistula. METHODS: This was a randomized, double-blind, placebo-controlled trial. Adults with kidney disease undergoing creation of a radiocephalic fistula (RCF) or brachiocephalic fistula were randomized to treatment with placebo (n = 51), PRT-201 at 10 µg (n = 51), or PRT-201 at 30 µg (n = 49). The primary efficacy measure was unassisted primary patency (PP) over 1 year. Secondary efficacy measures were secondary patency (SP), unassisted maturation by ultrasound interrogation, use for hemodialysis, and hemodynamically significant lumen stenosis. RESULTS: Median PP was 224 days for placebo and >365 days for the PRT-201 groups. At 1 year, 45%, 54%, and 53% of placebo, 10-µg, and 30-µg patients retained PP. The risk of PP loss was nonsignificantly reduced for 10 µg (hazard ratio [HR], 0.69; P = .19) and 30 µg (HR, 0.67; P = .17) vs placebo. In the subset (44% of patients) with a RCF, the median PP was 125 days for placebo and >365 days for the PRT-201 groups. At 1 year, 31%, 50%, and 63% of placebo, 10-µg, and 30-µg RCFs retained PP. The risk of RCF PP loss was nonsignificantly reduced by 10 µg (HR, 0.59; P = .18) and significantly reduced by 30 µg (HR, 0.37; P = .02) vs placebo. At 1 year, 77%, 81%, and 83% of placebo, 10-µg, and 30-µg patients retained SP. The risk of SP loss was nonsignificantly reduced for 10 µg (HR, 0.79; P = .61) and 30 µg (HR, 0.76; P = .55) vs placebo. In the subset with RCFs, 65%, 82%, and 90% of placebo, 10-µg, and 30-µg patients retained SP at 1 year. The risk of RCF SP loss was nonsignificantly reduced for 10 µg (HR, 0.45; P = .19) and 30 µg (HR, 0.27; P = .08) vs placebo. At month 3, 67%, 87% (P = .03), and 92% (P < .01) of the placebo, 10-µg, and 30-µg group fistulas had unassisted maturation by ultrasound interrogation. At month 3 in the subset with an RCF, 47%, 74% (P = .17), and 93% (P < .01) of placebo, 10-µg, and 30-µg group fistulas had unassisted maturation by ultrasound interrogation. Adverse event reports were not meaningfully different between groups. CONCLUSIONS: PRT-201 appeared safe. The primary efficacy end point was not met. However, both PRT-201 doses were associated with improved unassisted maturation. The 30-µg dose was associated with increased PP in the subset with RCF.

Neurite consolidation is an active process requiring constant repression of protrusive activity.

During development, neurons extend projections that pathfind to reach their appropriate targets. These projections are composed of two distinct domains: a highly dynamic growth cone and a stable neurite shaft, which is considered to be consolidated. Although the regulation of these domains is critical to the appropriate formation of neural networks, the molecular mechanisms that regulate neurite shape remain poorly understood. Here, we show that calpain protease activity localizes to the neurite shaft, where it is essential for the repression of protrusive activity by limiting cortactin levels and inhibiting actin polymerization. Correspondingly, inhibition of calpain by branching factors induces the formation of new growth cones along the neurite shaft through cAMP elevation. These findings demonstrate that neurite consolidation is an active process requiring constant repression of protrusive activity. We also show that sprouting is, at least in part, accomplished by turning off the mechanism of consolidation.

Adult expression of Semaphorins and Plexins is essential for motor neuron survival.

Axon guidance cues direct the growth and steering of neuronal growth cones, thus guiding the axons to their targets during development. Nonetheless, after axons have reached their targets and established functional circuits, many mature neurons continue to express these developmental cues. The role of axon guidance cues in the adult nervous system has not been fully elucidated. Using the expression pattern data available on FlyBase, we found that more than 96% of the guidance genes that are expressed in the Drosophila melanogaster embryo continue to be expressed in adults. We utilized the GeneSwitch and TARGET systems to spatiotemporally knockdown the expression of these guidance genes selectively in the adult neurons, once the development was completed. We performed an RNA interference (RNAi) screen against 44 guidance genes in the adult Drosophila nervous system and identified 14 genes that are required for adult survival and normal motility. Additionally, we show that adult expression of Semaphorins and Plexins in motor neurons is necessary for neuronal survival, indicating that guidance genes have critical functions in the mature nervous system.

Transmembrane semaphorin5B is proteolytically processed into a repulsive neural guidance cue.

Developing neuronal growth cones respond to a number of post-transcriptionally modified guidance cues to establish functional neural networks. The Semaphorin family has well-established roles as both secreted and transmembrane guidance cues. Here, we describe the first evidence that a transmembrane Semaphorin, Semaphorin 5B (Sema5B), is proteolytically processed from its transmembrane form and can function as a soluble growth cone collapsing guidance cue. Over-expression of A Disintegrin and Metalloprotease (ADAM)-17, results in an enhanced release of the Sema5B ectodomain, while removal of a predicted ADAM-17 cleavage site prevents its release. In contrast, knockdown of ADAM-17 does not significantly reduce Sema5B release, indicating there are additional unknown compensating proteases. This modulation of the transmembrane Sema5B to a diffusible cue represents a sophisticated method to regulate neuronal guidance in vivo.

How I Teach: Ultrasound-guided Peripheral Venous Access.

Ultrasound-guided peripheral intravenous (IV) placement is often required for patients with difficult IV access and is associated with a reduction in central line placement. Despite the importance, there is no standardized technical approach, and there is limited ability to attain mastery through simulation. We describe our step-by-step approach for teaching ultrasound-guided IV placement at the bedside using short-axis dynamic guidance, with emphasis on advancing the needle and catheter device almost entirely into the vessel before threading the catheter. Our teaching approach allows the opportunity for trainees to maximize the learning potential of a single insertion experience, which includes focused preprocedure hands-on practice, instruction with real-time feedback at the bedside, and a post-procedure debrief with reinforcement of concepts.

The naked truth: a comprehensive clarification and classification of current 'myths' in naked mole-rat biology.

The naked mole-rat (Heterocephalus glaber) has fascinated zoologists for at least half a century. It has also generated considerable biomedical interest not only because of its extraordinary longevity, but also because of unusual protective features (e.g. its tolerance of variable oxygen availability), which may be pertinent to several human disease states, including ischemia/reperfusion injury and neurodegeneration. A recent article entitled 'Surprisingly long survival of premature conclusions about naked mole-rat biology' described 28 'myths' which, those authors claimed, are a 'perpetuation of beautiful, but falsified, hypotheses' and impede our understanding of this enigmatic mammal. Here, we re-examine each of these 'myths' based on evidence published in the scientific literature. Following Braude et al., we argue that these 'myths' fall into four main categories: (i) 'myths' that would be better described as oversimplifications, some of which persist solely in the popular press; (ii) 'myths' that are based on incomplete understanding, where more evidence is clearly needed; (iii) 'myths' where the accumulation of evidence over the years has led to a revision in interpretation, but where there is no significant disagreement among scientists currently working in the field; (iv) 'myths' where there is a genuine difference in opinion among active researchers, based on alternative interpretations of the available evidence. The term 'myth' is particularly inappropriate when applied to competing, evidence-based hypotheses, which form part of the normal evolution of scientific knowledge. Here, we provide a comprehensive critical review of naked mole-rat biology and attempt to clarify some of these misconceptions.

Myosin-Va-interacting protein, RILPL2, controls cell shape and neuronal morphogenesis via Rac signaling.

Neuronal morphology plays an essential role in neuronal function. The establishment and maintenance of neuronal morphology is intimately linked to the actin cytoskeleton; however, the molecular mechanisms that regulate changes in neuronal morphology are poorly understood. Here we identify a novel myosin-Va (MyoVa)-interacting protein, RILPL2, which regulates cellular morphology. Overexpression of this protein in young or mature hippocampal neurons results in an increase in the number of spine-like protrusions. By contrast, knockdown of endogenous RILPL2 in neurons by short hairpin RNA (shRNA) interference results in reduced spine-like protrusions, a phenotype rescued by overexpression of an shRNA-insensitive RILPL2 mutant, suggesting a role for RILPL2 in both the establishment and maintenance of dendritic spines. Interestingly, we demonstrate that RILPL2 and the Rho GTPase Rac1 form a complex, and that RILPL2 is able to induce activation of Rac1 and its target, p21-activated kinase (Pak). Notably, both RILPL2-mediated morphological changes and activation of Rac1-Pak signaling were blocked by expression of a truncated tail form of MyoVa or MyoVa shRNA, demonstrating that MyoVa is crucial for proper RILPL2 function. This might represent a novel mechanism linking RILPL2, the motor protein MyoVa and Rac1 with neuronal structure and function.

Smoking-dependent reprogramming of alveolar macrophage polarization: implication for pathogenesis of chronic obstructive pulmonary disease.

When exposed to a specific microenvironment, macrophages acquire either M1- or M2-polarized phenotypes associated with inflammation and tissue remodeling, respectively. Alveolar macrophages (AM) directly interact with environmental stimuli such as cigarette smoke, the major risk factor for chronic obstructive pulmonary disease (COPD), a disease characterized by lung inflammation and remodeling. Transcriptional profiling of AM obtained by bronchoalveolar lavage of 24 healthy nonsmokers, 34 healthy smokers, and 12 COPD smokers was performed to test the hypothesis whether smoking alters AM polarization, resulting in a disease-relevant activation phenotype. The analysis revealed that AM of healthy smokers exhibited a unique polarization pattern characterized by substantial suppression of M1-related inflammatory/immune genes and induction of genes associated with various M2-polarization programs relevant to tissue remodeling and immunoregulation. Such reciprocal changes progressed with the development of COPD, with M1-related gene expression being most dramatically down-regulated (p < 0.0001 vs healthy nonsmokers, p < 0.002 vs healthy smokers). Results were confirmed with TaqMan real-time PCR and flow cytometry. Among progressively down-regulated M1-related genes were those encoding type I chemokines CXCL9, CXCL10, CXCL11, and CCL5. Progressive activation of M2-related program was characterized by induction of tissue remodeling and immunoregulatory genes such as matrix metalloproteinase (MMP)2, MMP7, and adenosine A3 receptor (ADORA3). Principal component analysis revealed that differential expression of polarization-related genes has substantial contribution to global AM phenotypes associated with smoking and COPD. In summary, the data provide transcriptome-based evidence that AM likely contribute to COPD pathogenesis in a noninflammatory manner due to their smoking-induced reprogramming toward M1-deactivated, partially M2-polarized macrophages.

Lung Ultrasound: The Essentials.

Although US of the lungs is increasingly used clinically, diagnostic radiologists are not routinely trained in its use and interpretation. Lung US is a highly sensitive and specific modality that aids in the evaluation of the lungs for many different abnormalities, including pneumonia, pleural effusion, pulmonary edema, and pneumothorax. This review provides an overview of lung US to equip the diagnostic radiologist with knowledge needed to interpret this increasingly used modality. Supplemental material is available for this article. © RSNA, 2021.

Semaphorin 5B is a novel inhibitory cue for corticofugal axons.

Neuronal connectivity is generated by the precise guidance of neuronal growth cones in response to the spatiotemporal distribution of molecular guidance cues in the developing embryo. Here we show that the class 5 semaphorin, Semaphorin 5B, is expressed in regions of the cortex and subcortex flanking the projection of and avoided by descending cortical axons, suggesting a role as a repulsive guidance cue in the formation of the internal capsule. Axons from cortical explants cultured in vitro with Semaphorin 5B-expressing cells exhibited characteristic avoidance behaviors. In organotypic slices, ectopic Semaphorin 5B expression along the presumptive internal capsule was sufficient to cause cortical axons to avoid their normal trajectory, resulting in either stalling at the boundary of Semaphorin 5B or turning into inappropriate areas of the cortex. In contrast, thalamocortical axons were not inhibited either in vitro or in slice culture by ectopic Semaphorin 5B. To further examine the function of Semaphorin 5B in situ, we knocked down its expression in the ventricular zone (VZ) at the corticostriatal angle. We found that labeled cortical fibers aberrantly navigated into the VZ where Semaphorin 5B expression was reduced. We propose that Semaphorin 5B functions to prevent corticofugal axons from abnormally projecting into germinal regions as they project to their subcortical targets.

Down-regulation of the notch pathway in human airway epithelium in association with smoking and chronic obstructive pulmonary disease.

RATIONALE: The airway epithelium of smokers is subject to a variety of mechanisms of injury with consequent modulation of epithelial regeneration and disordered differentiation. Several signaling pathways, including the Notch pathway, control epithelial differentiation in lung morphogenesis, but little is known about the role of these pathways in adults. OBJECTIVES: We tested the hypotheses that Notch-related genes are expressed in the normal nonsmoker small airway epithelium of human adults, and that Notch-related gene expression is down-regulated in healthy smokers and smokers with chronic obstructive pulmonary disease (COPD). METHODS: We used microarray technology to evaluate the expression of 55 Notch-related genes in the small airway epithelium of nonsmokers. We used TaqMan quantitative polymerase chain reaction (PCR) to confirm the expression of key genes and we used immunohistochemistry to assess the expression of Notch-related proteins in the airway epithelium. Changes in expression of Notch genes in healthy smokers and smokers with COPD compared with nonsmokers were evaluated by PCR. MEASUREMENTS AND MAIN RESULTS: Microarray analysis demonstrated that 45 of 55 Notch-related genes are expressed in the small airway epithelium of adults. TaqMan PCR confirmed the expression of key genes with highest expression of the ligand DLL1, the receptor NOTCH2, and the downstream effector HES1. Immunohistochemistry demonstrated the expression of Jag1, Notch2, Hes1, and Hes5 in airway epithelium. Several Notch ligands, receptors, and downstream effector genes were down-regulated in smokers, with more genes down-regulated in smokers with COPD than in healthy smokers. CONCLUSIONS: These observations are consistent with the hypothesis that the Notch pathway likely plays a role in the human adult airway epithelium, with down-regulation of Notch pathway gene expression in association with smoking and COPD.

Multi-model functionalization of disease-associated PTEN missense mutations identifies multiple molecular mechanisms underlying protein dysfunction.

Functional variomics provides the foundation for personalized medicine by linking genetic variation to disease expression, outcome and treatment, yet its utility is dependent on appropriate assays to evaluate mutation impact on protein function. To fully assess the effects of 106 missense and nonsense variants of PTEN associated with autism spectrum disorder, somatic cancer and PTEN hamartoma syndrome (PHTS), we take a deep phenotypic profiling approach using 18 assays in 5 model systems spanning diverse cellular environments ranging from molecular function to neuronal morphogenesis and behavior. Variants inducing instability occur across the protein, resulting in partial-to-complete loss-of-function (LoF), which is well correlated across models. However, assays are selectively sensitive to variants located in substrate binding and catalytic domains, which exhibit complete LoF or dominant negativity independent of effects on stability. Our results indicate that full characterization of variant impact requires assays sensitive to instability and a range of protein functions.

Quality control in microarray assessment of gene expression in human airway epithelium.

BACKGROUND: Microarray technology provides a powerful tool for defining gene expression profiles of airway epithelium that lend insight into the pathogenesis of human airway disorders. The focus of this study was to establish rigorous quality control parameters to ensure that microarray assessment of the airway epithelium is not confounded by experimental artifact. Samples (total n = 223) of trachea, large and small airway epithelium were collected by fiberoptic bronchoscopy of 144 individuals and hybridized to Affymetrix microarrays. The pre- and post-chip quality control (QC) criteria established, included: (1) RNA quality, assessed by RNA Integrity Number (RIN) > or = 7.0; (2) cRNA transcript integrity, assessed by signal intensity ratio of GAPDH 3' to 5' probe sets < or = 3.0; and (3) the multi-chip normalization scaling factor < or = 10.0. RESULTS: Of the 223 samples, all three criteria were assessed in 191; of these 184 (96.3%) passed all three criteria. For the remaining 32 samples, the RIN was not available, and only the other two criteria were used; of these 29 (90.6%) passed these two criteria. Correlation coefficients for pairwise comparisons of expression levels for 100 maintenance genes in which at least one array failed the QC criteria (average Pearson r = 0.90 +/- 0.04) were significantly lower (p < 0.0001) than correlation coefficients for pairwise comparisons between arrays that passed the QC criteria (average Pearson r = 0.97 +/- 0.01). Inter-array variability was significantly decreased (p < 0.0001) among samples passing the QC criteria compared with samples failing the QC criteria. CONCLUSION: Based on the aberrant maintenance gene data generated from samples failing the established QC criteria, we propose that the QC criteria outlined in this study can accurately distinguish high quality from low quality data, and can be used to delete poor quality microarray samples before proceeding to higher-order biological analyses and interpretation.

Disparate oxidant gene expression of airway epithelium compared to alveolar macrophages in smokers.

BACKGROUND: The small airway epithelium and alveolar macrophages are exposed to oxidants in cigarette smoke leading to epithelial dysfunction and macrophage activation. In this context, we asked: what is the transcriptome of oxidant-related genes in small airway epithelium and alveolar macrophages, and does their response differ substantially to inhaled cigarette smoke? METHODS: Using microarray analysis, with TaqMan RT-PCR confirmation, we assessed oxidant-related gene expression in small airway epithelium and alveolar macrophages from the same healthy nonsmoker and smoker individuals. RESULTS: Of 155 genes surveyed, 87 (56%) were expressed in both cell populations in nonsmokers, with higher expression in alveolar macrophages (43%) compared to airway epithelium (24%). In smokers, there were 15 genes (10%) up-regulated and 7 genes (5%) down-regulated in airway epithelium, but only 3 (2%) up-regulated and 2 (1%) down-regulated in alveolar macrophages. Pathway analysis of airway epithelium showed oxidant pathways dominated, but in alveolar macrophages immune pathways dominated. CONCLUSION: Thus, the response of different cell-types with an identical genome exposed to the same stress of smoking is different; responses of alveolar macrophages are more subdued than those of airway epithelium. These findings are consistent with the observation that, while the small airway epithelium is vulnerable, alveolar macrophages are not "diseased" in response to smoking. TRIAL REGISTRATION: ClinicalTrials.gov ID: NCT00224185 and NCT00224198.