The Renin-Angiotensin-Aldosterone System Regulates Sarcoidosis Granulomatous Inflammation.

Rationale: Sarcoidosis is a granulomatous disorder of unclear cause notable for abnormal elevation of blood and tissue angiotensin converting enzyme 1 (ACE1) levels and activity. ACE1 regulates the renin-angiotensin-aldosterone system (RAAS), the terminal product of which is aldosterone, which selectively engages mineralocorticoid receptors (MR) to promote inflammation. Objectives: We sought to determine whether the RAAS promotes sarcoidosis granuloma formation and related inflammatory responses. Methods: Using an established ex vivo model, we first determined whether aldosterone was produced by sarcoidosis granulomas and verified the presence of CYP11B2, the enzyme required for its production. We then evaluated the effects of selective inhibitors of ACE1 (captopril), angiotensin type 1 receptor (losartan) and MR (spironolactone, eplerenone) on granuloma formation, reflected by computer image analysis-generated granuloma area, and selected cytokines incriminated in sarcoidosis pathogenesis. Measurements and Main Results: Aldosterone was spontaneously produced by sarcoidosis PBMCs, and both intra- and extracellular levels steadily increased during granuloma formation. In parallel, PBMCs were shown to express more CYP11B2 during granuloma formation. Significant inhibition of sarcoidosis granulomas and related cytokines (TNFα, IL-1ß, IFNγ, IL-10) was observed in response to pretreatments with captopril, losartan, spironolactone or eplerenone, comparable to that of prednisone. Conclusions: The RAAS is intact in sarcoidosis granulomas and contributes significantly to early granuloma formation and to related inflammatory mediator responses with important implications for clinical management.

Investigating cryopreserved PBMC functionality in an antigen-induced model of sarcoidosis granuloma formation.

Sarcoidosis, a systemic inflammatory disease, poses challenges in understanding its etiology and variable clinical courses. Despite ongoing uncertainty about causative agents and genetic predisposition, granuloma formation remains its hallmark feature. To address this, we developed a validated in vitro human granuloma model using patient-derived peripheral blood mononuclear cells (PBMCs), offering a dynamic platform for studying early granuloma formation and sarcoidosis pathogenesis. However, a current limitation of this model is its dependence on freshly isolated PBMCs obtained from whole blood. While cryopreservation is a common method for long-term sample preservation, the biological effects of freezing and thawing PBMCs on granuloma formation remain unclear. This study aimed to assess the viability and functionality of cryopreserved sarcoidosis PBMCs within the granuloma model, revealing similar granulomatous responses to fresh cells and highlighting the potential of cryopreserved PBMCs as a valuable tool for studying sarcoidosis and related diseases.

Leveraging in vitro and pharmacokinetic models to support bench to bedside investigation of XTMAB-16 as a novel pulmonary sarcoidosis treatment.

Background: Sarcoidosis is a chronic, multisystem inflammatory disorder characterized by non-caseating epithelioid granulomas; infiltration of mononuclear cells; and destruction of microarchitecture in the skin, eye, heart, and central nervous system, and the lung in >90% of cases. XTMAB-16 is a chimeric anti-tumor necrosis factor alpha (TNFα) antibody, distinct from other anti-TNF antibodies based on its molecular structure. The efficacy of XTMAB-16 has not been clinically demonstrated, and it is still undergoing clinical development as a potential treatment for sarcoidosis. The current study demonstrates the activity of XTMAB-16 in a well-established in vitro sarcoidosis granuloma model, although XTMAB-16 is not yet approved by the United States Food and Drug Administration (FDA) for treatment of sarcoidosis, or any other disease. Objective: To provide data to guide safe and efficacious dose selection for the ongoing clinical development of XTMAB-16 as a potential treatment for sarcoidosis. Methods: First, XTMAB-16 activity was evaluated in an established in vitro model of granuloma formation using peripheral blood mononuclear cells from patients with active pulmonary sarcoidosis to determine a potentially efficacious dose range. Second, data obtained from the first-in-human study of XTMAB-16 (NCT04971395) were used to develop a population pharmacokinetic (PPK) model to characterize the pharmacokinetics (PK) of XTMAB-16. Model simulations were performed to evaluate the sources of PK variability and to predict interstitial lung exposure based on concentrations in the in vitro granuloma model. Results: XTMAB-16 dose levels of 2 and 4 mg/kg, once every 2 weeks (Q2W) or once every 4 weeks (Q4W) for up to 12 weeks, were supported by data from the non-clinical, in vitro secondary pharmacology; the Phase 1 clinical study; and the PPK model developed to guide dose level and frequency assumptions. XTMAB-16 inhibited granuloma formation and suppressed interleukin-1ß (IL-1ß) secretion in the in vitro granuloma model with a half maximal inhibitory concentration (IC50) of 5.2 and 3.5 µg/mL, respectively. Interstitial lung concentrations on average, following 2 or 4 mg/kg administered Q2W or Q4W, are anticipated to exceed the in vitro IC50 concentrations. Conclusion: The data presented in this report provide a rationale for dose selection and support the continued clinical development of XTMAB-16 for patients with pulmonary sarcoidosis.

Mucociliary Clearance Differs in Mild Asthma by Levels of Type 2 Inflammation.

BACKGROUND: Although mucus plugging is a well-reported feature of asthma, whether asthma and type 2 inflammation affect mucociliary clearance (MCC) is unknown. RESEARCH QUESTION: Does type 2 inflammation influence mucus clearance rates in patients with mild asthma who are not receiving corticosteroids? STUDY DESIGN AND METHODS: The clearance rates of inhaled radiolabeled particles were compared between patients with mild asthma with low (n = 17) and high (n = 18) levels of T2 inflammation. Fraction exhaled nitric oxide (Feno) was used to prospectively segregate subjects into T2 Lo (Feno < 25 ppb) and T2 Hi (Feno > 35 ppb) cohorts. Bronchial brush samples were collected with fiber-optic bronchoscopy, and quantitative polymerase chain reaction was performed to measure expression of genes associated with T2 asthma. MCC rate comparisons were also made with a historical group of healthy control subjects (HCs, n = 12). RESULTS: The T2 Lo cohort demonstrated increased MCC when compared with both T2 Hi and historic HCs. MCC within the T2 Hi group varied significantly, with some subjects having low or zero clearance. MCC decreased with increasing expression of several markers of T2 airway inflammation (CCL26, NOS2, and POSTN) and with Feno. MUC5AC and FOXJ1 expression was similar between the T2Lo and T2Hi cohorts. INTERPRETATION: Increasing T2 inflammation was associated with decreasing MCC. High rates of MCC in T2 Lo subjects may indicate a compensatory mechanism present in mild disease but lost with high levels of inflammation. Future studies are required to better understand mechanisms and whether impairments in MCC in more severe asthma drive worse clinical outcomes.

Phagosome-regulated mTOR signalling during sarcoidosis granuloma biogenesis.

INTRODUCTION: Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to Mycobacterium tuberculosis antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to M. tuberculosis antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis. METHODS: A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established in vitro human granuloma model wherein peripheral blood mononuclear cells were exposed to M. tuberculosis antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation via phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin). RESULTS: In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction. CONCLUSIONS: Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.

Current Sarcoidosis Models and the Importance of Focusing on the Granuloma.

The inability to effectively model sarcoidosis in the laboratory or in animals continues to hinder the discovery and translation of new, targeted treatments. The granuloma is the signature pathological hallmark of sarcoidosis, yet there are significant knowledge gaps that exist with regard to how granulomas form. Significant progress toward improved therapeutic and prognostic strategies in sarcoidosis hinges on tractable experimental models that recapitulate the process of granuloma formation in sarcoidosis and allow for mechanistic insights into the molecular events involved. Through its inherent representation of the complex genetics underpinning immune cell dysregulation in sarcoidosis, a recently developed in vitro human granuloma model holds promise in providing detailed mechanistic insight into sarcoidosis-specific disease regulating pathways at play during early stages of granuloma formation. The purpose of this review is to critically evaluate current sarcoidosis models and assess their potential to progress the field toward the goal of improved therapies in this disease. We conclude with the potential integrated use of preclinical models to accelerate progress toward identifying and testing new drugs and drug combinations that can be rapidly brought to clinical trials.

Evaluation of Peptide-Based Probes toward In Vivo Diagnostic Imaging of Bacterial Biofilm-Associated Infections.

The clinical management of bacterial biofilm infections represents an enormous challenge in today's healthcare setting. The NIH estimates that 65% of bacterial infections are biofilm-related, and therapeutic outcomes are positively correlated with early intervention. Currently, there is no reliable imaging technique to detect biofilm infections in vivo, and current clinical protocols for accurate and direct biofilm identification are nonexistent. In orthopedic implant-associated biofilm infections, for example, current detection methods are based on nonspecific X-ray or radiolabeled white blood cell imaging, coupled with peri-prosthetic tissue or fluid samples taken invasively, and must be cultured. This approach is time-consuming and often fails to detect biofilm bacteria due to sampling errors and a lack of sensitivity. The ability to quantify bacterial biofilms by real-time noninvasive imaging is an urgent unmet clinical need that would revolutionize the management and treatment of these devastating types of infections. In the present study, we assembled a collection of fluorescently labeled peptide candidates to specifically explore their biofilm targeting properties. We evaluated these fluorescently labeled peptides using various in vitro assays for their ability to specifically and nondestructively target biofilms produced by model bacterial pathogen Pseudomonas aeruginosa. The lead candidate that emerged, 4Iphf-HN17, demonstrated rapid biofilm labeling kinetics, a lack of bactericidal activity, and biofilm targeting specificity in human cell infection models. In vivo fluorescently labeled 4Iphf-HN17 showed enhanced accumulation in biofilm-infected wounds, thus warranting further study.

Multiprobe Nuclear Imaging of the Cystic Fibrosis Lung as a Biomarker of Therapeutic Effect.

Background: Nuclear imaging biomarkers illustrate unique aspects of lung physiology and are useful for assessing therapeutic effects in cystic fibrosis (CF) lung disease. We have developed a multiprobe method to simultaneously measure mucociliary clearance (MCC) and paracellular absorption (ABS). MCC is a direct measure of mucus clearance. ABS has been related to airway surface liquid (ASL) absorption through previous in vitro studies. Methods: We describe baseline factors affecting MCC and ABS using data from a retrospective baseline group (n = 22) and the response of the measures to inhaled 7% hypertonic saline (HS) and dry powder mannitol using data from a prospective response group (n = 7). A retrospective healthy control group (n = 15) is also described. The baseline and control groups performed single measurements of MCC/ABS. The response group performed baseline measurements of MCC/ABS and measurements after each intervention. Results: ABS was correlated (Spearman's ρ = 0.51, p = 0.06) to sweat chloride, a systemic measure of cystic fibrosis transmembrane conductance regulator (CFTR) function, whereas MCC was not. Baseline MCC was depressed after Pseudomonas aeruginosa infection as we have previously described. MCC provided a more sensitive indication of therapeutic effect and indicated improved clearance with mannitol compared with HS. Conclusion: MCC provides a useful and well-established means of testing therapies directed at improving mucus clearance in the lung. ABS may provide a means of detecting local changes in ASL absorption and CFTR function in the lung. Both are useful tools for studying the key aspects of CF lung pathophysiology (ASL hyperabsorption and MCC depression) that link the basic genetic defects of CF to disease manifestations in the lung.

IL-13-regulated Macrophage Polarization during Granuloma Formation in an In Vitro Human Sarcoidosis Model.

The mechanisms underlying abnormal granuloma formation in patients with sarcoidosis are complex and remain poorly understood. A novel in vitro human granuloma model was used to determine the molecular mechanisms of granuloma genesis in patients with sarcoidosis in response to putative disease-causing mycobacterial antigens. Peripheral blood mononuclear cells (PBMCs) from patients with active sarcoidosis and from normal, disease-free control subjects were incubated for 7 days with purified protein derivative-coated polystyrene beads. Molecular responses, as reflected by differential expression of genes, extracellular cytokine patterns, and cell surface receptor expression, were analyzed. Unbiased systems biology approaches were used to identify signaling pathways engaged during granuloma formation. Model findings were compared with human lung and mediastinal lymph node gene expression profiles. Compared with identically treated PBMCs of control subjects (n = 5), purified protein derivative-treated sarcoidosis PBMCs (n = 6) were distinguished by the formation of cellular aggregates resembling granulomas. Ingenuity Pathway Analysis of differential expression gene patterns identified molecular pathways that are primarily regulated by IL-13, which promotes alternatively activated (M2) macrophage polarization. M2 polarization was further demonstrated by immunohistochemistry performed on the in vitro sarcoidosis granuloma-like structures. IL-13-regulated gene pathways were confirmed in human sarcoidosis lung and mediastinal lymph node tissues. The in vitro human sarcoidosis granuloma model provides novel insights into early granuloma formation, particularly IL-13 regulation of molecular networks that regulate M2 macrophage polarization. M2 macrophages are predisposed to aggregation and multinucleated giant cell formation, which are characteristic features of sarcoidosis granulomas. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

Effect of ivacaftor on mucociliary clearance and clinical outcomes in cystic fibrosis patients with G551D-CFTR.

BACKGROUND: The ability to restore cystic fibrosis transmembrane regulator (CFTR) function with effective small molecule modulators in patients with cystic fibrosis provides an opportunity to study relationships between CFTR ion channel function, organ level physiology, and clinical outcomes. METHODS: We performed a multisite, prospective, observational study of ivacaftor, prescribed in patients with the G551D-CFTR mutation. Measurements of lung mucociliary clearance (MCC) were performed before and after treatment initiation (1 and 3 months), in parallel with clinical outcome measures. RESULTS: Marked acceleration in whole lung, central lung, and peripheral lung MCC was observed 1 month after beginning ivacaftor and was sustained at 3 months. Improvements in MCC correlated with improvements in forced expiratory volume in the first second (FEV1) but not sweat chloride or symptom scores. CONCLUSIONS: Restoration of CFTR activity with ivacaftor led to significant improvements in MCC. This physiologic assessment provides a means to characterize future CFTR modulator therapies and may help to predict improvements in lung function. TRIAL REGISTRATION: ClinicialTrials.gov, NCT01521338. FUNDING: CFF Therapeutics (GOAL11K1).

Use of a leukocyte-targeted peptide probe as a potential tracer for imaging the tuberculosis granuloma.

Granulomas are the histopathologic hallmark of tuberculosis (TB), both in latency and active disease. Diagnostic and therapeutic strategies that specifically target granulomas have not been developed. Our objective is to develop a probe for imaging relevant immune cell populations infiltrating the granuloma. We report the binding specificity of Cyanine 3 (Cy3)-labeled cFLFLFK-PEG12 to human leukocytes and cellular constituents within a human in vitro granuloma model. We also report use of the probe in in vivo studies using a mouse model of lung granulomatous inflammation. We found that the probe preferentially binds human neutrophils and macrophages in human granuloma structures. Inhibition studies showed that peptide binding to human neutrophils is mediated by the receptor formyl peptide receptor 1 (FPR1). Imaging the distribution of intravenously administered cFLFLFK-PEG12-Cy3 in the mouse model revealed probe accumulation within granulomatous inflammatory responses in the lung. Further characterization revealed that the probe preferentially associated with neutrophils and cells of the monocyte/macrophage lineage. As there is no current clinical diagnostic imaging tool that specifically targets granulomas, the use of this probe in the context of latent and active TB may provide a unique advantage over current clinical imaging probes. We anticipate that utilizing a FPR1-targeted radiopharmaceutical analog of cFLFLFK in preclinical imaging studies may greatly contribute to our understanding of granuloma influx patterns and the biological roles and consequences of FPR1-expressing cells in contributing to disease pathogenesis.

Overnight delivery of hypertonic saline by nasal cannula aerosol for cystic fibrosis.

AIM: Inhaled hypertonic saline increases mucociliary clearance, improves pulmonary function, and decreases exacerbations in cystic fibrosis (CF) but contributes to the already significant treatment burden of CF. Overnight delivery of inhaled medications via a specially designed nasal cannula-aerosol device (Trans-nasal Pulmonary Aerosol Delivery [tPAD]) is an alternative approach. Here, we test whether overnight inhalation of hypertonic saline via tPAD improves mucociliary clearance and assess the tolerability of the device. METHOD: In this study, 12 CF subjects inhaled 7% hypertonic saline (HS) for 8 h overnight using the tPAD system. Safety and tolerability were assessed and measurements of mucociliary and absorptive clearance (MCC/ABS) were performed after the treatment. Comparisons were made versus sham treatment where the same subjects wore the nasal cannula overnight but did not receive aerosol. RESULTS: Both the HS and sham treatments were well-tolerated. Only one subject did not complete the overnight HS treatment. There were no significant differences in MCC associated with HS inhalation at any time point (90 min, 3 h, 6 h) in any lung zone. Changes in FEV1 on both study days were similar. There were no differences in quality of sleep between HS and sham nights as assessed with the modified Leeds Sleep Evaluation Questionnaire (mLSEQ). Sino-Nasal Outcome Test (SNOT-14) questionnaires demonstrated significant increases (worsening) in 2/14 symptom categories with HS. CONCLUSIONS: The most likely cause for the failure to accelerate MCC was under-dosing of HS relative to the active transport of salt from the airways.

A Novel In Vitro Human Granuloma Model of Sarcoidosis and Latent Tuberculosis Infection.

Many aspects of pathogenic granuloma formation are poorly understood, requiring new relevant laboratory models that represent the complexity (genetics and diversity) of human disease. To address this need, we developed an in vitro model of granuloma formation using human peripheral blood mononuclear cells (PBMCs) derived from patients with active sarcoidosis, latent tuberculosis (TB) infection (LTBI), or normal healthy control subjects. PBMCs were incubated for 7 days with uncoated polystyrene beads or beads coated with purified protein derivative (PPD) or human serum albumin. In response to PPD-coated beads, PBMCs from donors with sarcoidosis and LTBI formed robust multicellular aggregates resembling granulomas, displaying a typical T-helper cell type 1 immune response, as assessed by cytokine analyses. In contrast, minimal PBMC aggregation occurred when control PBMCs were incubated with PPD-coated beads, whereas the response to uncoated beads was negligible in all groups. Sarcoidosis PBMCs responded to human serum albumin-coated beads with modest cellular aggregation and inflammatory cytokine release. Whereas the granuloma-like aggregates formed in response to PPD-coated beads were similar for sarcoidosis and LTBI, molecular profiles differed significantly. mRNA expression patterns revealed distinct pathways engaged in early granuloma formation in sarcoidosis and LTBI, and they resemble molecular patterns reported in diseased human tissues. This novel in vitro human granuloma model is proposed as a tool to investigate mechanisms of early granuloma formation and for preclinical drug discovery research of human granulomatous disorders. Clinical trial registered with www.clinicaltrials.gov (NCT01857401).

Pseudomonas infection and mucociliary and absorptive clearance in the cystic fibrosis lung.

Airway surface liquid hyperabsorption and mucus accumulation are key elements of cystic fibrosis lung disease that can be assessed in vivo using functional imaging methods. In this study we evaluated experimental factors affecting measurements of mucociliary clearance (MCC) and small-molecule absorption (ABS) and patient factors associated with abnormal absorption and mucus clearance.Our imaging technique utilises two radiopharmaceutical probes delivered by inhalation. Measurement repeatability was assessed in 10 adult cystic fibrosis subjects. Experimental factors were assessed in 29 adult and paediatric cystic fibrosis subjects (51 scans). Patient factors were assessed in a subgroup with optimal aerosol deposition (37 scans; 24 subjects). Paediatric subjects (n=9) underwent initial and 2-year follow-up scans. Control subjects from a previously reported study are included for comparison.High rates of central aerosol deposition influenced measurements of ABS and, to a lesser extent, MCC. Depressed MCC in cystic fibrosis was only detectable in subjects with previous Pseudomonas aeruginosa infection. Cystic fibrosis subjects without P. aeruginosa had similar MCC to control subjects. Cystic fibrosis subjects had consistently higher ABS rates.We conclude that the primary experimental factor affecting MCC/ABS measurements is central deposition percentage. Depressed MCC in cystic fibrosis is associated with P. aeruginosa infection. ABS is consistently increased in cystic fibrosis.

A physiologically-motivated compartment-based model of the effect of inhaled hypertonic saline on mucociliary clearance and liquid transport in cystic fibrosis.

BACKGROUND: Cystic Fibrosis (CF) lung disease is characterized by liquid hyperabsorption, airway surface dehydration, and impaired mucociliary clearance (MCC). Herein, we present a compartment-based mathematical model of the airway that extends the resolution of functional imaging data. METHODS: Using functional imaging data to inform our model, we developed a system of mechanism-motivated ordinary differential equations to describe the mucociliary clearance and absorption of aerosolized radiolabeled particle and small molecules probes from human subjects with and without CF. We also utilized a novel imaging metric in vitro to gauge the fraction of airway epithelial cells that have functional ciliary activity. RESULTS: This model, and its incorporated kinetic rate parameters, captures the MCC and liquid dynamics of the hyperabsorptive state in CF airways and the mitigation of that state by hypertonic saline treatment. CONCLUSIONS: We postulate, based on the model structure and its ability to capture clinical patient data, that patients with CF have regions of airway with diminished MCC function that can be recruited with hypertonic saline treatment. In so doing, this model structure not only makes a case for durable osmotic agents used in lung-region specific treatments, but also may provide a possible clinical endpoint, the fraction of functional ciliated airway.

Quantitative imaging of airway liquid absorption in cystic fibrosis.

New measures are needed to rapidly assess emerging treatments for cystic fibrosis (CF) lung disease. Using an imaging approach, we evaluated the absorptive clearance of the radiolabeled small molecule probe diethylene triamine penta-acetic acid (DTPA) as an in vivo indicator of changes in airway liquid absorption. DTPA absorption and mucociliary clearance rates were measured in 21 patients with CF (12 adults and nine children) and nine adult controls using nuclear imaging. The effect of hypertonic saline on DTPA absorption was also studied. In addition, in vitro studies were conducted to identify the determinants of transepithelial DTPA absorption. CF patients had significantly increased rates of DTPA absorption compared with control subjects but had similar mucociliary clearance rates. Treatment with hypertonic saline resulted in a decrease in DTPA absorption and an increase in mucociliary clearance in 11 out of 11 adult CF patients compared with treatment with isotonic saline. In vitro studies revealed that ∼ 50% of DTPA absorption can be attributed to transepithelial fluid transport. Apically applied mucus impedes liquid and DTPA absorption. However, mucus effects become negligible in the presence of an osmotic stimulus. Functional imaging of DTPA absorption provides a quantifiable marker of immediate response to treatments that promote airway surface liquid hydration.

Obesity accentuates circadian variability in breathing during sleep in mice but does not predispose to apnea.

Obesity is a primary risk factor for the development of obstructive sleep apnea in humans, but the impact of obesity on central sleep apnea is less clear. Given the comorbidities associated with obesity in humans, we developed techniques for long-term recording of diaphragmatic EMG activity and polysomnography in obese mice to assess breathing patterns during sleep and to determine the effect of obesity on apnea generation. We hypothesized that genetically obese ob/ob mice would exhibit less variability in breathing across the 24-h circadian cycle, be more prone to central apneas, and be more likely to exhibit patterns of increased diaphragm muscle activity consistent with obstructive apneas compared with lean mice. Unexpectedly, we found that obese mice exhibited a greater circadian impact on respiratory rate and diaphragmatic burst amplitude than lean mice, particularly during rapid eye movement (REM) sleep. Central apneas were more common in REM sleep (42 ± 17 h(-1)) than non-REM (NREM) sleep (14 ± 5 h(-1)) in obese mice (P < 0.05), but rates were not different between lean and obese mice in either sleep state. Even after experimentally enhancing central apnea generation by acute withdrawal of hypoxic chemoreceptor activation during sleep, central apnea rates remained comparable between lean and obese mice. Last, we were unable to detect patterns of diaphragmatic burst activity suggestive of obstructive apnea events in obese mice. In summary, obesity does not predispose mice to increased occurrence of central or obstructive apneas during sleep, but does lead to a more pronounced circadian variability in respiration.

Glucose regulation of load-induced mTOR signaling and ER stress in mammalian heart.

BACKGROUND: Changes in energy substrate metabolism are first responders to hemodynamic stress in the heart. We have previously shown that hexose-6-phosphate levels regulate mammalian target of rapamycin (mTOR) activation in response to insulin. We now tested the hypothesis that inotropic stimulation and increased afterload also regulate mTOR activation via glucose 6-phosphate (G6P) accumulation. METHODS AND RESULTS: We subjected the working rat heart ex vivo to a high workload in the presence of different energy-providing substrates including glucose, glucose analogues, and noncarbohydrate substrates. We observed an association between G6P accumulation, mTOR activation, endoplasmic reticulum (ER) stress, and impaired contractile function, all of which were prevented by pretreating animals with rapamycin (mTOR inhibition) or metformin (AMPK activation). The histone deacetylase inhibitor 4-phenylbutyrate, which relieves ER stress, also improved contractile function. In contrast, adding the glucose analogue 2-deoxy-d-glucose, which is phosphorylated but not further metabolized, to the perfusate resulted in mTOR activation and contractile dysfunction. Next we tested our hypothesis in vivo by transverse aortic constriction in mice. Using a micro-PET system, we observed enhanced glucose tracer analog uptake and contractile dysfunction preceding dilatation of the left ventricle. In contrast, in hearts overexpressing SERCA2a, ER stress was reduced and contractile function was preserved with hypertrophy. Finally, we examined failing human hearts and found that mechanical unloading decreased G6P levels and ER stress markers. CONCLUSIONS: We propose that glucose metabolic changes precede and regulate functional (and possibly also structural) remodeling of the heart. We implicate a critical role for G6P in load-induced mTOR activation and ER stress.

Liquid hyper-absorption as a cause of increased DTPA clearance in the cystic fibrosis airway.

BACKGROUND: Airway liquid hyper-absorption is a key pathophysiological link between the genetic mutations of cystic fibrosis (CF) and the development of lung disease. Here we consider whether the clearance of radiolabeled diethylene triamine pentaacetic acid (DTPA) might be used to detect changes in airway liquid absorption. METHODS: Tc99m-DTPA was added to the apical (luminal) surface of primary human bronchial epithelial cell cultures from CF and non-CF lungs. Liquid absorption rates were assessed using an optical method and compared to DTPA absorption rates. Measurements of transepithelial electrical resistance (TER) were made to determine the effect of epithelial permeability. DTPA absorption was assessed after stimuli known to influence liquid absorption (volume addition and osmotic gradients) and in cultures containing different proportions of CF and non-CF cells. RESULTS: DTPA absorption rate was increased in CF cultures matching previous in vivo studies in individuals with CF. DTPA and liquid absorption rates were proportional. There was no relationship between TER and DTPA absorption rate when measured in individual cultures. Apical volume addition increased both DTPA and liquid absorption rates. DTPA absorption increased in a dose-dependent manner after basolateral mannitol addition was used to create transepithelial osmotic gradients favoring liquid absorption. Conversely, apical mannitol (a candidate therapy) slowed DTPA absorption in CF cultures. CONCLUSIONS: These results imply that DTPA absorption is directly related to liquid absorption, consistent with increased rates of airway surface liquid absorption in the CF airway, and that modification of liquid absorption from osmotic therapies might be detectable through DTPA absorption measurements in vivo. TRIAL REGISTRATION: none.