Deletion of airway cilia results in noninflammatory bronchiectasis and hyperreactive airways.

The mechanisms for the development of bronchiectasis and airway hyperreactivity have not been fully elucidated. Although genetic, acquired diseases and environmental influences may play a role, it is also possible that motile cilia can influence this disease process. We hypothesized that deletion of a key intraflagellar transport molecule, IFT88, in mature mice causes loss of cilia, resulting in airway remodeling. Airway cilia were deleted by knockout of IFT88, and airway remodeling and pulmonary function were evaluated. In IFT88(-) mice there was a substantial loss of airway cilia on respiratory epithelium. Three months after the deletion of cilia, there was clear evidence for bronchial remodeling that was not associated with inflammation or apparent defects in mucus clearance. There was evidence for airway epithelial cell hypertrophy and hyperplasia. IFT88(-) mice exhibited increased airway reactivity to a methacholine challenge and decreased ciliary beat frequency in the few remaining cells that possessed cilia. With deletion of respiratory cilia there was a marked increase in the number of club cells as seen by scanning electron microscopy. We suggest that airway remodeling may be exacerbated by the presence of club cells, since these cells are involved in airway repair. Club cells may be prevented from differentiating into respiratory epithelial cells because of a lack of IFT88 protein that is necessary to form a single nonmotile cilium. This monocilium is a prerequisite for these progenitor cells to transition into respiratory epithelial cells. In conclusion, motile cilia may play an important role in controlling airway structure and function.

Loss of primary cilia upregulates renal hypertrophic signaling and promotes cystogenesis.

Primary cilia dysfunction alters renal tubular cell proliferation and differentiation and associates with accelerated cyst formation in polycystic kidney disease. However, the mechanism leading from primary ciliary dysfunction to renal cyst formation is unknown. We hypothesize that primary cilia prevent renal cyst formation by suppressing pathologic tubular cell hypertrophy and proliferation. Unilateral nephrectomy initiates tubular cell hypertrophy and proliferation in the contralateral kidney and provides a tool to examine primary cilia regulation of renal hypertrophy. Conditional knockout of the primary cilia ift88 gene leads to delayed, adult-onset renal cystic disease, which provides a window of opportunity to conduct unilateral nephrectomy and examine downstream kinetics of renal hypertrophy and cyst formation. In wild-type animals, unilateral nephrectomy activated the mTOR pathway and produced appropriate structural and functional hypertrophy without renal cyst formation. However, in ift88 conditional knockout animals, unilateral nephrectomy triggered increased renal hypertrophy and accelerated renal cyst formation, leading to renal dysfunction. mTOR signaling also increased compared with wild-type animals, suggesting a mechanistic cascade starting with primary ciliary dysfunction, leading to excessive mTOR signaling and renal hypertrophic signaling and culminating in cyst formation. These data suggest that events initiating hypertrophic signaling, such as structural or functional loss of renal mass, may accelerate progression of adult polycystic kidney disease toward end-stage renal disease.

A Photographic Case of Frostbite Treated with Delayed Hyperbaric Oxygen Therapy.

Davis, Ashlee, Brent Sinopoli, Nathaniel Mann, and Antine E. Stenbit. A photographic case of frostbite treated with delayed hyperbaric oxygen therapy. High Alt Med Biol. 23:194-197, 2022.-A 50-year-old female presented to the emergency department in June 2019 5 days after summiting Denali. Temperature on summit day was -25°F, and she developed frostbite on multiple digits of both hands. Eight digits demonstrated severe frostbite based on the grading set forth by Cauchy et al. Five of these had grade III injuries. She received three hyperbaric dives in Alaska before flying to her home state. On day 5 she received aspirin, aloe wraps, and underwent twenty-seven 90-minute dives at 2 atmospheres absolute. Unique in current literature, each dive was accompanied by photographs detailing clinical progression, which may assist providers in providing expectations in care. She eventually underwent amputation at, or distal to, the distal interphalangeals of bilateral third and fourth digits and the left fifth digit. By January 2020, she had returned to near-normal range of motion. Hyperbaric oxygen therapy represents a potential adjunct therapeutic option for frostbite, even in delayed manner, but its use as a treatment modality warrants further prospective analysis.

Pulmonary exacerbations in cystic fibrosis.

PURPOSE OF REVIEW: The chronic infection and inflammation of cystic fibrosis (CF) lung disease causes a progressive decline of lung function resulting in daily symptoms such as cough and sputum production. There are intermittent episodes of acute worsening of symptoms, more commonly referred to as pulmonary exacerbations. Despite this being a common event, there is still no standardized definition of an exacerbation. A recent set of guidelines from the CF Foundation Pulmonary Therapies Committee on the treatment of exacerbations noted the paucity of data supporting commonly used therapies. This review describes our current understanding of pulmonary exacerbations and the therapies used to treat them. RECENT FINDINGS: The treatment of an exacerbation is intended to resolve the worsened symptoms and to restore the lung function that is commonly lost in the acute presentation. A most striking finding is the observation that for many patients there is no restoration of lung function, suggesting we either need better therapies to prevent exacerbations or better treatment of exacerbations. SUMMARY: We have established recommendations on specific treatment of a pulmonary exacerbation and have outlined the areas where we need better information on appropriate therapies. Once we have a standardized definition of an exacerbation, we can proceed with clinical trials of therapies specific for its treatment.

Efficacy of therapeutic plasma exchange in the treatment of penn class 3 and 4 cytokine release syndrome complicating COVID-19.

OBJECTIVES: Cytokine release syndrome (CRS) is a potentially severe complication of COVID-19 most commonly resulting in respiratory failure. This ten-patient study was designed to determine the efficacy of therapeutic plasma exchange (TPE) in improving oxygenation and in reducing the cytokine load in a critically ill subset of patients. METHODS: Five single volume plasma exchanges over eight days within a 14-day study period. In mechanically ventilated patients, oxygenation was measured via the PaO2/FiO2 (P/F) ratio and the oxygenation index (OI) daily for 14 days. Supplemental oxygen requirements were tracked daily for non-ventilated patients. RESULTS: Non-ventilated patients were liberated from supplemental oxygen after TPE. The response was rapid with an 87% average reduction in oxygenation requirements following and average time to return to room air of 5.25 days. All mechanically ventilated patients demonstrated improvement in oxygenation with a 78% average improvement in the P/F ratio and a 43% improvement in OI. C-reactive protein (CRP) and serum levels of IL-6, IL-8, IL-10, TNFα, IFNγ and GM-CSF, were measured daily with immediate post TPE levels drawn on days 1, 2, 4, 6 and 8. All patients demonstrated significant reductions in CRP, IL-6, IL-10 and TNFα. CONCLUSIONS: In the majority of patients with Penn class 3 and 4 CRS complicating COVID-19, TPE demonstrated a prompt improvement in oxygenation and reduction in cytokine load without compromising patient safety. As this pilot study was envisioned to be hypothesis generating, expanded trials using TPE alone and in conjunction with novel pharmacologic agents are warranted. REGISTRATION: ClinicalTrials.gov NCT04374149.

alpha-E-catenin inactivation disrupts the cardiomyocyte adherens junction, resulting in cardiomyopathy and susceptibility to wall rupture.

BACKGROUND: alpha-E-catenin is a cell adhesion protein, located within the adherens junction, thought to be essential in directly linking the cadherin-based adhesion complex to the actin cytoskeleton. Although alpha-E-catenin is expressed in the adherens junction of the cardiomyocyte intercalated disc, and perturbations in its expression are observed in models of dilated cardiomyopathy, its role in the myocardium remains unknown. METHODS AND RESULTS: To determine the effects of alpha-E-catenin on cardiomyocyte ultrastructure and disease, we generated cardiac-specific alpha-E-catenin conditional knockout mice (alpha-E-cat cKO). alpha-E-cat cKO mice displayed progressive dilated cardiomyopathy and unique defects in the right ventricle. The effects on cardiac morphology/function in alpha-E-cat cKO mice were preceded by ultrastructural defects in the intercalated disc and complete loss of vinculin at the intercalated disc. alpha-E-cat cKO mice also revealed a striking susceptibility of the ventricular free wall to rupture after myocardial infarction. CONCLUSIONS: These results demonstrate a clear functional role for alpha-E-catenin in the cadherin/catenin/vinculin complex in the myocardium in vivo. Ablation of alpha-E-catenin within this complex leads to defects in cardiomyocyte structural integrity that result in unique forms of cardiomyopathy and predisposed susceptibility to death after myocardial stress. These studies further highlight the importance of studying the role of alpha-E-catenin in human cardiac injury and cardiomyopathy in the future.

Altered hepatic and muscle substrate utilization provoked by GLUT4 ablation.

Studies were conducted to explore altered substrate utilization and metabolism in GLUT4 null mice. Liver fatty acid synthase mRNA and fatty acid synthesis rates were dramatically increased in GLUT4 null mice compared with control mice and were supported by increased rates of the pentose phosphate pathway oxidative phase and sterol regulatory binding protein mRNA expression. Increased GLUT2 protein content, glucokinase mRNA, and glucose-6-phosphate in GLUT4 null mice may provide substrate for the enhanced fatty acid synthesis. Increased fatty acid synthesis, however, did not lead to hepatic triglyceride accumulation in GLUT4 null mice because of increased hepatic triglyceride secretion rates. GLUT4 null mice rapidly cleared orally administered olive oil, had reduced serum triglyceride concentrations in the fed and the fasted state, and increased skeletal muscle lipoprotein lipase when compared with controls. Oleate oxidation rates were increased in GLUT4 null skeletal muscle in association with mitochondrial hyperplasia/hypertrophy. This study demonstrated that GLUT4 null mice had increased hepatic glucose uptake and conversion into triglyceride for subsequent use by muscle. The ability of GLUT4 null mice to alter hepatic carbohydrate and lipid metabolism to provide proper nutrients for peripheral tissues may explain (in part) their ability to resist diabetes when fed a normal diet.

Timing of inhaled tobramycin affects assessment of intravenous tobramycin pharmacokinetic monitoring.

BACKGROUND: Aerosolized tobramycin inhalation solution (TIS) may be absorbed and result in measurable serum concentrations. We assessed the significance of TIS dosing in the latter portion of the IV dosing interval on the calculation of pharmacokinetic (PK) parameters and dosing. METHODS: Twenty adult CF patients admitted to the hospital for treatment of a pulmonary exacerbation were enrolled. PK parameters of tobramycin were calculated before and after introduction of TIS, which was given 5-9 h after the IV dose. RESULTS: Nine patients had a clinically significant change in tobramycin trough concentration. Fourteen patients had a reduced calculated elimination rate constant after TIS administration, which may be misinterpreted as a decreased clearance of IV tobramycin. CONCLUSION: Trough tobramycin concentrations were significantly influenced in some CF patients (45%), suggesting that timing of the inhaled dose should be considered when interpreting PK measures of IV tobramycin dosing.

A roadmap to the brittle bones of cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disorder which despite advances in medical care continues to be a life-limiting and often fatal disease. With increase in life expectancy of the CF population, bone disease has emerged as a common complication. Unlike the osteoporosis seen in postmenopausal population, bone disease in CF begins at a young age and is associated with significant morbidity due to fractures, kyphosis, increased pain, and decreased lung function. The maintenance of bone health is essential for the CF population during their lives to prevent pain and fractures but also as they approach lung transplantation since severe bone disease can lead to exclusion from lung transplantation. Early recognition, prevention, and treatment are key to maintaining optimal bone health in CF patients and often require a multidisciplinary approach. This article will review the pathophysiology, current clinical practice guidelines, and potential future therapies for treating CF-related bone disease.

Frequency of mitochondrial 12S ribosomal RNA variants in an adult cystic fibrosis population.

In adult cystic fibrosis patient populations, gram-negative bacteria, particularly Pseudomonas aeruginosa, frequently require aggressive therapy including systemic antibiotics, bronchodilators and airway clearance techniques. Aminoglycosides including tobramycin are used frequently to control these chronic airway infections. They, however, cause important nephrotoxic and ototoxic effects that can significantly alter the quality of life. We investigated the genetic predisposition to aminoglycoside ototoxicity in a typical unscreened North American cystic fibrosis population by screening for variants in mitochondrial 12S ribosomal RNA and noted several polymorphisms occurred at higher frequencies than expected and were associated with clinically significant cases of hearing loss. In the population studied, both patients possessing the 1555A>G transition exhibited profound ototoxicity after nontoxic dosing of tobramycin. We also identified new homoplasmic genetic variations in the mitochondrial 12S ribosomal RNA, several of which occurred in highly conserved regions of the gene and were present in patients with moderate-to-severe ototoxicity after exposure to aminoglycosides.

Increased expression of SERCA in the hearts of transgenic mice results in increased oxidation of glucose.

While several transgenic mouse models exhibit improved contractile characteristics in the heart, less is known about how these changes influence energy metabolism, specifically the balance between carbohydrate and fatty acid oxidation. In the present study we examine glucose and fatty acid oxidation in transgenic mice, generated to overexpress sarco(endo)plasmic reticulum calcium-ATPase (SERCA), which have an enhanced contractile phenotype. Energy substrate metabolism was measured in isolated working hearts using radiolabeled glucose and palmitate. We also examined oxygen consumption to see whether SERCA overexpression is associated with increased oxygen utilization. Since SERCA is important in calcium handling within the cardiac myocyte, we examined cytosolic calcium transients in isolated myocytes using indo-1, and mitochondrial calcium levels using pericam, an adenovirally expressed, mitochondrially targeted ratiometric calcium indicator. Oxygen consumption did not differ between wild-type and SERCA groups; however, we were able to show an increased utilization of glucose for oxidative metabolism and a corresponding decreased utilization of fatty acids in the SERCA group. Cytosolic calcium transients were increased in myocytes isolated from SERCA mice, and they show a faster rate of decay of the calcium transient. With these observations we noted increased levels of mitochondrial calcium in the SERCA group, which was associated with an increase in the active form of the pyruvate dehydrogenase complex. Since an increase in mitochondrial calcium levels leads to activation of the pyruvate dehydrogenase complex (the rate-limiting step for carbohydrate oxidation), the increased glucose utilization observed in isolated perfused hearts in the SERCA group may reflect a higher level of mitochondrial calcium.

Hepatic response to restoration of GLUT4 in skeletal muscle of GLUT4 null mice.

Expression of GLUT4 in fast-twitch skeletal muscle fibers of GLUT4 null mice (G4-MO) normalized glucose uptake in muscle and restored peripheral insulin sensitivity. GLUT4 null mice exhibit altered carbohydrate and lipid metabolism in liver and skeletal muscle. To test the hypothesis that increased glucose utilization by G4-MO muscle would normalize the changes seen in the GLUT4 null liver, serum metabolites and hepatic metabolism were compared in control, GLUT4 null, and G4-MO mice. The fed serum glucose and triglyceride levels of G4-MO mice were similar to those of control mice. In addition, the alternations in liver metabolism seen in GLUT4 nulls including increased GLUT2 expression and fatty acid synthesis accompanied by an increase in the oxidative arm of the pentose phosphate pathway were absent in G4-MO mice. The transgene used for GLUT4 restoration in muscle was specific for fast-twitch muscle fibers. The mitochondria hypertrophy/hyperplasia in all GLUT4 null skeletal muscles was absent in transgene-positive extensor digitorum longus muscle but present in transgene-negative soleus muscle of G4-MO mice. Results of this study suggest that the level of muscle GLUT4 expression influences mitochondrial biogenesis. These studies also demonstrate that the type and amount of substrate that muscle takes up and metabolizes, determined in part by GLUT4 expression levels, play a major role in directing hepatic carbohydrate and lipid metabolism.

Systems analysis of PKA-mediated phosphorylation gradients in live cardiac myocytes.

Compartmentation and dynamics of cAMP and PKA signaling are important determinants of specificity among cAMP's myriad cellular roles. Both cardiac inotropy and the progression of heart disease are affected by spatiotemporal variations in cAMP/PKA signaling, yet the dynamic patterns of PKA-mediated phosphorylation that influence differential responses to agonists have not been characterized. We performed live-cell imaging and systems modeling of PKA-mediated phosphorylation in neonatal cardiac myocytes in response to G-protein coupled receptor stimuli and UV photolysis of "caged" cAMP. cAMP accumulation was rate-limiting in PKA-mediated phosphorylation downstream of the beta-adrenergic receptor. Prostaglandin E1 stimulated higher PKA activity in the cytosol than at the sarcolemma, whereas isoproterenol triggered faster sarcolemmal responses than cytosolic, likely due to restricted cAMP diffusion from submembrane compartments. Localized UV photolysis of caged cAMP triggered gradients of PKA-mediated phosphorylation, enhanced by phosphodiesterase activity and PKA-mediated buffering of cAMP. These findings indicate that combining live-cell FRET imaging and mechanistic computational models can provide quantitative understanding of spatiotemporal signaling.