Determinants of SARS-CoV-2 receptor gene expression in upper and lower airways.

The recent outbreak of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), has led to a worldwide pandemic. One week after initial symptoms develop, a subset of patients progresses to severe disease, with high mortality and limited treatment options. To design novel interventions aimed at preventing spread of the virus and reducing progression to severe disease, detailed knowledge of the cell types and regulating factors driving cellular entry is urgently needed. Here we assess the expression patterns in genes required for COVID-19 entry into cells and replication, and their regulation by genetic, epigenetic and environmental factors, throughout the respiratory tract using samples collected from the upper (nasal) and lower airways (bronchi). Matched samples from the upper and lower airways show a clear increased expression of these genes in the nose compared to the bronchi and parenchyma. Cellular deconvolution indicates a clear association of these genes with the proportion of secretory epithelial cells. Smoking status was found to increase the majority of COVID-19 related genes including ACE2 and TMPRSS2 but only in the lower airways, which was associated with a significant increase in the predicted proportion of goblet cells in bronchial samples of current smokers. Both acute and second hand smoke were found to increase ACE2 expression in the bronchus. Inhaled corticosteroids decrease ACE2 expression in the lower airways. No significant effect of genetics on ACE2 expression was observed, but a strong association of DNA- methylation with ACE2 and TMPRSS2- mRNA expression was identified in the bronchus.

Repaired oesophageal atresia: respiratory morbidity and pulmonary function in adults.

Although after oesophageal atresia (OA) repair in infancy, respiratory problems are common, their natural history remains unclear. We assessed morbidity, pulmonary function (PF), and bronchial hyperresponsiveness (BHR) in adults with repaired OA respiratory. 588 patients who underwent surgery for OA during 1947-1985 were identified and those 262 who were alive and had their native oesophagus were included. Respiratory symptoms and respiratory symptom-related quality of life (RSRQoL) were assessed by questionnaire and interview, and the patients underwent spirometry, a histamine challenge test, and an exhaled nitric oxide test. For the questionnaires, we added 287 carefully matched general population-derived controls. Among the 101 (58 male) patients, median age 36 yrs (range 22-56 yrs), respiratory morbidity was significantly increased compared to controls. Patients had more respiratory symptoms and infections, as well as asthma and allergies, and more often impaired RSRQoL (p<0.001 for all). PF tests revealed restrictive ventilatory defect in 21 (21%) patients, obstructive ventilatory defect in 21 (21%) patients, and both in 36 (36%) patients. A total of 41 (41%) had BHR, and in 15 (15%), it was consistent with asthma. The most significant risk factors for restrictive ventilatory defect were thoracotomy-induced rib fusions (OR 3.4, 95% CI 1.3-8.7; p = 0.01) and oesophageal epithelial metaplasia (OR 3.0, 95% CI 1.0-8.9; p = 0.05). After repair of OA, respiratory-related morbidity, restrictive ventilatory defect and BHR extended into adulthood. Nearly half the patients had BHR and over half had a restrictive ventilatory defect. Thoracotomy-induced rib fusions and gastro-oesophageal reflux-associated oesophageal epithelial metaplasia were the strongest risk factors for restrictive ventilatory defect.

Bronchial response pattern of antigen presenting cells and regulatory T cells in children less than 2 years of age.

BACKGROUND: In early childhood, the ability to mount protective immune responses in the airways is impaired, with increased risk of allergic sensitisation to inhaled allergens. Antigen presenting cells (APC) and regulatory T cells (Treg) are important modifiers of T cell immunity but little is known about their distribution in bronchial mucosa at this age. Here the subset distribution of APC and the appearance of Foxp3(+) Treg and bronchus associated lymphoid tissue (BALT) were examined immunohistochemically in children less than 2 years of age with chronic asthma-like symptoms of the lower airways. METHODS: Immunophenotyping was performed in situ on bronchial biopsy specimens obtained from 45 infants, 4-23 months of age, under investigation for airway disease. RESULTS: A well developed HLA-DR(+) network of APC was present in all samples, approximately 50% of the cells being CD68(+) macrophages and the remainder various subsets of dendritic cells. The density of HLA-DR(+) cells increased significantly with age but was not related to atopy, clinical symptoms or lung function. Comparing the density of APC subsets and clinical parameters, only the number of intraepithelial CD1a(+) dendritic cells was significantly increased in infants who had recently suffered a respiratory infection. BALT structures were identified in 22 children, with no relation to lung function, atopic status or human rhinovirus positivity. Plasmacytoid dendritic cells and Foxp3(+) Treg were located primarily within these isolated lymphoid follicles. CONCLUSION: A bronchial network of dendritic cells and macrophages develops quite rapidly after birth, apparently independent of clinical symptoms or atopy. The high frequency of BALT structures containing putative tolerogenic dendritic cells and Treg suggests that these lymphoid follicles play an important role in bronchial immune homeostasis during infancy.

Ultrastructure of the reticular basement membrane in asthmatic adults, children and infants.

Reticular basement membrane (RBM) thickening in asthma is considered to be the result of subepithelial fibrosis. Thus, the RBM in asthma should contain an excess of fibrils identified as interstitial collagen and the ratio of fibril to matrix should be increased above normal levels. Electron micrographs of the RBM were compared with those of interstitial collagen deeper in the bronchial wall using endobronchial biopsy specimens from adult asthmatics (aged 18-41 yrs (n = 10)), children with difficult asthma (aged 6-16 yrs (n = 10)), wheezy infants with reversible airflow limitation (aged 0.3-2 yrs (n = 10)) and age-matched nonasthmatic controls: 10 adults, nine children and nine symptomatic infants with normal lung function. Fibrils in the RBM were significantly thinner (median (range) width 39 (30-52) nm versus 59 (48-73) nm), and fewer fibrils were banded than in the interstitial collagen (ratio of banded to non-banded fibrils 0.08 (0-0.17) versus 0.22 (0-1.3)). The ratio of fibrils to matrix in the thickened RBM of asthmatics did not differ from that of their respective controls (1.34 (0.63-2.49) versus 1.18 (0.31-2.6)). The ratio of fibril to matrix in the thickened reticular basement membrane of asthmatics is normal, and, contrary to what is expected in fibrosis, the fibrils do not resemble those of interstitial collagen.

Montelukast for treating seasonal allergic rhinitis: a randomized, double-blind, placebo-controlled trial performed in the spring.

BACKGROUND: Cysteinyl leukotrienes are important proinflammatory mediators believed to have a role in allergic rhinitis. OBJECTIVE: This multicentre, randomized, double-blind, placebo- and active-controlled trial evaluated the effectiveness and tolerability of montelukast, a cysteinyl leukotriene receptor antagonist, for treating patients with seasonal allergic rhinitis. METHODS: After a 3- to 5-day, single-blind placebo run-in period, 1302 male and female patients (aged 15-81 years) with active allergic rhinitis symptoms were randomly assigned to receive montelukast 10 mg (n = 348), loratadine 10 mg (n = 602), or placebo (n = 352) administered once daily at bedtime for 2 weeks during the spring allergy season. RESULTS: Mean patient characteristics and symptom scores at baseline were similar for the three treatment groups. The primary end-point, daytime nasal symptoms score (mean of nasal congestion, rhinorrhea, nasal pruritus, and sneezing scores; 0-3 scale), improved from baseline during treatment by (least squares mean, 95% confidence interval) - 0.37 (- 0.43, - 0.31), - 0.47 (- 0.52, - 0.43), and - 0.24 (- 0.29, - 0.18) in the montelukast, loratadine, and placebo groups, respectively (P < or = 0.001 comparing each active treatment with placebo). Mean changes from baseline in all other diary-based scores, including night-time and eye symptom scores, were significantly greater for each active treatment than for placebo. The rhinoconjunctivitis quality of life overall score improved significantly with montelukast and with loratadine as compared with placebo. Montelukast and loratadine showed a safety profile comparable to that of placebo. CONCLUSION: Montelukast is well tolerated and provides improvements in daytime and night-time symptoms, as well as quality of life parameters, for patients with seasonal allergic rhinitis.

Concomitant montelukast and loratadine as treatment for seasonal allergic rhinitis: a randomized, placebo-controlled clinical trial.

BACKGROUND: Nasal challenge studies have suggested histamine and cysteinyl leukotrienes are important proinflammatory mediators in allergic rhinitis. This study was designed to determine the efficacy of montelukast, a cysteinyl leukotriene receptor antagonist, administered alone or concomitantly with loratadine, an H(1)-receptor antagonist, in seasonal allergic rhinitis. OBJECTIVE: The purpose of this study was to determine the effect of concomitant use of montelukast and loratadine in the treatment of seasonal allergic rhinitis. METHODS: In this multicenter (N = 12) double-blind, randomized, parallel-group, placebo-controlled 2-week trial, 460 men and women, aged 15 to 75 years, with spring seasonal allergic rhinitis were randomly allocated to receive 1 of the following 5 treatments: montelukast 10 or 20 mg, loratadine 10 mg, montelukast 10 mg with loratadine 10 mg, or placebo, once daily in the evening. The primary end point was daytime nasal symptoms score (average of congestion, rhinorrhea, itching, and sneezing). Other end points were eye symptoms, nighttime symptoms, individual daytime nasal symptoms, global evaluations (patient's and physician's), and rhinoconjunctivitis quality-of-life scores. RESULTS: Concomitant montelukast with loratadine improved the primary end point significantly (P <.001) compared with placebo and each agent alone. Compared with placebo, montelukast with loratadine also significantly improved eye symptoms, nighttime symptoms, individual daytime nasal symptoms, global evaluations, and quality of life. Montelukast alone and loratadine alone caused modest improvements in rhinitis end points. All treatments were similarly well tolerated. CONCLUSIONS: Concomitant montelukast with loratadine provided effective treatment for seasonal allergic rhinitis and associated eye symptoms with a safety profile comparable with placebo.

Effect of montelukast on single-dose theophylline pharmacokinetics.

The effect of montelukast (MK-0476), a cysteinyl leukotriene receptor antagonist in development for treatment of asthma, on single-dose theophylline plasma concentrations was studied in three separate clinical trials. Montelukast was evaluated at 10 mg once daily (the clinical dosage), 200 mg once daily, and 600 mg (200 mg three times daily). At the clinical dosage, montelukast did not change single-dose theophylline plasma concentration in a clinically important manner. The geometric mean ratios for theophylline area under the plasma concentration versus time curve (AUC0-->infinity ) (0.92) and maximal plasma concentration (Cmax ) (1.04) were well within the predefined and generally accepted bioequivalence range of 0.80 and 1.25. Montelukast decreased theophylline Cmax by 12% and 10%, AUC0-->infinity by 43% and 44%, and elimination half-time by 44% and 39% at 200 mg/d (oral and intravenous, respectively), and at 600 mg/d, montelukast decreased theophylline Cmax by 25%, AUC0-->infinity by 66%, and elimination half-time by 63%. These results show that montelukast at the clinical dosage did not change theophylline pharmacokinetics in a clinically important manner, but at 20- to 60-fold higher dosages, montelukast significantly reduced the theophylline pharmacokinetics parameters; an apparent dosage dependence is suggested.

Use of radiation suicide to isolate constitutive and temperature-sensitive conditional Chinese hamster ovary cell mutants with defects in the endocytosis of low density lipoprotein.

A radiation suicide procedure was used to isolate cells with either constitutive or temperature-sensitive (ts) defects in the receptor-mediated endocytosis of low density lipoprotein (LDL). Mutagen-treated Chinese hamster ovary cells maintained at 34 degrees C (permissive temperature) were shifted to 39.5 degrees C (nonpermissive temperature) for 14-26 h and incubated at 39.5 degrees C for an additional 6-8 h with [3H]cholesteryl linoleate LDL. Wild-type cells internalized this lipoprotein via LDL receptors and accumulated [3H]cholesteryl linoleate (1.5-2 dpm/cell). Radiolysis during 80 days of frozen storage killed most of these cells (radiation suicide). Receptor-deficient cells were identified by screening the surviving cells for their inability to internalize and accumulate 125I-LDL using a replica plating assay. From 3.6 x 10(7) tritium-labeled cells, two clones fell into previously defined constitutive and ts complementation groups (ldlA and ldlG, respectively). Another constitutive and two other ts mutants defined two new complementation groups, ldlI (constitutive) and ldlH (ts). This increases to nine the current number of recessive, LDL receptor-deficient, Chinese hamster ovary complementation groups. All of the mutants with ts defects in LDL endocytosis exhibited ts conditional-lethal phenotypes. At the nonpermissive temperature, the rates of loss of LDL receptor activity (t 1/2 = 10-14 h) were significantly faster than the rates of loss of protein synthesis (t 1/2 greater than 24 h), suggesting that the temperature sensitivity of receptor activity was not simply due to the metabolic collapse of dying cells. Detailed analysis of these new classes of mutants should help define gene products and functions required for LDL receptor activity.

Intracellular cascades in the parathyroid-hormone-dependent regulation of Na+/phosphate cotransport in OK cells.

Parathyroid hormone (PTH) increased intracellular cyclic AMP and reduces Na+/phosphate cotransport activity in OK cells [Malmström & Murer (1986) Am. J. Physiol. 251, C23-C31; Caverzasio, Rizzoli & Bonjour (1986) J. Biol. Chem. 261, 3233-3237]. It was also shown that PTH activates phosphoinositide metabolism in OK cells [Hruska, Moskowitz, Esprit, Civitelli, Westbrook & Huskey (1987) J. Clin. Invest. 79, 230-239]. In the present paper we show that tumour-promoting phorbol esters are effective in reducing Na+/phosphate cotransport. The Ca2+ ionophores A23187 and ionomycin had only a small effect on Na+/phosphate cotransport; added together, A23187 and phorbol esters showed a synergistic action. Phorbol esters and phorbol esters plus ionomycin stimulated prostaglandin synthesis as well as cyclic AMP production; acetylsalicylic acid prevented phorbol-ester-induced prostaglandin synthesis and cyclic AMP production, but had no effect on inhibition of Na+/phosphate cotransport. In suspensions of OK cells, PTH and thrombin produced a rise in intracellular Ca2+. In contrast with PTH, thrombin did not elevate cellular cyclic AMP in suspended OK cells. PTH and thrombin reduced Na+/phosphate cotransport in suspended OK cells. It is suggested that two regulatory cascades are involved in PTH action on Na+/phosphate cotransport: cyclic AMP/kinase A and Ca2+/diacylglycerol/kinase C.

Freeze-thaw and high-voltage discharge allow macromolecule uptake into ileal brush-border vesicles.

High-voltage discharge or one cycle of freeze-thawing are shown to transiently permeabilize rabbit ileal brush-border membrane vesicles to macromolecules. Uptake of the radiolabeled macromolecule dextran, mol wt 70,000, used as a marker for vesicle permeability, was determined by a rapid filtration technique, with uptake defined as substrate associated with the vesicle and releasable after incubation of vesicles with 0.1% saponin. Dextran added immediately after electric shock (2,000 V) or at the beginning of one cycle of freeze-thawing was taken up approximately eightfold compared with control; with both techniques, the concentration of dextran after being taken up into the vesicles was similar to that in the incubation medium, suggesting attainment of equilibrium. ATP also was taken up into freeze-thawed vesicles, whereas there was no significant uptake into control vesicles. The increase in vesicle permeability was reversible, based on Na-dependent D-glucose uptake being decreased when studied 5 but not 15 min after electric shock, and was not significantly decreased after completion of one cycle of freeze-thawing. In addition, adenosine 3',5'-cyclic monophosphate and Ca2+-calmodulin-dependent protein kinase activity were similar in control vesicles and vesicles exposed to high-voltage discharge or freeze-thawing. Also, vesicles freeze-thawed with [32P]ATP demonstrated increased phosphorylation compared with nonfrozen vesicles, while freeze-thawing did not alter vesicle protein as judged by Coomassie blue staining. These techniques should allow intestinal membrane vesicles to be used for studies of intracellular control of transport processes, for instance, studies of protein kinase regulation of transport.

Mechanism of NaCl transport-stimulated prostaglandin formation in MDCK cells.

Recently we have found that stimulation of NaCl transport in high-resistance MDCK cells enhances their prostaglandin formation. In the present study, we investigated the mechanisms by which prostaglandin formation could be linked to the ion transport in these cells. We found that stimulation of transport caused a transient stimulation of prostaglandin formation lasting 5-10 min. The rise in prostaglandin formation was paralleled by a rise of free intracellular arachidonic acid. Analysis of membrane lipids revealed that the rise of free arachidonic acid was paralleled by a loss of arachidonic acid from polyphosphoinositides. We failed to obtain indications for the stimulation of calcium-dependent phospholipase A2. However, we did obtain evidence that the incorporation of arachidonic acid into phospholipids was diminished during stimulation of ion transport, indicating a decreased rate of reesterification. Despite the fact that there was no significant fall in total cellular ATP on stimulation of ion transport, we found a high and transient rise of lactate production of the cells on stimulation of the ion transport indicating an alteration of the ADP/ATP ratio. Moreover, prostaglandin formation and lactate formation were linearly correlated in this situation. When glucose utilization was inhibited by mannoheptulose, the rise in lactate formation was abolished, whereas that of PG formation was unaltered, indicating that lactate formation and prostaglandin formation were not causally linked on stimulation of ion transport. Our results suggest that an increase in the rate of sodium chloride transport by MDCK cells stimulates formation by an inhibition of reesterification of free arachidonic acid.(ABSTRACT TRUNCATED AT 250 WORDS)

Intracellular regulatory cascades: examples from parathyroid hormone regulation of renal phosphate transport.

The knowledge about intracellular regulatory cascades in hormone action has increased considerably over the last few years. Receptor occupation at the plasma membrane level results in a production of intracellular messengers, such as cyclic nucleotides (cAMP, cGMP), inositoltrisphosphate (IP3), diacylglycerol (DAG) and a rise in cytosolic calcium concentration. These messengers control the activity of different regulatory mechanisms which operate either in sequence or in parallel to generate the final biological response. In PTH-dependent regulation of renal phosphate transport, cAMP-dependent and calcium-dependent mechanisms are involved: Recent experiments with cultured renal epithelial cells have confirmed that activation of adenylate cyclase is the initial event. However, the cAMP signal can be bypassed and direct activation of protein kinase C seems to mimic PTH induced inhibition of phosphate transport. The final event in the regulatory cascade is most likely a removal of the phosphate transport system followed by a degradation.

Parathyroid hormone inhibits phosphate transport in OK cells but not in LLC-PK1 and JTC-12.P3 cells.

Na+-dependent phosphate transport and its response to parathyroid hormone (PTH) has been investigated in three continuous cell lines of renal epithelial origin (LLC-PK1, JTC-12.P3, and OK). The apparent Km for phosphate was similar, but the maximal transport rate (Vmax) was markedly different in the three cell lines. PTH and forskolin produced an increase of cellular adenosine 3',5'-cyclic monophosphate (cAMP) in all cell lines, but Na+-dependent phosphate transport was inhibited exclusively in the OK cells (a threefold reduction of influx after 4 h of exposure to 10(-10) M PTH). The change in phosphate transport is accounted for by a lowered Vmax (30.8 +/- 5.3 vs. 10.2 +/- 1.1 pmol X mg-1 X 3 min-1). The reduction in phosphate transport was reversible, such that 5 h after removal of PTH the Vmax had increased threefold over the inhibited state. Addition of PTH did not alter Na+-dependent L-alanine influx in the OK cells. Experiments with apical membrane vesicles showed that the change in Vmax occurred at the membrane level. It is concluded that the regulatory event responsible for PTH-reduced phosphate transport is beyond cAMP. Of the cell lines studied, only OK cells have a complete regulatory cascade.

Erythropoietin production by fetal mouse liver cells in response to hypoxia and adenylate cyclase stimulation.

This study was done to investigate aspects of control of extrarenal erythropoietin (Ep) production. To this end we studied the effects of three stimuli of renal Ep production in the adult, i.e. hypoxia, cobalt, and activation of adenylate cyclase on Ep generation by cultured fetal mouse liver cells. The fetal liver was taken as a model for extrarenal Ep production because this organ is considered the predominant site of extrarenal Ep production. We found that Ep production by the cells increased as the oxygen concentration was decreased in the incubation atmosphere from 20% to 1%. Cobalt (10(-4)-10(-5) M) had no effect on Ep production. Activation of adenylate cyclase by forskolin (10(-5) M) or isoproterenol (10(-5) M) greatly enhanced Ep production. These findings indicate that the Ep-stimulating effect of cobalt is specific for the kidney. However, oxygen depletion and activation of adenylate cyclase seem to be more general stimuli in Ep-producing cells. Furthermore we found that Ep production in hypoxia correlated with lactate formation in the cultured liver cells. This finding suggests that Ep production in fetal livers under hypoxic conditions parallels the shift from aerobic to anaerobic cellular energy metabolism.

Ca2+-dependent protein phosphorylation in brush border membranes of rat kidney proximal tubules.

Ca2+-dependent protein phosphorylation was studied in isolated brush border membrane vesicles from rat kidney cortex. Phosphorylation of about 20 target proteins with gamma-32P-ATP was observed after opening the vesicles transiently by detergent treatment or by hypotonic shock indicating that phosphorylation takes place from the cytoplasmic side. Five of these polypeptides (Mr 12, 19, 29, 97 and 138 kD) showed increased phosphorylation in the presence of micromolar calcium. Addition of exogenous calmodulin did not enhance the calcium induced phosphorylation significantly, nor did trifluoperazine depress it, suggesting that Ca2+-calmodulin-dependent protein kinase is not involved in the studied Ca2+-induced phosphorylation. 12-O-tetradecanoylphorbol-13-acetate (TPA) minimized the Ca2+ dependency for the 12 and 97 kD polypeptides. Cytotoxin I inhibited the incorporation of phosphate into the 12 and 97 kD polypeptides in a dose-dependent manner. Excess phosphatidylserine could reverse this inhibition but stimulated also the phosphorylation of the 19 kD polypeptide. These findings suggest that at least the 12 and the 97 kD polypeptides are substrates for an endogenous protein kinase C. When studied under conditions where Ca2+ increases membrane phosphorylation, no effects could be observed on the kinetic parameters (Km and Vmax) of the sodium-dependent phosphate uptake.

Phosphorylation of rat kidney proximal tubular brush border membranes. Role of c-AMP dependent protein phosphorylation in the regulation of phosphate transport.

A possible correlation between cyclic-AMP dependent protein phosphorylation and altered sodium dependent transport of inorganic phosphate was analyzed in isolated rat renal proximal tubular brush border membrane vesicles. In transiently opened vesicles (opened by an osmotic shock), the addition of gamma-32P-ATP leads to 32P-incorporation into several membrane proteins. The simultaneous addition of cyclic-AMP leads to increased phosphorylation of several proteins (e.g. apparent molecular weights: 40 kD, 46 kD, 55 kD). The addition of ATP, GTP and ITP to the osmotic shock medium leads to an (non-specific) inhibition of the sodium gradient dependent phosphate uptake. No further inhibition of the sodium dependent phosphate transport was observed when membrane vesicles were phosphorylated by ATP in the presence of cyclic-AMP. These data show a lack of correlation between cyclic-AMP dependent protein phosphorylation and altered sodium gradient dependent phosphate transport. Thus, there is no experimental support for the involvement of cyclic-AMP dependent protein phosphorylation as one of the final events in the regulation of phosphate transport across the rat renal proximal tubular brush border membrane.