Digital proximity tracing on empirical contact networks for pandemic control.

Digital contact tracing is a relevant tool to control infectious disease outbreaks, including the COVID-19 epidemic. Early work evaluating digital contact tracing omitted important features and heterogeneities of real-world contact patterns influencing contagion dynamics. We fill this gap with a modeling framework informed by empirical high-resolution contact data to analyze the impact of digital contact tracing in the COVID-19 pandemic. We investigate how well contact tracing apps, coupled with the quarantine of identified contacts, can mitigate the spread in real environments. We find that restrictive policies are more effective in containing the epidemic but come at the cost of unnecessary large-scale quarantines. Policy evaluation through their efficiency and cost results in optimized solutions which only consider contacts longer than 15-20 minutes and closer than 2-3 meters to be at risk. Our results show that isolation and tracing can help control re-emerging outbreaks when some conditions are met: (i) a reduction of the reproductive number through masks and physical distance; (ii) a low-delay isolation of infected individuals; (iii) a high compliance. Finally, we observe the inefficacy of a less privacy-preserving tracing involving second order contacts. Our results may inform digital contact tracing efforts currently being implemented across several countries worldwide.

First Spectroscopy of the Near Drip-line Nucleus

One of the most exotic light neutron-rich nuclei currently accessible for experimental study is ^{40}Mg, which lies at the intersection of the nucleon magic number N=28 and the neutron drip line. Low-lying excited states of ^{40}Mg have been studied for the first time following a one-proton removal reaction from ^{41}Al, performed at the Radioactive Isotope Beam Factory of RIKEN Nishina Center with the DALI2 γ-ray array and the ZeroDegree spectrometer. Two γ-ray transitions were observed, suggesting an excitation spectrum that shows unexpected properties as compared to both the systematics along the Z=12, N≥20 Mg isotopes and available state-of-the-art theoretical model predictions. A possible explanation for the observed structure involves weak-binding effects in the low-lying excitation spectrum.

Protein tyrosine phosphatase 1B negatively regulates S100A9-mediated lung damage during respiratory syncytial virus exacerbations.

Protein tyrosine phosphatase 1B (PTP1B) has anti-inflammatory potential but PTP1B responses are desensitized in the lung by prolonged cigarette smoke exposure. Here we investigate whether PTP1B expression affects lung disease severity during respiratory syncytial viral (RSV) exacerbations of chronic obstructive pulmonary disease (COPD). Ptp1b(-/-) mice infected with RSV exhibit exaggerated immune cell infiltration, damaged epithelial cell barriers, cytokine production, and increased apoptosis. Elevated expression of S100A9, a damage-associated molecular pattern molecule, was observed in the lungs of Ptp1b(-/-) mice during RSV infection. Utilizing a neutralizing anti-S100A9 IgG antibody, it was determined that extracellular S100A9 signaling significantly affects lung damage during RSV infection. Preexposure to cigarette smoke desensitized PTP1B activity that coincided with enhanced S100A9 secretion and inflammation in wild-type animals during RSV infection. S100A9 levels in human bronchoalveolar lavage fluid had an inverse relationship with lung function in healthy subjects, smokers, and COPD subjects. Fully differentiated human bronchial epithelial cells isolated from COPD donors cultured at the air liquid interface secreted more S100A9 than cells from healthy donors or smokers following RSV infection. Together, these findings show that reduced PTP1B responses contribute to disease symptoms in part by enhancing S100A9 expression during viral-associated COPD exacerbations.

Hydrogen peroxide stimulation of CFTR reveals an Epac-mediated, soluble AC-dependent cAMP amplification pathway common to GPCR signalling.

BACKGROUND AND PURPOSE: H2 O2 is widely understood to regulate intracellular signalling. In airway epithelia, H2 O2 stimulates anion secretion primarily by activating an autocrine PGE2 signalling pathway via EP4 and EP1 receptors to initiate cytic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion. This study investigated signalling downstream of the receptors activated by H2 O2 . EXPERIMENTAL APPROACH: Anion secretion by differentiated bronchial epithelial cells was measured in Ussing chambers during stimulation with H2 O2 , an EP4 receptor agonist or ß2 -adrenoceptor agonist in the presence and absence of inhibitors of ACs and downstream effectors. Intracellular calcium ([Ca(2+) ]I ) changes were followed by microscopy using fura-2-loaded cells and PKA activation followed by FRET microscopy. KEY RESULTS: Transmembrane adenylyl cyclase (tmAC) and soluble AC (sAC) were both necessary for H2 O2 and EP4 receptor-mediated CFTR activation in bronchial epithelia. H2 O2 and EP4 receptor agonist stimulated tmAC to increase exchange protein activated by cAMP (Epac) activity that drives PLC activation to raise [Ca(2+) ]i via Ca(2+) store release (and not entry). Increased [Ca(2+) ]i led to sAC activation and further increases in CFTR activity. Stimulation of sAC did not depend on changes in [HCO3 (-) ]. Ca(2+) -activated apical KCa 1.1 channels and cAMP-activated basolateral KV 7.1 channels contributed to H2 O2 -stimulated anion currents. A similar Epac-mediated pathway was seen following ß2 -adrenoceptor or forskolin stimulation. CONCLUSIONS AND IMPLICATIONS: H2 O2 initiated a complex signalling cascade that used direct stimulation of tmACs by Gαs followed by Epac-mediated Ca(2+) crosstalk to activate sAC. The Epac-mediated Ca(2+) signal constituted a positive feedback loop that amplified CFTR anion secretion following stimulation of tmAC by a variety of stimuli.

Steroid sensitivity of norepinephrine uptake by human bronchial arterial and rabbit aortic smooth muscle cells.

We have shown that an inhaled glucocorticosteroid (GS) causes alpha(1)-adrenergic antagonist-blockable, rapid, and transient bronchial vasoconstriction in healthy and asthmatic subjects. Steroids inhibit norepinephrine (NE) uptake by non-neuronal cells, thereby increasing NE concentration at alpha-adrenergic receptor sites. This could explain the GS-induced bronchial vasoconstriction. We therefore studied expression of the steroid-sensitive extraneuronal monoamine transporter (EMT) and steroid sensitivity of NE uptake in human bronchial artery and rabbit aorta (as a substitute for the limited supply of human bronchial artery). NE uptake was measured using a semiquantitative, sucrose-potassium phosphate-glyoxylic acid fluorescence method that we newly adapted for use in single cells. Both human bronchial arteries and rabbit aorta expressed messenger RNA for EMT, and steroids blocked NE uptake into freshly dissociated human bronchial arterial and rabbit aortic smooth-muscle cells (SMCs). In the latter, inhibition of NE uptake by steroids was not altered, either by a protein synthesis inhibitor (cycloheximide) or by a transcription inhibitor (actinomycin D), and corticosterone made membrane-impermeant by conjugation to bovine serum albumin inhibited NE uptake equipotently. These data show that NE uptake into bronchial arterial and rabbit aortic SMCs is sensitive to steroids, possibly mediated by EMT, and suggest a mechanism for GS-induced bronchial vasoconstriction.

Hyaluronan serves a novel role in airway mucosal host defense.

Enzymes secreted onto epithelial surfaces play a vital role in innate mucosal defense, but are believed to be steadily removed from the surface by mechanical actions. Thus, the amount and availability of enzymes on the surface are thought to be maintained by secretion. In contrast to this paradigm, we show here that enzymes are retained at the apical surface of the airway epithelium by binding to surface-associated hyaluronan, providing an apical enzyme pool 'ready for use' and protected from ciliary clearance. We have studied lactoperoxidase, which prevents bacterial colonization of the airway, and kallikrein, which mediates allergic bronchoconstriction that limits the inhalation of noxious substances. Binding to hyaluronan inhibits kallikrein, which is needed only in certain situations, whereas lactoperoxidase, useful at all times, does not change its activity. Hyaluronan itself interacts withthe receptor for hyaluronic acid-mediated motility (RHAMM or CD168) that is expressed at the apex of ciliated airway epithelial cells. Functionally, hyaluronan binding to RHAMM stimulates ciliary beating. Thus, hyaluronan plays a previously unrecognized pivotal role in mucosal host defense by stimulating ciliary clearance of foreign material while simultaneously retaining enzymes important for homeostasis at the apical surface so that they cannot be removed by ciliary action.

Agonist-stimulated calcium decreases in ovine ciliated airway epithelial cells: role of mitochondria.

1. In ovine ciliated tracheal epithelial cells, acetylcholine (ACh) activates signal transduction pathways that not only transiently increase cytoplasmic Ca2+ ([Ca2+]i) but also actively lower [Ca2+]i. The pathway for decreasing [Ca2+]i is clearly revealed after depletion of intracellular Ca2+ stores by thapsigargin (Tg), 2,5-di-(tert-butyl)-1,4-benzohydroquinone or NiCl2. Measurements with microinjected fura-2 excluded a [Ca2+] measurement artefact. 2. A four-compartment model to simulate calcium transients in non-excitable cells (consisting of a plasma membrane Ca2+ pump and channel; Ca2+ store with pump and channel; and cytosolic Ca2+ buffer) could not account for the observed [Ca2+]i decrease. We therefore explored, by simulation and experimentation, several different mechanisms that could account for it. 3. The ACh-stimulated [Ca2+]i decrease was not due to an inhibition of Ca2+ influx (Ca2+ channel blockers or absence of extracellular calcium had no effect), activation of a plasma membrane Ca2+-ATPase (two inhibitors, vanadate (30 mM) and lanthanum (10 mM), had no effect) or inhibition of the Na+-Ca2+ exchanger (replacing extracellular Na+ with N-methylglucamine had no effect). 4. The application of mitochondrial uncouplers (5 microM CCCP or 5 microM FCCP), eliminated the ACh-induced [Ca2+]i decrease. Addition of CCCP at the nadir of the decrease restored intracellular calcium levels of Tg-treated cells to baseline faster than controls not exposed to mitochondrial uncouplers. CCCP application to naïve cells did not block the ACh-induced transient increase in [Ca2+]i. 5. These data suggest that ACh-induced [Ca2+]i decreases in ciliated cells are caused by stimulated Ca2+ uptake into mitochondria.

Hyaluronic acid in cultured ovine tracheal cells and its effect on ciliary beat frequency in vitro.

Hyaluronic acid (hyaluronan, or HA) is secreted by submucosal glands, but its function in airway secretions other than influencing the rheology of mucus is not fully understood. HA is known to modulate cell behavior and to enhance sperm motility. Because sperm tails and cilia have the same microtubular structure, we studied the effect of HA on ciliary beat frequency (CBF) in vitro. CBF of cultured ovine airway epithelial cells was measured continuously by digital video microscopy. After removal of endogenous HA by hyaluronidase, cells were exposed to 50 to 100 microg/mL of HA at different times in culture. No change in CBF in response to HA was seen in cells cultured less than 7 days. After 7 days, however, 6 of 10 measured cells (from three different sheep) showed a transient CBF increase from a baseline of 6.4 +/- 0.3 Hz (mean +/- SE) to 7.4 +/- 0.4 Hz or 16% above baseline (p < 0.05). At these time points (but not before), cytochemical staining was positive for endogenous HA using a biotinylated HA-binding protein. These data suggest that HA can increase CBF of tracheal epithelial cells only late in culture when HA is able to bind to an unspecified cell surface structure. Because this binding has a physiological effect, we hypothesize that it is an HA-binding receptor, that is either transiently expressed late in culture or initially destroyed by the protease treatment for cell dispersion.

Lack of nitric oxide involvement in cholinergic modulation of ovine ciliary beat frequency.

Ciliary beat frequency (CBF) is regulated, at least in part, by the cytoplasmic calcium concentration ([Ca(2+)](i)). Because Ca(2+) can stimulate nitric oxide (NO) production by nitric oxide synthase (NOS) and NO has been implicated in the regulation of CBF in some species, we examined whether NOS is present in cultured ovine ciliated epithelial cells and whether NO plays a role in the Ca(2+)-mediated muscarinic stimulation of CBF. Dissociated ovine tracheal epithelial cells were grown in culture for 2 to 14 days. Frequency from a single cilium was measured by on-line Fourier transform methods using video microscopy. [Ca(2+)](i) was determined with fura-2 using fluorescence ratio imaging from the same single cells. Ciliated cells contained NOS in culture as indicated by NADPH-diaphorase staining. Acetylcholine (ACh) increased CBF and [Ca(2+)](i) transiently as previously shown. Measurements with 2',7'-dichlorofluorescin diacetate indicated that reactive oxygen/nitrogen species were produced in these cells on ACh exposure. NOS inhibitors N(G)-nitro-L-arginine methyl ester (< or =10 mM), N(G)-nitro-L-arginine (< or =10 mM), and 7-nitro indazole (1 microM) were unable to block the CBF or [Ca(2+)](i) response to ACh. Furthermore, the NO donors sodium nitroprusside and S-nitroso-N-acetylpenicillamine (< or =1 mM) did not change CBF or [Ca(2+)](i). Above these concentrations, they both lead to a reversible decrease in CBF. The membrane-permeable cyclic guanosine monophosphate analogue 8-bromo-cyclic guanosine monophosphate had no effect on CBF, whereas 8-bromo-cyclic adenosine monophosphate stimulated CBF. Taken together, these results suggest that NO does not play a role in mediating the ACh-induced increase in CBF through [Ca(2+)](i). The role and targets for NO in ovine ciliated cells remains to be determined.

The lactoperoxidase system functions in bacterial clearance of airways.

Airway mucus is a complex mixture of secretory products that provides a multifaceted defense against pulmonary infection. Mucus contains antimicrobial peptides (e.g., defensins) and enzymes (e.g., lysozyme) although the contribution of these to airway sterility has not been tested in vivo. We have previously shown that an enzymatically active, heme-containing peroxidase comprises 1% of the soluble protein in sheep airway secretions, and it has been hypothesized that this airway peroxidase may function as a biocidal system. In this study, we show that sheep airway peroxidase is identical to milk lactoperoxidase (LPO) and that sheep airway secretions contain thiocyanate (SCN(-)) at concentrations necessary and sufficient for a functional peroxidase system that can protect against infection. We also show that airway LPO, like milk LPO, produces the biocidal compound hypothiocyanite (OSCN(-)) in vitro. Finally, we show that in vivo inhibition of airway LPO in sheep leads to a significant decrease in bacterial clearance from the airways. The data suggest that the LPO system is a major contributor to airway defenses. This discovery may have significant implications for chronic airway colonization seen in respiratory diseases such as cystic fibrosis.

Mode of Ca2+ action on ciliary beat frequency in single ovine airway epithelial cells.

1. We analysed the kinetics of coupling between cytoplasmic calcium ([Ca2+]i) and ciliary beat frequency (CBF) using simultaneous single cilium recording and single cell [Ca2+]i measurements from cultured ovine tracheal epithelial cells. 2. CBF and [Ca2+]i (indicated by fura-2) were measured at rest and in response to activation of the G-protein coupled M3 muscarinic receptor by 10 microM acetylcholine (ACh). 3. Fourier transform analysis of 3 s data segments of light intensity from phase-contrast microscopy showed no significant delay between changes in [Ca2+]i and CBF during a 2 min exposure to ACh and subsequent washout. 4. CBF time resolution was improved by computing instantaneous beat frequency. This revealed that CBF lagged the rapid increase in [Ca2+]i in response to ACh with a delay of less than 1 beat cycle (143 ms at 7 Hz). When CBF was estimated by an improved Fourier method, this delay was observed to be 70 +/- 30 ms (mean +/- s.e.m.; n = 20 cilia). During the slower return to baseline, a lag of 8 +/- 3.2 s was observed, indicative of hysteresis. 5. While calmodulin inhibitors (calmidazolium and W-7; each n = 5) decreased baseline CBF by an average of 1.1 +/- 0.1 Hz, they did not alter the kinetic relationship between [Ca2+]i and CBF. Similarly, phosphatase inhibitors (okadaic acid and cyclosporin A; each n = 5), changed neither baseline CBF nor the kinetic coupling between [Ca2+]i and CBF. 6. These data suggest that the timing of Ca2+ action on CBF in ovine airway epithelial cells, is unlikely to be determined by phosphorylation reactions involving calmodulin or kinase/phosphatase reactions. 7. A simple model for Ca2+ stimulation of CBF is presented. Fits of the model to the data suggest four or more Ca2+ ions bind cooperatively to speed up CBF.

Isolation and characterization of a peroxidase from the airway.

Sheep airway mucus can potently scavenge hydrogen peroxide, an important mediator of airway inflammation. Here, the scavenging activity was identified as a peroxidase produced by goblet cells of the airway epithelium and secreted into the airway lumen. Ovine airway peroxidase activity was purified approximately 100-fold from airway lavage fluid in two steps, using cation exchange and lectin affinity chromatography, yielding an apparently homogeneous 82-kD glycoprotein. Ovine airway peroxidase represented about 1% of the total protein in airway mucus and thus was an abundant enzyme in airway secretions. The absorbance spectrum of the purified peroxidase showed a major peak at 412 nm indicative of a hemoprotein. The ratio of A412/A280 of the purified enzyme was 0.86. The absorption spectrum of ovine airway peroxidase, its ability to oxidize halides, its sensitivity to inhibitors and its apparent molecular mass on sodium dodecyl sulfate gels showed that airway peroxidase was similar to lactoperoxidase but distinguished from myeloperoxidase, eosinophil peroxidase as well as from glutathione peroxidases. Based on these observations, ovine airway peroxidase is a newly isolated and abundant enzyme of airway mucus which may function to control reactive oxygen species in the airway and to prevent infection by catalyzing the formation of biocidal compounds.

Muscarinic signaling in ciliated tracheal epithelial cells: dual effects on Ca2+ and ciliary beating.

To examine cholinergic signal transduction pathways that modulate ciliary beat frequency (CBF), cultured ovine tracheal epithelial cells were imaged using a combination of phase-contrast (CBF) and fluorescence (Ca2+) microscopy techniques. In single cells, acetylcholine (ACh) transiently increased CBF and intracellular Ca2+ concentration ([Ca2+]i), mainly by Ca2+ release from internal stores, with a small delayed contribution from Ca2+ influx. Nicotinic agonists did not alter CBF or [Ca2+]i, whereas atropine blocked the ACh-stimulated transients, consistent with the involvement of muscarinic receptors. 4-Diphenylacetoxy-N-methylpiperidine methiodide was approximately 100 times more potent than pirenzepine in inhibiting the ACh-induced [Ca2+]i peaks, suggesting that the receptor is a pharmacologically defined (M3) subtype. Interestingly, after depletion of intracellular Ca2+ stores by thapsigargin, ACh caused a rapid transient decrease in both CBF and [Ca2+]i, again with an antagonist profile of M3 receptors. We conclude that activation of M3 muscarinic receptors initiates specific signaling pathways that act simultaneously to increase and decrease [Ca2+]i and CBF.

Endothelial and epithelial sources of endothelin-1 in sheep bronchi.

Airway smooth muscle tone and growth is regulated by endothelin-1 (ET-1), but the sources of ET-1 in the airway wall have not been clearly defined. We therefore wished to estimate the relative contributions of the epithelium and endothelium to ET-1 production in the bronchi (mean ID 1.7-4.9 mm) of mature normal sheep. Morphometric assessment of bronchial cross-sections revealed a number of epithelial cells three times greater than endothelial cells by direct cell count. In contrast, the overall cell surface density was five to six times greater, and the airway smooth muscle-centered cell surface density was three to four times greater for endothelial cells than for epithelial cells. The expression of preproendothelin-1 mRNA was detected in cultured aortic endothelial cells (as a substitute for bronchial endothelial cells) but not in cultured bronchial epithelial cells, and the former secreted seven times more immunoreactive ET-1 than the latter. These findings show that topographically the endothelium is better positioned for the regulation of ovine bronchial smooth muscle than the epithelium. Furthermore, the findings suggest greater constitutive ET-1 secretion by cultured endothelium than by epithelium.

Hydrogen peroxide-scavenging properties of sheep airway mucus.

Reactive oxygen species released from luminal phagocytes in the airway can potentially injure the airway epithelium. Naturally occurring oxygen radical scavengers must therefore exist to protect the epithelium. This study was designed to determine whether the high-molecular-weight fraction of normal sheep tracheal mucus has hydrogen peroxide (H2O2)-scavenging activity. Lyophilized mucus from 10 sheep was reconstituted in phosphate-buffered saline (PBS) or Krebs-Henseleit buffer. H2O2 was added to these mucus samples to a final concentration of 15 microM, and the level of H2O2 remaining was measured over a 10 min period. From a zero-time level of 17 +/- 1.8 microM (mean +/- SD), the H2O2 concentration fell within 10 min to 8 +/- 1.7 microM in 0.05%; to 3.9 +/- 2.2 microM in 0.1%; to 2.6 +/- 2.4 microM in 0.2%; and to 1.2 +/- 1.5 microM in 0.4% mucus reconstituted in PBS. The results obtained in Krebs-Henseleit buffer were similar. The disappearance of H2O2 was not due to the transformation into hydroxyl radicals. Heat and acid denaturation and cleavage of carbohydrate-free peptides from glycoproteins by pronase E treatment abolished the scavenging potential. Fractionation of 0.4% mucus samples according to molecular weight by gel filtration revealed that only one fraction with proteins of M(r) > 110 kD contained the active scavenger. Polyacrylamide gel electrophoresis and lectin blotting with Ulex europaeus I (UEAI) showed that both the whole mucus and the actively scavenging gel filtration fraction contained a glycoprotein that comigrated with a 205 kD molecular weight marker.(ABSTRACT TRUNCATED AT 250 WORDS)

Coupling of [Ca2+]i and ciliary beating in cultured tracheal epithelial cells.

The molecular mechanisms responsible for the regulation of ciliary beating frequency (CBF) are only partially characterized. To determine whether elevation of intracellular Ca2+ ([Ca2+]i) can cause an increase in CBF, we measured CBF and Ca2+ in single cells. Ovine tracheal epithelial cells, obtained by dissociation with protease, were grown in primary culture for 1 to 28 days in a mucus-free system. CBF of a single cilium was measured by digital video phase-contrast microscopy and on-line Fourier-transform analysis. Changes in [Ca2+]i from single cells were determined with fura-2, using ratio imaging video microscopy. Activation of a muscarinic pathway with 10 microM ACh (acetylcholine) increased [Ca2+]i from 53 +/- 9 nM (mean +/- s.e.m.) to 146 +/- 12 nM or to 264 +/- 51% above initial baseline. In the same cells, ACh increased CBF from a baseline of 7 +/- 0.5 Hz to 9 +/- 0.2 Hz or to 31 +/- 2.8% above baseline (n = 14). The elevations of both [Ca2+]i and CBF were transient and relaxed back to an elevated plateau (10/14 cells) as long as ACh was present. To elevate [Ca2+]i by mechanisms independent of a G-protein-coupled receptor, we measured [Ca2+]i and CBF of the same cells in extracellular solutions with either 0 Ca2+ (+ 1 mM EGTA) or 10 mM Ca2+. Both signals rose and fell with similar kinetics in response to changing [Ca2+]0, suggesting that changes in [Ca2+]i alone can modulate CBF. In a second independent manipulation, cells were treated with 1 microM thapsigargin, an irreversible inhibitor of the endoplasmic reticulum Ca(2+)-ATPase. Upon thapsigargin addition, 37 of 42 cells showed a transient [Ca2+]i increase and, as measured in different experiments, 8 of 9 cells showed a transient increase in CBF. Interestingly, application of ACh after cells were treated with thapsigargin produced decreases in both [Ca2+]i and CBF in 8/8 cells. Lastly, after 1-3 days in culture, addition of 10 microM ACh often produced [Ca2+]i oscillations rather than transients in [Ca2+]i. Measurements of CBF in these cells showed frequency modulation of CBF with the same peak-to-peak time interval as the Ca2+ oscillation. These results show that: (1) CBF can be measured from a single cilium and monitored on-line to track changes; (2) CBF and [Ca2+]i can be measured in the same single cell; (3) transient changes in [Ca2+]i (induced by 4 different manipulations) are associated with kinetically similar changes in CBF; and (4) [Ca2+]i oscillations are coupled to frequency modulation of ciliary beating.(ABSTRACT TRUNCATED AT 400 WORDS)