SPX-101 is stable in and retains function after exposure to cystic fibrosis sputum

Background: In healthy lungs, epithelial sodium channel (ENaC) is regulated by short, palate, lung, and nasal clone 1 (SPLUNC1). In cystic fibrosis (CF), ENaC is hyperactivated in part due to a loss of SPLUNC1 function. We have developed SPX-101 to replace the lost function of SPLUNC1 in the CF lung. Methods: Expression of SPLUNC1 was determined in sputum from healthy and CF donors. Stability of SPLUNC1, S18 (the ENaC regulatory domain of SPLUNC1), and SPX-101 was determined in sputum from CF donors and towards neutrophil elastase. Activity of SPX-101 after exposure to CF sputum was determined in airway epithelial cells from CF donors and in the βENaC transgenic mouse model. Results: SPLUNC1 protein expression is significantly reduced in CF as compared to healthy sputum. SPLUNC1 is rapidly degraded in CF sputum as well as by a number of individual proteases known to be found in the sputum. SPX-101, but not S18, is stable in CF sputum. Finally, SPX-101 retains its ability to internalize ENaC, regulate airway surface liquid height, and increase survival of βENaC mice after exposure to CF sputum. Conclusions: Our results demonstrate that SPX-101, but not SPLUNC1 or S18, is stable in CF sputum. These results support the therapeutic development of SPX-101 for the treatment of cystic fibrosis.Fil: Sesma, Juliana. Spyryx Biosciences; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Wu, Bryant. Spyryx Biosciences; Estados UnidosFil: Stuhlmiller, Timothy J.. Spyryx Biosciences; Estados UnidosFil: Scott, David W.. Spyryx Biosciences; Estados Unido

Tobacco exposure inhibits SPLUNC1-dependent antimicrobial activity

Background: Tobacco smoke exposure impairs the lung´s innate immune response, leading to an increased risk of chronic infections. SPLUNC1 is a secreted, multifunctional innate defense protein that has antimicrobial activity against Gram negative organisms. We hypothesize that tobacco smoke-induced SPLUNC1 dysfunction contributes to the observed defect in innate immunity in tobacco smokers and that this dysfunction can be used as a potential biomarker of harm. Methods: We collected sputum from never-smokers and otherwise healthy smokers. We performed Western blotting to determine SPLUNC1 levels and determined antimicrobial activity against nontypeable Haemophilus influenzae. An in vitro exposure model was utilized to measure the effect of tobacco exposure on human bronchial epithelial culture (HBEC) antimicrobial activity against H. influenzae. The direct effects of cigarette and little cigar smoke exposure on SPLUNC1 function was determined using 24 h growth measurements and LPS binding assays. Results: H. influenzae growth in cigarette smoker´s sputum was significantly greater compared to never-smokers sputum over 24 h. HBEC supernatants and lysates contained significantly higher numbers of H. influenzae following chronic cigarette and little cigar smoke exposure compared to air-exposed controls. Furthermore, SPLUNC1´s antimicrobial activity and LPS-binding capability against both H. influenzae and P. aeruginosa was attenuated following cigarette and little cigar exposure. Conclusions: These data suggest that cigarette and little cigar exposure impairs SPLUNC1´s antimicrobial ability and that this inhibition may serve as a novel biomarker of harm that can be used to assess the toxicity of commercial tobacco products.Fil: Moore, Patrick J.. University of North Carolina; Estados UnidosFil: Sesma, Juliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; ArgentinaFil: Alexis, Neil E.. University of North Carolina; Estados UnidosFil: Tarran, Robert. University of North Carolina; Estados Unido

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR

IntroductionThe declaration of the end of the Public Health Emergency for COVID-19 on May 11th, 2023, has shifted the global focus led by WHO and CDC towards monitoring the evolution of SARS-CoV-2. Augmenting these international endeavors with local initiatives becomes crucial to not only track the emergence of new variants but also to understand their spread. We present a cost-effective digital PCR-based pooled sample testing methodology tailored for early variant surveillance.MethodsUsing 1200 retrospective SARS-CoV-2 positive samples, either negative or positive for Delta or Omicron, we assessed the sensitivity and specificity of our detection strategy employing commercial TaqMan variant probes in a 1:9 ratio of variant-positive to variant-negative samples.ResultsThe study achieved 100% sensitivity and 99% specificity in 10-sample pools, with an Area Under the Curve (AUC) exceeding 0.998 in ROC curves, using distinct commercial TaqMan variant probes.DiscussionThe employment of two separate TaqMan probes for both Delta and Omicron establishes dual validation routes, emphasizing the method’s robustness. Although we used known samples to model realistic emergence scenarios of the Delta and Omicron variants, our main objective is to demonstrate the versatility of this strategy to identify future variant appearances. The utilization of two divergent variants and distinct probes for each confirms the method’s independence from specific variants and probes. This flexibility ensures it can be tailored to recognize any subsequent variant emergence, given the availability of its sequence and a specific probe. Consequently, our approach stands as a robust tool for tracking and managing any new variant outbreak, reinforcing our global readiness against possible future SARS-CoV-2 waves

Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR

Detection of SARS-CoV-2 has created an enormous workload for laboratories worldwide resulting in a restriction at the time of massive testing. Pool testing is a strategy that reduces time and costs. However, beyond the detection of infectious diseases in blood banks, this approach is rarely implemented in routine laboratories. Therefore, what was learned from the SARS-CoV-2 pool testing should represent an opportunity to increase diagnostic capabilities. The present work, carried out in the context of a diagnostic laboratory of a public hospital during the COVID-19 pandemic, represents a contribution to this end. The main limitation of pool testing is the risk of false negatives that could have been identified by individual tests. These limitations are the dilution of samples with a low virus load during pooling and that the integrity of the sample may be affected by the quality of the sample collection. Fortunately, both limitations coincide with the main strengths of droplet digital PCR (ddPCR). ddPCR is a third-generation PCR that splits the amplification into thousands of droplets that work in parallel, increasing sensitivity and resistance to inhibitors. Therefore, ddPCR is particularly useful for pool testing. Here we show how to factor between test sensitivity and savings in test time and resources. We have identified and optimized critical parameters for pool testing. The present study, which analyzed 1000 nasopharyngeal samples, showed that the pool testing could detect even a single positive sample with a CT value of up to 30 in pools of 34 samples. This test was performed using three different standard extraction methods, the simplest being heating only, which resulted in substantial savings of extraction reagents in addition to PCR reagents. Moreover, we show that pooling can be extended to use saliva, which is less invasive and allows self-collection, reducing the risk for health personnel.Heckel S, Pacini A, Paredes F, Petreli M.V, Perez M, Adriani N, et al. (2022) Practical considerations to establish a validated platform for pooled detection of SARS-CoV-2 by droplet digital PCR. PLoS ONE 17(11): e0271860. https://doi.org/ 10.1371/journal.pone.0271860Fil: Heckel, Sofía. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Pacini, Antonella. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Paredes, Franco. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Petreli, María Victoria. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Perez, Marilina. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Adriani, Natalia. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Ibarra, Guadalupe. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Sesma, Juliana. Hospital Provincial de Rosario. Molecular Biology Department; Argentina.Fil: Heckel, Sofía. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET); Argentina.Fil: Pacini, Antonella. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET); Argentina.Fil:. Sesma, Juliana. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER-CONICET); Argentina.Fil: Heckel, Sofía. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Paredes, Franco. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Petreli, María Victoria. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Ibarra, Guadalupe. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Menzella, Hugo G. Instituto de Procesos Biotecnológicos y Químicos Rosario (IPROByQ); Argentina.Fil: Menzella, Hugo G. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.Fil: Colaneri, Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET); Argentina.Fil: Sesma, Juliana. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina

UDP-glucose promotes neutrophil recruitment in the lung

In addition to their role in glycosylation reactions, UDP-sugars are released from cells and activate widely distributed cell surface P2Y 14 receptors (P2Y 14 R). However, the physiological/pathophysiological consequences of UDP-sugar release are incompletely defined. Here, we report that UDP-glucose levels are abnormally elevated in lung secretions from patients with cystic fibrosis (CF) as well as in a mouse model of CF-like disease, the βENaC transgenic (Tg) mouse. Instillation of UDP-glucose into wild-type mouse tracheas resulted in enhanced neutrophil lung recruitment, and this effect was nearly abolished when UDP-glucose was co-instilled with the P2Y 14 R antagonist PPTN [4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl-2-naphthoic acid]. Importantly, administration of PPTN to βENaC-Tg mice reduced neutrophil lung inflammation. These results suggest that UDP-glucose released into the airways acts as a local mediator of neutrophil inflammation

Similarities between UDP-Glucose and Adenine Nucleotide Release in Yeast: Involvement of the Secretory Pathway

Extracellular UDP-glucose is a natural purinergic receptor agonist, but its mechanisms of cellular release remain unclear. We studied these mechanisms in Saccharomyces cerevisiae, a simple model organism that releases ATP, another purinergic agonist. Similar to ATP, UDP-glucose was released by S. cerevisiae at a rate that was linear over time. However, unlike ATP release, UDP-glucose release was not dependent on glucose stimulation. This discrepancy was resolved by demonstrating the apparent glucose stimulation of ATP release reflected glucose-dependent changes in the intracellular pattern of adenine nucleotides, with AMP release dominating in the absence of glucose. Indeed, total adenine nucleotide release, like UDP-glucose release, did not vary with glucose concentration over the short term. The genetic basis of UDP-glucose release was explored through analysis of deletion mutants, aided by development of a novel bioassay for UDP-glucose based on signaling through heterologously expressed human P2Y14 receptors. Using this assay, an elevated rate of UDP-glucose release was demonstrated in mutants lacking the putative Golgi nucleotide sugar transporter YMD8. An increased rate of UDP-glucose release in ymd8Δ was reduced by deletion of the YEA4 UDP-N-acetylglucosamine or the HUT1 UDP-galactose transporters, and overexpression of YEA4 or HUT1 increased the rate of UDP-glucose release. These findings suggest an exocytotic release mechanism similar to that of ATP, a conclusion supported by decreased rates of ATP, AMP, and UDP-glucose release in response to the secretory inhibitor Brefeldin A. These studies demonstrate the involvement of the secretory pathway in nucleotide and nucleotide sugar efflux in yeast and offer a powerful model system for further investigation

Inflammation Promotes Airway Epithelial ATP Release via Calcium-Dependent Vesicular Pathways

ATP in airway surface liquid (ASL) controls mucociliary clearance functions via the activation of airway epithelial purinergic receptors. However, abnormally elevated ATP levels have been reported in inflamed airways, suggesting that excessive ATP in ASL contributes to airway inflammation. Despite these observations, little is known about the mechanisms of ATP accumulation in the ASL covering inflamed airways. In this study, links between cystic fibrosis (CF)–associated airway inflammation and airway epithelial ATP release were investigated. Primary human bronchial epithelial (HBE) cells isolated from CF lungs exhibited enhanced IL-8 secretion after 6 to 11 days, but not 28 to 35 days, in culture, compared with normal HBE cells. Hypotonic cell swelling–promoted ATP release was increased in 6- to 11-day-old CF HBE cells compared with non-CF HBE cells, but returned to normal values after 28 to 35 days in culture. The exposure of non-CF HBE cells to airway secretions isolated from CF lungs, namely, sterile supernatants of mucopurulent material (SMM), also caused enhanced IL-8 secretion and increased ATP release. The SMM-induced increase in ATP release was sensitive to Ca2+ chelation and vesicle trafficking/exocytosis inhibitors, but not to pannexin inhibition. Transcript levels of the vesicular nucleotide transporter, but not pannexin 1, were up-regulated after SMM exposure. SMM-treated cultures displayed increased basal mucin secretion, but mucin secretion was not enhanced in response to hypotonic challenge after the exposure of cells to either vehicle or SMM. We propose that CF airway inflammation up-regulates the capacity of airway epithelia to release ATP via Ca2+-dependent vesicular mechanisms not associated with mucin granule secretion

Rho Signaling Regulates Pannexin 1-mediated ATP Release from Airway Epithelia

ATP released from airway epithelial cells promotes purinergic receptor-regulated mucociliary clearance activities necessary for innate lung defense. Cell swelling-induced membrane stretch/strain is a common stimulus that promotes airway epithelial ATP release, but the mechanisms transducing cell swelling into ATP release are incompletely understood. Using knockdown and knockout approaches, we tested the hypothesis that pannexin 1 mediates ATP release from hypotonically swollen airway epithelia and investigated mechanisms regulating this activity. Well differentiated primary cultures of human bronchial epithelial cells subjected to hypotonic challenge exhibited enhanced ATP release, which was paralleled by the uptake of the pannexin probe propidium iodide. Both responses were reduced by pannexin 1 inhibitors and by knocking down pannexin 1. Importantly, hypotonicity-evoked ATP release from freshly excised tracheas and dye uptake in primary tracheal epithelial cells were impaired in pannexin 1 knockout mice. Hypotonicity-promoted ATP release and dye uptake in primary well differentiated human bronchial epithelial cells was accompanied by RhoA activation and myosin light chain phosphorylation and was reduced by the RhoA dominant negative mutant RhoA(T19N) and Rho and myosin light chain kinase inhibitors. ATP release and Rho activation were reduced by highly selective inhibitors of transient receptor potential vanilloid 4 (TRPV4). Lastly, knocking down TRPV4 impaired hypotonicity-evoked airway epithelial ATP release. Our data suggest that TRPV4 and Rho transduce cell membrane stretch/strain into pannexin 1-mediated ATP release in airway epithelia

Clonal chromosomal mosaicism and loss of chromosome Y in elderly men increase vulnerability for SARS-CoV-2

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, COVID-19) had an estimated overall case fatality ratio of 1.38% (pre-vaccination), being 53% higher in males and increasing exponentially with age. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, we found 133 cases (1.42%) with detectable clonal mosaicism for chromosome alterations (mCA) and 226 males (5.08%) with acquired loss of chromosome Y (LOY). Individuals with clonal mosaic events (mCA and/or LOY) showed a 54% increase in the risk of COVID-19 lethality. LOY is associated with transcriptomic biomarkers of immune dysfunction, pro-coagulation activity and cardiovascular risk. Interferon-induced genes involved in the initial immune response to SARS-CoV-2 are also down-regulated in LOY. Thus, mCA and LOY underlie at least part of the sex-biased severity and mortality of COVID-19 in aging patients. Given its potential therapeutic and prognostic relevance, evaluation of clonal mosaicism should be implemented as biomarker of COVID-19 severity in elderly people. Among 9578 individuals diagnosed with COVID-19 in the SCOURGE study, individuals with clonal mosaic events (clonal mosaicism for chromosome alterations and/or loss of chromosome Y) showed an increased risk of COVID-19 lethality

Anales del III Congreso Internacional de Vivienda y Ciudad "Debate en torno a la nueva agenda urbana"

Acta de congresoEl III Congreso Internacional de Vivienda y Ciudad “Debates en torno a la NUEVa Agenda Urbana”, ha sido una apuesta de alto compromiso por acercar los debates centrales y urgentes que tensionan el pleno ejercicio del derecho a la ciudad. Para ello las instituciones organizadoras (INVIHAB –Instituto de Investigación de Vivienda y Hábitat y MGyDH-Maestría en Gestión y Desarrollo Habitacional-1), hemos convidado un espacio que se concretó con potencia en un debate transdisciplinario. Convocó a intelectuales de prestigio internacional, investigadores, académicos y gestores estatales, y en una metodología de innovación articuló las voces académicas con las de las organizaciones sociales y/o barriales en el Foro de las Organizaciones Sociales que tuvo su espacio propio para dar voz a quienes están trabajando en los desafíos para garantizar los derechos a la vivienda y los bienes urbanos en nuestras ciudades del Siglo XXI