Achieving Health Security and Threat Reduction through Sharing Sequence Data.

With the rapid development and broad applications of next-generation sequencing platforms and bioinformatic analytical tools, genomics has become a popular area for biosurveillance and international scientific collaboration. Governments from countries including the United States (US), Canada, Germany, and the United Kingdom have leveraged these advancements to support international cooperative programs that aim to reduce biological threats and build scientific capacity worldwide. A recent conference panel addressed the impacts of the enhancement of genomic sequencing capabilities through three major US bioengagement programs on international scientific engagement and biosecurity risk reduction. The panel contrasted the risks and benefits of supporting the enhancement of genomic sequencing capabilities through international scientific engagement to achieve biological threat reduction and global health security. The lower costs and new bioinformatic tools available have led to the greater application of sequencing to biosurveillance. Strengthening sequencing capabilities globally for the diagnosis and detection of infectious diseases through mutual collaborations has a high return on investment for increasing global health security. International collaborations based on genomics and shared sequence data can build and leverage scientific networks and improve the timeliness and accuracy of disease surveillance reporting needed to identify and mitigate infectious disease outbreaks and comply with international norms. Further efforts to promote scientific transparency within international collaboration will improve trust, reduce threats, and promote global health security.

Purinergic signaling underlies CFTR control of human airway epithelial cell volume.

BACKGROUND: Loss of cystic fibrosis transmembrane conductance regulator (CFTR) function in cystic fibrosis (CF) causes dysregulation of multiple ion channels, water channels, and acid-base transporters in epithelia. As such, we hypothesized that dysregulation of many critical ion channels and transporters may cause defects in human airway epithelial cell volume regulation. METHODS: Cell volume, regulatory volume decrease, and its regulation was assessed in real-time via Coulter Counter Multisizer III-driven electronic cell sizing in non-CF, CF, and CFTR-complemented CF human airway epithelial cells. SPQ halide fluorescence assay of hypotonicity-induced chloride efflux provided indirect validation of the cell volume assays. RESULTS: CFTR, via autocrine ATP signaling, governs human airway epithelial cell volume regulation. Non-CF cells and wild-type (WT)-CFTR-transfected CF cells had normal regulatory volume decrease (RVD) responses that were attenuated by blockade of autocrine and paracrine purinergic signaling. In contrast, parental IB3-1 CF cells or IB3-1 cells expressing CFTR mutants (DeltaF508, G551D, and S1455X) failed to RVD. CF cell RVD was rescued by agonists to P2Y G protein-coupled receptors and, more robustly, by agonists to P2X purinergic receptor channels. CONCLUSIONS: Loss of CFTR and CFTR-driven autocrine ATP signaling may underlie defective cell volume regulation and dysregulated ion, water, and acid-base transport in CF airway epithelia.

Extracellular zinc and ATP restore chloride secretion across cystic fibrosis airway epithelia by triggering calcium entry.

Cystic fibrosis (CF) is caused by defective cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator Cl(-) channels. Thus, CF epithelia fail to transport Cl(-) and water. A postulated therapeutic avenue in CF is activation of alternative Ca(2+)-dependent Cl(-) channels. We hypothesized that stimulation of Ca(2+) entry from the extracellular space could trigger a sustained Ca(2+) signal to activate Ca(2+)-dependent Cl(-) channels. Cytosolic [Ca(2+)](i) was measured in non-polarized human CF (IB3-1) and non-CF (16HBE14o(-)) airway epithelial cells. Primary human CF and non-CF airway epithelial monolayers as well as Calu-3 monolayers were used to assess anion secretion. In vivo nasal potential difference measurements were performed in non-CF and two different CF mouse (DeltaF508 homozygous and bitransgenic gut-corrected but lung-null) models. Zinc and ATP induced a sustained, reversible, and reproducible increase in cytosolic Ca(2+) in CF and non-CF cells with chemistry and pharmacology most consistent with activation of P2X purinergic receptor channels. P2X purinergic receptor channel-mediated Ca(2+) entry stimulated sustained Cl(-) and HCO(3)(-) secretion in CF and non-CF epithelial monolayers. In non-CF mice, zinc and ATP induced a significant Cl(-) secretory response similar to the effects of agonists that increase intracellular cAMP levels. More importantly, in both CF mouse models, Cl(-) permeability of nasal epithelia was restored in a sustained manner by zinc and ATP. These effects were reversible and reacquirable upon removal and readdition of agonists. Our data suggest that activation of P2X calcium entry channels may have profound therapeutic benefit for CF that is independent of cystic fibrosis transmembrane conductance regulator genotype.

Activation of chloride secretion in cystic fibrosis cells and tissues by the substituted imidazole SRI 2931.

Recent interest in nucleotides and related agents as part of clinical trials in cystic fibrosis (CF) therapy have elicited efforts to identify novel compounds capable of activating transepithelial chloride (Cl(-)) transport in CF cells and tissues. From a library of nucleosides, bases, and other substituted heterocycles, 341 compounds were screened for their ability to activate anion transport in CF cells grown on permeable supports. One compound, SRI 2931, was found to confer prolonged and potent activity when administered to the apical surfaces of CF pancreatic epithelial cells, primary CF nasal epithelial cells, non-CF human colonic epithelial cells, and intact tissue taken from mouse models for CF. Concentrations of SRI 2931 (20 microM), which activated Cl(-) transport, had minimal effect on cell proliferation. SRI 2931 was not calcium (Ca(2+)) or cAMP dependent, suggesting important differences from conventional chloride secretagogues. The compound selectively released ATP from the apical, but not basolateral, surfaces of CF cells grown on permeable supports. The magnitude, longevity, and mechanism of action of the response provide a tool for dissecting pathways of epithelial ATP extracellular signaling and Cl(-) permeability.

Plasma membrane CFTR regulates RANTES expression via its C-terminal PDZ-interacting motif.

Despite the identification of 1,000 mutations in the cystic fibrosis gene product CFTR, there remains discordance between CFTR genotype and lung disease phenotype. The study of CFTR, therefore, has expanded beyond its chloride channel activity into other possible functions, such as its role as a regulator of gene expression. Findings indicate that CFTR plays a role in the expression of RANTES in airway epithelia. RANTES is a chemokine that has been implicated in the regulation of mucosal immunity and the pathogenesis of airway inflammatory diseases. Results demonstrate that CFTR triggers RANTES expression via a mechanism that is independent of CFTR's chloride channel activity. Neither pharmacological inhibition of CFTR nor activation of alternative chloride channels, including hClC-2, modulated RANTES expression. Through the use of CFTR disease-associated and truncation mutants, experiments suggest that CFTR-mediated transcription factor activation and RANTES expression require (i) insertion of CFTR into the plasma membrane and (ii) an intact CFTR C-terminal PDZ-interacting domain. Expression of constructs encoding wild-type or dominant-negative forms of the PDZ-binding protein EBP50 suggests that EBP50 may be involved in CFTR-dependent RANTES expression. Together, these data suggest that CFTR modulates gene expression in airway epithelial cells while located in a macromolecular signaling complex at the plasma membrane.

Autocrine extracellular purinergic signaling in epithelial cells derived from polycystic kidneys.

ATP and its metabolites are potent autocrine agonists that act extracellularly within tissues to affect epithelial function. In polycystic kidneys, renal tubules become dilated and/or encapsulated as cysts, creating abnormal microenvironments for autocrine signaling. Previously, our laboratory has shown that high-nanomolar to micromolar quantities of ATP are released from cell monolayers in vitro and detectable in cyst fluids from microdissected human autosomal dominant polycystic kidney (ADPKD) cysts. Here, we show enhanced ATP release from autosomal recessive polycystic kidney (ARPKD) and ADPKD epithelial cell models. RT-PCR and immunoblotting for P2Y G protein-coupled receptors and P2X purinergic receptor channels show expression of mRNA and/or protein for multiple subtypes from both families. Assays of cytosolic Ca(2+) concentration and secretory Cl(-) transport show P2Y and P2X purinergic receptor-mediated stimulation of Cl(-) secretion via cytosolic Ca(2+)-dependent signaling. Therefore, we hypothesize that autocrine purinergic signaling may augment detrimentally cyst volume expansion in ADPKD or tubule dilation in ARPKD, accelerating disease progression.