Early Elemental and Ionic Changes in Cultured Cells After Stimulation with Epidermal Growth Factor

Stimulation of A43 l cells (a human vulval epidermal cell line) with 50 ng/ml of epidermal growth factor (EGF) in the presence of 1. 7 mM extracellular calcium produced a sharp and sustained rise in intracellular ionic Ca2+, increased elemental Na, decreased K and a rise in Ca. In the absence of extracellular calcium, the initial Ca2+ rise remained but the sustained elevation of intracellular Ca2+ was abolished, Na and K fluxes were variable and the Ca did not change. Increased Na and decreased K was marked at 2 minutes and returned to the control value after 60 minutes. The increase in Ca was an early event. Cells stimulated with EGF showed a pronounced morphological disruption, especially the mitochondria. The response of NR6/SA3 and NR6/DC7 cells (genetically engineered rodent fibroblast cell lines) to EGF stimulation was higher than that of the A431 cells, as was the resting cytoplasmic Ca2+. Untreated NR6/SA3 and NR6/DC7 cells possessed an increased Na/K ratio when compared with A431 cells

P2X Receptor-Dependent Modulation of Mast Cell and Glial Cell Activities in Neuroinflammation

From MDPI via Jisc Publications RouterHistory: accepted 2021-08-30, pub-electronic 2021-09-02Publication status: PublishedFunder: Biotechnology and Biological Sciences Research Council; Grant(s): BB/R505699/1Localisation of mast cells (MCs) at the abluminal side of blood vessels in the brain favours their interaction with glial cells, neurons, and endothelial cells, resulting in the activation of these cells and the release of pro-inflammatory mediators. In turn, stimulation of glial cells, such as microglia, astrocytes, and oligodendrocytes may result in the modulation of MC activities. MCs, microglia, astrocytes, and oligodendrocytes all express P2X receptors (P2XRs) family members that are selectively engaged by ATP. As increased concentrations of extracellular adenosine 5′-triphosphate (ATP) are present in the brain in neuropathological conditions, P2XR activation in MCs and glial cells contributes to the control of their communication and amplification of the inflammatory response. In this review we discuss P2XR-mediated MC activation, its bi-directional effect on microglia, astrocytes and oligodendrocytes and role in neuroinflammation

Enhanced release of IgE-dependent early phase mediators from nasal polyp tissue

Background: The mast cell is a crucial effector cell in allergic rhinitis and other inflammatory diseases. During the acute allergic reaction preformed mediators such as histamine, but also de novo produced mediators such as leukotrienes (LTC4/D-4/E-4) and prostaglandins (PGD(2)) are released. Mast cells represent targets for therapeutic intervention, and thus a human ex-vivo model to stimulate mast cells taken from mucosal sites would be instrumental for drug intervention studies. We have aimed to activate mast cells within ex-vivo human nasal tissue by IgE/anti-IgE specific (epsilon chain specific) stimulations and in this respect to test the usability of nasal polyps versus inferior turbinates Methods: Biopsy samples were collected from patients with nasal polyps and inferior turbinates from patients who underwent sinus or septal surgery. Tissue fragments were primed with IgE 1 mu g/ml for 60 minutes and then stimulated for 30 minutes with tissue culture medium (negative control), anti-IgE 10 mu g/ml, anti-IgE 30 mu g/ml and ionomycin 10 mu M (positive control). Histamine, leukotrienes and PGD2 were measured in supernatants. To help provide an understanding of the extent of the response, the number of tryptase and Fc epsilon RI alpha positive cells was evaluated by means of immunohistochemistry and the Fc epsilon RI alpha-chain was measured by means of quantitative PCR in the nasal polyp and inferior turbinate tissues. Finally, the correlation between IgE concentrations in the nasal tissue and the release of mediators was analysed. Results: Stimulations with anti-IgE on IgE-primed nasal tissue fragments lead to a concentration-dependent release of histamine, leukotrienes and PGD(2). The release of these early phase mediators was significantly higher in nasal polyps compared to inferior turbinates, although tryptase, Fc epsilon RI alpha positive cells and Fc epsilon RI alpha-chain transcripts were equally present in both groups. No correlation was found between baseline concentrations of IgE, and the release of histamine, LTC4/LTD4/LTE4 and PGD2 after stimulation. Conclusion: This human nasal challenge model mimics the allergic early phase reaction. The release of histamine, cys-leukotrienes and PGD(2) was significantly higher in nasal polyps versus inferior turbinates, however, this observation could not be explained by differences in mast cell or Fc epsilon RI+ cell numbers

Dynamical Correlation Functions using the Density Matrix Renormalization Group

The density matrix renormalization group (DMRG) method allows for very precise calculations of ground state properties in low-dimensional strongly correlated systems. We investigate two methods to expand the DMRG to calculations of dynamical properties. In the Lanczos vector method the DMRG basis is optimized to represent Lanczos vectors, which are then used to calculate the spectra. This method is fast and relatively easy to implement, but the accuracy at higher frequencies is limited. Alternatively, one can optimize the basis to represent a correction vector for a particular frequency. The correction vectors can be used to calculate the dynamical correlation functions at these frequencies with high accuracy. By separately calculating correction vectors at different frequencies, the dynamical correlation functions can be interpolated and pieced together from these results. For systems with open boundaries we discuss how to construct operators for specific wavevectors using filter functions.Comment: minor revision, 10 pages, 15 figure

Systematic 1/S study of the 2D Hubbard model at half-filling

The 2D Hubbard model is extended by placing 2S orbitals at each lattice site and studied in a systematic 1/S expansion. The 1/S results for the magnetic susceptibility and the spectra of spin-wave excitations at half-filling are consistent with the large S calculations for the Heisenberg antiferromagnet. The 1/S corrections to the fermionic spectrum lift the degeneracy along the edge of the magnetic Brillouin zone yielding minima at (+- pi/2, +- pi/2). Relation to previous papers on the subject is discussed.Comment: 18 pages, emTex version 3.

Identification of asthma associated microRNAs in bronchial biopsies

Background Changes in microRNA (miRNA) expression can contribute to the pathogenesis of many diseases, including asthma. We aimed to identify miRNAs that are differentially expressed between asthma patients and healthy controls, and explore their association with clinical and inflammatory parameters of asthma. Methods Differentially expressed miRNAs were determined by small RNA sequencing on bronchial biopsies of 79 asthma patients and 82 healthy controls using linear regression models. Differentially expressed miRNAs were associated with clinical and inflammatory asthma features. Potential miRNA-mRNA interactions were analysed using mRNA data available from the same bronchial biopsies, and enrichment of pathways was identified with Enrichr and g:Profiler. Results In total, 78 differentially expressed miRNAs were identified in bronchial biopsies of asthma patients compared with controls, of which 60 remained differentially expressed after controlling for smoking and inhaled corticosteroid treatment. We identified several asthma-associated miRNAs, including miR-125b-5p and miR-223-3p, based on a significant association with multiple clinical and inflammatory asthma features and their negative correlation with genes associated with the presence of asthma. The most enriched biological pathway(s) affected by miR-125b-5p and miR-223-3p were inflammatory response and cilium assembly/organisation. Of interest, we identified that lower expression of miR-26a-5p was linked to more severe eosinophilic inflammation as measured in blood, sputum as well as bronchial biopsies. Conclusion Collectively, we identified miR-125b-5p, miR-223-3p and miR-26a-5p as potential regulators that could contribute to the pathogenesis of asthma

Epithelial Interleukin‐1 Receptor‐Like‐1 Activation Is Contingent on Interleukin‐33 Isoforms and Asthma‐Related Receptor Variation

IntroductionThe interleukin-33/interleukin-1 receptor-like-1 (IL-33/IL1RL1) signalling pathway is implicated in asthma pathogenesis, with IL1RL1 nonsynonymous genetic polymorphisms associated with disease risk. We aimed to determine these variants' effect on IL1RL1 signalling induced by different IL33 isoforms thought to be elevated in the asthmatic airway.MethodIn a project funded by GSK plc, which has developed an IL-33 receptor inhibitor for asthma treatment, human embryonic kidney 293 (HEK293) cells expressing secreted embryonic alkaline phosphatase (SEAP) driven by a nuclear factor kappa-beta (NF-κB) promoter, were transiently transfected with IL1RL1, containing one of four extracellular and Toll/interleukin 1 receptor (TIR) domain haplotypes. Cells were stimulated with seven different splice and proteolytic-generated IL-33 isoforms (0.001–50 ng/mL) for 24 h. Supernatant SEAP activity and interleukin-8 (IL-8) levels were determined. Primary human bronchial epithelial cells (HBECs) representing different genotype carriers were stimulated with IL-33112–270 (50 ng/mL) and induced IL-8 mRNA expression measured.ResultsHEK293 cells carrying both asthma extracellular and TIR domain IL1RL1 risk haplotypes presented maximal IL33-driven signalling, with minimal signalling after IL-33 activation in other protective haplotypes. All IL-33 isoforms activated IL1RL1 but with differing magnitudes. Proteolytically cleaved IL3395–270 and IL33106–270 had the greatest effect and the IL33113–270, and Exon 3,4 deletion isoform exhibited the lowest. The effect of extracellular and TIR domain genetic variants on receptor signalling was replicated in primary HBECs. Maximal IL1RL1 signalling was observed in cells carrying both extracellular and TIR signalling domain risk haplotypes.ConclusionsOverall, our study suggests asthma patients carrying the extracellular and TIR domain risk haplotype and have a lung microenvironment that promotes elevated levels of cleaved IL33, particularly where IL3395–270 and IL33106–270 may be more amenable to IL33/IL1RL1 targeting

Human ClCa1 modulates anionic conduction of calcium-dependent chloride currents

Proteins of the CLCA gene family including the human ClCa1 (hClCa1) have been suggested to constitute a new family of chloride channels mediating Ca2+-dependent Cl− currents. The present study examines the relationship between the hClCa1 protein and Ca2+-dependent Cl− currents using heterologous expression of hClCa1 in HEK293 and NCIH522 cell lines and whole cell recordings. By contrast to previous reports claiming the absence of Cl− currents in HEK293 cells, we find that HEK293 and NCIH522 cell lines express constitutive Ca2+-dependent Cl− currents and show that hClCa1 increases the amplitude of Ca2+-dependent Cl− currents in those cells. We further show that hClCa1 does not modify the permeability sequence but increases the Cl− conductance while decreasing the GSCN−/GCl− conductance ratio from ∼2–3 to ∼1. We use an Eyring rate theory (two barriers, one site channel) model and show that the effect of hClCa1 on the anionic channel can be simulated by its action on lowering the first and the second energy barriers. We conclude that hClCa1 does not form Ca2+-dependent Cl− channels per se or enhance the trafficking/insertion of constitutive channels in the HEK293 and NCIH522 expression systems. Rather, hClCa1 elevates the single channel conductance of endogenous Ca2+-dependent Cl− channels by lowering the energy barriers for ion translocation through the pore

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707