Cultured Alveolar Epithelial Cells From Septic Rats Mimic In Vivo Septic Lung

Sepsis results in the formation of pulmonary edema by increasing in epithelial permeability. Therefore we hypothesized that alveolar epithelial cells isolated from septic animals develop tight junctions with different protein composition and reduced barrier function relative to alveolar epithelial cells from healthy animals. Male rats (200–300g) were sacrificed 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Alveolar epithelial cells were isolated and plated on fibronectin-coated flexible membranes or permeable, non-flexible transwell substrates. After a 5 day culture period, cells were either lysed for western analysis of tight junction protein expressin (claudin 3, 4, 5, 7, 8, and 18, occludin, ZO-1, and JAM-A) and MAPk (JNK, ERK, an p38) signaling activation, or barrier function was examined by measuring transepithelial resistance (TER) or the flux of two molecular tracers (5 and 20 Å). Inhibitors of JNK (SP600125, 20 µM) and ERK (U0126, 10 µM) were used to determine the role of these pathways in sepsis induced epithelial barrier dysfunction. Expression of claudin 4, claudin 18, and occludin was significantly lower, and activation of JNK and ERK signaling pathways was significantly increased in 2CLP monolayers, relative to sham monolayers. Transepithelial resistance of the 2CLP monolayers was reduced significantly compared to sham (769 and 1234 ohm-cm2, respectively), however no significant difference in the flux of either tracer was observed. Inhibition of ERK, not JNK, significantly increased TER and expression of claudin 4 in 2CLP monolayers, and prevented significant differences in claudin 18 expression between 2CLP and sham monolayers. We conclude that alveolar epithelial cells isolated from septic animals form confluent monolayers with impaired barrier function compared to healthy monolayers, and inhibition of ERK signaling partially reverses differences between these monolayers. This model provides a unique preparation for probing the mechanisms by which sepsis alters alveolar epithelium

MAPk Activation Modulates Permeability of Isolated Rat Alveolar Epithelial Cell Monolayers Following Cyclic Stretch

We cultured (5 days) rat alveolar epithelial cells to investigate the role of mitogen-activated protein kinase (MAPk) signaling in ventilator induced epithelial barrier dysfunction. Cells were stretched to a magnitude of 12% or 37% change in surface area at a rate of 0.25 Hz with and without pretreatment with either the JNK inhibitor SP600125 or the ERK inhibitor U0126. Following stretch (0, 10, 30, or 60 min), MAPk phosphorylation was examined, monolayer permeability to the uncharged tracer carboxyfluorescein measured (0, 10, 60 min of stretch), and occludin expression determined (0 and 60 min of stretch). Stretch to 12%, previously shown not to increase monolayer permeability, did not alter phosphorylation of any MAPk or occludin expression at any time point. Following stretch to 37%, phosphorylation of JNK, ERK, and p38 was significantly higher by 10 minutes than in unstretched monolayers. Phosphorylation of JNK and p38 subsided as stretch continued, and by 30 minutes returned to unstretched levels. Phosphorylation of ERK remained significantly elevated compared to unstretched levels at all stretch durations. Epithelial permeability increased significantly by 10 minutes of stretch compared to unstretched controls, with further significant increases by 60 minutes. Inhibition with U0126 and SP600125 prevented stretch-induced phosphorylation increases of ERK and JNK, respectively, however neither prevented increases in permeability following 10 minutes. Separately, inhibition of JNK or ERK prevented subsequent additional permeability increases as stretch continued to 60 minute time points. Inhibition of JNK, not ERK, prevented loss of occludin, and minimized loss of cell-cell contact following 60 minutes of stretch. These data suggest that stretch-induced JNK signaling modulates epithelial permeability through regulation tight junction protein expression, and is a potential target for clinical treatments during mechanical ventilation

Sepsis Enhances Epithelial Permeability with Stretch in an Actin Dependent Manner

Ventilation of septic patients often leads to the development of edema and impaired gas exchange. We hypothesized that septic alveolar epithelial monolayers would experience stretch-induced barrier dysfunction at a lower magnitude of stretch than healthy alveolar epithelial monolayers. Alveolar epithelial cells were isolated from rats 24 hours after cecal ligation and double puncture (2CLP) or sham surgery. Following a 5-day culture period, monolayers were cyclically stretched for 0, 10, or 60 minutes to a magnitude of 12% or 25% change in surface area (ΔSA). Barrier function, MAPk and myosin light chain (MLC) phosphorylation, tight junction (TJ) protein expression and actin cytoskeletal organization were examined after stretch. Significant increases in epithelial permeability were observed only in 2CLP monolayers at the 12% ΔSA stretch level, and in both 2CLP and sham monolayers at the 25% ΔSA stretch level. Increased permeability in 2CLP monolayers was not associated with MAPk signaling or alterations in expression of TJ proteins. 2CLP monolayers had fewer actin stress fibers before stretch, a more robust stretch-induced actin redistribution, and reduced phosphorylated MLCK than sham monolayers. Jasplakinolide stabilization of the actin cytoskeleton in 2CLP monolayers prevented significant increases in permeability following 60 minutes of stretch to 12% ΔSA. We concluded that septic alveolar epithelial monolayers are more susceptible to stretch-induced barrier dysfunction than healthy monolayers due to actin reorganization

Vaccine efficacy of ALVAC-HIV and bivalent subtype C gp120–MF59 in adults

BACKGROUND : A safe, effective vaccine is essential to eradicating human immunodeficiency virus (HIV) infection. A canarypox–protein HIV vaccine regimen (ALVAC-HIV plus AIDSVAX B/E) showed modest efficacy in reducing infection in Thailand. An analogous regimen using HIV-1 subtype C virus showed potent humoral and cellular responses in a phase 1–2a trial in South Africa. Efficacy data and additional safety data were needed for this regimen in a larger population in South Africa. METHODS : In this phase 2b–3 trial, we randomly assigned 5404 adults without HIV-1 infection to receive the vaccine (2704 participants) or placebo (2700 participants). The vaccine regimen consisted of injections of ALVAC-HIV at months 0 and 1, followed by four booster injections of ALVAC-HIV plus bivalent subtype C gp120–MF59 adjuvant at months 3, 6, 12, and 18. The primary efficacy outcome was the occurrence of HIV-1 infection from randomization to 24 months. RESULTS : In January 2020, prespecified criteria for non-efficacy were met at an interim analysis; further vaccinations were subsequently halted. The median age of the trial participants was 24 years; 70% of the participants were women. The incidence of adverse events was similar in the vaccine and placebo groups. During the 24-month followup, HIV-1 infection was diagnosed in 138 participants in the vaccine group and in 133 in the placebo group (hazard ratio, 1.02; 95% confidence interval, 0.81 to 1.30; P = 0.84). CONCLUSIONS : The ALVAC–gp120 regimen did not prevent HIV-1 infection among participants in South Africa despite previous evidence of immunogenicity.Supported by grants (HHSN272201300033C and HHSN272201600012C) to Novartis Vaccines and Diagnostics (now part of the GlaxoSmithKline [GSK] Biologicals) by the National Institute of Allergy and Infectious Diseases (NIAID) of the National Institutes of Health (NIH) for the selection and process development of the two gp120 envelope proteins TV1.C and 1086.C; by the Bill and Melinda Gates Foundation Global Health Grant (OPP1017604) and NIAID for the manufacture and release of the gp120 clinical grade material; and by U.S. Public Health Service Grants — UM1 AI068614 to the HIV Vaccine Trials Network (HVTN), UM1 AI068635 to the HVTN Statistical and Data Management Center, and UM1 AI068618 to the HVTN Laboratory Center — from the NIAID. GSK Biologicals contributed financially to the provision of preexposure prophylaxis to trial participants. The South African Medical Research Council supported its affiliated research sites.http://www.nejm.orgam2022School of Health Systems and Public Health (SHSPH

Area of sham and 2CP cells plated on fibronectin coated glass slides was analyzed on day 2.

<p>Images (60× objective) were taken of phalloidin stained actin to visualize the cell boundary. No differences in epithelial size was observed, indicated that the growth rates of sham and 2CLP cells was not significantly different. (Scale bar = 10 µm, N≥18 cells, μ ± SE).</p

Transepithelial resistance of sham and 2CLP monolayers following treatment with either U0126 or SP600125 to inhibit ERK or JNK activation respectively.

<p>Monolayers were serum deprived for 2 hours prior to the application of the inhibitor (time = 0). Measurements were taken at 1, 1.5, and 2 hours following treatment. 2CLP monolayers have significantly lower TER values than sham, and sham monolayer TER was unaffected by any of the treatments. Only U0126 significantly improved TER in 2CLP monolayers above initial values by 1.5 hours, and this persisted to 2 hours (*, p<0.05). (μ ± SE) Western blots of lysate obtained following 2 hours of treatment demonstrating inhibition of JNK and ERK are also shown.</p

Analysis of Complete Blood Count and Bronchoalveolar lavage fluid.

<p>(Left) Complete Blood Count (CBC) data (normalized per vol or %). Elevated hematocrit (HCT) is likely related to dehydration, while decreased levels of platelets (PLT) and lymphocytes are observed with sepsis. (Right) Bronchoalveolar lavage (BAL) fluid data (expressed as % total cells). The total number of cells in the BAL was not different between sham and 2CLP, however more neutrophils and less macrophages were observed in 2CLP lungs than sham lungs. Significance (-) is defined as p<0.05 as determined by a Mann-Whitney nonparametric test.</p

Serum cytokine levels in septic (2CLP) rats normalized to sham controls (value of 1) as determined by microarray.

<p>Increased concentrations of Cinc-2, IL-6, IL-10, LIX, MCP-1, MIP-3, and TIMP-1 show activation of the immune response to the bacterial infection. (N = 6, μ±SE).</p

Western analysis of MAPk signaling in 2CLP and sham monolayers.

<p>We found that activation (ratio of phospho-MAPk to total MAPk) of the JNK and ERK kinases were significantly elevated in the 2CLP monolayers compared to sham (*, p<0.05, N≥12). We include representative western blots showing the phosphorylated bands as well as their respective totals. (mean ± SE).</p