Information is scarce on human responses to high pressure exposures out of water, such as related to tunnel construction workers. We hypothesized that differences in the longer durations of exposures for tunnel workers versus underwater divers results in greater inflammatory responses linked to the pathophysiology of decompression sickness (DCS). Blood was analyzed from 15 tunnel workers (36.1 ± 10.5 (SD) years old, 6 women) exposed to 142-156 kPa pressure for 4.1-4.9 h compared to 8 SCUBA divers (39.3 ± 13.3 (SD) years old, 6 women) exposed to 149 kPa for 0.61 hours. Despite differences in pressure duration between groups, elevations were the same for blood microparticles (MPs) (128 ± 28% MPs/μl) and intra-MPs interleukin (IL-1β) (376 ± 212% pg/million MPs), and for decreases of plasma gelsolin (pGSN, 31 ± 27% μg/mL). The number of circulating CD66b + neutrophils and evidence of cell activation, insignificant for divers, increased in tunnel workers. Across 3 exposures, the mean neutrophil count increased 150 ± 11%. Neutrophil activation increased by 1 to 2% of cells expressing cell surface CD18, myeloperoxidase, platelet-specific CD41, and decrease of cell bound pGSN. We conclude that MPs elevations occur rapidly in humans and reach steady state in minutes with pressure exposures and neutrophil activation requires significantly longer exposure times.
© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.

CD20 monoclonal antibodies (mAbs) eliminate B cells in several clinical contexts. At least two of these Abs, obinutuzumab (OBI) and rituximab (RTX), induce quick elimination of targets and put cancer patients at risk of tumor lysis syndrome (TLS) within 12-24 h of the first dose. The mechanisms of killing can require the recruiting of effector mechanisms from the patient's immune system, but they can induce direct killing as well. This can be more rapid than recruiting cellular effectors and/or complement. We showed here that OBI and RTX induce quick (<1 h) and high (up to 60% for OBI) killing of two different B cell lines. This was unveiled by using two different techniques that circumvent cell centrifugation steps: a Muse® Cell Analyzer-based approach and a direct examination of the cells' physical properties by using forward scatter (FS) area and side scatter (SS) area by flow cytometry. These results excluded the presence of aggregates and were also confirmed by developing a normalized survival ratio based on the co-incubation of RTX- and OBI-sensitive cells with MOLM-13, an insensitive cell line. Finally, this normalized survival ratio protocol confirmed the RTX- and OBI-direct killing on primary tumor B cells from B cell chronic lymphocytic leukemia (B-CLL) and Non-Hodgkin's lymphoma (NHL) patients. Moreover, we unveiled that direct killing is higher than previously expected and absent in patients' samples at relapse. We also observed that these mAbs, prior to increasing intracellular calcium levels, decrease calcium entry, although manipulating calcium levels did not affect their cytotoxicity. Altogether, our results show that direct killing is a major mechanism to induce cell death by RTX and OBI mAbs.

We investigated the cytolytic and mechanistic activity of anti-CD19 chimeric antigen receptor natural killer (CD19.CAR.NK92) therapy in lymphoma cell lines (diffuse large B-cell, follicular, and Burkitt lymphoma), including rituximab- and obinutuzumab-resistant cells, patient-derived cells, and a human xenograft model. CD19.CAR.NK92 therapy significantly increased cytolytic activity at E:T ratios (1:1-10:1) via LDH release and prominent induction of apoptosis in all cell lines, including in anti-CD20 resistant lymphoma cells. The kinetics of CD19.CAR.NK92 cell death measured via droplet-based single cell microfluidics analysis showed that most lymphoma cells were killed by single contact, with anti-CD20 resistant cell lines requiring significantly longer contact duration with NK cells. In addition, systems biology transcriptomic analyses of flow-sorted lymphoma cells co-cultured with CD19.CAR.NK92 revealed conserved activation of IFNγ signaling, execution of apoptosis, ligand binding, and immunoregulatory and chemokine signaling pathways. Furthermore, a 92-plex cytokine panel analysis showed increased secretion of granzymes, increased secretion of FASL, CCL3, and IL10 in anti-CD20 resistant SUDHL4 cells with induction of genes relevant to mTOR and G2/M checkpoint activation, which were noted in all anti-CD20 resistant cells co-cultured with CD19.CAR.NK92 cells. Collectively, CD19.CAR.NK92 was associated with potent anti-lymphoma activity across a host of sensitive and resistant lymphoma cells that involved distinct immuno-biologic mechanisms of cell death.

Treatment with anti-CD20 antibodies is only moderately efficient in chronic lymphocytic leukemia (CLL), a feature which has been explained by the inherently low CD20 expression in CLL. It has been shown that CD20 is epigenetically regulated and that histone deacetylase inhibitors (HDACis) can increase CD20 expression in vitro in CLL. To assess whether HDACis can upregulate CD20 also in vivo in CLL, the HDACi valproate was given to three del13q/NOTCH1wt CLL patients and CD20 levels were analysed (the PREVAIL study). Valproate treatment resulted in expected global activating histone modifications suggesting HDAC inhibitory effects. However, although valproate induced expression of CD20 mRNA and protein in the del13q/NOTCH1wt I83-E95 CLL cell line, no such effects were observed in the patients studied. In contrast to the cell line, in patients valproate treatment resulted in transient recruitment of the transcriptional repressor EZH2 to the CD20 promoter, correlating to an increase of the repressive histone mark H3K27me3. This suggests that valproate-mediated induction of CD20 may be hampered by EZH2 mediated H3K27me3 in vivo in CLL. Moreover, valproate treatment resulted in induction of EZH2 and global H3K27me3 in patient cells, suggesting transcriptionally repressive effects of valproate in CLL. Our results suggest new in vivo mechanisms of HDACis which may have implications on the design of future clinical trials in B-cell malignancies.

We present a quantitative method for comparing the brightness of antibody-dye reagents and estimating antibodies bound per cell. The method is based on complementary binding of test and fill reagents to antibody capture microspheres. Several aliquots of antibody capture beads are stained with varying amounts of the test conjugate. The remaining binding sites on the beads are then filled with a second conjugate containing a different fluorophore. Finally, the fluorescence of the test conjugate compared to the fill conjugate is used to measure the relative brightness of the test conjugate. The fundamental assumption of the test-fill method is that if it takes X molecules of one test antibody to lower the fill signal by Y units, it will take the same X molecules of any other test antibody to give the same effect. We apply a quadratic fit to evaluate the test-fill signal relationship across different amounts of test reagent. If the fit is close to linear, we consider the test reagent to be suitable for quantitative evaluation of antibody binding. To calibrate the antibodies bound per bead, a PE conjugate with 1 PE molecule per antibody is used as a test reagent and the fluorescence scale is calibrated with Quantibrite PE beads. When the fluorescence per antibody molecule has been determined for a particular conjugate, that conjugate can be used for measurement of antibodies bound per cell. This provides comparisons of the brightness of different conjugates when conducted on an instrument whose statistical photoelectron (Spe) scales are known. © 2016 by John Wiley & Sons, Inc.
Copyright © 2016 John Wiley & Sons, Inc.