Validity of heart rate derived core temperature estimation during simulated firefighting tasks.

Rectal core temperature monitoring can help fire services mitigate heat injury but can be invasive and impractical. EQ02 + LifeMonitor provides a non-invasive estimation of core temperature. Therefore, the primary purpose of this study was to determine the validity of the EQ02 + LifeMonitor compared to the gold standard rectal thermometer core temperature assessment, as well as the potential influence of turnout gear on the estimated and physiological strain experienced during these activities. Thirteen participants completed simulated firefighting tasks with and without turnout gear, involving four rounds of a 5-min walk on a treadmill at 2.8 mph/2.5% grade and 20 deadlifts over 5 min in an environmental chamber set to 40.6 °C; 50% humidity. During each trial participants wore both an EQ02 + LifeMonitor and DataTherm II rectal thermometer. The results from the devices were statistically equivalent (p < 0.001), yet there was a statistically significant difference in the value (~ 0.1 °C; p < 0.001). There was a significant effect of devices [p < 0.001] and time [p < 0.001], but no interaction effect [p = 0.70] on core temperature drift. Estimated core temperature was marginally different from that measured via the DataTherm II. The EQ02 on average overestimated core temperature. Heart rate, rating of perceived exertion, and area under the curve of core temperature were significantly elevated due to turnout gear [ps < 0.025], but not core temperature skin temperature, or ventilatory rate [ps > 0.372]. These results suggest the EQ02 + LifeMonitor may be a viable, non-invasive alternative for assessing core temperature compared to rectal temperature monitoring, especially during rigorous, intermittent activities. Turnout gear does however increase heart rate, cumulative core temperature, and perceived exertion. Additionally, the validity of the estimated core temperature is not impacted by the use turnout gear. This is likely due to significant changes in heart rate, which allowed the heart-rate derived estimate of core temperature to remain consistent with changes in DataTherm II rectal temperatures.

Long-term voluntary wheel running effects on markers of long interspersed nuclear element-1 in skeletal muscle, liver, and brain tissue of female rats.

We sought to determine the effects of long-term voluntary wheel running on markers of long interspersed nuclear element-1 (L1) in skeletal muscle, liver, and the hippocampus of female rats. In addition, markers of the cGAS-STING DNA-sensing pathway that results in inflammation were interrogated. Female Lewis rats (n = 34) were separated into one of three groups including a 6-mo-old group to serve as a young comparator group (CTL, n = 10), a group that had access to a running wheel for voluntary wheel running (EX, n = 12), and an age-matched group that did not (SED, n = 12). Both SED and EX groups were carried out from 6 mo to 15 mo of age. There were no significant differences in L1 mRNA expression for any of the tissues between groups. Methylation of the L1 promoter in the soleus and hippocampus was significantly higher in SED and EX than in CTL group (P < 0.05). ORF1p expression was higher in older SED and EX rats than in CTL rats for every tissue (P < 0.05). There were no differences between groups for L1 mRNA or cGAS-STING pathway markers. Our results suggest there is an increased ORF1 protein expression across tissues with aging that is not mitigated by voluntary wheel running. In addition, although previous data imply that L1 methylation changes may play a role in acute exercise for L1 RNA expression, this does not seem to occur during extended periods of voluntary wheel running.

Systematic review and meta-analyses on the effects of whole-body vibration on bone health.

OBJECTIVE: To determine whole body vibration influence on human bone density and bone biomarkers. METHODS: We identified studies in Medline, Web of Science, Cumulative Index of Nursing and Allied Health, SPORTDiscus, Embase and Cochrane from inception to November 2021. Human randomized controlled trials involving commercially available whole body vibration platforms were included. Outcomes included bone density mean difference and serum concentrations of biomarkers (Procollagen type 1 N-terminal Propeptides, Osteocalcin, Bone specific alkaline phosphatase, and C-terminal Telopeptide of type 1 collagen). Random effects model (Hedges' g effect-size metric and 95% confidence-intervals) compared whole body vibration effect on bone density and bone biomarkers. Moderator analyses assessed health status, age, menopausal status, vibration type, vibration frequency, and study duration influence. RESULTS: Meta-analysis of 30 studies revealed bone density improvement after whole body vibration (Hedges' g = 0.11; p = 0.05; 95% CI = 0.00, 0.22). Whole body vibration improved bone density in healthy (Hedges' g = 0.10; p = 0.01; 95% CI = 0.02, 0.17) and postmenopausal women (Hedges' g = 0.09; p = 0.02; 95% CI = 0.01, 0.18). Bone density also increased following side-alternating whole body vibration intervention (Hedges' g = 0.21; p = 0.02; 95% CI = 0.04, 0.37). Whole body vibration had no significant effect on either bone formation biomarkers (Hedges' g = 0.22; p = 0.01; 95% CI = 0.05, 0.40) or bone resorption biomarkers (Hedges' g = 0.03; p = 0.74; 95% CI = -0.17, 0.23). CONCLUSION: Whole body vibration may be clinically useful as non-pharmacological/adjunct therapy to mitigate osteoporosis risk in healthy postmenopausal females. Additional studies are needed to determine the underlying mechanisms.

Design and Efficacy of a Monovalent Bispecific PD-1/CTLA4 Antibody That Enhances CTLA4 Blockade on PD-1+ Activated T Cells.

The clinical benefit of PD-1 blockade can be improved by combination with CTLA4 inhibition but is commensurate with significant immune-related adverse events suboptimally limiting the doses of anti-CTLA4 mAb that can be used. MEDI5752 is a monovalent bispecific antibody designed to suppress the PD-1 pathway and provide modulated CTLA4 inhibition favoring enhanced blockade on PD-1+ activated T cells. We show that MEDI5752 preferentially saturates CTLA4 on PD-1+ T cells versus PD-1- T cells, reducing the dose required to elicit IL2 secretion. Unlike conventional PD-1/CTLA4 mAbs, MEDI5752 leads to the rapid internalization and degradation of PD-1. Moreover, we show that MEDI5752 preferentially localizes and accumulates in tumors providing enhanced activity when compared with a combination of mAbs targeting PD-1 and CTLA4 in vivo. Following treatment with MEDI5752, robust partial responses were observed in two patients with advanced solid tumors. MEDI5752 represents a novel immunotherapy engineered to preferentially inhibit CTLA4 on PD-1+ T cells. SIGNIFICANCE: The unique characteristics of MEDI5752 represent a novel immunotherapy engineered to direct CTLA4 inhibition to PD-1+ T cells with the potential for differentiated activity when compared with current conventional mAb combination strategies targeting PD-1 and CTLA4. This molecule therefore represents a step forward in the rational design of cancer immunotherapy.See related commentary by Burton and Tawbi, p. 1008.This article is highlighted in the In This Issue feature, p. 995.

Spontaneous Secretion of the Citrullination Enzyme PAD2 and Cell Surface Exposure of PAD4 by Neutrophils.

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.

Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.

Versatility of homogeneous time-resolved fluorescence resonance energy transfer assays for biologics drug discovery.

Identification of potential lead antibodies in the drug discovery process requires the use of assays that not only measure binding of the antibody to the target molecule but assess a wide range of other characteristics. These include affinity ranking, measurement of their ability to inhibit relevant protein-protein interactions, assessment of their selectivity for the target protein, and determination of their species cross-reactivity profiles to support in vivo studies. Time-resolved fluorescence resonance energy transfer is a technology that offers the flexibility for development of such assays, through the availability of donor and acceptor fluorophore-conjugated reagents for detection of multiple tags or fusion proteins. The time-resolved component of the technology reduces potential assay interference, allowing screening of a range of different crude sample types derived from the bacterial or mammalian cell expression systems often used for antibody discovery projects. Here we describe the successful application of this technology across multiple projects targeting soluble proteins and demonstrate how it has provided key information for the isolation of potential therapeutic antibodies with the desired activity profile.

Application of the mirrorball high-sensitivity cytometer to multiplexed assays for antibody drug discovery.

Highly sensitive, high-throughput assay technologies are required for the identification of antibody therapeutics. Multiplexed assay systems are particularly advantageous because they allow evaluation of several parameters within 1 well, increasing throughput and reducing hands-on laboratory time. The mirrorball (TTP Labtech), using high-throughput fluorometric microvolume assay technology, offers simultaneous scanning with up to 3 lasers as well as laser scatter detection. This makes the mirrorball especially suitable for the development of highly sensitive and multiplexed assays. We have developed bead- and cell-based binding assays that demonstrate how the multilaser capability of the mirrorball can be exploited to enhance assay sensitivity. In addition, using the multilaser simultaneous scanning capability, we have established multiplexed cytokine quantitation assays and antibody-cell binding assays. Our results demonstrate the potential utility of this technology to improve the sensitivity and efficiency of biologics screening, resulting in streamlining of the lead antibody selection process.

Phage display and hybridoma generation of antibodies to human CXCR2 yields antibodies with distinct mechanisms and epitopes.

Generation of functional antibodies against integral membrane proteins such as the G-protein coupled receptor CXCR2 is technically challenging for several reasons, including limited epitope accessibility, the requirement for a lipid environment to maintain structure and their existence in dynamic conformational states. Antibodies to human CXCR2 were generated by immunization in vivo and by in vitro selection methods. Whole cell immunization of transgenic mice and screening of phage display libraries using CXCR2 magnetic proteoliposomes resulted in the isolation of antibodies with distinct modes of action. The hybridoma-derived antibody fully inhibited IL-8 and Gro-α responses in calcium flux and ß-arrestin recruitment assays. The phage-display derived antibodies were allosteric antagonists that showed ligand dependent differences in functional assays. The hybridoma and phage display antibodies did not cross-compete in epitope competition assays and mapping using linear and CLIPS peptides confirmed that they recognized distinct epitopes of human CXCR2. This illustrates the benefits of using parallel antibody isolation approaches with different antigen presentation methods to successfully generate functionally and mechanistically diverse antagonistic antibodies to human CXCR2. The method is likely to be broadly applicable to other complex membrane proteins.