Development of an experimental technique to determine the barrier performance of medical gloves when stretched.

Protective clothing standards, such as test methods published by ASTM International, play an integral role in ensuring the performance of personal protective equipment. The standard tests are not without limitations and are periodically reviewed and often updated. Some tests may not be reflective of in-use conditions. A new test cell was designed using sanitary fixtures to evaluate the effect of glove stretch on barrier performance using fluorescein solution as the challenge agent for enhanced visualization and fluorometer detection. Domed-shaped and flat screens were developed to permit and limit glove stretch within the test cell. The barrier performance of glove swatches was evaluated for both stretched and unstretched states. Latex, nitrile, and vinyl glove models of various thicknesses were evaluated. The tests were conducted following pressure and time parameters specified in ASTM F903, ASTM F1670, and ASTM F1671. Fluorescein solution movement, which may occur through penetration, was measured using a fluorometer. Glove stretch caused a reduction in glove thickness ranging from 16% to 40%. Overall, 21 sample failures were found (16.7%; n = 126) regardless of test condition. Nitrile gloves provided better barrier efficacy with the lowest failure rates (2.38%; 1 failure out of 42) compared to latex (19.4%; 7 failures out of 36) and vinyl gloves (27.1%; 13 failures out of 48). Differences in failure rates between stretched and unstretched gloves were insignificant; however, the latex material showed a 2.5 times increase in failures when stretched compared to unstretched. The new test apparatus was able to differentiate between the barrier performance of different glove materials. The use of a domed screen allowed the gloves to stretch, a condition that better represents the state of gloves when in use. Analysis of samples collected from the glove surface opposite to the exposure may provide a way to assess chemical permeation in addition to penetration.

Assessment of glove stretch and storage temperature on fentanyl permeation: Implications for standard test methods and PPE recommendations.

The National Institute for Occupational Safety and Health recommends the use of nitrile gloves with a minimum thickness of 5.0 ± 2.0 mil [0.127 ± 0.051 millimeters] in situations where it is suspected or known that fentanyl or other illicit drugs are present. However, there is limited data available on fentanyl permeation through gloves. Current test methods used to measure fentanyl permeation do not consider the effect of glove fit and flexion. Furthermore, first responders need to have PPE readily available in the field, and storage conditions may affect the protective performance of the gloves. The objective of this study was to evaluate the effects of glove stretch and storage temperatures on glove durability and barrier performance against fentanyl. Nine nitrile glove models previously shown to be resistant to fentanyl permeation were selected for this investigation. These nine models were stretched 25% in one linear direction, to consider glove fit and flexion, and tested against fentanyl hydrochloride permeation. Additionally, four of the nine glove models were stored at 48 °C, 22 °C, and -20 °C, and evaluated for tensile strength, ultimate elongation, and puncture resistance after up to 16 wk of storage and fentanyl permeation after up to 8 wk of storage. At least one sample for six of the nine tested models had maximum permeation over the test method fail threshold when stretched. The tested storage temperatures showed no effect on glove tensile strength, ultimate elongation, and puncture resistance. The findings of this study can be used to inform PPE recommendations, with consideration to storage practices and proper sizing for first responders with potential exposure to fentanyl and other illicit drugs. The results of this study can be used to assess the need for new standard test methods to evaluate the barrier performance of gloves and shelf-life determination with consideration to glove fit.

Imbalance of APOB Lipoproteins and Large HDL in Type 1 Diabetes Drives Atherosclerosis.

BACKGROUND: Individuals with type 1 diabetes (T1D) generally have normal or even higher HDL (high-density lipoprotein)-cholesterol levels than people without diabetes yet are at increased risk for atherosclerotic cardiovascular disease (CVD). Human HDL is a complex mixture of particles that can vary in cholesterol content by >2-fold. To investigate if specific HDL subspecies contribute to the increased atherosclerosis associated with T1D, we created mouse models of T1D that exhibit human-like HDL subspecies. We also measured HDL subspecies and their association with incident CVD in a cohort of people with T1D. METHODS: We generated LDL receptor-deficient (Ldlr-/-) mouse models of T1D expressing human APOA1 (apolipoprotein A1). Ldlr-/-APOA1Tg mice exhibited the main human HDL subspecies. We also generated Ldlr-/-APOA1Tg T1D mice expressing CETP (cholesteryl ester transfer protein), which had lower concentrations of large HDL subspecies versus mice not expressing CETP. HDL particle concentrations and sizes and proteins involved in lipoprotein metabolism were measured by calibrated differential ion mobility analysis and targeted mass spectrometry in the mouse models of T1D and in a cohort of individuals with T1D. Endothelial transcytosis was analyzed by total internal reflection fluorescence microscopy. RESULTS: Diabetic Ldlr-/-APOA1Tg mice were severely hyperglycemic and hyperlipidemic and had markedly elevated plasma APOB levels versus nondiabetic littermates but were protected from the proatherogenic effects of diabetes. Diabetic Ldlr-/-APOA1Tg mice expressing CETP lost the atheroprotective effect and had increased lesion necrotic core areas and APOB accumulation, despite having lower plasma APOB levels. The detrimental effects of low concentrations of larger HDL particles in diabetic mice expressing CETP were not explained by reduced cholesterol efflux. Instead, large HDL was more effective than small HDL in preventing endothelial transcytosis of LDL mediated by scavenger receptor class B type 1. Finally, in humans with T1D, increased concentrations of larger HDL particles relative to APOB100 negatively predicted incident CVD independently of HDL-cholesterol levels. CONCLUSIONS: Our results suggest that the balance between APOB lipoproteins and the larger HDL subspecies contributes to atherosclerosis progression and incident CVD in the setting of T1D and that larger HDLs exert atheroprotective effects on endothelial cells rather than by promoting macrophage cholesterol efflux.

The IRG1-itaconate axis protects from cholesterol-induced inflammation and atherosclerosis.

Atherosclerosis is fueled by a failure to resolve lipid-driven inflammation within the vasculature that drives plaque formation. Therapeutic approaches to reverse atherosclerotic inflammation are needed to address the rising global burden of cardiovascular disease (CVD). Recently, metabolites have gained attention for their immunomodulatory properties, including itaconate, which is generated from the tricarboxylic acid-intermediate cis-aconitate by the enzyme Immune Responsive Gene 1 (IRG1/ACOD1). Here, we tested the therapeutic potential of the IRG1-itaconate axis for human atherosclerosis. Using single-cell RNA sequencing (scRNA-seq), we found that IRG1 is up-regulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and in mouse models of atherosclerosis, where it is primarily expressed by plaque monocytes, macrophages, and neutrophils. Global or hematopoietic Irg1-deficiency in mice increases atherosclerosis burden, plaque macrophage and lipid content, and expression of the proatherosclerotic cytokine interleukin (IL)-1ß. Mechanistically, absence of Irg1 increased macrophage lipid accumulation, and accelerated inflammation via increased neutrophil extracellular trap (NET) formation and NET-priming of the NLRP3-inflammasome in macrophages, resulting in increased IL-1ß release. Conversely, supplementation of the Irg1-itaconate axis using 4-octyl itaconate (4-OI) beneficially remodeled advanced plaques and reduced lesional IL-1ß levels in mice. To investigate the effects of 4-OI in humans, we leveraged an ex vivo systems-immunology approach for CVD drug discovery. Using CyTOF and scRNA-seq of peripheral blood mononuclear cells treated with plasma from CVD patients, we showed that 4-OI attenuates proinflammatory phospho-signaling and mediates anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing IRG1-itaconate axis supplementation as a therapeutic approach for atherosclerosis in humans.

Clonal Expansion in Cardiovascular Pathology.

Clonal expansion refers to the proliferation and selection of advantageous "clones" that are better suited for survival in a Darwinian manner. In recent years, we have greatly enhanced our understanding of cell clonality in the cardiovascular context. However, our knowledge of the underlying mechanisms behind this clonal selection is still severely limited. There is a transpiring pattern of clonal expansion of smooth muscle cells and endothelial cells-and, in some cases, macrophages-in numerous cardiovascular diseases irrespective of their differing microenvironments. These findings indirectly suggest the possible existence of stem-like vascular cells which are primed to respond during disease. Subsequent clones may undergo further phenotypic changes to adopt either protective or detrimental roles. By investigating these clone-forming vascular cells, we may be able to harness this inherent clonal nature for future therapeutic intervention. This review comprehensively discusses what is currently known about clonal expansion across the cardiovascular field. Comparisons of the clonal nature of vascular cells in atherosclerosis (including clonal hematopoiesis of indeterminate potential), pulmonary hypertension, aneurysm, blood vessel injury, ischemia- and tumor-induced angiogenesis, and cerebral cavernous malformations are evaluated. Finally, we discuss the potential clinical implications of these findings and propose that proper understanding and specific targeting of these clonal cells may provide unique therapeutic options for the treatment of these cardiovascular conditions.

ß-Carotene accelerates the resolution of atherosclerosis in mice.

ß-Carotene oxygenase 1 (BCO1) catalyzes the cleavage of ß-carotene to form vitamin A. Besides its role in vision, vitamin A regulates the expression of genes involved in lipid metabolism and immune cell differentiation. BCO1 activity is associated with the reduction of plasma cholesterol in humans and mice, while dietary ß-carotene reduces hepatic lipid secretion and delays atherosclerosis progression in various experimental models. Here we show that ß-carotene also accelerates atherosclerosis resolution in two independent murine models, independently of changes in body weight gain or plasma lipid profile. Experiments in Bco1-/- mice implicate vitamin A production in the effects of ß-carotene on atherosclerosis resolution. To explore the direct implication of dietary ß-carotene on regulatory T cells (Tregs) differentiation, we utilized anti-CD25 monoclonal antibody infusions. Our data show that ß-carotene favors Treg expansion in the plaque, and that the partial inhibition of Tregs mitigates the effect of ß-carotene on atherosclerosis resolution. Our data highlight the potential of ß-carotene and BCO1 activity in the resolution of atherosclerotic cardiovascular disease.

ANGPTL3 deficiency impairs lipoprotein production and produces adaptive changes in hepatic lipid metabolism.

Angiopoietin-like protein 3 (ANGPTL3) is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein (LDL) cholesterol. Although ANGPTL3 modulates the metabolism of circulating lipoproteins, its role in triglyceride-rich lipoprotein assembly and secretion remains unknown. CRISPR-associated protein 9 (CRISPR/Cas9) was used to target ANGPTL3 in HepG2 cells (ANGPTL3-/-) whereupon we observed ∼50% reduction of apolipoprotein B100 (ApoB100) secretion, accompanied by an increase in ApoB100 early presecretory degradation via a predominantly lysosomal mechanism. Despite defective particle secretion in ANGPTL3-/- cells, targeted lipidomic analysis did not reveal neutral lipid accumulation in ANGPTL3-/- cells; rather ANGPTL3-/- cells demonstrated decreased secretion of newly synthesized triglycerides and increased fatty acid oxidation. Furthermore, RNA sequencing demonstrated significantly altered expression of key lipid metabolism genes, including targets of peroxisome proliferator-activated receptor α, consistent with decreased lipid anabolism and increased lipid catabolism. In contrast, CRISPR/Cas9 LDL receptor (LDLR) deletion in ANGPTL3-/- cells did not result in a secretion defect at baseline, but proteasomal inhibition strongly induced compensatory late presecretory degradation of ApoB100 and impaired its secretion. Additionally, these ANGPTL3-/-;LDLR-/- cells rescued the deficient LDL clearance of LDLR-/- cells. In summary, ANGPTL3 deficiency in the presence of functional LDLR leads to the production of fewer lipoprotein particles due to early presecretory defects in particle assembly that are associated with adaptive changes in intrahepatic lipid metabolism. In contrast, when LDLR is absent, ANGPTL3 deficiency is associated with late presecretory regulation of ApoB100 degradation without impaired secretion. Our findings therefore suggest an unanticipated intrahepatic role for ANGPTL3, whose function varies with LDLR status.

The Effect of Diet Composition on the Post-operative Outcomes of Roux-en-Y Gastric Bypass in Mice.

PURPOSE: Roux-en-Y gastric bypass (RYGB) leads to the improvement of many obesity-associated conditions. The degree to which post-operative macronutrient composition contributes to metabolic improvement after RYGB is understudied. METHODS: A mouse model of RYGB was used to examine the effects of diet on the post-operative outcomes of RYGB. Obese mice underwent either Sham or RYGB surgery and were administered either chow or HFD and then monitored for an additional 8 weeks. RESULTS: After RYGB, reductions to body weight, fat mass, and lean mass were similar regardless of diet. RYGB and HFD were independently detrimental to bone mineral density and plasma vitamin D levels. Independent of surgery, HFD accelerated hematopoietic stem and progenitor cell proliferation and differentiation and exhibited greater myeloid lineage commitment. Independent of diet, systemic iron deficiency was present after RYGB. In both Sham and RYGB groups, HFD increased energy expenditure. RYGB increased fecal energy loss, and HFD after RYGB increased fecal lipid content. RYGB lowered fasting glucose and liver glycogen levels but HFD had an opposing effect. Indices of insulin sensitivity improved independent of diet. HFD impaired improvements to dyslipidemia, NAFLD, and fibrosis. CONCLUSION: Post-operative diet plays a significant role in determining the degree to which RYGB reverses obesity-induced metabolic abnormalities such as hyperglycemia, dyslipidemia, and NAFLD. Diet composition may be targeted in order to assist in the treatment of post-RYGB bone mineral density loss and vitamin D deficiency as well as to reverse myeloid lineage commitment. HFD after RYGB continues to pose a significant multidimensional health risk.

ß-carotene accelerates the resolution of atherosclerosis in mice.

ß-carotene oxygenase 1 (BCO1) catalyzes the cleavage of ß-carotene to form vitamin A. Besides its role in vision, vitamin A regulates the expression of genes involved in lipid metabolism and immune cell differentiation. BCO1 activity is associated with the reduction of plasma cholesterol in humans and mice, while dietary ß-carotene reduces hepatic lipid secretion and delays atherosclerosis progression in various experimental models. Here we show that ß-carotene also accelerates atherosclerosis resolution in two independent murine models, independently of changes in body weight gain or plasma lipid profile. Experiments in Bco1-/- mice implicate vitamin A production in the effects of ß-carotene on atherosclerosis resolution. To explore the direct implication of dietary ß-carotene on regulatory T cells (Tregs) differentiation, we utilized anti-CD25 monoclonal antibody infusions. Our data show that ß-carotene favors Treg expansion in the plaque, and that the partial inhibition of Tregs mitigates the effect of ß-carotene on atherosclerosis resolution. Our data highlight the potential of ß-carotene and BCO1 activity in the resolution of atherosclerotic cardiovascular disease.

FITM2 deficiency results in ER lipid accumulation, ER stress, reduced apolipoprotein B lipidation, and VLDL triglyceride secretion in vitro and in mouse liver.

Objectives: Triglyceride (TG) association with apolipoprotein B100 (apoB100) serves to form very low density lipoproteins (VLDL) in the liver. The repertoire of factors that facilitate this association is incompletely defined. FITM2, an integral endoplasmic reticulum (ER) protein, was originally discovered as a factor participating in cytoplasmic lipid droplets (LDs) in tissues that do not form VLDL. We hypothesized that in the liver, in addition to promoting cytosolic LD formation, FITM2 would also transfer TG from its site of synthesis in the ER membrane to nascent VLDL particles within the ER lumen. Methods: Experiments were conducted using a rat hepatic cell line (McArdle-RH7777, or McA cells), an established model of mammalian lipoprotein metabolism, and mice. FITM2 expression was reduced using siRNA in cells and by liver specific cre-recombinase mediated deletion of the Fitm2 gene in mice. Effects of FITM2 deficiency on VLDL assembly and secretion in vitro and in vivo were measured by multiple methods, including density gradient ultracentrifugation, chromatography, mass spectrometry, simulated Raman spectroscopy (SRS) microscopy, sub-cellular fractionation, immunoprecipitation, immunofluorescence, and electron microscopy. Main findings: 1) FITM2-deficient hepatic cells in vitro and in vivo secrete TG-depleted VLDL particles, but the number of particles is unchanged compared to controls; 2) FITM2 deficiency in mice on a high fat diet (HFD) results in decreased plasma TG levels. The number of apoB100-containing lipoproteins remains similar, but shift from VLDL to LDL density; 3) Both in vitro and in vivo , when TG synthesis is stimulated and FITM2 is deficient, TG accumulates in the ER, and despite its availability this pool is unable to fully lipidate apoB100 particles; 4) FITM2 deficiency disrupts ER morphology and results in ER stress. Principal conclusions: The results suggest that FITM2 contributes to VLDL lipidation, especially when newly synthesized hepatic TG is in abundance. In addition to its fundamental importance in VLDL assembly, the results also suggest that under dysmetabolic conditions, FITM2 may be a limiting factor that ultimately contributes to non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH).

Colchicine promotes atherosclerotic plaque stability independently of inflammation.

Atherosclerosis is a chronic inflammatory disease which is driven in part by the aberrant trans -differentiation of vascular smooth muscle cells (SMCs). No therapeutic drug has been shown to reverse detrimental SMC-derived cell phenotypes into protective phenotypes, a hypothesized enabler of plaque regression and improved patient outcome. Herein, we describe a novel function of colchicine in the beneficial modulation of SMC-derived cell phenotype, independent of its conventional anti-inflammatory effects. Using SMC fate mapping in an advanced atherosclerotic lesion model, colchicine induced plaque regression by converting pathogenic SMC-derived macrophage-like and osteoblast-like cells into protective myofibroblast-like cells which thickened, and thereby stabilized, the fibrous cap. This was dependent on Notch3 signaling in SMC-derived plaque cells. These findings may help explain the success of colchicine in clinical trials relative to other anti-inflammatory drugs. Thus, we demonstrate the potential of regulating SMC phenotype in advanced plaque regression through Notch3 signaling, in addition to the canonical anti-inflammatory actions of drugs to treat atherosclerosis.

HDL regulates TGFß-receptor lipid raft partitioning, restoring contractile features of cholesterol-loaded vascular smooth muscle cells.

Background: Cholesterol-loading of mouse aortic vascular smooth muscle cells (mVSMCs) downregulates miR-143/145, a master regulator of the contractile state downstream of TGFß signaling. In vitro, this results in transitioning from a contractile mVSMC to a macrophage-like state. This process likely occurs in vivo based on studies in mouse and human atherosclerotic plaques. Objectives: To test whether cholesterol-loading reduces VSMC TGFß signaling and if cholesterol efflux will restore signaling and the contractile state in vitro and in vivo. Methods: Human coronary artery (h)VSMCs were cholesterol-loaded, then treated with HDL (to promote cholesterol efflux). For in vivo studies, partial conditional deletion of Tgfßr2 in lineage-traced VSMC mice was induced. Mice wild-type for VSMC Tgfßr2 or partially deficient (Tgfßr2+/-) were made hypercholesterolemic to establish atherosclerosis. Mice were then treated with apoA1 (which forms HDL). Results: Cholesterol-loading of hVSMCs downregulated TGFß signaling and contractile gene expression; macrophage markers were induced. TGFß signaling positively regulated miR-143/145 expression, increasing Acta2 expression and suppressing KLF4. Cholesterol-loading localized TGFß receptors into lipid rafts, with consequent TGFß signaling downregulation. Notably, in cholesterol-loaded hVSMCs HDL particles displaced receptors from lipid rafts and increased TGFß signaling, resulting in enhanced miR-145 expression and decreased KLF4-dependent macrophage features. ApoA1 infusion into Tgfßr2+/- mice restored Acta2 expression and decreased macrophage-marker expression in plaque VSMCs, with evidence of increased TGFß signaling. Conclusions: Cholesterol suppresses TGFß signaling and the contractile state in hVSMC through partitioning of TGFß receptors into lipid rafts. These changes can be reversed by promotion of cholesterol efflux, consistent with evidence in vivo.

Total outward leakage of half-mask respirators and surgical masks used for source control.

Both respirators and surgical masks (SM) are used as source control devices. During the COVID-19 pandemic, there was much interest in understanding the extent of particle total outward leakage (TOL) from these devices. The objective of this study was to quantify the TOL for five categories of devices: SMs, National Institute for Occupational Safety and Health (NIOSH) Approved N95 filtering facepiece respirators (FFRs) without exhalation valves, NIOSH Approved N95 FFRs with exhalation valves (N95 FFRV), NIOSH Approved elastomeric half-mask respirators (EHMRs) with exhalation valves, and NIOSH Approved EHMRs with an SM covering the exhalation valve (EHMRSM). A benchtop test system was designed to test two models of each device category. Each device was mounted on a headform at three faceseal levels (0% faceseal, 50% faceseal, and 100% faceseal). At each faceseal level, the TOL was assessed at three flow rates of minute ventilations of 17, 28, and 39 L/min. The experimental design was a split-split-plot configuration. Device type, faceseal level, flow rate, and the interaction of device type and faceseal level were found to have a significant effect (p-value < 0.05) on the TOL. This study found that the N95 FFRs without exhalation valves had the lowest mean TOL. The SMs had about three times higher TOL than the N95 FFRs without exhalation valves. The TOL of the N95 FFRV was comparable to that of the SM at 0% and 50% faceseal on average overall conditions, but the N95 FFRV had a significantly higher TOL than the SM at a 100% faceseal. The EHMRs had the highest TOL because of the exhalation valve. Using an SM to cover the exhalation valve did not improve the EHMRs' efficiency in mitigating the TOL. Caution should be exercised when using N95 FFRVs as a source control measure against respiratory activities with heavy work rates, such as performing CPR. Results of this study showed that reduced faceseal leakage for N95 FFRs and SMs improves source control.