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The glycosaminoglycan hyaluronan (HA) serves a variety of crucial physiological functions in vertebrates. Synthesized at the plasma membrane and secreted into the extracellular environment, HA polymers span a wide range of molecular weights (MW) that define their activity through a notable size-function relationship. Analytical technologies for determining HA MW distributions typically require selective extraction from complex biofluids or tissues. A common method for achieving this is immunoprecipitation-like pull-down using specific HA-binding proteins bound to magnetic beads. Here, we present a systematic investigation of experimental variables involved in this process, leading to an affinity extraction protocol that enables iterative bead reuse and reagent lifetime maximization, thereby enhancing the efficiency of the HA extraction process. Our methods provide a framework for general optimization of immunoprecipitation in other contexts with heterogenous analyte sizes., Competing Interests: A.R.H, P.L.D, and E.R are listed as inventors on a patent covering SSNP analysis of HA., (© 2024 The Author(s). Proteoglycan Research published by Wiley Periodicals LLC.)

Abstract: Background: Death due to hemorrhagic shock, particularly, noncompressible truncal hemorrhage, remains one of the leading causes of potentially preventable deaths. Automated partial and intermittent resuscitative endovascular balloon occlusion of the aorta (i.e., pREBOA and iREBOA, respectively) are lifesaving endovascular strategies aimed to achieve quick hemostatic control while mitigating distal ischemia. In iREBOA, the balloon is titrated from full occlusion to no occlusion intermittently, whereas in pREBOA, a partial occlusion is maintained. Therefore, these two interventions impose different hemodynamic conditions, which may impact coagulation and the endothelial glycocalyx layer. In this study, we aimed to characterize the clotting kinetics and coagulopathy associated with iREBOA and pREBOA, using thromboelastography (TEG). We hypothesized that iREBOA would be associated with a more hypercoagulopathic response compared with pREBOA due to more oscillatory flow. Methods: Yorkshire swine (n = 8/group) were subjected to an uncontrolled hemorrhage by liver transection, followed by 90 min of automated pREBOA, iREBOA, or no balloon support (control). Hemodynamic parameters were continuously recorded, and blood samples were serially collected during the experiment (i.e., eight key time points: baseline (BL), T0, T10, T30, T60, T90, T120, T210 min). Citrated kaolin heparinase assays were run on a TEG 5000 (Haemonetics, Niles, IL). General linear mixed models were employed to compare differences in TEG parameters between groups and over time using STATA (v17; College Station, TX), while adjusting for sex and weight. Results: As expected, iREBOA was associated with more oscillations in proximal pressure (and greater magnitudes of peak pressure) because of the intermittent periods of full aortic occlusion and complete balloon deflation, compared to pREBOA. Despite these differences in acute hemodynamics, there were no significant differences in any of the TEG parameters between the iREBOA and pREBOA groups. However, animals in both groups experienced a significant reduction in clotting times (R time: P < 0.001; K time: P < 0.001) and clot strength (MA: P = 0.01; G: P = 0.02) over the duration of the experiment. Conclusions: Despite observing acute differences in peak proximal pressures between the iREBOA and pREBOA groups, we did not observe any significant differences in TEG parameters between iREBOA and pREBOA. The changes in TEG profiles were significant over time, indicating that a severe hemorrhage followed by both pREBOA and iREBOA can result in faster clotting reaction times (i.e., R times). Nevertheless, when considering the significant reduction in transfusion requirements and more stable hemodynamic response in the pREBOA group, there may be some evidence favoring pREBOA usage over iREBOA., Competing Interests: L.P.N. and T.K.W. are cofounders and shareholders of Certus Critical Care, Inc. NTPP served as a consultant for Certus Critical Care, Inc. The other authors report no conflicts of interest., (Copyright © 2024 by the Shock Society.)

Abstract: Background: Critical care management of shock is a labor-intensive process. Precision Automated Critical Care Management (PACC-MAN) is an automated closed-loop system incorporating physiologic and hemodynamic inputs to deliver interventions while avoiding excessive fluid or vasopressor administration. To understand PACC-MAN efficacy, we compared PACC-MAN to provider-directed management (PDM). We hypothesized that PACC-MAN would achieve equivalent resuscitation outcomes to PDM while maintaining normotension with lower fluid and vasopressor requirements. Methods : Twelve swine underwent 30% controlled hemorrhage over 30 min, followed by 45 min of aortic occlusion to generate a vasoplegic shock state, transfusion to euvolemia, and randomization to PACC-MAN or PDM for 4.25 h. Primary outcomes were total crystalloid volume, vasopressor administration, total time spent at hypotension (mean arterial blood pressure <60 mm Hg), and total number of interventions. Results : Weight-based fluid volumes were similar between PACC-MAN and PDM; median and IQR are reported (73.1 mL/kg [59.0-78.7] vs. 87.1 mL/kg [79.4-91.8], P = 0.07). There was no statistical difference in cumulative norepinephrine (PACC-MAN: 33.4 μg/kg [27.1-44.6] vs. PDM: 7.5 [3.3-24.2] μg/kg, P = 0.09). The median percentage of time spent at hypotension was equivalent (PACC-MAN: 6.2% [3.6-7.4] and PDM: 3.1% [1.3-6.6], P = 0.23). Urine outputs were similar between PACC-MAN and PDM (14.0 mL/kg vs. 21.5 mL/kg, P = 0.13). Conclusion : Automated resuscitation achieves equivalent resuscitation outcomes to direct human intervention in this shock model. This study provides the first translational experience with the PACC-MAN system versus PDM., Competing Interests: AJ, LPN, and TKW are co-founders and shareholders of Certus Critical Care, Incorporated. The authors report no conflicts of interest., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Shock Society.)

The glycosaminoglycan hyaluronan (HA) plays important roles in diverse physiological functions where the distribution of its molecular weight (MW) can influence its behavior and is known to change in response to disease conditions. During inflammation, HA undergoes a covalent modification in which heavy chain subunits of the inter-alpha-inhibitor family of proteins are transferred to its structure, forming heavy chain-HA (HC•HA) complexes. While limited assessments of HC•HA have been performed previously, determining the size distribution of its HA component remains a challenge. Here, we describe a selective method for extracting HC•HA from mixtures that yields material amenable to MW analysis with a solid-state nanopore sensor. After demonstrating the approach in vitro, we validate extraction of HC•HA from osteoarthritic human synovial fluid as a model complex biological matrix. Finally, we apply our technique to pathophysiology by measuring the size distributions of HC•HA and total HA in an equine model of synovitis.

Introduction: The pressure-volume (P-V) relationships of the left ventricle are the classical benchmark for studying cardiac mechanics and pumping function. Perturbations in the P-V relationship (or P-V loop) can be informative and guide the management of heart failure, hypovolemia, and aortic occlusion. Traditionally, P-V loop analyses have been limited to a single-beat P-V loop or an average of consecutive P-V loops (e.g., 10 cardiac cycles). While there are several algorithms to obtain single-beat estimations of the end-systolic and end-diastolic pressure-volume relations (i.e., ESPVR and EDPVR, respectively), there remains a need to better evaluate the variations in P-V relationships longitudinally over time. This is particularly important when studying acute and transient hemodynamic and cardiac events, such as active hemorrhage or aortic occlusion. In this study, we aim to investigate the variability in P-V relationships during hemorrhagic shock and aortic occlusion, by leveraging on a previously published porcine hemorrhage model., Methods: Briefly, swine were instrumented with a P-V catheter in the left ventricle of the heart and underwent a 25% total blood volume hemorrhage over 30 min, followed by either Zone 1 complete aortic occlusion (i.e., REBOA), Zone 1 endovascular variable aortic control (EVAC), or no occlusion as a control, for 45 min. Preload-independent metrics of cardiac performance were obtained at predetermined time points by performing inferior vena cava occlusion during a ventilatory pause. Continuous P-V loop data and other hemodynamic flow and pressure measurements were collected in real-time using a multi-channel data acquisition system., Results: We developed a custom algorithm to quantify the time-dependent variance in both load-dependent and independent cardiac parameters from each P-V loop. As expected, all pigs displayed a significant decrease in the end-systolic pressures and volumes (i.e., ESP, ESV) after hemorrhage. The variability in response to hemorrhage was consistent across all three groups. However, upon introduction of REBOA, we observed significantly high levels of variability in both load-dependent and independent cardiac metrics such as ESP, ESV, and the slope of ESPVR ( E es ). For instance, pigs receiving REBOA experienced a 342% increase in ESP from hemorrhage, while pigs receiving EVAC experienced only a 188% increase. The level of variability within the EVAC group was consistently less than that of the REBOA group, which suggests that the EVAC group may be more supportive of maintaining healthier cardiac performance than complete occlusion with REBOA., Discussion: In conclusion, we successfully developed a novel algorithm to reliably quantify the single-beat and longitudinal P-V relations during hemorrhage and aortic occlusion. As expected, hemorrhage resulted in smaller P-V loops, reflective of decreased preload and afterload conditions; however, the cardiac output and heart rate were preserved. The use of REBOA and EVAC for 44 min resulted in the restoration of baseline afterload and preload conditions, but often REBOA exceeded baseline pressure conditions to an alarming level. The level of variability in response to REBOA was significant and could be potentially associated to cardiac injury. By quantifying each P-V loop, we were able to capture the variability in all P-V loops, including those that were irregular in shape and believe that this can help us identify critical time points associated with declining cardiac performance during hemorrhage and REBOA use., Competing Interests: MJ, TW, and LN are affiliated with Certus Critical Care™ Inc., which develops endovascular technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Mobin, Renaldo, Carrasco Perez, Jordan, Neff, Williams, Johnson and Rahbar.)

Background: Partial and intermittent resuscitative endovascular balloon occlusion of the aorta (pREBOA and iREBOA, respectively) are lifesaving techniques designed to extend therapeutic duration, mitigate ischemia, and bridge patients to definitive hemorrhage control. We hypothesized that automated pREBOA balloon titration compared with automated iREBOA would reduce blood loss and hypotensive episodes over a 90-minute intervention phase compared with iREBOA in an uncontrolled liver hemorrhage swine model., Methods: Twenty-four pigs underwent an uncontrolled hemorrhage by liver transection and were randomized to automated pREBOA (n = 8), iREBOA (n = 8), or control (n = 8). Once hemorrhagic shock criteria were met, controls had the REBOA catheter removed and received transfusions only for hypotension. The REBOA groups received 90 minutes of either iREBOA or pREBOA therapy. Surgical hemostasis was obtained, hemorrhage volume was quantified, and animals were transfused to euvolemia and then underwent 1.5 hours of automated critical care., Results: The control group had significantly higher mortality rate (5 of 8) compared with no deaths in both REBOA groups, demonstrating that the liver injury is highly lethal ( p = 0.03). During the intervention phase, animals in the iREBOA group spent a greater proportion of time in hypotension than the pREBOA group (20.7% [16.2-24.8%] vs. 0.76% [0.43-1.14%]; p < 0.001). The iREBOA group required significantly more transfusions than pREBOA (21.0 [20.0-24.9] mL/kg vs. 12.1 [9.5-13.9] mL/kg; p = 0.01). At surgical hemostasis, iREBOA had significantly higher hemorrhage volumes compared with pREBOA (39.2 [29.7-44.95] mL/kg vs. 24.7 [21.6-30.8] mL/kg; p = 0.04)., Conclusion: Partial REBOA animals spent significantly less time at hypotension and had decreased transfusions and blood loss. Both pREBOA and iREBOA prevented immediate death compared with controls. Further refinement of automated pREBOA is necessary, and controller algorithms may serve as vital control inputs for automated transfusion., Level of Evidence: Therapeutic/Care Management; Level III., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Abstract: Background: The endothelial glycocalyx layer (EGL) is a complex meshwork of glycosaminoglycans and proteoglycans that protect the vascular endothelium. Cleavage or shedding of EGL-specific biomarkers, such as hyaluronic acid (HA) and syndecan-1 (SDC-1, CD138) in plasma, have been shown to be associated with poor clinical outcomes. However, it is unclear whether levels of circulating EGL biomarkers are representative of the EGL injury within the tissues. The objective of the present feasibility study was to describe a pathway for plasma and tissue procurement to quantify EGL components in a cohort of surgical patients with intra-abdominal sepsis. We sought to compare differences between tissue and plasma EGL biomarkers and to determine whether EGL shedding within the circulation and/or tissues correlated with clinical outcomes. Methods: This was a prospective, observational, single-center feasibility study of adult patients (N = 15) with intra-abdominal sepsis, conducted under an approved institutional review boards. Blood and resected tissue (pathologic specimen and unaffected peritoneum) samples were collected from consented subjects at the time of operation and 24-48 hours after surgery. Endothelial glycocalyx layer biomarkers (i.e., HA and SDC-1) were quantified in both tissue and plasma samples using a CD138 stain and ELISA kit, respectively. Pairwise comparisons were made between plasma and tissue levels. In addition, we tested the relationships between measured EGL biomarkers and clinical status and patient outcomes. Results: Fifteen patients with intra-abdominal sepsis were enrolled in the study. Elevations in EGL-specific circulating biomarkers (HA, SDC-1) were positively correlated with postoperative SOFA scores and weakly associated with resuscitative volumes at 24 hours. Syndecan-1 levels from resected pathologic tissue significantly correlated with SOFA scores at all time points ( R = 0.69 and P < 0.0001) and positively correlated with resuscitation volumes at 24 hours ( R = 0.41 and P = 0.15 for t = 24 hours). Tissue and circulating HA and SDC-1 positively correlated with SOFA >6. Conclusions: Elevations in both circulating and tissue EGL biomarkers were positively correlated with postoperative SOFA scores at 24 hours, with resected pathologic tissue EGL levels displaying significant correlations with SOFA scores at all time points. Tissue and circulating EGL biomarkers were positively correlated at higher SOFA scores (SOFA > 6) and could be used as indicators of resuscitative needs within 24 hours of surgery. The present study demonstrates the feasibility of tissue and plasma procurement in the operating room, although larger studies are needed to evaluate the predictive value of these EGL biomarkers for patients with intra-abdominal sepsis., (Copyright © 2023 by the Shock Society.)

Background: Resuscitative endovascular balloon occlusion of the aorta (REBOA) is a lifesaving intervention for major truncal hemorrhage. Balloon-tipped arterial catheters are inserted via the femoral artery to create a temporary occlusion of the aorta, which minimizes the rate of internal bleeding until definitive surgery can be conducted. There is growing concern over the resultant hypoperfusion and potential damage to tissues and organs downstream of REBOA. To better understand the acute hemodynamic changes imposed by REBOA, we developed a three-dimensional computational fluid dynamic (CFD) model under normal, hemorrhage, and aortic occlusion conditions. The goal was to characterize the acute hemodynamic changes and identify regions within the aortic vascular tree susceptible to abnormal flow and shear stress. Methods: Hemodynamic data from established porcine hemorrhage models were used to build a CFD model. Swine underwent 20% controlled hemorrhage and were randomized to receive a full or partial aortic occlusion. Using CT scans, we generated a pig-specific aortic geometry and imposed physiologically relevant inlet flow and outlet pressure boundary conditions to match in vivo data. By assuming non-Newtonian fluid properties, pressure, velocity, and shear stresses were quantified over a cardiac cycle. Results: We observed a significant rise in blood pressure (∼147 mmHg) proximal to REBOA, which resulted in increased flow and shear stress within the ascending aorta. Specifically, we observed high levels of shear stress within the subclavian arteries (22.75 Pa). Alternatively, at the site of full REBOA, wall shear stress was low (0.04 ± 9.07E-4 Pa), but flow oscillations were high (oscillatory shear index of 0.31). Comparatively, partial REBOA elevated shear levels to 84.14 ± 19.50 Pa and reduced flow oscillations. Our numerical simulations were congruent within 5% of averaged porcine experimental data over a cardiac cycle. Conclusion: This CFD model is the first to our knowledge to quantify the acute hemodynamic changes imposed by REBOA. We identified areas of low shear stress near the site of occlusion and high shear stress in the subclavian arteries. Future studies are needed to determine the optimal design parameters of endovascular hemorrhage control devices that can minimize flow perturbations and areas of high shear., Competing Interests: TTW and LN are founders and stockholders of Certus Critical Care, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The views expressed in this manuscript are those of the authors and do not reflect the official policy of the US Government, the Department of Defense, the National Institutes of Health, or Wake Forest University., (Copyright © 2022 Renaldo, Lane, Shapiro, Mobin, Jordan, Williams, Neff, Gayzik and Rahbar.)

Background: We sought to determine the magnitude of the inherent inter-animal physiologic variability by automating a porcine Resuscitative Endovascular Balloon Occlusion of the Aorta (REBOA) protocol to minimize external influences that might alter physiology and confound experimental results., Methods: Swine (n = 42) underwent a controlled 30% blood volume hemorrhage followed by 30 minutes of REBOA (ie, ischemic phase). The animals were weaned from REBOA autonomously over 15 minutes, beginning the reperfusion phase, while continuing to provide partial flow balloon support to maintain a target proximal mean arterial pressure (pMAP) of 65 mmHg. Simultaneously, shed blood was re-transfused as part of the resuscitation efforts. Physiologic data were continuously recorded, and serum samples were serially collected. Baseline characteristics, variance in vital signs, and 8-isoprostane levels were quantified during hemorrhage, REBOA, and reperfusion phases., Results: There was no significant difference in baseline physiology across animals ( P > .05). Hemodynamic variability was highest for pMAP during the ischemic phase ( P = .001) and for distal mean arterial pressure (dMAP) during the weaning/reperfusion phase ( P = .001). The latter finding indicated the variable physiologic response to ischemia-reperfusion injury, as the automated balloon support required by each animal to maintain pMAP was highly variable. Circulating 8-isoprostane variance was significantly higher following the start of reperfusion compared to baseline levels ( P = .001)., Discussion: Despite subjecting animals to a highly consistent ischemia-reperfusion injury through automation, we noted significant variability in the hemodynamic and biochemical response. These findings illustrate the inherent physiologic variability and potential limitations of porcine large animal models for the study of shock.

The carbohydrate hyaluronan (or hyaluronic acid, HA) is found in all human tissues and biofluids where it has wide-ranging functions in health and disease that are dictated by both its abundance and size. Consequently, hyaluronan evaluation in physiological samples has significant translational potential. Although the analytical tools and techniques for probing other biomolecules such as proteins and nucleic acids have become standard approaches in biochemistry, those available for investigating hyaluronan are less well established. In this review, we survey methods related to the assessment of native hyaluronan in biological specimens, including protocols for separating it from biological matrices and technologies for determining its concentration and molecular weight.

Hyaluronan (HA) is an essential carbohydrate in vertebrates that is a potentially robust bioindicator due to its critical roles in diverse physiological functions in health and disease. The intricate size-dependent function that exists for HA and its low abundance in most biological fluids have highlighted the need for sensitive technologies to provide accurate and quantitative assessments of polysaccharide molecular weight and concentration. We have demonstrated that solid state (SS-) nanopore technology can be exploited for this purpose, given its molecular sensitivity and analytical capacity, but there remains a need to further understand the impacts of experimental variables on the SS-nanopore signal for optimal interpretation of results. Here, we use model quasi-monodisperse HA polymers to determine the dependence of HA signal characteristics on a range of SS-nanopore measurement conditions, including applied voltage, pore diameter, and ionic buffer asymmetry. Our results identify important factors for improving the signal-to-noise ratio, resolution, and sensitivity of HA analysis with SS-nanopores., (© 2022. The Author(s).)

The Modern Western Diet has been associated with the rise in metabolic and inflammatory diseases, including obesity, diabetes, and cardiovascular disease. This has been attributed, in part, to the increase in dietary omega-6 polyunsaturated fatty acid (PUFA) consumption, specifically linoleic acid (LA), arachidonic acid (ARA), and their subsequent metabolism to pro-inflammatory metabolites which may be driving human disease. Conversion of dietary LA to ARA is regulated by genetic variants near and within the fatty acid desaturase (FADS) haplotype block, most notably single nucleotide polymorphism rs174537 is strongly associated with FADS1 activity and expression. This variant and others within high linkage disequilibrium may potentially explain the diversity in both diet and inflammatory mediators that drive chronic inflammatory disease in human populations. Mechanistic exploration into this phenomenon using human hepatocytes is limited by current two-dimensional culture models that poorly replicate in vivo functionality. Therefore, we aimed to develop and characterize a three-dimensional hepatic construct for the study of human PUFA metabolism. Primary human hepatocytes cultured in 3D hydrogels were characterized for their capacity to represent basic lipid processing functions, including lipid esterification, de novo lipogenesis, and cholesterol efflux. They were then exposed to control and LA-enriched media and reproducibly displayed allele-specific metabolic activity of FADS1, based on genotype at rs174537. Hepatocytes derived from individuals homozygous with the minor allele at rs174537 (i.e., TT) displayed the slowest metabolic conversion of LA to ARA and significantly reduced FADS1 and FADS2 expression. These results support the feasibility of using 3D human hepatic cultures for the study of human PUFA and lipid metabolism and relevant gene-diet interactions, thereby enabling future nutrition targets in humans., Competing Interests: All authors report no competing interest or financial conflicts of interest related to this work.

Current in vitro three-dimensional (3D) models of liver tissue have been limited by the inability to study the effects of specific extracellular matrix (ECM) components on cell phenotypes. This is in part due to limitations in the availability of chemical modifications appropriate for this purpose. For example, hyaluronic acid (HA), which is a natural ECM component within the liver, lacks key ECM motifs (e.g. arginine-glycine-aspartic acid (RGD) peptides) that support cell adhesion. However, the addition of maleimide (Mal) groups to HA could facilitate the conjugation of ECM biomimetic peptides with thiol-containing end groups. In this study, we characterized a new crosslinkable hydrogel (i.e. HA-Mal) that yielded a simplified ECM-mimicking microenvironment supportive of 3D liver cell culture. We then performed a series of experiments to assess the impact of physical and biochemical signaling in the form of RGD peptide incorporation and transforming growth factor ß (TGF- ß ) supplementation, respectively, on hepatic functionality. Hepatic stellate cells (i.e. LX-2) exhibited increased cell-matrix interactions in the form of cell spreading and elongation within HA-Mal matrices containing RGD peptides, enabling physical adhesions, whereas hepatocyte-like cells (HepG2) had reduced albumin and urea production. We further exposed the encapsulated cells to soluble TGF- ß to elicit a fibrosis-like state. In the presence of TGF- ß biochemical signals, LX-2 cells became activated and HepG2 functionality significantly decreased in both RGD-containing and RGD-free hydrogels. Altogether, in this study we have developed a hydrogel biomaterial platform that allows for discrete manipulation of specific ECM motifs within the hydrogel to better understand the roles of cell-matrix interactions on cell phenotype and overall liver functionality., (© 2022 IOP Publishing Ltd.)

Background: TNF-α-stimulated gene 6 (TSG-6) protein, a TNF-α-responsive hyaladherin, possesses enzymatic activity that can catalyze covalent crosslinks of the polysaccharide hyaluronic acid (HA) to another protein to form heavy chain-hyaluronic acid (HC-HA) complexes in pathological conditions such as osteoarthritis (OA). Here, we examined HA synthase and inflammatory gene expression; synovial fluid HA, TNF-α, and viscosity; and TSG-6-mediated HC-HA complex formation in an equine OA model. The objectives of this study were to (1) evaluate the TNF-α-TSG-6-HC-HA signaling pathway across multiple joint tissues, including synovial membrane, cartilage, and synovial fluid, and (2) determine the impact of OA on synovial fluid composition and biophysical properties., Methods: HA and inflammatory cytokine concentrations (TNF-α, IL-1β, CCL2, 3, 5, and 11) were analyzed in synovial fluid from 63 OA and 25 control joints, and HA synthase (HAS1-3), TSG-6, and hyaluronan-degrading enzyme (HYAL2, HEXA) gene expression was measured in synovial membrane and cartilage. HA molecular weight (MW) distributions were determined using agarose gel electrophoresis and solid-state nanopore measurements, and HC-HA complex formation was detected via immunoblotting and immunofluorescence. SEC-MALS was used to evaluate TSG-6-mediated HA crosslinking, and synovial fluid and HA solution viscosities were analyzed using multiple particle-tracking microrheology and microfluidic measurements, respectively., Results: TNF-α concentrations were greater in OA synovial fluid, and TSG6 expression was upregulated in OA synovial membrane and cartilage. TSG-6-mediated HC-HA complex formation was greater in OA synovial fluid and tissues than controls, and HC-HA was localized to both synovial membrane and superficial zone chondrocytes in OA joints. SEC-MALS demonstrated macromolecular aggregation of low MW HA in the presence of TSG-6 and inter-α-inhibitor with concurrent increases in viscosity., Conclusions: Synovial fluid TNF-α concentrations, synovial membrane and cartilage TSG6 gene expression, and HC-HA complex formation were increased in equine OA. Despite the ability of TSG-6 to induce macromolecular aggregation of low MW HA with resultant increases in the viscosity of low MW HA solutions in vitro, HA concentration was the primary determinant of synovial fluid viscosity rather than HA MW or HC-HA crosslinking. The TNF-α-TSG-6-HC-HA pathway may represent a potential therapeutic target in OA., (© 2021. The Author(s).)

Background: Nutrition in the intensive care unit is vital for patient care; however, immunomodulatory diets rich in PUFAs like γ-linolenic acid (GLA), EPA, and DHA remain controversial for patients with acute respiratory distress syndrome. We postulate that genetic variants impacting PUFA metabolism contribute to mixed responses to PUFA-rich diets., Objectives: In this study, we aimed to test the effects of single nucleotide polymorphism (SNP) rs174537 on differential responses to PUFA-rich diets., Methods: We performed a secondary analysis of the OMEGA trial (NCT00609180) where 129 subjects received placebo control diets and 143 received omega-oil. DNA was extracted from buffy coats and used to genotype rs174537; plasma was used to quantitate PUFAs. We tested for SNP-diet interactions on PUFA concentrations, inflammatory biomarkers, and patient outcomes., Results: We observed that all individuals receiving omega-oil displayed significantly higher concentrations of GLA, EPA, and DHA (all P  < 0.0001), but they did not vary by genotype at rs174537. Statistically significant SNP-diet interactions were observed on circulating DHA concentrations in African Americans. Specifically, African American T-allele carriers on placebo illustrated elevated DHA concentrations. Additionally, all individuals receiving omega-oil had higher concentrations of EPA-derived urinary F3-isoprostane (Caucasians: P  = 0.0011; African Americans: P  = 0.0002). Despite these findings, we did not detect any significant SNP-diet interactions on pulmonary functional metrics, clinical outcomes, and mortality., Conclusions: This study highlights the importance of genetic and racial contributions to PUFA metabolism and inflammation. In particular, rs174537 had a significant impact on circulating DHA and urinary isoprostane concentrations. Given our relatively small sample size, further investigations in larger multiethnic cohorts are needed to evaluate the impact of rs174537 on fatty acid metabolism and downstream inflammation., (© The Author(s) 2020. Published by Oxford University Press on behalf of American Society for Nutrition.)

Traumatic brain injury (TBI) is a leading cause of death and disability in persons under age 45. The hallmark secondary injury profile after TBI involves dynamic interactions between inflammatory and metabolic pathways including fatty acids. Omega-3 polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) have been shown to provide neuroprotective benefits by minimizing neuroinflammation in rodents. These effects have been less conclusive in humans, however. We postulate genetic variants influencing PUFA metabolism in humans could contribute to these disparate findings. Therefore, we sought to (1) characterize the circulating PUFA response and (2) evaluate the impact of rs174537 on inflammation after TBI. A prospective, single-center, observational pilot study was conducted to collect blood samples from Level-1 trauma patients (N = 130) on admission and 24 h post-admission. Plasma was used to quantify PUFA levels and inflammatory cytokines. Deoxyribonucleic acid was extracted and genotyped at rs174537. Associations between PUFAs and inflammatory cytokines were analyzed for all trauma cases and stratified by race (Caucasians only), TBI (TBI: N = 47; non-TBI = 83) and rs174537 genotype (GG: N = 33, GT/TT: N = 44). Patients with TBI had higher plasma DHA levels compared with non-TBI at 24 h post-injury ( p  = 0.013). The SNP rs174537 was associated with both PUFA levels and inflammatory cytokines ( p  < 0.05). Specifically, TBI patients with GG genotype exhibited the highest plasma levels of DHA (1.33%) and interleukin-8 (121.5 ± 43.3 pg/mL), which were in turn associated with poorer outcomes. These data illustrate the impact of rs174537 on the post-TBI response. Further work is needed to ascertain how this genetic variant directly influences inflammation after trauma.

Tailoring interfaces with polymer brushes is a commonly used strategy to create functional materials for numerous applications. Existing methods are limited in brush thickness, the ability to generate high-density brushes of biopolymers, and the potential for regeneration. Here we introduce a scheme to synthesize ultra-thick regenerating hyaluronan polymer brushes using hyaluronan synthase. The platform provides a dynamic interface with tunable brush heights that extend up to 20 microns - two orders of magnitude thicker than standard brushes. The brushes are easily sculpted into micropatterned landscapes by photo-deactivation of the enzyme. Further, they provide a continuous source of megadalton hyaluronan or they can be covalently-stabilized to the surface. Stabilized brushes exhibit superb resistance to biofilms, yet are locally digested by fibroblasts. This brush technology provides opportunities in a range of arenas including regenerating tailorable biointerfaces for implants, wound healing or lubrication as well as fundamental studies of the glycocalyx and polymer physics.

Background: Clomiphene citrate (CC) is one of the widely used drugs as an ovulation stimulant, but its adverse effects on the endometrium results in lowering down the pregnancy rate. Endometrium CD98 is also important in the process of implantation., Objective: To evaluate the immunohistochemistry expression levels of endometrial CD98 following injection of CC with and without Human chorionic gonadotropin (HCG) in ovariectomized and non-ovariectomized rats., Materials and Methods: Seventy two (12-14 wk old) female Wistar rats were randomly divided into two groups (n = 36): (a) ovariectomized and (b) non-ovariectomized. Each group was further divided into six subgroups (n = 6/each): (1) CC 10 mg/kg, (2) CC 20 mg/kg, (3) HCG, (4) CC 10 mg/kg with HCG, (5) CC 20 mg/kg with HCG, and (6) control. The experimental subgroups received a single dose of CC (daily, five days) and HCG (after the last injection of CC) alone or in combination. Immunohistochemistry staining was performed on paraffin-embedded endometrial tissues to evaluate the expression levels of CD98., Results: Animals undergoing ovariectomy presented a significantly lower expression level of endometrial CD98 (p < 0.001) when compared with non-ovariectomized in the same condition that groups were subdivided. There was also a dose-dependent reduction (p < 0.001) in the expression of CD98 in non-ovariectomized subgroups when compared with control group. In addition, injection of HCG following treatment with CC improved its expression., Conclusion: It was concluded that CC impairs CD98 expression in endometrium and this impairment is intensified with the removal of the ovary. Also, an injection of HCG following treatment with CC can slightly improve the expression of CD98., Competing Interests: The authors declare that they have no conflict of interests.

Red blood cell (RBC) hemolysis is one of the most common storage lesions in packed RBCs (pRBC). Older units of pRBCs, especially those > 21 days old, have increasing levels of hemolysis leading to increased oxidative stress and premature platelet activation. This effect can mostly be attributed to the increase of cell-free hemoglobin (Hb). Therefore, removal of cell-free Hb from pRBCs prior to transfusion could mitigate these deleterious effects. We propose a new method for the removal of Hb from pRBCs using zinc beads. Prepared Hb solutions and pRBCs were treated with zinc beads using two different protocols. UV-Vis spectrophotometry was used to determine Hb concentrations, before and after treatment. Experiments were run in triplicate and paired t tests were used to determine significant differences between groups. Zinc beads removed on average 94% of cell-free Hb within 15 min and 78% Hb from pRBCs (p < 0.0001), demonstrating a maximum binding capacity ~ 66.2 ± 0.7 mg Hb/mL beads. No differences in RBC morphology or deformability were observed after treatment. This study demonstrates the feasibility of using zinc beads for the rapid and targeted removal of Hb from pRBC units. Further investigation is needed to scale this method for large volume removal.

Background: Nitrite is reduced by heme-proteins and molybdenum-containing enzymes to form the important signaling molecule nitric oxide (NO), mediating NO signaling. Substantial evidence suggests that deoxygenated hemoglobin within red blood cells (RBCs) is the main erythrocytic protein responsible for mediating nitrite-dependent NO signaling. In other work, infrared and far red light have been shown to have therapeutic potential that some attribute to production of NO. Here we explore whether a combination of nitrite and far red light treatment has an additive effect in NO-dependent processes, and whether this effect is mediated by RBCs., Methods and Results: Using photoacoustic imaging in a rat model as a function of varying inspired oxygen, we found that far red light (660 nm, five min. exposure) and nitrite feeding (three weeks in drinking water at 100 mg/L) each separately increased tissue oxygenation and vessel diameter, and the combined treatment was additive. We also employed inhibition of human platelet activation measured by flow cytometry to assess RBC-dependent nitrite bioactivation and found that far red light dramatically potentiates platelet inhibition by nitrite. Blocking RBC-surface thiols abrogated these effects of nitrite and far-red light. RBC-dependent production of NO was also shown to be enhanced by far red light using a chemiluminescence-based nitric oxide analyzer. In addition, RBC-dependent bioactivation of nitrite led to prolonged lag times for clotting in platelet poor plasma that was enhanced by exposure to far red light., Conclusions: Our results suggest that nitrite leads to the formation of a photolabile RBC surface thiol-bound species such as an S-nitrosothiol or heme-nitrosyl (NO-bound heme) for which far red light enhances NO signaling. These findings expand our understanding of RBC-mediated NO production from nitrite. This pathway of NO production may have therapeutic potential in several applications including thrombosis, and, thus, warrants further study., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

Objectives: To investigate which surgical technique orthodontists prefer for exposing palatally impacted canines (PICs), and to compare closed exposure and glass-ionomer open exposure (GOPEX) with regard to pre- and post-surgical orthodontic variables., Materials and Methods: A questionnaire with 19 questions and three cases visualising superficial, deep, or medial PICs was sent to 48 orthodontists working in a Swedish county. Sixty case records for patients with unilateral PICs from two centres were analysed; 30 patients having GOPEX (Centre A), and 30 undergoing closed exposure (Centre B). Pre- and post-surgical orthodontic variables were collected from the dental records., Results: The response rate was 81 per cent. There was an equal distribution of preference between open and closed exposure. Glass-ionomer cement (GIC) was predominately used as surgical packing in open exposure. No active traction was initiated until the canine erupted spontaneously. In the closed exposure cases, traction started shortly after exposure. The clinicians mentioned similar advantages of choosing one technique over the other and the main basis for the decision was the clinician's preference and not the location of the canine. There were no differences regarding post-exposure complications between the techniques. The overall treatment time was the same but there were fewer appointments and significantly shorter active treatment time with traction of the PIC in the GOPEX group., Limitations: Despite the homogeneity of the baseline patient characteristics, pre- and post-surgical orthodontic variables were analysed retrospectively, therefore, it is difficult to assess what impact these confounding factors may have had on the treatment time., Conclusions: The choice of exposure technique depends on the clinician's preferences. The active treatment time is shorter and the number of appointments fewer with open exposure when GIC is used as surgical packing.

Background: To address deficiencies associated with the classic definition of massive transfusion (MT), critical administration threshold (CAT) and resuscitation intensity (RI) were developed to better quantify the overall severity of illness and predict the need for transfusions and early mortality. We sought to evaluate these as more appropriate replacements for MT in defining mortality risk in patients undergoing major transfusions., Methods: Patients predicted to receive MT at 12 Level I trauma centers were randomized in the Pragmatic, Randomized Optimal Platelet and Plasma Ratios (PROPPR) trial. MT of 10 U or greater red blood cell (RBC) in 24 hours; CAT+, 3 U or greater RBC in the first hour; and RI, total products in the first 30 minutes (1 U RBC, 1 U plasma, 1000 mL crystalloid, 500 mL colloid each valued at 1 U). Resuscitation intensity was evaluated as a continuous variable and dichotomized as RI4+, where RI is 4 U or greater. Each metric was evaluated for its ability to predict mortality at 3 hours, 6 hours, and 24 hours, and at 30 days., Results: Of the 680 patients, 301 patients met MT definition, 521 were CAT+, and 445 were RI4+. Of those that died, 23% never reached MT threshold, but all were captured by CAT+ and RI4+. The 3-hour (9% vs. 9%), 6-hour (14% vs. 14%), 24-hour (17% vs. 18%), and 30-day mortality rates (28% vs. 29%) were similar between CAT+ and RI4+ patients. When RI was evaluated as a continuous variable, each unit increase was associated with a 20% increase in hemorrhage-related mortality (odds ratio, 1.20; 95% confidence interval, 1.15-1.29; p < 0.05)., Conclusion: Both RI and CAT are valid surrogates for early mortality in patients undergoing major transfusion, capturing patients omitted by the MT definition. The CAT+ showed the best sensitivity; RI4+ demonstrated better specificity and good positive predictive values and negative predictive values. While CAT+ may be suited for patients receiving an RBC-dominant resuscitation, RI4+ is more comprehensive. RI can also be used as a continuous variable to provide quantitative as well as qualitative risk of death., Level of Evidence: Prognostic, level III.

Background: Genetic variants within the fatty acid desaturase ( FADS ) gene cluster (human Chr11) are important regulators of long-chain (LC) polyunsaturated fatty acid (PUFA) biosynthesis in the liver and consequently have been associated with circulating LC-PUFA levels. More recently, epigenetic modifications such as DNA methylation, particularly within the FADS cluster, have been shown to affect LC-PUFA levels. Our lab previously demonstrated strong associations of allele-specific methylation (ASM) between a single nucleotide polymorphism (SNP) rs174537 and CpG sites across the FADS region in human liver tissues. Given that epigenetic signatures are tissue-specific, we aimed to evaluate the methylation status and ASM associations between rs174537 and DNA methylation obtained from human saliva, CD4+ cells and total leukocytes derived from whole blood. The goals were to (1) determine if DNA methylation from these peripheral samples would display similar ASM trends as previously observed in human liver tissues and (2) evaluate the associations between DNA methylation and circulating LC-PUFAs., Results: DNA methylation at six CpG sites spanning FADS1 and FADS2 promoter regions and a putative FADS enhancer region were determined in two Caucasian cohorts of healthy volunteers: leukocytes in cohort 1 ( n  = 89, median age = 43, 35% male) and saliva and CD4+ cells in cohort 2 ( n  = 32, median age = 41, 41% male). Significant ASM between rs174537 and DNA methylation at three CpG sites located in the FADS2 promoter region (i.e., chr11:61594865, chr11:61594876, chr11:61594907) and one CpG site in the putative enhancer region (chr11:61587979) were observed with leukocytes. In CD4+ cells, significant ASM was observed at CpG sites chr11:61594876 and chr11:61584894. Genotype at rs174537 was significantly associated with DNA methylation from leukocytes. Similar trends were observed with CD4+ cells, but not with saliva. DNA methylation from leukocytes and CD4+ cells also significantly correlated with circulating omega-6 LC-PUFAs., Conclusions: We observed significant ASM between rs174537 and DNA methylation at key regulatory regions in the FADS region from leukocyte and CD4+ cells. DNA methylation from leukocytes also correlated with circulating omega-6 LC-PUFAs. These results support the use of peripheral whole blood samples, with leukocytes showing the most promise for future nutrigenomic studies evaluating epigenetic modifications affecting LC-PUFA biosynthesis in humans., Competing Interests: Healthy adult subjects (21–65 years old) were recruited by mechanisms approved by the Wake Forest School of Medicine Institutional Review Board (IRB#s 00016822 and 00006156). All participants provided written and informed consent for their respective study, as well as for future research use of their archived biospecimens.Not applicableThe authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Hyaluronan (or hyaluronic acid, HA) is a ubiquitous molecule that plays critical roles in numerous physiological functions in vivo, including tissue hydration, inflammation, and joint lubrication. Both the abundance and size distribution of HA in biological fluids are recognized as robust indicators of various pathologies and disease progressions. However, such analyses remain challenging because conventional methods are not sufficiently sensitive, have limited dynamic range, and/or are only semi-quantitative. Here we demonstrate label-free detection and molecular weight discrimination of HA with a solid-state nanopore sensor. We first employ synthetic HA polymers to validate the measurement approach and then use the platform to determine the size distribution of as little as 10 ng of HA extracted directly from synovial fluid in an equine model of osteoarthritis. Our results establish a quantitative method for assessment of a significant molecular biomarker that bridges a gap in the current state of the art.

Genetic variants near and within the fatty acid desaturase (FADS) cluster are associated with polyunsaturated fatty acid (PUFA) biosynthesis, levels of several disease biomarkers and risk of human disease. However, determining the functional mechanisms by which these genetic variants impact PUFA levels remains a challenge. Utilizing an Illumina 450K array, we previously reported strong allele-specific methylation (ASM) associations (p = 2.69×10-29) between a single nucleotide polymorphism (SNP) rs174537 and DNA methylation of CpG sites located in the putative enhancer region between FADS1 and FADS2, in human liver tissue. However, this array only featured 20 CpG sites within this 12kb region. To better understand the methylation landscape within this region, we conducted bisulfite sequencing of the region between FADS1 and FADS2. Liver tissues from 50 male subjects (27 European Americans, 23 African Americans) were obtained from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study, and used to ascertain the genotype at rs174537 and methylation status across the region of interest. Associations between rs174537 genotype and methylation status of 136 CpG sites were determined. Age-adjusted linear regressions were used to assess ASM associations with rs174537 genotype. The majority of CpG sites (117 out of 136, 86%) exhibited high levels of methylation with the greatest variability observed at three key regulatory regions-the promoter regions for FADS1 and FADS2 and a putative enhancer site between the two genes. Eight CpG sites within the putative enhancer region displayed significant (FDR p <0.05) ASM associations with rs174537. These data support the concept that both genetic and epigenetic factors regulate PUFA biosynthesis, and raise fundamental questions as to how genetic variants such as rs174537 impact DNA methylation in distant regulatory regions, and ultimately the capacity of tissues to synthesize PUFAs.

Sickle cell disease is caused by a mutant form of hemoglobin that polymerizes under hypoxic conditions, increasing rigidity, fragility, calcium influx-mediated dehydration, and adhesivity of red blood cells. Increased red cell fragility results in hemolysis, which reduces nitric oxide (NO) bioavailability, and induces platelet activation and inflammation leading to adhesion of circulating blood cells. Nitric Oxide inhibits adhesion and platelet activation. Nitrite has emerged as an attractive therapeutic agent that targets delivery of NO activity to areas of hypoxia through bioactivation by deoxygenated red blood cell hemoglobin. In this study, we demonstrate anti-platelet activity of nitrite at doses achievable through dietary interventions with comparison to similar doses with other NO donating agents. Unlike other NO donating agents, nitrite activity is shown to be potentiated in the presence of red blood cells in hypoxic conditions. We also show that nitrite reduces calcium associated loss of phospholipid asymmetry that is associated with increased red cell adhesion, and that red cell deformability is also improved. We show that nitrite inhibits red cell adhesion in a microfluidic flow-channel assay after endothelial cell activation. In further investigations, we show that leukocyte and platelet adhesion is blunted in nitrite-fed wild type mice compared to control after either lipopolysaccharide- or hemolysis-induced inflammation. Moreover, we demonstrate that nitrite treatment results in a reduction in adhesion of circulating blood cells and reduced red blood cell hemolysis in humanized transgenic sickle cell mice subjected to local hypoxia. These data suggest that nitrite is an effective anti-platelet and anti-adhesion agent that is activated by red blood cells, with enhanced potency under physiological hypoxia and in venous blood that may be useful therapeutically., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

In comparative effectiveness studies of multicomponent, sequential interventions like blood product transfusion (plasma, platelets, red blood cells) for trauma and critical care patients, the timing and dynamics of treatment relative to the fragility of a patient's condition is often overlooked and underappreciated. While many hospitals have established massive transfusion protocols to ensure that physiologically optimal combinations of blood products are rapidly available, the period of time required to achieve a specified massive transfusion standard (e.g. a 1:1 or 1:2 ratio of plasma or platelets:red blood cells) has been ignored. To account for the time-varying characteristics of transfusions, we use semiparametric rate models for multivariate recurrent events to estimate blood product ratios. We use latent variables to account for multiple sources of informative censoring (early surgical or endovascular hemorrhage control procedures or death). The major advantage is that the distributions of latent variables and the dependence structure between the multivariate recurrent events and informative censoring need not be specified. Thus, our approach is robust to complex model assumptions. We establish asymptotic properties and evaluate finite sample performance through simulations, and apply the method to data from the PRospective Observational Multicenter Major Trauma Transfusion study.

Background: The early use of blood products has been associated with improved patient outcomes following severe hemorrhage or traumatic injury. We aimed to investigate the influence of pre-hospital blood products (i.e. plasma and/or RBCs) on admission hemostatic properties and patient outcomes. We hypothesized that pre-hospital plasma would improve hemostatic function as evaluated by rapid thrombelastography (rTEG)., Methods: We conducted a prospective observational study recruiting 257 trauma patients admitted to a Level I trauma center having received either blood products pre-hospital or in-hospital within 6 hours of admission. Clinical data on patient demographics, blood biochemistry, injury severity score and mortality were collected. Admission rTEG was conducted to characterize the coagulation profile and hemostatic function., Results: 75 patients received pre-hospital plasma and/or RBCs (PH group; nearly half received both RBCs and plasma) whereas 182 patients only received in-hospital blood products (RBCs, Plasma and Platelets) within 6 hours of admission (IH group). PH patients had lower Glasgow coma scale (GCS) scores, more penetrating injuries, lower systolic blood pressures, lower hemoglobin levels, lower platelet counts and greater acidosis upon ED admission than the IH group (all p < 0.05). Despite differences in type of injury and admission vitals indicating that the PH group had more signs of bleeding than the IH group, there were no significant differences in in-hospital mortality (PH 26.7% vs. IH 20.9% p = 0.31). When comparing rTEG variables between PH patients transfused with 0, 1 or 2 units of plasma, more pre-hospital plasma transfusion was tendency towards improved rTEG variables. When adjusting for pre-hospital RBC, pre-hospital plasma was associated with significantly higher rTEG MA (p = 0.012) at hospital admission., Discussion: After adjusting for pre-hospital RBCs, pre-hospital plasma transfusion was independently associated with increased rTEG MA, as well as arrival indices of shock and hemodynamic instability. Besides more severe injury and worse clinical presentation, the group that received pre-hospital transfusion had early and late mortality similar to patients not transfused pre-hospital., Conclusions: These data suggest that early administration of plasma can provide significant hemostatic and potential survival benefit to severely hemorrhaging trauma patients.

Background: In vitro and experimental animal studies have demonstrated that high levels of omega-6 (n-6) polyunsaturated fatty acids (PUFAs) and high ratios of n-6 to omega-3 (n-3) PUFAs are strongly associated with the development and progression of prostate cancer (PCA). However, epidemiological studies in humans have demonstrated inconsistent findings linking dietary PUFAs and PCA risk. We hypothesize that genetic and epigenetic variations within the fatty acid desaturase (FADS) gene cluster produce gene-diet interactions that may explain these disparate findings. This study tested the relationship of the genotype of a single nucleotide polymorphism, rs174537, and the methylation status of a CpG site, cg27386326, with PUFA composition, and markers of PUFA biosynthesis in PCA tissue., Methods: Sixty PCA specimens from patients undergoing radical prostatectomy were genotyped, pyrosequenced and quantitated for fatty acids (FAs)., Results: Long-chain (LC)-PUFAs, such as arachidonic acid (ARA), were abundant in these specimens, with ARA accounting for 15.8% of total FAs. In addition, there was a positive association of the G allele at rs174537 with concentrations of ARA and adrenic acid and ratios of products to precursors within the n-6 PUFA pathway such that specimens from homozygous G individuals exhibited increasingly higher values as compared to specimens from heterozygous individuals and homozygous T individuals. Finally, the methylation status of cg27386326 was inversely correlated with tissue concentrations of LC-PUFAs and markers of LC-PUFA biosynthesis., Conclusions: These data reveal that genetic and epigenetic variations within the FADS cluster are highly associated with LC-PUFA concentrations and LC-PUFA biosynthetic capacity in PCA tissue. They also raise the potential that gene-PUFA interactions play an important role in PCA risk and severity. Prostate 76:1182-1191, 2016. © 2016 The Authors. The Prostate published by Wiley Periodicals, Inc., (© 2016 The Authors. The Prostate published by Wiley Periodicals, Inc.)

Objective: Transfusion research seeks to improve survival for severely injured and hemorrhaging patients using optimal plasma and platelet ratios over red blood cells (RBCs). However, most published studies comparing different ratios are plagued with serious bias and ignore time-varying effects. We applied joint recurrent event frailty models to increase validity and clinical utility., Study Design and Setting: Using the PRospective Observational Multicenter Major Trauma Transfusion study data, our joint random-effects models estimated the association of (1) clinical covariates with transfusion rate intensities and (2) varying plasma:RBC and platelet:RBC ratios with survival over the 24 hours after hospital admission. Along with survival time, baseline patient vital signs, laboratory values, and longitudinal data on types and volumes of transfusions were included., Results: Baseline systolic blood pressure, heart rate, pH, and hemoglobin were significantly associated with RBC transfusion rates. Increased transfusion rates (per hour) of plasma (P = 0.05), platelets (P < 0.001), or RBCs were associated with increased 24-hour mortality. Higher ratios of plasma:RBC (P = 0.107) and platelet:RBC (P < 0.001) were associated with reduced mortality in a time-varying pattern (P < 0.001)., Conclusions: The proposed joint analysis of transfusion rates and ratios offers a more valid statistical approach to evaluate survival effects in the presence of informative censoring by early death., Competing Interests: Conflict of Interest/Financial Disclosure We have no issues to report with respect to confict of interest or financial disclosure., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Gamma-linolenic acid (GLA, 18:3n-6) is an omega-6 (n-6), 18 carbon (18C-) polyunsaturated fatty acid (PUFA) found in human milk and several botanical seed oils and is typically consumed as part of a dietary supplement. While there have been numerous in vitro and in vivo animal models which illustrate that GLA-supplemented diets attenuate inflammatory responses, clinical studies utilizing GLA or GLA in combination with omega-3 (n-3) PUFAs have been much less conclusive. A central premise of this review is that there are critical metabolic and genetic factors that affect the conversion of GLA to dihommo-gamma linolenic acid (DGLA, 20:3n-6) and arachidonic acid (AA, 20:4n-6), which consequently affects the balance of DGLA- and AA- derived metabolites. As a result, these factors impact the clinical effectiveness of GLA or GLA/(n-3) PUFA supplementations in treating inflammatory conditions. Specifically, these factors include: 1) the capacity for different human cells and tissues to convert GLA to DGLA and AA and to metabolize DGLA and AA to bioactive metabolites; 2) the opposing effects of DGLA and AA metabolites on inflammatory processes and diseases; and 3) the impact of genetic variations within the fatty acid desaturase (FADS) gene cluster, in particular, on AA/DGLA ratios and bioactive metabolites. We postulate that these factors influence the heterogeneity of results observed in GLA supplement-based clinical trials and suggest that "one-size fits all" approaches utilizing PUFA-based supplements may no longer be appropriate for the prevention and treatment of complex human diseases., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Thrombelastography (TEG) maximal amplitude (mA) has also been shown to reflect hypercoagulability and increased venous thromboembolism (VTE) risk in adult trauma patients. Based on these previous works, we sought to identify when children become adults with respect to TEG mA values and whether this correlated with VTE risk., Methods: We evaluated all trauma patients admitted from January 2010 to December 2013 who were highest-level activations. Age was evaluated as a continuous variable, followed by a categorical evaluation. TEG mA values were evaluated as continuous and dichotomous (hypercoagulable, mA ≥ 65 mm). Logistic regression was then constructed controlling for age categories, sex, and injury severity to assess the association with TEG mA values and VTE risk., Results: A total of 7,194 Level 1 trauma patients were admitted during this time frame (819 were <18 years of age). The likelihood of mA equal to or greater than 65 mm remained at 35% to 37% through age 30 years with significant increases observed at ages 31 years to 35 years (45%) and 46 years to 50 years (49%), both p < 0.01. When controlling for injury severity, race, and sex, logistic regression demonstrated that every 5-year increase in age (after age 30 years) was associated with a 16% increased likelihood of hypercoagulability at admission. Beginning with age 1 year, VTE risk remained at 1.5% or less until age 13 years where it increased to 2.3%, increasing again at age 15 years to 5.1%. Two additional significant increases were identified between ages 31 years and 35 years (5.5%) as well as 46 years and 50 years (7.6%), both p < 0.001. Logistic regression demonstrated a 3.4-fold increased risk for VTE among those aged 31 years to 50 years compared with those who are younger than 30 years. The same model noted a 2.3-fold increased risk compared with those who are older than 50 years., Conclusion: Beginning at age 13 years, children transition toward adult hypercoagulability, as evidenced by elevated TEG mA values and VTE risk. However, the greatest VTE risk (and highest likelihood of hypercoagulable mA) is among those adults 31 years to 50 years of age., Level of Evidence: Prognostic and epidemiologic study, level III.

Deficiencies in omega-3 (n-3) long chain polyunsaturated fatty acids (LC-PUFAs) and increases in the ratio of omega-6 (n-6) to n-3 LC-PUFAs in brain tissues and blood components have been associated with psychiatric and developmental disorders. Most studies have focused on n-3 LC-PUFA accumulation in the brain from birth until 2years of age, well before the symptomatic onset of such disorders. The current study addresses changes that occur in childhood and adolescence. Postmortem brain (cortical gray matter, inferior temporal lobe; n=50) and liver (n=60) from vervet monkeys fed a uniform diet from birth through young adulthood were collected from archived tissues. Lipids were extracted and fatty acid levels determined. There was a marked reduction in the ratio of n-6 LC-PUFAs, arachidonic acid (ARA) and adrenic acid (ADR), relative to the n-3 LC-PUFA, docosahexaenoic acid (DHA), in temporal cortex lipids from birth to puberty and then a more gradual decrease though adulthood. This decrease in ratio resulted from a 3-fold accumulation of DHA levels while concentrations of ARA remained constant. Early childhood through adolescence appears to be a critical period for DHA accretion in the cortex of vervet monkeys and may represent a vulnerable stage where lack of dietary n-3 LC-PUFAs impacts development in humans., (Copyright © 2015 Elsevier Inc. All rights reserved.)

There is no clear classification rule to rapidly identify trauma patients who are severely hemorrhaging and may need substantial blood transfusions. Massive transfusion (MT), defined as the transfusion of at least 10 units of red blood cells within 24 h of hospital admission, has served as a conventional surrogate that has been used to develop early predictive algorithms and establish criteria for ordering an MT protocol from the blood bank. However, the conventional MT rule is a poor proxy, because it is likely to misclassify many severely hemorrhaging trauma patients as they could die before receiving the 10th red blood cells transfusion. In this article, we propose to use a latent class model to obtain a more accurate and complete metric in the presence of early death. Our new approach incorporates baseline patient information from the time of hospital admission, by combining respective models for survival time and usage of blood products transfused within the framework of latent class analysis. To account for statistical challenges, caused by induced dependent censoring inherent in 24-h sums of transfusions, we propose to estimate an improved standard via a pseudo-likelihood function using an expectation-maximization algorithm with the inverse weighting principle. We evaluated the performance of our new standard in simulation studies and compared with the conventional MT definition using actual patient data from the Prospective Observational Multicenter Major Trauma Transfusion study. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Objective: The current study leveraged data from the Early Whole Blood (EWB) trial to explore the effects of modified whole blood (mWB) versus component (COMP) transfusions on coagulation parameters over time using longitudinal statistical methods., Study Design and Methods: The EWB study was a single-center randomized controlled trial, approved by the local IRB. Adult patients at highest-level trauma activations were randomized into mWB or COMP groups. Coagulation status was evaluated (at times 0, 3, 6, 12, and 24 h postadmission) using thrombelastography, platelet aggregometry, and calibrated automated thrombograms. Longitudinal statistical analyses with generalized estimating equations (GEE) were used to evaluate the effects of group, time, transfusion types, and their respective interactions on changes in measured coagulation markers., Results: A total of 59 patients were enrolled and adhered to protocol in the EWB trial, 25 in the mWB group, and 34 in the COMP group. Patients in both the mWB and COMP groups demonstrated a significant decline in their thrombelastography parameters during the first 3-6 h, specifically K-time, α-angle, maximum amplitude, G, and LY30. Patients receiving mWB exhibited improved thrombin potential than those receiving COMP. Platelet count and function declined over time in both mWB and COMP groups; however, platelet aggregation in response to ristocetin in the mWB group was significantly improved at 12 h compared with the COMP group. The longitudinal GEE model revealed significant group-time interactive effects on the changes in coagulation markers and significant effect of platelet transfusions on improvements in coagulation profile., Conclusions: We observed significant interactive group-time effects, indicating that the types of transfusion as well as the time of transfusion significantly affect the patient's coagulation status. Our pilot data suggest that there is an improvement in platelet function with mWB, but further studies are needed. Regardless, platelet transfusions were associated with improvements in coagulation over time in both the groups.

Background: In trauma research, "massive transfusion" (MT), historically defined as receiving ≥10 units of red blood cells (RBCs) within 24 h of admission, has been routinely used as a "gold standard" for quantifying bleeding severity. Due to early in-hospital mortality, however, MT is subject to survivor bias and thus a poorly defined criterion to classify bleeding trauma patients., Methods: Using the data from a retrospective trauma transfusion study, we applied a latent-class (LC) mixture model to identify severely hemorrhaging (SH) patients. Based on the joint distribution of cumulative units of RBCs and binary survival outcome at 24 h of admission, we applied an expectation-maximization (EM) algorithm to obtain model parameters. Estimated posterior probabilities were used for patients' classification and compared with the MT rule. To evaluate predictive performance of the LC-based classification, we examined the role of six clinical variables as predictors using two separate logistic regression models., Results: Out of 471 trauma patients, 211 (45 %) were MT, while our latent SH classifier identified only 127 (27 %) of patients as SH. The agreement between the two classification methods was 73 %. A non-ignorable portion of patients (17 out of 68, 25 %) who died within 24 h were not classified as MT but the SH group included 62 patients (91 %) who died during the same period. Our comparison of the predictive models based on MT and SH revealed significant differences between the coefficients of potential predictors of patients who may be in need of activation of the massive transfusion protocol., Conclusions: The traditional MT classification does not adequately reflect transfusion practices and outcomes during the trauma reception and initial resuscitation phase. Although we have demonstrated that joint latent class modeling could be used to correct for potential bias caused by misclassification of severely bleeding patients, improvement in this approach could be made in the presence of time to event data from prospective studies.

Background: Previous investigators have demonstrated that postinjury thrombocytosis is associated with an increase in thromboembolic (TE) risk. Increased rates of thrombocytosis have been found specifically in patients after splenectomy for trauma. We hypothesized that patients undergoing splenectomy (1) would demonstrate a more hypercoagulable profile during their hospital stay and (2) that this hypercoagulable state would be associated with increased TE events., Methods: This was a 14-month, prospective, observational trial evaluating serial rapid thrombelastography (rTEG) at 3 American College of Surgeons-verified, level 1 trauma centers. Inclusion criteria were highest-level trauma activation and arrival within 6 hours of injury. Exclusion criteria were <18 years of age, incarcerated, and burns>20% total body surface area. Serial rTEG (activated clotting time, k-time, α-angle, MA, lysis) and traditional coagulation testing (prothrombin time, partial thromboplastin time, fibrinogen and platelet count) were obtained at admission and then at 3, 6, 12, 24, 48, 72, 96, and 120 hours. Thromboembolic complications were defined as the development of deep-vein thrombosis, pulmonary embolism, acute myocardial infarction, or ischemic stroke during hospitalization. Patients were stratified into splenectomy versus nonsplenectomy cohorts. Univariate analysis was then conducted followed by longitudinal analysis using generalized estimating equations to evaluate the effects of time, splenectomy, and group-time interactions on changes in rTEG and traditional coagulation testing. We used an adjusted generalized estimating equation model to control for age, sex, ISS, admission blood pressure, base deficit, and hemoglobin., Results: A total of 1,242 patients were enrolled; 795 had serial rTEG data. Of these, 605 had serial values >24 hours and made up the study population. Splenectomy patients were younger, more hypotensive, and in shock on arrival. Although there was no difference in 24-hour or 30-day mortality, splenectomy patients were more likely to develop TE events. Using the GEE model, we found that α-angle and MA in splenectomy patients were lesser (more hypocoagulable) within the first 6 hours; however, they became substantially greater (more hypercoagulable) at 48, 72, 96, and 120 hours; all P < .05. In addition, platelet counts were greater in the splenectomy group beginning at 72 hours and continuing through 120 hours; P < .05., Conclusion: This multicenter, prospective study demonstrates that patients undergoing splenectomy have a more hypercoagulable state than other trauma patients. This hypercoagulable state (identified by greater α-angle and mA values) begins at approximately 48 hours after injury and continues through at least day 5. Moreover, this hypercoagulable state is associated with increased risk of TE complications., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The endothelial glycocalyx layer (EGL) is a key regulator of vascular permeability, cell adhesion, and inflammation. The EGL is primarily composed of syndecan-1, hyaluronic acid (HA), heparan sulfate (HS) and chondroitin sulfate (CS). While many studies have observed increased shedding of syndecan-1 during hemorrhagic shock, little is known about the shedding of other EGL components, and their effects on altered permeability and coagulation. We characterized shedding of all four primary components of the EGL, as well as the plasma's effect on permeability and thrombin generation in a cohort of trauma patients., Methods: Plasma samples were collected from 5 healthy consented volunteers and 22 severely injured trauma patients upon admission to the emergency department. ELISA assays were performed to quantify shed HA, HS, CS and syndecan-1 in plasma. A colloid osmometer and Electric Cell-substrate Impedance Sensing (ECIS) system were used to measure plasma colloid osmotic pressure (COP) and cell permeability, respectively. Thrombin generation was measured using a calibrated automated thrombogram (CAT). Initial vital signs, routine laboratory values, and injury severity scores (ISS) were recorded. Non-parametric statistical tests were used to compare differences between groups., Results: We observed increased shedding of all four proteins in trauma patient plasma compared to healthy controls: 31.7 vs. 21.2 U/L of CS, 175.8 vs. 121.9 ng/ml of HS, 946.7 vs. 618.6 ng/ml of HA and 245.8 vs. 31.6 ng/ml of syndecan-1 (all p<0.05). Patients with low plasma COP (≤16 mmHg) had significantly increased syndecan-1 and HA compared to those with normal COP, which corresponded to increased cell permeability via ECIS. CS and HS did not vary between COP groups. Lastly, patients with low COP displayed reduced peak thrombin generation of less than 250 nM on average (p<0.05)., Conclusions: Glycocalyx components were shed more in trauma patients compared to healthy controls in this cohort. However, only syndecan-1 and HA shedding were significantly higher in patients with reduced plasma COP. Thrombin generation was impaired in patients with low plasma COP. These data suggest that low plasma COP correlates well to glycocalyx degradation and thrombin loss following trauma, which consequently affect permeability and coagulation.

Background: Thrombin is the central coagulation protease that activates clotting proteins, triggers platelet aggregation, and converts fibrinogen to fibrin. Relationships between thrombin generation (TG) and clinical outcomes have not been defined following trauma. We hypothesize that TG is predictive of transfusion requirements and patient outcomes., Methods: Plasma was collected from 406 highest-level activation trauma patients upon admission and 29 healthy donors. Standard coagulation tests were performed, and TG was measured by calibrated automated thrombogram. Mann-Whitney U-tests were used to compare healthy versus trauma patients, and subgroup analyses were used to compare hypocoagulable versus nonhypocoagulable patients. Hypocoagulability was determined by area under the receiver operating characteristic curve analysis and was defined as peak TG of less than 250 nM. Multiple logistic regressions were used to assess the ability of TG to predict massive transfusion and mortality., Results: The median (interquartile range) age was 39 years (28-52 years), with an Injury Severity Score (ISS) of 17 (9-26). The trauma patients had greater TG (peak, 316.2 nM [270.1-355.5 nM]) compared with the healthy controls (124.6 nM [91.1-156.2 nM]), p < 0.001. The overall rate of hypocoagulability was 17%. The patients with peak TG of less than 250 nM were more severely injured (ISS, 25 [13-30] vs. 16 [9-25], p = 0.003); required more transfusions of red blood cells (p = 0.02), plasma (p = 0.003), and platelets (p = 0.006); had fewer hospital-free days (p = 0.001); and had increased mortality (10% vs. 3% at 24 hours, p = 0.006, and 29% vs. 11% at 30 days, p = 0.0004). Peak TG of less than 250 nM was predictive of massive transfusion (odds ratio, 4.18; p = 0.01) and 30-day mortality (odds ratio, 2.78; p = 0.02). Finally, peak TG was inversely correlated with standard coagulation tests., Conclusion: While the physiologic response to injury is to upregulate plasma procoagulant activity, the patients with reduced TG required more transfusions and had poorer outcomes. Measuring TG may provide an exquisitely sensitive tool for detecting disturbances in the enzymatic phases of coagulation in critically injured patients., Level of Evidence: Prognostic/epidemiologic study, level III.

Objective: To assess lymphatic flow adaptations to edema, we evaluated lymph transport function in rat mesenteric lymphatics under normal and increased fluid volume (edemagenic) conditions in situ., Methods: Twelve rats were infused with saline (intravenous infusion, 0.2 mL/min/100 g body weight) to induce edema. We intravitally measured mesenteric lymphatic diameter and contraction frequency, as well as lymphocyte velocity and density before, during, and after infusion., Results: A 10-fold increase in lymphocyte velocity (0.1-1 mm/s) and a sixfold increase in flow rate (0.1-0.6 μL/min), were observed post infusion, respectively. There were also increases in contraction frequency and fractional pump flow one minute post infusion. Time-averaged wall shear stress increased 10 fold post infusion to nearly 1.5 dynes/cm(2) . Similarly, maximum shear stress rose from 5 to 40 dynes/cm(2) ., Conclusions: Lymphatic vessels adapted to edemagenic stress by increasing lymph transport. Specifically, the increases in lymphatic contraction frequency, lymphocyte velocity, and shear stress were significant. Lymph pumping increased post infusion, though changes in lymphatic diameter were not statistically significant. These results indicate that edemagenic conditions stimulate lymph transport via increases in lymphatic contraction frequency, lymphocyte velocity, and flow. These changes, consequently, resulted in large increases in wall shear stress, which could then activate NO pathways and modulate lymphatic transport function., (© 2014 John Wiley & Sons Ltd.)

Objective: Hemorrhagic shock is the leading cause of traumatic deaths; many could be potentially prevented with appropriate resuscitation. However, to initiate resuscitation, one must identify patients with hemorrhagic shock early. In this article, we determined the associations between plasma colloid osmotic pressure (COP) and clinical outcomes in severely injured trauma patients., Methods: Plasma samples were collected from 104 trauma patients upon admission to the emergency department and 10 healthy volunteers to serve as control subjects. Plasma osmolality, COP, and serum protein were measured and correlated to clinical data. Thrombelastography and impedance aggregometry were performed to assess coagulopathy. Commercial enzyme-linked immunosorbent assays were used to quantify syndecan 1., Results: Plasma COP was significantly reduced in trauma patients compared to control subjects 17.7 ± 2.6 vs. 20.7 ± 2.1 mmHg (P < 0.05) and strongly correlated to serum protein values (R = 0.7). We divided our cohort into low (COP ≤16.5 mmHg) and normal (COP >16.5 mmHg) subgroups, illustrating significantly higher Injury Severity Score scores in patients with low COP (21 vs. 10, P = 0.007), despite no differences in vital signs. Patients with low COP received more red blood cells, plasma, and platelets (4 vs. 0 total units, P = 0.0005) within 24 h of admission. Syndecan 1 levels were significantly higher (184 vs. 52 ng/mL, P = 0.027) in patients with low COP., Conclusions: Reduced plasma COP and serum protein in trauma patients are indicative of injury severity. In the absence of significant alterations in vital signs, plasma COP levels were associated with increased requirements for blood products and increased syndecan 1 shedding. We believe that plasma COP provides new insight in guiding resuscitation.

Background: We hypothesized that patients with pelvic fractures and hemorrhage admitted during daytime hours were undergoing interventional radiology (IR) earlier than those admitted at night and on weekends, thereby establishing two standards of time to hemorrhage control., Methods: The trauma registry (January 2008 to December 2011) was reviewed for patients admitted with pelvic fractures, hemorrhagic shock, and transfusion of at least 1 U of blood. The control group (DAY) was admitted from 7:30 AM to 5:30 PM Monday to Friday, while the study group (after hours [AHR]) was admitted from 5:30 PM to 7:30 AM, on weekends or holidays., Results: A total of 191 patients met the criteria (45 DAY, 146 AHR); 103 died less than 24 hours and without undergoing IR (29% DAY group vs. 62% AHR, p < 0.001). Sixteen patients (all in AHR group) died while awaiting IR (p = 0.032). Eighty-eight patients (32 DAY, 56 AHR) survived to receive IR. Among these, the AHR group were younger (median, 30 years vs. 54 years; p = 0.007), more tachycardic (median pulse, 119 beats/min vs. 90 beats/min; p = 0.001), and had more profound shock (median base, -10 vs. -6; p = 0.006) on arrival. Time from admission to IR (median, 301 minutes vs. 193 minutes; p < 0.001) and computed tomographic scan to IR (176 minutes vs. 87 minutes, p = 0.011) were longer in the AHR group. There was no difference in the 30-day mortality by univariate analysis. However, after controlling for age, arrival physiology, injury severity, and degree of shock, the AHR group had a 94% increased risk of mortality., Conclusion: The current study demonstrated that patients admitted at night and on weekends have a significant increase in time to angioembolization compared with those arriving during the daytime and during the week. Multivariate regression noted that AHR management was associated with an almost 100% increase in mortality. While this is a single-center study and retrospective in nature, it suggests that we are currently delivering two standards of care for pelvic trauma, depending on the day and time of admission., Level of Evidence: Therapeutic study, level II.

Exposure to 3, 4-methylenedioxymethamphetamine (MDMA) leads to spatial memory impairment and hippocampal cell death. In the present study we have examined the protective effects of N-acetyl-L-cysteine (NAC) on MDMA-induced neurotoxicity. A total of 56 male Sprague Dawley rats (200-250 g) received twice daily intraperitoneal (IP) injections of 5, 10 or 20 mg/kg MDMA plus NAC (100 mg/kg). Rectal temperatures were recorded before and after daily treatment. We used a Morris water maze (MWM) to assess spatial learning and memory. At the end of the study rats' brains were removed, cells were counted and the level of Bcl-2, Bax and caspase-3 expression in the hippocampi were measured. NAC pretreatment significantly reduced MDMA-induced hyperthermia. In the MWM, NAC significantly attenuated the MDMA-induced increase in distance traveled; however the observed increase in escape latency was not significant. The decrease in time spent in the target quadrant in MDMA animals was significantly attenuated (p < 0.001, all groups). NAC protected against MDMA-induced cell death and the up -regulation of Bax and Caspase-3, in addition to the down-regulation of Bcl-2. This data suggested a possible benefit of NAC in the treatment of neurotoxicity among those who use MDMA.