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.
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.
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.
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.
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.
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).
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.
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.
Secondary lymphatic valves are essential for minimizing backflow of lymph and are presumed to gate passively according to the instantaneous trans-valve pressure gradient. We hypothesized that valve ...gating is also modulated by vessel distention, which could alter leaflet stiffness and coaptation. To test this hypothesis, we devised protocols to measure the small pressure gradients required to open or close lymphatic valves and determine if the gradients varied as a function of vessel diameter. Lymphatic vessels were isolated from rat mesentery, cannulated, and pressurized using a servo-control system. Detection of valve leaflet position simultaneously with diameter and intraluminal pressure changes in two-valve segments revealed the detailed temporal relationships between these parameters during the lymphatic contraction cycle. The timing of valve movements was similar to that of cardiac valves, but only when lymphatic vessel afterload was elevated. The pressure gradients required to open or close a valve were determined in one-valve segments during slow, ramp-wise pressure elevation, either from the input or output side of the valve. Tests were conducted over a wide range of baseline pressures (and thus diameters) in passive vessels as well as in vessels with two levels of imposed tone. Surprisingly, the pressure gradient required for valve closure varied >20-fold (0.1-2.2 cmH(2)O) as a passive vessel progressively distended. Similarly, the pressure gradient required for valve opening varied sixfold with vessel distention. Finally, our functional evidence supports the concept that lymphatic muscle tone exerts an indirect effect on valve gating.
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.
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.
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.
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.
Prognostic/epidemiologic study, level III.
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.
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.
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.
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.
Therapeutic study, level II.
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.
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.
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.
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.
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.
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