In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

BACKGROUND: High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. METHODS: In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. RESULTS: The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. CONCLUSIONS: HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

Linking Computational Fluid Dynamics Modeling to Device-Induced Platelet Defects in Mechanically Assisted Circulation.

Thrombotic and bleeding events are the most common hematologic complications in patients with mechanically assisted circulation and are closely related to device-induced platelet dysfunction. In this study, we sought to link computational fluid dynamics (CFD) modeling of blood pumps with device-induced platelet defects. Fresh human blood was circulated in circulatory loops with four pumps (CentriMag, HVAD, HeartMate II, and CH-VAD) operated under a total of six clinically representative conditions. Blood samples were collected and analyzed for glycoprotein (GP) IIb/IIIa activation and receptor shedding of GPIbα and GPVI. In parallel, CFD modeling was performed to characterize the blood flow in these pumps. Numerical indices of platelet defects were derived from CFD modeling incorporating previously derived power-law models under constant shear conditions. Numerical results were correlated with experimental results by regression analysis. The results suggested that a scalar shear stress of less than 75 Pa may have limited contribution to platelet damage. The platelet defect indices predicted by the CFD power-law models after excluding shear stress <75 Pa correlated excellently with experimentally measured indices. Although numerical prediction based on the power-law model cannot directly reproduce the experimental data. The power-law model has proven its effectiveness, especially for quantitative comparisons.

In-Vivo Evaluation of a Novel Integrated Pediatric Pump Lung in a 30 Day Ovine Animal Model.

Recently there has been increased use of mechanical circulatory support in pediatric patients as a bridge to cardiopulmonary recovery or transplantation. However, there are few devices that are optimized and approved for use in pediatric patients. We designed and prototyped a novel integrated pediatric pump lung (PPL) that underwent 30 day in-vivo testing in seven juvenile Dorset Hybrid sheep. Devices were implanted in a right atrial to pulmonary artery configuration. Six of seven sheep survived with a device functioning for 25-35 days. The device flow rate was maintained at 2.08 ± 0.34 to 2.54 ± 0.16 L/min with oxygen transfer of 109.8 ± 24.8 to 151.2 ± 26.2 ml/min over the study duration. Aside from a postoperative drop in hematocrit, all hematologic and blood chemistry test values returned to normal ranges after 1-2 weeks postoperatively. Similarly, lactate dehydrogenase increased postoperatively and returned to baseline. In two sheep, there were early device failures due to oxygenator thrombosis on postoperative days zero and five; they then had oxygenator exchanges with subsequent devices performing stably for 30 days. This study demonstrated that the integrated PPL device exhibited stable performance and acceptable biocompatibility in a 30 day ovine model.

Investigation of the role of von Willebrand factor in shear-induced platelet activation and functional alteration under high non-physiological shear stress.

BACKGROUND: von Willebrand factor (vWF) plays a crucial role in physiological hemostasis through platelet and subendothelial collagen adhesion. However, its role in shear-induced platelet activation and functional alteration under non-physiological conditions common to blood-contacting medical devices (BCMDs) is not well investigated. METHODS: Fresh healthy human blood was treated with an anti-vWF antibody to block vWF-GPIbα interaction. Untreated blood was used as a control. They were exposed to three levels of non-physiological shear stress (NPSS) (75, 125, and 175 Pa) through a shearing device with an exposure time of 0.5 s to mimic typical shear conditions in BCMDs. Flow cytometric assays were used to measure the expression levels of PAC-1 and P-Selectin and platelet aggregates for platelet activation and the expression levels of GPIbα, GPIIb/IIIa, and GPVI for receptor shedding. Collagen/ristocetin-induced platelet aggregation capacity was characterized by aggregometry. RESULTS: The levels of platelet activation and aggregates increased with increasing NPSS in the untreated blood. More receptors were lost with increasing NPSS, resulting in a decreased capacity of collagen/ristocetin-induced platelet aggregation. In contrast, the increase in platelet activation and aggregates after exposure to NPSS, even at the highest level of NPSS, was significantly lower in treated blood. Nevertheless, there was no notable difference in receptor shedding, especially for GPIIb/IIIa and GPVI, between the two blood groups at the same level of NPSS. The block of vWF exacerbated the decreased capacity of collagen/ristocetin-induced platelet aggregation. CONCLUSIONS: High NPSS activates platelets mainly by enhancing the vWF-GPIbα interaction. Platelet activation and receptor shedding induced by high NPSS likely occur through different pathways.

Increased phagocytosis capacity of circulating neutrophils in patients on continuous flow ventricular assist device support.

BACKGROUND: Neutrophils take part in the innate immune response, phagocytosis, and pro-inflammatory cytokine release. The phagocytic capacity of circulating neutrophils in patients on continuous flow (CF) ventricular assist device (VAD) has not been well studied. METHODS: Blood samples from 14 patients undergoing CF-VAD implantation were collected and analyzed preoperatively (at baseline) and on postoperative days (POD) 3, 7, 14, and 28. Flow cytometry was used to assess the surface expression levels of CD62L, CD162, and macrophage antigen-1 (MAC-1) and neutrophil phagocytic capacity. Interleukin 1 (IL1), IL6, IL8, TNF-α, neutrophil elastase, and myeloperoxidase in plasma were measured using enzyme-linked immunosorbent assays. RESULTS: Among the 14 patients, seven patients had preoperative bridge device support. Relative to baseline, patients with no bridge device had elevated leukocyte count and neutrophil elastase by POD3 which normalized by POD7. Neutrophil activation level, IL6, IL8, and TNF-α increased by POD3 and sustained elevated levels for 7-14 days postoperatively. Elevated neutrophil phagocytic capacity persisted even until POD28. Similar patterns were observed in patients on a preoperative bridge device. CONCLUSIONS: Neutrophil activation and phagocytic capacity increased in response to VAD support, while inflammatory cytokines remain elevated for up to 2 weeks postoperatively. These findings may indicate that VAD implantation elicits circulating neutrophils to an abnormal preemptive phagocytotic phenotype.

Neutrophil Structural and Functional Alterations After High Mechanical Shear Stress Exposure.

Patients on mechanical circulatory support are prone to infections, increasing morbidity and mortality. These circulatory support devices generate high mechanical shear stress (HMSS) that can causes trauma to blood. When leukocytes become damaged, their immune response function may be impaired or weakened, leading to increased infection vulnerability. This study examined neutrophil structural and functional alterations after exposure to 75, 125, and 175 Pa HMSS for 1 second. Human blood was exposed to three levels of HMSS using a blood shearing device. Neutrophil morphological alteration was characterized by examining blood smears. Flow cytometry assays were used to analyze expression levels of CD62L and CD162 receptors, activation level (CD11b), and aggregation (platelet-neutrophil aggregates). Neutrophil phagocytosis and rolling were examined via functional assays. The results show neutrophil structure (morphology and surface receptors) and function (activation, aggregation, phagocytosis, rolling) were significantly altered after HMSS exposure. These alterations include cell membrane damage, loss of surface receptors (CD62L and CD162), initiation of activation and aggregation, upregulation of phagocytic ability and increased rolling speed. The alterations were the most severe after 175 Pa exposure. HMSS caused damage and activation of neutrophils, potentially impairing normal neutrophil function, leading to weakened immune defense and increasing a patient's vulnerability to infections.

Knock-out of N-glycolylneuraminic acid attenuates antibody-mediated rejection in xenogenically perfused porcine lungs.

BACKGROUND: Antibody-mediated rejection has long been known to be one of the major organ failure mechanisms in xenotransplantation. In addition to the porcine α1,3-galactose (α1,3Gal) epitope, N-Glycolylneuraminic acid (Neu5Gc), a sialic acid, has been identified as an important porcine antigen against which most humans have pre-formed antibodies. Here we evaluate GalTKO.hCD46 lungs with an additional cytidine monophospho-N-acetylneuraminic acid hydroxylase (CMAH) gene knock-out (Neu5GcKO) in a xenogeneic ex vivo perfusion model METHODS: Eleven GalTKO.hCD46.Neu5GcKO pig lungs were perfused for up to 6 h with fresh heparinized human blood. Six of them were treated with histamine (H) blocker famotidine and 1-thromboxane synthase inhibitor Benzylimidazole (BIA) and five were left untreated. GalTKO.hCD46 lungs without Neu5GcKO (n = 18: eight untreated and 10 BIA+H treated) served as a reference. Functional parameters, blood, and tissue samples were collected at pre-defined time points throughout the perfusion RESULTS: All but one Neu5GcKO organs maintained adequate blood oxygenation and "survived" until elective termination at 6 h whereas two reference lungs failed before elective termination at 4 h. Human anti-Neu5Gc antibody serum levels decreased during the perfusion of GalTKO.hCD46 lungs by flow cytometry (∼40% IgM, 60% IgG), whereas antibody levels in Neu5GcKO lung perfusions did not fall (IgM p = .007; IgG p < .001). Thromboxane elaboration, thrombin generation, and histamine levels were significantly reduced with Neu5GcKO lungs compared to reference in the untreated groups (p = .007, .005, and .037, respectively); treatment with BIA+H masked these changes. Activation of platelets, measured as CD62P expression on circulating platelets, was lower in Neu5GcKO experiments compared to reference lungs (p = .023), whereas complement activation (as C3a rise in plasma) was not altered. MCP-1 and lactotransferin level elevations were blunted in Neu5GcKO lung perfusions (p = .007 and .032, respectively). Pulmonary vascular resistance (PVR) rise was significantly attenuated and delayed in untreated GalTKO.hCD46.Neu5GcKO lungs in comparison to the untreated GalTKO.hCD46 lungs (p = .003) CONCLUSION: Additional Neu5GcKO in GalTKO.hCD46 lungs significantly reduces parameters associated with antibody-mediated inflammation and activation of the coagulation cascade. Knock-out of the Neu5Gc sialic acid should be beneficial to reduce innate immune antigenicity of porcine lungs in future human recipients.

Role of thrombin to non-physiological shear stress induced platelet activation and function alternation.

OBJECTIVE: Non-physiological shear stress (NPSS) and thrombin have two distinct mechanisms for activating platelets. NPSS in mechanically assisted circulation (MAC) devices can cause platelet dysfunction, e.g., by shedding its key receptors. In addition, patients with heart failure have increased levels of thrombin generation, which may further affect the NPSS-induced platelet dysfunction, resulting in device-associated complications. This study aimed to assess the combined effect of NPSS and thrombin in platelet activation, expression of adhesion receptors on the platelet surface, and alterations of platelet aggregation. METHODS: Fresh human blood from healthy donors was divided into two groups; one group was treated by adding 0.01 U/mL thrombin, and another group not treated with thrombin served as a control comparison. They were then pumped through a novel blood shearing device which produces similar shear stress conditions to those in the MAC devices. Three levels of NPSS (i.e., 75, 125, and 175 Pa) with a 1.0 s exposure time were selected for the shearing conditions. Expression of platelet activation markers (PAC-1, activated GPIIb/IIIa and CD62P, platelet surface P-selectin) were investigated along with the shedding of platelet receptors (GPIb, GPIIb/IIIa, and GPVI), generation of platelet microparticles, and Phosphatidylserine (PS)-positive platelets detected by flow cytometry. Platelet aggregation (induced by collagen/ristocetin) was measured by Lumi-aggregometry. RESULTS: Platelet receptors were shed after exposure to NPSS showing a positive correlation with the level of shear stress. The generation of platelet microparticles and PS-positive platelets also increased with greater NPSS. Elevated NPSS decreased the platelet aggregation capacity. Platelet activation level increased with greater NPSS. Being treated by thrombin can further exacerbate these characteristics under same level of NPSS, except that platelet activation level drastically dropped after the exposure to 175 Pa NPSS in the thrombin-treated blood. CONCLUSION: After being treated by thrombin, platelets became more susceptible to NPSS, resulting in more receptor shedding, platelet microparticles, and PS-positive platelets, thus limiting platelet aggregation capacity after exposure to NPSS. Platelet activation, in terms of PAC-1 and P-selectin, is an interim status competing between the expression and shedding of these makers/receptors. When platelets have reached a saturation level of activation, exposure to excessive NPSS can potentially impair activation.

Acquired platelet defects are responsible for nonsurgical bleeding in left ventricular assist device recipients.

BACKGROUND: Left ventricular assist devices (LVADs) have been used as a standard treatment option for patients with advanced heart failure. However, these devices are prone to adverse events. Nonsurgical bleeding (NSB) is the most common complication in patients with continuous flow (CF) LVADs. The development of acquired von Willebrand syndrome (AVWS) in CF-LVAD recipients is thought to be a key factor. However, AVWS is seen across a majority of LVAD patients, not just those with NSB. The purpose of this study was to examine the link between acquired platelet defects and NSB in CF-LVAD patients. METHODS: Blood samples were collected from 62 CF-LVAD patients at pre- and 4 post-implantation timepoints. Reduced adhesion receptor expression (GPIbα and GPVI) and activation of platelets (GPIIb/IIIa activation) were used as markers for acquired platelet defects. RESULTS: Twenty-three patients experienced at least one NSB episode. Significantly higher levels of platelet activation and receptor reduction were seen in the postimplantation blood samples from bleeders compared with non-bleeders. All patients experienced the loss of high molecular weight monomers (HMWM) of von Willebrand Factor (vWF), but no difference was seen between the two groups. Multivariable logistic regression showed that biomarkers for reduced platelet receptor expression (GPIbα and GPVI) and activation (GPIIb/IIIa) have more predictive power for NSB, with the area under curve (AUC) values of 0.72, 0.68, and 0.62, respectively, than the loss of HMWM of vWF (AUC: 0.57). CONCLUSION: The data from this study indicated that the severity of acquired platelet defects has a direct link to NSB in CF-LVAD recipients.

Human erythrocyte fragmentation during ex-vivo pig organ perfusion.

Platelet sequestration is a common process during organ reperfusion after transplantation. However, instead of lower platelet counts, when using traditional hemocytometers and light microscopy, we observed physiologically implausible platelet counts in the course of ex-vivo lung and liver xenograft organ perfusion studies. We employed conventional flow cytometry (FC) and imaging FC (AMINS ImageStream X) to investigate the findings and found platelet-sized fragments in the circulation that are mainly derived from red blood cell membranes. We speculate that this erythrocyte fragmentation contributes to anemia during in-vivo organ xenotransplant.

An ex vivo comparison of partial thromboplastin time and activated clotting time for heparin anticoagulation in an ovine model.

BACKGROUND: Sheep are a primary model of mechanical circulatory support (MCS) with heparin anticoagulation therapy frequently being monitored by activated clotting time (ACT) due to ease and cost. In patients undergoing long-term heparin therapy, other anticoagulation monitoring strategies, such as activated partial thromboplastin time (aPTT), have proven to be more reliable indicators for the adequacy of anticoagulation, frequently determined by heparin concentration. As there is a paucity of similar studies in sheep, we sought to investigate the correlation between heparin concentration and ACT and aPTT using whole sheep blood in an ex vivo model. METHODS: Fresh whole blood was serially drawn from an adult female Dorset-hybrid sheep and aliquots were placed into tubes containing heparin saline solutions with concentrations ranging from 0 to 7.81 U heparin per mL of whole blood. ACT and aPTT values were measured on each of the samples. The experiment was performed four times with the same animal. A simple linear regression was performed to determine correlation, and subgroup analysis was performed on low versus high heparin concentrations typically seen in human patients on long-term MCS, such as extracorporeal membrane oxygenation (ECMO), versus cardiopulmonary bypass, respectively. RESULTS: aPTT measurements versus the heparin concentration had an R2  = 0.7295. ACT measurements versus the heparin concentration had a R2  = 0.4628. aPTT measurements versus the ACT measurements had a R2  = 0.2974. The strength of the correlation between aPTT and heparin concentration increased at low heparin concentrations (R2  = 0.8392). CONCLUSION: aPTT had a more reliable correlation to heparin concentration and thus anticoagulation level than ACT. This was particularly true at lower heparin concentrations, similar to ranges seen for patients on ECMO. The correlation between aPTT and ACT values was poor. Further in vivo studies should be performed to confirm our results.

Neutrophil dysfunction due to continuous mechanical shear exposure in mechanically assisted circulation in vitro.

OBJECTIVE: Leukocytes play an important role in the body's immune system. The aim of this study was to assess alterations in neutrophil phenotype and function in pump-assisted circulation in vitro. METHODS: Human blood was circulated for four hours in three circulatory flow loops with a CentriMag blood pump operated at a flow of 4.5 L/min at three rotational speeds (2100, 2800, and 4000 rpm), against three pressure heads (75, 150, and 350 mm Hg), respectively. Blood samples were collected hourly for analyses of neutrophil activation state (Mac-1, CD62L, CD162), neutrophil reactive oxygen species (ROS) production, apoptosis, and neutrophil phagocytosis. RESULTS: Activated neutrophils indicated by both Mac-1 expression and decreased surface expression of CD62L and CD162 receptors increased with time in three loops. The highest level of neutrophil activation was observed in the loop with the highest rotational speed. Platelet-neutrophil aggregates (PNAs) progressively increased in two loops with lower rotational speeds. PNAs peaked at one hour after circulation and decreased subsequently in the loop with the highest rotational speed. Neutrophil ROS production dramatically increased at one hour after circulation and decreased subsequently in all three loops with similar levels and trends. Apoptotic neutrophils increased with time in all three loops. Neutrophil phagocytosis capacity in three loops initially elevated at one hour after circulation and decreased subsequently. Apoptosis and altered phagocytosis were dependent on rotational speed. CONCLUSIONS: Our study revealed that the pump-assisted circulation induced neutrophil activation, apoptosis, and functional impairment. The alterations were strongly associated with pump operating condition and duration.

Neutrophil injury and function alterations induced by high mechanical shear stress with short exposure time.

High mechanical shear stresses (HMSS) can cause damage to blood, which manifests as morphologic changes, shortened life span, biochemical alterations, and complete rupture of blood cells and proteins, leading to the alterations of normal blood function. The aim of this study is to determine the state of neutrophil activation and function alterations caused by HMSS with short exposure time relevant to ventricular assist devices. Blood from healthy donors was exposed to three levels of HMSS (75Pa, 125Pa, and 175Pa) for a short exposure time (0.5 s) using our Couette-type blood-shearing device. Neutrophil activation (Mac-1, platelet-neutrophil aggregates) and surface expression levels of two key functional receptors (CD62L and CD162) on neutrophils were evaluated by flow cytometry. Neutrophil phagocytosis and transmigration were also examined with functional assays. Results showed that the expression of Mac-1 on neutrophils and platelet-neutrophil aggregates increased significantly while the level of CD62L expression on neutrophils decreased significantly after the exposure to HMSS. The Mac-1 expression progressively increased while the CD62L expression progressively decreased with the increased level of HMSS. The level of CD162 expression on neutrophils slightly increased after the exposure to HMSS, but the increase was not significant. The phagocytosis assay data revealed that the ability of neutrophils to phagocytose latex beads coated with fluorescently labeled rabbit IgG increased significantly with the increased level of HMSS. The transmigration ability of neutrophils slightly increased after the exposure to HMSS, but did not reach a significant level. In summary, HMSS with a short exposure time of 0.5 seconds could induce neutrophil activation, platelet-neutrophil aggregation, shedding of CD62L receptor, and increased phagocytic ability. However, the exposure to the three levels of HMSS did not cause a significant change in neutrophil transmigration capacity and shedding of CD162 receptor on neutrophils.

5-Hydroxymethylcytosine signature in circulating cell-free DNA as a potential diagnostic factor for early-stage colorectal cancer and precancerous adenoma.

Approximately 85% colorectal cancers (CRCs) are thought to evolve through the adenoma-to-carcinoma sequence associated with specific molecular alterations, including the 5-hydroxymethylcytosine (5hmC) signature in circulating cell-free DNA (cfDNA). To explore colorectal disease progression and evaluate the use of cfDNA as a potential diagnostic factor for CRC screening, here, we performed genome-wide 5hmC profiling in plasma cfDNA and tissue genomic DNA (gDNA) acquired from 101 samples (63 plasma and 38 tissues), collected from 21 early-stage CRC patients, 21 AD patients, and 21 healthy controls (HC). The gDNA and cfDNA 5hmC signatures identified in gene bodies and promoter regions in CRC and AD groups were compared with those in HC group. All the differential 5hmC-modified regions (DhMRs) were gathered into four clusters: Disease-enriched, AD-enriched, Disease-lost, and AD-lost, with no overlap. AD-related clusters, AD-enriched and AD-lost, displayed the unique 5hmC signals in AD patients. Disease-enriched and Disease-lost clusters indicated the general 5hmC changes when colorectal lesions occurred. Cancer patients with a confirmable adenoma history segmentally gathered in AD-enriched clusters. KEGG functional enrichment and GO analyses determined distinct differential 5hmC-modified profiles in cfDNA of HC individuals, AD, and CRC patients. All patients had comprehensive 5hmC signatures where Disease-enriched and Disease-lost DhMR clusters demonstrated similar epigenetic modifications, while AD-enriched and AD-lost DhMR clusters indicated complicated subpopulations in adenoma. Analysis of CRC patients with adenoma history showed exclusive 5hmC-gain characteristics, consistent with the 'parallel' evolution hypothesis in adenoma, either developed through the adenoma-to-carcinoma sequence or not. These findings deepen our understanding of colorectal disease and suggest that the 5hmC modifications of different pathological subtypes (cancer patients with or without adenoma history) could be used to screen early-stage CRC and assess adenoma malignancy with large-scale follow-up studies in the future.

Sphingosine Kinase Blockade Leads to Increased Natural Killer T Cell Responses to Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive subtype of non-Hodgkin's lymphoma. Despite being responsive to combination chemotherapy, median survival remains around 5 years due to high rates of relapse. Sphingolipid metabolism regulates MCL survival and proliferation and we found that sphingosine-1-phosphate (S1P) is upregulated in MCL cells. Therapeutic targeting of the S1P1 receptor or knockdown of sphingosine kinase 1 (SK1), the enzyme responsible for generating S1P, in human MCL cells results in a significant increase in Natural Killer T (NKT) cell activation. NKT cells recognize glycolipid antigens presented on CD1d and can reduce MCL tumor burden in vivo. Lipidomic studies identified cardiolipin, which has been reported to bind to CD1d molecules, as being upregulated in SK1 knockdown cells. We found that the pretreatment of antigen presenting cells with cardiolipin leads to increased cytokine production by NKT cell hybridomas. Furthermore, the ability of cardiolipin to activate NKT cells was dependent on the structure of its acyl chains. Collectively, these studies delineate novel pathways important for immune recognition of malignant cells and could lead to the development of new treatments for lymphoma.

Impact of high mechanical shear stress and oxygenator membrane surface on blood damage relevant to thrombosis and bleeding in a pediatric ECMO circuit.

The roles of the large membrane surface of the oxygenator and the high mechanical shear stress (HMSS) of the pump in the extracorporeal membrane oxygenation (ECMO) circuit were examined under a pediatric support setting. A clinical centrifugal pump and a pediatric oxygenator were used to construct the ECMO circuit. An identical circuit without the oxygenator was constructed for comparison. Fresh human blood was circulated in the two circuits for 4 hours under the identical pump speed and flow. Blood samples were collected hourly for blood damage assessment, including platelet activation, generation of platelet-derived microparticles (PDMP), losses of key platelet hemostasis receptors (glycoprotein (GP) Ibα (GPIbα) and GPVI), and high molecular weight multimers (HMWM) of von Willebrand factor (VWF) and plasma free hemoglobin (PFH). Platelet adhesion on fibrinogen, VWF, and collagen was further examined. The levels of platelet activation and generation of PDMP and PFH exhibited an increasing trend with circulation time while the expression levels of GPIbα and GPVI receptors on the platelet surface decreased. Correspondingly, the platelets in the blood samples exhibited increased adhesion capacity to fibrinogen and decreased adhesion capacities on VWF and collagen with circulation time. Loss of HMWM of VWF occurred in both circuits. No statistically significant differences were found in all the measured parameters for blood damage and platelet adhesion function between the two circuits. The results indicate that HMSS from the pump played a dominant role in blood damage associated with ECMO and the impact of the large surface of the oxygenator on blood damage was insignificant.

Amelioration of nonalcoholic fatty liver disease by swertiamarin in fructose-fed mice.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver disease. Swertia bimaculata (Sieb. et Zucc.) Hook. Thoms.ex Clarke, a glabrous or procumbent perennial herb, is a traditional herb medicine. Swertiamarin, a secoiridoid glycoside, is a representative ingredient in this medical plant crude extract and shows antidiabetic and antihyperlipidaemic activities and protective effect against hepatic injury. PURPOSE: The present study aimed to determine whether swertiamarin can attenuate NAFLD in fructose-fed mice. METHODS: Healthy male mice freely drank water containing 10% fructose for 12 consecutive weeks, whereas animals in those swertiamarin tested groups received different doses of swertiamarin (25, 50 and 100 mg/kg) by intragastric administration once a day from the ninth week to the twelfth week. RESULTS: At the end of the experiment, fructose-fed mice administrated with swertiamarin showed low levels of serum glucose, triglycerides, uric acid, alanine aminotransferase and aspartate transaminase. Histological examinations suggested the alleviation of hepatic ballooning degeneration and steatosis by swertiamarin treatment. Moreover, swertiamarin administration mitigated hepatic oxidative stress along with decreases of hepatic pro-inflammation cytokines, which was associated with decrease of hepatic xanthine oxidase (XO) activity and enhancements of anti-oxidant defense system enzymes, as well as activation of nuclear factor E2-related factor 2 (Nrf2) in fructose-fed mice. In addition, swertiamarin down-regulated expression of sterol-regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC1) in liver of fructose-fed mice. CONCLUSION: The present study demonstrates that swertiamarin alleviates NAFLD and metabolic alterations in fructose-fed mice.

Simple Entropy Terms for End-Point Binding Free Energy Calculations.

We introduce a number of computationally inexpensive modifications to the MM/PBSA and MM/GBSA estimators for binding free energies, which are based on average receptor-ligand interaction energies in simulations of a noncovalent complex, to improve the treatment of entropy: second- and higher-order terms in a cumulant expansion and a confining potential on ligand external degrees of freedom. We also consider a filter for snapshots where ligands have drifted from the initial binding pose. The variations were tested on six sets of systems for which binding modes and free energies have previously been experimentally determined. For some data sets, none of the tested estimators led to results significantly correlated with measured free energies. In data sets with nontrivial correlation, a ligand RMSD cutoff of 3 Å and a second-order truncation of the cumulant expansion was found to be comparable or better than the average interaction energy by several statistical metrics.

Black Sesame Seeds Ethanol Extract Ameliorates Hepatic Lipid Accumulation, Oxidative Stress, and Insulin Resistance in Fructose-Induced Nonalcoholic Fatty Liver Disease.

The aim of the present study was to investigate the effect of black sesame seeds ethanol extract (BSSEE) against nonalcoholic fatty liver disease (NAFLD) in fructose-fed mice. Mice were fed a standard diet without or with 30% fructose in drinking water for 8 consecutive weeks, while mice in three BSSEE tested groups received different doses of BSSEE (0.5, 1, and 2 mL/kg) once a day from the fifth week to the eighth week. Administration of BSSEE dose-dependently exerted antiobesity and protective effect against metabolism disorder in fructose-fed mice. Histological examinations indicated that administration of BSSEE obviously reduced hepatic lipid accumulation. Insulin tolerance test (ITT) and glucose tolerance test (GTT) along with decreases of serum insulin and glucose levels by BSSEE treatment suggested the improvement of body insulin resistance, and administration of 1 and 2 mL/kg BSSEE mitigated liver insulin resistance as the evidence of downregulated expression of phospho-JNK1/2/3, phospho-NF-κB p65, phospho-IRS1, and phospho-IKK alpha/beta, up-regulated XBP1 expression, and reductions of TNF-α and IL-6 levels. In addition, BSSEE treatment ameliorated hepatic oxidative stress through increasing GSH, vitamin C, and Nrf2 levels, decreasing MDA and NO levels, and enhancing SOD, CAT, and GSH-Px activities. These results demonstrated that BSSEE showed protective effects against NAFLD-related metabolic diseases in fructose-fed mice. Therefore, BSSEE may be a potent dietary supplement to ameliorate the diseases.

Silver ion chromatography for peak resolution enhancement: Application to the preparative separation of two sesquiterpenes using online heart-cutting LC-LC technique.

Silver ion chromatography, utilizing columns packed with silver ions bonded to silica gel, has proved to be an invaluable technique for the analysis of some positional isomers. In this work, silver ion chromatography by combination with online heart-cutting LC-LC technique for the preparative separation of two sesquiterpenes positional isomers from a natural product was investigated. On the basis of the evaluation that silver ion content impacts on the separation, the laboratory-made silver ion columns, utilizing silica gel impregnated with 15% silver nitrate as column packing materials, were used for peak resolution improvement of these two isomers and the preparative separation of them in heart-cutting LC-LC. The relationship among the maximal sample load, flow rate and peak resolution in the silver ion column were optimized, and the performance of the silver ion column was compared with conventional C18 column and silica gel column. Based on the developed chromatographic conditions, online heart-cutting LC-LC chromatographic separation system in combination with a silica gel column and a silver ion column that was applied to preparative separation of these two isomers from a traditional Chinese medicine, Inula racemosa Hook.f., was established. The results showed that the online heart-cutting LC-LC technique by combination of a silica gel column and a silver ion column for the preparative separation of these two positional isomers from this natural plant was superior to the preparative separation performed on a single-column system with C18 column or silica gel column.