Go Shine Huang

Expression of thrombomodulin on monocytes is associated with early outcomes in patients with coronary artery bypass graft surgery.

Thrombomodulin (TM) mediates blood coagulation and inflammation and is expressed constitutively on resting monocytes. This expression might be a key regulator of monocyte-related inflammation. Conventional cardiopulmonary bypass (CPB), beating-heart CPB, and off-pump techniques have been used widely in cardiac surgery. Although beating-heart CPB and off-pump techniques have reduced postoperative inflammation significantly, the underlying mechanisms remain unclear. Whether CPB affects the expression of TM and changes the actual immune capacity of monocytes is also unknown. In this study, we analyzed TM expression on monocytes and in plasma among patients undergoing elective coronary artery bypass graft surgery. The days spent in an intensive care unit (ICU) and incidence of fever in the ICU were significantly lower in the beating-heart CPB and off-pump groups than in the conventional CPB group. Enzyme-linked immunosorbent assay showed a significant increase in TM at 30 min after the commencement of CPB and at the end of surgery in the conventional CPB group, whereas the level increased less markedly in the beating-heart CPB group. Flow cytometry showed that conventional CPB markedly reduced the expression of TM on monocytes. Based on monocyte chemotaxis analysis and an actin polymerization assay, we propose that TM expression on monocytes is associated with systemic inflammation. We conclude that the beating-heart CPB and off-pump techniques have a lower impact on patients than conventional CPB. The reduced incidence of fever and shorter ICU stay seem to be associated predominantly with the lower concentration of TM in plasma and with a higher expression of TM on monocytes.

Poincaré plot indexes of heart rate variability detect dynamic autonomic modulation during general anesthesia induction

PURPOSE Beat-to-beat heart rate variability (HRV) is caused by the fluctuating balance of sympathetic and parasympathetic tone. The Poincaré plot has been used to evaluate HRV. In this study, we validate that this new method may qualitatively and quantitatively assess the sympathovagal fluctuation in patients during induction of anesthesia with sevoflurane. METHODS Twenty-eight young patients were allocated for the study. The patients received a tilt test and on the next day they sustained anesthesia induced with inhaled anesthetics. Electrocardiography signals from the patients were relayed to an analogue-digital converter. The Poincaré plot is quantified by measuring SD1, SD2, and SD1/SD2. Power spectral analyses were performed and LF, HF and HF/LF were calculated. RESULTS The LF power and the SD2 of the Poincaré plot increased while subjects were tilt-up from the supine position. Additionally, a significant correlation were found between LF and SD2, HF and SD1 (p < 0.05), and LF/HF and SD2/SD1 (p < 0.01). Sevoflurane inhalation for 10 minutes had no effect on heart rate, but diminished LF, total power and SD1, SD2 of the Poincaré plot respectively. However, the LF, SD2 and LF/HF increased; the HF, SD1 and SD1/SD2 ratio decreased after intubation stimulation. CONCLUSION Poincaré plot and power spectral analysis of HRV during tilt test and sevoflurane induction significantly correlate. Poincaré plot analysis is easier and more sensitive at evaluating the sympathovagal balance and observing the beat-to-beat HRV.

TNF-α inhibits toll-like receptor 4 expression on monocytic cells via tristetraprolin during cardiopulmonary bypass

Toll-like receptor 4 (TLR4) plays a major role in regulating the innate immune response, which is related to postoperative complications. Although inflammatory capacity and TNF-&agr; synthesis were altered on monocytes after cardiopulmonary bypass (CPB), whether the CPB and the CPB-induced TNF-&agr; affect TLR4 expression on monocytes have not yet clarified. We speculate that the changing of TNF-&agr; level during CPB may be involved in monocytic TLR4 expression. As previous report, our enzyme-linked immunosorbent assay showed that CPB elevated the plasma level of TNF-&agr;, whereas off-pump cardiac surgery does not. Flow cytometry reported decreased levels of monocytic TLR4 in patients undergoing CPB but not undergoing off-pump cardiac surgery. To elucidate whether the CPB-induced TNF-&agr; is related to TLR4 down-regulation, we used human monocytic THP-1 cells. Actinomycin D chase experiments demonstrated that TNF-&; decreased TLR4 expression and TLR4 mRNA stability on THP-1. Confocal microscopy and real-time polymerase chain reaction showed that TNF-&agr; induced intracellular tristetraprolin (TTP) expression. Transfection with TTP siRNA reversed the down-regulation of TLR4 in TNF-&agr;-stimulated THP-1. Treatment with ERK1/2 inhibitor and SAPK/JNK inhibitor decreased TNF-&agr;-induced TTP expression. Immunoprecipitation and Western blot analysis showed that the TNF-&agr;-mediated activation of TTP might be inhibited by p38 mitogen-activated protein kinase inhibitor and by PD98059. We also demonstrated in clinical samples with confocal microscopy and flow cytometry that CPB led to an elevation of TTP in monocytes. In conclusion, CPB and TNF-&agr; decrease TLR4 expression on monocytes; TTP expression and mitogen-activated protein kinase-signaling pathways play critical roles in CPB- and TNF-&agr;-mediated decreases of TLR4 on monocytes. Our results suggest that using TTP to control cytokine message decay rate may be a promising approach for controlling system inflammation and preventing post-CPB complications.

Lidocaine priming reduces ADP-induced P-selectin expression and platelet-leukocyte aggregation

BACKGROUND Activation of platelets, which plays an important role in inflammation, has recently been reported to enhance platelet P-selectin expression and form platelet-leukocyte aggregation (PLA). Platelet P-selectin expression and PLA formation have been reported to be potential markers of inflammatory diseases such as sepsis, thrombosis, myocardial ischemic disorders and stroke. Lidocaine, one of the most commonly used anesthetics, is known to inhibit platelet function, but its effect on platelet P-selectin expression and PLA remains unclear. METHODS To determine the effect of lidocaine on platelet activation, and on platelet activation-related septic condition (lipopolysaccharide-induced), we treated platelets with lidocaine (0.03-3 mM) and then measured platelet P-selectin expression and PLA. Whole blood for in vitro study was obtained from healthy men aged 27 to 33 years who had not taken any medication for at least 15 days. RESULTS All samples were analyzed by flow cytometry. We found that lidocaine produced a concentration-dependent inhibition of P-selectin expression and PLA. Moreover, in lipopolysaccharide-challenged samples, lidocaine at concentrations of 1-3 mM inhibited PLA. CONCLUSION Our findings may help to elucidate the inhibitory role of lidocaine on platelet P-selectin expression and PLA and infer possible therapeutic targets in the treatment of inflammatory diseases. However, further investigations are needed to determine whether the observed attenuation of excessive inflammatory responses has clinical implications. These results suggest that lidocaine might have potential clinical application in the modulation of excessive platelet activation, inflammatory response and septic condition.

Amniotic fluid induces platelet-neutrophil aggregation and neutrophil activation

OBJECTIVE Amniotic fluid embolism syndrome is a fatal disease in pregnant women. The exact role of platelets and neutrophils in amniotic fluid embolism syndrome is not clear. We examined whether amniotic fluid could affect platelet-neutrophil aggregation and activation and the possible mechanisms. STUDY DESIGN Blood samples from the pregnant women were pretreated ex vivo with their own amniotic fluid. Flow cytometry was used to measure platelet-neutrophil aggregation and activation. Neutrophil-mediated activity of p38 mitogen-activated protein kinase and extracellular signal-regulated protein kinases 1 and 2 was analyzed by Western blotting. RESULTS Amniotic fluid significantly induced platelet-neutrophil aggregation, neutrophil CD11b expression, and reactive oxygen species production. Amniotic fluid induced minimal platelet P-selectin expression. The increase of intracellular calcium level of neutrophils and the activity of p38 mitogen-activated protein kinase were enhanced by amniotic fluid stimulation. CONCLUSION Amniotic fluid was able to induce neutrophil activation and platelet-neutrophil aggregation with minimal effect on platelet activation. These findings may provide a new insight in the understanding of the pathophysiologic condition of amniotic fluid embolism syndrome.

Phosphoproteomics and Bioinformatics Analyses of Spinal Cord Proteins in Rats with Morphine Tolerance

Introduction Morphine is the most effective pain-relieving drug, but it can cause unwanted side effects. Direct neuraxial administration of morphine to spinal cord not only can provide effective, reliable pain relief but also can prevent the development of supraspinal side effects. However, repeated neuraxial administration of morphine may still lead to morphine tolerance. Methods To better understand the mechanism that causes morphine tolerance, we induced tolerance in rats at the spinal cord level by giving them twice-daily injections of morphine (20 µg/10 µL) for 4 days. We confirmed tolerance by measuring paw withdrawal latencies and maximal possible analgesic effect of morphine on day 5. We then carried out phosphoproteomic analysis to investigate the global phosphorylation of spinal proteins associated with morphine tolerance. Finally, pull-down assays were used to identify phosphorylated types and sites of 14-3-3 proteins, and bioinformatics was applied to predict biological networks impacted by the morphine-regulated proteins. Results Our proteomics data showed that repeated morphine treatment altered phosphorylation of 10 proteins in the spinal cord. Pull-down assays identified 2 serine/threonine phosphorylated sites in 14-3-3 proteins. Bioinformatics further revealed that morphine impacted on cytoskeletal reorganization, neuroplasticity, protein folding and modulation, signal transduction and biomolecular metabolism. Conclusions Repeated morphine administration may affect multiple biological networks by altering protein phosphorylation. These data may provide insight into the mechanism that underlies the development of morphine tolerance.

Hypertonic saline, mannitol and hydroxyethyl starch preconditioning of platelets obtained from septic patients attenuates CD40 ligand expression in vitro

BACKGROUND Because platelet CD40 ligand (CD40L) expression plays an important role in inflammatory conditions, reduction of CD40L expression may be beneficial for patients with sepsis. Although hypertonic saline, mannitol, and hydroxyethyl starch (HES) solutions have been shown to modulate inflammatory responses, their effects on platelet CD40L expression are unclear. We assessed the effects of hypertonic saline, mannitol, and HES solutions on platelet CD40L expression. METHODS Platelet-rich plasma samples were obtained from septic patients and diluted to 1%, 2.5%, 5%, or 7.5% (vol/vol) with 7.5% saline, 3% saline, 0.9% saline, 20% mannitol, 10% HES (200/0.5), or Ringers solution. Twenty-five samples were used per dilution. To determine platelet CD40L expression, platelet samples were stimulated with thrombin (0.1 U/mL), incubated with fluorochrome-conjugated platelet antibodies, and analyzed using flow cytometry. RESULTS Preconditioning of platelet-rich plasma with hypertonic saline, mannitol, and HES attenuated CD40L expression at dilution ratios of 5%, 1%, and 1%, respectively. The decreases were concentration dependent. The effects of mannitol and HES on CD40L expression were almost identical and were superior to those of 3% saline. In contrast, 0.9% saline and Ringers solution had no effect on CD40L expression. CONCLUSIONS Our data show that resuscitation fluids, such as hypertonic saline, mannitol, and HES, inhibit agonist-induced CD40L expression on platelets. These resuscitation fluids may have an anti-inflammatory action when administered to septic patients.

Matrix Metalloproteinase-9 Production following Cardiopulmonary Bypass Was Not Associated with Pulmonary Dysfunction after Cardiac Surgery

Background. Cardiopulmonary bypass (CPB) causes release of matrix metalloproteinase- (MMP-) 9, contributing to pulmonary infiltration and dysfunction. The aims were to investigate MMP-9 production and associated perioperative variables and oxygenation following CPB. Methods. Thirty patients undergoing elective cardiac surgery were included. Arterial blood was sampled at 6 sequential points (before anesthesia induction, before CPB and at 2, 4, 6, and 24 h after beginning CPB) for plasma MMP-9 concentrations by ELISA. The perioperative laboratory data and variables, including bypass time, PaO2/FiO2, and extubation time, were also recorded. Results. The plasma MMP-9 concentrations significantly elevated at 2–6 h after beginning CPB (P < 0.001) and returned to the preanesthesia level at 24 h (P = 0.23), with predominant neutrophil counts after surgery (P < 0.001). The plasma MMP-9 levels at 4 and 6 h were not correlated with prolonged CPB time and displayed no association with postoperative PaO2/FiO2, regardless of reduced ratio from preoperative 342.9 ± 81.2 to postoperative 207.3 ± 121.3 mmHg (P < 0.001). Conclusion. Elective cardiac surgery with CPB induced short-term elevation of plasma MMP-9 concentrations within 24 hours, however, without significant correlation with CPB time and postoperative pulmonary dysfunction, despite predominantly increased neutrophils and reduced oxygenation.

MicroRNA-125a expression in isolated lymphocytes and decreased regulated on activation, normal T-cell expressed and secreted production during cardiac surgery with cardiopulmonary bypass

Background: Cardiopulmonary bypass (CPB) induces postoperative immunosuppression, including decreased T-cells and lower plasma regulated on activation, normal T-cell expressed and secreted (RANTES) concentrations. MicroRNA-125a negatively regulates RANTES expression in activated T-cells. The aims were to investigate microRNA-125a expression in T-cells and RANTES production following CPB. Materials and Methods: Twenty-eight patients undergoing elective cardiac surgery were included in this study. Arterial blood was sampled at six sequential points (before anesthesia induction, before CPB, at 2, 4, 6, and 24 h after beginning CPB) for plasma RANTES concentrations by enzyme-linked immunosorbent assay. T-lymphocytes were isolated from whole blood at four points (before anesthesia, before CPB, at 2 and 4 h after beginning CPB) for intracellular microRNA-125a expression by quantitative real-time reverse transcription polymerase chain reaction in 14 patients. Perioperative laboratory data and variables were also recorded. Results: The plasma RANTES concentrations decreased significantly at 2-24 h after beginning CPB, with concurrent reduction of postoperative lymphocyte counts, as compared with the preanesthesia level (P < 0.001). Intra-T-cell microRNA-125a expression was activated at 2 and 4 h, however, without significance (P = 0.078 and 0.124, respectively). The plasma RANTES levels at 4 h were not correlated with CPB time (P = 0.671), anesthesia time (P = 0.305), postoperative extubation time (P = 0.508), and Intensive Care Unit (ICU) stay (P = 0.756). Three patients expired with pneumonia- or mediastinitis-related septic shock in the ICU. Conclusion: Plasma RANTES concentrations were depressed till 24 h following CPB, with reduced lymphocytes after cardiac surgery. MicroRNA-125a expression in T-lymphocytes was not correlated with perioperative variables and its role in downregulation of RANTES production needs to be determined.

Can hypertonic saline influence platelet P selectin expression and platelet-leukocyte aggregation?☆

OBJECTIVES Part of platelet function involves aggregation and activation. Activation leads to platelet P selectin expression and platelet-leukocyte aggregation. Hypertonic saline inhibits platelet aggregation, although the effects of hypertonic saline on platelet activation are not known. We evaluated the effects of hypertonic saline on platelet activation as measured by platelet P selectin expression and platelet-leukocyte aggregation. METHODS Blood samples from healthy volunteers (n = 6) were treated in vitro with various solutions including 23.5%, 7.5%, 3%, and 0.9% saline; Ringers solution; 5% dextrose in water; and 10% hydroxyethyl starch. Blood was diluted with each type of solution to 2.5%, 5%, 10%, 20%, and 30% (vol/vol) dilution. All blood samples were activated with adenosine diphosphate (20 micromol/L), stained with fluorochrome-conjugated antibodies, and analyzed by flow cytometry to measure platelet P selectin expression and platelet-leukocyte aggregation. RESULTS The 23.5% saline solution reduced P selectin expression at 20% and 30% dilutions and platelet-leukocyte aggregation at 10%, 20%, and 30% dilutions. The 7.5% solution saline had no effect on P selectin expression and significantly inhibited platelet-leukocyte aggregation only at 30% dilution. Other solutions had no effect on platelet P selectin expression or platelet-leukocyte aggregation. CONCLUSIONS Our data suggest that hypertonic saline does not affect platelet P selectin expression or platelet-leukocyte aggregation at therapeutic plasma concentrations but that an inhibitory effect occurs at supratherapeutic doses. Dilutions of other solutions caused the least disturbance of platelet activation.