Leukocyte- and Platelet-Derived Microvesicle Interactions following In Vitro and In Vivo Activation of Toll-Like Receptor 4 by Lipopolysaccharide

BACKGROUND: Pro-coagulant membrane microvesicles (MV) derived from platelets and leukocytes are shed into the circulation following receptor-mediated activation, cell-cell interaction, and apoptosis. Platelets are sentinel markers of toll-like receptor 4 (TLR4) activation. Experiments were designed to evaluate the time course and mechanism of direct interactions between platelets and leukocytes following acute activation of TLR4 by bacterial lipopolysaccharide (LPS). METHODOLOGY/PRINCIPAL FINDINGS: Blood from age-matched male and female wild type (WT) and TLR4 gene deleted (dTLR4) mice was incubated with ultra-pure E. coli LPS (500 ng/ml) for up to one hour. At designated periods, leukocyte antigen positive platelets, platelet antigen positive leukocytes and cell-derived MV were quantified by flow cytometry. Numbers of platelet- or leukocyte-derived MV did not increase within one hour following in vitro exposure of blood to LPS. However, with LPS stimulation numbers of platelets staining positive for both platelet- and leukocyte-specific antigens increased in blood derived from WT but not dTLR4 mice. This effect was blocked by inhibition of TLR4 signaling mediated by My88 and TRIF. Seven days after a single intravenous injection of LPS (500 ng/mouse or 20 ng/gm body wt) to WT mice, none of the platelets stained for leukocyte antigen. However, granulocytes, monocytes and apoptotic bodies stained positive for platelet antigens. CONCLUSIONS/SIGNIFICANCE: Within one hour of exposure to LPS, leukocytes exchange surface antigens with platelets through TLR4 activation. In vivo, leukocyte expression of platelet antigen is retained after a single exposure to LPS following turn over of the platelet pool. Acute expression of leukocyte antigen on platelets within one hour of exposure to LPS and the sustained expression of platelet antigen on leukocytes following a single acute exposure to LPS in vivo explains, in part, associations of platelets and leukocytes in response to bacterial infection and changes in thrombotic propensity of the blood

Evaluation and treatment for ovotesticular disorder of sex development (OT-DSD) - experience based on a Chinese series

Abstract Background The aim of this study is to review and present the clinical features and process of evaluation and treatment for OT-DSD in a single center in recent years in China. Methods Sixteen patients with OT-DSD during the past 4 years underwent the evaluation and treatment in a single center. The clinical characteristics and outcomes of surgery were analyzed. Results The surgical age ranged from 17 months to 66 months with a mean age of 20 months, and the mean follow-up was 30 months (4 months to 56 months). The presentation in 11 patients was ambiguous genitalia, and the rest 5 patients were suspected to have DSD in preoperative examination before hypospadias repair. The karyotypes were 46, XX in 11 patients, 46, XX/46, XY in 3, 46, XX/47, XXY in 1, and 46, XY in 1. Initial reared sex was male in 14 patients, female in 1, and undetermined in 1. After surgery, genders were reassigned in 3 patients, while 15 patients were raised as male with testicular tissue left. Only 1 patient with ovarian tissue left was raised as female. Repair was completed in 11 males and 1 female, and stage I urethroplasty was done in 4 males. No further surgery to remove the gonads was needed for inconsonance of gender assignment. No gonadal tumors were detected. Conclusions OT-DSD is a rare and complex deformity with few systematic reports in China. It’s important to establish a regular algorithm for evaluation and treatment of OT-DSD

Influencing factors analysis of anammox bacteria cultured by mixing denitrifying-anammox

In order to get the optimal growth conditions of anammox bacteria, the mature-cultured anammox granule sludge is used to investigate the influencing factors. The effects of temperature, pH value, COD and influent substrate (NO-2-N and NH+4-N) on anammox bacteria activity are investigated. The results demonstrate that the optimal temperature is 40 ℃ and the optimal pH value is between 7.0~8.0 for anammox bacteria. The anammox bacteria activity is not inhibited severely when COD concentration is lower than 100 mg/L, while the denitrifying bacteria is dominant and inhibits the anammox bacteria activity when COD concentration is higher than 100 mg/L. When the influent NH+4-N and NO-2-N concentration are lower than 1 540 mg/L and 140 mg/L respectively, the anammox bacteria activity is not inhibited severely. Controlling the optimal growth conditions of anammox technical helps the rapid growth of anammox bacteria, which establishes the foundation for the rapid start-up of anammox reactor

Up-Regulatory Effects of Curcumin on Large Conductance Ca2+-Activated K+ Channels.

Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms

Curcumin inhibits transforming growth factor-β1-induced EMT via PPARγ pathway, not Smad pathway in renal tubular epithelial cells.

Tubulointerstitial fibrosis (TIF) is the final common pathway in the end-stage renal disease. Epithelial-to-mesenchymal transition (EMT) is considered a major contributor to the TIF by increasing the number of myofibroblasts. Curcumin, a polyphenolic compound derived from rhizomes of Curcuma, has been shown to possess potent anti-fibrotic properties but the mechanism remains elusive. We found that curcumin inhibited the EMT as assessed by reduced expression of α-SMA and PAI-1, and increased E-cadherin in TGF-β1 treated proximal tubular epithelial cell HK-2 cells. Both of the conventional TGF-β1/Smad pathway and non-Smad pathway were investigated. Curcumin reduced TGF-β receptor type I (TβR-I) and TGF-β receptor type II (TβR II), but had no effect on phosphorylation of Smad2 and Smad3. On the other hand, in non-Smad pathway curcumin reduced TGF-β1-induced ERK phosphorylation and PPARγ phosphorylation, and promoted nuclear translocation of PPARγ. Further, the effect of curcumin on α-SMA, PAI-1, E-cadherin, TβR I and TβR II were reversed by ERK inhibitor U0126 or PPARγ inhibitor BADGE, or PPARγ shRNA. Blocking PPARγ signaling pathway by inhibitor BADGE or shRNA had no effect on the phosphorylation of ERK whereas the suppression of ERK signaling pathway inhibited the phosphorylation of PPARγ. We conclude that curcumin counteracted TGF-β1-induced EMT in renal tubular epithelial cells via ERK-dependent and then PPARγ-dependent pathway

Implementation of an Integrated Dielectrophoretic and Magnetophoretic Microfluidic Chip for CTC Isolation

Identification of circulating tumor cells (CTCs) from a majority of various cell pools has been an appealing topic for diagnostic purposes. This study numerically demonstrates the isolation of CTCs from blood cells by the combination of dielectrophoresis and magnetophoresis in a microfluidic chip. Taking advantage of the label-free property, the separation of red blood cells, platelets, T cells, HT-29, and MDA-231 was conducted in the microchannel. By using the ferromagnet structure with double segments and a relatively shorter distance in between, a strong gradient of the magnetic field, i.e., sufficiently large MAP forces acting on the cells, can be generated, leading to a high separation resolution. In order to generate strong DEP forces, the non-uniform electric field gradient is induced by applying the electric voltage through the microchannel across a pair of asymmetric orifices, i.e., a small orifice and a large orifice on the opposite wall of the channel sides. The distribution of the gradient of the magnetic field near the edge of ferromagnet segments, the gradient of the non-uniform electric field in the vicinity of the asymmetric orifices, and the flow field were investigated. In this numerical simulation, the effects of the ferromagnet structure on the magnetic field, the flow rate, as well as the strength of the electric field on their combined magnetophoretic and dielectrophoretic behaviors and trajectories are systemically studied. The simulation results demonstrate the potential of both property- and size-based cell isolation in the microfluidic device by implementing magnetophoresis and dielectrophoresis

Anticoagulant Activities of Indobufen, an Antiplatelet Drug

Indobufen is a new generation of anti-platelet aggregation drug, but studies were not sufficient on its anticoagulant effects. In the present study, the anticoagulant activity of indobufen was determined by monitoring the activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) in rabbit plasma. We evaluated the anticoagulant mechanisms on the content of the platelet factor 3,4 (PF3,4), and the coagulation factor 1, 2, 5, 8, 10 (FI, II, V, VIII, X) in rabbits, as well as the in vivo bleeding time and clotting time in mice. The pharmacodynamic differences between indobufen and warfarin sodium, rivaroxaban, and dabigatran were further studied on thrombus formation and the content of FII and FX in rats. Animal experiments showed that intragastric-administrated indobufen can significantly reduce the APTT, PT, TT, PF3, FI, II, V, VIII, and X plasma contents. Its inhibitory effect on plasma FII was better than thrombin inhibitor dabigatran with effect on FX better than FXa inhibitor rivaroxaban. These results suggest that indobufen has some anticoagulant effects as strong as some conventional anticoagulants. The mechanism may be related to both exogenous and endogenous coagulation system

Assessment of psychosocial functioning and its risk factors in children with pectus excavatum

Abstract Background Psychosocial functioning is poor in patients with pectus excavatum (PE). However, a comprehensive understanding of this issue does not exist. The aim of this study was to assess the severity of psychosocial problems as associated with PE, as well as to identify its risk factors. Methods A comparative study was performed at the Sichuan Academy of Medical Sciences/Sichuan Provincial People's Hospital in Chengdu, China. Patients age 6 to 16 who admitted to the outpatient department for the evaluation or treatment for PE were included in the study. In addition to parental reports of child psychosocial problems on the Achenbach Child Behavior Checklist (CBCL), parents also filled in other structured questionnaires, including socio-demographic variables, patients' medical and psychological characteristics. The severity of malformation was assessed by CT scan. For comparison, an age- and gender- matched control group was recruited from the general population. The socio-demographic and scores on CBCL were compared between patients and control subjects. Univariate and multivariate analysis were performed to examine risk factors for psychosocial problems in patients. Results No statistically significant differences were found with respect to social-demographic variables between children with PE and control subjects. Compared with control subjects, children with PE displayed higher prevalence of psychosocial problems in the different scales of the CBCL questionnaire such as 'withdraw', 'anxious-depressed', 'social problems' and 'total problems'. Both univariate and multivariate analyses suggested that age, severity of malformation, and being teased about PE were significantly associated with patients' psychosocial problems. Conclusions The information derived from this study supports the opinion that children with PE have more psychosocial problems than children from the general population. Multiple medical and psychosocial factors were associated with patients' impairment of psychosocial functioning.</p