Phase Ib Study of Bavituximab With Carboplatin and Pemetrexed in Chemotherapy-Naive Advanced Nonsquamous Non–Small-Cell Lung Cancer

Introduction

Bavituximab is an immunomodulatory chimeric monoclonal antibody that inhibits phosphatidylserine signaling, which promotes innate and adaptive immune responses. In this phase Ib trial we evaluated the safety, tolerability, and preliminary antitumor activity of pemetrexed, carboplatin, bavituximab in advanced non–small-cell lung cancer (NSCLC).

Novel roles for TIM-1 in immunity and infection

T cell, immunoglobulin domain and mucin domain-1 (TIM-1) is the nominant member of a small family of related proteins that regulate immune cell activities. TIM-1 was initially characterized in a mouse congenic analysis of Th2 T cell responses and related pathology. Data accumulated to date suggest that TIM-1 regulates effector T cell function, and may play distinct roles in the activities of B cells, invariant NKT cells and epithelial cells. In addition, a variety of ligands for TIM-1 have been proposed. In this review I discuss recent data that have accumulated on the function of TIM-1, propose a model to explain how TIM-1 regulates effector T cell activity through recognition of distinct ligands, and review others functions of this increasingly fascinating protein. Of considerable interest are the novel findings that TIM-1 mediates virus entry and virulence.     

正常血细胞表面磷脂酰丝氨酸的表达及其促凝功能

目的 探讨正常成人外周血细胞表面磷脂酰丝氨酸(PS)的表达及其在凝血过程中的作用.方法 取10名正常成人外周血(每份5ml),分离血小板、中性粒细胞、淋巴细胞和红细胞,用乳黏素和组织因子(TF)抗体标记流式细胞仪检测PS和TF的表达.通过凝血复钙时间和内源性或外源性凝血因子Xa以及凝血酶的生成实验测定各细胞组分的促凝活性,并用乳黏素和TF抗体封闭PS和TF,观察其促凝血拮抗作用.结果 正常成人外周血细胞表面有少量PS的表达,阳性率分别为:血小板9.1％、中性粒细胞5.4％、淋巴细胞3.9％、红细胞3.2％;凝血复钙时间试验示血小板、中性粒细胞、淋巴细胞和红细胞分别缩短凝血复钙时间47％、36.5％、25％和12.5％;内源性或外源性凝血因子Xa以及凝血酶生成实验结果显示四种正常血细胞成份使活化酶产量增加13％～26％.结论 正常血细胞表面具有不同程度的PS表达,并在体内凝血过程中起到一定的促凝作用.     

Anticoagulant effects of an antidiabetic drug on monocytes in vitro

Introduction

Monocyte- and microparticle (MP)-associated tissue factor (TF) is upregulated in diabetes. Lipopolysaccharide (LPS) induces expression of TF and alternatively spliced TF (asTF) and increases MP release from monocytes. Using LPS-stimulated TF-bearing human monocytes, we examined whether glibenclamide, a sulfonylurea used to treat diabetes type 2, might possess anticoagulant properties.

Methods

We studied the effects of glibenclamide on cell- and supernatant-associated procoagulant activity (Factor Xa-generating assay and clot formation assay), on expression of TF and asTF (flow cytometry, RT-qPCR, western blot) and on cell viability and MP release (flow cytometry).

Results

Glibenclamide dose-dependently decreased procoagulant activity of cells and supernatants. The reduction in cellular procoagulant activity coincided with reduced expression of TF and asTF in cells, whereas cell viability remained almost unchanged. The glibenclamide-induced reduction in procoagulant activity of supernatants appeared to be associated with a decreased number of released MPs.

Conclusions

Reduction of monocyte- and supernatant-associated procoagulant activity by glibenclamide is associated with decreased expression of TF and asTF and possibly with a reduced MP number. Our data indicate that glibenclamide reduces the prothrombotic state in LPS-stimulated monocytes in vitro. Glibenclamide might therefore also have an anticoagulant effect in vivo, but this needs to be further evaluated.     

Accelerated suicidal erythrocyte death in Klotho-deficient mice

Klotho, a membrane protein mainly expressed in parathyroid glands, kidney, and choroid plexus, counteracts aging and increases the life span. Accordingly, life span is significantly shorter in Klotho-deficient mice (klotho ^−/− ) than in their wild-type littermates (klotho ^+/+ ). The pleotropic effects of Klotho include inhibition of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) formation. Vitamin D-deficient diet reverses the shortening of life span in klotho ^−/− mice. In a variety of cells, 1,25(OH)₂D₃ stimulates Ca²⁺ entry. In erythrocytes, increased Ca²⁺ entry stimulates suicidal erythrocyte death, which is characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. The present study explored the putative impact of Klotho on eryptosis. According to Fluo3 fluorescence, cytosolic Ca²⁺ concentration was significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. According to annexin V-binding, phosphatidylserine exposure was significantly enhanced, and according to forward scatter, cell volume significantly decreased in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Energy depletion (13 h glucose depletion) and oxidative stress (35 min 1 mM tert-butyl-hydroxyl-peroxide [tert-BOOH]) increased phosphatidylserine exposure to values again significantly larger in klotho ^−/− erythrocytes as compared to klotho ^+/+ erythrocytes. Reticulocyte number was significantly increased in klotho ^−/− mice, pointing to enhanced erythrocyte turnover. Vitamin D-deficient diet reversed the enhanced Ca²⁺ entry and annexin V-binding of klotho ^−/− erythrocytes. The present observations reveal a novel function of Klotho, i.e., the at least partially vitamin D-dependent regulation of cytosolic Ca²⁺ activity in and suicidal death of erythrocytes.     

红细胞磷脂酰丝氨酸外露在腹膜透析患者贫血中的意义初探

目的探讨腹膜透析患者红细胞磷脂酰丝氨酸(PS)外露在贫血发生中的作用。方法选择北京大学第一医院67例临床稳定的腹膜透析患者。采用荧光标记的AnnexinV方法结合流式细胞仪测定红细胞PS外露水平,并分析其与贫血的关系。结果多元Logistic回归分析提示红细胞PS外露水平是贫血发生的危险因素之一(Hazardsratio=-0.421,P<0.05)。偏相关分析提示红细胞PS外露水平与血红蛋白水平呈负相关(r=-0.2601,P<0.05)(控制促红细胞生成素因素)。结论红细胞磷脂酰丝氨酸外露增加是腹膜透析患者贫血发生的危险因素,红细胞磷脂酰丝氨酸外露水平越高,患者越容易出现贫血。     

红细胞衰亡与红细胞膜磷脂酰丝氨酸外翻机制研究进展

背景 因成熟红细胞的死亡方式不同于有核细胞,故提出红细胞衰亡概念,描述红细胞的自发性程序性死亡.目的 了解红细胞最终衰亡是否主要通过红细胞膜磷脂酰丝氨酸(phosphatidylserine,PS)外翻暴露,进而被吞噬细胞表面PS受体识别、吞噬和降解清除实现. 内容 综述红细胞衰亡的多种途径,以及这些途径是如何导致红细胞膜PS外翻暴露,进而介导红细胞凋亡及被吞噬降解过程. 趋向 明确两者之间关系可对预防红细胞破坏提供新的思路和方法.     

Inhibition of Rho-ROCK signaling induces apoptotic and non-apoptotic PS exposure in cardiomyocytes via inhibition of flippase

Subsequent to myocardial infarction, cardiomyocytes within the infarcted areas and border zones expose phosphatidylserine (PS) in the outer plasma membrane leaflet (flip-flop). We showed earlier that in addition to apoptosis, this flip-flop can be reversible in cardiomyocytes. We now investigated a possible role for Rho and downstream effector Rho-associated kinase (ROCK) in the process of (reversible) PS exposure and apoptosis in cardiomyocytes. In rat cardiomyoblasts (H9c2 cells) and isolated adult ventricular rat cardiomyocytes Clostridium difficile Toxin B (TcdB), a Rho GTPase family inhibitor, C3 transferase (C3), a Rho(A,B,C) inhibitor and the ROCK inhibitors Y27632 and H1152 were used to inhibit Rho-ROCK signaling. PS exposure was assessed via flow cytometry and fluorescent digital imaging microscopy using annexin V. Akt expression and phosphorylation were analyzed via Western blot, and Akt activity was inhibited by wortmannin. The cellular concentration activated caspase 3 was determined as a measure of apoptosis, and flippase activity was assessed via flow cytometry using NBD-labeled PS. TcdB, C3, Y27632 and H1152 all significantly increased PS exposure. TcdB, Y27632 and H1152 all significantly inhibited phosphorylation of the anti-apoptotic protein Akt and Akt inhibition by wortmannin lead to increased PS exposure. However, only TcdB and C3, but not ROCK- or Akt inhibition led to caspase 3 activation and thus apoptosis. Notably, pancaspase inhibitor zVAD only partially inhibited TcdB-induced PS exposure indicating the existence of apoptotic and non-apoptotic PS exposure. The induced PS exposure coincided with decreased flippase activity as measured with NBD-labeled PS flip-flop. In this study, we show a regulatory role for a novel signaling route, Rho-ROCK-flippase signaling, in maintaining asymmetrical membrane phospholipid distribution in cardiomyocytes.     

Suicidal death of erythrocytes in recurrent hemolytic uremic syndrome

Hemolytic uremic syndrome (HUS) is characterized by hemolytic anemia with fragmented erythrocytes, thrombocytopenia, and acute renal failure. Lack of complement inactivating factor H predisposes to the development of atypical HUS. Little is known about mechanisms linking complement activation with loss of erythrocyte integrity during HUS. Recent studies disclosed that increased cytosolic Ca²⁺ activity and cellular ceramide trigger programmed erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. In the present study, we investigated whether eryptosis occurs during the course of HUS. To this end, erythrocytes from healthy volunteers were exposed to plasma from a patient with severe idiopathic recurrent HUS secondary to factor H depletion. Phosphatidylserine exposure (Annexin binding), cell volume (forward scatter), cytosolic Ca²⁺ activity (Fluo3 fluorescence), and ceramide formation [anti-ceramide antibody and enzymatic (diacylgycerol kinase) analysis] were determined. Exposure of erythrocytes to plasma from the patient, but not to plasma from healthy individuals, triggered Annexin binding. The effect of plasma on erythrocyte Annexin binding was abolished by plasmapheresis or filtration at 30 kDa. It was paralleled by formation of ceramide and increase of cytosolic Ca²⁺ activity. Enhanced Annexin binding of erythrocytes from healthy individuals was observed after exposure to plasma from three other patients with HUS. The proeryptotic effect of patient plasma was mimicked by exposure to the Ca²⁺ ionophore ionomycin, and eryptosis was potentiated in the presence of cell membrane-permeable C₆-ceramide. Furthermore, in vitro complement activation similarly triggered erythrocyte phosphatidylserine exposure, an effect which was blunted by the addition of factor H. In conclusion, our present observations disclose a novel, pathophysiological, factor-H dependent mechanism leading to injury of erythrocytes during the course of hemolytic uremic syndrome.     

Betanodavirus induces phosphatidylserine exposure and loss of mitochondrial membrane potential in secondary necrotic cells, both of which are blocked by bongkrekic acid

In this study, we show how the red spotted grouper nervous necrosis virus (RGNNV) causes loss of mitochondrial membrane potential and promotes host secondary apoptotic necrosis. RGNNV viral proteins such as protein alpha (42 kDa) and protein A (110 kDa) were quickly expressed between 12 h and 24 h postinfection (p.i.) in GL-av cells. Annexin V staining revealed that the NNV infection of GL-av cells induced phosphatidylserine (PS) externalization and development of bulb-like vesicles (bleb formation) at 24 h p.i. NNV infection also induced DNA fragmentation detectable by TUNEL assay between 12 h (8%) and 72 h (32%) p.i. Bongkrekic acid (1.6 microM; BKA) blocked permeability of the mitochondrial permeability transition pore, but cyclosporine A (CsA) did not block secondary necrosis. Finally, secondary necrotic cells were not engulfed by neighboring cells. Our data suggest that RGNNV induces apoptotic death via opening the mitochondrial permeability transition pore thereby triggering secondary necrosis in the mid-apoptotic phase.