Apoptosis driven infection

Professional phagocytes like polymorphonuclear neutrophil granulocytes (PMN) and macrophages (MF) kill pathogens as the first line of defense. These cells possess numerous effector mechanisms to eliminate a threat at first contact. However, several microorganisms still manage to evade phagocytic killing, survive and retain infectivity. Some pathogens have developed strategies to silently infect their preferred host phagocytes. The best example of an immune silencing phagocytosis process is the uptake of apoptotic cells. Immune responses are suppressed by the recognition of phosphatidylserine (PS) on the outer leaflet of their plasma membrane. Taking Leishmania major as a prototypic intracellular pathogen, we showed that these organisms can use the apoptotic “eat me” signal PS to silently enter PMN. PS-positive and apoptotic parasites, in an altruistic way, enable the intracellular survival of the viable parasites. Subsequently these pathogens again use PS exposition, now on infected PMN, to silently invade their definitive host cells, the MF. In this review, we will focus on L. major evasion strategies and discuss other pathogens and their use of the apoptotic “eat me” signal PS to establish infection.     

Adenosine protects against suicidal erythrocyte death

Suicidal death of erythrocytes or eryptosis is characterized by cell shrinkage and cell membrane scrambling leading to phosphatidylserine exposure at the erythrocyte surface. The cell membrane scrambling is triggered by an increase in cytosolic Ca²⁺ activity and activation of protein kinase C (PKC). Phosphatidylserine exposure fosters adherence of affected erythrocytes to the vascular wall. Thus, microcirculation in ischemic tissues may be impaired by the appearance of eryptotic erythrocytes. Ischemia leads to release of adenosine, which in most tissues leads to vasodilation and protects against cell injury. The present experiments explored whether adenosine influences mechanisms underlying eryptosis. Erythrocyte phosphatidylserine exposure was estimated from annexin V binding, cell volume from forward scatter and cytosolic Ca²⁺ activity from Fluo3 fluorescence. Glucose depletion (for 24 or 48 h) significantly increased annexin binding and decreased forward scatter, effects partially reversed by adenosine. The protective effect of adenosine reached statistical significance (s.d.) at ≥30 μM. Low Cl⁻ solution (Cl⁻ exchanged by gluconate for 24 h) similarly increased annexin binding and decreased forward scatter, effects again reversed by adenosine (s.d. at ≥10 and 30 μM, respectively). Similarly, phosphatase inhibitor okadaic acid (OA, 1 μM) and PKC activator phorbol 12-myristate-13-acetate (PMA, 3 μM) significantly enhanced annexin binding and decreased forward scatter. Adenosine significantly blunted the effects of OA and PMA on annexin V binding (s.d. at ≥30 and 10 μM, respectively) and the effect of OA on forward scatter (s.d. at ≥10 μM). In conclusion, adenosine inhibits eryptosis by a mechanism presumably effective downstream of PKC. The effect may participate in the maintenance of microcirculation in ischemic tissue.     

Platelet Storage Media Change the Expression Characteristics of the Platelet-Derived Microparticles

Activated platelets shed microparticles in vivo and definitely in vitro upon aging under storage. Studies about the platelet-derived microparticles (PMPs) produced in different storage media of PC were very limited. The aim of this research was to compare some surface molecules of these microvesicles in dissimilar microenvironments; plasma and the candidate medium for the platelet concentrate, Composol. Thirty units of PCs were prepared from Iranian Blood Transfusion Organization. Each unit was divided into two portions. In one of the portions, plasma was replaced with Composol using a connecting device instrument. MPs were isolated from PC and the levels of PS exposure (the annexin-binding capacity) and binding to vWF were surveyed on their surface using ELISA and flow cytometry techniques. The levels of PS exposure were increased on MPs during 7 days storage in the both media but the differences were not significant (P value >0.05). In addition, binding of PMP to vWF was declined during storage. The binding capabilities of PMP were significantly higher in Composol than that of plasma at the day 4 or 7 of storage (P value = 001). It seemed that the binding of PMPs to vWF was affected from the storage media of PC (plasma and Composol) but PS exposure was not affected from the type of storage media.     

MR细胞凋亡成像研究进展

检测细胞凋亡对于早期发现可能病变及评价疾病早期治疗效果具有重要意义。近年出现的许多基于MRI的新型凋亡探针改善了MR成像质量。通过合成针对细胞膜上外翻的磷脂酰丝氨酸和显示半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)活性的尺寸更小的探针,提高了探针组织穿透力;为提高探针与靶点结合的特异性,优化了许多原有探针分子结构;基于MR成像新技术,合成了一些新型探针如Caspase-3敏感的纳米聚合性MR探针(C-SNAM),在一定程度上达到了提高信号强度的目的。     

Microbubble-Assisted Ultrasound-Induced Transient Phosphatidylserine Translocation

Microbubble-assisted ultrasound (sonopermeabilization) results in reversible permeabilization of the plasma membrane of cells. This method is increasingly used in vivo because of its potential to deliver therapeutic molecules with limited cell damage. Nevertheless, the effects of sonopermeabilization on the plasma membrane remain not fully understood. We investigated the influence of sonopermeabilization on the transverse mobility of phospholipids, especially on phosphatidylserine (PS) externalization. We performed studies using optical imaging with Annexin V and FM1-43 probes to monitor PS externalization of rat glioma C6 cells. Sonopermeabilization induced transient membrane permeabilization, which is positively correlated with reversible PS externalization. This membrane disorganization was temporary and not associated with loss of cell viability. Sonopermeabilization did not induce PS externalization via activation of the scramblase. We hypothesize that acoustically induced membrane pores may provide a new pathway for PS migration between both membrane leaflets. During the membrane-resealing phase, PS asymmetry may be re-established by amino-phospholipid flippase activity and/or endocytosis, along with exocytosis processes.     

Caspase inhibition decreases both platelet phosphatidylserine exposure and aggregation: caspase inhibition of platelets

Apoptosis of nucleated cells is regulated by caspases, a group of cysteine proteases, and is characterized by phosphatidylserine expression on the outer leaflet of the plasma membrane. Reports indicate that platelets contain caspases. However, the role of caspases in platelet function is not well understood. When platelets become activated, they express phosphatidylserine (PS) on the outer leaflet of the plasma membrane. In addition, platelets aggregate when activated. The aims of this study were to determine if caspase inhibition (using the pan-caspase inhibitor zVAD-fmk): (1) decreased PS expression and (2) decreased platelet aggregation following activation. Flow cytometry was used to determine PS expression and a platelet aggregometer was used to assess aggregation. We found that platelets treated with zVAD-fmk significantly decreased both A23187-induced PS exposure (total fluorescence index, TFI: A23187=791.42±174; zVAD+A23187=92.97±57, p≤0.05) and ADP-induced PS exposure (TFI: ADP=669.24±145, zVAD+ADP=174.6±151, p≤0.05). Further, treatment with zVAD-fmk significantly decreased ADP-induced platelet aggregation (%: UNTREATED=80±1.5, zVAD TREATED=69±3.0, p≤0.05). These results indicate that caspases play a role in platelet activation, suggesting a unique physiologic role for these proteases.     

Evidence of a Phospholipid Binding Species within Human Fibrinogen Preparations

Fibrinogen has been reported to interact with phospholipid; however, the properties of this binding interaction have not been characterized. Purified preparations of human fibrinogen bound to small unilamellar vesicles containing phosphatidylserine (PS) as measured by light scattering and radioisotope filtration. Binding to 100% PS was saturable (apparent K_d=5 μM, B_max=1.9 g protein/g lipid), reversible, and involved a minor subfraction of the fibrinogen preparation (3–6% of total protein). Fibrinogen interacted minimally with phosphatidylinositol, and not at all with pure phosphatidylcholine (PC) or PC vesicles containing 5% glycosphingolipid (lactosylceramide, ganglioside GM3, ganglioside GD3). Binding efficiency decreased as the PS content of vesicles was diluted with PC. Calcium chloride (2 mM) enhanced protein binding to PS, which was reversed by EDTA. Fibrin clot formation almost quantitatively precipitated the PS binding activity. PS, but not PC, increased the final turbidity of fibrin clots. Computerized sequence analysis of fibrinogen revealed three candidate acidic phospholipid binding motifs located at position 143–210 in the alpha chain, and positions 59–77 and 101–139 in the beta chain. Further study of the PS binding activity of fibrinogen may lead to new insights about fibrinogen function.     

灯盏花素预处理对缺血再灌注心肌细胞凋亡的影响

目的：观察灯盏花素预处理对大鼠缺血再灌注心肌细胞凋亡的影响。方法：40只SD大鼠随机分为假手术组、模型组、灯盏花素预处理Ⅰ组和Ⅱ组。灯盏花素预处理组大鼠于术前1周腹腔注射不同剂量的灯盏花素（25、50mg／kg／d）;采用结扎大鼠左冠状动脉前室间支的方法制备心肌缺血再灌注模型,结扎30min,再灌注2h;采用Annexin V／PI双染,流式细胞仪检测心肌细胞的凋亡情况。结果：灯盏花素预处理可明显降低缺血再灌注大鼠心肌细胞的凋亡率（P〈0．05,P〈0．01）。结论：灯盏花素预处理可抑制心肌细胞凋亡,对缺血再灌注心肌细胞具有保护作用。     

Neuronal Specific Increase of Phosphatidylserine by Docosahexaenoic Acid

Phosphatidylserine (PS), the major acidic phospholipid class in eukaryotic biomembranes, plays an important role in various signaling pathways. We have previously demonstrated that docosahexaenoic acid (DHA, 22:6n-3) positively modulates PS biosynthesis and accumulation in neuronal cells, promoting survival. In this paper, we demonstrate that the increase of PS levels upon DHA enrichment is not a universal mechanism, but specific to neuronal cells. When cells were enriched with 20 muM DHA, 18:0, 22:6-PS increased in both neuronal (Neuro 2A) and non-neuronal cells (Chinese hamster ovary K1 cells, NIH-3T3, and human embryonic kidney cells). However, the increase of the total PS level was observed only in Neuro 2A cells because of the fact that other PS species, such as 18:0, 18:1-PS and 18:1, 18:1-PS decreased significantly in non-neuronal cells, compensating for the increase of 18:0, 22:6-PS. DHA enrichment did not affect the messenger RNA levels of PS synthase 1 (PSS1) and PSS2. Over-expression of genes encoding PSS1 or PSS2 altered neither the PS level nor the effect of DHA on PS increase in both neuronal and non-neuronal cells. From these results, it is concluded that the PS increase by DHA, specifically observed in neuronal cells, may represent a unique mechanism for expanding the PS pool so far known in mammalian cells.