Effect of bacterial peptidoglycan on erythrocyte death and adhesion to endothelial cells

Peptidoglycans, bacterial wall components, have previously been shown to trigger eryptosis, the suicidal erythrocyte death, characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine exposing erythrocytes adhere to the vascular wall at least partially by interaction of erythrocytic phosphatidylserine with endothelial CXC chemokine ligand 16 (CXCL16). The present study explored whether peptidoglycan exposure fosters the adhesion of erythrocytes to human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were treated for 48 h with peptidoglycan (10 μg/ml) and CXCL16 abundance determined by confocal microscopy and FACS analysis. Moreover, human erythrocytes were exposed for 48 h to peptidoglycan (10 μg/ml) and phosphatidylserine exposure estimated from binding of fluorescent annexin-V, cell volume from forward scatter in FACS analysis and erythrocyte adhesion to human umbilical vein endothelial cells (HUVEC) from trapping of labeled erythrocytes in a flow chamber. As a result, bacterial peptidoglycan exposure was followed by increased CXCL16 expression in HUVEC as well as erythrocyte shrinkage, phosphatidylserine exposure and adhesion to HUVEC under flow conditions at arterial shear rates. The adhesion was significantly attenuated but not abrogated in the presence of either, erythrocyte phosphatidylserine-coating annexin-V (5 μl/ml) or CXCL16 neutralizing antibody directed against endothelial CXCL16 (4 μg/ml). In conclusion, exposure to peptidoglycan increases endothelial CXCL16 expression and leads to eryptosis followed by phosphatidylserine- and CXCL16-mediated adhesion of eryptotic erythrocytes to vascular endothelial cells.     

A role of phosphatidylserine externalization in clearance of erythrocytes exposed to stress but not in eliminating aging populations of erythrocyte in mice

Age dependent changes in phosphatidylserine (PS) externalization were studied in mouse erythrocytes of different age groups (range 1-55 days) by using a newly developed double in vivo biotinylation (DIB) technique. Around 3-4% of the erythrocytes freshly released in the circulation were PS(+) but this proportion fell rapidly to 1% or less and did not increase at later time points. Blocking erythrocyte clearance from the circulation by in vivo depletion of macrophages (by treatment with clodronate loaded liposomes) for up to 7 days did not result in accumulation of PS(+) erythrocytes in the circulation indicating that the low percentage of PS(+) cells within old erythrocytes (age >40 days) was not related to the clearance of PS(+) erythrocytes by macrophages. In vitro treatment with stress inducing agents like deoxyglucose or Ca(++)/calcium ionophore resulted in a marked induction of PS externalization in mouse erythrocytes and this effect was most prominent in the youngest erythrocyte population (age <10 days). Kinetics of clearance of different age groups of stress exposed erythrocytes after intravenous infusion into recipient mice indicated that the young erythrocytes were cleared at fastest rate from the circulation as compared to erythrocytes of older age groups. Within young erythrocytes exposed to stress, PS(+) erythrocytes were preferentially cleared. Taken together our results suggest that PS externalization is unlikely to have a role in the removal of old erythrocytes from blood circulation but may have a role in the clearance of stressed and damaged young erythrocytes in blood circulation.     

Induction of apoptotic erythrocyte death by rotenone

The pesticide rotenone stimulates apoptosis and rotenone intoxication has been considered a cause of Parkinson's disease. Rotenone further sensitizes tumor cells to cytotoxic drugs. The apoptotic effect of rotenone is at least partially due to mitochondrial injury. Even though lacking mitochondria and nuclei, erythrocytes may undergo eryptosis, an apoptosis-like suicidal death characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine-exposure at the cell surface. Triggers of eryptosis include increase of cytosolic Ca(2+)-activity ([Ca(2+)](i)) and enhanced ceramide formation. The present study explored, whether rotenone elicits eryptosis. To this end, [Ca(2+)](i) was estimated utilizing Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine-exposure from annexin-V-binding, ceramide utilizing fluorescence antibodies and hemolysis from hemoglobin release. A 48 h exposure to rotenone significantly increased Fluo3-fluorescence(i) (≥1 μM), increased ceramide abundance (10 μM), decreased forward scatter (≥2.5 μM) and increased annexin-V-binding (≥ 1 μM). Rotenone exposure was further followed by slight but significant hemolysis. Rotenone-induced cell membrane scrambling was significantly blunted, but not completely abrogated by removal of extracellular Ca(2+). The present observations disclose a novel effect of rotenone, i.e. triggering of erythrocyte shrinkage and cell membrane scrambling, an effect paralleled by and partially dependent on Ca(2+)-entry.     

Arsenic-induced suicidal erythrocyte death

Environmental exposure to arsenic has been associated with anemia, which could result from suicidal erythrocyte death or eryptosis, characterized by cell shrinkage and phosphatidylserine exposure at the erythrocyte surface. Eryptosis is triggered by increase in cytosolic Ca²⁺ concentration, ceramide and energy depletion. The present experiments explored, whether arsenic stimulates eryptosis. According to annexin V-binding, arsenic trioxide (7 μM) within 48 h significantly increased phosphatidylserine exposure of human erythrocytes without inducing hemolysis. According to forward scatter, arsenic trioxide (7 μM) significantly decreased cell volume. Moreover, Fluo3-fluorescence showed that arsenic (10 μM) significantly increased cytosolic Ca²⁺ concentration. According to binding of respective fluorescent antibodies, arsenic trioxide (10 μM) significantly increased ceramide formation. Arsenic (10 μM) further lowered the intracellular ATP concentration. Removal of extracellular Ca²⁺ or inhibition of the Ca²⁺-permeable cation channels with amiloride blunted the effects of arsenic on annexin V-binding and cell shrinkage. In conclusion, arsenic triggers suicidal erythrocyte death by increasing cytosolic Ca²⁺ concentration, by stimulating the formation of ceramide and by decreasing ATP availability.     

Damage to the cell antioxidative system in human erythrocytes incubated with idarubicin and glutaraldehyde

Encapsulation of antineoplastic drugs within erythrocytes is one of the studied strategies to diminish the toxic side effects of anthracycline antibiotics. Glutaraldehyde is often used as crosslinking agent to link the drugs, including idarubicin (IDA) to the cells. The previous studies indicated that in glutaraldehyde-treated human erythrocytes the elevated level of drug was observed but also the various changes in the organization of the red cells were noted.In this study, we continue our investigations and now we concentrate on the effect of these compounds on antioxidative system in erythrocytes. We determined reactive oxygen species (ROS) production, glutathione content and alterations in the activity of enzymes responsible for maintaining glutathione in reduced form in human erythrocytes. Measurements of both reduced and total glutathione levels and the activity of glutathione reductase and glucose-6-phosphate dehydrogenase were performed spectrophotometrically. The results show that ROS were produced in erythrocytes treated with IDA and with IDA and glutaraldehyde. IDA at a concentration of 10 μg/ml did not cause any changes in total or reduced glutathione levels. When IDA-preincubated erythrocytes were treated with glutaraldehyde, significant changes in the determined parameters were observed in a glutaraldehyde concentration dependent manner. It was correlated with decreased activity of glutathione reductase (GR) and glucose-6-phosphate dehydrogenase (G6PD). Together with the significant changes in reduced form of glutathione (GSH)/total glutathione ratio, the exposure of phosphatidylserine at the cell surface was also observed.     

A critical role for phagocytosis in resistance to malaria in iron-deficient mice

Both iron-deficient anemia (IDA) and malaria remain a threat to children in developing countries. Children with IDA are resistant to malaria, but the reasons for this are unknown. In this study, we addressed the mechanisms underlying the protection against malaria observed in IDA individuals using a rodent malaria parasite, Plasmodium yoelii (Py). We showed that the intra-erythrocytic proliferation and amplification of Py parasites were not suppressed in IDA erythrocytes and immune responses specific for Py parasites were not enhanced in IDA mice. We also found that parasitized IDA cells were more susceptible to engulfment by phagocytes in vitro than control cells, resulting in rapid clearance of parasitized cells and that protection of IDA mice from malaria was abrogated by inhibiting phagocytosis. One possible reason for this rapid clearance might be increased exposure of phosphatidylserine at the outer leaflet of parasitized IDA erythrocytes. The results of this study suggest that parasitized IDA erythrocytes are eliminated by phagocytic cells, which sense alterations in the membrane structure of parasitized IDA erythrocytes.     

Loss of phospholipid asymmetry and elevated brain apoptotic protein levels in subjects with amnestic mild cognitive impairment and Alzheimer disease

Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process.The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro- and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.     

结肠直肠癌中PISD的表达及其与临床病理的关系

目的:检测Wnt通路靶基因PISD在结肠直肠细胞及组织标本中的表达情况及其与临床病理的关系。方法:应用Western印迹法测定结肠直肠癌细胞株中PISD的蛋白表达水平,应用免疫组化法测定PISD在组织标本中的表达情况及其与临床病理的关系。结果:Western印迹法结果显示PISD在7种结肠直肠癌细胞株中均呈阳性表达,免疫组化结果显示PISD在结肠直肠癌组织中表达阳性,灵敏度为62.9%。结论:PISD在结肠直肠癌细胞及组织中表达上调,但与肿瘤的转移无明显相关。     

A case of hyperhemolysis syndrome in sickle cell disease and concomitant COVID-19

BackgroundHyperhemolysis syndrome (HHS) is an uncommon transfusion reaction described in several hematologic disorders, including sickle cell disease (SCD). HHS is characterized by a decline in hemoglobin (Hb) values below pre-transfusion levels following transfusion of red blood cells (RBCs), coupled with laboratory markers consistent with hemolysis. The proposed pathophysiologic mechanisms underlying HHS include increased phosphatidylserine expression, macrophage activation, and complement dysregulation. Many pathophysiologic mechanisms thought to contribute to HHS have been similarly described in cases of severe COVID-19.Case reportA 28-year-old male with a history of HbSS presented with shortness of breath, right-sided chest pain, and a two-day history of fever. Polymerase chain reaction (PCR) detected SARS-CoV-2 infection with the omicron variant. The patient required an RBC transfusion (pre-transfusion hemoglobin [Hb]5.8 g/dL) with an immediate post-transfusion Hb of 6.3 g/dL. However, Hb rapidly declined to 1.7 g/dL, and lactate dehydrogenase (LDH) rose to 8701 u/L. The absolute reticulocyte count of 538 × 10⁹/L correspondingly fell to 29 × 10⁹/L. Despite additional RBC transfusions and initiation of immunosuppressive therapy, he expired on Day 9(D9).ConclusionGiven the similarities in their proposed pathophysiology, patients with SCD and concomitant SARS-CoV-2 infection may be predisposed to developing HHS.     

Early antithrombotic therapy for another highly lethal viral pneumonia pandemic

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