Two-way trafficking of Annexin V positive cells between mother and fetus: determination of apoptosis at delivery

2007 ◽  
Vol 27 (4) ◽  
pp. 348-351 ◽  
Author(s):  
A. Kolialexi ◽  
G.Th Tsangaris ◽  
A. Anagnostopoulos ◽  
D. Chondros ◽  
V. Bagiokos ◽  
...  
Keyword(s):  
Annexin V

scholarly journals Stimulation of Erythrocyte Cell Membrane Scrambling by C-Reactive Protein

2017 ◽  
Vol 41 (2) ◽  
pp. 806-818 ◽  
Author(s):  
Majed Abed ◽  
Christian Thiel ◽  
Syeda T. Towhid ◽  
Kousi Alzoubi ◽  
Sabina Honisch ◽  
...  
Keyword(s):  
Plasma Concentration ◽  
Healthy Volunteers ◽  
Annexin V ◽  
Caspase Activation ◽  
C Reactive Protein ◽  
Forward Scatter ◽  
Reactive Protein ◽  
Phosphatidylserine Translocation ◽  
Binding Cell

Background: Eryptosis, the suicidal erythrocyte death characterized by cell shrinkage and phosphatidylserine-translocation, is triggered by fever and inflammation. Signaling includes increased cytosolic Ca2+-activity ([Ca2+]i), caspase activation, and ceramide. Inflammation is associated with increased plasma concentration of C-reactive protein (CRP). The present study explored whether CRP triggers eryptosis. Methods: Phosphatidylserine abundance at the cell surface was estimated from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide abundance and caspase-3-activity utilizing FITC-conjugated antibodies. Moreover, blood was drawn from patients with acute appendicitis (9♀,11♂) and healthy volunteers (10♀,10♂) for determination of CRP, blood count and phosphatidylserine. Results: A 48h CRP treatment significantly increased the percentage of annexin-V-binding cells (≥5µg/ml), [Ca2+]i (≥5µg/ml), ceramide (20µg/ml) and caspase-activity (20µg/ml). Annexin-V-binding was significantly blunted by caspase inhibitor zVAD (10µM). The percentage of phosphatidylserine-exposing erythrocytes in freshly drawn blood was significantly higher in appendicitis patients (1.83±0.21%) than healthy volunteers (0.81±0.09%), and significantly higher following a 24h incubation of erythrocytes from healthy volunteers to patient plasma than to plasma from healthy volunteers. The percentage of phosphatidylserine-exposing erythrocytes correlated with CRP plasma concentration. Conclusion: C-reactive protein triggers eryptosis, an effect at least partially due to increase of [Ca2+]i, increase of ceramide abundance and caspase activation.

Antiphospholipid Antibody Syndrome: Annexin V Competitive Flow Cytometric Assay for Determination of Anti-Platelet Phospholipid Autoantibodies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4126-4126
Author(s):  
Aaron Tomer ◽  
Shira Barlev ◽  
Mahmoud Abu-Shakra ◽  
Boris Shenkman
Keyword(s):  
Lupus Erythematosus ◽  
Annexin V ◽  
Antiphospholipid Antibody ◽  
Antiphospholipid Antibody Syndrome ◽  
Flow Cytometric Assay ◽  
Platelet Phospholipid ◽  
Standard Equipment ◽  
Anionic Phospholipids ◽  
Flow Cytometric

Abstract A flow cytometric assay was developed for the determination of autoantibodies directed against platelet anionic-phospholipids in antiphospholipid syndrome (APS). The method is based on demonstrable competition between the placental anticoagulant protein I, annexin V, and the patients’ autoantibodies on the platelet anionic-phospholipids (the binding site for the prothrombinase complex; prothrombin, factors Va and Xa). The method is practical and rapid, uses readily available reagents, and standard equipment. Ninety-two plasma samples, 41 from patients with clinical diagnosis of APS, 27 from patients with systemic lupus erythematosus (SLE) and 24 from healthy individuals were analyzed. Thirty-five (85%) of patients with APS (either with or without SLE), 15 (94%) of patients with APS and SLE, and 20 (80%) of APS patients without SLE were positive. Nineteen (70%) out of 27 patients with SLE alone were also positive; of whom 15 (58%) were positive for either anti-cardiolipin antibody (ACL) or lupus anti-coagulant (LAC). Comparison with ACL showed 40 (93%) out of 43 patients positive for ACL were also positive by flow cytometry (FCM). However, 13 (48%) out of 27 patients negative for ACL were found positive by FCM. Seven of these patients have primary APS and 6 have SLE; 7 of whom were positive for LAC. Three (13%) out of the 24 control samples, all negative by FCM, were positive for ACL. Comparison with LAC showed 32 (91%) out of 35 patients positive for LAC also positive by FCM. Of 18 patients negative for LAC, 7 (39%) were negative and 11 (61%) were positive by FCM. Four of the 11 patients have a diagnosis of APS and 7 of SLE; 6 of whom were positive for ACL. None of the controls, all negative by FCM, was positive for LAC. In conclusion, the annexin V competitive flow cytometric assay for the determination of anti-platelet phospholipid autoantibodies maybe useful for the laboratory diagnosis of APS.

Caffeic Acid Phenethyl Ester Induces Apoptosis in Acute Myeloid Leukemia Cells - Possible Role of Chemokine/G-Protein Signaling.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4802-4802
Author(s):  
Priscila S. Scheucher ◽  
Guilherme Augusto Silva dos Santos ◽  
Hamilton Luiz G Teixeira ◽  
Roberto P. Falcao ◽  
Eduardo Magalhaes Rego
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Cell Lines ◽  
Annexin V ◽  
Caffeic Acid Phenethyl Ester ◽  
Nb4 Cells ◽  
Induction Of Apoptosis ◽  
Induced Apoptosis

Abstract Abstract 4802 Caffeic acid phenethyl ester (CAPE) is an active phenolic compound present in propolis obtained from honeybee hives. It is reported to present a spectrum of biological activities including antioxidant, anti-inflammatory and antitumoral. The antitumoral activity of CAPE as evaluated by several studies in vitro and in vivo seems to be related to distinct effects like inhibition of angiogenesis, invasion and metastasis and induction of apoptosis or differentiation of cancer cells. In the scenario of AML the demonstration of CAPE-induced apoptosis or cellular differentiation is restricted to the HL-60 cell line. Our aim was to evaluate the effects of CAPE treatment on primary AML samples as well as APL cell lines NB4 and NB4-R2 (a cell resistant to ATRA-induced differentiation) and on AML cell line Kasumi-1 (representative of core binding factor leukemia with AML1-ETO rearrangement). Proliferation and viability was evaluated by cell count with tripan blue in Neubauer chamber at fixed time intervals. Differentiation was evaluated by flow cytometer determination of CD11b expression. Apoptotic cells were defined as sub-G0 fraction and were evaluated by flow cytometer determination of propidium iodide- DNA fluorescence. Also apoptosis was detected by the annexin-V method. Leishman stained cytospins were used to confirm apoptosis or differentiation. CAPE did not induce differentiation in the cell lines NB4, NB4-R2 or Kasumi-1 and did not alter the differentiation induced by ATRA in NB4 cells. CAPE inhibited the proliferation of AML cell lines in a time and dose dependent fashion. The ED50 in 24h treatment for NB4 cell line (tripan blue) was 32.1 mcg/ml. ED50 (at 24h) for induction of apoptosis in the more sensitive assay using annexin-V in NB4 cells after 24h was 7.5mcg/ml and for Kasumi-1 was 10.2mcg/ml. CAPE (32 mcg/ml) significantly induced apoptosis after 24h in cells from AML patients (n=10), mean (IC95%) of 40.5% (29.26 – 51.76) versus control treated cells 18.16% (12.27 – 24.05); p=0.0004 In order to evaluate the mechanisms of CAPE-induced apoptosis in NB4 cells we performed a microarray analysis after 12 hours treatment (32mcg/ml). The majority of downregulated genes fall into two categories: positive cell cycle regulators and ribosomal genesis / protein traduction. In the other hand, upregulated genes fall into several categories, we point out chemokines and G- protein signalization genes. (Table 1 and 2) The role of IL-8 and Gro chemokines, that signaling by G-protein coupled receptors, has been determined in tumor progression and invasiveness. We are currently investigating the possibility that CAPE exerts an inhibitory effect in chemokine signaling in APL. In conclusion, CAPE-induced apoptosis in AML is associated with the regulation of specific genes. These properties are interesting and need further investigation. Disclosures: No relevant conflicts of interest to declare.

Novel murine anti-HER2 monoclonal antibodies to induce apoptosis and regulate miR-21 in breast cancer cell.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3081-3081
Author(s):  
Alice Y. Kim ◽  
Cindy Zer ◽  
John C. Williams ◽  
Michael F. Press ◽  
Jinha Mark Park
Keyword(s):  
Breast Cancer ◽  
Cell Proliferation ◽  
Intracellular Signaling ◽  
Epitope Mapping ◽  
Annexin V ◽  
Apoptotic Cells ◽  
Antibody Treatment ◽  
Steady Level ◽  
Proliferation Assays

3081 Background: MicroRNAs play critical roles in biological and pathological processes. Of these, miR-21 is frequently observed to be overexpressed in various cancers. However, treatment of breast cancer cells with trastuzumab enhances the level of miR-21 by a relative fold of 2.26 (Ichikawa, PLos ONE, 2012, p.5). In this study, we demonstrate that two murine anti-HER2 mAbs, 10H8 and 8H11, while inhibiting cell proliferation and inducing apoptosis, do not perturb the level of miR-21 in the same way as trastuzumab. To understand the differential effects of trastuzmab and the two mAbs, epitope mapping studies were also conducted. Methods: SKBR3 cells were used to confirm HER2 binding and determine the antibodies’ impact on proliferation, apoptosis, and miRNA expression. Cell proliferation upon a 3-day antibody treatment (10 to 15 μg/ml) was measured using MTS and Crystal Violet Assays. Apoptotic cells were identified using Annexin V-FITC flow cytometry. To determine their epitope mapping, cells were grown on coverslips overnight, fixed, blocked, and incubated in AF488-mAb or AF555-mAb, and images were captured with IX81. QRT-PCR was used to measure the relative levels of miR-21, using miR-16 as the endogenous control Results: Cell proliferation assays showed that the treatment of 10H8 resulted in a better growth inhibition in SKBr3 cells than that of trastuzumab (ex: 80.2% vs 84%, respectively). Combining 10H8 and 8H11 produced less viable cells and more early- and post-apoptotic cells than those treated with trastuzumab alone. In addition, whereas trastuzumab increased the relative quantity of the known oncogene, both 10H8 and 8H11 maintained a steady level of miR-21 (0.99 and 1.2, respectively). Conclusions: Initial results indicate that 10H8 and 8H11 inhibit cell proliferation and induce apoptosis in HER2-overexpressing cells. However, unlike trastuzumab, 10H8 and 8H11 do not involve miR-21 up-regulation. Moreover, the three mAbs do not have overlapping epitopes on HER2. Further investigations of the antibodies’ impact on intracellular signaling pathways and the structural determination of the HER2 binding sites would provide further understanding of the therapeutic potentials of these two mAbs.

scholarly journals Effects of Temperature and Water-Filtered Infrared-A Alone or in Combination on Healthy and Glyoxal-Stressed Fibroblast Cultures

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Lilla Knels ◽  
Monika Valtink ◽  
Jamlec De la Vega Marin ◽  
Gerald Steiner ◽  
Cora Roehlecke ◽  
...  
Keyword(s):  
Annexin V ◽  
Vital Staining ◽  
Irradiation Damage ◽  
3T3 Fibroblasts ◽  
Resazurin Assay ◽  
Glycation End Products ◽  
Different Temperatures ◽  
Effects Of Temperature ◽  
Exact Temperature

Water-filtered infrared-A (wIRA) radiation has been described as supportive for tissue regeneration. We sought to investigate in detail the wIRA effect at different temperatures in 3T3 fibroblasts that were treated with glyoxal to induce formation of advanced glycation end products (AGEs). Nonirradiated and nonglyoxal-treated cells served as controls. Experiments were carried out over a range of 37∘–45∘C with exact temperature monitoring to distinguish between temperature and wIRA effects. Metabolic activity was assessed by resazurin assay. Mitochondrial membrane potential was assessed by JC-1 vital staining. Apoptotic changes were determined by vital staining with annexin V and YO-PRO-1 and determination of subG1 DNA content. Temperature had a dominant effect overriding effects exerted by wIRA or glyoxal treatment. The number of apoptotic cells was significantly higher at 45∘C, while the percentage of healthy cells was significantly lower at 45∘C. WIRA irradiation itself or in combination with glyoxal treatment exerted no damaging effects on the fibroblasts at physiological (37∘–40∘C) or higher (42∘–45∘C) temperatures compared to untreated controls. Temperatures of 45∘C, which can occur during inappropriate application of infrared irradiation, damage cells even in the absence of wIRA or glyoxal application.

scholarly journals Phospholipid profiles and annexin V content in villous chorion from women with spontaneous miscarriage associated with cytomegalovirus infection

2021 ◽  
pp. 72-79
Author(s):  
N. A. Ishutina ◽  
I. A. Andrievskaya ◽  
N. G. Prikhodko
Keyword(s):  
Spontaneous Abortion ◽  
Cytomegalovirus Infection ◽  
Thrombus Formation ◽  
Annexin V ◽  
Medical Abortion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Control Group ◽  
Layer Chromatography ◽  
Chloroform Methanol

Aim. To carry out a comparative analysis of the phospholipid profiles and annexin V content in the villous chorion obtained from women with spontaneous abortion associated with exacerbation of cytomegalovirus infection (CMVI) and the physiological course of pregnancy.Materials and methods. 66 patients were randomly selected at a gestational age of 6-8 weeks. A control group was identified in the amount of 32 healthy pregnant women with medical abortion. The main study group consisted of 34 women with spontaneous abortion associated with exacerbation of cytomegalovirus CMVI. The material for the study was the villous chorion, peripheral blood and urine. Type-specific antibodies to CMV immunoglobulins M and G class and the avidity index were determined in blood serum by the method of enzyme-linked immunosorbent assay. Lipids in the villous chorion homogenate were extracted with a chloroform-methanol mixture, phospholipids were fractionated by thin layer chromatography. The quantitative determination of annexin V was carried out by the method of enzyme immunoassay.Results. The phospholipid profile of the villous chorion in the main group was characterized by a decrease in the fraction of phosphatidylcholine to 22,39±0,04% (control group – 28,50±0,052%; p<0,001), phosphatidylethanolamine to 27,63±0,011% (control group – 30,11±0,073%; p<0,001), phosphatidylinositol up to 14,24±0,021% (control group – 16,17±0,018%; p<0,001), an increase in lysophosphatidylcholine up to 2,25±0,032% (control group – 1,07±0,022%; p<0,001), phosphatidylserine up to 14,57±0,075% (control group – 10,02±0,012%; p<0,001), sphingomyelin up to 18,92±0,012% (control group – 14,13±0,012%; p<0,001). At the same time, an increase in the content of annexin V was revealed up to 43,12±0,50 U/mL (control group – 20,21±0,50 U/mL; p<0,001).Conclusion. The results of the study showed that in the villous chorion from women with spontaneous abortion associated with exacerbation of CMVI, there was a change in the profile and ratio of phospholipids, as well as in the concentration of annexin V. The established increase in the content of phosphatidylserine and the associated annexin V in the villous chorion indicates a violation of the hemostatic system and microcirculation due to increased thrombus formation, which supports local inflammation and apoptosis of the trophoblast, which leads to embryonic demise and spontaneous abortion.

scholarly journals Impact of Cyclin-Dependent Kinase CDK4 Inhibition on Eryptosis

2015 ◽  
Vol 37 (3) ◽  
pp. 1178-1186 ◽  
Author(s):  
Elisabeth Lang ◽  
Christine Zelenak ◽  
Matthias Eberhard ◽  
Rosi Bissinger ◽  
Anand Rotte ◽  
...  
Keyword(s):  
Organic Anion ◽  
Annexin V ◽  
Human Erythrocytes ◽  
Cyclin Dependent Kinase ◽  
Cell Shrinkage ◽  
Forward Scatter ◽  
Altered Gene Expression ◽  
Altered Gene ◽  
Cdk4 Inhibition

Background/Aims: The cyclin-dependent kinase 4 (CDK4) participates in the regulation of apoptosis of nucleated cells by altering transcriptional regulation of genes governing cell proliferation and cell death. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, which is characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine (PS) exposure at the cell surface. As mature erythrocytes lack nuclei, acute stimulation of eryptosis cannot result from altered gene expression. Eryptosis is triggered by isotonic cell shrinkage following Cl- removal (replacement with the impermeant organic anion gluconate) or by oxidative stress (exposure to 0.3 mM tertbutyl-hydroperoxide [tBOOH]). The present study explored whether CDK4 is expressed in erythrocytes and whether the CDK4 inhibitors II (NSC625987) and III (ryuvidine) influence eryptosis. Methods: Western blotting was utilized for determination of the presence of CDK4 protein in human erythrocytes, and FACS analysis to determine Fluo3 fluorescence (reflecting cytosolic Ca2+), annexin-V-binding (reflecting PS-exposure) and forward scatter (reflecting cell volume). Results: CDK4 protein was present in human erythrocytes. Cl- removal was followed by decrease of forward scatter and increase of both annexin-V-binding and Fluo3 fluorescence, an effect significantly curtailed by CDK4 inhibitors II and III. Furthermore, CDK4 inhibition blunted enhanced PS-exposure elicited by tBOOH treatment. Conclusions: The present observations disclose the presence of CDK4 protein in human erythrocytes and the suppression of suicidal erythrocyte death by pharmacological inhibition of CDK4.

scholarly journals Satellite cells attract monocytes and use macrophages as a support to escape apoptosis and enhance muscle growth

2003 ◽  
Vol 163 (5) ◽  
pp. 1133-1143 ◽  
Author(s):  
Bénédicte Chazaud ◽  
Corinne Sonnet ◽  
Peggy Lafuste ◽  
Guillaume Bassez ◽  
Anne-Cécile Rimaniol ◽  
...  
Keyword(s):  
Muscle Growth ◽  
Annexin V ◽  
Precursor Cells ◽  
Monocyte Chemoattractant Protein 1 ◽  
Proliferation And Differentiation ◽  
Monocyte Recruitment ◽  
Chemotactic Factors ◽  
Urokinase System

Once escaped from the quiescence niche, precursor cells interact with stromal components that support their survival, proliferation, and differentiation. We examined interplays between human myogenic precursor cells (mpc) and monocyte/macrophages (MP), the main stromal cell type observed at site of muscle regeneration. mpc selectively and specifically attracted monocytes in vitro after their release from quiescence, chemotaxis declining with differentiation. A DNA macroarray–based strategy identified five chemotactic factors accounting for 77% of chemotaxis: MP-derived chemokine, monocyte chemoattractant protein-1, fractalkine, VEGF, and the urokinase system. MP showed lower constitutive chemotactic activity than mpc, but attracted monocytes much strongly than mpc upon cross-stimulation, suggesting mpc-induced and predominantly MP-supported amplification of monocyte recruitment. Determination of [3H]thymidine incorporation, oligosomal DNA levels and annexin-V binding showed that MP stimulate mpc proliferation by soluble factors, and rescue mpc from apoptosis by direct contacts. We conclude that once activated, mpc, which are located close by capillaries, initiate monocyte recruitment and interplay with MP to amplify chemotaxis and enhance muscle growth.

scholarly journals PHARMACOLOGICAL EFFECT OF AMINOFERROCENE IN MICE WITH L1210 LEUKEMIA

2015 ◽  
Vol 37 (2) ◽  
pp. 120-125 ◽  
Author(s):  
V F Chekhun ◽  
A Mokhir ◽  
S Daum ◽  
I Todor ◽  
N Yu Lukianova ◽  
...  
Keyword(s):  
Biological Effects ◽  
Leukemia Cell ◽  
Annexin V ◽  
Active Species ◽  
L1210 Leukemia ◽  
Lymphoid Leukemia ◽  
Thiol Compounds ◽  
Free Iron ◽  
Nonprotein Thiol

Aim: To study the cytostatic and some biological effects of aminoferrocene using mice with L1210 lymphoid leukemia. Materials and Methods: Experiments were performed on BDF1 male mice (DBA/2, female × C57Bl/6, male) with transplantable L1210 lymphoid leukemia. Determination of antitumor activity of Benzyl-Fc Boron (Bn), it was injected intraperitoneally 6 times daily, starting on day 2 after L1210 leukemia cell transplantation. Doses of Bn such as 26; 260 and 2600 μg/kg were used. The determination of intracellular content of cardiolipin, thiols, reactive oxygen species (ROS) and also analysis of Annexin V positivity and mitochondrial transmembrane potential (JC-1 staining) were performed with use of flow cytometry. The levels of “free iron” complexes, transferrin active forms and the rate of NO generation were measured by EPR-specroscopy. Results: Six daily injections of Bn at a dose of 26 μg/kg resulted in an increased survival of mice with L1210 leukemia by 28% (p < 0.05). Bn led to an increase of apoptotic cells number and ROS amount in leukemia cells. Besides, Bn caused a decrease of cardiolipin and nonprotein thiol compounds content. The membrane electrochemical potential of cell mitochondria was decreased also after Bn administration. Studies using EPR-spectroscopy revealed a significant increase in a level of “free iron”, content of transferrin active species and generation rate of NO by inducible NO-synthase in L1210 cells after aminoferrocene administration. Conclusion: Our data indicate that Benzyl-Fc Boron can be promising candidate for realizing a new strategy of anticancer therapy with the use of ROS-inducing agents.

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