PURPOSE: To survive in an ecological environment, an individual must develop immunity to various antigens. Therefore, populations of peripheral blood mononuclear cells (PBMC) in humans change continuously with growth. The object of this study is to evaluate the apoptosis of peripheral blood mononuclear cells (PBMC) in normal children of different ages. METHODS: PBMC were isolated from the study groups. Ten cord blood samples of normal babies, 10 blood samples of normal children each from 4 different age groups (0-1, 2-5, 6-10, 11-15 year- old and adult), and 20 from normal adults were included in this study. After 24 and 48 hrs incubation in RPMI1640 media containing 10% fetal calf serum, cells were stained with Annexin V and PI and then analyzed with FACScan flowcytometer. RESULTS: Cord blood mononuclear cells showed the lowest percentage of apoptosis compared to other age groups. PBMC isolated from the 0-1 year-old normal children showed the highest percentage of apoptosis, and the percentage of apoptosis decreased with increase of age. After the age of 10, the percentage of PBMC apoptosis was the same as that of adults. CONCLUSION: The differences in the percentage of PBMC apoptosis with different age groups might be from immunologically different state of the hosts with different age. This result could be a useful reference data for the study of apoptosis in pediatric disease in the future.
Adult ; Annexin A5 ; Apoptosis* ; Child* ; Fetal Blood ; Humans

PURPOSE: Thawed stem cells should be infused as early as possible because delay of infusion leads to decrease of cell viability and formation of DNA clumping. The procedure of 10% Dimethyl sulfoxide(DMSO) removal and a long distance from the thawing location to the patient are the main causes of delay of infusion that results in the loss of cell viability and apoptosis. Therefore, we investigated the changes of cell viability and apoptosis after thawing with lapse of time. METHODS: Five samples of mobilized peripheral blood were evaluated. We measured cell viability, colony forming unit(CFU) and apoptosis at 0, 1, 2, 4, and 24 hours after thawing. The state of stem cells were divided into live, apoptotic and dead with double staining using annexin V and 7-aminoactinomycin D(7-AAD) in flow cytometry. RESULTS: Viability of the total cells after thawing was 77.3(53.3-97.7)%. The percentage which recovered to initial CFU at 1, 2, 4 and 24 hours after thawing decreased to 63.9%, 50.2%, 45.8% and 11.6%, respectively. The proportion of apoptotic cells among CD34+ cells after thawing were increased from 0.2% at 0 hour to 16.5% at 1 hour, 21.9% at 2 hours, and then decreased to 15.0% at 4 hours, 2.7% at 24 hours because they were replaced by dead cells. CONCLUSION: Thawed cells changed to apoptotic and had less colony forming capacity from 1 hour after thawing, and were then replaced by dead cells from 4 hours after thawing.
Annexin A5 ; Apoptosis* ; Cell Survival ; DNA ; Flow Cytometry ; Humans ; Stem Cells*

BACKGROUND: Because of a lack of substances for platelet (PLT) metabolism and preservation, normal saline (NS) washed PLTs can only be stored for short lengths of time. However, the use of platelet additive solutions (PAS) could help solve this problem. In this study, the in vitro quality of NS washed platelets (wPLTs) stored in two types of PAS were compared with those of wPLTs stored in NS. METHODS: Five units of NS washed apheresis platelets were pooled aseptically and separated into five aliquots for storage in NS only as well as T-PAS+ (Terumo BCT, Lakewood, CO, USA) and CompoSol PS (Fenwal, Lake Zurich, IL, USA) with or without 15 mM glucose. The parameters of wPLTs quality were assessed up to 48 hrs after washing and the whole experiment was repeated 10 times independently. RESULTS: wPLTs in two kinds of PAS had better quality than wPLTs in NS, and wPLTs in T-PAS+ showed better quality than those in CompoSol PS. PAS-stored wPLTs with added glucose maintained stable CD62P and Annexin V expression during storage, but exhibited increased lactate accumulation. Evaluation of in vitro quality revealed that all wPLTs had a rating of 4 immediately after washing. However, only T-PAS+-stored wPLTs with glucose maintained a rating of 4 up to 48 hrs of post-washing. CONCLUSION: Using PAS storage for wPLTs may be beneficial compared to NS. The results presented herein suggest that T-PAS+ containing glucose has the potential to extend storage time by up to 48-hours.
Annexin A5 ; Blood Component Removal ; Blood Platelets* ; Blood Preservation ; Glucose ; In Vitro Techniques ; Lactic Acid ; Lakes ; Metabolism

PURPOSE: To investigate the effect of ROCK inhibitor Y27632 on the human corneal endothelial cell proliferation in vitro and in vivo. METHODS: Using corneal endothelial cells isolated and cultured from human donor cornea, we compared the effect of Y27632 (10 microM) on the proliferation in vitro by flow cytometry analysis. For the evaluation of the effect of Y27632 (10 mM) in vivo, corneal thickness and wound area were analyzed for the corneal endothelial wound rabbit model induced by transcorneal freezing. RESULTS: Ki67 positive cells were increased in the Y27632 group (9.1 +/- 4.1%) than the control group (8.0 +/- 5.9%), whereas annexin V positive cells in the Y27632 group (2.9 +/- 1.0%) were decreased compared to the control group (4.2 +/- 2.2%). However these were not statistically significant. Wound area after Y27632 application in animal model is concerned, the control group showed significant smaller area (45.6 +/- 0.6 mm2) compared to the Y27632 group (49.3 +/- 0.8 mm2; p = 0.029, Mann-Whitney U test), however, these were not significantly different from the baseline. Corneal thickness was not different between the two groups. CONCLUSIONS: Different from other reports for the effect of Y27632, no significant effect on the proliferation in vitro and wound healing in vivo, regarding human corneal endothelial cell, were found in this study.
Amides ; Annexin A5 ; Cornea ; Endothelial Cells ; Flow Cytometry ; Humans ; Models, Animal ; Pyridines ; Tissue Donors ; Wound Healing

BACKGROUND AND OBJECTIVES: Annexin V is known to bind to the phosphatidylserine (PS) of damaged cell membranes. We recently demonstrated that annexin V binds to oxidized red blood cells (oxRBC). The aim of this study was to find whether annexin V binds to oxidized lipids or to the PS of oxRBC. MATERIALS AND METHODS: Red blood cells (RBC) were oxidized by the addition of CuSO4, and the degree of oxidation evaluated using the semiquantitative measurement of thiobarbituric acid reactive substance (TBARS). The binding of annexin V to oxRBC was evaluated by flow cytometry. RESULTS: Annexin V was found to bind to oxRBC, but not to native RBC. The percentage of RBC binding to annexin V was closely correlated with the degree of oxidation, as measured using TBARS (r=0.99, p=0.000) in relation to the concentration of CuSO4. The binding of annexin V to oxRBC was attenuated in the presence of oxidized low density lipoprotein (oxLDL), with these phenomena also being dosedependent. The binding was reduced by 71.0+/-3.0% in the presence of 100 microgram/mL oxLDL. LDL had no influence on the binding of annexin V to oxRBC. CONCLUSION: These findings suggest that annexin V may bind to the oxidized lipids of cell membranes. Further studies will be required to evaluate the relative importance between oxidized lipids and PS, and to find the characteristics of oxidized lipids in the binding of annexin V to damaged cell membranes.
Annexin A5* ; Cell Membrane ; Erythrocytes* ; Flow Cytometry ; Lipid Peroxidation ; Lipoproteins ; Thiobarbituric Acid Reactive Substances

BACKGROUND: The quality of cord blood largely depends on cell viability. Viability assessments using trypan blue or 7-aminoactinomycin (7-AAD) staining, which are commonly used methods, may not reflect early apoptosis of cord blood cells. We aimed to investigate early apoptosis in cord blood cells following elapsed time after collection using double staining with annexin V and 7-AAD and to compare the result with that of viability evaluation using trypan blue or 7-AAD staining. METHODS: Umbilical cord blood samples were obtained from 30 pregnant women at the time of delivery between July 2012 and March 2013. Viability of cord blood cells was determined at 0 (T0), 24, and 48 hr after collection by using trypan blue exclusion assay, 7-AAD staining, and 7-AAD/annexin V staining. RESULTS: Viabilities defined by 7-AAD/annexin V staining at T0, 24, and 48 hr after collection were respectively as follows: total nucleated cells, 92.8+/-4.5%, 78.4+/-7.8%, and 65.5+/-8.1%; mononuclear cells, 94.4+/-1.7%, 90.8+/-4.2%, and 84.2+/-6.7%; and CD34-positive cells, 92.4+/-3.0%, 90.7+/-4.7%, and 89.3+/-7.0%. The viability using trypan blue was more than 90% until 48 hr after collection. CONCLUSIONS: The mean viability of total nucleated cells using 7-AAD/annexin V staining decreased to less than 80% at 24 hr after collection; however, the viability of CD34-positive cells was more than 85% until 48 hr. Our study's data will provide useful information for the assessing the quality of cord blood products.
Annexin A5 ; Apoptosis ; Cell Survival* ; Female ; Fetal Blood* ; Humans ; Methods ; Pregnant Women ; Trypan Blue ; Umbilical Cord*

PURPOSE: To evaluate radiation sensitivity of dendritic cells in comparison with lymphocytes. MATERIALS AND METHODS: T lymphocytes captured from peripheral blood were irradiated by 0 Gy, 10 Gy, 30 Gy. Apoptosis was measured by flowcytometry for staining of Annexin V 4 hours after irradiation. Immature and mature dendritic cells processed from blood hematopoietic stem cell were irradiated by 0 Gy, 10 Gy, 30 Gy, 100 Gy respectively and apoptosis was measured by flowcytometry with time difference as 4h, 24h and 48h after irradiation. Morphometric analysis by percent nucleus was measured in three cell groups, also. RESULTS: Lymphocytes showed radiation sensitivity by increasing apoptotic fraction according to radiation dose. However, both mature and immature dendritic cells showed consistent fraction of apoptosis in spite of increasing radiation dose. Percent nucleus ratio is significantly higher in lymphocytes than that of mature or immature dendritic cells. Stimulation of T-cell by dendritic cells was not changed after irradiation. CONCLUSION: Dendritic cells showed radioresistance which was associated with small size of nucleus in comparison with lymphocytes and this result would be used as a basal data of radio-labelling for the cellular trafficking studies in nuclear medicine fields.
Annexin A5 ; Apoptosis ; Dendritic Cells* ; Hematopoietic Stem Cells ; Lymphocytes ; Nuclear Medicine ; Radiation Tolerance ; T-Lymphocytes

PURPOSE: To evaluate the effect of variable ceramides on the apoptosis of corneal endothelial cell and then, if ceramide induce the apoptosis in endothelial cells, via which pathway apoptosis occur. METHODS: Corneal endothelial cells were isolated from fresh rabbit cornea and cultured. Cultured corneal endothelial cells were exposed to 10, 20, 40 and 80 micro M of ceramide type II, VI and phytoceramide type II, VI. And then, apoptosis was evaluated with Hoechst staining and flow cytometric analysis with Annexin V for evaluation of apoptotic response. Corneal endothelial cells were preincubated in various concentrations of CPP32-like protease inhibitor (Z-VAD-FMK(R)), specific caspase-8 inhibitor(IETD-CHO(R)) and specific caspase-9 inhibitor (Z-LEHD-FMK(R)), then treated with 20 M of 4 types of ceramide. 12 hours later, LDH assay was done. Cytochrome c immunostaining was done after exposure to 4 types of ceramide. RESULTS: Shrinkage of cytoplasm, formation of apoptotic bodies, and nuclear fragmentation were observed on Hoechst staining. In flow cytometric analysis, early apoptotic responses were identified. Apoptotic response increased significantly at the concentration of 10M and more 12 hours later. CPP32-like protease inhibitor, caspase-8, 9 inhibitor reduced the LDH activity. Apoptotic endothelial cells induced by ceramide were stained with cytochrome c antibody. CONCLUSIONS: Ceramide induced apoptosis in cultured corneal endothelial cells. This apoptosis developed via caspase and mitochondrial pathway.
Annexin A5 ; Apoptosis* ; Caspase 8 ; Caspase 9 ; Ceramides ; Cornea ; Cytochromes c ; Cytoplasm ; Endothelial Cells* ; Protease Inhibitors

Apoptosis plays a role in the pathophysiology of many kinds of diseases and in the response of treatment. Compared to the necrosis, the apoptosis is a genetically controlled and energy-dependent process which removes the unwanted cells from the body; programmed cell death or cell suicide. During the apoptosis, phosphatidylserine is expressed in the cytoplasmic outer membrane in the early phase. Annexin V, an endogenous human protein (MW=35 kD), has an affinity of about 10 (-9) M for the phosphatidylserine exposed on the outer membrane of apoptotic cells. Annexin V can be radiolabeled with 99mTc by HYNIC or EC chelators, which can be used as an radiotracer for the in vivo imaging of apoptosis. In this article, we reviewed the apoptosis, radiolabeling of annexin V, and the experimental and clinical data using annexin V imaging.
Annexin A5 ; Apoptosis* ; Cell Death ; Chelating Agents ; Cytoplasm ; Humans ; Membranes ; Necrosis ; Suicide

OBJECTIVEApoptosis contributes to the instability of the atherosclerotic (AS) lesions. The vulnerable plaque was identified in vivo by detecting the apoptosis with radiolabeled annexin V in an atherosclerotic rabbit model.

METHODSEight male New Zealand white rabbits on 2% cholesterol diet for 2 weeks had abdominal aortic balloon injury and fed a 2% cholesterol diet for another 15 weeks (AS group), 3 rabbits fed a normal rabbit chow for 17 weeks without balloon injury served as controls. Annexin V labeled with (99)Tc(m) was then intravenously administered and planar whole-body images were captured using a gamma camera in the left lateral position. The entire length of the abdominal aorta was explanted for ex vivo imaging with gamma camera. The aorta then was divided into several segments according to the severity of AS. The segments were separated weighted and counted in an gamma counter for the absorptive dose of annexin per gram of tissue. Histology examinations were made on specimens.

RESULTSAt 2 hours post annexin V injection, clear delineation of radiolabel within the abdominal aorta could be evidenced in vivo gamma imaging. After explanation of the aorta, ex vivo imaging showed a robust uptake of radiotracer in the infradiaphragmatic aorta corresponding to the in vivo images and conforming to the macroscopic distribution of atherosclerotic lesions. The uptake of radiolabel was absent in areas without grossly visible atherosclerotic lesions. The in vivo and ex vivo images identified plaque areas were identical and corresponded histological results on the explanted specimen. The aortic specimen was divided into 18 segments on lesions. The magority of the lesions (14/18) manifested as type IV or type V lesions of AHA classification (vulnerable lesions), except segments 1 - 4, which manifested as type I or type II lesions. The thickness of fibrous cap (TFC) and the ratio of cap and lipid nuclear (RCN) were significantly reversely correlated to the unit radioactivity counts, and the correlation between RCN and the unit radioactivity counts was more significant than that between TFC and the unit radioactivity counts (r = -0.904, P < 0.01, and r = -0.8, P < 0.01). Apoptosis detection (TUNEL): annexin V intake in plaques was positively correlated to apoptotic index(r = 0.651, P = 0.012).

CONCLUSIONNoninvasive Annexin V imaging could be used to detect vulnerable atherosclerotic plaques in vivo.