Use Of Annexin Research Articles

EGR1 mediates MDR1 transcriptional activity regulating gemcitabine resistance in pancreatic cancer

BackgroundGemcitabine is a cornerstone drug for the treatment of all stages of pancreatic cancer and can prolong the survival of patients with pancreatic cancer, but resistance to gemcitabine in pancreatic cancer patients hinders its efficacy. The overexpression of Early growth response 1(EGR1) in pancreatic ductal adenocarcinoma as a mechanism of gemcitabine chemoresistance in pancreatic cancer has not been explored. The major mechanisms of gemcitabine chemoresistance are related to drug uptake, metabolism, and action. One of the common causes of tumor multidrug resistance (MDR) to chemotherapy in cancer cells is that transporter proteins increase intracellular drug efflux and decrease drug concentrations by inducing anti-apoptotic mechanisms. It has been reported that gemcitabine binds to MDR1 with high affinity. The purpose of this research was to investigate the potential mechanisms by which EGR1 associates with MDR1 to regulate gemcitabine resistance in pancreatic cancer cells.MethodsThe following in vitro and in vivo techniques were used in this research to explore the potential mechanisms by which EGR1 binds to MDR1 to regulate gemcitabine resistance in pancreatic cancer cells. Cell culture; in vitro and in vivo study of EGR1 function by loss of function analysis. Binding of EGR1 to the MDR1 promoter was detected using the ChIP assay. qRT-PCR, Western blot assays to detect protein and mRNA expression; use of Annexin V apoptosis detection assay to test apoptosis; CCK8, Edu assay to test cell proliferation viability. The animal model of pancreatic cancer subcutaneous allograft was constructed and the tumours were stained with hematoxylin eosin and Ki-67 expression was detected using immunohistochemistry.FindingsWe revealed that EGR1 expression was increased in different pancreatic cancer cell lines compared to normal pancreatic ductal epithelial cells. Moreover, gemcitabine treatment induced upregulation of EGR1 expression in a dose- and time-dependent manner. EGR1 is significantly enriched in the MDR1 promoter sequence.Upon knockdown of EGR1, cell proliferation was impaired in CFPAC-1 and PANC-1 cell lines, apoptosis was enhanced and MDR1 expression was decreased, thereby partially reversing gemcitabine chemoresistance. In animal experiments, knockdown of EGR1 enhanced the inhibitory effect of gemcitabine on tumor growth compared with the sh-NC group.ConclusionsOur study suggests that EGR1 may be involved in the regulation of MDR1 to enhance gemcitabine resistance in pancreatic cancer cells. EGR1 could be a novel therapeutic target to overcome gemcitabine resistance in pancreatic cancer.

Genistein Enhances TRAIL-Mediated Apoptosis Through the Inhibition of XIAP and DcR1 in Colon Carcinoma Cells Treated with 5-Fluorouracil.

Colorectal cancer is one of the most common cancers worldwide. However, surgical intervention and chemotherapy provide only limited benefits for the recovery and survival of patients. The anticarcinogenic effect of genistein has attracted attention because epidemiological studies have shown that soybean consumption is associated with a decrease in the incidence of cancer. There are limited studies on the effects of genistein in colorectal carcinoma cells. We aimed to investigate the cytotoxic, genotoxic, and apoptotic effects of genistein in SW480 and SW620 colon adenocarcinoma cells treated with 5-fluorouracil, the basis of chemotherapy, and the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) ligand, the mediator of apoptosis, both alone and in combination. Cytotoxicity and genotoxicity were determined by MTT and comet assays, respectively. The apoptotic effects were evaluated by reverse transcription-polymerase chain reaction assay, with the additional use of Annexin V FITC, mitochondrial membrane potential (MMP), caspase 3, 8, and 9 activity, and reactive oxygen species (ROS) assay kits. According to our findings, genistein, 5-fluorouracil, and TRAIL had synergistic apoptotic effects because of DR5 upregulation, ROS production, and DNA damage, which were mediated by increased caspase-8, and -9 activity and decreased MMP. The applied combinations of these compounds may contribute to the resistance problem that may occur in treating colorectal cancer, with a decrease in DcR1 and XIAP genes.

Abstract 1403: Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss

Abstract Triple negative breast cancer (TNBC) accounts for about 20% of all breast cancers. TNBC is an aggressive subtype of breast cancer that lacks the expression of estrogen and progesterone receptors and HER2 protein. This lack of functioning receptors results in limited treatment options, usually resulting in the use of taxanes, platinums, and anthracyclines. Although TNBC often starts as being sensitive to traditional chemotherapies, tumor relapse and/or resistance often develops. Paclitaxel (PTX) is a commonly used taxane for TNBC, well known to induce mitotic arrest through stabilization of microtubules (MT) leading to an interference with the treadmilling function of MT through the activation of the spindle assembly checkpoint. We have previously shown that loss of the Adenomatous Polyposis Coli (APC) tumor suppressor significantly increases resistance to PTX in APC knockdown (APCKD) cells generated in two human TNBC cell lines, MDA-MB-157 and MDA-MB-231. Given this striking PTX resistance, we were interested in seeing if resistance in APCKD can be seen using other non-taxane MT targeting agents. To accomplish this, we have used an epothilone (Epothilone B) and a vinca alkaloid (Vinblastine), that function by stabilizing and destabilizing the MTs, respectively. Our previous investigations demonstrated that APC loss results in increased CDK1 and CDK6, so these have been used as markers of cell cycle progression after drug treatment. In addition, changes in apoptosis were measured through the use of Annexin V and PI staining, and cleaved caspase 3 expression. Examined together, a better understanding of how APC loss influences chemoresistance in TNBC can lead to more efficient treatments for TNBC patients resistant to taxanes. Given the critical interaction between APC and MTs, this may be an avenue for targeted therapies to overcome taxane resistance. Citation Format: Pamela D. Arenas, Sara M. Maloney, Jenifer R. Prosperi. Efficacy of microtubule targeting agents on triple negative breast cancer cells influenced by adenomatous polyposis coli loss [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1403.

Calophyllum brasiliense Extracts Induced Apoptosis in Human Breast Adenocarcinoma Cells

Aims: Apoptosis, or programmed cell death, is linked to several mechanisms of cell growth control. The present work aimed at evaluating the induction of apoptosis in MCF-7 human breast adenocarcinoma cell by Calophyllum brasiliense.
 Study design: The tests were performed in triplicates in the apoptosis assays and sextuplicates in the cytotoxic assays, to each group, and the data expressed the mean +/- standard deviations. The cytotoxicity IC50s were obtained based on nonlinear regression curve fit. Two-way ANOVA and Tukey’s tests were applied in the apoptosis analyses.
 Place and duration of study: The work was done at the Center for Research in Biodiversity (Cell Culture Laboratory, and Phytochemistry Laboratory), and Research Center (Molecular Biology Laboratory), Paulista University, between Jan 2019 and Dec 2019.
 Methodology: Two aqueous extracts, obtained from the stem (STE) and from the leaves (LFE) of Calophyllum brasiliense by a 24-h maceration, were submitted to a cytotoxic assay against MCF-7 breast cancer cell lines at the concentrations of 0.01 µg/ml, 0.1 µg/ml, 1.0 µg/ml, 10 µg/ml and 100 µg/ml. They were also subjected to the evaluation of apoptosis and necrosis cell death induction at concentrations of 50, 100 and 200 μg/ml, after 6 h, 12 h and 24 h. Curcumin was used as a reference drug for both cytotoxic (50 mM, 5.0 mM, 0.5 mM, 0.05 mM and 0.005 mM ) and apoptosis/necrosis (12.5 μM, 25 μM and 50 μM / 6 h, 12 h and 24 h) assays. Apoptosis and necrosis were accessed by the use of annexin V and 7-AAD, in a two-channel flow cytometer.
 Results: In terms of the cytotoxic activity, STE (IC50 7.86 µg/ml) was more toxic than LFE (IC50 74.35 µg/ml), and curcumin IC50 was 0.00159 µg/ml. STE induced 21.19 % and LFE, 20.63 %, in comparison to 13.4% of apoptosis induction by curcumin. The results of apoptosis induction in the cancer cells were achieved at 24 h, extract concentrations at 100 µg/ml.
 Conclusion: Both the extracts, STE and LFE, were cytotoxic against MCF-7 breast cancer cell line, and induced more apoptosis in MCF-7 cells than curcumin, suggesting that they are high potential sources of natural product-inducing apoptosis agents to be used against adenocarcinoma breast cells.

Annexin V and gaussia luciferase fusion protein studies for bioluminescence apoptosis detection

Programmed cellular death, otherwise known as apoptosis, is a hallmark of many prominent diseases ranging from cancerous to neurodegenerative. Due to this common cellular fate found in a variety of diseases, it has become a goal to develop and construct a mechanism through which apoptotic cells can be detected with a high selectivity. Herein, we describe the development of a class of biosensors are capable of identifying these cells destined for apoptosis through the use of Annexin V alongside a bioluminescent reporter expressed as a single fusion protein. In this work, the bioluminescent protein used is Gaussia luciferase (GLuc), natively expressed by the copepod Gaussia princeps. Annexin V is a protein innately found in the human body that will bind to phosphatidylserine (PS), a membrane phospholipid that shifts towards the outer membrane when the cell has become apoptotic, a key marker of regulated cell death. By combining Annexin V with Gaussia luciferase, we developed a a fusion protein (AgFP), and demonstrated it to be stable, show correlating results when testing for bioluminescence. Analyses through in vitro studies show the potency that the fusion protein, AgFP, does not inhibit either component of the construct from either binding PS or generating bioluminescent signals. Future prognosis of this Annexin‐Gaussia fusion protein includes testing using in vivo models with certain pathologies and determining whether bioluminescence is seen in the target area at a level of affinity and selectivity that can be statistically significant in detecting apoptosis. End stage applications of the fusion protein construct would be an effective and reliable way to screen various areas of the human body for abnormal rates of apoptosis and possibly intervene before some of these lethal diseases become too advanced.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Fluorimetric Techniques for the Assessment of Sperm Membranes.

Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.

Apoptosis of CD19+ chimeric antigen receptor T cells after treatment with chemotherapeutic agents.

The use of chemotherapeutic agents prior to treatment with infusion of cluster of differentiation (CD)19-chimeric antigen receptor (CAR)-T cells is important for the efficacy of clinical therapies against hematological malignancies. However, the effect of chemotherapeutic agents on CD19-CAR-T cells and the associated underlying mechanisms remain unknown. The first aim of the present study was to determine the effect of chemotherapeutic agents on CAR-T cells using the in vitro Cell Counting kit 8 assay. The second aim was to evaluate the abilities of fludarabine (FDR) and mafosfamide (MFA; a metabolite of cyclophosphamide) to induce apoptosis of CD19-CAR-T cells via the use of Annexin V/propidium iodide double staining. In addition, a JC-1 fluorescent probe was used to detect alterations in cell membrane potential, and flow cytometry analysis was used to measure concentrations of caspase-3/7 to identify apoptotic pathways of CD19-CAR-T cells. The data of the present study suggested that FDR and MFA inhibit the activities of CD19-CAR-T cells. Alterations to the mitochondrial membrane potential and an increase in the concentration of caspase-3/7 indicated early apoptosis of FDR- and MFA-treated CD19-CAR-T cells. The present study laid a theoretical foundation for the development of programs for clinical treatment.

La selección espermática mediante técnica de separación magnética de células activadas (MACS) en las técnicas de reproducción asistida

The sperm integrity is essential to ensure reproductive competence. Several studies have shown that ejaculated sperm exhibit changes associated with apoptosis, in particular externalisation of phosphatidylserine, decreased MMP integrity, and DNA fragmentation.The classification of magnetically activated cells with Annexin V microspheres recognises phosphatidylserine residues externalised on the surface of apoptotic spermatozoa. In addition, the use of Annexin V microspheres to select non-apoptotic spermatozoa reduces the percentage of altered cells with DNA fragmentation and aneuploidy, improving the possibility of pregnancy after assisted reproduction techniques.In this review, our objective is to evaluate the possible value of activated cell magnetic classification technology in infertile patients undergoing assisted reproduction techniques.

Use of Annexin V based Sperm Selection in Assisted Reproduction

Innovative methods to select sperm subpopulations with the best fertilizing ability are needed in assisted reproductive techniques (ART) in order to improve fertilization and pregnancy rates, while also considering possible epigenetic effects on the offspring. Molecular based selection methods are searched for, under the premise that they could be an improvement over classical selection by morphology and movement. One of these methods sustains the elimination of sperm that can bind to annexin A5 (ANX V), coupled to paramagnetic beads, through the phosphatidyl-serine exposed on their membranes upon apoptosis. Although reports accumulate about the use of this method, controversy persists as to the benefits of ANX V based sperm selection in ART. In this review we consider the arguments in favour and against this method and conclude that to the moment the evidence does not support MACS regular use in ART.

Utilidad de la separación magnética mediante columnas de anexina V en el procesado de muestras seminales para inseminación intrauterina

Infertile men show a higher proportion of apoptotic spermatozoa in ejaculate, and have a negative impact on the assisted reproduction techniques (ART) results. An adequate sperm selection which eliminates the apoptotic spermatozoa could improve the results. The objective of the study was to evaluate the effect of eliminating apoptotic spermatozoa in the ejaculate by density gradient (DG) and Magnetic Activated Cell Sorting (MACS) processing of sperm on the pregnancy rate (PR) and new born rate (NBR) after intrauterine insemination (IUI).A prospective observational and analytical study was performed on 566 cycles of IUI in 326 couples, from June 2012 to March 2014 in the Reproductive Medicine Service of Dexeus Women's Health comparing a study group (MACS) and a control group (Non-MACS). The preparation of the semen sample in both groups was by DG, and in the MACS group, the post capacitation fraction was processed using annexin V columns.The PR and NBR obtained in MACS group were 16.2% and 12.9%, respectively versus 17.7% and 15.3% in Non-MACS group. No significant differences were observed.Our results suggest that MACS used in IUI without a clinical indication does not improve the results in either the pregnancy rate or new born rate. The use of annexin V columns in other techniques, or specific indications cannot be ruled out. Further prospective randomised studies are needed in order to determine the advantages of MACS in ART.

Use of annexin v- macs in ICSI infertile couples: effect on fertilization rate (FR), pregnancy rate (PR) and embryo quality

To compare FR, PR and embryo quality in ICSI procedures when density gradient centrifugation (DGC) and MACS is used in teratozoospermic and apoptotic sperm samples vs. DGC applied in teratozoospermic samples.

Case report: the use of annexin V coupled with magnetic activated cell sorting in cryopreserved spermatozoa from a male cancer survivor: healthy twin newborns after two previous ICSI failures

The aim of the study is to report successful outcome (live births) after sperm sorting with annexin V-MACS on cryopreserved spermatozoa with high level of sperm DNA fragmentation from a cancer patient survivor. Cryopreserved spermatozoa were sorted with annexin V-MACS prior to ICSI. Sperm DNA fragmentation was evaluated by SCSA(®) and TUNEL. The couple had two previous IVF/ICSI cycles failures using sperm cryopreserved before cancer treatment. On third ICSI cycle attempt results were as follow: pre-annexin V-MACS sperm quality: 10 × 10(6)/ml, 3.3 % progressive motility, 1 % normal forms, TUNEL: 72.5 % positive cells, SCSA(®): 76.6 % DFI. Post-annexin V-MACS sperm quality: 2.8 × 10(6)/ml, 10 % progressive motility, TUNEL: 58.8 % positive cells. Eight metaphase II oocytes were collected, 4 fertilized, 2 embryos were transferred on day 3 and healthy twins were born (1 boy, 1 girl). Annexin V-MACS technique could be a potential tool to improve sperm quality on cryopreserved spermatozoa of cancer patient and improve ICSI outcome.

Down-Regulation of BMI-1 Is a New Marker of Sensitivity to Mdm2 Inhibition in B-Acute Lymphoblastic Leukemia.

AbstractAbstract 2522 Introduction:Although p53 gene mutations are relatively infrequent in cases of B-ALL, the CDKN2A locus is deleted or inactivated in nearly half of all cases, especially Ph+ B-ALL (Mullighan et al., 2008; Iacobucci et al., 2011), contributing to a worse prognosis. In testing novel therapeutic approaches activating p53, we investigated the preclinical activity of the MDM2 antagonist Nutlin-3a in leukemic cell line models and primary B-ALL patient samples. Methods:TP53 mutation screening was performed by Sanger sequencing of exons 4 to 11; copy number status of CDKN2A was determined by MLPA kit P335-A2 ALL-IKZF1 (MRC Holland); cellular viability was assessed by using a colorimetric assay based on mitochondrial dehydrogenase cleavage of WST-1 reagent (Roche); apoptosis was assessed by use of Annexin V/Propidium Iodide (PI); gene expression profile was performed using Affymetrix GeneChip Human Gene 1.0 ST platform (Affymetrix). Mdm2 inhibitor (Mdm2i) Nutlin-3a was provided by Roche. Results:BCR-ABL1-positive (BV-173, SUPB-15) and negative (NALM19, REH) ALL cell lines were investigated for TP53 mutations and CDKN2A deletion. A p53 mutation (R181C) was identified in REH cells, whereas all the remaining cell lines resulted p53 wild-type but they were deleted in the locus containing CDKN2A. Leukemia cell lines were incubated with increasing concentrations of Nutlin-3a (0.005–2 μM) for 24, 48 and 72 hours (hrs). Mdm2 inhibition resulted in a dose and time-dependent cytotoxicity with IC50 at 24 hrs ranging from around 1.5 μM for BV-173 and SUPB-15 to 3.7 μM for NALM-19. By contrast, no significant changes in cell viability were observed in RHE p53-mutated cells after incubation with Mdm2i. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after 24 hrs in sensitive cell lines and in primary leukemia blasts, whereas no apoptosis was observed in REH cells. To examine the possible mechanisms underlying Mdm2i-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased as soon as 24 hrs of incubation with Mdm2i. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis was next performed, comparing sensitive cell lines at 24 hrs of incubation with concentrations equal to the IC50 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). We found a strong down-regulation of GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells. Both genes are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. Additionally, experimental reduction of BMI1 protein levels results in apoptosis in tumor cells and increases susceptibility to cytotoxic agents and radiation therapy (Wu et al., 2011). Given the importance of BMI in the control of apoptosis, we investigated by western blot its pattern in treated and untreated cells, confirming a marked decrease as soon as 24 hrs of exposure to MDM2i both in leukemia cell lines and primary blast samples. Noteworthy, the BMI-1 levels remained constant in resistant cells. Conclusions:Inhibition of Mdm2 efficiently activates the p53 pathway promoting apoptosis. BMI-1 expression is markedly reduced in sensitive cells and it may be used as a biomarker of response. Evaluation of its expression before and after treatment in clinical settings will better gain insight into its role. Supported by:ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione – Università 2007 – 2009, INPDAP. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:BMS: Consultancy, Honoraria, Speakers Bureau; NOVARTIS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Are the annexin V microbeads, a real solution to sperm DNA fragmentation?

ObjectiveThe aim of our study was to assess the effect of annexin V microbeads on cycles with abnormal sperm DNA fragmentation (>20%), in fertilization, embryo quality and success rate.DesignAnalytic Retrospective Observational study.Materials and MethodsWe analyzed 88 cycles of Assisted Reproduction treatments.All patients had abnormal sperm DNA fragmentation and previously had undergone one or more unsuccessful cycles of IVF/ICSI.The cycles were divided into two groups:Group 1: unsuccessful cycles of IVF/ICSI.Group 2: cycles with sperm sorting with annexin V columns (36 cycles) from these same partners.The sperm DNA fragmentation was assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling).The embryos were evaluated according to criteria of the Spanish Association for Reproductive Biology (ASEBIR). The transfer was performed on day 3 of embryo development.ResultsNo significant differences were found between groups or age, nor in the rates of fertilization (P>0.05). However, embryo quality did show statistically significant differences, being higher the number of top quality embryos (A, P=0.004. A+B, P=0.02) in group 2.ConclusionThe results suggest that the use of annexin V columns improves embryo quality in couples with previous failure in IVF/ICSI. So it could be used, as a useful tool, in patients with previous IVF failures. The fertilization rate shows no significant differences. This may be because sperm with fragmented DNA were capable of fertilizing the egg as effectively as sperm without DNA fragmentation. ObjectiveThe aim of our study was to assess the effect of annexin V microbeads on cycles with abnormal sperm DNA fragmentation (>20%), in fertilization, embryo quality and success rate. The aim of our study was to assess the effect of annexin V microbeads on cycles with abnormal sperm DNA fragmentation (>20%), in fertilization, embryo quality and success rate. DesignAnalytic Retrospective Observational study. Analytic Retrospective Observational study. Materials and MethodsWe analyzed 88 cycles of Assisted Reproduction treatments.All patients had abnormal sperm DNA fragmentation and previously had undergone one or more unsuccessful cycles of IVF/ICSI.The cycles were divided into two groups:Group 1: unsuccessful cycles of IVF/ICSI.Group 2: cycles with sperm sorting with annexin V columns (36 cycles) from these same partners.The sperm DNA fragmentation was assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling).The embryos were evaluated according to criteria of the Spanish Association for Reproductive Biology (ASEBIR). The transfer was performed on day 3 of embryo development. We analyzed 88 cycles of Assisted Reproduction treatments.All patients had abnormal sperm DNA fragmentation and previously had undergone one or more unsuccessful cycles of IVF/ICSI.The cycles were divided into two groups: Group 1: unsuccessful cycles of IVF/ICSI. Group 2: cycles with sperm sorting with annexin V columns (36 cycles) from these same partners. The sperm DNA fragmentation was assessed by TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling).The embryos were evaluated according to criteria of the Spanish Association for Reproductive Biology (ASEBIR). The transfer was performed on day 3 of embryo development. ResultsNo significant differences were found between groups or age, nor in the rates of fertilization (P>0.05). However, embryo quality did show statistically significant differences, being higher the number of top quality embryos (A, P=0.004. A+B, P=0.02) in group 2. No significant differences were found between groups or age, nor in the rates of fertilization (P>0.05). However, embryo quality did show statistically significant differences, being higher the number of top quality embryos (A, P=0.004. A+B, P=0.02) in group 2. ConclusionThe results suggest that the use of annexin V columns improves embryo quality in couples with previous failure in IVF/ICSI. So it could be used, as a useful tool, in patients with previous IVF failures. The fertilization rate shows no significant differences. This may be because sperm with fragmented DNA were capable of fertilizing the egg as effectively as sperm without DNA fragmentation. The results suggest that the use of annexin V columns improves embryo quality in couples with previous failure in IVF/ICSI. So it could be used, as a useful tool, in patients with previous IVF failures. The fertilization rate shows no significant differences. This may be because sperm with fragmented DNA were capable of fertilizing the egg as effectively as sperm without DNA fragmentation.

Healthy baby born after reduction of sperm DNA fragmentation using cell sorting before ICSI

Magnetic activated cell sorting (MACS) with annexin V microbeads recognizes externalized phosphatidylserine (PS) residues on the surface of apoptotic spermatozoa. The successful use of this novel technique applied to a highly apoptotic semen sample before performing intracytoplasmic sperm injection (ICSI) is reported here. The use of annexin V microbeads for selecting non-apoptotic spermatozoa seems to reduce the percentage of altered cells, improving the chance of pregnancy after ICSI.

Apoptosis in chondrogenesis of human mesenchymal stem cells: effect of serum and medium supplements

Apoptosis is an inevitable process during development and is evident in the formation of articular cartilage and endochondral ossification of growth plate. Mesenchymal stem cells (MSCs) can serve as alternative sources for cell therapy in focal chondral lesions or diffuse osteoarthritis. But there are few, if any, studies investigating apoptosis during chondrogenesis by MSCs. The aim of this study was to find the better condition to prevent apoptosis during chondrogenesis by MSCs. Apoptosis were evaluated in MSCs induced in different chondrogenic media by the use of Annexin V, TUNEL staining, lysosomal labeling with lysotracker and immunostaining of apoptotic markers. We found apparent apoptosis was demonstrated by Annexin V, TUNEL staining and lysosomal labeling during chondrogenesis. Meanwhile, the degree of apoptosis was related to the reagents of the defined chondrogenic medium. Adding serum in medium increased apoptosis, however, TGF-beta1 inhibited apoptosis. The apoptosis was associated with the activation of caspase-3, the increase in the Bax/Bcl-2 ratio, the loss of lysosomal integrity, and the increase of PARP-cleavage. Pro-inflammatory cytokines, IL-1alpha, IL-1beta and TNFalpha did not induce any increase in apoptosis. Interestingly, the inhibition of apoptosis by serum free medium supplemented with ITS was also associated with an increase in the expression of type II collagen, and a decrease in the expression of type X collagen, Runx2, and other osteogenic genes, while TGF-beta1 increased the expression of Sox9, type II and type X collagen and decreased the expression of osteogenic genes. These data suggest apoptosis occurs during chondrogenesis by MSCs by cell death intrinsic pathway activation and this process may be modulated by culture conditions.

Lidocaine Potentiates the Chondrotoxicity of Methylprednisolone

This study examined the viability of bovine articular chondrocytes after exposure to methylprednisolone, methylprednisolone with lidocaine, and methylprednisolone in a simulated inflammatory environment. Bovine articular chondrocytes were suspended in alginate beads and cultured in Dulbecco's modified Eagle's medium/F-12 for 1 week before experimentation. Suspended chondrocytes were exposed to 0.9% saline solution (negative control), methylprednisolone (4, 8, and 16 mg/mL), methylprednisolone (8 mg/mL) with 1% lidocaine, or methylprednisolone (8 mg/mL) and saline solution in a simulated inflammatory environment (interleukin [IL] 1beta exposure, 10 ng/mL) for 15, 30, and 60 minutes. Flow cytometry was performed 1 day, 4 days, and 7 days after exposure by use of annexin V and propidium iodide to assess chondrocyte viability. Chondrocyte viability decreased from 84% in saline solution to 62%, 38%, and 2.4% 1 day after 60 minutes of exposure to 4, 8, and 16 mg/mL of methylprednisolone, respectively (n = 7, P < .05). Chondrotoxicity increased with increasing time of exposure to methylprednisolone and with increasing time after exposure. In IL-1beta-activated chondrocytes, viability decreased from 76% in saline solution to 2.9% after 60 minutes of methylprednisolone exposure (8 mg/mL) (n = 4, P < .05). The combination of 8 mg/mL of methylprednisolone and 1% lidocaine further reduced viability to 1.0% after 60 minutes (n = 4, P < .05). These results show a dose- and time-dependent decrease in chondrocyte viability after exposure to clinically relevant doses of methylprednisolone. The combination of methylprednisolone and lidocaine was toxic, with virtually no cells surviving after treatment. In addition, methylprednisolone did not mitigate the inflammatory effects of IL-1beta; rather, it further potentiated the chondrotoxicity. Intra-articular injections of corticosteroids and local anesthetics are widely used in clinical practice. This in vitro study provides information on the potential effects of these drugs on articular cartilage.

Measurement of phosphatidylserine exposure during storage of platelet concentrates using the novel probe lactadherin: a comparison study with annexin V

Annexin V binding to platelets (PLTs) is considered the gold standard for monitoring phosphatidylserine (PS) exposure. However, recent comparison of annexin V with the new calcium-independent PS probe lactadherin revealed that annexin V requires a certain threshold of PS exposure (2%-8%) for binding to occur. The aim of this study was to compare annexin V and lactadherin labeling of PLTs in PLT concentrates (PCs). Optimal labeling conditions for lactadherin and annexin V were established and then compared in either resting or calcium ionophore (CI)-activated PLTs from normal whole blood. Furthermore, 40 PCs (20 apheresis-derived and 20 pooled buffy coat-derived) were stored under standard blood bank conditions and PLT activation was monitored by measuring PS exposure with annexin V and lactadherin along with CD42b, CD61, and CD62P by flow cytometry on Days 1, 3, 5, and 7. Lactadherin reported a higher exposure of PS than did annexin V in normal PLTs at submaximal doses of CI. PLTs from both types of concentrate, as expected, demonstrated evidence of increased activation during storage using annexin V, lactadherin, CD42b, or CD62P. However, a significantly higher percentage of PS-positive PLTs was found with lactadherin than annexin V. PS exposure on the surface of stored PLTs has been previously underestimated due to the wide use of annexin V. Lactadherin provides a truer reflection of the degree of PS exposure and offers a new calcium-independent approach to studying PLT activation and/or apoptosis.

Calcium, leukocyte cell death and the use of annexin V: fatal encounters

One hallmark of programmed cell death (PCD) is redistribution of phosphatidylserine (PS) to the plasma membrane's outer leaflet. Annexin V is widely used in cell death research due to its calcium-dependent ability to bind phosphatidylserine, thus marking apoptotic cells. However, calcium is invariably used at high concentrations in annexin V staining, at doses that can induce cell death. We used flow cytometric annexin V staining, together with propidium iodide and TMRM for determination of dissipation of mitochondrial potential, with a variety of calcium concentrations, cell media, and incubation times, to identify a possible bias in PCD determination of human primary leukocytes. Here we show that measurements of PCD in human monocytes, polymorphonuclear cells, and monocyte-derived dendritic cells using annexin V may be dramatically affected by calcium concentration, time of incubation on ice, and media choice. We propose a method that enables accurate and unbiased annexin V staining, without affecting results.

TGF-β inhibits Akt-induced transformation in intestinal epithelial cells

During the early stages of colorectal carcinogenesis, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is activated, enabling the transformed cells to survive and grow in the absence of anchorage to extracellular matrix. Transforming growth factor beta (TGF-beta) is an important tumor suppressor in the colon, and it is inactivated during later stages of colorectal carcinogenesis. The purpose of this study was to determine whether TGF-beta inhibits Akt-induced anchorage-independent growth and resistance to anoikis in gut epithelial cells. Rat intestinal epithelial cells (RIE-1) were infected with a retrovirus containing pLXSN-mAkt, and three independent clones were selected. Anchorage-independent growth was examined by colony formation in soft agar and cell counting in ultralow attachment plates. Anoikis was analyzed with the use of Annexin V staining. All three clones of RIE-1/mAkt formed colonies in soft agar, which were decreased by TGF-beta. TGF-beta induced anoikis and treatment with a general caspase inhibitor, zVAD-fluoromethyl ketone, blocked TGF-beta-mediated decrease in colony formation. TGF-beta attenuated Akt-induced anchorage-independent growth in RIE-1 cells in part by enhancing anoikis. Our data demonstrate a novel tumor-suppressor activity of TGF-beta and provide the molecular justification for the required activation of the PI3K/Akt pathway and the subsequent inactivation of TGF-beta signaling during colorectal carcinogenesis.