scholarly journals In vitro and in vivo efficacy of non-psychoactive cannabidiol in neuroblastoma

2016 ◽  
Vol 23 ◽  
pp. 15 ◽  
Author(s):  
T. Fisher ◽  
H. Golan ◽  
G. Schiby ◽  
S. PriChen ◽  
R. Smoum ◽  
...  
Keyword(s):  
Folk Medicine ◽  
Annexin V ◽  
Cell Cycle Distribution ◽  
Cell Accumulation ◽  
Pharmacologic Activity ◽  
Induced Apoptosis ◽  
Annexin V Assay

Background Neuroblastoma (nbl) is one of the most common solid cancers in children. Prognosis in advanced nbl is still poor despite aggressive multimodality therapy. Furthermore, survivors experience severe long-term multi-organ sequelae. Hence, the identification of new therapeutic strategies is of utmost importance. Cannabinoids and their derivatives have been used for years in folk medicine and later in the field of palliative care. Recently, they were found to show pharmacologic activity in cancer, including cytostatic, apoptotic, and antiangiogenic effects.Methods We investigated, in vitro and in vivo, the anti-nbl effect of the most active compounds in Cannabis, Δ9-tetrahydrocannabinol (thc) and cannabidiol (cbd). We set out to experimentally determine the effects of those compounds on viability, invasiveness, cell cycle distribution, and programmed cell death in human nbl SK-N-SH cells.Results Both compounds have antitumourigenic activity in vitro and impeded the growth of tumour xenografts in vivo. Of the two cannabinoids tested, cbd was the more active. Treatment with cbd reduced the viability and invasiveness of treated tumour cells in vitro and induced apoptosis (as demonstrated by morphology changes, sub-G1 cell accumulation, and annexin V assay). Moreover, cbd elicited an increase in activated caspase 3 in treated cells and tumour xenografts.Conclusions Our results demonstrate the antitumourigenic action of cbd on nbl cells. Because cbd is a non psychoactive cannabinoid that appears to be devoid of side effects, our results support its exploitation as an effective anticancer drug in the management of nbl.

scholarly journals A Higher Frequency Administration of the Nontoxic Cycloartane-Type Triterpene Argentatin A Improved Its Anti-Tumor Activity

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1780 ◽  
Author(s):  
Zaira Tavarez-Santamaría ◽  
Nadia J. Jacobo-Herrera ◽  
Leticia Rocha-Zavaleta ◽  
Alejandro Zentella-Dehesa ◽  
Beatriz del Carmen Couder-García ◽  
...  
Keyword(s):  
Waste Product ◽  
Industrial Process ◽  
Annexin V ◽  
Control Group ◽  
Healthy Control ◽  
Hct 116 ◽  
Hct 116 Cells ◽  
Induced Apoptosis

Parthenium argentatum (Gray), commonly known as guayule, has been used to obtain natural rubber since the beginning of the 20th century. Additionally, the so called “resin” is a waste product derived from the industrial process. The cycloartane-type triterpene Argentatin A (AA) is one of the main constituents of the industrial waste resin. In this study we evaluated the AA anticancer activity both in vitro and in vivo in the HCT116 colon cancer cells. The apoptosis promotion of AA was assessed by the annexin V/propidium iodide (PI) assay. The senescence was evaluated for SA-β-galactosidase, and PCNA was used as a marker of proliferation. Its antitumor activity was evaluated using a xenograft mouse model. The results indicated that AA-induced apoptosis in HCT-116 cells and was positively stained for SA-β-galactosidase. In the xenografted mice test, the administration of AA at the dose of 250 mg/kg three times a week for 21 days reduced tumor growth by 78.1%. A comparable tumor reduction was achieved with cisplatin at the dose of 2 mg/kg administered three times a week for 21 days. However, nude mice treated with AA did not lose weight, as they did remarkably when treated with cisplatin. Furthermore, the animals treated with AA showed similar blood profiles as the healthy control group. These data indicate the low toxicity of AA compared to that shown by cisplatin.

scholarly journals A Novel High-Throughput Technique for Identifying Monoclonal Antibodies capable of Death Receptor Induced Apoptosis

2009 ◽  
Vol 2 ◽  
pp. JCD.S3660
Author(s):  
Hang Fai Kwok ◽  
Julie A. Gormley ◽  
Christopher J. Scott ◽  
James A. Johnston ◽  
Shane A. Olwill
Keyword(s):  
Cell Death ◽  
High Throughput ◽  
High Throughput Screening ◽  
False Negative ◽  
Death Receptor ◽  
Annexin V ◽  
Fas Receptor ◽  
Induced Apoptosis

The study of death receptor family induced apoptosis has gained momentum in recent years with the knowledge that therapeutic antibodies targeting DR4 and DR5 (death receptor's 4 and 5) have proved efficacious in multiple clinical trials. The therapeutic rationale is based on targeting and amplifying a tumour tissues normal cell death programme (apoptosis). While advances in the targeting of DR4 and DR5 have been successful the search for an agonistic antibody to another family member, the Fas receptor, has proven more elusive. This is partly due to the differing in vitro and in vivo characteristics of individual antibodies. In order to induce Fas targeted cell death an antibody must be capable of binding to and trimerising the receptor. It has been shown that antibodies capable of performing this function in vivo, with the assistance of tumour associated cells, do not always induce apoptosis in vitro. As a result the use of current methodologies to detect functional antibodies in vitro may have dismissed potential therapeutic candidates ('false negative'). Here we report a novel high throughput screening technique which artificially cross-links antibodies bound to the Fas receptor. By combining this process with Annexin-V and Prodidium Iodide (PI) staining we can select for antibodies which have the potential to induce apoptosis in vivo.

Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 866-866
Author(s):  
Carolina Berger ◽  
Michael C. Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Annexin V ◽  
T Cell Memory ◽  
T Cell Clones ◽  
Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for >30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

NPI-2358, a Novel Vascular Disrupting Agent Blocks Growth and Angiogenesis in Multiple Myeloma Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3842-3842
Author(s):  
Dharminder Chauhan ◽  
Ajita V. Singh ◽  
Madhavi Bandi ◽  
Noopur Raje ◽  
Robert L Schlossman ◽  
...  
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Vascular Endothelial ◽  
Caspase 9 ◽  
Migration Assays ◽  
Induced Apoptosis ◽  
Rpmi 8226 ◽  
Anti Tumor Activity

Abstract Abstract 3842 Poster Board III-778 Background and Rationale Vascular disrupting agents (VDAs) act via selectively disrupting established tumor vasculature and have shown remarkable clinical success as anti-cancer therapies. NPI-2358 is a novel VDA with a distinct structure and mechanism of action from other available VDAs. NPI-2358 binds to the colchicine-binding site of beta-tubulin preventing polymerization and disrupting the cytoplasmic microtubule network, thereby causing loss of vascular endothelial cytoskeletal function, and inducing cytotoxicity in cancer cells. Here, we examined the anti-angiogenic and anti-tumor activity of NPI-2358 in multiple myeloma (MM) cells using both in vitro and in vivo model systems. Material and Methods We utilized MM.1S, MM.1R, RPMI-8226, U266, and INA-6 human MM cell lines, as well as purified tumor cells from MM patients relapsing after prior anti-MM therapies. Cell viability/apoptosis assays were performed using MTT, trypan blue exclusion, and Annexin V/PI staining. Angiogenesis was measured in vitro using Matrigel capillary-like tube structure formation assays: Since human vascular endothelial cells (HUVECs) plated onto Matrigel differentiate and form capillary-like tube structures similar to in vivo neovascularization, this assay measures anti-angiogenic effects of drugs/agents. Migration assays were performed using transwell insert assays. Immunoblot analysis was performed using antibodies to caspase-8, caspase-9, caspase-3, PARP, Bcl-2, Bax, pJNK and GAPDH. Statistical significance was determined using a Student t test. Results Treatment of MM.1S, RPMI-8226, MM.1R, INA-6, and KMS-12BM with NPI-2358 for 24h induces a dose-dependent significant (P < 0.005) decrease in viability of all cell lines (IC50 range: 5-8 nM; n=3). To determine whether NPI-2358-induced decrease in viability is due to apoptosis, MM cell lines were treated with NPI-2358 for 24h; harvested, and analyzed for apoptosis using Annexin V/PI staining. A significant increase in NPI-2358-induced apoptosis was observed in all MM cell lines (% Annexin V+/PI- apoptotic cells: MM.1S, 48 ± 2.3%; MM.1R, 46.6 ± 3.1%; RPMI-8226, 61.7 ± 4.5%; and INA-6, 59.9 ± 3.2%; P < 0.05; n=3). Importantly, NPI-2358 decreased viability of freshly isolated MM cells from patients (IC50 range: 3-7 nM; P < 0.005), without affecting the viability of normal peripheral blood mononuclear cells, suggesting specific anti-MM activity and a favorable therapeutic index for NPI-2358. Examination of in vitro angiogenesis using capillary-like tube structure formation assay showed that even low doses of NPI-2358 (7 nM treatment for 12h; IC50: 20 nM at 24h) significantly decreased tubule formation in HUVECs (70-80% decrease; P < 0.05). Transwell insert assays showed a marked reduction in serum-dependent migration of NPI-2358-treated MM cells (42 ± 2.1% inhibition in NPI-2358-treated vs. control; P < 0.05). NPI-2358 at the concentrations tested (5 nM for 12h) in the migration assays did not affect survival of MM cells (> 95% viable cells). A similar anti-migration activity of NPI-2358 was noted against HUVEC cells (48 ± 1.7% decrease in migration; P < 0.05). Mechanistic studies showed that NPI-2358-induced apoptosis was associated with activation of caspase-8, caspase-9, caspase-3 and PARP. Importantly, treatment of MM.1S cells with NPI-2358 (5 nM) triggered phosphorylation of c-Jun amino-terminal kinase (JNK), a classical stress response protein, without affecting Bcl-2 family members Bax and Bcl-2. Blockade of JNK using dominant negative strategy markedly abrogated NPI-2358-induced apoptosis. Conclusion Our preclinical data provide evidence for remarkable anti-angiogenic and anti-tumor activity of NPI-2358 against MM cells, without significant toxicity in normal cells. Ongoing studies are examining in vivo anti-MM activity of NPI-2358 in animal models. Importantly, a Phase-1 study of NPI-2358 as a single agent in patients with advanced malignancies (lung, prostrate and colon cancer) has already established a favorable pharmacokinetic, pharmacodynamic and safety profile; and, a Phase-2 study of the combination of NPI-2358 and docetaxel in non-small cell lung cancer showed encouraging safety, pharmacokinetic and activity data. These findings, coupled with our preclinical studies, provide the framework for the development of NPI-2358-based novel therapies to improve patient outcome in MM. Disclosures: Chauhan: Nereus Pharmaceuticals, Inc: Consultancy. Lloyd:Nereus Pharmaceuticals, In: Employment. Palladino:Nereus Pharmaceuticals, Inc: Employment. Anderson:Nereus Pharmaceuticals, Inc: Consultancy.

scholarly journals Metallothionein 3 Is a Hypoxia-Upregulated Oncogene Enhancing Cell Invasion and Tumorigenesis in Human Bladder Carcinoma Cells

2019 ◽  
Vol 20 (4) ◽  
pp. 980 ◽  
Author(s):  
Ke-Hung Tsui ◽  
Chen-Pang Hou ◽  
Kang-Shuo Chang ◽  
Yu-Hsiang Lin ◽  
Tsui-Hsia Feng ◽  
...  
Keyword(s):  
Bladder Carcinoma ◽  
Annexin V ◽  
Carcinoma Cells ◽  
Gene Expressions ◽  
T24 Cells ◽  
Apoptosis Assay ◽  
Induced Apoptosis ◽  
Bladder Carcinoma Cells

Metallothioneins have been viewed as modulators in a number of biological regulations regarding cancerous development; however, the function of metallothionein 3 (MT3) in bladder cancer is unexplored. We determined the regulatory mechanisms and potential function of MT3 in bladder carcinoma cells. Real-Time Reverse Transcriptase-Polymerase Chain Reaction (RT-qPCR) assays revealed that TSGH-8301 cells expressed more MT3 levels than RT-4, HT1376, and T24 cells. Immunoblot and RT-qPCR assays showed that arsenic (AS2O3) treatments enhanced the gene expression of MT3. Hypoxia induced HIF-1α, HIF-2α, and MT3 expression; furthermore, HIF-2α-knockdown attenuated hypoxic activation on MT3 expression. Ectopic overexpression of MT3 increased cell proliferation, invasion, and tumorigenesis significantly in T24 and HT1376 cells in vitro and in vivo; however, MT3-knockdown in TSGH-8301 cells had the reverse effect. Moreover, knockdown of MT3 enhanced arsenic-induced apoptosis determined by the Annexin V-FITC apoptosis assay. MT3-overexpression downregulated the gene expressions of N-myc downstream regulated gene 1 (NDRG1), N-myc downstream regulated gene 2 (NDRG2), and the mammary serine protease inhibitor (MASPIN) in HT1376 and T24 cells, whereas MT3-knockdown in TSGH-8301 cells had the opposite effect. The experiments indicated that MT3 is an arsenic- and hypoxia-upregulated oncogene that promotes cell growth and invasion of bladder carcinoma cells via downregulation of NDRG1, NDRG2, and MASPIN expressions.

scholarly journals Suppression of CHOP Reduces Neuronal Apoptosis and Rescues Cognitive Impairment Induced by Intermittent Hypoxia by Inhibiting Bax and Bak Activation

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Linhao Xu ◽  
Yanli Bi ◽  
Yizhou Xu ◽  
Yihao Wu ◽  
Xiaoxue Du ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Synaptic Plasticity ◽  
Signaling Pathway ◽  
Intermittent Hypoxia ◽  
Specific Chemical ◽  
Activating Transcription Factor ◽  
Induced Apoptosis

Our previous study showed that growth arrest- and DNA damage-inducible gene 153 (GAD153/CHOP) plays an important role in intermittent hypoxia- (IH-) induced apoptosis and impaired synaptic plasticity. This study is aimed at determining which signaling pathway is activated to induce CHOP and the role of this protein in mitochondria-dependent apoptosis induced by IH. In the in vivo study, mice were placed in IH chambers for 8 h daily over a period of 2 weeks; the IH chambers had oxygen (O2) concentrations that oscillated between 10% and 21%, cycling every 90 s. In the in vitro study, PC12 cells were exposed to 21% O2 (normoxia) or 8 IH cycles (25 min at 21% O2 and 35 min at 0.1% O2 for each cycle). After 2 weeks of IH treatment, we observed that the expression levels of phosphorylated protein kinase-like endoplasmic reticulum kinase (p-PERK), activating transcription factor 4 (ATF-4) and phosphorylated eukaryotic initiation factor 2 alpha (p-elf2α), were increased, but the levels of activating transcription factor 6 (ATF-6) and inositol-requiring enzyme 1 (IRE-1) were not increased. GSK2606414, a specific chemical inhibitor of the PERK pathway, reduced the expression of p-PERK, ATF-4, p-elf2α, and CHOP and rescued ER structure. In addition, Bax and Bak accumulated in the mitochondria after IH treatment, which induced cytochrome c release and initiated apoptosis. These effects were prevented by GSK2606414 and CHOP shRNA. Finally, the impaired long-term potentiation and long-term spatial memory in the IH group were rescued by GSK2606414. Together, the data from the in vitro and in vivo experiments indicate that IH-induced apoptosis and impaired synaptic plasticity were mediated by the PERK-ATF-4-CHOP pathway. Suppressing PERK-ATF-4-CHOP signaling pathway attenuated mitochondria-dependent apoptosis by reducing the expression of Bax and Bak in mitochondria, which may serve as novel adjunct therapeutic strategy for ameliorating obstructive sleep apnea- (OSA-) induced neurocognitive impairment.

scholarly journals Methyl gallate, gallic acid-derived compound, inhibit cell proliferation through increasing ROS production and apoptosis in hepatocellular carcinoma cells

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0248521
Author(s):  
Chien-Yu Huang ◽  
Yu-Jia Chang ◽  
Po-Li Wei ◽  
Chin-Sheng Hung ◽  
Weu Wang
Keyword(s):  
Hepatocellular Carcinoma ◽  
Western Blot ◽  
Gallic Acid ◽  
Annexin V ◽  
Inhibitory Effect ◽  
Ros Production ◽  
Methyl Gallate ◽  
Induced Apoptosis

Hepatocellular carcinoma (HCC) is a global health problem. Currently, there is no effective therapeutic strategy for HCC. Methyl gallate (MG), from plant-derived phenolic gallic acid, has exhibited antitumor efficacy. However, the effect of MG on HCC is unclear. In vitro growth activity was detected by a sulforhodamine assay. A zebrafish xenotransplantation was applied to evaluate the inhibitory effect of MG. Reactive oxygen species (ROS) production, autophagy, and lysosome formation were detected by specific dyes. Finally, apoptosis was examined using annexin V-FITC/PI staining and western blot was performed to determine the molecular mechanism. It was demonstrated that MG treatment inhibited the proliferation of Hep3B, Mahlavu, and HepJ5 cells. Xenotransplantation also showed that MG inhibited the growth of Hep3B and HepJ5 cells. MG treatment increased cellular levels of superoxide and oxidative stress. Increases in autophagy and lysosome formation were found after MG treatment. The western blot analysis showed that MG activated cleavage of caspase-3 and poly (SDP ribose) polymerase (PARP), modulated levels of the Bcl2, Bax, and Bad ligands, and induced apoptosis. MG induced autophagy with notable activation of beclin-1, autophagy related 5+12 (ATG5+12), and conversion of light chain 3-I (LC3-I) to II. Our study showed that MG exposure inhibited HCC proliferation both in vitro and in vivo. And blocking autophagy enhanced MG-induced cytotoxicity in HCC cells. These findings suggested MG might serve as a powerful therapeutic supplement for human HCC patients.

scholarly journals MicroRNA-29c Increases the Chemosensitivity of Pancreatic Cancer Cells by Inhibiting USP22 Mediated Autophagy

2018 ◽  
Vol 47 (2) ◽  
pp. 747-758 ◽  
Author(s):  
Limin Huang ◽  
Chaoquan Hu ◽  
Hui Cao ◽  
Xiaoliang Wu ◽  
Rongpin Wang ◽  
...  
Keyword(s):  
Pancreatic Cancer ◽  
Flow Cytometry ◽  
Annexin V ◽  
Luciferase Reporter ◽  
Pancreatic Cancer Cells ◽  
Reporter Assays ◽  
Direct Target ◽  
Induced Apoptosis

Background/Aims: Pancreatic cancer (PC) is an aggressive malignancy with a poor survival rate. Despite advances in the treatment of PC, the efficacy of therapy is limited by the development of chemoresistance. Here, we examined the role of microRNA-29c (miR-29c) and the involvement of autophagy and apoptosis in the chemoresistance of PC cells in vivo and in vitro. Methods: We employed qRT-PCR, western blot and immunofluorescence to examine the expression level of miR-29c, USP22 and autophagy relative protein. In addition, we used MTT assay to detect cell proliferation and transwell assay to measure migration and invasiveness. The apoptosis was determined using annexin V-FITC/PI apoptosis detection kit by flow cytometry. Luciferase reporter assays confirmed the relationship between USP22 and miR-29c. Results: miR-29c overexpression in the PC cell line PANC-1 enhanced the effect of gemcitabine on decreasing cell viability and inducing apoptosis and inhibited autophagy, as shown by western blotting, immunofluorescence staining, colony formation assays, and flow cytometry. Ubiquitin specific peptidase (USP)-22, a deubiquitinating enzyme known to induce autophagy and promote PC cell survival, was identified as a direct target of miR-29c. USP22 knockdown experiments indicated that USP22 suppresses gemcitabine-induced apoptosis by promoting autophagy, thereby increasing the chemoresistance of PC cells. Luciferase reporter assays confirmed that USP22 is a direct target of miR-29c. A xenograft mouse model demonstrated that miR-29c increases the chemosensitivity of PC in vivo by downregulating USP22, leading to the inhibition of autophagy and induction of apoptosis. Conclusions: Taken together, these findings reveal a potential mechanism underlying the chemoresistance of PC cells mediated by the regulation of USP22-mediated autophagy by miR-29c, suggesting potential targets and therapeutic strategies in PC.

scholarly journals Apoptosis in T-Lymphocyte Subsets in Human Immunodeficiency Virus-Infected Children Measured Immediately Ex Vivo and following In Vitro Activation

2001 ◽  
Vol 8 (1) ◽  
pp. 74-78 ◽  
Author(s):  
Tim Niehues ◽  
Thomas W. McCloskey ◽  
Jennifer Ndagijimana ◽  
Gerd Horneff ◽  
Volker Wahn ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Ex Vivo ◽  
Annexin V ◽  
Cd4 Cells ◽  
T Lymphocyte Subsets ◽  
Cd8 Cells ◽  
Immunodeficiency Virus ◽  
Annexin V Assay

ABSTRACT Phosphatidylserine molecules are translocated to the outer plasma membrane of lymphocytes undergoing apoptosis and can be detected by the binding of fluorochrome-conjugated annexin V. Using the annexin V assay, we examined CD4 and CD8 T cells from human immunodeficiency virus (HIV)-infected children for apoptosis upon isolation or following in vitro culture. Immediate ex vivo analysis or overnight culture showed significantly higher levels of apoptosis in CD8 cells than in CD4 cells. Following culture with the activating stimulus phytohemagglutinin or anti-CD3 monoclonal antibody, we observed an increase in the percentage of apoptotic CD4 cells, whereas there was no change in the rate of CD8 cell death. These results demonstrate that in HIV-infected children, CD8 apoptosis may occur at a greater rate than CD4 apoptosis in vivo; greater CD4 depletion may be observed due to more efficient mechanisms for peripheral lymphocyte replacement in the CD8 compartment. Furthermore, our data suggest that CD8 lymphocytes may be maximally activated in vivo, a condition which may lead to the exhaustion of CD8-mediated immunity. These findings clarify the differences between the CD4 and CD8 apoptotic responses to HIV.

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