OBJECTIVEEpstein-Barr virus (EBV) is a common causative agent of infectious mononucleosis (IM) and capable of efficiently immortalizing primary B cells into continuously growing lymphoblastoid cells in vitro. As B cell activation antigen, CD23 expression is induced by EBV infection of B cells and remains constitutively expressed at high levels in virtually all EBV-immortalized cells, which have been strongly linked to the development of B-cell lymphoproliferative disease and lymphoma. Whereas previous studies were performed in vivo in animals or ex vivo cultures, the present study aimed to explore the role of EBV-immortalized cells (CD23(+)/CD19(+)) in vivo analysis of children with EBV-IM.

METHODSIn a prospective trial, a group of 30 patients with IM (18 boys and 12 girls) with mean age of 3.9 +/- 1.3 years (range 6 months to 8 years) were enrolled. Clinical diagnosis of IM was confirmed based on fever, lymphadenopathy, splenomegaly, lymphocytosis (> 50%), atypical lymphocytes (> 10%) in blood smears and the elevated levels of IgM antibody against EBV capsid antigen. The day of onset of fever was recognized as day 1 of illness. Blood samples taken during acute (3 - 5 days), early convalescent (about 11 - 15 days) and convalescent phase (about 30 - 45 days) were analyzed for expressions of CD19(+)/CD23(+), CD23, CD19 on peripheral blood mononuclear cells by flow cytometry (FCM) and was compared with those of control group.

RESULTS(1) The levels of CD23(+)/CD19(+) and CD23 expressions were markedly decreased in acute stage [CD23(+)/CD19(+) (2.22 +/- 1.47)%, (132 +/- 91)/mm(3); CD23 (3.12 +/- 1.88)%, (195 +/- 102)/mm(3)] and in early convalescent stage [CD23(+)/CD19(+) (4.51 +/- 2.25)%, (166 +/- 85)/mm(3); CD23 (5.55 +/- 2.76)%, (231 +/- 130)/mm(3)] in patients with IM as compared with those of the healthy controls [CD23(+)/CD19(+) (6.71 +/- 2.25)%, (215 +/- 68)/mm(3); CD23 (7.85 +/- 3.09)%, (249 +/- 86)/mm(3), respectively]. The earlier the history was, the lower the expressive levels were. The levels of CD23(+)/CD19(+) expressions returned to, but those of CD23 expressions exceeded, normal level in convalescent stage [CD23(+)/CD19(+) (6.72 +/- 2.16)%, (213 +/- 108)/mm(3); CD23 (9.46 +/- 2.73)%, (366 +/- 200)/mm(3)]. (2) There was a positive correlation in the expressions of CD23(+)/CD19(+) and CD23 among the three stages (P < 0.01). The positive correlation between the expressions of CD23(+)/CD19(+) and CD19 only occurred during acute stage (P < 0.01). There was no correlation between the expressions of CD23 and CD19 (P > 0.05).

CONCLUSIONEBV-immortalized cells and CD23(+) cells were inhibited effectively during the acute and early convalescent stage of IM. With the recovery of the disease, they gradually recovered and the levels of CD23 expressions exceeded normal level in convalescent stage.

B-Lymphocytes ; metabolism ; Cell Culture Techniques ; Cell Survival ; Child ; Child, Preschool ; Female ; Herpesvirus 4, Human ; pathogenicity ; Humans ; Infant ; Infectious Mononucleosis ; metabolism ; Male ; Receptors, IgE ; metabolism

OBJECTIVEInfectious mononucleosis (IM) is a lymphoproliferative disease caused primarily by the Epstein-Barr virus (EBV) infection. The initial viral infection by EBV occurs in B lymphocytes and is followed by an extensive proliferation of T lymphocytes. Previous studies on immunity to EBV (including IM) have mainly focused on activation of peripheral blood T cells, which are responsible for the lymphocytosis in blood during acute IM. B cells, regarding CD23 as their activation marker, are the target cells of EBV infection. There are few reports on their effect in patients with IM. The role of them during acute IM is not known yet. The present study aimed to explore the action of B cells in patients with IM.

METHODSIn a prospective trial, a group of subjects comprised 22 patients with IM (14 boys and 8 girls) with mean age of 3.48 +/- 0.81 years (range 7 months to 8 years). Clinical diagnosis of IM was confirmed based on fever, lymphadenopathy, splenomegaly, lymphocytosis (> 50%), atypical lymphocytes (> 10%) in blood smears and the elevated levels of IgM antibody against EBV capsid antigen. The day of onset of fever was recognized as day 1 of illness. Blood samples taken during acute (3 - 5 days) and convalescent phase (about 15 days) were analyzed for expressions of CD19, CD19(+)/CD23(+) on PBMC by flow cytometry (FCM) and was compared with those of control group. The number of the days with fever was recorded.

RESULTS(1) The levels of CD19 and CD19(+)/CD23(+) expressions were markedly decreased in acute stage [CD19 (5.63 +/- 2.91)%, (387 +/- 178)/mm(3), CD19(+)/CD23(+) (2.45 +/- 1.87)%, (160 +/- 99)/mm(3)] and in convalescent stage [CD19 (12.49 +/- 5.70)%, (428 +/- 156)/mm(3), CD19(+)/CD23(+) (5.05 +/- 2.79)%, (172 +/- 78)/mm(3)] in patients with IM as compared with those of the healthy controls [CD19 (16.20 +/- 2.80)%, (545 +/- 150)/mm(3); CD19(+)/CD23(+) (7.08 +/- 2.78)%, (249 +/- 136)/mm(3)]. The earlier the specimens were taken after onset, the lower the expressed levels were. (2) There was a positive correlation of the expressions of CD19 and CD19(+)/CD23(+) between acute and convalescent stage (P < 0.01);there was also a positive correlation between the expressions of CD19 and CD19(+)/CD23(+) during acute and convalescent stage (P < 0.01). (3) A negative correlation was found between the duration of fever and the level of CD19 and CD19(+)/CD23(+) in acute stage (P < 0.01).

CONCLUSIONThe results indicate that B cells and CD23(+) B cells were significantly inhibited during the onset of IM in the patients, that with the recovery of the disease, the condition was gradually improved, and that the more evidently the CD19 and CD19(+)/CD23(+) decreased, the more serious the clinical symptoms were and the longer time the recovery needed. The levels of CD19 and CD19(+)/CD23(+) expressions may be useful in diagnosis and predicting the severity.

Antigens, CD19 ; immunology ; B-Lymphocytes ; immunology ; Child ; Child, Preschool ; Female ; Herpesvirus 4, Human ; Humans ; Infant ; Infectious Mononucleosis ; diagnosis ; immunology ; virology ; Male ; Prospective Studies ; Receptors, IgE ; immunology ; T-Lymphocytes ; immunology

To study the molecular mechanisms of nitric oxide donor sodium nitroprusside (SNP) -induced apoptosis in K562 human leukemia cell line, the different concentrations of SNP and different time of culture were used to treat K562 cell. At the same time, potassium ferricyamide (PFC) was used as control, blank was designed in experiment. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantify in situ cell apoptosis. Reactive oxygen species (ROS) in cells and mitochondrial transmembrane potential (DeltaPsim) were labeled by dihydrorhodamin 123, 2', 7'-dichlorodihydrofluorescein diacetate and rhodamin 123/PI. bcl-2, bax, bad, p53 gene proteins and mitochondrial membrane protein were analysed by flow cytometry. The results showed that the K562 cell apoptosis was confirmed by typical cell morphology, DNA fragment, sub-G(1) phase, TUNEL and annexin-V/PI labeling. A majority of K562 cells were arrested in G(0)/G(1) phase. During the process of SNP-induced apoptosis in K562 cell, the mean fluorescence intensity of ROS in cells was significantly higher than those in blank and PFC control, while the DeltaPsim reduced. The expression of p53, bax, bad, Fas protein and mitochondrial membrane protein increased and bcl-2 protein decreased after SNP treatment. It is concluded that SNP induces K562 cell apoptosis through increasing ROS in cells, expressing the p53, bax, bad, Fas protein and mitochondrial membrane protein and decreasing bcl-2 protein, opening the mitochondrial permeability transition pore and reducing DeltaPsim. Furthermore, the Fas was activated during the apoptosis process.
Apoptosis ; drug effects ; Dose-Response Relationship, Drug ; Humans ; In Situ Nick-End Labeling ; K562 Cells ; Membrane Potential, Mitochondrial ; drug effects ; Nitric Oxide Donors ; pharmacology ; Nitroprusside ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Reactive Oxygen Species ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism ; fas Receptor ; biosynthesis

This study was aimed to investigate the changes of reactive oxygen species and antioxidative capacity on nitric oxide induced apoptosis in HL-60 cells. By means of in vitro incubation of HL-60 cells with sodium nitroprusside (SNP), the growth inhibition was detected by MTT assay. Cell morphology was observed by transmission electronmicroscopy and light microscopy. The apoptosis was analyzed by DNA agarose gel electrophoresis, DNA content and Annexin-V/PI labeling method. Reactive oxygen species (ROS) labeled with dihydrorhodamin 123 in cells was determinated by flow cytometry. The SNP-treated cells were examined for glutathione (GSH) level and activity of catalase (CAT), glutathione S-transferase (GST) and glutathione peroxidase (GPX). The results indicated that SNP could inhibit HL-60 cell growth. Cell apoposis was confirmed by typical cell morphology, DNA fragment, sub-G(1) phase and Annexin-V/PI labeling method. HL-60 cell apoptosis was induced by SNP in a dosage- and time-dependent manner. After exposing to SNP at the concentration of 0.5 - 3.0 mmol/L for 48 hours, the mean fluorescence intensity of ROS in cells was significantly higher than those in groups control and potassium ferricyanide (PFC). During the apoptosis process, level of ROS in cells increased, levels of GSH, CAT, GPTand GPX decreased. The significant dose-effect relationship existed between the levels of ROS, CAT, GST, GPX and SNP dose. It is concluded that change of intracellular reactive oxygen species and antioxidative capacity are an important factors during the process of SNP-induced apoptosis in HL-60 cell.
Antioxidants ; metabolism ; Apoptosis ; drug effects ; Cell Nucleus ; drug effects ; ultrastructure ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; HL-60 Cells ; Humans ; Microscopy, Electron ; Nitric Oxide Donors ; pharmacology ; Nitroprusside ; pharmacology ; Reactive Oxygen Species ; metabolism

This study is to explore the mechanism and effect of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation and apoptosis of A549 cells in vitro and on A549 xenograft tumor in nude mice. With different concentrations of ZGDHu-1 at different times were used to treat A549 cells in vitro. The proliferation was determined by living cell count, SRB assay and Brdu-ELISA. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin V/PI and Hoechst 33258 labeling method. The nude mice model of A549 xenograft tumor was established by subcutaneous inoculation. The suppression activity of ZGDHu-1 by intraperitoneal injection on xenograft mice model was detected. The expressions of bcl-2, bax and p53 gene and protein were analyzed by RT-PCR and flow cytometry. ZGDHu-1 can inhibit A549 cell proliferation viability within a certain range of treating time and does, and a majority of A549 cells were arrested in G2-M phase. The A549 cells apoptosis was confirmed by typical cell morphology, DNA fragment, Sub G1 phase, Hoechst 33258 and Annexin V/PI labeling method with a time and dose related manner. When the xenograft tumor mice model were treated with 10, 20 and 40 mg x kg(-1) ZGDHu-1 for 14 days, the tumor growth inhibition rate were 43.7%, 56.9% and 60.0%, respectively. The expression of bax, bax/bcl-2 and p53 gene and protein increased significantly and bcl-2 decreased slightly by the treatment of ZGDHu-1. ZGDHu-1 can significantly suppress the growth of A549 xenograft tumor in vivo and inhibited proliferation by inducing tumor cell apoptosis in vitro. The mechanism may associate with its up-regulation of bax and p53 during the apoptosis process.
Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Female ; Flow Cytometry ; Heterocyclic Compounds, 1-Ring ; pharmacology ; Humans ; Lung Neoplasms ; metabolism ; pathology ; prevention & control ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; Xenograft Model Antitumor Assays ; bcl-2-Associated X Protein ; biosynthesis ; genetics

The aim of study was to investigate the mechanism of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) inducing apoptosis in SHI-1 human leukemia cell line. Different concentrations of ZGDHu-1 and different times of culture were used to treat SHI-1 cells; the apoptosis of SHI-1 cells was analyzed by morphology, DNA agarose gel electrophoresis, DNA content detection, Annexin-V/PI and Hoechst33258 labeling method, the mitochondrial transmembrane potential (Delta Psi m) were measured by dihydrorhodamin 123, and expressions of bcl-2, bax, Fas, p53 and mitochondrial membrane protein were analyzed by flow cytometry, while the bcl-2, bax and p53 gene were analyzed by RT-PCR. The transcriptional level of hTERT-mRNA was measured by real-time fluorescence quantitative RT-PCR. The results showed that after exposure to ZGDHu-1, SHI-1 cells were induced to apoptosis in a time-and does-dependent manner. SHI-1 cell apoptosis was confirmed by typical cell morphology, DNA fragmentation, sub-G(1) phase, Hoechst33258 and Annexin-V/PI labeling etc. The expression of bax, bax/bcl-2, p53 and Fas gene significantly increased and bcl-2 slightly decreased. ZGDHu-1 could increased the expression of mitochondrial membrane protein in a dose-dependent manner while Delta Psi m reduced. The expression of hTERT-mRNA significantly decreased. It is concluded that ZGDHu-1 can up-regulate the expression of p53, bax and bax/bcl-2. The mitochondrial pathway mediated by descent of mitochondrial transmembrane potential may be one of the mechanisms inducing apoptosis by ZGDHu-1, in which Fas gene also participates. Telomerase may be an effective gene target for anti-tumour effect of ZGDHu-1.
Animals ; Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Heterocyclic Compounds, 1-Ring ; pharmacology ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Mice ; Mice, Nude ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; metabolism ; Telomerase ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

The purpose of this study was to explore the effect of N, N'-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation, differentiation and apoptosis in NB4 human leukemia cell line and its possible mechanism. Different concentrations of ZGDHu-1 and the different time of cultivation were used to treat NB4 cells. The proliferation inhibition of NB4 cells was analysed by cell counting, alive cell count, MTT assay. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin-V/PI and Hoechst 33258 labeling method. The analysis of cell morphological change, expression of CD11b, CD13 and NBT reduction were performed to evaluate the differentiation of NB4 cells. The expressions of bcl-2, bax and phosphorylated p38MAPK or STAT3 were detected by flow cytometry. While the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that ZGDHu-1 could inhibit NB4 cell proliferation viability within a certain range of treating time and does, IC(50) values at 48 and 72 hours were 450 ng/ml and 200 ng/ml respectively. A majority of NB4 cells were arrested in G(2/M) phase and a progressive decline of cells was seen in G(0/1). The NB4 cells apoptosis was confirmed by cell typical cell morphology, DNA fragments and sub-G(1) phase peak as well as Hoechst33258 and Annexin-V/PI labeling method with a time-dose-related manner. The morphology of NB4 cells cultured in the presence of 2 - 100 ng/ml ZGDHu-1 for three days was more mature with higher NBT positivity and expressions of CD11b and CD13 than those in control. The expression of phosphor-p38MAPK and bax was increased while phosphor-STAT3 and bcl-2 were unchanged by the treatment of ZGDHu-1. ZGDHu-1 could decrease the expression of hTERT-mRNA in a dose-dependent manner. It is concluded that ZGDHu-1 can inhibit proliferation, induce differentiation and apoptosis of NB4 cells. The mechanism may be associated with up-regulation of bax expression, enhancement of phosphor-p38MAPK activation and inhibition of hTERT-mRNA.
Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cell Transformation, Neoplastic ; drug effects ; Heterocyclic Compounds, 1-Ring ; pharmacology ; Humans ; Leukemia, Promyelocytic, Acute ; pathology ; Tumor Cells, Cultured