A therapeutic anti-CD4 monoclonal antibody inhibits T cell receptor signal transduction in mouse autoimmune cardiomyopathy / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>T cell immune abnormalities in patients with dilated cardiomyopathy (DCM) has been intensively studied over the past 10 years. Our previous study has suggested that immunization of mice with the peptides derived from human adenine nucleotide translocator (ANT) result in the production of autoantibodies against the ANT and histopathological changes similar to those in human DCM. The ANT peptides can induce autoimmune cardiomyopathy like DCM in Balb/c mice. In this study we aimed to focus on the molecular mechanism of T cells in the autoimmune cardiomyopathy mouse model by detecting the expression of the two T cell signaling molecules.</p><p><b>METHODS</b>The ANT peptides were used to cause autoimmune cardiomyopathy in Balb/c mice. Anti-L3T4 or rat anti-mouse IgG was administered to the mice (n = 6 in each group) simultaneously immunized with ANT. ELISA analysis was used to detect autoantibodies against the ANT peptides and the percentages of interferon-gamma and interleukin-4 producing cells among splenic CD4(+) lymphocytes was determined by using flow cytometry analysis. The expression of CD45 in spleen T cells was determined by immunohistochemistry and the mRNAs of T cell signaling molecules were detected by real-time PCR.</p><p><b>RESULTS</b>Treatment of ANT immunized Balb/c mice with anti-CD4 mAb caused a reduction in the gene expression of P56lck and Zap-70 and a lower level of CD45 expression by spleen T cells. Also, a reverse of the Th1/Th2 ratio that results in the reduced production of antibodies against ANT was found in the anti-CD4 monoclonal antibodies (mAb) group. Whereas irrelevant antibody (rat anti-mouse IgG) did not suppress T cell signaling molecules nor inhibit CD45 expression, and control-antibody mice did not show any significant differences compared with the DCM group.</p><p><b>CONCLUSION</b>The results show that anti-CD4 mAb is a powerful inhibitor of the early initiating events of T cell receptor (TCR) signal transduction in mouse autoimmune dilated cardiomyopathy.</p>

Immunophenotypes in 115 patients with acute myeloid leukemia by multi-color flow cytometry / 中国实验血液学杂志

Immunophenotyping has become common in the diagnosis and classification of leukemia. To evaluate the immunophenotype of acute myeloid leukemia (AML), multiparameter flow cytometry and CD45/SSC gating were used to analyze the surface and cytoplasmic antigen expressions in 115 cases of AML. The results were compared with the French-American-British (FAB) Cooperative Group classification to help define the best use and role of multiparameter flow cytometry in the diagnosis and proper classification of AML. The results showed that CD38, CD38 and CD13 were the most commonly expressed antigen (94.8%, 91.3% and 89.6%, respectively). CD7 was the most commonly expressed lymphoid antigen (20.2%), followed by CD19 (16.5%) and CD2 (15%). Some immunophenotypes correlated with FAB type, including increased frequency of CD2 in M(3); lack of HLA-DR, CD34 and CD56 expression in M(3); increased frequency of CD19 in M(2), CD14 and CD56 in M(5) and lack of MPO in M(0). In conclusion, multiparameter flow cytometry is a reliable technique in the diagnosis of AML, and some immunophenotypes correlate with FAB type.

Protein kinase C inhibitor Gö6976 sensitizes arsenic trioxide-induced cell apoptosis in chronic myeloid leukemic cells / 中国实验血液学杂志

To investigate the As(2)O(3)-chemosensitization of Gö6976 in K562 cells by its abrogation of As(2)O(3)-induced G(2)/M cell cycle arrest, K562 cells were treated with As(2)O(3) (5 micromol/L) and Gö6976 with various concentrations, the distributions of cell cycles were detected by flow cytometry, the cell viability was observed by trypan blue exclusion test and cell proliferation was tested by MTT assay. The results indicated that having treated by As(2)O(3) for 24 h and 48 h, the proportion of K562 cells in G(2)/M phase were (38.02 +/- 7.70)% and (32.58 +/- 7.43)% respectively, and no obvious cell apoptosis appeared. 50 nmol/L Gö6976 combined with As(2)O(3) decrease the proportion of cells in G(2)/M phase to (23.24 +/- 2.93)% and (16.18 +/- 1.60)% respectively and increase the proportion of cells in subG(1) phase to (11.82 +/- 2.31)% and (27.80 +/- 2.89)% respectively. Gö6976 abrogated G(2)/M cell cycle arrest induced by As(2)O(3) and increased cell apoptosis in a concentration- and time-dependent manner. Additionally, comparing to the control group, Gö6976 combined with As(2)O(3) decreased the cell viability and depressed the cell proliferation, but Gö6976 alone showed no same effect on them. In conclusion, the effects of AS(2)O(3)-chemosensitization of Gö6976 on K562 cells is associated with its abrogation of As(2)O(3)-induced G(2)/M cell cycle arrest.

Effects of PLK1 gene silence on apoptosis of K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the effects of PLK1 gene silence by short hairpin RNA (shRNA) on PLK1 expression and apoptosis in K562 cells, and explore the role of PLK1 in the pathogenesis of leukemia.</p><p><b>METHODS</b>The shRNA fragment targeting at 1416-1436 bp of PLK1 mRNA was synthesized and cloned into pEGFP-H1 vector, named as pEGFP-H1/PLK1. The empty control, pEGFP-H1 and pEGFP-H1/PLK1 were transfected into K562 cells respectively via electroporation. 24 h or 48 h after transfection, gene and protein expression of PLK1 in the cells were assayed by RT-PCR and Western blot analysis respectively, cells viability by MTT assay, caspase-3 activity by colorimetry, cell cycle and apoptosis by FACS.</p><p><b>RESULTS</b>24 and 48 h after transfection, PLK1 expression in K562 cells was 1.25 +/- 0.07 for control group, 0.52 +/- 0.04 and 0.25 +/- 0.02 for pEGFP-H1/PLK1 group, and 1.24 +/- 0.08 and 1.23 +/- 0.09 for pEGFP-H1 group respectively. The alteration status of PLK1 protein levels were similar to that of PLK mRNA levels. The apoptosis rate was (8.3 +/- 0.6)% in control group, (8.7 +/- 0.7)% in pEGFP-H1 group and (49.7 +/- 3.8)% and (82.3 +/- 6.9)% in pEGFP-H1/PKLK1 group at 24 and 48 h, respectively. In addition, cell fraction at G(2)/M phase was increased obviously compared with control and pEGFP-H1-transfected group.</p><p><b>CONCLUSION</b>The constructed shRNA can remarkably inhibit PLK1 expression and transfected K562 cell proliferation, increase apoptosis and block cell-cycle, suggesting that PLK1 play important roles in apoptosis and cell-cycle control of leukemia cells.</p>

Experimental study of inhibiting CTLL-2 cell apoptosis and enhancing cytotoxicity to Yac-1 cell by hammerhead ribozyme / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the inhibition role of anti-Fas hammerhead ribozyme on Fas expression and Fas-mediated apoptosis in mouse cytotoxic T lymphocyte (CTL) cell line--CTLL-2 cells, and explore a novel approach to enhance the ability of T cells against leukemia in donor lymphocytes infusion (DLI).</p><p><b>METHODS</b>A hammerhead ribozyme targeting the Fas mRNA was synthesized and transfected into CTLL-2 cells by electroporation. Fas expression in CTLL-2 cells was detected by using RT-PCR, Western blot and flow cytometry, CTLL-2 cells viability was measured by MTT assay, caspase-3 proteolytic activity by caspase-3 detection kit, and cell apoptosis by flow cytometry. Killing activity of CTLL-2 was detected by LDH releasing assay in vitro.</p><p><b>RESULTS</b>Expression of Fas mRNA and protein in CTLL-2 cells was reduced to 50% after transfection with anti-Fas ribozyme. Being treated with anti-Fas antibody (JO(2)), compared with control and mock-transfected cells, viability of CTLL-2 cells transfected with anti-Fas ribozyme increased by 1-fold, caspase-3 activity and apoptosis rate of ribozyme-transfected cells decreased to 50% and 37%, respectively, and cell killing activity was enhanced by 2-fold.</p><p><b>CONCLUSION</b>Anti-Fas ribozyme can cleave Fas efficiently and inhibit Fas-mediated apoptosis of CTLL-2 cells, resulting in improvement of their viability.</p>

Experimental study on activating antileukemic T cells by vaccination with dendritic cells pulsed with survivin / 中国实验血液学杂志

The objective of this study is to investigate the effect of vaccination with dendritic cells pulsed with survivin antigen on activation of antileukemic T cells, and inhibiting proliferation of leukemic cells. The expression of survivin on acute leukemic cells were detected by cofocal microscopy and immunoprecipitation-Western blot. DCs collected from peripheral blood mononuclear cells were pulsed with survivin purified proteins. Stimulation index (SI) and antileukemia CTL induction were analyzed with (3)H-TdR incorporation and (51)Cr releasing assay, respectively. The phenotype of T cells and DCs were identified by flow cytometry. By immunofluorescence of bone marrow and peripheral blood mononuclear cells, survivin expression was detected in 16 out of 19 AML cases (84.2%). The results showed that survivin fluorescence distribution was in cytoplasm. DCs from peripheral blood mononuclear cells were successfully induced, with typical DC morphologic characteristic. The vaccination with dendritic cells pulsed with survivin antigen dramatically stimulated the proliferation of T cells. The DCs loading survivin activated T cells with higher CD4(+) T(H) ratio as compared with DCs group, T cells activated with DCs expressed CD8 and CD56. Survivin DCs significantly inhibited the growth of leukemic cells in vitro. In conclusion, survivin antigen expressed in the cytoplasm of leukemic cells, leukemic vaccination with DCs pulsed with survivin antigen in vitro inhibited the proliferation of leukemic cells, that may be a pathway for therapy of leukemia.

Cloning of expression vector of human tissue factor gene and its expression in human ovarian cancer cell line / 中国实验血液学杂志

The aim was to construct the expressive vector of human tissue factor (TF), and determine its expressive level in stable-transfected human ovarian cancer cell line. The human TFcDNA was obtained from human placenta by RT-PCR and then inserted into eukaryotic expressive vector pcDNA3 to obtain the TF-pcDNA3 recombinant. This recombinant gene was introduced into human ovarian cell line A2780 through transfection mediated by lipofectamine. Stable-transfected cells were screened by G418. The TF expressive levels were detected by RT-PCR and flow cytometry. The results showed that: (1) the constructed product was identified as TF-pcDNA3 recombinant by sequencing. (2) TF was highly expressed not only at transcriptional level in the stable-transfected A2780 cell (transfected cell 3.99 +/- 0.15, untransfected cell 0.97 +/- 0.23, P < 0.01), but also on the membrane of the cell surface [transfected cell (48.56 +/- 9.53)%, untransfected cell (2.73 +/- 1.15)%, P < 0.01]. It was concluded that TF gene was successfully cloned, and was introduced into human ovarian cancer cell, and the subline A2780/TF which stably expresses TF at high level was obtained. It will provide good experimental basis for investigating new mechanisms of tumor growth, invasion, metastasis, hypercoagulability, and for exploring a new strategy of gene therapy.