OBJECTIVELeukemia is the most common hematopoietic malignancies in children. Chemotherapy is currently the primary modality of treatment for this fatal disease. Although chemotherapy is very effective in terms of cell killing, severe side effects such as severe infections, intracranial hemorrhage etc. are frequently encountered due to its poor selective damage between normal and malignant cells or tissues. Thus, a new therapy with highly selective killing of malignant cells which leaves the normal cells unaffected is desperately desired. The aim of this study was to investigate the targeting efficacy in vitro with a new clone of anti-human CD19 antibody immunotoxin 2E8-Genistein on B lineage leukemia cell line Nalm-6 cells and its mechanisms in order to provide the evidence of target therapy on B lineage leukemia and lymphoma.

METHODS2E8-Genistein immunotoxin was generated by conjugating Mab 2E8 with a tyrosine kinase inhibitor, Genistein (Gen) via the Sulfo-SANPAH, an ultra-violet sensitive reagent. Nalm-6, a CD19+ B cell leukemia cell line, was used as target cells, while Molt-3, a CD19-T cell leukemia cell line, was taken as the negative control. The morphology of the cells was observed under the reverted reversed light microscope and the viability was checked with either trypan blue exclusion or MTT methods. Two-color flow cytometry was applied to study the mechanism of cell killing.

RESULTSAfter 24 hours of culture, 2E8-Genistein showed marked target killing on Nalm-6 cells at nine different concentrations from 20 nmol/L through 100 nmol/L with cell survival rates from (71.8 +/- 7.9)% down to (16.6 +/- 12.9)%, respectively (n = 3), which were all significantly lower than that of control group (100 +/- 13.9)% (P < 0.05). The killing effect was even more significant when the concentration was over 80 nmol/L. The growth inhibition rates of this immunotoxin on Nalm-6 cells were 82%, 84% and 94%, respectively at 24, 48 and 72 hours of culture in a time dependent manner. Significant difference was observed between the cell growth curve of Nalm-6 cultured with 100 nmol/L of 2E8-Gen and those of Nalm-6 cultured with medium (blank), PBS (negative control) or the same concentration of pure 2E8 antibody (negative control) groups (F = 152.15, P = 2.15 x 10(-7)), but there was no significant difference among the three control groups (F = 1.51, P = 0.29). When Molt-3 cells were used as target cells, the cell growth curves of Molt-3 cultured with 2E8-Gen (100 nmol/L) and with negative control of blank did not show any significant difference (F = 0.34, P = 0.59). PI/FITC Annexin V double staining analysis with flow cytometry showed that the positive rate (33.45 +/- 8.77)% of early apoptosis on Nalm-6 cells induced by 100 nmol/L of 2E8-Genistein was significantly higher than that of negative control of blank (10.44% +/- 1.28%, t = -4.39, P = 0.001) at 24 hours of culture.

CONCLUSION2E8-Genistein immunotoxin can significantly target the Nalm-6 cells in vitro in a time response manner and the apoptosis induction is involved in the course of this killing effect.

Antibodies, Monoclonal ; immunology ; pharmacology ; Antigens, CD19 ; Apoptosis ; drug effects ; Cell Line, Tumor ; Flow Cytometry ; Genistein ; immunology ; pharmacology ; Humans ; Immunotoxins ; immunology ; pharmacology ; Leukemia, B-Cell ; immunology

OBJECTIVESTo study the effect of monoclonal antibody (McAb) against helicobacter pylori (Hp) ureB, 1F11 on platelet aggregation and activation, and its mechanism.

METHODSThe relativity between human platelet glycoproteins (GPs) and Hp ureB was identified by Western blot and FCM. Platelet aggregation was measured by turbidimetry, and P-selectin and TXB2 assay by ELISA.

RESULTS1F11 could bind to platelet GPIIIa, and ADP-induced platelet aggregation was inhibited by 1F11 in a dose-dependent manner. However, 1F11 had no effect on plasma P-selectin and TXB2 induced by ADP. The FCM results show that the positive rates of platelet binding to FITC-SZ21 was decreased from 99.5% to 77.4% after addition of 1F11.

CONCLUSIONMcAb against Hp ureB 1F11 inhibits platelet aggregation through binding to platelet GPIIIa but does not block platelet activation. There might be crossed-epitopes on Hp ureB and platelet GPIIIa, and Hp infection might be involved in ITP immunopathology.

Antibodies, Bacterial ; pharmacology ; Antibodies, Monoclonal ; pharmacology ; Bacterial Proteins ; immunology ; metabolism ; Helicobacter pylori ; immunology ; Humans ; Integrin beta3 ; immunology ; P-Selectin ; immunology ; Platelet Activation ; drug effects ; Platelet Aggregation ; drug effects ; Urease ; immunology ; Urokinase-Type Plasminogen Activator ; immunology

The objective of this study was to investigate the synergistic effect of soluble human recombinant tumor necrosis factor related apoptosis inducing ligand (TRAIL) protein combined with anti-vascular endothelial growth factor (anti-VEGF) antibody on inducing apoptosis of leukemia K562 cells. The inhibitory rates and apoptotic rates of K562 cells treated with TRAIL and anti-VEGF antibody alone and their combination for 48 hours were examined by CCK-8 assay and flow cytometry respectively. The results indicated that the apoptotic rates of K562 cells induced with 75, 100 and 150 ng/ml TRAIL after culture for 48 hours were (4.26±0.67)%, (8.91±0.55)% and (11.71±0.78)% respectively. The apoptotic rates of K562 cells induced with 2.5, 5 and 7.5 µg/ml anti-VEGF antibody after culture for 48 hours were (3.95±0.69)%, (7.98±0.74)% and (10.26±0.83)% respectively. The apoptotic rates of K562 cells treated with combination use of 2.5 µg/ml anti-VEGF antibody and 75 ng/ml TRAIL, 5 µg/ml anti-VEGF antibody and 100 ng/ml TRAIL, and 7.5 µg/ml anti-VEGF antibody and 150 ng/ml TRAIL for 48 hours were (22.16±0.93)%, (36.32±1.31)% and (49.19±0.71)% respectively. The combined use of above mentioned agents induced significantly higher apoptosis and cytotoxicity than that of TRAIL or anti-VEGF antibody alone (p<0.05). It is concluded that the combination use of TRAIL and anti-VEGF antibody can significantly increase the sensitivity of K562 cells to apoptosis.
Antibodies, Monoclonal ; pharmacology ; Apoptosis ; drug effects ; Humans ; K562 Cells ; TNF-Related Apoptosis-Inducing Ligand ; pharmacology ; Vascular Endothelial Growth Factor A ; immunology

This study sought to clarify the distribution of HMGN2 in HeLa cells. The recombinant eukaryotic expression vectors pcDNA3. 1-myc-his-HMGN2 and pEGFP-N1-HMGN2 were constructed, and then were transfected into HeLa cells. immunocytochemistry staining indicated that HMGN2 were present not only in HeLa nucleus but also in the cytoplasm. The presence of HMGN2 was also detected in the culture supernatant by ELISA with rabbit anti-serum against HMGN2 and mouse anti-His6 monoclonal antibodies. The confocal microscope observation showed the same subcellular localization as that of immunocytochemistry staining. There results suggested that HMGN2 could be present in the nucleus and cytoplasm of HeLa cell as well as in the extracellular environment.
Animals ; Antibodies, Monoclonal ; HMGN2 Protein ; immunology ; metabolism ; pharmacology ; HeLa Cells ; Humans ; Mice ; Rabbits ; Recombinant Fusion Proteins ; biosynthesis ; pharmacology ; Transfection

OBJECTIVETo study the regulation of Fc receptor expression by immune complexes (ICs) on neutrophils and U937 cells.

METHODSIgA ICs, IgG1 ICs, IgG2 ICs, IgG3 ICs, IgG4 ICs, and IgM ICs were incubated with neutrophils or U937 cells for 1 h. Then their surface Fc receptors were stained by anti-Fc gammaR I, anti-Fc gammaR II , anti-Fc gammaR III, and anti-Fc alphaR I monoclonal antibodies and analyzed by fluorescent activated cell sorting (FACS).

RESULTSIgG1 ICs and IgG3 ICs up-regulated Fc gammaR II and Fc gammaR III on U937 cells, Fc gammaR I and Fc alphaR I on neutrophils. Almost all ICs down-regulated Fc gammaR II on neutrophils.

CONCLUSIONSICs can regulate Fc receptor expression on neutrophils and U937 cells, among which IgG1 ICs and IgG3 ICs are most effective.

Antibodies, Anti-Idiotypic ; immunology ; Antibodies, Monoclonal ; pharmacology ; Antigen-Antibody Complex ; immunology ; metabolism ; Antigens, CD ; immunology ; Humans ; Immunoglobulin A ; classification ; immunology ; Immunoglobulin G ; chemistry ; classification ; immunology ; metabolism ; Neutrophils ; metabolism ; Receptors, Fc ; biosynthesis ; genetics ; Receptors, IgG ; immunology ; U937 Cells ; immunology

In order to study how to activate T cells and their immunological characteristics, the anti-CD3 and anti-CD28 McAbs were used to stimulate PBMNC, then their related immunological changes, such as lymphocyte transformation function, the percentage of CD8(+)CD25(+) cells and TGF-beta expression were deleted by lymphocyte transformation assay, flow cytometry and RT-PCR respectively. The results showed that in costimulation with anti-CD28 antibody stimulation, the activity of anti-CD3 antibody was significantly enhanced, the ratio of CD8(+)CD25(+) cells of T cells was obviously increased, while TGF-beta expression was down-regulated. It was concluded that the anti-CD28 antibody costimulation could provide stimulatory signal II, which make T cells more active, while the expression of TGF-beta significantly down-regulated.
Antibodies, Monoclonal ; pharmacology ; CD28 Antigens ; immunology ; CD3 Complex ; immunology ; Down-Regulation ; Humans ; Leukocytes, Mononuclear ; immunology ; metabolism ; Lymphocyte Activation ; drug effects ; T-Lymphocytes ; immunology ; Transforming Growth Factor beta ; biosynthesis

In order to investigate the effect of anti-TGF-beta1 monoclonal antibody on the expansion of cord blood CD34(+) cells, the purified cord blood CD34(+) cells were divided into three groups: blank control group: purified cord blood CD34(+) cells cultured on day 0; control group: cells cultured for 3 days in the culture system, containing SCF, IL-3, IL-6 and FLT3-L; test group: cells cultured for 3 days in the same culture system as control group, but with anti-TGF-beta1 monoclonal antibody. The mononuclear cell counting (MNC), the expression of CD34 and c-kit, and CFU-GEMM, BFU-E and CFU-GM counting were detected in all three groups. The result showed that the expansion of MNCs, CD34(+) cells and CD34(+)c-kit(+) cells in test group [(2.35 +/- 0.25) x 10(5), (1.16 +/- 0.29) x 10(5), (1.09 +/- 0.26) x 10(5)] was significantly higher than that in control group [(1.25 +/- 0.13) x 10(5), (0.55 +/- 0.19) x 10(5), (0.51 +/- 0.2) x 10(5)](p < 0.01). The expansion of more primitive CD34(+)c-kit(-) subpopulation in test group [(12.95 +/- 3.17) x 10(3)] was even significantly higher than in control group (1.71 +/- 0.83) x 10(3) (p < 0.01). Colony forming assay showed that the number of earlier progenitor colony CFU-GEMM, BFU-E in test group [(16.3 +/- 4.72) x 10(3), (60.5 +/- 20.96) x 10(3)] was higher than that in control group [(5.0 +/- 2.58) x 10(3), (16.25 +/- 7.93) x 10(3)] (p < 0.01). The number of relatively mature CFU-GM between test group and control group was not statistical significance [(6.33 +/- 2.85) x 10(3) vs (4.0 +/- 2.28) x 10(3)](p > 0.05), but both were higher than that in blank group (0.75 +/- 0.29) x 10(3). These results demonstrated that anti-TGF-beta1 monoclonal antibody promoted the expansion of MNC and CD34(+) cells, and even more marked expansion of the more primitive progenitor cells-CD34(+)c-kit(-) cells. Meanwhile, it enhanced the output of more immature colony CFU-GEMM and BFU-E, but had no evident influence on the mature myeloid colony CFU-GM. It is concluded that the anti-TGF-beta1 monoclonal antibody can synergize other cytokines to enhance the proliferation of cord blood CD34(+) progenitor cells effectively, and it is more important that can reserve more primitive progenitor cells.
Antibodies, Monoclonal ; immunology ; pharmacology ; Antigens, CD34 ; immunology ; metabolism ; Cell Division ; drug effects ; Cells, Cultured ; Fetal Blood ; cytology ; drug effects ; immunology ; Humans ; Transforming Growth Factor beta1 ; immunology

OBJECTIVETo investigate the specific anti-breast cancer immune response induced by dendritic cells (DC) loaded with trastuzumab and apoptotic Her-2+ breast cancer cells.

METHODSDCs were generated from healthy peripheral blood mononuclear cells (PBMCs) in the presence of recombinant cytokines GM-CSF, IL-4 and TNF-alpha. Mature DCs were harvested after 7 days' co-culture of PBMCs and trastuzumab-treated apoptotic SKBr3 cells. The morphologic characteristics and ultrastructure of the DC were observed under the inverted phase-contrast microscope and transmission electron microscope (TEM), respectively. Flow cytometry (FCM) was used to check the expression of several DC specific markers: CD14, CD1a, CD64, CD80, CD83, CD86, HLA-ABC and HLA-DR. DC-cytokine induced killer (DC-CIK) cells were prepared by co-culture of DCs and peripheral blood lymphocytes in the presence of anti-CD3 antibodies and human IL-2 at an appropriate concentration. The number of antigen-specific T cells was analyzed by human interferon gamma enzyme linked immunospot (ELISPOT) assay. MTT assay was employed to assess the lysis of breast cancer cell line induced by DC-CIK cells.

RESULTS5 minutes after the adding of DCs to SKBr3 cells pretreated with trastuzumab, the apoptotic SKBr3 cells were found to be circled by DCs. 48 hours later, many membrane-wrapped organelles of the apoptotic target cells in the cytoplasm of DCs were found by TEM. The majority of the organelles were degraded. Fewer organelles from the apoptotic cells were found in DCs without Herceptin. More than 60% in every group of DCs expressed a high-affinity receptor for IgG (FcgammaRI or CD64). CD14 expression on the mature DCs were comparatively lower, and HLA-DR and HLA-ABC expressions were higher in the trastuzumab group. The expression of CD1a, CD80, CD83 and CD86 in trastuzumab group were higher than those in immature DCs group (P < 0.05). ELISPOT assay suggests that the spot number of antigen-specific T cells were higher in trastuzumab group than that in the antigen unloaded DCs group (P < 0.05). The lysis of SKBr3 cells induced by the SKBr3 antigen loaded DC-CIK cells were 1.7 times higher than that of CIK.

CONCLUSIONThe lysis of SKBr3 cells induced by DC-CIK was increased after that DCs were combined with trastuzumab to capture antigen from SKBr3 cells. These findings support further investigation into the use of combination immunotherapy of the humanized monoclonal antibody, DC vaccines and immunological effector cells.

Antibodies, Monoclonal ; pharmacology ; Antibodies, Monoclonal, Humanized ; Apoptosis ; Cell Line, Tumor ; Coculture Techniques ; Cytokine-Induced Killer Cells ; immunology ; Cytokines ; metabolism ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; cytology ; immunology ; metabolism ; ultrastructure ; Humans ; Receptor, ErbB-2 ; metabolism ; Receptors, IgG ; metabolism ; Trastuzumab

OBJECTIVETo study the biological response of B-cell lymphoma cells positive for CD20 expression to (131)I-labeled rituximab.

METHODSAnti-CD20 monoclonal antibody rituximab was labeled with (131)I by means of IODO-GEN method, and its effects on apoptosis of Raji cells were determined by Annexin-V/PI double-labeled cytometry. Its effects on the cell cycles was evaluated by cytometry with PI staining.

RESULTSThe cell apoptosis rate measured by Annexin v-FITC/PI was 51.99% in (131)I-rituximab group, significantly higher than that in (131)I group, rituximab group and control group (42.71%, 29.42% and 26.17%, respectively, P<;0.05). The apoptosis rate by flow cytometry with PI staining was 4.32% in (131)I-rituximab group, also significantly higher than that in the other 3 groups (1.47%, 1.39% and 0.37%, respectively, P<0.05). Cell cycle alteration of Raji cells occurred in (131)I-rituximab group, and the majority of cells were arrested at G(1)/G(2) stage.

CONCLUSION(131)I-rituximab can regulate the cell cycle of Raji cells and induce their apoptosis to inhibit their proliferation.

Antibodies, Monoclonal ; immunology ; pharmacology ; Antibodies, Monoclonal, Murine-Derived ; Antigens, CD20 ; immunology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Flow Cytometry ; Humans ; Iodine Radioisotopes ; Lymphoma, B-Cell ; immunology ; pathology ; physiopathology ; Radioimmunotherapy

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo™ Luciferase Assay System using Jurkat/NFAT-luc+FcγRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation: y = (A-D)/[1 + (X/C)(B)] + D. The optimized parameters of the method were determined including the antibodies diluted concentration (18,000 ng·mL(-1)), dilution rate (1:5), the ratio of effector cell and target cell (6:1), and induction time (6 h). The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of (44.39±3.93)%, (72.74±2.78)%, (128.28±7.01)% and (168.19±2.70)% respectively, with a variation coefficient of less than 10%, and the recoveries of (88.78±7.85)%, (96.99±3.70)%, (102.63±5.61)% and (112.12±1.80)% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.
Antibodies, Monoclonal, Murine-Derived ; pharmacology ; Antibody-Dependent Cell Cytotoxicity ; Antigens, CD20 ; immunology ; Genes, Reporter ; Humans ; Reproducibility of Results ; Rituximab