The present study was attempted to compare the Neospora caninum (N. caninum) antigenic bands recognized by different bovine immunoglobulin classes. A total 10, 5, 2, and 6 antigenic bands were exhibited on immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. A 46 kDa band was probed as a common antigenic band except IgA; 69 kDa band was bovine IgM and IgE; 33, 37, 55, and 79 kDa bands were bovine IgM and IgG; 72 kDa band was found IgM and IgA profiles. Based on the analysis, it appeared that different immunoglobulin classes recognizing different antigenic molecules were cooperating to cope with neosporosis.
Animals ; Antigens, Protozoan/immunology ; Cattle ; Cattle Diseases/diagnosis/immunology/*parasitology ; Coccidiosis/diagnosis/immunology/parasitology/*veterinary ; Female ; Immunoblotting ; Immunoglobulin Idiotypes/diagnostic use/*immunology ; Neospora/*immunology

INTRODUCTIONMultiple myeloma (MM), a malignancy of plasma cells, accounts for 10% of all haematological malignancies and is currently incurable. Although it can be treated, the disease tends to relapse after several years and becomes increasingly resistant to conventional therapy. Investigations into using humoral therapy for MM are now underway with a view that novel therapeutic agents may provide a more targeted therapy for MM.

MATERIALS AND METHODSHere, phage display, a faster and more efficient method compared to classical hybridoma fusion technology, was used as a proof-of-concept to isolate several single-chain Fragment variables (scFv) against Ku86.

RESULTSAnti-Ku86 polyclonal scFvs biopanning was successful where third round scFvs (A(450)~1.1) showed a 1/3 increase in binding as compared to the fi rst round scFvs (A(450)~0.4) with 100 microg/mL of antigen (purified human Ku86). Subsequent selection and verification of monoclonal antibodies using third round biopanning revealed 4 good affinity binding clones ranging from A(450)~0.1 to A450~0.15 on 12.5 microg/mL of antigen as compared to low binders (A(450)~0.07) and these antibodies bind to Ku86 in a specific and dose-dependent manner. Comparative studies were also performed with commercially available murine antibodies and results suggest that 2 of the clones may bind close to the following epitopes aa506-541 and aa1-374.

CONCLUSIONSThese studies using phage display provide an alternative and viable method to screen for antibodies quickly and results show that good affinity antibodies against Ku86 have been successfully isolated and they can be used for further studies on MM and form the basis for further development as anti-cancer therapeutic agents.

Antibodies, Monoclonal ; isolation & purification ; Antibody Affinity ; Cell Line ; DNA Helicases ; immunology ; Humans ; Immunoglobulin Idiotypes ; immunology ; isolation & purification ; Immunoglobulin Variable Region ; isolation & purification ; Ku Autoantigen ; Multiple Myeloma ; immunology ; Peptide Library ; Recombinant Proteins

OBJECTIVEAn anti-idiotypic minibody with optimal antigenicity which mimicking ovarian cancer antigen was used for therapeutic research in mice model bearing ovarian cancer.

METHODSUsing gene engineering technique, prokaryotic expression vector was constructed by genetic fusion of 6B11scFv to human IgG1 hinge and CH3 region. The fusion protein named minibody was induced with IPTG in E. coli and analyzed with Western blot and inhibition ELISA tests respectively. Twenty human-PBL-SCID mice bearing i.p. Skov3.ip1 cells were divided into two groups (10 per-group), 10 mice were immunized repeatedly by minibody every two weeks for three times. Indirect ELISA test was employed for analyzing the characterization of anti-anti-idiotypic scFv (Ab3). The latent period of ascites growth and the mean survival time were observed respectively. CD4+ and CD8+ T cells from the spleen of immunized mice were assayed by flow cytometry.

RESULTSSDS-PAGE gel electrophoresis showed that a protein band with molecular weight of 50,000 appeared as the expected size after transformation and induction the host bacteria BL21 (DE3). The expressed minibody could be reacted with COC166-9 (Ab1 of 6B11) and binding goat anti-human IgG1 antibody in Western blot. Inhibition ELISA showed minibody had the capacity of binding ovarian cancer monoclonal antibody COC166-9 instead of primal antigen. Ab3 could be detected in the sera of immunized mice with minibody by ELISA test. Ab3 reached the highest at the 14th day after last vaccination and lasted for 6 weeks. The ratio of CD4+/CD8+ was the highest at the 13th day after last vaccination. The latent period of ascites growth were (37.7 +/- 5.5) days and (48.6 +/- 14.3) days (P = 0.04) respectively; while the mean survival time were (42.5 +/- 1.8) days and (59.4 +/- 16.8) days (P = 0.011) in the control and minibody group respectively.

CONCLUSIONSThese results demonstrate the successful construction and expression minibody with good immune activities of 6B11scFv and human IgG1 molecules function. Antigenicity is increased without adjuvants and partial humanization is realized. Minibody can induce humoral anti-idiotypic immunity responses against ovarian carcinoma in vivo. When ascites formation was delayed or prevented and the survival was prolonged in minibody group. We expect that minibody may be used as tumor vaccine to ovarian carcinoma in the future clinical trails.

Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Neoplasm ; therapeutic use ; Cancer Vaccines ; Cystadenocarcinoma, Papillary ; immunology ; therapy ; Female ; Humans ; Immunoglobulin Idiotypes ; therapeutic use ; Immunotherapy ; Mice ; Mice, SCID ; Ovarian Neoplasms ; immunology ; therapy ; Tumor Cells, Cultured

OBJECTIVETo confirm the therapeutic effect of dendritic cell (DC) vaccine on treatment for mice with lymphoma and the protective effect of DC vaccine loaded with different antigens on the tumor-bearing BAL B/c mice.

METHODSFirstly, a mouse tumor model was set up by s. c. inoculation of 1 x 10(6)/mouse A20 tumor cells. Then different DC vaccines were injected, respectively, and the tumor size and survival time were observed. Secondly, the immunized mice with DC vaccines were challenged with A20 tumor cells, and observed whether a new tumor occurred in the mice and the time of survival.

RESULTSThe tumor of mice immunized with Id-DC vaccines grew slower than the controls (mean time of survival was 40.4 days vs. 33.4 days), but statistically not significantly different. The tumor of mice injected with CPP-Id-DC vaccines grew slower than that injected with Id-DC vaccines and controls, and one of 5 mice got CR and the tumor in another one mouse became stable. The median survival time was 70.8 days during a 90-days observation period. The difference was significant (P<0.01). The mice injected with Id-DC vaccines were challenged with A20 tumor cells showed new tumor occurred at 7 - 12 days, and 1 of the 5 mice survived for 60 days. The mice injected with CPP-Id-DC vaccines had no tumor.

CONCLUSIONThe DC loaded with CPP-Id was better than that loaded with Id alone in treating B cell lymphoma, and It can enhance their antitumor responses and prolong the survival time of the A20 tumor animal models. The vaccine of DC loaded with CPP-Id can protect mice from A20 tumor cell challenge.

Animals ; Cancer Vaccines ; immunology ; therapeutic use ; Cell Line, Tumor ; Cells, Cultured ; Dendritic Cells ; immunology ; Female ; Immunoglobulin Idiotypes ; immunology ; Lymphoma ; immunology ; pathology ; therapy ; Mice ; Mice, Inbred BALB C ; Neoplasm Transplantation ; Peptide Fragments ; therapeutic use ; Peptides ; therapeutic use ; Random Allocation

OBJECTIVETo prepare the tumor antigen peptide complex (HSP70-1d) of HSP70 and idiotype (Id) from SmIg ScFv fragment in patients with Chronic B cell leukemia (B-CLL), and to study the anti-tumor effect of cytotoxic T lymphocyte (CTL) induced by HSP70-Id complex-modified dendritic cell (DC) in vitro and explore their immune mechanism.

METHODSPurified HSP70 was combined into peptide complex (HSP70-Id) with the prepared Id-ScFv from B-CLL cells in vitro by using biochemical technique. The plastic-adherent monocytes from human peripheral blood were cultured and induced into DC with rhGM-CSF and rhIL-4 using cell culture and separation technique. The cultured DC were harvested and pulsed by HSP70-Id complex. DC morphology was observed under converted phase microscope and its phenotype was characterized by FCM on 8th day as well as their secreting cytokines were measured. Host lymphocytes were stimulated by DC loaded with HSP70-Id complex and co-cultured in the medium containing IL-2. The activation and proliferation of lymphocytes were examined by MTr test, which was also used to assay cytotoxicity of CTL elicited by modified DC to Daudi, K562 and HepG2 tumor cells, and FCM analyzed the changes of T lymphocyte subsets.

RESULTSMature DCs were obtained successfully, showing typical morphology and phenotypic properties, the expression ratio of cellular surface molecules, CD1a was 20% - 30%, CD83 was more than 72% , both CD86 and HLA-DR over-expressed obviously in the complex-loaded DC group secreting cytokines of Thl type, IL-12 and TNF-alpha. The culturing lymphocytes that were activated by modified DC could more effectively and specifically kill Daudi (71. 24%), but not K562 and HepG2 tumor cells. Results of FCM assay demonstrated that percentage of CD4+ and CD8+ T lymphocytes cocultured with complex-modified DC increased notably to 56.51% and 70.21%, respectively. CD4+ T/ CD8+ T proportion was changed from 1.49 to 0.81. The dose of peptide would be reduced to 1/50 if specific CTL induced by complex-modified DC instead of directly by peptide complex.

CONCLUSIONDCs modified by HSP70-Id complex exhibit powerful biological activities, and could induce CTL to specific cytotoxicity against carcinoma cells. It might be produced by cooperation of CD4+ T, CD8+ T lymphocytes and DC. The results also suggested that DC modified by HSP70-Id complex can present antigen and induce CTL with high efficacy and specificity.

Cell Line, Tumor ; Cells, Cultured ; Cytotoxicity, Immunologic ; Dendritic Cells ; cytology ; drug effects ; immunology ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HSP70 Heat-Shock Proteins ; pharmacology ; Humans ; Immunoglobulin Idiotypes ; pharmacology ; Immunoglobulin Variable Region ; pharmacology ; Interleukin-4 ; genetics ; pharmacology ; K562 Cells ; Lymphocyte Activation ; Monocytes ; cytology ; drug effects ; immunology ; Recombinant Proteins ; pharmacology ; T-Lymphocytes, Cytotoxic ; immunology

The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.
Amino Acid Sequence ; Animals ; Cancer Vaccines ; biosynthesis ; genetics ; immunology ; Chemokine CCL7 ; biosynthesis ; genetics ; immunology ; Genetic Vectors ; Humans ; Immunoglobulin Idiotypes ; immunology ; Immunoglobulin Variable Region ; biosynthesis ; genetics ; immunology ; Lymphoma, B-Cell ; immunology ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; Plasmids ; genetics ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Vaccines, DNA ; biosynthesis ; genetics ; immunology