OBJECTIVETo elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen.

METHODSThe ABO blood group antigens on red blood cells were identified by monoclonal antibodies, the ABO antibodies in serum were detected by standard A, B, O cells and the activity of the B glycosyltransferase was analyzed. The full-length sequence and 5'-untranslated region (5'-UTR) sequence of ABO gene were amplified by polymerase chain reaction (PCR) respectively and direct sequencing. The alternative splicing isoforms of ABO cDNA were obtained by reverse transcription-PCR(RT-PCR) and analyzed with cloning and sequencing techniques. The level of methylation of the CpG island in ABO gene promoter was analyzed by bisulfite sequencing method.

RESULTSThe serological characteristic of the donor showed that the B antigen was decreased obviously without anti-B antibodies in serum and the B glycosyltransferase activity was decreased as well. The genotype of the donor was B101/O01 without any other mutations in the full-length coding sequences and splice receptor sites. The nucleotide characteristics of the 5'-UTR was consistent with B101/O01 and no any abnormity was identified in the promoter, enhancer and the negative regulatory sequence regions. The integrative cDNA transcript of ABO gene was obtained and no new splicing isoform was found. Compared with the normal B phenotype, a number of methylated CpG sites were found near the promoter of ABO gene in this sample.

CONCLUSIONThe methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.

ABO Blood-Group System ; genetics ; immunology ; Alleles ; Antibodies, Monoclonal ; immunology ; Blood Donors ; CpG Islands ; genetics ; Erythrocytes ; immunology ; Gene Expression Regulation ; immunology ; Humans ; Phenotype ; Reverse Transcriptase Polymerase Chain Reaction

The full-length P32 gene and the truncated P32 gene (MP-32) were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction (PCR) and cloned into pcDNA3. 1(+) and pcDNA3.1-CpG respectively. The recombinant plasmids (pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3. 1-CpG-MP32) were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay (IFA). The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4+ and CD8+ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post im munization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4+ /CD8+ T cells was significantly higher than that of the control. In conclusion, these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.
Animals ; Antibodies, Viral ; blood ; Capripoxvirus ; genetics ; immunology ; Cell Line ; CpG Islands ; Cricetinae ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; immunology ; T-Lymphocyte Subsets ; immunology ; Vaccines, Synthetic ; immunology ; Viral Envelope Proteins ; immunology ; Viral Vaccines ; immunology

Adjuvants, Immunologic ; Animals ; Antibodies, Anti-Idiotypic ; genetics ; immunology ; CpG Islands ; Hepatitis B ; prevention & control ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis B Vaccines ; administration & dosage ; immunology ; Mice ; Mice, Inbred C57BL ; Mice, Transgenic ; Oligodeoxyribonucleotides ; genetics ; immunology ; Th1 Cells ; immunology ; Vaccination

OBJECTIVETo construct the eukaryotic expression plasmid containing lvgA gene flanked with CpG motifs of Legionella pneumophila for its expression in NIH3T3 cells.

METHODSlvgA gene flanked with CpG motifs of Legionella pneumophila was amplified by PCR. The PCR products was inserted into the eukaryotic expression plasmid pcDNA3.1/myc-his(+) to construct the recombinant plasmid pclvgA/CpG, which was subsequently transfected into NIH3T3 cells via lipofection. Immunofluorescence analysis was carried out to detect the transient expression of the plasmid in the cells.

RESULTSSequence analysis showed that the recombinant plasmid pclvgA/CpG contained the lvgA/CpG fragment with a length of 657 bp, encoding a protein of 27.7 Ku. Immunofluorescence analysis identified the transient expression of the recombinant plasmid pclvgA/CpG in NIH3T3 cells.

CONCLUSIONThe lvgA gene flanked with CpG motifs of Legionella pneumophila has been constructed successfully, and the transient expression of the recombinant plasmid pclvgA/CpG can be detected in NIH3T3 cells.

Animals ; Bacterial Vaccines ; genetics ; CpG Islands ; genetics ; Legionella pneumophila ; genetics ; Mice ; NIH 3T3 Cells ; Plasmids ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Transfection ; Virulence Factors ; biosynthesis ; genetics

OBJECTIVE: To explore the effect of unmethylated CpG motif containing oligodeoxynucleotides (CpG ODN) on the function of dendritic cells (DCs) in patients with chronic hepatitis B (CHB). METHODS: DCs were obtained from peripheral blood mononuclear cells (PBMCs) of 15 CHB patients, 12 hepatitis B virus (HBV) carriers, and 10 healthy controls. The expressions of HLA-DR, CD80, and CD86 on DCs were determined by fluorescence activated cell sorting (FACS). The IL-12 level in supernatant of the culture medium was measured by ELISA, and the morphological changes of DCs were observed under transmission electron microscope. RESULTS: Compared with the controls, DCs stimulated with CpG ODN represented enrichment in cell surface protrusions and rough endoplasmic reticulum, decreased or disappeared vacuole. The expressions of HLA-DR, CD86, and CD80 were much higher in DCs stimulated with CpG ODN than those in complete medium control (P<0.05). When culturing in complete medium, the expressions of HLA-DR, CD86, and CD80 were much lower in CHB patients and HBV carriers than healthy controls (P<0.05). The expressions of HLA-DR and CD86 stimulated with CpG ODN were much lower in CHB patients than HBV carriers and healthy controls (P<0.05). The expressions of CD80 were much lower in CHB patients and HBV carriers than healthy controls (P<0.05). The levels of IL-12 were much higher in DCs stimulated with CpG ODN than that in complete medium controls (P<0.05). The levels of IL-12 in complete medium or medium added with CpG ODN were much lower in CHB patients and HBV carriers than in healthy controls (P<0.05). CONCLUSION CpG ODN could significantly promote the maturation of dendritic cells in peripheral blood in CHB patients.
Adult ; B7-1 Antigen ; metabolism ; B7-2 Antigen ; metabolism ; Carrier State ; immunology ; Case-Control Studies ; CpG Islands ; Dendritic Cells ; drug effects ; immunology ; Female ; HLA-DR Antigens ; metabolism ; Hepatitis B, Chronic ; immunology ; Humans ; Male ; Oligodeoxyribonucleotides ; pharmacology

Animals ; Antigens, Neoplasm ; chemistry ; immunology ; Cancer Vaccines ; chemistry ; therapeutic use ; Cell Line, Tumor ; CpG Islands ; Cytotoxicity, Immunologic ; Dendritic Cells ; immunology ; metabolism ; Humans ; In Vitro Techniques ; Lactic Acid ; chemistry ; Leukemia ; immunology ; therapy ; Lymphocytes ; cytology ; immunology ; Nanoparticles ; chemistry ; Peptides ; chemistry ; immunology ; therapeutic use ; Polyglycolic Acid ; chemistry ; Polylactic Acid-Polyglycolic Acid Copolymer ; WT1 Proteins ; chemistry ; immunology

Cytomegalovirus (CMV) infection is a dangerous complication in patients with chronic graft versus host disease (cGVHD). CMV-specific immunity depends on the activity of T cells. This study was aimed to investigate the effect of CMV pp65 gene modified dendritic cells (DCs) on activation of autologous T cells. Lentivirus system was utilized to introduce the CMV full-length pp65 gene into mouse DCs; CpG-DNA was used to induce mature DCs; flow cytometry and immunofluorescence were used to determine the expression of antigen and IFNgamma in T lymphocytes. The results showed that the DCs were infected with lentivirus at a multiplicity of infection (MOI) of 50 with optimal infectious efficiency of 30%-40%; mature DCs expressing pp65 gene could stimulate autologous naive T cells to express CD69 specifically; mature DCs expressing PP65 could stimulate autologous CD4+ or CD8+ T cells to produce IFNgamma. It is concluded that CMV pp65-modified and CpG-DNA-induced mature DCs can activate CMV-specific T lymphocytes in vitro.
Animals ; Antigens, CD ; genetics ; metabolism ; Antigens, Differentiation, T-Lymphocyte ; genetics ; metabolism ; Antigens, Viral ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; CD8-Positive T-Lymphocytes ; immunology ; CpG Islands ; genetics ; Cytomegalovirus ; immunology ; DNA ; genetics ; Dendritic Cells ; cytology ; immunology ; metabolism ; Humans ; Interferon-gamma ; genetics ; metabolism ; Lectins, C-Type ; Lentivirus ; genetics ; metabolism ; Mice ; Phosphoproteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism

This study examined the adjuvant effects of dimethyl dioctadecyl ammonium bromide (DDA), CpG oligodeoxynucleotides (CpG-ODN), and chicken interferon-gamma (ChIFN-gamma) on a DNA vaccine (pcDNA-VP243) against the infectious bursal disease virus (IBDV). A plasmid encoding chicken IFN-atilde was constructed. Twice at 2-week intervals, twoweek-old chickens were injected intramuscularly and intraperitoneally with either a DNA vaccine alone or a DNA vaccine together with the respective adjuvants. On week 2 after the second immunization, the chickens were orally challenged with the highly virulent IBDV. The groups that received the DNA vaccines plus either DDA or CpG-ODN showed significantly lower survival rates than the group that received the DNA vaccine alone. However, the survival rates for the DNA vaccine alone and for the DNA vaccine plus ChIFN-gamma were similar. The chickens had no detectable antibodies to the IBDV before the challenge but all the surviving chickens in all groups except for the normal control group showed the induction of antibodies to the IBDV at day 10 after the challenge. As judged by the lymphocyte proliferation assays using the a WST-8 solution performed on the peripheral blood and splenic lymphocytes, the stimulation indices (SI) of the peripheral blood lymphocytes in all groups except for the normal control group were similar immediately before the challenge. At 10 days post-challenge, the SI for DNA vaccine plus either CpG-ODN or ChIFN-gamma was similar to that of the DNA vaccine control group. For splenic lymphocytes, the SI in the DNA vaccine plus CpG-ODN and DNA vaccine plus ChIFN-gamma groups were higher than for the DNA vaccine control. These results suggest that DDA actually compromises the protection against the IBDV by DNA vaccine, and CpG-ODN and IFN-gamma had no significant effect.
Adjuvants, Immunologic ; Animals ; Antibodies, Viral/blood ; Birnaviridae Infections/*immunology/*prevention & control/virology ; Bursa of Fabricius/immunology/virology ; Cell Proliferation ; Chickens ; CpG Islands/immunology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Immunization/methods/*veterinary ; Infectious bursal disease virus/*immunology ; Interferon-gamma/immunology/therapeutic use ; Lymphocytes/cytology/immunology ; Oligonucleotides/immunology ; Poultry Diseases/immunology/*prevention & control/*virology ; Specific Pathogen-Free Organisms ; Vaccines, DNA/immunology/therapeutic use ; Viral Vaccines/*immunology/therapeutic use

OBJECTIVETo investigate whether CpG ODN can affect the antitumor responses of DC-tumor cell vaccine against Lewis lung cancer.

METHODSCpG oligonucleotides 1826 (ODN 1826) were used to promote maturation of DCs in vitro. By fusing DCs with Lewis lung carcinoma L3-8 cells, DC-based tumor cell vaccines were developed. To determine the immune responses to the vaccines, T cell proliferation and cytotoxicity were done in vitro. Therapeutic and prophylactic immunization with DC vaccines were performed in C57BL/6 mice bearing Lewis lung carcinoma.

RESULTSBone marrow cells cultured in the presence of GM-CSF and IL-4 plus additional ODN 1862 appeared typical morphology of DCs. FACS analyses showed that the mean fluorescence index (MFI) of CD40 expression of DCs stimulated with and without CpG ODN was 24 and 11, respectively, and that of CD86 expression was 75 and 33, respectively. IL-12 secreted by DCs cultured with ODN 1826 was 10-fold as high as that without ODN 1826. Significant T-cell proliferation and T cell-mediated cytotoxicity against L3-8 was induced in vitro. Marked inhibition of tumor growth in L3-8 bearing mice was observed upon prophylactic and therapeutic immunizations with the vaccine.

CONCLUSIONCpG ODN can enhance the antitumor responses of DC vaccine by promoting DC maturation.

Animals ; Antigens, CD ; metabolism ; B7-2 Antigen ; CD40 Antigens ; metabolism ; Cancer Vaccines ; administration & dosage ; immunology ; therapeutic use ; Carcinoma, Lewis Lung ; pathology ; prevention & control ; Cell Fusion ; Cell Line, Tumor ; CpG Islands ; immunology ; Dendritic Cells ; immunology ; transplantation ; Female ; Interleukin-12 ; metabolism ; Membrane Glycoproteins ; metabolism ; Mice ; Neoplasm Transplantation ; Oligodeoxyribonucleotides ; administration & dosage ; immunology ; therapeutic use

OBJECTIVETo develop a safe and novel immunoadjuvant to enhance the immunity and resistance of animals against E. coli infection.

METHODSAn 88-base immunostimulatory oligodeoxynuleotide containing eleven CpG motifs (CpG ODN) was synthesized and amplified by PCR. The chitosan nanoparticle (CNP) was prepared by ion linking method to entrap the CpG ODN that significantly promotes the proliferation of lymphocytes of pig in vitro. Then the CpG-CNP was inoculated into 21-day old Kunming mice, which were orally challenged with virulent K88/K99 E. Coli 35 days after inoculation. Blood was collected from the tail vein of mice on days 0, 7, 14, 21, 28, 35, 42, and 49 after inoculation to detect the changes and content of immunoglobulins, cytokines and immune cells by ELISA, such as IgG, IgA, IgM, IL-2, IL-4, and IL-6.

RESULTSThe CpG provoked remarkable proliferation of lymphocytes of pig in vitro in comparison with that of control group (P < 0.05). The inoculation with CpG-CNP significantly raised the content of IgG, IgM, and IgA in the sera of immunized mice (P < 0.05). The levels of IL-2, IL-4, and IL-6 in the mice significantly increased in comparison with those in controls (P < 0.05), so was the number of white blood cells and lymphocytes in immunized mice. The humoral and cellular immunities were significantly enhanced in immunized mice, which resisted the infection of E. coli and survived, while the control mice manifested evident symptoms and lesions of infection.

CONCLUSIONSCpG-CNP can significantly promote cellular and humoral immunity and resistance of mice against E. coil infection, and can be utilized as an effective adjuvant to improve the immunoprotection and resistance of porcine against infectious disease.

Adjuvants, Immunologic ; administration & dosage ; Animals ; Antibodies, Bacterial ; blood ; Biocompatible Materials ; administration & dosage ; Chitosan ; administration & dosage ; CpG Islands ; Escherichia coli ; pathogenicity ; Escherichia coli Infections ; immunology ; microbiology ; prevention & control ; Female ; Immunity, Cellular ; Interleukins ; biosynthesis ; Lymphocyte Activation ; Mice ; Nanoparticles ; Oligodeoxyribonucleotides ; administration & dosage ; Swine ; Vaccination