ABSTRACT
Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.
Subject(s)
Cell Line , Collagenases , Distemper Virus, Canine , Embryonic Structures , Epithelial Cells , Family Characteristics , Fibroblasts , Kidney , Madin Darby Canine Kidney Cells , Retroviridae , TelomeraseABSTRACT
The maintenance of peripheral immune tolerance and prevention of chronic inflammation and autoimmune disease require CD4+CD25+ T cells (regulatory T cells). The transcription factor Foxp3 is essential for the development of functional, regulatory T cells, which plays a prominent role in self-tolerance. Retroviral vectors can confer high level of gene transfer and transgene expression in a variety of cell types. Here we observed that following retroviral vector-mediated gene transfer of Foxp3, transductional Foxp3 expression was increased in the liver, lung, brain, heart, muscle, spinal cord, kidney and spleen. One day after vector administration, high levels of transgene and gene expression were observed in liver and lung. At 2 days after injection, transductional Foxp3 expression level was increased in brain, heart, muscle and spinal cord, but kidney and spleen exhibited a consistent low level. This finding was inconsistent with the increase in both CD4+CD25+ T cell and CD4+Foxp3+ T cell frequencies observed in peripheral immune cells by fluorescence-activated cell-sorting (FACS) analysis. Retroviral vector-mediated gene transfer of Foxp3 did not lead to increased numbers of CD4+CD25+ T cell and CD4+Foxp3+ T cell. These results demonstrate the level and duration of transductional Foxp3 gene expression in various tissues. A better understanding of Foxp3 regulation can be useful in dissecting the cause of regulatory T cells dysfunction in several autoimmune diseases and raise the possibility of enhancing suppressive functions of regulatory T cells for therapeutic purposes.
Subject(s)
Animals , Mice , Autoimmune Diseases , Brain , Gene Expression , Heart , Immune Tolerance , Inflammation , Kidney , Liver , Lung , Muscles , Spinal Cord , Spleen , T-Lymphocytes , T-Lymphocytes, Regulatory , Transcription Factors , Transgenes , ZidovudineABSTRACT
Piroplasms are tick-transmitted, intracellular, hemoprotozoan parasites that cause anorexia, fever, anemia, and icterus. Theileriosis is caused by Theileria sergenti and causes major economic losses in grazing cattle in Japan and Korea. In May 2003, we examined the antigenic diversity of the major piroplasm surface protein (MPSP) gene in 35 healthy Jeju black cattle that were born and raised at the National Institute of Subtropical Agriculture. On microscopic examination of Giemsa-stained blood smears, 9 of 35 cattle had intra-erythrocytic piroplasms. Hematological data were within normal range for all 35 cattle. Amplification of DNA from all blood samples using universal MPSP gene primers showed mixed infections with C, I, and B type Theileria spp. Type C was identified in 20 of 35 blood samples, and type B was identified in 17 samples. Allelic variation was seen in type B.
Subject(s)
Animals , Cattle , Antigens, Protozoan/genetics , Base Sequence , DNA Primers/genetics , Genetic Variation , Korea , Molecular Sequence Data , Phylogeny , Protozoan Proteins/genetics , Sequence Analysis, DNA , Theileria/genetics , Theileriasis/parasitologyABSTRACT
Fucoidan is a sulfated polysaccharide purified from brown algae including Fucus vesiculosus and has a variety of biological effects including mobilization of hematopoietic progenitor cells. Recently, we demonstrated that fucoidan stimulates the antigen-presenting functions of dendritic cells. In this study, we investigated the radioprotective effects of fucoidan on bone marrow cells (BMCs), which are the main cellular reservoir for the hematopoietic and immune system. To evaluate the effects of fucoidan, we assayed cell viability and immune responses. In a viability assay, fucoidan significantly increased the viability of BMCs. Based on the results of flow cytometric analysis, the increased viability of fucoidan-treated BMCs was attributed to the inhibition of radiation-induced apoptosis. Furthermore, fucoidan altered the production of immune-related cytokines from BMCs and increased the capability of BMCs to induce proliferation of allogeneic splenocytes. Taken together, our study demonstrated that fucoidan has radioprotective effects on BMCs with respect to cell viability and immunoreactivity. These results may provide valuable information, useful in the field of radiotherapy.
Subject(s)
Animals , Female , Mice , Bone Marrow Cells/drug effects , Cell Death/drug effects , Cell Proliferation , Cell Survival/drug effects , Cells, Cultured , Gamma Rays/adverse effects , Mice, Inbred BALB C , Mice, Inbred C57BL , Polysaccharides/pharmacology , Radiation-Protective Agents/pharmacology , Spleen/cytologyABSTRACT
Five monoclonal antibodies (MAbs) and chicken immunoglobulin (IgY) were developed by immunizing with flagella purified from Listeria monocytogenes 4b and the five MAbs have been confirmed to be specific against three different epitopes of flagellin. The antibodies showed specific reaction to Listeria genus and no cross-reactivity with other bacteria tested in this experiment including E.coli O157:H7 and Salmonella enteritidis. Sandwich enzyme-linked immunosorbent assays (ELISA) using the MAbs and IgY were developed to detect Listeria species and the sensitivity and specificity of the developed ELISA have been analyzed. The detection limit of ELISA using MAb 2B1 and HRP labeled IgY was 1 x105cells/0.1 ml at 22degrees C and 1x106 cells/0.1 ml at 30degrees C. ELISA using the pair of MAbs (MAbs 2B1 and HRP labeled MAbs 7A3) detected up to 104cells/0.1 ml at 22degrees C and 30degrees C. Detection limit of sandwich ELISA using IgY was 10 times lower than MAb pair. Using the developed ELISA, we could detect several Listeria contaminated in food samples after 48 h-culturing. In conclusion, both MAbs and IgY have been proved to be highly specific to detect Listeria flagella and the developed sandwich ELISA using these antibodies would be useful tool for screening Listeria spp. in food.
Subject(s)
Animals , Antibodies, Bacterial/chemistry , Antibodies, Monoclonal , Antibody Specificity , Antigens, Bacterial/analysis , Enzyme-Linked Immunosorbent Assay/methods , Flagella/genetics , Food Microbiology , Immunoglobulins/analysis , Listeria/classification , Meat/microbiology , Milk/microbiology , Sensitivity and Specificity , SwineABSTRACT
We immunized sixty two healthy subjects with the five different viral titers (300, 500, 1000, 1500 and 2000 plaque forming unit; pfu) of the MAV/06 strain of live attenuated Varicella-zoster virus (VZV) in order to gain sufficient information on safety and immuogenicity as a vaccine strain. Humoral immunity of all vaccine recipients was tested by the fluorescent antibody to membrane antigen (FAMA) assay and Enzyme-linked immunosorbent assay (ELISA) for the quantitative detection of IgG antibody. We tested neutralized antibody in 62 subjects by plaque reduction neutralization test (PRNT50). All of thirty two subjects with initial seronegative response had antibody by FAMA method at four weeks after immunization with four different preparations of dosage. The geometric mean titers (GMTs) of VZV antibody to membrane antigen was 160.9 in 6 subjects with 1500 pfu group; 83.3 in 14 subjects with 1000 pfu group: 116.2 in 7 subjects with 500 pfu groups and 72.0 in 6 subjects with 300 pfu group. Thirty subjects who had VZV antibody at the time before immunization demonstrated elevated antibody titer by FAMA assay and PRNT50 test. Side reactions of the vaccination was not demonstrated in all cases.
Subject(s)
Chickenpox , Enzyme-Linked Immunosorbent Assay , Herpesvirus 3, Human , Immunity, Humoral , Immunization , Immunoglobulin G , Membranes , Neutralization Tests , VaccinationABSTRACT
No abstract available.
Subject(s)
Animals , Humans , Cell Line , Guinea Pigs , Guinea , Hantaan virusABSTRACT
No abstract available.