Various primary cells and an established cell line were cultured in roller tubes and in suspension to evaluate their potential roles as host cells to support the growth of M. leprae in vitro. The primary cells originated from the organs of chipmunks, mice and humans. Phagocytic ability of those cells except for macrophages was found to be low and did not vary much according to their origin. However, when macrophages from mice peritonial exudate were exposed to the bacteria, the phagocytic efficiency was higher than 47%. In spite of those good primary results, the macrophages are not cells which can adapt well in vitro for long term culture, which is essential for the growth of such a slow growing M. leprae. Thus, somatic cell hybridization between the macrophages and HeLa was made by fusing them with polyethylene glycole. Those hybrids appeared to have both the characteristics of the parent cells, which can provide a natural intracellular environment such as the macrophages and the infinite growth capability of the HeLa cells in vitro.
Animal ; Culture Media ; Hela Cells ; Human ; Hybrid Cells ; Macrophages ; Mice ; Mycobacterium leprae/growth & development* ; Sciuridae

Various primary cells and an established cell line were cultured in roller tubes and in suspension to evaluate their potential roles as host cells to support the growth of M. leprae in vitro. The primary cells originated from the organs of chipmunks, mice and humans. Phagocytic ability of those cells except for macrophages was found to be low and did not vary much according to their origin. However, when macrophages from mice peritonial exudate were exposed to the bacteria, the phagocytic efficiency was higher than 47%. In spite of those good primary results, the macrophages are not cells which can adapt well in vitro for long term culture, which is essential for the growth of such a slow growing M. leprae. Thus, somatic cell hybridization between the macrophages and HeLa was made by fusing them with polyethylene glycole. Those hybrids appeared to have both the characteristics of the parent cells, which can provide a natural intracellular environment such as the macrophages and the infinite growth capability of the HeLa cells in vitro.
Animal ; Culture Media ; Hela Cells ; Human ; Hybrid Cells ; Macrophages ; Mice ; Mycobacterium leprae/growth & development* ; Sciuridae

BACKGROUND: Mutations in the human Cu, Zn-superoxide dismutase (SOD1) gene have been identified in some cases of familial amyotrophic lateral sclerosis (ALS). Neuronal cells with mutant SOD1 gene promoted cell death during differentiation by dibutyryl cAMP and aphidicolin. The aim of this study is to delineate if there is an impairment of the neural differentiation process in mutant SOD1 cells. METHODS: We studied the motoneuron-neuroblastoma hybrid cells (VSC 4.1) expressing wild-type or mutant SOD1 (G93A) during the differentiation by dibutyryl cAMP and aphidicolin. RESULTS: Mutant SOD1 cell (G93A) showed an impairment in the neurite formation. Western blot analysis revealed that the amount of neurofilament decreased before differentiation. A decrease in the amount of MAP-2 is observed during differentiation. CONCLUSIONS: Our results suggest that the impairment in the neurite formation of mutant SOD1 cell (G93A) is a differentiation failure and is associated with neuronal cell death.
Amyotrophic Lateral Sclerosis ; Aphidicolin ; Blotting, Western ; Cell Death ; Humans ; Hybrid Cells ; Neurites* ; Neurons

In order to expand gene resources and improve Brassica napus cultivars, protoplasts isolated from hypocotyls of Brassica napus cv. Huayou No. 3 and Eruca sativa were fused by PEG-high Ca2+-high pH. Fusion frequency was up to 18.2% when fusion system contained 5 x 10(5) protoplasts/mL, and when PEG concentration of fusion agents were 35% and when fusion time was 25 min. Then the fused protoplasts were cultured by the method of thin liquid layer at the density of 1 x 10(5) protoplasts/mL in improved KM8p medium supplemented with 1.0 mg/L 2,4-D, 0.5 mg/L NAA, 0.5 mg/L 6-BA, 200 mg/L inositol, 300 mg/L protein hydrolysate, and the combinations of 0.1 mol/L sucrose and 0.2 mol/L glucose and 0.2 mol/L mannitol for osmotic regulator, the frequency of callus regeneration was up to 6.8%. When the micro-calli transferred to the proliferation medium that contained B5 salts, 0.087 mol/L sucrose, 0.2 mg/L 2,4-D, 0.5 mg/L NAA, 0.2 mg/L 6-BA and 0.5% Agar, pH 5.8, have grown up to 3-5 mm of diameter, the calli were transferred to the differentiation medium that contained MS salts, 0.087 mol/L sucrose, 0.1 mg/L IAA, 0.8 mg/L 6-BA, 0.8% Agar, pH5.8, the shoots were regenerated in 4 weeks and its frequency was up to 32.8%. Then 2-3 cm shoots were transferred to 1/2 MS medium with 0.5 mg/L IBA+0.2mg/L 6-BA, plantlets were obtained in 14 days and the plantlet frequency was up to 88%. When the protoplasts of Eruca sativa were treated with UV radiation for 2 minutes calli and plantlets have been regenerated, treated for 4 min only calli have been regenerated, and treated for more than 5 min calli have not been regenerated. The callus regeneration and callus proliferation and plant regeneration from symmetric fusion were more than from asymmetric fusion. 16 hybrid plantlets have been regenerated on 21 piece of hybrid calli identified by cytology method.
Brassica ; genetics ; Brassicaceae ; genetics ; Cell Fusion ; Hybrid Cells ; Hybridization, Genetic ; Protoplasts ; Regeneration ; Ultraviolet Rays

Alcohol Oxidoreductases ; genetics ; isolation & purification ; Cloning, Molecular ; Hep G2 Cells ; Humans ; Two-Hybrid System Techniques

This perspective article highlights the leukocyte-cancer cell hybrid theory as a mechanism for cancer metastasis. Beginning from the first proposal of the theory more than a century ago and continuing today with the first proof for this theory in a human cancer, the hybrid theory offers a unifying explanation for metastasis. In this scenario, leukocyte fusion with a cancer cell is a secondary disease superimposed upon the early tumor, giving birth to a new, malignant cell with a leukocyte-cancer cell hybrid epigenome.
Animals ; Bone Marrow Cells ; cytology ; pathology ; Cell Fusion ; Humans ; Hybrid Cells ; pathology ; Neoplasm Metastasis ; Neoplasms ; pathology ; Neoplastic Stem Cells ; pathology

BACKGROUND: Vascular endothelial growth factor (VEGF) is an angiogenic peptide that enhances microvascular perfusion. Recently, VEGF is known to have neurotrophic effect and rescues neurons from cell death induced by serum deprivation. To investigate the serial changes in VEGF expression and neuroprotective properties of VEGF during acute ischemia. METHODS: Human cortical-neuroblastoma hybrid cell line (A1G11), human neuroglioma cell line (H4), and human vascular endothelial cell line (ECV304) were placed in the glucose/serum free media and incubated in the hypoxic chamber (94% N2/5% CO2/1% room air) at 37 degrees C. Cell viability was determined by MTT assay. Western blot analysis was performed to detect VEGF and its receptor (VEGFR) expression. To test the protective effect of VEGF, human recombinant VEGF165 was used. RESULTS: Morphological changes and the decrease of cell viability were observed following 6 hr ischemia. In A1G11 cells, VEGF expression was not noted until 3 hr ischemia, but was induced after 6hr and continued to 12 hr and then diminished. In H4 and ECV304, the change of VEGF expression was not observed. VEGFR-2/Flk-1 expression was induced from 6 hr (peak level) to 12 hr in A1G11, and induced after 3 hr and continued to 12hr in ECV304. Administration of VEGF increased cell viability in A1G11 cells at 6 hr, 12 hr and 18 hr ischemia (p=0.009, p=0.01 p=0.013), but not in H4 or ECV304 cells ( p>0.05). CONCLUSION: Ischemia induces VEGF production in neurons and VEGF may exert a direct neuron-specific protective effect through VEGFR-2/Flk receptors during the acute phase of ischemic neuronal injury.
Blotting, Western ; Cell Death ; Cell Line ; Cell Survival ; Endothelial Cells ; Humans ; Hybrid Cells ; Ischemia* ; Neurons ; Perfusion ; Vascular Endothelial Growth Factor A*

Breast cancer is among the most common malignancies affecting women and reproductively intact female dogs, resulting in death from metastatic disease if not treated effectively. To better manage the disease progression, canine mammary tumor (CMT) cells derived from malignant canine mammary cancers were fused to autologous dendritic cells (DCs) to produce living hybrid-cell fusion vaccines for canine patients diagnosed with spontaneous mammary carcinoma. The high-speed sorting of rare autologous canine patient DCs from the peripheral blood provides the autologous component of fusion vaccines, and fusion to major histocompatibility complex-unmatched CMT cells were produced at high rates. The vaccinations were delivered to each patient following a surgical resection 3 times at 3-week intervals in combination with immuno-stimulatory oligonucleotides and Gemcitabine adjunct therapy. The immunized patient animals survived 3.3-times longer (median survival 611 days) than the control patients (median survival 184 days) and also appeared to exhibit an enhanced quality of life. A comparison of vaccinated patients diagnosed with inflammatory mammary carcinoma resulted in a very short median survival (42 days), suggesting no effect of vaccination. The data showed that the development of autologous living DC-based vaccine strategies in patient animals designed to improve the management of canine mammary carcinoma can be successful and may allow an identification of the antigens that can be translatable to promote effective immunity in canine and human patients.
Animals ; Breast Neoplasms ; Breast ; Dendritic Cells ; Disease Progression ; Dogs ; Female ; Histocompatibility ; Humans ; Hybrid Cells ; Oligonucleotides ; Quality of Life ; Vaccination ; Vaccines

OBJECTIVETo screen the protein interacting with retinoic acid receptor variant protein (RAR alpha-V) via the yeast two-hybrid technique (YTHT) and to find out the targets protein and study its biological function.

METHODSThe bait vector of pGBKT7-RAR alpha-V was constructed for screening proteins interacting with RAR alpha-V in K562 cell cDNA expression library via YTHT. The protein-protein interaction was confirmed with re-transformation in yeast and GST pull-down in vitro.

RESULTSThe bait vector was successfully constructed without toxicity, leakage and self-activation. Sixteen proteins were screened by YTHT and eight positive clones were identified by re-transformation in yeast. The interaction between RAR alpha-V and JTV-1 was confirmed by GST pull-down in vitro.

CONCLUSIONSThere are some kinds of proteins interacting with RAR alpha-V in cell. The biological dysfunction caused by certain protein-protein interaction may be involved in the pathogenesis of leukemia.

Gene Library ; Humans ; K562 Cells ; Protein Interaction Mapping ; Receptors, Retinoic Acid ; metabolism ; Retinoic Acid Receptor alpha ; Two-Hybrid System Techniques

OBJECTIVETo construct dendritomas by fusion of human dendritic cells with HLE cells, a human hepatocellular carcinoma cell line.

METHODSHLE cells were cultured in RPMI 1640 with 15% FCS. Human dendritic cells (DCs) were obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4 for 7 days, matured with TNF-alpha and PGE(2) for 2 days. The DCs and HLE cells were labeled with green fluorescence dye PKH67-GL and red fluorescence dye PKH26-GL, respectively, and fused in 50% polyethylene glycol (PEG) + 10% dimethyl sulfoxide (DMSO) to generate dendritomas for rapid fluorescence-activated cell sorting (FACS).

RESULTSDendritomas with dual red-green fluorescence were constructed successfully, and FACS analysis showed the effective fusion rate was 16.8%.

CONCLUSIONWith fluorescence dyes PKH67-GL and PKH26-GL as fusion markers, dendritomas for rapid fluorescence-activated cell sorting are constructed, which may throw new light on immunotherapy of hepatocellular carcinoma.

Cancer Vaccines ; biosynthesis ; Carcinoma, Hepatocellular ; pathology ; Cell Fusion ; methods ; Cell Line, Tumor ; Cells, Cultured ; Dendritic Cells ; cytology ; Humans ; Hybrid Cells ; Liver Neoplasms ; pathology