Cell culture

About: Cell culture is a research topic. Over the lifetime, 133361 publications have been published within this topic receiving 5364150 citations. The topic is also known as: cell culture techniques.

Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity.

Abstract: NKp46 has been shown to represent a novel, natural killer (NK) cell-specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3zeta chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs. Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.

Tumor Cells Abrogates Tumorigenicity and Induces Protective Immunity

Abstract: Summary To study the effects oflocalized secretion ofcytokines on tumor progression, the gene for human interleukin 2 (IL2)was introduced via retroviral vectors intoCMS-5 cells, a weakly immunogenic mouse fibrosarcoma cell line of BALB/c origin. Secretion of low levels of 11,2 from the tumor cells abrogated their tumorigenicity and induced a long-lasting protectiveimmune response against a challenge with a tumorigenic dose of parental CMS-5 cells . Co-injection of IL2-producing CMS-5 cells with unmodified tumor cells inhibited tumor formation evenwhen highly tumorigenic doses of CMS-5 cells were used . Cytolytic activity in mice injected with parental CMS-5 cells was transient and was greatly diminished 3 wk after injection, as commonly observed in tumorbearing animals. However, in mice injected with IL-2-producing cells, tumor-specific cytolytic activity persisted at high levels for the duration of the observation period (at least 75 d) . High levels oftumor-specific cytolytic activity could also be detected in parental CMS-5 tumor-bearing animals 18 d after inoculation with tumor cells, if 11,2-producing CMS-5 cells but not unmodified parental tumor cells were used as targets . These studies highlight the potential advantages of localized secretion of cytokines mediated via gene transfer to induce potent anti-tumor immune responses.

Functional correlation between cell adhesive properties and some cell surface proteins.

Abstract: The adhesive properties of Chinese hamster V79 cells were analyzed and characterized by various cell dissociation treatments. The comparisons of aggregability among cells dissociated with EDTA, trypsin + Ca2+, and trypsin + EDTA, revealed that these cells have two adhesion mechanisms, a Ca2+-independent and a Ca2+-dependent one. The former did not depend on temperature, whereas the latter occurred only at physiological temperatures. Both mechanisms were trypsin sensitive, but the Ca2+-dependent one was protected by Ca2+ against trypsinization. In morphological studies, the Ca2+-independent adhesion appeared to be a simple agglutination or flocculation of cells, whereas the Ca2+-dependent adhesion seemed to be more physiological, being accompanied by cell deformation resulting in the increase of contact area between adjacent cells. Lactoperoxidase-catalyzed iodination of cell surface proteins revealed that several proteins are more intensely labeled in cells with Ca2+-independent adhesiveness than in cells without that property. It was also found that a cell surface protein with a molecular weight of approximately 150,000 is present only in cells with Ca2+-dependent adhesiveness. The iodination and trypsinization of this protein were protected by Ca2+, suggesting its reactivity to Ca2+. Possible mechanisms for each adhesion property are discussed, taking into account the correlation of these proteins with cell adhesiveness.

Tumour necrosis factor (TNF) gene polymorphism influences TNF-alpha production in lipopolysaccharide (LPS)-stimulated whole blood cell culture in healthy humans.

Abstract: TNF-alpha is involved in infectious and immuno-inflammatory diseases. Different individuals may have different capacities for TNF-alpha production. This might determine a predisposition to develop some complications or phenotypes of these diseases. The aims of our study were to assess the inter-individual variability of TNF-alpha production and to correlate this variability to a single base pair polymorphism located at position -308 in TNF gene. We studied 62 healthy individuals. TNF-alpha production after LPS stimulation was evaluated using a whole blood cell culture model. The TNF gene polymorphism was studied by an allele-specific polymerase chain reaction. Other cytokines produced in the culture, soluble CD14 concentrations and expression of CD14 on blood cells were also measured. Among the 62 individuals, 57 were successfully genotyped. There were 41 TNF1 homozygotes and 16 TNF1/TNF2 heterozygotes. TNF-alpha production after LPS stimulation of whole blood cell culture was higher among TNF2 carriers than among TNFI homozygotes (929pg/ml (480-1473pg/ml) versus 521 pg/ ml (178-1307 pg/ml); P<0.05). This difference was even more significant after correction of TNF-alpha production for CD14 expression on blood cells. In conclusion, the single base pair polymorphism at position -308 in the TNF gene may influence TNF-alpha production in healthy individuals.

The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells.

Abstract: CD44 is a polymorphic integral membrane protein which recognizes hyaluronate and whose proposed roles encompass lymphocyte activation, matrix adhesion and the attachment of lymphocytes to lymph node high endothelial venules (HEVs) Immunochemical and RNA blot data have supported the existence of two forms of CD44: a hematopoietic form expressed by cells of mesodermal origin (and by some carcinoma cell lines) and an epithelial form weakly expressed by normal epithelium but highly expressed by carcinomas This report describes the isolation of a cDNA encoding a distinct CD44 polypeptide expressed by epithelial cells Re-expression of each form of CD44 in a B cell line allowed cells transfected with the hematopoietic but not the epithelial form to bind to viable rat lymph node HEV cells in primary culture