Subject(s)
Antibodies, Monoclonal/immunology , Bacteriophages , Genetic Vectors , Protein Engineering , Recombinant Fusion Proteins/immunology , Amino Acid Sequence , Animals , Bacteriophages/genetics , Bacteriophages/immunology , Base Sequence , Biosensing Techniques , Genes, Immunoglobulin , Humans , Immunoglobulin Fragments/analysis , Immunoglobulin Variable Region/genetics , Mice , Mice, SCID , Molecular Sequence Data , Mutagenesis , Random AllocationABSTRACT
The kinetics of lymphokine-specific DNA transcription during in vitro immunization of human peripheral blood lymphocytes and splenocytes were studied using the polymerase chain reaction. The levels of specific mRNA were shown to be down regulated by cytolytic L-leucyl-leucine methyl ester-sensitive lymphocytes. In in vitro immunizations using L-leucyl-leucine methyl ester-treated human PBL or splenocytes, the lymphokine mRNA expression pattern indicated an active gene transcription during the entire stimulation period, especially for the IL-2 and IL-5 genes. Transcription of IL-6 and TNF beta started on day 4, whereas IFN gamma mRNA reached its maximum level on day 4. In vitro immunizations of cells not treated with L-leucyl-leucine methyl ester revealed a transient transcription of lymphokine DNA that was declining already after day 2. Exogenously added recombinant IL-2, IL-4, and IL-6 all exhibited a positive immunoregulatory effect on Ig secretion, whereas IL-5 was not found to have any effect on immunoglobulin secretion during the in vitro culture. These results present the first information useful for designing in vitro immunization systems based on recombinant lymphokines and antisense DNA for gene regulation.
