Enhancement of monoclonal antibody production after single and combination treatment of the hybridoma cells with all-trans retinoic acid and docosahexaenoic acid: An in vitro and in vivo study.

Murine hybridoma cells can produce monoclonal antibody (MAb) and the production of these antibodies in culture and peritoneum can be affected by different factors, including stimulants, inhibitors and supplements. Among these factors, the impact of micronutrients on the production of MAbs by mouse hybridoma cells has not fully been explored. In this study the murine hybridoma cells, M3C5, were cultured and treated with different concentrations of ATRA and DHA, alone, in combinations, and at different time of exposure. Then, changes in the production of MAb in culture medium were evaluated using ELISA. The hybridoma cells after single and combined treatment with ATRA, DHA and vehicles were IP injected to Balb/c mice and the changes in production of MAb in ascites were determined by ELISA. The results showed that single and combined treatment of ATRA and DHA elevated the production of MAb by hybridoma cells in both in vivo and in vitro. The production of MAb following in vitro single treatment with 1 µM of ATRA and 10 µM of DHA for 2 days was significantly increased. The in vitro effects of ATRA on increase of MAb production was obtained more than DHA. The MAb productions in combined treatment with 0.5 µΜ of ATRA plus 5 µΜ of DHA were significantly increased in in vivo and in vitro. However, the effect of DHA was obtained more significant in in vivo conditions. The results of this study showed for the first time that in vitro and in vivo treatments of ATRA and DHA could increase the production of MAb in mouse M3C5 hybridoma cells.

The role of interleukin-23 in stability of in vitro T helper-17 cells.

Interleukin (IL)-17-producing T helper (Th)-17 cells have recently been explained as a distinct population of CD4+ T cells which play an important role in immunity against infectious agents. Establishment of persistent phenotype of Th17 cells and recognition of lineage-deviating factors are of most attractive goals in modern researches in immunology. Although IL-6 and TGF-ß are frequently used to differentiate naive T cells to Th17 phenotype in mouse models, the application of IL-23 and its importance in preventing cells from plasticity needs to be more investigated. Our main objective was to evaluate the role of IL-23 in Th17 to Th1 plasticity. In this research project, we generated in vitro Myelin oligodendrocyte glycoprotein (MOG)-specific Th17 cells in the presence of TGF-ß, IL-6, IL-23 and peptide MOG35-55. Th17 development was confirmed by assessment of relevant transcription factors and secreted cytokines by flowcytometry and ELISA, respectively. Th17 to Th1 plasticity was monitored by consecutive samplings in different time points without any extra supplementation of IL-23. Cell culture supernatant was evaluated for Interferon (IFN)-γ secretion and cells were evaluated for intracellular expression of this cytokine. Our results showed that the employed method was relatively convenient in developing antigen-specific Th17 cells. We also showed that IL-23 deprivation which happens by prolongation of culture period, can convert IL-17 producing cells to IFN-γ secreting Th1 phenotype. IL-23 can be considered as a Th17 phenotype stabilizing factor for in-vitro developed lineages.

Specificity and isotype of Rh specific antibodies produced by human B-cell lines established from alloimmunized Rh negative women.

Despite the successful outcome of anti-D prophylaxis program, alloimmunization still occurs. The aim of this study was to examine the specificity and isotype of anti-Rh antibodies in plasma samples of Rh negative alloimmunized individuals and to study the same parameters in lymphoblastoid cell lines (LCLs) generated from the same donors. Specificity of anti-Rh antibodies was determined in plasma of nine alloimmunized subjects by direct hemagglutination using a panel of known RBC genotypes and isotype of specific antibodies were identified by an antigen specific ELISA. Similar methods were employed to determine specificity and isotype of antibodies produced by Rh specific LCLs established from four donors. LCLs were generated by Epstein-Barr virus transformation of peripheral blood mononuclear cells isolated from each donor followed by their culture over a feeder of human fetal fibroblasts. Upon emergence of lymphoblastoid cells, culture supernatants were assayed for presence of Rh specific antibody by hemagglutination assay. Anti-D was the predominant antibody in both plasma samples and among the 128 established LCLs; however, antibodies to other Rh specificities namely C and E were also produced. The isotype of anti-Rh antibody in all plasma samples was found to be IgG, predominantly IgG1, combined in 7 samples with IgM. Similarly 76%, 9.2% and 14.8% of LCLs were determined to produce antibody of IgG, IgM and of both isotypes, respectively. The data supported that the D antigen is the immunodominant component of the Rh system as indicated by the in vitro and in vivo profiles of Rh specificities in our alloimmunized subjects.