PhD (Doctor of Philosophy)
Date of Award
Activated lymphocytes secrete many products including the lymphokine human lymphocyte mitogenic factor (HLMF). In preliminary experiments lymphocytes from peripheral blood and palatine tonsils were evaluated as possible sources of HLMF by evaluating their level of activation through screening their spontaneous and concanavalin A (con A)-induced blastogenic responses. Tonsil lymphocytes (TL) were found to have high spontaneous proliferation as compared to peripheral blood lymphocytes (PBL). Cells from both sources responded to con A by undergoing a typical blastogenic response. Because TL must be obtained septically, they are frequently cultured in the presence of the antimycotic agent, Amphotericin B (Am B). Since the primary and induced blastogenesis of TL were greatly inhibited by even low concentrations of Am B, those lymphocytes were considered unacceptable sources of HLMF. In contrast to TL the induced blastogenic responses of PBL were found to be augmented by concentrations of Am B less than 5 (mu)g/ml, but the drug appeared to provide no beneficial effect on the quantity of HLMF produced by the cells. HLMF appeared to be produced optimally in the first 48 hr of culture by 10('7) PBL/ml, cultured in Neuman-Tytell serumless medium which had been adjusted to 5 x 10('-5) M 2-mercaptoethanol, and 5-35 (mu)g con A/ml. Stability of the HLMF activity could best be maintained by immediate dialysis against 0.05 M NH(,4)HCO(,3) solution, followed by lyophilization and storage of the dried material at -80(DEGREES)C until use. Activity was retained at -80(DEGREES)C for greater than 3 months. The activity was diminished after exposure to 56(DEGREES)C for 30 min, and completely lost after treatment at 80(DEGREES)C for 10 min or 100(DEGREES)C for 5 min. HLMF was insensitive to trypsin and exposure to pH ranges 2-7. Separation of HLMF and con A blastogenic activities was accomplished by addition of ovalbumin followed by Bio-Gel P-100 column Chromatography. HLMF activity eluted in the 12,000-20,000 d and 30,000-50,000 d ranges. The lower molecular weight material was active in the pH range 3.4-4.6 as demonstrated by isoelectric focusing. The larger molecular weight fractions had a pI of 4.14 (+OR-) 0.97. HLMF activated T cells, B cells and unfractionated PBL in assay, with the T cell response being generally, but not always greater. The factor behaved in a dose dependent fashion when assayed against unfractionated PBL.
Dissertation - Open Access
Seay, Thomas E., "Generation, Isolation and Assay Methods for Human Lymphocyte Mitogenic Factor" (1982). Electronic Theses and Dissertations. Paper 2791. http://dc.etsu.edu/etd/2791