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Immunological responses to oxidized ferredoxin and its chemically modified derivatives Gregerson, Dale

Abstract

Guinea pig lymph node and spleen cells responded in vitro to con-canavalin A (con A), lipopolysaccharide (LPS), and a specific antigen, oxidized ferredoxin, in serum free medium, medium with mercaptoethanol (ME), medium with foetal calf serum (FCS), and medium with both FCS and ME. The addition of ME to serum free medium supported a mixed leucocyte reaction as did media with FCS or FCS and ME. Nylon wool fractionation of the cells eliminated the LPS response and treatment with anti-immunoglobulin (alg) and complement (C) reduced the LPS response indicating that LPS may be a B lymphocyte mitogen. The con A response was enhanced by the alg and Cl treatment. The specificities of the humoral and cellular immune responses to the amino terminal heptapeptide antigenic determinant of performic acid oxidized ferredoxin (O-Fd) were tested and compared using several synthetic peptides and analogues of the determinant in leucocyte migration inhibition tests with guinea pig spleen cells and inhibition of complement fixation between O-Fd and homologous rabbit antiserum. A tetrapeptide comprising the four amino acids at the carboxy terminal of the test determinant was able to produce significant inhibition of leucocyte migration. Modifications of amino acids within the tetrapeptide resulted in a loss of migration inhibition. This same peptide would only inhibit the complement fixation reaction if a hydrophobic group was attached to it. Otherwise a hexapeptide was required to produce significant inhibition of' complement fixation. Guinea pigs sensitized with a conjugate of the heptapeptide determinant and bovine serum albumin gave positive immediate and delayed skin reactions to conjugates of the heptapeptide with other proteins indicating that the heptapeptide functions as both a hapten and carrier. Also, migration of spleen cells from these animals was inhibited by O-Fd. The abilities of several chemically modified forms of ferredoxin to stimulate DNA synthesis in vitro in spleen cells from mice sensitized to O-Fd and to fix complement (C') in the presence of rabbit anti-O-Fd sera were tested. Of all the ,ferredoxins, only O-Fd, native ferredoxin (native-Fd) and N-ethylmaleimide alkylated ferredoxin (NEM-vFd) were able to stimulate DNA synthesis. O-Fd, native-Fd, acid precipitated ferredoxin (TCA-Fd) and dinitrophenylated O-Fd (DNP-O-Fd) fixed C' while methylated ferredoxin (meth-O-Fd) did not. NEM-Fd and carboxymethylated ferredoxin (CM-Fd) fixed C' weakly. Only native-Fd, O-Fd and NEM-Fd were found to be immunogenic in mice when assayed for lymphocyte stimulation by all of the ferredoxins., The 24 hour DNA synthetic response was sensitive to treatment with anti-mouse immunoglobulin and C' and anti-brain associated 0 and C1 while the 120 hour response was only sensitive to the'anti-O and C'.

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