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6-0-(2-Tetradecylhexadecanoyl)-N-acetylmuramyl-L-alanyl-D-isoglutamine (B30-MDP) and 6-0-(3hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) were examined for immunopotentiating activity in stimulating the primary immune responses of guinea pigs to ovalbumin and for adverse reactions at the injection site and in the regional lymph nodes when administered in combination with several nonirritating vehicles. B30-MDP was found to potently stimulate both humoral and cellular immune responses, the latter by induction of delayed-type hypersensitivity, irrespective of the administration vehicle examined (liposomes, a squalene in water emulsion, Intralipid, phosphate-buffered saline, and Nikkol HCO-60 glucose solution), although the minimum effective dose was dependent on the vehicle used. Reactions in the footpad receiving the injection were negligible. Noticeable local reaction consisted of swelling of the lymph nodes in the region of the injection, but the swelling was only noted with higher doses and subsided 3 to 4 weeks after immunization, unlike the persistent swelling caused by the administration of water-in-mineral oil emulsions with or without B30-MDP. BH48-MDP and its L-serine analog, BH48-MDP(L-Ser), which has L-serine in place of L-alanine in MDP, in combination with the squalene-in-water emulsion, also intensely stimulated both cellular and humoral antiovalbumin immune responses, but the effects of these compounds seemed to be influenced to a greater degree by the vehicles than by B30-MDP. Thus, B30-MDP was chosen from a series of a 6-0-acyl derivatives of MDP as the most promising candidate for possible application in practical vaccines.In an earlier study (6), we compared the immunopotentiating ability of 13 6-0-acyl derivatives of N-acetylmuramyl-L-alanyl-D-isoglutamine (6-0-acyl-MDP) to stimulate primary immune responses (humoral and cellular) of guinea pigs to ovalbumin when administered in combination with various vehicles. Among them, 6-0-(2-tetradecylhexadecanoyl)-MDP (B30-MDP) and, to a lesser extent, 6-0-(3hydroxy-2-docosylhexacosanoyl)-MDP (BH48-MDP) were found to exert strong adjuvant activity in both the induction of delayed-type hypersensitivity and the stimulation of circulating precipitating antibody levels when combined with nonirritating vehicles such as liposomes, squalene-in-water emulsion, and phosphate-buffered saline. These two compounds have a-branched higher fatty acid groups at the 6-position of the muramic acid residue and, when administered intravenously, have no detectable pyrogenicity, which is one of the harmful bioactivities of the parent molecule, MDP.Thus, we focused our attention on B30-MDP and BH48-MDP (or its L-serine analog) and investigated their effects on primary immune responses, both humoral and cell mediated, and the local injurious reactions caused when it was administered to guinea pigs at graded doses with ovalbumin in combination with several vehicles. MATERIALS AND METHODS B30-MDP, BH48-MDP, and BH48-MDP(L-Ser). The structures and chemical names of the acyl portio...

When the peripheral blood lymphocytes from patients with drug-induced allergic hepatitis were stimulated with a specific drug in vitro in the presence of a liver cytosol fraction containing liver specific antigen, lymphocyte transformation was seen in eight out of 11 patients. The macrophage activating factor (MAF), a kind of lymphokines, was also detectable in the culture medium of activated lymphocytes from seven out of eight patients who showed positive blastogenesis evaluated the uptake of 3H-glucosamine into macrophages. MAF-activated macrophages exhibited a cytotoxic effect on separated liver cells resulting in a marked inhibition of albumin synthesis. This macrophage-mediated cytotoxicity was also observed in eight out of 11 patients who showed positive lymphocyte transformation. These observations suggest that macrophage-mediated cytotoxicity may play some role in the pathogenesis of drug-induced allergic hepatitis.

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The possible involvement of cell-mediated immune response to liver specific lipoprotein (LSP) in the pathogenesis of liver injury was investigated. The subjects were consisted of one patient with acute hepatitis, five cases with chronic active hepatitis and one case with chronic failure inactive hepatitis. When peripheral blood lymphocytes from these patients were cultured in the presence of LSP and lymphocyte transformation was determined by measuring the uptake of [3H]-thymidine into the acid-insoluble materials, positive blastogenesis was seen in three cases with chronic active hepatitis. Furthermore, when peripheral blood lymphocytes from halothane-induced cholestatic hepatitis were cultured with offending drug in the presence of LSP and lymphocyte transformation was determined, a positive blastogenesis was seen. The factor which activated Kupffer cells (KCAF), a kind of lymphokine, was also detectable in the culture medium of activated lymphocytes from four patients who showed positive blastogenesis by estimating [3H]-glucosamine incorporation into Kupffer cells. The macrophage activating factor (MAF) was detectable in culture medium of activated lymphocytes from three patients who showed positive blastogenesis by estimating [3H]-glucosamine incorporation into macrophages. Furthermore, the KCAF-activated Kupffer cells and MAF-activated macrophages were shown to be cytotoxic for the isolated liver cells causing marked inhibition of albumin synthesis. The observations suggest that Kupffer cell-mediated cytotoxicity and macrophage-mediated cytotoxicity play a role in the pathogenesis of liver disease.