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The ability of staphylococcal toxins to stimulate large numbers of T cells has led to their designation as a superantigen. Previous studies have indicated that activation of T cells bearing particular V beta elements may be responsible for the toxic effects of these bacterial products. However, the widespread expression of functionally similar proteins by unrelated bacterial species suggests the possibility that these products may represent a successful microbial strategy for subversion of the host antibacterial response. We have examined the effects of the staphylococcal enterotoxin B (SEB) on T cell clones that express different V beta elements. We note that SEB stimulates clones bearing previously defined V beta elements to proliferate and to produce cytokines. In addition, we demonstrate that an interaction between SEB and the TCR of clones that express additional V beta elements, including V beta 2 and V beta 6, results in a sterile form of immunological activation. This activation phenotype is characterized by proliferation without detectable cytokine production and is followed by profound immunological unresponsiveness in vitro and in vivo. We propose that reduced levels of antibacterial responses resulting from this form of T cell unresponsiveness may account for the highly conserved expression of superantigens by diverse bacterial species.

CD4+ T cells are equipped to detect two major classes of ligands. Infectious microbial agents, including bacteria and retroviruses, carry a class of proteins termed superantigens that are recognized by the T-cell receptor in association with class II products of the major histocompatibility complex. Proteins expressed by other cells and organisms are processed by macrophages into peptides that are presented to CD4+ T cells by class II molecules. We have examined CD4+ T-cell clones that proliferate vigorously in response both to conventional peptide antigens and to bacterial or retroviral superantigens. The response to peptide antigen is characterized by a rapid and sustained increase in the levels of intracellular free Ca2+ and a vigorous cytokine response. In contrast, the proliferative response of these clones to bacterial or retroviral superantigen is not accompanied by detectable increases in intracellular Ca2+ or by significant cytokine production. Further analysis of T-cell activation indicates that interaction of a single T-cell receptor with the two types of ligand may be coupled to functionally distinct signaling pathways that interact in a synergistic fashion to achieve T-cell activation.

1. The metabolism of methapyrilene (MPH) by rat, hamster and mouse liver microsomes in vitro was investigated together with the binding of 14C-MPH to calf thymus DNA after metabolic activation. 2. Both quantitative and qualitative differences in MPH metabolism were observed in these three species. Mouse liver microsomes catalyse the formation of two novel isomers of hydroxypyrdylmethapyrilene (hydroxypyridyl-MPH) as determined by mass spectral analysis. N,N'-Didesmethylmethapyrilene (didesmethyl-MPH) was formed in detectable quantities only when hamster liver microsomes were used. 3. Incubation of liver microsomes from all three species catalysed the binding of 14C-MPH to exogenous DNA, which was quantitatively similar for all three species. The effect of the cytochrome P-450 inhibitor, 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), and methimazole, a flavin-dependent monooxygenase inhibitor, on binding differed significantly for the three species studied.

The lipids of mammalian stratum corneum are known to be important regulators of skin permeability. Since the human stratum corneum displays remarkable regional variations in skin permeability, we assessed the total lipid concentration, the distribution of all major lipid species, and the fatty acid composition in Bligh-Dyer extracts from four skin sites (abdomen, leg, face, and sole) that are known to display widely disparate permeability. Statistically significant differences in lipid weight were found at the four sites that were inversely proportional to their known permeability. In all four sites, among the polar lipids, the stratum corneum contained negligible phospholipids, but substantially more cholesterol sulfate (1-7%) than previously appreciated. As in the stratum corneum from other mammals, the bulk of the lipids consisted of neutral (60-80%) and sphingolipids (15-35%). Of the neutral lipids, free sterols (4- to 5-times greater than esterified sterols), free fatty acids, triglycerides, and highly nonpolar species (n-alkanes and squalene) predominated. n-Alkanes, which were present in greater quantities than previously appreciated, comprised a homologous series of odd- and even-chained compounds ranging from C19 to C34. The sphingolipids comprised over 80% ceramides vs. lesser quantities of glycosphingolipids. In all four sites, the sphingolipids were the major repository of long-chain, saturated fatty acids. The neutral lipid:sphingolipid ratio generally was proportional to the known permeability of each site: higher neutral lipids and lower sphingolipids generally were associated with superior barrier properties. These studies provide: 1) the first detailed, quantitative analysis of human stratum corneum lipids and 2) information about the variability in lipid composition at four skin sites with known differences in permeability. The latter results suggest that variations in neutral lipids, rather than sphingolipids, may underlie local variations in skin permeability.

Rats were fed 100 or 1000 p.p.m. methapyrilene (MPH) in their diet for 1, 2, 4, 8 or 16 weeks. Liver microsomes were prepared from both control and treated rats. After incubation with 1 mM MPH, eight metabolites were detected and six were quantitated. Five of the metabolites have been previously identified as 2-hydroxymethyl-thiophene (ThM), thiophene-2-carboxylic acid (ThCA), N-(2-thienylmethyl)-2-aminopyridine (TMAP), N-2-pyridyl-N',N'-dimethylethylenediamine (PMED), and 2-aminopyridine (AP). Three other metabolites have been tentatively identified based on their mass spectral fragmentation patterns as normethapyrilene (N-MPH), (5-hydroxypyridyl)methapyrilene (HP-MPH), and methapyrileneamide (MPH-A). The same metabolites were found in both control and treated animals, the most abundant being N-MPH and PMED. Pretreatment with MPH resulted in inhibition of both consumption of MPH and formation of some metabolites. However increases in the formation of all of the metabolites also occurred under different treatment conditions. In both control and treated tissue, the preliminary mass balance was less than 55%, except in incubations with tissue from rats treated with 1000 p.p.m. for 8 or 16 weeks where it was 92 and 89%, respectively. Dramatic increases in the fraction of TMAP, MPH-A, N-MPH, and HP-MPH relative to MPH consumed account for the increase in the mass balance after 8 weeks pretreatment with 1000 p.p.m. MPH, and increases in the amounts of PMED, HP-MPH and ThCA account for the higher mass balance after 16 weeks. The toxicological consequences of these complex metabolic changes may be important in the induction of cancer by MPH.

Methapyrilene ([14C]MPH) was found to bind to calf thymus DNA only after activation by both rat liver microsomes and NADPH. The cytochrome P-450 inhibitors 2,4-dichloro-6-phenylphenoxyethylamine, 2-diethylaminoethyl-2,2-diphenylvalerate and metyrapone inhibited binding, but methimazole, a flavin-dependent monooxygenase inhibitor, had no effect. However, 1,2-epoxy-3,3,3-trichloropropane, an epoxide hydrolase inhibitor, decreased binding by 30%. Pre-treatment of rats with isosafrole, pregnenolone-16 alpha-carbonitrile or phenobarbital had little or no effect on binding while 3-methylcholanthrene pretreatment decreased binding by 37%. Incubations in the presence of either N-acetylcysteine, glutathione, catalase or glutathione-peroxidase decreased binding to DNA while superoxide dismutase had no effect. These data suggest that MPH is metabolically activated to a species which binds to DNA and that this activation may be mediated by cytochrome P-450 isozymes.

1. The metabolism of methapyrilene (I), was examined in vivo by g.l.c. and g.l.c.-mass spectrometric analysis of rat urinary extracts. 2. Dosing the animals with tetradeuterium-labelled I helped identify 7 different metabolites of I in the urine, including (5-hydroxylpyridyl)-methapyrilene, which was identified by comparison with a synthetic reference standard. 3. After 4 weeks of treatment with I, rats also excrete detectable amounts of the 3- and (6-hydroxylpyridyl)-methapyrilene metabolites suggesting that pretreatment with I alters the metabolism of the pyridine ring. 4. Metabolic removal of the 2-thienylmethylene moiety is also facile, as large amounts of N'-(2-pyridyl)-N,N-dimethylethylenediamine and its metabolite N'-[2(5-hydroxylpyridyl)]-N,N-dimethylethylenediamine are excreted under all dosing regimens. 5. Urinary concn of both I and metabolites decline with time, despite continuous dosing, indicating a change in absorption, metabolism, and/or excretion of I on repeated dosing.

The in vitro metabolism of 3-(p-chlorophenyl)-3-(2-pyridyl)-N, N-dimethylpropylamine (chlorpheniramine, I) by rabbit liver microsomes was examined. The metabolites, tentatively identified by gas-liquid chromatography-mass spectrometry, included the mono- and didemethyl metabolites, the aldehyde that results from deamination, and further metabolites of this aldehyde including its intramolecular cyclization product, an indolizine, and its reduction product, the alcohol. Inhibition of metabolism of I by N2, CO, SKF-525A, 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), or deletion of NADPH implies some involvement of cytochrome P-450 in the metabolic reactions. Quantitation of metabolism in these studies accounted for only 69% of the dose, so that binding and/or other undetected metabolic pathways were operative.