ABSTRACT
Defining the pattern of lymphokine production associated with Brucella abortus is critical for advancing the development of B. abortus as a vaccine carrier. In the present study we investigated the ability of heat-inactivated B. abortus or lipopolysaccharide from B. abortus to induce lymphokine production from purified human T cells in vitro. Gamma interferon (IFN-gamma), interleukin-2 (IL-2), IL-4, and IL-5 induction was assayed by mRNA-specific PCR and by enzyme-linked immunosorbent assay and bioassay for protein production. Following depletion of monocytes and B cells, B. abortus increased IFN-gamma and IL-2 mRNA expression in purified T cells compared with expression in unstimulated cells. In contrast, no IL-5 mRNA expression and only transient low-level IL-4 mRNA expression and no IL-4 protein secretion were detected. Phytohemagglutinin or phorbol myristate acetate plus ionomycin induced mRNA and protein for all these cytokines. Similar results were obtained with LPS purified from B. abortus. Removal of NK cells did not reduce lymphokine production, and enriched NK cells did not express IFN-gamma mRNA or secrete IFN-gamma protein in response to B. abortus, indicating that NK cells were not the responding population. Both CD4+ and CD8+ populations produced IFN-gamma and IL-2 in response to B. abortus. Preincubation of resting T cells with B. abortus or LPS from B. abortus for 7 days induced their differentiation into Th1-like cells as judged by their subsequent lymphokine response to phorbol myristate acetate plus ionomycin. These results suggest that B. abortus can induce differentiation of Th0 into Th1-type cells.
Subject(s)
Brucella abortus/immunology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cytokines/metabolism , Killer Cells, Natural/immunology , T-Lymphocyte Subsets/immunology , Th1 Cells/immunology , Base Sequence , DNA Primers/chemistry , Gene Expression/drug effects , Hot Temperature , Humans , Interferon-gamma/metabolism , Interleukin-2/genetics , Interleukin-4/biosynthesis , Ionomycin/pharmacology , Lipopolysaccharides/immunology , Molecular Sequence Data , RNA, Messenger/genetics , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
A general procedure is presented for the isolation of several liver microsomal target proteins of the reactive trifluoroacetyl halide metabolite of halothane. It was found that most of these proteins could be selectively extracted from microsomes with 0.1% sodium deoxycholate and separated into partially purified fractions by DEAE-Sepharose anion-exchange chromatography. Using this method, we describe the isolation and identification of a 63-kDa target protein of halothane in rat liver. Amino acid sequences of the N-terminal and of several internal peptides of the protein, as well as the deduced amino acid sequence of a nearly full-length rat liver cDNA clone of the protein, showed 98% identity with a reported murine cDNA that encodes for calreticulin, a major calcium-binding protein of the lumen of endoplasmic reticulum. Although it remains to be determined what role calreticulin has in the development of halothane hepatitis, this study has shown that calreticulin can be a target of reactive metabolites of xenobiotics.
Subject(s)
Calcium-Binding Proteins/metabolism , Fluoroacetates , Halothane/pharmacology , Liver/metabolism , Amino Acid Sequence , Animals , Calcium-Binding Proteins/isolation & purification , Calreticulin , Chromatography, High Pressure Liquid , Halothane/metabolism , Liver/drug effects , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Sequence Data , Rats , Rats, Inbred Strains , Trifluoroacetic Acid/metabolismABSTRACT
Adult male Fischer 344 rats were fed inadequate, adequate, and excessive quantities of dietary protein (8, 12, and 22%, respectively) for a period of 14 days. An increase in dietary protein did not increase liver weight but resulted in an increase in cytochrome P-450 content and 7-ethoxycoumarin O-deethylase activity. No significant difference in glutathione S-transferase activity was observed at the three protein levels. The in vivo hepatotoxicity of bromobenzene increased with an elevation in dietary protein intake from 12 to 22%. These data from mature rats follow trends similar to findings we have reported previously with juvenile rats.
Subject(s)
Dietary Proteins/pharmacology , Mixed Function Oxygenases/metabolism , Pharmaceutical Preparations/metabolism , Animals , Biotransformation , Body Weight/drug effects , Bromobenzenes/pharmacology , Dietary Proteins/metabolism , Liver/drug effects , Male , Organ Size/drug effects , Rats , Rats, Inbred F344ABSTRACT
Diets containing protein levels of 8, 12, and 22% were fed for 14 days to Fischer 344 male weanling rats. Enzyme activities in liver and kidney were measured using several substrates for each of the following enzyme systems: Cytochrome P-450-dependent monooxygenase, ester hydrolysis, and conjugations with sulfate, glutathione, and glucuronic acid. Enzyme activities, for the various mechanisms, decreased from 15 to 65% with decreased dietary protein, with the exception of glucuronide and sulfate conjugation, which increased with decreased dietary protein. In vivo effects were evaluated by measuring hexobarbital sleeping time, procaine paralysis time, and bromobenzene hepatotoxicity. Increased dietary protein shortened hexobarbital sleeping time and procaine paralysis time, and increased procaine lethality, whereas low protein intake protected against bromobenzene hepatotoxicity. These data suggest that typical laboratory diets (22-25% protein) may provide artificially altered activities of xenobiotic biotransformation in rats, relative to nutritionally adequate (12% protein) diets.