Human anti-endoplasmic reticulum antibodies in sera of patients with halothane-induced hepatitis are directed against a trifluoroacetylated carboxylesterase. Proc Natl Acad Sci USA

Laboratory of Chemical Pharmacology, National Heart, Lung and Blood Institute, Bethesda, MD 20892.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 02/1989; 86(1):322-6. DOI: 10.1073/pnas.86.1.322
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Previous studies have demonstrated that patients with halothane-induced hepatitis have serum antibodies that are directed against novel liver microsomal neoantigens and have suggested that these neoantigens may play an immunopathological role in development of the patients' liver damage. These investigations have further revealed that the antibodies are directed against distinct polypeptide fractions (100 kDa, 76 kDa, 59 kDa, 57 kDa, 54 kDa) that have been covalently modified by the reactive trifluoroacetyl halide metabolite of halothane. In this paper, the trifluoroacetylated (TFA) 59-kDa neoantigen (59-kDa-TFA) recognized by the patients' antibodies was isolated from liver microsomes of halothane-treated rats by chromatography on an immunoaffinity column of anti-TFA IgG. Antibodies were raised against the 59-kDa-TFA protein and were used to purify the native protein from liver microsomes of untreated rats. Based upon its apparent monomeric molecular mass, NH2-terminal amino acid sequence, catalytic activity, and other physical properties, the protein has been identified as a previously characterized microsomal carboxylesterase (EC A similar strategy may be used to purify and characterized neoantigens associated with other drug toxicities that are believed to have an immunopathological basis.

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Available from: Brian Michael Martin, Apr 02, 2014
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    • "Ample evidence suggests that halothane hepatitis is caused by an immune reaction against endogenous proteins that have been covalently modified by the trifluoroacetyl chloride (TFA) metabolite of halothane (Kenna et al., 1988;Pohl, 1990). Sera from patients afflicted with halothane hepatitis contain specific antibodies recognizing TFA-modified liver proteins (Vergani et al., 1980;Satoh et al., 1989). TFA-protein adducts have also been detected in liver biopsies obtained from halothane hepatitis patients (Pohl et al., 1989). "
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    ABSTRACT: Immune-mediated adverse drug reactions (IADRs) represent a significant problem in clinical practice and drug development. Studies of the underlying mechanisms of IADRs have been hampered by the lack of animal models. Halothane causes severe allergic hepatitis with clinical features consistent with an IADR. Our ultimate goal is to develop a mouse model of halothane hepatitis. Evidence suggests that adaptive immune responses targeting liver protein adducts of the reactive metabolite (trifluoroacetyl (TFA)) play an important role in the pathogenesis. The present study demonstrated that the combination of an anti-CD40 antibody (Ab) and a Toll-like receptor (TLR) agonist served as a potent adjuvant in generating TFA-specific T cell responses in mice. Both CD4(+) and CD8(+) subsets of T cells were activated and the TFA-specific responses were detected not only in the spleen but also in the liver of mice immunized with mouse serum albumin adducts of TFA (TFA-MSA) plus the combined CD40/TLR agonist. Whereas all three TLR agonists examined were effective in eliciting TFA-specific immune responses in BALB/cByJ mice, only polyI:C was effective in DBA/1 mice and none of the TLR agonists could aid the generation of TFA-specific T cells in C57BL/6J mice. This result, combined with our previous finding that BALB/cByJ mice were the most susceptible to halothane-induced acute liver injury, provides the basis for employing this strain in future studies. Collectively, our data demonstrated the successful completion of a crucial first step in the development of a murine model of halothane hepatitis.
    Toxicology Letters 02/2010; 194(3):79-85. DOI:10.1016/j.toxlet.2010.02.009 · 3.26 Impact Factor
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    • "Among structurally similar anesthetics, which include halothane, isoflurane, and desflurane, the likelihood of causing IDR appears to correlate with the degree of reactive metabolite formation. Twenty percent of halothane is metabolized to trifloroacetyl chloride (TFA), and it is associated with the highest incidence of allergic hepatitis compared with isoflurane and desflurane, which are metabolized to TFA to much lesser degrees (Njoku et al. 1997; Satoh et al. 1989; Vergani et al. 1980). The sites of reactive metabolite formation often correlate with the major tissue targets of IDR. "
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    ABSTRACT: Idiosyncratic drug reactions (IDR) account for approximately 6%-10% of all adverse drug reactions. The unpredictable and serious nature of these reactions makes them a significant economic burden and safety concern to the health care community and the pharmaceutical industry. Clinical and laboratory evidence suggests that adverse immune responses against drug-protein adducts play a role in the pathogenesis of IDR. However, it remains unclear why only a small percentage of patients are susceptible to developing these reactions. We hypothesized that most patients develop immunological tolerance against drug-protein adducts as a default mechanism, and that IDRs can only occur when this tolerance is deficient or abrogated in susceptible individuals. Using a murine model of 2,4-dinitrochlorobenzene (DNCB)-induced delayed type hypersensitivity (DTH) reaction, our previously published data demonstrated that intravenous pretreatment of mice with dinitrophenyl-bovine serum albumin (DNP-BSA) induced immunological tolerance to subsequent DNCB sensitization, and that hepatic macrophages (Kupffer cells, KC) played an important role in mediating such tolerance. Further mechanistic investigation revealed that KC, acting as incompetent antigen-presenting cells, cannot elicit strong T cell reactions, and that they actively suppress T cell activation through production of prostaglandins. These findings suggest that KCs may play a critical role in regulating immune reactions within the liver and contributing to liver-mediated systemic immune tolerance.
    Toxicologic Pathology 02/2009; 37(1):12-7. DOI:10.1177/0192623308329475 · 2.14 Impact Factor
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    • "For some xenobiotics, reactive intermediates may indirectly lead to cellular damage through the triggering of immunological responses against altered cellular macromolecules (reviewed in Park et al. 1998). Antibodies against endoplasmic reticulum stress proteins have been identified in patients with idiosyncratic reactions to sulfonamides and halothane (Satoh et al.1989; Cribb et al.1997) and antibodies against the endoplasmic reticulum resident cytochrome P450 enzymes have been identified as well. The endoplasmic reticulum thus appears to be one of the targets of reactive intermediate-mediated damage. "
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    ABSTRACT: Prior induction of an endoplasmic reticulum stress response has been associated with an increased tolerance to cellular toxins in in vitro systems, primarily involving renal and neuronal cells. Reactive intermediates are involved in toxicity in many tissues, therefore, we wished to determine if cytoprotection after induction of an endoplasmic reticulum stress response was a general phenomenon in other cell types. A stress response was induced by tunicamycin in a human hepatocyte cell line (HepG2), a rat hepatocyte cell line (H4IIE), a porcine kidney cell line (LLC-PK1), and a human lymphocyte cell line (K562). Induction of the endoplasmic reticulum stress proteins GRP78, GRP94, calreticulin and protein disulfide isomerase was assessed by immunoblotting. Cytotoxicity was assessed 24 hr after a 3 hr exposure to iodoacetamide, tert-butylhydrogenperoxide, menadione, or sulfamethoxazole hydroxylamine, or after a 2 hr exposure to N-acetyl-p-benzoquinoneimine, the reactive metabolite of acetaminophen. Induction of endoplasmic reticulum stress proteins in LLC-PK1 cells resulted in a 2-6 times increase in the concentration of all the cytotoxins required to cause a 50% decrease in cell viability at 24 hr. In contrast, tunicamycin pretreatment only resulted in a 1.7-times increase for iodo-acetamide in HepG2 cells and a 2.2-times increase for N-acetyl-p-benzoquinoneimine in the H4IIE cells, but had no effect on the other toxins tested. Induction of endoplasmic reticulum stress proteins in K562 cells did not alter susceptibility to any toxins tested. Our results indicate that protection afforded by the induction of an endoplasmic reticulum stress response is dependent on the cell type and may be toxin specific. These results suggest that either the molecular pathways of cell death for individual toxins are different between cell types and toxins, or that the function of endoplasmic reticulum stress proteins are dependent on the cell type.
    Basic &amp Clinical Pharmacology &amp Toxicology 04/2004; 94(3):124-31. DOI:10.1111/j.1742-7843.2004.pto940305.x · 2.38 Impact Factor
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