Genetic association studies in drug-induced liver injury

A large number of case-control association studies on genetic susceptibility to drug-induced liver injury, involving both candidate gene and genome-wide association approaches, have now been reported. The strongest associations have been observed for human leukocyte antigen (HLA) class I and II genes and N-acetyltransferase 2 (NAT2). The associations with HLA class I and II genes are drug specific, though some apparently unrelated compounds show genetic associations with the same alleles. The underlying mechanism for the HLA association is likely to involve T-cell responses to either drug-protein adducts or to drug alone, but needs further investigation. The NAT2 association relates to liver injury induced by isoniazid, with most published studies finding an increased risk of injury in slow acetylators lacking NAT2 enzyme activity, presumably because of the accumulation of toxic metabolites. Other associations with genes relevant to drug disposition, innate immunity, oxidative stress, and mitochondrial function have also been reported, though these still need to be confirmed by replication in independent cohorts.     

儿童急性淋巴细胞性白血病人白细胞抗原表达及免疫功能探讨

目的 通过研究儿童急性淋巴细胞性白血病患者的白血病细胞及完全缓解后患者外周血人白细胞（HLA）Ⅰ类、Ⅱ类抗原表达情况,探讨儿童急性淋巴细胞性白血病发病机制及机体免疫状态.方法 采用流式细胞术检测儿童急性淋巴细胞性白血病初发患者42例、完全缓解期患者36例、对照组34例患者HLA-B、HLA-DQ、HLA-DR抗原表达.结果 （1）初发患者组的白血病细胞表面HLA-B抗原表达明显下调,初发患者组与完全缓解患者组或对照组比较,差异有显著意义（P＜0.01）.（2）初发患者组白血病细胞表面HLA-DQ、HLA-DR抗原表达明显升高,尤以HLA-DR升高更为明显,初发患者组与完全缓解组或对照组比较,差异有显著意义（P＜0.01）.结论 （1）急性淋巴细胞白血病人白血病细胞表面HLA-B抗原表达下调,可能是白血病细胞逃脱机体免疫监视的主要机制之一.（2）急性淋巴细胞白血病人白血病细胞表面HLA-DQ和HLA-DR抗原表达异常升高,尤其HLA-DR升高更为明显;使白血病细胞处于去分化状态,不能刺激机体产生免疫应答,可能也是白血病细胞逃脱机体免疫监视机制之一.     

Effect of endotoxin on the angiotensin II receptor in cultured vascular smooth muscle cells.

1. In some tissues, a decrease in the number of cell surface receptors and alterations of the receptor coupling have been proposed as possible mechanisms mediating the deleterious effects of bacterial endotoxin in septic shock. 2. The effects of bacterial lipopolysaccharide (Escherichia coli 0111-B4; LPS) on vascular angiotensin II and vasopressin receptors have been examined in cultured aortic smooth muscle cells (SMC) of the rat by use of radioligand binding techniques. 3. In vascular SMC exposed to 1 micrograms ml-1 endotoxin for 24 h, a significant increase in angiotensin II binding was found. The change in [125I]-angiotensin II binding corresponded to an increase in the number of receptors whereas the affinity of the receptors was not affected by LPS. In contrast, no change in [3H]-vasopressin binding was observed. 4. The pharmacological characterization of angiotensin II binding sites in control and LPS-exposed cells demonstrated that LPS induced an increase in the AT1 subtype of the angiotensin II receptors. Receptor coupling as evaluated by measuring total inositol phosphates was not impaired by LPS. 5. The effect of LPS on the angiotensin II receptor was dose-, time- and protein-synthesis dependent and was associated with an increased expression of the receptor gene. 6. The ability of LPS to increase angiotensin II binding in cultured vascular SMC was independent of the endotoxin induction of NO-synthase. 7. These results suggest that, besides inducing factors such as cytokines and NO-synthase, endotoxin may enhance the expression of cell surface receptors. The surprising increase in angiotensin II binding in LPS exposed VSM cells may represent an attempt by the cells to compensate for the decreased vascular responsiveness. It may also result from a non-specific LPS-related induction of genes.     

Effect of monoamine reuptake inhibiting antidepressants on major histocompatibility complex expression on macrophages in normal rats and rats with experimental allergic neuritis (EAN)

This study examined the modulation of IFN-γ induced MHC class I and II expression on normal Lewis rats and rats with EAN peritoneal macrophages cultured in the absence of presence of 10⁻⁴−10⁻⁸ M of the 5-HT reuptake inhibiting antidepressents zimeldine, and its metabolites norzimeldine and cpp200 oxalate as well as the antidepressants clomipramine and imipramine, in addition amitriptyline, nortriptyline and maprotiline inEAN rats. In normal rats, MHC class I expression was suppressed by the antidepressants zimeldine, norzimeldine and cpp200 oxalate at concentrations up to 10⁻⁵ M. At concentrations between 10⁻⁶ to 10⁻⁸ M, the same drugs significantly enhanced MHC class expression. Clomipramine at 10⁻⁸ M and imipramine at 10⁻⁶−10⁻⁷ M enhanced MHC class I expression, while the MHC class II expression was not significantly influenced by concentrations ≤ 10⁻⁵ M of these two drugs. In EAN rats, MHC class I expression was enhanced by zimeldine, cpp200, imipramine, and nortriptyline at 10⁻⁵−10⁻⁸ M, amitriptyline at 10⁻⁵−10⁻⁷ M as well as by norzimeldine and clomipramine at 10⁻⁶ M−10⁻⁸ M. However, maprotiline at 10⁻⁴−10⁻⁶ M suppressed class I expression in the presence of 0.5 U/ml and 1.0 U/ml of IFN-γ. MHC class II expression was suppressed by cpp200 and clomipramine at 10⁻⁴−10⁻⁵ M in presence of 0.5 U/ml of IFN-γ. At concentrations < 10⁻⁵ M most tested drugs significantly enhanced IFN-γ induced MHC class II expression. Compared to the results in normal rats, drug effects on EAN macrophages were more pronounced and reached higher levels of significance. The 5-HT reuptake inhibiting antidepressants also exerted a modulatory effect on MHC class I and II in EAN rat macrophages even in the absence of IFN-γ. The modulatory effect of antidepressant drugs on IFN-γ induced MHC class I and II expression may contribute to their influence on demyelinating autoimmune diseases, and may have implications for their clinical use.     

Glucocorticoids inhibit lipopolysaccharide-induced up-regulation of arginase in rat alveolar macrophages

1. As arginase by limiting nitric oxide (NO) synthesis may play a role in airway hyperresponsiveness and glucocorticoids are known to induce the expression of arginase I in hepatic cells, glucocorticoid effects on arginase in alveolar macrophages (AM Phi) were studied. 2. Rat AM Phi were cultured in absence or presence of test substances. Thereafter, nitrite accumulation, arginase activity, and the expression pattern of inducible NO synthase, arginase I and II mRNA (RT - PCR) and proteins (immunoblotting) were determined. 3. Lipopolyssacharides (LPS, 20 h) caused an about 2 fold increase in arginase activity, whereas interferon-gamma (IFN-gamma), like LPS a strong inducer of NO synthesis, had no effect. 4. Dexamethasone decreased arginase activity by about 25% and prevented the LPS-induced increase. Mifepristone (RU-486) as partial glucocorticoid receptor agonist inhibited LPS-induced increase by 45% and antagonized the inhibitory effect of dexamethasone. 5. Two different inhibitors of the NF-kappa B-pathway also prevented LPS-induced increase in arginase activity. 6. Rat AM Phi expressed mRNA and protein of arginase I and II, but arginase I expression was stronger. Arginase I mRNA and protein was not affected by IFN-gamma, but increased by LPS and this effect was prevented by dexamethasone. Both, LPS and IFN-gamma enhanced the levels of arginase II mRNA and protein, effects also inhibited by dexamethasone. As IFN-gamma did not affect total arginase activity, arginase II may represent only a minor fraction of total arginase activity. 7. In rat AM Phi glucocorticoids inhibit LPS-induced up-regulation of arginase activity, an effect which may contribute to the beneficial effects of glucocorticoids in the treatment of inflammatory airway diseases.     

Human leukocyte antigen typing, response to neuroleptics, and clozapine-induced agranulocytosis in jewish Israeli schizophrenic patients.

The atypical antipsychotic agent clozapine is known to be effective in schizophrenic patients refractory to other medications; however, it induces agranulocytosis in approximately 1-2%. In Jews, this complication is associated with the haplotype HLA B38,DR4,DQ3. The aim of the present study was to determine which human leukocyte antigen (HLA) antigens are involved in clozapine-induced agranulocytosis. We performed HLA typing in 88 Jewish Israeli schizophrenic patients and in 127 ethnically matched healthy individuals. Thirty-eight patients responsive to standard antipsychotic medications were treated with haloperidol, and 50 refractory patients received clozapine. A trend was noted for elevated rates of HLA B38 among control individuals and clozapine-treated patients of Ashkenazi origin compared to individuals of non-Ashkenazi origin, but the findings failed to reach statistical significance. No association was found between HLA class I antigens and the response to haloperidol or clozapine. Neutropenia developed in two clozapine-treated patients and agranulocytosis in one. Two of these three patients were of Ashkenazi origin, and both demonstrated the HLA B38 phenotype. Although the findings did not reach a statistical significance because of the small number of patients, they may support an association between clozapine-induced neutropenia/agranulocytosis and Ashkenazi origin and the HLA B38 phenotype. The rate of agranulocytosis in our sample (2%) is similar to the usual cumulative risk of agranulocytosis but in contrast to its high frequency among Jewish American patients. One possible explanation for this difference is the high rate of Ashkenazi patients in the American sample and the preponderance of non-Ashkenazi patients in our population.     

Post-UV Survival and Mutagenesis in DNA Repair-proficient and -deficient Strains of Escherichia coli K-12 Grown in 5-Azacytidine to Inhibit DNA Cytosine Methylation: Evidence for Mutagenic Excision Repair

Abstract— Inhibition of cytosine methylation by growth in 5-azacytidine (5-azaC), did not affect the sensitivities to DNA damage induced by exposure to ultraviolet light (UV) of Escherichia coli K-12 strains AB1157 dcm⁺, which is fully DNA repair-proficient, LR68 (a dcm derivative of ABU57), JC3890 dcm⁺ uvrB, deficient in error-free excision repair, TK702 dcm⁺ umuC, deficient in error-prone repair, or TK501 dcm⁺ uvrB umuC, which lacks both excision repair and error-prone repair. However, growth in 5-azaC increased the post-UV survival of strains AB2463 recA(Def), AB2470 recB and AB2494 lexA(Ind^?), which are deficient in the induction or expression of recombination repair or error-prone repair of DNA. Spontaneous mutation frequencies were increased in strains LR68, AB2463, AB2470 and AB2494 by growth in 5-azaC, but remained unaltered in strains AB1157, JC3890, TK702 or TK501. Growth in 5-azaC significantly increased UV-induced mutation frequencies in strains AB2463 and AB2470, significantly reduced UV-induced mutation in strain JC3890, but had little effect on UV-induced mutation in the other strains. The results suggest that 5-azaC may induce a normally error-free DNA repair pathway to become error-prone and therefore genotoxic.     

Macrophage function during the development of adjuvant disease

Using rat peritoneal macrophages and blood monocytes we examined the relationship of developing adjuvant disease (AD) with the expression of class I and II antigens, release of interleukin-1 (IL-1) and production of prostaglandin E2 (PGE2). We observed that class I and II antigens initially decreased; class II remained low throughout, whereas class I returned to normal. PGE2 and IL-1 gradually increased. Treatment in vivo with the NSAID Na-diclofenac lowered PGE2 and IL-1 release and partly reversed the observed reduction of class I and II antigens. Also, exposure of the cells in vitro to lipopolysaccharide increased class I antigen expression.     

Identification of a new anti-LPS agent,geniposide, from Gardenia jasminoides Ellis,and its ability of direct binding and neutralization of lipopolysaccharide in vitro and in vivo

Lipopolysaccharide (LPS/endotoxin) is a key pathogen recognition molecule for sepsis. Currently, one of the therapeutic approaches for severe sepsis is focusing on the neutralization of LPS, and clinical trials have shown a lot of traditional Chinese herbs possess anti-sepsis function. Herein, to elucidate the bioactive components of traditional Chinese herbs that can neutralize LPS, the lipid A-binding abilities of sixty herbs were tested using affinity biosensor technology. The aqueous extract of Gardenia jasminoides Ellis, traditionally used to treat inflammation in Asian countries for centuries, was further investigated. Subsequently, a monomer, identified as geniposide, was isolated. In vitro, geniposide was found to directly bind LPS and neutralize LPS. It dose-dependently inhibited cytokines release from RAW264.7 cells induced by LPS without affecting the cell viability, and inhibited TNF-α mRNA expression up-regulated by LPS. However, geniposide did not decrease TNF-α release induced by CpG DNA, Poly I:C or IL-1β. Significantly, geniposide dose-dependently down-regulated TLR4 mRNA expression up-regulated by LPS, and suppressed the phosphorylations of p38 MAKP induced by LPS but not by IL-1β. In vivo, geniposide (40 mg/kg) could significantly protect mice challenge with lethal heat-killed E. coli, and dose-dependently decreased the level of serum endotoxin which was tightly associated with the cytokine levels in endotoxemia mice. In summary, we successfully isolated geniposide from G. jasminoides Ellis. Geniposide directly bound LPS and neutralized LPS in vitro, and significantly protected sepsis model mice. Therefore, geniposide could be as a useful lead compound for anti-sepsis drug development.     

Cancer vaccines,a critical review--Part I

Cancer vaccines are more properly referred to as 'active specific immunotherapy', and are used to treat cancers rather than to prevent them, at least at present. Vaccines augment already established tumor immunity, are far more specific against the tumor than cytokines, have little or no toxicity, and thus may easily be combined with other types of immunotherapy. They also elicit immunological memory, which may check recurrence of the tumor. Melanoma vaccines have received the most attention thus far. Among the several vaccines in clinical trials are whole cell lysates, such as Melacine, hapten-treated autologous melanoma cells (M-Vax) and irradiated allogeneic cells (CancerVax). Regressions of metastatic nodules have been noted with each preparation. Controlled trials of Melacine indicate prolongation of survival in patients with resected stage IIB disease, particularly those with one or more of the following HLA class I alleles: HLA-A2 or -A28 (-A6802), HLA-B12, -44 or -45, and HLA-C3. A combination of interferon-alpha2b and Melacine appears to enhance the anti-tumor response in advanced (stage IV) disease, and is being tested in a large randomized controlled trial in resected stage III disease. An irradiated autologous colon carcinoma vaccine has improved relapse-free survival in resected stage II disease (Dukes B) in a controlled trial. Second-generation whole cell vaccines include those incorporating genes such as GM-CSF or CD80 (B7-1) to improve immunogenicity, and the use of immunogenic cell membranes such as large multivalent immunogen (LMI). Upregulation of HLA class II molecules and concomitant inhibition of the Ii molecule are also being explored as a strategyfor improved presentation of tumor-associated antigens in vaccines. Complex whole cell-derived vaccines have given clinically superior responses compared to vaccines containing well-defined antigens, such as peptides or gangliosides; however, well-defined vaccines are theoretically more desirable because of their reproducibility.     

Inhibitory effects of a new iridoid, patridoid II and its isomers, on nitric oxide and TNF-alpha production in cultured murine macrophages

Patridoids I, II and IIA, are new iridoids isolated from the whole plants of Patrinia saniculaefolia. These compounds were examined by assessing their effects on the production of tumor necrosis factor-alpha (TNF-alpha) as well as by investigating the expression of two enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), in the lipopolysaccharide (LPS)-stimulated murine macrophage-like cell line, Raw 264.7. Among them, patridoid II consistently inhibited nitric oxide (NO) and TNF-alpha production in a dose-dependent manner, with IC (50) values of 14.1 and 17.6 microM, respectively. Western Blotting probed with specific anti-iNOS antibodies showed that the decrease in the quantity of the NO product was accompanied by a decrease in the iNOS protein level. However, all three patridoids did not affect the COX-2 protein expression level in the LPS-stimulated macrophages. In addition, the C-5 isomer of patridoid II, patridoid I, only slightly affected the production of NO. Moreover, the C-3 isomer of patridoid II, patridoid IIA, did not inhibit proinflammatory cytokines and NO production. These results suggest that the orientations of the C-3 and C-5 methoxy groups in the patridoids have a significant role in the expression of their biological activity.     

Suppression of MyD88- and TRIF-dependent signaling pathways of Toll-like receptor by (-)-epigallocatechin-3-gallate, a polyphenol component of green tea

Toll-like receptors (TLRs) play an important role in recognition of microbial components and induction of innate immunity. The microbial components trigger the activation of two downstream signaling pathways of TLRs; MyD88- and/or TRIF-dependent pathways leading to activation of NF-kappaB. (-)-Epigallocatechin-3-gallate (EGCG), a flavonoid found in green tea, is known to inhibit NF-kappaB activation induced by many pro-inflammatory stimuli. EGCG was shown to inhibit the activity of IKKbeta which is the key kinase in the canonical pathway for NF-kappaB activation in MyD88-dependent pathway of TLRs. However, it is not known whether EGCG inhibits TRIF-dependent pathway through which more than 70% of lipopolysaccharide (LPS)-induced genes are regulated. Therefore, we attempted to identify the molecular target of EGCG in TRIF-dependent pathways of TLR3 and TLR4. EGCG inhibited the activation of IFN regulatory factor 3 (IRF3) induced by LPS, poly[I:C], or the overexpression of TRIF. The inhibition of IRF3 activation by EGCG was mediated through the suppression of the kinase activity of TBK1. However, EGCG did not inhibit activation of IRF3 induced by overexpression of constitutively active IRF3. These results suggest that the molecular target of EGCG is TBK1 in TRIF-dependent signaling pathways of TLR3 and TLR4. Therefore, our results suggest that green tea flavonoids can modulate both MyD88- and TRIF-dependent signaling pathways of TLRs and subsequent inflammatory target gene expression.     

Induction of arginase II in livers of bile duct-ligated rats

Nitric oxide (NO) has been implicated in playing a role in liver cirrhosis, but the regulatory mechanisms are still unclear. As arginase shares a common substrate with NO synthase (NOS), the aim of this study was to investigate the expression of arginase I and II in cirrhotic liver. Liver cirrhosis was induced in rats by chronic bile duct ligation (BDL). Controls were sham-operated. Competitive polymerase chain reaction was performed to assay the expression of messenger RNA of arginase I and II. Protein expression was detected by immunohistochemistry and western-blotting. The level of arginine in plasma was lower in BDL rats, while the ornithine level in plasma was correspondingly higher (r= -0.96, P<0.0001). Arginase I messenger RNA was reduced significantly in BDL rats (3.34+/-0.32 vs. 1.32+/-0.21 x 10(4) attomole/microg of total RNA, sham vs. BDL, P<0.001), as well as arginase I protein. In contrast, arginase II mRNA was induced in the livers of BDL rats, with negligible expression in controls (0.35+/-0.11 vs. 3.64+/-0.54 attomole/microg of total RNA, sham vs. BDL, P<0.001). Arginase II protein was localized in some hepatocytes and hyperplastic bile ductular epithelial cells of cirrhotic livers but not in control livers. In conclusion, arginase II was induced in BDL livers, while the expression of arginase I was down-regulated. These data suggest that arginase I and II are regulated differently and may have different functions in the livers of BDL rats. Reduction of arginase I in BDL livers may be responsible for the lowering of arginine levels in the plasma, while induction of arginase II could be important in regulating NO synthesis as well as other important mechanisms involved in liver cirrhosis.     

Role of nitric oxide in downregulation of cytochrome P450 1a1 and NADPH: Quinone oxidoreductase 1 by tumor necrosis factor-alpha and lipopolysaccharide

We previously demonstrated that tumor necrosis factor alpha (TNF-alpha) and lipopolysaccharide (LPS) downregulate aryl hydrocarbon receptor (AhR)-regulated genes, such as cytochrome P450 1a1 (Cyp1a1) and NADPH: quinone oxidoreductase 1 (Nqo1) gene expression, yet the mechanisms involved remain unknown. The correlation between the inflammation-mediated suppression of AhR-regulated genes and the TNF-alpha or LPS-induced nitric oxide (NO) production especially in murine hepatoma Hepa 1c1c7 cells has been questioned; therefore we investigated whether NO is involved in the modulation of Cyp1a1 and Nqo1 by TNF-alpha or LPS in Hepa 1c1c7 cells. A significant dose-dependent increase in the inducible nitric oxide synthase (NOS2) expression and NO production were observed by various concentrations of TNF-alpha (1, 5, and 10 ng/mL) and LPS (1 and 5 microg/mL) which was completely inhibited by a NOS2 inhibitor, L-N6-(1-iminoethyl) lysine (L-NIL) (1 mM). Furthermore, TNF-alpha and LPS significantly induced NOS2 expression. Both TNF-alpha and LPS repressed the beta-naphthoflavone (betaNF)-mediated induction of Cyp1a1 and Nqo1 at mRNA and activity levels. The downregulation of Cyp1a1, but not Nqo1, was significantly prevented by L-NIL. However, proxynitrite decomposer, iron tetrakis (N-methyl-4'-pyridyl) porphyrinato (FeTMPyP) (5 microM) did not affect TNF-alpha- and LPS-mediated downregulation of Cyp1a1 and Nqo1 at mRNA and activity levels. These results show that NO, but not peroxynitrite, may be involved in TNF-alpha- and LPS-mediated downregulation of Cyp1a1 without affecting the downregulation of Nqo1.     

An in vitro test to screen skin sensitizers using a stable THP-1-derived IL-8 reporter cell line, THP-G8

Several studies have suggested that interleukin (IL)-8 can serve as a biomarker for discrimination of skin sensitizers from nonsensitizers. We established a stable THP-1-derived IL-8 reporter cell line, THP-G8, which harbors SLO and SLR luciferase genes under the control of IL-8 and glyceraldehyde 3-phosphate dehydrogenase promoters, respectively. After 6 h treatment with chemicals, normalized SLO luciferase activity (nSLO-LA) was calculated by dividing SLO-LA by SLR-LA, and the fold induction of nSLO-LA (FInSLO-LA) was calculated by dividing nSLO-LA of chemically treated cells by that of nontreated cells. The nSLO-LA of THP-G8 cells increased in response to lipopolysaccharide (LPS) and several sensitizers. The FInSLO-LA in THP-G8 cells induced by LPS or sensitizers positively correlated with their induction of IL-8 messenger RNA in THP-1 cells. The nSLO-LA value of THP-G8 cells was significantly increased (FInSLO-LA ≥ 1.4) by 13 of the 15 sensitizers as well as by 5 of the 7 nonsensitizers. Interestingly, pretreatment with N-acetylcysteine suppressed the increase in FInSLO-LA induced by all sensitizers (inhibition index (II) ≤ 0.8) but did not suppress that induced by most of the nonsensitizers. We then evaluated the performance of this assay using values of FInSLO-LA ≥ 1.4 and II ≤ 0.8 in at least two of three independent experiments as the criteria of a sensitizer, which resulted in test accuracies of 82% for the 22 chemicals used and of 88% for the chemicals proposed by European Center for the Validation of Alternative Methods. This newly developed assay is a candidate replacement for animal tests of skin sensitization because of its accuracy, convenience, and high throughput performance.     

粒细胞—巨噬细胞集落刺激因子和干扰素—γ诱导脐血嗜碱粒细胞表达人类白细胞抗原Ⅱ类分子

目的:探讨嗜碱粒细胞是否可表达人类白细胞抗原(HLA)Ⅱ类分子.方法:采用密度梯度离心和免疫磁珠方法分离和纯化脐血嗜碱粒细胞,通过体外培养及粒细胞-巨噬细胞集落刺激因子(GM-CSF)和干扰素(IFN)-γ刺激20-60小时后,用流式细胞仪分析 HLA Ⅱ类分子表达的情况.结果:嗜碱粒细胞的纯度 ≥83.5％,在 GM-CSF10 μg/L或 IFN-γ100kU/L刺激后,其膜表面可表达HLA Ⅱ类分子,表达百分率在刺激后20小时较高,分别为10.2％±2.1％和11.3％±1.0％.结论:嗜碱粒细胞具有表达HLA Ⅱ类分子的潜在能力.