Concomitant occurence of multiple autoantibodies against human cytochromes P450.

Cytochromes P450 (CYPs) are a large superfamily of heme-containing enzymes that are essential for the metabolism of a variety of endogenous and xenobiotic compounds. The role and the possible diagnostic or prognostic value of the occurrence of anti-CYP autoantibodies (aAbs) in cancer patients are essentially unclear. Recently we reported the monitoring of aAbs against CYP4Z1 and CYP19A1 in breast cancer patients and healthy controls. In the present study, we extended this investigation by screening the sera of 47 lung cancer patients (17 female and 30 male; age range 49-84) and 119 healthy controls (60 female and 59 male; age range 21-72) for the presence of aAbs directed against CYP2D6, CYP4Z1, or CYP17A1, respectively. Determination of anti-CYP aAb levels was done using our previously established ELISA method. Most sera gave low signals while a small fraction showed stronger responses; however, there were no statistically significant differences between the different test groups. Also, there was no significant difference in aAb signals between the various subtypes of lung cancer. Unexpectedly, sera from two female lung cancer patients (age 67 (adenocarcinoma) and 70 (small cell carcinoma)) and from four healthy controls (one female and three male; age range 34-48) showed significantly elevated signals for more than one of the three CYPs tested. These findings corroborate earlier reports that anti-CYP aAbs occur with low frequency in the general population and, moreover, suggest that the simultaneous presence of multiple aAbs targeting different CYPs should be taken into consideration when evaluating anti-CYP aAbs as biomarkers.

Effects of adenovirus-induced hepatocyte damage on chronic bile duct inflammation in a sclerosing cholangitis mouse model.

BACKGROUND & AIMS: Four major autoimmune diseases target the liver. They develop because of bile duct destruction, leading to chronic cholestasis or result from hepatocyte damage like autoimmune hepatitis (AIH). Interestingly, some patients simultaneously show features of both cholangitis and AIH. Our goal was to mimic such concurrent characteristics in a mouse model that would help deciphering mechanisms possibly involved in an inflammatory crosstalk between cholestatic disease and hepatitis. METHODS: Mdr2-/- mice, which spontaneously develop sclerosing cholangitis because of accumulation of toxic bile salts, were infected with adenovirus (Ad) encoding human Cytochrome P4502D6 (hCYP2D6), the major target autoantigen in type-2 AIH, to trigger hepatocyte injury. Wild type FVB mice were controls. RESULTS: Resulting Ad-Mdr2-/- mice presented with cholangitis, fibrosis and cellular infiltrations that were higher than in Mdr2-/- or Ad-FVB mice. Increased levels of anti-neutrophil cytoplasmic antibodies but similar anti-hCYP2D6 antibody titres were detected in Ad-Mdr2-/- compared to Mdr2-/- and Ad-FVB mice respectively. IFNγ-expressing hCYP2D6-specific CD4 T cells declined, whereas hCYP2D6-specific CD8 T cells increased in Ad-Mdr2-/- compared to Ad-FVB mice. The overall T cell balance in Ad-Mdr2-/- mice was a combination of a type 17 T cell response typically found in Mdr2-/- mice with a type 1 dominated T cell response characteristic for Ad-FVB mice. Simultaneously, the type 2 T cell compartment was markedly reduced. CONCLUSIONS: Experimental hepatitis induction in a mouse with sclerosing cholangitis results in a disorder which represents not simply the sum of the individual characteristics but depicts a more complex entity which urges on further analysis.

Non-alcoholic fatty liver disease (NAFLD) potentiates autoimmune hepatitis in the CYP2D6 mouse model.

Non-alcoholic fatty liver disease (NAFLD) and its more severe development non-alcoholic steatohepatitis (NASH) are increasing worldwide. In particular NASH, which is characterized by an active hepatic inflammation, has often severe consequences including progressive fibrosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). Here we investigated how metabolic liver injury is influencing the pathogenesis of autoimmune hepatitis (AIH). We used the CYP2D6 mouse model in which wild type C57BL/6 mice are infected with an Adenovirus expressing the major liver autoantigen cytochrome P450 2D6 (CYP2D6). Such mice display several features of human AIH, including interface hepatitis, formation of LKM-1 antibodies and CYP2D6-specific T cells, as well as hepatic fibrosis. NAFLD was induced with a high-fat diet (HFD). We found that pre-existing NAFLD potentiates the severity of AIH. Mice fed for 12 weeks with a HFD displayed increased cellular infiltration of the liver, enhanced hepatic fibrosis and elevated numbers of liver autoantigen-specific T cells. Our data suggest that a pre-existing metabolic liver injury constitutes an additional risk for the severity of an autoimmune condition of the liver, such as AIH.

Retinoic acid stabilizes antigen-specific regulatory T-cell function in autoimmune hepatitis type 2.

Imbalance between effector and regulatory T-cells (Treg) underlies the loss of immune-tolerance to self-antigens in autoimmune disease. In autoimmune hepatitis type 2 (AIH-2), effector CD4 T-cell immune responses to cytochrome P450IID6 (CYP2D6) are permitted by numerically and functionally impaired Treg. Restoration of CYP2D6-specific Treg in AIH-2 would enable control over effectors sharing the same antigen specificity, leading to re-establishment of immune-tolerance. We have previously developed a protocol for generating antigen-specific Treg through co-culture with semi-mature dendritic cells presenting CYP2D6 peptides. In this study, we aimed to explore phenotypic and functional features of patient Treg compared to health, to test Treg stability under pro-inflammatory conditions, and to investigate the potential benefit of supplementation with all-trans-retinoic acid (RA) or rapamycin (RP), agents proven to enhance Treg function. We show that antigen-specific Treg from patients have comparable phenotypic and functional features to those from healthy controls, suppressing both proliferation and pro-inflammatory cytokine production by effector cells. Treg exposure to inflammatory challenge results in decreased suppressive function and up-regulation of Th1/Th2/Th17 transcription factors both in health and AIH-2. The increase of Th1 and Th17 transcription factors is limited by addition of RA in controls and Th1 expression is decreased by RP in patients. Importantly, inflammation-induced decrease in Treg function is also abrogated by RA/RP in health and RA in patients. Our data provide important information for the optimization of protocols aiming at generating antigen-specific Treg for treatment of autoimmune disease and for understanding their biology upon pro-inflammatory challenge and RP/RA supplementation.

Mechanism of autoimmune hepatic fibrogenesis induced by an adenovirus encoding the human liver autoantigen cytochrome P450 2D6.

Autoimmune hepatitis type 2 (AIH-2) is a severe autoimmune liver disease with unknown etiology. We recently developed the CYP2D6 mouse model for AIH-2, in which mice are challenged with an adenovirus (Ad-2D6) expressing human cytochrome P450 2D6 (hCYP2D6), the major autoantigen in AIH-2. Such mice develop chronic hepatitis with cellular infiltrations and generation of hCYP2D6-specific antibodies and T cells. Importantly, the CYP2D6 model represents the only model displaying chronic fibrosis allowing for a detailed investigation of the mechanisms of chronic autoimmune-mediated liver fibrogenesis. We found that hCYP2D6-dependent chronic activation of hepatic stellate cells (HSC) resulted in an increased extracellular matrix deposition and elevated expression of α-smooth muscle actin predominantly in and underneath the liver capsule. The route of Ad-2D6 infection dramatically influenced the activation and trafficking of inflammatory monocytes, NK cells and hCYP2D6-specific T cells. Intraperitoneal Ad-2D6 infection caused subcapsular fibrosis and persistent clustering of inflammatory monocytes. In contrast, intravenous infection caused an accumulation of hCYP2D6-specific CD4 T cells throughout the liver parenchyma and induced a strong NK cell response preventing chronic HSC activation and fibrosis. In summary, we found that the location of the initial site of inflammation and autoantigen expression caused a differential cellular trafficking and activation and thereby determined the outcome of AIH-2-like hepatic damage and fibrosis.

Molecular mimicry rather than identity breaks T-cell tolerance in the CYP2D6 mouse model for human autoimmune hepatitis.

In our novel mouse model for autoimmune hepatitis (AIH), wildtype FVB mice infected with an Adenovirus (Ad) expressing the major AIH autoantigen human cytochrome P450 2D6 (hCYP2D6) show persistent histological and immunological features associated with AIH, including the generation of anti-hCYP2D6 antibodies with an epitope specificity identical to LKM-1 autoantibodies in AIH-patients. Since FVB mice do not express hCYP2D6, the immune response was directed against mouse CYP (mCYP) homologues. Additional expression of hCYP2D6 in transgenic mice resulted in amelioration of the liver disease. In the present study we used the CYP2D6 model to assess why tolerance breakdown and induction of autoimmune liver disease is more efficient if the triggering antigen is similar but not identical to the target autoantigen. We found that in contrast to the specificity and magnitude of anti-hCYP2D6 antibody responses, T-cell responses differ profoundly between wildtype and transgenic mice. Detailed T-cell epitope mapping studies show a robust, antigen-specific T-cell reactivity in FVB mice largely directed against one CD4 and three CD8 epitopes, activating a total of approximately 1% CD4 and 10% CD8 T-cells, respectively, while infected hCYP2D6 mice generated almost no hCYP2D6-specific T-cells. The frequency of hCYP2D6-specific T-cells was approximately 3-fold higher in the liver compared with the spleen. Amino acid sequence comparison revealed that the immunodominant epitopes were located in hCYP2D6-segments of intermediate homology between hCYP2D6 and its mCYP homologues. Our data indicate that self/non-self molecular mimicry, rather than molecular identity, is a prerequisite for breaking T-cell tolerance in the liver.

The CYP2D6 animal model: how to induce autoimmune hepatitis in mice.

Autoimmune hepatitis is a rare but life threatening autoimmune disease of the liver of unknown etiology(1,2). In the past many attempts have been made to generate an animal model that reflects the characteristics of the human disease (3-5). However, in various models the induction of disease was rather complex and often hepatitis was only transient(3-5). Therefore, we have developed a straightforward mouse model that uses the major human autoantigen in type 2 autoimmune hepatitis (AIH-2), namely hCYP2D6, as a trigger(6). Type 1 liver-kidney microsomal antibodies (LKM-1) antibodies recognizing hCYP2D6 are the hallmark of AIH-2(7,8). Delivery of hCYP2D6 into wildtype FVB or C57BL/6 mice was by an Adenovirus construct (Ad-2D6) that ensures a direct delivery of the triggering antigen to the liver. Thus, the ensuing local inflammation generates a fertile field(9) for the subsequent development of autoimmunity. A combination of intravenous and intraperitoneal injection of Ad-2D6 is the most effective route to induce a long-lasting autoimmune damage to the liver (section 1). Here we provide a detailed protocol on how autoimmune liver disease is induced in the CYP2D6 model and how the different aspects of liver damage can be assessed. First, the serum levels of markers indicating hepatocyte destruction, such as aminotransferases, as well as the titers of hCYP2D6 antibodies are determined by sampling blood retroorbitaly (section 2). Second, the hCYP2D6-specific T cell response is characterized by collecting lymphocytes from the spleen and the liver. In order to obtain pure liver lymphocytes, the livers are perfused by PBS via the portal vein (section 3), digested in collagen and purified over a Percoll gradient (section 4). The frequency of hCYP2D6-specific T cells is analyzed by stimulation with hCYP2D6 peptides and identification of IFNγ-producing cells by flow cytometry (section 5). Third, cellular infiltration and fibrosis is determined by immunohistochemistry of liver sections (section 6). Such analysis regimen has to be conducted at several times after initiation of the disease in order to prove the chronic nature of the model. The magnitude of the immune response characterized by the frequency and activity of hCYP2D6-specific T and/or B cells and the degree of the liver damage and fibrosis have to be assessed for a subsequent evaluation of possible treatments to prevent, delay or abrogate the autodestructive process of the liver.

Different sites of xenoantigen delivery lead to a virally induced late-onset hepatitis in mice through molecular mimicry.

BACKGROUND: Epidemiological and laboratory evidences led to the hypothesis that molecular mimicry between viruses and self-proteins could be linked to the onset of autoimmune hepatitis (AIH). Hepatotropic viruses could be good candidates, as a pro-inflammatory environment may facilitate the development of AIH. AIMS: The aims of this study were to test a virus ability to induce an AIH through molecular mimicry and the influence of hepatic inflammation in this process. METHODS: C57BL/6 mice were injected i.v. or i.m. with recombinant adenoviral vectors (RecAdV) encoding for human type 2 AIH antigens to target xenoantigens expression in the liver and to create a transient hepatitis (i.v.) or for 'peripheral' xenoantigens expression (i.m.). Liver injury and B-cell response were evaluated. RESULTS: Late-onset hepatitis was observed 8 months after i.v. or i.m. RecAdV injections, despite presence or absence of an initial transient hepatitis. Intensity of B-cell response was similar for both type of injections, but the Ig isotypes produced were different. B-cell autoimmune response spread to several liver proteins. CONCLUSIONS: Liver autoimmune response can be initiated using molecular mimicry over a long period of time, validating the hit-and-run hypothesis. Initial liver inflammatory injury is neither necessary, nor detrimental to the development of AIH. These results highlight the significance of initial events on the pathogenesis of autoimmune liver injury.

Epitope spreading of the anti-CYP2D6 antibody response in patients with autoimmune hepatitis and in the CYP2D6 mouse model.

Autoimmune hepatitis (AIH) is a serious chronic inflammatory disease of the liver with yet unknown etiology and largely uncertain immunopathology. The hallmark of type 2 AIH is the generation of liver kidney microsomal-1 (LKM-1) autoantibodies, which predominantly react to cytochrome P450 2D6 (CYP2D6). The identification of disease initiating factors has been hampered in the past, since antibody epitope mapping was mostly performed using serum samples collected late during disease resulting in the identification of immunodominant epitopes not necessarily representing those involved in disease initiation. In order to identify possible environmental triggers for AIH, we analyzed for the first time the spreading of the anti-CYP2D6 antibody response over a prolonged period of time in AIH patients and in the CYP2D6 mouse model, in which mice infected with Adenovirus-human CYP2D6 (Ad-h2D6) develop antibodies with a similar specificity than AIH patients. Epitope spreading was analyzed in six AIH-2-patients and in the CYP2D6 mouse model using SPOTs membranes containing peptides covering the entire CYP2D6 protein. Despite of a considerable variation, both mice and AIH patients largely focus their humoral immune response on an immunodominant epitope early after infection (mice) or diagnosis (patients). The CYP2D6 mouse model revealed that epitope spreading is initiated at the immunodominant epitope and later expands to neighboring and remote regions. Sequence homologies to human pathogens have been detected for all identified epitopes. Our study demonstrates that epitope spreading does indeed occur during the pathogenesis of AIH and supports the concept of molecular mimicry as a possible initiating mechanism for AIH.

Primary biliary cirrhosis and autoimmune hepatitis: apotopes and epitopes.

Autoimmune liver diseases (ALDs) represent a wide spectrum of chronic inflammatory diseases that are characterized by an immune-mediated attack against either hepatocytes (in the case of autoimmune hepatitis types 1 and 2, AIH-1, 2) or cholangiocytes (in primary biliary cirrhosis, PBC). PBC is considered a model autoimmune disease due to the homogeneity of patients, the high specificity of antimitochondrial antibodies (AMAs), and the specificity of biliary epithelial cell (BEC) destruction. It ensues from a multi-lineage loss of tolerance to the E2 component of the pyruvate dehydrogenase complex (PDC-E2). One of the major unanswered questions in the pathogenesis of PBC is the specificity of small intrahepatic bile duct attack while PDC-E2 is present in mitochondria of nucleated cells. Recent findings suggest that the apoptosis of BECs may be of considerable importance for understanding PBC, and that they are more than simply an innocent victim of an immune attack. Rather, they attract immune attack by virtue of the unique biochemical mechanisms by which they handle PDC-E2. The role of apoptotic cells in AIH is not well defined, but advances in the study of autoreactive T cells stem mostly from AIH type 2, where the main autoantigen (CYP2D6) is known, enabling the characterization of antigen-specific immune responses. This review article is intended to provide a critical overview of current evidence on tissue specificity in ALDs, as well as the characteristics of the relevant epitopes and apotopes and their biological and clinical significance.

Cytochrome P450 2D6 as a model antigen.

Cytochrome P450 2D6 (CYP2D6) has been identified as the major autoantigen in type 2 autoimmune hepatitis (AIH). However, because of a lack of appropriate animal models, the etiology of AIH is still poorly understood. We generated a mouse model for AIH using the human CYP2D6 as a triggering molecule for autoimmunity. We infected wild-type FVB mice with an adenovirus expressing human CYP2D6 (Ad-2D6) to break self-tolerance to the mouse CYP2D6 homologues. Ad-2D6-infected mice showed persistent features of liver damage including hepatic fibrosis, cellular infiltrations, focal-to-confluent necrosis and generation of anti-CYP2D6 antibodies, which predominantly recognized the identical immunodominant epitope recognized by LKM-1 antibodies from AIH patients. Interestingly, Ad-2D6 infection of transgenic mice expressing the human CYP2D6 (CYP2D6 mice) resulted in delayed kinetics and reduced severity of liver damage. However, the quantity and quality of anti-CYP2D6 antibodies was only moderately reduced in CYP2D6 mice. In contrast, the frequency of CYP2D6-specific CD4 and CD8 T cells was dramatically decreased in CYP2D6 mice, indicating the presence of a strong T cell tolerance to human CYP2D6 established in CYP2D6 mice, but not in wild-type mice. CYP2D6-specific T cells reacted to human CYP2D6 peptides with intermediate homology to the mouse homologues, but not to those with high homology, indicating that molecular mimicry rather than molecular identity breaks tolerance and subsequently causes severe persistent autoimmune liver damage. The CYP2D6 model provides a platform to investigate mechanisms involved in the immunopathogenesis of autoimmune-mediated chronic hepatic injury and evaluate possible ways of therapeutic interference.

Breaking tolerance to the natural human liver autoantigen cytochrome P450 2D6 by virus infection.

Autoimmune liver diseases, such as autoimmune hepatitis (AIH) and primary biliary cirrhosis, often have severe consequences for the patient. Because of a lack of appropriate animal models, not much is known about their potential viral etiology. Infection by liver-tropic viruses is one possibility for the breakdown of self-tolerance. Therefore, we infected mice with adenovirus Ad5 expressing human cytochrome P450 2D6 (Ad-2D6). Ad-2D6-infected mice developed persistent autoimmune liver disease, apparent by cellular infiltration, hepatic fibrosis, "fused" liver lobules, and necrosis. Similar to type 2 AIH patients, Ad-2D6-infected mice generated type 1 liver kidney microsomal-like antibodies recognizing the immunodominant epitope WDPAQPPRD of cytochrome P450 2D6 (CYP2D6). Interestingly, Ad-2D6-infected wild-type FVB/N mice displayed exacerbated liver damage when compared with transgenic mice expressing the identical human CYP2D6 protein in the liver, indicating the presence of a stronger immunological tolerance in CYP2D6 mice. We demonstrate for the first time that infection with a virus expressing a natural human autoantigen breaks tolerance, resulting in a chronic form of severe, autoimmune liver damage. Our novel model system should be instrumental for studying mechanisms involved in the initiation, propagation, and precipitation of virus-induced autoimmune liver diseases.

Cytochrome P450IID6-specific CD8 T cell immune responses mirror disease activity in autoimmune hepatitis type 2.

UNLABELLED: Autoimmune hepatitis type 2 (AIH-2) is a severe organ-specific disorder characterized by liver kidney microsomal antibody type 1 targeting cytochrome P4502D6 (CYP2D6). Growing evidence implicates the involvement of CD8 T cell immune responses in its pathogenesis. We investigated CYP2D6-specific CD8 T cell human leukocyte antigen (HLA)-A2 restricted responses in AIH-2 (20 patients, 11 HLA-A2+). Binding affinity of CYP2D6 peptides to HLA-A2 was predicted by the algorithm SYFPEITHI and assessed in vivo by T2 cell assays. CD8 T cell interferon (IFN)-gamma production was assessed via intracellular cytokine staining, cytotoxicity via chromium release assay, and frequency of circulating and intrahepatic CYP2D6-specific CD8 T cells via tetramer staining. CYP2D6-specific CD8 T cell reactivity was tested at diagnosis and during treatment and correlated with indices of disease activity. Seven CYP2D6 peptides with high HLA-A2 binding affinity colocalizing with known B cell or CD4 T cell epitopes were selected. Five sequences inducing high levels of IFN-gamma were used for HLA-A2 tetramer construction. Frequency, IFN-gamma production, and cytotoxicity of CYP2D6-specific CD8 T cells were higher at diagnosis than during treatment. Intensity of CYP2D6-specific CD8 T cell responses correlated with disease activity. Immune responses to CYP2D6(245-254) were the strongest both at diagnosis and during treatment. CONCLUSION: HLA-A2-restricted, CYP2D6-specific CD8 T cell immune responses vary according to disease stage and correlate with hepatocyte damage. CD8 T cell targets on CYP2D6-in particular CYP2D6(245-254)-may be the focus of novel immune intervention in AIH-2. (HEPATOLOGY 2007.).

Type 2 autoimmune hepatitis murine model: the influence of genetic background in disease development.

Genetic predisposition is recognized as an important factor for the development of autoimmune hepatitis (AIH). To assess the potential contribution of MHC and non-MHC genes, type 2 AIH was reproduced in three mice strains, taking advantage of their different genetic makeup with regard to MHC and non-MHC genes. Mice (C57BL/6, 129/Sv and BALB/c) were DNA vaccinated with a pCMV-CTLA4-CYP2D6-FTCD plasmid coding for the extracellular region of CTLA-4 and for the antigenic region of the CYP2D6 and FTCD, and with pCMV-IL12. ALT and total IgG levels, liver histology, FACS analysis and liver T-cell cytotoxicity assays were monitored up to 8 months post-injection. C57BL/6 mice showed elevated serum ALT levels, autoantibodies, antigen-specific cytotoxic T-cells and lobular and periportal inflammatory infiltrate. The 129/Sv mice showed slightly elevated ALT levels, sparse liver lobular infiltrate and cytotoxic T-cells. The BALB/c mice showed no liver inflammation. All mice had elevated total serum IgG levels. This murine model of type 2 AIH shows that MHC and non-MHC genes contribute to the susceptibility to autoimmune hepatitis. The understanding of the genetic determinants implicated in AIH development will be a major advance in the study of its pathogenesis and could lead to a better diagnostic approach and preventive strategies.

Characterization of human cytochrome P450 isoenzymes involved in the metabolism of vinorelbine.

Vinorelbine (VRL) (IV Navelbine) is a semi-synthetic vinca alkaloid, used in therapeutics for the treatment of non-small-cell lung cancer and advanced breast cancer. The aim of this study was to characterize the cytochrome P450 (CYP) isoenzymes involved in VRL metabolism. VRL was incubated at 1.28 x 10(-5) m for 90 min with human hepatic microsomes prepared from 14 donors (one woman and 13 men aged from 27 to 76 years old) and characterized for CYP1A2, CYP2D6, CYP2E1 and CYP3A4 activities. A four-combined-approach study was performed, including correlation between CYP activities and VRL metabolism among the 14 batches of microsomes, inhibition of VRL biotransformation by isoform-selective substrates and by specific inhibitory antibodies, and incubation with supersomes. Analysis of unchanged VRL and its metabolites was performed using an HPLC method coupled with both radioactive and UV detections. No correlation between CYP1A2 or CYP2E1 and VRL metabolism was observed in the 14 batches of microsomes used. A correlation was shown between VRL metabolism and CYP3A4 activity as determined with the dextromethorphan N-demethylase and nifedipine oxidase activities (r(2)=0.80 and 0.59, respectively). These results were strengthened by a correlation between the metabolism extent of VRL and CYP3A4 protein content determined by immunoblotting (r(2)=0.75). Furthermore, VRL biotransformation was inhibited by troleandomycine, the CYP3A4-specific inhibitor substrate (80% of inhibition) and by anti-CYP3A antibodies (36% of inhibition). On the contrary, a low correlation with CYP2D6 activity as determined by dextrometorphan O-demethylation (r(2)=0.31) was established. CYP2D6 supersomes did not metabolize the drug whereas 63.4% of VRL were metabolized by microsomes overexpressing CYP3A4 isoform. These data indicated that CYP3A4 is the main enzyme involved in the hepatic metabolism of VRL in human, whereas CYP2D6 is not involved.

Relative contributions of the five major human cytochromes P450, 1A2, 2C9, 2C19, 2D6, and 3A4, to the hepatic metabolism of the proteasome inhibitor bortezomib.

VELCADE (bortezomib, PS-341), reversibly inhibits the 20S proteasome and exhibits cytotoxic and antitumor activities. Pretreatment of cancer cells with bortezomib increases the chemosensitivity of these cells, suggesting that bortezomib may be used in combination chemotherapy. The relative contributions of the five major human cytochromes P450 (P450s), 1A2, 2C9, 2C19, 2D6, and 3A4 (the focus of the present study), to the metabolism of bortezomib are an important aspect of potential drug interactions. Relative activity factor (RAF), chemical inhibition, and immunoinhibition using monoclonal antibodies were three approaches employed to determine the relative contributions of the major human P450s to the net hepatic metabolism of bortezomib. RAFs for the P450 isoform-selective substrates were determined; the ratio of the rate of metabolism of bortezomib with cDNA-expressed P450s versus rate of metabolism with human liver microsomes was normalized with respect to the RAF for each P450 isoform to determine the percentage contributions of the P450s to the net hepatic metabolism of bortezomib. CYP3A4 followed by CYP2C19 were determined to be the major contributors to the metabolism of bortezomib. Chemical inhibition and immunoinhibition confirmed that CYP3A4 and CYP2C19 were the major P450s responsible for the hepatic metabolism of bortezomib. The studies were conducted with 2 muM bortezomib, and the disappearance of bortezomib, rather than appearance of a specific metabolite, was quantified to determine the contributions of the P450s to the overall hepatic metabolism of bortezomib in humans.

Astroglial CYP1B1 up-regulation in inflammatory/oxidative toxic conditions: IL-1beta effect and protection by N-acetylcysteine.

The present work aims to determine the relevance of an astrocytoma cell line U373 MG, for assessing the role of some astroglial cytochrome P450 in neurotoxicity and neuroprotection. CYP1B1, CYP2C8, CYP2C9, CYP2D6, CYP2J2, CYP2E1 and CYP4A11 mRNA were detected by reverse transcriptase-polymerase chain reaction in control U373 MG cell cultures. Among them we focused on CYP1B1 expression. After 48 h treatment with a range of concentrations of interleukin-1beta (1, 5, 10 ng/ml) used to simulate stress conditions, CYP1B1 mRNA expression was enhanced in a dose-dependent way. This increased expression was followed 24 h later by an increase in protein level, determined by Western-blot. N-acetylcysteine (NAC) partially inhibited this effect both on the mRNA and protein levels. As CYP1B1 activates procarcinogenic compounds to reactive metabolites, an increase in this P450 isoform will participate to toxic consequences of an inflammatory/oxidative stress. NAC will prevent this deleterious effect.

Hepatitis C virus infection associated with liver-kidney microsomal antibody type 1 (LKM1) autoantibodies in children.

OBJECTIVE: To evaluate the clinical pattern and evolution of chronic hepatitis C in children with liver/kidney microsomal antibody type 1 autoantibodies (LKM1). STUDY DESIGN: A multicenter, retrospective study, including the following groups of children with hepatitis C virus infection: (1). 21 consecutive LKM1-positive patients, (2). 42 age- and sex- matched LKM1-negative patients, and (3). 4 interferon-induced LKM1-positive cases. LKM1 reactivity to human microsomes and recombinant cytochrome P450IID6 (CYP2D6) was assayed by immunoblotting. RESULTS: Clinical and biochemical features overlapped in LKM1-positive and LKM1-negative children, but a fibrosis score >3 (range 0-6) was significantly more frequent (P =.04) in the former. Reactivity to microsomal protein and CYP2D6 was significantly (P =.02) associated with LKM1 titers >or=1:320 and was found in 39% of patients, including severe cases and both children (of 4 treated) who achieved a sustained alanine aminotransferase (ALT) normalization after steroid treatment. Five of 7 LKM1-positive children treated with interferon had an ALT exacerbation. CONCLUSIONS: LKM1-positive hepatitis C in children is characterized by a wide spectrum of biochemical, serologic, and histologic features. Whether autoimmunity may contribute to liver damage in a subgroup of patients with more severe liver disease, high LKM1 titers, and reactivity to CYP2D6 is a question deserving further investigation.