The CYP autoantibodies show no correlation to the administration of calcium blockers, blockers and loop diuretic furosemide used for management of nephrotic syndrome and renal function, and there were no drug interactions that affect bioavailability of immunosuppressants (result not shown). Table 3 Increased drug metabolism of paediatric kidney and liver disease patients on immunosuppressants is age-dependent Open in a separate window DISCUSSION There are to our knowledge no previous reports of antibodies to intact CYPs among subjects on immunosuppressive drugs. (= 16), and 21% of kidney and or liver transplant patients on FK506 (= 14). In contrast, the frequency of reactive immunoblots was only 85% among the normal paediatric controls (= 25) and 7% among adult kidney transplant patients on CyA or FK506 (= 30). The CYP2C9+ sera were able to immunoprecipitate translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection. = 2), extrahepatic biliary atresia (= 5), chronic cholestasis (= 1), acute fulminant hepatitis (= 2), glycogenosis (= 3), BuddCChiari syndrome with hepatic vein occlusion (= 1) and other congenital or inherited hepatic dysfunction (= 6), were given postoperative treatments of either CyA, mean dosage 5 mg/kg per day, dosage range 3C9 mg/kg per day, or FK506, mean dosage 013mg/kg per day, dosage range 005C023 mg/kg per day, to maintain 12h trough values in the range of 100C300 ng/ml and 4C9 ng/ml, respectively. The sera of the 24 control paediatric subjects had no drug treatments and were normal donors (13 boys and 11 girls; age range 05C3 years, mean age 2 years). The sera of 30 kidney transplant recipients (24 males and eight females), age range 15C62 years (mean 38 years), on postoperative CyA, mean dose 7 mg/kg per day (range 27C16 mg/kg per day), to maintain mean trough values of 187 ng/ml (range 42C316 ng/ml) or FK506 mean 012 mg/kg per day (range 003C02 mg/day) and mean trough values of 12 ng/ml were kindly provided by Professor M. I. Lorber (Section of Organ Transplantation and Immunology, Department of Surgery, Yale University School of Medicine, New Haven, CT). Sera of eight adult primary biliary cirrhosis (PBC) patients without immunosuppressive treatments were kindly provided by Dr U. Broome (Department BQCA of Medicine, Huddinge Hospital, Karolinska BQCA Institutet) and Dr E. Eliasson (Division of Molecular Toxicology, Karolinska Institutet). This study was carried out with consent of the BQCA human subjects and Rabbit Polyclonal to MLH1 approval by the Swedish Ethics Committee. Human proteins The human recombinant CYP2E1, CYP3A4, CYP1A2 and CYP2C9 were produced from over-expressing plasmid in at Panvera Ltd (Madison, WI). The N-terminal modifications were as previously described [19,20]. Microsomes derived from AHH-1 TK+/? human lymphoblastoid B cell line co-expressed full-length cDNA of human CYPs: CYP2E1, CYP3A4, CYP1A2, CYP2C9 or CYP2D6 and the human cytochrome P450 reductase. Microsomes derived from baculovirus-infected insect cell system co-expressed cDNA of CYP3A4 or CYP3A5 with cytochrome P450 reductase or the cytochrome P450 reductase alone. All microsome preparations were produced from GENTEST Corp. (Woburn, MA). The BQCA human recombinant FK-binding protein 12 (FKBP12) was expressed in (Sigma Chemical Co., St Louis, MO). Antigens Rat CYP3A1 and NADPH reductase-cytochrome P450 was purified from microsomal fractions of rat liver, and human liver microsomes were from frozen stocks prepared as described previously [21]. Functional assays Studies of drug bioactivation were performed by additions of CyA in DMSO or FK506 in ethanol at final concentrations of 100 m drug and 05% v/v solvent to baculovirus microsomes containing 50 pmol of either CYP3A4 or CYP3A5 in 50 mm potassium phosphate buffer and 50 mm KCl pH 74 as described previously [22]. The above reaction mixtures were incubated for 1 h at 37C in the presence of NADPH generating system; 1 mm NADP+, 50 mm glucose-6-phosphate and 05 U/ml glucose-6-phosphate dehydrogenase. CYP3A-dependent 6 hydroxylation of 14C testosterone was monitored as described previously [21]. The activity of NADPH-cytochrome P450 reductase was measured spectrophotometrically using cytochrome c as an electron receptor [23]. ELISA ELISA was performed in polystyrene microwell plates (Sigma) as described previously [12]. In vitro coupled transcription-translation of human CYP3A4his6 Full-length human.