Passive immunization against nicotine attenuates somatic nicotine withdrawal syndrome in the rat.

INTRODUCTION: Nicotine immunization is under consideration as an intervention for smoking cessation. Therefore, it was of interest to evaluate the effects of nicotine antibodies on the withdrawal syndrome following termination of chronic nicotine administration. METHODS: Experiment 1 determined whether passive immunization following continuous nicotine infusion would alter the intensity of nicotine withdrawal syndrome in the rat. Fourteen rats were rendered nicotine dependent by 7 days of subcutaneous nicotine bitartrate infusion. On the final day, seven rats received 150 mg intraperitoneal (i.p.) of immune gamma globulin (IgG) raised against 3'-aminomethylnicotine-recombinant Pseudomonas aeruginosa exoprotein A (NicVAX, Nabi Biopharmaceuticals, Rockville, MD) and seven rats received normal IgG. Rats were observed under blind conditions for somatically expressed nicotine abstinence signs immediately prior to drug termination and at 12, 24, and 36 hr afterward. In Experiment 2, similarly treated rats were observed at 6- and 72-hr postwithdrawal, to test the possibility that immunization altered the time course rather than the intensity of withdrawal syndrome. Experiment 3 tested whether immunized rats were still nicotine dependent. Without pump removal, each rat was challenged by 1/mg/kg mecamylamine HCl and observed for precipitated withdrawal syndrome. RESULTS: In Experiment 1, there was no premature withdrawal syndrome during nicotine infusion. After termination, the immunized group had significantly fewer withdrawal signs than controls. Experiment 2 showed that immunization did not simply alter the timing of the nicotine abstinence syndrome since immunization did not increase signs before or after the usual withdrawal timeframe. In Experiment 3, rats immunized on the final day of infusion were still nicotine dependent since they exhibited a vigorous mecamylamine-precipitated withdrawal syndrome. DISCUSSION: Nicotine antibodies did not precipitate a withdrawal syndrome, and they markedly reduced the severity of spontaneous nicotine withdrawal. The present data suggests that this may be most readily explained by their reported delay of nicotine clearance.

Development of a second generation anti-Müllerian hormone (AMH) ELISA.

AMH is a glycoprotein dimer composed of two 72kDa monomers linked by disulfide bridges. It belongs to the transforming growth factor-ß family. AMH performs various physiological functions. In males, AMH is secreted by the Sertoli cells of the testis. During embryonic development, AMH is responsible for Müllerian duct regression. AMH continues to be produced by the testicles until puberty and then decreases slowly to residual post-puberty values. In females, AMH is produced in small amounts by ovarian granulosa cells after birth, until menopause, and then becomes undetectable. A two-step, sandwich-type enzymatic microplate assay has been developed to measure AMH levels in 20 µL of sample in less than 3h. AMH calibrators range from 0.2 to 28 ng/mL. The antibodies used in the assay bind to the mature region of AMH, which is more stable to proteolysis compared to prohormone region. The AMH Gen II assay (Beckman Coulter, Inc., Webster, Texas) was standardized to the Immunotech (IOT, Beckman Coulter, Inc., Marseilles, France) AMH assay. AMH Gen II, when compared to IOT using 120 serum samples in the range of 0-20.4 ng/mL yielded a correlation coefficient of 0.98 and a slope of 1.0. Total imprecision, calculated on four samples over 40 runs, four replicates per run, between two lots using CLSI EP5-A guidelines, was 5.7% at 3.8 ng/mL, 7.7% at 4.4 ng/mL, 5.8% at 14 ng/mL and 5.3% at 16.4 ng/mL. The average analytical sensitivity calculated by the interpolation of the mean plus two standard deviations of 16 replicates of the zero calibrator on two independent lots was 0.08ng/mL. Dilution and spiking studies showed an average recovery of 91-110%. Lot-to-lot comparison of two independent lots testing 38 serum samples (1.5-33 ng/mL range) yielded a slope of 1.01, intercept of -0.08 ng/mL and r of 0.99. When potential interferents (hemoglobin, triglycerides, and bilirubin) were added at two times the physiological concentrations, AMH concentrations were within ± 10% of the control. A highly specific and reproducible microplate AMH Gen II assay has been developed to standardize the measurement of AMH between methods. The performance of the AMH Gen II assay is ideal for investigation into the physiologic roles of AMH in men and women.