Repeated nicotine vapor inhalation induces behavioral sensitization in male and female C57BL/6 mice.

Electronic cigarette use has significantly increased over the past decade. However, there is limited preclinical research on the behavioral and abuse-related effects of nicotine vapor inhalation in rodents. The present study evaluates the effects of repeated nicotine vapor inhalation in male and female mice using a nicotine behavioral sensitization model. Male and female C57BL/6 mice were administered vaporized nicotine (0-10.0 mg/ml) or the positive control of intraperitoneally administered nicotine (0.5 mg/kg) once daily for 5 days, and locomotor activity was assessed. Body temperatures were measured before and after nicotine vapor inhalation to assess hypothermia. Nicotine vapor inhalation (1.0-3.0 mg/ml) produced a dose-dependent behavioral sensitization effect and produced hypothermia in male and female mice. Nicotine (0.5 mg/kg) also produced significant behavioral sensitization. No sex differences were found for nicotine behavioral sensitization with either route of administration. Pretreatment with the nonselective nicotinic antagonist mecamylamine blocked the behavioral sensitization produced by 1.0 mg/ml of nicotine vapor inhalation. These results established that nicotine vapor inhalation produces behavioral sensitization in an inverted U-shaped curve that is similar to the effects of injected nicotine across several behavioral models. Additionally, pretreatment with mecamylamine demonstrated that nicotinic receptor activation was responsible for the behavioral sensitization produced by nicotine vapor inhalation and was not a conditioned response to the vapor. The methods used in the present study provide an additional behavioral approach for evaluating the behavioral effects of repeated nicotine vapor inhalation that allows the manipulation of several variables, including e-liquid oil blend, e-liquid flavors, puff duration, etc.

Characterization and comparative performance of lentiviral vector preparations concentrated by either one-step ultrafiltration or ultracentrifugation.

Gene therapy utilizing lentiviral vectors (LVs) constitutes a real therapeutic alternative for many inherited monogenic diseases. Therefore, the generation of functional vectors using fast, non-laborious and cost-effective strategies is imperative. Among the available concentration methods for VSV-G pseudotyped lentiviruses to achieve high therapeutic titers, ultracentrifugation represents the most common approach. However, the procedure requires special handling and access to special instrumentation, it is time-consuming, and most importantly, it is cost-ineffective due to the high maintenance expenses and consumables of the ultracentrifuge apparatus. Here we describe an improved protocol in which vector stocks are prepared by transient transfection using standard cell culture media and are then concentrated by ultrafiltration, resulting in functional vector titers of up to 6×10(9) transducing units per millilitre (TU/ml) without the involvement of any purification step. Although ultrafiltration per se for concentrating viruses is not a new procedure, our work displays one major novelty; we characterized the nature and the constituents of the viral batches produced by ultrafiltration using peptide mass fingerprint analysis. We also determined the viral functional titer by employing flow cytometry and evaluated the actual viral particle size and concentration in real time by using laser-based nanoparticle tracking analysis based on Brownian motion. Vectors generated by this production method are contained in intact virions and when tested to transduce in vitro either murine total bone marrow or human CD34(+) hematopoietic stem cells, resulted in equal transduction efficiency and reduced toxicity, compared to lentiviral vectors produced using standard ultracentrifugation-based methods. The data from this study can eventually lead to the improvement of protocols and technical modifications for the clinical trials for gene therapy.