Cannabinoid distribution and postmortem redistribution in rabbits following controlled cannabis administration

Millions of people in the world use Cannabis sativa for its mood-altering properties. The main psychoactive component of cannabis, delta^9-tetrahydrocannabinol (THC) is commonly detected in forensic toxicology laboratories handling motor vehicle and plane crash fatalities. As few studies characterized cannabinoid distribution and postmortem redistribution, we developed a study to investigate postmortem cannabinoid concentrations in rabbits following controlled cannabis administration via a smoking machine. Five rabbits were exposed to cannabis smoke and a broad array of biological specimens were collected immediately upon death. High THC concentrations were observed in lungs, moderate concentrations were seen in the brain, heart, and kidneys, and low concentrations were noted in the liver. A physiologically based pharmacokinetic (PBPK) model was constructed to describe blood and tissue THC concentrations in rabbits following the administration of smoked cannabis by inhalation. The results showed similar THC concentrations in blood and tissues between the predicted and experimental data. Building upon the disposition of cannabinoids in various postmortem fluids and tissues in the rabbits, we evaluated time- and temperature-dependent changes in the concentrations of cannabinoids after rabbit carcasses were stored under room temperature or refrigerated conditions for various times after death. No significant differences in THC concentrations were observed between rabbits stored at room temperature versus under refrigerated conditions. When THC concentrations from various postmortem intervals (PMI) were compared to those observed at PMI 0 h, significant THC concentration changes in heart blood and peripheral blood were noted only in refrigerated samples collected at 2 and 16 h after death. Mean central:peripheral and liver:peripheral ratios for THC were 1.6 and 2.9, respectively. These data indicate that THC was not prone to substantial postmortem redistribution in the rabbit. This was the first study to consider either cannabinoid distribution or postmortem redistribution after inhaled cannabis administration using an animal model. The findings add substantially to our understanding of postmortem cannabinoid concentrations and can aid forensic toxicologists in the interpretation of cannabinoid findings in death investigations

Single-cell RNA-seq reveals transcriptomic heterogeneity mediated by host-pathogen dynamics in lymphoblastoid cell lines

Lymphoblastoid cell lines (LCLs) are generated by transforming primary B cells with Epstein-Barr virus (EBV) and are used extensively as model systems in viral oncology, immunology, and human genetics research. In this study, we characterized single-cell transcriptomic profiles of five LCLs and present a simple discrete-time simulation to explore the influence of stochasticity on LCL clonal evolution. Single-cell RNA sequencing (scRNA-seq) revealed substantial phenotypic heterogeneity within and across LCLs with respect to immunoglobulin isotype; virus-modulated host pathways involved in survival, activation, and differentiation; viral replication state; and oxidative stress. This heterogeneity is likely attributable to intrinsic variance in primary B cells and host-pathogen dynamics. Stochastic simulations demonstrate that initial primary cell heterogeneity, random sampling, time in culture, and even mild differences in phenotype-specific fitness can contribute substantially to dynamic diversity in populations of nominally clonal cells

Modeling flow cytometry data for cancer vaccine immune monitoring

Flow cytometry (FCM) is widely used in cancer research for diagnosis, detection of minimal residual disease, as well as immune monitoring and profiling following immunotherapy. In all these applications, the challenge is to detect extremely rare cell subsets while avoiding spurious positive events. To achieve this objective, it helps to be able to analyze FCM data using multiple markers simultaneously, since the additional information provided often helps to minimize the number of false positive and false negative events, hence increasing both sensitivity and specificity. However, with manual gating, at most two markers can be examined in a single dot plot, and a sequential strategy is often used. As the sequential strategy discards events that fall outside preceding gates at each stage, the effectiveness of the strategy is difficult to evaluate without laborious and painstaking back-gating. Model-based analysis is a promising computational technique that works using information from all marker dimensions simultaneously, and offers an alternative approach to flow analysis that can usefully complement manual gating in the design of optimal gating strategies. Results from model-based analysis will be illustrated with examples from FCM assays commonly used in cancer immunotherapy laboratories

Multivariate analysis of FcR-mediated NK cell functions identifies unique clustering among humans and rhesus macaques

Rhesus macaques (RMs) are a common pre-clinical model used to test HIV vaccine efficacy and passive immunization strategies. Yet, it remains unclear to what extent the Fc-Fc receptor (FcR) interactions impacting antiviral activities of antibodies in RMs recapitulate those in humans. Here, we evaluated the FcR-related functionality of natural killer cells (NKs) from peripheral blood of uninfected humans and RMs to identify intra- and inter-species variation. NKs were screened for FcγRIIIa (human) and FcγRIII (RM) genotypes (FcγRIII(a)), receptor signaling, and antibody-dependent cellular cytotoxicity (ADCC), the latter mediated by a cocktail of monoclonal IgG1 antibodies with human or RM Fc. FcγRIII(a) genetic polymorphisms alone did not explain differences in NK effector functionality in either species cohort. Using the same parameters, hierarchical clustering separated each species into two clusters. Importantly, in principal components analyses, ADCC magnitude, NK contribution to ADCC, FcγRIII(a) cell-surface expression, and frequency of phosphorylated CD3ζ NK cells all contributed similarly to the first principal component within each species, demonstrating the importance of measuring multiple facets of NK cell function. Although ADCC potency was similar between species, we detected significant differences in frequencies of NK cells and pCD3ζ+ cells, level of cell-surface FcγRIII(a) expression, and NK-mediated ADCC (P<0.001), indicating that a combination of Fc-FcR parameters contribute to overall inter-species functional differences. These data strongly support the importance of multi-parameter analyses of Fc-FcR NK-mediated functions when evaluating efficacy of passive and active immunizations in pre- and clinical trials and identifying correlates of protection. The results also suggest that pre-screening animals for multiple FcR-mediated NK function would ensure even distribution of animals among treatment groups in future preclinical trials

Breaking the communication barrier: guidelines to aid communication within pair programming

Pair programming is a software development technique with many cited benefits in learning and teaching. However, it is reported that novice programmers find several barriers to pairing up, typically due to the added communication that is required of this approach. This paper will present a literature review discussing the issue of communication, and through a series of observations with industry-based pairs, will derive a set of guidelines which aim to help novice pairs experience better communication within their pairs. An evaluation of the guidelines with undergraduate students is then reported, showing that exposure to these guidelines improved the self-perceived communication experience of novice pairs