Glutamate, N-acetyl aspartate and psychotic symptoms in chronic ketamine users

Rationale: Ketamine, a non-competitive NMDA receptor antagonist, induces acute effects resembling the positive, negative and cognitive symptoms of schizophrenia. Chronic use has been suggested to lead to persistent schizophrenia-like neurobiological changes. Objectives: This study aims to test the hypothesis that chronic ketamine users have changes in brain neurochemistry and increased subthreshold psychotic symptoms compared to matched poly-drug users. Methods: Fifteen ketamine users and 13 poly-drug users were included in the study. Psychopathology was assessed using the Comprehensive Assessment of At-Risk Mental State. Creatine-scaled glutamate (Glu/Cr), glutamate + glutamine (Glu + Gln/Cr) and N-acetyl aspartate (NAA/Cr) were measured in three brain regions—anterior cingulate, left thalamus and left medial temporal cortex using proton magnetic resonance spectroscopy. Results: Chronic ketamine users had higher levels of subthreshold psychotic symptoms (p < 0.005, Cohen’s d = 1.48) and lower thalamic NAA/Cr (p < 0.01, d = 1.17) compared to non-users. There were no differences in medial temporal cortex or anterior cingulate NAA/Cr or in Glu/Cr or Glu + Gln/Cr in any brain region between the two groups. In chronic ketamine users, CAARMS severity of abnormal perceptions was directly correlated with anterior cingulate Glu/Cr (p < 0.05, r = 0.61—uncorrected), but NAA/Cr was not related to any measures of psychopathology. Conclusions: The finding of lower thalamic NAA/Cr in chronic ketamine users may be secondary to the effects of ketamine use compared to other drugs of abuse and resembles previous reports in individuals at genetic or clinical risk of schizophrenia

Brain volume in chronic ketamine users - Relationship to sub-threshold psychotic symptoms and relevance to schizophrenia

RATIONALE: Ketamine may model aspects of schizophrenia arising through NMDA receptor activity deficits. Although acute ketamine can induce effects resembling both positive and negative psychotic symptoms, chronic use may be a closer model of idiopathic psychosis. OBJECTIVES: We tested the hypotheses that ketamine users had lower brain volumes, as measured using MRI, and greater sub-threshold psychotic symptoms relative to a poly-drug user control group. METHODS: Ketamine users (n = 17) and poly-drug using controls (n = 19) were included in the study. All underwent volumetric MRI imaging and measurement of sub-threshold psychotic symptoms using the Comprehensive Assessment of At-Risk Mental State (CAARMS). Freesurfer was used to analyse differences in regional brain volume, cortical surface area and thickness between ketamine users and controls. The relationship between CAARMS ratings and brain volume was also investigated in ketamine users. RESULTS: Ketamine users were found to have significantly lower grey matter volumes of the nucleus accumbens, caudate nucleus, cerebellum and total cortex (FDR p \u3c 0.05; Cohen\u27s d = 0.36-0.75). Within the cortex, ketamine users had significantly lower grey matter volumes within the frontal, temporal and parietal cortices (Cohen\u27s d 0.7-1.31; FDR p \u3c 0.05). They also had significantly higher sub-threshold psychotic symptoms (p \u3c 0.05). Frequency of ketamine use showed an inverse correlation with cerebellar volume (p \u3c 0.001), but there was no relationship between regional brain volumes and sub-threshold psychotic symptoms. CONCLUSIONS: Chronic ketamine use may cause lower grey matter volumes as well as inducing sub-threshold psychotic symptoms, although these likely arise through distinct mechanisms

Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors : assessment of immunophenotype and differentiation capacity in vitro

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studiesSupplementary Figure 1 Gaussian distribution curve of GFP positive transduced cells. Adherent ASCs (48 000 cells) were transduced with different dilutions of lentiviral vector stock solutions and GFP expression was measured over 10 post-transduction passages using flow cytometry. The following amounts of lentivirus vector stock solution - 0-, 5-, 10-, 25-, 50-, 100-, 150-, 200-, 250- and 300 µl – were utilized to determine the optimal titer for ASC transduction. Data at all post transduction passages (T), with the exception of T0 and T3 fits with the Gaussian distribution curve. The interpolated value for 100 % transduction was found to be 363.18 µl lentiviral vector stock solution. (TIFF 130 kb)Supplementary Figure 2 Overlaying plots comparing the unstained control, non-transduced and transduced GFP positive cells for individual markers. The plots are from one biological replicate at a specific post-transduction passage. Each plot displays the expression of an individual antibody marker or GFP expression in the cell cytoplasm. (A) CD73 BV510; (B) CD90 PC5; (C) CD105 PE; (D) CD34 PC; (E) CD45 KO; and (F) GFP. (TIFF 388 kb)http://link.springer.com/journal/10616hb2016Immunolog

Human adipose derived mesenchymal stromal cells transduced with GFP lentiviral vectors : assessment of immunophenotype and differentiation capacity in vitro

Adipose derived mesenchymal stromal/stem cells (ASCs) are a heterogeneous population characterized by (a) their ability to adhere to plastic; (b) immunophenotypic expression of certain cell surface markers, while lacking others; and (c) the capacity to differentiate into lineages of mesodermal origin including osteocytes, chondrocytes and adipocytes. The long-term goal is to utilize these cells for clinical translation into cell-based therapies. However, preclinical safety and efficacy need to be demonstrated in animal models. ASCs can also be utilized as biological vehicles for vector-based gene delivery systems, since they are believed to home to sites of inflammation and infection in vivo. These factors motivated the development of a labelling system for ASCs using lentiviral vector-based green fluorescent protein (GFP) transduction. Human ASCs were transduced with GFP-expressing lentiviral vectors. A titration study determined the viral titer required to transduce the maximum number of ASCs. The effect of the transduced GFP lentiviral vector on ASC immunophenotypic expression of surface markers as well as their ability to differentiate into osteocytes and adipocytes were assessed in vitro. A transduction efficiency in ASC cultures of approximately 80 % was observed with an MOI of ~118. No significant immunophenotypic differences were observed between transduced and non-transduced cells and both cell types successfully differentiated into adipocytes and osteocytes in vitro. We obtained >80 % transduction of ASCs using GFP lentiviral vectors. Transduced ASCs maintained plastic adherence, demonstrated ASC immunophenotype and the ability to differentiate into cells of the mesodermal lineage. This GFP-ASC transduction technique offers a potential tracking system for future pre-clinical studiesSupplementary Figure 1 Gaussian distribution curve of GFP positive transduced cells. Adherent ASCs (48 000 cells) were transduced with different dilutions of lentiviral vector stock solutions and GFP expression was measured over 10 post-transduction passages using flow cytometry. The following amounts of lentivirus vector stock solution - 0-, 5-, 10-, 25-, 50-, 100-, 150-, 200-, 250- and 300 µl – were utilized to determine the optimal titer for ASC transduction. Data at all post transduction passages (T), with the exception of T0 and T3 fits with the Gaussian distribution curve. The interpolated value for 100 % transduction was found to be 363.18 µl lentiviral vector stock solution. (TIFF 130 kb)Supplementary Figure 2 Overlaying plots comparing the unstained control, non-transduced and transduced GFP positive cells for individual markers. The plots are from one biological replicate at a specific post-transduction passage. Each plot displays the expression of an individual antibody marker or GFP expression in the cell cytoplasm. (A) CD73 BV510; (B) CD90 PC5; (C) CD105 PE; (D) CD34 PC; (E) CD45 KO; and (F) GFP. (TIFF 388 kb)http://link.springer.com/journal/10616hb2016Immunolog

Novel flow cytometric approach for the detection of adipocyte subpopulations during adipogenesis

The ability of mesenchymal stromal cells (MSCs) to differentiate into adipocytes provides a cellular model of human origin to study adipogenesis in vitro. One of the major challenges in studying adipogenesis is the lack of tools to identify and monitor the differentiation of various subpopulations within the heterogeneous pool of MSCs. Cluster of differentiation (CD)36 plays an important role in the formation of intracellular lipid droplets, a key characteristic of adipocyte differentiation/maturation. The objective of this study was to develop a reproducible quantitative method to study adipocyte differentiation by comparing two lipophilic dyes [Nile Red (NR) and Bodipy 493/503] in combination with CD36 surface marker staining. We identified a subpopulation of adiposederived stromal cells that express CD36 at intermediate/high levels and show that combining CD36 cell surface staining with neutral lipidspecific staining allows us to monitor differentiation of adiposederived stromal cells that express CD36 during adipocyte differentiation in vitro. The gradual increase of CD36 NR cells during the 21 day adipogenesis induction period correlated with upregulation of adipogenesisassociated gene expression.http://www.jlr.org2017-04-30hb2016Immunolog

Implications of direct-to-consumer whole-exome sequencing in South Africa

This editorial examines a number of vitally important ethical, legal and scientific concerns that have to be addressed to ensure proper and ethical implementation of direct-to-consumer whole-exome sequencing in South Africa. Individuals taking part in this endeavour must be fully informed of the positive and negative sequelae

An in vitro and in vivo study on the properties of hollow polycaprolactone cell-delivery particles

The field of dermal fillers is evolving rapidly and numerous products are currently on the market. Biodegradable polymers such as polycaprolactone (PCL) have been found to be compatible with several body tissues, and this makes them an ideal material for dermal filling purposes. Hollow PCL spheres were developed by the Council for Scientific and Industrial Research (CSIR) to serve both as an anchor point and a ªtissue harbourº for cells. Particles were tested for cytotoxicity and cell adherence using mouse embryo fibroblasts (MEF). MEFs adhered to the particles and no significant toxic effects were observed based on morphology, cell growth, cell viability and cell cycle analysis, suggesting that the particles are suitable candidates for cell delivery systems in an in vivo setting. The objective of providing a ªtissue harbourº was however not realized, as cells did not preferentially migrate into the ported particles. In vivo studies were conducted in BALB/c mice into whom particles were introduced at the level of the hypodermis. Mice injected with PCL particles (ported and nonported; with or without MEFs) showed evidence of local inflammation and increased adipogenesis at the site of injection, as well as a systemic inflammatory response. These effects were also observed in mice that received apparently inert (polystyrene) particles. Ported PCL particles can therefore act as a cell delivery system and through their ability to induce adipogenesis, may also serve as a dermal bulking agent.S1 File. Figure A: Chronic inflammation in the test animals over the trial period. Figure B: Acute inflammation in the test animals over the trial period. Figure C: Tissue necrosis in the test animals over the trial period. Figure D: Fibrosis in the test animals over the trial period. Figure E: Granulomatous/foreign body response in the test animals over the trial period. Figure F: Representative TEMs of skin biopsies of particles group (A) and particles+MEFs group (B) in the in vivo experiment injecting particles+MEFs. Particles could be identified in skin biopsies of both the particles and particles+MEFs groups. The aim of the TEM investigation was to determine if any cells could be detected inside the particles. No cells were present inside the particles in either group. These results reflect the conclusion that was made after the light microscopy study, indicating that cells did not migrate into the ported PCL particles. Bar in A = 5μm and in B = 10μm.S2 File. In vitro and in vivo data. Table A: Groups of rats used in the biotoxicity trial. Table B: Observations on mice in the in vivo experiment assessing the effect of ported PCL particles and cells. Table C: Statistical comparisons preformed between the various white blood cell types assessed from blood smears of experimental mice injected with ported PCL particles with or without MEFs. Table D: Schedule of the in vivo experiment assessing the effect of ported and non-ported PCL as well as polystyrene (PS) particles. Table E: Overview of the animals, tests and procedures performed in the in vivo experiment assessing the effect of ported and non-ported PCL as well as polystyrene (PS) particles in BALB/c mice.S3 File. All data underlying the findings of the study.The Council for Scientific and Industrial Research, South Africa, by the Institute for Cellular and Molecular Medicine of the University of Pretoria and by the South African Medical Research Council (Flagship Award SAMRC-RFA-UFSP-01-2013/ STEM CELLS and the SAMRC Extramural Unit for Stem Cell Research and Therapy).http://www.plosone.orgam2019ImmunologyPhysiolog