Datasets:

TaylorAI

/

pubmed_author_manuscripts

like 3

Follow

Taylor 16

PMC004xxxxxx/PMC4396961.txt

LICENSE: This file is available for text mining. It may also be used consistent with the principles of fair use under the copyright law. 101313252 34584 J Vis Exp J Vis Exp Journal of visualized experiments : JoVE 1940-087X 25549203 4396961 10.3791/52432 NIHMS831093 Article Macrophage Cholesterol Depletion and Its Effect on the Phagocytosis of Cryptococcus neoformans Bryan Arielle M. 1* Farnoud Amir M. *1 Mor Visesato 1 Del Poeta Maurizio 1 1 Department of Molecular Genetics and Microbiology, Stony Brook University Correspondence to: Maurizio Del Poeta at maurizio.delpoeta@stonybrook.edu * These authors contributed equally 21 11 2016 19 12 2014 19 12 2014 28 11 2016 94 10.3791/52432This file is available for text mining. It may also be used consistent with the principles of fair use under the copyright law. Cryptococcosis is a life-threatening infection caused by pathogenic fungi of the genus Cryptococcus. Infection occurs upon inhalation of spores, which are able to replicate in the deep lung. Phagocytosis of Cryptococcus by macrophages is one of the ways that the disease is able to spread into the central nervous system to cause lethal meningoencephalitis. Therefore, study of the association between Cryptococcus and macrophages is important to understanding the progression of the infection. The present study describes a step-by-step protocol to study macrophage infectivity by C. neoformansin vitro. Using this protocol, the role of host sterols on host-pathogen interactions is studied. Different concentrations of methyl--cyclodextrin (MCD) were used to deplete cholesterol from murine reticulum sarcoma macrophage-like cell line J774A.1. Cholesterol depletion was confirmed and quantified using both a commercially available cholesterol quantification kit and thin layer chromatography. Cholesterol depleted cells were activated using Lipopolysacharide (LPS) and Interferon gamma (IFNγ) and infected with antibody-opsonized Cryptococcus neoformans wild-type H99 cells at an effector-to-target ratio of 1:1. Infected cells were monitored after 2 hr of incubation with C. neoformans and their phagocytic index was calculated. Cholesterol depletion resulted in a significant reduction in the phagocytic index. The presented protocols offer a convenient method to mimic the initiation of the infection process in a laboratory environment and study the role of host lipid composition on infectivity. Immunology Issue 94 Infection phagocytosis Cryptococcus cholesterol cyclodextrin macrophages Introduction Phagocytosis is a process by which extracellular entities are internalized by host cells. It is a key weapon in the immune system’s arsenal to defend against pathogens, but the process may often be subverted by pathogens to allow for internalization and spreading throughout the body1. Phagocytosis is mediated by several signaling events that result in attachment and engulfment via rearrangements of the host cell’s cytoskeleton. ‘Professional’ phagocytes are able to recognize and bind to opsonins on the surface of the invading pathogen to signal for attachment and the formation of lamellipodia, which engulf the pathogen and form a phagosome2. Among the so-called ‘professional’ phagocytes are macrophages. Macrophages are highly specialized cells that carry out protective functions that include seeking out and eliminating disease causing agents, repairing damaged tissues, and mediating inflammation, most of these through the process of phagocytosis1,2. Cryptococcus neoformans is a species of pathogenic yeast that causes a serious disease known as Cryptococcosis. Cryptococcus spores are inhaled by the host and result in a pulmonary infection that is usually asymptomatic. It is thought that exposure is extremely prevalent; a sample of 61 children from the Pediatric Infectious Diseases Clinic at the Bronx-Lebanon Hospital Center found that all those surveyed had antibodies to the cryptococcal polysaccharide glucuronoxylomannan and other studies have shown prevalence in both human immunodeficiency virus (HIV) uninfected and infected adults3,4. Alveolar macrophages are the first line of response to the pulmonary infection and in most cases successfully clear the pathogen. However, in immunocompromised individuals (e.g., HIV and AIDS patients) the yeast is able to survive within the macrophages. In these cases, the macrophages can serve as a niche for the replication of the pathogen and may facilitate its dissemination to the central nervous system (CNS) where the disease becomes fatal5–8. It is thought that macrophages may even deliver the yeast directly into the meninges, helping the yeast to cross the blood brain barrier via the “Trojan horse” model3,9–11. Thus, it is important to understand the process of phagocytosis and the factors that affect it, especially in cryptococcal infections. Previous work in other pathogen systems point to cholesterol and lipid rafts formed by cholesterol as having an important role to play in phagocytosis12–15. Cholesterol is the most abundant lipid species in mammalian cells and comprises 25 – 50% of the mammalian cell membrane16. It has been found to play a role in modulating the biophysical properties of membranes by changing their rigidity17. Cholesterol and sphingolipids together form lipid microdomains within the membrane known as lipid rafts. Lipid rafts have been found to be involved in the formation of caveolae, as well as providing an isolated domain for certain types of signaling16–18. Due to their small size, it is difficult to study lipid rafts in vivo. One useful way to study the role of lipid rafts is to alter their constituents. Methyl-β-cyclodextrin (MβCD) is a compound that has been found to deplete cholesterol from mammalian membranes and is commonly used to study the role of lipid rafts18. In this protocol, we present a method to deplete cholesterol from host cell membranes and quantify the effect of the depletion on the ability of the host cells to phagocytose C. neoformans in vitro. This procedure makes use of cell culture techniques on an immortalized macrophage like cell line (J774A.1) as a model for infection. Cholesterol depletion was accomplished by exposure to MβCD, which has a hydrophobic core specific to the size of sterols and is able to act as a sink for cholesterol to draw it out of the membrane19. Cholesterol depletion was measured quantitatively using a commercially available kit and qualitatively using a modified Bligh-Dyer lipid extraction followed by thin layer chromatography (TLC)20. Phagocytosis was measured by infecting the cell line with a culture of opsonized yeast mixed with a cocktail of interferon-γ and lipopolysaccharide for activating the macrophages. Cryptococcus was opsonized using a glucuronoxylomannan (GXM) antibody21–23. Staining and microscopy experiments allowed for visualization of the cells and calculation of the phagocytic index to assess the degree of phagocytosis. Taken together, this protocol describes a basic method that integrates the alteration of lipid composition with a physiological process. Protocol 1. Cholesterol Depletion of J774A.1 Cells with MβCD In a sterile biosafety cabinet, seed 105 J774A.1 macrophage-like cells per well on a 96-well cell culture plate in 200 μl of Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). Incubate at 37 °C and 5% CO2 O/N. Remove media from the cell monolayer and wash the cells twice with 1x phosphate buffered saline (PBS) that has been filtered or autoclaved. Add 200 μl of MβCD solution at the desired concentration (10 mM or 30 mM in PBS) or 1x PBS as a control and incubate for 30 min at 37 °C with shaking. Remove supernatant and reserve at RT for quantitative analysis with commercially available kit immediately following the procedure. Wash the cells two to three times with 1x PBS or serum free DMEM and continue with infection or lyse cells by pipetting two to three times with deionized H2O for analysis with thin layer chromatography or with a kit. NOTE: Following cholesterol depletion a commercially available cholesterol quantification kit can be used. See materials section for details. Follow manufacturer’s instructions as written. 2. Observation of Cholesterol Content by Thin Layer Chromatography (TLC) Wash a TLC tank twice with acetone and once with a solution of petroleum ether:diethyl ether:acetic acid (65:30:1 by volume). Saturate the tank with the petroleum ether:diethyl ether:acetic acid (65:30:1 by volume) solution and leave O/N. NOTE: Organic solvents should always be used under a fume hood to prevent inhalation of vapor. Gloves and lab coat should be worn at all times. Acetic acid is a strong acid and should be used with caution. In a sterile biosafety cabinet, seed 106 J774A.1 macrophage-like cells per well on a 6-well cell culture plate in a volume of 5 ml of warm DMEM supplemented with 10% FBS and 1% P/S. Incubate at 37 °C, 5% CO2 O/N. Deplete macrophages of cholesterol by following steps 1.2 – 1.4, substituting 1 ml for 200 μl where applicable to account for the larger well size. Add 500 μl of Trypsin-EDTA to each well, incubate for 3 min at 37 °C, and gently scrape cells with a cell scraper. Transfer into a microfuge tube and add an additional 500 μl of warm DMEM supplemented with 10% FBS and 1% P/S. Spin the cells for 5 min at 300 × g and remove the supernatant. Add an additional 500 μl of warm DMEM supplemented with 10% FBS and 1% P/S to the cell pellet and resuspend carefully by pipetting up and down. Remove 10 μl of cells and count cells on a hemocytometer. Normalize the cell concentrations from each sample and add an equal number of cells to glass tubes. NOTE: At this step, one may choose to combine cells from the same treatment groups to obtain a more concentrated final lipid extract. Centrifuge cells at 300 × g for 5 min at RT and remove media. Add 2 ml of methanol and vortex. Then, add 1 ml of chloroform and vortex. Check the phase status to make sure the solution in monophasic. NOTE: Tubes can be stored at 4 °C O/N. Centrifuge at 1,700 × g for 10 min at RT and transfer the supernatant to a new tube Add an additional 1 ml of chloroform followed by 1 ml of dH2O. Vortex twice for 30 sec. Centrifuge at 1,700 × g for 5 min at RT. Weigh a glass tube on a sensitive balance and use a glass Pasteur pipette to transfer the lower phase into the glass tube of known weight. Dry down lipids in a centrifugal evaporator until dry (approximately 2 hr). Weigh tube with dried lipids and calculate the dry lipid weight. NOTE: Dry lipids can be stored in −20 °C until ready to perform TLC. Dilute dried lipids in enough chloroform to normalize the concentration of lipid (usually 20 – 50 μl) and load 20 μl of the diluted lipid on a silica TLC plate. Load 20 μg of cholesterol diluted in 20 μl of chloroform as a standard. Add dried TLC plate to the saturated TLC tank and allow solvent to migrate up to 1 cm before plate edge. Remove TLC plate from tank and allow it to dry about 5 min. Visualize lipids by placing in an iodine vapor tank to check migration. Remove and allow spots to fade for about 10 – 15 min under the hood. NOTE: Iodine is an inhalation hazard. Always use under a fume hood. Prepare a solution for neutral lipid staining by combining 60 ml of methanol with 60 ml of deionized H2O, 4 ml of sulfuric acid, and 630 mg of manganese chloride. Carefully and slowly dip the TLC plate into the neutral lipid staining solution in a tray and remove without sloughing off the silica layer. NOTE: The neutral lipid staining solution can be reused several times and so it can be retrieved from the tray and placed back in a bottle for later use. Allow plate to dry under the hood at RT until all bubbles has disappeared. Heat the TLC plate on a heat block set to 160 °C and char to the desired color. NOTE: A densitometry program such as Vision Works LS can be used to quantify the charred bands to compare lipid samples. 3. Infection of Macrophages with C. neoformans (H99) In a sterile biosafety cabinet, seed 105 macrophage-like cells per well on a 96-well cell culture plate in 200 μl of DMEM supplemented with 10% FBS and 1% P/S. Incubate at 37 °C, 5% CO2, 5% CO2 O/N. NOTE: Infection can also be done in glass bottomed confocal dishes for easier imaging; all amounts remain the same. Grow a culture of C. neoformans (H99) by inoculating 10 ml of YNB with one colony obtained from a struck plate and incubating it O/N at 30 °C with shaking. Wash and count C. neoformans (H99) cells. Centrifuge C. neoformans O/N culture at 1,700 × g for 10 min at 4 °C. Remove media and discard. Wash cells with 5 ml of 1x PBS. Centrifuge at 1,700 × g for 10 min at 4 °C. Remove PBS and wash with 5 ml of filtered 1x PBS. Centrifuge at 1,700 × g for 10 min at 4 °C. Repeat this step 2 more times. Remove PBS and resuspend in 5 ml of 1x PBS. Make a serial dilution in PBS to obtain a 1:500 dilution of the washed culture. Add 100 μl of the original sample to 900 μl of 1x PBS to obtain a 1:10 dilution. Add 100 μl of 1:10 diluted sample to 900 μl of 1x PBS to obtain a 1:100 dilution. Add 200 μl of the 1:100 diluted sample to 800 μl of 1x PBS to obtain a 1:500 dilution Take 10 μl of 1:500 dilution and count on hemocytometer to calculate the number of cells. Prepare working solution for activating macrophages and opsonizing C. neoformans. Dilute LPS and IFNγ 100x from stock solutions by adding 10 μl to 990 μl. NOTE: LPS and IFNγ are used to enhance phagocytic uptake but are not required for phagocytosis. If there is an interest in fungicidal activity of macrophages, perform activation O/N at 37 °C with shaking prior to infection. Per sample combine 7.5 μl of diluted LPS, 1.25 μl of diluted IFNγ, 1.25 μl of GXM antibody and the volume of the C. neoformans culture that gives 1.25 × 105 cells. Bring volume up to 250 μl multiplied by number of samples with DMEM supplemented with 10% FBS and 1% P/S. Vortex and incubate solution for 20 min at 37 °C with shaking. NOTE: Cholesterol depletion (steps 1.2 – 1.4) can be done concurrently with the opsonization step. Be sure to treat macrophages prior to combining the working solution, as the opsonized cells should optimally be used no longer than 20 min following the step 3.4.3 incubation. Infect Macrophages Wash macrophages twice with serum free DMEM and add 200 μl of opsonized C. neoformans working solution to each well. Incubate for 2 hr at 37 °C. Fix and Stain Cells Remove media and wash cells 2 times with DMEM. Air dry the cell monolayer for 10 min and add 200 μl of ice cold methanol to fix the cells. Incubate for 15 min at RT and remove any remaining methanol. Add 200 μl of 10x Giemsa and incubate for 5 min at RT. Wash 2 – 3 times with deionized water and dry O/N with cap off. NOTE: Imaging can be done the next day or up to a week following staining. Visualize and Count Using a microscope, count 300 cells per data point (if there are 2 of the same treatment count 150 per well) and note the number of infected macrophages and number of engulfed Cryptococcus cells. NOTE: To ensure even sampling per data point use 2 plates per condition, choose 3 non-overlapping areas per plate and count 50 cells per area. Calculate phagocytic index by multiplying the percentage of infected macrophages by the mean number of C. neoformans per macrophage. Normalize phagocytic index by expressing values as a percentage of the 1x PBS treated control. After several trials calculate the mean value and the standard deviation of the mean to determine trends in phagocytic index. Use student t-test to determine significance. Take micrographs of cells at 1,000X or 400X magnification. 4. Trypan Blue Assay In a sterile biosafety cabinet, seed 106 macrophage-like cells per well on a 6-well cell culture plate in a volume of 5 ml of Dulbecco’s minimal essential medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Incubate at 37 °C, 5% CO2 O/N. Deplete macrophages of cholesterol by following steps 1.2 – 1.4 substituting 2 ml for 200 μl where applicable to account for the larger well size. NOTE: Be sure to include a control treated with 1x PBS as well as a control that is scraped prior to any treatment. Add 500 μl of 1x PBS to each well and gently scrape cells with a cell scraper. Transfer into a microfuge tube and suspend the cells by gently pipetting up and down. Remove 10 μl of cells and stain with 1 μl of 4% Trypan blue. Count cells on a hemocytometer and calculate viability using the following equation: % viability = [1 − (Blue cells/total cells)] × 100. Normalize values to the control that was untreated. After several trials calculate the mean value and the standard deviation to determine trends in viability. Use student t-test to determine significance. Representative Results Cholesterol Depletion Analysis of the supernatant reserved in step 1.3 of the protocol by following the manufacturer’s instructions in the Amplex Red Cholesterol Assay kit yields an elevated concentration of cholesterol in MβCD treated sample as compared to the 1x PBS control. Depending on cell type and MβCD concentration used cholesterol depletion may vary. For J774 treated with 10 mM MβCD, a depletion of approximately 50% was observed. Depletion can be calculated using values obtained from the supernatant and cell lysate collected in step 1.4 (Figure 1). Cell lysate analyzed using TLC shows a marked decrease in staining of cholesterol in cells treated with increasing concentration of MβCD (Figure 2A). Densitometry analysis of the TLC shows a similar trend to the quantitative assay (Figure 2B). The Bligh-Dyer method gives a crude extract of total lipids and it is essential to allow for adequate separation of lipids in order to identify the correct band utilizing the cholesterol standard. Infection After following the infection procedure, cells remain adhered and intact. Cell morphology remains unchanged between treatment groups. A control group that has not been exposed to C. neoformans serves as a checkpoint (Figure 3). It is possible to obtain suboptimal results and may manifest as lysis of cells and other abnormal morphologies. The most likely cause is contamination of the cell line or reagents used in the procedure. Micrographs of optimally infected cells clearly show C. neoformans engulfed within the mammalian cells. Differences in number of phagocytized yeast may be noted by observation between treatment groups (Figure 4). After calculating phagocytic index from 300 macrophage cells per treatment group a reduction in phagocytic index is found in cholesterol depleted cells (Figure 5). The reduction in the phagocytic index does not appear to be dependent on potential differences in macrophage activation, although they may occur. Performing the infection in the absence of macrophage activators, but after treatment with MCD results in a similar reduction of phagocytic index (data not shown). Trypan Blue Trypan Blue staining is used to assess the viability of cells after cholesterol depletion. No change in viability is observed between PBS treated and 10mM MCD treated cells. Viability appears to drop off slightly after treatment with 30 mM MCD, which may be expected due to the approximately 75% depletion in cholesterol (an essential lipid) observed in the densitometry analysis (Figure 6 and Figure 2B). Discussion In working with this protocol it is important to obtain accurate cell counts when plating mammalian cells and opsonizing C. neoformans cells. This minimizes variation between trials and ensures an accurate 1:1 target to effector ratio throughout the study. It is also critical to coordinate the timing of the cholesterol depletion and infection to prevent the opsonized yeast cells or treated macrophage cells from resting at RT in between the procedures. Long waiting periods could lead to loss of antibody opsonization or the replenishing of depleted cholesterol before infection can begin. If experiments are done with precision the data analysis allows for conclusions to be discerned about the role of cholesterol in phagocytosis. The limitations of the technique prevent any conclusions as to the specific mechanism by which cholesterol depletion lowers the phagocytic index of macrophage like cells, and it is unclear whether the effect is directly due to cholesterol or due to a secondary mechanism. Further work along this vein investigates other constituents of lipid rafts such as sphingolipids or proteins known to function in phagocytosis such as the Fcγ receptor and the complement receptor 3 2. Modifying this technique to use either antibody opsonization or complement alone could help distinguish a role for cholesterol in one or both of these known pathways. It is also important to remember that MβCD extracts cholesterol based on its hydrophobicity and size, thus sterols of a similar size may also be depleted and will migrate at a similar rate as cholesterol on a TLC. It is also important to note that cholesterol depletion can partially affect macrophage activation, this is unlikely to be responsible for the difference observed in uptake as performing the infection in the absence of IFN- and LPS shows the same reduction in the uptake of C. neoformans (data not shown), but it is of interest when modifying this technique to study the anti-fungal activities of macrophages and the role of activation. This method also does not allow us to discern whether cholesterol depletion has any therapeutic implications in fungal infection. Further work in vivo with cholesterol-lowering drugs and epidemiological studies of patients using cholesterol-lowering drugs could further elucidate a role for cholesterol depletion in the treatment of the disease and may offer a more selective way to inhibit cholesterol accumulation. This procedure could easily be used to study uptake of other pathogens or solid particles (i.e., glass beads) being phagocytized and allow for the study of basic biology of phagocytosis. Modifications could allow for the study of other aspects of phagocytosis by treating the macrophage cells with enzymes to selectively degrade other membrane components or various drugs and inhibitors which may be of interest. It should also be noted that flow cytometry may present a more accurate and quantitative way to characterize phagocytosis and could be used to replace the direct microscopic count24. Altogether, this is a fairly simple technique that can be used as a starting point for more in depth studies that answer questions about how lipids may play an important part in infection and immune response. This work was supported by NIH grants AI56168, AI71142, AI87541 and AI100631 to MDP. Maurizio Del Poeta is Burroughs Wellcome Investigator in Infectious Diseases. Figure 1 Cholesterol content of supernatant after treatment Quantification of cholesterol in the supernatant collected from treated cells shows enrichment in MCD when compared to 1x PBS. Cholesterol depletion is 50 ± 5% calculated from total cholesterol in 1x PBS (supernatant + cell lysate). Error bars show standard deviation (n = 5). Figure 2 TLC of cholesterol in cell lysate and densitometry Image of developed TLC plate visualized with MnCl2 charring. A marked decrease in cholesterol is seen after MβCD treatment (A). Densitometry analysis of bands as compared to the PBS treated control (shown as 100%) confirms trend found in Cholesterol quantification assay (B). Please click here to view a larger version of this figure. Figure 3 Uninfected control micrographs of treated J774 macrophages Images of uninfected J774 cells taken at 200X magnification. Scale bar is 50 μm. 1x PBS (A), 10 mM MβCD (B), and 30 mM MβCD (C) treated cells show no change. Please click here to view a larger version of this figure. Figure 4 Infection of J774 macrophages with C. neoformans. Images of infected J774 cells taken at 400X (top row A.1 – C.1) and 1,000X (bottom row A.2 – C.2) magnification are shown. Internalized C. neoformans cells appear as blue-violet spheres with a lighter ring surrounding them. Cells treated with 1x PBS (A), 10 mM MβCD (B), and 30 mM MβCD (C) show differences in C. neoformans uptake. Please click here to view a larger version of this figure. Figure 5 Phagocytic index Phagocytic index is shown with respect to the control group that was treated with PBS (Marked at 100 for comparison). Phagocytic index was reduced by 25% by 10 mM MβCD treatment and by almost 55% by 30 mM treatment. Error bars show standard deviation of the mean (n = 4). Figure 6 Cell viability Variations in cell viability by trypan blue assay show little variation when comparing all three treatment groups. There is a slight drop off in viability in the 30 mM MβCD treatment group, which can be expected from depletion of such a major component of the membrane. Error bars show standard deviation (n = 4). Video Link The video component of this article can be found at http://www.jove.com/video/52432/ Disclosures The authors have nothing to disclose. 1 Sarantis H Grinstein S Subversion of phagocytosis for pathogen survival Cell Host & Microbe 12 4 419 31 10.1016/J.Chom.2012.09.001 2012 23084912 2 Rougerie P Miskolci V Cox D Generation Of Membrane Structures During Phagocytosis And Chemotaxis Of Macrophages: Role And Regulation Of The Actin Cytoskeleton Immunological Reviews 256 1 222 39 10.1111/Imr.12118 2013 24117824 3 Liu T Perlin DS Xue C Molecular Mechanisms Of Cryptococcal Meningitis Virulence 3 173 181 10.4161/Viru.18685 2012 22460646 4 Abadi J Pirofski L A Antibodies Reactive With The Cryptococcal Capsular Polysaccharide Glucuronoxylomannan Are Present In Sera From Children With And Without Human Immunodeficiency Virus Infection The Journal Of Infectious Diseases 180 3 915 9 10.1086/314953 1999 10438394 5 Kechichian TB Shea J Del Poeta M Depletion Of Alveolar Macrophages Decreases The Dissemination Of A Glucosylceramide-Deficient Mutant Of Cryptococcus Neoformans In Immunodeficient Mice Infection And Immunity 75 10 4792 8 10.1128/IAI.00587-07 2007 17664261 6 Casadevall A Cryptococci At The Brain Gate: Break And Enter Or Use A Trojan Horse? The Journal Of Clinical Investigation 120 5 1389 92 10.1172/JCI42949 2010 20424319 7 Chrétien F Pathogenesis Of Cerebral Cryptococcus Neoformans Infection After Fungemia The Journal Of Infectious Diseases 186 4 522 30 10.1086/341564 2002 12195380 8 Luberto C Martiñez-Marino B Identification Of App1 As A Regulator Of Phagocytosis And Virulence Of Cryptococcus Neoformans The Journal Of Clinical Investigation 112 7 1080 94 10.1172/JCI18309 2003 14523045 9 Mcquiston TJ Williamson PR Paradoxical Roles Of Alveolar Macrophages In The Host Response To Cryptococcus Neoformans Journal Of Infection And Chemotherapy: Official Journal Of The Japan Society Of Chemotherapy 18 1 1 9 10.1007/S10156-011-0306-2 2012 22045161 10 García-Rodas R Zaragoza O Catch Me If You Can: Phagocytosis And Killing Avoidance By Cryptococcus Neoformans. FEMS Immunology And Medical Microbiology 64 2 147 61 10.1111/J.1574-695X.2011.00871.X 2012 22029633 11 Coelho C Bocca AL Casadevall A The Intracellular Life Of Cryptococcus Neoformans Annual Review Of Pathology 9 219 38 10.1146/Annurev-Pathol-012513-104653 2014 12 Rao M Peachman KK Alving CR Rothwell SW Depletion Of Cellular Cholesterol Interferes With Intracellular Trafficking Of Liposome-Encapsulated Ovalbumin Immunology And Cell Biology 81 415 423 10.1046/J.1440-1711.2003.01192.X 2003 14636238 13 Sein KK Aikawa M The Prime Role Of Plasma Membrane Cholesterol In The Pathogenesis Of Immune Evasion And Clinical Manifestations Of Falciparum Malaria Medical Hypotheses 51 2 105 110 10.1016/S0306-9877(98)90102-5 1998 9881815 14 Pucadyil TJ Tewary P Madhubala R Chattopadhyay A Cholesterol Is Required For Leishmania Donovani Infection: Implications In Leishmaniasis Molecular And Biochemical Parasitology 133 145 152 10.1016/J.Molbiopara.2003.10.002 2004 14698427 15 Tagliari L Membrane Microdomain Components Of Histoplasma Capsulatum Yeast Forms, And Their Role In Alveolar Macrophage Infectivity Biochimica Et Biophysica Acta 1818 3 458 66 10.1016/J.Bbamem.2011.12.008 2012 22197503 16 Crane JM Tamm LK Role Of Cholesterol In The Formation And Nature Of Lipid Rafts In Planar And Spherical Model Membranes Biophysical Journal 86 5 2965 79 10.1016/S0006-3495(04)74347-7 2004 15111412 17 Brown DA London E Functions Of Lipid Rafts In Biological Membranes Annual Review Of Cell And Developmental Biology 14 111 36 10.1146/Annurev.Cellbio.14.1.111 1998 18 Simons K Toomre D Lipid Rafts And Signal Transduction Nature Reviews Molecular Cell Biology 1 1 31 9 10.1038/35036052 2000 11413487 19 Ilangumaran S Hoessli DC Effects Of Cholesterol Depletion By Cyclodextrin On The Sphingolipid Microdomains Of The Plasma Membrane The Biochemical Journal 335 Pt 2 433 40 1998 9761744 20 Bligh EG Dyer WJ A Rapid Method Of Total Lipid Extraction And Purification Canadian Journal Of Biochemistry And Physiology 37 8 911 917 10.1139/O59-099 1959 13671378 21 Mukherjee S Lee S Casadevall A Antibodies To Cryptococcus Neoformans Glucuronoxylomannan Enhance Antifungal Activity Of Murine Macrophages Infect Immun 63 2 573 579 1995 7822024 22 Deshaw M Pirofski LA Antibodies To The Cryptococcus Neoformans Capsular Glucuronoxylomannan Are Ubiquitous In Serum From HIV+ And HIV− Individuals Clinical And Experimental Immunology 99 3 425 32 1995 7882565 23 Tripathi K Mor V Bairwa NK Del Poeta M Mohanty BK Hydroxyurea Treatment Inhibits Proliferation Of Cryptococcus Neoformans In Mice Frontiers In Microbiology 3 5 187 10.3389/Fmicb.2012.00187 2012 22783238 24 Chaka W Quantitative Analysis Of Phagocytosis And Killing Of Cryptococcus Neoformans By Human Peripheral Blood Mononuclear Cells By Flow Cytometry Clin Diagn Lab Immunol 2 6 753 759 1995 8574842

PMC004xxxxxx/PMC4969178.txt

LICENSE: This file is available for text mining. It may also be used consistent with the principles of fair use under the copyright law. 101242342 32406 Contemp Clin Trials Contemp Clin Trials Contemporary clinical trials 1551-7144 1559-2030 27282117 4969178 10.1016/j.cct.2016.06.002 NIHMS794281 Article Interim Methadone and Patient Navigation in Jail: Rationale and Design of a Randomized Clinical Trial Schwartz Robert P. M.D. 1* Kelly Sharon M. Ph.D. 1 Mitchell Shannon G. Ph.D. 1 Dunlap Laura Ph.D. 2 Zarkin Gary A. Ph.D. 2 Sharma Anjalee M.S.W. 1 O’Grady Kevin E. Ph.D. 3 Jaffe Jerome H. M.D. 14 1* Friends Research Institute, Baltimore, MD USA 2 RTI International, Research Triangle Park, NC USA 3 Department of Psychology, University of Maryland, College Park, College Park, MD USA 4 Department of Psychiatry, University of Maryland School of Medicine, Baltimore, MD USA * Please address correspondence to Robert P. Schwartz, M.D., Friends Research Institute, Inc., 1040 Park Avenue, Suite 103, Baltimore, MD 21201 USA; Voice: 410-837-3977 x276; Fax: 410-752-4218; rschwartz@friendsresearch.org 14 6 2016 07 6 2016 7 2016 01 7 2017 49 2128 This file is available for text mining. It may also be used consistent with the principles of fair use under the copyright law. Background Methadone maintenance is an effective treatment for opioid dependence but is rarely initiated in US jails. Patient navigation is a promising approach to improve continuity of care but has not been tested in bridging the gap between jail- and community-based drug treatment programs. Methods This is an open-label randomized clinical trial among 300 adult opioid dependent newly-arrested detainees that will compare three treatment conditions: methadone maintenance without routine counseling (termed Interim Methadone; IM) initiated in jail v. IM and patient navigation v. enhanced treatment-as-usual. The two primary outcomes will be: (1) the rate of entry into treatment for opioid use disorder within 30 days from release and (2) frequency of opioid positive urine tests over the 12-month follow-up period. An economic analysis will examine the costs, cost-effectiveness, and cost-benefit ratio of the study interventions. Results We describe the background and rationale for the study, its aims, hypotheses, and study design. Conclusions Given the large number of opioid dependent detainees in the US and elsewhere, initiating IM at the time of incarceration could be a significant public health and clinical approach to reducing relapse, recidivism, HIV-risk behavior, and criminal behavior. An economic analysis will be conducted to assist policy makers in determining the utility of adopting this approach. methadone treatment interim methadone patient navigation criminal justice jail Introduction and background Opioid use disorder is a serious problem among the millions of annual arrestees throughout much of the developed world [1, 2]. In the US, individuals with heroin and/or other opioid misuse are overrepresented in jails compared to the general population with 8% of the US sentenced jail population reporting such misuse [3]. Although methadone maintenance treatment (MMT) in the community is an effective treatment for opioid use disorder [4, 5], such treatment is rarely initiated in US jails [6]. Given the increased risk of relapse, re-arrest, and overdose death after brief periods of imposed abstinence [7–9], there is considerable public health and economic interest in demonstrating cost-effective approaches to engage arrestees in effective treatment. Jails house arrestees awaiting trial and those serving sentences up to one year. There are about 3,163 jails in the US [10]. While some US jails provide methadone to pregnant women [11] and a few (e.g., in Albuquerque, NM, Baltimore, MD, New Haven, CT, and New York, NY) continue MMT for patients receiving treatment in the community at the time of arrest [12], the jail at Rikers Island in New York City is one of the only US jails that has reported initiating MMT for those of its inmates who request it [13]. Barriers to providing MMT in jails include security concerns related to methadone storage, stigma, unfamiliarity or philosophical opposition to opioid maintenance, lack of sufficient medical staff, and logistical and cost issues [14, 15]. An additional barrier is the cost of counseling needed under Federal and State methadone regulations. This barrier could be overcome by providing methadone without routine counseling. Such care is delivered throughout many European and Australian communities [16, 17]. In the US, methadone treatment without routine counseling (termed Interim Methadone [IM]) has been shown to be as effective as methadone with routine counseling in suppressing illicit opioid use [18–20]. These findings provide support for offering methadone without routine counseling to out-of-treatment arrestees who are opioid-dependent at the time of incarceration. Reports from the Rikers Island MMT indicate that many inmates started on MMT do not enter treatment upon release [21]. This led to the suggestion of providing help to patients, such as obtaining tokens for public transportation, ID cards, Medicaid, and negotiating the treatment entry process [6]. Patient navigation (PN), is a form of strengths-based case management that has been shown to be beneficial in increasing cancer screening and follow-up rates, improving entry and adherence to HIV treatment, and increasing the likelihood of drug treatment entry [22–26]. It is reasonable to assume that patient navigation might be useful to assist inmates in maintaining continuity of care in MMT upon release from incarceration. Because patient navigation was originally developed to assist women to receive medical services [27] and women seeking to enter substance abuse treatment face particular barriers including social stigma, childcare concerns [28], gender might be a moderating factor in response to navigation. The present study will compare the effectiveness and cost-effectiveness of three conditions: methadone maintenance without routine counseling (termed Interim Methadone; IM) initiated in jail v. IM and patient navigation v. enhanced treatment-as-usual for newly-arrested adults in the Baltimore City Detention Center. This study is part of the National Institute on Drug Abuse-funded SOMATICS cooperative study that is examining approaches to delivering FDA-approved pharmacotherapies to recently arrested adults with opioid dependence. The other two studies, which are examining extended-release naltrexone (XR-NTX), are led by University of California Los Angeles and New York University and are described elsewhere in this journal. 2. Research Design and Study Population 2.1 Study Design This study is a parallel three-group randomized effectiveness trial that examines: (1) interim methadone maintenance (IM) initiated in jail v. (2) IM and patient navigation (PN) v. (3) enhanced treatment-as-usual (ETAU) that includes a brief methadone detoxification. The participants will be 300 opioid-dependent newly-arrested detainees receiving treatment for opioid withdrawal in the Baltimore City Detention Center. 2.2 Research questions and hypotheses The primary research aims are to determine the relative effectiveness and IM+PN v. IM alone v. ETAU delivered to a newly-arrested adult population with an opioid use disorder in regard to: (1) increasing the likelihood of post-release treatment entry and retention in community-based methadone treatment; (2) reducing the likelihood of post-release opioid and cocaine use; and, (3) reducing HIV-risk and criminal behavior, arrest, and days of incarceration. We hypothesize that IM+PN and IM Alone Conditions will have superior outcomes compared to ETAU, and IM+PN will have superior outcomes to IM alone. Secondary research aims are to determine the relationship between gender and effectiveness and to examine the cost, cost effectiveness, and cost-benefit of the three study Conditions from a societal perspective. 2.3 Study site The study is being conducted by Friends Research Institute in Baltimore. The recruitment and during-detention methadone treatment site is in the Baltimore City Pretrial and Detention Services, a division of Maryland’s Department of Public Safety and Correctional Services (DPSCS). DPSCS operates a methadone treatment program that provides methadone detoxification and maintains methadone treatment for arrestees who were enrolled in a methadone program at the time of arrest. Prior to the present study, this program did not initiate methadone maintenance treatment for arrestees with an opioid use disorder with the exception of pregnant women. The site does not provide buprenorphine or extended-release naltrexone to arrestees. The four community Opioid Treatment Programs agreed to receive new patients who were released from the Detention Center on methadone because these programs did not have waiting lists. 2.4 Inclusion/Exclusion Criteria To be included in the study, detainees must: (1) meet DSM-5 criteria for opioid use disorder; (2) be detained for at least 48 hours (because those detainees who are released quickly are most often released within 48 hours and hence would not have time to receive services provided through the study); (3) be receiving opioid withdrawal treatment at the men’s and women’s Pre-trial Facilities; (4) be able and willing to provide informed consent in English; (5) be detained for a charge that, if found guilty, will likely result in a sentence of less than 1 year; (6) plan to reside in Baltimore (city or county) upon release; and (7) be 18 years of age or older. Individuals are excluded if they: (1) are enrolled in opioid agonist treatment (methadone or buprenorphine treatment) in the community at the time of arrest; (2) have a medical or psychiatric condition that would make participation unsafe in the judgment of the medical staff or the PI; (3) are pregnant; (4) are allergic to methadone; or (5) require treatment for moderate or severe alcohol or sedative hypnotic withdrawal. 2.5 Recruitment The Medical Staff of the Detention Center refers newly-detained adults who are receiving opioid detoxification to speak with the Friends Research Institute’s Research Assistant (RA) about the study. Nursing coordinates the RA’s visit for informed consent and baseline assessment and the pre-screening medical eligibility visit with the methadone program physician. 2.6 Informed Consent During the research visit, the RA describes the study, reviews the informed consent form and reviews the risks and benefits of participation. The RA underlines the points that participation is completely voluntary and will not impact their criminal justice status. Individuals who decline participation are informed about alternative options including completing the detoxification that they are receiving and attending drug abuse treatment in the community. The RA administers a consent quiz on which individuals must receive a perfect score within three attempts to be deemed eligible. 2.7 Screening, Randomization, and Follow-up Procedures After the individual provides written informed consent, the RA administers the baseline instruments described below. The RA completes the eligibility checklist and obtains the prescreen eligibility checklist and physical exam from the physician. The PI reviews the eligibility checklists and source documents (RA eligibility checklists, consent quiz, informed consent, physician pre-screen eligibility checklist and physical exam and pregnancy test results [which are collected for all women upon detention]) and then enrolls eligible individuals. Following enrollment, the RA meets with the participant to provide standardized information about drug abuse, HIV prevention, overdose prevention, and information on how to contact Baltimore’s substance abuse treatment helpline for an intake and referral to treatment in the community. The RA opens the next randomization envelope and informs the participant of his/her assigned study Condition. Participants are assigned to Conditions using a random permutation procedure, such that, within gender, for each block of 3, 6, or 9 participants, one-third will be assigned at random to the IM+PN Condition, one-third to the IM alone Condition, and one-third to the ETAU Condition. Random block sizes are used to thwart any attempt by the RA or others to deduce the random assignment procedure. The Project Manager provides the RA with sealed opaque envelopes based on this random permutation procedure. Participants do not receive compensation for the baseline interview during incarceration in order not to have the appearance of monetary coercion for study enrollment, but they will receive $30 for each of the four follow-up interviews regardless of whether they are in the community or re-incarcerated. 2.8 Data Management RAs complete baseline study assessments on paper teleform (including only the participant’s study ID number and no other identifiers) and upload PDFs of the teleforms to the UCLA Data Management Center (DMC). Paper forms are necessary because the RAs do not have internet access in the Detention Center. Data from follow-up visits are entered by the RA using UCLA DMC’s web-based data entry system. 3.0 Approvals and Data and Safety Monitoring 3.1 Approvals The Friends Research Institute (FRI) Institutional Review Board (IRB) approved the study. The US Office of Human Research Protections approved the study protocol and agreed with the IRB that it met the conditions for ethical conduct of research among prisoners under 45 CFR 46.306(a)(2)(iv). The study was registered at ClinicalTrials.gov (NCT 02334215). A federal Certificate of Confidentiality was obtained to protect the confidentiality of the participants’ data. 3.2 Data and Safety Monitoring The study is being monitored by a Data and Safety Monitoring Board (DSMB) at UCLA. The FRI IRB, the UCLA DSMB, and NIDA (the study sponsor) monitor recruitment, retention, and study safety. All Serious Adverse Events are reported to the IRB, DSMB, and NIDA medical monitor regardless of their possible relationship to study procedures. 4.0 Interventions 4.1 Enhanced Treatment-as-Usual (ETAU) Individuals assigned to ETAU receive opioid withdrawal treatment with a methadone detoxification over about one week. In addition, Research Assistants (RAs) provided these participants with standardized information used in all three SOMATICS studies on the harms associated with drug abuse, referral information to substance abuse treatment in the community, and HIV and overdose prevention information. 4.2 Interim Methadone Maintenance Alone (IM Alone) Individuals assigned to IM Alone receive an individualized gradual dose induction and administered methadone under direct observation through the Pretrial and Detention Service’s Methadone Treatment Program. They do not receive routine counseling but are able to receive mental health treatment at their request (as would any other inmate). They remain on methadone during their sojourn in the Detention Center until their release, unless they request to discontinue treatment or are transferred to another facility (e.g., due to sentencing or for serious rule infractions such as attempted diversion of methadone). In these cases, whenever possible, the participant undergoes a gradual dose reduction under medical supervision. The RA asks the participant at enrollment which of four participating methadone maintenance treatment (MMT) programs he/she would like to attend in the community. The Pre-trial and Detention Services Methadone Program nursing staff will arrange for transfer to the community programs upon release. Participants are told by the RAs to report to their MMT program on the day following release (or within three days, at the most) to ensure admission and continuity of care. The nursing staff arranges “courtesy dosing” at one of the four cooperating community-based MMT programs (as would occur on a trip to another state) until the participant can be seen by the receiving MMT program for intake and admission. 4.3 Interim Methadone plus Patient Navigation (IM+PN) In addition to interim methadone described above, participants assigned to the IM+PN Condition are seen by the study’s Patient Navigator once while detained for an assessment of community reentry needs (focused on barriers to MMT entry). The navigator is available to the participant approximately weekly for up to three months post-release. During this time, following a patient navigation manual developed for the project based on the work of Sorensen and colleagues [6], the navigator seeks to meet the participant at the MMT program to increase the likelihood of a smooth admission process and reaches out to those participants who do not attend their first visit or who subsequently drop out of treatment in the community following release. Using strengths-based case management and motivational techniques, the navigator helps the participant obtain needed services such as an ID card, reduced fare bus passes, health insurance, and medical or psychiatric appointments. The navigator has a small amount of funds available (an average of approximately $40 per participant) to assist the participant in obtaining ID cards, bus passes, and other related items. 5.0 Assessments Assessments are conducted by trained RAs. The RAs are blind to study Condition at baseline but it will not be possible to blind RAs at follow-up visits conducted at 1, 3, 6, and 12 months post-release because follow-up interviews for those in treatment are conducted at the MMT. All participants will be sought for follow-up interviews regardless of whether they remained in treatment. Addiction Severity Index (ASI) - Lite is a 30–45 minute self-report interview covering seven domains over the participant’s past 30 days, including days of heroin and cocaine use, and days committing illegal activities [29]. The ASI will be administered at baseline and all follow-up points. Urine Drug Screening Urine samples will be collected by project staff at 1, 3, 6, and 12 months post-release and tested by an approved rapid drug screen test card for opiates, oxycodone, methadone, buprenorphine, cocaine, marijuana, amphetamine, and benzodiazepines. Methadone or buprenorphine positive tests will not be treated as “illicit” drugs for the purposes of analysis if the participants are enrolled in a treatment program. Modified Composite International Diagnostic Interview, version 2 (CIDI-2) for Substance Use Disorders The modified CIDI-2 [30] will be used to determine whether individuals met the DSM-5 criteria for opioid and cocaine use disorders or remission in the 12 months prior to baseline and during the one-month period prior to the 3, 6, and 12 month study assessments. The CIDI-2 was recommended by an expert panel of the NIDA Clinical Trials Network (CTN) for gauging DSM-IV remission in clinical studies [31]. It has been shown to have excellent reliability in diagnosing individuals with drug dependence [32]. Arrests and Incarcerations In addition to self-reports, the official arrest and incarceration records will be obtained for 1 year prior and 1 year post-study enrollment from the Maryland Department of Public Safety and Correctional Services. Risk Assessment Battery (RAB) is a 45-item questionnaire covering substance use and sexual HIV-risk behaviors that has been extensively used with drug-dependent populations [33]. It will be administered at baseline, 6 and 12 month follow-up. The scale’s drug- and sex-risk scores will be used as secondary outcome measures. World Health Organization Quality of Life (WHOQOL-BREF) is a brief 32-item instrument developed by the World Health Organization that has been used in a wide variety of populations internationally [34–36] and has been found to have strong psychometric properties [37, 38]. The WHOQOL-BREF produces scores in four QoL domains: physical, psychological, social, and environmental. The WHOQOL-BREF also contains a single item, which is not incorporated into any of the four scale scores, asking participants to rate their overall QoL on a 5-point Likert-type scale from very poor to very good. It will be administered at baseline, 1, 3, 6 and 12 month follow-up. Methadone Dose Higher methadone maintenance doses in jail have been shown to be associated with higher rates of treatment entry following release from incarceration [39] and improved retention rates in community-based treatment [5]. As such, methadone dose will be recorded at release from the Detention Center and at each follow-up to permit an examination of the relationship between methadone dose and outcomes by treatment Condition. Methadone Treatment Exposure Questionnaire This brief 7 item questionnaire was devised for the present study and inquires whether and when the participant entered methadone treatment following release as well as the number of days of methadone treatment in the community. It will be administered at baseline, 1, 3, 6 and 12 month follow-up. We will validate the self-report with available Opioid Treatment Program records. Economic Form 90 survey was originally designed to collect alcohol use and economic outcome data for alcohol treatment studies [40, 41]. It will be modified to collect data on patients’ residential drug treatment, outpatient, emergency room, and inpatient hospital utilization; and criminal behavior, including number of arrests, severity of offense, and nights incarcerated. Also collected will be labor market information, including employment status, current wage, average hours worked per week, and amount of money received from government sources (e.g., Social Security) and off-the-books earnings. It will be administered at baseline, 3, 6 and 12 month follow-up. Overdose Adverse Event Form A brief questionnaire devised for this study will be administered at 1,3, 6 and 12 month follow-up to determine the number and type of non-fatal overdoses that occur. Substance Abuse Services Cost Analysis Program (SASCAP) Provider costs for each of the study conditions will be estimated using an activity-based costing approach which will allow cost estimation at the service level for study participants. To collect activity-level resource use and cost data, we will modify the SASCAP [42] to collect resource use and cost data for identified activities that are performed within each study condition. Activities will comprise clinically relevant treatment activities (including related support activities), and we will exclude research-related activities from the cost estimation. The SASCAP consists of a provider questionnaire administered once during the intervention phase to collect activity-level resource use and cost data for provider staff and non-labor resources such as contracted services, building space, supplies and materials, and other miscellaneous resources (e.g., utilities). It has been used to reliably estimate the costs of specific treatment activities and the total cost per patient [43–46]. 5.1 Primary Outcomes The study has two primary outcomes: (1) the rate of entry into treatment for opioid use disorder within 30 days from release from incarceration measured by self-report on the methadone treatment exposure questionnaire and (2) frequency of opioid positive urine test results over the 12-month follow-up period, determined from study administered urine screening at each of the four follow-up points. 5.2 Secondary Outcomes The study has a number of secondary outcomes measured as change over time across the four follow-up interviews. These include the presence of opioid use and cocaine use disorder, the drug- and sex-risk scores on the RAB, the specific domain and overall scores on the WHOQOL-BREF [35], the number of days in treatment for opioid use disorder, self-reported illicit opioid and cocaine use and criminal behavior, the number of arrests and incarcerations, and health care utilization. The cost of substance use services, health care utilization, and incarceration will be calculated over the 12 month post-release follow-up period. 5.3 Hypotheses There are several study hypotheses. Hypothesis 1A is that the IM+PN and IM Alone Conditions will have higher rates of treatment entry and greater retention, and concomitant lower rates of illicit opioid and cocaine use and of meeting DSM-5 criteria for opioid and cocaine use disorders, HIV-risk behavior, criminal behavior, arrests, and days of incarcerations than the ETAU Condition. We are not aware of any randomized trial comparing methadone treatment entry rates for jail-based methadone maintenance, with or without PN, compared to treatment as usual. The rationale for this hypothesis stems from a longitudinal non-random assignment study from the Rikers Island MMT Program that has shown better treatment entry rates post-release for inmates started on MMT rather than provided with detoxification in jail [6]. Hypothesis 1B is that the IM+PN condition will have superior outcomes compared to the other study conditions. This hypothesis is supported by research showing that PN is associated with higher rates of treatment entry than no PN in non-jail and non-prison samples of opioid-addicted adults [22]. Hypothesis 2A is that women in the IM+PN condition will have superior outcomes to men in the IM+PN condition, while hypothesis 2B is that women in the IM alone and ETAU will have poorer outcomes than men in those two conditions. These hypotheses stem from the few studies of reentry from jail for men and women treated with methadone that have shown that women are less likely to enter and remain in treatment [6]. In contrast, the PN literature shows that women respond well to this intervention [47, 48]. Therefore, we hypothesize that women will respond better than men to IM+PN. 6.0 Statistical Analysis 6.1 Explanatory Variables There will be a single treatment variable with three conditions: Intervention Condition [IM+PN v. IM Alone v. ETAU); a single moderator variable: Participant Gender, and three predictor variables – participant age, prior methadone maintenance treatment (yes v. no), and self-reported cocaine use at baseline. The predictor variables were chosen because of their association with outcomes in prior research in community based methadone treatment [49–53]. Finally, the “repeated factor” for all outcome variables measured repeatedly (see Table 1) will be assessment Time point, allowing for evaluation of both differential course and impact of the interventions. 6.2 Planned Contrasts It is possible to construct two orthogonal, single degree of freedom planned contrasts that directly test Hypothesis 1A and 1B, respectively. Contrast 1A will compare the two IM treatment conditions (IM+PN and IM Alone, pooled) to the ETAU condition, directly addressing the question of the differential effectiveness of some form of IM treatment in comparison to ETAU. Contrast 1B will compare the IM+PN condition to the IM alone condition, directly addressing the question of the relative effectiveness of adding PN to IM treatment. Similarly, it is possible to construct two orthogonal, single df planned contrasts that directly test Hypothesis 2A and 2B, respectively. Contrast 2A will compare males and females in the IM+PN condition, directly addressing the question of the differential effectiveness of IM+PN treatment for males and females. Contrast 2B will compare males and females in the IM Alone and ETAU conditions, pooled, directly addressing the question of the differential effectiveness of non-PN treatments for males and females. Given that both the course and impact of treatment are important to evaluate, planned contrasts can be examined both as each interacts with the “repeated factor” of Time (when there is a repeated factor), and as a “simple effect” at a given time point, in the event either Planned Contrast X Time interaction subeffect proves to be significant. 6.3 Economic Evaluation Using the collected cost data, we will derive total costs and costs per participant, and costs for specific services for each intervention. The total provider cost for each of the interventions will be the sum across each activity of: (1) staff labor costs (e.g., time spent performing intervention activities); (2) costs of building space; (3) costs of any equipment; (4) costs of medication; (5) costs of any supplies or materials; and (6) costs of any other miscellaneous resources used in the intervention. Taking the mean across participants for a given intervention will yield the mean cost per participant of that intervention. Following our cost estimation, we will conduct a cost-effectiveness analysis of the interventions from the provider perspective. We will combine the estimated provider costs of delivering the interventions with selected intervention effects. Our cost-effectiveness method will follow the approach described in the literature (e.g.,[44, 54]). Starting with the intervention with the smallest cost (or effectiveness), cost-effectiveness ratios will be computed for each intervention relative to the next most expensive option after eliminating intervention options that are dominated by other interventions [54]. An intervention may be either strictly dominated (higher cost and lower effectiveness than another option) or weakly dominated (higher cost-effectiveness ratio than a more effective option). Separate cost-effectiveness analyses will be performed for each selected outcome. Primary outcomes for the cost-effectiveness analysis include: (1) percentage of participants without opioid use disorder diagnosis at 12-month follow-up (based on the DSM-5 Opioid Use Disorder Diagnosis; and (2) number of days of opioid use in the past 30 days assessed at the 12-month follow-up. Secondary outcomes that we will also examine include: (1) number of days incarcerated; and (2) percentage of participants not engaging in HIV-related risky behavior (as measured by the RAB [33]). Finally, we will conduct a cost-benefit analysis to estimate the economic benefits associated with reductions in criminal activity and criminal justice system costs, improved employment, and reduced health care use (e.g., ER visits, hospitalizations). The difference between the monetized economic benefits and intervention costs represents the net economic benefits of the interventions (or cost savings). 6.4 Sample Size, Power, and Effect Size Power was estimated according to a procedure for general linear models outlined by Stroup [55, 56] as well as by the set correlation method [57, 58], assuming the primary outcome measures follow a normal distribution. Assuming a Type I error rate of .01 and a sample size of 300, the general linear model power estimates (1 – β, where β is the Type II error rate) associated with the hypotheses exceeded .81 in all cases, assuming “small” (.2 of a standard deviation [57]) differences associated with each such effect, even assuming an unstructured covariance matrix for those outcomes measured repeatedly. For the set correlation approach, assuming a Type I error rate of .01 and a sample size of 270 due to attrition in order to remain conservative, an effect size f2=.045 for a Planned Contrast effect for an outcome measured only once, and effect sizes f2=.045, .059, and .064 with a Planned Contrast X Time subeffect for outcomes measured two, four, or five times, respectively, for the hypotheses would yield a power of .9 for that subeffect, where f2 is defined as the ratio of the variance of the means relative to the variance of the observations [57]. These effect sizes, f2, all fall in the “small-to-medium” range, with f2=.02 considered a “small” effect and f2=.15 a “medium” effect [57]. In other words, and imprecisely, under the assumption that the effect in the population was ≥ .045 for a Planned Contrast or ≥ .045, .059, or .064 for the Treatment Condition X Time subeffects, for outcomes measured two, four, or five times, respectively, there is an 90% chance of concluding that effect is significant if α is set to .01 and 270 participants are assessed at 12-month follow-up. 8.0 Discussion Despite the large number of adults with opioid use disorder (OUD) arrested in the US every year, pharmacotherapy for OUD is rarely provided in the over 3,000 US detention centers. After almost 50 years of experience with methadone, only one US jail (in New York City) has reported initiating opioid agonist treatment pharmacotherapy for arrestees with OUD. As mentioned briefly in the introduction, there are numerous barriers to initiating pharmacotherapy for OUD in jail settings. Methadone treatment, unlike buprenorphine or extended release naltrexone, requires special federal and state program licenses. A critical mass of patients is desired to make operating a methadone program in a correctional institution practical, thus making it less likely to open such programs in smaller jails or those with small numbers of opioid dependent inmates. There are also philosophic barriers to providing opioid agonist medications to inmates. Additionally, budgetary considerations are barriers to providing any of these treatments. Corrections budgets may not reap most of the potential benefit of a post-release reduction in criminal behavior and health care costs. The lack of strong cost-benefit economic data of these programs makes it somewhat difficult to convince some policymakers of the utility of these programs. Developing a research database for these treatments with prisoners has been difficult, although not impossible, because of the legacy of concerns regarding human subject protection in vulnerable populations and that it requires approval from the federal Office of Human Research Protection. Despite all of these challenges, the number of correctional institutions providing opioid agonist treatment is increasing throughout the world. To date, prisons in most countries of the European Union, Australia, Canada, China, Iran, Indonesia, Moldova, and Kyrgyzstan have such programs [59]. The rationale for these programs has been the principle of making treatment in the community available to incarcerated individuals, the goal of reducing the spread of HIV infection, and the hopes of reducing recidivism. The present study, by examining the use of methadone without counseling, will provide a potentially practical solution for detention centers that may not have the funds to provide counseling but that already have existing medical infrastructure to administer methadone. Unlike the other FDA-approved medications for treating opioid dependence, methadone itself costs pennies per day. Thus, these cost-considerations might provide some support for the use of interim methadone treatment in jails. Because the low range of reported rates of entry into community-based MMT programs by inmates initiated on methadone at the Rikers Island program (ranging from 14% to 50%) leaves considerable room for improvement, we have chosen to compare IM alone to IM+PN. We expect that patient navigation, by reducing barriers to continued treatment in the community and re-engaging those who drop out over the first 3 months of MMT, will result in the IM+PN condition having higher rates of treatment entry, longer retention in treatment, and thereby have superior treatment outcomes. In designing the present trial, we considered alternative designs. First, we considered using buprenorphine or extended-release naltrexone. Both of these medications have some advantages because they can be used with less regulatory and storage security burdens than methadone. However, they both are considerably more expensive to purchase than methadone. Buprenorphine appears to be much easier to divert during treatment than methadone because it must be administered sublingually, and if diversion is to be minimized, the patient must be observed for 5–10 minutes until the dose is absorbed (in tablet or film form). A study using buprenorphine in a Maryland prison with sentenced prisoners expected to be released within several months found that more than 10% of participants attempted to divert the medication and hence were discontinued from receiving the medication [60]. This rate of attempted buprenorphine diversion was similar to that found by Magura and colleagues in a study at Rikers Island comparing methadone to buprenorphine treatment for sentenced jail inmates [21]. Although extended-release naltrexone has the advantage of being a non-controlled substance, it is relatively expensive and not yet widely available in the community. Nevertheless, there are certainly advantages and disadvantages of these three medications and conceptually it would be advantageous to patients to have all three as available in jails as they are in the community. We decided to include an enhanced treatment-as-usual (ETAU) arm as a comparison because virtually all jails in the US (including in Baltimore) use detoxification with non-opioid medications (or less often with methadone) as their “treatment as usual.” ETAU also includes substance use, overdose, and HIV-prevention information, and a referral to community treatment services, thus exceeding the normal provision of service to this population. These latter elements of ETAU are shared by the other two SOMATICS studies. The economic analysis may be of benefit to policy makers and correctional and public health officials. We anticipate that IM alone will have greater treatment costs than ETAU and that IM+PN will have greater costs than IM alone. However, the greater costs for the two experimental conditions may yield superior outcomes and thereby prove more cost-effective than ETAU. Similarly, cost offset in terms of reduced hospital and criminal justice costs associated with superior outcomes for the two experimental conditions may yield greater net benefits for these conditions compared with ETAU. There are a number of limitations to the study design. First, because the efficacy of methadone treatment was not in question, the use of a placebo was deemed inappropriate, and therefore the Patient Navigation intervention is being compared to an enhanced version of treatment as usual. Second, the study is being conducted in a "real world" jail in an urban community with both a substantial number of arrests and a relatively high prevalence of illicit opioid use. Therefore, findings may not generalize to other jail systems in other localities where the number of arrestees or the prevalence of illicit opioid use or the availability of maintenance treatment in the community could make the utilization of methadone maintenance impractical. For the same reason, the economic analysis may not generalize to other localities. However, in jail facilities where there is a lower prevalence of illicit opioid use among arrestees there may be sufficient time to supervise the use of buprenorphine. Initiating buprenorphine maintenance for opioid dependent arrestees might result in fewer deaths and continued treatment following discharge. We would like to thank the medical team at Wexford Health: Kelly Blizzard, RN, Tabethia Pardoe, RN, Kara Hope, RN and Drs. Woreta, Goodwin, Tessias, Getachew and Luka and the Maryland Department of Public Safety and Correctional Services including Drs. Sharon Baucom and Adaora Odunze; and the Daybreak, Glenwood Life, Man Alive, and REACH Opioid Treatment Programs, for their assistance in making this study possible. This study is being funded by the National Institute on Drug Abuse grant number 2U01DA013636 (PI: Schwartz). ClinicalTrials.gov: NCT02334215 Table 1 Data Collection Schedule and Measures Measures Baseline 1-month 3-month 6-month 12-month Treatment Entry (Methadone Treatment Exposure Form) ♦ Days in Treatment (self-report from EF-90) ♦ ♦ ♦ Illicit Opioid and Cocaine Use (ASI and urine drug testing) ♦ ♦ ♦ ♦ ♦ DSM-5 Criteria of opioid and cocaine use disorder (modified CIDI-2 SAM) ♦ ♦ ♦ ♦ Criminal Behavior (Addiction Severity Index) ♦ ♦ ♦ ♦ ♦ Arrests and Incarcerations: (Self report) ♦ ♦ ♦ ♦ HIV Risk Behavior (Risk Assessment Battery) ♦ ♦ ♦ Overdose Adverse Event Form ♦ ♦ ♦ ♦ Methadone Treatment Exposure Questionnaire ♦ ♦ ♦ ♦ Quality of Life (WHOQOL-BREF) ♦ ♦ ♦ ♦ ♦ Economic Form 90 (Health care utilization) ♦ ♦ ♦ ♦ Substance Abuse Services Cost Analysis Program (SASCAP) † Arrest and incarceration (Criminal Justice Records) ♦ ♦ Note: ASI = Addiction Severity Index; CIDI-2 SAM = Modified Composite International Diagnostic Interview 2 Substance Abuse Module; WHOQOL-BREF = World Health Organization Quality of Life-Brief Form * Participants transitioning directly to other community Methadone Maintenance Programs or buprenorphine treatment will be considered still in treatment. † Consistent with prior research, the SASCAP will be administered once during the treatment phase. Table 2 Consent Quiz INSTRUCTIONS: Please circle T for True or F for False. T or F 1. This study is comparing three ways to treat opiate addiction in the Detention Center. T or F 2. If I am in the study, I can choose which study group I am in. T or F 3. Methadone has no side effects. T or F 4. If I get the methadone detox group I may have a greater chance of overdosing if I relapse     to heroin use. T or F 5. I can drop out of the study once I start. T or F 6. The study does not provide drug abuse counseling services while I am in the Detention     Center. T or F 7. The patient navigator will help every participant in the study. T or F 8. Because I am in the Detention Center, I have to be in the study. T or F 9. Possible side effects of methadone include drowsiness. T or F 10. There are risks to being in the study. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Reference 1 Dolan K Khoei EM Brentari C Stevens A Prisons and drugs: A global review of incarceration, drug use and drug services 2007 Oxford Beckley Foundation 2 Fazel S Bains P Doll H Substance abuse and dependence in prisoners: a systematic review Addiction (Abingdon, England) 2006 101 2 181 191 3 James DJ Profile of Jail Inmates, 2002. Special Report. US Department of Justice Programs Bureau of Statistics 2004 accessed May 12, 2016 http://www.bjs.gov/content/pub/ascii/pji02.txt 4 Johnson RE Chutuape MA Strain EC Walsh SL Stitzer ML Bigelow GE A comparison of levomethadyl acetate, buprenorphine, and methadone for opioid dependence The New Engl. J Med 2000 343 18 1290 1297 11058673 5 Strain EC Bigelow GE Liebson IA Stitzer ML Moderate- vs high-dose methadone in the treatment of opioid dependence: a randomized trial JAMA 1999 281 11 1000 1005 10086434 6 Magura S Rosenblum A Lewis C Joseph H The effectiveness of in-jail methadone maintenance J Drug Issues 1993 23 75 99 7 Office of National Drug Control Policy 2013 Annual report, arrestee drug abuse monitoring program II, Executive Office of the President 2014 Washington, DC 8 Bird SM Hutchinson SJ Male drugs-related deaths in the fortnight after release from prison: Scotland, 1996–99 Addiction (Abingdon, England) 2003 98 2 185 190 9 Farrell M Marsden J Acute risk of drug-related death among newly released prisoners in England and Wales Addiction (Abingdon, England) 2008 103 2 251 255 10 A.J. Assocation Statistics of Note accessed March 10, 2015 https://members.aja.org/About/StatisticsOfNote.aspx 11 Nunn A Zaller N Dickman S Trimbur C Nijhawan A Rich JD Methadone and buprenorphine prescribing and referral practices in US prison systems: results from a nationwide survey Drug Alcohol Depend 2009 105 1–2 83 88 19625142 12 Trigg BG Dickman SL Medication-assisted therapy for opioid-dependent incarcerated populations in New Mexico: statewide efforts to increase access Substance abuse : official publication of the Association for Medical Education and Research in Substance Abuse 2012 33 1 76 84 13 Center for Substance Abuse Treatment, Substance Abuse Treatment for Adults in the Criminal Justice System, Substance Abuse and Mental Health Services Administration (Treatment Improvement Protocol [TIP] Series, No. 44) DHHS Publication No. (SMA) 05-4056 2005 Rockville, MD 14 McKenzie M Nunn A Zaller ND Bazazi AR Rich JD Overcoming obstacles to implementing methadone maintenance therapy for prisoners: implications for policy and practice J Opioid Manag 2009 5 4 219 227 19736902 15 Rich JD McKenzie M Shield DC Wolf FA Key RG Poshkus M Clarke J Linkage with methadone treatment upon release from incarceration: a promising opportunity J Addict. Disease 2005 24 3 49 59 16186082 16 European Monitoring Centre for Drugs and Drug Addiction (EMCDDA), Annual report: The State of the drugs problem in Europe 2010 Portugal EMCDDA accessed March 17, 2015 http://www.emcdda.europa.eu/online/annual-report/2010 archived by Webcite at http://www.webcitation.org/5yG72bp1G 17 Farrell M Ward J Mattick R Hall W Stimson GV des Jarlais D Gossop M Strang J Methadone maintenance treatment in opiate dependence: a review BMJ 1994 309 6960 997 1001 7950725 18 Schwartz RP Kelly SM O'Grady KE Gandhi D Jaffe JH Interim methadone treatment compared to standard methadone treatment: 4-Month findings J Subst. Abuse Treat 2011 41 1 21 29 [PMCID: PMC3110526] 21353445 19 Schwartz RP Kelly SM O'Grady KE Gandhi D Jaffe JH Randomized trial of standard methadone treatment compared to initiating methadone without counseling: 12-month findings Addiction (Abingdon, England) 2012 107 5 943 952 PMCID: PMC3319854 20 Gruber VA Delucchi KL Kielstein A Batki SL A randomized trial of 6-month methadone maintenance with standard or minimal counseling versus 21-day methadone detoxification Drug Alcohol Depend 2008 94 1–3 199 206 18243585 21 Magura S Lee JD Hershberger J Joseph H Marsch L Shropshire C Rosenblum A Buprenorphine and methadone maintenance in jail and post-release: a randomized clinical trial Drug Alcohol Depend 2009 99 1–3 222 230 18930603 22 Gardner LI Metsch LR Anderson-Mahoney P Loughlin AM del Rio C Strathdee S Sansom SL Siegal HA Greenberg AE Holmberg SD A.T.A.A.S.S. Group Efficacy of a brief case management intervention to link recently diagnosed HIV-infected persons to care AIDS (London, England) 2005 19 4 423 431 23 Sorensen JL Masson CL Delucchi K Sporer K Barnett PG Mitsuishi F Lin C Song Y Chen T Hall SM Randomized trial of drug abuse treatment-linkage strategies J. Consult. Clin. Psychol 2005 73 6 1026 1035 16392976 24 Bradford JB Coleman S Cunningham W HIV System Navigation: an emerging model to improve HIV care access AIDS patient care and STDs 21 Suppl 2007 1 S49 S58 25 Mejta C Bokos PJ Mickenberg J Maslar ME Senay E Improving substance abuse treatment access and retention using a case management approach J Drug Issues 1997 27 2 329 340 26 Scott CK Dennis ML Foss MA Utilizing Recovery Management Checkups to shorten the cycle of relapse, treatment reentry, and recovery Drug Alcohol Depend 2005 78 3 325 338 15893164 27 Freeman HP Muth BJ Kerner JF Expanding access to cancer screening and clinical follow-up among the medically underserved Cancer practice 1995 3 1 19 30 7704057 28 Copeland J A qualitative study of barriers to formal treatment among women who self-managed change in addictive behaviours J Subst Abuse Treat 1997 14 2 183 190 9258863 29 McLellan AT Kushner H Metzger D Peters R Smith I Grissom G Pettinati H Argeriou M The Fifth Edition of the Addiction Severity Index J Subst Abuse Treat 1992 9 3 199 213 1334156 30 Andrews G Peters L The psychometric properties of the Composite International Diagnostic Interview Social psychiatry and psychiatric epidemiology 1998 33 2 80 88 9503991 31 Forman RF Svikis D Montoya ID Blaine J Selection of a substance use disorder diagnostic instrument by the National Drug Abuse Treatment Clinical Trials Network J Subst Abuse Treat 2004 27 1 1 8 15223087 32 Horton J Compton W Cottler LB Reliability of substance use disorder diagnoses among African-Americans and Caucasians Drug Alcohol Depend 2000 57 3 203 209 10661671 33 Metzer DS Navaline HA Woody GE Assessments of substance abuse: HIV risk assessment battery (RAB), Encyclopedia of Drugs Alcohol, and Addictive Behavior 2001 34 Noerholm V Groenvold M Watt T Bjorner JB Rasmussen NA Bech P Quality of life in the Danish general population--normative data and validity of WHOQOL-BREF using Rasch and item response theory models Quality of life research : an international journal of quality of life aspects of treatment, care and rehabilitation 2004 13 2 531 540 35 The World Health Organization Development of the World Health Organization WHOQOL-BREF quality of life assessment The WHOQOL Group, Psychological medicine 1998 28 3 551 558 9626712 36 The World Health Organization Quality of Life (WHOQOL) - Bref 2004 Geneva, Switzerland World Health Organization 37 O'Carroll RE Smith K Couston M Cossar JA Hayes PC A comparison of the WHOQOL-100 and the WHOQOL-BREF in detecting change in quality of life following liver transplantation Quality of life research : an international journal of quality of life aspects of treatment, care and rehabilitation 2000 9 1 121 124 38 Skevington SM Lotfy M O'Connell KA W. Group The World Health Organization's WHOQOL-BREF quality of life assessment: psychometric properties and results of the international field trial. A report from the WHOQOL group Quality of life research : an international journal of quality of life aspects of treatment, care and rehabilitation 2004 13 2 299 310 39 Wickersham JA Zahari MM Azar MM Kamarulzaman A Altice FL Methadone dose at the time of release from prison significantly influences retention in treatment: implications from a pilot study of HIV-infected prisoners transitioning to the community in Malaysia Drug Alcohol Depend 2013 132 1–2 378 382 23414931 40 Miller WR Del Boca FK Measurement of drinking behavior using the Form 90 family of instruments J. Stud. Alcohol 1994 12 112 118 41 Scheurich A Muller MJ Anghelescu I Lorch B Dreher M Hautzinger M Szegedi A Reliability and validity of the Form 90 interview European Addict Res 2005 11 1 50 56 15608472 42 Zarkin GA Dunlap LJ Homsi G The Substance Abuse Services Cost Analysis Program (SASCAP): A new method for estimating drug treatment services costs Eval Program Plann 2004 27 1 35 43 43 Zarkin GA Bray JW Mitra D Cisler RA Kivlahan DR Cost methodology of COMBINE J Stud Alcohol 2005 15 50 55 discussion 33 44 Zarkin GA Bray JW Aldridge A Mitra D Mills MJ Couper DJ Cisler RA Cost and cost-effectiveness of the COMBINE study in alcohol-dependent patients Arch Gen Psychiatry 2008 65 10 1214 1221 18838638 45 Zarkin GA Bray JW Davis KL Babor TF Higgins-Biddle JC The costs of screening and brief intervention for risky alcohol use J Stud Alcohol 2003 64 6 849 857 14743949 46 Dunlap LJ Zarkin GA Bray JW Mills M Kivlahan DR McKay JR Latham P Tonigan JS Revisiting the cost-effectiveness of the COMBINE study for alcohol dependent patients: the patient perspective Medical care 2010 48 4 306 313 20355261 47 Burhansstipanov L Dignan MB Schumacher A Krebs LU Alfonsi G Apodaca CC Breast screening navigator programs within three settings that assist undeserved women J Cancer Educat 2010 25 2 247 252 48 Han HR Lee H Kim MT Kim KB Tailored lay health worker intervention improves breast cancer screening outcomes in non-adherent Korean-American women Health Educ Res 2009 24 2 318 329 18463411 49 Schwartz RP Kelly SM O'Grady KE Mitchell SG Brown BS Antecedents and correlates of methadone treatment entry: a comparison of out-of-treatment and in-treatment cohorts Drug Alcohol Depend 2011 115 1–2 23 29 PMCID: PMC3059350 21126830 50 Saxon AJ Wells EA Fleming C Jackson TR Calsyn DA Pre-treatment characteristics, program philosophy and level of ancillary services as predictors of methadone maintenance treatment outcome Addiction (Abingdon, England) 1996 91 8 1197 1209 51 Deck D Carlson MJ Retention in publicly funded methadone maintenance treatment in two Western States J Behav Health Serv Res 2005 32 1 43 60 15632797 52 Shah NG Celentano DD Vlahov D Stambolis V Johnson L Nelson KE Strathdee SA Correlates of enrollment in methadone maintenance treatment programs differ by HIV-serostatus AIDS (London, England) 2000 14 13 2035 2043 53 Booth RE Corsi KF Mikulich SK Improving entry to methadone maintenance among out-of-treatment injection drug users J Subst. Abuse Treat 2003 24 4 305 311 12867204 54 Drummond MF Sculpher MJ Torrance GW O'Brien BJ Stoddart GL Methods for the Economic Evaluation of Health Care Programmes 2005 Third New York Oxford University Press 55 Stroup WW Mixed model procedures to assess power, precision, and sample size in the design of experiments 1999 Lincoln, NE University of Nebraska, American Statistical Association 19 24 56 Littell RC Millike GA Stroup WW Wolfinger RD Schabenberger O SAS for mixed models 2006 Cary, NC SAS Institute, Inc. 57 Cohen J Statistical power analysis for the behavioral sciences 1988 Second Hillsdale, NJ LEA 58 Borenstein M Cohen J Statistical power analysis 1988 Hillsdale, NY Erlbaum 59 Kastelic A Pont J Stöver H Opioid Substitution Treatment in Custodial Settings A Practical Guide 2008 Oldenburg, BIS-Verlag 60 Gordon MS Kinlock TW Schwartz RP Fitzgerald TT O'Grady KE Vocci FJ A randomized controlled trial of prison-initiated buprenorphine: prison outcomes and community treatment entry Drug Alcohol Depend 2014 142 33 40 24962326

PMC005xxxxxx/PMC5068189.txt

LICENSE: This file is available for text mining. It may also be used consistent with the principles of fair use under the copyright law. 101489144 35623 Circ Cardiovasc Genet Circ Cardiovasc Genet Circulation. Cardiovascular genetics 1942-325X 1942-3268 27625337 5068189 10.1161/CIRCGENETICS.116.001431 EMS69925 Article Combination of Whole Genome Sequencing, Linkage and Functional Studies Implicates a Missense Mutation in Titin as a Cause of Autosomal Dominant Cardiomyopathy with Features of Left Ventricular Non-Compaction Hastings Robert MBChB DPhil MRCP 1 de Villiers Carin PhD 1 Hooper Charlotte PhD 1 Ormondroyd Liz PhD, MSc 1 Pagnamenta Alistair PhD 23 Lise Stefano PhD 3 Salatino Silvia PhD 3 Knight Samantha JL PhD, CBiol, MSB, FRCPath 23 Taylor Jenny C. PhD 23 Thomson Kate L. BSc, FRCPath 14 Arnold Linda MSc 1 Chatziefthimiou Spyros D. PhD 5 Konarev Petr V. PhD 56 Wilmanns Matthias PhD 5 Ehler Elisabeth PhD 7 Ghisleni Andrea MSc 7 Gautel Mathias MD, PhD 7 Blair Edward BMSc, MBChB 4 Watkins Hugh MD, PhD, FRCP 1 Gehmlich Katja PhD 1 1 Division of Cardiovascular Medicine in the Radcliffe Department of Medicine, University of Oxford; BHF Centre of Research Excellence 2 NIHR Biomedical Research Centre Oxford, University of Oxford, Oxford, United Kingdom 3 Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, United Kingdom 4 Department of Clinical Genetics, Churchill Hospital, Oxford University NHS Trust, Oxford, United Kingdom 5 European Molecular Biology Laboratory, Hamburg, Germany 6 Laboratory of Reflectometry and Small-Angle Scattering, A.V.Shubnikov Institute of Crystallography, Russian Academy of Sciences, Moscow, Russian Federation 7 Randall Division of Cell and Molecular Biophysics and Cardiovascular Division, King’s College London BHF Centre of Research Excellence, London, United Kingdom Correspondence: Katja Gehmlich, Division of Cardiovascular Medicine, Radcliffe Department of Medicine, University of Oxford, Level 6, West Wing John Radcliffe Hospital, Headley Way, Oxford OX3 9DU, United Kingdom, Tel: ++44 1865 234902, Fax: ++44 1865 234681, katja.gehmlich@cardiov.ox.ac.uk 3 10 2016 13 9 2016 10 2016 20 10 2016 9 5 426435 This file is available to download for the purposes of text mining, consistent with the principles of UK copyright law. Background High throughput next generation sequencing techniques have made whole genome sequencing accessible in clinical practice, however, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging. Methods and Results Here we combine whole genome sequencing with linkage analysis in a three-generation family affected by cardiomyopathy with features of autosomal dominant left-ventricular non-compaction cardiomyopathy. A missense mutation in the giant protein titin is the only plausible disease-causing variant that segregates with disease amongst the eight surviving affected individuals, with interrogation of the entire genome excluding other potential causes. This A178D missense mutation, affecting a conserved residue in the second immunoglobulin-like domain of titin, was introduced in a bacterially expressed recombinant protein fragment and biophysically characterised in comparison to its wild-type counterpart. Multiple experiments, including size exclusion chromatography, small angle X-ray scattering and circular dichroism spectroscopy suggest partial unfolding and domain destabilisation in the presence of the mutation. Moreover, binding experiments in mammalian cells show that the mutation markedly impairs binding to the titin ligand telethonin. Conclusions Here we present genetic and functional evidence implicating the novel A178D missense mutation in titin as the cause of a highly penetrant familial cardiomyopathy with features of left-ventricular non-compaction. This expands the spectrum of titin’s roles in cardiomyopathies. It furthermore highlights that rare titin missense variants, currently often ignored or left un-interpreted, should be considered to be relevant for cardiomyopathies and can be identified by the approach presented here. genetics basic science research left ventricular noncompaction cardiomyopathy whole genome sequencing titin telethonin missense mutation Introduction Cardiomyopathies (CM) are a diverse group of diseases affecting the heart muscle 1; many of them are inherited and transmitted in autosomal dominant patterns. The first cardiomyopathy genes were identified by genome-wide linkage analysis in large families 2. In practice, however, the small size of most families, or even the availability of members of larger families, often limits the power of linkage analysis. Recently, high throughput next generation sequencing (NGS) techniques have become widely accessible, making whole genome sequencing (WGS) cost- and time-effective. However, the abundance of variation in the human genome 3 makes it difficult to distinguish rare benign variants from rare disease-causing mutations in an isolated individual, even with growing knowledge of variants in population cohorts (e.g. >60,000 sequenced exomes in the ExAC database, http://exac.broadinstitute.org/). NGS poses, therefore, a significant clinical challenge: the capability to assess variants as pathogenic lags significantly behind variant identification, especially for non-synonymous point mutations 4, 5. Algorithmic predictors are currently unable to accurately assess their exact impact on protein-protein interactions or even on protein folding. Experimental validation of genetic variants is therefore an increasingly indispensable component of NGS discoveries. In the current study, we combine WGS with linkage analysis in a medium-sized family affected by cardiomyopathy with features of left-ventricular non-compaction cardiomyopathy (LVNC). By performing WGS in two family members, filtering against variants seen in normal population cohorts and using linkage information derived from single nucleotide polymorphism (SNP) arrays of 13 family members, we could identify a missense variant in the titin gene (TTN) as the most plausible cause of disease in the family. Functional data, generated from biophysical and protein binding experiments on this titin missense variant provide further support of a causative role in cardiomyopathy through domain misfolding and destabilisation, resulting in impaired binding to the ligand telethonin (also known as t-cap). Methods Clinical Evaluation The study was approved by the Oxfordshire Research Ethics Committee B (REC Ref 09/H0605/3) and all subjects gave informed consent. A three-generational family with history of cardiomyopathy was recruited. Clinical assessment and genetic studies were performed in available family members, who had clinical examination, ECG, echocardiography (with contrast agent where appropriate) and cardiac MRI if possible. Diagnosis of cardiomyopathy was based on established criteria. The diagnosis of LVNC was based on published criteria from echocardiographic and/or cardiac MR imaging 6, 7: the compaction ratio (CR), i.e. the ratio of the thickness of non-compacted to compacted myocardium >2.3 measured on MRI in diastole, or >2.0 on echocardiography in systole was used to diagnose LVNC. Genetic Studies SNP array genotyping was performed using the Illumina HumanCytoSNP-12v1 BeadChip (Illumina, San Diego, CA), containing nearly 300,000 genetic markers, according to the manufacturer’s protocols. A refined subset of roughly 24,000 SNPs in approximate linkage equilibrium was generated using the software PLINK v1.07 8 and the HapMap genotype file available from the PLINK website (http://pngu.mgh.harvard.edu/purcell/plink/). Linkage analysis of the SNP subset was performed using MERLIN v1.1.2 9, specifying an autosomal dominant disease model. Genomic intervals with LOD scores > 0, compatible with segregation of variants in these regions, were selected for downstream analyses. WGS was performed on genomic DNA extracted from peripheral blood as part of the WGS500 project as described previously 10. Sequence reads from the affected individuals were mapped to the human reference genome (hs37d5 version of build 37) using STAMPY 11. Duplicate reads were removed with PICARD (http://broadinstitute.github.io/picard/). The software Platypus (version 0.8.1, default parameters) 12 was used jointly on the two .bam files in order to call SNPs and short (< 50 bp) indels across both samples. All the 5,946,161 identified variants were annotated with an in-house pipeline based on the Variant Effect Predictor (VEP) Ensembl framework (version 77) 13. A number of additional databases were used to integrate the information provided by VEP (Table S1). Known associations with diseases were screened using HGMD (http://www.hgmd.cf.ac.uk/ac/index.php) and ClinVar 14. Variants were filtered by in-house Python scripts based on criteria outlined in Table S1 (steps 1-10), followed by manual inspection (steps 11-13). The variants remaining after step 10 are documented in Results and in Tables S2, S3. Confirmatory Sanger sequencing was performed with the primers listed in Table S4. Both SNP and WGS data were interrogated also for clinically relevant copy number variants (CNVs) using Nexus Copy Number 7.5.2 Discovery Edition (BioDiscovery, Hawthorne, CA; see Supplementary methods). Functional characterisation of the titin missense variant The mutation was introduced into human titin Z1Z2 constructs (amino acids 1-196, accession no. ACN81321.1) for bacterial and mammalian expression using Quikchange II XL (Agilent) with primers given in Table S4. Bacterial expression and purification was performed as previously described 15. Size exclusion chromatography - Tridetector analysis (light scattering, refractive index, and UV absorbance), small angle X-ray scattering (SAXS) experiments, circular dichroism spectroscopy, and thermolysin digests were essentially performed as described 15–18 and experimental details are given in Suppl. Material. NRC cultures were established and transfected 16 using hemagglutinin(HA)-tagged expression constructs and counter-stained for titin T12 epitope 19 or telethonin (mouse monoclonal antibody, Santa Cruz) 48 hrs post transfection and analysed by confocal microscopy. GST pulldown assays were performed as described 20 using mammalian expression constructs for telethonin amino acids 1-90 and 1-167 fused to GST, and titin Z1Z2 fused to GFP (pEGFP-N1, Clontech) in transfected COS-1 cells. Förster Resonance Energy Transfer (FRET) experiments from transfected COS-1 cells and the assessment of reduced protein stability in NRC and COS-1 cells are described in the Suppl. Material. Results The proband was a 20yr old male (II-3 in Figure 1A) who died suddenly in hospital in 1970 having presented with rapidly decompensating congestive heart failure; at post mortem his heart (680 g) had evidence of dilatation and both macroscopic and microscopic hypertrophy but no myocyte disarray. His brother (II-4) was later found to have an enlarged heart with wall thickness at the upper limit of normal and marked hypertrabeculation. The proband’s sister (II-2) presented with a non ST-elevation myocardial infarct due to coronary embolus at the age of 61. LVNC with mild LV dilatation and apical hypertrophy was diagnosed at this time (Figure 1B, C). Cascade screening identified the same condition in further family members with consistent clinical features of adult onset cardiomyopathy with features of LVNC. Five affected family members had sufficient non-compaction to meet the diagnostic criteria for LVNC while three others with early or mild disease had lesser extent of hypertrabeculation but clear evidence of cardiomyopathy with LV dilatation and/or systolic dysfunction (Figures 1A, S1, Table 1). Aside from the proband who had advanced congestive failure, there were no arrhythmic features in any affected family member, nor were there any extra-cardiac (e.g. neuromuscular) manifestations. Identification of TTN mutation A178D segregating with disease Affected first cousins III-1 and III-4 were selected for WGS. Sequencing was performed by Illumina Cambridge as 100bp paired-end reads to a mean coverage of 56.9x and 52.0x respectively, such that 99 % of the genome was covered at 20x or more in both samples, identifying 5,946,161 variants shared by the two individuals. In addition, SNP arrays were performed on all individuals of the family (except II-3 and III-2, Figure 1A). Neither the SNP array nor WGS data revealed likely causative CNVs. Genomic regions identical by descent were identified through linkage analysis (see Methods, Figure S2) and out of the 100,789 candidate variants within the three linkage regions (on chromosomes 2, 9 and 16), potentially pathogenic ones were selected based on an autosomal dominant model, caused by a rare heterozygous mutation. Variants were filtered accordingly by in-house Python scripts and the remaining six variants were manually inspected (Table S2). Four of them were excluded: one is assumed to be an artefact due to an incorrect transcript being present in Ensembl and another variant did not segregate with disease in the family; two splice variants were predicted to be silent (at positions -5 and -3 of a 3' splice junction, respectively, for details see Table S3). Only two final candidate variants were considered conceivably linked to the phenotype: missense changes in PDP2 and TTN, respectively (Table S2). PDP2 codes for pyruvate dehyrogenase phosphatase catalytic subunit 2 and has low expression levels in the heart. Although the change E316K is predicted to be damaging by Polyphen and SIFT algorithms (Table S2), a heterozygous loss-of-function in this enzyme would not be expected to produce a phenotype, and indeed heterozygous loss-of-function mutations in PDP1 are clinically silent 21. The variant is not plausible as a cause of a penetrant dominant disorder because it is found six times in 121,412 alleles in the ExAC database. Six instances would equal at least 10 % of all expected LVNC cases in ExAC, assuming a maximal prevalence of 1:1,000 for the disease 22. This appears an implausibly high percentage for a novel, unpublished disease-causing variant. In support, in the two largest clinical cardiomyopathy cohorts published to date, the most common reported pathogenic variant (MYBPC3, p.Arg502Trp) detected in 104 out of 6179 HCM cases (1.7%, 95CI 1.4-2.0%), was only observed 3 times in ExAC (3/120,674) with all other pathogenic variants for HCM or DCM being present 0 or 1 times only 23. The second variant is found in TTN, the gene which codes for titin, an abundant skeletal muscle and heart specific protein with crucial functions 24, 25 (and reviewed in 26). Mutations in titin have been associated with CM and skeletal myopathy (reviewed in 27). The identified missense variant c.533C>A in TTN, which codes for a p.A178D change at the amino-acid level, is absent in ExAC. Sanger sequencing confirmed the co-segregation of the heterozygous mutation with disease in all affected individuals of the family (Figures 1, S3A, LOD score 2.1). Thus comprehensive whole genome analysis reveals this as the most plausible causative mutation in the family. Functional studies Prediction of deleterious effects of the mutation Each single molecule of the giant protein titin spans half a sarcomere from the Z-disk to the M-band 28. The first two Immunoglobulin-like (Ig) domains (Z1Z2) of titin are located in the Z-disk and form a super-stable complex with telethonin 29. The A178 position is evolutionarily very well conserved back to zebrafish and lamprey. Additionally, A178 is located in a highly conserved structural section (Figure S3B), the β-strand F of the second Ig-domain of Z1Z2, neighbouring the β-strand G of titin Z2 which forms a strong and extended interaction with the β-strands of telethonin (30, Figure 2A). The A178D mutation is predicted to directly affect the β-strands B and C as well as the loop connecting the β-strands B and C due to steric hindrance of D178 with V127 and P133 respectively (Figure 2B). Thus, the insertion of a charged residue in this position is likely to have significant impact on the secondary structure of this domain and could potentially cause misfolding of the protein. Altered protein characteristics of purified titin Z1Z2 A178D recombinant fragment To assess how the A178D mutation affects the folding and stability of the protein, recombinant titin Z1Z2 WT and A178D were expressed in E. coli and purified under native conditions. Of note, the yield of the soluble protein fraction was consistently lower for A178D compared to WT preparations, despite equal total expression levels (data not shown). Circular dichroism (CD) spectroscopy demonstrated a typical β-sheet signature for WT Z1Z2 (Figure 3A). In contrast, the spectrum for Z1Z2 A178D differs significantly: Although the characteristic negative band at 216 nm is still present, but slightly shifted, there was no significant positive band at around 200 nm. The absence of this band, associated with β-sheet conformation, and the presence of a negative peak at around 198 nm, characteristic of random coil structures, indicate that the Z1Z2 A178D mutant is partially unfolded. In support, thermal denaturation experiments for Z1Z2 A178D showed high fluorescence signal already at low temperatures, suggesting solvent exposed hydrophobic residues due to partial unfolding. No melting temperature can be deducted for titin Z1Z2 A178D, in contrast to the WT protein, which has a melting temperature of 62 ºC, typical for Ig domains (Figure S4). SAXS experiments confirmed the presence of unfolded parts/flexible domains in Z1Z2 A178D, as shown by the Kratky plot (Figure S5A), whereas Z1Z2 WT displays a typical profile for folded structures. The domain destabilisation as a consequence of partial unfolding is evidenced by the formation of higher oligomers (approx. 20-mers) for the Z1Z2 A178D mutant in vitro: Size exclusion chromatography and Tridetector analysis revealed that in contrast to the monomeric Z1Z2 WT, the A178D mutant eluted in two peaks, corresponding predominantly to higher molecular aggregates and to a lesser extent to dimeric protein (Figure 3B, Table 2). SAXS measurements also confirmed that Z1Z2 WT is monomeric, whereas Z1Z2 A178D is found in a higher oligomeric state (Figure S5B, Table 2). In conclusion, the mutation A178D leads to partial misfolding of bacterially expressed Z1Z2 protein fragment. Reduced stability of titin Z1Z2 A178D as a consequence of the partial misfolding When performing denaturing gel electrophoresis, a degradation product was observed exclusively for Z1Z2 A178D preparations (arrowhead in Figure 4A) and upon thermolysin treatment, only Z1Z2 A178D showed rapid degradation, while Z1Z2 WT was resistant to the protease treatment (Figure 4B). In addition, Z1Z2 A178D showed reduced stability when expressed in neonatal rat cardiomyocytes and COS-1 cells (Figures 4C, S6), suggesting that the mutation destabilises Z1Z2 also in a physiological, cellular environment. However, formation of large aggregates was not observed in transfected cells expressing Z1Z2 A178D (Figures 4D, S7). Impaired binding to telethonin Localisation of transfected Z1Z2 was not altered in the presence of the A178D mutation (Figure 4D). To assess the consequences of the mutation on binding telethonin, semi-quantitative GST-pulldown assays were performed with titin Z1Z2 and telethonin co-expressed in mammalian cells. Z1Z2 A178D showed impaired binding to two telethonin constructs (Figure 5A, B). The interaction between titin and telethonin was further quantified in FRET experiments, where close proximity of proteins in a complex allows energy transfer from Cyan Florescent Protein (CFP) to Yellow Fluorescent Protein (YFP) between two fusion protein constructs 31. By introducing the A178D mutation into a Z1Zr3-CFP construct, FRET efficiency to telethonin-YFP was almost abolished (Figure 5C, D), validating and quantifying the observation that A178D impairs binding to telethonin in the cellular context. Taken together, our functional data suggest that the A178D mutant may affect protein folding, stability and impairs binding to telethonin, thus supporting its pathogenic potential. Discussion In this study, we present a three-generation family with multiple individuals affected by cardiomyopathy with features of LVNC, systolic impairment and an autosomal dominant inheritance pattern. Of note, the affected family members show a consistent phenotype with prominent hypertrabeculation as the main abnormality in the majority; this is relatively unusual as it is more typical to see LVNC in individual members of families with other forms of cardiomyopathy. We employed a combination of WGS in two affected individuals and linkage analysis in 13 family members; this approach identified only two rare candidate variants across the whole genome that segregated with the autosomal dominant cardiomyopathy. Since one of the identified genes (PDP2) is barely expressed in the heart, and the variant appears in implausible high numbers in the ExAC database, it is extremely unlikely to be disease-causative. In contrast, titin, the gene affected by the other missense variant (TTN p.A178D), has crucial functions in the heart and is a known disease gene for cardiomyopathies (see below). Despite the fact that the family is too small for traditional genome-wide linkage analysis to identify the genetic cause of the disease (the LOD score of 2.1, i.e. odds ratio 1:125, is well below the threshold of 3.0, i.e. odds ratio 1:1000), interrogation of the entire genome adds substantial weight to a likely causative role of the titin missense mutation for disease: no other plausible mutations, including larger genomic re-organisations (CNVs), were detected in any other genes in the linkage regions and the remainder of the genome is excluded by negative LOD scores. Titin has been implicated in cardiac and skeletal muscle disease, occasionally involving a combination of both. Mutations in this gene have been described in various forms of CM, such as Dilated CM, Arrhythmogenic Right Ventricular CM, Hypertrophic CM and Restrictive CM (reviewed in 27). Truncating variants in titin (TTNtv) are the most frequent genetic finding in idiopathic Dilated CM, being present in 15-25 % of the cases 32 and are also frequent in Peripartum CM (15 %) 33. However, penetrance appears to be low, as TTNtv are also found in approx. 1 % of normal populations and hence the large majority of carriers do not manifest with disease 34. More recent work 35 showed that Dilated CM causing TTNtv are enriched in the sarcomeric A-band region, whereas TTNtv found in control cohorts tend to spare the A-band region and are in exons with low usage in cardiac transcripts. An internal promotor in titin rescuing TTNtv N-terminally of the A-band region may explain this phenomenon 36. Titin missense mutations have been identified in Dilated and Hypertrophic CM cohorts 4, 37, 38. A causative role for TTN p. W976R in Dilated CM is well supported by co-segregation within a large family and functional data 39, 40. However, generally, titin missense mutations are challenging to interpret, as rare benign variants are common in normal population cohorts. In the ExAC database, more than a third of the individuals carry a rare missense variant in titin (21,939 missense variants with <0.01 % allelic frequency in 58,687 exomes), and although a proportion of these may represent recessive pathogenic alleles 27, only a very small fraction will be disease-causing with dominant inheritance. Hence, clinical practitioners require co-segregation information to assign causality as bioinformatic prediction tools can only give probabilistic data 4, 37. As we document here, interrogation of the entire genome combined with linkage analysis can help to narrow down lists of potential causative variants, even in small families. Our finding of TTN p.A178D in a family with features of LVNC expands the spectrum of titinopathies: to our knowledge, this is the first report of a titin missense mutation implicated in cardiomyopathy with predominant features of LVNC and one of the first titin missense mutations supported by robust genome-wide genetics and detailed functional data. The latter suggests a likely pathogenic role of titin A178D by a) evidence of protein degradation, partial unfolding and domain destabilisation in vitro, b) protein destabilisation in two cellular systems and c) altered binding properties to the ligand telethonin. Although extrapolations from such in vitro experiments on isolated domains to the full length giant protein are not without uncertainty, such parameters will be useful complements in the future studies of other TTN missense variants. It is currently unclear how this particular mutation leads to this distinct phenotype, and more insight into the biology of Z-disk titin is needed to understand the underlying disease pathways. This will be addressed with the help of model organisms 36, 41 or patient-derived induced pluripotent stem cell derived cardiomyocytes 40, focussing on the titin-telethonin complex 29 and its downstream signalling targets 42 in future work. Supplementary Material Supplemental Material Acknowledgments” We thank Stephan Lange (UCSD) for a titin Z1Z2 expression constructs. SAXS data were collected at the beamline P12, operated by EMBL, Hamburg unit, at the PETRA III storage ring (DESY, Hamburg, Germany). We gratefully thank Dmitry Svergun and his group for help with the SAXS data, the SPC facility at EMBL Hamburg for technical support and Annabel Parret for her help with the Tridetector Analysis. Sources of Funding: KG is supported by British Heart Foundation Grants (FS/12/40/29712, PG/15/113/31944). KG, RH and HW acknowledge support from the BHF Centre of Research Excellence, Oxford (grant codes HSRNWBY, HSRNWB11 and RE/13/1/30181). KLT is the recipient of a National Institute for Health Research (NIHR) doctoral fellowship (NIHR-HCS-D13-04-006). This publication includes independent research supported also by the NIHR Biomedical Research Centre, Oxford. The work was supported also by funding from the Wellcome Trust Core Award Grant Number 090532/Z/09/Z. The views expressed are those of the authors and not necessarily those of the Department of Health or Wellcome Trust. MG and AG were supported by the EU MUZIC network, the MRC and the Leducq Foundation. MG holds the BHF Chair of Molecular Cardiology. Clinical Perspective High throughput next generation sequencing techniques have made whole genome sequencing accessible and are increasingly applied in clinical practice. However, the abundance of variation in the human genomes makes the identification of a disease-causing mutation on a background of benign rare variants challenging. To illustrate, more than one third of individuals in normal population cohorts carry a rare missense variant in the giant protein titin (coded by the gene TTN), but only a very small fraction of these will be disease-causing with dominant inheritance. Hence titin missense variants are currently often ignored or left un-interpreted when found in cardiomyopathy patients. Here we combine whole genome sequencing with linkage analysis in a three-generation family affected by cardiomyopathy with features of autosomal dominant left-ventricular non-compaction cardiomyopathy. A missense mutation in titin (TTN p. A178D) is the only plausible disease-causing variant that segregates with disease amongst affected individuals of the family, with interrogation of the entire genome excluding other potential causes. Functional studies on this missense mutation demonstrate domain misfolding and destabilisation, resulting in paired binding to the ligand telethonin/t-cap,and hence supporting its highly likely causative role. Our report expands the spectrum of titin’s roles in cardiomyopathies and furthermore highlights that rare titin missense variants should be considered to be relevant for cardiomyopathies and can be identified by combining whole genome sequencing with linkage analysis in medium-sized cardiomyopathy families. Figure 1 A – Pedigree of the family, males depicted as squares, females as circles, slanted symbols deceased individuals. Clinically affected individuals are marked in grey, unaffected are shown in white, “?” means unclassified clinical status. The presence of the TTN p.A178D mutation is indicated (“+”present, “-“absent, ND not determined.) Individuals selected for WGS are marked with thicker symbols (III-1 and III-4). B – Echocardiogram images showing the characteristic 'spongy' appearance of non-compaction in individual II-2 with and without contrast. C – Echocardiogram image from individual II-4 showing significant dilatation, but maintaining a thickened myocardium and preserved ejection fraction. Figure 2 A – Position of the TTN p. A178D on a structural model (pdb: 1YA5) of the titin Z1Z2 domains (purple) in complex with telethonin (pink). B – Close-up of the site of mutation. The red discs show van der Waals overlaps or steric clashing that A178D is predicted to cause with valine127 and proline133. The figures of the crystal structure were generated by pymol (http://www.pymol.org). Figure 3 A – CD spectroscopy of purified titin Z1Z2 fragments (WT solid line and A178D dashed line). B – Size exclusion chromatography for titin Z1Z2 fragments (WT solid line and A178D dashed line). Z1Z2 WT elutes as monomeric protein ($), whereas peaks corresponding to dimer (*) and higher molecular aggregates (#) are observed for Z1Z2 A178D. Figure 4 Destabilisation of the titin Z1Z2 fragment in the presence of the A178D mutation: A – Denaturing gel-electrophoresis of purified titin Z1Z2 fragments (WT and A178D) expressed in E. coli. The WT fragment is detected as a single band of 23 kD (arrow). Only for Z1Z2 A178D a degradation product (arrowhead) is observed. The position of marker proteins and their size (in kD) is indicated. B – Titin Z1Z2 protein fragments (WT – left, A178D – right) were incubated with protease thermolysin for the length indicated. Control: titin Z1Z2 protein sample without thermolysin. A stable degradation fragment of approx. 15 kD is observed for the mutant titin Z1Z2. The position of marker proteins and their size (in kD) is indicated. C – Decreased stability of titin Z1Z2 A178D in NRC: NRC were infected in duplicates with adenoviral particles for HA-tagged titin Z1Z2 (WT or A178D, MOI 5). Infection with parental empty vector (GFP) and non-infected cells (NI) served as controls. Steady-state titin fragment protein amount was assayed by Western blotting for the HA-tag. Probing for hrGFP served as infection control and probing for endogenous GAPDH served as loading control. Despite equal infection rates (for confirmatory control experiments see Figure S6A), less titin A178D protein fragment was detected, indicating reduced stability in NRC. D – Localisation of titin Z1Z2 in NRC: Cells were transfected with constructs coding for HA-tagged titin Z1Z2 WT (top, first row) or titin Z1Z2 A178D (bottom, first row) mutant protein fragment and counterstained for endogenous titin with T12 antibody (middle row, Z-disk proximal epitope, but not recognising the transfected titin Z1Z2 protein fragment). Merged images are shown in the third row, HA shown in red, endogenous titin in green. Scale bar represents 10 microns. Figure 5 Functional implications of the titin Z1Z2 A178D mutation. A – Semi-quantitative GST-pulldown assays using telethonin fragments fused to GST (left aa 1-90, right full length) and titin Z1Z2 fragments (WT and A178D as indicated) as GFP fusions expressed in COS-1 cells. Bound titin-GFP fragments are detected by Western blotting (top row), input lysate controls are shown in the second row. Pulled down GST-telethonin fragments are shown in row three, as well as lysate controls (bottom row). B – Quantification of GST-pulldown experiments from panel A visualises reduced binding of titin Z1Z2 to telethonin in the presence of the A178D mutation, values are expressed as bound protein relative to lysate, with the first WT experiment set to 100 % (n = 2 per group, values expressed as mean with standard deviation for error bars; one representative experiment of three independent ones is shown). Due to the semi-quantitative nature of the experiments, no statistical test was performed. C – FRET experiments using COS-1 cells co-transfected with telethonin (aa 1-90) fused to YFP (first row) and titin Z1Zr3 fused to CFP (second row). Images pre- and post-bleach are shown, FRET ratios are shown in the third row. Inserts show magnification of indicated area. Scale bar represents 10 microns. D – Quantification of FRET efficiency for titin Z1Zr3 WT/A178D and telethonin pairs. The A178D mutation reduces the FRET efficiency from approx. 0.15 to 0.01 indicating a dramatic loss of binding ability (WT n=20 and A178D n=26 cells; * p < 0.0001 unpaired Student’s t-test). Table 1 Summary of clinical findings. Symbol Sex Age* Clinical status† Genetic status Trabeculation compaction ratio (CR)‡ IVS D PW D LVED D LVES D EF ECG Comments I-1 M 87 affected TTN A178D 2.5 (echo) 24 13 40 35 42 left axis deviation, anteroseptal Q wave Marked ASH with low EF, CVA, hypertension; meets diagnostic criteria for LVNC (echo). I-2 F 72 unaffected WT 1.8 (echo) 12 11 48 30 76 AF, paced LVNC excluded (echo), structurally normal heart at age 70, moderate concentric LVH by age 85 II-2 F 61 affected TTN A178D 2.0 (echo) 9 10 53 36 47 left axis deviation Mildly thickened apical segments, cardiac embolus at 61y; meets diagnostic criteria for LVNC (echo). II-3 M 20 assumed affected no DNA Rapidly progressive HF with sudden death at 20y (1970); hypertrophy and dilatation at post mortem. II-4 M 37 affected TTN A178D <2 (MRI) 12 12 64 44 57 normal Dilated LV, late Gd on MRI, hypertension. II-6 M 42 unaffected WT 11 9 50 30 78 normal III-1 M 27 affected TTN A178D 2.5 (echo) 12 11 64 42 63 QRS 120 msec Mild regional systolic dysfunction; meets diagnostic criteria for LVNC (echo). III-2 M 21 unclassified no DNA 0.8 (echo) 13 13 54 37 68 normal Mild concentric LVH III-3 M 32 affected TTN A178D 2.8 (MRI) 11 13 52 35 68 normal Hypertension; meets diagnostic criteria for LVNC (MRI) III-4 F 23 affected TTN A178D 2.6 (MRI) 8 9 46 32 58 normal Meets diagnostic criteria for LVNC (MRI) III-5 M 25 affected TTN A178D 2.0-2.5 (MRI) 10 10 46 27 51 inferior T-wave inversion Hypokinesia apical LV incl. septum; borderline for diagnostic criteria for LVNC (MRI) III-6 M 23 affected TTN A178D 1.5 (MRI) 8 9 53 34 48 Q wave & T-wave inversion in lead III Mild DCM, faint late Gd, borderline dilated LV with mildly impaired function, inferior hypokinesia III-7 M 21 unclassified WT 1.6 (MRI) 9 7 55 38 59 normal Documented myocarditis at 21y (MRI) Cardiac dimensions (IVSD – Interventricular Septal Thickness at Diastole, PWD –Posterior Wall Thickness at Diastole, LVEDD – Left Ventricular End Diastolic Diameter, LVESD – Left Ventricular End Systolic Diameter) are given in mm Abbreviations: EF – Ejection Fraction (in %). ASH – Asymmetric Septal Hypertrophy, CVA – Cerebrovascular Accident, LVH – Left Ventricular Hypertrophy, Gd – Gadolinium, HF – Heart Failure, AF – Atrial Fibrillation, DCM – Dilated CM. Blank cells indicate no data available. * Age of diagnosis or first clinical assessment, however parameters of most recent cardiac assessment are given (with exception of I-2 where data at first assessment aged 70 are given and II-7, where the last assessment before myocarditis is shown). † Clinical status ‘affected’ means affected by cardiomyopathy. Whether individuals meet diagnostic criteria for LVNC is shown in the Comments column (in brackets shown whether MRI or echo criteria have been used). ‡ For the definition of trabeculation compaction ratio (CR) see Methods section, in brackets the mode of imaging is indicated. Representative MRI images are shown in Figure S1. Table 2 Biophysical characterisation of recombinant Z1Z2 WT and A178D protein fragments Titin Z1Z2 WT Titin Z1Z2 A178D Calculated molecular weight (kDa) 22.7 22.8 Size exclusion chromatography Retention time (mL) 15.7 9.9 (1st peak) 14.4 (2nd peak) Static Light Scattering Molecular weight (kDa) 21 ± 2 452 ± 45 (1st peak) 45 ± 4 (2nd peak) Small Angle X-ray Scattering Vp excluded volume of the hydrated particle (nm3) 40 ± 5 305 ± 20 Rg radius of gyration (nm) 3.10 ± 0.05 6.8 ± 0.1 Dmax maximum particle size (nm) 10.5 ± 0.5 25.0 ± 1.0 Normalized Kratky plot folded partly unfolded/ flexible domains Disclosures: None. 1 Watkins H Ashrafian H Redwood C Inherited cardiomyopathies N Engl J Med 2011 364 1643 1656 21524215 2 Jarcho JA McKenna W Pare JA Solomon SD Holcombe RF Dickie S Mapping a gene for familial hypertrophic cardiomyopathy to chromosome 14q1 N Engl J Med 1989 321 1372 1378 2811944 3 1000 Genomes Project Consortium Abecasis GR Auton A Brooks LD DePristo MA Durbin RM An integrated map of genetic variation from 1,092 human genomes Nature 2012 491 56 65 23128226 4 Haas J Frese KS Peil B Kloos W Keller A Nietsch R Atlas of the clinical genetics of human dilated cardiomyopathy Eur Heart J 2015 36 1123 1135a 25163546 5 Watkins H Assigning a causal role to genetic variants in hypertrophic cardiomyopathy Circ Cardiovasc Genet 2013 6 2 4 23424253 6 Jenni R Oechslin E Schneider J Attenhofer Jost C Kaufmann PA Echocardiographic and pathoanatomical characteristics of isolated left ventricular non-compaction: a step towards classification as a distinct cardiomyopathy Heart 2001 86 666 671 11711464 7 Petersen SE Selvanayagam JB Wiesmann F Robson MD Francis JM Anderson RH Left ventricular non-compaction: insights from cardiovascular magnetic resonance imaging J Am Coll Cardiol 2005 46 101 105 15992642 8 Purcell S Neale B Todd-Brown K Thomas L Ferreira MA Bender D PLINK: a tool set for whole-genome association and population-based linkage analyses Am J Hum Genet 2007 81 559 575 17701901 9 Abecasis GR Cherny SS Cookson WO Cardon LR Merlin--rapid analysis of dense genetic maps using sparse gene flow trees Nat Genet 2002 30 97 101 11731797 10 Taylor JC Martin HC Lise S Broxholme J Cazier JB Rimmer A Factors influencing success of clinical genome sequencing across a broad spectrum of disorders Nat Genet 2015 47 717 726 25985138 11 Lunter G Goodson M Stampy: a statistical algorithm for sensitive and fast mapping of Illumina sequence reads Genome Res 2011 21 936 939 20980556 12 Rimmer A Phan H Mathieson I Iqbal Z Twigg SR Consortium WGS Integrating mapping-, assembly- and haplotype-based approaches for calling variants in clinical sequencing applications Nat Genet 2014 46 912 918 25017105 13 McLaren W Pritchard B Rios D Chen Y Flicek P Cunningham F Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor Bioinformatics 2010 26 2069 2070 20562413 14 Landrum MJ Lee JM Riley GR Jang W Rubinstein WS Church DM ClinVar: public archive of relationships among sequence variation and human phenotype Nucleic Acids Res 2014 42 D980 985 24234437 15 Zou P Gautel M Geerlof A Wilmanns M Koch MH Svergun DI Solution scattering suggests cross-linking function of telethonin in the complex with titin J Biol Chem 2003 278 2636 2644 12446666 16 Geier C Gehmlich K Ehler E Hassfeld S Perrot A Hayess K Beyond the sarcomere: CSRP3 mutations cause hypertrophic cardiomyopathy Hum Mol Genet 2008 17 2753 2765 18505755 17 Reinhard L Mayerhofer H Geerlof A Mueller-Dieckmann J Weiss MS Optimization of protein buffer cocktails using Thermofluor Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 69 209 214 18 Shaya D Kreir M Robbins RA Wong S Hammon J Bruggemann A Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins Proc Natl Acad Sci U S A 2011 108 12313 12318 21746903 19 Furst DO Osborn M Nave R Weber K The organization of titin filaments in the half-sarcomere revealed by monoclonal antibodies in immunoelectron microscopy: a map of ten nonrepetitive epitopes starting at the Z line extends close to the M line J Cell Biol 1988 106 1563 1572 2453516 20 Gehmlich K Asimaki A Cahill TJ Ehler E Syrris P Zachara E Novel missense mutations in exon 15 of desmoglein-2: role of the intracellular cadherin segment in arrhythmogenic right ventricular cardiomyopathy? Heart Rhythm 2010 7 1446 1453 20708101 21 Cameron JM Maj M Levandovskiy V Barnett CP Blaser S Mackay N Pyruvate dehydrogenase phosphatase 1 (PDP1) null mutation produces a lethal infantile phenotype Hum Genet 2009 125 319 326 19184109 22 Carrilho-Ferreira P Almeida AG Pinto FJ Non-compaction cardiomyopathy: prevalence, prognosis, pathoetiology, genetics, and risk of cardioembolism Curr Heart Fail Rep 2014 11 393 403 25239435 23 Walsh R Thomson KL Ware JS Funke BH Woodley J McGuire KJ Reassessment Of Mendelian Gene Pathogenicity Using 7,855 Cardiomyopathy Cases And 60,706 Reference Samples Genet Med 2016 8 17 10.1038/gim.2016.90 [Epub ahead of print] 24 Labeit S Kolmerer B Linke WA The giant protein titin. Emerging roles in physiology and pathophysiology Circ Res 1997 80 290 294 9012751 25 Wang K McClure J Tu A Titin: major myofibrillar components of striated muscle Proc Natl Acad Sci U S A 1979 76 3698 3702 291034 26 Gerull B The Rapidly Evolving Role of Titin in Cardiac Physiology and Cardiomyopathy Can J Cardiol 2015 31 1351 1359 26518445 27 Chauveau C Rowell J Ferreiro A A rising titan: TTN review and mutation update Hum Mutat 2014 35 1046 1059 24980681 28 Hidalgo C Granzier H Tuning the molecular giant titin through phosphorylation: role in health and disease Trends Cardiovasc Med 2013 23 165 171 23295080 29 Bertz M Wilmanns M Rief M The titin-telethonin complex is a directed, superstable molecular bond in the muscle Z-disk Proc Natl Acad Sci U S A 2009 106 13307 133310 19622741 30 Zou P Pinotsis N Lange S Song YH Popov A Mavridis I Palindromic assembly of the giant muscle protein titin in the sarcomeric Z-disk Nature 2006 439 229 233 16407954 31 Truong K Ikura M The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo Curr Opin Struct Biol 2001 11 573 578 11785758 32 Herman DS Lam L Taylor MR Wang L Teekakirikul P Christodoulou D Truncations of titin causing dilated cardiomyopathy N Engl J Med 2012 366 619 628 22335739 33 Ware JS Li J Mazaika E Yasso CM DeSouza T Cappola TP Shared Genetic Predisposition in Peripartum and Dilated Cardiomyopathies N Engl J Med 2016 374 233 241 26735901 34 Watkins H Tackling the achilles' heel of genetic testing Sci Transl Med 2015 7 270fs1 35 Roberts AM Ware JS Herman DS Schafer S Baksi J Bick AG Integrated allelic, transcriptional, and phenomic dissection of the cardiac effects of titin truncations in health and disease Sci Transl Med 2015 7 270ra6 36 Zou J Tran D Baalbaki M Tang LF Poon A Pelonero A An internal promoter underlies the difference in disease severity between N- and C-terminal truncation mutations of Titin in zebrafish Elife 2015 4 e09406 26473617 37 Begay RL Graw S Sinagra G Merlo M Slavov D Gowan K Role of Titin Missense Variants in Dilated Cardiomyopathy J Am Heart Assoc 2015 4 pii: e002645 38 Lopes LR Zekavati A Syrris P Hubank M Giambartolomei C Dalageorgou C Genetic complexity in hypertrophic cardiomyopathy revealed by high-throughput sequencing J Med Genet 2013 50 228 239 23396983 39 Gerull B Gramlich M Atherton J McNabb M Trombitas K Sasse-Klaassen S Mutations of TTN, encoding the giant muscle filament titin, cause familial dilated cardiomyopathy Nat Genet 2002 30 201 204 11788824 40 Hinson JT Chopra A Nafissi N Polacheck WJ Benson CC Swist S HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy Science 2015 349 982 986 26315439 41 Gramlich M Pane LS Zhou Q Chen Z Murgia M Schotterl S Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy EMBO Mol Med 2015 7 562 576 25759365 42 Knoll R Linke WA Zou P Miocic S Kostin S Buyandelger B Telethonin deficiency is associated with maladaptation to biomechanical stress in the mammalian heart Circ Res 2011 109 758 769 21799151

PMC005xxxxxx/PMC5111163.txt

LICENSE: This file is available for text mining. It may also be used consistent with the principles of fair use under the copyright law. 8907828 20554 J Psychopharmacol J. Psychopharmacol. (Oxford) Journal of psychopharmacology (Oxford, England) 0269-8811 1461-7285 24327452 5111163 10.1177/0269881113515061 NIHMS828453 Article Psychiatric profiles of mothers who take Ecstasy/MDMA during pregnancy: Reduced depression 1 year after giving birth and quitting Ecstasy Turner John JD 1 Parrott Andrew C 2 Goodwin Julia 1 Moore Derek G 1 Fulton Sarah 3 Min Meeyoung O 3 Singer Lynn T 3 1 University of East London, London, UK 2 Swansea University, Swansea, UK 3 Case Western Reserve University, Cleveland, USA Corresponding author: John JD Turner, School of Psychology, University of East London, London E15 4LZ, UK. j.j.d.turner@uel.ac.uk 9 11 2016 10 12 2013 1 2014 16 11 2016 28 1 5561 This file is available for text mining. It may also be used consistent with the principles of fair use under the copyright law. Background The recreational drug MDMA (3,4-methylenedioxymethamphetamine) or ‘Ecstasy’ is associated with heightened psychiatric distress and feelings of depression. The Drugs and Infancy Study (DAISY) monitored the psychiatric symptom profiles of mothers who used Ecstasy/MDMA while pregnant, and followed them over the first year post-partum. Methods We compared 28 young women whom took MDMA during their pregnancy with a polydrug control group of 68 women who took other psychoactive drugs while pregnant. The Brief Symptom Inventory (BSI) was completed for several periods: The first trimester of pregnancy; and 1, 4 and 12 months after childbirth. Recreational drug use was monitored at each time point. Results During the first trimester of pregnancy, MDMA-using mothers reported higher depression scores than the polydrug controls. At 1 year after childbirth, their BSI depression scores were significantly lower, now closer to the control group values. At the same time point, their self-reported use of MDMA became nearly zero, in contrast to their continued use of Cannabis/marijuana, nicotine and alcohol. We found significant symptom reductions in those with BSI obsessive-compulsive and interpersonal sensitivity, following Ecstasy/MDMA cessation. Conclusions The findings from this unique prospective study of young recreational drug-using mothers are consistent with previous reports of improved psychiatric health after quitting MDMA. Cessation depression drug addiction Ecstasy MDMA middle class mother post-partum pregnancy quitting recreational drugs Introduction ‘Ecstasy’ or 3,4-methylenedioxymethamphetamine (MDMA) is used as an illicit drug by subgroups of adolescents and young adults. Its recreational use is mainly associated with dance clubs, all-night ‘raves’ and house parties (Parrott et al., 2008; Winstock et al., 2001). Population surveys in the US reveal usage levels as high as 9.5% in college students (Johnston et al., 2005; Singer et al., 2004). In the American National Survey on drug use and health, Ecstasy/MDMA is found to be used more by young women than men (Wu et al., 2010). Neuroimaging studies of abstinent MDMA users reveal significantly lower levels of the serotonin transporter (SERT) (Erritizoe et al., 2011; Kish et al., 2010), and are widely interpreted as suggesting serotonergic neurotoxicity (Benningfield and Cowan, 2013; Parrott, 2013a; Puerta et al., 2009; Ricaurte et al., 2000). Recreational use of MDMA is also associated with various neuropsychobiological problems, including memory deficits (Montgomery et al., 2010; Rogers et al., 2009; Zakzanis and Campbell, 2006), impairments in higher cognitive processing (Fox et al, 2002; Parrott, 2012, 2013b; Reay et al., 2006), sleep apnea (McCann et al., 2009), raised cortisol levels (Parrott, 2009; Parrott et al., 2012), psychosocial impairment (Topp et al., 1999) and various psychiatric problems (Briere et al., 2012; MacInnes et al., 2000; Milani et al., 2004; Morgan et al., 2002; Schifano et al., 1998; Singer et al., 2004; Verheyden et al., 2003). Laboratory animal studies show adverse effects of MDMA upon the developing foetus (Adori et al., 2010; Skelton et al., 2008), raising concerns about potentially damaging effects when taken by female recreational users during pregnancy. To date, there has been no controlled empirical data addressing this question, although there is some evidence of adverse birth consequences (McElhatton et al., 1999; Singer et al., 2012b). To investigate the potential effects of foetal MDMA exposure on development, the US National Institute on Drug Abuse (NIDA) funded the Drugs and Infancy Study (DAISY). This prospective study monitored a group of mothers whom took recreational Ecstasy/MDMA while pregnant, and a control group of pregnant females, the other ‘polydrug’ users. The two groups were followed over time, in order to monitor the physical development and psychobiological well-being of their children. Over the first year of life, the children of MDMA-using mothers displayed significantly poorer gross psychomotor skills than control group children (Singer et al., 2012a, 2012b). The DAISY study also assessed maternal well-being, using the Brief Symptom Inventory, a self-reporting measure of psychiatric health for non-clinical populations, derived from the earlier Symptom Check List-90 (Derogatis and Nelisaratos, 1983). This psychiatric measure was included, because previous research shows higher symptom profiles in abstinent Ecstasy/MDMA users. Soar et al. (2001) reviews the medical case study literature, which indicated an increased risk of several psychiatric disorders, including depression and psychosis, in MDMA users. Schifano et al. (1998) noted that regular Ecstasy/MDMA users are at increased risk of developing various psychiatric problems, the most frequent being depression. MacInnes et al. (2000) found significantly raised Beck Depression Inventory (BDI) scores in a non-clinical sample of abstinent regular Ecstasy/MDMA users. Singer et al. (2004) found that abstinent Ecstasy users reported significantly higher BSI scores for anxiety, depression and obsessive-compulsive disorder than non-user controls. Milani et al. (2004) reported significant gender effects, with female Ecstasy/MDMA users reporting higher levels of BSI anxiety, depression and somatization scores. Verheyden et al. (2003) investigated the reasons for quitting Ecstasy/MDMA: They found that most users in their large survey reported improved mental health after drug cessation. In the current DAISY, the BSI allowed us to prospectively monitor the psychiatric health of our pregnant mothers and to investigate how any changes in drug usage were associated with their report of psychological distress on the BSI. Based on previous findings, it might be predicted that elevated psychiatric symptoms would be evident in mothers who are continuing MDMA users, whilst those who discontinue use may show improvements; however, given its uniqueness, and the additional biopsychosocial changes associated with pregnancy and motherhood, the aims of the study were largely exploratory. Methods Experimental design The data in the current report were collected as part of the maternal assessment component of DAISY, a prospective study primarily exploring the effects of recreational drug use, notably MDMA/Ecstasy, on infant social and cognitive development (Moore et al., 2010; Singer et al., 2012a, 2012b). In a mixed design, mothers who used MDMA/Ecstasy during pregnancy (MDMA/Ecstasy users) were compared with those who used other drugs, but not MDMA/Ecstasy (Polydrug user controls), across measures of drug use and symptoms of mental distress, at four distinct time periods: the first trimester of pregnancy and at 1, 4 and 12 months post-partum. Participants We prospectively recruited 96 pregnant women from the UK through midwife referrals, leaflets describing the study at prenatal clinics and advertisements in commercial pregnancy magazines. We sought pregnant women whom were using recreational drugs during pregnancy, listing ecstasy, tobacco, Cannabis, alcohol and cocaine as examples. The majority of participants were therefore recreational ‘polydrug’ users. Exclusionary factors included: positive HIV status, moderate or severe intellectual disability, chronic medical disorder or psychiatric diagnosis. In total, there were 28 mothers in the MDMA-exposed group, who used MDMA (and other substances) during pregnancy, and 68 non-MDMA controls (some of whom used substances during pregnancy, but not MDMA). The majority of the sample were white, married or with a partner, and educated to a UK degree level. Their mean ages at the birth of their infants were 30.3 (SD 6.4) years of age in the MDMA-exposed group and 28.4 (SD 6.2) in the controls. The groups did not differ on basic demographic profiles. Participants were informed of data confidentiality and they gave written informed consent. The study protocol was approved by ethics committees from the University of East London, UK; Case Western Reserve University, US; and the National Health Service, UK. For a fuller description of the participant sample and screening procedures, see Singer et al. (2012a). Drug usage All women were individually interviewed about their substance use by fully trained female research assistants. The interview was an adaptation of the Maternal Post-Partum Interview, which was developed for earlier studies of maternal cocaine exposure (Singer et al., 2002). Interview questions covered substances commonly used in the UK and were based on the University of East London Recreational Drug Usage Questionnaire (Parrott et al., 2001). The list of drugs included tobacco/cigarettes, alcohol, Cannabis, Ecstasy/MDMA, amphetamine, cocaine, LSD, benzodiazepines, hallucinogenic mushrooms, ketamine and opiates. It may be noted that mephedrone (m-cathinone or ‘m-cat’) was not on this list, since the DAISY study was undertaken before ‘m-cat’ was used as a recreational drug (Schifano et al., 2011). Mean usage for each drug per week was calculated by multiplying the frequency of use with the amount taken per occasion. The MDMA user group comprised women who reported taking MDMA during pregnancy or in the month prior to pregnancy. Those who reported MDMA use prior to this time were categorized as non-users, because the study was designed to assess foetal drug exposure. Assessment battery The study included a comprehensive battery of assessment measures, covering various aspects of child behaviour and physical health indices, maternal activities and psychological well-being (Singer et al., 2012a,2012b). This report describes the findings from the Brief Symptom Inventory (BSI) (Derogatis and Nelisaratos, 1983). This questionnaire comprises 53 self-rating questions across nine psychiatric subscales, for: depression, anxiety, phobic anxiety, hostility, somatic complaints, obsessive-compulsive behavior, interpersonal sensitivity, paranoid ideation and psychosis/schizophrenia. The summary measure, the General Severity Index (GSI), provided a general index for overall psychiatric distress. The assessments covered four occasions: first trimester of pregnancy, 1 month post-partum, 4 months postpartum and 12 months post-partum. Statistical analyses Data that were positively skewed were transformed using natural logarithm, prior to analysis; however, the means and SDs are reported for the untransformed scores. Bivariate correlations were employed to calculate the inter-relationships between variables. Multicollinearity was assessed using tolerance and variance inflation factor. We implemented repeated measures Analysis of Variance (ANOVA), using a mixed model approach, by SAS Proc Mixed with maximum estimation method, to compare the substance use for both groups, MDMA-users during pregnancy (n = 28) and non-users of MDMA during pregnancy (n = 68), at the four different assessment times (during pregnancy, 4 weeks after birth, 12 weeks and 52 weeks). As noted earlier, both groups contained polydrug users of various substances, both legal (tobacco and/or alcohol), and illegal (Cannabis, amphetamine and/or cocaine) (Moore et al., 2010). Because the dependent variables were repeated measures and correlated within subjects, we used an unstructured covariance matrix to account for these correlated responses. We included interaction terms between drug groups and time, to test for homogeneity of MDMA effects over time. For all BSI outcome measures, we employed repeated measures Analysis of Covariance (ANCOVA). The covariates included other substance usage that differed by MDMA status at p < 0.10, and were correlated with the given outcome at p < 0.10 on at least two time points: They were then entered into the longitudinal model. Different sets of covariates were adjusted on each psychological outcome, and included demographic variables and use of all other drugs. Results The socioeconomic and educational profiles of mothers enrolled in the study are described more fully elsewhere (Singer et al., 2012a). In brief, the cohort was primarily white, married or in a stable relationship, and represented a wide range of socioeconomic backgrounds that included many from middle and higher psychosocial groupings. The MDMA-using mothers and polydrug control mothers were well matched on most variables (Singer et al., 2012a). Table 1 describes the group mean weekly rates of usage for the five main types of drug used: alcohol, nicotine/cigarettes, Cannabis/marijuana, cocaine and Ecstasy/MDMA. Other psychoactive drugs were taken by a few individuals, and those data are described more fully elsewhere (Moore et al., 2010). A mixed ANOVA was conducted with group as the between-conditions factor and time as the within-conditions factor. The between-groups ANOVA revealed that the two groups did not differ in overall use of alcohol, cigarettes, Cannabis nor cocaine; although the cocaine group effect was statistically borderline (Table 1, where the group effect for Ecstasy was not calculated, because it was used to define these two groups). The ANOVA for the time factor was significant for all five drugs (all p = 0.01 or smaller), with lower rates of usage during the weeks after giving birth. The ANOVA grouped by time interactions were significant for alcohol and cigarettes (F[3,88] = 4.06; p < 0.005 and F[3,88] = 3.61; p < 0.02 respectively), with the MDMA mothers using slightly more than the controls during the first trimester of pregnancy, but slightly less than controls across all the other time periods (Table 1). The group × time interaction was not significant for Cannabis, though Ecstasy/MDMA-using mothers appeared to be taking slightly more Cannabis than controls, across all time points (Table 1). The group × time interaction was significant for cocaine (F[3,88] = 3.48; p < 0.05), with the most usage during the first session by Ecstasy users (Table 1). The Ecstasy-using mothers reported taking an average of 0.84 Ecstasy tablets/week during the first trimester of pregnancy. In terms of previous lifetime usage (Singer et al., 2012a), they reported first using Ecstasy at a mean age of 20.2 years (range 14 – 29 years), had taken it on an average of 171 times/lifetime (range 6 – 936 times), and typically ingested an average of 3 tablets per occasion (range 1 – 8 tablets), with an average maximum usage per occasion of 7.4 Ecstasy tablets (range 2 – 20 tablets). Turning to their usage around the time of pregnancy, the mean total amount of MDMA used during pregnancy and in the month prior was 25 tablets (range 0.45 – 180 tablets). Within the polydrug control group, several women had used ecstasy/MDMA previously, but were currently non-users (Singer et al., 2012a). The Brief Symptom Inventory findings are summarized in Table 2. The main focus of interest here is the difference in psychiatric well-being between the first and last sessions. Over that time period, the control group mothers showed a significant decline in BSI symptoms for somatization (p < 0.001) and anxiety (p < 0.05). Over the same period, the Ecstasy/MDMA subgroup mothers showed significant declines in BSI symptoms for (Table 2): somatization (p < 0.001), depression (p < 0.05), interpersonal sensitivity (p < 0.05) and obsessive-compulsive disorder (p < 0.05). Discussion The young mothers in the DAISY study provided a unique cohort in several respects. Although recreational polydrug users, they were predominantly middle class with middle socioeconomic status, and in stable interpersonal relationships; hence, unlike many studies of illicit drug users, they were not socially disadvantaged. The study covered an extended time period of nearly 2 years, and is to our knowledge the first study of pregnant Ecstasy/MDMA users. The cohort of almost 100 mothers was comparatively large, especially for a prospective study with repeated assessments. One of the main aims of the DAISY study was to investigate the effects of recreational Ecstasy/MDMA usage during pregnancy on subsequent child development. The main findings were that the children of Ecstasy/MDMA using mothers displayed significant psychomotor problems in comparison to control group children, as described elsewhere (Singer et al., 2012a, 2012b). The study design allowed us to monitor changes in maternal reports of psychological well-being over time, in particular any alterations in their psychiatric status from the first to the last assessment. In this respect, both groups of mothers showed significantly higher somatization scores during the first trimester of pregnancy, when compared to 12 months post-partum (Table 2). The control group mothers also showed a significant reduction in BSI symptoms of anxiety, while the MDMA subgroup showed a very similar trend (Table 2). The first trimester of pregnancy is a period of pronounced somatic body changes, and so intuitively explains the higher somatization scores in both groups of women. Thus, the reduced BSI somatization scores 1 year post-partum may reflect a return to physical normality in both groups of women. The first trimester of pregnancy is also a period of general anxiety, with natural concerns and worries over becoming pregnant. This may help to explain the comparatively higher BSI anxiety scores during the first trimester, and the reduced scores at the final session (Table 2). The Ecstasy/MDMA-using mothers showed a different pattern of change, compared to the controls, on three BSI subscales, for: depression, obsessive compulsive disorder and interpersonal sensitivity (Table 2). The Ecstasy subgroup mothers reported feeling more depressed than control mothers at the first time point, with a statistically borderline between-group difference (p = 0.058, two-tail). At 1 year post-partum, the depression scores for the MDMA group had reduced significantly (p < 0.05), to become almost identical to the control group (Figure 1). The BSI depression scores for the control group mothers remained broadly unchanged over this period. The MDMA group also showed significant BSI reductions for interpersonal sensitivity and obsessive-compulsive disorder (Table 2). In order to examine the potential reasons for these changes, the changing patterns of drug usage over time should be noted. The Ecstasy/MDMA-group mothers had reduced their usage of Ecstasy to near-zero after giving birth (Table 1); hence, 1 year post-partum they had become former MDMA users. Their BSI improvement may reflect this cessation of Ecstasy/MDMA use. There is extensive empirical literature demonstrating higher rates of psychiatric distress in current Ecstasy/MDMA users and psychiatric gains following drug cessation. Schifano et al. (1998) gave structured psychiatric interviews to young Ecstasy/MDMA users at an addiction centre in Italy, reporting that around one-half the sample reported symptoms of psychiatric distress, especially depression, but also psychotic disorder, impulse control disorder, bulimia and panic disorder. MacInnes et al. (2000) compared young Ecstasy/MDMA users and polydrug controls, with participants screened to exclude anyone with a prior psychiatric history. On the BDI, Ecstasy users displayed significantly higher depression scores than the non-MDMA-user controls. In a survey of over 700 young people from the UK and Italy, the SCL-90 symptom profiles of the Ecstasy polydrug users were significantly higher than the non-MDMA-user controls (Parrott et al., 2001). In a US study of abstinent MDMA users compared to non-user controls who visited raves (Singer et al., 2004), the Ecstasy/MDMA users reported significantly higher BSI depression, anxiety and obsessive-compulsive disorder than the controls. Brière et al. (2012) prospectively found that taking up recreational Ecstasy/MDMA in Canadian schoolchildren led to increased depression 1 year later. There are also indications that psychiatric health can improve after quitting. Morgan et al. (2002) report that current Ecstasy/MDMA users have elevated scores on many SCL-90 subscales, whereas former Ecstasy users have scores intermediate between the current Ecstasy users and the non-user controls. Verheyden et al. (2003) interviewed former users about their reasons for quitting Ecstasy/MDMA. Over one-half reported that ‘mental health problems due to MDMA’ were the main reason for quitting drug use: That using Ecstasy led to feelings of anxiety and depression, and that they feared for their mental health in the longer-term. Over 70% of those participants report ‘improved mental health’ after quitting. An important potential confounder for Ecstasy/MDMA research is the use of other recreational drugs, because many Ecstasy users take a range of psychoactive drugs (Parrott et al., 2001; Parrott et al., 2007; Sala and Braida, 2004; Scholey et al., 2004). In the DAISY study, we collected systematic drug usage data at all four time points. As noted above, the use of Ecstasy/MDMA was largely restricted to the first trimester of pregnancy. In contrast, the use of alcohol, tobacco and Cannabis continued throughout the study. There is some indication of a decline in all drug use in the Ecstasy/MDMA group, with significant group/time interactions for alcohol and cigarettes, especially. As such, it could be argued that the depression effect in the Ecstasy/MDMA users was in part due to changes in alcohol and/or cigarette use, as both have been linked to higher depression scores (Munafo and Araya, 2010; Raimo and Schuckit, 1998); however, usage rates at baseline were broadly similar to 1 year post-partum, in both groups (Table 1). Hence, the changes in psychiatric status noted here (Table 2) cannot easily be attributed to alcohol, tobacco, nor Cannabis usage; however, the usage pattern for cocaine was very similar to Ecstasy/MDMA, with almost total cessation after the first trimester (Table 1). Thus, the selective reductions in particular psychiatric symptoms may reflect the cessation of Ecstasy/MDMA and/or cocaine usage. There are several ways in which central nervous system (CNS) stimulant drugs like MDMA can enhance psychiatric distress. In acute terms, MDMA is a powerful mood intensifier, but it can boost positive and negative-feeling states; thus, increased levels of happiness and euphoria are often accompanied by emotional tension. This intensification of both positive and negative moods is reported in studies of recreational users and in placebo-controlled laboratory studies (Kirkpatrick et al., 2012; Parrott et al., 2011). It is also noted in the psychotherapeutic situation: Two clients undergoing ‘MDMA-assisted psychotherapy’ experienced a resurgence of previous psychiatric problems following acute MDMA administration, with one client needing psychotherapy for a year afterwards, to resolve the MDMA-induced problems (Greer and Tolbert, 1986; Parrott, 2007). In sub-acute terms, MDMA use is typically followed by a period of neurochemical recovery, when low moods and feelings of depression predominate; indeed, the ‘mid-week blues’ can often last for several days and may reach clinical levels in some individuals (Curran and Travill, 1997). Because the positive mood intensification under MDMA is brief (several hours), and the post-MDMA period of mood recovery is more prolonged (several days), the average weekly mood of Ecstasy users will often be lower than in non-users (Parrott and Lasky, 1998). Such effects are supported in the animal literature by the acute and subacute impact of MDMA on 5-HT, notably, delays in recovery of this transmitter in brain regions regulating emotion (Colado et al., 1999); and similar pattern reductions in other functional serotonergic factors, such as SERT and tryptophan hydroxylase (Adori et al., 2011). In addition, in chronic terms, abstinent Ecstasy/MDMA users report higher levels of stress and lower levels of happiness than non-user controls (Scholey et al., 2011). When used repeatedly, sympathomimetic drugs such as amphetamine, cocaine and MDMA can adversely affect the hypothalamic pituitary adrenal (HPA) axis and impair homeostatic control via the stress hormone cortisol (Seyle, 1955). Indeed, acute MDMA use can increase cortisol levels by 800% in young dance club attendees (Parrott et al., 2008). While sub-chronically, recent Ecstasy/MDMA users display a 400% increase of cortisol in 3-month hair samples (Parrott et al., 2012); hence, recreational MDMA is both an acute and chronic stressor for the HPA axis (Parrott, 2009). There is also evidence that premorbid factors may heighten the likelihood of clinical problems in disadvantaged individuals; this interactive ‘diathesis-stress’ model for recreational Ecstasy/MDMA is described more fully elsewhere (Parrott, 2006). The possible causative factors (including neurotoxicity, recovery and/or HPA axis changes) for the effects observed here in the current data, and in much of the literature, still need considerable further empirical investigation. There are several limitations to the DAISY study. We relied on self-reported drug use and cannot therefore be certain that ‘Ecstasy’ comprised ‘MDMA’; however, data collection occurred during 2003 – 2006, which corresponded with a period of high MDMA purity in the UK. This was apparent in another study we undertook during 2006, which shows very high concordance between self-rated Ecstasy and MDMA use as detected in saliva samples (Parrott et al., 2008). The second weakness was the absence of a non-user control group, because many studies have found that polydrug users are more impaired than non-users (Morgan et al., 2002; Parrot et al., 2001). Thirdly, although the DAISY study was designed as a prospective study, this was only partially achieved (Moore et al., 2010); hence, missing data point’s retrospective ratings were sometimes required (Singer et al., 2012a). Finally, the overall BSI difference scores were not large (Table 2); however, we were not expecting strong drug effects, because our participants were psychiatrically normal and their use of most drugs was similar at the first and last time points. Furthermore, although the group mean reduction of 0.2 on the BSI depression subscale may have been comparatively slight, it would still be beneficial for the individual user. It would also reduce the likelihood of individuals with prior vulnerability factors from developing more severe psychiatric problems (Parrott, 2006). In summary, recreational stimulant drugs such as MDMA, cocaine and amphetamine, are well-known to be associated with enhanced psychiatric distress. The DAISY study found that women who took Ecstasy/MDMA during their first trimester of pregnancy reported slightly higher psychiatric symptom profiles than a control group of polydrug-using mothers. One year after giving birth, their psychiatric symptom profiles improved to values near the control group (Table 2 and Figure 1). The main explanatory factor proposed for this gain in psychiatric well-being was the cessation of Ecstasy/MDMA usage, coupled with the parallel reduction in cocaine use. Hence, this study confirmed that a reduction in stimulant drug usage can have beneficial effects on well-being. Finally, we should also note that the DAISY study investigated the effects of MDMA use during pregnancy on the child’s subsequent development. It reveals that the children of MDMA-using mothers have various impairments in gross psychomotor skill (Singer et al., 2012a,2012b); hence, an important message for young females and their partners is to stop taking MDMA before pregnancy. This will protect the developing child and enhance maternal well-being. Acknowledgements We would like to thank all the mothers who gave of their time and patience. Many thanks also to Fleur Braddick, Emma Axelsson, Stephanie Lynch, Helena Ribeiro, Caroline Frostick, Alice Toplis and Helen Fox, for undertaking the data collection and scoring. Funding This work (DAISY study) was funded by the National Institute on Drug Abuse in America (grant number DA-14910-05). Figure 1 Brief Symptom Inventory ratings of depression during the first trimester and at 12 months post-partum, in women reporting MDMA/Ecstasy use during pregnancy, and in control women taking other recreational drugs during pregnancy. *p < 0.05; Error bars indicate ±1 SE. MDMA: ‘Ecstasy’ or 3,4-methylenedioxymethamphetamine. Table 1 Ecstasy/MDMA, alcohol, cigarettes, marijuana/Cannabis and cocaine usage patterns for 28 mothers whom took Ecstasy/MDMA during pregnancy and a control group of 68 polydrug users during pregnancy. Drug values represent mean weekly rates of usage, during first trimester of pregnancy and three times up to 1 year post-partum. Drug type Maternal group First trimester of pregnancy 1 month post-partum 4 months post- partum 12 months post-partum ANOVA Group Time (G×T) Ecstasy (tablets) Polydrug controls 0.00 +/− 0.00 0.00 +/− 0.00 0.02 +/− 0.16 0.01 +/− 0.02 No between- group analysis < .0001 - Ecstasy users 0.82 +/− 1.57 0.01 +/− 0.03 0.03 +/− 0.13 0.06 +/− 0.09 Alcohol (units) Polydrug controls 6.94 +/− 16.90 3.11 +/− 10.66 6.48 +/−10.89 13.75 +/−24.02 n.s .0001 .02 Ecstasy users 12.07 +/− 16.62 1.33 +/− 1.80 5.30 +/− 5.70 6.01 +/− 5.99 Cigarette (numbers) Polydrug controls 28.15 +/−48.10 23.45 +/− 50.13 27.27 +/− 40.02 32.88 +/−48.14 n.s .0001 .003 Ecstasy users 44.78 +/− 49.50 17.88 +/−30.79 17.59 +/− 22.23 28.68 +/−34.37 Cannabis (joints) Polydrug controls 7.44 +/− 19.24 3.36 +/− 7.87 3.12 +/−7.51 5.26 +/−12.95 n.s .0001 n.s. Ecstasy users 10.28 +/− 20.81 6.86 +/− 17.36 6.20 +/− 16.12 7.35 +/−15.46 Cocaine (grams) Polydrug controls 0.02 +/−0.18 0.001 +/− 0.01 0.01 +/− 0.07 0.02 +/− 0.14 .057 .013 .03 Ecstasy users 0.23 +/− 0.85 0.01 +/− 0.04 0.02 +/− 0.06 0.02 +/− 0.05 MDMA: ‘Ecstasy’ or 3,4-methylenedioxymethamphetamine. Note: Units refer to UK units of alcohol (1 unit = 10ml or 7.9 grams of alcohol); n.s.= non-significant. Table 2 Psychiatric symptoms on the Brief Symptom Inventory during and after pregnancy for 28 mothers whom took Ecstasy/MDMA during pregnancy and for a non-user control group of 68 mothers whom took other drugs during pregnancy (polydrug controls). Group Time 1: Early-mid Pregnancy Time 2: Postpartum 1 month Time 3: Postpartum 4 months Time 4: Postpartum 12 months Paired comparison, Time 1 vs. 4 General symptoms Polydrug controls 0.61 0.51 0.54 0.50 - Ecstasy users 0.79 0.71 0.81 0.56 - Depression Polydrug controls 0.50 0.45 0.57 0.50 - Ecstasy users 0.87 0.74 0.80 0.51 p < 0.05 Anxiety Polydrug controls 0.55 0.46 0.48 0.34 p < 0.05 Ecstasy users 0.74 0.68 0.69 0.56 - Hostility Polydrug controls 0.71 0.65 0.66 0.59 - Ecstasy users 0.74 0.80 1.24 0.55 - Psychoticism Polydrug controls 0.31 0.25 0.36 0.30 - Ecstasy users 0.52 0.51 0.62 0.42 - Somatization Polydrug controls 0.58 0.36 0.27 0.32 p < 0.001 Ecstasy users 0.78 0.50 0.51 0.39 p < 0.01 Paranoid ideation Polydrug controls 0.61 0.48 0.64 0.66 - Ecstasy users 0.75 0.70 066 0.73 - Obsessive-compulsive Polydrug controls 1.08 1.10 0.98 0.89 - Ecstasy users 1.20 1.23 1.31 0.82 p < 0.05 Interpersonal sensitivity Polydrug controls 0.74 0.64 0.64 0.73 - Ecstasy users 0.92 0.85 0.96 0.57 p < 0.05 Phobic anxiety Polydrug controls 0.27 0.20 0.30 0.20 - Ecstasy users 0.47 0.39 0.52 0.37 - MDMA: ‘Ecstasy’ or 3,4-methylenedioxymethamphetamine. Conflict of interest The authors declare no conflict of interest. References Ádori C Andó RD Szekeres M Recovery and aging of serotonergic fibers after single and intermittent MDMA treatment in dark agouti rat J Comp Neurol 2011 519 2353 2378 21456018 Ádori C Zelena D Tímár J Intermittent prenatal MDMA exposure alters physiological but not mood related parameters in adult rat offspring Behav Brain Res 2010 206 299 309 19782105 Benningfield MM Cowan RL Brain serotonin function in MDMA (ecstasy) users: Evidence for persisting neurotoxicity Neuropsychopharmacol 2013 38 253 255 Brière FN Fallu JS Janosz M Prospective associations between meth/amphetamine (speed) and MDMA (ecstasy) use and depressive symptoms in secondary school students J Epidemiol Commun Health 2012 66 990 994 Colado MI Granados R O’Shea E The acute effect in rats of 3, 4-methylenedioxyethamphetamine (MDEA, ‘Eve’) on body temperature and long term degeneration of 5-HT neurones in brain: A comparison with MDMA (‘Ecstasy’) Pharmacol Toxicol 1999 84 261 266 10401727 Curran HV Travill RA Mood and cognitive effects of 3,4-methylenedioxymethamphetamine (MDMA, ‘ecstasy’): weekend ‘high’ followed by mid-week ‘low’ Addiction 1997 92 821 831 9293041 Derogatis L Nelisaratos N The Brief Symptom Inventory: An introductory report Psycholog Med 1983 13 595 605 Erritizoe D Frokjaer VG Holst KK In vivo imaging of cerebral serotonin transporter and serotonin (2A) receptor binding in 3,4-methylenedioxymethamphetamine (MDMA or ‘ecstasy’) and hallucinogen users Arch Gen Psychiatr 2011 68 562 576 21646575 Fox HC McLean A Turner JJD Neuropsychological evidence of a relatively selective profile of temporal dysfunction in drug-free MDMA (‘ecstasy’) polydrug users Psychopharmacol 2002 162 203 214 Greer G Tolbert R Subjective Reports of the Effects of MDMA in a Clinical Setting Journal of Psychoactive Substances 1986 18 319 327 Johnston LD O’Malley PM Brackman JG Monitoring the future national survey on drug abuse 1975 – 2004: Volume 2; College students and adults aged 19 – 45 Report for the US National Institute of Health, no. 05–5728 2005 National Institute on Drug Abuse Bethesda Kirkpatrick MG Gunderson EW Perez AY A direct comparison of the behavioral and physiological effects of methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA) in humans Psychopharmacol 2012 219 109 122 Kish SJ Lerch J Furukawa Y Decreased cerebral cortical serotonin transporter binding in ecstasy users: A positron emission tomography/[(11)C]DASB and structural brain imaging study Brain 2010 133 1779 1797 20483717 McCann UD Sgambati FP Schwartz AR Sleep apnea in young abstinent recreational MDMA (‘ecstasy’) consumers Neurology 2009 73 2011 2017 19955499 McElhatton PR Bateman DN Evans C Congenital abnomalies after prenatal ecstasy exposure Lancet 1999 354 1441 1442 10543673 MacInnes N Handley SL Harding GFA Former chronic methylenedioxymethamphetamine (MDMA or ecstasy) users report mild depressive symptoms J Psychopharmacol 2001 15 181 186 11565625 Milani RM Parrott AC Turner JJD Gender differences in self-reported anxiety, depression and somatization among ecstasy/MDMA polydrug users, alcohol/tobacco users and nondrug users Addict Behav 2004 29 965 971 15219343 Montgomery C Hatton NP Fisk JE Assessing the functional significance of ecstasy-related memory deficits using a virtual reality paradigm Hum Psychopharmacol 2010 25 318 325 20521322 Moore DG Turner JD Parrott AC During pregnancy, recreational drug-using women stop taking ecstasy (3,4-methylenedioxy-N-methylamphetamine) and reduce alcohol consumption, but continue to smoke tobacco and Cannabis: initial findings from the Development and Infancy Study J Psychopharmacol 2010 24 1403 1410 19939863 Morgan MJ McFie L Fleetwood LH Ecstasy (MDMA): Are the psychological problems associated with its use reversed by prolonged abstinence? Psychopharmacol 2002 159 294 303 Munafo MR Araya R Cigarette smoking and depression: A question of causation Brit J Psychiatry 2010 196 425 426 20513848 Parrott AC MDMA in humans: Factors which affect the neuropsychobiological profiles of recreational Ecstasy users, the integrative role of bio-energetic stress J Psychopharmacol 2006 20 147 163 16510474 Parrott AC The psychotherapeutic potential of MDMA (3,4-methylenedioxymethamphetamine): an evidence-based review Psychopharmacology 2007 191 181 193 17297639 Parrott AC Cortisol and MDMA (3,4-methylenedioxymethamphetamine): Neurohormonal aspects of bioenergetic-stress in Ecstasy users Neuropsychobiol 2009 60 148 158 Parrott AC MDMA and serotonergic neurotoxicity: Empirical evidence for adverse effects in humans - no need for translation Brit J Pharmacol 2012 166 1518 1520 22404300 Parrott AC MDMA neurotoxicity: the functional implications of serotonin loss in recreational ecstasy users Neurosci Biobehav Revs 2013a 37 1466 1486 23660456 Parrott AC Human psychobiology of MDMA or ‘Ecstasy’: An overview of 25 years of empirical research Hum Psychopharmacol 2013b 28 289 307 23881877 Parrott AC Gibbs A Scholey AB MDMA and methamphetamine: some paradoxical negative and positive mood changes in an acute dose laboratory study Psychopharmacology 2011 215 527 36 21318566 Parrott AC Lasky J Ecstasy (MDMA) effects upon mood and cognition; before, during, and after a Saturday night dance Psychopharmacol 1998 139 261 268 Parrott AC Jones L Sands HR High cortisol levels in recent Ecstasy/MDMA users: Preliminary findings from the Swansea, Westminster and Dresden collaborative study British Psychological Society Annual Psychobiology Conference UK 3–5 September 2012 2012 Conference Abstract p.16 Parrott AC Lock J Conner AC Dance clubbing on-MDMA and during abstinence from MDMA: Prospective neuroendocrine and psychobiological changes Neuropsychobiol 2008 57 165 180 Parrott AC Milani RM Gouzoulis-Mayfrank E Cannabis and Ecstasy/MDMA (3,4-methylenedioxymethamphetamine): An analysis of their neuropsychobiological interactions in recreational users J Neural Transmiss 2007 114 959 968 Parrott AC Milani RM Parmar R Recreational Ecstasy/MDMA and other drug users from the UK and Italy: Psychiatric symptoms and psychobiological problems Psychopharmacol 2001 159 77 82 Puerta E Hervias I Aguirre N On the mechanisms underlying 3,4-methylenedioxymethamphetamine toxicity: the dilemma of the chicken and the egg Neuropsychobiology 2009 60 119 129 19893329 Raimo EB Schuckit MA Alcohol dependence and mood disorders Addict Behav 1998 23 933 946 9801727 Reay JL Hamilton C Kennedy DO MDMA polydrug users show process-specific central executive impairments coupled with impaired social and emotional judgment processes J Psychopharmacol 2006 20 385 388 16574712 Ricaurte GA McCann UD Szaboc Z Scheffelc U Toxicodynamics and long-term toxicity of the recreational drug, 3,4-methylenedioxymethamphetamine (MDMA, ‘Ecstasy’) Toxicology Letters 2000 112–113 143 146 Rogers G Elston J Garside R The harmful health effects of recreational ecstasy: A systematic review of observational evidence Health Technol Assess 2009 13 1 315 Sala M Braida D Endocannabinoids and 3,4-methylenedioxymethamphetamine (MDMA) interaction Pharmacol Biochem Behav 2005 81 407 416 15927242 Schifano F Albanese A Fergus S Mephedrone (4-methylmethcathinone; ‘meow meow’): chemical, pharmacological and clinical issues Psychopharmacol 2011 214 593 602 Schifano F Di Furia L Forza G MDMA (‘ecstasy’) consumption in the context of polydrug abuse: A report on 150 patients Drug Alc Depend 1998 52 85 90 Scholey AB Owen L Gates J Hair MDMA samples are consistent with reported Ecstasy use: Findings from a study investigating effects of Ecstasy on mood and memory Neuropsychobiology 2011 63 15 21 20962543 Scholey AB Parrott AC Buchanan T Increased intensity of Ecstasy and polydrug usage in the more experienced recreational Ecstasy/MDMA users: a WWW study Addict Behav 2004 29 743 52 15135556 Singer LT Salvator A Arendt RE Effects of cocaine/polydrug exposure and maternal psychological distress on infant birth outcomes Neurotoxicol Teratol 2002 24 127 135 11943500 Singer LT Linares TJ Ntiri S Psychosocial profiles of older adolescent MDMA users Drug Alc Depend 2004 74 245 252 Singer LT Moore DG Fulton S Neurobehavioral outcomes of infants exposed to MDMA (Ecstasy) and other recreational drugs during pregnancy Neurotoxicol Teratol 2012a 34 303 310 22387807 Singer LT Moore DG Min MO One-year outcomes of prenatal exposure to MDMA and other recreational drugs Pediatrics 2012b 130 407 413 22908109 Skelton MR Williams MT Vorhees CV Developmental effects of 3,4-methylenedioxymethamphetamine: A review Behav Pharmacol 2008 19 91 111 18332674 Soar K Turner JJD Parrott AC Psychiatric disorders in recreational Ecstasy (MDMA) users: A literature review focusing upon personal predisposition factors and drug histories Hum Psychopharmacol 2001 16 641 646 12404545 Topp L Hando J Dillon P Ecstasy use in Australia: Patterns of use and associated harm Drug Alc Depend 1999 55 105 115 Verheyden SL Maidment R Curran HV Quitting ecstasy: An investigation of why people stop taking the drug and their subsequent mental health J Psychopharmacol 2003 17 371 378 14870948 Winstock AR Griffiths P Stewart D Drugs and the dance music scene: A survey of current drug use patterns among a sample of dance music enthusiasts in the UK Drug Alc Depend 2001 64 9 17 Wu P Liu X Pham TH Ecstasy use among US adolescents from 1999 to 2008 Drug Alc Depend 2010 112 33 38 Zakzanis KK Campbell Z Memory impairment in now abstinent MDMA users and continued users: A longitudinal follow-up Neurology 2006 66 740 741 16534114

PMC005xxxxxx/PMC5111169.txt

"LICENSE: This file is available for text mining. It may also be used consistent with the principles(...TRUNCATED)

PMC005xxxxxx/PMC5111634.txt

"LICENSE: This file is available for text mining. It may also be used consistent with the principles(...TRUNCATED)

PMC005xxxxxx/PMC5111811.txt

"LICENSE: This file is available for text mining. It may also be used consistent with the principles(...TRUNCATED)

PMC005xxxxxx/PMC5111812.txt

"LICENSE: This file is available for text mining. It may also be used consistent with the principles(...TRUNCATED)

PMC005xxxxxx/PMC5111816.txt

"LICENSE: This file is available for text mining. It may also be used consistent with the principles(...TRUNCATED)

PMC005xxxxxx/PMC5112117.txt

"LICENSE: This file is available for text mining. It may also be used consistent with the principles(...TRUNCATED)