Role of marijuana components on the regenerative ability of stem cells.

Stem cell therapy promotes tissue regeneration and wound healing. Efforts have been made to prime stem cells to enhance their regenerative abilities. Certain marijuana components, namely the non-psychoactive cannabidiol (CBD) and psychoactive tetrahydrocannabinol (THC), are defined as immunomodulators.9 We test whether two sources of stem cells, primed with CBD or THC, would demonstrate improved regenerative abilities. Human adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (BMDSCs), not obtained from the same individual, were treated with low (300 nM) or high (3 µM) concentration CBD. Porcine ASCs and BMDSCs were isolated from a single pig, and treated with either low or high concentrations of CBD or THC. Transwell migration and MTT proliferation assays were performed on the human ASCs and BMDSCs. Also, transwell migration assay was performed on the porcine ASCs and BMDSCs. Finally, a wound healing scratch assay in porcine primary fibroblasts (PFs) was performed, co-cultured with the cannabinoid-treated ASCs. CBD priming at low concentration induces migration by 180% (P < .01) in porcine ASCs, and by only 93% (P < .02) in porcine BMDSCs. In porcine stem cells, THC priming at low concentration induces migration by 91.6% (P < .01) in ASCs but by only 44.3% (P < .03) in BMDSCs. Compared to PFs co-cultured with untreated ASCs, PFs co-cultured with low CBD-primed ASCs had 75% faster wound closure at 18 hours (P < .01). CBD and THC priming of ASCs and BMDSCs, particularly at lower doses, enhances a number of regenerative parameters, suggesting that these major marijuana components may improve stem cell-based therapies. SIGNIFICANCE OF THE STUDY: Our study demonstrates that cannabinoids can enhance the regenerative capacity of two major sources of stem cells, adipose- and bone marrow-derived, from human and porcine donors. Stem cell isolation and expansion is invasive, costly and time consuming. Stem cells with improved regenerative properties may be effective in the treatment of acute or chronic wounds. This is the first study to compare the priming potential of two sources of stem cells from the same animal, with the same genetic and epigenetic profile, as well as the first to prime with THC.

Long Noncoding RNA AW112010 Promotes the Differentiation of Inflammatory T Cells by Suppressing IL-10 Expression through Histone Demethylation.

Long noncoding RNAs (lncRNAs) have been demonstrated to play important regulatory roles in gene expression, from histone modification to protein stability. However, the functions of most identified lncRNAs are not known. In this study, we investigated the role of an lncRNA called AW112010. The expression of AW112010 was significantly increased in CD4+ T cells from C57BL/6J mice activated in vivo with myelin oligodendrocyte glycoprotein, Staphylococcal enterotoxin B, or in vitro with anti-CD3 anti-CD28 mAbs, thereby demonstrating that activation of T cells leads to induction of AW112010. In contrast, anti-inflammatory cannabinoids such as cannabidiol or δ-9-tetrahydrocannabinol decreased the expression of AW112010 in T cells. Interestingly, the expression of AW112010 was high in in vitro-polarized Th1 and Th17 cells but low in Th2 cells, suggesting that this lncRNA may regulate inflammation. To identify genes that might be regulated by AW112010, we used chromatin isolation by RNA purification, followed by sequencing. This approach demonstrated that AW112010 regulated the transcription of IL-10. Additionally, the level of IL-10 in activated T cells was low when the expression of AW112010 was increased. Use of small interfering RNA to knock down AW112010 expression in activated T cells led to increased IL-10 expression and a decrease in the expression of IFN-γ. Further studies showed that AW112010 interacted with histone demethylase KDM5A, which led to decreased H3K4 methylation in IL-10 gene locus. Together, these studies demonstrate that lncRNA AW112010 promotes the differentiation of inflammatory T cells by suppressing IL-10 expression through histone demethylation.

Step toward Roadside Sensing: Noninvasive Detection of a THC Metabolite from the Sweat Content of Fingerprints.

The sudden increase in states legalizing marijuana has forced law enforcement into a situation where the use and consumption are legal, but there are no limitations for what is acceptable for driving or operating machinery. Using ultraviolet-visible (UV-vis) spectroscopy, fingerprints from volunteers who had used marijuana were analyzed via a competitive immunoassay for the detection of Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive component of marijuana, and 11-nor-9-carboxy-THC (THC-COOH), one of the main metabolites produced in the body following the use/consumption of THC-related products. In this research, the THC-COOH metabolite and the enzyme-labeled conjugate compete against each other as the antigens for the system. The antibody used in this assay has a greater affinity for the metabolite; so, as its concentration increases, the absorbance of the system decreases due to less binding of the enzyme-labeled conjugate.

Disposable electrochemical immunosensor array for the multiplexed detection of the drug metabolites morphine, tetrahydrocannabinol and benzoylecgonine.

Heroin, marijuana and cocaine are widely abused drugs. Their use can be readily detected by analyzing urine for the metabolites morphine (MOR), tetrahydrocannabinol (THC) or benzoylecgonine (BZC). A multiplex immunosensor is described here for detection of MOR, THC and BZC using screen printed carbon array electrodes modified with gold nanoparticles. Antibodies against MOR, THC and BZC were immobilized on eight electrodes in a sensor array simultaneously, and a competitive assay was used for the detection. The free analytes in the sample compete with bovine serum albumin-conjugated analytes for the immobilized antibodies on the sensor surface. The array is capable of detecting the three drugs simultaneously within 20-40 min. The method has a high sensitivity, with detection limits as low as 1.2, 7.0, and 8.0 pg.mL-1 for MOR, THC and BZC, respectively. Cross reactivity testing was preformed to monitor any nonspecific binding. The results revealed good selectivity. Urine samples were spiked with the 3 drugs and tested with the multiplexed immunosensor. Recovery percentages ranged between 88 to 115%. Graphical abstract Schematic presentation of the multiplexed immunosensor for drugs of abuse,viz. tetrahydrocannabinol (THC), morphine (MOR), and benzoylecgonine (BZC)) by using an array of modified electrodes.

A Novel Tetrahydrocannabinol Electrochemical Nano Immunosensor Based on Horseradish Peroxidase and Double-Layer Gold Nanoparticles.

In the current study, a novel double-layer gold nanoparticles-electrochemical immunosensor electrode immobilized with tetrahydrocannabinol (THC) antibody derived from Balb/c mice was developed. To increase the fixed quantity of antibodies and electrochemical signals, an electrochemical biosensing signal amplification system was utilized with gold nanoparticles-thionine-chitosan absorbing horseradish peroxidase (HRP). In addition, a transmission electron microscope (TEM) was used to characterize the nanogold solution. To evaluate the quality of the immunosensor, the amperometric I-t curve method was applied to determine the THC in PBS. The results showed that the response current had a good linear correlation with the THC concentration range from 0.01~10³ ng/mL with a correlation coefficient of 0.9986. The lowest detection limit for THC was 3.3 pg/mL (S/N = 3). Moreover, it was validated with high sensitivity and reproducibility. Apparently, the immunosensor may be a very useful tool for monitoring the THC.

Small Molecule Detection in Saliva Facilitates Portable Tests of Marijuana Abuse.

As medical and recreational use of cannabis, or marijuana, becomes more prevalent, law enforcement needs a tool to evaluate whether drivers are operating vehicles under the influence of cannabis, specifically the psychoactive substance, tetrahydrocannabinol (THC). However, the cutoff concentration of THC that causes impairment is still controversial, and current on-site screening tools are not sensitive enough to detect trace amounts of THC in oral fluids. Here we present a novel sensing platform that employs giant magnetoresistive (GMR) biosensors integrated with a portable reader system and smartphone to detect THC in saliva using competitive assays. With a simple saliva collection scheme, we have optimized the assay to measure THC in the range from 0 to 50 ng/mL, covering most cutoff values proposed in previous studies. This work facilitates on-site screening for THC and shows potential for testing of other small molecule drugs and analytes in point-of-care (POC) settings.

A Retrospective Analysis of Urine Drugs of Abuse Immunoassay True Positive Rates at a National Reference Laboratory.

Urine drug screens are commonly performed to identify drug use or monitor adherence to drug therapy. The purpose of this retrospective study was to evaluate the true positive and false positive rates of one of our in-house urine drug screen panels. The urine drugs of abuse panel studied consists of screening by immunoassay then positive immunoassay results were confirmed by mass spectrometry. Reagents from Syva and Microgenics were used for the immunoassay screen. The screen was performed on a Beckman AU5810 random access automated clinical analyzer. The percent of true positives for each immunoassay was determined. Agreement with previously validated GC-MS or LC-MS-MS confirmatory methods was also evaluated. There were 8,825 de-identified screening results for each of the drugs in the panel, except for alcohol (N = 2,296). The percent of samples that screened positive were: 10.0% for amphetamine/methamphetamine/3,4-methylenedioxy-methamphetamine (MDMA), 12.8% for benzodiazepines, 43.7% for opiates (including oxycodone) and 20.3% for tetrahydrocannabinol (THC). The false positive rate for amphetamine/methamphetamine was ∼14%, ∼34% for opiates (excluding oxycodone), 25% for propoxyphene and 100% for phencyclidine and MDMA immunoassays. Based on the results from this retrospective study, the true positive rate for THC drug use among adults were similar to the rate of illicit drug use in young adults from the 2013 National Survey; however, our positivity rate for cocaine was higher than the National Survey.

Chronic Δ-9-tetrahydrocannabinol administration increases lymphocyte CXCR4 expression in rhesus macaques.

Cannabinoids have been reported to produce various immunomodulatory effects, which could potentially impact the host response to bacterial or viral infection. We have recently demonstrated that chronic Δ-9-tetrahydrocannabinol (THC; 0.32 mg/kg i.m., BID) decreased early mortality in rhesus macaques infected with simian immunodeficiency virus (SIV). However, the possibility that prolonged THC administration affects lymphocyte counts, phenotype, and proliferation indices has not been addressed. We examined expression of proliferative and phenotypic markers in circulating lymphocytes of male young adult rhesus macaques chronically-treated with THC (i.m. twice daily 0.32 mg/kg) for 12 months. Chronic THC administration did not alter lymphocyte subtypes, naïve and memory subsets, proliferation, or apoptosis of T lymphocytes when compared to time-matched vehicle-treated controls. However, chronic THC increased T lymphocyte CXCR4 expression on both CD4+ and CD8+ T lymphocytes compared to control. These results show that chronic THC administration produces changes in T cell phenotype, which can potentially contribute to host immunomodulation to infectious challenges.

Immunochemical approach using monoclonal antibody against Δ(9)-tetrahydrocannabinolic acid (THCA) to discern cannabis plants and to investigate new drug candidates.

A monoclonal antibody (MAb-4A4) against Δ9- tetrahydrocannabinolic acid (THCA) showing extensive cross-reactivity against various cannabinoids was prepared. Using this antibody, a competitive enzyme-linked immunoassay (ELISA) was developed to detect Δ9-THCA in the range of 1 to 100 mg/ml. Various cannabinoids including Δ9-THC (Δ9-tetrahydrocannabinolic acid), Δ8-THCA (Δ8-tetrahydrocannabinolic acid), Δ8-THC (Δ8-tetrahydrocannabinol), CBD (cannabidiol), and CBN (cannabinol) were recognized by MAb-4A4, and their cross-reactivities were 55-1600% compared with Δ9-THCA (100%). This novel characteristic of this MAb enabled detection of marijuana residues in biological samples by detection of residual cannabinoids. The ELISA using MAb-4A4 was found to be applicable even for withered samples which contained only trace amounts of Δ9-THCA and Δ9-THC. In addition, this method using MAb-4A4 could be useful in forensic analysis since the MAb-4A4 also shows cross-reactivities against cannabinoid metabolites in body fluids. As well as forensic applications using this MAb, an investigation of new drug candidates focusing on cannabinoid metabolites arising from biotransformation in plant tissue was performed using immunochemical screening. The resulting new drug candidates were cannabinoid glycosides biotransformed by Pinellia ternata whose bioactivity is as yet unidentified. Our results indicate the utility of the application of ELISA using MAb-4A4 for further experiments involving marijuana and cannabinoids not only in the forensic field but also in the context of drug discovery.

A structural insight into the molecular recognition of a (-)-Delta9-tetrahydrocannabinol and the development of a sensitive, one-step, homogeneous immunocomplex-based assay for its detection.

(-)-Delta9-tetrahydrocannabinol (THC) is the main psychoactive compound found in cannabis. In this study, an anti-THC Fab fragment, designed T3, was isolated from a display library cloned from the spleen cells of a mouse immunized with a THC-bovine serum albumin conjugate, and the crystal structures of the T3 Fab in its free form and in complex with THC were determined at 1.9 A and 2.0 A resolution, respectively. The THC binding site of the T3 Fab is a narrow cavity: the n-pentyl group of THC protrudes deep into the interface area between the variable domains and the C(10) monoterpene moiety of the hapten is partially exposed to solvent. The metabolites of THC, with modifications in the C(10) monoterpene moiety, 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol and 11-hydroxy-?(9)-tetrahydrocannabinol, are bound by the T3 Fab with a higher affinity than THC. The crystal structures suggest that Ser52H and Arg53H of the T3 Fab are able to make hydrogen bonds with the metabolites, which leads to an increased binding against these metabolites. By developing a T3 Fab-Delta(9)-THC immunocomplex binding antibody from a naïve antibody phage display library, the specificity of the Delta(9)-THC binding is highly increased, which allows a one-step, homogeneous, fluorescence resonance energy transfer-based sensitive immunoassay, with a detection limit of 20 ng/ml from saliva samples.

Validation of an enzyme immunoassay for detection and semiquantification of cannabinoids in oral fluid.

BACKGROUND: Oral fluid (OF) is gaining prominence as an alternative matrix for monitoring drugs of abuse in the workplace, criminal justice, and driving under the influence of drugs programs. It is important to characterize assay performance and limitations of screening techniques for Delta(9)-tetrahydrocannabinol (THC) in OF. METHODS: We collected OF specimens by use of the Quantisal OF collection device from 13 daily cannabis users after controlled oral cannabinoid administration. All specimens were tested with the Immunalysis Sweat/OF THC Direct ELISA and confirmed by 2-dimensional GC-MS. RESULTS: The limit of detection was <1 microg/L THC equivalent, and the assay demonstrated linearity from 1 to 50 microg/L, with semiquantification to 200 microg/L. Intraplate imprecision (n = 7) ranged from 2.9% to 7.7% CV, and interplate imprecision (n = 20) was 3.0%-9.1%. Cross-reactivities at 4 microg/L were as follows: 11-hydroxy-THC, 198%; Delta(8)-tetrahydrocannabinol (Delta(8)-THC), 128%; 11-nor-9-carboxy-THC (THCCOOH), 121%; THC (target), 98%; cannabinol, 87%; THCCOOH-glucuronide, 11%; THC-glucuronide, 10%; and cannabidiol, 2.4%. Of 499 tested OF specimens, 52 confirmed positive (THC 2.0-290 microg/L), with 100% diagnostic sensitivity at the proposed Substance Abuse and Mental Health Services Administration screening cutoff of 4 microg/L cannabinoids and GC-MS cutoff of 2 microg/L THC. Forty-seven specimens screened positive but were not confirmed by 2D-GC-MS, yielding 89.5% diagnostic specificity and 90.6% diagnostic efficiency. Thirty-one of 47 unconfirmed immunoassay positive specimens were from 1 individual and contained >400 ng/L THCCOOH, potentially contributing to cross-reactivity. CONCLUSIONS: The Immunalysis Sweat/OF THC Direct ELISA is an effective screening procedure for detecting cannabinoids in OF.

Attenuation of experimental autoimmune hepatitis by exogenous and endogenous cannabinoids: involvement of regulatory T cells.

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. Natural cannabinoids such as Delta(9)-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases. We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury. Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3+ T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC. The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels. In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation.

Delta9-tetrahydrocannabinol immunochemical studies: haptens, monoclonal antibodies, and a convenient synthesis of radiolabeled delta9-tetrahydrocannabinol.

Immunopharmacotherapy as an approach to combat drugs of abuse has become an active area of investigation. Marijuana is the most commonly used illicit drug in the U.S. The main active chemical in marijuana is delta9-tetrahydrocannabinol (delta9-THC); hence, monoclonal antibodies with high affinity and specificity for delta9-tetrahydrocannabinol could be valuable immunopharmacotherapeutic intervention and diagnostic tools. We have synthesized immunoconjugates that induce an effective immune response to delta9-THC and describe a convenient synthesis of radiolabeled delta9-THC. We demonstrate the value and use of this probe to select anti-delta9-THC antibodies that bind delta9-THC with good affinity. The synthetic route to radiolabeled delta9-THC has enabled the correct assessment of the affinity of these antibodies to their ligand and may facilitate future binding studies between delta9-THC and its analogues and the cannabinoid receptors.

Cannabinoid influence on cytokine profile in multiple sclerosis.

Cannabinoids have been suggested as possessing immunomodulatory properties, and cannabinoid receptors are present on leucocytes. Clinically, there is some evidence that cannabinoids may be therapeutically useful in treating multiple sclerosis, which is generally believed to be an autoimmune condition. This paper reports data derived from the Cannabinoids in MS (CAMS) study, which was the largest randomized controlled trial yet conducted to evaluate the therapeutic efficacy of cannabinoids. We found no evidence for cannabinoid influence on serum levels of interferon (IFN)-gamma, interleukin (IL)-10, IL-12 or C-reactive protein as measured using enzyme-linked immunosorbent assay (ELISA), in comparison to control values. Mitogenic stimulation experiments also failed to demonstrate any significant reduction in percentage of CD3+, IFN-gamma producing cells after exposure to cannabinoids in vivo, although numbers were small. Further work is needed to establish the functional significance of cannabinoid receptors on immune cells.

Delta 9-Tetrahydrocannabinol regulates Th1/Th2 cytokine balance in activated human T cells.

Human leukocytes express cannabinoid (CB) receptors, suggesting a role for both endogenous ligands and Delta 9-tetrahydrocannabinol (THC) as immune modulators. To evaluate this, human T cells were stimulated with allogeneic dendritic cells (DC) in the presence or absence of THC (0.625-5 microg/ml). THC suppressed T cell proliferation, inhibited the production of interferon-gamma and shifted the balance of T helper 1 (Th1)/T helper 2 (Th2) cytokines. Intracellular cytokine staining demonstrated that THC reduced both the percentage and mean fluorescence intensity of activated T cells capable of producing interferon-gamma, with variable effects on the number of T cells capable of producing interleukin-4. Exposure to THC also decreased steady-state levels of mRNA encoding for Th1 cytokines, while increasing mRNA levels for Th2 cytokines. The CB2 receptor antagonist, SR144528, abrogated the majority of these effects. We conclude that cannabinoids have the potential to regulate the activation and balance of human Th1/Th2 cells by a CB2 receptor-dependent pathway.

Hu 210: a potent tool for investigations of the cannabinoid system.

The synthetic compound HU 210 displays a multiplicity of biochemical, pharmacological, and behavioral effects, most of which have been demonstrated to be dependent on a selective agonistic activity at CB(1) and CB(2) cannabinoid receptors and to involve the main neurotransmitter systems. Results obtained in various studies suggest a potential clinical application of this highly potent drug (e.g., as antipyretic, antiinflammatory, analgesic, antiemetic, and antipsychotic agent) as well as its usefulness in research aimed to develop a better understanding of the involvement of the endogenous cannabinoid system in a number of physiopathological functions.

[Impact of delta-9-tetrahydrocannabinol and its metabolites on the immune system]. / Impact du delta-9-tetrahydrocannabinol et ses métabolites sur le système immunitaire.

Many studies obvious impact of cannabinoids on the immune system. These studies follow the rapid advanced researches led in the immunology field. D9 Tetrahydrocannabinol and their metabolites decrease production of tumoral necrosis factor alpha. This decrease has for consequence a decrease of the apoptosis. Recent discovery of implication of cytokines in the phenomena of dependence, make the cannabis and their metabolites promoting agent induced dependence in association with drug abuse. The withdrawal of these products necessitates a intact immune system. D9 Tetrahydrocannabinnol and their metabolites inhibit production of IL-1 and gamma interferon. This inhibition has for consequence a decrease of 33% of the lymphocytes activity and an inhibition of 66% of the lymphocytes adenyl cyclase activity. The consumption of cannabis decreases immunological competence of macrophages, and alterate their essential role of trophicity of the nervous central system. Furthermore, inhibiting actions of cannabinoids on the cyclo-oxygenase, promote production of arachidonic acid degradation products. This compounds mimic the action of histamine, and inducing a raise of the vascular permeability and bronchospasm. These inolecules contributes at delayed reaction of anaphylaxia. However these actions of cannabinoids on the immune system promote their pull-back in cure of new pathology likes AIDS.

Monoclonal antibody against tetrahydrocannabinolic acid distinguishes Cannabis sativa samples from different plant species.

The cross-reaction of anti-delta 1-THCA MAb against other cannabinoids was very wide. However, other naturally occurring and synthetic phenolics including opium alkaloids did not react to the MAb. Using this ELISA, this paper reports application of the competitive ELISA for detection of marijuana samples. The ELISA described here was very sensitive to the ether extracts of marijuana samples when compared to those of other plants. The assay provided a sensitive method useful for the judge of marijuana samples.

Monoclonal antibody based enzyme immunoassay for marihuana (cannabinoid) compounds.

MAb against delta 1-THCA was produced by fusing hybridoma with splenocytes immunized with delta 1-THCA-BSA conjugate and hypoxanthine, aminopterine, thymidine-sensitive mouse myeloma cell line, P3-X63-Ag8-653. The cross-reaction of anti-delta 1-THCA antibody against other cannabinoids was very wide, thus many cannabinoids and a spiro-compound were reactive suggesting that 2'-hydroxyl, 6'-hydroxyl or 6'-O-alkyl, 4'-alkylbenzene ring moiety is necessary for its reactivity. It became evident that this ELISA was able to be applied to the biotransformation experiments of cannabinoids in plant tissue culture system. The metabolites of delta 6-THC such as two major metabolites, 7-oxo-delta 6-THC and 7-hydroxyl-delta 6-THC were also detectable by this ELISA.