Assessment of Risks and Benefits of Using Antibiotics Resistance Genes in Mesenchymal Stem Cell-Based Ex-Vivo Therapy.

Recently, ex-vivo gene therapy has emerged as a promising approach to enhance the therapeutic potential of mesenchymal stem cells (MSCs) by introducing functional genes in vitro. Here, we explored the need of using selection markers to increase the gene delivery efficiency and evaluated the potential risks associated with their use in the manufacturing process. We used MSCs/CD that carry the cytosine deaminase gene (CD) as a therapeutic gene and a puromycin resistance gene (PuroR) as a selection marker. We evaluated the correlation between the therapeutic efficacy and the purity of therapeutic MSCs/CD by examining their anti-cancer effect on co-cultured U87/GFP cells. To simulate in vivo horizontal transfer of the PuroR gene in vivo, we generated a puromycin-resistant E. coli (E. coli/PuroR) by introducing the PuroR gene and assessed its responsiveness to various antibiotics. We found that the anti-cancer effect of MSCs/CD was directly proportional to their purity, suggesting the crucial role of the PuroR gene in eliminating impure unmodified MSCs and enhancing the purity of MSCs/CD during the manufacturing process. Additionally, we found that clinically available antibiotics were effective in inhibiting the growth of hypothetical microorganism, E. coli/PuroR. In summary, our study highlights the potential benefits of using the PuroR gene as a selection marker to enhance the purity and efficacy of therapeutic cells in MSC-based gene therapy. Furthermore, our study suggests that the potential risk of horizontal transfer of antibiotics resistance genes in vivo can be effectively managed by clinically available antibiotics.

Cell Type-specific Knockout with Gli1-mediated Cre Recombination in the Developing Cerebellum.

The inducible Cre-loxP system provides a useful tool for inducing the selective deletion of genes that are essential for proper development and enables the study of gene functions in properly developed animals. Here, we show that inducible Cre-loxP driven by the Gli1-promoter can induce cell-type-specific deletion of target genes in cerebellar cortical neurons. We used reporter mice containing the YFP (yellow fluorescence protein) gene at the Gt(ROSA)26Sor locus with a loxP-flanked transcriptional stop sequence, in which successful Cre-mediated excision of the stop sequence is indicated by YFP expression in Cre-expressing cells. Administration of tamoxifen during early postnatal days (P4~7) induces Cre-dependent excision of stop sequences and allows YFP expression in proliferating neuronal progenitor cells in the external granule layer and Bergmann glia in the Purkinje cell layer. A substantial number of YFP-positive progenitor cells in the external granule layer migrated to the internal granule cell layer and became granule cell neurons. By comparison, injection of tamoxifen during late postnatal days (P19~22) induces YFP expression only in Bergmann glia, and most granule cell neurons were devoid of YFP expression. The results indicate that the Gli1 promoter is temporarily active in progenitor cells in the external granule layer during the early postnatal period but constitutively active in Bergmann glia. We propose that the Gli1-mediated CreER system can be applied for the conditional deletion of genes of interest from cerebellar granule cell neurons and/or Bergmann glia.

Blockade of TRPV1 Inhibits Methamphetamine-induced Rewarding Effects.

Methamphetamine (MAP) is the most widely used psychostimulant in the world, but the exact mechanisms underlying MAP addiction are not yet fully understood. Recent studies have identified the distribution of TRPV1 in several brain regions that are related to drug addiction, including nucleus accumbens (NAc) and dorsal striatum (DSt). In the present study, we performed conditioned place preference (CPP) and self-administration tests to examine the effects of capsazepine (CPZ) and SB366791 (SB) on MAP reward. We found that both CPZ and SB significantly inhibited MAP-induced CPP and self-administration; in contrast, TRPV1 knock-out (KO) mice did not develop MAP-induced CPP. Real-time RT-PCR, Western blot and quantitative autoradiographic tests showed up-regulation of TRPV1 mRNA and protein expression in the NAc and/or DSt regions of mice exhibiting MAP-induced CPP. In addition, an in vivo microdialysis experiment showed that CPZ dramatically reduced dopamine (DA) levels in the NAc region of MAP-treated mice. Furthermore, attenuated dopamine transporter (DAT) binding levels in the NAc and DSt regions of MAP-induced CPP mice were reversed by CPZ. Together, these data suggest that TRPV1 plays an important role in MAP reward via the modulation of DA release and DAT density, thereby providing a novel therapeutic target for MAP addiction.

The role of striatal Gαq/11 protein in methamphetamine-induced behavioral sensitization in mice.

Gαq/11 protein transduces signals from neurotransmitter receptors and has been implicated in several functions of the central nervous system. In this study, the role of Gαq/11 protein in methamphetamine (METH)-induced behavioral sensitization was investigated using neurochemical and behavioral approaches. Repeated treatment with METH (2mg/kg, intraperitoneally) significantly increased behavioral sensitization as well as Gαq/11 protein expression and Gα protein activity in the striata of mice, while a single treatment of METH at the same dose did not affect these parameters. Repeated intrastriatal injections of a Gαq/11 inhibitor, [D-Trp7,9,10]-substance P, significantly reduced behavioral sensitization and striatal dopamine (DA) level in response to METH, with no effect on striatal tyrosine hydroxylase expression. These results suggest that Gαq/11 protein facilitates METH-induced behavioral sensitization by modulating DA release in the mouse striatum.

Potential for Dependence on Lisdexamfetamine - In vivo and In vitro Aspects.

Although lisdexamfetamine is used as a recreational drug, little research exists regarding its potential for dependence or its precise mechanisms of action. This study aims to evaluate the psychoactivity and dependence profile of lisdexamfetamine using conditioned place preference and self-administration paradigms in rodents. Additionally, biochemical techniques are used to assess alterations in the dopamine levels in striatal synaptosomes following administration of lisdexamfetamine. Lisdexamfetamine increased both conditioned place preference and self-administration. Moreover, after administration of the lisdexamfetamine, dopamine levels in the striatal synaptosomes were significantly increased. Although some modifications should be made to the analytical methods, performing high performance liquid chromatography studies on synaptosomes can aid in predicting dependence liability when studying new psychoactive substances in the future. Collectively, lisdexamfetamine has potential for dependence possible via dopaminergic pathway.

5-(2-Aminopropyl)benzofuran and phenazepam demonstrate the possibility of dependence by increasing dopamine levels in the brain.

Although 5-(2-aminopropyl)benzofuran (5-APB) and 7-bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (phenazepam) are being used as recreational drugs, research on their dependence liability or mechanisms of action is lacking. The present study aimed to evaluate the behavioral effects and dependence liability of these drugs using conditioned place preference and self-administration paradigms in rodents. Additionally, biochemical techniques were used to assess the substance-induced alterations in synaptosome-released dopamine. While both of the tested substances elicited increases in conditioned place preference and dopamine, neither of them facilitated self-administration, suggesting that 5-APB and phenazepam have rewarding effects, rather than reinforcing effects.

Dependence potential of tramadol: behavioral pharmacology in rodents.

Tramadol is an opioid analgesic agent that has been the subject of a series of case reports suggesting potential for misuse or abuse. However, it is not a controlled substance and is not generally considered addictive in Korea. In this study, we examined the dependence potential and abuse liability of tramadol as well as its effect on the dopaminergic and serotonergic systems in rodents. In animal behavioral tests, tramadol did not show any positive effects on the experimental animals in climbing, jumping, and head twitch tests. However, in the conditioned place preference and self-administration tests, the experimental animals showed significant positive responses. Taken together, tramadol affected the neurological systems related to abuse liability and has the potential to lead psychological dependence.

Dependence Potential of the Synthetic Cannabinoids JWH-073, JWH-081, and JWH-210: In Vivo and In Vitro Approaches.

Synthetic cannabinoids (CBs) such as the JWH series have caused social problems concerning their abuse liability. Because the JWH series produces euphoric and hallucinogenic effects, they have been distributed illegally under street names such as "Spice" and "Smoke". Many countries including Korea have started to schedule some of the JWH series compounds as controlled substances, but there are a number of JWH series chemicals that remain uncontrolled by law. In this study, three synthetic CBs with different binding affinities to the CB1 receptor (JWH-073, 081, and 210) and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) were evaluated for their potential for psychological dependence. The conditioned place preference test (unbiased method) and self-administration test (fixed ratio of 1) using rodents were conducted. Ki values of the three synthetic cannabinoids were calculated as supplementary data using a receptor binding assay and overexpressed CB1 protein membranes to compare dependence potential with CB1 receptor binding affinity. All mice administered JWH-073, 081, or 210 showed significantly increased time spent at unpreferred space in a dose-dependence manner in the conditioned place preference test. In contrast, all tested substances except Δ(9)-THC showed aversion phenomenon at high doses in the conditioned place preference test. The order of affinity to the CB1 receptor in the receptor binding assay was JWH-210 > JWH-081 >> JWH-073, which was in agreement with the results from the conditioned place preference test. However, no change in self-administration was observed. These findings suggest the possibility to predict dependence potential of synthetic CBs through a receptor binding assay at the screening level.

A large polypoid vascular ectasia removed by using a polypectomy with a detachable snare in an asymptomatic patient.

Vascular ectasia is a well-known cause of lower gastrointestinal bleeding in the elderly. Endoscopically, it usually appears as a flat or elevated bright red lesion. We report on an extremely rare case of a large, pedunculated, polypoid vascular ectasia in an asymptomatic patient. A large pedunculated polypoid mass in the sigmoid colon was observed on colonoscopy during a regular health check-up, and a polypectomy was performed using a detachable snare. In histology, vessels with massive dilation were found mainly in the submucosa, which was consistent with vascular ectasia.

7-nitroindazole, nitric oxide synthase inhibitor, attenuates physical dependence on butorphanol in rat.

Butorphanol is a synthetic opioid agonist/antagonist analgesic agent that mainly exerts its effects through kappa-opioid receptors. It has been demonstrated that kappa-opioid receptors preferentially mediate the development of physical dependence upon butorphanol and the associated withdrawal syndrome. However, it is not fully understood whether or not nNOS-containing neurons in the various brain regions play an important role in butorphanol withdrawal. Therefore, this study was conducted to determine whether the selective nNOS inhibitor, 7-NI, modifies the development of butorphanol withdrawal and changes of nNOS expressions in different brain regions in physically butorphanol-dependent rats. The first part of the study focused on withdrawal behaviors in male Sprague-Dawley rats. Physical dependence was induced by a 72-h i.c.v. infusion with butorphanol (26 nmol/mixrol/h) and withdrawal was subsequently precipitated by i.c.v. challenge with naloxone (48 nmol/5 microl/rat) 2 h after termination of the butorphanol infusion. The butorphanol/7-NI coadministration group showed a significant decrease in several signs of withdrawal such as teeth chattering, as compared with the butorphanol-treated group. In the second part of the study, immunohistochemical analysis was performed to determine the expression of nNOS in the various brain regions. In the butorphanol/7-NI coadministration group, the number of cells labeled for nNOS was significantly lower in the various brain regions (including the caudate putamen, nucleus accumbens, and hippocampus) than in the butorphanol group. Therefore, 7-NI decreased in butorphanol-induced physical dependence and nNOS expression. Taken together, these findings suggest that the nNOS system is involved in the development of butorphanol-induced physical dependence, and 7-NI has potential clinical application as a candidate for the treatment of opioid withdrawal syndrome.

Involvement of 5-HT receptors in the development and expression of methamphetamine-induced behavioral sensitization: 5-HT receptor channel and binding study.

Methamphetamine (MAP) is one of the most commonly abused drugs in Asia, and previous studies suggest that serotonin 3 receptors (5-HT(3)) are involved in MAP-induced locomotion and reward. However, little is known about the role of 5-HT(3) receptors in MAP-induced behavioral sensitization. Here, we measured the effects of MDL 72222, a 5-HT(3) antagonist, and SR 57227 A, a 5-HT(3) agonist, on the development and expression of MAP-induced behavioral sensitization, and alternations of 5-HT(3) receptor binding labeled with the 5-HT(3)-selective antagonist, [(3)H]GR65630, in mice. In addition, we investigated the effects of MAP on 5-HT(3A) receptor channel activity in Xenopus laevis oocytes expressing 5-HT(3A) receptors. We found that MDL 72222 attenuated both the development and expression of behavioral sensitization to MAP (1.0 mg/kg, i.p.), and that this attenuating effect of MDL 72222 was reversed by pre-treatment with SR 57227 A. In oocytes expressing 5-HT(3A) receptor, MAP exhibited a dual modulation of 5-HT(3A) receptor channel activity, i.e. pre-treatment with a low dose of MAP (0.1 microm) enhanced 5-HT-induced inward peak current (I(5-HT)) but a high dose of MAP (100 microm) inhibited I(5-HT). The acute administration of MDL 72222 with MAP decreased [(3)H]GR65630 binding versus MAP alone in the mouse striatum. Our results suggest that MDL 72222 attenuates MAP-induced behavioral sensitization via 5-HT(3) receptors in the caudate putamen, and that 5-HT(3) receptor antagonists like MDL 72222 have potential as novel anti-psychotic agents for the treatment of MAP dependence and psychosis.