A Mechanistic Approach to the Development of Gene Therapy for Chronic Pain.

Treatment of chronic pain has created a "silent epidemic," a term that describes the serious public health problem of the abuse of opioid painkillers and other prescription drugs. Conventional pharmacotherapy is limited by the loss of effectiveness in the long-term and by potentially lethal side effects. Efforts need to be focused on the development of nonpharmacological approaches. As significant progress is made in the viral vector technology, gene therapy involving recombinant viruses as vehicles may become a viable alternative for treatment of severe pain. Virus-based gene therapy has several advantages: (1) the transfer of a therapeutic gene to produce/release bioactive therapeutic molecules in a specific location in the nervous system thus minimizing the risks of off-target side effects, and (2) sustained long-term production of the therapeutic agent. This review compiles recently developed strategies for gene therapy targeting specific mechanisms of specific chronic pain conditions. A few successful studies on animal models of chronic pain have been translated to human clinical trials.

Reappraisal of bovril as a source of arginine in the arginine stimulation test for growth hormone deficiency.

The purpose of this case study is to report the use of oral Bovril (a food supplement which contains arginine) as an alternative test for growth hormone stimulation test. We performed oral Bovril test in 3 patients -- one with suspected growth hormone deficiency in whom insulin tolerance test could not be performed (subject A), one sex-matched control (subject B), and one with confirmed growth hormone deficiency (subject C). 14g/m(2) of oral Bovril was mixed with 150ml of warm water and was given to all three subjects. Blood for growth hormone was taken at baseline, and every 30 minutes till 150 minutes after ingestion of oral Bovril. The ingestion of oral Bovril showed a positive response in subjects A and B, with highest growth hormone levels of 28.4mIU/L and 42.0mIU/L respectively at 150 minutes. Subject C had suppressed growth hormone throughout the test. Oral Bovril is readily available and is a safe alternative for standard growth hormone stimulation test.

ß-arrestin protects neurons by mediating endogenous opioid arrest of inflammatory microglia.

Microglial activation worsens neuronal loss and contributes to progressive neurological diseases like Parkinson's disease (PD). This inflammatory progression is countered by dynorphin (Dyn), the endogenous ligand of the kappa-opioid receptor (KOR). We show that microglial ß-arrestin mediates the ability of Dyn/KOR to limit endotoxin-elicited production of pro-inflammatory effectors and cytokines, subsequently protecting neurons from inflammation-induced neurotoxicity. Agonist-activated KOR enhances the interaction of ß-arrestin2 with transforming growth factor-beta-activated kinase 1 (TAK1)-binding protein 1 (TAB1), disrupting TAK1-TAB1 mediated pro-inflammatory gene expression. We reveal a new physiological role for ß-arrestin in neuroprotection via receptor internalization-triggered blockade of signal effectors of microglial inflammatory neurotoxicity. This result offers novel drug targets in the convergent KOR/ß-arrestin2 and inflammatory pathways for treating microglial inflammatory neuropathologies like PD.

Acute renal failure and posterior reversible encephalopathy syndrome following multiple wasp stings: a case report.

Wasp stings can present in various ways, ranging from mild self-limiting illness to severe multi organ failure with a potentially fatal outcome. We report a case of multiple wasp stings leading to acute renal failure needing prolonged dialysis support and posterior reversible encephalopathy syndrome.

Intrathecal delivery of a mutant micro-opioid receptor activated by naloxone as a possible antinociceptive paradigm.

Direct injection of double-stranded adeno-associated virus type 2 (dsAAV2) with a mu-opioid receptor (MOR) mutant [S4.45(196)A], and a reporter protein (enhanced green fluorescent protein) into the spinal cord (S2/S3) dorsal horn region of ICR mice resulted in antinociceptive responses to systemic injection of opioid antagonist naloxone without altering the acute agonist morphine responses and no measurable tolerance or dependence development during subchronic naloxone treatment. To develop further such mutant MORs into a therapeutic agent in pain management, a less invasive method for virus delivery is needed. Thus, in current studies, the dsAAV2 was locally injected into the subarachnoid space of the spinal cord by intrathecal administration. Instead of using the MORS196A mutant, we constructed the dsAAV2 vector with the MORS196ACSTA mutant, a receptor mutant in which naloxone has been shown to exhibit full agonistic properties in vitro. After 2 weeks of virus injection, naloxone (10 mg/kg s.c.) elicited antinociceptive effect (determined by tail-flick test) without tolerance (10 mg/kg s.c., b.i.d. for 6 days) and significant withdrawal symptoms. On the other hand, subchronic treatment with morphine (10 mg/kg s.c., b.i.d.) for 6 days induced significant tolerance (4.8-fold) and withdrawal symptoms. Furthermore, we found that morphine, but not naloxone, induced the rewarding effects (determined by conditioned place preference test). These data suggest that local expression of MORS196ACSTA in spinal cord and systemic administration of naloxone has the potential to be developed into a new strategy in the management of pain without addiction liability.

Epigenetic regulation of kappa opioid receptor gene in neuronal differentiation.

The gene of mouse kappa opioid receptor (KOR) utilizes two promoters, P1 and P2. P1 is active in various brain areas and constitutively in P19 mouse embryonal carcinoma cells. P2 is active in limited brain stem areas of adult animals and only in late differentiated cells of P19 induced for neuronal differentiation in the presence of nerve growth factor (NGF). NGF response of P2 was found to be mediated by a specific binding site for transcription factor activation protein 2 (AP2) located in P2. Electrophoretic gel shift assay showed specific binding of this AP2 site by AP2beta, but not AP2alpha. Knockdown of endogenous AP2beta with siRNA abolished the stimulating effect of NGF on the expression of transcripts driven by P2. Binding of endogenous AP2beta on the endogenous KOR P2 chromatin region was also confirmed by chromatin immunoprecipitation. The effect of NGF was inhibited by LY2942002 (phosphatidylinositol 3-kinase, PI3K inhibitor), suggesting that PI3K was involved in signaling pathway mediating the effect of NGF stimulation on KOR P2. The chromatin of P2 in P19 was found to be specifically modified following NGF stimulation, which included demethylation at Lys9 and dimethylation at Lys4 of histone H3 and was consistent with the increased recruitment of RNA polymerase II to this promoter. This study presents the first evidence for epigenetic changes occurred on a specific KOR promoter triggered by NGF in cells undergoing neuronal differentiation. This epigenetic change is mediated by recruited AP2beta to this promoter and involves the PI3K system.

dsAAV type 2-mediated gene transfer of MORS196A-EGFP into spinal cord as a pain management paradigm.

We previously reported that mutations in the mu-opioid receptor (MOR), S196L or S196A, rendered MOR responsive to the opioid antagonist naloxone without altering the agonist phenotype. Subsequently, a mouse strain carrying the S196A mutation exhibited in vivo naloxone antinociceptive activity without the development of tolerance. In this study we investigated the possibility of combining the in vivo site-directed delivery of MORS196A and systemic naloxone administration as a paradigm for pain management. Double-stranded adenoassociated virus type 2 (dsAAV2) was used to deliver MORS196A-EGFP by injecting the virus into the spinal cord (S2/S3) dorsal horn region of ICR mice. MORS196A-EGFP fluorescence colocalized with some calcitonin gene-related peptide and neuron-specific protein immunoreactivity in the superficial layers of the dorsal horn 1 week after injection and lasted for at least 6 months. In mice injected with the mutant receptor, morphine induced similar antinociceptive responses and tolerance development or precipitated withdrawal symptoms and reward effects, similar to those in the control mice (saline injected into the spinal cord). Conversely, in the dsAAV2-injected mice, naloxone produced antinociceptive effects at the spinal level but not at the supraspinal level, whereas naloxone had no measurable effect on the control mice. Furthermore, the chronic administration of naloxone to mice injected with dsAAV2-MORS196A-EGFP did not induce tolerance, dependence, or reward responses. Thus, our current approach to activate a mutant receptor, but not the endogenous receptor, with an opioid antagonist represents an alternative to the use of traditional opioid agonists for pain management.

Insights into the receptor transcription and signaling: implications in opioid tolerance and dependence.

Drug addiction has great social and economical implications. In order to resolve this problem, the molecular and cellular basis for drug addiction must be elucidated. For the past three decades, our research has focused on elucidating the molecular mechanisms behind morphine tolerance and dependence. Although there are many working hypotheses, it is our premise that cellular modulation of the receptor signaling, either via transcriptional or post-translational control of the receptor, is the basis for morphine tolerance and dependence. Thus, in the current review, we will summarize our recent work on the transcriptional and post-translational control of the opioid receptor, with special emphasis on the mu-opioid receptor, which is demonstrated to mediate the in vivo functions of morphine.

mu-Opioid receptor gene expression: the role of NCAM.

We have investigated the mechanisms regulating the expression of the mu-opioid receptor, using P19 mouse embryonal carcinoma cells, which normally lack this receptor, but which can be induced to express it in aggregated cells by retinoic acid treatment. The expression level of mu-opioid receptor mRNA was found to be closely correlated with aggregation status, and more specifically by cell to cell interaction requiring neural cell adhesion molecules (NCAM). We showed that NCAM activates the mu-opioid receptor gene through a pathway involving phospholipase C-arachidonic acid-calcium channel-calcium/calmodulin kinase II. A similar pathway was previously shown to promote neurite outgrowth, however, with distinct specificity, including the role of calcium channels. Activation of L-type calcium channels elevated mu-opioid receptor expression, while N-type-channel activity had the opposite effect. The effect of anti-NCAM-antibody treatment was not due to retardation of general neural development and was specific to the mu-opioid receptor gene. Our results indicate that the P19 system is an useful model to study the expression of the mu-opioid receptor gene.

Morphine modulates lymph node-derived T lymphocyte function: role of caspase-3, -8, and nitric oxide.

The major objective of this paper is to characterize the mechanism by which morphine modulates lymphocyte function and if these effects are mediated through the mu-opioid receptor. We evaluated the in vitro effects of morphine on lymphocytes that were freshly isolated from lymph nodes from wild type (WT) and mu-opioid receptor knock-out (MORKO) mice. Results show that morphine inhibits Con A-induced lymph node T-cell proliferation and IL-2 and IFN-gamma synthesis in a dose-dependent manner. This effect was abolished in lymph node cells isolated from MORKO mice. The inhibition of T-cell function with low-dose morphine was associated with an increase in caspase-3- and caspase-8-mediated apoptosis. The inhibition of T-cell function with high-dose morphine was associated with an increase in the inducible NO synthase mRNA expression. N(G)-nitro-L-arginine methyl ester (L-NAME) antagonized the apoptosis induced by high-dose morphine. Our results suggest that low-dose morphine, through the mu-opioid receptor, can induce lymph node lymphocyte apoptosis through the cleavage activity of caspase-3 and caspase-8. Morphine at high doses induces NO release. This effect of morphine is also mediated through the mu-opioid receptor present on the surface of macrophages.

Lack of mu-opioid receptor leads to an increase in the NMDA receptor subunit mRNA expression and NMDA-induced convulsion.

The present study investigated in situ hybridization of N-methyl-D-aspartate (NMDA) receptor (NR) subunit mRNA and convulsion induced by intracerebroventricular injection of NMDA, in order to examine changes in NMDA receptor function in mu-opioid receptor gene knockout mice. Levels of NR1 and NR2A subunit mRNA were significantly increased in the parietal cortex (8.4 and 10.6%, respectively) and hypothalamus (8.7 and 15.2%, respectively) in mu-opioid receptor knockout mice. Levels of NR2B subunit mRNA were noted to be increased in the parietal cortex (9.1%), thalamus (7.7%), and hypothalamus (10.4%) in mu-opioid receptor knockout mice. The ED(50) for NMDA-induced convulsion in wild-type mice was 0.20 microg/10 microl/mouse. The ED(50) in mu-opioid receptor knockout mice was 0.14 microg/10 microl/mouse. There is a significant difference in the potency ratio of wild-type mice versus knockout mice (potency ratio: 1.44, P < 0.05). These results indicate that mu-opioid receptor knockout mice are more sensitive to NMDA-induced convulsion. Therefore, these results suggest that absence of mu-opioid receptor gene is accompanied by changes in the NMDA receptor system which can modulate the synaptic excitability in the process such as convulsion or epilepsy.

Differential effects of morphine, DPDPE, and U-50488 on apomorphine-induced climbing behavior in mu-opioid receptor knockout mice.

The present study examined the hypothesis whether the opioid receptors (mu, delta, and kappa) contribute to a behavioral dopaminergic activation produced by dopamine receptor agonist, apomorphine, by comparing responses in wild type and mu-opioid receptor knockout mice. The data suggest that expression of mu-opioid receptors plays an important role in the enhancement of climbing behavior induced by apomorphine. Compared to wild type mice, a response in the dopaminergic behavior by treatment with delta-receptor agonist, DPDPE, is more sensitive to the mice lacking mu-opioid receptor. Treatment with kappa-receptor agonist, U-50488, is potentiated the apomorphine-induced climbing behavior in wild type and mu-opioid receptor knockout mice. These responses may be independent of that through mu-opioid receptors. Therefore, the our results show that dopaminergic activation measured by climbing behavior in mu-opioid receptors knockout mice are differently regulated by mu-, delta-, and kappa-opioid receptor agonists.

Transcriptional regulation of mouse delta-opioid receptor gene: role of Ets-1 in the transcriptional activation of mouse delta-opioid receptor gene.

Previously, we identified a minimum core promoter of the mouse delta-opioid receptor (DOR) gene. The DOR promoter contains an E-box that binds upstream stimulatory factor and is crucial for the DOR promoter activity in NS20Y cells, a mouse neuronal cell line that constitutively expresses DOR. In the present study, we further analyzed the DOR promoter in NS20Y cells and have demonstrated that transcription factor Ets-1 binds to an Ets-1-binding site overlapping the E-box and trans-activates the DOR promoter by synergizing with upstream stimulatory factor in specific DNA binding. In addition, the Ets-1 DNA-binding domain is sufficient to play the functional role of Ets-1 in trans-activating the DOR promoter. Furthermore, through in vivo cross-linking assays and Northern blot analyses, we have demonstrated that Ets-1 binds to the DOR promoter in the neonatal mouse brain and that overexpressed Ets-1 can significantly enhance the expression of DOR mRNA in primary neonatal mouse neuronal cells. Collectively, our data suggest that Ets-1 functions as a trans-activator of the DOR promoter in the neonatal mouse brain and thus may contribute to the development of the mouse brain DOR system.

Opioid and cannabinoid modulation of precipitated withdrawal in delta(9)-tetrahydrocannabinol and morphine-dependent mice.

The goal of the present study was to elucidate the relationship between cannabinoid and opioid systems in drug dependence. The CB(1) cannabinoid receptor antagonist SR 141716A precipitated both paw tremors and head shakes in four different mouse strains that were treated repeatedly with Delta(9)-tetrahydrocannabinol (Delta(9)-THC). SR 141716A-precipitated Delta(9)-THC withdrawal was ameliorated in mu-opioid receptor knockout mice compared with the wild-type control animals and failed to occur in mice devoid of CB(1) cannabinoid receptors. An acute injection of morphine in Delta(9)-THC-dependent mice undergoing SR 1417161A-precipitated withdrawal dose dependently decreased both paw tremors, antagonist dose 50 (AD(50)) (95% CL) = 0.035 (0.03--0.04), and head shakes, AD(50) (95% CL) = 0.07 (0.04--0.12). In morphine-dependent mice, the opioid antagonist naloxone precipitated head shakes, paw tremors, diarrhea, and jumping. As previously reported, naloxone-precipitated morphine withdrawal failed to occur in mu-opioid knockout mice and was significantly decreased in CB(1) cannabinoid receptor knockout mice. Acute treatment of Delta(9)-THC in morphine-dependent mice undergoing naloxone-precipitated withdrawal blocked paw tremors, AD(50) (95% CL) = 0.5 (0.3--1.0), and head shakes AD(50) (95% CL) = 0.6 (0.57--0.74) in dose-dependent manners, but failed to diminish the occurrence of diarrhea or jumping. Finally, naloxone and SR 141716A failed to elicit any overt effects in Delta(9)-THC-dependent and morphine-dependent mice, respectively. These findings taken together indicate that the mu-opioid receptor plays a modulatory role in cannabinoid dependence, thus implicating a reciprocal relationship between the cannabinoid and opioid systems in dependence.

Role of hypothalamic-pituitary axis in morphine-induced alteration in thymic cell distribution using mu-opioid receptor knockout mice.

Mu-opioid receptor knockout mice (MORKO), were used to address two questions: (1) if morphine induced decrease in thymic weight and cell distribution is mediated by the mu-opioid receptor and (2) the role of corticosteroids in morphine mediated alteration in thymic cell distribution. Our result show that morphine mediated increase in plasma corticosterone is mediated by the mu-opioid receptor since morphine at doses as high as 25 mg/kg-body weight does not increase plasma corticosterone levels in the MORKO. In addition, we have also shown that morphine treatment results in the differentiation of CD4+CD8+ (double positive cells) to single positive CD4+ cells while dexamethasone treatment results in the deletion of CD4+CD8+ (double positive) cells.

delta-Opioid receptor gene: effect of Sp1 factor on transcriptional regulation in vivo.

delta-Opioid receptor (DOR) promoter exhibited a cell-type-specific expression pattern. Protein-DNA interactions in this promoter were identified by dimethyl sulfate in vivo footprinting analysis of NG108-15 cells, expressing endogenous DOR. Complete protection of the putative Sp1 cis-element and partial protection of the sequence defined as X-NotI in the basal promoter were observed only in the G0/G1 phase of the cell cycle. No protection was detected in Neuro2A cells that do not express DOR. In vivo formaldehyde cross-linking confirmed Sp1 factor binding to its cis-acting element during the G0/G1 phase. The functional significance of these Sp1 and X-NotI sites was evaluated by transient transfection analysis. Northern blot analysis and nuclear run-off assays revealed maximum DOR mRNA level and transcription rate, respectively, during the G0/G1 phase of NG108-15 cells. In summary, the protein-DNA interactions at the Sp1 and X-NotI sites are necessary for cell cycle-dependent and cell-type-specific up-regulated DOR gene expression.

Affinity labels as tools for the identification of opioid receptor recognition sites.

Affinity labels have proven to be useful tools in opioid research. We review experiments carried out with the mu opioid receptor affinity label, beta-funaltrexamine (2), that support the concept of different recognition sites for mu opioid agonists and antagonists. The data are interpreted in the context of a dimeric receptor that contains two allosterically coupled binding sites: one that binds endogenous agonist, and the second that functions as an inhibitory modulator of agonism. It is proposed that exogenous antagonists bind selectively to the second site. The first of a new class of affinity labels, PGNA (5), that contains the phthaldehyde moiety attached to an opioid antagonist pharmacophore, is described. This class of ligands has been named 'reporter affinity labels' because covalent association leads to the formation of a fluorescent isoindole that is diagnostic for cross-linking of lysine and cysteine residues. PGNA binds opioid receptors covalently, as suggested by (a) irreversible binding to cloned opioid receptors, (b) irreversible opioid antagonism in the guinea pig ileum preparation, and (c) ultra-long opioid antagonism in mice. Since flow cytometry experiments revealed specific enhancement of fluorescence in cloned mu receptors after a 1 min exposure to 5, it is concluded that covalent binding has occurred via the formation of an isoindole, presumably by cross-linking neighboring lysine and cysteine residues in the vicinity of the receptor recognition site.

Effects of morphine on pentobarbital-induced responses in mu-opioid receptor knockout mice.

Effects of morphine on the potentiation of pentobarbital-induced responses were investigated using mu-opioid receptor knockout mice. The duration of loss of righting reflex, hypothermia, and loss of motor coordination induced by pentobarbital were measured after pretreatment with either morphine or saline. Morphine pretreatment failed to show potentiation of both pentobarbital-induced loss of righting reflex and hypothermia in mu-opioid receptor knockout mice, while it significantly potentiated these responses in the wild-type controls. For motor incoordination test, morphine potentiated pentobarbital-induced motor incoordination in the wild-type mice. However, morphine may have opposite effects in the mu-opioid receptor knockout mice. These results demonstrate that synergism between morphine and pentobarbital is not detected in mu-opioid receptor knockout mice and that potentiation of pentobarbital-induced loss of righting reflex and hypothermia by morphine is mediated through mu-opioid receptor. It was interesting to note that pentobarbital-induced decrease in body temperature was less severe in mu-opioid receptor knockout mice than in wild-type mice.