Paradoxical effects of the cannabinoid CB2 receptor agonist GW405833 on rat osteoarthritic knee joint pain.

OBJECTIVE: The present study examined whether local administration of the cannabinoid-2 (CB(2)) receptor agonist GW405833 could modulate joint nociception in control rat knee joints and in an animal model of osteoarthritis (OA). METHOD: OA was induced in male Wistar rats by intra-articular injection of sodium monoiodo-acetate with a recovery period of 14 days. Immunohistochemistry was used to evaluate the expression of CB(2) and transient receptor potential vanilloid channel-1 (TRPV1) receptors in the dorsal root ganglion (DRG) and synovial membrane of sham- and sodium mono-iodoacetate (MIA)-treated animals. Electrophysiological recordings were made from knee joint primary afferents in response to rotation of the joint both before and following close intra-arterial injection of different doses of GW405833. The effect of intra-articular GW405833 on joint pain perception was determined by hindlimb incapacitance. An in vitro neuronal release assay was used to see if GW405833 caused release of an inflammatory neuropeptide (calcitonin gene-related peptide - CGRP). RESULTS: CB(2) and TRPV1 receptors were co-localized in DRG neurons and synoviocytes in both sham- and MIA-treated animals. Local application of the GW405833 significantly reduced joint afferent firing rate by up to 31% in control knees. In OA knee joints, however, GW405833 had a pronounced sensitising effect on joint mechanoreceptors. Co-administration of GW405833 with the CB(2) receptor antagonist AM630 or pre-administration of the TRPV1 ion channel antagonist SB366791 attenuated the sensitising effect of GW405833. In the pain studies, intra-articular injection of GW405833 into OA knees augmented hindlimb incapacitance, but had no effect on pain behaviour in saline-injected control joints. GW405833 evoked increased CGRP release via a TRPV1 channel-dependent mechanism. CONCLUSION: These data indicate that GW405833 reduces the mechanosensitivity of afferent nerve fibres in control joints but causes nociceptive responses in OA joints. The observed pro-nociceptive effect of GW405833 appears to involve TRPV1 receptors.

The clinical effectiveness and cost of repetitive transcranial magnetic stimulation versus electroconvulsive therapy in severe depression: a multicentre pragmatic randomised controlled trial and economic analysis.

OBJECTIVE: To investigate if repetitive transcranial magnetic stimulation (rTMS) was as effective as electroconvulsive therapy (ECT) in treating major depressive episodes and to perform a cost-effectiveness analysis. DESIGN: A single-blind pragmatic multicentre randomised controlled trial (RCT) with 6 months of follow-up to test equivalence of rTMS with ECT. SETTING: The South London and Maudsley NHS Trust and Pembury Hospital in the Invicta Mental Health Trust in Kent. PARTICIPANTS: Right-handed adult patients referred for ECT for treatment of a major depressive episode (DSM-IV) were assessed. During the 2.5-year trial period, 260 patients were referred for ECT, of whom 46 entered the trial. The main reason for not entering the trial was not consenting to ECT while being formally treated under the UK Mental Health Act 1983. INTERVENTIONS: Patients were randomised to receive a 15-day course of rTMS of the left dorsolateral prefrontal cortex (n = 24) or a course of ECT (n = 22). MAIN OUTCOME MEASURES: Patients were assessed before randomisation, at end of treatment and at the 6-month follow-up. Primary outcome measures were the 17-item Hamilton Rating Scale for Depression (HRSD) and proportion of remitters (defined as HRSD score

Presynaptic nicotinic receptors modulating dopamine release in the rat striatum.

The modulation of striatal dopamine release by presynaptic nicotinic acetylcholine receptors is well documented for both synaptosomes and slices. Because the latter retain local anatomical integrity, we have compared [3H]dopamine release evoked by the nicotinic receptor agonists (-)-nicotine and (+/-)-anatoxin-a from striatal synaptosome and slice preparations in parallel. At higher agonist concentrations, mecamylamine-sensitive [3H]dopamine release was greater from slices, indicative of an additional component, and this increase was abolished by glutamate receptor antagonists. To begin to examine the localisation of specific nicotinic acetylcholine receptor subtypes in the striatum, immunogold electron microscopy was undertaken with the beta2-specific monoclonal antibody 270. In striatal sections, gold particles were associated with symmetric synapses (dopaminergic) but were absent from asymmetric synapses (glutamatergic). Surface labelling of striatal synaptosomes with gold particles was also demonstrated. Taken together, these results are consistent with dopamine release mediated by beta2-containing nicotinic acetylcholine receptors on dopamine terminals, while non-beta2-containing nicotinic acetylcholine receptors may enhance dopamine release indirectly by releasing glutamate from neighbouring terminals.

UV-stimulated recombination in mammalian cells is not dependent upon DNA replication.

The ability of UV light to stimulate recombination between viruses in mammalian cells is well established. Here we provide evidence that UV can also stimulate recombination between irradiated plasmids transfected into mammalian COS-7 cells. As the plasmids used, pSVOcat and pRSVcat(amb38), are incapable of replication in mammalian cells it is likely that the stimulatory effect of UV on recombination is due to a direct effect on the DNA rather than a consequence of the accumulation of nicked and gapped DNA-replication intermediates.

Altered pharmacology of native rodent spinal cord TRPV1 after phosphorylation.

BACKGROUND AND PURPOSE: Evidence suggests that phosphorylation of TRPV1 is an important component underlying its aberrant activation in pathological pain states. To date, the detailed pharmacology of diverse TRPV1 receptor agonists and antagonists has yet to be reported for native TRPV1 under phosphorylating conditions. Our goal was to optimize a relatively high-throughput methodology to allow pharmacological characterization of the native TRPV1 receptor using a spinal cord neuropeptide release assay under naive and phosphorylating states. EXPERIMENTAL APPROACH: Herein, we describe characterization of rodent TRPV1 by measurement of CGRP release from acutely isolated lumbar (L1-L6) spinal cord using a 96-well technique that combines use of native, adult tissue with quantitation of CGRP release by ELISA. KEY RESULTS: We have studied a diverse panel of TRPV1 agonists and antagonists under basal and phosphorylating conditions. We show that TRPV1-mediated CGRP release is evoked, in a temperature-dependent manner, by a PKC activator, phorbol 12,13-dibutyrate (PDBu); and that treatment with PDBu increases the potency and efficacy of known TRPV1 chemical agonists, in an agonist-specific manner. We also show that the pharmacological profile of diverse TRPV1 antagonists is dependent on whether the stimulus is PDBu or capsaicin. Of note, HPPB was identified as an antagonist of capsaicin-evoked, but a potentiator of PDBu-evoked, CGRP release. CONCLUSIONS AND IMPLICATIONS: Our findings indicate that both TRPV1 agonist and antagonist profiles can be differentially altered by PKC activation. These findings may offer new insights for targeting TRPV1 in pain states.

A randomized controlled trial with 4-month follow-up of adjunctive repetitive transcranial magnetic stimulation of the left prefrontal cortex for depression.

BACKGROUND: Effectiveness of repetitive transcranial magnetic stimulation (rTMS) for major depression is unclear. The authors performed a randomized controlled trial comparing real and sham adjunctive rTMS with 4-month follow-up. METHOD: Fifty-nine patients with major depression were randomly assigned to a 10-day course of either real (n=29) or sham (n=30) rTMS of the left dorsolateral prefrontal cortex (DLPFC). Primary outcome measures were the 17-item Hamilton Depression Rating Scale (HAMD) and proportions of patients meeting criteria for response (50% reduction in HAMD) and remission (HAMD8) after treatment. Secondary outcomes included mood self-ratings on Beck Depression Inventory-II and visual analogue mood scales, Brief Psychiatric Rating Scale (BPRS) score, and both self-reported and observer-rated cognitive changes. Patients had 6-week and 4-month follow-ups. RESULTS: Overall, Hamilton Depression Rating Scale (HAMD) scores were modestly reduced in both groups but with no significant groupxtime interaction (p=0.09) or group main effect (p=0.85); the mean difference in HAMD change scores was -0.3 (95% CI -3.4 to 2.8). At end-of-treatment time-point, 32% of the real group were responders compared with 10% of the sham group (p=0.06); 25% of the real group met the remission criterion compared with 10% of the sham group (p=0.2); the mean difference in HAMD change scores was 2.9 (95% CI -0.7 to 6.5). There were no significant differences between the two groups on any secondary outcome measures. Blinding was difficult to maintain for both patients and raters. CONCLUSIONS: Adjunctive rTMS of the left DLPFC could not be shown to be more effective than sham rTMS for treating depression.

Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside antibiotics G-418 and paromomycin.

Aminoglycoside antibiotics in Escherichia coli and yeast can cause ribosomes to read through stop codons during translation. This can result in the phenotypic suppression of nonsense mutations. We show here for the first time that the aminoglycosides G-418 and paromomycin have similar effects in monkey (COS-7) cells in vivo. Suppression of an amber mutation (TAG) by aminoglycosides can restore the activity of a mutant gene transfected into COS-7 cells to almost 20% of wild type levels.

Construction of a vector, pRSVcatamb38, for the rapid and sensitive assay of amber suppression in human and other mammalian cells.

We describe the generation of an amber mutation in the chloramphenicol acetyltransferase (cat) gene of the mammalian cell transfection vector pRSVcat (Gorman et.al. (1982), Proc.Natl.Acad.Sci. 79 6777-6791). We have demonstrated the in vivo suppression of this amber mutation in monkey and human cells by co-transfection with a synthetic Xenopus suppressor tRNATyr under the control of the late SV40 promoter. The vector, pRSVcatamb38, may be used to quantitate amber suppression in various mammalian cells.

Histochemical staining of clonal mammalian cell lines expressing E. coli beta galactosidase indicates heterogeneous expression of the bacterial gene.

An evaluation has been made of the E. coli beta-galactosidase (beta-gal) gene for use as a reporter gene in mammalian cells in culture. We have adopted a histochemical procedure which enables identification of those cells within a population that express the introduced bacterial gene. Data is presented concerning the sensitivity of the histochemical method relative to an immunological method of detection. It has been found that several clonal cell lines generated after transfection of human 293 cells with a Rous sarcoma virus (RSV) long terminal repeat (LTR) promoter-beta-gal construction are mosaic for expression of the introduced mini-gene. Furthermore, after treatment of these clonal cell lines with the nucleoside analog 5-aza-cytidine (5-aza-C), an increase in production of beta-gal under control of this promoter element was observed.

Aminoglycoside suppression at UAG, UAA and UGA codons in Escherichia coli and human tissue culture cells.

We have compared the suppression of nonsense mutations by aminoglycoside antibiotics in Escherichia coli and in human 293 cells. Six nonsense alleles of the chloramphenicol acetyl transferase (cat) gene, in the vector pRSVcat, were suppressed by growth in G418 and paromomycin. Readthrough at UAG, UAA and UGA codons was monitored with enzyme assays for chloramphenicol acetyl transferase (CAT), in stably transformed bacteria and during transient expression from the same plasmid in human 293 tissue culture cells. We have found significant differences in the degree of suppression amongst three UAG codons and two UAA codons in different mRNA contexts. However, the pattern of these effects are not the same in the two organisms. Our data suggest that context effects of nonsense suppression may operate under different rules in E. coli and human cells.

Protective efficacy of a recombinant protective antigen against Bacillus anthracis challenge and assessment of immunological markers.

The efficacy of recombinant Bacillus anthracis Protective Antigen (rPA) produced in Bacillus subtilis and formulated in Alhydrogel or MPL-TDM-CWS (Ribi adjuvant) has been tested and compared to the licensed UK human vaccine in guinea pigs challenged by the aerosol route with the Ames strain of B. anthracis. rPA combined with the Ribi adjuvant was found to be the only formulation to provide 100% protection from challenge. Analysis of immunological parameters in the individual animals revealed significant differences between the rPA/Ribi vaccine group and rPA/Alhydrogel and human vaccine groups for antigen specific lymphocyte proliferation, PA neutralisation and antigen specific IgG2 levels, but indicated no significant differences in PA-specific IgG1 levels. rPA formulated in Alhydrogel induced a mainly IgG1 response whilst the rPA/Ribi vaccine produced a predominantly IgG2 response.