Clonal variation in interferon response determines the outcome of oncolytic virotherapy in mouse CT26 colon carcinoma model.

In our earlier studies, Semliki Forest virus vector VA7 completely eliminated type I interferon (IFN-I)-unresponsive human U87-luc glioma xenografts, whereas interferon-responsive mouse gliomas proved refractory. Here, we describe in two clones of CT26 murine colon carcinoma, opposed patterns of IFN-I responsiveness and sensitivity to VA7. Both CT26WT and CT26LacZ clones secreted biologically active interferon in vitro upon virus infection but only CT26WT cells were protected. Focal infection of CT26WT cultures was self-limiting but could be rescued using IFN-I pathway inhibitor Ruxolitinib or antibody against IFNß. Whole transcriptome sequencing (RNA-Seq) and protein expression analysis revealed that CT26WT cells constitutively expressed 56 different genes associated with pattern recognition and IFN-I signaling pathways, spanning two reported anti-RNA virus gene signatures and 22 genes with reported anti-alphaviral activity. Whereas CT26WT tumors were strictly virus-resistant in vivo, infection of CT26LacZ tumors resulted in complete tumor eradication in both immunocompetent and severe combined immune deficient mice. In double-flank transplantation experiments, CT26WT tumors grew despite successful eradication of CT26LacZ tumors from the contralateral flank. Tumor growth progressed uninhibited also when CT26LacZ inoculums contained only a small fraction of CT26WT cells, demonstrating dominance of IFN responsiveness when heterogeneous tumors are targeted with interferon-sensitive oncolytic viruses.

Unusually Strong Temperature Dependence of P2X3 Receptor Traffic to the Plasma Membrane.

ATP-gated P2X3 receptors are expressed by nociceptive neurons and participate in transduction of pain. Responsiveness of P2X3 receptors is strongly reduced at low temperatures, suggesting a role for these receptors in analgesic effects of cooling. Since sustained responsiveness depends on receptor trafficking to the plasma membrane, we employed total internal reflection fluorescence (TIRF) microscopy to highlight perimembrane pool of DsRed-tagged P2X3 receptors and studied the effects of temperature on perimembrane turnover of P2X3-DsRed. Patch-clamp recordings confirmed membrane expression of functional, rapidly desensitizing P2X3-DsRed receptors. By combining TIRF microscopy with the technique of fluorescence recovery after photobleaching (FRAP), we measured the rate of perimembrane turnover of P2X3-DsRed receptors expressed in hippocampal neurons. At room temperature, the P2X3-DsRed perimembrane turnover as measured by TIRF-FRAP had a time constant of ∼2 min. At 29°C, receptor turnover was strongly accelerated (0.6 min), yielding an extremely high temperature dependence coefficient Q(10) ∼4.5. In comparison, AMPA receptor turnover measured with TIRF-FRAP was only moderately sensitive to temperature (Q(10) ∼1.5). The traffic inhibitor Brefeldin A selectively decelerated P2X3-DsRed receptor turnover at 29°C, but had no effect at 21°C (Q(10) ∼1.0). This indicates that receptor traffic to plasma membrane is the key temperature-sensitive component of P2X3 turnover. The selective inhibitor of the RhoA kinase Y27632 significantly decreased the temperature dependence of P2X3-DsRed receptor turnover (Q(10) ∼2.0). In summary, the RhoA kinase-dependent membrane trafficking of P2X3 receptors to plasma membrane has an exceptionally high sensitivity to temperature. These findings suggest an important role of P2X3 receptor turnover in hypothermia-associated analgesia.

The expression of heat shock protein 27 in retinal ganglion and glial cells in a rat glaucoma model.

The heat shock protein 27 kDa (HSP27) is a member of proteins that are highly inducible under various forms of cellular stress. This study describes constitutive HSP27 expression in rat retina and stress-associated expression of HSP27 in an experimental rat glaucoma model. Glaucoma was induced unilaterally using laser photocoagulation of the episcleral and limbal veins. Three and seven days after the elevation of intraocular pressure (IOP), groups of rats were killed. The second laser treatment was performed for those rats killed 14 and 21 days after the first laser treatment. The RGCs were labeled with a retrograde tracer 7 days before kill. The expression of HSP27 was analyzed by Western blotting in retinas of rats killed on day 14 after the first laser treatment. Retinal astrocytes, Müller cells and HSP27-positive cells were visualized using immunohistochemical methods both from retinal whole-mounts and paraffin sections. The total number of retrogradely labeled RGCs decreased by 23.2% after 7 days, 28% after 14 days, and 29.3% after 21 days of elevated IOP when compared with controls. A significant decrease of glial fibrillary acidic protein (GFAP)-immunoreactive retinal astrocytes in laser-treated eyes was observed compared with the controls (accounted for 44.9%, 38.2% and 35% of the control values in the 7-day, 14-day and 21-day groups, respectively). The expression of HSP27 in RGCs and retinal astrocytes was also increased in laser-treated eyes when compared with controls in all groups. However, glycinergic and cholinergic cells in the inner nuclear layer and the highest number of RGCs and astrocytes that expressed HSP27 were found in the 14-day group of rats. The constitutive expression of HSP27 was observed only in retinal astrocytes and Müller cells. This study suggests that constitutive HSP27 expression is a cell-type specific phenomenon in the rat retina. However, at the same time, HSP27 might be considered as a marker for neuronal injury in the rat glaucoma model.

Electrophysiologic changes in the lateral and basal amygdaloid nuclei in temporal lobe epilepsy: an in vitro study in epileptic rats.

The functional consequences of neuronal loss during epileptogenesis in the lateral and basal amygdaloid nuclei are poorly understood. The present study tested the hypothesis that electrical responsiveness varies in different amygdaloid nuclei in the chronically epileptic amygdala. Further, we examined the amygdaloid region most prone to seizure initiation. Epileptogenesis was triggered in 20 rats by inducing status epilepticus (SE) with electrical stimulation of the lateral nucleus of the amygdala. Electrode-implanted non-stimulated rats served as controls. The occurrence and duration of spontaneous seizures were monitored with video-electroencephalography (EEG) at 8-9 weeks after SE. Thereafter, animals were killed and extracellular recordings were made from slices of both amygdalas. In the lateral nucleus of epileptic animals, the frequency of spontaneous responses was reduced compared with controls (P < 0.05). The amplitudes of evoked field responses were reduced (P < 0.01), whereas paired pulse (PP) facilitation was enhanced (P < or = 0.05). In the basal nucleus of the epileptic animals, PP facilitation was enhanced (P < 0.05) and sensitivity to 4-aminopyridine (4-AP)-induced epileptiform activity was increased compared with controls (P < 0.05). In the epileptic animals, the basal nucleus was also more sensitive than the lateral nucleus to 4-AP-induced epileptiform activity (P < 0.05). Correlation analysis indicated that longer SE duration was associated with longer half widths (P = 0.001) and smaller slopes (P < 0.05) of evoked responses as well as with attenuated PP facilitation (P<0.01). Moreover, a higher frequency of spontaneous seizures was associated with longer half widths (P < 0.05) and smaller slopes (P < 0.05) of evoked responses as well as with enhanced PP facilitation (P < 0.05). These data suggest that there is a reduced release of glutamate and reduced inhibition in the lateral and basal amygdaloid nuclei in epileptic animals. Further, the basal nucleus is more prone to epileptic activity than the lateral nucleus. Finally, the severity of SE and spontaneous seizures in vivo is associated with electrophysiologic alterations in vitro.

Selegiline reduces N-methyl-D-aspartic acid induced perturbation of neurotransmission but it leaves NMDA receptor dependent long-term potentiation intact in the hippocampus.

This study examined the effects of monoamine oxidase (MAO) inhibitors on N-methyl-D-aspartic acid (NMDA)-induced perturbation of neurotransmission and normal NMDA-receptor dependent function (long-term potentiation, LTP) in the CA1 field of hippocampus. During baseline recording, neurotransmission was unaffected by long-term bath perfusion with MAO inhibitors (selegiline, pargyline). After NMDA (100 microM) infusion, the presence of selegiline (1 microM) promoted the recovery rate and increased the size of recovered extracellular field excitatory postsynaptic potentials (fEPSPs). Selegiline (1 microM) also prevented the NMDA-induced increase in paired pulse facilitation (PPF). The induction and maintenance of LTP were normal with this same concentration of selegiline. The presence of lower concentration (10 nM) of selegiline or pargyline (1 microM) did not improve the recovery process. These results suggest that selegiline partially protects the function of CA3-CA1 hippocampal connections against overactivation of NMDA receptors. Further, the same concentration of selegiline does not interfere with the physiological function of NMDA receptors in the CA1 field of the hippocampus. The exact mechanism of action remains to be determined, but it is apparently downstream to the overactivation of NMDA receptors.

Diminution of N-methyl-D-aspartate-induced perturbation of neurotransmission by dexmedetomidine in the CA1 field of rat hippocampus in vitro.

The effects of alpha(2)-adrenoceptor activation on N-methyl-D-aspartic acid (NMDA)-induced perturbation of neurotransmission and normal NMDA-receptor dependent function (long-term potentiation, (LTP)) were investigated in the hippocampal CA1 field in vitro. Bath perfusion of dexmedetomidine hydrochloride (50 nM), which was initiated before NMDA (100 microM) exposure, enhanced the extent of recovery of extracellular field excitatory postsynaptic potentials after NMDA infusion. On the other hand, the induction and early maintenance of LTP was normal in the presence of dexmedetomidine. Thus, dexmedetomidine can diminish acute NMDA-induced perturbation of neurotransmission while the same dose of this drug does not influence the normal activation of NMDA receptors.

Activation of muscarinic M3-like receptors and beta-adrenoceptors, but not M2-like muscarinic receptors or alpha-adrenoceptors, directly modulates corticostriatal neurotransmission in vitro.

The aim of this study was to characterize the modulation of synaptic transmission in the glutamatergic corticostriatal pathway by cholinergic and adrenergic receptors. In coronal slices of mouse brain, negative-going field potentials were recorded in the dorsal striatum in response to stimulation of the overlying white matter, and their susceptibility to various pharmacological manipulations was studied. The responses were mediated by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors, since they were augmented by aniracetam (0.5-1.5 mM), a positive modulator of AMPA-type glutamate receptors, and blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (> or = 10 microM), a selective antagonist of AMPA receptors. Carbachol (10 microM), a muscarinic agonist, reduced the size of responses and abolished paired-pulse depression; these effects being consistent with previous studies indicating that muscarinic activation inhibits release of glutamate in the corticostriatal pathway. Muscarinic antagonists could block the effect of carbachol. Their rank order was: 10 microM scopolamine (a non-selective muscarinic antagonist) > or = 1 microM 4-diphenylacetoxy-N-methyl-piperidine (M3/M1 antagonist)>1 microM pirenzepine (M1 antagonist)>10 microM methoctramine (M2 antagonist). McN-A-343 (1-10 microM), an M1 muscarinic agonist, was ineffective in this preparation. In contrast, isoproterenol (10-30 microM), a beta-adrenergic agonist, slightly increased the synaptic responses, but it did not affect paired-pulse depression. None of alpha-adrenergic agents (30 nM-1.0 microM dexmedetomidine, an alpha2-adrenergic agonist, 0.3 microM atipamezole, an alpha2-adrenergic antagonist or 30 microM phenylephrine, an alpha1-adrenergic agonist) influenced the size of the responses; neither did these drugs alter paired-pulse depression. These results indicate that the activation of striatal M3-like muscarinic receptors and beta-adrenoceptors, but not M2-like muscarinic receptors and alpha-adrenoceptors, modulates directly corticostriatal glutamatergic neurotransmission.

Systemic administration of atipamezole, a selective antagonist of alpha-2 adrenoceptors, facilitates behavioural activity but does not influence short-term or long-term memory in trimethyltin-intoxicated and control rats.

The present study used trimethyltin (TMT)-intoxicated rats as a model for the behavioural syndrome seen after neuronal damage to the limbic system. Behavioural assessments indicated increased locomotor activity and reduced number of groomings in an open-arena task in TMT-intoxicated (6.6 mg/kg as a free base) rats, as has been found previously. A novel finding was the severe deficit in swimming to a visible platform in the water maze task, with reduced swimming speed at the beginning of the training period. During the reacquisition phase of a radial arm maze task, TMT-intoxicated rats made more short-term and long-term memory errors, and their behavioural activity was increased in comparison with controls. The administration of atipamezole (300 micrograms/kg), a selective antagonist of alpha 2-adrenoceptors, enhanced locomotor activity compared to saline-treated rats, but these effects did not differ between the TMT group and their controls. Atipamezole did not enhance short-term or long-term memory in either TMT or control groups. Taken together, the present data indicate that TMT intoxication is a model for global dementia rather than for a specific loss of relational memory. Previous studies on the neurochemical effects of TMT and the alleviation or prevention of neurotoxicity of TMT are reviewed.

Systemic administration of atipamezole, an alpha 2-antagonist, can reduce scopolamine-induced hyperactivity in rats.

The present study investigated whether an alpha 2-adrenoceptor antagonist (atipamezole) can influence hyperactivity induced by the systemic administration of scopolamine. In the water maze (WM) task, scopolamine (scop) 0.25 mg/kg treatment significantly increased swimming speed during the acquisition phase of this task. Atipamezole (ati) 30 micrograms/kg slightly reduced swimming speed both in saline- and in scop-treated rats. Ati 300 micrograms/kg slightly reduced swimming speed in saline-treated rats, and it prevented the scop-induced increase in swimming speed, because ati300-scop treated rats swam more slowly than the saline-saline group. In addition, ati 300 micrograms/kg reduced the total distance swum in scop-treated rats during a free swim trial (the platform was removed from the pool) performed 1 day after the acquisition phase of the WM test, even though it did not affect this parameter when administered alone. In the open arena task, which assessed the ambulation of rats when the pool was covered with a solid floor, scopolamine dose-dependently increased locomotor activity. The rats ambulated more when treated with scop 0.50 mg/kg compared to vehicle treatment, whereas the effect of scop 0.25 mg/kg treatment did not reach significance. In a test investigating the effects of ati 300 micrograms/kg and scop 0.50 mg/kg given singly or combined, the rats ambulated more during both ati 300 micrograms/kg and scop 0.50 mg/kg treatments given alone, but when combined, the rats ambulated less than during scop-saline treatment even though this was more than during control (saline-saline) treatment. These results indicate that the systemic administration of an alpha 2-antagonist can reduce hyperactivity induced by scopolamine.

Salmonella and mammalian-cell mutagenicity of 3-chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone.

3-Chloro-4-(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF), a chlorine disinfection by-product in drinking water, was mutagenic in the Salmonella/his (Ames) assay for both base-pair substitution strains (TA1535, TA100, TA102) and frameshift strains (TA97, TA98) with the highest mutagenic response observed in strain TA100 (1292 revertants/microgram). The presence in TA100 of pKM101 plasmid, which enhances error-phone DNA repair, greatly increased susceptibility to CMCF mutagenicity relative to the isogenic strain TA1535 lacking pKM101. In the Chinese hamster ovary (CHO) cell hprt (6-thioguanine resistance) locus assay, the mutagenicity of CMCF (1.04 mutants/10(6) clonable cells per microgram/ml) was barely detectable because of the low mutagenicity/cytotoxicity ratio. From the present experiments it appears that CMCF acts in a manner similar to that of another drinking water mutagen, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX). However, CMFC appears to be a less potent mutagen in vitro than MX.

Lack of uniformity in the mutational spectra of chlorohydroxyfuranones in Salmonella typhimurium strain TA100.

The mutational specificity of three chlorohydroxyfuranones found in chlorinated drinking water, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), 3-chloro-4(chloromethyl)-5-hydroxy-2(5H)-furanone (CMCF) and 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MCA), was examined in Salmonella typhimurium strain TA100. DNA colony-hybridization of TA100 revertants showed that MX and CMCF both induced predominantly G:C-->T:A transversions (87 and 75% of total, respectively) with a 3:1 preference for the second position of the hisG46 (CCC) target codon. By contrast, MCA produced primarily G:C-->A:T transitions (66% of the total) with a 4:1 preference for the second position of the CCC codon. The mutational specificity of MCA is consistent with the idea that chloroacetaldehyde, a degradation product of MCA, is responsible for the observed mutations. The chemical mechanism by which either MX or CMCF induces G:C-->T:A transversions remains unknown.

Mutagenicity in vitro of 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid), a chlorine disinfection by-product in drinking water.

The mutagenicity of chlorinated humic drinking waters is accounted for mainly by a single contaminant, 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), as assessed in Salmonella typhimurium strain TA100. In the present study 3,4-dichloro-5-hydroxy-2(5H)-furanone (mucochloric acid, MA), another drinking water contaminant much less potent as a mutagen in TA100 than MX, was tested in Chinese hamster ovary (CHO) cells for the induction of mutation at the hypoxanthine phosphoribosyl transferase (hprt) locus to 6-thioguanine resistance (TGr). Unexpectedly, MA induced TGr mutants in CHO cells with a potency comparable to that reported previously for MX. In subsequent experiments with S. typhimurium, the presence of pKM101 plasmid in strain TA100 increased susceptibility to the mutagenicity of MA, but much less than to that of MX, relative to the parental strain TA1535 lacking pKM101. The difference between the two compounds in TA100 thus appears to be due to a higher enhancement of the mutagenicity of MX than that of MA by pKM101 mediated error-prone DNA repair.