The synthesis and pharmacological activity of a new series of hexahydro-2H-pyrano[3,2-c]quinoline... more The synthesis and pharmacological activity of a new series of hexahydro-2H-pyrano[3,2-c]quinoline derivatives as potent σ1 receptor (σ1R) ligands are reported. This family, which does not contain the highly basic amino group usually present in other σ1R ligands, showed high selectivity over the σ2 receptor (σ2R). The activity was shown to reside in only one of the four possible diastereoisomers, which exhibited a perfect match with known σ1R pharmacophores. A hit to lead program based on a high-throughput screening hit (8a) led to the identification of compound 32c, with substantially improved activity and physicochemical properties. Compound 32c also exhibited a good ADMET (absorption, distribution, metabolism, excretion, toxicity) profile and was identified as a σ1R antagonist on the basis of its analgesic activity in the mouse capsaicin and formalin models of neurogenic pain.
Theiler's murine encephalomyelitis virus (TMEV) disease is induced following intracerebral in... more Theiler's murine encephalomyelitis virus (TMEV) disease is induced following intracerebral inoculation of TMEV, a member of picornavirus family, in susceptible animals. The pathogenesis of paralytic syndrome is associated with a chronic progressive demyelinating disease characterized by perivascular of immune inflammatory cells. Although TMEV induced demyelinating disease (TMEV IDD) is initiated by virus specific CD4+ T cells targeting CNS persistent virus, CD4+ T cell responses against self myelin epitopes activated via epitope spreading contribute to chronic disease pathogenesis. In the present report we delineated possible pathogenic mechanisms related with inflammatory process, leading to demyelination and axonal loss. The importance of proinflammatory cytokines in sustaining the inflammatory process and cause direct oligodendrotoxicity is emphasized. Different approaches in therapeutic strategies affecting cytokines are also presented.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2002
Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory respo... more Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory responses and the enhancement of neuronal survival after injury. Although cannabinoid receptors are distributed widely in brain, their presence has not been investigated previously in oligodendrocytes. This study examined the expression of cannabinoid type 1 (CB1) receptors in rat oligodendrocytes in vivo and in culture and explored their biological function. Expression of CB1 receptors by oligodendrocytes was demonstrated immunocytochemically in postnatal and in adult white matter as well as in oligodendrocyte cultures. Reverse transcription-PCR and Western blotting further confirmed the presence of CB1 receptors. Oligodendrocyte progenitors undergo apoptosis with the withdrawal of trophic support, as determined by TUNEL assay and caspase-3 activation, and both the selective CB1 agonist arachidonyl-2'-chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA) and the n...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2003
Theiler's virus infection of the CNS induces an immune-mediated demyelinating disease in susc... more Theiler's virus infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids may act as immunosuppressive compounds that have shown therapeutic potential in chronic inflammatory disorders. Using the Theiler's murine encephalomyelitis virus model, we report here that treatment with the synthetic cannabinoids WIN 55,212-2, ACEA, and JWH-015 during established disease significantly improved the neurological deficits in a long-lasting way. At a histological level, cannabinoids reduced microglial activation, abrogated major histocompatibility complex class II antigen expression, and decreased the number of CD4+ infiltrating T cells in the spinal cord. Both recovery of motor function and diminution of inflammation paralleled extensive remyelination. Overall, the data presented may have potential therapeutic implications in demyelinating pathologies such as...
American journal of physiology. Gastrointestinal and liver physiology, 2003
Numerous genes expressed by intestinal epithelial cells are developmentally regulated, and the in... more Numerous genes expressed by intestinal epithelial cells are developmentally regulated, and the influence that adaptive (AI) and passive (PI) immunity have in controlling their expression has not been evaluated. In this study, we tested the hypothesis that both PI and AI influenced enterocyte gene expression by developing a breeding scheme that used T and B cell-deficient recombination-activating gene (RAG) mice. RNA was isolated from the liver and proximal/distal small intestine at various ages, and the steady-state levels of six different transcripts were evaluated by RNase protection assay. In wild-type (WT) pups, all transcripts [Fc receptor of the neonate (FcRn), polymeric IgA receptor (pIgR), GLUT5, lactase-phlorizin hydrolase (lactase), apical sodium-dependent bile acid transporter (ASBT), and Na+/glucose cotransporter (SGLT1)] studied were developmentally regulated at the time of weaning, and all transcripts except ASBT had the highest levels of expression in the proximal sma...
Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic... more Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tol...
Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor's response to radiation ... more Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor's response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tu...
We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivativ... more We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivatives, compound N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1piperazinehexanamide (LP-211) previously identified as a high-affinity brain penetrant ligand for 5-hydroxytryptamine (serotonin) type 7 (5-HT 7 ) receptors, and its analog N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide (MEL-9). Both ligands exhibited competitive displacement of [ 3 H]-(2R)-1-[(3-hydroxyphenyl) sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine ([ 3 H]-SB-269970) radioligand binding and insurmountable antagonism of 5-carboxamidotryptamine (5-CT)-stimulated cyclic adenosine monophosphate (cAMP) signaling in human embryonic kidney (HEK293) cells stably expressing human 5-HT 7 receptors. They also inhibited forskolin-stimulated adenylate cyclase activity in 5-HT 7expressing HEK293 cells but not in the parental cell line. The compounds elicited long-lasting (at least 24 h) concentration-dependent inhibition of radioligand binding at 5-HT 7 -binding sites in whole-cell radioligand binding assays, after pretreatment of the cells with the compounds and subsequent compound removal. In cAMP assays, pretreatment of cells with the compounds rendered 5-HT 7 receptors unresponsive to 5-CT and also rendered 5-HT 7 -expressing HEK293 cells unresponsive to forskolin. Compound 1-(2-biphenyl)piperazine (RA-7), a known active metabolite of LP-211 present in vivo, was able to partially inhibit 5-HT 7 radioligand binding in a long-lasting irreversible manner. Hence, LP-211 and MEL-9 were identified as high-affinity long-acting inhibitors of human 5-HT 7 receptor binding and function in cell lines. The detailed in vitro characterization of these two pharmacological tools targeting 5-HT 7 receptors may benefit the study of 5-HT 7 receptor function and it may lead to the development of novel selective pharmacological tools with defined functional properties at 5-HT 7 receptors.
The therapeutic potential of S1RA (E-52862), a selective sigma-1 receptor (r 1 R) antagonist, has... more The therapeutic potential of S1RA (E-52862), a selective sigma-1 receptor (r 1 R) antagonist, has been explored in experimental neuropathic pain, but not in inflammatory pain models. The present study investigated the effect of the intraperitoneal administration of S1RA on the hind paw withdrawal response to thermal and mechanical stimulation following an intraplantar injection of carrageenan (CARR) and complete Freund's adjuvant (CFA), which are two well-characterized models of acute and chronic inflammatory pain, respectively. S1RA fully reversed both mechanical [dose of drug that produced half of its maximal response (ED 50 ) = 35.9 and 42.1 mg/kg for CARR-induced and CFA-induced pain, respectively] and thermal (ED 50 = 27.9 mg/kg, CARR) hypersensitivity, whereas ibuprofen (CARR, mechanical allodynia) and celecoxib (CARR, thermal hyperalgesia; CFA, mechanical allodynia) failed to reach maximum efficacy. Morphine also showed maximum efficacy in all tests. Unlike celecoxib and ibuprofen, which decreased paw volume significantly, CARR-induced paw oedema was not reduced by S1RA and morphine, thus suggesting that the antinociceptive effect of S1RA does not involve a major anti-inflammatory (antioedema) action. S1RA was devoid of efficacy when administered to r 1 R knockout mice, thus suggesting the involvement of r 1 R in the antinociceptive effects exerted by S1RA. We conclude that S1RA represents a promising novel analgesic therapy for inflammatory pain.
The in vivo pharmacology of the sigma 1 receptor (r1R) is certainly complex; however, r1R antagon... more The in vivo pharmacology of the sigma 1 receptor (r1R) is certainly complex; however, r1R antagonists are of therapeutic interest, because they enhance mu-opioid receptor (MOR)-mediated antinociception and reduce neuropathic pain. Thus, we investigated whether the r1R is involved in the negative control that glutamate N-methyl-d-aspartate acid receptors (NMDARs) exert on opioid antinociception. Results: The MOR C terminus carries the histidine triad nucleotide-binding protein 1 (HINT1) coupled to the regulator of Gprotein signaling RGSZ2-neural nitric oxide synthase assembly. Activated MORs stimulate the production of nitric oxide (NO), and the redox zinc switch RGSZ2 converts this signal into free zinc ions that are required to recruit the redox sensor PKCc to HINT1 proteins. Then, PKCc impairs HINT1-RGSZ2 association and enables r1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. The inhibition of NOS or the absence of r1Rs prevents HINT1-PKCc interaction, and MOR-NMDAR cross-regulation fails. The r1R antagonists transitorily remove the binding of r1Rs to NR1 subunits, facilitate the entrance of negative regulators of NMDARs, likely Ca 2+ -CaM, and prevent NR1 interaction with HINT1, thereby impairing the negative feedback of glutamate on opioid analgesia. Innovation: A redox-regulated process situates MOR signaling under NMDAR control, and in this context, the r1R binds to the cytosolic C terminal region of the NMDAR NR1 subunit. Conclusion: The r1R antagonists enhance opioid analgesia in naïve mice by releasing MORs from the negative influence of NMDARs, and they also reset antinociception in morphine tolerant animals. Moreover, r1R antagonists alleviate neuropathic pain, probably by driving the inhibition of up-regulated NMDARs. Antioxid. Redox Signal. 00, 000-000.
The spinal cord is a prime site of action for analgesia. Here we characterize the effects of esta... more The spinal cord is a prime site of action for analgesia. Here we characterize the effects of established analgesics on segmental spinal reflexes. The aim of the study was to look for the pattern of action or signature of analgesic effects on these reflexes. We used a spinal cord in vitro preparation of neonate mice to record ventral root responses to dorsal root stimulation. Pregabalin, clonidine, morphine and duloxetine and an experimental sigma-1 receptor antagonist (S1RA) were applied to the preparation in a cumulative concentration protocol. Drug effects on the wind-up produced by repetitive stimulation of C-fibres and on responses to single A- and C-fibre intensity stimuli were analysed. All compounds produced a concentration-dependent inhibition of total spikes elicited by repetitive stimulation. Concentrations producing ∼50% reduction in this parameter were (in μM) clonidine (0.01), morphine (0.1), pregabalin (1), duloxetine (10) and S1RA (30). At these concentrations clonidine, pregabalin and S1RA had significant effects on the wind-up index and little depressant effects on responses to single stimuli. Morphine and duloxetine did not depress wind-up index and showed large effects on responses to single stimuli. None of the compounds had strong effects on the amplitude of the non-nociceptive monosynaptic reflex. morphine and duloxetine had general depressant effects on spinal reflexes, whereas the effects of clonidine, pregabalin and S1RA appeared to be restricted to signals originated by strong repetitive activation of C-fibres. Results are discussed in the context of reported behavioural effects of the compounds studied.
Approximately 10-15% of individuals infected with Helicobacter pylori will develop ulcer disease ... more Approximately 10-15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl a-glucosides in H. pylori cell wall by a1,4-GlcNAc-capped mucin Oglycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl a-glucosyltransferase (aCgT), which forms cholesteryl a-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of aCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl a-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of aCgT showing a range of enzymatic activity. However, cholesteryl a-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active aCgT into iNKT cell-deficient (Ja18 2/2 ) or wild-type mice, bacterial recovery significantly increased in Ja18 2/2 compared to wildtype mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Ja18 2/2 compared to wild-type mice. These findings demonstrate that cholesteryl a-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.
Sigma-1 receptor (sigma(1)R) is expressed in key CNS areas involved in nociceptive processing but... more Sigma-1 receptor (sigma(1)R) is expressed in key CNS areas involved in nociceptive processing but only limited information is available about its functional role. In the present study we investigated the relevance of sigma(1)R in modulating nerve injury-evoked pain. For this purpose, wild-type mice and mice lacking the sigma(1)R gene were exposed to partial sciatic nerve ligation and neuropathic pain-related behaviors were investigated. To explore underlying mechanisms, spinal processing of repetitive nociceptive stimulation and expression of extracellular signal-regulated kinase (ERK) were also investigated. Sensitivity to noxious heat of homozygous sigma(1)R knockout mice did not differ from wild-type mice. Baseline values obtained in sigma(1)R knockout mice before nerve injury in the plantar, cold-plate and von Frey tests were also indistinguishable from those obtained in wild-type mice. However, cold and mechanical allodynia did not develop in sigma(1)R null mice exposed to partial sciatic nerve injury. Using isolated spinal cords we found that mice lacking sigma(1)R showed reduced wind-up responses respect to wild-type mice, as evidenced by a reduced number of action potentials induced by trains of C-fiber intensity stimuli. In addition, in contrast to wild-type mice, sigma(1)R knockout mice did not show increased phosphorylation of ERK in the spinal cord after sciatic nerve injury. Both wind-up and ERK activation have been related to mechanisms of spinal cord sensitization. Our findings identify sigma(1)R as a constituent of the mechanisms modulating activity-induced sensitization in pain pathways and point to sigma(1)R as a new potential target for drugs designed to alleviate neuropathic pain.
This work aimed to evaluate the potential role of the 5-HT(7) receptor in nociception secondary t... more This work aimed to evaluate the potential role of the 5-HT(7) receptor in nociception secondary to a sensitizing stimulus in mice. For this purpose, the effects of relevant ligands (5-HT(7) receptor agonists: AS-19, MSD-5a, E-55888; 5-HT(7) receptor antagonists: SB-258719, SB-269970; 5-HT(1A) receptor agonist: F-13640; 5-HT(1A) receptor antagonist: WAY-100635) were assessed on capsaicin-induced mechanical hypersensitivity, a pain behavior involving hypersensitivity of dorsal horn neurons (central sensitization). For the 5-HT(7) receptor agonists used, binding profile and intrinsic efficacy to stimulate cAMP formation in HEK-293F cells expressing the human 5-HT(7) receptor were also evaluated. AS-19 and E-55888 were selective for 5-HT(7) receptors. E-55888 was a full agonist whereas AS-19 and MSD-5a behaved as partial agonists, with maximal effects corresponding to 77% and 61%, respectively, of the cAMP response evoked by the full agonist 5-HT. Our in vivo results revealed that systemic administration of 5-HT(7) receptor agonists exerted a clear-cut dose-dependent antinociceptive effect that was prevented by 5-HT(7) receptor antagonists, but not by the 5-HT(1A) receptor antagonist. The order of efficacy (E-55888>AS-19>MSD-5a) matched their in vitro efficacy as 5-HT(7) receptor agonists. Contrary to agonists, a dose-dependent promotion of mechanical hypersensitivity was observed after administration of 5-HT(7) receptor antagonists, substantiating the involvement of the 5-HT(7) receptor in the control of capsaicin-induced mechanical hypersensitivity. These findings suggest that serotonin exerts an inhibitory role in the control of nociception through activation of 5-HT(7) receptors, and point to a new potential therapeutic use of 5-HT(7) receptor agonists in the field of analgesia.
Theiler's murine encephalomyelitis virus (TMEV) causes an acute encephalomyelitis followed by a p... more Theiler's murine encephalomyelitis virus (TMEV) causes an acute encephalomyelitis followed by a persistent infection of the central nervous system (CNS) resulting in a chronic inflammation and axonal demyelination in susceptible strains of mice. The pathogenesis of TMEV-induced demyelinating disease remains unknown, but infection of brain glial cells is a critical factor for virus persistence in the CNS. In the present study we investigated the effects of the anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) on the production of inflammatory mediators, such as prostaglandins, after infection of primary astroglial SJL/J murine cultures with TMEV. This infection resulted in a time-dependent transcription of the gene encoding cyclooxygenase-2 (COX-2) and an increased production of prostaglandin E2 (PGE 2 ). Both, IL-4 but mainly, IL-10 (1 and 10 ng/ml) decreased the TMEV-induced expression of COX-2 as well as the synthesis of PGE 2 . Interestingly, treatment with IL-10 completely abrogated COX-2 induction. The molecular mechanisms involved in the regulation of COX-2 expression by TMEV are unknown, but the effects of anti-inflammatory cytokines may involve the inhibition of the transcription factor nuclear factor kB activity and lead to strategies capable of interrupting the inflammatory cascade triggered by TMEV in brain glial cells. q
Increasing evidence associates schizophrenia with prenatal exposure to infection. Impaired abilit... more Increasing evidence associates schizophrenia with prenatal exposure to infection. Impaired ability to "gate out" sensory and cognitive information is considered to be a central feature of schizophrenia and is manifested, among others, in disrupted prepulse inhibition (PPI) of the acoustic startle reflex. We analyzed the effect of a prenatal immune challenge-peripheral administration of bacterial endotoxin lipopolysaccharide (LPS) to pregnant female rats-upon PPI and immune function in adult offspring. Prenatal LPS treatment disrupted PPI which was reversed by antipsychotics. Serum levels of interleukin-2 and interleukin-6 were increased. In addition, histopathological features in brain areas related with PPI circuitry were observed. These results illustrate the critical influence of prenatal immune events upon adult CNS functioning in association with the putative role of the immune system in the etiopathogenesis of schizophrenia.
Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells e... more Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.
Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inf... more Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases, but remarkably little is known about its effect on oligodendroglial cells. In this study we explored the role of IL-1 in oligodendrocyte progenitors and differentiated oligodendrocytes. The effects of IL-1 were compared to those of IL-1 receptor antagonist, the specific inhibitor of IL-1 activity, since progenitors and differentiated oligodendrocytes produce IL-1 and express IL-1 receptors. Unlike other proinflammatory cytokines (TNF␣ and IFN␥), IL-1 was not toxic for oligodendrocyte lineage cells. However, this cytokine inhibited proliferation of oligodendrocyte progenitors in the presence of growth factors (PDGF plus bFGF). This was evidenced by a significant decrease in both cells incorporating bromodeoxyuridine (45%) and total cell numbers (57%) after 6 days of treatment. Interestingly, IL-1 blocked proliferation at the late progenitor/prooligodendrocyte (O4؉) stage but did not affect proliferation of early progenitors (A2B5؉). Inhibition of proliferation paralleled with promotion of differentiation, as revealed by the increased percentage of R-mab؉ cells (6.7-fold). Moreover, when oligodendrocyte progenitors were allowed to differentiate in the absence of growth factors, treatment with IL-1 promoted maturation to the MBP؉ stage (4.2-fold) and survival of differentiating oligodendrocytes (2.1-fold). Regarding intracellular signaling, IL-1 activated the p38 mitogen-activated protein kinase (MAPK) but not the p42/p44 MAPK and, when combined with growth factors, intensified p38 activation but inhibited the growth-factor-induced p42/p44 activation. IL-1 also induced a time-dependent inhibition of PFGF-R␣ gene expression. These results support a role for IL-1 in promoting mitotic arrest and differentiation of oligodendrocyte progenitors as well as maturation and survival of differentiating oligodendrocytes.
The synthesis and pharmacological activity of a new series of hexahydro-2H-pyrano[3,2-c]quinoline... more The synthesis and pharmacological activity of a new series of hexahydro-2H-pyrano[3,2-c]quinoline derivatives as potent σ1 receptor (σ1R) ligands are reported. This family, which does not contain the highly basic amino group usually present in other σ1R ligands, showed high selectivity over the σ2 receptor (σ2R). The activity was shown to reside in only one of the four possible diastereoisomers, which exhibited a perfect match with known σ1R pharmacophores. A hit to lead program based on a high-throughput screening hit (8a) led to the identification of compound 32c, with substantially improved activity and physicochemical properties. Compound 32c also exhibited a good ADMET (absorption, distribution, metabolism, excretion, toxicity) profile and was identified as a σ1R antagonist on the basis of its analgesic activity in the mouse capsaicin and formalin models of neurogenic pain.
Theiler's murine encephalomyelitis virus (TMEV) disease is induced following intracerebral in... more Theiler's murine encephalomyelitis virus (TMEV) disease is induced following intracerebral inoculation of TMEV, a member of picornavirus family, in susceptible animals. The pathogenesis of paralytic syndrome is associated with a chronic progressive demyelinating disease characterized by perivascular of immune inflammatory cells. Although TMEV induced demyelinating disease (TMEV IDD) is initiated by virus specific CD4+ T cells targeting CNS persistent virus, CD4+ T cell responses against self myelin epitopes activated via epitope spreading contribute to chronic disease pathogenesis. In the present report we delineated possible pathogenic mechanisms related with inflammatory process, leading to demyelination and axonal loss. The importance of proinflammatory cytokines in sustaining the inflammatory process and cause direct oligodendrotoxicity is emphasized. Different approaches in therapeutic strategies affecting cytokines are also presented.
The Journal of neuroscience : the official journal of the Society for Neuroscience, Jan 15, 2002
Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory respo... more Cannabinoids exert pleiotropic actions in the CNS, including the inhibition of inflammatory responses and the enhancement of neuronal survival after injury. Although cannabinoid receptors are distributed widely in brain, their presence has not been investigated previously in oligodendrocytes. This study examined the expression of cannabinoid type 1 (CB1) receptors in rat oligodendrocytes in vivo and in culture and explored their biological function. Expression of CB1 receptors by oligodendrocytes was demonstrated immunocytochemically in postnatal and in adult white matter as well as in oligodendrocyte cultures. Reverse transcription-PCR and Western blotting further confirmed the presence of CB1 receptors. Oligodendrocyte progenitors undergo apoptosis with the withdrawal of trophic support, as determined by TUNEL assay and caspase-3 activation, and both the selective CB1 agonist arachidonyl-2'-chloroethylamide/(all Z)-N-(2-cycloethyl)-5,8,11,14-eicosatetraenamide (ACEA) and the n...
The Journal of neuroscience : the official journal of the Society for Neuroscience, 2003
Theiler's virus infection of the CNS induces an immune-mediated demyelinating disease in susc... more Theiler's virus infection of the CNS induces an immune-mediated demyelinating disease in susceptible mouse strains and serves as a relevant infection model for human multiple sclerosis (MS). Cannabinoids may act as immunosuppressive compounds that have shown therapeutic potential in chronic inflammatory disorders. Using the Theiler's murine encephalomyelitis virus model, we report here that treatment with the synthetic cannabinoids WIN 55,212-2, ACEA, and JWH-015 during established disease significantly improved the neurological deficits in a long-lasting way. At a histological level, cannabinoids reduced microglial activation, abrogated major histocompatibility complex class II antigen expression, and decreased the number of CD4+ infiltrating T cells in the spinal cord. Both recovery of motor function and diminution of inflammation paralleled extensive remyelination. Overall, the data presented may have potential therapeutic implications in demyelinating pathologies such as...
American journal of physiology. Gastrointestinal and liver physiology, 2003
Numerous genes expressed by intestinal epithelial cells are developmentally regulated, and the in... more Numerous genes expressed by intestinal epithelial cells are developmentally regulated, and the influence that adaptive (AI) and passive (PI) immunity have in controlling their expression has not been evaluated. In this study, we tested the hypothesis that both PI and AI influenced enterocyte gene expression by developing a breeding scheme that used T and B cell-deficient recombination-activating gene (RAG) mice. RNA was isolated from the liver and proximal/distal small intestine at various ages, and the steady-state levels of six different transcripts were evaluated by RNase protection assay. In wild-type (WT) pups, all transcripts [Fc receptor of the neonate (FcRn), polymeric IgA receptor (pIgR), GLUT5, lactase-phlorizin hydrolase (lactase), apical sodium-dependent bile acid transporter (ASBT), and Na+/glucose cotransporter (SGLT1)] studied were developmentally regulated at the time of weaning, and all transcripts except ASBT had the highest levels of expression in the proximal sma...
Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic... more Understanding immune tolerance mechanisms is a major goal of immunology research, but mechanistic studies have generally required the use of mouse models carrying untargeted or targeted antigen receptor transgenes, which distort lymphocyte development and therefore preclude analysis of a truly normal immune system. Here we demonstrate an advance in in vivo analysis of immune tolerance that overcomes these shortcomings. We show that custom superantigens generated by single chain antibody technology permit the study of tolerance in a normal, polyclonal immune system. In the present study we generated a membrane-tethered anti-Igkappa-reactive single chain antibody chimeric gene and expressed it as a transgene in mice. B cell tolerance was directly characterized in the transgenic mice and in radiation bone marrow chimeras in which ligand-bearing mice served as recipients of nontransgenic cells. We find that the ubiquitously expressed, Igkappa-reactive ligand induces efficient B cell tol...
Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor's response to radiation ... more Cyclooxygenase 2 (COX-2) inhibitors have been shown to enhance tumor's response to radiation in several animal models. The strong association of COX-2 and angiogenesis suggests that the tumor vasculature may be involved in this process. The current study investigated whether treatment with the COX-2 inhibitor E-6087 could influence response to local radiation in orthotopically growing murine gliomas and aimed to analyze the involvement of the tumor vasculature. GL261 glioma cells were injected into the cerebrum of C57bl/6 mice. From day 7 after tumor cell injection, mice were treated with COX-2 inhibitor at 50 mg/kg i.p. every third day. Radiation consisted of three fractions of 2 Gy given daily from day 9 to day 11. Mice were killed at day 21. The COX-2 inhibitor significantly enhanced the response to radiation, reducing mean volume to 32% of tumors treated with radiation only. The combination treatment neither increased apoptosis of tumor cells or stromal cells nor affected tu...
We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivativ... more We performed a detailed in vitro pharmacological characterization of two arylpiperazine derivatives, compound N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1piperazinehexanamide (LP-211) previously identified as a high-affinity brain penetrant ligand for 5-hydroxytryptamine (serotonin) type 7 (5-HT 7 ) receptors, and its analog N-benzyl-4-(2-diphenyl)-1-piperazinehexanamide (MEL-9). Both ligands exhibited competitive displacement of [ 3 H]-(2R)-1-[(3-hydroxyphenyl) sulfonyl]-2-[2-(4-methyl-1-piperidinyl)ethyl]pyrrolidine ([ 3 H]-SB-269970) radioligand binding and insurmountable antagonism of 5-carboxamidotryptamine (5-CT)-stimulated cyclic adenosine monophosphate (cAMP) signaling in human embryonic kidney (HEK293) cells stably expressing human 5-HT 7 receptors. They also inhibited forskolin-stimulated adenylate cyclase activity in 5-HT 7expressing HEK293 cells but not in the parental cell line. The compounds elicited long-lasting (at least 24 h) concentration-dependent inhibition of radioligand binding at 5-HT 7 -binding sites in whole-cell radioligand binding assays, after pretreatment of the cells with the compounds and subsequent compound removal. In cAMP assays, pretreatment of cells with the compounds rendered 5-HT 7 receptors unresponsive to 5-CT and also rendered 5-HT 7 -expressing HEK293 cells unresponsive to forskolin. Compound 1-(2-biphenyl)piperazine (RA-7), a known active metabolite of LP-211 present in vivo, was able to partially inhibit 5-HT 7 radioligand binding in a long-lasting irreversible manner. Hence, LP-211 and MEL-9 were identified as high-affinity long-acting inhibitors of human 5-HT 7 receptor binding and function in cell lines. The detailed in vitro characterization of these two pharmacological tools targeting 5-HT 7 receptors may benefit the study of 5-HT 7 receptor function and it may lead to the development of novel selective pharmacological tools with defined functional properties at 5-HT 7 receptors.
The therapeutic potential of S1RA (E-52862), a selective sigma-1 receptor (r 1 R) antagonist, has... more The therapeutic potential of S1RA (E-52862), a selective sigma-1 receptor (r 1 R) antagonist, has been explored in experimental neuropathic pain, but not in inflammatory pain models. The present study investigated the effect of the intraperitoneal administration of S1RA on the hind paw withdrawal response to thermal and mechanical stimulation following an intraplantar injection of carrageenan (CARR) and complete Freund's adjuvant (CFA), which are two well-characterized models of acute and chronic inflammatory pain, respectively. S1RA fully reversed both mechanical [dose of drug that produced half of its maximal response (ED 50 ) = 35.9 and 42.1 mg/kg for CARR-induced and CFA-induced pain, respectively] and thermal (ED 50 = 27.9 mg/kg, CARR) hypersensitivity, whereas ibuprofen (CARR, mechanical allodynia) and celecoxib (CARR, thermal hyperalgesia; CFA, mechanical allodynia) failed to reach maximum efficacy. Morphine also showed maximum efficacy in all tests. Unlike celecoxib and ibuprofen, which decreased paw volume significantly, CARR-induced paw oedema was not reduced by S1RA and morphine, thus suggesting that the antinociceptive effect of S1RA does not involve a major anti-inflammatory (antioedema) action. S1RA was devoid of efficacy when administered to r 1 R knockout mice, thus suggesting the involvement of r 1 R in the antinociceptive effects exerted by S1RA. We conclude that S1RA represents a promising novel analgesic therapy for inflammatory pain.
The in vivo pharmacology of the sigma 1 receptor (r1R) is certainly complex; however, r1R antagon... more The in vivo pharmacology of the sigma 1 receptor (r1R) is certainly complex; however, r1R antagonists are of therapeutic interest, because they enhance mu-opioid receptor (MOR)-mediated antinociception and reduce neuropathic pain. Thus, we investigated whether the r1R is involved in the negative control that glutamate N-methyl-d-aspartate acid receptors (NMDARs) exert on opioid antinociception. Results: The MOR C terminus carries the histidine triad nucleotide-binding protein 1 (HINT1) coupled to the regulator of Gprotein signaling RGSZ2-neural nitric oxide synthase assembly. Activated MORs stimulate the production of nitric oxide (NO), and the redox zinc switch RGSZ2 converts this signal into free zinc ions that are required to recruit the redox sensor PKCc to HINT1 proteins. Then, PKCc impairs HINT1-RGSZ2 association and enables r1R-NR1 interaction with MOR-HINT1 complexes to restrain opioid signaling. The inhibition of NOS or the absence of r1Rs prevents HINT1-PKCc interaction, and MOR-NMDAR cross-regulation fails. The r1R antagonists transitorily remove the binding of r1Rs to NR1 subunits, facilitate the entrance of negative regulators of NMDARs, likely Ca 2+ -CaM, and prevent NR1 interaction with HINT1, thereby impairing the negative feedback of glutamate on opioid analgesia. Innovation: A redox-regulated process situates MOR signaling under NMDAR control, and in this context, the r1R binds to the cytosolic C terminal region of the NMDAR NR1 subunit. Conclusion: The r1R antagonists enhance opioid analgesia in naïve mice by releasing MORs from the negative influence of NMDARs, and they also reset antinociception in morphine tolerant animals. Moreover, r1R antagonists alleviate neuropathic pain, probably by driving the inhibition of up-regulated NMDARs. Antioxid. Redox Signal. 00, 000-000.
The spinal cord is a prime site of action for analgesia. Here we characterize the effects of esta... more The spinal cord is a prime site of action for analgesia. Here we characterize the effects of established analgesics on segmental spinal reflexes. The aim of the study was to look for the pattern of action or signature of analgesic effects on these reflexes. We used a spinal cord in vitro preparation of neonate mice to record ventral root responses to dorsal root stimulation. Pregabalin, clonidine, morphine and duloxetine and an experimental sigma-1 receptor antagonist (S1RA) were applied to the preparation in a cumulative concentration protocol. Drug effects on the wind-up produced by repetitive stimulation of C-fibres and on responses to single A- and C-fibre intensity stimuli were analysed. All compounds produced a concentration-dependent inhibition of total spikes elicited by repetitive stimulation. Concentrations producing ∼50% reduction in this parameter were (in μM) clonidine (0.01), morphine (0.1), pregabalin (1), duloxetine (10) and S1RA (30). At these concentrations clonidine, pregabalin and S1RA had significant effects on the wind-up index and little depressant effects on responses to single stimuli. Morphine and duloxetine did not depress wind-up index and showed large effects on responses to single stimuli. None of the compounds had strong effects on the amplitude of the non-nociceptive monosynaptic reflex. morphine and duloxetine had general depressant effects on spinal reflexes, whereas the effects of clonidine, pregabalin and S1RA appeared to be restricted to signals originated by strong repetitive activation of C-fibres. Results are discussed in the context of reported behavioural effects of the compounds studied.
Approximately 10-15% of individuals infected with Helicobacter pylori will develop ulcer disease ... more Approximately 10-15% of individuals infected with Helicobacter pylori will develop ulcer disease (gastric or duodenal ulcer), while most people infected with H. pylori will be asymptomatic. The majority of infected individuals remain asymptomatic partly due to the inhibition of synthesis of cholesteryl a-glucosides in H. pylori cell wall by a1,4-GlcNAc-capped mucin Oglycans, which are expressed in the deeper portion of gastric mucosa. However, it has not been determined how cholesteryl a-glucosyltransferase (aCgT), which forms cholesteryl a-glucosides, functions in the pathogenesis of H. pylori infection. Here, we show that the activity of aCgT from H. pylori clinical isolates is highly correlated with the degree of gastric atrophy. We investigated the role of cholesteryl a-glucosides in various aspects of the immune response. Phagocytosis and activation of dendritic cells were observed at similar degrees in the presence of wild-type H. pylori or variants harboring mutant forms of aCgT showing a range of enzymatic activity. However, cholesteryl a-glucosides were recognized by invariant natural killer T (iNKT) cells, eliciting an immune response in vitro and in vivo. Following inoculation of H. pylori harboring highly active aCgT into iNKT cell-deficient (Ja18 2/2 ) or wild-type mice, bacterial recovery significantly increased in Ja18 2/2 compared to wildtype mice. Moreover, cytokine production characteristic of Th1 and Th2 cells dramatically decreased in Ja18 2/2 compared to wild-type mice. These findings demonstrate that cholesteryl a-glucosides play critical roles in H. pylori-mediated gastric inflammation and precancerous atrophic gastritis.
Sigma-1 receptor (sigma(1)R) is expressed in key CNS areas involved in nociceptive processing but... more Sigma-1 receptor (sigma(1)R) is expressed in key CNS areas involved in nociceptive processing but only limited information is available about its functional role. In the present study we investigated the relevance of sigma(1)R in modulating nerve injury-evoked pain. For this purpose, wild-type mice and mice lacking the sigma(1)R gene were exposed to partial sciatic nerve ligation and neuropathic pain-related behaviors were investigated. To explore underlying mechanisms, spinal processing of repetitive nociceptive stimulation and expression of extracellular signal-regulated kinase (ERK) were also investigated. Sensitivity to noxious heat of homozygous sigma(1)R knockout mice did not differ from wild-type mice. Baseline values obtained in sigma(1)R knockout mice before nerve injury in the plantar, cold-plate and von Frey tests were also indistinguishable from those obtained in wild-type mice. However, cold and mechanical allodynia did not develop in sigma(1)R null mice exposed to partial sciatic nerve injury. Using isolated spinal cords we found that mice lacking sigma(1)R showed reduced wind-up responses respect to wild-type mice, as evidenced by a reduced number of action potentials induced by trains of C-fiber intensity stimuli. In addition, in contrast to wild-type mice, sigma(1)R knockout mice did not show increased phosphorylation of ERK in the spinal cord after sciatic nerve injury. Both wind-up and ERK activation have been related to mechanisms of spinal cord sensitization. Our findings identify sigma(1)R as a constituent of the mechanisms modulating activity-induced sensitization in pain pathways and point to sigma(1)R as a new potential target for drugs designed to alleviate neuropathic pain.
This work aimed to evaluate the potential role of the 5-HT(7) receptor in nociception secondary t... more This work aimed to evaluate the potential role of the 5-HT(7) receptor in nociception secondary to a sensitizing stimulus in mice. For this purpose, the effects of relevant ligands (5-HT(7) receptor agonists: AS-19, MSD-5a, E-55888; 5-HT(7) receptor antagonists: SB-258719, SB-269970; 5-HT(1A) receptor agonist: F-13640; 5-HT(1A) receptor antagonist: WAY-100635) were assessed on capsaicin-induced mechanical hypersensitivity, a pain behavior involving hypersensitivity of dorsal horn neurons (central sensitization). For the 5-HT(7) receptor agonists used, binding profile and intrinsic efficacy to stimulate cAMP formation in HEK-293F cells expressing the human 5-HT(7) receptor were also evaluated. AS-19 and E-55888 were selective for 5-HT(7) receptors. E-55888 was a full agonist whereas AS-19 and MSD-5a behaved as partial agonists, with maximal effects corresponding to 77% and 61%, respectively, of the cAMP response evoked by the full agonist 5-HT. Our in vivo results revealed that systemic administration of 5-HT(7) receptor agonists exerted a clear-cut dose-dependent antinociceptive effect that was prevented by 5-HT(7) receptor antagonists, but not by the 5-HT(1A) receptor antagonist. The order of efficacy (E-55888>AS-19>MSD-5a) matched their in vitro efficacy as 5-HT(7) receptor agonists. Contrary to agonists, a dose-dependent promotion of mechanical hypersensitivity was observed after administration of 5-HT(7) receptor antagonists, substantiating the involvement of the 5-HT(7) receptor in the control of capsaicin-induced mechanical hypersensitivity. These findings suggest that serotonin exerts an inhibitory role in the control of nociception through activation of 5-HT(7) receptors, and point to a new potential therapeutic use of 5-HT(7) receptor agonists in the field of analgesia.
Theiler's murine encephalomyelitis virus (TMEV) causes an acute encephalomyelitis followed by a p... more Theiler's murine encephalomyelitis virus (TMEV) causes an acute encephalomyelitis followed by a persistent infection of the central nervous system (CNS) resulting in a chronic inflammation and axonal demyelination in susceptible strains of mice. The pathogenesis of TMEV-induced demyelinating disease remains unknown, but infection of brain glial cells is a critical factor for virus persistence in the CNS. In the present study we investigated the effects of the anti-inflammatory cytokines interleukin-4 (IL-4) and interleukin-10 (IL-10) on the production of inflammatory mediators, such as prostaglandins, after infection of primary astroglial SJL/J murine cultures with TMEV. This infection resulted in a time-dependent transcription of the gene encoding cyclooxygenase-2 (COX-2) and an increased production of prostaglandin E2 (PGE 2 ). Both, IL-4 but mainly, IL-10 (1 and 10 ng/ml) decreased the TMEV-induced expression of COX-2 as well as the synthesis of PGE 2 . Interestingly, treatment with IL-10 completely abrogated COX-2 induction. The molecular mechanisms involved in the regulation of COX-2 expression by TMEV are unknown, but the effects of anti-inflammatory cytokines may involve the inhibition of the transcription factor nuclear factor kB activity and lead to strategies capable of interrupting the inflammatory cascade triggered by TMEV in brain glial cells. q
Increasing evidence associates schizophrenia with prenatal exposure to infection. Impaired abilit... more Increasing evidence associates schizophrenia with prenatal exposure to infection. Impaired ability to "gate out" sensory and cognitive information is considered to be a central feature of schizophrenia and is manifested, among others, in disrupted prepulse inhibition (PPI) of the acoustic startle reflex. We analyzed the effect of a prenatal immune challenge-peripheral administration of bacterial endotoxin lipopolysaccharide (LPS) to pregnant female rats-upon PPI and immune function in adult offspring. Prenatal LPS treatment disrupted PPI which was reversed by antipsychotics. Serum levels of interleukin-2 and interleukin-6 were increased. In addition, histopathological features in brain areas related with PPI circuitry were observed. These results illustrate the critical influence of prenatal immune events upon adult CNS functioning in association with the putative role of the immune system in the etiopathogenesis of schizophrenia.
Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells e... more Natural killer T cells (NKT cells) recognize glycolipid antigens presented by CD1d. These cells express an evolutionarily conserved, invariant T cell antigen receptor (TCR), but the forces that drive TCR conservation have remained uncertain. Here we show that NKT cells recognized diacylglycerol-containing glycolipids from Streptococcus pneumoniae, the leading cause of community-acquired pneumonia, and group B Streptococcus, which causes neonatal sepsis and meningitis. Furthermore, CD1d-dependent responses by NKT cells were required for activation and host protection. The glycolipid response was dependent on vaccenic acid, which is present in low concentrations in mammalian cells. Our results show how microbial lipids position the sugar for recognition by the invariant TCR and, most notably, extend the range of microbes recognized by this conserved TCR to several clinically important bacteria.
Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inf... more Interleukin-1 (IL-1) is a pleiotropic cytokine expressed during normal CNS development and in inflammatory demyelinating diseases, but remarkably little is known about its effect on oligodendroglial cells. In this study we explored the role of IL-1 in oligodendrocyte progenitors and differentiated oligodendrocytes. The effects of IL-1 were compared to those of IL-1 receptor antagonist, the specific inhibitor of IL-1 activity, since progenitors and differentiated oligodendrocytes produce IL-1 and express IL-1 receptors. Unlike other proinflammatory cytokines (TNF␣ and IFN␥), IL-1 was not toxic for oligodendrocyte lineage cells. However, this cytokine inhibited proliferation of oligodendrocyte progenitors in the presence of growth factors (PDGF plus bFGF). This was evidenced by a significant decrease in both cells incorporating bromodeoxyuridine (45%) and total cell numbers (57%) after 6 days of treatment. Interestingly, IL-1 blocked proliferation at the late progenitor/prooligodendrocyte (O4؉) stage but did not affect proliferation of early progenitors (A2B5؉). Inhibition of proliferation paralleled with promotion of differentiation, as revealed by the increased percentage of R-mab؉ cells (6.7-fold). Moreover, when oligodendrocyte progenitors were allowed to differentiate in the absence of growth factors, treatment with IL-1 promoted maturation to the MBP؉ stage (4.2-fold) and survival of differentiating oligodendrocytes (2.1-fold). Regarding intracellular signaling, IL-1 activated the p38 mitogen-activated protein kinase (MAPK) but not the p42/p44 MAPK and, when combined with growth factors, intensified p38 activation but inhibited the growth-factor-induced p42/p44 activation. IL-1 also induced a time-dependent inhibition of PFGF-R␣ gene expression. These results support a role for IL-1 in promoting mitotic arrest and differentiation of oligodendrocyte progenitors as well as maturation and survival of differentiating oligodendrocytes.
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Papers by Jose Vela