RESUMEN
The vanilloid receptor 1 (TRPV1) is activated by capsaicin, several endogenous lipids, acidic pH and elevated temperatures. Inflammatory mediators (BK, substance P) also modulate TRPV1 activity. In this study we investigated the effect of TRPV1 agonists and elevated temperatures on neuronal membrane excitability by electrophysiological techniques using freshly isolated rat dorsal root ganglion neurons (DRGs). Focal application of heated solutions demonstrated that the normal threshold (approximately 42 degrees C) of TRPV1 activation was reduced in the presence of capsaicin (1 microM) to approximately 30 degrees C. In current-clamp recordings, increasing the temperature of the solution resulted in larger membrane depolarizations and significantly altered the pattern and onset of the action potential train evoked by 1 microM capsaicin. These effects were blocked by the TRPV1 antagonist capsazepine (10 microM). In contrast to capsaicin, anandamide (10 microM) alone did not evoke action potentials, but it did alter the excitability of neurons to subsequent applications of heat (50 degrees C). Together these results provide evidence that a synergistic interaction of TRPV1 ligands and elevated temperature activates TRPV1 receptors and results in profound effects on membrane excitability.
Asunto(s)
Ganglios Espinales/fisiología , Canales Catiónicos TRPV/fisiología , Potenciales de Acción/efectos de los fármacos , Animales , Ácidos Araquidónicos/farmacología , Capsaicina/farmacología , Endocannabinoides , Ganglios Espinales/efectos de los fármacos , Masculino , Fosforilación , Alcamidas Poliinsaturadas/farmacología , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/agonistas , TemperaturaRESUMEN
Growing evidence supports a role for the immune system in the induction and maintenance of chronic pain. ATP is a key neurotransmitter in this process. Recent studies demonstrate that the glial ATP receptor, P2X7, contributes to the modulation of pathological pain. To further delineate the endogenous mechanisms that are involved in P2X7-related antinociception, we utilized a selective P2X7 receptor antagonist, A-438079, in a series of in vivo and in vitro experiments. Injection of A-438079 (10-300 micromol/kg, i.p.) was anti-allodynic in three different rat models of neuropathic pain and it attenuated formalin-induced nocifensive behaviors. Using in vivo electrophysiology, A-438079 (80 micromol/kg, i.v.) reduced noxious and innocuous evoked activity of different classes of spinal neurons (low threshold, nociceptive specific, wide dynamic range) in neuropathic rats. The effects of A-438079 on evoked firing were diminished or absent in sham rats. Spontaneous activity of all classes of spinal neurons was also significantly reduced by A-438079 in neuropathic but not sham rats. In vitro, A-438079 (1 microM) blocked agonist-induced (2,3-O-(4-benzoylbenzoyl)-ATP, 30 microM) current in non-neuronal cells taken from the vicinity of the dorsal root ganglia. Furthermore, A-438079 dose-dependently (0.3-3 microM) decreased the quantity of the cytokine, interleukin-1beta, released from peripheral macrophages. Thus, ATP, acting through the P2X7 receptor, exerts a wide-ranging influence on spinal neuronal activity following a chronic injury. Antagonism of the P2X7 receptor can in turn modulate central sensitization and produce antinociception in animal models of pathological pain. These effects are likely mediated through immuno-neural interactions that affect the release of endogenous cytokines.
Asunto(s)
Piridinas/farmacología , Receptores Purinérgicos P2/fisiología , Ciática/metabolismo , Ciática/fisiopatología , Tetrazoles/farmacología , Potenciales de Acción/efectos de los fármacos , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Analgésicos/farmacología , Analgésicos/uso terapéutico , Animales , Astrocitoma , Conducta Animal/efectos de los fármacos , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ganglios Espinales , Humanos , Interleucina-1beta/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Neuronas , Dimensión del Dolor/métodos , Agonistas del Receptor Purinérgico P2 , Antagonistas del Receptor Purinérgico P2 , Piridinas/uso terapéutico , Ratas , Ratas Sprague-Dawley , Receptores Purinérgicos P2X7 , Ciática/tratamiento farmacológico , Tetrazoles/uso terapéutico , Factores de TiempoRESUMEN
Vanilloid receptor type 1 (TRPV1) is a ligand-gated nonselective cation channel that is considered to be an important integrator of various pain stimuli such as endogenous lipids, capsaicin, heat, and low pH. In addition to expression in primary afferents, TRPV1 is also expressed in the CNS. To test the hypothesis that the CNS plays a differential role in the effect of TRPV1 antagonists in various types of pain, the analgesic effects of two TRPV1 antagonists with similar in vitro potency but different CNS penetration were compared in vivo. Oral administration of either A-784168 (1-[3-(trifluoromethyl)pyridin-2-yl]-N-[4-(trifluoromethylsulfonyl)phenyl]-1,2,3,6-tetrahydropyridine-4-carboxamide) (good CNS penetration) or A-795614 (N-1H-indazol-4-yl-N'-[(1R)-5-piperidin-1-yl-2,3-dihydro-1H-inden-1-yl]urea) (poor CNS penetration) blocked capsaicin-induced acute pain with the same potency. In complete Freund's adjuvant (CFA)-induced chronic inflammatory pain, oral administration of either compound blocked thermal hyperalgesia with similar potency. Furthermore, intraplantar or intrathecal administration of A-784168 blocked CFA-induced thermal hyperalgesia, suggesting that both peripheral and CNS TRPV1 receptors may play a role in inflammatory thermal hyperalgesia. The effects of the two TRPV1 antagonists were further assessed in models presumably mediated by central sensitization, including CFA- and capsaicin-induced mechanical allodynia and osteoarthritic pain. In these models, the potency of the two compounds was similar after intrathecal administration. However, when administered orally, A-784168, with good CNS penetration, was much more potent than A-795614. Together, these results demonstrate that TRPV1 receptors in the CNS play an important role in pain mediated by central sensitization. In addition, these results demonstrate that significant CNS penetration is necessary for a TRPV1 antagonist to produce broad-spectrum analgesia.
Asunto(s)
Analgésicos/farmacocinética , Sistema Nervioso Central/efectos de los fármacos , Nociceptores/efectos de los fármacos , Dolor/tratamiento farmacológico , Canales Catiónicos TRPV/antagonistas & inhibidores , Administración Oral , Analgésicos/metabolismo , Animales , Artralgia/tratamiento farmacológico , Artralgia/metabolismo , Artralgia/fisiopatología , Calcio/metabolismo , Capsaicina/antagonistas & inhibidores , Línea Celular , Células Cultivadas , Sistema Nervioso Central/metabolismo , Modelos Animales de Enfermedad , Humanos , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Hiperalgesia/fisiopatología , Indazoles/farmacología , Mediadores de Inflamación/antagonistas & inhibidores , Inyecciones Espinales , Masculino , Nociceptores/metabolismo , Dolor/metabolismo , Dolor/fisiopatología , Piridinas/farmacología , Ratas , Ratas Sprague-Dawley , Sulfonas/farmacología , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , Resultado del Tratamiento , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
The focus of the present study is the molecular and functional characterization of four splice variants of the human Nav1.3 alpha subunit. These subtypes arise due to the use of alternative splice donor sites of exon 12, which encodes a region of the alpha subunit that resides in the intracellular loop between domains I and II. This region contains several important phosphorylation sites that modulate Na+ channel kinetics in related sodium channels, i.e. Nav1.2. While three of the four Nav1.3 isoforms, 12v1, 12v3 and 12v4 have been previously identified in human, 12v2 has only been reported in rat. Herein, we evaluate the distribution of these splice variants in human tissues and the functional characterization of each of these subtypes. We demonstrate by reverse transcriptase-polymerase chain reaction (RT-PCR) that each subtype is expressed in the spinal cord, thalamus, amygdala, cerebellum, adult and fetal whole brain and heart. To investigate the functional properties of these different splice variants, each alpha subunit isoform was cloned by RT-PCR from human fetal brain and expressed in Xenopus oocytes. Each isoform exhibited functional voltage-dependent Na+ channels with similar sensitivities to tetrodotoxin (TTX) and comparable current amplitudes. Subtle shifts in the V 1/2 of activation and inactivation (2-3 mV) were observed among the four isoforms, although the functional significance of these differences remains unclear. This study has demonstrated that all four human splice variants of the Nav1.3 channel alpha subunit are widely expressed and generate functional TTX-sensitive Na+ channels that likely modulate cellular excitability.
Asunto(s)
Membrana Celular/metabolismo , Sistema Nervioso Central/metabolismo , Proteínas del Tejido Nervioso/genética , Canales de Sodio/genética , Empalme Alternativo/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Encéfalo/metabolismo , Membrana Celular/genética , Femenino , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Canal de Sodio Activado por Voltaje NAV1.3 , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Oocitos , Isoformas de Proteínas/genética , Isoformas de Proteínas/aislamiento & purificación , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Ratas , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/aislamiento & purificación , Canales de Sodio/metabolismo , Médula Espinal/metabolismo , XenopusRESUMEN
The objective of this study was to use a new high throughput method to compare nicotinic acetylcholine receptor (nAChR)-mediated [(3)H]-dopamine (DA) release from slices of rat striatum and cortex. (-)Nicotine, (-)-cytisine, 1,1-dimethyl-4-phenyl-piperazinium (DMPP), and (+/-)-epibatidine evoked release of striatal [(3)H]-DA with pEC(50) values of 6.7, 8.25, 5.11, and 9.08, respectively. The same agonists evoked release of cortical [(3)H]-DA with pEC(50) values of 6.98, 8.06, 5.58, and 9.59. Relative to (-)-nicotine, (-)-cytisine was a partial agonist in both tissues. In contrast, the maximal response evoked by DMPP differed between the two tissues. The rank order of potency for antagonists to block DA release was the same (mecamylamine (Mec)>dihydro-beta-erythroidine (DHbetaE)>hexamethonium (Hex)>D-tubocurarine (D-TC)); however, the pIC(50) values varied between the two regions. Whereas Mec potently antagonized (-)-nicotine-evoked DA release similarly from striatum and cortex, with pIC(50) values of 6.07 and 6.53 respectively, the values obtained for DHbetaE, D-TC and Hex differed. Additionally, the present study was able to distinguish exocytotic vesicular-mediated from transporter-mediated DA release, by altering temperature of the incubation and exclusion of calcium. Assays carried out under these conditions indicate that approximately 60% of nicotine-evoked cortical DA release was likely mediated through the DA transporter. In contrast, under the same conditions only 15%-20% of striatal release appeared to be transporter-mediated. We conclude that the relative contributions of the mechanisms by which (-)-nicotine evokes DA release differ between striatum and cortex. In addition, the data suggest that the subtypes of nAChRs involved in regulating [(3)H]-DA release may be somewhat different in the two tissues.
Asunto(s)
Corteza Cerebral/metabolismo , Dopamina/metabolismo , Neostriado/metabolismo , Receptores Nicotínicos/metabolismo , Alcaloides/farmacología , Animales , Azocinas , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Corteza Cerebral/efectos de los fármacos , Yoduro de Dimetilfenilpiperazina/farmacología , Exocitosis/fisiología , Masculino , Neostriado/efectos de los fármacos , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , Piridinas/farmacología , Quinolizinas , Ratas , Ratas Sprague-Dawley , Receptores Nicotínicos/efectos de los fármacos , TemperaturaRESUMEN
It has been proposed that the clinical utility of methotrexate (MTX) in the treatment of rheumatoid arthritis may be due, in part, to inhibition of 5-amino imidazole-4-carboxamide ribonucleotide formyltransferase (AICARFT) by polyglutamated forms of MTX. AICARFT is the second folate dependent enzyme in de novo purine biosynthesis. In this study, the effects of MTX on de novo purine biosynthesis as well as total nucleotide pools were evaluated in both the human T cell line, CEM, and phytohemagglutinin-activated normal human T lymphocytes. De novo synthesized purines were metabolically labeled with 14C-glycine after MTX treatment and analyzed by HPLC. In normal T cells, MTX produced a dose-dependent reduction in de novo adenosine and guanosine pools with maximal effects (>50%) at 1 microM MTX. In CEM cells, de novo purine synthesis was almost completely blocked by 1 microM MTX. Total purine pools were also reduced in both cell types after MTX treatment. Since 1 microM MTX caused almost complete growth inhibition in CEM cells, we evaluated whether growth could be reconstituted with exogenous purine bases and pyrimidine nucleosides which can be utilized via salvage pathways. The combination of hypoxanthine and thymidine substantially reversed growth inhibition with 1 microM MTX in CEM cells. Taken together, these results demonstrate that MTX inhibits de novo nucleotide synthesis in T cells and suggest that AICARFT inhibition may be one aspect of the multi-site mechanism of MTX action in the treatment of rheumatoid arthritis.
Asunto(s)
Antimetabolitos Antineoplásicos/farmacología , Metotrexato/farmacología , Nucleótidos/metabolismo , Linfocitos T/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Separación Celular , Cromatografía Líquida de Alta Presión , Glicina/metabolismo , Humanos , Técnicas In Vitro , Marcaje Isotópico , Fitohemaglutininas/farmacología , Nucleótidos de Purina/biosíntesis , Linfocitos T/química , Linfocitos T/efectos de los fármacosRESUMEN
Signal transduction by interleukin-12 (IL-12) requires phosphorylation and activation of STAT4. Direct interaction of the SH2 domain of STAT4 with a phosphotyrosine residue in the IL-12 receptor has been proposed to be required for the subsequent STAT4 phosphorylation. The IL-12 receptor beta2 subunit contains three tyrosine residues in its cytoplasmic domain. To test the hypothesis that one of these tyrosines is involved in binding STAT4, phosphopeptides were synthesized according to the amino acid sequences surrounding each of these tyrosine residues. Only the phosphopeptide containing pTyr800 strongly bound to STAT4 in a cell-free binding assay. When this phosphopeptide was introduced into TALL-104 cells, it blocked IL-12-induced STAT4 phosphorylation by competing with the IL-12 receptor for binding to STAT4. A series of alanine replacements was performed in this phosphopeptide to elucidate which amino acids surrounding the pTyr800 residue are critical for STAT4 binding. To summarize, the site on the IL-12 receptor which binds STAT4 can be described as -T-X-X-G-pY(800)-L-, where the core G-pY(800)-L motif is critical for the binding; the threonine at the pY-4 position has only a minor contribution and X represents amino acids not critical for the binding. These results demonstrate that only a small region of the IL-12 receptor is critically involved in binding STAT4 and suggest the feasibility that small molecule inhibitors could be identified which interfere with IL-12 signal transduction for treatment of autoimmune diseases.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores de Interleucina/metabolismo , Transactivadores/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Proteínas de Unión al ADN/química , Humanos , Técnicas In Vitro , Fosfopéptidos/química , Fosfopéptidos/metabolismo , Fosforilación , Receptores de Interleucina/química , Receptores de Interleucina-12 , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Factor de Transcripción STAT4 , Transducción de Señal , Transactivadores/química , Tirosina/química , Dominios Homologos srcRESUMEN
Lck encodes a 56-kDa protein-tyrosine kinase, predominantly expressed in T lymphocytes, crucial for initiating T cell antigen receptor (TCR) signal transduction pathways, culminating in T cell cytokine gene expression and effector functions. As a consequence of a high-throughput screen for selective, novel inhibitors of p56(lck), an isothiazolone compound was identified, methyl-3-(N-isothiazolone)-2-thiophenecarboxylate(A-125800), which inhibits p56(lck) kinase activity with IC50 = 1-7 microM. Under similar assay conditions, the isothiazolone compound was equipotent in blocking the ZAP-70 tyrosine kinase activity but was 50 to 100 times less potent against the catalytic activities of p38 MAP kinase and c-Jun N-terminal kinase 2alpha. A-125800 blocked activation-dependent TCR tyrosine phosphorylation and intracellular calcium mobilization in Jurkat T cells (IC50 = 35 microM) and blocked T cell proliferation in response to alloantigen (IC50 = 14 microM) and CD3/CD28-induced IL-2 secretion (IC50 = 2.2 microM) in primary T cell cultures. Inhibition of p56(lck )by A-125800 was dose- and time-dependent and was irreversible. A substitution of methylene for the sulfur atom in the isothiazolone ring of the compound completely abrogated the ability to inhibit p56(lck) kinase activity and TCR-dependent signal transduction. Incubation with thiols such as beta-ME or DTT also blocked the ability of the isothiazolone to inhibit p56(lck) kinase activity. LC/MS analysis established the covalent modification of p56(lck) at cysteine residues 378, 465, and 476. Together these data support an inhibitory mechanism, whereby cysteine -SH groups within the p56(lck) catalytic domain react with the isothiazolone ring, leading to ring opening and disulfide bond formation with the p56(lck) enzyme. Loss of p56(lck) activity due to -SH oxidation has been suggested to play a role in the pathology of AIDS. Consequently, a similar mechanism of sulfhydryl oxidation leading to p56(lck) inhibition, described in this report, may occur in the intact T cell and may underlie certain T cell pathologies.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/antagonistas & inhibidores , Tiazoles/farmacología , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/efectos de los fármacos , Catálisis/efectos de los fármacos , Línea Celular , Cisteína/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Datos de Secuencia Molecular , Fosforilación/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/efectos de los fármacos , Compuestos de Sulfhidrilo/farmacología , Tiazoles/metabolismo , Factores de TiempoRESUMEN
We investigated the ability of human recombinant interleukin-7 (IL-7) to enhance cytotoxic T lymphocyte (CTL) activity in vivo using mice infected with herpes simplex virus type-1 (HSV-1). IL-7 or interleukin-2 (IL-2) was administered twice daily to immune naive mice subjected to adoptive transfer of immune T cells after infection with HSV-1. The immunotherapeutic effect was measured by detecting the virus recovered from pinna. Administration of HSV-1 immune T cells to naive mice significantly increased their ability to clear the virus. Twice-daily injections of IL-7 at 200 IU provided an additional 20-fold reduction in virus load, compared with T cell therapy alone (P < 0.0005). Combining IL-2 and T cell therapy provided about a sevenfold reduction compared with T cell therapy alone (P < 0.0009). IL-7 also enhanced the antiviral effects of T cell therapy against HSV-1 through the enhancement of CD8+ CTLs, as observed with IL-2. These results indicate that IL-7 may be used adjunct to adoptive T lymphocyte therapy in modulating human viral diseases and cancer through enhanced immune T cell activities.
Asunto(s)
Adyuvantes Inmunológicos/uso terapéutico , Herpes Simple/terapia , Herpesvirus Humano 1/inmunología , Inmunoterapia Adoptiva , Interleucina-2/uso terapéutico , Interleucina-7/uso terapéutico , Animales , Humanos , Ratones , Proteínas Recombinantes/uso terapéutico , Linfocitos T Citotóxicos/inmunología , Carga ViralRESUMEN
In this report, we describe the discovery and characterization of a novel biarylhydrazone series of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors typified by the prototype WIN 41662 (3-phenyl-N1-[1-(4-pytidyl)pyrimidine]hydrazone). WIN 41662 inhibited PDGF-stimulated autophosphorylation of PDGF receptors from human vascular smooth muscle cells (hVSMC) with an IC50 value of 60 nM. The inhibitor appeared to be competitive with respect to substrate (Mn(2+)-ATP), having a calculated Ki of 15 +/- 5 nM. WIN 41662 was approximately 500-fold more potent in inhibiting the PDGF receptor tyrosine kinase than the p56lck tyrosine kinase. It was inactive against other serine/threonine and tyrosine kinases tested. WIN 41662 produced concentration-dependent inhibition of PDGF-stimulated receptor autophosphorylation in intact hVSMC with an IC50 < 100 nM. Intracellular Ca2+ mobilization and cell proliferation were events that occurred in hVSMC subsequent to PDGF receptor activation. WIN 41662 inhibited PDGF-stimulated Ca2+ mobilization and cell proliferation ([3H]TdR incorporation) with IC50 values of 430 nM and 2.3 microM, respectively. These effects appeared to be specifically related to PDGF receptor tyrosine kinase inhibition since WIN 41662 was not cytotoxic (in vitro) and since WIN 72039, a close structural analog that does not inhibit PDGF receptor tyrosine kinase, also did not inhibit PDGF-stimulated receptor autophosphorylation, Ca2+ mobilization, or hVSMC proliferation. Thus, WIN 41662 is representative of a novel class of selective PDGF receptor tyrosine kinase inhibitors that inhibit PDGF-regulated secondary events in intact cells.
Asunto(s)
Aorta/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Proteínas Tirosina Quinasas/efectos de los fármacos , Piridinas/farmacología , Pirimidinas/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/efectos de los fármacos , Adenosina Trifosfato/farmacología , Calcio/metabolismo , Células Cultivadas/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana EdadRESUMEN
A non-myristylated form (LCK M) of the human T-lymphocyte-specific protein tyrosine kinase (LCK) was produced at high levels in a baculovirus expression system (BVES) using two strategies. First, LCK M was produced by direct expression of a Gly2 --> Ala mutant of LCK. Second, LCK was produced as a glutathione S-transferase (GST) fusion, and LCK M was derived from the fusion protein by cleavage with thrombin. Both recombinant proteins (re-proteins) were produced at 5% of the total protein of infected Spodoptera frugiperda (Sf9) cells and were purified to >95% homogeneity. The enzymatic properties of the re-proteins and their inhibition by protein kinase inhibitors were comparable to the native enzyme (LCK N) derived from Jurkat cells and wild-type LCK derived from the BVES. The high production levels will facilitate the recovery of large quantities of re-protein for use in biochemical and biophysical studies.
Asunto(s)
Baculoviridae/genética , Expresión Génica/genética , Vectores Genéticos/genética , Proteínas Tirosina Quinasas/genética , Linfocitos T/enzimología , Familia-src Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Datos de Secuencia Molecular , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citología , Familia-src Quinasas/química , Familia-src Quinasas/metabolismoRESUMEN
Damnacanthal, an anthraquinone isolated from a plant extract, was found to be a potent, selective inhibitor of p56lck tyrosine kinase activity. The structure, potency, and selectivity of damnacanthal were confirmed by independent synthesis and testing. Damnacanthal exhibited an IC50 of 17 nM for inhibition of p56lck autophosphorylation and an IC50 of 620 nM for phosphorylation of an exogenous peptide by p56lck. Damnacanthal had > 100-fold selectivity for p56lck over the serine/threonine kinases, protein kinase A and protein kinase C, and > 40-fold selectivity for p56lck over four receptor tyrosine kinases. It also demonstrated modest (7-20-fold), but highly statistically significant, selectivity for p56lck over the homologous enzymes p60src and p59fyn. Mechanistic studies demonstrated that damnacanthal was competitive with the peptide binding site, but mixed noncompetitive with the ATP site. Although damnacanthal contains a potentially reactive aldehyde moiety, equilibrium dialysis experiments demonstrated that significant amine formation between damnacanthal and amines occurred only at high concentrations of reactants. However, damnacanthal appeared to bind nonspecifically to membrane lipids and was not active in whole cell tyrosine kinase assays. Damnacanthal is the most potent, selective inhibitor of p56lck tyrosine kinase activity described to date and may represent the starting point for the identification of novel, selective inhibitors of p56lck which are active in whole cell as well as in cell-free systems.
Asunto(s)
Antraquinonas/farmacología , Inhibidores Enzimáticos/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Alcaloides/farmacología , Antraquinonas/síntesis química , Antraquinonas/metabolismo , Relación Dosis-Respuesta a Droga , Cinética , Lípidos/farmacología , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Péptidos/metabolismo , Fosforilación/efectos de los fármacos , Polilisina/metabolismo , Proteínas Serina-Treonina Quinasas/efectos de los fármacos , Proteínas Tirosina Quinasas/efectos de los fármacos , Ribonucleasa Pancreática/metabolismo , Estaurosporina , Familia-src Quinasas/metabolismoRESUMEN
Murine reconstitution assays were used to investigate the effects of recombinant human interleukin-7 (rhIL-7) on myeloid and lymphoid precursors and on bone marrow engraftment. Reconstitution with bone marrow from rhIL-7-treated mice results in a 3.4-fold decrease in total colony-forming unit-spleen (CFU-S) activity (day 9) and an 18.1- and 11.9-fold decrease in its ability to generate thymocytes and splenic B lineage cells, respectively. In contrast, after reconstitution with splenocytes from rhIL-7-treated mice, CFU-S activity increased 23.6-fold (day 9) and the thymocyte and splenic B lineage cell regenerative capacity increased by 4.0- and 3.2-fold, respectively. In addition, CD43low+, B220low+ cells that contain pre-pro-B cells and pro-B cells were expanded two- to threefold and Ig mu-, B220+, CD2- and Ig mu-, B220+, CD2+ B lineage cells were expanded approximately 10-fold and 10- to 45-fold (depending on the tissue examined), respectively, after rhIL-7 treatment. Administration of rhIL-7 to irradiated mice transplanted with bone marrow resulted in accelerated T cell and B cell reconstitution by up to 2-4 weeks. Thus, rhIL-7 administration affects the distribution of myeloid and lymphoid precursors. Moreover, rhIL-7 administration accelerates murine bone marrow cell engraftment and therefore may be useful in reducing the engraftment time in bone marrow transplant patients.
Asunto(s)
Linfocitos B/inmunología , Trasplante de Médula Ósea/fisiología , Interleucina-7/farmacología , Linfocitos/inmunología , Animales , Subgrupos de Linfocitos B/efectos de los fármacos , Subgrupos de Linfocitos B/inmunología , Linfocitos B/citología , Trasplante de Médula Ósea/inmunología , Diferenciación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Humanos , Transfusión de Linfocitos , Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/farmacología , Regeneración , Bazo/efectos de los fármacos , Bazo/inmunología , Factores de TiempoRESUMEN
OBJECTIVE: Since interleukin-7 (IL-7) has been shown to induce monocyte production of IL-1, IL-6, IL-8, and tumor necrosis factor-alpha, we investigated plasma and synovial fluid levels of IL-7 in patients with juvenile rheumatoid arthritis (JRA). METHODS: IL-7 levels were measured using an ELISA in 60 patients with JRA and in 25 patients with other rheumatic disorders (ORD). RESULTS: In patients with systemic JRA, but not in patients with polyarticular or pauciarticular JRA or with ORD, plasma IL-7 levels were significantly higher (p < 0.0001) than those of healthy controls. IL-7 was undetected in all synovial fluid tested. Among patients with systemic JRA, those with persistent systemic symptoms had IL-7 levels significantly higher (p < 0.01) than those of patients in which systemic symptoms subsided. CONCLUSION: Plasma IL-7 levels are elevated in patients with systemic JRA and are associated with the presence and severity of systemic symptoms.
Asunto(s)
Artritis Juvenil/sangre , Interleucina-7/sangre , Adolescente , Artritis Juvenil/metabolismo , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Humanos , Interleucina-6/sangre , Interleucina-7/análisis , Enfermedades Reumáticas/sangre , Enfermedades Reumáticas/metabolismo , Líquido Sinovial/químicaRESUMEN
The T cell antigen receptor complex (CD3/Ti) plays a role in specific antigen recognition as well as in signal transduction, with its surface expression required for the function of several other structurally distinct receptor systems, including CD2, Ly-6(TAP), and Thy-1. In this communication, evidence is presented suggesting an association between the surface expression of CD3/Ti and that of the type 1 interferon (IFN) receptor in a CD4+ murine T cell clone. We tested the proliferative responses and their capacity to be inhibited by type 1 IFN with the wild-type, CD3/Ti-positive T cell clone and its CD3/Ti-negative variants did not respond to specific antigen or anti-CD3 antibody stimulation but they did respond to T cell growth factor (TCGF), stimulation as did the wild-type parental cells. Therefore, the type 1 IFN inhibition of TCGF-stimulated proliferative responses of wild-type and variant cells were compared. Both natural and recombinant type 1 IFNs inhibited TCGF-induced tritiated thymidine (3H-TdR) incorporation in the wild-type T cell clone, with a ID50 of 60-80 U/ml. By contrast, the variants required much higher doses of type 1 IFN. The ID50 with natural murine IFN-beta was 10,000 U/ml, but this same dose of human IFN-alpha A/D gave only a marginal inhibitory effect. Accompanying the loss of IFN responsiveness, these variants also exhibited a loss of high-affinity type 1 IFN receptors. Taken together, these data suggest that the CD3/Ti complex plays a role in the surface expression of the type 1 IFN receptor in a CD4+ T cell clone.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Linfocitos T CD4-Positivos/química , Interferón Tipo I/sangre , Receptores de Antígenos de Linfocitos T/análisis , Receptores de Interferón/análisis , Animales , Línea Celular , Ratones , Ratones Endogámicos A , Valores de ReferenciaRESUMEN
A new p56lck tyrosine kinase inhibitor WIN 61651 [1,4-dihydro-7-(4-methyl-1-piperizinyl)-1-(4-(4-methyl-1-piperi zinyl))phenyl- 4-oxo-3-quinolinecarboxamide) is described. WIN 61651, which is competitive with ATP, demonstrates selectivity for the lymphoid restricted tyrosine kinase p56lck over serine/threonine kinases, such as protein kinase C and protein kinase A, and over some other tyrosine kinases, including erbB2, epidermal growth factor receptor, and insulin receptor; however, it is equipotent for inhibition of p56lck and the platelet derived growth factor receptor tyrosine kinases. WIN 61651 inhibits p56lck activity in cell-free assays, tyrosine kinase activity in a T lymphocytic cell line, and T cell activation, as measured by IL-2 production by purified CD4 positive peripheral blood T lymphocytes and the mixed lymphocyte reaction. WIN 61651 constitutes a new tool for studies on the role for tyrosine kinases in lymphocyte function.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Piperazinas/farmacología , Quinolinas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/enzimología , Familia-src Quinasas/antagonistas & inhibidores , Anticuerpos/farmacología , Complejo CD3/inmunología , Antígenos CD4/inmunología , Células Cultivadas , Humanos , Cinética , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Fosforilación , Linfocitos T/fisiología , Tirosina/metabolismoRESUMEN
p56lck, which is a lymphoid-restricted tyrosine kinase, plays a significant role both in activation of mature T lymphocytes and in T cell development. Recombinant human p56lck was expressed in a baculovirus system and purified to > 95% purity. Purified recombinant p56lck was found to have enzymatic characteristics indistinguishable from those of the native enzyme expressed in the Jurkat human T lymphocytic cell line. The comparisons of recombinant and native p56lck were performed using an assay in which the enzyme was immobilized with antibodies onto the wells of a microtiter plate, enabling in situ purification of the native enzyme. Further characterization of the purified recombinant p56lck revealed significantly different enzyme concentration curves for peptide phosphorylation by immobilized and soluble p56lck. In particular, there was very low activity at low concentrations of enzyme assayed in solution. The activity at low enzyme concentrations could be substantially increased by preincubation of high concentrations of enzyme with ATP prior to dilution for the peptide phosphorylation reaction. We examined the relationship between autophosphorylation and activation for peptide phosphorylation. As for peptide phosphorylation, nonlinear enzyme concentration curves were also observed for autophosphorylation in solution, with very low levels of autophosphorylation at low enzyme concentrations. There was a correlation between the enzyme concentration dependency for autophosphorylation and for preincubation with ATP for maximal enhancement of subsequent peptide phosphorylation. The results suggest that autophosphorylation activates p56lck for phosphorylation of exogenous substrates and that high enzyme concentrations accelerate intermolecular autophosphorylation and enzyme activation.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Baculoviridae/genética , Células Cultivadas , Activación Enzimática , Humanos , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Linfocitos/enzimología , Datos de Secuencia Molecular , Mariposas Nocturnas/citología , Fosforilación , Proteínas Tirosina Quinasas/genética , Proteínas Recombinantes/metabolismoRESUMEN
We investigated the ability of human recombinant interleukin-7 (IL-7) to enhance the immune responses of mice vaccinated with either the alum-associated or liposome-formulated recombinant human immunodeficiency virus (HIV)-envelope protein, env-2-3SF2 (a nonglycosylated denatured gp 120 of HIV-1SF2 produced in genetically engineered yeast). Pathogen-free (C3H) mice were vaccinated on days 0, 14, and 28 with 10 micrograms of either the alum-associated env-2-3SF2 or liposome-formulated env-2-3SF2, both containing a lipophylic muramyl tripeptide, MTP-PE. Liposome-formulated IL-7 (5 micrograms/mouse) or empty liposomes were given on days 7, 14, 21, and 28. Antibody response against the immunized antigen, evaluated on day 21 and day 35 or 42, showed that liposome-formulated antigen induced higher antibody titer than did alum-associated antigen, and these antibody responses can be enhanced by concurrent administration of IL-7 liposomes. Spleen cells were harvested on day 21 and day 35 or 42 to evaluate cytotoxic T lymphocyte responses directed against autologous cells infected with vaccinia virus-expressing HIV-envelope protein. Mice treated with liposome-formulated antigen expressed the highest cytotoxic t-lymphocyte (CTL) activity, regardless of whether IL-7 liposome was given as an immune potentiator. In contrast, spleen cells from mice vaccinated with alum-associated antigen exhibited minimal CTL response, which was enhanced by concurrent IL-7 liposome treatment. Collectively, IL-7 liposome treatment enhanced the antibody production of the alum-associated or liposome-formulated env-2-3SF2, whereas its enhancement of CTL activity was detected only in mice vaccinated with alum-associated antigen.
Asunto(s)
Vacunas contra el SIDA/inmunología , Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adyuvantes Inmunológicos , Productos del Gen env/inmunología , VIH-1/inmunología , Interleucina-7/inmunología , Fosfatidiletanolaminas/inmunología , Vacunas contra el SIDA/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/administración & dosificación , Acetilmuramil-Alanil-Isoglutamina/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Citotoxicidad Inmunológica , Portadores de Fármacos , Anticuerpos Anti-VIH/biosíntesis , Interleucina-7/administración & dosificación , Liposomas , Ratones , Ratones Endogámicos C3H , Fosfatidiletanolaminas/administración & dosificación , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Organismos Libres de Patógenos Específicos , Linfocitos T Citotóxicos/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
These studies investigate the effects of exogenously administered recombinant human IL-7 (rhIL-7) on mouse leukocyte subsets in vivo in normal and tumor-bearing mice. The administration of rhIL-7 to normal mice caused a pronounced leukocytosis (three- to fivefold increase over background) in the spleen and lymph nodes, with B-lineage and T cells, NK cells, and macrophages all being increased. CD8+ T cells increased disproportionately, such that the CD4 to CD8 ratio decreased dramatically. The rhIL-7-induced effects were dose-dependent, increased with duration of treatment, and were reversible after cessation of rhIL-7 administration. T cell number increases after rhIL-7 treatment were primarily a result of an expansion of the peripheral T cell population. Importantly, splenocytes from rhIL-7-treated mice have enhanced proliferative responses to various T cell stimuli in vitro and were able to potentiate an allogeneic CTL response in vivo. The rhIL-7-induced changes in T cell number and the CD4 to CD8 ratio also were observed in mice bearing early Renca renal adenocarcinoma pulmonary metastases, and these changes coincided with up to a 75% reduction in pulmonary metastases. Overall, these results demonstrate that the administration of rhIL-7 to mice profoundly increases the number of B and T cells, and reduces the number of pulmonary metastases. The results also suggest that IL-7 may be useful for restoring lymphoid subsets in immunosuppressed hosts and in enhancing T cell-mediated immune responses. Such effects may be useful in the treatment of microbial diseases and cancer.
Asunto(s)
Linfocitos B/inmunología , Interleucina-7/farmacología , Metástasis de la Neoplasia/inmunología , Subgrupos de Linfocitos T/inmunología , Animales , Antígenos de Diferenciación/metabolismo , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/secundario , Carcinoma de Células Renales/terapia , Citotoxicidad Inmunológica , Relación Dosis-Respuesta Inmunológica , Humanos , Interleucina-7/administración & dosificación , Neoplasias Renales/inmunología , Neoplasias Renales/secundario , Neoplasias Renales/terapia , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fenotipo , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/farmacología , Bazo/inmunologíaRESUMEN
We investigated the biological activity of human recombinant interleukin-7 (rhIL-7) on guinea-pig cells in vitro and in vivo. RhIL-7 can stimulate plastic non-adherent cells of guinea-pigs to proliferate in vitro at a degree comparable to that of human cells. Administration of rhIL-7 to guinea-pigs significantly increased their white blood cell concentration, specifically the lymphocyte population. The effect of rhIL-7 on circulating blood cells was dose dependent, in that 14 doses of twice daily subcutaneous injections at 8 x 10(5) U kg-1 and 2.4 x 10(6) U kg-1 rhIL-7 increased peripheral blood lymphocyte concentrations by 38% (p = 0.047) and 139% (p = 0.0005), respectively, compared with the placebo group. Two weekly doses of rhIL-7 liposome (5.6 x 10(6) U kg-1 dose, equivalent to 11.2 x 10(6) U kg-1 total dose) elicited a similar effect on the lymphocyte population, comparable to that of the twice daily administrations of 8 x 10(5) U kg-1 soluble rhIL-7 for 7 days (7 days x two doses/day x 8 x 10(5) U kg-1 = 11.2 x 10(6) U kg-1 total dose). However, the increased lymphocyte count induced by rhIL-7 administration did not protect guinea-pigs from the primary herpes simplex virus (HSV)-2 infection. Only when rhIL-7 was given in association with HSV-antigen gD in an antigen-specific mode did it exhibit an enhanced protective effect against the sublethal dose of genital HSV-2 challenge.(ABSTRACT TRUNCATED AT 250 WORDS)