RESUMO
Mitogen activation of human T-lymphocytes induces a morphine-binding site. Morphine binding is displaceable by beta-endorphin (1--31) and (--)-naloxone but not DAMGO. This site is not stereoselective for (--)-morphine. T-lymphocytes, expressing this binding site, were assayed by reverse-transcription polymerase chain reaction (RT-PCR) for expression of hMOR-1 mRNA. Several primer sets were used and each assay compared with cells known to express human or mouse MOR-1 mRNA. Neither hMOR-1 nor any homologous receptor was detected in human T-lymphocytes. Therefore, the morphine-binding site on mitogen-activated T-lymphocytes is unlikely to be closely related to hMOR-1.
Assuntos
Morfina/metabolismo , Entorpecentes/metabolismo , Receptores Opioides mu/metabolismo , Linfócitos T/metabolismo , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Cálcio/metabolismo , Primers do DNA/genética , Primers do DNA/imunologia , Primers do DNA/metabolismo , Humanos , Interleucina-2/farmacocinética , Morfina/imunologia , Entorpecentes/imunologia , RNA Mensageiro/genética , RNA Mensageiro/imunologia , RNA Mensageiro/metabolismo , Receptores Opioides mu/genética , Receptores Opioides mu/imunologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sódio/metabolismo , Linfócitos T/imunologiaRESUMO
The direct actions of opiates on the mammalian immune system depend on the existence of ligand binding sites either on the surface of the affected cell or in the interior of the cell. With the cloning of various opiate receptors from neuronal tissue, numerous researchers have screened leukocyte cDNA libraries for the expression of these receptors with some positive results. However, the pattern of expression of neuronal opiate receptors in the cellular immune system does not completely explain the biological action of opiates there. Several possibilities could account for this non-congruence including differential expression of the receptors as determined by such factors as cell population or prior history of the cells; the existence of sequence modified versions of the neuronal receptors such that the amplification methods miss their presence; or the opiates act by a different, non-receptor mechanism in the cellular immune system.
Assuntos
Sistema Imunitário/química , Morfina/imunologia , Entorpecentes/imunologia , Neuroimunomodulação/imunologia , Receptores Opioides/imunologia , Animais , Sítios de Ligação/imunologia , Regulação da Expressão Gênica/imunologia , Humanos , Morfina/metabolismo , Entorpecentes/metabolismo , Receptores Opioides/genética , Receptores Opioides/metabolismoRESUMO
The Huntington disease (HD) phenotype is associated with expansion of a trinucleotide repeat in the IT15 gene, which is predicted to encode a 348-kDa protein named huntington. We used polyclonal and monoclonal anti-fusion protein antibodies to identify native huntingtin in rat, monkey, and human. Western blots revealed a protein with the expected molecular weight which is present in the soluble fraction of rat and monkey brain tissues and lymphoblastoid cells from control cases. In lymphoblastoid cell lines from juvenile-onset heterozygote HD cases, both normal and mutant huntingtin are expressed, and increasing repeat expansion leads to lower levels of the mutant protein. Immunocytochemistry indicates that huntingtin is located in neurons throughout the brain, with the highest levels evident in larger neurons. In the human striatum, huntingtin is enriched in a patch-like distribution, potentially corresponding to the first areas affected in HD. Subcellular localization of huntingtin is consistent with a cytosolic protein primarily found in somatodendritic regions. Huntingtin appears to particularly associate with microtubules, although some is also associated with synaptic vesicles. On the basis of the localization of huntingtin in association with microtubules, we speculate that the mutation impairs the cytoskeletal anchoring or transport of mitochondria, vesicles, or other organelles or molecules.
Assuntos
Química Encefálica , Células-Tronco Hematopoéticas/química , Doença de Huntington , Linfócitos/química , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas Nucleares/isolamento & purificação , Animais , Encéfalo/citologia , Compartimento Celular , Linhagem Celular , Citosol/química , Haplorrinos , Humanos , Proteína Huntingtina , Imuno-Histoquímica , Microscopia Imunoeletrônica , Proteínas do Tecido Nervoso/imunologia , Neurônios/química , Proteínas Nucleares/imunologia , Ratos , Proteínas Recombinantes de Fusão/imunologia , Distribuição TecidualAssuntos
Ativação Linfocitária/efeitos dos fármacos , Mitógenos/farmacologia , Morfina/farmacologia , Receptores Opioides/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Analgésicos/farmacologia , Células Cultivadas , Meios de Cultura , Endorfinas/farmacologia , Ala(2)-MePhe(4)-Gly(5)-Encefalina , Encefalinas/farmacologia , Humanos , Interleucina-2/farmacologia , Ligantes , Naloxona/farmacologia , Fito-Hemaglutininas/farmacologia , beta-Endorfina/farmacologiaRESUMO
Nonneuronal cells, such as the human T lymphocyte, react directly with opiates in vitro causing significant alterations in the metabolism of these cells. Morphine and cocaine, for example, can modulate the repair of DNA damage caused by ultraviolet light (UVC)--morphine in the negative direction and cocaine in the positive. The mechanism by which these drugs cause these metabolic changes is not yet known, but a simple receptor mechanism such as is found in the central nervous system (CNS) can not be demonstrated. Binding studies using lymphocyte membrane preparations or whole cells do not support the premise that T lymphocytes have opiate binding sites with specificities comparable to those identified in the CNS--the mu, delta and kappa receptors. Even without knowing the mechanism for the opiate-induced metabolic changes, the alterations can be used as the basis for a test of the genetics of opiate metabolism. If the interindividual variation in the opiate-induced repair response is greater than the intraindividual variation as assessed by repeated measures on the same subject, it may be possible to utilize this assay in the classic sorts of family or twin studies to determine the genetic component of the response to opiates.
Assuntos
Transtornos Relacionados ao Uso de Opioides/genética , Receptores Opioides/genética , Linfócitos T/efeitos dos fármacos , Linhagem Celular , Cocaína/farmacologia , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Humanos , Ativação Linfocitária/efeitos dos fármacos , Entorpecentes/farmacologia , Transtornos Relacionados ao Uso de Substâncias/genéticaAssuntos
Cromossomos/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Tolerância Imunológica/efeitos dos fármacos , Entorpecentes/farmacologia , Animais , Formação de Anticorpos/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Dano ao DNA , Suscetibilidade a Doenças/induzido quimicamente , Suscetibilidade a Doenças/imunologia , Feminino , Infecções por HIV/complicações , Dependência de Heroína/complicações , Dependência de Heroína/imunologia , Humanos , Síndromes de Imunodeficiência/induzido quimicamente , Recém-Nascido , Ativação Linfocitária/efeitos dos fármacos , Macaca mulatta , Testes de Mutagenicidade , Entorpecentes/efeitos adversos , Neoplasias/etiologia , Gravidez , Receptores Opioides/efeitos dos fármacos , Troca de Cromátide Irmã/efeitos dos fármacosAssuntos
Cocaína/farmacologia , Dependência de Heroína/imunologia , Heroína/farmacologia , Sistema Imunitário/efeitos dos fármacos , Formação de Roseta , Transtornos Relacionados ao Uso de Substâncias/imunologia , Antígenos CD/biossíntese , Relação CD4-CD8/efeitos dos fármacos , Interações Medicamentosas , Etanol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Heroína/antagonistas & inibidores , Dependência de Heroína/complicações , Humanos , Morfina/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Subpopulações de Linfócitos T/imunologiaRESUMO
To refine previous studies of chromosome damage (CD) and sister-chromatid exchanges (SCE) in heroin addicts, we applied new methods developed in our laboratory to enhance detection of the cytogenetic effects of low-level radiation exposure in hospital workers. For CD analysis, we applied our thymidine-fluorodeoxyuridine-caffeine (TFC) enhancement procedure in which cells at setup receive 1 x 10(-7) M fluorodeoxyuridine to inhibit thymidylate synthetase and 4 X 10(-5) M thymidine to satisfy the induced requirement, and then in G2 receive 2.2 mM caffeine to modulate DNA repair. For SCE enhancement, caffeine treatment was initiated in G1 at 19 h before harvest. Using both standard and enhanced procedures for CD and SCE analysis, blood samples were evaluated from 20 street heroin addicts and 22 controls. Standard 2-day CD and 3-day SCE assays showed small, insignificant genotoxic increases in addicts while the enhanced CD and SCE assays showed highly significant increases. Most CD events were in the form of chromatid and chromosome breaks. There were no rings and only a few dicentrics were observed in the TFC-enhanced cultures. Although quadriradials are rare, 10 were found in addict TFC-cultures and 3 in control TFC-cultures. With the standard CD assay, the mean number of chromosome breaks per 100 cells was 0.727 for controls and 1.056 for addicts (not significant). With the TFC-enhanced assay, the same measure showed 1.483 chromosome breaks for controls and 5.143 for addicts (highly significant, ANOVA: p less than 0.0001). A highly significant difference was also observed for chromatid-type damage with the TFC-enhanced assay (chromatid breaks per 100 cells: 16.793 for controls; 48.191 for addicts). The SCE data also showed significant differences with the enhanced assay. Scoring 25 cells/condition, standard SCE cultures showed 10.892 SCE/cell for controls and 11.732 SCE/cell for addicts (not significant). With CAF enhancement there were 13.08 SCE/cell for controls and 17.05 SCE/cell for addicts (ANOVA: p less than 0.008). These findings indicate that detection of CD and SCE effects can be significantly enhanced by the use of these new procedures. The finding of greatly increased chromatid damage in the addicts with the TFC procedure suggests that at least part of the CD detected occurred in vitro and is not a product of prior in vivo damage. Therefore exposure to this drug and perhaps other environmental agents may not only leave a residue of DNA or chromosome damage but may also induce a sensitivity to further genotoxic damage that is revealed by using the enhanced procedures.
Assuntos
Aberrações Cromossômicas , Dano ao DNA , Dependência de Heroína/genética , Troca de Cromátide Irmã , Análise de Variância , Células Cultivadas , Dependência de Heroína/sangue , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/patologia , Valores de ReferênciaRESUMO
Our laboratories have shown that opiate addicts have higher chromosome damage and sister chromatid exchange frequencies, and that these effects are more pronounced in the addicts when we employ enhanced culturing assays developed in our laboratory. We have also demonstrated that opiates diminish DNA repair capacity and reduce immunoresponsiveness as measured by T-cell E-rosetting and other assays. These interactions of opiates with T-lymphocytes may regulate cell metabolism and could thereby be responsible for the sensitivity of cells from opiate addicts to both genotoxic damage and immunological effects.
Assuntos
Anormalidades Induzidas por Medicamentos/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Entorpecentes/efeitos adversos , Transtornos Relacionados ao Uso de Opioides/genética , Receptores Opioides/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Feminino , Dependência de Heroína/genética , Humanos , Tolerância Imunológica/genética , Recém-Nascido , Gravidez , Fatores de Risco , Troca de Cromátide Irmã/efeitos dos fármacosRESUMO
Naloxone binding to T lymphocytes seems to occur both at the outer cell surface and in the interior of the cell. The binding sites on the outer membrane appear from previous work to be of low affinity and to be displaced by morphine only at very high concentrations. Permeable cells seem to express both high and low affinity binding sites in their interiors. Morphine readily displaces naloxone from this high affinity site, suggesting that this site may be the long sought after morphine receptor responsible for morphine's naloxone reversible actions on the human T lymphocyte. Do these results suggest that the opiates need to enter the lymphocyte before finding a receptive binding site to initiate their biological activities? Not necessarily. In isolating the lymphocytes, the surface opiate binding sites may be lost because of the exhaustive washing procedures needed for cell purification. The internal receptors may just represent new receptors on their way to the cell surface or used receptors remaining after endocytosis. Or they may indeed represent the true site of action of the opiates on the lymphocyte. Fractionation experiments are currently underway to distinguish between these possibilities.
Assuntos
Sistema Imunitário/metabolismo , Receptores Opioides/metabolismo , Animais , Humanos , Sistema Imunitário/citologia , Naloxona/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
Incubation of human T lymphocytes with saturating concentrations of combinations of certain anti-CD2 and -CD4 mAb results in reciprocal down-regulation of the cell surface density expression of the respective CD molecules. Such reciprocal down-regulation occurs at 0 degrees C in the presence of sodium azide and appears selective for CD2 and CD4 molecules because mAb identifying various other CD T cell surface molecules (anti-Leu2a, -OK-CLL, -W6/32, -beta 2-microglobulin, -4B4) do not modulate CD2 or CD4 R density, and because anti-CD2 mAb (anti-OKT11 and -D66 clone-1) do not alter CD8 R density (anti-OKT8, -Leu2a) and vice versa. Down-regulation of CD2 by mAb specific to CD4 is epitope-specific but does not vary on the basis of the antibody isotype used. The anti-CD4 mAb, Leu3a, was the strongest CD2 down-regulator examined followed by OKT4F. mAb specific to other CD4 epitopes (B, C, D, and E) caused only slight down-regulation of CD2 expression whereas anti-OKT4 and -OKT4A mAb had no significant regulatory effect. Also, mAb specific to the 9.6 (anti-OKT11) and D66 (anti-D66 clone 1) epitopes of the CD2 molecule down-regulated CD4 density detectable with Leu3a, OKT4, and OKT4A anti-CD4 mAb. Down-regulation of CD2 by anti-CD4 mAb also occurred with the transformed T cell line, KE-37, which demonstrates that such effects can occur without mononuclear phagocytic accessory cells. From these data it can be concluded that important T cell immunoregulatory signals may be transmitted intramembranally between CD2 and CD4 glycoproteins.
Assuntos
Anticorpos Monoclonais/fisiologia , Antígenos de Diferenciação de Linfócitos T/análise , Citometria de Fluxo , Linfócitos T/classificação , Adulto , Reações Antígeno-Anticorpo , Humanos , Linfócitos T/análise , Linfócitos T Citotóxicos/análise , Linfócitos T Auxiliares-Indutores/análise , Linfócitos T Reguladores/análiseRESUMO
Purified T lymphocytes have a specific binding site for naloxone, the opiate antagonist. The KD for the site was 50.6 +/- 2.4 nM, while the Hill coefficient (n) was 1.67 +/- 0.16, indicating a degree of positive cooperativity of ligand binding. The bound naloxone was partially displaceable by various opiate agonists including morphine (56%), beta-endorphin (61%), met5- and leu5-enkephalin (40% each), [D-ala2, D-leu5]-enkephalin (78%) and [D-ala2, D-leu5]-enkephalinamide (66%). Virtually all of the binding capacity was recovered in the particulate membrane fraction after sonic lysis of the cells. There was great interindividual variability in Bmax between samples, suggesting a possible mechanistic basis for the variability in drug action seen between different individuals.
Assuntos
Naloxona/metabolismo , Receptores Opioides/metabolismo , Linfócitos T/metabolismo , Ligação Competitiva , Plaquetas/metabolismo , Meios de Cultura , Encefalina Leucina/metabolismo , Encefalina Metionina/metabolismo , Glutationa/farmacologia , Humanos , Cinética , Morfina/metabolismo , Sonicação , beta-Endorfina/metabolismoAssuntos
Antígenos de Superfície/imunologia , Entorpecentes/farmacologia , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Transtornos Relacionados ao Uso de Substâncias/imunologia , Humanos , Capeamento Imunológico/efeitos dos fármacos , Modelos Biológicos , Morfina/farmacologia , Receptores de Antígenos de Linfócitos T/fisiologia , Formação de Roseta , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologiaRESUMO
Simultaneous and independent use of cocaine and alcohol by heroin addicts was shown to variably modulate the ability of their T cells to form E-rosettes with sheep erythrocytes (E). As reported previously, the percentages of E-rosette-forming T cells of both active and total types were depressed in association with heroin addiction. We show here that the kinetic curve of the rate of E-rosette formation is also depressed by heroin use and that the use of cocaine but not alcohol by heroin addicts reverses depression of E-rosette formation by heroin. The percentages of E-rosette-forming T cells from the bloods of heroin addicts who used both alcohol and cocaine, as well as the kinetic rate curves of E-rosette formation, were intermediate between the essentially normal levels found for heroin addicts who used cocaine and the severely depressed levels evident for users of heroin alone or heroin plus alcohol. Modulation of the levels of E-rosette formation by alcohol used in conjunction with cocaine and/or heroin was variably dose dependent. Polydrug effects evident by analyses of E-rosette formation were not seen when the percentages of lymphocytes reactive with LYT-3 (anti-E-receptor, 9.6 epitope) and OKT-3 (anti-total T cell) monoclonal antibodies were assessed cytofluorometrically, although the data suggested that subnormal percentages of LYT-3+ T cells were present when heroin addicts also used cocaine. These findings are relevant to basic understanding of T-cell physiology from a neuroimmunological perspective and also suggest ways that addictive drugs may modulate the immunocompetence of drug addicts.
Assuntos
Alcoolismo/imunologia , Cocaína , Dependência de Heroína/imunologia , Transtornos Relacionados ao Uso de Substâncias/imunologia , Linfócitos T/imunologia , Adulto , Antígenos de Diferenciação de Linfócitos T , Antígenos de Superfície/análise , Interações Medicamentosas , Humanos , Imunidade Celular , Formação de RosetaAssuntos
Reparo do DNA/efeitos dos fármacos , Dependência de Heroína/genética , Entorpecentes/efeitos adversos , Adoção , DNA/efeitos da radiação , Dano ao DNA , Doenças em Gêmeos/genética , Ligação Genética , Dependência de Heroína/imunologia , Dependência de Heroína/terapia , Humanos , Metadona/uso terapêutico , Entorpecentes/farmacologia , Formação de Roseta , Troca de Cromátide Irmã/efeitos dos fármacos , Raios UltravioletaRESUMO
Two kinetic assays were developed to assess opiate effects on rates of T cell E-rosetting. The first adopted the thermal conditions of active E-rosetting assays (varying between 37 and 23 C) whereas the second incorporated cooler thermal conditions (varying between 0 and 29 C). In vitro treatment of lymphocytes with morphine depressed E-receptor levels and E-rosetting in both assays. With the 0-29 C procedure early stages of E-rosette formation were characterized by phase transition kinetics indicative of sequential gain and loss of E-rosettes. Assay thermal and erythrocyte (E) to T cell contact conditions, and the inclusion of morphine during E-rosetting, were independent variables that coordinately modulated the expression of phase transitions. Phase transitions were also noted during capping of total T cell E-rosettes at 37 C. The reason for phase transitions appears to be that T cells undergo sequential cycling of E-receptors, increasing because of the new expression of dormant E-receptors as the result of E-receptor microdisplacement and decreasing because E-rosettes are lost owing to patching and capping processes. According to this construction of the E-rosetting process, morphine inhibits E-rosetting and modulates expression of phase transitions by interfering with E-receptor microdisplacement processes. Presumably this interference by morphine is mediated through alteration of membrane fluidity and promotion of E-receptor coupling (and/or inhibition of uncoupling) to a transducer-effector component within the cell membrane. These findings and conclusions are specifically relevant to immunoregulatory processes and are also helpful for understanding the general nature of biological and physiological responses associated with receptor-ligand interactions.
Assuntos
Imunossupressores/farmacologia , Morfina/farmacologia , Formação de Roseta , Linfócitos T/imunologia , Eritrócitos/metabolismo , Humanos , Capeamento Imunológico/efeitos dos fármacos , Cinética , Receptores de Antígenos/efeitos dos fármacos , Formação de Roseta/métodos , Linfócitos T/efeitos dos fármacosRESUMO
Ethanol use by pregnant women increased, in a dose-dependent manner, the rate of sheep erythrocyte rosette (E-rosette) formation with T lymphocytes. The time curve for E-rosette formation by T cells from nondrinking subjects was biphasic, with a rapid formation of half the E-rosettes within the first 16 min, followed by a much slower rate for E-rosette formation until the maximal T-cell percentage was reached overnight. For pregnant drinkers, greater than 85% of the E-rosettes formed during the initial rate period, with a concomitant smaller number forming during the overnight incubation. Despite the faster initial rate of E-rosette formation in the drinking subjects, the total percentage T cells was the same for both groups. Other demographic factors, like tobacco or marijuana use, or trimester, did not significantly contribute to the observed differences. An increase in the rate of E rosetting was also obtained by incubating lymphocytes from nondrinkers overnight in physiologically attainable concentrations of ethanol (less than or equal to 0.1%). These results demonstrate that drinking by pregnant women, even at relatively moderate levels (2 oz/week absolute ethanol), causes alterations in their cellular immune systems. With the ability of ethanol to cross the placental barrier and persist in utero, it is apparent that these levels of ethanol have the potential to affect the developing fetal immune system.