Induction and activation of mitogen-activated protein kinases of human lymphocytes as one of the signaling pathways of the immunomodulatory effects of morphine sulfate.

Morphine sulfate causes immunomodulatory and immunosuppressive effects in human. In this study, the signaling pathway involved in these morphine effects was studied. Addition of morphine sulfate to human CEMx174 lymphocytic cells resulted in increased expression of mitogen-activated protein kinase cascade proteins. Morphine enhanced the cellular levels of ERK1 (44 kDa), ERK2 (42 kDa), a 54-kDa ERK, MEK1 (45 kDa), and MEKK (78 kDa). A time-dependent increase in the activated (Thr and Tyr dually phosphorylated) state of ERK1 and ERK2 was also observed. Naloxone, a morphine antagonist, reversed the observed morphine effects, implicating a micro opioid receptor-mediated process. These findings suggest that mitogen-activated protein kinases are important intermediates in signal transduction pathways initiated by morphine receptors in immune cells.

SIV infection of macaques: a model for studying AIDS and drug abuse.

Rhesus monkeys (Macaca mulatta) and the simian immunodeficiency virus (SIV) were used as an animal model system to evaluate longitudinally the effects of opioid dependence on the development of AIDS. Results have shown that in addition to weakening the host's T cell and PMN functions, chronic opioid treatment of SIVmac239-infected animals caused (a) an increased virus replication rate, (b) an increased rate of viral mutation, (c) a nascent humoral (antibody) response against mutated autologous strains of virus, (d) an alteration of CD8 + cell-mediated immunity toward the mutant virus, (e) an increased tolerance of AZT among the infectious virus and (f) a shorter life span for the infected animals. These results suggest that chronic administration of opioids alters general fundamental aspects of the AIDS viremia.

Mu opioid receptor gene expression in immune cells.

We have previously demonstrated that administering morphine sulfate to rhesus monkeys alters the cell-mediated as well as humoral immune responses of these primates. Furthermore, morphine treatment greatly reduces the chemotactic and phagocytotic activities of primate polymorphonuclear (PMN) cells. The present study describes the identification and isolation of mRNA encoding the mu opioid receptor gene sequence from human and monkey immune cells. Through the use of primer sequences designed from the human brain mu opioid receptor cDNA sequence, specific opioid receptor segments in mRNA transcripts were amplified, cloned, and sequenced. The mu opioid receptor gene was therefore found expressed in the following cell types: CEM x174 (a hybrid of human T and B cells), Raji (human B cells), human CD4+ cells, human monocytes/macrophages, human PMN, monkey peripheral blood mononuclear cells (PBMC), and monkey PMN. These studies present the first evidence to demonstrate that cells of human and monkey immune systems constitutively express mu opioid receptor mRNA.

Expression of kappa opioid receptors in human and monkey lymphocytes.

mRNA encoding the kappa opioid receptor gene sequence was identified and isolated from various human lymphocytic cells: CEM x174 (a hybrid of T and B origin) cells, Jurkat-T4 cells, human peripheral blood mononuclear cells (PBMC) and purified CD4+ cells. Analyzing the cDNA sequences of RNA transcripts spanning the putative second extracellular loop, which has reported dynorphin specificity, and the seventh transmembrane domain revealed a 100% and 95% homology in amino acid sequence to corresponding kappa opioid receptor sequences in human placenta and rat brain, respectively. Expression of a similar kappa opioid receptor sequence could be detected in normal monkey PBMC but not in monkey PBMC in which the CD4+/CD8+ cell ratio (or CD4+ cell number) was significantly reduced due to prolonged SIV infection. These findings suggest that human and monkey lymphocytes constitutively express kappa opioid receptor mRNA.

Inhibition of simian immunodeficiency virus (SIV) replication by CD8+ cells of SIV-infected rhesus macaques: implications for immunopathogenesis.

The ability of the CD8+ cells from simian immunodeficiency virus (SIV)-infected rhesus macaques to inhibit SIV replication was investigated. Inhibition was produced by a heat-stable soluble factor of molecular size greater than 10kDa. CD8+ supernatants from some macaques were found not only to suppress SIV growth but also to be cytolytic toward both infected and uninfected CD4+ cells. Such indiscriminate CD8+ cell-mediated cell killing may therefore account for DC4+ cell depletion in certain SIV-infected macaques.

Delta opioid receptor gene expression in lymphocytes.

Previous studies have shown that morphine stimulates simian immunodeficiency virus (SIV) replication in SIV-infected human CEM x174 cells as well as in monkey lymphocytes through a mechanism of delaying the lysis of infected cells (Biochem. Biophys. Res. Commun. 195:1165-1173, 1993). The present study describes the identification of brain-like opioid receptor sequences in RNA transcripts of both CEM x174 cells and monkey lymphocytes. Study of the gene sequence of a lymphocyte opioid receptor encompassing the third transmembrane domain and the third cytoplasmic loop indicates a 96% homology in amino acid composition to the delta opioid receptor in brain cells. Expression of such an opioid receptor sequence in lymphocytic cells is constitutive, since it could be detected in both saline-treated and morphine-treated monkeys as well as in morphine-treated monkeys after detoxification.

Emergence of antigenic variants of simian immunodeficiency virus (SIVmac) in a seronegative macaque after SIVmac239 infection.

Infection with the macaque strain of the simian immunodeficiency virus (SIVmac) induces simian immunodeficiency syndrome in rhesus macaques. This report describes the isolation and identification of antigenic variants of SIVmac in one of the infected monkeys (macaque #22803). Eight naive rhesus monkeys were inoculated with a titered viral stock of the molecularly cloned SIVmac239. Standard serological analysis revealed that all but two were seroconverted. Western blot analysis confirmed the seronegativity of macaque #22803. In addition, sera recovered from this monkey were not able to neutralize the parent SIVmac239. However, virus could be isolated from all of the infected animals, including macaque #22803. Sera recovered were reactive to the autologous virus. The results suggest that the virus from macaque #22803 may have undergone extensive antigenic shift in vivo. To test this hypothesis, a portion of the gag gene was amplified by the polymerase chain reaction (PCR), cloned, and sequenced. Sequence analysis revealed amino acid changes that were clustered between amino acids 200-245. Evaluation of the possible selective pressures contributing to the observed viral mutation revealed that in comparison with the other SIVmac239-infected monkeys, macaque #22803 produced an unusually high T cell proliferative response toward mitogen stimulation before infection, and continued to display a persistently high plasma viremia titer after infection.

Increased replication of simian immunodeficiency virus in CEM x174 cells by morphine sulfate.

Infection with simian immunodeficiency virus induces cytopathic effects on CEM x174 cells in vitro. Syncytium formation of SIV-infected CEM x174 cells was significantly enhanced in the presence of morphine sulfate, with a concomitant increase in the activity of cellular reverse transcriptase and in the expression of SIV p27 core antigen. Parallel establishment of the viability of the morphine-treated cells indicates that the short-acting opioid protects against cell lysis induced by SIV so that replication and production of SIV particles continued and exceeded those without morphine treatment. This delayed cell lysis induced by morphine as seen in vitro correlated with an in vivo observation that peripheral blood mononuclear cells isolated from morphine-treated rhesus macaques displayed a less degree of programmed cell death by apoptosis during early stages of SIVmac infection. These studies suggest that the modification of the biological properties of HIV-infected cells by morphine sulfate may be one of the mechanisms by which opioids exacerbate the progression of HIV in drug abusers.

Inhibition of the replication of native and 3'-azido-2',3'-dideoxythymidine (AZT)-resistant simian immunodeficiency virus (SIV) by 3-nitrosobenzamide.

The 3-nitrosobenzamide (NOBA) drug abolishes SIV replication sharply at 20 microM concentration when CEM x 174 cells are preincubated for 1 h with the drug prior to viral infection. Treatment of CEM x 174 cells with 20 microM NOBA resulted in the inhibition of the synthesis of the DNA sequence coding for the gag gene, as determined by the PCR technique. Cell viability was directly proportional to the antiviral action of NOBA. Replication of AZT-resistant SIV 23740 in MMU 23740 cells in vitro was suppressed by NOBA in a concentration-dependent manner without significant effects on cell viability. Reverse transcriptase activity of SIVmac239 was unaffected by NOBA up to 800 microM concentration. Preincubation of two SIV strains with NOBA completely abolished their infectivity in human PHA-PBL cells. Replication of two strains of SIV in PHA-PBL cells was also inhibited by NOBA.

Opioid dependency and T-helper cell functions in rhesus monkey.

Administration of morphine sulfate to rhesus monkeys may activate the quiescent lymphocyte for proliferation, induce a transient increase in the T cell proliferative response to mitogens, and cause an enhanced interleukin-2 release from the mitogen-stimulated lymphocytes. However, longitudinal studies of the animals dependent upon morphine or L-a-acetyl-methadol, a long-acting opioid, revealed an overall immunosuppression of T helper functions. In vitro studies using morphine and its antagonist naloxone suggested that the immunosuppression was not a result of a direct interaction between the opioids and conventional opiate receptors which might have been present on the lymphocytes. The studies also showed the importance of measuring [3H]thymidine incorporation rather than its uptake into cells to assess T cell activation.

Effects of in vivo and in vitro administration of morphine sulfate upon rhesus macaque polymorphonuclear cell phagocytosis and chemotaxis.

The effects of morphine administration were evaluated upon the polymorphonuclear (PMN) cell activities of rhesus macaques. Sixteen animals were used in the study. Seven macaques were treated with saline and nine animals were administered morphine in the form of morphine sulfate, following a control period of saline injections. The dosage regime was initiated at 1 mg of morphine sulfate per kg of b.wt., 3 times daily, with incremental steps to a stable dose of 5 mg/kg, 3 times daily. PMN cells prepared from morphine-treated animals showed a marked, transient reduction in their ability to kill ingested yeast blastospores, although the ingestive capacity of the PMN cells was not impaired. PMN cells prepared from three of the nine morphine-treated animals showed a transient reduction in chemotaxis toward a complement-derived chemotactic factor. In vitro studies on PMN cells prepared from morphine-naive animals suggested that the killing activity and the chemotaxis of the cells were reduced by treatment with 50 pM morphine sulfate, but not with 50 fmol of morphine sulfate.

Detection of simian immunodeficiency virus RNA from infected rhesus macaques by the polymerase chain reaction.

A rapid method for the detection of simian immunodeficiency virus (SIV) RNA from peripheral blood mononuclear cells (PBMC) of experimentally infected rhesus macaques by the polymerase chain reaction (PCR) is reported. The PCR was carried out with a complementary DNA (cDNA) template using 3 pairs of primers that were designed to anneal to homologous sequences in conserved regions of 3 molecular clones of SIVmac. The specificity of the primers was confirmed by performing the PCR with template DNA from the 3 molecular clones. SIV-specific RNA was detected from 30 and 50 infected PBMC/6.25 x 10(5) PBMC of two animals.

Evidence for neurotransmitter abnormalities related to seizure activity in the epileptic baboon.

The experimental evidence that indicates that alterations in neurotransmitter activity exacerbate or diminish seizure responses to intermittent light stimulation in the Papio papio model of epilepsy is reviewed. Studies of the effects of drugs known to modify the synthesis, storage release, or activity of known or putative neurotransmitters are included. Catecholamines, to a lesser extent the indolamine, serotonin, and the inhibitory transmitter gamma-aminobutyric acid all alter seizures as do less well understood intrinsic hormones and pentapeptides. The anticonvulsant profile of the model is briefly reviewed. It is concluded that there is as yet no concrete evidence of an intrinsic deficit in any of the neurotransmitters to which the genetic photosensitivity of this model can be attributed.