David A. Shafer

Binding of naloxone to human T lymphocytes

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.

Coordinate and independent effects of heroin, cocaine, and alcohol abuse on T-cell E-rosette formation and antigenic marker expression

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.

A Comparison of the Karyotypes of Six Species of the Genus Macaca and a Species of the Genus Cercocebus

Karyotypes from 72-hour whole blood cultures were compared for six species of macaques (Macaca arctoides, M. fascicularis, M. mulatta, M. nemestrina, M. nigra, and M. radiata) and one species of mangabey (Cercocebus atys). G-bands, sequential C-bands, and late replication patterns were studied. Results showed a variation in a single chromosome pair which differentiated C. atys from the macaques. Heteromorphic variation in silver stained nucleolar organizing regions was seen between and within individuals. This data supports previous work showing the highly conserved nature of the chromosomes of the subfamily Cercopithecus.

Parallel increases in sister-chromatid exchanges at base level and with UV treatment in human opiate users.

The SCE base level frequency and SCE levels induced by far-UV (254 nm) treatment of cells in early G1 and early S phases of the cell cycle were significantly higher in leukocytes from heroin addicts as compared to controls. The increased SCE levels in addicts was greatest at base level and smallest after UV irradiation of cells in S phase. These results corroborate and extend our previous findings of increased chromosome damage and reduced DNA-repair synthesis in heroin users. Since opiates do not directly damage DNA, the elevated cytogenetic effects associated with opiate use probably arise from secondary promotional effects related to opiate-mediated alterations in leukocyte metabolism.

Biogenetic effects of opiates

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.

Enhanced assays detect increased chromosome damage and sister-chromatid exchanges in heroin addicts

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.

Opiates as Immunosuppressive and Genotoxic Agents

From a historical prospective our immunologic investigations started as a natural consequence of our findings that opiates reduced the genetic integrity of human lymphocytes indicated by evidence of an increased frequency of chromosome damage and a deficiency in DNA repair capacity. Such changes in genetic structure and function are expected to indicate mutagenic and/or carcinogenic outcomes (1).

Seven Novel Probe Systems for Real-Time PCR Provide Absolute Single-Base Discrimination, Higher Signaling, and Generic Components

We have developed novel probe systems for real-time PCR that provide higher specificity, greater sensitivity, and lower cost relative to dual-labeled probes. The seven DNA Detection Switch (DDS)-probe systems reported here employ two interacting polynucleotide components: a fluorescently labeled probe and a quencher antiprobe. High-fidelity detection is achieved with three DDS designs: two internal probes (internal DDS and Flip probes) and a primer probe (ZIPR probe), wherein each probe is combined with a carefully engineered, slightly mismatched, error-checking antiprobe. The antiprobe blocks off-target detection over a wide range of temperatures and facilitates multiplexing. Other designs (Universal probe, Half-Universal probe, and MacMan probe) use generic components that enable low-cost detection. Finally, single-molecule G-Force probes employ guanine-mediated fluorescent quenching by forming a hairpin between adjacent C-rich and G-rich sequences. Examples provided show how these probe technologies discriminate drug-resistant Mycobacterium tuberculosis mutants, Escherichia coli O157:H7, oncogenic EGFR deletion mutations, hepatitis B virus, influenza A/B strains, and single-nucleotide polymorphisms in the human VKORC1 gene.

T-cell E-receptor Modulation by Opiates, Opioids and Other Behaviorally Active Substances

Homeostasis has been defined as a state of metastable equilibrium wherein the constancy of the internal milieu is forever being challenged 1,2 Using this definition, any antihomestatic stimulus may be regarded as a stressor2. As reviewed eleswhere 3,4, the neural and immune systems share many basic physiologic processes in response to environmental stressors. The ultimate purpose of the neuroimmune system is maintenance of homeostasis. Neurons and lymphocytes are pluripotent sensory cells5 capable of eliciting and responding to various hormone and hormonelike peptides that have systemic and local effects characteristic of endocrine, exocrine, and paracrine functions. The primary physiologic basis for the adaptability of the neuroimmue system resides in a complex network of receptor-response systems. This review focuses on the means by which opiates and other behaviorally active substances, as examples of environmental stressors of known behavioral modifying capacity, can modulate receptor activity on T-cell lymphocytes. This capacity for receptor modulation appears to be a primary means by which opiates and like factors evoke both neurobiological and immunobiological changes in host physiology and responsive behavior.

Influence of Demographic Factors on Scheduled DNA Synthesis as Measured in Human Peripheral Leukocytes and Fibroblasts

In a population study of leukocytes from 140 human subjects, far UV-induced unscheduled DNA synthesis (UDS) variation correlated with at least three demographic factors — smoking tobacco, season of sample obtainment and “street opiate” addiction. The ability to correlate UDS variability with these factors demonstrates the importance of environmental influences in control of DNA repair processes and the need to include the effect of demographics in genetic risk assessment models. In addition, mutagen-induced UDS patterns for mononuclear leukocytes from a single subject did not match the UDS pattern for fibroblasts grown from a forearm skin biopsy from the same individual which suggests that UDS may be tissue specific.