Barbara M. Bayer - US grants

Amino acid transport through the "A" system increases at an early stage of cell division or differentiation as indicated by studies in cell culture lines as well as in isolated hepatocytes and lymphocyte preparations. Recent studies suggest that this increase is due specifically to the emergence of a high affinity (HA), Na+- and energy-dependent component of the "A" system. As the emergence of the HA component is selectively suppressed by nonsteroidal anti-inflammatory drugs (NSAID) in a dose dependent manner, by a mechanism that appears to diminish the cell's ability to produce functional carriers (Vmax is decreased, without impairment of the carriers' ability to bind substrate or Na+), the possibility that this is one mechanism by which these drugs inhibit blast transformation will be investigated. Initial studies will determine whether emergence of the HA component and its inhibition by the NSAID is common to all lymphocyte populations during mitogenic stimulation. The question of whether corticosteroids have similar or more selective actions than the NSAID on this process will also be investigated. Secondly, experiments will be devised to see if emergence of the HA system is a primary event by blockade of subsequent biochemical changes during transformation (i.e., use of ornithine decarboxylase inhibitors and inhibitors of RNA/DNA synthesis) and looking at the effects of these inhibitions, if any, on amino acid transport. Lastly the possible mechanisms of the inhibition of the HA system will be sought primarily by investigation of the actions of the NSAID on thermodynamic parameters of amino acid uptake and on various aspects of cellular energy metabolism. These studies will be undertaken with freshly isolated lymphocytes from thymus and spleen, use of common mitogenic stimulants and measurement of the rate of uptake of labelled nonmetabolizable amino acids. Other parameters of blast transformation such as thymidine incorporation into cellular DNA, activation of ornithine decarboxylase and changes in cyclic AMP concentrations will be monitored.

stress; leukocyte activation /transformation; morphine;

DESCRIPTION: Applicant's Abstract The overall goal of these studies is to determine the mechanism(s) underlying morphine-induced immunosuppression and to further examine the factors contributing to an increased immune sensitivity following repetitive morphine exposure to stress. Specifically, the experiments in this proposal will test the hypotheses that the inhibition of immune cell activities by morphine: 1) is mediated through several discrete central nuclei which modulate the activity of the paraventricular nucleus (PVN) of the hypothalamus; 2) is peripherally mediated through stimulation of sympathetic neuronal outflow and/or alteration of circulating cytokine levels; 3) produces an apparent 'tolerant' state which is accompanied by an increased immune sensitivity to stress or drug withdrawal. The specific aims which will test these hypotheses include: Aim # 1: Determine the central pathways involved in morphine-induced suppression of immune cell function. It is hypothesized that morphine suppresses lymphocyte responses by modulating the autonomic outflow from the PVN from multiple sites in the brain, including the periaqueductal gray (PAG), dorsal raphe nucleus (DR), bed nucleus of the stria terminalis (BNST) and the anterior hypothalamus (AH). These studies will 1) establish the site specificity for morphine in the regulation of immune cell activity for each of these areas, 2) pharmaco-logically characterize the opioid receptor involved, 3) evaluate the role of the PVN as a coordinator of opioid-induced immune cell alterations and 4) begin to evaluate the interactions of opioids with other neurotransmitter systems utilized by these selected tissues which innervate the PVN. Aim #2: Identify the peripheral mechanisms involved in central opioid receptor modulation of immune responses. An underlying hypothesis of these studies is that the suppression of lymphocyte proliferation following morphine is mediated through opioid stimulation of the autonomic nervous system (ANS). To more definitively test this hypothesis, these studies will examine the effects of 1) lesioning descending neuronal output, 2) neuronal stimulating selective target peripheral tissues and 3) increasing IL-6 and potentially other cytoyines on morphine-induced changes in immune cell activity. Aim #3: Examine mechanisms contributing to the increased sensitivity of morphine-tolerant rats to the immunosuppressive effects of stress. These studies will test the hypothesis that the prolonged stimulation of adrenal secretion of glucocorticoids and/or the activity of the ANS, which accompany chronic morphine administration, results in an increased vulnerability of the immune system to exposure to stress and spontaneous withdrawal of drug. The fact that intravenous drug users represent a major risk group for ANS makes it imperative to understand the central action and peripheral mechanisms by which abusive drugs and stress interact to modulate the immune system.

DESCRIPTION: (adapted from applicant's abstract) Preliminary experiments demonstrate that prenatal exposure to cocaine both prior to and during gestation results in significant suppression of in vitro and in vivo measures of cellular immunity. The overall goal of the proposed studies is to further examine these effects and determine whether prenatal cocaine exposure increases the susceptibility of offspring to other drugs and stressors. To meet this goal the following specific aims will be carried out: Specific Aim #1: To determine the effects of prenatal cocaine exposure on in vitro and in vivo immune parameters in offspring under controlled conditions. These studies will examine in vitro (lymphocyte phenotype expression, mitogen-induced proliferation and natural killer (NK) cell activity) and in vivo (antigen-elicited humoral and cellular immune responses) at 50 days postnatal in offspring born of dams exposed to cocaine prior to and during gestation. Such effects will be compared to pups born of pairfed and vehicle- treated dams to control for the effects (if any) of nutrition and handling on similar immunological indices. The contribution of changes in maternal behaviors of cocaine-treated dams to subsequent immune reactivity in pups will also be examined by comparing immune reactivity of fostered and nonfostered pups. Both male and female offspring will be examined to determine gender effects (if any) in immune reactivity. Finally, sampling from individual litters will be limited to control for specific litter effects that might confound interpretation of effects of prenatal exposure. Specific Aim #2: To characterize the temporal relationship of the effects of prenatal cocaine exposure on in vitro and in vivo immune parameters in offspring. Although prenatal cocaine exposure reduces immune responses 50 days postnatally, it is unknown when these effects first appear and how long they will persist. Therefore, following prenatal exposure to cocaine various immune indicators will be examined at postnatal days 10, 20, 50 and 100. Specific Aim #3: To determine the exposure period sufficient for the production of in vitro and in vivo immune changes in prenatally exposed offspring. Although prenatal cocaine exposure both prior to and during gestation decreases cellular immunity, it is unknown whether the immunosupression in the offspring is a function of exposure period. Therefore, measures of immunity will be measured 50 days postnatally in offspring born of dams exposed to cocaine prior to, during and both prior to and during gestation. Specific Aim #4: To examine in vitro immune sensitivity of offspring prenatally exposed to cocaine to subsequent drug and stress challenges. A number of recreational drugs, including cocaine, affect immune responsitivity in adult animals with no history of prenatal cocaine exposure. In these studies, the effects of prenatal cocaine exposure on immune functioning and how such exposure may alter the vulnerability of the immune system to subsequent challenges such as cocaine, nicotine, morphine, restraining stress or endotoxin (LPS) will be determined.

DESCRIPTION (provided by applicant): This is a revised application for the continued support of an Institutional Training Grant in the Neurobiology of Drug Abuse. The long range goal of this program is to prepare and develop scientists who will be in a position to continue to define mechanisms underlying the effects of drugs of abuse using interdisciplinary approaches. The 12 core training faculty and 12 supporting faculty are drawn from 2 clinical and 7 basic science departments on the Main Campus and in the Medical Center. Their NIDA relevant research is focused on several areas including the 1) role of stress in augmenting the adverse effects of drug addiction, 2) structure and function of nicotinic receptors and genetic predisposition to addiction and 3) the involvement of excitatory amino acids in CMS disorders and their participation in the neurotoxic effects of addictive drugs. These focused research areas are integrated with faculty who have expertise in 1) cognition and computational neuroscience, 2) the role of role of neurotrophic factors in development and 3) receptor pharmacology and mechanisms of signal transduction. The training environment is excellent for collaborative, multidisciplinary research efforts of both faculty and trainees. Many of the faculty have previous or ongoing collaborations and students are encouraged to seek co-mentorship between faculty with complementary research interests. We are requesting support for 6 predoctoral trainees during the first 2 years of their training and course work. Thereafter, students are encouraged to apply for fellowships and nearly 80% of the trainees have been successful in obtaining awards from NIH or DoD to complete their thesis research. An aggressive recruitment of students in underrepresented racial and ethnic groups is a top priority of Georgetown and has been a particular strength of this program. The predoctoral trainees will receive their Ph.D. from either the Pharmacology or the Interdisciplinary Program in Neurosciences at Georgetown. The training program includes broad-based didactic coursework, as well as rotations in laboratories of the training faculty. In addition to their formal course work, the trainees participate in a number of ongoing seminar series, joint Neurology, Psychiatry and Neuroscience grand rounds, national professional meetings and a journal club with special emphasis of the neurobiology of drugs of abuse. Outside experts in the field are invited to lecture in a required course dedicated to the most recent advances in the effects of addictive drugs. Trainees are also required to take courses related to improving professional skills (writing and reviewing manuscripts, grantsmanship, mentorship, teaching, conflict resolution, career choices, oral presentations) and understanding the ethical issues surrounding biomedical research.