Martin W. Adler - US grants

The primary objective of this project continues to be the in vivo study of the receptors involved in the actions of opioids and endogenous opioids. The focus is on the roles played by the endogenous opioid systems in normal homeostasis and in perturbed homeostasis, including opioid tolerance and dependence. Our previous research has allowed us to begin to delineate the roles of the endogenous opioid system in thermoregulation, analgesia, and cerebral excitability. Using selective agonists and antagonists, we will continue our efforts by examining how the opioid receptor subsystems interact with each other to affect these roles. In addition, because it is our belief that the interplay of neuropeptides is crucial for most physiological systems, we will investigate the interaction between the opioid systems and selected other neuropeptide systems. Dose-response curves will be generated in all cases and routes of administration will be chosen based on the pharmacokinetics of the drugs being tested and the questions being asked. Chronic administration of drugs will be used to study how the usual roles of the receptors and the interactions with other neuropeptide systems are modified by repeated administration of drugs. The opioid receptor types that will receive primary attention are mu and kappa, although other types of opioid receptors, especially delta, will be studied when appropriate. Neuropeptides to be examined include cholecystokinin, substance P, somatostatin, neurotensin, and TRH. Measurements will be made of body temperature, antinociception, and seizure thresholds. Techniques to be used include calorimetry, in vivo microdialysis, RIA, and HPLC. The ultimate goal of these studies is to understand the functions of the endogenous opioid systems, the mechanisms involved in those actions, and how chronic administration of opioids affects those roles. Such knowledge will not only provide greater insight into the consequences of chronic use of opioids, but may well lead to improved therapies for pathological states involving pain, disruption of thermoregulation, or altered cerebral excitability.

There is a rapidly emerging literature showing not only interactive feedback between the neural, endocrine and immune systems, but also that the cells of the immune system produce and have receptors for opioids. A limited number of published studies have shown immunosuppressive effects of opioids in vitro. As chronic drug abusers are at increased risk of acquiring infectious diseases, including HIV, it is of interest to explore the hypothesis that drug usage may predispose to infection by immunosuppression. It is proposed to carry out a systematic investigation of the effects of opioids on parameters of immune responsiveness and resistance to infection in rodents. As our preliminary studies indicate that opioids are immunosuppressive, it is further proposed to assess which cells of the immune system are targets for modification by opioids; to determine with selective ligands which of the opioid receptor types is/are involved in alteration in immune system function, and to determine if there is an interaction among the various opioid receptor systems in terms of alteration in immune function. Such interactions could be additive, greater than additive, or antagonistic. Both in vivo and in vitro assays of immune function will be used, as well as in vivo administration of drugs and in vitro assay of immune responses. In vitro assays will permit extensive dose response and drug interaction studies as well as identification of target cells, identification of mechanisms of immunosuppression, and of receptor types. In vivo assays will allow evaluation of the significance of observations made in vitro, which may be particularly important in light of the feedback interactions between the immune and other homeostatic systems. These studies are of significance in that they can provide a scientific basis for understanding how opioids adversely affect immune competence and susceptibility to infectious diseases. Such studies are especially timely in view of the connection between IV drug abuse and AIDS.

The purpose of this program continues to be to support the training of pre- and post-doctoral fellows in several biomedical aspects of drugs of abuse. The principal focus of the program is interdisciplinary mentoring by the faculty in the Departments of Pharmacology and Microbiology and Immunology. The training will focus on the interrelationships among drugs of abuse and endogenous substances such as neuropeptide and cytokines. In addition to their classical effects, the drugs will be studied for their actions on the immune system and various aspects of behavior, including the mechanisms responsible for the effects. Members of participating departments will bring their expertise and specialized techniques to the program. Members of other departments will bring their expertise and specialized techniques to the program. Members of other departments will bring their expertise and specialized techniques to the program. Members of other departments also contribute in areas such as behavior, signal transduction, second messenger receptor systems, neural mechanisms, and genetics. Current research by members of our program involves opioids, alcohol cannabinoids, and cocaine, as well as opioid and other rain neuropeptides. Techniques include in vitro and in vivo approaches and methods that range from molecular to behavioral. For acceptance into the program, predoctoral trainees will have completed at least 1 year of graduate training. They will be exposed to a rigorous program for both didactic and laboratory experiences, with the aim of producing independent researchers highly knowledgeable and expert in the broad field of drugs of abuse. Postdoctoral trainees with the Ph.D. degree will have received their doctorate or defended their thesis as part of a well-recognized graduate program prior to beginning training in this program; trainees with the M.D. degree will have completed at least 1 year of residency. Postdoctoral trainees will concentrate on research during their training but will be required to audit or take courses related to the field of drug abuse, should their background indicate that such courses are desirable. All trainees will receive instruction in pharmacology, immunology, and behavior. In addition, all trainees will receive instruction in teaching, presentation of data at meetings and seminars, preparation and evaluation of manuscripts and preparation of grants. We expect them to become careful, independent, and productive researchers in the drug abuse field and/or the related fields of neuropeptides and neuroimmunology. The primary facilities in this program will be in the Departments of Pharmacology and Microbiology and Immunology at the Temple University School of Medicine. We are requesting funds to continue and expand on the accomplishments of the first 9 years of this program.

This application is for a NIDA P30 Center titled "Intersystem Regulation by Drugs of Abuse." We have evolved a strong group of funded investigators working on a variety of aspects of drug abuse and addiction. It has become apparent that a P30 Center would enhance the overall productivity, synergy, and integration of the interdisciplinary research and enable the recruitment of additional faculty into the field of drugs of abuse and addiction. The themes of the proposed Center are: 1) to determine the role of abused and therapeutically used opioids and the endogenous opioid system and its ligands, in regulation of pain, body temperature, the immune system, and infection, including HIV; 2) to determine the basic mechanisms responsible for these changes; 3) to compare effects produced by acutely administered drugs with those of chronically administered drugs. A corollary to these themes will be the determination of the similarities and differences in the mechanisms involved in these systems relative to the opioid and endogenous systems associated with abused drugs, and the relationships that exist among these systems relative to the opioid and endogenous systems associated with abused drugs, and the relationships that exist among these systems. In light of the fact that individuals rarely, if ever, abuse only one drug, it is particularly important to determine whether or not interactions occur between opioids and other abused drugs such as cocaine and marijuana. Both in vivo and in vitro approaches will be used, ranging form whole- animal measurements of behavior and activity, to biochemical and molecular techniques, to assess the overall mechanisms of action of opioids and other drugs of abuse in the brain and the periphery, and the feedback loops that integrate these systems. The potential for synergy among many investigators having diverse expertise and using multiple approaches and techniques is enormous. In order to carry out the aims of the Center, we are requesting 6 Cores: Administrative, Animal, Biochemical Pharmacology, Cell and Immunology, Integrative Pharmacology, and Molecular Biology. Members of the Center come from several departments, and have expertise in such areas as neuropharmacology, molecular pharmacology, quantitative and theoretical pharmacology, behavioral pharmacology, microbiology (bacteriology and virology), immunology, and biochemistry. Faculty from both basic science and clinical departments will be involved in the NIDA Center. A Center will be the glue that holds together and attracts others to this wide-ranging group of outstanding scientists. Temple University School of Medicine has made a major commitment in providing the funds and space necessary to form a Center. The NIDS Center will provide the Core facilities to foster the use of techniques and methods not currently available to the individual research. It will provide cost savings, and raise the quality of the science and the vision of future of research. It will truly provide the environment that will result in the highest quality, innovative, state-of-the-art research. It will provide what is necessary for our group to become a national resource in terms of several aspects of drug abuse research, especially drug interactions and neuroimmunopharmacology.

Opioids and cannabinoids affect not only the nervous, but also the immune system in humans and rodents. Mu, kappa, and delta opioid receptors and CB1 and CB2 cannabinoid receptors have been demonstrated in the immune system. In addition, in rodents, products of the immune system, such as cytokines, have been shown to alter neural function and to modify the effects of opioids on body temperature and analgesia. Chemokines, a subclass of cytokines, are produced by microglia in the brain. In human peripheral blood mononuclear cells, it has been shown that there is an interaction between opioid receptors and chemokine receptors, both being G-protein coupled, seven transmembrane receptors. Selected ligands for each class of receptor can cross-desensitize the other by cross-phosphorylation. Our preliminary results using transfected cell lines support this conclusion as do our recent novel and very exciting results showing that chemokines injected into rat brain PAG block opioid- induced analgesia. The findings suggest the chemokines may alter opioid function in the brain. We have also found super- and sub- additive effects of opioid receptor-selective ligands (mu and delta2) on immune cell function, as well as similar effects between opioids and cannabinoids. Taken together, these results suggest that the opioid, cannabinoid, and chemokine systems interact in the immune system and in the nervous system. It is the purpose of the proposed studies to document the nature of the interactions among these three classes of ligands and their receptors in both the immune and nervous systems. Specifically, we propose to 1) determine the interactions that occur among the classical opioid receptor types and between opioids and cannabinoids in the immune system of mice; 2) determine the cross-regulation of opioid, cannabinoid, and chemokine receptor function in primary human cells and transfected cell lines, and the effect of combinations of these classes of ligands on HIV replication; and 3) examine the effects of Chemokines given supraspinally to rats and mice on morphine- and cannabinoid- mediated analgesia and hypothermia. These studies will explore basic interactions between abused drugs and immune function, and between products of the immune system and these drugs on nervous system function, whether the drugs are used therapeutically or non-therapeutically. Common cellular targets and mechanisms will be sought to elucidate drug interaction pathways. As drug abusers usually use more than one drug, the studies also have public health relevance. Because chemokine receptors are co- receptors for HIV, the studies of how opioids and cannabinoids affect levels and function of chemokine receptors have implications for the effects of drugs of abuse, particularly when taken in combination, on HIV progression.

Opioids and cannabinoids affect not only the nervous, but also the immune system in humans and rodents. Mu, kappa, and delta opioid receptors and CB1 and CB2 cannabinoid receptors have been demonstrated in the immune system. In addition, in rodents, products of the immune system, such as cytokines, have been shown to alter neural function and to modify the effects of opioids on body temperature and analgesia. Chemokines, a subclass of cytokines, are produced by microglia in the brain. In human peripheral blood mononuclear cells, it has been shown that there is an interaction between opioid receptors and chemokine receptors, both being G-protein coupled, seven transmembrane receptors. Selected ligands for each class of receptor can cross-desensitize the other by cross-phosphorylation. Our preliminary results using transfected cell lines support this conclusion as do our recent novel and very exciting results showing that chemokines injected into rat brain PAG block opioid- induced analgesia. The findings suggest the chemokines may alter opioid function in the brain. We have also found super- and sub- additive effects of opioid receptor-selective ligands (mu and delta2) on immune cell function, as well as similar effects between opioids and cannabinoids. Taken together, these results suggest that the opioid, cannabinoid, and chemokine systems interact in the immune system and in the nervous system. It is the purpose of the proposed studies to document the nature of the interactions among these three classes of ligands and their receptors in both the immune and nervous systems. Specifically, we propose to 1) determine the interactions that occur among the classical opioid receptor types and between opioids and cannabinoids in the immune system of mice; 2) determine the cross-regulation of opioid, cannabinoid, and chemokine receptor function in primary human cells and transfected cell lines, and the effect of combinations of these classes of ligands on HIV replication; and 3) examine the effects of Chemokines given supraspinally to rats and mice on morphine- and cannabinoid- mediated analgesia and hypothermia. These studies will explore basic interactions between abused drugs and immune function, and between products of the immune system and these drugs on nervous system function, whether the drugs are used therapeutically or non-therapeutically. Common cellular targets and mechanisms will be sought to elucidate drug interaction pathways. As drug abusers usually use more than one drug, the studies also have public health relevance. Because chemokine receptors are co- receptors for HIV, the studies of how opioids and cannabinoids affect levels and function of chemokine receptors have implications for the effects of drugs of abuse, particularly when taken in combination, on HIV progression.

effectiveness and efficiency;it provides the necessary services for the interactions among the Cores;it coordinates the use of shared resources;it coordinates the seminar series and discussion series;it runs informal sessions to acquaint members of the Center with new techniques that bear on the research conducted by the members;it insures that there are mechanisms in place to foster collaborations;it houses a small library and conference room facility for members of the Center;its personnel and facilities aid in the preparation of grants and reports;it processes orders for all supplies and equipment related to the Cores of the Center;it provides coordination of meetings to discuss research results prior to presentation at local, national, or international meetings;it insures quality control and promotes scientific excellence throughout the Center. In short, this Core brings together all the activities that create centeredness. The Director, Co-Director, and Scientific Coordinator have worked together for over 17 years in a manner that insures a coordinated and efficient means of meeting the goals of the Center.

DESCRIPTION (provided by applicant): In order to carry out the aims of the NIDA PSOCenter, we are requesting a 5-year competing renewal with support for the 5 current Cores (Administrative, Animal, Biochemical Pharmacology, Cell and Immunology, Integrative Pharmacology, and Molecular Biology) and the addition of a new Core, the Analytical Core. The Center has enhanced productivity, synergy, and interdisciplinary research, and enabled recruitment of new faculty into the field of drugs of abuse. Major integrative themes include 1) Study of drugs of abuse and their endogenous ligands, particularly opioids and cannabinoids, in regulation of pain, body temperature, and the immune system; research addresses and compares basic mechanisms responsible for changes in these systems produced by both acute and chronic administration, and 2) Drug interactions-as individuals rarely abuse a single drug, it is important to determine whether interactions occur among abused drugs such as opioids, cocaine, and marijuana, and to assess if the types of interactions are similar in the different systems. Experimental results are built on evolving theories of drug interactions. The scope of the ongoing projects in the Center marries in vivo and in vitro approaches that examine effects of drugs at the cellular, biochemical, molecular and whole-organism levels. Integration of information ranging from measurements of behavior to gene activation has stimulated collaborations leading to novel hypotheses and results. The Cores of the P30 Center have fostered multidisciplinary interactions resulting in a true intellectual integration leading to hypothesis building and implementation of lines of experimentation never previously conceived. While the focus of the NIDA P30 Center is basic research, clinical faculty have become interested in the potential for translational research in the area of drug abuse. Temple University has made major commitments that will sustain growth in the area of drug abuse research in the future. The Cores are providing the technical expertise to facilitate this transition in a cost-efficient way. The P30 Center is the glue that holds together and attracts others to our group of outstanding scientists. Work fostered by the Center has given us national visibility in the areas of drug interactions and neuroimmunopharmacology.

[unreadable] DESCRIPTION (provided by applicant): There is a well-recognized intersection between drugs of abuse and AIDS or other states of compromised immune function, but the mechanisms behind this interaction are just beginning to be revealed. Cross-talk between opioids or cannabinoids and chemokines (small proteins produced by both microglia and neurons in the brain) occurs both in vitro and in vivo. For example, heterologous cross-desensitization, in terms of reduced chemotactic and antinociceptive activities, occurs between opioids and chemokines that are involved in HIV entry into cells. Our findings since the last competing renewal demonstrate unequivocally that the opioid, cannabinoid, and chemokine systems interact in both the immune system and the nervous system. Our results, with those from other laboratories, have led us to propose that the chemokine system in the brain is a major communication system between neurons, and between glia and neurons, thus joining neuropeptides and neurotransmitters subserving this function in normal and pathological states. It is the purpose of the proposed studies to document the nature of the interactions among these three classes of ligands and their receptors. Specifically, we propose to 1) explore the functional and biological consequences of injection of selected chemokines into various brain areas in terms of their altering the actions of drugs of abuse, including analgesia, thermoregulation, tolerance,and dependence; 2) examine the reciprocal effect of opioids and cannabinoids on the activity of chemokines in the brain, and 3) assess neurophysiological effects of the chemokines alone and with opioids or cannabinoids in brain slices. The drugs chosen alter levels of HIV when added to human lymphoid, monocytic, or microglial cultures in vitro and interact with the chemokine receptors involved in HIV infection. The chemokines to be studied are ligands for HIV chemokine co-receptors. The implications for public health include altering treatment of pain associated with neuroinflammatory states, treatment of neuropathies associated with HIV, altering the ability of HIV to enter cells, altering actions of neurotransmitter/neuropeptide systems involved in the effects of drugs abused or used therapeutically, and potentially bringing new approaches to prevention and treatment of drug abuse and neurodegenerative diseases and AIDS through affecting the actions of chemokines and their receptors in the nervous system. [unreadable] [unreadable] [unreadable]

The molecular core facility provides services to investigators that involve either short- or long-term use of molecular techniques. It is our experience that two types of investigators are interested in our services. The first type of investigator is one who is not currently equipped to conduct experiments involving molecular biology, but recognize the advantages that can be derived by studying their experimental system on a molecular level. Investigators in this group may not be able or interested in making the investment in the equipment and supplies that are required to utilize molecular techniques. For example, this investigator may need to evaluate gene expression in their experimental system. The second type of investigator is one who is already active in molecular biology, but appreciates the advantage in both time and research funds to take advantage of our services. This type of investigator may recognize that it is possible to utilize the Molecular Core and continue to carry out other molecular studies without the need to stop to prepare molecular constructs, or measure gene expression, with precious limited resources of their own. An example would be an investigator who would like to stably express an epitope-tagged form of one of the opioid receptors in a convenient cell line. An additional example would be an investigator who is interested in measuring gene expression in vivo following drug treatment, and would like to have information about the activity of a gene which is likely to be involved in a regulatory signal transduction pathway. We provide services for investigators who span a variety of disciplines, and we carry out our work either in direct collaboration with the investigator (and their colleagues), or we carry out our work independently and pass along the product of our work to the investigator (e.g., in the form of a molecular construct, or in the form of a stably-transfected cell line).

The Pilot Project Core will provide critical support for new initiatives and feasibility studies to test new hypotheses and generate novel data in the field of substance abuse, pain and addiction research, and the intersection of drugs of abuse with HIV. Research that is supported ranges over a variety of disciplines and techniques including molecular, pharmacological, physiological, immunological, and behavioral. Projects involve a variety of drug classes including opioids, cannabinoids, psychostimulants, alcohol and nicotine. The amount of support is sufficient to allow the testing of new ideas and accumulating data that can be used to form the basis for an application for external funding. In addition to the actual dollars supporting the project, the investigators are encouraged to make use of the expertise and the facilities of the Research Support Cores of the NIDA P30 Center. Use of the Research Support Cores allows those carrying out a Pilot Project to expand their areas of expertise by receiving assistance with new techniques and approaches with which they may be unfamiliar, and to receive mentoring from the Core faculty. In the previous funding period, three projects/year were supported by the Pilot Projects Core, resulting in a recently awarded NIDA R01 as well as other submitted grant applications, several publications, and presentations at national and regional meetings. Potential applicants are solicited by general and targeted announcements to faculty at Temple University and to faculty of other institutions through professional list-serves. Applications for pilot project support are evaluated by a sub-committee of P30 Center faculty and are judged on their innovation and significance of the research question and approach, the probability that sufficient outcomes will be obtained to serve as preliminary data for an independent grant application, and the strength of the integration with the aims of the NIDA P30 Center. Special consideration is given to early career investigators and to investigators new to the field of substance abuse research. Progress is evaluated via informal and frequent discussions of progress and formal reports from awardees. It is anticipated that three pilot projects will be supported directly by the Pilot Project Core each year, including at least two new projects per year.