William J McBride, Ph.D. - US grants

The dose of therapeutic radiation that can be delivered to the brain is limited primarily by the risk of delayed brain necrosis which is debilitating and frequently fatal. Clinical and experimental dose-response data for this complication are limited. They are particularly inadequate for predicting how the brain will respond to dose fractionation in particular at doses of less than or equal to 2 Gy. Traditional isoeffect formulae are almost certainly inappropriate for the brain and newer formulations are suspect at low doses per fraction. Experimental studies on the response of brain to radiation have been limited to histology and lethality and no quantitative data are available on responses to clinically relevant radiation doses and fractionation schemes. We intend to measure the response of mouse brain to different single and multifractionated doses of radiation (down to 1 Gy per fraction) and to derive accurate isoeffect dose relationships. In addition, the effect of varying the interfraction interval on responses will be measured. "Remembered" dose will also be assessed as will the potential usefulness of radioprotectors. We will use sensitive immunological (ELISA) methods to measure radiation-induced changes in brain cell type-specific proteins in brain and body fluids. In this way we will measure responses by different brain cell populations and establish the kinetics of their damage and recovery. Alterations in brain cell-specific proteins as a function of time and dose will be corroborated by direct histological, immunohistochemical and autoradiographical examination. We believe that the isoeffect relationships that are established should serve as a guide in the establishment of new radiotherapeutic regimens and lead to better understanding of the biological bases for brain responses to radiation. In addition, in the future, the assays will be clinically applicable to the measurement of brain damage caused by radiation and other cytotoxic agents.

The major hypothesis in this proposal is that a cause of clinically significant heterogeneity in radioresistance between tumors lies in their radiation response modifiers. This hypothesis will be investigated utilizing melanoma and renal cell carcinoma lines. A cause and effect relationship as well as a correlation will be sought between radiation resistance and cytokine expression. One of several investigative approaches will be to genetically manipulate selected human tumor cell lines using retrovirus vectors so that they express specific cytokines and determine whether this changes cellular radioresistance. Mechanisms of radioresistance will be examined within these well-defined tumor cell systems, with initial emphasis on certain free radical scavengers, cell cycle effects, and recovery from potentially lethal radiation damage since there is evidence that these mechanisms are subject to modification by cytokines. The significance of this study on cytokine-directed radioresistance is 3-fold: 1) if it is possible to correlate cytokine profile and/or phenotypic marker expression with radioresistance, then these measures could be used to predict radiation response. 2) Cytokines or cytokine receptors could act as targets and/or vehicles for enhancing radiosensitivity prior to and during radiation therapy, as well as for independent therapeutic attack. 3) Knowing the importance of endogenous cytokines to radioresponsiveness, and the pathways by which they operate, will improve our understanding of the basic radiobiology of cell killing and of the nature of "intrinsic" tumor radioresistance.

The ventral tegmental area (VTA) dopamine (DA) and dorsal raphe nucleus (DRN) serotonin (5-HT) pathways to the nucleus accumbens (ACC) have been implicated in mediating the reinforcing properties of alcohol. The ACC and VTA receive 5-HT innervations from the DRN and there is evidence that 5-HT can influence DA release in the VTA and ACC. Data also indicate that ethanol-induced DA release in the ACC may be mediated by 5-HT3 receptors. The hypotheses to be tested are that (1) the VTA DA neurons to the ACC are regulated by the DRN 5-HT system via 5-HT3 receptors, and (2) this 5-HT system is involved in mediating the actions of alcohol on the mesolimbic DA system. The neurochemical experimental approach will utilize a microdialysis technique coupled with a small-bore HPLC-EC procedure to collect and measure extracellular levels of DA and 5-HT in the ACC of unanesthetized, freely moving, adult male Wistar rats as a measure for determining changes in neuronal activity. The experimental design will establish if (1) local application of 5-HT3 agonists into the VTA and ACC stimulates the release of DA in the ACC; (2) local application of 5-HT3 antagonists into the VTA and ACC inhibits DA release induced by activation of the DRN 5-HT neurons; (3) local administration of 5-HT3 antagonists into the VTA and ACC attenuates the alcohol-stimulated release of DA in the ACC following i.p. ethanol administration; and (4) the activity of the DRN 5-HT neuronal system alters the effects of i.p. alcohol on the VTA DA pathway to the ACC. The data from this proposal will provide valuable information on the involvement of the DRN 5-HT system in regulating the activity of VTA DA neurons projecting to the ACC and mediating the actions of alcohol on this DA pathway. Such knowledge may aid in the development of specific pharmacological agents for treatment of alcoholism and alcohol abuse.

The overall long-range objectives of this proposal are to identify CNS neurotransmitter systems that are involved in mediating the rewarding properties of ethanol in order to understand better some of the neurobiological factors underlying excessive alcohol drinking behavior. The hypothesis to be tested is that the ventral tegmental area (VTA) dopamine (DA) neuronal system projecting to the nucleus accumbens (Acb) is a major neuronal pathway involved in initiating and maintaining the rewarding actions of alcohol. Since alcohol drinking behavior can be significantly influenced by genetic factors, experiments will be conducted with the alcohol-preferring P line and the high-alcohol drinking HAD line of rate. Results will be compared with data obtained with the alcohol-nonpreferring NP line and the low-alcohol drinking LAD line as well as with data obtained from stock rate (Wistar, N/Nih). The technique of intracranial self administration(ICSA) will be used to determine if the VTA, Acb or medial prefrontal (MPF) cortex is a potential site for the rewarding actions of alcohol. An electrolytic microinjection transducer (EMIT) system will be used to deliver the alcohol (0-200 mg%/100 nl artificial CSF over 5 seconds) into the 3 brain sites. Animals will be stereotaxically implanted with unilateral guide cannulas in either the VTA, Acb or MPF cortex. Following recovery from surgery, rate will be transferred to 2-lever operant chambers and allowed to self-train for the ICSA of ethanol by pressing an active lever which will deliver the ethanol solution directly into the brain site. These infusions are initially delivered on a FRl schedule of reinforcement and are paired with a red cue-light stimulus. Responses on the active and inactive levers, and the number of reinforcements are recorded throughout the sessions. The dose-response effects of the ICSA of ethanol into the 3 regions will be compared among the selectively bred lines and the stock rats to determine if sites other than the VTA will support ICSA of ethanol and if only the P and HAD lines will significantly self-administer ethanol. Experiments will also establish if the VTA DA system is involved in the ICSA of ethanol into the VTA by determining the effects of (a) D-1 and D-2 receptor antagonists on self-administration; (b) 6-hydroxy dopamine lesions in the VTA on self-administration; and (c) effects of self-administration into VTA on DA release in the Acb or MPF cortex. Finally, experiments will also determine if the GABA-A, 5-HT3 or opioid receptors are involved in mediating the ICSA of ethanol into the VTA. Overall, the information obtained from the proposed studies will hopefully provide a better understanding of some of the neurobiological mechanisms involved in human alcohol addiction.

The major hypothesis in this proposal is that a cause of clinically significant heterogeneity in radioresistance between tumors lies in their radiation response modifiers. This hypothesis will be investigated utilizing melanoma and renal cell carcinoma lines. A cause and effect relationship as well as a correlation will be sought between radiation resistance and cytokine expression. One of several investigative approaches will be to genetically manipulate selected human tumor cell lines using retrovirus vectors so that they express specific cytokines and determine whether this changes cellular radioresistance. Mechanisms of radioresistance will be examined within these well-defined tumor cell systems, with initial emphasis on certain free radical scavengers, cell cycle effects, and recovery from potentially lethal radiation damage since there is evidence that these mechanisms are subject to modification by cytokines. The significance of this study on cytokine-directed radioresistance is 3-fold: 1) if it is possible to correlate cytokine profile and/or phenotypic marker expression with radioresistance, then these measures could be used to predict radiation response. 2) Cytokines or cytokine receptors could act as targets and/or vehicles for enhancing radiosensitivity prior to and during radiation therapy, as well as for independent therapeutic attack. 3) Knowing the importance of endogenous cytokines to radioresponsiveness, and the pathways by which they operate, will improve our understanding of the basic radiobiology of cell killing and of the nature of "intrinsic" tumor radioresistance.

The ventral tegmental area (VTA) dopamine (DA) and dorsal raphe nucleus (DRN) serotonin (5-HT) pathways to the nucleus accumbens (ACC) have been implicated in mediating the reinforcing properties of alcohol. The ACC and VTA receive 5-HT innervations from the DRN and there is evidence that 5-HT can influence DA release in the VTA and ACC. Data also indicate that ethanol-induced DA release in the ACC may be mediated by 5-HT3 receptors. The hypotheses to be tested are that (1) the VTA DA neurons to the ACC are regulated by the DRN 5-HT system via 5-HT3 receptors, and (2) this 5-HT system is involved in mediating the actions of alcohol on the mesolimbic DA system. The neurochemical experimental approach will utilize a microdialysis technique coupled with a small-bore HPLC-EC procedure to collect and measure extracellular levels of DA and 5-HT in the ACC of unanesthetized, freely moving, adult male Wistar rats as a measure for determining changes in neuronal activity. The experimental design will establish if (1) local application of 5-HT3 agonists into the VTA and ACC stimulates the release of DA in the ACC; (2) local application of 5-HT3 antagonists into the VTA and ACC inhibits DA release induced by activation of the DRN 5-HT neurons; (3) local administration of 5-HT3 antagonists into the VTA and ACC attenuates the alcohol-stimulated release of DA in the ACC following i.p. ethanol administration; and (4) the activity of the DRN 5-HT neuronal system alters the effects of i.p. alcohol on the VTA DA pathway to the ACC. The data from this proposal will provide valuable information on the involvement of the DRN 5-HT system in regulating the activity of VTA DA neurons projecting to the ACC and mediating the actions of alcohol on this DA pathway. Such knowledge may aid in the development of specific pharmacological agents for treatment of alcoholism and alcohol abuse.

The overall objectives of this proposal are to determine using animal models, neurobiological factors involved in promoting high alcohol drinking behavior in adolescents and to determine the long range consequences of adolescent alcohol drinking. The hypotheses to be tested are that (1) neurobiological factors associated with excessive alcohol drinking in adults are also present at adolescence and contribute to high alcohol consumption in the young; (2) experience with the rewarding effects of alcohol during adolescence in vulnerable individuals exacerbates these reinforcing actions in adulthood and promotes further excessive alcohol consumption; and (3) chronic alcohol drinking during adolescence produces long-range behavioral consequences in adulthood. These hypotheses will be tested using genetic animal models, i.e., the selectively bred alcohol-preferring P and -nonpreferring NP rats and the high-alcohol drinking HAD and low-alcohol drinking LAD replicate lines. All measurements will be conducted on both male and female animals. The first specific aim will determine if there are differences in the serotonin (5-HT) and dopamine (DA) systems between P and NP rats and between HAD and LAD rats at the age of onset of adolescent alcohol drinking by (a) using HPLC-EC analysis to measure the regional CNS contents of 5-HT, DA and their metabolites in animals at 25 days of age; (b) using membrane preparations from selected CNS regions to determine K/D and B/max values for binding of [3H]8-OH-DPAT to 5-HT/1A sites and [3H]Sulpiride to D2 sites; and (c) autoradiography procedures to determine the densities of 5-HT2 sites (labelled with [3H]Ketanserin) and 5-HT/1A sites in selected CNS subregions. The second specific aim will examine the development of alcohol drinking after weaning (day 22 of age) and if this development can be altered by (a) housing conditions; (b) pharmacological interventions; and (c) taste aversion conditioning. The third specific aim will examine the presence of behavioral factors which might be associated with early onset of alcohol drinking, such as (a) low-dose ethanol stimulation of locomotor activity; (b) difference in sensitivity to high-dose ethanol; (c) rapid development of acute ethanol tolerance; and (d) differences in measures of emotionality and cognitive function (e.g., Plus Maze, Morris Water Maze, etc.) between P and HAD versus NP and LAD rats. The fourth specific aim will examine the long range consequences of adolescent alcohol drinking on adult behaviors (i.e., changes in emotionality and cognitive function, changes in response to the reinforcing and aversive actions of ethanol). These studies with animal models are significant since they will provide important information on (a) neurobiological factors which might contribute to adolescent alcohol drinking; (b) methods which might reduce adolescent alcohol drinking; (c) possible behavioral features associated with adolescent alcohol drinking; and (d) potential long range consequences of adolescent alcohol drinking. Information gained from these animal studies will hopefully provide greater insight into the causes and prevention of adolescent alcohol drinking.

DESCRIPTION: (Adapted from the Investigator's Abstract) The long-range goals of this project are to identify CNS sites, neurotransmitter systems and receptors involved in mediating the actions of ethanol (E) and promoting high alcohol-seeking behavior. The overall hypothesis to be tested is that there are innate neurobiological differences within key CNS limbic structures which underlie genetic vulnerability to high alcohol drinking behavior. The objectives of this proposal are to determine (1) the effects of chronic alcohol consumption on neuronal activity within limbic and other important CNS structures; (2) the effects of E drinking conditions on altering neuronal activity within limbic and other important CNS structures; (3) the involvement of ventral tegmental area (VTA) dopamine (DA) neurons, and (5-HT) serotonin transmission and 5-HT3 receptors in alcohol-seeking behavior; and (4) differences between rat lines, selectively bred for disparate alcohol drinking behaviors, in GABAA and 5-HT3 receptor regulation of VTA mediated reinforcement processes. The overall hypothesis will be tested in the alcohol-preferring P, alcohol-nonpreferring NP, high alcohol-drinking HAD and low alcohol-drinking LAD lines. The [14C]-2-deoxyglucose (2-DG) uptake procedure coupled to quantitative autoradiography will be employed to assess changes in regional CNS neuronal activity of P and HAD rats that may be associated with chronic alcohol drinking and alcohol drinking conditions. In vivo microdialysis will be used to measure changes in extracellular levels of DA and 5-HT in the VTA and/or nucleus accumbens (ACB) to examine the effects of local E and 5-HT3 agonist administration, and alcohol drinking conditions on DA and 5-HT neuronal activity. The intracranial self-administration (ICSA) procedure will be utilized to examine line differences in reinforcement processes mediated by GABAA and 5-HT3 receptors within the VTA. The results of this project will provide fundamental information toward understanding the neurobiological mechanisms underlying high alcohol-seeking behavior. Such information would be important for developing pharmacotherapies.

behavioral /social science research tag

radiopharmacology; technetium; gonadotropin releasing factor; image enhancement; breast neoplasm /cancer diagnosis; chemical synthesis; diagnosis design /evaluation; breast neoplasms; neoplasm /cancer radiodiagnosis; xenotransplantation; athymic mouse;

A series of anti-CEA x anti-indium-DTPA (hMN-14 x 734) bispecific antibodies (bsAbs) will be prepared and characterized. These bsAbs will be compared for tumor uptake and clearance pattern in GW-39 tumor bearing mice. The bsAb with the highest tumor uptake and high tumor/non-tumor (T/NT) ratio will be studied further to develop into an imaging system. The tumors will be targeted with the bsAb. After allowing a sufficient time for tumor accretion and a high T/NT ratio, 99m/Tc-IMP-192, will be injected. IMP-192, a peptidyl Tc-chelator, is formulated with excess indium and lyophilized into a single vial for radiolabeling with Tc-99m. Imp-192 contains two DTPA molecules for antibody recognition and clears rapidly from circulation. The dose of the bsAb and the Tc-99m labeled peptide will be optimized for pretargeted imaging. Reduced background is expected with this pretargeted approach. This optimized pretargeted approach will then later be applicable to pretargeted radioimmunotherapy using Re-188 or Re-186 instead of Tc-99m. Re-188 radiolabeling will require reformulation of the peptide. PROPOSED COMMERCIAL APPLICATION: Final product is envisaged as a two-vial imaging kit for the diagnosis of a variety of cancers. The first vial will contain the bispecific antibody to target the tumors. The second vial will contain a lyophilized peptidyl Tc-99m chelator for instant radiolabeling with Tc-99m. For therapy, Tc-99m will be switched to Re-1898 and peptide will be reformulated.

Alcohol drinking is influenced by sensitivity to the rewarding effects of alcohol, with increased sensitivity to the low-dose stimulating effects of ethanol (EtOH) being associated with high alcohol preference. Understanding neuronal mechanisms underlying the reinforcing effects of EtOH will greatly advance our knowledge of CMS neurobiological factors that promote high alcohol drinking behavior. The goals of this project are to better understand how neuronal circuits within the meso-cortico-limbic dopamine (DA) system are involved in regulating the reinforcing effects of EtOH. The ventral tegmental area (VTA) dopamine (DA) system has a critical role in the brain reward system, and in mediating the actions of most drugs of abuse, including alcohol. The overall hypothesis to be tested is that "neuronal circuits within the meso-cortico-limbic DA system mediate the response-contingent behavior for the self-infusion of EtOH into the posterior VTA." This hypothesis will be tested using site selective microinjection, intra-cranial self-administration (ICSA), and in vivo microdialysis techniques. Male Wistar rats will be used in this project. ICSA findings indicate that the posterior VTA is a neuro-anatomical site supporting the reinforcing actions of EtOH, and that activation of DA neurons may underlie these reinforcing effects. The first aim will extend current studies to examine the involvement of serotonin-2 (5-HT-2) and nicotinic (NIC) receptors in mediating EtOH self-infusion into the posterior VTA. The second aim will use the combination of ICSA and microdialysis to determine the effects of infusions of EtOH into the posterior VTA on the release of DA in four VTA target sites (i.e., nucleus accumbens shell, central nucleus of the amygdala, medial prefrontal cortex and ventral pallidum). The third aim will use microinjections of DA antagonists into VTA target sites in combination with ICSA of EtOH to determine the involvement of DA receptors in mediating the response-contingent behavior for the self- infusion of EtOH into the posterior VTA. Overall, the results of this project will provide important information on neuronal mechanisms underlying the rewarding actions of alcohol, which could greatly aid the development of pharmaco-therapies for the treatment of alcoholism and alcohol abuse.

This is an exploratory (R21) grant to examine the involvement of acetaldehyde (ACD) and tetrahydroisoquinolines (THIQs) in alcohol addiction. The overall hypothesis to be tested is that the formation of ACD and THIQs contribute to alcohol addiction. The goals of the present application are to examine the reinforcing effects of ACD and salsolinol (SAL) within the mesolimbic dopamine (DA) system of the rat. The following hypotheses will be tested in the present proposal: (a) ACID and SAL are reinforcing within the ventral tegmental area (VTA) of alcohol-preferring (P) rats; (b) SAL is reinforcing within the nucleus accumbens (NAC) shell of P rats; and (c) ACD and SAL potentiate ethanol reinforcement within the VTA of P rats. These hypotheses will be tested using the intracranial self-administration (ICSA) technique to examine the reinforcing actions of ACD and/or SAL within the VTA and/or NAC of P rats. Preliminary data indicate that female P rats self- administer 6 - 90 NM ACD directly into the posterior VTA. The present application will extend these findings using male P and Wistar rats. Specific aim 1 will examine the dose-response effects of ACD within the anterior VTA, as well as in regions adjacent to the VTA, to establish the site for the reinforcing effects of ACD. This aim will also examine extinction and reinstatement for the ICSA of ACD in the posterior VTA. In addition, specific aim 1 will compare the dose-response effects for the ICSA of ACD in the posterior VTA of P and Wistar rats. Specific aim 2 will examine the dose-response effects of SAL within the VTA and NAC of P rats. Specific aim 3 will examine the effects of ACD or SAL on the reinforcing effects of ethanol in the posterior VTA of P rats. In addition, this aim will examine whether conversion of ethanol to ACD may under- lie the ICSA of ethanol into the posterior VTA of P rats by co-infusing a catalase inhibitor with ethanol. The results of this study will provide information on the effects of ACD and SAL within the mesolimbic system, which could indicate a direct reinforcing effect of these compounds themselves and/or a potentiating effect of these compounds on the reinforcing properties of ethanol. Such results could provide valuable information on novel mechanisms underlying the addictive properties of alcohol and would provide a firm scientific foundation for future neurobiological studies.

This application is a competing renewal for an institutional research training program on the "Genetic Aspects of Alcoholism." Among the institutional training grants funded by NIAAA, our training program is one of five devoted to genetics of alcoholism. The main focus of research training is on the genetic, biological, and molecular basis of high alcohol-seeking behavior. Major topics of research include: neuronal mechanisms responsible for the reinforcing effects of ethanol; the genetics of alcohol metabolizing enzymes and receptors related to neurotransmission; the genetics (including QTL and finer mapping analyses) of alcohol preference and correlated phenotypes in selectively bred rat and mouse lines; studies of factors that regulate the expression of genes relevant to alcoholism; analysis of the extent that genetically-influenced biobehavioral factors such as disinhibition/impulsivity and acute tolerance/insensitivity contribute to alcoholism risks in selectively bred rodents and in human populations; and the use of an alcohol clamp method to assess the effects of genetics (MZ twins and FHP vs FHN subjects) and recent drinking history on electrophysiologic, neuroendocrine, and subjective assessment of alcohol experience. Other topics of interest include; structure and function of mutated forms of alcohol and aldehyde dehydrogenases; the relationship of ethanol exposure to retinoid metabolism, the effect of ethanol on lipoprotein metabolism; and neurodevelopmental abnormalities of fetal alcohol syndrome in mouse and rat models. One major strength of our training program is the long history of collaboration with both M.D.S. and PhDs in research and research training and the involvement of multiple scientific disciplines. A number of important resources are available to foster research training; the selective bred P/NP, HAD/LAD, HARF/LARF rat lines and the HAP/LAP mouse lines that differ in alcohol preference, a large collection of DNA samples extracted from different human populations relevant for genetic studies of alcoholism and alcohol-related end-organ injuries, and NIAAA-funded Alcohol Research Center (Molecular Biology Core, Animal Production Core, Structural and Cellular Biology Core, Mouse Selective Breeding Component, and two Human Genetics Cores, a group of IRPG grants (they replaced a former Program Project grant) that focuses on identification of genes and neurobiological mechanisms underlying high alcohol seeking behavior through studies of the HAD/LAD and P/NP rats, and the participation as one of six sites for the "Collaborative Studies on the Genetics of Alcoholism (COGA)." The trainees for each year will include five predoctoral, five postdoctoral fellows (with one M.D. and four Ph.D. trainees), and six undergraduate students on short-term training during their summer or "off quarter". Training will be through an apprenticeship mode but seminars, research conferences and hands-on method courses are amply available.

DESCRIPTION (provided by applicant): The long-range goals of this 5-year proposal are to understand, in the CNS of genetically vulnerable subjects, the complex neuronal alterations that occur as a result of alcohol exposure, which promote high alcohol drinking behavior and relapse. The overall hypothesis is that, in individuals with a genetic predis-position for high alcohol drinking behavior, certain CNS neuronal circuits are susceptible to alterations by alcohol and, as a result, high alcohol drinking is initiated and maintained, and alcohol relapse drinking is promoted. The goals of the present proposal are to determine the long-term changes in gene and protein expression produced by voluntary alcohol drinking that may be associated with high alcohol drinking and relapse. Inbred adult male alcohol preferring (iP), high-alcohol-drinking (iliAD), alcohol-non-preferring (iNP) and low-alcohol-drinking (iLAD) rats will be used for these studies. The microchip expression array will be used to examine changes in gene expression, and 2-dimensional gel electrophoresis procedures will be used to examine changes in protein expression. The first 2 specific aims will compare innate differences between iP and iNP rats and between replicate iliAD and iLAD rats, and determine the effects of voluntary alcohol drinking by iP and iliAD rats on changes in gene and protein expression in the nucleus accumbens, prefrontal cortex and posterior hippocampus. The second 2 specific aims will examine the effects of operant oral alcohol self-administration, extinction training and reinstatement on gene and protein expression in these limbic regions of the iP and iliAD rats. This research component integrates well with the Center's theme of studying genetic determinants of alcohol ingestion. The proposed project interacts directly with the following cores: Administrative, Animal Production, Genomics and Molecular Biology, and Proteomics. This rat genetic component complements the 2 research components dealing with Human Genetics, and scientifically interacts with the Mouse Selective Breeding and Genetics Component. The results of this project will provide valuable information on mechanisms underlying alcohol addiction, which could lead to the development of pharmacotherapies for the treatment of alcoholism and alcohol abuse.

DESCRIPTION (provided by applicant): Our primary interest is to develop an imaging method based on bispecific antibody (bsMAb) pretargeting used in combination with F-18 labeled peptides. Such a method would improve the specificity for PET imaging based on the bsMAb's reactivity with either tumor antigens or other markers. This imaging method would take advantage of excellent sensitivity of PET and the high tumor to non-tumor ratios provided by bsMab pretargeting. We have previously described a novel bsMAb pretargeting system based on the hapten binding specificity of an antibody directed to histamine-succinyl-glycine (HSG; 32). Using this system, radiolabeled peptides can be prepared with a variety of radioisotopes for use in imaging and therapy. This pretargeting system has excellent tumor uptake and high tumor/nontumor ratios within 1-3 hours of the peptide injection, and has substantially less renal uptake than directly radiolabeled antibody fragments (e.g., Fab' or scFv). Given the proven success of this pretargeting system, the primary goal of this Phase I application is to examine methods for preparing peptides suitable for use with F-18, the most commonly used radionuclide for PET imaging. The main focus will be to choose a linker to attached the F-18 to a peptide through the F-18 labeled molecule 4-[F-18]fluorobenzaldehyde. Several potential synthetic pathways will be examined with respect to the facile use, yield of product, specific activity, stability, retention of binding to the anti-hapten component of the bsMAb and ease of purification. The targeting peptide will carry two HSG haptens to stabilize binding on the tumor cell surface. Most of the preclinical work has been done with a bsMab that binds to the colo-rectal tumor marker carcinoembryonic antigen and HSG. The proof of principal for this work will be tested using a bsMAb that targets the human pancreatic tumor antigen, MUC1 and HSG. The goal is to help improve the early detection and diagnosis of pancreatic cancer as well as determine the extent of the disease. It is anticipated that following successful synthesis a SBIR Phase II grant would support the optimization, automation and the scale-up of the 4-[F-18]fluorobenzaldehyde conjugation to the peptide to a commercial scale. It would also support the preclinical targeting, synthesis of new peptide derivatives to optimize excretion, serum stablity and safety studies in animals that would be needed before this agent goes into the clinic.

alcoholism /alcohol abuse; mass spectrometry; two dimensional gel electrophoresis

DESCRIPTION (provided by applicant): The long-range goals of this proposal are to better understand brain mechanisms underlying high alcohol- seeking behavior. The overall hypothesis to be tested is that the ventral tegmental area (VTA), nucleus accumbens (ACB), medial prefrontal cortex (MPF), basolateral amygdala (BLA) and ventral subiculum (vSUB) are involved in mediating alcohol-seeking behavior. Alcohol-seeking behavior is a result of chronic ethanol self-administration, which is expressed in the absence of alcohol, in response to cues, priming or stress. Different brain mechanisms may underlie drug-self-administration and drug-seeking behavior (reviewed in Kalivas and McFarland, 2003), although little has been done to address this difference with regard to ethanol (EtOH). Recent data from our laboratory and from other studies (Samson and Chappell 2004;Liu and Weiss 2004) support the idea that different CNS mechanisms may be regulating EtOH self- administration and EtOH-seeking behavior. The overall hypothesis will be tested using mainly the alcohol- preferring (P) line of rats;however, comparative studies will be conducted with high-alcohol-drinking (HAD) and Wistar rats. The P rat meets the criteria proposed by Cicero (1979) as a suitable animal model of alcoholism, and these rats express robust contextual cue-induced EtOH-seeking behavior (Rodd-Henricks et al, 2002a,b). Operant techniques will be used to measure EtOH self-administration and EtOH-seeking behavior. In AIM 1, c-fos and fos-B immunocytochemistry will be used to identify CNS sites and neuronal populations involved in EtOH-seeking behavior. In AIM 2, microinjection techniques in combination with operant measures of cue-induced EtOH-seeking behavior will be used to identify discrete CNS sub-regions and receptors (primarily dopamine and glutamate) involved in the expression of EtOH-seeking behavior. In AIM 3, no-net-flux and conventional microdialysis techniques will be used to assess long-term effects of chronic EtOH self-administration on dopamine (DA) neurotransmission in different VTA pathways, and the relationship between the activity of these DA systems and expression of EtOH-seeking behavior. The results of this project will provide important and novel information on CNS sub-regions, neuronal pathways, and receptors involved in EtOH-seeking behavior, which could provide new avenues for developing unique pharmacotherapeutic treatments for reducing craving and preventing alcohol relapse.

[unreadable] DESCRIPTION (provided by applicant): The long-range goals of this proposal are to better understand the molecular neurobiological events that underlie the development of excessive alcohol drinking, and contribute to the long-range consequences of excessive alcohol drinking. The overall hypothesis to be tested is that changes in the expression of genes involved in neurotransmission, neuroplasticity, and intracellular signaling pathways within discrete regions of the extended amydala (E-AMYG) contribute to the development of excessive alcohol drinking. Excessive drinking is defined as sustainable blood alcohol concentrations (BACs) in the range of 100-150 mg% that are repeatedly attained over a chronic period. The overall hypothesis will be tested using selectively bred alcohol-preferring (P) and high-alcohol-drinking (HAD) rats. Micro-punch techniques will be used to obtain samples containing the nucleus accumbens shell (ACB-sh) and central nucleus of the amygdala (CeA). Changes in gene expression will be determined using Affymetrix microarrays. RT-PCR and in situ hybridization will be used to verify key findings from the microarray experiments. The excessive alcoholdrinking paradigm to be used will be the 'drinking in the dark multiple scheduled access' (DID-MSA) procedure. Time-course changes in gene expression will be determined following a drinking episode, and during the development of excessive alcohol drinking. The specific aims will be designed to determine changes in gene expression within the ACB-sh and CeA of P and HAD rats prior to, during initiation of, and following development of excessive alcohol drinking. The effects of chronic alcohol exposure and the development of excessive alcohol drinking are influenced by multiple genetic and environmental factors. This project will provide important molecular neurobiological information in discrete regions of the E-AMYG of genetically vulnerable subjects that could more comprehensively describe the complex inter- and intracellular events leading to the development, maintenance, and consequences of excessive alcohol drinking. Such information would be critically important for developing pharmacotherapeutic strategies to treat alcoholism and alcohol abuse. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): This application is a competing renewal for an Institutional Ruth L. Kirschstein NRSA on the Genetic Aspects of Alcoholism. The objective of this training program is to provide high level predoctoral and postdoctoral research training on various aspects of the genetics of alcoholism, and mechanisms underlying high alcohol drinking behavior. The main focus of the research training is on the genetic, biological and molecular basis of high alcohol -drinking and -seeking behaviors. Major topics of research include neuronal mechanisms underlying high alcohol-drinking and -seeking behavior; the genetics of alcohol preference in selectively bred rodent lines; studies of factors that regulate the expression of genes relevant to alcoholism; analysis of the extent that genetically-influenced biobehavioral factors such as disinhibition-impulsivity and tolerance contribute to alcoholism risks in human populations; effects of genetics and alcohol drinking on protein and gene expression in selectively bred rodent lines; neuropsycho-pharmacological and neuroimaging studies on alcohol craving in humans and rodents; co-morbidity of alcohol addiction and schizophrenia in rodent models; mechanisms and genetics underlying co-abuse of alcohol and nicotine; and neurodevelopmental abnormalities of fetal alcohol syndrome in rodent models. The rationale for the research training program is that we do not yet fully understand how genetics influence alcohol drinking behavior. This is a very important topic and the field needs highly trained research investigators proficient in the newest genetic, molecular and behavioral neuroscience approaches. In addition, our program offers translational research training, encompassing both human and animal studies. The program is designed to give the trainee exposure to and participation in high- powered research projects in which state-of-the-art methodologies are used. We expect to support 7 predoctoral trainees (after their first 2 years of graduate study), and 3 postdoctoral trainees (usually with 0-1 year of post-graduate experience). We anticipate supporting trainees for at least 3 years (some predoctoral trainees may take 1 or 2 additional years). RELEVANCE: There is convincing evidence that alcoholism runs in families. Identifying the genes and neurobiological systems that contribute to alcohol use and abuse would greatly contribute toward understanding and treating alcohol use disorders.

[unreadable] DESCRIPTION (provided by applicant): The long-range goals of this proposal are to better understand the molecular neurobiological events that underlie the development of excessive alcohol drinking, and contribute to the long-range consequences of excessive alcohol drinking. The overall hypothesis to be tested is that changes in the expression of genes involved in neurotransmission, neuroplasticity, and intracellular signaling pathways within discrete regions of the extended amydala (E-AMYG) contribute to the development of excessive alcohol drinking. Excessive drinking is defined as sustainable blood alcohol concentrations (BACs) in the range of 100-150 mg% that are repeatedly attained over a chronic period. The overall hypothesis will be tested using selectively bred alcohol-preferring (P) and high-alcohol-drinking (HAD) rats. Micro-punch techniques will be used to obtain samples containing the nucleus accumbens shell (ACB-sh) and central nucleus of the amygdala (CeA). Changes in gene expression will be determined using Affymetrix microarrays. RT-PCR and in situ hybridization will be used to verify key findings from the microarray experiments. The excessive alcoholdrinking paradigm to be used will be the 'drinking in the dark multiple scheduled access' (DID-MSA) procedure. Time-course changes in gene expression will be determined following a drinking episode, and during the development of excessive alcohol drinking. The specific aims will be designed to determine changes in gene expression within the ACB-sh and CeA of P and HAD rats prior to, during initiation of, and following development of excessive alcohol drinking. The effects of chronic alcohol exposure and the development of excessive alcohol drinking are influenced by multiple genetic and environmental factors. This project will provide important molecular neurobiological information in discrete regions of the E-AMYG of genetically vulnerable subjects that could more comprehensively describe the complex inter- and intracellular events leading to the development, maintenance, and consequences of excessive alcohol drinking. Such information would be critically important for developing pharmacotherapeutic strategies to treat alcoholism and alcohol abuse. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Our primary interest is to develop an imaging method based on bispecific antibody (bsMAb) pretargeting used in combination with F-18 labeled peptides. Such a method would improve the specificity for PET imaging based on the bsMAb's reactivity with either tumor antigens or other markers. This imaging method would take advantage of excellent sensitivity of PET and the high tumor to non-tumor ratios provided by bsMab pretargeting. We have previously described a novel bsMAb pretargeting system based on the hapten binding specificity of an antibody directed to histamine-succinyl-glycine (HSG). Using this system, radiolabeled peptides can be prepared with a variety of radioisotopes for use in imaging and therapy. This pretargeting system has excellent tumor uptake and high tumor/nontumor ratios within 1-3 hours of the peptide injection, and has substantially less renal uptake than directly radiolabeled antibody fragments (e.g., Fab'or scFv). Given the proven success of this pretargeting system, the primary goal of this Phase II application is to examine methods of attaching the most commonly used radionuclide for PET imaging, F-18, to a peptide. The main focus in the first year will be to further develop the novel method of attaching F-18 to a peptide that was disclosed in the Phase I report. Several modifications of the method will be examined with respect to the facile use, yield of product, specific activity, stability and ease of purification. The targeting peptide will carry two HSG haptens to stabilize binding on the tumor cell surface (11). Most of the preclinical work has been done with a bsMab that binds to the colo-rectal tumor marker carcinoembryonic antigen and HSG. The proof of principal for this work will be tested using a bsMAb (TF10) that targets the human pancreatic tumor antigen, MUC1 and HSG. The goal is to help improve the early detection and diagnosis of pancreatic cancer as well as determine the extent of the disease. PUBLIC HEALTH RELEVANCE: The first goal of this work is to develop a general, simple method of attaching the PET imaging isotope, F-18, to peptides. The second goal of this work is to deliver the F-18 labeled peptide specifically to a tumor while at the same time delivering minimal activity to normal tissues by first administering a novel, genetically engineered, trivalent bispecific antibody that binds specifically to the target tissue of interest (in this case pancreatic cancer), allowing the unbound antibody to clear from the blood (days) and then injecting the F-18 labeled peptide, which contains two groups that bind to the targeted antibody. The injected peptide rapidly localizes to the bispecific antibody on the tumor surface and the portion of the peptide that does not bind to the tumor is rapidly (minutes) cleared from the blood, minimizing uptake in normal tissues thus increasing the contrast between the tumor and normal tissues.

DESCRIPTION (provided by applicant): Project Summary/Abstract: This is a GO application to ddress "Mechanisms of Alcohol and Nicotine Co-Dependence" as the area of scientific priority. The GO mechanism is used because this application requires a multi-lab approach to accelerate current and future research. The objective of this proposal is to provide a better understanding of the mechanisms underlying the interactions of the reinforcing effects of ethanol (EtOH) and nicotine (NIC) in promoting co-abuse. The overall hypothesis to be tested is that administration of EtOH or NIC will produce cross-sensitization to the reinforcing effects of the other, which promotes the potential for their co-abuse. The ventral tegmental area (VTA) is a site supporting the reinforcing actions of EtOH and NIC, and is an excellent starting point for studying the interactions of EtOH and NIC. Since genetic factors contribute to the abuse potential of drinking and smoking, an animal model that has demonstrated both EtOH and NIC abuse independently will be used, i.e., the selectively bred alcohol-preferring (P) rat. Operant techniques will be used for the intra-cranial self-administration and i.v. NIC self-administration experiments. Microdialysis-HPLC procedures will be used to measure extracellular levels of dopamine (DA) and glutamate. Targeted gene expression changes will be measured with quantitative RT-PCR. The aims are designed to determine the effects of EtOH and NIC administration on the sensitivity of the mesolimbic system to the reinforcing effects of the other drug, if these reinforcing effects are associated with increased response of DA neurons to the drug, and whether EtOH and NIC interact synergistically. Another aim is designed to establish an animal model of co-abuse of EtOH and NIC, and to determine the effects of co-abuse of EtOH and NIC on the expression of genes for addiction-associated receptors or receptor subunits for DA, GABA, glutamate, 5-HT and nicotinic systems. Another aim is designed to identify changes in the activity of DA and glutamatergic neuronal pathways within the mesolimbic system that are associated with EtOH and NIC co-abuse. Overall, the results of this project will provide a better understanding of the neurobiological basis for the interactions of EtOH and NIC that contribute to their co-abuse. This is an important initial step toward developing treatment strategies to reduce their use and co-abuse. Since alcohol drinking and smoking affect millions of people, this project has potential far reaching impact on improving the health and lives of many individuals. PUBLIC HEALTH RELEVANCE: The co-use of alcohol and tobacco is common and affects millions of people. When used together, alcohol and tobacco compound the health risks found with their individual use. The long-range goals of this project are to better understand the brain mechanisms underlying the co-abuse of alcohol and nicotine, so that treatment strategies can be developed to reduce or prevent their use.

DESCRIPTION (provided by applicant): Alcoholics go through multiple detoxifications with each episode being more severe and the likelihood of relapse higher with each episode. In addition, cues associated with alcohol drinking can enhance 'craving' and precipitate relapse drinking. The overall goals of this project are to determine neuronal mechanisms underlying cue-induced ethanol (EtOH)-seeking behavior and the effects that repeated deprivation cycles have on these mechanisms. The overall hypothesis is that increased activities of dopamine (DA) and glutamate (Glu) pathways within the meso-cortico-limbic system underlie cue-induced EtOH-seeking behavior and the activities of these pathways are further enhanced with repeated deprivation cycles. A Pavlovian Spontaneous Recovery (PSR) test will be used to measure expression of EtOH-seeking behavior in alcohol-preferring (P) rats following prolonged abstinence. Repeated 2-week cycles of abstinence and operant ethanol access will be used to evaluate the effects of repeated deprivations on cue-induced EtOH-seeking behavior. No-net-flux (NNF) and conventional in vivo microdialysis techniques will be used to examine the involvement of DA, Glu and GABA systems in EtOH-seeking behavior, and changes in these systems associated with distinct cues and repeated deprivations. Microinjection procedures will be used to identify pathways and receptors involved in regulating EtOH-seeking behavior. Aim 1 will determine the interactions of odor cues and repeated deprivations on expression of EtOH-seeking behavior and transmitter release. Aim 2 will assess the effects of repeated deprivations on basal neurotransmission of DA, Glu and GABA within the meso-limbic system that persists in the absence of EtOH. Aim 3 will use microinjection techniques to assess the involvement of different receptors in regulating cue-induced EtOH-seeking behavior. Overall, this project will provide important information on neuronal mechanisms involved in regulating cue-induced EtOH-seeking behavior and the impact of repeated deprivations on these neuronal systems.