Norbert Kaminski - US grants

Derived from the plant Cannabis sativa, marijuana is a widely used drug that is primarily known for its psychoactive properties. The primary psychoactive component, delta-9-tetrahydrocannabinol (delta-9-THC), is responsible for its many behavioral and physiological effects which include analgesia as well as changes in memory, cognition, psychomotor skills, mood, perception and immunosuppression. Structure activity relationship (SAR) studies combined with the recent cloning of a G protein-coupled cannabinoid receptor identified in regions of the brain suggests that cannabimimetic agents mediate their CNS effects through specific receptor- ligand interactions. The overall goal of this project is to investigate the mechanisms by which cannabinoid compounds produce immune inhibition by testing the following hypothesis: Immunosuppression by cannabimimetic agents is mediated through a G-protein coupled cannabinoid receptor present on lymphoid cells which when stimulated by cannabimimetic agents produces an intracellular increase in cAMP. The basis for this proposal are five key observations from work in our laboratory supporting the role for a cannabinoid receptor in immunosuppression: (1) [3H]-CP-55,940, demonstrates a high degree of specific binding to lymphoid cells in radioligand binding studies; (2) SAR studies demonstrate that synthetic cannabinoid compounds have a similar rank order of immunoinhibitory potency to that reported with respect to their CNS related effects; (3) cannabinoids demonstrate enantioselective immunosuppression; (4) delta-9- THC and CP-55,940 markedly enhance forskolin-induced intracellular cAMP in lymphoid cells; (5) T-cells are more sensitive to immune inhibition by delta-9-THC than B-cells and macrophages which supports a cAMP-associated mechanism of immunosuppression. Our hypothesis will be tested using the following specific aims: (1) Changes in the phenotypic composition of murine spleen cells isolated from delta-9-THC-treated mice will be characterized by fluorescence activated cell sorting (FACS) analysis; (2) Enantioselective immune inhibition by cannabinoid agents will be further characterized; (3) Cannabinoid receptors on mouse and human lymphoid cells will be verified for the presence of unique regions of mRNA associated with the putative receptor as well as the expression of this receptor using radioligand studies; (4) Modulation of intracellular cAMP through a G protein-coupled mechanism by cannabimimetic agents will be characterized in mouse spleen cells; and (5) the effects of cannabinoids on human lymphoid cell immune function and modulation of cAMP will be investigated.

Pharmacodynamic tolerance, which is characterized by adaptive changes within affected systems, has been widely described for many structurally diverse agents among which drugs of abuse have been the most extensively studies. Cannabinoids, including the primary psychoactive component of marihuana, delta9-THC, are agents which have been established to readily produce this phenomenon. Despite the fact that tolerance has been described ina variety of experimental systems, the mechanisms responsible for these adaptive changes are not well understood. The overall goal of this 4 year research plan is to utilize=e an immunologic experimental model system to investigate cellular elements responsible for signal transduction through the cannabinoid receptor to identify their role in mediating cannabinoid-induced tolerance by testing the following Hypothesis: Tolerance to immune suppression by delta9-THC is mediated through a decrease in signaling through the cannabinoid receptor present on lymphocytes. This diminution in receptor associated signal transduction is mediated either through desensitization and/or down regulation of cannabinoid receptors following extended receptor agonist stimulation. This hypothesis is based on the following critical observations made from work conducted in our laboratory: (1) Mouse splenocytes possess a functional G-protein coupled cannabinoid receptor which is responsible for mediating the immunosuppressive effects associated with exposure to cannabimimetic agents; (2) Characterization thus far of this receptor on spleen cells strongly suggests that it is very similar if not identical to that present in neuronal tissue; (3) Immune suppression by cannabinoids is at least in part mediated through inhibition of adenylate cyclase activity in mouse spleen cells; (4) Tolerance of splenocytes to immune inhibition by delta9-THC is readily induced by chronic delta9-THC treatment of mice or through the incubation of spleen cells in vitro in the presence of delta9- THC; and (5) Immune tolerance to delta9-THC is paralleled by a loss in the ability of cannabinoids to inhibit adenylate cyclase in splenocytes. Based on these observation, our hypothesis as it relates to cannabinoid-induced tolerance will be tested using the following specific aims; (1) Compare changes in receptor function in tolerized and nontolerized splenocytes, specifically, (a) cannabinoid receptors number (Kd); (b) affinity of receptor-ligand binding (Bmax); and (c0 cannabinoid receptor mRNA; (2) Investigate the mechanism responsible for the loss of adenylate cyclase inhibition by delta9-THC following chronic delta9-THC exposure in murine lymphocytes; (30 Identify differences in Gi protein function between tolerized and nontolerized lymphocytes; and (40 Identify differences in protein kinase A activity between tolerized and nontolerized lymphocytes in the presence and absence of cannabinoids. One of the greatest strengths of this proposal is the model system which; (1) capitalizes on the use of isolated cells in primary culture, rather than cell-lines; (2) possess a functional receptor which is coupled to an identified second messenger system; and (3) can be manipulated both in vivo and in vitro in order to correlate biochemical changes to functional changes as they relate to the mechanisms of tolerance.

The overall goals of this Project are to determine how selected Superfund chemicals influence immunity and inflammation and to understand the mechanisms and potential health consequences of such effects. The aims for the next granting period were driven by a need for knowledge of interactions among chemicals and the specific and nonspecific components of the immune system. Specific hypotheses to be tested derive from the recent findings of the investigators of the three subprojects. This Subproject is a new addition based on the observation that administration to animals of certain hepatotoxicants at doses that cause modest liver pathology results in release of a serum factor that is immunosuppressive. Recently, this factor has been identified as transforming growth factor-beta(TGF-beta). During the next granting period, a battery of functional assays will be used to determine whether alterations in immunocompetence are mediated by TGF-beta alone or involve other factors, such as overproduction of interleukin-2 by helper T-cells. The studies proposed in this Project will employ cellular, isolated organ and whole animal models and entail molecular, biochemical and morphologic methods that include techniques such as cell imaging and flow cytometry. Accomplishing goals of this Project as well as aiding in the toxicologic evaluation of products of remediation will involve considerable interaction not only among Project investigators but also with investigator of other projects and cores. Results from these studies will increase understanding of the mechanisms and potential health consequences of interactions of Superfund chemicals with the immune system.

Previous results from our laboratory indicate that immune suppression by cannabinoids is mediated through cannabinoid receptors expressed on immunocompetent cells, most notably T-cells which we have found to exhibits a marked sensitivity. This is indicated by an inhibition of T-cell mediated responses and Il-2 expression in the presence of cannabinoids. We have also shown that ligand binding to cannabinoid receptors inhibits cAMP signal transduction in T-cells as evidenced by decreased; (i) cAMP formation; (ii) protein kinase A activity; and (iii) binding by CREB/ATF transcription factors to their cognate DNA binding site, cAMP responsive element (CRE). CREB/ATF regulatory proteins also dimerize with Fos and Jun family members to help regulate gene transcription at AP-1 sites (e.g., IL- 2). Our studies suggest that the inhibition of cAMP signaling is responsible for immune inhibition by cannabinoids based on three critical observations: 91) membrane permeable cAMP analogs reversed the immunoinhibitory effects produced by cannabinoids; (ii) pertussis toxin pretreatment of splenocytes abrogated cannbinoid-induced immune suppression and inhibition of adenylate cyclase; and (iii) glucagon, which stimulates adenylate cyclase activity through interaction with its own G-protein coupled receptor, reversed the inhibitory effects produced by cannabinoids. Based on the observations described above our present investigation will have two objectives; (1) to identify which specific cAMP-regulated transcription factors are inhibited by cannabinoids during T-cell activation; and (2) to identify specific cAMP regulated genes that exhibit altered expression during T-cell activation in athe presence of cannabinoids. In SA#1, we will identify the DNA binding proteins within the CREB/ATF family of transcription regulators that are involved in T-cell activation and which concomitantly exhibit decreased DNA binding in the presence of cannabinoids. In SA#2, we will characterize the specific time course and magnitude of transcriptional dysregulation at CRE and AP-1 DNA regulatory sites by cannabinoids following T-cell activation using CRE and AP-1 luciferase reporter constructs. In SA#3, we will characterize cannabinoid-mediated inhibition of IL-2 gene expression using I-2 luciferase reporter constructs. In SA#4, we will identify cAMP-regulated genes involved in T-cell activation whose expression is altered by cannabinoid-treatment. Lastly, in SA#5 we will, characterize genes involved in T-cell activation that exhibit sensitivity to dysregulation by cannabinoids.

The overall goal of this five year research plan is to test the Hypothesis: TCDD acts directly on B-cells to suppress immunoglobulin secretion. This suppression is mediated by the AhR which adversely regulates immunologically relevant genes possessing DREs in their 5' regulatory regions. Toward characterizing the putative role by the AhR in TCDD-mediated B-cell dysfunction, our results have shown that mouse leukocytes and purified B-cells express functional Ah receptor and ARNT as evidenced by (a) Western blot; (b) increased DRE binding in the presence of TCDD, and (c) induction of cytochrome-P450 1A1 (Cyp1A1) gene expression. Moreover, we demonstrated that activation of mouse splenocytis, or primary B-cells, produced a marked increase in AhR mRNA and protein following activation. The significance of AhR up-regulation is unclear but may explain the sensitivity of immunocompetent cells to TCDD. Identical results were observed in the CH12.LX B-cell line following LPS activation. In the presence of TCDD CH12.LX cells exhibited inhibition in LPS-induced immunoglobulin secretion and induction of Cyp1A1. A second B-cell line, BCL-1, possesses ARNT but is AhR-deficient. BCL-1 cells in the presence of TCDD exhibited no inhibition of LPS-induced immunoglobulin secretion and no induction of Cyp1A1. Most recently, we have also demonstrated that TCDD induced a marked increase in the tumor suppressor gene and transcription factor, p53 which we believe plays a critical role in B-cell dysfunction by TCDD. In light of the above findings. We will test our hypothesis using the following specific aims (SA): In SA number1, we will characterize the structure activity relationship between TCDD-mediated inhibition of immunoglobulin secretion and heavy chain mRNA expression versus the regulation of gene transcription through the DRE. In SA number2, we will determine whether transfection of the AhR into an AhR-deficient cell-line (BCL-1) can confer sensitivity to TCDD. In SA number3, we will determine whether TCDD treatment of B-cells interferes with the ability of the transcription factor, B-cell specific activator protein (BSAP), in binding to its consensus recognition sequence. In, SA number4, we will characterize the role of premature p53 upregulation by TCDD in B-cells on cell cycle progression and immunoglobulin secretion. Lastly, in SA number5 we will determine whether up-regulation of p53 by TCDD in B-cells is mediated through the binding of the AhR/ARNT complex to a DRE-like site in the p53 promotor.

The overall goals of this 4 year research plan are two-fold: (a) to characterize the role of cannabinoid receptors CB1 and CB2 in the modulation of T-cell function by endogenous eicosinoid-like cannabinoids; and (b) to elucidate the signal transduction pathways by which these endocannabinoids disrupt T-cell activation. The significance of this goal is that it will provide direct insight into the role of cannabinoid receptors, and their naturally occurring ligands, in the regulation of both immunologic homeostasis and effector function. To date a number of putative eicosinoid-like cannabinoids have been identified by competitive binding as endogenous cannabinoid receptor ligands. The most extensively characterized with respect to immune modulation are arachidonylethanolamide (anandamide; Ana) and 2-arachidonyl- glycerol (2-Ara-Gl). Both ligands bind to CB1 and CB2 and induce a variety of cannabimimetic responses. At the biochemical level, the most significant similarities include inhibition of adenylate cyclase, repression of DNA binding by certain specific transcription factors critical for leukocyte activation, and the inhibition of IL-2 gene expression. At the cellular level, similarities to plant-derived cannabinoids include suppression of a variety of immunologic responses. Specifically, inhibition of T- cell and B-cell proliferation, decreased recognition of class II MHC (mixed lymphocyte response), and the inhibition of IL-2 secretion. Based on the observations described above, our present investigation will test the hypothesis: Modulation of T-cell activity by endogenous cannabinoid receptor ligands, anandamide and 2-arachidonyl-glycerol, is mediated directly through cannabinoid receptors, CB1 and/or CB2, resulting in disruption T-cell activation. We will test our hypothesis using the following specific aims (SA): In SA#l we will characterize the role of CB l and CB2 in the inhibition of IL-2 expression by Ana and 2-Ara-Gl in activated T-cells. In SA#2 we will characterize the direct effects of Ana and 2-Ara-Gl on the regulation of NF-AT in activated T- cells. In SA#3 we will characterize, in the context of defined activation stimuli: (a) the inhibition of T-cell activation by endocannabinoids; and (b) the temporal relationship between exposure to endocannabinoids and inhibition of T-cell activation. In SA#4 we will characterize the direct effects of Ana and 2-Ara-Gl on PKC and MAP kinase activity.

Polychlorinated biphenyls (PCBs) modulate signal transductions cascades leading to diverse functional change with a variety of cells. The pathways activated or inhibited by PCBs are common among cell types and different between classes of congeners (i.e., coplanar vs. non-coplanar). It is generally accepted that coplanar PCBs mediate their effects through the aryl hydrocarbon receptor (AhR). However, non-coplanar PCBs are low affinity ligands for the AhR, and also exert biological effects including activation or disruption of signal transduction pathways leading to altered cell function. Previous results and preliminary data are discussed which suggest that immediate early response by terminally differentiated polymorphonuclear neutrophils (PMNs) are modulated by PCBs primarily in an AhR-independent manner. Moreover, the response by PMNs to PCB treatment is characterized by cellular activation. Conversely, B-cells, which are not terminally differentiated cells, are marked sensitivity to inhibition by PCBs. Moreover, the profile of PCB- mediated B-cell inhibition follows a structure-activity relationship concordant with AhR binding affinity. Collectively, these findings indicate that PCBs activate multiple signaling cascades in leukocytes and that the toxicity exerted by any given congener is cell type-dependent. In light of these findings, the overall goal of this 5 year research plan is to test the following HYPOTHESIS: In leukocytes, PCBs activate multiple signal transduction cascades dependently and independently of the AhR. Immediately-early leukocyte responses are altered by PCBs through the rapid modulation of protein kinases (i.e., Src and MAP kinases) both dependently and independently of the AhR; whereas, delayed responses are AhR-dependent and mediated transcriptionally through the regulation of genes under the control of DREs. A multifaceted approach will be used to test this hypothesis using the following specific aims (SA): In SA#1, we will determine the role of c-Src in PCB-mediated alterations of leukocyte function. In SA#2, we will characterize the role of MAP kinases in PCB-mediated alterations or leukocyte function. In SA#3, we will characterize the modulation of critical genes by PCBs as a putative mechanism of altered leukocyte function. Lastly, in SA#4 we will characterize the mechanism responsible for the antagonistic interactions between coplanar and non-coplanar PCB congeners in alterations of leukocyte function. We believe that the successful completion of these specific aims will provide important new insight into the basic mechanism(s) by which PCBs modulate the immune system.

DESCRIPTION (provided by applicant): Previous results from this laboratory demonstrate that T cells are markedly sensitive to modulation by cannabinoids as evidenced by altered interleukin-2 (IL-2) gene expression. Our results suggest that the molecular mechanism for IL-2 modulation by cannabinoids is complex and involves multiple signaling cascades whose modulation is both cannabinoid receptor-dependent and -independent. The following key observations support this premise: (a) cannabinoid-treatment induced a marked, sustained and concentration dependent elevation in intracellular ([Ca+2]i) that was attenuated by cannabinoid receptor antagonists; (b) concordant with elevated [Ca+ cannabinoid treatment disrupted the activity of the downstream effectors, NF-AT, AP-1, ERK MAP kinases and calcium/calmodulin-dependent kinase, CaM KII; and (c) cannabinoid treatment induced rapid nuclear translocation and DNA binding of glucocorticoid receptors (GR). Based on these and other findings, the overall goal of this five year research plan is to test the Hypothesis: Cannabinoid-induced inhibition of T cell activation, as assessed by IL-2 gene expression, is mediated concomitantly through cannabinoid receptor-dependent and -independent mechanisms involving sustained intracellular calcium elevation and activation of glucocorticoid receptors, respectively. Our hypothesis will be test using four specific aims (SA). In SA#1, we will characterize cannabinoid-mediated [Ca+ elevation and the role of CB 1 and/or CB2; In SA#2 we will characterize the activation of CaM KII by cannabinoid- mediated enhancement in [Ca2]. In SA#3 we will characterize the downstream consequences of cannabinoid-mediated enhanced of [Ca2] and CaM KII activation on IL-2 regulation. Lastly, in SA#4 we will characterize the role of cannabinoid-mediated GR activation in T cell dysfunction. The significance of the results from these studies is that they will provide important new insights into the molecular mechanism(s) responsible for cannabinoid-mediated immune suppression.

Support is requested to continue human health-oriented research on risks from exposure to chemicals commonly found in Superfund sites and on new clean-up technologies to eliminate the potential for exposure to chemicals from thos3e sites. The pollutants under investigation include relatively water soluble chemicals that contaminate groundwater such as trichloroethylene and environmentally persistent lipid soluble chemicals such as PCBs and PAHs. A highly integrated, multi-disciplinary research program is proposed consisting of nine research projects and four supporting core units. The research team of 24 investigators include faculty at nine research projects and four supporting core units. The research team of 24 investigators include faculty at Michigan State University (12), The University of Michigan (8), Stanford (2), Rutgers (1) ant the Oak Ridge National Laboratory (1). The major thrusts of the research are in four areas: 1) understanding the mechanisms of factors controlling the environmental persistence and bioavailability of contaminants to microbes involved in biodegradation, 3) determination of the effectiveness of purposefully altered (engineered) subsurface environments on the rates of biodegradation and 4) evaluation of chemical products produced from environments on the rates of biodegradation and 4) evaluation of chemical products produced from bioremediation for toxicity potential, 3) determination of the effectiveness of purposefully altered (engineered) subsurface environments on the rates of biodegradation and 4) evaluation of chemical products produced from bioremediation on the rate of biodegradation and 4) evaluation of chemical products produced from bioremediation for toxicity potential. The research projects classified as biomedical include studies examining the effects of PCBs to cause immunotoxicity, reproductive and developmental toxicity, and developmental neurotoxicity, Site remediation research includes reproductive bioremediation of groundwater contaminants, the synthesis and development of novel macrochemical to participate in retention of chemicals to participate in retention of chemicals in groundwater plumes containing halogenated solvents and metals, and an examination of factors that control the rate of groundwater and soil bioremediation. A core laboratory will coordinate the testing of the intermediate and end products generated from biodegradation of PCBs and other contaminants commonly found in mixed waste. This testing will permit modification of experimental remediation processes to maximize the detoxification of toxic mixtures found in the environment. A postdoctoral training program will involve cross training experiences to improve the ability of recent Ph.D. graduates to perform in multi-disciplinary research teams. An Outreach Core will partner with the community assistance component of an EPA Hazardous Substance Research Center at the University of Michigan to produce information materials for use in working with communities impacted by the threat of exposure to hazardous chemicals.

DESCRIPTION (provided by applicant): The overall goal of this 5 year research plan is to elucidate the molecular mechanism responsible for the modulation of T cell function and interleukin-2 (IL-2) deregulation by the structurally-related endocannabinoids, anandamide (AEA) and 2-arachidonyl glycerol (2-AG). Presently, the teleological role of the endocannabinoid system is unknown but there is a growing body of evidence suggesting that it may significantly contribute to the maintenance of immunologic homeostasis. Numerous studies have demonstrated profound effects on biological systems by AEA and 2-AG, with the immune system representing one of the most extensively characterized. The significance of the current proposed studies is that they will provide direct mechanistic insight into the molecular mechanism by which endocannabinoids modulate T cell function, specifically IL-2 regulation. Novel preliminary results are presented demonstrating that IL-2 suppression by both AEA and 2-AG are dependent on COX-2 metabolism leading to the activation of the nuclear receptor, peroxisome proliferator activated receptor gamma (PPARgamma), independently of CB1 and CB2. Additional results are present suggesting that the specific mechanism involves the disruption of the nuclear factor of activated T cells (NFAT) by PPARgamma activation. Based on the observations described above and other preliminary data presented in the proposal, our present investigation will test the hypothesis: Suppression of IL-2 by the endocannabinoids, AEA and 2-AG, is mediated through disruption of NFAT regulation by two distinct cannabinoid receptor-independent mechanisms: (a) altered intracellular calcium regulation; and (b) activation of PPARgamma following COX-2-mediated conversion of AEA and 2-AG into PPARgamma agonists. We will test our hypothesis using the following specific aims (SA): SA1 is to characterize the role altered intracellular calcium regulation by AEA and 2-AG plays in deregulation of NFAT and, consequently, suppression of IL-2 gene expression; SA2 is to characterize the role of COX-2 on the deregulation of NFAT and suppression of IL-2 by AEA and 2-AG; SA3 is to characterize the role of PPARgamma activation by AEA and 2-AG treatment in altered NFAT regulation and suppression of IL-2; and SA4 is to identify and characterize the bioactive forms of AEA and 2-AG responsible for PPARgamma activation and to elucidate its contribution to IL- 2 suppression.

5'Untranslated Regions; Ablation; Antibodies; Aryl Hydrocarbon Receptor; Attenuated; attenuation; B cell differentiation; B lymphocyte-induced maturation protein 1; B-Lymphocytes; base; Binding (Molecular Function); Boxing; c-myc Genes; Cell physiology; Cities; Complex; Cytochrome P450; Dioxins; DNA Binding; DNA-Binding Proteins; Elements; Employee Strikes; Enhancers; Environmental Pollution; Exhibits; Face; Funding; Genes; Genetic Transcription; Goals; graduate student; Heavy-Chain Immunoglobulins; Human Resources; Immune response; Immunoglobulin Domain; Immunoglobulin M; Immunoglobulins; Instruction; Isoenzymes; JUN gene; Laboratories; Ligands; Light; Mature B-Lymphocyte; Mediating; Messenger RNA; Michigan; Molecular; Mutation; Names; Pharmaceutical Preparations; plasma cell differentiation; Plasma Cells; Postdoctoral Fellow; Principal Investigator; Printing; programs; Promotor (Genetics); Proteins; Receptor Activation; Regulation; Repression; Research; Research Personnel; Research Project Grants; response; Response Elements; Role; Signal Transduction; Site; Specialist; Testing; Tetrachlorodibenzodioxin; transcription factor; Transcription Factor AP-1; Universities; Up-Regulation (Physiology)

The overall goal of this 5 year research plan is two-fold: (a) to characterize the molecular mechanism for[unreadable] impairment of B cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like[unreadable] compounds; and (b) to develop a computational model describing the biochemical pathways that regulate B[unreadable] cell differentiation and the interaction of this pathway with the aryl hydrocarbon reception (AhR). Previous[unreadable] studies have established the B cell as a sensitive cellular target for TCDD as evidenced by suppression of[unreadable] immunoglobulin (lg)M through a direct effect on B cells involving the AhR. Moreover, suppression of the IgM[unreadable] response by TCDD is mediated at the level of transcription and, in part, occurs through AhR binding to dioxin[unreadable] response elements (ORE) in regulatory domains within the Ig heavy chain (IgH) 3'a enhancer. Importantly,[unreadable] in addition to IgH suppression, the Ig kappa light chain (Igk), IgM joining chain (J chain) and X-box protein-1[unreadable] (XBP-1), which are essential for IgM assembly and secretion, are also markedly suppressed by TCDD[unreadable] suggesting the involvement of additional targets other than just the IgH 3'a enhancer. Moreover, TCDD[unreadable] alters the levels of B lymphocyte.-induced maturation protein-1 (Blimp-1), a master regulatory of B cell[unreadable] differentiation and its downstream target, Pax5, a transcriptional represser of B cell differentiation, which[unreadable] represses IgH, Igk, J chain and XBP-1. We also show that TCDD treatment of B cells: (a) altered the[unreadable] magnitude of DNA methylation and MRNA levels of DNA methylating enzymes, Dnmt3b, which putatively[unreadable] influences the expression of genes crucial to B cell differentiation, including Pax5; (b) is functionally[unreadable] antagonized by IFNg; and (c) rapidly induces the suppressor of cytokine signaling-2 (SOCS-2), a protein[unreadable] that negatively regulates signaling through cytokine receptors coupled to the JAK/STAT pathway, such as[unreadable] the IFNg receptor (IFNgR). The project objective is to test the hypothesis: Suppression of the primary[unreadable] humoral immune response by AhR agonists is mediated through changes in the B cell differentiation[unreadable] program via a mechanism that is blocked by IFNg. A computational description of the biochemical pathway[unreadable] of B cell differentiation and the direct interactions of AhR agonists on this pathway, will provide a mechanistic[unreadable] approach for predicting the effects of AhR agonists, alone and in combination as complex mixtures, on the[unreadable] pathway and on humoral immune responses.

[unreadable] DESCRIPTION (provided by applicant) [unreadable] Support is requested to continue human health-oriented research on risks from exposure to chemicals commonly found in Superfund sites and on remediation technologies to eliminate the potential for exposure to chemicals from those sites. The pollutants under investigation are a subclass of chemicals belonging to the halogenated aromatic hydrocarbon family that bind and activate the aryl hydrocarbon receptor (AhR).These chemicals, which include chlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls and polycyclic aromatic hydrocarbons, are environmentally persistent lipid soluble compounds that accumulate in the food chain and lead to human and wildlife exposure. A highly integrated, multidisciplinary research program is proposed consisting of seven research projects and four supporting core units. The research team of 27 investigators includes faculty at Michigan State University (20), CUT Centers for Health Research (4), Rutgers (2), and Purdue University (1). The central overarching theme of the proposed new program is to define specific aspects of environmental, microbial and mammalian biomolecular responses to environmental contaminants that act as ligands for the AhR. The major research thrusts are in three areas: (1) characterizing the diversity of dehalogenases and oxygenase gene sequences in microbial populations indigenous to soils, sediments and groundwater that metabolize AhR ligands; (2) defining the geochemical parameters governing adsorbtion, bioavailability and long-term fate of AhR ligands through interactions with clays; and(3) elucidation and computational modeling of the biochemical pathways and their interactions with the ligand-activated AhR, which cause altered responses in the liver and the immune system, specifically in Bcells. Two major goals of the new program are to characterize molecular mechanisms of AhR ligand interactions with specific abiotic and biotic processes and to develop new tools that can be used to more accurately estimate the fate, microbial biotransformation and human risk associated with AhR ligands contaminating the environment. Two support core facilities will assist the biomedical projects, one in the areas of bioinformatics and a second in developing dynamic computational models of mammalian biological responses induced by AhR ligands. A third core facility will provide support for the non-biomedical projects in three areas related to analysis of microbial-derived enzymes: microarray development and enhancement, automated bioinformatics analysis of PCR product sequences and biodegradative gene clusters, and high throughput screening and sequencing. In addition, a Research Translation core will communicate important research findings and outcomes emanating from the program to appropriate target audiences in government, industry and academia. [unreadable] [unreadable] [unreadable]

The overall goal of this 5 year research plan is two-fold: (a) to characterize the molecular mechanism for[unreadable] impairment of B cell differentiation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like[unreadable] compounds; and (b) to develop a computational model describing the biochemical pathways that regulate B[unreadable] cell differentiation and the interaction of this pathway with the aryl hydrocarbon reception (AhR). Previous[unreadable] studies have established the B cell as a sensitive cellular target for TCDD as evidenced by suppression of[unreadable] immunoglobulin (lg)M through a direct effect on B cells involving the AhR. Moreover, suppression of the IgM[unreadable] response by TCDD is mediated at the level of transcription and, in part, occurs through AhR binding to dioxin[unreadable] response elements (ORE) in regulatory domains within the Ig heavy chain (IgH) 3'a enhancer. Importantly,[unreadable] in addition to IgH suppression, the Ig kappa light chain (Igk), IgM joining chain (J chain) and X-box protein-1[unreadable] (XBP-1), which are essential for IgM assembly and secretion, are also markedly suppressed by TCDD[unreadable] suggesting the involvement of additional targets other than just the IgH 3'a enhancer. Moreover, TCDD[unreadable] alters the levels of B lymphocyte.-induced maturation protein-1 (Blimp-1), a master regulatory of B cell[unreadable] differentiation and its downstream target, Pax5, a transcriptional represser of B cell differentiation, which[unreadable] represses IgH, Igk, J chain and XBP-1. We also show that TCDD treatment of B cells: (a) altered the[unreadable] magnitude of DNA methylation and MRNA levels of DNA methylating enzymes, Dnmt3b, which putatively[unreadable] influences the expression of genes crucial to B cell differentiation, including Pax5; (b) is functionally[unreadable] antagonized by IFNg; and (c) rapidly induces the suppressor of cytokine signaling-2 (SOCS-2), a protein[unreadable] that negatively regulates signaling through cytokine receptors coupled to the JAK/STAT pathway, such as[unreadable] the IFNg receptor (IFNgR). The project objective is to test the hypothesis: Suppression of the primary[unreadable] humoral immune response by AhR agonists is mediated through changes in the B cell differentiation[unreadable] program via a mechanism that is blocked by IFNg. A computational description of the biochemical pathway[unreadable] of B cell differentiation and the direct interactions of AhR agonists on this pathway, will provide a mechanistic[unreadable] approach for predicting the effects of AhR agonists, alone and in combination as complex mixtures, on the[unreadable] pathway and on humoral immune responses.

DESCRIPTION (provided by applicant) Support is requested to continue human health-oriented research on risks from exposure to chemicals commonly found in Superfund sites and on remediation technologies to eliminate the potential for exposure to chemicals from those sites. The pollutants under investigation are a subclass of chemicals belonging to the halogenated aromatic hydrocarbon family that bind and activate the aryl hydrocarbon receptor (AhR).These chemicals, which include chlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls and polycyclic aromatic hydrocarbons, are environmentally persistent lipid soluble compounds that accumulate in the food chain and lead to human and wildlife exposure. A highly integrated, multidisciplinary research program is proposed consisting of seven research projects and four supporting core units. The research team of 27 investigators includes faculty at Michigan State University (20), CUT Centers for Health Research (4), Rutgers (2), and Purdue University (1). The central overarching theme of the proposed new program is to define specific aspects of environmental, microbial and mammalian biomolecular responses to environmental contaminants that act as ligands for the AhR. The major research thrusts are in three areas: (1) characterizing the diversity of dehalogenases and oxygenase gene sequences in microbial populations indigenous to soils, sediments and groundwater that metabolize AhR ligands;(2) defining the geochemical parameters governing adsorbtion, bioavailability and long-term fate of AhR ligands through interactions with clays;and(3) elucidation and computational modeling of the biochemical pathways and their interactions with the ligand-activated AhR, which cause altered responses in the liver and the immune system, specifically in Bcells. Two major goals of the new program are to characterize molecular mechanisms of AhR ligand interactions with specific abiotic and biotic processes and to develop new tools that can be used to more accurately estimate the fate, microbial biotransformation and human risk associated with AhR ligands contaminating the environment. Two support core facilities will assist the biomedical projects, one in the areas of bioinformatics and a second in developing dynamic computational models of mammalian biological responses induced by AhR ligands. A third core facility will provide support for the non-biomedical projects in three areas related to analysis of microbial-derived enzymes: microarray development and enhancement, automated bioinformatics analysis of PCR product sequences and biodegradative gene clusters, and high throughput screening and sequencing. In addition, a Research Translation core will communicate important research findings and outcomes emanating from the program to appropriate target audiences in government, industry and academia.

DESCRIPTION (provided by applicant): The two most important viral pathogens, based on mortality in the United States are HIV and influenza. The overall goal of this 5 year research plan is to test the hypothesis: ?9-tetrahydrocannbinol (?9-THC) attenuates antiviral responses against HIV and influenza virus through impairment of CD4+ T cell activation and function, and elicitation of antiviral specific CD8+ T cell effectors through CB1/CB2-dependent and -independent mechanisms. Our findings show that ?9-THC markedly impairs: (a) host resistance to influenza infection as evidenced by increased lung viral burden and decreased CD4+ and CD8+ T cell effectors;and (b) CD8+ T cell function, cytotoxic T lymphocyte activity and interferon 3 productions in vitro, in response to HIV gp120 and influenza-associated PB1. In addition, we show that cannabinoid treatment suppresses T cell function by impairing T cell activation via a mechanism involving rapid and sustained elevation in intracellular calcium [Ca+2]i, leading to T cell anergy. The rise in [Ca+2]i levels causes deregulation of the nuclear factor of activated T cells (NFAT) and impairs transcription of interleukin-2 and other NFAT-regulated cytokines. Additional results show that CB1-/-/CB2-/- mice are markedly more efficient in clearing influenza virus than wild type mice implicating a role for CB1 and/or CB2 in viral host resistance. Based on the above findings we will test our hypothesis using novel cell-based models with the following specific aims (SA): SA1 is to characterize impairment by ?9-THC, and the involvement of CB1/CB2, on the elicitation of antigen-specific multifunctional CD8+ T cells in response to HIV gp120 and influenza-associated PB1;SA2 is to characterize the impairment by ?9-THC, and the involvement of CB1/CB2, on CD4+ T cell activation and function induced by HIV gp120 and influenza-associated PB1;SA3 is in an in vivo surrogate model of HIV and influenza challenge, to characterize the impairment by ?9-THC, and involvement of CB1/CB2, on the elicitation of antigen-specific multifunctional CD8+ T cell responses to HIV gp120 and influenza-associated PB1;SA4 is in an in vivo surrogate model of HIV and influenza challenge, to characterize the impairment by ?9-THC, and involvement of CB1/CB2, on the CD4+ T cell response to HIV gp120 and influenza PB1;and SA5 is to determine the effect of 9-THC on the generation of multifunctional human peripheral blood (HPB) CD8+ T cells in response to HIV gp120 and influenza-associated M1. The significance of the proposed studies is that in their immunocompromised state, those infected with HIV or cancer patients are especially susceptible to infectious pathogens including influenza. Moreover, HIV and cancer patients are well known users of cannabis for stimulating appetite to alleviate the wasting syndrome that accompanies AIDS and as an antiemetic to relieve the nausea produced by cancer chemotherapy. In spite of the many HIV and cancer patients utilizing cannabis multiple times daily, an important data gap concerns the extent to which this practice may lead to a further diminution in immune competence and a more rapid progression of disease or mortality due to secondary infections. PUBLIC HEALTH RELEVANCE: Patients suffering from AIDS and/or cancer are immune compromised and are also well-established users of cannabis, an illicit drug that is immunosuppressive;HIV patients to stimulate appetite and alleviate AIDS wasting syndrome and cancer patients to relieve nausea produced by cancer chemotherapy. This project assesses the consequences of cannabis on immunity against HIV and the common pathogen, influenza, which afflicts immune compromised individuals at a higher incidence.

Approximately 6 billion pounds of bisphenol A (BPA) is synthesized each year making it one of the highest volume chemicals produced worldwide. The greatest applications of BPA are as a starting material in the manufacturing of polycarbonate plastic and as a component in the resin that lines beverage and food cans. BPA is also a constituent of plastics other than polycarbonates including polynil chloride and polyethylene terephthalate, which are also widely used. It is now well established that BPA can leach from polycarbonate. Human exposure to BPA occurs through a variety of sources with consumption of contaminated food products being the most important. BPA exposure is virtually ubiquitous as evidenced by its detection in 95% of urine samples tested in the US. BPA has also been detected in human breast milk, amniotic fluid and cord blood. Because BPA possesses estrogenic activity by binding to estrogen receptors (ER), estrogen related receptors (ERR), and has been shown to induce non-genomic events at the cellular level through GPR30, there is concern that exposure to this compound can alter or interfere with endocrine signaling pathways, even at low doses, including those affecting immune system development and function. The overall goal of this four-year research plan is to evaluate the effects of BPA on immune competence. Specifically we will test the hypothesis: Chronic low dose BPA exposure, beginning in utero, results in altered immune development and immune competence in the adult, which is mediated, in part, through changes in leukocyte composition, function and through changes in estrogen receptor (ER), estrogen related receptor (ERR) and/or estrogen related receptor (ERR) or GPR30 expression by leukocytes. This hypothesis will be tested using four specific aims (SA). SA1 is to determine the effects of chronic BPA exposure on the relative number and proportion of leukocyte subpopulations in the spleen. SA2 is to characterize the effect of chronic BPA treatment on leukocyte function by quantification of immune responses to defined stimuli. SAS is to determine the effect of chronic BPA exposure on estrogen receptor (ERa and ERp), estrogen-related receptor (ERRy) and GPR30 levels in leukocyte subpopulations. SA4 will be to define the effect of chronic BPA exposure on a selected suite of estrogen sensitive genes known to be involved in leukocyte function. The successful completion of the aforementioned specific aims will provide critical information on the putative role of long-term stimulation of estrogen receptors by BPA on immune development and competence.

DESCRIPTION (provided by applicant): Epidemiologic studies have established an association between 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure and altered B cell function including, suppression of humoral immunity and increased incidence of non-Hodgkin's lymphoma. TCDD is ubiquitous, highly stable, lipophilic environmental contaminants that impair plasma cell formation. The overall goal of this five-year research plan is to test the Hypothesis: Impaired human B cell function by TCDD is mediated through sustained elevation of BCL6 in activated B cells. This sustained elevation in BCL6 promotes anergy and cell death in the majority of TCDD-affected B cells and impaired differentiation in a smaller population of TCDD-affected but surviving B cells. Next to induction of certain cytochrome P-450 drug metabolizing isozymes, suppression of primary humoral immune responses by impairment of plasma cell formation is one of the most sensitive sequela produced by TCDD, and has been demonstrated in virtually every animal species investigated. During the existing project period, one of the most significant advancement has been the demonstration that in the majority of human donors evaluated, human primary B (HPB) cells exhibited similar sensitivity to TCDD as B cells from TCDD responsive mouse strains (e.g., C57Bl/6) in spite of the fact that the human aryl hydrocarbon receptor (AHR) has approximately ten-fold lower affinity for TCDD than the mouse AHR. The direct effects of TCDD on HPB cell function represents a longstanding data gap, which we have begun to address and will continue in this competing renewal application. Moreover our results show, that in addition to decreased numbers of IgM secreting cells, TCDD- treatment caused elevated and sustained levels of BCL6, and decreased levels of CD80, CD69, pERK, p- p65/NF?B, and p-cJun, which are hallmarks of impaired B cell activation. Based on the above findings, a multifaceted cellular and molecular approach will be used to test our hypothesis using human primary B cells with the following specific aims (SA): SA1 is to determine the molecular mechanism responsible for impaired BCL6 down-regulation by TCDD in activated HPB cells; SA2 is to determine the role of TCDD-mediated induction of SHP-1 on suppression of B cell activation and function; SA3 is to establish the role of NFkB, AP-1 and BCL6 in TCDD-mediated impairment of CD80 up-regulation, a hallmark of altered B cell activation; and SA4 is to determine whether TCDD induces B cell anergy or an anergy-like phenotype. The significance of the proposed studies is that they will for the first time provide new and important information concerning the molecular mechanisms by which TCDD impairs human primary B cell function as well as offer new insights on how TCDD might contribute to the increased incidence of non-Hodgkin's lymphoma. Finally, the proposed experiments will provide new knowledge concerning the fundamental role of the AHR in B cell immunobiology.

Suppression of humoral immunity by the ubiquitous environmental contaminant and prototypical aryl hydrocarbon receptor (AhR) ligand, 2,3,7,8-tetrachlorodiobenzo-p-dioxin (TCDD), has been demonstrated in virtually every animal species tested. Unfortunately, quantification of the effects of AhR ligands on the human immune system, including B cell function, has been sparse. The overall goal of this research is to define the molecular mechanism(s) responsible for AhR agonist-mediated suppression of antibody production by human primary B cells. Results from the current funding period demonstrate that human B cells are sensitive to IgM suppression by TCDD, and exhibit important species-related mechanistic differences compared to mouse B cells. The most striking being that mouse B cells maintain viability under conditions of suboptimal activation (due to weak stimulation or xenobiotic impaired activation), whereas suboptimal activation of human B cells resulted in cell death. These data suggest the existence of a minimal activation threshold that human B cell must attain in order to survive. Based on this critical observation, we will test the hypothesis: Suppression of the human primary antibody response by AhR agonists is a multievent process involving impairment of B cell activation, which in turn impedes attainment of a minimum activation threshold requisite for B cell survival. The magnitude of this impediment in activation strength is dictated by AHR polymorphisms and determines B cell sensitivity to AhR agonist-mediated suppression of plasma cell formation. Four specific aims (SA) will test the central hypothesis. SAI is to determine the magnitude of AhR activation required to impair cellular activation and cell survival. SA2 is to determine the magnitude of AhR activation required to impair signaling initiated through ligation of CD40 vs. cytokine receptors. SA3 is to determine the role of epigenetic alterations induced by AhR activation in disruption of the biochemical pathway controlling human primary B cell activation. SA4 is to define the role of AHR polymorphisms on human B cell sensitivity to AhR-mediated IgM suppression. Completion of the aforementioned specific aims will provide critical new mechanistic information on; (1) the magnitude of AhR activation required to suppress human B cell function; (2) whether the B cell impairment exhibits threshold like properties; and (3) the role specific AHR polymorphisms in human B cell play in determine sensitivity to AhR ligands, which if understood could be used as a biomarker of susceptibility (or lack of) to humoral immune suppression by dioxin-like compounds.

DESCRIPTION (provided by applicant): Support is requested to continue human health-oriented research on risks from exposure to chemicals commonly found in Superfund sites and on remediation technologies to eliminate the potential for exposure to chemicals from those sites. The pollutants under investigation are a subclass of chemicals belonging to the halogenated aromatic hydrocarbon family that bind and activate the aryl hydrocarbon receptor (AhR). These chemicals, which include chlorinated dibenzo-p-dioxins, dibenzofurans, biphenyls and polyaromatic hydrocarbons, are environmentally persistent, lipid soluble that accumulate in the food chain leading to human and wildlife exposure. A highly integrated, multidisciplinary research program is proposed consisting of six research projects and six supporting core units. The research team of 25 investigators includes faculty at Michigan State University (18), the Hamner Institutes for Health Sciences (4), Rutgers University (2), Purdue University (1) and United States Environmental Protection Agency (1). The central overarching theme of the proposed program is to define specific aspects of environmental, microbial and mammalian biomolecular responses to environmental contaminants that act as ligands for the AhR. The major research thrusts will provide new mechanistic information in three areas: (1) characterizing the diversity and physiogenomic responses of (chloro)dioxin degrading microbial populations indigenous to soils, sediments and groundwater; (2) defining the geochemical parameters governing adsorption, bioavailability and long-term fate of AhR ligands through interactions with geosorbent compositions in soils and sediment components; and (3) elucidation and computational modeling of the interactions of specific biochemical pathways with the ligand-activated AhR which cause altered responses in the liver and immune system with a particular emphasis on responses by human primary cells. One research core will assist the biomedical projects, in developing dynamic computational models of mammalian responses induced by AhR ligands. A second research core will provide bioinformatics support and custom bioinformatics tool development, along with development of a novel molecular method for enriching metagenomes and metatranscriptomes in functional genes of interest to project 4 (biomedical), 5 and 6 (environmental). In addition, a Research Translation Core will communicate research findings to appropriate target audiences in government, industry and academia, a Community Engagement Core will communicate with community target audiences, and a Training Core will provide cross-disciplinary training to pre- and postdoctoral students.

PROJECT SUMMARY (See instructions): The Community Engagement Core will partner with Michigan State University (MSU) Extension in the Michigan Tri-Cities region (Saginaw, Midland, and Bay City) to strengthen social networks, enhance community knowledge about the state-of-the-science including knowledge about health risks associated with exposure to dioxin and dioxin-like compounds, and facilitate decision-making around reducing environmental health risks. We propose identifying key actors and stakeholders in the area, then holding a series of workshops between these key actors and MSU SRP researchers. The workshops have three main objectives: 1) To bring diverse stakeholders together in a face-to-face, interactive setting in order to strengthen relationships and community capacity to address local environmental health problems; 2) to provide education about the scientific method, and how dioxin and dioxin-like compounds interact in the body and in the environment; and 3) to increase MSU SRP researchers' understanding of community questions and concerns about health risks associated with dioxin exposure and the forces beyond science that shape public policy. Each annual series of workshops will cumulate in a white paper or MSU Extension factsheet identifying common questions and summarizing concerns and possible solutions. The white papers and/or factsheets will be published online via the MSU SPR website and the MSU Extension website. Printed versions will be made available in district libraries, county public health offices, and MSU Extension offices. Summaries of these papers also will be available through a mobile phone app developed by the MSU SRP RTC. Long-term outcomes of these workshops may include community identification of research strategies for addressing dioxins at the whole system, community, and human health levels, as well as agreement on cleanup criteria.

PROJECT SUMMARY An estimated 37 million people worldwide are infected with human immunodeficiency virus (HIV). Combined antiretroviral therapy (ART) has turned HIV into a chronic infection with significantly longer life expectancy. New health issues have surfaced as HIV patients live longer. Specifically, 50% of HIV-infected (HIV+) individuals exhibit neurocognitive impairment, termed HIV-associated neurocognitive disorders (HAND). A key event leading to HAND is persistent low-level chronic neuroinflammation resulting in neuronal damage and cell death. A major contributor to HIV-induced neuroinflammation is activated monocytes, which are significantly elevated in patients? with HIV-associated dementia. Activated, CD16+ monocytes are infected by HIV in the periphery and migrate across the blood-brain barrier (BBB) to release HIV virions and neurotoxic and proinflammatory factors. Before entering the CNS, resting (CD16?) monocytes transition into the CD16+ through poorly understood mechanisms including elevated interferon alpha (IFN?), a potent antiviral cytokine produced by plasmacytoid dendritic cells (pDC), an observation consistent with the IFN? gene signature in monocytes from HIV patients. Chronic IFN? production in HIV patients has been linked to neurocognitive impairment. In parallel, IFN? also activates CD8+ T cells, which are recruited from systemic circulation to cross the BBB. Once in the perivascular space, activated monocytes and CD8+ T cells interact with astrocytes to drive a chronic neuroinflammatory response leading to destruction of neurons and declining cognative function. Interestingly, cannabis, which has constituents (e.g., ?9-tetrahydrocannabinol (THC)) possessing immune suppressive and anti-inflammatory activity, is widely used (approximately 25-37%) by HIV patients. The beneficial vs. deleterious effects of cannabinoid therapy in HIV patients remains unknown and understudied. Preliminary results show that THC suppresses IFN?-mediated CD16? to CD16+ monocyte transition as well as IL-7 receptor upregulation on CD8+ T cells. Moreover, HIV+ marijuana-users (MJ+) have fewer circulating CD16+ monocytes compared to HIV+MJ-. Preliminary data is also presented using a novel all human coculture system demonstrating that both monocytes and CD8+ T cells, when cocultured with astrocytes significant drive the secretion of astrocyte-derived inflammatory mediators, including IL-6 and IP-10, a response suppressed by THC. Mechanistic studies are proposed to test the hypothesis: Cannabinoids suppress interferon-?-mediated monocyte and CD8+ T cell activation in the periphery and their detrimental effects on astrocyte function, all of which are key processes in HIV-associated chronic neuroinflammation.

PROJECT SUMMARY/ABSTRACT An estimated 37 million people worldwide are infected with human immunodeficiency virus (HIV). Combined antiretroviral therapy (ART) has turned HIV into a chronic infection with significantly longer life expectancy. New health issues have surfaced as HIV patients live longer. Specifically, 50% of HIV-infected (HIV+) individuals exhibit neurocognitive impairment, termed HIV-associated neurocognitive disorder (HAND). A key event leading to HAND is persistent low-level chronic inflammation resulting in neuronal damage and cell death. A major contributor to HIV-induced neuroinflammation is activated monocytes, which are significantly elevated in patients? with HIV-associated dementia (HAD). A hallmark of HIV infection is chronic, systemic inflammation in part through translocation of microbial derived products from the gut which, activates monocytes and promote migration across the blood-brain barrier (BBB). Upon entry, activated monocytes release neurotoxic and proinflammatory factors (e.g., IL-1?), which are thought to contribute to HAND. Recently, evidence has emerged implicating the importance of inflammasome activation as a contributing mechanism to neuroinflammation and HAND. Preliminary results presented in this application show that inflammasome activation in monocytes, as evidenced by IL-1? secretion, promotes astroglial cell inflammation. These findings support a critical role for inflammasome activation in monocytes as a mechanism driving neuroinflammation. In particular, immunohistocytochemistry of brain tissue from post-mortem HIV patients with HAD showed significant infiltration of proinflammatory CD16+ monocytes. By contrast, cannabinoid exposure displays anti-inflammatory properties in HIV-infected patients, which we and others have reported. The anti- inflammatory properties of cannabis are attributed largely to the canonical ligand, ?9-tetrahydrocannabinol (THC), which exerts psychotropic activity through cannabinoid receptor (CB) 1 and the non-psychotropic- mediating CB2, while cannabidiol (CBD) does not act through CB1/CB2. Likewise, the CB2 selective agonist, JWH-015, represents a potential strategy for understanding the role of CB2 in limiting HIV-associated neuroinflammation. In fact, preliminary results demonstrate that a CB2 selective agonist impairs monocyte secretion of IL-1?, a hallmark of inflammation and inflammasome activation. Mechanistic studies are proposed to test the hypothesis: CD16+ monocytes from non-cannabis using HIV+ subjects exhibit greater inflammasome formation and subsequent astrocyte activation compared to cannabis using HIV+ subjects, which is associated with the frequency of cannabis use.