开题报告内容：（包括拟研究或解决的问题、采用的研究手段及文献综述，不少于2000字）1. BackgroundIn 2012, approximately 35 million people worldwide were infected with HIV. HIV enters the CNS within 8 days after initial infection, primarily infecting macrophages/microglia. Infected cells produce new virus and act as viral reservoirs within the CNS. Infected macrophages/microglia also release neurotoxic viral proteins and host factors that contribute to neuroinflammation and HIV-associated neurocognitive disorders (HAND). Drug abuse is a significant comorbidity in HIV infection, and HIV drug abusers are especially vulnerable to the accelerated development of HIV-associated neuropathogenesis. Approximately 10-20% of HIV individuals around the world use drugs, and drug abuse is fueling the fast-developing HIV epidemics in Eastern Europe and Asia.In addition to producing virus, these cells are long-lived and can act as viral reservoirs, as well as release inflammatory viral proteins and host factors. Thus, infection of CNS macrophages is essential to the development of neuropathogenesis and HAND. HIV-associated neuropathogenesis is exacerbated by drugs of abuse. In the US, approximately 80% of HIV individuals have used illicit drugs. All addictive substances, including cocaine, heroin, cannabis, and alcohol, as well as a number of therapeutics, increase extracellular CNS dopamine, either through direct interaction with the dopaminergic system or indirect effects on the reward pathway.2. Aim of our studyTo optimize the dopamine concentrations that increase macrophages susceptibility to HIV infection. To determine the relationship between dopamine-increased HIV infection and CCR5. To determine which pathway that dopamine may mediate its effects in macrophages.3. Methods3.1 Western BlotBasic Western Blot procedure. Here we use (phosphate protein/total protein)/total protein stain to analyze. Then we use 2 mins in 700 channel and 0.5 min in 800 channel to image total protein stain and 0.5 min in 700 channel and 10 mins in 800 channel to image phosphate protein and total protein.3.2 Cell CultureCell isolation and culture Human peripheral blood mononuclear cells (PBMC) were separated from blood obtained from healthy donors (New York Blood Center, Long Island City, New York) by Ficoll-Paque (GE Healthcare, Piscataway, NJ) gradient centrifugation. Monocytes present in the PBMC were determined by flow cytometry of CD14 cells in the PBMC using a FACS Canto II flow cytometer (Becton-Dickinson, Franklin Lakes, NJ). Monocyte derived macrophages (MDM) were obtained by adherence isolation, through culture in macrophage media (RPMI-1640 with 10% FCS, 5% human AB serum, 10 mM HEPES, 1% P/S, and 10 ng/mL MCSF) for 3 days, washing and culturing another 35 days. After 6 8 total days in culture the cells were considered to be mature MDM. Flp-In T-Rex HEK 293 cells (HEK 293 cells, Life Technologies) were maintained in DMEM supplemented with 10% FBS and 2 mM L-glutamine (Life).3.3 Statistics Normally distributed were analyzed using a two-tailed Students T-test and data that were not normally distributed were analyzed with Wilcoxen Matched Pairs Signed Rank Test. P ＜0.05 was considered significant.4. Literature review With the development of cART, HIV has become a manageable, chronic disease. HIV-infected individuals are living longer while suppressing their infection, but no current treatment regimen has been able to fully eliminate the virus. The major impediment to complete eradication of the virus is the existence of cell-based and anatomical reservoirs in which the virus is protected. One of these reservoirs is in CNS. Within the CNS, HIV targets primarily myeloid cells such as perivascular macrophages and microglia. These long-lived cells are the principle constituents of the CNS reservoir, and can maintain low-level replication even in the presence of CNS-penetrant cART. Therefore, factors that increase the infection of myeloid cells can reduce the effectiveness of therapy by expanding the CNS reservoir. One such factor is dopamine. Macrophages in the CNS are exposed to dopamine, a kind of catecholamine neurotransmitter that is increased by illicit drugs like cocaine and methamphetamine , as well as by legal therapeutics s. Studies in SIV-infected rhesus macaques demonstrate that increases in extracellular dopamine correlate with increased CNS viral loads as well. With high levels of dopamine, HIV-infected individuals show exacerbated neuropathology in regions of the brain, just like the basal ganglia and substantia nigra. Dopamine acts principally through dopamine receptors (DR), G-protein coupled receptors (GPCR) that are divided into D1-like DR (D1R and D5R) and D2-like DR (D2R, D3R and D4R) depending upon whether they activate (D1-like DR) or inhibit (D2-like DR) adenylyl cyclase. Studies show that DR also activate alternative pathways, including mobilization of calcium from the endoplasmic reticulum. The effects of dopamine on macrophage function, and the signaling pathways by which these effects are mediated, have not been studied extensively.Previous studies showed that dopamine increases HIV replication in human macrophages, and this increase through activation of DR, increasing the total number of infected cells. The mechanisms of which are unclear, but one possibility is by increasing HIV entry into macrophages. HIV entry is complex, and in macrophages, it is mediated by the interaction of the viral envelope protein gp120 with the surface receptor CD4 and coreceptor CCR5.The early events in HIV entry are well characterized, in which the viral envelope protein, gp120gp 41, engages with the primary receptor CD4 on target cell. This interaction permits conformational changes within gp120, which allow additional binding to a co-receptor, usually CXCR4 for T-lymphocyte tropic strains, or CCR5 for macrophage tropic isolates. Co-receptor binding is the trigger for virus entry, during which the fusion peptide gp41, inserts into the cellular membrane to drive the fusion event. The lipid composition of the host cell is important for HIV entry.Also, the previous studies demonstrated the critical importance of cholesterol for entry of HIV-1 into macrophages. First, they showed that productive virus entry is significantly reduced when the macrophage target cells are treated with Mbeta;CD, a cholesterol-depleting drug. By incubating cells with this drug before the addition of virus, only cholesterol from the target cells would be removed allowing virus particle cholesterol and infectivity to remain intact. The addition of exogenous cholesterol to macrophage membranes directly after Mbeta;CD treatment substantially restored HIV infection, indicating that the decreased infectivity was at least in part due to the depletion of cholesterol from the cell membrane. Secondly, they modified the properties of cholesterol-rich macrophage membranes using nystatin and filipin complex to sequester cholesterol into large aggregates. Treatment of macrophages with these drugs significantly inhibited productive HIV entry in a concentration-dependent manner. Thirdly, depletion of macrophage cholesterol using Lovastatin, a compound that inhibits HMG-CoA reductase, the rate limiting enzyme in cholesterol biosynthesis, significantly inhibited HIV productive entry after a prolonged 4 day treatment. Incubation of macrophages with mevalonate, whose production is prevented by Lovastatin, partially restored productive HIV entry into macrophages. Therefore, modification of macrophage membrane cholesterol content using 4 different pharmacological inhibitors acting on cholesterol by different mechanisms all significantly inhibited HIV entry.Moreover, there were results demonstrate for the first time that dopamine increases HIV replication in human macrophages and that the mechanism by which dopamine mediates this change is by increasing the total number of HIV-infected macrophages. This increase in HIV replication is mediated by activation of dopamine receptor 2. These findings suggest a common mechanism by which drugs of abuse enhance HIV replication in macrophages and indicate that the drug abuse-heightened levels of central nervous system dopamine could increase viral replication, thereby accelerating the development of HAND.Furthermore, there was clear evidence that psychostimulants, opiates, ethanol, cannabinoids and nicotine increase dopamine transmission in limbic nuclei, and the nucleus accumbens in particular. Psychostimulants such as amphetamine and cocaine increase extracellular dopamine levels in limbic nuclei by interacting with the dopamine transporter (DAT). Opiates stimulate mu; opioid receptors on GABAergic interneurons in the VTA, which disinhinits dopaminergic neuronal activity resulting in increased dopamine release in the nucleus accumbens. Ethanol disinhibits dopaminergic neuronal activity in the VTA by positive modulation of GABAAreceptors located on GABAergic interneurons. Ethanol also increases the firing rates of dopaminergic neurons by decreasing potassium currents in these cells. Cannabinoids increase dopaminergic neuronal activity in the VTA through inhibition of GABA release following stimulation of presynaptic CB1 cannabinoid receptors in the VTA. Nicotine excites dopaminergic neurons in the VTA, through activation of nicotinic acetylcholine receptors located on dopaminergic cells in addition to increasing glutamate and decreasing GABA transmission in the VTA. Thus, these five classes of abused drugs all increase dopaminergic neuronal activity and/or dopamine release via interactions with a number of ionotropic receptors, metabotropic receptors, ion channels and transporters.Although the presence of CD4 and CCR5 in detergent-resistant membrane microdomains has been demonstrated, the distribution of CD4, CCR5, and CXCR4 receptors in T-cell lipid rafts remains largely unknown. Recent studies, using immunogold electron microscopy, have clearly shown the existence of homogenous microclusters of CD4 and chemokine receptors separated by distances of less than 100 nm, suggesting that these human immunodeficiency viruses type 1 (HIV-1) receptors may colocalize in rafts.Besides, there were findings support an important role for DA in HIV infection of macrophages, demonstrating that MDMs inoculated with HIV in the presence of DA generate increased levels of viral replication relative to MDMs inoculated in the absence of DA. In infection model, MDMs were exposed to 20 mu;mol/L DA during the initial 24-hour inoculation with HIV and the significant increase in HIV replication induced by DA was seen on days 3, 4, 5, and 6 after the initial infection. These DA-induced increases in HIV replication were shown in infections of MDMs derived from different donors with different concentrations of two isolates of HIV, indicating that the effects of DA are independent of donor, viral strain, and the concentration of the inoculating virus.There are a number of possible mechanisms by which DA could mediate this increase in HIV replication. DA treatment could increase the number of macrophages initially infected by HIV, resulting in higher levels of viral replication due to virion production from a greater number of infected cells. Results demonstrate that on days 4, 5, and 6 postinoculation, there is a significant increase in the number of HIV infected MDMs when HIV inoculation is performed in the presence of DA. These data suggest that the increase in viral replication is due, at least in part, to an increase in the number of macrophages infected by HIV in the presence of DA. Additionally, DA could modulate production of HIV particles, enabling cells treated with DA to produce more virions per cell. The mechanisms mediating the DA-induced increase in HIV replication in MDMs are likely multifactorial and are the subject of ongoing research.It is important to note that because of the similarities between some DR subtypes, DR agonists often lack a high degree of specificity for distinguishing between DRs of the same subtype; however, they do distinguish very well between the D1-like and D2-like classes of DRs. Quinpirole is widely used as a specific D2R agonist, and has a higher affinity for D2R than other D2-like DRs, but studies have shown it to have activity against other D2-like DRs. Similarly, SKF 82958 acts on D1-like receptors, primarily against D1R, but there are no agonists that distinguish well between D1R and D5R. Another caveat is that despite the D2-like specificity of the agonists involved in the increased HIV replication, the potency of both quinpirole and DA is lower than expected. Given the apparently relatively low levels of D2R in macrophages, it is possible the majority of D2R on the cell surface might be present as heterodimers with another receptor that allosterically modulates the potency of signaling by D2R agonists. There is an increasing amount of data showing that DRs can participate in G protein-coupled receptor heterodimers, resulting in modulation of their pharmacological properties (Han Y, Moreira I, Urizar E, Weinstein H, and Javitch JA, Nature Chemical Biology, in press). Therefore, it is possible that the lower potency of both DA and quinpirole as agonists is due to modulation of D2R by heterodimer partners. This is an ongoing area of study. This project will define the mechanisms by which dopamine increases HIV entry intomacrophages, and we will also use a primate model of acute HIV infection in the CNS to examine the impact of dopamine on macrophage infection in vivo and characterize the early neuropathogenic sequelae of these effects.5.References:1. Carter GC, Bernstone L, Sangani D, Bee JW, Harder T, James W: HIV entry in macrophages is dependent on intact lipid rafts. Virology 2009, 386:192-2022. van Wilgenburg B, Moore MD, James WS, Cowley SA: The productive entry pathway of HIV-1 in macrophages is dependent on endocytosis through lipid rafts containing CD4. PloS one 2014, 9:e86071.3. Arthos J, Rubbert A, Rabin RL, Cicala C, Machado E, Wildt K, Hanbach M, Steenbeke TD, Swofford R, Farber JM, Fauci AS: CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes. Journal of virology 2000, 74:6418-244. Di Chiara G, Imperato A: Drugs abused by humans preferentially increase synaptic dopamine concentrations in the mesolimbic system of freely moving rats. Proceedings of the National Academy of Sciences of the United States of America 1988, 85:5274-8.5. Gaskill PJ, Calderon TM, Luers AJ, Eugenin EA, Javitch JA, Berman JW: Human immunodeficiency virus (HIV) infection of human macrophages is increased by dopamine: a bridge between HIV-associated neurologic disorders and drug abuse. The American journal of pathology 2009, 175:1148-596. Gaskill PJ, Yano HH, Kalpana GV, Javitch JA, Berman JW: Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages. PloS one 2014, 9:e108232.7. Hinkin CH, Barclay TR, Castellon SA, Levine AJ, Durvasula RS, Marion SD, Myers HF, Longshore D: Drug use and medication adherence among HIV-1 infected individuals. AIDS and behavior 2007, 11:185-94.8. 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