Rationale for the Use of Immunotoxins in the Treatment of HIV-infected Humans

Abstract

The first step in the replication of human immunodeficiency virus (HIV) is selective binding of the envelope glycoprotein (gp120) to CD4 receptors on T cells or macrophages. After penetration in these cells, the genome of the virus is integrated in the human genome. HIV-infection causes depletion of CD4-positive cells resulting in a severe immunosuppression. It is believed that eliminating HIV-infected cells is crucial in limiting further reduction of CD4-positive cells and thus, preventing disease progression. The most commonly used drugs, such as zidovudine (AZT), appeared to be not completely effective. Therefore many investigators are searching for alternative treatment modalities. The use of immunotoxins (ITs) to eliminate HIV-infected cells is discussed.

ITs are chimeric molecules in which cell-binding ligands are coupled to toxins and can specifically eliminate undesired cells. The cell-binding carriers of anti-HIV ITs have been directed against different regions of the HIV envelope glycoprotein (gp120 and gp41) and surface antigens (e.g CD4, CD25). The ITs have been composed of different ribosome-inactivating proteins (RIPs) like pokeweed antiviral protein (PAP), Pseudomonas exotoxin (PE), Diphtheria toxin (DT), or ricin. In in vitro studies, several of these ITs have been shown to be effective and specific in killing acute and persistently HIV-infected cells. The ITs were effective at concentrations (ID50 range from 10^-9 M to 10^-12 M) that were not toxic to uninfected cells or cells without the antigen. The IT CD4(178)PE40, a fusion protein directed against the CD4 binding site of gp120, has been investigated in two in vivo trials. The results were disappointing considering die antiviral activity in vitro. This was thought to be due to the rapid clearance of the IT and the differential resistance of clinical HIV isolates. Use of a panel of ITs is likely to be more effective because multiple approaches cover the intrinsic variability of HIV and the presence of IT-resistant or latently infected cells, as well as the blocking presence of neutralizing anti-HIV antibodies and the immunogenicity of most ITs.

It may be possible to control the virus completely with a panel of ITs in combination with other antiviral or immunosuppressive agents such as RT inhibitors (e.g AZT), interferon α, or cyclosporine. More research will be necessary to develop such a combined therapy.     

Drug Targeting Using Conjugates: The Importance of Pharmaceutical Chemistry

Background: Leishmania parasite is an obligate intracellular parasite of the mammalian host and lives inside resident macrophages of liver and spleen. A high dose of paromomycin (PM) is required for the treatment.

Purpose: Preparation and in vitro evaluation of PM loaded albumin microspheres (MS) (of size ≤ 5?µm) to target macrophages for treatment of visceral leishmaniasis.

Methods: PM loaded MS were prepared by spray-drying method using albumin as a polymer matrix and stabilized using heat treatment. These MS were evaluated for product yield, encapsulation efficiency, particle size, size distribution, contact angle, drug–polymer interactions, and for in vitro drug release. Fluorescent labeling and in vitro uptake of these MS was assessed in RAW 264.7 cell line.

Results: PM loaded albumin MS were prepared with a mean particle size ≈3?µm. Free albumin content and contact angle study confirmed the stabilization of these MS. Release studies showed biphasic release pattern. Interaction studies ruled out any possibility of drug–polymer interaction. Uptake study in macrophage confirmed the suitability of prepared MS for macrophage targeting.

Conclusion: The proposed drug-delivery system was found suitable for targeting macrophages in vitro and may serve as an optimum carrier to target macrophages where Leishmania parasite resides.     

Core-shell nanoparticles for targeted and combination antiretroviral activity in gut-homing T cells

A major sanctuary site for HIV infection is the gut-associated lymphoid tissue (GALT). The α4β7 integrin gut homing receptor is a promising therapeutic target for the virus reservoir because it leads to migration of infected cells to the GALT and facilitates HIV infection. Here, we developed a core-shell nanoparticle incorporating the α4β7 monoclonal antibody (mAb) as a dual-functional ligand for selectively targeting a protease inhibitor (PI) to gut-homing T cells in the GALT while simultaneously blocking HIV infection. Our nanoparticles significantly reduced cytotoxicity of the PI and enhanced its in vitro antiviral activity in combination with α4β7 mAb. We demonstrate targeting function of our nanocarriers in a human T cell line and primary cells isolated from macaque ileum, and observed higher in vivo biodistribution to the murine small intestines where they accumulate in α4β7+ cells. Our LCNP shows the potential to co-deliver ARVs and mAbs for eradicating HIV reservoirs.     

Reversal of LPS induced endothelial cell TNF synthesis and increased permeability with microencapsulated antisense oligomers to NF-κB

Endothelial cells form the barrier between the circulation and interstitial space. Changes in permeability of endothelial cells allow penetration of inflammatory cells such as polymorphonuclear cells and macrophages to respond to infections and other inflammatory stimuli. Endothelial cells have also been shown to be phagocytic and produce pro-inflammatory cytokines such as TNF. It is the purpose of this study to evaluate endothelial cell phagocytosis of albumin microspheres containing antisense oligonucluetide to NF-κB (MASO), the effect of MASO on TNF synthesis after LPS stimulation and the effect of TNF inhibition on the permeability of endothelial cells in vitro. Results were (1) endothelial cells avidly phagocytozed albumin miocrospheres 1.0 and 1.7?µm in size, (2) phagocytosis of microspheres was potentiated by LPS, (3) TNF is synthesized by endothelial cells in cell culture with the peak concentrations occurring 4?h after stimulation with LPS, (4) MASO results in high intracellular concentration of oligomer, (5) MASO inhibits TNF synthesis to a greater extent than equivalent amounts of NF-κB antisense in solution and (6) the inhibition of TNF by MASO significantly decreases the permeability of albumin through endothelial cells in vitro.     

商陆皂甙甲抑制小鼠巨噬细胞和抗体生成

商陆皂甙甲(EsA)体外在0.01～10μmol·L^-1范围内,对小鼠腹腔巨噬细胞吞噬中性红和LPS诱导的巨噬细胞合成及释放白介素1都呈明显的抑制作用。体内给药2.5～5mg·kg^-1可显著减少绵羊红细胞致敏的小鼠血清溶血素的含量。提示EsA的抗炎作用可能主要通过抑制巨噬细胞的吞噬和分泌功能。EsA间接作用于B细胞,抑制抗体生成。     

The Role of Cohort Studies in Drug Development: Clinical Evidence of Antiviral Activity of Serotonin Reuptake Inhibitors and HMG-CoA Reductase Inhibitors in the Central Nervous System

Background Effective antiretroviral therapy (ART) has reduced the incidence of HIV-associated neurocognitive impairment (HNCI) but its prevalence remains high. Clinical trials have yet to identify a consistently effective treatment for HNCI, other than ART, but in vitro data support that some drugs approved by the Food and Drug Administration (FDA) for other indications might benefit individuals with HNCI. Some of these drugs, such as serotonin reuptake inhibitors (SRIs) and HMG-CoA reductase inhibitors (statins), may do so by reducing HIV replication in the CNS and are already widely used by HIV-infected individuals. Methods Six-hundred fifty-eight HIV-infected participants of the CHARTER cohort had a baseline assessment, which included comprehensive neuropsychological (NP) testing and HIV RNA measurements in plasma and cerebrospinal fluid (CSF). Four-hundred sixty-seven (71%) subjects used ART, 195 (30%) used SRIs, and 63 (10%) used statins. Results SRI users were less likely to have HIV RNA levels in CSF above 50 copies (c)/mL (29 vs. 37% in non-SRI users, OR 0.69, p = 0.05). This association was most evident for three of the seven SRIs (citalopram, sertraline, and trazodone, or “antiviral” SRIs, combined 25 vs. 38% in non-SRI users, OR 0.56, p = 0.01) and was strongest in those not taking concomitant ART (61 vs. 83%, OR 0.31, p = 0.01). “Antiviral” SRI users also performed better on NP tests (median global deficit score 0.37 vs. 0.47, p = 0.04). Statin users were also less likely to have HIV RNA levels in CSF above 50 c/mL (16 vs. 37%, p < 0.001) but, in contrast to SRIs, the association was strongest in those taking ART (2 vs. 18%, p < 0.001). Statin use was not associated with better NP performance. Multivariate analyses indicated that the use of “antiviral” SRIs—but not statins—was associated with undetectable HIV RNA levels in CSF and better NP performance. Conclusions SRIs may reduce HIV replication in CSF and improve NP performance. This was particularly true for three SRIs—supporting differences in antiviral efficacy between drugs—in individuals who were not taking ART. In contrast, statins were not associated with lower HIV replication in CSF in multivariate analyses and were not associated with better NP performance. These analyses support the value of large observational cohort studies in identifying FDA-approved drugs that may be worth further investigation.     

Formulation,pharmacokinetics and biodistribution of Ofloxacin-loaded albumin microparticles and nanoparticles

Albumin microparticles containing Ofloxacin (Fluoroquinolone derivative commonly used in hospitals) were formulated by the spray dry method. By decreasing the pump feed rate or the total polymer concentration, a mixture of albumin/hypromellose acetate succinate (HPMCAS) microparticles and nanoparticles (MP/NP), containing Ofloxacin, were formulated. MP/NP were characterized, in vitro (particle size, zeta potential, and encapsulation efficiency). A comparison of the pharmacokinetics and biodistribution of aqueous Ofloxacin and Ofloxacin-loaded MP/NP, in Balb/c mice, revealed that peak concentrations were reduced in the serum, liver, spleen and brain, and a more sustained release was observed in serum and all of the organs tested for Ofloxacin MP/NP, compared to aqueous Ofloxacin. The MP/NP formulation allowed extended release by 24?h in the liver and more than 48?h in the brain. In serum, the elimination rate of Ofloxacin MP/NP is slower, the half life is longer, area under the plasma concentration time curve is decreased and volume of distribution is increased.     

Is there a role for immunoconjugates in the treatment of AIDS?

The use of antibodies to deliver therapeutic agents to specific cells is a well-established concept in the treatment of malignancy. Therapeutic effects have been shown in both animal models and human clinical trials. By analogy, antibodies and other ligands may be used to treat AIDS by targeting cells that are actively producing HIV and spreading the infection. Immunoconjugates with specificity for HIV antigens or structures on the surface of HIV-infected cells have been made. These agents have undergone extensive in vitro testing. In tissue culture they can eliminate infected cells and halt the production of HIV. A number of agents have been shown to enhance the efficacy of anti-HIV immunoconjugates. Because animal models of HIV-infection are problematic, there has been little preclinical testing. Nevertheless, several clinical trials have begun.     

Sustained and specific in vitro inhibition of HIV-1 replication by a protease inhibitor encapsulated in gp120-targeted liposomes

Selective delivery of antiretrovirals to human immunodeficiency virus (HIV) infected cells may reduce toxicities associated with long-term highly active antiretroviral therapy (HAART), may improve therapeutic compliance and delay the emergence of resistance. We developed sterically stabilized pegylated liposomes coated with targeting ligands derived from the Fab’ fragment of HIV-gp120-directed monoclonal antibody F105, and evaluated these liposomes as vehicles for targeted delivery of a novel HIV-1 protease inhibitor. We demonstrated that the immunoliposomes were selectively taken up by HIV-1-infected cells and localized intracellularly, enabling the establishment of a cytoplasmic reservoir of protease inhibitor. In antiviral experiments, the drug delivered by the immunoliposomes showed greater and longer antiviral activity than comparable concentrations of free drug or drug encapsulated in non-targeted liposomes. In conclusion, by combining a targeting moiety with drug-loaded liposomes, efficient and specific uptake by non-phagocytic HIV-infected cells was facilitated, resulting in drug delivery to infected cells. This approach to targeted delivery of antiretroviral compounds may enable the design of drug regimens for patients that allow increased therapeutic adherence and less toxic treatment of HIV infection.     

Bisphosphonate-coated BSA nanoparticles lack bone targeting after systemic administration

A polymeric conjugate of polyethyleneimine-graft-poly(ethylene glycol) and 2-(3-mercaptopropylsulfanyl)-ethyl-1,1-bisphosphonic acid (PEI-PEG-thiolBP) was prepared and used for surface coating of bovine serum albumin (BSA) nanoparticles (NPs) designed for bone-specific delivery of bone morphogenetic protein-2 (BMP-2). The NP coating was achieved with a dialysis and an evaporation method, and the obtained NPs were characterized by particle size, ζ-potential, morphology, and cytotoxicity in vitro. The particle size and surface charge of the NPs could be effectively tuned by the PEG and thiolBP substitution ratios of the conjugate, the coating method, and the polymer concentration used for coating. The PEG modification on PEI reduced the toxicity of PEI and the coated NPs, based on in vitro assessment with human C2C12 cells and rat bone marrow stromal cells. On the basis of an alkaline phosphatase (ALP) induction assay, the NP-encapsulated BMP-2 displayed full retention of its bioactivity, except for BMP-2 in PEI-coated NPs. By encapsulating ¹²⁵I-labeled BMP-2, the polymer-coated NPs were assessed for hydroxyapatite (HA) affinity; all NP-encapsulated BMP-2 showed significant affinity to HA as compared with free BMP-2 in vitro, and the PEI-PEG-thiolBP coated NPs improved the in vivo retention of BMP-2 compared with uncoated NPs. However, the biodistribution of NPs after intravenous injection in a rat model indicated no beneficial effects of thiolBP-coated NPs for bone targeting. Our results suggested that the BP-conjugated NPs are useful for localized delivery of BMP-2 in bone repair and regeneration, but they are not effective for bone targeting after intravenous administration.     

Targeting dexamethasone to macrophages

Abstract

Dexamethasone-21-phosphate (Dex-21-P) was encapsulated in human erythrocytes by a procedure of hypotonic hemolysis and isotonic resealing in a wide range of concentrations (0.2 to 8.5 mm). The erythrocyte membrane was then modified by the addition of Zn and bis(sulfosuccinimidyl)suberate inducing the formation of stable antigenic sites that are recognized by autologous immunoglobulins with subsequent complement deposition (C3b). Dexamethasone-21-phosphateloaded erythrocytes, modified as above, are readily recognized by macrophages through their Fe and C3b receptors with consequent phagocytosis and delivery of Dex-21-P from erythrocytes to the macrophages. In vitro targeting of Dex-21-P-loaded erythrocytes to human monocyte-derived macrophages was found to re-duce by more than 60% the phorbol 12-myristate 13-acetate-stimulated O₂^- release. Thus, the targeting of glucocorticoids to macrophages is feasible and results in the inhibition of respiratory burst.     

Submicron-size biodegradable polymer-based didanosine particles for treating HIV at early stage: an in vitro study

Human immunodeficiency viruses (HIV) hide themselves in macrophages at the early stage of infection. Delivering drug in a sustained manner from polymeric nanoparticles in those cells could control the disease effectively. The study was intended to develop poly(d,l-lactic-co-glycolic acid)-based nanoparticles containing didanosine and to observe their uptake by macrophages in vitro. Various physicochemical evaluations related to nanoparticles, such as drug–excipient interaction, surface morphology, particle size, zeta potential, polydispersity index, drug loading, in vitro drug release and nanoparticle-uptake by macrophages in vitro were determined. Homogenising speeds and drug–polymer ratio varied drug loading and polydispersity index of nanoparticles, providing sustained drug release. Dimethyl sulphoxide/polyethylene glycol improved drug loading predominantly. Nanoparticle-uptake by macrophages was concentration dependent. Experimental nanoparticles successfully transported didanosine to macrophages in vitro, suggesting reduction of dose, thus minimising toxicity and side effects. Developed nanoparticle may control HIV infection effectively at an early stage.     

Anti-tumor activity of paclitaxel through dual-targeting lipoprotein-mimicking nanocarrier

In the present study, we devised a strategy that paclitaxel (PTX) with lipid and octadecylamine were prepared to lipid nanoparticle (PTX-LNP) with positive charge, folic acid-modified bovine serum albumin (FB)-coated surface of PTX-LNP through electrostatic attraction and generated the lipoprotein-mimicking nanocomplex (FB-PTX-LNP) for dual-targeting therapy. Bovine serum albumin (BSA) was used as the protein model due to its specific targeting to tumor by increased transendothelial gp60-mediated transport and increased intratumoral accumulation as a result of the secreted protein, acidic and rich in cysteine (SPARC)–albumin interaction. The further conjugating folic acid to BSA achieved the dual active targeting. In vitro cytotoxicity tests suggested FB-PTX-LNP and BSA-PTX-LNP exhibited significantly higher cytotoxic activity against MCF-7 and HepG2 cells compared to PTX-LNP. The cellular uptake experiments indicated that FB-coumarin-6-LNP modified with dual-targeting had a faster and greater cellular uptake when compared to BSA-coumarin-6-LNP and coumarin-6-LNP by MCF-7 cells. Thus, both BSA and FA did play roles in in vitro cytotoxicity and cellular uptake. Furthermore, the targeting ability and therapeutic efficacy of FB-PTX-LNP were assessed in vivo. FB-PTX-LNP produced very marked targeting ability and anti-tumor activity in MDA-MB-231 tumor-bearing mice. These results indicate the protein–lipid nanocomplex FB-PTX-LNP is a potential nanocarrier for Paclitaxel dual-targeting to tumor.     

Erythrocytes as carriers of antisense PNA addressed against HIV-1 gag-pol transframe domain

PNA_PR2 is a peptide nucleic acid (PNA) complementary to a sequence of the viral protease-encoding gene, effective in blocking HIV release, when used at high doses. Erythrocytes (RBC) were used to target PNA_PR2 to the macrophage compartment. The antiviral activity was assessed in human HIV-infected macrophages both as inhibition of p24 production and reduction of HIV DNA content. PNA_PR2, either added to the medium at a concentration of 100 μM or loaded into RBC at about 40 μM, inhibited p24 production ~80% compared with infected samples and reduced HIV DNA content by 83% and 90%, respectively. The results show that (1) a stronger anti-HIV effect is achievable with higher doses of PNA_PR2, both when given free and encapsulated into RBC; (2) the antiviral effect obtained by free PNA_PR2 at a concentration of 100 μM is achievable by encapsulating it into RBC at a concentration of 40 μM, suggesting that RBC can be used as a delivery system to increase the antisense effect of PNA_PR2.     

Dimethyl Fumarate Prevents HIV-Induced Lysosomal Dysfunction and Cathepsin B Release from Macrophages

HIV-associated neurocognitive disorders (HAND) are prevalent despite combined antiretroviral therapy, affecting nearly half of HIV-infected patients worldwide. During HIV infection of macrophages secretion of the lysosomal protein, cathepsin B, is increased. Secreted cathepsin B has been shown to induce neurotoxicity. Oxidative stress is increased in HIV-infected patients, while antioxidants are decreased in monocytes from patients with HIV-associated dementia (HAD). Dimethyl fumarate (DMF), an antioxidant, has been reported to decrease HIV replication and neurotoxicity mediated by HIV-infected macrophages. Thus, we hypothesized that DMF will decrease cathepsin B release from HIV-infected macrophages by preventing oxidative stress and enhancing lysosomal function. Monocyte-derived macrophages (MDM) were isolated from healthy donors, inoculated with HIV-1_ADA, and treated with DMF following virus removal. After 12 days post-infection, HIV-1 p24 and total cathepsin B levels were measured from HIV-infected MDM supernatants using ELISA; intracellular reactive oxygen and nitrogen species (ROS/RNS) were measured from MDM lysates, and functional lysosomes were assessed using a pH-dependent lysosomal dye. Neurons were incubated with serum-free conditioned media from DMF-treated MDM and neurotoxicity was determined using TUNEL assay. Results indicate that DMF reduced HIV-1 replication and cathepsin B secretion from HIV-infected macrophages in a dose-dependent manner. Also, DMF decreased intracellular ROS/RNS levels, and prevented HIV-induced lysosomal dysfunction and neuronal apoptosis. In conclusion, the improvement in lysosomal function with DMF treatment may represent the possible mechanism to reduce HIV-1 replication and cathepsin B secretion. DMF represents a potential therapeutic strategy against HAND.     

Poly (propyleneimine) dendrimer based nanocontainers for targeting of efavirenz to human monocytes/macrophages in vitro

Cells of the mononuclear phagocytic system, in particular monocytes/macrophages (Mo/Mac) serve as a reservoir for human immunodeficiency virus (HIV) and are believed to be responsible for its dissemination throughout the body and especially into the brain. Treatment of HIV infection, therefore, must reach these cells in addition to the lymphocytes. The purpose of the present study is to develop poly(propyleneimine) (PPI) dendrimer-based nanocontainers for targeting of efavirenz (EFV) to Mo/Mac. Fifth generation PPI dendrimer, t-Boc–glycine conjugated PPI dendrimer (TPPI) and mannose conjugated dendrimers were synthesized and characterized. While the haemolytic activity and cytotoxicity of PPI dendrimer was found to be very high, the toxicity of t-Boc–glycine conjugated dendrimer and mannose conjugated dendrimers were found to be negligible. The entrapment efficiency of mannose conjugated dendrimer was found to be 47.4%, followed by that of PPI dendrimer (32.15%) and t-Boc–glycine conjugated dendrimer (23.1%). The in vitro drug release profile shows that while PPI dendrimer releases the drug by 24 h, the dendrimer-based nanocontainers prolong the release rate up to 144 h (83 ± 0.4% in case of t-Boc–glycine conjugated dendrimer and 91 ± 0.3% in mannose conjugated dendrimer). The cellular uptake of EFV was found to be both concentration and time dependent. Significant increase in cellular uptake of EFV by Mo/Mac cells were observed in case of mannose conjugated dendrimer which is 12 times higher than that of free drug and 5.5 times higher than that of t-Boc–glycine conjugated dendrimer. While mannose conjugated dendrimer was taken up by the lectin receptors of the cells, phagocytosis of t-Boc–glycine conjugated dendrimer might be responsible for its enhanced uptake. Results suggest that the proposed carriers hold potential to increase the efficacy and reduce the toxicity of antiretroviral therapy.     

Anti-HIV drugs,lopinavir/ritonavir and atazanavir,modulate innate immune response triggered by Leishmania in macrophages: The role of NF-κB and PPAR-γ

This study evaluated the influence of HIV protease inhibitors lopinavir/ritonavir (LPV/RTV) and atazanavir (ATV) on macrophage functions during their first interaction with Leishmania. Macrophages from BALB/c mice treated for 10 days with LPV/RTV and ATV, infected or not in vitro with L. (L.) amazonensis, were used to investigate the effects of these drugs on infection index, leishmanicidal capacity, cytokine production and PPAR-γ and RelB expression. LPV/RTV and ATV treatments significantly increased the infection index and the percentage of Leishmania-infected macrophages compared to untreated infected macrophages. There was no correlated increase in the production of NO and H₂O₂ leishmanicidal molecules. Promastigotes derived from Leishmania-infected macrophages from LPV/RTV and ATV-treated BALB/c mice had an in vitro growth 45.1% and 56.4% higher in groups treated with LPV/RTV and ATV than with PBS in culture. ATV treatment reduced IL-12p70 and IL-10 secretion in Leishmania-infected macrophages, but had no effect on IL-23 and TNF production. LPV reduced IL-10 and had no effect on IL-12p70, TNF and IL-23 secretion. ATV treatment decreased PPAR-γ expression in Leishmania-infected macrophages compared to untreated infected macrophages. In addition, LPV/RTV, but not ATV, reduced RelB cytoplasm-to-nucleus translocation in Leishmania-infected macrophages. Results showed that LPV/RTV and ATV HIV protease inhibitors were able to modulate innate defense mechanisms against Leishmania via different intracellular pathways. Although HIV protease inhibitors are highly efficient to control the Human Immunodeficiency Virus, these drugs might also influence the course of leishmaniasis in HIV-Leishmania-co-infected individuals.     

Adenosine Triphosphate Released from HIV-Infected Macrophages Regulates Glutamatergic Tone and Dendritic Spine Density on Neurons

Despite wide spread use of combination antiretroviral therapy (cART) in developed countries, approximately half of HIV-infected patients will develop impairments in cognitive function. Accumulating evidence suggests that neuronal dysfunction can be precipitated by HIV-infection of macrophages by mechanisms that involve alterations in innate and adaptive immune responses. HIV-infection of macrophages is known to increase the release of soluble neurotoxins. However, the composition of products released from infected macrophages is complex and not fully known. In this study we provide evidence that ATP and other immuno-/neuromodulatory nucleotides are exported from HIV-infected macrophages and modify neuronal structure. Supernatants collected from HIV-infected macrophages (HIV/MDM) contained large amounts of ATP, ADP, AMP and small amounts of adenosine, in addition to glutamate. Dilutions of these supernatants that were sub-threshold for glutamate receptor activation evoked rapid calcium flux in neurons that were completely inhibited by the enzymatic degradation of ATP, or by blockade of calcium permeable purinergic receptors. Applications of these highly diluted HIV/MDM onto neuronal cultures increased the amount of extracellular glutamate by mechanisms dependent on purinergic receptor activation, and downregulated spine density on neurons by mechanisms dependent on purinergic and glutamate receptor activation. We conclude from these data that ATP released from HIV-infected macrophages downregulates dendritic spine density on neurons by a mechanism that involves purinergic receptor mediated modulation of glutamatergic tone. These data suggest that neuronal function may be depressed in HIV infected individuals by mechanisms that involve macrophage release of ATP that triggers secondary effects on glutamate handling.