HbAHP-25 attenuates HIV-1 gp120 mediated inflammation and barrier dysfunction.

OBJECTIVES: Development of HbAHP-25, a peptide that prevents HIV-1 entry into cells by blocking gp120-CD4 interaction, as a topical anti-HIV drug, necessitates that it is first tested for toxic or abrasive effects on genital epithelial cells and also on the vaginal microbiome. The present study was, therefore, undertaken to investigate whether: (1) HbAHP-25 has any adverse effect on growth and membrane integrity of various cell lines, and (2) HbAHP-25 neutralizes gp120 mediated insults on genital epithelial cells. METHODS: MTT and trans-epithelial resistance (TER) assays were performed to assess the viability and integrity of epithelial cells. Real-time PCR and Immunofluorescence/Western blotting were used to decipher the expression of tight junction proteins, at the mRNA and protein levels, respectively. A multiplex cytokine assay was performed to quantify the cytokines. RESULTS: HbAHP-25 had no adverse effect on the viability of VK2/E6E7, End1/E6E7, Ect1/E6E7 and HEC-1A cells, and also on growth of lactobacilli. The barrier integrity of HbAHP-25-treated cells remained unaltered. Expression of tight junction proteins, Claudin-1 and ZO-1, at transcript and protein levels, remained unaltered in HbAHP-25-treated HEC-1A cells. Interestingly, HbAHP-25 treatment prevented the breach of barrier integrity caused by gp120. Further, HbAHP-25 did not elicit the expression of inflammatory cytokines. Instead, the in vitro induction of inflammatory cytokines by gp120 was also abrogated in the presence of HbAHP-25. CONCLUSION: HbAHP-25 is exceedingly safe to genital epithelial cells and attenuates HIV-1 gp120-mediated barrier dysfunction by limiting excessive inflammation. This study provides significant evidences in the favor of HbAHP-25's potential as a topical anti-HIV agent.

Effects of nanoparticle-encapsulated curcumin on HIV-gp120-associated neuropathic pain induced by the P2X3 receptor in dorsal root ganglia.

HIV-1 envelope glycoprotein (Glycoprotein 120, gp120) can directly stimulate primary sensory afferent neurons and cause chronic neuropathic pain. The P2X3 receptor in the dorsal root ganglia (DRG) is associated with the transmission of neuropathic pain. Curcumin isolated from the herb Curcuma rhizome has anti-inflammatory and anti-tumor effects. The water solubility, targeting and bioavailability of curcumin can be improved by nanoparticle encapsulation. In this study, we sought to explore the effects of nanoparticle-encapsulated curcumin (nano curcumin) on HIV-gp120-induced neuropathic pain mediated by the P2X3 receptor in DRG neurons. The results showed that mechanical and thermal hyperalgesia in rats treated with gp120 were increased compared to those in the control group. The expression levels of P2X3 mRNA and protein in rats treated with gp120 were higher than those in the control group. Nano curcumin treatment decreased mechanical hyperalgesia and thermal hyperalgesia and upregulated the expression levels of P2X3 mRNA and protein in rats treated with gp120. Nano curcumin treatment also reduced the ERK1/2 phosphorylation levels in gp120-treated rat DRG. In addition, P2X3 agonist α,ß-methylene ATP (α,ß-meATP)-induced currents in DRG neurons cultured with gp120 significantly decreased after co-treatment with nano curcumin. Therefore, nano curcumin treatment may inhibit P2X3 activation, decrease the sensitizing DRG primary afferents and relieve mechanical hyperalgesia and thermal hyperalgesia in gp120-treated rats.

The Low-Cost Compound Lignosulfonic Acid (LA) Exhibits Broad-Spectrum Anti-HIV and Anti-HSV Activity and Has Potential for Microbicidal Applications.

OBJECTIVES: Lignosulfonic acid (LA), a low-cost lignin-derived polyanionic macromolecule, was extensively studied for its anti-HIV and anti-HSV activity in various cellular assays, its mechanism of viral inhibition and safety profile as potential microbicide. RESULTS: LA demonstrated potent inhibitory activity of HIV replication against a wide range of R5 and X4 HIV strains and prevented the uptake of HIV by bystander CD4+ T cells from persistently infected T cells in vitro (IC50: 0.07 - 0.34 µM). LA also inhibited HSV-2 replication in vitro in different cell types (IC50: 0.42 - 1.1 µM) and in rodents in vivo. Furthermore, LA neutralized the HIV-1 and HSV-2 DC-SIGN-mediated viral transfer to CD4+ T cells (IC50: ~1 µM). In addition, dual HIV-1/HSV-2 infection in T cells was potently blocked by LA (IC50: 0.71 µM). No antiviral activity was observed against the non-enveloped viruses Coxsackie type B4 and Reovirus type 1. LA is defined as a HIV entry inhibitor since it interfered with gp120 binding to the cell surface of T cells. Pretreatment of PBMCs with LA neither increased expression levels of cellular activation markers (CD69, CD25 and HLA-DR), nor enhanced HIV-1 replication. Furthermore, we found that LA had non-antagonistic effects with acyclovir, PRO2000 or LabyA1 (combination index (CI): 0.46 - 1.03) in its anti-HSV-2 activity and synergized with tenofovir (CI: 0.59) in its anti-HIV-1 activity. To identify mechanisms of LA resistance, we generated in vitro a mutant HIV-1 NL4.3LAresistant virus, which acquired seven mutations in the HIV-1 envelope glycoproteins: S160N, V170N, Q280H and R389T in gp120 and K77Q, N113D and H132Y in gp41. Additionally, HIV-1 NL4.3LAresistant virus showed cross-resistance with feglymycin, enfuvirtide, PRO2000 and mAb b12, four well-described HIV binding/fusion inhibitors. Importantly, LA did not affect the growth of vaginal Lactobacilli strains. CONCLUSION: Overall, these data highlight LA as a potential and unique low-cost microbicide displaying broad anti-HIV and anti-HSV activity.

DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers.

UNLABELLED: Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. IMPORTANCE: Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans.

3D visualization of HIV virions by cryoelectron tomography.

The structure of the human immunodeficiency virus (HIV) and some of its components have been difficult to study in three-dimensions (3D) primarily because of their intrinsic structural variability. Recent advances in cryoelectron tomography (cryo-ET) have provided a new approach for determining the 3D structures of the intact virus, the HIV capsid, and the envelope glycoproteins located on the viral surface. A number of cryo-ET procedures related to specimen preservation, data collection, and image processing are presented in this chapter. The techniques described herein are well suited for determining the ultrastructure of bacterial and viral pathogens and their associated molecular machines in situ at nanometer resolution.

Characterizing the anti-HIV activity of papuamide A.

Papuamide A is representative of a class of marine derived cyclic depsipeptides, reported to have cytoprotective activity against HIV-1 in vitro. We show here that papuamide A acts as an entry inhibitor, preventing human immunodeficiency virus infection of host cells and that this inhibition is not specific to R5 or X4 tropic virus. This inhibition of viral entry was determined to not be due to papuamide A binding to CD4 or HIV gp120, the two proteins involved in the cell-virus recognition and binding. Furthermore, papuamide A was able to inhibit HIV pseudotype viruses expressing envelope glycoproteins from vesicular stomatitis virus or amphotropic murine leukemia virus indicating the mechanism of viral entry inhibition is not HIV-1 envelope glycoprotein specific. Time delayed addition studies with the pseudotyped viruses show that papuamide A inhibits viral infection only at the initial stage of the viral life cycle. Additionally, pretreatment studies revealed that the virus, and not the cell, is the target of papuamide A's action. Together, these results suggest a direct virucidal mechanism of HIV-1 inhibition by papuamide A. We also demonstrate here that the other papuamides (B-D) are able to inhibit viral entry indicating that the free amino moiety of 2,3-diaminobutanoic acid residue is not required for the virucidal activity.

The tyrosine kinase inhibitor genistein blocks HIV-1 infection in primary human macrophages.

Binding of HIV-1 envelope glycoprotein (Env) to its cellular receptors elicits a variety of signaling events, including the activation of select tyrosine kinases. To evaluate the potential role of such signaling, we examined the effects of the tyrosine kinase inhibitor, genistein, on HIV-1 entry and infection of human macrophages using a variety of assays. Without altering cell viability, cell surface expression of CD4 and CCR5 or their abilities to interact with Env, genistein inhibited infection of macrophages by reporter gene-encoding, beta-lactamase containing, or wild type virions, as well as Env-mediated cell-fusion. The observation that genistein blocked virus infection if applied before, during or immediately after the infection period, but not 24h later; coupled with a more pronounced inhibition of infection in the reporter gene assays as compared to both beta-lactamase and p24 particle entry assays, imply that genistein exerts its inhibitory effects on both entry and early post-entry steps. These findings suggest that other exploitable targets, or steps, of the HIV-1 infection process may exist and could serve as additional opportunities for the development of new therapeutics.

The appealing story of HIV entry inhibitors : from discovery of biological mechanisms to drug development.

Current therapeutic intervention in HIV infection relies upon 20 different drugs. Despite the impressive efficacy shown by these drugs, we are confronted with an unexpected frequency of adverse effects, such as mitochondrial toxicity and lipodystrophy, and resistance, not only to individual drugs but to entire drug classes.Thus, there is now a great need for new antiretroviral drugs with reduced toxicity, increased activity against drug-resistant viruses and a greater capacity to reach tissue sanctuaries of the virus. Two different HIV molecules have been selected as targets of drug inhibition so far: reverse transcriptase and protease. Drugs that target the interactions between the HIV envelope and the cellular receptor complex are a 'new entry' into the scenario of HIV therapy and have recently raised great interest because of their activity against multidrug-resistant viruses. There are several compounds that are at different developmental stages in the pipeline to counter HIV entry, among them: (i) the attachment inhibitor dextrin-2-sulfate; (ii) the inhibitors of the glycoprotein (gp) 120/CD4 interaction PRO 542, TNX 355 and BMS 488043; (iii) the co-receptor inhibitors subdivided in those targeting CCR5 (SCH 417690 [SCH D], UK 427857 GW 873140, PRO 140, TAK 220, AMD 887) and those targeting CXCR4 (AMD 070, KRH 2731); and (iv) the fusion inhibitors enfuvirtide (T-20) and tifuvirtide (T-1249). The story of the first of these drugs, enfuvirtide, which has successfully completed phase III clinical trials, has been approved by the US FDA and by the European Medicines Agency, and is now commercially available worldwide, is an example of how the knowledge of basic molecular mechanisms can rapidly translate into the development of clinically effective molecules.

HIV-chemotherapy and -prophylaxis: new drugs, leads and approaches.

In recent years, significant progress has been made towards the chemotherapy (and prophylaxis) of HIV infections. This progress is situated at three different levels. (i) New anti-HIV drugs have been approved for clinical use and have entered the market: the virus entry inhibitor enfuvirtide (Fuzeon), the nucleoside reverse transcriptase inhibitor (NRTI) emtricitabine (Emtriva), the nucleotide reverse transcriptase inhibitor (NtRTI) tenofovir disoproxil fumarate (Viread trade mark ) and the HIV protease inhibitor (PI) atazanavir (Reyataz trade mark ). (ii) Other compounds have proceeded through preclinical and/or clinical development: CXCR4 antagonists (i.e. AMD070), CCR5 antagonists (i.e. SCH-C), NRTIs (such as amdoxovir), NNRTIs (such as etravirine), integrase inhibitors (such as S-1360) and PIs (such as tipranavir). (iii) Yet other compounds, acting by novel mechanisms, have recently been identified as anti-HIV agents that seem worthy of further (pre)clinical development: cell receptor CD4 down-modulators (i.e. cyclotriazadisulfonamides), viral envelope gp120-binding agents such as plant lectins and glycopeptide antibiotics, HIV integrase inhibitors such as the pyranodipyrimidine V-165, and two new classes of compounds (i.e. N-aminoimidazoles and pyridine oxide derivatives) which seem to interfere with a post-integration, transcription transactivation event. Taken together, it is obvious that the approaches for the treatment of HIV infections in recent years have become both more diverse and more efficient.

Epigallocatechin gallate, the main component of tea polyphenol, binds to CD4 and interferes with gp120 binding.

BACKGROUND: Epigallocatechin gallate (EGCG), the major component of tea polyphenol, has been reported to have various physiologic modulatory activities. Several reports also have shown that catechin has a protective effect against HIV infection, part of which is mediated by inhibiting virions to bind to the target cell surface. OBJECTIVE: We investigated the effect of EGCG on the expression of CD4 molecules and on its ability to bind gp120, an envelope protein of HIV-1. METHODS: Peripheral blood CD4+ T cells were incubated in the presence of EGCG, and the expression of CD4 was evaluated by means of flow cytometry. The effect of EGCG on the antibody binding to CD4 was investigated by using a sandwich ELISA, and the effect on the gp120 binding to CD4 was analyzed by means of flow cytometry. RESULTS: EGCG efficiently inhibited binding of anti-CD4 antibody to its corresponding antigen. This effect was mediated by the direct binding of EGCG to the CD4 molecule, with consequent inhibition of antibody binding, as well as gp120 binding. CONCLUSION: The present results suggest a potential preventive effect of EGCG on HIV-1 infection by modulating binding to CD4.

The bicyclam AMD3100 story.

The discovery and development of the bicyclam AMD3100--a chemokine receptor antagonist--has highlighted the therapeutic potential of such compounds in HIV infection, inflammatory diseases, cancer and stem-cell mobilization. Here, I describe the development process of AMD3100, which began about 15 years ago with the isolation of an impurity, and the basis for the clinical application of AMD3100 and its congeners.

The fusion of IGF I with stromal cell-derived factor I or alpha1 proteinase inhibitor alters their mitogenic or chemotactic activities while keeping their ability to inhibit HIV-1-gp120 binding.

It has been previously reported that insulin-like growth factor I (IGF I) decreases in AIDS patients with wasting, a condition that is partially prevented by combined IGF I growth hormone therapy. By generating bifunctional proteins of IGF I and stromal cell-derived factor 1alpha (SDF-1alpha) or alpha1 proteinase inhibitor (API), two proteins known to prevent HIV infection, it may be possible to improve the therapeutic effectiveness of these compounds for the treatment of AIDS-mediated wasting. SDF-1alpha or the M351E-M358L mutant of API were attached at the C-terminal end of IGF I and synthesized by a stable insect cell expression technique. The IGF I-SDF-1alpha chimera reduced the enhancement of thymidine incorporation into bovine fetal erythroid cells observed in the presence of insect cell produced IGF I alone. It also decreased the SDF-1 and IGF I-stimulated hematopoietic cell migration, without losing the capacity to compete with the binding of HIV-1 (IIIB)-surface glycoprotein gp120. The IGF I-API chimera displayed the same mitogenic activity and a similar, but lower chemotactic activity than IGF I in the assays mentioned above. It had a comparable anti-elastase activity to that observed with a previously described IGF II-API fusion protein with the single mutation M351E. The binding of gp120 to a murine hematopoietic cell line was stimulated by human neutrophil elastase (25-100 nM) and inhibited by IGF I-API. In conclusion, the linkage of IGF I with SDF-1 or API can alter some biological functions of the single components of the chimera while keeping their ability to compete with HIV-1-gp120 binding.

New anti-HIV agents and targets.

Virtually all the compounds that are currently used or are subject of advanced clinical trials for the treatment of HIV infections, belong to one of the following classes: (i) nucleoside reverse transcriptase inhibitors (NRTIs): i.e., zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine and nucleotide reverse transcriptase inhibitors (NtRTIs) (i.e., tenofovir disoproxil fumarate); (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs): i.e., nevirapine, delavirdine, efavirenz, emivirine; and (iii) protease inhibitors (PIs): i.e., saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, and lopinavir. In addition to the reverse transcriptase and protease reaction, various other events in the HIV replicative cycle can be considered as potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulfates, polysulfonates, polycarboxylates, polyoxometalates, polynucleotides, and negatively charged albumins); (ii) viral entry, through blockade of the viral coreceptors CXCR4 (i.e., bicyclam (AMD3100) derivatives) and CCR5 (i.e., TAK-779 derivatives); (iii) virus-cell fusion, through binding to the viral envelope glycoprotein gp41 (T-20, T-1249); (iv) viral assembly and disassembly, through NCp7 zinc finger-targeted agents [2,2'-dithiobisbenzamides (DIBAs), azadicarbonamide (ADA)]; (v) proviral DNA integration, through integrase inhibitors such as 4-aryl-2,4-dioxobutanoic acid derivatives; (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (flavopiridol, fluoroquinolones). Also, various new NRTIs, NNRTIs, and PIs have been developed that possess, respectively: (i) improved metabolic characteristics (i.e., phosphoramidate and cyclosaligenyl pronucleotides by-passing the first phosphorylation step of the NRTIs), (ii) increased activity ["second" or "third" generation NNRTIs ( i.e., TMC-125, DPC-083)] against those HIV strains that are resistant to the "first" generation NNRTIs, or (iii), as in the case of PIs, a different, modified peptidic (i.e., azapeptidic (atazanavir)) or non-peptidic scaffold (i.e., cyclic urea (mozenavir), 4-hydroxy-2-pyrone (tipranavir)). Non-peptidic PIs may be expected to inhibit HIV mutant strains that have become resistant to peptidomimetic PIs.

Inhibitors of protein-disulfide isomerase prevent cleavage of disulfide bonds in receptor-bound glycoprotein 120 and prevent HIV-1 entry.

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.

New developments in anti-HIV chemotherapy.

Virtually all the compounds that are currently used, or are subject of advanced clinical trials, for the treatment of human immunodeficiency virus (HIV) infections, belong to one of the following classes: (i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs): i.e. zidovudine (AZT), didanosine (ddI), zalcitabine (ddC), stavudine (d4T), lamivudine (3TC), abacavir (ABC), emtricitabine [(-)FTC], tenofovir disoproxil fumarate; (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs): i.e. nevirapine, delavirdine, efavirenz, emivirine; and (iii) protease inhibitors (PIs): i.e. saquinavir, ritonavir, indinavir, nelfinavir, amprenavir and lopinavir. In addition to the reverse transcriptase (RT) and protease reaction, various other events in the HIV replicative cycle can be considered as potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120 (polysulfates, polysulfonates, polycarboxylates, polyoxometalates, polynucleotides, and negatively charged albumins); (ii) viral entry, through blockade of the viral coreceptors CXCR4 [bicyclam (AMD3100) derivatives] and CCR5 (TAK-779 derivatives); (iii) virus-cell fusion, through binding to the viral envelope glycoprotein gp41 (T-20, T-1249); (iv) viral assembly and disassembly, through NCp7 zinc finger-targeted agents [2,2'-dithiobisbenzamides (DIBAs), azadicarbonamide (ADA)]; (v) proviral DNA integration, through integrase inhibitors such as 4-aryl-2,4-dioxobutanoic acid derivatives; (vi) viral mRNA transcription, through inhibitors of the transcription (transactivation) process (flavopiridol, fluoroquinolones). Also, various new NRTIs, NNRTIs and PIs have been developed that possess, respectively: (i) improved metabolic characteristics (i.e. phosphoramidate and cyclosaligenyl pronucleotides by-passing the first phosphorylation step of the NRTIs), (ii) increased activity ["second" or "third" generation NNRTIs (i.e. TMC-125, DPC-083)] against those HIV strains that are resistant to the "first" generation NNRTIs, or (iii) as in the case of PIs, a different, nonpeptidic scaffold [i.e. cyclic urea (mozenavir), 4-hydroxy-2-pyrone (tipranavir)]. Nonpeptidic PIs may be expected to inhibit HIV mutant strains that have become resistant to peptidomimetic PIs. Given the multitude of molecular targets with which anti-HIV agents can interact, one should be cautious in extrapolating the mode of action of these agents from cell-free enzymatic assays to intact cells. Two examples in point are L-chicoric acid and the nonapeptoid CGP64222, which were initially described as an integrase inhibitor or Tat antagonist, respectively, but later shown to primarily act as virus adsorption/entry inhibitors, the latter through blockade of CXCR4.

Ethanol pre-exposure suppresses HIV-1 glycoprotein 120-induced neuronal degeneration by abrogating endogenous glutamate/Ca2+-mediated neurotoxicity.

The neurotoxic mechanism of HIV-1 envelope glycoprotein 120 (gp120) involves glutamatergic (NMDA) receptor/Ca2+-dependent excitotoxicity, mediated in part via glia. Pro-inflammatory cytokines also may have roles. We have reported that pre-exposure of brain cultures to 'physiological' ethanol concentrations (20-30 mM) protects against neuronal damage from HIV-1 gp120, but not from the direct receptor agonist, NMDA. Using lactate dehydrogenase assays and propidium iodide staining of rat organotypic hippocampal-entorhinal cortical slice cultures we determined that ethanol's suppression of gp120 neurotoxicity required at least 4 days of pretreatment. The gp120-induced neurotoxicity was accompanied by interleukin-6 elevations that were not affected by the pretreatment. However, gp120 induced substantial, early increases in extracellular glutamate levels that were blocked by ethanol pretreatment, conceivably abrogating excitotoxicity. Consistent with abrogation of excitotoxic pathways, fura-2 imaging showed selective deficits in gp120-dependent intracellular Ca2+ responses in ethanol-pretreated slices. Gp120 is believed to increase glutamate levels by both stimulating release and inhibiting (re)uptake. Results with a labeled glutamate analog, D-[3H]aspartate, revealed that gp120's inhibition of glutamate uptake, rather than its stimulation of release, was abolished after ethanol. Further studies indicated that two converging effects of ethanol pretreatment may underlie the abolishment of gp120-mediated glutamate uptake inhibition: (a) blockade of gp120-induced release (ostensibly from glia) of arachidonic acid, an inhibitor of astroglial glutamate reuptake, and (b) modest proliferation and activation of astroglia upon gp120 stimulation--which are likely to augment glutamate transporters. Thus, as with gp120 itself, glia and glutamate/arachidonic acid regulation appear to be important targets for ethanol. Since moderate ethanol consumption is as common among HIV-infected individuals as in the general population, this newly recognized neuroprotective (and apparently anti-excitotoxic) effect of ethanol withdrawal in vitro could be important, but it requires further study before its significance, if any, is understood.

New developments in anti-HIV chemotherapy.

Virtually all the compounds that are currently used, or under advanced clinical trial, for the treatment of HIV infections, belong to one of the following classes: (i) nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs): i.e. zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, emtricitabine, tenofovir (PMPA) disoproxil fumarate; (ii) non-nucleoside reverse transcriptase inhibitors (NNRTIs): i.e. nevirapine, delavirdine, efavirenz, emivirine; and (iii) protease inhibitors (PIs): i.e. saquinavir, ritonavir, indinavir, nelfinavir and amprenavir. In addition, various other events in the HIV replicative cycle are potential targets for chemotherapeutic intervention: (i) viral adsorption, through binding to the viral envelope glycoprotein gp120; (ii) viral entry, through blockade of the viral coreceptors CXCR4 and CCR5; (iii) virus-cell fusion; (iv) viral assembly and disassembly; (v) proviral DNA integration; (vi) viral mRNA transcription. Also, new NRTIs, NNRTIs and PIs have been developed that possess respectively improved metabolic characteristics, or increased activity against NNRTI-resistant HIV strains or, as in the case of PIs, a different, non-peptidic scaffold. Given the multitude of molecular targets with which anti-HIV agents can interact, one should be cautious in extrapolating from cell-free enzymatic assays to the mode of action of these agents in intact cells.