Penetration of CD4 T cells by HIV-1. The CD4 receptor does not internalize with HIV, and CD4-related signal transduction events are not required for entry.

Receptor binding of HIV to the CD4 molecule is required for efficient infection of T cells, but the post-binding steps that result in penetration of HIV are not well understood. CD4 is induced to internalize upon T cell activation, and mAb to CD4 modify signal transduction and T cell activation as does HIV in some systems. It is not known whether HIV binding triggers CD4 endocytosis or whether signal transduction events are required for penetration. Selected inhibitors of signal transduction were evaluated for their effects on penetration using two assays that are dependent on penetration. After short term exposure to inhibitor and HIV, cells were analyzed for reverse-transcribed HIV DNA (DNA amplification assay), or productive infection is monitored (infectivity assay). Viral penetration was tested in the presence of H7 (protein kinase C inhibition), EGTA (extracellular Ca2+ chelation), cyclosporine A (inhibition of Ca2+/calmodulin-dependent activation), or pertussis toxin (inhibition of G protein function). All agents were used at concentrations that were inhibitory for their respective signal transduction pathways. None of the inhibitors affected viral penetration. We tracked the CD4 molecule with fluorescent probes that do not interfere with HIV binding in a system where CD4 T cells were saturated with HIV and the penetration event was relatively synchronized. Under conditions where detection of CD4 was more sensitive than the detection of HIV, HIV internalization was readily detected but CD4 internalization was not.     

Nelfinavir induces TRAIL receptor upregulation in ovarian cancer cells

HIV protease inhibitors are currently being discussed to be useful as new and alternative anti-cancer agents, especially as second line treatments for chemo-resistant human cancer types. Among three clinically applied HIV protease inhibitors tested, we found a high efficacy of nelfinavir on ovarian cancer cells, accompanied by apoptosis (annexin binding) and necrosis (propidium iodide permeability). In vitro, at concentrations used to induce cell death in ovarian cancer cells, nelfinavir had no effect on the cellular viability of fibroblasts or peripheral blood mononuclear leukocytes. Nelfinavir sensitized ovarian cancer cells to treatment with an apoptosis-inducing TRAIL receptor antibody due to upregulation of the TRAIL receptor DR5 as shown by RT-PCR and FACScan analysis. We conclude that nelfinavir, an already approved drug, is a highly efficient agent against ovarian cancer cells and could sensitize ovarian cancer cells to TRAIL treatment, either therapeutically applied or endogenously produced by cells of the immune system.     

HIV and chemokines: ligands sharing cell-surface receptors

At the cell surface, chemokine receptors and CD4 act in concert to bind to human immunodeficiency virus (HIV) and trigger its entry into and infection of cells. Several different chemokine receptors can act as co-receptors for HIV entry, although either CCR5 or CXCR4 is used by all HIV-1 strains studied so far. The capacity of different HIV strains to exploit different chemokine receptors influences their cell tropism, cytopathicity and pathogenicity. Chemokines, the natural ligands for these receptors, can block HIV entry and are thus potential starting points for the design of novel therapeutic agents against HIV infection.     

Microbicides and other topical strategies to prevent vaginal transmission of HIV

The HIV epidemic is, by many criteria, the worst outbreak of infectious disease in history. The rate of new infections is now approximately 5 million per year, mainly in the developing world, and is increasing. Women are now substantially more at risk of infection with HIV than men. With no cure or effective vaccine in sight, a huge effort is required to develop topical agents (often called microbicides) that, applied to the vaginal mucosa, would prevent infection of these high-risk individuals. We discuss the targets for topical agents that have been identified by studies of the biology of HIV infection and provide an overview of the progress towards the development of a usable agent.     

Assessment of developmental toxicity of antiretroviral drugs using a rat whole embryo culture system

BACKGROUND: Previous guidelines for HIV-infected pregnant women have recommended zidovudine (ZDV) monotherapy during the second and third trimesters of pregnancy to prevent fetal HIV infection. New guidelines suggest that women should continue or be offered combination antiretroviral therapy (including protease inhibitors) during pregnancy. Nevertheless, little animal or human toxicity data underlie these recommendations. METHODS: We used an in vitro rat whole embryo culture system to assess the embryo toxicity of various nucleoside analogues, namely, ZDV, dideoxyinosine (ddI), and 2', 3'-dideoxycytidine (ddC), and the HIV-1 protease inhibitor, indinavir, both alone and in combination. RESULTS: Although human fetal concentrations of these compounds are unknown, no gross abnormalities were detected after incubation with these agents, either alone or in combination at concentrations that would be expected to be achievable in human maternal serum (1-50 microM). ZDV in combination with ddC at >100 microM, resulted in severe growth retardation and morphologic abnormalities not seen with either agent singly. CONCLUSIONS: We conclude that the combination of ZDV/ddC results in severe concentration-dependent embryo toxicity. No growth retardation or gross morphologic abnormalities were found for any of the agents, either singly or in combination, at clinically relevant concentrations.     

Rapid antibody quantification and generation of whole proteome antibody response profiles using LIPS (luciferase immunoprecipitation systems)

The application of LIPS to the rapid quantification of antibody responses to infectious agents is described. Chimeric genes encoding pathogen antigens fused to Renilla luciferase are expressed in mammalian cells; crude extracts are prepared and, without purification, employed in immunoprecipitation assays to quantify pathogen-specific antibodies. In cross-sectional and longitudinal studies, antibody levels to the MSG-14 antigen of Pneumocystis jirovecii measured by this assay correlated well with levels previously obtained with an optimized ELISA. We also correctly predicted Hepatitis B (HBV), Hepatitis C (HCV), and HIV infection status in all but 2 of 99 assays analyzing 33 patient sera. We then used 15 HIV-encoded proteins comprising the whole HIV proteome to generate antibody response profiles for these 33 sera. Each HIV antigen was recognized by antibodies in serum from at least one HIV-infected individual. Data generated with these simple, quantitative antibody-detection assays have both clinical and research applications.     

Parameters of human immunodeficiency virus infection of human cervical tissue and inhibition by vaginal virucides

Heterosexual transmission of human immunodeficiency virus (HIV) is the most frequent mode of infection worldwide. However, the immediate events between exposure to infectious virus and establishment of infection are still poorly understood. This study investigates parameters of HIV infection of human female genital tissue in vitro using an explant culture model. In particular, we investigated the role of the epithelium and virucidal agents in protection against HIV infection. We have demonstrated that the major target cells of infection reside below the genital epithelium, and thus HIV must cross this barrier to establish infection. Immune activation enhanced HIV infection of such subepithelial cells. Furthermore, our data suggest that genital epithelial cells were not susceptible to HIV infection, appear to play no part in the transfer of infectious virus across the epithelium, and thus may provide a barrier to infection. In addition, experiments using a panel of virucidal agents demonstrated differential efficiency to block HIV infection of subepithelial cells from partial to complete inhibition. This is the first demonstration that virucidal agents designed for topical vaginal use block HIV infection of genital tissue. Such agents have major implications for world health, as they will provide women with a mechanism of personal and covert protection from HIV infection.     

Cysteine protects Na,K-ATPase and isolated human lymphocytes from silver toxicity.

Metal-binding proteins are important components of retroviruses such as human immunodeficiency virus (HIV). Therefore, metals could be used as antiviral agents. However, most metals are toxic for humans with the exception of silver which is toxic only to prokaryotic cells and viruses. In addition, HIV infection causes a decrease in body cysteine. We formed a complex of silver and cysteine, named silver-cysteine. Healthy human lymphocytes were incubated with silver-nitrate or silver-cysteine. Negligible cell survival was seen at 50 microM silver-nitrate. However, in presence of 1 mM cysteine, the viability remained unaffected up to 1 mM of silver. Further, silver inhibition of isolated Na,K-ATPase was easily reversed by cysteine. Thus, non-toxic silver-cysteine could be used as an anti-viral and cysteine-replenishing agent.     

1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide (EICAR) markedly potentiate the inhibitory effect of 2',3'-dideoxyinosine on human immunodeficiency virus in peripheral blood lymphocytes.

Ribavirin and EICAR are two antiviral agents that share a similar antiviral activity spectrum and are targeted at inosine 5'-monophosphate (IMP) dehydrogenase. Neither ribavirin nor EICAR inhibit the replication of human immunodeficiency virus (HIV) in peripheral blood lymphocyte cells (PBL) at subtoxic concentrations. However, both compounds markedly potentiate the anti-HIV activity of 2',3'-dideoxyinosine (DDI) in PBL cells without a marked increase of toxicity. Both the increased IMP levels and the decreased guanine nucleotide levels caused by ribavirin and EICAR may be responsible for their potentiating effect on the anti-HIV activity of DDI.     

Carbovir: A Carbocyclic Nucleoside with Potent and Selective Activity Against Human Immunodeficiency Virus (HIV) in Vitro

Abstract

Carbocyclic 2′, 3′-didehydro-2′,3′-dideoxyquanosine (carbovir), a novel nucleoside analog, emerged as a potent and selective anti-HIV agent from a primary screen of a large number of carbocyclic nucleosides.¹ Carbovir inhibited the infectivity and replication of HIV in T-cells at concentrations 200 to 400-fold below toxicity to host cells. Carbovir was also evaluated for its Inhibitory effects on the expression of viral antigen in HIV-infected CEM cells. Production of p 24 core antigen at optimal inhibitory concentrations of the antiviral agents indicated comparable results for AZT, ddA and carbovir.     

Ultra-Fast Analysis of Plasma and Intracellular Levels of HIV Protease Inhibitors in Children: A Clinical Application of MALDI Mass Spectrometry

HIV protease inhibitors must penetrate into cells to exert their action. Differences in the intracellular pharmacokinetics of these drugs may explain why some patients fail on therapy or suffer from drug toxicity. Yet, there is no information available on the intracellular levels of HIV protease inhibitors in HIV infected children, which is in part due to the large amount of sample that is normally required to measure the intracellular concentrations of these drugs. Therefore, we developed an ultra-fast and sensitive assay to measure the intracellular concentrations of HIV protease inhibitors in small amounts of peripheral blood mononuclear cells (PBMCs), and determined the intracellular concentrations of lopinavir and ritonavir in HIV infected children. An assay based on matrix-assisted laser desorption/ionization (MALDI) - triple quadrupole mass spectrometry was developed to determine the concentrations of HIV protease inhibitors in 10 µL plasma and 1×10⁶ PBMCs. Precisions and accuracies were within the values set by the FDA for bioanalytical method validation. Lopinavir and ritonavir did not accumulate in PBMCs of HIV infected children. In addition, the intracellular concentrations of lopinavir and ritonavir correlated poorly to the plasma concentrations of these drugs. MALDI-triple quadrupole mass spectrometry is a new tool for ultra-fast and sensitive determination of drug concentrations which can be used, for example, to assess the intracellular pharmacokinetics of HIV protease inhibitors in HIV infected children.     

Use of a focal infectivity assay for testing susceptibility of HIV to antiviral agents

A highly sensitive and quantitative focal immunoassay has been developed for detecting the human immunodeficiency virus (HIV). The assay can be used to measure cell-free virus or the production of HIV by virus-infected cells. Both laboratory-adapted strains of HIV and patient isolates can be studied with this assay. In this communication, we demonstrate the utility of this assay for measuring the effects of anti-HIV agents on viral isolates. We show that the anti-viral effects of such diverse agents as azidothymidine, interferon-alpha, immunotoxins, soluble CD4 and antibody can be accurately quantified. This assay may be used in the discovery and evaluation of new anti-HIV therapies or may be adapted for use in testing the sensitivity of patient isolates to standard therapeutic agents.     

HIV protease inhibitors modulate apoptosis signaling <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>

HIV protease inhibitors are an integral part of effective anti-HIV therapy. The drugs block HIV protease, prevent proper packaging of HIV virions, and decrease the HIV viral burden in the peripheral blood of infected individuals. In addition to direct anti-viral effects, the HIV protease inhibitors also modulate apoptosis. A growing body of work demonstrates the anti-apoptotic effects of HIV protease inhibitors on CD4+ and CD8+ T cells during HIV infection. The mechanism of this apoptosis inhibition is supported by several proposed hypotheses for how they alter the fate of the cell, including preventing adenine nucleotide translocator pore function, which consequently prevents loss of mitochondrial transmembrane potential. More recently, the anti-apoptotic effects of the HIV protease inhibitors have been tested in non-HIV, non-immune cell, whereby protease inhibitors prevent apoptosis, and disease in animal models of sepsis, hepatitis, pancreatitis and stroke. Interestingly, when HIV protease inhibitors are used at supra-therapeutic concentrations, they exert pro-apoptotic effects. This has been demonstrated in a number of tumor models. Although it is unclear how HIV protease inhibitors can induce apoptosis at increased concentrations, future research will define the targets of the immunomodulation and reveal the full clinical potential of this intriguing class of drugs.     

The antiproliferative agents trans-bis(resorcylaldoximato)copper(II) and trans-bis(2,3,4-trihydroxybenzaldoximato)copper(II) and cytopathic effects of HIV

trans-Bis(resorcylaldoximato)copper(II) and trans-bis-(2,3,4-trihydroxybenzaldoximato)copper(II) (CuRES2 and CuTRI2, respectively) have been tested for antiviral properties against HIV, using an in vitro assay that measures the ability of the test compounds to prevent the killing of susceptible human cells by HIV. In the case of CuTRI2, T4 lymphocytes (CEM-V and CEM-Z cell lines) were exposed to HIV at a virus to cell ratio approx. 0.05 in microtiter plates. In the case of CuRES2, a human leukemia cell line (MT-2) was used instead. The tetrazolium salt XTT was added to all wells, and the cultures were incubated and analyzed spectrophotometrically to quantitate formazan production and viewed microscopically for detection of viable cells. In spite of their antiproliferative properties, neither agent had any detectable ability to prevent the cytopathic effects of HIV in cultures of the target cells used. Because the test system employed was constructed in such a way as to detect antiviral agents acting at any stage of the virus reproductive cycle, the results obtained strongly suggest that neither studied agent has any value as the direct prevention of the cell destruction caused by HIV is concerned.     

Identification of the benzodiazepines as a new class of antileishmanial agent

The continual increase in drug resistance; the lack of new chemotherapeutic agents; the toxicity of existing agents and the increasing morbidity with HIV co-infection mean the search for new antileishmanial agents has never been more urgent. We have identified the benzodiazepines as a structural class for antileishmanial hit optimisation, and demonstrated that their in vitro activity is comparable with the clinically used drug, sodium stibogluconate, and that the compounds are not toxic to macrophages.     

Analytic results for quantifying HIV infectivity

In a separate paper, we developed a mathematical model describing HIV infection and used it to suggest experiments for quantifying characteristic viral parameters. In this paper we generalize the model to any well-mixed assay system. We also present complete and rigorous derivations of fundamental results needed for the design and analysis of HIV infectivity assays. The model is applicable to infectious agents with multiple receptors for their target cell (e.g. HIV, Epstein-Barr virus and Plasmodium), and to blockers (both reversible and irreversible), as long as blocker and target cells are the same diffusion compartment.     

Correlation of in vitro chemopreventive efficacy data from the human epidermal cell assay with animal efficacy data and clinical trial plasma levels

The human epidermal cell (HEC) assay, which uses carcinogen exposed normal skin keratinocytes to screen for cancer prevention efficacy, was used to screen possible preventive agents. The endpoints measured were inhibition of carcinogen-induced growth and induction of involucrin, an early marker of differentiation. Sixteen of twenty agents (apigenin, apomine, budesonide, N-(2-carboxyphenyl)retinamide, ellagic acid, ibuprofen, indomethacin, melatonin, (-)-2-oxo-4-thiazolidine carboxylic acid, polyphenon E, resveratrol, beta-sitosterol, sulfasalazine, vitamin E acetate, and zileuton) were positive in at least one of the two assay endpoints. Four agents (4-methoxyphenol, naringenin, palmitoylcarnitine chloride, and silymarin) were negative in the assay. Nine of the sixteen agents were positive for both endpoints. Agents that showed the greatest response included: ellagic acid > budesonide, ibuprofen > apigenin, and quinicrine dihydrochloride. Fifty-eight of sixty-five agents that have been evaluated in the HEC assay have also been evaluated in one or more rodent bioassays for cancer prevention and several are in clinical trials for cancer prevention. The assay has an overall predictive accuracy of approximately 91.4% for efficacy in rodent cancer prevention irrespective of the species used, the tissue model, or the carcinogen used. Comparison of the efficacious concentrations in vitro to plasma levels in clinical trials show that concentrations that produced efficacy in the HEC assay were achieved in clinical studies for 31 of 33 agents for which plasma levels and/or C(max) levels were available. For two agents, 9-cis-retinoic acid (RA) and dehydroepiandrosterone (DHEA), the plasma levels greatly exceeded the highest concentration (HC) found to have efficacy in vitro. Thus, the HEC assay has an excellent predictive potential for animal efficacy and is responsive at clinically achievable concentrations.