Negative regulation of the NFAT1 factor by CD45: implication in HIV-1 long terminal repeat activation.

HIV-1 gene regulation is greatly dependent on the presence of the -104/-81 enhancer region which is regulated by both NF-kappaB and NFAT transcription factors. We have found that a greater induction in HIV-1 long terminal repeat-driven gene expression was observed upon PMA/ionomycin (Iono) stimulation of a CD45-deficient cell line (J45.01) in comparison to the parental Jurkat cells. Unlike NF-kappaB which was not affected by the absence of CD45, NFAT showed a much greater augmentation in nuclear translocation and transcriptional activity in J45.01 cells upon PMA/Iono stimulation. PMA/Iono-induced NFAT activation, NFAT translocation and calcium influx peaked at similar time points for both Jurkat and J45.01 cell lines. The NFAT-dependent promoters from the IL-2 and TNF-alpha genes were also more potently activated by PMA/Iono in J45.01 cells. Interestingly, higher levels of intracellular calcium were consistently demonstrated in PMA/Iono-induced CD45-deficient cell lines (J45.01 and HPB45.0). Furthermore, PMA/Iono induction of calcium mobilization in both Jurkat and J45.01 cell lines was observed to be EGTA-sensitive. Mechanistic studies revealed that CD3zeta and ZAP-70 were more heavily tyrosine phosphorylated in J45.01 cells than Jurkat cells. Analysis of the HIV-1 enhancer by EMSAs demonstrated that the bound NFAT complex was present at higher levels in J45.01 nuclear extracts and that the NFAT1 member was predominant. In conclusion, our results indicate that NFAT activation by stimuli acting in a more distal fashion from the TCR-mediated signaling pathway can be down-regulated by CD45 and that this CD45-dependent regulation in turn affects HIV-1 long terminal repeat activation.

Human herpesvirus 8 viral FLICE-inhibitory protein inhibits Fas-mediated apoptosis through binding and prevention of procaspase-8 maturation.

Viral FLICE-inhibitory proteins (v-FLIPs) encoded by several herpesviruses and poxviruses share the ability to inhibit apoptosis after engagement of death receptors. In the current article, we provide insights into the mechanisms by which the v-FLIP of human herpesvirus 8 (HHV-8) (also referred to as Kaposi's sarcoma-associated virus) protects cells from apoptosis after Fas-induced signaling. Using v-FLIP expression vectors, our results clearly show that HHV-8 v-FLIP reduces the cleavage of procaspase-8 into its active p18 and p10 protease subunits upon Fas-induced cell death. These results were confirmed by lower caspase-8 and caspase-3 protease activities in extracts of HeLa cells expressing HHV-8 v-FLIP. Coimmunoprecipitation studies further indicate that HHV-8 v-FLIP physically interacts with procaspase-8, but not with Fas-associated protein with death domain in the cellular cytoplasm. These results suggest that binding of HHV-8 v-FLIP to procaspase-8 affects the recruitment and the activation of the latter at the death-induced signaling complex, resulting in diminished apoptotic cascade initiation. Because cellular FLIP was recently reported to modulate promoter containing NF-kappaB motifs and that both HHV-8 and human immunodeficiency virus type 1 (HWV-1) can infect monocytes, we studied the effects of v-FLIP on HIV-1 gene expression. Cotransfection experiments indicated that v-FLIP expression is associated with activation of HIV long terminal repeats: events that were strictly dependent on the presence of NF-kappaB consensus elements. In conclusion, HHV-8 v-FLIP can possibly contribute to the pathogenesis of both HHV-8 and HIV-1 through impaired Fas-dependent killing of infected cells by cytotoxic T cells and through activation of HIV gene expression.

Sodium lauryl sulfate abrogates human immunodeficiency virus infectivity by affecting viral attachment.

The microbicidal activity of sodium lauryl sulfate (SLS) against human immunodeficiency virus type 1 (HIV-1) was studied in cultured cells. Pretreatment of HIV-1(NL4-3) with SLS decreased, in a concentration-dependent manner, its infectivity when using 1G5 as target cells. In the absence of a viral pretreatment period or when 1G5 cells were pretreated with SLS, the surfactant-induced inactivation of viral infectivity was less pronounced, especially at concentrations between 375 and 550 microM. SLS had no effect on HIV-1 when the virus was adsorbed to 1G5 cells by a 2-h incubation period. SLS almost completely inhibited the fusion process by decreasing the attachment of HIV-1 to target cells. SLS also inhibited the infectivity of HIV-1-based luciferase reporter viruses pseudotyped with the amphotropic murine leukemia virus envelope (which enters cells in a CD4-, CCR5-, and CXCR4-independent manner), indicating that SLS may inactivate other envelope viruses. In contrast, no effect was seen with vesicular stomatitis virus envelope glycoprotein G (which enters cells through receptor-mediated endocytosis) pretreated with up to 700 microM SLS. SLS also decreased, in a dose-dependent manner, the HIV-1-dependent syncytium formation between 1G5 and J1.1 cells after a 24-h incubation. The reduction of luciferase activity was more pronounced when J1.1 cells (which express HIV-1 proteins on their surface) were pretreated with SLS rather than 1G5 cells. Taken together, our results suggest that SLS could represent a candidate of choice for use in vaginal microbicides to prevent the sexual transmission of HIV and possibly other pathogens causing sexually transmitted diseases.

Thermoreversible gel as a candidate barrier to prevent the transmission of HIV-1 and herpes simplex virus type 2.

BACKGROUND: Sexually transmitted diseases (STDs) caused by HIV, herpes simplex virus (HSV), and other pathogens are spreading dramatically. The need to develop active products and vehicles to reduce this epidemic is urgent. GOAL: The efficacy of a thermoreversible gel formulation as a possible barrier to prevent the transmission of pathogens causing STDs was evaluated. STUDY DESIGN: This evaluation investigated the ability of the gel formulation to prevent infection of susceptible cells by HIV-1 and HSV-2 in vitro, the diffusion of radiolabeled herpes virus and micelles of polymer through an insertion membrane, and the electron microscopic appearance of herpes virus and gel alone or mixed together. RESULTS: The gel formulation prevents infection of susceptible cells by HIV-1 and HSV-2. It acts as an effective artificial physical barrier against the herpes virus within the first 4 hours of incubation. Herpes virus is coated by the gel or entrapped within micelles of the gel, which could hinder its attachment to target cells and inhibit its infectivity. CONCLUSION: This thermoreversible gel formulation represents an attractive matrix for the incorporation of microbicides to prevent the spread of STDs.

Neuraminidase from a bacterial source enhances both HIV-1-mediated syncytium formation and the virus binding/entry process.

Neuraminidases, also termed sialidases, which catalyze the removal of sialic acid residues from various glycoconjugates, have been previously reported to modulate HIV-1 replication. Given that some of the known opportunistic microbes found in patients infected with HIV-1 harbor neuraminidase (NA) activity, we speculated that pathogen-derived NA might be envisaged as an important factor in the pathogenesis of this retroviral infection. In the present study, we have monitored the putative modulation of HIV-1-mediated syncytium formation and virus replication by highly purified bacterial-derived NA from Arthrobacter ureafaciens. Taking advantage of a luciferase-based syncytium quantitative assay, we demonstrate here that the level of HIV-1-mediated syncytium formation is enhanced in the presence of NA and that it necessitates interaction between gp120 and CD4/chemokine coreceptor. By using pseudotyped recombinant luciferase-encoding HIV-1 particles, we found that NA treatment of human CD4-positive target cells (i.e., T lymphoid, monocytoid, and peripheral blood mononuclear cells) significantly augmented single-round infection by T- and macrophage-tropic isolates of HIV-1. The observed increase in HIV-1 infection was linked with an enhancement in the initial steps of the virus replicative cycle as monitored by viral binding and entry assays. Interestingly, NA treatment also enhances infectivity of HIV-1 pseudotypes with envelope glycoprotein from the amphotropic murine leukemia virus or the vesicular stomatitis virus. Taken together, our results provide useful information regarding the possible contribution of microbial agents carrying NA activity to HIV-1 pathogenesis.

Inhibition of HIV-1-mediated syncytium formation and virus replication by the lipophosphoglycan from Leishmania donovani is due to an effect on early events in the virus life cycle.

Previous findings have indicated that the major surface molecule of Leishmania, lipophosphoglycan (LPG), could abrogate HIV-1-induced syncytium formation and virus replication. In the present work, we were interested in characterizing this inhibitory process. Data from a new luciferase-based semiquantitative assay for syncytium formation, relying on the coincubation of a T-cell line containing an HIV-1 LTR-driven luciferase construct with a cell line chronically infected with HIV-1, confirmed that LPG was indeed a strong inhibitor of HIV-1-dependent syncytium formation and that this inhibition was dose-dependent. As determined by flow cytometric analyses, this inhibition was not apparently due to downregulation of CD4, CXCR4 or LFA-1, three distinct surface glycoproteins known to be important in HIV-1 mediated syncytium formation. Furthermore, LPG did not seem to affect signal transduction pathways in T cells as judged by measurement of HIV-1 LTR-driven reporter gene activity upon treatment with different stimuli. However, pretreatment of either of the cell lines used in the assay with LPG led to a significant decrease of virus-mediated syncytium formation, which was further accentuated when both cell lines were pretreated. LPG inhibition of HIV-1 replication was next assessed. When measuring either infection with luciferase-encoding recombinant HIV-1 particles or multinucleated giant cell formation following an acute virus infection, we again observed that LPG was efficient at blocking HIV-1 replication. Specific assays probing different steps of viral entry demonstrated that attachment was not hindered by LPG but that viral entry was modulated, suggesting that LPG targets a postbinding step. Hence, incorporation of LPG into a target cell membrane could influence its fluidity and diminish both the virus-cell and cell-to-cell fusion processes initiated by HIV-1.

Regulation of nuclear factor of activated T cells by phosphotyrosyl-specific phosphatase activity: a positive effect on HIV-1 long terminal repeat-driven transcription and a possible implication of SHP-1.

Although protein tyrosine phosphatase (PTP) inhibitors used in combination with other stimuli can induce interleukin 2 (IL-2) production in T cells, a direct implication of nuclear factor of activated T cells (NFAT) has not yet been demonstrated. This study reports that exposure of leukemic T cells and human peripheral blood mononuclear cells to bis-peroxovanadium (bpV) PTP inhibitors markedly induce activation and nuclear translocation of NFAT. NFAT activation by bpV was inhibited by the immunosuppressive drugs FK506 and cyclosporin A, as well as by a specific peptide inhibitor of NFAT activation. Mobility shift assays showed specific induction of the NFAT1 member by bpV molecules. The bpV-mediated NFAT activation was observed to be important for the up-regulation of the human immunodeficiency virus 1 (HIV-1) long terminal repeat (LTR) and the IL-2 promoter; NFAT1 was demonstrated to be particularly important in bpV-dependent positive action on HIV-1 LTR transcription. The active participation of p56(lck), ZAP-70, p21(ras), and calcium in the bpV-mediated signaling cascade leading to NFAT activation was confirmed, using deficient cell lines and dominant-negative mutants. Finally, overexpression of wild-type SHP-1 resulted in a greatly diminished activation of NFAT by bpV, suggesting an involvement of SHP-1 in the regulation of NFAT activation. These data were confirmed by constitutive NFAT translocation observed in Jurkat cells stably expressing a dominant-negative version of SHP-1. The study proposes that PTP activity attenuates constitutive kinase activities that otherwise would lead to constant NFAT activation and that this activation is participating in HIV-1 LTR stimulation by PTP inhibition.

Intercellular adhesion molecule-1 (ICAM-1) gene expression in human T cells is regulated by phosphotyrosyl phosphatase activity. Involvement of NF-kappaB, Ets, and palindromic interferon-gamma-responsive element-binding sites.

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in adhesion phenomena involved in the immune response. The strength of adhesion has been shown to be modulated by changes in ICAM-1 gene expression. In T cells, signaling pathways are intimately regulated by an equilibrium between protein-tyrosine kinases and protein tyrosine phosphatases (PTP). The use of bis-peroxovanadium (bpV) compounds, a class of potent PTP inhibitors, enabled us to investigate the involvement of phosphotyrosyl phosphatases in the regulation of ICAM-1 gene expression in human T cells. Here, we demonstrate for the first time that inhibition of PTP results in an increase of ICAM-1 surface expression on both human T lymphoid and primary mononuclear cells. The crucial role played by the NF-kappaB-, Ets-, and pIgammaRE-binding sites in bpV[pic]-mediated activation of ICAM-1 was demonstrated using various 5' deletion and site-specific mutants of the ICAM-1 gene promoter driving the luciferase reporter gene. Co-transfection experiments with trans-dominant mutants and electrophoretic mobility shift assays confirmed the importance of constitutive and inducible transcription factors that bind to specific responsive elements in bpV-dependent up-regulation of ICAM-1 surface expression. Altogether, these observations suggest that expression of ICAM-1 in human T cells is regulated by phosphotyrosyl phosphatase activity through NF-kappaB-, Ets-, and STAT-1-dependent signaling pathways.

Attachment of human immunodeficiency virus-1 (HIV-1) particles bearing host-encoded B7-2 proteins leads to nuclear factor-kappa B- and nuclear factor of activated T cells-dependent activation of HIV-1 long terminal repeat transcription.

Previous studies have shown that human immunodeficiency virus type-1 (HIV-1) can incorporate several surface proteins of host origin. Recent findings indicate that host-encoded cell surface constituents retain their functionality when found embedded into the viral envelope. The primary objective of the current study was to define whether interaction between some specific virion-bound host proteins with their natural cognate ligands present on target cells could mediate intracellular signaling cascade(s). For this purpose, we have generated a whole series of isogenic virus stocks (NL4-3 backbone) bearing or not bearing on their surface foreign CD28, CD54 (ICAM-1), CD80 (B7-1) or CD86 (B7-2) proteins. Our results indicate that incubation of human T lymphoid cells with virions bearing host-derived B7-2 proteins and anti-CD3 antibody can potently activate HIV-1 long terminal repeat-driven gene expression. This up-regulating effect necessitates the involvement of nuclear factor-kappa B (NF-kappa B) and nuclear factor of activated T cells (NFAT) as revealed by the use of vectors coding for dominant negative versions of both transcription factors (i.e. I kappa B alpha S32A/36A and dnNFAT) and band shift assays. The increase of NF-kappa B activity was abolished when infection with B7-2-bearing HIV-1 particles was performed in the presence of the fusion protein CTLA-4 Ig suggesting that the interaction between virally embedded B7-2 and CD28 on the target cell is responsible for the observed NF-kappa B induction. The findings presented here provide the first demonstration that host-encoded proteins acquired by HIV-1 can mediate signal transduction events.

Mono Mac 1: a new in vitro model system to study HIV-1 infection in human cells of the mononuclear phagocyte series.

Throughout the years, most researchers have used continuous cell lines as in vitro models to evaluate the immunopathogenesis of human immunodeficiency virus type-1 (HIV-1) infection. Unfortunately, the most commonly used monocytoid malignant cells have not been shown to adequately mimic primary human monocyte-derived macrophages, at least with respect to HIV-1 infection. The Mono Mac 1 cell line has been defined as a model system for studying biochemical, immunological, and genetic functions of human cells of the monocyte/macrophage lineage. In this study, we have investigated whether Mono Mac 1 represents an in vitro culture system for HIV-1 infection. Flow cytometric analyses revealed that Mono Mac 1 are positive for the HIV-1 primary receptor (CD4), as well as for the coreceptors (CXCR4, CCR5, and CCR3). Infectivity experiments conducted with recombinant luciferase-encoding and fully infectious viruses demonstrated that Mono Mac 1 can support a highly productive infection with both macrophage- and dual-tropic isolates of HIV-1. Furthermore, differentiation of such cells led to a marked increase in virus production. Data from semiquantitative polymerase chain reaction analysis and mobility shift assays indicated that enhanced virus production in differentiated Mono Mac 1 cells was most likely related to an increase in nuclear translocation of NF-kappaB. Mono Mac 1 can thus be considered as a human monocytoid cell line representing a proper in vitro system for studying the complex interactions between HIV-1 and cells of the monocyte/macrophage lineage.

Episomal and stable expression of the luciferase reporter gene for quantifying Leishmania spp. infections in macrophages and in animal models.

We have expressed the reporter firefly luciferase gene (LUC) in Leishmania donovani and Leishmania major either as part of episomal vectors or integrated into the parasite genome under the control of their respective ribosomal promoter regions. An excellent linear correlation between parasite number and luciferase activity was observed with all the transfectants. LUC-expressing recombinant parasites were useful to monitor Leishmania spp. infections in macrophages or in animal models. For prolonged growth in absence of drug selection, such as within animal models, quantitation of parasites is more reliable when the reporter gene LUC is stably integrated in the parasite genome. These recombinant strains should be useful tools to monitor Leishmania growth under a number of conditions.

Sterically stabilized liposomes bearing anti-HLA-DR antibodies for targeting the primary cellular reservoirs of HIV-1.

The ability of liposomes bearing anti-HLA-DR Fab' fragments at the end termini of polyethyleneglycol chains (sterically stabilized immunoliposomes) to target HLA-DR expressing cells and increase the accumulation of liposomes into lymphoid organs has been evaluated and compared to that of conventional liposomes, sterically stabilized liposomes and conventional immunoliposomes after a single subcutaneous injection to mice. The accumulation of sterically stabilized liposomes in lymph nodes was higher than that of conventional liposomes. Sterically stabilized immunoliposomes accumulated much better than conventional immunoliposomes in all tissues indicating that the presence of PEG has an important effect on the uptake of immunoliposomes by the lymphatic system. Fluorescence microscopy studies showed that sterically stabilized liposomes are mainly localized in macrophage-rich areas such as the subcapsular region of lymph nodes and in the red pulp and marginal zone of the spleen. In contrast, sterically stabilized immunoliposomes mostly accumulated in the cortex in which follicles are located and in the white pulp of the spleen. As the human HLA-DR determinant of the major histocompatibility complex class II is expressed on activated CD4+ T lymphocytes and antigen presenting cells such as monocyte/macrophages and dendritic cells, known as the cellular reservoirs of HIV-1, liposomes bearing anti-HLA-DR antibodies constitute an attractive approach to concentrate drugs in HIV-1 reservoirs and improve their therapeutic effect.

EBV suppresses prostaglandin E2 biosynthesis in human monocytes.

It is well known that EBV has developed strategies to evade immune surveillance. Previously, EBV was shown to bind specifically to monocytes and regulate expression of proinflammatory mediators such as IL-1, IL-6, TNF-alpha, and leukotrienes. EBV was also found to affect phagocytosis of monocytes. In this study, we show that in addition to these effects, EBV suppresses the biosynthesis of PGE2, a pleiotropic immunomodulatory molecule that is synthesized by the dioxygenation of arachidonic acid via the cyclooxygenase (COX) pathway. This down-regulation of PGE2 formation involved the inhibition of the inducible COX-2 isoform expression both at the transcriptional and translational levels, whereas expression of the constitutive COX-1 isoform was unaltered. Furthermore, exposure of monocytes to EBV was found to impact on the NF-kappaB activation pathway, which plays an essential role in the induction of COX-2 in monocytes. The inhibition of PGE2 biosynthesis was relieved when the experiments were conducted in presence of phosphonoacetic acid, an inhibitor of herpesviruses DNA polymerase, indicating that viral replication and/or neosynthesized viral proteins were involved in this process. Thus, inhibition of PGE2 biosynthesis in monocytes may represent an additional mechanism underlying EBV pathogenicity.

Interaction between virion-bound host intercellular adhesion molecule-1 and the high-affinity state of lymphocyte function-associated antigen-1 on target cells renders R5 and X4 isolates of human immunodeficiency virus type 1 more refractory to neutralization.

The oligomeric nature of the viral envelope proteins has been partly held responsible for the observed differences in neutralization sensitivity between primary and laboratory-adapted strains of human immunodeficiency virus type 1 (HIV-1). However, recent evidence suggests that host factors can also modify the sensitivity of HIV-1 particles to neutralization. Having previously demonstrated that the acquisition of host-encoded intercellular adhesion molecule (ICAM)-1 proteins by newly formed viruses has a functional significance for the life cycle of HIV-1, we investigated whether the acquisition of host-derived ICAM-1 by HIV-1 could affect the virus sensitivity to neutralization. In this study, we have first shown that the physical presence of host cell membrane ICAM-1 on HIV-1 was not modifying virus sensitivity to neutralization by either two different anti-gp120 monoclonal antibodies (0.5beta and 4.8D) or soluble CD4. However, the ability of the F105 anti-gp120 monoclonal antibody (specific for the CD4-binding site) to neutralize ICAM-1-bearing virions was diminished when target cells were pretreated with an lymphocyte function-associated antigen-1 (LFA-1)-activating antibody. Interestingly, ICAM-1/POS progeny viruses were found to be slightly more resistant to neutralization by individual human sera in target cells expressing a low-affinity form of LFA-1 than viruses devoid of host-encoded ICAM-1 proteins. This resistance was markedly enhanced when target cells expressed an activated LFA-1 form on their surface. These results suggest that the interaction between virally embedded host ICAM-1 and target cell surface LFA-1 should be considered a factor modulating neutralization sensitivity of HIV-1 by human sera from HIV-1-infected individuals.

Targeting cell-free HIV and virally-infected cells with anti-HLA-DR immunoliposomes containing amphotericin B.

OBJECTIVE: To evaluate the ability of liposomes bearing anti-HLA-DR Fab' fragments (immunoliposomes) and containing amphotericin B (AmB) to target and neutralize cell-free HIV-1 particles and virally-infected cells. METHODS: The effect of AmB on the attachment and fusion of HIV-1(NL4-3) to Jurkat E6.1 cells has been evaluated using a p24 enzymatic assay. The ability of AmB to inhibit HIV-1-based luciferase reporter viruses pseudotyped with HXB2, AML-V and VSV-G envelopes has been evaluated in Jurkat E6.1 cells. The efficacy of free and immunoliposomal AmB to inhibit cell-free HIV, that have incorporated or not HLA-DR molecules, has been evaluated in HLA-DR/negative (NEG) 1G5 T cells and HLA-DR/positive (POS) Mono Mac 1 cells. RESULTS: AmB inhibited HIV infectivity independently of the nature of viral envelope proteins. Pretreatment of HIV with AmB had no major effect on viral attachment and fusion process to Jurkat E6.1 cells. Immunoliposomal AmB (0.5 microg/ml) led to a 77% inhibition of replication of HLA-DR/POS HIV-1 with no cell toxicity, whereas free AmB had no significant antiviral activity at this concentration. A complete inhibition of viral replication was observed following incubation of viruses with immunoliposomal AmB (2.5 microg/ml). Anti-HLA-DR immunoliposomes containing AmB had no effect on the infectivity of HLA-DR/NEG HIV-1 particles in HLA-DR/NEG T lymphoid cells but completely inhibited replication of viruses in an HLA-DR/POS monocytic cell line. CONCLUSION: The incorporation of neutralizing agents in anti-HLA-DR immunoliposomes could represent a novel therapeutic strategy to specifically target cell-free HIV particles and virally-infected cells to treat HIV infection more efficiently.

p56(lck), ZAP-70, SLP-76, and calcium-regulated effectors are involved in NF-kappaB activation by bisperoxovanadium phosphotyrosyl phosphatase inhibitors in human T cells.

This study investigates the second messengers involved in NF-kappaB activation by the bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors. We first initiated a time course analysis of bpV-mediated activation of the human immunodeficiency virus type-1 long terminal repeat- and NF-kappaB-driven reporter gene. Our results showed a slower and more transient activation of both kappaB-regulated luciferase-encoding vectors by bpV compounds when compared with the action of tumor necrosis factor-alpha (TNF). Time course analyses of NF-kappaB translocation by shift assay experiments further confirmed these results, hence implying distinct pathways of NF-kappaB activation for bpV compounds and TNF. Attempts to characterize the bpV-dependent signaling cascade revealed that the src family protein tyrosine kinase p56(lck) was critical for NF-kappaB induction by bpV. Furthermore, p56(lck) interaction with the intracytoplasmic tail of CD4 markedly enhanced such induction. Optimal activation of NF-kappaB following bpV treatment necessitated downstream effectors of p56(lck) such as the syk family protein tyrosine kinase ZAP-70 and the molecular adaptor SLP-76. Importantly, reduced NF-kappaB activation was observed when capacitative calcium entry was deficient but also upon pharmacological inhibition of calmodulin and calcineurin. Altogether, these results suggest that induction of NF-kappaB by phosphotyrosyl phosphatase bpV inhibitors necessitates both proximal and distal effectors of T cell activation.

Targeting lymph nodes with liposomes bearing anti-HLA-DR Fab' fragments.

The ability of liposomes bearing anti-HLA-DR Fab' fragments to target cells expressing the human HLA-DR determinant of the major histocompatibility complex class II (MHC-II) has been evaluated and compared to that of conventional liposomes. Anti-HLA-DR immunoliposomes did not bind to HLA-DR-negative cells. In contrast, a high level of binding was observed following incubation of immunoliposomes with cells bearing important levels of human HLA-DR. The accumulation of conventional and murine anti-HLA-DR immunoliposomes in different tissues has been investigated following a single subcutaneous injection given in the upper back of C3H mice. Anti-HLA-DR immunoliposomes resulted in a much better accumulation in the cervical and brachial lymph nodes when compared to conventional liposomes. The accumulation in the liver was similar for both liposomal preparations, whereas an approximately twofold decrease in accumulation was observed for immunoliposomes in the spleen. Given that HLA-DR surface marker is expressed on monocyte/macrophages and activated CD4+ T lymphocytes, the primary cellular reservoirs of the human immunodeficiency virus (HIV), the use of liposomes bearing surface-attached anti-HLA-DR could constitute a convenient strategy to more efficiently treat this debilitating retroviral disease. Moreover, the reported incorporation of high amounts of host-encoded HLA-DR proteins by HIV particles renders the use of liposomes bearing anti-HLA-DR antibodies even more attractive.

Reducing chorioretinal viral counts with intravitreal foscarnet injections in a rabbit model of Herpes simplex virus type-1 retinitis.

The efficacy of intravitreal foscarnet injections was evaluated in a rabbit model of Herpes simplex virus type-1 (HSV-1) retinitis. In untreated infected animals, viral titration revealed that the optic chiasm, vitreous and chorioretina were positive for HSV-1. On the other hand, foscarnet treatment significantly decreased the viral count in the chorioretina when compared to the untreated group. Immunolocalization of HSV in untreated infected animals clearly showed infected cells in the outer and inner layers of the retina and also in the ciliary body of the eye. Clinical examination by indirect ophthalmoscopy indicated an absence of optic nerve congestion and a lower level of vitritis in foscarnet treated animals compared to the untreated group. It is concluded that intravitreal injections of foscarnet reduced the viral titer in the chorioretina in a rabbit model of HSV-1 retinitis. This route of administration might be valuable for the treatment of CMV retinitis in AIDS patients with sight threatening lesions or intolerance to intravenous anti-CMV drugs.

Role of the leukocyte function antigen-1 conformational state in the process of human immunodeficiency virus type 1-mediated syncytium formation and virus infection.

Human immunodeficiency virus type 1 (HIV-1)-mediated syncytium formation is recognized as being highly dependent on intercellular adhesion molecule (ICAM)-1-leukocyte function-associated molecule 1 (LFA)-1 interaction, whereas the process of infection with cell-free virions is independent of such complementary interaction. Our group has recently demonstrated that an antibody-mediated induction of the high affinity state of LFA-1 for ICAM-1 renders target T cells more prone to HIV-1-dependent syncytium formation and infection by ICAM-1-bearing virions. To further substantiate these results, we made use of mutant cell lines expressing LFA-1 in either a low (parental HPB-ALL and HAmut) or a high affinity state for ICAM-1 (HAP4) and compared syncytium formation and virus infection. Cells expressing the activated form of LFA-1 were found to be more susceptible to HIV-1-induced syncytium formation and to infection by ICAM-1-bearing HIV-1 particles. The observed increase was solely due to the LFA-1 activation state because it was abrogated by anti-LFA-1 or anti-ICAM-1 antibodies and not due to variations in surface expression of LFA-1, CD4, or the chemokine coreceptor CXCR4. However, a linear relation was seen between the level of CXCR4 surface expression and susceptibility to syncytium formation/virus infection when ICAM-1-LFA-1 interaction was either absent (i.e., infection with ICAM-1-negative virions) or abrogated (treatment with anti-LFA-1 or anti-ICAM-1 antibodies). These results emphasize the important role of the LFA-1 activation state with respect to virus-induced syncytium formation and HIV-1 infection.

Protective effect of a thermoreversible gel against the toxicity of nonoxynol-9.

BACKGROUND AND OBJECTIVES: One major problem associated with the use of nonoxynol-9 is that it can induce local inflammation and ulceration of the vaginal and cervical mucosa that might favor the entry of pathogens. With the aim of developing a gel formulation that could be effective in preventing sexually transmitted infections, the authors have evaluated the capacity of a polyoxypropylene/polyoxyethylene polymer to reduce or eliminate the toxicity of nonoxynol-9. STUDY DESIGN: The cytotoxicity of nonoxynol-9 alone or incorporated into the gel was investigated in human cervical and colon epithelial cells and after daily intravaginal application for 2 weeks in rabbits. RESULTS: In vitro experiments showed that nonoxynol-9 was highly toxic to human cervical and colon epithelial cells in a dose-dependent manner. However, the incorporation of the spermicide into the gel markedly reduced its toxicity under the same experimental conditions. In vivo studies showed that in animals treated with nonoxynol-9, the spermicide was very toxic to the vaginal and cervical mucosa as evidenced by the presence of bleeding, irritation, epithelial disruption, necrosis, the accumulation of leukocytes in the submucosa, and the loss of integrity of the epithelial cells. Of prime importance, the incorporation of nonoxynol-9 into the gel markedly reduced the toxicity of this potent spermicide/microbicide. CONCLUSION: The gel formulation could be used as an interesting approach to eliminate the toxicity of potent spermicides/microbicides such as nonoxynol-9.