Quinolinic acid/tryptophan ratios predict neurological disease in SIV-infected macaques and remain elevated in the brain under cART.

Activation of the kynurenine pathway (KP) of tryptophan catabolism likely contributes to HIV-associated neurological disorders. However, KP activation in brain tissue during HIV infection has been understudied, and the effect of combination antiretroviral therapy (cART) on KP induction in the brain is unknown. To examine these questions, tryptophan, kynurenine, 3-hydroxykynurenine, quinolinic acid, and serotonin levels were measured longitudinally during SIV infection in the striatum and CSF from untreated and cART-treated pigtailed macaques. Messenger RNA (mRNA) levels of KP enzymes also were measured in the striatum. In untreated macaques, elevations in KP metabolites coincided with transcriptional induction of upstream enzymes in the KP. Striatal KP induction was also temporally associated-but did not directly correlate-with serotonin losses in the brain. CSF quinolinic acid/tryptophan ratios were found to be the earliest predictor of neurological disease in untreated SIV-infected macaques, outperforming other KP metabolites as well as the putative biomarkers interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). Finally, cART did not restore KP metabolites to control levels in the striatum despite the control of the virus, though CSF metabolite levels were normalized in most animals. Overall, these results demonstrate that cerebral KP activation is only partially resolved with cART and that CSF QUIN/TRP ratios are an early, predictive biomarker of CNS disease.

Elevated brain monoamine oxidase activity in SIV- and HIV-associated neurological disease.

We recently demonstrated direct evidence of increased monoamine oxidase (MAO) activity in the brain of a simian immunodeficiency virus (SIV) model of human immunodeficiency virus (HIV)-associated central nervous system (CNS) disease, consistent with previously reported dopamine deficits in both SIV and HIV infection. In this study, we explored potential mechanisms behind this elevated activity. MAO B messenger RNA was highest in macaques with the most severe SIV-associated CNS lesions and was positively correlated with levels of CD68 and GFAP transcripts in the striatum. MAO B messenger RNA also correlated with viral loads in the CNS of SIV-infected macaques and with oxidative stress. Furthermore, in humans, striatal MAO activity was elevated in individuals with HIV encephalitis, compared with activity in HIV-seronegative controls. These data suggest that the neuroinflammation and oxidative stress caused by SIV infection in the CNS may provide the impetus for increased transcription of MAO B and that MAO, and more broadly, oxidative stress, have significant potential as therapeutic targets in CNS disease due to HIV.

ELISA-based detection of immunoglobulins against extracellular vesicles in blood plasma.

Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable safety profiles and low immunogenicity make EVs an attractive alternative to synthetic nanoparticles. We recently showed that repeated intravenous administration of human cell-derived EVs into pig-tailed macaques unexpectedly elicited antibody responses after three or more injections. This coincided with decreasing EV circulation time, and may thus hamper successful EV-mediated cargo delivery into tissues. Here, we share the custom ELISA protocol that we used to measure such antibody responses. This protocol may help other researchers evaluate immune responses to EV-based therapies in preclinical studies.

Simultaneous Protein and RNA Analysis in Single Extracellular Vesicles, Including Viruses.

The individual detection of human immunodeficiency virus (HIV) virions and resolution from extracellular vesicles (EVs) during analysis is a difficult challenge. Infectious enveloped virions and nonviral EVs are released simultaneously by HIV-infected host cells, in addition to hybrid viral EVs containing combinations of HIV and host components but lacking replicative ability. Complicating the issue, EVs and enveloped virions are both delimited by a lipid bilayer and share similar size and density. The feature that distinguishes infectious virions from host and hybrid EVs is the HIV genomic RNA (gRNA), which allows the virus to replicate. Single-particle analysis techniques, which provide snapshots of single biological nanoparticles, could resolve infectious virions from EVs. However, current single-particle analysis techniques focus mainly on protein detection, which fail to resolve hybrid EVs from infectious virions. A method to simultaneously detect viral protein and internal gRNA in the same particle would allow resolution of infectious HIV from EVs and noninfectious virions. Here, we introduce SPIRFISH, a high-throughput method for single-particle protein and RNA analysis, combining single particle interferometric reflectance imaging sensor with single-molecule fluorescence in situ hybridization. Using SPIRFISH, we detect HIV-1 envelope protein gp120 and genomic RNA within single infectious virions, allowing resolution against EV background and noninfectious virions. We further show that SPIRFISH can be used to detect specific RNAs within EVs. This may have major utility for EV therapeutics, which are increasingly focused on EV-mediated RNA delivery. SPIRFISH should enable single particle analysis of a broad class of RNA-containing nanoparticles.

Longitudinal characterization of circulating extracellular vesicles and small RNA during simian immunodeficiency virus infection and antiretroviral therapy.

OBJECTIVES: Latent infection by HIV hinders viral eradication despite effective antiretroviral treatment (ART). Among proposed contributors to viral latency are cellular small RNAs that have also been proposed to shuttle between cells in extracellular vesicles. Thus, we profiled extracellular vesicle small RNAs during different infection phases to understand the potential relationship between these extracellular vesicle associated small RNAs and viral infection. DESIGN: A well characterized simian immunodeficiency virus (SIV)/macaque model of HIV was used to profile extracellular vesicle enriched blood plasma fractions harvested during preinfection, acute infection, latent infection/ART treatment, and rebound after ART interruption. METHODS: Measurement of extracellular vesicle concentration, size distribution, and morphology was complemented with qPCR array for small RNA expression, followed by individual qPCR validations. Iodixanol density gradients were used to separate extracellular vesicle subtypes and virions. RESULTS: Plasma extracellular vesicle particle counts correlated with viral load and peaked during acute infection. However, SIV gag RNA detection showed that virions did not fully explain this peak. Extracellular vesicle microRNAs miR-181a, miR-342-3p, and miR-29a decreased with SIV infection and remained downregulated in latency. Interestingly, small nuclear RNA U6 had a tight association with viral load peak. CONCLUSION: This study is the first to monitor how extracellular vesicle concentration and extracellular vesicle small RNA expression change dynamically in acute viral infection, latency, and rebound in a carefully controlled animal model. These changes may also reveal regulatory roles in retroviral infection and latency.

Replication-competent simian immunodeficiency virus (SIV) Gag escape mutations archived in latent reservoirs during antiretroviral treatment of SIV-infected macaques.

In response to pressure exerted by major histocompatibility complex (MHC) class I-mediated CD8(+) T cell control, human immunodeficiency virus (HIV) escape mutations often arise in immunodominant epitopes recognized by MHC class I alleles. While the current standard of care for HIV-infected patients is treatment with highly active antiretroviral therapy (HAART), suppression of viral replication in these patients is not absolute and latently infected cells persist as lifelong reservoirs. To determine whether HIV escape from MHC class I-restricted CD8(+) T cell control develops during HAART treatment and then enters latent reservoirs in the periphery and central nervous system (CNS), with the potential to emerge as replication-competent virus, we tracked the longitudinal development of the simian immunodeficiency virus (SIV) Gag escape mutation K165R in HAART-treated SIV-infected pigtailed macaques. Key findings of these studies included: (i) SIV Gag K165R escape mutations emerged in both plasma and cerebrospinal fluid (CSF) during the decaying phase of viremia after HAART initiation before suppression of viral replication, (ii) SIV K165R Gag escape mutations were archived in latent proviral DNA reservoirs, including the brain in animals receiving HAART that suppressed viral replication, and (iii) replication-competent SIV Gag K165R escape mutations were present in the resting CD4(+) T cell reservoir in HAART-treated SIV-infected macaques. Despite early administration of aggressive antiretroviral treatment, HIV immune escape from CD8(+) T cell control can still develop during the decaying phases of viremia and then persist in latent reservoirs, including the brain, with the potential to emerge if HAART therapy is interrupted.

Cigarette smoke-induced extracellular vesicles from dendritic cells alter T-cell activation and HIV replication.

Despite decreased rates of tobacco smoking in many areas, cigarette smoking remains a major contributor to many health problems. Cigarette smoking can reduce immune system functioning while concurrently increasing inflammation. Dendritic cells in the lung exposed to cigarette smoke become stimulated and go on to activate T-cells. Extracellular vesicles (EVs) are nano-sized particles released by cells. They participate in intercellular communication by transferring functional proteins and nucleic acids to recipient cells and have been implicated in immune responses. For example, they can display MHC-peptide complexes to activate T-cells. In the current study, we sought to understand the role of cigarette smoke extract (CSE) on dendritic cell-derived EVs and their capacity to activate and differentiate T-cells. Primary human dendritic cells (iDCs) were exposed to CSE and EVs were separated and characterized. We exposed autologous primary CD4 + T-cells to iDC-EVs and observed T helper cell populations skewing towards Th1 and Th17 phenotypes. As HIV + individuals are disproportionately likely to be current smokers, we also examined the effects of iDC-EVs on acutely infected T-cells as well as on a cell model of HIV latency (ACH-2). We found that in most cases, iDC-CSE EVs tended to reduce p24 release from the acutely infected primary T-cells, albeit with great variability. We did not observe large effects of iDC-EVs or direct CSE exposure on p24 release from the ACH-2 cell line. Together, these data suggest that iDC-CSE EVs have the capacity to modulate the immune responses, in part by pushing T-cells towards Th1 and Th17 phenotypes.

Pharmacokinetics and biodistribution of extracellular vesicles administered intravenously and intranasally to Macaca nemestrina.

Extracellular vesicles (EVs) have potential in disease treatment since they can be loaded with therapeutic molecules and engineered for retention by specific tissues. However, questions remain on optimal dosing, administration, and pharmacokinetics. Previous studies have addressed biodistribution and pharmacokinetics in rodents, but little evidence is available for larger animals. Here, we investigated the pharmacokinetics and biodistribution of Expi293F-derived EVs labelled with a highly sensitive nanoluciferase reporter (palmGRET) in a non-human primate model (Macaca nemestrina), comparing intravenous (IV) and intranasal (IN) administration over a 125-fold dose range. We report that EVs administered IV had longer circulation times in plasma than previously reported in mice and were detectable in cerebrospinal fluid (CSF) after 30-60 minutes. EV association with PBMCs, especially B-cells, was observed as early as one minute post-administration. EVs were detected in liver and spleen within one hour of IV administration. However, IN delivery was minimal, suggesting that pretreatment approaches may be needed in large animals. Furthermore, EV circulation times strongly decreased after repeated IV administration, possibly due to immune responses and with clear implications for xenogeneic EV-based therapeutics. We hope that our findings from this baseline study in macaques will help to inform future research and therapeutic development of EVs.

Characterization of extracellular vesicles and synthetic nanoparticles with four orthogonal single-particle analysis platforms.

We compared four orthogonal technologies for sizing, counting, and phenotyping of extracellular vesicles (EVs) and synthetic particles. The platforms were: single-particle interferometric reflectance imaging sensing (SP-IRIS) with fluorescence, nanoparticle tracking analysis (NTA) with fluorescence, microfluidic resistive pulse sensing (MRPS), and nanoflow cytometry measurement (NFCM). EVs from the human T lymphocyte line H9 (high CD81, low CD63) and the promonocytic line U937 (low CD81, high CD63) were separated from culture conditioned medium (CCM) by differential ultracentrifugation (dUC) or a combination of ultrafiltration (UF) and size exclusion chromatography (SEC) and characterized by transmission electron microscopy (TEM) and Western blot (WB). Mixtures of synthetic particles (silica and polystyrene spheres) with known sizes and/or concentrations were also tested. MRPS and NFCM returned similar particle counts, while NTA detected counts approximately one order of magnitude lower for EVs, but not for synthetic particles. SP-IRIS events could not be used to estimate particle concentrations. For sizing, SP-IRIS, MRPS, and NFCM returned similar size profiles, with smaller sizes predominating (per power law distribution), but with sensitivity typically dropping off below diameters of 60 nm. NTA detected a population of particles with a mode diameter greater than 100 nm. Additionally, SP-IRIS, MRPS, and NFCM were able to identify at least three of four distinct size populations in a mixture of silica or polystyrene nanoparticles. Finally, for tetraspanin phenotyping, the SP-IRIS platform in fluorescence mode was able to detect at least two markers on the same particle, while NFCM detected either CD81 or CD63. Based on the results of this study, we can draw conclusions about existing single-particle analysis capabilities that may be useful for EV biomarker development and mechanistic studies.

miRNA profiling of primate cervicovaginal lavage and extracellular vesicles reveals miR-186-5p as a potential antiretroviral factor in macrophages.

Cervicovaginal secretions, or their components collected, are referred to as cervicovaginal lavage (CVL). CVL constituents have utility as biomarkers and play protective roles in wound healing and against HIV-1 infection. However, several components of cervicovaginal fluids are less well understood, such as extracellular RNAs and their carriers, for example, extracellular vesicles (EVs). EVs comprise a wide array of double-leaflet membrane extracellular particles and range in diameter from 30 nm to over one micron. The aim of this study was to determine whether differentially regulated CVL microRNAs (miRNAs) might influence retrovirus replication. To this end, we characterized EVs and miRNAs of primate CVL during the menstrual cycle and simian immunodeficiency virus (SIV) infection of macaques. EVs were enriched by stepped ultracentrifugation, and miRNA profiles were assessed with a medium-throughput stem-loop/hydrolysis probe qPCR platform. Whereas hormone cycling was abnormal in infected subjects, EV concentration correlated with progesterone concentration in uninfected subjects. miRNAs were present predominantly in the EV-depleted CVL supernatant. Only a small number of CVL miRNAs changed during the menstrual cycle or SIV infection, for example, miR-186-5p, which was depleted in retroviral infection. This miRNA inhibited HIV replication in infected macrophages in vitro. In silico target prediction and pathway enrichment analyses shed light on the probable functions of miR-186-5p in hindering HIV infections via immunoregulation, T-cell regulation, disruption of viral pathways, etc. These results provide further evidence for the potential of EVs and small RNAs as biomarkers or effectors of disease processes in the reproductive tract.

Cytotoxicity of aqueous cigarette smoke extract is affected by properties of pipettes used to prepare the extract.

Cigarette smoke contains a host of molecules including toxins and carcinogens, most of which have not been well studied. Aqueous cigarette smoke extract (CSE) is one of various cigarette smoke derivatives that can be used for in vitro studies, and the influence of different method parameters on CSE composition and toxicity remains incompletely understood. Herein, we prepared CSE by bubbling cigarette smoke through mammalian cell culture medium, varying the type of pipette inserted into the recipient medium. Changing this one component of the preparation apparatus had a marked effect on the toxicity of the resulting CSE. Since many other parameters can also be varied in CSE preparation, these results stress the importance of standardization within and between studies.

Acetylcholinesterase is not a generic marker of extracellular vesicles.

Acetylcholinesterase (AChE) activity is found in abundance in reticulocytes and neurons and was developed as a marker of reticulocyte EVs in the 1970s. Easily, quickly, and cheaply assayed, AChE activity has more recently been proposed as a generic marker for small extracellular vesicles (sEV) or exosomes, and as a negative marker of HIV-1 virions. To evaluate these proposed uses of AChE activity, we examined data from different EV and virus isolation methods using T-lymphocytic (H9, PM1 and Jurkat) and promonocytic (U937) cell lines grown in culture conditions that differed by serum content. When EVs were isolated by differential ultracentrifugation, no correlation between AChE activity and particle count was observed. AChE activity was detected in non-conditioned medium when serum was added, and most of this activity resided in soluble fractions and could not be pelleted by centrifugation. The serum-derived pelletable AChE protein was not completely eliminated from culture medium by overnight ultracentrifugation; however, a serum "extra-depletion" protocol, in which a portion of the supernatant was left undisturbed during harvesting, achieved near-complete depletion. In conditioned medium also, only small percentages of AChE activity could be pelleted together with particles. Furthermore, no consistent enrichment of AChE activity in sEV fractions was observed. Little if any AChE activity is produced by the cells we examined, and this activity was mainly present in non-vesicular structures, as shown by electron microscopy. Size-exclusion chromatography and iodixanol gradient separation showed that AChE activity overlaps only minimally with EV-enriched fractions. AChE activity likely betrays exposure to blood products and not EV abundance, echoing the MISEV 2014 and 2018 guidelines and other publications. Additional experiments may be merited to validate these results for other cell types and biological fluids other than blood.

Induction of HIF-1α by HIV-1 Infection in CD4+ T Cells Promotes Viral Replication and Drives Extracellular Vesicle-Mediated Inflammation.

Chronic immune activation and inflammation are hallmarks of HIV-1 infection and a major cause of serious non-AIDS events in HIV-1-infected individuals on antiretroviral treatment (ART). Herein, we show that cytosolic double-stranded DNA (dsDNA) generated in infected CD4+ T cells during the HIV-1 replication cycle promotes the mitochondrial reactive oxygen species (ROS)-dependent stabilization of the transcription factor hypoxia-inducible factor 1α (HIF-1α), which in turn, enhances viral replication. Furthermore, we show that induction of HIF-1α promotes the release of extracellular vesicles (EVs). These EVs foster inflammation by inducing the secretion of gamma interferon by bystander CD4+ T cells and secretion of interleukin 6 (IL-6) and IL-1ß by bystander macrophages through an HIF-1α-dependent pathway. Remarkably, EVs obtained from plasma samples from HIV-1-infected individuals also induced HIF-1α activity and inflammation. Overall, this study demonstrates that HIF-1α plays a crucial role in HIV-1 pathogenesis by promoting viral replication and the release of EVs that orchestrate lymphocyte- and macrophage-mediated inflammatory responses.IMPORTANCE Human immunodeficiency virus type 1 (HIV-1) is a very important global pathogen that preferentially targets CD4+ T cells and causes acquired immunodeficiency syndrome (AIDS) if left untreated. Although antiretroviral treatment efficiently suppresses viremia, markers of immune activation and inflammation remain higher in HIV-1-infected patients than in uninfected individuals. The hypoxia-inducible factor 1α (HIF-1α) is a transcription factor that plays a fundamental role in coordinating cellular metabolism and function. Here we show that HIV-1 infection induces HIF-1α activity and that this transcription factor upholds HIV-1 replication. Moreover, we demonstrate that HIF-1α plays a key role in HIV-1-associated inflammation by promoting the release of extracellular vesicles which, in turn, trigger the secretion of inflammatory mediators by noninfected bystander lymphocytes and macrophages. In summary, we identify that the coordinated actions of HIF-1α and extracellular vesicles promote viral replication and inflammation, thus contributing to HIV-1 pathogenesis.

Vaginal transmission of cell-associated HIV-1 in the mouse is blocked by a topical, membrane-modifying agent.

Because both HIV-1 virions and HIV-infected cells are present in the semen and cervical mucus of infected individuals, HIV-1 prevention strategies must consider both cell-free and cell-associated virus. Antibodies that target HIV-1 virions have been shown to prevent vaginal transmission of cell-free virus in macaques, but since cell-associated transmission has not been reliably demonstrated in this model system, no strategies to prevent such transmission have been tested. We have employed a mouse model in which SCID mice carry human peripheral blood leukocytes (HuPBLs). In these mice, vaginal transmission of cell-associated, but not cell-free, HIV-1 transmission occurs, mediated by transepithelial migration of HIV-infected cells. Topical application of beta-cyclodextrin (beta-CD), a cholesterol-sequestering agent that interferes with cell migration and budding of virus from lipid rafts, blocks transmission of cell-associated HIV-1. The HuPBL-SCID model of vaginal HIV-1 transmission should prove useful for investigating cell-associated HIV-1 transmucosal HIV-1 transmission, as well as for screening reagents for their potential efficacy in preventing sexual HIV-1 transmission.

Serum extracellular vesicle depletion processes affect release and infectivity of HIV-1 in culture.

Extracellular vesicles (EVs) are involved in intercellular communication and affect processes including immune and antiviral responses. Blood serum, a common cell culture medium component, is replete with EVs and must be depleted prior to EV-related experiments. The extent to which depletion processes deplete non-EV particles is incompletely understood, but depleted serum is associated with reduced viability and growth in cell culture. Here, we examined whether serum depleted by two methods affected HIV-1 replication. In cell lines, including HIV-1 latency models, increased HIV-1 production was observed, along with changes in cell behavior and viability. Add-back of ultracentrifuge pellets (enriched in EVs but possibly other particles) rescued baseline HIV-1 production. Primary cells were less sensitive to serum depletion processes. Virus produced under processed serum conditions was more infectious. Finally, changes in cellular metabolism, surface markers, and gene expression, but not miRNA profiles, were associated with depleted serum culture. In conclusion, depleted serum conditions have a substantial effect on HIV-1 production and infectivity. Dependence of cell cultures on "whole serum" must be examined carefully along with other experimental variables, keeping in mind that the effects of EVs may be accompanied by or confused with those of closely associated or physically similar particles.

Characterization of an anti-lymphocyte function-associated antigen-1 antibody in a simian immunodeficiency virus-pig-tailed macaque (Macaca nemestrina) model.

The simian immunodeficiency virus (SIV)/pig-tailed macaque (Macaca nemestrina) model of acquired immune deficiency syndrome (AIDS) is a powerful system in which to study cell adhesion molecules and retroviral pathogenesis in vivo. Preliminary experiments were conducted to examine the role of lymphocyte function-associated antigen 1 (LFA-1) in early SIV infection in vivo by using an LFA-1 monoclonal antibody (MHM.23) specific to human LFA-1. In vitro studies revealed that at concentrations of > or = 20 microg/ml, MHM.23 blocked LFA-1-mediated adhesion and T-cell activation (>90%) of pig-tailed macaque peripheral blood mononuclear cells (PBMCs). In addition, SIVmac239 infection of macaque cells was inhibited in a dose-dependant manner by MHM.23. Administration of MHM.23 to pig-tailed macaques inhibited LFA-1-ICAM-1-mediated activity in vivo and maintained binding on macaque cells for < or = 4 d. Our in vitro studies indicated that at an MHM.23 concentration of 20 microg/ml, macaque PBMCs were completely saturated. Our in vivo studies determined that 5 mg/kg MHM.23 intravenously every 24 h was required to maintain saturating levels and inhibit LFA-1-ICAM-1 function in pig-tailed macaques.

Lipid rafts and HIV pathogenesis: virion-associated cholesterol is required for fusion and infection of susceptible cells.

We have shown that HIV budding occurs at cholesterol-rich membrane microdomains called lipid rafts (Nguyen and Hildreth, J Virol 2000;74:3264-3272). This observation prompted us to examine the role in HIV entry of cholesterol in the membrane of cells. We recently reported that host cell cholesterol is required for HIV infection (Liao et al., AIDS Res Hum Retroviruses 2001;17:1009-1019). In the present study we examined the role of virion-associated cholesterol in HIV infection by modulating the cholesterol content of virions and infected cells with 2-hydoxypropyl-beta-cyclodextrin (beta-cyclodextrin). Our results show that removal of cholesterol from the membrane of HIV-infected cells dramatically lowered virus release and that virions released from cholesterol-depleted cells are minimally infectious. Exposure of infectious HIV particles to beta-cyclodextrin resulted in a dose-dependent inactivation of the virus. In both cases, the effect was attributable to loss of cholesterol and could be reversed by replenishing cholesterol. beta-Cyclodextrin-treated, noninfectious HIV retained its ability to bind cells. Western blot, p24 core ELISA, and reverse transcription assays indicated that virions remained intact after treatment with beta-cyclodextrin at concentrations that abolished infectivity. Electron microscopy revealed that beta-cyclodextrin-treated HIV had a morphology very similar to that of untreated virus. R18 fluorescence dequenching studies showed that beta-cyclodextrin-treated HIV did not fuse to the membrane of susceptible cells. Dequenching was restored by replenishing virion-associated cholesterol. The results indicate that cholesterol in HIV particles is strictly required for fusion and infectivity. These observations in combination with those of past studies indicate beta-cyclodextrin to be an excellent candidate for use as a chemical barrier for AIDS prophylaxis.

Attenuation of pathogenic immune responses during infection with human and simian immunodeficiency virus (HIV/SIV) by the tetracycline derivative minocycline.

HIV immune pathogenesis is postulated to involve two major mechanisms: 1) chronic innate immune responses that drive T cell activation and apoptosis and 2) induction of immune regulators that suppress T cell function and proliferation. Both arms are elevated chronically in lymphoid tissues of non-natural hosts, which ultimately develop AIDS. However, these mechanisms are not elevated chronically in natural hosts of SIV infection that avert immune pathogenesis despite similarly high viral loads. In this study we investigated whether minocycline could modulate these pathogenic antiviral responses in non-natural hosts of HIV and SIV. We found that minocycline attenuated in vitro induction of type I interferon (IFN) and the IFN-stimulated genes indoleamine 2,3-dioxygenase (IDO1) and TNF-related apoptosis inducing ligand (TRAIL) in human plasmacytoid dendritic cells and PBMCs exposed to aldrithiol-2 inactivated HIV or infectious influenza virus. Activation-induced TRAIL and expression of cytotoxic T-lymphocyte antigen 4 (CTLA-4) in isolated CD4+ T cells were also reduced by minocycline. Translation of these in vitro findings to in vivo effects, however, were mixed as minocycline significantly reduced markers of activation and activation-induced cell death (CD25, Fas, caspase-3) but did not affect expression of IFNß or the IFN-stimulated genes IDO1, FasL, or Mx in the spleens of chronically SIV-infected pigtailed macaques. TRAIL expression, reflecting the mixed effects of minocycline on activation and type I IFN stimuli, was reduced by half, but this change was not significant. These results show that minocycline administered after infection may protect against aspects of activation-induced cell death during HIV/SIV immune disease, but that in vitro effects of minocycline on type I IFN responses are not recapitulated in a rapid progressor model in vivo.

Cervicovaginal evaluation in macaques used as a model for topical microbicide safety studies.

INTRODUCTION: Macaques are a commonly used non-human primate (NHP) model to evaluate safety and efficacy of topically applied vaginal microbicides. Cervicovaginal evaluation for topical microbicide safety studies requires proper technique, equipment, supplies, and sequence of sample collection. MATERIALS AND METHODS: Eleven rhesus macaques received a comprehensive sequential cervicovaginal examination under sedation before treatment and 24 hours post-instillation of test material. Examination was initiated with colposcopy, followed by diagnostics including vaginal culture, pH determination, cervicovaginal lavage, and cervicovaginal biopsy. RESULTS: Overall, the methods performed yielded samples that were appropriate for diagnostic evaluation and interpretation, and the macaques experienced minimal discomfort and complications. DISCUSSION: This paper provides a descriptive summary of compiled techniques required to conduct a safety evaluation for topically applied vaginal microbicides. This novel method-based approach should be methodically executed when evaluating a vaginally-applied, topical microbicide candidate.

Enhancement of cell mediated immune responses using lipid depleted lentivirus as immunogen: a novel approach for inducing recognition of new viral epitopes.

We tested the hypothesis that removal of viral lipids using diisopropylether can enhance antigenicity of SIVmac251. DIPE delipidation removed cholesterol from SIVmac251 without significant loss of viral protein or RNA. Mice immunized with the same SIV preparation but boosted with delipidated SIVmac251 exhibited significantly broader and higher cellular and humoral immune responses compared to live or AT-2-inactivated virus. As little as 1microg (total protein) of delipidated virus was sufficient to induce such enhanced immune responses. Thus, solvent treated lentivirus may provide a novel strategy to generate immune responses to additional viral epitopes.