Cerebrospinal Fluid Biomarkers of Simian Immunodeficiency Virus Encephalitis : CSF Biomarkers of SIV Encephalitis.

Antiretroviral therapy has led to increased survival of HIV-infected patients but also increased prevalence of HIV-associated neurocognitive disorders. We previously identified YKL40 as a potential cerebrospinal fluid (CSF) biomarker of lentiviral central nervous system (CNS) disease in HIV-infected patients and in the macaque model of HIV encephalitis. The aim of this study was to define the specificity and sensitivity along with the predictive value of YKL40 as a biomarker of encephalitis and to assess its relationship to CSF viral load. CSF YKL40 and SIV RNA concentrations were analyzed over the course of infection in 19 SIV-infected pigtailed macaques and statistical analyses were performed to evaluate the relationship to encephalitis. Using these relationships, CSF alterations of 31 neuroimmune markers were studied pre-infection, during acute and asymptomatic infection, at the onset of encephalitis, and at necropsy. YKL40 CSF concentrations above 1122 ng/ml were found to be a specific and sensitive biomarker for the presence of encephalitis and were highly correlated with CSF viral load. Macaques that developed encephalitis had evidence of chronic CNS immune activation during early, asymptomatic, and end stages of infection. At the onset of encephalitis, CSF demonstrated a rise of neuroimmune markers associated with macrophage recruitment, activation and interferon response. CSF YKL40 concentration and viral load are valuable biomarkers to define the onset of encephalitis. Chronic CNS immune activation precedes the development of encephalitis while some responses suggest protection from CNS lentiviral disease.

Oral administration of the nucleoside EFdA (4'-ethynyl-2-fluoro-2'-deoxyadenosine) provides rapid suppression of HIV viremia in humanized mice and favorable pharmacokinetic properties in mice and the rhesus macaque.

Like normal cellular nucleosides, the nucleoside reverse transcriptase (RT) inhibitor (NRTI) 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) has a 3'-hydroxyl moiety, and yet EFdA is a highly potent inhibitor of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) replication with activity against a broad range of clinically important drug-resistant HIV isolates. We evaluated the anti-HIV activity of EFdA in primary human cells and in HIV-infected humanized mice. EFdA exhibited excellent potency against HIVJR-CSF in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs), with a 50% inhibitory concentration of 0.25 nM and a selectivity index of 184,000; similar antiviral potency was found against 12 different HIV clinical isolates from multiple clades (A, B, C, D, and CRF01_AE). EFdA was readily absorbed after oral dosing (5 mg/kg of body weight) in both mice and the rhesus macaque, with micromolar levels of the maximum concentration of drug in serum (Cmax) attained at 30 min and 90 min, respectively. Trough levels were at or above 90% inhibitory concentration (IC90) levels in the macaque at 24 h, suggesting once-daily dosing. EFdA showed reasonable penetration of the blood-brain barrier in the rhesus macaque, with cerebrospinal fluid levels at approximately 25% of plasma levels 8 h after single oral dosing. Rhesus PBMCs isolated 24 h following a single oral dose of 5 mg/kg EFdA were refractory to SIV infection due to sufficiently high intracellular EFdA-triphosphate levels. The intracellular half-life of EFdA-triphosphate in PBMCs was determined to be >72 h following a single exposure to EFdA. Daily oral administration of EFdA at low dosage levels (1 to 10 mg/kg/day) was highly effective in protecting humanized mice from HIV infection, and 10 mg/kg/day oral EFdA completely suppressed HIV RNA to undetectable levels within 2 weeks of treatment.

Isolation, characterization, and functional analysis of ferret lymphatic endothelial cells.

The lymphatic endothelium (LE) serves as a conduit for transport of immune cells and soluble antigens from peripheral tissues to draining lymph nodes (LNs), contributing to development of host immune responses and possibly dissemination of microbes. Lymphatic endothelial cells (LECs) are major constituents of the lymphatic endothelium. These specialized cells could play important roles in initiation of host innate immune responses through sensing of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs), including toll-like receptors (TLRs). LECs secrete pro-inflammatory cytokines and chemokines to create local inflammatory conditions for recruitment of naïve antigen presenting cells (APCs) such as dendritic cells (DCs) to sites of infection and/or vaccine administration. In this study, we examined the innate immune potential of primary LEC populations derived from multiple tissues of an animal model for human infectious diseases - the ferret. We generated a total of six primary LEC populations from lung, tracheal, and mesenteric LN tissues from three different ferrets. Standard RT-PCR characterization of these primary LECs showed that they varied in their expression of LEC markers. The ferret LECs were examined for their ability to respond to poly I:C (TLR3 and RIG-I ligand) and other known TLR ligands as measured by production of proinflammatory cytokine (IFNα, IL6, IL10, Mx1, and TNFα) and chemokine (CCL5, CCL20, and CXCL10) mRNAs using real time RT-PCR. Poly I:C exposure induced robust proinflammatory responses by all of the primary ferret LECs. Chemotaxis was performed to determine the functional activity of CCL20 produced by the primary lung LECs and showed that the LEC-derived CCL20 was abundant and functional. Taken together, our results continue to reveal the innate immune potential of primary LECs during pathogen-host interactions and expand our understanding of the roles LECs might play in health and disease in animal models.

Resistance to simian immunodeficiency virus low dose rectal challenge is associated with higher constitutive TRIM5α expression in PBMC.

BACKGROUND: At least six host-encoded restriction factors (RFs), APOBEC3G, TRIM5α, tetherin, SAMHD1, schlafen 11, and Mx2 have now been shown to inhibit HIV and/or SIV replication in vitro. To determine their role in vivo in the resistance of macaques to mucosally-acquired SIV, we quantified both pre-exposure (basal) and post-exposure mRNA levels of these RFs, Mx1, and IFNγ in PBMC, lymph nodes, and duodenum of rhesus macaques undergoing weekly low dose rectal exposures to the primary isolate, SIV/DeltaB670. RESULTS: Repetitive challenge divided the monkeys into two groups with respect to their susceptibility to infection: highly susceptible (2-3 challenges, 5 monkeys) and poorly susceptible (≥6 challenges, 3 monkeys). Basal RF and Mx1 expression varied among the three tissues examined, with the lowest expression generally detected in duodenal tissues, and the highest observed in PBMC. The one exception was A3G whose basal expression was greatest in lymph nodes. Importantly, significantly higher basal expression of TRIM5α and Mx1 was observed in PBMC of animals more resistant to mucosal infection. Moreover, individual TRIM5α levels were stable throughout a year prior to infection. Post-exposure induction of these genes was also observed after virus appearance in plasma, with elevated levels in PBMC and duodenum transiently occurring 7-10 days post infection. They did not appear to have an effect on control of viremia. Interestingly, minimal to no induction was observed in the resistant animal that became an elite controller. CONCLUSIONS: These results suggest that constitutively expressed TRIM5α appears to play a greater role in restricting mucosal transmission of SIV than that associated with type I interferon induction following virus entry. Surprisingly, this association was not observed with the other RFs. The higher basal expression of TRIM5α observed in PBMC than in duodenal tissues emphasizes the understated role of the second barrier to systemic infection involving the transport of virus from the mucosal compartment to the blood. Together, these observations provide a strong incentive for a more comprehensive examination of the intrinsic, variable control of constitutive expression of these genes in the sexual transmission of HIV.

Macaque paneth cells express lymphoid chemokine CXCL13 and other antimicrobial peptides not previously described as expressed in intestinal crypts.

CXCL13 is a constitutively expressed chemokine that controls migration of immune cells to lymphoid follicles. Previously, we found CXCL13 mRNA levels increased in rhesus macaque spleen tissues during AIDS. This led us to examine the levels and locations of CXCL13 by detailed in situ methods in cynomolgus macaque lymphoid and intestinal tissues. Our results revealed that there were distinct localization patterns of CXCL13 mRNA compared to protein in germinal centers. These patterns shifted during the course of simian immunodeficiency virus (SIV) infection, with increased mRNA expression within and around follicles during AIDS compared to uninfected or acutely infected animals. Unexpectedly, CXCL13 expression was also found in abundance in Paneth cells in crypts throughout the small intestine. Therefore, we expanded our analyses to include chemokines and antimicrobial peptides (AMPs) not previously demonstrated to be expressed by Paneth cells in intestinal tissues. We examined the expression patterns of multiple chemokines, including CCL25, as well as α-defensin 6 (DEFA6), ß-defensin 2 (BDEF2), rhesus θ-defensin 1 (RTD-1), and Reg3γ in situ in intestinal tissues. Of the 10 chemokines examined, CXCL13 was unique in its expression by Paneth cells. BDEF2, RTD-1, and Reg3γ were also expressed by Paneth cells. BDEF2 and RTD-1 previously have not been shown to be expressed by Paneth cells. These findings expand our understanding of mucosal immunology, innate antimicrobial defenses, homeostatic chemokine function, and host protective mechanisms against microbial translocation.

Simian immunodeficiency virus infection potently modulates chemokine networks and immune environments in hilar lymph nodes of cynomolgus macaques.

BACKGROUND: Chemokines provide critical immune cell homing and activation signals that if altered could affect the inflammatory milieu and cellular composition of lymphoid tissues. During HIV-1 and simian immunodeficiency virus (SIV)-infection, the virus triggers an increase in inflammation or activation, leading to immunodeficiency and development of opportunistic infections, such as in the lungs-a massive interface between the host and the environment. METHODS: Chemokine, cytokine, and chemokine receptor expression profiles were determined using real-time reverse transcriptase-polymerase chain reaction and in situ hybridization in hilar lymph nodes (HiLNs) from cynomolgus macaques at different stages after infection with SIV/DeltaB670. Immunostaining of tissue sections and flow cytometric analysis of cryopreserved cells were used to examine cellular compositions of lymph nodes. RESULTS: Interferon-gamma, type 1 chemokine, and cognate chemokine receptor mRNAs were upregulated, whereas type 2 and homeostatic chemokine and chemokine receptor mRNAs were down-regulated in HiLNs after SIV infection. Local SIV and interferon-gamma levels were positively correlated with type 1 chemokine levels but negatively correlated with type 2 and homeostatic chemokine levels. Using in situ hybridization, Pneumocystis carinii rRNA was detected in lung-draining lymph nodes from animals with P. carinii pneumonia. Changes in the cellular composition of HiLNs included decreased proportions of CD4 cells and dendritic cells and increased proportions of CD8, CXCR3, and CCR5 cells. CONCLUSIONS: SIV infection of cynomolgus macaques dramatically alters the cellular homing signals of lung-draining lymph nodes, which correlated with changes in the immune cellular composition. These changes could contribute to the loss of immune function that defines AIDS.

Response of simian immunodeficiency virus to the novel nucleoside reverse transcriptase inhibitor 4'-ethynyl-2-fluoro-2'-deoxyadenosine in vitro and in vivo.

Nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) are essential components in first-line therapy for human immunodeficiency virus (HIV) infection. However, long-term treatment with existing NRTIs can be associated with significant toxic side effects and the emergence of drug-resistant strains. The identification of new NRTIs for the continued management of HIV-infected people therefore is paramount. In this report, we describe the response of a primary isolate of simian immunodeficiency virus (SIV) to 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA) both in vitro and in vivo. EFdA was 3 orders of magnitude better than tenofovir (TFV), zidovudine (AZT), and emtricitabine (FTC) in blocking replication of SIV in monkey peripheral blood mononuclear cells (PBMCs) in vitro, and in a preliminary study using two SIV-infected macaques with advanced AIDS, it was highly effective at treating SIV infection and AIDS symptoms in vivo. Both animals had 3- to 4-log decreases in plasma virus burden within 1 week of EFdA therapy (0.4 mg/kg of body weight, delivered subcutaneously twice a day) that eventually became undetectable. Clinical signs of disease (diarrhea, weight loss, and poor activity) also resolved within the first month of treatment. No detectable clinical or pathological signs of drug toxicity were observed within 6 months of continuous therapy. Virus suppression was sustained until drug treatment was discontinued, at which time virus levels rebounded. Although the rebound virus contained the M184V/I mutation in the viral reverse transcriptase, EFdA was fully effective in maintaining suppression of mutant virus throughout the drug treatment period. These results suggest that expanded studies with EFdA are warranted.

Therapeutic DNA vaccine induces broad T cell responses in the gut and sustained protection from viral rebound and AIDS in SIV-infected rhesus macaques.

Immunotherapies that induce durable immune control of chronic HIV infection may eliminate the need for life-long dependence on drugs. We investigated a DNA vaccine formulated with a novel genetic adjuvant that stimulates immune responses in the blood and gut for the ability to improve therapy in rhesus macaques chronically infected with SIV. Using the SIV-macaque model for AIDS, we show that epidermal co-delivery of plasmids expressing SIV Gag, RT, Nef and Env, and the mucosal adjuvant, heat-labile E. coli enterotoxin (LT), during antiretroviral therapy (ART) induced a substantial 2-4-log fold reduction in mean virus burden in both the gut and blood when compared to unvaccinated controls and provided durable protection from viral rebound and disease progression after the drug was discontinued. This effect was associated with significant increases in IFN-γ T cell responses in both the blood and gut and SIV-specific CD8+ T cells with dual TNF-α and cytolytic effector functions in the blood. Importantly, a broader specificity in the T cell response seen in the gut, but not the blood, significantly correlated with a reduction in virus production in mucosal tissues and a lower virus burden in plasma. We conclude that immunizing with vaccines that induce immune responses in mucosal gut tissue could reduce residual viral reservoirs during drug therapy and improve long-term treatment of HIV infection in humans.

HIV-1 Vaccine Trials: Evolving Concepts and Designs.

An effective prophylactic HIV-1 vaccine is needed to eradicate the HIV/AIDS pandemic but designing such a vaccine is a challenge. Despite many advances in vaccine technology and approaches to generate both humoral and cellular immune responses, major phase-II and -III vaccine trials against HIV/AIDS have resulted in only moderate successes. The modest achievement of the phase-III RV144 prime-boost trial in Thailand re-emphasized the importance of generating robust humoral and cellular responses against HIV. While antibody-directed approaches are being pursued by some groups, others are attempting to develop vaccines targeting cell-mediated immunity, since evidence show CTLs to be important for the control of HIV replication. Phase-I and -IIa multi-epitope vaccine trials have already been conducted with vaccine immunogens consisting of known CTL epitopes conserved across HIV subtypes, but have so far fallen short of inducing robust and consistent anti-HIV CTL responses. The concepts leading to the development of T-cell epitope-based vaccines, the outcomes of related clinical vaccine trials and efforts to enhance the immunogenicity of cell-mediated approaches are summarized in this review. Moreover, we describe a novel approach based on the identification of SIV and FIV antigens which contain conserved HIV-specific T-cell epitopes and represent an alternative method for developing an effective HIV vaccine against global HIV isolates.

Pulmonary vascular lesions are common in SIV- and SHIV-env-infected macaques.

The lack of animal models of HIV-related pulmonary arterial hypertension (HIV-PAH) severely limits investigation of this serious disease. While histological evidence of HIV-PAH has been demonstrated in macaques infected with simian immunodeficiency virus (SIV) as well as with chimeric simian/human immunodeficiency virus (SHIV) containing HIV-1-derived Nef protein, other primate models have not been studied. The objective was to document and describe the development of pulmonary vascular changes in macaques infected with SIV or with SIV containing HIV-1-derived envelope protein (SHIV-env). Lung tissue was obtained at necropsy from 13 SHIV (89.6P)-env-infected macaques and 10 SIV (ΔB670)-infected macaques. Pulmonary arterial pathology, including arterial hyperplasia and the presence of plexiform lesions, was compared to normal monkey lung. Pulmonary artery hyperplasia was present in 8 of 13 (62%) SHIV-env-infected macaques and 4/10 (36%) SIV-infected macaques. The most common histopathological lesions were intimal and medial hyperplasia of medium and large pulmonary arteries. Hyperplastic lesions were predominantly due to smooth muscle cell hyperplasia. This is the first report of pulmonary vascular lesions in SHIV-env-infected macaques and confirms prior reports of pulmonary vasculopathy in SIV-infected macaques. The finding of pulmonary arteriopathy in monkeys infected with SHIV not containing HIV-nef suggests that other factors might also be important in the development of HIV-PAH. This SHIV-env model provides a new means to investigate HIV-PAH.

Simian immunodeficiency virus infection alters chemokine networks in lung tissues of cynomolgus macaques: association with Pneumocystis carinii infection.

Infection by HIV-1 frequently leads to pulmonary complications, including alterations to local immune environments. To better understand these alterations, we have examined in detail the patterns and levels of expression of chemokine, cytokine, and chemokine receptor mRNAs in lung tissues from 16 uninfected or simian immunodeficiency virus (SIV)/DeltaB670 infected cynomolgus macaques at different stages of infection. Among the most up-regulated immune genes were interferon (IFN)-gamma, IFN-gamma-inducible CXCR3 ligands, and CCR5 ligands, as well as the cognate chemokine receptors. These changes were greatest in animals with clear Pneumocystis carinii coinfection. Immunohistochemistry and in situ hybridization revealed monocytes/macrophages to be the predominant type of cell infiltrating into lung tissues and serving as the major cellular source of chemokines. To explore the causes of chemokine alterations, we treated macaque lung cells with IFN-gamma, lipopolysaccharide, Poly(I:C), and P. carinii in vitro, and results revealed that these stimuli can induce the expression of CXCR3 ligand and/or CCR5 ligand mRNAs. Taken together, these studies provide a comprehensive definition of the chemokine networks available to modulate cellular recruitment to lung tissues during SIV infection and implicate both cytokines (IFN-gamma) and pathogens (SIV and P. carinii) as contributors to increased expression of pro-inflammatory chemokines.

GM-CSF increases mucosal and systemic immunogenicity of an H1N1 influenza DNA vaccine administered into the epidermis of non-human primates.

BACKGROUND: The recent H5N1 avian and H1N1 swine-origin influenza virus outbreaks reaffirm that the threat of a world-wide influenza pandemic is both real and ever-present. Vaccination is still considered the best strategy for protection against influenza virus infection but a significant challenge is to identify new vaccine approaches that offer accelerated production, broader protection against drifted and shifted strains, and the capacity to elicit anti-viral immune responses in the respiratory tract at the site of viral entry. As a safe alternative to live attenuated vaccines, the mucosal and systemic immunogenicity of an H1N1 influenza (A/New Caledonia/20/99) HA DNA vaccine administered by particle-mediated epidermal delivery (PMED or gene gun) was analyzed in rhesus macaques. METHODOLOGY/PRINCIPAL FINDINGS: Macaques were immunized at weeks 0, 8, and 16 using a disposable single-shot particle-mediated delivery device designed for clinical use that delivers plasmid DNA directly into cells of the epidermis. Significant levels of hemagglutination inhibiting (HI) antibodies and cytokine-secreting HA-specific T cells were observed in the periphery of macaques following 1-3 doses of the PMED HA DNA vaccine. In addition, HA DNA vaccination induced detectable levels of HA-specific mucosal antibodies and T cells in the lung and gut-associated lymphoid tissues of vaccinated macaques. Importantly, co-delivery of a DNA encoding the rhesus macaque GM-CSF gene was found to significantly enhance both the systemic and mucosal immunogenicity of the HA DNA vaccine. CONCLUSIONS/SIGNIFICANCE: These results provide strong support for the development of a particle-mediated epidermal DNA vaccine for protection against respiratory pathogens such as influenza and demonstrate, for the first time, the ability of skin-delivered GM-CSF to serve as an effective mucosal adjuvant for vaccine induction of immune responses in the gut and respiratory tract.

Boron-Doped Diamond Microelectrodes Reveal Reduced Serotonin Uptake Rates in Lymphocytes from Adult Rhesus Monkeys Carrying the Short Allele of the 5-HTTLPR.

Uptake resolved by high-speed chronoamperometry on a second-by-second basis has revealed important differences in brain serotonin transporter function associated with genetic variability. Here, we use chronoamperometry to investigate variations in serotonin transport in primary lymphocytes associated with the rhesus serotonin transporter gene-linked polymorphism (rh5-HTTLPR), a promoter polymorphism whose orthologs occur only in higher order primates including humans. Serotonin clearance by lymphocytes is Na(+)-dependent and inhibited by the serotonin-selective reuptake inhibitor paroxetine (Paxil®), indicative of active uptake by serotonin transporters. Moreover, reductions in serotonin uptake rates are evident in lymphocytes from monkeys with one or two copies of the short 's' allele of the rh5-HTTLPR (s/s

YKL-40, a marker of simian immunodeficiency virus encephalitis, modulates the biological activity of basic fibroblast growth factor.

Human immunodeficiency virus encephalitis causes dementia in acquired immune deficiency syndrome patients. Using proteomic analysis of postmortem cerebrospinal fluid (CSF) and brain tissue from the simian immunodeficiency virus primate model, we demonstrate here a specific increase in YKL-40 that was tightly associated with lentiviral encephalitis. Longitudinal analysis of CSF from simian immunodeficiency virus-infected pigtailed macaques showed an increase in YKL-40 concentration 2 to 8 weeks before death from encephalitis. This increase in YKL-40 correlated with an increase in CSF viral load; it may therefore represent a biomarker for the development of encephalitis. Analysis of banked human CSF from human immunodeficiency virus-infected patients also demonstrated a correlation between YKL-40 concentration and CSF viral load. In vitro studies demonstrated increased YKL-40 expression and secretion by macrophages and microglia but not by neurons or astrocytes. We found that YKL40 displaced extracellular matrix-bound basic fibroblast growth factor (bFGF) as well as inhibited the mitogenic activity of both fibroblast growth factor receptor 1-expressing BaF3 cells and bFGF-induced axonal branching in hippocampal cultures. Taken together, these findings demonstrate that during lentiviral encephalitis, YKL-40 may interfere with the biological activity of bFGF and potentially of other heparin-binding growth factors and chemokines that can affect neuronal function or survival.

Longitudinal in vivo positron emission tomography imaging of infected and activated brain macrophages in a macaque model of human immunodeficiency virus encephalitis correlates with central and peripheral markers of encephalitis and areas of synaptic degeneration.

Human immunodeficiency virus encephalitis is characterized by infiltration of the brain with infected and activated macrophages; however, it is not known why disease occurs after variable lengths of infection in 25% of immunosuppressed acquired immune deficiency syndrome patients. We determined in vivo correlates (in peripheral blood and the central nervous system) for the development and progression of lentiviral encephalitis by longitudinally following infected and activated macrophages in the brain using positron emission tomography (PET). Using human postmortem brain tissues from both lentivirus-infected encephalitic patients and cell culture systems, we showed that the PET ligand [(3)H](R)-PK11195 bound specifically to virus-infected and activated macrophages. We longitudinally imaged infected and activated brain macrophages in a cohort of macaques infected with simian immunodeficiency virus using [(11)C](R)-PK11195. [(11)C](R)-PK11195 retention in vivo in the brain correlated with viral burden in the brain and cerebrospinal fluid, and with regions of both presynaptic and postsynaptic damage. Finally, longitudinal changes in [(11)C](R)-PK11195 retention in the brain in vivo correlated with changes in circulating monocytes as well as in both natural killer and memory CD4(+) T cells in the periphery. Our results suggest that development and progression of simian immunodeficiency virus encephalitis in vivo correlates with changes in specific cell subtypes in the periphery. A combination of PET imaging and the assessment of these peripheral immune parameters may facilitate longitudinal assessment of lentiviral encephalitis in living patients as well as evaluation of therapeutic efficacies.

Chemokine and cytokine mediated loss of regulatory T cells in lymph nodes during pathogenic simian immunodeficiency virus infection.

Regulatory T cells (T(reg)) play key roles in immune regulation through multiple modes of suppression. The effects of HIV-1 infection on T(reg) levels in lymphoid tissues remain incompletely understood. To explore this issue, we have measured the levels of forkhead box protein 3 (FOXP3)-positive cells and associated immunomodulatory genes in a pathogenic simian immunodeficiency virus/macaque model and found that a loss of T(reg) in lymph nodes occurred following simian immunodeficiency virus infection. Changes in expression of the ligands for CXCR3, CCR4, and CCR7 and the cytokines TGF-beta and IL-2 were all linked to this loss of T(reg), which in turn was linked with increased levels of cellular activation. Our findings identify three mechanisms that likely contribute to SIV-driven loss of T(reg), including reduced levels of cytokines associated with T(reg) differentiation and altered expression of agonist and antagonist chemokines. The loss of T(reg) and the associated cellular activation in lymphoid tissues is consistent with the events in HIV-1-infected individuals and suggest that components of the T(reg) differentiation and trafficking network could be targets for therapeutic intervention.

Systemic and brain macrophage infections in relation to the development of simian immunodeficiency virus encephalitis.

The brains of individuals with lentiviral-associated encephalitis contain an abundance of infected and activated macrophages. It has been hypothesized that encephalitis develops when increased numbers of infected monocytes traffic into the central nervous system (CNS) during the end stages of immunosuppression. The relationships between the infection of brain and systemic macrophages and circulating monocytes and the development of lentiviral encephalitis are unknown. We longitudinally examined the extent of monocyte/macrophage infection in blood and lymph nodes of pigtailed macaques that did or did not develop simian immunodeficiency virus encephalitis (SIVE). Compared to levels in macaques that did not develop SIVE, more ex vivo virus production was detected from monocyte-derived macrophages and nonadherent peripheral blood mononuclear cells (PBMCs) from macaques that did develop SIVE. Prior to death, there was an increase in the number of circulating PBMCs following a rise in cerebrospinal fluid viral load in macaques that did develop SIVE but not in nonencephalitic macaques. At necropsy, macaques with SIVE had more infected macrophages in peripheral organs, with the exception of lymph nodes. T cells and NK cells with cytotoxic potential were more abundant in brains with encephalitis; however, T-cell and NK-cell infiltration in SIVE and human immunodeficiency virus encephalitis was more modest than that observed in classical acute herpes simplex virus encephalitis. These findings support the hypothesis that inherent differences in host systemic and CNS monocyte/macrophage viral production are associated with the development of encephalitis.

Association between decreased CXCL12 and CCL25 expression and increased apoptosis in lymphoid tissues of cynomolgus macaques during SIV infection.

BACKGROUND: Chemokines likely play multiple roles in HIV-1 and SIV pathogenesis. To examine potential associations between chemokine expression levels and apoptosis of cells in lymphoid tissues during SIV infection, we measured chemokine and cytokine mRNA levels in multiple lymphoid tissues compartments from uninfected and SIV-infected cynomolgus macaques (Macaca fascicularis). METHODS: Real-time RT-PCR was used to measure host mRNA levels in macaque lymphoid tissues. Proliferating or apoptotic cells were identified in lymphoid tissues by immunohistochemistry. RESULTS: We found that CXCL12 and CCL25 mRNAs in SIV-infected lymphoid tissues were decreased and their levels were negatively correlated with the numbers of proliferating and apoptotic cells. In vitro analyses revealed that CXCL12 and CCL25 were capable of reducing apoptosis induced by SIV infection. CONCLUSIONS: These findings suggest that increased apoptosis in lymphoid tissues due to reduced levels of anti-apoptotic chemokines might be a mechanism that contributes to loss of immune function following pathogenic SIV infection.

Virologic and immunologic events in hilar lymph nodes during simian immunodeficiency virus infection: development of polarized inflammation.

Lymphoid tissues are sites of soluble and cell-associated antigen sampling of peripheral tissues, and they are key compartments for the generation of cellular and humoral immune responses. Hilar lymph nodes (HiLNs), which drain the lungs, were examined to understand the effects of simian immunodeficiency virus (SIV) infection on this compartment of the immune system. Histologic and messenger RNA (mRNA) expression profiling approaches were used to determine the numbers, types, and distributions of SIV viral RNA cells and to identify differentially expressed genes in HiLNs during SIV infection. SIV RNA cells were found to be primarily CD68 and localized to paracortical and medullary regions early in infection, whereas they resided mainly in paracortex during AIDS. As SIV infection progressed, CXCL9, CXCL10, interferon-gamma, and Toll-like receptor 3 levels all increased. In contrast, CCL19 increased early in infection but decreased during AIDS, whereas CCL21 decreased progressively throughout infection. Finally, local levels of cellular activation were increased throughout infection. Taken together, these findings indicate that SIV infection leads to an inflammatory environment in lung-draining lymph nodes that is characterized by type 1 cytokines and chemokines and likely has an impact on the nature and strength of immune responses to pulmonary pathogens.

Effects of monotherapy with (R)-9-(2-phosphonylmethoxypropyl)adenine (PMPA) on the evolution of a primary Simian immunodeficiency virus (SIV) isolate.

Determining the impact of antiretroviral therapy on virus evolution could advance the development of improved therapeutics/vaccines against HIV. Toward this goal, we analyzed virus burden, quasispecies complexity, and T cell responses in SIV/DeltaB670-infected rhesus macaques+/-treatment for 7 months with PMPA (2-30 weeks postinfection). Treatment divided the animals into two groups: poor responders (a reduction of < or =1 log) and responders (> or =2 log reduction) in virus burden. Virus evolution in poor responders and untreated controls was characterized by expression of a complex quasispecies that evolved as the disease progressed. This included the universal loss of a viral genotype selected against by in vitro passage in monkey cells and selected for by propagation in human cells. In contrast, a good response to PMPA was characterized by infection with a less complex quasispecies that evolved more slowly. Interestingly, in 2 of the best responders, the human-preferred genotype persisted until the study was discontinued (89 weeks p.i.). Neither virus burden nor the magnitude of the T cell response at 2 weeks postinfection predicted PMPA responsiveness. However, responders expressed a less complex quasispecies than nonresponders prior to treatment. These data suggest a role for intrinsic host factors in treatment responsiveness, and lend support for therapeutic vaccination as an adjunct to effective therapy.