Human Beta-Defensin 2 and 3 Inhibit HIV-1 Replication in Macrophages.

Human beta-defensins (hBDs) are broad-spectrum antimicrobial peptides, secreted by epithelial cells of the skin and mucosae, and astrocytes, which we and others have shown to inhibit HIV-1 in primary CD4+ T cells. Although loss of CD4+ T cells contributes to mucosal immune dysfunction, macrophages are a major source of persistence and spread of HIV and also contribute to the development of various HIV-associated complications. We hypothesized that, besides T cells, hBDs could protect macrophages from HIV. Our data in primary human monocyte-derived macrophages (MDM) in vitro show that hBD2 and hBD3 inhibit HIV replication in a dose-dependent manner. We determined that hBD2 neither alters surface expression of HIV receptors nor induces expression of anti-HIV cytokines or beta-chemokines in MDM. Studies using a G-protein signaling antagonist in a single-cycle reporter virus system showed that hBD2 suppresses HIV at an early post-entry stage via G-protein coupled receptor (GPCR)-mediated signaling. We find that MDM express the shared chemokine-hBD receptors CCR2 and CCR6, albeit at variable levels among donors. However, cell surface expression analyses show that neither of these receptors is necessary for hBD2-mediated HIV inhibition, suggesting that hBD2 can signal via additional receptor(s). Our data also illustrate that hBD2 treatment was associated with increased expression of APOBEC3A and 3G antiretroviral restriction factors in MDM. These findings suggest that hBD2 inhibits HIV in MDM via more than one CCR thus adding to the potential of using ß-defensins in preventive and therapeutic approaches.

The macrophage and HIV: basic concepts and methodologies.

Along with CD4+ T-lymphocytes, macrophage lineage cells serve as primary hosts for HIV replication in vivo. In some tissues such as brain, where T-cell infection is essentially absent, the development of HIV-associated disease is mediated through infection of macrophages. This fact underscores the importance of experimental methods that yield results and conclusions that accurately reflect the mechanisms operational in vivo. Unfortunately, our understanding of key aspects of HIV-macrophage interactions, most notably, features of viral entry, replication, latency and persistence, lags behind that of T-cell infection. While some questions are best approached by direct examination of patient specimens using methods such as immunohistochemistry and phylogenetics, experiments based on HIV infection of macrophages in vitro can, necessarily, identify and elucidate the events, molecular mechanisms, and pathological consequences associated with this infection. In addition, macrophage culture methods can provide for the isolation of infectious HIV from patient blood monocytes and tissue macrophages, as well as subsequent continued propagation of these isolates in their host cell of origin. Maintenance of the host cell pedigree limits the possibility of alteration of viral properties such as chemokine coreceptor usage that may then no longer reflect the situation in vivo. This chapter focuses on HIV infection of macrophages. We describe methods for the cultivation of human blood monocyte-derived macrophages, their infection with HIV and subsequent maintenance, and the isolation of infectious HIV from them. Also included is a protocol using accutase for macrophage detachment. Accutase is a relatively new dissociation medium, used primarily in stem cell research. In our laboratory, it has far out-performed all other methods by providing for the gentle, yet thorough, detachment of macrophages without the need for scraping, and without loss of surface antigens or viability.

De novo generation of cells within human nurse macrophages and consequences following HIV-1 infection.

Nurse cells are defined as those that provide for the development of other cells. We report here, that in vitro, human monocyte-derived macrophages can behave as nurse cells with functional capabilities that include de novo generation of CD4+ T-lymphocytes and a previously unknown small cell with monocytoid characteristics. We named these novel cells "self-renewing monocytoid cells" (SRMC), because they could develop into nurse macrophages that produced another generation of SRMC. SRMC were not detectable in blood. Their transition to nurse behavior was characterized by expression of CD10, a marker of thymic epithelium and bone marrow stroma, typically absent on macrophages. Bromodeoxyuridine labeling and immunostaining for cdc6 expression confirmed DNA synthesis within nurse macrophages. T-cell excision circles were detected in macrophages, along with expression of pre-T-cell receptor alpha and recombination activating gene 1, suggesting that genetic recombination events associated with generation of the T-cell receptor were occurring in these cells. SRMC expressed CCR5, the coreceptor for R5 HIV-1 isolates, and were highly susceptible to HIV-1 entry leading to productive infection. While expressing HIV-1, SRMC could differentiate into nurse macrophages that produced another generation of HIV-1-expressing SRMC. The infected nurse macrophage/SRMC cycle could continue in vitro for multiple generations, suggesting it might represent a mechanism whereby HIV-1 can maintain persistence in vivo. HIV-1 infection of nurse macrophages led to a decline in CD4+ T-cell production. There was severe, preferential loss of the CCR5+ CD4+ T-cell subpopulation. Confocal microscopy revealed individual HIV-1-expressing nurse macrophages simultaneously producing both HIV-1-expressing SRMC and non-expressing CD3+ cells, suggesting that nurse macrophages might be a source of latently infected CD4+ T-cells. Real-time PCR experiments confirmed this by demonstrating 10-fold more HIV-1-genome-harboring T-cells, than virus-expressing ones. These phenomena have far-reaching implications, and elicit new perspectives regarding HIV pathogenesis and T-cell and hematopoietic cell development.

Characterization of the follicular dendritic cell reservoir of human immunodeficiency virus type 1.

Throughout the natural course of human immunodeficiency virus (HIV) infection, follicular dendritic cells (FDCs) trap and retain large quantities of particle-associated HIV RNA in the follicles of secondary lymphoid tissue. We have previously found that murine FDCs in vivo could maintain trapped virus particles in an infectious state for at least 9 months. Here we sought to determine whether human FDCs serve as an HIV reservoir, based on the criteria that virus therein must be replication competent, genetically diverse, and archival in nature. We tested our hypothesis using postmortem cells and tissues obtained from three HIV-infected subjects and antemortem blood samples obtained from one of these subjects. Replication competence was determined using coculture, while genetic diversity and the archival nature of virus were established using phylogenetic and population genetics methods. We found that FDC-trapped virus was replication competent and demonstrated greater genetic diversity than that of virus found in most other tissues and cells. Antiretrovirus-resistant variants that were not present elsewhere were also detected on FDCs. Furthermore, genetic similarity was observed between FDC-trapped HIV and viral species recovered from peripheral blood mononuclear cells obtained 21 and 22 months antemortem, but was not present in samples obtained 4 and 18 months prior to the patient's death, indicating that FDCs can archive HIV. These data indicate that FDCs represent a significant reservoir of infectious and diverse HIV, thereby providing a mechanism for viral persistence for months to years.

Molecular programming of endothelin-1 in HIV-infected brain: role of Tat in up-regulation of ET-1 and its inhibition by statins.

Human Immune Deficiency Virus-1 (HIV-1) infection can induce severe and debilitating neurological problems, including behavioral abnormalities, motor dysfunction, and dementia. HIV can persistently infect astrocytes, during which viral accessory proteins are produced that are unaffected by current antiretroviral therapy. The effect of these proteins on astrocyte function remains unknown. Astrocytes are the predominant cells within the brain; thus, disruption of astrocyte function could influence the neuropathogenesis of HIV infection. To explore further these effects, we constitutively expressed HIV-Tat protein in astrocytes. Since the nuclear presence of Tat protein leads to alteration of host gene expression, we further analyzed the effects of Tat on host gene transcripts. Endothelin-1 (ET-1) was a significantly elevated transcript as verified by reverse transcription-polymerase chain reaction (RT-PCR), and it was subsequently released extracellularly in Tat-expressing and HIV-infected astrocytes. ET-1 expression was also prominent in reactive astrocytes and neurons in brain tissues from basal ganglia and frontal lobes of HIV encephalitic patients. HIV-Tat regulated ET-1 at the transcriptional level through NF-kappaB (NF-kappaB)-responsive sites in the ET-1 promoter. Intriguingly, simvastatin (10 microM) down-regulated HIV-Tat-induced ET-1 and also inhibited activation of NF-kappaB in astrocytes. Our findings suggest that ET-1 may be critical in mediating the neuropathogenesis of HIV dementia and that statins may have therapeutic potential in these patients.

In vitro modeling of the HIV-macrophage reservoir.

Macrophages are recognized as a putative reservoir for HIV-1, but whether HIV can establish latent infection in this cell type is not known. An in vitro model using long-term cultured primary human monocyte-derived macrophages (MDM) infected with an M-tropic, enhanced green fluorescent protein (EGFP) tagged reporter virus was developed to test the hypothesis that HIV can establish a latent infection of this cell type. The EGFP-IRES-Nef cassette allowed detection of early gene transcription. The expression of GFP+ MDM was followed with time and the GFP- population was purified and analyzed for evidence of latent infection. Interestingly, in MDM cultures propagated for over two months, distinct subpopulations of infected GFP+ cells were observed and quantitated. In particular, infected MDM that displayed a high level of transcription, characterized as the GFP hi group, yet produced low levels of the late viral gene product, p24, increased with time and represented 10% of the GFP+ population in long-term cultures. The high level production of early genes such as Nef, a protein that can facilitate viral immune escape, but low level of structural proteins such as p24 in the GFP hi population suggests that a subset of infected MDM can exhibit an alternative mode of replication. The GFP- MDM population obtained by a two-step purification protocol using flow cytometry and laser ablation contained integrated provirus as assessed by Alu-LTR real-time PCR analyses. A subset of these, were replication competent as shown by their ability to express GFP and/or p24 antigen after reactivation with IL-4.

Increased vulnerability of ApoE4 neurons to HIV proteins and opiates: protection by diosgenin and L-deprenyl.

Human immunodeficiency virus (HIV) infection continues to rise in drug-abusing populations and causes a dementing illness in a subset of individuals. Factors contributing to the development of dementia in this population remain unknown. We found that HIV-infected individuals with the E4 allele of Apolipoprotein E (ApoE) or history of intravenous drug abuse had increased oxidative stress in the CNS. In vitro studies showed that HIV proteins, gp120 and Tat, Tat + morphine but not tumor necrosis factor-alpha (TNF-alpha), caused increased neurotoxicity in human neuronal cultures with ApoE4 allele. Microarray analysis showed a differential alteration of transcripts involved in energy metabolism in cultures of ApoE3 and 4 neurons upon treatment with Tat + morphine. This was confirmed using assays of mitochondrial function and exposure of the neurons to Tat + morphine. Using this in vitro model, we screened a number of novel antioxidants and found that only L-deprenyl and diosgenin protected against the neurotoxicity of Tat + morphine. Furthermore, Tat-induced oxidative stress impaired morphine metabolism which could also be prevented by diosgenin. In conclusion, opiate abusers with HIV infection and the ApoE4 allele may be at increased risk of developing dementia. L-deprenyl and a plant estrogen, diosgenin, may have therapeutic potential in this population.

HIV-1-based defective lentiviral vectors efficiently transduce human monocytes-derived macrophages and suppress replication of wild-type HIV-1.

BACKGROUND: Human monocytes play an important role in mediating human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS), and monocytes-derived macrophages (MDM) represent a major viral reservoir within the brain and other target organs. Current gene transduction of MDM is hindered by a limited efficiency. In this study we established a lentiviral vector-based technique for improved gene transfer into human MDM cultures in vitro and demonstrated significant protection of transduced MDM from super-infection with wild-type HIV-1. METHODS: HIV-1-based lentiviral vector stocks were prepared in 293T cells by the established calcium phosphate transfection method. Human monocytes were isolated from donors' blood by Ficoll-Paque separation and cultured in vitro. To establish an effective technique for vector-mediated gene transfer, primary cultures of human MDM were transduced at varying multiplicities of infection (MOI) and at a range of time points following initial isolation of cells (time-in-culture). Transduced cells were then examined for transgene (green fluorescent protein (GFP)) expression by fluorescent microscopy and reverse transcription polymerase chain reaction (RT-PCR). These cultures were then exposed to wild-type HIV-1, and viral replication was quantitated by p24 assay; production of neurotoxic effector molecules by the transduced MDM was also examined, using indicator neurons. RESULTS: We have demonstrated that primary human MDM could be efficiently transduced (>50%) with concentrated HIV-1-based defective lentiviral vectors (DLV). Furthermore, DLV-mediated gene transduction was stable, and the transduced cells exhibited no apparent difference from normal MDM in terms of their morphology, viability and neurotoxin secretion. Challenge of DLV-transduced MDM cultures with HIV-1(Ba-L) revealed a 4- to 5-fold reduction in viral replication, as measured by p24 antigen production. This effect was associated with the mobilization of the GFP-expressing DLV construct by the wild-type virus. CONCLUSIONS: These data demonstrate the inhibition of HIV-1 replication in primary MDM, by a DLV vector that lacks any anti-HIV-1 transgene. These findings lay the initial groundwork for future studies on the ability of DLV-modified monocytes to introduce anti-HIV-1 genes into the CNS. Lentiviral vector-mediated gene delivery to the CNS by monocytes/macrophages is a promising, emerging strategy for treating neuro-AIDS.

HLA-A2 down-regulation on primary human macrophages infected with an M-tropic EGFP-tagged HIV-1 reporter virus.

Multiple mechanisms are used by the human immunodeficiency virus type 1 (HIV-1) to interfere with host-cell immune effector functions. The 27-kD Nef protein has been shown to down-modulate specific genes of the major histocompatibility complex class I (MHC-I) on the surface of infected primary T cells, facilitating their escape from lysis by cytolytic T lymphocytes. Macrophages, as the other major immune cell type targeted by the virus, also contribute to the transmission, persistence, and pathogenesis of HIV-1. Yet, whether Nef modulates MHC-I expression on HIV-infected primary macrophages remains unclear. Currently available infectious HIV-1 molecular clones, which express a reporter gene, only infect T cells and/or do not express Nef. To overcome these limitations, we generated macrophage-tropic green fluorescent protein (GFP)-tagged HIV-1 viruses, which express the complete viral genome, and used these to assess the expression of human leukocyte antigen (HLA)-A2 on the surface of productively infected macrophages. The reporter viral genomes were replication-competent and stable, as Nef, p24 antigen, and GFP expression could be detected by immunostaining of infected, monocyte-derived macrophages (MDM) after more than 2 months postinfection. Fluorescence-activated cell sorter analyses of infected macrophages and T cells revealed that although wild-type reporter virus infection induced a statistically significant decrease in the density of surface HLA-A2, down-regulation of HLA-A2 was not seen in cells infected with reporter viruses encoding a frameshift or a single point mutation in Nef at prolines 74P and P80. The impact of Nef on HLA-A2 surface expression in MDM was also confirmed by confocal microscopy. These results suggest that the mechanisms of HLA-A2 down-modulation are similar in primary T cells and macrophages.

Circulating proviral HIV DNA and HIV-associated dementia.

OBJECTIVE: Individuals continue to develop HIV-1-associated dementia (HAD) despite treatment with highly active antiretroviral therapy (HAART). Monocytes/macrophages (M/MPhi) can harbor proviral DNA that is not eradicated by HAART. To determine if HAD is associated with the level of HIV-1 infection within circulating leukocytes, we quantified HIV-1 DNA copy number in peripheral blood mononuclear cells (PBMC), and in PBMC subsets. DESIGN: Cross-sectional analysis within the Hawaii Aging with HIV Cohort comparing participants with HAD to those with normal cognition (NC). METHODS: Real-time PCR assays assessing HIV DNA copy number/1 x 10 cells were performed on PBMC and subsets. RESULTS: Individuals with HAD (n = 27) had a median (interquartile range) of 9.11 (37.20) HIV DNA per 1 x 10 PBMC compared to 0.49 (0.89) HIV DNA per 1 x 10 PBMC in individuals with NC (n = 22). Using a univariate analysis in the subset of individuals with undetectable viral load (HAD, n = 11; NC, n = 13), the odds of HAD attributable to HIV DNA copy number was 2.76 (1.28-5.94), P < 0.01. Preliminary analysis of a small subset of patients (n = 5) suggested that the primary source of HIV DNA may be the activated M/MPhi (CD14/CD16) subset. CONCLUSIONS: These findings suggest a potentially important association between circulating provirus and HAD.

Amyloid precursor protein expression in circulating monocytes and brain macrophages from patients with HIV-associated cognitive impairment.

We examined amyloid precursor protein (APP) surface expression on circulating leukocytes and in brain tissues from normal individuals and HIV+ subjects with cognitive impairment. Most monocytes, and a subset of B-lymphocytes, expressed APP, while T-lymphocytes, granulocytes, and natural killer (NK) cells did not. CD14bright/CD16+ monocytes expressed the highest levels, and CD14dim/CD16+ cells were negative, suggesting a relationship with activation. Higher APP+ monocyte levels correlated with increased numbers of CD16+ monocytes, but not with the degree of cognitive impairment. Treatment of monocytes with M-CSF, but not LPS, upregulated APP expression. In the brain, APP appeared as axonal immunoreactivity and diffuse plaques, and APP+ perivascular macrophages were seen in cases with severe dementia. APP may facilitate monocyte entry into the brain.

High-affinity interaction between HIV-1 Vpr and specific sequences that span the C/EBP and adjacent NF-kappaB sites within the HIV-1 LTR correlate with HIV-1-associated dementia.

Numerous host and viral factors likely participate in the onset and progression of HIV-1-associated dementia (HIVD). Previous studies have suggested that viral gene expression in resident central nervous system (CNS) cells of monocyte/macrophage lineage play a central role in the production of neurotoxic viral proteins and infectious virus, deregulation of cellular gene expression, and/or dysfunction of glial and neuronal cell populations. HIV-1 replication is regulated, in part, by interactions between cellular transcription factors and the viral trans-activators, Tat and viral protein R (Vpr), with cis-acting promoter elements within the LTR. We have previously demonstrated that Vpr binds with high affinity to selected sequence configurations within CCAAT/enhancer binding protein (C/EBP) site I and downstream sequences immediately adjacent to this site. Studies reported herein establish a correlation between the diagnosis of HIVD and the increased prevalence of HIV-1 LTRs containing a C/EBP binding site I that exhibits high affinity for Vpr. To this end, the interaction of Vpr with C/EBP site I variants in 47 LTRs from three nondemented patients and 96 LTRs from seven demented patients was examined. Competition electrophoretic mobility shift (EMS) analyses were utilized to examine Vpr binding to oligonucleotide probes containing C/EBP site I variants. We demonstrated that 89% of LTRs derived from patients exhibiting clinical dementia contained C/EBP site I configurations that displayed a high relative affinity for Vpr, while only 11% of LTRs contained C/EBP site I configurations that exhibited a low relative affinity Vpr binding phenotype. In contrast, examination of LTRs derived from patients lacking clinically evident dementia revealed that only 53% of brain-derived LTRs contained C/EBP site I configurations that displayed a high relative affinity for Vpr, while 47% of LTRs contained C/EBP site I configurations that exhibited a low relative affinity Vpr binding phenotype. We propose that sequence-specific interactions between cis-acting elements in the LTR, members of the C/EBP family of transcription factors, and the virion-associated trans-activator protein Vpr play important roles in the pathogenesis of HIVD.

Assessment of HIV-1 DNA copies per cell by real-time polymerase chain reaction.

Measurements of HIV-1 DNA and plasma RNA levels represent unique entities, thus clinically and molecularly, data obtained from each can be used independently in assessing therapy or experiments. Plasma HIV-1 RNA levels are used to make clinical decisions regarding treatment strategies, but viral DNA can still be detectable when plasma RNA levels are undetectable. At the molecular level, accurate assessment of HIV-1 DNA copies/cell could increase the ability to target specific tissues for further analysis such as identification of site-specific integration of HIV in cellular DNA. Using real-time polymerase chain reaction (PCR), HIV-1 copies/cell were determined in peripheral blood mononuclear cells (PBMC), bone marrow (BM), and tissue. Duplicate specimens were analyzed for plasma HIV-1 RNA levels and for viral DNA copies/cell from 24 HIV-1 infected individuals. DNA from an additional 58 PBMC and 34 other tissue specimens were also assayed with the results reported as a log of HIV-1 DNA copies/cell. The log viral DNA copies/cell of the 24 matched specimens ranged from -2.699 to 0.278 with no correlation to the plasma HIV-1 RNA levels (range 52 to 2 X 105 copies/mL). Similar range in log HIV-1 DNA copies/cell was found in the other specimens. Real-time PCR assay for viral DNA copies/cell provides a rapid assessment of HIV-1 copies/cell in specimens independent of plasma HIV-1 RNA levels. From selected cases with relatively high HIV-1 DNA copies/cell, inverse PCR successfully identified viral integration. This type of assay could facilitate further studies when relatively high viral copies/cell are needed for screening.

Region-specific distribution of human immunodeficiency virus type 1 long terminal repeats containing specific configurations of CCAAT/enhancer-binding protein site II in brains derived from demented and nondemented patients.

Previous studies have shown that two CCAAT/enhancer binding protein (C/EBP) binding sites (sites I and II) within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) are critically important for efficient virus replication within cells of the monocyte lineage, a primary cell type infected by HIV-1. Sequence variation at C/EBP sites I and II has been shown to alter the affinity of C/EBP factors to these sites. Specifically, sequence variation within C/EBP binding site II has been shown to alter binding of purified C/EBP beta protein and basal activity of the HIV-1 LTR. We have previously demonstrated that the C/EBP site II consensus cladeB (ConB) variant was highly conserved in brain- and peripheral blood-derived LTRs of individuals with advanced HIV-1 disease. Given these important observations, the regional distribution of LTRs containing the C/EBP site II ConB variant derived from brain tissues of patients with and without HIV-1-associated dementia (HIVD) was examined. A statistically significant difference was found in the distribution of LTRs containing the C/EBP site II ConB variant in brain regions derived from patients with and without HIVD. In addition, we have previously shown that LTRs containing C/EBP site II 4C and 6G variants (designated according to the position at which nucleotide change occurred relative to ConB, followed by the actual nucleotide found at the variant position) were only found in brain tissue of patients with HIVD. As an extension of these observations, the regional distribution of LTRs containing C/EBP site II 4C or 6G variants derived from the brains of patients with HIVD was examined and a statistically significant difference was observed. We have shown that LTRs containing a low-affinity C/EBP site II 4C variant accumulated in the cerebellum. LTRs containing the 4C site variant in conjunction with the consensus cladeB (ConB) site I exhibited the lowest basal LTR activity of any of the LTRs examined. These results suggest that LTRs containing the C/EBP site II 4C configuration may promote the establishment of a latent provirus in the cerebellum, a region of the HIVD brain that exhibits little viral gene expression. Furthermore, LTRs containing a high affinity C/EBP site II 6G variant accumulated in the mid-frontal gyrus, a site of highly productive replication. In addition, LTRs containing the C/EBP site II 6G variant with the ConB at site I exhibited the highest basal LTR activity. In conclusion, distinct LTR populations with specific C/EBP site II configurations were found in different neuroanatomical regions of the brain, potentially due to differences in the molecular architecture of the LTR, viral entry pathways, and/or brain microenvironments.

Human immunodeficiency virus-associated dementia: an evolving disease.

This article reviews the changing epidemiology of HIV-associated dementia, current concepts of the different patterns of dementia under the influence of highly active antiretroviral therapy, and reviews therapeutic aspects.

Structural and functional evolution of human immunodeficiency virus type 1 long terminal repeat CCAAT/enhancer binding protein sites and their use as molecular markers for central nervous system disease progression.

The appearance and progression of human immunodeficiency virus type 1 (HIV-1)-associated pathogenesis in the immune and central nervous systems is dependent on the ability of the virus to replicate in these compartments, which is, in turn, controlled by numerous factors, including viral binding and entry, receptor and coreceptor usage, and regulation of viral expression by the long terminal repeat (LTR). The LTR promotes viral expression in conjunction with viral and cellular regulatory proteins, including members of the CCAAT/enhancer binding protein (C/EBP) family, which modulate LTR activity through at least two cis-acting binding sites. Previous studies have shown that these sites are necessary for HIV-1 replication in cells of the monocyte/macrophage lineage, but dispensable in T lymphocytes. To establish potential links between this important family of transcription factors and HIV-1-associated pathogenesis, C/EBP site I and II sequence variation in peripheral blood mononuclear cell (PBMC)-derived LTRs from HIV-1-infected patients with varying degrees of disease severity was examined. A high prevalence of C/EBP site variants 3T (site I) and consensus B (site II) within PBMC-derived HIV-1 LTRs was shown to correlate with late stage disease in HIV-1-infected patients. These results suggest that the increased prevalence in the PBMCs of HIV-1 LTRs containing the 3T C/EBP site I variant and the consensus B site II variant may serve as a molecular marker for disease progression within the immune system. The relative low or high binding affinity of C/EBP beta to sites I and II in electrophoretic mobility shift (EMS) analyses correlated with low or high LTR activity, respectively, in transient expression analyses during both early and late disease stages. The 3T C/EBP site I was the only variant examined that was not found in LTRs derived from PBMCs of patients at early stages of HIV-1 disease, but was found at increasing frequencies in patients with late stage disease. Furthermore, the 3T C/EBP site I was not found in brain-derived LTRs of patients without HIV-1-associated dementia (HIVD), but was found in increasing numbers in brain-derived LTRs from patients diagnosed with HIVD. The C/EBP site I 3T variant appears to be exclusive to patients progressing to increasingly severe HIV-1-associated immunologic and neurologic disease.

Follicular dendritic cell-mediated up-regulation of CXCR4 expression on CD4 T cells and HIV pathogenesis.

Follicular dendritic cells (FDCs) represent a major reservoir of HIV, and active infection occurs surrounding these cells, suggesting that this microenvironment is highly conducive to virus transmission. Because CD4 T cells around FDCs in germinal centers express the HIV coreceptor, CXCR4, whereas CD4 lymphocytes in many other sites do not, it prompted the hypothesis that FDCs may increase CXCR4 expression on CD4 T cells, thereby facilitating infection. To test this, HIV receptor/coreceptor expression was determined on CD4 T cells cultured with or without FDCs, and its consequence to infection was assessed by measuring virus binding and entry. FDCs had little effect on CCR5 or CD4 expression but increased CXCR4 expression on CD4 T cells. FDC-mediated up-regulation of CXCR4 on CD4 T cells occurred by 24 h and was sustained for at least 96 h in vitro, and FDC-CD4 T cell contact was necessary. Importantly, increased CXCR4 expression directly correlated with increased binding and entry of HIV-1 X4 isolates. Furthermore, CD4(+)CD57(+) germinal center T cells expressed high levels of CXCR4 and supported enhanced entry of X4 HIV compared with other CD4 T cells from the same tissue. Thus, in addition to serving as a reservoir of infectious virus, FDCs render surrounding germinal center T cells highly susceptible to infection with X4 isolates of HIV-1.

Follicular dendritic cell contributions to HIV pathogenesis.

Early after infection, large quantities of HIV are trapped on follicular dendritic cells (FDCs) thus establishing a potent reservoir of infectious virus adjacent to highly susceptible CD4-bearing T lymphocytes. Throughout much of the disease course, active HIV infection is largely confined to sites surrounding FDCs suggesting that this microenvironment is highly conducive to infection. FDCs maintain HIV infectivity and trapped virus can cause infection even in the presence of neutralizing antibody. FDCs also contribute signaling to the germinal center microenvironment that appears to increase HIV infection and replication. This article discusses these FDC contributions to HIV pathogenesis.

Proliferating cellular nuclear antigen expression as a marker of perivascular macrophages in simian immunodeficiency virus encephalitis.

Brain perivascular macrophages are a major target of simian immunodeficiency virus (SIV) infection in rhesus macaques and HIV infection in humans. Perivascular macrophages are distinct from parenchymal microglia in their location, morphology, expression of myeloid markers, and turnover in the CNS. In contrast to parenchymal microglia, perivascular macrophages are continuously repopulated by blood monocytes, which undergo maturation to macrophages on entering the central nervous system (CNS). We studied differences in monocyte/macrophages in vivo that might account for preferential infection of perivascular macrophages by SIV. In situ hybridization for SIV and proliferating cellular nuclear antigen (PCNA) immunohistochemistry demonstrated that SIV-infected and PCNA-positive cells were predominantly found in perivascular cuffs of viremic animals and in histopathological lesions that characterize SIV encephalitis (SIVE) in animals with AIDS. Multilabel techniques including double-label immunohistochemistry and combined in situ hybridization and immunofluorescence confocal microscopy revealed numerous infected perivascular macrophages that were PCNA-positive. Outside the CNS, SIV-infected, PCNA-expressing macrophage subpopulations were found in the small intestine and lung of animals with AIDS. While PCNA is used as a marker of cell proliferation it is also strongly expressed in non-dividing cells undergoing DNA synthesis and repair. Therefore, more specific markers for cell proliferation including Ki-67, topoisomerase IIalpha, and bromodeoxyuridine (BrdU) incorporation were used which indicated that PCNA-positive cells within SIVE lesions were not proliferating. These observations are consistent with perivascular macrophages as terminally differentiated, non-dividing cells and underscores biological differences that could potentially define mechanisms of preferential, productive infection of perivascular macrophages in the rhesus macaque model of neuroAIDS. These studies suggest that within CNS and non-CNS tissues there exist subpopulations of macrophages that are SIV-infected and express PCNA.

Insights into the role of immune activation in HIV neuropathogenesis.

How does HIV infection lead to the development of central nervous system disease? Central to this question is identification of the relative contributions of (1) the virus, (2) its host cells, and (3) secondary or downstream events to the pathological process. These are re-examined in this brief review. Also, a greater appreciation for the role of systemic events in neuroinflammation is emerging, with likely relevance to HIV-associated dementia. We propose here a model for HIV neuropathogenesis that highlights the role of systemic monocyte activation and subsequent neuroinvasion in initiating the disease.