Harris Goldstein

CCL2/Monocyte Chemoattractant Protein-1 Mediates Enhanced Transmigration of Human Immunodeficiency Virus (HIV)-Infected Leukocytes across the Blood–Brain Barrier: A Potential Mechanism of HIV–CNS Invasion and NeuroAIDS

Encephalitis and dementia associated with acquired immunodeficiency syndrome (AIDS) are characterized by leukocyte infiltration into the CNS, microglia activation, aberrant chemokine expression, blood–brain barrier (BBB) disruption, and eventual loss of neurons. Little is known about whether human immunodeficiency virus 1 (HIV-1) infection of leukocytes affects their ability to transmigrate in response to chemokines and to alter BBB integrity. We now demonstrate that HIV infection of human leukocytes results in their increased transmigration across our tissue culture model of the human BBB in response to the chemokine CCL2, as well as in disruption of the BBB, as evidenced by enhanced permeability, reduction of tight junction proteins, and expression of matrix metalloproteinases (MMP)-2 and MMP-9. HIV-infected cells added to our model did not transmigrate in the absence of CCL2, nor did this condition alter BBB integrity. The chemokines CXCL10/interferon-gamma-inducible protein of 10 kDa, CCL3/macrophage inflammatory protein-1α, or CCL5/RANTES (regulated on activation normal T-cell expressed and secreted) did not enhance HIV-infected leukocyte transmigration or BBB permeability. The increased capacity of HIV-infected leukocytes to transmigrate in response to CCL2 correlated with their increased expression of CCR2, the chemokine receptor for CCL2. These data suggest that CCL2, but not other chemokines, plays a key role in infiltration of HIV-infected leukocytes into the CNS and the subsequent pathology characteristic of NeuroAIDS.

Methamphetamine inhibits antigen processing, presentation, and phagocytosis.

Methamphetamine (Meth) is abused by over 35 million people worldwide. Chronic Meth abuse may be particularly devastating in individuals who engage in unprotected sex with multiple partners because it is associated with a 2-fold higher risk for obtaining HIV and associated secondary infections. We report the first specific evidence that Meth at pharmacological concentrations exerts a direct immunosuppressive effect on dendritic cells and macrophages. As a weak base, Meth collapses the pH gradient across acidic organelles, including lysosomes and associated autophagic organelles. This in turn inhibits receptor-mediated phagocytosis of antibody-coated particles, MHC class II antigen processing by the endosomal–lysosomal pathway, and antigen presentation to splenic T cells by dendritic cells. More importantly Meth facilitates intracellular replication and inhibits intracellular killing of Candida albicans and Cryptococcus neoformans, two major AIDS-related pathogens. Meth exerts previously unreported direct immunosuppressive effects that contribute to increased risk of infection and exacerbate AIDS pathology.

Enhancement of HIV-1 infection by the capsular polysaccharide of Cryptococcus neoformans

Patients with AIDS who become infected with Cryptococcus neoformans have a poor prognosis. We speculated that the presence of cryptococcal capsular polysaccharide may enhance HIV-1 infection. In an in-vitro study, the presence of cryptococcal polysaccharide significantly increased (p less than 0.05) production of p24 antigen after infection of H9 cells with HIV-1-infected H9 cells. We also found similar results when lymphocytes from an HIV-1-infected patient were co-cultured with mononuclear cells from an uninfected individual. Our findings suggest a new pathogenic role for the capsular polysaccharide--namely, the capacity to enhance HIV-1 infectivity.

Lentiviral vectors encoding human immunodeficiency virus type 1 (HIV-1)-specific T-cell receptor genes efficiently convert peripheral blood CD8 T lymphocytes into cytotoxic T lymphocytes with potent in vitro and in vivo HIV-1-specific inhibitory activity.

ABSTRACT The human immunodeficiency virus type 1 (HIV-1)-specific CD8 cytotoxic T-lymphocyte (CTL) response plays a critical role in controlling HIV-1 replication. Augmenting this response should enhance control of HIV-1 replication and stabilize or improve the clinical course of the disease. Although cytomegalovirus (CMV) or Epstein-Barr virus (EBV) infection in immunocompromised patients can be treated by adoptive transfer of ex vivo-expanded CMV- or EBV-specific CTLs, adoptive transfer of ex vivo-expanded, autologous HIV-1-specific CTLs had minimal effects on HIV-1 replication, likely a consequence of the inherently compromised qualitative function of HIV-1-specific CTLs derived from HIV-1-infected individuals. We hypothesized that this limitation could be circumvented by using as an alternative source of HIV-1-specific CTLs, autologous peripheral CD8+ T lymphocytes whose antigen specificity is redirected by transduction with lentiviral vectors encoding HIV-1-specific T-cell receptor (TCR) α and β chains, an approach used successfully in cancer therapy. To efficiently convert peripheral CD8 lymphocytes into HIV-1-specific CTLs that potently suppress in vivo HIV-1 replication, we constructed lentiviral vectors encoding the HIV-1-specific TCR α and TCR β chains cloned from a CTL clone specific for an HIV Gag epitope, SL9, as a single transcript linked with a self-cleaving peptide. We demonstrated that transduction with this lentiviral vector efficiently converted primary human CD8 lymphocytes into HIV-1-specific CTLs with potent in vitro and in vivo HIV-1-specific activity. Using lentiviral vectors encoding an HIV-1-specific TCR to transform peripheral CD8 lymphocytes into HIV-1-specific CTLs with defined specificities represents a new immunotherapeutic approach to augment the HIV-1-specific immunity of infected patients.

Inhibition of In Vivo HIV Infection in Humanized Mice by Gene Therapy of Human Hematopoietic Stem Cells with a Lentiviral Vector Encoding a Broadly Neutralizing Anti-HIV Antibody

ABSTRACT Due to the inherent immune evasion properties of the HIV envelope, broadly neutralizing HIV-specific antibodies capable of suppressing HIV infection are rarely produced by infected individuals. We examined the feasibility of utilizing genetic engineering to circumvent the restricted capacity of individuals to endogenously produce broadly neutralizing HIV-specific antibodies. We constructed a single lentiviral vector that encoded the heavy and light chains of 2G12, a broadly neutralizing anti-HIV human antibody, and that efficiently transduced and directed primary human B cells to secrete 2G12. To evaluate the capacity of this approach to provide protection from in vivo HIV infection, we used the humanized NOD/SCID/γcnull mouse model, which becomes populated with human B cells, T cells, and macrophages after transplantation with human hematopoietic stem cells (hu-HSC) and develops in vivo infection after inoculation with HIV. The plasma of the irradiated NOD/SCID/γcnull mice transplanted with hu-HSC transduced with the 2G12-encoding lentivirus contained 2G12 antibody, likely secreted by progeny human lymphoid and/or myeloid cells. After intraperitoneal inoculation with high-titer HIV-1JR-CSF, mice engrafted with 2G12-transduced hu-HSC displayed marked inhibition of in vivo HIV infection as manifested by a profound 70-fold reduction in plasma HIV RNA levels and an almost 200-fold reduction in HIV-infected human cell numbers in mouse spleens, compared to control hu-HSC-transplanted NOD/SCID/γcnull mice inoculated with equivalent high-titer HIV-1JR-CSF. These results support the potential efficacy of this new gene therapy approach of using lentiviral vectors encoding a mixture of broadly neutralizing HIV antibodies for the treatment of HIV infection, particularly infection with multiple-drug-resistant isolates.

A novel action of minocycline: Inhibition of human immunodeficiency virus type 1 infection in microglia

Human immunodeficiency virus type 1 (HIV-1) infection of the brain produces a characteristic disease called acquired immunodeficiency syndrome (AIDS) dementia in which productive infection and inflammatory activation of microglia and macrophages play a central role. In this report, the authors demonstrate that minocycline (MC), a second-generation tetracycline with proven safety and penetration to the central nervous system, potently inhibited viral production from microglia. Inhibition of viral release was sustained through the entire course of infection and even when the drug exposure was limited to the first day of infection. Minocycline was effective even at low viral doses, and against R5- and X4R5-HIV, as well as in single-cycle reporter virus assays. Electrophoretic mobility shift analysis showed that minocycline inhibited nuclear factor (NF)-κB activation in microglia. HIV-1 long terminal repeat (LTR)-promoter activity in U38 cells was also inhibited. These results, combined with recently demonstrated in vivo anti-inflammatory effects of MC on microglia, suggest a potential utility for MC as an effective adjunct therapy for AIDS dementia.

Human Immunodeficiency Virus Type 1 Infection Increases the In Vivo Capacity of Peripheral Monocytes To Cross the Blood-Brain Barrier into the Brain and the In Vivo Sensitivity of the Blood-Brain Barrier to Disruption by Lipopolysaccharide

ABSTRACT Human immunodeficiency virus type 1 (HIV-1), introduced into the brain by HIV-1-infected monocytes which migrate across the blood-brain barrier (BBB), infects resident macrophages and microglia and initiates a process that causes HIV-1-associated neurocognitive disorders. The mechanism by which HIV-1 infection circumvents the BBB-restricted passage of systemic leukocytes into the brain and disrupts the integrity of the BBB is not known. Circulating lipopolysaccharide (LPS), which can compromise the integrity of the BBB, is significantly increased in HIV-1-infected individuals. We hypothesized that HIV-1 infection increases monocyte capacity to migrate across the BBB, which is further facilitated by a compromise of BBB integrity mediated by the increased systemic LPS levels present in HIV-1-infected individuals. To investigate this possibility, we examined the in vivo BBB migration of monocytes derived from our novel mouse model, JR-CSF/EYFP mice, which are transgenic for both a long terminal repeat-regulated full-length infectious HIV-1 provirus and ROSA-26-regulated enhanced yellow fluorescent protein. We demonstrated that JR-CSF/EYFP mouse monocytes displayed an increased capacity to enter the brain by crossing either an intact BBB or a BBB whose integrity was partially compromised by systemic LPS. We also demonstrated that the JR-CSF mouse BBB was more susceptible to disruption by systemic LPS than the control wild-type mouse BBB. These results demonstrated that HIV-1 infection increased the ability of monocytes to enter the brain and increased the sensitivity of the BBB to disruption by systemic LPS, which is elevated in HIV-1-infected individuals. These mice represent a new in vivo system for studying the mechanism by which HIV-1-infected monocytes migrate into the brain.

CD4-Specific Transgenic Expression of Human Cyclin T1 Markedly Increases Human Immunodeficiency Virus Type 1 (HIV-1) Production by CD4+ T Lymphocytes and Myeloid Cells in Mice Transgenic for a Provirus Encoding a Monocyte-Tropic HIV-1 Isolate

ABSTRACT Human immunodeficiency virus type 1 (HIV-1)-encoded Tat provides transcriptional activation critical for efficient HIV-1 replication by interacting with cyclin T1 and recruiting P-TEFb to efficiently elongate the nascent HIV transcript. Tat-mediated transcriptional activation in mice is precluded by species-specific structural differences that prevent Tat interaction with mouse cyclin T1 and severely compromise HIV-1 replication in mouse cells. We investigated whether transgenic mice expressing human cyclin T1 under the control of a murine CD4 promoter/enhancer cassette that directs gene expression to CD4+ T lymphocytes and monocytes/macrophages (hu-cycT1 mice) would display Tat responsiveness in their CD4-expressing mouse cells and selectively increase HIV-1 production in this cellular population, which is infected primarily in HIV-1-positive individuals. To this end, we crossed hu-cycT1 mice with JR-CSF transgenic mice carrying the full-length HIV-1JR-CSF provirus under the control of the endogenous HIV-1 long terminal repeat and demonstrated that human cyclin T1 expression is sufficient to support Tat-mediated transactivation in primary mouse CD4 T lymphocytes and monocytes/macrophages and increases in vitro and in vivo HIV-1 production by these stimulated cells. Increased HIV-1 production by CD4+ T lymphocytes was paralleled with their specific depletion in the peripheral blood of the JR-CSF/hu-cycT1 mice, which increased over time. In addition, increased HIV-1 transgene expression due to human cyclin T1 expression was associated with increased lipopolysaccharide-stimulated monocyte chemoattractant protein 1 production by JR-CSF mouse monocytes/macrophages in vitro. Therefore, the JR-CSF/hu-cycT1 mice should provide an improved mouse system for investigating the pathogenesis of various aspects of HIV-1-mediated disease and the efficacies of therapeutic interventions.

Stromal-Derived Factor 1 Expression in the Human Thymus

Stromal-derived factor-1 (SDF-1), the only known ligand for the chemokine receptor CXCR4, is broadly expressed in cells of both the immune and central nervous systems, and it can induce the migration of resting leukocytes and hemopoietic progenitors. SDF-1 mRNA was previously detected in human thymus-derived stromal cells, but its role in thymopoiesis was unknown. Here we show that SDF-1 is expressed in medullar epithelial cells forming Hassall’s corpuscles (HC). In search of the cell type that may be attracted by SDF-1+ cells in the medulla, we determined that dendritic cells (DC) could be found in situ in close proximity to SDF-1+ epithelial cells in HC. In HIV-1-infected SCID-hu thymuses, DC contained apoptotic cells and were located within enlarged HC. It was further demonstrated that uptake of apoptotic thymocytes by immature DC induced an increase in CXCR4 expression and SDF-1-mediated chemotaxis. Our data suggest a role for SDF-1 in the elimination of apoptotic thymocytes.

GM-CSF and M-CSF modulate β-chemokine and HIV-1 expression in microglia

Significant numbers of patients with acquired immunodeficiency syndrome (AIDS) develop CNS infection primarily in macrophages and microglial cells. Therefore, the regulation of human immunodeficiency virus type 1 (HIV‐1) infection and activation of the brain mononuclear phagocytes subsequent to infection are important areas of investigation. In the current report, we studied the role of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and macrophage‐CSF (M‐CSF) in the expression of antiviral β‐chemokines and HIV‐1 p24 in cultures of primary human fetal microglia. We found that stimulation with GM‐CSF or M‐CSF induced macrophage inflammatory proteins (MIP‐1α and MIP‐1β) and augmented RANTES expression, after HIV‐1 infection of microglia. This was not due to the effect of GM‐CSF on viral expression because GM‐CSF was neither necessary nor stimulatory for viral infection, nor did GM‐CSF enhance the expression of env‐pseudotyped reporter viruses. Blocking GM‐CSF‐induced microglial proliferation by nocodazole had no effect on β‐chemokine or p24 expression. The functional significance of the GM‐CSF‐induced β‐chemokines was suggested by the finding that, in the presence of GM‐CSF, exogenous β‐chemokines lost their anti‐HIV‐1 effects. We further show that although HIV‐1‐infected microglia produced M‐CSF, they failed to produce GM‐CSF. In vivo, GM‐CSF expression was localized to activated astrocytes and some inflammatory cells in HIV‐1 encephalitis, suggesting paracrine activation of microglia through GM‐CSF. Our results demonstrate a complex interplay between CSFs, chemokines, and virus in microglial cells and may have bearing on the interpretation of data derived in vivo and in vitro. GLIA 39:174–183, 2002.