rAAV-mediated overexpression of FGF-2 promotes cell proliferation, survival, and alpha-SMA expression in human meniscal lesions.

Meniscal tears are a common problem in sports medicine. Direct application of therapeutic vectors derived from the adeno-associated virus might be beneficial to enhance meniscal repair. We tested the hypothesis that overexpression of fibroblast growth factor 2 (FGF-2) through recombinant adeno-associated virus (rAAV) vectors leads to detectable metabolic changes in human meniscal fibrochondrocytes and in human meniscal defects. rAAV-mediated gene transfer was investigated for its ability to promote FGF-2 secretion in human meniscal fibrochondrocytes in vitro, in intact human meniscal explants in situ, and in experimentally created human meniscal lesions. Effects of the treatment on cell proliferation and survival, extracellular matrix synthesis, and expression of the alpha-smooth muscle actin (alpha-SMA) contractile marker were monitored using biochemical, immunohistochemical, histological, and histomorphometric analyses. Efficient production of FGF-2 through rAAV could be achieved in vitro and in situ, both in the intact and injured meniscus. Application of the candidate FGF-2 vector allowed for enhanced cell proliferation and survival compared with control transduction, in particular in areas with poor healing capacity and in sites of injury, consistent with the mitogenic activities of the growth factor. Remarkably, a significant reduction of the amplitude of meniscal tears was noted after FGF-2 treatment, with increased levels of alpha-SMA expression. In contrast, there was no significant stimulation of synthesis of the major extracellular matrix components when the candidate vector was applied and instead, a decrease in the matrix/DNA contents was reported, in good agreement with the properties of FGF-2. Such a direct gene-based approach may have value in options aiming at treating human meniscal defects.

The calcium sensing receptor is directly involved in both osteoclast differentiation and apoptosis.

Intracellular transduction pathways that are dependent on activation of the CaR by Ca(o)2+ have been studied extensively in parathyroid and other cell types, and include cytosolic calcium, phospholipases C, A2, and D, protein kinase C isoforms and the cAMP/protein kinase A system. In this study, using bone marrow cells isolated from CaR-/- mice as well as DN-CaR-transfected RAW 264.7 cells, we provide evidence that expression of the CaR plays an important role in osteoclast differentiation. We also establish that activation of the CaR and resultant stimulation of PLC are involved in high Ca(o)2+-induced apoptosis of mature rabbit osteoclasts. Similar to RANKL, Ca(o)2+ (20 mM) appeared to trigger rapid and significant nuclear translocation of NF-kappaB in a CaR- and PLC-dependent manner. In summary, our data suggest that stimulation of the CaR may play a pivotal role in the control of both osteoclast differentiation and apoptosis in the systems studied here through a signaling pathway involving activation of the CaR, phospholipase C, and NF-kappaB.

Extracellular calcium sensing in rat aortic vascular smooth muscle cells.

Extracellular calcium (Ca(2+)(o)) can act as a first messenger in many cell types through a G protein-coupled receptor, calcium-sensing receptor (CaR). It is still debated whether the CaR is expressed in vascular smooth muscle cells (VSMCs). Here, we report the expression of CaR mRNA and protein in rat aortic VSMCs and show that Ca(2+)(o) stimulates proliferation of the cells. The effects of Ca(2+)(o) were attenuated by pre-treatment with MAPK kinase 1 (MEK1) inhibitor, as well as an allosteric modulator, NPS 2390. Furthermore, stimulation of the VSMCs with Ca(2+)(o)-induced phosphorylation of ERK1/2, but surprisingly did not cause inositol phosphate accumulation. We were not able to conclusively state that the CaR mediates Ca(2+)(o)-induced cell proliferation. Rather, an additional calcium-sensing mechanism may exist. Our findings may be of importance with regard to atherosclerosis, an inflammatory disease characterized by abnormal proliferation of VSMCs and high local levels of calcium.

Selective gene expression in brain microglia mediated via adeno-associated virus type 2 and type 5 vectors.

Microglia represent a crucial cell population in the central nervous system, participating in the regulation and surveillance of physiological processes as well as playing key roles in the etiologies of several major brain disorders. The ability to target gene transfer vehicles selectively to microglia would provide a powerful new approach to investigations of mechanisms regulating brain pathologies, as well as enable the development of novel therapeutic strategies. In this study, we evaluate the feasibility of specifically and efficiently targeting microglia relative to other brain cells, using vectors based on two different serotypes of adeno-associated virus (AAV) carrying cell-type-specific transcriptional elements to regulate gene expression. Among a set of promoter choices examined, an element derived from the gene for the murine macrophage marker F4/80 was the most discriminating for microglia. Gene expression from vectors controlled by this element was highly selective for microglia, both in vitro and in vivo. To our knowledge, this is the first demonstration of selective expression of transferred genes in microglia using AAV-derived vectors, as well as the first utilization of recombinant AAV-5 vectors in any macrophage lineage. These results provide strong encouragement for the application of these vectors and this approach for delivering therapeutic and other genes selectively to microglia.

Calcium-sensing receptor stimulates PTHrP release by pathways dependent on PKC, p38 MAPK, JNK, and ERK1/2 in H-500 cells.

Elevated extracellular calcium ([Ca2+]o) and other agonists potentially acting via the calcium-sensing receptor (CaR) increase parathyroid hormone-related peptide (PTHrP) release from H-500 Leydig cells. Here, we provide strong evidence for the CaR's involvement by using a dominant negative CaR that attenuates high [Ca2+]o-induced PTHrP release. This effect is likely transcriptional, because high [Ca2+]o upregulates the PTHrP transcript, an effect that is abolished by actinomycin D. Regulation of PTHrP release by the CaR involves activation of PKC as well as ERK1/2, p38 MAPK, and JNK pathways. However, we show for the first time that high [Ca2+]o-induced activation of the stress-activated protein kinase SEK1 is PKC independent, because there is an additive effect of a PKC inhibitor in combination with the JNK inhibitor on [Ca2+]o-stimulated PTHrP release. Furthermore, high [Ca2+]o, in a PKC-independent fashion, induces phosphorylation of ERK1/2, SEK1, p38 MAPK, and its downstream transcription factor ATF-2. We conclude that CaR regulation of PTHrP release in H-500 cells involves activation of PKC as well as the ERK1/2, p38 MAPK, and JNK pathways.

Facilitation of affiliation and pair-bond formation by vasopressin receptor gene transfer into the ventral forebrain of a monogamous vole.

Behaviors associated with monogamy, including pair-bond formation, are facilitated by the neuropeptide vasopressin and are prevented by a vasopressin receptor [V1a receptor (V1aR)] antagonist in the male prairie vole. The neuroanatomical distribution of V1aR dramatically differs between monogamous and nonmonogamous species. V1aR binding is denser in the ventral pallidal region of several unrelated monogamous species compared with nonmonogamous species. Because the ventral pallidum is involved in reinforcement and addiction, we hypothesize that V1aR activation in this region promotes pair-bond formation via a mechanism similar to conditioning. Using an adeno-associated viral vector to deliver the V1aR gene, we increased the density of V1aR binding in the ventral pallial region of male prairie voles. These males exhibited increased levels of both anxiety and affiliative behavior compared with control males. In addition, males overexpressing the V1aR in the ventral pallidal region, but not control males, formed strong partner preferences after an overnight cohabitation, without mating, with a female. These data demonstrate a role for ventral pallidal V1aR in affiliation and social attachment and provide a potential molecular mechanism for species differences in social organization.

Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy.

Antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial targeting, an absence of drug resistance. To promote tumor regression and to maintain dormancy, antiangiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. As a step toward this goal, we have generated recombinant adeno-associated virus (rAAV) vectors that carry genes coding for angiostatin, endostatin, and an antisense mRNA species against vascular endothelial growth factor (VEGF). These rAAVs efficiently transduced three human tumor cell lines tested. Transduction with an rAAV-encoding antisense VEGF mRNA inhibited the production of endogenous tumor cell VEGF. Conditioned media from cells transduced with this rAAV or with rAAV-expressing endostatin or angiostatin inhibited capillary endothelial cell proliferation in vitro. Antiangiogenic rAAVs may offer a novel gene therapy approach to undermining tumor neovascularization and cancer progression.

Adeno-associated virus vector-mediated transgene integration into neurons and other nondividing cell targets.

The site-specific integration of wild-type adeno-associated virus (wtAAV) into the human genome is a very attractive feature for the development of AAV-based gene therapy vectors. However, knowledge about integration of wtAAV, as well as currently configured recombinant AAV (rAAV) vectors, is limited. By using a modified Alu-PCR technique to amplify and sequence the vector-cellular junctions, we provide the first direct evidence both in vitro and in vivo of rAAV-mediated transgene integration in several types of nondividing cells, including neurons. This novel technique will be highly useful for further delineating the mechanisms underlying AAV-mediated integration, including issues of frequency, site preference, and DNA rearrangement in human as well as animal cells. Results from these studies should be beneficial for the development of the next generation of gene delivery vectors.

Potent inhibition of human immunodeficiency virus type 1 in primary T cells and alveolar macrophages by a combination anti-Rev strategy delivered in an adeno-associated virus vector.

The rate of viral replication appears to play a pivotal role in human immunodeficiency virus type 1 (HIV-1) pathogenesis and disease progression as it outstrips the capacity of the immune system to respond. Important cellular sites for HIV-1 production include T lymphocytes and tissue macrophages. Antiviral strategies, including newer treatment modalities such as gene therapy of HIV-1-susceptible cell populations, must be capable of engendering durable inhibitory effects to HIV-1 replication in both of these primary cell types in order to be effective. Among the potential genetic targets for intervention in the HIV-1 life cycle, the Rev regulatory system, consisting of Rev and its binding site, the Rev-responsive element (RRE), stands out as particularly attractive. Rev is essential for maintaining the stability of the viral genomic RNA as well as viral mRNAs encoding key structural and regulatory proteins. Moreover, it exhibits favorable threshold kinetics, in that Rev concentrations must rise above a critical level to exert their effect. To disable Rev function, primary T cells or macrophages were transduced with anti-Rev single-chain immunoglobulin (SFv) or RRE decoy genes either singly or in combination by employing adeno-associated virus vectors and then challenged with HIV-1. By directing both a protein and a nucleic acid against the normal interaction between Rev and the RRE, this genetic antiviral strategy effectively inhibited infection by either clinical or laboratory virus isolates. These results provide a framework for novel interventions to reduce virus production in the infected host.

Differential effects of interleukin-13 on cytomegalovirus and human immunodeficiency virus infection in human alveolar macrophages.

Alveolar macrophages, which form a principal line of defense against a variety of pulmonary pathogens, may themselves be infected by viruses like human immunodeficiency virus-1 (HIV-1), which impair their defensive functions. Interleukin-13 (IL-13), a multifunctional cytokine, has been considered for therapeutic use based on its potent inhibition of HIV-1 in these cells. We have further examined the effects of IL-13 on alveolar macrophages under conditions that reflect those seen in acquired immune deficiency syndrome, where this cell type is often infected by the opportunistic pathogen human cytomegalovirus (HCMV). Alveolar macrophages exposed to both HCMV and HIV-1 consistently exhibited higher levels of HIV-1 replication than cells exposed to HIV-1 alone. HIV-1 production was strongly suppressed in alveolar macrophages treated with IL-13 regardless of whether or not the cultures were coinfected with HCMV. However, IL-13 treatment markedly enhanced the expression of HCMV in otherwise latently infected macrophages in a dose dependent manner. These unexpected differential effects of IL-13 on host-virus interactions are important considerations in guiding its potential therapeutic applications.

Characterization of an envelope mutant of HIV-1 that interferes with viral infectivity.

A mutant human immunodeficiency virus (HIV-1) provirus encoding an envelope (Env) protein with a truncated transmembrane protein cytoplasmic domain was defective for replication. Coexpression of the mutant with a wild-type (wt) HIV-1 provirus potently inhibited the production of infectious virus. The maximum inhibitory effect was reached when the ratio of mutant to wt proviral DNA was 2:1. This transdominant defect in infectivity conferred by the mutant Env did not appear to involve the late steps of virus replication, since the synthesis, precursor processing, and intracellular transport of the Env proteins were not blocked; nor did it prevent the incorporation of the envelope proteins into virions or the subsequent release of the virus. Although the mutant Env protein still retained syncytia-forming ability, the truncated protein was unable to mediate cell-to-cell transmission of the virus. Moreover, coexpression with the mutant effectively inhibited the ability of the wt Env to mediate cell-to-cell transmission. The mutant Env protein formed a complex with the wt protein when they were coexpressed, producing heterooligomeric structures which appeared to be severely defective in an early, post-CD4 binding step of the virus life cycle despite the inclusion of wt Env in the complexes.

Direct cytotoxicity of HIV-1 envelope protein gp120 on human NT neurons.

A new in vitro system comprising a pure population of neurons, human NT cells, was used to characterize the direct neurotoxic effect of HIV-1 envelope protein gp120. Cytotoxicity was monitored by a quantitative assay after exposure to recombinant gp120 in the presence or absence of other reagents. Treatment of mature NT neurons with various doses of gp120 for 24 h caused a decrease of up to 27% in the number of viable cells. This neurotoxicity was abolished by co-treatment with either D-2-amino-5-phosphonopentanoic acid (APV), MK801 or nimodipine, or by culturing cells in a Ca(2+)-free environment. Taken together, these data indicate that gp120 exerts a direct neurotoxic effect by acting through NMDA receptors and Ca2+ channels.

Efficient transduction of human neurons with an adeno-associated virus vector.

An adeno-associated virus vector containing a lacZ gene driven by a CMV immediate-early promoter (AAV beta-gal) was evaluated with respect to its transduction efficiency and integration ability in nondividing human NT neurons. A dose-dependent pattern in transduction efficiency of the AAV beta-gal was demonstrated immunocytochemically, with up to 100% of the neurons expressing the gene product. No neurotoxic effects of the vector were detected. Quantitative PCR analyses of high molecular weight cellular DNA from the transduced neurons indicated that the copy number of the AAV beta-gal genome increased gradually in a time dependent manner, suggesting a slow but progressive rate of vector integration over a period of approximately 1 week following transduction. Equal or greater transduction efficiency of the AAV beta-gal into NT neurons than into a standard target cell line indicated that the NT neurons were readily susceptible to AAV-mediated gene transfer. This study demonstrates that AAV-based vectors can efficiently transduce and stably express a foreign gene in post-mitotic human neurons.

HIV-1 Tat modulates invasion by a bacterial enteric pathogen into a human intestinal cell line.

OBJECTIVE: To explore the possibility that an HIV-1 gene product may modulate entry of an invasive enteric pathogen into a terminally differentiated human intestinal cell line. HIV-1 Tat was selected for investigation because of its unique ability to cross cell membranes. METHODS: After transient transfection of HT29-C1 cells with plasmids containing HIV-1 long terminal repeat (LTR)-lacZ plus a Tat expression cassette, or with a pSR-lacZ control plasmid, bacterial invasion assays were performed on both groups of cells utilizing a clinical Salmonella isolate. Assays were performed concurrently on a control group of non-transfected cells. A second series of experiments compared bacterial invasion into cells transfected with the Tat expression vector alone versus cells transfected with either an isogenic expression vector that did not make Tat, or with pSR-lacZ. Finally, the ability of exogenous Tat protein to transactivate an HIV-1 LTR-chloramphenicol acetyltransferase (CAT) plasmid which had been transfected into HT29-C1 cells and to modulate Salmonella invasion was also assessed. RESULTS: HT29-C1 cells transfected with a Tat expression vector, either alone or in combination with another plasmid, were significantly less susceptible to bacterial invasion than cells that either did not undergo transfection, were transfected with an otherwise isogenic expression vector without Tat, or transfected with an unrelated plasmid. Duplicate experiments also demonstrated that exogenous purified Tat protein transactivated an HIV-1 LTR-CAT plasmid which had been transfected into HT29-C1 cells and inhibited Salmonella invasion compared with unexposed cells. CONCLUSION: HIV-1 Tat inhibits Salmonella invasion of a human enterocyte cell line whether the protein is expressed intracellularly or provided exogenously.

Characterization of a Human Stromal Cell Line Supporting Hematopoietic Progenitor Cell Proliferation: Effect of HIV Expression.

Our objective was to determine the role that bone marrow-derived stromal cells have on human hematopoiesis in HIV infection. In particular, we dissected the heterogeneous bone marrow microenvironment to study the effect HIV expression might have on the cell population capable of producing the cytokines which will support human CD34+ cell differentiation. A stromal cell line, Lof(11-10), was established from human bone marrow by transfecting a plasmid containing the SV40 large T-antigen and isolating foci exhibiting a transformed phenotype. The Lof(11-10) cell line was characterized to determine its susceptibility to HIV infection, to identify its cytokine production profile, and to test the ability of conditioned media from this line to support CD34+ cell differentiation in the presence and absence of HIV expression. Nine cytokines were detected by RT-PCR and ELISA analysis. Conditioned media obtained from the Lof(11-10) cell line was able to support CD34+ celle differentiation. However, because the Lof(11-10) cells are not infectible by HIV, molecular clones of HIV were introduced into these cells by transfection. There was no qualitative difference in the levels of cytokine production between HIV-expressing and control Lof(11-10) cells. Furthermore, conditioned media derived from HIV-expressing and control Lof(11-10) cells added to bone marrow-derived CD34+ progenitor cells yielded similar colony formation in methylcellulose assays. Our data suggest that HIV infection of the cytokine-producing cells within the bone marrow microenvironment, as represented by the Lof(11-10) cell line, results in both normal cytokine production and hematopoiesis in spite of HIV expression. This report adds to the evidence against stromal cells being a significant target of HIV and establishes a system for comparison with more relevant models. Copyright 1995 S. Karger AG, Basel

Biology of HIV-1 and treatment strategies.

Effective vaccines and treatments for HIV-1, the retrovirus responsible for AIDS, continue to be elusive. Many of the obstacles to more effective intervention can be traced to special properties of the virus itself. Although great progress has been made in our understanding of the biology of the virus, important gaps remain regarding how HIV-1 functions as an agent of disease. The poor track record of traditional approaches to date has also spurred the development and testing of novel strategies, such as gene therapy, for the treatment of HIV-1 disease.

Allelic variation in the effects of the nef gene on replication of human immunodeficiency virus type 1.

The effects of the viral gene nef on human immunodeficiency virus type 1 (HIV-1) replication in culture were investigated using nef alleles of the HIV-1 IIIB and ELI strains. The results demonstrate significant allelic variation in the effect of nef on virus replication in both an established human CD4+ T-cell line and primary human lymphocytes. In the context of the HXB2 virus, the ELI nef allele but not the IIIB nef allele permits initiation of efficient low-multiplicity infection in primary peripheral blood mononuclear cells, including unfractionated peripheral blood lymphocytes, T cells, and monocyte/macrophages. Within the same genetic context, the IIIB nef allele slightly retards replication of the virus in a T-cell line, whereas the ELI nef allele accelerates replication of the virus. Sequences in the IIIB and ELI genomes outside of nef also moderate the effects of nef on HIV-1 replication. nef did not appear to determine the host-cell preference of the virus. These studies may help to reconcile apparently conflicting reports on the role of nef in HIV-1 replication and suggest that HIV-1 nef may play an important role in viral pathogenesis.

Replication of human immunodeficiency virus type 1 in primary dendritic cell cultures.

The ability of the human immunodeficiency virus type 1 (HIV-1) to replicate in primary blood dendritic cells was investigated. Dendritic cells compose less than 1% of the circulating leukocytes and are nondividing cells. Highly purified preparations of dendritic cells were obtained using recent advances in cell fractionation. The results of these experiments show that dendritic cells, in contrast to monocytes and T cells, support the active replication of all strains of HIV-1 tested, including T-cell tropic and monocyte/macrophage tropic isolates. The dendritic cell cultures supported much more virus production than did cultures of primary unseparated T cells, CD4+ T cells, and adherent as well as nonadherent monocytes. Replication of HIV-1 in dendritic cells produces no noticeable cytopathic effect nor does it decrease total cell number. The ability of the nonreplicating dendritic cells to support high levels of replication of HIV-1 suggests that this antigen-presenting cell population, which is also capable of supporting clonal T-cell growth, may play a central role in HIV pathogenesis, serving as a source of continued infection of CD4+ T cells and as a reservoir of virus infection.

Mechanisms of infectivity and replication of HIV-1 and implications for therapy.

Human immunodeficiency virus type 1 (HIV-1), a retrovirus, is the etiologic agent of AIDS. Like all retroviruses, the viral genes are carried in the viral particle in the form of single-stranded RNA. Once inside a susceptible host cell, this RNA template is reverse-transcribed by virally supplied enzyme functions into a DNA copy, which becomes integrated permanently into the host's own genetic material. The genome of HIV-1, comprising approximately 10,000 bases, is much more complex than those of classic retroviruses, encoding a minimum of six gene products in addition to the gag, pol, and env genes characteristic of all retroviruses. These genes encode regulatory functions that act at diverse points in the virus life cycle. Together, they provide HIV-1 with an exceptional ability to modulate its replication depending on its host environment. This characteristic is reflected in the different stages presented by the disease and the diverse behaviors of the virus in different types of host cells. A greater understanding of the mechanics of this regulation and the factors that influence it may someday permit therapeutic intervention in the disease process that will halt virus replication and the progression of pathology in infected individuals.

Identification of HIV-1 vpr product and function.

To investigate the role of vpr (viral protein R) in the replication and cytopathicity of human immunodeficiency virus type 1 (HIV-1), infectious proviruses were constructed that were isogenic except for the ability to produce the protein product of vpr. The experiments described here demonstrate that vpr encodes a 96 amino acid 15 kDa protein. The vpr product increases the rate of replication and accelerates the cytopathic effect of the virus in T cells. Vpr acts in trans to increase levels of viral protein expression. The stimulatory effect of vpr is observed to act on the HIV-1 LTR as well as on several heterologous promoters.