Regulation of nitric oxide synthase activity in human immunodeficiency virus type 1 (HIV-1)-infected monocytes: implications for HIV-associated neurological disease.

Mononuclear phagocytes (monocytes, macrophages, and dendritic cells) play major roles in human immunodeficiency virus (HIV) persistence and disease pathogenesis. Macrophage antigen presentation and effector cell functions are impaired by HIV-1 infection. Abnormalities of macrophage effector cell function in bone marrow, lung, and brain likely result as a direct consequence of cellular activation and HIV replication. To further elucidate the extent of macrophage dysfunction in HIV-1 disease, a critical activation-specific regulatory molecule, nitric oxide (NO.), which may contribute to diverse pathology, was studied. Little, if any, NO. is produced by uninfected human monocytes. In contrast, infection with HIV-1 increases NO. production to modest, but significant levels (2-5 microM). Monocyte activation (with lipopolysaccharide, tumor necrosis factor alpha, or through interactions with astroglial cells) further enhances NO. production in HIV-infected cells, whereas its levels are diminished by interleukin 4. These results suggest a possible role for NO. in HIV-associated pathology where virus-infected macrophages are found. In support of this hypothesis, RNA encoding the inducible NO synthase (iNOS) was detected in postmortem brain tissue from one pediatric AIDS patient with advanced HIV encephalitis. Corresponding iNOS mRNA was not detected in brain tissue from five AIDS patients who died with less significant brain disease. These results demonstrate that HIV-1 can influence the expression of NOS in both cultured human monocytes and brain tissue. This newly described feature of HIV-macrophage interactions suggests previously unappreciated mechanisms of tissue pathology that result from productive viral replication.

Oxidative stress and neuroAIDS: triggers, modulators and novel antioxidants.

Neurological disorders represent one of the most common disturbances accompanying HIV infection. In the past few years, highly antiretroviral active therapy has significantly reduced the incidence of HIV-related diseases. However, neurological dysfunction in AIDS patients still remains an unresolved problem. Oxidative stress, which occurs in brain tissues of patients undergoing HIV infection and is implicated in cell death of both astroglia and neurones, has recently been suggested to play a role in the pathogenesis of neuroAIDS. Thus, a better understanding of the processes that trigger and modulate free radical formation in brain tissues of AIDS patients might help in a successful therapeutic approach to the neuropathogenesis of HIV infection.

Induction of cyclooxygenase-2 expression during HIV-1-infected monocyte-derived macrophage and human brain microvascular endothelial cell interactions.

Human immunodeficiency virus type-1 (HIV-1)-associated dementia (HAD) is a neurodegenerative disease characterized by HIV infection and replication in brain tissue. HIV-1-infected monocytes overexpress inflammatory molecules that facilitate their entry into the brain. Prostanoids are lipid mediators of inflammation that result from cyclooxygenase-2 (COX-2) activity. Because COX-2 is normally induced during inflammatory processes, the aim of this study was to investigate whether COX-2 expression is up-regulated during monocyte-brain endothelium interactions. In vitro cocultures of HIV-infected macrophages and brain endothelium showed an up-regulation of COX-2 expression by both cell types. This up-regulation occurs via an interleukin-1beta (IL1beta)-dependent mechanism in macrophages and via an IL-1beta-independent mechanism in endothelial cells. Thus, interactions between HIV-infected monocytes and brain endothelium result in COX-2 expression and, as such, might contribute to the neuropathogenesis of HIV infection.

Cellular aspects of HIV-1 infection of macrophages leading to neuronal dysfunction in in vitro models for HIV-1 encephalitis.

HIV-1 is a hematogenously spread virus that most likely gains entry into the brain within blood-derived macrophages. Indeed, productive viral replication selectively occurs within perivascular and parenchymal blood-derived macrophages and microglia and HIV-infected macrophages have increased potential to bind and transmigrate through the blood-brain barrier. Once inside the brain, HIV-infected macrophages secrete a variety of pro-inflammatory mediators that display neuromodulatory and neurotoxic activities in several in vitro models for HIV-1 encephalitis. The final outcome regarding neuronal function and cell loss is regulated through intercellular interactions between these virus-infected cells and astrocytes. In this regard, both HIV-induced intracellular events in macrophages and interactions between HIV-infected macrophages and brain cells are reviewed as factors that might lead to neuronal injury in in vitro model systems for HIV-1 encephalitis.

The neuropathogenesis of HIV-1 infection.

HIV infection in brain revolves around productive viral replication in cells of mononuclear phagocyte lineage, including brain macrophages, microglia, and multinucleated giant cells [1-4]. Together, they are the investigators for cellular and viral neurotoxic activities [5-10]. Several published reports show that viral and/or cellular products produced from HIV-1-infected macrophages injure neurons and induce glial proliferation during advancing central nervous system (CNS) infection [11-18]. These findings are supported by the apparent discrepancy between the distribution and numbers of virus-infected cells and concomitant brain tissue pathology [5, 19]. Whether these soluble factors are indirectly responsible for neuronal damage remains undefined. The identification and regulation of neurotoxins produced from HIV-infected macrophages are central to uncovering how HIV mediates CNS disease. The authors who contributed to this work represent laboratories with overlapping areas of expertise. Broad-based complementary hypotheses regarding HIV neuropathogenesis are now provided.

Role for oxygen radicals in self-sustained HIV-1 replication in monocyte-derived macrophages: enhanced HIV-1 replication by N-acetyl-L-cysteine.

N-acetyl-L-cysteine (NAC) has been proposed as a therapeutic agent for AIDS patients because it reduces human immunodeficiency virus type 1 (HIV-1) replication in stimulated T cells. However, NAC and glutathione enhanced acute HIV-1 replication in monocyte-derived macrophages. Buthionine sulfoximine did not affect NAC-mediated enhanced HIV-1 replication, indicating that the NAC-mediated effects are glutathione-independent. Superoxide dismutase and the hydroxyl radical scavengers dimethylthiourea and thiourea, but not urea, inhibited acute HIV-1 replication in macrophages. NAC reduced ferricytochrome c and increased dose-dependently Fe(III)-citrate and Fe(III)-EDTA-catalyzed hydroxyl radical formation in a system using glucose and glucose oxidase. Dimethylthiourea and thiourea, but not urea and superoxide dismutase, dose-dependently inhibited NAC-mediated enhancement of HIV-1 replication. These data suggest that oxygen radicals play an important role in self-sustained HIV-1 replication in macrophages and that oxygen radical scavengers other than NAC should be considered as therapeutic agents for AIDS patients.

N-acetyl-L-cysteine-induced up-regulation of HIV-1 gene expression in monocyte-derived macrophages correlates with increased NF-kappaB DNA binding activity.

Nuclear factor kappaB (NF-kappaB) is an important cellular regulator of human immunodeficiency virus (HIV) gene expression. In T cells, N-acetyl-L-cysteine (NAC) inhibits the induction of NF-kappaB and transcription of HIV-1. However, NAC up-regulates HIV-1 replication in monocyte-derived macrophages (MDM). In this study we demonstrate that NAC treatment of MDM transfected with a chloramphenicol acetyltransferase (CAT) construct under transcriptional control of the HIV-1 long terminal repeat resulted in an up-regulation of CAT activity. Furthermore, MDM transfected with a HIV-1-NF-kappaB-CAT construct also produced increased CAT activity after NAC treatment. In addition, electrophoretic mobility shift assays revealed that nuclei of NAC-treated MDM contained increased binding activity to wild-type, but not mutant, kappaB oligonucleotides. Components of the binding activity were identified with antibodies as the NF-kappaB subunits p50 and p65. These data indicate that NAC-induced enhancement of HIV-1 replication in MDM is regulated at the level of viral gene expression and mediated by NF-kappaB.

Enhancement of HIV-1 replication in peripheral blood mononuclear cells by Cryptococcus neoformans is monocyte-dependent but tumour necrosis factor-independent.

OBJECTIVE: To investigate the possible role of Cryptococcus neoformans in HIV-1 pathogenesis. DESIGN: An in vitro system was developed to study HIV-1 replication in freshly HIV-1-infected peripheral blood mononuclear cells (PBMC) incubated with whole azide-killed C. neoformans. METHODS: Human PBMC or peripheral blood lymphocytes were infected with lymphocytotropic HIV-1 and incubated with azide-killed encapsulated or non-encapsulated C. neoformans for 10 days. Viral replication was followed by HIV-1 p24 enzyme-linked immunosorbent assay and median tissue culture infective dose determination. Tumour necrosis factor (TNF) release by PBMC, induced by C. neoformans, was measured. Anti-TNF monoclonal antibodies or pentoxifylline were used to inhibit TNF bioactivity. RESULTS: Both encapsulated and non-encapsulated C. neoformans enhanced HIV-1 replication in PBMC but not in peripheral blood lymphocytes. C. neoformans induced TNF release by PBMC. Inhibition of TNF bioactivity did not block C. neoformans-enhanced HIV-1 replication in PBMC. CONCLUSIONS: C. neoformans can enhance HIV-1 replication in T cells only in the presence of monocytic cells. This enhancement is not dependent on encapsulation nor can it be attributed to TNF release.

Antibodies and complement enhance binding and uptake of HIV-1 by human monocytes.

OBJECTIVE: To characterize antibody- and complement-mediated binding and uptake of HIV-1 by human monocytes. DESIGN: The first step in the infection of the monocyte by HIV-1 is binding of the virus to the susceptible cell. Procedures were designed to assess the influence of anti-HIV-1 antibodies and complement on this binding, and to study the process of internalization following binding. METHODS: Human monocytes were incubated with fluorescein-labelled purified HTLV-IIIB virions and human sera with high-titre anti-HIV-1 antibodies and/or complement. Binding and uptake of virus by the monocytes was measured as fluorescence per cell by flow cytometry. RESULTS: Binding of purified HIV-1 to monocytes was increased by complement and, to a lesser extent, by anti-HIV-1 antibodies. Uptake of HIV-1 bound to the monocyte appeared to be mediated by antibodies and was increased further by the presence of complement. Complement alone, however, resulted in the uptake of only a small part of the bound virus. CONCLUSIONS: Complement significantly increases the binding of HIV-1 to human monocytes, and a combination of antibodies and complement efficiently mediates uptake of HIV-1 by monocytes.

Enhanced expression of fractalkine in HIV-1 associated dementia.

The CX(3)C chemokine fractalkine was found to be up-regulated in the brain during inflammatory processes. In this study, we tried to assess the role of fractalkine in HIV-1-associated dementia. Fractalkine expression is up-regulated in the brains of AIDS patients with HAD. Fractalkine immunoreactivity was mainly detected in astrocytes. In addition, fractalkine expression was found to be up-regulated in cocultures of astrocytes and HIV-infected macrophages. This up-regulation was dependent on cell-cell contact. We propose that fractalkine produced during interactions between astrocytes and HIV-infected macrophages plays a role in HAD by regulating the trafficking of monocytic cells in the brain parenchyma.

Overexpression of nerve growth factor and basic fibroblast growth factor in AIDS dementia complex.

Although neurotrophic factors are currently considered as treatment for neurodegenerative diseases, little is still known about their presence in the central nervous system under pathological conditions. We investigated the expression of the neurotrophic molecules NGF, bFGF, BDNF and IGF-1 in brain tissue of patients suffering from AIDS dementia complex. In contrast to IGF-1 and BDNF, NGF and bFGF mRNA levels were significantly elevated. Strong NGF immunoreactivity was found in perivascular areas and was colocalized with infiltrating macrophages, whereas intense bFGF staining was found in cells with characteristic astrocytic morphology. These data suggest that the induction of NGF and bFGF alone appears to be insufficient as a compensatory mechanism to prevent ADC.

Intracellular pathways involved in TNF-alpha and superoxide anion release by Abeta(1-42)-stimulated primary human macrophages.

In this study, the intracellular signal transduction pathways leading to the production of TNF-alpha and superoxide anions by amyloid-beta-stimulated primary human monocyte-derived macrophages was investigated. Using Western blotting and specific inhibitors it is shown that both ERK 1/2 and p38 MAPK signal transduction pathways as well as PKC are involved in the amyloid-beta-stimulated superoxide anion production. In contrast, only ERK 1/2 MAPK seems to be involved in TNF-alpha production: questioning the connection between PKC and ERK 1/2 activation. Our results suggest the use of ERK 1/2 MAPK inhibitors in the prevention of macrophage activation in the context of Alzheimer's disease.

The amino-terminus of the amyloid-beta protein is critical for the cellular binding and consequent activation of the respiratory burst of human macrophages.

Here, we show that amyloid-beta (Abeta) is capable to prime and activate the respiratory burst of human macrophages. Previously, the N-terminus of Abeta(1-42) has been shown to contain a cell binding domain that is implicated in eliciting neuropathogenic microglia in vitro. To evaluate the role of this domain in the Abeta(1-42)-induced respiratory burst activity, the effect of Abeta subfragments on the Abeta(1-42)-induced superoxide release were studied. On the basis of the antagonistic properties of Abeta(1-16), it is concluded that the N-terminal region of Abeta is critical for the cellular binding and consequent activation of the respiratory burst of human phagocytes.

Anti-HIV effect of iron chelators: different mechanisms involved.

BACKGROUND: Drugs for the treatment of AIDS have been directed to specific events in the human immunodeficiency virus (HIV-1) life cycle, aimed to stop viral replication by inhibition of reverse transcriptase or protease activity. Studies showing that oxidative stress and iron may be important in the activation of HIV-1 have focused attention on the potential therapeutic use of iron chelators. OBJECTIVES: The goal of this review is to describe several possibilities as to how iron is involved in the replication of HIV and how iron chelation may interfere in this process. STUDY DESIGN: First some physico-chemical properties of iron concerning solubility, oxidation-reduction potential, catalysis, and chelation will be discussed. In the second part, the role of iron in various biochemical systems is explained. RESULTS: Nuclear factor kappa B (NF-kappaB) activation, regulating proviral transcription, can be influenced by iron through the production of reactive oxygen species. A second route by which iron chelation could influence HIV replication, is by inhibition of DNA synthesis through inactivation of iron-dependent ribonucleotide reductase. Another strategy which can be employed in targeting iron chelators against HIV-1, is direct oxidative viral RNA/DNA attack. This could be achieved by bleomycin, a cytostatic agent with the ability to form a complex with DNA and RNA. CONCLUSION: Chelation may withhold iron from viral metabolism but on the other hand may also favor catalysis of reactive oxygen species directed to viral constituents. In combination with existing antivirals, iron chelation could add to improve the treatment of HIV-disease.

Potential role of CCR5 polymorphism in the development of AIDS dementia complex.

The chemokine receptor CCR5 and to a lesser extent CCR2b and CCR3 have been shown to serve as coreceptors for HIV-1 entry into macrophages. Individuals that are homozygous for a defective CCR5 allele (DeltaCCR5) are highly, but not fully, resistant to infection with HIV-1. Here, we want to emphasize the importance of DeltaCCR5 in in vitro as well as in vivo studies. We provide data that suggest that CCR5 polymorphism may affect the onset of AIDS dementia complex in vivo and data that show that HIV-1 replication is influenced by the DeltaCCR5 allele in vitro. Knowing the CCR5 genotype of an individual will help to better interpret research results and may even provide new information about mechanisms of disease.

The chemotherapeutic agent bleomycin in a two-drug combination with zidovudine, ritonavir or indinavir synergistically inhibits HIV Type-1 replication in peripheral blood lymphocytes.

It has been suggested that the combination of cancer chemotherapy with antiviral therapy is helpful for the containment of lymphomas in HIV-infected patients. Since we have recently shown that the nucleic acid binding chemotherapeutic agent bleomycin in itself has antiviral properties, we looked to see if there was any possible synergy with current anti-HIV agents. Combinations of zidovudine, indinavir or ritonavir with bleomycin, synergistically inhibited HIV-1(AT) replication in stimulated peripheral blood lymphocytes (combination index at 50% virus inhibition was 0.427, 0.604 and 0.535, respectively) and this synergism was not accompanied by any synergistic effects on cytotoxicity. We conclude from these data that further studies to investigate the clinical efficacy of combinations of antiviral and cancer chemotherapeutic agents are warranted in relation to viral load improvement.

Role of the pro-inflammatory cytokines TNF-alpha and IL-1beta in HIV-associated dementia.

Human immunodeficiency virus-1 (HIV-1)-infected and immune-activated macrophages and microglia secrete neurotoxins. Two of these neurotoxins are the pro-inflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), which are thought to play a major role in inducing neuronal death. Both TNF-alpha and IL-1beta increase the permeability of the blood-brain barrier, through which subsequently HIV-infected monocytes can enter the brain. They both induce over-stimulation of the NMDA-receptor via several pathways, resulting in a lethal neuronal increase in Ca(2+) levels. Additionally, TNF-alpha co-operates with several other proinflammatory mediators to enhance their toxic effects. Although most research has focused on the neurotoxic effects of TNF-alpha and IL-1beta in HAD, there is also evidence that these cytokines can be neuroprotective. In this paper the effect of TNF-alpha and IL-1beta on neuronal life and death in HAD is discussed.

Interactions between macrophages and brain microvascular endothelial cells: role in pathogenesis of HIV-1 infection and blood - brain barrier function.

Monocytes have been shown to infiltrate in brain tissue during various neurological disorders including AIDS dementia complex. The presence of an excess of activated macrophages in brain tissue is accompanied by tissue damage resulting in a loss in neuronal function and viability. Therapeutic options against such neurological disorders could therefore be aimed at the prevention of monocyte infiltration across the blood - brain barrier. Therefore, a better understanding of these processes is needed. Recent insights in cellular processes between monocytes/macrophages and brain microvascular endothelial cells in the neuropathogenesis of HIV-1 infection demonstrate that monocytes roll on endothelial cells via the inducible endothelial adhesion molecule E-selectin. Binding of these cells are mainly mediated via the endothelial adhesion molecule vascular cell adhesion molecule-1. The transmigration through the blood - brain barrier is facilitated by both endothelial and monocyte/macrophage-derived nitric oxide and by the increased production of gelatinase B activity by HIV-infected monocytes/macrophages. Chemokines produced within the brain regulate the traffic of the infiltrating monocytes through the brain parenchyma. In addition, endothelial cells also produce monocyte attracting chemokines during their first interactions with HIV-infected monocytes/macrophages thus promoting additional influx of phagocytes into the brain. Furthermore, excessive infiltration of monocytes is accompanied by endothelial damage resulting in the loss of tight junctions. Thus, in toto, brain microvascular endothelial cells might contribute to the neuropathogenesis of HIV-1 infection.

Unraveling the neuroimmune mechanisms for the HIV-1-associated cognitive/motor complex.

Infection of the brain with human immunodeficiency virus 1 (HIV-1) often leads to the devastating loss of mental faculties. Surprisingly, HIV-1 elicits such brain dysfunction without significantly infecting neurons, astrocytes and oligodendroglia. The target for HIV-1 in the brain is the macrophage, which usually functions as a phagocytic, antigen-presenting and immune-regulatory cell. How can these cells produce such serious cognitive and motor brain impairments? Here, Hans Nottet and Howard Gendelman propose that HIV-1 penetrates the blood-brain barrier inside differentiating macrophages, which become immune activated once inside the brain, and secrete high levels of neurotoxins. Chronic, subclinical disease results by astrocyte regulation of macrophage effector functions. Ultimately, endogenous control mechanisms break down, leading to motor and mental impairments in some affected subjects.

Christiaan Eijkman. First bacteriologist at Utrecht University, Nobel laureate for his work on vitamins.

One century ago, Christiaan Eijkman was appointed Professor of Bacteriology at the Utrecht University, The Netherlands. Despite his appointment to teach bacteriology, Christiaan Eijkman made his main contribution to medical science not in bacteriology but in nutrition. He discovered that Beri-Beri was not an infection but a nutritient deficiency (later called vitamin-deficiency) and was awarded the Nobel Prize for Medicine in 1929 for these observations. These landmark studies were made in the former Dutch East Indies. Interestingly, the results of his studies were presented in the Dutch language, in the medical journal of the Dutch East Indies. As a professor of Bacteriology, his work was the beginning of an important school in biochemistry in The Netherlands.