Dominique Dormont

Obtention and Characterization of Primary Astrocyte and Microglial Cultures From Adult Monkey Brains

Simple methods for obtention of primary cultures of isolated astrocytes and microglia from adult simian brain have been developed. Characterization of these two glial cell populations were performed by morphological observations and by immunocytochemistry. The astroglial cultures were obtained by an indirect method. After L‐leucine methyl‐ester treatment and trypsinizations, more than 99% of cells expressed glial fibrillary acidic protein (GFAP), whereas no macrophages or microglia could be detected. Likely, the 1% remaining cells were immature astrocytes or cells that lost their GFAP expression. Cultured simian astrocytes expressed vimentin, laminin, and fibronectin. We also found a constitutively low expression of major histocompatibility complex (MHC) class II by cultured astrocytes which was significantly enhanced by lipopolysaccharide (LPS), interferon gamma (IFN‐γ), or tumor necrosis factor alpha (TNF‐α) treatments. Microglial cultures were obtained by a direct method of isolation using Percoll gradient separations and compared to simian monocyte‐derived macrophages or alveolar macrophages. Microglial cells differed from macrophages by their proliferation upon granulocyte‐macrophage colony stimulating factor (GM‐CSF) treatment and by their typical morphology when observed by scanning electron microscopy. As macrophages, they expressed in vitro CD68, CD64, CD14, CD11b, MHC class II, and fibronectin. However, contrary to macrophages, simian cultured microglia expressed laminin. This observation suggests that microglia represent a new potential source of this extracellular matrix protein in the brain. J. Neurosci. Res. 49:576–591, 1997.

Modulations of Cytokine Expression in Pregnant Women

PROBLEM: Although the overall anti‐infectious and anti‐parasitic immunity of parous women appears normal, several aspects of maternal cell‐mediated and humoral immunity are altered during pregnancy. This has been suggested to occur via preferential local and systemic secretion of Th‐2 type cytokines, which down‐regulate or prevent secretion/action of Th‐1 type cytokines, in animals as well as in humans.

Expression of excitatory amino acid transporter‐2 (EAAT‐2) and glutamine synthetase (GS) in brain macrophages and microglia of SIVmac251‐infected macaques

Na+‐dependent transporters for glutamate (excitatory amino acid transporters, EAATs) clear extracellular glutamate in the brain and prevent excitotoxic neuronal damage. Glutamine synthetase (GS) provides metabolic support for neurones by producing the neurotrophic amino acid glutamine. EAAT and GS expression has recently been demonstrated in macrophages and microglial cells inu2003vitro, and in two models of acute inflammation inu2003vivo. This observation might modify our current understanding of brain inflammation, which considers activated microglia and brain macrophages as the main neurotoxic cells through their production of a variety of neurotoxins, including glutamate. EAAT and GS expression by these cells would entail neuroprotective and neurotrophic properties, counterbalancing the deleterious consequences of microglial activation.

Regulated Expression of Sodium-dependent Glutamate Transporters and Synthetase: a Neuroprotective Role for Activated Microglia and Macrophages in HIV Infection?

It is now widely accepted that neuronal damage in HIV infection results mainly from microglial activation and involves apoptosis, oxidative stress and glutamate‐mediated neurotoxicity. Glutamate toxicity acts via 2 distinct pathways: an excitotoxic one in which glutamate receptors are hyperactivated, and an oxidative one in which cystine uptake is inhibited, resulting in glutathione depletion and oxidative stress. A number of studies show that astrocytes normally take up glutamate, keeping extracellular glutamate concentration low in the brain and preventing excitotoxicity. This action is inhibited in HIV infection, probably due to the effects of inflammatory mediators and viral proteins. Other in vitro studies as well as in vivo experiments in rodents following mechanical stimulation, show that activated microglia and brain macrophages express high affinity glutamate transporters. These data have been confirmed in chronic inflammation of the brain, particularly in SIV infection, where activated microglia and brain macrophages also express glu‐tamine synthetase. Recent studies in humans with HIV infection show that activated microglia and brain macrophages express the glutamate transporter EAAT‐1 and that expression varies according to the disease stage. This suggests that, besides their recognized neurotoxic properties in HIV infection, these cells also have a neuroprotective function, and may partly make up for the inhibited astrocytic function, at least temporarily. This hypothesis might explain the discrepancy between microglial activation which occurs early in the disease, and neuronal apoptosis and neuronal loss which is a late event. In this review article, we discuss the possible neuro‐protective and neurotrophic roles of activated microglia and macrophages that may be generated by the expression of high affinity glutamate transporters and glutamine synthetase, 2 major effectors of glial glutamate metabolism, and the implications for HIV‐induced neuronal dysfunction, the underlying cause of HIV dementia.

Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brains.

In the brain, granulocyte‐macrophage colony stimulating factor (GM‐CSF) may be released by infiltrated cells of the immune system including T and B lymphocytes and mononuclear phagocytes, but also by nervous system resident cells such as microglia and astrocytes. Astrocyte‐secreted GM‐CSF may play an important role in enhancing the local inflammatory response to central nervous system (CNS) injury and in recruting microglia and activated macrophages. In this study, we demonstrated that GM‐CSF, as TNFα and IL 6, stimulates in vitro proliferation of simian astrocytes in primary cultures. Results were confirmed by blocking experiments performed with a specific neutralizing mAb directed against GM‐CSF. Furthermore, we demonstrated that GM‐CSF mediates its effect on these cells through the α subunit of the GM‐CSF receptor which is constitutively expressed at the membrane of the cultured simian astrocytes as assessed by immunofluorescence. GM‐CSF effects on astrocytes could be involved in astrocytosis, a hallmark of various neurological injuries and in inflammatory processes in an autocrine manner.

Prion diseases: pathogenesis and public health concerns.

Transmissible spongiform encephalopathy (TSE) agents or prions induce neurodegenerative fatal diseases in humans and in some mammalian species. Human TSEs include Creutzfeldt–Jakob disease (CJD), Gerstmann–Sträussler–Scheinker syndrome, kuru and fatal familial insomnia. In animals, scrapie in sheep and goats, feline spongiform encephalopathy, transmissible mink encephalopathy, chronic wasting disease in wild ruminants, and bovine spongiform encephalopathy (BSE), which appeared in the UK in the mid‐1980s [Wells, G.A.H. et al. (1987) Vet. Rec. 121, 419–420], belong to the TSE group. Prions have biological and physicochemical characteristics that differ significantly from those of other microorganisms; for example, they are resistant to inactivation processes that are effective against conventional viruses, including those that alter nucleic acid structure or function. Alternatively, infectivity is highly susceptible to procedures that modify protein conformation. Today, the exact nature of prions remains unknown even though it is likely that they consist of protein only. At the biochemical level, TSEs are characterised by the accumulation, within the central nervous system of the infected individual, of an abnormal isoform of a particular protein from the host, the prion protein [Prusiner, S.B. (1982) Science 216, 136–144]. TSEs are transmissible among their species of origin, but they can also cross the species barrier and induce chronic infection and/or disease in other species. Transmissibility has been proven in natural situations such as the outbreak of CJD among patients treated with pituitary‐derived hormones and the appearance of BSE that affected UK cattle in the mid‐1980s.

Simian immunodeficiency virus mac251 infection of astrocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes has been demonstrated in the brains of patients with AIDS dementia complex (ADC) and may play an important role in neuropathological pathways of HIV-related encephalopathy. SIVmac-infected monkeys develop an acquired immunodeficiency syndrome (AIDS) with CNS involvement which is quite similar to that seen in human AIDS. We investigated the in vitro infection of primary astrocytes derived from adult macaques with SIVmac251 or an isogenic virus that expresses a non-functional Nef protein (SIVmac251-DeltaNef). In both cases we observed that viral expression was mostly limited to early regulatory genes after a transient phase of late viral gene expression (i.e. env and gag), as reported for HIV-1-infected astrocytes in vivo. Late viral gene expression could be reactivated by TNF-alpha, GM-CSF and IFN-gamma treatment of SIVmac251-infected astrocytes but not by similarly treated SIVmac251-DeltaNef-infected cells. Our findings suggest that Nef is not involved in the restricted expression of SIV in astrocytes, but may be important for astrocytes to function as a viral reservoir in the CNS. In additional experiments, we demonstrated Rev and Nef expression in 17 of 27 primary astrocyte cultures derived from macaques infected by SIVmac251. Nef was located in the cytoplasm of astrocytes infected by SIVmac251 in vivo, but displayed perinuclear localisation after infection in vitro. Attempts to activate late viral gene expression by astrocytes infected in vivo using cytokines or by coculture with human cord blood mononuclear cells were unsuccessful.

PMS-601, a New Platelet-Activating Factor Receptor Antagonist That Inhibits Human Immunodeficiency Virus Replication and Potentiates Zidovudine Activity in Macrophages

ABSTRACT We assessed the anti-human immunodeficiency virus (anti-HIV) activity in vitro of new platelet-activating factor (PAF) receptor antagonists, as PAF and viral replication are thought to be involved in HIV neuropathogenesis. We found that PMS-601 inhibited proinflammatory cytokine synthesis and HIV replication in macrophages and potentiated the antiretroviral activity of zidovudine. These results suggest that PMS-601 is of potential value as an adjuvant treatment for HIV infection.

Mechanisms of opsonized HIV entry in normal B lymphocytes

Using our in vitro model of normal B cell infection that functions with low doses of HIV but requires virus opsonization by seropositive patient serum, and complement, we analyzed what receptors allowed virus entry. Here, we show that HIV infection of B cells occurs through 2 major receptors: the CD4 antigen and the CR1/CR2 complex. These 2 pathways work independently since a complete inhibition of virus entry requires both CD4 and CD21/CD35 blockade on CD4dim tonsillar B cells whereas only the latter is critical on CD4‐negative B cells.

Nitric oxide synthesis during acute SIVmac251 infection of macaques

Summary During HIV1 infection, nitric oxide (NO) could significantly contribute to immune dysregulation by its multiple effects on the modulation of the host immune response. The in vivo regulation of NO production is attributable to several nitric oxide synthases, one of which is a cytokine-inducible enzyme (iNOS). In vitro experiments suggest that iNOS expression in macrophages may be directly modulated by HIV infection. Acute infection of macaques with a pathogenic strain of the simian immunodeficiency virus (SIV) represents a relevant animal model for the in vivo study of the relationships between iNOS expression and lentiviral replication. Indeed, acute infection in this model is characterized by high rates of viral replication associated with early cytokine dysregulations, in the absence of opportunistic infection. In our experiment, two cynomolgus macaques were inoculated intravenously with a pathogenic isolate of SIVmac251, and iNOS gene expression was investigated ex vivo during acute infection in mononuclear cells obtained from bronchoalveolar lavage (BALMCs). An enhancement of this gene expression was observed as early as the second week of infection, at the time of peak of systemic viraemia, and increased until day 31 p.i. This overexpression was concomitant with a marked linear increase in IFNγ expression in BALMCs. At the time of systemic viral load peak, the production of NO in plasma of these two monkeys was evidenced by the detection of large amounts of nitrate.