Detection of HIV-1 Gene Sequences in Hippocampal Neurons Isolated from postmortem AIDS Brains by Laser Capture Microdissection

University of Miami, كورال غيبلز، فلوريدا, Florida, United States
Journal of Neuropathology and Experimental Neurology (Impact Factor: 3.8). 09/2001; 60(9):885-92. DOI: 10.1093/jnen/60.9.885
Source: PubMed


We employed laser capture microdissection to remove individual pyramidal neurons from the CA1, CA3, and CA4 regions of formalin-fixed, paraffin-embedded hippocampus from 8 AIDS brains and 2 HIV-1-seronegative normal brains. We amplified HIV-1 gag and nef gene sequences using separate, double round PCR reactions for each of the primer sets. In all 3 hippocampal regions, amplification efficiency was best with sequence length between 284 and 324 bp; HIV-1 nef gene sequences were more common than HIV-1 gag sequences; and rank order for percent positive amplification was CA3 > CA4 > CA1 samples. These results are the first to detect HIV-1 gene sequences in microdissected human tissue. They indicate that brain neurons in vivo contain HIV-1 DNA sequences consistent with latent infection by this virus, and suggest that neurons display a selective vulnerability for HIV infection. Neuronal HIV infection could contribute to neuronal injury and death or act as a potential viral reservoir if reactivated.

Download full-text


Available from: Jorge E Torres-Muñoz
  • Source
    • "Human immunodeficiency virus (HIV) infection within the central nervous system (CNS) causes a variety of neuropathological changes. Neurons are not generally thought to be infected by HIV, although latent neuron infection has been reported (Trillo-Pazos et al. 2003, Torres-Munoz et al. 2001). Neuropathology is instead mediated by direct neurotoxic actions of released viral proteins, or secondarily, through toxic effects orchestrated by glial cells (Kaul et al. 2001, Gendelman et al. 1994, Persidsky & Gendelman 2003, Hauser et al. 2007, Brack-Werner 1999, Kramer-Hammerle et al. 2005b). "
    [Show abstract] [Hide abstract]
    ABSTRACT: J. Neurochem. (2010) 114, 97–109. Human immunodeficiency virus (HIV)-1 neuropathology results from collective effects of viral proteins and inflammatory mediators on several cell types. Significant damage is mediated indirectly through inflammatory conditions promulgated by glial cells, including microglia that are productively infected by HIV-1, and astroglia. Neural and glial progenitors exist in both developing and adult brains. To determine whether progenitors are targets of HIV-1, a multi-plex assay was performed to assess chemokine/cytokine expression after treatment with viral proteins transactivator of transcription (Tat) or glycoprotein 120 (gp120). In the initial screen, ten analytes were basally released by murine striatal progenitors. The beta-chemokines CCL5/regulated upon activation, normal T cell expressed and secreted, CCL3/macrophage inflammatory protein-1α, and CCL4/macrophage inflammatory protein-1β were increased by 12-h exposure to HIV-1 Tat. Secreted factors from Tat-treated progenitors were chemoattractive towards microglia, an effect blocked by 2D7 anti-CCR5 antibody pre-treatment. Tat and opiates have interactive effects on astroglial chemokine secretion, but this interaction did not occur in progenitors. gp120 did not affect chemokine/cytokine release, although both CCR5 and CXCR4, which serve as gp120 co-receptors, were detected in progenitors. We postulate that chemokine production by progenitors may be a normal, adaptive process that encourages immune inspection of newly generated cells. Pathogens such as HIV might usurp this function to create a maladaptive state, especially during development or regeneration, when progenitors are numerous.
    Full-text · Article · Jul 2010 · Journal of Neurochemistry
  • Source
    • "HAND (Antinori et al. 2007) and its most severe manifestation HIV-1 dementia (HAD) persist at a high rate in spite of the increasingly common use of antiretroviral therapies (McArthur 2004; Anthony et al. 2005; Ellis et al. 2007). Although the core pathophysiological defects of HAD are neuronal damage (Masliah et al. 1992; Everall et al. 1993; Navia and Price 1998), neurons rarely show evidence of HIV-1 infection (Wiley et al. 1986; Takahashi et al. 1996; Torres-Muñoz et al. 2001a). HIV-1 replicates in the brain in microglia and infiltrating macrophages (Gartner et al. 1986; Genis et al. 1992), and virus genome and proteins can also be found in a small and variable fraction of astrocytes in vivo, particularly in advanced brain disease (Saito et al. 1994; Tornatore et al. 1994; Ranki et al. 1995; Takahashi et al. 1996; An et al. 1999; Trillo-Pazos et al. 2003). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Astrocytes are the major cellular component of the central nervous system (CNS), and they play multiple roles in brain development, normal brain function, and CNS responses to pathogens and injury. The functional versatility of astrocytes is linked to their ability to respond to a wide array of biological stimuli through finely orchestrated changes in cellular gene expression. Dysregulation of gene expression programs, generally by chronic exposure to pathogenic stimuli, may lead to dysfunction of astrocytes and contribute to neuropathogenesis. Here, we review studies that employ functional genomics to characterize the effects of HIV-1 and viral pathogenic proteins on cellular gene expression in astrocytes in vitro. We also present the first microarray analysis of primary mouse astrocytes exposed to HIV-1 in culture. In spite of different experimental conditions and microarray platforms used, comparison of the astrocyte array data sets reveals several common gene-regulatory changes that may underlie responses of these cells to HIV-1 and its proteins. We also compared the transcriptional profiles of astrocytes with those obtained in analyses of brain tissues of patients with HIV-1 dementia and macaques infected with simian immunodeficiency virus (SIV). Notably, many of the gene characteristics of responses to HIV-1 in cultured astrocytes were also altered in HIV-1 or SIV-infected brains. Functional genomics, in conjunction with other approaches, may help clarify the role of astrocytes in HIV-1 neuropathogenesis.
    Full-text · Article · Mar 2010 · Journal of Neuroimmune Pharmacology
  • Source
    • "Recent reports have combined LMD with quantitative real-time PCR (Burbach et al., 2003, 2004; Kerman et al., 2006; Prosniak et al., 2003; Valerie and Vincent, 2002). These have included genome-wide gene expression analyses using DNA microarrays (Hemby et al., 2002), as well as expression analyses of individual genes using RT-PCR (Kamme et al., 2003; Lu et al., 2004; Luo, 1999; Mutsuga et al., 2004; Torres-Munoz et al., 2001; Ye et al., 2003). This combination has made it possible to study the characteristics of mRNA expression in a specific cell population (Burbach et al., 2003). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The selective involvement of a subset of neurons in many psychiatric disorders, such as gamma-aminobutyric acid (GABA)-ergic interneurons in schizophrenia, creates a significant need for in-depth analysis of these cells. Here we introduce a combination of techniques to examine the relative gene expression of N-methyl-d-aspartic acid (NMDA) receptor subtypes in GABAergic interneurons from the rat prefrontal cortex. Neurons were identified by immunostaining, isolated by laser microdissection and RNA was prepared for reverse transcription polymerase chain reaction (RT-PCR) and real-time PCR. These experimental procedures have been described individually; however, we found that this combination of techniques is powerful for the analysis of gene expression in individual identified neurons. This approach provides the means to analyze relevant molecular mechanisms that are involved in the neuropathological process of a devastating brain disorder.
    Full-text · Article · Oct 2008 · Journal of Neuroscience Methods
Show more