Katherine A. Thompson

Improvements in lipoatrophy, mitochondrial Dna levels and fat apoptosis after replacing stavudine with abacavir or zidovudine

Objective:To determine if stavudine (α4T)-associated mitochondrial toxicity could be reversed by substitution with another nucleoside reverse transcriptase inhibitor. As apoptosis and dysfunction of electron transport chain (ETC) activities may underlie mitochondrial toxicity, these parameters were also evaluated. Design:The 16 participants (on d4T for >3 years; with lipoatrophy and/or hyperlactatemia) substituted abacavir or zidovudine for stavudine in their antiretroviral regimen. Key parameters including dual-energy X-ray absorptiometry (DEXA) scans, fat apoptosis, mitochondrial DNA (mtDNA) content in peripheral blood mononuclear cells (PBMC), skeletal muscle and fat, as well as skeletal muscle mitochondrial ETC activities were evaluated at study entry and at 48 weeks after the substitution. Methods:Quantitative PCR was used to evaluate mtDNA levels and the presence of deletions/rearrangements; CLIA-validated methods for ETC activities; terminal deoxynucleotidyl transferase dUTP-digoxigenin nick-end labeling assays to evaluate adipocyte apoptosis; and DEXA scans to measure changes in body fat. Results:MtDNA was depleted at study entry in muscle, adipose tissue and PBMC but levels rebounded with respective mean increases of 141%, 146%, and 369% at week 48. Corresponding fat improvements were noted with DEXA increases of 21%, 11%, and 16% in arm, leg, and trunk, respectively. Quantitative adipocyte apoptosis were significantly increased at baseline (P < 0.01 versus HIV-negative controls), with a significant reduction at week 48 (P < 0.05 versus baseline). Mean values for seven mitochondrial enzyme activities assays at entry indicated substantial loss of function (48% to 85% of controls) with significant improvement of complex I activity by week 48. Conclusions:Substitution of stavudine with abacavir or zidovudine improves mitochondrial indices and fat apoptosis in the setting of lipoatrophy.

Correlation between neurological progression and astrocyte apoptosis in HIV-associated dementia

The pathogenesis of HIV‐associated dementia (HIVD) has been postulated to be due to the indirect effects of HIV infection, including the aberrant central nervous system production of cytokines and other neurotoxins. A correlation between the severity of dementia and production of neurotoxins in HIVD has been demonstrated. We have previously identified nonproductive HIV infection of astrocytes. Because astrocytes participate in the inactivation of neurotoxins, we hypothesize that HIV nonproductive infection of astrocytes may lead to an environment in which there is a significant level of astrocyte apoptosis and a consequent increase in the levels of neurotoxins and that this results in more rapidly progressing dementia. Postmortem brain tissue was examined from clinically well‐characterized HIV‐positive demented patients, HIV‐positive nondemented patients, and HIV‐seronegative nondemented control subjects. The HIVD group was further categorized into subjects with rapid and those with slow progression of dementia. Tissue was paraformaldehyde fixed and paraffin embedded, and 6‐μm sections from the basal ganglia and mid‐frontal gyrus were processed to detect apoptosis by in situ transferase dUTP nick end labeling. Astrocytes were co‐localized using immunohistochemical techniques. In situ polymerase chain reaction (PCR) techniques were utilized to detect HIV DNA in astrocytes. The density of apoptotic astrocytes was significantly greater in the HIV‐positive groups than in the HIV‐negative group (p < 0.01). The HIVD rapid progressors had a significantly greater number of apoptotic astrocytes in the basal ganglia than did the HIVD slow progressors (p < 0.05). In addition, there were a greater number of HIV DNA–positive astrocytes, as demonstrated by in situ PCR, in the HIVD rapid progressors than in the slow progressor and HIV‐nondemented groups. These data suggest that there is an increased rate of astrocyte loss in the subjects with rapidly progressive dementia, in association with an increased number of HIV DNA–positive astrocytes. The results emphasize the importance of understanding more completely the role of HIV infection of astrocytes in the neuropathogenesis of HIVD.

Brain Cell Reservoirs of Latent Virus in Presymptomatic HIV-Infected Individuals

We detected HIV-1 DNA in pure populations of perivascular macrophages, parenchymal microglia, and astrocytes, isolated using laser microdissection from brain tissue of five untreated individuals who died in the presymptomatic stage of infection from non-HIV causes. HIV-1 DNA was detected in the three cell populations, most consistently in perivascular macrophages, without evidence of productive infection. The percentage of PCR reactions detecting HIV-1 DNA in perivascular macrophages correlated inversely with peripheral blood CD4 counts. These findings demonstrate that brain cell reservoirs of latent HIV-1 exist before pathological HIV encephalitis and suggest that perivascular macrophage trafficking of latent virus into the brain increases with immunosuppression.

Use of laser capture microdissection to detect integrated HIV-1 DNA in macrophages and astrocytes from autopsy brain tissues

The importance of astrocytes as a reservoir of human immunodeficiency virus type 1 (HIV-1) in the brain remains elusive. By combining immunohistochemistry, laser capture microdissection, and triple-nested Alu-PCR, we demonstrate integrated HIV-1 in astrocytes and macrophages isolated directly from autopsy brain tissues of HIV-1-infected subjects. The ability of HIV-1 to integrate in terminally differentiated astrocytes suggests a permanent reservoir of provirus in brain that will impact the development and likely success of strategies aimed at eradicating HIV-1.

Astrocyte specific viral strains in HIV dementia.

We molecularly characterized human immunodeficiency virus type 1 (HIV‐1) present in pure populations of astrocytes, macrophages, and multinucleated giant cells isolated using laser capture microdissection from brain tissue of two patients who died with HIV‐associated dementia. The V3 region of the HIV‐1 envelope (env) gene was amplified from the pure‐cell populations, and multiple clones were sequenced. In both patients, the V3 env sequences were distinct in astrocytes compared with neighboring macrophages or multinucleated giant cells and were characteristic of CCR5‐using (R5) HIV‐1. These results demonstrate cell‐specific compartmentalization of distinct R5‐like viral strains in the central nervous system microenvironment. Ann Neurol 2004

Herpes simplex replication and dissemination is not increased by corticosteroid treatment in a rat model of focal Herpes encephalitis.

Neurological damage in Herpes simplex type 1 encephalitis results from neuronal cell death secondary to viral invasion, and from inflammatory changes and cerebral oedema secondary to the immune response to the virus. Corticosteroids could have an important role in the management of Herpes simplex encephalitis because their anti-inflammatory action reduces cerebral oedema. However their use has been limited by concerns that their immunosuppressive actions could increase viral replication and spread. The present study examined this issue in a rat model in which injection of HSV-1 into the cervical vagus nerve produced a well-defined focal encephalitis, characterised by an orderly progression of the virus through central neural pathways connected with vagal afferent termination sites in the medulla oblongata. After injection of HSV-1, rats were treated twice a day, either with vehicle (saline, 400 microl i.p.), with acyclovir (30 mg/kg i.p.), with dexamethasone (5 mg/kg i.p.), or with both acyclovir and dexamethasone. Animals were sacrificed after 72 h, and viral load in different brain regions was quantified by computer-assisted measurement of the area occupied by immunohistochemical reaction product. Treatment with acyclovir reduced viral load to 17 +/- 5% of the saline value (P < 0.01). After dexamethasone treatment, the viral load (63 +/- 13% of the saline value) was also reduced (P < 0.05). Treatment with both acyclovir and dexamethasone reduced viral load to 26 +/- 8% of the saline value (P < 0.01 compared with saline, and P > 0.05 compared to acyclovir alone). Our results confirm the effectiveness of acyclovir in a new model of HSV-1 infection, and provide evidence that corticosteroids do not inhibit the antiviral action of acyclovir. In addition corticosteroids may decrease the extent of infection in their own right. The acute time course studied in our model parallels the time course of acute Herpes simplex encephalitis in humans. Our data suggests that corticosteroids are not detrimental when combined with acyclovir in the management of this condition.

Immunopathogenesis of HIV-associated dementia.

This review provides a subjective analysis of the advances in our understanding of the immunopathogenesis of HIV-associated dementia that have occurred over the past 12 months. The review will focus on the following areas: (i) the role of chemokines and cytokines; (ii) the role of astrocytes, astrocyte cell death and non-productive infection of astrocytes; (iii) a model of the neuropathogenesis of HIV-associated dementia and its impact on treatment paradigms and future research. The requirements for the development of HIV-associated dementia are immunosuppression, the loss of macrophage regulation, central nervous system HIV infection of microglia and macrophages with a neurovirulent HIV strain, restricted HIV infection of astrocytes, and astrocyte cell death, all of which lead to an intracellular milieu that is neurotoxic. This cascade can be prevented and probably reversed by the use of highly active antiretroviral therapy, which controls viral replication both systemically and centrally. However, for those patients who have resistant virus and persistently high levels of replication, or who develop resistance or toxicity, other treatment strategies need to be developed. The control of excessive microglial and macrophage activation or a diminution of astrocyte and neuronal apoptosis could have benefits in terms of cognitive function. We therefore need to develop further our understanding of the immunopathogenesis of HIV-associated dementia so that we can control a number of other steps in the cascade rather than simply controlling the viral replication.

Decreased Neurotropism of nef Long Terminal Repeat ( nef /LTR)-Deleted Simian Immunodeficiency Virus

Simian immunodeficiency virus (SIV) infection of macaques results in neurological abnormalities similar to those of human immunodeficiency virus (HIV)-associated dementia in humans and is a valuable system for the identification of viral neurotropic and neurovirulence factors. The authors recently established an SIV-macaque model where macaques can be infected with wild-type or nef/LTR-deleted SIVmac239 via administration of purified proviral DNA. In this study, the ability of wild-type and nef/LTR-deleted SIV infections to enter the cerebral spinal fluid (CSF) and brain was analyzed. In situ polymerase chain reaction (PCR) readily detected SIV gag DNA-positive cells in the mid-frontal gyrus and basal ganglia of the wild-type SIV-infected macaques, but not in nef/LTR-deleted SIV-infected or SIV-uninfected macaques. PCR on extracted DNA confirmed the in situ results, with multiple brain regions of the wild-type SIV-infected macaques positive for both gag and wild-type nef, whereas in the nef/LTR-deleted SIV-infected macaques, nef/LTR and gag DNA were undetectable. Further, macaques infected with nef/LTR-deleted SIV, which later became superinfected with wild-type SIV, also remained negative for SIV DNA in the brain by both in situ and extracted DNA techniques, despite having high levels of SIV RNA both in the CSF and plasma. This study provides evidence of the inability of nef/LTR-deleted SIV to initiate central nervous system (CNS) infection and suggests that, in the brain regions examined, nef/LTR-deleted viruses have either diminished neurotropism or insufficient systemic viral replication for entry into the CNS.

Cell-specific temporal infection of the brain in a simian immunodeficiency virus model of human immunodeficiency virus encephalitis

Increasing evidence supports early brain infection by human immunodeficiency virus (HIV). Definitive temporal studies determining when and within which brain cells viral DNA is present are lacking. This study utilized simian immunodeficiency virus (SIV)-infected macaques sacrificed at days 10, 21, 56, and 84 post inoculation. Laser-microdissection isolated pure perivascular macrophage, parenchymal microglia, and astrocyte populations. Nested polymerase chain reaction (PCR) and sequencing determined the presence and characteristics of SIV V3 and V1 env DNA from each population. At day 10, SIV DNA was detected in perivascular macrophage and astrocytes but not parenchymal microglia. gp41 expression was restricted to perivascular macrophage. At day 21, SIV DNA was not detected in any cell type. At day 56, SIV DNA was detectable in perivascular macrophage from one of two macaques, with no gp41 expression detected. At day 84 (morphologic and clinical encephalitis), SIV DNA was detected in all cell types, gp41 was only detected in perivascular macrophage and parenchymal microglia. The neurovirulent molecular clone, SIV/17E-Fr, was the only genotype identified in the brain cell populations. Early, productive brain SIV infection was transient and restricted to trafficking perivascular macrophage. During the nonencephalitic stage, there was a period of time when no SIV DNA could be detected in the brain cell populations. SIV was then seen to reenter the brain via infected perivascular macrophage, leading to productive infection of brain parenchymal macrophage/microglia with a terminal phase of encephalitis. These data challenge current notions of a HIV reservoir within latently infected, semipermanent brain cells and has significant implications for the timing and design of therapies to prevent HIV encephalitis (HIVE).