Neuronal damage induced by ongoing human immunodeficiency virus type 1 (HIV-1) infection was investigated in humanized NOD/scid-IL-2Rγ(c)(null) mice transplanted at birth with human CD34-positive hematopoietic stem cells. Mice infected at 5 months of age and followed for up to 15 weeks maintained significant plasma viral loads and showed reduced numbers of CD4(+) T-cells. Prospective serial proton magnetic resonance spectroscopy tests showed selective reductions in cortical N-acetyl aspartate in infected animals. Diffusion tensor imaging revealed structural changes in cortical gray matter. Postmortem immunofluorescence brain tissue examinations for neuronal and glial markers, captured by multispectral imaging microscopy and quantified by morphometric and fluorescence emission, showed regional reduction of neuronal soma and synaptic architectures. This was evidenced by loss of microtubule-associated protein 2, synaptophysin, and neurofilament antigens. This study is the first, to our knowledge, demonstrating lost neuronal integrity after HIV-1 infection in humanized mice. As such, the model permits studies of the relationships between ongoing viral replication and virus-associated neurodegeneration.
Metabolites are biomarkers for a broad range of central nervous system disorders serving as molecular drivers and byproducts of disease pathobiology. However, despite their importance, routine measures of brain tissue metabolomics are not readily available based on the requirements of rapid tissue preservation. They require preservation by microwave irradiation, rapid freezing or other methods designed to reduce post mortem metabolism. Our research on human immunodeficiency virus type one (HIV-1) infection has highlighted immediate needs to better link histology to neural metabolites. To this end, we investigated such needs in well-studied rodent models. First, the dynamics of brain metabolism during ex vivo tissue preparation was shown by proton magnetic resonance spectroscopy in normal mice. Second, tissue preservation methodologies were assessed using liquid chromatography tandem mass spectrometry and immunohistology to measure metabolites and neural antigens. Third, these methods were applied to two animal models. In the first, immunodeficient mice reconstituted with human peripheral blood lymphocytes then acutely infected with HIV-1. In the second, NOD scid IL2 receptor gamma chain knockout mice were humanized with CD34+ human hematopoietic stem cells and chronically infected with HIV-1. Replicate infected animals were treated with nanoformulated antiretroviral therapy (nanoART). Results from chronic infection showed that microgliosis was associated with increased myoinostitol, choline, phosphocholine concentrations and with decreased creatine concentrations. These changes were partially reversed with nanoART. Metabolite responses were contingent on the animal model. Taken together, these studies integrate brain metabolomics with histopathology towards uncovering putative biomarkers for neuroAIDS.
Abstract Potent antiretroviral activities and a barrier to viral resistance characterize the human immunodeficiency virus type one (HIV-1) integrase strand transfer inhibitor dolutegravir (DTG). Herein, a long-acting parenteral DTG was created through chemical modification to improve treatment outcomes. A hydrophobic and lipophilic modified DTG prodrug is encapsulated into poloxamer nanoformulations (NMDTG) and characterized by size, shape, polydispersity, and stability. Retained intracytoplasmic NMDTG particles release drug from macrophages and attenuate viral replication and spread of virus to CD4+ T cells. Pharmacokinetic tests in Balb/cJ mice show blood DTG levels at, or above, its inhibitory concentration 90 of 64 ng/mL for 56 days, and tissue DTG levels for 28 days. NMDTG protects humanized mice from parenteral challenge of the HIV-1 ADA strain for two weeks. These results are a first step towards producing a long-acting DTG for human use by affecting drug apparent half-life, cell and tissue drug penetration, and antiretroviral potency.
Increasing evidence suggests that tau aggregates spread and replicate via cell-to-cell transmission, with the uptake of pathological tau causing misfolded aggregations of monomeric tau in recipient cells. A single intracerebral inoculation of extracellular vesicles containing tau into murine brains was shown to induce tau phosphorylation and soluble tau oligomer formation. Thus, we hypothesize that tau-containing exosomes derived from Alzheimer's affected human brains can serve as a seed for the spread of tauopathy in recipient animal brains. Exosome-enriched fractions were isolated from unfixed frozen human brain samples from Alzheimer's disease (AD) and control (CTRL) cases, as well as from tau knockout (TKO) mouse brains. Tau oligomer epitopes were determined by dot blot using antibodies against T22, T18, TOMA1, TOMA2, TOMA3 and TOMA4 for all of the exosome fractions that were used as injectates. Two-month old C57BL/6 mice were inoculated with human brain exosomes containing tau, TKO exosomes, or saline, into the right dorsal hippocampus. After injection, the brains were incubated for 18 weeks. The brains were then subjected to immunohistochemistry for phosphorylated-tau (p-tau) epitopes. Each of the human-derived exosome samples has its own unique fingerprint of tau oligomer expression by dot blot. Surprisingly, mice that were injected with one of the AD and with one of the CTRL exosome samples showed remarkable AT8-immunoreactivity in the hilus and subgranular zone regions of the hippocampus, while this was absent in the 9 other injectate groups including TKO-injected mice. Interestingly, one of these two positive exosome samples was only positive for TOMA2, while the other was positive for T22, T18 and TOMA3. Alzheimer's brain derived tau-exosomes accelerate pathological tau phosphorylation in relation to the specific type of tau oligomer(s) included within the exosomes.
Abstract Human immunodeficiency virus type 1 (HIV‐1) infection of the central nervous system is associated with characteristic virological, clinical, and neuropathological findings in adults and children. Productive infection in the brain and spinal cord occurs in blood‐derived macrophages, resident microglia, and multinucleated giant cells. Previous work implicated indirect mechanisms for neurotoxicity by HIV‐1 gene products or by factors secreted from HIV‐1‐infected macrophages. However, this cannot explain the paradox between the small numbers of infected cells and the widespread tissue pathology. Based on recent studies from our laboratories, we suggest that HIV‐1‐infected macrophages can initiate neurotoxicity, which is then amplified through cell‐to‐cell interactions with astrocytes. Macrophageastrocyte interactions produce cytokines tumor necrosis factor‐α and interleukin‐1b̃ and arachidonic metabolites that cause astroglial proliferation and neuronal injury. Inevitably, the astrogliosis serves to amplify these cellular processes while brain infection maintains itself in macrophage and microglia and possibly in astrocytes (by restricted infection). These findings, taken together, provide fresh insights into how low numbers of productively infected cells could elicit progressive and devastating neurological impairment during HIV‐1 disease, and suggest therapeutic strategies to interrupt the pathological process.
A human immunodeficiency virus type 1 (HIV)—seropositive, antiretroviral-naive patient presented with significant cognitive dysfunction. Neuropsychologic, neuroradiologic, immunologic, and virologic studies confirmed HIV-associated dementia (HAD). After 12 weeks of highly active antiretroviral therapy (HAART) with ibuprofen, dramatic improvements were demonstrated in neurologic function and were sustained for >1 year. HIV-1 RNA in cerebrospinal fluid (CSF) decreased from 105 to 104 copies/mL after 4 weeks. After 20 weeks of therapy, plasma viremia decreased from 106 copies/mL to undetectable (<96 copies/mL). Assays of neurotoxins (tumor necrosis factor-α, quinolinic acid, and nitric oxide) in plasma and CSF were considerably elevated at presentation and significantly decreased after therapy. Baseline plasma and CSF demonstrated neurotoxic activities in vitro, which also reduced markedly. These data, taken together, support the notion that HAD is a reversible metabolic encephalopathy fueled by viral replication. HAART used with nonsteroidal antiinflammatory agents leads to the suppression of inflammatory neurotoxins and can markedly improve neurologic function in HAD.
