Human endogenous retrovirus-K(II) envelope induction protects neurons during HIV/AIDS.

Human endogenous retroviruses (HERVs) are differentially expressed depending on the cell type and physiological circumstances. HERV-K has been implicated in the pathogenesis of several diseases although the functional consequences of its expression remain unknown. Human immunodeficiency virus (HIV) infection causes neuroinflammation with neuronal damage and death. Herein, we investigated HERV-K(II)/(HML-2) envelope (Env) expression and its actions in the brain during HIV/AIDS. HERV-K(II) Env expression was assessed in healthy brain tissues, autopsied HIV HIV- infected (HIV+) and uninfected (HIV-) brains and in neural cell cultures by real time RT-PCR, massively parallel (deep) sequencing, immunoblotting and immunohistochemistry. Neuronal and neural stem cells expressing HERV-K(II) Env were analyzed in assays of host responses including cellular viability, immune responses and neurobehavioral outcomes. Deep sequencing of human brain transcriptomes disclosed that RNA sequences encoded by HERV-K were among the most abundant HERV sequences detected in human brain. Comparison of different cell types revealed that HERV-K(II) env RNA abundance was highest in cultured human neurons but was suppressed by epidermal growth factor exposure. HERV-K(II) Env immunoreactivity was increased in the cerebral cortex from persons with HIV/AIDS, principally localized in neurons. Human neuronal cells transfected with HERV-K(II) Env exhibited increased NGF and BDNF expression. Expression of HERV-K(II) Env in neuronal cells increased cellular viability and prevented neurotoxicity mediated by HIV-1 Vpr. Intracerebral delivery of HERV-K(II) Env expressed by neural stem cells suppressed TNF-α expression and microglial activation while also improving neurobehavioral deficits in vpr/RAG1-/- mice. HERV-K(II) Env was highly expressed in human neurons, especially during HIV/AIDS, but in addition exerted neuroprotective effects. These findings imply that HERV gene products might exert adaptive effects in circumstances of pathophysiological stress, perhaps underlying the conservation of HERVs within the human genome.

Rapid inflammasome activation in microglia contributes to brain disease in HIV/AIDS.

BACKGROUND: Human immunodeficiency virus type 1(HIV-1) infects and activates innate immune cells in the brain resulting in inflammation and neuronal death with accompanying neurological deficits. Induction of inflammasomes causes cleavage and release of IL-1ß and IL-18, representing pathogenic processes that underlie inflammatory diseases although their contribution HIV-associated brain disease is unknown. RESULTS: Investigation of inflammasome-associated genes revealed that IL-1ß, IL-18 and caspase-1 were induced in brains of HIV-infected persons and detected in brain microglial cells. HIV-1 infection induced pro-IL-1ß in human microglia at 4 hr post-infection with peak IL-1ß release at 24 hr, which was accompanied by intracellular ASC translocation and caspase-1 activation. HIV-dependent release of IL-1ß from a human macrophage cell line, THP-1, was inhibited by NLRP3 deficiency and high extracellular [K+]. Exposure of microglia to HIV-1 gp120 caused IL-1ß production and similarly, HIV-1 envelope pseudotyped viral particles induced IL-1ß release, unlike VSV-G pseudotyped particles. Infection of cultured feline macrophages by the related lentivirus, feline immunodeficiency virus (FIV), also resulted in the prompt induction of IL-1ß. In vivo FIV infection activated multiple inflammasome-associated genes in microglia, which was accompanied by neuronal loss in cerebral cortex and neurological deficits. Multivariate analyses of data from FIV-infected and uninfected animals disclosed that IL-1ß, NLRP3 and caspase-1 expression in cerebral cortex represented key molecular determinants of neurological deficits. CONCLUSIONS: NLRP3 inflammasome activation was an early and integral aspect of lentivirus infection of microglia, which was associated with lentivirus-induced brain disease. Inflammasome activation in the brain might represent a potential target for therapeutic interventions in HIV/AIDS.

HIV-1 Nef expression in microglia disrupts dopaminergic and immune functions with associated mania-like behaviors.

BACKGROUND: Neuropsychiatric disorders during HIV/AIDS are common although the contribution of HIV-1 infection within the brain, and in particular individual HIV-1 proteins, to the development of these brain disorders is unknown. Herein, an in vivo transgenic mouse model was generated in which the HIV-1 Nef protein was expressed in microglia cells, permitting investigation of neurobehavioral phenotypes and associated cellular and molecular properties. METHODS: Transgenic (Tg) mice that expressed full length HIV-1 nef under the control of the c-fms promoter and wildtype (Wt) littermates were investigated using different measures of neurobehavioral performance including locomotory, forced swim (FST), elevated plus maze (EPM) and T-maze tests. Host gene and transgene expression were assessed by RT-PCR, immunoblotting, enzymatic activity and immunohistochemistry. Biogenic amine levels were measured by HPLC with electrochemical detection. RESULTS: Tg animals exhibited Nef expression in brain microglia and cultured macrophages. Tg males displayed hyperactive behaviors including augmented locomotor activity, decreased immobility in the FST and increased open-arm EPM exploration compared to Wt littermates (p<0.05). Tg animals showed increased CCL2 expression with concurrent IFN-α suppression in striatum compared with Wt littermates (p<0.05). Dopamine levels, MAO activity and the dopamine transporter (DAT) expression were reduced in the striatum of Tg animals (p<0.05). CONCLUSIONS: HIV-1 Nef expression in microglia induced CCL2 expression together with disrupting striatal dopaminergic transmission, resulting in hyperactive behaviors which are observed in mania and other psychiatric comorbidities among HIV-infected persons. These findings emphasize the selective effects of individual viral proteins in the brain and their participation in neuropathogenesis.

Inflammasome induction in Rasmussen's encephalitis: cortical and associated white matter pathogenesis.

BACKGROUND: Rasmussen's encephalitis (RE) is an inflammatory encephalopathy of unknown cause defined by seizures with progressive neurological disabilities. Herein, the pathogenesis of RE was investigated focusing on inflammasome activation in the brain. METHODS: Patients with RE at the University of Alberta, Edmonton, AB, Canada, were identified and analyzed by neuroimaging, neuropsychological, molecular, and pathological tools. Primary human microglia, astrocytes, and neurons were examined using RT-PCR, enzyme-linked immunosorbent assay (ELISA), and western blotting. RESULTS: Four patients with RE were identified at the University of Alberta. Magnetic resonance imaging (MRI) disclosed increased signal intensities in cerebral white matter adjacent to cortical lesions of RE patients, accompanied by a decline in neurocognitive processing speed (P <0.05). CD3ϵ, HLA-DRA, and TNFα together with several inflammasome-associated genes (IL-1ß, IL-18, NLRP1, NLRP3, and CASP1) showed increased transcript levels in RE brains compared to non-RE controls (n = 6; P <0.05). Cultured human microglia displayed expression of inflammasome-associated genes and responded to inflammasome activators by releasing IL-1ß, which was inhibited by the caspase inhibitor, zVAD-fmk. Major histocompatibility complex (MHC) class II, IL-1ß, caspase-1, and alanine/serine/cysteine (ASC) immunoreactivity were increased in RE brain tissues, especially in white matter myeloid cells, in conjunction with mononuclear cell infiltration and gliosis. Neuroinflammation in RE brains was present in both white matter and adjacent cortex with associated induction of inflammasome components, which was correlated with neuroimaging and neuropsychological deficits. CONCLUSION: Inflammasome activation likely contributes to the disease process underlying RE and offers a mechanistic target for future therapeutic interventions.

Differential type 1 interferon-regulated gene expression in the brain during AIDS: interactions with viral diversity and neurovirulence.

The lentiviruses, human and feline immunodeficiency viruses (HIV-1 and FIV, respectively), infect the brain and cause neurovirulence, evident as neuronal injury, inflammation, and neurobehavioral abnormalities with diminished survival. Herein, different lentivirus infections in conjunction with neural cell viability were investigated, concentrating on type 1 interferon-regulated pathways. Transcriptomic network analyses showed a preponderance of genes involved in type 1 interferon signaling, which was verified by increased expression of the type 1 interferon-associated genes, Mx1 and CD317, in brains from HIV-infected persons (P<0.05). Leukocytes infected with different strains of FIV or HIV-1 showed differential Mx1 and CD317 expression (P<0.05). In vivo studies of animals infected with the FIV strains, FIV(ch) or FIV(ncsu), revealed that FIV(ch)-infected animals displayed deficits in memory and motor speed compared with the FIV(ncsu)- and mock-infected groups (P<0.05). TNF-α, IL-1ß, and CD40 expression was increased in the brains of FIV(ch)-infected animals; conversely, Mx1 and CD317 transcript levels were increased in the brains of FIV(ncsu)-infected animals, principally in microglia (P<0.05). Gliosis and neuronal loss were evident among FIV(ch)-infected animals compared with mock- and FIV(ncsu)-infected animals (P<0.05). Lentiviral infections induce type 1 interferon-regulated gene expression in microglia in a viral diversity-dependent manner, representing a mechanism by which immune responses might be exploited to limit neurovirulence.

Brain microbial populations in HIV/AIDS: α-proteobacteria predominate independent of host immune status.

The brain is assumed to be a sterile organ in the absence of disease although the impact of immune disruption is uncertain in terms of brain microbial diversity or quantity. To investigate microbial diversity and quantity in the brain, the profile of infectious agents was examined in pathologically normal and abnormal brains from persons with HIV/AIDS [HIV] (n = 12), other disease controls [ODC] (n = 14) and in cerebral surgical resections for epilepsy [SURG] (n = 6). Deep sequencing of cerebral white matter-derived RNA from the HIV (n = 4) and ODC (n = 4) patients and SURG (n = 2) groups revealed bacterially-encoded 16 s RNA sequences in all brain specimens with α-proteobacteria representing over 70% of bacterial sequences while the other 30% of bacterial classes varied widely. Bacterial rRNA was detected in white matter glial cells by in situ hybridization and peptidoglycan immunoreactivity was also localized principally in glia in human brains. Analyses of amplified bacterial 16 s rRNA sequences disclosed that Proteobacteria was the principal bacterial phylum in all human brain samples with similar bacterial rRNA quantities in HIV and ODC groups despite increased host neuroimmune responses in the HIV group. Exogenous viruses including bacteriophage and human herpes viruses-4, -5 and -6 were detected variably in autopsied brains from both clinical groups. Brains from SIV- and SHIV-infected macaques displayed a profile of bacterial phyla also dominated by Proteobacteria but bacterial sequences were not detected in experimentally FIV-infected cat or RAG1⁻/⁻ mouse brains. Intracerebral implantation of human brain homogenates into RAG1⁻/⁻ mice revealed a preponderance of α-proteobacteria 16 s RNA sequences in the brains of recipient mice at 7 weeks post-implantation, which was abrogated by prior heat-treatment of the brain homogenate. Thus, α-proteobacteria represented the major bacterial component of the primate brain's microbiome regardless of underlying immune status, which could be transferred into naïve hosts leading to microbial persistence in the brain.

Neurosteroid-mediated regulation of brain innate immunity in HIV/AIDS: DHEA-S suppresses neurovirulence.

Neurosteroids are cholesterol-derived molecules synthesized within the brain, which exert trophic and protective actions. Infection by human and feline immunodeficiency viruses (HIV and FIV, respectively) causes neuroinflammation and neurodegeneration, leading to neurological deficits. Secretion of neuroinflammatory host and viral factors by glia and infiltrating leukocytes mediates the principal neuropathogenic mechanisms during lentivirus infections, although the effect of neurosteroids on these processes is unknown. We investigated the interactions between neurosteroid-mediated effects and lentivirus infection outcomes. Analyses of HIV-infected (HIV(+)) and uninfected human brains disclosed a reduction in neurosteroid synthesis enzyme expression. Human neurons exposed to supernatants from HIV(+) macrophages exhibited suppressed enzyme expression without reduced cellular viability. HIV(+) human macrophages treated with sulfated dehydroepiandrosterone (DHEA-S) showed suppression of inflammatory gene (IL-1ß, IL-6, TNF-α) expression. FIV-infected (FIV(+)) animals treated daily with 15 mg/kg body weight. DHEA-S treatment reduced inflammatory gene transcripts (IL-1ß, TNF-α, CD3ε, GFAP) in brain compared to vehicle-(ß-cyclodextrin)-treated FIV(+) animals similar to levels found in vehicle-treated FIV(-) animals. DHEA-S treatment also increased CD4(+) T-cell levels and prevented neurobehavioral deficits and neuronal loss among FIV(+) animals, compared to vehicle-treated FIV(+) animals. Reduced neuronal neurosteroid synthesis was evident in lentivirus infections, but treatment with DHEA-S limited neuroinflammation and prevented neurobehavioral deficits. Neurosteroid-derived therapies could be effective in the treatment of virus- or inflammation-mediated neurodegeneration.

Metagenomic and metabolomic characterization of rabies encephalitis: new insights into the treatment of an ancient disease.

Rabies virus (RV) infection is a fatal nervous system disorder. We describe a patient who died of rabies despite a neuroprotective intervention. Neuropathology showed neuronal loss with abundant RV antigen, genome, and Negri bodies, accompanied by intense neuroinflammation, including by CD8(+) T lymphocyte infiltrates. Deep sequencing and real-time reverse-transcription polymerase chain reaction revealed RNA encoding a bat RV strain together with inflammatory gene induction. RV-infected brain demonstrated reduced neuronal metabolites with an anaerobic metabolic profile by nuclear magnetic resonance (NMR) spectroscopy. These multiplatform studies highlight the extent of ongoing viral replication coupled with inflammation in treated rabies, indicative of a neurological immune reconstitution inflammatory syndrome.

Proteinase-activated receptor-1 mediates dorsal root ganglion neuronal degeneration in HIV/AIDS.

Distal sensory polyneuropathy is a frequent complication of lentivirus infections of the peripheral nervous system including both human immunodeficiency virus and feline immunodeficiency virus. Proteinase-activated receptors are G protein-coupled receptors implicated in the pathogenesis of neuroinflammation and neurodegeneration. Proteinase-activated receptor-1 is expressed on different cell types within the nervous system including neurons and glia, but little is known about its role in the pathogenesis of inflammatory peripheral nerve diseases, particularly lentivirus-related distal sensory polyneuropathy. Herein, the expression and functions of proteinase-activated receptor-1 in the peripheral nervous system during human immunodeficiency virus and feline immunodeficiency virus infections were investigated. Proteinase-activated receptor-1 expression was most evident in autopsied dorsal root ganglion neurons from subjects infected with human immunodeficiency virus, compared with the dorsal root ganglia of uninfected subjects. Human immunodeficiency virus or feline immunodeficiency virus infection of cultured human or feline dorsal root ganglia caused upregulation of interleukin-1ß and proteinase-activated receptor-1 expression. In the human immunodeficiency virus- or feline immunodeficiency virus-infected dorsal root ganglia, interleukin-1ß activation was principally detected in macrophages, while neurons showed induction of proteinase-activated receptor-1. Binding of proteinase-activated receptor-1 by the selective proteinase-activated receptor-1-activating peptide resulted in neurite retraction and soma atrophy in conjunction with cytosolic calcium activation in human dorsal root ganglion neurons. Interleukin-1ß exposure to feline or human dorsal root ganglia caused upregulation of proteinase-activated receptor-1 in neurons. Exposure of feline immunodeficiency virus-infected dorsal root ganglia to the interleukin-1 receptor antagonist prevented proteinase-activated receptor-1 induction and neurite retraction. In vivo feline immunodeficiency virus infection was associated with increased proteinase-activated receptor-1 expression on neurons and interleukin-1ß induction in macrophages. Moreover, feline immunodeficiency virus infection caused hyposensitivity to mechanical stimulation. These data indicated that activation and upregulation of proteinase-activated receptor-1 by interleukin-1ß contributed to dorsal root ganglion neuronal damage during lentivirus infections leading to the development of distal sensory polyneuropathy and might also provide new targets for future therapeutic interventions.

Impaired neurosteroid synthesis in multiple sclerosis.

High-throughput technologies have led to advances in the recognition of disease pathways and their underlying mechanisms. To investigate the impact of micro-RNAs on the disease process in multiple sclerosis, a prototypic inflammatory neurological disorder, we examined cerebral white matter from patients with or without the disease by micro-RNA profiling, together with confirmatory reverse transcription-polymerase chain reaction analysis, immunoblotting and gas chromatography-mass spectrometry. These observations were verified using the in vivo multiple sclerosis model, experimental autoimmune encephalomyelitis. Brains of patients with or without multiple sclerosis demonstrated differential expression of multiple micro-RNAs, but expression of three neurosteroid synthesis enzyme-specific micro-RNAs (miR-338, miR-155 and miR-491) showed a bias towards induction in patients with multiple sclerosis (P < 0.05). Analysis of the neurosteroidogenic pathways targeted by micro-RNAs revealed suppression of enzyme transcript and protein levels in the white matter of patients with multiple sclerosis (P < 0.05). This was confirmed by firefly/Renilla luciferase micro-RNA target knockdown experiments (P < 0.05) and detection of specific micro-RNAs by in situ hybridization in the brains of patients with or without multiple sclerosis. Levels of important neurosteroids, including allopregnanolone, were suppressed in the white matter of patients with multiple sclerosis (P < 0.05). Induction of the murine micro-RNAs, miR-338 and miR-155, accompanied by diminished expression of neurosteroidogenic enzymes and allopregnanolone, was also observed in the brains of mice with experimental autoimmune encephalomyelitis (P < 0.05). Allopregnanolone treatment of the experimental autoimmune encephalomyelitis mouse model limited the associated neuropathology, including neuroinflammation, myelin and axonal injury and reduced neurobehavioral deficits (P < 0.05). These multi-platform studies point to impaired neurosteroidogenesis in both multiple sclerosis and experimental autoimmune encephalomyelitis. The findings also indicate that allopregnanolone and perhaps other neurosteroid-like compounds might represent potential biomarkers or therapies for multiple sclerosis.

Interactions between human immunodeficiency virus (HIV)-1 Vpr expression and innate immunity influence neurovirulence.

BACKGROUND: Viral diversity and abundance are defining properties of human immunodeficiency virus (HIV)-1's biology and pathogenicity. Despite the increasing availability of antiretroviral therapy, HIV-associated dementia (HAD) continues to be a devastating consequence of HIV-1 infection of the brain although the underlying disease mechanisms remain uncertain. Herein, molecular diversity within the HIV-1 non-structural gene, Vpr, was examined in RNA sequences derived from brain and blood of HIV/AIDS patients with or without HIV-associated dementia (HAD) together with the ensuing pathobiological effects. RESULTS: Cloned brain- and blood-derived full length vpr alleles revealed that amino acid residue 77 within the brain-derived alleles distinguished HAD (77Q) from non-demented (ND) HIV/AIDS patients (77R) (p < 0.05) although vpr transcripts were more frequently detected in HAD brains (p < 0.05). Full length HIV-1 clones encoding the 77R-ND residue induced higher IFN-α, MX1 and BST-2 transcript levels in human glia relative to the 77Q-HAD encoding virus (p < 0.05) but both viruses exhibited similar levels of gene expression and replication. Myeloid cells transfected with 77Q-(pVpr77Q-HAD), 77R (pVpr77R-ND) or Vpr null (pVpr(-))-containing vectors showed that the pVpr77R-ND vector induced higher levels of immune gene expression (p < 0.05) and increased neurotoxicity (p < 0.05). Vpr peptides (amino acids 70-96) containing the 77Q-HAD or 77R-ND motifs induced similar levels of cytosolic calcium activation when exposed to human neurons. Human glia exposed to the 77R-ND peptide activated higher transcript levels of IFN-α, MX1, PRKRA and BST-2 relative to 77Q-HAD peptide (p < 0.05). The Vpr 77R-ND peptide was also more neurotoxic in a concentration-dependent manner when exposed to human neurons (p < 0.05). Stereotaxic implantation of full length Vpr, 77Q-HAD or 77R-ND peptides into the basal ganglia of mice revealed that full length Vpr and the 77R-ND peptide caused greater neurobehavioral deficits and neuronal injury compared with 77Q-HAD peptide-implanted animals (p < 0.05). CONCLUSIONS: These observations underscored the potent neuropathogenic properties of Vpr but also indicated viral diversity modulates innate neuroimmunity and neurodegeneration.

Inflammation and epithelial cell injury in AIDS enteropathy: involvement of endoplasmic reticulum stress.

Immunosuppressive lentivirus infections, including human, simian, and feline immunodeficiency viruses (HIV, SIV, and FIV, respectively), cause the acquired immunodeficiency syndrome (AIDS), frequently associated with AIDS enteropathy. Herein, we investigated the extent to which lentivirus infections affected mucosal integrity and intestinal permeability in conjunction with immune responses and activation of endoplasmic reticulum (ER) stress pathways. Duodenal biopsies from individuals with HIV/AIDS exhibited induction of IL-1ß, CD3ε, HLA-DRA, spliced XBP-1(Xbp-1s), and CHOP expression compared to uninfected persons (P<0.05). Gut epithelial cells exposed to HIV-1 Vpr demonstrated elevated TNF-α, IL-1ß, spliced Xbp-1s, and CHOP expression (P<0.05) together with calcium activation and disruption of epithelial cell monolayer permeability. In addition to reduced blood CD4(+) T lymphocyte levels, viral loads in the gut and plasma were high in FIV-infected animals (P<0.05). FIV-infected animals also exhibited a failure to gain weight and increased lactulose/mannitol ratios compared with uninfected animals (P<0.05). Proinflammatory and ER stress gene expression were activated in the ileum of FIV-infected animals (P<0.05), accompanied by intestinal epithelial damage with loss of epithelial cells and leukocyte infiltration of the lamina propria. Lentivirus infections cause gut inflammation and ensuing damage to intestinal epithelial cells, likely through induction of ER stress pathways, resulting in disruption of gut functional integrity.

Hepatitis C virus core protein induces neuroimmune activation and potentiates Human Immunodeficiency Virus-1 neurotoxicity.

BACKGROUND: Hepatitis C virus (HCV) genomes and proteins are present in human brain tissues although the impact of HIV/HCV co-infection on neuropathogenesis remains unclear. Herein, we investigate HCV infectivity and effects on neuronal survival and neuroinflammation in conjunction with HIV infection. METHODOLOGY: Human microglia, astrocyte and neuron cultures were infected with cell culture-derived HCV or exposed to HCV core protein with or without HIV-1 infection or HIV-1 Viral Protein R (Vpr) exposure. Host immune gene expression and cell viability were measured. Patch-clamp studies of human neurons were performed in the presence or absence of HCV core protein. Neurobehavioral performance and neuropathology were examined in HIV-1 Vpr-transgenic mice in which stereotaxic intrastriatal implants of HCV core protein were performed. PRINCIPAL FINDINGS: HCV-encoded RNA as well as HCV core and non-structural 3 (NS3) proteins were detectable in human microglia and astrocytes infected with HCV. HCV core protein exposure induced expression of pro-inflammatory cytokines including interleukin-1ß, interleukin-6 and tumor necrosis factor-α in microglia (p<0.05) but not in astrocytes while increased chemokine (e.g. CXCL10 and interleukin-8) expression was observed in both microglia and astrocytes (p<0.05). HCV core protein modulated neuronal membrane currents and reduced both ß-III-tubulin and lipidated LC3-II expression (p<0.05). Neurons exposed to supernatants from HCV core-activated microglia exhibited reduced ß-III-tubulin expression (p<0.05). HCV core protein neurotoxicity and interleukin-6 induction were potentiated by HIV-1 Vpr protein (p<0.05). HIV-1 Vpr transgenic mice implanted with HCV core protein showed gliosis, reduced neuronal counts together with diminished LC3 immunoreactivity. HCV core-implanted animals displayed neurobehavioral deficits at days 7 and 14 post-implantation (p<0.05). CONCLUSIONS: HCV core protein exposure caused neuronal injury through suppression of neuronal autophagy in addition to neuroimmune activation. The additive neurotoxic effects of HCV- and HIV-encoded proteins highlight extrahepatic mechanisms by which HCV infection worsens the disease course of HIV infection.

Clinical outcomes and immune benefits of anti-epileptic drug therapy in HIV/AIDS.

BACKGROUND: Anti-epileptic drugs (AEDs) are frequently prescribed to persons with HIV/AIDS receiving combination antiretroviral therapy (cART) although the extent of AED use and their interactions with cART are uncertain. Herein, AED usage, associated toxicities and immune consequences were investigated. METHODS: HIV replication was analysed in proliferating human T cells during AED exposure. Patients receiving AEDs in a geographically-based HIV care program were assessed using clinical and laboratory variables in addition to assessing AED indication, type, and cumulative exposures. RESULTS: Valproate suppressed proliferation in vitro of both HIV-infected and uninfected T cells (p <0.05) but AED exposures did not affect HIV production in vitro. Among 1345 HIV/AIDS persons in active care between 2001 and 2007, 169 individuals were exposed to AEDs for the following indications: peripheral neuropathy/neuropathic pain (60%), seizure/epilepsy (24%), mood disorder (13%) and movement disorder (2%). The most frequently prescribed AEDs were calcium channel blockers (gabapentin/pregabalin), followed by sodium channel blockers (phenytoin, carbamazepine, lamotrigine) and valproate. In a nested cohort of 55 AED-treated patients receiving cART and aviremic, chronic exposure to sodium and calcium channel blocking AEDs was associated with increased CD4+ T cell levels (p <0.05) with no change in CD8+ T cell levels over 12 months from the beginning of AED therapy. CONCLUSIONS: AEDs were prescribed for multiple indications without major adverse effects in this population but immune status in patients receiving sodium or calcium channel blocking drugs was improved.

Regulation of lentivirus neurovirulence by lipopolysaccharide conditioning: suppression of CXCL10 in the brain by IL-10.

Lentivirus infections including HIV and feline immunodeficiency virus (FIV) cause neurovirulence, which is largely mediated by innate immunity. To investigate the interactions between neurovirulence and repeated conditioning by innate immune activation, models of lentivirus infection were exposed to LPS. Gene expression in HIV-infected (HIV+) and control (HIV-) patient brains was compared by real time RT-PCR and immunocytochemistry. Supernatants from mock and HIV-infected monocyte-derived macrophages exposed to LPS were applied to human neurons. FIV-infected (FIV+) and control (FIV-) animals were exposed repeatedly to LPS postinfection together with concurrent neurobehavioral testing, viral load, and host gene analyses. Brains from HIV+ individuals exhibited induction of CD3epsilon, CXCL10, and granzyme A expression (p < 0.05). Supernatants from HIV+ monocyte-derived macrophages induced CXCL10 expression in neurons, which was diminished by IL-10 treatment (p < 0.05). LPS-exposed FIV+ animals demonstrated lower plasma and brain viral loads (p < 0.05). Neuronal CXCL10 expression was increased in FIV+ animals but was suppressed by LPS exposure, together with reduced brain CD3epsilon and granzyme A expression (p < 0.05). In conjunction with preserved NeuN-positive neuronal counts in parietal cortex (p < 0.05), FIV+ animals exposed to LPS also showed less severe neurobehavioral deficits (p < 0.05). Repeated LPS exposures suppressed CXCL10 in the brain and ensuing T cell infiltration with a concomitant reduction in neurovirulence. Thus, innate immune chronic conditioning exerted beneficial effects on neurovirulence through suppression of a specific chemotactic factor, CXCL10, mediated by IL-10, leading to reduced leukocyte infiltration and release of neurotoxic factors.

Neurobehavioral performance in feline immunodeficiency virus infection: integrated analysis of viral burden, neuroinflammation, and neuronal injury in cortex.

Human immunodeficiency virus (HIV) infection causes motor and neurocognitive abnormalities affecting >50% of children and 20% of adults with HIV/AIDS (acquired immunodeficiency syndrome). The closely related lentivirus, feline immunodeficiency virus (FIV), also causes neurobehavioral deficits. Herein, we investigated the extent to which FIV infection affected specific motor and cognitive tasks in conjunction with viral burden and immune responses within the brain. Neonatal animals were infected with a neurovirulent FIV strain (FIV-Ch) and assessed in terms of systemic immune parameters, viral burden, neurobehavioral performance, and neuropathological features. FIV-infected animals displayed less weight gain and lower blood CD4(+) T-cell levels than mock-infected animals (p < 0.05). Gait analyses disclosed greater gait width with increased variation in FIV-infected animals (p < 0.05). Maze performance showed that FIV-infected animals were slower and made more navigational errors than mock-infected animals (p < 0.05). In the object memory test, the FIV-infected group exhibited fewer successful steps with more trajectory errors compared with the mock-infected group (p < 0.05). Performance on the gait, maze, and object memory tests was inversely correlated with F4/80 and CD3 epsilon expression (p < 0.05) and with viral burden in parietal cortex (p < 0.05). Amino acid analysis in cortex showed that D-serine levels were reduced in FIV-infected animals, which was accompanied by diminished kainate and AMPA receptor subunit expression (p < 0.05). The neurobehavioral findings in FIV-infected animals were associated with increased gliosis and reduced cortical neuronal counts (p < 0.05). The present studies indicated that specific motor and neurocognitive abilities were impaired in FIV infection and that these effects were closely coupled with viral burden, neuroinflammation, and neuronal loss.

West Nile virus-induced neuroinflammation: glial infection and capsid protein-mediated neurovirulence.

West Nile virus (WNV) infection causes neurological disease at all levels of the neural axis, accompanied by neuroinflammation and neuronal loss, although the underlying mechanisms remain uncertain. Given the substantial activation of neuroinflammatory pathways observed in WNV infection, we hypothesized that WNV-mediated neuroinflammation and cell death occurred through WNV infection of both glia and neurons, which was driven in part by WNV capsid protein expression. Analysis of autopsied neural tissues from humans with WNV encephalomyelitis (WNVE) revealed WNV infection of both neurons and glia. Upregulation of proinflammatory genes, CXCL10, interleukin-1beta, and indolamine-2',3'-deoxygenase with concurrent suppression of the protective astrocyte-specific endoplasmic reticulum stress sensor gene, OASIS (for old astrocyte specifically induced substance), was evident in WNVE patients compared to non-WNVE controls. These findings were supported by increased ex vivo expression of these proinflammatory genes in glia infected by WNV-NY99. WNV infection caused endoplasmic reticulum stress gene induction and apoptosis in neurons but did not affect glial viability. WNV-infected astrocytic cells secreted cytotoxic factors, which caused neuronal apoptosis. The expression of the WNV-NY99 capsid protein in neurons and glia by a Sindbis virus-derived vector (SINrep5-WNVc) caused neuronal death and the release of neurotoxic factors by infected astrocytes, coupled with proinflammatory gene induction and suppression of OASIS. Striatal implantation of SINrep5-WNV(C) induced neuroinflammation in rats, together with the induction of CXCL10 and diminished OASIS expression, compared to controls. Moreover, magnetic resonance neuroimaging showed edema and tissue injury in the vicinity of the SINrep5-WNVc implantation site compared to controls, which was complemented by neurobehavioral abnormalities in the SINrep5-WNVc-implanted animals. These studies underscore the important interactions between the WNV capsid protein and neuroinflammation in the pathogenesis of WNV-induced neurological disorders.

Phosphorylation of Cdc25C by pp90Rsk contributes to a G2 cell cycle arrest in Xenopus cycling egg extracts.

In unfertilized Xenopus eggs, the p42 mitogen activated protein kinase (p42MAPK) pathway is known to maintain cell cycle arrest at metaphase of meiosis II. However, constitutive activation of p42MAPK in post-meiotic, cycling Xenopus egg extracts can lead to either a G2 or M-phase arrest of the cell cycle, depending on the timing of p42MAPK activation. Here, we examined the molecular mechanism by which activation of the p42MAPK pathway during interphase leads to cell cycle arrest in G2. When either a recombinant wild type Cdc25C(WT) or a mutated form of Cdc25C, in which serine 287 was replaced by an alanine (S287A), was added to cycling egg extracts, S287A accelerated entry into M-phase. Furthermore, the addition of S287A overcame the G2 arrest caused by p42MAPK, driving the extract into M-phase. p90Rsk a kinase that is the target of p42MAPK, was phosphorylated and activated (pp90Rsk) in the G2-arrested egg extracts, and was able to phosphorylate WT but not S287A in vitro. 14-3-3 proteins were associated with endogenous Cdc25C in G2-arrested extracts. Cdc25C(WT) that had been phosphorylated by pp90(Rsk) bound 14-3-3zeta, whereas S287A could not. These data suggest that the link between the p42MAPK signaling pathway and Cdc25C involves the activation of pp90Rsk and its phosphorylation of Cdc25C at S287, causing the binding of 14-3-3 proteins. We propose that the binding of 14-3-3 proteins to pp90Rsk phosphorylated-Cdc25C results in a G2 arrest in a manner similar to the cell cycle delays induced by differentiation signals that occur later in embryonic development.