Jenae Limoges

Microglial and Astrocyte Chemokines Regulate Monocyte Migration through the Blood-Brain Barrier in Human Immunodeficiency Virus-1 Encephalitis

The numbers of immune-activated brain mononuclear phagocytes (MPs) affect the progression of human immunodeficiency virus (HIV)-1-associated dementia (HAD). Such MPs originate, in measure, from a pool of circulating monocytes. To address the mechanism(s) for monocyte penetration across the blood-brain barrier (BBB), we performed cross-validating laboratory, animal model, and human brain tissue investigations into HAD pathogenesis. First, an artificial BBB was constructed in which human brain microvascular endothelial and glial cells-astrocytes, microglia, and/or monocyte-derived macrophages (MDM)-were placed on opposite sides of a matrix-coated porous membrane. Second, a SCID mouse model of HIV-1 encephalitis (HIVE) was used to determine in vivo monocyte blood-to-brain migration. Third, immunohistochemical analyses of human HIVE tissue defined the relationships between astrogliosis, activation of microglia, virus infection, monocyte brain infiltration, and beta-chemokine expression. The results, taken together, showed that HIV-1-infected microglia increased monocyte migration through an artificial BBB 2 to 3.5 times more than replicate numbers of MDM. In the HIVE SCID mice, a marked accumulation of murine MDM was found in areas surrounding virus-infected human microglia but not MDM. For human HIVE, microglial activation and virus infection correlated with astrogliosis, monocyte transendothelial migration, and beta-chemokine expression. Pure cultures of virus-infected and activated microglia or astrocytes exposed to microglial conditioned media produced significant quantities of beta-chemokines. We conclude that microglial activation alone and/or through its interactions with astrocytes induces beta-chemokine-mediated monocyte migration in HAD.

An analysis of HIV-1-associated inflammatory products in brain tissue of humans and SCID mice with HIV-1 encephalitis

The human immunodeficiency virus type 1 (HIV)-associated dementia complex (ADC) is a neuroimmunological disorder fueled by viral replication in mononuclear phagocytes (MP) (brain macrophages and microglia). The elucidation of MP inflammatory factors involved in neurological dysfunction is pivotal for unraveling pathogenic mechanisms and in developing new therapies for this disease. Recent advances in animal model systems for ADC and its associated encephalitis have provided important insights into how virus-infected macrophages cause brain injury. Indeed, the stereotactic inoculation of HIV infected monocytes into the basal ganglia/cortex of mice with severe combined immunodeficiency disease (SCID) results in pathological features similar to those of human HIV-1 encephalitis (HIVE). We used this SCID model to study the roles of macrophage secretory factors in HIVE. The expression of interleukin-1 (IL-1 beta, IL-6, IL-10), tumor necrosis factors-alpha (TNF alpha), vascular endothelial growth factor (VEGF), and adhesion molecules (E-selectin, intracellular cell adhesion molecule (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1)) in encephalitic brains of mice and humans was evaluated by semi-quantitative polymerase chain reaction (PCR). In SCID mice with HIVE, human and mouse TNF alpha, and mouse IL-6, VEGF, VCAM-1 and E-selectin were expressed at high levels. These results paralleled, to a great extent, those in HIVE brain tissues. Laser scanning confocal microscopy performed to assess the associated neuronal damage showed that microtubule associated protein-2 (MAP-2) immunoreactive dendrites were significantly reduced in both the ipsilateral and contralateral hemispheres of encephalitic mice. These results demonstrate the importance of macrophage inflammatory products in the pathogenesis of HIVE and further validates this model of viral encephalitis in SCID mice.

Reduction in glial immunity and neuropathology by a PAF antagonist and an MMP and TNFα inhibitor in SCID mice with HIV-1 encephalitis

The effects of anti-inflammatory drugs on glial immunity and neuropathology were determined in a severe combined immune deficiency (SCID) mouse model of HIV-1 encephalitis. HIV-1-infected human monocyte-derived macrophages (MDM) are stereotactically inoculated into basal ganglia resulting in a multinucleated giant cell encephalitis. A platelet activating factor antagonist and a matrix metalloproteinase inhibitor, which also inhibits tumor necrosis factor alpha release, were administered to animals at the time of the MDM inoculation. The drugs administered in combination markedly reduced brain inflammation, astrogliosis and microglia activation. These findings demonstrate that reduction of brain inflammatory responses, independent of viral replication, can affect HIVE pathology in an animal model system of disease.

Evaluation of antiretroviral drug efficacy for HIV-1 encephalitis in SCID mice

Objectives: To compare the efficacy of the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir, zidovudine (AZT), lamivudine (3TC), didanosine (ddI), and stavudine (d4T) to inhibit viral replication in brain macrophages. A severe combined immunodeficiency (SCID) mouse model of HIV-1 encephalitis (HIVE) was used to monitor spreading viral infection in the CNS. Background: The development of antiretroviral therapies with CNS efficacy against neuroinvasive virus is important if eradication of HIV-1 can be achieved within critical “hidden reservoirs.” Methods: HIV-1–infected human monocyte–derived macrophages (MDMs) (after a single round of viral replication) were inoculated into the caudate and putamen of SCID mice. This resulted in the spreading of viral infection with a concomitant multinucleated giant cell encephalitis (astrogliosis, microglial activation, and neuronal injury). NRTIs were administered to animals at the time of intracerebral MDM inoculations and continued until the time of sacrifice. Antiretroviral effects were assessed by viral load and percentages of infected MDMs. Results: In brains of SCID mice with HIVE, abacavir and lamivudine reduced HIV-1 p24 antigen–positive cells by 80% and 95%, respectively, whereas both decreased viral load by ∼1 log. Zidovudine, didanosine, and stavudine showed variable effects. Conclusions: Abacavir and lamivudine showed significant antiretroviral activity in SCID mice with HIVE when compared with other NRTIs. The extrapolation of these results to humans with HIV-1 dementia awaits future investigations.

Molecular Characterization of a Putative Antiretroviral Transcriptional Factor, OTK18

Elucidation of the factors involved in host defense against human immunodeficiency viral infection remains pivotal if viral control may be achieved. Toward these ends, we investigated the function of a putative antiretroviral factor, OTK18, isolated by differential display of mRNA from HIV type 1-infected primary human monocyte-derived macrophages. Molecular and immunohistochemical analyses showed that the OTK18 nucleotide sequence contains 13 adjacent C2H2-type zinc finger motifs, a Krüppel-associated box, and is localized to both cytosol and nucleus. Mutational analyses revealed that both the Krüppel-associated box and zinc finger regions of OTK18 are responsible for the transcriptional suppressive activities of this gene. OTK18 was copiously expressed in macrophages following HIV type I infection and diminished progeny virion production. A mechanism for this antiretroviral activity was by suppression of HIV type 1 Tat-induced viral long terminal repeat promoter activity. Our findings suggest that one possible function of OTK18 is as a HIV type 1-inducible transcriptional suppresser.

Peripheral nerve induces macrophage neurotrophic activities: regulation of neuronal process outgrowth, intracellular signaling and synaptic function

Rat cortical neurons cultured in conditioned media from human monocyte-derived macrophages (MDM) show increased neuronal protein synthesis, neurite outgrowth, mitogen-activating protein kinase activity, and synaptic function. Neurotrophic properties of human MDM-conditioned media are significantly enhanced by human peripheral nerve and to a more limited extent by CD40 ligand pre-stimulation. Such positive effects of MDM secretions on neuronal function parallel the secretion of brain-derived neurotrophic factor (BDNF). MDM activation cues may serve to balance toxic activities produced during neurodegenerative diseases and thus, under certain circumstances, mitigate neuronal degeneration.

Dexamethasone Therapy Worsens the Neuropathology of Human Immunodeficiency Virus Type 1 Encephalitis in SCID Mice

Human immunodeficiency virus (HIV) dementia is a late complication of viral infection. Cognitive dysfunction revolves around the secretion of neurotoxins from immunologically competent virus-infected brain macrophages and microglia. Such macrophage neurotoxins are inflammatory factors that produce selective neuronal dysfunction and ultimately cell death. To evaluate the potential efficacy of antiinflammatory therapy for HIV dementia, dexamethasone was administered to severe combined immunodeficient mice with HIV-1 encephalitis. Mice were given therapeutic doses of dexamethasone before intracerebral inoculation with HIV-1-infected human monocytes. Histochemical evaluation showed a worsening of neuropathology after treatment, with astrogliosis and increased apoptosis of neurons. Laboratory investigation of the mechanisms for the dexamethasone effects revealed increased viability of HIV-infected macrophages and incomplete suppression of neurotoxic inflammatory secretions. The results suggest the need for caution in administering glucocorticoids for treatment of HIV encephalitis in humans.

OTK18 expression in brain mononuclear phagocytes parallels the severity of HIV-1 encephalitis

OTK18 was isolated by mRNA differential display of human monocyte-derived macrophages (MDM) infected with human immunodeficiency virus type one (HIV-1). Northern blot and real-time reverse transcription polymerase chain reaction showed low levels of OTK18 expression in human tissue, which markedly increased during advanced HIV-1 encephalitis (HIVE). Immunocytochemistry, using rabbit polyclonal antisera, showed OTK18 localized to brain mononuclear phagocytes (MP) in moderate to severe HIV-1 encephalitis. OTK18 expression was selective and not found in HIV-1-infected brain tissue with limited neuropathological abnormalities, nor in cytomegalovirus encephalitis, multiple sclerosis, Alzheimers disease, or uninfected control brains. Thus, OTK18 expression in brain mononuclear phagocytes is a signature for advanced HIV-1 encephalitis.

Levels of human immunodeficiency virus type 1 (HIV-1) replication in macrophages determines the severity of murine HIV-1 encephalitis

The presence of specific neuroinvasive strains and necessity for brain viral replication for disease progression remain controversial issues in neuro-AIDS research. To investigate these questions, the authors injected human monocyte-derived macrophages (MDMs) infected with diverse viral strains were injected into the caudate and putamen of severe combined immunodeficient (SCID) mice. Independent of viral strain, infected MDMs became immunologically activated and elicited profound inflammatory reactions in brain areas most affected in humans. The intensity of neuropathologic changes, including microglial reactions, paralleled levels of viral infection and numbers of infected MDMs. The data suggest that HIV-1-associated neurological disease is related to the level of productive viral infection in activated macrophages. Virus infection, per se, may affect the ability of macrophages to respond to immune stimuli by overproduction of proinflammatory factors and neurotoxins, leading to neuronal dysfunction.

Inflammatory responses in SCID mice with HIV-1 encephalitis and drug development for HIV-1 neurological disorders

377 Induction of Cytokines and Anti-cytokine Autoantibodies in Cerebro-spinal Fluid During Experimental Bacterial Meningitig M_ Mustafa, A. Diab, J. Zhu, L. Lindquist, M. Bakhiet, Karolinska Institute, Sweden 380 Retrovirus, Cytotoxic Molecules Including Superantigen and Multiple Sclerosis: Epiphcnomenon or New Avenue of Research H. Perron, F. Pradel, G. Baccala-Paranhos, F. Bedin, u1~1o3 CNRS/BioMerisux, Lyon, France, A. Paraz-Ounanian, Joseph Fourier Univ., Grenoble, France, C. JolivetRaynaud, UMR I 03 CNRS/BioMerieux, Lyon, France