William R. Tyor

Oral simvastatin treatment in relapsing-remitting multiple sclerosis.

Many drugs have been approved for relapsing forms of multiple sclerosis but are only partly effective, are injected, and are expensive. We aimed to investigate use of of oral simvastatin (80 mg) in 30 individuals with relapsing-remitting multiple sclerosis. The mean number of gadolinium-enhancing lesions at months 4, 5, and 6 of treatment was compared with the mean number of lesions noted on pretreatment brain MRI scans. Number and volume of Gd-enhancing lesions declined by 44%, (p<0.0001) and 41% (p=0.0018), respectively. Treatment was well tolerated. Oral simvastatin might inhibit inflammatory components of multiple sclerosis that lead to neurological disability.

Unifying hypothesis for the pathogenesis of HIV-associated dementia complex, vacuolar myelopathy, and sensory neuropathy

Neurological diseases associated with HIV infection include dementia, vacuolar myelopathy, and sensory neuropathy. Although in vitro studies suggest that other nervous system cell types could harbor HIV, immunohistochemical and in situ hybridization studies have indicated that only macrophages/microglia are significantly infected in the central nervous system. In the peripheral nervous system, even HIV-infected macrophages are rare. Therefore, theories regarding the pathogenesis of HIV-associated neurologic disorders have centered around the elaboration of substances that may be toxic to neurons, oligodendrocytes or myelin. These potential toxins include HIV proteins, cellular metabolites, and cytokines. In this review we present evidence that there are large numbers of macrophages/microglia present in the nervous system of patients with these diseases and that they produce tumor necrosis factor (TNF)-alpha. The large increase in macrophage activity late in HIV infection may be due to the diminution in production by CD4-positive T cells of cytokines such as interleukin (IL)-4 and IL-10 which are inhibitors of macrophage activities. We hypothesize that HIV-associated dementia complex, vacuolar myelopathy, and sensory neuropathy are directly or indirectly related to the increased numbers of macrophages found in brain, spinal cord, and peripheral nerve. Future therapies may be directed towards inhibition of macrophage functions.

The role of antibody in recovery from alphavirus encephalitis

Alphaviruses infect neurons in the brain and spinal cord and cause acute encephalomyelitis in a variety of mammals. The outcome of infection is determined by whether the neurons survive infection and this, in turn, is determined by the virulence of the virus and the age of the host at the time of infection. We have been studying Sindbis virus (SV) infection i of mice as a model system for alphavirus‐induced encephalomyelitis. Investigation of intracerebral infection of weanling mice with two different strains of SV bas allowed us to analyze the role of the immune response in protection from fatal disease virulent NSV strain) and in CLEARANCE of virus from the nervous system during non‐fatal disease (less virulent SV AR339 strain). Neutralizing and non‐neutralizing antibodies to the El and E2 surface glycoproteins can protect mice from fatal NSV infection when given before or after infection, while T cells are not protective, The mechanism of antibody‐mediated protection is not known, but it is likely that more than one mechanism Is involved and that different mechanisms are involved in pre‐infection and post‐infection treatment protection. Clearance of infectious virus from the nervous system of mice during recovery from non‐fatal disease is accomplished by antibodies to the E2 glycoprotein. The process does not involve damage to the infected neurons and is independent of complement and mononuclear cells. Bivalent antibody is required and binds to the surface of the infected cell. Initially, release of virus by budding from the cell surface is prevented and, subsequently, intracellular virus replication is inhibited possibly through antiviral mechanisms induced in co‐operation with interferon. This non lytic mechanism for control of virus infection results in the prolonged presence of viral RNA in tissue and the need for prolonged intrathecal synthesis of antiviral antibody by B cells within the central nervous system.

HIV-1 clade-specific differences in the induction of neuropathogenesis

Human immunodeficiency virus (HIV)-associated dementia (HAD) is common among clade B HIV-infected individuals, but less common and less severe among individuals infected with clade C HIV-1, suggesting clade-specific differences in neuropathogenicity. Although differences in neuropathogenicity have been investigated in vitro using viral proteins responsible for HAD, to date there are no virological studies using animal models to address this issue. Therefore, we investigated neuropathogenesis induced by HIV-1 clades using the severe combined immune deficiency (SCID) mouse HIV encephalitis model, which involves intracranial injection of macrophages infected with representative clade B (HIV-1ADA) or clade C (HIV-1Indie-C1) HIV-1 isolates into SCID mice. In cognitive tests, mice exposed to similar inputs of HIV-1 clade C made fewer memory errors than those exposed to HIV-1 clade B. Histopathological analysis of mice exposed to clade B exhibited greater astrogliosis and increased loss of neuronal network integrity. In vitro experiments revealed differences in a key characteristic of HIV-1 that influences HAD, increased monocyte infiltration. HIV-1Indie-C1-infected macrophages recruited monocytes poorly in vitro compared with HIV-1ADA-infected macrophages. Monocyte recruitment was HIV-1 Tat and CCL2 dependent. This is the first demonstration, ever since HIV neuropathogenesis was first recognized, that viral genetic differences between clades can affect disease severity and that such studies help identify key players in neuropathogenesis by HIV-1.

Cytokine expression of macrophages in HIV‐1‐associated vacuolar myelopathy

Macrophages are frequently present within the periaxonal and intramyelinic vacuoles that are located primarily in the posterior and lateral funiculi of the thoracic spinal cord in HIV-associated vacuolar myelopathy. But the role of these macrophages in the formation of the vacuoles is unclear. One hypothesis is that cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor (TNF)-α, are produced locally by macrophages and have toxic effects on myelin or oligodendrocytes. The resulting myelin damage eventually culminates in the removal of myelin by macrophages and vacuole formation. We studied thoracic spinal cord specimens taken at autopsy from HIV-positive (+) and HIV-negative individuals. The predominant mononuclear cells present in HIV+ spinal cords are macrophages. They are located primarily in the posterior and lateral funiculi regardless of the presence or absence of vacuolar myelopathy. Macrophages and microglia are more frequent in HIV+ than HIV-negative individuals and these cells frequently stain for class I and class II antigens, IL-1, and TNF-α. Activated macrophages positive for IL-1 and TNF-α are greatly increased in the posterior and lateral funiculi of HIV+ individuals with and without vacuolar myelopathy, suggesting they are present prior to the development of vacuoles. Cytokines, such as TNF-α, may be toxic for myelin or oligodendrocytes, leading to myelin damage and removal by macrophages and vacuole formation.

Treatment of spinal cord impact injury in the rat with transforming growth factor-β

To investigate the contribution of cytokines, proinflammatory TNF-alpha and inhibitory TGF-beta, to spinal cord injury (SCI) in a rat model, two studies were performed using adult Sprague-Dawley rats which were injured at T9/T10. In the first study, rats were sacrificed at 1, 6, 24, 96 and 168 h after SCI for immunocytochemistry of coronal sections for the presence of mononuclear phagocytes, astrocytes, TNF-alpha and TGF-beta, among other markers. From intervening frozen sections, RNA was extracted for semiquantitative polymerase chain reaction (RT-PCR) analysis of TNF-alpha and TGF-beta. In the second experiment, rats were treated with intravenous TGF-beta 30 min after injury and sacrificed at 6 and 48 h after injury. Spinal cord sections were immunocytochemically stained and RNA extracted for semiquantitative PCR as mentioned above, as well as quantitation of lesion volume. There were increases in mononuclear phagocytes and astrocytes, as early as 1 h after SCI, with steady progression over 168 h after injury. TNF-alpha and TGF-beta was produced locally by mononuclear phagocytes and astrocytes. There was an 18-h delay in peak mRNA production of TGF-beta compared to TNF-alpha. The treatment of SCI rats with TGF-beta reduced lesion volume by 50% at 48 h and this was associated with decreased accumulation of mononuclear phagocytes in and around the injury site. This reduction of mononuclear phagocyte numbers around the site of trauma would reduce their contribution to secondary injury.

Molecular Targets of Opiate Drug Abuse in NeuroAIDS

Opiate drug abuse, through selective actions at μ opioid receptors (MOR), exacerbates the pathogenesis of human immunodeficiency virus-1 (HIV-1) in the CNS by disrupting glial homeostasis, increasing inflammation, and decreasing the threshold for pro-apoptotic events in neurons. Neurons are affected directly and indirectly by opiate-HIV interactions. Although most opiates drugs have some affinity for κ (KOR) and/or δ (DOR) opioid receptors, their neu-rotoxic effects are largely mediated through MOR. Besides direct actions on the neurons themselves, opiates directly affect MOR-expressing astrocytes and microglia. Because of their broad-reaching actions in glia, opiate abuse causes widespread metabolic derangement, inflammation, and the disruption of neuron-glial relationships, which likely contribute to neuronal dysfunction, death, and HIV encephalitis. In addition to direct actions on neural cells, opioids modulate inflammation and disrupt normal intercellular interactions among immu-nocytes (macrophages and lymphocytes), which on balance further promote neuronal dysfunction and death. The neural pathways involved in opiate enhancement of HIV-induced inflammation and cell death, appear to involve MOR activation with downstream effects through PI3-kinase/Akt and/or MAPK signaling, which suggests possible targets for therapeutic intervention in neuroAIDS.

Interferon-α Causes Neuronal Dysfunction in Encephalitis

Interferon-α (IFNα) is a pleomorphic cytokine produced by nucleated cells in response to viral infection. In patients, treatment with IFNα has side effects including cognitive impairment resembling subcortical dementia, which is a hallmark of human immunodeficiency virus (HIV)-associated dementia (HAD). IFNα is increased in the CSF of HAD patients compared with HIV patients without dementia. In this study, blocking IFNα in a HIV encephalitis (HIVE) mouse model with intraperitoneal injections of IFNα neutralizing antibodies (NAbs) significantly improved cognitive function compared with untreated or control antibody-treated HIVE mice during water radial arm maze behavioral testing. Treatment with IFNα NAbs significantly decreased microgliosis and prevented loss of dendritic arborization in the brains of HIVE mice. Furthermore, treatment of primary neuron cultures with IFNα resulted in dose-dependent loss of dendritic arborization that was blocked with IFNα NAb treatment and partially blocked with NMDA antagonists [AP5 and MK801 (dizocilpine maleate)] indicating glutamate signaling is involved in IFNα-mediated neuronal damage. These results show that IFNα has a major role in the pathogenesis of HIVE in mice and is likely important in the development neurocognitive dysfunction in humans with HIV. Blocking IFNα could be important in improving cognitive and pathological developments in HAD patients and may be clinically important in other neuroinflammatory diseases as well.

SCID mice with HIV encephalitis develop behavioral abnormalities

Severe combined immunodeficient (SCID) mice inoculated intracerebrally (i.c.) with HIV-infected human monocytes develop brain pathology similar to that in humans with HIV encephalitis. This includes HIV-positive macrophages and multinucleated giant cells, astrogliosis, microglial nodules, and neuronal dropout. These xenografts survive about 1 month. To develop a model of chronic HIV encephalitis and to assay the resulting behavioral abnormalities, we reinoculated SCID mice i.c. every 4 weeks for 3 months with either HIV-infected human monocytes (n = 5) or uninfected human macrophages (n = 4) or administered no inoculation (n = 6); these three groups were monitored for behavioral abnormalities. Tests of cognitive function in a Morris water maze 3.5 months after the first inoculation suggested that HIV-infected mice performed poorly compared with controls. Following testing in the water maze on days 4 and 5 of acquisition, motor activity of infected mice was reduced in comparison with that of controls. Retention of goal location when tested 1 week later was impaired in HIV-infected mice compared with controls. Histopathologic analysis of brains revealed significant astrogliosis and strongly suggested higher numbers of major histocompatibility complex (MHC) class II-positive multinucleated macrophages in HIV-infected compared with control mice. Thus, our preliminary studies indicate that SCID mice with HIV encephalitis develop behavioral abnormalities reminiscent of human disease. These behavioral abnormalities are associated with significantly increased astrogliosis, the presence of HIV, and probably multinucleated giant cells. These studies further support the use of this SCID animal model system for studies of the pathogenesis of HIV encephalitis and for drug interventions.

Upregulation of calpain correlates with increased neurodegeneration in acute experimental auto-immune encephalomyelitis.

Although calpain up‐regulation is well established in experimental auto‐immune encephalomyelitis (EAE), a link between increased calpain expression and activity and neurodegeneration has not been examined. Therefore, spinal cord tissue from Lewis rats with EAE was examined to test the hypothesis that increased calpain expression in neurons would correlate with increased cell death and axonal damage in a time‐dependent manner following EAE induction. We found that increased calpain expression in EAE corresponded to increased TUNEL‐positive neurons and to increased expression of dephosphorylated neurofilament protein, markers of cell death and axonal degeneration, respectively. An increase in internucleosomal DNA fragmentation in EAE spinal cord suggested that cell death was, at least partially, due to apoptosis. Axonal damage was further demonstrated in EAE spinal cord compared with control via morphological analysis, revealing granular degeneration of filament and microtubule integrity, loss of myelin, and mitochondrial damage. Calcium (Ca2+) influx, which is required for calpain activation, was also increased in EAE spinal cord. From these findings, we conclude that increases in Ca2+‐induced calpain activity may play a crucial role in neurodegeneration in acute EAE.