The Animal Models Unit has employed a murine model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE), to focus on the mechanisms driving pathogenic autoreactivity and triggers of autoimmunity in the CNS as well as investigatingnew therapeutic approaches to treat autoimmune demyelination in the CNS using EAE. The first approach targets a reduction in the inflammatory component of the disease. In collaboration with John Hallenbeck of the Stroke Branch, NINDS, we have thus far shown that intranasal administration of the human cell adhesion molecule E selectin results in a tolerization process to this molecule. In the active induction model of EAE, E selectin tolerization pretreatment can reduce the incidence and severity of clinical symptoms once disease is induced. Moreover, preliminary studies also show a reduction in the severity of disease when E selectin is administered once disease has been established, emphasizing the therapeutic potential of this approach. Studies to identify the tolerogenic populations generated and their mechanisms of action in this model are ongoing. Thus far, E selectin-specific populations have been identified and antibodies to E selectin have been characterized. Transfer of T cells from E selectin tolerized animals modulate disease, supporting a role for E selectin-specific regulatory populations. A patent has been filed for this work. An extension of this work has been intranasal administration of E selectin in models of atherosclerosis and plaque rupture. Thus far, E selectin tolerized animals show less lipid deposition in the aortic arch compared to PBS tolerized animals. Histopathological and array analyses are currently underway, supported by an NINDS/NHLBI Bench to Bedside Proposal. A second study in collaboration with James Mitchell, NCI has been investigating the potential for a well characterized anti-oxidant to prevent or treat autoimmune demyelination in the EAE model. Studies suggest this antioxidant is protective against induction of EAE. Furthermore, transfer of myelin-reactive T cells into animals on anti-oxidant feed develop less severe disease compared to animals on control feed. This reduction in disease is also observed when animals are administered anti-oxidant feed at the onset of clinical symptoms, supporting both an immunomodulatory and neuroprotective role for antioxidants in treating autoimmune-driven CNS disease. A patent has been filed describing this work and studies are ongoing to determine the precise mechanisms involved. We have also successfully established two models of EAE a modelin the marmoset. In these models, either recombinant human myelin oligodendrocyte glycoprotein (MOG) or human white matter homogenate is used as the auto-antigen to drive disease. Using these methods we are able to follow clinical and MRI parameters that will allow us to test new disease modifying therapies. Using the 7T MRI, we now have a MRI protocol optimized to follow lesion development and quantitate lesion loads in affected marmosets. We are currently working to expand the MRI protocol in collaboration with Afonso Silva in NINDS to obtain images of the spinal cord in the marmosets, to better track disease development. We are also working to establish another model of demyelination using cuprizone treatment. This has never been done in the marmoset and will allow us to follow demyelination, remyelination, and axonal damage by MRI in a well-defined system. Magnetic resonance spectroscopy (MRS) protocols are currently in development for the analysis of the cuprizone model as well.
The aims of this study are to find MRI parameters that may act as markers of demyelination, remyelination, or axonal damage. This could result in the development of improved MRI techniques that can be transferred to human MRI providing more clinically relevant information in a non-invasive way and thus improve clinical care. We are also conducting studies to examine the role of human viruses in CNS disease, focusing on the role of human herpesvirus 6. HHV-6 is associated with a variety of neurologic diseases including multiple sclerosis, encephalitis, epilepsy, and brain cancer. The virus is ubiquitous and has two variants HHV-6A and HHV-6B. Exposure to HHV-6B occurs at a young age and is the etiologic agent of Roseola. Given the ubiquitous nature of the virus and the early exposure time, it is difficult to prove causation of neurologic disease by the virus. The generation of an animal model of HHV-6 infection would allow studies on the potential of this virus to cause neurologic disease. We initially inoculated marmosets with HHV-6A, HHV-6B, or vehicle control intravenously and followed disease development clinically, radiologically, and immunologically. Marmosets were exposed to virus every 30 days for a total of 4 exposures. None of the virus inoculated animals showed signs of a primary infection;however shortly after the second exposure HHV-6A inoculated marmosets developed signs of neurologic disease. The neurologic disease was seen in 3 of 4 exposed marmosets and was characterized primarily by sensory deficits. We saw no changes in MRI in the brains of affected marmosets during the initial disease development. We were able to observe transient areas of hyperintensity in the corpus callosum on T2 weighted images 25 weeks post-inoculation in one marmoset. Marmosets exposed to HHV-6A generated a rapid antibody response to HHV-6 generating both IgM and IgG antibodies against the virus. This was not seen in the HHV-6B inoculated marmosets only 2 of the 4 animals generated HHV-6 specific IgG antibodies and no IgM antibodies were detected. We detected very little HHV-6 DNA, using nested PCR, in the PBMC, plasma, and saliva of virus exposed marmosets. Currently we are developing methods to look at cellular immune responses to HHV-6 in cells from the virus-exposed marmosets. The route of HHV-6 exposure may also influence the disease development seen as a result of viral infection. Human exposure to HHV-6 occurs most likely through mucous membranes, and we have recently shown that the nasal mucous is a reservoir for the virus. To examine a more physiologic route of exposure we inoculated another group of marmosets with HHV-6A intranasally. In this group of marmosets we did not observe signs of neurologic disease or detect HHV-6 specific antibody responses. We do however consistently find HHV-6 DNA in the PBMC, plasma, and saliva of exposed marmosets. Experiments in this cohort of marmosets are ongoing and data is still being collected and analyzed. We intend to continue optimizing and defining the HHV-6 infection model in the marmosets. With this model we have generated a system in which to study independently the biology of the 2 HHV-6 variants. This is an ongoing problem in studying HHV-6 due to the high homology between the variants and early exposure time to HHV-6B. Reagents to measure immune responses to the individual variants are not available and the marmoset infection models provide a system to develop these reagents. The marmoset infection model also provides a system in which to test anti-herpesvirus drugs to improve current anti-viral therapies. We are also beginning experiments to examine whether exposure to HHV-6 can alter the disease pathogenesis of EAE, to look at whether the virus may be a cofactor in the development of CNS autoimmunity.

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Leibovitch, Emily C; Jacobson, Steven (2018) Viruses in chronic progressive neurologic disease. Mult Scler 24:48-52
Lee, Nathanael J; Ha, Seung-Kwon; Sati, Pascal et al. (2018) Spatiotemporal distribution of fibrinogen in marmoset and human inflammatory demyelination. Brain 141:1637-1649
Absinta, Martina; Sati, Pascal; Schindler, Matthew et al. (2016) Persistent 7-tesla phase rim predicts poor outcome in new multiple sclerosis patient lesions. J Clin Invest 126:2597-609
Luciano, Nicholas J; Sati, Pascal; Nair, Govind et al. (2016) Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning. J Vis Exp :
Guy, Joseph R; Sati, Pascal; Leibovitch, Emily et al. (2016) Custom fit 3D-printed brain holders for comparison of histology with MRI in marmosets. J Neurosci Methods 257:55-63
Lin, Cheng-Te Major; Leibovitch, Emily C; Almira-Suarez, M Isabel et al. (2016) Human herpesvirus multiplex ddPCR detection in brain tissue from low- and high-grade astrocytoma cases and controls. Infect Agent Cancer 11:32
Leibovitch, Emily C; Jacobson, Steven (2016) Vaccinations for Neuroinfectious Disease: A Global Health Priority. Neurotherapeutics 13:562-70
Leibovitch, Emily C; Jacobson, Steven (2015) Human Herpesvirus 6 as a Viral Trigger in Mesial Temporal Lobe Epilepsy. J Infect Dis 212:1011-3
Virtanen, J O; Wohler, J; Fenton, K et al. (2014) Oligoclonal bands in multiple sclerosis reactive against two herpesviruses and association with magnetic resonance imaging findings. Mult Scler 20:27-34
Leibovitch, Emily C; Brunetto, Giovanna S; Caruso, Breanna et al. (2014) Coinfection of human herpesviruses 6A (HHV-6A) and HHV-6B as demonstrated by novel digital droplet PCR assay. PLoS One 9:e92328

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