Neuropilin-1 modulates interferon-γ-stimulated signaling in brain microvascular endothelial cells.

Inflammatory response of blood-brain barrier (BBB) endothelial cells plays an important role in pathogenesis of many central nervous system inflammatory diseases, including multiple sclerosis; however, the molecular mechanism mediating BBB endothelial cell inflammatory response remains unclear. In this study, we first observed that knockdown of neuropilin-1 (NRP1), a co-receptor of several structurally diverse ligands, suppressed interferon-γ (IFNγ)-induced C-X-C motif chemokine 10 expression and activation of STAT1 in brain microvascular endothelial cells in a Rac1-dependent manner. Moreover, endothelial-specific NRP1-knockout mice, VECadherin-Cre-ERT2/NRP1flox/flox mice, showed attenuated disease progression during experimental autoimmune encephalomyelitis, a mouse neuroinflammatory disease model. Detailed analysis utilizing histological staining, quantitative PCR, flow cytometry and magnetic resonance imaging demonstrated that deletion of endothelial NRP1 suppressed neuron demyelination, altered lymphocyte infiltration, preserved BBB function and decreased activation of the STAT1-CXCL10 pathway. Furthermore, increased expression of NRP1 was observed in endothelial cells of acute multiple sclerosis lesions. Our data identify a new molecular mechanism of brain microvascular endothelial inflammatory response through NRP1-IFNγ crosstalk that could be a potential target for intervention of endothelial cell dysfunction in neuroinflammatory diseases.

In vivo detection of connectivity between cortical and white matter lesions in early MS.

BACKGROUND: The relationship between cortical lesions (CLs) and white matter lesions (WMLs) in multiple sclerosis (MS) is poorly understood. Pathological studies support a topographical association between CLs and underlying subcortical WMLs and suggest CLs may play a role in both disease initiation and progression. We hypothesized that cortical MS lesions are physically connected to white matter MS lesions via axonal connections. OBJECTIVE: To assess the presence of CL-WML connectivity utilizing novel magnetic resonance imaging (MRI) methodology. METHODS: In all, 28 relapsing-remitting MS patients and 25 controls received 3 T MRI scans, including double inversion recovery (DIR) for CL detection coupled with diffusion tensor imaging (DTI). CL and WML maps were created, and DTI was used to calculate inter-lesional connectivity and volumetric connectivity indices. RESULTS: All patients showed inter-lesional WML connectivity (median 76% of WMLs connected to another WML; interquartile range (IQR), 58%-88%). On average, 52% of detected CLs per patient were connected to at least one WML (IQR, 42%-71%). Volumetric connectivity analysis showed significantly elevated cortical lesion ratios in MS patients (median, 2.3; IQR, 1.6-3.3) compared to null MS and healthy control datasets ( p < 0.001). CONCLUSION: These findings provide strong evidence of inter-lesional connectivity between CLs and WMLs, supporting our hypothesis of intrinsic CL-WML connectivity.

Clinical and pathological insights into the dynamic nature of the white matter multiple sclerosis plaque.

OBJECTIVE: An extensive analysis of white matter plaques in a large sample of multiple sclerosis (MS) autopsies provides insights into the dynamic nature of MS pathology. METHODS: One hundred twenty MS cases (1,220 tissue blocks) were included. Plaque types were classified according to demyelinating activity based on stringent criteria. Early active, late active, smoldering, inactive, and shadow plaques were distinguished. A total of 2,476 MS white matter plaques were identified. Plaque type distribution was analyzed in relation to clinical data. RESULTS: Active plaques were most often found in early disease, whereas at later stages, smoldering, inactive, and shadow plaques predominated. The presence of early active plaques rapidly declined with disease duration. Plaque type distribution differed significantly by clinical course. The majority of plaques in acute monophasic and relapsing-remitting MS (RRMS) were active. Among secondary progressive MS (SPMS) cases with attacks, all plaque types could be distinguished including active plaques, in contrast to SPMS without attacks, in which inactive plaques predominated. Smoldering plaques were frequently and almost exclusively found in progressive MS. At 47 years of age, an equilibrium was observed between active and inactive plaques, whereas smoldering plaques began to peak. Men displayed a higher proportion of smoldering plaques. INTERPRETATION: Disease duration, clinical course, age, and gender contribute to the dynamic nature of white matter MS pathology. Active MS plaques predominate in acute and early RRMS and are the likely substrate of clinical attacks. Progressive MS transitions to an accumulation of smoldering plaques characterized by microglial activation and slow expansion of pre-existing plaques. Whether current MS therapeutics impact this pathological driver of disease progression remains uncertain.

MR microscopy of formalin fixed paraffin embedded histology specimens.

PURPOSE: Archived formalin-fixed paraffin-embedded (FFPE) tissue collections represent a valuable informational resource for numerous studies. However, there is no NMR signal from FFPE specimens at room temperature. To obtain MR images and enable comparison of magnetic resonance microscopy (MRM) and histology studies we propose to image FFPE tissue at elevated temperature. METHODS: A FFPE tissue block was imaged at 7 Tesla (T) and 16.4T at 70-80°C using T2 -weighting methods. The only difference from standard MR microscopy (MRM) is elevated temperature at which the embedding medium melts. RESULTS: Using FFPE murine brain tissue, we were able to demonstrate the feasibility of tissue MRM from paraffin embedded specimens. Histology images taken from the specimen after MRM demonstrate that keeping the FFPE specimen in paraffin melt at 70-80°C for dozens of hours does not affect subsequent histology analysis. CONCLUSION: MRM of FFPE tissue from paraffin melt opens new avenues for analyzing archived histological specimens (biopsies, post mortem, etc.) and for correlating MR images with histology (optical microscopy). In addition, 3D MRM of FFPE specimens could guide histology in search for appropriate regions of interest and subsequently minimize occurrences of false negatives in tissue analysis.

Theiler's murine encephalomyelitis virus as an experimental model system to study the mechanism of blood-brain barrier disruption.

Theiler's murine encephalomyelitis virus is a widely used model to study the initiation and progression of multiple sclerosis. Many researchers have used this model to investigate how the immune system and genetic factors contribute to the disease process. Current research has highlighted the importance of cytotoxic CD8 T cells and specific major histocompatibility complex (MHC) class I alleles. Our lab has adopted this concept to create a novel mouse model to study the mechanism of blood-brain barrier (BBB) disruption, an integral feature of numerous neurological disorders. We have demonstrated that epitope-specific CD8 T cells cause disruption of the tight junction architecture and ensuing CNS vascular permeability in the absence of neutrophil support. This CD8 T cell-initiated BBB disruption is dependent on perforin expression. We have also elucidated a potential role for hematopoietic factors in this process. Despite having identical MHC class I molecules, similar inflammation in the CNS, and equivalent ability to utilize perforin, C57BL/6 mice are highly susceptible to this condition, while 129 SvIm mice are resistant. This susceptibility is transferable with the bone marrow compartment. These findings led us to conduct a comprehensive genetic analysis which has revealed a list of candidate genes implicated in regulating traits associated with BBB disruption. Future studies will continue to define the underlying molecular mechanism of CD8 T cell-initiated BBB disruption and may assist in the development of potential therapeutic approaches to ameliorate pathology associated with BBB disruption in neurological disorders.

CD8 T cell-initiated blood-brain barrier disruption is independent of neutrophil support.

Blood-brain barrier (BBB) disruption is a common feature of numerous neurologic disorders. A fundamental question in these diseases is the extent inflammatory immune cells contribute to CNS vascular permeability. We have previously shown that CD8 T cells play a critical role in initiating BBB disruption in the peptide-induced fatal syndrome model developed by our laboratory. However, myelomonocytic cells such as neutrophils have also been implicated in promoting CNS vascular permeability and functional deficit in murine models of neuroinflammatory disease. For this reason, we evaluated neutrophil depletion in a murine model of CD8 T cell-initiated BBB disruption by employing traditionally used anti-granulocyte receptor-1 mAb RB6-8C5 and Ly-6G-specific mAb 1A8. We report that CNS-infiltrating antiviral CD8 T cells express high levels of granulocyte receptor-1 protein and are depleted by treatment with RB6-8C5. Mice treated with RB6-8C5, but not 1A8, display: 1) intact BBB tight junction proteins; 2) reduced CNS vascular permeability visible by gadolinium-enhanced T1-weighted magnetic resonance imaging; and 3) preservation of motor function. These studies demonstrate that traditional methods of neutrophil depletion with RB6-8C5 are broadly immune ablating. Our data also provide evidence that CD8 T cells initiate disruption of BBB tight junction proteins and CNS vascular permeability in the absence of neutrophil support.

Quantitative trait loci analysis reveals candidate genes implicated in regulating functional deficit and CNS vascular permeability in CD8 T cell-initiated blood-brain barrier disruption.

BACKGROUND: Blood-brain barrier (BBB) disruption is an integral feature of numerous neurological disorders. However, there is a relative lack of knowledge regarding the underlying molecular mechanisms of immune-mediated BBB disruption. We have previously shown that CD8 T cells and perforin play critical roles in initiating altered permeability of the BBB in the peptide-induced fatal syndrome (PIFS) model developed by our laboratory. Additionally, despite having indistinguishable CD8 T cell responses, C57BL/6J (B6) mice are highly susceptible to PIFS, exhibiting functional motor deficits, increased astrocyte activation, and severe CNS vascular permeability, while 129S1/SvImJ (129S1) mice remain resistant. Therefore, to investigate the potential role of genetic factors, we performed a comprehensive genetic analysis of (B6 x 129S1) F2 progeny to define quantitative trait loci (QTL) linked to the phenotypic characteristics stated above that mediate CD8 T cell-initiated BBB disruption. RESULTS: Using single nucleotide polymorphism (SNP) markers and a 95% confidence interval, we identified one QTL (PIFS1) on chromosome 12 linked to deficits in motor function (SNP markers rs6292954, rs13481303, rs3655057, and rs13481324, LOD score = 3.3). In addition we identified a second QTL (PIFS2) on chromosome 17 linked to changes in CNS vascular permeability (SNP markers rs6196216 and rs3672065, LOD score = 3.7). CONCLUSIONS: The QTL critical intervals discovered have allowed for compilation of a list of candidate genes implicated in regulating functional deficit and CNS vascular permeability. These genes encode for factors that may be potential targets for therapeutic approaches to treat disorders characterized by CD8 T cell-mediated BBB disruption.

Potassium channel complex autoimmunity induced by inhaled brain tissue aerosol.

OBJECTIVE: To test the hypothesis that autoimmunity induced by inhalation of aerosolized brain tissue caused outbreaks of sensory-predominant polyradiculoneuropathy among swine abattoir employees in the Midwestern United States. METHODS: Mice were exposed intranasally, 5 days per week, to liquefied brain tissue. Serum from exposed mice, patients, and unaffected abattoir employees were analyzed for clinically pertinent neural autoantibodies. RESULTS: Patients, coworkers, and mice exposed to liquefied brain tissue had an autoantibody profile dominated by neural cation channel immunoglobulin Gs (IgGs). The most compelling link between patients and exposed mice was magnetic resonance imaging (MRI) evidence of grossly swollen spinal nerve roots. Autoantibody responses in patients and mice were dose-dependent and declined after antigen exposure ceased. Autoantibodies detected most frequently, and at high levels, bound to detergent-solubilized macromolecular complexes containing neuronal voltage-gated potassium channels ligated with a high affinity Kv1 channel antagonist, 125I-α-dendrotoxin. Exposed mice exhibited a behavioral phenotype consistent with potassium channel dysfunction recognized in drosophila with mutant ("shaker") channels: reduced sensitivity to isoflurane-induced anesthesia. Pathological and electrophysiological findings in patients supported peripheral nerve hyperexcitability over destructive axonal loss. The pain-predominant symptoms were consistent with sensory nerve hyperexcitability. INTERPRETATION: Our observations establish that inhaled neural antigens readily induce neurological autoimmunity and identify voltage-gated potassium channel complexes as a major immunogen.

Onset of progressive phase is an age-dependent clinical milestone in multiple sclerosis.

BACKGROUND: It is unclear if all patients with relapsing-remitting multiple sclerosis (RRMS) ultimately develop progressive MS. Onset of progressive disease course seems to be age- rather than disease duration-dependent. Some forms of progressive MS (e.g. primary progressive MS (PPMS)) are uncommon in population-based studies. Ascertainment of patients with PPMS from clinic-based populations can facilitate a powerful comparison of age at progression onset between secondary progressive MS (SPMS) and PPMS but may introduce unclear biases. OBJECTIVE: Our aim is to confirm that onset of progressive disease course is more relevant to the patient's age than the presence or duration of a pre-progression relapsing disease course in MS. METHODS: We studied a population-based MS cohort (n=210, RRMS n=109, progressive MS n=101) and a clinic-based progressive MS cohort (n=754). Progressive course was classified as primary (PPMS; n=322), single attack (SAPMS; n=112) and secondary progressive (SPMS; n=421). We studied demographics (chi(2) or t-test), age-of-progression-onset (t-test) and time to Expanded Disability Status Scale of 6 (EDSS6) (Kaplan-Meier analyses). RESULTS: Sex ratio (p=0.58), age at progression onset (p=0.37) and time to EDSS6 (p=0.16) did not differ between the cohorts. Progression had developed before age 75 in 99% of patients with known progressive disease course; 38% with RRMS did not develop progression by age 75. Age at progression onset did not differ between SPMS (44.9±9.6), SAPMS (45.5±9.6) and PPMS (45.7±10.8). In either cohort, only 2% of patients had reached EDSS6 before onset of progression. CONCLUSIONS: Patients with RRMS do not inevitably develop a progressive disease course. Onset of progression is more dependent on age than the presence or duration of a pre-progression symptomatic disease course. Moderate disability is sustained predominantly after the onset of a progressive disease course in MS.

Preserved vascular integrity and enhanced survival following neuropilin-1 inhibition in a mouse model of CD8 T cell-initiated CNS vascular permeability.

BACKGROUND: Altered permeability of the blood-brain barrier (BBB) is a feature of numerous neurological conditions including multiple sclerosis, cerebral malaria, viral hemorrhagic fevers and acute hemorrhagic leukoencephalitis. Our laboratory has developed a murine model of CD8 T cell-initiated central nervous system (CNS) vascular permeability in which vascular endothelial growth factor (VEGF) signaling plays a prominent role in BBB disruption. FINDINGS: In this study, we addressed the hypothesis that in vivo blockade of VEGF signal transduction through administration of peptide (ATWLPPR) to inhibit neuropilin-1 (NRP-1) would have a therapeutic effect following induction of CD8 T cell-initiated BBB disruption. We report that inhibition of NRP-1, a co-receptor that enhances VEGFR2 (flk-1) receptor activation, decreases vascular permeability, brain hemorrhage, and mortality in this model of CD8 T cell-initiated BBB disruption. We also examine the expression pattern of VEGFR2 (flk-1) and VEGFR1 (flt-1) mRNA expression during a time course of this condition. We find that viral infection of the brain leads to increased expression of flk-1 mRNA. In addition, flk-1 and flt-1 expression levels decrease in the striatum and hippocampus in later time points following induction of CD8 T cell-mediated BBB disruption. CONCLUSION: This study demonstrates that NRP-1 is a potential therapeutic target in neuro-inflammatory diseases involving BBB disruption and brain hemorrhage. Additionally, the reduction in VEGF receptors subsequent to BBB disruption could be involved in compensatory negative feedback as an attempt to reduce vascular permeability.

A hematopoietic contribution to microhemorrhage formation during antiviral CD8 T cell-initiated blood-brain barrier disruption.

BACKGROUND: The extent to which susceptibility to brain hemorrhage is derived from blood-derived factors or stromal tissue remains largely unknown. We have developed an inducible model of CD8 T cell-initiated blood-brain barrier (BBB) disruption using a variation of the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis. This peptide-induced fatal syndrome (PIFS) model results in severe central nervous system (CNS) vascular permeability and death in the C57BL/6 mouse strain, but not in the 129 SvIm mouse strain, despite the two strains' having indistinguishable CD8 T-cell responses. Therefore, we hypothesize that hematopoietic factors contribute to susceptibility to brain hemorrhage, CNS vascular permeability and death following induction of PIFS. METHODS: PIFS was induced by intravenous injection of VP2121-130 peptide at 7 days post-TMEV infection. We then investigated brain inflammation, astrocyte activation, vascular permeability, functional deficit and microhemorrhage formation using T2*-weighted magnetic resonance imaging (MRI) in C57BL/6 and 129 SvIm mice. To investigate the contribution of hematopoietic cells in this model, hemorrhage-resistant 129 SvIm mice were reconstituted with C57BL/6 or autologous 129 SvIm bone marrow. Gadolinium-enhanced, T1-weighted MRI was used to visualize the extent of CNS vascular permeability after bone marrow transfer. RESULTS: C57BL/6 and 129 SvIm mice had similar inflammation in the CNS during acute infection. After administration of VP2121-130 peptide, however, C57BL/6 mice had increased astrocyte activation, CNS vascular permeability, microhemorrhage formation and functional deficits compared to 129 SvIm mice. The 129 SvIm mice reconstituted with C57BL/6 but not autologous bone marrow had increased microhemorrhage formation as measured by T2*-weighted MRI, exhibited a profound increase in CNS vascular permeability as measured by three-dimensional volumetric analysis of gadolinium-enhanced, T1-weighted MRI, and became moribund in this model system. CONCLUSION: C57BL/6 mice are highly susceptible to microhemorrhage formation, severe CNS vascular permeability and morbidity compared to the 129 SvIm mouse. This susceptibility is transferable with the bone marrow compartment, demonstrating that hematopoietic factors are responsible for the onset of brain microhemorrhage and vascular permeability in immune-mediated fatal BBB disruption.

Biogenesis and significance of central nervous system myelin.

The central nervous system is composed of neurons and glia cells. Although neurons have long been considered the functionally important cells, an ever-expanding body of research has revealed many critical functions of neuroglia. Among these, the myelin sheath elaborated by oligodendrocytes acts as a dynamic partner to the axons it enwraps and can no longer be considered as an inert membrane. In addition to its best known roles of providing insulation and optimizing conduction velocity, myelination modulates the maturation, survival, and regenerative capacity of axons through trophic support and signaling molecules. Myelin is produced through a complex process involving cell differentiation, biosynthesis of specialized lipids and proteins, interaction with environmental signals, and coordinated changes in cell morphology. Understanding the pathophysiology of primary myelin disorders, and the challenges faced in treating them, is facilitated through understanding of the structure, function, and generation/regeneration of myelin.

Advances in understanding gray matter pathology in multiple sclerosis: are we ready to redefine disease pathogenesis?

The purpose of this special issue in BMC Neurology is to summarize advances in our understanding of the pathological, immunological, imaging and clinical concepts of gray matter (GM) pathology in patients with multiple sclerosis (MS). Review articles by Lucchinetti and Popescu, Walker and colleagues, Hulst and colleagues and Horakova and colleagues summarize important recent advances in understanding GM damage and its implications to MS pathogenesis. They also raise a number of important new questions and outline comprehensive approaches to addressing those questions in years to come. In the last decade, the use of immunohistochemistry staining methods and more advanced imaging techniques to detect GM lesions, like double inversion recovery, contributed to a surge of studies related to cortical and subcortical GM pathology in MS. It is becoming more apparent from recent biopsy studies that subpial cortical lesions in early MS are highly inflammatory. The mechanisms responsible for triggering meningeal inflammation in MS patients are not yet elucidated, and they should be further investigated in relation to their role in initiating and perpetuating the disease process. Determining the role of antigens, environmental and genetic factors in the pathogenesis of GM involvement in MS is critical. The early involvement of cortical and subcortical GM damage in MS is very intriguing and needs to be further studied. As established in numerous cross-sectional and longitudinal studies, GM damage is a better predictor of physical disability and cognitive impairment than WM damage. Monitoring the evolution of GM damage is becoming an important marker in predicting future disease course and response to therapy in MS patients.

CD8 T cell-initiated vascular endothelial growth factor expression promotes central nervous system vascular permeability under neuroinflammatory conditions.

Dysregulation of the blood-brain barrier (BBB) is a hallmark feature of numerous neurologic disorders as diverse as multiple sclerosis, stroke, epilepsy, viral hemorrhagic fevers, cerebral malaria, and acute hemorrhagic leukoencephalitis. CD8 T cells are one immune cell type that have been implicated in promoting vascular permeability in these conditions. Our laboratory has created a murine model of CD8 T cell-mediated CNS vascular permeability using a variation of the Theiler's murine encephalomyelitis virus system traditionally used to study multiple sclerosis. Previously, we demonstrated that CD8 T cells have the capacity to initiate astrocyte activation, cerebral endothelial cell tight junction protein alterations and CNS vascular permeability through a perforin-dependent process. To address the downstream mechanism by which CD8 T cells promote BBB dysregulation, in this study, we assess the role of vascular endothelial growth factor (VEGF) expression in this model. We demonstrate that neuronal expression of VEGF is significantly upregulated prior to, and coinciding with, CNS vascular permeability. Phosphorylation of fetal liver kinase-1 is significantly increased early in this process indicating activation of this receptor. Specific inhibition of neuropilin-1 significantly reduced CNS vascular permeability and fetal liver kinase-1 activation, and preserved levels of the cerebral endothelial cell tight junction protein occludin. Our data demonstrate that CD8 T cells initiate neuronal expression of VEGF in the CNS under neuroinflammatory conditions, and that VEGF may be a viable therapeutic target in neurologic disease characterized by inflammation-induced BBB disruption.

Iron deposition and inflammation in multiple sclerosis. Which one comes first?

Whether iron deposition is an epiphenomenon of the multiple sclerosis (MS) disease process or may play a primary role in triggering inflammation and disease development remains unclear at this time, and should be studied at the early stages of disease pathogenesis. However, it is difficult to study the relationship between iron deposition and inflammation in early MS due to the delay between the onset of symptoms and diagnosis, and the poor availability of tissue specimens. In a recent article published in BMC Neuroscience, Williams et al. investigated the relationship between inflammation and iron deposition using an original animal model labeled as "cerebral experimental autoimmune encephalomyelitis", which develops CNS perivascular iron deposits. However, the relative contribution of iron deposition vs. inflammation in the pathogenesis and progression of MS remains unknown. Further studies should establish the association between inflammation, reduced blood flow, iron deposition, microglia activation and neurodegeneration. Creating a representative animal model that can study independently such relationship will be the key factor in this endeavor.

Rapid formation of extended processes and engagement of Theiler's virus-infected neurons by CNS-infiltrating CD8 T cells.

A fundamental question in neuroimmunology is the extent to which CD8 T cells actively engage virus-infected neurons. In the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis, an effective central nervous system (CNS)-infiltrating antiviral CD8 T cell response offers protection from this demyelinating disease. However, the specific CNS cell types engaged by these protective CD8 T cells in TMEV-resistant strains remains unknown. We used confocal microscopy to visualize the morphology, migration, and specific cellular interactions between adoptively transferred CD8 T cells and specific CNS cell types. Adoptively transferred GFP+ CD8+ splenocytes migrated to the brain and became 93% specific for the immunodominant virus epitope D(b):VP2(121-130). These CD8 T cells also polarized T cell receptor, CD8 protein, and granzyme B toward target neurons. Furthermore, we observed CD8 T cells forming cytoplasmic processes up to 45 µm in length. Using live tissue imaging, we determined that these T cell-extended processes (TCEPs) could be rapidly formed and were associated with migratory behavior through CNS tissues. These studies provide evidence that antiviral CD8 T cells have the capacity to engage virus-infected neurons in vivo and are the first to document and measure the rapid formation of TCEPs on these brain-infiltrating lymphocytes using live tissue imaging.

Multiple sclerosis: pathogenesis and MR imaging features of T1 hypointensities in a [corrected] murine model.

PURPOSE: To prospectively determine how T1 hypointensities (T1 black holes) on brain magnetic resonance (MR) images are generated by the immune system by using a Theiler murine encephalitis virus-induced model of multiple sclerosis and high-field-strength MR imaging. MATERIALS AND METHODS: All animal protocols and experiments were approved by the institutional animal care and use committee. Volumetric MR imaging studies were conducted at 7 T in six C57BL/6 mice and in immune differentiation marker (recombination activation gene [RAG]-1)-, immune cell (CD4, CD8)-, and immune effector molecule (Fas ligand, perforin)-deficient mice (six mice in each group) to determine which immune cell types and effector molecules lead to T1 hypointensities. The main outcome measure was the total T1 black hole volume per animal, as determined with volumetric analysis, and was analyzed statistically by using software. RESULTS: Compared with C57BL/6 mice, RAG-1-deficient mice showed a significant (P = .003) decrease in total T1 black hole volume, suggesting a clear role for the adaptive immune system. While CD4-deficient mice did not show a significant decrease in T1 black hole volume (P = .33), CD8-deficient mice did (P = .003). Perforin-deficient mice showed a significant reduction of T1 black hole volume (P = .002), whereas Fas ligand-deficient mice did not (P = .77). CONCLUSION: The data suggest that CD8 T cells utilizing perforin effector molecules are responsible for T1 black hole formation.

Acute hemorrhagic demyelination in a murine model of multiple sclerosis.

Acute hemorrhagic leukoencephalomyelitis (AHLE) is a rare neurological condition characterized by the development of acute hemorrhagic demyelination and high mortality. The pathomechanism of AHLE, as well as potential therapeutic approaches, have remained elusive due to the lack of suitable animal models. We report the first murine model of AHLE using a variation of the Theiler's Murine Encephalitis Virus (TMEV) MS model. During acute TMEV infection, C57BL/6 mice do not normally undergo demyelination. However, when 7 day TMEV infected C57BL/6 mice are intravenously administered the immunodominant CD8 T cell peptide, VP2121-130, animals develop characteristics of human AHLE based on pathologic, MRI and clinical features including microhemorrhages, increased blood-brain barrier permeability, and demyelination. The animals also develop severe disability as assessed using the rotarod assay. This study demonstrates the development of hemorrhagic demyelination in TMEV infected C57BL/6 mice within 24 hours of inducing this condition through intravenous administration of CD8 T cell restricted peptide. This study is also the first demonstration of rapid demyelination in a TMEV resistant non-demyelinating strain without transgenic alterations or pharmacologically induced immunosuppression.

A translatable molecular approach to determining CD8 T-cell epitopes in TMEV infection.

Defining the epitope specificity of CD8+ T cells is an important goal in autoimmune and immune-mediated disease research. We have developed a translational molecular approach to determine the epitope specificity of CD8+ T cells using the Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis (MS). TMEV-specific CD8+ T cells were isolated from brains and spleens of 7-day TMEV-infected C57BL/6J mice and stimulated by Cos-7 cells that were co-transfected with expression vectors encoding the D(b) class I molecule along with overlapping segments of the TMEV genome. Both brain-infiltrating and spleen-derived CD8+ T cells expressed IFN-gamma when Cos-7 cells were co-transfected with D(b) class I molecule and the TMEV genomic segment that encoded the immunodominant TMEV epitope. This demonstrated that peripheral and brain-infiltrating CD8+ T-cell responses were focused on peptide epitope(s) encoded by the same region of the TMEV genome. We propose that a similar molecular approach could also be used to determine the antigen specificity of suppressor CD8 T cells by the measurement of transforming growth factor-beta (TGF-beta) production. In addition, with a randomly generated library and peripheral blood or isolated CSF CD8+ T cells, this would be an effective method of predicting the epitope specificity of CD8+ T cells in human inflammatory CNS diseases, in animal models of MS or other organ-specific inflammatory diseases with a protective or pathogenic role of CD8 T cells.

A potential role for CD8+ T-cells as regulators of CNS vascular permeability.

The role of immune cells in promoting central nervous system (CNS) vascular permeability is poorly understood. In recent years, there is a growing body of literature that suggests CD8+ T-cells are potent mediators of vascular permeability in peripheral viral infections as well as in immune mediated neurological diseases. This review outlines the recent advances in tissue culture and animal models used to study vascular permeability. In addition, we put forth our hypothesis that CD8+ T-cells promote the opening of tight junctions between cerebral endothelial cells, enabling the infiltration of white blood cells and in certain models even leading to microhemorrhages in the CNS. Determining the mechanism by which CD8+ T-cells and other immune cells promote CNS vascular permeability in animal models could define new targets for immune mediated neurological conditions characterized by vascular permeability.