Gamez, Jeffrey D.

Abstract 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.

AbstractThe clinical management of amyloidosis is based on the treatment of the underlying etiology, and accurate identification of the protein causing the amyloidosis is of paramount importance. Current methods used for typing of amyloidosis such as immunohistochemistry have low specificity and sensitivity. In this study, we report the development of a highly specific and sensitive novel test for the typing of amyloidosis in routine clinical biopsy specimens. Our approach combines specific sampling by laser microdissection (LMD) and analytical power of tandem mass spectrometry (MS)–based proteomic analysis. We studied 50 cases of amyloidosis that were well-characterized by gold standard clinicopathologic criteria (training set) and an independent validation set comprising 41 cases of cardiac amyloidosis. By use of LMD/MS, we identified the amyloid type with 100% specificity and sensitivity in the training set and with 98% in validation set. Use of the LMD/MS method will enhance our ability to type amyloidosis accurately in clinical biopsy specimens.

ABSTRACT Epidemiological studies suggest that an environmental factor (possibly a virus) acquired early in life may trigger multiple sclerosis (MS). The virus may remain dormant in the central nervous system but then becomes activated in adulthood. All existing models of MS are characterized by inflammation or demyelination that follows days after virus infection or antigen inoculation. While investigating the role of CD4 + T cell responses following Theiler's virus infection in mice deficient in STAT4 or STAT6, we discovered a model in which virus infection was followed by demyelination after a very prolonged incubation period. STAT4−/− mice were resistant to demyelination for 180 days after infection, but developed severe demyelination after this time point. Inflammatory cells and up‐regulation of Class I and Class II MHC antigens characterized these lesions. Virus antigen was partially controlled during the early chronic phase of the infection even though viral RNA levels remained high throughout infection. Demyelination correlated with the appearance of virus antigen expression. Bone marrow reconstitution experiments indicated that the mechanism of the late onset demyelination was the result of the STAT4−/− immune system. Thus, virus infection of STAT4−/− mice results in a model that may allow for dissection of the immune events predisposing to late‐onset demyelination in MS.

ABSTRACT The human monoclonal antibody rHIgM22 enhances remyelination following spinal cord demyelination in a virus‐induced murine model of multiple sclerosis. Using three‐dimensional T2‐weighted in vivo spinal cord magnetic resonance imaging (MRI), we have therefore assessed the extent of spinal cord demyelination, before and after 5 weeks of treatment with rHIgM22, to determine whether antibody enhanced remyelination can be detected by MRI. A significant decrease was seen in T2 high signal lesion volume following antibody treatment. Histologic examination of the spinal cord tissue reveals that this decrease in lesion volume correlates with antibody promoted remyelination. To show that rHIgM22 enters the spinal cord and colocalizes with demyelinating lesions, we used ultrasmall superparamagnetic iron oxide particle (USPIO)‐labeled antibodies. This may be considered as additional evidence to the hypothesis that rHIgM22 promotes remyelination by local effects in the lesions, likely by binding to CNS cells. The reduction in high signal T2‐weighted lesion volume may be an important outcome measure in future clinical trials in humans.

ABSTRACT We developed a novel MRI technique to image immune cell location and homing in vivo to the central nervous system (CNS). Superparamagnetic antibodies specific for cell surface markers allowed imaging of CD4+ T cells, CD8+ T cells, and Mac1+ cells in the CNS of mice infected with Theiler's murine encephalomyelitis virus (TMEV) and in mice with experimental autoimmune encephalomyelitis (EAE). Superparamagnetic antibodies have excellent T2, T2∗, and good T1 relaxation properties, which makes them ideal MRI contrast materials. Immunohistochemistry of corresponding sections confirmed the specificity of the technique to detect immune cell types in the CNS. This powerful technique has potential to image any cell with unique surface antigens. Because superparamagnetic antibodies similar to those used in the study are approved for human use, the in vivo MRI technique we have described could be developed for human use.

ABSTRACTWe evaluated the role of gamma interferon (IFN-γ) in protecting neurons from virus-induced injury following central nervous system infection. IFN-γ−/−and IFN-γ+/+mice of the resistant major histocompatibility complex (MHC)H-2bhaplotype and intracerebrally infected with Theiler's murine encephalomyelitis virus (TMEV) cleared virus infection from anterior horn cell neurons. IFN-γ+/+H-2bmice also cleared virus from the spinal cord white matter, whereas IFN-γ−/−H-2bmice developed viral persistence in glial cells of the white matter and exhibited associated spinal cord demyelination. In contrast, infection of IFN-γ−/−mice of the susceptibleH-2qhaplotype resulted in frequent deaths and severe neurologic deficits within 16 days of infection compared to the results obtained for controls. Morphologic analysis demonstrated severe injury to spinal cord neurons in IFN-γ−/−H-2qmice during early infection. More virus RNA was detected in the brain and spinal cord of IFN-γ−/−H-2qmice than in those of control mice at 14 and 21 days after TMEV infection. Virus antigen was localized predominantly to anterior horn cells in infected IFN-γ−/−H-2qmice. IFN-γ deletion did not affect the humoral response directed against the virus. However, the level of expression of CD4, CD8, class I MHC, or class II MHC in the central nervous system of IFN-γ−/−H-2qmice was lower than those in IFN-γ+/+H-2qmice. Finally, in vitro analysis of virus-induced death in NSC34 cells and spinal motor neurons showed that IFN-γ exerted a neuroprotective effect in the absence of other aspects of the immune response. These data support the hypothesis that IFN-γ plays a critical role in protecting spinal cord neurons from persistent infection and death.