Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

Brain atrophy is a common feature of numerous neurologic diseases in which the role of neuroinflammation remains ill-defined. In this study, we evaluated the contribution of major histocompatibility complex class I molecules to brain atrophy in Theiler's murine encephalomyelitis virus (TMEV)-infected transgenic FVB mice that express the Db class I molecule. FVB/Db and wild-type FVB mice were evaluated for changes in neuroinflammation, virus clearance, neuropathology, and development of brain atrophy via T2-weighted MRI and subsequent 3-dimensional volumetric analysis. Significant brain atrophy and hippocampal neuronal loss were observed in TMEV-infected FVB/Db mice, but not in wild-type FVB mice. Brain atrophy was observed at 1 mo postinfection and persisted through the 4-mo observation period. Of importance, virus-infected FVB/Db mice elicited a strong CD8 T-cell response toward the immunodominant Db-restricted TMEV-derived peptide, VP2121-130, and cleared TMEV from the CNS. In addition, immunofluorescence revealed CD8 T cells near virus-infected neurons; therefore, we hypothesize that class I restricted CD8 T-cell responses promote development of brain atrophy. This model provides an opportunity to analyze the contribution of immune cells to brain atrophy in a system where persistent virus infection and demyelination are not factors in long-term neuropathology.-Huseby Kelcher, A. M., Atanga, P. A., Gamez, J. D., Cumba Garcia, L. M., Teclaw, S. J., Pavelko, K. D., Macura, S. I., Johnson. A. J. Brain atrophy in picornavirus-infected FVB mice is dependent on the H-2Db class I molecule.

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.

Effective Treatment of Established GL261 Murine Gliomas through Picornavirus Vaccination-Enhanced Tumor Antigen-Specific CD8+ T Cell Responses.

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA257-264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific Kb:OVA257-264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA257-264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.

Correlation of Brain Atrophy, Disability, and Spinal Cord Atrophy in a Murine Model of Multiple Sclerosis.

BACKGROUND: Disability progression in multiple sclerosis (MS) remains incompletely understood. Unlike lesional measures, central nervous system atrophy has a strong correlation with disability. Theiler's murine encephalomyelitis virus infection in SJL/J mice is an established model of progressive MS. We utilized in vivo MRI to quantify brain and spinal cord atrophy in this model and analyzed the temporal relationship between atrophy and disability. METHODS: Infected and control mice were followed for 12 months. Disability was assessed periodically using rotarod assay. Volumetric MRI datasets were acquired at 7 Tesla. Ventricular volume and C4-5 spinal cord cross-sectional area measurements were performed using Analyze 10. RESULTS: At 3 months, brain atrophy reached statistical significance (P = .005). In contrast, disability did not differ until 4 months post-infection (P = .0005). Cord atrophy reached significance by 9 months (P = 0.009). By 12 months, brain atrophy resulted in 111.8% increased ventricular volume (P = .00003), while spinal cord cross-sectional area was 25.6% reduced (P = .001) among cases. CONCLUSIONS: Our results suggest that significant brain atrophy precedes and predicts the development of disability, while spinal cord atrophy occurs late and correlates with severe disability. The observed temporal relationship establishes a framework for mechanisms of disability progression and enables further investigations of their underlying substrate.

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.

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.

Contrasting roles for CD4 vs. CD8 T-cells in a murine model of virally induced "T1 black hole" formation.

MRI is sensitive to tissue pathology in multiple sclerosis (MS); however, most lesional MRI findings have limited correlation with disability. Chronic T1 hypointense lesions or "T1 black holes" (T1BH), observed in a subset of MS patients and thought to represent axonal damage, show moderate to strong correlation with disability. The pathogenesis of T1BH remains unclear. We previously reported the first and as of yet only model of T1BH formation in the Theiler's murine encephalitis virus induced model of acute CNS neuroinflammation induced injury, where CD8 T-cells are critical mediators of axonal damage and related T1BH formation. The purpose of this study was to further analyze the role of CD8 and CD4 T-cells through adoptive transfer experiments and to determine if the relevant CD8 T-cells are classic epitope specific lymphocytes or different subsets. C57BL/6 mice were used as donors and RAG-1 deficient mice as hosts in our adoptive transfer experiments. In vivo 3-dimensional MRI images were acquired using a 7 Tesla small animal MRI system. For image analysis, we used semi-automated methods in Analyze 9.1; transfer efficiency was monitored using FACS of brain infiltrating lymphocytes. Using a peptide depletion method, we demonstrated that the majority of CD8 T-cells are classic epitope specific cytotoxic cells. CD8 T-cell transfer successfully restored the immune system's capability to mediate T1BH formation in animals that lack adaptive immune system, whereas CD4 T-cell transfer results in an attenuated phenotype with significantly less T1BH formation. These findings demonstrate contrasting roles for these cell types, with additional evidence for a direct pathogenic role of CD8 T-cells in our model of T1 black hole formation.

MRI in rodent models of brain disorders.

Magnetic resonance imaging (MRI) is a well-established tool in clinical practice and research on human neurological disorders. Translational MRI research utilizing rodent models of central nervous system (CNS) diseases is becoming popular with the increased availability of dedicated small animal MRI systems. Projects utilizing this technology typically fall into one of two categories: 1) true "pre-clinical" studies involving the use of MRI as a noninvasive disease monitoring tool which serves as a biomarker for selected aspects of the disease and 2) studies investigating the pathomechanism of known human MRI findings in CNS disease models. Most small animal MRI systems operate at 4.7-11.7 Tesla field strengths. Although the higher field strength clearly results in a higher signal-to-noise ratio, which enables higher resolution acquisition, a variety of artifacts and limitations related to the specific absorption rate represent significant challenges in these experiments. In addition to standard T1-, T2-, and T2*-weighted MRI methods, all of the currently available advanced MRI techniques have been utilized in experimental animals, including diffusion, perfusion, and susceptibility weighted imaging, functional magnetic resonance imaging, chemical shift imaging, heteronuclear imaging, and (1)H or (31)P MR spectroscopy. Selected MRI techniques are also exclusively utilized in experimental research, including manganese-enhanced MRI, and cell-specific/molecular imaging techniques utilizing negative contrast materials. In this review, we describe technical and practical aspects of small animal MRI and provide examples of different MRI techniques in anatomical imaging and tract tracing as well as several models of neurological disorders, including inflammatory, neurodegenerative, vascular, and traumatic brain and spinal cord injury models, and neoplastic diseases.

Detection of C4d deposition in cardiac allografts: a comparative study of immunofluorescence and immunoperoxidase methods.

CONTEXT: Complement activation, evidenced by deposition of C4d, is important in the diagnosis of antibody-mediated rejection of cardiac allografts. C4d deposition can be assessed by either immunofluorescence (IF)- or immunoperoxidase (IP)-based methods. The use of methods varies considerably among institutions, but there are few data addressing their diagnostic equivalence. OBJECTIVE: To compare IF and IP C4d staining on paired endomyocardial biopsy samples from a large number of heart transplant patients. DESIGN: Retrospectively selected paired frozen and paraffin-embedded samples from the same biopsy were stained for C4d by IF and IP methods. Capillary staining was scored by using a 0, 1+, 2+, 3+ scale. RESULTS: A total of 296 biopsy pairs from 70 patients were studied. There were two hundred forty-three cases that were scored 0, twenty-four scored 1+, sixteen scored 2+, and thirteen scored 3+ by IF. Two hundred thirty-one cases scored 0, forty scored 1+, ten scored 2+, and fifteen scored 3+ by IP. Complete agreement was seen in 81% of cases. Among discrepant cases, 89% (n  =  51) were minor (±1) and 11% (n  =  6) were major (±2). Five of the 6 major discrepancy biopsies came from 2 patients, both of whom had concordant (IF and IP) 3+ results on prior biopsies. The weighted κ value for the entire sample set was 0.78 and for the first biopsy only set (to correct for bias introduced by multiple biopsies from the same patient) the weighted κ value was 0.88. CONCLUSIONS: Immunofluorescence and IP C4d staining methods are highly comparable and are both viable options for antibody-mediated rejection surveillance in transplant heart biopsies.

Mass spectrometry-based proteomic diagnosis of renal immunoglobulin heavy chain amyloidosis.

BACKGROUND AND OBJECTIVES: Amyloidosis is a group of disorders characterized by accumulation of extracellular deposition of proteins as insoluble aggregates. The clinical management of amyloidosis is based on identifying the underlying etiology and accurate typing of the amyloid. Ig heavy chain amyloid involving the kidney is poorly recognized and often poses a diagnostic dilemma. DESIGN, SETTING, PARTICIPANTS, & MEASURES: In this study, we describe the use of laser microdissection (LMD) and mass spectrometry (MS)-based proteomic analysis for the accurate typing of 14 cases of amyloidosis. We also describe the clinicopathologic findings of four problematic cases of renal Ig heavy chain amyloidosis that required LMD/MS proteomic analysis for accurate typing of the amyloid. RESULTS: LMD/MS proteomic data of four cases of Ig heavy chain renal amyloidosis showed Ig heavy chains with or without light chains. The break up of the Ig heavy chains was as follows: one case showed Igγ1 chain constant region and λ light chains, one case showed Igα chain constant region and κ light chains variable and constant regions, whereas two cases showed Igγ3 chain constant region and heavy chains variable region I and/or III without light chains. We compare the LMD/MS proteomic data of Ig heavy chain renal amyloid with that of other types of amyloid, including Ig light chains, serum amyloid A, fibrinogen A-α chain renal amyloid, and transthyretin amyloid. CONCLUSIONS: We conclude that LMD/MS is a sensitive and specific tool for diagnosis and accurate typing of renal amyloidosis, including Ig heavy chain amyloid.

Classification of amyloidosis by laser microdissection and mass spectrometry-based proteomic analysis in clinical biopsy specimens.

The 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.

Clinicopathologic features of CDK6 translocation-associated B-cell lymphoproliferative disorders.

Cyclin-dependent protein kinase 6 (CDK6), in cooperation with cyclin Ds, drives cell cycle progression from G1 to S phase through phosphorylation and subsequent inactivation of retinoblastoma 1 protein. Alteration of this pathway results in both nonhematologic and hematologic malignancies, which include a small subset of B-cell lymphoproliferative disorders (BLPDs). We identified 5 cases of BLPD that carried CDK6 chromosomal translocations and characterized their clinical, pathologic, immunophenotypic, and genetic features. Common clinical characteristics included marked neoplastic lymphocytosis, systemic lymphadenopathy, splenomegaly, and bone marrow involvement. Three patients were diagnosed with low-grade B-cell lymphoma and had an indolent clinical course, and 2 patients (one who transformed to large B-cell lymphoma, and the other who was initially diagnosed with a high-grade B-cell lymphoma) had an aggressive clinical course. Immunophenotypically, the neoplastic B cells expressed CD5, CDK6, and cytoplasmic retinoblastoma 1 protein in all cases, expressed phospho-RB, p27kip1, and cyclin D2 in most cases, and uniformly lacked expression of all other cyclins. In 4 cases, the CDK6 translocation partner was kappa immunoglobulin light-chain gene; and in the fifth case, the CDK6 translocation partner was unknown. These distinct clinicopathologic and cytogenetic features distinguish the CDK6 translocation-associated BLPDs (CDK6-BLPDs) from other mature B-cell lymphomas.

Glomeruli of Dense Deposit Disease contain components of the alternative and terminal complement pathway.

Dense Deposit Disease (DDD), or membranoproliferative glomerulonephritis type II, is a rare renal disease characterized by dense deposits in the mesangium and along the glomerular basement membranes that can be seen by electron microscopy. Although these deposits contain complement factor C3, as determined by immunofluorescence microscopy, their precise composition remains unknown. To address this question, we used mass spectrometry to identify the proteins in laser microdissected glomeruli isolated from paraffin-embedded tissue of eight confirmed cases of DDD. Compared to glomeruli from five control patients, we found that all of the glomeruli from patients with DDD contain components of the alternative pathway and terminal complement complex. Factor C9 was uniformly present as well as the two fluid-phase regulators of terminal complement complex clusterin and vitronectin. In contrast, in nine patients with immune complex-mediated membranoproliferative glomerulonephritis, glomerular samples contained mainly immunoglobulins and complement factors C3 and C4. Our study shows that in addition to fluid-phase dysregulation of the alternative pathway, soluble components of the terminal complement complex contribute to glomerular lesions found in DDD.

Immunoglobulin derived depositions in the nervous system: novel mass spectrometry application for protein characterization in formalin-fixed tissues.

Proteinaceous deposits are occasionally encountered in surgically obtained biopsies of the nervous system. Some of these are amyloidomas, although the precise nature of other cases remains uncertain. We studied 13 cases of proteinaceous aggregates in clinical specimens of the nervous system. Proteins contained within laser microdissected areas of interest were identified from tryptic peptide sequences by liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS). Immunohistochemical studies for immunoglobulin heavy and light chains and amyloidogenic proteins were performed in all cases. Histologically, the cases were classified into three groups: 'proteinaceous deposit not otherwise specified' (PDNOS) (n=6), amyloidoma (n=5), or 'intracellular crystals' (n=2). LC-MS/MS demonstrated the presence of lambda, but not kappa, light chain as well as serum amyloid P in all amyloidomas. lambda-Light-chain immunostaining was noted in amyloid (n=5), although demonstrable monotypic lymphoplasmacytic cells were seen in only one case. Conversely, in PDNOS kappa, but not lambda, was evident in five cases, both light chains being present in a single case. In three cases of PDNOS, a low-grade B-cell lymphoma consistent with marginal zone lymphoma was present in the brain specimen (n=2) or spleen (n=1). Lastly, in the 'intracellular crystals' group, the crystals were present within CD68+ macrophages in one case wherein kappa-light chain was found by LC-MS/MS only; the pathology was consistent with crystal-storing histiocytosis. In the second case, the crystals contained immunoglobulin G within CD138+ plasma cells. Our results show that proteinaceous deposits in the nervous system contain immunoglobulin components and LC-MS/MS accurately identifies the content of these deposits in clinical biopsy specimens. LC-MS/MS represents a novel application for characterization of these deposits and is of diagnostic utility in addition to standard immunohistochemical analyses.

CD33 detection by immunohistochemistry in paraffin-embedded tissues: a new antibody shows excellent specificity and sensitivity for cells of myelomonocytic lineage.

A new monoclonal antibody to CD33 that reacts in paraffin-embedded tissue samples was evaluated. The expected reactivity in granulocytic and monocytic cells was found in a tissue microarray composed of multiple tissue sites. There was no unexpected reactivity found in a wide variety of hematolymphoid and nonhematolymphoid disorders. In cases of acute leukemia, the CD33 antibody showed equivalent results by immunohistochemical analysis compared with flow cytometric analysis. The CD33 antibody was also found to be a useful marker in the workup of myeloid sarcomas. This anti-CD33 antibody will be a useful marker in the workup of acute leukemias and myeloid sarcomas on paraffin-embedded tissue samples.

STAT4- and STAT6-signaling molecules in a murine model of multiple sclerosis.

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.

A 40-cM region on chromosome 14 plays a critical role in the development of virus persistence, demyelination, brain pathology and neurologic deficits in a murine viral model of multiple sclerosis.

Theiler virus persists and induces immune-mediated demyelination in susceptible mice and serves as a model of multiple sclerosis. Previously, we identified 4 markers--D14Mit54, D14Mit60, D14Mit61, and D14Mit90--in a 40-cM region of chromosome 14 that are associated with demyelination in a cross between susceptible DBA/2 and resistant B10.D2 mice. We generated congenic-inbred mice to examine the contribution of this 40-cM region to disease. DBA Chr.14B10 mice, containing the chromosomal segment marked by the microsatellite polymorphisms, developed less spinal cord demyelination than did DBA/2 mice. More demyelination was found in the reciprocal congenic mouse B10.D2 Chr.14D2 than in the B10.D2 strain. Introduction of the DBA/2 chromosomal region onto the B10.D2 genetic background resulted in more severe disease in the striatum and cortex relative to B10.D2 mice. The importance of the marked region of chromosome 14 is indicated by the decrease in neurological performance using the Rotarod test during chronic disease in B10.D2 Chr.14D2 mice in comparison to B10.D2 mice. Viral replication was increased in B10.D2 Chr.14D2 mice as determined by quantitative real-time RT-PCR. These results indicate that the 40-cM region on chromosome 14 of DBA/2 mice contributes to viral persistence, subsequent demyelination, and loss of neurological function.