Reactive Oxygen Species Are Key Mediators of Demyelination in Canine Distemper Leukoencephalitis but not in Theiler's Murine Encephalomyelitis.

(1) Background: Canine distemper virus (CDV)-induced demyelinating leukoencephalitis (CDV-DL) in dogs and Theiler's murine encephalomyelitis (TME) virus (TMEV)-induced demyelinating leukomyelitis (TMEV-DL) are virus-induced demyelinating conditions mimicking Multiple Sclerosis (MS). Reactive oxygen species (ROS) can induce the degradation of lipids and nucleic acids to characteristic metabolites such as oxidized lipids, malondialdehyde, and 8-hydroxyguanosine. The hypothesis of this study is that ROS are key effector molecules in the pathogenesis of myelin membrane breakdown in CDV-DL and TMEV-DL. (2) Methods: ROS metabolites and antioxidative enzymes were assessed using immunofluorescence in cerebellar lesions of naturally CDV-infected dogs and spinal cord tissue of TMEV-infected mice. The transcription of selected genes involved in ROS generation and detoxification was analyzed using gene-expression microarrays in CDV-DL and TMEV-DL. (3) Results: Immunofluorescence revealed increased amounts of oxidized lipids, malondialdehyde, and 8-hydroxyguanosine in CDV-DL while TMEV-infected mice did not reveal marked changes. In contrast, microarray-analysis showed an upregulated gene expression associated with ROS generation in both diseases. (4) Conclusion: In summary, the present study demonstrates a similar upregulation of gene-expression of ROS generation in CDV-DL and TMEV-DL. However, immunofluorescence revealed increased accumulation of ROS metabolites exclusively in CDV-DL. These results suggest differences in the pathogenesis of demyelination in these two animal models.

Dynamic changes and molecular analysis of cell death in the spinal cord of SJL mice infected with the BeAn strain of Theiler's murine encephalomyelitis virus.

Theiler's murine encephalomyelitis (TME) is caused by the TME virus (TMEV) and represents an important animal model for multiple sclerosis (MS). Oligodendroglial apoptosis and reduced apoptotic elimination of encephalitogenic leukocytes seem to participate in autoimmune demyelination in MS. The present study quantified apoptotic cells in BeAn-TMEV-induced spinal cord white matter lesions at 14, 42, 98, and 196 days post infection (dpi) using immunostaining. Apoptotic cells were identified by transmission electron microscopy and double-immunofluorescence. The mRNA expression of apoptosis-related genes was investigated using microarray analysis. Oligodendroglial apoptosis was already detected in the predemyelinating phase at 14 dpi. Apoptotic cell numbers peaked at 42 dpi and decreased until 196 dpi partly due to reduced T cell apoptosis. In addition to genes involved in the classical pathways of apoptosis induction, microarray analysis detected the expression of genes related to alternative mechanisms of cell death such as pyroptosis, necroptosis, and endoplasmic reticulum stress. Consequently, oligodendroglial apoptosis is involved in the initiation of the TME demyelination process, whereas the development of apoptosis resistance of T cells potentially favors the maintenance of inflammation and myelin loss.

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro.

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as "respiratory burst", whereas M2-polarization was associated with processes such as "mitosis". Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for future studies upon the role of macrophage polarization in spontaneous diseases of the dog, a species that has emerging importance for translational research.

Microarray-Based Gene Expression Analysis for Veterinary Pathologists: A Review.

High-throughput, genome-wide transcriptome analysis is now commonly used in all fields of life science research and is on the cusp of medical and veterinary diagnostic application. Transcriptomic methods such as microarrays and next-generation sequencing generate enormous amounts of data. The pathogenetic expertise acquired from understanding of general pathology provides veterinary pathologists with a profound background, which is essential in translating transcriptomic data into meaningful biological knowledge, thereby leading to a better understanding of underlying disease mechanisms. The scientific literature concerning high-throughput data-mining techniques usually addresses mathematicians or computer scientists as the target audience. In contrast, the present review provides the reader with a clear and systematic basis from a veterinary pathologist's perspective. Therefore, the aims are (1) to introduce the reader to the necessary methodological background; (2) to introduce the sequential steps commonly performed in a microarray analysis including quality control, annotation, normalization, selection of differentially expressed genes, clustering, gene ontology and pathway analysis, analysis of manually selected genes, and biomarker discovery; and (3) to provide references to publically available and user-friendly software suites. In summary, the data analysis methods presented within this review will enable veterinary pathologists to analyze high-throughput transcriptome data obtained from their own experiments, supplemental data that accompany scientific publications, or public repositories in order to obtain a more in-depth insight into underlying disease mechanisms.

Reduced angiogenic gene expression in morbillivirus-triggered oncolysis in a translational model for histiocytic sarcoma.

Histiocytic sarcoma represents a rare malignant tumour with a short survival time, indicating the need of novel treatment strategies including oncolytic virotherapy. The underlying molecular mechanisms of viral oncolysis are largely unknown. As cancer in companion animals shares striking similarities with human counterparts, we chose a permanent canine histiocytic sarcoma cell line (DH82 cells) to identify global transcriptome changes following infection with canine distemper virus (CDV), a paramyxovirus closely related to human measles virus. Microarray analysis identified 3054 differentially expressed probe sets (DEPs), encoding for 892 up- and 869 down-regulated unique canine genes, respectively, in DH82 cells persistently infected with the vaccine strain Onderstepoort of CDV (DH82-Ond-pi), compared to non-infected DH82 cells. Up-regulated genes were predominantly related to immune processes, as demonstrated by functional enrichment analysis. Moreover, there was substantial enrichment of genes characteristic for classically activated M1 and alternatively activated M2 macrophages in DH82-Ond-pi; however, significant polarization into either of both categories was lacking. 'Angiogenesis' was the dominant enriched functional term for the down-regulated genes, highlighting decreased blood vessel generation as a potential mechanism of paramyxovirus-induced oncolysis in DH82 cells. The anti-angiogenic effect of infection was verified by immunohistochemistry, which revealed a lower blood vessel density in an in vivo mouse model, xenotransplanted with DH82-Ond-pi, compared to mice transplanted with non-infected DH82 cells. Reduction in angiogenesis appears to be an important oncolytic mechanism of CDV in DH82 cells, suggesting that similar mechanisms might account for human histiocytic sarcoma and maybe other tumours in conjunction with measles virus.

Persistent Morbillivirus Infection Leads to Altered Cortactin Distribution in Histiocytic Sarcoma Cells with Decreased Cellular Migration Capacity.

Histiocytic sarcomas represent rare but fatal neoplasms in humans. Based on the absence of a commercially available human histiocytic sarcoma cell line the frequently affected dog displays a suitable translational model. Canine distemper virus, closely related to measles virus, is a highly promising candidate for oncolytic virotherapy. Therapeutic failures in patients are mostly associated with tumour invasion and metastasis often induced by misdirected cytoskeletal protein activities. Thus, the impact of persistent canine distemper virus infection on the cytoskeletal protein cortactin, which is frequently overexpressed in human cancers with poor prognosis, was investigated in vitro in a canine histiocytic sarcoma cell line (DH82). Though phagocytic activity, proliferation and apoptotic rate were unaltered, a significantly reduced migration activity compared to controls (6 hours and 1 day after seeding) accompanied by a decreased number of cortactin mRNA transcripts (1 day) was detected. Furthermore, persistently canine distemper virus infected DH82 cells showed a predominant diffuse intracytoplasmic cortactin distribution at 6 hours and 1 day compared to controls with a prominent membranous expression pattern (p ≤ 0.05). Summarized, persistent canine distemper virus infection induces reduced tumour cell migration associated with an altered intracellular cortactin distribution, indicating cytoskeletal changes as one of the major pathways of virus-associated inhibition of tumour spread.

Immunohistochemical and transcriptome analyses indicate complex breakdown of axonal transport mechanisms in canine distemper leukoencephalitis.

INTRODUCTION: CDV-DL (Canine distemper virus-induced demyelinating leukoencephalitis) represents a spontaneously occurring animal model for demyelinating disorders. Axonopathy represents a key pathomechanism in this disease; however, its underlying pathogenesis has not been addressed in detail so far. This study aimed at the characterization of axonal cytoskeletal, transport, and potential regenerative changes with a parallel focus upon Schwann cell remyelination. METHODS: Immunohistochemistry of canine cerebellar tissue as well as a comparative analysis of genes from an independent microarray study were performed. RESULTS: Increased axonal immunoreactivity for nonphosphorylated neurofilament was followed by loss of cytoskeletal and motor proteins. Interestingly, a subset of genes encoding for neurofilament subunits and motor proteins was up-regulated in the chronic stage compared to dogs with subacute CDV-DL. However, immunohistochemically, hints for axonal regeneration were restricted to up-regulated axonal positivity of hypoxia-inducible factor 1 alpha, while growth-associated protein 43, erythropoietin and its receptor were not or even down-regulated. Periaxin-positive structures, indicative of Schwann cell remyelination, were only detected within few advanced lesions. CONCLUSIONS: The present findings demonstrate a complex sequence of axonal cytoskeletal breakdown mechanisms. Moreover, though sparse, this is the first report of Schwann cell remyelination in CDV-DL. Facilitation of these very limited endogenous regenerative responses represents an important topic for future research.

A Novel RAF Kinase Inhibitor with DFG-Out-Binding Mode: High Efficacy in BRAF-Mutant Tumor Xenograft Models in the Absence of Normal Tissue Hyperproliferation.

BI 882370 is a highly potent and selective RAF inhibitor that binds to the DFG-out (inactive) conformation of the BRAF kinase. The compound inhibited proliferation of human BRAF-mutant melanoma cells with 100× higher potency (1-10 nmol/L) than vemurafenib, whereas wild-type cells were not affected at 1,000 nmol/L. BI 882370 administered orally was efficacious in multiple mouse models of BRAF-mutant melanomas and colorectal carcinomas, and at 25 mg/kg twice daily showed superior efficacy compared with vemurafenib, dabrafenib, or trametinib (dosed to provide exposures reached in patients). To model drug resistance, A375 melanoma-bearing mice were initially treated with vemurafenib; all tumors responded with regression, but the majority subsequently resumed growth. Trametinib did not show any efficacy in this progressing population. BI 882370 induced tumor regression; however, resistance developed within 3 weeks. BI 882370 in combination with trametinib resulted in more pronounced regressions, and resistance was not observed during 5 weeks of second-line therapy. Importantly, mice treated with BI 882370 did not show any body weight loss or clinical signs of intolerability, and no pathologic changes were observed in several major organs investigated, including skin. Furthermore, a pilot study in rats (up to 60 mg/kg daily for 2 weeks) indicated lack of toxicity in terms of clinical chemistry, hematology, pathology, and toxicogenomics. Our results indicate the feasibility of developing novel compounds that provide an improved therapeutic window compared with first-generation BRAF inhibitors, resulting in more pronounced and long-lasting pathway suppression and thus improved efficacy.

Central Nervous System Demyelination and Remyelination is Independent from Systemic Cholesterol Level in Theiler's Murine Encephalomyelitis.

High dietary fat and/or cholesterol intake is a risk factor for multiple diseases and has been debated for multiple sclerosis. However, cholesterol biosynthesis is a key pathway during myelination and disturbances are described in demyelinating diseases. To address the possible interaction of dyslipidemia and demyelination, cholesterol biosynthesis gene expression, composition of the body's major lipid repositories and Paigen diet-induced, systemic hypercholesterolemia were examined in Theiler's murine encephalomyelitis (TME) using histology, immunohistochemistry, serum clinical chemistry, microarrays and high-performance thin layer chromatography. TME-virus (TMEV)-infected mice showed progressive loss of motor performance and demyelinating leukomyelitis. Gene expression associated with cholesterol biosynthesis was overall down-regulated in the spinal cord of TMEV-infected animals. Spinal cord levels of galactocerebroside and sphingomyelin were reduced on day 196 post TMEV infection. Paigen diet induced serum hypercholesterolemia and hepatic lipidosis. However, high dietary fat and cholesterol intake led to no significant differences in clinical course, inflammatory response, astrocytosis, and the amount of demyelination and remyelination in the spinal cord of TMEV-infected animals. The results suggest that down-regulation of cholesterol biosynthesis is a transcriptional marker for demyelination, quantitative loss of myelin-specific lipids, but not cholesterol occurs late in chronic demyelination, and serum hypercholesterolemia exhibited no significant effect on TMEV infection.

Transcriptional analysis of glial cell differentiation in the postnatal murine spinal cord.

Postnatal murine spinal cord represents a good model system to study mammalian central nervous system myelination in vivo as a basis for further studies in demyelinating diseases. Transcriptional changes were analyzed in SJL/J mice on postnatal day 0, 14, 49 and 231 (P0, P14, P49, P231) employing Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Additionally, marker gene signatures for astrocyte and oligodendrocyte lineage-stages were defined to study their gene expression in more detail. In addition, immunohistochemistry was used to quantify the abundance of commonly used glial cell markers. 6092 differentially regulated genes (DEGs) were identified. The up-regulated DEGs at P14, P49 and P231 compared to P0 exhibited significantly enriched associations to gene ontology terms such as myelination and lipid metabolic transport and down-regulated DEGs to neurogenesis and axonogenesis. Expression values of marker gene signatures for neural stem cells, oligodendrocyte precursor cells, and developing astrocytes were constantly decreasing, whereas myelinating oligodendrocyte and mature astrocyte markers showed a steady increase. Molecular findings were substantiated by immunohistochemical observations. The transcriptional changes observed are an important reference for future analysis of degenerative and inflammatory conditions in the spinal cord.

Identification of specific mRNA signatures as fingerprints for carcinogenesis in mice induced by genotoxic and nongenotoxic hepatocarcinogens.

Long-term rodent carcinogenicity studies for evaluation of chemicals and pharmaceuticals concerning their carcinogenic potential to humans are currently receiving critical revision. Additional data from mechanistic studies can support cancer risk assessment by clarifying the underlying mode of action. In the course of the IMI MARCAR project, a European consortium of EFPIA partners and academics, which aims to identify biomarkers for nongenotoxic carcinogenesis, a toxicogenomic mouse liver database was generated. CD-1 mice were orally treated for 3 and 14 days with 3 known genotoxic hepatocarcinogens: C.I. Direct Black 38, Dimethylnitrosamine and 4,4'-Methylenedianiline; 3 nongenotoxic hepatocarcinogens: 1,4-Dichlorobenzene, Phenobarbital sodium and Piperonyl butoxide; 4 nonhepatocarcinogens: Cefuroxime sodium, Nifedipine, Prazosin hydrochloride and Propranolol hydrochloride; and 3 compounds that show ambiguous results in genotoxicity testing: Cyproterone acetate, Thioacetamide and Wy-14643. By liver mRNA expression analysis using individual animal data, we identified 64 specific biomarker candidates for genotoxic carcinogens and 69 for nongenotoxic carcinogens for male mice at day 15. The majority of genotoxic carcinogen biomarker candidates possess functions in DNA damage response (eg, apoptosis, cell cycle progression, DNA repair). Most of the identified nongenotoxic carcinogen biomarker candidates are involved in regulation of cell cycle progression and apoptosis. The derived biomarker lists were characterized with respect to their dependency on study duration and gender and were successfully used to characterize carcinogens with ambiguous genotoxicity test results, such as Wy-14643. The identified biomarker candidates improve the mechanistic understanding of drug-induced effects on the mouse liver that result in hepatocellular adenomas and/or carcinomas in 2-year mouse carcinogenicity studies.

Dynamic Changes of Microglia/Macrophage M1 and M2 Polarization in Theiler's Murine Encephalomyelitis.

Microglia and macrophages play a central role for demyelination in Theiler's murine encephalomyelitis (TME) virus infection, a commonly used infectious model for chronic-progressive multiple sclerosis. In order to determine the dynamic changes of microglia/macrophage polarization in TME, the spinal cord of Swiss Jim Lambert (SJL) mice was investigated by gene expression profiling and immunofluorescence. Virus persistence and demyelinating leukomyelitis were confirmed by immunohistochemistry and histology. Electron microscopy revealed continuous myelin loss together with abortive myelin repair during the late chronic infection phase indicative of incomplete remyelination. A total of 59 genes out of 151 M1- and M2-related genes were differentially expressed in TME virus-infected mice over the study period. The onset of virus-induced demyelination was associated with a dominating M1 polarization, while mounting M2 polarization of macrophages/microglia together with sustained prominent M1-related gene expression was present during the chronic-progressive phase. Molecular results were confirmed by immunofluorescence, showing an increased spinal cord accumulation of CD16/32(+) M1-, arginase-1(+) M2- and Ym1(+) M2-type cells associated with progressive demyelination. The present study provides a comprehensive database of M1-/M2-related gene expression involved in the initiation and progression of demyelination supporting the hypothesis that perpetuating interaction between virus and macrophages/microglia induces a vicious circle with persistent inflammation and impaired myelin repair in TME.

STAT3 represents a molecular switch possibly inducing astroglial instead of oligodendroglial differentiation of oligodendroglial progenitor cells in Theiler's murine encephalomyelitis.

AIMS: Insufficient oligodendroglial differentiation of oligodendroglial progenitor cells (OPCs) is suggested to be responsible for remyelination failure and astroglial scar formation in Theiler's murine encephalomyelitis (TME). The aim of the present study is to identify molecular key regulators of OPC differentiation in TME, and to dissect their mechanism of action in vitro. METHODS: TME virus (TMEV) infected SJL/J-mice were evaluated by rotarod analysis, histopathology, immunohistology and gene expression microarray analysis. The STAT3 pathway was activated using meteorin and inhibited using STAT3 inhibitor VII in the glial progenitor cell line BO-1 and in primary rat OPCs in vitro. RESULTS: As expected, immunohistology demonstrated progressively decreasing myelin basic protein-positive white matter in TME. In contrast, intralesional NG2-positive OPCs as well as GFAP-positive astrocytes were increased. Gene Set Enrichment Analysis revealed 26 Gene Ontology terms including 'JAK-STAT cascade' to be significantly positively correlated with the density of NG2-positive OPCs. Immunohistology revealed an increased amount of activated, phosphorylated STAT3-expressing astrocytes, OPCs, and microglia/macrophages within the lesions. Meteorin-induced activation of STAT3-signalling in BO-1 cells and primary rat OPCs resulted in an enhanced GFAP and reduced CNPase expression. In contrast, an oppositional result was observed in BO-1 cells treated with STAT3 inhibitor VII. CONCLUSIONS: The STAT3 pathway is a key regulator of OPC-differentiation, suggested to shift their differentiation from an oligodendroglial towards an astrocytic fate, thereby inducing astrogliosis and insufficient remyelination in TME.

Transcriptional profiling predicts overwhelming homology of Schwann cells, olfactory ensheathing cells, and Schwann cell-like glia.

Schwann cells (SCs), olfactory ensheathing cells (OECs), and central nervous system Schwann cell-like glia (SG) represent a group of nerve growth factor receptor p75 (NGFR)-positive cells, originating from different tissues. Because of their pro-regenerative capacities, these cells are subjects in experimental transplantation-based therapies of spinal cord trauma. The objective of this study was to compare the transcriptomes of uninfected and canine distemper virus-infected OECs, SCs, SG and fibroblasts (FBs) derived from four beagle dogs and cultured under identical conditions in vitro, employing canine genome 2.0 arrays (Affymetrix). Here, we observed a complete lack of transcriptional differerences between OECs and SG, a high similarity of OECs/SG to SCs, and a marked difference of SCs and OECs/SG towards FBs. Differentially expressed genes possibly involved in the maintenance of cell type-specific identity included an up-regulation of HOXD8 and HOXC4 in SCs, and an up-regulation of CNTNAP2 and EFEMP1 in OECs/SG. We identified cell type-specific biomarkers employing supervised clustering with a K-nearest-neighbors algorithm and correlation-based feature selection. Thereby AQP1 and SCRG1 were predicted to be the most powerful biomarkers distinguishing SCs from OECs/SG. Immunofluorescence confirmed a higher expression of SCRG1 in OECs and SG, and conversely a higher expression of AQP1 in SCs in vitro. Furthermore, canine and murine olfactory nerves showed SCRG1-positive, AQP1-negative OECs and/or axons, whereas sciatic nerves displayed multifocal non-myelinated, AQP1-positive, SCRG1-negative cells. Conclusively, OECs/SG are suggested to be a uniform cell type differing only in the tissue of origin and highly related to SCs.

Transcriptional changes in canine distemper virus-induced demyelinating leukoencephalitis favor a biphasic mode of demyelination.

Canine distemper virus (CDV)-induced demyelinating leukoencephalitis in dogs (Canis familiaris) is suggested to represent a naturally occurring translational model for subacute sclerosing panencephalitis and multiple sclerosis in humans. The aim of this study was a hypothesis-free microarray analysis of the transcriptional changes within cerebellar specimens of five cases of acute, six cases of subacute demyelinating, and three cases of chronic demyelinating and inflammatory CDV leukoencephalitis as compared to twelve non-infected control dogs. Frozen cerebellar specimens were used for analysis of histopathological changes including demyelination, transcriptional changes employing microarrays, and presence of CDV nucleoprotein RNA and protein using microarrays, RT-qPCR and immunohistochemistry. Microarray analysis revealed 780 differentially expressed probe sets. The dominating change was an up-regulation of genes related to the innate and the humoral immune response, and less distinct the cytotoxic T-cell-mediated immune response in all subtypes of CDV leukoencephalitis as compared to controls. Multiple myelin genes including myelin basic protein and proteolipid protein displayed a selective down-regulation in subacute CDV leukoencephalitis, suggestive of an oligodendrocyte dystrophy. In contrast, a marked up-regulation of multiple immunoglobulin-like expressed sequence tags and the delta polypeptide of the CD3 antigen was observed in chronic CDV leukoencephalitis, in agreement with the hypothesis of an immune-mediated demyelination in the late inflammatory phase of the disease. Analysis of pathways intimately linked to demyelination as determined by morphometry employing correlation-based Gene Set Enrichment Analysis highlighted the pathomechanistic importance of up-regulated genes comprised by the gene ontology terms "viral replication" and "humoral immune response" as well as down-regulated genes functionally related to "metabolite and energy generation".

CNS Schwann cells display oligodendrocyte precursor-like potassium channel activation and antigenic expression in vitro.

Central nervous system (CNS) injury triggers production of myelinating Schwann cells from endogenous oligodendrocyte precursors (OLPs). These CNS Schwann cells may be attractive candidates for novel therapeutic strategies aiming to promote endogenous CNS repair. However, CNS Schwann cells have been so far mainly characterized in situ regarding morphology and marker expression, and it has remained enigmatic whether they display functional properties distinct from peripheral nervous system (PNS) Schwann cells. Potassium channels (K+) have been implicated in progenitor and glial cell proliferation after injury and may, therefore, represent a suitable pharmacological target. In the present study, we focused on the function and expression of voltage-gated K+ channels Kv(1-12) and accessory ß-subunits in purified adult canine CNS and PNS Schwann cell cultures using electrophysiology and microarray analysis and characterized their antigenic phenotype. We show here that K+ channels differed significantly in both cell types. While CNS Schwann cells displayed prominent K D-mediated K+ currents, PNS Schwann cells elicited K(D-) and K(A-type) K+ currents. Inhibition of K+ currents by TEA and Ba2+ was more effective in CNS Schwann cells. These functional differences were not paralleled by differential mRNA expression of Kv(1-12) and accessory ß-subunits. However, O4/A2B5 and GFAP expressions were significantly higher and lower, respectively, in CNS than in PNS Schwann cells. Taken together, this is the first evidence that CNS Schwann cells display specific properties not shared by their peripheral counterpart. Both Kv currents and increased O4/A2B5 expression were reminiscent of OLPs suggesting that CNS Schwann cells retain OLP features during maturation.

Transcriptomic meta-analysis of multiple sclerosis and its experimental models.

BACKGROUND: Multiple microarray analyses of multiple sclerosis (MS) and its experimental models have been published in the last years. OBJECTIVE: Meta-analyses integrate the information from multiple studies and are suggested to be a powerful approach in detecting highly relevant and commonly affected pathways. DATA SOURCES: ArrayExpress, Gene Expression Omnibus and PubMed databases were screened for microarray gene expression profiling studies of MS and its experimental animal models. STUDY ELIGIBILITY CRITERIA: Studies comparing central nervous system (CNS) samples of diseased versus healthy individuals with n >1 per group and publically available raw data were selected. MATERIAL AND METHODS: Included conditions for re-analysis of differentially expressed genes (DEGs) were MS, myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (EAE) in rats, proteolipid protein-induced EAE in mice, Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD), and a transgenic tumor necrosis factor-overexpressing mouse model (TNFtg). Since solely a single MS raw data set fulfilled the inclusion criteria, a merged list containing the DEGs from two MS-studies was additionally included. Cross-study analysis was performed employing list comparisons of DEGs and alternatively Gene Set Enrichment Analysis (GSEA). RESULTS: The intersection of DEGs in MS, EAE, TMEV-IDD, and TNFtg contained 12 genes related to macrophage functions. The intersection of EAE, TMEV-IDD and TNFtg comprised 40 DEGs, functionally related to positive regulation of immune response. Over and above, GSEA identified substantially more differentially regulated pathways including coagulation and JAK/STAT-signaling. CONCLUSION: A meta-analysis based on a simple comparison of DEGs is over-conservative. In contrast, the more experimental GSEA approach identified both, a priori anticipated as well as promising new candidate pathways.

A toxicogenomic approach for the prediction of murine hepatocarcinogenesis using ensemble feature selection.

The current strategy for identifying the carcinogenicity of drugs involves the 2-year bioassay in male and female rats and mice. As this assay is cost-intensive and time-consuming there is a high interest in developing approaches for the screening and prioritization of drug candidates in preclinical safety evaluations. Predictive models based on toxicogenomics investigations after short-term exposure have shown their potential for assessing the carcinogenic risk. In this study, we investigated a novel method for the evaluation of toxicogenomics data based on ensemble feature selection in conjunction with bootstrapping for the purpose to derive reproducible and characteristic multi-gene signatures. This method was evaluated on a microarray dataset containing global gene expression data from liver samples of both male and female mice. The dataset was generated by the IMI MARCAR consortium and included gene expression profiles of genotoxic and nongenotoxic hepatocarcinogens obtained after treatment of CD-1 mice for 3 or 14 days. We developed predictive models based on gene expression data of both sexes and the models were employed for predicting the carcinogenic class of diverse compounds. Comparing the predictivity of our multi-gene signatures against signatures from literature, we demonstrated that by incorporating our gene sets as features slightly higher accuracy is on average achieved by a representative set of state-of-the art supervised learning methods. The constructed models were also used for the classification of Cyproterone acetate (CPA), Wy-14643 (WY) and Thioacetamid (TAA), whose primary mechanism of carcinogenicity is controversially discussed. Based on the extracted mouse liver gene expression patterns, CPA would be predicted as a nongenotoxic compound. In contrast, both WY and TAA would be classified as genotoxic mouse hepatocarcinogens.

Matrix metalloproteinase-12 deficiency ameliorates the clinical course and demyelination in Theiler's murine encephalomyelitis.

Matrix metalloproteinases (MMPs) are a family of extracellular proteases involved in the pathogenesis of demyelinating diseases like multiple sclerosis (MS). The aim of the present study was to investigate whether MMPs induce direct myelin degradation, leukocyte infiltration, disruption of the blood-brain barrier (BBB), and/or extracellular matrix remodeling in the pathogenesis of Theiler's murine encephalomyelitis (TME), a virus-induced model of MS. During the demyelinating phase of TME, the highest transcriptional upregulation was detected for Mmp12, followed by Mmp3. Mmp12 (-/-) mice showed reduced demyelination, macrophage infiltration, and motor deficits compared with wild-type- and Mmp3 knock-out mice. However, BBB remained unaltered, and the amount of extracellular matrix deposition was similar in knock-out mice and wild-type mice. Furthermore, stereotaxic injection of activated MMP-3, -9, and -12 into the caudal cerebellar peduncle of adult mice induced a focally extensive primary demyelination prior to infiltration of inflammatory cells, as well as a reduction in the number of oligodendrocytes and a leakage of BBB. All these results demonstrate that MMP-12 plays an essential role in the pathogenesis of TME, most likely due to its primary myelin- or oligodendrocyte-toxic potential and its role in macrophage extravasation, whereas there was no sign of BBB damage or alterations to extracellular matrix remodeling/deposition. Thus, interrupting the MMP-12 cascade may be a relevant therapeutic approach for preventing chronic progressive demyelination.

Distinct spatio-temporal extracellular matrix accumulation within demyelinated spinal cord lesions in Theiler's murine encephalomyelitis.

The accumulation of extracellular matrix (ECM) and glial scar formation are considered important factors for the failure of regeneration in central nervous system (CNS) injury and multiple sclerosis. Theiler's murine encephalomyelitis (TME) as a model of multiple sclerosis served to evaluate the spatio-temporal course of ECM alterations in demyelinating conditions. Microarray analysis revealed only mildly upregulated gene expression of ECM molecules, their biosynthesis pathways and pro-fibrotic factors, while upregulation of matrix remodeling enzymes was more prominent. Immunohistochemistry demonstrated progressive accumulation of chondroitin sulfate proteoglycans, glycoproteins and collagens within demyelinated TME lesions, paralleling the development of astrogliosis. Deposition of collagen IV, laminin, perlecan and tenascin-C started 28 days postinfection (dpi), collagen I, decorin, entactin and neurocan accumulated from 56 dpi on, and fibronectin from 98 dpi on. The basement membrane (BM) molecules collagen IV, entactin, fibronectin, laminin and perlecan showed perivascular and parenchymal deposition, while the non-BM components collagen I, decorin, neurocan and tenascin-C only accumulated in a nonvascular pattern in demyelinated areas. Contrary, phosphacan expression progressively decreased during TME. The immunoreactivity of aggrecan and brevican remained unchanged. The spatio-temporal association of matrix accumulation with astrogliosis suggests a mainly astrocytic origin of ECM deposits, which in turn may contribute to remyelination failure in TME.