Encephalitis is inflammation of brain tissue, and in humans and other animals, it is usually accompanied by inflammation of the adjacent meninges (meningoencephalitis). Although many CNS inflammatory disorders have a bacterial, viral, fungal, or protozoal cause, other inflammatory responses do not have such clear etiologies. In particular, there are many situations in which clinical signs and lesions suggest viral involvement, yet an agent is not identified. In humans, only between 30% and 50% of suspected cases of viral encephalitis are confirmed by detection of the etiologic agent in CSF samples via PCR assay.1,2 In addition, in several encephalitic conditions, in which the histologic features together with clinical, epidemiological, and pathological observations support a viral etiology, the causative agent has yet to be identified in birds, mink, cattle, fish, and humans.3–8
Determining the causes of novel encephalitic conditions is important. The site and severity of the inflammatory response usually have devastating effects on the host, potentially leading to long-term loss of neurologic function or death. In addition, the global movement of humans, animals, and goods has provided for the spread, genetic modification, and emergence of infectious agents, many of which are viral and manifest as encephalitic disorders (eg, West Nile virus, Nipah virus, and Hendra virus).9–12
Despite the known viral etiology of many encephalitic conditions in dogs, vigilant histologic surveillance for CNS diseases has revealed conditions for which there is no known etiologic agent.13,14 These conditions include granulomatous meningoencephalitis and breed-associated encephalitic conditions in Pointers, Pugs, Maltese dogs, and Yorkshire Terriers.13 In Greyhounds, a fatal, nonsuppurative meningoencephaltitis centering on the cerebral gray matter and rostal aspect of the brainstem is considered breed associated.14,15 Whereas the lesions of breed-associated encephalitic conditions are characterized by a nonsuppurative meningoencephalitis with varying degrees of tissue necrosis, the conditions differ among breeds in lesion topography, allowing them to be distinguished histologically.15–17 To date, studies performed to identify the cause of these conditions have been limited and, moreover, have failed to yield evidence of many of the known encephalitis-inducing agents such as canine distemper virus, canine herpes virus, rabies virus, canine parvovirus, adenoviruses, Borna disease virus, louping ill virus, and tick-borne encephalitis virus.13,15–21 Although the outcomes of such studies did not categorically rule out the involvement of microbial agents, they did highlight the potential role of autoimmune processes and genetic predispositions in the development of disease.21–24
Gene expression analysis with species-specific microarray technology is a powerful tool used in rodents, dogs, and humans to elucidate mechanisms underlying disease pathogenesis and identify biomarkers predicting clinical outcome.25–29 The purpose of the study reported here was to obtain additional insight into the cause of Greyhound meningoencephalitis and, in particular, to explore the potential mechanisms at the gene level that contribute to the induction of severe, fatal meningoencephalitis by use of a canine-specific microarray.
Materials and Methods
Dogs—Three Greyhounds with meningoencephalitis and 3 juvenile Greyhounds with no evidence of meningoencephalitis were used in the study. The 3 unrelated, diseased dogs (2 males and 1 female) were 8, 10, and 15 months of age; originated from 3 kennels in southern Ireland; and had been euthanized because of their illness. The control Greyhounds were approximately 10 months of age, were from a fourth kennel in southern Ireland, and had been euthanized because of poor compliance in early stages of racing training. All dogs were euthanized by IV administration of an overdose of pentobarbitone sodium,a in accordance with the guidelines of the University College Dublin Animal Research Ethics Committee.
Specimen collection—For each dog, a postmortem examination was performed immediately after euthanasia, and specimens of brain and spinal cord tissue were collected. Tissue specimens were fixed in neutral-buffered 10% formalin and processed for paraffin embedding. Sections were cut at a thickness of approximately 4 μm and stained with H&E to allow microscopic examination of the cerebrum, cerebellum, brainstem, medulla, pons, choroid plexus, mid portion of the cervical spinal cord, caudal portion of the thoracic spinal cord, cranial portion of the lumbar spinal cord, dorsal root ganglia, meningeal tissue, and optic nerves within the optic chiasm. In addition, cerebral tissue was immediately snap frozen in liquid nitrogen and stored at −80°C. A portion of cerebral cortex, the classic location of inflammation in Greyhound meningoencephalitis, was collected for mRNA extraction.
Microarray analysis of gene expression—Methods previously established for a canine-specific microarray were used for development of a gene expression profile.28,30 The canine-specific microarrayb contained 9,070 sequences from the Celera canine genomic database derived from a 1.5× Poodle shotgun sequence.31 An additional 6,545 sequences from the public canine-expressed sequence tags database were included. Several human and mouse gene sequences were also included in the microarray to facilitate cross-species annealing.
Total RNA was isolated by use of an isolating reagentc and purified with a purification columnd in accordance with the manufacturer's directions. Reverse transcription was performed with 12 μg of total RNA,e the resulting cDNA was purified, and biotinylated cRNA was generated.f Finally, cRNA samples were fragmented for hybridization with the canine microarray as described elsewhere.30
Arrays were washed and stained in accordance with the manufacturer's protocol. Briefly, wash cycles were completed within the fluidics station as follows: 10 cycles of wash buffer A at 25°C, 4 cycles of wash buffer B at 50°C, followed by staining of the probe array for 30 minutes at 25°C with streptavidin phycoerythrin solution. The final wash consisted of 10 cycles of wash buffer A at 25°C. Data analysis of microarray results was performed with commercially available software.g Data were filtered by use of the data mining tool for signal log ratio (-1 < signal log ratio > 1) and for the presence or absence of a given gene. Gene expression changes (n = 55 changes) were reported on the basis of probe sets recognized as present on all chips (6 chips) analyzed. Pairwise statistical analysis was performed by fitting data to an ANOVA model to compare specimens from diseased animals with control specimens. Fold change in gene expression was calculated as the ratio of the mean signals of diseased versus control specimens; when the fold change was < 1, the ratio was reversed (ie, mean signals for control vs treated specimens) and a negative sign was added. To control the likelihood of a false-positive test result attributable to testing the expression change of thousands of genes simultaneously, the false discovery rate was estimated by use of an algorithm derived from one reported elsewhere.32 Overall gene expression scores comprised the total degree of gene expression as measured via microarray assay or by use of the qRT-PCR assay. Upregulated gene expression in diseased dogs was defined as a higher fold change in comparison with gene expression in control dogs, whereas downregulation reflected a lower fold change in comparison with that in control animals.
Quantitative PCR assay—Although the validation of microarray expression scores obtained for specific genes by use of independent techniques is still considered a desirable component of any microarray experiment, the genes selected for validation a priori are usually identified from the microarray data.33 Therefore, to confirm transcriptional changes detected with the canine-specific microarray, a subset of transcripts was selected for qRT-PCR assay on the basis of the 5 genes representing the most significant expression changes as determined with the microarray. These genes included those for IgC, MHC class II HLA-DRB1, CCL5, MHC class I DLA-64, and MHC class I DLA-88. Gene-specific primers were designed with the aid of commercially available software.h From the total RNA isolates described previously, 3.2-μg aliquots were removed, treated with DNA endonuclease,i and reverse transcribed.j For the PCR assay, 100μM forward and reverse primers, PCR mix,k and cDNA template were used. Each assay, including a standard curve and negative control specimen, was performed in triplicate with reaction conditions as follows: 50°C for 2 minutes, 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds, and 60°C for 1 minute. The size of the resulting amplification product was confirmed by use of agarose electrophoresis prior to quantitative fluorescence-based PCR detection.l Data were analyzed by use of the comparative threshold method of relative quantitation, and the constitutively expressed glyceraldehyde 3-phosphate dehydrogenase transcript was used as the internal standard.
Statistical analysis—Mean ± SD microarray expression scores as well as fold change in the degree of expression were determined for each gene. The Student t test was used to evaluate differences between expression scores in nervous-tissue specimens from control and diseased dogs. A value of P ≤ 0.05 was considered significant.
Results
Dogs—Histologic evaluation of nervous tissue from the 3 Greyhounds with meningoencephalitis confirmed the classic features of Greyhound meningoencephalitis. Features included patchy nodular gliosis and gemistocytosis of the rostal portion of the cerebrocortical gray matter and caudate nucleus, accompanied by perivascular accumulation of lymphocytes and plasma cells and infiltration of the meninges by the same cell types.13 No microscopic CNS abnormalities were detected in the 3 control dogs.
Microarray of gene expression—The canine-specific arrays, in which expression in > 22,000 genes was evaluated, revealed 31 genes that had been significantly upregulated or downregulated in Greyhounds with versus without meningoencephalitis (Figure 1). Thirty-one genes had significant changes in expression. Ten of these genes were proprietary sequences, gene sequences that have not yet been annotated in dogs, or pseudo-genes and were therefore not investigated further. Of the remaining 21 annotated sequences with significant expression changes, 18 had upregulated expression values and 3 had downregulated expression values. Of those upregulated genes, 8 were immune-related sequences, 2 were ligand-binding sequences, 3 were signal-transduction sequences, and 3 were transcription regulatory sequences. The remaining 2 upregulated genes were involved in protein structure and transport.
Photograph of a region of particular interest on a canine oligo microarray, revealing visibly intense differences in gene expression between 3 control Greyhounds without meningoencephalitis (C1-C3) and 3 Greyhounds with meningoencephalitis (M1-M3). The orange-to-yellow color range indicates an increasingly higher degree of gene expression, and the blue-to-black color range indicates an increasingly lower degree of gene expression, compared with that in control dogs. For example, in diseased dogs, gene expression of MHC class I DLA-88 and CCL5 is upregulated in comparison with expression in control dogs, whereas gene expression of MHC class I DLA-12 is upregulated in both groups and that for MHC class II DLA-DQB is low in both groups.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.547
Expression of immune-related genes was significantly upregulated in diseased dogs, compared with expression in control dogs (Figure 2). Genes with downregulated expression included those also involved in ligand binding (n = 2) and a gene (PRSS2) involved in enzyme function. Genes involved in transcription regulation and signal transduction were collectively upregulated. However, expression of each gene was independently responsive to the encephalitic condition (Figure 3). A significant and strong correlation between disease status and gene expression was detected by use of robust multichip analysis (r = 0.89; P < 0.05) and micro-array analysis (r = 0.92; P < 0.05).
Mean ± SD gene expression scores (fluorescence) as determined via microarray for immune system-related proteins MHC class I DLA-88, MHC class I DLA-64, IgC, MHC class II HLA-DRB1, CCL5, Mx1, Mx2, and MHC class I DLA-12 in nervous tissue from Greyhounds with (n = 3; black bars) and without (3; gray bars) meningoencephalitis. Values differ significantly (P < 0.05) between dog groups for all genes.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.547
Mean ± SD gene expression scores (fluorescence) as determined via microarray for genes involved in ligand binding (MAP1B, OGT, LCP1, and TTRl, signal transduction (COL1A2, C2, and SERPING1), and taxon regulation (MBNL2, CD37, and S100A11) and for enzymes (PRSS2), structural proteins (COL1A2), and transport proteins (CHML)) in nervous tissue from Greyhounds with (n = 3; black bars) and without (3; gray bars) meningoencephalitis. See Figure 2 for remainder of key.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.547
qRT-PCR assay—Overall, results of the qRT-PCR assay verified the visual differences observed in micro-array experiments. Gene expression for CCL5, IgC, MHC class I HLA-DRB1, MHC class I DLA-64, and MHC class I DLA-88 was increased in all diseased dogs, compared with expression in control dogs. Furthermore, for each dog, the relative degree of change in expression among the genes was also similar. However, the changes in expression based on microarray analysis did not agree with results of the qRT-PCR assay. Specifically, results of the microarray analysis indicated large differences in degrees of gene expression between diseased and control dogs. Although the qRT-PCR assays also revealed significant differences, those differences were much smaller than those detected by use of the microarray.
Overall degree of change in expression of each gene evaluated also differed between the microarray analysis and qRT-PCR assay. For example, results of micro-array analysis indicated that MHC class I DL-88 had the greatest degree of gene expression among immune-related genes, whereas results of the qRT-PCR assay suggested that CCL5 had the highest degree and MHC class I DL-88 had the lowest degree of gene expression among the 5 genes tested.
Mean ± SD gene expression scores (fluorescence) as determined via qRT-PCR assay for CCL5, IgC, HLA-DRB1, DLA-64, and DLA-88, in nervous tissue from Greyhounds with (n = 3; black bars) and without (3; gray bars) meningoencephalitis. *Values differ significantly (P < 0.05) between dog groups for indicated genes.
Citation: American Journal of Veterinary Research 71, 5; 10.2460/ajvr.71.5.547
Discussion
The cause of Greyhound meningoencephalitis is undetermined but is postulated to be multifactorial, potentially involving viruses, autoimmune processes, and genetics.14,15,21 In the present study, a canine-specific cDNA microanalysis technique was used to help elucidate the pathogenic mechanisms leading to this fatal brain inflammatory process. To date, canine-specific cDNA microarrays have had limited applications but have been used to characterize the spectrum of gene expression associated with particular canine genetic, neoplastic, degenerative, and inflammatory conditions. 28,29,34–36 Use of microarray technologies in the evaluation of CNS disease processes has been confined to the evaluation of brain tumors in dogs, although in other species including humans, microarray analyses have been undertaken to evaluate CNS inflammatory and degenerative responses.35,37,38
In the study reported here, we compared the degree of expression of > 22,000 genes in nervous tissue from Greyhounds with (n = 3) and without (3) meningoencephalitis. Microarray analysis revealed significant alterations in 21 genes, 18 of which had upregulated expression in diseased Greyhounds, compared with the expression in control Greyhounds. This finding was consistent among the diseased dogs, even though they were not closely related, were bred at different locations, and represented males and females of ages between 8 and 15 months. Such findings strengthened prior observations that meningoencephalitis in Greyhounds has a common etiology and pathogenesis.15,21 The greatest number of genes with upregulated expression related to immune function (8/18), with the remaining genes involved in ligand binding, signal transduction, transcriptional regulation, and the formation and transportation of protein including enzymes.
In the present study, as in others, genes with similar microarray expression scores were unlikely to have similar qRT-PCR assay results, reflecting the different hybridization kinetics of the probe sets for each gene. Thus, it is generally not feasible to predict the true degree of expression of a given gene on the basis of the microarray expression score alone. In the present study, microarray expression scores and qRT-PCR assay results indicated a significant correlation for many genes (at least by robust multichip analysis) with microarray expression scores of < 100 (approx log2 100 = 6.64), which is at the lower end of the range of microarray scores obtained in our study (range, 6 to 23,000). This finding indicated that the exclusion of genes with low microarray expression scores (eg, < 100) from further analysis, as has been practiced in the past, may not be justified. Determining fold changes in the degree of gene expression between subsets of interest is often a critical aim of microarray studies. Our findings indicated that the direction of change in degrees of gene expression (ie, either upward or downward) between diseased and control dogs was accurately predicted by comparison of mean microarray expression scores. Again, the fold-change correlations observed were similar, irrespective of the normalization procedure used. Consistent with the results of other researchers,39 fold changes in gene expression determined by use of the qRT-PCR assay were significantly greater than fold-change values assessed for the same genes by use of microarray analysis.
Results of the present study suggested the upregulation of genes involved in MHC class I (DLA-12, DLA-64, and DLA-88) and class II (HLA-DRB1) molecule expression in Greyhounds with meningoencephalitis, supporting the hypothesis that the associated neuropathologic change reflects an intense inflammatory response.40 In general terms, the MHC gene products interact with bound peptides of intracellular and extracellular origin and present these as antigens to T cells via interactions with T-cell receptors. Furthermore, MHC class I involvement is supported by results of previous preliminary immunohistochemical analysis of the inflammatory response, which revealed the upregulation of MHC class II molecule expression coupled with a strong, but not exclusive, CD8+ T-cell response that would also favor involvement of MHC class I molecules.41 Although increased production of MHC class I and II molecules may be expected with inflammatory diseases associated with intracellular and extracellular infectious agents, this type of increase is not specific; it also occurs in autoimmune diseases.40 Therefore, such findings provide the opportunity to search for potential gene polymorphisms between diseased and nondiseased dogs, which may explain potential genetic susceptibilities, if indeed autoimmunity or augmented susceptibilities to infection are suspected.42,43 Polymorphisms are common in genes for DLA-88 and have also been detected in genes for DLA-12 and DLA-64.44 Similarly, the HLA-DRB1 gene is highly polymorphic.42
In the present study, the detected upregulation of the gene for IgC, which encodes for the IgCs that form components of immunoglobulins, was also noteworthy, as was the upregulation of genes for T-cell receptors, CD1 surface glycoproteins, and MHC II and MHC I proteins (in the context of encoding a (β2 microglobulin precursor). In some viral infections, most notably human herpesvirus-7, and some cancers, it has been postulated that the downregulated expression of MHC I and II and (β2 microglobulin genes is a strategy to avoid immune-system recognition,45 but our results suggested that this mechanism is not a feature of Greyhound meningoencephalitis.
Expression of the CCL5 gene, also known as RANTES, was upregulated in diseased Greyhounds in our study. The associated protein is a recognized chemokine associated with neuroinflammatory processes, in which it is secreted by migrating inflammatory cells (T cells and monocytes) and involved in cell migration across the blood-brain barrier.46 Interferon is a potent stimulator of CCL5 production, which increases in viral infections. However, an increase in CCL5 production is not exclusive to viral infections; it has also been detected in models of autoimmune neuroinflammation.47
Specific upregulation of genes encoding Mx proteins 1 and 2 in nervous tissue from Greyhounds with meningoencephalitis was an interesting finding in the present study. The Mx proteins, components of the innate immune response, are considered important antiviral proteins and are produced in response to interferon, which is commonly produced during many viral infections.48,49 Immune protection by Mx1 and Mx2 proteins against Orthomyxoviridae and Rhabdoviridae, respectively, has been reported,50,51 but the proteins do not protect against all viruses. The importance of the upregulation we detected could support a viral cause for the disease; however, as with CCL5, the trigger for protein production is interferon, which may occur in response to infectious agents and also during autoimmune processes.52
Expression of 4 genes involved in protein binding was either upregulated or downregulated in the diseased Greyhounds in our study. Expression of the OGT gene encoding the catalytic subunit of O-linked N-acetylglucosamine transferase was upregulated, and this protein is involved in cell cycle, transcription, and protein transport.53 Although within any immune response there is a critical need for protein-protein interactions, the importance of the upregulation in the context of this finding was unclear but may emerge as the genomic analysis of more neuroinflammatory disorders is undertaken. The second gene with increased expression was the gene for lymphocyte cytosolic protein (L-plastin), which is expressed in cells of the hematopoietic system. In that system, the protein is involved in the regulation of leukocyte integrin-adhesion function, in particular polymorphonuclear neutrophils and T cells. Its role in T-cell function is more likely to be applicable to Greyhound meningoencephalitis, in which the inflammatory reaction is rich in lymphocytes and devoid of neutrophils.15,54,55 In contrast, the genes for microtubule-associated protein-1B and transthyretin were downregulated. Interestingly, whereas microtubule-associated protein-1B is associated with neural development, it was recently reported that the gene is downregulated in HIV encephalitis.56–58 The transthyretin gene encodes for transthyretin protein, which is involved in thyroid hormone and retinal protein transport. The protein is primarily synthesized in the choroid plexus and liver, and in the liver, its rate of synthesis decreases in response to acute inflammation.39 Similar patterns of reductions in gene expression reportedly occur in the choroid plexus in response to systemic inflammatory responses.60
The upregulated expression of genes responsible for encoding complement proteins Clq and C2 (C1QA and C2, respectively) in the present study indicated the activation of the complement cascade by the classic pathway, which is mediated through the cross-linkage of IgG and IgM to microbial or tissue antigens.61 Therefore, this finding would not allow distinction between an autoimmune or microbial etiology for Greyhound meningoencephalitis.62 Interestingly, however, biosynthesis of complement Clq by microglia is a prominent feature in simian immunodeficiency virus infection, Borna disease virus infection in rats, and experimentally induced allergic encephalomyelitis in rats.63,64 The upregulated expression of the complement-associated genes was accompanied by upregulated expression of the gene encoding serpin peptidase inhibitor (SERPING1), which inhibits C1 activation, presumably providing a defense mechanism in controlling complement activation.65
Microarray analysis also highlighted a significant increase in expression of 3 genes involved in transcription regulation. The gene for muscleblind-like protein 2 (MBNL2) encodes proteins involved in splicing to induce protein diversity and has been associated with cell and tissue development. Expression of this gene occurs in healthy brain tissue, but its role in the context of neuro-inflammation is unknown.66 Upregulated expression of the gene CD37, which encodes for a leukocyte-specific protein involved in T- and B-cell interactions, has a role in T-cell proliferation by influencing early events of T-cell receptor signaling.67 Much of the research on the gene S100A11, the expression of which was also upregulated in our study, has focused on the gene's role in cancer progression, although it also has been associated with inflammatory processes and healthy brain tissue.68,69
Three additional genes had different degrees of expression between dogs with and without meningoencephalitis, and the importance of these findings could not be linked to the location and nature of the neural changes. Expression of the gene PRSS2, which encodes a trypsinogen, was downregulated in Greyhounds with meningoencephalitis. Expression of the gene COL1A2, which encodes collagen type I, was upregulated in this group of dogs, as was expression of the CHML gene, which encodes for proteins involved in Rab protein function within the retina.
In the study reported here, gene microarray analysis of nervous tissue from Greyhounds with meningoencephalitis revealed a unique gene signature, with elements common to viral and autoimmune pathological processes. These findings provide a basis for the evaluation of tissues from a larger numbers of dogs, potentially with a more focused approach on specific lesions through laser-capture microdissection. In addition, the findings provide a basis with which to compare other canine neuroinflammatory disorders of infectious or autoimmune origin.
ABBREVIATIONS
| CCL5 | Small inducible cytokine A5 precursor |
| DLA | Dog leukocyte antigen |
| HLA | Human leukocyte antigen |
| IgC | Immunoglobulin domain constant region |
| MHC | Major histocompatibility complex |
| Mx | Myxovirus (influenza virus) resistance |
| qRT-PCR | Quantitative reverse transcription PCR |
Merial, Harlow, Essex, England.
Affymetrix Microarray, Viagen Inc, Austin, Tex.
RNA Stat60, TelTest, Friendswood, Tex.
Qiagen RNeasy columns, Valencia, Calif.
SuperScript II, Invitrogen, Carlsbad, Calif.
BioArray T-7 polymerase, Enzo, Farmingdale, NY.
Affymetrix Microarray Suite 5.0 and associated Data Mining Tool, University of California, Los Angeles, Calif.
Primer Express, version 1.5, Applied Biosystems, Foster City, Calif.
DNA-free Kit, Ambion Inc, Applied Biosystems, Foster City, Calif.
Omniscript reverse transcriptase, Qiagen, Valencia, Calif.
SYBR Green PCR Master Mix, Applied Biosystems, Foster City, Calif.
Prism 7900HT Sequence Detection System, Applied Biosystems, Foster City, Calif.
References
- 1.
Kennedy PG. Viral encephalitis. J Neurol 2005;252:268–272.
- 2.
Steiner I, Budka H, Chaudhuri A, et alViral encephalitis: a review of diagnostic methods and guidelines for management. Eur J Neurol 2005;12:331–343.
- 3.
Theil D, Fatzer R, Schiller I, et alNeuropathological and aetiological studies of sporadic non-suppurative meningoencephalitis of cattle. Vet Rec 1998;143:244–249.
- 4.
Pennycott TW, Gough RE, Wood AM, et alEncephalitis of unknown aetiology in young starlings (Sturnus vulgaris) and house sparrows (Passer domesticus). Vet Rec 2002;151:213–214.
- 5.
Gavier-Widen D, Brojer C, Dietz HH, et alInvestigations into shaking mink syndrome: an encephalomyelitis of unknown cause in farmed mink (Mustela vison) kits in Scandinavia. J Vet Diagn Invest 2004;16:305–312.
- 6.
Oleksyk TK, Goldfarb LG, Sivtseva T, et alEvaluating association and transmission of eight inflammatory genes with Viliuisk encephalomyelitis susceptibility. Eur J Immunogenet 2004;31:121–128.
- 7.
Watson PJ, Scholes SF. Polioencephalomyelitis of unknown aetiology in a heifer. Vet Rec 2004;154:766–777.
- 8.
Monette S, Dallaire AD, Mingelbier M, et alMassive mortality of common carp (Cyprinus carpio carpio) in the St. Lawrence River in 2001: diagnostic investigation and experimental induction of lymphocytic encephalitis. Vet Pathol 2006;43:302–310.
- 9.↑
Daszak P, Cunningham AA, Hyatt AD. Emerging infectious diseases of wildlife—threats to biodiversity and human health. Science 2000;287:443–449.
- 10.
McCormack JG. Hendra, Menangle and Nipah viruses. Aust N Z J Med 2000;30:9–10.
- 11.
Johnson RT. Emerging viral infections of the nervous system. J Neurovirol 2003;9:140–147.
- 12.
Kennedy PG. Viral encephalitis: causes, differential diagnosis, and management. J Neurol Neurosurg Psychiatry 2004;75(suppl 1):10–15.
- 13.↑
Summers BA, Cummings JF, deLahunta A. Inflammatory diseases of the central nervous system. In: Summers BA, Cummings JF, deLahunta A, eds. Veterinary neuropathology. St Louis: Mosby, 1995;95–188.
- 14.
Braund KG. Inflammatory diseases of the central nervous system. In: Braund KG, ed. Clinical neurology in small animals—localization, diagnosis and treatment. Ithaca, NY: International Veterinary Information Services, 2003.
- 15.
Callanan JJ, Mooney CT, Mulcahy G, et alA novel nonsuppurative meningoencephalitis in young Greyhounds in Ireland. Vet Pathol 2002;39:56–65.
- 16.
Cordy DR, Holliday TA. A necrotizing meningoencephalitis of pug dogs. Vet Pathol 1989;26:191–194.
- 17.
Tipold A, Fatzer R, Jaggy A, et alNecrotizing encephalitis in Yorkshire terriers. J Small Anim Pract 1993;34:623–628.
- 18.
Stalis IH, Chadwick B, Dayrell-Hart B, et alNecrotizing meningoencephalitis of Matlese dogs. Vet Pathol 1995;32:230–235.
- 19.
Kuwamura M, Adachi T, Yamate J, et alNecrotizing encephalitis in the Yorkshire terrier: a case report and literature review. J Small Anim Pract 2002;43:459–463.
- 20.
Schatzberg SJ, Haley NJ, Barr C, et alPolymerase chain reaction screening for DNA viruses in paraffin-embedded brains from dogs with necrotizing meningoencephalitis, necrotizing leukoencephalitis, and granulomatous meningoencephalitis. J Vet Intern Med 2005;19:553–559.
- 21.
Daly P, Drudy D, Chalmers WSK, et alGreyhound meningoencephalitis: PCR-based detection methods highlight an absence of the most likely primary inducing agents. Vet Microbiol 2006;118:189–200.
- 22.
Kipar A, Baumgartner W, Vogl C, et alImmunohistochemical characterization of inflammatory cells in brains of dogs with granulomatous meningoencephalitis. Vet Pathol 1998;35:43–52.
- 23.
Uchida K, Hasegawa T, Ikeda M, et alDetection of an autoantibody from pug dogs with necrotizing encephalitis (Pug dog encephalitis). Vet Pathol 1999;36:301–307.
- 24.
Matsuki N, Fujiwara K, Tamahara S, et alPrevalence of autoantibodies in cerebrospinal fluids from dogs with various CNS diseases. J Vet Med Sci 2004;66:295–297.
- 25.
Pennie WD. Use of cDNA microarrays to probe and understand the toxicological consequences of altered gene expression. Toxicol Lett 2000;112–113:473–477.
- 26.
Ulrich R, Friend SH. Toxicogenomics and drug discovery: will new technologies help us produce better drugs. Nat Rev Drug Discov 2002;1:84–88.
- 27.
Baker TK, Higgins MA, Carfagna MA, et alCharacterisation of hepatocytes amd their use as a model of toxicogenomics. In: Burczynski ME, ed. Introduction to toxicogenomics. Boca Raton, Fla: CRC Press, 2003;117–143.
- 28.
Greer KA, Higgins MA, Cox ML, et alGene expression analysis in a canine model of X-linked Alport syndrome. Mamm Genome 2006;17:976–990.
- 29.
Shelton GD, Hoffman EP, Ghimbovschi S, et alImmunopathogenic pathways in canine inflammatory myopathies resemble human myositis. Vet Immunol Immunopathol 2006;113:200–214.
- 30.↑
Higgins MA, Berridge BR, Mills BJ, et alGene expression analysis of the acute phase response using canine microarray. Toxicol Sci 2003;74:470–484.
- 31.↑
Kirkness EF, Bafna V, Halpern AL, et alThe dog genome: survey sequencing and comparative analysis. Science 2003;301:1898–1903.
- 32.↑
Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Series B Stat Method 1995;57:289–300.
- 33.↑
Dallas PB, Gottardo NG, Firth MJ, et alGene expression levels assessed by oligonucleotide microarray analysis and quantitative real-time RT-PCR—how well do they correlate? BMC Genomics 2005;6:59.
- 34.
Clark LA, Wahl JM, Steiner JM, et alLinkage analysis and gene expression profile of pancreatic acinar atrophy in the German Shepherd Dog. Mamm Genome 2005;16:955–962.
- 35.
Thomson SA, Kennerly E, Olby N, et alMicroarray analysis of differentially expressed genes of primary tumors in the canine central nervous system. Vet Pathol 2005;42:550–558.
- 36.
Ruiz P, Witt H. Microarray analysis to evaluate different animal models for human heart failure. J Mol Cell Cardiol 2006;40:13–15.
- 37.
Sui Y, Potula R, Pinson D, et alMicroarray analysis of cytokine and chemokine genes in the brains of macaques and SHIV-encephalitis. J Med Primatol 2003;32:229–239.
- 38.
Gebicke-Haerter PJ. Microarrays and expression profiling in microglia research and in inflammatory brain disorders. J Neurosci Res 2005;81:327–341.
- 39.↑
Yuen T, Ebersole BJ, Zhang W, et alMonitoring G-protein-coupled receptor signaling with DNA microarrays and real-time polymerase chain reaction. Methods Enzymol 2002;345:556–569.
- 40.↑
Wagner JL. Molecular organization of the canine major histocompatibility complex. J Hered 2003;94:23–26.
- 41.↑
Coates S, Brady J, Worrell S, et alImmunohistochemical characterization of the CNS cell infiltrations in greyhound meningoencephalitis, in Proceedings. 24th Annu Meet Eur Soc Vet Pathol 2006;94–95.
- 42.↑
Wagner JL, Burnett RC, Storb R. Organization of the canine major histocompatibility complex: current perspectives. J Hered 1999;90:35–38.
- 43.
Klein J, Sato A. The HLA system. N Engl J Med 2000;343:782–786.
- 44.↑
Graumann MB, DeRose SA, Ostrander E, et alPolymorphism analysis of four canine class I genes. Tissue Antigens 1998;51:374–381.
- 45.↑
Mirandola P, Sponzilli I, Solenghi E, et alDown-regulation of human leukocyte antigen class I & II and β2-microglobulin expression in human herpesvirus-7-infected cells. J Infect Dis 2006;193:917–926.
- 46.↑
Ubogu EE, Callahan MK, Tucky BH, et alDeterminants of CCL-5 driven mononuclear cell migration across the blood-brain barrier. Implications for therapeutically modulation neuroinflammation. J Neuroimmunol 2006;179:132–144.
- 47.↑
Ransohoff RM. The chemokine system in neuroinflammation: an update. J Infect Dis 2002;186(suppl 2):S152–S156.
- 48.
Zhou A, Paranjape JM, Der SD, et alInterferon action in triply deficient mice reveals the existence of alternative antiviral pathways. Virology 1999;258:435–440.
- 49.
Lee S-H, Vidal SM. Functional diversity of Mx proteins: variations on a theme of host resistance to infection. Genome Res 2002;12:527–530.
- 50.
Arnheiter H, Frese M, Kambadu R, et alMx transgenic mice: animal models of health. Curr Top Microbiol Immunol 1996;206:119–147.
- 51.
Anton LC, Schubert U, Bacik I, et alIntracellular localization of proteasomal degradation of a viral antigen. J Cell Biol 1999;146:113–124.
- 52.↑
Selmi C, lleo A, Zuin M, et alInterferon alpha and its contribution to autoimmunity. Curr Opin Investig Drugs 2006;7:451–456.
- 53.↑
Kreppel LK, Hart GW. Regulation of a cytosolic and nuclear O-GlcNAc transferase. J Biol Chem 1999;274:32015–32022.
- 54.
Jones SL, Wang J, Turck CW, et alA role for the actin-binding protein L-plastin in the regulation of leukocyte integrin function. Proc Natl Acad Sci U S A 1998;95:9331–9336.
- 55.
Wabnitz GH, Kocher T, Lohneis P, et alCostimulation induced phosphorylation of L-plastin facilitates surface transport of the T cell activation molecules CD69 and CD25. Eur J Immunol 2007;37:649–662.
- 56.
Meixner A, Haverkamp S, Wassle H, et alMAP1B is required for axon guidance and is involved in the development of the central and peripheral nervous system. J Cell Biol 2000;151:1169–1178.
- 57.
Masliah E, Roberts ES, Langford D, et alPatterns of gene dysregulation in the fronal cortex of patients with HIV encephalitis. J Neuroimmunol 2004;157:163–175.
- 58.
Everall I, Salaria S, Roberts E, et alMethamphetamine stimulates interferon-inducible genes in HIV infected brain. J Neuroimmunol 2005;170:158–171.
- 59.
Dickson PW, Howlett GJ, Schreiber G. Rat transthyretin (prealbumin). J Biol Chem 1985;260:8214–8219.
- 60.↑
Marques F, Sousa JC, Correia-Neves M, et alThe choroid plexus response to peripheral inflammatory stimulus. Neuroscience 2007;144:424–430.
- 61.↑
Francis K, van Beek J, Canova C, et alInnate immunity and brain inflammation: the key role of complement. Expert Rev Mol Med 2003;5:1–19.
- 62.↑
Ackermann MR. Acute inflammation. In: McGavin MD, Zachery JF, eds. Pathologic basis of veterinary disease. 4th ed. St Louis: Mosby Elsvier, 2007;124.
- 63.
Dietzschold B, Schwaeble W, Schäfer MK, et alExpression of C1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental allergic encephalomyelitis. J Neurol Sci 1995;130:11–16.
- 64.
Depboylu C, Schafer MK-H, Schwaeble WJ, et alIncrease of C1q biosynthesis in brain microglia and macrophages during lentivirus infection in the rhesus macaque is sensitive to antiretroviral treatment with 6-chloro-2′,3′-dideoxyguanosine. Neurobiol Dis 2005;20:12–26.
- 65.↑
Liu D, Lu F, Qin G, et alC1 inhibitor-mediated protection from sepsis. J Immunol 2007;179:3966–3972.
- 66.↑
Pascual M, Vicente M, Monferrer L, et alThe muscleblind family of proteins: an emerging class of regulators of developmentally programmed alternative splicing. Differentiation 2006;74:65–80.
- 67.↑
van Spriel AB, Puls KL, Sofi M, et alA regulatory role for CD37 in T cell proliferation. J Immunol 2004;172:2953–2961.
- 68.
Zimmer DB, Chaplin J, Baldwin A, et alS100-mediated signal transduction in the nervous system and neurological diseases. Cell Mol Biol 2005;51:201–214.
- 69.
Heizmann CW, Ackermann GE, Galichet A. Pathologies involving the S100 proteins and RAGE. Subcell Biochem 2007;45:93–138.
