Brain, Vol. 124, No. 10, 1897-1899,
October 2001
© 2001 Oxford University Press
Editorial |
Gene microarrays and experimental demyelinating disease: a tool to enhance serendipity
Department of Neurological Sciences, Beckman Center for Molecular Medicine, Stanford University, USA
A few years ago a new technology for monitoring gene transcription arrived and profiling thousands of gene transcripts simultaneously from small amounts of tissue became possible. In this issue of Brain, Ibrahim and colleagues applied this technology to monitor gene transcription in a model of multiple sclerosis. They found that 213 genes were differentially regulated, including increased expression of immune-related molecules and genes involved in the movement of cells such as metalloproteases and adhesion molecules (Ibrahim et al., 2001
).
Several different types of microarrays have been developed based on cDNA clones, PCR products or oligonucleotides immobilized on a solid support. The Affymetrix Genechip (Affymetrix, Santa Clara, Calif., USA) consists of a high-density oligonucleotide array synthesized directly onto glass slides by photochemical methods. The expression arrays measure 1.28 x 1.28 cm and contain 64 000 synthesis features. Each 50 x 50 µm `synthesis feature' or `probe cell' contains >107 copies of a particular 25-mer DNA oligonucleotide sequence. Newer versions of the chips have as many as 400 000 20 x 20 µm `synthesis features'. The `synthesis features' are organized in pairs, consisting of a perfect match (PM) oligonucleotide and a mismatch oligonucleotide (MM) immediately below.
Samples are prepared by isolation of polyA+ mRNA, which is made into cDNA and then converted back into biotinylated cRNA. The labelled cRNA is fragmented and hybridized to the array. After washing and staining, the array is scanned with a confocal laser microscope. The presence or absence of a particular RNA is determined from the hybridization pattern using PM/MM differences and ratios. The signal intensity is proportional to the amount of bound RNA. The relative concentrations of RNA in a population can then be estimated. The hybridization signal is determined by averaging 20–60 probe pairs, rather than using a single datum point as for other types of arrays. The chips have high sensitivity and can detect message at an abundance of 1 : 100 000, which corresponds to ~3–5 copies/cell.
Experimental autoimmune encephalomyelitis (EAE) serves as the prototypic model for T-cell-mediated autoimmunity. EAE has striking similarities with the human disease, acute disseminated encephalomyelitis, a complication seen with vaccination and after certain viral infections. EAE has been used as a model to help understand the pathogenesis of multiple sclerosis, and to help identify potential therapeutic candidates for this disease (Table 1).
EAE now comes in a few varieties: there is acute EAE, relapsing–remitting EAE and chronic EAE. Over 4500 papers have been published on the EAE model since it was first described (Weiner, 2001). Zamvil and colleagues first showed that Th1 T-cell clones could induce EAE (Zamvil et al., 1985). LaFaille and Tonegawa advanced this story demonstrating that CD4+, myelin specific T cells induced EAE predominantly, but not always, via production of Th1 cytokines (Lafaille et al., 1997); indeed, they showed that Th2 T cells could trigger EAE. Such Th2 myelin specific T cells can also cause anaphylaxis, creating a new version of `horror autotoxicus' with `allergy to self' (Pedotti et al., 2001). Thus, EAE has been a durable model, and may indeed come in at least two forms: experimental autoimmune encephalomyelitis and experimental allergic encephalomyelitis (Pedotti et al., 2001). Not only are there roles for Th1 and Th2 T cells, and not only are there autoimmune and allergic forms of EAE, but we must now deal with at least two versions of T cells, those bearing the CD4 molecule and those bearing the CD8 molecule, for both of them can induce this model disease (Huseby et al., 2001; Sun et al., 2001).
One of the areas where EAE has failed us is in predicting the outcome of new therapeutics. Copaxone was chosen in the first place in preclinical studies where it suppressed EAE. It is now a licensed drug for treatment of multiple sclerosis and reduces relapses by about one-third. The beta interferons were not really tested in EAE until after they were approved for use in multiple sclerosis. Other promising approaches such as antibody to alpha 4 integrin and altered peptide ligands, which are highly successful in EAE, are now advancing beyond phase II trials in multiple sclerosis patients. However, some approaches which succeed in EAE, e.g. administration of systemic gamma interferon, make multiple sclerosis worse (Panitch et al., 1987). The same is true for blockade of TNF-
or its receptor (Lenercept Multiple Sclerosis Study Group, 1999). Treatment with the same anti-TNF antibody that is beneficial in rheumatoid arthritis and in Crohn's disease makes multiple sclerosis worse, as does the soluble TNF–receptor–immunoglobulin construct, known as Lenercept, which actually increases the relapse rate in relapsing–remitting multiple sclerosis (Lenercept Multiple Sclerosis Study Group, 1999).
In the Ibrahim investigation only one form of EAE was studied, acute EAE induced by myelin oligodendroglial glycoprotein in complete Freund's adjuvant plus Bordetella pertussis toxin, which serves as an additional immune adjuvant. The Freund's adjuvant and pertussis toxin may have contributed to the alterations in gene transcription. Furthermore, only two time points on Day 16 and Day 22 post-immunization at the onset and peak of acute clinical disease were examined. It would be useful in the future not only to study a model of acute EAE, but also one of the several other varieties of this disease.
Most importantly this work has to be extrapolated to studies on multiple sclerosis brain tissue. Only one study using cDNA microarrays analysing two plaques from one brain has been published to date (Whitney et al., 1999). Another study using a combination of custom-made oligonucleotide microarrays and large-scale sequencing of mRNA transcripts from multiple sclerosis plaques has identified osteopontin as a critical gene in the progression of multiple sclerosis (Chabas et al., 2001). It will be important to compare genes elevated in multiple sclerosis brains at autopsy with those gene transcripts modulated in EAE. However, identifying transcriptional changes can only take us so far: it will also be vital to validate potential new candidates in the pathogenesis of multiple sclerosis by using methods to block or deliver the products of such genes, and to see how they modulate EAE. If protection is seen in an EAE model, after such an identification is made, someone might be bold enough to use this as a basis for testing the approach in a human clinical trial (Steinman, 1999). The gene microarray will be a tremendous tool for enhancing serendipity and leading us to new participants in the pathogenesis of demyelinating disease.
|
|
References
Chabas D, Baranzini SE, Mitchell D, Bernard CCA, Rittling SR, Denhardt DT, et al. The influence of osteopontin on autoimmune demyelinating disease. Science. In press 2001.
Huseby E, Liggitt D, Brabb T, Schnabel B, Ohlen C, Goverman J. A pathogenic role for CD8+ T cells in a model for multiple sclerosis. J Exp Med. In press 2001.
Ibrahim SM, Mix E, Böttcher T, Koczan D, Gold R, Rolfs A, Thiesen H–J. Gene-expression profiling of the nervous system in murine experimental autoimmune encephalomyelitis. Brain 2001; 124: 1927–1938.
Lafaille JJ, Keere FV, Hsu AL, Baron JL, Hass W, Raine CS, et al. Myelin basic protein-specific T helper 2 (Th2) cells cause experimental autoimmune encephalomyelitis in immunodeficient hosts rather than protect them from disease. J Exp Med 1997; 186: 307–12.
Lenercept Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group. TNF neutralization in MS: results of a randomized, placebo-controlled multicenter study. Neurology 1999; 53: 457–65.
Panitch HS, Hirsch RL, Schindler J, Johnson KP. Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system. Neurology 1987; 37: 1097–102.
Pedotti R, Mitchell D, Wedemeyer J, Karpuj M, Chabas D, Hattab EM, et al. An unexpected version of horror autotoxicus: anaphylactic shock to a self-peptide. Nat Immunol 2001; 2: 216–22.[Web of Science][Medline]
Steinman L. Assessment of the utility of animal models for multiple sclerosis and demyelinating disease in the design of rational therapy. [Review]. Neuron 1999; 24: 511–14.[Web of Science][Medline]
Sun D, Whitaker JN, Huang Z, Liu D, Coleclough C, Wekerle H, Raine CS. Myelin antigen-specific CD8+T cells are encephalitogenic and produce severe disease in C57BL/6 mice. J Immunol 2001; 166: 7579–87.
Weiner HL. The fine line between autoimmune and allergic encephalomyelitis [letter]. Nat Immunol 2001; 2: 193–4.[Medline]
Whitney LW, Becker KG, Tressler NJ, Caballero-Ramos CI, Munson PJ, Prabhu VV, et al. Analysis of gene expression in multiple sclerosis lesions using cDNA microarrays. Ann Neurol 1999; 46: 425–8.[Web of Science][Medline]
Zamvil S, Nelson P, Trotter J, Mitchell D, Knobler R, Fritz R et al.. T cell clones specific for myelin basic protein induce chronic relapsing paralysis and demyelination. Nature 1985; 317: 355–8.[Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  R P Lisak, J A Benjamins, B Bealmear, B Yao, S Land, L Nedelkoska, and D Skundric Differential effects of Th1, monocyte/macrophage and Th2 cytokine mixtures on early gene expression for immune-related molecules by central nervous system mixed glial cell cultures Multiple Sclerosis, April 1, 2006; 12(2): 149 - 168. [Abstract] [PDF]
|
|
|
|
 |
|
 B. Weinstock-Guttman, D. Badgett, K. Patrick, L. Hartrich, R. Santos, D. Hall, M. Baier, J. Feichter, and M. Ramanathan Genomic Effects of IFN-{beta} in Multiple Sclerosis Patients J. Immunol., September 1, 2003; 171(5): 2694 - 2702. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Mycko, R. Papoian, U. Boschert, C. S. Raine, and K. W. Selmaj cDNA microarray analysis in multiple sclerosis lesions: detection of genes associated with disease activity Brain, May 1, 2003; 126(5): 1048 - 1057. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L.-M. Sturla, A. Fernandez-Teijeiro, and S. L. Pomeroy Application of Microarrays to Neurological Disease Arch Neurol, May 1, 2003; 60(5): 676 - 682. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||