Genomic Approaches

Although immunocytochemistry and in situ hybridization have provided important clues to the pathogenesis of the MS lesion, these techniques are largely qualitative and static, and as such are limited in their ability to provide dynamic information on disease evolution and cellular interactions. More recently, DNA microarray technology and high throughput sequencing of cDNA have been applied to the analysis of MS brain tissue, in an attempt to identify genes that contribute to lesion pathology. These approaches allow for the simultaneous measurement of expression of thousands of genes and the identification of gene activation patterns in tissue at specific time points. Several studies have analyzed gene expression in postmortem MS brain tissue compared to non-MS control tissue. Whitney et al. (159) did one of the earliest studies using custom printed microarrays, and compared gene expression in normal white matter with that in acute lesions in brain tissue from one MS patient. They identified many genes that were either up- or downregulated in MS plaques. Arachidonate 5-lipoxygenase, a key enzyme in the biosynthetic pathway of leukotrienes, is overex-pressed in MS lesions, but since it is also expressed in other CNS diseases associated with macrophages and monocyte activation, its pathogenic relevance to MS is still uncertain. A study by Lock et al. (160) using Affymetrix GeneChip microarrays to study acute active MS lesions and chronic silent MS lesions revealed granulocyte colony-stimulating factor was highly expressed in acute lesions, and not in silent lesions, whereas transcripts encoding the IgG Fc receptor I were found over-expressed in silent lesions. These two candidate target genes were further studied in the EAE model. EAE was associated with less severe acute disease and absent chronic disease in mice deficient for the immunoglobulin Fc-receptor, whereas granulocyte-colony stimulating factor decreased the severity of early EAE. Another study revealed that MS lesions are associated with the upregulation of several known immune-related genes, as well as unique transcripts which may be relevant to MS pathogenesis (161). Comparative analysis of differential gene expression of chronic active and inactive lesions revealed significant differences in the transcriptional profiles of these two lesions, both within the lesion center and the lesion margin. Active lesion margins and centers showed upregulation of genes associated with inflammation (e.g., TNF, IL-6), compared to an under-representation in inactive lesions. Inactive lesions, however, contained many apoptosis-related genes such as bcl-x, growth factor receptor-bound protein 2, and stress-induced proteins such as hsp90A and hsp60.

Studies utilizing large-scale sequence analysis of cDNA libraries, generated from brain tissue of MS patients, have identified a number of cDNAs that are over-represented in MS, compared to those from control brain tissue (162). Among these was osteopontin (OPN), a cytokine with pleiotropic functions, including a role in inflammation and immunity to infectious diseases. Immunocytochemistry has revealed expression of OPN adjacent to both MS and EAE lesions. Since the induction and severity of EAE, and the expression of inflammatory cytokines by T-cells were greatly reduced in mice lacking the OPN gene, OPN may be a good target for future anti-inflammatory therapy.

Although gene profiling of tissue samples using microchip arrays seems powerful, easy, and plausible, there are a number of limitations that may contribute to a lack of reproducibility in published data. A primary issue relates to the quality of the sampled material, and although fresh frozen unfixed tissue is most suitable for mRNA analysis, this is rare in most brain banks, and requires careful preservation by snap-freezing. Even under the best circumstances, the structural integrity of the tissue may be impaired, leading to problems in both classifying and characterizing the sample. The nonspecific binding of antibodies to frozen tissue further limits the ability to reliably characterize the lesions. Although formaldehyde-fixed, paraffin-embedded tissue is abundant in archival brain banks, the fixation and postmortem delay can impact both the success and quality of immunocytochemistry.

Another limitation relates to the complexity of the sampled material. Since the MS lesion results from multiple inflammatory, degenerative, and reparative events typically occurring simultaneously within the acute lesion, it is difficult to relate changes in transcriptional patterns to individual cell components. Laser capture microdissection of single cells from a stained microscopic tissue slide offers a potential solution. Single-cell microdissection produces highly specific results, but the amount of mRNA/cDNA obtained is minute, and usually insufficient to be combined with microarray analyses. Furthermore, the quality of extracted mRNA is a factor limiting microdissection of human material. mRNA is often degraded in human postmortem material to a degree that excludes polymerase chain reaction amplification and scanning of the transcriptome.

Another limitation is the information technology resources required to store the enormous amount of information generated, as well as to analyze and discriminate the individual signals from noise, in order to integrate the results into coherent gene clusters. Once a candidate gene is identified, its relevance must be examined by more conventional biological testing in the postgenomic phase. The identification of additional genes requires verification of the candidate in tissue samples using real-time polymerase chain reaction, in situ hybridization, or immunocytochemistry, as well as cell culture studies, transfection experiments, or the construction of transgenic mice.

Cure Tennis Elbow Without Surgery

Cure Tennis Elbow Without Surgery

Everything you wanted to know about. How To Cure Tennis Elbow. Are you an athlete who suffers from tennis elbow? Contrary to popular opinion, most people who suffer from tennis elbow do not even play tennis. They get this condition, which is a torn tendon in the elbow, from the strain of using the same motions with the arm, repeatedly. If you have tennis elbow, you understand how the pain can disrupt your day.

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