Pmog level 7 tool

The experiment was repeated twice with the same results. The observed differences between protein and mRNA levels in splenocytes from pMOG-IFNbeta-treated rats may be caused by expansion of another cell type during restimulation in this group which would reduce the mRNA expression relative to housekeeping genes. This issue can be specifically addressed as soon as anti-rat IL antibodies for intracellular staining becomes available. The observed auto fluorescence was caused by the restimulation ex vivo and could not be further reduced by gating of the cells.

This is likely caused by clonal expansion of Ag-specific Th17 and Th1 cells ex vivo , which reduces the frequency of other Th cell types. Herein we report that Th17 cell responses during the peak of disease are dramatically impaired in DNA vaccinated rats compared to in controls.

IL and IL are both expressed by Th17 cells but exert different functions, IL being thought to be an effector molecule whereas IL potentiates the Th17 cell response [29] , [30]. The reduced Th17 cell responses reported herein could be caused by enhanced activation of induced Treg during DNA vaccination. Probably the differentiation of Th17 cells is specifically impaired because Th1 and Th2 responses do not correlate with protection in our model [3] , [7] , [11].

In contrast, we observed reduced IL responses after DNA vaccination, which is in agreement with our previous findings that IL is not enhanced by the pMOG vaccination [3] , [7] , [11]. Critical differences may be that we study suppression of Th17 responses in vivo whereas Guo et al study these responses in vitro ; that IL is expressed at an earlier timepoint in our system or that we use different species. Further analyses of T cell responses in DNA vaccinated rats are warranted and are ongoing in our laboratory.

However, the pathways that are activated by the DNA vaccine and which led to impaired Ag-specific Th17 immune responses have not been elucidated. The reason for this could be: a clonal expansion of another cell type in the pMOG-IFNbeta-treated group that skews the IL mRNA expression relative to a housekeeping gene, or b there are additional, unknown mechanisms involved.

Four-to-five wk old locally bred LEW. Oligonucleotides coding for the sense strand of a scrambled siRNA sequence, a loop sequence, and the antisense strand of a scrambled siRNA sequence were hybridized and ligated into pSilencer 1.

Plasmid DNA was prepared using the Qiagen plasmid preparation protocol. Membranes were incubated for 1 h in blocking buffer TBS Rat splenocytes were isolated from five-to-six wk old female DA rats. Four-to-five wk old LEW. Three wks after DNA vaccination rats were injected s. The symptoms were scored as follows: grade 1; tail weakness or tail paralysis, grade 2; hind leg paraparesis, grade 3; hind leg paralysis, grade 4; complete paralysis, moribund state or death.

The lower limit of detection is 3. Q-PCR to quantify levels of cytokines has been previously described [40] , [41]. Samples were analyzed as previously described [7]. The generation, use and analysis of microarrays representing cDNAs has been described previously [42] — [44]. Labelling and hybridization protocols have been previously described [43]. The data series is available online at GEO at www. Data was normalized by Lowess normalization using the statistical software R.

The significance of the expression ratios was then estimated using the Significance Analysis of Microarray SAM statistical technique [45]. A q value was assigned for all detectable genes. The q value represents the lowest false discovery rate at which the differential expression of the gene is considered significant.

In an additional step, only genes with a ratio of 1. Because rat genes are not as well annotated as human genes, the rat gene identities were mapped to human locuslink numbers of orthologues genes. These were then used to search in the GOchart program. Monocytes were obtained from heparinized blood of 4—5 rats using standard Lymphoprep density gradient centrifugation Nycomed, NY.

Monocytes were further enriched by magnetic beads conjugated to RT1 Ab Miltenyi Biotec, Germany following the instructions provided by the manufacturer. After three d cells were harvested and used for further analysis. To measure differences between gene expression and cytokine levels, we first tested if the groups were normally distributed.

If they were, we analyzed differences using unpaired t-test. If the groups were not normally distributed, we analyzed the differences with Mann-Whitney U test or Kruskal-Wallis test to compare three or more groups using Graphpad Prism 4. Competing Interests: The authors have declared that no competing interests exist. Lobell , Petrus and Augusta Hedlunds foundation A.

Lobell and O. Lobell , Tore Nilson foundation A. Lobell , Karolinska Institutet funds A. Lobell , Socialstyrelsen funds A. Lobell , the Swedish Association for Neurologically disabled A. Lobell and the Swedish Research Council O. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. There are no financial, personal, or professional interests that have influenced the paper.

National Center for Biotechnology Information , U. PLoS One. Published online Nov Robert A. Jean Kanellopoulos, Editor. Author information Article notes Copyright and License information Disclaimer. Received May 19; Accepted Oct Copyright Andersson et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

This article has been cited by other articles in PMC. Open in a separate window. Figure 1. Impaired Th17 cell responses after pMOG vaccination. Foxp3 expression is reduced in splenocytes from DNA vaccinated rats Induced regulatory T cells Treg have been implicated in the protective mechanism of DNA vaccination against other organ-specific autoimmune diseases such as murine Experimental Autoimmune Uveitis [18].

Figure 2. Figure 3. Table 1 Differentially expressed genes in DNA vaccinated rats compared to controls. Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Transfection of splenocytes Rat splenocytes were isolated from five-to-six wk old female DA rats. Statistical analysis of microarray data Data was normalized by Lowess normalization using the statistical software R. Isolation and culture of monocytes Monocytes were obtained from heparinized blood of 4—5 rats using standard Lymphoprep density gradient centrifugation Nycomed, NY.

Acknowledgments A. Footnotes Competing Interests: The authors have declared that no competing interests exist. References 1. Multiple sclerosis: deeper understanding of its pathogenesis reveals new targets for therapy. Annu Rev Neurosci. Combination of gene delivery and DNA vaccination to protect from and reverse Th1 autoimmune disease via deviation to the Th2 pathway.

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