Additional file 3: Table S2. of Allelic imbalance of multiple sclerosis susceptibility genes IKZF3 and IQGAP1 in human peripheral blood

Details of excluded measurements per investigated gene. (PDF 82 kb

Modular fulltext search for MySQL

An objective of the project is to develop a modular fulltext search engine using MySQL database server. The search engine should operate with the Czech language's specific attributes. There is no endeavor to develop high quality modules solving linguistic problems. Project should provide interface and ability to plug-in (plug-out) next modules. Project's software platform is Unix, programming language C++

Additional file 4: Figure S2. of Allelic imbalance of multiple sclerosis susceptibility genes IKZF3 and IQGAP1 in human peripheral blood

Allele-specific expression analyses of IKZF3 and IQGAP1 in samples from healthy controls. Allele-specific expression of the genes was normalised to the mean of all genomic DNA for (a) rs907091 in IKZF3 and (b) rs11609 in IQGAP1. Each bar represents five replicate measurements. Data are presented as the normalized change in Ct between the two alleles (nΔCt). nΔCt values above zero represent lower expression of the MS risk allele, whereas nΔCt values below zero represents higher expression of the MS risk allele. Error bars represent the standard error of the mean. A two-tailed unpaired Student’s t-test was used to compare each column with the gDNA measurement, P-values <0.05 are indicated with *. A > B = allele A expressed higher than B, A < B = allele A expressed lower than B. (TIF 84 kb

Additional file 1: of Friedreich ataxia in Norway – an epidemiological, molecular and clinical study

Meiotic instability of GAA repeats analyzed by differences in GAA repeat expansion sizes in parents and children. (DOCX 22 kb

Additional file 2: of Friedreich ataxia in Norway – an epidemiological, molecular and clinical study

ROC curves based on measuring frataxin from whole blood in FRDA patients, carriers and healthy controls. (DOCX 38 kb

<i>SH2D2A</i>-deficient mice have increased numbers of Id-specific TCR–transgenic SP CD4+ thymocytes.

<p>The graphs represents from the left; “UC” –unchallenged mice, i.e. control Id-specific TCR-transgenic mice that did not receive tumor cells, wild-type (<i>SH2D2A</i>+/+) and <i>SH2D2A</i> deficient (<i>SH2D2A</i>−/−) mice that had to be sacrificed due to development of large tumors (tumor volume > 1 cm³) (T = tumor) or that was tumor free at the end of the experiment 70 days after tumor inoculation (TF = tumor free). These tumor-free mice never had a palpable tumor during the course of the experiment, or they had a tumor that never reached 1 cm³ and that disappeared prior to the end of the experiment. The median values are shown as lines in the diagrams. (A, B) Thymocytes from mice with indicated genotypes were labeled with anti Id-TCR (GB113), anti-CD4 and anti-CD8 mAbs and their expressions were monitored by flow cytometry. The total number of Id-specific TCR-transgenic (GB113+) double positive (DP, CD4+ CD8+) and single positive CD4+ (SP, CD4+ CD8−) thymocytes is indicated for each genotype. (C) The diagram display the number of GB113+ CD4+ T cells in the tumor-draining lymph node of the same mice as in A and B. Of note is that the number of GB113+ CD4+ T cells in unchallenged mice is very low due to the small size of the draining lymph node in the absence of tumor challenge. (D) The GB113+ CD4+ T cells from the draining lymph node were labeled with anti-CD69 mAb, and their expression was monitored by flow cytometry. The diagram shows the median fluorescence intensity (MFI) of CD69 divided by the MFI of isotype control in GB113+ CD4+ T cells. P values were calculated with two-tailed Mann-Whitney U test. Ns = non-significant.</p

TCR transgenic <i>SH2D2A</i>-deficient mice are also resistant towards tolerizing amounts of myeloma.

<p>Id-specific TCR transgenic mice with (+/+, n = 11) or without (-/-, n = 15) <i>SH2D2A</i> expression were injected subcutaneously with high dose (2×10⁶) MOPC315 cells. Tumor development was followed by palpation (A–C) and M315 serum myeloma protein concentration was measured by ELISA 7, 21 and 35 days after tumor inoculation (D). (A) The plot display survival of Id-specific TCR-transgenic mice with and without <i>SH2D2A</i>. Mice with large tumors (tumor volume > 1 cm³) were euthanized. (B) The graphs display the tumor growth in the individual Id-specific TCR transgenic mice with (+/+, left diagram) and without (−/−, right diagram) <i>SH2D2A</i> expression. The grey lines highlight established tumors that were rejected. The horizontal lines at 0 mm³ visualize mice that did not develop a palpable tumor during the course of the experiment. (C) The plot displays tumor take of <i>SH2D2A</i>+/+ and <i>SH2D2A</i>−/− Id-specific TCR-transgenic mice. A tumor-free mouse was defined as a mouse that has not had a palpable tumor during the course of the experiment. (D) The plots present the M315 serum level 7, 21 and 35 days after tumor cell inoculation. Lines represent the median values of the M315 measurements. The dots below the dashed line (at 2 ng/µl) are below the detection limit of the ELISA assay. P values were calculated with two-tailed log rank test (A, C) and one-tailed Mann-Whitney U test (D). Ns = non-significant.</p

Small changes in thymocyte proportions in <i>SH2D2A</i>-deficient mice.

<p>(A) The graphs show the mean number of thymocytes (upper panel) and splenocytes (lower panel) with SD from <i>SH2D2A</i>-deficient C57BL/6 (7–16 weeks) and BALB/c (11–20 weeks) mice and corresponding age-matched controls. Student’s paired t-test showed no significant differences between the groups. (B, C) Thymocytes (B) and splenocytes (C) from C57BL/6 and BALB/c mice with indicated genotypes were labeled with anti-CD4 and -CD8 mAbs and their expression were monitored by flow cytometry. (B) The percentage of double negative (DN – upper left), double positive (DP – lower left), single positive (SP) CD4+ (upper right) and CD8+ (lower right) thymocytes is indicated for each genotype. (C) The percentage of CD4+ and CD8+ splenocytes is indicated in each group. (B, C) The median values are shown as lines in the diagrams P-values were calculated by the Mann-Whitney U test, p-values are indicated only when significant (p < 0.05). (D) Freshly isolated splenic CD4+ T cells were labeled with indicated mAbs prior to flow cytometry analysis. One representative experiment of at least four experiments performed on splenic CD4+ T cells from C57BL/6 mice (8–16 weeks old) is shown.</p

TCR transgenic <i>SH2D2A</i> −<b>/</b>− mice are resistant towards transplanted myeloma.

<p>Non-transgenic BALB/c and Id-specific TCR-transgenic (Id-TCR) BALB/c mice with (+/+) or without (−/−) <i>SH2D2A</i> expression were injected subcutaneously with low dose MOPC315 cells (160 000 cells). Tumor development was followed by palpation. (A, B) A tumor-free mouse was defined as a mouse that did not have a palpable tumor during the course of the experiment. The plots display tumor-take for (A) non-transgenic BALB/c mice with (n = 9) or without (n = 11) <i>SH2D2A</i> expression and (B) Id-specific TCR-transgenic BALB/c mice with (n = 23) or without (n = 23) <i>SH2D2A</i> expression. P values were calculated with two-tailed log rank test. Ns = non-significant.(C, D) Splenic CD4+ T cells were isolated from surviving tumor-free mice and stimulated <i>in vitro</i> with Id-positive F9 cells. Cells were labeled with GB113, recognizing the Id-specific transgenic TCR, and anti-CD69, CD44, CD62L or CD25 mAbs, prior to and after 24, 48 and 72 hours in culture with Id-positive cells. (C) FACS plots gated on GB113+ CD4+ T cells from one representative experiment are shown. (D) The diagram show the median value of the MFI (median fluorescent intensity) of CD69 at the indicated time points (n = 11) with SD. P-values were calculated with two-tailed, unpaired student t test, * indicate significant differences (at 48 hours: p = 0,003; at 72 hours: p = 0,03).</p

Characterization of <i>SH2D2A</i>-deficient Id-specific TCR-transgenic CD4+ T cells.

<p>(A) Thymocytes from Id-specific TCR-transgenic mice with indicated genotypes (9–15 weeks old) were labeled with anti-transgenic TCR (GB113), anti-CD4 and -CD8 mAbs. Expression of CD4 and CD8 on GB113+ thymocytes was monitored by flow cytometry. The mean average percentage with SD of double positive (DP), double negative (DN), single positive (SP) GB113+ CD4+ and CD8+ thymocytes is indicated for each genotype (n = 3). (B) Splenocytes from normal (<i>SH2D2A</i>+/+) and age-matched <i>SH2D2A-</i>deficient (<i>SH2D2A</i>−/−) mice (6–14 weeks old) were labeled with anti-CD4 and GB113 and expression was monitored by flow cytometry. Diagrams show the frequency of CD4+ T cells (left diagram) and the percentage of CD4+ T cells that express the Id-specific transgenic TCR (GB113+) (right diagram). The median values are shown as lines in the diagrams. (C) CFSE labeled CD4+ T cells from TCR transgenic <i>SH2D2A</i>+/+ (upper diagram) and <i>SH2D2A</i>−/− (lower diagram) were harvested after 24, 48 and 72 hours in culture with Id-positive F9, labeled with GB113 antibody. The CFSE dilution in GB113+ CD4+ T cells was measured by flow cytometry; grey area - CFSE profiles after 24 hours, open area (black line) - CFSE profiles after 48 hours and open area (stippled line) - CFSE profiles after 72 hours. One representative experiment out of four is shown. (D) Unlabeled CD4+ T cells from <i>SH2D2A</i>+/+ and <i>SH2D2A</i>−/− mice were cultivated as in C. Prior to and after 24, 48 and 72 hours, cells were stained with GB113 in addition to CD69, CD44, CD62L or CD25 mAbs to assess T cell activation by flow cytometry. FACS plots gated on GB113+ CD4+ T cells from one representative experiment of at least three are shown.</p