Data_Sheet_1_Concentration and Subclass Distribution of Anti-ADAMTS13 IgG Autoantibodies in Different Stages of Acquired Idiopathic Thrombotic Thrombocytopenic Purpura.PDF

Background<p>The acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying deficiency of the ADAMTS13 protease is caused by autoantibodies, predominantly of the IgG isotype. Certain HLA-DR-DQ haplotypes were associated with the risk of developing TTP.</p>Objectives<p>To investigate the development of the ADAMTS13-specific antibody response during the course of the disease, we analyzed the concentration, subclass distribution, and inhibitory potential of anti-ADAMTS13 IgG autoantibodies in samples of TTP patients drawn during the first acute phase, in remission, and during relapse. Additionally, we compared the anti-ADAMTS13 IgG levels between patients carrying and not carrying risk and protective HLA-DR-DQ haplotypes.</p>Patients and Methods<p>We determined the anti-ADAMTS13 IgG concentration and subclass distribution in 101 antibody-positive samples of 81 acquired TTP patients by ELISA methods. The presence and semi-quantitative amount of anti-ADAMTS13 inhibitors were determined in 97 of 100 deficient samples, and the specific inhibitory potential of anti-ADAMTS13 autoantibodies was determined in 49 selected samples, by mixing ADAMTS13-activity assays. HLA-DR-DQ typing and haplotype prediction were performed in 70 of the above patients.</p>Results<p>We found that IgG1 and IgG4 were the predominant subclasses, present in almost all samples. While IgG1 was the dominant subclass in almost half of the samples taken during the first acute episode, IgG4 was dominant in all samples taken during or following a relapse. The inhibitory potential of the samples correlated with levels of the IgG4 subclass. Anti-ADAMTS13 antibodies of IgG4-dominant samples had higher specific inhibitory potentials than IgG1-dominant samples, independently of disease stage. Interestingly, we found that patients carrying the protective DR7-DQ2 and DR13-DQ6 haplotypes had higher anti-ADAMTS13 IgG levels.</p>Conclusion<p>Our results indicate that IgG4 becomes the dominant subtype at some point of the disease course, apparently before the first relapse, parallel to the increase in inhibitory potential of the anti-ADAMTS13 autoantibodies. Furthermore, we found an association between the genetic background and the antibody response in TTP.</p

Quantitative determination of ABCG2 expression in the erythrocyte membrane by flow cytometry.

<p>Anticoagulated blood samples of healthy volunteers were fixed in paraformaldehyde, stained with monoclonal antibodies recognizing human ABCG2, and subjected to flow cytometry (see Online Methods). Antibody staining was performed by BXP34 (Panel B), BXP21 (Panel C) and 5D3 (Panel D) mAbs specific for ABCG2, or the respective IgG control antibodies, followed by staining with PE-labeled secondary antibodies. In Panel E cells were stained with a FITC-conjugated anti-Glycophorin A mAb. Intact erythrocytes and erythrocyte ghost were gated based on the forward scatter (FSC) and side scatter (SSC) parameters (Panel A).</p

Pedigrees of two families carrying different ABCG2 premature stop mutations – co-segregation of the heterozygous mutation with reduced erythrocyte ABCG2 expression levels.

<p>Blood samples obtained from the 14 family members of the two healthy volunteer probands, carrying the premature stop mutations (see Fig. 3 -indicated with arrowheads) were analyzed for ABCG2 expression and the respective mutations. The RBC-G2 factor values, reflecting ABCG2 expression in erythrocytes, are shown in parentheses. Family members not available for blood donation are labeled by N.A.</p

ABCG2 is differentially expressed in the red blood cells of individuals carrying homozygous wild-type, heterozygous polymorphic or premature stop codon mutant ABCG2 alleles.

<p>Boxplot presentation showing the median and the 25–75^th percentiles, whiskers represent 10–90^th percentiles. ABCG2 expression is calculated based on the combined reactivity of anti-ABCG2 mAbs (RBC-G2 factor – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048423#s2" target="_blank">Methods</a>). Labels: individuals carrying wild-type ABCG2 (WT), polymorphic (Q141K, V12M) ABCG2 alleles, or a heterozygous stop mutation (STOP).</p

Expression Levels of the ABCG2 Multidrug Transporter in Human Erythrocytes Correspond to Pharmacologically Relevant Genetic Variations

<div><p>We have developed a rapid, simple and reliable, antibody-based flow cytometry assay for the quantitative determination of membrane proteins in human erythrocytes. Our method reveals significant differences between the expression levels of the wild-type ABCG2 protein and the heterozygous Q141K polymorphic variant. Moreover, we find that nonsense mutations on one allele result in a 50% reduction in the erythrocyte expression of this protein. Since ABCG2 polymorphisms are known to modify essential pharmacokinetic parameters, uric acid metabolism and cancer drug resistance, a direct determination of the erythrocyte membrane ABCG2 protein expression may provide valuable information for assessing these conditions or for devising drug treatments. Our findings suggest that erythrocyte membrane protein levels may reflect genotype-dependent tissue expression patterns. Extension of this methodology to other disease-related or pharmacologically important membrane proteins may yield new protein biomarkers for personalized diagnostics.</p> </div

Detection of ABCG2 expression level by specific monoclonal antibodies.

<p>The Figure shows the correlation of the ABCG2 expression level detected by the 5D3 monoclonal antibody in the fixed whole cells, recognizing the ABCG2 protein on an extracellular epitope, and the weighed average of BXP21 and BXP34 antibodies (BXP34/3+BXP21)/2, recognizing intracellular epitopes of ABCG2. The correlation is linear; the value of the correlation coefficient R is 0.859.</p

Gene set enrichment results for estrogen receptor (ER)-positive breast tumors

Results are presented as in Figure 2. Proliferation set. Genomic grade index. ER-associated genes (probe set n = 201). Mutant p53 gene signature (probe set n = 25). Heat maps corresponding to these plots are provided in Supplementary Figure 2. pCR, pathologic complete response; RD, residual disease.<p><b>Copyright information:</b></p><p>Taken from "Evaluation of biological pathways involved in chemotherapy response in breast cancer"</p><p>http://breast-cancer-research.com/content/10/2/R37</p><p>Breast Cancer Research : BCR 2008;10(2):R37-R37.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2397539.</p><p></p

Distribution of values computed from the unequal variance test in patients with estrogen receptor (ER)-negative and ER-positive tumors

Gene expressions were compared between ER-negative tumors that had pathologic complete response and those that had a lesser response to preoperative chemotherapy. The resulting values for all comparisons were modeled as beta-uniform mixture. The straight line indicates the contribution of the uniform component, and the curved line is the fitted beta-distribution from the observed values. Deviation above the straight line indicates values that may represent true discovery. Distribution of values in patients with ER-positive tumors.<p><b>Copyright information:</b></p><p>Taken from "Evaluation of biological pathways involved in chemotherapy response in breast cancer"</p><p>http://breast-cancer-research.com/content/10/2/R37</p><p>Breast Cancer Research : BCR 2008;10(2):R37-R37.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2397539.</p><p></p

Gene set enrichment results for estrogen receptor-negative breast tumors

Running enrichment scores (RESs) and the location of each probe set within the complete rank-ordered gene list for each gene set. The dotted line on the left indicates the position of the maximum RES, and the dotted line on the right indicates the zero position of the ranking metric score. Proliferation set (probe set n = 74). Genomic grade index (probe set n = 242). E2F3 pathway (probe set n = 173). Heat maps corresponding to these plots are provided in Supplementary Figure 1. pCR, pathologic complete response; RD, residual disease.<p><b>Copyright information:</b></p><p>Taken from "Evaluation of biological pathways involved in chemotherapy response in breast cancer"</p><p>http://breast-cancer-research.com/content/10/2/R37</p><p>Breast Cancer Research : BCR 2008;10(2):R37-R37.</p><p>Published online 29 Apr 2008</p><p>PMCID:PMC2397539.</p><p></p

Molecular genetics of the <i>ABCB6</i> gene.

<p>Abbreviations: DUH: dyschromatosis universalis hereditaria; del: deletion; dup: duplication; fs: frameshift; ins: insertion; N.D.:not determined; rs: RefSNP.</p><p>Molecular genetics of the <i>ABCB6</i> gene.</p