Signaling pathways analysis results.

<p>Signaling pathways analysis results.</p

Sotos Syndrome Is Associated with Deregulation of the MAPK/ERK-Signaling Pathway

<div><p>Sotos syndrome (SoS) is characterized by tall stature, characteristic craniofacial features and mental retardation. It is caused by haploinsufficiency of the <em>NSD1</em> gene. In this study, our objective was to identify downstream effectors of NSD1 and to map these effectors in signaling pathways associated with growth. Genome-wide expression studies were performed on dermal fibroblasts from SoS patients with a confirmed <em>NSD1</em> abnormality. To substantiate those results, phosphorylation, siRNA and transfection experiments were performed. A significant association was demonstrated with the Mitogen-Activated Protein Kinase (MAPK) pathway. Members of the fibroblast growth factor family such as <em>FGF4</em> and <em>FGF13</em> contributed strongly to the differential expression in this pathway. In addition, a diminished activity state of the MAPK/ERK pathway was demonstrated in SoS. The Ras Interacting Protein 1 (RASIP1) was identified to exhibit upregulated expression in SoS. It was shown that RASIP1 dose-dependently potentiated bFGF induced expression of the MAPK responsive SBE reporter providing further support for a link between NSD1 and the MAPK/ERK signaling pathway. Additionally, we demonstrated <em>NSD1</em> expression in the terminally differentiated hypertrophic chondrocytes of normal human epiphyseal growth plates. In short stature syndromes such as hypochondroplasia and Noonan syndrome, the activation level of the FGF-MAPK/ERK-pathway in epiphyseal growth plates is a determining factor for statural growth. In analogy, we propose that deregulation of the MAPK/ERK pathway in SoS results in altered hypertrophic differentiation of <em>NSD1</em> expressing chondrocytes and may be a determining factor in statural overgrowth and accelerated skeletal maturation in SoS.</p> </div

Protein phosphorylation.

<p>The results are shown for the phosphorylation levels for the MEK1 (A), ERK1/ERK2 (B), ERK2 (C), p38MAPK (D), cJUN (E), ATF2 (F), JNK (G), HSP27 (H) and p90RSK kinases (I). Bar heights depict the mean fluorescence intensity levels measured (MFI) and the p-values for the difference between SoS and control (after correction for total protein levels) are shown above the bars.</p

Geneplots of the probe sets influencing the MAPK pathway.

<p>Geneplots are shown for the 50 most influential probe sets from the KEGG MAPK pathway (A) and for the GO-term MAPKKK cascade (B) after stimulation with RA that contribute to the differential pathway expression in SoS and control. Probe sets are scaled to unit standard deviations and the height of the bars are the number of standard deviations above the cut-off level of 0.7. Higher bars indicate higher influence on the pathway. Probe sets with the highest influence on the pathway (i.e. FGF13 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049229#pone-0049229-g001" target="_blank">Figure 1A</a> and TNIK in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049229#pone-0049229-g001" target="_blank">Figure 1B</a>) are depicted on the left. Corresponding gene names are written below each bar.</p

bFGF induced SBE reporter activation is potentiated by RASIP1.

<p>Values are expressed as fold induction compared to control. Cells were stimulated with bFGF for 24 hours. Control was not stimulated with bFGF and no RASIP1 was co-transfected. Co-tranfection of RASIP1 did not affect basal reporter activity. bFGF (10 ng/ml) significantly stimulated SBE reporter activity (indicated with #; p<0.05). RASIP1 enhanced bFGF induced reporter dose-dependently (indicated with *; p<0.05).</p

Differentially expressed probe sets in SoS in basal situation.

<p>Differentially expressed probe sets in SoS in basal situation.</p

Differentially expressed probe sets in SoS after stimulation with RA.

<p>Differentially expressed probe sets in SoS after stimulation with RA.</p

Generation of human IgG1 and IgG3 TA99 mAb with histidine-arginine rearrangements in Fc domains at amino acid position 435.

<p>Human IgG1 was mutated to contain an arginine at position 435 (IgG1 H435R), whereas the arginine at position 435 in human IgG3 was changed to histidine (IgG3 R435H). (<b>A</b>) Specific anti-human IgG1 or (<b>B</b>) anti-human IgG3 ELISA confirmed the correct isotype of mAbs. Staining of B16F10-gp75 with (<b>C</b>) different human TA99 mAb and (<b>D</b>) mouse TA99 IgG2a confirmed binding to surface gp75 and equal binding efficiency to gp75 of all human TA99 mAb. Concentration curves of human IgG1 and IgG3 mAb (<b>E</b>) and mouse IgG2a TA99 (<b>F</b>) on B16F10-gp75. Of note, scales of human (<b>E</b>) and mouse (<b>F</b>) antibodies are different.</p

Cytotoxicity assays using murine macrophages and B16F10-gp75 tumour cells.

<p>(<b>A</b>) Remaining B16F10-gp75 cells after a 24 hour incubation with macrophages and 2 μg/ml TA99 mAbs (different isotypes). (<b>B</b>) FACS analysis of co-cultures of DiO labelled murine macrophages (FL1) and DiI labelled B16F10-gp75 (FL2) tumour cells after 24 hours of treatment with 1 μg/ml mouse IgG2a or human IgG1 or IgG3 TA99 mAb. Macrophages, which have phagocytosed B16F10-gp75 tumour cells are encircled in FACS plots. (<b>C</b>) Percentage of remaining viable tumour cells and (<b>D</b>) increase in number of macrophages, which have phagocytosed B16F10-gp75 tumour cells after treatment of co-cultures with different concentrations of mAb. Percentages of tumour cells after culture with isotype antibodies were set at 100%. Double-positive macrophages were depicted relative to the co-cultures with isotype antibodies (set to 1), as described previously [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177736#pone.0177736.ref004" target="_blank">4</a>]. Mouse MG4 or human HEPC mAb were used as isotypes controls, which were set to 100%. *P<0.05, **P<0.01, ***p<0.001, ****p<0.0001.</p

DataSheet_1_Cellular surface plasmon resonance-based detection of anti-HPA-1a antibody glycosylation in fetal and neonatal alloimmune thrombocytopenia.pdf

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) can occur due to maternal IgG antibodies targeting platelet antigens, causing life-threatening bleeding in the neonate. However, the disease manifests itself in only a fraction of pregnancies, most commonly with anti-HPA-1a antibodies. We found that in particular, the core fucosylation in the IgG-Fc tail is highly variable in anti-HPA-1a IgG, which strongly influences the binding to leukocyte IgG-Fc receptors IIIa/b (FcγRIIIa/b). Currently, gold-standard IgG-glycoanalytics rely on complicated methods (e.g., mass spectrometry (MS)) that are not suited for diagnostic purposes. Our aim was to provide a simplified method to quantify the biological activity of IgG antibodies targeting cells. We developed a cellular surface plasmon resonance imaging (cSPRi) technique based on FcγRIII-binding to IgG-opsonized cells and compared the results with MS. The strength of platelet binding to FcγR was monitored under flow using both WT FcγRIIIa (sensitive to Fc glycosylation status) and mutant FcγRIIIa-N162A (insensitive to Fc glycosylation status). The quality of the anti-HPA-1a glycosylation was monitored as the ratio of binding signals from the WT versus FcγRIIIa-N162A, using glycoengineered recombinant anti-platelet HPA-1a as a standard. The method was validated with 143 plasma samples with anti-HPA-1a antibodies analyzed by MS with known clinical outcomes and tested for validation of the method. The ratio of patient signal from the WT versus FcγRIIIa-N162A correlated with the fucosylation of the HPA-1a antibodies measured by MS (r=-0.52). Significantly, FNAIT disease severity based on Buchanan bleeding score was similarly discriminated against by MS and cSPRi. In conclusion, the use of IgG receptors, in this case, FcγRIIIa, on SPR chips can yield quantitative and qualitative information on platelet-bound anti-HPA-1a antibodies. Using opsonized cells in this manner circumvents the need for purification of specific antibodies and laborious MS analysis to obtain qualitative antibody traits such as IgG fucosylation, for which no clinical test is currently available.</p