Experimental workflow.

<p>In the first stage, a label free approach was used to determine candidate proteins that are differentially expressed in PBMCs between responder (R) and non-responder (NR) RA patients before initiation of etanercept in the population 1. This approach was used to quantify the relative abundance of S100 proteins. To independently confirm these results, label free approach by mass spectrometry and absolute quantification by ELISA were used to quantify S100 proteins in sera samples from R and NR RA patient before etanercept initiation in the population 2.</p

Thresholds of serum S100A9 concentration able to identify non-responders patients.

<p>Absolute quantification by ELISA of serum S100A9 protein in responders versus non-responders prior to MTX/ETA initiation. The calculated thresholds resulting from ROC analyses are also given with the corresponding sensitivities (Sen) and specificities (Spe). Circles and squares represent individual points for R (n = 12) and NR (n = 10) patients respectively.</p

Baseline expression of S100A8 and S100A9 in PBMCs from RA patients according to the response/non-response status to the ETA/MTX combination in the first population.

<p>Relative quantification by mass spectrometry of S100A9 protein (A) and S100A8 protein (B) at baseline in responders (n = 3) versus non-responders (n = 3) to MTX/etanercept (significant difference is noted by asterisk, p<0.05; Mann-Whitney non-parametric test). Both S100A9 and S100A8 proteins accumulated in R patient (p-value  = 0.0022). The upper and lower bounds of each box indicate the 25^th and 75^th percentile respectively and heavy lines within the box represent the median. Whiskers are drawn to the min and max values.</p

Demographic, clinical and biological data of RA patients at baseline.

<p>Demographic, clinical and biological data of RA patients at baseline.</p

Relationship between baseline levels of several potential markers measured prior to MTX/ETA initiation and the degree of response over the 6 months follow-up in the second population.

<p>Relationship between baseline levels of several potential markers measured prior to MTX/ETA initiation and the degree of response over the 6 months follow-up in the second population.</p

Over-expression of the S100A9 protein in baseline serum from responder patients to the MTX/ETA combination according to 2 different approaches in the second population.

<p>Relative quantification of serum S100A9 protein at baseline in R (n = 12) versus NR (n = 10) by mass spectrometry (A). This result showed an over-expression of S100A9 protein in R patient (p-value  = 0.0022). Serum ELISA absolute quantification of S100A9 (B), S100A8 (C) and calprotectin (D) at baseline in responders versus non-responders. No significant difference in the expression of S100A8 protein and calprotectin was observed between R and NR (p-value  = 0.75 and 0.32, respectively). Only S100A9 showed an over-expression in R patients (p-value  = 0.023).</p

Opthalmological evaluation of control and mutant mice.

<p>Similar gross morphology of the anterior segment in control (<b>A, A’</b>) and <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutant littermates at P90 (<b>B, B’</b>). In some cases pupillary ectopia was observed in the mutants (<b>C, C’</b>). Fundus photographs of control (<b>D, F</b>) and mutant eyes (<b>E, G</b>) at P60 (<b>D, E</b>) and P180 (<b>F, G</b>) respectively show chorioretinal atrophy and peripapillary staphyloma. (<b>H</b>) Comparisons of intraocular pressure between control and mutant littermates at the indicated postnatal ages. Two-way ANOVA post hoc Tukey test was used, ***<i>P</i><0.001, ns: not statistically significant, values are mean ± SEM of 10 animals per age and genotype.</p

MRI analysis of post-natal eye growth in control and <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants over the first year of life.

<p>High resolution sagittal slices through the optical axis of the right eye of each mouse were acquired (<b>A-H</b>). A variety of optical parameters were extracted from the MRI images collected from groups of control and mutant mice at the ages indicated (<b>I-N</b>). Growth rate of axial length and vitreous chamber depth (<b>O</b>, <b>P</b>). A two-way ANOVA post hoc Tukey test was used, <i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, ns: not statistically significant, values are mean ± SEM of 4 animals per age and genotype. Scale bars: 1500 μm in A-H.</p

Lrp2-deficient eyes are abnormally enlarged.

<p>Sagittal cryosections through the developing eye (<b>A-D</b>). Lrp2 is expressed in the developing neuroretina (nr) at E12.5 (<b>A</b>). From E15.5 onward the signal is restricted in the lens (L) facing inner layer of the ciliary body (cb) epithelium, a low expression is also seen in the outer layer of the CB and in the retinal pigmented epithelium (rpe) (<b>B</b>). Loss of Lrp2 signal in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants at E12.5 and E15.5 (<b>C, D</b>). Sagittal cryosections of control and mutant retinas at P60 showing retinal thinning and the presence of a posterior staphyloma in the mutant (<b>E</b>, <b>F</b>). Reconstruction of the mouse face using MRI at P60 (<b>G</b>, <b>H</b>). The <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants display bilateral eye enlargement, through the anterior-posterior axis and the equatorial diameter (double headed arrows in <b>G, H</b>). Horizontal MRI images showed that the retrobulbar space, between the orbit and the eyeball, (double-headed arrows in <b>I, J</b>) is decreased in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutants. The corpus callosum (arrow in <b>I</b>) is not formed in the mutants (asterisk in J). (vz) ventricular zone. Scale bars: 25 μm in A-D; 600 μm in E, F.</p

Scleral modifications in <i>Lrp2</i>^{<i>FoxG1</i>.<i>cre-KO</i>} mutant eyes.

<p>Nissl staining of retinal sections in control (<b>A</b>) and mutant (<b>B</b>) eyes shows reduced scleral thickness at P90. The choroid and occasionally the RPE appear thicker at the posterior pole of the mutant eyes. Transmission electron micrographs of the posterior sclera wall (<b>C, D</b>) shows that the collagen fibrils form well-organized lamellae in the control sclera (<b>C</b>); in the mutant fibril-poor areas and impaired packing are evident (<b>D</b>). Contrary to the control sclera the collagen fibril density is lower in all layers of the mutant sclera (<b>E</b>). Transmission electron micrographs showed fibril collagen organization within a lamella of the posterior sclera (<b>F, H</b>) and in localized areas (<b>G, I</b>) of control (<b>F, G</b>) and mutant (<b>H, I</b>). Fibrils were morphologically abnormal with irregular contours and heterogeneous diameters in the mutants. Measurements of cross-sectional diameters of fibrils from the inner, middle and outer posterior sclera in control (<b>J</b>) and mutant eyes (<b>K</b>). The mean fibril diameter distribution is modified in the mutants in all three layers; the frequency of very small as well large diameter fibrils is increased. (ch) choroid, (fb) fibroblast, (Lg) longitudinal and (Tr) transversal orientation of the cross-sectioned fibrils. Two-tailed unpaired <i>t</i> test was used. ***<i>P</i><0.001, Values are mean ± SEM of 3 animals per genotype; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129518#sec009" target="_blank">methods</a>. Scale bars: 50 μm in A, B; 3.5 μm in C, D; 1.2 μm in F, H; 300 nm in G, I.</p