How do systemic sclerosis manifestations influence patients’ lives? Results from a survey on patients and caregivers

To investigate the patient- and caregiver-reported impact of systemic sclerosis (SSc) manifestations (hand/feet/joint involvement and pulmonary complications) on the diagnostic and therapeutic journey, working productivity, and social life. Two questionnaires (one for the patients, n = 260 and one for the caregivers, n = 47) were designed in collaboration with the patients’ association Gruppo Italiano per la Lotta alla Sclerodermia (GILS). Validated questionnaires were combined with specific questions relevant to the Italian scenario. Pulmonary fibrosis and hand/feet/joint involvement have a major impact on patient’s working status: (85.3% of patients with pulmonary fibrosis and 72.6% with hand/feet/joint involvement report loss of job/job change due to SSc. Productivity was affected as well: 60.6% of the patients (75% of those with fibrosis) reported that working productivity in the previous 4 weeks was restricted by physical limitations. The disease has a significant impact on patients’ life, limiting the ability to conduct common activities, especially those related to movement, such as object manipulation (61.1%), doing small manual jobs (44.0%), writing (38.9%), and an increased impact in case of pulmonary fibrosis and hands/feet/joints involvement. Half of the patients also present some difficulties in eating-related activities a Patients also experience poorer social life, personal relationships, and sexual life. Caregivers are also deeply influenced by the manifestations of SSc. Pulmonary fibrosis and hand/feet/joint involvement represent an additional challenge. Pulmonary fibrosis and hand/feet/joint involvement are extremely burdensome complications for both SSc patients and caregivers, decreasing work productivity, limiting relationship and social life, and impacting psychological status and everyday activities.</p

<i>In silico</i> representation of the subcloned <i>ALK</i> regulatory region.

<p>A) In the upper section of the figure, a portion of the <i>ALK</i> gene, as shown by the Vista Browser, is reported: the <i>ALK</i> coding region (dark grey), the predicted 5′ untranslated region (light grey), part of the 5′ regulatory region (black) and their levels of phylogenetic conservation (dark bars) are represented. In the lower part, the sequence corresponds to the part of the gene subcloned into the pGL3basic vector, containing 672 bp upstream the putative transcriptional start site (indicated with +1) and 384 bp of the 5′ untranslated region (shown in Italic). Moreover, the five ATTA sites are boxed and progressively named from 1 to 5. Arrows represent the start point of the deletion constructs. B) Activity of the pGL3basic-<i>ALK</i> promoter construct (grey bars) in HeLa and IMR-32 cell lines, expressed as fold induction with respect to the empty pGL3basic vector (black bars). Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. C) <i>ALK</i> promoter induction in HeLa and IMR-32 cells co-transfected with the PHOX2B expression plasmid and expressed as fold induction of the Luciferase activity with respect to cells transfected with the empty vector (pcDNA3.1, arbitrary value = 1) (*: <i>P</i><0.05).</p

<i>In vitro</i> interaction of PHOX2B with the <i>ALK</i> promoter.

<p>A) EMSAs were performed using probes containing one of the ATTA sites of the region under analysis (ATTA 1, ATTA 2, ATTA 3 and the complex ATTA 4/5). Each labeled probe was incubated in the absence of nuclear extracts (lane 1), with IMR-32 nuclear extracts (lanes 2–4) or the <i>in vitro</i> expressed PHOX2B-Myc fusion protein (lanes 5–7). As negative control the oligonucleotides were also incubated with the <i>in vitro</i> reaction performed using the empty vector pcDNA3.1 M/H (lane 8). The competition experiments were performed in the presence of a molar excess of the unlabeled oligonucleotides (lanes 3 and 6). The anti-PHOX2B or the anti-c-Myc antibodies were added to the samples run in lanes 4 and 7, respectively. On the left, the arrows indicate the specific retarded bands detected; on the right, one or two asterisks indicate the supershifted complexes containing PHOX2B obtained by incubation of IMR-32 nuclear extracts with the anti-PHOX2B antibody (*) or the <i>in vitro</i> expressed protein with the anti-cMyc antibody (**), respectively. The free probes are shown at the bottom of the gels. B) ChIP assay. Chromatin extracted from IMR-32 cells was immunoprecipitated using the antibody against PHOX2B; pre-immune chicken IgY and the anti-acetylated histone H4 antibodies were used as negative and positive controls, respectively. The input represent 0,5% of the total chromatin extract. The precipitated DNA has undergone PCR amplification by using primers bordering the ATTA 3 and the ATTA 4/5 boxes in the <i>ALK</i> promoter.</p

siRNA-mediated silencing of <i>ALK, PHOX2B</i> and <i>PHOX2A</i> in NB cells.

<p>Effects on the transcription level of the <i>ALK</i> (left side graphs), <i>PHOX2B</i> (middle graphs) and <i>PHOX2A</i> (right side graphs) genes after knock-down of the same genes in SHSY-5Y (A), IMR-32 (B) and HTLA-230 (C) cells. Gene-specific knock-down, evaluated 48 hours post-transfection by real-time RT-qPCR analysis, is very effective but also <i>PHOX2</i>-directed siRNAs are able to downregulate <i>ALK</i> at a similar extent (**: <i>P</i><0.01; ***: <i>P</i><0.001). Values are the mean ± s.d. of N = 3 independent experiments performed in duplicate. (D) Gene silencing was confirmed at 72 hours post-transfection by Western blot.</p

Effects of mutagenesis of ATTA 3 and ATTA 4/5 on the PHOX2B-mediated <i>ALK</i> trans-activation.

<p>Left side: schematic representation of the three constructs carrying all the ATTA boxes functional (wt, all four black circles), the ATTA 3 disrupted (ATTA 3 mut, one white circle) or both the ATTA 4 and 5 disrupted (ATTA 4/5 mut, two white circles). Right side: induction of the <i>ALK</i> promoter containing the mutant ATTA 3 and ATTA 4/5 in HeLa cells co-transfected with the PHOX2B expression plasmid are expressed as percentage of the Luciferase activity obtained by cells co-transfected with PHOX2B and the <i>ALK</i> promoter (wt) vectors (wt, arbitrary value = 100). Values are the mean ± s.d. of N = 3 independent experiments (*: <i>P</i><0.05).</p

Effect of ATTA 4/5 disruption in competition of PHOX2B binding.

<p>IMR32 nuclear extracts were incubated with the ATTA4/5 probe (lane 2) and competition obtained by adding an excess of: a wild type (wt) probe (lane 3), a probe mutated in both ATTA 4 and ATTA 5 sites (lane 4) or in each of them (lanes 5–6). Incubation without nuclear extracts was regarded as negative control (lane 1).</p

Oligonucleotides used in EMSA^*.

<p>*Original or mutated ATTA sequences are underlined in each oligonucleotide.</p

Gene expression analysis in NB and HeLa cells and correlation analysis.

<p>(A) Relative gene expression analysis of the <i>ALK</i>, <i>PHOX2B</i> and <i>PHOX2A</i> genes, carried out in a panel of NB and in HeLa cell lines by real-time RT-qPCR using a pool of normal tissue RNAs as reference sample (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013108#s4" target="_blank">Methods</a>), shows over-expression of the three genes in all but two NB cell lines tested (GI-ME-N and ACN). (B) X-Y Plots showing a significant correlation between the expression level of <i>PHOX2B</i> and <i>PHOX2A vs.ALK</i> (left) and <i>PHOX2Avs. PHOX2B</i> (right) genes in the analyzed cell lines. Pearson's correlation coefficient indicates a very significant correlation of the three transcription levels <i>vs.</i> each other. Values are the mean ± s.d. of N = 3 independent RT-qPCR analyses performed in triplicate.</p

Analysis of Mda-9/syntenin protein expression in uveal melanoma cell lines.

<p><b>A:</b> Immunostaining of fixed and permeabilized cell lines (left) and primary cultures (right). Original magnification 400×. The insets show the negative control performed by the use of non-immune rabbit Ig.</p

Silencing of <i>SDCBP</i> by siRNA inhibits uveal melanoma cell migration.

<p><b>A:</b> Western blot analysis of MEL 270 and 92.1 cell lines upon 72 hrs treatment with scrambled siRNA (C), and <i>SDCBP</i> targeting siRNA (Synt). <b>B:</b> wound-healing assay on MEL 270 and 92.1 cell lines treated with scrambled siRNA (C) or with <i>SDCBP</i> targeting siRNA (Synt). Mean of migration distance of MEL 270 cells (<b>C</b>) and 92.1 (<b>D</b>) treated with scrambled siRNA (black bars) or with <i>SDCBP</i> targeting siRNA (grey bars), P values are indicated.</p