Continuous mass spectrometry assay for proDer p 3 maturation by Der p 1.

<p>Electrospray mass spectra (positive ion mode) for 16 µM proDer p 3 activation by 0.16 µM Der p 1 in 25 mM ammonium acetate, pH 7.4, containing 1 mM DTT, obtained after 5 min (<b>A</b>) and 40 min (<b>B</b>). ProD, D and prop refer to proDer p 3, Der p 3 and propeptide, respectively. <b>C</b>: The decrease in the proDer p 3 and the increase in the Der p 3 relative concentrations were calculated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068014#s2" target="_blank">Material and Methods</a>.</p

Continuous mass spectrometry assays for proDer p 3 mutants maturation by Der p 1.

<p><b>A</b>: P2A, <b>B</b>: P5A, <b>C</b>: P8A and <b>D</b>: P-A mutants. Evolution of relative protein concentrations during the maturation of 16 µM zymogens by 0.16 µM Der p 1 in 25 mM ammonium acetate, pH 7.4, containing 1 mM DTT. The proteins were characterized as follows: entire proDer p 3 zymogen, rDer p 3, A₂ILPAS- form, S₇PQAT- form, A₅ASPQA- form, A₆SPQAT- form, QAT- form, AT- form, SAQAT- form and AQAT- form. Forms that are not described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0068014#pone-0068014-t001" target="_blank">Table 1</a> appeared during the desalting process.</p

Apparent T_m values of the different proDer p 3 zymogens.

<p>Apparent T_m values of the different proDer p 3 zymogens.</p

Heat-induced unfolding transitions monitored by fluorescence emission at 320 nm.

<p>The fractions of unfolded proDer p 3 wild type and mutants as a function of temperature.</p

Continuous enzymatic assay for proDer p 3 maturation by Der p 1.

<p><b>A</b>: proDer p 3 (black) and P2A (gray), <b>B</b>: P5A and <b>C</b>: P8A (black) and P-A (gray) mutants. Substrate hydrolysis (10 µM IEGR-MCA) versus time curves showing the activation of 12.5 nM zymogens by Der p 1 with the indicated concentrations in 50 mM polybuffer 2 at 37°C.</p

Additional file 1 of Oral supplementation with fish cartilage hydrolysate in an adult population suffering from knee pain and function discomfort: results from an innovative approach combining an exploratory clinical study and an ex vivo clinical investigation

Figure S1. Mean evolution (± SE) of the KOOS sub-scores over time: (a) KOOS Pain score over time; (b) KOOS Symptoms score over time; (c) KOOS Daily living activities score over time; (d) KOOS Sport and recreation function score over time; (e) Quality of life score over time ? FAS population. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. Figure S2. Mean evolution (± SE) of the SF-36 sub-scores over time: (a) SF-36 Physical functioning over time; (b) SF-36 Role limitations due to physical health over time; (c) SF-36 Energy/Fatigue over time; (d) SF-36 Pain over time; (e) SF-36 General health over time ? FAS population. ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05; ns, not significant. Table S1. Subjects? knee discomfort history description at the inclusion in the study ? FAS population. Table S2. Results of the repeated measures ANOVA models for KOOS global score and each subscale, and mean difference and effect size between baseline and 3 months of follow-up ? PP population ? N=24. Table S3. Results of the repeated measures ANOVA models for SF-36 global score and each subscale ? PP population ? N=24. Table S4. Results of the repeated measures ANOVA models for knee pain at rest and while walking and PGA using VAS scale, and mean difference and effect size between baseline and 3 months of follow-up ? PP population ? N=24. Table S5. Comparison of patients? treatment response between the first and the second follow-up ? PP population ? N=24. Table S6. Results of the comparison between the first and the second follow-up for compliance? FAS population. Table S7. Comparisons of subjects satisfaction between the two follow-up visits ? FAS population. Table S8. Results of the analysis of the time evolution for pain killer use and frequency of intake ? FAS population. Table S9: Listing of AE and SAE by decreasing order of frequency ? Safety population ? N=28 adverse events. Table S10. Distribution of link with FCH and action taken in response to AE ? Safety population ? N=28 adverse events. Table S11. Distribution of subjects at each visit ? Safety population ? N=32 patients. Table S12. Complete list of exclusion criteria. Figure S3. Primary human chondrocytes subjected to ex vivo procedures for validation of cell viability in human serum. Cell viability was measured with an XTT-based assay upon either FCS or human serum incubation (H-NAIVE for human naive serum and H-FCH for human serum enriched with circulating FCH metabolites) for 24 h and 48 h (A and B). Measures were performed in quadruplicates per condition/volunteer (n=10 volunteers). Values are presented as mean ± SD. The differences were considered statistically significant at p < 0.05 with ** for p < 0.01 and **** for p < 0.0001

Pseudo first order rate constants (k_obs) for the maturation of the different proDer p 3 zymogens by 11 nM Der p 1.

<p>Pseudo first order rate constants (k_obs) for the maturation of the different proDer p 3 zymogens by 11 nM Der p 1.</p

Inter-molecular activation of proDer p 3 zymogens by Der p 1.

<p><b>A</b>: proDer p 3, <b>B</b>: P5A mutant and <b>C</b>: P8A mutant. SDS-PAGE (15%) analysis: 2.5 µM proDer p 3, P5A or P8A (lanes I) was incubated at 37°C in 50 mM PBS, pH 7.4, containing 5 mM DTT and 5 mM EDTA with 30 nM Der p 1. Lanes II: 2.2 µM Der p 1 as reference. The protein molecular mass marker (St) was from Fermentas. <b>D</b>: Der p 3 activities corresponding to the activations of (green) proDer p 3, (yellow) P2A, (red) Δ1–2, (pink) P5A, (blue) Δ1–5, (gray) P8A, (cyan) Δ1–8 or (dark red) P-A by 30 nM Der p 1 for increasing times of 0 to 24 min. Proteins were diluted 2000-fold, and Der p 3 activity was measured using IEGR-MCA as the substrate (150 µM).</p

k_cat values of 12.5 nM proDer p 3 zymogens determined with the use of 150 µM IEGR-MCA in 50 M polybuffer 1 at 37°C.

<p>k_cat values of 12.5 nM proDer p 3 zymogens determined with the use of 150 µM IEGR-MCA in 50 M polybuffer 1 at 37°C.</p

DataSheet_1_Multipotent mesenchymal stromal cells as treatment for poor graft function after allogeneic hematopoietic cell transplantation: A multicenter prospective analysis.pdf

IntroductionPoor graft function (PGF) is a rare but serious complication of allogeneic hematopoietic cell transplantation (alloHCT). Due to their hematopoietic supporting properties and immune regulatory effects, multipotent mesenchymal stromal cells (MSC) could be considered a good candidate to help to restore bone marrow (BM) niches homeostasis and facilitate hematopoiesis after alloHCT.MethodsWe prospectively assessed the efficacy and safety of ex-vivo expanded BM-derived MSC from third-party donor in a series of 30 patients with prolonged severe cytopenia and PGF after alloHCT. This multicenter trial was registered at www.clinicaltrials.gov (#NTC00603330).ResultsWithin 90 days post-MSC infusion, 53% (95% CI, 35 – 71%) of patients improved at least one cytopenia (overall response, OR) and 37% (95% CI, 19 - 54%) achieved a complete hematological response (CR: absolute neutrophil count, ANC >0.5 x 109/L, Hb > 80g/L and platelet count > 20 x 109/L with transfusion independence). Corresponding response rates increased to 67% (95% CI, 50 - 84%) OR and 53% (95% CI, 35 - 71%) CR within 180 days after MSC infusion. A significant decrease in red blood cells and platelets transfusion requirement was observed after MSC (median of 30-days transfusion requirement of 0.5 and 0 from d90-120 post-MSC versus 5 and 6.5 before MSC, respectively, p ≤0.001). An increase in ANC was also noted by day +90 and +180, with 3/5 patients with severe neutropenia having recovered an ANC > 1 x 109/L within the 90-120 days after MSC infusion. Overall survival at 1 year post-MSC was 70% (95% CI, 55.4 – 88.5), with all but one of the patients who achieved CR being alive. A single infusion of third-party MSC appeared to be safe, with the exception of one deep vein thrombotic event possibly related to the intervention.DiscussionIn conclusion, a single i.v. infusion of BM-derived MSC from third party donor seemed to improve hematological function after alloHCT, although spontaneous amelioration cannot be excluded. Comparative studies are warranted to confirm these encouraging results.</p