Data_Sheet_1.PDF

<p>Pentraxin 3 (PTX3) is an inflammatory mediator acting as a fluid-phase pattern recognition molecule and playing an essential role in innate immunity and matrix remodeling. Inflammatory mediators also contribute to skeletal homeostasis, operating at multiple levels in physiological and pathological conditions. This study was designed to investigate the role of PTX3 in physiological skeletal remodeling and bone healing. Micro-computed tomography (μCT) and bone histomorphometry of distal femur showed that PTX3 gene-targeted female and male mice (ptx3^−/−) had lower trabecular bone volume than their wild-type (ptx3^+/+) littermates (BV/TV by μCT: 3.50 ± 1.31 vs 6.09 ± 1.17 for females, p < 0.0001; BV/TV 9.06 ± 1.89 vs 10.47 ± 1.97 for males, p = 0.0435). In addition, μCT revealed lower trabecular bone volume in second lumbar vertebra of ptx3^−/− mice. PTX3 was increasingly expressed during osteoblast maturation in vitro and was able to reverse the negative effect of fibroblast growth factor 2 (FGF2) on osteoblast differentiation. This effect was specific for the N-terminal domain of PTX3 that contains the FGF2-binding site. By using the closed transversal tibial fracture model, we found that ptx3^−/− female mice formed significantly less mineralized callus during the anabolic phase following fracture injury compared to ptx3^+/+ mice (BV/TV 17.05 ± 4.59 vs 20.47 ± 3.32, p = 0.0195). Non-hematopoietic periosteal cells highly upregulated PTX3 expression during the initial phase of fracture healing, particularly CD51⁺ and αSma⁺ osteoprogenitor subsets, and callus tissue exhibited concomitant expression of PTX3 and FGF2 around the fracture site. Thus, PTX3 supports maintenance of the bone mass possibly by inhibiting FGF2 and its negative impact on bone formation. Moreover, PTX3 enables timely occurring sequence of callus mineralization after bone fracture injury. These results indicate that PTX3 plays an important role in bone homeostasis and in proper matrix mineralization during fracture repair, a reflection of the function of this molecule in tissue homeostasis and repair.</p

FACS analysis of PTX3 binding to <i>Neisseria meningitidis</i>.

<p>Live bacteria were incubated with different doses of human bPTX3 for 1h at room temperature. Results were assessed by flow cytometry. a) Histograms from one out of three experiments performed, showing the negative control (Streptavidin-PE) and two doses of bPTX3 (22 nM and 222 nM); b) Dose response of human bPTX3 (2.2–2200 nM) binding to Nm. Results are MFI ± SD from three independent experiments. Preincubation with cold human PTX3 (1.1 μM—10 min at room temperature) before addition of bPTX3 (222 nM) results in a 86.5% reduction of MFI.</p

Interaction of PTX3 with OMV.

<p>Microtiter plates were coated with OMV (1 μg/well) and binding of PTX3 (a, b) or CRP (c) is presented as mean absorbance at 450 nm (A₄₅₀) ± SD of triplicate wells. For each experimental setting data are from one out of three independent experiments. a) dose response of bPTX3; b) binding of bPTX3 to coated OMV in the presence or absence of calcium; c) Interaction of CRP with OMV. Microtiter plates were coated with 1 μg/well of OMV or Histone H1 (used as positive control for CRP binding), and incubated with different doses of CRP.</p

Interaction of PTX3 with recombinant proteins from <i>Neisseria meningitidis</i>.

<p>Binding was evaluated on plastic-immobilized proteins and expressed as mean A₄₅₀ ± SD from triplicate wells. a) Plastic wells were coated with the different recombinant proteins (1 μg/well) and incubated with human or murine PTX3, human CRP and SAP (22 pmoles of all proteins) for 1 h at 37°C before addition of the different antibodies. Results are from one out of three independent experiments. b) plastic wells were coated with the indicated Nm antigens (1 μg/well) and incubated with specific polyclonal antibodies (all diluted 1:1000) against the different Nm proteins. c) Plates were coated with recombinant PTX3 (1 μg/well) and binding was evaluated incubating with the indicated Nm antigens (1 μg/well) followed by incubation with specific antibodies. As background control, incubation with Neisserial antigens was omitted while wells were incubated with the specific antibodies.</p

Characterization of PTX3 interaction with recombinant proteins from <i>Neisseria meningitidis</i>.

<p>a) Microtiter plate assay of the binding of different doses of bPTX3 to recombinant GNA0667, GNA1030 and GNA2091 (each used at 1 μg/well). b) Binding was performed in the presence or not of calcium. Results are from four independent experiments (mean ± SD of triplicate wells). c) Microtiter plate assay of the binding of recombinant N-terminal or C-terminal PTX3 domains (22 pmoles) to GNA0667, GNA1030 and GNA2091. Data are expressed as percentage of binding compared to recombinant full length PTX3 (mean ± SD from three independent experiments). d) Affinity of the interaction between GNA0667, GNA1030 and GNA2091, and various amount of bPTX3: specific binding was measured in accordance with a standard curve of bPTX3 with non-linear fitting analysis. Data are representative of three experiments.</p

Antibody titer in mice immunized with OVA.

<p>Immune response to OVA was analyzed in WT and <i>ptx3-/-</i> mice. a) OVA was immobilized on plastic wells and presence of coated protein was confirmed by incubation with anti-OVA antibody. b) Binding to immobilized OVA or OMV as control (both at 1 μg/well) was analysed using human bPTX3. Results are reported as A₄₅₀ (mean ± SD of triplicate wells). c) WT and <i>ptx3-/-</i> mice were immunized im with OVA (100 μg/mice ± 2 μg PTX3; each group n = 5) following the same setting used for OMV. Antibody titer was evaluated by ELISA.</p

Protective effect of PTX3 in the infant rat model.

<p>Infant rats were infected ip with 4x10⁴ CFU MenB strain 2996 in association with 0.2 or 2 mg/Kg of human recombinant PTX3 (n = 16 and n = 9 respectively) or vehicle (n = 13). Blood CFU were evaluated 18 hrs after challenge. * p<0.05 (unpaired Student’s <i>t</i> test).</p

Bactericidal activity of serum from mice immunized with OMV.

<p>WT and <i>ptx3-/-</i> mice were immunized by ip or im treatment with OMV and sera were collected two weeks after the last immunization. Data are presented as mean SBA titres ± SD a) comparison of log SBA titres in WT and <i>ptx3-/-</i> mice (** p<0.01, paired Student’s t test; pooled data from ip immunized animals). b) mean SBA titres in representative experiments performed in WT and <i>ptx3-/-</i> mice immunized ip (WT n = 8 <i>ptx3-/-</i> n = 6) or im (WT n = 15, <i>ptx3-/-</i> n = 16) with 0.05 (ip) or 0.5 (im) μg OMV. c) mean SBA titres in WT and <i>ptx3-/-</i> mice immunized with 0.05 μg OMV ± 2 μg PTX3 [one out of three experiments; WT(OMV), n = 8; WT(OMV+PTX3), n = 7; <i>ptx3-/-</i>(OMV), n = 6; <i>ptx3-/-</i>(OMV+PTX3), n = 8]. *p<0.5; ** p<0.01 (Student’s <i>t</i> test).</p

Table_1_A cytokine/PTX3 prognostic index as a predictor of mortality in sepsis.docx

BackgroundEarly prognostic stratification of patients with sepsis is a difficult clinical challenge. Aim of this study was to evaluate novel molecules in association with clinical parameters as predictors of 90-days mortality in patients admitted with sepsis at Humanitas Research Hospital.MethodsPlasma samples were collected from 178 patients, diagnosed based on Sepsis-3 criteria, at admission to the Emergency Department and after 5 days of hospitalization. Levels of pentraxin 3 (PTX3), soluble IL-1 type 2 receptor (sIL-1R2), and of a panel of pro- and anti-inflammatory cytokines were measured by ELISA. Cox proportional-hazard models were used to evaluate predictors of 90-days mortality.ResultsCirculating levels of PTX3, sIL-1R2, IL-1β, IL-6, IL-8, IL-10, IL-18, IL-1ra, TNF-α increased significantly in sepsis patients on admission, with the highest levels measured in shock patients, and correlated with SOFA score (PTX3: r=0.44, pConclusionThese data suggest that a prognostic index based on selected cytokines, PTX3 and clinical parameters, and hence easily adoptable in clinical practice, performs in predicting 90-days mortality better than SOFA. An independent validation is required.</p

Image_1_A cytokine/PTX3 prognostic index as a predictor of mortality in sepsis.tiff

BackgroundEarly prognostic stratification of patients with sepsis is a difficult clinical challenge. Aim of this study was to evaluate novel molecules in association with clinical parameters as predictors of 90-days mortality in patients admitted with sepsis at Humanitas Research Hospital.MethodsPlasma samples were collected from 178 patients, diagnosed based on Sepsis-3 criteria, at admission to the Emergency Department and after 5 days of hospitalization. Levels of pentraxin 3 (PTX3), soluble IL-1 type 2 receptor (sIL-1R2), and of a panel of pro- and anti-inflammatory cytokines were measured by ELISA. Cox proportional-hazard models were used to evaluate predictors of 90-days mortality.ResultsCirculating levels of PTX3, sIL-1R2, IL-1β, IL-6, IL-8, IL-10, IL-18, IL-1ra, TNF-α increased significantly in sepsis patients on admission, with the highest levels measured in shock patients, and correlated with SOFA score (PTX3: r=0.44, pConclusionThese data suggest that a prognostic index based on selected cytokines, PTX3 and clinical parameters, and hence easily adoptable in clinical practice, performs in predicting 90-days mortality better than SOFA. An independent validation is required.</p