Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 1. Pulmonary and plasma osteopontin concentrations are elevated during pneumococcal pneumonia. Osteopontin concentrations in (A) lung and (B) plasma before and 6, 24, and 48 h after infection with 104 colony-forming units of Streptococcus pneumoniae. Data are expressed as mean 6 standard error of the mean (SEM); n 5 8 mice per group. Asterisk, P , .05; double asterisk, P , .01; triple asterisk, P , .001, compared with t 5 0. Osteopontin concentrations in culture supernatants after incubation of (C) MH-S cells and (D) primary alveolar macrophages with medium or growth- arrested S. pneumoniae (multiplicity of infection, 1:6 and 1:60 for MH-S cells; 1:20 and 1:200 for primary alveolar macrophages) for 4 h (MH-S cells) or 20 h (primary alveolar macrophages). Data are expressed as mean 6 SEM; n 5 3 per group. Asterisk, P ,.05, compared with medium. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Clinical Proteomics, Infection, Incubation

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 2. Prolonged survival and reduced bacterial growth in osteopontin knockout (KO) mice. A, Percentage survival of wild-type (WT) mice (filled symbols) and osteopontin KO mice (open symbols) after intranasal infection with 104 colony-forming units (CFU) of Streptococcus pneumoniae (n 5 14 mice per group). P value indicates the difference between groups. WT (gray) and osteopontin KO (white) mice were infected with 104 CFU of S. pneumoniae, and bacterial loads were determined 6, 24, and 48 h after infection in (B) lung, (C) blood, and (D) spleen. Data are expressed as box-and- whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group; asterisk, P ,.05; double asterisk, P ,.01; triple asterisk, P ,.001, compared with WT mice. Note to panels C and D: none of the mice in either group displayed positive blood or spleen culture results 6 h after infection; at 24 h, S. pneumoniae could be cultured from samples of the blood of only 3 of 8 osteopontin KO mice, compared with 7 of 8 WT mice and from the spleen tissue of only 1 of 7 osteopontin KO mice, compared with 7 of 8 WT mice (P , .05 and P , .01, respectively). OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Infection, Whisker Assay, Cell Culture

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 3. Decreased lung histopathology in osteopontin knockout (KO) mice. Representative lung histology of wild-type (WT) (A, D, G) and osteopontin KO (B, E, H ) mice at 6 h (A–C ), 24 h (D–F ), and 48 h (G–I) after intranasal infection with 104 CFU of Streptococcus pneumoniae. The lung sections are representative for 8 mice per group per time point. Hematoxilin and eosin staining, original magnification, 310. Inflammation scores are expressed as mean 6 standard error of the mean (WT mice, black bars; osteopontin KO mice, white bars; n 5 8 mice per group). Double asterisk, P ,.01, compared with WT mice. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Histopathology, Knock-Out, Infection, Staining

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 4. Osteopontin stabilizes Streptococcus pneumoniae viability in vitro. A, S. pneumoniae in saline (106 colony-forming units [CFU]/mL) was incubated with increasing doses (0.8–800 ng/mL) of recombinant osteopontin (black symbols) or saline (white symbols), and the viability of S. pneumoniae was determined over 6 h at 37C. B, S. pneumoniae in saline (106 CFU/mL) was incubated with 800 ng/mL recombinant osteopontin (filled squares), 800 ng/mL boiled recombinant osteopontin (open squares), 800 ng/mL bovine serum albumin (triangles), or saline (circles), and the viability of S. pneumoniae was determined over 6 h at 37C. Dashed lines depict detection limits. C, Osteopontin binds to S. pneumoniae. Enzyme-linked immunosorbent assay plates were coated or not coated with 1 3 108 CFU/mL S. pneumoniae type 3 (ATCC 6303) or serotype 2 (D39); coating with anti-osteopontin IgG was used as positive control. Binding was assessed using biotin-labeled recombinant mouse osteopontin. Data are means 6 standard error (n 5 4–6). Double asterisk, P , .01 vs buffer. OPN, osteopontin.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: In Vitro, Saline, Incubation, Recombinant, Enzyme-linked Immunosorbent Assay, Positive Control, Binding Assay, Labeling

Journal: The Journal of infectious diseases

Article Title: Osteopontin impairs host defense during pneumococcal pneumonia.

doi: 10.1093/infdis/jir185

Figure Lengend Snippet: Figure 5. Similar bacterial growth during pneumococcal sepsis. Bacterial loads in (A) blood, (B) lung, (C) liver, and (D) spleen from wild-type (WT; gray) and osteopontin knockout (OPN KO; white) mice at 24 and 48 h after intravenous injection with 105 colony-forming units (CFU) of Streptococcus pneumoniae. Data are expressed as box-and-whisker diagrams depicting the smallest observation, lower quartile, median, upper quartile, and largest observation; n 5 8 mice per group. Dashed line depicts detection limit.

Article Snippet: Effect of Osteopontin on S. pneumoniae Viability, Phagocytosis, and Phagolysosomal Fusion S. pneumoniae or Staphylococcus (S.) aureus (Newman strain) (1 3 106 bacteria/mL) was incubated in sterile normal saline in the presence of 0.8–800 ng/mL recombinant mouse osteopontin (rOPN;,1.0 endotoxin units per 1 lg as determined by the LAL assay; R&D Systems), 800 ng/mL boiled rOPN (30 min at 100 C), 800 ng/mL bovine serum albumin (BSA), or normal saline only at 37 C for 6 h. At indicated time points the number of bacteria was determined.

Techniques: Knock-Out, Injection, Whisker Assay

Journal: International immunopharmacology

Article Title: Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

doi: 10.1016/j.intimp.2024.111584

Figure Lengend Snippet: Fig. 5. Increased production of osteopontin (OPN) and prostaglandin E2 (PGE2) in EL4luc2 cells and associated tumor model. (a and b), Expression of angiogenic factors and cytokines produced by EL4luc2 cells was screened using proteome profiler kits for mouse angiogenesis factors (a) and cytokines/chemokines (b). (c-e), levels of OPN and PGE2, and IL-17 were measured in the control culture medium (white bars: c, d, e) EL4 cells-derived cell culture medium (black bars: c, d, e) and sera from tumor free (control) mice (white bars: c, d, e), and EL4luc2 tumor-bearing mice (black bars: c, d, e), respectively. Statistical analysis was performed using the Mann-Whitney U test (*P ≤0.05).

Article Snippet: The levels of PGE2, OPN and IL-17 in the cell culture supernatants and serum samples were quantified using commercially available ELISA kits (Mouse Prostaglandin E2 (PGE2) ELISA Kit, MyBioSource, Inc. San Diego, CA, USA, Mouse/Rat Osteopontin (OPN) Quantikine ELISA Kit, R&D Systems, Abingdon, UK, and ELISA Flex: Mouse IL-17A (HRP), Mabtech AB, Nacka Strand, Sweden), in accordance with the manufacturers’ instructions.

Techniques: Expressing, Produced, Control, Derivative Assay, Cell Culture, MANN-WHITNEY

Journal: International immunopharmacology

Article Title: Tumor cell derived osteopontin and prostaglandin E2 synergistically promote the expansion of myeloid derived suppressor cells during the tumor immune escape phase.

doi: 10.1016/j.intimp.2024.111584

Figure Lengend Snippet: Fig. 6. Osteopontin (OPN) and prostaglandin E2 (PGE2) synergistically induce myelopoiesis with immunosuppressive activity. (a-d) Freshly flushed bone marrow (BM) cells were cultured in a 24-well cell culture plate for 72 h in the presence of various combinations of PGE2, OPN, and IL-17. Subsequently, cell viability (a and d) and immunophenotypic analysis of myeloid cells (b and e) flow cytometry analyses were conducted on the cultured BM cells using trypan blue exclusion test and flow cytometry, respectively. Representative photographs of myeloid cell populations are displayed above respective bars (b and e). c) Conditioned medial levels of arginase were quantified using colorimetric assays after treating BM cells with PGE2 and OPN. f) Effect of pioglitazone on the inhibition of proliferation of EL4 cells. Statistical analysis was performed using ANOVA test (*P ≤0.05, **P < 0.01, ***P < 0.01).

Article Snippet: The levels of PGE2, OPN and IL-17 in the cell culture supernatants and serum samples were quantified using commercially available ELISA kits (Mouse Prostaglandin E2 (PGE2) ELISA Kit, MyBioSource, Inc. San Diego, CA, USA, Mouse/Rat Osteopontin (OPN) Quantikine ELISA Kit, R&D Systems, Abingdon, UK, and ELISA Flex: Mouse IL-17A (HRP), Mabtech AB, Nacka Strand, Sweden), in accordance with the manufacturers’ instructions.

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Inhibition

Journal: Smart Materials in Medicine

Article Title: Eggshell-derived amorphous calcium phosphate: Synthesis, characterization and bio-functions as bone graft materials in novel 3D osteoblastic spheroids model

doi: 10.1016/j.smaim.2023.04.001

Figure Lengend Snippet: Fig. 10. Confocal microscopic inspection of OPN and collagen I expression of MC-3T3-E1 cells in 3D spheroids with ACP particles embedded and 2D culture system, respectively. (A) Left panel: OPN and collagen I expression in different culture media in 3D reconstructed images; Right panel: maximal fluorescent projection of OPN and collagen I in 3D spheroids as shown in left penal; (B) OPN and collagen I expression in different culture media in a 2D culture system. Scale bar ¼ 100 μm.

Article Snippet: In brief, cells and spheroids were cultured for 7 days and then fixed with 4 wt % PFA for 20 min. After washing in PBS thrice, samples were treated with 1% (v/v) TritionX-100 (X100, Sigma-Aldrich, USA) for 20 min, followed by blocking in 1 wt % BSA/PBS solution for 1 h. Then, the samples were incubated in goat-anti-mouse OPN primary antibody (Ab) (AF808, R&D systems, USA) and rabbit-anti-mouse collagen I Ab (ab21286, Abcam, USA) at 4 C overnight.

Techniques: Expressing

Journal: Kidney international

Article Title: Vitamin D receptor agonists increase klotho and osteopontin while decreasing aortic calcification in mice with chronic kidney disease fed a high phosphate diet

doi: 10.1038/ki.2012.322

Figure Lengend Snippet: (A) Arterial medial osteopontin (OPN) levels were increased by VDRA treatment. (i) OPN expression was low but detectable in the aortic media of CKD mice fed a high phosphate diet (CKD+HP). VDRA treatment increased smooth muscle cell expression of OPN in the aortic media in (ii) CKD+HP+C30 animal; and (iii) CKD+HP+P300 animal. Arrows point to aortic medial cells expressing OPN. Scale bars are 30 μm and objective is 40X. (B) Quantitation of OPN immunostaining showed no OPN expression in aortas from non-CKD controls, weak staining in CKD high phosphate-fed mice, and increased levels in VDRA-treated CKD mice (mean ± s.e.m., n =3 for all except CKD+HP+paricalcitol, n =5 where P100 and P300 samples were grouped). (C) Treatment of cultured VSMCs with 50 nM paricalcitol increased OPN levels in the media (significantly higher levels by ELISA at 48hr compared to time zero, ** P <0.001). Klotho (2 ng/mL) with/without FGF23 (2 ng/mL) did not upregulate OPN secretion. Three wells were sampled per time-point per treatment group, data are mean ± s.e.m. EtOH = ethanol control, KL = klotho, P50 = paricalcitol 50 nM.

Article Snippet: The following assays were used: QuantiChromTM Urea Assay Kit (BioAssay Systems, Hayward, CA) for BUN; o -cresolphthalein complexone kit from Teco Diagnostics (Anaheim, CA) for calcium; standard bioanalyzer at Phoenix Central Laboratory (Everett, WA) for phosphate; mouse FGF23 C-terminus ELISA kit (Immutopics, San Clemente, CA); DuoSet mouse osteopontin ELISA kit (R&D Systems, Minneapolis, MN); mouse intact PTH enzyme-linked immunosorbent assay kits (ALPCO Diagnostics, Salem, NH and Immutopics).

Techniques: Expressing, Quantitation Assay, Immunostaining, Staining, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: Journal of structural biology

Article Title: Nanostructure of mouse otoconia.

doi: 10.1016/j.jsb.2020.107489

Figure Lengend Snippet: Figure 6. Immunogold labeling of OPN in otoconia. (a, c) In wild-type mice, otoconial voids shown by TEM and SEM, respectively, after immunolabeling for OPN (aqueous procedures dissolve the calcitic otoconia). Inset: Intact wild-type mouse otoconia (without aqueous exposure) shown by SEM. By TEM (b) and SEM (d), immunogold labeling for OPN shows gold particles (arrows) at the surface of otoconial voids. (e, g) In OPN-deficient mice, otoconial voids shown by TEM and SEM, respectively, after immunolabeling for OPN. Inset: Intact otoconia from OPN-deficient mice (without aqueous exposure) shown by SEM. By TEM (f) and SEM (h), as expected in this negative control, immunogold labeling for OPN was absent.

Article Snippet: Otoconia-containing thin sections and block faces were incubated with anti-mouse OPN antibody (R&D Systems Oakville, Canada) for 1 h followed by washing and incubation with protein A-colloidal gold complex (14-nm gold particles, from G. Posthuma, University of Utrecht) for 1 h to detect the immunolabeling reactions.

Techniques: Labeling, Immunolabeling, Negative Control