pgrn sheep mab  (R&D Systems)

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 1. PGRN levels are elevated in clinical and experimental influenza/S. pneumoniae coinfection. (A) ELISA detection of PGRN levels in the sera collected from pediatric patients with IAV infection (n = 47), S. pneumoniae infection (n = 53), and influenza/S. pneumoniae coinfection (n = 38). Healthy subjects served as controls (n = 30). (B) Groups of C57BL/6 WT mice were infected intranasally with A/Puerto Rico/8/1934 H1N1 (PR8; 400 PFU) alone, S. pneumoniae (19F; 1 3 108 CFU) alone, or PR8+19F coinfection. Secondary 19F infection was performed 3 d after influenza infection. Mice inoculated with PBS served as controls. The sera, lung homogenates, and sections of mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (C and D) PGRN levels in the lung homogenates and sera were measured by ELISA kits (n = 4–5). (E) The expression and distribution of PGRN in lung tissues were detected by immunohistochemical staining with mouse monoclonal PGRN Ab. Each dot represents one patient or mouse. PGRN quantification in (A), (C), and (D); these experiments were performed once. Experiments in (E) were performed twice with representative data presented. **p , 0.01, ***p , 0.001 by Mann–Whitney U test, compared between groups.

Article Snippet: For immunohistochemical analysis, lung sections were incubated with mouse monoclonal PGRN Ab (R&D Systems) or cleaved caspase-3 [Asp175] Ab (5A1E; Cell Signaling Technology) overnight at 4 ̊C.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Expressing, Immunohistochemical staining, Staining, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 3. PGRN deficiency impairs bacterial clearance of mice in influenza/S. pneumoniae coinfection. Lungs and nasal washes of mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (A and B) Viral loads in lungs were determined by PR8 matrix protein gene copy detected with quantitative PCR (n = 3). (C–F) Lung homogenates and nasal washes were inoculated on blood agar plates, and bacterial loads were de- termined by CFU analysis (n = 4–5). The label CFU/lung or CFU/nasal wash indicates total number of CFU in each lung homogenate or nasal wash sample. Data are representative of at least three independent experiments. *p , 0.05 based on Mann–Whitney U test. ns, not significant.

Article Snippet: For immunohistochemical analysis, lung sections were incubated with mouse monoclonal PGRN Ab (R&D Systems) or cleaved caspase-3 [Asp175] Ab (5A1E; Cell Signaling Technology) overnight at 4 ̊C.

Techniques: Infection, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 2. PGRN deficiency increases mortality of mice in influenza/S. pneumoniae coinfection. WT and PGRN2/2 mice were infected with PR8 alone, 19F alone, or PR8+19F coinfection. Mice were monitored for 2 wk after primary PR8 or 19F infection. (A) Kaplan–Meier survival curves were assessed by log-rank (Mantel–Cox) test for significance (n = 8 for single infection groups and n = 18 for coinfection groups). *p , 0.05. (B–D) Body weight loss of mice was recorded daily and analyzed by two-way ANOVA test for significance. ***p , 0.001. (E and F) Coinfected WT and PGRN2/2 mice were i.p. injected with 20 mg rPGRN at 3 and 5 d after influenza (0 and 2 d after 19F coinfection); the survival (E) and weights (F) were monitored for 2 wk after influenza. Statistical analyses of survival and weight loss were performed in the same way (n = 10 per group, compared between groups). *p , 0.05. Survival and weight loss curves are shown by combining two independent experiments.

Article Snippet: For immunohistochemical analysis, lung sections were incubated with mouse monoclonal PGRN Ab (R&D Systems) or cleaved caspase-3 [Asp175] Ab (5A1E; Cell Signaling Technology) overnight at 4 ̊C.

Techniques: Infection, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 4. PGRN deficiency aggravates lung inflammatory damage in influenza/S. pneumoniae coinfection. Lungs and BALF samples from mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (A) Lung sections were stained with H&E for histopathology assay. (B and C) The LDH activity and total protein levels were tested by LDH and BCA protein assay kits, respectively (n = 4–5). (D) The wet/dry ratios of lungs were determined to evaluate lung edema of mice in different infection scenarios (n = 4–5). (E) Levels of proinflammatory cytokines IL-6, TNF-a, and IL-1b in lungs were detected by ELISA kits (n = 4–5). Data are representative of at least three independent experiments. *p , 0.05, **p , 0.01 based on unpaired Student t test. ns, not significant.

Article Snippet: For immunohistochemical analysis, lung sections were incubated with mouse monoclonal PGRN Ab (R&D Systems) or cleaved caspase-3 [Asp175] Ab (5A1E; Cell Signaling Technology) overnight at 4 ̊C.

Techniques: Infection, Staining, Histopathology, Activity Assay, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 5. PGRN deficiency increases immune cell infiltration in lungs during influenza/S. pneumoniae coinfection. Cells from BALF were counted, purified, and identified by flow cytometry. Neutrophils were determined by gating on CD11b+ Ly6G+ cells, whereas macrophages were determined by gating on CD11b+ F4/80+ cells. (A) Percentages of neutrophils and macrophages. (B–D) The absolute numbers of total cells, neutrophils, and macrophages (n = 5–7). *p , 0.05, **p , 0.01, ***p , 0.001 based on Mann–Whitney U test. (E) Levels of chemokines CXCL1, CCL2, and CXCL10 in lungs were measured by ELISA kits (n = 5). Data are representative of at least three independent experiments. *p , 0.05, based on unpaired Student t test. ns, not significant.

Article Snippet: For immunohistochemical analysis, lung sections were incubated with mouse monoclonal PGRN Ab (R&D Systems) or cleaved caspase-3 [Asp175] Ab (5A1E; Cell Signaling Technology) overnight at 4 ̊C.

Techniques: Cytometry, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Progranulin Decreases Susceptibility to Streptococcus pneumoniae in Influenza and Protects against Lethal Coinfection.

doi: 10.4049/jimmunol.1900248

Figure Lengend Snippet: FIGURE 6. PGRN deficiency increases ERS-mediated apoptosis in mice during influenza and coinfection. Lungs and BALF from mice were collected 3 d after PR8 infection and 1 d after 19F primary infection or coinfection. (A) Apoptosis of cells collected from BALF was analyzed by flow cytometry with the staining of annexin V and propidium iodide. Annexin V–positive cells were identified as apoptotic and necrotic cells. Apoptotic cell numbers were calculated from total cell numbers by multiplying their respective apoptosis rates (n = 5–6). **p , 0.01, ***p , 0.001 based on Mann–Whitney U test. ns, not significant. (B) TUNEL staining of lung sections was performed with the use of In Situ Cell Death Detection Kit according to manufacturer instructions (original magnification, 3200). (C) Immunohistochemical assay of cleaved caspase-3 expression in lung tissues. (D) The expressions of XBP1s, CHOP, phospho-JNK, and cleaved caspase-3 in lungs were detected by Western blotting. Data are representative of at least three independent experiments.

Article Snippet: For immunohistochemical analysis, lung sections were incubated with mouse monoclonal PGRN Ab (R&D Systems) or cleaved caspase-3 [Asp175] Ab (5A1E; Cell Signaling Technology) overnight at 4 ̊C.

Techniques: Infection, Cytometry, Staining, MANN-WHITNEY, TUNEL Assay, In Situ, Immunohistochemical staining, Expressing, Western Blot

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Affinity comparison of mAbs derived from chicken immunizations and other sources for 2 unrelated model antigens, PCSK9 and PGRN. (A) Biacore binding curves and global fits of select anti-PCSK9 mAbs from chicken immunization showing a diverse set of kinetic profiles. The colored curves represent the measured binding responses of hPCSK9 when injected at concentrations of 5 nM (red) and 50 nM (green), with the global fit overlaid in black. (B) Isoaffinity plot comparing anti-PCSK9 mAbs generated from chicken immunization (olive green) with those from human phage display libraries (blue). (C) Isoaffinity plot comparing anti-PGRN mAbs generated from immunizations in chicken (olive green) and mouse (purple). The red dotted line indicates the k d limit of 5.70 × 10 −5 (1/s) that was placed on interactions which showed < 5 % signal decay within the allowed dissociation phase of 15 min, also known as the “5 % rule” (see Methods).

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Comparison, Derivative Assay, Binding Assay, Injection, Generated

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Solution affinity determination of 2 high affinity clones obtained from chicken immunization toward their respective serum antigens. (A) Schematic representation of the KinExA assay set-up. The mAb of interest is titrated into human serum and the equilibrated mixtures are injected over beads absorption-coated with a competing mAb to capture the free antigen. The bead-captured antigen is then detected using a Dylight-labeled sandwiching mAb. (B) Global analysis of mAb C34 binding serum PCSK9. (C) Global analysis of mAb C25 binding serum PGRN. In each case, the reported apparent K D value is the best fit and 95 % confidence interval of the fit.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Clone Assay, Injection, Labeling, Binding Assay

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Chicken-mouse merged binning results for PGRN. (A) Heat map showing binning assignments for 32 mAbs generated from chicken immunization (olive green) and 20 mAbs generated from mouse immunization (purple). SPR-derived K D values toward human PGRN are reported, along with their cross-reaction toward mouse (m) PGRN, and their GEP subdomain assignment (just for the human-specific clones; n/d = not determined). (B) Blocking network plot, colored by mAb library, with GEP assignments indicated (determined empirically, or inferred). (C) Dendrogram of the antibody sequence lineages for the chicken mAbs alongside the binning heat map for these clones (drawn from panel A, transposed, and resorted). (D) Blocking network plot for the chicken clones, colored by bin, with GEP assignments indicated. See Table S2.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Generated, Derivative Assay, Clone Assay, Blocking Assay, Sequencing

Journal: mAbs

Article Title: Assessing kinetic and epitopic diversity across orthogonal monoclonal antibody generation platforms

doi: 10.1080/19420862.2015.1118596

Figure Lengend Snippet: Comparison of the epitope coverage observed for anti-PGRN mAbs raised via the immunization of chickens or mouse . *Of the 14 chicken mAbs that showed human/mouse crossreactivity, 8 were mapped to a GEP subdomain (by inferring from the binning data shown in Fig. 4B ) and are included in the A – P tally, and 6 were not assigned to a GEP subdomain.

Article Snippet: Mouse anti-PGRN mAbs were generated via standard hybridoma technology using a single Balb/c mouse that was immunized with 50 μg recombinant hPGRN (R&D systems, 2420-PG) mixed with Gerbu adjuvant with weekly intraperitoneal (i.p.) boosts for 5 boosts total.

Techniques: Comparison

Journal: Experimental and Therapeutic Medicine

Article Title: Upregulated expression level of the growth factor, progranulin, is associated with the development of primary Sjögren’s syndrome

doi: 10.3892/etm.2014.1981

Figure Lengend Snippet: Quantitative polymerase chain reaction (qPCR) and immunoblot analysis. Peripheral blood mononuclear cells were separated from 26 healthy controls and 26 pSS patients prior to and following treatment with prednisone, and the total mRNA and protein were isolated. (A) Expression levels of PGRN mRNA were examined by qPCR and normalized against the expression of the GAPDH gene. (B) Protein expression levels of PGRN were examined by immunoblotting, where GAPDH was used as the loading control. Each sample was determined in triplicate and a representative blot is shown for one of the 26 patients. * P<0.05, vs. healthy controls; # P<0.05, vs. pre-treated pSS group. pSS, primary Sjögren’s syndrome; PGRN, progranulin.

Article Snippet: The primary mouse anti-human PGRN monoclonal antibody (clone 296628) was purchased from R&D Systems (Minneapolis, MN, USA), while the primary mouse anti-GAPDH monoclonal and secondary horseradish peroxidase-conjugated goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Isolation, Expressing, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Upregulated expression level of the growth factor, progranulin, is associated with the development of primary Sjögren’s syndrome

doi: 10.3892/etm.2014.1981

Figure Lengend Snippet: Comparison of serum levels of PGRN and IL-6 by ELISA.

Article Snippet: The primary mouse anti-human PGRN monoclonal antibody (clone 296628) was purchased from R&D Systems (Minneapolis, MN, USA), while the primary mouse anti-GAPDH monoclonal and secondary horseradish peroxidase-conjugated goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Comparison, Enzyme-linked Immunosorbent Assay, Control

Journal: Experimental and Therapeutic Medicine

Article Title: Upregulated expression level of the growth factor, progranulin, is associated with the development of primary Sjögren’s syndrome

doi: 10.3892/etm.2014.1981

Figure Lengend Snippet: Spearman’s rank correlation analyses of PGRN and IL-6 levels in pSS patients (A) prior to and (B) following treatment with prednisone. P<0.05 indicates a statistically significant difference. PGRN, progranulin; IL-6, interleukin-6; pSS, primary Sjögren’s syndrome.

Article Snippet: The primary mouse anti-human PGRN monoclonal antibody (clone 296628) was purchased from R&D Systems (Minneapolis, MN, USA), while the primary mouse anti-GAPDH monoclonal and secondary horseradish peroxidase-conjugated goat anti-mouse antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: