Journal: PLoS ONE

Article Title: Plasmin Plays an Essential Role in Amplification of Psoriasiform Skin Inflammation in Mice

doi: 10.1371/journal.pone.0016483

Figure Lengend Snippet: ( A ) A significant reduction in the expression of PLG in PBMC of psoriasis patients was detected by flow cytometry. ( B ) Cryosections from lesional skin of psoriasis patients or normal volunteers were stained with a PLG mAb (red). ( C ) Lysates from lesional skin of psoriasis patients or normal skin of volunteers were subjected to western blotting for PLG expression. Human PLG was used as a positive control (right panel). Actin - loading control. ( D, E ) Cryosections from lesional skin of psoriasis patients or normal volunteers were stained with uPA or tPA mAbs (green) for its expression. Original magnification for immunofluorescent staining, ×400. e, epidermis; d, dermis. Dotted lines indicate the border between epidermis and dermis.

Article Snippet: The following antibodies were used for flow cytometry, all in conjunction with AlexaFluor 555 (Caltag Laboratories): anti-human PLG (Santa Cruz), anti-human uPA, anti-human uPA (R&D), and anti-human annexin A2 (ABGENT).

Techniques: Expressing, Flow Cytometry, Staining, Western Blot, Positive Control

Journal: Frontiers in Immunology

Article Title: Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation

doi: 10.3389/fimmu.2022.755694

Figure Lengend Snippet: Schematic diagram of the homologous modeling structure of FHR1*A and FHR1*B. The SCR3-SCR5 of FHR1*A (A) and FHR1*B (B) are shown in green and yellow, respectively. The mutant amino acids are shown in red. The aligned view of SCR3-SCR5 of FHR1*A and FHR1*B showed some differences (C) . The structures of SCR4-SCR5 are identical between FHR1*A and FHR1*B, while SCR3 of FHR1*B is more prone to SCR4-5 than that of FHR1*A. The representative angle (∠p.139-p.189-p.194) of CFHR1 *B (E) is smaller than that of CFHR1 *A (D) , with a maximum angle difference of 8.9°.

Article Snippet: And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich).

Techniques: Mutagenesis

Journal: Frontiers in Immunology

Article Title: Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation

doi: 10.3389/fimmu.2022.755694

Figure Lengend Snippet: The included angles of SCR3 to SCR4-5 in FHR1*A and FHR1*B.

Article Snippet: And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich).

Techniques:

Journal: Frontiers in Immunology

Article Title: Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation

doi: 10.3389/fimmu.2022.755694

Figure Lengend Snippet: Interaction of the two FHR1 isoforms with C3b. Equal concentrations of two immobilized isoforms of FHR1 (0.078 μg/ml-2.5 μg/ml) bound C3b (2 μg/ml) in a dose-dependent manner. The binding-C3b was finally detected. FHR1*B showed a higher capacity to bind C3b at a concentration of 1.25 μg/ml (A) . In a reverse setting, equal concentrations of two isoforms of FHR1 (1.25 μg/ml-10 μg/ml) bound immobilized C3b (100nM) in a dose-dependent manner. The bound FHR1 was detected at last. FHR1*B also showed higher binding capacity to C3b (B) . Moreover, the 100nM C3b were added onto the microplates, overspread with the Saccharomyces cerevisiae , and equal concentrations of two isoforms of FHR1 (1.25 μg/ml-5 μg/ml) was added. The surface-bound C3b was detected by anti-C3c antibody (C) , and the binding of FHR1 to surface-bound C3b was detected with anti-FHR1 antibody (D) . The representative figures from three independent experiments are shown. The results are representative of three independent experiments. The data are presented as the means ± SDs. The symbol ns indicates nonsignificance.

Article Snippet: And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich).

Techniques: Binding Assay, Concentration Assay

Journal: Frontiers in Immunology

Article Title: Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation

doi: 10.3389/fimmu.2022.755694

Figure Lengend Snippet: Binding of the FHR1 isoforms to necrotic cells. Necrosis cells was evaluated by PI and Annexin V staining. Double positive for both Annexin V and PI cells were considered as necrotic cells (A) . The representative figures for blank control, isotype control and sample incubated with FHR1 protein were showed (B) . FHR1 proteins (0.078 μg/ml-0.313 μg/ml) bound to necrotic HUVECs in a dose-dependent manner. The representative figures for samples incubated with both FHR1*A and FHR1*B protein were showed (C) . In addition, compared to FHR1*A, FHR1*B presented significantly increased capacity to bind to necrotic HUVECs at concentrations between 0.078 μg/ml and 0.313 μg/ml (D) . The results are representative of three independent experiments. The data are presented as the means ± SDs. MFI, Mean fluorescence intensity.

Article Snippet: And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich).

Techniques: Binding Assay, Staining, Incubation, Fluorescence

Journal: Frontiers in Immunology

Article Title: Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation

doi: 10.3389/fimmu.2022.755694

Figure Lengend Snippet: Competition assays of FHR1 with FH to C3b and C3b convertase assays. FHR1*A and FHR1*B compete with FH for binding C3b in a dose-dependent manner, and FHR1*B binds more C3b than FHR1*A at concentrations of 0.078 μg/ml-2.5 μg/ml (A) . FH inhibited the C3bBb assembling, while both isoforms of FHR1 competed with FH and reverse the inhibition to some extent. Moreover, FHR1*B showed a more powerful deregulation effect on FH mediated regulation of the solid-phase C3 convertase (B) . The results are representative of three independent experiments. The data are presented as the means ± SDs.

Article Snippet: And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich).

Techniques: Binding Assay, Inhibition

Journal: Frontiers in Immunology

Article Title: Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation

doi: 10.3389/fimmu.2022.755694

Figure Lengend Snippet: Effect of the FHR1 isoforms on the cofactor activity of FH. Compared to FH (positive control, lane 9), both FHR1*A (lanes 1-3) and FHR1*B (lanes 4-6) at concentrations of 90-11.25 ug/ml showed no or little FI cofactor activity (A) . When incubated with C3b, FI and FH together, the FHR1 groups (lanes 1-3, 4-6, 7-9, 10-12 were technical replicates, respectively; (C) generated fewer C3b cleavage products than the group without FHR1 (no-FHR1 control, lanes 13-14, technical replicates; (C) . Furthermore, the FHR1*B groups generated more C3b cleavage products than the FHR1*A groups at the same concentrations (C, D) . The data are presented as the means ± SDs.

Article Snippet: And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich).

Techniques: Activity Assay, Positive Control, Incubation, Generated

Journal: Frontiers in Immunology

Article Title: Atypical Hemolytic Uremic Syndrome-Associated FHR1 Isoform FHR1*B Enhances Complement Activation and Inflammation

doi: 10.3389/fimmu.2022.755694

Figure Lengend Snippet: FHR1 induces monocytes to secrete inflammatory cytokines. Compared to BSA, both FHR1*A and FHR1*B significantly increased IL-1β (A) and IL-6 (B) secretion by monocytes. In addition, FHR1*B induced the secretion of higher levels of IL-1β (A) and IL-6 (B) by monocytes than FHR1*A. The data are presented as the means ± SDs. The results are representative of three independent experiments. The data are presented as the means ± SDs.

Article Snippet: And mouse anti-human FHR1 antibody (R&D, cross-reacts with FH) was added as primary antibody, followed by adding AP-conjugated goat anti-mouse IgG antibody (Sigma-Aldrich).

Techniques:

Journal: PLoS ONE

Article Title: Plasmin Plays an Essential Role in Amplification of Psoriasiform Skin Inflammation in Mice

doi: 10.1371/journal.pone.0016483

Figure Lengend Snippet: ( A ) A significant reduction in the expression of PLG in PBMC of psoriasis patients was detected by flow cytometry. ( B ) Cryosections from lesional skin of psoriasis patients or normal volunteers were stained with a PLG mAb (red). ( C ) Lysates from lesional skin of psoriasis patients or normal skin of volunteers were subjected to western blotting for PLG expression. Human PLG was used as a positive control (right panel). Actin - loading control. ( D, E ) Cryosections from lesional skin of psoriasis patients or normal volunteers were stained with uPA or tPA mAbs (green) for its expression. Original magnification for immunofluorescent staining, ×400. e, epidermis; d, dermis. Dotted lines indicate the border between epidermis and dermis.

Article Snippet: The following antibodies were used for flow cytometry, all in conjunction with AlexaFluor 555 (Caltag Laboratories): anti-human PLG (Santa Cruz), anti-human uPA, anti-human uPA (R&D), and anti-human annexin A2 (ABGENT).

Techniques: Expressing, Flow Cytometry, Staining, Western Blot, Positive Control