Journal: Frontiers in Immunology

Article Title: The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus

doi: 10.3389/fimmu.2022.1021370

Figure Lengend Snippet: GC block fails to prevent development of autoantibodies and PB/PC expansion in lupus mice. (A) Overview of experimental setup. Bcl-6 flx/flx (Cre-, purple) and Aicda-Cre Bcl-6 flx/flx (Cre+, blue) mice were left untreated (dark color, n=7 and n=8, respectively) or treated with R848 (light color, n=6 and n=8, respectively). (B) Spleen weights. (C) Anti-dsDNA IgG2c. (D) Total IgG2c. (E) Total IgG1. (F) Total IgG3. (G) Representative confocal micrograph of spleen from Cre- untreated animal, stained for CD169 (red), IgD (blue) and Ki67 (green). Scale bar is 400 µm. (H) As G, but for Cre- treated animal. (I) As G, but for Cre+ untreated animal. (J) As G, but for Cre+ treated animal. (K) High-resolution image of GC from Cre- treated mouse. Scale bar=100 µm. (L) GC/follicle in spleen. (M) Flow cytometry analyses of monocyte frequencies (Ly6CG int ) in IngLN, MesLN, and spleen. (N) As M, but neutrophil frequencies (Ly6CG hi ). (O) As M, but B cell frequencies (B220 + CD4 - CD8 - ). (P) As M, but GCB frequencies (CD95 hi CD38 lo ). (Q) As M, but PB and PC frequencies (CD138 hi ). Data pooled from two independent experiments. Bar graphs show mean ± SD. ns = p≥0.05, *p < 0.05, **p < 0.01, ***p < 0.001, based on two-way ANOVA with Holm-Sidak’s post-test.

Article Snippet: In brief, antibody [either “14D12” rat IgG2a to mouse MBL-C (Hycult Biotech), or “RTK2758” rat IgG2a isotype control (Abcam)] was concentrated in antibody preparation buffer to a concentration of 2 mg/mL or above.

Techniques: Blocking Assay, Staining, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: The extrafollicular response is sufficient to drive initiation of autoimmunity and early disease hallmarks of lupus

doi: 10.3389/fimmu.2022.1021370

Figure Lengend Snippet: GC block causes increased PB/PCs levels in adolescent 564Igi mice. (A) Schematic overview of experimental setup: 564Igi-Bcl-6 flx/flx (Cre-, dark green, n=11) and 564Igi-Aicda-Cre Bcl-6 flx/flx (Cre+, light green, n=8) mice. (B) Anti-dsDNA IgG2a. (C) Anti-dsDNA IgG2b. (D) Anti-dsDNA total Ig. (E) Total IgG1. (F) Total IgG2a. (G) Total IgG3. Unpaired t-test or Mann-Whitney’s test was used was used to analyze TRIFMA data. (H) Flow cytometry analyses of B cell frequencies (B220 + CD4 - CD8 - of live, singlet lymphocytes) in blood, IngLN, MesLN, and spleen. (I) As H, but CD4 T cells (CD4 + B220 - ). (J) As H, but CD8 T cells (CD8 + B220 - ). (K) As H, but 9D11 B cell frequencies (9D11 + ). (L) As H, but GCB frequencies (CD95 hi CD38 lo ). (M) As H, but PB and PC frequencies (CD138 hi ), (N) As H, but PB frequencies (CD138 hi B220 hi ), (O) As H, but PC frequencies (CD138 hi B220 lo ). Data are pooled from two independent experiments. Bar graphs show mean ± SD. Two-way ANOVA with Holm-Sidak’s post hoc test was used to analyze the data. ns = p≥0.05, *p < 0.05, ***p < 0.001.

Article Snippet: In brief, antibody [either “14D12” rat IgG2a to mouse MBL-C (Hycult Biotech), or “RTK2758” rat IgG2a isotype control (Abcam)] was concentrated in antibody preparation buffer to a concentration of 2 mg/mL or above.

Techniques: Blocking Assay, Flow Cytometry

Journal: PLoS ONE

Article Title: Human L-ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus pneumoniae

doi: 10.1371/journal.pone.0082583

Figure Lengend Snippet: Serial dilutions of NMS or NHS in BBS were incubated in ELISA plates pre-coated with PLY or specific ligands for LP recognition molecules. The binding of specific LP recognition molecules to PLY was subsequently detected using antibodies directed against MBL, ficolins and CL-11. The binding of murine MBL-A and MBL-C (A), murine ficolin-A (C), murine CL-11 (E), human MBL (B), human FCN2 & 3 (D) and human CL-11 (F) were assessed by ELISA. In contrast to mouse ficolin-A (which has no binding affinity to PLY) human L-ficolin (FCN2) (the human orthologoue of murine ficolin-A) showed strong binding to PLY. Results are means (±SEM) of three independent experiments.

Article Snippet: Plates were washed and bound proteins were detected using monoclonal rat anti-mouse MBL-A (Hycult), rat anti-mouse MBL-C (Hycult), rabbit anti-mouse ficolin-A, rabbit anti-human L-ficolin (Sigma), mouse anti-human H-ficolin (Hycult), mouse anti-human CL-11, rat anti-mouse CL-11 mAb, rabbit anti-human C1q.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: PLoS ONE

Article Title: Human L-ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus pneumoniae

doi: 10.1371/journal.pone.0082583

Figure Lengend Snippet: Serial dilutions of NMS or NHS in BBS were incubated in ELISA plates pre-coated with PLY or specific ligands for LP recognition molecules. The binding of specific LP recognition molecules to PLY was subsequently detected using antibodies directed against MBL, ficolins and CL-11. The binding of murine MBL-A and MBL-C (A), murine ficolin-A (C), murine CL-11 (E), human MBL (B), human FCN2 & 3 (D) and human CL-11 (F) were assessed by ELISA. In contrast to mouse ficolin-A (which has no binding affinity to PLY) human L-ficolin (FCN2) (the human orthologoue of murine ficolin-A) showed strong binding to PLY. Results are means (±SEM) of three independent experiments.

Article Snippet: Plates were washed and bound proteins were detected using monoclonal rat anti-mouse MBL-A (Hycult), rat anti-mouse MBL-C (Hycult), rabbit anti-mouse ficolin-A, rabbit anti-human L-ficolin (Sigma), mouse anti-human H-ficolin (Hycult), mouse anti-human CL-11, rat anti-mouse CL-11 mAb, rabbit anti-human C1q.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Binding Assay