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: Frontiers in Immunology

Article Title: Complement Initiation Varies by Sex in Intestinal Ischemia Reperfusion Injury

doi: 10.3389/fimmu.2021.649882

Figure Lengend Snippet: Intestinal IR-induced complement initiation transcription differs between sexes. C57B6L/J mice were subjected to Sham and IR treatment with 15, 30, 60, or 120 min reperfusion. Mid-jejunum of (A) C1q, (B) MBL-C, and (C) Factor B, RNA expression was determined by RT-PCR analysis. Samples were normalized to 18S followed by fold change compared to Sham treatment. *, indicates significant difference between IR treatment and Sham within the same sex (P ≤ 0.05), as determined by 1-way ANOVA followed by Newman Keuls post-hoc test; Ф, indicates significant difference between sexes in the same time point and same treatment (P ≤ 0.05), as determined by unpaired t-test. Each bar is representative of 7-12 mice per group.

Article Snippet: Deposition of C3b, C1q, MBL-C, Factor B, and IgM deposition on the tissue sections was determined by staining with a purified rat-anti-mouse anti-C3b antibody (Hycult Biotechnologies, cat. # HM1065), anti-C1q or anti-MBL-C antibody (Cedarlane, cat. # CL7501AP and #CL7303AP, respectively), or rabbit-anti-mouse anti-Factor B antibody (Abcam, ab231072) followed by an Alexa Flour 594 conjugated donkey-anti-rat IgG secondary antibody (Jackson ImmunoResearch, cat. # 712586153) or FITC-green conjugated donkey-anti-rabbit IgG secondary (Jackson ImmunoResearch, cat. # 711096152).

Techniques: RNA Expression, Reverse Transcription Polymerase Chain Reaction

Journal: Frontiers in Immunology

Article Title: Complement Initiation Varies by Sex in Intestinal Ischemia Reperfusion Injury

doi: 10.3389/fimmu.2021.649882

Figure Lengend Snippet: Intestine tissue C1q, MBL-C, Factor B, deposition differs between sexes. C57B6L/J mice were subjected to Sham and IR treatment with 30 or 120 min reperfusion. Intestinal sections were stained for (A) C1q, (B) MBL-C, (C) Factor B, by immunohistochemistry. Bar = 40 µm. The image analyses for each animal were determined by digital densitometry recognition using computer-aided ImageJ (NIH). Microphotographs (200X) are representative of at least 3-4 mice stained in at least 3 independent experiments.

Article Snippet: Deposition of C3b, C1q, MBL-C, Factor B, and IgM deposition on the tissue sections was determined by staining with a purified rat-anti-mouse anti-C3b antibody (Hycult Biotechnologies, cat. # HM1065), anti-C1q or anti-MBL-C antibody (Cedarlane, cat. # CL7501AP and #CL7303AP, respectively), or rabbit-anti-mouse anti-Factor B antibody (Abcam, ab231072) followed by an Alexa Flour 594 conjugated donkey-anti-rat IgG secondary antibody (Jackson ImmunoResearch, cat. # 712586153) or FITC-green conjugated donkey-anti-rabbit IgG secondary (Jackson ImmunoResearch, cat. # 711096152).

Techniques: Staining, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Complement Initiation Varies by Sex in Intestinal Ischemia Reperfusion Injury

doi: 10.3389/fimmu.2021.649882

Figure Lengend Snippet: MBL-C or Factor P deficiency decreases serum IgM, C3b and C5a levels in both sexes following intestinal IR. (A) Serum from untreated C57B6L/J WT, C1q - / - , MBL - / - , and P - / - male (blue) and female (yellow) mice was analyzed by ELISA for IgM. Sham treatment is indicated by light colors whereas IR is indicated by dark colors. Each bar represents 2-3 pools of 3-5 mice. (B) C3b deposition on intestines from male (blue) and female (yellow) C57B6L/J WT, C1q - / - , MBL - / - , and P - / - mice was examined after Sham and IR treatment with 120 min reperfusion. Each bar represents 3-4 animals in at least 3 independent experiments (C) Serum C5a from C57B6L/J WT, C1q - / - , MBL - / - , and P - / - mice was determined after Sham and IR treatment with 120 min reperfusion. Sham treatment represents pooled analysis of all strains. Each bar is representative of 7-12 mice per group. Serum IgM and C5a were measured with sandwich ELISA. C3b deposition was quantitated by Image J analysis after IHC. * indicates significant difference between IR treatment and Sham within the same sex (P ≤0.05), as determined by 1-way ANOVA with Newman Keuls post-hoc test; Ф indicates significant difference between sexes in the same time point same treatment (P ≤ 0.05), as determined by unpaired t-test; τ indicates significant difference between same sex IR compared to WT IR (P ≤ 0.05), as determined by 1-way ANOVA with Newman Keuls post-hoc test.

Article Snippet: Deposition of C3b, C1q, MBL-C, Factor B, and IgM deposition on the tissue sections was determined by staining with a purified rat-anti-mouse anti-C3b antibody (Hycult Biotechnologies, cat. # HM1065), anti-C1q or anti-MBL-C antibody (Cedarlane, cat. # CL7501AP and #CL7303AP, respectively), or rabbit-anti-mouse anti-Factor B antibody (Abcam, ab231072) followed by an Alexa Flour 594 conjugated donkey-anti-rat IgG secondary antibody (Jackson ImmunoResearch, cat. # 712586153) or FITC-green conjugated donkey-anti-rabbit IgG secondary (Jackson ImmunoResearch, cat. # 711096152).

Techniques: Enzyme-linked Immunosorbent Assay, Sandwich ELISA

Journal: Acta Neuropathologica Communications

Article Title: Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role

doi: 10.1186/s40478-020-01041-1

Figure Lengend Snippet: Summary table

Article Snippet: The brain coronal sections were incubated overnight at 4 °C with primary monoclonal antibody anti-mouse MBL-C (1 µg/ml; Hycult Biotechnology, Uden, The Netherlands) followed by a secondary biotinylated antibody against rat IgG.

Techniques:

Journal: Acta Neuropathologica Communications

Article Title: Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role

doi: 10.1186/s40478-020-01041-1

Figure Lengend Snippet: Experimental design. a WT or KOs mice (including: MASP-2 −/− , MBL −/− , FCN-A −/− , CL-11 −/− , MBL-C −/− , MASP-1/3 −/− and MBL-A −/− ) were subjected to TBI or sham injury. Sensorimotor deficits were assessed weekly by neuroscore and beam walk test. The sum of 4-week performances of each mouse genotype has been used to calculate the health score. b MBL brain presence and residual LP activity in plasma was assed in MASP-2 −/− and WT TBI mice 30′ after surgery. Histopathological analysis was done for MASP-2 −/− and WT mice at 6 weeks after TBI

Article Snippet: The brain coronal sections were incubated overnight at 4 °C with primary monoclonal antibody anti-mouse MBL-C (1 µg/ml; Hycult Biotechnology, Uden, The Netherlands) followed by a secondary biotinylated antibody against rat IgG.

Techniques: Activity Assay

Journal: Acta Neuropathologica Communications

Article Title: Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury, with MASP-2 playing a major role

doi: 10.1186/s40478-020-01041-1

Figure Lengend Snippet: Brain MBL-C deposition and plasmatic LP activation 30′ after TBI in WT or MASP-2 −/− mice. a Representative low-magnification images of MBL-C immunolabeling at 30′ after TBI or sham surgery (the cortical edge is outlined in yellow). MBL-C quantification was done over an area of 350 µm from the contusion edge (Fig. ). Scale bars 50 μm. b MBL-C deposition in brains of MASP-2 −/− mice was similar to that of WT. Data is shown as a scatter dot plot, line at mean ± SEM ( n = 2–4). Two-way Anova followed by Sidak’s post hoc test, ** p <0.01 compared with Sham MASP-2 −/− , *** p < 0.001 compared with Sham WT. c In vitro assay for MBL-driven LP activation on mannan—plasma from MASP-2 −/− lack C4 convertase activity, resulting in minimal C4b deposition compared to WT mice. The data is shown as a scatter dot plot, line at mean ± SEM ( n = 2–4), Two-way Anova followed by Sidak’s post hoc test, *** p <0.001 compared with Sham or TBI WT

Article Snippet: The brain coronal sections were incubated overnight at 4 °C with primary monoclonal antibody anti-mouse MBL-C (1 µg/ml; Hycult Biotechnology, Uden, The Netherlands) followed by a secondary biotinylated antibody against rat IgG.

Techniques: Activation Assay, Immunolabeling, In Vitro, Activity Assay