standard purified human complement components  (Merck KGaA)

Journal: JCI Insight

Article Title: IgM anti-ACE2 autoantibodies in severe COVID-19 activate complement and perturb vascular endothelial function

doi: 10.1172/jci.insight.158362

Figure Lengend Snippet: ( A ) Anti-ACE2 IgM antibodies from patient CV-1 or control do not inhibit ACE2 activity. Positive and negative controls were ACE2 alone, and ACE2 plus ACE2 inhibitor, respectively (see ). ( B ) IgM antibodies to ACE2 activate complement. Purified IgMs from anti-ACE2 IgM–positive COVID-19 patients ( n = 8) and healthy controls ( n = 11) was used for C1q binding assays. Values are means from 2 independent experiments performed on different days. *** P < 0.0001, Mann-Whitney test. (C–G) Anti-ACE2 IgM affects the pulmonary endothelium. ( C ) Phase image of a pulmonary microvessel (top). Fluorescence images of microvessels exposed to anti-ACE2–negative IgM (HC) or anti-ACE2–positive IgM (CV) after perfusion with 10 kDa dextran (lower). Representative images across n = 3 to 6 independent experiments for each IgM condition are shown. ( D ) ACE2 and CD31 microvessel staining following 24-hour perfusion with IFN-α/γ. Representative images across n = 3 independent experiments are shown. ( E ) C3c staining after perfusion with IFN and anti-ACE2–positive or control IgM (3.33 μg/mL). Representative images across n = 3 independent experiments are shown. ( F ) Permeability of microvessels perfused with IFN and anti-ACE2–positive IgM (CV) or anti-ACE2 negative IgM (HC) (100 μg/mL). D.L., detection limit. A linear mixed effects model was used to test the effect of anti-ACE2–positive IgM on permeability. n = 3 to 5 for each IgM condition; each dot represents an independent replicate. ( G ) Inhibition of microvessel permeability to 10 kDa dextran in response to anti-ACE2 IgM is IFN dependent. Low CV-1: 3.33 μg/mL; affinity-purified anti-ACE2 IgM: 100 ng/mL. To compare each condition to control, a Kruskal-Wallis test followed by Dunn’s multiple-comparison test was performed. n = 4 to 6 for each IgM condition; each dot represents an independent replicate.

Article Snippet: Seventy-two hours after initial cell seeding (24 hours after incubation with IFNs), microvessels were treated with 100 μg/mL patient IgM in EC medium supplemented with 10% normal human complement (Quidel) for 30 minutes under low-flow conditions (~0.3 dyne/cm 2 ).

Techniques: Control, Activity Assay, Purification, Binding Assay, MANN-WHITNEY, Fluorescence, Staining, Permeability, Inhibition, Affinity Purification, Comparison

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a Map of complement-mediated bactericidal activity. b The proportion of gut bacteria after 16s rRNA analyses of colon contents in normal CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice (pooled samples from six mice). Also see Fig. . c The proportion of gut bacteria after 16s rRNA analyses in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS. Also see Fig. . d The proportion of E. coli (Escherichia-Shigella) in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS ( n = 5). Also see Fig. S3C. e Flow cytometry of feces bacteria after staining using anti-LPS antibodies in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CDKO) mice after 2% DSS. f QPCR of colonic content (upper) and colonic tissues (lower) in CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS ( n = 6). g FISH of E. coli in the gut tissues of CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice after 2% DSS. One representative from six mice. Scale bar, 40 μM; Scale bar in bottom panel, 5 μM. Two side student’s t test in e and f ; * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Activity Assay, Bacteria, Flow Cytometry, Staining

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a E. coli clones in the normal human sera (N. Sera, Quidel), C3deficient sera (C3defi, Quidel), C1 deficient sera (C1 defi, Quidel) and inactive sera at different time points. *C3deficint sera vs normal sera. b E. coli clones in the normal sera (N. Sera, Quidel), CFD-deficient sera (CFD defi, Quidel) inactive sera at different time points. *CFD-deficient sera vs normal sera. c E. coli clones in the normal human sera after adding LPS (containing mannose), mannose or peptidoglycan. *LPS vs normal sera. d E. coli clones in the normal human sera after adding rReg4, rREG4 or mutated rREG4. *Reg4 vs normal sera. Only mrReg4 and only hrREG4, E.coli clones in inactive complement serum after adding mrReg4 or hrREG4. e E. coli clones in the normal human sera after adding different concentrations of rReg4. f E. coli clones in the normal human sera (N. sera) or CFD-deficient (CFD defi) sera after adding rReg4 (N. sera/rReg4 or CFDdefi/rReg4). *Normal sera/Reg4 vs normal sera. g E. coli clones in the normal human sera after adding rREG4, IgA or IgA+REG4. *IgA+Reg4 vs Reg4. Around 5 × 10 5 in a – c or 1 × 10 6 in d – g E. coli CFUs were added into 1 ml 10% sera. Bacterial suspension were diluted and plated on MacConkey agar plates. Plates were incubated overnight at 37 °C, and CFU were counted. Analysis of variance test used in a – g . * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. One representative of three independent experiments.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Clone Assay, Suspension, Incubation

Journal: Communications Biology

Article Title: Reg4 and complement factor D prevent the overgrowth of E. coli in the mouse gut

doi: 10.1038/s42003-020-01219-2

Figure Lengend Snippet: a Immunostaining of C3b, C5-9, Reg4, IgA, MASP1/3 and MASP2 on GFP-labeled E. coli in 2% DSS-treated GFP-labeled E. coli infused CFD fl/fl pvillin-cre T (CFDKO/0160) mice (Left) and statistic analyses (right, three images/slide, three slides/mouse, n = 6). Scale bar, 40 μM. No, number; Iso, isotypic antibod. b Flow cytometry of Reg4, IgA, MASP1/3 and MASP2 on the GFP-labeled E. coli in untreated GFP-labeled E. coli infused CFD fl/fl pvillin-cre w (WT) and CFD fl/fl pvillin-cre T (CFDKO) mice and statistic analyses ( n = 3). c ELISA for detecting binding of Reg4 with LPS (left), Reg4 with IgA (middle) or IgA with LPS (right) in different concentration of Reg4 (left and middle) or IgA (right) coated plates d ELISA of C3 in different concentration of Reg4 (left) or IgA (right) with LPS coated plates. For complement resources, 5% normal human sera were added. e ELISA of C3b in the LPS, Reg4, IgA, LPS + Reg4 (LPS/Reg4), LPS + IgA (LPS/IgA), Reg4+IgA (Reg4/IgA) and LPS + Reg4+IgA (LPS/Reg4/IgA) coated plates. For complement resources, 5% normal human sera were added. Mann–Whitney U test in a ; Two side student’s t test in b ; ANOVA plus post-Bonferroni analysis in e ; Analysis of variance test in c and d . * P < 0.05; ** P < 0.01; *** P < 0.001; NS no significance. In c – e , one representative of three independent experiments.

Article Snippet: 5 × 10 5 or 1 × 10 6 CFU was added into PBS that contains 10% Normal Human Complement Standard (Quidel), C1q-depleted serum (Quidel) or C3 depleted and CFD depleted serum (Quidel).

Techniques: Immunostaining, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, MANN-WHITNEY