Journal: Investigative Ophthalmology & Visual Science

Article Title: Complement-Mediated Regulation of Apolipoprotein E in Cultured Human RPE Cells

doi: 10.1167/iovs.16-20083

Figure Lengend Snippet: Cell-associated ApoE accumulation was dependent on MAC deposition. RPE cells were primed with or without S58 (1.2 mg/mL) for 30 minutes and then treated with 6% serum for 30 minutes ( A ), 6 hours ( B , C ), or 5 hours ( D , E ). ( A ) Induction of MAC accumulation on RPE cells from a 62-year-old donor with ApoE phenotype E3/E3 and CFH YY402 variant. After serum treatments in the presence or absence of anti-C5 antibody (10 μg/mL), cells were fixed in 4% PFA for 15 minutes and stained with mouse anti-human C5b-9 (aE11) antibody. Data are representative of two separate experiments in two donors with similar results. Red stain indicates MAC deposition. Blue stain corresponds to 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei. Scale bar : 100 μm. ( B ) Accumulation of ApoE was prevented when C5 was blocked by anti-C5 antibody. Total proteins (15 μg) obtained from RPE cells of a 61-year-old donor with ApoE phenotype E3/E4 and CFH YH402 variant were separated by SDS-PAGE 6 hours post complement challenge in the presence or absence of anti-C5 antibody (10 μg/mL). ( C ) The quantity of ApoE relative to GAPDH shown in ( B ) was determined by densitometry. * P = 0.001 vs. C1q-Dep and C5 Ab+C1q-Dep. ** P = 0.002 vs. S58+C1q-Dep. Data are representative of three separate experiments in three donors with similar results. ( D ) Accumulation of ApoE was blocked by absence of C6. Total proteins (30 μg) obtained from RPE cells of a 51-year-old donor with ApoE phenotype E3/E3 and CFH HH402 variant were separated by SDS-PAGE after treatment with C6-Dep in the presence or absence of C6 protein at 7.3 or 65 μg/mL. ( E ) The quantity of ApoE relative to GAPDH shown in ( D ) was determined by densitometry. * P < 0.05 vs. C6q-Dep and S58+C6-Dep. Data are representative of two separate experiments in two donors with similar results.

Article Snippet: C1q-depleted human serum (C1q-Dep), C6-depleted human serum (C6-Dep), and purified C6 protein were purchased from Quidel Corp. (San Diego, CA, USA).

Techniques: Variant Assay, Staining, SDS Page

Journal: Frontiers in immunology

Article Title: Clinical Isolates of Acinetobacter spp. Are Highly Serum Resistant Despite Efficient Recognition by the Complement System.

doi: 10.3389/fimmu.2022.814193

Figure Lengend Snippet: FIGURE 1 | Survival of Acinetobacter isolates in human serum and blood, and recognition by the complement system. (A) Survival of Acinetobacter isolates in the presence of 30% NHS. Survival of isolates (B) A. baumannii DF1000, (C) A. baumannii DF1011, (D) A. baumannii DF1013 and (E) A. baumannii KR792 in human blood. Survival of bacteria was analyzed as CFU/mL, bars represent mean ± SD of at least 3 independent experiments. Horizontal dotted line refers to the starting number of bacteria used in the assay. One-way ANOVA with Dunnett’s post-test was used to analyze results from bactericidal activity assays, considering input as a control sample. Two-way ANOVA with Dunnett’s post-test was used to analyze results from blood killing assay, considering sample with full blood as a control sample. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001. Recognition of Acinetobacter by human (F) IgG and (G) IgM antibodies. (H) Deposition of C1q on bacterial surface. (F–H) Bars represent gMFI ± SD of at least 3 independent experiments; horizontal dotted line represents gMFI (average + SD) measured in human IgG/IgM- depleted serum or human C1q-depleted serum acting as a binding/deposition control.

Article Snippet: Heat-inactivated normal human serum was obtained after incubation of NHS at 56°C for 30 min. Human C1q-depleted serum was purchased from Quidel (A509) or prepared in house during the purification of C1q protein.

Techniques: Bacteria, Activity Assay, Control, Binding Assay

Journal: Frontiers in immunology

Article Title: Clinical Isolates of Acinetobacter spp. Are Highly Serum Resistant Despite Efficient Recognition by the Complement System.

doi: 10.3389/fimmu.2022.814193

Figure Lengend Snippet: FIGURE 2 | Complement deposition on Acinetobacter isolates via all pathways (GVB++ buffer). (A) Deposition of C4b on the bacterial surface in NHS. (B) Deposition of C4b on bacterial surface in human C1q-depleted serum. (C) Deposition of C4b on bacterial surface in NHS, human C1q-depleted serum, and human MBL- depleted serum. (D) Deposition of C4b on bacterial surface in the presence of C1-INH. Two-way ANOVA with Dunnett’s post-test was used to analyze results from C4b deposition assays in depleted sera, considering sample with NHS as a control sample. *p < 0.05, **p < 0.01. Deposition of (E) C3b and (F) MAC on bacterial surface in NHS. Bars represent gMFI ± SD of at least 3 independent experiments; horizontal dotted line represents gMFI (average + SD) measured in human sera with EDTA acting as a deposition control.

Article Snippet: Heat-inactivated normal human serum was obtained after incubation of NHS at 56°C for 30 min. Human C1q-depleted serum was purchased from Quidel (A509) or prepared in house during the purification of C1q protein.

Techniques: Control

Journal: Frontiers in immunology

Article Title: Clinical Isolates of Acinetobacter spp. Are Highly Serum Resistant Despite Efficient Recognition by the Complement System.

doi: 10.3389/fimmu.2022.814193

Figure Lengend Snippet: FIGURE 4 | Survival and complement system activation on A. baumannii mutant strains. (A) Survival of wt and capsule mutant strains in 30% NHS. Binding of human (B) IgG and (C) IgM. (D) Deposition of C1q. Deposition of C4b in (E) NHS, (F) human C1q-depleted serum, (G) NHS, human C1-depleted serum and MBL- depleted serum, (H) the presence of C1-INH. Deposition via all pathways of (I) C3b and (J) MAC and deposition via alternative pathway of (K) iC3b, (L) C5b and (M) MAC. (B–M) Bars represent gMFI ± SD of at least 3 independent experiments; horizontal dotted line represents gMFI (average + SD) measured in human sera with EDTA acting as a deposition control. One-way ANOVA with Dunnett’s post-test was used to analyze results from bactericidal activity assays and complement deposition assays, considering the parental AB307-0294 wt strain as control sample. Two-way ANOVA with Dunnett’s post-test was used to analyze results from C4b deposition assays in depleted sera, considering sample with NHS as a control sample. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.0001.

Article Snippet: Heat-inactivated normal human serum was obtained after incubation of NHS at 56°C for 30 min. Human C1q-depleted serum was purchased from Quidel (A509) or prepared in house during the purification of C1q protein.

Techniques: Activation Assay, Mutagenesis, Binding Assay, Control, Activity Assay