Journal: medRxiv

Article Title: Loss of Factor H family proteins associates with meningococcal disease severity

doi: 10.1101/2021.02.05.21251142

Figure Lengend Snippet: (a , b) Differences in FH, FHR-1/1 homodimers, FHR-1/2 heterodimers, FHR-2/2 homodimers, FHR-3, FHR-4A and FHR-5 homodimers as assessed at the acute stage (samples obtained during the first or second day of hospitalization, n = 106) compared to levels at convalescence ( n = 91). Two children appeared to carry a homozygous CFHR3/CFHR1 deletion, as evidenced by the lack of either protein in their convalescent sample. They were excluded from the analysis for FHR-1/1, FHR-1/2, FHR-2/2 and FHR-3 ( n = 104 and n = 89 for acute stage and convalescence). Acute serum samples comprised 88 samples drawn at day 1 and 18 samples drawn at day 2 of hospitalization, for patients of whom no day 1 sample was available. Levels of FHR-2/2 were calculated based on FHR-1/1 and FHR-1/2 levels. Shaded area indicates 95% range in healthy patients, with dashed line indicating the median. Scatter dot plots depict median and interquartile range (IQR) as red lines. Statistical significance was tested using a Mann-Whitney test. (c) Correlations ( r ) between the relative decreases of FH family proteins (ratios between acute and convalescent levels, by dividing acute levels over convalescent levels) were assessed using Pearson’s measure of association, followed by the Benjamini-Hochberg procedure to control for the false discovery rate (FDR, set to 0.05). Blue shades indicate, from light to dark: p < 0.05; p < 0.01; p < 0.001; and p < 0.0001. ( d ) Examples of correlations in ( c ), showing relative decrease in FH levels versus relative decrease in FHR levels. A:C ratio: acute:convalescent ratio; Conv.: convalescent.

Article Snippet: FHR-1/1 homodimers and FHR-1/2 heterodimers were captured using clone anti-FH.02 (Sanquin Research), and detected with biotinylated anti-FH.02 (Sanquin Research) or anti-FHR-2 (clone MAB5484, R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: MANN-WHITNEY

Journal: medRxiv

Article Title: Loss of Factor H family proteins associates with meningococcal disease severity

doi: 10.1101/2021.02.05.21251142

Figure Lengend Snippet: Serum levels of FH, FHR-1/1, FHR-1/2, FHR-2/2, FHR-3, FHR-4A and FHR-5 at the acute stage of patients who did ( n = 15) or did not ( n = 90) receive renal support. Both surviving and non-surviving patients are included here. Statistical significance was tested using a Mann-Whitney test. Lines depict median and IQR. ****: p < 0.0001; ***: p < 0.001; **: p < 0.01; *: p < 0.05; ns = not significant.

Article Snippet: FHR-1/1 homodimers and FHR-1/2 heterodimers were captured using clone anti-FH.02 (Sanquin Research), and detected with biotinylated anti-FH.02 (Sanquin Research) or anti-FHR-2 (clone MAB5484, R&D Systems, Minneapolis, MN, USA), respectively.

Techniques: MANN-WHITNEY

Journal: Nature Communications

Article Title: Elevated plasma complement factor H related 5 protein is associated with venous thromboembolism

doi: 10.1038/s41467-023-38383-y

Figure Lengend Snippet: a Overview of the VEBIOS ER discovery cohort. 756 HPA antibodies, targeting 408 candidate proteins, were used to analyse plasma samples using affinity proteomics. Log fold changes in antibody MFI (median fluorescent intensity) signal were calculated between VTE cases ( n = 48) and controls ( n = 48) in ( b ) citrate or ( c ) EDTA anticoagulated plasma; coloured circles indicate antibodies that generated signals significantly associated with VTE in both. MFI signals generated by these antibodies for controls ( n = 48) and cases ( n = 48) in ( d ) citrate plasma and ( e ) EDTA plasma. f Immunocapture-mass spectrometry identification of protein targets of HPA059937 ( n = 3 of independent biological replicates). g Dual binder assays were developed using an anti-CFHR5 detection antibody, combined with HPA059937 (raised against SULF1), anti-CFHR5 HPA073894 or anti-CFHR5 HPA072446 as capture antibodies. Monoclonal anti-CFHR5 (MAB3845) was applied as detection antibody in the three combinations. CFHR5 levels in the citrated plasma samples were re-analysed, using the respective dual binder assays, to determine levels (MFI) in controls ( n = 48) vs . cases ( n = 48) and the correlation between the signal and those generated by the original single binder assay using HPA059937 [all p < 1E-04]. Dual binder assay using capture antibody HPA072446 with a recombinant protein standard and MAB3845 as detection antibody, was used for absolute quantification of CFHR5 in samples from: ( h ) VEBIOS ER and ( i ) VEBIOS Coagulation (controls = 135, cases = 142). CFHR5 concentration was measured in controls and cases, with associated OR (odds ratio per 1 standard deviation increase [ h and i , left]) or used to determine the correlation with C-reactive protein (CRP), or D-dimer concentration [ h and i , right panels]. All data in dot plots (1 d – i ) are represented as median value with 95% CI (confidence interval). Case and control groups was compared using a linear model adjusting for age and sex in d , e , g , h and i .***** p < 0.00001, **** p < 0.0001, *** p < 0.001, ** p < 0.01. Two-sided Spearman´s correlation analysis for h and i . For summary statistics see Supplementary data 1 [Tab_2, Panel A] . Source data are provided as a Source Data file ( b – i ).

Article Snippet: IC-MS was performed in triplicate of pooled plasma, as previously described using the HPA059937 antibody (Atlas Antibodies) or MAB3845 (R&D systems, clone:390513) and rabbit or mouse immunoglobulin G (rIgG, AB-105-C [R&D] and PMP01X [Biorad], respectively) as respective negative controls.

Techniques: Clinical Proteomics, Generated, Mass Spectrometry, Recombinant, Quantitative Proteomics, Coagulation, Concentration Assay, Standard Deviation, Control

Journal: Frontiers in Pharmacology

Article Title: Latent Transforming Growth Factor-β Binding Protein-2 Regulates Lung Fibroblast-to-Myofibroblast Differentiation in Pulmonary Fibrosis via NF-κB Signaling

doi: 10.3389/fphar.2021.788714

Figure Lengend Snippet: LTBP2 silencing inhibits TGFβ1-induced differentiation of fibroblasts in vitro . (A) Quantitative RT-PCR and western blot analysis of α-SMA, fibronectin, and collagen I in HFL1 cells transfected with scrambled shRNA or LTBP2 shRNA followed by PBS or TGF-β1 stimulation. GAPDH was detected as the internal control. ** p < 0.01 vs. scr shRNA, # p < 0.05, ## p < 0.01 vs. scr shRNA + TGFβ1 (B) Representative immunofluorescence staining of α-SMA (red) in HFL1 cells. Nuclei are stained with DAPI (blue). Scale bar = 20 μm. (C) Representative immunofluorescence staining of collagen I (red) in HFL1 cells. Nuclei are stained with DAPI (blue). Scale bar = 20 μm. Data are represented as means ± SD, n = 3. Statistical analysis was performed by one-way ANOVA.

Article Snippet: HFL1 cells were pretreated with BAY 11-7082 (MedChem Express, NJ, United States) for 1 h followed by transfection with LTBP2 plasmids.

Techniques: In Vitro, Quantitative RT-PCR, Western Blot, Transfection, shRNA, Control, Immunofluorescence, Staining

Journal: Frontiers in Pharmacology

Article Title: Latent Transforming Growth Factor-β Binding Protein-2 Regulates Lung Fibroblast-to-Myofibroblast Differentiation in Pulmonary Fibrosis via NF-κB Signaling

doi: 10.3389/fphar.2021.788714

Figure Lengend Snippet: LTBP2 silencing suppresses the activation of NF-κB signaling induced by TGFβ1. (A) Representative immunofluorescence staining of phospho-NF-κB p65 (ser536) (red) in HFL1 cells transfected with scrambled shRNA or LTBP2 shRNA followed by PBS or TGF-β1 stimulation. Nuclei are stained with DAPI (blue). Scale bar = 20 μm. (B) Western blot analysis of nuclear NF-κB p65 and nuclear phospho-NF-κB p65 (ser536) in HFL1 cells. Lamin B was detected as the internal control. ** p < 0.01 vs. scr shRNA, # p < 0.05, ## p < 0.01 vs. scr shRNA + TGFβ1. Data are represented as means ± SD, n = 3. Statistical analysis was performed by one-way ANOVA.

Article Snippet: HFL1 cells were pretreated with BAY 11-7082 (MedChem Express, NJ, United States) for 1 h followed by transfection with LTBP2 plasmids.

Techniques: Activation Assay, Immunofluorescence, Staining, Transfection, shRNA, Western Blot, Control

Journal: Frontiers in Pharmacology

Article Title: Latent Transforming Growth Factor-β Binding Protein-2 Regulates Lung Fibroblast-to-Myofibroblast Differentiation in Pulmonary Fibrosis via NF-κB Signaling

doi: 10.3389/fphar.2021.788714

Figure Lengend Snippet: LTBP2 overexpression-induced lung fibroblast-to-myofibroblast differentiation depends on NF-κB phosphorylation and nuclear translocation in vitro . Western blot analysis of α-SMA, fibronectin, and collagen I in HFL1 cells transfected with LTBP2 cDNA and/or BAY 11-7082 or vector. GAPDH was detected as the internal control. ## p < 0.01 vs. vector. * p < 0.05, ** p < 0.01 vs. OE-LTBP2. Data are represented as means ± SD, n = 3. Statistical analysis was performed by one-way ANOVA.

Article Snippet: HFL1 cells were pretreated with BAY 11-7082 (MedChem Express, NJ, United States) for 1 h followed by transfection with LTBP2 plasmids.

Techniques: Over Expression, Phospho-proteomics, Translocation Assay, In Vitro, Western Blot, Transfection, Plasmid Preparation, Control

Journal: Frontiers in Immunology

Article Title: Protective role of complement factor H against the development of preeclampsia

doi: 10.3389/fimmu.2024.1351898

Figure Lengend Snippet: (A, B) Measurement of circulating Factor H (FH) levels in sera of normal (CTRL) and preeclamptic (PE) women, collected during third (A) or first (B) trimester, by ELISA. During both the first trimester and the third trimester lower levels of FH were observed in PE pregnancies compared to CTRL, showing a highly significant difference ( n = 26 PE vs n =31 CTRL sera were compared for third trimester of pregnancy; n = 20 PE vs n = 20 CTRL for first trimester of pregnancy). * p < 0.05, **** p < 0.0001 (t-test). (C, D) Evaluation of FH deposition on placental STBMs via Western blot. STBMs derived from PE ( n = 4) and CTRL ( n = 4) were solubilized and separated by SDS-PAGE under reducing conditions. After transfer, the membrane was probed with anti-FH antibody and IRDye 800CW secondary antibody. Signal intensity was detected using an Odyssey CLx near-infrared scanner (LI-COR Biosciences, Lincoln, NE, USA). Image acquisition, processing and data analysis were performed with Image Studio 5.2 (LI-COR Biosciences). Histograms represent the mean ± SD of two independent experiments performed in duplicate. Beta-actin (ACTB) was used to normalize the results. * p < 0.05 (Mann-Whitney test).

Article Snippet: Human Complement FH DuoSet ELISA kit (#DY4779; R&D Systems, Inc., Minneapolis, Canada) was used to quantify FH in serum samples, following the manufacturer's instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, SDS Page, Membrane, MANN-WHITNEY