Journal: Pathogens

Article Title: Relationship Between Cell Surface Viral Glycoprotein Expression and Resistance of Parainfluenza Virus Persistently Infected Cells to Complement-Mediated Lysis

doi: 10.3390/pathogens14080815

Figure Lengend Snippet: C′ regulators are not significantly upregulated in H1975 and HEp2 PI cells. ( A , B ) RNA samples from H1975 ( A ) and HEp2 ( B ) cells were collected as described in the legend to . Samples were analyzed via RT-qPCR to determine gene expression of three C′ regulators: CD46, CD55, and CFH. Values were normalized to expression of β-actin. C′ inhibitor expression in mock-infected cell cultures was set equal to 1 and expression in AI and PI cel cultures is expressed as fold change relative to mock. ( C ) Mock-infected, AI, and PI HEp2 cells were stained with an anti-CD55 antibody and processed via flow cytometry. MFI; Mean Fluorescence Intensity. ns stands for not significant, **** indicates p -value < 0.0001.

Article Snippet: CD55 surface expression was monitored using an AF405 conjugated anti-human CD55 antibody (R&D Systems, FAB20091V).

Techniques: Quantitative RT-PCR, Gene Expression, Expressing, Infection, Staining, Flow Cytometry, Fluorescence

Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: ADR induces PLAD-dependent cleavage of DAF. (A and B) Representative images (A) and distribution of DAF expression (B) quantified in hiPod exposed to vehicle, ADR (0.3 µg/ml), PLADi (1 µM), or ADR + PLADi (0.3 µg/ml in 1 µM) for 24 h. DAF IF signal was normalized to actin expression pixel by pixel, and the MFI for each cell was computed. Results are representative of two independent experiments with similar results. (C and D) Representative blots (C) and densitometric analysis (D) of PLAD expression in hiPod cell lysates previously exposed to vehicle or ADR for 24 h. (E and F) Representative blots (E) and densitometric analysis (F) of DAF in the supernatants of hiPod exposed to ADR for 24 h with or without PLADi (WB). (G) Representative blot of DAF in the urine from BALB/c male mice at 2 wk after treatment with vehicle or ADR compared with recombinant mouse DAF (rDAF). In each group, we pooled and concentrated urine samples from eight mice (see Materials and methods). All experimental data were verified in at least three independent experiments. *P < 0.05; n.s., not significant. Scale bars: 50 µm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Expressing, Recombinant

Journal: The Journal of Experimental Medicine

Article Title: Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis

doi: 10.1084/jem.20191699

Figure Lengend Snippet: FSGS in humans is associated with DAF down-regulation and complement activation. (A–D) C3 (A), C3aR (B), C5aR (C), and DAF mRNA (D) expression in glomeruli of human biopsy specimens with pathological diagnosis of FSGS or diabetic kidney disease compared with normal kidneys. Data are from previously published microarray studies by and were subjected to further analysis using Nephroseq. (E–H) Representative renal staining and data quantification for C3d (IF; E and F) and DAF (immunohistochemistry; G and H) in patients with FSGS ( n = 18) and in kidneys from healthy renal donors ( n = 10). (I) Correlation between protein and C3a in urine samples from 27 patients with FSGS taken at the time of kidney biopsy (before therapy). (J and K) Differences in proteinuria (J) and urinary C3a (K) measured before versus 3–6 mo after steroid therapy in a subset of 13 patients with FSGS. (L) Correlation between the change in proteinuria and change in urinary C3a before and after therapy for each of the same 13 patients. (M) Representative blot of DAF in the urine from healthy control individuals and patients with FSGS compared with recombinant human DAF (rDAF). In each group, we pooled and concentrated urine samples from five and five subjects, respectively (see Materials and methods). *P ≤ 0.05. Scale bars: 25 μm. Error bars are SEM.

Article Snippet: The urine from mouse and human samples was separated on 4–20% precast Protean TGX gels (Bio-Rad Laboratories) with recombinant mouse (55 μg/ml, 5490-CD-050; R&D Systems) or human (15 μg/ml, SRP6437; Sigma-Aldrich) CD55 protein, respectively.

Techniques: Activation Assay, Expressing, Biomarker Discovery, Microarray, Staining, Immunohistochemistry, Control, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy.

doi: 10.4049/jimmunol.175.9.6219

Figure Lengend Snippet: FIGURE 1. TaqMan RT-PCR amplification plots of DAF/CD55. A and C, DAF1 (A) and DAF2 (C) expression in skeletal muscle (M. quad- riceps) of a 30-wk-old SJL/J mouse compared with a C57BL/6 control mouse of the same age (Ctrl). E, DAF/CD55 expression in skeletal mus- cle of a patient with LGMD2B (patient 4, Table III) compared with a healthy control. Housekeep- ing genes porphobilinogen deaminase (B and D) and 2-microglobulin (F) were used as internal standards.

Article Snippet: Murine DAF was detected with polyclonal rat anti-mouse Ab (MDI) (17); human CD55 was detected with SM1141PS (Acris Antibodies).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy.

doi: 10.4049/jimmunol.175.9.6219

Figure Lengend Snippet: FIGURE 2. DAF/CD55 protein expression in dysferlin deficiency. Im- munofluorescence staining using anti-DAF/CD55 Abs. A–D, Murine skel- etal muscle. A–D have the same scale as indicated in B. E and F, Murine cardiac muscle. E and F have the same scale as indicated in F. G and H, Human skeletal muscle. G and H have the same scale as indicated in H. A, SJL/J, wk 28; B, A/J wk 16; C, Dysf /, wk 16; D, C57BL/6; E, SJL/J; F, C57BL/6; G, LGMD2B patient 2 (Table III); H, control skeletal muscle without detectable neuromuscular disorder.

Article Snippet: Murine DAF was detected with polyclonal rat anti-mouse Ab (MDI) (17); human CD55 was detected with SM1141PS (Acris Antibodies).

Techniques: Expressing, Staining, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Increased susceptibility to complement attack due to down-regulation of decay-accelerating factor/CD55 in dysferlin-deficient muscular dystrophy.

doi: 10.4049/jimmunol.175.9.6219

Figure Lengend Snippet: FIGURE 3. Complement lysis assay and binding of C5b9-MAC to nonnecrotic muscle cells. A, Quantification of PI uptake of myotubes after exposure to complement (ratio of PI-posi- tive cells after exposure to complement to Vero- nal buffer control). n, number of wells counted. Normal human (B and C) and dysferlin-deficient (D and E) human myoblasts after exposure to complement (B and D) and after preincubation with anti-CD55 Ab and subsequent exposure to complement (C and E). F and G, Serial sections of quadriceps muscle in LGMD2B (patient 1), demonstrating dystrophic changes with increase in connective tissue and pathological variation in fiber size (Gomori-TriChrome stain). There was sarcolemmal expression of C5b9-MAC on non- necrotic muscle fibers. Staining was performed with anti-C5b9 mAb and Cy3-labeled donkey anti-mouse Ab.

Article Snippet: Murine DAF was detected with polyclonal rat anti-mouse Ab (MDI) (17); human CD55 was detected with SM1141PS (Acris Antibodies).

Techniques: Lysis, Binding Assay, Control, Staining, Expressing, Labeling