Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Sequences of the primers used for reverse transcription polymerase chain reaction.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Reverse Transcription, Sequencing

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: The expression of CCL3 and its receptors in cornea after alkali injury. A : Semi-quantitative RT–PCR was performed to assess mRNA expression of CCL3 and its receptors, CCR1 and CCR5 , and the ratios of target gene expression to β-actin were determined. All values represent the mean±SEM of three to five independent measurements. B : Whole eyes were obtained at 0, 2, 4, and 7 days after alkali injury and processed for immunohistochemical analysis using anti-CCL3 (upper panels) or anti-CCR1 antibodies (lower panels). Representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Targeted Gene Expression, Immunohistochemical staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Alkali-induced corneal neovascularization and macrophage infiltration. A : The macroscopic appearances of WT, CCL3-KO, CCR1-KO, and CCR5-KO mouse eyes two weeks after alkali injury are illustrated. Representative results from at least 10 animals in each group are shown here. B : Corneal tissues were obtained from WT, CCR1-KO, and CCL3-KO mice two weeks after the injury. Tissues were stained with hematoxylin and eosin (left panels) or immunostained with anti-CD31 antibodies (right panels), and representative results from five individual animals are shown. Original magnifications, 400X. Scale bar, 50 μm. C : CNV numbers per mm 2 in hot spots (upper panel) and % CNV areas in hot spots (lower panel) were determined on corneas obtained from WT or KO mice two weeks after the injury. Each value represents the mean and SEM (n=5 animals). An asterisk represents a p<0.05 and that the value was obtained comparing WT and CCL3-KO mice. D : The numbers of infiltrated F4/80 positive macrophages were determined two and four days after the injury. Each value represents the mean and SEM (n=5). The double asterisk indicates a p<0.01 and that the value was obtained comparing WT and CCL3-KO mice.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Angiogenic factor expression. A : RT–PCR analysis of pro-angiogenic and anti-angiogenic gene expressions in the injured corneas of WT and CCL3-KO mice. RT–PCR analysis was performed on total RNAs extracted from eyes 0, 2, 4, and 7 days after alkali injury, and then the ratios of VEGF to β-actin, bFGF to β-actin, and TSP-1 to β-actin of WT (black bars) and CCL3-KO mice (open bars) were determined. All values represent the mean and SEM (n=3-5 animals). The asterisk denotes a p<0.05; the hash mark denotes a p<0.01 and that the value was obtained comparing WT and KO mice. The effects of CCL3 on VEGF expression by murine peritoneal macrophages is shown in B and C . B : RT–PCR was performed on macrophages incubated with the indicated concentrations of CCL3 for 12 h, and the ratio of VEGF to β-actin was calculated. Each value represents the mean and SEM (n=3). C : Murine macrophages were stimulated with either 0, 10, or 100 ng/ml of CCL3 for 24 h. VEGF concentrations in the supernatants were determined with ELISA as described in Methods. The representative results from three independent experiments are shown. The asterisk denotes a p<0.05 and the double asterisk denotes a p<0.01 when compared to untreated.

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: Intracorneal CCR5 positive cell infiltration. A : A double-color immunofluorescence analysis of CCR5-expressing cells is illustrated. Corneas were obtained from WT mice 0 and 4 days after the injury. The samples were immunostained with a combination of anti-CCR5 and anti-CCR2 antibodies as described in Methods and observed with fluorescence microscopy (original magnification, 400X). Signals were digitally merged in the right panels. Arrows indicate the double, positively stained cells. Representative results from three independent experiments are shown. B : Corneal tissues from WT mice (left panel) or CCL3-KO mice (right panel) obtained four days after the injury were stained with anti-CCR5 Ab. Scale bar, 100 μm. C : The numbers of intracorneal CCR5 positive cells four days after the injury were determined as described in Methods, and the mean and SEM are shown here (n=5).

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques: Immunofluorescence, Expressing, Fluorescence, Microscopy, Staining

Journal: Molecular Vision

Article Title: Essential contribution of CCL3 to alkali-induced corneal neovascularization by regulating vascular endothelial growth factor production by macrophages

doi:

Figure Lengend Snippet: The effects of topical CCL3 application on corneal neovascularization. A : Macroscopic appearances of WT, CCL3-KO mice, and CCL3-KO mice topically applied with CCL3 two weeks after alkali injury are shown. Representative results from five animals from each group are shown here. B : Corneal tissues were obtained two weeks after the injury from WT, CCL3-KO, and CCL3-KO mice topically applied with CCL3 and were immunostained with anti-CD31 antibodies. Representative results from five individual mice from each group are shown. Original magnification, 400X. Scale bar, 50 μm. C : The CNV numbers per mm 2 in hot spots (left panel) and % CNV areas in hot spots (right panel) were determined. Each value represents the mean and SEM (n=5 animals). D : The number of infiltrated F4/80 positive macrophages was determined on WT, CCL3-KO, and CCL3-KO, which were all treated with CCL3, two days after the injury. Each value mean represents both the mean and SEM (n=5). The asterisk denotes a p<0.05, and the double asterisk means a p<0.01 when compared with CCL3-KO (this applies to both C and D ).

Article Snippet: Recombinant CCL3/MIP-1α (270-LD) and goat anti-mouse CCL3 antibodies were obtained from R&D Systems (Minneapolis, MN).

Techniques:

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 1. Binding and in vitro activity of murine 18V4F hybridoma antibody. (A) ELISA binding of original 18V4F hybridoma antibody to a panel of chemokines (determined in triplicate, shown as mean +/2 standard deviation). Directly coated chemokines were used here for direct comparisons, however in other experiments CCL3 showed a significantly enhanced signal when biotinylated and coated on streptavidin plates. (B) Chemotaxis inhibition by 18V4F hybridoma antibody of CCR5-transfected Ba/F3 cells to 5 ng/mL of CCL3, CCL4, and CCL5. Data are representative of at least three similar experiments. All chemotaxis data are represented as a percent of maximum migration in the absence of inhibitors and is fit using a standard four parameter dose-response model (GraphPad). doi:10.1371/journal.pone.0043332.g001

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Binding Assay, In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Chemotaxis Assay, Inhibition, Transfection, Migration

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 2. Diagram of phage display selection strategy. Individual CDR libraries were sequentially panned against CCL3, CCL4 and CCL5. In step 1, phage libraries were combined with biotinylated CCL3 and bound to streptavidin beads. Bound phage were eluted, amplified, and subjected to panning against biotinylated CCL4 and CCL5 in steps 2 and 3, respectively. This process was repeated 4–5 times with increasing stringency to yield sequences with improved affinities. doi:10.1371/journal.pone.0043332.g002

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Selection, Amplification

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 3. Chemotaxis inhibition by affinity matured 18V4F variants. Chemotaxis inhibition by humanized 18V4F Fab, d5 variant, d7 variant, d5d7, and a negative control Fab of CCR5-transfected Ba/F3 cells to 5 ng/mL of (A) CCL3, (B) CCL4, and (C) CCL5. Data are representative of at least two similar experiments. A loss in potency of humanized 18V4F Fab was observed compared with the 18V4F hybridoma shown in Figure 1b and is likely a result of both the humanization process and loss in avidity caused by switching from full IgG to Fab fragment. doi:10.1371/journal.pone.0043332.g003

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Chemotaxis Assay, Inhibition, Variant Assay, Negative Control, Transfection

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 4. Comparison of vCCI and d5d7 binding epitopes. Competitive binding ELISA examining molecules that can disrupt the d5d7-CCL3 binding interaction using d5d7 as a homologous competitor and vCCI-Fc, commercial anti-CCL3 antibody, and control IgG as heterologous competitors. Data are representative of at least two similar experiments. Competition experiments were also completed to analyze the d5d7-CCL4 and d5d7-CCL5 binding interactions and similar binding competition was observed between d5d7 and vCCI-Fc (data not shown). doi:10.1371/journal.pone.0043332.g004

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 5. Inhibition of chemotaxis induced with mixtures of chemokines by MAb d5d7. Inhibition of chemotaxis of (A) CCR5 transfectants to a pool of recombinant CCL3, CCL4, and CCL5 and (B) CCR1 transfectants to a pool of CCL3 and CCL5, by MAb d5d7 antibody, vCCI-Fc, individual commercial anti-chemokine antibodies (anti-CCL3, anti-CCL4, and anti-CCL5), and IgG controls. Chosen chemokine concentrations were those that produced 50% maximal chemotaxis when tested individually (a pool of 3 ng/mL CCL3, 10 ng/mL CCL4, and 3 ng/mL CCL5 was used in CCR5 experiments and a mixture of 20 ng/ mL CCL3 and 5 ng/mL CCL5 was used in CCR1 experiments). doi:10.1371/journal.pone.0043332.g005

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Inhibition, Chemotaxis Assay, Recombinant, Produced

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 8. SCID-hu mouse model of leukocyte migration. (A) NSG (NOD/SCID/IL2r-cnull) mice were injected i.v. with human PBMC and allowed to engraft for 10 d. MAb d5d7 was administered i.v. just before chemokines were injected s.c. in Matrigel. After 7 d the skin sites were harvested and single cell suspensions were generated. Human leukocytes were tagged with specific antibodies and analyzed by flow cytometry. (B) Inhibition by MAb d5d7 of skin leukocyte migration into chemokine-embedded Matrigel plugs in NSG mice engrafted with human PBMC. The negative control group consisted of animals treated with s.c. injection of Matrigel + PBS and i.v. administration of control IgG. All other groups had s.c. injections of Matrigel containing CCL3, CCL4, and CCL5 (400 ng each) with i.v. administration of PBS, control IgG, or MAb d5d7 antibody. Data were analyzed using a student t test. doi:10.1371/journal.pone.0043332.g008

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Migration, Injection, Generated, Flow Cytometry, Inhibition, Negative Control, Control