Journal: The Journal of Immunology Author Choice

Article Title: A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

doi: 10.4049/jimmunol.1202578

Figure Lengend Snippet: Erythrocytes infected by P. falciparum parasites expressing a DC4-containing PfEMP1 protein adhere to ICAM-1. Surface expression of DC4-containing PfEMP1 proteins on erythrocytes infected by P. falciparum 3D7, BM021, or BM057 before (DC4−) and after (DC4+) repeated selection for reactivity with DC4-specific antisera (A). The ability of these IEs to bind to wild-type (CHO-wt), CD36-transfected (CHO-CD36), and ICAM-1–transfected (CHO-ICAM1) CHO cells before (−) and after (+) selection for surface expression of DC4 (3D7, BM021, and BM057) or the DC4-negative PfEMP1 PF11_0008 (3D7 only) (B). Mean adhesion (three independent experiments) relative to the adhesion of PDF1235w+ 3D7-IEs to ICAM-1-transfected CHO cells in (B) is shown. Error bars indicate SD.

Article Snippet: Recombinant ICAM-1–Fc chimera (0.2 μg/well) was added (1 h; room temperature), followed by washing and detection of bound chimera by anti-human HRP-IgG (1:3000 in blocking buffer; 1 h) or by mouse anti-human ICAM-1 (clone RR1/1; AH Diagnostics; 1:400) followed by anti-mouse HRP-IgG (DakoCytomation; 1:1000) ( 39 ).

Techniques: Infection, Expressing, Selection, Transfection

Journal: The Journal of Immunology Author Choice

Article Title: A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

doi: 10.4049/jimmunol.1202578

Figure Lengend Snippet: Binding of recombinant PfEMP1 domains to ICAM-1. ICAM-1 binding of recombinant PFD1235w domains (A). Concentration-dependent ICAM-1 binding of recombinant PfEMP1 domains within (filled symbols) or outside (open symbols) DC4 (B). Concentration-dependent inhibition of PFD1235W-DBLβ3_D4 binding to ICAM-1 by recombinant PfEMP1 domains within (filled symbols) or outside (open symbols) DC4 (C). Error bars (A) indicate SD. Data are representative of a minimum of three independent experiments.

Article Snippet: Recombinant ICAM-1–Fc chimera (0.2 μg/well) was added (1 h; room temperature), followed by washing and detection of bound chimera by anti-human HRP-IgG (1:3000 in blocking buffer; 1 h) or by mouse anti-human ICAM-1 (clone RR1/1; AH Diagnostics; 1:400) followed by anti-mouse HRP-IgG (DakoCytomation; 1:1000) ( 39 ).

Techniques: Binding Assay, Recombinant, Concentration Assay, Inhibition

Journal: The Journal of Immunology Author Choice

Article Title: A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

doi: 10.4049/jimmunol.1202578

Figure Lengend Snippet: Phylogeny of ICAM-1–binding and –nonbinding DBLβ domains. Maximum-likelihood phylogram of 16 ICAM-1–binding DBLβ domains (red) and 19 DBLβ domains not binding to ICAM-1 (blue). The shaded area indicates DBLβ domains in DC4. The edge numbers indicate the bipartition bootstrap support (%), whereas the UpsA, UpsB, or UpsC group that each gene belongs to is indicated by an uppercase letter within a box next to the gene names. Scale bar indicates amino acid substitutions per site.

Article Snippet: Recombinant ICAM-1–Fc chimera (0.2 μg/well) was added (1 h; room temperature), followed by washing and detection of bound chimera by anti-human HRP-IgG (1:3000 in blocking buffer; 1 h) or by mouse anti-human ICAM-1 (clone RR1/1; AH Diagnostics; 1:400) followed by anti-mouse HRP-IgG (DakoCytomation; 1:1000) ( 39 ).

Techniques: Binding Assay

Journal: The Journal of Immunology Author Choice

Article Title: A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

doi: 10.4049/jimmunol.1202578

Figure Lengend Snippet: Mapping the ICAM-1–binding site in PFD1235w-DBLβ3_D4. The modeled structure of the PFD1235w domains DBLβ3_D4 and DBLβ3_D5, showing the N-terminal (orange), central (magenta), and C-terminal (green) parts of the domains (A). Schematic representation of the PFD1235w domains DBLβ3_D4 (gray) and DBLβ3_D5 (white) and hybrids (H1–H6) of their N-terminal (A), central (B), and C-terminal (C) parts (B). A version of DBLβ3_D4 truncated at the C-terminal end (T1) is also shown in (B). ICAM-1 binding of PFD1235w-DBLβ3_D4 (D4, T1), PFD1235w-DBLβ3_D5 (D5), and their hybrids (H1–H6) (C). The ability of PFD1235w-DBLβ3_D4, PFD1235w-DBLβ3_D5, and their hybrids (H1–H6) to inhibit the ICAM-1 binding of PFD1235w-DBLβ3_D4 (D), Dd2var32-DBLβ3_D4 (E), and IT4var16-DBLβ5_D4 (F). The reactivity of antisera specific for hybrids H3, H5, and H6 with erythrocytes infected by PFD1235w+ IEs (G). Data are representative of a minimum of three independent experiments.

Article Snippet: Recombinant ICAM-1–Fc chimera (0.2 μg/well) was added (1 h; room temperature), followed by washing and detection of bound chimera by anti-human HRP-IgG (1:3000 in blocking buffer; 1 h) or by mouse anti-human ICAM-1 (clone RR1/1; AH Diagnostics; 1:400) followed by anti-mouse HRP-IgG (DakoCytomation; 1:1000) ( 39 ).

Techniques: Binding Assay, Infection

Journal: The Journal of Immunology Author Choice

Article Title: A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

doi: 10.4049/jimmunol.1202578

Figure Lengend Snippet: PFD1235W-DBL3β_D4-specificity of IE adhesion-inhibitory Abs. The ability of pooled immune serum to inhibit binding of PFD1235w-DBL3β_D4 to ICAM-1 before (−) and after (+) depletion of Abs reactive with PFD1235w-DBL3β_D4 (D4), PFD1235w-DBL3β_D5 (D5), or hybrids thereof (H3, H4). The subdomain composition of the recombinant constructs is as in Fig. 6. Data are representative of a minimum of three independent experiments.

Article Snippet: Recombinant ICAM-1–Fc chimera (0.2 μg/well) was added (1 h; room temperature), followed by washing and detection of bound chimera by anti-human HRP-IgG (1:3000 in blocking buffer; 1 h) or by mouse anti-human ICAM-1 (clone RR1/1; AH Diagnostics; 1:400) followed by anti-mouse HRP-IgG (DakoCytomation; 1:1000) ( 39 ).

Techniques: Binding Assay, Recombinant, Construct

Journal: The Journal of Immunology Author Choice

Article Title: A Novel Domain Cassette Identifies Plasmodium falciparum PfEMP1 Proteins Binding ICAM-1 and Is a Target of Cross-Reactive, Adhesion-Inhibitory Antibodies

doi: 10.4049/jimmunol.1202578

Figure Lengend Snippet: Cross-reactivity and acquisition of Abs to DC4-containing PfEMP1 proteins. ELISA reactivity of pooled immune plasma IgG with PfEMP1 proteins (coating Ag) containing (red boxes) or not containing DC4 after depletion of reactivity with the same proteins (competing Ag). The reduction in reactivity by depletion is indicated by shading: black (>75%), dark gray (51–75%), light gray (26–50%), and white (0–25%) (A). Ab-mediated inhibition of ICAM-1 binding of DBLβ3 domains from PfEMP1 proteins containing DC4 (PFD1235w, BM021, BM028, BM048, BM057, and BM066) or not containing DC4 (Dd2var32, IT4var16) (B). The inhibitory capacity of an immune plasma pool, Abs from this pool purified on PFD1235w-DBLβ3_D4, Dd2var32-DBLα1.7-DBLβ1, IT4var16-DBLβ5, or PFD1235w-DBLβ3_D5, respectively, as well as a nonimmune plasma pool (Hu IgG) are shown. The inhibitory capacity is indicated by shading as in (A). The ability of plasma samples from P. falciparum–exposed children aged 2–4 and 5–9 y to interfere with binding of PFD1235w-DBLβ3_D4 to ICAM-1 (C). Binding relative to binding in the absence of Abs is shown for one experiment representative of a minimum of three independent experiments. Medians (center line), central 50% (shaded boxes), central 80% (whiskers), and outliers are indicated.

Article Snippet: Recombinant ICAM-1–Fc chimera (0.2 μg/well) was added (1 h; room temperature), followed by washing and detection of bound chimera by anti-human HRP-IgG (1:3000 in blocking buffer; 1 h) or by mouse anti-human ICAM-1 (clone RR1/1; AH Diagnostics; 1:400) followed by anti-mouse HRP-IgG (DakoCytomation; 1:1000) ( 39 ).

Techniques: Enzyme-linked Immunosorbent Assay, Inhibition, Binding Assay, Purification