Prophylactic administration schedule for 6F6 in <i>cfh−/−</i> mice.

<p>Prophylactic administration schedule for 6F6 in <i>cfh−/−</i> mice.</p

Total Aβ 1–42 levels in <i>cfh−/−</i> mouse sera after prophylactic administration regime.

<p>Concentration of total Aβ 1–42 in serum samples treated systemically are shown as geometric means with standard deviation, (A) and with 95% Confidence Intervals from statistical analysis (B). Data is shown at the end of the prophylactic regime, (6 months, after 3 months treatment), for n = 5 mice per treatment group, 6F6 dosed unless stated. Note the substantial increases in serum total Aβ 1–42 after 6F6 dosing, (see text for details)<b>.</b> Statistical significance, p<0.0001, (FDR adjusted), was reached for all doses of 6F6 over PBS controls, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065518#pone.0065518.s013" target="_blank">Table S7</a>.</p

Primary antibodies used for IHC detection in <i>cfh−/−</i> mouse retinae.

<p>Primary antibodies used for IHC detection in <i>cfh−/−</i> mouse retinae.</p

Retinal imaging and immunohistochemical analysis of 12 month old <i>cfh−/−</i> mice.

<p>(A) Scanning laser ophthalmoscope image of the retinae: Large clusters of hyperfluorescent foci (showing as autofluorescent point sources are observed. (B–D) Immunohistochemically labelled sections of the fluorescent debris/deposits, the majority of which may be containined within macrophages, often occur simultaneously on either side of the RPE. (B) acts as a negative control for Aβ detection by IHC (background signal from an isotype specific control antibody). The signal for fluorescent debris, (yellow), was dampened by use of Sudan Black in all cases. RPE-associated foci, show cross-reactivity, (green, highlighted with white arrows), to activated complement C3, (C3b+, detected with clone 2/11, #HM1065, Hycult Biotech, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065518#pone-0065518-t003" target="_blank">Table 3</a>), (C), and Aβ, (4G8+), (D). A- scalebar = 200 µm; B–D scalebar = 25 µm. Blue label is DAPI, (4', 6-diamidino-2-phenylindole), a nuclear stain. CHO = choroid, RPE = retinal pigment epithelial cells.</p

Longitudinal study of Aβ deposition along Bruch’s membrane in <i>wt</i> and <i>cfh−/−</i> mice.

<p>Aβ positive staining, (4G8+), is highlighted in green along the boundary of the retinal pigment epithelial, cells and Bruch’s membrane, (highlighted with a green arrow head), at the base of the retinae in the cfh−/− mouse eye. Aβ deposition increases with time in both <i>cfh–/−</i>, (B) 3 months, (D), 6 months and (F) 12 months, and the age­matched control (<i>wt</i>), retinae, (A) 3 months, (C), 6 months and (E) 12 months. Aβ deposition in <i>cfh−/–</i> mouse is elevated compared to wild type (<i>wt</i>) at similar ages. Note that there are some autofluorescent deposits, (yellow), interspersed below the RPE with the 4G8+ staining in the aged cfh−/− samples: (D) & (F). Blue label is DAPI, (4′,6-diamidino-2-phenylindole), a nuclear stain. Scalebar = 25 µm.</p

Secondary antibodies used for IHC detection in <i>cfh−/−</i> mouse retinae.

<p>Secondary antibodies used for IHC detection in <i>cfh−/−</i> mouse retinae.</p

Summary of peripheral amyloid beta detection assays.

<p>Summary of peripheral amyloid beta detection assays.</p

Immunohistochemical analysis of <i>cfh−/−</i> mice retinae of after the therapeutic regime.

<p>Aβ (4G8+, red) and activated complement C3 (detected with clone 2/11+, #HM1065, Hycult Biotech., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065518#pone-0065518-t003" target="_blank">Table 3</a>, green), cross reactivity to retinal sections at 9 months, (after 3 months dosing): (A) negative control (PBS treated), 6F6, at 60 µg dose (B) and 600 µg dose (C). Scalebar = 25 µm), PR = phototreceptors/outer segments, RPE = retinal pigment epithelium, CHO = choroid, DAPI = 4',6-diamidino-2-phenylindole. Left hand panel (A), (B), (C) is stained with DAPI, right hand panel (A), (B), (C), is without DAPI staining. Aβ (D), and activated complement C3, (E), deposition in the RPE/Bruch’s membrane of <i>cfh−/−</i> mice, are plotted as differences from vehicle control with 95% confidence intervals (CI) after therapeutic treatment. Data in graphs (D) and (E) show data at an intermediate time point, after 7 months of age (7MO), 1 month (+4w) after treatment as well as at the end of the regime at 9 months of age (9MO), 3 months (+12w) after treatment. Data in graphs (D) and (E) also shows wild type (WT) mice treated with 6F6 (WT6F6) after 7 months of age (7MO), 1 month (+4w) after treatment. Mice dosed with 6F6 show a dose dependent significant reduction, (asterisks, see text for levels of significance), in Aβ deposition at 60 µg and 600 µg and in activated C3 deposition at 600 µg compared compared to those treated with vehicle, (D) at 9 months of age (9MO), 3 months (+12w) after treatment.</p

GSK933776 plasma pharmacodynamics.

<p>A) Geometric mean plasma Aβ concentration–time plots over the three dosing intervals (semi-log plot). Plasma levels of total Aβ42 and Aβ increased whereas plasma levels of free Aβ decreased in dose-dependent manner. Peak:trough ratios for Aβ decreased with increasing dose of GSK933776. B) Week 12 ratio to baseline for CSF Aβ (Aβ1–42 and AβX–42) concentrations. Presented as individual values and mean (95%CI). There were no significant changes from baseline for Aβ1–42 or AβX–42.</p

GSK933776 plasma pharmacokinetics.

<p>Time-course of plasma concentrations of GSK933776 by dose: medians (lines) and individual data (dots). LLQ is 100 ng/mL for the 0.1 mg/kg dose and 5 μg/mL for the 1, 3, and 6 mg/kg doses. SD = single dose; RD = repeat dose. Maximum plasma concentrations increased with dose.</p