Journal: bioRxiv

Article Title: Early Binding of Anti-Amyloid Antibodies to CAA Drives Complement Activation, Inflammation and ARIA in Mice

doi: 10.64898/2026.03.04.709591

Figure Lengend Snippet: a, Schematic of anti-Aβ immunization with 500 µg/wk 3D6 IgG2a mAb and an IgG2a isotype control for 13 weeks from 17 to 20 months in aged male APP/PS1dE9;hAPOE4 AD-like mouse model. For baseline data, baseline control mice were used. For spontaneous hemorrhage detection, 1x PBS-injected aged controls were used. b, T2*-weighted FLASH MRI of 20-month-old male APP/PS1dE9;hApoE4 mice after 13 weeks of passive immunization with 3D6 mAb (500 µg/week), showing cerebral microbleeds as hypointense spots (arrowheads), compared to 1× PBS–treated aged controls. c, Frequency of cerebral microbleeds detected by T2* MRI in 3D6–treated (n = 3) and PBS-treated (n = 4) mice. d, Quantification of Prussian blue–positive (% hemosiderin-stained) area in IgG2a and 3D6–treated mice. Unpaired t-test: **p < 0.005, ***p < 0.0005. Data represent mean ± SEM. e, Representative Prussian blue–stained sections showing hemosiderin deposits (arrowheads, with selected examples in insets) in cortex, hippocampus, thalamus, and cerebellum from 1× PBS (n=4), IgG2a isotype control (n = 5), and 3D6 IgG2a–treated mice (n = 5). Scale bar: 200 µm. f, Immunofluorescence staining in the cerebellum showing 3D6 mAb localization (anti-Ms IgG2a) and C1q deposition at sites of vascular and parenchymal amyloid labeled with Amylo-Glo (AG). g, Detection of activated complement fragments C3b/iC3b/C3c at vascular amyloid sites associated with 3D6 mAb. h, Localization of membrane attack complex (MAC/C5b-9) along vascular amyloid (arrows) and plaques (asterisk) in 3D6–treated mice. Minimal complement staining is observed in IgG2a-treated controls. Scale bars: f–h, 25 µm.

Article Snippet: Following washes in PBS, sections were blocked either in 10% normal goat serum (Gibco, #16210064), or heat inactivated goat serum (Novus Biologicals #S13110H), or normal donkey serum (Sigma, #S30-M) for 1 hour at room temperature followed by overnight 4°C incubation with primary antibodies against C1q (1:500, Abcam #ab182451), C3b/iC3b/C3c (1:300, HycultBiotech #HM1078), C5b-9 (1:300, Bioss #bs-2673R), CD31 (1:500, R&D Systems #AF3628), C3 (1:200, HycultBiotech #HM1045), GFAP (1:1000, Abcam # ab5804), MMR/CD206 (1:300,R&D #AF2535) MMP-9 (1:500, Bioss #bs-0397R), Iba-1 (1:1000; Dako, Wako #019-19741), Iba-1 (1:500, Synaptic Systems, #234308), CD68 (1:500, Bio-Rad #MCA1957), S97 Aβ (pan-Aβ rabbit polyclonal, 1:2000, gift Dr. Dominic Walsh, BWH, USA), CD31 (1:500, R&D #AF3628), Collagen-IV (1:300, Abcam #6586), Alexa Fluor 488 anti-β-Amyloid, aggregated antibody (1: 200, clone A17171C, Biolegend # 856508), Von Willebrand Factor Antibody (1:500, Novus Biologicals #NB600-586).

Techniques: Control, Injection, Staining, Immunofluorescence, Labeling, Membrane

Journal: bioRxiv

Article Title: Early Binding of Anti-Amyloid Antibodies to CAA Drives Complement Activation, Inflammation and ARIA in Mice

doi: 10.64898/2026.03.04.709591

Figure Lengend Snippet: a, Representative immunofluorescence staining in the cerebellum area showing activated C3 fragments (C3b/iC3b/C3c) and TCC/MAC complex (C5b-9) along with fibrillar amyloid (Amylo-Glo dye, AG) in IgG2a isotype and 3D6 mAb passively immunized mice. Arrows indicate iC3b or C5b-9 immunoreactivity along CAA + vessels stained with AG. Scale bar 50 µm. Quantification of % area immunoreactivity for b, iC3b c, C5b-9 per brain section. Approximately 22 to 25 Amylo-Glo + CAA vessels were outlined to quantify d, CAA + iC3b + colocalization & e, CAA + C5b9 + colocalization area. f, Serum TCC/MAC levels measured by ELISA g, % CAA estimated by AG + CD31 + colocalization data. h, Representative double IHC staining for iC3b (DAB), S97 Aβ (Vector red), with Prussian blue combination for microhemorrhages detection in the 3D6 treated mice showed iC3b-associated microhemorrhages in leptomeningeal or penetrating vessels (arrows) and signs of vascular amyloid clearance (arrowheads) in vessels with complement deposition. In IgG2a-treated mice, vascular amyloid/plaque staining is prominent with minimal complement and Prussian blue staining (arrows), Scale bar=20 µm. N=5/group, Parametric unpaired t-test, *p<0.05 and **p<0.005, ****p<0.00005. Data are expressed as mean ± SEM.

Article Snippet: Following washes in PBS, sections were blocked either in 10% normal goat serum (Gibco, #16210064), or heat inactivated goat serum (Novus Biologicals #S13110H), or normal donkey serum (Sigma, #S30-M) for 1 hour at room temperature followed by overnight 4°C incubation with primary antibodies against C1q (1:500, Abcam #ab182451), C3b/iC3b/C3c (1:300, HycultBiotech #HM1078), C5b-9 (1:300, Bioss #bs-2673R), CD31 (1:500, R&D Systems #AF3628), C3 (1:200, HycultBiotech #HM1045), GFAP (1:1000, Abcam # ab5804), MMR/CD206 (1:300,R&D #AF2535) MMP-9 (1:500, Bioss #bs-0397R), Iba-1 (1:1000; Dako, Wako #019-19741), Iba-1 (1:500, Synaptic Systems, #234308), CD68 (1:500, Bio-Rad #MCA1957), S97 Aβ (pan-Aβ rabbit polyclonal, 1:2000, gift Dr. Dominic Walsh, BWH, USA), CD31 (1:500, R&D #AF3628), Collagen-IV (1:300, Abcam #6586), Alexa Fluor 488 anti-β-Amyloid, aggregated antibody (1: 200, clone A17171C, Biolegend # 856508), Von Willebrand Factor Antibody (1:500, Novus Biologicals #NB600-586).

Techniques: Immunofluorescence, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Plasmid Preparation

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identification of potent siRNA targeting complement C5 and its robust activity in pre-clinical models of myasthenia gravis and collagen-induced arthritis

doi: 10.1016/j.omtn.2023.01.005

Figure Lengend Snippet: Treatment with mouse/rat C5-siRNA/LNP suppresses disease symptoms and loss of neuromuscular junctions in experimental MG model in rats Disease severity (A), body weight change (B), hemolysis (C), and complement C5 expression levels in the liver (D) of MG rats (n = 3/group) treated with PBS, Ctrl-siRNA/LNP, or 0.01, 0.03, or 0.1 mg/kg of mouse/rat (m/r) C5-siRNA/LNP are shown. (A) Rats received i.v. administration of PBS, 0.1 mg/kg Ctrl-siRNA/LNP, or 0.01–0.1 mg/kg of m/r C5-siRNA/LNP on day 0. On day 7, anti-AChR mAb (mAb35, 1 mg/kg) was i.v. administered to rats to induce the symptoms of MG. Disease severity was monitored on days 8 and 9. (B) Body weights of rats treated with PBS, Ctrl-siRNA/LNP, or rat C5-siRNA/LNP were expressed as a ratio of the initial body weight (day 0). (C and D) Blood samples were collected from the jugular vein on days 3, 7, 8, and 9 and livers were collected on day 9 to evaluate the complement hemolytic activity in the serum and liver complement C5 mRNA expression levels (rat C5/rat GAPDH). (E) Soleus muscle sections were stained with Alexa Fluor 488-labeled α-BTX, and fluorescence signal was quantified for at least 50 or more neuromuscular junctions in each treated rat. Data are presented as the mean ± SEM. n = 3 in each group; ∗∗p < 0.01 versus Ctrl-siRNA/LNP.

Article Snippet: Cryosections were blocked in Block Ace (DS Pharma Biomedical, Osaka, Japan) at room temperature for 1 h and incubated with Alexa Fluor 488-conjugated α-BTX (no. B-13422, Life Technologies) and mouse anti-rat C5b-9 monoclonal antibody (no. HM3033, Hycult) at room temperature for 2 h. The sections were washed three times with 0.1% Triton X-100 in PBS and then incubated at room temperature for 1 h with Cy3-conjugated donkey anti-mouse IgG (no. 715-166-151, Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Activity Assay, Staining, Labeling, Fluorescence

Journal: Molecular Therapy. Nucleic Acids

Article Title: Identification of potent siRNA targeting complement C5 and its robust activity in pre-clinical models of myasthenia gravis and collagen-induced arthritis

doi: 10.1016/j.omtn.2023.01.005

Figure Lengend Snippet: Mouse/rat C6-siRNA/LNP treatment suppresses MG symptoms in rats Disease severity (A), body weight change (B), hemolysis (C), and complement C6 expression levels in the liver (D) of MG rats (n = 3/group) treated with PBS, Ctrl-siRNA/LNP, or 0.1 and 1 mg/kg mouse/rat (m/r) C6-siRNA/LNP are shown. (A) Rats received i.v. administration of PBS, 0.1 mg/kg of Ctrl-siRNA/LNP, or 0.01 or 0.1 mg/kg m/r C6-siRNA/LNP on day 0. On day 7, anti-AChR mAb (Mab35, 1 mg/kg) was i.v. administered to rats to induce the symptoms of MG. Disease severity was monitored on days 8 and 9 (B). Body weights of rats treated with PBS, Ctrl-siRNA/LNP, or rat C6-siRNA/LNP were expressed as a ratio of the initial body weight (day 0). (C and D) Blood samples were collected from the jugular vein on days 3, 7, 8, and 9 and livers were collected on day 9 to evaluate the complement hemolytic activity in serum and liver complement C6 mRNA expression levels (rat C6/rat GAPDH). Data are presented as the mean ± SEM. n = 4 in each group; ∗p < 0.05 and ∗∗p < 0.01 versus Ctrl-siRNA/LNP.

Article Snippet: Cryosections were blocked in Block Ace (DS Pharma Biomedical, Osaka, Japan) at room temperature for 1 h and incubated with Alexa Fluor 488-conjugated α-BTX (no. B-13422, Life Technologies) and mouse anti-rat C5b-9 monoclonal antibody (no. HM3033, Hycult) at room temperature for 2 h. The sections were washed three times with 0.1% Triton X-100 in PBS and then incubated at room temperature for 1 h with Cy3-conjugated donkey anti-mouse IgG (no. 715-166-151, Jackson ImmunoResearch Laboratories).

Techniques: Expressing, Activity Assay