Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: (A–D) qPCR of CFB (A and C) and C3 (B and D) expression in BMDMs or THP1s treated with 20 nM of Pam3Cys4. (E–I) IHC of C3d (purple) in healthy non-disease donor (E and F) and AMD donor eyes (G–I). (J and K) Black arrow and black asterisk denote C3d in CC and basal laminar deposits in AMD donor eye (representative of n = 4 non-disease donor eyes, n = 5 AMD donor eyes). qPCR of CFB (J) and C3 (K) in ARPE-19 cells treated with 20 nm of Pam3Cys4. Data shown are mean ± SD for a representative of three separate experiments. CC, choriocapillaris; RPE, retinal pigment epithelium; BM, Bruch’s membrane. (L) Generation and chemical structure of CEP-adduct from DHA. (M and N) qPCR of (M) C3 and (N) CFB in hfRPE cells treated with 0.1 µg of IgG or anti-TLR2 antibody prior to 10 µM of CEP-HSA for 24 h. (O) IHC of TLR2 (purple) in a healthy donor. Bottom panel (black box) is photobleached to illustrate apical and basolateral RPE immunoreactivity (n = 4 non-disease donor eyes). (P) Secreted CFB at 24 h and C3 at 48 h in hfRPE cells treated with 17.5 µM or 35 µM of CEP-HSA and 20 nM of Pam3Cys4, mean ± SD representative of three independent experiments: *p < 0.05; **p < 0.01; ***p < 0.001. See also and .

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Expressing

Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: (A–D) Three micrograms of anti-TLR2 or anti-IgG was injected IVT into C57Bl6 mice that were then exposed to 100K lux light for 7 days. (A) Quantification of photoreceptor cell rows in anti-IgG versus anti-TLR2 groups. (B and C) Immunofluorescence (IF) of C3 (green) and nuclear DAPI (blue) in mice injected with (B) anti-IgG versus (C) anti-TLR2 (scale bars represent 20 µm). (D) Quantification of outer retinal C3 positive cells/deposits detected in ONL and subretinal space. Data shown are mean ± SEM. The p value was determined by nonparametric t test, p < 0.05. n = 9–10 per experiment, *p < 0.05. (E and F) BMDMs from WT, TLR2 –/– , Mal –/– , or MyD88 –/– mice were treated for 3, 6, and 24 h with 20 nM of Pam3Cys4; expression of (E) C3 and (F) CFB was assayed by RT-PCR. (G and H) HEK293-TLR2 cells were transfected for 24 h with C3 promoter-luciferase (100 ng), Renilla -luciferase (40 ng), and empty vector (EV) or plasmid expressing (G) Mal or (H) MyD88 at 10, 50, and 80 ng. Results are normalized for Renilla luciferase activity and represented as relative stimulation over the non-stimulated EV control, mean ± SD for triplicate determinations p value determined by one-way ANOVA and Tukey post test: *p < 0.05, **p < 0.01, ***p < 0.001. (I and J) Secreted C3 expression in (I) BMDMs and (J) primary mouse microglia treated with 20 nM of Pam3Cys4 for 6 and 24 or 48 h.

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Injection, Immunofluorescence, Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Luciferase, Plasmid Preparation, Activity Assay

Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: (A–E) Polarized hfRPE cells (A and B) or ARPE-19 cells (C–E) were treated with 10% Hi-NHS or NHS in combination with HSA or CEP-HSA for 24 h, phase transmission, presence of MAC (red), Phalloidin (green), and DAPI (blue), with representative images from three separate experiments. (B and E) Quantification of MAC+ specks in three 303 frames, data mean ± SD, one-way ANOVA followed by Tukey post-test to determine significance between groups; ***p < 0.001. (F–I) IF of MAC (red), Phalloidin (green), and DAPI (blue) in ARPE-19 cells treated with (F and G) 0.1 µg of IgG or anti-TLR2 antibody for 1 h or (H and I) with DMSO or 40 µm of Mal peptide inhibitor for 2 h prior to CEP-HSA and 10% NHS for 24 h. (G and I) quantification of MAC+ specks in four 20× frames, mean ± SD; p value determined by nonparametric t test; p < 0.05. (J–O) WT and TLR2 –/– mice injected IV, with NaIO 3 (50 mg/kg). (J) Quantification of MAC fluorescent intensity (scale bars represent 20 µm). (K–O) Representative IF images of MAC at 72 h, (K and O) depicts MAC staining in the whole retina section, (L–N) show higher magnifications, (L) shows MAC in the IS and OS, (M) shows MAC in OS and on RPE, (N) shows MAC on RPE. (P and R) Lysed tissue was assayed by western blot for expression of CFB(Bb). C9 and C9b at (P) 24 h and (R) 72 h. (Q and S) Mean pixel density for C9b was quantified at (Q) 24 h and (S) 72 h using the software ImageJ. See also and . *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Transmission Assay, Injection, Staining, Western Blot, Expressing, Software

Journal: Cell reports

Article Title: Toll-like Receptor 2 Facilitates Oxidative Damage-Induced Retinal Degeneration

doi: 10.1016/j.celrep.2020.01.064

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Antibodies for CFB (Atlas Antibodies Sigma) 1:250, C3 (MP Biomedicals-855444), C3d 1:1000 (Dako), ZO-1 (Invitrogen) 1:1000 and C5b-9 (Santa Cruz) 1:500 were incubated overnight at 4°C.

Techniques: Plasmid Preparation, Isolation, Recombinant, In Situ, Enzyme-linked Immunosorbent Assay, RNA Extraction, Viability Assay, Luciferase, Software

Journal: Seminars in Immunopathology

Article Title: Expanding horizons in complement drug discovery: challenges and emerging strategies

doi: 10.1007/s00281-017-0655-8

Figure Lengend Snippet: The current complement drug development landscape. This schematic illustrates drug which are being developed for clinical use, either approved (bullseye, yellow) or in the pipeline (remainder of target; clinical trial, black-red; preclinical, white). Complement drug discovery is a rapidly moving field and, inevitably, some compounds will progress, others will fail and new drugs will emerge to take their positions in the landscape in the coming years. Antibodies (whole Ig or fragments) in development or approved include eculizumab (Alexion), a blockbuster drug approved for treatment of PNH and aHUS, and its follow-up molecule ‘next-generation eculizumab’ ALXN1210 (phase 3). Lampalizumab (Genentech/Roche) is in phase 3 for geographic atrophy, IFX-1 (InflaRx) is an anti-neo C5a, and LFG316 and CLG561 are specific for C5 and properdin (both developed by Novartis). Other antibodies which are pathway-specific include OMS721 and OMS906 (Omeros) against lectin pathway proteins, TNT009 (True North therapeutics) specific for C1s, PRO-02 (Prothix) against C2, and ANX005 (Annexon Biosciences) against C1q. Novelmed have antibodies against CFB (bikaciomab) and properdin (NM9401) in preclinical development. Other antibodies block C5 or MAC and include SKY59 (Chugai/Roche)—a sweeping antibody against C5, ALXN5500 (Alexion) specific for C5, Regenemab—an antibody specific for C6 (Regenesance) and Mubodina (Adienne Pharma & Biotech), an antibody against C5 being developed for typical HUS. Cinryze, Berinert and Ruconest are all C1 inhibitor, either native (Cinryze, Berinert) or recombinant (Ruconest)—approved for HAE. Various other biologics target the alternative pathway including TT30 (Alexion), Mirococept (King’s College London/MRC) and AMY-201 (Amyndas). SOBI-005 (SOBI) is an affibody and Coversin is a recombinant tick protein, both biologics which block C5. Peptide-based agents include the alternative pathway blockers, all derived from Compstatin: AMY-101 (Amyndas), APL-1 and APL-2 (both Apellis Pharmaceuticals), and the C5-blocking peptide from RaPharma, RA101495. Small molecules which target complement have been slow to develop but success is now evident with CCX-168 (ChemoCentryx; C5a-receptor blocker) and ACH-4471 (CFD blocker, Achillion Pharmaceuticals); trials of the SM inhibitors of both CFD and CFB (Novartis) are anticipated. Small molecule inhibitors of properdin (Novelmed) have been described although recent development is not reported. There are a small number of nucleic acid-based therapies which have been developed which bind target and block function, these include Zimura (Ophthotech), an aptamer which blocks C5, NOX-D19-D21 which bind C5a (Noxxon Pharmaceuticals) and DF-2593A (Dompé) which binds C5a receptor and blocks via an allosteric mechanism. Finally, various agents which function at the gene expression level are under development and these include antisense for CFB (IONIS-FB-LRx; Ionis Pharmaceuticals), RNAi for C5 (ALN-CC5, Alnylam Pharmaceuticals), and an agent under development by Regenesance based on locked nucleic acid (LNA) technology, which prevents C6 expression. This figure is adapted from Morgan BP, Harris CL. Nat Rev. Drug Discov. 2015 Dec;14(12):857-77; ‘Complement, a target for therapy in inflammatory and degenerative diseases’

Article Snippet: Novelmed have antibodies against CFB (bikaciomab) and properdin (NM9401) in preclinical development.

Techniques: Drug discovery, Blocking Assay, Recombinant, Derivative Assay, Gene Expression, Expressing