Journal: Regenerative biomaterials

Article Title: Role of integrin β1 and tenascin C mediate TGF-SMAD2/3 signaling in chondrogenic differentiation of BMSCs induced by type I collagen hydrogel.

doi: 10.1093/rb/rbae017

Figure Lengend Snippet: Figure 5. (A) H&E staining. (B) Gene expression levels of ACAN, COL2A1 and SOX9. (C) Western blot detect the protein expression of COL2A1. (D) Semi- quantitation of gray level in (C). (E) Immunofluorescence staining for COL2A1. (F) Semi-quantitation of fluorescence intensity in (E) (scale bars: 170 lm. mean± SD, n ¼ 3; P 0.05, P 0.01, , ###P 0.001. represents the comparison between the experimental group and the control group, and # represents the comparison between the experimental groups).

Article Snippet: After blocking with normal goat serum, primary antibodies against TNC (1:100; Proteintech, Wuhan, China), ITGB1 (1:100; Proteintech, Wuhan, China), COL2A1 (1:100; Proteintech, Wuhan, China), COL1A1 (1:100; Proteintech, Wuhan, China), p-SMAD2/3 (1:100; Cell Signaling, USA) and SMAD2/3 (1:100; Cell Signaling, USA) were added and incubated overnight at 4�C.

Techniques: Staining, Gene Expression, Western Blot, Expressing, Quantitation Assay, Immunofluorescence, Fluorescence, Comparison, Control

Journal: Regenerative biomaterials

Article Title: Role of integrin β1 and tenascin C mediate TGF-SMAD2/3 signaling in chondrogenic differentiation of BMSCs induced by type I collagen hydrogel.

doi: 10.1093/rb/rbae017

Figure Lengend Snippet: Figure 8. Immunohistochemical staining for COL1A1 (A) and COL2A1 (B) in repaired cartilage after 4 and 8 weeks post-surgery (scale bars: 200 and100 lm).

Article Snippet: After blocking with normal goat serum, primary antibodies against TNC (1:100; Proteintech, Wuhan, China), ITGB1 (1:100; Proteintech, Wuhan, China), COL2A1 (1:100; Proteintech, Wuhan, China), COL1A1 (1:100; Proteintech, Wuhan, China), p-SMAD2/3 (1:100; Cell Signaling, USA) and SMAD2/3 (1:100; Cell Signaling, USA) were added and incubated overnight at 4�C.

Techniques: Immunohistochemical staining, Staining

Journal: Regenerative biomaterials

Article Title: Role of integrin β1 and tenascin C mediate TGF-SMAD2/3 signaling in chondrogenic differentiation of BMSCs induced by type I collagen hydrogel.

doi: 10.1093/rb/rbae017

Figure Lengend Snippet: Figure 9. Collagen hydrogel acts on ITGB1, followed by activating the ITGB1, then induces phosphorylation of SMAD2/3, the p-SMAD2/3 enters into the nucleus and binds to the promoter regions of TNC to initiate its transcription [58]. TNC increases the expression of ITGB1 and further induces activation of ITGB1 and SMAD2/3. p-SMAD2/3 promotes the stability and accumulation of SOX9 protein and forms a transcriptional complex with SOX9, promoting the expression of cartilage-related genes, like COL2A1, etc.

Article Snippet: After blocking with normal goat serum, primary antibodies against TNC (1:100; Proteintech, Wuhan, China), ITGB1 (1:100; Proteintech, Wuhan, China), COL2A1 (1:100; Proteintech, Wuhan, China), COL1A1 (1:100; Proteintech, Wuhan, China), p-SMAD2/3 (1:100; Cell Signaling, USA) and SMAD2/3 (1:100; Cell Signaling, USA) were added and incubated overnight at 4�C.

Techniques: Phospho-proteomics, Expressing, Activation Assay

Journal: bioRxiv

Article Title: Anti-citrullinated protein antibodies with diverse specificities ameliorate collagen antibody-induced arthritis in a time-dependent manner

doi: 10.1101/2023.03.08.531560

Figure Lengend Snippet: A. C57BL/6 mice were injected with different doses of anti-CII/mouse: 5 mg (recommended dose), 3 mg, and 2 mg. B. DBA/1J mice were injected with different doses of anti-CII/mouse: 1.5 mg (recommended dose), 1 mg, and 0.75 mg. Naïve wild-type (WT) controls were not injected with anti-CII antibodies. Timelines depict mean arthritis scores throughout the experiment (maximum score:16). Animals were sacrificed on day 14. In A , all doses were significantly different from WT controls from day 7 onwards and there were no significant differences between sub-optimal doses throughout the experiment (purple asterisks). In B , the 1 mg aCII/mouse group was significantly different from WT controls from day 9 onwards, while the 0.75 mg aCII/mouse group was never significantly different from WT controls (purple asterisks). 2-way ANOVAs were used for statistical analysis, colored asterisks indicate significant differences between sub-optimal doses and the corresponding optimal dose for the strain, while black asterisks represent significant differences between sub-optimal doses. n =3-5 mice/group.

Article Snippet: The collagen antibody induced arthritis (CAIA) was induced by retro-orbital injection of a cocktail of 5 monoclonal antibodies (mAbs) against mouse collagen type II (anti-CII cocktail, Chondrex Inc), followed by intraperitoneal injection of lipopolysaccharide (LPS) (50 μg/mouse) three days later.

Techniques: Injection

Journal: bioRxiv

Article Title: Anti-citrullinated protein antibodies with diverse specificities ameliorate collagen antibody-induced arthritis in a time-dependent manner

doi: 10.1101/2023.03.08.531560

Figure Lengend Snippet: A. Suboptimal CAIA was induced by injecting C57BL/6 mice with 2.5 mg anti-CII/mouse. Recombinant antibodies were injected on days 7 and 10 post-induction, and animals were sacrificed on day 14. ACPA-treatment groups were formed as follows: (cit-)PAD4+ (RA64, RA65, RA71); PAD4- (RA66, RA74, RA75); and Polyspecific (RA78, RA80, RA82). Control groups: naïve wild-type (WT); CAIA isotype control (IC); Optimal-dose CAIA not injected with recombinant antibodies (NoAb). n =3-6 mice/group B. The clinical scores of paw inflammation and the hind paw thickness were reduced in all ACPA-treated groups, as early as 2 days post-antibody injection. Compared to isotype controls, mean paw thickness was significantly reduced in all ACPA-treated groups. All ACPA groups were never significantly different from each other and had similar p values when compared to controls. By the experiment endpoint, they were not significantly different from WT controls C. Accumulated change in hind paw thickness following the first antibody injection. All ACPA-treated groups were significantly different from CAIA controls and showed signs of active amelioration (negative %change). All ACPA groups were not significantly different from each other and had similar p values when compared to controls D. CAIA was induced by injecting 5 mg anti-CII/animal in C57BL/6 mice. Recombinant antibodies were injected on days 7 and 10 post-induction and animals were sacrificed on day 14. A single ACPA, representative of the combinations tested in A , was injected per group. n =3-5 mice/group E. The clinical scores of paw inflammation and the hind paw thickness were significantly reduced in all ACPA-treated groups compared to both isotype and no-antibody control animals, and as early as 2 days post-antibody injection. All ACPA groups were never significantly different from each other and had similar p values when compared to controls. By the experiment endpoint, they were not significantly different from WT controls F. Accumulated change in hind paw thickness following the first antibody injection. All ACPA-treated groups were significantly different from CAIA controls and showed signs of active amelioration (negative %change), suggesting a treatment effect. All ACPA groups were not significantly different from each other and had similar p values when compared to controls. B & E. Only scores for hind legs are shown (1 value/animal, max score: 8). Group mean scores per timepoint +SD are plotted. Both hind paws were included for calculating mean paw thickness (2 values/animal). Group mean paw thickness per timepoint (mm) +SD is plotted. A mixed-effects model with matched values was used for statistical comparisons. C & F. Both hind paws were included (2 values/animal). The mean accumulated % change per group +/-SD is plotted. Dots represent individual paws. 1-way ANOVA was used for statistical comparisons. B, C, E, F. We only show statistical comparisons for one of the ACPA groups (PAD4-, RA74) vs controls. * = p≤0.05; ** = p≤0.01; *** = p≤0.001

Article Snippet: The collagen antibody induced arthritis (CAIA) was induced by retro-orbital injection of a cocktail of 5 monoclonal antibodies (mAbs) against mouse collagen type II (anti-CII cocktail, Chondrex Inc), followed by intraperitoneal injection of lipopolysaccharide (LPS) (50 μg/mouse) three days later.

Techniques: Recombinant, Injection, Control

Journal: bioRxiv

Article Title: Anti-citrullinated protein antibodies with diverse specificities ameliorate collagen antibody-induced arthritis in a time-dependent manner

doi: 10.1101/2023.03.08.531560

Figure Lengend Snippet: CAIA was induced in DBA/1J mice by injecting 1.5 mg anti-CII cocktail/mouse. Recombinant ACPA (RA74) and isotype control (IC) antibodies were injected on days 10 and 13. For RA74, two doses were tested: 1.5 mg/mouse/injection and 0.5 mg/mouse/injection. The isotype control antibody was injected at 1.5 mg/mouse/injection. No antibody-treated CAIA controls (No Ab) were injected with vehicle, while wild-type (WT) naïve controls did not receive any antibody injection. Timelines of mean clinical scores (A) and mean paw thickness (B) are shown. After the first antibody injection (day 10), there were no significant differences between the two ACPA doses tested, for both parameters here displayed. Of note, both ACPA groups (0.5 and 1.5 mg/mouse) showed sharper reductions of their clinical scores and paw thickness after the first antibody injection (day 10) compared to CAIA controls, though not significantly. No differences were observed after the second antibody injection (day 13). 2-way ANOVAs were used for statistical analysis. n =4 mice/group.

Article Snippet: The collagen antibody induced arthritis (CAIA) was induced by retro-orbital injection of a cocktail of 5 monoclonal antibodies (mAbs) against mouse collagen type II (anti-CII cocktail, Chondrex Inc), followed by intraperitoneal injection of lipopolysaccharide (LPS) (50 μg/mouse) three days later.

Techniques: Recombinant, Control, Injection

Journal: bioRxiv

Article Title: Anti-citrullinated protein antibodies with diverse specificities ameliorate collagen antibody-induced arthritis in a time-dependent manner

doi: 10.1101/2023.03.08.531560

Figure Lengend Snippet: A. Co-injection of ACPAs and anti-CII cocktail. Sub-optimal CAIA was induced by injecting 1 mg anti-CII/mouse in DBA/1J mice. Recombinant antibodies were injected on day 0, alongside the anti-CII antibody cocktail, and animals were sacrificed on day 14. Two recombinant ACPAs were tested in this and subsequent CAIA experiments: RA66 and RA74 (both PAD4-ACPAs). Control groups included: naïve wild-type (WT), CAIA isotype control (IC), and CAIA not injected with recombinant antibodies (No Ab). n =4 mice/group. Co-injection of ACPAs completely abolished the development of joint inflammation, as depicted in the clinical scores ( B ) and paw thickness ( C ) timelines. ACPA-injected groups did not show any clinical signs of CAIA throughout the experiment. Paw thickness was significantly reduced for RA66-injected animals, compared to both isotype control and no-antibody CAIA groups, from day 10 onwards. Repeated measures 2-way ANOVA was used for statistical analysis D . Accumulated change in paw thickness following the first antibody injection. ACPA-treated groups were significantly different from CAIA controls and showed no accumulated paw inflammation. They were not significantly different from WT controls. Kruskal-Wallis tests were used for comparisons E. ACPAs in early-phase CAIA. CAIA was induced by injecting 1.5 mg anti-CII/mouse in DBA/1J mice. Recombinant antibodies were injected on days 3 and 7 post-induction and animals were sacrificed on day 14. n=4 mice/group. As shown in the clinical scores ( F ) and paw thickness ( G ) timelines, ACPAs fully prevented the development of joint inflammation when injected at early stages of CAIA. ACPA-treated groups had significantly lower clinical scores and paw thickness than both isotype and no-antibody control animals from day 7 onwards, and they were always undistinguishable from WT controls. Repeated measures 2-way ANOVA was used for statistical analysis H . Accumulated change in paw thickness following the first antibody injection. All ACPA-treated groups were significantly different from CAIA controls and showed no signs of paw inflammation. They were not significantly different from WT controls. 1-way ANOVA was used for statistical comparisons I . ACPAs in late-stage CAIA. CAIA was induced by injecting 1.5 mg anti-CII/mouse in DBA/1J mice. Recombinant antibodies were injected on days 10 and 13 post-induction and animals were sacrificed on day 18. n=4 mice/group. Clinical scores ( J ) and paw thickness ( K ) were decreased after injection of ACPAs on day 10, compared to no antibody and to isotype controls, though only significantly for the clinical scores of RA66-treated mice. Injection of ACPAs on day 13 did not alter the resolution phase of CAIA in any capacity. ACPA-treated groups were never significantly different from each other, but they remained significantly different from WT controls for most of the experiment. A mixed-effects model with matched values was used for statistical comparisons L . Accumulated change in paw thickness following the first antibody injection. All CAIA groups experienced reductions in their accumulated paw thickness, regardless of treatment, indicating a recovery phase for CAIA. ACPA-treated groups were not significantly different from CAIA controls. Kruskal-Wallis tests were used for comparisons. B,F,J All paws were included in the calculation of mean clinical scores (1 value/animal, max score: 16). Group mean scores per timepoint +SD are plotted. C,G,K All paws were included for calculating mean paw thickness (4 values/animal). Group mean paw thickness per timepoint (mm) +SD is plotted. D,H,L All paw were included for calculating accumulated % change in paw thickness (4 values/animal). The mean accumulated % change per group +/-SD is plotted. Dots represent individual paws. B, C, D, F, G, H, J, K, L. We only show statistical comparisons for one of the ACPA groups (RA74) vs controls. * = p≤0.05; ** = p≤0.01; *** = p≤0.001

Article Snippet: The collagen antibody induced arthritis (CAIA) was induced by retro-orbital injection of a cocktail of 5 monoclonal antibodies (mAbs) against mouse collagen type II (anti-CII cocktail, Chondrex Inc), followed by intraperitoneal injection of lipopolysaccharide (LPS) (50 μg/mouse) three days later.

Techniques: Injection, Recombinant, Control