Journal: Arthritis Research & Therapy

Article Title: Effect of Porphyromonas gingivalis infection on gut dysbiosis and resultant arthritis exacerbation in mouse model

doi: 10.1186/s13075-020-02348-z

Figure Lengend Snippet: Effect of PgPAD on the onset of joint arthritis. The effect of PgPAD on the onset of arthritis was determined. In this experiment, PgPAD deletion mutant 33277 strain (⊿ pad ) and Pg 33277 wild-type (WT) strain were used, unless Pg W83 strain was specifically described in caption of Figures. ABL of the upper jaw in each group was measured ( a ). Ctrl, PBS inoculation; WT, Pg wild-type inoculation; ⊿ pad , Pg pad knockout strain inoculation. To evaluate inflammation of the gingival tissue 6 weeks after Pg inoculation, IL-6 production in gingival tissue of SKG mice was measured by ELISA ( b ). AS was measured 3 and 6 weeks after Pg inoculation with LA i.p. injection ( c ). To assess CP synthesis, ELISA was performed ( d gingival tissue, e joint tissue, f small intestine, g large intestine). The titer of serum ACPA 6 weeks after Pg inoculation with LA i.p. injection in mice from each group was measured by ELISA ( h ). The production of endogenous PADI2 and PADI4 in joint tissue was determined by Western blotting, and then the density of each band was measured by NIH Image-J ( i and j ). Data represent mean ± SD ( b – j ) of 5 mice per group. Statistical analyses were performed using the Tukey-Kramer test and Bonferroni-corrected Mann-Whitney U test for multiple comparisons (* P < 0.05, ** P < 0.01)

Article Snippet: As a control, the amount of Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was detected by anti-GAPDH antibody (10 μg/ml, HRP-60004, PROTEINTECH JAPAN, Japan) The density of target PADI2 and PADI4 bands was measured by NIH Image-J software.

Techniques: Mutagenesis, Knock-Out, Enzyme-linked Immunosorbent Assay, Injection, Western Blot, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: In situ citrullination of human fibrinogen. (A) Fibrinogen (Fib) or citrullinated fibrinogen (cFib) was immobilized to enzyme-linked immunosorbent assay (ELISA) plates at a concentration of 1 μg/ml. Increasing concentrations of the anti-cFib monoclonal antibody (mAb; clone 20B2) were added, followed by addition of enzyme-conjugated rabbit anti-mouse immunoglobulin antibodies and o -phenylenediamine substrate. All data points represent means and ranges of duplicate measurements of optical density at 490 nm. (B) ELISA plates were coated with 1.0 μg/ml human fibrinogen and incubated with citrullination buffer including recombinant human peptidylarginine deiminase (rhPAD) enzyme, ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), MQPAD4 (49 mU/ml) or rhPAD4 (1,500 ng/ml). Shown is the activity as a percentage of the maximal activity obtained for each enzyme, corresponding to the activity at 4 hours. Anti-cFib mAb was used at a concentration of 0.5 μg/ml, as determined from the graph shown in (A). Symbols and error bars represent means and ranges of duplicate measurements.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: In Situ, Enzyme-linked Immunosorbent Assay, Concentration Assay, Incubation, Recombinant, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: Comparison between enzymatic activities of peptidylarginine deiminases 2 and 4. Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen. (A) Recombinant human peptidylarginine deiminase 2 (rhPAD2) or rhPAD4 raised in-house were used for citrullination at mass concentrations ranging from 0.1 ng/ml to 15 μg/ml. (B) Commercially available ModiQuest (MQ) PAD2 or MQPAD4 enzymes were used in units ranging from 0.1 mU/ml to 100 mU/ml. Following a 3-hour citrullination period, the anti-cFib mAb (0.5 μg/ml) was used for detection of citrullinated fibrinogen. Duplicate measurements of optical density (OD) at 490 nm are shown as mean and range.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: Calcium dependency of peptidylarginine deiminases. Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml). Dithiothreitol was added at a concentration of 1.0 mM, and CaCl 2 was added at concentrations ranging from 50 μM to 10 mM. Following 3 hours of incubation, citrullination was measured using mouse anti-cFib monoclonal antibody (0.5 μg/ml). Shown is the activity as a percentage of maximal activity for each enzyme. Symbols and error bars represent means and ranges of duplicate measurements.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Concentration Assay, Activity Assay

Journal: Arthritis Research & Therapy

Article Title: Demonstration of extracellular peptidylarginine deiminase (PAD) activity in synovial fluid of patients with rheumatoid arthritis using a novel assay for citrullination of fibrinogen

doi: 10.1186/s13075-014-0498-9

Figure Lengend Snippet: Peptidylarginine deiminases activity in synovial fluid samples from rheumatoid arthritis patients. (A) Synovial fluid (SF) samples from five rheumatoid arthritis patients were applied on enzyme-linked immunosorbent assay plates coated with fibrinogen (1 μg/ml) after 1:3 dilution into Tris buffer with 1 mM dithiothreitol (DTT; black columns), Tris buffer containing 1 mM DTT and 10 mM CaCl 2 (grey columns) or Tris buffer containing 1 mM DTT and 10 mM ethylenediaminetetraacetic acid (EDTA; white columns). Following a 3-hour incubation period, the monoclonal mouse anti-citrullinated fibrinogen (anti-cFib) (0.5 μg/ml) was used for detection of citrullinated fibrinogen. Peptidylarginine deiminase (PAD) activity is presented as optical density (OD) at 490 nm of duplicated measurements. (B) Association between PAD2 concentration and PAD activity in the five SF samples diluted 1:3 in citrullination buffer containing 10 mM CaCl 2 (grey circles, solid line) or no additive calcium (black circles, dotted line). Pearson’s correlation coefficients ( r ) and levels of significance are shown.

Article Snippet: Enzyme-linked immunosorbent assay plates were coated with 1.0 μg/ml human fibrinogen and incubated with four different recombinant human peptidylarginine deiminase (rhPAD) preparations: ModiQuest (MQ) PAD2 (8 mU/ml), rhPAD2 (42 ng/ml), rhPAD4 (1,500 ng/ml) or MQPAD4 (49 mU/ml).

Techniques: Activity Assay, Enzyme-linked Immunosorbent Assay, Incubation, Concentration Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: PAD activity in NAWM of MS patients. (A) Quantification of PAD2 protein in white matter from normal and MS brain by immunoslot blot ( n =5, P <0.0001). (B) Citrullinated protein in white matter from normal and MS brains by immunoslot blot as pixel density ( n =4, P <0.01). (C) PAD enzyme activity in normal and MS tissue, with or without pre-incubation with 2CA ( n =5, P <0.05). Each dot represents one patient analysed three times. The means (horizontal bar) for all the MS patients were compared with the normal.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Incubation

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: Interaction of PAD with 2CA. (A) PAD2 and PAD4 inhibition curves in the presence of increasing 2CA concentrations. Insert: PAD1-4 enzymes contain a common C-terminal active-site cysteine residue (Cys656) bound by 2CA, confirmed by ESI mass spectrometry of tryptic digests of PAD2-acetamidine adducts. (B) Schematic of the nucleophilic reaction between 2CA and the Cys656 residue in the active site of PAD2 . (C) Tabular summary of peptide fragment atomic masses for 2CA-modified and native PAD2.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Inhibition, Residue, Mass Spectrometry, Modification

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: mAb4E12 (anti-2CA adduct) immunogold labeled optic nerve cryosections from control and 2CA-treated PAD2 transgenic mice. Minimal labeling in untreated mice (black arrows). Numerous gold particles (white arrows) in nuclei (N) and cytoplasm of oligodendrocytes, myelin and axonoplasm (Ax) of 2CA-treated mice. Scale bars: 500 nm.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Labeling, Control, Transgenic Assay

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates demyelinating disease in ND4 mice. (A) ND4 mice treated with PBS, 2CA (5 mg/kg), or 2CA+B12 (5 mg/kg and 10 mg/kg) starting at 2 months before disease onset ( n =5, P <0.0001). (B) ND4 mice treated at disease onset ( n =4, P <0.0001). (C) Stopping 2CA, but continuing B12 at 3.5 months in ND4 mice ( n =5, P <0.0001) demonstrates that B12 alone does not attenuate disease. (D) PAD activity in brains from animals shown in ( n =5, P <0.05). The first four bars represent results from animals at 6 months of age, whereas the post-treatment animals were 8 months of age. (E) PAD2 RT PCR in white matter extracts from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice ( n =9, P <0.05). (F) LFB and hematoxylin stain of cerebella from normal, PBS-, 2CA- and 2CA+B12-treated ND4 mice (40×). WM, white matter; GM, grey matter.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Activity Assay, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Disease Models & Mechanisms

Article Title: Inhibition of peptidyl-arginine deiminases reverses protein-hypercitrullination and disease in mouse models of multiple sclerosis

doi: 10.1242/dmm.010520

Figure Lengend Snippet: 2CA attenuates PAD2 overexpressor. (A) Demyelinating disease in PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12 starting at 6 months of age ( n =5, P <0.0001). (B) PAD activity in brain extracts of PAD2 transgenic mice treated with PBS, 2CA or 2CA+B12, and non-transgenic littermates ( n =4, P <0.05). PAD activity is reduced to normal levels by treatment.

Article Snippet: Recombinant PAD2 (5 μg)was reacted with 2CA in the presence of Ca 2+ in HEPES buffer pH 7.6 (BioShop Canada, Burlington, ON) at 52°C for one hour, after which it was dialyzed and lyophilized.

Techniques: Transgenic Assay, Activity Assay