Journal: Regenerative Therapy

Article Title: Multiple intra-articular injections with adipose-derived stem cells for knee osteoarthritis cause severe arthritis with anti-histone H2B antibody production

doi: 10.1016/j.reth.2023.06.007

Figure Lengend Snippet: MASCOT search results. The silver-stained band corresponding to 15 kDa was excised and digested with trypsin. The peptide mixture was analyzed in duplicate with MALDI-TOF MS. MALDI-TOF spectra of peptide ion masses from the protein digest were used to predict the protein sequence. Mascot was used to compare the observed spectra with a database of known proteins and determine the most likely matches. The results showed histone H2B as the candidate protein corresponding to the immunoprecipitated autoantigen. Protein sequence coverage was 8%.

Article Snippet: In short, Recombinant Human Histone H2B protein (NBP3-07913, Novus Biologicals, Centennial, CO, USA) was electrophoretically separated by SDS-PAGE using a 4–15% Mini-PROTEAN® TGXTM gel (Bio-Rad, Hercules, CA, USA) and later transferred to a polyvinylidene fluoride (PVDF) membrane.

Techniques: Staining, Sequencing, Immunoprecipitation

Journal: Regenerative Therapy

Article Title: Multiple intra-articular injections with adipose-derived stem cells for knee osteoarthritis cause severe arthritis with anti-histone H2B antibody production

doi: 10.1016/j.reth.2023.06.007

Figure Lengend Snippet: Immunoblot analysis of histone H2B protein as the corresponding autoantigen. (A) Ponceau S-stained PVDF membrane. Recombinant human histone H2B protein was separated electrophoretically by SDS-PAGE using a 4–15% Mini-PROTEAN® TGXTM gel, transferred to a PVDF membrane, and stained with Ponceau S to confirm protein transfer. (B) After cutting the each lane, the membranes were reacted with the anti-15 kDa-positive synovial fluid, anti-15 kDa-negative synovial fluid, commercial anti-histone H2B antibody, and anti-histone H2B-positive serum from a patient with SLE. Lane 1: molecular weight marker; lane 2: commercial anti-histone H2B antibody; lanes 3–6: anti-15 kDa-positive synovial fluid; lanes 7–9: anti-15 kDa-negative synovial fluid; and lane 10: anti-histone H2B-Ab-positive serum from a patient with SLE.

Article Snippet: In short, Recombinant Human Histone H2B protein (NBP3-07913, Novus Biologicals, Centennial, CO, USA) was electrophoretically separated by SDS-PAGE using a 4–15% Mini-PROTEAN® TGXTM gel (Bio-Rad, Hercules, CA, USA) and later transferred to a polyvinylidene fluoride (PVDF) membrane.

Techniques: Western Blot, Staining, Membrane, Recombinant, SDS Page, Molecular Weight, Marker

Journal: Regenerative Therapy

Article Title: Multiple intra-articular injections with adipose-derived stem cells for knee osteoarthritis cause severe arthritis with anti-histone H2B antibody production

doi: 10.1016/j.reth.2023.06.007

Figure Lengend Snippet: Patients who experienced knee pain and presence of anti-human histone H2A-H2B-DNA autoantibody (HHDNA) in their synovial fluid before and after treatment.

Article Snippet: In short, Recombinant Human Histone H2B protein (NBP3-07913, Novus Biologicals, Centennial, CO, USA) was electrophoretically separated by SDS-PAGE using a 4–15% Mini-PROTEAN® TGXTM gel (Bio-Rad, Hercules, CA, USA) and later transferred to a polyvinylidene fluoride (PVDF) membrane.

Techniques: Injection

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) Identification of the cognate autoantigen of A6. Schematic view shows the workflow to identify atherosclerosis autoantigens using diseased artery-detecting antibodies. A6-loaded protein A beads were incubated with tissue lysates prepared from diseased mouse arteries followed by elution of proteins from the beads to undergo autoantigen screening of candidates by mass spectrometry (MS) analyses. Several autoantigen-antibody interaction assays were used to confirm protein-protein interaction in vitro including western blots, ELISA, biolayer interferometry (BLI) and surface plasmon resonance (SPR) analyses using various expression-cloned autoantibodies derived from RLNs and ATLO GCs. (B) A6 reacts with mouse aorta-associated atherosclerotic plaques; co-staining of A6, macrophages/DCs and nuclei in Apoe -/- atherosclerosis plaque sections. A6 (red), CD11c (green) and nuclei (DAPI, blue). Bars 100 μm. (C) A6 reacts with human aorta or carotid artery atherosclerotic plaques; staining of A6 in human carotid atherosclerosis plaques by IHC. Bars 50 µm. (D) A6 identifies a 10-15 kDa protein in mouse diseased artery lysates; western blot (WB) shows A6 staining of aorta tissues obtained from 3 individual aged Apoe -/- mice. (E) Immunoprecipitation (IP) combined with MS analyses to identify autoantigen candidates. The ATLO GC-derived A6 antibody was used as a bait to capture autoantigens from protein lysates of diseased aorta of 78-weeks aged Apoe -/- mice. Beads without primary antibody was used as control. Label-free quantitation (LFQ) values of A6 were compared to controls. A6 antibody-enriched candidates are shown as a Venn diagram from three aorta samples from three individual aged Apoe -/- mice. The molecular weight (MW) of each candidate protein is shown. (F) A6 antibody captured histone 2b (H2B) protein in the diseased aorta. The ATLO GC-derived A6 antibody was used as a bait to capture autoantigens from protein lysates of diseased aorta of 32-weeks Apoe -/- mice. An isotype control antibody and the ATLO-GC-derived A11 antibody were used as controls. The peptide-spectrum matches (PSM) values of H2B are shown. Each dot represents one individual biological repeat. Three 32-weeks old Apoe -/- aortas were combined as one sample. (G) A6 binding to recombinant H2B by WB; recombinant H2a, H2b, H4 were used for comparison. (H) A6 reacts with human recombinant H2B at high-affinity; binding affinity of A6 to human recombinant H2B by SPR and BLI analyses. (I) A6 acquires high-affinity binding to the H2B autoantigens via SHM from its unmutated ancestor; binding of ATLO GC-derived A6 antibody to H2B determined by ELISA: Mutated A6 (red line), the unmutated parent of A6 (unmutated-A6, blue line), and the isotype control antibody (black line). (J) Summary figure: A6 acquires high-affinity binding to H2B via ATLO GC responses indicating that negative selection of GC autoreactive BCRs to prevent high-affinity autoantibody generation is compromised in ATLOs.

Article Snippet: In order to determine the titer of anti-histone H2b antibodies in the circulation of humans and mice, 100 μl purified recombinant histone H2b proteins (Active motif company, Cat: 31892) at 2 μg/ml concentration in the carbonate-bicarbonate buffer (Sigma, C3041-50CAP) were immobilized on high binding capacity microliter plates (Costar, 9018) overnight at 4 °C.

Techniques: Immunopeptidomics, Incubation, Mass Spectrometry, In Vitro, Western Blot, Enzyme-linked Immunosorbent Assay, SPR Assay, Expressing, Clone Assay, Derivative Assay, Staining, Immunoprecipitation, Control, Quantitation Assay, Molecular Weight, Binding Assay, Recombinant, Comparison, Selection

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) Anti-H2B autoantibody titers increase in Apoe -/- mice during aging. Anti-H2B IgG1 serum antibody titers of WT and Apoe -/- mice during aging were examined by ELISA; each dot represents one mouse. n= 8-weeks WT (n = 11), Apoe -/- (n = 12); 32-weeks WT (n = 12), Apoe -/- (n = 11); 78-weeks WT (n = 13), Apoe -/- (n = 16). Two-tailed student T test. (B) H2B vaccination promotes atherosclerosis. Vaccination using recombinant H2B in young Apoe -/- mice. Apoe -/- mice were vaccinated with recombinant H2B with adjuvant (termed as H2B vaccine), adjuvant alone, or PBS alone at the age of 8 weeks with a boost at the age of 12 weeks. The experiment was completed at 32 weeks. Blood was collected at different time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm. for PBS control (n = 7 mice), adjuvant control (n = 7), H2B + adjuvant (n = 8). (C-D) H2B vaccination induced long-lasting T cell dependent autoantibody responses in Apoe -/- mice. Serum anti-H2B total antibody and anti-H2B IgG1 antibody titers were determined by ELISA. n = 7 PBS, 7 adjuvant, and 8 H2B + adjuvant. (E) A6 antibody transfer accelerated atherosclerosis in chow diet-fed Apoe -/- mice. 100 µg A6 antibody was transferred via intraperitoneal (i.p.) injection at 10 weeks, and injections were repeated every 10 days. The experiment was completed at 32 weeks. Blood was collected at multiple time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm for PBS control (n = 17 mice), isotype antibody control (n = 12), A6 antibody (n = 18). (F) A6 antibody transfer accelerated atherosclerosis in high fat diet-fed Apoe -/- mice. 100 µg A6 antibody was transferred via i.p. injection at 10 weeks, and injections were repeated every 7 days. The experiment was completed at 14 weeks. Blood was collected at several time points before and after the injection; en face staining for whole aorta. Atherosclerotic plaques were quantified, as described in Methods. Scale bar 5 mm for PBS control (n = 8 mice), irrelevant isotype antibody control (n = 7), A6 antibody (n = 9).

Article Snippet: In order to determine the titer of anti-histone H2b antibodies in the circulation of humans and mice, 100 μl purified recombinant histone H2b proteins (Active motif company, Cat: 31892) at 2 μg/ml concentration in the carbonate-bicarbonate buffer (Sigma, C3041-50CAP) were immobilized on high binding capacity microliter plates (Costar, 9018) overnight at 4 °C.

Techniques: Enzyme-linked Immunosorbent Assay, Two Tailed Test, Recombinant, Adjuvant, Injection, Staining, Control

Journal: bioRxiv

Article Title: Self-Reactive B Cells in Artery Tertiary Lymphoid Organs Encode Pathogenic High Affinity Autoantibody in Atherosclerosis

doi: 10.1101/2025.07.10.664251

Figure Lengend Snippet: (A) study design of the clinical cohort. A cross-sectional cohort of 495 individuals of the general population aged between 30 - 70 years who underwent routine medical screening of preventive health care was studied. Exclusion criteria included patients with previously diagnosed cardiovascular diseases, strokes, autoimmune diseases requiring systemic treatment, active malignancies, and severe kidney or liver dysfunctions. 84.4% (418 out of 495) cases voluntarily underwent chest 3-dimentional computed tomography (CT) scans as part of the preventive health screening program. Serum anti-H2B antibody titers were examined. (B) Serum anti-H2B antibody titers is an independent risk factor for aorta and coronary artery calcification. Anti-H2B antibody titers were logarithmically transformed before entering the model to approximate Gaussian distribution. Odds ratios and 95% confidence intervals (CI) are calculated by binary logistic regression with simultaneous entry of all variables (Cox & Snell R 2 0.296, P <0.001). eGFR, estimated glomerular filtration rate; HbA1c, glycated hemoglobin A1c; DBP, diastolic blood pressure; SBP, systolic blood pressure; BMI, body mass index; (C) Serum anti-H2B antibody is an independent risk factor for atherosclerosis.

Article Snippet: In order to determine the titer of anti-histone H2b antibodies in the circulation of humans and mice, 100 μl purified recombinant histone H2b proteins (Active motif company, Cat: 31892) at 2 μg/ml concentration in the carbonate-bicarbonate buffer (Sigma, C3041-50CAP) were immobilized on high binding capacity microliter plates (Costar, 9018) overnight at 4 °C.

Techniques: Computed Tomography, Transformation Assay, Filtration