Journal: Arthritis Research & Therapy

Article Title: Identification of monoclonal antibodies against human renal glomerular endothelial cells in lupus nephritis that induce endothelial interferon-alpha production

doi: 10.1186/s13075-021-02552-5

Figure Lengend Snippet: IFN-α in SLE patients and its production by monoclonal antibody-treated HRGEC. a Serum levels of IFN-α between SLE patients with LN, SLE patients without LN, and healthy controls. The mean and SD are given. * p < 0.05. b The levels of IFN-α in the supernatants of HRGEC cultured alone and co-cultured with LN1–4 or IgG1/IgG3 isotype controls at a concentration of 100 μg/ml. The mean and SD are given. ** p < 0.001. c The levels of IFN-α in the supernatants of HRGEC treated with LN1, LN2, and IgG3 isotype control at the indicated concentrations. The mean and SD are given. * p < 0.05, ** p < 0.001. d The levels of IFN-α between the supernatants of cultured HRGEC and DNAse I-treated HRGEC that were co-cultured with the same monoclonal antibodies (LN1, LN2, or LN3) at the same concentration of 100 μg/ml. The mean and SD are given

Article Snippet: Twenty-four hours later, the supernatants were collected for the analysis of interleukin (IL)-1, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-γ, and IFN-α (IL-1, 6, 8; MCP-1; and IFN-γ detected by DuoSet ELISA Kits, R&D Systems, Inc., Minneapolis, USA; IFN-α detected by Matched Antibody Pair Kit, Eugene, OR, USA).

Techniques: Cell Culture, Concentration Assay, Control, Bioprocessing

Journal: Arthritis Research & Therapy

Article Title: Identification of monoclonal antibodies against human renal glomerular endothelial cells in lupus nephritis that induce endothelial interferon-alpha production

doi: 10.1186/s13075-021-02552-5

Figure Lengend Snippet: The summary of some IgG DNA-reactive anti-HRGEC antibodies in lupus nephritis inducing DNA-independent production of IFN-α by HRGEC

Article Snippet: Twenty-four hours later, the supernatants were collected for the analysis of interleukin (IL)-1, IL-6, IL-8, monocyte chemoattractant protein (MCP)-1, interferon (IFN)-γ, and IFN-α (IL-1, 6, 8; MCP-1; and IFN-γ detected by DuoSet ELISA Kits, R&D Systems, Inc., Minneapolis, USA; IFN-α detected by Matched Antibody Pair Kit, Eugene, OR, USA).

Techniques:

Journal: PLOS Pathogens

Article Title: The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKK α and IKK β through the recruitment of TOLLIP

doi: 10.1371/journal.ppat.1011580

Figure Lengend Snippet: The protein levels of IL-1 β (A), TNF- α (B), and IFN- α (C) in the serum samples were measured by commercial ELISA kits. Serum antibodies against p72 (D) or p30 (E) in the inoculated pigs were detected by ELISA. The results were shown as a blocking percentage. For the anti-p72 antibodies, the blocking rate above 50% was considered positive, while below 40% was considered negative; for the anti-p30 antibodies, the blocking rate below 40% was considered positive, while above 50% was considered negative. Samples with blocking between 40 and 50% were considered doubtful.

Article Snippet: The infected cells were cultured in the fresh RPMI 1640 medium for 12 and 20 h. The concentrations of IL-1 β (Ray Biotech, ELP-IL1b-1), TNF- α (Ray Biotech, ELP-TNFa-1), and IFN- α (Ray Biotech, ELP-IFNa-1) in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay

Journal: Experimental and Therapeutic Medicine

Article Title: TlR2 and TlR4 are involved in the treatment of rheumatoid arthritis synovial fibroblasts with a medicated serum of asarinin through inhibition of T h 1/T h 17 cytokines

doi: 10.3892/etm.2020.8557

Figure Lengend Snippet: Effect of asarinin on cytokine expression of rheumatoid synoviocytes. (A) Left: Fibroblast-like synovial cell morphology of third generation osteoarthritic synoviocytes. Right: third generation of the fibroblast-like synovial cell morphology of rheumatoid synoviocytes (magnification, x40). (B) Changes in the expression of IL-17A, TNF-α, IFN-γ and IL-6 were determined by performing reverse transcription-quantitative PCR. (C) Protein expression of cytokines in rheumatoid synoviocytes. Upper panel: Expression of IL-17A, TNF-α, IFN-γ and IL-6 was analyzed using western blotting with β-actin as a loading control. Lower panel: Density histogram data from three separate western blot analyses (mean ± standard deviation), which represent the relative expression of IL-17A, TNF-α, IFN-γ and IL-6. (D) Quantitative analyses of IL-17A, TNF-α, IFN-γ and IL-6 using ELISA. * P<0.05 and ** P<0.01 vs . control cells. IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; RASF, rheumatoid arthritis synovial fibroblast; OASF, osteoarthritis synovial fibroblast.

Article Snippet: The ELISA kit for IL-17A (cat. no. D1700) was obtained from R&D Systems, Inc. Primary antibodies for TNF-α (cat. no. TA808184), IL-17A (cat. no. TA337063), IL-6 (cat. no. TA500067) and IFN-γ (cat. no. TA353236) were from OriGene Technologies, Inc. Peptidoglycan (PGN) and lipopolysaccharide (LPS) were purchased from Sigma-Aldrich.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation, Enzyme-linked Immunosorbent Assay

Journal: Clinical and Experimental Immunology

Article Title: Characterization of T-bet expressing B cells in lupus patients indicates a putative prognostic and therapeutic value of these cells for the disease

doi: 10.1093/cei/uxaf008

Figure Lengend Snippet: ( A-C ) T-bet + B cells diminish, following treatment with HCQ, fasudil and/or anifrolumab. Data have emerged from three independent experiments, MEAN ± SD are presented ➔ Unst: 2.85 ± 0.9 vs St: 13.64 ± 2.8 vs HCQ 1 : 3.64 ± 0.47 vs HCQ 10 : 3.15 ± 1.34 vs HCQ 50 : 2.69 ± 2.15 vs FAS 1 : 1.65 ± 0.46 vs FAS 10 : 1.57 ± 0.72 vs FAS 50 : 1.56 ± 0.74 vs ANIF 2 : 5.2 ± 0.62 vs ANIF 10 : 2.54 ± 0.68 vs ANIF 30 : 2.31 ± 1. Statistical significance was evaluated via Student’s t test. *<0.05 **<0.01 Unst: Unstimulated, St: Stimulated, HCQ: hydroxychloroquine, FAS: fasudil, ANIF:anifrolumab ( D ) Cell culture supernatant IFNα levels are elevated in stimulated samples, according to ELISA conducted. Data have emerged from 3 independent experiments, MEAN ± SD are presented ➔ Unst: 0.6 ± 0.01 vs St:181.3 ± 15.8. Statistical significance was evaluated via Student’s t test. *<0.05 **<0.01 Unst: Unstimulated, St: Stimulated

Article Snippet: Human IFNA1 Sandwich ELISA Kit (Proteintech, cat. #KE00044) was used for IFNα estimation and AuthentiKine TM Human TNF-alpha Sandwich ELISA Kit (Proteintech, cat. #KE00154) was used for TNFα estimation.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKK α and IKK β through the recruitment of TOLLIP

doi: 10.1371/journal.ppat.1011580

Figure Lengend Snippet: (A) PAMs were mock infected or infected with Del2R or ASFV-WT (MOI = 5) for 12 and 20 h for RNA-seq analysis. Volcano plot of gene changes in Del2R-infected PAMs compared with the expression in ASFV-WT-infected PAMs. Red dots and blue dots denote upregulated or downregulated differentially expressed genes (DEGs), respectively. (B) Gene ontology (GO) category functional enrichment by 3 categories (Biological process, Molecular function, and Cell component) and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis was performed for up-regulated genes in the group of Del2R versus ASFV-WT. (C) The heatmaps show the expression levels of DEGs in the NF- κ B signaling pathway induced by Del2R versus ASFV-WT at 12 and 20 hours post-infection (hpi). (D and E) PAMs were either mock infected or infected with Del2R or ASFV-WT (MOI = 5). At 12 and 20 hpi, the mRNA levels of IL-1 β and TNF- α (D) in the cell lysates were determined by RT-qPCR, and the production of IL-1 β and TNF- α (E) in the cell culture supernatants were detected by commercial ELISA kits.

Article Snippet: The infected cells were cultured in the fresh RPMI 1640 medium for 12 and 20 h. The concentrations of IL-1 β (Ray Biotech, ELP-IL1b-1), TNF- α (Ray Biotech, ELP-TNFa-1), and IFN- α (Ray Biotech, ELP-IFNa-1) in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

Techniques: Infection, RNA Sequencing Assay, Expressing, Functional Assay, Quantitative RT-PCR, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKK α and IKK β through the recruitment of TOLLIP

doi: 10.1371/journal.ppat.1011580

Figure Lengend Snippet: (A) HEK293T cells were transfected with increasing amounts of the expressing plasmid pHA-MGF300-2R (0.5, 1, and 1.5 μ g) for 24 h and then mock-treated or treated with TNF- α (10 ng/mL) for 8 h. The protein expression of MGF300-2R was examined by western blotting and the luciferase activity was measured at 24 hours post-transfection (hpt). (B) HEK293T cells were transfected with the pFlag-MGF300-2R or empty plasmid and then treated with or without TNF- α (10 ng/mL) for 1 h. At 24 hpt, the cells were fractionated into cytoplasmic and nuclear fractions and analyzed by immunoblotting with the indicated antibodies. The p65 in the nuclear and cytoplasmic compartments was checked by western blotting. Lamin B1 and tubulin were used as nuclear and cytosolic markers, respectively. (C) PAMs were mock-infected or infected with ASFV-WT or Del2R (MOI = 5). At 20 hpi, PAMs were treated with or without TNF- α (10 ng/mL) for 1 h, the separation of cellular fractions of ASFV-infected PAMs was performed as described above. (D) Analysis of the phosphorylation levels of I κ B α and p65 in PAMs mock-infected or infected with ASFV-WT, or Del2R (MOI = 5) by western blotting at 2 and 6 hpi. (E) HEK293T cells were transfected with a plasmid encoding HA-MGF300-2R along with a plasmid encoding Flag-IKK α , Flag-IKK β , or Flag-NEMO as indicated. The cells were lysed and whole cell lysates (WCL) were immunoprecipitated with anti-HA mAb at 36 hpt. The immunoprecipitates were examined by western blotting with the indicated antibodies. (F) HEK293T cells were transfected with expressing plasmids pFlag-IKK α or pFlag-IKK β for 36 h and lysed with NP-40 buffer. The purified GST or GST-MGF300-2R protein was used to pull down the IKK α or IKK β in the lysates and analyzed by western blotting with the indicated antibodies. (G and H) HEK293T cells were transfected with pHA-MGF300-2R alone and cotransfected with pFlag-IKK α or pFlag-IKK β in combination with pHA-MGF300-2R. IKK α , IKK β , and MGF300-2R were analyzed by laser confocal microscopy. Scale bar, 10 μ m. The colocalization of MGF300-2R and IKK α or IKK β was analyzed by the Coloc2 tool of ImageJ/FIJI and shown as Pearson’s correlation coefficients.

Article Snippet: The infected cells were cultured in the fresh RPMI 1640 medium for 12 and 20 h. The concentrations of IL-1 β (Ray Biotech, ELP-IL1b-1), TNF- α (Ray Biotech, ELP-TNFa-1), and IFN- α (Ray Biotech, ELP-IFNa-1) in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

Techniques: Transfection, Expressing, Plasmid Preparation, Western Blot, Luciferase, Activity Assay, Infection, Immunoprecipitation, Purification, Confocal Microscopy

Journal: PLOS Pathogens

Article Title: The MGF300-2R protein of African swine fever virus is associated with viral pathogenicity by promoting the autophagic degradation of IKK α and IKK β through the recruitment of TOLLIP

doi: 10.1371/journal.ppat.1011580

Figure Lengend Snippet: The protein levels of IL-1 β (A), TNF- α (B), and IFN- α (C) in the serum samples were measured by commercial ELISA kits. Serum antibodies against p72 (D) or p30 (E) in the inoculated pigs were detected by ELISA. The results were shown as a blocking percentage. For the anti-p72 antibodies, the blocking rate above 50% was considered positive, while below 40% was considered negative; for the anti-p30 antibodies, the blocking rate below 40% was considered positive, while above 50% was considered negative. Samples with blocking between 40 and 50% were considered doubtful.

Article Snippet: The infected cells were cultured in the fresh RPMI 1640 medium for 12 and 20 h. The concentrations of IL-1 β (Ray Biotech, ELP-IL1b-1), TNF- α (Ray Biotech, ELP-TNFa-1), and IFN- α (Ray Biotech, ELP-IFNa-1) in the cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Blocking Assay