Journal: Cell reports

Article Title: Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions

doi: 10.1016/j.celrep.2020.107633

Figure Lengend Snippet: (A) HEK293T cells were transfected with a plasmid encoding the various ISG15 variants: luciferase (Luc), wild-type ISG15 (WT), c.310G>A, c.352C>T, c.83T>A, c.284del ISG15, and c.297_313del. Relative mRNA levels for ISG15 assessed by qRT-PCR, performed three times for each variant, with technical triplicates; the data for a representative experiment (n = 3) are shown. (B) HEK293T cells were transfected with a plasmid encoding the various ISG15 variants: Luciferase (Luc), wild-type ISG15 (ISG15), c.310G>A, or c.352C>T ISG15. Cell lysates were analyzed by western blotting for ISG15; a representative experiment is shown. (C) HEK293T cells were transfected with a plasmid encoding the various ISG15 variants: Luciferase (Luc), wild-type ISG15 (ISG15), c.83T>A, or c.284del ISG15. Cell lysates were analyzed by western blotting for ISG15; a representative experiment is shown. (D) HEK293T cells were transfected with a plasmid encoding the various ISG15 variants: Luciferase (Luc), wild-type ISG15 (ISG15), or c.297_313del ISG15. Cell lysates were analyzed by western blotting for ISG15; a representative experiment is shown. (E) RNA was isolated from P2 and P3 and subjected to 3’RACE analysis. Schematic diagram of the ISG15 amplicons obtained on 3’RACE RT-PCR. (F) 3′ RACE amplicons were inserted into a TOPO vector for the quantification of each splicing variant. In total, 26 colonies per patient were grown and sequenced. The graph shows the percentage of each of the three splicing variants identified. (G) hTert-immortalized fibroblasts from a control donor (Control), P2, or P3 were treated with 1,000 U/mL of IFN-α2b for 24 h. Cell lysates were analyzed by western blotting with the indicated antibodies; data for a representative experiment are shown. (H) HEK293T cells were transfected with a constant amount of wild-type USP18 (WT) together with various amounts of the different variants of ISG15 (WT or c.310G>A). Cell lysates were analyzed by western blotting with the indicated antibodies. (I) HEK293T cells were transfected with a constant amount of wild-type USP18 (WT) together with various amounts of the different variants of ISG15 (WT, c.284del, or c.83T>A). Cell lysates were analyzed by western blotting with the indicated antibodies. (J) Epstein-Barr virus (EBV)-transformed B cells from a control donor or P1 were stimulated with 1,000 U/mL IFN-α2b for 24 h. Cell lysates were analyzed by western blotting with the indicated antibodies; data from a representative experiment are shown. (K) hTert-immortalized fibroblasts from a control or an ISG15-deficient patient were transduced with luciferase, WT ISG15, or c.310G>A ISG15 and sorted. These fibroblasts were treated with 1,000 U/mL IFN-α2bfor 12 h, washed with PBS, and left to rest for 36 h, after which relative mRNA levels were determined for MX1, three times for each individual, with technical triplicates; a representative experiment is shown. Bars represent the mean ± SD.

Article Snippet: Transduced hTERT fibroblasts were primed by incubation with 0, 10, 100, or 1,000 IU ml −1 IFN-α2b (Merck IntronA) in normal growth medium for 12 h.The IFN-α2b was then eliminated by thorough washing with PBS and the cells were allowed to rest for 36 h in normal medium.

Techniques: Transfection, Plasmid Preparation, Luciferase, Quantitative RT-PCR, Variant Assay, Western Blot, Isolation, Reverse Transcription Polymerase Chain Reaction, Transformation Assay, Transduction

Journal: Cell reports

Article Title: Systemic Type I IFN Inflammation in Human ISG15 Deficiency Leads to Necrotizing Skin Lesions

doi: 10.1016/j.celrep.2020.107633

Figure Lengend Snippet: (A) CRISPR was used to knock out ISG15 in HaCaT cells. The cells were stimulated with 1,000 U/mL IFN-α2b for 24 h. Cells were lysed, and the lysates were analyzed by western blotting with the indicated antibodies; the results of a representative experiment are shown. (B) WT and ISG15-KO HaCaT cells were stimulated with 1,000 U/mL IFN-α2b for 12 and 24 h. Relative mRNA levels for IFIT1, MX1, OAS1, and CXCL10 were assessed by qRT-qPCR. Bars represent the mean ± standard error of the mean (SEM). (C) Human WT and ISG15-deficient iPSCs were differentiated to develop into endothelial cells and stimulated with 1,000 U/mLIFN-α2b for 24 h. Cell lysates were analyzed by western blotting with the indicated antibodies; data from a representative experiment are shown. (D) WT and ISG15-deficient endothelial cells were stimulated with 1,000 U/mL IFN-α2b for 12 and 24 h. Relative mRNA levels for IFIT1, MX1, OAS1, and CXCL10 were determined by qRT-qPCR. Bars represent the mean ± SEM. (E) Representative H&E staining and staining for pSTAT1 (purple) and CD68 (brown) immunohistochemistry (IHC) results for skin biopsies from P1 and a healthy control. Original magnification: 10× (a and e), 40× (b-d, f, and g). Arrows indicate pSTAT1 + cells. Asterisks indicate CD68 + cells (macrophage infiltration).

Article Snippet: Transduced hTERT fibroblasts were primed by incubation with 0, 10, 100, or 1,000 IU ml −1 IFN-α2b (Merck IntronA) in normal growth medium for 12 h.The IFN-α2b was then eliminated by thorough washing with PBS and the cells were allowed to rest for 36 h in normal medium.

Techniques: CRISPR, Knock-Out, Western Blot, Staining, Immunohistochemistry

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: STAT3/5-Dependent IL9 Overexpression Contributes to Neoplastic Cell Survival in Mycosis Fungoides

doi: 10.1158/1078-0432.CCR-15-1784

Figure Lengend Snippet: Patients under photo(chemo)therapy treatment exhibit reduced IL9 production. Samples from the archived materials from patients with MF (ST2) were analyzed for IL9 (A) and IL9r (B) expression at baseline and after 12 weeks at clinical remission (micrographs from patient A1 are shown). The average of positive cells in three 40× fields per sample is presented: adjusted to the unit of area (mm2). Biopsies from the PUVA trial (ST3) were analyzed by qPCR for expression of IL9 (C) and STAT3 (D) at baseline and 6 weeks after start of treatment. E, MyLa2000 cells were transfected with siSTAT5 or siCTRL 24 hours before treated with PUVA in vitro (1 µmol/L 8-MOP, 0.4 J/cm2). F, MyLa2000 cells were given PUVA followed by incubation in media supplemented with IFNα2b (1,500 U/mL), goat polyclonal anti-human IL9 antibody (20 µg/mL) or the two combined. E and F, viability was assessed by Annexin V/PI incorporation after 48 hours of culture. Experiments were done in triplicates; statistical significance was assessed by Student paired t test (A–D), or Dunnet post test (E and F); P values are shown.

Article Snippet: Cells were treated with 1 μmol/L of 8-Methoxypsoralen (8-MOP) 2 hours prior to UVA irradiation, a sublethal dose of UVA light (0.4 or 0.8 J/cm 2 ) was given and the cells were incubated in DMEM supplemented with 1,500 U/mL of IFNα2b (Merck-Millipore, GF417), 20 μg/mL of goat polyclonal anti-human IL9 antibodies (R&D Systems, AB-209-NA).

Techniques: Expressing, Transfection, In Vitro, Incubation

Journal: mAbs

Article Title: A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus

doi: 10.1080/19420862.2015.1055443

Figure Lengend Snippet: Binding activity of AIA22 to different antigens. All the antigens were coated at 5 μg/ml. The optical density (OD) at 492 nm reduced in a dose-dependent manner with decreasing amounts of AIA22 when binding to IFN-α1b and IFN-α2b. No cross-reactivity was observed when AIA22 was incubated with other type I IFN (IFN-β, IFN-ω), type II IFN (IFN-γ), cytokines (VEGF, TNF), and human serum albumin (HSA).

Article Snippet: Specificity and binding activity ELISAs Ninety-six-well microtiter plates (Costar, 9018) were coated with recombinant IFN-α1b, IFN-α2b, IFN-β (Abnova, P3623), IFN-ω (gifted from Professor Wei Chen), IFN-γ, or control antigens in carbonate buffer (pH9.6) and incubated overnight at 4°C.

Techniques: Binding Assay, Activity Assay, Incubation

Journal: mAbs

Article Title: A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus

doi: 10.1080/19420862.2015.1055443

Figure Lengend Snippet: Affinity constant of AIA22 and sifalimumab

Article Snippet: Specificity and binding activity ELISAs Ninety-six-well microtiter plates (Costar, 9018) were coated with recombinant IFN-α1b, IFN-α2b, IFN-β (Abnova, P3623), IFN-ω (gifted from Professor Wei Chen), IFN-γ, or control antigens in carbonate buffer (pH9.6) and incubated overnight at 4°C.

Techniques:

Journal: mAbs

Article Title: A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus

doi: 10.1080/19420862.2015.1055443

Figure Lengend Snippet: Predicted interaction mechanism of AIA22 with IFN-α2b and IFN-α1b. (A) The AIA22/IFN-α2b complex model. Variable region of AIA22 light chain (VL): green, variable region of heavy chain (VH): marine, IFN-α2b: pink. (B) The recognition region mapping of AIA22 (Green), IFNAR2 (Blue), and their overlapping region (Red) on IFN-α2b surface. (C) The predicted interaction interface between AIA22 and IFN-α2b. H1∼H3 and L1∼L3 represent the 6 CDR regions of heavy chain and light chain respectively. IFN-α2b and its residues are labeled as pink. The dotted lines between resides indicate hydrogen-bonds and solid lines indicate π interactions. (D) The predicted interaction interface between AIA22 and IFN-α1b. IFN-α1b and its residues were labeled as yellow. The atoms of Tyr99 (CDR H3) are displayed as CPK scale to clearly reveal its clash with IFN-α1b backbone residues Leu30 and Met31.

Article Snippet: Specificity and binding activity ELISAs Ninety-six-well microtiter plates (Costar, 9018) were coated with recombinant IFN-α1b, IFN-α2b, IFN-β (Abnova, P3623), IFN-ω (gifted from Professor Wei Chen), IFN-γ, or control antigens in carbonate buffer (pH9.6) and incubated overnight at 4°C.

Techniques: Labeling

Journal: mAbs

Article Title: A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus

doi: 10.1080/19420862.2015.1055443

Figure Lengend Snippet: The evaluation results of variants derived from AIA22

Article Snippet: Specificity and binding activity ELISAs Ninety-six-well microtiter plates (Costar, 9018) were coated with recombinant IFN-α1b, IFN-α2b, IFN-β (Abnova, P3623), IFN-ω (gifted from Professor Wei Chen), IFN-γ, or control antigens in carbonate buffer (pH9.6) and incubated overnight at 4°C.

Techniques: Derivative Assay, Activity Assay

Journal: mAbs

Article Title: A fully human monoclonal antibody with novel binding epitope and excellent neutralizing activity to multiple human IFN-α subtypes: A candidate therapy for systemic lupus erythematosus

doi: 10.1080/19420862.2015.1055443

Figure Lengend Snippet: Neutralizing activities of monoclonal antibodies to multiple IFN-α subtypes

Article Snippet: Specificity and binding activity ELISAs Ninety-six-well microtiter plates (Costar, 9018) were coated with recombinant IFN-α1b, IFN-α2b, IFN-β (Abnova, P3623), IFN-ω (gifted from Professor Wei Chen), IFN-γ, or control antigens in carbonate buffer (pH9.6) and incubated overnight at 4°C.

Techniques: Concentration Assay, Activity Assay