Journal: Cells

Article Title: Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

doi: 10.3390/cells11060942

Figure Lengend Snippet: Treatment of keratinocytes with hAK23 and 4B3 antibodies results in Dsg3 depletion, and 4B3 abrogates FcRn protein level. ( a ) hTert cells were plated on 6-well plates, grown in KGM2 medium with 0.05 mM CaCl 2 until confluent, and then switched to KGM2 with 2 mM CaCl 2 for 24 h. Thereafter, the cells were treated for 24 h with the recombinant antibodies hAK23 (12.5 µg/mL) or 4B3 (50 µg/mL). As controls, mock incubation (untreated), an isotype-matched hIgG (12.5 µg/mL IgG4, or 50 µg/mL hIgG1), and mAK23 (75 µg/mL) were used. The cells were lysed, and the level of Dsg3, Dsg1 and FcRn was analyzed by Western blot. GAPDH was included as a loading control. A representative experiment is shown. ( b – d ) Western blot signals were quantified using ImageJ software, normalized against GAPDH, and expressed as relative values compared to the untreated control. The error bars represent the SD of values obtained from four independent experiments. Statistical analysis was done using one-way analysis of variance (ANOVA) with Dunnett´s post-test. Statistically significant differences are indicated by ** = p ≤ 0.01; *** = p ≤ 0.001.

Article Snippet: For visualization of the pathogenic effect, the cells were incubated for 24 h at 37 °C with Alexa Fluor 488-coupled hAK23 (12.5 μg/mL) or 4B3 (50 μg/mL) or an isotype matched human IgG4 (12.5 μg/mL, Cat. DDXCHO1A488-100, Novus Biologicals/Bio-Techne, Wiesbaden, Germany) or IgG1 (50 μg/mL, Cat. DDXCHO4A488-100, Novus Biologicals) antibodies.

Techniques: Recombinant, Incubation, Western Blot, Control, Software

Journal: Cells

Article Title: Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

doi: 10.3390/cells11060942

Figure Lengend Snippet: Efgartigimod treatment protects keratinocyte monolayers against dissociation induced by PV IgG. hTert keratinocytes were plated on 24-well plates, grown in KGM2 medium with 0.05 mM CaCl 2 until confluent, and then switched to KGM2 with 2 mM CaCl 2 for 24 h. The cells were left untreated, or efgartigimod (25 µg/mL) was applied for 30 min prior to the 24 h treatment with hAK23 (12.5 µg/mL), control IgG4 (12.5 µg/mL), PV IgG (150 µg/mL) or human control IgG (150 µg/mL). Monolayer dissociation assay was performed in triplicates, and the number of fragments was quantified using ImageJ software. A representative experiment is shown. The error bars show the SD of the mean values obtained from five independent experiments. Statistical analysis was done using two-way ANOVA with Bonferroni’s post-test. Statistically significant differences are indicated by *** = p ≤ 0.001.

Article Snippet: For visualization of the pathogenic effect, the cells were incubated for 24 h at 37 °C with Alexa Fluor 488-coupled hAK23 (12.5 μg/mL) or 4B3 (50 μg/mL) or an isotype matched human IgG4 (12.5 μg/mL, Cat. DDXCHO1A488-100, Novus Biologicals/Bio-Techne, Wiesbaden, Germany) or IgG1 (50 μg/mL, Cat. DDXCHO4A488-100, Novus Biologicals) antibodies.

Techniques: Control, Software

Journal: Cells

Article Title: Stabilization of Keratinocyte Monolayer Integrity in the Presence of Anti-Desmoglein-3 Antibodies through FcRn Blockade with Efgartigimod: Novel Treatment Paradigm for Pemphigus?

doi: 10.3390/cells11060942

Figure Lengend Snippet: Efgartigimod does not rescue the aberrant Dsg3 staining pattern induced by hAK23 and 4B3. hTert keratinocytes were cultured on coverslips in KGM2 medium with 0.05 mM CaCl 2 for at least three days, and then switched to KGM2 with 2 mM CaCl 2 for 24 h. The cells were either mock-treated, or efgartigimod (25 µg/mL) was applied for 30 min, after which a 24 h treatment with the recombinant antibodies hAK23 (12.5 µg/mL), 4B3 (50 µg/mL), an isotype-matched human IgG4 (12.5 µg/mL) or IgG1 control (50 µg/mL) coupled to Alexa Fluor 488 (green) was initiated. After methanol fixation, the control IgG-treated cells were stained with the 5H10 anti-Dsg3 antibody, detected with a fluorochrome coupled secondary antibody (anti-mouse Alexa Fluor 546, red). Representative images from one out of at least three independent experiments are shown. Scale bar: 20 µm.

Article Snippet: For visualization of the pathogenic effect, the cells were incubated for 24 h at 37 °C with Alexa Fluor 488-coupled hAK23 (12.5 μg/mL) or 4B3 (50 μg/mL) or an isotype matched human IgG4 (12.5 μg/mL, Cat. DDXCHO1A488-100, Novus Biologicals/Bio-Techne, Wiesbaden, Germany) or IgG1 (50 μg/mL, Cat. DDXCHO4A488-100, Novus Biologicals) antibodies.

Techniques: Staining, Cell Culture, Recombinant, Control

Journal: Brain

Article Title: Imaging protein aggregates in the serum and cerebrospinal fluid in Parkinson’s disease

doi: 10.1093/brain/awab306

Figure Lengend Snippet: Aggregates with pathogenic β-sheet structure detected using AD-PAINT following immunodepletion of α-syn and amyloid-β protein aggregates in Parkinson’s disease ( n = 11) and healthy control ( n = 10) serum samples from two independent cohorts pooled together. ( A ) Quantification of the relative content (%) of β-sheet α-syn and amyloid-β aggregates in Parkinson’s disease (PD) versus healthy control (HC) serum samples. The percentage of α-syn or amyloid-β aggregates in each sample was determined as the difference in the number of detected aggregates between the neat and α-syn or amyloid-β immunodepleted serum normalized to their sum (α-syn + amyloid-β). In turn, the data for neat, IgG1 control, α-syn and amyloid-β immunodepleted serum of each subject were normalized to the sum of the aggregate number in α-syn and amyloid-β immunodepleted samples (α-syn + amyloid-β ImDep). ( B ) Quantification of the β-sheet α-syn/amyloid-β ratio retrieved from A for the same serum samples. In A and B , different subjects are indicated by a specific empty symbol for the first independent cohort and a specific filled symbol for the second cohort of samples (legend). The data are shown as mean ± SD, and the lower and upper boundaries of the box indicate the 25th and 75th percentiles, respectively. The statistical significance for the difference in the serum aggregate composition between Parkinson’s disease and healthy control groups was established by the permutation (exact) test. * P < 0.05, ** P < 0.01. ( C ) ROC analysis for disease status classification by relative β-sheet α-syn content (optimal threshold = 0.41) as well as the β-sheet α-syn/amyloid-β ratio (optimal threshold = 0.7) showing high performance of each tested biomarker (AUC = 98.2% for each). ( D ) Examples of super-resolved α-syn and amyloid-β aggregates present in Parkinson’s disease and healthy control serum. Scale bar = 100 nm.

Article Snippet: Immunodepletion of α-syn and amyloid-β proteins from serum samples ( n = 11 Parkinson’s disease and 10 control subjects, assayed in duplicate) was performed using the following immunoprecipitation protocol: 5 μg of purified mouse anti-α-syn antibody (1:10, 250 μg/ml, 15–123, SYN-1, BD Transduction Laboratories) or purified mouse anti-amyloid-β antibody (1:40, 1 mg/ml, 1–16, 6E10, BioLegend) or mouse IgG1 isotype control antibody (1:40, 1 μg/ml, COLIS69A, Kingfisher Biotech) (a negative control) were incubated with 1.5 mg magnetic Dynabeads ® protein G (30 mg/ml, Invitrogen) in a total volume of 200 μl 0.02% Tween-20 (PBST) in separate 1.5 ml Protein LoBind Tubes (Eppendorf AG) on a rotating wheel at 4°C for 2 h. After spinning down the immunoprecipitated antibody-bead complexes, the Eppendorf tubes were placed on a magnetic rack and the supernatants were aspirated.

Techniques: Immunodepletion, Control, Biomarker Discovery

Journal: Frontiers in Immunology

Article Title: Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells

doi: 10.3389/fimmu.2023.1229341

Figure Lengend Snippet: Characterization of the isolated recombinant sPD-1 protein. (A) Gel-based proteomic analysis of purified sPD-1. Left panel: Coomassie staining of the isolated recombinant sPD-1 revealed a band of approximately 20 kDa corresponding to sPD-1 molecular weight. BSA sample was used as a control. Right panel: western blot analysis allowed detection of the recombinant sPD-1 protein with both the anti-Streptavidin-HRP and anti-PD-1 antibodies. (B) SKNAS and IMR-32 cell lines were analysed for PD-L1 and PD-L2 expression. Only PD-L1 could be detected on the surface of both cell lines. (C) Analysis of recombinant sPD-1 binding to PD-L1 ligand. Flow cytometry analysis demonstrated that recombinant sPD-1 was able to bind PD-L1 expressed by SKNAS and IMR-32 cell lines. (D) SKNAS were incubated with PBS, IgG1 Isotype or Atezolizumab and then analysed for PD-L1 expression. Abrogation of PD-L1 signal occurred only when tumor cells were treated with Atezoliumab (left panel). Binding of sPD-1 to tumor expressed PD-L1 was abrogated when NB cells were previously incubated with Atezolizumab. (E) Incubation of PD-L1 + SKNAS and IMR-32 cell lines with recombinant sPD-1 resulted in a decreased PD-L1 signal. MFI from ctrl (not stained) samples was subtracted from their corresponding stained samples. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **; p<0.05 *.

Article Snippet: Binding of Nivolumab (A2002, Selleckchem) to PD-1 was performed incubating stimulated NK cells with IgG4 control Isotype (DDXCH04P-100, Novus Biological) or Nivolumab for 30 min at 4°C and then with anti-PD1 for 30 min at 4°C.

Techniques: Isolation, Recombinant, Purification, Staining, Molecular Weight, Control, Western Blot, Expressing, Binding Assay, Flow Cytometry, Incubation

Journal: Frontiers in Immunology

Article Title: Analysis of the mechanisms regulating soluble PD-1 production and function in human NK cells

doi: 10.3389/fimmu.2023.1229341

Figure Lengend Snippet: Recombinant sPD-1 can modulate NK cell effector function. (A) Control and stimulated NK cells were analysed for membrane PD-1 and PD-L1 expression. Membrane-bound PD-1 was detected only in stimulated NK cells compared to control cells. Conversely, while Ctrl NK cells expressed PD-L1, it was barely detectable on stimulated NK cells. One representative experiment has been shown. Quantifications of four different experiments have been reported. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **; p< 0.001 ***. (B) NB cell lines were incubated with stimulated NK cells, expressing PD-1, with or without recombinant sPD-1. An increase in cytotoxicity toward both SKNAS (n=7) and IMR-32 (n=8) cell lines at different Effector (E) Target (T) ratios was observed only when recombinant sPD-1 was present in the co-culture. Data have been compared using paired T test, p< 0.05 *; p< 0.01 **, p< 0.001 ***. (C) Similarly, in the presence of recombinant sPD-1 an increase in degranulation (left and middle panels) and accumulation of IFN-γ (right panel) of NK cells were observed. Values are mean ± SEM. Statistical significance has been determined by Paired T test, p< 0.01 **, p< 0.001 ***. (D) Comparison of sPD-1 and Nivolumab treatments on cytotoxicity of stimulated NK cells. Incubation with Nivolumab abrogated PD-1 signal compared to Ctrl and IgG4 samples (left panel). Increase of cytotoxicity toward SKNAS cell line at different E:T ratio was observed in both the analysed conditions (treated or not with Nivolumab) only in the presence of sPD-1. Values are mean ± SEM. Data have been compared using paired T test, p< 0.05 *; p< 0.01 **, p< 0.001 ***.

Article Snippet: Binding of Nivolumab (A2002, Selleckchem) to PD-1 was performed incubating stimulated NK cells with IgG4 control Isotype (DDXCH04P-100, Novus Biological) or Nivolumab for 30 min at 4°C and then with anti-PD1 for 30 min at 4°C.

Techniques: Recombinant, Control, Membrane, Expressing, Incubation, Co-Culture Assay, Comparison