Journal: OncoImmunology

Article Title: IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer

doi: 10.1080/2162402x.2021.1880687

Figure Lengend Snippet: Figure 4. Evaluation of IgG4 expression in CRC tumor site. (a) One representative immunohistochemistry staining of formalin fixed paraffin embedded colon cancer slides against IgG4 (brown spots). (b) Highlighted area in red was magnified and IgG4 (brown spots) are marked with white arrows. (c) Quantification of IgG4 cell positivity in ten different tumor slides. We found significantly more IgG4 positive cells in the stroma site than in the tumor site microenvironment (Paired two tails Student’s t test, p < .0001). (d) One representative IF staining of formalin fixed paraffin embedded colon cancer slides against CD38 positive B cells (green), against IgG4 (red), and against CD68 positive macrophages cells (white). (e) Highlighted area in red was magnified to show the close contact between CD38+ green B cell expressing IgG4 (red spots) and CD68+ (white) macrophages.

Article Snippet: Alternatively, a fluorescent triple staining was performed using: 1) mouse anti-human IgG4 clone HP6025 1:500 (Genway Biotech); 2) rabbit antihuman CD38 clone EPR4106 dilution 1:100 (Abcam); 3) goat anti-human CD68 clone C-18: sc-7082 dilution 1:100 (Santa Cruz Biotechnology).

Techniques: Expressing, Immunohistochemistry, Staining, Formalin-fixed Paraffin-Embedded

Journal: OncoImmunology

Article Title: IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer

doi: 10.1080/2162402x.2021.1880687

Figure Lengend Snippet: Figure 5. Production of M2b-like cytokines/chemokines by M2a macrophages treated with mIgG1 or mIgG4 antibodies. The supernatants of monocyte-derived macrophages (MDM) from the PBMCs of healthy donors or primary tumor patients or metastatic tumor patients were polarized ex vivo for 72 h. M2a macrophages treated ex vivo with plate-bound myeloma IgG1 (mIgG1) or plate-bound myeloma IgG1 (mIgG4) or left untreated were evaluated by cytokine ELISA for the detection of CCL-1, IL-10 and IL-6 production. Our results showed an increase of CCL-1, IL-10 and IL-6 production by M2a macrophages treated with mIgG4, similarly to M2b-like cytokine and chemokine production. These findings were true for all PBMC donor groups. The z-normalization of pg/ml concentration was performed for each marker and each donor before the statistical validation. Gray bars: unstimulated M2a cells; white bars: M2a cells + mIgG1; black bars: M2a cells + mIgG4. The results from thirty- one independent experiments were combined for statistical analysis. One-way ANOVA with Tukey post-hoc test was performed. n.s. P > .05, * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: Alternatively, a fluorescent triple staining was performed using: 1) mouse anti-human IgG4 clone HP6025 1:500 (Genway Biotech); 2) rabbit antihuman CD38 clone EPR4106 dilution 1:100 (Abcam); 3) goat anti-human CD68 clone C-18: sc-7082 dilution 1:100 (Santa Cruz Biotechnology).

Techniques: Derivative Assay, Ex Vivo, Enzyme-linked Immunosorbent Assay, Concentration Assay, Marker, Biomarker Discovery

Journal: OncoImmunology

Article Title: IgG4 induces tolerogenic M2-like macrophages and correlates with disease progression in colon cancer

doi: 10.1080/2162402x.2021.1880687

Figure Lengend Snippet: Figure 6. Anti-human EGFR-IgG1 or IgG4 subclass effects on the ADCP and ADCC of tumor cell lines by human U937 monocytes. U937 in vitro mediated mainly ADCP (a) and lower levels of ADCC (b) of EGFR differently expressing A431, HT-29 and CaCo2 tumor cell lines (c). The percentage of tumor dead cells was evaluated by flow cytometric analysis following the gating strategy represented on Repository figure 2. Black bars: A431 cells; white bars: HT-29 cells; gray bars: CaCo2 cells. The results from three independent experiments were combined for statistical analysis. One-way ANOVA with Tukey post-hoc test was performed. ** P < .01.

Article Snippet: Alternatively, a fluorescent triple staining was performed using: 1) mouse anti-human IgG4 clone HP6025 1:500 (Genway Biotech); 2) rabbit antihuman CD38 clone EPR4106 dilution 1:100 (Abcam); 3) goat anti-human CD68 clone C-18: sc-7082 dilution 1:100 (Santa Cruz Biotechnology).

Techniques: In Vitro, Expressing

Journal: Comprehensive Physiology

Article Title: Porcine Fetal Hypothyroidism Induces Temporal and Tissue‐Specific Alterations in the Insulin‐Like Growth Factor System

doi: 10.1002/cph4.70028

Figure Lengend Snippet: IGFBP levels in fetal serum. (A) Example Western Ligand Blot showing human recombinant protein standards in the left lane, with the right lane showing the six bands detected in fetal porcine serum. Relative density of (B) a 26 kDa IGFBP, (C) a 28 kDa IGFBP, (D) IGFBP2, (E) the 40 kDa IGFBP3, and (F) IGFBP4 in fetal porcine serum derived from fetuses ( n = 11–12/group) at various gestational timepoints following 21 days of maternal CON or MMI treatment. Relative density was calculated relative to a consistent reference sample run on each gel, with unique letter superscripts denoting statistically significant ( p < 0.05) changes in density in the CON group over time, and statistical differences between the CON and MMI group within each timepoint denoted as * p < 0.05, ** p < 0.01, and *** p < 0.001. Data are presented as the estimated marginal means with 95% confidence intervals, as extracted from a linear model including gestational day, treatment group, and the interaction.

Article Snippet: In addition to samples of interest, each blot contained a lane of pooled recombinant human protein standards representing IGFBPs ‐1, ‐2, ‐3, and ‐4 (R&D Systems), which were used as reference points for identification of bands of interest, as well as a lane of pooled fetal serum which was used to calculate the relative density of each band of interest for analysis.

Techniques: Western Blot, Recombinant, Derivative Assay