Journal:

Article Title: Extended Analysis of the In Vitro Tropism of Porcine Endogenous Retrovirus

doi:

Figure Lengend Snippet: Relative susceptibility of nonhuman cell lines to infection by PERV-NIH-2°-derived PERV pseudotypes

Article Snippet: 293 human embryonic kidney (ATCC CRL-1573), MMK Mus musculus molossinus kidney (ATCC CRL-6439; no longer available), and SC-1 mouse embryo epithelial (ATCC CRL-6450) cells, MDTF Mus dunni tail fibroblasts (kindly provided by Olivier Danos), NRK normal rat kidney cells (ATCC CRL-6509), Rat-2 rat embryo fibroblasts (ATCC CRL-1764), SIRC rabbit corneal fibroblasts (ATCC CCL-60), D17 canine oseosarcoma (ATCC CRL-6248), MDBK bovine kidney epithelium (ATCC CCL-22), FRhK4 rhesus monkey kidney (ATCC CRL-1688), Mv1Lu mink lung epithelial (ATCC CCL-64), and MiCL.1 (S+L−) mink lung (ATCC CCL-64.1) cells, Fc3TG feline tongue fibroblasts (ATCC CCL 176), and AK-D feline lung epithelial (ATCC CCL-150), CRFK feline kidney epithelial (ATCC CCL-94), CaKi-1 clear cell kidney carcinoma (ATCC HTB-46), HeLa cervical adenocarcinoma (ATCC CCL-2), HOS human osteosarcoma (ATCC CRL-1543), and CaCO-2 colorectal adenocarcinoma (ATCC HTB-37) cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, 1 mM sodium pyruvate, 100 U of penicillin per ml, and 100 μg of streptomycin per ml.

Techniques: Infection, Control

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: L1CAM promotes the recruitment of Tregs by upregulating CCL22. (A) Volcano plot of differentially expressed genes (DEGs), on the basis of the median value of the L1CAM mRNA expression level. (B) The upregulated DEGs were used to perform GO enrichment analysis. (C) The secretion of cytokines and chemokines in the supernatants of shNC and shL1CAM-EC1 cells (up) or scramble and OE-L1CAM-KYSE450 cells (down) cultured for 48 h was assessed with a human multiplex bead-based kit. The expression of CCL22 was detected by qRT-PCR (D) and ELISA (E) between shNC and shL1CAM-EC1 cells, or scramble and OE-L1CAM-KYSE450 cells. (F) Expression of L1CAM and CCL22 mRNAs in the different groups tumor samples derived from Figure 2H . (G) IHC of tumor tissues in different groups derived from Figure 2H . Representative images are shown (200×). (H) The IHC scores of L1CAM and CCL22 in different groups. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: On days 23 and 26 after tumor inoculation, the nude mice were randomly selected for treatment via intraperitoneal injection with the IgG control (IgG2b: R&D Systems) or CCL22 neutralizing antibody (500 ng/mL; R&D, MAB336).

Techniques: Expressing, Cell Culture, Multiplex Assay, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: L1CAM promotes the recruitment of Tregs through CCL22 in vivo . (A) The correlation between FOXP3 and CCL22 in the ESCC TCGA database, n = 94. (B) The purity of CD4 + CD25 + CD127 - Tregs sorted from the peripheral blood of patients with ESCC, according to FACS. (C) The numbers of migrating Tregs obtained from ESCC patient blood samples was calculated for the shNC, shL1CAM, shL1CAM + recombinant protein CCL22 (100 ng/mL), shL1CAM + PBS, scramble, OE-L1CAM, OE-L1CAM + CCL22 antibody (500 ng/mL), and OE-L1CAM + IgG groups. (D) Flow chart of the in vivo experiments. (E) mRNA expression of FOXP3 and CCR4 in the indicated groups. (F) Percentage of CD4 + FOXP3 + Tregs and FOXP3 + CCR4 + Tregs recruited to tumor sites, as analyzed by flow cytometry. (G) Expression of FOXP3 in tumor tissues of different groups, determined by IHC (200×). (H) The expression of p-Akt, Akt, p-NF-κB, NF-κB, and CCL22 was detected by Western blot between shL1CAM and OE-L1CAM cells treated with or without p-Akt inhibitor (RF-04691502) and p-NF-κB inhibitor (QNZ). β-actin expression was used as a loading control. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: On days 23 and 26 after tumor inoculation, the nude mice were randomly selected for treatment via intraperitoneal injection with the IgG control (IgG2b: R&D Systems) or CCL22 neutralizing antibody (500 ng/mL; R&D, MAB336).

Techniques: In Vivo, Recombinant, Expressing, Flow Cytometry, Western Blot

Journal: Cancer Biology & Medicine

Article Title: L1CAM overexpression promotes tumor progression through recruitment of regulatory T cells in esophageal carcinoma

doi: 10.20892/j.issn.2095-3941.2020.0182

Figure Lengend Snippet: High expression of CCL22 predicts poor survival in patients with ESCC. (A–B) qRT-PCR analysis of CCL22 expression in paired fresh tissues from 47 patients with ESCC. GAPDH was used to normalize the data, which were analyzed with the 2 -ΔΔCt method. (C) Kaplan–Meier survival analysis of overall survival on the basis of high ( n = 24) and low ( n = 23) CCL22 expression by qRT-PCR. (D) The percentage of CD4 + FOXP3 + Tregs in tumor tissues obtained from patients with ESCC. Correlations between CD4 + FOXP3 + Tregs and L1CAM (E), CD4 + FOXP3 + Tregs, and CCL22 (F), L1CAM, and CCL22 in the tumor tissues of patients with ESCC (G) were evaluated with linear regression analysis. (H) Schematic diagram of the proposed molecular mechanism: L1CAM promotes the recruitment of Tregs into the tumor site through PI3K/Akt/NF-κB-CCL22-CCR4. Tregs produce TGF-β, which further induces L1CAM upregulation in ESCC tumor cells. ** P < 0.01.

Article Snippet: On days 23 and 26 after tumor inoculation, the nude mice were randomly selected for treatment via intraperitoneal injection with the IgG control (IgG2b: R&D Systems) or CCL22 neutralizing antibody (500 ng/mL; R&D, MAB336).

Techniques: Expressing, Quantitative RT-PCR

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Impact of prolonged stimulation by Tumor CM on the release of inflammatory chemokines by the resulting CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or MCF-7 cells (B) over a prolonged period of time (~30 days; MSCs + MDA CM or MSCs + MCF-7 CM, respectively). Twenty-four hours after medium exchange to fresh Tumor CM, cell supernatants were collected and the expression of CCL2 (A1, B1), CXCL8 (A2, B2) and CCL5 (A3, B3) was determined in comparison with supernatants of MSCs that were not supplemented with CM (MSCs) and with the original Tumor CM of MDA-MB-231 or MCF-7 cells alone (MDA CM or MCF-7 CM, respectively). Chemokine expression was determined by ELISA, in the linear range of absorbance. (A1), (A2), (B1) Representatives of n = 3 independent experiments that have shown similar results. (A3), (B2), (B3) Ratios between MSCs and MSCs + Tumor CM were not consistent in different experimental repeats. Therefore, in these panels, the findings are presented as mean ± standard deviation of normalized values (MSCs were given the value of 1) obtained in relevant experimental repeats (at least n = 3).

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Cell Culture, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α induces potent elevation in inflammatory traits in MSCs and Tumor-CM-generated CAFs. Human BM-derived MSCs were cultured with Tumor CM from MDA-MB-231 cells (MDA) (A) or from MCF-7 cells (B) over a prolonged period of time (~30 days). TNF-α (50 ng/ml) or its vehicle (in control cells) was added for the last 24 hours. Expression of CCL2, CXCL8 and CCL5 was then determined in supernatants of the cells. (A) Expression of the chemokines in the four experimental groups included in the study: MSCs grown in culture for ~30 days without any additional stimulus (MSCs); MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells (MSCs + MDA CM); MSCs grown in culture for ~30 days and stimulated by TNF-α at the last 24 hours of culture (MSCs + TNF-α); and MSCs grown in culture for ~30 days in the presence of Tumor CM derived from MDA-MB-231 cells and stimulated by TNF-α at the last 24 hours of culture (MSCs + MDA CM + TNF-α). Expression of CCL2 (A1), CXCL8 (A2) and CCL5 (A3) was determined by ELISA, in the linear range of absorbance. (B) Experimental design as in (A), but with MCF-7-derived CM. In each panel, the findings are representatives of at least n = 3 experiments that have shown similar results. In comparisons between MSCs and all other groups: * P <0.05, ** P ≤0.01, *** P ≤0.001. NS, not significant. # Differences between the two indicated groups have shown variability in n ≥ 3 independent experiments and overall were not significant.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Generated, Derivative Assay, Cell Culture, Control, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Basal and cytokine-induced release of inflammatory chemokines by patient CAFs. CAFs were isolated from lung metastasis of a breast cancer patient (CAFs #1) or from a primary tumor of a different breast cancer patient (CAFs #2). (A1, B1) Expression of the pro-malignancy chemokines CCL2, CXCL8 and CCL5 was determined by ELISA, in the linear range of absorbance. (A2, B2) Expression of CCL5 was determined following TNFa and IL-1β stimulation (TNF-α, 50 ng/ml; IL-1β, 500 pg/ml; 48 hours). Control cells were stimulated by vehicle. Expression of the chemokines was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Isolation, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: TNF-α upregulates the inflammatory profile of MSCs via receptors TNF-RI and TNF-RII. (A) Relative induction of CCL2, CXCL8 and CCL5 in MSCs by TNF-α. Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 24 hours and the expression of the inflammatory chemokines CCL2, CXCL8 and CCL5 in supernatants of MSCs was determined by ELISA, in the linear range of absorbance. Results are from an experiment in which all three chemokines were analyzed in parallel, and the ratios between the three chemokines are representatives of the values obtained in many experimental repeats. (B) Human BM-derived MSCs were exposed to neutralizing antibodies against tumor necrosis factor receptors TNF-RI and TNF-RII or nonrelevant isotype-matched control antibodies (Control Ab) 1 hour prior to stimulation with TNF-α (50 ng/ml), and in the course of 24 hours of stimulation by the cytokine. Control, cells stimulated with vehicle. Expression of CCL2 was determined by ELISA, in the linear range of absorbance. The findings are representatives of at least n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Expressing, Enzyme-linked Immunosorbent Assay, Control

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: CCL2 is expressed by CAFs localized in vicinity to breast cancer cells in tumors of invasive ductal carcinoma (IDC) patients. CCL2 expression was determined by immunohistochemistry in biopsy sections of patients diagnosed with IDC: (A) IDC #1, (B) IDC #2. CCL2 staining was detected in cancer cells and in adjacent fibroblasts. Some of the CCL2-expressing CAFs are indicated by arrows.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Expressing, Immunohistochemistry, Staining

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: Induction of CCL2 and CLXL8 in TNFα-stimulated MSCs is not mediated via the AP-1 pathway. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 5 and 10 minutes. Control cells were treated by the vehicle of TNF-α. c-Jun levels and phosphorylation were determined by western blot (WB) analyses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Human BM-derived MSCs were transiently transfected by small interfering RNA (siRNA) to c-Jun or by control siRNA. (B1) c-Jun expression was determined by WB analyses. β-Tubulin was used as loading control. (B2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; in this part of the study we used a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 24 hours. Expression levels of CCL2 and CXCL8 in the supernatants of the cells were determined by ELISA, in the linear range of absorbance. # siRNA to c-Jun has yielded minor increases or reductions in CCL2 and CXCL8 secretion in different experiments (see Results and discussion), and thus overall there was no significant effect on CCL2 and CXCL8 secretion. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Phospho-proteomics, Western Blot, Transfection, Small Interfering RNA, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: NF-κB is essential in mediating TNF-α-induced release of chemokines by MSCs. (A) Human BM-derived MSCs were stimulated by TNF-α (50 ng/ml) for 15 minutes. The levels of IκBα (the negative regulator of the NF-κB pathway) were determined by WB analyses. GAPDH was used as a loading control throughout. (B) CAFs were generated by culturing MSCs with Tumor CM from MDA-MB-231 (MDA) or MCF-7 breast tumor cells over a prolonged period of time (~30 days). TNF-α (50 ng/ml) was added for the last 24 hours to MSCs + Tumor CM cells and IκBα levels were determined by WB analyses. (C) CAF #1 cells were stimulated for 48 hours by TNF-α (50 ng/ml). IκBα levels were determined by WB analyses. (D) Human BM-derived MSCs were stimulated with TNF-α (50 ng/ml) for 10 minutes. p65 phosphorylation was determined by WB analyses. (E) Human BM-derived MSCs were transiently transfected by siRNA to p65 or by control siRNA. (E1) p65 expression was determined by WB analyses. (E2) Following siRNA transfection, the cells were stimulated by TNF-α (25 ng/ml; a suboptimal concentration of TNF-α in order to facilitate detection of inhibitory effects) for 48 hours. Expression of CCL2 and CXCL8 in the supernatants of the cells was determined by ELISA, in the linear range of absorbance. In all panels, the findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Derivative Assay, Control, Generated, Phospho-proteomics, Transfection, Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Stem Cell Research & Therapy

Article Title: Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-α and the NF-κB pathway

doi: 10.1186/s13287-015-0080-7

Figure Lengend Snippet: MSCs promote monocyte migration through TNF-α-induced secretion of CCL2. Supernatants (sups) were collected from human BM-derived MSCs that were stimulated by TNF-α (50 ng/ml) or by its vehicle for 24 hours. Thereafter, the sups were incubated with neutralizing antibodies for CCL2 or with nonrelevant isotype-matched control antibody (I.C.) for 30 minutes. The migration of monocytic cells in response to control medium (without chemokine), to recombinant human CCL2 (rhCCL2; 100 ng/ml) or to sups from TNF-α-stimulated or TNF-α nonstimulated MSCs was determined by Boyden chamber migration assays. Cells were counted under a high-power field (HPF). The findings are representatives of n = 3 independent experiments that have shown similar results.

Article Snippet: Supernatants from control and from stimulated cells were then collected and divided into groups as follows: supernatants from untreated control MSCs; supernatants from TNF-α-stimulated MSCs, treated by neutralizing antibodies against CCL2 (2 μg/ml, #MAB679; R&D Systems); and supernatants from TNF-α-stimulated MSCs, treated by isotype-matched nonrelevant antibody (2 μg/ml, #401201; BioLegend).

Techniques: Migration, Derivative Assay, Incubation, Control, Recombinant

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: (A) Melanoma cells commonly metastasize to the lung, generating surface lesions detectable as pigmented spots. (B) In human melanoma, Treg are detectable by nuclear FoxP3 expression (red) and membrane CD3 staining (blue) resulting in double stained cells (purple arrows) that can be enumerated as a fraction of total T cells (C) Vaccination using plasmid DNA encoding Ccl22 is applied to ventral murine skin to provide proof of concept for (D) diverting Treg away from the circulation and towards skin.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Expressing, Membrane, Staining, Plasmid Preparation

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: (A) Expression of melanoma marker TRP-1 in a spinal metastasis is inversely related to (B) FoxP3 expression detected just outside a melanoma lesion whereas (C) such FoxP3 expression colocalizes with CCR4 chemokine receptor expression and (D) faint CCL22 expression detected in the same area. (E) CCR8 chemokine receptor expression and TRP-1 expression are likewise readily mutually exclusive whereas (F) CCL1 expression again colocalizes with its receptor CCR8 in areas surrounding the metastasis. In metastatic lesions of the (G) lung, (H) lymph node and (I) brain, similar distributions of CCL22-expressing cells are observed. Under (J) we quantified CCL22 expression in 3 melanoma in situ samples compared to 4 unaffected adult skin samples, detecting 5-fold upregulated chemokine expression whereas (K) an 8-fold upregulation of CCL1 expression was observed.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Expressing, Marker, In Situ

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: To demonstrate that Ccl22 vaccination increases the fraction of skin Treg without converting T cells into Treg, we exposed 3 samples of T cells from a FoxP3 reporter mouse to Ccl22 or to TGF-β overnight. Splenocytes were gated for CD3+ T cells and exposed to CD4 and CD25 immunostaining. (A) FoxP3+CD25+ Treg in a representative untreated sample, (B) Treg abundance is increased in a representative sample of TGFβ treated cells whereas (C) exposure to Ccl22 does not induce a Treg phenotype in vitro. (D) Tregs are quantified, demonstrating a significant increase in response to 2 and 20 ng/mL TGF-β of up to 60%. (E) In a representative transwell migration assay among 3 performed, Treg abundance is increased among splenocytes migrating towards Ccl22, whereas (F) preferential Treg migration is not observed towards Ccl1.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Immunostaining, In Vitro, Transwell Migration Assay, Migration

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: (A) In a representative experiment of 2 performed, 5 mice/group were gene gun vaccinated with Ccl22-encoding or empty vector DNA as shown. (B) At euthanasia, lung tissues were collected to identify tumors (C) Significantly reduced pulmonary tumor counts were observed in Ccl22 vaccinated mice as quantified for 5 mice/group (P<0.05). (D) In therapeutic experiments, mice were tumor challenged 3 days before vaccination applied every 5 days as shown (E) Examples of pulmonary tissues from the Ccl22 (n=7) and empty vector control (n=8) groups. (F) Trimmed means (highest and lowest values removed from each group) were used to calculate that significantly reduced portions of the lung surface were covered by tumor in Ccl22 mice (representative experiment of 2 performed).

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Plasmid Preparation

Journal: Cancer research

Article Title: CCL22 DIVERTS T REGULATORY CELLS AND CONTROLS THE GROWTH OF MELANOMA

doi: 10.1158/0008-5472.CAN-16-0618

Figure Lengend Snippet: Treg in formalin-fixed cryosections by immunofluorescent double staining. CD3 shown in green and FoxP3 staining in red. DAPI counterstaining was used. Shown is a sequence representing (A) FoxP3 stained and (B) CD3 stained lung tissue as well as (C) an overlay, in an untreated mouse, including DAPI counterstaining and (D) the number of Treg was quantified as a ratio to total infiltrating T cells in pulmonary tissue, revealing a marked reduction in Ccl22 vaccinated mice (n=3, *P<0.05), representative experiment of 2 evaluated. The overall abundance of pulmonary (E) T cells and (F) NK cells was not affected. (G) Example TUNEL stainings of lung tissue from empty vector (top) and Ccl22 (bottom) treated mice in a prophylactic experiment. (H) The number of Treg is elevated in the skin of vaccinated mice. (I) Tregs in a sample of Ccl22 vaccinated skin.

Article Snippet: Primary antibodies include clones Ta99 to TRP-1 (Covance, Madison, WI), polyclonal rabbit antiserum to mouse and human FoxP3 (ThermoFisher, Rockford, IL) or MF-14 PE rat monoclonal to mouse FoxP3 (Biolegend, San Diego, CA), 145-2C11 bio, FITC or PE to mouse CD3ε (BD Biosciences, Franklin Lakes, NJ) or HIT3A FITC to human CD3 (BD Biosciences), PK138 Alexa Fluor 488 to mouse NK1.1 (BioLegend, San Diego, CA), 8P16-N4-L21 to human CCL1 (Enzo Life Sciences, Farmingdale, NY), 57203 to human CCL22 (R&D Systems, Minneapolis, MN) and MAB4391 to mouse Ccl22 (R&D Systems), TG6/CCR4 to human CCR4 (BioLegend, San Diego, CA) and 191704 to human CCR8 (R&D Systems).

Techniques: Double Staining, Staining, Sequencing, TUNEL Assay, Plasmid Preparation

Journal: Xenobiotica; the fate of foreign compounds in biological systems

Article Title: 4,4’-Methylene Diphenyl Diisocyanate Exposure Induces Expression of Alternatively Activated Macrophage-Associated Markers and Chemokines Partially Through Krüppel-Like Factor 4 Mediated Signaling in Macrophages

doi: 10.1080/00498254.2023.2284867

Figure Lengend Snippet: (A) Experimental timelines showing MDI exposure time points, routes. Total RNA was isolated from BALCs from either dermal exposed or dermal/aerosol exposed mice by miRVana™ miR isolation kit, reverse transcribed, and subjected to TaqMan RT-qPCR. M2 macrophage-associated markers and chemokine mRNA expression of (B) Chil3, (C) Chil4 (D) Rentlb, (E) Clec7a, (F) Clec10a, (G) Ccl17, (H) Ccl22, (I) Cd206, (J) Cd273 and (K) Tgm2 were determined. (N=3; bars, s.e.m) (*P<0.05, **P<0.01, ***P<0.001). Ctl: Control; ACN: Acetone; MDI: 4,4’-methylene diphenyl diisocyanate. D−/M−: BALC RNA isolated from animals with neither MDI dermal exposure nor MDI-aerosol exposure, D+/M-: BALC RNA isolated from animals with MDI dermal exposure on days 1, 2, 3, 14, 15, 16 followed by 1 h house-air control exposure on day 21. D+/M+24h: Total RNA isolated from BALCs collected 24h post-exposure to 1h MDI-aerosol exposure on day 21 with prior MDI dermal exposure on days 1, 2, 3, 14, 15, 16.

Article Snippet: Gene expression assays used in this study were acquired from Thermo Fisher Scientific and include: mouse Chil3 (Cat#4331182; Assay ID: Mm00657889_mH) , Chil4 (Mm00840870_m1) , Retnlb (Mm00445845_m1) , Clec7a (Mm01183349_m1) , Clec10a (Mm00546125_g1) , Cd163 (Mm00474091_m1) , Mrc1/Cd206 (Mm01329359_m1) , Pdcd1lg2/Cd273 (Mm00451734_m1) , Tgm2 (Mm00436979_m1), Klf4 (Mm00516104_m1), Pparg (Mm00440940_m1), Stat6 (Mm01160477_m1), Irf4 (Mm00516431_m1), Spi1 (Mm00488140_m1), Cebpb (Mm07294206_s1), Ccl17 (Mm01244826_g1) , Ccl22 (Mm00436439_m1) , Ccl24 (Mm00444701_m1), and B2m (Mm00437762_m1); human CD206 (Hs00267207_m1), TGM2 (Hs01096681_m1), KLF4 (Hs00358836_m1), PPARG (Hs01115513_m1), STAT6 (Hs00180031_m1), IRF4 (Hs00180031_m1), SPI1 (Hs02786711_m1), CEBPB (Hs00942496_s1), CCL17 (Hs00171074_m1) , CCL22 (Hs01574247_m1) , CCL24 (Hs00171082_m1), and B2M (Hs00187842_m1).

Techniques: Isolation, Aerosol, Reverse Transcription, Quantitative RT-PCR, Expressing, Control

Journal: Xenobiotica; the fate of foreign compounds in biological systems

Article Title: 4,4’-Methylene Diphenyl Diisocyanate Exposure Induces Expression of Alternatively Activated Macrophage-Associated Markers and Chemokines Partially Through Krüppel-Like Factor 4 Mediated Signaling in Macrophages

doi: 10.1080/00498254.2023.2284867

Figure Lengend Snippet: Total RNA was isolated from MDI-GSH conjugate treated THP-1 macrophages by miRVana™ miR isolation kit, reverse transcribed, and subjected to TaqMan RT-qPCR. Endogenous mRNA expressions of (A) CD206 (B) TGM2, (D) CCL17, (E) CCL22, and (F) CCL24 were determined at 24 h post exposure (N=3; bars, s.e.m). (C) Endogenous M2 markers CD206 and TGM2 protein expression of THP-1 macrophages treated with MDI-GSH conjugate were determined by immunoblot analysis. β-actin served as a loading control. The secreted chemokine levels of (G) CCL17, (H) CCL22 and (I) CCL24 in conditioned media collected from MDI-GSH conjugate treated THP-1 macrophages were determined by ELISA according to manufacturer’s instructions. MDI: 4,4’-methylene diphenyl diisocyanate. GSH: Glutathione. (*P<0.05, **P<0.01, ***P<0.001).

Article Snippet: Gene expression assays used in this study were acquired from Thermo Fisher Scientific and include: mouse Chil3 (Cat#4331182; Assay ID: Mm00657889_mH) , Chil4 (Mm00840870_m1) , Retnlb (Mm00445845_m1) , Clec7a (Mm01183349_m1) , Clec10a (Mm00546125_g1) , Cd163 (Mm00474091_m1) , Mrc1/Cd206 (Mm01329359_m1) , Pdcd1lg2/Cd273 (Mm00451734_m1) , Tgm2 (Mm00436979_m1), Klf4 (Mm00516104_m1), Pparg (Mm00440940_m1), Stat6 (Mm01160477_m1), Irf4 (Mm00516431_m1), Spi1 (Mm00488140_m1), Cebpb (Mm07294206_s1), Ccl17 (Mm01244826_g1) , Ccl22 (Mm00436439_m1) , Ccl24 (Mm00444701_m1), and B2m (Mm00437762_m1); human CD206 (Hs00267207_m1), TGM2 (Hs01096681_m1), KLF4 (Hs00358836_m1), PPARG (Hs01115513_m1), STAT6 (Hs00180031_m1), IRF4 (Hs00180031_m1), SPI1 (Hs02786711_m1), CEBPB (Hs00942496_s1), CCL17 (Hs00171074_m1) , CCL22 (Hs01574247_m1) , CCL24 (Hs00171082_m1), and B2M (Hs00187842_m1).

Techniques: Isolation, Reverse Transcription, Quantitative RT-PCR, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay

Journal: Xenobiotica; the fate of foreign compounds in biological systems

Article Title: 4,4’-Methylene Diphenyl Diisocyanate Exposure Induces Expression of Alternatively Activated Macrophage-Associated Markers and Chemokines Partially Through Krüppel-Like Factor 4 Mediated Signaling in Macrophages

doi: 10.1080/00498254.2023.2284867

Figure Lengend Snippet: Macrophages were transfected with 2.5 mg of either pCMV6-Entry-KLF4 or pCMV6-Entry vector plasmids for 48 h. Total RNA was isolated from plasmid transfected THP-1 macrophages by miRVana™ miR isolation kit, reverse transcribed, and subjected to TaqMan stem-loop RT-qPCR. Endogenous M2 markers of (A) CD206, (B), TGM2, (D) CCL17 (E) CCL22, and (F) CCL24 mRNA levels were determined (N=3; bars, s.e.m). (D) The endogenous CD206 and TGM2 proteins of THP-1 macrophages transfected with either pCMV6-Entry-KLF4 or pCMV6-Entry vector plasmids was analyzed by immunoblot. β-actin served as a loading control. MDI: 4,4’-methylene diphenyl diisocyanate. GSH: Glutathione. (*P<0.05, ***P<0.001)

Article Snippet: Gene expression assays used in this study were acquired from Thermo Fisher Scientific and include: mouse Chil3 (Cat#4331182; Assay ID: Mm00657889_mH) , Chil4 (Mm00840870_m1) , Retnlb (Mm00445845_m1) , Clec7a (Mm01183349_m1) , Clec10a (Mm00546125_g1) , Cd163 (Mm00474091_m1) , Mrc1/Cd206 (Mm01329359_m1) , Pdcd1lg2/Cd273 (Mm00451734_m1) , Tgm2 (Mm00436979_m1), Klf4 (Mm00516104_m1), Pparg (Mm00440940_m1), Stat6 (Mm01160477_m1), Irf4 (Mm00516431_m1), Spi1 (Mm00488140_m1), Cebpb (Mm07294206_s1), Ccl17 (Mm01244826_g1) , Ccl22 (Mm00436439_m1) , Ccl24 (Mm00444701_m1), and B2m (Mm00437762_m1); human CD206 (Hs00267207_m1), TGM2 (Hs01096681_m1), KLF4 (Hs00358836_m1), PPARG (Hs01115513_m1), STAT6 (Hs00180031_m1), IRF4 (Hs00180031_m1), SPI1 (Hs02786711_m1), CEBPB (Hs00942496_s1), CCL17 (Hs00171074_m1) , CCL22 (Hs01574247_m1) , CCL24 (Hs00171082_m1), and B2M (Hs00187842_m1).

Techniques: Transfection, Plasmid Preparation, Isolation, Reverse Transcription, Quantitative RT-PCR, Western Blot, Control

Journal: Xenobiotica; the fate of foreign compounds in biological systems

Article Title: 4,4’-Methylene Diphenyl Diisocyanate Exposure Induces Expression of Alternatively Activated Macrophage-Associated Markers and Chemokines Partially Through Krüppel-Like Factor 4 Mediated Signaling in Macrophages

doi: 10.1080/00498254.2023.2284867

Figure Lengend Snippet: Cell-free conditioned media were obtained from THP-1 macrophages transfected with either KLF4 overexpression plasmid or empty vector for 48 h or else collected 24 h after differentiated THP-1 macrophages subjected with KLF4 siRNAs or nontargeting siRNA control transfection as indicated where the siRNA transfected macrophages were treated with or without 10 mM MDI-GSH conjugates. The secreted protein levels of (A&E) CCL17 (B&F) CCL22, and (C&G) CCL24 in conditioned media from either KLF4 overexpressed THP-1 macrophages or KLF4-knockdown macrophages treated with MDI-GSH conjugates were determined by ELISA according to manufacturer’s instructions. The isolated conditioned media were used as chemoattractant to attract (D&H) Jurkat T-cells clone E6–1 or differentiated HL-60 C_15 eosinophils. T-cells and eosinophils migration responding to the conditioned media from either (D) KLF4 overexpression or (H) KLF4-knockdown macrophages was measured after 6 h. Percent of cells migrated towards the bottom chamber are shown. MDI: 4,4’-methylene diphenyl diisocyanate. GSH: Glutathione. (*P<0.05, **P<0.01, ***P<0.001, when compared to nontargeting siRNA control (siCtl); #p < 0.05, ##p < 0.01, ###p < 0.001, when compared to macrophages transfected with siCtl followed by 10 mM MDI-GSH conjugate exposure.)

Article Snippet: Gene expression assays used in this study were acquired from Thermo Fisher Scientific and include: mouse Chil3 (Cat#4331182; Assay ID: Mm00657889_mH) , Chil4 (Mm00840870_m1) , Retnlb (Mm00445845_m1) , Clec7a (Mm01183349_m1) , Clec10a (Mm00546125_g1) , Cd163 (Mm00474091_m1) , Mrc1/Cd206 (Mm01329359_m1) , Pdcd1lg2/Cd273 (Mm00451734_m1) , Tgm2 (Mm00436979_m1), Klf4 (Mm00516104_m1), Pparg (Mm00440940_m1), Stat6 (Mm01160477_m1), Irf4 (Mm00516431_m1), Spi1 (Mm00488140_m1), Cebpb (Mm07294206_s1), Ccl17 (Mm01244826_g1) , Ccl22 (Mm00436439_m1) , Ccl24 (Mm00444701_m1), and B2m (Mm00437762_m1); human CD206 (Hs00267207_m1), TGM2 (Hs01096681_m1), KLF4 (Hs00358836_m1), PPARG (Hs01115513_m1), STAT6 (Hs00180031_m1), IRF4 (Hs00180031_m1), SPI1 (Hs02786711_m1), CEBPB (Hs00942496_s1), CCL17 (Hs00171074_m1) , CCL22 (Hs01574247_m1) , CCL24 (Hs00171082_m1), and B2M (Hs00187842_m1).

Techniques: Transfection, Over Expression, Plasmid Preparation, Control, Knockdown, Enzyme-linked Immunosorbent Assay, Isolation, Migration

Journal: Xenobiotica; the fate of foreign compounds in biological systems

Article Title: 4,4’-Methylene Diphenyl Diisocyanate Exposure Induces Expression of Alternatively Activated Macrophage-Associated Markers and Chemokines Partially Through Krüppel-Like Factor 4 Mediated Signaling in Macrophages

doi: 10.1080/00498254.2023.2284867

Figure Lengend Snippet: (A) Illustration of two siRNA target sites in KLF4 coding sequences (CDS). Endogenous KLF4 transcripts were determined using total RNA isolated from differentiated macrophages transfected with 25 nM of either two different KLF4 siRNAs or nontargeting siRNA controls for 48 h (N=3; bars, s.e.m). The endogenous KLF4 protein expression of THP-1 macrophages treated with MDI-GSH conjugate was analyzed by immunoblot. β-actin served as a loading control. Endogenous M2 macrophage-associated transcription factor of (B) KLF4, markers of (C) CD206, (D), TGM2, and chemokines (E) CCL17 (F) CCL22, and (G) CCL24 mRNA levels were determined in total RNA isolated from differentiated macrophages transfected with KLF4 siRNAs for 48h following treatments with or without 10 mM MDI-GSH conjugates for 24h (N=3; bars, s.e.m). Total RNA was isolated from siRNA transfected THP-1 macrophages by miRVana™ miR isolation kit, reverse transcribed, and subjected to TaqMan stem-loop RT-qPCR. MDI: 4,4’-methylene diphenyl diisocyanate. GSH: Glutathione. CDS: coding sequences (*P<0.05, ***P<0.001, when compared to nontargeting siRNA control (siCtl); #p < 0.05, ##p < 0.01, ###p < 0.001, when compared to macrophages transfected with siCtl followed by 10 mM MDI-GSH conjugate exposure.)

Article Snippet: Gene expression assays used in this study were acquired from Thermo Fisher Scientific and include: mouse Chil3 (Cat#4331182; Assay ID: Mm00657889_mH) , Chil4 (Mm00840870_m1) , Retnlb (Mm00445845_m1) , Clec7a (Mm01183349_m1) , Clec10a (Mm00546125_g1) , Cd163 (Mm00474091_m1) , Mrc1/Cd206 (Mm01329359_m1) , Pdcd1lg2/Cd273 (Mm00451734_m1) , Tgm2 (Mm00436979_m1), Klf4 (Mm00516104_m1), Pparg (Mm00440940_m1), Stat6 (Mm01160477_m1), Irf4 (Mm00516431_m1), Spi1 (Mm00488140_m1), Cebpb (Mm07294206_s1), Ccl17 (Mm01244826_g1) , Ccl22 (Mm00436439_m1) , Ccl24 (Mm00444701_m1), and B2m (Mm00437762_m1); human CD206 (Hs00267207_m1), TGM2 (Hs01096681_m1), KLF4 (Hs00358836_m1), PPARG (Hs01115513_m1), STAT6 (Hs00180031_m1), IRF4 (Hs00180031_m1), SPI1 (Hs02786711_m1), CEBPB (Hs00942496_s1), CCL17 (Hs00171074_m1) , CCL22 (Hs01574247_m1) , CCL24 (Hs00171082_m1), and B2M (Hs00187842_m1).

Techniques: Isolation, Transfection, Expressing, Western Blot, Control, Reverse Transcription, Quantitative RT-PCR