Journal: Journal of Interferon & Cytokine Research

Article Title: Identification of 2′-5′-Oligoadenylate Synthetase-Like Gene in Goose: Gene Structure, Expression Patterns, and Antiviral Activity Against Newcastle Disease Virus

doi: 10.1089/jir.2015.0167

Figure Lengend Snippet: GoOASL mRNA expression levels in PBMCs stimulated by virus pathogens (A) and agonists (B). The mRNA levels of goOASL were quantified by RT-qPCR. GAPDH was used as housekeeping gene. Data were represented as the mean ± SD (n = 3). The statistical analysis was performed in GraphPad Prism using unpaired 2-tailed t-tests: *P < 0.05; **P < 0.01; ***P < 0.001. The cell density was adjusted as 1 × 106/mL, Virus pathogens were GPV (10−6.6 EID50/0.2 mL), H9N2 AIV (7.14 × 1012.64 copies/mL), and DTMUV (6.3 × 10−6 TCID50/mL), with 50 μL to stimulate cells for 6 h, respectively. Agonists, including LPS, R848, Poly (I: C), and ODN2006, the final concentration of stimulus were 25, 5, 30, and 50 μg/mL correspondingly.

Article Snippet: Agonists are R848 (MedChem Express), LPS (Invivogen), Poly (I: C) (Sigma), and ODN2006 (Sangon).

Techniques: Expressing, Virus, Quantitative RT-PCR, Concentration Assay

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Peripheral blood immune populations in healthy donors (HD; N = 25) and metastatic melanoma (MM) at baseline (T0; N = 29).

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques:

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Frequency of interferon alpha (IFN-α) and CXCL10-producing plasmacytoid dendritic cells (pDCs) in chemo-naïve MM patients and HD. Representative dot plots of R848-stimulated IFN-α + and CXCL10 + pDC subsets obtained from HD and MM patients are shown ( A ). PBMCs were isolated from peripheral blood of HD ( n = 25) and MM patients ( n = 29). Total PBMCs were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 and stimulated with R848 or IMQ for 2 h ( B , D ) and 6 h ( C , E ), and with CpG-ODN 2216 for 6 h ( B – E ). IFN-α ( B , D ) and CXCL10 ( C , E ) were analyzed by intracellular flow cytometry staining. Scatter dot plot graphs illustrate the percentages of positive pDCs evaluated on BDCA-2 + /CD123 + cells. Subgroup analysis of the MM cohort illustrating the frequency of IFN-α + and CXCL10 + pDCs in M1a-c categories ( D , E ). Median and IQR are shown in ( B , C ). Mean and SD are shown in ( D , E ). The statistical significance was calculated by Wilcoxon–Mann–Whitney test ( B , C ) and by a Student’s t -test ( D , E ). * p < 0.05; ** p < 0.01; **** p < 0.0001.

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques: Isolation, Cell Culture, Flow Cytometry, Staining, MANN-WHITNEY

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Peripheral blood immune populations and lactate dehydrogenase (LDH) level in MM patients cohort at baseline (T0; N = 29).

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques:

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Correlation between tumor burden (mm) and peripheral blood immune cells of the MM patient cohort at baseline (T0; N = 29).

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques:

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: In vitro lactic acidosis affects IFN-α production by pDCs. pDCs purified from buffy coats of HD were cultured in RPMI 1640 medium supplemented with 10% FBS and IL-3 plus lactic acid (10 mM; 15 mM; 20 mM) ( n = 7; ( A )) or hydrochloric acid (pH = 7.4; 7.0; 6.5; 6.0; 5.5) ( n = 7; ( B )) for 24 h. pDCs were stimulated with R848 for 2 h. Intracellular IFN-α was analyzed by flow cytometry. Aligned dot plot graphs show the percentages of IFN-α + pDCs evaluated on BDCA-2 + /CD123 + cells. Bars represent the mean of biological replicates. The statistical significance was calculated by two-sample paired sign test. * p < 0.05.

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques: In Vitro, Purification, Cell Culture, Flow Cytometry

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Univariate and multivariate Cox regression models for OS in MM patients at baseline (T0; N = 29).

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques:

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Variation of the peripheral blood immune populations at 1 month (T1; N = 12) and 4 months (T2; N = 9) from therapy’s initiation compared to the baseline (T0; N = 16) in the group of BRAF V600+ MM patients.

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques:

Journal: Cancers

Article Title: Plasmacytoid Dendritic Cell Impairment in Metastatic Melanoma by Lactic Acidosis

doi: 10.3390/cancers12082085

Figure Lengend Snippet: Frequency of peripheral blood immune cells and IFN-α + pDCs in BRAF V600+ MM patients over therapy administration. Cell counts ( A ) and flow cytometry ( B , C ) analysis were performed on whole blood from BRAF V600+ MM patients before therapy initiation (T0; n = 16), after 30 days (T1; n = 12), and after 120 days (T2; n = 9) from therapy administration ( A – C ). Total PBMCs isolated from peripheral blood of MM patients were cultured in RPMI 1640 medium and stimulated with R848 for 2 h ( D ). IFN-α was analyzed by intracellular flow cytometry staining ( D ). Before–after graphs illustrate the frequency of lymphocytes ( A ), pDCs ( B ), and mDCs ( C ) on total PBMCs, and the frequency of IFN-α + pDCs on BDCA-2 + /CD123 + cells ( D ) for each subject. Bold black lines represent the median values. The statistical significance was calculated by Wilcoxon signed-rank test. * p < 0.05.

Article Snippet: A total of 20 ng/mL human IL-3 (Miltenyi Biotec, Bergisch Gladbach, Germany) was added to the pDC culture medium. pDCs were treated with 1 μM of the BRAF Inhibitor PLX4032 (Selleck Biochem, Houston, TX), with or without 12.5 μM of the MEK inhibitor U0126 (Merck Millipore, Darmstadt, Germany), and the 0.2% of DMSO in RPMI 1640 medium was used as vehicle control. pDCs were stimulated with 5 μg/mL of R848 or IMQ (Invivogen) and 6 μg/mL of CpG-ODN 2216 (Miltenyi-Biotec) for 2 h and 6 h. Brefeldin A (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA) was added after 1 h and 4 h, respectively.

Techniques: Flow Cytometry, Isolation, Cell Culture, Staining

Journal: Science advances

Article Title: Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.

doi: 10.1126/sciadv.aay1057

Figure Lengend Snippet: Fig. 1. Binding of IRF5-CPPs to IRF5. (A) The crystal structure of dimeric IRF5 highlights the importance of interactions between Helix 2 and Helix 5 of different mono- mers for dimerization. One monomer is shown in blue, and the other is shown in brown. (B) Polarity and hydrophobicity plot of CPPs. Two CPP templates, mPrP (1–28) and YLK, were selected for testing with IRF5 sequences. Green- and yellow-shaded regions denote good to moderate cell penetration, respectively, while pink denotes no penetration. (C) Individual curves generated from time-resolved fluorescence resonance energy transfer (TR-FRET) using fluorescein isothiocyanate (FITC)–labeled IRF5-CPPs, the YLK CPP control FITC-CPP7 and His-tagged IRF5. All six IRF5-CPP peptides bound to IRF5 (222 to 425) with submicromolar potencies, while the negative YLK CPP control did not. (D) Representative Native gel electrophoresis showing effect of IRF5-CPP2 and IRF5-CPP5 on R848-induced IRF5 homodimerization in THP-1 cells. Stimulation with 1 M R848 for 1 hour induced intracellular IRF5 homodimerization (lane 2). Preincubation with IRF5-CPP5 provided a dose-dependent reduction in R848-induced IRF5 homodimerization. (E) Same as (D) except scrambled negative control IRF5-CPP8 and IRF5-CPP9 were examined by cellular IRF5 homodimerization assay. (F) Quantification of IRF5 homodimerization from (D) and (E) is shown after normalization to -actin. One-way analysis of variance (ANOVA) with Bonferroni’s multiple comparison test was performed. ***P < 0.001, ****P < 0.0001. (G) THP-1 cells were preincubated with FITC-CPP2, FITC-CPP5, FITC-CPP8, or FITC-CPP9 for 1 hour, followed by permeabilization and staining for intracellular IRF5 with tetramethyl rhodamine isothiocyanate (TRITC)–conjugated antibodies. FRET units were calculated from fluo- rescence emissions (see Materials and Methods). (H) Representative cellular images of in-cell FRET from 10,000 acquired events by imaging flow cytometry is shown. (I) Percentage of THP-1 cells from (H) showing FRET signal by FITC-TRITC similarity score. Data in (D) to (I) are representative of three independent experiments performed in triplicate with SD shown in (F), (G), and (I).

Article Snippet: For NF-B and IRF7, 4 × 106 PBMCs were stimulated with R848 or CpGA, surface-stained with anti-CD14 or anti-CD123 and anti-BDCA2 antibodies to detect monocytes and pDCs, respectively, fixed, and permeabilized for intracellular staining with preconjugated anti–NF-B and antiIRF7 antibodies (Santa Cruz Biotechnology).

Techniques: Binding Assay, Generated, Fluorescence, Förster Resonance Energy Transfer, Labeling, Control, Nucleic Acid Electrophoresis, Negative Control, Comparison, Staining, Imaging, Flow Cytometry

Journal: Science advances

Article Title: Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.

doi: 10.1126/sciadv.aay1057

Figure Lengend Snippet: Fig. 3. IRF5-CPPs inhibit IL12 production from human PBMCs and IRF5 nuclear translocation in a concentration-dependent manner. (A) Human PBMCs were pretreated for 30 min with various concentrations of IRF5-CPPs and stimulated overnight with 1 M R848. IL12p40 levels in supernatant were measured by enzyme-linked immunosorbent assay (ELISA) and normalized to values from wells stimulated with 1 M R848 and peptide vehicle [0.05% dimethyl sulfoxide (DMSO) and 5% water]. Summarized data are from n = 4 healthy donors performed in triplicate; reported errors indicate SEM. Percentage of CD14+ monocytes (B) and CD19+ B cells (C) with nuclear-localized IRF5. PBMCs were preincubated with the indicated concentrations of IRF5-CPP2 or IRF5-CPP5, stimulated with 1 M R848 for 2 hours, stained for IRF5 and nuclear DRAQ5 (deep red anthraquinone 5), and then subjected to imaging flow cytometry. Nuclear translocation was defined as cells with a similarity score of IRF5 and DRAQ5 of ≥1.5. Data are from n = 4 independent donors; reported errors indicate SD. (D) Representative images of CD19+ B cells and CD14+ monocytes from (B) and (C). One-way ANOVA with Bonferroni’s multiple comparison test was performed.

Article Snippet: For NF-B and IRF7, 4 × 106 PBMCs were stimulated with R848 or CpGA, surface-stained with anti-CD14 or anti-CD123 and anti-BDCA2 antibodies to detect monocytes and pDCs, respectively, fixed, and permeabilized for intracellular staining with preconjugated anti–NF-B and antiIRF7 antibodies (Santa Cruz Biotechnology).

Techniques: Translocation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Staining, Imaging, Flow Cytometry, Comparison

Journal: Science advances

Article Title: Inhibition of IRF5 cellular activity with cell-penetrating peptides that target homodimerization.

doi: 10.1126/sciadv.aay1057

Figure Lengend Snippet: Fig. 6. IRF5-CPPs inhibit SLE serum–induced nuclear translocation of pIRF5 and TLR-mediated proinflammatory cytokine expression from SLE PBMCs. (A) Rep- resentative kinetic analysis of pIRF5 from imaging flow cytometry analysis. PBMCs were stimulated with CpGA over a time course and percentage of BDCA2+CD123+ pDCs with nuclear-localized pIRF5 plotted. Data are representative of three independent donors. (B) Similar to (A) except the effect of IRF5-CPP2 on CpGA-induced pIRF5 expression, measured as mean fluorescence intensity (MFI), is shown at 2 hours after stimulation. Data are from n = 3 independent healthy donors with SD. (C) Same as (B) except pIRF5 nuclear translocation is shown. (D) PBMCs were pretreated with IRF5-CPP2 or IRF5-CPP5 and stimulated with SLE serum for 2 hours. The percentage of CD14+ monocytes (D) and CD19+ B cells (E) with nuclear-localized IRF5 is shown. Data are from n = 3 independent healthy donors with SD. (F) Same as (A) except PBMCs were stimulated with SLE serum. Data are representative of three independent donors. (G) Similar to (B) except pIRF5 mean fluorescence intensity was measured in the presence or absence of IRF5-CPP2 and IRF5-CPP5 after 1-hour stimulation with SLE serum. Data are from n = 3 independent healthy donors with SD. (H) Same as (G) except nuclear-localized pIRF5 is shown. (I to K) SLE PBMCs were pretreated with IRF5-CPP2 or IRF5-CPP5 at various concentrations or 1 M hydroxychloroquine (HCQ) and stimulated with 0.5 M CpGA or 1 M R848. IFN (I), IL6 (J), and TNF (K) levels in supernatant were measured and normalized to values obtained from wells stimulated with TLR ligand and peptide vehicle. Summarized data are from n = 3 SLE donors performed in triplicate; reported errors indicate SEM. One-way ANOVA with Bonferroni’s multiple comparison test was performed.

Article Snippet: For NF-B and IRF7, 4 × 106 PBMCs were stimulated with R848 or CpGA, surface-stained with anti-CD14 or anti-CD123 and anti-BDCA2 antibodies to detect monocytes and pDCs, respectively, fixed, and permeabilized for intracellular staining with preconjugated anti–NF-B and antiIRF7 antibodies (Santa Cruz Biotechnology).

Techniques: Translocation Assay, Expressing, Imaging, Flow Cytometry, Fluorescence, Comparison

Journal: NPJ Vaccines

Article Title: A KIF20A-based thermosensitive hydrogel vaccine effectively potentiates immune checkpoint blockade therapy for hepatocellular carcinoma

doi: 10.1038/s41541-024-01060-2

Figure Lengend Snippet: a TEM image of K/R Lip . Scalebar: 100 nm. b Hydrated diameter of K/R Lip and ICG-K/R Lip . c Zeta potentials of K/R Lip and ICG- K/R Lip . d Hydrated diameter and Zeta potentials K/R Lip on the 1, 3, 5 and 7 days. e R848 release rate of K/R Lip at the pH of 7.4 and 5.6. f KIF20A release rate of K/R Lip at the pH of 7.4 and 5.6. g Photographs of K/R Lip @Gel at 25 °C and 37 °C. h ESEM image of K/R Lip @Gel at 37 °C. i Liposomes (K/R Lip ) detected with ESEM (Red arrows). j Rheological analysis quantifying storage modulus (G’) and loss modulus (G”) of K/R Lip @Gel at different temperatures. k Rheological analysis of gel stability for 6 min. l Absorbance spectra of ICG and ICG-K/R Lip @Gel. m Fluorescence spectra of ICG and ICG- K/R Lip @Gel. n NIR-II fluorescence images of mice injected with small molecule ICG, nanoscale ICG-K/R Lip and hydrogel ICG-K/R Lip @Gel. o Quantification of NIR-II fluorescence intensity at different time points after injections.

Article Snippet: Then, 2 mg R848 (Aladdin Scientific, China) was added into 10 mL stock solution.

Techniques: Liposomes, Fluorescence, Injection

Journal: eLife

Article Title: Macrophage innate training induced by IL-4 and IL-13 activation enhances OXPHOS driven anti-mycobacterial responses

doi: 10.7554/eLife.74690

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , Resiquimod/R848 , InvivoGen , Cat# tlrl-r848 , .

Techniques: Cell Isolation, Irradiation, Blocking Assay, Sequencing, Recombinant, Bicinchoninic Acid Protein Assay, Isolation, Enzyme-linked Immunosorbent Assay, Software, Flow Cytometry, Confocal Microscopy, Cell Culture, Staining, Modification, Radio Immunoprecipitation, Lysis, Random Hexamer Labeling