Journal: Frontiers in Immunology

Article Title: Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

doi: 10.3389/fimmu.2016.00458

Figure Lengend Snippet: MPLA-tDCs from rheumatoid arthritis patients and healthy controls display a tolerogenic phenotype that remains unaffected under pro-inflammatory conditions . (A) Monocytes from patients with rheumatoid arthritis (RA) ( black closed symbols ) and from healthy control (HC) subjects ( gray open symbols ) were differentiated into DCs in the presence of GM-CSF and IL-4 within 5 days. For tolerization, DCs were conditioned with dexamethasone (Dex) during 48 h and were additionally activated with MPLA for the last 24 h of culture (MPLA-tDCs). Surface expression levels of CD86, CD40, CD83, HLA-DR, and TLR2 were assessed by flow cytometry. Dot plots show all data points and median values are indicated as a line. (B) MPLA-tDCs from RA patients were incubated for 72 h in the presence of synovial fluid (200 μg/ml) or TNFα (10 ng/ml), and the expression of phenotypic markers was analyzed. Untreated iDCs were used as controls. Statistical differences were calculated using either Kruskal–Wallis test (comparison between HC and RA) or Friedman test (comparison between culture conditions) and Dunn’s multiple comparison was used as post test (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: At days 3 and 4, cells were modulated with 1 μM dexamethasone (tDCs) (Sigma-Aldrich, St. Louis, MO, USA) and activated with 1 μg/ml cGMP-grade MPLA (MPLA-tDCs) (Avanti, Alabaster, AL, USA).

Techniques: Expressing, Flow Cytometry, Incubation

Journal: Frontiers in Immunology

Article Title: Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

doi: 10.3389/fimmu.2016.00458

Figure Lengend Snippet: MPLA-tDCs from rheumatoid arthritis patients exhibit an anti-inflammatory cytokine profile . Monocyte-derived DC (moDC) subsets from rheumatoid arthritis (RA) patients ( black closed symbols ) and healthy control subjects (HC; gray open symbols ) were cultured with CD40L-transfected NIH3T3 cells at 1:1 ratio for 24 h. (A) Concentrations of IL-10, IL-12, IL-23, TNF-α, and active TGF-β1 were determined in culture supernatants of moDCs by ELISA. (B) Bar chart with values of IL-10/IL-12 ratio of different moDC subsets from RA patients. Dot plots show all data points and horizontal lines indicate median values. Statistical differences were calculated using Kruskal–Wallis test (comparison of HC and RA) or Friedman test (comparison of DC types) and Dunn’s multiple comparison post test (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: At days 3 and 4, cells were modulated with 1 μM dexamethasone (tDCs) (Sigma-Aldrich, St. Louis, MO, USA) and activated with 1 μg/ml cGMP-grade MPLA (MPLA-tDCs) (Avanti, Alabaster, AL, USA).

Techniques: Derivative Assay, Cell Culture, Transfection, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

doi: 10.3389/fimmu.2016.00458

Figure Lengend Snippet: MPLA-tDCs from rheumatoid arthritis patients display the capacity to migrate toward CCR7 and CXCR4 ligands . After 5 days of culture, the migratory capacity of MPLA-tDC ( dark gray ), tDCs ( white ) and mDCs ( light gray ) from rheumatoid arthritis (RA) patients was studied in vitro . (A) The percentage of CD11c+ DCs expressing CXCR4 or CCR7 was determined by flow cytometry ( n = 15). Fold expression changes on receptor expression on mDCs, tDCs and MPLA-tDCs compared to untreated iDCs are shown. (B) Transwell assays were used to assess migration of DCs in response to 250 ng/ml of CXCL12 or CCL19 ( n = 12). Migration index indicates the quotient of cells that migrated toward a specific chemokine divided by cells that migrated toward medium alone. Box plots show median, 25- and 75%-quartile, minimum and maximum values, respectively. Wilcoxon signed-rank test was used to evaluate statistical differences between MPLA-tDCs, tDCs, and mDCs (* P < 0.05; ** P < 0.01; *** P < 0.001).

Article Snippet: At days 3 and 4, cells were modulated with 1 μM dexamethasone (tDCs) (Sigma-Aldrich, St. Louis, MO, USA) and activated with 1 μg/ml cGMP-grade MPLA (MPLA-tDCs) (Avanti, Alabaster, AL, USA).

Techniques: In Vitro, Expressing, Flow Cytometry, Migration

Journal: Frontiers in Immunology

Article Title: Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

doi: 10.3389/fimmu.2016.00458

Figure Lengend Snippet: MPLA-tDCs modulate CD4+ T cell responses to synovial antigens . Monocytes ( n = 6–9) were differentiated into mDCs and MPLA-tDCs as described in the Section “ .” At day 4 of DC generation, 4 h prior to stimulation with MPLA, mDCs and MPLA-tDCs were loaded with 1 μg/ml tuberculin purified protein derivative (PPD), 200 μg/ml synovial fluid (SF), or left unloaded for further 24 h. For the assessment of their T cell-stimulatory capacity, antigen-loaded or unloaded mDCs and MPLA-tDCs were co-cultured with autologous CFSE-labeled CD4+ T cells in a ratio of 1:2 (DC:T cell) for 6 days. To detect IFN-γ intracellularly, cells were stimulated with 50 ng/ml phorbol-12-myriastate-13-acetate and 1 μg/ml ionomycin in the presence of 1 μg/ml brefeldin-A. Proliferation-associated CFSE dilution and IFN-γ production of CD4+ T cells were analyzed by flow cytometry. (A) Representative dot plots and (B) graphic representation of the percentage of IFN-γ-producing proliferating (CFSE low ) CD4+ T cells are shown. (C) IL-17A secretion was measured in supernatants of co-cultures by ELISA. (B,C) , Box plots show median, 25- and 75%-quartiles and both extreme values (* P < 0.05).

Article Snippet: At days 3 and 4, cells were modulated with 1 μM dexamethasone (tDCs) (Sigma-Aldrich, St. Louis, MO, USA) and activated with 1 μg/ml cGMP-grade MPLA (MPLA-tDCs) (Avanti, Alabaster, AL, USA).

Techniques: Purification, Cell Culture, Labeling, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Treatment with Dexamethasone and Monophosphoryl Lipid A Removes Disease-Associated Transcriptional Signatures in Monocyte-Derived Dendritic Cells from Rheumatoid Arthritis Patients and Confers Tolerogenic Features

doi: 10.3389/fimmu.2016.00458

Figure Lengend Snippet: Conditioning with dexamethasone and MPLA induces similar transcriptional profiles on monocyte-derived DCs from rheumatoid arthritis patients and healthy controls and counterpoints disease-associated effects on gene expression . (A) Venn diagram displaying numbers of differentially expressed (DE) transcripts due to differentiation protocol (iDCs, mDCs, tDCs, or MPLA-tDCs), presence of disease (healthy controls: HC, or rheumartoid arthritis: RA) or interaction of protocol and disease. (B) Principal component analysis using the first two components separates samples according to their variance. (C,D) Pairwise comparison between moDCs from RA patients and HC were performed for each differentiation protocol separately using t -test. (C) 44 DE genes found exclusively in iDCs were hierarchically clustered. Genes with known or potential association to RA are highlighted in red color. (D) Changes in means of expression of selected RA-associated DE genes in iDCs vs. MPLA-tDCs were shown for HC and RA patients. iDCs, unstimulated immature DCs; tDCs, dexamethasone-modulated TolDCs; mDCs, MPLA-matured DCs; MPLA-tDCs, dexamethasone-modulated MPLA-activated TolDCs.

Article Snippet: At days 3 and 4, cells were modulated with 1 μM dexamethasone (tDCs) (Sigma-Aldrich, St. Louis, MO, USA) and activated with 1 μg/ml cGMP-grade MPLA (MPLA-tDCs) (Avanti, Alabaster, AL, USA).

Techniques: Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Gelsolin Counteracts ER Stress-Driven Inflammatory Circuits in Psoriasis-like Dermatitis

doi: 10.1101/2025.11.20.689413

Figure Lengend Snippet: a, WT and MyD88-deficient BMDCs were pre-stained with Fluo-8, followed by IMQ stimulation. Ca 2+ influx was monitored by fluorescent microscopy. Left panel shows representative Fluo-8 fluorescent intensity images. Right panel shows violin plots ( n = 20). b, BMDCs were pre-stained with Rhod-2-AM reagent, followed by LPS stimulation. Mitochondrial Ca 2+ was analyzed by flow cytometry ( n = 4). Left panel shows a representative flow cytometry image. c, BMDCs were pretreated with 2-APB, followed by IMQ stimulation. Intracellular ROS was analyzed by flow cytometry ( n = 3). d, e, f, BMDCs were pretreated with LPS, followed by 2-APB treatment, and stimulated with IMQ. IL-1β concentrations in cell culture supernatant were measured by ELISA (d) ( n = 3). Cells were stained with PI and Annexin V, and cell death was analyzed by flow cytometry (e) ( n = 3). Cell lysates were analyzed by immunoblotting for cleaved caspase-1 (p20) (f). Data are representative immunoblot analysis images of two independent experiments. g, BMDCs were stimulated with IMQ or RSQ, stained with JC-1 reagent, and mitochondrial damage was analyzed by flow cytometry ( n = 3). Left panel shows representative flow cytometry images. h, BMDCs were pre-stained with MitoSOX red reagent, stimulated with IMQ or RSQ, and mitochondrial ROS was analyzed by flow cytometry ( n = 4). Left panel shows representative flow cytometry images. i, BMDCs were pretreated with LPS, followed by treatment with the indicated anti-oxidants, stimulated with IMQ, and IL-1β concentrations in cell culture supernatants were measured by ELISA ( n = 3). j, BMDCs were pretreated with LPS, followed by treatment with the indicated anti-oxidants, stimulated with IMQ, stained with PI and Annexin V, and cell death was analyzed by flow cytometry ( n = 3). k, BMDCs were pretreated with mitoquinone (MitoQ), followed by IMQ stimulation, and IL-1β concentrations in cell culture supernatants were measured by ELISA ( n = 3). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P- values were calculated by one-way ANOVA with Tukey’s test (c-e and g-k) or Student’s t -test (a and b). (*: p > 0.05, ***: p > 0.001).

Article Snippet: For IL-1β measurements, cells were preincubated with 20 ng/ml of LPS for 3 h and stimulated with 50 μg/ml IMQ, 50 μg/ml RSQ, 5 mM ATP, or 5 μM nigericin for 30 min or 3 h. In the case of pDCs, the cells were seeded onto 96-well plates at a density of 2.0 × 10 cells per well and stimulated with ligands for 24 h. The cytokine levels of IL-6, TNF-α, and IL-1β in the culture supernatant were measured using a mouse IL-6 DuoSet ELISA (R&D Systems), mouse TNF-α ELISA kit (Invitrogen), and mouse IL-1β ELISA kit (Invitrogen), respectively, according to the manufacturer’s instructions.

Techniques: Staining, Microscopy, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: Gelsolin Counteracts ER Stress-Driven Inflammatory Circuits in Psoriasis-like Dermatitis

doi: 10.1101/2025.11.20.689413

Figure Lengend Snippet: a, BMDCs were pretreated with LPS, followed by IMQ stimulation. DNA was harvested from cytosolic and mitochondrial fractions, and the amount of mitochondrial DNA and nuclear DNA (nDNA) were measured by qPCR ( n = 3). b, BMDCs were pretreated with LPS and 2-APB, followed by IMQ stimulation. DNA was harvested from cytosolic fraction, and the amount of mitochondrial DNA was measured by qPCR ( n = 3). c, BMDCs were pretreated with LPS, followed by IMQ stimulation. DNA was harvested from cytosolic fraction, and oxidized DNA was detected by dot blot analysis. Data are representative images of two independent experiments. d, BMDCs were stimulated with IMQ, and mitochondria-enriched fractions were collected from the cell culture supernatants and lysed. Western blot analysis was performed using an anti-TOM20 antibody to visualize mitochondrial content in the extracellular fraction. Data are representative images of two independent experiments. e, WT and NLRP3-deficient BMDCs were pretreated with LPS, followed by IMQ stimulation. DNA in cell culture supernatants was harvested and the amount of mitochondrial DNA was measured by qPCR ( n = 3). f, Bone marrow-derived pDCs were stimulated with DNA isolated from the cytosolic fraction of BMDCs stimulated with IMQ, and mBD14. Total RNA was extracted from the pDCs and the expression of Il6 and Tnf mRNA was measured by qPCR ( n = 4). g, BMDCs were stimulated with synthesized mtDNA or oxidized mtDNA with or without recombinant mBD14. The expression of Tnf and Il6 mRNA was measured by qPCR ( n = 3) (upper panel). The concentration of TNF-α and IL-6 in cell culture supernatants was measured by ELISA ( n = 3) (bottom panel). h, WT and TLR9-deficient bone marrow-derived pDCs were stimulated with mtDNA or oxidized mtDNA with or without mBD14. The expression of Il6 mRNA was measured by qPCR ( n = 3). i , BMDCs were stimulated with oxidized double stranded-DNA synthesized from mitochondrial or genomic DNA. The expression of Il6 mRNA was measured by qPCR ( n = 3). j, HEK293T cells were transfected with FLAG-mBD14 or FLAG-hBD3 expression plasmids. Cell lysates were subjected to DNA immunoprecipitation with anti-FLAG antibody and immunoprecipitants were analyzed by qPCR ( n = 3). Data are presented as the mean ± s.e.m. Each circle indicates an independent biological sample. P- values were calculated by one-way ANOVA with Tukey’s test (e-j) or Student’s t -test (a and b). (N.S.: not significant, *: p > 0.05, **: p > 0.01, ***: p > 0.001).

Article Snippet: For IL-1β measurements, cells were preincubated with 20 ng/ml of LPS for 3 h and stimulated with 50 μg/ml IMQ, 50 μg/ml RSQ, 5 mM ATP, or 5 μM nigericin for 30 min or 3 h. In the case of pDCs, the cells were seeded onto 96-well plates at a density of 2.0 × 10 cells per well and stimulated with ligands for 24 h. The cytokine levels of IL-6, TNF-α, and IL-1β in the culture supernatant were measured using a mouse IL-6 DuoSet ELISA (R&D Systems), mouse TNF-α ELISA kit (Invitrogen), and mouse IL-1β ELISA kit (Invitrogen), respectively, according to the manufacturer’s instructions.

Techniques: Dot Blot, Cell Culture, Western Blot, Derivative Assay, Isolation, Expressing, Synthesized, Recombinant, Concentration Assay, Enzyme-linked Immunosorbent Assay, Transfection, Immunoprecipitation

Journal: The Journal of Neuroscience

Article Title: Active Membrane Properties and Signal Encoding in Graded Potential Neurons

doi: 10.1523/JNEUROSCI.18-19-07972.1998

Figure Lengend Snippet: a, Expected coherence functions as determined from the signal and noise spectra shown in Figure ​Figure6.6. Because of the highest signal-to-noise ratio for HS-cells at rest the expected coherence was highest too, for this experimental condition.b, Nonlinearity as defined by the difference between the expected and the measured coherence. This nonlinearity is highest when the cells were permanently hyperpolarized, producing full-blown action potentials in response to visual stimulation. The error bars at 1.5 Hz show the SEM for a single representative frequency.

Article Snippet: Stimulation Stimuli were generated on Tektronix 608 monitors by an image synthesizer (Picasso, Innisfree) and consisted of a one-dimensional grating of 14° spatial wavelength and 87% contrast displayed at a frame rate of 200 Hz.

Techniques:

Journal:

Article Title: Auditory Cortical Responses Elicited in Awake Primates by Random Spectrum Stimuli

doi:

Figure Lengend Snippet: Example random spectrum stimuli (RSS) from the same set. Two RSS from the same stimulus set are shown plotted as bin levels (top), tone levels (middle), and amplitude spectra (bottom). The bins have a level SD of 10 dB over the entire set, indicated by horizontal dotted lines. All of the stimuli span two octaves of frequency from 4 to 16 kHz with 20 bins per octave. Each bin contains three logarithmically spaced tones of the same level for a density of 60 tones per octave. This set contained an additional 41 stimuli, including the stimulus with all bin levels at the mean value (i.e., the flat-spectrum stimulus).

Article Snippet: Random spectrum stimulus generation Acoustic stimuli used for these experiments were adapted from similar stimuli devised by E. D. Young (Johns Hopkins University School of Medicine) to study subcortical auditory neurons ( Yu and Young, 2000 ).

Techniques: