Journal: Nature cancer

Article Title: Single-cell RNA sequencing reveals compromised immune microenvironment in precursor stages of multiple myeloma.

doi: 10.1038/s43018-020-0053-3

Figure Lengend Snippet: (A) Mean activity of MHC II gene signature (M-sig2) and mean expression of these genes across samples, grouped by stage of progression. Significance was tested by a BH-corrected two-tailed t-test with df=19.7 on n=30 patient samples with at least 10 CD14+ monocytes. (B) HLA-DR cell surface protein expression measured by CyTOF across samples. Significantly lower levels of HLA-DR molecules were observed on the surface of CD14+ monocytes/macrophages in patients (SMM and MM samples) as compared to healthy donors (NBM), with median value of 26.7 for NBM, 10.9 and 7.9 for SMM and MM samples respectively. Significant difference was tested with two-tailed t-test, bars representing SD. (C) Myeloma cells significantly downregulate surface representation of MHC II on CD14+ monocytes after direct co-culture. Human CD14+ monocytes were isolated from blood of healthy donors and co-cultured with CD19+ B cells or MM1.S and RPMI8226 (RPMI) myeloma cells. FACS analysis was performed on day 3 of co-culture. Median values for surface expression on CD14+ cells co-cultured with CD19+ cells or with RPMI cells in transwell or direct co-culture were 94.2, 60.4 and 31.3 for HLA-DR; 64.6, 35.9 and 1.7 for HLA-DP respectively. Median values of 97.0, 97.2 and 91.2 for HLA-DR; 75.8, 86.2 and 6.4 for HLA-DP were detected on the surface of CD14+ cells after similar experimental settings with MM1.S cells. The median proportion of CD14+ intracellularly expressing HLA-DR was 97.8, 96.4 and 98.0; 96.7, 95.9 and 96.0 for HLA-DP in co-culture with RPMI; 97.9, 98.6 and 91.9 for HLA-DR, 96.8, 97.8 and 90.9 for HLA-DP in co-culture with MM1.S cells respectively. Experiment was performed with three independent donors/2 different cell lines in triplicates. (Significance was derived from technical replicates, but different donors demonstrate similar loss of HLA-DR expression as compared to controls, t-test 2-sided; error bars indicate SD) (D) Immunofluorescence staining of tissue microarrays from MGUS patients demonstrates intracellular accumulation of HLA-DR (green) in CD14-expressing monocytes (red) as compared to membrane-bound localization of HLA-DR in healthy bone marrow monocytes (BM tissue microarrays of MGUS, SMM and MM patients (n=45, performed in triplicates, total of 135 BM sections, yellow arrows point on cells with HLA-DR localized to the cell membrane, white arrows point on cells with HLA-DR accumulated in the cytoplasm). (E) Upregulated MARCH-1 expression and decreased VAPA levels in CD14+ cells from BM of MM patients, compared to healthy donors. Violin plots show minimum, medium, and maximum values, and a BH-corrected two-tailed t-test was performed on n=27 patient samples with >=50 CD14+ monocytes.

Article Snippet: Human CD14 + monocytes or CD19 + B-cells were obtained from fresh PB cells using CD14 and CD19 Microbeads (Miltenyi Biotec).

Techniques: Activity Assay, Expressing, Two Tailed Test, Co-Culture Assay, Isolation, Cell Culture, Derivative Assay, Immunofluorescence, Staining, Membrane

Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: PSMD14 acts as a major regulator for SLC7A11 stability in response to DEX stimulation. A) Schematic illustration of the preparation of SLC7A11‐binding protein sample in MLO‐Y4 cells for LC‐MS/MS analysis to identify the deubiquitinase. B) After proofreading using the UbiBrowser library, the SLC7A11‐bound deubiquitinases were ranked according to sequence coverage. C) LC‐MS/MS analysis of SLC7A11‐bound deubiquitinase eluted from MLO‐Y4 cell lysate. PSM: Peptide Spectrum Match. D) Immunoprecipitation of SLC7A11 or control IgG was performed on MLO‐Y4 cells treated with PBS or DEX (100 µM) for 8 h. E) Immunoprecipitation of PSMD14 or control IgG was performed on MLO‐Y4 cells. F) Immunoprecipitation of the interaction between SLC7A11 and PSMD14 was performed on plasmid‐transfected MLO‐Y4 cells with the indicated antibodies. After transfection was completed, MLO‐Y4 cells were treated with MG132 (10 µM) for 8 h while adding PBS or DEX (100 µM). G) Western blot and quantitative analysis of Flag‐PSMD14 and SLC7A11 protein was performed on MLO‐Y4 cells transfected with indictaed plasmid (n = 5 per group). H) Western blot and quantitative analysis of PSMD14 protein was performed on MLO‐Y4 cells transfected with si‐RNA (50 nM si‐NC or si‐PSMD14 #1‐3 for 24 h) (n = 5 per group). I) Western blot and quantitative analysis of PSMD14 and SLC7A11 proteins were performed on siRNA‐transfected MLO‐Y4 cells (50 nM si‐NC or si‐PSMD14 for 24 h). After transfection was completed, MLO‐Y4 cells were treated with PBS or DEX (100 µM) for 8 h (n = 5 per group). J) Immunoprecipitation of SLC7A11 ubiquitination and its binding to PSMD14 were performed on siRNA‐transfected MLO‐Y4 cells (50 nM si‐NC or si‐PSMD14 for 24 h). After transfection was completed, MLO‐Y4 were treated with MG132 (10 µM) for 8 h while adding PBS or DEX (100 µM). K) Predicted binding complex model of SLC7A11 and PSMD14 (left). The picture showed the hydrogen bonds in the protein interaction region and the corresponding amino acid residues (right). L) Binding energy analysis of the PSMD14‐SLC7A11 complex and the PSMD14‐SLC7A11‐DEX complex based on molecular dynamics simulations. Data are expressed as mean ± SD, with biologically individual data points shown. p values were determined by one‐way ANOVA test with Tukey's multiple comparisons (H) and two‐way ANOVA test with Tukey's multiple comparisons (G,I), * p < 0.05, ** p < 0.01.

Article Snippet: The recombinant mouse PSMD14 protein was synthesized by MedChemExpress.

Techniques: Binding Assay, Liquid Chromatography with Mass Spectroscopy, Sequencing, Immunoprecipitation, Control, Plasmid Preparation, Transfection, Western Blot, Ubiquitin Proteomics

Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: PSMD14 maintains SLC7A11 expression by cleaving K48‐linked polyubiquitin chains from SLC7A11. A) Schematic diagram of the PSMD14‐SLC7A11 protein complex and the PSMD14 mutant plasmids (residues 1–233 and 234–310) used in subsequent immunoprecipitation assays. B) Immunoprecipitation of the interaction between SLC7A11 and each PSMD14 fragment was performed on plasmid‐transfected MLO‐Y4 cells. C) Immunoprecipitation of the interaction between SLC7A11 and PSMD14 (234‐310) fragment was performed on plasmid‐transfected MLO‐Y4 cells. After transfection was completed, MLO‐Y4 cells were treated with PBS or DEX (100 µM) for 8 h. D,E) Immunoprecipitation was performed to identify the type of polyubiquitination of SLC7A11 in MLO‐Y4 cell. MLO‐Y4 cells were cotransfected with Myc‐SLC7A11 plasmid, Flag‐PSMD14 plasmid and HA‐Ub K48/K63 or HA‐Ub K48R/K63R plasmid. After transfection was completed, MLO‐Y4 cells were treated with MG132 (10 µM) for 8 h. F) Immunoprecipitation of SLC7A11 ubiquitination and its binding to PSMD14 was HA‐Ub K48/K48R plasmid‐treated MLO‐Y4 cells. After transfection was completed, MLO‐Y4 cells were treated with MG132 (10 µM) and supplemented with DEX (100 µM) and/or THL (2 µM) for 8 h. G) Stable PSMD14 knockout MLO‐Y4 cells were constructed by CRISPR‐Cas9 strategy. H) Deubiquitination of SLC7A11 requires the intact PSMD14 fragment. PSMD14 knockout MLO‐Y4 cells treated with HA‐Ub K48 plasmid, Myc‐SLC7A11 plasmid, and PSMD14 full‐length or deletion mutant plasmids upon MG132 (10 µM) treatment for 8 h. I) Deubiquitination of SLC7A11 requires the intact PSMD14 fragment. Stable PSMD14 knockout MLO‐Y4 cells were treated with HA‐Ub K48 plasmid, Myc‐SLC7A11 plasmid, and Flag‐PSMD14 or PSMD14 mutants upon MG132 (10 µM) treatment for 8 h. The experiments were repeated three times independently with similar results.

Article Snippet: The recombinant mouse PSMD14 protein was synthesized by MedChemExpress.

Techniques: Expressing, Mutagenesis, Immunoprecipitation, Plasmid Preparation, Transfection, Ubiquitin Proteomics, Binding Assay, Knock-Out, Construct, CRISPR

Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: Activation of PSMD14 with Pantethine suppresses osteocyte ferroptosis and bone loss. A) Schematic diagram of screening and identification of PSMD14 agonist. B) Docking scores of the top 20 candidates based on virtual screening. C) CCK‐8 assay was performed on MLO‐Y4 cells treated with different 20 candidates (10 µM) and DEX for 48 h (n = 5 per group). D) Chemical structures of five drug candidates (CA, TA, TB, PT and PC). E) Western blot and quantitative analysis of GPX4 protein were performed on MLO‐Y4 cells treated with DEX (100 µM) supplemented with or wihtout candidates (CA, TA, TB, PT or PC 10 µM) for 48 h. F) Cystine uptake assay was performed on MLO‐Y4 cells treated with DEX (100 µM) supplemented with or wihtout candidates (CA, TA, TB, PT or PC 10 µM) for 48 h (n = 9 per group). G) Immunoprecipitation of SLC7A11 ubiquitination and its binding to PSMD14 were performed on MG132 and DEX‐exposed MLO‐Y4 cells (MG132 10µM and DEX 100 µM). MG132 and DEX‐expousred MLO‐Y4 cells were treated with PT (10 µM) and/or THL (2 µM) for 8 h. H) Binding affinity of PT with recombinant PSMD14 was determined using an SPR assay (K D = 5.14 µM). I) Recombinant PSMD14 were incubated with PT, followed by the measurement of the absorbance at OD 445 nm to detect PSMD14 activity using Ubiquitin‐AMC assay (n = 5 per group). J) CCK‐8 assay was performed on DEX‐exposed MLO‐Y4 cells treated with PT (0–100 µM) and/or THL (2 µM) for 48 h (n = 5 per group). K–N) Western blot and quantitative analysis of GPX4 protein (K), MDA concentration detection (L), C11‐BODIPY 581/591 staining (M) and quantitative analysis (N) were performed on DEX‐exposed MLO‐Y4 cells treated with PT (100 µM) and/or THL (2 µM) for 48 h (n = 5 per group). O) Schematic showing the experimental protocol for 8‐weeks of PT / PT + THL injections in GIOP mice. P) Micro‐CT 3D restruction and H&E staining of the distal femur of mice in each group. The processing details of each group are shown in (O). Q) Distal femur BV/TV, Tb.Th, Tb.Sp, Tb.N, and BMD of mice in each group were measured by micro‐CT (n = 6 per group). Quantitative analysis of the empty lacunae in cortical bone (Number of empty lacunae with respect to bone area, N. Empt. Lc./B. Ar. per mm 2 ) based on H&E staining (n = 6 per group). R) GPX4, SLC7A11 and PSMD14 IHC staining of the distal femur of mice in each group. The processing details of each group are shown in (O). S) Quantification of GPX4, SLC7A11 and PSMD14‐positive osteocytes in mouse cortical femurs based on IHC staining (n = 6 per group). T) MDA content in tibia tissue of mice in each group (n = 6 per group). U) Maximum load and Maximum deflection of femoral cortical bone evaluated by the three‐point bending test (n = 6 per group). Data are expressed as mean ± SD, with biologically individual data points shown. p values were determined by one‐way ANOVA test with Tukey's multiple comparisons (C,E,F,H,I,L,N,Q,S–U) and two‐way ANOVA test with Tukey's multiple comparisons (J,K), ns, p > 0.05, * p < 0.05, ** p < 0.01.

Article Snippet: The recombinant mouse PSMD14 protein was synthesized by MedChemExpress.

Techniques: Activation Assay, CCK-8 Assay, Western Blot, Immunoprecipitation, Ubiquitin Proteomics, Binding Assay, Recombinant, SPR Assay, Incubation, Activity Assay, Ub-AMC Assay, Concentration Assay, Staining, Micro-CT, Immunohistochemistry

Journal: Advanced Science

Article Title: PSMD14 Stabilizes SLC7A11 to Ameliorate Glucocorticoid‐Induced Osteoporosis by Suppressing Osteocyte Ferroptosis

doi: 10.1002/advs.202414902

Figure Lengend Snippet: Schematic diagram illustrating the mechanism of GC‐mediated osteocyte ferroptosis. Top: Under physiological conditions, PSMD14 binds to and deubiquitinates SLC7A11 to stabilize SLC7A11 expression and the cystine uptake capacity of osteocytes, thereby ensuring the GSH content and GPX4 activity in osteocytes to maintain cellular function and vitality. Down: During DEX exposure, SLC7A11 is degraded due to limited binding with PSMD14, leading to insufficient cystine in osteocytes and triggering ferroptosis. Combined with virtual screening, we identified PT as a PSMD14 agonist that stabilizes SLC7A11 expression against DEX‐mediated ferroptosis.

Article Snippet: The recombinant mouse PSMD14 protein was synthesized by MedChemExpress.

Techniques: Expressing, Activity Assay, Cell Function Assay, Binding Assay

Journal: Journal of Neuroimmune Pharmacology

Article Title: Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

doi: 10.1007/s11481-021-09994-3

Figure Lengend Snippet: 2CdA treatment reduces T- and B-cell viability. Flow cytometry analysis of viability of ( a ) CD4 + T cells and (b) CD19 + B cells treated as indicated for 48 and 72 h, using Annexin V/propidium iodide labeling of apoptotic cells and necrotic cells, respectively. Data are presented for cells from 8 (CD4 + T cells) or 5 (CD19 + B cells) healthy donors assayed in independent experiments; * P < 0.05, ** P < 0.01, **** P < 0.0001. Unstim. = unstimulated cells; Unstim. + 2CdA = unstimulated cells treated with 2CdA (500 nM); Stim. = cells stimulated with anti-CD3/CD28 beads (CD4 + T cells) or IL-15/CpG (CD19 + B cells); Stim. + 2CdA = cells (CD4 + T cells or CD19 + B cells) stimulated as before and treated with 2CdA (500 nM)

Article Snippet: CD4 + T cells and CD19 + B cells were isolated by negative selection from freshly isolated PBMC using Human CD4 + T- and CD19 + B-cell Isolation Kits (Milteny, Germany) according to manufacturer’s instructions.

Techniques: Flow Cytometry, Labeling

Journal: Journal of Neuroimmune Pharmacology

Article Title: Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

doi: 10.1007/s11481-021-09994-3

Figure Lengend Snippet: DCK and NT5C2 mRNA and/or protein expression are upregulated by 2CdA in T and B cells. ( a ) DCK and ( b ) NT5C2 mRNA expression measured by Real-time PCR in CD4 + T cells stimulated with anti-CD3/CD28 beads for 48 and 72 h in the presence or absence of 2CdA (500 nM). The mean ΔCT + SD baseline levels of expression of DCK and NT5C2 mRNA in unstimulated cells were 9.98 + 0.41 and 8.5 + 0.13, respectively. Data are presented for T cells isolated from 8 healthy donors assayed in independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001. ( c ) Representative Western blot (left panel) and quantification (right panel) of dCK in stimulated CD4 + T cells exposed or not to 2CdA (500 nM) for 72 h. Data were normalized to loading control protein, VCN. Fold changes were calculated using Unstim. controls as reference. Data are presented for T cells isolated from 5 healthy donors assayed in independent experiments; * P < 0.05, ** P < 0.01. Unstim. = unstimulated cells;. Stim. = CD4 + T cells stimulated with anti-CD3/CD28 beads; Stim. + 2CdA = CD4 + T cells stimulated with anti-CD3/CD28 beads and treated with 2CdA (500 nM). ( d ) DCK and NT5C2 mRNA expression measured by Real-time PCR in CD19 + B cells stimulated anti-IL-15/CpG for 72 h in the presence or absence of 2CdA. The mean ΔCT + SD baseline levels of expression of DCK and NT5C2 mRNA in unstimulated cells were 10.5 + 0.40 and 7.475 + 0.12, respectively. Data are presented for B cells isolated from 5 healthy donors assayed in independent experiments; * P < 0.05. Stim. = CD19 + B cells activated with IL-15/CpG; Stim. + 2CdA = CD19 + B cells stimulated with IL-15/CpG treated with 2CdA (500 nM). GAPDH mRNA was used as endogenous control to normalize the expression data in the PCR analyses

Article Snippet: CD4 + T cells and CD19 + B cells were isolated by negative selection from freshly isolated PBMC using Human CD4 + T- and CD19 + B-cell Isolation Kits (Milteny, Germany) according to manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Control

Journal: Journal of Neuroimmune Pharmacology

Article Title: Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

doi: 10.1007/s11481-021-09994-3

Figure Lengend Snippet: B- and T-cell activation increases dCK activity but exposure to 2CdA abrogates such activity in stimulated B cells. ( a ) Validation of dCK activity assay on different amounts of dCK in enriched preparation from PBMC. dCK activity is presented as RLUs), corresponding to the residual ATP from kinase reaction. Data are presented for PBMC isolated from 3 healthy donors assayed in independent experiments; ( b ) dCK activity in CD4 + T cells stimulated with anti-CD3/CD28 beads for 72 h in the presence or absence of 2CdA (500 nM). Data are presented for T cells isolated from 6 healthy donors assayed in independent experiments. Unstim. = unstimulated CD4 + T cells; Unstim. + 2CdA = unstimulated CD4 + T cells treated with 2CdA (500 nM); Stim. = CD4 + T cells stimulated with anti-CD3/CD28 beads; Stim. + 2CdA = CD4 + T cells stimulated with anti-CD3/CD28 beads and treated 2CdA (500 nM). ( c ) dCK enzymatic activity in CD19 + B cell stimulated with anti-IL-15/CpG for 72 h in the presence or absence of 2CdA (500 nM). Data are presented for B cells isolated from 6 healthy donors assayed in independent experiments. Unstim. = unstimulated CD19 + B cells; Unstim. + 2CdA = unstimulated CD19 + B cells treated with 2CdA (500 nM); Stim. = CD19 + B cells activated with IL-15/CpG; Stim.+ 2CdA = CD19 + B cells activated with IL-15/CpG treated with 2CdA (500 nM). * P < 0.05, ** P < 0.01, **** P < 0.0001

Article Snippet: CD4 + T cells and CD19 + B cells were isolated by negative selection from freshly isolated PBMC using Human CD4 + T- and CD19 + B-cell Isolation Kits (Milteny, Germany) according to manufacturer’s instructions.

Techniques: Activation Assay, Activity Assay, Biomarker Discovery, Isolation

Journal: Journal of Neuroimmune Pharmacology

Article Title: Different Susceptibility of T and B Cells to Cladribine Depends On Their Levels of Deoxycytidine Kinase Activity Linked to Activation Status

doi: 10.1007/s11481-021-09994-3

Figure Lengend Snippet: Antibody and fluorochrome panels to identify CLP and CMP from bone-marrow mononuclear cells

Article Snippet: CD4 + T cells and CD19 + B cells were isolated by negative selection from freshly isolated PBMC using Human CD4 + T- and CD19 + B-cell Isolation Kits (Milteny, Germany) according to manufacturer’s instructions.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Ectopic germinal centers are rare in Sjogren's syndrome salivary glands and do not exclude autoreactive B cells.

doi: 10.4049/jimmunol.0803588

Figure Lengend Snippet: FIGURE 2. Microdissection of GC-like struc- tures from salivary glands and tonsils before quan- titative-RT-PCR analysis. A, Sections were stained with HistoGene to locate infiltrates, and with FITC-anti-CD19 to recognize B cells. FDCs were found in real GCs, as well as in aggregates, through their staining with CD21 and CD35. B, The levels of mRNAs for activation-induced cytidine deami- nase (AICDA) were determined by quantitative RT-PCR and normalized relative to GAPDH. All of the four real GC-containing structures expressed AID, compared with none of the seven aggregate- containing structures.

Article Snippet: In parallel, anti-CD19 mAb was combined with rabbit anti-BR3, anti-Bcl-6 (both from ProSci), or anti-IRF-4 (Abcam) Abs.

Techniques: Laser Capture Microdissection, Reverse Transcription Polymerase Chain Reaction, Staining, Activation Assay, Quantitative RT-PCR

Journal: International Journal of Nanomedicine

Article Title: Effect of buckminsterfullerenes on cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cells

doi: 10.2147/IJN.S33773

Figure Lengend Snippet: The effect of varying concentrations (0.08–800 ng/mL = 0.1–100 nM) of poly-C60 and nepo-C60 on the proliferative response of PBMC from 20 healthy volunteers measured by the lymphocyte transformation test. Notes: Individuals values are given. Statistically significant differences on the whole group level comparing the spontaneous and the fullerene-induced proliferation are indicated: * P < 0.05; ** P < 0.01. Abbreviations: SI, stimulation index; 0,00, spontaneous proliferation without antigen; –, median.

Article Snippet: Antibody cocktails were used for the demonstration of activated PBMC subpopulations (activated CD4+ T cells: FastImmuneTM CD4/CD69/CD3, activated, CD8+ T-cells: FastImmuneTM CD8/CD69/CD3; activated CD19+ B-cells: FastImmune TMCC D19/CD69/CD45); activated CD56+ NK-cells: FastImmuneTM CD56/CD69/CD45; Becton-Dickinson [San Jose, CA]).

Techniques: Transformation Assay

Journal: International Journal of Nanomedicine

Article Title: Effect of buckminsterfullerenes on cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cells

doi: 10.2147/IJN.S33773

Figure Lengend Snippet: The effect of varying concentrations (0.08–800 ng/mL = 0.1–100 nM) of poly-C60 and nepo-C60 on the IFN-γ production by PBMC from 20 healthy volunteers measured by ELISA. Notes: Individual values are given. There were no statistical differences on the whole group level comparing the spontaneous and the fullerene-induced IFN-γ production. Abbreviations: 0, spontaneous IFN-γ production without antigen; –, median.

Article Snippet: Antibody cocktails were used for the demonstration of activated PBMC subpopulations (activated CD4+ T cells: FastImmuneTM CD4/CD69/CD3, activated, CD8+ T-cells: FastImmuneTM CD8/CD69/CD3; activated CD19+ B-cells: FastImmune TMCC D19/CD69/CD45); activated CD56+ NK-cells: FastImmuneTM CD56/CD69/CD45; Becton-Dickinson [San Jose, CA]).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Effect of buckminsterfullerenes on cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cells

doi: 10.2147/IJN.S33773

Figure Lengend Snippet: The effect of varying concentrations (0.08–800 ng/mL = 0.1–100 nM) of poly-C60 and nepo-C60 on the IL-10 production by PBMC from 18 healthy volunteers measured by ELISA. Notes: Two individuals producing already spontaneously high levels of IL-10 were omitted. Individual values are given. There were no statistical differences on the whole group level comparing the spontaneous and the fullerene-induced IL-10 production. Abbreviations: 0, spontaneous IL-10 production without antigen; –, median.

Article Snippet: Antibody cocktails were used for the demonstration of activated PBMC subpopulations (activated CD4+ T cells: FastImmuneTM CD4/CD69/CD3, activated, CD8+ T-cells: FastImmuneTM CD8/CD69/CD3; activated CD19+ B-cells: FastImmune TMCC D19/CD69/CD45); activated CD56+ NK-cells: FastImmuneTM CD56/CD69/CD45; Becton-Dickinson [San Jose, CA]).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Effect of buckminsterfullerenes on cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cells

doi: 10.2147/IJN.S33773

Figure Lengend Snippet: The effect of varying concentrations (0.08–800 ng/mL = 0.1–100 nM) of poly-C60 and nepo-C60 on the IL-6 production by PBMC from 17 healthy volunteers measured by ELISA. Notes: Three individuals producing already spontaneously high levels of IL-6 were omitted. Individual values are given. Statistically significant differences on the whole group level comparing the spontaneous and the fullerene-induced IL-6 production are indicated: * P < 0.05. Abbreviations: 0, spontaneous IL-6 production without antigen; –, median.

Article Snippet: Antibody cocktails were used for the demonstration of activated PBMC subpopulations (activated CD4+ T cells: FastImmuneTM CD4/CD69/CD3, activated, CD8+ T-cells: FastImmuneTM CD8/CD69/CD3; activated CD19+ B-cells: FastImmune TMCC D19/CD69/CD45); activated CD56+ NK-cells: FastImmuneTM CD56/CD69/CD45; Becton-Dickinson [San Jose, CA]).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Effect of buckminsterfullerenes on cells of the innate and adaptive immune system: an in vitro study with human peripheral blood mononuclear cells

doi: 10.2147/IJN.S33773

Figure Lengend Snippet: The effect of varying concentrations (0.08–800 ng/mL = 0.1–100 nM) of poly-C60 and nepo-C60 on activation of NK-cells measured by CD69 expression by flow cytometry with PBMC from 20 healthy volunteers. Notes: Individual values are given. Statistically significant differences on the whole group level comparing the spontaneous and the fullerene-induced CD69 expression are indicated: * P < 0.05; ** P < 0.01. Abbreviations: 0, spontaneous CD69 expression without antigen; –, median.

Article Snippet: Antibody cocktails were used for the demonstration of activated PBMC subpopulations (activated CD4+ T cells: FastImmuneTM CD4/CD69/CD3, activated, CD8+ T-cells: FastImmuneTM CD8/CD69/CD3; activated CD19+ B-cells: FastImmune TMCC D19/CD69/CD45); activated CD56+ NK-cells: FastImmuneTM CD56/CD69/CD45; Becton-Dickinson [San Jose, CA]).

Techniques: Activation Assay, Expressing, Flow Cytometry

Journal: Molecular Therapy

Article Title: AAV8 Can Induce Innate and Adaptive Immune Response in the Primate Eye

doi: 10.1016/j.ymthe.2017.08.018

Figure Lengend Snippet: Activation Assays of PBMC Fractions

Article Snippet: Antibody cocktails were used for the demonstration of activated PBMC subpopulations (activated CD4 + T cells, FastImmune CD4/CD69/CD3; activated CD8 + T cells, FastImmune CD8/CD69/CD3; activated CD19 + B cells, FastImmune CD19/CD69/CD45; activated CD56 + natural killer [NK] cells, FastImmune CD56/CD69/CD45; Becton Dickinson, San Jose, CA).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Whole Transcriptome Analysis Reveals Heterogeneity in B Cell Memory Populations in Patients With Juvenile Idiopathic Arthritis-Associated Uveitis

doi: 10.3389/fimmu.2020.02170

Figure Lengend Snippet: Blood CD19 + B cell transcriptomics in juvenile idiopathic arthritis-associated uveitis. (A) Deconvolution-based estimation of immune cell fractions using the RNA-sequencing data and CIBERSORT . The estimated cell fraction for each immune cell type in each sample is depicted in a stacked barplot (using the LM22 as signature sets to estimate the cell fractions) in the disease groups and controls. (B) The estimated cell fraction for naive and memory B cells for each of the disease groups and Kruskal–Wallis test statistic (KWt). (C) Principal component analysis of 614 DEGs at nominal significance (likelihood ratio test P < 0.05). (D) Pathway enrichment analysis of the 614 DEGs using the R package clusterProfiler with the Reactome database. (E) Hierarchical clustering of the 614 DEGs from the (C) using Euclidean distance and Ward’s method. Cluster 1–3 are indicated by different colors. A selection of genes associated to each cluster is indicated by the corresponding color (full list of genes and clusters are depicted in , ).

Article Snippet: FACS purified CD19 + B cells were immediately taken up in lysis buffer (RLT plus, Qiagen, Venlo, Netherlands) containing 1% β-mercaptoethanol and subjected to RNA extraction using the AllPrep Universal Kit (Qiagen) on the QIAcube according to the manufacturer’s instructions.

Techniques: RNA Sequencing Assay, Selection