Journal: Frontiers in Microbiology

Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

doi: 10.3389/fmicb.2018.02765

Figure Lengend Snippet: Analysis of expression of activation surface markers in PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Activation was measured by flow cytometry using mAbs against surface molecules (CD69, CD86). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. ∗ P

Article Snippet: Evaluation of Surface Markers of Peripheral Blood Mononuclear Cells (PBMCs) Induced by OMVs Formalin-killed bacteria (FKB) was obtained as follows: A. hydrophila ATCC® 7966TM was cultured 24 h onto TSA plates, then a cell suspension was prepared in sterile phosphate-buffered saline (PBS 0.1 M, pH 7.3).

Techniques: Expressing, Activation Assay, Cell Culture, Flow Cytometry, Cytometry

Journal: Frontiers in Microbiology

Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

doi: 10.3389/fmicb.2018.02765

Figure Lengend Snippet: Cytokine quantification from PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. Inflammatory cytokines were determined by CBA cytometric assay at different time points. A. hydrophila OMVs induced high levels of inflammatory cytokines such as IL-8, IL-1β, IL-6, and TNFα. ∗ P

Article Snippet: Evaluation of Surface Markers of Peripheral Blood Mononuclear Cells (PBMCs) Induced by OMVs Formalin-killed bacteria (FKB) was obtained as follows: A. hydrophila ATCC® 7966TM was cultured 24 h onto TSA plates, then a cell suspension was prepared in sterile phosphate-buffered saline (PBS 0.1 M, pH 7.3).

Techniques: Crocin Bleaching Assay

Journal: Frontiers in Microbiology

Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

doi: 10.3389/fmicb.2018.02765

Figure Lengend Snippet: Analysis of expression of inhibition surface markers in PBMCs stimulated with A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with OMVs from A. hydrophila . Inhibition was measured by flow cytometry using mAbs against surface molecules (PD-1, PD-L1). Monocyte (CD91+) and lymphocytes (CD3+, CD19+) were gated to analyze individually the expression of surface molecules. FKB were used as control for the expression of surface markers. ∗ P

Article Snippet: Evaluation of Surface Markers of Peripheral Blood Mononuclear Cells (PBMCs) Induced by OMVs Formalin-killed bacteria (FKB) was obtained as follows: A. hydrophila ATCC® 7966TM was cultured 24 h onto TSA plates, then a cell suspension was prepared in sterile phosphate-buffered saline (PBS 0.1 M, pH 7.3).

Techniques: Expressing, Inhibition, Cell Culture, Flow Cytometry, Cytometry

Journal: Frontiers in Microbiology

Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

doi: 10.3389/fmicb.2018.02765

Figure Lengend Snippet: Apoptosis and DNA damage of PBMCs induced by A. hydrophila ATCC ® 7966 TM OMVs. PBMCs from healthy donor were co-cultured with different concentrations of A. hydrophila OMVs during 24 h. Apoptosis and DNA damaged was evaluated by flow cytometry with mAbs anti-PARP and anti-H2AX. CCCP was used as control for apoptosis induction. ∗ P

Article Snippet: Evaluation of Surface Markers of Peripheral Blood Mononuclear Cells (PBMCs) Induced by OMVs Formalin-killed bacteria (FKB) was obtained as follows: A. hydrophila ATCC® 7966TM was cultured 24 h onto TSA plates, then a cell suspension was prepared in sterile phosphate-buffered saline (PBS 0.1 M, pH 7.3).

Techniques: Cell Culture, Flow Cytometry, Cytometry

Journal: Frontiers in Microbiology

Article Title: The Outer Membrane Vesicles of Aeromonas hydrophila ATCC® 7966TM: A Proteomic Analysis and Effect on Host Cells

doi: 10.3389/fmicb.2018.02765

Figure Lengend Snippet: Morphological alterations induced by A. hydrophila ATCC ® 7966 TM OMVs in PBMCs. (A) After 2 h post-stimulation with A. hydrophila OMVs, no morphology changes of the PBMCs were observed compared with PBMCs after 24 h post-stimulation with vesicles, after 24 h post-stimulation conglomerated cells were observed (arrowheads) (B) . Giemsa staining revealed differences in the affinity of the dye to the nucleus and cytoplasm, bigger cells, and vacuolization of the stimulated PBMCs with A. hydrophila OMVs (arrowheads) (C) . Confocal microscopy, showed defective tubulin polymerization in PBMCs stimulated with OMVs (D) . bars = 50 μm.

Article Snippet: Evaluation of Surface Markers of Peripheral Blood Mononuclear Cells (PBMCs) Induced by OMVs Formalin-killed bacteria (FKB) was obtained as follows: A. hydrophila ATCC® 7966TM was cultured 24 h onto TSA plates, then a cell suspension was prepared in sterile phosphate-buffered saline (PBS 0.1 M, pH 7.3).

Techniques: Staining, Confocal Microscopy

Journal: Cells

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

doi: 10.3390/cells7050045

Figure Lengend Snippet: Qualification of HCMV-specific T-cell IFN-γ ImmunoSpot® testing of PBMC. ( A ) Establishing the CD4+/CD8+ lineage of HCMV-specific memory T cells. CD4+ or CD8+ T-cell depleted PBMC or unseparated PBMC of Donor 1 were tested in an IFN-γ ImmunoSpot® assay. The numbers of I-HCMV or HCMVpp65-induced IFN-γ SFU were measured for each condition, and the mean and SD for three replicate measurements of each are shown. ( B ) PBMC cell-number dependence of SFU. PBMC of Donor 1 were plated in the specified cell numbers per well and I-HCMV or HCMVpp65 (specified by color) was added at 50 μg/mL or 1 μg/mL, respectively. The numbers of IFN-γ SFU were established. Means and SD are shown for triplicate wells. The result of regression analysis for the experimental data approaching linearity is specified for both conditions as the R 2 value in color. ( C ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by HCMVpp65 peptides. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different peptide concentrations specified. The number of IFN-γ SFU was measured in single wells. ( D ) Establishing the optimal antigen dose for stimulation of HCMV-specific T cells by I-HCMV. PBMC of Donor 1 were plated at 3 × 10 5 cells per well along with the different antigen concentrations specified. The number of IFN-γ SFU was measured in single wells.

Article Snippet: Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes.

Techniques:

Journal: Cells

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

doi: 10.3390/cells7050045

Figure Lengend Snippet: Establishing the specificity of I-HCMV- and HCMVpp65-induced IFN-γ ImmunoSpot® formation. Donor 1 was seropositive for HCMV, Donor 72 was seronegative. PBMC from both donors were tested in an IFN-γ ImmunoSpot® assay in medium alone as the negative control (“MEDIUM”), or in the presence of the HCMVpp65 peptide pool (HCMVpp65) or inactivated HCMV virions (I-HCMV), as specified. As positive controls for eliciting memory T-cell responses, a pool of 32 peptides of HCMV, EBV, and influenza virus were used (CEFpp). The test was performed as specified in Materials and Methods, with three replicate wells for each condition. A representative image of one of the replicates is shown for each condition.

Article Snippet: Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes.

Techniques: Negative Control

Journal: Cells

Article Title: Direct Detection of T- and B-Memory Lymphocytes by ImmunoSpot® Assays Reveals HCMV Exposure that Serum Antibodies Fail to Identify

doi: 10.3390/cells7050045

Figure Lengend Snippet: Qualification of detecting HCMV-specific B cells by ImmunoSpot® testing of PBMC. ( A ) Establishing the specificity of the assay. Membranes were coated with either I-HCMV, or with anti-kappa/lambda capture antibody as a positive control, as specified. PBMC of Donor 1 of Donor 72 were added after polyclonal stimulation and plate-bound IgG was detected as described in Materials and Methods. Representative wells are shown. ( B ) Correlation between cell numbers plated and HCMV SFU detected. Preactivated PBMC of Donor 1 were plated in the specified cell numbers and the IgG SFU were counted. The result of regression analysis for the experimental data approaching linearity is specified by the R 2 value. ( C ) Identifying the IgG subclasses of HCMV-specific B cells. Wells were coated with I-HCMV antigen followed by plating of preactivated PBMC of Donor 1. The plate-bound antibodies were visualized using detection antibodies specific for pan-IgG, IgG1, IgG2, and IgG3 as specified.

Article Snippet: Eighty-two HCMV seronegative individuals were identified, and their peripheral blood mononuclear cells (PBMC) were tested in ImmunoSpot® assays for the presence of HCMV-specific T- and B-memory lymphocytes.

Techniques: Positive Control

Journal: Cancer research

Article Title: GSK 3 inhibition drives maturation of NK cells and enhances their antitumor activity

doi: 10.1158/0008-5472.CAN-17-0799

Figure Lengend Snippet: Inhibition of GSK3 with CHIR99021 enhances NK cell cytokine production and anti-tumor function in vitro CD3/19-depleted PBMCs from CMV seropositive donors were cultured overnight with 10 ng/ml IL-15, for 7 days with 10 ng/ml IL-15 and DMSO or for 7 days with 10 ng/ml IL-15 and 5 μM CHIR99021 prior to functional analysis. A , NK cells cultured in the conditions above were incubated with K562 cells at the indicated effector:target ratios followed by the addition of Caspase-3/7 Green Detection Reagent. FACS was used to determine the percentage of K562 cells undergoing apoptosis ( n = 15). Results are from 3 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells cultured in the conditions above were incubated with K562 cells at a 1:1 ratio, and intracellular FACS was used to measure the percentage of CD3 − CD56 + NK cells positive for B , TNF and C , IFN-γ (right panel) ( n = 7). Results are from 4 independent experiments and are shown as mean ± SEM. Paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01. NK cells ( n = 3) cultured in the conditions above were incubated with D , A549 cells, E , SKOV-3 cells and F , PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of a control anti-CD20 antibody. Parallel assays with NK cells ( n = 3) cultured in the conditions above were performed with G , A549 cells, H, SKOV-3 cells and I, PANC-1 cells expressing NucLight Red fluorescent protein at a 3:1 ratio with the addition of either anti-Her2 or anti-EGFR antibodies. An IncuCyte Imaging System was used to measure target cell killing over 48 hours. Results are from 3 independent experiments and are shown as mean ± SEM. Two-way ANOVA with Tukey’s multiple comparisons tests was used to determine significance.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from healthy CMV seropositive donors were obtained from Memorial Blood Bank (Minneapolis, MN), AllCells Inc. (Alameda, CA) and Key Biologics (Memphis, TN).

Techniques: Inhibition, In Vitro, Cell Culture, Functional Assay, Incubation, FACS, Expressing, Imaging

Journal: Cancer research

Article Title: GSK 3 inhibition drives maturation of NK cells and enhances their antitumor activity

doi: 10.1158/0008-5472.CAN-17-0799

Figure Lengend Snippet: NK cells expanded with CHIR99021 exhibit elevated expression of transcription factors associated with late-stage NK cell maturation CD3/19-depleted PBMCs from CMV seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either DMSO or 5 μM CHIR99021 ( n = 9). CD3 − CD56 + NK cells were then isolated from each culture condition and analyzed by A , qRT-PCR for expression of TBX21 , ZEB2 , PRDM1A , PRDM1B and EOMES mRNA. Shown are cumulative data of relative fold expression values of cells cultured with CHIR99021 relative to cells cultured with DMSO. All data are normalized against ACTB . B , CD3 − CD56 + NK cells from these cultures were also analyzed by intracellular staining and FACS for expression levels of TBX21, ZEB2, BLIMP-1 and EOMES (as determined by mean fluorescence intensity). Shown are cumulative relative fold expression values for each transcription factor in cells cultured with CHIR99021 relative to cells cultured with DMSO. Results are from 2 independent experiments. Data is shown as mean ± SEM, and paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from healthy CMV seropositive donors were obtained from Memorial Blood Bank (Minneapolis, MN), AllCells Inc. (Alameda, CA) and Key Biologics (Memphis, TN).

Techniques: Expressing, Cell Culture, Isolation, Quantitative RT-PCR, Staining, FACS, Fluorescence

Journal: Cancer research

Article Title: GSK 3 inhibition drives maturation of NK cells and enhances their antitumor activity

doi: 10.1158/0008-5472.CAN-17-0799

Figure Lengend Snippet: Addition of CHIR99021 to NK cells expanded with autologous monocytes enriches for NK cells with a mature phenotype CD3/19-depleted PBMCs from CMV seropositive donors were cultured for 7 days with 10 ng/ml IL-15 and either DMSO or 5 μM CHIR99021. A , Shown is fold NK cell expansion after 7 days in each culture condition relative to day 0 ( n = 15). B , Representative FACS scatter plots of CD57 and NKG2C expression on gated CD3 − CD56 + NK cells from before and after culture (upper panel). Cumulative ( n = 15) data showing the percentages of CD57 + NK cells and the mean fluorescence intensity of CD57 gated on CD57 + NK cells as well as the percentages of adaptive CD57 + NKG2C + NK cells within the total CD3 − CD56 + NK cell population before and after culture (lower panel). Results are from 5 independent experiments. C , Representative dot plots or FACS histogram plots for each receptor or cytotoxic granule component on gated CD3 − CD56 + NK cells from each culture condition (upper panel). Cumulative ( n = 6) data showing the relative median fluorescence intensity for each receptor or cytotoxic granule component on gated CD3 − CD56 + NK cells from DMSO and CHIR99021 cultures relative to the pre-culture phenotype (lower panel). All results are from 3–5 independent experiments. All cumulative data is shown as mean ± SEM, and paired Student’s t tests were used for statistical analyses. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Peripheral blood mononuclear cells (PBMCs) from healthy CMV seropositive donors were obtained from Memorial Blood Bank (Minneapolis, MN), AllCells Inc. (Alameda, CA) and Key Biologics (Memphis, TN).

Techniques: Cell Culture, FACS, Expressing, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Progranulin and a Five Transmembrane Domain-Containing Receptor-like Gene Are the Key Components in Receptor Activator of Nuclear Factor κB (RANK)-dependent Formation of Multinucleated Osteoclasts *

doi: 10.1074/jbc.M114.608786

Figure Lengend Snippet: Effects of PGRN on the differentiation of human OCs and PGRN as a predictor of osteoporosis. A , HBMCs and PBMCs were differentiated with RANKL in the absence or presence of PGRN. TRAP + MNCs were counted as described above. B , four sh-RNAs targeting human

Article Snippet: Human bone marrow cells (HBMCs) and peripheral blood mononuclear cells (PBMCs) were obtained from healthy volunteers and were separated by density gradient centrifugation using Ficoll-Histopaque (Sigma-Aldrich).

Techniques:

Journal: Frontiers in Immunology

Article Title: Complement Protein C1q Interacts with DC-SIGN via Its Globular Domain and Thus May Interfere with HIV-1 Transmission

doi: 10.3389/fimmu.2016.00600

Figure Lengend Snippet: HIV transfer assay mediated by DC-SIGN . Cell surface DC-SIGN expressing HEK (DC-HEK) cells were grown in a 12-well plate to form a confluent layer. Different concentrations of proteins were added to the cells and incubated for 2 h for binding. Unbound proteins were removed and cells were challenged with 2.5 ng/mL p24 of HIV-1 (SF-162 strain) for 1 h. Unbound virus was washed off and cells were co-cultured with PHA-activated PBMCs for 24 h. PBMCs were separated from the DC-HEK monolayer and cultured separately for 7 days to determine viral titer of the supernatants collected on days 4 and 7. (A) C1q, ghA, ghB, ghC, and ghABC; (B) gC1qR in presence of C1q, ghA, ghB, ghC, and ghABC. Data represent mean ± SD. P

Article Snippet: HIV-1 Transfer Assay with DC-HEK Cells and Pooled Peripheral Blood Mononuclear Cell (PBMC) Pooled peripheral blood mononuclear cells (HiMedia Laboratories, India) were cultured in RPMI 1640 medium (Sigma Aldrich) containing 10% FBS, 1% Penicillin–Streptomycin (Complete RPMI medium), and stimulated with 5 µg/mL phytohemaglutinin (PHA) and 10 U/Ml of recombinant-human IL-2 (Gibco) for 24 h. PHA/IL-2 was washed off and activated PBMCs were cultured further for 3 days in complete RPMI 1640 medium.

Techniques: Expressing, Incubation, Binding Assay, Cell Culture

Journal: PLoS ONE

Article Title: Evidence for a Role for Interleukin-17, Th17 Cells and Iron Homeostasis in Protective Immunity against Tuberculosis in Cynomolgus Macaques

doi: 10.1371/journal.pone.0088149

Figure Lengend Snippet: Comparison of differentially expressed gene lists across experimental groups. Microarray analysis was used to identify differentially expressed genes in PPD-stimulated PBMCs isolated at 6 weeks post-challenge with M. tuberculosis from vaccinated controllers (n = 6), unvaccinated controllers (n = 3) and unvaccinated progressors (n = 3) compared with expression when animals were naïve. Key genes of interest in each list are indicated. Underlined genes were down-regulated in expression from naïve time-points.

Article Snippet: Isolation and Stimulation of PBMCs Peripheral blood mononuclear cells (PBMC) were isolated from heparin-anticoagulated blood by Ficoll-Hypaque Plus (GE Healthcare, Buckinghamshire, UK) density gradient separation using standard procedures.

Techniques: Microarray, Isolation, Expressing

Journal: PLoS ONE

Article Title: Evidence for a Role for Interleukin-17, Th17 Cells and Iron Homeostasis in Protective Immunity against Tuberculosis in Cynomolgus Macaques

doi: 10.1371/journal.pone.0088149

Figure Lengend Snippet: Expression of iron regulatory genes in PBMCs isolated from unvaccinated controllers and progressors at post-mortem. Gene expression of a) ferritin heavy chain b) solute carrier family 11 member A1, and c) solute carrier family 11 member A2, d) transferrin receptor 1 and e) heme oxygenase 1 in unstimulated PBMCs isolated at post-mortem. The fold change from naïve baseline levels was determined using RT-PCR. To determine statistically significant differences of relative gene expression, a two-sample t-test was performed where * and ** represent P-values of

Article Snippet: Isolation and Stimulation of PBMCs Peripheral blood mononuclear cells (PBMC) were isolated from heparin-anticoagulated blood by Ficoll-Hypaque Plus (GE Healthcare, Buckinghamshire, UK) density gradient separation using standard procedures.

Techniques: Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

Journal: Vaccine

Article Title: Cancer immunotherapy using a potent immunodominant CTL epitope

doi: 10.1016/j.vaccine.2014.09.021

Figure Lengend Snippet: Systemic immune response to LCMV-GP33 peptide C57BL/6 mice (5 mice/group) were challenged subcutaneously with TC-1 tumor cells and subsequently treated with various combinations of cisplatin, CpG and GP33 peptide as shown. The PBMCs were analyzed 1 week after the last antigen delivery. The presence of GP33-specific CD8+ T cells in PBMCs was analyzed using intracellular cytokine staining for IFN-γ and CD8+ staining followed by flow cytometry analysis. A. Representative flow cytometry contour plot depicting the frequency of IFN-γ-secreting CD8+ T cells among PBMCs after being pulsed with GP33 peptide. B. Bar graph depicting the number of IFN-γ+ CD8+ T cells among PBMCs (mean ±S.E).

Article Snippet: To detect HPV16 E7-specific and LCMV GP33-specific CD8+ T cell responses by IFN-γ intracellular staining, peripheral blood mononuclear cells (PBMCs), single-cell suspended splenocytes or tumor infiltrating lymphocytes were stimulated with either HPV E7aa49-57 or LCMV GP33 peptide (1μg/ml) in the presence of Golgiplug (BD Pharmingen, San Diego, CA) at 37°C overnight.

Techniques: Mouse Assay, Staining, Flow Cytometry, Cytometry