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  • 99
    AllCells LLC pbmcs
    HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. <t>NSG-hu</t> mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in <t>PBMCs</t> at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P
    Pbmcs, supplied by AllCells LLC, used in various techniques. Bioz Stars score: 99/100, based on 135 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Astarte Biologics pbmcs
    HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. <t>NSG-hu</t> mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in <t>PBMCs</t> at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P
    Pbmcs, supplied by Astarte Biologics, used in various techniques. Bioz Stars score: 99/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Becton Dickinson pbmc
    Proliferation of lymphocytes in <t>LNs</t> and <t>PBMCs</t> as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.
    Pbmc, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 7353 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    BioreclamationIVT pbmcs
    Proliferation of lymphocytes in <t>LNs</t> and <t>PBMCs</t> as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.
    Pbmcs, supplied by BioreclamationIVT, used in various techniques. Bioz Stars score: 96/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore pbmcs
    Cytokine induced <t>CIS</t> mRNA expression and STAT5 phosphorylation according to CISH haplotypes. a CIS mRNA expression of <t>PBMCs</t> from healthy adult donors with distinct haplotypes (circles: homozygous H1 carriers (H1/H1), triangles: H2 carriers (H1/H2, H2/H2), squares: H3 carriers (H1/H3)) treated with IL-2 (left graph) or IL-7 (right graph) for 1 and 2 h. CIS mRNA levels—calculated in comparison to the housekeeping gene GAPDH—are normalized against time point 0 for each donor. Median and IQR of 2 −ΔΔCT values are indicated. p value for effect of IL-2/IL-7 stimulation on CIS mRNA levels considering all genotypes (Friedman test) is indicated as *** for p
    Pbmcs, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 15970 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Sanquin pbmc
    Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all <t>HLA-A2</t> positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) <t>PBMC</t> from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.
    Pbmc, supplied by Sanquin, used in various techniques. Bioz Stars score: 97/100, based on 142 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    STEMCELL Technologies Inc pbmc
    Induction of HIV by LRAs in a primary <t>CD4+</t> T-cell direct infection latency model. CD4 + T-cells were isolated from <t>PBMC</t> by negative selection, cultured with CCL19 and then magnetofected with HIV, or mock infected (no virus). Two days later, cells were depleted of activated cells. We defined activated cells as those expressing at least one of CD69, HLA-DR, or CD25 based on a study that defined CD4+ T-cells with the triple-negative phenotype as quiescent[ 29 ]. In parallel, CD4 + T-cells were activated using anti-CD3/anti-CD28 antibodies and infected with HIV (productively infected). A . Depletion of activated cells by anti-PE microbeads. Shown are flow cytometry data gated on CD3 + CD4 + lymphocytes (95% pure) and depicting CD25/CD69/HLA-DR staining (all pooled on PE channel)–y-axis, by HIV-Gag–x-axis. The left and middle panels represent pre- and post-depletion of activated cells, respectively to generate latently-infected cells. The right panel shows productively-infected target cells. B . Latently infected or mock-infected resting CD4 + T-cells were prepared as in A and then either stimulated with 1.5 nM IL-15, with 2.6 μM prostratin, or left unstimulated for 36 hours. Shown are flow cytometry data, gated on lymphocytes (SSC/FSC) and depicting CD4 –y-axis, by HIV-Gag–x-axis. C . In a separate experiment analogous to B latently-infected or mock-infected cells were treated with the indicated concentrations of IL-15 for 16 hours. The percentage of HIV-Gag + cells (gated as in B ) is plotted against the concentration of IL-15 (red circles = infected, green squares = uninfected). D. Latently-infected cells were stimulated with the indicated LRAs for 36 hours and HIV p24 in supernatants was quantified by ELISA. Shown are background-subtracted mean ± SEM values (duplicates). P-values were calculated by ANOVA with Holm-Sidak’s multiple comparison test (comparing each condition with no treatment [No Tx]) * p
    Pbmc, supplied by STEMCELL Technologies Inc, used in various techniques. Bioz Stars score: 99/100, based on 1807 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pbmcs
    In vivo and in vitro associations between endotoxin (LPS) levels and TLR4 mRNA expression in <t>PBMCs.</t> (A) In samples obtained directly from HIV uninfected subjects, an inverse relationship between plasma LPS levels and TLR4 expression was apparent (R = −0.35, P = 0.053, Spearman Rank correlation). However, in HIV infected subjects no correlation or trend was seen (R = 0.029, P = 0.83). Additionally, plasma HIV <t>RNA</t> levels were positively associated with increased TLR4 mRNA levels (Spearman R = 0.3260, P = 0.049). (B) In a similar pattern, overnight culture with 0.1 ug/l LPS reduced TLR4 mRNA expression in PBMC of HIV uninfected (n = 70, P = 0.009), but not infected subjects (n = 21, P = 0.627). Isolated HIV ssRNA40 increased TLR4 mRNA expression in PBMC from HIV uninfected subjects (n = 70, P = 0.011), but not infected subjects (n = 21, P = 0.808). Spearman rank sum and Wilcoxon signed rank tests were used, two-tailed ns = not significant, t = trend(P
    Pbmcs, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 30379 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 30379 article reviews
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    pbmc  (ZenBio)
    90
    ZenBio pbmc
    EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. <t>CD4+</t> T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells <t>(PBMC),</t> CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.
    Pbmc, supplied by ZenBio, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Cellular Technology Ltd pbmc
    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The <t>PBMC</t> and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, <t>non-cryopreserved</t> PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.
    Pbmc, supplied by Cellular Technology Ltd, used in various techniques. Bioz Stars score: 97/100, based on 167 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbmc/product/Cellular Technology Ltd
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    96
    Focus Biomolecules pbmc
    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The <t>PBMC</t> and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, <t>non-cryopreserved</t> PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.
    Pbmc, supplied by Focus Biomolecules, used in various techniques. Bioz Stars score: 96/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Miltenyi Biotec pbmcs
    Bryostatin-1 treatment induces an increase in exhaustion marker expression in stimulated <t>CD8</t> + T cells. Eight HIV-negative donors' <t>PBMCs</t> were treated with LRAs and examined for PD-1 expression on CD8 + T cells, and four donors' PBMCs were examined for TIM-3, 2B4, and CD160 expression. A) PD-1 expression in CD8 + T cells treated with drug for 6 h and then incubated with anti-CD3/CD28 antibodies for an additional two days. B) 2B4 expression. C) CD160 expression. D) TIM-3 expression. The level of significance indicated is in comparison to no treatment. * p
    Pbmcs, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 99/100, based on 21773 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbmcs  (Roche)
    99
    Roche pbmcs
    Bryostatin-1 treatment induces an increase in exhaustion marker expression in stimulated <t>CD8</t> + T cells. Eight HIV-negative donors' <t>PBMCs</t> were treated with LRAs and examined for PD-1 expression on CD8 + T cells, and four donors' PBMCs were examined for TIM-3, 2B4, and CD160 expression. A) PD-1 expression in CD8 + T cells treated with drug for 6 h and then incubated with anti-CD3/CD28 antibodies for an additional two days. B) 2B4 expression. C) CD160 expression. D) TIM-3 expression. The level of significance indicated is in comparison to no treatment. * p
    Pbmcs, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 1435 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    SLIT2 LTD pbmcs
    Exogenous <t>Slit2</t> inhibits HIV-1 replication in T cells by binding to Robo1 receptor (a) Slit2 expression in partially purified conditioned media from Slit2-myc-transfected human embryonic kidney (HEK) cells (Slit2) or vector-transfected cells (VCs) was determined by WB analysis (left panel). HIV-1 IIIB replication in MT4 cells (middle panel) or PHA-stimulated peripheral blood mononuclear cells <t>(PBMCs)</t> from normal donors (right panel) in the presence of partially purified conditioned medium containing Slit2 (approximately 6 nmol/l) or conditioned medium from control HEK293 cells was determined by analyzing HIV-1 p24 antigen levels in the supernatants by ELISA. In the case of MT4 cells, the supernatants were collected after 48 h. Virus production in the positive control (HIV-1-infected MT4 cells cultured only with medium): 12.1 ng/ml p24 Ag. * P
    Pbmcs, supplied by SLIT2 LTD, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Ficoll-Paque Pharmacia pbmcs
    Semiquantitative <t>PCR</t> analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in <t>PBMC</t> from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.
    Pbmcs, supplied by Ficoll-Paque Pharmacia, used in various techniques. Bioz Stars score: 99/100, based on 242 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Lifeline Cell Technology pbmcs
    Semiquantitative <t>PCR</t> analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in <t>PBMC</t> from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.
    Pbmcs, supplied by Lifeline Cell Technology, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    3H Biomedical pbmc
    Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, <t>PMN,</t> <t>PBMC,</t> THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
    Pbmc, supplied by 3H Biomedical, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Pfizer Inc pbmc
    Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, <t>PMN,</t> <t>PBMC,</t> THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.
    Pbmc, supplied by Pfizer Inc, used in various techniques. Bioz Stars score: 94/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbmcs  (4Gene)
    92
    4Gene pbmcs
    <t>C(−280)NFAT-binding</t> activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human <t>PBMC</t> extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.
    Pbmcs, supplied by 4Gene, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hemacare pbmcs
    <t>C(−280)NFAT-binding</t> activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human <t>PBMC</t> extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.
    Pbmcs, supplied by Hemacare, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sysmex Corporation pbmcs
    <t>C(−280)NFAT-binding</t> activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human <t>PBMC</t> extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.
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    96
    Cytoskeleton Inc pbmcs
    Analysis of expression of activation surface markers in <t>PBMCs</t> stimulated with A. hydrophila ATCC ® 7966 TM <t>OMVs.</t> 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
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    GraphPad Prism Inc pbmc
    Analysis of expression of activation surface markers in <t>PBMCs</t> stimulated with A. hydrophila ATCC ® 7966 TM <t>OMVs.</t> 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
    Pbmc, supplied by GraphPad Prism Inc, used in various techniques. Bioz Stars score: 97/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega pbmc
    Analysis of expression of activation surface markers in <t>PBMCs</t> stimulated with A. hydrophila ATCC ® 7966 TM <t>OMVs.</t> 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
    Pbmc, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    10X Genomics pbmcs
    Biologically meaningful DE results for the <t>10x</t> Genomics <t>PBMC</t> dataset. a Scatterplot of the first two t-SNE dimensions obtained from the first ten principal components. Cells are color-coded by clusters found using the SEURAT graph-based clustering method on the first ten principal components. Pseudo-color images on the right display normalized enrichment scores after gene set enrichment analysis for cell types related to CD4+ T cells (see “ Methods ”), for clustering based on b the first ten principal components and c W from ZINB-WaVE with K =20. For dimensionality reduction, ZINB-WaVE was fitted with X = 1 n , V = 1 J , K =20 for W (based on the Akaike information criterion), common dispersion, and ε =10 12 . To compute the weights for differential expression analysis, ZINB-WaVE was fitted with intercept and cell-type covariate in X , V = 1 J , K =0 for W , common dispersion, and ε =10 12 . Normalized enrichment scores for more cell types are shown in Additional file 1 : Figure S17. PCA principal component analysis
    Pbmcs, supplied by 10X Genomics, used in various techniques. Bioz Stars score: 99/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HiMedia Laboratories pbmcs
    Biologically meaningful DE results for the <t>10x</t> Genomics <t>PBMC</t> dataset. a Scatterplot of the first two t-SNE dimensions obtained from the first ten principal components. Cells are color-coded by clusters found using the SEURAT graph-based clustering method on the first ten principal components. Pseudo-color images on the right display normalized enrichment scores after gene set enrichment analysis for cell types related to CD4+ T cells (see “ Methods ”), for clustering based on b the first ten principal components and c W from ZINB-WaVE with K =20. For dimensionality reduction, ZINB-WaVE was fitted with X = 1 n , V = 1 J , K =20 for W (based on the Akaike information criterion), common dispersion, and ε =10 12 . To compute the weights for differential expression analysis, ZINB-WaVE was fitted with intercept and cell-type covariate in X , V = 1 J , K =0 for W , common dispersion, and ε =10 12 . Normalized enrichment scores for more cell types are shown in Additional file 1 : Figure S17. PCA principal component analysis
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    pbmcs  (Lonza)
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    Lonza pbmcs
    Ex vivo cytotoxicity <t>Cryopreserved</t> <t>PBMCs</t> were thawed and incubated with GFP + K562 target cells and CD107a PE-Cy5 Ab overnight at 37°C. Co-cultures were stained the following day for CD3/CD56 and analyzed for ( A ) killing and ( B ) CD107a incorporation indicative of degranulation. Killing was measured as the percentage of K562 cells that were lost based on a condition with target cells only ( A , top left). CD107a gating was set with conditions lacking target cells. Left panels summarize group data while right panels correspond to a representative example. ( C ) The absolute numbers of degranulating (CD107a + ) CD56 bright and CD56 dim NK cells per 1000 lymphocytes are depicted for each time point (B, T1 and T2). Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. ( D ) The absolute numbers of degranulating CD56 bright NK cells are positively correlated with the target killing using Spearman’s correlation. * denotes p
    Pbmcs, supplied by Lonza, used in various techniques. Bioz Stars score: 99/100, based on 1429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    79
    Cellgenix pb mncs
    Ex vivo cytotoxicity <t>Cryopreserved</t> <t>PBMCs</t> were thawed and incubated with GFP + K562 target cells and CD107a PE-Cy5 Ab overnight at 37°C. Co-cultures were stained the following day for CD3/CD56 and analyzed for ( A ) killing and ( B ) CD107a incorporation indicative of degranulation. Killing was measured as the percentage of K562 cells that were lost based on a condition with target cells only ( A , top left). CD107a gating was set with conditions lacking target cells. Left panels summarize group data while right panels correspond to a representative example. ( C ) The absolute numbers of degranulating (CD107a + ) CD56 bright and CD56 dim NK cells per 1000 lymphocytes are depicted for each time point (B, T1 and T2). Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. ( D ) The absolute numbers of degranulating CD56 bright NK cells are positively correlated with the target killing using Spearman’s correlation. * denotes p
    Pb Mncs, supplied by Cellgenix, used in various techniques. Bioz Stars score: 79/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore pbmc ficoll
    Ex vivo cytotoxicity <t>Cryopreserved</t> <t>PBMCs</t> were thawed and incubated with GFP + K562 target cells and CD107a PE-Cy5 Ab overnight at 37°C. Co-cultures were stained the following day for CD3/CD56 and analyzed for ( A ) killing and ( B ) CD107a incorporation indicative of degranulation. Killing was measured as the percentage of K562 cells that were lost based on a condition with target cells only ( A , top left). CD107a gating was set with conditions lacking target cells. Left panels summarize group data while right panels correspond to a representative example. ( C ) The absolute numbers of degranulating (CD107a + ) CD56 bright and CD56 dim NK cells per 1000 lymphocytes are depicted for each time point (B, T1 and T2). Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. ( D ) The absolute numbers of degranulating CD56 bright NK cells are positively correlated with the target killing using Spearman’s correlation. * denotes p
    Pbmc Ficoll, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Advanced BioScience Laboratories Inc rhesus pbmc
    Ex vivo cytotoxicity <t>Cryopreserved</t> <t>PBMCs</t> were thawed and incubated with GFP + K562 target cells and CD107a PE-Cy5 Ab overnight at 37°C. Co-cultures were stained the following day for CD3/CD56 and analyzed for ( A ) killing and ( B ) CD107a incorporation indicative of degranulation. Killing was measured as the percentage of K562 cells that were lost based on a condition with target cells only ( A , top left). CD107a gating was set with conditions lacking target cells. Left panels summarize group data while right panels correspond to a representative example. ( C ) The absolute numbers of degranulating (CD107a + ) CD56 bright and CD56 dim NK cells per 1000 lymphocytes are depicted for each time point (B, T1 and T2). Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. ( D ) The absolute numbers of degranulating CD56 bright NK cells are positively correlated with the target killing using Spearman’s correlation. * denotes p
    Rhesus Pbmc, supplied by Advanced BioScience Laboratories Inc, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Becton Dickinson pbmc isolation
    ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of <t>HIV-1</t> infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with <t>PBMC</t> of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.
    Pbmc Isolation, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 99/100, based on 117 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Miltenyi Biotec cp pbmcs
    IFN-α enhances the suppressive capacity of VP and ES <t>CD8</t> + T cells without inhibiting HIV-specific IFN-γ responses of CP CD8 + T cells. (A) <t>PBMCs</t> from 8 CP were pulsed with either medium alone (baseline) or 100 U/ml of IFN-α for
    Cp Pbmcs, supplied by Miltenyi Biotec, used in various techniques. Bioz Stars score: 97/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. NSG-hu mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in PBMCs at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P

    Journal: Blood

    Article Title: Inhibitory effect of HIV-specific neutralizing IgA on mucosal transmission of HIV in humanized mice

    doi: 10.1182/blood-2012-04-422303

    Figure Lengend Snippet: HIV-1 infection in humanized mice passively infused with b12-IgA or b12-IgG isotype. (A) Neutralization activity of anti-HIV human mAb b12-IgA. Reporter cell line TZM-bl cells that express CD4, CXCR4, CCR5, and a Tat-responsive reporter gene for luciferase were infected with 200 TCID 50 of replication-defective pseudovirus containing Env (SF162.LS) in the presence of various concentrations of anti-HIV monoclonal antibodies. Neutralization activity was measured by the reduction in luciferase reporter gene expression after a single round of pseudovirus infection in TZM-bl cells in triplicate. b12-IgG1 and the recombinant b12-IgA2 were compared at indicated concentrations. (B) Antibody level in circulation after passive transfer. NSG-hu mice were injected intravenously with various concentrations of purified b12-IgA2 (pIgA: mIgA = 1:1, mass ratio) or b12-IgG1 and the blood was collected after 4 hours. The plasma antibody concentrations were measured using ELISA. (n = 4-6, mean ± SEM). (C-D) Concentrations of b12 antibodies in plasma (C) or genital secretions (D) at the time of challenge. NSG-hu mice were injected intravenously with either 200 μg of b12-IgA2 or 20 μg of b12-IgG1 per mouse. Blood and genital secretions were collected after 4 hours. (E-I) Peripheral CD4 + T cell loss after HIV-1 challenge in NSG-hu mice injected with different b12 antibody isotypes; (E) purified human IgG/κ control antibody (hIgG/κ); (F) b12-IgA2 includes both monomeric and polymeric IgA as described in (B), (b12IgA2 [M+P]); (G) b12-IgG1; (H) b12-IgA2 monomer only (b12IgA2 [M]). Mice were challenged intravaginally with HIV-1 JR-CSF . (I) Average percent CD4 + T cells in CD3 T cells in PBMCs at each time points (n = 5-9). Statistical analysis was performed using unpaired t test. (* P = .0313 and *** P

    Article Snippet: For passive antibody transfer experiments, NOD.Cg- Prkd scid IL2rg tm1Wjl /SzJ (NOD/SCID IL2r γ−/− , NSG) mice were transplanted intraperitoneally with 3 × 106 human PBMCs (AllCells) that were activated by PHA (5 μg/mL) for 4 days before transplantation (NSG-hu mice).

    Techniques: Infection, Mouse Assay, Neutralization, Activity Assay, Luciferase, Expressing, Recombinant, Injection, Purification, Enzyme-linked Immunosorbent Assay

    UC-MSCs reversed the impaired suppressive function of Tregs from MS A . Bar graph showing that Tregs purified from naïve PBMCs (without UCMSC-priming) of MS patients failed to reduce the proliferation of allogeneic Teffs from healthy controls while Tregs purified from UCMSC-primed PBMCs of the same MS significantly reduced the proliferation index of allogeneic Teffs from healthy donors. No significant difference in the proliferation index of allogeneic Teffs was detected in Tregs purified from UCMSC-modulated PBMCs of MS and Tregs purified from naïve PBMCs of HC, suggesting that UC-MSC-priming may have reversed the functional deficits of Tregs from MS in the co-culture. Tregs:Teffs =1:10. **p

    Journal: Oncotarget

    Article Title: Umbilical cord-derived mesenchymal stem cells reversed the suppressive deficiency of T regulatory cells from peripheral blood of patients with multiple sclerosis in a co-culture – a preliminary study

    doi: 10.18632/oncotarget.12345

    Figure Lengend Snippet: UC-MSCs reversed the impaired suppressive function of Tregs from MS A . Bar graph showing that Tregs purified from naïve PBMCs (without UCMSC-priming) of MS patients failed to reduce the proliferation of allogeneic Teffs from healthy controls while Tregs purified from UCMSC-primed PBMCs of the same MS significantly reduced the proliferation index of allogeneic Teffs from healthy donors. No significant difference in the proliferation index of allogeneic Teffs was detected in Tregs purified from UCMSC-modulated PBMCs of MS and Tregs purified from naïve PBMCs of HC, suggesting that UC-MSC-priming may have reversed the functional deficits of Tregs from MS in the co-culture. Tregs:Teffs =1:10. **p

    Article Snippet: Co-cultures of UC-MSCs and PBMCs PBMCs obtained from patients with relapsing-remitting multiple sclerosis (RRMS, n=12, female=8, male=4), mean age 53.75 years old (49 years to 64 years old) and healthy donors (n=10, female=4, male=6), mean age 28.38 years old (22 years to 39 years old) were purchased from AllCells Inc (Emeryville, CA) or StemExpress (Placerville, CA).

    Techniques: Mass Spectrometry, Purification, Functional Assay, Co-Culture Assay

    UC-MSCs significantly improved the frequency of Tregs among resting CD4 + T cells from MS A , B , C . Representative dot plot for CD25 + CD127 low/− Tregs among resting CD4+ T cells in cultured PBMCs of healthy donors (A), MS patients (B) following 3-day co-culture without UC-MSCs and (C) with UC-MSCs (UC-MSCs:PBMCs=1:5). D . Bar graph showing that the frequency of CD4 + CD25 + CD127 low/− Tregs among resting CD4+ T cells in PBMCs of MS was significantly increased following a 3-day co-culture with UC-MSCs compared to controls without UC-MSCs. No significant differences in the frequency of CD4 + CD25 + CD127 low/− Tregs were detected in naïve healthy donors and naïve MS patients. The data were expressed as mean ± SD of four experiments. **p

    Journal: Oncotarget

    Article Title: Umbilical cord-derived mesenchymal stem cells reversed the suppressive deficiency of T regulatory cells from peripheral blood of patients with multiple sclerosis in a co-culture – a preliminary study

    doi: 10.18632/oncotarget.12345

    Figure Lengend Snippet: UC-MSCs significantly improved the frequency of Tregs among resting CD4 + T cells from MS A , B , C . Representative dot plot for CD25 + CD127 low/− Tregs among resting CD4+ T cells in cultured PBMCs of healthy donors (A), MS patients (B) following 3-day co-culture without UC-MSCs and (C) with UC-MSCs (UC-MSCs:PBMCs=1:5). D . Bar graph showing that the frequency of CD4 + CD25 + CD127 low/− Tregs among resting CD4+ T cells in PBMCs of MS was significantly increased following a 3-day co-culture with UC-MSCs compared to controls without UC-MSCs. No significant differences in the frequency of CD4 + CD25 + CD127 low/− Tregs were detected in naïve healthy donors and naïve MS patients. The data were expressed as mean ± SD of four experiments. **p

    Article Snippet: Co-cultures of UC-MSCs and PBMCs PBMCs obtained from patients with relapsing-remitting multiple sclerosis (RRMS, n=12, female=8, male=4), mean age 53.75 years old (49 years to 64 years old) and healthy donors (n=10, female=4, male=6), mean age 28.38 years old (22 years to 39 years old) were purchased from AllCells Inc (Emeryville, CA) or StemExpress (Placerville, CA).

    Techniques: Mass Spectrometry, Cell Culture, Co-Culture Assay

    UC-MSCs significantly improved the production of cytokines in the co-cultures Bar graph showing that UC-MSCs significantly augmented the production of TGF-β1 ( A ), PGE2 ( B ), and IL-10 ( C ) while reducing the production of IFN-γ ( D ) in co-cultures of UC-MSCs and PBMCs from MS patients. Each experiment was performed in triplicate. Data were presented as mean ± SD of four independent experiments. *p

    Journal: Oncotarget

    Article Title: Umbilical cord-derived mesenchymal stem cells reversed the suppressive deficiency of T regulatory cells from peripheral blood of patients with multiple sclerosis in a co-culture – a preliminary study

    doi: 10.18632/oncotarget.12345

    Figure Lengend Snippet: UC-MSCs significantly improved the production of cytokines in the co-cultures Bar graph showing that UC-MSCs significantly augmented the production of TGF-β1 ( A ), PGE2 ( B ), and IL-10 ( C ) while reducing the production of IFN-γ ( D ) in co-cultures of UC-MSCs and PBMCs from MS patients. Each experiment was performed in triplicate. Data were presented as mean ± SD of four independent experiments. *p

    Article Snippet: Co-cultures of UC-MSCs and PBMCs PBMCs obtained from patients with relapsing-remitting multiple sclerosis (RRMS, n=12, female=8, male=4), mean age 53.75 years old (49 years to 64 years old) and healthy donors (n=10, female=4, male=6), mean age 28.38 years old (22 years to 39 years old) were purchased from AllCells Inc (Emeryville, CA) or StemExpress (Placerville, CA).

    Techniques: Mass Spectrometry

    Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Detection of SMN Protein in SMNA Type 1 PBMCs. SMA Type I patient samples (N = 4) were tested in the SMN ELISA at dilutions of 1∶4 with 1.25×10 6 and 2.5×10 6 PBMCs. SMN protein signal was detected in all samples, with an average of 8.32 pg SMN protein per 10 6 cells. Based on the average of 70.2 pg SMN protein per 10 6 cells calculated for adult normal donor PBMCs, the amount of SMN protein in PBMCs of Type I SMA patients in this patient cohort is 88% less than normal. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Enzyme-linked Immunosorbent Assay

    Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Quantitation of SMN by PBMC subtype. There was no significant difference in SMN protein levels between human lymphocytes and monocytes fractions separated from PBMCs. SMN protein was detectable in the majority of samples for both cell types, ranging from 6.8–25.8 pg/10 6 cells for lymphocytes and 8–17.5 pg/10 6 cells for monocytes. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Quantitation Assay

    ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: ELISA Performance, parallelism and detection of SMN in human PBMCs. A : The recombinant hSMN standard curve data developed from N = 6 curves were highly reproducible with standard deviations of about ±0.23 OD unit variations. The dotted lines surrounding the dose-response curve represent 2 standard deviations. B : Comparison of SMN signal detection between recombinant human SMN (dilutions from 1∶4 to 1∶256) and SMN extracted from HeLa cell lysates (1∶4–1∶32) revealed a high degree of parallelism between reagents, allowing for accurate evaluation of native SMN protein using the recombinant SMN ELISA standard. C : The SMN ELISA detected protein in adult donor PBMCs; values ranged from 86 to 229 pg SMN per well at a dilution of 1∶4 and an average of 70.2 pg SMN protein per 10 6 cells. 10 7 PBMCs diluted 1∶4 through 1∶32 produced linear SMN protein levels. Even with only N = 6 normal samples, SMN levels in PBMCs varied by nearly 3-fold. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Produced

    Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.

    Journal: PLoS ONE

    Article Title: Utility of Survival Motor Neuron ELISA for Spinal Muscular Atrophy Clinical and Preclinical Analyses

    doi: 10.1371/journal.pone.0024269

    Figure Lengend Snippet: Smn protein levels by tissue in WT mice. Smn protein levels are distinct across the an array of tissues and regions of the brain. A : Smn levels varied by as much as 10-fold across tissue types in adult FVB mice (14 weeks old). SMN levels by tissue were distributed on a basis loosely ordered by having lesser to greater dividing cell populations. Smn levels for tissues are represented as Smn pg/mg total protein. PBMCs are represented as Smn pg/mg total protein in the lysis buffer extract. On a per cell basis the average Smn level was 67.2 pg/10 6 PBMCs. B : Smn protein in the brain, spinal cord, sciatic nerve and quadriceps muscle of wildtype mice shows marked variations, with spinal cord having 6-fold greater levels than muscle and nerve. In the brain, Smn protein signal in the thalamus was nearly 2.5-fold less than hippocampal levels. Error bars represent standard deviations.

    Article Snippet: Non-human primate PBMC lysates were run alongside lysates from normal human PBMCs (from AllCells) and mouse PBMCs harvested from 8 week old FVBn animals at PharmOptima.

    Techniques: Mouse Assay, Lysis

    Proliferation of lymphocytes in LNs and PBMCs as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.

    Journal: Journal of Virology

    Article Title: [18F]-Fluorodeoxyglucose Uptake in Lymphoid Tissue Serves as a Predictor of Disease Outcome in the Nonhuman Primate Model of Monkeypox Virus Infection

    doi: 10.1128/JVI.00897-17

    Figure Lengend Snippet: Proliferation of lymphocytes in LNs and PBMCs as measured by flow cytometry. (A) Ki-67 immunostaining of lymphocytes isolated from LNs of healthy animals on day −18 preexposure ( n = 2) and from LNs of surviving animals ( n = 4) on days 9 and 22 p.i. (B to D) Ki-67 immunostaining of CD4 + T cells (B), CD8 + T cells (C), and CD20 + B cells (D) in PBMCs in treated and untreated survivors versus moribund NHPs. Abbreviations: LN, lymph node; NHP, nonhuman primate; PBMCs, peripheral blood mononuclear cells; p.i., postinfection.

    Article Snippet: PBMC samples and lymphocytes isolated from LNs were surface stained with a custom antibody cocktail (BD Biosciences) for CD3, CD4, CD8, CD14, CD16, CD20, CD45, and Ki-67.

    Techniques: Flow Cytometry, Cytometry, Immunostaining, Isolation

    Cytokine induced CIS mRNA expression and STAT5 phosphorylation according to CISH haplotypes. a CIS mRNA expression of PBMCs from healthy adult donors with distinct haplotypes (circles: homozygous H1 carriers (H1/H1), triangles: H2 carriers (H1/H2, H2/H2), squares: H3 carriers (H1/H3)) treated with IL-2 (left graph) or IL-7 (right graph) for 1 and 2 h. CIS mRNA levels—calculated in comparison to the housekeeping gene GAPDH—are normalized against time point 0 for each donor. Median and IQR of 2 −ΔΔCT values are indicated. p value for effect of IL-2/IL-7 stimulation on CIS mRNA levels considering all genotypes (Friedman test) is indicated as *** for p

    Journal: Molecular and Cellular Pediatrics

    Article Title: CISH promoter polymorphism effects on T cell cytokine receptor signaling and type 1 diabetes susceptibility

    doi: 10.1186/s40348-018-0080-7

    Figure Lengend Snippet: Cytokine induced CIS mRNA expression and STAT5 phosphorylation according to CISH haplotypes. a CIS mRNA expression of PBMCs from healthy adult donors with distinct haplotypes (circles: homozygous H1 carriers (H1/H1), triangles: H2 carriers (H1/H2, H2/H2), squares: H3 carriers (H1/H3)) treated with IL-2 (left graph) or IL-7 (right graph) for 1 and 2 h. CIS mRNA levels—calculated in comparison to the housekeeping gene GAPDH—are normalized against time point 0 for each donor. Median and IQR of 2 −ΔΔCT values are indicated. p value for effect of IL-2/IL-7 stimulation on CIS mRNA levels considering all genotypes (Friedman test) is indicated as *** for p

    Article Snippet: For measurement of CIS mRNA expression, PBMCs were stimulated with IL-2 (100 IU/ml, Sigma-Aldrich) or IL-7 (10 ng/ml, Sigma-Aldrich) for 1 or 2 h. mRNA was then isolated and reverse transcribed.

    Techniques: Expressing, Chromogenic In Situ Hybridization

    Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

    Journal: Journal of Translational Medicine

    Article Title: Exploring dendritic cell based vaccines targeting survivin for the treatment of head and neck cancer patients

    doi: 10.1186/1479-5876-11-152

    Figure Lengend Snippet: Spontaneously arising survivin specific T cells in patients. A ) Direct ex vivo tetramer staining of T cells derived from HNSCC patients. The patients (1 thru 5) were all HLA-A2 positive and the CD8 positive T cells were stained with HLA-A2 tetramers containing either the survivin 5 (light grey bars), survivin 96 (dark grey bars) or the modified survivin 96M (black bars) epitope. The percentage tetramer positive T cells in incidated on the Y-axis. B ) PBMC from HNSCC patients were stained with HLA-A2 tetramers containing the survivin 5 epitope and subsequently sorted by flow cytometry. Cells were plated out at a concentration of 0.2 cells per well and stimulated with feeder mix weekly. The left hand panel shows the forward scatter (X-axis; FSC) and side scatter (Y-axis; SSC) of the cells after four weeks of stimulation with feeder mix. A boxed live gate is indicated. The right hand panel shows tetramer staining of the cells in the live gates. Survivin 5 tetramer labeled with either APC (X-axis) or PE (Y-axis) were used to stain the T cells. The percentage of double tetramer positive cells is shown in the upper right quadrant. C ) Draining lymph nodes were obtained from a patient with locally advanced breast cancer, who had received neo-adjuvant chemotherapy before surgery. The spontaneously arising T cells were tested in an ELIspot assay against a number of different HLA-A2 binding peptides representing CTL epitopes; Survivin(95–104), CEA(571–579), Her-2/Neu(369–377) and Her-2/Neu(654–662) respectively and HPV16E7-(11–20) peptide as a negative control. Indicated are the number of spots per 100.000 T cells.

    Article Snippet: T cell activation and survivin protein detection Isolation of resting CD8β positive T cells from HLA-A2 positive, healthy donor PBMC (Sanquin) was performed by positive selection using a MACS column (Miltenyi Biotec).

    Techniques: Ex Vivo, Staining, Derivative Assay, Modification, Flow Cytometry, Cytometry, Concentration Assay, Labeling, Enzyme-linked Immunospot, Binding Assay, CTL Assay, Negative Control

    Induction of HIV by LRAs in a primary CD4+ T-cell direct infection latency model. CD4 + T-cells were isolated from PBMC by negative selection, cultured with CCL19 and then magnetofected with HIV, or mock infected (no virus). Two days later, cells were depleted of activated cells. We defined activated cells as those expressing at least one of CD69, HLA-DR, or CD25 based on a study that defined CD4+ T-cells with the triple-negative phenotype as quiescent[ 29 ]. In parallel, CD4 + T-cells were activated using anti-CD3/anti-CD28 antibodies and infected with HIV (productively infected). A . Depletion of activated cells by anti-PE microbeads. Shown are flow cytometry data gated on CD3 + CD4 + lymphocytes (95% pure) and depicting CD25/CD69/HLA-DR staining (all pooled on PE channel)–y-axis, by HIV-Gag–x-axis. The left and middle panels represent pre- and post-depletion of activated cells, respectively to generate latently-infected cells. The right panel shows productively-infected target cells. B . Latently infected or mock-infected resting CD4 + T-cells were prepared as in A and then either stimulated with 1.5 nM IL-15, with 2.6 μM prostratin, or left unstimulated for 36 hours. Shown are flow cytometry data, gated on lymphocytes (SSC/FSC) and depicting CD4 –y-axis, by HIV-Gag–x-axis. C . In a separate experiment analogous to B latently-infected or mock-infected cells were treated with the indicated concentrations of IL-15 for 16 hours. The percentage of HIV-Gag + cells (gated as in B ) is plotted against the concentration of IL-15 (red circles = infected, green squares = uninfected). D. Latently-infected cells were stimulated with the indicated LRAs for 36 hours and HIV p24 in supernatants was quantified by ELISA. Shown are background-subtracted mean ± SEM values (duplicates). P-values were calculated by ANOVA with Holm-Sidak’s multiple comparison test (comparing each condition with no treatment [No Tx]) * p

    Journal: PLoS Pathogens

    Article Title: A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes

    doi: 10.1371/journal.ppat.1005545

    Figure Lengend Snippet: Induction of HIV by LRAs in a primary CD4+ T-cell direct infection latency model. CD4 + T-cells were isolated from PBMC by negative selection, cultured with CCL19 and then magnetofected with HIV, or mock infected (no virus). Two days later, cells were depleted of activated cells. We defined activated cells as those expressing at least one of CD69, HLA-DR, or CD25 based on a study that defined CD4+ T-cells with the triple-negative phenotype as quiescent[ 29 ]. In parallel, CD4 + T-cells were activated using anti-CD3/anti-CD28 antibodies and infected with HIV (productively infected). A . Depletion of activated cells by anti-PE microbeads. Shown are flow cytometry data gated on CD3 + CD4 + lymphocytes (95% pure) and depicting CD25/CD69/HLA-DR staining (all pooled on PE channel)–y-axis, by HIV-Gag–x-axis. The left and middle panels represent pre- and post-depletion of activated cells, respectively to generate latently-infected cells. The right panel shows productively-infected target cells. B . Latently infected or mock-infected resting CD4 + T-cells were prepared as in A and then either stimulated with 1.5 nM IL-15, with 2.6 μM prostratin, or left unstimulated for 36 hours. Shown are flow cytometry data, gated on lymphocytes (SSC/FSC) and depicting CD4 –y-axis, by HIV-Gag–x-axis. C . In a separate experiment analogous to B latently-infected or mock-infected cells were treated with the indicated concentrations of IL-15 for 16 hours. The percentage of HIV-Gag + cells (gated as in B ) is plotted against the concentration of IL-15 (red circles = infected, green squares = uninfected). D. Latently-infected cells were stimulated with the indicated LRAs for 36 hours and HIV p24 in supernatants was quantified by ELISA. Shown are background-subtracted mean ± SEM values (duplicates). P-values were calculated by ANOVA with Holm-Sidak’s multiple comparison test (comparing each condition with no treatment [No Tx]) * p

    Article Snippet: Productively-infected cell killing assays Primary CD4+ T-cells were enriched from PBMC by negative selection (Easysep, Stemcell Technologies) and then activated for 48 hours with 1 μg/ml each of anti-CD3 (OKT3, ebioscience) and anti-CD28 (CD28.2, ebioscience) in RPMI-10 supplemented with 50 U/ml IL-2.

    Techniques: Infection, Isolation, Selection, Cell Culture, Expressing, Flow Cytometry, Cytometry, Staining, Concentration Assay, Enzyme-linked Immunosorbent Assay

    ALT-803 induces HIV transcription from both a primary cell model of latency and from ex vivo patient samples. Primary CD4 + T-cells from healthy donor PBMC were activated, infected with a luciferase reporter HIV virus, and allowed to return to a resting state. This is similar to a post-activation latency model that has been previously described [ 17 ] with additional minor modifications (see Methods ). After 1 week, infected cells were treated with the agents at concentrations indicated for 2 days. A. Shown are luminescence values from luciferase, indicating dose-dependent HIV reactivation by each of the compounds tested. B. Compound associated cytotoxicity was determined in latently-infected cells in parallel with the virus activation assay using Cell Titer Glo (CTG) Dose dependent cytotoxicity was observed with SAHA and romidepsin but not with IL-15 or ALT-803. C and D. PBMC were isolated from HIV-infected subjects who had been suppressed by ART for at least 2 years. Cultures were maintained in the presence of ARVs. Either 1 nM of ALT-803 or 5 ng/ml PMA + 500 ng/ml ionomycin were added for 7 days. HIV activation was measured by quantitating viral RNA in cell-free supernatant using COBAS qPCR. C. The geometric mean of viral copies/ml for each donor are indicated for control (DMSO) and ALT-803 treated wells, following 7 days of treatment. D . The fold HIV activations of all donors are plotted as the ratio of the viral copies/ml for ALT-803 treated or PMA/ionomycin treated to control wells. These results indicate that ALT-803 reactivates virus from the natural patient reservoir, though to a lesser degree than PMA/ionomycin. E-I. Cryopreserved CD4 + T-cells from ARV-treated HIV-infected subjects were thawed, CFSE-labeled, and treated with either 1 nM ALT-803 or 200 ng/ml of PHA for 7 days in the presence of ARVs. E . Activation of CD4 + T-cells within PBMCs was measured at day 7 by flow cytometry, gating on CD3 + CD4 + T-cells and assessing CD69 expression. Each line represents a different subject. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. F . Proliferation of CD4+ T-cells within PBCs was measured at day 7 by flow, gating on CD3 + CD4 + T-cells and assessing proliferation as %CFSE dim cells. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. G-I. Cell associated DNA was isolated from purified CD4 + T-cells following 6 days of stimulation. Absolute copies of HIV-Gag and RPP30 were determined by droplet digital PCR and used to calculate copies of HIV per 10 6 CD4 + T-cells. Shown are results from three different subjects. Quantifications were determined in triplicate ( G ) or quadruplicate ( H I ). Means and SEM are shown and the P value was calculated by one-way ANOVA with Holm-Sidak’s multiple comparison test.

    Journal: PLoS Pathogens

    Article Title: A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4+ T-Cells to Recognition by Cytotoxic T-Lymphocytes

    doi: 10.1371/journal.ppat.1005545

    Figure Lengend Snippet: ALT-803 induces HIV transcription from both a primary cell model of latency and from ex vivo patient samples. Primary CD4 + T-cells from healthy donor PBMC were activated, infected with a luciferase reporter HIV virus, and allowed to return to a resting state. This is similar to a post-activation latency model that has been previously described [ 17 ] with additional minor modifications (see Methods ). After 1 week, infected cells were treated with the agents at concentrations indicated for 2 days. A. Shown are luminescence values from luciferase, indicating dose-dependent HIV reactivation by each of the compounds tested. B. Compound associated cytotoxicity was determined in latently-infected cells in parallel with the virus activation assay using Cell Titer Glo (CTG) Dose dependent cytotoxicity was observed with SAHA and romidepsin but not with IL-15 or ALT-803. C and D. PBMC were isolated from HIV-infected subjects who had been suppressed by ART for at least 2 years. Cultures were maintained in the presence of ARVs. Either 1 nM of ALT-803 or 5 ng/ml PMA + 500 ng/ml ionomycin were added for 7 days. HIV activation was measured by quantitating viral RNA in cell-free supernatant using COBAS qPCR. C. The geometric mean of viral copies/ml for each donor are indicated for control (DMSO) and ALT-803 treated wells, following 7 days of treatment. D . The fold HIV activations of all donors are plotted as the ratio of the viral copies/ml for ALT-803 treated or PMA/ionomycin treated to control wells. These results indicate that ALT-803 reactivates virus from the natural patient reservoir, though to a lesser degree than PMA/ionomycin. E-I. Cryopreserved CD4 + T-cells from ARV-treated HIV-infected subjects were thawed, CFSE-labeled, and treated with either 1 nM ALT-803 or 200 ng/ml of PHA for 7 days in the presence of ARVs. E . Activation of CD4 + T-cells within PBMCs was measured at day 7 by flow cytometry, gating on CD3 + CD4 + T-cells and assessing CD69 expression. Each line represents a different subject. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. F . Proliferation of CD4+ T-cells within PBCs was measured at day 7 by flow, gating on CD3 + CD4 + T-cells and assessing proliferation as %CFSE dim cells. P values were calculated by RM one-way ANOVA with Holm-Sidak’s multiple comparison test. G-I. Cell associated DNA was isolated from purified CD4 + T-cells following 6 days of stimulation. Absolute copies of HIV-Gag and RPP30 were determined by droplet digital PCR and used to calculate copies of HIV per 10 6 CD4 + T-cells. Shown are results from three different subjects. Quantifications were determined in triplicate ( G ) or quadruplicate ( H I ). Means and SEM are shown and the P value was calculated by one-way ANOVA with Holm-Sidak’s multiple comparison test.

    Article Snippet: Productively-infected cell killing assays Primary CD4+ T-cells were enriched from PBMC by negative selection (Easysep, Stemcell Technologies) and then activated for 48 hours with 1 μg/ml each of anti-CD3 (OKT3, ebioscience) and anti-CD28 (CD28.2, ebioscience) in RPMI-10 supplemented with 50 U/ml IL-2.

    Techniques: Ex Vivo, Infection, Luciferase, Activation Assay, CTG Assay, Isolation, Real-time Polymerase Chain Reaction, Labeling, Flow Cytometry, Cytometry, Expressing, Purification, Digital PCR

    CD4+ T-cell proliferative responses to G1m1 allotype-containing Fc fragments. The G1m1-containing Fc fragment purified from infliximab after papain cleavage (closed symbols) and the nG1m1-containing Fc fragment purified from bevacizumab after papain cleavage (open symbols) were co-cultured with CD4+ T cell-enriched PBMC from G1m1 homozygous (circles) or nG1m1 homozygous (squares) donors. ( a ) Fc fragments were titered into cultures as soluble proteins. ( b ) Fc proteins were heat-denatured for 5 min at 95 °C then cooled to room temperature. Proliferation was assessed by tritiated thymidine incorporation on day 6. s.e.m. is only shown on the infliximab Fc fragment proliferative responses for clarity.

    Journal: Genes and Immunity

    Article Title: The human G1m1 allotype associates with CD4+ T-cell responsiveness to a highly conserved IgG1 constant region peptide and confers an asparaginyl endopeptidase cleavage site

    doi: 10.1038/gene.2010.68

    Figure Lengend Snippet: CD4+ T-cell proliferative responses to G1m1 allotype-containing Fc fragments. The G1m1-containing Fc fragment purified from infliximab after papain cleavage (closed symbols) and the nG1m1-containing Fc fragment purified from bevacizumab after papain cleavage (open symbols) were co-cultured with CD4+ T cell-enriched PBMC from G1m1 homozygous (circles) or nG1m1 homozygous (squares) donors. ( a ) Fc fragments were titered into cultures as soluble proteins. ( b ) Fc proteins were heat-denatured for 5 min at 95 °C then cooled to room temperature. Proliferation was assessed by tritiated thymidine incorporation on day 6. s.e.m. is only shown on the infliximab Fc fragment proliferative responses for clarity.

    Article Snippet: Autologous CD4+ T cells were isolated by negative selection from thawed PBMC samples (Stem Cell Technologies, Vancouver, British Columbia, Canada) then co-cultured with irradiated dendritic cells and peptides (5 μg ml−1 ) in AIM V media for 5 days.

    Techniques: Purification, Cell Culture

    In vivo and in vitro associations between endotoxin (LPS) levels and TLR4 mRNA expression in PBMCs. (A) In samples obtained directly from HIV uninfected subjects, an inverse relationship between plasma LPS levels and TLR4 expression was apparent (R = −0.35, P = 0.053, Spearman Rank correlation). However, in HIV infected subjects no correlation or trend was seen (R = 0.029, P = 0.83). Additionally, plasma HIV RNA levels were positively associated with increased TLR4 mRNA levels (Spearman R = 0.3260, P = 0.049). (B) In a similar pattern, overnight culture with 0.1 ug/l LPS reduced TLR4 mRNA expression in PBMC of HIV uninfected (n = 70, P = 0.009), but not infected subjects (n = 21, P = 0.627). Isolated HIV ssRNA40 increased TLR4 mRNA expression in PBMC from HIV uninfected subjects (n = 70, P = 0.011), but not infected subjects (n = 21, P = 0.808). Spearman rank sum and Wilcoxon signed rank tests were used, two-tailed ns = not significant, t = trend(P

    Journal: PLoS ONE

    Article Title: HIV-1 RNA Dysregulates the Natural TLR Response to Subclinical Endotoxemia in Kenyan Female Sex-WorkersCross signaling between Toll-like receptors dysregulates the response to subclinical endotoxemia in HIV-1 infection

    doi: 10.1371/journal.pone.0005644

    Figure Lengend Snippet: In vivo and in vitro associations between endotoxin (LPS) levels and TLR4 mRNA expression in PBMCs. (A) In samples obtained directly from HIV uninfected subjects, an inverse relationship between plasma LPS levels and TLR4 expression was apparent (R = −0.35, P = 0.053, Spearman Rank correlation). However, in HIV infected subjects no correlation or trend was seen (R = 0.029, P = 0.83). Additionally, plasma HIV RNA levels were positively associated with increased TLR4 mRNA levels (Spearman R = 0.3260, P = 0.049). (B) In a similar pattern, overnight culture with 0.1 ug/l LPS reduced TLR4 mRNA expression in PBMC of HIV uninfected (n = 70, P = 0.009), but not infected subjects (n = 21, P = 0.627). Isolated HIV ssRNA40 increased TLR4 mRNA expression in PBMC from HIV uninfected subjects (n = 70, P = 0.011), but not infected subjects (n = 21, P = 0.808). Spearman rank sum and Wilcoxon signed rank tests were used, two-tailed ns = not significant, t = trend(P

    Article Snippet: TLR expression Total RNA was extracted from TRIzol samples containing approximately 1×106 PBMCs as per manufacturer instructions (Invitrogen).

    Techniques: In Vivo, In Vitro, Expressing, Infection, Isolation, Two Tailed Test

    In vitro endotoxin (LPS) ‘tolerance’ and TLR ‘priming’ of cytokine responses by single-stranded RNA. Data for triplicate experiments in PBMCs from two HIV uninfected subjects are shown in (A and B) using 0.1 ug/ml of LPS, 1 ug/ml ssRNA40, or media alone for initial priming and subsequent stimulation steps (e.g. ‘media-LPS’). In the first subject, pre-incubating PBMCs with LPS dramatically reduced subsequent LPS-induced TNF-α responses (P

    Journal: PLoS ONE

    Article Title: HIV-1 RNA Dysregulates the Natural TLR Response to Subclinical Endotoxemia in Kenyan Female Sex-WorkersCross signaling between Toll-like receptors dysregulates the response to subclinical endotoxemia in HIV-1 infection

    doi: 10.1371/journal.pone.0005644

    Figure Lengend Snippet: In vitro endotoxin (LPS) ‘tolerance’ and TLR ‘priming’ of cytokine responses by single-stranded RNA. Data for triplicate experiments in PBMCs from two HIV uninfected subjects are shown in (A and B) using 0.1 ug/ml of LPS, 1 ug/ml ssRNA40, or media alone for initial priming and subsequent stimulation steps (e.g. ‘media-LPS’). In the first subject, pre-incubating PBMCs with LPS dramatically reduced subsequent LPS-induced TNF-α responses (P

    Article Snippet: TLR expression Total RNA was extracted from TRIzol samples containing approximately 1×106 PBMCs as per manufacturer instructions (Invitrogen).

    Techniques: In Vitro

    DNA damage assessment in cryopreserved PBMCs. qPCR measurement of UVC-induced ( A ) nDNA and( B ) mtDNA damage in revived PBMCs following cryopreservation over 12 weeks ( n = 3). Results are presented as mean ± SE. * p

    Journal: Biology

    Article Title: Development of a qPCR Method to Measure Mitochondrial and Genomic DNA Damage with Application to Chemotherapy-Induced DNA Damage and Cryopreserved Cells

    doi: 10.3390/biology5040039

    Figure Lengend Snippet: DNA damage assessment in cryopreserved PBMCs. qPCR measurement of UVC-induced ( A ) nDNA and( B ) mtDNA damage in revived PBMCs following cryopreservation over 12 weeks ( n = 3). Results are presented as mean ± SE. * p

    Article Snippet: Cryopreservation of PBMCs PBMCs were resuspended in sterile filtered 50% FCS (Gibco, USA), 40% RPMI (Gibco, USA) and 10% DMSO (Merck, Darmstadt, Germany) in 1.8 mL Nunc™ conical sterile polypropylene cryo vials (Thermo Scientific, Waltham, MA, USA) and placed overnight in a CoolCell® (Biocision, Larkspur, CA, USA) at −80 °C for controlled cooling.

    Techniques: Real-time Polymerase Chain Reaction

    EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. CD4+ T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells (PBMC), CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.

    Journal: Oncotarget

    Article Title: Simultaneous targeting of Eph receptors in glioblastoma

    doi: 10.18632/oncotarget.10978

    Figure Lengend Snippet: EphA3 co-stains with macrophage/leukocyte markers A. Immunofluorescent staining of CD68, GFAP and Von Willebrand factor in a GBM specimen. B. Immunofluorescent staining of EphA3 (red) and CD31, GFAP, CD68, CD163, and CD206 on consecutive frozen sections of BTCOE4443 human GBM specimen. Nuclei are stained with DAPI (blue). Selected areas were magnified (last column on the right). C. Confocal immunofluorescent staining of EphA3 and CD68 in a GBM specimen using stacked 2D images. D, E. -stacked 2D images, and F. Confocal microscopy of co-staining of EphA3 and CD45 in GBM specimens. G. CD4+ T cells do not express the EphA3 receptor. Western blot analysis of peripheral blood mononuclear cells (PBMC), CD4+ and CD4+ activated T cells were probed for EphA3 immunoreactivity.

    Article Snippet: Isolation and activation of CD4+ T cells Total CD4+ T Cells were isolated from PBMC (ZenBio, Durham, NC) using the Dynabeads Untouched human CD4+ T-Cell Kit (Invitrogen) according to the manufacturer's instructions.

    Techniques: Staining, Confocal Microscopy, Western Blot

    CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: CD8 cells respond to CEF peptides ( A ), and CD4 cells to mumps antigen ( B ): these responses seen ex vivo are preserved after freeze thawing ( C D ). To determine T cell subsets responding to the CEF and mumps antigens, magnetic affinity bead-based separation was performed depleting more than 95% of CD4 or CD8 T cells, as specified. The PBMC and cell fractions were stimulated with CEF peptides ( A ) or mumps antigen ( B ). For each condition, triplicate wells were tested with the mean and standard deviation shown. The data shown are from one of two experiments performed with similar results. Freshly isolated, non-cryopreserved PBMC ( ex vivo ) and cryopreserved PBMC that were tested directly without resting following thawing (fresh) were tested in an IFN-γ ELISPOT assay against CEF peptides ( C ) and mumps antigen ( D ). Data points obtained for individual donors are connected with a line. Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or freeze-thawed, the medium background was less than 10 spots per well. Nonparametric Wilcoxon signed-rank test was used to compare matched ex vivo vs . fresh responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques: Ex Vivo, Standard Deviation, Isolation, Enzyme-linked Immunospot

    Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of mumps antigen elicited IFN-γ in freshly thawed and rested cryopreserved PBMC for low ( A ) and high responder subjects ( B ). Each data point represents the mean of triplicate antigen-stimulated wells. Standard deviations are not shown being between 5 and 20% of the mean. For each sample, fresh or rested, the medium background was less than 10 spots per well. Non-parametric Wilcoxon signed-rank test was used to compare matched fresh vs . rested responses with a p -value ≤ 0.05 being considered significant.

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques:

    Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Journal: Cells

    Article Title: Resting of Cryopreserved PBMC Does Not Generally Benefit the Performance of Antigen-Specific T Cell ELISPOT Assays

    doi: 10.3390/cells1030409

    Figure Lengend Snippet: Comparison of CEF responses elicited by freshly thawed (“fresh”) and rested cryopreserved PBMC in ELISPOT assays. ( A ) CEF-specific responses among “low responders” who were defined as subjects who had fewer than 50 SFU in freshly thawed PMBC. ( B ) CEF-specific responses among “high responders” who were defined as subjects who had greater than 50 spots forming units (SFU) in freshly thawed PMBC. Data points obtained from the same donor before and after resting are connected by a line. Each data point represents the mean of triplicate antigen-stimulated wells. For each donor and antigen tested, the standard deviation was

    Article Snippet: Thawing and Resting of Cryopreserved PBMC Cryopreserved human PBMC of 25 donors were acquired from a library of PBMC (ePBMC, CTL‑CP1) offered by Cellular Technologies Ltd. (CTL, Shaker Heights, OH, USA).

    Techniques: Enzyme-linked Immunospot, Standard Deviation

    Bryostatin-1 treatment induces an increase in exhaustion marker expression in stimulated CD8 + T cells. Eight HIV-negative donors' PBMCs were treated with LRAs and examined for PD-1 expression on CD8 + T cells, and four donors' PBMCs were examined for TIM-3, 2B4, and CD160 expression. A) PD-1 expression in CD8 + T cells treated with drug for 6 h and then incubated with anti-CD3/CD28 antibodies for an additional two days. B) 2B4 expression. C) CD160 expression. D) TIM-3 expression. The level of significance indicated is in comparison to no treatment. * p

    Journal: EBioMedicine

    Article Title: The Effect of Latency Reversal Agents on Primary CD8 + T Cells: Implications for Shock and Kill Strategies for Human Immunodeficiency Virus Eradication

    doi: 10.1016/j.ebiom.2016.04.019

    Figure Lengend Snippet: Bryostatin-1 treatment induces an increase in exhaustion marker expression in stimulated CD8 + T cells. Eight HIV-negative donors' PBMCs were treated with LRAs and examined for PD-1 expression on CD8 + T cells, and four donors' PBMCs were examined for TIM-3, 2B4, and CD160 expression. A) PD-1 expression in CD8 + T cells treated with drug for 6 h and then incubated with anti-CD3/CD28 antibodies for an additional two days. B) 2B4 expression. C) CD160 expression. D) TIM-3 expression. The level of significance indicated is in comparison to no treatment. * p

    Article Snippet: When applicable, CD8 + T cells were obtained via positive selection from PBMCs (CD8 microbeads, Miltenyi Biotech) prior to any negative selection performed in experiments described below.

    Techniques: Marker, Expressing, Incubation

    CD69 expression is upregulated due to treatment with PKC agonists. Eight HIV-negative donors' PBMCs were treated with LRAs for 6 h before being washed and then cultured in non-stimulating media for up to three days and examined for CD69 expression on CD8 + T cells. Mean expression ± standard error is indicated for each treatment. Bryostatin-1 treatment at 10 nM and 1 nM and bryostatin-1 (10 nM)/romidepsin treatment all significantly upregulate CD69 for all four timepoints ( p

    Journal: EBioMedicine

    Article Title: The Effect of Latency Reversal Agents on Primary CD8 + T Cells: Implications for Shock and Kill Strategies for Human Immunodeficiency Virus Eradication

    doi: 10.1016/j.ebiom.2016.04.019

    Figure Lengend Snippet: CD69 expression is upregulated due to treatment with PKC agonists. Eight HIV-negative donors' PBMCs were treated with LRAs for 6 h before being washed and then cultured in non-stimulating media for up to three days and examined for CD69 expression on CD8 + T cells. Mean expression ± standard error is indicated for each treatment. Bryostatin-1 treatment at 10 nM and 1 nM and bryostatin-1 (10 nM)/romidepsin treatment all significantly upregulate CD69 for all four timepoints ( p

    Article Snippet: When applicable, CD8 + T cells were obtained via positive selection from PBMCs (CD8 microbeads, Miltenyi Biotech) prior to any negative selection performed in experiments described below.

    Techniques: Expressing, Cell Culture

    Bryostatin-1 treatment induces an increase in exhaustion marker expression in unstimulated CD8 + T cells. Eight HIV-negative donors' PBMCs were treated with LRAs and examined for PD-1 expression on CD8 + T cells, and four donors' PBMCs were examined for TIM-3, 2B4, and CD160 expression. A) PD-1 expression in CD8 + T cells treated with drug for 6 h and then incubated in non-stimulating media for an additional three days. B) TIM-3 expression. C) 2B4 expression. D) CD160 expression. The level of significance indicated is in comparison to no treatment. * p

    Journal: EBioMedicine

    Article Title: The Effect of Latency Reversal Agents on Primary CD8 + T Cells: Implications for Shock and Kill Strategies for Human Immunodeficiency Virus Eradication

    doi: 10.1016/j.ebiom.2016.04.019

    Figure Lengend Snippet: Bryostatin-1 treatment induces an increase in exhaustion marker expression in unstimulated CD8 + T cells. Eight HIV-negative donors' PBMCs were treated with LRAs and examined for PD-1 expression on CD8 + T cells, and four donors' PBMCs were examined for TIM-3, 2B4, and CD160 expression. A) PD-1 expression in CD8 + T cells treated with drug for 6 h and then incubated in non-stimulating media for an additional three days. B) TIM-3 expression. C) 2B4 expression. D) CD160 expression. The level of significance indicated is in comparison to no treatment. * p

    Article Snippet: When applicable, CD8 + T cells were obtained via positive selection from PBMCs (CD8 microbeads, Miltenyi Biotech) prior to any negative selection performed in experiments described below.

    Techniques: Marker, Expressing, Incubation

    Bryostatin-1 and bryostatin-1/romidepsin combination treatments cause an increase in CD8 + T cell death. Eight HIV negative donors' PBMCs were treated with LRAs and examined for annexin V expression on CD8 + T cells. A) Annexin V expression in CD8 + T cells treated with drug for 6 h and then cultured for three days post-treatment in non-stimulating media. B) Annexin V expression in CD8 + T cells treated with drug for 6 h and then stimulated with anti-CD3/CD28 for either one or two days afterward. C) Annexin V and 7-AAD expression in CD8 + T cells treated with drug for 6 h and then stimulated with anti-CD3/CD28 for either one or two days afterward. One-way repeated measures ANOVAs were used to calculate significance, and the level of significance indicated is in comparison to the no treatment condition. * p

    Journal: EBioMedicine

    Article Title: The Effect of Latency Reversal Agents on Primary CD8 + T Cells: Implications for Shock and Kill Strategies for Human Immunodeficiency Virus Eradication

    doi: 10.1016/j.ebiom.2016.04.019

    Figure Lengend Snippet: Bryostatin-1 and bryostatin-1/romidepsin combination treatments cause an increase in CD8 + T cell death. Eight HIV negative donors' PBMCs were treated with LRAs and examined for annexin V expression on CD8 + T cells. A) Annexin V expression in CD8 + T cells treated with drug for 6 h and then cultured for three days post-treatment in non-stimulating media. B) Annexin V expression in CD8 + T cells treated with drug for 6 h and then stimulated with anti-CD3/CD28 for either one or two days afterward. C) Annexin V and 7-AAD expression in CD8 + T cells treated with drug for 6 h and then stimulated with anti-CD3/CD28 for either one or two days afterward. One-way repeated measures ANOVAs were used to calculate significance, and the level of significance indicated is in comparison to the no treatment condition. * p

    Article Snippet: When applicable, CD8 + T cells were obtained via positive selection from PBMCs (CD8 microbeads, Miltenyi Biotech) prior to any negative selection performed in experiments described below.

    Techniques: Expressing, Cell Culture

    CD3 expression is decreased due to treatment with bryostatin-1, prostratin, and the combination of bryostatin-1/romidepsin. Eight HIV-negative donors' PBMCs were treated with LRAs for at least 6 h and examined for CD3 expression on CD8 + T cells. A) Mean fluorescence intensity (MFI) of CD3 expression in CD8 + T cells treated with drug for 6 or 18 h normalized to no treatment. One-way repeated measures ANOVAs were used to calculate significance for 6-hour and 18-hour treatments separately. B) Mean fluorescence intensity (MFI) of CD3 expression in CD8 + T cells treated with drug for 6 h and then incubated in non-stimulating media for one day afterward normalized to no treatment. A one-way repeated measures ANOVA was used to calculate significance. The level of significance indicated is in comparison to vehicle (DMSO). * p

    Journal: EBioMedicine

    Article Title: The Effect of Latency Reversal Agents on Primary CD8 + T Cells: Implications for Shock and Kill Strategies for Human Immunodeficiency Virus Eradication

    doi: 10.1016/j.ebiom.2016.04.019

    Figure Lengend Snippet: CD3 expression is decreased due to treatment with bryostatin-1, prostratin, and the combination of bryostatin-1/romidepsin. Eight HIV-negative donors' PBMCs were treated with LRAs for at least 6 h and examined for CD3 expression on CD8 + T cells. A) Mean fluorescence intensity (MFI) of CD3 expression in CD8 + T cells treated with drug for 6 or 18 h normalized to no treatment. One-way repeated measures ANOVAs were used to calculate significance for 6-hour and 18-hour treatments separately. B) Mean fluorescence intensity (MFI) of CD3 expression in CD8 + T cells treated with drug for 6 h and then incubated in non-stimulating media for one day afterward normalized to no treatment. A one-way repeated measures ANOVA was used to calculate significance. The level of significance indicated is in comparison to vehicle (DMSO). * p

    Article Snippet: When applicable, CD8 + T cells were obtained via positive selection from PBMCs (CD8 microbeads, Miltenyi Biotech) prior to any negative selection performed in experiments described below.

    Techniques: Expressing, Fluorescence, Incubation

    Exogenous Slit2 inhibits HIV-1 replication in T cells by binding to Robo1 receptor (a) Slit2 expression in partially purified conditioned media from Slit2-myc-transfected human embryonic kidney (HEK) cells (Slit2) or vector-transfected cells (VCs) was determined by WB analysis (left panel). HIV-1 IIIB replication in MT4 cells (middle panel) or PHA-stimulated peripheral blood mononuclear cells (PBMCs) from normal donors (right panel) in the presence of partially purified conditioned medium containing Slit2 (approximately 6 nmol/l) or conditioned medium from control HEK293 cells was determined by analyzing HIV-1 p24 antigen levels in the supernatants by ELISA. In the case of MT4 cells, the supernatants were collected after 48 h. Virus production in the positive control (HIV-1-infected MT4 cells cultured only with medium): 12.1 ng/ml p24 Ag. * P

    Journal: AIDS (London, England)

    Article Title: A novel role for Slit2/Robo1 axis in modulating HIV-1 replication in T cells

    doi: 10.1097/QAD.0b013e32834bab86

    Figure Lengend Snippet: Exogenous Slit2 inhibits HIV-1 replication in T cells by binding to Robo1 receptor (a) Slit2 expression in partially purified conditioned media from Slit2-myc-transfected human embryonic kidney (HEK) cells (Slit2) or vector-transfected cells (VCs) was determined by WB analysis (left panel). HIV-1 IIIB replication in MT4 cells (middle panel) or PHA-stimulated peripheral blood mononuclear cells (PBMCs) from normal donors (right panel) in the presence of partially purified conditioned medium containing Slit2 (approximately 6 nmol/l) or conditioned medium from control HEK293 cells was determined by analyzing HIV-1 p24 antigen levels in the supernatants by ELISA. In the case of MT4 cells, the supernatants were collected after 48 h. Virus production in the positive control (HIV-1-infected MT4 cells cultured only with medium): 12.1 ng/ml p24 Ag. * P

    Article Snippet: We also demonstrated Slit2 expression in PBMCs from normal donors ( , right panel).

    Techniques: Binding Assay, Expressing, Purification, Transfection, Plasmid Preparation, Western Blot, Enzyme-linked Immunosorbent Assay, Positive Control, Infection, Cell Culture

    Overexpression and downregulation of Slit2 modulates HIV-1 replication (a) Slit2 expression in lysates from MT4 cells and Jurkat T cells loaded at different concentrations (left panel) and from peripheral blood mononuclear cells (PBMCs) isolated from three normal donors (right panel) was determined by western blot (WB) analysis. Equal protein loading was determined by probing the same blot with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. (b) Slit2 expression in Jurkat T cells transfected with nontargeting (NT) or Slit2-specific small inhibitor RNA (siRNA) was analyzed by WB analysis (left panel). Intracellular p24 staining of the transfected cells infected with HIV-1 IIIB for 48 h was determined by flow cytometric analysis (right panel). * P

    Journal: AIDS (London, England)

    Article Title: A novel role for Slit2/Robo1 axis in modulating HIV-1 replication in T cells

    doi: 10.1097/QAD.0b013e32834bab86

    Figure Lengend Snippet: Overexpression and downregulation of Slit2 modulates HIV-1 replication (a) Slit2 expression in lysates from MT4 cells and Jurkat T cells loaded at different concentrations (left panel) and from peripheral blood mononuclear cells (PBMCs) isolated from three normal donors (right panel) was determined by western blot (WB) analysis. Equal protein loading was determined by probing the same blot with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. (b) Slit2 expression in Jurkat T cells transfected with nontargeting (NT) or Slit2-specific small inhibitor RNA (siRNA) was analyzed by WB analysis (left panel). Intracellular p24 staining of the transfected cells infected with HIV-1 IIIB for 48 h was determined by flow cytometric analysis (right panel). * P

    Article Snippet: We also demonstrated Slit2 expression in PBMCs from normal donors ( , right panel).

    Techniques: Over Expression, Expressing, Isolation, Western Blot, Transfection, Staining, Infection, Flow Cytometry

    Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

    Journal: Journal of Virology

    Article Title: Bovine Leukemia Virus Structural Gene Vectors Are Immunogenic and Lack Pathogenicity in a Rabbit Model

    doi:

    Figure Lengend Snippet: Semiquantitative PCR analysis to evaluate differences in proviral load. (A) PCR to detect BLV pol in PBMC from treated rabbits. Rabbit PBMC were harvested at 1, 4, and 10 months postinoculation and subjected to PCR with BLV pol primers KB560 and KB561 (10 × 10 4 PBMC) or β- actin to control for sample variation (2 × 10 4 PBMC). Each panel is labeled with month of sample harvest. Lanes are labeled with the source of PBMC DNA by rabbit number and treatment, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). In lower panels, the corresponding rabbit samples are designated by the matching parallel black lines. Positions of BLV pol amplicon (591 bp) and β- actin amplicon (594 bp) are designated. *, not determined (ND) because sample was not available; †, not determined because the animal died before harvest. (B) PCR standard curve. PBMC lysate harvested at 1 month postinoculation from BLV rabbit 44-10 was serially diluted in a range of 5 × 10 2 to 5 × 10 4 PBMC and subjected to PCR with BLV pol primers KB560 and KB561. Each lane is labeled with the number of cells used for PCR amplification, FLK(BLV) (positive control DNA [100 ng] from BLV-producing fetal lamb kidney cells), or marker (pGem DNA size standard [Promega]). The arrow on the left indicates the position of BLV pol amplicon (591 bp), and the lines on the right indicate positions of 676- and 571-bp DNA size markers.

    Article Snippet: To prepare PBMCs samples for PCR, PBMCs were isolated by Ficoll-Paque (Pharmacia) gradient centrifugation and washed three times with phosphate-buffered saline.

    Techniques: Polymerase Chain Reaction, Labeling, Positive Control, Marker, Amplification

    Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Journal: BMC Molecular Biology

    Article Title: Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

    doi: 10.1186/1471-2199-10-75

    Figure Lengend Snippet: Expression of different uPAR splice variant mRNAs in different tissues/cell types normalised to total uPAR . A series of real-time PCR assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to total uPAR and relative to a suitable plasmid control (designated 100%). (A) total classical uPAR (exon 7), (B) classical uPAR exon 6 deletion, (C) classical uPAR exons 5+6 deletion, (D) classical uPAR exon 3 deletion, (E) total alternative uPAR (exon7b), (F) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Article Snippet: RNA and lysates from PMN and PBMC were also obtained commercially (3 H Biomedical, Uppsala, Sweden).

    Techniques: Expressing, Variant Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation

    Expression of uPAR splice variant mRNA in different tissues/cell types . A series of real-time PCR (TaqMan) assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to HPRT1 and relative to a suitable plasmid positive control containing the specific splice variant cDNA (designated 100%). (A) total uPAR, (B) total classical uPAR (exon 7), (C) classical uPAR exon 6 deletion, (D) classical uPAR exons 5+6 deletion, (E) classical uPAR exon 3 deletion, (F) total alternative uPAR (exon7b), (G) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Journal: BMC Molecular Biology

    Article Title: Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

    doi: 10.1186/1471-2199-10-75

    Figure Lengend Snippet: Expression of uPAR splice variant mRNA in different tissues/cell types . A series of real-time PCR (TaqMan) assays was used to measure the expression of different splice variants of uPAR in an extended panel of tissues/cell types (lung, brain, HASM, undifferentiated HBEC, BEAS2B, PMN, PBMC, THP1). Expression of each variant is shown as mean + SEM of three PCR replicates, for two donors or biological replicates as appropriate. Data are shown as 2 -ΔCt normalised to HPRT1 and relative to a suitable plasmid positive control containing the specific splice variant cDNA (designated 100%). (A) total uPAR, (B) total classical uPAR (exon 7), (C) classical uPAR exon 6 deletion, (D) classical uPAR exons 5+6 deletion, (E) classical uPAR exon 3 deletion, (F) total alternative uPAR (exon7b), (G) alternative uPAR exon 4+5 deletion. Classical uPAR exon 5 and 4+5 deletions and alternative uPAR exon 5 deletion were not detected.

    Article Snippet: RNA and lysates from PMN and PBMC were also obtained commercially (3 H Biomedical, Uppsala, Sweden).

    Techniques: Expressing, Variant Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Plasmid Preparation, Positive Control

    Expression of uPAR protein in different cell types . Western blotting of cell lysates was performed for an extended panel of cell types (HASM, undifferentiated HBEC, BEAS2B, THP1, PMN, PBMC) plus recombinant uPAR (ruPAR) using domain I specific (A) and domain II specific (B) antibodies to identify different variants. A β-actin antibody was used as a loading control (C). An ELISA assay, with a sensitivity of 30 pg/mL, was used to detect soluble uPAR in the culture supernatant where appropriate (D).

    Journal: BMC Molecular Biology

    Article Title: Characterisation of urokinase plasminogen activator receptor variants in human airway and peripheral cells

    doi: 10.1186/1471-2199-10-75

    Figure Lengend Snippet: Expression of uPAR protein in different cell types . Western blotting of cell lysates was performed for an extended panel of cell types (HASM, undifferentiated HBEC, BEAS2B, THP1, PMN, PBMC) plus recombinant uPAR (ruPAR) using domain I specific (A) and domain II specific (B) antibodies to identify different variants. A β-actin antibody was used as a loading control (C). An ELISA assay, with a sensitivity of 30 pg/mL, was used to detect soluble uPAR in the culture supernatant where appropriate (D).

    Article Snippet: RNA and lysates from PMN and PBMC were also obtained commercially (3 H Biomedical, Uppsala, Sweden).

    Techniques: Expressing, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay

    C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

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

    Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter

    doi:

    Figure Lengend Snippet: C(−280)NFAT-binding activity does not depend on AP-1 cooperativity and has a higher affinity than NFAT sites with AP-1 cooperativity. a, NFAT binding to C(−280)NFAT is independent of AP-1. AP-1 sequence failed to compete for binding by C(−280)NFAT in EMSA using human PBMC extracts and vice versa. C(−280)NFAT failed to compete against AP-1 binding. *C-NFAT probe, C(−280)NFAT; *AP-1 probe, AP-1 oligonucleotide. b, NFAT-binding complex does not contain AP-1 components. Abs to Jun-B or c-Fos when incubated with PBMC extracts in EMSAs do not alter binding to the C(−280)NFAT probe. c, NFAT DNA-binding kinetic analysis. NFAT binding to C(−280)NFAT is more stable than to NFAT:AP-1 sites from the human and murine IL-2 promoter in PBMC extracts. Excess competitor for the indicated sites was added and the duration of binding was measured by EMSA. Right panel, The absorbance (OD) of the binding complex after addition of excess competitor. d, NFAT promoter sequence comparison. C(−280)NFAT sequence has four adenosine bases (shadowed) in common when aligned with NFAT IL-2 promoter sequences. Underlined adenosine bases are critical for DNA binding as determined by EMSA ( e ). Sp = species; h = human; m = mouse; D = A,T; N = A,C,T,G; X = A,T,C; a = noncoding sequence; b = EMSA competitor. e, Mutation analysis of C(−280)NFAT to identify nucleotides important for DNA binding. Single-base pair substitutions (C↔A and G↔T) across the C(−280)NFAT sequence were introduced into the indicated nucleotide positions and the corresponding double-stranded oligonucleotides containing mutations were used as competitor sequence in EMSA.

    Article Snippet: To study the dependence of CTLA-4 expression on NFAT activity in PBMCs, cell-permeable inhibitors that affect NFAT specifically were added to PBMCs and CTLA-4 gene expression was measured.

    Techniques: Binding Assay, Activity Assay, Sequencing, Incubation, Mutagenesis

    Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

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

    Article Title: Induction of the CTLA-4 Gene in Human Lymphocytes Is Dependent on NFAT Binding the Proximal Promoter

    doi:

    Figure Lengend Snippet: Identifying NFAT as the factor that binds to the proximal CTLA-4 promoter. a, Sequence of the proximal CTLA-4 promoter important for transcription and potential transcription factor recognition motifs predicted by Transcription ESS is shown from nucleotides −264 to −297 from the translational start site. This sequence has consensus sites for NFAT, c-Myb, and SRE and is defined as C(−280)NFAT. b, NFAT sequences compete away DNA binding to C(−280)NFAT probe. EMSA results with γ - 32 P-labeled dsC(−280)NFAT probe, with extracts prepared from stimulated human PBMCs. Competition assays were performed using excess double-stranded unlabeled oligonucleotides for C(−280)NFAT = C-NFAT, human IL-2 NFAT = hNFAT, Sp-1, or GAS sites. c, C(−280)NFAT oligonucleotide interacts with DNA-binding activity for NFAT and is competed by other NFAT sequences (hIL-2 NFAT and mIL-2 NFAT). Competition with excess c-Myb or SRE oligonucleotide does not alter DNA binding to the C(−280)NFAT probe. d, NFAT1 binds to C(−280)NFAT. EMSA performed in the presence of Abs specific to NFAT1 yields a supershifted band (arrow) whereas nonspecific Ab (Ns Ab) or anti-NFAT2 does not.

    Article Snippet: To study the dependence of CTLA-4 expression on NFAT activity in PBMCs, cell-permeable inhibitors that affect NFAT specifically were added to PBMCs and CTLA-4 gene expression was measured.

    Techniques: Sequencing, Binding Assay, Labeling, Activity Assay

    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

    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: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

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

    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

    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: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

    Techniques: Crocin Bleaching Assay

    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

    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: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

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

    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

    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: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

    Techniques: Cell Culture, Flow Cytometry, Cytometry

    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.

    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: Effect of OMVs on PBMCs Cytoskeleton PBMCs were fixed onto cover slips by duplicated and permeabilized with Triton X-100 0.5% in PBS.

    Techniques: Staining, Confocal Microscopy

    Biologically meaningful DE results for the 10x Genomics PBMC dataset. a Scatterplot of the first two t-SNE dimensions obtained from the first ten principal components. Cells are color-coded by clusters found using the SEURAT graph-based clustering method on the first ten principal components. Pseudo-color images on the right display normalized enrichment scores after gene set enrichment analysis for cell types related to CD4+ T cells (see “ Methods ”), for clustering based on b the first ten principal components and c W from ZINB-WaVE with K =20. For dimensionality reduction, ZINB-WaVE was fitted with X = 1 n , V = 1 J , K =20 for W (based on the Akaike information criterion), common dispersion, and ε =10 12 . To compute the weights for differential expression analysis, ZINB-WaVE was fitted with intercept and cell-type covariate in X , V = 1 J , K =0 for W , common dispersion, and ε =10 12 . Normalized enrichment scores for more cell types are shown in Additional file 1 : Figure S17. PCA principal component analysis

    Journal: Genome Biology

    Article Title: Observation weights unlock bulk RNA-seq tools for zero inflation and single-cell applications

    doi: 10.1186/s13059-018-1406-4

    Figure Lengend Snippet: Biologically meaningful DE results for the 10x Genomics PBMC dataset. a Scatterplot of the first two t-SNE dimensions obtained from the first ten principal components. Cells are color-coded by clusters found using the SEURAT graph-based clustering method on the first ten principal components. Pseudo-color images on the right display normalized enrichment scores after gene set enrichment analysis for cell types related to CD4+ T cells (see “ Methods ”), for clustering based on b the first ten principal components and c W from ZINB-WaVE with K =20. For dimensionality reduction, ZINB-WaVE was fitted with X = 1 n , V = 1 J , K =20 for W (based on the Akaike information criterion), common dispersion, and ε =10 12 . To compute the weights for differential expression analysis, ZINB-WaVE was fitted with intercept and cell-type covariate in X , V = 1 J , K =0 for W , common dispersion, and ε =10 12 . Normalized enrichment scores for more cell types are shown in Additional file 1 : Figure S17. PCA principal component analysis

    Article Snippet: 10x Genomics PBMC dataset We analyzed a dataset of PBMCs that is freely available from 10x Genomics ( https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/pbmc3k ) [ ].

    Techniques: Expressing

    Comparison of differential expression methods on simulated scRNA-seq datasets. Differential expression methods are compared based on FDP-TPR curves for data simulated from a 10x Genomics PBMC single-cell RNA-seq dataset ( n =1200). Zoomed versions of the FDP-TPR curves are shown here and full curves are in Additional file 1 : Figure S12. Circles represent working points on a nominal 5% FDR level and are filled if the empirical FDR (i.e., FDP) is below the nominal FDR. 10x Genomics sequencing typically involves high-throughput and massive multiplexing, resulting in very shallow sequencing depths and thus, low counts, making it extremely difficult to identify excess zeros. Unweighted and ZINB-WaVE-weighted EDGER are tied for best performance, followed by ZINB-WaVE-weighted DESEQ2 . In general, bulk RNA-seq methods perform well in this simulation, probably because the extremely high zero abundance in combination with low counts can be reasonably accommodated by the negative binomial distribution. The behavior in the lower half of the curve for NODES is due to a smooth increase in true positives with an identical number of false positives over a range of low FDR cut-offs. FDP false discovery proportion, FDR false discovery rate, PBMC peripheral blood mononuclear cell, TPR true positive rate

    Journal: Genome Biology

    Article Title: Observation weights unlock bulk RNA-seq tools for zero inflation and single-cell applications

    doi: 10.1186/s13059-018-1406-4

    Figure Lengend Snippet: Comparison of differential expression methods on simulated scRNA-seq datasets. Differential expression methods are compared based on FDP-TPR curves for data simulated from a 10x Genomics PBMC single-cell RNA-seq dataset ( n =1200). Zoomed versions of the FDP-TPR curves are shown here and full curves are in Additional file 1 : Figure S12. Circles represent working points on a nominal 5% FDR level and are filled if the empirical FDR (i.e., FDP) is below the nominal FDR. 10x Genomics sequencing typically involves high-throughput and massive multiplexing, resulting in very shallow sequencing depths and thus, low counts, making it extremely difficult to identify excess zeros. Unweighted and ZINB-WaVE-weighted EDGER are tied for best performance, followed by ZINB-WaVE-weighted DESEQ2 . In general, bulk RNA-seq methods perform well in this simulation, probably because the extremely high zero abundance in combination with low counts can be reasonably accommodated by the negative binomial distribution. The behavior in the lower half of the curve for NODES is due to a smooth increase in true positives with an identical number of false positives over a range of low FDR cut-offs. FDP false discovery proportion, FDR false discovery rate, PBMC peripheral blood mononuclear cell, TPR true positive rate

    Article Snippet: 10x Genomics PBMC dataset We analyzed a dataset of PBMCs that is freely available from 10x Genomics ( https://support.10xgenomics.com/single-cell-gene-expression/datasets/1.1.0/pbmc3k ) [ ].

    Techniques: Expressing, RNA Sequencing Assay, Sequencing, High Throughput Screening Assay, Multiplexing

    Ex vivo cytotoxicity Cryopreserved PBMCs were thawed and incubated with GFP + K562 target cells and CD107a PE-Cy5 Ab overnight at 37°C. Co-cultures were stained the following day for CD3/CD56 and analyzed for ( A ) killing and ( B ) CD107a incorporation indicative of degranulation. Killing was measured as the percentage of K562 cells that were lost based on a condition with target cells only ( A , top left). CD107a gating was set with conditions lacking target cells. Left panels summarize group data while right panels correspond to a representative example. ( C ) The absolute numbers of degranulating (CD107a + ) CD56 bright and CD56 dim NK cells per 1000 lymphocytes are depicted for each time point (B, T1 and T2). Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. ( D ) The absolute numbers of degranulating CD56 bright NK cells are positively correlated with the target killing using Spearman’s correlation. * denotes p

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

    Article Title: An IL-2 Paradox; Blocking CD25 on T Cells Induces IL-2-driven Activation of CD56bright NK Cells 1

    doi: 10.4049/jimmunol.0902238

    Figure Lengend Snippet: Ex vivo cytotoxicity Cryopreserved PBMCs were thawed and incubated with GFP + K562 target cells and CD107a PE-Cy5 Ab overnight at 37°C. Co-cultures were stained the following day for CD3/CD56 and analyzed for ( A ) killing and ( B ) CD107a incorporation indicative of degranulation. Killing was measured as the percentage of K562 cells that were lost based on a condition with target cells only ( A , top left). CD107a gating was set with conditions lacking target cells. Left panels summarize group data while right panels correspond to a representative example. ( C ) The absolute numbers of degranulating (CD107a + ) CD56 bright and CD56 dim NK cells per 1000 lymphocytes are depicted for each time point (B, T1 and T2). Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. ( D ) The absolute numbers of degranulating CD56 bright NK cells are positively correlated with the target killing using Spearman’s correlation. * denotes p

    Article Snippet: Cryopreserved PBMCs were thawed and plated in X-vivo media (Lonza, Walkersville, MD) at 1×105 cells/well in a 96-well plate.

    Techniques: Ex Vivo, Incubation, Staining

    Ex vivo Treg proliferation and CD127 expression Cryopreserved PBMCs were stained for intranuclear transcription factor FoxP3 ( A ) and proliferation marker Ki67 ( B C ) at baseline and treatment time points (B, T1 T2). Prior to permeabilization, they were stained for CD25 ( D ) and CD127 ( E ) expression, displayed here by mean fluorescence intensity (MFI) of the FoxP3 + Treg population. Furthermore, the MFI of Foxp3 expression on CD4 + CD25 + Tregs is displayed ( F ) for baseline and subsequent treatment time points. Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. * denotes p

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

    Article Title: An IL-2 Paradox; Blocking CD25 on T Cells Induces IL-2-driven Activation of CD56bright NK Cells 1

    doi: 10.4049/jimmunol.0902238

    Figure Lengend Snippet: Ex vivo Treg proliferation and CD127 expression Cryopreserved PBMCs were stained for intranuclear transcription factor FoxP3 ( A ) and proliferation marker Ki67 ( B C ) at baseline and treatment time points (B, T1 T2). Prior to permeabilization, they were stained for CD25 ( D ) and CD127 ( E ) expression, displayed here by mean fluorescence intensity (MFI) of the FoxP3 + Treg population. Furthermore, the MFI of Foxp3 expression on CD4 + CD25 + Tregs is displayed ( F ) for baseline and subsequent treatment time points. Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. * denotes p

    Article Snippet: Cryopreserved PBMCs were thawed and plated in X-vivo media (Lonza, Walkersville, MD) at 1×105 cells/well in a 96-well plate.

    Techniques: Ex Vivo, Expressing, Staining, Marker, Fluorescence

    Ex vivo IL-2 and IL-7 signaling Cryopreserved PBMCs from baseline and therapy time points (B, T1 T2) were thawed and rested for an hour at 37°C in X-vivo media. They were subsequently pulsed with IL-2 ( A C ) or IL-7 ( B ) for 10 minutes and then fixed in formaldehyde, washed, and permeabilized with methanol. They were then stained for surface markers and phosphorylated STAT5 (pSTAT5). Gating was set from control wells pulsed with X-vivo media lacking additional cytokines. For CD56 bright NK cells ( C ), both the proportion (left panel) and absolute numbers (calculated per 1000 lymphocytes; right panel) of pSTAT5 + cells are displayed. Again, patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. Means are marked with a thick black line. Raw data FACS plots are taken from a representative patient.

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

    Article Title: An IL-2 Paradox; Blocking CD25 on T Cells Induces IL-2-driven Activation of CD56bright NK Cells 1

    doi: 10.4049/jimmunol.0902238

    Figure Lengend Snippet: Ex vivo IL-2 and IL-7 signaling Cryopreserved PBMCs from baseline and therapy time points (B, T1 T2) were thawed and rested for an hour at 37°C in X-vivo media. They were subsequently pulsed with IL-2 ( A C ) or IL-7 ( B ) for 10 minutes and then fixed in formaldehyde, washed, and permeabilized with methanol. They were then stained for surface markers and phosphorylated STAT5 (pSTAT5). Gating was set from control wells pulsed with X-vivo media lacking additional cytokines. For CD56 bright NK cells ( C ), both the proportion (left panel) and absolute numbers (calculated per 1000 lymphocytes; right panel) of pSTAT5 + cells are displayed. Again, patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. Means are marked with a thick black line. Raw data FACS plots are taken from a representative patient.

    Article Snippet: Cryopreserved PBMCs were thawed and plated in X-vivo media (Lonza, Walkersville, MD) at 1×105 cells/well in a 96-well plate.

    Techniques: Ex Vivo, Staining, FACS

    In vitro NK proliferation ( A ) IL-2 consumption by T cells was measured by ELISA from supernatants of activated T cells cultured with either daclizumab (Dac) or an anti-CD25 control Ab which does not bind to the IL-2 binding tac epitope (M-A251): T cells were polyclonally stimulated for 3 days, extensively washed and reseeded in equal numbers with exogenously added IL-2 (20 IU/ml) and Dac or M-A251 Ab (10μg/ml each). Supernatants were collected in 24–48h and the amount of IL-2 remaining was measured by ELISA. Samples were taken from 4 patients (2 patients with purified (negatively-selected) T cells and 2 with NK-depleted PBMC). ( B ) CFSE dilution of NK cells was measured after 5 days in culture with increasing ratios of daclizumab-treated CD3/CD28 stimulated T cells (n=4). ( C D ) Transwell experiments: Negatively-selected T cells (or NK-depleted PBMC) were polyclonally activated for 24–72h and 1×10 6 activated T cells were seeded in the lower compartment of transwells in the presence of control Ab M-A251, daclizumab (Dac), IL-2-neutralizing Ab (α-IL-2) or 20IU IL-2. Autologous NK cells were negatively selected from either fresh or cryopreserved apheresis samples 24 hours before co-culture with activated T cells, they were CFSE stained and rested overnight before adding 1×10 5 NK cells into the transwell inserts. In 3–5 days, NK cell proliferation was assessed by CFSE dilution and their absolute numbers were proportionally enumerated between conditions by normalizing per 1000 fluorescent beads added prior to acquisition by flow cytometry. Because we observed that activated NK cells were able to migrate through 3μm pores in the transwell inserts to the T cell compartment, we present total cultures from both the upper compartment of the transwell (top row) and the lower compartment of the transwell (bottom row): ( C ) Representative raw data FACS plots (with numbers indicating absolute numbers of CD56 bright (red) and CD56 dim (blue) NK cells) and ( D ) enumeration of proportion and absolute numbers of proliferating CD56 bright NK cells. Data are representative of 4 independent patients/experiments (2 for both purified T cells and 2 for NK-depleted PBMCs). ( E F ) Supernatant-transfer experiments: Negatively-selected T cells were polyclonally activated for 24–72h in the presence/absence of control Ab M-A251, daclizumab or IL-2-neutralizing Ab (α-IL-2). Supernatant from these cultures were collected 24–48 hours post-activation and cryopreserved until testing. NK cells were isolated by negative selection from fresh or cryopreserved apheresis samples, CFSE stained and cultured for 3 days with collected supernatant from activated T cells: ( E ) FACS plots representative of NK cell proliferation (absolute numbers of CD56 bright and CD56 dim NK cells are depicted in the gates) and ( F ) enumeration of proportion and absolute numbers of proliferating CD56 bright NK cells. Data are representative of 2 independent patients/experiments. * denotes p

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

    Article Title: An IL-2 Paradox; Blocking CD25 on T Cells Induces IL-2-driven Activation of CD56bright NK Cells 1

    doi: 10.4049/jimmunol.0902238

    Figure Lengend Snippet: In vitro NK proliferation ( A ) IL-2 consumption by T cells was measured by ELISA from supernatants of activated T cells cultured with either daclizumab (Dac) or an anti-CD25 control Ab which does not bind to the IL-2 binding tac epitope (M-A251): T cells were polyclonally stimulated for 3 days, extensively washed and reseeded in equal numbers with exogenously added IL-2 (20 IU/ml) and Dac or M-A251 Ab (10μg/ml each). Supernatants were collected in 24–48h and the amount of IL-2 remaining was measured by ELISA. Samples were taken from 4 patients (2 patients with purified (negatively-selected) T cells and 2 with NK-depleted PBMC). ( B ) CFSE dilution of NK cells was measured after 5 days in culture with increasing ratios of daclizumab-treated CD3/CD28 stimulated T cells (n=4). ( C D ) Transwell experiments: Negatively-selected T cells (or NK-depleted PBMC) were polyclonally activated for 24–72h and 1×10 6 activated T cells were seeded in the lower compartment of transwells in the presence of control Ab M-A251, daclizumab (Dac), IL-2-neutralizing Ab (α-IL-2) or 20IU IL-2. Autologous NK cells were negatively selected from either fresh or cryopreserved apheresis samples 24 hours before co-culture with activated T cells, they were CFSE stained and rested overnight before adding 1×10 5 NK cells into the transwell inserts. In 3–5 days, NK cell proliferation was assessed by CFSE dilution and their absolute numbers were proportionally enumerated between conditions by normalizing per 1000 fluorescent beads added prior to acquisition by flow cytometry. Because we observed that activated NK cells were able to migrate through 3μm pores in the transwell inserts to the T cell compartment, we present total cultures from both the upper compartment of the transwell (top row) and the lower compartment of the transwell (bottom row): ( C ) Representative raw data FACS plots (with numbers indicating absolute numbers of CD56 bright (red) and CD56 dim (blue) NK cells) and ( D ) enumeration of proportion and absolute numbers of proliferating CD56 bright NK cells. Data are representative of 4 independent patients/experiments (2 for both purified T cells and 2 for NK-depleted PBMCs). ( E F ) Supernatant-transfer experiments: Negatively-selected T cells were polyclonally activated for 24–72h in the presence/absence of control Ab M-A251, daclizumab or IL-2-neutralizing Ab (α-IL-2). Supernatant from these cultures were collected 24–48 hours post-activation and cryopreserved until testing. NK cells were isolated by negative selection from fresh or cryopreserved apheresis samples, CFSE stained and cultured for 3 days with collected supernatant from activated T cells: ( E ) FACS plots representative of NK cell proliferation (absolute numbers of CD56 bright and CD56 dim NK cells are depicted in the gates) and ( F ) enumeration of proportion and absolute numbers of proliferating CD56 bright NK cells. Data are representative of 2 independent patients/experiments. * denotes p

    Article Snippet: Cryopreserved PBMCs were thawed and plated in X-vivo media (Lonza, Walkersville, MD) at 1×105 cells/well in a 96-well plate.

    Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Cell Culture, Binding Assay, Purification, Co-Culture Assay, Staining, Flow Cytometry, Cytometry, FACS, Activation Assay, Isolation, Selection

    Ex vivo CD56 bright NK cell proliferation Cryopreserved PBMCs were stained prior to permeabilization for NK cell marker CD56, T cell marker CD3 ( A ) and following permeabilization, stained for intranuclear proliferation marker Ki67 ( B ) at baseline and treatment time points (B, T1 T2). Ki67 gating was set individually on CD56 bright NK cells only. Both proportion (left panel) and absolute numbers of proliferating CD56 bright NK cells per 1000 gated lymphocytes (right panel) are depicted. Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. Means are marked with a thick black line. Raw data FACS plots are taken from a representative patient.

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

    Article Title: An IL-2 Paradox; Blocking CD25 on T Cells Induces IL-2-driven Activation of CD56bright NK Cells 1

    doi: 10.4049/jimmunol.0902238

    Figure Lengend Snippet: Ex vivo CD56 bright NK cell proliferation Cryopreserved PBMCs were stained prior to permeabilization for NK cell marker CD56, T cell marker CD3 ( A ) and following permeabilization, stained for intranuclear proliferation marker Ki67 ( B ) at baseline and treatment time points (B, T1 T2). Ki67 gating was set individually on CD56 bright NK cells only. Both proportion (left panel) and absolute numbers of proliferating CD56 bright NK cells per 1000 gated lymphocytes (right panel) are depicted. Patients from the monotherapy trial are indicated by open squares and patients from the combination therapy trial are indicated by black squares. Means are marked with a thick black line. Raw data FACS plots are taken from a representative patient.

    Article Snippet: Cryopreserved PBMCs were thawed and plated in X-vivo media (Lonza, Walkersville, MD) at 1×105 cells/well in a 96-well plate.

    Techniques: Ex Vivo, Staining, Marker, FACS

    ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of HIV-1 infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with PBMC of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: ( a ) p24 viral antigen levels 10 days post-infection in the supernatant of HIV-1 infected PBMCs treated with TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Results obtained with PBMC of 20 different healthy donors are shown. Median values and statistical significance is presented. ( b ) Inhibition (%) of viral replication in PBMC of 20 different healthy donors infected in vitro with HIV-1 in the presence of TIZ- (10 μg/mL) or RM-4848- (10 μg/mL). Median values and statistical significance is presented. ( c ) p24 viral antigen levels in PBMCs infected with HIV-1 in the presence of RM-4848 throughout infection (RM-4848)(10 μg/mL) or in medium alone (no treatment). In addition to these two situations, results of 3 other experimental conditions are shown: 1) PBMCs treated with RM-4848 before being infected (pre-infection); 2) RM-4848 present during the 24 hours infection period (during infection) and then washed away; 3) RM-4848 added 24 hours after infection (post infection). Mean values ± standard errors are shown.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Infection, Inhibition, In Vitro

    Cytokines (IL-2, IFNγ) and chemokines (MCP-1, MIP-1α, MIP-1β, RANTES) production (pg/ml) by PBMCs of healthy donors. PBMCs were either uninfected (white bars) or HIV-1-infected (grey bars) and were cultured in medium alone (no drug), or in medium containing either TIZ- (10 μg/mL) ( a ) or RM-4848- (10 μg/mL) ( b ). Mean values 7 days post-infection ± standard errors and statistical differences are shown.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: Cytokines (IL-2, IFNγ) and chemokines (MCP-1, MIP-1α, MIP-1β, RANTES) production (pg/ml) by PBMCs of healthy donors. PBMCs were either uninfected (white bars) or HIV-1-infected (grey bars) and were cultured in medium alone (no drug), or in medium containing either TIZ- (10 μg/mL) ( a ) or RM-4848- (10 μg/mL) ( b ). Mean values 7 days post-infection ± standard errors and statistical differences are shown.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Infection, Cell Culture

    mRNA expression of 84 genes that are part of the HIV-1 life cycle and known to directly obstacle HIV infectivity has been assessed by real-time quantitative RT-PCR, calculated relative to five housekeeping genes and shown as fold-change expression from the untreated sample. Gene expression (nfold) is shown as a color scale from green to red (−2 to +9) (MEV multiple experiment viewer software). Results were obtained in HIV1-infected PBMCs at day 7 post infection, cultured in medium containing TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Only targets showing > 2-fold modulation are considered significant and are shown in tab.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: mRNA expression of 84 genes that are part of the HIV-1 life cycle and known to directly obstacle HIV infectivity has been assessed by real-time quantitative RT-PCR, calculated relative to five housekeeping genes and shown as fold-change expression from the untreated sample. Gene expression (nfold) is shown as a color scale from green to red (−2 to +9) (MEV multiple experiment viewer software). Results were obtained in HIV1-infected PBMCs at day 7 post infection, cultured in medium containing TIZ (10 μg/mL) or RM-4848 (10 μg/mL). Only targets showing > 2-fold modulation are considered significant and are shown in tab.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Software, Cell Culture

    mRNA expression of genes involved in cholesterol metabolism and efflux in PBMCs of healthy donors. ( a ) Uninfected PBMCs cultured in medium containing TIZ (10 μg/mL); ( b ) Uninfected PBMCs cultured in medium containing RM-4848 (10 μg/mL); ( c ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing TIZ (10 μg/mL); ( d ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing RM-4848 (10 μg/mL). Gene expression genes is assessed by quantitative RT-PCR and shown as fold-change expression from the untreated sample. Only targets showing > 2-fold modulation are considered significant. Mean values ± S.D. and p values are indicated.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: mRNA expression of genes involved in cholesterol metabolism and efflux in PBMCs of healthy donors. ( a ) Uninfected PBMCs cultured in medium containing TIZ (10 μg/mL); ( b ) Uninfected PBMCs cultured in medium containing RM-4848 (10 μg/mL); ( c ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing TIZ (10 μg/mL); ( d ) HIV-1–infected PBMCs (day 7 post infection) cultured in medium containing RM-4848 (10 μg/mL). Gene expression genes is assessed by quantitative RT-PCR and shown as fold-change expression from the untreated sample. Only targets showing > 2-fold modulation are considered significant. Mean values ± S.D. and p values are indicated.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Expressing, Cell Culture, Infection, Quantitative RT-PCR

    p24 viral antigen concentration in HIV-1 - infected PBMCs cultured in medium alone (no drug) or in the presence of TO-901317 (1 μM), an LXR agonist. Results were obtained with PBMCs of 20 different healthy donors 10 days post-infection. Mean values ± standard errors and statistical significance are shown.

    Journal: Scientific Reports

    Article Title: Thiazolides Elicit Anti-Viral Innate Immunity and Reduce HIV Replication

    doi: 10.1038/srep27148

    Figure Lengend Snippet: p24 viral antigen concentration in HIV-1 - infected PBMCs cultured in medium alone (no drug) or in the presence of TO-901317 (1 μM), an LXR agonist. Results were obtained with PBMCs of 20 different healthy donors 10 days post-infection. Mean values ± standard errors and statistical significance are shown.

    Article Snippet: PBMC isolation and HIV-1 infection Whole blood was collected from 20 healthy volunteers by venipuncture in Vacutainer tubes containing EDTA (Becton Dickinson, Franklin Lakes, NJ), and PBMCs were separated on lymphocyte separation medium (Organon Teknica, Malvern, PA).

    Techniques: Concentration Assay, Infection, Cell Culture

    IFN-α enhances the suppressive capacity of VP and ES CD8 + T cells without inhibiting HIV-specific IFN-γ responses of CP CD8 + T cells. (A) PBMCs from 8 CP were pulsed with either medium alone (baseline) or 100 U/ml of IFN-α for

    Journal: Journal of Virology

    Article Title: Interferon Alpha Enhances NK Cell Function and the Suppressive Capacity of HIV-Specific CD8+ T Cells

    doi: 10.1128/JVI.01541-18

    Figure Lengend Snippet: IFN-α enhances the suppressive capacity of VP and ES CD8 + T cells without inhibiting HIV-specific IFN-γ responses of CP CD8 + T cells. (A) PBMCs from 8 CP were pulsed with either medium alone (baseline) or 100 U/ml of IFN-α for

    Article Snippet: Specifically, CD8+ T cells were isolated from CP PBMCs (Miltenyi Biotec) and pulsed with either medium or various concentrations of IFN-α diluted in medium as before.

    Techniques: