Journal:

Article Title: Protein Kinase D1: A New Component in Toll-Like Receptor 9 Signaling

doi:

Figure Lengend Snippet: CpG-B DNA, not CpG-A DNA, induces phosphorylation of PKD proteins in murine splenic B cells, cDCs, and pDCs, and human PBMCs

Article Snippet: Because, in addition to macrophages, other types of cells, including B cells and DCs, also express TLR9 (the CpG motif containing DNA receptor) and respond to CpG motif containing DNA, we further investigated whether CpG motif containing DNA can induce activation of PKD family members in B cells, pDCs, cDCs, and human PBMCs.

Techniques:

Journal: Molecular Therapy Oncolytics

Article Title: Regression of established renal cell carcinoma in nude mice using lentivirus-transduced human T cells expressing a human anti-CAIX chimeric antigen receptor

doi: 10.1038/mto.2014.3

Figure Lengend Snippet: Antibody-dependent cellular cytotoxicity of carbonic anhydrase IX (CAIX)-specific Antibodies. 1 µg/ml CAIX-specific scFv-Fc minibodies were added to the target tumor cells in the presence of human peripheral blood mononucleated cell (E:T 25:1). Similar results were obtained in two experiments. Irrelevant anti-SARS scFv-Fc (11A) and anti-CCR4 scFv-Fc (48) minibodies were used as negative controls. ( a ) CAIX + sk-rc-09 cells; ( b ) CAIX + sk-rc-52 cells; ( c ) CAIX- sk-rc-59 cells.

Article Snippet: Human peripheral blood mononucleated cells were isolated by ficoll density gradient separation and were activated with 2 µg/ml phytohemagglutinin (PHA) (Sigma) plus 100 IU/ml human IL-2 for 4 days.

Techniques:

Journal: International Immunology

Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

doi: 10.1093/intimm/dxt061

Figure Lengend Snippet: EAT-2 over-expression enhances human DC maturation. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-Null. At 72 hpi, cells were stained for surface expression of CD86 (A), HLA-DR (B), CD80 (C) and CD83 (D) and FACS analysis was performed. Data are representative of at least three independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

Techniques: Over Expression, Isolation, Infection, Staining, Expressing, FACS

Journal: International Immunology

Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

doi: 10.1093/intimm/dxt061

Figure Lengend Snippet: Transduction of human innate and adaptive immune cells by GFP-expressing rAd5 vector. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-GFP. FACS analysis for GFP expression at 48h following rAd5-GFP infection of human NK cells (A), CD4 + and CD8 + T cells (B), CD14 + monocytes and CD1a + cells (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

Techniques: Transduction, Expressing, Plasmid Preparation, Isolation, Infection, FACS, Flow Cytometry, Cytometry, Software

Journal: International Immunology

Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

doi: 10.1093/intimm/dxt061

Figure Lengend Snippet: Intracellular staining analysis for EAT-2 expression in human immune cells following rAd5-EAT2 infection. Human PBMCs were isolated from fresh buffy coat material and were either mock infected or infected with MOI of 10000 with rAd5-EAT2 or rAd5-EAT2(R31Q). Intracellular staining analysis for EAT-2 expression at 48h following Ad infection of human NK cells (A), CD14 + monocytes (B) and CD1a + DCs (C) is shown. Data were collected using a BD LSRII flow cytometer and analyzed using FlowJo software.

Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

Techniques: Staining, Expressing, Infection, Isolation, Flow Cytometry, Cytometry, Software

Journal: International Immunology

Article Title: Manipulation of EAT-2 expression promotes induction of multiple beneficial regulatory and effector functions of the human innate immune system as a novel immunomodulatory strategy

doi: 10.1093/intimm/dxt061

Figure Lengend Snippet: EAT-2 over-expression enhances the cytolytic activity of human NK cells. Human PBMCs isolated from fresh buffy coat material were either mock infected or infected with MOI of 10000 with rAd5-EAT2, rAd5-EAT2(R31Q) or rAd5-Null for 72h and then co-cultured with CFSE-labeled K562 cells for an additional 48h at an E:T ratio of 1:10. Viability of K562 cells was assessed by either PI (A and B) or ViViD (C). Data are representative of two (A) or three (B) independent experiments from three different blood donors with similar results. Samples were plated in quadruplicates. Bars represent mean ± SD. Statistical analysis was completed using a one way ANOVA with a Student–Newman–Keuls post-hoc test. P

Article Snippet: To identify human immune cells transduced by Ad-mediated EAT-2 or GFP gene transduction, human PBMCs (2×106 ) were infected with multiplicity of infection (MOI) of 10000 of Ad5-EAT-2 or Ad5-GFP for 48h and were then stained with the following antibodies: APC-Cy7-CD14, PE-CD1a, Alexa Fluor 700-CD8a, V450-CD4, APC-CD65 and PerCpCy5.5-CD3 (all at 4 µg ml−1 ; BD Biosciences).

Techniques: Over Expression, Activity Assay, Isolation, Infection, Cell Culture, Labeling

Journal: Journal for Immunotherapy of Cancer

Article Title: Agonist redirected checkpoint, PD1-Fc-OX40L, for cancer immunotherapy

doi: 10.1186/s40425-018-0454-3

Figure Lengend Snippet: The human PD1-Fc-OX40L ARC has functional activity in vitro and ex vivo in both tumor co-culture and SEB super-antigen assays. a Schematic of the tumor/T cell co-culture assay. CD3+ human T cells stimulated for 48 h with suboptimal levels of CD3/CD28/IL-2, were plated on mitomycin-c treated PD-L1 low (PC3) and PD-L1 high (HCC827) tumor cells ± the PD1-Fc-OX40L ARC, for an additional 3–5 days (days 5–7 of the entire time-course). b On day 6 of the assay, culture supernatant was collected and analyzed by human IL-2 ELISA. c On days 5 (top) and 7 (bottom) of the assay, floating T cells were collected and subjected to extra- and intra-cellular flow cytometry in order to assess proliferation (Ki67) and markers of T cell activation (IFNγ TNFα). d Workflow of the SEB super-antigen assay. Total primary human PBMCs were harvested and treated with Staphylococcal enterotoxin B ± the PD1-Fc-OX40L ARC and benchmark antibody controls. Culture supernatants were collected 3 days later and assessed for secreted levels of IL-2 by ELISA

Article Snippet: Additionally, the SEB assay was performed in human PBMCs depleted of CD4, CD8, or both CD4/CD8 cells, using magnetic positive selection kits (StemCell Technologies).

Techniques: Functional Assay, Activity Assay, In Vitro, Ex Vivo, Co-Culture Assay, Co-culture Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cytometry, Activation Assay, Antigen Assay

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

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

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

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

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Autoantibodies to IgG/HLA class II complexes are associated with rheumatoid arthritis susceptibility

doi: 10.1073/pnas.1401105111

Figure Lengend Snippet: IgGH is transported to the cell surface by HLA-DR. ( A ) The secreted forms of IgGHs cloned from human PBMCs that have different V regions were cotransfected with HLA-DRα, DRB1*04:04 (HLA-DR4), and GFP. Cell surface IgG on GFP-expressing cells was

Article Snippet: IgGHs were amplified from human PBMCs (3H Biomedical) using sense primers for V regions (5′-GTCTTGTCCCAGGTCACCTTGAAGGAG-3′ for clone 1 and 3, 5′-GCCCACTCCCAGGTGCAGCTGGTGCAG-3′ for clone 2, 5′-GTGCAGCTGGTGCAGTCTGGAGCAGAG-3′ for clone 4, 5′-GTCCAGTGTGAAGTGCAGCTGGTGGAG-3′ for clone 5), and antisense primer for constant region of secreted IgG (5′-TCATTTACCCGGAGACAGGGAG-3′) and cloned into pME18S expression vector containing a CD150 signal sequence.

Techniques: Clone Assay, Expressing

Journal: International Journal of Nanomedicine

Article Title: Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production

doi: 10.2147/IJN.S166259

Figure Lengend Snippet: The evaluation of the immunostimulatory effects of NIPAM-hemin. Abbreviations: NIPAM, N -isopropylacrylamide; hemin, ferriprotoporphyrin IX chloride; IFN-γ, interferon-γ; IL, interleukin; NK, natural killer; PBMCs, peripheral blood mononuclear cells; ELISA, enzyme-linked immunosorbent assay; TLRs, toll-like receptors; SEAP, secreted embryonic alkaline phosphatase.

Article Snippet: Stimulation of human PBMCs with NIPAM-hemin Commercially available frozen PBMCs were purchased from Cellular Technology Limited (Shaker Heights, OH, USA).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: International Journal of Nanomedicine

Article Title: Synthesis of a hemin-containing copolymer as a novel immunostimulator that induces IFN-gamma production

doi: 10.2147/IJN.S166259

Figure Lengend Snippet: NIPAM-hemin induced the production of IFN-γ and IL-6, and had a lesser effect on the induction of IL-1β, in PBMCs. PBMCs were stimulated with NIPAM-hemin, hemin, NIPAM, or poly-NIPAM (MW=66,400). The levels of ( A ) IFN-γ, ( B ) IL-6, and ( C ) IL-1β were determined after 48 h. The concentration of each stimulant was 500 μg/mL. Hemin was dissolved in DMSO, while the other compounds were dissolved in sterilized deionized water. Notes: Data are expressed as the mean±SD (n=3). * P

Article Snippet: Stimulation of human PBMCs with NIPAM-hemin Commercially available frozen PBMCs were purchased from Cellular Technology Limited (Shaker Heights, OH, USA).

Techniques: Concentration Assay

Journal: Nature Communications

Article Title: ASK1 restores the antiviral activity of APOBEC3G by disrupting HIV-1 Vif-mediated counteraction

doi: 10.1038/ncomms7945

Figure Lengend Snippet: AZT induces ASK1 and promotes the antiviral activity of A3G. ( a , b ) PBMC or H9 cells were treated with the indicated agents (0–10 μM) for 24 h. The expression of ASK1 was confirmed by western blotting. APV, amprenavir; ATV, atazanavir; AZT, zidovudine; DRV, darunavir; IDV, indinavir; LPV, lopinavir; NFV, nelfinavir; NVP, nevirapine; SQV, saquinavir; 3TC, lamivudine. ( c ) H9 cells were transfected with control siRNA or ASK1-targeted siRNA for 48 h before infection of HIV-1. At 2 days after infection, cells were washed and additionally cultured for 24 h in the presence or absence of AZT (10 μM). Cells and supernatants were then harvested and analysed by western blotting against the indicated antibodies. We used AZT-resistant virus to avoid the effects of AZT carry-over from the culture supernatant of producer cells during the reverse transcription step in target cells. ( d ) TZM-bl reporter cells were infected with harvested and normalized virus to measure viral infectivity ( n =3, mean±s.d.). Full images for all western blots analysis are shown in Supplementary Fig. 6 .

Article Snippet: CEM, CEMSS, H9, M8166 (NIH AIDS Reagent Program) and human PBMCs (purchased from Kurabo, Osaka, Japan) were cultured in RPMI containing 10% fetal bovine serum.

Techniques: Activity Assay, Expressing, Western Blot, Transfection, Infection, Cell Culture

Journal: Journal of Virology

Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

doi: 10.1128/JVI.02507-14

Figure Lengend Snippet: (A) Transcriptome analysis of KSHV during de novo infection of human PBMCs. Total RNAs extracted from de novo -infected PBMCs harvested at 4 h, 24 h, 48 h, 72 h, and 120 h postinfection were subjected to cDNA library preparation using a TrueSeq RNA-seq

Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

Techniques: Infection, cDNA Library Assay, RNA Sequencing Assay

Journal: Journal of Virology

Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

doi: 10.1128/JVI.02507-14

Figure Lengend Snippet: Transcriptome analysis of KSHV in de novo -infected PBMCs, TIVE cells, and CD14 + cells. RPKM values, calculated based the number of reads for each gene, were used for analyzing relative expression of KSHV genes as heat maps. Hierarchal clustering of genes

Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

Techniques: Infection, Expressing

Journal: Journal of Virology

Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

doi: 10.1128/JVI.02507-14

Figure Lengend Snippet: Viral genes transcribed at 4 hpi and 24 hpi, identified by a nascent RNA capture approach. Human PBMCs infected with KSHV virions were incubated with EdU (alkyne) at 4 hpi and 24 hpi to label the newly transcribing RNA. Total RNA extracted from these

Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

Techniques: Infection, Incubation

Journal: Journal of Virology

Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

doi: 10.1128/JVI.02507-14

Figure Lengend Snippet: KSHV viral transcripts are abundantly present during the primary infection of human PBMCs, CD14+ , and TIVE cells.

Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

Techniques: Infection

Journal: Journal of Virology

Article Title: Transcriptome Analysis of Kaposi's Sarcoma-Associated Herpesvirus during De Novo Primary Infection of Human B and Endothelial Cells

doi: 10.1128/JVI.02507-14

Figure Lengend Snippet: KSHV genome copies exponentially increase after infection. (A) Approximately 8 × 10 7 human PBMCs were infected with KSHV isolated from reactivated TRExBCBL1-RTA, with a multiplicity of infection (MOI) of 10. De novo -infected PBMCs were harvested

Article Snippet: To determine the profiles of the viral transcripts expressed during the primary infection of B and endothelial cells (natural target cells of KSHV), purified virions were used for the de novo infection of human PBMCs, CD14+ monocytes, and endothelial (TIVE) cells.

Techniques: Infection, Isolation