pbmcs  (Roche)


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    Structured Review

    Roche pbmcs
    pDC-mediated suppression of <t>HIV</t> replication in autologous CD4 + T cells. pDCs were purified from HIV-infected individuals and plated overnight at 5×10 4 cells/well with or without 1 µg/ml CpG ODN 2216. For targets, autologous <t>PBMCs</t> were stimulated overnight with anti-CD3 and IL-2. On day 0, CD4 + T cells were purified and plated at 1.2×10 6 cells/well alone or with 5×10 4 stimulated or unstimulated pDCs, or with 1.2×10 5 purified autologous CD8 + T cells. Neutralizing antibody against IFN-α receptor was added at 5 µg/ml to some wells. Culture supernatants were collected at days 3, 5, and 7 and assayed for HIV p24 by ELISA. All data shown are from day 7. A) A representative example of data from a viremic patient. CpG ODN 2216 had no effect on CD4 + T cells alone, and the degree of viral suppression by activated pDCs was comparable to that by CD8 + T cells. B) Effect of pDCs on HIV replication in autologous CD4 + T cells. The degree of HIV suppression by pDCs was expressed as the difference of log HIV p24 production between the culture containing CD4 + T cells alone and the co-culture of CD4 + T cells and pDCs (Δlog p24 = log HIV p24[CD4 cells alone]-log HIV p24[CD4+pDC]). Data are shown for unstimulated pDCs (left panel) and CpG-stimulated pDCs (right panel). Horizontal lines represent median values. n.s., not significant.
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    Images

    1) Product Images from "Impact of HIV on Cell Survival and Antiviral Activity of Plasmacytoid Dendritic Cells"

    Article Title: Impact of HIV on Cell Survival and Antiviral Activity of Plasmacytoid Dendritic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0000458

    pDC-mediated suppression of HIV replication in autologous CD4 + T cells. pDCs were purified from HIV-infected individuals and plated overnight at 5×10 4 cells/well with or without 1 µg/ml CpG ODN 2216. For targets, autologous PBMCs were stimulated overnight with anti-CD3 and IL-2. On day 0, CD4 + T cells were purified and plated at 1.2×10 6 cells/well alone or with 5×10 4 stimulated or unstimulated pDCs, or with 1.2×10 5 purified autologous CD8 + T cells. Neutralizing antibody against IFN-α receptor was added at 5 µg/ml to some wells. Culture supernatants were collected at days 3, 5, and 7 and assayed for HIV p24 by ELISA. All data shown are from day 7. A) A representative example of data from a viremic patient. CpG ODN 2216 had no effect on CD4 + T cells alone, and the degree of viral suppression by activated pDCs was comparable to that by CD8 + T cells. B) Effect of pDCs on HIV replication in autologous CD4 + T cells. The degree of HIV suppression by pDCs was expressed as the difference of log HIV p24 production between the culture containing CD4 + T cells alone and the co-culture of CD4 + T cells and pDCs (Δlog p24 = log HIV p24[CD4 cells alone]-log HIV p24[CD4+pDC]). Data are shown for unstimulated pDCs (left panel) and CpG-stimulated pDCs (right panel). Horizontal lines represent median values. n.s., not significant.
    Figure Legend Snippet: pDC-mediated suppression of HIV replication in autologous CD4 + T cells. pDCs were purified from HIV-infected individuals and plated overnight at 5×10 4 cells/well with or without 1 µg/ml CpG ODN 2216. For targets, autologous PBMCs were stimulated overnight with anti-CD3 and IL-2. On day 0, CD4 + T cells were purified and plated at 1.2×10 6 cells/well alone or with 5×10 4 stimulated or unstimulated pDCs, or with 1.2×10 5 purified autologous CD8 + T cells. Neutralizing antibody against IFN-α receptor was added at 5 µg/ml to some wells. Culture supernatants were collected at days 3, 5, and 7 and assayed for HIV p24 by ELISA. All data shown are from day 7. A) A representative example of data from a viremic patient. CpG ODN 2216 had no effect on CD4 + T cells alone, and the degree of viral suppression by activated pDCs was comparable to that by CD8 + T cells. B) Effect of pDCs on HIV replication in autologous CD4 + T cells. The degree of HIV suppression by pDCs was expressed as the difference of log HIV p24 production between the culture containing CD4 + T cells alone and the co-culture of CD4 + T cells and pDCs (Δlog p24 = log HIV p24[CD4 cells alone]-log HIV p24[CD4+pDC]). Data are shown for unstimulated pDCs (left panel) and CpG-stimulated pDCs (right panel). Horizontal lines represent median values. n.s., not significant.

    Techniques Used: Purification, Infection, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

    pDC killing in HIV-infected individuals. A) Plasmacytoid dendritic cell numbers are reduced in HIV-infected versus uninfected individuals. PBMCs were stained with anti-BDCA-2 (FITC) and anti-CD123 (PE) to measure the levels of pDCs. Horizontal lines represent median values. B) pDCs are susceptible to Fas-mediated apoptosis. Fas was crosslinked on the surface of PBMCs, and resultant pDC apoptosis was measured by Annexin V staining. n.s., not significant. C–E) pDC cell death induced by HIV-infected cells. 2×10 6 CEM cells chronically infected with HIV (CEM-IIIB) were incubated for 8 h with 10 7 PBMCs from HIV-infected donors. The levels of apoptosis and necrosis were assessed by Annexin V and 7-AAD staining. Cells within the pDC (BDCA-2 + /CD123 + ) population that were double-positive for both Annexin V and 7-AAD were considered necrotic, while those positive for Annexin V and negative for 7-AAD were considered apoptotic. In some cultures, 100 µg/ml T20 was added. C) Cells expressing HIV induce pDC death. The percent of pDCs that were necrotic (Annexin V + /7-AAD + ) is indicated on the y-axis. PBMCs alone versus PBMCs+CEM-IIIB are shown. As indicated, CEM-IIIB cells showed a significant induction of cell death vs. cells alone. D) Representative examples of T20-mediated blockade of CEM-IIIB-induced pDC cell death. E) Cell death is blocked by T20. Cells were incubated with CEM cells alone (left panel), CEM-IIIB, or CEM-IIIB+T20, and the percentage of pDCs in the apoptotic or necrotic quadrants was quantified. No cell death above baseline was induced by CEM cells alone. Both types of cell death induced by CEM-IIB cells were significantly reduced (p values indicated) in the presence of T20.
    Figure Legend Snippet: pDC killing in HIV-infected individuals. A) Plasmacytoid dendritic cell numbers are reduced in HIV-infected versus uninfected individuals. PBMCs were stained with anti-BDCA-2 (FITC) and anti-CD123 (PE) to measure the levels of pDCs. Horizontal lines represent median values. B) pDCs are susceptible to Fas-mediated apoptosis. Fas was crosslinked on the surface of PBMCs, and resultant pDC apoptosis was measured by Annexin V staining. n.s., not significant. C–E) pDC cell death induced by HIV-infected cells. 2×10 6 CEM cells chronically infected with HIV (CEM-IIIB) were incubated for 8 h with 10 7 PBMCs from HIV-infected donors. The levels of apoptosis and necrosis were assessed by Annexin V and 7-AAD staining. Cells within the pDC (BDCA-2 + /CD123 + ) population that were double-positive for both Annexin V and 7-AAD were considered necrotic, while those positive for Annexin V and negative for 7-AAD were considered apoptotic. In some cultures, 100 µg/ml T20 was added. C) Cells expressing HIV induce pDC death. The percent of pDCs that were necrotic (Annexin V + /7-AAD + ) is indicated on the y-axis. PBMCs alone versus PBMCs+CEM-IIIB are shown. As indicated, CEM-IIIB cells showed a significant induction of cell death vs. cells alone. D) Representative examples of T20-mediated blockade of CEM-IIIB-induced pDC cell death. E) Cell death is blocked by T20. Cells were incubated with CEM cells alone (left panel), CEM-IIIB, or CEM-IIIB+T20, and the percentage of pDCs in the apoptotic or necrotic quadrants was quantified. No cell death above baseline was induced by CEM cells alone. Both types of cell death induced by CEM-IIB cells were significantly reduced (p values indicated) in the presence of T20.

    Techniques Used: Infection, Staining, Incubation, Expressing

    2) Product Images from "Abnormal telomere shortening of peripheral blood mononuclear cells and granulocytes in patients with chronic idiopathic neutropenia"

    Article Title: Abnormal telomere shortening of peripheral blood mononuclear cells and granulocytes in patients with chronic idiopathic neutropenia

    Journal: Haematologica

    doi: 10.3324/haematol.2011.053983

    Relative telomere length values of PBMCs in CIN patients and age/sex-matched healthy controls. (A-B) Correlation (regression line ±95% confidence limits) between the relative telomere length values of PBMCs and age in the healthy controls and
    Figure Legend Snippet: Relative telomere length values of PBMCs in CIN patients and age/sex-matched healthy controls. (A-B) Correlation (regression line ±95% confidence limits) between the relative telomere length values of PBMCs and age in the healthy controls and

    Techniques Used:

    Relative telomere length values of PBMCs and granulocytes in CIN patients and age- and sex-matched healthy individuals. The bars represent the mean (+ 1SD) relative telomere length in PBMCs and granulocytes of healthy individuals (A) and CIN patients
    Figure Legend Snippet: Relative telomere length values of PBMCs and granulocytes in CIN patients and age- and sex-matched healthy individuals. The bars represent the mean (+ 1SD) relative telomere length in PBMCs and granulocytes of healthy individuals (A) and CIN patients

    Techniques Used:

    3) Product Images from "Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one"

    Article Title: Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one

    Journal: Immunogenetics

    doi: 10.1007/s00251-013-0744-3

    Preparation and sequencing of samples. a) Whole blood from each animal is separated using Ficoll-Paque centrifugation and PBMCs are collected. b) Total RNA is isolated from PBMCs using standard RNA isolation methods and cDNA is synthesized. c) The MHC
    Figure Legend Snippet: Preparation and sequencing of samples. a) Whole blood from each animal is separated using Ficoll-Paque centrifugation and PBMCs are collected. b) Total RNA is isolated from PBMCs using standard RNA isolation methods and cDNA is synthesized. c) The MHC

    Techniques Used: Sequencing, Centrifugation, Isolation, Synthesized

    4) Product Images from "Low autocrine interferon beta production as a gene therapy approach for AIDS: Infusion of interferon beta-engineered lymphocytes in macaques chronically infected with SIVmac251"

    Article Title: Low autocrine interferon beta production as a gene therapy approach for AIDS: Infusion of interferon beta-engineered lymphocytes in macaques chronically infected with SIVmac251

    Journal: Retrovirology

    doi: 10.1186/1742-4690-1-29

    Evolution of immuno-virological parameters in SIVmac251 chronically infected macaques from the control group. Immunological and virological parameters were followed in macaques that received their own cells transduced by the deleted form of the retroviral construct. (A) Absolute number of circulating CD4+ lymphocytes was followed by immunophenotyping and flow cytometry. (B) Cell-associated viral load was estimated in PBMCs by a quantitative PCR method based on the specific amplification of the SIV gag gene. (C) Plasma viral load was estimated by a quantitative branched-DNA method based on the specific amplification of the SIV genome. Y axis split X axis at the first reinfusion date (D0) whereas black arrows indicate the second and third reinfusion dates.
    Figure Legend Snippet: Evolution of immuno-virological parameters in SIVmac251 chronically infected macaques from the control group. Immunological and virological parameters were followed in macaques that received their own cells transduced by the deleted form of the retroviral construct. (A) Absolute number of circulating CD4+ lymphocytes was followed by immunophenotyping and flow cytometry. (B) Cell-associated viral load was estimated in PBMCs by a quantitative PCR method based on the specific amplification of the SIV gag gene. (C) Plasma viral load was estimated by a quantitative branched-DNA method based on the specific amplification of the SIV genome. Y axis split X axis at the first reinfusion date (D0) whereas black arrows indicate the second and third reinfusion dates.

    Techniques Used: Infection, Construct, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Amplification

    Evolution of immuno-virological parameters in SIVmac251 chronically infected macaques from the IFN group. Immunological and virological parameters were followed in macaques that received their own cells transduced by the retroviral construct allowing expression of the biologically active form of IFN-β. (A) Absolute number of circulating CD4+ lymphocytes was followed by immunophenotyping and flow cytometry. (B) Cell-associated viral load was estimated in PBMCs by a quantitative PCR method based on the specific amplification of the SIV gag gene. (C) Plasma viral load was estimated by a quantitative branched-DNA method based on the specific amplification of the SIV genome. Y axis split X axis at the first reinfusion date (D0) whereas black arrows indicate the second and third reinfusion dates.
    Figure Legend Snippet: Evolution of immuno-virological parameters in SIVmac251 chronically infected macaques from the IFN group. Immunological and virological parameters were followed in macaques that received their own cells transduced by the retroviral construct allowing expression of the biologically active form of IFN-β. (A) Absolute number of circulating CD4+ lymphocytes was followed by immunophenotyping and flow cytometry. (B) Cell-associated viral load was estimated in PBMCs by a quantitative PCR method based on the specific amplification of the SIV gag gene. (C) Plasma viral load was estimated by a quantitative branched-DNA method based on the specific amplification of the SIV genome. Y axis split X axis at the first reinfusion date (D0) whereas black arrows indicate the second and third reinfusion dates.

    Techniques Used: Infection, Construct, Expressing, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction, Amplification

    5) Product Images from "Primary Isolates of Human Immunodeficiency Virus Type 1 Are Usually Dominated by the Major Variants Found in Blood ▿"

    Article Title: Primary Isolates of Human Immunodeficiency Virus Type 1 Are Usually Dominated by the Major Variants Found in Blood ▿

    Journal:

    doi: 10.1128/JVI.01035-07

    Changes in viral populations associated with coculture. Samples from three HIV-positive subjects (QA203, QB374, and QA210) were used to establish parallel cocultures with PBMCs from three HIV-negative donors, randomly chosen from four available donors
    Figure Legend Snippet: Changes in viral populations associated with coculture. Samples from three HIV-positive subjects (QA203, QB374, and QA210) were used to establish parallel cocultures with PBMCs from three HIV-negative donors, randomly chosen from four available donors

    Techniques Used:

    Comparison of V1/V2 genotyping of proviral DNAs isolated from infected subjects by GeneScan analysis and by sequencing. PBMC DNAs from eight HIV-infected subjects (QA203 through QD385) were analyzed by both GeneScan analysis and sequencing of single-copy
    Figure Legend Snippet: Comparison of V1/V2 genotyping of proviral DNAs isolated from infected subjects by GeneScan analysis and by sequencing. PBMC DNAs from eight HIV-infected subjects (QA203 through QD385) were analyzed by both GeneScan analysis and sequencing of single-copy

    Techniques Used: Isolation, Infection, Sequencing

    6) Product Images from "Mutations in the selenocysteine insertion sequence-binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans"

    Article Title: Mutations in the selenocysteine insertion sequence-binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI43653

    Abnormal cytokine production from PBMCs. ( A ) IL-6 secretion in response to LPS, Pam2, and Pam3 exposure (24 h), and TNF-α secretion in response to LPS and LPS plus IFN-γ exposure (12 h), were assayed in supernatants of PBMCs from P1 and an age-matched control subject. ( B ) Whole cell lysates from CD14 + monocytes were Western blotted for SELS (arrow), with actin as a loading control. The control subject was age- and sex-matched. ( C ) PBMCs of P1 and an age-matched control subject were stimulated either by PHA or by IL-12 and IL-18, and IFN-γ responses were assayed at 48 h. ( D and E ) Intracellular IFN-γ levels in CD3 + and CD4 + cell populations derived from PBMCs exposed to PMA and ionomycin were determined by FACS analysis (see Methods) in P1 and a group of age- and sex-matched control subjects. Each symbol denotes 1 individual; red symbol denotes P1.
    Figure Legend Snippet: Abnormal cytokine production from PBMCs. ( A ) IL-6 secretion in response to LPS, Pam2, and Pam3 exposure (24 h), and TNF-α secretion in response to LPS and LPS plus IFN-γ exposure (12 h), were assayed in supernatants of PBMCs from P1 and an age-matched control subject. ( B ) Whole cell lysates from CD14 + monocytes were Western blotted for SELS (arrow), with actin as a loading control. The control subject was age- and sex-matched. ( C ) PBMCs of P1 and an age-matched control subject were stimulated either by PHA or by IL-12 and IL-18, and IFN-γ responses were assayed at 48 h. ( D and E ) Intracellular IFN-γ levels in CD3 + and CD4 + cell populations derived from PBMCs exposed to PMA and ionomycin were determined by FACS analysis (see Methods) in P1 and a group of age- and sex-matched control subjects. Each symbol denotes 1 individual; red symbol denotes P1.

    Techniques Used: Western Blot, Derivative Assay, FACS

    7) Product Images from "Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group"

    Article Title: Genotypic testing on HIV-1 DNA as a tool to assess HIV-1 co-receptor usage in clinical practice: results from the DIVA study group

    Journal: Infection

    doi: 10.1007/s15010-013-0510-3

    Rate of successful V3 sequencing. a Rate of successful V3 sequencing based on different levels of total HIV-1 DNA quantification. b Rate of successful V3 sequencing in patients for which DNA was extracted from whole blood ( WB ) or peripheral blood mononuclear cells ( PBMCs ). The quantification of total HIV-DNA is expressed in copies/10 6 cells
    Figure Legend Snippet: Rate of successful V3 sequencing. a Rate of successful V3 sequencing based on different levels of total HIV-1 DNA quantification. b Rate of successful V3 sequencing in patients for which DNA was extracted from whole blood ( WB ) or peripheral blood mononuclear cells ( PBMCs ). The quantification of total HIV-DNA is expressed in copies/10 6 cells

    Techniques Used: Sequencing, Western Blot

    8) Product Images from "A Randomized Double-Blinded, Placebo-Controlled Trial Investigating the Effect of Fish Oil Supplementation on Gene Expression Related to Insulin Action, Blood Lipids, and Inflammation in Gestational Diabetes Mellitus-Fish Oil Supplementation and Gestational Diabetes"

    Article Title: A Randomized Double-Blinded, Placebo-Controlled Trial Investigating the Effect of Fish Oil Supplementation on Gene Expression Related to Insulin Action, Blood Lipids, and Inflammation in Gestational Diabetes Mellitus-Fish Oil Supplementation and Gestational Diabetes

    Journal: Nutrients

    doi: 10.3390/nu10020163

    Effect of 6-week supplementation with omega-3 or placebo on expression ratio of LDLR gene in PBMCs of GDM women.
    Figure Legend Snippet: Effect of 6-week supplementation with omega-3 or placebo on expression ratio of LDLR gene in PBMCs of GDM women.

    Techniques Used: Expressing

    Effect of 6-week supplementation with omega-3 or placebo on expression ratio of IL-1, IL-8, and TNF-α gene in PBMCs of GDM women.
    Figure Legend Snippet: Effect of 6-week supplementation with omega-3 or placebo on expression ratio of IL-1, IL-8, and TNF-α gene in PBMCs of GDM women.

    Techniques Used: Expressing

    Effect of 6-week supplementation with omega-3 or placebo on expression ratio of PPAR-γ gene in PBMCs of GDM women.
    Figure Legend Snippet: Effect of 6-week supplementation with omega-3 or placebo on expression ratio of PPAR-γ gene in PBMCs of GDM women.

    Techniques Used: Expressing

    9) Product Images from "Antiviral activity of Ladania067, an extract from wild black currant leaves against influenza A virus in vitro and in vivo"

    Article Title: Antiviral activity of Ladania067, an extract from wild black currant leaves against influenza A virus in vitro and in vivo

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2014.00171

    Immune cells are not affected by Ladania067 . (A) 3 H-thymidine incorporation assay was performed with fresh human isolated PBMCs. Cells were treated for 6 days with different concentrations of Ladania067 (800, 80, 8, 0.8, 0.08 μg/ml). PWM, (black bar; 40 μg/ml) was used as a non-specific positive control. After incubation cells were pulsed for 16 h with 3 H-thymidine. Stimulation index were calculated by dividing treated cells to spontaneous release. (B) Flow cytometry analysis of different activated immune cell subtypes, CD4 + (B) and CD8 + T lymphocytes (C) , B cells (D) and NK cells (E) after 1 day Ladania067 treatment (120, 12, 1.2, 0.12, 0.012 μg/ml). Values are given as percent of total gated. All results represent three different donors.
    Figure Legend Snippet: Immune cells are not affected by Ladania067 . (A) 3 H-thymidine incorporation assay was performed with fresh human isolated PBMCs. Cells were treated for 6 days with different concentrations of Ladania067 (800, 80, 8, 0.8, 0.08 μg/ml). PWM, (black bar; 40 μg/ml) was used as a non-specific positive control. After incubation cells were pulsed for 16 h with 3 H-thymidine. Stimulation index were calculated by dividing treated cells to spontaneous release. (B) Flow cytometry analysis of different activated immune cell subtypes, CD4 + (B) and CD8 + T lymphocytes (C) , B cells (D) and NK cells (E) after 1 day Ladania067 treatment (120, 12, 1.2, 0.12, 0.012 μg/ml). Values are given as percent of total gated. All results represent three different donors.

    Techniques Used: Thymidine Incorporation Assay, Isolation, Positive Control, Incubation, Flow Cytometry, Cytometry

    Ladania067 treatment shows no cytotoxic effects in vitro . The cytotoxic concentration 50% (CC 50 ) of Ladania067 was determined in MDCK (A) , A549 (B) , HeLa (C) , and PBMCs (D) . Cells were incubated with Ladania067 (0–1 mg/ml) for 24 h. After incubation, cell viability was measured by WST-1 assay regarding manufactory guidelines ( n = 3).
    Figure Legend Snippet: Ladania067 treatment shows no cytotoxic effects in vitro . The cytotoxic concentration 50% (CC 50 ) of Ladania067 was determined in MDCK (A) , A549 (B) , HeLa (C) , and PBMCs (D) . Cells were incubated with Ladania067 (0–1 mg/ml) for 24 h. After incubation, cell viability was measured by WST-1 assay regarding manufactory guidelines ( n = 3).

    Techniques Used: In Vitro, Concentration Assay, Incubation, WST-1 Assay

    10) Product Images from "Porcine circovirus type 2 (PCV2) infection decreases the efficacy of an attenuated classical swine fever virus (CSFV) vaccine"

    Article Title: Porcine circovirus type 2 (PCV2) infection decreases the efficacy of an attenuated classical swine fever virus (CSFV) vaccine

    Journal: Veterinary Research

    doi: 10.1186/1297-9716-42-115

    The effect of porcine circovirus type 2 (PCV2) on the classical swine fever virus (CSFV)-specific cell proliferation reponse of peripheral blood mononuclear cells (PBMCs) in various ex vivo treatments . The PBMCs were collected from pigs that had been vaccinated with Lapinized Philippines Coronel (LPC) vaccine twice, followed by challenge with wild-type CSFV (ALD) 6 weeks later. The PBMCs were pre-inoculated with 0.1, 0.05 or 0.01 MOI of PCV2, 0.1 MOI of UV-inactivated PCV2 or Con A at 5 μg/mL followed by inoculation with 1 MOI of wild-type CSFV 18 h later. This ex vivo CSFV-specific cell proliferation response was done in triplicates and assayed 4 days after wild-type CSFV stimulation by commercial kits as indicated in the material and methods. The values are shown as mean ± SD of five different pigs. a-d Values with different superscripts indicate that the differences among groups are statistically significant ( P
    Figure Legend Snippet: The effect of porcine circovirus type 2 (PCV2) on the classical swine fever virus (CSFV)-specific cell proliferation reponse of peripheral blood mononuclear cells (PBMCs) in various ex vivo treatments . The PBMCs were collected from pigs that had been vaccinated with Lapinized Philippines Coronel (LPC) vaccine twice, followed by challenge with wild-type CSFV (ALD) 6 weeks later. The PBMCs were pre-inoculated with 0.1, 0.05 or 0.01 MOI of PCV2, 0.1 MOI of UV-inactivated PCV2 or Con A at 5 μg/mL followed by inoculation with 1 MOI of wild-type CSFV 18 h later. This ex vivo CSFV-specific cell proliferation response was done in triplicates and assayed 4 days after wild-type CSFV stimulation by commercial kits as indicated in the material and methods. The values are shown as mean ± SD of five different pigs. a-d Values with different superscripts indicate that the differences among groups are statistically significant ( P

    Techniques Used: Ex Vivo

    11) Product Images from "Induction of CD8+ Cells Able To Suppress CCR5-Tropic Simian Immunodeficiency Virus SIVmac239 Replication by Controlled Infection of CXCR4-Tropic Simian-Human Immunodeficiency Virus in Vaccinated Rhesus Macaques ▿"

    Article Title: Induction of CD8+ Cells Able To Suppress CCR5-Tropic Simian Immunodeficiency Virus SIVmac239 Replication by Controlled Infection of CXCR4-Tropic Simian-Human Immunodeficiency Virus in Vaccinated Rhesus Macaques ▿

    Journal:

    doi: 10.1128/JVI.01475-07

    SIVmac239 replication in vitro in the absence or the presence of CD8 + cells in macaques R00-017 (A), R00-020 (B), R00-023 (C), and R00-024 (D). PBMC-derived CD8 − (target) cells infected with SIVmac239 were cultured alone (no CD8) or cocultured
    Figure Legend Snippet: SIVmac239 replication in vitro in the absence or the presence of CD8 + cells in macaques R00-017 (A), R00-020 (B), R00-023 (C), and R00-024 (D). PBMC-derived CD8 − (target) cells infected with SIVmac239 were cultured alone (no CD8) or cocultured

    Techniques Used: In Vitro, Derivative Assay, Infection, Cell Culture

    SIVGP1-specific CD8 + T-cell frequencies in macaques before and after SHIV89.6PD challenge. Frequencies of CD8 + T cells showing SIVGP1-specific IFN-γ induction per total CD8 + T cells in PBMCs are shown. The first time point
    Figure Legend Snippet: SIVGP1-specific CD8 + T-cell frequencies in macaques before and after SHIV89.6PD challenge. Frequencies of CD8 + T cells showing SIVGP1-specific IFN-γ induction per total CD8 + T cells in PBMCs are shown. The first time point

    Techniques Used:

    12) Product Images from "Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection"

    Article Title: Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003885

    NLRP3/NLRP12 containing inflammasomes and caspase-1 activation in PBMCs from P. vivax malaria patients. ( A ) PBMCs derived from P. vivax malaria patients and healthy donors were lysed, cross-linked by treatment with disuccinimidyl suberate [70] , and ASC oligomerization assessed by Western blot analysis. PBMCs from a healthy donor stimulated with LPS and nigericin were used as positive control. . ( B ) NLRP3, NLRP12 and AIM2 containing inflammasomes (specks) in monocytes from P. vivax malaria patients were visualized in a confocal microscope. ( C ) The bar graphs show the frequency of specks in monocytes derived from P. vivax malaria patients. We saw no specks on cells from healthy donors or cells from malaria patients incubated with the secondary antibody only. See also Figure S6 .
    Figure Legend Snippet: NLRP3/NLRP12 containing inflammasomes and caspase-1 activation in PBMCs from P. vivax malaria patients. ( A ) PBMCs derived from P. vivax malaria patients and healthy donors were lysed, cross-linked by treatment with disuccinimidyl suberate [70] , and ASC oligomerization assessed by Western blot analysis. PBMCs from a healthy donor stimulated with LPS and nigericin were used as positive control. . ( B ) NLRP3, NLRP12 and AIM2 containing inflammasomes (specks) in monocytes from P. vivax malaria patients were visualized in a confocal microscope. ( C ) The bar graphs show the frequency of specks in monocytes derived from P. vivax malaria patients. We saw no specks on cells from healthy donors or cells from malaria patients incubated with the secondary antibody only. See also Figure S6 .

    Techniques Used: Activation Assay, Derivative Assay, Western Blot, Positive Control, Microscopy, Incubation

    13) Product Images from "Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8+ responses"

    Article Title: Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8+ responses

    Journal: Journal of Clinical Investigation

    doi:

    Association between HIV-1 epitope–specific ELISPOT responses (see text for definition of an HIV-1 epitope–specific response) and levels of sex work in persistently HIV-1–seronegative Kenyan sex workers. HIV-1–specific ELISPOT responses are shown on the y axis as SFU/10 6 PBMCs. Solid lines represent IFN-γ release in response to HIV-1 CTL peptide epitopes, and the dashed lines represent the IFN-γ response to R10 medium alone. Assay date is shown on the x axis, together with the reported number of daily clients and percentage of condom use. Stopping sex work completely was generally associated with loss of HIV-1–specific ELISPOT responses, demonstrated by ML 1192 ( a ) and ML 851 ( b ), although ML 1732 maintained a response for over a year after stopping sex work ( c ). A temporary break (≥2 months) from sex work was also associated with the loss of HIV-1–specific responses (ML 1437) ( d ), and responses were detected again 1–12 months after resuming sex work in ML 1250 ( e ), ML 1749 ( f ), and ML 851 (before retirement) ( b ). HIV-1–specific responses tended to be maintained in the absence of a break from sex work, shown by ML 1671 ( g ) and ML 887 ( h ).
    Figure Legend Snippet: Association between HIV-1 epitope–specific ELISPOT responses (see text for definition of an HIV-1 epitope–specific response) and levels of sex work in persistently HIV-1–seronegative Kenyan sex workers. HIV-1–specific ELISPOT responses are shown on the y axis as SFU/10 6 PBMCs. Solid lines represent IFN-γ release in response to HIV-1 CTL peptide epitopes, and the dashed lines represent the IFN-γ response to R10 medium alone. Assay date is shown on the x axis, together with the reported number of daily clients and percentage of condom use. Stopping sex work completely was generally associated with loss of HIV-1–specific ELISPOT responses, demonstrated by ML 1192 ( a ) and ML 851 ( b ), although ML 1732 maintained a response for over a year after stopping sex work ( c ). A temporary break (≥2 months) from sex work was also associated with the loss of HIV-1–specific responses (ML 1437) ( d ), and responses were detected again 1–12 months after resuming sex work in ML 1250 ( e ), ML 1749 ( f ), and ML 851 (before retirement) ( b ). HIV-1–specific responses tended to be maintained in the absence of a break from sex work, shown by ML 1671 ( g ) and ML 887 ( h ).

    Techniques Used: Enzyme-linked Immunospot, CTL Assay

    14) Product Images from "A mutation in the human Uncoordinated 119 gene impairs TCR signaling and is associated with CD4 lymphopenia"

    Article Title: A mutation in the human Uncoordinated 119 gene impairs TCR signaling and is associated with CD4 lymphopenia

    Journal: Blood

    doi: 10.1182/blood-2011-04-350686

    Proliferation of patient's (P1) CD4 T cells. PBMCs from the patient P1 and healthy controls (C) were labeled with CFSE and stimulated with plastic-bound anti-CD3 and anti-CD28 Abs for 72 hours. Cells were then stained with an allophycocyanin-labeled anti-CD4
    Figure Legend Snippet: Proliferation of patient's (P1) CD4 T cells. PBMCs from the patient P1 and healthy controls (C) were labeled with CFSE and stimulated with plastic-bound anti-CD3 and anti-CD28 Abs for 72 hours. Cells were then stained with an allophycocyanin-labeled anti-CD4

    Techniques Used: Labeling, Staining

    15) Product Images from "Upregulation of OLR1 and IL17A genes and their association with blood glucose and lipid levels in femoropopliteal artery disease"

    Article Title: Upregulation of OLR1 and IL17A genes and their association with blood glucose and lipid levels in femoropopliteal artery disease

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.4081

    Expression of (A) OLR1 and (B) IL17A mRNA in peripheral blood mononuclear cells of patients with FP artery disease compared with healthy controls. OLR1, oxidized low-density lipoprotein receptor 1; IL17A, interleukin 17A; FP, femoropopliteal.
    Figure Legend Snippet: Expression of (A) OLR1 and (B) IL17A mRNA in peripheral blood mononuclear cells of patients with FP artery disease compared with healthy controls. OLR1, oxidized low-density lipoprotein receptor 1; IL17A, interleukin 17A; FP, femoropopliteal.

    Techniques Used: Expressing

    16) Product Images from "STAT3-Mediated Transcriptional Regulation of Osteopontin in STAT3 Loss-of-Function Related Hyper IgE Syndrome"

    Article Title: STAT3-Mediated Transcriptional Regulation of Osteopontin in STAT3 Loss-of-Function Related Hyper IgE Syndrome

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01080

    (A) Representative flow cytometry histogram of STAT3 protein expression in PC-3 and LNCaP cells. PC-3 cells had no expression of STAT3 protein. (B) STAT3 mRNA expression in PC3, LNCaP, hyper-IgE syndrome (HIES) subjects and healthy controls (HCs) by conventional PCR. PC-3 cells showed no expression of STAT3 mRNA. (C) Agarose gel pictures showing either absence or non-specific/extremely faint bands of STAT3 amplification (exons 9–22) in PC-3 cells. Gel 1 (Exons 9–10/12–24/15 and 16–17), gel 2 (exon 11), and gel 3 (exons 18-20/21/22-23) have been grouped together for representation purpose. (D) Scatter plot showing per cent STAT3 phosphorylation in HCs, HIES subjects, LNCaP, and PC3 cells; mean values are shown as horizontal bars. Bar graphs showing quantitative real time PCR (qRT-PCR) of SOCS3 mRNA expression in HC peripheral blood mononuclear cells stimulated with IL-6 in a (E) time-dependent manner at 30 ng/ml and (F) dose-dependent manner for 2 h. Data are shown as mean ± SD for three independent experiments. (G) Bar graphs showing qRT-PCR results of SOCS3 expression from HCs, HIES subjects, LNCaP, and PC-3 cells stimulated with IL-6. The primary transcript for each gene were assayed at least in duplicate and normalized to β-actin expression. (Data are represented as mean ± SD, * p
    Figure Legend Snippet: (A) Representative flow cytometry histogram of STAT3 protein expression in PC-3 and LNCaP cells. PC-3 cells had no expression of STAT3 protein. (B) STAT3 mRNA expression in PC3, LNCaP, hyper-IgE syndrome (HIES) subjects and healthy controls (HCs) by conventional PCR. PC-3 cells showed no expression of STAT3 mRNA. (C) Agarose gel pictures showing either absence or non-specific/extremely faint bands of STAT3 amplification (exons 9–22) in PC-3 cells. Gel 1 (Exons 9–10/12–24/15 and 16–17), gel 2 (exon 11), and gel 3 (exons 18-20/21/22-23) have been grouped together for representation purpose. (D) Scatter plot showing per cent STAT3 phosphorylation in HCs, HIES subjects, LNCaP, and PC3 cells; mean values are shown as horizontal bars. Bar graphs showing quantitative real time PCR (qRT-PCR) of SOCS3 mRNA expression in HC peripheral blood mononuclear cells stimulated with IL-6 in a (E) time-dependent manner at 30 ng/ml and (F) dose-dependent manner for 2 h. Data are shown as mean ± SD for three independent experiments. (G) Bar graphs showing qRT-PCR results of SOCS3 expression from HCs, HIES subjects, LNCaP, and PC-3 cells stimulated with IL-6. The primary transcript for each gene were assayed at least in duplicate and normalized to β-actin expression. (Data are represented as mean ± SD, * p

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

    17) Product Images from "Relevance of Vitamin D Receptor Target Genes for Monitoring the Vitamin D Responsiveness of Primary Human Cells"

    Article Title: Relevance of Vitamin D Receptor Target Genes for Monitoring the Vitamin D Responsiveness of Primary Human Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0124339

    Range of mRNA expression changes in PBMCs of VitDbol study participants. qPCR was used to determine the change in mRNA expression of 16 most relevant VDR target genes in PBMCs isolated from 4–5 samples of the VitDbol study, where healthy adults were supplemented once with a high vitamin D 3 dose (2,000 μg). Gene expression was measured at days 0, 1 and 2 and fold changes were calculated between days 1 and 0 (1) and days 2 and 0 (2). The top, fine central and bottom lines of the box plots represent the 75th, 50th and 25th percentiles, respectively, of the expression changes and the bars indicate standard deviations. The prominent blue central line within the box plots marks the means. The genes were sorted from highest to lowest induction at day 1.
    Figure Legend Snippet: Range of mRNA expression changes in PBMCs of VitDbol study participants. qPCR was used to determine the change in mRNA expression of 16 most relevant VDR target genes in PBMCs isolated from 4–5 samples of the VitDbol study, where healthy adults were supplemented once with a high vitamin D 3 dose (2,000 μg). Gene expression was measured at days 0, 1 and 2 and fold changes were calculated between days 1 and 0 (1) and days 2 and 0 (2). The top, fine central and bottom lines of the box plots represent the 75th, 50th and 25th percentiles, respectively, of the expression changes and the bars indicate standard deviations. The prominent blue central line within the box plots marks the means. The genes were sorted from highest to lowest induction at day 1.

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Isolation

    18) Product Images from "Cytochrome P450s in human immune cells regulate IL-22 and c-Kit via an AHR feedback loop"

    Article Title: Cytochrome P450s in human immune cells regulate IL-22 and c-Kit via an AHR feedback loop

    Journal: Scientific Reports

    doi: 10.1038/srep44005

    Inhibition of CYP1 activity induces c-Kit on CD3 + PBMCs. Human PBMCs were activated with anti-CD3/28 antibodies and treated with increasing concentrations of the CYP1 inhibitor 1-PP (0.1 nM–10 μM) with or without FICZ (0.5 nM), respectively. ( a and b ) Concentration-dependent induction of c-Kit on CD3 + PBMCs at 48 h with means ± s.e.m. of eight different subjects are shown. *p
    Figure Legend Snippet: Inhibition of CYP1 activity induces c-Kit on CD3 + PBMCs. Human PBMCs were activated with anti-CD3/28 antibodies and treated with increasing concentrations of the CYP1 inhibitor 1-PP (0.1 nM–10 μM) with or without FICZ (0.5 nM), respectively. ( a and b ) Concentration-dependent induction of c-Kit on CD3 + PBMCs at 48 h with means ± s.e.m. of eight different subjects are shown. *p

    Techniques Used: Inhibition, Activity Assay, Concentration Assay

    Inhibition of CYP1 activity regulates AHR pathway related targets in human PBMCs. Human PBMCs were activated with anti-CD3/28 antibodies and treated with the AHR agonist FICZ (0.5 nM), the CYP1 inhibitor 1-PP (1 μM) and the AHR antagonist CH-223191 (3 μM) for 5 days. Inhibition of CYP1 activity increased RNA levels of ( a ) CYP1A1 , ( b ) CYP1B1, ( d ) NQO1 , and the ( e ) KIT genes, and slightly down-regulated ( c ) AHR transcription levels. CYP1 inhibition also increased IL-22, but decreased IL-17 on RNA ( f and g ), and on protein ( h and i ) level. Supplementation with the AHR antagonist inverted CYP1-induced effects. RNA expression levels were analyzed by qRT-PCR. Expression values (C t values) were normalized by the housekeeping gene EF1A and related to the target gene expression in the medium control (ΔΔC t ). Data were expressed as 2 −( ΔΔCt ) indicating relative fold regulation. Boxplots show medians, interquartile ranges and ranges of seven different subjects (*p
    Figure Legend Snippet: Inhibition of CYP1 activity regulates AHR pathway related targets in human PBMCs. Human PBMCs were activated with anti-CD3/28 antibodies and treated with the AHR agonist FICZ (0.5 nM), the CYP1 inhibitor 1-PP (1 μM) and the AHR antagonist CH-223191 (3 μM) for 5 days. Inhibition of CYP1 activity increased RNA levels of ( a ) CYP1A1 , ( b ) CYP1B1, ( d ) NQO1 , and the ( e ) KIT genes, and slightly down-regulated ( c ) AHR transcription levels. CYP1 inhibition also increased IL-22, but decreased IL-17 on RNA ( f and g ), and on protein ( h and i ) level. Supplementation with the AHR antagonist inverted CYP1-induced effects. RNA expression levels were analyzed by qRT-PCR. Expression values (C t values) were normalized by the housekeeping gene EF1A and related to the target gene expression in the medium control (ΔΔC t ). Data were expressed as 2 −( ΔΔCt ) indicating relative fold regulation. Boxplots show medians, interquartile ranges and ranges of seven different subjects (*p

    Techniques Used: Inhibition, Activity Assay, RNA Expression, Quantitative RT-PCR, Expressing

    Inhibition of CYP1 activity amplifies c-Kit and IL-22 in human CD3 + PBMCs. Human PBMCs were activated with anti-CD3/28 antibodies and treated with the AHR agonist FICZ (0.5 nM), the CYP1 inhibitor 1-PP (1 μM or 0.1 μM for c-Kit + /IL-22 + PBMCs) and the AHR antagonist CH-223191 (3 μM) for 5 days. Inhibition of CYP1 activity induced c-Kit on ( a ) CD8 + Tc cells and on ( b ) CD4 + Th cells. ( c and d ) The frequency of CD4 + Th cells that co-express c-Kit and IL-22 was also augmented in the 1-PP and FICZ co-treatment. ( e–g ) Absolute cell numbers of IL-22 + and c-Kit + cells during 1-PP (0.1 μM) and FICZ (0.5 nM) co-treatment. ( h–k ) c-Kit is up-regulated on ( h ) complete CD3 − cells, and in detail on ( i–k ) CD3 − CD56 + and CD3 − CD56 − PBMCs. All reactions were dependent on AHR activation. Boxplots indicate medians, interquartile ranges and ranges of seven different subjects (*p
    Figure Legend Snippet: Inhibition of CYP1 activity amplifies c-Kit and IL-22 in human CD3 + PBMCs. Human PBMCs were activated with anti-CD3/28 antibodies and treated with the AHR agonist FICZ (0.5 nM), the CYP1 inhibitor 1-PP (1 μM or 0.1 μM for c-Kit + /IL-22 + PBMCs) and the AHR antagonist CH-223191 (3 μM) for 5 days. Inhibition of CYP1 activity induced c-Kit on ( a ) CD8 + Tc cells and on ( b ) CD4 + Th cells. ( c and d ) The frequency of CD4 + Th cells that co-express c-Kit and IL-22 was also augmented in the 1-PP and FICZ co-treatment. ( e–g ) Absolute cell numbers of IL-22 + and c-Kit + cells during 1-PP (0.1 μM) and FICZ (0.5 nM) co-treatment. ( h–k ) c-Kit is up-regulated on ( h ) complete CD3 − cells, and in detail on ( i–k ) CD3 − CD56 + and CD3 − CD56 − PBMCs. All reactions were dependent on AHR activation. Boxplots indicate medians, interquartile ranges and ranges of seven different subjects (*p

    Techniques Used: Inhibition, Activity Assay, Activation Assay

    19) Product Images from "Prostaglandin E2–Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti–PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection"

    Article Title: Prostaglandin E2–Induced Immune Exhaustion and Enhancement of Antiviral Effects by Anti–PD-L1 Antibody Combined with COX-2 Inhibitor in Bovine Leukemia Virus Infection

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1900342

    Quantification of EP4 , EP2, and HPGD mRNA expression in cattle infected with BLV. ( A – C ) Quantification of EP4 [BLV (−), n = 7; AL, n = 7; PL, n = 7), EP2 [BLV (−), n = 6; AL, n = 6; PL, n = 7), and HPGD [BLV (−), n = 4; AL, n = 6; PL, n = 6) mRNA expression in PBMCs of BLV-uninfected and BLV-infected cattle by qPCR. ( D and E ) Negative correlation between proviral load or the serum concentration of PGE 2 and HPGD expression in PBMCs of cattle infected with BLV ( n = 12). Each symbol represents relative expression level of each animal; pooled data from several experiments were analyzed by the Steel–Dwass test (A–C). Correlation statistic was analyzed using the Spearman correlation (D and E). BLV (−), BLV-uninfected cattle.
    Figure Legend Snippet: Quantification of EP4 , EP2, and HPGD mRNA expression in cattle infected with BLV. ( A – C ) Quantification of EP4 [BLV (−), n = 7; AL, n = 7; PL, n = 7), EP2 [BLV (−), n = 6; AL, n = 6; PL, n = 7), and HPGD [BLV (−), n = 4; AL, n = 6; PL, n = 6) mRNA expression in PBMCs of BLV-uninfected and BLV-infected cattle by qPCR. ( D and E ) Negative correlation between proviral load or the serum concentration of PGE 2 and HPGD expression in PBMCs of cattle infected with BLV ( n = 12). Each symbol represents relative expression level of each animal; pooled data from several experiments were analyzed by the Steel–Dwass test (A–C). Correlation statistic was analyzed using the Spearman correlation (D and E). BLV (−), BLV-uninfected cattle.

    Techniques Used: Expressing, Infection, Real-time Polymerase Chain Reaction, Concentration Assay

    Activation of BLV-specific responses by the combination of meloxicam and anti–PD-L1 Ab. ( A – D ) PBMCs from BLV-infected cattle (A–C, n = 12; D, n = 8) were cultured with meloxicam in the presence of FLK–BLV. CD4 + (A) and CD8 + (B) cell proliferations were assayed by flow cytometry. IFN-γ (C) and TNF-α (D) productions were determined by ELISA. ( E – G ) PBMCs from BLV-infected cattle were incubated with meloxicam and anti–PD-L1 mAb in the presence of FLK or FLK–BLV. CD4 + (E: FLK, n = 7; FLK–BLV, n = 15) and CD8 + (F: FLK, n = 8; FLK–BLV, n = 24) cell proliferations were assayed by flow cytometry. IFN-γ production was determined by ELISA (G, FLK, n = 7; FLK–BLV, n = 20). ( H – J ) PBMCs from BLV-infected cattle were incubated with meloxicam and anti–PD-L1 chAb in the presence of FLK or FLK–BLV (H and I, n = 16; J, n = 18). CD4 + (H) and CD8 + (I) cell proliferations were assayed by flow cytometry. IFN-γ production was determined by ELISA (J). Symbols represent mean from single-culture wells of different individuals; pooled data from several experiments were analyzed by the Wilcoxon signed-rank test (A–D) and the Steel­–Dwass test (E–J). Cont Ig, rat IgG (for E–G), or bovine IgG (for H–J); αPD-L1 chAb, anti–PD-L1 chAb (Boch4G12); αPD-L1 mAb: anti–PD-L1 mAb (4G12).
    Figure Legend Snippet: Activation of BLV-specific responses by the combination of meloxicam and anti–PD-L1 Ab. ( A – D ) PBMCs from BLV-infected cattle (A–C, n = 12; D, n = 8) were cultured with meloxicam in the presence of FLK–BLV. CD4 + (A) and CD8 + (B) cell proliferations were assayed by flow cytometry. IFN-γ (C) and TNF-α (D) productions were determined by ELISA. ( E – G ) PBMCs from BLV-infected cattle were incubated with meloxicam and anti–PD-L1 mAb in the presence of FLK or FLK–BLV. CD4 + (E: FLK, n = 7; FLK–BLV, n = 15) and CD8 + (F: FLK, n = 8; FLK–BLV, n = 24) cell proliferations were assayed by flow cytometry. IFN-γ production was determined by ELISA (G, FLK, n = 7; FLK–BLV, n = 20). ( H – J ) PBMCs from BLV-infected cattle were incubated with meloxicam and anti–PD-L1 chAb in the presence of FLK or FLK–BLV (H and I, n = 16; J, n = 18). CD4 + (H) and CD8 + (I) cell proliferations were assayed by flow cytometry. IFN-γ production was determined by ELISA (J). Symbols represent mean from single-culture wells of different individuals; pooled data from several experiments were analyzed by the Wilcoxon signed-rank test (A–D) and the Steel­–Dwass test (E–J). Cont Ig, rat IgG (for E–G), or bovine IgG (for H–J); αPD-L1 chAb, anti–PD-L1 chAb (Boch4G12); αPD-L1 mAb: anti–PD-L1 mAb (4G12).

    Techniques Used: Activation Assay, Infection, Cell Culture, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Incubation

    Upregulation on the gene expression of gp51 , G4 , and R3 by PGE 2 . ( A – F ) PBMCs from BLV-infected cattle were incubated with PGE 2 , EP2 agonist, or EP4 agonist, and the gene expression of gp51 , G4 , and R3 was quantitated by qPCR (A–C, n = 6; D–F, n = 9). Symbols represent mean from single-culture wells of different individuals; pooled data from several experiments were analyzed by the Wilcoxon signed-rank test (A–C) and the Steel–Dwass test (D–F).
    Figure Legend Snippet: Upregulation on the gene expression of gp51 , G4 , and R3 by PGE 2 . ( A – F ) PBMCs from BLV-infected cattle were incubated with PGE 2 , EP2 agonist, or EP4 agonist, and the gene expression of gp51 , G4 , and R3 was quantitated by qPCR (A–C, n = 6; D–F, n = 9). Symbols represent mean from single-culture wells of different individuals; pooled data from several experiments were analyzed by the Wilcoxon signed-rank test (A–C) and the Steel–Dwass test (D–F).

    Techniques Used: Expressing, Infection, Incubation, Real-time Polymerase Chain Reaction

    Kinetic analysis of PGE 2 in cattle infected with BLV. ( A ) Plasma PGE 2 concentration in cattle uninfected ( n = 6) or infected with BLV (AL, n = 7; PL, n = 6) were determined using ELISA. ( B – E ) Positive correlation between the lymphocyte number ( n = 13), proviral load (C: Japanese black cattle and first filial of Japanese black/Holstein cattle, n = 95; D: Holstein cattle, n = 30) or the percentages of PD-L1 + cells in IgM + cells and plasma PGE 2 concentration in cattle infected with BLV. ( F – H ) Quantification of COX2 mRNA expression in PBMCs [BLV (−), n = 7; AL, n = 7; PL, n = 7] and subpopulations of CD21 + [BLV (−), n = 4; AL, n = 7; PL, n = 6) and CD14 + [BLV (−), n = 4; AL, n = 5; PL, n = 6) cells derived from BLV-uninfected and BLV-infected cattle by qPCR. ( I ) Quantitation of PGE 2 production in PBMCs, CD21 + cells, and CD14 + cells by ELISA ( n = 8). Each symbol represents PGE 2 concentration or relative expression level of each animal; pooled data from several experiments were analyzed by the Steel–Dwass test (A and F–I). Correlation statistic was analyzed using the Spearman correlation (B–E). BLV (−), BLV-uninfected cattle.
    Figure Legend Snippet: Kinetic analysis of PGE 2 in cattle infected with BLV. ( A ) Plasma PGE 2 concentration in cattle uninfected ( n = 6) or infected with BLV (AL, n = 7; PL, n = 6) were determined using ELISA. ( B – E ) Positive correlation between the lymphocyte number ( n = 13), proviral load (C: Japanese black cattle and first filial of Japanese black/Holstein cattle, n = 95; D: Holstein cattle, n = 30) or the percentages of PD-L1 + cells in IgM + cells and plasma PGE 2 concentration in cattle infected with BLV. ( F – H ) Quantification of COX2 mRNA expression in PBMCs [BLV (−), n = 7; AL, n = 7; PL, n = 7] and subpopulations of CD21 + [BLV (−), n = 4; AL, n = 7; PL, n = 6) and CD14 + [BLV (−), n = 4; AL, n = 5; PL, n = 6) cells derived from BLV-uninfected and BLV-infected cattle by qPCR. ( I ) Quantitation of PGE 2 production in PBMCs, CD21 + cells, and CD14 + cells by ELISA ( n = 8). Each symbol represents PGE 2 concentration or relative expression level of each animal; pooled data from several experiments were analyzed by the Steel–Dwass test (A and F–I). Correlation statistic was analyzed using the Spearman correlation (B–E). BLV (−), BLV-uninfected cattle.

    Techniques Used: Infection, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Derivative Assay, Real-time Polymerase Chain Reaction, Quantitation Assay

    20) Product Images from "Dipeptidyl Peptidase 2 apoptosis assay determines the B-cell activation stage and predicts prognosis in chronic lymphocytic leukemia"

    Article Title: Dipeptidyl Peptidase 2 apoptosis assay determines the B-cell activation stage and predicts prognosis in chronic lymphocytic leukemia

    Journal: Experimental hematology

    doi: 10.1016/j.exphem.2010.08.008

    Identification of two subsets of CLL – sensitive and resistant to DPP2 inhibition-induced apoptosis. PBMCs from CLL patients were incubated with either 10 µM/L VbP or 30 µM/L AX8819 for 16 h, stained with CD19, PI and Annexin and
    Figure Legend Snippet: Identification of two subsets of CLL – sensitive and resistant to DPP2 inhibition-induced apoptosis. PBMCs from CLL patients were incubated with either 10 µM/L VbP or 30 µM/L AX8819 for 16 h, stained with CD19, PI and Annexin and

    Techniques Used: Inhibition, Incubation, Staining

    21) Product Images from "Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma"

    Article Title: Chromatin structure, transcriptional activity and DNA repair efficiency affect the outcome of chemotherapy in multiple myeloma

    Journal: British Journal of Cancer

    doi: 10.1038/bjc.2014.410

    The induction of the apoptotic pathway in BMCPs and PBMCs following melphalan treatment. Vertical bar charts show mRNA synthesis in malignant BMPCs ( A ) and in PBMCs ( B ). R=responders; NR=non-responders; H=healthy controls. ( C ) Western blots show the accumulation of S15P-p53 from one healthy volunteer (HV), one MM patient responder and one MM patient non-responder, in PBMCs representative of both tissues analysed. β -Actin was used as normalisation/loading control. Box plots showing the statistical distribution of the lowest concentration of melphalan required to induce ( D ) detectable levels of S15P-p53 and ( F ) detectable change in the levels of apoptosis (defined as a three-fold increase relative to controls) are presented. The horizontal lines within the boxes represent the median value and the vertical lines extending above and below the box indicate maximum and minimum values, respectively. ( E ) The correlation between the lowest concentration of melphalan required to induce detectable levels of S15P-p53 in malignant BMPCs and in PBMCs from the same patients. ( G ) The correlation between the lowest concentration of melphalan required to induce detectable change in the levels of apoptosis in malignant BMPCs and in PBMCs from the same patients. Finally, the correlation between the lowest melphalan concentrations inducing phosphorylation of p53 at serine 15 and those inducing apoptosis in malignant BMPCs ( H ) and in PBMCs ( I ) are presented. The lines represent the theoretical line for a perfect correlation between the two sets of data.
    Figure Legend Snippet: The induction of the apoptotic pathway in BMCPs and PBMCs following melphalan treatment. Vertical bar charts show mRNA synthesis in malignant BMPCs ( A ) and in PBMCs ( B ). R=responders; NR=non-responders; H=healthy controls. ( C ) Western blots show the accumulation of S15P-p53 from one healthy volunteer (HV), one MM patient responder and one MM patient non-responder, in PBMCs representative of both tissues analysed. β -Actin was used as normalisation/loading control. Box plots showing the statistical distribution of the lowest concentration of melphalan required to induce ( D ) detectable levels of S15P-p53 and ( F ) detectable change in the levels of apoptosis (defined as a three-fold increase relative to controls) are presented. The horizontal lines within the boxes represent the median value and the vertical lines extending above and below the box indicate maximum and minimum values, respectively. ( E ) The correlation between the lowest concentration of melphalan required to induce detectable levels of S15P-p53 in malignant BMPCs and in PBMCs from the same patients. ( G ) The correlation between the lowest concentration of melphalan required to induce detectable change in the levels of apoptosis in malignant BMPCs and in PBMCs from the same patients. Finally, the correlation between the lowest melphalan concentrations inducing phosphorylation of p53 at serine 15 and those inducing apoptosis in malignant BMPCs ( H ) and in PBMCs ( I ) are presented. The lines represent the theoretical line for a perfect correlation between the two sets of data.

    Techniques Used: Western Blot, Concentration Assay

    Region-specific repair of melphalan-induced damage along the active N-ras and p53 genes in PBMCs. Box plots are showing the statistical distribution of melphalan-induced N- alkylpurine levels in healthy volunteers (HV) and MM patients, responders (R) and non-responders (NR), in different regions of the TS ( A , D ) and the NTS ( B , E ) of the N-ras ( A , B ) and p53 ( D , E ) genes. The horizontal lines within the boxes represent the median value and the vertical lines extending above and below the boxes indicate maximum and minimum values, respectively. Correlation between DNA damage in malignant BMPCs and DNA damage in PBMCs from the same patients in the N-ras ( C ) and p53 ( F ) genes. Abbreviations: melph=melphalan; nucl=nucleotides.
    Figure Legend Snippet: Region-specific repair of melphalan-induced damage along the active N-ras and p53 genes in PBMCs. Box plots are showing the statistical distribution of melphalan-induced N- alkylpurine levels in healthy volunteers (HV) and MM patients, responders (R) and non-responders (NR), in different regions of the TS ( A , D ) and the NTS ( B , E ) of the N-ras ( A , B ) and p53 ( D , E ) genes. The horizontal lines within the boxes represent the median value and the vertical lines extending above and below the boxes indicate maximum and minimum values, respectively. Correlation between DNA damage in malignant BMPCs and DNA damage in PBMCs from the same patients in the N-ras ( C ) and p53 ( F ) genes. Abbreviations: melph=melphalan; nucl=nucleotides.

    Techniques Used:

    Transcriptional activity and chromatin condensation in untreated primary cells. Representative autoradiograms showing RNA slot blots ( A ) in malignant BMPCs and ( B ) in PBMCs from one healthy volunteer (HV), one MM patient responder (R) and one non-responder (NR) to melphalan therapy. β -Actin was used as normalisation/loading control. ( C ) The gene loci where chromatin condensation and region-specific repair were evaluated. Black boxes, transcribed regions of the genes. ( D ) Box plots showing statistical distribution of the steady-state RNA levels in non-responders as a ratio to that of responders in the N-ras , p53 and d-globin genes, in the untreated PBMCs and BMPCs. The horizontal lines within the boxes represent the median value and the vertical lines extending above and below the boxes indicate maximum and minimum values, respectively. Representative autoradiograms showing chromatin condensation in the malignant BMPCs along the active N-ras ( E ) and p53 ( F ) genes in all subjects, the active d-globin gene in 11 out of 15 MM patients ( G ) and the silent d-globin gene in 4 out of 15 MM patients ( H ). Moreover, representative autoradiograms showing chromatin condensation in the malignant BMPCs from MM patients responders and non-responders in the FN4 fragment of the active N-ras ( I ), in the FP7 of the active p53 ( J ), in the FD3 of the active d-globin ( K ) and in the FD3 fragment of the silent d-globin genes ( L ). Symbols M, D, Tri and Tet represent the positions of nucleosome monomer, dimmer, trimer and tetramer, respectively.
    Figure Legend Snippet: Transcriptional activity and chromatin condensation in untreated primary cells. Representative autoradiograms showing RNA slot blots ( A ) in malignant BMPCs and ( B ) in PBMCs from one healthy volunteer (HV), one MM patient responder (R) and one non-responder (NR) to melphalan therapy. β -Actin was used as normalisation/loading control. ( C ) The gene loci where chromatin condensation and region-specific repair were evaluated. Black boxes, transcribed regions of the genes. ( D ) Box plots showing statistical distribution of the steady-state RNA levels in non-responders as a ratio to that of responders in the N-ras , p53 and d-globin genes, in the untreated PBMCs and BMPCs. The horizontal lines within the boxes represent the median value and the vertical lines extending above and below the boxes indicate maximum and minimum values, respectively. Representative autoradiograms showing chromatin condensation in the malignant BMPCs along the active N-ras ( E ) and p53 ( F ) genes in all subjects, the active d-globin gene in 11 out of 15 MM patients ( G ) and the silent d-globin gene in 4 out of 15 MM patients ( H ). Moreover, representative autoradiograms showing chromatin condensation in the malignant BMPCs from MM patients responders and non-responders in the FN4 fragment of the active N-ras ( I ), in the FP7 of the active p53 ( J ), in the FD3 of the active d-globin ( K ) and in the FD3 fragment of the silent d-globin genes ( L ). Symbols M, D, Tri and Tet represent the positions of nucleosome monomer, dimmer, trimer and tetramer, respectively.

    Techniques Used: Activity Assay

    22) Product Images from "STING-dependent sensing of self-DNA drives silica-induced lung inflammation"

    Article Title: STING-dependent sensing of self-DNA drives silica-induced lung inflammation

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07425-1

    Circulating DNA and CXCL10 in the airways of silicosis patients, STING, and type I IFN pathway activation in ILD patient lungs. a , b Presence of ( a ) dsDNA in the plasma and ( b ) CXCL10 in the sputum of patients with silicosis (ILO 5–12, see Table 1 ), as compared with healthy individuals (ILO 0). c – f Human lung tissue samples of ILD patients treated or not with cortisone (see Table 2 ). c Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 50 µm. Bars, 20 µm for zoomed regions. d Immunoblots of phosphorylated-STING (p-STING), STING, TBK1, phospho-TBK1, IRF3, phospho-IRF3, and β-actin. STING dimers quantification. e CXCL10 levels. f Correlation between STING dimers and epithelial damage scored on human histological tissue sections for necrotic cells, desquamation or denudation, and flattening of the epithelial barrier (indicated by arrows). Control lung tissue (patient G) and fibrotic area (patient D). Bars, 100 µm. g – n Human PBMCs were stimulated with silica microparticles (250 µg/mL) for 18 h, transfected with c-di-AMP (6 µg/mL) or unstimulated. g Extracellular dsDNA in cell supernatant, h IFNβ transcript, i correlation between IFNβ transcripts and released dsDNA. ( j ) CXCL10. ( k ) Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 5 µm. l Immunoblots of phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, and β-actin. m Flow cytometry annexin V/PI analysis gated on singlet cells. n Correlations between dead cells and extracellular dsDNA or IFNβ transcripts. * p
    Figure Legend Snippet: Circulating DNA and CXCL10 in the airways of silicosis patients, STING, and type I IFN pathway activation in ILD patient lungs. a , b Presence of ( a ) dsDNA in the plasma and ( b ) CXCL10 in the sputum of patients with silicosis (ILO 5–12, see Table 1 ), as compared with healthy individuals (ILO 0). c – f Human lung tissue samples of ILD patients treated or not with cortisone (see Table 2 ). c Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 50 µm. Bars, 20 µm for zoomed regions. d Immunoblots of phosphorylated-STING (p-STING), STING, TBK1, phospho-TBK1, IRF3, phospho-IRF3, and β-actin. STING dimers quantification. e CXCL10 levels. f Correlation between STING dimers and epithelial damage scored on human histological tissue sections for necrotic cells, desquamation or denudation, and flattening of the epithelial barrier (indicated by arrows). Control lung tissue (patient G) and fibrotic area (patient D). Bars, 100 µm. g – n Human PBMCs were stimulated with silica microparticles (250 µg/mL) for 18 h, transfected with c-di-AMP (6 µg/mL) or unstimulated. g Extracellular dsDNA in cell supernatant, h IFNβ transcript, i correlation between IFNβ transcripts and released dsDNA. ( j ) CXCL10. ( k ) Confocal images of DNA dye Draq5 (cyan) and STING (red). Bars, 5 µm. l Immunoblots of phospho-STING, STING, phospho-TBK1, TBK1, phospho-IRF3, IRF3, and β-actin. m Flow cytometry annexin V/PI analysis gated on singlet cells. n Correlations between dead cells and extracellular dsDNA or IFNβ transcripts. * p

    Techniques Used: Activation Assay, Western Blot, Transfection, Flow Cytometry, Cytometry

    23) Product Images from "Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection"

    Article Title: Malaria-Induced NLRP12/NLRP3-Dependent Caspase-1 Activation Mediates Inflammation and Hypersensitivity to Bacterial Superinfection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003885

    NLRP3/NLRP12 containing inflammasomes and caspase-1 activation in PBMCs from P. vivax malaria patients. ( A ) PBMCs derived from P. vivax malaria patients and healthy donors were lysed, cross-linked by treatment with disuccinimidyl suberate [70] , and ASC oligomerization assessed by Western blot analysis. PBMCs from a healthy donor stimulated with LPS and nigericin were used as positive control. . ( B ) NLRP3, NLRP12 and AIM2 containing inflammasomes (specks) in monocytes from P. vivax malaria patients were visualized in a confocal microscope. ( C ) The bar graphs show the frequency of specks in monocytes derived from P. vivax malaria patients. We saw no specks on cells from healthy donors or cells from malaria patients incubated with the secondary antibody only. See also Figure S6 .
    Figure Legend Snippet: NLRP3/NLRP12 containing inflammasomes and caspase-1 activation in PBMCs from P. vivax malaria patients. ( A ) PBMCs derived from P. vivax malaria patients and healthy donors were lysed, cross-linked by treatment with disuccinimidyl suberate [70] , and ASC oligomerization assessed by Western blot analysis. PBMCs from a healthy donor stimulated with LPS and nigericin were used as positive control. . ( B ) NLRP3, NLRP12 and AIM2 containing inflammasomes (specks) in monocytes from P. vivax malaria patients were visualized in a confocal microscope. ( C ) The bar graphs show the frequency of specks in monocytes derived from P. vivax malaria patients. We saw no specks on cells from healthy donors or cells from malaria patients incubated with the secondary antibody only. See also Figure S6 .

    Techniques Used: Activation Assay, Derivative Assay, Western Blot, Positive Control, Microscopy, Incubation

    24) Product Images from "Functions, structure, and read-through alternative splicing of feline APOBEC3 genes"

    Article Title: Functions, structure, and read-through alternative splicing of feline APOBEC3 genes

    Journal: Genome Biology

    doi: 10.1186/gb-2008-9-3-r48

    Expression analysis of feline A3C, A3H and A3CH. (a,b) Analysis of expression of feline A3C, A3H and A3CH by RT-PCR of total RNA from feline cell lines (CrFK, MYA-1, KE-R) and feline PBMCs (a) and expression of A3CH in PBMCs of lion, puma, Sumatra-tiger (tiger), and lynx (b). H 2 O indicates PCRs using primers specific for the A3s without template cDNA added. (c) Analysis of expression of cat A3CH by immunoblot using rabbit serum against the sequence flanked by the C- and H-domains in cat A3C (linker) using 293T cells transfected with A3 expression plasmid or empty vector as indicated and CrFK and MyA-1 cells (two independent cultures each).
    Figure Legend Snippet: Expression analysis of feline A3C, A3H and A3CH. (a,b) Analysis of expression of feline A3C, A3H and A3CH by RT-PCR of total RNA from feline cell lines (CrFK, MYA-1, KE-R) and feline PBMCs (a) and expression of A3CH in PBMCs of lion, puma, Sumatra-tiger (tiger), and lynx (b). H 2 O indicates PCRs using primers specific for the A3s without template cDNA added. (c) Analysis of expression of cat A3CH by immunoblot using rabbit serum against the sequence flanked by the C- and H-domains in cat A3C (linker) using 293T cells transfected with A3 expression plasmid or empty vector as indicated and CrFK and MyA-1 cells (two independent cultures each).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Plasmid Preparation

    25) Product Images from "Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein"

    Article Title: Lymphocryptovirus Infection of Nonhuman Primate B Cells Converts Destructive into Productive Processing of the Pathogenic CD8 T Cell Epitope in Myelin Oligodendrocyte Glycoprotein

    Journal: The Journal of Immunology Author Choice

    doi: 10.4049/jimmunol.1600124

    qPCR of cathepsin transcripts in MNC subsets from rhesus monkey and marmosets. Total RNA was extracted from rhesus monkey ( A ) and marmoset ( B ) PBMCs and spleen MNCs. Also, CD20 + and CD20 − fractions were isolated from rhesus monkey samples. cDNA
    Figure Legend Snippet: qPCR of cathepsin transcripts in MNC subsets from rhesus monkey and marmosets. Total RNA was extracted from rhesus monkey ( A ) and marmoset ( B ) PBMCs and spleen MNCs. Also, CD20 + and CD20 − fractions were isolated from rhesus monkey samples. cDNA

    Techniques Used: Real-time Polymerase Chain Reaction, Isolation

    RNA sequence data. High-throughput Illumina single-end sequencing was performed on three replicates of BLC, CD20 + , CD20 − , and PBMC samples. ( A ) PCA was done on the samples, and the first and second components are depicted as a scatter plot. (
    Figure Legend Snippet: RNA sequence data. High-throughput Illumina single-end sequencing was performed on three replicates of BLC, CD20 + , CD20 − , and PBMC samples. ( A ) PCA was done on the samples, and the first and second components are depicted as a scatter plot. (

    Techniques Used: Sequencing, High Throughput Screening Assay

    26) Product Images from "Expression of HERV-Fc1, a Human Endogenous Retrovirus, Is Increased in Patients with Active Multiple Sclerosis"

    Article Title: Expression of HERV-Fc1, a Human Endogenous Retrovirus, Is Increased in Patients with Active Multiple Sclerosis

    Journal: Journal of Virology

    doi: 10.1128/JVI.06723-11

    Sensitivity and specificity evaluation of the SYBR green assay for HERV-Fc1 DNA copy number detection in PBMCs and detection of extracellular viral RNA in plasma.
    Figure Legend Snippet: Sensitivity and specificity evaluation of the SYBR green assay for HERV-Fc1 DNA copy number detection in PBMCs and detection of extracellular viral RNA in plasma.

    Techniques Used: SYBR Green Assay

    Sensitivity and specificity evaluation of the SYBR green assay for HERV-Fc1 DNA copy number detection in PBMCs and detection of extracellular viral RNA in plasma.
    Figure Legend Snippet: Sensitivity and specificity evaluation of the SYBR green assay for HERV-Fc1 DNA copy number detection in PBMCs and detection of extracellular viral RNA in plasma.

    Techniques Used: SYBR Green Assay

    27) Product Images from "Global Gene Transcriptome Analysis in Vaccinated Cattle Revealed a Dominant Role of IL-22 for Protection against Bovine Tuberculosis"

    Article Title: Global Gene Transcriptome Analysis in Vaccinated Cattle Revealed a Dominant Role of IL-22 for Protection against Bovine Tuberculosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003077

    BCG-vaccinated and control cattle samples mapped to ifn-γ and il-22 genes. Visualization by IGB of RNA sequencing reads of representative PPD-B stimulated PBMC from vaccinated-protected, vaccinate-un-protected and non-vaccinated control cattle. Y-axis shows the number of reads covering each base along the transcript in RPKM expression values for each sample. Black track: unvaccinated control cattle; red track: vaccinated/un-protected cattle and blue track: vaccinated/protected cattle. The schematic representation of transcript for (A) ifn-γ and (B) il-22 is show in green at the bottom of the each figure; the boxes show the exons of the gene.
    Figure Legend Snippet: BCG-vaccinated and control cattle samples mapped to ifn-γ and il-22 genes. Visualization by IGB of RNA sequencing reads of representative PPD-B stimulated PBMC from vaccinated-protected, vaccinate-un-protected and non-vaccinated control cattle. Y-axis shows the number of reads covering each base along the transcript in RPKM expression values for each sample. Black track: unvaccinated control cattle; red track: vaccinated/un-protected cattle and blue track: vaccinated/protected cattle. The schematic representation of transcript for (A) ifn-γ and (B) il-22 is show in green at the bottom of the each figure; the boxes show the exons of the gene.

    Techniques Used: RNA Sequencing Assay, Expressing

    Results of RNA-Seq analysis. A. Signficantly modulated genes in the three treatment groups. B. Venn diagrams of genes significantly up-regulated and (C) down-regulated genes after vaccination but prior to M. bovis challenge. A. Fold change compared to unstimulated PBMC (medium controls) of PPD-B stimulated PBMC compared to medium controls from unvaccinated, naïve calves (group 1), vaccinated/non-protected (group 2), and vaccinated/protected calves (group 3).
    Figure Legend Snippet: Results of RNA-Seq analysis. A. Signficantly modulated genes in the three treatment groups. B. Venn diagrams of genes significantly up-regulated and (C) down-regulated genes after vaccination but prior to M. bovis challenge. A. Fold change compared to unstimulated PBMC (medium controls) of PPD-B stimulated PBMC compared to medium controls from unvaccinated, naïve calves (group 1), vaccinated/non-protected (group 2), and vaccinated/protected calves (group 3).

    Techniques Used: RNA Sequencing Assay

    Protection and in vitro IFN-γ responses prior to challenge. A. Individual pathology scores are shown for the animals used in this study. Naïve animals = no vaccination, Vacc/NP = vaccinated calves that were not protected; Vacc/P = vaccinated calves that were protected. B. Correlation of IFN-γ protein production in culture supernatants measured by Bovigam ELISA (y-axis) and ifn-γ gene expression as determined by deep sequencing (x-axis). Data are shown from PPD-B stimulated PBMC from individual animals. Supernatants and RNA were prepared after 24 h culture.
    Figure Legend Snippet: Protection and in vitro IFN-γ responses prior to challenge. A. Individual pathology scores are shown for the animals used in this study. Naïve animals = no vaccination, Vacc/NP = vaccinated calves that were not protected; Vacc/P = vaccinated calves that were protected. B. Correlation of IFN-γ protein production in culture supernatants measured by Bovigam ELISA (y-axis) and ifn-γ gene expression as determined by deep sequencing (x-axis). Data are shown from PPD-B stimulated PBMC from individual animals. Supernatants and RNA were prepared after 24 h culture.

    Techniques Used: In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Sequencing

    Gene expression in PPDB-stimulated PBMC from BCG vaccinated and control cattle. A. Protective efficacy after M. bovis challenge determined by pathology scoring. Results are expressed as total pathology scores [5] . Filled circles, unvaccinated control calves; open triangles, BCG vaccinated calves. (B, C). Transcription of the genes expressing IFN-γ (B) and IL-22 (C) following in vitro stimulation. PBMC were collected from BCG vaccinated (filled symbols) and controls (open symbols) before challenge (week -1) and after challenge with M. bovis at weeks 2, 4 and 8, and stimulated with PPD-B for 24 hours. cDNA was prepared and gene expression determined by RT-qPCR. Data are expressed as log10 relative expression levels compared to non-stimulated cells. Statistical analysis: 2-way ANOVA with Bonferroni post test, * P
    Figure Legend Snippet: Gene expression in PPDB-stimulated PBMC from BCG vaccinated and control cattle. A. Protective efficacy after M. bovis challenge determined by pathology scoring. Results are expressed as total pathology scores [5] . Filled circles, unvaccinated control calves; open triangles, BCG vaccinated calves. (B, C). Transcription of the genes expressing IFN-γ (B) and IL-22 (C) following in vitro stimulation. PBMC were collected from BCG vaccinated (filled symbols) and controls (open symbols) before challenge (week -1) and after challenge with M. bovis at weeks 2, 4 and 8, and stimulated with PPD-B for 24 hours. cDNA was prepared and gene expression determined by RT-qPCR. Data are expressed as log10 relative expression levels compared to non-stimulated cells. Statistical analysis: 2-way ANOVA with Bonferroni post test, * P

    Techniques Used: Expressing, In Vitro, Quantitative RT-PCR

    28) Product Images from "Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome"

    Article Title: Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome

    Journal: Journal of Medical Genetics

    doi: 10.1136/jmg.2009.067553

    Complex I assembly is altered in patient PBMC. Mitochondria were isolated from PBMC of wild-type (A/A), carriers (A/C) and a patient (C/C) donors, solubilised in 1% Triton X-100, and subjected to BN-PAGE and Western immunoblot analysis and quantified. Complex I detected by an antibody to the 39 kDa subunit (NDUFA9) or ND1 subunit is at approximately 880 kDa, and complex II detected by an antibody to the 70 kDa subunit is at approximately 130 kDa. (A) After 10–30 s exposure time. (B) Histogram of A showing the density volumes of complex I normalised by complex II as percentage of the wild-type (IV:10) donor, with error bars representing the SEM of three or four gels. *p
    Figure Legend Snippet: Complex I assembly is altered in patient PBMC. Mitochondria were isolated from PBMC of wild-type (A/A), carriers (A/C) and a patient (C/C) donors, solubilised in 1% Triton X-100, and subjected to BN-PAGE and Western immunoblot analysis and quantified. Complex I detected by an antibody to the 39 kDa subunit (NDUFA9) or ND1 subunit is at approximately 880 kDa, and complex II detected by an antibody to the 70 kDa subunit is at approximately 130 kDa. (A) After 10–30 s exposure time. (B) Histogram of A showing the density volumes of complex I normalised by complex II as percentage of the wild-type (IV:10) donor, with error bars representing the SEM of three or four gels. *p

    Techniques Used: Isolation, Polyacrylamide Gel Electrophoresis, Western Blot

    29) Product Images from "HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells"

    Article Title: HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells

    Journal: Retrovirology

    doi: 10.1186/1742-4690-8-40

    Analysis of CD4, CXCR4 and CCR5 expression in MSCs. Analysis of CD4, CXCR4, CCR5 and β-actin mRNA expression by qualitative real time RT-PCR in MSCs (A). A typical gel electrophoresis of qualitative real time RT-PCR is shown. As positive controls, total RNAs extracted from PBMC were employed. The total RNAs extracted from NK-92 cells (for CD4) and E. coli total RNA (for CXCR4 and CCR5) served as negative control. Panel B displays a typical flow cytometry analysis of CCR5, CXCR4 and CD4 staining in MSCs. Unshadowed areas represent MSCs treated with FITC-conjugated specific mAb, whereas the negative control (MSCs stained by FITC-conjugated irrelevant isotype matched mAb) is represented by shadowed areas. Three experiments were performed in duplicate.
    Figure Legend Snippet: Analysis of CD4, CXCR4 and CCR5 expression in MSCs. Analysis of CD4, CXCR4, CCR5 and β-actin mRNA expression by qualitative real time RT-PCR in MSCs (A). A typical gel electrophoresis of qualitative real time RT-PCR is shown. As positive controls, total RNAs extracted from PBMC were employed. The total RNAs extracted from NK-92 cells (for CD4) and E. coli total RNA (for CXCR4 and CCR5) served as negative control. Panel B displays a typical flow cytometry analysis of CCR5, CXCR4 and CD4 staining in MSCs. Unshadowed areas represent MSCs treated with FITC-conjugated specific mAb, whereas the negative control (MSCs stained by FITC-conjugated irrelevant isotype matched mAb) is represented by shadowed areas. Three experiments were performed in duplicate.

    Techniques Used: Expressing, Quantitative RT-PCR, Nucleic Acid Electrophoresis, Negative Control, Flow Cytometry, Cytometry, Staining

    HIV-­‐1 proviral DNA and p24 protein detection in MSCs infected by HIV-­‐1 strains. Analysis of HIV-1 proviral DNA by qualitative real time PCR (A): agarose gel electrophoresis of MSC infected by HIV-1 IIIb and HIV-1 ada at days 3 and 7 post-infection. Positive control was activated PBMC at day 3 and negative control was mock-infected MSCs. All experiments were performed using 5 × 10 5 MSC or activated PBMCs infected or not with HIV-1 IIIb and HIV-1 ada (5 ng/ml p24). Panel B shows DNA integrated proviral HIV-1. The total DNA extracted from 5 × 10 5 MSC or activated PBMC cells was run in agarose gel electrophoresis and, after the purification of cellular DNA as previously described ( 51 ), a nested Alu -PCR was performed. The MSCs challenged by HIV-1 strains were analyzed at day 7. Activated PBMCs infected with the two HIV-1 strains served as positive controls. A specific LTR 100 bp band is detectable in HIV-1 infected MSCs and positive controls. Panel C displays the cell supernatant p24 analysis. ELISA p24 kit was employed to analyze the p24 content in cell supernatant. This assay exhibits a sensitive limit at 3 pg/ml. The amount of p24 in MSCs challenged with HIV-1 strains was very low and, in these experimental conditions, slowly declined at later tested times. The positive controls were performed by activated PBMC infected with HIV-1 strains.
    Figure Legend Snippet: HIV-­‐1 proviral DNA and p24 protein detection in MSCs infected by HIV-­‐1 strains. Analysis of HIV-1 proviral DNA by qualitative real time PCR (A): agarose gel electrophoresis of MSC infected by HIV-1 IIIb and HIV-1 ada at days 3 and 7 post-infection. Positive control was activated PBMC at day 3 and negative control was mock-infected MSCs. All experiments were performed using 5 × 10 5 MSC or activated PBMCs infected or not with HIV-1 IIIb and HIV-1 ada (5 ng/ml p24). Panel B shows DNA integrated proviral HIV-1. The total DNA extracted from 5 × 10 5 MSC or activated PBMC cells was run in agarose gel electrophoresis and, after the purification of cellular DNA as previously described ( 51 ), a nested Alu -PCR was performed. The MSCs challenged by HIV-1 strains were analyzed at day 7. Activated PBMCs infected with the two HIV-1 strains served as positive controls. A specific LTR 100 bp band is detectable in HIV-1 infected MSCs and positive controls. Panel C displays the cell supernatant p24 analysis. ELISA p24 kit was employed to analyze the p24 content in cell supernatant. This assay exhibits a sensitive limit at 3 pg/ml. The amount of p24 in MSCs challenged with HIV-1 strains was very low and, in these experimental conditions, slowly declined at later tested times. The positive controls were performed by activated PBMC infected with HIV-1 strains.

    Techniques Used: Infection, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Negative Control, Purification, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

    30) Product Images from "Endoscopic biopsy of islet transplants in the gastric submucosal space provides evidence of islet graft rejection in diabetic pigs"

    Article Title: Endoscopic biopsy of islet transplants in the gastric submucosal space provides evidence of islet graft rejection in diabetic pigs

    Journal: Islets

    doi: 10.1080/19382014.2016.1149283

    Suppression of T cell proliferation and activation by BMX-001 PBMCs from pigs (n = 4) and humans (n = 3) were stimulated with PHA for 72h with/without 10µM BMX-001. BMX-001 significantly reduced proliferation of both pig (A) and human (D) PBMCs
    Figure Legend Snippet: Suppression of T cell proliferation and activation by BMX-001 PBMCs from pigs (n = 4) and humans (n = 3) were stimulated with PHA for 72h with/without 10µM BMX-001. BMX-001 significantly reduced proliferation of both pig (A) and human (D) PBMCs

    Techniques Used: Activation Assay

    31) Product Images from "Polyfunctional CD4+ T-Cell Induction in Neutralizing Antibody-Triggered Control of Simian Immunodeficiency Virus Infection ▿"

    Article Title: Polyfunctional CD4+ T-Cell Induction in Neutralizing Antibody-Triggered Control of Simian Immunodeficiency Virus Infection ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00145-09

    Anti-SIV efficacy in vitro of CD8 + cells. PBMC-derived CD8 − (target) cells infected with SIVmac239 were cultured alone (no CD8) or cocultured with autologous PBMC-derived CD8 + (effector) cells obtained prechallenge (pre) or at week 3 postchallenge (wk 3) at an E/T ratio of 1:4. The results were obtained from three unimmunized controls and four NAb-immunized macaques. The ratios of p27 concentrations in the culture supernatants after 8 days of coculture with pre-CD8 + or week 3 CD8 + cells to those without CD8 + cells (CD8 negative) are shown. The coculture with either R03-011 week 3 CD8 + , R03-020 week 3 CD8 + , and R03-013 week 3 CD8 + cells showed undetectable or marginal SIV p27 production after 8 days.
    Figure Legend Snippet: Anti-SIV efficacy in vitro of CD8 + cells. PBMC-derived CD8 − (target) cells infected with SIVmac239 were cultured alone (no CD8) or cocultured with autologous PBMC-derived CD8 + (effector) cells obtained prechallenge (pre) or at week 3 postchallenge (wk 3) at an E/T ratio of 1:4. The results were obtained from three unimmunized controls and four NAb-immunized macaques. The ratios of p27 concentrations in the culture supernatants after 8 days of coculture with pre-CD8 + or week 3 CD8 + cells to those without CD8 + cells (CD8 negative) are shown. The coculture with either R03-011 week 3 CD8 + , R03-020 week 3 CD8 + , and R03-013 week 3 CD8 + cells showed undetectable or marginal SIV p27 production after 8 days.

    Techniques Used: In Vitro, Derivative Assay, Infection, Cell Culture

    32) Product Images from "IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease"

    Article Title: IL-1R3 blockade broadly attenuates the functions of six members of the IL-1 family, revealing their contribution to models of disease

    Journal: Nature immunology

    doi: 10.1038/s41590-019-0467-1

    IL-1R3 blockade results in a broader anti-inflammatory phenotype compared to primary pathway inhibition. (a+b) Inhibition of hk. C. albicans (0.5 ×10 6 /mL, 24hrs) induced IL-6 (-a) or IFN-γ (−b) production in PBMCs, using equal concentrations MAB-hR3, sIL-1R4 and IL-36Ra (69nM) (ns: p=0.052) (c-i) Percent change in cytokines measured in the supernatants of two-way MLRs (5-day culture). MLR range of IFN-γ (1.9–10.7 ng/mL, IL-13 (312–1808 pg/mL), IL-17 (48.6–189 ng/mL), IL-22 (143–2696 pg/mL), TNF (168−1712 pg/mL), IL-6 (254–8361 pg/mL) and IL-10 (107–1302 pg/mL) (ns: p=0.07). Inhibitors compared to stimulation or MLR alone. *p
    Figure Legend Snippet: IL-1R3 blockade results in a broader anti-inflammatory phenotype compared to primary pathway inhibition. (a+b) Inhibition of hk. C. albicans (0.5 ×10 6 /mL, 24hrs) induced IL-6 (-a) or IFN-γ (−b) production in PBMCs, using equal concentrations MAB-hR3, sIL-1R4 and IL-36Ra (69nM) (ns: p=0.052) (c-i) Percent change in cytokines measured in the supernatants of two-way MLRs (5-day culture). MLR range of IFN-γ (1.9–10.7 ng/mL, IL-13 (312–1808 pg/mL), IL-17 (48.6–189 ng/mL), IL-22 (143–2696 pg/mL), TNF (168−1712 pg/mL), IL-6 (254–8361 pg/mL) and IL-10 (107–1302 pg/mL) (ns: p=0.07). Inhibitors compared to stimulation or MLR alone. *p

    Techniques Used: Inhibition

    33) Product Images from "Xenogeneic Graft-Versus-Host Disease in Humanized NSG and NSG-HLA-A2/HHD Mice"

    Article Title: Xenogeneic Graft-Versus-Host Disease in Humanized NSG and NSG-HLA-A2/HHD Mice

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2018.01943

    Histological analyses in NSG and NSG-HLA-A2/HHD mice transplanted with HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice received sublethal total body irradiation (2.5 Gy) and were infused 24 h later with 2 × 10 6 human PBMCs intravenously. PBMCs were isolated from HLA-A2 + healthy donors. Mice were sacrificed at day 14 to collect their organs. (A) Histological quantification of human CD3 + cells (stained in brown) infiltration in lungs and liver of NSG and NSG-HLA-A2/HHD mice. Panels on the right show the comparison of 7–8 mice/group. (B) Histopathological evaluation of GVHD score in lungs (top panel) and liver (bottom panel). Data show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles.
    Figure Legend Snippet: Histological analyses in NSG and NSG-HLA-A2/HHD mice transplanted with HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice received sublethal total body irradiation (2.5 Gy) and were infused 24 h later with 2 × 10 6 human PBMCs intravenously. PBMCs were isolated from HLA-A2 + healthy donors. Mice were sacrificed at day 14 to collect their organs. (A) Histological quantification of human CD3 + cells (stained in brown) infiltration in lungs and liver of NSG and NSG-HLA-A2/HHD mice. Panels on the right show the comparison of 7–8 mice/group. (B) Histopathological evaluation of GVHD score in lungs (top panel) and liver (bottom panel). Data show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles.

    Techniques Used: Mouse Assay, Irradiation, Isolation, Staining

    Histological analyses in NSG and NSG-HLA-A2/HHD mice transplanted with non-HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice received sublethal total body irradiation (2.5 Gy) and were infused 24 h later either with 2 × 10 6 human PBMCs intravenously. PBMCs were isolated from HLA-A2 − healthy donors and experiment was repeated twice with different donors of PBMCs. Mice were sacrificed at day 14 to collect their organs. (A) Histopathological evaluation of GVHD score in lungs and liver. Black arrows show apoptotic bodies, red arrows show mitosis and orange arrows show bile plugs. Panels on the right show the comparison of 7–8 mice/group. (B) Histological quantification of human CD3 + cells (stained in brown) infiltration in lungs and liver of NSG and NSG-HLA-A2/HHD mice. Panels on the right show the comparison of 7–8 mice/group. Data show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles.
    Figure Legend Snippet: Histological analyses in NSG and NSG-HLA-A2/HHD mice transplanted with non-HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice received sublethal total body irradiation (2.5 Gy) and were infused 24 h later either with 2 × 10 6 human PBMCs intravenously. PBMCs were isolated from HLA-A2 − healthy donors and experiment was repeated twice with different donors of PBMCs. Mice were sacrificed at day 14 to collect their organs. (A) Histopathological evaluation of GVHD score in lungs and liver. Black arrows show apoptotic bodies, red arrows show mitosis and orange arrows show bile plugs. Panels on the right show the comparison of 7–8 mice/group. (B) Histological quantification of human CD3 + cells (stained in brown) infiltration in lungs and liver of NSG and NSG-HLA-A2/HHD mice. Panels on the right show the comparison of 7–8 mice/group. Data show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles.

    Techniques Used: Mouse Assay, Irradiation, Isolation, Staining

    Differentiation and phenotype of human T cells in NSG and NSG-HLA-A2/HHD mice transplanted with non-HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice were transplanted with HLA-A2 − PBMCs and were sacrificed at day 14 to collect their organs and perform flow cytometry analyzes. (A) CD4/CD8 ratio in indicated organs (11 mice/group). (B) Frequency of Treg in the different organs (11 mice/group). (C) Representative FACS plots and gating strategy of the data shown in panel D . (D) Mean frequency of naive (TN, CD45RA + CD27 + ), effector (TE, CD45RA − CD27 − ), central memory (TCM, CD45RA − CD27 + CD62L + ) and effector memory (TEM, CD45RA − CD27 + CD62L − ) T cells (11 mice/group). (E) Frequency of PD-1 + T cells (4–5 mice/group). (F) Frequency of IFNγ + , TNF-α + , IFNγ + TNF-α + , IL-4 + , and IL-17 + T cells in spleen (11 mice/group). (G) Serum concentration of IFNγ, TNF-α and IL-17 cytokines, assessed by bioplex (6–7 mice/group). (H) Representative FACS plots and gating strategy of the data shown in panel I . (I) Mean frequency of IFNγ − TNF-α + , IFNγ + TNF-α + , IFNγ + TNF-α − , IFNγ − TNF-α − , and IL-17 + IFNγ + T cells (4–5 mice/group). Data in A, B, E, F , and G show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles.
    Figure Legend Snippet: Differentiation and phenotype of human T cells in NSG and NSG-HLA-A2/HHD mice transplanted with non-HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice were transplanted with HLA-A2 − PBMCs and were sacrificed at day 14 to collect their organs and perform flow cytometry analyzes. (A) CD4/CD8 ratio in indicated organs (11 mice/group). (B) Frequency of Treg in the different organs (11 mice/group). (C) Representative FACS plots and gating strategy of the data shown in panel D . (D) Mean frequency of naive (TN, CD45RA + CD27 + ), effector (TE, CD45RA − CD27 − ), central memory (TCM, CD45RA − CD27 + CD62L + ) and effector memory (TEM, CD45RA − CD27 + CD62L − ) T cells (11 mice/group). (E) Frequency of PD-1 + T cells (4–5 mice/group). (F) Frequency of IFNγ + , TNF-α + , IFNγ + TNF-α + , IL-4 + , and IL-17 + T cells in spleen (11 mice/group). (G) Serum concentration of IFNγ, TNF-α and IL-17 cytokines, assessed by bioplex (6–7 mice/group). (H) Representative FACS plots and gating strategy of the data shown in panel I . (I) Mean frequency of IFNγ − TNF-α + , IFNγ + TNF-α + , IFNγ + TNF-α − , IFNγ − TNF-α − , and IL-17 + IFNγ + T cells (4–5 mice/group). Data in A, B, E, F , and G show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles.

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, FACS, Transmission Electron Microscopy, Concentration Assay

    Differentiation and phenotype of human T cells in NSG and NSG-HLA-A2/HHD mice transplanted with HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice were transplanted with HLA-A2 + PBMCs and were sacrificed at day 14 to collect their organs and perform flow cytometry analyzes. (A) CD4/CD8 ratio in indicated organs (11–12 mice/group). (B) Frequency of Treg in the different organs (11–12 mice/group). (C) Mean frequency of naive (TN, CD45RA + CD27 + ), effector (TE, CD45RA − CD27 − ), central memory (TCM, CD45RA − CD27 + CD62L + ) and effector memory (TEM, CD45RA − CD27 + CD62L − ) T cells (11–12 mice/group). (D) Frequency of PD-1 + T cells in spleen (5 mice/group). (E) Frequency of IFNγ + , TNF-α + , IFNγ + TNF-α + , IL-4 + , and IL-17 + T cells in spleen (11 mice/group). (F) Mean frequency of IFNγ − TNF-α + , IFNγ + TNF-α + , IFNγ + TNF-α − , IFNγ − TNF-α − , and IL-17 + IFNγ + T cells (5 mice/group). Data in A, B, D , and E show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles (* p
    Figure Legend Snippet: Differentiation and phenotype of human T cells in NSG and NSG-HLA-A2/HHD mice transplanted with HLA-A2 PBMCs. NSG and NSG-HLA-A2/HHD (NSG.A2 in figure) mice were transplanted with HLA-A2 + PBMCs and were sacrificed at day 14 to collect their organs and perform flow cytometry analyzes. (A) CD4/CD8 ratio in indicated organs (11–12 mice/group). (B) Frequency of Treg in the different organs (11–12 mice/group). (C) Mean frequency of naive (TN, CD45RA + CD27 + ), effector (TE, CD45RA − CD27 − ), central memory (TCM, CD45RA − CD27 + CD62L + ) and effector memory (TEM, CD45RA − CD27 + CD62L − ) T cells (11–12 mice/group). (D) Frequency of PD-1 + T cells in spleen (5 mice/group). (E) Frequency of IFNγ + , TNF-α + , IFNγ + TNF-α + , IL-4 + , and IL-17 + T cells in spleen (11 mice/group). (F) Mean frequency of IFNγ − TNF-α + , IFNγ + TNF-α + , IFNγ + TNF-α − , IFNγ − TNF-α − , and IL-17 + IFNγ + T cells (5 mice/group). Data in A, B, D , and E show the median, 25 and 75th percentiles of the distribution (boxes), and whiskers extend to the 5 and 95th percentiles (* p

    Techniques Used: Mouse Assay, Flow Cytometry, Cytometry, Transmission Electron Microscopy

    34) Product Images from "Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease"

    Article Title: Prostaglandin E2 Induction Suppresses the Th1 Immune Responses in Cattle with Johne's Disease

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00910-17

    Functional analysis of the COX-2 inhibitor. PBMCs from uninfected cattle ( n = 9 [a and b] or 8 [c]) were cultured with meloxicam in the presence of anti-CD3 and anti-CD28 MAbs. CD8 + cell proliferation was assayed by flow cytometry (a). IFN-γ (b) and TNF-α (c) production was determined by ELISA in duplicate. Statistical significance was determined by the Wilcoxon signed-rank test.
    Figure Legend Snippet: Functional analysis of the COX-2 inhibitor. PBMCs from uninfected cattle ( n = 9 [a and b] or 8 [c]) were cultured with meloxicam in the presence of anti-CD3 and anti-CD28 MAbs. CD8 + cell proliferation was assayed by flow cytometry (a). IFN-γ (b) and TNF-α (c) production was determined by ELISA in duplicate. Statistical significance was determined by the Wilcoxon signed-rank test.

    Techniques Used: Functional Assay, Cell Culture, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    Kinetic analysis of PGE 2 in cattle infected with M. avium subsp. paratuberculosis . (a) Serum PGE 2 concentrations in cattle uninfected ( n = 16) or naturally infected with M. avium subsp. paratuberculosis ( n = 8) were determined by ELISA. (b) The expression of COX-2 mRNA in PBMCs from cattle experimentally infected with M. avium subsp. paratuberculosis ( n = 7) cultured with J-PPD was assayed by real-time PCR. (c) PBMCs of the uninfected ( n = 6) and experimentally infected ( n = 7) cattle were incubated with J-PPD. The concentration of PGE 2 in culture supernatants was determined by ELISA in duplicate. Statistical significance was determined by the Wilcoxon signed-rank test (a and b) or the Steel-Dwass test (c). N.S., no stimulation.
    Figure Legend Snippet: Kinetic analysis of PGE 2 in cattle infected with M. avium subsp. paratuberculosis . (a) Serum PGE 2 concentrations in cattle uninfected ( n = 16) or naturally infected with M. avium subsp. paratuberculosis ( n = 8) were determined by ELISA. (b) The expression of COX-2 mRNA in PBMCs from cattle experimentally infected with M. avium subsp. paratuberculosis ( n = 7) cultured with J-PPD was assayed by real-time PCR. (c) PBMCs of the uninfected ( n = 6) and experimentally infected ( n = 7) cattle were incubated with J-PPD. The concentration of PGE 2 in culture supernatants was determined by ELISA in duplicate. Statistical significance was determined by the Wilcoxon signed-rank test (a and b) or the Steel-Dwass test (c). N.S., no stimulation.

    Techniques Used: Infection, Enzyme-linked Immunosorbent Assay, Expressing, Cell Culture, Real-time Polymerase Chain Reaction, Incubation, Concentration Assay

    Upregulation of PD-L1 expression by J-PPD. PBMCs from cattle experimentally infected with M. avium subsp. paratuberculosis ( n = 6) were incubated with J-PPD, and the expression of PD-L1 in PBMCs (a), CD4 + T cells (c), CD8 + T cells (d), IgM + B cells (e), and CD14 + cells (f) was assayed by flow cytometry. PBMCs of uninfected cattle ( n = 5) were incubated with J-PPD, and PD-L1 expression in PBMCs was assayed by flow cytometry (a). Expression of PD-L1 mRNA in PBMCs of the experimentally infected cattle ( n = 7) was assayed by real-time PCR in duplicate (b). Statistical significance was determined by the Wilcoxon signed-rank test. N.S., no stimulation.
    Figure Legend Snippet: Upregulation of PD-L1 expression by J-PPD. PBMCs from cattle experimentally infected with M. avium subsp. paratuberculosis ( n = 6) were incubated with J-PPD, and the expression of PD-L1 in PBMCs (a), CD4 + T cells (c), CD8 + T cells (d), IgM + B cells (e), and CD14 + cells (f) was assayed by flow cytometry. PBMCs of uninfected cattle ( n = 5) were incubated with J-PPD, and PD-L1 expression in PBMCs was assayed by flow cytometry (a). Expression of PD-L1 mRNA in PBMCs of the experimentally infected cattle ( n = 7) was assayed by real-time PCR in duplicate (b). Statistical significance was determined by the Wilcoxon signed-rank test. N.S., no stimulation.

    Techniques Used: Expressing, Infection, Incubation, Flow Cytometry, Cytometry, Real-time Polymerase Chain Reaction

    Immunosuppressive effects of PGE 2 . (a to d) PBMCs from uninfected cattle ( n = 6 [a to c] or 8 [d]) were cultured with PGE 2 in the presence of anti-CD3 and anti-CD28 MAbs for 72 h. The proliferation of CD4 + cells (a) and CD8 + cells (b) was assayed by flow cytometry. IFN-γ (c) and TNF-α (d) production was determined by ELISA. (e to k) PBMCs from uninfected cattle ( n = 6 [e to i] or 7 [j and k]) were cultured with PGE 2 for 24 h. Real-time PCR was performed in duplicate to quantitate the mRNA expression of IFN-γ (e), IL-2 (f), TNF-α (g), IL-10 (h), STAT3 (i), and PD-L1 (j). The expression of PD-L1 protein was measured by flow cytometry (k). Statistical significance was determined by the Steel-Dwass test (a to c) or the Wilcoxon signed-rank test (d to k).
    Figure Legend Snippet: Immunosuppressive effects of PGE 2 . (a to d) PBMCs from uninfected cattle ( n = 6 [a to c] or 8 [d]) were cultured with PGE 2 in the presence of anti-CD3 and anti-CD28 MAbs for 72 h. The proliferation of CD4 + cells (a) and CD8 + cells (b) was assayed by flow cytometry. IFN-γ (c) and TNF-α (d) production was determined by ELISA. (e to k) PBMCs from uninfected cattle ( n = 6 [e to i] or 7 [j and k]) were cultured with PGE 2 for 24 h. Real-time PCR was performed in duplicate to quantitate the mRNA expression of IFN-γ (e), IL-2 (f), TNF-α (g), IL-10 (h), STAT3 (i), and PD-L1 (j). The expression of PD-L1 protein was measured by flow cytometry (k). Statistical significance was determined by the Steel-Dwass test (a to c) or the Wilcoxon signed-rank test (d to k).

    Techniques Used: Cell Culture, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Expressing

    Activation of T-cell responses by the COX-2 inhibitor or anti-PD-L1 MAb. (a to c) PBMCs from cattle experimentally infected with M. avium subsp. paratuberculosis ( n = 7 [a and b] or 6 [c]) were cultured with meloxicam in the presence of J-PPD. CD8 + cell proliferation (a) was assayed by flow cytometry. IFN-γ (b) and TNF-α (c) production was determined by ELISA in duplicate. (d and e) PBMCs from the infected cattle ( n = 7) were cultured with anti-PD-L1 MAb in the presence of J-PPD. CD8 + cell proliferation (d) was assayed by flow cytometry. IFN-γ production (e) was determined by ELISA in duplicate. Statistical significance was determined by the Wilcoxon signed-rank test. Cont Ig, rat control IgG.
    Figure Legend Snippet: Activation of T-cell responses by the COX-2 inhibitor or anti-PD-L1 MAb. (a to c) PBMCs from cattle experimentally infected with M. avium subsp. paratuberculosis ( n = 7 [a and b] or 6 [c]) were cultured with meloxicam in the presence of J-PPD. CD8 + cell proliferation (a) was assayed by flow cytometry. IFN-γ (b) and TNF-α (c) production was determined by ELISA in duplicate. (d and e) PBMCs from the infected cattle ( n = 7) were cultured with anti-PD-L1 MAb in the presence of J-PPD. CD8 + cell proliferation (d) was assayed by flow cytometry. IFN-γ production (e) was determined by ELISA in duplicate. Statistical significance was determined by the Wilcoxon signed-rank test. Cont Ig, rat control IgG.

    Techniques Used: Activation Assay, Infection, Cell Culture, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay

    35) Product Images from "A Randomized Double-Blinded, Placebo-Controlled Trial Investigating the Effect of Fish Oil Supplementation on Gene Expression Related to Insulin Action, Blood Lipids, and Inflammation in Gestational Diabetes Mellitus-Fish Oil Supplementation and Gestational Diabetes"

    Article Title: A Randomized Double-Blinded, Placebo-Controlled Trial Investigating the Effect of Fish Oil Supplementation on Gene Expression Related to Insulin Action, Blood Lipids, and Inflammation in Gestational Diabetes Mellitus-Fish Oil Supplementation and Gestational Diabetes

    Journal: Nutrients

    doi: 10.3390/nu10020163

    Effect of 6-week supplementation with omega-3 or placebo on expression ratio of PPAR-γ gene in PBMCs of GDM women.
    Figure Legend Snippet: Effect of 6-week supplementation with omega-3 or placebo on expression ratio of PPAR-γ gene in PBMCs of GDM women.

    Techniques Used: Expressing

    Effect of 6-week supplementation with omega-3 or placebo on expression ratio of IL-1, IL-8, and TNF-α gene in PBMCs of GDM women.
    Figure Legend Snippet: Effect of 6-week supplementation with omega-3 or placebo on expression ratio of IL-1, IL-8, and TNF-α gene in PBMCs of GDM women.

    Techniques Used: Expressing

    Effect of 6-week supplementation with omega-3 or placebo on expression ratio of LDLR gene in PBMCs of GDM women.
    Figure Legend Snippet: Effect of 6-week supplementation with omega-3 or placebo on expression ratio of LDLR gene in PBMCs of GDM women.

    Techniques Used: Expressing

    36) Product Images from "Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma"

    Article Title: Interferon Alpha Signalling and Its Relevance for the Upregulatory Effect of Transporter Proteins Associated with Antigen Processing (TAP) in Patients with Malignant Melanoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0146325

    IFNα-2b (IntronA) stimulates TAP1 expression in peripheral blood mononuclear cells (PBMC) of patients (n = 18) with malignant melanoma receiving adjuvant high-dose immunotherapy. (A) The actual administered dose of IFNα-2b was about 20 million IU/m 2 dependent on the clinically observed side effects. Only days of IFNα treatment are shown. (B) mRNA expression of TAP1 in PBMCs of patients treated with adjuvant high-dose IFNα-2b. Statistical analysis of TAP expression was performed using the Statistical Analysis System of the SAS Institute Inc. (Cary, NC, USA).
    Figure Legend Snippet: IFNα-2b (IntronA) stimulates TAP1 expression in peripheral blood mononuclear cells (PBMC) of patients (n = 18) with malignant melanoma receiving adjuvant high-dose immunotherapy. (A) The actual administered dose of IFNα-2b was about 20 million IU/m 2 dependent on the clinically observed side effects. Only days of IFNα treatment are shown. (B) mRNA expression of TAP1 in PBMCs of patients treated with adjuvant high-dose IFNα-2b. Statistical analysis of TAP expression was performed using the Statistical Analysis System of the SAS Institute Inc. (Cary, NC, USA).

    Techniques Used: Expressing

    37) Product Images from "Differential Escape Patterns within the Dominant HLA-B*57:03-Restricted HIV Gag Epitope Reflect Distinct Clade-Specific Functional Constraints"

    Article Title: Differential Escape Patterns within the Dominant HLA-B*57:03-Restricted HIV Gag Epitope Reflect Distinct Clade-Specific Functional Constraints

    Journal: Journal of Virology

    doi: 10.1128/JVI.03303-13

    Median viral loads of KF11 responders versus nonresponders in B clade virus-infected individuals expressing HLA-B*57:01. PBMCs from B clade virus-infected, ART-naive individuals expressing HLA-B*57:01 were analyzed by IFN-γ ELISPOT assay for responses
    Figure Legend Snippet: Median viral loads of KF11 responders versus nonresponders in B clade virus-infected individuals expressing HLA-B*57:01. PBMCs from B clade virus-infected, ART-naive individuals expressing HLA-B*57:01 were analyzed by IFN-γ ELISPOT assay for responses

    Techniques Used: Infection, Expressing, Enzyme-linked Immunospot

    38) Product Images from "ISO-66, a novel inhibitor of macrophage migration inhibitory factor, shows efficacy in melanoma and colon cancer models"

    Article Title: ISO-66, a novel inhibitor of macrophage migration inhibitory factor, shows efficacy in melanoma and colon cancer models

    Journal: International Journal of Oncology

    doi: 10.3892/ijo.2014.2551

    ISO-66 enhances NK and LAK cell cytotoxicity. (A) PBMCs were incubated for 3 days with ISO-66 (1 mM-1 μM), NK cells were purified and tested as effectors against K562, at an E:T ratio of 10:1. * p
    Figure Legend Snippet: ISO-66 enhances NK and LAK cell cytotoxicity. (A) PBMCs were incubated for 3 days with ISO-66 (1 mM-1 μM), NK cells were purified and tested as effectors against K562, at an E:T ratio of 10:1. * p

    Techniques Used: Incubation, Purification

    39) Product Images from "IN VITRO TESTING OF AN ANTI-CD40 MONOCLONAL ANTIBODY, CLONE 2C10, IN PRIMATES AND PIGS"

    Article Title: IN VITRO TESTING OF AN ANTI-CD40 MONOCLONAL ANTIBODY, CLONE 2C10, IN PRIMATES AND PIGS

    Journal: Transplant immunology

    doi: 10.1016/j.trim.2015.09.007

    The combination of 2C10R4 with belatacept increased suppression of PBMC proliferation after stimulation with pAECs
    Figure Legend Snippet: The combination of 2C10R4 with belatacept increased suppression of PBMC proliferation after stimulation with pAECs

    Techniques Used:

    40) Product Images from "Functions, structure, and read-through alternative splicing of feline APOBEC3 genes"

    Article Title: Functions, structure, and read-through alternative splicing of feline APOBEC3 genes

    Journal: Genome Biology

    doi: 10.1186/gb-2008-9-3-r48

    Expression analysis of feline A3C, A3H and A3CH. (a,b) Analysis of expression of feline A3C, A3H and A3CH by RT-PCR of total RNA from feline cell lines (CrFK, MYA-1, KE-R) and feline PBMCs (a) and expression of A3CH in PBMCs of lion, puma, Sumatra-tiger (tiger), and lynx (b). H 2 O indicates PCRs using primers specific for the A3s without template cDNA added. (c) Analysis of expression of cat A3CH by immunoblot using rabbit serum against the sequence flanked by the C- and H-domains in cat A3C (linker) using 293T cells transfected with A3 expression plasmid or empty vector as indicated and CrFK and MyA-1 cells (two independent cultures each).
    Figure Legend Snippet: Expression analysis of feline A3C, A3H and A3CH. (a,b) Analysis of expression of feline A3C, A3H and A3CH by RT-PCR of total RNA from feline cell lines (CrFK, MYA-1, KE-R) and feline PBMCs (a) and expression of A3CH in PBMCs of lion, puma, Sumatra-tiger (tiger), and lynx (b). H 2 O indicates PCRs using primers specific for the A3s without template cDNA added. (c) Analysis of expression of cat A3CH by immunoblot using rabbit serum against the sequence flanked by the C- and H-domains in cat A3C (linker) using 293T cells transfected with A3 expression plasmid or empty vector as indicated and CrFK and MyA-1 cells (two independent cultures each).

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Sequencing, Transfection, Plasmid Preparation

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    In Vitro:

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    XTT Assay:

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    Enzyme-linked Immunosorbent Assay:

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    Staining:

    Article Title: Comparative Investigation of Frankincense Nutraceuticals: Correlation of Boswellic and Lupeolic Acid Contents with Cytokine Release Inhibition and Toxicity against Triple-Negative Breast Cancer Cells
    Article Snippet: .. Analysis of Antiproliferative and Cytotoxic Effects In Vitro To verify that the concentrations of the FNs and pure BAs and LAs used, are not cytotoxic to PBMC but cytotoxic to cancer cells, analysis of lactate dehydrogenase (LDH) release (Roche, Basel, Switzerland), XTT assay of cell viability and proliferation (Roche), and analysis of cell membrane integrity by propidium iodide staining [ ] were carried out. .. Triple-negative human breast cancer cells MDA-MB-231 (ATCC, Rockville, MD, USA), CAL-51 and MDA-MB-453 (both from DSMZ, Braunschweig, Germany), and PBMC were analyzed.

    Polymerase Chain Reaction:

    Article Title: Abnormal telomere shortening of peripheral blood mononuclear cells and granulocytes in patients with chronic idiopathic neutropenia
    Article Snippet: .. Telomerase activity in extracts of PBMCs and granulocytes of CIN patients and healthy controls was determined using the highly sensitive TeloTAGGG Telomerase PCR ELISA kit (Roche Diagnostics Scandinavia AB, Stockholm, Sweden), a specific photometric enzyme immunoassay based on the Telomeric Repeat Amplification Protocol (TRAP)., Reactions were performed with 50 μg of total cell protein for each sample according to the manufacturer’s instructions. .. Immortalized telomerase-expressing human kidney cells were used as positive control.

    Sonication:

    Article Title: Mutations in the selenocysteine insertion sequence-binding protein 2 gene lead to a multisystem selenoprotein deficiency disorder in humans
    Article Snippet: .. Skin tissue or PBMCs from P1, his sibling, and normal controls were homogenized in buffer (50 mM Tris; 1 mM EDTA; protease inhibitor cocktail, Roche Diagnostics), sonicated, and then centrifuged to generate supernatants. .. TrxR activity in cell lysates was measured by a sensitive method, based on assay of thioredoxin-dependent reduction of fluorescent-labeled insulin substrate ( ).

    Recombinant:

    Article Title: Inhibition of Simian Immunodeficiency Virus (SIV) Replication by CD8+ T Lymphocytes from Macaques Immunized with Live Attenuated SIV
    Article Snippet: .. For lectin stimulation, PBMC were incubated with ConA at 5 μg/ml for 2 days, washed, and resuspended in R-10 medium supplemented with 10 to 20 U of recombinant human IL-2 (donated by M. Gately, Hoffman-La Roche). .. CD8+ and CD4+ T lymphocytes were isolated by negative selection of rhesus macaque PBMC.

    Isolation:

    Article Title: Full-length novel MHC class I allele discovery by next-generation sequencing: two platforms are better than one
    Article Snippet: .. RNA was isolated from frozen PBMCs using the Roche MagNA Pure instrument and RNA High Performance kit (Roche, Indianapolis, IN, USA) following manufacturer's protocols ( ). cDNA was synthesized using Oligo (dt) primers and Superscript III reverse transcriptase (Life Technologies, Grand Island, NY, USA) with equivalent concentrations of RNA for each sample ( ). .. The entire ∼1.2kb MHC class I coding region was amplified using Phusion high fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) and the following primers shown in Online Resource 1 and as previously published: TiFL-MHC1-F_MIDXX (5'UTR), TiFL-MHC1-FL_MIDXX (5' Leader), TiFL-MHC3-Ra_MIDXX, TiFL-MHC3-Rb_MIDXX, and TiFL-MHC3-Rb2_MIDXX ( ).

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    Roche pbmc proliferation assays
    Tumor cell derived <t>MVs</t> promote the proliferative impact of anti-CD3/CD28 mAb stimulation on <t>PBMC.</t> Freshly isolated human PBMC were kept for 3 days in the presence of CD3/CD28, CD3/CD28/MVs and MVs alone. As control untreated cells were used. For the last 14-18 h of cell culture the BrdU compound (Roche) was added. Then the cell proliferation assay was performed according to the manufacturer’s protocol. Experiments were performed in triplicates and results presented are expressed as means of OD 450 nm ± SD. The data show one representative result of four independent repeats of the experiment. The difference between the control group and the CD3/CD28 and CD3/CD28/MVs treated groups as well as the CD3/CD28 and CD3/CD28/MVs treated groups were determined by student t-test (*p≤0.005 and **p≤0.0065, respectively; n=3).
    Pbmc Proliferation Assays, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbmcs  (Roche)
    94
    Roche pbmcs
    pDC-mediated suppression of <t>HIV</t> replication in autologous CD4 + T cells. pDCs were purified from HIV-infected individuals and plated overnight at 5×10 4 cells/well with or without 1 µg/ml CpG ODN 2216. For targets, autologous <t>PBMCs</t> were stimulated overnight with anti-CD3 and IL-2. On day 0, CD4 + T cells were purified and plated at 1.2×10 6 cells/well alone or with 5×10 4 stimulated or unstimulated pDCs, or with 1.2×10 5 purified autologous CD8 + T cells. Neutralizing antibody against IFN-α receptor was added at 5 µg/ml to some wells. Culture supernatants were collected at days 3, 5, and 7 and assayed for HIV p24 by ELISA. All data shown are from day 7. A) A representative example of data from a viremic patient. CpG ODN 2216 had no effect on CD4 + T cells alone, and the degree of viral suppression by activated pDCs was comparable to that by CD8 + T cells. B) Effect of pDCs on HIV replication in autologous CD4 + T cells. The degree of HIV suppression by pDCs was expressed as the difference of log HIV p24 production between the culture containing CD4 + T cells alone and the co-culture of CD4 + T cells and pDCs (Δlog p24 = log HIV p24[CD4 cells alone]-log HIV p24[CD4+pDC]). Data are shown for unstimulated pDCs (left panel) and CpG-stimulated pDCs (right panel). Horizontal lines represent median values. n.s., not significant.
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    Roche pbmc discovery cohort
    CD200R potentiates IFNG <t>mRNA</t> production in systemic lupus erythematosus patients. ( A, B ) IFNG mRNA production relative to GAPDH (A) or B2M (B) by healthy control and SLE patients’ <t>PBMC</t> in two cohorts. PBMC were seeded on an isotype control (open circles) or an agonistic anti-CD200R antibody (filled circles) prior to stimulation with R848 for 4h. Each connected dot represents one patient/control. The color of the connecting arrow indicates: inhibition by CD200R ligation (blue, > 15% inhibition compared to isotype control); potentiation by CD200R ligation (red, > 15% potentiation compared to isotype control); or no effect of CD200R ligation (grey,
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    91
    Roche pbmc dna
    Frequency of T-cell subpopulations according to changes in HIV-1 <t>DNA</t> copies at 1 month from vaccination. HIV-1 DNA copies/10 6 peripheral blood mononuclear cells <t>(PBMCs)</t> were measured in PBMCs from 22 HIV-1-infected children (A) . In the group “decrease,” 12 children are included who displayed > 10% decrease in the number of HIV-1 DNA copies at 1 month from last vaccination as compared with BL value; in the group “stable,” 5 children who displayed minor variations (
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    Tumor cell derived MVs promote the proliferative impact of anti-CD3/CD28 mAb stimulation on PBMC. Freshly isolated human PBMC were kept for 3 days in the presence of CD3/CD28, CD3/CD28/MVs and MVs alone. As control untreated cells were used. For the last 14-18 h of cell culture the BrdU compound (Roche) was added. Then the cell proliferation assay was performed according to the manufacturer’s protocol. Experiments were performed in triplicates and results presented are expressed as means of OD 450 nm ± SD. The data show one representative result of four independent repeats of the experiment. The difference between the control group and the CD3/CD28 and CD3/CD28/MVs treated groups as well as the CD3/CD28 and CD3/CD28/MVs treated groups were determined by student t-test (*p≤0.005 and **p≤0.0065, respectively; n=3).

    Journal: PLoS ONE

    Article Title: Tumor and Endothelial Cell-Derived Microvesicles Carry Distinct CEACAMs and Influence T-Cell Behavior

    doi: 10.1371/journal.pone.0074654

    Figure Lengend Snippet: Tumor cell derived MVs promote the proliferative impact of anti-CD3/CD28 mAb stimulation on PBMC. Freshly isolated human PBMC were kept for 3 days in the presence of CD3/CD28, CD3/CD28/MVs and MVs alone. As control untreated cells were used. For the last 14-18 h of cell culture the BrdU compound (Roche) was added. Then the cell proliferation assay was performed according to the manufacturer’s protocol. Experiments were performed in triplicates and results presented are expressed as means of OD 450 nm ± SD. The data show one representative result of four independent repeats of the experiment. The difference between the control group and the CD3/CD28 and CD3/CD28/MVs treated groups as well as the CD3/CD28 and CD3/CD28/MVs treated groups were determined by student t-test (*p≤0.005 and **p≤0.0065, respectively; n=3).

    Article Snippet: PBMC proliferation assays The proliferation effect of MVs was studied using a BrdU Proliferation Kit (Roche) according to the manufacturers’ protocol.

    Techniques: Derivative Assay, Isolation, Cell Culture, Proliferation Assay

    CEACAM1-positive MVs significantly increase the anti-CD3 and anti-CD3/CD28 mAb triggered T-cell proliferation. Freshly isolated human PBMC were labeled with CFSE and cultured for 4 days in the presence of anti CD3 and anti CD3/CD28 with and without CHO- and CHO-CEACAM1 derived MVs (A). B) CFSE labeled PBMC were cultured for 4 days in the presence and absence of antiCD3 and antiCD3/CD28 with and without HT29-derived MVs. Untreated treated cells served as control. In indicated cases samples were co-cultured with antiCEACAM1 mAb18/20 (50 µg/ml) or isotype matched control IgG (50 µg/ml). Then PBMCs were analyzed utilizing the Accuri C6 flow cytometer system. The histograms depict PBMCs that have divided 1-3 times based on CFSE dilution peaks and reflex the cell proliferation rate given in %. The data shown are representative for three independent repeats of the experiment.

    Journal: PLoS ONE

    Article Title: Tumor and Endothelial Cell-Derived Microvesicles Carry Distinct CEACAMs and Influence T-Cell Behavior

    doi: 10.1371/journal.pone.0074654

    Figure Lengend Snippet: CEACAM1-positive MVs significantly increase the anti-CD3 and anti-CD3/CD28 mAb triggered T-cell proliferation. Freshly isolated human PBMC were labeled with CFSE and cultured for 4 days in the presence of anti CD3 and anti CD3/CD28 with and without CHO- and CHO-CEACAM1 derived MVs (A). B) CFSE labeled PBMC were cultured for 4 days in the presence and absence of antiCD3 and antiCD3/CD28 with and without HT29-derived MVs. Untreated treated cells served as control. In indicated cases samples were co-cultured with antiCEACAM1 mAb18/20 (50 µg/ml) or isotype matched control IgG (50 µg/ml). Then PBMCs were analyzed utilizing the Accuri C6 flow cytometer system. The histograms depict PBMCs that have divided 1-3 times based on CFSE dilution peaks and reflex the cell proliferation rate given in %. The data shown are representative for three independent repeats of the experiment.

    Article Snippet: PBMC proliferation assays The proliferation effect of MVs was studied using a BrdU Proliferation Kit (Roche) according to the manufacturers’ protocol.

    Techniques: Isolation, Labeling, Cell Culture, Derivative Assay, Flow Cytometry, Cytometry

    pDC-mediated suppression of HIV replication in autologous CD4 + T cells. pDCs were purified from HIV-infected individuals and plated overnight at 5×10 4 cells/well with or without 1 µg/ml CpG ODN 2216. For targets, autologous PBMCs were stimulated overnight with anti-CD3 and IL-2. On day 0, CD4 + T cells were purified and plated at 1.2×10 6 cells/well alone or with 5×10 4 stimulated or unstimulated pDCs, or with 1.2×10 5 purified autologous CD8 + T cells. Neutralizing antibody against IFN-α receptor was added at 5 µg/ml to some wells. Culture supernatants were collected at days 3, 5, and 7 and assayed for HIV p24 by ELISA. All data shown are from day 7. A) A representative example of data from a viremic patient. CpG ODN 2216 had no effect on CD4 + T cells alone, and the degree of viral suppression by activated pDCs was comparable to that by CD8 + T cells. B) Effect of pDCs on HIV replication in autologous CD4 + T cells. The degree of HIV suppression by pDCs was expressed as the difference of log HIV p24 production between the culture containing CD4 + T cells alone and the co-culture of CD4 + T cells and pDCs (Δlog p24 = log HIV p24[CD4 cells alone]-log HIV p24[CD4+pDC]). Data are shown for unstimulated pDCs (left panel) and CpG-stimulated pDCs (right panel). Horizontal lines represent median values. n.s., not significant.

    Journal: PLoS ONE

    Article Title: Impact of HIV on Cell Survival and Antiviral Activity of Plasmacytoid Dendritic Cells

    doi: 10.1371/journal.pone.0000458

    Figure Lengend Snippet: pDC-mediated suppression of HIV replication in autologous CD4 + T cells. pDCs were purified from HIV-infected individuals and plated overnight at 5×10 4 cells/well with or without 1 µg/ml CpG ODN 2216. For targets, autologous PBMCs were stimulated overnight with anti-CD3 and IL-2. On day 0, CD4 + T cells were purified and plated at 1.2×10 6 cells/well alone or with 5×10 4 stimulated or unstimulated pDCs, or with 1.2×10 5 purified autologous CD8 + T cells. Neutralizing antibody against IFN-α receptor was added at 5 µg/ml to some wells. Culture supernatants were collected at days 3, 5, and 7 and assayed for HIV p24 by ELISA. All data shown are from day 7. A) A representative example of data from a viremic patient. CpG ODN 2216 had no effect on CD4 + T cells alone, and the degree of viral suppression by activated pDCs was comparable to that by CD8 + T cells. B) Effect of pDCs on HIV replication in autologous CD4 + T cells. The degree of HIV suppression by pDCs was expressed as the difference of log HIV p24 production between the culture containing CD4 + T cells alone and the co-culture of CD4 + T cells and pDCs (Δlog p24 = log HIV p24[CD4 cells alone]-log HIV p24[CD4+pDC]). Data are shown for unstimulated pDCs (left panel) and CpG-stimulated pDCs (right panel). Horizontal lines represent median values. n.s., not significant.

    Article Snippet: HIV p24 assays 2–4×108 total PBMCs from HIV-infected individuals were stimulated overnight with anti-CD3 antibody and rIL-2 (Roche).

    Techniques: Purification, Infection, Enzyme-linked Immunosorbent Assay, Co-Culture Assay

    pDC killing in HIV-infected individuals. A) Plasmacytoid dendritic cell numbers are reduced in HIV-infected versus uninfected individuals. PBMCs were stained with anti-BDCA-2 (FITC) and anti-CD123 (PE) to measure the levels of pDCs. Horizontal lines represent median values. B) pDCs are susceptible to Fas-mediated apoptosis. Fas was crosslinked on the surface of PBMCs, and resultant pDC apoptosis was measured by Annexin V staining. n.s., not significant. C–E) pDC cell death induced by HIV-infected cells. 2×10 6 CEM cells chronically infected with HIV (CEM-IIIB) were incubated for 8 h with 10 7 PBMCs from HIV-infected donors. The levels of apoptosis and necrosis were assessed by Annexin V and 7-AAD staining. Cells within the pDC (BDCA-2 + /CD123 + ) population that were double-positive for both Annexin V and 7-AAD were considered necrotic, while those positive for Annexin V and negative for 7-AAD were considered apoptotic. In some cultures, 100 µg/ml T20 was added. C) Cells expressing HIV induce pDC death. The percent of pDCs that were necrotic (Annexin V + /7-AAD + ) is indicated on the y-axis. PBMCs alone versus PBMCs+CEM-IIIB are shown. As indicated, CEM-IIIB cells showed a significant induction of cell death vs. cells alone. D) Representative examples of T20-mediated blockade of CEM-IIIB-induced pDC cell death. E) Cell death is blocked by T20. Cells were incubated with CEM cells alone (left panel), CEM-IIIB, or CEM-IIIB+T20, and the percentage of pDCs in the apoptotic or necrotic quadrants was quantified. No cell death above baseline was induced by CEM cells alone. Both types of cell death induced by CEM-IIB cells were significantly reduced (p values indicated) in the presence of T20.

    Journal: PLoS ONE

    Article Title: Impact of HIV on Cell Survival and Antiviral Activity of Plasmacytoid Dendritic Cells

    doi: 10.1371/journal.pone.0000458

    Figure Lengend Snippet: pDC killing in HIV-infected individuals. A) Plasmacytoid dendritic cell numbers are reduced in HIV-infected versus uninfected individuals. PBMCs were stained with anti-BDCA-2 (FITC) and anti-CD123 (PE) to measure the levels of pDCs. Horizontal lines represent median values. B) pDCs are susceptible to Fas-mediated apoptosis. Fas was crosslinked on the surface of PBMCs, and resultant pDC apoptosis was measured by Annexin V staining. n.s., not significant. C–E) pDC cell death induced by HIV-infected cells. 2×10 6 CEM cells chronically infected with HIV (CEM-IIIB) were incubated for 8 h with 10 7 PBMCs from HIV-infected donors. The levels of apoptosis and necrosis were assessed by Annexin V and 7-AAD staining. Cells within the pDC (BDCA-2 + /CD123 + ) population that were double-positive for both Annexin V and 7-AAD were considered necrotic, while those positive for Annexin V and negative for 7-AAD were considered apoptotic. In some cultures, 100 µg/ml T20 was added. C) Cells expressing HIV induce pDC death. The percent of pDCs that were necrotic (Annexin V + /7-AAD + ) is indicated on the y-axis. PBMCs alone versus PBMCs+CEM-IIIB are shown. As indicated, CEM-IIIB cells showed a significant induction of cell death vs. cells alone. D) Representative examples of T20-mediated blockade of CEM-IIIB-induced pDC cell death. E) Cell death is blocked by T20. Cells were incubated with CEM cells alone (left panel), CEM-IIIB, or CEM-IIIB+T20, and the percentage of pDCs in the apoptotic or necrotic quadrants was quantified. No cell death above baseline was induced by CEM cells alone. Both types of cell death induced by CEM-IIB cells were significantly reduced (p values indicated) in the presence of T20.

    Article Snippet: HIV p24 assays 2–4×108 total PBMCs from HIV-infected individuals were stimulated overnight with anti-CD3 antibody and rIL-2 (Roche).

    Techniques: Infection, Staining, Incubation, Expressing

    CD200R potentiates IFNG mRNA production in systemic lupus erythematosus patients. ( A, B ) IFNG mRNA production relative to GAPDH (A) or B2M (B) by healthy control and SLE patients’ PBMC in two cohorts. PBMC were seeded on an isotype control (open circles) or an agonistic anti-CD200R antibody (filled circles) prior to stimulation with R848 for 4h. Each connected dot represents one patient/control. The color of the connecting arrow indicates: inhibition by CD200R ligation (blue, > 15% inhibition compared to isotype control); potentiation by CD200R ligation (red, > 15% potentiation compared to isotype control); or no effect of CD200R ligation (grey,

    Journal: bioRxiv

    Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

    doi: 10.1101/2020.02.06.933739

    Figure Lengend Snippet: CD200R potentiates IFNG mRNA production in systemic lupus erythematosus patients. ( A, B ) IFNG mRNA production relative to GAPDH (A) or B2M (B) by healthy control and SLE patients’ PBMC in two cohorts. PBMC were seeded on an isotype control (open circles) or an agonistic anti-CD200R antibody (filled circles) prior to stimulation with R848 for 4h. Each connected dot represents one patient/control. The color of the connecting arrow indicates: inhibition by CD200R ligation (blue, > 15% inhibition compared to isotype control); potentiation by CD200R ligation (red, > 15% potentiation compared to isotype control); or no effect of CD200R ligation (grey,

    Article Snippet: mRNA isolation, cDNA synthesis and qPCR analysis mRNA from the PBMC discovery cohort was isolated using an mRNA capture kit (Roche) and cDNA was synthesized with the Reverse transcriptase kit (Promega) according to manufacturer protocol. mRNA from all other PBMC cohorts was isolated using QIAGEN mRNA micro isolation kits, followed by iScript (Biorad) cDNA synthesis according to manufacturer protocol. qPCR was performed on a QuantStudio 12K Flex or a StepOnePlus Realtime PCR system (AB Instruments) with SYBR Select Master Mix (Life Technologies), or with TaqMan, where indicated, with 5ng cDNA input and 400nM of each primer using the fast protocol with melt curve as is suggested for SYBR Select Master Mix.

    Techniques: Inhibition, Ligation

    Exposure to type I IFN reverses CD200R function in PBMC ( A ) Cytokine secretion by moDC stimulated with R848 for 6h in presence of control, Akt-inhibitor AktVIII or Mek-inhibitor U0126. IL-1β: n=5, IL-8: n=8. Significance was determined by one-sample t test. ( B, C ) Phosphorylation of p-Akt473 and p-rpS6 in HC moDC. Fresh replated (n=8, 3 samples without IFN-counterpart) or IFNα-stimulated (16h, n=5, paired with fresh samples) moDCs were seeded on plates coated with control (recombinant human CD200R1, rhCD200R1) or recombinant human CD200 (rhCD200). After 2h pre-incubation, the moDC were stimulated with 1µg/ml R848 for 30 minutes. Phosphorylation was assessed by flow cytometry. Depicted is the ratio of phosphorylation in CD200-stimulated conditions over control stimulated conditions. Significance between the effect of CD200R on fresh and IFNα stimulated cells was determined by t test. Significance of the effect of CD200R in medium or IFNα stimulated cells was determined by one-sample t test. ( D, E ) mRNA (n=11) and protein (n=6) data on R848-induced IFNG or IFNγ production in PBMC. Donors with blue lines are matched between mRNA and protein data. Significance was determined by Wilcoxon test. ND = not detectable. ( F ) The ratio of R848 induced IFNG mRNA production of PBMC stimulated with anti-CD200R over PBMC stimulated with isotype. IFNG mRNA production was measured in healthy control PBMC that were used fresh after isolation (Fr., n=14), or have been cultured for 16h in medium (Med, n=20), 10 (n=8), 100 (n=8) or 1000 (n=20) U/ml of IFNα prior to ligation of CD200R with an agonistic antibody and stimulation of TLR7/8 with R848 for 4h. Significance was determined by a paired t test with Welch correction.

    Journal: bioRxiv

    Article Title: Inhibitory CD200-receptor signaling is rewired by type I interferon

    doi: 10.1101/2020.02.06.933739

    Figure Lengend Snippet: Exposure to type I IFN reverses CD200R function in PBMC ( A ) Cytokine secretion by moDC stimulated with R848 for 6h in presence of control, Akt-inhibitor AktVIII or Mek-inhibitor U0126. IL-1β: n=5, IL-8: n=8. Significance was determined by one-sample t test. ( B, C ) Phosphorylation of p-Akt473 and p-rpS6 in HC moDC. Fresh replated (n=8, 3 samples without IFN-counterpart) or IFNα-stimulated (16h, n=5, paired with fresh samples) moDCs were seeded on plates coated with control (recombinant human CD200R1, rhCD200R1) or recombinant human CD200 (rhCD200). After 2h pre-incubation, the moDC were stimulated with 1µg/ml R848 for 30 minutes. Phosphorylation was assessed by flow cytometry. Depicted is the ratio of phosphorylation in CD200-stimulated conditions over control stimulated conditions. Significance between the effect of CD200R on fresh and IFNα stimulated cells was determined by t test. Significance of the effect of CD200R in medium or IFNα stimulated cells was determined by one-sample t test. ( D, E ) mRNA (n=11) and protein (n=6) data on R848-induced IFNG or IFNγ production in PBMC. Donors with blue lines are matched between mRNA and protein data. Significance was determined by Wilcoxon test. ND = not detectable. ( F ) The ratio of R848 induced IFNG mRNA production of PBMC stimulated with anti-CD200R over PBMC stimulated with isotype. IFNG mRNA production was measured in healthy control PBMC that were used fresh after isolation (Fr., n=14), or have been cultured for 16h in medium (Med, n=20), 10 (n=8), 100 (n=8) or 1000 (n=20) U/ml of IFNα prior to ligation of CD200R with an agonistic antibody and stimulation of TLR7/8 with R848 for 4h. Significance was determined by a paired t test with Welch correction.

    Article Snippet: mRNA isolation, cDNA synthesis and qPCR analysis mRNA from the PBMC discovery cohort was isolated using an mRNA capture kit (Roche) and cDNA was synthesized with the Reverse transcriptase kit (Promega) according to manufacturer protocol. mRNA from all other PBMC cohorts was isolated using QIAGEN mRNA micro isolation kits, followed by iScript (Biorad) cDNA synthesis according to manufacturer protocol. qPCR was performed on a QuantStudio 12K Flex or a StepOnePlus Realtime PCR system (AB Instruments) with SYBR Select Master Mix (Life Technologies), or with TaqMan, where indicated, with 5ng cDNA input and 400nM of each primer using the fast protocol with melt curve as is suggested for SYBR Select Master Mix.

    Techniques: Recombinant, Incubation, Flow Cytometry, Isolation, Cell Culture, Ligation

    Frequency of T-cell subpopulations according to changes in HIV-1 DNA copies at 1 month from vaccination. HIV-1 DNA copies/10 6 peripheral blood mononuclear cells (PBMCs) were measured in PBMCs from 22 HIV-1-infected children (A) . In the group “decrease,” 12 children are included who displayed > 10% decrease in the number of HIV-1 DNA copies at 1 month from last vaccination as compared with BL value; in the group “stable,” 5 children who displayed minor variations (

    Journal: Frontiers in Immunology

    Article Title: Hepatitis B Virus Vaccination in HIV-1-Infected Young Adults: A Tool to Reduce the Size of HIV-1 Reservoirs?

    doi: 10.3389/fimmu.2017.01966

    Figure Lengend Snippet: Frequency of T-cell subpopulations according to changes in HIV-1 DNA copies at 1 month from vaccination. HIV-1 DNA copies/10 6 peripheral blood mononuclear cells (PBMCs) were measured in PBMCs from 22 HIV-1-infected children (A) . In the group “decrease,” 12 children are included who displayed > 10% decrease in the number of HIV-1 DNA copies at 1 month from last vaccination as compared with BL value; in the group “stable,” 5 children who displayed minor variations (

    Article Snippet: Measurement of Total PBMC HIV-1 DNA The PBMC DNA was obtained by manual extraction with the High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland).

    Techniques: Infection

    Antibody titers to hepatitis B virus vaccine and HIV-1 DNA copies in peripheral blood mononuclear cells (PBMCs) of HIV-1-infected children pre-vaccination and postvaccination. Antibodies to the HBsAg were detected in plasma (A) and HIV-1 DNA copies/10 6 PBMCs were measured in PBMCs (B) from 22 HIV-1-infected children pre-vaccination and at 1 month postvaccination. ns, not significant; BL, baseline; M1, month 1.

    Journal: Frontiers in Immunology

    Article Title: Hepatitis B Virus Vaccination in HIV-1-Infected Young Adults: A Tool to Reduce the Size of HIV-1 Reservoirs?

    doi: 10.3389/fimmu.2017.01966

    Figure Lengend Snippet: Antibody titers to hepatitis B virus vaccine and HIV-1 DNA copies in peripheral blood mononuclear cells (PBMCs) of HIV-1-infected children pre-vaccination and postvaccination. Antibodies to the HBsAg were detected in plasma (A) and HIV-1 DNA copies/10 6 PBMCs were measured in PBMCs (B) from 22 HIV-1-infected children pre-vaccination and at 1 month postvaccination. ns, not significant; BL, baseline; M1, month 1.

    Article Snippet: Measurement of Total PBMC HIV-1 DNA The PBMC DNA was obtained by manual extraction with the High Pure Viral Nucleic Acid Kit (Roche, Basel, Switzerland).

    Techniques: Infection