Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: Patient information.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques:

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: ZIKV neutralizing antibody titers detectable within days after ZIKV infection. ( A ) ZIKV-specific plasma IgM and IgG titers for patient ZK016 at 3 and 7 DPO and for ZK018 at 8 DPO. Titers were determined by ZIKV IgM antibody capture enzyme linked immunosorbent assay (MAC-ELISA) and modified IgG ELISA. The values are represented as ratios of the optical densities of patient plasma to healthy control plasma. For IgM, a ratio >3 = positive (dark gray), 2–3 = equivocal (light gray), >2 = negative (white) and for IgG, a ratio >1.5 = positive (dark gray), 1.2–1.5 = equivocal (light gray), >1.2 = negative (white). ( B ) Plasma endpoint IgG binding titers to ZIKV lysate as determined by ELISA. The dotted line indicates the starting plasma dilution of 1:100. ( C ) The Focus Reduction Neutralization Test (FRNT) titers of plasma from patients against ZIKV and DENV1-4. The values plotted are the plasma dilutions required for 50% neutralization of ZIKV foci, termed FRNT50. The FRNT assay for each sample was repeated in two or more independent experiments, and the mean values are plotted. The dotted line represents the starting plasma dilution of 1:30.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Infection, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Modification, Control, Binding Assay, Neutralization

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: Virus-specific plasmablasts appear in circulation early after ZIKV infection. ( A ) Flow cytometry plots showing patient plasmablast (CD3− CD19+ CD20−/low CD38high CD27high) frequencies as a fraction of total CD19+ B cells. PBMCs were isolated from whole blood collected from patient ZK016 (7 DPO) and patient ZK018 (8 DPO) for flow cytometry analysis and plasmablast sorting. The workflow chart follows the generation and characterization of mAbs from single-cell sorted plasmablasts. ( B ) Representative ELISpot showing ZIKV E protein and NS1 protein-specific antibody secreting cells (ASCs). The wells shown contain 7 DPO PBMCs from patient ZK016. The percentages beside the wells represent the frequency of antigen-specific ASCs relative to the total number of IgG + ASCs.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Virus, Infection, Flow Cytometry, Isolation, Enzyme-linked Immunospot

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: Clonality and mutational characteristics of the ZIKV plasmablast response. ( A ) Clonal relatedness of heavy chain variable region (VH) sequences amplified from patient plasmablasts. The number at the center of each pie chart is the total number of heavy chain sequences analyzed, including unpaired VH sequences that were not pursued for mAb synthesis. The numbers within the pie slices denote the number of clones with a shared VH rearrangement and junction. The percentage of total clonal sequences per donor is shown to the bottom right of the pie charts. ( B ) Average per donor VH mutation frequencies of the two ZIKV patients compared to historical data. Each circle represents the average number of VH nucleotide mutations per donor. Somatic hypermutation frequencies in naive, memory and germinal center B cells, and peripheral B cells from influenza and dengue infected donors were derived from previously published data [ , ]. ( C ) The range of VH mutation frequencies in the two ZIKV patients. Each circle represents the number of VH mutations per mAb sequence. The binding specificities of the mAbs to ZIKV and DENV are color coded: Cross-reactive mAbs are shown in blue, ZIKV-reactive in green and mAbs of undetermined specificity in orange. ( D ) VH CDR3 amino acid length distribution of all mAb sequences per patient.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Amplification, Clone Assay, Mutagenesis, Infection, Derivative Assay, Sequencing, Binding Assay

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: The recall ZIKV plasmablast response is highly cross-reactive against DENV. Heat map showing the binding potency of the ZK016 and ZK018 mAbs against ZIKV and DENV. The mAbs generated from single-cell sorted plasmablasts were tested for reactivity against ZIKV and DENV1-4 recombinant E protein (E) and whole virion by ELISA. The mAbs that are fusion loop (FL)-specific are indicated with asterisks. In terms of scale, brown represents the highest and white the lowest potency of binding based on minimum effective concentration. The minimum effective concentration is the minimum mAb concentration required to obtain three times the background signal. The results plotted are mean values from two or more independent ELISA experiments.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: Plasmablast-derived mAbs from dengue-experienced individual preferentially neutralize DENV compared to ZIKV. The mAbs generated from patient ZK016 ( left panels) and ZK018 ( right panels) were tested for neutralization activity against ZIKV (white bar) and DENV2 (black bar). FL-specific mAbs are indicated by asterisks. The values plotted represent mAb concentration required for 50% reduction of viral foci, termed FRNT50. The vertical dotted lines indicate the maximum concentrations of mAb tested (10 μg/mL). The horizontal dotted line separates the positive control, dengue plasmablast-derived mAb 33.3A06 reported previously . The results plotted are mean values from two or more independent experiments.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Derivative Assay, Generated, Neutralization, Activity Assay, Concentration Assay, Positive Control

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: ZIKV plasmablast responses enhance DENV2 infection in vitro. ( A ) Representative flow cytometry plots comparing ADE activity of plasma from ZK016 vs. healthy control. Boxed population represents DENV2-infected cells based on positive 4G2 staining. Plots shown display ADE at plasma dilution of 1:3000. ( B ) DENV2 ADE activities of plasma from ZK016, ZK018 and a flavivirus-naïve healthy donor (dotted line). ( C ) DENV2 ADE activities of FL-specific and non FL-specific mAbs from ZK016 and ZK018.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Infection, In Vitro, Flow Cytometry, Activity Assay, Clinical Proteomics, Control, Staining

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: ZIKV infection drives activation of NS1-specific plasmablast responses. ( A ) Binding curves of ZK016 and ZK018 NS1-specific mAbs. Antibodies were tested for binding to ZIKV and DENV2 recombinant NS1 proteins by ELISA. The dotted lines represent three times the background signal obtained with plain blocking buffer. ( B ) Binding activity of mAbs to ZIKV (white bar) or DENV2 (black bar) recombinant NS1 protein by ELISA. The values plotted represent the minimum concentrations required to obtain three times the background signal. The dotted line indicates the maximum concentration of mAbs tested in ELISA (10 μg/mL). Results plotted are representative of two or more independent ELISA experiments.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Infection, Activation Assay, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Blocking Assay, Activity Assay, Concentration Assay

Journal: Molecular Cancer Therapeutics

Article Title: DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis

doi: 10.1158/1535-7163.mct-07-2383

Figure Lengend Snippet: Figure 1. CD44 antisense transfected DA3 parental cells: generation and tumorigenecity. Schematic presentation of the CD44 gene, primers, and plasmid. Arrowheads, positions of the 5¶ and 3¶ ORF primers-sequence. Hatched columns, 5¶ and 3¶ constant exons (excluding introns); open columns, variant exons; lines connecting the exons, intron sequences. The purified CD44v4-v10 PCR product was digested with EcoRI and ligated into the pcDNA3.1 vector further digested with BamHI to assess insert orientation (A). Antisense transfected parental DA3 cell clones (as7 and as17) with CD44-specific antisense plasmid reduced the expression of their CD44 receptor when compared with the parental-DA3P-TRo cells as assessed by mean fluorescence intensity (B). Decrease of local tumor growth and lung metastases following i.m. inoculation (C). Note that the size of the local tumor growth is associated with its metastatic capacity. **, P < 0.01; *, P < 0.05, Mann-Whitney versus DA3P cells.

Article Snippet: © 2008 American Association for Cancermct.aacrjournals.org Downloaded from mAbs and Other Reagents Rat KM81 (IgG2b) anti-mouse pan CD44 mAb (constant region-specific) was generated from the American Type Culture Collection hybridomas (18).

Techniques: Transfection, Plasmid Preparation, Sequencing, Variant Assay, Purification, Clone Assay, Expressing, Fluorescence, MANN-WHITNEY

Journal: Molecular Cancer Therapeutics

Article Title: DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis

doi: 10.1158/1535-7163.mct-07-2383

Figure Lengend Snippet: Figure 2. DA3hi cells express CD44 epitopes more intensively than DA3lo

Article Snippet: © 2008 American Association for Cancermct.aacrjournals.org Downloaded from mAbs and Other Reagents Rat KM81 (IgG2b) anti-mouse pan CD44 mAb (constant region-specific) was generated from the American Type Culture Collection hybridomas (18).

Techniques:

Journal: Molecular Cancer Therapeutics

Article Title: DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis

doi: 10.1158/1535-7163.mct-07-2383

Figure Lengend Snippet: Figure 3. DA3hi cells transfected with CD44-specific antisense plasmid down-regulate their CD44 receptor and display reduced local tumor growth and fewer lung metastases. Flow cytometry analysis of three different DA3hi cloned cells: DA3hi-as2, DA3hi-as3, and DA3hi-as5 (A, 2-4) transfected with CD44-specific antisense plasmids displayed gradually reduced expression of CD44 when their mean fluorescence intensities were compared with those of the parental DA3hi cells (A, 1). Following i.m. inoculation, cloned DA3hi cells containing antisense plasmids showed gradually reduced local tumor growth (B, 2-4) when compared with those of DA3hi cells (B, 1). Lung metastases assessed by measuring lung weight at day 21 (C). ***, P < 0.001; **, P < 0.01; *, P < 0.05, Mann- Whitney versus DA3hi-TRo cells. Pearson linear correlation between local tumor growth or metastasis formation (D). A positive correlation was observed between CD44 expression and local tumor growth (R = 0.98) or metastatic capacity (R = 0.93).

Article Snippet: © 2008 American Association for Cancermct.aacrjournals.org Downloaded from mAbs and Other Reagents Rat KM81 (IgG2b) anti-mouse pan CD44 mAb (constant region-specific) was generated from the American Type Culture Collection hybridomas (18).

Techniques: Transfection, Plasmid Preparation, Flow Cytometry, Clone Assay, Expressing, Fluorescence, MANN-WHITNEY

Journal: Molecular Cancer Therapeutics

Article Title: DNA vaccination with CD44 variant isoform reduces mammary tumor local growth and lung metastasis

doi: 10.1158/1535-7163.mct-07-2383

Figure Lengend Snippet: Figure 6. Namalwa cells expressing human CD44 molecules interact with blood serum obtained from mice immunized with VLN containing hCD44v3-v10 plasmids. Serum obtained from mice immunized with VLN loaded with hCD44v-cDNA (pcDNAv4-10), but not with hCD44s-cDNA (pcDNAs) or empty vector (pcDNA3.1), enabled flow cytometry detection, over background, of hCD44v-transfected Namalwa cells but not of the hCD44s-transfected Namalwa cells, indicating the presence of anti-human CD44v antibodies in these mice. Immunization with VLN loaded with CD44s cDNA generated lower level of antibodies detected with Namalwa cells transfected with CD44v, but not with CD44s, cDNA, suggesting better exposure of constant epitope(s) on the former cells. Background staining of the transfected Namalwa cells after immunization with VLN containing an empty vector (pcDNA3.1) is marked by X. The relative fluorescence intensity of the stained cells following their interaction with the fluorescein- conjugated second antibody is shown. * P < 0.05, Mann-Whitney.

Article Snippet: © 2008 American Association for Cancermct.aacrjournals.org Downloaded from mAbs and Other Reagents Rat KM81 (IgG2b) anti-mouse pan CD44 mAb (constant region-specific) was generated from the American Type Culture Collection hybridomas (18).

Techniques: Expressing, Plasmid Preparation, Flow Cytometry, Transfection, Generated, Staining, Fluorescence, MANN-WHITNEY

Journal: Clinical and Translational Radiation Oncology

Article Title: 18 F-DCFPyL (PSMA) PET as a radiotherapy response assessment tool in metastatic prostate cancer

doi: 10.1016/j.ctro.2023.100583

Figure Lengend Snippet: PSMA expression in prostate cancer cell lines and its modulation by radiation treatment. (A.) Immunoblotting analysis of PSMA, neuron-specific enolase (NSE) and GAPDH in untreated and irradiated (0, 2 or 8 Gy) LNCAp, 22RV1, DU145, and PC3 cells. Representative immunoblots of three independent experiments are shown. (B.) Immunoblot densitometric analysis of the experiments in (A.) for PSMA and NSE. Densitometry values for PSMA and NSE in each sample were normalized to GAPDH. Individual values were then normalized to the mean value of control (mock-radiated) samples. Graph shows Mean ± SE of 3 independent experiments; * p < 0.05, *** p < 0.001; two-way ANOVA test). (C.) RT-qPCR analysis of PSMA (FOLH1 gene). ΔCT values were obtained by normalization to 18S ribosomal RNA. Graph shows Mean ± SE values for LNCAp and 22RV1 cells treated with 0 Gy (mock) or 5 Gy RT of 3 independent experiments (differences were not statistically significant).

Article Snippet: TaqMan probes used: Catalog ID: 18 s: Hs03003631_g1 and FOLH1 Hs00379515_m (Life Technologies, CA).

Techniques: Expressing, Western Blot, Irradiation, Control, Quantitative RT-PCR

Journal: Clinical and Translational Radiation Oncology

Article Title: 18 F-DCFPyL (PSMA) PET as a radiotherapy response assessment tool in metastatic prostate cancer

doi: 10.1016/j.ctro.2023.100583

Figure Lengend Snippet: PSMA expression in radiation-resistant clones of prostate cancer cells and xenografts. (A.) Representative immunoblots of PSMA, neuron-specific enolase (NSE) and GAPDH expression in parental 22RV1 (22RV1-P), radio-resistant 22RV1 (22RV1-RR), parental DU145 (DU145-P), radio-resistant of DU145 (DU145-RR). (B.) Densitometric analysis of PSMA and NSE immunoblots (Mean ± SE of 3 independent experiments; * p < 0.05, **** p < 0.0001; one-way ANOVA). (C.) RT-qPCR analysis of PSMA gene (FOLH1) expression in 22RV1-P and 22RV1-RR cells. ΔCT values were obtained by normalization to 18S ribosomal RNA (Mean ± SE of 3 independent experiments; * p < 0.05; unpaired T-test). (D.) Representative images of IHC analysis of 22RV1-P and 22RV1-RR xenografts (40x magnification) (tumor growth curves are shown in Fig.s2, 6 animals per treatment group). PSMA and NSE staining are shown for mock- (0 Gy) or radiation-treated (1 × 5 Gy) tumors. (E.) H-score analysis of PSMA staining in mock-treated or irradiated xenografts above (Mean ± SE of 3 xenografts analyzed × 10 high power fields quantified per xenograft). (F.) RT-qPCR analysis of PSMA (FOLH1 gene) expression in 22RV1-P and 22RV1-RR xenografts. ΔCT values were obtained by normalization to 18S ribosomal RNA. (Mean ± SE, 6 xenograft tumors per group; * p < 0.05, **** p < 0.0001; unpaired T-test).

Article Snippet: TaqMan probes used: Catalog ID: 18 s: Hs03003631_g1 and FOLH1 Hs00379515_m (Life Technologies, CA).

Techniques: Expressing, Clone Assay, Western Blot, Quantitative RT-PCR, Staining, Irradiation, Gene Expression

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: Virus-specific plasmablasts appear in circulation early after ZIKV infection. ( A ) Flow cytometry plots showing patient plasmablast (CD3− CD19+ CD20−/low CD38high CD27high) frequencies as a fraction of total CD19+ B cells. PBMCs were isolated from whole blood collected from patient ZK016 (7 DPO) and patient ZK018 (8 DPO) for flow cytometry analysis and plasmablast sorting. The workflow chart follows the generation and characterization of mAbs from single-cell sorted plasmablasts. ( B ) Representative ELISpot showing ZIKV E protein and NS1 protein-specific antibody secreting cells (ASCs). The wells shown contain 7 DPO PBMCs from patient ZK016. The percentages beside the wells represent the frequency of antigen-specific ASCs relative to the total number of IgG + ASCs.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Virus, Infection, Flow Cytometry, Isolation, Enzyme-linked Immunospot

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: Clonality and mutational characteristics of the ZIKV plasmablast response. ( A ) Clonal relatedness of heavy chain variable region (VH) sequences amplified from patient plasmablasts. The number at the center of each pie chart is the total number of heavy chain sequences analyzed, including unpaired VH sequences that were not pursued for mAb synthesis. The numbers within the pie slices denote the number of clones with a shared VH rearrangement and junction. The percentage of total clonal sequences per donor is shown to the bottom right of the pie charts. ( B ) Average per donor VH mutation frequencies of the two ZIKV patients compared to historical data. Each circle represents the average number of VH nucleotide mutations per donor. Somatic hypermutation frequencies in naive, memory and germinal center B cells, and peripheral B cells from influenza and dengue infected donors were derived from previously published data [ , ]. ( C ) The range of VH mutation frequencies in the two ZIKV patients. Each circle represents the number of VH mutations per mAb sequence. The binding specificities of the mAbs to ZIKV and DENV are color coded: Cross-reactive mAbs are shown in blue, ZIKV-reactive in green and mAbs of undetermined specificity in orange. ( D ) VH CDR3 amino acid length distribution of all mAb sequences per patient.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Amplification, Clone Assay, Mutagenesis, Infection, Derivative Assay, Sequencing, Binding Assay

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: The recall ZIKV plasmablast response is highly cross-reactive against DENV. Heat map showing the binding potency of the ZK016 and ZK018 mAbs against ZIKV and DENV. The mAbs generated from single-cell sorted plasmablasts were tested for reactivity against ZIKV and DENV1-4 recombinant E protein (E) and whole virion by ELISA. The mAbs that are fusion loop (FL)-specific are indicated with asterisks. In terms of scale, brown represents the highest and white the lowest potency of binding based on minimum effective concentration. The minimum effective concentration is the minimum mAb concentration required to obtain three times the background signal. The results plotted are mean values from two or more independent ELISA experiments.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Binding Assay, Generated, Recombinant, Enzyme-linked Immunosorbent Assay, Concentration Assay

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: Plasmablast-derived mAbs from dengue-experienced individual preferentially neutralize DENV compared to ZIKV. The mAbs generated from patient ZK016 ( left panels) and ZK018 ( right panels) were tested for neutralization activity against ZIKV (white bar) and DENV2 (black bar). FL-specific mAbs are indicated by asterisks. The values plotted represent mAb concentration required for 50% reduction of viral foci, termed FRNT50. The vertical dotted lines indicate the maximum concentrations of mAb tested (10 μg/mL). The horizontal dotted line separates the positive control, dengue plasmablast-derived mAb 33.3A06 reported previously . The results plotted are mean values from two or more independent experiments.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Derivative Assay, Generated, Neutralization, Activity Assay, Concentration Assay, Positive Control

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: ZIKV plasmablast responses enhance DENV2 infection in vitro. ( A ) Representative flow cytometry plots comparing ADE activity of plasma from ZK016 vs. healthy control. Boxed population represents DENV2-infected cells based on positive 4G2 staining. Plots shown display ADE at plasma dilution of 1:3000. ( B ) DENV2 ADE activities of plasma from ZK016, ZK018 and a flavivirus-naïve healthy donor (dotted line). ( C ) DENV2 ADE activities of FL-specific and non FL-specific mAbs from ZK016 and ZK018.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Infection, In Vitro, Flow Cytometry, Activity Assay, Clinical Proteomics, Control, Staining

Journal: Viruses

Article Title: Pre-Existing Dengue Immunity Drives a DENV-Biased Plasmablast Response in ZIKV-Infected Patient

doi: 10.3390/v11010019

Figure Lengend Snippet: ZIKV infection drives activation of NS1-specific plasmablast responses. ( A ) Binding curves of ZK016 and ZK018 NS1-specific mAbs. Antibodies were tested for binding to ZIKV and DENV2 recombinant NS1 proteins by ELISA. The dotted lines represent three times the background signal obtained with plain blocking buffer. ( B ) Binding activity of mAbs to ZIKV (white bar) or DENV2 (black bar) recombinant NS1 protein by ELISA. The values plotted represent the minimum concentrations required to obtain three times the background signal. The dotted line indicates the maximum concentration of mAbs tested in ELISA (10 μg/mL). Results plotted are representative of two or more independent ELISA experiments.

Article Snippet: ZIKV E-specific mAbs that target the conserved FL domain in the ZIKV E protein were determined by competing them against FL-specific mouse mAbs 4G2 (ATCC; Manassas, VA, USA HB-112) [ ] and E18 (BEI Resources; Manassas, VA, USA; NR-10135) [ ] using an ELISA-based assay.

Techniques: Infection, Activation Assay, Binding Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Blocking Assay, Activity Assay, Concentration Assay

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: (A—B) U937 cell macrophages containing either an shRNA targeting TRIF (TRIF KD) or a non-targeting scramble shRNA (SCM) were infected with strain 130b (A) or Philadelphia-1 (B) at a multiplicity of infection (MOI) of 0.5, and the levels of IL-6 in culture supernatants at 12, 24, and 48 h post infection (HPI) were then determined by single-cytokine ELISA. (C) SCM and TRIF KD cells were infected with strain 130b at a MOI of 20, and the levels of IL-6 (left) and TNFα (right) in culture supernatants after 9 h of infection were ascertained by single-cytokine ELISA. (D) PBMC-derived macrophages containing either an siRNA targeting TRIF (TRIF KD) or a non-targeting scramble siRNA (SCM) were infected with strain 130b at a MOI of 20 for 9 h, and then the levels of secreted IL-6 (left) and TNFα (right) determined by ELISA. The graphs in (A–D) show the average cytokine levels pooled from three (A -C) or two (D) independent experiments, each done in technical triplicate, with standard errors. The cytokine levels (pg/ml) were calculated relative to serial dilution of recombinant cytokine controls. Asterisks indicate points at which the values for samples from KD cells were significantly different from those for samples from SCM cells (** P < 0.01, *** P < 0.001, by Student’s t test).

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: shRNA, Infection, Enzyme-linked Immunosorbent Assay, Derivative Assay, Serial Dilution, Recombinant

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: (A) U937 cell macrophages containing either a mutation in the TRIF gene (TRIF KO1, TRIF KO2) or MyD88 gene (MyD88 KO1, MyD88 KO2) or a non-targeting CRISPR Guide plasmid (CTL) were infected with strain 130b at MOI of 20, and levels of IL-6 (top) and TNFα (bottom) in culture supernatants obtained at 0, 1, 3, 6, 9, 12, 24, and 48 h (as indicated) were determined by ELISA. The cytokine levels (pg/ml) were calculated relative to serial dilution of recombinant cytokine controls, and the presented graphs show the average cytokine levels (n = 3) pooled from three independent experiments, with standard errors. Asterisks indicate points at which the values for samples from KO cells were significantly different from those for samples from control cells (* P < 0.05, ** P < 0.01, *** P < 0.001, by Student’s t test). (B) CTL, TRIF KO, and MyD88 KO U937 cells were infected with L . pneumophila strain 130b at a MOI of 20, and the secreted levels of IL-6, IL-1β, IL-10, TNFα, and IL-8 at 9 h post infection were then ascertained by multiplex ELISA. Cytokine levels resulting from infection of the control were set to a value of 1, and the levels from the various infected KO cells were normalized to the value of the control. Multiplex represents analysis of three pooled biological replicates ( n = 3 ) each done in technical triplicate.

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: Mutagenesis, CRISPR, Plasmid Preparation, Infection, Enzyme-linked Immunosorbent Assay, Serial Dilution, Recombinant, Control, Multiplex Assay

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: U937 cells expressing a non-targeting CRISPR guide plasmid (CTL, black bars) and independent clones of U937 cells containing a CRISPR/Cas9-generated mutation (KO1, KO2; purple and magenta bars) in either TLR3 (A), TLR4 (B), TLR2 (C), TLR5 (D), TLR9 (E), TRAF6 (F), or TRAM (G) were infected with strain 130b at a MOI of 20, and the levels of secreted IL-6 and TNFα at 9 h post infection were then determined by ELISA. The cytokine levels (pg/ml) were calculated relative to serial dilution of recombinant cytokine controls. Graphs show the average cytokine levels ( n = 3) pooled from three independent experiments, done in technical triplicate, with standard errors. Asterisks indicate points at which the values for samples from KO cells were significantly different from those for samples from CTL cells (* P < 0.05, ** P < 0.01, *** P < 0.001, by Student’s t test).

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: Expressing, CRISPR, Plasmid Preparation, Clone Assay, Generated, Mutagenesis, Infection, Enzyme-linked Immunosorbent Assay, Serial Dilution, Recombinant

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: (A) U937 cells and (B) human PBMC-derived macrophages were pre-treated with either the TLR3 inhibitor Cu CPT 4a (T3i), TLR4 inhibitor TAK 242 (T4i), or vehicle control, DMSO (CTL). The macrophages were then exposed to either Poly I:C, E . coli LPS, or flagellin for 11 h, or infected for 9 h with strain 130b inoculated at a MOI 20 and levels of IL-6 and TNFα determined by ELISA. The cytokine levels (pg/ml) were calculated relative to serial dilution of recombinant cytokine controls. Graphs show the average cytokine levels ( n = 3 ) pooled from three independent experiments, done in technical triplicate, with standard errors. Asterisks indicate points at which the values for samples from the inhibitor-treated cells were significantly different from those samples from CTL cells (* P < 0.05, ** P < 0.01 *** P < 0.001, Student’s t test).

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: Derivative Assay, Control, Infection, Enzyme-linked Immunosorbent Assay, Serial Dilution, Recombinant

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: (A) U937 expressing a non-targeting CRISPR guide plasmid were either not infected (UI) or infected with strain 130b at a MOI of 20 (CTL), and the levels of sixteen different cytokines in culture supernatants were ascertained after 9 h by multiplex ELISA analysis, with the pg/ml levels presented using the indicated color scheme. U937 cells containing a KO of TLR3 or TLR4 (TLR3 KO, TLR4 KO) were similarly infected and assayed for cytokine production. Multiplex represents the analysis of three pooled biological replicates ( n = 3 ) each done in technical triplicate. (B) Neutrophil-like HL-60 cells were exposed to the conditioned tissue culture media obtained from each one of the four macrophage populations noted above and then allowed to migrate across a monolayer of A549 cells into the lower chamber of a 3μm-transwell apparatus. The number of HL-60 cells crossing following exposure to the supernatants obtained from the CTL infection was set to a value of 1, and the levels of migration from the other treatments were normalized to the value of this control. The graph shows the average migration levels ( n = 3) pooled from three independent experiments, done in technical triplicate, with standard errors. Asterisks indicate points at which the values were significantly different from those for samples from the CTL (* P < 0.05, ** P < 0.01 *** P < 0.001, Student’s t test).

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: Expressing, CRISPR, Plasmid Preparation, Infection, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Migration, Control

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: (A) U937 cell macrophages containing either an shRNA targeting TBK1 (TBK1 KD), cGAS (cGAS KD), or STING (STING KD) or a non-targeting scramble shRNA (SCM) were infected with strain 130b at a MOI of 20, and the levels of IL-6 (left panel) and TNFα (right panel) in culture supernatants at 9 h post infection were then determined by single-cytokine ELISA. The cytokine levels (pg/ml) were calculated relative to serial dilution of recombinant cytokine controls. (B) U937 cells expressing a non-targeting CRISPR guide plasmid (CTL, black bars) and U937 cells containing a CRISPR/Cas9-generated mutation in DNA-PK (purple bars) were infected with strain 130b for 9 h following a MOI of 20 and the levels of IL-6 (left) and TNFα (right) in culture supernatants were ascertained by ELISA. The cytokine levels (pg/ml) were calculated relative to serial dilution of recombinant cytokine controls. The graphs show the average cytokine levels ( n = 3 ) pooled from three independent experiments, each done in technical triplicate, with standard errors. Asterisks indicate points at which the values for samples from KD or KO cells were significantly different from those for samples from SCM or CTL cells (* P < 0.05, ** P < 0.01, by Student’s t test).

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: shRNA, Infection, Enzyme-linked Immunosorbent Assay, Serial Dilution, Recombinant, Expressing, CRISPR, Plasmid Preparation, Generated, Mutagenesis

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: (A) U937 cells were pre-treated with either the MALT1 inhibitor Z-VRPR-FMK (MALT1i, yellow bars) or the vehicle control, DMSO (CTL, black bars). They were then exposed to either depleted zymosan (DZ) or E . coli LPS for 12 h (left panel), or infected for 9, 12, 24, or 48 h with strain 130b ( Lpn ) inoculated at a MOI 20 (for 9-h infection; left panel) or 0.5 (for 12, 24, and 48-h infections; right panel), and levels of secreted IL-6 (top) and TNFα (bottom) determined by ELISA. (B) U937 cells expressing a non-targeting CRISPR guide plasmid (CTL, black bars) and two independent clones of U937 cells containing a CRISPR/Cas9-generated MCL mutation (KO1, KO2; purple and magenta bars) were infected for 9, 12, 24, or 48 h with strain 130b ( Lpn ) inoculated at a MOI 20 (for 9-h infection; left panel) or 0.5 (for 12, 24, and 48-h infections; right panel), and levels of secreted IL-6 (top) and TNFα (bottom) determined by ELISA. Cytokine levels (pg/ml) secreted during infection were calculated relative to serial dilution of the recombinant cytokine standard. The graphs show the average cytokine levels ( n = 3) pooled from three independent experiments, done in technical triplicate, with standard errors. Asterisks indicate points at which the values for samples from inhibited cells were significantly different from those for samples from CTL cells (** P < 0.01, *** P < 0.001, by Student’s t test).

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: Control, Infection, Enzyme-linked Immunosorbent Assay, Expressing, CRISPR, Plasmid Preparation, Clone Assay, Generated, Mutagenesis, Serial Dilution, Recombinant

Journal: PLoS Pathogens

Article Title: Human macrophages utilize a wide range of pathogen recognition receptors to recognize Legionella pneumophila, including Toll-Like Receptor 4 engaging Legionella lipopolysaccharide and the Toll-like Receptor 3 nucleic-acid sensor

doi: 10.1371/journal.ppat.1009781

Figure Lengend Snippet: (A) U937 macrophages expressing a non-targeting CRISPR guide plasmid (CTL) and U937 cells containing a CRISPR-generated KO of either TLR4, CD14, MyD88, TRIF, TLR2 or TLR3 were treated with E . coli LPS for 12 h and then levels of secreted IL-6 determined by ELISA. (B) CTL U937 cells were treated with L . pneumophila LPS purified from either log phase (Log) or early-stationary phase (ES) cultures of wildtype strain 130b (WT), and then secreted IL-6 levels determined as above. (C) CTL U937 cells and the various KO U937 cells noted in (A) were treated for 12 h with LPS obtained from ES cultures of strain 130b and then IL-6 determined by ELISA. (D—E) CTL, TLR3 KO, and/or TLR4 KO U937 cells were either exposed to LPS purified from ES WT or lxpP mutant bacteria for 12 h (D) or infected with WT or lxpP mutant bacteria for 9 h following inoculation with a MOI of 20 (E), and their levels of secreted IL-6 were then determined by ELISA. All graphs show the average cytokine levels ( n = 3) pooled from three independent experiments, done in technical triplicate with standard errors. Asterisks indicate either the values for samples from KO cells that were significantly different from those for samples from CTL cells (A, C), the values for samples from the lxpP mutant that were different from those of the WT (D, E), or the significant difference between the samples from log vs. ES phase LPS (B) (* P < 0.05, ** P < 0.01 Student’s t test).

Article Snippet: Specifically, this included the human U937 cell line (ATCC CRL-1593.2), human HEK-293 cell line (ATCC CRL-11268), human A549 cell line (ATCC CCL-185), human HL-60 cell line (ATCC CCL-240), human primary macrophages derived from peripheral blood mononuclear cells (PBMC) (ATCC, #PCS-800-011), and murine primary macrophages derived from either the PBMCs or bone marrow (BMDMs) obtained from 6- to 8-week-old female A/J mice (Jackson Laboratory).

Techniques: Expressing, CRISPR, Plasmid Preparation, Generated, Enzyme-linked Immunosorbent Assay, Purification, Mutagenesis, Bacteria, Infection