Journal: Journal of Fungi

Article Title: Development of a Simple and Robust Whole Blood Assay with Dual Co-Stimulation to Quantify the Release of T-Cellular Signature Cytokines in Response to Aspergillus fumigatus Antigens

doi: 10.3390/jof7060462

Figure Lengend Snippet: Comparison of Aspergillus -induced cytokine release in patients with Aspergillus -associated lung pathologies and other chronic lung diseases. WB from 9 patients with Aspergillus -associated lung pathologies (2 CF with A. fumigatus sensitization, 3 ABPA, and 4 CPA) and a control group of 5 patients with other chronic lung diseases was stimulated with AfuLy or Aspf4 using the RPMI-supplemented, α-CD28-, and α-CD49d-containing stimulation tubes, as described in the Materials and Methods Section. Cytokine concentrations in plasma supernatants were quantified using a 21-plex Luminex assay. ( a , b ) Heat maps indicating median background-adjusted cytokine concentrations in the ABPA/CF/CPA patient cohort “A” and control cohort “C” depending on the antigen used for stimulation. The numeric value in the MMR column represents the median-to-median ratio between the two cohorts, with values > 1.0 indicating greater median cytokine concentrations in the ABPA/CF/CPA cohort. Δ denotes infinite median-to-median ratios (median = 0 pg/mL in the control cohort). ◊ denotes undefined median-to-median ratios (median = 0 pg/mL in both cohorts). ( c , d ) Heat maps summarizing individual cytokine response to AfuLy in all 14 patients. ( e ) Comparison of individual AfuLy-induced concentrations of selected T-helper cell signature cytokines. Medians and interquartile ranges for patients with ABPA/CF/CPA (“A”, red) and controls (“C”, grey) are indicated by black bars and colored boxes, respectively. Two-sided Mann–Whitney U test. Abbreviations: AfuLy = A. fumigatus mycelial lysate, ABPA = allergic bronchopulmonary aspergillosis, CF = cystic fibrosis, CPA = chronic pulmonary aspergillosis, Fractalk. = fractalkine, WB = whole blood.

Article Snippet: The Whole Blood Column Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) was applied to a MidiMACSTM cell separator (Miltenyi Biotec, Bergisch Gladbach, Germany) and rinsed with PBS (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 0.5% human AB serum (Sigma-Aldrich, St. Louis, MO, USA).

Techniques: Comparison, Control, Clinical Proteomics, Luminex, MANN-WHITNEY

Journal:

Article Title: Preclinical and Clinical Performance of the Efoora Test, a Rapid Test for Detection of Human Immunodeficiency Virus-Specific Antibodies

doi: 10.1128/JCM.43.5.2399-2406.2005

Figure Lengend Snippet: Efoora HIV rapid test preclinical testing locations and information

Article Snippet: Specimens tested at sites 2 and 4 were predominantly, if not exclusively, from patients whose infection originated in the United States and thus were probably subtype B. table ft1 table-wrap mode="anchored" t5 TABLE 2. caption a7 Site Date No. of specimens tested Reference test(s) a Specimen type(s) U.S. East Coast March 1999 1,931 Genetic Systems HIV-1/2, Bio-Rad WB Serum February 2000 291 Genetic Systems HIV-1/2, Genetic Systems rLAV EIA, Bio-Rad WB Fresh whole blood February 2001 528 Genetic Systems HIV-1/2, Bio-Rad WB Serum, plasma, whole blood U.S. Midwest June 1999 1,076 Genetic Systems HIV-1/2, Abbott HIV-1/2, Bio-Rad WB Serum, plasma Ivory Coast, West Africa June 1999 962 Enzygnost Plus, immunocapture HIV Serum, plasma South Africa March 2000 150 Genetic Systems HIV-1/2 Serum, plasma, whole blood Open in a separate window a rLAV, recombinant lymphadenopathy-associated virus (HIV-1).

Techniques:

Journal: Scientific Reports

Article Title: Cyto-Feature Engineering: A Pipeline for Flow Cytometry Analysis to Uncover Immune Populations and Associations with Disease

doi: 10.1038/s41598-020-64516-0

Figure Lengend Snippet: Feature engineering identifies CD3+ phenotypes and correlates identified populations. ( a ) The heatmaps show the CD3+ phenotypes that constitute greater than 0.5% of the live leukocytes in at least one sample. Green indicates positive expression, and blue indicates negative expression of all 18 markers used for analysis in the flow cytometry experiment on the x-axis. The plots are separated by four different CD3+ lineages based on CD4 and CD8 expression (double negative immune cells, T helper cells, cytotoxic T cells, and double positive T cells). The “cell” column classifies cells as either central memory, effector, T stem-cell like memory, or unknown cells that need to be explored further. The “resident” column indicates if the population is a resident cell, as determined by expression of CD103. ( b ) The correlation across study samples between the percentage of cells in each population can be used to see the similarities and differences between different populations. Yellow indicates high positive correlation and purple is high negative correlation. Populations are grouped by cell lineages, and each number on the x-axis and y-axis identifies a separate cell population, corresponding to the population numbers in Fig. 3a.

Article Snippet: Thirty microliters of whole blood cells suspended in 100 μL eBioscience Flow Cytometry Staining Buffer (Cat# 00-4222-26) and stained with fluorochrome-conjugated mouse anti-human 0.5 μL CD45-PE-Cy7 (Clone: HI30), 5 μL CD3-PE (Clone: OKT3), 7 μL CD19-Alexa Fluor 488 (Clone: HIB19) and 5 μL CD27-APC (Clone: M-T271) monoclonal antibodies for twenty minutes at room temperature.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: Primary human monocytes isolated from PBMCs from healthy donors were pretreated with water solvent (H 2 O) or 15 μM P7-Pen for 30 min, followed by stimulation with CL075, R837 (1 μg/ml) or K12 LPS (100 ng/ml) for indicated time, followed by collection of supernatants and cell lysis. ( A ) Quantification of IFNβ , TNF and IL-1β mRNA expression by qRT-PCR in primary human monocytes. Data presented as relative fold change when compared to unstimulated sample pretreated by water (H 2 O), mean ± SEM (n = 6–11). ( B, C ) Cytokine secretion was addressed by ELISA for IFNβ and by BioPlex for other cytokines and graphed as mean ± SEM (n = 5–8). ( C ). P7-Pen effect on CL075- or LPS-mediated secretion of all tested cytokines shown as Volcano plots generated using multiple paired t-test analysis, controlled for a False Discovery Rate (FDR) of 1%. ( D ) Cell viability was addressed by LDH content in supernatants using LDH cytotoxicity assay and presented as % of dead cells, mean ± SD. ( A, B, D ) Statistical testing was done by two-way ANOVA or mixed effects model on log-transformed data (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Isolation, Solvent, Lysis, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Generated, LDH Cytotoxicity Assay, Transformation Assay

Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: ( A ) Whole-blood assay for samples of healthy donors with lepirudin as an anticoagulation reagent. Blood samples were pretreated with water solvent or 20 μM P7-Pen peptide for 30 min, followed by addition of CL075, R837 (2 μg/ml), K12 LPS (100 ng/ml), E. coli particles (2x10 6 /ml), or S. aureus particles (4x10 6 /ml) for 5 h before the collection of plasma samples. Plasma samples were probed for IFNβ, TNF and IL-6 secretion by ELISA. Data presented as mean ± SEM, statistical significance evaluated using Wilcoxon matched-pairs signed-rank test. ( B, C ) Quantification of phagocytosis based on flow cytometry for primary human monocytes pretreated by a solvent (water, H 2 O) or 15 μM P7-Pen, or 3 μM CytoD for 30 min and incubated with E. coli ( B ) or S. aureus ( C ) pHrodo particles for indicated time. Median pHrodo fluorescence intensity MFI and percentage of pHrodo-positive cells shown on graphs ( B, C ). Data presented as mean ± SEM, statistical significance evaluated by 2way ANOVA or mixed effect analysis. Representative images of flow cytometry results for one out of 3-5 donors are shown on the right. ( D ) Results of live bacteria phagocytosis assays for DH5α E. coli and S. aureus Wood strain by PMA-differentiated THP-1 monocytic cells. Data presented as mean ± SD, statistical significance evaluated by unpaired t-test ( A-D ) Significance levels: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, nonsignificant if not shown otherwise; ND, not detected.

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Whole Blood Assay, Solvent, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Incubation, Fluorescence, Bacteria

Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: BMDMs ( A ) and iDCs ( B ) were generated from BM cells from C57BL/6 mice. ( A-C ) M-CSF-differentiated BMDMs ( A ), bone marrow derived iDCs (GM-CSF and IL-4-differentiated) ( B ) or immortalized murine macrophages ( C ) were pretreated with water solvent (H 2 O) or 10 μM P7-Pen for 30 min and stimulated with R848 (1 μg/ml) for indicated time, followed by cell lysis, RNA isolation and RT-qPCR analysis of Ifnβ, Tnf and Il-1β mRNA expression. Data presented as relative fold change when compared to unstimulated sample pretreated by water (H 2 O), mean ± SEM (n = 3). ( D ) Immortalized murine macrophages were kept untreated or stimulated with B4 LPS (100 ng/ml) or R848 (1 μg/ml) for indicated time, followed by cell lysis and IP assays. Endogenous TIRAP was immunoprecipitated from WCL of stimulated cells (1 mg protein/IP). LPS stimulation was applied as a positive control for TIRAP recruitment to MyD88. Cellular lysates were analyzed in parallel as input control (5%), with WB performed for total levels of MyD88 and TIRAP in WCLs and IPs, and for levels of phosphorylated p38 MAPK and IKKα/β in WCLs as a positive control for cell activation by TLR ligands. A representative experiment is shown from a total of three consecutive experiments.

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Generated, Derivative Assay, Solvent, Lysis, Isolation, Quantitative RT-PCR, Expressing, Immunoprecipitation, Positive Control, Control, Activation Assay

Journal: bioRxiv

Article Title: A novel TIRAP-MyD88 inhibitor blocks TLR7 and TLR8-induced type I IFN responses

doi: 10.1101/2025.04.25.650557

Figure Lengend Snippet: Recruitment of TIRAP to the TLR8-Myddosome complex 45–60 minutes after TLR8 activation (late complex) promotes Akt activation, IRF5 dimerization and nuclear translocation, and the expression and secretion of IFNβ and IL-12. The P7-Pen peptide disrupts TIRAP recruitment to the TLR8-MyD88 complex, thereby inhibiting phosphorylation of Akt and IKKα/β, IRF5 dimerization, and nuclear translocation, ultimately leading to reduced expression of IRF5-regulated genes.

Article Snippet: The following TaqMan® Gene Expression Assays (Applied Biosys-tems®, ThermoFisher Scientific, Waltham, MA, USA) were used: IFNβ (Hs01077958_s1), TNF (Hs00174128_m1), TBP (Hs00427620_m1), IL-6 (Hs00985639_m1), IL-1β (Hs01555410_m1) for human cells; Ifnβ (Mm00439552_s1), Tnf (Mm00443258_m1), Il-1β (Mm00434228_m1), and Tbp (Mm01277042_m1) for murine cells.

Techniques: Activation Assay, Translocation Assay, Expressing