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: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Study Design Under BSL-4 containment, we collected blood samples from a total of 21 rhesus monkeys at multiple days post-EBOV inoculation, extracted peripheral blood mononuclear cells (PBMCs), and profiled single-cell transcriptomes and 42 protein markers using Seq-Well and CyTOF. Seq-Well quantifies both host (black) and viral (red) RNA expression, allowing comparisons between infected and bystander cells. Daily clinical parameters (body temperature, clinical signs, and body weight) were also collected for each animal, and complete blood counts were obtained for each blood draw. See also Figure S1 A and .

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: RNA Expression, Infection

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Quantification of Cytokine Expression and Enrichment of Response Signatures, Related to and ( A ) Average expression values (log e TP10K) of literature-annotated cytokines (columns) across cell types and stages of acute EVD (rows). Values are plotted as a ratio relative to the maximum across cell types and stages. Values that are statistically different from baseline (p < 0.05) are indicated with a blue star. ( B ) Heatmap of rank-sum test statistics for comparison of differential expression log fold-changes of genes in a gene set (rows) compared to genes not in the set. The log fold-changes were defined from differential expression profiles of each cell type at each EVD stage (columns) relative to baseline. Five gene sets were tested — three from the Hallmark database (IFN ALPHA, IFN GAMMA, and TNF ALPHA VIA NFKB) ( Liberzon et al., 2015 ) and 2 constructed from the hallmark sets, as uniquely IFNα-regulated genes in “IFN ALPHA” but not “IFN GAMMA” (“IFN ALPHA - GAMMA”), and vice versa for uniquely IFNγ-regulated (“IFN GAMMA - ALPHA”). See also . ( C ) Fold change (log 2 scale) in average HLA-DR CyTOF intensity on B cells at each DPI relative to baseline for each PBMC sample. Colored lines connect serial samples from the same NHP.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Expressing, Comparison, Quantitative Proteomics, Construct

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: ISG Suppression, Co-expression of CD14 and CD16, and Expression of Macrophage Genes Are Associated with Monocyte Infectivity (A) Differential expression between infected and bystander monocytes from DPI 5–8. Genes are colored by membership in sets of genes (Mac. Up/Down = up- or downregulated during in vitro differentiation of monocytes into macrophages). See also . (B) UMAP embedding of monocyte gene expression data, colored by (left-to-right) DPI, CD16 expression (log e TP10K), CD14 expression (log e TP10K), and percentage of cellular transcripts mapping to EBOV. (C) Smoothed expression (log e TP10K) of CD14 and CD16 for monocytes during EVD. Boxes: CD14 + , CD16 + , DN, and DP subsets described in the text; numbers: percentage of cells in each subset at that EVD stage. See also A and S5B. (D) CD14 and CD16 protein expression (CyTOF intensity) on monocytes at each DPI. Bivariate kernel density plot with 200 randomly sampled cells is overlaid as a scatterplot. See also Figure S5 C. (E) CD14 and CD16 protein expression (CyTOF intensity) on monocytes in a case of human EVD, colored by Ki67 protein expression for multiple days after symptom onset. See also Figure S5 D. (F) Percentage of assignment of NHP CD14/CD16 subsets at each EVD stage to human myeloid reference populations (BM-MP: bone marrow monocyte progenitors, PBMC-CD16 + : circulating CD16 + monocytes, PBMC-CD14 + : circulating CD14 + monocytes). See also E–S5K. (G) Percentage of infected monocytes in each CD14/CD16 subset in late EVD. Error bars: 95% CI on the mean based on 1,000 bootstraps. (H) Association between macrophage score (x axis) and percentage of infected cells (left y axis, red) and expression of the differentiation marker NR1H3 (right y axis, blue, log e TP10K). We ordered monocytes from late EVD by macrophage score, and averaged percentage of infected cells and NR1H3 expression within 400-cell sliding windows. See also A–S6C. (I) MX1 expression (log e TP10K) in monocytes at baseline, and uninfected bystanders or infected cells in late infection. Boxes: median and interquartile range; whiskers: 2.5 th and 97.5 th percentiles. Statistical significance was assessed by rank-sum test. See also Figure S6 D. (J) Scatterplot of ISG score (y axis) versus percentage of cellular transcripts mapping to EBOV (x axis) for infected monocytes in late EVD (DPI 6–8). Statistical significance was assessed by Spearman ρ.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Expressing, Infection, Quantitative Proteomics, In Vitro, Gene Expression, Marker

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Extended Characterization of Interferon and Double-Negative CD14 – CD16 – Monocytes, Related to Figure 5 ( A ) Clustermap of pairwise Pearson correlations between cell type clusters at baseline and late EVD. Correlations are computed on average log e TP10K expression values of overdispersed genes. DN and DP monocytes at late EVD are more similar to monocytes (including baseline CD14+s) than other cell types. ( B ) Scatterplot of MAGIC-smoothed expression values (log e TP10K) of CD14 and CD16 for monocytes in baseline, early, mid, and late disease stages. Cells are colored by smoothed expression levels of MKI67 (the gene coding for Ki67 protein). Boxes: CD14+, CD16+, DN, and DP subsets described in the text; numbers: percentage of cells falling into each subset. ( C ) Scatterplot of protein expression (CyTOF intensity) of CD14 and CD16 for 1,000 randomly sampled monocytes at each DPI. Cells are colored by Ki67 expression. Boxes: CD14+, CD16+, DN, and DP subsets described in the text; numbers: percentage of cells falling into each subset. ( D ) Scatterplot of protein expression (CyTOF intensity) of CD14 and CD16 for monocytes during human EVD. Left: monocytes from healthy human controls. Right: monocytes from 3 EVD cases (S1, S2, and S3) at various days post symptom onset. Cells are colored by Ki67 marker intensity. Boxes: CD14+, CD16+, DN, and DP subsets described in the text; numbers: percentage of cells falling into each subset. ( E ) UMAP embedding of healthy human PBMCs dataset, colored by annotated cluster assignment, based on known marker genes. (Plasma.: Plasmablast). ( F ) UMAP embedding of healthy bone marrow cells, colored by cluster assignment, based on marker genes. (HSC: hematopoietic stem cell, Plasma.: Plasmablast, Megakar.: Megakaryocyte, Mono/DC: monocyte and dendritic cell, BM-Macro: bone marrow macrophage). ( G ) UMAP embedding of sub-clustered HSC and monocyte/dendritic lineage cells. (BM: bone marrow, MP: monocyte progenitor) ( H ) Same UMAP embedding as Figure S5 G, but colored by the cluster identity of their nearest neighbor in the human PBMC dataset ( Figure S5 E). ( I ) UMAP embedding of the merged reference dataset of healthy bone marrow HSCs and monocyte lineage cells and PBMCs. Left sub-panel is colored by cluster assignment. Right sub-panels are colored by marker gene expression (log e TP10K). ( J ) Expression profiles of selected genes for human bone marrow monocyte progenitors (BM-MPs) and human circulating monocytes (PBMC-Monos). Circle area: percentage of cells in which the gene was detected; color: average expression ( Z -normalized log e TP10K). ( K ) Expression profiles of selected genes for NHP monocyte subsets at baseline or late EVD for orthologs of the genes in (J). Circle area: percentage of cells in which the gene was detected; color: average expression level ( Z -normalized log e TP10K). CD34 is grayed out because it is detected in <10 cells.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Expressing, Marker, Clinical Proteomics, Gene Expression

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: Viral Transcriptional Dynamics of Infected Monocytes In Vivo and Ex Vivo (A) Schematic of EBOV challenge of PBMCs ex vivo . See also Figure S7 . (B and C) Percentage of cellular transcripts derived from EBOV (intracellular viral load) in monocytes from PBMCs inoculated with live virus ex vivo (B) or from PBMCs of NHPs infected in vivo (C). See also A–S8D. (D) Schematic of EBOV transcription. The viral RNA-directed RNA-polymerase transcribes each gene sequentially but occasionally releases the genomic RNA template, ending transcription. As a result, transcription frequency decreases from NP to L . (E and F) Proportion of each EBOV gene versus viral load (log 10 scale), ex vivo (E) or in vivo (F). We ordered infected monocytes by viral load and averaged the percentage of each viral gene over 50-cell sliding windows. Bands: mean ± 1 SD. See also E and S8F.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Infection, In Vivo, Ex Vivo, Derivative Assay, Virus

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet: EBOV Infection Downregulates Host Antiviral Genes and Upregulates Putative Pro-viral Genes (A and B) Association between host gene expression and viral load within infected monocytes from PBMCs 24 HPI treated with live virus ex vivo (A) or from PBMCs of NHPs in vivo on DPI 5–8 (B). See also . (C and D) Select negatively (C) and positively (D) associated genes in monocytes from ex vivo infections. We ordered infected cells by viral load and averaged gene expression (log e TP10K) over 100-cell sliding windows; Spearman correlation (ρ) is given in the legend. Boxplots show gene expression in uninfected cells (boxes: median and interquartile range; whiskers: 2.5 th and 97.5 th percentiles). See also G and S8H.

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Infection, Gene Expression, Virus, Ex Vivo, In Vivo

Journal: Cell

Article Title: Single-Cell Profiling of Ebola Virus Disease In Vivo Reveals Viral and Host Dynamics

doi: 10.1016/j.cell.2020.10.002

Figure Lengend Snippet:

Article Snippet: Human healthy PBMC scRNA-Seq , 10X , https://support.10xgenomics.com/single-cell-gene-expression/datasets “Aggregate of 8 Chromium Connect channels and 8 manual channels,” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor (Next GEM),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (v3 chemistry),” “5k Peripheral blood mononuclear cells (PBMCs) from a healthy donor with cell surface proteins (Next GEM),” “10k PBMCs from a Healthy Donor - Gene Expression and Cell Surface Protein,” “10k PBMCs from a Healthy Donor (v3 chemistry)”.

Techniques: Virus, Recombinant, Lysis, Electron Microscopy, Infection, Gene Expression, Sequencing, Software

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neutrophil-macrophage crosstalk via NETs–IL-17/VEGF/S100A9 axis promotes hepatocellular carcinoma progression

doi: 10.1186/s13046-025-03618-x

Figure Lengend Snippet: The levels of NETs increase in HCC patients, especially in advanced and metastatic stages. A A heatmap illustrating the differential expression patterns of NETs-related genes in normal and HCC samples based on TCGA database analysis. B Box plots depicting the expression differences of NETs-related genes between normal and HCC samples from the TCGA database. C , D Representative images of HE (Hematoxylin and eosin) and IF staining for CD66b (C) and CitH3 (D) in tissue sections from HCs and HCC patients. The mean IOD of CD66b or CitH3 was quantified (Image Pro Plus) from three random regions per section (HC, n = 5; HCC, n = 5) and shown as fold change relative to HCs. E Representative images of IF staining for CitH3 in peripheral blood neutrophils from HC and HCC patient F Western blot analysis of CitH3 expression in peripheral blood neutrophils from HCs ( n = 8) and HCC ( n = 8) patients, with band intensities normalized to β-actin and shown as fold change relative to controls G ELISA analysis of serum MPO-DNA levels in HCs and HCC patients. H ELISA analysis of serum MPO-DNA levels in HCC patients at different TNM stages (I–IV). I ELISA analysis of serum MPO-DNA levels in HCC patients with and without metastasis. J ELISA analysis of serum MPO-DNA levels in HCC patients with intrahepatic metastasis and extrahepatic metastasis. K Western blot analysis of CitH3 expression in peripheral blood neutrophils from HCC patients with ( n = 6) and without ( n = 6) extrahepatic metastasis. Band intensities were normalized to β-actin and quantified relative to the non-metastasis group White and black scale bars: 50 μm. Data are presented as median (IQR) (G-J) and other data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Student’s t test (C, D, F, K), Mann–Whitney U test (B, G, I, J), Kruskal–Wallis test (H)

Article Snippet: Neutrophils were isolated from the peripheral blood of healthy volunteers and HCC patients using the Human Peripheral Blood Neutrophil Isolation Kit (P9040, Solarbio, China), according to the manufacturer’s instructions.

Techniques: Quantitative Proteomics, Expressing, Staining, Western Blot, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Neutrophil-macrophage crosstalk via NETs–IL-17/VEGF/S100A9 axis promotes hepatocellular carcinoma progression

doi: 10.1186/s13046-025-03618-x

Figure Lengend Snippet: Activation of IL-17R/NF-κB signaling by NETs-attached IL-17 is responsible for NETs-induced M2 macrophage polarization. A Schematic illustration of the transcriptome sequencing protocol for THP1-M0 under three treatment conditions. The volcano plot shows significantly DEGs for Group B vs. A, Group C vs. A, and Group C vs. B. B KEGG pathway enrichment analysis of DEGs was conducted using the DAVID tool. C FCM analysis of the proportion of CD68 + CD206 + M2 derived from THP-1-M0 following 48-hour co-culture of HuH-7 cells with NETs and (or) AMG827. Quantification of the fold change in the proportion from three independent experiments is shown in the right panel. D Western blot analysis of IL-10 and VEGF expression in cell lysates following the same treatments as in C. Densitometric values were normalized to β-actin and compared to the control group, with quantification shown in the right panel E Western blot analysis of IL-17 expression in extracted and purified NETs from peripheral blood neutrophils of HCs ( n = 8) and HCC patients ( n = 8). F ELISA analysis of IL-17 levels carried by NETs standardized to 50 ng/µL in HCs ( n = 8) and patients with HCC ( n = 8). G Western blot analysis of IL-17 expression in peripheral blood neutrophils from HCs ( n = 8) and HCC ( n = 8) patients. Densitometric values were normalized to β-actin and shown as a fold change relative to HCs. H Representative IF images of IL-17 and NE staining in peripheral blood neutrophils with (lower panel) or without PMA stimulation (upper panel) for 4 h from HCs and HCC patients. I Western blot analysis was conducted to assess the expression levels of p-65 and p-p65(S536) in THP-1-M0 following 48 h co-culture of HuH-7 cells with NETs and (or) AMG827. Densitometric values were normalized to β-actin and shown as fold changes relative to control in the right panel. J FCM was used to analyze the proportion of CD68 + CD206 + M2 derived from THP-1-M0 following 48-hour co-culture of HuH-7 cells with NETs and (or) BAY11-7082. Quantification of the fold change in the proportion from three independent experiments is shown in the right panel. K IF analysis of CD206 + M2 macrophage from the above co-culture systems of J treatment for 48 h. L ELISA analysis of secretion levels of M2d-related cytokines VEGF, IL-10 and TGF-β, in the culture supernatant from the above co-culture systems of J treatment for 48 h White scale bars: 20 μm. Data are presented as mean ± SD. Ns, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001. One-way ANOVA followed by the Newman-Keuls multiple comparison test (C, D, I, J, L); Student’s t test (F, G)

Article Snippet: Neutrophils were isolated from the peripheral blood of healthy volunteers and HCC patients using the Human Peripheral Blood Neutrophil Isolation Kit (P9040, Solarbio, China), according to the manufacturer’s instructions.

Techniques: Activation Assay, Sequencing, Derivative Assay, Co-Culture Assay, Western Blot, Expressing, Control, Purification, Enzyme-linked Immunosorbent Assay, Staining, Comparison