Journal: Pathogens and Immunity

Article Title: Immune Dysregulation in Acute SARS-CoV-2 Infection

doi: 10.20411/pai.v7i2.537

Figure Lengend Snippet: Analysis of ACE2 inhibition by anti-S1(A) and anti-RBD (B) IgG antibodies by disease severity. Quantitative plasma anti-S had a correlation of r = 0.885 ( P < 0.001) and anti-RBD had a correlation of r = 0.75 ( P < 0.001) to ACE2 neutralization.

Article Snippet: The detection antibody used for this assay is R&D Systems Human ACE2 Biotinylated Antibody, (catalog BAF933).

Techniques: Inhibition, Clinical Proteomics, Neutralization

Journal: Pathogens and Immunity

Article Title: Immune Dysregulation in Acute SARS-CoV-2 Infection

doi: 10.20411/pai.v7i2.537

Figure Lengend Snippet: Proinflammatory dysregulation between cytokine microenvironment and antibody neutralization capacity. Correlation analysis of inflammatory marker plasma concentration and anti-spike antibody ACE2 neutralization disease severity. Elevations in ICAM ( P < 0.0001), IL-1β ( P = 0.0233), IL-4 ( P < 0.0001), IL-6 ( P = 0.0001), TNFα ( P = 0.001), and Syndecan ( P = 0.00) all showed statistically significant correlations to antibody neutralization capacity amongst PCR-positive COVID-19 disease cohorts.

Article Snippet: The detection antibody used for this assay is R&D Systems Human ACE2 Biotinylated Antibody, (catalog BAF933).

Techniques: Neutralization, Marker, Clinical Proteomics, Concentration Assay

Journal: Cell reports

Article Title: Evaluation of Single-Cell Cytokine Secretion and Cell-Cell Interactions with a Hierarchical Loading Microwell Chip

doi: 10.1016/j.celrep.2020.107574

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: To prepare functionalized beads to detect IFN-γ or IL-2, a biotinylated antibody against human IFN-γ (Thermo Fisher, M701B) or a biotinylated antibody against human IL-2 (R&D Systems, BAF202) was conjugated to streptavidin polystyrene beads (mean size: 18.4 μm, Spherotech, SVP-200–4) or streptavidin magnetic beads (mean size: 21.7 μm, Spherotech, SVM-200–4) by shaking beads in 50 μg/mL IFN-γ antibody or IL-2 antibody solution at 4°C overnight in PBS, respectively.

Techniques: Recombinant, Labeling, Software

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Trem2 Y38C variant and Trem2 deficient mice affects microglial morphology. a CRISPR/Cas9 strategy to generate the tyrosine to cystine point mutation (red) in exon 2 of murine Trem2 to create the Trem2 Y38C/Y38C mouse model. The sequences for the reference genome, guide RNA, and homology directed repair (HDR), which indicates the Y38C variant (red) and a silent mutation (blue) to ablate the protospacer adjacent motif (PAM), are shown. b Quantification of Trem2 mRNA levels in cortical lysates showed similar Trem2 expression in WT (black) and Trem2 Y38C/Y38C (green) mice and no expression in Trem2 −/− (red) mice. Data are presented as mean ± SEM fold change normalized gene expression relative to WT. N = 8 (4 males and 4 females for each group) c Immunohistochemistry for TREM2 (green) and Iba-1 (red) is shown in cortices of 6 month WT, Trem2 Y38C/Y38C , and Trem2 −/− mice. Merged imaged indicates colocalization of TREM2 staining with Iba-1+ (red) cell bodies in WT and Trem2 Y38C/Y38C mice. No TREM2 staining was observed in Trem2 −/− mice. (representative images from females). d Quantification of total TREM2 in WT, Trem2 Y38C/Y38C , and Trem2 −/− cortical lysates by ELISA indicates slightly higher levels of TREM2 in Trem2 Y38C/Y38C as compared to the WT mice. e sTREM2 levels, quantified in TBS soluble cortical extracts, were similar in both Trem2 Y38C/Y38C and WT. f Quantification of the number of Iba-1 positive cells showed similar counts in cortices of 6 months old WT, Trem2 Y38C/Y38C and Trem2 −/− mice. g Quantification of Iba-1 immunoreactive area showed increased staining in Trem2 Y38C/Y38C and Trem2 −/− mice. f-g Data represented as mean ± SEM, N = 6 (3 males and 3 females for each group; n = 56 fields for WT, n = 89 fields for Trem2 Y38C/Y38C , and n = 112 fields for Trem2 −/− ). h Skeletonized reconstructions (representative reconstructions from females), i quantification of number of branches and j number of junctions in microglia from the cortices of 6 months old WT, Trem2 Y38C/Y38C and Trem2 −/− mice. h-j Data represented as mean ± SEM, N = 6 (3 males and 3 females for each group, each data point represents microglia analyzed). b,d-i One-way ANOVA with Tukey’s post hoc test. * P < 0.05, *** P < 0.001, **** P < 0.0001

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Variant Assay, CRISPR, Mutagenesis, Expressing, Gene Expression, Immunohistochemistry, Staining, Enzyme-linked Immunosorbent Assay

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Loss of functional TREM2 alters gene expression associated with oligodendrocyte/myelin and neuronal function. a Volcano plot showing differentially expressed genes in 6 month Trem2 Y38C/Y38C versus WT cortices and Trem2 −/− versus WT cortices. Dashed line represents P < 0.005. Significantly altered genes are shown in green with downregulated and upregulated genes above |logFC| > 0.58 threshold of in green. b-c Enrichr pathway analysis of the shared downregulated genes between Trem2 Y38C/Y38C and Trem2 −/− mice (ranked by P value; Fischer’s exact test, P-adjusted, Benjamini-Hochberg method for correction for multiple hypotheses testing). b Tissue enrichment analysis showing significant enrichment in brain regions. Dashed line represents P < 0.005. c Enrichment analysis for specific cellular compartments showing myelin-related enrichment. Dashed line represents P < 0.005. d Top 10 pathways significantly altered using IPA pathway analysis. Analysis based on significantly differentially expressed genes between Trem2 Y38C/Y38C versus WT mice and Trem2 −/− vs WT mice. Yellow line represents P < 0.05 yellow line (ranked by P value, Fischer’s extact test). e Top 5 predicted upstream regulators of differentially expressed genes in Trem2 Y38C/Y38C versus WT mice and Trem2 −/− vs WT mice using IPA Upstream Regulator analysis. Activated (black) and inhibited (grey) upstream regulators are shown. P value, Fisher’s exact test. Sample sizes: WT mice, N = 8 (4 males, 4 females); Trem2 Y38C/Y38C mice, N = 8 (4 males, 4 females); Trem2 −/− mice, N = 5 (1 male, 4 females)

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Functional Assay, Gene Expression

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Oligodendrocyte/Myelin impairments in adult Trem2 Y38C/Y38C and Trem2 −/− mice. a-c Mbp, Mobp and Olig2 transcript levels were determined by quantitative-PCR. a Mbp transcripts were reduced in both Trem2 Y38C/Y38C and Trem2 −/− cortical lysates and b-c Mobp and Olig2 mRNA reduced significantly in Trem2 −/− cortical lysates. d Western blot analysis and e Quantification of myelin proteins CNPase and MBP. CNPase levels were reduced significantly in Trem2 −/− mice and Mbp levels were significantly reduced in both Trem2 Y38C/Y38C and Trem2 −/− mice. a-e Data represented as mean ± SEM. N = 4 (2 males, 2 females); One-way ANOVA with Tukey’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Decreased synaptic elements in adult Trem2 Y38C/Y38C and Trem2 −/− mice. Microdissected a,b cortices and c,d hippocampi of WT, Trem2 Y38C/Y38C and Trem2 −/− mice were analyzed using western blot with antibodies against PSD95 and synaptophysin. a,b Trem2 Y38C/Y38C and Trem2 −/− mice showed significant reduction in synaptophysin but not in PSD-95 protein levels. c,d PSD95 and synaptophysin protein levels were significantly reduced in hippocampi of Trem2 Y38C/Y38C and Trem2 −/− mice. e Comparison of PSD95 and synaptophysin protein expression in hippocampal lysates of WT, Trem2 Y38C/Y38C and Trem2 −/− mice at P20 and 6 months. Comparing P20 to 6 months, PSD95 levels increase significantly (black asterisk) in WT mice (black), while both Trem2 Y38C/Y38C (green) and Trem2 −/− (red) mice show comparatively less increase resulting in reduced PSD95 levels at 6 months as compared to WT. Synaptophysin levels were maintained in WT mice (black) and Trem2 Y38C/Y38C (green), while Trem2 −/− (red) mice showed significantly lower levels at 6 months as compared to P20 (red asterisk) indicating loss of presynaptic elements in adult mice. Sample sizes for each group: N = 6–8 (3–4 each, males and females). Data are shown as mean ± SEM. b,d One-way ANOVA with Tukey’s post hoc test; * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. e Two-way ANOVA with Bonferroni’s post hoc test; * P < 0.05, WT mice at 6 months versus P20 (black) and Trem2 −/− mice at 6 months versus P20 (red)

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: Western Blot, Comparison, Expressing

Journal: Molecular Neurodegeneration

Article Title: Trem2 Y38C mutation and loss of Trem2 impairs neuronal synapses in adult mice

doi: 10.1186/s13024-020-00409-0

Figure Lengend Snippet: Hippocampal synaptic plasticity is reduced in Trem2 Y38C/Y38C and Trem2 −/− mice. a Input-output curves were similar in Trem2 Y38C/Y38C , Trem2 −/− , and WT mice. b,c Hippocampal Schaffer collateral-CA1 LTP is significantly reduced in Trem2 Y38C/Y38C (green) and Trem2 −/− (red) mice as compared to WT mice (black) at 6 months of age. b Superimposed representative traces before (black) and 60 min after high-frequency stimulation (gray) in each genotype. c Mean time course of fEPSP slope in hippocampal slices from WT (black), Trem2 Y38C/Y38C (green) and Trem2 −/− (red). Arrow indicates time of high frequency stimulation. d Average of normalized fEPSP slope for final 10 min of recording (60–70 min) relative to 10-min baseline average (dotted line). Data are shown as mean ± SEM. Sample sizes: WT mice, N = 15 (7 females and 8 males, n = 33 recordings); Trem2 Y38C/Y38C mice, N = 7 (3 females and 4 males, n = 20 recordings); Trem2 −/− mice, N = 8 (4 males and 4 females, n = 15 recordings). One-way ANOVA with Tukey’s post hoc test. *** P < 0.001, **** P < 0.0001

Article Snippet: Wells were washed 4 times with 0.05% Tween in PBS and incubated with 0.25μg/ml of the TREM2 biotinylated detection antibody (R&D Systems, BAF1729) for 1 h at RT.

Techniques: