Journal: Stroke and Vascular Neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Human serum amyloid A1 (SAA1) protein levels in plasma and extracellular vesicles correlated (EVs) with clinical and laboratory assessments. ( A, B ) Bar graph indicating the concentration of SAA1 protein in both EVs and plasma for the ICH group and the healthy controls group. Unit: ng/mL in EVs and μg/mL in plasma. **p<0.01, ***p<0.001 by the two-tailed unpaired Student’s t-test and data are presented as means±SEM. ( A ) n=6, 15 in the control and ICH group, ( B ) n=13, 19 in the control and the ICH group. ( C, D ) Linear dependence graph revealing the relationship between neutrophils or leucocytes and the SAA1 protein in EVs (n=15). Correlation was analysed by spearman correlation analysis and the dashed line indicates the 95% CI. ( E–H ) Correlation analysis between the EVs-derived, plasma-derived SAA1 levels and the clinical assessments, including NIHSS and haemorrhage volume, through a linear dependence graph (n=15). Correlation was analysed by spearman correlation analysis and the dashed line indicates the 95% CI. ICH, intracerebral haemorrhage; NIHSS, National Institutes of Health Stroke Scale.

Article Snippet: To evaluate the exogenous effect of SAA1 on the mouse brain, we administered varying doses (0 ng, 83 ng, 250 ng, 750 ng) of SAA1 protein (CSB-EP020656MO, CUSABIO, China) dissolved in 1 μL of saline via intracerebral injection.

Techniques: Clinical Proteomics, Concentration Assay, Two Tailed Test, Control, Derivative Assay

Journal: Stroke and Vascular Neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Exogenous SAA1 upregulates the cell counts of microglia and astrocytes. ( A ) Schematic representation of exogenous SAA1 inserting into the basal ganglia region at varying dosages (750 ng, 250 ng, 83 ng and 0 ng). Images of immunostaining of microglia and astrocyte activation on day three are provided. ( B, D ) Images of immunostaining ( B ) and quantification ( D ) of microglia (stained by a specific marker, Iba1) around the ipsilateral basal ganglia region at different doses of exogenous SAA1. Microglia counts per mm 2 : 9.3±2.2, 41.5±3.9, 64.0±4.0, 88.3±8.8 for 0 ng, 83 ng, 250 ng, and 750 ng, respectively. ( C, E ) Images of immunostaining of GFAP (glial fibrillar acidic protein, known as the astrocyte active marker) representing astrocytes ( C ) and quantification of astrocytes ( E ) at different doses of exogenous SAA1 in the ipsilateral basal ganglia region. Astrocyte counts per mm 2 : 36.0±3.8, 93.5±6.1, 134.0±3.5, 176.5±11.6 for 0 ng, 83 ng, 250 ng, and 750 ng, respectively. Scale bars=100 µm, inset scale bars=20 µm, n=4 in each group. ***p<0.001 by the Kruskal-Wallis test. Data are presented as means±SEM.

Article Snippet: To evaluate the exogenous effect of SAA1 on the mouse brain, we administered varying doses (0 ng, 83 ng, 250 ng, 750 ng) of SAA1 protein (CSB-EP020656MO, CUSABIO, China) dissolved in 1 μL of saline via intracerebral injection.

Techniques: Immunostaining, Activation Assay, Staining, Marker

Journal: Stroke and Vascular Neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Blocking SAA1 promotes microglia reactivity and leucocyte infiltration. ( A ) Bar graph indicating the elevation of plasma SAA1 levels in ICH mice as compared with the sham group. n= 5 mice in the ICH group and n=10 mice in the sham group. **p<0.01 by two-tailed unpaired Student’s t-test. ( B ) Schematic diagram depicting intracerebral haemorrhage induction followed by intravenous administration of anti-SAA1 mAb or IgG 1 hour later. Immune cell populations were assessed using flow cytometry on days 1 and 3 post-ICH initiation. ( C ) Flow cytometry gating strategy depicting immune cell populations in mice brain treated with anti-SAA1 antibody or IgG on days 1 and day three post-ICH induction. The graph illustrates CD45 high leucocytes, including CD3 + CD19 - T lymphocytes and its subtypes: CD4 + CD8 − T and CD4 - CD8 + T lymphocytes, CD3 − CD19 + B lymphocytes, CD11b + Ly6G + neutrophils, and CD11b + F4/80 + macrophages. It also illustrates CD45 int CD11b + microglia, including its subtypes: CD86 + microglia and CD206 + microglia. All gates were set using fluorescence-minus-one (FMO) controls. ( D ) Bar graph indicates the number of microglia and their subtypes in ICH mice with anti-SAA1 antibody or IgG treatment from days 1 to 3. ( E ) Bar graph shows major brain infiltrated leucocytes, involving CD8 + T lymphocytes, B lymphocytes, and neutrophils in ICH mice with anti-SAA1 antibody or IgG treatment from days 1 to 3. Int, intermediate. n=5, 6, 5 on day 1 and n=5, 13, 10 on day 3 for sham, IgG and mAb group. n=5, 9, 7 on day 3 for CD86 + microglia group in sham, IgG and mAb group specially. *p<0.05, **p<0.01, ***p<0.001 by one-way ANOVA and Tukey’s test. Data are presented as means±SEM. ANOVA, analysis of variance; ICH, intracerebral haemorrhage.

Article Snippet: To evaluate the exogenous effect of SAA1 on the mouse brain, we administered varying doses (0 ng, 83 ng, 250 ng, 750 ng) of SAA1 protein (CSB-EP020656MO, CUSABIO, China) dissolved in 1 μL of saline via intracerebral injection.

Techniques: Blocking Assay, Clinical Proteomics, Two Tailed Test, Flow Cytometry, Fluorescence

Journal: Stroke and Vascular Neurology

Article Title: Extracellular vesicles bearing serum amyloid A1 exacerbate neuroinflammation after intracerebral haemorrhage

doi: 10.1136/svn-2024-003525

Figure Lengend Snippet: Anti-SAA1 mAb administration alleviates brain injury in mice with ICH. ( A ) Schematic diagram illustrating the ICH followed by intravenous injection of anti-SAA1 mAb or IgG 1 hour later. Subsequently, mice underwent MRI and neurological evaluations on days 1 and 3 after ICH induction. ( B ) Neurological scores of the sham group and the ICH group treated with anti-SAA1 mAb or IgG on day 1 and day 3. The modified Neurological Severity Score (mNSS) and rotarod test were used to measure the neurological deficit. n=6, 12, 7 for sham, IgG and mAb group. *p<0.05 by two-way ANOVA. ( C ) MRI of lesion volume (red) and perihaemorrhagic oedema (PHE) volume (yellow) on day 1 and day 3 post-ICH. ( D, E ) Quantification of lesion volume and PHE volume using the MRI. n=4, 6 for IgG and mAb group on day 1, n=5, 8 for IgG and mAb group on day 3. *p<0.05 by two-tailed unpaired Student’s t-test. Data are presented as means±SEM. ANOVA, analysis of variance; ICH, intracerebral haemorrhage.

Article Snippet: To evaluate the exogenous effect of SAA1 on the mouse brain, we administered varying doses (0 ng, 83 ng, 250 ng, 750 ng) of SAA1 protein (CSB-EP020656MO, CUSABIO, China) dissolved in 1 μL of saline via intracerebral injection.

Techniques: Injection, Modification, Two Tailed Test

Journal: The Journal of Clinical Investigation

Article Title: SAA1/FPR2 signaling between keratinocytes and neutrophils sustains chronic inflammation in Sweet syndrome

doi: 10.1172/JCI193566

Figure Lengend Snippet: ( A ) Ligand-receptor analysis reveals keratinocyte- and neutrophil-specific interactions. Keratinocytes expressed SAA1 transcripts and neutrophils expressed the FPR2 receptor (red dot, right-most column). ( B ) Dot plot demonstrating predominantly cell-specific expression of SAA1 and FPR2 transcripts. The dot size reflects the percentage of cells expressing the gene, and the color illustrates the level of gene expression. ( C ) Representative immunofluorescence staining images and quantification from 5 diseased and 5 control samples, confirming the expression of SAA1 and FPR2 in keratinocytes and neutrophils, respectively. Scale bars: 100 μm. ( D ) Dot plot comparing keratinocyte SAA1 and the control gene DEFB1 in different inflammatory skin conditions. The dot size reflects the percentage of cells expressing the gene, and the color illustrates the level of gene expression. ( E ) SAA1 secretion measured by ELISA in healthy neutrophils or healthy neutrophils exposed to keratinocytes. ( F ) Antibodies blocking SAA1 and FPR2 restored long-lived neutrophil lifespan to WT neutrophil levels. ( G ) Recombinant human SAA1 increased neutrophil survival at 72 hours ( n = 3 independent donors). Data indicate the mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001, by 2-tailed, unpaired Student’s t test ( E and G ) and 1-way ANOVA with individual comparisons ( F ).

Article Snippet: For immunofluorescence microscopy, the following antibodies were used: CD3 (MCA1477, Bio-Rad), CD8 (170306S, Cell Signaling Technology), CD20 (14-0202-82, Thermo Fisher Scientific), CD68 (14-0688-82, Thermo Fisher Scientific), FCN1 (PA5-51552, Thermo Fisher Scientific), S100A8 (33254S, Cell Signaling Technology), S100A9 (34425S, Cell Signaling Technology), MHC-II (68258S, Cell Signaling Technology), IgG (66362S, Cell Signaling Technology), NE (AB68672, Abcam), ELANE (LS-B4244, LS-Bio), FPR2 (NLS1878, Novus), SAA1 (16721-1-AP, Proteintech), and MPO (sc-52707, Santa Cruz Biotechnology).

Techniques: Expressing, Gene Expression, Immunofluorescence, Staining, Control, Enzyme-linked Immunosorbent Assay, Blocking Assay, Recombinant

Journal: Nature communications

Article Title: Mid-old cells are a potential target for anti-aging interventions in the elderly.

doi: 10.1038/s41467-023-43491-w

Figure Lengend Snippet: Fig. 3 | The Effect of SAA1 on old-aged tissues. a Schematic drawing of the effect of SAA1 on tissue. b Young fibroblasts or c smooth muscle cells (PASMCs) were treated with rhSAA1 for 24 h and SASP expression was analyzed using real-time PCR (left panel). MMP9 protein expression was analyzed by ELISA (right panel). d Serially dissected human colon tissues were subjected to IHC analysis for SAA1 and MMP9. Corresponding quantification data is shown in the right panel. IHC results were presented as the percentage of positive cells in the stromal region. e PASMCs were treated with rhSAA1 at the indicated concentration for 24 h and real-time PCR was performed using primers for atrophy-related genes. f IHC ana- lysis for SAA1 in colon tissue from young and elderly subjects, and muscular mucosa thickness was measured. g IHC analysis for type IV collagen of colon tissues from young and elderly subjects is shown. “1” and “2” indicate high-magnification views of the original figure (left panel). Each protein expression was presented as weak, moderate, and strong (right upper panel). mRNA expression level of COL4A1 and COL4A2 in fibroblasts/smooth muscle cells was analyzed between young and old subjects in scRNA-seq data set (GSE178341)34 (right lower panel). h COL4A1,

Article Snippet: Mouse Serum Amyloid A (SAA) Quantikine ELISA Kit (MSAA00, R&D systems) was used for quantitative measurement of circulating SAA levels according to the manufacturer’s instructions.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Concentration Assay, Lysis

Journal: Nature communications

Article Title: Mid-old cells are a potential target for anti-aging interventions in the elderly.

doi: 10.1038/s41467-023-43491-w

Figure Lengend Snippet: Fig. 6 | SLIT2 has an impact on mid-old cells. a Mid-old cells were treated for 24 h with rhSLIT2, and inflammatory gene expression was analyzed using real-time PCR (upper panel). IL1β and SAA1 protein level was analyzed by ELISA in rhSLIT2-treated mid-old cells (lower panel). b Mid-old cells were treated with rhSLIT2 for the indi- cated times and analyzed for Pyk2-NFκB signaling by western blot analysis (left panel). The number of nuclear p-NFκB-positive cells was quantified in mid-old cells using IF staining (right panel). c SLIT2-expressing lentivirus infected into mid-old fibroblasts, and inflammation related genes expression including SAA1 and IL1β were analyzed using real-time PCR (right lower panel). IL1β and SAA1 protein level was analyzed by ELISA in SLIT2-overexpressing mid-old cells (left lower panel). d The morphology and cell size of mid-old cells were analyzed after treating rhSLIT2 for 20 days. Cell growth rate was analyzed every 4 days for 20 days by counting the number of cells. e The expression of p53 and p21Waf1 were measured in mid-old cells after administration of rhSLIT2 at the indicated concentration for 2 days using real-time PCR (upper panel) and western blot analysis (lower panel). f The changes in gene expression of p53 and p21Waf1-regulating genes were eval- uated after treating cells with rhSLIT2 using RNA-seq FPKM count (left panel). The mRNA expression of SOX2 and OCT4 were analyzed using real-time PCR (right

Article Snippet: Mouse Serum Amyloid A (SAA) Quantikine ELISA Kit (MSAA00, R&D systems) was used for quantitative measurement of circulating SAA levels according to the manufacturer’s instructions.

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Western Blot, Staining, Expressing, Infection, Concentration Assay, RNA Sequencing