Journal: bioRxiv

Article Title: A genome-wide screen in the mouse liver reveals sex-specific and cell non-autonomous regulation of cell fitness

doi: 10.1101/2021.01.30.428976

Figure Lengend Snippet: a, Table of values used to estimate number of hepatocytes in postnatal day one livers. Liver volume was measured by volume displacement and percent hepatocytes and hepatocyte volume were measured by immunostaining and microscopy. b, Images of endogenous mCherry and mTurq2 fluorescence in livers from mice four days or 14 weeks after injection of 2.5 × 10 7 TU of an equal mixture of sgAAVS1-mCherry and sgAAVS1-mTurq2 lentiviruses. Scale bars, 100 μm. c, Percent mCherry-, mTurq2-, and double-positive hepatocytes in livers from mice four days or 14 weeks after injection of 2.5 × 10 7 TU of an equal mixture of sgAAVS1-mCherry and sgAAVS1-mTurq2 lentiviruses. n = 3 mice per time point and 200 hepatocytes per mouse. Error bars indicate standard deviation. d, Images of livers from LSL-Cas9 mice injected with 0 or 2 × 10 11 GC of AAV-Cre on postnatal day five and harvested four days thereafter immunostained for ASGR1 (hepatocyte marker, magenta) and Cas9 (green) and counterstained with Hoechst (blue). Scale bars, 15 μm. e, Percent Cas9-positive hepatocytes in livers from LSL-Cas9 mice injected with varying doses of AAV-Cre on postnatal day five and harvested four days thereafter as determined by Cas9 immunostaining. n = 1 male and 1 female mouse per dose and 200 hepatocytes per mouse. Error bars indicate standard deviation. f, Images of livers from LSL-Cas9 mice injected with sgLmnb2-mCherry followed by PBS or AAV-Cre immunostained for mCherry (magenta) and lamin B2 (green) and counterstained with Hoechst (blue). Scale bars, 45 μm. g, Nuclear lamin B2 intensity per μm in mCherry-positive and mCherry-negative hepatocytes from LSL-Cas9 mice injected with sgLmnb2-mCherry followed by PBS or AAV-Cre. n = 1 male and 1 female mouse per condition and 25 cells per mouse. Closed and open circles represent values from male and female mouse, respectively. h, Images of livers from unperturbed postnatal day 12 LSL-Cas9 mice or postnatal day 12 LSL-Cas9 mice injected with 1.25 × 10 7 TU of sgMaob-mCherry or sgLmnb2-mCherry lentivirus on postnatal day 1 followed by AAV-Cre on postnatal day 5 immunostained for CD45 (green) and mCherry (magenta) and counterstained for Hoechst (blue). Scale bars, 45 μm. i, Number of CD45-positive cells per 40X field in unperturbed postnatal day 12 LSL-Cas9 mice or postnatal day 12 LSL-Cas9 mice injected with 1.25 × 10 7 TU of sgMaob-mCherry or sgLmnb2-mCherry lentivirus on postnatal day 1 followed by AAV-Cre on postnatal day 5 as determined by CD45 immunostaining. n = 2 male and 2 female mice per condition and five fields per mouse. Closed and open circles represent values from male and female mice, respectively. j, Images of livers from unperturbed postnatal day 12 LSL-Cas9 mice or postnatal day 12 LSL-Cas9 mice injected with 1.25 × 10 7 TU of sgMaob-mCherry or sgLmnb2-mCherry lentivirus on postnatal day 1 followed by AAV-Cre on postnatal day 5 immunostained for Ki67 (white), mCherry (magenta) and actin (green) and counterstained for Hoechst (blue). Scale bars, 45 μm. k, Percent Ki67-positive hepatocytes in livers from unperturbed postnatal day 12 LSL-Cas9 mice or postnatal day 12 LSL-Cas9 mice injected with 1.25 × 10 7 TU of sgMaob-mCherry or sgLmnb2-mCherry lentivirus on postnatal day 1 followed by AAV-Cre on postnatal day 5 as determined by Ki67 immunostaining. n = 2 male and 2 female mice per condition and 50 cells per mouse. Closed and open circles represent values from male and female mice, respectively.

Article Snippet: The following primary antibodies were used: Cas9 (1:200, clone 7A9-3A3, Abcam ab191468), asialoglycoprotein receptor 1 (ASGR1) (1:500, clone 114, Sino Biological 50083-R114), mCherry (1:500, clone 16D7, ThermoFisher Scientific M11217), monoamine oxidase B (MAO-B) (1:1,000, Novus Biologicals NBP1-87493), lamin B2 (1:1,000, clone EPR9701(B), Abcam ab151735), actin (1:250, clone AC-74, Sigma Aldrich A2228), CD45 (1:500, Abcam ab10558), and Ki-67 (1:200, clone SP6, Abcam ab16667).

Techniques: Immunostaining, Microscopy, Fluorescence, Injection, Standard Deviation, Marker

Journal: Science Advances

Article Title: Targeted inhibition of hepatic de novo ceramide synthesis ameliorates MASH

doi: 10.1126/sciadv.adx2681

Figure Lengend Snippet: ( A ) Whole-body IVIS imaging of mice injected with nonlabeled LNP-siRNA (control group) or LNPs loaded Cy5-labeled siRNA. ( B ) Representative IVIS image illustrating the distribution of LNPs across various organs. ( C ) Flow cytometry analysis showing cellular uptake of Cy5-siRNA in hepatocytes. ASGR1 was used as a hepatocyte specific marker. ( D ) Distribution of Cy5-labeled siRNA among different liver cell populations in healthy mice and MASH mice fed an MCD diet for 8 weeks. Data were presented as the percentage of Cy5-positive cells within each cell type based on FACS analysis. EC, endothelial cells; HSCs, hepatic stellate cells; DCs, dendritic cells. ( E to G ) Time-course analysis of hepatic Sptlc2 mRNA levels and SPTLC2 protein levels following a single intravenous dose of LNP-siRNA (0.3 mg/kg) in MASH mice fed an MCD diet for 4 weeks. Glyceraldehyde-3-phosphate dehydrogenase was used as a loading control for Western blot analysis, and protein expression levels were normalized to that on day 2 postdosing of LNP-Scrambled siRNA (control). ( H ) Time-course analysis of hepatic Sptlc2 mRNA levels in healthy mice following siRNA treatment at 0.3 mg/kg per dose, administered either once or twice weekly. The once-weekly group received doses on days 0 and 7, while the twice-weekly group received doses on days 0, 3, 7, and 10. N = 3 mice per group. * P < 0.05, ** P < 0.01, and *** P < 0.001; one-way ANOVA.

Article Snippet: Cells were stained with an ASGR1 antibody (Proteintech, catalog no. CL488-11739, Illinois, USA) for 30 min at 4°C in the dark.

Techniques: Imaging, Injection, Control, Labeling, Flow Cytometry, Marker, Western Blot, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide.

doi: 10.4049/jimmunol.174.3.1580

Figure Lengend Snippet: FIGURE 4. T cell proliferation in mice immunized with the epitope vaccine PADRE-A1–15-MAP and other control Ags. Splenocytes from individual mice were restimulated in vitro with the indicated peptides. Only the epitope vaccine PADRE- A1–15-MAP (A) induced the activa- tion of PADRE-specific, but not A-specific, T cells. In contrast, A1–33-MAP (B), fibrillar A42 (C), and A1–33 (D) Ags stimulated anti- A T cell responses.

Article Snippet: Detection of CD4 T cells expressing IL-18R (Th1) or T1/ST2 (Th2) molecules Spleens from mice immunized with different immunogens or control animals were depleted of CD8 cells using MACS depletion kit (Miltenyi Biotec), and the remaining splenocytes were restimulated in HL-1 medium with the same peptide that was used for in vivo immunization.

Techniques: Control, In Vitro

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide.

doi: 10.4049/jimmunol.174.3.1580

Figure Lengend Snippet: FIGURE 6. Expression of IL-18R (Th1) and T1/ST2 (Th2) molecules on the surface of CD4 T cells. Spleno- cytes from all groups were depleted by CD8 T cells and restimulated with the same peptides that were used for in vivo injections, except splenocytes from mice immunized with A42 were activated in vitro with A40. Splenocytes were analyzed before in vitro activation (0 day) and after 7 days of activation (7 day). For details, see Materials and Methods.

Article Snippet: Detection of CD4 T cells expressing IL-18R (Th1) or T1/ST2 (Th2) molecules Spleens from mice immunized with different immunogens or control animals were depleted of CD8 cells using MACS depletion kit (Miltenyi Biotec), and the remaining splenocytes were restimulated in HL-1 medium with the same peptide that was used for in vivo immunization.

Techniques: Expressing, In Vivo, In Vitro, Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide.

doi: 10.4049/jimmunol.174.3.1580

Figure Lengend Snippet: FIGURE 5. Detection of splenocytes producing IFN- ((Th1), IL-4 (Th2), and TNF- in mice immunized with A1–33-MAP, PADRE-A1–15 MAP, fibrillar A42, or linear A1–15. The ELISPOT technique was used, as described in Materials and Methods.

Article Snippet: Detection of CD4 T cells expressing IL-18R (Th1) or T1/ST2 (Th2) molecules Spleens from mice immunized with different immunogens or control animals were depleted of CD8 cells using MACS depletion kit (Miltenyi Biotec), and the remaining splenocytes were restimulated in HL-1 medium with the same peptide that was used for in vivo immunization.

Techniques: Enzyme-linked Immunospot

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP gating strategy and in vitro results. (A) Transmission electron microscopy images of EP. (B) The EP gate was defined using 0.5 µm and 1.53 µm calibration beads. EP population shift was observed after staining with ASGR1 antibodies. (C) In vitro , TNF-α induced human hepatocyte–derived EP. Primary human hepatocytes following liver resection were isolated, cultured overnight, and stimulated with 10 and 20 ng/ml TNF-α. EP surface antigens were stained, and absolute AnnV + , CD130 + , Cx43 + , ASGR1 + EP were analyzed. The ordinary one-way ANOVA with Tukey’s post hoc test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: In Vitro, Transmission Assay, Electron Microscopy, Staining, Derivative Assay, Isolation, Cell Culture

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Time course of EP after LT (n = 11). (A) Total EP (/µl) (B) AnnV + , CD130 + , CD31 + EP (/µl) (C) Cx43 + , ASGR1 + , MDR3 + EP (/µl) were stained and analyzed. The Wilcoxon matched-pairs signed ranked test was used. The plots are indicated by the median, and all error bars indicate the interquartile range (IQR). A single asterisk indicates significance at p < 0.05 (D) Patients at risk for acute rejection. EP dynamics from preoperative state to POD 1 of the total, AnnV + , CD130 + , Cx43 + , ASGR1 + , MDR3 + , CD31 + EP (/µl) and their fold change were analyzed. Absolute EP were non-normally distributed; the two-tailed Mann-Whitney U test was used. The plots are indicated by the mean, and all error bars indicate the SD.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Staining, Two Tailed Test, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: EP profiles during histology-proven ACR. EP were determined in the blood plasma of patients with concomitant LBs. The patients were then classified as histology-proven ACR or non-ACR. EP surface antigens (A) AnnV + , (B) CD130 + , (C) Cx43 + , (D) ASGR1 + , (E) MDR3 + , (F) CD31 + EP were stained, and relative EP (%) were analyzed. (G) Example FSC-A (forward scatter) histograms of MDR3 + EP (%) (H) demonstrating the highest receiver operating characteristic (ROC) of all single antigens. One-way analysis of variance followed by Tukey’s post hoc test was used. The plots are indicated by the median, and all error bars indicate the IQR.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Clinical Proteomics, Staining

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Clustering and color coding with FlowSOM and viSNE for identifying EP subpopulations via density plots. (A) Qualitative analysis and population identification were performed using viSNE and FlowSOM on samples of patient 52, who had episodes of graft dysfunction caused by ACR and non-ACR. Several LBs were performed over time, and their concomitant blood samples presented the following data. t-SNE (t-distributed stochastic neighbor embedding), an unsupervised nonlinear dimensionality reduction algorithm, was used to fit six-dimensional data into two dimensions. All clusters were created via FlowSOM analysis. Clusters were formed based on FACS channels. The arrow indicates the novel ASGR1 + CD130 + AnnV + EP population. Coordinates for each t-SNE dimension (t-SNE1 and t-SNE2) were calculated for each microparticle after dimensionality reduction. (B) Color-coded t-SNE density plots showing different antigen expression levels by the channel of above-mentioned samples.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Analysis of ASGR1 + CD130 + AnnV + EP (%) population discovered by viSNE maps and FlowSOM algorithm. (A) The particular EP subpopulation was identified in the plasma of patients with concomitant LBs. The patients were then classified as histology-proven ACR or non-ACR. ROC was constructed by comparing (B) ACR vs non-ACR and (C) ACR vs control. One-way analysis of variance followed by Tukey’s post hoc test was used. ROC curves and AUC were generated for relative EP. The plots are indicated by the median, and all error bars indicate the IQR.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Clinical Proteomics, Construct, Control, Generated

Journal: Frontiers in Immunology

Article Title: Sensing Acute Cellular Rejection in Liver Transplant Patients Using Liver-Derived Extracellular Particles: A Prospective, Observational Study

doi: 10.3389/fimmu.2021.647900

Figure Lengend Snippet: Diagnosis sensitivity and specificity of ASGR1 + Annexin V + CD130 + EP (%) for ACR in liver transplant.

Article Snippet: Each sample containing 50 μl supernatant EP and 5 μL 10x AnnV binding buffer was subsequently incubated with antibodies: APC Alexa 700-conjugated Cx43 (clone: FAB7737N, R&D Systems, Minneapolis, MN, USA), BV421-conjugated CD130 (clone: AM64, BD Biosciences, San Jose, CA, USA), and PE-conjugated AnnV (Cat. No. 640908, BioLegend, San Diego, CA, USA), and FITC-conjugated ASGR1 (clone: REA608, Miltenyi Biotec, Auburn, CA, USA) or FITC-conjugated MDR3 (Cat. No. LS-C694886, LifeSpan BioSciences, Seattle, WA, USA) or FITC-conjugated CD31 (Cat. No. 303104, BioLegend).

Techniques: Biomarker Discovery