Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: Characteristics of rRABV expressing GitrL in vitro. ( A ) Schematic diagrams for the construction of rLBNSE and rLBNSE-GitrL. The vector pLBNSE was derived from the SAD-B19 strain with pseudogene deletion. BsiW I and Nhe I were introduced between the G and L genes. N , P , M , G , and L represent RABV nucleoprotein, phosphoprotein, matrix, glycoprotein, and polymerase genes, respectively. ( B ) Amino acid sequence alignment of the GitrL gene, the red area indicates the amino acid sequence was consistent with the reference sequence of the GitrL gene. ( C ) Viral titers from the fourth passage to the tenth passage on BSR cells. ( D , E ) The virus titers of different rRABVs-infected cells with MOI = 0.01 and MOI = 5 were determined at 1, 2, 3, 4, and 5 dpi, respectively. On this basis, the multi-step growth curves ( D ) and one-step growth curves ( E ) of rLBNSE- and rLBNSE-GitrL-infected BSR cells were plotted. ( F , G ) Morphology and size of viral fluorescent spots formed by different rRABVs in BSR cells. ( H ) The expression level of GitrL in the supernatant of infected cells was detected by ELISA. ( I ) Cell viability of different rRABVs-infected cells with MOI = 0.01 at 1, 2, 3, and 4 dpi, respectively. The error bar represented the mean ± standard deviation (SD, n = 3). The following symbols are used to indicate significant differences between groups: ns, not significant.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Expressing, In Vitro, Plasmid Preparation, Derivative Assay, Sequencing, Virus, Infection, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: The pathogenicity of rRABV expressing GitrL. ( A ) Body weight changes in female 6-week-old ICR mice (n = 7) infected with 5 × 10 6 FFU of rLBNSE, rLBNSE-GitrL, or DMEM via i.c. pathways. ( B ) Survival rate of 5-day-old mice (n = 18) i.c. injected with 100 FFU of the above rRABVs. ( C ) N mRNA levels, ( D ) vRNA levels, ( E ) GitrL mRNA levels in the infected BALB/c mouse brain (n = 6) were quantified using RT-qPCR. The error bar indicates the mean ± standard error of the mean (SEM). The following symbols are used to indicate significant differences between groups: *, p < 0.05; ns, not significant.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Expressing, Infection, Injection, Quantitative RT-PCR

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: rLBNSE-GitrL promotes activation of DCs in vitro. ( A ) Isolation and cultivation of BMDCs. ( B ) DCs were cultured and stimulated with DMEM as the control group. ( C ) DCs were cultured and stimulated with rLBNSE. ( D ) DCs were cultured and stimulated with rLBNSE-GitrL. The white arrow indicated clustered DCs. ( E ) Gating strategies for the detection of the mature CD11C + CD80 + BMDCs, CD11C + CD86 + BMDCs, and CD11C + MHCII + BMDCs. ( F ) Representative flow cytometry plots of activated DCs. ( G ) Percentage of activated DCs (CD11C + CD80 + ) per 10 5 BMDCs after 24 h of incubation. ( H ) Percentage of activated DCs (CD11C + CD86 + ) per 10 5 bone BMDCs after 24 h of incubation. ( I ) Percentage of activated DCs (CD11C + MHCII + ) per 10 5 BMDCs after 24 h of incubation. ( J ) The expression level of IFN-α in the supernatant of activated DCs was detected by ELISA. ( K ) The expression level of IL-12p40 in the supernatant of activated DCs was detected by ELISA. The error bar represents the mean ± SD (n = 3). The following symbols are used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Activation Assay, In Vitro, Isolation, Cell Culture, Control, Flow Cytometry, Incubation, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: rLBNSE-GitrL increases the number of activated DCs in vivo. ( A ) Cell populations and gating strategies for DCs by flow cytometry within LNs. ( B ) Representative flow cytometry plots of activated DCs. ( C ) Total number of activated DCs (CD11C + CD80 + ) per 10 5 draining LNs cells at 3 and 6 dpi. ( D ) Total number of activated DCs (CD11C + CD86 + ) per 10 5 draining LNs cells at 3 and 6 dpi. ( E ) Total number of activated DCs (CD11C + MHCII + ) per 10 5 draining LNs cells at 3 and 6 dpi. The error bar indicates the mean ± SEM (n = 3). The following symbols are used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: In Vivo, Flow Cytometry

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: Transcriptome sequencing and differentially expressed gene analysis. ( A ) Comparative bar chart of differential genes. ( B ) Volcano plot of the differentially expressed genes between immunized mice of rLBNSE and rLBNSE-GitrL. ( C ) Gene expression heatmap. ( D ) Validation of the CXCL9. ( E ) Validation of the IL-6. ( F ) Validation of the IL-17d. ( G ) Validation of the MDP1. ( H ) Validation of the MMP2. ( I ) Validation of the STAT1. ( J ) Validation of the TIMP1. ( K ) Validation of the TNF-α. ( L ) Bar chart of KEGG enrichment pathway of the differentially expressed genes between rLBNSE and rLBNSE-GitrL. ( M ) KEGG enrichment pathway scatter plot of the difference expression genes between rLBNSE and rLBNSE-GitrL. The error bar indicates the mean ± SEM (n = 3). The following symbols are used to indicate significant differences between groups: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Sequencing, Gene Expression, Biomarker Discovery, Expressing

Journal: Viruses

Article Title: Overexpression of GitrL in Recombinant Rabies Virus rLBNSE-GitrL Enhances Innate Immunity by Activating Dendritic Cells and Innate Immune-Related Pathways and Genes

doi: 10.3390/v17101354

Figure Lengend Snippet: Overexpression of GitrL improves antibody response and protection against pathogenic RABV challenge. ICR mice (n = 10) were i.m. vaccinated with 100 µL volume containing 10 6 FFU rLBNSE or rLBNSE-GitrL, or an equivalent amount of DMEM (as mock). Serum samples were collected at 3 and 6 dpi, and VNA titers ( A ) and GMT ( B ) were measured by FAVN assay. ( C , D ) The OD values of RABV G-specific IgG ( C ) and IgM ( D ) in the serum of immunized mice were determined by ELISA. ( E ) At 3 dpi, groups of immunized ICR mice (n = 15) were challenged with 50 LD50 of CVS-24 via i.c. route, and then monitored for another 20 days, and survivorship was recorded. The error bar indicates the mean ± SEM. The following symbols were used to indicate significant differences between groups: **, p < 0.01; ***, p < 0.001.

Article Snippet: The expression of GitrL in the supernatants of infected cells was quantified using a Mouse GitrL ELISA kit (RayBiotech, Peachtree Corners, GA, USA), following the manufacturer’s instructions.

Techniques: Over Expression, Enzyme-linked Immunosorbent Assay

Journal: Platelets

Article Title: Lymphatic blood filling in CLEC-2-deficient mouse models

doi: 10.1080/09537104.2020.1734784

Figure Lengend Snippet: CLEC-2 deficiency during chronic, but not acute, dermal vascular remodeling can lead to localized lymphatic vessel blood filling. Pf4-Cre CLEC-2 deficient mice and littermate wildtype controls were subjected to cutaneous reverse passive Arthus reaction (rpA) and skin biopsies were collected 4 h after reaction initiation. In neither genotype could blood-filled lymphatic vessels be identified by gross or microscopic assessment. (Ai) Representative images of rpA-challenged skin from Pf4-Cre CLEC-2 deficient mice and littermate wildtype controls. (Aii) Representative images of rpA-challenged skin sections from Pf4-Cre CLEC-2 deficient mice and littermate wildtype controls stained for podoplanin (brown) and hematoxylin (blue). Lymphatic vessels indicated by yellow arrows. Scale bar = 100 μm. Wildtype mice rendered thrombocytopenic by treatment of 2 μg/g of anti-GpIba rat anti-mouse monoclonal antibody mixture (R300) 18 h before rpA initiation had red blood cells in lymphatic vessels upon microscopic inspection. (Bi) Representative images of non-inflamed and inflamed areas of rpA-challenged skin sections from thrombocytopenic wildtype mice stained for podoplanin (brown) and hematoxylin (blue) and red blood cells (yellow). Lymphatic vessels indicated by yellow arrows. Scale bar = 50 μm. n = 4. (Bii) Quantification of the presence of red blood cells in podoplanin positive lymphatic vessels in rpA-challenged skin sections from thrombocytopenic wildtype mice. Pf4-Cre CLEC-2 deficient mice and littermate wildtype controls received an intradermal injection of B16F10 melanoma cells into back skin. After 12 days of tumor growth, skin was harvested. (C) Representative images of melanoma-challenged skin from Pf4-Cre CLEC-2 deficient mice and littermate wildtype controls and quantification of blood-filled tumor-associated lymphatic vessels (yellow arrows) in Pf4-Cre CLEC-2 deficient mice. n = 10.

Article Snippet: Anti-CLEC-2 rat anti-mouse monoclonal antibody (17D9, used for flow cytometry) and rat anti-mouse control monoclonal antibody were from BioRad.

Techniques: Staining, Injection