Journal: Vaccines

Article Title: Immunological Analysis of a CCHFV mRNA Vaccine Candidate in Mouse Models

doi: 10.3390/vaccines7030115

Figure Lengend Snippet: ELISPOT assays. ( A ) Splenocyte cells from immunized C57BL/6 were obtained on week four after immunization. IFN-gamma secretion was analyzed by ELISPOT assay. The number of IFN-gamma specific spots in the booster dose group was higher than for the single dose. ( B ) The number of IL-4-specific spots was similar to that for IFN-gamma. This indicates that the booster dose had a stronger effect than the single dose. The data was analyzed using the log-rank test (* P < 0.05; ** P < 0.01; and *** P < 0.001) and presented as mean ± SD. Error bars indicate standard deviations (SD).

Article Snippet: The ELISPOT assays were performed by ELISPOT Mouse IFN-gamma and IL-4 kits (R&D Systems, Minneapolis, MN, USA) based on the established protocol of the manufacturer.

Techniques: Enzyme-linked Immunospot

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

Article Title: The C5 Anaphylatoxin Receptor (C5aR1) Protects against Listeria monocytogenes Infection by Inhibiting Type 1 Interferon Expression

doi: 10.4049/jimmunol.1401750

Figure Lengend Snippet: WT and C5aR1−/− mice were injected i.v. with 1 × 105 CFU of L. monocytogenes or PBS, and serum was isolated from the mice 24 h. (A) IFN-α and IFN-β were measured by ELISA. Sera from PBS injected animals had no detectable type 1 IFN (data not shown). Data from two independent experiments were combined and are presented as mean pg/ml ± SEM. n = 9–11 per genotype, *** p ≤ 0.0004 by t test. (B and C) WT and C5aR1−/− mice were treated with PBS or infected with L. monocytogenes and their spleens removed at 72 h. Splenocytes were stained with the viability dye DAPI, TRAIL-PE, and NK1.1-APC to determine the percentage of live TRAIL+ NK cells. Representative plots of TRAIL expression in DAPI-, NK1.1+ cells are shown (B). The percentage of live TRAIL+ NK cells in mice from two independent experiments are depicted (C). n = 6 per group, ** p = 0.0055, *** p = 0.0001 by ANOVA with the Tukey post-test.

Article Snippet: Serum IFN-α and IFN-β levels at 24 h were measured using the VeriKine Mouse IFN Alpha ELISA kit and VeriKine Mouse IFN Beta ELISA kit (R&D Systems), respectively, as per manufacturer’s instructions.

Techniques: Injection, Isolation, Enzyme-linked Immunosorbent Assay, Infection, Staining, Expressing

Journal: Frontiers in Immunology

Article Title: Structure-guided design of a bivalent SARS-CoV-2 mRNA vaccine with NTD stabilizing mutations enhances broad immunity

doi: 10.3389/fimmu.2025.1718740

Figure Lengend Snippet: In silico design of spike mRNA vaccine induces potent cellular and humoral immune responses against variant of concerns in BALB/c mice. (a) Immunization and bleed schedule for BALB/c mice. Groups of mice (n=8) were vaccinated intramuscularly with two doses of Control spike, in silico spike, or PBS buffer at 3-week intervals. (b) Total splenocytes of immunized mice were collected 2 weeks after the booster vaccination. Cells were re-stimulated with the ancestral spike peptide pool, and splenocytes secreting IFN-γ were measured using ELISpot assay. P -values were determined using two-way ANOVA and Turkey’s multiple comparison, ns, not significant, *p < 0.05, **p < 0.01. (c) Sera of the vaccinated mice were collected 2 weeks after the second vaccination and the neutralizing antibody titers in the sera were analyzed via a plaque reduction neutralization test (PRNT) using the ancestral, Delta, BA.5, and BN.1 SARS-CoV-2 viruses. P-values were determined using one-way ANOVA with Dunnett’s test, ns, not significant, *p < 0.05, **p < 0.01.

Article Snippet: IFN-γ–secreting cells were detected using the mouse IFN-gamma ELISpot kit (XEL485, R&D System).

Techniques: In Silico, Variant Assay, Control, Enzyme-linked Immunospot, Comparison, Plaque Reduction Neutralization Test

Journal: Frontiers in Immunology

Article Title: Structure-guided design of a bivalent SARS-CoV-2 mRNA vaccine with NTD stabilizing mutations enhances broad immunity

doi: 10.3389/fimmu.2025.1718740

Figure Lengend Snippet: Design and optimization of a omicron-based mosaic spike antigens with NTD stabilizing mutations. (a) Schematic of the Omi S and Omi_dsg S spike antigen constructs. Omi S is a mosaic antigen composed of NTD and RBD sequences from Omicron variants and an S2 region from Css_dsg S. Omi_dsg S was further optimized for structural stability and codon usage. (b) Western blot analysis of spike protein expression in HEK293T cells transfected with in vitro -transcribed Omi S or Omi_dsg S mRNA. Omi_dsg S showed enhanced and prolonged expression at 48 h after transfection. (c) Relative spike protein expression levels with quantitative values of western blot analysis. The intensity of the bands in (b) was quantified to the expression of Omi S at 12 h using ImageJ software. (d) Schematic of the mouse immunization schedule. BALB/c mice were immunized with PBS, Omi S, or Omi_dsg S mRNA vaccines. (e) IFN-γ ELISpot analysis of splenocytes from vaccinated mice. (f) Neutralizing antibody titers measured against SARS-CoV-2 variants at 2 weeks after immunization. Omi_dsg S induced significantly higher neutralization titers than Omi S. P-values were determined using one-way ANOVA with Dunnett’s test, ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (g) Western blot analysis of spike protein expression from JN.1-based constructs transfected into HEK293T cells. Q173S and S256Q mutations, alone or in combination, showed increased expression levels compared to wild-type JN.1 spike. Omission of these mutations from a 12-mutation NTD design reduced expression, supporting their functional role as key stabilizing residues. (h) , Relative spike protein expression levels with quantitative values of western blot analysis. The intensity of the bands in (f) was quantified to the expression of JN.1 WT Spike using ImageJ software.

Article Snippet: IFN-γ–secreting cells were detected using the mouse IFN-gamma ELISpot kit (XEL485, R&D System).

Techniques: Construct, Western Blot, Expressing, Transfection, In Vitro, Software, Vaccines, Enzyme-linked Immunospot, Neutralization, Mutagenesis, Functional Assay

Journal: Leukemia & lymphoma

Article Title: Chidamide enhances T-cell-mediated anti-tumor immune function by inhibiting NOTCH1/NFATC1 signaling pathway in ABC-type diffuse large B-cell lymphoma.

doi: 10.1080/10428194.2024.2328227

Figure Lengend Snippet: Figure 2. Effects of chidamide on PDL1, NOTCH1, and NFATC1 signaling pathways in human DLBCL cells and effects of interfering NOTCH1 expression on NFATC1 protein expression and cell proliferation in human DLBCL cells. The expression of the cytoplasmic protein PDL1, NOTCH1, and nuclear protein NFATC1 was detected by treating OCI-LY3, OCI-LY10, and HBL-1 with a concentration gradient (A, B) and a time gradient (C, D) of chidamide, respectively. (E) Flow cytometry was used to detect the expression of cell NOTCH1; (F) cell culture supernatant was collected and IL-10 secretion levels were detected by ELISA (n = 3; *p < .05; **p < .01; ***p < .001; ****p < .0001; unmarked means p > .05, no statistical difference). (G–I) NOTCH1-shRNA transfection inhibits NFATC1 protein expression in OCI-LY10 cells. (H, I) After the transfection of NOTCH1-shRNA into OCI-LY3 cells, the growth viability of DLBCL cells with the interference of NOTCH1 expression was significantly inhibited with the extension of culture time (H). After 72 h of culture, the OD value of the NOTCH1-sh group was (0.7527 ± 0.0817), and the cell survival rate of this group was 47.75% of that of the NC group (negative control group), with a significant difference (p < .0001) (I). Compared with the NC group (negative control group), the cell proliferation viability of the NOTCH1-sh group decreased with time, p < .0001. The experiment was repeated three times independently.

Article Snippet: Annexin V-FITC/PI Apoptosis Detection Kit (cat. no. E-CK-A211), Human IL-10 ELISA Kit (cat. no. E-EL-0162c), Mouse IL-2 ELISA Kit (cat. no. E-EL-M0042c), Mouse IFN-γ ELISA Kit (cat. no. E-EL-M0048c), and Mouse TNF-α ELISA Kit (cat. no. E-EL-M3063) were obtained from Elabscience Biotechnology (Wuhan, China).

Techniques: Protein-Protein interactions, Expressing, Concentration Assay, Flow Cytometry, Cell Culture, Enzyme-linked Immunosorbent Assay, shRNA, Transfection, Negative Control

Journal: Leukemia & lymphoma

Article Title: Chidamide enhances T-cell-mediated anti-tumor immune function by inhibiting NOTCH1/NFATC1 signaling pathway in ABC-type diffuse large B-cell lymphoma.

doi: 10.1080/10428194.2024.2328227

Figure Lengend Snippet: Figure 6. Serum levels of IL-2, IFN-γ, and TNF-α in mice. After the completion of treatment with chidamide, mouse serum was taken and IFN-γ (A) and IL-2 (B), TNF-α (C) were detected using the ELISA method, using GraphPad Prism 9.0 (La Jolla, CA) to display statistical charts of relative cytokine secretion (n = 5; **p < .01; ***p < .001; unmarked means p > .05, no statistical difference).

Article Snippet: Annexin V-FITC/PI Apoptosis Detection Kit (cat. no. E-CK-A211), Human IL-10 ELISA Kit (cat. no. E-EL-0162c), Mouse IL-2 ELISA Kit (cat. no. E-EL-M0042c), Mouse IFN-γ ELISA Kit (cat. no. E-EL-M0048c), and Mouse TNF-α ELISA Kit (cat. no. E-EL-M3063) were obtained from Elabscience Biotechnology (Wuhan, China).

Techniques: Enzyme-linked Immunosorbent Assay