Journal: Frontiers in Cell and Developmental Biology

Article Title: Partial Inhibition of the 6-Phosphofructo-2-Kinase/Fructose-2,6-Bisphosphatase-3 (PFKFB3) Enzyme in Myeloid Cells Does Not Affect Atherosclerosis

doi: 10.3389/fcell.2021.695684

Figure Lengend Snippet: Decreased Pfkfb3 expression and decreased glycolysis in Pfkfb3 fl/fl bone marrow-derived macrophages. (A) Pfkfb3 mRNA expression in Pfkfb3 fl/fl (FL) relative to Pfkfb3 wt/wt (WT) bone marrow-derived macrophages (BMDM). (B) Extracellular acidification rate (ECAR, percentage of baseline) of Pfkfb3 fl/fl and Pfkfb3 wt/wt BMDMs after 10 mM glucose stimulus, as assessed with a Seahorse XF Analyzer. (C) Glucose and (D) lactate levels in Pfkfb3 fl/fl and Pfkfb3 wt/wt BMDM culture media after 26 h of culture. (E) Il10 mRNA expression in unstimulated Pfkfb3 fl/fl relative to Pfkfb3 wt/wt BMDMs. (F) Pfkfb3 mRNA expression in Pfkfb3 fl/fl relative to Pfkfb3 wt/wt BMDMs after pro-inflammatory stimulation with LPS and IFN-γ. (G) Il6 and (H) Il12b mRNA expression in Pfkfb3 fl/fl and Pfkfb3 wt/wt BMDMs cultured without additions (ctrl; control) or stimulated with LPS and IFN-γ. Expression is relative to unstimulated Pfkfb3 wt/wt BMDMs. (I) Pfkfb3 mRNA expression in Pfkfb3 fl/fl relative to Pfkfb3 wt/wt bone marrow-derived dendritic cells. The graphs represent mean ± SEM. * p < 0.05, ** p < 0.01. Data were analyzed using Mann-Whitney U test. ECAR; extracellular acidification rate.

Article Snippet: For pro-inflammatory polarization of BMDMs, cells were incubated with LPS (10 ng/ml, L2880, Sigma) and IFN-γ (100 units/ml, HC1020, Hycult Biotech) for 24 h after overnight attachment.

Techniques: Expressing, Derivative Assay, Cell Culture, MANN-WHITNEY

Journal: Nature Communications

Article Title: Small molecule APOL1 inhibitors as a precision medicine approach for APOL1-mediated kidney disease

doi: 10.1038/s41467-024-55408-2

Figure Lengend Snippet: A Assessment of urinary albumin-to-creatinine ratio (UACR) in response to hydrodynamic injection of IFNγ plasmid in APOL1 G2 sc transgenic mice over 14 days. n = 12 (Vehicle) and n = 11 (Compound 3). Vehicle group had 5 F and 7 M; Compound 3 group had 6 F and 5 M. B UACR area under the curve (AUC) showed reduced proteinuria in Compound 3-treated mice. One male Vehicle mouse was excluded from UACR AUC analysis due to premature removal from study because of disease progression. n = 11 mice in both groups. Percent reduction in UACR AUC with compound 3 treatment was calculated. UACR AUC data were log-transformed and analyzed by unpaired t -test. Significance was set at p ≤ 0.05. **** p < 0.0001. p < 0.0001 for Vehicle vs. treatment group. Results are presented as arithmetic mean ± SEM with each symbol representing an individual mouse. Representative data of n = 2 experiments. Source data are provided as a Source Data file.

Article Snippet: In APOL1 G2 hom copy mice, increased proteinuria (mean urinary albumin-to-creatinine ratio, UACR) was induced by a single intraperitoneal (IP) injection of 150 μg/kg of murine IFNγ (R&D Systems, Catalog# 485-MI/CF) in a volume of 10 mL/kg.

Techniques: Injection, Plasmid Preparation, Transgenic Assay, Biomarker Discovery, Transformation Assay

Journal: Advanced Science

Article Title: An Adenoviral‐Vectored Tp0326 Vaccine Elicits Robust Functional Antibodies to Prevent Treponema pallidum Dissemination in a Rabbit Model

doi: 10.1002/advs.78078

Figure Lengend Snippet: The immunological activity of antibodies in rabbit Sera immunized with recombinant adenovirus and protein vaccines. (a) Flow chart of immunization and challenge plan for New Zealand rabbits ( n = 3, respectively). (b) Detection of specific antibody titers at the immune endpoint in the serum of rabbits immunized with different vaccine groups. (c) The concentrations of interleukin‐4 (IL‐4), interleukin‐17A (IL‐17A), and interferon‐gamma (IFN‐γ) in the sera of each vaccine immunization group. (d) The antigen sensitivity of antibodies in each vaccine group and the detection of T.pallidum lysate. (e) Detection of the effects of different sera on the adhesion of T.pallidum to cells by indirect immunofluorescence. (f) Detection of the effects of antisera from each group on the adhesion of T.pallidum to cells by dark‐field microscopy. (g) Bactercidal of antibodies from different groups on the growth of T.pallidum cultivation in vitro. (h) The phagocytosis rate of each group was statistically analyzed according to the number of T.pallidum phagocytized by macrophage and separate tests were conducted on the Nichols reference strain and clinical strains. n.s.: no significance.

Article Snippet: ELISA kits for Mouse IL‐4, Mouse IL‐17a, Mouse IFN‐γ, Mouse IFN‐γ were procured from RaybioTech Co., Ltd. (China).

Techniques: Activity Assay, Recombinant, Vaccines, Sensitivity, Immunofluorescence, Microscopy, In Vitro

Journal: Advanced Science

Article Title: An Adenoviral‐Vectored Tp0326 Vaccine Elicits Robust Functional Antibodies to Prevent Treponema pallidum Dissemination in a Rabbit Model

doi: 10.1002/advs.78078

Figure Lengend Snippet: Protective immune responses in rabbits immunized via different routes with a recombinant adenovirus vaccine against T.pallidum following a low‐dose challenge (1 × 10 2 T. pallidum per site). (a) Flow chart of immunization, challenge, and RIT plan for New Zealand rabbits ( n = 3, respectively). (b) Detection of specific antibody titers at the immune endpoint in the serum of rabbits immunized with different vaccine groups. (c) The concentrations of interleukin‐4 (IL‐4), interleukin‐17A (IL‐17A), and interferon‐gamma (IFN‐γ) in the sera of each vaccine immunization group. (d) Changes in the diameter of chancres following intradermal T.pallidum challenge in rabbits (X = 8/rabbit, respectively). (e) Specific and non‐specific antibodies induced by T.pallidum infection were detected using the T.pallidum particle agglutination assay (TPPA) and the Rapid Plasma Reagin (RPR) test, respectively. (f) The polA gene was amplified by quantitative polymerase chain reaction (qPCR) to measure the T.pallidum burden in the skin at the local site. n.s.: no significance.

Article Snippet: ELISA kits for Mouse IL‐4, Mouse IL‐17a, Mouse IFN‐γ, Mouse IFN‐γ were procured from RaybioTech Co., Ltd. (China).

Techniques: Recombinant, Infection, Agglutination, Clinical Proteomics, Amplification, Real-time Polymerase Chain Reaction

Journal: Scientific Reports

Article Title: Cholesterol-dependent cytolysins impair pro-inflammatory macrophage responses

doi: 10.1038/s41598-018-24955-2

Figure Lengend Snippet: Activation receptors are shed during intrinsic repair. ( A ) BMDM were unchallenged (control) or challenged with 500 HU/mL SLO WT, PFO or an equivalent mass of SLO ML for 10 min at 37 °C and centrifuged at 2000 × g for 5 min to yield cell pellet (C). Supernatants were spun at 100,000xg for 40 min at 4 °C to collect the high speed supernatant (S) and microvesicle pellet (M). Samples were solubilized at 95 °C in SDS-sample buffer, resolved by SDS-PAGE and transferred to nitrocellulose. Portions of the blot were probed with 6D11 anti-SLO, anti-IFNγR1, 4B4F12 anti-CD14, 76B357.1 anti-TLR4, O91B8 anti-MyD88, 1H4B01 anti-Trif, EPR4477 anti-Alkaline Phosphatase, MANLAC-4A7 anti-Lamin A/C, and AC-15 anti-β-Actin antibodies followed by relevant secondary antibodies and ECL. Full-length blots are presented in Supplementary Figure . ( B ) BMDM were challenged with 500 HU/mL SLO WT or SLO N402C, SLO ML at equivalent mass to SLO WT, or SLO N402E at equivalent mass to SLO N402C for 10 min. Cell pellets (C), high speed supernatants (S) and microvesicles (M) were isolated as in ( A ) and probed with 6D11 anti-Streptolysin O, 76B357.1 anti-TLR4, O91B8 anti-MyD88, 1H4B01 anti-Trif, EPR4477 anti-Alkaline Phosphatase, MANLAC-4A7 anti-Lamin A/C, and AC-15 anti-β-Actin antibodies. Full-length blots are presented in Supplementary Figure . The blots are representative of at least 3 independent experiments.

Article Snippet: Anti-IFNγR1 goat polyclonal antibody (Catalog: 1026-GR) was from R&D Systems (Minneapolis, MN).

Techniques: Activation Assay, Control, SDS Page, Isolation