Journal: Science Advances

Article Title: Biofunctional lipid nanoparticles for precision treatment and prophylaxis of bacterial infections

doi: 10.1126/sciadv.adk9754

Figure Lengend Snippet: ( A ) Expression levels of CD80 + and CD86 + in BMDCs (CD11c + ) and ( B ) production of IL-6 and TNF-α of the cell supernatant measured by ELISA after different treatments (concentration, 2 μg ml −1 NPs) for 24 hours ( n = 3). ( C ) Schematics of experimental design to evaluate the in vivo short-term immune responses activated by a single dose of various LNPs (20 μg/20 g) via subcutaneous injection. ( D ) Percentage of CD80 + and ( E ) CD86 + cells (gated on CD11c + cells) in the LNs at day 3 after immunization ( n = 6). ( F ) Percentage of CD3 + T cells, ( G ) CD8 + T cells (gated on CD3 + T cells), and ( H ) CD4 + T cells (gated on CD3 + T cells) in the spleen at day 3 after immunization ( n = 6). Naive mice without immunization were used as control. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Fluorescein isothiocyanate (FITC)–conjugated anti-mouse CD11c, phycoerythrin (PE)–conjugated anti-mouse CD80, PE-conjugated anti-mouse CD86, allophycocyanin (APC)–conjugated anti-mouse CD3, PE-conjugated anti-mouse CD8a, and FITC-conjugated anti-mouse CD4 were purchased from Tonbo Biosciences, USA.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay, In Vivo, Injection, Control

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

Article Title: Cholera toxin promotes the induction of regulatory T cells specific for bystander antigens by modulating dendritic cell activation.

doi: 10.4049/jimmunol.171.5.2384

Figure Lengend Snippet: FIGURE 9. CT enhances CD80 and CD86, but inhibits CD40 and ICAM-1 expression on DC. DC were stimulated with medium alone, CT (1 g/ml), LPS (10 ng/ml), or CT plus LPS in the presence or the absence of anti-IL-10 (10 g/ml). After 24-h incubation, cells were washed and stained with Abs specific for CD80, CD86, OX40, CCR5, ICAM-1, and CD40 or with isotype-matched control Abs. Immunofluorescence is shown for treated (black line) compared with untreated (gray histograms) DC. The numbers on the right of each histogram refer to the mean fluorescence intensity of the treated cells; the value for cells treated with medium alone is presented on the left of the first histogram in each case. Profiles are shown for a single experiment and are representative of five experiments.

Article Snippet: The expression of DC surface markers was assessed using anti-mouse CD40 (rat IgG2a, clone 3/23), I-A d (mouse IgG2b, clone AMS-32.1), CD80 (hamster IgG2, clone 16-10A1), CD86 (rat IgG2a, clone GL1), CD11c (hamster IgG1, clone HL3), and CD134 (OX40; rat IgG1, clone OX86; Serotec, Oxford, U.K.).

Techniques: Expressing, Incubation, Staining, Control

Journal: Frontiers in Immunology

Article Title: Targeting immunosuppressive Ly6C+ classical monocytes reverses anti-PD-1/CTLA-4 immunotherapy resistance

doi: 10.3389/fimmu.2023.1161869

Figure Lengend Snippet: Anti-PD-1/CTLA-4/Ly6C treatment reduces tumor growth and increases CD8 T cells/dendritic cells. (A) 344SQ tumor bearing 129/Sv mice were weekly treated with anti-PD-1_CTLA-4 (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 per mouse), anti-Ly6C alone (200 µg of anti-Ly6C per mouse), anti-PD-1_CTLA-4_Ly6C (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 plus 200 µg of anti-Ly6C per mouse), or their IgG control mixture (IgG control) beginning on week 2 after a subcutaneous cancer cell injection (1 x 10 6 cells per mouse; n = 5) for 4 weeks. Mice received total of 4 treatments starting at week 2 post tumor cell implantation. (B) tSNE CD45 plots from 344SQ tumors treated with IgG control, anti-PD-1_CTLA-4, or anti-PD-1_CTLA-4_Ly6C from (A) . Tumors from (A) were harvested to prepare single cell suspensions for FACS analysis. (C) Percentages of CD8 T cells (left), Effector/memory CD8 T cells (middle), and dendritic cells (right). (D) Percentage of Ly6C- (left) and Ly6C+ (right) CD14+CD115+ monocytes. Kras LSL-G12D /p53 fl/fl mice generated through intratracheal administration of adenovirus expressing Cre recombinase were treated with either anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C for 8 weeks (E) Micro-CT images shown at week 0 (baseline) and week 8 (endpoint) for anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C. Dashed yellow circles indicate lung tumors. H indicates of heart. (F) Percentage change of tumor area was calculated taking into account prior time point and normalized to the baseline measurement. (G) H&E stained lung sections at week 8 from anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C treated mice from (E) . bar= 5 mm. (H) Number of lung tumors on H&E sections from (G) treated with anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C weekly for 8 weeks. (I) IHC of CD8 stained lung tumors from (F) week 8. Percentage of CD8 T cells found in the lung tumors from Kras LSL-G12D /p53 fl/fl mice treated for 8 weeks with anti-PD-1/CTLA-4 or anti-PD-1/CTLA-4/Ly6C. (J) The dendritic cells from CD11c-DTR mice were transferred into C57BL/6 mice to generate the chimerical mice, after 8-week stabilization, the mice were treated with diphtheria toxin twice a week to maintain the depletion of dendritic cells. The B16 melanoma-bearing mice were treated weekly with anti-PD-1_CTLA-4_Ly6C for 3 weeks staring on week 1 after tumor cells inoculation. (K) 344SQ tumor bearing 129/Sv mice were treated weekly with blockade of PD-1, CTLA-4, and Ly6C (200 µg of anti-PD-1 plus 200 µg of anti-CTLA-4 plus 200 µg of anti-Ly6C per mouse) or their IgG control mixture (IgG control) beginning on day 7 after a subcutaneous cancer cell injection (0.1 x 10 6 cells per mouse; n = 5) for 4 weeks. For blocking B7 signal, antibodies (anti-B7: 300 μg of anti-CD80 and 300 μg of anti-CD86 per mouse) were intraperitoneally administered 1 day before the first dose of therapy, and then once a week to maintain the blockade. ANOVA test was used to analyze the data. n.s., not significance; *p < 0.05; **p < 0.01; ***p< 0.001; ****p < 0.0001.

Article Snippet: Anti-mouse PD-1 (clone RMP1-14), anti-mouse CTLA-4 (clone 9D9), anti-mouse CD80 (clone 16-10A1), anti-mouse CD86 (clone GL-1), anti-mouse Ly6C (clone Monts 1), anti-mouse CCL2 (clone 2H5), and the isotype-matched IgG controls were purchased from BioXCell.

Techniques: Control, Injection, Generated, Expressing, Micro-CT, Staining, Blocking Assay

Journal: Advanced Science

Article Title: Mucous Permeable Nanoparticle for Inducing Cuproptosis‐Like Death In Broad‐Spectrum Bacteria for Nebulized Treatment of Acute Pneumonia

doi: 10.1002/advs.202408580

Figure Lengend Snippet: Activation of macrophage response by Cu 2 O‐BSO NPs in vitro. a) Typical scatter plots of MH‐S surface markers CD86 (M1 macrophage marker) and CD206 (M2 macrophage marker) detected by flow cytometry. b) Typical images of MH‐S engulfing a bacterium. The green fluorescence represents GFP‐MRSA, the red fluorescence represents MH‐S, and the blue fluorescence represents the nucleus. The stronger the green fluorescence, the stronger the phagocytosis of MH‐S. c,d) Representative SPM photographs and CFU count of MRSA engulfed by MH‐S. e–g) ELISA results indicating levels of cytokines (IL‐1β, IL‐6, and TNF‐α) secreted by MH‐S in different groups.

Article Snippet: Each group of cells was centrifuged, washed, and resuspended in 500 μL of PBS, which contained an anti‐mouse CD86 antibody coupled with FITC (Elabscience) and an anti‐mouse CD206 antibody coupled with PE (Elabscience).

Techniques: Activation Assay, In Vitro, Marker, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Pharmacology

Article Title: Huanshaodan regulates microglial glucose metabolism reprogramming to alleviate neuroinflammation in AD mice through mTOR/HIF-1α signaling pathway

doi: 10.3389/fphar.2024.1434568

Figure Lengend Snippet: Expression levels of CD86, Arg1 and IL-1β protein in hippocampus and cortex of SAMP8 mice detected by Western-blot, the expression levels of IL-6 and TNF-α in hippocampus and cortex of SAMP8 mice by ELISA. (A,E) Blotting of CD86, Arg1 and IL-1β protein in hippocampus and cortex of mice in each group; (B–D) Expression of CD86, Arg1 and IL-1β protein in hippocampus of mice in each group; (F–H) Expression of CD86, Arg1 and IL-1β protein in cortex of mice in each group; (I–L) Expression of IL-6 and TNF-α protein in hippocampus and cortex of mice in each group. Model group compared with the control group * p < 0.05, ** p < 0.01, *** p < 0.001, Donepezil group compared with the model group # p < 0.05, ## p < 0.01, ### p < 0.001, HSD group compared with the model group $ p < 0.05, $$ p < 0.01, $$$ p < 0.001.

Article Snippet: The main materials that were used in this study were LPS (L8880, Solarbio), MHY1485 (MHY) (M9050-5MG, AbMole), RIPA buffer (high) (R0010, Solarbio), primary antibodies β-Actin and CD86 (42kDa, BM0627; 70kDa, BM4121, BOSTER), Arg1 and mTOR (40kDa, GB11285-100; 289kDa, GB111839-100, Servicebio), p-mTOR and HIF-1α (289kDa, 5536T; 120kDa, 48085S, Cell Signaling Technology), IL-1β (31kDa, 16806-1-AP, Proteintech), Horseradish peroxidase (HRP)-linked secondary antibodies against rabbit and mouse IgG of the primary antibodies (ZB-2301; ZB-2305, Beijing Zhong Shan-Golden Bridge Biological Technology), Glucose (GLU) Assay Kit, Lactic Acid (LD) assay kit, ATP assay kit, Lactate dehydrogenase (LDH) assay kit (A154-1-1; A019-2-1; A095-1-1; A020-2-2, Nanjing Jiancheng Bioengineering Institute), Pyruvate Dehydrogenase (PDH) Activity Assay Kit (E-BC-K650-M, Elabscience), BCA Protein Assay Kit (PC0020, Solarbio), ELISA Kit for Hexokinase 2 (HK2) activity based on enzyme standard of HK2, ELISA Kit for Tumor Necrosis Factor alpha (TNF-α) (MM-45418M1; MM-0132M1, MEIMIAN), ELISA Kit for Interleukin 6 (IL-6) (E-MSEL-M0001, Elabscience) and ELISA Kit for Pyruvate kinase isozymes M2 (PKM2) (SEA588Mu, Cloud Clone Corp), Cell Staining Buffer, PE Anti-Mouse F4/80 Antibody [CI:A3-1], PE/Cyanine7 Anti-Mouse CD86 Antibody [GL-1], Intracellular Fixation/Permeabilization Buffer Kit and APC Anti-Mouse CD206 Antibody (E-CK-A107; E-AB-F0995D; E-AB-F0994H; E-CK-A109; E-AB-F1135E, Elabscience).

Techniques: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Control

Journal: Frontiers in Pharmacology

Article Title: Huanshaodan regulates microglial glucose metabolism reprogramming to alleviate neuroinflammation in AD mice through mTOR/HIF-1α signaling pathway

doi: 10.3389/fphar.2024.1434568

Figure Lengend Snippet: The expression levels of p-mTOR/mTOR ratio, HIF-1α, and IL-1β in BV2 cells tested by western blot, the levels of GLU, LD, ATP, LDH and PDH quantified by biochemical test kit and the levels of polarization biomarkers of CD86 and CD206 detected by flow cytometry, the expression levels of IL-6 and TNF-α in BV2 cells tested by ELISA. (A) Blotting of p-mTOR, mTOR and HIF-1α protein in BV2 cells in each group; (B, C) Expression levels of p-mTOR/mTOR ratio and HIF-1α in BV2 cells in each group. (D) GLU content analysis chart; (E) LD content analysis chart; (F) ATP content analysis chart; (G) . LDH activity analysis graph; (H) PDH activity analysis graph. (I) Flow cytometry analysis charts; (J) Flow cytograms of BV2 cells in each group; (K) Blotting of IL-1β protein in BV2 cells in each group; (L) Expression level of IL-1β in BV2 cells in each group. (M, N) Expression level of IL-6 and TNF-α in BV2 cells in each group. Model group compared with control group * p < 0.05, ** p < 0.01, *** p < 0.001, HSD-containing serum-treated group compared with the model group $ p < 0.05, $$ p < 0.01, $$$ p < 0.001, Reverse validation group compared with the HSD-containing serum-treated group & p < 0.05, && p < 0.01, &&& p < 0.001.

Article Snippet: The main materials that were used in this study were LPS (L8880, Solarbio), MHY1485 (MHY) (M9050-5MG, AbMole), RIPA buffer (high) (R0010, Solarbio), primary antibodies β-Actin and CD86 (42kDa, BM0627; 70kDa, BM4121, BOSTER), Arg1 and mTOR (40kDa, GB11285-100; 289kDa, GB111839-100, Servicebio), p-mTOR and HIF-1α (289kDa, 5536T; 120kDa, 48085S, Cell Signaling Technology), IL-1β (31kDa, 16806-1-AP, Proteintech), Horseradish peroxidase (HRP)-linked secondary antibodies against rabbit and mouse IgG of the primary antibodies (ZB-2301; ZB-2305, Beijing Zhong Shan-Golden Bridge Biological Technology), Glucose (GLU) Assay Kit, Lactic Acid (LD) assay kit, ATP assay kit, Lactate dehydrogenase (LDH) assay kit (A154-1-1; A019-2-1; A095-1-1; A020-2-2, Nanjing Jiancheng Bioengineering Institute), Pyruvate Dehydrogenase (PDH) Activity Assay Kit (E-BC-K650-M, Elabscience), BCA Protein Assay Kit (PC0020, Solarbio), ELISA Kit for Hexokinase 2 (HK2) activity based on enzyme standard of HK2, ELISA Kit for Tumor Necrosis Factor alpha (TNF-α) (MM-45418M1; MM-0132M1, MEIMIAN), ELISA Kit for Interleukin 6 (IL-6) (E-MSEL-M0001, Elabscience) and ELISA Kit for Pyruvate kinase isozymes M2 (PKM2) (SEA588Mu, Cloud Clone Corp), Cell Staining Buffer, PE Anti-Mouse F4/80 Antibody [CI:A3-1], PE/Cyanine7 Anti-Mouse CD86 Antibody [GL-1], Intracellular Fixation/Permeabilization Buffer Kit and APC Anti-Mouse CD206 Antibody (E-CK-A107; E-AB-F0995D; E-AB-F0994H; E-CK-A109; E-AB-F1135E, Elabscience).

Techniques: Expressing, Western Blot, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Activity Assay, Control, Biomarker Discovery