Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Probiomimetics-Novel Lactobacillus-Mimicking Microparticles Show Anti-Inflammatory and Barrier-Protecting Effects in Gastrointestinal Models.

doi: 10.1002/smll.202003158

Figure Lengend Snippet: Figure 6. Cytokine production in inflamed macrophage-like dTHP-1 cells as well as in enterocyte-like Caco-2 cells. Cells were stimulated with lipopoly- saccharides (10 µg mL-1) and cotreated with probiomimetics or MVs. Supernatants were harvested after 6 or 24 h, and the protein content was analyzed using ELISA. To exclude the effects of different MV concentrations during the assembly of the probiomimetics, all results were normalized to the protein content. a) ELISA measurement of proinflammatory TNF-α released in dTHP-1 cells after 6 h. b) Release of anti-inflammatory IL-10 after 6 h. c) ELISA measurement of proinflammatory TNF-α released in dTHP-1 cells after 24 h. d) Release of anti-inflammatory IL-10 after 24 h. e) Release of proinflam- matory IL-8 in enterocyte-like Caco-2 cells after 24 h. Values represent the mean of 3–9 biological replicates with standard deviations.

Article Snippet: The supernatants were thawed and the concentrations of IL-10 and TNF-α were analyzed using Human IL-10 ELISA Set (Diaclone, Besançon, France), Human TNF-α ELISA Set (Diaclone, Besançon, France), and Human IL-8 ELISA Set (Diaclone, Besançon, France), respectively, according to the supplier’s protocols.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: ( A , B ) TAM isolated from ovarian cancer ascites were cultured in Glc low Gln low medium containing GM-CSF and IFNγ supplemented with Glc or lactic acid. Glycogen ( A ; n = 6–8) and relative mRNA expression of PCK2 ( B ; n = 5–13) were quantified in freshly purified TAM and after 3 days of culture. The boxplots display a median line, interquartile range (IQR) boxes, min to max whiskers. ( C ) Immunohistochemical analysis of CD163 expression in lung adenocarcinoma. On the same tissue section, glycogen was detected by PAS staining, after treatment or not with amylase; scale bar, 100 µm. ( D ) Lactic acid and glycogen quantification by FTIR spectroscopic imaging in lung adenocarcinoma. Left panel: unstained bright field image; right panel, glycogen and lactic acid maps; scale bar, 200 µm; linear correlation between lactic acid and glycogen contents in infiltrating lung adenocarcinoma was calculated (results are representative 1 out of 2 biologically independent experiments). ( E ) Day 3 TAM were treated or not with CP-91149 for 15 min before a 6 h stimulation with LPS. TNFα, VEGF and G-CSF were quantified by ELISA ( n = 3). Values are represented as the mean ± SEM, each dot represents a different donor. Statistical significance was determined by Welch’s ANOVA test followed by Dunnett’s multiple comparison post hoc test ( A ), or by two-tailed unpaired Welch t test ( B ) or by paired t test ( E ). * P < 0.01, ** P < 0.005, *** P < 0.001, **** P < 0.0001. .

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Isolation, Cell Culture, Expressing, Purification, Immunohistochemical staining, Staining, Imaging, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: ( A , B ) Day 5 M1 and M2 cells (generated in conventional medium or CM) were treated or not for 15 min with 50 µM CP-91149 before stimulation with 100 ng/mL LPS or with E. coli at a multiplicity of infection of 10. ( A ) Glycogen was quantified after 24 h activation ( n = 3). ( B ) Cytokines were quantified in the supernatants by ELISA after 24 h (IL-12p70) or 6 h (IL-10) stimulation ( n = 5). ( C ) GM-CSF-Mφ were switched on day 2 to Glc low Gln low medium supplemented or not with Gln, lactic acid or glycerol and treated or not with CP-91149 15 min before LPS stimulation. TNFα and IL-6 (2 h), or IL-12p70 (24 h) were quantified by ELISA in stimulated day 5 M1 cells culture supernatants ( n = 6–8). ( D ) M2 cells were pretreated with 50 µM CP-91149 or 10 µM cytochalasin B (CytoB) for 15 min before addition of 0.2 mg/mL pHrodo-conjugated E. coli BioParticles for 2 h at 37 °C. Fluorescence was determined by flow cytometry and expressed in MFI values ( n = 3). ( E ) G6PC1-3 mRNA expression was determined by RT-qPCR in monocytes and day 5 Mφ ( n = 6). Liver cells were used as a positive control. Values are represented as the mean ± SEM, each dot represents a different donor. Statistical significance was determined by paired t test ( A , B ) or by two-tailed unpaired Welch t test ( C , D ). * P < 0.01, ** P < 0.005, *** P < 0.001, **** P < 0.0001.

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Generated, Infection, Activation Assay, Enzyme-linked Immunosorbent Assay, Fluorescence, Flow Cytometry, Expressing, Quantitative RT-PCR, Positive Control, Two Tailed Test

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: ( A ) Day 5 M1 cells were cultured in CM or in Glc low medium and treated or not with CP-91149 or 6AN, 15 min before LPS stimulation. Cytokines were quantified by ELISA in the M1 cells culture supernatants after 2 h (TNFα and IL-6), 6 h (IL-1β) or 24 h (IL-12p70) activation with LPS ( n = 5–14). ( B , C ) Day 2 GM-CSF-Mφ were incubated either with siRNA targeting PYGL, PYGB, G6PD or a control siRNA. Cells were then stimulated with LPS at day 5, and cytokines were quantified by ELISA in M1 cell culture supernatants after 2 h (TNFα) or 6 h (IL-1β) ( B ) PYGL, PYGB and G6PD mRNA expression were determined by RT-qPCR in day 5 macrophages ( n = 4) ( C ). ( D ) Day 5 M2 cells were cultured in CM or in Glc low medium and treated or not with CP-91149, 15 min before LPS stimulation. Cytokines were quantified by ELISA in the M2 cells culture supernatants after 2 h (IL-6), 6 h (IL-10) activation with LPS ( n = 5–14). Phagocytosis was assessed by flow cytometry after 3 h LPS stimulation ( n = 6). ( E , F ) Lactate was quantified in 6 h LPS-stimulated M1 ( E ) and M2 ( F ) cells culture supernatants and oxygen consumption rate (OCR) of M1 and M2 cells was monitored after 2 h ( n = 4–6). Values are represented as the mean ± SEM, each dot represents a different donor. Statistical significance was determined by Welch’s ANOVA test followed by Dunnett’s multiple comparison post hoc test or two-tailed unpaired Welch t test for phagocytosis assay in ( D ). * P < 0.01, ** P < 0.005, *** P < 0.001, **** P < 0.0001. .

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Incubation, Control, Expressing, Quantitative RT-PCR, Flow Cytometry, Comparison, Two Tailed Test, Phagocytosis Assay

Journal: EMBO Reports

Article Title: Glycogenesis and glyconeogenesis from glutamine, lactate and glycerol support human macrophage functions

doi: 10.1038/s44319-024-00278-4

Figure Lengend Snippet: Reagents and tools table

Article Snippet: IL-6 (human) ELISA set , Diaclone , 851 520 020.

Techniques: Sequencing, Control, Magnetic Beads, Recombinant, Colorimetric Assay, Staining, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Software, Microscopy, Cytometry, Mass Spectrometry, Imaging, Spectrophotometry