Journal: Frontiers in Pharmacology

Article Title: Evidence for the existence of facilitatory interactions between the dopamine D2 receptor and the oxytocin receptor in the amygdala of the rat. Relevance for anxiolytic actions

doi: 10.3389/fphar.2023.1251922

Figure Lengend Snippet: Detection of D2R–OXTR heteroreceptor complexes in the different subfields of rat amygdala by in situ proximity ligation assay ( in situ PLA). The nuclei are shown in blue (DAPI) and the positive PLA puncta/blobs (D2R–OXTR heteroreceptor complexes) in red. (Left panel) Quantitative analysis of the D2R–OXTR heteroreceptor complex expression in the CeA and BLA of the rat brain detected by means of the in situ PLA. PLA was quantified as PLA (red puncta/blobs) per nuclei per field by an experimenter blind to the experiment conditions. The analysis (means ± S.E.M.) was performed on images obtained from at least two slices per animal (four rats in total). Statistical analysis was performed by Student’s t-test with Bonferroni correction. The p -value of 0.05 and lower was considered significant: * p < 0.05. Bregma 1.00 mm, Scale bar is 30 μm. (Right panels) Representative confocal image of the in situ PLA at the level of the CeA and BLA. Some positive PLA clusters/blobs are indicated by an arrow, and the scale bar for this panel is indicated on the right bottom of the panel.

Article Snippet: Control experiments for in situ PLA were conducted on free-floating formalin-fixed rat brain sections using only one primary antibody (mouse monoclonal anti-D2R, MABN53, 1:600, Millipore, Sweden).

Techniques: In Situ, Proximity Ligation Assay, Expressing

Journal: bioRxiv

Article Title: The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

doi: 10.1101/2023.09.23.559132

Figure Lengend Snippet: (A) Diagrammatic representation of the experimental set-up. WT, Gla -KO, or A4galt -KO mice were immunized with NP-OVA adsorbed on alum. (B) FACS plots and percentages of centroblasts (CB) and centrocytes (CC) in the spleen on day 10 post immunization. Each dot represents one mouse (7 or 8 mice per group), and the experiment was repeated three times. (C) Immunoblot showing BCR and CD19 downstream signaling molecules and transcription factors. Anti-BCR antibodies (F(ab) 2 ) were used to stimulate FACS-sorted GC B cells for 2 or 5 minutes. U = unstimulated. (D) Proximity ligation assay (PLA) performed on GC B cells to probe for vicinity between CD81 and CD19 (top panel; blue = DAPI, red = CD19:CD81 PLA signal), and BCR and CD19 (bottom panel; blue = DAPI, red = CD19:BCR PLA signal). In all panels, experiments were repeated at least three times, and PLA signal on more than 30 cells in different fields was calculated for statistical analysis. (E) PLA performed on FACS-sorted GC B cells to probe for proximity between CD19 and Gb3 (blue = DAPI, red = CD19:Gb3 PLA signal). PLA signal was captured by confocal microscopy, and images were processed and analyzed by Image J software. Experiment was repeated at least three times, and PLA signal on more than 30 cells in different fields was calculated for statistical analysis. (F) Structures of different lipids and Gb3 analog used in the study. (G) Phospho-flow to examine the effect of lipid reconstitution on Akt phosphorylation in GC B cells from A4galt -KO mice. Histogram overlay and mean fluorescence intensity (MFI) of pAkt staining in GC B cells cultured with different lipids (bottom panel). FACS-sorted GC B cells from A4galt -KO mice were seeded with a complex of lipid and BSA for 2h at 37°C (see methods), and Akt phosphorylation was quantified after stimulation of GC B cells with F(ab) 2 . (H) Isothermal titration calorimetry (ITC) to measure the binding between Gb3 and CD19. CD19 and the Gb3 analog were dissolved in PBS, and thermodynamic analysis of their binding was carried out at 25°C on a MicroCal ITC 200 instrument. Top panel: x-axis depicts time, and y-axis represents rate of heat release (μcal/sec). Bottom panel: x-axis represents molar ratio between CD19 and Gb3-analog, and y-axis depicts change in enthalpy. (I) A mechanistic scheme of the effect of Gb3 on the CD19 translocation and BCR downstream signaling pathway. The p -values in all graphs were calculated by Kruskal-Wallis H test with Dunn’s multiple comparison test.

Article Snippet: To study the interaction between Gb3 and CD19, anti-mouse CD19 and anti-Gb3 antibodies were directly tagged to plus and minus oligo probes, using the PLA probe maker kit following the manufacturer’s guidelines (Millipore Sigma MA, USA), and fixed cells were directly labeled with tagged antibodies overnight.

Techniques: Western Blot, Proximity Ligation Assay, Confocal Microscopy, Software, Fluorescence, Staining, Cell Culture, Isothermal Titration Calorimetry, Binding Assay, Translocation Assay, Comparison

Journal: bioRxiv

Article Title: The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

doi: 10.1101/2023.09.23.559132

Figure Lengend Snippet: (A) Proximity ligation assay (PLA) performed on GC B cells to quantify the recruitment of PI3K to CD19 (blue = DAPI, red = CD19:PI3K PLA signal). FACS-sorted GC B cells were stimulated with anti-BCR antibodies (F(ab) 2 ) and were probed with specific antibodies. Experiment was repeated three times, and PLA signal on more than 30 cells in different fields was calculated for statistical analysis. U = unstimulated. (B) Protein lipid overlay assay to assess the CD19 binding capability of different lipids. Lipids were deposited on a PVDF membrane and their binding to CD19 (containing histidine) was tested by using anti-His-HRP secondary antibody using chemiluminescence. The p -values in graph were calculated by Kruskal-Wallis H test with Dunn’s multiple comparison test.

Article Snippet: To study the interaction between Gb3 and CD19, anti-mouse CD19 and anti-Gb3 antibodies were directly tagged to plus and minus oligo probes, using the PLA probe maker kit following the manufacturer’s guidelines (Millipore Sigma MA, USA), and fixed cells were directly labeled with tagged antibodies overnight.

Techniques: Proximity Ligation Assay, Protein-lipid Overlay Assay (PLOA), Binding Assay, Membrane, Comparison

Journal: bioRxiv

Article Title: The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

doi: 10.1101/2023.09.23.559132

Figure Lengend Snippet: (A) Experimental set-up depicting immunization and anti-IFNAR1 antibody treatment. Mice were immunized with NP-OVA adsorbed on alum, and blocking antibodies were administered on day 6 and 8 after immunization. (B) Frequencies of GC B cells in the spleen on day 10 post immunization. Each dot represents one mouse (4 mice per group), and the experiment was repeated at least three times. (C) Anti-NP-OVA immunoglobulin titers of indicated isotypes in the serum were measured by ELISA. Each dot represents one mouse (4 mice per group), and the experiment was repeated at least three times. (D) Experimental set-up depicting NP-OVA immunization of Gla - and Ifnar -deficient mice. (E) FACS plots and percentages of IgG1 + and IgG2c + GC B cells in the spleen were quantified by flow cytometry. Dots in bar graphs depict individual mice (n=5 mice per group). (F) Ratio of IgG2c to IgG1 titers in the serum. Bar graph shows the ratio of endpoint titers for IgG2c and IgG1, and each point in the graph depicts an individual mouse (n=5 mice per group). (G) PLA performed on GC B cells to probe for proximity between Gb3 and IFNAR1. Images were captured by confocal microscopy (blue = DAPI, red = IFNAR:Gb3 PLA signal). Experiments were repeated at least three times, and PLA signal on more than 30 cells in different fields was calculated for statistical analysis. (H) Immunoblot showing IFNAR1 downstream signaling. IFNα was used to stimulate B cells for 5 or 15 minutes. U = unstimulated. The p -values in graph B and G were calculated by Kruskal-Wallis H test with Dunn’s multiple comparison test, while two-sided Mann-Whitney U test was used for analysis in graph C, E, and F.

Article Snippet: To study the interaction between Gb3 and CD19, anti-mouse CD19 and anti-Gb3 antibodies were directly tagged to plus and minus oligo probes, using the PLA probe maker kit following the manufacturer’s guidelines (Millipore Sigma MA, USA), and fixed cells were directly labeled with tagged antibodies overnight.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Confocal Microscopy, Western Blot, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: The lipid Gb3 promotes germinal center B cell responses and anti-viral immunity

doi: 10.1101/2023.09.23.559132

Figure Lengend Snippet: (A) Representative FACS plots showing gating strategy to identify B cell subsets among human tonsil cells. GC B cells (CD38 + ); plasma cells (CD27 + , IgD - ). CD77-positive and CD77-negative GC B cells were sorted from human tonsils, and CD77-negative GC B cells were subsequently cultured with Gb3. FACS analysis shows Gb3-positivity after culture (bottom panel). CD77 = Gb3. (B) Proximity ligation assay (PLA) performed on GC B cells to probe for vicinity between Gb3 and CD19 (blue = DAPI, red = CD19:Gb3 PLA signal). CD77-negative, CD77-positive, and CD77-negative B cells reconstituted with Gb3 were used for PLA. (C) Immunoblot showing CD19 downstream signaling molecules (Akt) and transcription factors (Foxo1). CD77-negative, CD77-positive, and CD77-negative B cells reconstituted with Gb3 were used for immunoblot analysis. Anti-BCR antibodies (F(ab) 2 ) were used to stimulate B cells. U = unstimulated. (D) Workflow for stimulation of human tonsil organoids with antigens and adjuvants. (E) Graphs depicting GC B cell and plasma cell percentages from unstimulated or antigen/adjuvant-stimulated tonsil organoids on day 10 post treatment measured by flow cytometry. Each dot in bar graph represents a separate human tonsil donor (n=7). (F) IgG1 secretion from unstimulated or antigen/adjuvant-stimulated tonsil organoids on day 21 post treatment. Each dot in graph represents a different human tonsil donor (n=7). In all graphs, data were analyzed using Kruskal-Wallis H test with Dunn’s multiple comparison test. (G) Illustration depicts the regulation of the GC B cell response by Gb3 (left panel) and its translation as an adjuvant against influenza infection (right panel). Gb3 binds to CD19 to promote BCR downstream signaling and Foxo1 modulation, driving the cycling of GC B cells to the light zone. Moreover, Gb3 regulates MHC-II antigen presentation to Tfh cells to facilitate selection of antibodies against subdominant epitopes. Use of Gb3 as an adjuvant induces IgG2c class switch and selects antibodies recognizing the stalk region of hemagglutinin. Collectively, these mechanisms provide superior protection against heterologous influenza infection. GC = germinal center; Tfh = T follicular helper cell; TCR = T cell receptor; FDC = follicular dendritic cell; rHA = recombinant hemagglutinin; dLN = draining lymph node; H1N1/H3N2 = influenza virus strains.

Article Snippet: To study the interaction between Gb3 and CD19, anti-mouse CD19 and anti-Gb3 antibodies were directly tagged to plus and minus oligo probes, using the PLA probe maker kit following the manufacturer’s guidelines (Millipore Sigma MA, USA), and fixed cells were directly labeled with tagged antibodies overnight.

Techniques: Cell Culture, Proximity Ligation Assay, Western Blot, Adjuvant, Flow Cytometry, Comparison, Infection, Selection, Recombinant, Virus