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

Article Title: Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells.

doi: 10.4049/jimmunol.1001919

Figure Lengend Snippet: FIGURE 1. Effect of IL-18 on the growth of gd T cells and CD56brightCD11c+ cells. A, Effect of IL-18 (100 ng/ml) on expansion of gd T cells. PBMCs derived from a representative healthy adult individual were stimulated by ZOL (1 mM)/IL-2 (10 ng/ml) or 2M3BPP (100 nM–1 mM)/IL-2 (solid bar); ZOL/IL-2/IL-18 or 2M3BPP/IL-2/IL-18 (open bar); or ZOL/IL- 2/anti–IL-18Ra mAb (2 mg/ml; gray bar) or 2M3BPP/IL-2/anti–IL-18Ra mAb. The total number of gd T cells was quantified by trypan blue dye exclusion and flow cytometry. **p , 0.001. B, Massive cell aggregation induced by ZOL/IL-2/IL-18. Cell clusters were photo- graphed using a Nikon Digital Sight TE300- HM-2 (310) microscope (Nikon, Tokyo, Ja- pan) after 5 d of culture and analyzed by Lu- mina Vision Software (Mitani, Tokyo, Japan). C, Proportion of gd TCR-bearing cells in cul- ture on days 0 and 14. PBMCs were incubated in the presence of ZOL/IL-2/IL-18 for 14 d and analyzed for expression of CD3 and gd TCR. D, Phenotypic analysis of PBMCs in culture on days 0 and 14. PBMCs incubated with ZOL/ IL-2 or ZOL/IL-2/IL-18 were analyzed for expression of CD11c, CD3, and CD56 on days 0 and 14. CD56intCD11c2, CD56intCD11c+, CD56brightCD11c2, and CD56brightCD11c+ cells were gated, respectively. E, Time course of the expansion of gd T cells and CD56bright

Article Snippet: Depletion of CD3 +, CD56+, and CD14+ cells from PBMCs was conducted using LD columns and microbeads conjugated with mAbs to CD3, CD56, and CD14 (Miltenyi Biotec, Auburn, CA), respectively. gd T cells and CD14+ cells were purified by positive selection using MS columns (anti-TCR gd MicroBeads kit; Miltenyi Biotec).

Techniques: Derivative Assay, Cytometry, Microscopy, Software, Incubation, Expressing

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

Article Title: Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells.

doi: 10.4049/jimmunol.1001919

Figure Lengend Snippet: FIGURE 2. Effect of depletion of CD56+ or CD14+ cells on the expansion of gd T cells. Whole PBMCs and PMBCs depleted of CD56+ or CD14+

Article Snippet: Depletion of CD3 +, CD56+, and CD14+ cells from PBMCs was conducted using LD columns and microbeads conjugated with mAbs to CD3, CD56, and CD14 (Miltenyi Biotec, Auburn, CA), respectively. gd T cells and CD14+ cells were purified by positive selection using MS columns (anti-TCR gd MicroBeads kit; Miltenyi Biotec).

Techniques:

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

Article Title: Involvement of CD56brightCD11c+ cells in IL-18-mediated expansion of human γδ T cells.

doi: 10.4049/jimmunol.1001919

Figure Lengend Snippet: FIGURE 3. Expansion of CD56brightCD11+ cells by IL-2/IL-18 in CD3-depleted PBMCs. A, Expan- sion of CD56brightCD11c+ cells in CD3-depleted PBMCs. CD3-depleted PBMCs were incubated in the presence of ZOL/IL-2 or ZOL/IL-2/IL-18 and then analyzed for expression of CD3, CD11c, and CD56. B, Proliferation of PBMCs depleted of CD3+ cells. CD3-depleted PBMCs were incubated in the presence of various cyto- kines in combinations, as indicated, and the number of living cells was counted at indicated time points. C, Expansion of CD56brightCD11c+ cells in CD3- depleted PBMCs. The total number of CD56bright

Article Snippet: Depletion of CD3 +, CD56+, and CD14+ cells from PBMCs was conducted using LD columns and microbeads conjugated with mAbs to CD3, CD56, and CD14 (Miltenyi Biotec, Auburn, CA), respectively. gd T cells and CD14+ cells were purified by positive selection using MS columns (anti-TCR gd MicroBeads kit; Miltenyi Biotec).

Techniques: Incubation, Expressing

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Identification of anti-NCAM1 autoantibodies (A) Titers of anti-NCAM1 autoantibodies in serum by ELISA. ∗∗p < 0.01 (n = 201, healthy controls; n = 223, patients with schizophrenia; Mann-Whitney U test). (B) Immunocytochemistry using a commercial anti-NCAM1 antibody, serum and CSF from schizophrenia patient 1, and serum from healthy controls. NCAM1 and EGFP were expressed from a plasmid. Confocal images show antibodies bound to the membrane of EGFP-positive HeLa cells. Similar results were obtained for all anti-NCAM1 antibody-positive patients with schizophrenia ( Figure S1 B). Antibodies in serum did not react with EGFP because (1) they did not react with EGFP in the nucleus and (2) they did not react with cells transfected with an empty plasmid expressing only EGFP (data not shown). Scale bar: 10 μm. Ab, antibody; Sz, schizophrenia. (C) Titers of anti-NCAM1 autoantibodies in serum by cell-based assay. ∗∗p < 0.01 (n = 201, healthy controls; n = 223, patients with schizophrenia; Mann-Whitney U test). (D) ELISA analysis of serum-soluble NCAM in healthy controls (n = 201) and patients with schizophrenia (n = 223). ∗∗p < 0.01 (Mann-Whitney U test). (E) ELISA analysis of serum-soluble NCAM in schizophrenia patients with (n = 211) or without (n = 12) anti-NCAM1 autoantibodies defined by cell-based assay. ∗p < 0.05 (Mann-Whitney U test). (F) ELISA analysis of serum-soluble NCAM in healthy controls (n = 201) and schizophrenia patients without anti-NCAM1 autoantibodies (n = 211) defined by cell-based assay. ∗∗p < 0.01 (Mann-Whitney U test).

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Immunocytochemistry, Plasmid Preparation, Membrane, Transfection, Expressing, Cell Based Assay

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: The main epitope recognized by anti-NCAM1 antibodies in schizophrenia resides within the Ig1 domain (A) NCAM1 deletion constructs. (B) Immunocytochemistry using serum from patient 1 with schizophrenia, who was positive for anti-NCAM1 autoantibodies. NCAM1 deletion constructs and EGFP were expressed from a plasmid. Similar results were obtained from all anti-NCAM1 antibody-positive patients with schizophrenia. Scale bar: 10 μm. (C) Immunocytochemical confirmation of the expression of NCAM1ΔIg1 and NCAM1ΔIg1–5 using a commercial anti-NCAM1 antibody. Scale bar: 10 μm. (D) Western blot analysis of deletion constructs of NCAM1 transfected into HeLa cells revealed that the main epitope recognized by anti-NCAM1 autoantibodies is in the Ig1 domain.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Construct, Immunocytochemistry, Plasmid Preparation, Expressing, Western Blot, Transfection

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Anti-NCAM1 autoantibodies disrupt NCAM1-NCAM1 and NCAM1-GDNF interactions (A) Pull-down assay confirming that IgG purified from a patient with schizophrenia who was positive for anti-NCAM1 autoantibodies disrupts NCAM1-NCAM1 interactions. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose. (B) Pull-down assay showing that IgG purified from a patient with schizophrenia who was anti-NCAM1 autoantibody-positive disrupts the NCAM1-GDNF interaction. His-tagged proteins were pulled down by Ni-NTA-agarose, and GST-tagged proteins were pulled down by Glutathione Sepharose.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Pull Down Assay, Purification

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Injection of anti-NCAM1 autoantibodies from a patient with schizophrenia into mice (A) Experimental protocol for IgG injection. AAV1-SYN1-EGFP and AAV2-VAMP2-mCherry were injected into the frontal cortex of mice aged 6 weeks, and purified IgG was injected into the CSF of mice aged 8 weeks. Molecular, histological, two-photon microscopy, and behavioral analyses were performed in 9-week-old mice. IHC, immunohistochemistry; IP, immunoprecipitation; WB, western blot. (B) Immunoprecipitation analysis of tissue from the frontal cortex of mice revealed that the NCAM1-Fyn interaction was inhibited by anti-NCAM1 autoantibodies acquired from patients with schizophrenia. CT, computed tomography. (C) Effect of IgG purified from patient 1 with schizophrenia on FAK, MEK1, and ERK1 phosphorylation in the frontal cortex. Removal of anti-NCAM1 antibodies from purified IgG reversed the decrease in pFAK, pMEK, and pERK1. (D) Quantitative analyses of western blots with five mice per group. ∗∗p < 0.01 (n = 5, Tukey’s honest significant difference [HSD] test). Data are expressed as the mean ± SEM. (E) Two-photon microscopic images of dendritic spines in the first layer of the frontal cortex of mice injected with AAV1-SYN1-EGFP and IgG purified from patient 1 with schizophrenia or IgG purified from a healthy control. Removal of anti-NCAM1 antibodies from the purified IgG reversed the decrease in the number of spines. The graph on the right shows quantitative analysis of spine number. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. Scale bar: 5 μm. (F) Two-photon microscopic images showing contact between axon terminals and dendritic spines in the first layer of the frontal cortex of mice injected with AAV2-VAMP2-mCherry, AAV1-SYN1-EGFP, IgG purified from patient 1 with schizophrenia, or IgG from a healthy control. The graph on the right shows quantitative analysis of axon terminals merged with spines. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. Scale bar: 5 μm. (G) Alteration ratios in the Y maze test after injection of purified IgG from patient 1 with schizophrenia or from a healthy control. Removal of anti-NCAM1 antibodies from purified IgG reversed the decrease in the alteration ratios. ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM. (H) Pre-pulse inhibition rates of mice injected with IgG purified from patient 1 with schizophrenia or a healthy control. Removal of anti-NCAM1 antibodies from purified IgG reversed the deficiency in pre-pulse inhibition. ∗p < 0.05 and ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Injection, Purification, Microscopy, Immunohistochemistry, Immunoprecipitation, Western Blot, Computed Tomography, Control, Inhibition

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet: Anti-NCAM1 autoantibodies from patients with schizophrenia cause schizophrenia-related behavior and changes in synapse numbers in mice (A) Experimental protocol for IgG injection. AAV1-SYN1-EGFP and AAV2-VAMP2-mCherry were injected into the frontal cortex of mice aged 6 weeks, and purified IgG was injected into the CSF of mice aged 8 weeks. Two-photon microscopy and behavioral analyses were performed in 9-week-old mice. (B) Two-photon microscopic analysis of dendritic spines in the first layer of the frontal cortex of mice injected with AAV1-SYN1-EGFP and IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. (C) Two-photon microscopic analysis of axon terminals merged with spines in the first layer of the frontal cortex of mice injected with AAV2-VAMP2-mCherry, AAV1-SYN1-EGFP, and IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗∗p < 0.01 (n = 5 mice per group; 50 dendrites/mouse, 500 spines/mouse; Tukey’s HSD test). Data are expressed as the mean ± SEM. Scale bar: 5 μm. (D) Alteration ratios in the Y maze test after injection of IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM. (E) Pre-pulse inhibition rates of mice injected with IgG purified from a healthy control and patient 2 and patient 3 with schizophrenia. ∗p < 0.05 and ∗∗p < 0.01 (n = 9 mice per group; Tukey’s HSD test). Data are expressed as the mean ± SEM.

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Injection, Purification, Microscopy, Control, Inhibition

Journal: Cell Reports Medicine

Article Title: Autoantibodies against NCAM1 from patients with schizophrenia cause schizophrenia-related behavior and changes in synapses in mice

doi: 10.1016/j.xcrm.2022.100597

Figure Lengend Snippet:

Article Snippet: Polystyrene microtiter plates (3455, Thermo Scientific, Waltham, MA, USA) were coated with 100 μL (2 μg/mL) of NCAM1 recombinant protein (10673-H08H, Sino Biological) in TBS buffer and incubated overnight at 4°C.

Techniques: Virus, Plasmid Preparation, Recombinant, Labeling, Enzyme-linked Immunosorbent Assay, Software

Journal: Nutrients

Article Title: Korean Red Ginseng Enhances Immunotherapeutic Effects of NK Cells via Eosinophils in Metastatic Liver Cancer Model

doi: 10.3390/nu14010134

Figure Lengend Snippet: Detection of circulating NK cells in an experimental metastasis model. ( A ). CD56 levels measured via FACS analysis. ( B ). Ratio of CD56 dim+ cells. Data are expressed as the mean ± standard deviation ( n = 3/group). **, p < 0.01; ***, p < 0.005. (two-way ANOVA with Tukey’s post hoc test) ( C ). CD56 positive cells in liver tissues. Brown dots represent CD56 positive cells. Scale bar, 100 μm.

Article Snippet: The sections were stained with anti-NCAM1 (CD56) antibody (1:100; Cell signaling technology, MA, USA), anti-matrix metalloproteinase (MMP)2 antibody (1:200; Millipore, Burlington, MA, USA), anti-MMP7 antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), anti-MMP9 antibody (1:200; Abcam, Cambridge, UK), and anti-Ki67 antibody (1:200, Abcam) overnight at 4 °C.

Techniques: Standard Deviation