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: International Journal of Molecular Sciences

Article Title: Glycan Structures in Osteosarcoma as Targets for Lectin-Based Chimeric Antigen Receptor Immunotherapy

doi: 10.3390/ijms25105344

Figure Lengend Snippet: CD301-CARs display specific cytotoxicity against osteosarcoma cell lines. ( A ) Schematic representation of the CD301 LEC-CAR: ( B ) Expression of CD301 CAR at the cell surface of NK92 cells. NK92 cells were retrovirally transduced and sorted based on the GFP expression. The expression of the CD301 CAR construct was measured by flow cytometry using an antibody specific to the CD301 CRD. Color denotes areas of high and low population density. ( C ) Detection of CD301 ligands on target cells: osteosarcoma cell lines were stained with fluorescently labeled recombinant CD301 and analyzed by flow cytometry. ( D ) Cytotoxicity assay: NK92 cells expressing the CD301-CAR were cocultured for 3 h with calcein-labeled target cells with an effector-to-target ratio of 3:1 and 5:1, respectively, and measured as quadruplicates. Columns represent the median. Error bars show standard deviation. p < 0.01 is indicated by **, respectively.

Article Snippet: The degranulation of NK92 CAR and wild-type NK92 cells was induced upon interaction with target cells at an E:T ratio of 1:1 for 4 h at 37 °C and was assessed by measuring the expression of CD107a with a PE/Cy7-labeled anti-CD107a antibody (Biolegend, #328617) at the surface of FITC-labeled anti-CD56-stained NK92 cells (Miltenyi, #130-114-740).

Techniques: Expressing, Construct, Flow Cytometry, Staining, Labeling, Recombinant, Cytotoxicity Assay, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Glycan Structures in Osteosarcoma as Targets for Lectin-Based Chimeric Antigen Receptor Immunotherapy

doi: 10.3390/ijms25105344

Figure Lengend Snippet: Lytic activity of CD301-CAR-expressing NK92 cells correlates with interferon-gamma secretion and increased degranulation. ( A ) CAR expression leads to enhanced interferon-gamma secretion of NK92 cells upon engagement with osteosarcoma cell lines. ( B ) CAR expression leads to enhanced degranulation of NK92 cells upon engagement with osteosarcoma cells. Columns represent the mean of three independent experiments measured in triplicates. Error bars show the standard deviation of the mean. p < 0.05, p < 0.01, and p < 0.001 are indicated by *, **, or *** respectively.

Article Snippet: The degranulation of NK92 CAR and wild-type NK92 cells was induced upon interaction with target cells at an E:T ratio of 1:1 for 4 h at 37 °C and was assessed by measuring the expression of CD107a with a PE/Cy7-labeled anti-CD107a antibody (Biolegend, #328617) at the surface of FITC-labeled anti-CD56-stained NK92 cells (Miltenyi, #130-114-740).

Techniques: Activity Assay, Expressing, Standard Deviation

Journal: International Journal of Molecular Sciences

Article Title: Glycan Structures in Osteosarcoma as Targets for Lectin-Based Chimeric Antigen Receptor Immunotherapy

doi: 10.3390/ijms25105344

Figure Lengend Snippet: Combination of immune checkpoint inhibition with CD301 CAR immunotherapy. ( A ) Flow cytometry analysis showing the surface expression of the TIGIT and PVRIG on NK92 cells. Respective antibody staining is shown as a red histogram, and the isotype control and unstained control are depicted as blue and gray histograms. ( B ) Target cell expression of PVR and PVRL2. Expression was analyzed by flow cytometry. The binding of anti-PVR and anti-PVRL2 antibodies are shown as orange histograms. The isotype and unstained control are shown as blue and gray histograms. ( C ) Cytotoxicity assay: NK92 cells expressing the CD301-CAR were cocultured for 3 h with calcein-labeled targets with an effector target ratio of 3:1, respectively, in the presence or absence of different amounts of the inhibitory anti-TIGIT antibody. Measurements were performed as quadruplicates. Columns represent the mean of three independent experiments. Error bars show the standard deviation of the mean. p < 0.05 and p < 0.01 are indicated by * and **, respectively.

Article Snippet: The degranulation of NK92 CAR and wild-type NK92 cells was induced upon interaction with target cells at an E:T ratio of 1:1 for 4 h at 37 °C and was assessed by measuring the expression of CD107a with a PE/Cy7-labeled anti-CD107a antibody (Biolegend, #328617) at the surface of FITC-labeled anti-CD56-stained NK92 cells (Miltenyi, #130-114-740).

Techniques: Inhibition, Flow Cytometry, Expressing, Staining, Control, Binding Assay, Cytotoxicity Assay, Labeling, Standard Deviation

Journal: The FASEB Journal

Article Title: Genes controlling the activation of natural killer lymphocytes are epigenetically remodeled in intestinal cells from germ-free mice

doi: 10.1096/fj.201800787R

Figure Lengend Snippet: Lower representation of CD56 + /CD45 + cells in duodenum, ileum, and colon from GF mice, compared with Conv-R mice. A , B ) Representative immunofluorescence images showing CD56 + /CD45 + cells in the colon from Conv-R ( A ) and GF ( B ) mice. Scale bars, 50 µm. C ) Total counts of cells expressing both CD56 and CD45 in Conv-R and GF mice. Conv-R, n = 5; GF, n = 5. Data represent means ± sd . * P < 0.05 (nonparametric Mann-Whitney test).

Article Snippet: Sections were blocked in 10% donkey serum for 1 h at room temperature, incubated with primary antibodies against CD56 (AF2408, dilution 1:1000; R&D Systems, Minneapolis, MN, USA) and CD45 (ab25386, dilution 1:250; Abcam, Cambridge, United Kingdom) overnight at 4°C, and washed in PBS-Tween.

Techniques: Immunofluorescence, Expressing, MANN-WHITNEY

Journal: iScience

Article Title: miRNA-194-3p represses NF-κB in gliomas to attenuate iPSC genes and proneural to mesenchymal transition

doi: 10.1016/j.isci.2023.108650

Figure Lengend Snippet:

Article Snippet: NCAM1 , Thermo Fisher , Cat#Hs00941830_m1.

Techniques: Recombinant, Protease Inhibitor, Transfection, Isolation, cDNA Synthesis, MTT Assay, Extraction, Transcription Factor Assay, Expressing, Plasmid Preparation, Reporter Assay, Staining, Negative Control, shRNA, Software

Journal: Cell Reports Medicine

Article Title: Merkel cell polyomavirus-specific and CD39 + CLA + CD8 T cells as blood-based predictive biomarkers for PD-1 blockade in Merkel cell carcinoma

doi: 10.1016/j.xcrm.2023.101390

Figure Lengend Snippet:

Article Snippet: 114 - CD56 (clone: REA196) , Miltenyi Biotec , Cat# 130-108-016, RRID: AB_2658728.

Techniques: Recombinant, Control, Sequencing, RNA Sequencing, Software