Journal: Acta Neuropathologica Communications

Article Title: Co-expression of NMDA-receptor subunits NR1, NR2A, and NR2B in dysplastic neurons of teratomas in patients with paraneoplastic NMDA-receptor-encephalitis: a retrospective clinico-pathology study of 159 patients

doi: 10.1186/s40478-020-00999-2

Figure Lengend Snippet: NMDAR subunit analysis in neurons of teratomas with/without anti-NMDARE

Article Snippet: All serum and CSF specimens were evaluated by indirect immunofluorescence using EU 90 cells transfected with the NMDAR1 subunit (NR1) of the NMDAR complex and immobilized on BIOCHIPs (Euroimmun AG, L €ubeck, Germany) as previously described [ ].

Techniques: Staining

Journal: Acta Neuropathologica Communications

Article Title: Co-expression of NMDA-receptor subunits NR1, NR2A, and NR2B in dysplastic neurons of teratomas in patients with paraneoplastic NMDA-receptor-encephalitis: a retrospective clinico-pathology study of 159 patients

doi: 10.1186/s40478-020-00999-2

Figure Lengend Snippet: Pathology findings of neural tissues and NMDAR staining in teratomas with anti-NMDARE. Pathology findings of neural tissues in teratomas and NMDAR staining from patients with anti-NMDARE. ( a ) HE staining showed degenerative neurons. ( b ) Normal neurons with enlarged nuclei. ( c ) Neural cells positive for MAP2. ( d ) Neuroglia positive for GFAP. ( e ) Neural cells positive for S100. ( f ) In case N2, IgG positivity is strong and abundant. ( g ) Plasma cells positive for CD138. ( h ) and ( i ) Nodules of lymphocytes positive for CD20 and less positive for CD3, respectively. ( j ) Less helper T lymphocytes positive for CD4. ( k ) Cytotoxic T lymphocytes positive for CD8. ( l ), Isotype control of case N6, ( m ) Mimicking floating-frog dysmorphic neurons showing positivity for NeuN. ( n - p ) Moderate NR1/NR2A positivity and strong NR2B positivity in serial sections to M. ( q ) In case N7, scattered dysmorphic neurons positive for MAP2. ( r - t ) Moderate NR1 positivity and strong NR2A/NR2B positivity in serial sections to Q. ( u ) In case N10,clusters dysmorphic neurons positive for NeuN. ( v - x ) Moderate NR1 positivity and strong NR2A/NR2B positivity in serial sections to U

Article Snippet: All serum and CSF specimens were evaluated by indirect immunofluorescence using EU 90 cells transfected with the NMDAR1 subunit (NR1) of the NMDAR complex and immobilized on BIOCHIPs (Euroimmun AG, L €ubeck, Germany) as previously described [ ].

Techniques: Staining, Clinical Proteomics, Control

Journal: Scientific Reports

Article Title: Viromers as carriers for mRNA-mediated expression of therapeutic molecules under inflammatory conditions

doi: 10.1038/s41598-020-72004-8

Figure Lengend Snippet: Spleen and bone marrow are major target sites for Viromer delivery. ( a ) Biodistribution of Viromer-mediated FLuc-mRNA delivery analysed by in vivo bioluminescence imaging. Images were taken 6 h and 24 h after i.v. injection of complexes. The lateral view highlights a substantial uptake of Viromer/FLuc by spleen cells, which is supported by the ventral view (no uptake into liver or lungs visible). Minor expression is visible in the upper backbone, tail root, vertebrae and bones. The expression of luciferase is transient. The maximum of FLuc signal was detected after 6 h and signals decrease afterwards. ( b ) Quantification of FLuc expression in different tissues after i.v. application of Viromer/FLuc-mRNA-complexes. Graphs displayed as floating bars (min to max). Line represents the mean. *** p < 0.001, two-way ANOVA, Sidak multiple comparison test. n = 1 (PBMC), n = 4 (bone marrow, control, spleen, liver). ( c ) FACS analysis of cells present in lavage after thioglycolate-induced peritonitis. Representative FACS plots of the groups injected with NaCl, Thioglycolate (Thio) and Thioglycolate and FLuc-mRNA (FLuc) are depicted. Markers Ly-6B.2 (granulocytes, monocytes) and Ly-6G (granulocytes) were used to separate the two populations. ( d , e ) Quantification of infiltrating monocytes ( d ) and granulocytes ( e ) using TruCount tubes based on counting 10.000 beads/tube. Mean ± SEM, one-way ANOVA, Dunnett test, * p < 0.05, ** p < 0.01, n = 2–3. ( f ) Biodistribution of FLuc in the model of thioglycolate-induced peritonitis after i.v. application of Viromer/FLuc complexes. Graphs displays floating bars (min to max) with line at mean. Two-way ANOVA, Sidak multiple comparison test. *** p < 0.001, n = 3. ( g ) Direct quantification of 7ND-mRNA (comparative) in different organs; Graphs displayed as box and whiskers plot (min to max). Line represents mean, n = 8.

Article Snippet: The maximum of FLuc signal was detected after 6 h and signals decrease afterwards. ( b ) Quantification of FLuc expression in different tissues after i.v. application of Viromer/FLuc-mRNA-complexes.

Techniques: In Vivo, Imaging, Injection, Expressing, Luciferase, Comparison, Control

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

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: We examined the biological impact of introducing BMDMs transfected with synthetic mRNA encoding CCL3 into the peritoneal cavity of mice. A: Experiment setup. In brief, we transfected synthetic mRNAs encoding either CCL2 or CCL3 into bone marrow-derived macrophage (BMDM) generated from CD45.1+ congenic mice. As control, we transfected BMDM with VR without mRNA. 8h after transfection, the cells were harvested, washed with PBS, and injected intraperitoneally into CD45.2+ congenic mice. 16h later, we collected the cells from the peritoneal cavity and analyzed them by flow cytometry. The use of the congenic CD45.1+/CD45.2+ mouse strains enabled us to discriminate between the donor BMDM transfected with the chemokine mRNAs and the resident tissue macrophages. B: Demonstration of the presence of CCL3 protein in the supernatant from BMDMS transfected with Ccl3 mRNA, as well as in the peritoneal fluid from mice inoculated with these BMDMs. C: Ccl3 synthetic mRNA transfected BMDM recruited a population of Ly6ChiCD11b+ monocytes (Gate 1), increased the number of Ly6CintCD11b+ monocytes (Gate 2), and decreased the number of F4/80hi large peritoneal macrophages. Experiments were repeated 3 times. Data showed representative flow plot from one experiment. D: The numbers of Ly6Chi and Ly6CintCD11b+ monocytes, F4/80hi large peritoneal macrophage and Ly6G+ neutrophil were quantified. E: As an additional control to demonstrate the recruitment phenotype was due to CCL3-encoding mRNA we compared the levels of cellular recruitment to VR alone, VR coated with Gfp mRNA and VR coated with Ccl3 mRNA. The recruitment of the cell population of significance, the Ly6Cint CD11b+ monocytes was clearly specific to the presence of the Ccl3 mRNA. Data represent mean ± SD. ns: p>0.05, **: p < 0.01, ***: p < 0.001, Unpaired t test with Wench correction.

Article Snippet: E: Quantification of iNOS + and RELM-α + number of monocyte recruited by Viromer Red complexed mRNAs.

Techniques: Transfection, Derivative Assay, Generated, Injection, Flow Cytometry

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

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: In order to demonstrate that the recruited cells can be polarized towards M1- and M2-like macrophage phenotypes we co-transfected BMDMs with cytokine-encoding mRNAs. A: Representative flow plots showing the expression of iNOS and RELM-α in recruited monocytes (Ly6C+CD11b+, upper panel) and in large peritoneal macrophages (F4/80hiCD11b+, lower panel) following their induction of Ccl3 mRNA in combination with Ifn-γ or Il-4 mRNA. B: The numbers of iNOS+ or RELM-α+ recruited monocytes and F4/80hi macrophages were quantified and shown to increase in the presence of appropriate cytokine-encoding mRNAs Experiments were repeated three times with comparable results. Data represent mean ± SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: E: Quantification of iNOS + and RELM-α + number of monocyte recruited by Viromer Red complexed mRNAs.

Techniques: Transfection, Expressing

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

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: To generate functional data demonstrating the polarization of the recruited macrophages we quantified the intensity of their superoxide burst. A: The kinetics and magnitude of superoxide burst generated by BMDMs treated with or without recombinant IFN-γ and IL-4. Activation with IFN-γ enhanced the superoxide burst (Oxyburst beads), while alternative activation with IL-4 resulted in its decrease. Analysis was performed on a Perkin Elmer Envision fluorescent plate reader and the experiment was repeated three times. B: The kinetics and magnitude of superoxide burst generated by peritoneal cells from mice (n=3) injected with BMDMs transfected with mRNAs encoding CCL3 alone and in combination with either Ifn-γ and Il-4 mRNAs. The experiment was repeated twice. C: Peritoneal cells from mice (n=3) injected with different mRNA transfected BMDMs were analyzed by flow cytometry. Representative flow plots showing the gating of positive Oxyburst signal. Oxyburst beads without cells served as the negative control (upper panel). The lower panels were gated on recruited monocytes (Ly6C+CD11b+) from 4 groups incubated with Oxyburst beads and harvested at different times (10min, 30min and 60min). The experiment was repeated three times. D: Quantification of fluorescent index of mean Oxyburst bead fluorescent intensity. The mean Oxyburst fluorescent intensity of all the recruited monocytes that were positive for calibration fluor were calculated. The number was divided by the mean calibration fluorescent intensity to generate a fluorescent index. The experiment was repeated three times with comparable results. Data represent mean ± SD. *: p < 0.05, **: p < 0.01, ***: p < 0.001, One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: E: Quantification of iNOS + and RELM-α + number of monocyte recruited by Viromer Red complexed mRNAs.

Techniques: Functional Assay, Generated, Recombinant, Activation Assay, Injection, Transfection, Flow Cytometry, Negative Control, Incubation

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

Article Title: Exploitation of synthetic mRNA to drive immune effector cell recruitment and functional re-programming in vivo .

doi: 10.4049/jimmunol.1800924

Figure Lengend Snippet: The potential of this approach for therapeutic applications is supported by the demonstration that the mRNA-coated VR particles can mediate comparable effects following direct inoculation. A: Representative flow plots of monocyte populations in the peritoneal cavity after injection of naked, Ccl3 mRNA or Ccl3 mRNA-coated Viromer Red particles. B: Representative flow plots of F4/80hi large peritoneal macrophage in the peritoneal cavity after injection of naked, Ccl3 mRNA or Ccl3 mRNA-coated Viromer Red particles. C: Quantification of the numbers of Ly6Chi and Ly6Cint monocytes, F4/80hi large peritoneal macrophage and Ly6G+ neutrophil. D: Representative flow plots of iNOS and RELM-α expression in these monocytes recruited by Viromer Red complexed mRNAs. E: Quantification of iNOS+ and RELM-α+ number of monocyte recruited by Viromer Red complexed mRNAs. Experiment was repeated two times. Data represent mean ± SD. ns: p>0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001, One-way ANOVA with Tukey’s multiple comparison test.

Article Snippet: E: Quantification of iNOS + and RELM-α + number of monocyte recruited by Viromer Red complexed mRNAs.

Techniques: Injection, Expressing

Journal: PLOS ONE

Article Title: Human BST2 inhibits rabies virus release independently of cysteine-linked dimerization and asparagine-linked glycosylation

doi: 10.1371/journal.pone.0292833

Figure Lengend Snippet: (A) HEK293T cells were transfected with plasmids expressing flag-hBST2. Cell lysates were prepared and incubated in the presence or absence of PNGase F and subjected to western blot analysis of hBST2. HEK293T cells were transfected with (B) 1 μg of flag-hBST2 plasmid and an increasing amount (0.5, 1 and 2 μg) of plasmids encoding RABV G-myc, M-myc, N-myc and P-myc or (C) 1 μg of plasmids encoding individual myc-tagged RABV proteins with an increasing amount of flaghBST2 plasmid (0.5, 1 and 2 μg). At 48 hpt, cell lysates were collected and analyzed by western blotting for RABV protein and hBST2 expression using rabbit α-myc and α-human BST2 antibodies, respectively. Arrow heads indicate glycosylated monomeric hBST2 of approximately 30 kDa. Arrows indicate a low molecular weight, glycosylated dimeric hBST2 of approximately 55 kDa. Asterisks indicate high molecular weight glycosylated dimeric hBST2 of approximately 65 kDa.

Article Snippet: HEK293T cells seeded in 10-cm 2 dishes were transfected with 6 μg of plasmid expressing RABV Gmyc and flaghBST2 complexed with Fugene ® HD transfection reagent (Promega).

Techniques: Transfection, Expressing, Incubation, Western Blot, Plasmid Preparation, Molecular Weight, High Molecular Weight

Journal: PLOS ONE

Article Title: Human BST2 inhibits rabies virus release independently of cysteine-linked dimerization and asparagine-linked glycosylation

doi: 10.1371/journal.pone.0292833

Figure Lengend Snippet: HEK293T cells were transfected with plasmids expressing RABV Gmyc and flaghBST2. At 24 hpt, cells were fixed and probed with mouse α-myc and rabbit α-human BST2 primary antibodies to detect RABV G and hBST2, respectively followed by Alexa Fluor ® 488-conjugated goat α-rabbit IgG H&L and Alexa Fluor ® 647-conjugated goat α-mouse IgG H&L secondary antibodies. Localization of the proteins were visualized by confocal microscopy.

Article Snippet: HEK293T cells seeded in 10-cm 2 dishes were transfected with 6 μg of plasmid expressing RABV Gmyc and flaghBST2 complexed with Fugene ® HD transfection reagent (Promega).

Techniques: Transfection, Expressing, Confocal Microscopy