Journal: Virology

Article Title: HIV-1 Gag p17 presented as virus-like particles on the E2 scaffold from Geobacillus stearothermophilus induces sustained humoral and cellular immune responses in the absence of IFN? production by CD4+ T cells

doi: 10.1016/j.virol.2010.08.026

Figure Lengend Snippet: C57BL/6 recipient mice (5 per group) of CFSE-labelled OT-II T cells were immunized with E2wt particles, OVA323-339 E2 particles or soluble ovalbumin (OVA) plus poly (I:C) or PBS. 3 days after immunization, mice were sacrificed and the spleen cells analysed by flow cytometry for CFSE content (A–B), or restimulated in vitro with OVA323-339 synthetic peptide for IFNγ production analysis (C–D).

Article Snippet: The OVA 257-264 and OVA 323-339 peptides from chicken ovalbumin were purchased from Primm srl.

Techniques: Flow Cytometry, In Vitro

Journal: EMBO Molecular Medicine

Article Title: Active poly‐GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model

doi: 10.15252/emmm.201910919

Figure Lengend Snippet: Experimental paradigm for vaccination of GA‐CFP mice and wild‐type littermates. Anti‐GA response in vaccinated mice measured by ELISA using GST‐(GA) 15 antigen. Tukey‐style box plot shows 25 th , 50 th , and 75 th percentiles, and whiskers extend to ± 1.5 interquartile range. Outliers depicted as dots. Number of mice per group as indicated. Two‐way repeated‐measure ANOVA (group x time) revealed a significant main effect of treatment ( F 5,57 = 478.2, P < 0.0001) and time ( F 6,342 = 296.1, P < 0.0001) and a significant interaction between factors ( F 30,342 = 119.5, P < 0.0001), followed by Tukey's post hoc test. P ‐values are indicated in Appendix Table S1 . Immunohistochemistry of sections from a C9orf72 patient and a healthy control. Monoclonal anti‐GA and antiserum from Ova‐GA‐vaccinated mice detect neuronal cytoplasmic inclusions (arrows) in the molecular layer of the cerebellum. Note that crude antiserum shows higher background than the monoclonal antibody. Scale bar indicates 20 μm. Quantitative analysis for poly‐GA‐positive inclusions per 100 neurons in (C). Bar graphs represents mean ± SD from 6 images. One‐way ANOVA, Tukey's post hoc test, F 3,15 = 2.676, P = 0.0846, C9: anti‐GA 1A12 vs. C9: TG‐Ova‐(GA) 10 serum P = 0.9483. Immunohistochemistry of sections from occipital cortex of a C9orf72 patient and a healthy control with monoclonal anti‐GA and antiserum preincubated with 0.1 mg/ml recombinant GST‐(GA) 15 or GST. Scale bar indicates 20 μm. Arrows depict poly‐GA positive inclusions.

Article Snippet: For the first immunization, 40 μg Ova‐(PEG) 3 ‐(GA) 10 or (GA) 15 (Peptide Specialty Laboratories GmbH, Heidelberg) was mixed with 5 nmol CpG ODN 1668 oligonucleotide (Enzo Life Science) in 200 μl PBS and 250 μl incomplete Freund's adjuvant and injected half‐half intraperitoneally (i.p.) and subcutaneously (s.c.) at 8 weeks of age.

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Recombinant

Journal: EMBO Molecular Medicine

Article Title: Active poly‐GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model

doi: 10.15252/emmm.201910919

Figure Lengend Snippet: Analysis of motor function in vaccinated GA‐CFP mice and wild‐type littermates in a beam walk assay. Average time to cross the beam from duplicate repeat measurements in consecutive weeks. Tukey‐style box plot shows 25 th , 50 th , and 75 th percentiles, and whiskers extend to ± 1.5 interquartile range. Outliers depicted as dots. Pairwise Wilcoxon rank sum test with Benjamini–Hochberg correction. All comparisons with WT‐PBS and the TG‐Ova‐(GA) 10 mice vs. TG‐PBS comparison are depicted with * P < 0.05, ** P < 0.01. Number of mice per group as in Fig B, exact P ‐values are indicated in Appendix Table S2 . Average number of drop‐downs per mouse from all aggregated runs. Number of mice per group as in Fig 1B. Bar graph represents mean ± SD. Kruskal–Wallis test with Dunn's multiple comparisons. * P < 0.05. TG‐Ova‐(GA) 10 vs. TG‐PBS P = 0.0188. Representative image of poly‐GA aggregates in the anterior horn in spinal cord using immunohistochemistry. Scale bar indicates 100 μm. Quantitative analysis of poly‐GA aggregates in the spinal cord using immunohistochemistry. Images from complete spinal cord sections at 1‐mm intervals from n = 3 animals. Dot plot represents mean ± SD. One‐way ANOVA, Tukey's post hoc test, F 5,12 = 1.625, P = 0.2270. *** P < 0.001, ns not significant. TG‐Ova‐(GA) 10 vs. TG‐PBS P < 0.0001; TG‐(GA) 15 vs. TG‐PBS P = 0.2460. Immunoassay of poly‐GA from the insoluble fraction of spinal cord lysate. Dot plot represents mean ± SD from n = 5‐6 animals per group. One‐way ANOVA, Tukey's post hoc test, F 5,24 = 3.098, P = 0.0269. ** P < 0.001, ns not significant. TG‐Ova‐(GA) 10 vs. TG‐PBS P < 0.0001; TG‐(GA) 15 vs. TG‐PBS P = 0.1975. Source data are available online for this figure.

Article Snippet: For the first immunization, 40 μg Ova‐(PEG) 3 ‐(GA) 10 or (GA) 15 (Peptide Specialty Laboratories GmbH, Heidelberg) was mixed with 5 nmol CpG ODN 1668 oligonucleotide (Enzo Life Science) in 200 μl PBS and 250 μl incomplete Freund's adjuvant and injected half‐half intraperitoneally (i.p.) and subcutaneously (s.c.) at 8 weeks of age.

Techniques: Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: Active poly‐GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model

doi: 10.15252/emmm.201910919

Figure Lengend Snippet: A Gene ontology analysis of differentially expressed genes in TG‐PBS mice comparing genes significantly rescued by Ova‐(GA) 10 immunization and non‐rescued genes (absolute log2‐fold change > 0.585, compare Datasets , , and ). The dot size and color represent the fraction of the differentially expressed genes in each category and adjusted P ‐values, respectively. Mouse number as indicated in Fig B. B Network of the genes dysregulated in TG‐PBS and significantly rescued in TG‐Ova‐(GA) 10 . C, D Analysis of microglia/macrophage activation using Iba1 immunohistochemistry from complete spinal cord sections at 1‐mm interval. Dot plot represents mean ± SD from n = 3 animals per group. One‐way ANOVA, Tukey's post hoc test. ** P < 0.01, * P < 0.05, ns not significant. Scale bar indicates 100 μm. F 5,12 = 0.6974, P = 0.6357; TG‐Ova‐(GA) 10 vs. TG‐PBS P = 0.0131; TG‐(GA) 15 vs. TG‐PBS P = 0.7650. TG‐PBS vs. WT‐PBS P = 0.0018. E The percentage of cells with partial cytoplasmic mislocalization of TDP‐43 (compare Fig E) was quantified from six images from spinal cord sections at 1‐mm intervals. Dot plot represents mean ± SD from n = 3 animals per group. One‐way ANOVA, Tukey's post hoc test. ** P < 0.01, *** P < 0.001, ns not significant. F 5,12 = 0.6533, P = 0.6650; TG‐Ova‐(GA) 10 vs. TG‐PBS P = 0.0096; TG‐(GA) 15 vs. TG‐PBS P = 0.4215. F Immunoassay of NFL level in cerebrospinal fluid. Dot plot represents mean ± SD from n = 4 animals per group. One‐way ANOVA, Tukey's post hoc test, F 3,11 = 1.911, P = 0.1862. ** P < 0.01. TG‐Ova‐(GA) 10 vs. TG‐PBS P = 0.0081. Source data are available online for this figure.

Article Snippet: For the first immunization, 40 μg Ova‐(PEG) 3 ‐(GA) 10 or (GA) 15 (Peptide Specialty Laboratories GmbH, Heidelberg) was mixed with 5 nmol CpG ODN 1668 oligonucleotide (Enzo Life Science) in 200 μl PBS and 250 μl incomplete Freund's adjuvant and injected half‐half intraperitoneally (i.p.) and subcutaneously (s.c.) at 8 weeks of age.

Techniques: Activation Assay, Immunohistochemistry

Journal: EMBO Molecular Medicine

Article Title: Active poly‐GA vaccination prevents microglia activation and motor deficits in a C9orf72 mouse model

doi: 10.15252/emmm.201910919

Figure Lengend Snippet: A Analysis of microglia activation using Iba1 immunohistochemistry by measuring the area of Iba1 staining from complete spinal cord sections at 1‐mm interval. Dot plot represents mean ± SD from n = 3 animals per group. One‐way ANOVA, Tukey's post hoc test. *** P < 0.001, ns not significant. F 5,12 = 1.201, P = 0.3655, TG‐Ova‐(GA) 10 vs. TG‐PBS P < 0.0001, TG‐(GA) 15 vs. TG‐PBS P = 0.8573. TG‐PBS vs. WT‐PBS P < 0.0001. B–D Automated analysis of microglia/macrophage morphology from 100‐μm spinal cord sections stained for Iba1. Example reconstructions in (B). Scale bar indicates 30 μm. Colors in scatter plot indicate the different mice (3–4 mice per groups), blue area covers 25 th to 75 th percentiles and horizontal lines indicates 25 th , 50 th , and 75 th percentiles respectively n = 35–95 number of microglia analyzed. Kruskal–Wallis test with Benjamini–Hochberg correction. ** P < 0.01, *** P < 0.001. (C) Kruskal–Wallis χ 2 = 29.917, df = 3, P = 0.0000014; TG‐Ova‐(GA) 10 vs. TG‐PBS P = 0.0003; WT‐PBS vs. TG‐PBS P = 0.000078. (D) Kruskal–Wallis χ 2 = 28.532, df = 3, P = 0.0000028; TG‐Ova‐(GA) 10 vs. TG‐PBS P = 0.00041; WT‐PBS vs. TG‐PBS P = 0.00032. E Representative immunofluorescence images of endogenous TDP‐43 in the anterior horn of the spinal cord. In TG mice, more neurons show partial cytoplasmic mislocalization of TDP‐43 (arrows). Scale bar indicates 20 μm. Analysis in Fig E.

Article Snippet: For the first immunization, 40 μg Ova‐(PEG) 3 ‐(GA) 10 or (GA) 15 (Peptide Specialty Laboratories GmbH, Heidelberg) was mixed with 5 nmol CpG ODN 1668 oligonucleotide (Enzo Life Science) in 200 μl PBS and 250 μl incomplete Freund's adjuvant and injected half‐half intraperitoneally (i.p.) and subcutaneously (s.c.) at 8 weeks of age.

Techniques: Activation Assay, Immunohistochemistry, Staining, Immunofluorescence