Journal: PLoS Genetics

Article Title: A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

doi: 10.1371/journal.pgen.1002455

Figure Lengend Snippet: (A) A chromosomal plot of P -values in GWAS for SLE. (B) A regional plot in the AFF1 locus. Diamond-shaped data points represent −log 10 ( P -values) of the SNPs. Large-sized points indicate the P -values of the landmark SNP, rs340630 (green for the combined study and red for the GWAS). Density of red color represents r 2 values with rs340630. Blue line represents recombination rates. Lower part indicates RefSeq genes. Gray dashed horizontal lines represent the threshold of P = 5.0×10 −8 . The plots were drawn using SNAP, version 2.1 .

Article Snippet: Specific probes (Hs01089428_m1) for transcript of AFF1 (NM_001166693) were used.

Techniques:

Journal: PLoS Genetics

Article Title: A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

doi: 10.1371/journal.pgen.1002455

Figure Lengend Snippet: Results of combined study for Japanese patients with SLE.

Article Snippet: Specific probes (Hs01089428_m1) for transcript of AFF1 (NM_001166693) were used.

Techniques:

Journal: PLoS Genetics

Article Title: A Genome-Wide Association Study Identified AFF1 as a Susceptibility Locus for Systemic Lupus Eyrthematosus in Japanese

doi: 10.1371/journal.pgen.1002455

Figure Lengend Snippet: (A) Correlation between rs340630 genotypes and transcript levels of AFF1 (NM_001166693) in EBV-transfected cell lines ( n = 62) stimulated with PMA. (B) Allele-specific quantification (ASTQ) of AFF1 transcripts. Allele specific-probes for rs340638 were used for quantification by qPCR. The ratios of A allele over G allele for the amounts of both cDNAs and DNAs were plotted in log scale for each cell line. (C) AFF1 expression in various tissues. Transcripts levels of AFF 1 were quantified by qPCR and were normalized by GAPDH levels.

Article Snippet: Specific probes (Hs01089428_m1) for transcript of AFF1 (NM_001166693) were used.

Techniques: Transfection, Expressing

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Titers, kinetics and reactivity of AFF 1-induced antibodies after repeated immunization in MBP-α-syn tg mice. (A) Titers of antibodies against human and murine α-syn elicited after immunization with vehicle, original C-terminal α-syn antigen or AFF 1 (B) IgG response towards the immunizing peptide AFF 1 (as BSA conjugate) as well as against recombinant human α-syn and β-syn from plasma taken at end point (C) Kinetics of the IgG responses to the immunizing peptide following vaccination with vehicle or AFF 1 (D) Kinetics of the IgG responses to recombinant human α-syn following vaccination with vehicle or AFF 1 (E) α-syn immune reactivity of AFF 1-induced antibodies in brain sections of naïve non-tg, mThy1-α-syn tg and MBP-α-syn tg mice. Plasma from AFF 1-immunized MBP-α-syn tg mice was used at a final dilution of 1:100. As positive control, the human α-syn-specific antibody LB509 was used. An anti-mouse IgG antibody was used as negative control. Cell nuclei were stained with DAPI (blue) (F) Immunoblot analysis of AFF 1-induced antibodies against aggregated (o) and monomeric (m) recombinant α-syn. The human α-syn-specific antibody LB509 was used as positive control. Aggregated (oligomeric) α-syn was obtained by 4-hydroxy-2-nonenal treatment. Titers are depicted as OD max/2 at 405 nm. PP, Preplasma; EP, end point plasma; P1-P5, plasma taken after each immunization. For titer calculations, n = 10 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Recombinant, Positive Control, Negative Control, Staining, Western Blot

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Trafficking of AFF 1-induced antibodies into the CNS of MBP-α-syn tg mice. (A) Monoclonal AFF 1-induced antibodies were tagged with Alexa-488 and administered to non-tg or MBP-α-syn mice. Alexa-488 tagged mAb-AFF 1 bounded α-syn within cell bodies (arrow-head) and blood vessels (bv). As negative control, a non-immune Alexa-488-tagged IgG1 was used. Scale bar = 5 μm (B) mAb-AFF 1 or non-immune IgG1 were tagged with Alexa-488 and administered to non-tg or MBP-α-syn mice. Time course analysis was performed every 24 h for 3 days, and fluorescence was only increased in brain sections of MBP-α-syn tg animals injected with Alexa-488-tagged mAb-AFF 1. Results are shown as corrected intensity values and expressed as average ± SEM. n = 3 animals per group and time point (C) AFF 1-induced antibodies were detected with and FITC-tagged anti-mouse antibody in brain sections of immunized MBP-α-syn tg mice (green), together with an antibody against Iba1 (microglia) or S100 (astrocytes) (red). Cell nuclei were stained with DAPI (blue). Colocalization was observed in microglial cell bodies and projections, but not in astroglial cells (arrows) (D) AFF 1-induced antibodies detected with and FITC-tagged anti-mouse antibody in brain sections of immunized MBP-α-syn tg mice (green), together with an antibody against p25 (oligodendrocytes) or NeuN (neurons) (red). Cell nuclei were stained with DAPI (blue). Colocalization was observed in oligodendroglial cell bodies, but not in neurons (arrows). Scale bar = 5 μm.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Negative Control, Fluorescence, Injection, Staining

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Immunization with AFF 1 reduces α-syn accumulation in MBP-α-syn tg mice. (A) α-syn immunoreactivity in neocortex and striatum of non-tg and MBP-α-syn tg mice immunized with vehicle, original C-terminal α-syn antigen, or AFF 1 (B) Cell counts of α-syn-positive cells in neocortex (C) Cell counts of α-syn-positive cells in striatum (D) Immunoblot analysis of total α-syn in the cytosolic and membrane fractions of protein extracts from non-tg and MBP-α-syn tg mice immunized with vehicle, α-syn antigen, or AFF 1. Significant results of two mice per group are shown (E) Densitometric analysis of the α-syn immunoreactive bands in the cytosolic fraction (F) Densitometric analysis of the α-syn immunoreactive bands in the membrane fraction. Results are expressed as average ± SEM. (#) p < 0.05 when comparing vehicle-treated tg animals vs. immunized tg animals by one-way ANOVA with post hoc Tukey-Kramer. For the vehicle-treated non-tg group, n = 5. For tg groups, n = 10 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Western Blot

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Immunization with AFF 1 promotes microglial activation in MBP-α-syn tg mice. Non-tg mice or MBP-α-syn tg mice were immunized with vehicle, original C-terminal α-syn antigen or AFF 1, and glial markers were analyzed by immunohistochemistry. (A) Immunostaining of the astroglial marker GFAP in neocortex and striatum. Scale bar = 25 μm (B) Optical density quantification of GFAP staining in neocortex (C) Optical density quantification of GFAP staining in striatum (D) Immunostaining of the microglial marker Iba1 in neocortex and striatum. Scale bar = 25 μm (E) Quantification of Iba1-positive cell counts in neocortex (F) Quantification of Iba1 cell counts in striatum. Results are expressed as average ± SEM. (*) p < 0.05 when comparing vehicle-treated non-tg animals to tg groups by one-way ANOVA with post hoc Dunnett; (#) p < 0.05 when comparing vehicle-treated tg animals with immunized tg groups by one-way ANOVA with post hoc Tukey-Kramer. For the non-tg group, animal numbers were n = 5 for vehicle and n = 3 for AFF 1. For tg groups, n = 10 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Activation Assay, Immunohistochemistry, Immunostaining, Marker, Staining

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Immunization with AFF-1 modulates cytokine levels in MBP-α-syn tg mice. Cytokine levels in the cytosolic fraction of non-tg or MBP-α-syn tg mice treated with vehicle or AFF 1 were analyzed using a proteomic array. Results are expressed as optical density relative to the non-tg vehicle condition. (A) IL-1Ra (B) IL-3 (C) IFNγ (D) IL-1α (E) GM-CSF. Results are expressed as average ± SEM. (*) p < 0.05, (**) p < 0.01 when comparing vehicle-treated non-tg animals with vehicle-treated tg animals by two-way ANOVA with post hoc Tukey. (#) p < 0.05, (##) p < 0.01 when comparing vehicle-treated tg animals with AFF 1-treated tg animals by two-way ANOVA with post hoc Tukey. n = 4 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques:

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Immunization with AFF 1 ameliorates the neurodegenerative pathology in MBP-α-syn tg mice. (A) Immunostaining for the synaptic marker MAP2 (green) in the neocortex and striatum of non-tg mice or MBP-α-syn tg mice immunized with vehicle, original C-terminal α-syn antigen or AFF 1. Cell nuclei were stained with DAPI (blue). Scale bar = 5 μm (B) Quantification of the percentage of the MAP2-positive area of neuropil in neocortex (C) Quantification of the percentage of the MAP2-positive area of neuropil in striatum (D) Immunostaining for the neuronal marker NeuN in the neocortex and striatum of non-tg mice or MBP-α-syn tg mice immunized with vehicle, original C-terminal α-syn antigen or AFF 1. Scale bar = 5 μm (E) Quantification of NeuN-positive cell counts per 0.1 mm 3 in neocortex (F) Quantification of NeuN-positive cell counts per 0.1 mm 3 in striatum. Results are expressed as average ± SEM. (*) p < 0.05 when comparing vehicle-treated non-tg animals to tg groups by one-way ANOVA with post hoc Dunnett; (#) p < 0.05 when comparing vehicle-treated tg animals with immunized tg groups by one-way ANOVA with post hoc Tukey-Kramer. For the non-tg group, animal numbers were n = 5 for vehicle and n = 3 for AFF 1. For tg groups, n = 10 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Immunostaining, Marker, Staining

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Immunization with AFF 1 reduces demyelination in MBP-α-syn tg mice. (A) LFB staining of myelin in the neocortex (and corpus callosum) and striatum of non-tg mice or MBP-α-syn tg mice immunized with vehicle, original C-terminal α-syn antigen or AFF 1. Scale bar = 250 μm (B) Quantification of LFB staining by optical density in neocortex (C) Quantification of LFB staining by optical density in striatum (D) Electron microscopy images of myelin sheaths in the corpus callosum of non-tg mice or MBP-α-syn tg mice immunized either with vehicle, α-syn antigen or AFF 1. Representative images taken with the transmission electron microscope (TEM) are shown at low magnification (5,000x) and high magnification (25,000x). Scale bars = 2.5 μm and 500 nm. vac, vacuola (E) Quantification of the number of myelinated axons in corpus callosum (F) Quantification of the average number of myelin layers per axon in corpus callosum. Results are expressed as average ± SEM. (*) p < 0.05 when comparing vehicle-treated non-tg animals to tg groups by one-way ANOVA with post hoc Dunnett; (#) p < 0.05 when comparing vehicle-treated tg animals with immunized tg groups by one-way ANOVA with post hoc Tukey-Kramer. For the non-tg group, animal numbers were n = 5 for vehicle and n = 3 for AFF 1. For tg groups, n = 10 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Staining, Electron Microscopy, Transmission Assay, Microscopy

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Immunization with AFF 1 reduces behavioral impairments and TH alterations in MBP-α-syn tg mice. (A) Performance in the transversal round beam test, measured as slips per 10 cm in non-tg or MBP-α-syn tg mice immunized with vehicle or AFF 1 (B) Speed of the animals in the transversal round beam test, measured as cm per second in non-tg or MBP-α-syn tg mice immunized with vehicle or AFF 1 (C) Quantification of TH staining by optical density in striatum. Results are expressed as average ± SEM. (*) p < 0.05 when comparing vehicle-treated non-tg animals to vehicle-treated tg animals by one-way ANOVA with post hoc Dunnett; (#) p < 0.05 when comparing vehicle-treated tg animals with AFF 1-treated tg animals by one-way ANOVA with post hoc Tukey-Kramer. For the non-tg group, animal numbers were n = 5 for vehicle and n = 3 for AFF 1. For tg groups, n = 10 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Staining

Journal: Molecular Neurodegeneration

Article Title: Active immunization against alpha-synuclein ameliorates the degenerative pathology and prevents demyelination in a model of multiple system atrophy

doi: 10.1186/s13024-015-0008-9

Figure Lengend Snippet: Immunization with AFF 1 increases microglial α-syn clearance in MBP-α-syn tg mice. (A) Double immunostaining for the microglial marker Iba1 (red) and α-syn (green) in vehicle- and AFF 1-immunized MBP-α-syn tg mice. The location of Iba1-positive cells is denoted by arrows and the location of α-syn-positive cells by asterisks. Cell nuclei were stained with DAPI (blue). Scale bar = 10 μm. (B) Quantification of the percentage of colocalization between Iba1 and α-syn (C) Double immunostaining for the oligodendroglial marker p25 (red) and α-syn (green) in vehicle- and AFF 1-immunized MBP-α-syn tg mice. The location of p25-positive cells is denoted by arrows. Cell nuclei were stained with DAPI (blue). Scale bar = 10 μm. (D) Quantification of the percentage of colocalization between p25 and α-syn (E) Double immunostaining for the astroglial marker S100 (red) and α-syn (green) in vehicle- and AFF 1-immunized MBP-α-syn tg mice. The location of S100-positive cells is denoted by arrows and the location of α-syn-positive cells by asterisks. Cell nuclei were stained with DAPI (blue). Scale bar = 10 μm. (F) Quantification of the percentage of colocalization between S100 and α-syn (G) Double immunostaining for the neuronal marker NeuN (red) and α-syn (green) in vehicle- and AFF 1-immunized MBP-α-syn tg mice. Cell nuclei were stained with DAPI (blue). Scale bar = 5 μm. (H) Quantification of the percentage of colocalization between NeuN and α-syn. Results are expressed as average ± SEM. (*) p < 0.05 when comparing vehicle-treated tg animals with AFF 1-treated tg animals by Student’s t -test. n = 10 animals per group.

Article Snippet: Briefly, AFF 1 was synthesized by FMOC solid phase peptide synthesis (EMC microcollections GmbH) conjugated to the carrier protein KLH (Biosyn GmbH) using N-gamma-Maleimidobutyryl-oxysuccinimide ester (Thermo Scientific) through an additional N-terminal cysteine residue.

Techniques: Double Immunostaining, Marker, Staining