Journal: Scientific Reports

Article Title: Perineuronal Net Protein Neurocan Inhibits NCAM/EphA3 Repellent Signaling in GABAergic Interneurons

doi: 10.1038/s41598-018-24272-8

Figure Lengend Snippet: Expression of neurocan and NCAM in postnatal and adult brain. ( A ) Immunoblot of total brain extracts for neurocan, NCAM, PSA-NCAM, and GAPDH (loading control). ( B ) Co-localization of tdTomato puncta (red) with the presynaptic marker GAD65 (green) around a neuronal soma (blue). Scale bar = 5 μm. ( C ) Immunofluorescent staining of neurocan (green) around PV + tdTomato-labeled interneurons (red) in mouse frontal cortex at P28 and P60. Scale bar = 50 μm. ( D ) Confocal images of neurocan (green) around perisomatic synaptic puncta (red) onto MATH-2 expressing pyramidal neurons (blue nucleus, outlined with white border). A magnified inset is indicated with a white box. Scale bars = 2.5 μm. ( E ) Electron micrograph of immunogold labeling for neurocan along the membrane of a neuronal soma (black arrows point to examples). Scale bar = 1 μm. ( F ) Electron micrograph of an inhibitory axon terminal onto a neuronal soma. Neurocan immunogold labeling is indicated with black arrows, and the axon terminal (AT) is labeled with a white arrowhead. Cytoplasm and nucleus of the soma are identified with text. Scale bar = 1 μm. ( G ) Verification of neurocan antibody specificity by immunostaining of COS7 cells transfected with Neurocan-AP (+control) or AP (−control), and a no primary antibody control. Scale bar = 50 μm.( H ) Immunofluorescent localization of NCAM and neurocan in GABA+ and GABA− cortical neuron cultures. Confocal images of GABA (green), NCAM (blue), and neurocan (red) in cortical neuron cultures. White arrow indicates a GABA-negative neuron positive for NCAM and neurocan, and the green neuron represents a GABAergic interneuron. Scale bar = 10 μm.

Article Snippet: Polyclonal antibodies were against neurocan (AF5800, R&D), EphA3 (C-19; Santa Cruz Biotechnology), NCAM (H300; Santa Cruz Biotechnology), GAPDH (IMG-3073, Imgenex), GABA (A2052; Sigma and ab17413; Abcam), GAD65 (GAD-6; Developmental Studies Hybridoma Bank), MATH-2 (ab85824; Abcam), and GFP (13970; Abcam).

Techniques: Expressing, Western Blot, Control, Marker, Staining, Labeling, Membrane, Immunostaining, Transfection

Journal: Frontiers in Neuroscience

Article Title: Striatal and Tegmental Neurons Code Critical Signals for Temporal-Difference Learning of State Value in Domestic Chicks

doi: 10.3389/fnins.2016.00476

Figure Lengend Snippet: Direct contacts of MSt terminals on DA-ergic neurons in the tegmentum. (A) Dense arborizations of MSt efferent fibers were found in the FRM and SN (sagittal plane); low magnification (a) , high magnification in the FRM (b) , and the SN (c) . The inlet figure shows the injection site in the MSt. (B) BDA/TH double labeling in the tegmentum; BDA, green; TH, red. (C) Confocal images of direct contacts between BDA-positive terminal boutons and TH-positive dendrites and soma. Reconstructed on 3 orthographic planes. Arrowheads and arrows indicate the close appositions. (D) BDA/GAD65 double labeling in the SN, indicating co-localization on the terminal boutons. Sagittal sections with laterality: L1.4 in (Aa–c) ; L0.9 in the (A) inlet; L1.4 in B(a–c) ; L1.5, 1.1, and 1.0 in C(a–c) ; L1.3 in D(a–c) .

Article Snippet: The sections were then processed with a primary antibody; rabbit anti-TH (1:1000, 4°C, overnight; AB152, Chemicon®, EMD Millipore Co., USA) or rabbit anti-GAD65 (1:1000, 4°C, 3 days; bs-0400R, Bioss Inc., USA).

Techniques: Injection, Labeling

Journal: PLoS Biology

Article Title: GAD2 on Chromosome 10p12 Is a Candidate Gene for Human Obesity

doi: 10.1371/journal.pbio.0000068

Figure Lengend Snippet: (A) Fine mapping of the Chromosome 10p locus between markers D10S548 and D10S220 in 188 nuclear families (620 individuals). Multipoint analysis for obesity phenotype. (B) SNP map of the GAD2 gene. Positions were assigned according the location to the A of the ATG. The 15 SNPs selected for association studies are indicated in red. See also .

Article Snippet: [ 35 S]Methionine-labeled GAD65 was produced by in vitro coupled transcription/translation with SP6 RNA polymerase and nuclease-treated rabbit reticulocyte lysate (Promega, Madison, Wisconsin, United States) as previously described ( ).

Techniques:

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: The role of spinal GABAergic circuits in the control of phrenic nerve motor output

doi: 10.1152/ajpregu.00244.2014

Figure Lengend Snippet: Immunohistochemical identification and firing patterns of respiratory interneurons recorded from C3–C5 ventral horn. A1–A3: one expiratory interneuron (light blue arrow; see B1 for firing pattern) and one inspiratory interneuron with tonic extended expiratory activity (red arrow; see C2 for firing pattern) double-labeled with neurobiotin (subpanel A1) and FITC-immunofluorescence secondary anti-GAD65/67 antibodies (subpanels A2 and A3). Scale bars: A1, 100 μm; A2 and A3, 60 μm. B1, B2, C1, C2: examples of recorded expiratory (top) and inspiratory interneurons (bottom) with extended expiratory activity. Averaged phrenic triggered integrated phrenic nerve activity (blue traces, arbitrary units, 100–120 sweeps, integration τ = 50 ms), cycle-triggered histograms (CTHs, red traces, 50–75 ms bin size) of analyzed interneurons (left scale, Hz), integrated phrenic nerve (PN) and spiking activity of recorded interneurons (low two black traces) are shown. Time bar = 250 ms, applied to B1–C2.

Article Snippet: Following this, sections were washed in PBS (3 × 10 min) and incubated in goat anti-rabbit biotinylated secondary antibodies (1:500; Vector Labs, cat. no. BA-1000) for 2 h. GAD65/67-positive sections were then processed with Vectastain ABC kit (Vector Labs, PK-6100), washed in PBS again (3 × 10 min), and visually explored using a microscope (Olympus-Bx51).

Techniques: Immunohistochemical staining, Activity Assay, Labeling, Immunofluorescence

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: The role of spinal GABAergic circuits in the control of phrenic nerve motor output

doi: 10.1152/ajpregu.00244.2014

Figure Lengend Snippet: Dynamics of phrenic nerve activity during anti-GAD65/67 siRNA microinjection into the phrenic nucleus. Raw and integrated ipsilateral (blue) and contralateral (black) PN activity at different time points relative to the beginning of anti-GAD65/67 siRNA microinjections. A: before (Control) microinjections, and after: 30 min (B); 45 min (C); 60 min (D); 75 min (E); 90 min (F); 105 min (G); and 120 min (H). H, traces: overlapping of integrated ipsilateral PN traces before (black) and after 2 h (blue) of siRNA microinjections. Integrated PN activity in arbitrary units; raw activity in mV (×105). Time bar = 250 ms.

Article Snippet: Following this, sections were washed in PBS (3 × 10 min) and incubated in goat anti-rabbit biotinylated secondary antibodies (1:500; Vector Labs, cat. no. BA-1000) for 2 h. GAD65/67-positive sections were then processed with Vectastain ABC kit (Vector Labs, PK-6100), washed in PBS again (3 × 10 min), and visually explored using a microscope (Olympus-Bx51).

Techniques: Activity Assay

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: The role of spinal GABAergic circuits in the control of phrenic nerve motor output

doi: 10.1152/ajpregu.00244.2014

Figure Lengend Snippet: Dynamic changes in phrenic nerve phasic (inspiratory) and tonic background (expiratory) activity during anti-GAD65/67 siRNA microinjection. Across-animal normalized (in % to control preinjection state) inspiratory (A, top) and expiratory (C, top) integrated PN activity ipsilateral to siRNA injection at 0 (black), 45 (blue), 60 (cyan), 75 (green), 90 (orange), 105 (pink) and 120 (red) min following siRNA injection. A, middle: same as top, but with control values subtracted. A and C, bottom: bin P values with significance level of P < 0.05, compared with control. B, same as A, except that expiratory background activity has been subtracted. For correct averaging, respiratory phases were divided in 15 bins to avoid their differences in length in individual animals and across animal set (see Electrophysiology).

Article Snippet: Following this, sections were washed in PBS (3 × 10 min) and incubated in goat anti-rabbit biotinylated secondary antibodies (1:500; Vector Labs, cat. no. BA-1000) for 2 h. GAD65/67-positive sections were then processed with Vectastain ABC kit (Vector Labs, PK-6100), washed in PBS again (3 × 10 min), and visually explored using a microscope (Olympus-Bx51).

Techniques: Activity Assay, Injection

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: The role of spinal GABAergic circuits in the control of phrenic nerve motor output

doi: 10.1152/ajpregu.00244.2014

Figure Lengend Snippet: Compressed record of phrenic nerve activity before and during anti-GAD65/67 siRNA monolateral microinjection into the phrenic nucleus. Traces, from top to bottom: integrated contralateral (contra, untreated right side, PNR) and ipsilateral (ipsi, treated left side, PNL) PN discharges. BP, blood pressure, TP, tracheal pressure, CO2, end-tidal CO2 level. Arrow indicates beginning of siRNA microinjection.

Article Snippet: Following this, sections were washed in PBS (3 × 10 min) and incubated in goat anti-rabbit biotinylated secondary antibodies (1:500; Vector Labs, cat. no. BA-1000) for 2 h. GAD65/67-positive sections were then processed with Vectastain ABC kit (Vector Labs, PK-6100), washed in PBS again (3 × 10 min), and visually explored using a microscope (Olympus-Bx51).

Techniques: Activity Assay

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: The role of spinal GABAergic circuits in the control of phrenic nerve motor output

doi: 10.1152/ajpregu.00244.2014

Figure Lengend Snippet: Distribution of GAD65/67-containing spinal interneurons after unilateral anti-GAD65/67 siRNA microinjection into the C4 ventral horn. A: C4 transverse section following a 2-h unilateral anti-GAD65/67 siRNA block (siRNA) vs. contralateral side (contra). B and C: magnified view of ventral horn on ipsi (B) and contralateral side (C) showing clearly labeled GAD-positive neurons (white arrows indicate some in focal plane on high power magnification panels B1 and C1).

Article Snippet: Following this, sections were washed in PBS (3 × 10 min) and incubated in goat anti-rabbit biotinylated secondary antibodies (1:500; Vector Labs, cat. no. BA-1000) for 2 h. GAD65/67-positive sections were then processed with Vectastain ABC kit (Vector Labs, PK-6100), washed in PBS again (3 × 10 min), and visually explored using a microscope (Olympus-Bx51).

Techniques: Blocking Assay, Labeling

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: The role of spinal GABAergic circuits in the control of phrenic nerve motor output

doi: 10.1152/ajpregu.00244.2014

Figure Lengend Snippet: Changes in GAD65/67-immunopositive cells after anti-GAD65/67 siRNA injection into the C4 ventral horn, and location of respiratory interneurons. A: C4 transverse spinal section taken from Paxinos and Watson stereotaxic rat brain atlas (55), where from VII to IX are Rexed's layers in C4 ventral horn and M, L, and VL are medial, lateral, and ventrolateral layer subdivisions, respectively. Grid spacing is 1 mm. B: normalized (% of saline-microinjected control set of animals) number of GAD-65/67-positive cells after anti-GAD65/67 siRNA injection into the C4 ventral horn (colors of bars corresponded to layers in A); asterisks (*) show significant (P < 0.05) difference in GAD-65/67 expression compared with control. C3–C5: distribution of respiratory interneurons recorded from C3–C5 ventral horn: squares, inspiratory with extended expiratory activity (I+E; n = 6); circles, decrementing inspiratory with extended expiratory activity (I-Dec+E; n = 5); pentagons, augmenting expiratory (E-Aug; n = 7); diamonds, slow decrementing expiratory (E-Dec; n = 5). GAD65/67-positive (n = 8) cells are indicated in red and GAD65/67-negative cells are indicated by other solid colors (I+E, black squares; I-Dec+E, green circles; E-Aug, violet pentagons; E-Dec, blue diamonds), respectively. Scale bar is 1 mm.

Article Snippet: Following this, sections were washed in PBS (3 × 10 min) and incubated in goat anti-rabbit biotinylated secondary antibodies (1:500; Vector Labs, cat. no. BA-1000) for 2 h. GAD65/67-positive sections were then processed with Vectastain ABC kit (Vector Labs, PK-6100), washed in PBS again (3 × 10 min), and visually explored using a microscope (Olympus-Bx51).

Techniques: Injection, Expressing, Activity Assay

Journal: Stem cells (Dayton, Ohio)

Article Title: Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking bone morphogenetic protein signaling.

doi: 10.1634/stemcells.2005-0110

Figure Lengend Snippet: Figure 6. Characterization of hESC-derived neural progenitors and neurons by immunocytochemistry. (A): Typical rosette-neural progenitor appeared at approximately 3 weeks of differentiation and is positive with nestin antibody staining. After disassociation, the neural progenitors are also positive for other neural progenitor markers, such as (C) musashi and (D) PSA-NCAM, in addition to (B) nestin. These neural progeni- tors are expandable, and the cells in the center of the rosette are positive for BrdU labeling (J). At P4, the cells are positive for early neuronal markers as well such as (E) MAP2 and (F) β-tubulin III. Most of the neurons differentiated from hESCs in the N2B27 medium are GABA neu- rons, indicated by their positive staining for (G) GABA and (H) GAD65 antibodies. TH neurons can be induced by exposure of neural progeni- tors at 4 weeks of differentiation to SHH and FGF8 (I). No GFAP-positive cells were detected (K) until long term in culture (L). Blue staining represents DAPI counterstain. Timelines are indicated at lower left corner. Scale bar = 75 μm in A, B, and I and 25 μm in the others. Abbrevia- tions: DAPI, 4,6-diamidino-2-phenylindole; GABA, γ-aminobutyric acid; GFAP, glial fibrilliary acidic protein; hESC, human embryonic stem cell; TH, tyrosine hydroxylase.

Article Snippet: The following primary antibodies were used in this study: mouse monoclonal antibodies against nestin, polysialic acid– neural cell adhesion molecule (PSA-NCAM), and TH (1:200; all from Chemicon, Temecula, CA, http://www.chemicon.com), β-tubulin III (1:1,000; Sigma), and α-fetoprotein (1:500; Sigma); rabbit polyclonal antibodies against nestin and musashi (1:200), GAD65 (1:500), MAP2 and Pax6 (1:1,000; all from Chemicon), GABA and Sox1 (1:500; Sigma), GATA6 (1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), and glial fibrilliary acidic protein (GFAP) (1:500; DakoCytomation, Glostrup, Denmark, http://www.dakocytomation.com).

Techniques: Derivative Assay, Immunocytochemistry, Staining, Labeling

Journal: Diabetes

Article Title: GAD65 Autoantibodies Detected by Electrochemiluminescence Assay Identify High Risk for Type 1 Diabetes

doi: 10.2337/db13-0534

Figure Lengend Snippet: Sera with positive GADA by radioassay from 14 subjects (7 ECL-GADA positive and 7 ECL-GADA negative) were incubated with different concentrations of unlabeled GAD65 protein and analyzed with standard NIDDK harmonized GADA radioassay. GADA negative by ECL-GADA assay (dotted line), compared with GADA positive by ECL-GADA assay (solid line), required higher concentrations of GAD65 protein for 50% maximal inhibition, which is consistent with low affinity. Results are expressed as percentage of signal not absorbed.

Article Snippet: The serum samples were diluted five times with PBS and mixed at a 1:1 ratio with SULFO-TAG–labeled GAD65 protein 32 ng/mL (Diamyd Medical, Pittsburgh, PA) and biotin-labeled GAD65 protein 1,000 ng/mL in PBS containing 5% BSA for overnight incubation at 4°C.

Techniques: Incubation, Inhibition

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: Tolerogenic DCs but not tolDCs-GAD65 prevent diabetes in the adoptive transfer model of non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) mice. Tolerogenic DCs (tolDCs) or glutamic acid decarboxylase 65 (GAD65) (2 µg/mL) loaded tolDCs (tolDCs-GAD65) were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and monophosphoryl lipid A. Dendritic cells (3 × 10 6 ) were resuspended in phosphate bovine saline (PBS) together with 5 × 10 6 diabetogenic splenocytes from 13-week-old prediabetic NOD females ( n = 8). Cells were then injected i.p. (left side of the belly) in a volume of 300 µL PBS to 8-week-old NOD-SCID female recipients ( n = 12). Diabetogenic splenocytes in PBS were used as the Control group. Data are presented as cumulative diabetes incidence in NOD-SCID recipients, p -values were compensated for multiple comparisons, tolDC vs. tolDC-GAD65: * p = 0.0159.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: Adoptive Transfer Assay, Generated, Injection

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: Antigen-loaded tolDCs fail to lower induction of diabetes in non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) mice by transfer of diabetogenic NOD splenocytes but also by transfer of splenic T cells. Diabetogenic splenocytes (5 × 10 6 per mouse) were isolated from 13-week-old prediabetic NOD females ( n = 11). T cells were enriched (cell purity >92%) by negative selection (EasySep T cell Enrichment kit, Stemcell Tech.), and equivalent of 33% of splenocytes, i.e., 1.65 × 10 6 T cells per mouse were used for diabetes induction in NOD-SCID recipients. Tolerogenic DCs, glutamic acid decarboxylase 65 (GAD65)- (1 µg/mL) or OVA- (1 µg/mL) loaded tolDCs were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and stabilized by monophosphoryl lipid A (MPLA). Diabetogenic splenocytes or enriched T cells (groups marked as T cell + ) were resuspended in phosphate bovine saline (PBS) together 3 × 10 6 tolDCs and injected i.p. (left side of the belly) in a volume of 300 µL PBS to 8-week-old NOD-SCID female recipients ( n = 8). Diabetogenic splenocytes in PBS were used as the Control group. Data are presented as cumulative diabetes incidence in NOD-SCID recipients, p -values were compensated for multiple comparisons.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: Isolation, Selection, Generated, Injection

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: Serum-free cultured tolDCs and GAD65-loaded tolDCs in diabetes prevention using the non-obese diabetes (NOD)-severe combined immunodeficiency (NOD-SCID) model of adoptive transfer of diabetes. Dendritic cells were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and final maturation with monophosphoryl lipid A (MPLA). (A) Tolerogenic DCs loaded with 1 µg/mL of glutamic acid decarboxylase 65 (GAD65) (tolDC-GAD65) or OVA (tolDC-OVA) were prepared in SF medium, whereas tolDCs were cultured in both serum-supplemented (10% fetal bovine serum RPMI-1640) and SF media (tolDC SF). (B) In another experiment, unloaded tolDCs were compared to tolDCs loaded with 1 µg/mL of the GAD65-immunodominant peptide no. 35 (tolDC-pept) and prepared in both serum-supplemented and SF media. Diabetogenic splenocytes (5 × 10 6 per mouse) from 12-week-old prediabetic NOD females ( n = 10) and above listed groups of tolDCs (3 × 10 6 ) were mixed and applied i.p. (left side of the belly) in a volume of 300 µL phosphate bovine saline (PBS) to 7-week-old NOD-SCID female recipients ( n = 8). Diabetogenic splenocytes in PBS were used as the Control group in both experiments. Data are presented as cumulative diabetes incidence in NOD-SCID recipients and p - values were compensated for multiple comparisons (A) Control PBS vs. tolDC SF: * p = 0.026.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: Cell Culture, Adoptive Transfer Assay, Generated

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: Diabetes-preventive effect of tolDCs but not antigen-loaded tolDCs in the spontaneous model of type 1 diabetes—the non-obese diabetes (NOD) mice. Tolerogenic DCs, GAD65- (1 µg/mL) or OVA- (1 µg/mL) loaded tolDCs were generated from bone marrows of 8- to 10-week-old NOD females by cultivation in serum-supplemented (10% fetal bovine serum RPMI-1640) medium in the presence of GM-CSF and IL-4 followed by additions of dexamethasone/vitamin D2 and stabilized by MPLA. Tolerogenic DCs were also prepared in SF media (tolDC SF). TolDCs (3 × 10 6 ) were resuspended in phosphate bovine saline (PBS) and injected i.p. (left side of the belly) in a volume of 200 µL to 4-week-old NOD females ( n = 16). i.p. application of 200 µL PBS alone was used for the Control group. Diabetes incidence was observed weekly (from week 12) until the age of 310 days. Data are presented as cumulative diabetes incidence, p -values were compensated for multiple comparisons.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: Generated, Injection

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: Phenotypic characteristics, anti-inflammatory cytokine profile, and stability of tolDCs and antigen-loaded tolDCs. (A) Expression of maturation markers CD40, CD80, CD86, and MHC II on immature bone marrow-derived dendritic cells (iDCs), control matured bone marrow-derived dendritic cells (cDCs), tolDCs, tolDCs-GAD65, tolDCs-OVA, and DCs loaded with 1 µg/mL of glutamic acid decarboxylase 65 (GAD65)-immunodominant peptide no. 35 (tolDCs-pept), cultured in serum-supplemented (RPMI-1640 with 10% fetal bovine serum) and SF conditions, was determined by surface staining of live cells and flow cytometry. Data are expressed as mean fluorescence intensities (MFI) from 4 to 5 (serum-supplemented conditions) or 2 to 3 (SF conditions) experiments ± SEM, * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Example dot plots from flow cytometry analyses of additional surface markers, i.e., CD103, CCR7, and IL-7Ra (CD127) on tolDCs and autoantigen-loaded tolDCs-GAD65. (C) Stability test of above listed types of DCs was carried out by additional 24 h culture (Control) or restimulation with 1 µg/mL lipopolysaccharide (LPS). Changes in expression of maturation markers CD40, CD80, CD86, and MHC II were assessed by flow cytometry and are displayed as MFI. (D) Interleukin 10 (IL-10) release after MPLA activation or 24 h LPS restimulation (stability test) by iDCs, cDCs, tolDCs, tolDCs-GAD65, and tolDCs-OVA. Data are expressed as means from two to four parallel cell cultures.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: Expressing, Derivative Assay, Cell Culture, Staining, Flow Cytometry, Fluorescence, Activation Assay

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: Allogeneic T cell proliferation and IFN-γ production by stimulation with tolDC vs. antigen-loaded tolDCs. (A) Allogeneic proliferative responses of immature bone marrow-derived dendritic cells (iDCs), control matured bone marrow-derived dendritic cells (cDCs), tolDCs, tolDCs-GAD65, tolDCs-OVA, and tolDC-pept were assessed by coculture of CFSE-labeled splenocytes (6- to 8-week-old C57BL/6 females) with DCs (8-week-old non-obese diabetes females) at 10:1 ratio for 3 and 5 days. Proliferation was measured as CFSE dilution in live CD3 + cells by flow cytometry. Splenocytes cultured alone were used as a control. All experiments were carried out in the serum-supplemented RPMI-1640 medium. Data are expressed as mean percentage of CFSElowCD3 + cells ± SEM of four experiments, ** p < 0.01, *** p < 0.001. (B) Example of proliferation analysis by the flow cytometry of CFSE-labeled CD3 + splenocytes. (C) Induction of INF-γ in allogeneic CD4 + CD3 + T cells was measured after 5 days of coculture with iDCs, cDCs, tolDCs, tolDCs-GAD65, tolDCs-OVA, and tolDCs-pept, following 4-h restimulation with phorbo-12-myristate-13-acetate/ionomycin by intracellular staining and flow cytometry analysis. Data are expressed as mean percentage of CD4 + CD3 + cells ± SEM of four experiments, ** p < 0.01, *** p < 0.001. (D) Example flow cytometry data of allogeneic induction of IFN-γ by DCs within CD3 + CD4 + splenocytes.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: Derivative Assay, Labeling, Flow Cytometry, Cell Culture, Staining

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: CD8 + and CD4 + T-cell-mediated in vitro killing of tolDCs vs. antigen-loaded tolDCs. (A) Equal number (5 × 10 6 ) of immature bone marrow-derived dendritic cells (iDCs), tolDCs, GAD65-, or OVA-loaded tolDCs was mixed 1:1 with CD4 + or CD8 + splenic T cells (enriched by negative magnetic selection) and cocultured for 4, 8, 12, and 24 h. Dendritic cells cultured without splenic T cells were used as controls. Percentage of live nonapotopic cells was measured by flow cytometry as DCs (gated according to the FSC, SSC and CD3 − CD11c + parameters) stained double negative for Hoechst33342 − and AnnexinV − . Data are expressed as mean ± SEM of two to three experiments, * p < 0.05, ** p < 0.01, *** p < 0.001. (B) Example of CD4 + and CD8 + T cell enrichment by negative magnetic selection. (C) Example of flow cytometry analyses of CD3 − CD11c + AnnexinV − Hoechst33342 − cells at the 4 h timepoint.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: In Vitro, Derivative Assay, Selection, Cell Culture, Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Antigen Loading (e.g., Glutamic Acid Decarboxylase 65) of Tolerogenic DCs (tolDCs) Reduces Their Capacity to Prevent Diabetes in the Non-Obese Diabetes (NOD)-Severe Combined Immunodeficiency Model of Adoptive Cotransfer of Diabetes As Well As in NOD Mice

doi: 10.3389/fimmu.2018.00290

Figure Lengend Snippet: In vivo migration of PKH26-labeled tolDCs and tolDCs-GAD65. Bone marrow-derived dendritic cells were prepared from 8-week-old non-obese diabetes (NOD) mice. Tolerogenic DCs and tolDCs-GAD65 (1 µg/mL) were labeled with fluorescent PKH26 dye and 5 × 10 6 cells were applied i.p. (left side of the belly) to 6-week-old NOD females. Unlabeled tolDCs were used as a negative control. FACS detection of PKH26 + cells was carried out on cell suspensions from spleen, mesenteric lymph nodes (MLNs), pancreatic lymph nodes (PLNs), and systemic inguinal lymph nodes (ILNs) after 3, 5, 7, 9, and 12 days (three mice per group for day 12, five mice per group for all other timepoints). Following doublets exclusion cells were gated according to the FSC-A and SSC-A parameters and dead cells were excluded by Hoechst 33258. PKH26 + tolDCs and tolDCs-GAD65 are displayed as percentage of live CD11c + cells (1–2 × 10 6 events per sample). Example of a larger (no. of time points) of two independent experiments.

Article Snippet: For antigen loading of tolDCs the mouse recombinant GAD65 was obtained from Sino Biological, Beijing, China, whereas Ovalbumin EndoGrade (OVA) was purchased from Hyglos, Regensburg, Germany and immunodominant peptide no. 35 of GAD65 sequence (purity 97%) from ThinkPeptides, Oxford, UK.

Techniques: In Vivo, Migration, Labeling, Derivative Assay, Negative Control