Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: CS protein specific immune responses increase in an Ad-CSP dose dependent manner. BALB/cJ mice (n = 3) were injected IM with Ad-CSP ranging from 1×10 7 to 1×10 9 vps/mouse, increasing by half logs. 14 days post injection splenocytes and plasma were collected. (A) ELISpot assays were performed to quantify IFNγ secreting cells from splenocytes stimulated with CS protein peptide, NYDNAGTNL, ex vivo . (B) IFNγ, TNFα, and IL-2 expression by splenocyte derived CD3 + CD8 + T cells was analyzed by flow cytometry following ex vivo stimulation with NYDNAGTNL. (C) Total IgG against CS protein was assessed by ELISA. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, *,**,*** denotes significance over naïve, p<0.05, p<0.01, p<0.001.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Clinical Proteomics, Enzyme-linked Immunospot, Ex Vivo, Expressing, Derivative Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: Co-vaccination with Ad-CSP and Ad-EAT2 dramatically increases IFNγ secreting splenocytes in response to stimulation with CS protein epitope, NYDNAGTNL. BALB/cJ mice were injected IM with either 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse Ad-GFP or 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse of either (A) Ad-GFP/rEA (n = 5) or (B) Ad-EAT2 (n = 6). Splenocytes were collected 14 days post co-injection. ELISpot were performed on the splenocytes of these mice stimulated with NYDNAGTNL peptide to assess the amount of IFNγ secreting cells. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test,* Denotes significance over naïve animals, p<0.05. Representative figures of two independent experiments.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Enzyme-linked Immunospot

Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: Co-vaccination with Ad-CSP and Ad-EAT2 resulted in increased NYDNAGTNL tetramer positive CD8 + T cells as well as improved IFNγ secretion from CD8 + T cells. BALB/cJ mice (n = 6) were co-injected IM with 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse of Ad-EAT2 or 5×10 7 vps/mouse of Ad-CSP and 5×10 7 vps/mouse of Ad-GFP. (A) Peripheral Blood Mononuclear Cells (PBMCs) were stained with CD8-Alexa Flour700, CD3-APC-Cy7, and CSP (NYD)-Tetramer. (B–C) Intracellular staining was performed on splenocytes after stimulation with NYDNAGTNL peptide. Cells were stained with CD8-Alexa Flour700, CD3-APC-Cy7, ViViD, IFNg-APC, and Perforin-PE antibodies. The bars represent mean ± SD. Statistical analysis was completed using One Way ANOVA with a Student-Newman-Keuls post-hoc test, *, **, *** denotes significance over naïve animals, p<0. 05, p<0. 01, p<0.001.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Staining

Journal: PLoS ONE

Article Title: Vaccine Platforms Combining Circumsporozoite Protein and Potent Immune Modulators, rEA or EAT-2, Paradoxically Result in Opposing Immune Responses

doi: 10.1371/journal.pone.0024147

Figure Lengend Snippet: Co-vaccination of mice with Ad-CSP and Ad-EAT2 increased specific killing cells pulsed with CS protein peptides. BALB/cJ mice (n = 4) were co-injected IM with either 1×10 8 vps/mouse Ad-CSP and 1×10 8 vps/mouse Ad-GFP or 1×10 8 vps/mouse Ad-CSP and 1×10 8 vps/mouse Ad-EAT2 on Day 0. Day 14 splenocytes were collected from naïve mice and pulsed with either NYDNAGTNL peptide or an irrelevant peptide. NYDNAGTNL pulsed splenocytes were stained with a high concentration of CFSE while splenocytes pulsed with irrelevant peptide were stained with a low concentration of CFSE. Stained splenocytes were then combined in equivalent doses. 8 million cells were then injected IV into naïve, Ad-CSP+Ad-GFP co-vaccinated, or Ad-CSP+Ad-EAT2 co-vaccinated mice. After 18 hrs splenocytes from these mice were collected and analyzed by flow cytometry to assess the amount of NYDNAGTNL specific killing. % Specific killing = 1-((%CFSE High /%CFSE Low ) immunized /(%CFSE High /CFSE Low ) non-immunized ). * denotes significant difference between treatments p<0.05.

Article Snippet: Splenocytes were stimulated ex vivo with 4 μg/mL of the >98% pure CS protein immunodominant peptide NYDNAGTNL (amino acids 43–51 of the CS protein sequence) (GenScript Piscataway, NJ) .

Techniques: Injection, Staining, Concentration Assay, Flow Cytometry

Journal: The Journal of Neuroscience

Article Title: Polarization-Sensitive Descending Neurons in the Locust: Connecting the Brain to Thoracic Ganglia

doi: 10.1523/JNEUROSCI.3624-10.2011

Figure Lengend Snippet: Morphology and response characteristics of ipsilaterally descending brain neurons. A, Reconstruction of arborizations in the brain (posterior view), SOG, and the three thoracic ganglia (dorsal view). Arborizations in all ganglia are confined to the ipsilateral hemispheres. Dendritic ramifications are in the posterior protocerebrum at the level of the posterior optic tubercle (arrow; POTu, gray shaded; see also G). The antennal mechanosensory and motor center (dotted line, asterisk; see also H) is invaded by beaded arborizations. An axonal fiber descends through the SOG with ramifications extending laterally and medially around the axon. In the Pro-TG, two characteristic areas of ramification extend laterally. The axon passes through the mesothoracic (Meso-TG) and metathoracic (Meta-TG) ganglion, with only sparse side branches, and could not be traced beyond the fused first abdominal ganglion. B, C, Circular diagrams of mean frequencies of action potentials plotted against E-vector orientation during dorsal stimulation with a rotating polarizer. Background activity is indicated by a black circle within each plot. B, During counterclockwise rotations of the polarizer, E-vector tuning (Φmax) was at 142° (n = 7; error bars indicate SD; bin size, 10°; p = 1.9 × 10−12). C, The preferred E-vector orientation during clockwise rotations (n = 7; error bars indicate SD; bin size, 10°; p = 2.2 × 10−8) had a Φmax of 111° and differed significantly from Φmax during counterclockwise rotations (n = 7; p = 0.009, Student's t test for paired probes). D, Circular diagram showing normalized mean activities from all recordings (n = 6) to dorsal stimulation through a rotating polarizer with Φmax set to 0°. Background activity is indicated by a black circle. E, Spike train (bottom trace) and mean spiking frequency (top trace; gliding average with bin size of 1 s) from the same neuron as in B and C during a counterclockwise (360–0°) and a clockwise (0–360°) rotation of the polarizer. F, Responses to a moving black and white grating presented in front of the animal. A small cardboard with black and white stripes was moved by hand from left to right and vice versa under unpolarized light condition (black bar). Black arrows indicate movement direction of the pattern as seen by the locust. The neuron with the soma in the left brain hemisphere was excited by movement from right to left and shows inhibition to moving bars from left to right. G, H, Morphological details in the brain. Maximum intensity views of optical sections (frontal plane; thickness, 2 μm) from confocal image stacks showing anti-synapsin staining (blue), anti-PDH immunostaining (green), and ramifications of the Neurobiotin-stained neuron (red). G, Maximum intensity projection of 78 optical sections showing ramifications around the posterior optic tubercle (POTu), which is marked with the anti-PDH antiserum. H, Maximum intensity projection of two combined stacks (76 optical sections), showing varicose arborizations in the antennal mechanosensory and motor center (AMMC). Some dendritic arborizations are visible dorsally from the antennal mechanosensory and motor center. Scale bars: A, 200 μm; G, H, 40 μm.

Article Snippet: Their tips were either filled with a 3% aqueous solution of fluorescent dextran (Alexa Fluor 488 or 647; 10,000 molecular weight, anionic, fixable; Invitrogen) or with 4% Neurobiotin (Vector Laboratories) in 1 m KCl and backed up with 1 m KCl.

Techniques: Plasmid Preparation, Activity Assay, Inhibition, Staining, Immunostaining

Journal: The Journal of Neuroscience

Article Title: Polarization-Sensitive Descending Neurons in the Locust: Connecting the Brain to Thoracic Ganglia

doi: 10.1523/JNEUROSCI.3624-10.2011

Figure Lengend Snippet: Confocal images of 130 μm sections showing parts of Neurobiotin-stained descending SOG neurons (red) combined with immunostaining using antisera against FMRFamide, serotonin, Lom-TK II, AST-A, or GABA (green). The Neurobiotin-stained somata and ramifications are shown in A–E, the immunostainings are shown in A′–E′, and the combined illustrations are shown in A‴–E‴. None of the tested neuroactive substance is localized in the stained neurons. A, Soma and part of the primary neurite in the SOG. FMRFamide immunostaining is not present in the soma or axon of the neuron. B, Dendritic ramifications in the SOG and presynaptic terminals from the Pro-TG (inset). The labeled neuron does not show serotonin immunostaining. C, D, In the Pro-TG, presynaptic terminals with bleb-like endings do not show immunostaining for Lom-TK II (C) or AST-A (D). E, The soma of the SOG neuron does not exhibit GABA immunostaining. Scale bars, 40 μm.

Article Snippet: Their tips were either filled with a 3% aqueous solution of fluorescent dextran (Alexa Fluor 488 or 647; 10,000 molecular weight, anionic, fixable; Invitrogen) or with 4% Neurobiotin (Vector Laboratories) in 1 m KCl and backed up with 1 m KCl.

Techniques: Staining, Immunostaining, Labeling