Journal: PLoS Medicine

Article Title: PD-L1 checkpoint inhibition and anti-CTLA-4 whole tumor cell vaccination counter adaptive immune resistance: A mouse neuroblastoma model that mimics human disease

doi: 10.1371/journal.pmed.1002497

Figure Lengend Snippet: (A) PD-L1 expression on the surface of mouse Neuro2a was analyzed by flow cytometry and is up-regulated in a dose-dependent manner (mean fluorescent intensity [MFI]). The expression of PD-L1 on human neuroblastoma cell lines SY5Y and SK-N-SH (non-NMYC amplified) had similar changes with exposure to IFNγ. (B) CD3 and PD-L1 expression in mouse neuroblastoma tumors following receipt of Id2kd vaccine with or without checkpoint blockade therapy. Representative tumors were dissected from naïve mice (I, V, IX, and XIII) and from mice after receipt of Id2kd vaccine (II, VI, X, and XIV), Id2kd plus PD-L1 antibody (III, VII, XI, and XV), and Id2kd plus CTLA-4 antibody (IV, VIII, XII, and XVI). Hematoxylin and eosin (H&E) staining (I–VIII) and immunofluorescence double staining (IX–XVI) for CD3 (green) and PD-L1 (red) were performed. The nuclei were stained with DAPI (blue). Representative micrographs from each cohort are shown. Areas of necrotic tissues are marked with black broken lines and coincide with areas of inflammatory cell infiltrates. Panel 1 (I–IV) and panel 3 (IX–XII) are 40× and 100× original magnification, respectively. The scale bar for (I–IV) is 500 μm and for (IX–XII) is 100 μm. The enlarged images in panel 2 (V–VIII) and panel 4 (XIII–XVI) are of 600× original magnification. The scale bar is 20 μm. (C) Expression of activation markers on the surface of CD8+ tumor-infiltrating lymphocytes isolated from shrinking tumors of mice treated with α-CTLA-4 plus Id2kd vaccine. Programmed cell death 1 (PD1), TIM3, and LAG3 were expressed on tumor-infiltrating lymphocytes (TILs) by flow cytometry compared with isotype controls. APC, antigen-presenting cell; FITC, fluorescein isothiocyanate; IgG, immunoglobulin G; PerCP, peridinin-chlorophyll-protein complex.

Article Snippet: The murine neuroblastoma cell line Neuro2a (N2a) is derived from an A/J mouse and was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, US).

Techniques: Expressing, Flow Cytometry, Amplification, Staining, Immunofluorescence, Double Staining, Activation Assay, Isolation

Journal: Development (Cambridge, England)

Article Title: P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea.

doi: 10.1242/dev.002279

Figure Lengend Snippet: Fig. 3. Analysis of P2X receptor expression in neonatal rat SGN. (A) Isolation of a single SGN from a P4 rat cochlear slice using a micropipette. (B) Example of an inward current response to the P2X3 and P2X2/3 receptor agonist ,MeATP (100 M) in a SGN using whole-cell voltage clamp (holding potential –60 mV). (C) Block of the ATP response (100 M, 5 seconds of focal application) by the P2X2/3 receptor-specific antagonist A- 317491 (500 nM, bath superfusion). (D) Real-time PCR amplification plot showing detection of P2X3 cDNA in a sample of individual SGN. (E) Average transcript copy number for each P2X receptor subunit and the housekeeping genes Nse and Gapdh in individual neurons. Note that the P2X3 transcript number was twice that of P2X2 (*P<0.01; P2X3 transcript number was significantly greater than the other P2X subunits; GAPDH transcript copy number was significantly greater than NSE transcript copy number). (F) Relative distribution of P2X receptor subunits in the population of SGN. This plot shows the normalized mRNA transcript copy number for each of the seven candidate P2X receptor subunits expressed by individual SGN. (G) Immunofluorescence labeling of P2X2, P2X3 and P2X4 in neonatal (P4) spiral ganglion tissue.

Article Snippet: P2X3 polyclonal rabbit antiserum (Neuromics, Bloomington, MN) was targeted to residues 383-397 (VEKQSTDSGAYSIGH), P2X4 polyclonal rabbit antiserum (Alomone Laboratories, Israel) was targeted to residues 370-388 (YVEDYEQGLSGEMNQ), and P2X2 was targeted to residues 96-113 (VSIITRIEVTPSQTLGTC) (Kanjhan et al., 1996).

Techniques: Expressing, Isolation, Blocking Assay, Real-time Polymerase Chain Reaction, Amplification, Immunofluorescence, Labeling

Journal: Development (Cambridge, England)

Article Title: P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea.

doi: 10.1242/dev.002279

Figure Lengend Snippet: Fig. 5. Pharmacology of the recombinant P2X2-3/3 receptor. (A) Injection of P2X2-3 and P2X3 mRNAs in a ratio of 1:2 into Xenopus oocytes resulted in expression of ATP-gated inward currents with a broad sensitivity to ATP agonists. (B) Concentration-response curves for ATP at pH 7.5 show an EC50 of 0.4 M for these ATP-gated ion channels. Acidification to pH 6.5 produced a leftward shift in the concentration-response curve, to reduce activation thresholds to <10 nM ATP. This is attributable to positive allosteric modulation by protons acting through the P2X2 subunit.

Article Snippet: P2X3 polyclonal rabbit antiserum (Neuromics, Bloomington, MN) was targeted to residues 383-397 (VEKQSTDSGAYSIGH), P2X4 polyclonal rabbit antiserum (Alomone Laboratories, Israel) was targeted to residues 370-388 (YVEDYEQGLSGEMNQ), and P2X2 was targeted to residues 96-113 (VSIITRIEVTPSQTLGTC) (Kanjhan et al., 1996).

Techniques: Recombinant, Injection, Expressing, Concentration Assay, Produced, Activation Assay

Journal: Development (Cambridge, England)

Article Title: P2X receptor signaling inhibits BDNF-mediated spiral ganglion neuron development in the neonatal rat cochlea.

doi: 10.1242/dev.002279

Figure Lengend Snippet: Fig. 6. Developmental profile of P2X receptor subunit expression in rat spiral ganglion tissue from P0-P14, determined by end- point RT-PCR. Note the downregulation of the P2X3 transcript by P14.

Article Snippet: P2X3 polyclonal rabbit antiserum (Neuromics, Bloomington, MN) was targeted to residues 383-397 (VEKQSTDSGAYSIGH), P2X4 polyclonal rabbit antiserum (Alomone Laboratories, Israel) was targeted to residues 370-388 (YVEDYEQGLSGEMNQ), and P2X2 was targeted to residues 96-113 (VSIITRIEVTPSQTLGTC) (Kanjhan et al., 1996).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease

doi: 10.1093/nar/gkn084

Figure Lengend Snippet: UV-LMD of individual neurons. ( A ) Left panel: cresylviolet (CV)-stained mouse coronal midbrain section indicating the area of the substantia nigra pars compacta (SNpc) before (left) and after (right) UV-laser-microdissection of the entire area. Right panel: gel electrophoresis results of qualitative reverse transcription (RT) multiplex-nested PCR products for a whole LMD SNpc. RT–PCR signals for all eight different dopaminergic (TH, DAT, Girk2, D2s/l) and nondopaminergic (GAD, Girk1, GFAP, CB) marker-genes, detected in the heterogeneous cellular mixture of dopaminergic, GABAergic and glial cells. (DNA-ladder: 100-bp marker). ( B ) Top: CV-stained coronal midbrain section after UV-laser-microdissection of 15 individual DA neurons. Middle: selection and laser-microdissection of an individual neuron from upper section. Lower: after UV-LMD of individual neuron—section (left) and cap-control (right) demonstrating successful isolation. ( C ) PCR products after gel electrophoresis of qualitative RT multiplex-nested PCR for individual LMD SN DA neurons and small SN DA pools. Upper and second panel: similar gene-expression profile of dopaminergic marker genes (TH, DAT, Girk2, D2; not Girk2, GAD67, GFAP) in pools of 20 and single SN DA neurons illustrate sensitivity and specificity of the protocol. Third panel: expression profile of an individual calbindin-positive CB (+) SN DA neuron ( D and E ) Quantitative real-time RT–PCR analysis of Eno2 gene-expression using 10% of cDNA from pools of 15 mouse SN DA neurons as template. Neurons were UV-LMD-collected from either fresh or stored (1-week) and re-used tissue slices. (D) Representative real-time PCR traces testing for Eno2 expression, relative fluorescence levels on a logarithmic scale are plotted against PCR cycles (ΔRN: relative fluorescence, normalized to internal fluorescence marker ROX); threshold-line for determinations of threshold cycles (Ct) indicated by the green line (Δ RN 0.4). (E) No significant difference n.s. between Eno2 detection levels (Ct) in SN DA cell-pools from fresh and frozen tissue sections (fresh: 34.43 ± 0.51, n = 10; re-used: 34.62 ± 0.48 n = 9; p = 0.785).

Article Snippet: Applied Biosystems identifying assay IDs: human ENO2 assay B = ENO2-B: Hs00157360_m1, fragment size: 77 bp; human α-SYN: HS00240906_m1, fragment size: 62 bp.

Techniques: Staining, Laser Capture Microdissection, Nucleic Acid Electrophoresis, Reverse Transcription, Multiplex Assay, Nested PCR, Reverse Transcription Polymerase Chain Reaction, Marker, Selection, Control, Isolation, Gene Expression, Expressing, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Fluorescence

Journal: Nucleic Acids Research

Article Title: Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease

doi: 10.1093/nar/gkn084

Figure Lengend Snippet: Human postmortem midbrain samples—characteristics and gene-expression

Article Snippet: Applied Biosystems identifying assay IDs: human ENO2 assay B = ENO2-B: Hs00157360_m1, fragment size: 77 bp; human α-SYN: HS00240906_m1, fragment size: 62 bp.

Techniques: Control

Journal: Nucleic Acids Research

Article Title: Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease

doi: 10.1093/nar/gkn084

Figure Lengend Snippet: Significantly lower mean mRNA-levels of α-synuclein, tyrosine hydroxylase, and neuron-specific enolase in individual SN DA neurons for Parkinson's brains compared to control brains

Article Snippet: Applied Biosystems identifying assay IDs: human ENO2 assay B = ENO2-B: Hs00157360_m1, fragment size: 77 bp; human α-SYN: HS00240906_m1, fragment size: 62 bp.

Techniques: Control, Gene Assay

Journal: Nucleic Acids Research

Article Title: Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease

doi: 10.1093/nar/gkn084

Figure Lengend Snippet: Effects of RNA integrity and PCR-assay size on real-time quantitative PCR performance. Serial dilutions of high-quality whole brain cDNA (200, 20 and 2 pg; Clontech), and cDNA derived from partly degraded midbrain RNA (400, 40 and 4 pg; brain SN 2/02, RIN = 6.1) were used as templates for ENO2 real-time PCR, employing two different assays for ENO2 with large (ENO2-A, 108 bp) and small (ENO2-B, 77 bp) amplicon size ( A and C ) With high-quality cDNA as templates, amplification curves (A) and slopes (amplification-efficiencies) of standard curves were similar for both assays (Δ RN: relative fluorescence, normalized to internal fluorescence marker ROX) (C). ( B and D ) With cDNA from partly degraded RNA as templates, amplification curves (B) and slopes of standard curves (D) were similar as for high-quality cDNA only when using the small amplicon size assay ENO2-B. The larger ENO2-A assay did not allow the generation of a respective reliable standard curve and was not suited for RT–PCR quantification of SN 2/02 RNA sample.

Article Snippet: Applied Biosystems identifying assay IDs: human ENO2 assay B = ENO2-B: Hs00157360_m1, fragment size: 77 bp; human α-SYN: HS00240906_m1, fragment size: 62 bp.

Techniques: Real-time Polymerase Chain Reaction, Derivative Assay, Amplification, Fluorescence, Marker, Reverse Transcription Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Elevated α-synuclein mRNA levels in individual UV-laser-microdissected dopaminergic substantia nigra neurons in idiopathic Parkinson's disease

doi: 10.1093/nar/gkn084

Figure Lengend Snippet: LMD and mRNA-expression analysis of individual SN DA neurons from human PD and control postmortem brains. ( A and B ) Pools of neuromelanin-positive [NM(+)] neurons were isolated via LMD of cresylviolet-stained horizontal midbrain cryosections from PD (A) and control brains (B). Upper panel: PD (A) and control (B) cryosections after LMD of small pools of NM+ neurons from SNpc. Lower panels: Representative PD (A) and control (b) SNpc NM(+) neurons before (left) and after dissection (right). Insert: cap control after UV-LMD. Scale bars: 250 μm, 20 μm, respectively. ( C ) Scatter plot of α-synuclein gene-expression-levels in PD and control brains. α-Synuclein gene-expression of each pool of 15 NM(+) and TH(+) SNpc neurons is given as pg-equivalents of total cDNA derived from human SN-tissue per cell (standard curve quantification), determined via quantitative real-time PCR. Bars represent mean α-synuclein expression for SNpc pools of each brain ± SEM. ( D ) Plot of the mean α-synuclein cDNA levels (±SEM) against the RNA integrity number for each brain. Brain Bank codes are indicated next to each dot (see Figure 5C and ). ( E ) Linear regression between mean α-synuclein expression and RNA integrity number for all individual analyzed control and PD brains showed no positive correlation between higher RNA quality of the tissue and detected α-synuclein expression-levels. (controls: black dotted line, R = 0.0506; PD brains: red dotted line, R = 0.9950; all analyzed brains combined: black line, R = 0.4369). Please note that PD brains showed a strong inverse correlation between RNA-integrity and detected α-synuclein expression-levels (red dotted line, R = 0.9950). ( F ) Mean expression levels of α-SYN, TH and ENO2 were significantly higher in individual NM(+) SN DA neurons from PD brains compared to controls (see and ).

Article Snippet: Applied Biosystems identifying assay IDs: human ENO2 assay B = ENO2-B: Hs00157360_m1, fragment size: 77 bp; human α-SYN: HS00240906_m1, fragment size: 62 bp.

Techniques: Expressing, Control, Isolation, Staining, Dissection, Gene Expression, Derivative Assay, Real-time Polymerase Chain Reaction