Journal: The Journal of Neuroscience

Article Title: Genetic Deletion of Paired Immunoglobulin-Like Receptor B Does Not Promote Axonal Plasticity or Functional Recovery after Traumatic Brain Injury

doi: 10.1523/JNEUROSCI.3228-10.2010

Figure Lengend Snippet: Traumatic cortical injury in wild-type and PirB−/− mice. A, Schematic illustration of corticofugal projections from the primary motor cortex and the experimental procedures. Traumatic injury to the sensorimotor cortex disrupted both the CST and CRT (dotted lines). BDA was injected into the contralesional motor cortex 2 weeks after the injury. The black arrow shows axons of the intact CST or CRT sprouting and crossing the midline into the denervated side. B, Representative Nissl-stained coronal sections of brain injured by a pneumatic impactor. Sensorimotor cortex was specifically destroyed. Scale bar, 1 cm. C, Measurement of lesion volume in wild-type and PirB−/− mice. There were no significant differences between the groups. D, Immunostaining for PKCγ in the cervical spinal cord at 1 week after the cortical injury. PKCγ immunoreactivity completely disappeared in the right dorsal CST originating from the injured motor cortex. Arrowheads indicate the left intact CST from the uninjured motor cortex. Scale bar, 200 μm.

Article Snippet: For immunostaining of MAG, myelin basic protein (MBP), PirB, and protein kinase Cγ (PKCγ), sections (20 μm thick) were blocked with 5% bovine serum albumin (BSA; Sigma) and 0.1% Triton X-100 in PBS and incubated with mouse anti-MAG (1:200, Millipore Bioscience Research Reagents), rat anti-MBP (1:100, Abcam), rat anti-PirB (5 μg/ml, R&D Systems), or rabbit anti-PKCγ (1:500, Santa Cruz Biotechnology) antibodies overnight at 4°C.

Techniques: Injection, Staining, Immunostaining

Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: Cell surface expression of PIR molecules on cultured mast cells. Nonadherent cells from the bone marrow of (a) wild-type mice (C57BL/cJ) and (b) FcεRIα–/– mice and from the neonatal spleens of (c) wild-type mice (C3H/HeJ) and (d) me/me mice were cultured for 6 weeks with rIL-3. Cells were sequentially incubated with the PE-labeled 6C1 anti-PIR mAb, biotinylated ACK-2 anti–c-kit mAb, and APC-labeled streptavidin or with rat IgE anti-DNP mAb (dark histogram) and FITC-labeled goat anti-rat Ig antibodies before analysis by flow cytometry. Isotype-matched control rat mAb’s were used to set the quadrants for immunofluorescence analysis, and unstained background controls are indicated by open histograms.

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Expressing, Cell Culture, Incubation, Labeling, Flow Cytometry, Immunofluorescence

Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: PIR expression by IL-3–induced mast cells. (a) RT-PCR analysis of PIR-A, PIR-B, and actin gene expression by C3H/HeJ bone marrow–derived mast cells and the WEHI-3 myeloid cell line. The PCR products were electrophoresed in 1.5% agarose and stained with ethidium bromide. (b) Analysis of the PIR molecules on splenocytes, mast cells, and macrophages. Cell surface proteins were 125I-labeled, solubilized in 1% NP-40, and immunoadsorbed with 6C1 anti-PIR or an isotype-matched control mAb before analysis by SDS-10% PAGE under reducing conditions as described in Methods. (c) Analysis of PIR molecules on mast cells derived from adult bone marrow of FcεRIα–/– mice and littermate controls (upper panel) and from neonatal spleens of me/me mice and littermate controls (lower panel). Iodinated cell surface PIR proteins were resolved on SDS-10% PAGE under reducing conditions.

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Staining, Labeling

Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: PIR-B inhibitory activity in FcεRI-mediated Ca2+ mobilization. Indo-1/AM dye preloaded mast cells from wild-type mice (upper panel) and motheaten mice (lower panel) were analyzed by flow cytometry for intracellular Ca2+ levels in the presence or absence (data not shown) of extracellular Ca2+. Cells were stimulated with rat IgE anti-DNP (IgE), F(ab′)2 fragments of rat anti-PIR (anti-PIR), or both mAb’s (IgE + anti-PIR). F(ab′)2 fragments of rabbit anti-rat Ig antibodies were used as the cross-linking reagent.

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Activity Assay, Flow Cytometry

Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: PIR-B inhibitory activity in FcεRI-induced serotonin release. 3H-Serotonin preloaded mast cells from wild-type neonatal spleen (left) and motheaten mutant neonatal spleen (right) were incubated with rat IgE anti-DNP mAb plus various concentrations of F(ab′)2 fragments of rat mAb specific for PIR (filled circles) or unknown cell surface antigen (open circles). Antibody-primed cells were then triggered with F(ab′)2 fragments of rabbit anti-rat Ig antibody as a cross-linker, and the 3H-serotonin release was measured as described in Methods. The standard deviation from the mean (circle) is indicated by bars only for the points at which this value (1 SD) is greater than the circle radius.

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Activity Assay, Mutagenesis, Incubation, Standard Deviation

Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: Comparison of surface expression of FcγRIIB/PIR chimeric receptors among various stable transfectants. RBL-2H3 rat basophilic leukemia cells were transfected by electroporation with various types (YYYY, FYFF, YY, YF, and Δ712) of chimeric construct (FcγRIIB/PIR-B). These constructs encode the extracellular (EC), transmembrane (TM), and the first six amino acids of the cytoplasmic tail of the mouse FcγRIIB1 (open portions of bars) and fused with different intracytoplasmic regions (IC) of the mouse PIR-B (filled portions of bars) as depicted in the right panel. Four cytoplasmic tyrosine (Y) residues are also indicated. For the left panel, stably transfected cells were sequentially incubated with 2.4G2 rat mAb and FITC-labeled goat anti-rat Ig antibody (filled profile) or with BC4 mouse IgE mAb and FITC-labeled goat anti-mouse Ig antibody (thick line) in order to determine their cell surface levels of FcγRIIB/PIR-B chimeric receptors and endogenous FcεRI by flow cytometry. Irrelevant mouse IgG (thin line) was used as a control to obtain a background staining.

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Expressing, Transfection, Electroporation, Construct, Stable Transfection, Incubation, Labeling, Flow Cytometry, Staining

Journal:

Article Title: Inhibition of IgE-mediated mast cell activation by the paired Ig-like receptor PIR-B

doi:

Figure Lengend Snippet: Inhibitory activity of FcγRIIB/PIR-B chimeric receptors expressed by RBL transfected cells. RBL-2H3 cells transfected with the indicated FcγRIIB/PIR-B constructs were incubated with various dilutions of mouse IgE in the absence (filled circles) or presence (open circles) of 2.4G2 rat anti-FcγRII/III mAb (20 μg/ml). F(ab′)2 fragments of goat anti-murine Ig antibody (50 μg/ml) were used as a cross-linker. A representative result is shown as a mean ± 1 SD (if the SD values are greater than the radius of circles) from at least three independent experiments. Statistical analysis revealed that inhibition is significant for IgE at 1:1,000 and 1:300 dilutions in a, b, and d (data not shown).

Article Snippet: Briefly, mast cells were loaded with Indo-1/AM (1 μg/ml; Calbiochem-Novabiochem Corp., San Diego, California, USA) in the presence or absence of rat IgE anti-DNP mAb (1 μg/ml) for 30 minutes at 37°C, resuspended at 10 6 cells/ml either in HBSS containing 5% FCS or Ca 2+ -free HBSS containing 1 mM MgCl 2 and were incubated first with F(ab′) 2 fragments of the rat anti-PIR mAb (10.1 clone; γ2bκ; 20 μg/ml) and then with F(ab′) 2 fragments of rabbit anti-rat Ig antibody (50 μg/ml; Southern Biotechnology Associates) as a cross-linker.

Techniques: Activity Assay, Transfection, Construct, Incubation, Inhibition