Journal: Neurotherapeutics

Article Title: Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats

doi: 10.1007/s13311-019-00718-3

Figure Lengend Snippet: Preparation of recombinant pcDNA-GMCSF-NgR-PirB eukaryotic expression vector and its expression in CHO cells. (a) Diagram of the constructed pcDNA-GMCSF-NgR-PirB expression vector. (b) Colony PCR identification of recombinant vector of pcDNA-GMCSF-NgR-PirB. Lane 1-5: NgR positive PCR products of pcDNA-GMCSF-NgR-PirB; M: DNA marker DL2000. (c) Restriction enzymes analysis of recombinant vector of pcDNA-GMCSF-NgR-PirB. M: DNA marker DL10000; Lane 1: pcDNA3.1(+) digested with BamH I; Lane 2: pcDNA-GMCSF-NgR-PirB digested with BamH I; Lane 3: pcDNA-GMCSF-NgR-PirB digested with BamH I and Hind III; Lane 4: pcDNA-GMCSF-NgR-PirB digested with BamH I and Not I; Lane 5: pcDNA-GMCSF-NgR-PirB digested with Not I and Xba I; Lane 6: pcDNA-GMCSF-NgR-PirB digested with Hind III and Xba I. (d) The expression of NgR and PirB in GMCSF-NgR-PirB fusion protein in pcDNA-GMCSF-NgR-PirB transfected CHO cells was detected after transfected 24 hours by using immunofluorescence. Scale bar, 50 μm. (e) The expression of NgR and PirB in GMCSF-NgR-PirB fusion protein in pcDNA-GMCSF-NgR-PirB transfected CHO cells was detected after transfected 24 hours by using Western blot

Article Snippet: After incubation with a blocking buffer (10% normal goat serum, 0.3% Triton×100 in 0.01 M PBS) for 1 hour at room temperature, sections reacted with primary antibodies, including a rabbit anti-NgR polyclonal antibody (1:100, Santa Crue), a mouse anti-PirB monoclonal antibody (1:100, Sino Biological), or a rabbit anti-GAP-43 polyclonal antibody (1:100, Santa Crue) at 4°C overnight, and then with FITC conjugated goat anti-rabbit antibody or Cy3 conjugated goat anti-mouse antibody (1:100, Sigma) for 1 hour at 37°C.

Techniques: Recombinant, Expressing, Plasmid Preparation, Construct, Marker, Transfection, Immunofluorescence, Western Blot

Journal: Neurotherapeutics

Article Title: Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats

doi: 10.1007/s13311-019-00718-3

Figure Lengend Snippet: Detection of antibody response against NgR and PirB. (a) ELISA detection for specific antibody responses to NgR in the sera of immunized rats. (b) ELISA detection for specific antibody responses to PirB in the sera of immunized rats. The average antibody titers were shown as mean optical density (OD) at 450 nm measured with an ELISA reader, and the titer of rats immunized with pcDNA-GMCSF-NgR-PirB as well as pcDNA-NgR-PirB was significantly higher than that of unimmunized SCI rats, and the average antibody titer in rats immunized with pcDNA-GMCSF-NgR-PirB was significantly higher than that immunized with pcDNA-NgR-PirB. *P<0.05, **P<0.01, compared with the unimmunized SCI group; #P<0.05, ##P<0.01, compared with NgR-PirB IM group (n=6 per group)

Article Snippet: After incubation with a blocking buffer (10% normal goat serum, 0.3% Triton×100 in 0.01 M PBS) for 1 hour at room temperature, sections reacted with primary antibodies, including a rabbit anti-NgR polyclonal antibody (1:100, Santa Crue), a mouse anti-PirB monoclonal antibody (1:100, Sino Biological), or a rabbit anti-GAP-43 polyclonal antibody (1:100, Santa Crue) at 4°C overnight, and then with FITC conjugated goat anti-rabbit antibody or Cy3 conjugated goat anti-mouse antibody (1:100, Sigma) for 1 hour at 37°C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Neurotherapeutics

Article Title: Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats

doi: 10.1007/s13311-019-00718-3

Figure Lengend Snippet: The inhibitory effect of MAG on neurite outgrowth was reversed by antisera from GMCSF-NgR-PirB immunized rats. (a) Immunostaining with a mouse anti-β III tubulin monoclonal antibody for the neurite of SH-SY5Y cells, SH-SY5Y cells cultured with MAG-Fc, and SH-SY5Y cells cultured with MAG-Fc in the presence of pre-immune sera, sera from pcDNA3.1(+), pcDNA-GMCSF, pcDNA-NgR-PirB, and pcDNA-GMCSF-NgR-PirB immunized rats, and rabbit anti-NgR/PirB polyclonal antibody, rabbit anti-NgR or PirB polyclonal antibody, as positive controls. Scale bar, 50 μm. (b) Quantifications of the average neurite lengths of SH-SY5Y cells. Sera from GMCSF-NgR-PirB immunized rats significantly reversed the inhibition of MAG and promoted neurite outgrowth compared with pre-immune sera, as well as sera from GMCSF-NgR-PirB IM rats compared with that from NgR-PirB IM rats. **P<0.01, compared with pre-immune sera group; #P<0.05, ##P<0.01, compared with NgR-PirB IM group (n=6 per group)

Article Snippet: After incubation with a blocking buffer (10% normal goat serum, 0.3% Triton×100 in 0.01 M PBS) for 1 hour at room temperature, sections reacted with primary antibodies, including a rabbit anti-NgR polyclonal antibody (1:100, Santa Crue), a mouse anti-PirB monoclonal antibody (1:100, Sino Biological), or a rabbit anti-GAP-43 polyclonal antibody (1:100, Santa Crue) at 4°C overnight, and then with FITC conjugated goat anti-rabbit antibody or Cy3 conjugated goat anti-mouse antibody (1:100, Sigma) for 1 hour at 37°C.

Techniques: Immunostaining, Cell Culture, Inhibition

Journal: Neurotherapeutics

Article Title: Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats

doi: 10.1007/s13311-019-00718-3

Figure Lengend Snippet: Vaccination promoted functional recovery after SCI. (a) Except for the normal control, the BBB scores in pcDNA-NgR-PirB and pcDNA-GMCSF-NgR-PirB immunized rats were consistently higher than those in the other negative control groups including pcDNA-GMCSF, pcDNA3.1(+) immunized rats, and unimmunized SCI rats, and there was also significant change between the GMCSF-NgR-PirB IM group and the NgR-PirB IM group, except at 1 week and 4 weeks post SCI. (b) Legend of CatWalk. The following parameters were selected for analysis: stride length, base of support (BOS), swing (s), and stance (s). Stride length is the distance between successive placements of the same paw. Base of support (BOS) is the average width between either the front or hind paws. Stance (s) is the duration of the paw in contact with the glass plate during a step cycle. Swing (s) or swing phase is the duration of no contact of a paw with the glass plate in a step cycle. (c) The swing (s) of left hind paws (LH-swing) 6 weeks post SCI. (d) The stance (s) of left hind paws (LH-stance) 6 weeks post SCI. (e) The stride length of left hind paws (LH-stride length) 6 weeks post SCI. (f) The base of support (BOS) of hind paws (H-BOS) 6 weeks post SCI. *P<0.05, **P<0.01, compared with unimmunized SCI group; #P<0.05, ##P<0.01, compared with NgR-PirB IM group (n=6 per group)

Article Snippet: After incubation with a blocking buffer (10% normal goat serum, 0.3% Triton×100 in 0.01 M PBS) for 1 hour at room temperature, sections reacted with primary antibodies, including a rabbit anti-NgR polyclonal antibody (1:100, Santa Crue), a mouse anti-PirB monoclonal antibody (1:100, Sino Biological), or a rabbit anti-GAP-43 polyclonal antibody (1:100, Santa Crue) at 4°C overnight, and then with FITC conjugated goat anti-rabbit antibody or Cy3 conjugated goat anti-mouse antibody (1:100, Sigma) for 1 hour at 37°C.

Techniques: Functional Assay, Negative Control

Journal: Neurotherapeutics

Article Title: Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats

doi: 10.1007/s13311-019-00718-3

Figure Lengend Snippet: Vaccination promoted axon regeneration following SCI. (a) Staining of BDA-labeled axons in sagittal sections caudal to the lesion site 6 weeks post SCI. Scale bar, 100 μm. (b) NY-labeled neurons of transverse sections of the spinal cords at levels rostral to the lesion site 6 weeks post SCI. Scale bar, 100 μm. (c) Quantifications of mean optical density of BDA-labeled axons in sagittal sections caudal to the lesion site 6 weeks post SCI. (d) Quantifications of fluorescence intensity in neurons labeled with NY at sagittal sections rostral to the lesion site 6 weeks post SCI. **P<0.01, compared with unimmunized SCI group; ##P<0.01, compared with the NgR-PirB IM group (n=6 per group)

Article Snippet: After incubation with a blocking buffer (10% normal goat serum, 0.3% Triton×100 in 0.01 M PBS) for 1 hour at room temperature, sections reacted with primary antibodies, including a rabbit anti-NgR polyclonal antibody (1:100, Santa Crue), a mouse anti-PirB monoclonal antibody (1:100, Sino Biological), or a rabbit anti-GAP-43 polyclonal antibody (1:100, Santa Crue) at 4°C overnight, and then with FITC conjugated goat anti-rabbit antibody or Cy3 conjugated goat anti-mouse antibody (1:100, Sigma) for 1 hour at 37°C.

Techniques: Staining, Labeling, Fluorescence

Journal: Neurotherapeutics

Article Title: Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats

doi: 10.1007/s13311-019-00718-3

Figure Lengend Snippet: Histological analyses of spinal cord sections after SCI. (a) HE staining of the sections of spinal cord segment containing the rostrocaudal extension of the lesion cavity. Trabeculated cysts (*) in the left portion of the cord containing foamy macrophages; spared nervous tissue with microcysts (+). Scale bar, 100 μm. (b) The transverse sections 3-6 mm rostral to the injury site were analyzed for residual ventral horn motoneurons by Nissl stain. In the pcDNA-GMCSF-NgR-PirB immunized group, the majority of neurons presented undisturbed morphology (arrows), and some in the pcDNA-NgR-PirB immunized group. However, transverse sections taken from the SCI group were associated with the presence of ischemic neurons (arrowheads) in ventral horn rostral to the lesion site as well as the GMCSF and Blank IM group. Scale bar, 100 μm. (c) Quantifications for the percentage of spared spinal cord tissue in different groups by HE stain. (d) Quantifications for the number of surviving ventral horn neurons in spinal cord tissue of different groups by Nissl stain. *P<0.05, **P<0.01, compared with SCI; #P<0.05, ##P<0.01, compared with NgR-PirB IM group (n=6 per group)

Article Snippet: After incubation with a blocking buffer (10% normal goat serum, 0.3% Triton×100 in 0.01 M PBS) for 1 hour at room temperature, sections reacted with primary antibodies, including a rabbit anti-NgR polyclonal antibody (1:100, Santa Crue), a mouse anti-PirB monoclonal antibody (1:100, Sino Biological), or a rabbit anti-GAP-43 polyclonal antibody (1:100, Santa Crue) at 4°C overnight, and then with FITC conjugated goat anti-rabbit antibody or Cy3 conjugated goat anti-mouse antibody (1:100, Sigma) for 1 hour at 37°C.

Techniques: Staining, H&E Stain

Journal: Neurotherapeutics

Article Title: Nucleic Acid Vaccine Targeting Nogo-66 Receptor and Paired Immunoglobulin-Like Receptor B as an Immunotherapy Strategy for Spinal Cord Injury in Rats

doi: 10.1007/s13311-019-00718-3

Figure Lengend Snippet: Anti-NgR antibody, anti-PirB antibody and anti-GAP-43 antibody immunoreactivity in the injured spinal cords of control, immunized, and SCI rats. (a) Anti-NgR antibody immunoreactivity. Scale bar, 100 μm. (b) Anti-PirB antibody immunoreactivity. Scale bar, 100 μm. (c) Anti-GAP-43 antibody immunoreactivity. Scale bar, 100 μm. Sagittal sections of the spinal cord showed that the NgR (a) and PirB (b) immunoreactivity of GMCSF-NgR-PirB nucleic acid vaccine immunized rats was lower, and the expression of GAP-43 (c) in GMCSF-NgR-PirB nucleic acid vaccine immunized rats was much higher than that of the spinal cord-injured rats sacrificed at 6 weeks after SCI. The same change was found in the GMCSF-NgR-PirB IM group compared with the NgR-PirB IM group (a, b, c). (d) Quantifications of NgR, PirB and GAP-43 immunoreactivity in control, immunized, and SCI rats. *P<0.05, **P<0.01, compared with the SCI group; #P<0.05, ##P<0.01, compared with the NgR-PirB IM group (n=6 per group)

Article Snippet: After incubation with a blocking buffer (10% normal goat serum, 0.3% Triton×100 in 0.01 M PBS) for 1 hour at room temperature, sections reacted with primary antibodies, including a rabbit anti-NgR polyclonal antibody (1:100, Santa Crue), a mouse anti-PirB monoclonal antibody (1:100, Sino Biological), or a rabbit anti-GAP-43 polyclonal antibody (1:100, Santa Crue) at 4°C overnight, and then with FITC conjugated goat anti-rabbit antibody or Cy3 conjugated goat anti-mouse antibody (1:100, Sigma) for 1 hour at 37°C.

Techniques: Expressing