Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells

doi: 10.1016/j.omtm.2021.10.010

Figure Lengend Snippet: AAV-CAR Tregs suppress capsid-specific immune response in vivo (A) Expression of hAAT protein in the serum of animals injected i.m. with rh32.33-AAT followed by AAV-CAR Tregs (red), polyclonal Tregs (orange), or PBS (blue) as measured by ELISA (n = 6 for AAV-CAR Treg and PBS-treated groups, n = 5 for polyclonal Treg-treated group). (B) Anti-rh32.33 antibodies in the serum measured by ELISA. Anti-capsid antibodies increased in all groups over time (n = 6 for AAV-CAR Treg group, n = 7 for PBS group, n = 5 for polyclonal Treg-treated group). (C) Serum level of free active TGF-β1 measured by CBA assay over time. A significant increase in free active TGF-β1 was observed in AAV-CAR Treg group (red) at week 11 post injection compared with the PBS control group (blue). (D) Serum levels of IL-10 measured by CBA assay over time. Two-way ANOVA repeated measure with Tukey's multiple comparisons was used (n = 6 for AAV-CAR Treg and PBS-treated groups, n = 5 for polyclonal Treg treated group for C and D). (E) Quantification of number nuclei in right limb muscles of animals 2 weeks after AAV injection. Each dot represents one animal, which is the average of 10 images per animal (HPF, high-power field). One-way ANOVA, n = 3. Error bars are mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01. (F) Representative images of rh32.33-AAT muscles 2 weeks post injection stained for H&E for cellular infiltration. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. (G) Representative images of rh32.22-AAT-injected muscles stained by immunohistochemistry for AAT protein (brown), 26 weeks after AAV injection. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. ∗AAV-CAR Tregs compared with PBS; #AAV-CAR Tregs compared with polyclonal Tregs; ˆpolyclonal Tregs compared with PBS.

Article Snippet: After washing two times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (Rockland Immunochemicals, Limerick, PA) (1:5,000 diluted) was added and incubated for 1 h at room temperature.

Techniques: In Vivo, Expressing, Injection, Enzyme-linked Immunosorbent Assay, Control, Muscles, Staining, Immunohistochemistry

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells

doi: 10.1016/j.omtm.2021.10.010

Figure Lengend Snippet: AAV-CAR Tregs bystander suppress immune response to AAV-delivered transgene (A) Expression of OVA protein from animals i.m. injected with AAV1-OVA followed by i.v. AAV-CAR Tregs (red), polyclonal Tregs (orange), or PBS (blue) as measured by ELISA. Serum levels of OVA were significantly greater in AAV-CAR Treg-treated animals compared with PBS. (B) Anti-OVA antibodies detected in the serum by ELISA. (C) Anti-AAV1 antibodies in the serum measured by ELISA. (D) Serum levels of IL-10 measured by CBA assay. (E) Serum level of free active TGF-β1 measured by CBA assay over time. Two-way repeated-measure ANOVA with Tukey's multiple comparisons was used (n = 5 for AAV-CAR Treg and polyclonal Treg-treated groups, n = 3 for the PBS treated group for A, B, C, D, and E). (F) Quantification of number of nuclei in i.m. injected muscles. Each dot represents one animal, which is the average of 10 images per animal. (G) Representative images of H&E-stained limb muscles of mice 16 weeks post i.m. injection with AAV1-OVA. Substantial cellular infiltration (blue) was observed in animals treated with PBS or polyclonal Tregs. (Upper) scale bar, 527 μm; (lower) scale bar, 131 μm. one-way ANOVA, n = 2. Error bars are mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. ∗AAV-CAR Tregs compared with PBS.-CAR Tregs compared with PBS.

Article Snippet: After washing two times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (Rockland Immunochemicals, Limerick, PA) (1:5,000 diluted) was added and incubated for 1 h at room temperature.

Techniques: Expressing, Injection, Enzyme-linked Immunosorbent Assay, Muscles, Staining

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Modulating immune responses to AAV by expanded polyclonal T-regs and capsid specific chimeric antigen receptor T-regulatory cells

doi: 10.1016/j.omtm.2021.10.010

Figure Lengend Snippet: Injection of AAV-CAR Tregs reduced IFN-γ and increased IL-10 production in muscular but not splenic immune cells after ex vivo isolation and restimulation (A) Experimental schematic. (B) Levels of IFN-γ in muscle after ex vivo isolation and restimulation measured by CBA assay. (C) Levels of IL-4 in muscle after ex vivo isolation and restimulation measured by CBA assay. (D) Levels of IL-10 in muscle after ex vivo isolation and restimulation measured by CBA assay. (E) Levels of free active TGF-β1 in muscle after ex vivo isolation and restimulation measured by CBA assay. Two-way ANOVA with Tukey's multiple comparisons was used. Error bars are mean ± SEM; ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001 (n = 3 for AAV-CAR Treg and polyclonal Treg-treated groups, n = 2 for the PBS-treated group for B, C, D and E).

Article Snippet: After washing two times, peroxidase-conjugated rabbit anti-OVA polyclonal antibody (Rockland Immunochemicals, Limerick, PA) (1:5,000 diluted) was added and incubated for 1 h at room temperature.

Techniques: Injection, Ex Vivo, Isolation

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: Awd expression in tracheal cells. (A) Affinity-purified rabbit polyclonal antibody against Drosophila Awd recognizes a single band by Western blotting in whole embryo and Drosophila S2 cell lysates. (B) The same antibody was used to detect Awd expression (red) in vivo. The genotypes of the embryos are 1-eve-1 (wt); y, w; 1-eve-1, awdj2A4 (awd); and y, w; btl-GAL4, UAS-awd; 1-eve-1, awdj2A4 (UAS-awd + awd). Tracheal cells were visualized by mouse monoclonal anti-β-Gal staining (green). Arrows in the top left panel mark the anterior (a) and dorsal (d) sides of all the embryos shown. The top two rows of images are projections of five 1.5-μm confocal sections and the close-up images are single sections. The bottom two rows are projections of five 2-μm sections. The two channels were recorded sequentially to avoid bleed-through.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Expressing, Affinity Purification, Western Blot, In Vivo, Staining

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: awd mutant alleles display distinct tracheal phenotypes. Embryos were collected at 25°C and double stained using the 2A12 monoclonal antibody against a lumen antigen and a FITC-conjugated polyclonal antibody against β-Gal (red) (A–I), or double stained using the rat polyclonal anti-Trh antibody (green) and mouse monoclonal anti-β-GAL antibody (J–H). btl and the three awd mutants are homozygotes (see Materials and Methods). Stage 15–16 embryos are shown with anterior to the left. (A) Lateral view of a y, w embryo, representing wild type. Branches relevant to the following figures are marked. (DT) Dorsal trunk; (LT) lateral trunk; (DB) dorsal branch. (B) Dorsal view of a y, w embryo. (C) Lateral view of a btl embryo. There is very little tubule formation. (D) Lateral view of an awdj2A4 embryo. There is a general disruption of the tracheal network with ectopic branches emanating from the lateral trunk (marked between arrows in the enlarged section). Also, additional branches are seen sprouting out of the lateral region (bracket). (E) Dorsal–lateral view of an awdj2A4 embryo. Two ectopic branches are shown (arrowheads). (F) Lateral view of an awdKRS6 embryo. An ectopic “bulge” is indicated (arrowhead). (G) Dorsal–lateral view of an awdKRS6 embryo. The region exhibiting abnormal branching and looping is highlighted. (H) Dorsal–lateral view of an awdKRB embryo. One dorsal branch (DB) shows an ectopic loop (arrowhead) and two other dorsal branches form abnormal axial connection (arrow). (I) Dorsal–lateral view of an awdKRB embryo. The region exhibits ectopic branching and looping is indicated by a bracket. Also the dorsal trunk (DT) shows an abnormal 90° turn (arrow). (J) Lateral view of a y, w embryo showing nuclear expression of the Trh protein in all tracheal cells. (K) Lateral view of an awdj2A4 embryo. Arrows point to dorsal trunk (DT) cells that migrate ectopically (misallocated), resulting in gaps in dorsal trunk. (L) Lateral view of an awdKRS6 embryo. Arrows point to misallocated dorsal trunk (DT) cells and ectopic connection of two dorsal branches (DB). White bars, 50 μm; yellow bars, 20 μm.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Staining, Expressing

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: awd mutant exhibits ectopic tracheal cell migration in contrast to the btl phenotype. All embryos were collected at 25°C and stained with mouse monoclonal anti-β-Gal antibody. Identification of homozygous and heterozygous awd and btl is described in Materials and Methods. Unless noted, anterior is to the left and dorsal side is up. (A) Lateral view of a wild-type stage 14 embryo. One tracheal subunit is highlighted. Three tracheal branches relevant in the following figures are marked. (DB) Dorsal branches; (DT) dorsal trunk; (TC) transverse connectives. (B) Dorsal–lateral view of a wild-type stage 17 embryo showing fully extended tracheal tubes. (C) A dorsal close-up view of a wild-type stage 16 embryo showing connection of two dorsal branches across the dorsal midline (arrow). (D) Dorsal view of a stage 16 homozygous awdj2A4 embryo showing disrupted dorsal trunk (DT) and sprouts of ectopic branches (sharp arrows). The first compartment of the properly formed midgut can be seen in this view (the light, rounded internal structure in the middle). (E) Dorsal–lateral view of a stage 17 heterozygous awdj2A4 embryo. A segment of the dorsal trunk moves ventrally into the position of transverse connective (bracket) instead of connecting with the lateral neighbor. Ectopic small cellular processes are seen sprouting from the abnormal branches (sharp arrows in the insert). (F) Dorsal–lateral view of a stage 16 heterozygous awdj2A4 embryo. The highlighted region shows two dorsal branches fused at the base but separate at the tip (asterisk) as well as misconnection of two neighboring dorsal branches (sharp arrow), instead of crossing the dorsal midline. (G) Close-up view of dorsal branches of a wild-type embryo. Arrows point to the few filopodia extending from the tip cells of the dorsal branches. (H) Close-up view of dorsal branches of a heterozygous awdj2A4 embryo. Multiple, random cellular projections extend from the tip cells (sharp arrows). (I) Lateral view of a stage 14 homozygous btlH82Δ3 embryo. There is very little tubule migration as compared to the wild type. (J) Dorsal–lateral view of a stage 16 heterozygous btlH82Δ3 embryo. The highlighted region shows lack of dorsal branch formation. (K) Quantitation of the recorded phenotypes as shown in A–J. The awd and btl alleles are awdj2A4 and btlH82Δ3. Note that to avoid complications from early embryonic patterning defects, only embryos older than stage 13 that showed proper germband retraction and, after stage 15, proper gut formation, were tabulated. Construction of the btl-GAL4, UAS-awd rescue chromosome is described in Materials and Methods.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Migration, Staining, Quantitation Assay

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: shi mutant shows migration phenotype similar to awd. Progenies of shi1 homozygous females crossed with 1-eve-1 males (A–D) and of shi1 homozygous females crossed with 1-eve-1, awdj2A4/TM3, P{ry + t7.2 = HZ2.7}DB2, Sb males (E). The embryos were collected at 25°C for 7 h, then incubated for 7 h at 34°C and stained with anti-β-Gal antibody. Unless indicated, anterior is to the left and dorsal side is up. (A) Dorsal–lateral view. A sharp arrow points to the ectopic branching event. Also, abnormal cellular projections are seen extending from the dorsal branch tip cells (arrows in the inset). (B) Lateral view. A segment of the dorsal trunk is seen moving ventrally (bracket) into the position normally occupied by transverse connectives, instead of anteriorly to connect with the lateral neighbor. (C) Dorsal–lateral view. A group of tracheal cells move dorsally (bracket) at the expense of forming a proper dorsal trunk connection. Ectopic cellular extensions are also visible (arrow). (D) Lateral view. An extra loop of dorsal trunk is formed (bracket) with ectopic small projections (arrows). (E) Dorsal view of severely disrupted trachea. The enlarged section shows ectopic cellular projections and randomly migrating tracheal cells (arrows). (F) Quantitation of the recorded phenotypes as shown in A–E. There is a nearly 30-fold increase in the most severe phenotype (E) when shi is combined with heterozygous awd mutant. Only embryos older than stage 13 that appear normal in segmental patterns were tabulated as described in Figure 3K.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Migration, Incubation, Staining, Quantitation Assay

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: Overaccumulation of Btl/FGFR in awd mutant. The genotypes of the indicated embryos are y, w; btl-GAL4/+; 1-eve-1, UAS-btl-GFP/+ (wt) and y, w; btl-GAL4/+; 1-eve-1, UAS-btl-GFP, awdj4A2/+ (awd/+). Embryos were double stained with anti-β-Gal (red) and anti-GFP (green) antibodies. The two channels were recorded sequentially. Anterior is to the left. (A) Dorsal–lateral view of a wild-type stage 14 embryo. A projection of five 2-μm confocal sections. The Btl-GFP chimeric protein is barely detectable. In the close-up image (single confocal section), circles mark individual tracheal cells and sharp arrows point to examples of internalized Btl-GFP. (B) Lateral view of portion of a wild-type stage 12 tracheal subunit. Circles mark individual tracheal cells and sharp arrows point to examples of internalized Btl-GFP. Single confocal section. (C) Single confocal section of a wild-type stage 15 dorsal branch tip cell. Sharp arrows point to examples of internalized Btl-GFP. (D) Stage 13 awd/+ tracheal subunits. A projection of five 1-μm confocal sections. In the close-up image, arrows point to examples of large aggregates of Btl-GFP. (E) Lateral view of three dorsal branches from a stage 15 awd/+ embryo. A projection of five 1-μm confocal sections. Abnormal migration (sharp arrows), ectopic cellular projections (asterisk), and high level of Btl-GFP are seen. In the close-up images (single confocal section), Btl-GFP is seen outlining a very fine cellular projection (bracket). Note that the close-up section is only partially included in the projected image. (F) Dorsal view of a stage 17 awd/+ embryo. A projection of five 2-μm confocal sections. The total disruption of the tracheal system is rarely seen in the awd heterozygotes. The properly formed midgut can also be seen beneath the abnormal tracheal branches (red auto-fluorescence). The enlarged view (GFP stain only) shows high level of Btl-GFP expression and ectopic cellular projections (arrows). (G) Western blot quantitation of the Btl-GFP accumulation. Embryonic extracts from awd/+ and wt embryos as described above were Western blotted and probed with antibodies indicated. Two independent lines of 1-eve-1, UAS-btl-GFP, awdj2A4 were examined. Because the btl-GFP and the 1-eve-1 transgenes are located on the same chromosome, β-Gal was used as a loading control. Btl-GFP is up-regulated by approximately fivefold whereas there is no concomitant increase in EGFR (a slight decrease, in fact) or the total MAPK levels in the awd mutant.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Mutagenesis, Staining, Migration, Fluorescence, Expressing, Western Blot, Quantitation Assay

Journal:

Article Title: Drosophila awd , the homolog of human nm23 , regulates FGF receptor levels and functions synergistically with shi/dynamin during tracheal development

doi: 10.1101/gad.1096903

Figure Lengend Snippet: Ectopic activation of MAPK in awd mutant. 1-eve-1 (wt) and awdj2A4, 1-eve-1/1-eve-1 (awd/+) stage 12 and stage 15 embryos were double stained with anti-β-Gal-FITC (green) and anti-dp-MAPK (red) antibodies. The two channels were recorded sequentially. Anterior is to the left. (A) Tracheal subunits of a stage 12 wild-type embryo. A projection of five 1.5-μm confocal sections. Within the tracheal placodes, activated MAPK are detected in the two tips cells marked by circles. Lighter MAPK activation is also detected on the opposite tip (arrow). Activated MAPK-expressing cells surrounding the tracheal placodes are of mesodermal origin, including cardiac precursors. (B) One dorsal branch of a wild-type stage 15 embryo. A single confocal section. Only one cell at the tip contains activated MAPK. (C) Tracheal subunits of a stage 12 awd heterozygote. A projection of five 1.5-μm confocal sections. Multiple cells besides the migrating tips within the tracheal placodes express activated MAPK (arrows). (D) Two laterally fused dorsal branches of a stage 15 awd heterozygote. A projection of five 1.5-μm confocal sections. The stalks (brackets) ectopically express activated MAPK. Bars, 10 μm.

Article Snippet: Rabbit polyclonal anti-β-Gal-FITC conjugate (Rockland; at 1:500) was used for fluorescence costaining with anti-dp-MAPK.

Techniques: Activation Assay, Mutagenesis, Staining, Expressing