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Targeted Delivery of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand to Keratinocytes with a Pemphigus mAb  Michiyoshi Kouno, Chenyan Lin, Norman.

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Presentation on theme: "Targeted Delivery of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand to Keratinocytes with a Pemphigus mAb  Michiyoshi Kouno, Chenyan Lin, Norman."— Presentation transcript:

1 Targeted Delivery of Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand to Keratinocytes with a Pemphigus mAb  Michiyoshi Kouno, Chenyan Lin, Norman M. Schechter, Don Siegel, Xiaoping Yang, John T. Seykora, John R. Stanley  Journal of Investigative Dermatology  Volume 133, Issue 9, Pages (September 2013) DOI: /jid Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Recombinant production and immunofluorescent analysis of anti-desmoglein (Dsg) single-chain variable fragment (scFv) fused to green fluorescent protein (GFP). (a) Modular baculovirus vector with cDNA encoding scFv in an SfiI restriction site and enhanced GFP (EGFP) in a NotI restriction site. cDNA encoding histidine 6 (H6) and a hemaggultinin (HA)-epitope tag are on the 3′ end. The polyhedron promoter is a strong promoter in insect cells. The gp67 signal peptide allows secretion of the translated fusion protein. (b) PCR strategy for amplifying cDNA for TRAIL (or any other protein) to insert in the NotI site of the vector. (c) The Px44GFP fusion protein as injected intradermally into human skin in organ culture, which was harvested 18 hours after injection and microscopically examined for GFP fluorescence. GFP is present on the cell surface of keratinocytes. Marker=12μm. (d) The Px44GFP fusion protein was injected in the tail vein of a mouse that was killed 6 hours later. The ear (shown here en face) shows GFP fluorescence on the cell surface of keratinocytes. Marker=10μm. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Biochemical and immunological characterization of Px44–mouse tumor necrosis factor–related apoptosis-inducing ligand (mTRAIL). (a) Western blotting with anti-mTRAIL antibody (left panel), and Coomassie Blue staining (right panel) of SDS-PAGE of Px44-mTRAIL and commercial recombinant mTRAIL (rmTRAIL) protein show bands of expected approximate molecular weights. (b) Size-exclusion chromatography of Px44-mTRAIL. The peak slightly larger than the 150kDa (IgG) marker indicates trimer formation of Px44-mTRAIL. (c) Desmoglein 3 (Dsg3) ELISA by using anti-hemaggultinin (HA) antibody (left panel) and anti-mTRAIL antibody (right panel) shows binding of Px44-mTRAIL to Dsg3. Crbn Anhyd, carbonic anhydride; MOPS, 3-(N-morpholino)propanesulfonic acid; Trnfn, transferrin. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Biological activity of mouse tumor necrosis factor–related apoptosis-inducing ligand (mTRAIL) fused to Px44 determined by flow cytometry. (a) Px44-mTRAIL (with anti-hemaggultinin (HA) antibodies) induced apoptosis of Jurkat cells, as did recombinant mTRAIL (rmTRAIL). (b) Px44-mTRAIL (with anti-HA antibodies) inhibited IFN-γ production of living activated mouse CD4+ T cells. (Dead cells were gated out.) PBS, phosphate-buffered saline; PI, propidium iodide. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Target antigen–specific function of Px44–mouse tumor necrosis factor–related apoptosis-inducing ligand (mTRAIL). (a) Px44-mTRAIL bound to the cell surface of normal human epidermal keratinocytes in low calcium (0.4mM) (left panel), whereas control fusion protein AM3-13-mTRAIL did not (middle panel). Binding of Px44-mTRAIL was detected by anti-hemaggultinin (HA) antibody (left panel) and anti-mTRAIL antibody (right panel). (b) The normal human epidermal keratinocytes in low calcium (0.4mM) were treated with Px44-mTRAIL or AM3-13-mTRAIL. After 2 hours of incubation, cells were washed and then cultured for a further 16 hours, and the cells were analyzed by flow cytometry for apoptosis. Increased cell death was seen in Px44-mTRAIL-treated keratinocytes (upper left) compared with cells treated with AM3-13-mTRAIL. In the presence of recombinant desmoglein (rDsg3) during the 2-hour incubation with fusion proteins, the effect of Px44-mTRAIL-HA treatment was inhibited (lower left). In high calcium (1.2mM), keratinocytes were resistant to Px44-mTRAIL-induced apoptosis. PBS, phosphate-buffered saline; PI, propidium iodide. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Enforced trimerization by foldon enhanced and stabilized the biological activity of Px44–human tumor necrosis factor–related apoptosis-inducing ligand (hTRAIL). (a, b) Flow-cytometric analysis of the apoptosis-inducing activity on Jurkat cells of Px44-foldon-hTRAIL and Px44-hTRAIL after incubation at 37°C (before incubation with the cells). (a) Px44-hTRAIL’s ability to induce apoptosis decreased over time incubated at 37°C (lower panel) compared with Px44-foldon-hTRAIL (upper panel). (b) Px44-foldon-hTRAIL kept its activity even after 48 hours at 37°C (upper panels). The reduction of activity of Px44-hTRAIL was restored by anti-HA cross-linking (lower panels). (c) Size-exclusion chromatography of Px44-foldon-hTRAIL and Px44-hTRAIL before and after 37°C incubation for 48 hours. Px44-hTRAIL before incubation at 37°C and Px44-foldon-hTRAIL formed homotrimers (arrowhead). A lower-molecular-weight peak developed in Px44-hTRAIL after the 48-hour incubation at 37°C (arrow), indicating instability of the homotrimer with dissociation into smaller units. anti-HA, anti-hemaggultinin; PI, propidium iodide. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Px44–mouse tumor necrosis factor–related apoptosis-inducing ligand (mTRAIL) and Px44–foldon–human TRAIL (hTRAIL) diffuse across the basement membrane of mouse and human epidermis to bind to the cell surface of keratinocytes. (a) Px44-mTRAIL injected into neonatal mice binds to the cell surface of hair follicle and basal keratinocytes as detected by anti-hemaggultinin (HA) immunofluorescence. Area in white box is shown closer up to the right of its image. Marker=10 μm. (b) Px44-foldon-hTRAIL injected intradermally into human skin in organ culture binds to the cell surface of keratinocytes as detected by immunofluorescence with anti-HA and anti-hTRAIL. Marker=10μm. (c) Px44-foldon-hTRAIL injected intradermally into human skin xenografts on mice binds to the cell surface of keratinocytes as detected by immunofluorescence with anti-HA antibodies. Area in white box is shown closer up to the right of its image. Marker=10μm. PBS, phosphate-buffered saline. Journal of Investigative Dermatology  , DOI: ( /jid ) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

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