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• W2039533218 abstract "Reorganization of skin during wound healing, inflammatory disorders, or cancer growth is the result of expression changes of multiple genes associated with tissue morphogenesis. We wanted to identify proteins involved in skin remodeling and select those that may be targeted for agonistic or antagonist therapeutic approaches in various disease processes. Full-thickness human skin was grafted to severe combined immunodeficient mice and injected intradermally with 38 different adenoviral vectors inserted with 37 different genes coding for growth factors, cytokines, proteolytic enzymes and their inhibitors, adhesion receptors, oncogenes, and tumor suppressor genes. Responses were characterized for infiltration of inflammatory cells, vascular density, matrix formation, fibroblast-like cell proliferation, and epidermal hyperplasia. Of the 17 growth factor vectors, 16 induced histological changes in human skin. Members of the VEGF and angiopoietin families induced neovascularization. PDGFs and TGF-βs stimulated connective tissue formation, and the chemokines IL-8 and MCP-1 attracted inflammatory neutrophils and monocytes, respectively. The serine protease uPA induced a vascular response similar to that of VEGF. Vectors with adhesion receptors, oncogenes and tumor suppressor genes had, with few exceptions, little effects on skin architecture. The overall results suggest that adenoviral vectors can effectively remodel the architecture of human skin for studies in morphogenesis, inflammatory skin disorders, wound healing, and cancer development. Reorganization of skin during wound healing, inflammatory disorders, or cancer growth is the result of expression changes of multiple genes associated with tissue morphogenesis. We wanted to identify proteins involved in skin remodeling and select those that may be targeted for agonistic or antagonist therapeutic approaches in various disease processes. Full-thickness human skin was grafted to severe combined immunodeficient mice and injected intradermally with 38 different adenoviral vectors inserted with 37 different genes coding for growth factors, cytokines, proteolytic enzymes and their inhibitors, adhesion receptors, oncogenes, and tumor suppressor genes. Responses were characterized for infiltration of inflammatory cells, vascular density, matrix formation, fibroblast-like cell proliferation, and epidermal hyperplasia. Of the 17 growth factor vectors, 16 induced histological changes in human skin. Members of the VEGF and angiopoietin families induced neovascularization. PDGFs and TGF-βs stimulated connective tissue formation, and the chemokines IL-8 and MCP-1 attracted inflammatory neutrophils and monocytes, respectively. The serine protease uPA induced a vascular response similar to that of VEGF. Vectors with adhesion receptors, oncogenes and tumor suppressor genes had, with few exceptions, little effects on skin architecture. The overall results suggest that adenoviral vectors can effectively remodel the architecture of human skin for studies in morphogenesis, inflammatory skin disorders, wound healing, and cancer development. Human skin has a unique architecture and is very different from skin of laboratory animals, including mice. It has a multilayered epidermis with keratinocytes, melanocytes, and Langerhans cells, and a thick, well-vascularized dermis. By comparison, the mouse skin has only 3–4 keratinocyte layers and a thin dermis. Studies of tissue reorganization in wound healing, cancer and inflammation are difficult to compare between mice and humans. We therefore developed a human skin/SCID mouse chimera model in which full-thickness human skin is grafted to immunodeficient mice (Juhasz et al., 1993Juhasz I. Albelda S.M. Elder D.E. et al.Growth and invasion of human melanomas in human skin grafted to immunodeficient mice.Am J Pathol. 1993; 143: 528-537PubMed Google Scholar). The human epidermis remains unchanged, whereas the dermis becomes infiltrated by murine blood vessels and some murine inflammatory cells (Yan et al., 1993Yan H.C. Juhasz I. Pilewski J. Murphy G.F. Herlyn M. Albelda S.M. Human/severe combined immunodeficient mouse chimeras: An experimental in vivo model system to study the regulation of human endothelial cell-leukocyte adhesion molecules.J Clin Invest. 1993; 91: 986-996Crossref PubMed Scopus (96) Google Scholar). The human skin graft maintains its unique architecture throughout the lifetime of the animal, and it has successfully been used for studies of wound healing (Juhasz et al., 1993Juhasz I. Albelda S.M. Elder D.E. et al.Growth and invasion of human melanomas in human skin grafted to immunodeficient mice.Am J Pathol. 1993; 143: 528-537PubMed Google Scholar;Sylvester et al., 2000Sylvester K.G. Nesbit M. Radu A. Herlyn M. Adzick N.S. Crombleholme T.M. Adenoviral-mediated gene transfer in wound healing: Acute inflammatory response in human skin in the SCID mouse model.Wound Repair Regen. 2000; 8: 36-44Crossref PubMed Google Scholar), inflammation (Oka et al., 2000Oka M. Berking C. Nesbit M. et al.Interleukin-8 overexpression is present in pyoderma gangrenosum ulcers and leads to ulcer formation in human skin xenografts.Lab Invest. 2000; 80: 595-604Crossref PubMed Scopus (97) Google Scholar), carcinogenesis (Atillasoy et al., 1998Atillasoy E.S. Seykora J.T. Soballe P.W. Elenitsas R. Nesbit M. Elder D.E. Montone K.T. Sauter E. Herlyn M. UVB induces atypical melanocytic lesions and melanoma in human skin.Am J Pathol. 1998; 152: 1179-1186PubMed Google Scholar), tumor growth and metastasis (Sauter et al., 1999aSauter E.R. Nesbit M. Watson J.C. Klein-Szanto A. Litwin S. Herlyn M. Vascular endothelial growth factor is a marker of tumor invasion and metastasis in squamous cell carcinomas of the head and neck.Clin Cancer Res. 1999; 5: 775-782PubMed Google Scholar). Increasingly, this model is being used for pre-clinical studies to predict the outcome in human skin following therapeutic intervention. Pathological changes in skin are associated with profound changes in expression of a variety of genes that are associated with skin morphogenesis. Correlative studies testing for protein or mRNA expression have provided information of potentially important agents in skin remodeling processes. Injection of recombinant proteins into skin or their topical application to wounds has provided preliminary information on their potential for wound repair and skin remodeling. However, recombinant proteins are rapidly degraded and need frequent reapplications to have lasting effects. Therefore, gene therapy approaches are attractive alternatives for studies of protein functions. Adenoviral vectors readily infect most human skin cells and are widely accepted for efficient gene transduction (Setoguchi et al., 1994Setoguchi Y. Jaffe H.A. Danel C. Crystal R.G. Ex vivo and in vivo gene transfer to the skin using replication-deficient recombinant adenovirus vectors.J Invest Dermatol. 1994; 102: 415-421Abstract Full Text PDF PubMed Google Scholar). Adenovirus-mediated gene transfer results in protein expression within 24 h with a maximum at 72 h (Berking et al., 2001aBerking C. Takemoto R. Satyamoorthy K. Elenitsas R. Herlyn M. Basic fibroblast growth factor and UVB transform melanocytes in human skin.Am J Pathol. 2001; 158: 943-953Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, Berking et al., 2001bBerking C. Takemoto R. Schaider H. Showe L. Satyamoorthy K. Robbins P. Herlyn M. TGF-ß1 increases survival of human melanoma through stroma remodeling.Cancer Res. 2001; 61: 8306-8316PubMed Google Scholar;Nesbit et al., 2001aNesbit M. Schaider H. Andl T. et al.α5-Integrin gene transfer mimics PDGF-B transformed phenotypes in human dermal fibroblasts and human skin.Lab Invest. 2001; 166: 6483-6490Google Scholar;Nesbit et al., 2001bNesbit M. Schaider H. Miller T. Herlyn M. Low level MCP-1 stimulation of monocytes leads to tumor formation in non-tumorigenic melanoma cells.J Immunol. 2001; 199: 6483-6490Crossref Scopus (196) Google Scholar). Depending on the proliferation of transduced cells and a potential immune response, forced gene expression in human skin grafts may last up to 3 weeks. Wound healing, cutaneous inflammatory disorders such as psoriasis and scleroderma, or skin cancer formation are complex processes that are difficult to dissect into gene expression changes that cause tissue remodeling. However, investigations of these conditions are often hampered by the lack of adequate animal models, where single parameters can be experimentally modified. In this study, we have transduced human skin grafted to SCID mice with adenoviral vectors without prior wounding. We chose human skin for injection of adenoviral vectors instead of mouse skin because the mouse dermis is generally too thin for even small quantities of fluid requiring subdermal or subcutaneous deposition of the vector(s). The proteins under study were selected for their potential involvement in skin morphogenesis, wound healing, inflammation, and cancer. The full-thickness well-healed skin grafts were injected with adenoviral vectors for growth factors, chemokines, proteolytic enzymes, enzyme inhibitors, adhesion receptors, proto-oncogenes, tumor suppressor genes, several antagonists to growth factors, growth factor receptors or a receptor for a protease. The resulting remodeling of human skin demonstrates the utility of the presented approach for further in-depth studies of a variety of dermatological conditions. Inflammation, neovascularization, and connective tissue formation could be induced. These pathobiological reaction patterns are hallmarks of skin diseases such as psoriasis and scleroderma. The endogenous overexpression of genes produced strong responses. Serotype 5, replication defective adenovirus vectors were used in this study. The backbone of adenoviruses had either E1 or E1 and E3 deleted regions and all the transgenes were driven by the cytomegalyvirus promoter. The parental adenoviruses include pMJ17 (E1 deleted), d17001, Adeasy-1 and in340 (E1 and E3 deleted) viruses. The viruses included in the study are listed in Table 1. All viruses were prepared from 293 cells, purified by double CsCl density centrifugation (Graham and Prevec, 1995Graham F.L. Prevec L. Methods for construction of adenovirus vectors.Mol Biotechnol. 1995; 3: 207-220Crossref PubMed Scopus (427) Google Scholar) and stored in 50% glycerol containing 10 mM Tris, pH 8.0, 0.1 mM EDTA, and 0.1% BSA at – 80°C. Virus titers were determined by measuring absorbance at 260 nm and by plaque assay as described before (Satyamoorthy et al., 1997Satyamoorthy K. Soballe P.W. Soans F. Herlyn M. Adenovirus infection enhances killing of melanoma cells by a mitotoxin.Cancer Res. 1997; 57: 1873-1876PubMed Google Scholar). Virus preparations were dialyzed extensively against phosphate buffered saline before injection into animals. Expressions of all transgenes were measured as previously described (see references in Table 1) by Western analysis, ELISA, RIA, or Northern analysis wherever applicable. All animal experiments were approved by the IACUC (institutional review board for all animal experiments).Table 1Adenoviral Vectors For Over-Expression of Proteases, Inhibitors, Adhesion Receptors, Oncogenes, Tumor Suppressor Genes, and Miscellaneous Human GenesaCMV promoter was used for all adenoviral constructs.Gene groupAbbreviationAdenovirusDescriptionReference (source)cReferences refer to characterization of the adenoviral vector.Growth factors and cytokinesbAll genes were of human origin unless otherwise indicated.Basic fibroblast growth factor (18 kDa)bFGFdl7001E1−, E3−, Ad5Nesbit et al., 1999Nesbit M. Nesbit H.K. Bennett J. et al.Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes.Oncogene. 1999; 18: 6469-6476Crossref PubMed Scopus (97) Google Scholar,Berking et al., 2001aBerking C. Takemoto R. Satyamoorthy K. Elenitsas R. Herlyn M. Basic fibroblast growth factor and UVB transform melanocytes in human skin.Am J Pathol. 2001; 158: 943-953Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarBasic fibroblast growth factor (18, 22, 23, 24 kDa)bFGF(l)dl7001E1−, E3−, Ad5Nesbit et al., 1999Nesbit M. Nesbit H.K. Bennett J. et al.Basic fibroblast growth factor induces a transformed phenotype in normal human melanocytes.Oncogene. 1999; 18: 6469-6476Crossref PubMed Scopus (97) Google Scholar,Berking et al., 2001aBerking C. Takemoto R. Satyamoorthy K. Elenitsas R. Herlyn M. Basic fibroblast growth factor and UVB transform melanocytes in human skin.Am J Pathol. 2001; 158: 943-953Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarPlatelet-derived growth factor APDGF-Adl7001E1−, E3−, Ad5(Herlyn Lab)Platelet-derived growth factor BPDGF-Bdl7001E1−, E3−, Ad5Liechty et al., 1999Liechty K.W. Nesbit M. Herlyn M. Radu A. Adzick N.S. Crombleholme T.M. Adenoviral-mediated overexpression of platelet-derived growth factor-B corrects ischemic impaired wound healing.J Invest Dermatol. 1999; 113: 375-383Crossref PubMed Scopus (119) Google Scholar;Nesbit et al., 2001aNesbit M. Schaider H. Andl T. et al.α5-Integrin gene transfer mimics PDGF-B transformed phenotypes in human dermal fibroblasts and human skin.Lab Invest. 2001; 166: 6483-6490Google ScholarInsulin-like growth factor-1IGF-1AdEasy-1E1−, E3−, Ad5Satyamoorthy et al., 2001aSatyamoorthy K. Li G. Vaidya B. Patel D. Herlyn M. IGF-1 induces survival and growth in biologically early melanoma cells through both the MAP kinase and ß-catenin pathways.Cancer Res. 2001; 61: 7318-7324PubMed Google Scholar, Satyamoorthy et al., 2001bSatyamoorthy K. Muyers J.P. Meier F.E. Patel D. Herlyn M. Mel-CAM-specific genetic suppressor elements inhibit growth and invasion through loss of gap junctional communication.Oncogene. 2001; 20: 4676-4684Crossref PubMed Scopus (68) Google ScholarMonocyte chemoattractant protein-1MCP-1dl7001E1−, E3−, Ad5Nesbit et al., 2001bNesbit M. Schaider H. Miller T. Herlyn M. Low level MCP-1 stimulation of monocytes leads to tumor formation in non-tumorigenic melanoma cells.J Immunol. 2001; 199: 6483-6490Crossref Scopus (196) Google ScholarInterleukin-8IL-8dl7001E1−, E3−, Ad5Oka et al, 1999Vascular endothelial cell growth factor-121VEGFdl7001E1−, E3−, Ad5Herlyn LabVascular endothelial cell growth factor CVEGF-CAdEasy-1E1−, E3−, Ad5(Herlyn/Detmar Labs.)Vascular endothelial cell growth factor DVEGF-DAdEasy-1E1−, E3−, Ad5(Herlyn/Detmar Labs.)PleiotrophindReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.Pleiotrophindl7001E1−, E3−, Ad5Satyamoorthy et al., 2000Satyamoorthy K. Oka M. Herlyn M. An antisense strategy for inhibition of human melanoma growth targets the growth factor pleiotrophin.Pigment Cell Res Supplement. 2000; 8: 87-93Crossref Scopus (21) Google ScholarTransforming growth factor beta-1, active porcineTGF-β1E1−, E3−, Ad5Lee et al., 1998Lee W.C. Zhong C. Qian S. et al.Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-beta.Transplantation. 1998; 66: 1810-1817Crossref PubMed Scopus (98) Google Scholar,Berking et al., 2001aBerking C. Takemoto R. Satyamoorthy K. Elenitsas R. Herlyn M. Basic fibroblast growth factor and UVB transform melanocytes in human skin.Am J Pathol. 2001; 158: 943-953Abstract Full Text Full Text PDF PubMed Scopus (66) Google ScholarTransforming growth factor beta-3TGF-β3AdEasy-1E1−, E3−, Ad5(Cromblehome Lab.)Hepatocyte growth factor/Scatter factorHGF/SFdl7001E1−, E3−, Ad5Phaneuf et al., 2000Phaneuf D. Chen S.J. Wilson J.M. Intravenous injection of an adenovirus encoding hepatocyte growth factor results in liver growth and has a protective effect against apoptosis.Mol Med. 2000; 6: 96-103Crossref PubMed Google ScholarAngiopoietin-1Ang-1in340E1−, E3−, Ad5RegeneronEndothelin-3ET-3AdEasy-1E1−, E3−, Ad5(Herlyn Lab)Stem cell factorSCFAdEasy-1E1−, E3−, Ad5(Herlyn Lab)Proteolytic enzymes, inhibitors, and antagonistsMatrix metalloproteinase-9MMP-9dl7001E1−, E3-Ad5(Herlyn Lab.)Urokinase type plasminogen activatoruPApJM17E1−, Ad5Nagai et al., 1999Nagai N. De Mol M. Lijnen H.R. Carmeliet P. Collen D. Role of plasminogen system components in focal cerebral ischemic infarction: A gene targeting and gene transfer study in mice.Circulation. 1999; 99: 2440-2444Crossref PubMed Scopus (207) Google ScholarPlasminogen activator inhibitor-1PAI-1pJM17E1−, Ad5Carmeliet et al., 1997Carmeliet P. Moons L. Lijnen R. Janssens S. Lupu F. Collen D. Gerard R.D. Inhibitory role of plasminogen activator inhibitor-1 in arterial wound healing and neointima formation: A gene targeting and gene transfer study in mice.Circulation. 1997; 96: 3180-3191Crossref PubMed Scopus (219) Google ScholarAntisense urokinase type plasminogen activator receptorASuPARpJM17E1−, Ad5Mohan et al., 1999Mohan P.M. Chintala S.K. Mohanam S. et al.Adenovirus-mediated delivery of antisense gene to urokinase-type plasminogen activator receptor suppresses glioma invasion and tumor growth.Cancer Res. 1999; 59: 3369-3373PubMed Google ScholarTissue inhibitor of matrix metalloproteinase-1TIMP-1pJM17E1−, Ad5Baker et al., 1996Baker A.H. Wilkinson G.W. Hembry R.M. Murphy G. Newby A.C. Development of recombinant adenoviruses that drive high level expression of the human metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 and -2 genes: Characterization of their infection into rabbit smooth muscle cells and human MCF-7 adenocarcinoma cells.Matrix Biol. 1996; 15: 383-395Crossref PubMed Scopus (54) Google ScholarAdhesion receptorsN-CadherindReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.N-Cadherindl7001E1−, E3−, Ad5Li et al, 2001E-CadherindReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.E-Cadherindl7001E1−, E3−, Ad5Hsu et al., 2000Hsu M.Y. Meier F.E. Nesbit M. et al.E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.Am J Pathol. 2000; 56: 1515-1525Abstract Full Text Full Text PDF Scopus (262) Google Scholarβ3-IntegrindReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.β3dl7001E1−, E3−, Ad5Hsu et al., 1998Hsu M.Y. Shih D.T. Meier F.E. et al.Adenoviral gene transfer of beta3 integrin subunit induces conversion from radial to vertical growth phase in primary human melanoma.Am J Pathol. 1998; 153: 1435-1442Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholarα2 IntegrindReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.α2dl7001E1−, E3−, Ad5Nesbit et al., 2001aNesbit M. Schaider H. Andl T. et al.α5-Integrin gene transfer mimics PDGF-B transformed phenotypes in human dermal fibroblasts and human skin.Lab Invest. 2001; 166: 6483-6490Google Scholarα5 IntegrindReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.α5dl7001E1−, E3−, Ad5Nesbit et al., 2001aNesbit M. Schaider H. Andl T. et al.α5-Integrin gene transfer mimics PDGF-B transformed phenotypes in human dermal fibroblasts and human skin.Lab Invest. 2001; 166: 6483-6490Google ScholarMelanoma cell adhesion molecule (MUC18, CD146, S-Endo-1)eInduction of angiogenesis at 195±12 vessels/mm2.Mel-CAMAdEasy-1E1−, E3−, Ad5Satyamoorthy et al., 2001bSatyamoorthy K. Muyers J.P. Meier F.E. Patel D. Herlyn M. Mel-CAM-specific genetic suppressor elements inhibit growth and invasion through loss of gap junctional communication.Oncogene. 2001; 20: 4676-4684Crossref PubMed Scopus (68) Google ScholarOncogenes, tumor suppressor genes and othersHuman papillomavirus type 16-E6 oncogenedReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.HPV16-E6dl7001E1−, E3−, Ad5Prabhu et al., 1998Prabhu N.S. Somasundaram K. Satyamoorthy K. Herlyn M. El-Deiry W.S. p73beta, unlike p53, suppresses growth and induces apoptosis of human papillomavirus E6-expressing cancer cells.Int J Oncol. 1998; 13: 5-9PubMed Google ScholarHuman papillomavirus type 16-E7 oncogenedReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.HPV16-E7dl7001E1−, E3−, Ad5(Eck Lab.)Tumor suppressor protein p16INK4AdReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.p16INK4Adl7001E1−, E3−, Ad5Mitra et al., 1999Mitra J. Dai C.Y. Somasundaram K. El-Deiry W.S. Satyamoorthy K. Herlyn M. Enders G.H. Induction of p21 (WAF1/CIP1) and inhibition of Cdk2 mediated by the tumor suppressor p16 (INK4a).Mol Cell Biol. 1999; 19: 3916-3928Crossref PubMed Google ScholarTumor suppressor protein p21Waf1/cip1dReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.p21Waf1/cip1pJM17E1−, Ad5Prabhu et al., 1996Prabhu N.S. Blagosklonny M.V. Zeng Y.X. Wu G.S. Waldman T. El-Deiry W.S. Suppression of cancer cell growth by adenovirus expressing p21 (WAF1/CIP1) deficient in PCNA interaction.Clin Cancer Res. 1996; 2: 1221-1229PubMed Google ScholarTumor suppressor protein p53fInduction of hyperplasia at 0.17mm/mm2. c-myc/Ad5 also induced thrombosin at 75 vessels/mm2.p53pJM17E1−, Ad5Blagosklonny and El-Deiry, 1996Blagosklonny M.V. El-Deiry W.S. In vitro evaluation of a p53-expressing adenovirus as an anti-cancer drug.Int J Cancer. 1996; 6: 386-392Crossref Scopus (191) Google ScholarAntisense cyclin D1 (AS)dReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.Cyclin D1 (AS)dl7001E1−, E3−, Ad5Sauter et al., 1999bSauter E.R. Nesbit M. Litwin S. Klein-Szanto A.J. Cheffetz S. Herlyn M. Antisense cyclin D1 induces apoptosis and tumor shrinkage in human squamous carcinomas.Cancer Res. 1999; 59: 4876-4881PubMed Google ScholarCellular homologue of avian myelocytomatosis virus oncogenefInduction of hyperplasia at 0.17mm/mm2. c-myc/Ad5 also induced thrombosin at 75 vessels/mm2.c-mycpMJ17E1−, Ad5Mitchell and El-Deiry, 1999Mitchell K.O. El-Deiry W.S. Overexpression of c-Myc inhibits p21WAF1/CiP1 expression and induces S-phase entry in 12-O-tetradecanoylphorbol-13-acetate (TPA) -sensitive human cancer cells.Cell Growth Differ. 1999; 10: 223-230PubMed Google ScholarFibroblast growth factor binding proteinFGF-BPdl7001E1−, E3−, Ad5Sauter et al., 2001Sauter E.R. Nesbit M. Tichansky D. Palazzo J. Herlyn M. FGF-BP expression changes with disease progression in clinical and experimental human squamous epithelium.Int J Cancer. 2001; 92: 374-381Crossref PubMed Scopus (11) Google ScholarControl vectorsLacZLacZdl7001E1−, E3−, Ad5Satyamoorthy et al., 1997Satyamoorthy K. Soballe P.W. Soans F. Herlyn M. Adenovirus infection enhances killing of melanoma cells by a mitotoxin.Cancer Res. 1997; 57: 1873-1876PubMed Google ScholarGreen fluorescence proteindReactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.GFPdl7001E1−, E3−, Ad5De Martin et al., 1997De Martin R. Raidl M. Hofer E. Binder B.R. Adenovirus-mediated expression of green fluorescent protein.Gene Ther. 1997; 4: 493-495Crossref PubMed Scopus (57) Google Scholara CMV promoter was used for all adenoviral constructs.b All genes were of human origin unless otherwise indicated.c References refer to characterization of the adenoviral vector.d Reactivity in human skin was similar to the LacZ/Ad5 vector. Therefore, results in figures are not listed.e Induction of angiogenesis at 195±12 vessels/mm2.f Induction of hyperplasia at 0.17 mm/mm2. c-myc/Ad5 also induced thrombosin at 75 vessels/mm2. Open table in a new tab C.B.-17 SCID mice between 4 and 6 wk of age were used for grafting of human foreskins from newborns (Atillasoy et al., 1998Atillasoy E.S. Seykora J.T. Soballe P.W. Elenitsas R. Nesbit M. Elder D.E. Montone K.T. Sauter E. Herlyn M. UVB induces atypical melanocytic lesions and melanoma in human skin.Am J Pathol. 1998; 152: 1179-1186PubMed Google Scholar;Berking et al., 2001aBerking C. Takemoto R. Satyamoorthy K. Elenitsas R. Herlyn M. Basic fibroblast growth factor and UVB transform melanocytes in human skin.Am J Pathol. 2001; 158: 943-953Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, Berking et al., 2001bBerking C. Takemoto R. Schaider H. Showe L. Satyamoorthy K. Robbins P. Herlyn M. TGF-ß1 increases survival of human melanoma through stroma remodeling.Cancer Res. 2001; 61: 8306-8316PubMed Google Scholar). The 1.5–2 cm2 full-thickness skin specimens were placed into size-matched wound beds on the dorsal torso of the mice. Each mouse received a separate graft, which was allowed to heal for 4–6 wk prior to injection of adenoviral vectors. Human skin grafts were injected with 100 μL of virus suspension containing 5×108 plaque forming units (pfu) in phosphate-buffered saline (PBS). The viruses were injected intradermally with Hamilton gas-light syringes with 30-gauge needles (Reno, NY). Four to 15 grafts were injected with the same vector. For each set of 5 viruses one control lacZ group was included. Intradermal injection was confirmed by presence of an epidermal elevation. The area of injection was marked with indelible ink. Starting three d following injection, mice were sacrificed and grafts were harvested for histological analysis. The human skin grafts were dissected free from surrounding murine tissue and bisected perpendicular to the long axis of the graft. The grafts were fixed overnight in 10% neutral buffered formalin at 4°C, and half were paraffin embedded for histological assessment. The other half of the grafts were cryopreserved after dehydration in 20% sucrose overnight at 4°C. Serial sections (5 μm) from each of the formalin-fixed, paraffin-embedded grafts were cut onto slides and stained with hematoxylin and eosin (H&E). The degree of acute inflammatory response, vascular response, connective tissue and epidermal reaction to the adenovirus-mediated transgenes was assessed independently by 2 observers (C.J.G. and J.L.). Uniform staining of sections was evaluated at low magnification (X100). Individual counts for inflammatory cells such as granulocytes, lymphocytes and monocytes, vessels, and fibroblasts were then taken at high magnification (X400). Counts were quantified as the number of structures/mm2 (1 high power field=0.0496 mm2). Each count was performed independently on five representative high power fields by the two investigators. For evaluation of collagen in the skin tissues, Masson's trichrome stain was performed on formalin-fixed paraffin-embedded sections according to standard protocols. Collagen deposition was quantified using Image-ProÆ Plus analyzing software (Media Cybernetics, L.p., Silver Spring, MD). With the help of this image analyzing software, the collagen-specific color densitometric measurements were converted to optical density. Epidermal hyperplasia was evaluated by determining the number of papillae/mm epidermal surface and measuring epidermal thickness. Changes in skin were scored negative if both observers agreed the effect was below a prospectively set threshold for each evaluation criteria using skin injected with the lacZ as GFP adenoviral vectors and PBS (phosphate-buffered saline) as controls. To distinguish between human and mouse cells, sections were stained with Hoechst dye that provides characteristic staining for human vs. mouse cells (Atillasoy et al., 1998Atillasoy E.S. Seykora J.T. Soballe P.W. Elenitsas R. Nesbit M. Elder D.E. Montone K.T. Sauter E. Herlyn M. UVB induces atypical melanocytic lesions and melanoma in human skin.Am J Pathol. 1998; 152: 1179-1186PubMed Google Scholar;Berking et al., 2001aBerking C. Takemoto R. Satyamoorthy K. Elenitsas R. Herlyn M. Basic fibroblast growth factor and UVB transform melanocytes in human skin.Am J Pathol. 2001; 158: 943-953Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar,Berking et al., 2001bBerking C. Takemoto R. Schaider H. Showe L. Satyamoorthy K. Robbins P. Herlyn M. TGF-ß1 increases survival of human melanoma through stroma remodeling.Cancer Res. 2001; 61: 8306-8316PubMed Google Scholar). Statistical comparisons between test group and control groups, lacZ/Ad5 or GFP/Ad5-injected skin, were done by Student's t test. For β-Galactosidase histochemistry, cryosections on slides were fixed in 0.5% glutaraldehyde for 10 min, followed by rinsing twice in 1 mM MgCl2 for 10 min each. The slides were then incubated for 1 h in the dark at 37°C in 1 mM MgCl2, 20XKCl in PBS solution at pH 7.4, and β-galactosidase (5-bromo-4-chloro-3-indolyl β-galactopyranoside) at a final concentration of 1 mg/ml. They were then washed 3 times in tap water and coverslipped with mounting medium for microscopic evaluation. Immunohistochemistry was performed on serial sections using an avidin-biotin-peroxidase system kit (Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine tetrahydro-chloride (Sigma) or 3-amino-9-ethylcarbazole (Vector) as chromogens. Antigens in the formalin-fixed tissues were retrieved by trypsin digestion at 37°C or microwave heat treatment in citrate buffer. Cryostat sections cut between 6 and 8 μm on a slide were air-dried and fixed in ice-cold acetone for 10 min prior to incubation with the primary antibodies in a humidified chamber at 4°C overnight or at room temperature for 1–2 h. Non-specific binding was blocked with 10% normal horse or 10% normal goat serum. Primary monoclonal antibodies used in this study were: mouse anticollagen type IV, antimurine PECAM-1, and antismooth muscle actin antibodies. A mouse IgG1 isotype antibody (P3) was used as negative control for each staining. Between each incubation step, slides were rinsed twice in PBS for 3–5 min. A biotin-" @default.
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