Skin as a Potential Organ for Ectopic Monoclonal Antibody Production11The authors declared not to have a conflict of interest.
2002; Elsevier BV; Volume: 118; Issue: 2 Linguagem: Inglês
10.1046/j.0022-202x.2001.01625.x
ISSN1523-1747
AutoresDanièle Noël, Jean-Eudes Dazard, Mireia Pelegrín, Chantal Jacquet, Marc Piechaczyk,
Tópico(s)Viral Infectious Diseases and Gene Expression in Insects
ResumoThe therapeutic potential of monoclonal antibodies for treating a variety of severe or life-threatening diseases is high. Although intravenous infusion appears the simplest and most obvious mode of administration, it is not applicable to many long-term treatments. It might be advantageously replaced by gene/cell therapies, however, rendering treatments cost-effective and eliminating the short- and long-term side-effects associated with injection of massive doses of antibodies. We have tested whether skin can potentially be used as an organ for production and systemic delivery of ectopic antibodies. Normal human primary keratinocytes were shown to be capable of synthesis and secretion of a model monoclonal antibody directed against human thyroglobulin upon retroviral gene transduction in vitro. Neo- epidermis reconstructed in vitro, either in cell culture inserts or on dermal substrates, from such modified keratinocytes also produced the monoclonal antibody. Interestingly, the latter could cross the epidermis basal layer and be released in culture fluids. Finally, grafting of epidermis reconstituted in vitro on dermal substrates to SCID mice permitted sustained monoclonal antibody delivery into the bloodstream to be achieved. Our data thus show that genetically engineered keratinocytes can potentially be used for genetic antibody-based immunotherapies. They also indicate that proteins as big as 150 kDa, after release by engineered keratinocytes into skin intercellular spaces, can migrate to the general circulation, which is potentially important for a number of other gene-based therapies. The therapeutic potential of monoclonal antibodies for treating a variety of severe or life-threatening diseases is high. Although intravenous infusion appears the simplest and most obvious mode of administration, it is not applicable to many long-term treatments. It might be advantageously replaced by gene/cell therapies, however, rendering treatments cost-effective and eliminating the short- and long-term side-effects associated with injection of massive doses of antibodies. We have tested whether skin can potentially be used as an organ for production and systemic delivery of ectopic antibodies. Normal human primary keratinocytes were shown to be capable of synthesis and secretion of a model monoclonal antibody directed against human thyroglobulin upon retroviral gene transduction in vitro. Neo- epidermis reconstructed in vitro, either in cell culture inserts or on dermal substrates, from such modified keratinocytes also produced the monoclonal antibody. Interestingly, the latter could cross the epidermis basal layer and be released in culture fluids. Finally, grafting of epidermis reconstituted in vitro on dermal substrates to SCID mice permitted sustained monoclonal antibody delivery into the bloodstream to be achieved. Our data thus show that genetically engineered keratinocytes can potentially be used for genetic antibody-based immunotherapies. They also indicate that proteins as big as 150 kDa, after release by engineered keratinocytes into skin intercellular spaces, can migrate to the general circulation, which is potentially important for a number of other gene-based therapies. normal human epidermal primary keratinocytes The potential of therapeutic applications of monoclonal antibodies (MoAb) for treating pathologic manifestations as varied as cancer, viral infections, transplant rejection, cachexia, neuropathologies, autoimmune diseases, etc., is high (Chester and Hawkins, 1995Chester K.A. Hawkins R.E. Clinical issues in antibody design.Trends Biotechnol. 1995; 13: 294-300Abstract Full Text PDF PubMed Scopus (74) Google Scholar). The actual therapeutic value of MoAb has recently been illustrated by efficient injection-based treatment of non-Hodgkin's lymphomas (Grillo-Lopez et al., 1999Grillo-Lopez A.J. White C.A. Varns C. Shen D. Wei A. McClure A. Dallaire B.K. Overview of the clinical development of rituximab: first monoclonal antibody approved for the treatment of lymphoma.Semin Oncol. 1999; 26: 66-73PubMed Google Scholar;Maloney, 1999Maloney D.G. Preclinical and phase I and II trials of rituximab.Semin Oncol. 1999; 26: 74-78PubMed Google Scholar), acute myelogenous leukemia (Cheson et al., 2000Cheson B.D. Zwiebel J.A. Dancey J. Murgo A. Novel therapeutic agents for the treatment of myelodysplastic syndromes.Semin Oncol. 2000; 27: 560-577PubMed Google Scholar), breast cancers (Baselga, 2000aBaselga J. Clinical trials of single-agent trastuzumab (Herceptin).Semin Oncol. 2000; 27: 20-26PubMed Google Scholar;Baselga, 2000bBaselga J. Current and planned clinical trials with trastuzumab (Herceptin).Semin Oncol. 2000; 27: 27-32PubMed Google Scholar), acute rejection in renal transplantation (Ekberg et al., 2000Ekberg H. Backman L. Tufveson G. Tyden G. Nashan B. Vincenti F. Daclizumab prevents acute rejection and improves patient survival post transplantation: 1 year pooled analysis.Transpl Int. 2000; 13: 151-159https://doi.org/10.1007/s001470050677Crossref PubMed Google Scholar;Thistlethwaite et al., 2000Thistlethwaite Jr, J.R. Nashan B. Hall M. Chodoff L. Lin T.H. Reduced acute rejection and superior 1-year renal allograft survival with basiliximab in patients with diabetes mellitus. The Global Simulect Study Group.Transplantation. 2000; 70: 784-790Crossref PubMed Scopus (44) Google Scholar), Crohn's disease (Present et al., 1999Present D.H. Rutgeerts P. 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Along the same line, a total of eight MoAb have now been approved by the US Food and Drug Administration for clinical use and more than 70 MoAb are currently in clinical trials beyond phase I and phase II (Glennie and Johnson, 2000Glennie M.J. Johnson P.W. Clinical trials of antibody therapy.Immunol Today. 2000; 21: 403-410Abstract Full Text Full Text PDF PubMed Scopus (273) Google Scholar). In theory, intravenous injection is the simplest and most obvious mode of administration of purified antibodies to patients. Though satisfactory for short-term treatments, however, it is often not applicable for long-term treatments (which might involve up to grams of antibody per month and per patient for treating certain diseases) due to the restrictive cost of proteins produced in vitro and certified for human application. Moreover, many technical problems posed by industrial mass production have not yet been solved and still limit the availability of therapeutic antibodies. In addition, hour-long infusions require specialist expertise to administer and are often associated with mild to very severe side-effects. The latter can include vomiting, migraines, anemia, and thrombocytopenia and, in certain cases, coma or even myocarditis infarction (Maloney et al., 1997Maloney D.G. Grillo L.A. White C.A. et al.IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma.Blood. 1997; 90: 2188-2195PubMed Google Scholar;McLaughlin et al., 1998McLaughlin P. Grillo-Lopez A.J. Link B.K. et al.Rituximab chimeric anti-CD20 monoclonal antibody therapy for relapsed indolent lymphoma: half of patients respond to a four-dose treatment program.J Clin Oncol. 1998; 16: 2825-2833Crossref PubMed Scopus (2467) Google Scholar;Soykan et al., 2000Soykan I. Ertan C. Ozden A. Severe anaphylactic reaction to infliximab: report of a case.Am J Gastroenterol. 2000; 95 ([letter]): 2395-2396Crossref PubMed Scopus (42) Google Scholar). In other words, the large-scale clinical application of a number of MoAb – some with an already demonstrated therapeutic activity – is currently impossible or severely compromised. The in vivo production of therapeutic antibodies, after either genetic modification of the patients' own cells or implantation of exogenous antibody-producing cells, might thus advantageously replace regular intravenous injection by a single-step year-long treatment rendering long-term therapeutic antibody treatments cost-effective and eliminating side-effects associated with the infusion of large quantities of antibodies. In addition, continuous and sustained delivery of antibodies at a low, but therapeutic, level should also suppress, or at least delay, the onset of neutralizing anti-idiotypic immune responses, which can develop when massive doses of purified immunoglobulins are repeatedly injected into humans or animals (Isaacs, 1990Isaacs J.D. The antiglobulin response to therapeutic antibodies.Semin Immunol. 1990; 2: 449-456PubMed Google Scholar;Kuus-Reichel et al., 1994Kuus-Reichel K. Grauer L.S. Karavodin L.M. Knott C. Krusemeier M. Kay N.E. Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies?.Clin Diagn Lab Immunol. 1994; 1: 365-372PubMed Google Scholar). As a first step to the development of antibody-based gene/cell therapies, we have shown the technical feasibility of month- to year-long in vivo production and systemic delivery of MoAb in the mouse, as an animal model, using the grafting of genetically modified myoblasts (Noël et al., 1997Noël D. Pelegrin M. Marin M. Biard P.M. Ourlin J.C. Mani J.C. Piechaczyk M. In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.Hum Gene Ther. 1997; 8: 1219-1229Crossref PubMed Scopus (31) Google Scholar) and skin fibroblasts (Noël et al., 2000Noël D. Pelegrin M. Brockly F. Lund A.H. Piechaczyk M. Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts.J Invest Dermatol. 2000; 115: 740-745Crossref PubMed Scopus (15) Google Scholar) and the implantation of antibody-producing cells encapsulated in a matrix of cellulose sulfate (Pelegrin et al., 1998aPelegrin M. Marin M. Noel D. et al.Systemic long-term delivery of antibodies in immunocompetent animals using cellulose sulphate capsules containing antibody-producing cells.Gene Ther. 1998; 5: 828-834Crossref PubMed Scopus (55) Google Scholar, Pelegrin et al., 2000Pelegrin M. Marin M. Oates A. Noel D. Saller R. Salmons B. Piechaczyk M. Immunotherapy of a viral disease by in vivo production of therapeutic monoclonal antibodies.Hum Gene Ther. 2000; 11: 1407-1415https://doi.org/10.1089/10430340050057486Crossref PubMed Scopus (29) Google Scholar). Interestingly, in no case was an anti-idiotypic response against the ectopic antibody detected (Pelegrin et al., 1998bPelegrin M. Marin M. Noël D. Piechaczyk M. Genetically engineered antibodies in gene transfer and gene therapy.Hum Gene Ther. 1998; 9: 2165-2175Crossref PubMed Scopus (29) Google Scholar;Noël et al., 2000Noël D. Pelegrin M. Brockly F. Lund A.H. Piechaczyk M. Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts.J Invest Dermatol. 2000; 115: 740-745Crossref PubMed Scopus (15) Google Scholar). More recently, we have validated the therapeutic value of this novel therapeutic approach by efficiently protecting mice that were lethally infected with a neurodegeneration-inducing retrovirus by implantation of cellulose-sulfate-encapsulated cells producing a virus-neutralizing antibody (Pelegrin et al., 2000Pelegrin M. Marin M. Oates A. Noel D. Saller R. Salmons B. Piechaczyk M. Immunotherapy of a viral disease by in vivo production of therapeutic monoclonal antibodies.Hum Gene Ther. 2000; 11: 1407-1415https://doi.org/10.1089/10430340050057486Crossref PubMed Scopus (29) Google Scholar). Plasmocytes, which are the cells naturally specialized in antibody production, cannot be used for long-term genetic antibody-based therapies because they already secrete an antibody and are short-lived (half-life of a few days). Towards the aim of human application, a major issue is thus to identify tissues amenable to genetic modification for MoAb production and persisting for virtually the whole patient's life after grafting. Skin is obviously a good candidate for this purpose because (i) it is the most accessible tissue, (ii) keratinocytes can easily be expanded ex vivo, (iii) they can be transduced by retroviral vectors, (iv) normal human epidermal primary keratinocyte (NHEPK) populations contain stem cells with a high replicative potential (Barrandon and Green, 1987Barrandon Y. Green H. 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Out of Eden: stem cells and their niches.Science. 2000; 287: 1427-1430Crossref PubMed Scopus (1402) Google Scholar), (v) skin grafts can persist long-term, as is well established in the treatment of burn victims, (vi) efficiency of skin grafting can be monitored by direct visual examination, (vii) keratinocytes have a high protein secretion potential as evidenced from stratified cultures (seeKatz and Taichman, 1999Katz A.B. Taichman L.B. A partial catalog of proteins secreted by epidermal keratinocytes in culture.J Invest Dermatol. 1999; 112: 818-821Crossref PubMed Scopus (81) Google Scholar;Cao et al., 2000Cao T. Wang X.J. Roop D.R. Regulated cutaneous gene delivery: the skin as a bioreactor.Hum Gene Ther. 2000; 11: 2297-2300Crossref PubMed Scopus (37) Google Scholar), (viii) skin has already been demonstrated to be capable of systemic delivery of ectopic proteins though the latter were of small size (Fenjves et al., 1989Fenjves E.S. Gordon D.A. Pershing L.K. Williams D.L. 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Induction of keratinocyte proliferation and lymphocytic infiltration by in vivo introduction of the IL-6 gene into keratinocytes and possibility of keratinocyte gene therapy for inflammatory skin diseases using IL-6 mutant genes.J Immunol. 1998; 161: 5633-5639PubMed Google Scholar), and (ix) we have previously shown that the A431 keratinocytic cell line can produce MoAb showing the same thermodynamic and kinetic properties as the parental antibody produced by B cells in vitro (Noël et al., 1997Noël D. Pelegrin M. Marin M. Biard P.M. Ourlin J.C. Mani J.C. Piechaczyk M. In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.Hum Gene Ther. 1997; 8: 1219-1229Crossref PubMed Scopus (31) Google Scholar). Supporting the idea that engineered skin can potentially be used for antibody-based therapies, we report here the in vivo production and systemic delivery of a model MoAb directed against human thyroglobulin in an experimental setting consisting of the grafting to SCID mice of human epidermis reconstituted in vitro from genetically modified keratinocytes. The Tg10-antibody-expressing PM130 (Noël et al., 1997Noël D. Pelegrin M. Marin M. Biard P.M. Ourlin J.C. Mani J.C. Piechaczyk M. In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.Hum Gene Ther. 1997; 8: 1219-1229Crossref PubMed Scopus (31) Google Scholar) and the CD167 (Noël et al., 2000Noël D. Pelegrin M. Brockly F. Lund A.H. Piechaczyk M. Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts.J Invest Dermatol. 2000; 115: 740-745Crossref PubMed Scopus (15) Google Scholar) retroviral vectors were derived from the MoMuLV-based pLXPXSN (Morgan et al., 1992Morgan R.A. Couture L. Elroy-Stein O. Ragheb J. Moss B. Anderson W.F. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and application to human gene therapy.Nucl Acids Res. 1992; 20: 1293-1299Crossref PubMed Scopus (224) Google Scholar) and Akv-derived ptv-Akv-tRNApro (Lund et al., 1993Lund A.H. Duch M. Lovmand J. Jorgensen P. Pedersen F.S. Mutated primer binding sites interacting with different tRNAs allow efficient murine leukemia virus replication.J Virol. 1993; 67: 7125-7130PubMed Google Scholar) plasmids, respectively. PA130.2 (Noël et al., 1997Noël D. Pelegrin M. Marin M. Biard P.M. Ourlin J.C. Mani J.C. Piechaczyk M. In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.Hum Gene Ther. 1997; 8: 1219-1229Crossref PubMed Scopus (31) Google Scholar) and FlyA167.1 (Noël et al., 2000Noël D. Pelegrin M. Brockly F. Lund A.H. Piechaczyk M. Sustained systemic delivery of monoclonal antibodies by genetically modified skin fibroblasts.J Invest Dermatol. 2000; 115: 740-745Crossref PubMed Scopus (15) Google Scholar) are retrovirus-producing cell lines derived by transfection of PM130 and CD167 into PA317 (Miller and Buttimore, 1986Miller A.D. Buttimore C. Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production.Mol Cell Biol. 1986; 6: 2895-2902Crossref PubMed Scopus (1127) Google Scholar) and FlyA (Cosset et al., 1995Cosset F.-L. Takeuchi Y. Battini J.-L. Weiss R.A. Collins M.K.L. High-titer packaging cells producing recombinant retroviruses resistant to human serum.J Virol. 1995; 69Google Scholar) amphotrophic packaging cell lines, respectively. PA96.7 cells produce the void pLXPXSN vector, used as a negative control in our experiments, and are derived from PA317 helper cells. All cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) (Gibco BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS). Conditions for retrovirus production and assay have been described elsewhere (Noël et al., 1997Noël D. Pelegrin M. Marin M. Biard P.M. Ourlin J.C. Mani J.C. Piechaczyk M. In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.Hum Gene Ther. 1997; 8: 1219-1229Crossref PubMed Scopus (31) Google Scholar). Titers of retroviral supernatants were estimated to be 104 colony-forming units (cfu) per ml on NIH 3T3 fibroblast cells for the PA130.2 and FlyA167.1 cell lines and 105 cfu per ml for the PA96.7 cells. A431.130.2 cells were obtained by transfection of PM130 into A431 keratinocytic cells (Noël et al., 1997Noël D. Pelegrin M. Marin M. Biard P.M. Ourlin J.C. Mani J.C. Piechaczyk M. In vitro and in vivo secretion of cloned antibodies by genetically modified myogenic cells.Hum Gene Ther. 1997; 8: 1219-1229Crossref PubMed Scopus (31) Google Scholar) and were grown in RPMI 1640 medium (Gibco BRL) containing 10% FBS. NHEPK were freshly prepared from human neonatal foreskins. Briefly, foreskins were placed in Ca2+- and Mg2+-free phosphate-buffered saline (PBS; 150 mM NaCl, 10 mM Na3PO4 pH 7) containing 20 µg per ml of gentamycin (Gibco BRL) for 1 h. They were then incubated in the presence of 25 U per ml of dispase (Gibco BRL) at 4°C for 18 h and the epidermal layer containing the keratinocytes was peeled off from the dermis using forceps. Cell dissociation was obtained through three serial 5 min treatments at 37°C with a solution containing 0.05% trypsin and 0.53 mM ethylenediamine tetraacetic acid (EDTA) (Gibco BRL) under gentle agitation in a humid atmosphere with 5% CO2. Trypsin activity was blocked by addition of FBS and cells were gently resuspended in low calcium defined keratinocyte serum-free medium (DK-SFM) (Gibco BRL), supplemented with 15 ng per ml epidermal growth factor (Sigma, St. Louis, MO) final, 100 µg per ml streptomycin, and 100 U per ml penicillin. The culture medium was changed every 2–3 d until cells reached 80% confluence, at which point they were passaged. For retroviral gene transfer, four rounds of infection were carried out in the presence of 8 µg per ml polybrene (Sigma) for a period of 2 d in 60 mm diameter dishes using keratinocytes passaged twice and cultured to 50%-60% confluence. Typically, for each round of infection, 105 cells were placed in the presence of 1.5 ml of cell culture supernatant containing 104 infectious viruses at 37°C for 2 h, which means a multiplicity of infection of 0.15. Then, the medium was replaced by 3 ml of complete DK-SFM for further culture. As efficiency of infection by all three vectors could not be assayed directly after infection of keratinocytes, we resorted to an indirect assay. Duplicates of keratinocyte cultures were infected in parallel experiments with culture supernatants containing 0.5 × 104 cfu of a β-galactosidase-expressing amphotropic retroviral vector produced by the FlyA packaging cell line (Marin et al., 1996Marin M. Noël D. Valsesia-Wittman S. et al.Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single-chain antibody fragment-envelope fusion proteins.J Virol. 1996; 70: 2957-2962PubMed Google Scholar) plus 0.5 × 104 cfu of either PM130, CD167, or pLXPXSN vectors. Two days after the last infection round, keratinocytes were stained for β-galactosidase activity (Marin et al., 1996Marin M. Noël D. Valsesia-Wittman S. et al.Targeted infection of human cells via major histocompatibility complex class I molecules by Moloney murine leukemia virus-derived viruses displaying single-chain antibody fragment-envelope fusion proteins.J Virol. 1996; 70: 2957-2962PubMed Google Scholar;Bachrach et al., 2000Bachrach E. Marin M. Pelegrin M. Karavanas G. Piechaczyk M. Low amounts of envelope glycoprotein are sufficent for efficient cell infection by Moloney murine leukemia virus.J Virol. 2000; 74: 8480-8486Crossref PubMed Scopus (24) Google Scholar) and blue plaques were scored for calculation of the efficiency of transduction. Dermal substrates were prepared from normal human adult skin samples obtained from healthy volunteers undergoing plastic surgery after informed consent. For de-epidermization, skin specimens were heated at 56°C in 20 mM EDTA-containing PBS for 5 min and immediately transferred onto ice where epidermis was peeled off with forceps. De-epidermized dermis (DED) were placed into sterile cryotubes and mortified through 10 rounds of freezing and thawing in liquid nitrogen as described elsewhere (Régnier et al., 1981Régnier M. Pruniéras M. Woodley D. Growth and differentiation of adult human epidermal cells on dermal substrates.Front Matrix Biol. 1981; 9: 4-35Google Scholar). DED were stored at -80°C until use. Epidermis was reconstructed either on dermal substrates or on membrane inserts according to the well-established techniques ofPruniéras et al., 1983Pruniéras M. Régnier M. Woodley D. Methods of cultivation of keratinocytes with an air–liquid interface.J Invest Dermatol. 1983; 81: 289-294PubMed Google Scholar andRosdy and Clauss, 1990Rosdy M. Clauss L.C. Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air–liquid interface.J Invest Dermatol. 1990; 95: 409-414Abstract Full Text PDF PubMed Google Scholar, respectively. Cultures were performed in PR culture medium, which consists of 66% DMEM, 22% Ham F12, 10% FBS, 8.4 ng per ml cholera toxin (Sigma), 400 ng per ml hydrocortisone (Sigma), 20 ng per ml epidermal growth factor (Sigma), 5 µg per ml insulin (Sigma), 1.5 mM CaCl2, 1% non-essential amino acids (Gibco BRL), and 10-6 M L-isoproterenol; the medium was changed every 2 d. For culture on membrane inserts, 3 × 105 NHEPK were seeded per culture well (Font et al., 1994Font J. Braut-Boucher F. Pichon J. et al.A new three-dimensional culture of human keratinocytes: optimization of differentiation.Cell Biol Toxicol. 1994; 10: 353-359Crossref PubMed Scopus (15) Google Scholar) in the presence of 300 µl of PR medium in both the lower and the upper culture cell compartments. After 5 d, the upper phase culture medium was removed and NHEPK were further cultured at the air–liquid interface for the rest of the experiment. For culture on dermal substrates, 3 × 105 NHEPK, in a volume of 15 µl, were dropped on mortified DED, dermal side down, and cultured at the air–liquid interface. In both cases, fully reconstituted epidermis was obtained within 15 d of culture. The Tg10 antibody was assayed in culture supernatants and mouse sera by enzyme-linked immunosorbent assay (ELISA) as described byPiechaczyk et al., 1985Piechaczyk M. Chardes T. Cot M.-C. Pau B. Bastide J.-M. 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Human thyroglobulin (hTg) for coating plates was from Biogenesis (Poole, U.K.). Tg10 antibody purified to homogeneity from hybridoma supernatants was used as a standard. Immunohistochemical detection of Tg10 in human epidermis regenerated on dermal substrates was performed as follows: after 15 d in culture, reconstituted tissues were collected and embedded in OCT (Tissue-Tek, The Netherlands), frozen in liquid nitrogen, and stored at -80°C until use. Cryostat sections (7 µm) were prepared and fixed in 3.7% paraformaldehyde for 15 min, rinsed three times with PBS, and stored at -80°C or immediately processed for immunostaining at room temperature. They were treated with acetone for 20 s, rinsed three times in PBS, placed in PBS solution containing 1% bovine serum albumin (Sigma) for 30 min, incubated in PBS solution containing FITC-conjugated goat anti-mouse IgG and 1% Tween-20 for 30 min, and finally washed three times in PBS solution containing 1% Tween-20. Slides were mounted in DAPI-containing Vectashield (Biovalley, Marne la Vallée, France) before microscopic examination. Grafting of retrovirally transduced and regenerated epidermis was performed subcutaneously as previously described (Levy et al., 1998Levy L. Broad S. Zhu A.J. Carroll J.M. Khazaal I. Peault B. Watt F.M. Optimised retroviral infection of human epidermal keratinocytes: long-term expression of transduced integrin gene following grafting on to SCID mice.Gene Ther. 1998; 5: 913-922Crossref PubMed Scopus (57) Google Scholar) using 10–12-wk-old CB17/SCIDbg mice (Harlan, Gannat, France) as recipients. Briefly, mice were anesthetized using 0.01 ml per g of body weight of a solution containing 0.1% xylasine (Rompun, Bayer, Leverkusen, Germany) and 10 mg per ml ketamine (Imal
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