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Michele DE LUCA

Professore Ordinario
Dipartimento di Scienze della Vita sede ex-Scienze Biomediche

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2022 - Clonal analysis of human clonogenic keratinocytes [Articolo su rivista]
Enzo, E.; Cattaneo, C.; Consiglio, F.; Polito, M. P.; Bondanza, S.; De Luca, M.

Regenerative medicine has its roots in harnessing stem cells for permanent restoration of damaged or diseased tissues. The first procedure for the transplantation of epidermal cultures in massive full-thickness burns was established in the 1980s. Since then, epithelial stem cell-based therapies have been further developed in cell and gene therapy protocols aimed at restoring visual acuity in severe ocular burns and treating patients affected by genetic skin diseases, as Epidermolysis Bullosa. The clinical success of these Advanced Therapy Medicinal Products (ATMPs) requires the presence of a defined number of epithelial stem cells in the grafts, detected as holoclone-forming cells. To date, the most trustworthy method to identify and measure holoclones in a culture is the clonal analysis of clonogenic keratinocytes. Here we describe in detail how to perform such a clonal analysis and identify each epidermal clonal type.

2022 - Stairways to Advanced Therapies for Epidermolysis Bullosa [Articolo su rivista]
De Rosa, Laura; Enzo, Elena; Palamenghi, Michele; Sercia, Laura; De Luca, Michele

Epidermolysis bullosa (EB) is a devastating genetic skin disease typified by a plethora of different phenotypes and ranking from severe, early lethal, to mild localized forms. Although there is no cure for EB, recent progress in pharmacology and molecular and cellular biology is boosting the development of new advanced therapeutic strategies. Here we will focus on two main categories of such therapies: (1) those aimed at controlling inflammation and inducing reepithelialization of the wounds, and (2) those, perhaps more challenging and ambitious, that aim to permanently regenerate a fully functional epidermis, which requires targeting of epidermal stem cells. In both cases, the genetic variants underlying the different EB forms and factors, such as genetic background, modifier genes, comorbidities, and lifestyle, all of which impinge on EB genotype-phenotype correlation, need to be defined.

2022 - The joint battle to tackle epidermolysis bullosa through gene therapy [Articolo su rivista]
De Rosa, L.; De Luca, M.

Efforts are dedicated to definitively tackle skin lesions plaguing patients with epidermolysis bullosa (EB), a devastating genetic disorder affecting the integumentary system. Both in vivo gene therapy, as recently reported by Gurevich et al., and combined ex vivo cell and gene therapy strategies are under investigation. Here, we address the advantages and disadvantages of these different approaches.

2021 - Editorial: Gene, Cell and Protein Replacement Therapy for Genetic Muscle, Bone and Skin Disorders [Articolo su rivista]
Schneider, H.; De Luca, M.

2021 - Epigenetic and metabolic regulation of epidermal homeostasis [Articolo su rivista]
Wagner, R. N.; Pinon Hofbauer, J.; Wally, V.; Kofler, B.; Schmuth, M.; De Rosa, L.; De Luca, M.; Bauer, J. W.

Continuous exposure of the skin to environmental, mechanical and chemical stress necessitates constant self-renewal of the epidermis to maintain its barrier function. This self-renewal ability is attributed to epidermal stem cells (EPSCs), which are long-lived, multipotent cells located in the basal layer of the epidermis. Epidermal homeostasis – coordinated proliferation and differentiation of EPSCs – relies on fine-tuned adaptations in gene expression which in turn are tightly associated with specific epigenetic signatures and metabolic requirements. In this review, we will briefly summarize basic concepts of EPSC biology and epigenetic regulation with relevance to epidermal homeostasis. We will highlight the intricate interplay between mitochondrial energy metabolism and epigenetic events – including miRNA-mediated mechanisms – and discuss how the loss of epigenetic regulation and epidermal homeostasis manifests in skin disease. Discussion of inherited epidermolysis bullosa (EB) and disorders of cornification will focus on evidence for epigenetic deregulation and failure in epidermal homeostasis, including stem cell exhaustion and signs of premature ageing. We reason that the epigenetic and metabolic component of epidermal homeostasis is significant and warrants close attention. Charting epigenetic and metabolic complexities also represents an important step in the development of future systemic interventions aimed at restoring epidermal homeostasis and ameliorating disease burden in severe skin conditions.

2021 - Hologene 5: A Phase II/III Clinical Trial of Combined Cell and Gene Therapy of Junctional Epidermolysis Bullosa [Articolo su rivista]
De Rosa, L.; Enzo, E.; Zardi, G.; Bodemer, C.; Magnoni, C.; Schneider, H.; De Luca, M.

Epidermolysis bullosa (EB) is a group of devastating genetic diseases characterized by skin and mucosal fragility and formation of blisters, which develop either spontaneously or in response to minor mechanical trauma. There is no definitive therapy for any form of EB. Intermediate junctional EB (JEB) caused by mutations in the gene LAMB3 has been the first genetic skin disease successfully tackled by ex vivo gene therapy. Here, we present a multicenter, open-label, uncontrolled phase II/III study that aims at confirming the efficacy of Hologene 5, a graft consisting of cultured transgenic keratinocytes and epidermal stem cells and meant to combine cell and gene therapy for the treatment of LAMB3-related JEB. Autologous clonogenic keratinocytes will be isolated from patients’ skin biopsies, genetically corrected with a gamma-retroviral vector (γRV) carrying the full-length human LAMB3 cDNA and plated onto a fibrin support (144cm2). The transgenic epidermis will be transplanted onto surgically prepared selected skin areas of at least six JEB patients (four pediatric and two adults). Evaluation of clinical efficacy will include, as primary endpoint, a combination of clinical parameters, such as percentage of re-epithelialization, cellular, molecular, and functional parameters, mechanical stress tests, and patient-reported outcome (PRO), up to 12months after transplantation. Safety and further efficacy endpoints will also be assessed during the clinical trial and for additional 15years in an interventional non-pharmacological follow-up study. If successful, this clinical trial would provide a therapeutic option for skin lesions of JEB patients with LAMB3 mutations and pave the way to a combined cell and gene therapy platform tackling other forms of EB and different genodermatoses. Clinical Trial Registration: EudraCT Number: 2018-000261-36.

2021 - Single-keratinocyte transcriptomic analyses identify different clonal types and proliferative potential mediated by FOXM1 in human epidermal stem cells [Articolo su rivista]
Enzo, E.; Secone Seconetti, A.; Forcato, M.; Tenedini, E.; Polito, M. P.; Sala, I.; Carulli, S.; Contin, R.; Peano, C.; Tagliafico, E.; Bicciato, S.; Bondanza, S.; De Luca, M.

Autologous epidermal cultures restore a functional epidermis on burned patients. Transgenic epidermal grafts do so also in genetic skin diseases such as Junctional Epidermolysis Bullosa. Clinical success strictly requires an adequate number of epidermal stem cells, detected as holoclone-forming cells, which can be only partially distinguished from the other clonogenic keratinocytes and cannot be prospectively isolated. Here we report that single-cell transcriptome analysis of primary human epidermal cultures identifies categories of genes clearly distinguishing the different keratinocyte clonal types, which are hierarchically organized along a continuous, mainly linear trajectory showing that stem cells sequentially generate progenitors producing terminally differentiated cells. Holoclone-forming cells display stem cell hallmarks as genes regulating DNA repair, chromosome segregation, spindle organization and telomerase activity. Finally, we identify FOXM1 as a YAP-dependent key regulator of epidermal stem cells. These findings improve criteria for measuring stem cells in epidermal cultures, which is an essential feature of the graft.

2021 - Transgenic epidermal cultures for junctional epidermolysis bullosa — 5-year outcomes [Articolo su rivista]
Kueckelhaus, M.; Rothoeft, T.; de Rosa, L.; Yeni, B.; Ohmann, T.; Maier, C.; Eitner, L.; Metze, D.; Losi, L.; Seconetti, A. S.; de Luca, M.; Hirsch, T.

Inherited junctional epidermolysis bullosa is a severe genetic skin disease that leads to epidermal loss caused by structural and mechanical fragility of the integuments. There is no established cure for junctional epidermolysis bullosa. We previously reported that genetically corrected autologous epidermal cultures regenerated almost an entire, fully functional epidermis on a child who had a devastating form of junctional epidermolysis bullosa. We now report long-term clinical outcomes in this patient. (Funded by POR FESR 2014–2020 — Regione Emilia-Romagna and others.)

2020 - Toward combined cell and gene therapy for genodermatoses [Articolo su rivista]
De Rosa, L.; Latella, M. C.; Seconetti, A. S.; Cattelani, C.; Bauer, J. W.; Bondanza, S.; De Luca, M.

To date, more than 200 monogenic, often devastating, skin diseases have been described. Because of unmet medical needs, development of long-lasting and curative therapies has been consistently attempted, with the aim of correcting the underlying molecular defect. In this review, we will specifically address the few combined cell and gene therapy strategies that made it to the clinics. Based on these studies, what can be envisioned for the future is a patient-oriented strategy, built on the specific features of the individual in need. Most likely, a combination of different strategies, approaches, and advanced therapies will be required to reach the finish line at the end of the long and winding road hampering the achievement of definitive treatments for genodermatoses.

2019 - Advances in stem cell research and therapeutic development [Articolo su rivista]
De Luca, M.; Aiuti, A.; Cossu, G.; Parmar, M.; Pellegrini, G.; Robey, P. G.

Despite many reports of putative stem-cell-based treatments in genetic and degenerative disorders or severe injuries, the number of proven stem cell therapies has remained small. In this Review, we survey advances in stem cell research and describe the cell types that are currently being used in the clinic or are close to clinical trials. Finally, we analyse the scientific rationale, experimental approaches, caveats and results underpinning the clinical use of such stem cells.

2019 - Inherited epidermolysis bullosa: description of clinical and subclinical morphological features with optical coherence tomography [Articolo su rivista]
De Pace, B.; Fiorentini, C.; Ciardo, S.; Chester, J.; Kaleci, S.; Veltri, T.; De Luca, M.; Pellacani, G.


2019 - Laminin 332-Dependent YAP Dysregulation Depletes Epidermal Stem Cells in Junctional Epidermolysis Bullosa [Articolo su rivista]
De Rosa, L.; Secone Seconetti, A.; De Santis, G.; Pellacani, G.; Hirsch, T.; Rothoeft, T.; Teig, N.; Pellegrini, G.; Bauer, J. W.; De Luca, M.

Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and alpha 6 beta 4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB.

2019 - Look for methods, not conclusions [Articolo su rivista]
Bassi, R.; Bucci, E. M.; Calogero, R. A.; Carninci, P.; Ciliberto, G.; Conte, P.; De Luca, M.; Corbellini, G.; Giordano, A.; Marchionni, L.; Massaro Giordano, G.; Parini, A.; Sbardella, G.

2018 - Advances on potential therapeutic options for epidermolysis bullosa [Articolo su rivista]
De Rosa, L.; Koller, U.; Bauer, J. W.; De Luca, M.; Reichelt, J.

Introduction: Epidermolysis bullosa is a severe genodermatosis in which pain and wound management dominate patients’ lives leaving a high demand for more effective treatments. At present, gene therapy is the only treatment with the potential to cure inherited diseases. A small number of individuals with junctional and dystrophic forms of epidermolysis bullosa have received gene replacement therapies involving ex vivo viral transduction of intact transgenes into skin stem cells followed by autologous grafting of corrected epidermal sheets. Corrected stem cells ensure permanent regeneration of stable skin at the graft sites. Areas covered: Alternative therapeutic options are either RNA-based or involve the latest preclinical developments in the area of gene editing using designer nucleases such as TALEN or CRISPR/Cas9, which enable precise targeting of any desired locus and a potential traceless repair of mutations. Due to low targeting efficiencies and safety considerations regarding off-target effects, research in this area focuses on ex vivo approaches involving selection of correctly modified keratinocyte clones. Expert opinion: Although there is currently no single optimal therapy for epidermolysis bullosa, cell and gene therapy technologies are advancing rapidly holding great potential for modifying disease severity and improving quality of life for people living with this devastating disease.

2018 - Gentamicin induces LAMB3 nonsense mutation readthrough and restores functional laminin 332 in junctional epidermolysis bullosa [Articolo su rivista]
Lincoln, Vadim; Cogan, Jon; Hou, Yingping; Hirsch, Michaela; Hao, Michelle; Alexeev, Vitali; De Luca, Michele; De Rosa, Laura; Bauer, Johann W.; Woodley, David T.; Chen, Mei

Herlitz junctional epidermolysis bullosa (H-JEB) is an incurable, devastating, and mostly fatal inherited skin disease for which there is only supportive care. H-JEB is caused by loss-of-function mutations in LAMA3, LAMB3, or LAMC2, leading to complete loss of laminin 332, the major component of anchoring filaments, which mediate epidermal-dermal adherence. LAMB3 (laminin β3) mutations account for 80% of patients with H-JEB, and ∼95% of H-JEB–associated LAMB3 mutations are nonsense mutations leading to premature termination codons (PTCs). In this study, we evaluated the ability of gentamicin to induce PTC readthrough in H-JEB laminin β3-null keratinocytes transfected with expression vectors encoding eight different LAMB3 nonsense mutations. We found that gentamicin induced PTC readthrough in all eight nonsense mutations tested. We next used lentiviral vectors to generate stably transduced H-JEB cells with the R635X and C290X nonsense mutations. Incubation of these cell lines with various concentrations of gentamicin resulted in the synthesis and secretion of full-length laminin β3 in a dose-dependent and sustained manner. Importantly, the gentamicin-induced laminin β3 led to the restoration of laminin 332 assembly, secretion, and deposition within the dermal/epidermal junction, as well as proper polarization of α6β4 integrin in basal keratinocytes, as assessed by immunoblot analysis, immunofluorescent microscopy, and an in vitro 3D skin equivalent model. Finally, newly restored laminin 332 corrected the abnormal cellular phenotype of H-JEB cells by reversing abnormal cell morphology, poor growth potential, poor cell-substratum adhesion, and hypermotility. Therefore, gentamicin may offer a therapy for H-JEB and other inherited skin diseases caused by PTC mutations.

2018 - Living with Keratinocytes [Articolo su rivista]
Pellegrini, Graziella; De Luca, Michele

A feature distinguishing human hematopoietic and epithelial stem cells from other equally fascinating stem cells is perhaps their easier translation into a clinical setting. We have devoted nearly our entire scientific career in trying to turn our understanding of epithelial stem cell biology into something that could help people suffering from virtually untreatable diseases of squamous epithelia. We have done that as a team, together with our numerous students, postdocs, technicians and valuable collaborators, clinicians, regulators, and, lately, industrial partners. We had rewarding successes and burning failures, but we always did our best. This award, given by friends and colleagues deserving it more than us, has been the most important recognition of our work. Below, we summarize our story.

2018 - Navigating Market Authorization: The Path Holoclar Took to Become the First Stem Cell Product Approved in the European Union [Articolo su rivista]
Pellegrini, Graziella; Ardigò, Diego; Milazzo, Giovanni; Iotti, Giorgio; Guatelli, Paolo; Pelosi, Danilo; De Luca, Michele

Gene therapy, cell therapy, and tissue engineering have the potential to revolutionize the treatment of disease and injury. Attaining marketing authorization for such advanced therapy medicinal products (ATMPs) requires a rigorous scientific evaluation by the European Medicines Agency—authorization is only granted if the product can fulfil stringent requirements for quality, safety, and efficacy. However, many ATMPs are being provided to patients under alternative means, such as “hospital exemption” schemes. Holoclar (ex vivo expanded autologous human corneal epithelial cells containing stem cells), a novel treatment for eye burns, is one of the few ATMPs to have been granted marketing authorization and is the first containing stem cells. This review highlights the differences in standards between an authorized and unauthorized medicinal product, and specifically discusses how the manufacture of Holoclar had to be updated to achieve authorization. The result is that patients will have access to a therapy that is manufactured to high commercial standards, and is supported by robust clinical safety and efficacy data. Stem Cells Translational Medicine 2018;7:146–154.

2017 - Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells [Articolo su rivista]
Bauer, Johann; Koller, Josef; Murauer, Eva; DE ROSA, Laura; Enzo, Elena; Carulli, Sonia; Bondanza, Sergio; Recchia, Alessandra; Muss, Wolfgang; Diem, Anja; Mayr, Elisabeth; Schlager, Pamina; Gratz, Iris; Pellegrini, Graziella; DE LUCA, Michele

Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3,LAMB3,or LAMC2,which together encode laminin-332, a hetero-trimeric protein consisting ofa3,b3, andg2chain. In nonlethal generalized intermediate JEB, laminin-332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basement membrane upon minor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life. We report on a patient in whom gene-corrected epidermal sheets were transplanted onto a large nonhealing epidermal ulceration following a good manufacturing practice protocol

2017 - Correction of recessive dystrophic epidermolysis bullosa by transposon-mediated integration of COL7A1 in transplantable patient-derived primary keratinocytes. [Articolo su rivista]
Latella, Maria Carmela; Cocchiarella, Fabienne; De Rosa, Laura; Turchiano, Giandomenico; Gonçalves, Manuel; Larcher, Fernando; De Luca, Michele; Recchia, Alessandra

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by defects in type-VII collagen (C7), a protein encoded by the COL7A1 gene and essential for anchoring fibril formation at the dermal-epidermal junction. Gene therapy of RDEB is based on transplantation of autologous epidermal grafts generated from genecorrected keratinocytes sustaining C7 deposition at the dermal-epidermal junction. Transfer of the COL7A1 gene is complicated by its very large size and repetitive sequence. This article reports a gene delivery approach based on the Sleeping beauty transposon, which allows integration of a full-length COL7A1 cDNA and secretion of C7 at physiological levels in RDEB keratinocytes without rearrangements or detrimental effects on their clonogenic potential. Skin equivalents derived from gene-corrected RDEB keratinocytes were tested in a validated preclinical model of xenotransplantation on immunodeficient mice, where they showed normal deposition of C7 at the dermal-epidermal junction and restoration of skin adhesion properties. These results indicate the feasibility and efficacy of a transposon-based gene therapy approach to RDEB.

2017 - In vitro method for producing a flap of genetically modified cells on fibrin substrate [Brevetto]
Pellegrini, G.; Alessandrini, A.; De Luca, M.

2017 - Marketing of unproven stem cell-based interventions: A call to action [Articolo su rivista]
Sipp, D.; Caulfield, T.; Kaye, J.; Barfoot, J.; Blackburn, C.; Chan, S.; De Luca, M.; Kent, A.; Mccabe, C.; Munsie, M.; Sleeboom-Faulkner, M.; Sugarman, J.; Van Zimmeren, E.; Zarzeczny, A.; Rasko, J. E. J.

Commercial promotion of unsupported therapeutic uses of stem cells is a global problem that has proven resistant to regulatory efforts. Here, we suggest a coordinated approach at the national and international levels focused on engagement, harmonization, and enforcement to reduce the risks associated with direct-to-consumer marketing of unproven stem cell treatments.

2017 - Regeneration of the entire human epidermis using transgenic stem cells [Articolo su rivista]
Hirsch, Tobias; Rothoeft, Tobias; Teig, Norbert; Bauer, Johann W.; Pellegrini, Graziella; De Rosa, Laura; Scaglione, Davide; Reichelt, Julia; Klausegger, Alfred; Kneisz, Daniela; Romano, Oriana; SECONE SECONETTI, Alessia; Contin, Roberta; Enzo, Elena; Jurman, Irena; Carulli, Sonia; Jacobsen, Frank; Luecke, Thomas; Lehnhardt, Marcus; Fischer, Meike; Kueckelhaus, Maximilian; Quaglino, Daniela; Morgante, Michele; Bicciato, Silvio; Bondanza, Sergio; De Luca, Michele

Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, lifethreatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.

2016 - A discussion on cell therapy in Manchester [Articolo su rivista]
Cossu, Giulio; Buccione, Roberto; De Luca, Michele


2016 - Comparative assessment of cultures from oral and urethral stem cells for urethral regeneration [Articolo su rivista]
Corradini, F.; Zattoni, Michela; Barbagli, G.; Bianchi, Giampaolo; Giovanardi, M.; Serafini, Chiara; Genna, VINCENZO GIUSEPPE; Ribbene, A.; Balò, S.; Fidanza, Francesco Antonio; Lazzeri, M.; DE LUCA, Michele; Pellegrini, Graziella

Urethral reconstruction has received much attention in recent years, due to pathologies such as recurrence of urethral strictures after treatments. Various surgical techniques have been developed to obtain the best risk-benefit ratio, such as autologous grafts taken from the oral cavity. Tissue engineering and stem cells, growing tissue from a small biopsies, can further improve surgery, reducing invasiveness and morbidity. To determine whether urethra or other epithelia can be equally useful for urethra engineering, a comparison of clonogenic ability, proliferative potential and stem cell markers should be obtained. In this study, 19 biopsies from urethra, and 21 from oral mucosa were obtained from patients, during reconstructive surgery. Urethral and oral tissues were removed from the same donor, to develop primary cultures and cell characterization. The long term regenerative properties of both tissues was investigated in vitro by life span, clonal analysis and markers of different clonal types. Results revealed the same high proliferative potential for urethra and oral mucosa cultures, but maintenance of specific markers. Karyotype and growth factor dependence confirmed the normal phenotype of cultured cells. Clonal analysis of the proliferative compartment highlighted a very different proportion of stem and transient amplifying cells, characterised by dissimilar cell size profile and marker expression. In conclusion, both tissues can be cultured and preserve their stem cells in vitro. Few differences appeared in oral mucosa vs urethra, suggesting that they can be equally useful for tissue engineering of the urethral tract.

2016 - Holoclar: first of its kind in more ways than one [Articolo su rivista]
Milazzo, G; Ardigò, D; Toschi, M; Matuska, S; Rama, P; DE LUCA, Michele; Pellegrini, Graziella

This article describes the regulatory pathway that enabled the translation of academic research and clinical experience of an ex-vivo expanded autologous stem cell-based treatment for limbal stem cell deficiency (LSCD) into a pharmaceutical product compliant with the European Union regulations of Advanced Therapy Medicinal Products (ATMP). Holoclar® was originally developed in Italy as a surgical procedure and used in more than 200 patients. Following the establishment of the EU ATMP Regulation EC 1394/2007, Holoclar development required that manufacturing was as per current Good Manufacturing Practice requirements and collection of retrospective clinical data was ICH-E6 and E3 compliant. Holoclar is an ex-vivo expanded autologous human corneal epithelial cells containing stem cells and is classified as “tissue engineered product”. Based on the evidence of quality and control of the manufacturing process, safety, efficacy and on a positive benefit–risk balance in 104 patients (72.1%) of 148 patients treated, Holoclar received conditional Marketing Approval in the EU. Holoclar is the first medicine approved in the EU for this rare eye condition that can result in blindness.

2015 - Advances in Gene/Cell Therapy in Epidermolysis Bullosa [Articolo su rivista]
Murauer, Eva M; Koller, Ulrich; Pellegrini, Graziella; DE LUCA, Michele; Bauer, Johann W.

In the past few years, substantial preclinical and experimental advances have been made in the treatment of the severe monogenic skin blistering disease epidermolysis bullosa (EB). Promising approaches have been developed in the fields of protein and cell therapies, including allogeneic stem cell transplantation; in addition, the application of gene therapy approaches has become reality. The first ex vivo gene therapy for a junctional EB (JEB) patient was performed in Italy more than 8 years ago and was shown to be effective. We have now continued this approach for an Austrian JEB patient. Further, clinical trials for a gene therapy treatment of recessive dystrophic EB are currently under way in the United States and in Europe. In this review, we aim to point out that sustainable correction of autologous keratinocytes by stable genomic integration of a therapeutic gene represents a realistic option for patients with EB.

2015 - Amplicon-based Next Generation Sequencing: an effective approach to molecular diagnosis of Epidermolysis Bullosa [Abstract in Atti di Convegno]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, Antonio; Manfredini, Rossella; DE ROSA, Laura; Contin, Roberta; Pellacani, Giovanni; Giannetti, Alberto; Pagani, Jacopo; DE LUCA, Michele; Tagliafico, Enrico

Epidermolysis Bullosa (EB) is caused by mutations in genes encoding for proteins of the epidermal–dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, current methods in EB diagno- stics comprise immunohistochemistry on bioptic samples and transmission electron microscopy followed by single candidate gene Sanger Sequencing (SS) that therefore represents the final phase of a labour intensive and ex- pensive clinical pathway. Methods: Participants in a cross sectional study included individuals with Muenke syndrome (P250R mutation in FGFR3) and their mutation negative siblings. Participants completed validated assessments of executive functio- ning (Behavior Rating Inventory of Executive Function; BRIEF) and adaptive behavior skills (Adaptive Behavior Assessment System; ABAS-II). According to the recently published recommendations for diagnosis and treatment in EB, the assessment of mutational landscape is instead a fun- damental step to a comprehensive diagnosis path; Next Generation Sequen- cing (NGS), throughout parallel ultra-deep sequencing of many genes, would represent a proper method for reducing timing and costs in EB diagnostics. We developed an EB disease-comprehensive amplicon panel (AmpliSeq pa- nel), to accomplish NGS onto Ion Torrent PGM platform. The panel was dealt on ten patients with known genetic diagnosis, and then employed in eight family trios with unknown molecular footprinting. Results: Forty-four FGFR3 mutation positive individuals, median age 9, range 6 months to 52 years were evaluated with the BRIEF and ABAS-II. Additio- nally, 10 unaffected siblings were used as controls. For the General Executive Composite scale of the BRIEF, 32.1% of the cohort had scores greater than +1.5 SD, signifying “Potential Clinical Significance.” For the General Adaptive Composite of the ABAS-II, 28.2% of affected individuals scored in the “Ex- tremely Low” category” (3rd -8th percentile of normative population) and 53.9% were below the “Average” category (less than the 25th percentile). Multiple regression analysis showed that the presence of craniosynostosis was not a predictor (P = 0.7) of BRIEF and ABAS-II scores. The AmpliSeq panel, obtaining a proof of concept of the sensitivity, specificity, and accuracy of this kind of procedure, showed successful in finding the causative mutations in all the ten patients with known mutations, fully confirming SS data. Besides, showing consistent with the clinical diagnosis, it was effective in trios, identifying all the variants, even the ones SS missed or in case of de novo mutations. NGS

2015 - Amplicon-based next-generation sequencing: an effective approach for the molecular diagnosis of epidermolysis bullosa [Articolo su rivista]
Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, Antonio; De Rosa, Laura; Contin, Roberta; Manfredini, Rossella; Pellacani, Giovanni; Giannetti, Alberto; Pagani, J.; De Luca, Michele; Tagliafico, Enrico

Epidermolysis bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of biopsy samples and transmission electron microscopy followed by single-candidate gene Sanger sequencing (SS), which are labour-intensive and expensive clinical pathways.

Tenedini, Elena; Artuso, Lucia; Bernardis, Isabella; Artusi, Valentina; Percesepe, A; DE ROSA, Laura; Contin, Roberta; Manfredini, Rossella; Pellacani, Giovanni; Giannetti, A; DE LUCA, Michele; Tagliafico, Enrico

Background: Epidermolysis Bullosa (EB) is caused by mutations in genes that encode proteins belonging to the epidermal-dermal junction assembly. Due to the extreme clinical/genetic heterogeneity of the disease, the current methods available for diagnosing EB involve immunohistochemistry of bioptic samples and transmission electron microscopy followed by single candidate gene Sanger Sequencing (SS), which are labour intensive and expensive clinical pathways. Objectives: According to the recently published recommendations for the EB diagnosis and treatment, the assessment of the mutational landscape is now a fundamental step for developing a comprehensive diagnostic path. Next-Generation Sequencing (NGS) via the parallel ultra-deep sequencing of many genes represents a proper method for reducing the processing time and costs of EB diagnostics. Methods: We developed an EB disease-comprehensive AmpliSeq panel to accomplish the NGS on the Ion Torrent PGM platform. The panel was performed on ten patients with known genetic diagnoses and was then employed in eight family trios with unknown molecular footprints. Results: The panel was successful in finding the causative mutations in all ten of the patients with known mutations, fully confirming the SS data and providing proof of concept of the sensitivity, specificity, and accuracy of this procedure. In addition to being consistent with the clinical diagnosis, it was also effective in the trios, identifying all of the variants, including ones that the SS missed or de novo mutations. Conclusions: The NGS and AmpliSeq were shown to be an effective approach for the diagnosis of EB, resulting in a costand time-effective 72-hour procedure.

2014 - Concise review: hurdles in a successful example of limbal stem cell-based regenerative medicine [Articolo su rivista]
Pellegrini, Graziella; P., Rama; DI ROCCO, Antonio; Panaras, Athanasios; DE LUCA, Michele

Recent breakthroughs in regenerative medicine have generated enthusiasm and many efforts to explore new therapeutic potentials of both somatic and pluripotent stem cells. About 30 years passed since a discovery of a method of producing a great number of human epidermal keratinocytes by cultivation from a small skin biopsy, many possibilities are now envisaged for therapeutic application of different cultured cell types. The importance of stem cell content was proven for many tissues or organs in different pathologies. Ocular burns cause depletion of limbal stem cells, which lead to corneal opacification and visual loss. Most of available treatments are palliative and focused on the relief of the devastating clinical picture. This review is focused on recent developments cell based therapy of limbal stem cell deficiency. All findings can provide support for improvement and standardization of the cure for this disabling disease. Stem Cells 2013.

2014 - Corneal Bioengineering [Capitolo/Saggio]
Corradini, Francesca; Zattoni, Michela; Rama, Paolo; DE LUCA, Michele; Pellegrini, Graziella

The cornea is the main structure of the ocular surface and enables the transmission of light entering the eye. The cornea is covered by a nonkeratinized stratified epithelium, which is renewed by stem cells located in the basal layer of the limbus. Injury and disease, such as chemical and thermal burns, can destroy the limbus leading to a limbal stem cell deficiency (LSCD) with consequent visual loss. In 1997, Pellegrini et al. firstly described the use of ex vivo cultured limbal epithelial stem cell to treat LSCD. Since then, several reports describing alternative methods of the clinical use of this technology have been published but the retention of stem cells, which is the essential property of the graft, has not been investigated. The definition of a graftable limbal culture in light of the clinical performance must follow specific quality criteria, here discussed, which are relevant for the future use of any cultured cell type for clinical application.

2014 - Eyes on the prize: limbal stem cells and corneal restoration. [5YIF: 24,57; Citations: 5] [Articolo su rivista]
Pellegrini, Graziella; DE LUCA, Michele

Corneal diseases and blindness can result from deficiency in limbal stem cells, and autologous transplantation is often the only therapeutic option. Two recent studies in Nature have provided insights into regulation of limbal stem cell function and corneal regeneration and present new opportunities for clinical interventions in eye diseases.

2014 - Long-term stability and safety of transgenic cultured epidermal stem cells in gene therapy of junctional epidermolysis bullosa. [Articolo su rivista]
DE ROSA, Laura; Carulli, S; Cocchiarella, Fabienne; Quaglino, Daniela; Enzo, Elena; Franchini, Eleonora; Giannetti, A; DE SANTIS, Giorgio; Recchia, Alessandra; Pellegrini, Graziella; DE LUCA, Michele

We report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.

2014 - Stamina therapies: Let the record stand [Articolo su rivista]
Bianco, Paolo; Cattaneo, Elena; DE LUCA, Michele; Pani, Luca


2013 - Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells [Articolo su rivista]
Pellegrini, Graziella; Rama, P; Matuska, S; Lambiase, A; Bonini, S; Pocobelli, A; Colabelli, Rg; Spadea, L; Fasciani, R; Balestrazzi, E; Vinciguerra, P; Rosetta, P; Tortori, A; Nardi, M; Gabbriellini, G; Traverso, Ce; Macaluso, C; Losi, Lorena; Percesepe, Antonio; Venturi, Beatrice; Corradini, Francesca; Panaras, Athanasios; DI ROCCO, Antonio; Guatelli, P; DE LUCA, Michele

Aim: Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigated the biological parameters instrumental for their clinical success. Methods: We report a long-term multicenter prospective study on 152 patients carrying corneal destruction due to severe ocular burns, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3-J2 feeder cells. Clinical results were statistically evaluated both by parametric and nonparametric methods. Results: Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14 and 14.81% of eyes, respectively. The total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, the clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. No adverse effects related to the feeder layer have been observed and the regenerated epithelium was completely devoid of any 3T3‑J2 contamination. Conclusion: Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.

2013 - Isolation of human keratinocyte stem cells and high-throughput screening approach for their characterization [Abstract in Atti di Convegno]
Di Rocco, Antonio; Carulli, Sonia; Tenedini, Elena; Bianchi, Elisa; Tagliafico, Enrico; Manfredini, Rossella; Pellegrini, Graziella; DE LUCA, Michele

In the last three decades, regenerative medicine has opened new horizons for the in vitro reconstruction of epithelial tissues and gene therapy treatment of skin disorders involving the use of adult keratinocyte stem cells (KSCs). Although the ability to identify and isolate these cells represents an important prerequisite for the development of these approaches, molecular markers and their precise in vivo localization are still lacking. In order to define genes involved in the control of stemness and commitment of KSCs, we developed a non-invasive, stem cell-preserving magnetic micro beads based method in order to obtain a KSCs enriched population for high throughput screening experiments. After 3T3 murine fibroblast feeder layer depletion from our keratinocyte cultures, we isolated a subpopulation of basal epithelial cells on the basis of the different expression levels of the a6β4 integrin. By using different approaches, including clonal analysis and p63 bright cells quantification, we clearly showed that a6β4 integrin bright cells have greater growth potential and clonogenic capacity compared to the remaining cell fraction and they include the KSCs population. Comparing gene expression profile of a KSCs-enriched and a terminally differentiated cell population coming from the same original primary cell culture we defined a set of genes most probably involved in stemness maintenance. Ongoing gene profiling on single clone type will allow us to validate this gene signature and to start functional studies on selected genes. Extending this approach to different ectodermal derived tissues will provide a genome wide signature of the molecular pathways underlying self-renewal, commitment and differentiation of KSCs.

2013 - Regulation of stem cell therapies under attack in Europe: for whom the bell tolls [Articolo su rivista]
Paolo, Bianco; Roger, Barker; Oliver, Brüstle; Elena, Cattaneo; Hans, Clevers; George Q., Daley; DE LUCA, Michele; Lawrence, Goldstein; Olle, Lindvall; Christine, Mummery; Pamela G., Robey; Clara Sattler de Sousa e., Brito; Austin, Smith

At the time of writing, the Italian Parliament is debating a new law that would make it legal to practice an unproven stem cell treatment in public hospitals. The treatment, offered by a private non-medical organization, may not be safe, lacks a rationale, and violates current national laws and European regulations. This case raises multiple concerns, most prominently the urgent need to protect patients who are severely ill, exposed to significant risks, and vulnerable to exploitation. The scientific community must consider the context-social, financial, medical, legal-in which stem cell science is currently situated and the need for stringent regulation. Additional concerns are emerging. These emanate from the novel climate, created within science itself, and stem cell science in particular, by the currently prevailing model of 'translational medicine'. Only rigorous science and rigorous regulation can ensure translation of science into effective therapies rather than into ineffective market products, and mark, at the same time, the sharp distinction between the striving for new therapies and the deceit of patients.

2013 - The long and winding road that leads to a cure for epidermolysis bullosa [Articolo su rivista]
Carulli, Sonia; Contin, Roberta; DE ROSA, Laura; Pellegrini, Graziella; DE LUCA, Michele

Inherited epidermolysis bullosa (EB) is a family of rare genetic skin disorders characterized by structural and mechanical fragility of skin and mucosal membranes. The main feature of EB is the presence of recurrent skin blistering or erosions, which have a profound impact in the quality of life of EB patients and, in the most severe forms, cause early lethality. During the past two decades, it became possible to identify mutations in genes responsible for different types of EB and characterize the abnormalities of the related proteins. Nowadays, there is no cure for EB; all the treatments are palliative and focused on the relief of the devastating EB clinical picture. Recent advancements in molecular biology, stem cell biology and regenerative medicine have fostered new therapeutic approaches for EB. This review is focused on recent developments in gene therapy, protein replacement and cell-based therapy for EB, all aimed at finding a cure for this devastating disease.

2012 - Dormant and restless skin stem cells [Articolo su rivista]
De Rosa, Laura; De Luca, Michele

It has been unclear whether a uniform group of stem cells gives rise to most cells in the epidermis. A study reveals the presence of at least two stem-cell populations that have different proliferative abilities

2012 - Methods for characterization/manipulation of human corneal stem cells and their applications in regenerative medicine. [Capitolo/Saggio]
Corradini, Francesca; Venturi, Beatrice; Pellegrini, Graziella; DE LUCA, Michele

Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Speci fi cally, ocular burns cause depletion of limbal stem cells, which leads to corneal opaci fi cation and visual loss. Corneal stem cells are segregated in the basal layer of the limbus, which is the transitional zone of the epithelium located between the cornea and the bulbar conjunctiva. Autologous cultured limbal epithelial cells can restore damaged corneas. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets. We outline some quality criteria, which assure the clinical performance of keratinocyte culture: evaluation of the number of holoclones within a cultured epithelial graft, proportion of aborting colonies, and percentage of cells expressing high levels of D Np63 a .

2011 - Vision from the right stem. [5YIF: 9.93; Citations: 13] [Articolo su rivista]
Pellegrini, Graziella; P., Rama; DE LUCA, Michele

Cultures of limbal cells are a safe and effective treatmentfor the destruction of the human cornea owing to chemicalburns. The essential feature of the graft is the presenceof an adequate number of stem cells, which can bedetermined by the expression of the p63 transcriptionfactor. Here, we will discuss the general principles definingthe rigorous criteria for graftable limbal cultures inlight of their clinical performances. Such criteria mightprove relevant to the future therapeutic use of anycultured cell type.

2010 - Human embryonic stem cell-derived keratinocytes: how close to clinics? [5YIF: 24.57; Citations: 8] [Articolo su rivista]
Pellegrini, Graziella; De Luca, Michele

Recently in The Lancet, Guenou et al. (2009) demonstrate that human embryonic stem cells (hESCs) can differentiate into mature keratinocytes able to generate a pluristratified epithelium on immunodeficient mice. Their findings and the potential clinical use of hESC-derived keratinocytes will be discussed.

2010 - Limbal stem-cell therapy and long-term corneal regeneration. [Articolo su rivista]
Rama, P; Matuska, S; Paganoni, G; Spinelli, A; De Luca, Michele; Pellegrini, Graziella

BACKGROUND: Corneal renewal and repair are mediated by stem cells of the limbus, the narrow zone between the cornea and the bulbar conjunctiva. Ocular burns may destroy the limbus, causing limbal stem-cell deficiency. We investigated the long-term clinical results of cell therapy in patients with burn-related corneal destruction associated with limbal stem-cell deficiency, a highly disabling ocular disease.METHODS: We used autologous limbal stem cells cultivated on fibrin to treat 112 patients with corneal damage, most of whom had burn-dependent limbal stem-cell deficiency. Clinical results were assessed by means of Kaplan-Meier, Kruskal-Wallis, and univariate and multivariate logistic-regression analyses. We also assessed the clinical outcome according to the percentage of holoclone-forming stem cells, detected as cells that stain intensely (p63-bright cells) in the cultures.RESULTS: Permanent restoration of a transparent, renewing corneal epithelium was attained in 76.6% of eyes. The failures occurred within the first year. Restored eyes remained stable over time, with up to 10 years of follow-up (mean, 2.91+/-1.99; median, 1.93). In post hoc analyses, success--that is, the generation of normal epithelium on donor stroma--was associated with the percentage of p63-bright holoclone-forming stem cells in culture. Cultures in which p63-bright cells constituted more than 3% of the total number of clonogenic cells were associated with successful transplantation in 78% of patients. In contrast, cultures in which such cells made up 3% or less of the total number of cells were associated with successful transplantation in only 11% of patients. Graft failure was also associated with the type of initial ocular damage and postoperative complications.CONCLUSIONS: Cultures of limbal stem cells represent a source of cells for transplantation in the treatment of destruction of the human cornea due to burns.

2009 - Epithelial stem cells in corneal regeneration and epidermal gene therapy. [5YIF: 6.94; Citations: 68] [Articolo su rivista]
Pellegrini, Graziella; P., Rama; Mavilio, Fulvio; DE LUCA, Michele

Regenerative medicine refers to innovative therapies aimed at the permanent restoration of diseased tissues and organs. Regeneration of self-renewing tissues requires specific adult stem cells, which need to be genetically modified to correct inherited genetic diseases. Cultures of epithelial stem cells permanently restore severe skin and mucosal defects, and genetically corrected epidermal stem cells regenerate a normal epidermis in patients carrying junctional epidermolysis bullosa. The keratinocyte stem cell is therefore the only cultured stem cell used both in cell therapy and gene therapy clinical protocols. Epithelial stem cell identification, fate and molecular phenotype have been extensively reviewed, but not in relation to tissue regeneration. In this paper we focus on the localization and molecular characterization of human limbal stem cells in relation to corneal regeneration, and the gene therapy of genetic skin diseases by means of genetically modified epidermal stem cells.

2009 - Gene therapy of inherited skin adhesion disorders: a critical overview [Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella; Mavilio, Fulvio

Gene therapy has the potential to treat devastating inherited diseases for which there is little hope of finding a conventional cure. These include lethal diseases, like immunodeficiencies or several metabolic disorders, or conditions associated with a relatively long life expectancy but poor quality of life and expensive and life-long symptomatic treatments, such as muscular dystrophy, cystic fibrosis and thalassaemia. Skin adhesion defects belong to both groups. For the nonlethal forms, gene therapy, or transplantation of cultured skin derived from genetically corrected epidermal stem cells, represents a very attractive therapeutic option, and potentially a definitive treatment. Recent advances in gene transfer and stem cell culture technology are making this option closer than ever. This paper critically reviews the progress and prospects of gene therapy for epidermolysis bullosa, and the technical and nontechnical factors currently limiting its development.

2009 - In vitro evidence of nerve growth factor effects on human conjunctival epithelial cell differentiation and mucin gene expression. [5YIF: 3.67; Citations: 24] [Articolo su rivista]
Lambiase, A; Micera, A; Pellegrini, Graziella; Merlo, D; Rama, P; DE LUCA, Michele; Bonini, S; Bonini, S.

PURPOSE: Mucins released into the tear film are crucial to maintaining a healthy ocular surface. Alterations in goblet cell numbers and mucin secretion are observed in chronic ocular surface inflammatory diseases. Nerve growth factor (NGF) plays a crucial role in healing and inflammation of the ocular surface. The aim of this study was to evaluate in vitro the effect of NGF on conjunctival goblet cell differentiation and mucin production and secretion. METHODS: Human conjunctival epithelial cells were exposed to increasing NGF concentrations (1 to 250 ng/mL) and analyzed to quantify cell growth (MTT/Ki67/BrdU), goblet cell differentiation (PAS/MUC5AC confocal staining), and mucin mRNA expression (real-time PCR). Secreted and cellular MUC5AC were also analyzed by sandwich-ELISA and FACS, respectively. To confirm the biological effects of NGF, the same evaluations were performed on primary cultures, and changes in markers of stemness (p63) and commitment (14-3-3 sigma) were also investigated. RESULTS: In cell cultures, NGF induced a dose-dependent increase of goblet cell numbers, MUC5AC production, storage, and release. Additionally, in primary cultures, NGF induced an increase of abortive colonies and 14-3-3 sigma protein, and a decrease of p63 mRNA and protein, suggesting a differentiating effect of NGF on human conjunctival epithelium. CONCLUSIONS: These findings show that NGF might play a role in the complex mechanism leading to conjunctival epithelium differentiation and mucin secretion. In addition to the known roles of NGF in promoting ocular surface healing and sensitivity, its effects on conjunctival goblet cells support a rationale to investigate the therapeutic effectiveness of NGF in dry eye disease.

2009 - Isolation and Production of Cells Suitable for Human Therapy: Challenges Ahead [Articolo su rivista]
Ahrlund Richter, L; DE LUCA, Michele; Marshak, Dr; Munsie, M; Veiga, A; Rao, M.

Considerable practical hurdles must be overcome prior to the broad application of stem cell therapies. We outline challenges that may vary across different models of cell therapy, including the following broad concepts: issues related to the sourcing of material, and issues related to product manufacturing, shipping, storage and tracking, and standardization.

2008 - Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors [Articolo su rivista]
DI NUNZIO, Francesca; Maruggi, Giulietta; Stefano, Ferrari; Enzo Di, Iorio; Poletti, Valentina; Marta, Garcia; Marcela Del, Rio; DE LUCA, Michele; Fernando, Larcher; Pellegrini, Graziella; Mavilio, Fulvio

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer–restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.

2008 - Gene therapy of inherited skin adhesion disorders [Articolo su rivista]
De Luca, M.; Pellegrini, G.; Mavilio, F.

Gene therapy is a potential treatment for devastatinginherited diseases for which there is little hope offinding a conventional cure. These include lethal diseaseslike immunodeficiencies and metabolic disorders,and non-lethal conditions associated with poorquality of life and life-long symptomatic treatments,like muscular dystrophy, cystic fibrosis or thalassemia.Skin adhesion defects belong to both groups. For thenon-lethal forms, gene therapy, or transplantation ofcultured skin derived from genetically corrected epidermalstem cells, represents a very attractive therapeuticoption, and potentially a definitive treatment.Recent advances in gene transfer and stem cell culturetechnology are making this option closer than ever.This paper critically reviews the progress and prospectsof gene therapy for skin adhesion defects, andthe technical and non-technical factors currently limitingits development.

2008 - New ISSCR Guidelines Underscore Major Principles for Responsible Translational Stem Cell Research [Articolo su rivista]
Hyun, I; Lindvall, O; Ahrlund Richter, L; Cattaneo, E; Cavazzana Calvo, M; Cossu, G; DE LUCA, Michele; Fox, Ij; Gerstle, C; Goldstein, Ra; Hermerén, G; High, Ka; Kim, Ho; Lee, Hp; Levy Lahad, E; Li, L; Lo, B; Marshak, Dr; Mcnab, A; Munsie, M; Nakauchi, H; Rao, M; Rooke, Hm; Valles, Cs; Srivastava, A; Sugarman, J; Taylor, Pl; Veiga, A; Wong, Al; Zoloth, L; Daley, G. Q.

The International Society for Stem Cell Research (ISSCR) task force that developed new Guidelines for the Clinical Translation of Stem Cells discusses core principles that should guide the responsible transition of basic stem cell research into appropriate clinical applications.

2007 - C/EBPδ regulates cell cycle and self-renewal of human limbal stem cells. [Articolo su rivista]
Barbaro, V; Testa, Anna; DI IORIO, E; Mavilio, Fulvio; Pellegrini, Graziella; DE LUCA, Michele

Human limbal stem cells produce transit amplifying progenitors that migrate centripetally to regenerate the corneal epithelium. Coexpression of CCAAT enhancer binding protein (C/EBP), Bmi1, and Np63 identifies mitotically quiescent limbal stem cells, which generate holoclones in culture. Upon corneal injury, a fraction of these cells switches off C/EBP and Bmi1, proliferates, and differentiates into mature corneal cells. Forced expression of C/EBP inhibits the growth of limbal colonies and increases the cell cycle length of primary limbal cells through the activity of p27Kip1 and p57Kip2. These effects are reversible; do not alter the limbal cell proliferative capacity; and are not due to apoptosis, senescence, or differentiation. C/EBP, but not Np63, indefinitely promotes holoclone self-renewal and prevents clonal evolution, suggesting that self-renewal and proliferation are distinct, albeit related, processes in limbal stem cells. C/EBP is recruited to the chromatin of positively (p27Kip1 and p57Kip2) and negatively (p16INK4A and involucrin) regulated gene loci, suggesting a direct role of this transcription factor in determining limbal stem cell identity.

2007 - Towards therapeutic application of ocular stem cells [Articolo su rivista]
Pellegrini, Graziella; DE LUCA, Michele; Arsenijevic, Y.

The first example of cell therapy using cultured stem cells dates back to 1981, when it was demonstrated that human epidermis could be grown in the laboratory and transplanted onto burnt patients to reconstitute a functional epidermis [Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc Natl Acad Sci USA 1979;76(11):5665–8: [1]; Banks-Schlegel S, Kehinde O, Green H. Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1981;1:75–8: [2]; Gallico 3rd GG, O’Connor NEMJ, Compton CC, Kehinde O, Green H. Permanent coverage of large burn wounds with autologous cultured human epithelium. N Engl J Med 1984;311(7):448–51: [3]]. This was the onset of regenerative medicine, which is now being developed also in many other fields including ophthalmology.Emerging cell therapies for the restoration of sight have focused on two areas of the eye that are critical for visual function, the cornea and the retina. The relatively easy access of the cornea, the homogeneity of the cells forming the different layers of the corneal epithelium and the improvement of cell culture protocols are leading to considerable success in corneal epithelium restoration. Rebuilding the entire cornea is however still far from reality. The restoration of the retina has recently been achieved in different animal models of retinal degeneration using immature photoreceptors, and two other promising strategies have been demonstrated: transplantation of endothelial precursors to rescue retinal vessels and neurons, and transplantation of retinal pigmented epithelial cells to preserve vision over the long term. The relevance of these approaches will be discussed in function of the disease targeted.

2006 - Aberrant splicing in the ocular albinism type 1 gene (OA1/GPR143) is corrected in vitro by morpholino antisense oligonucleotides [Articolo su rivista]
F., Vetrini; R., Tammaro; S., Bondanza; Em, Surace; A., Auricchio; DE LUCA, Michele; A., Ballabio; Marigo, Valeria

An intronic point mutation was identified in the ocular albinism type 1 (OA1) gene (HUGO symbol, GPR143) in a family with the X-linked form of ocular albinism. Interestingly, the mutation creates a new acceptor splice site in intron 7 of the OA1 gene. In addition to low levels of normally spliced mRNA product of the OA1 gene, the patient samples contained also an aberrantly spliced mRNA with a 165 bp fragment of intron 7 (from position +750 to +914) inserted between exons 7 and 8. The abnormal transcript contained a premature stop codon and was unstable, as revealed by Northern blot analysis. We defined that mutation NC_000023.8:g.25288G > A generated a consensus binding motif for the splicing factor enhancer ASF/SF2, which most likely favored transcription of the aberrant mRNA. Furthermore, it activated a cryptic donor-splice site causing the inclusion between exons 7 and 8 of the 165 bp intronic fragment. Thus, the aberrant splicing is most likely explained by the generation of a de novo splicing enhancer motif. Finally, to rescue OA1 expression in the patient's melanocytes, we designed an antisense morpholino modified oligonucleotide complementary to the mutant sequence. The morpholino, oligonucleotide (MO) was able to rescue OA1 expression and restore the OA1 protein level in the patient's melanocytes through skipping of the aberrant inclusion. The use of MO demonstrated that the lack of OA1 was caused by the generation of a new splice site. Furthermore, this technique will lead to new approaches to correct splice site mutations that cause human diseases.

2006 - Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells. [Articolo su rivista]
Mavilio, Fulvio; Pellegrini, Graziella; Ferrari, S.; DI NUNZIO, Francesca; Di Iorio, E.; Recchia, Alessandra; Maruggi, Giulietta; Ferrari, G.; Provasi, E.; Bonini, C.; Capurro, S.; Conti, A.; Magnoni, Cristina; Giannetti, Alberto; DE LUCA, Michele

The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease.

2006 - Expression of VSX1 in human corneal keratocytes during differentiation into myofibroblasts in response to wound healing [Articolo su rivista]
V., Barbaro; E., Di Iorio; S., Ferrari; L., Bisceglia; A., Ruzza; DE LUCA, Michele; Pellegrini, Graziella

PURPOSE. To characterize the expression of the visual system homeobox gene (VSX1) in human corneal keratocytes both in vitro and in vivo. METHODS. The expression of VSX1 was evaluated through semi-quantitative RT-PCR, immunofluorescence and in situ hybridization both in corneas (either freshly obtained or wounded) and in collagenase/hyaluronidase-isolated keratocytes grown in the absence or presence of serum to promote keratocyte-to-myofibroblast differentiation. RESULTS. Quiescent or resting keratocytes normally residing in the corneal stroma or cultured in vitro in the absence of serum did not express VSX1. In wounded corneas or when cultured in the presence of serum to mimic wound-healing responses, keratocytes underwent fibroblastic transformation (with appearance of alpha-SMA and disappearance of CD-34 and keratocan signals) and started expressing VSX1. CONCLUSIONS. The results show that VSX1 is expressed in vitro and in vivo during human corneal wound healing, a process in which differentiation of corneal keratocytes into myofibroblasts occurs. These data may help to elucidate the role of VSX1 in cornea physiology suggesting a potential involvement in cornea-related diseases such as keratoconus.

2006 - Facciamo il punto sulla medicina rigenerativa della superficie oculare [Capitolo/Saggio]
Pellegrini, Graziella; DE LUCA, Michele

Medicina rigenerativa della superficie oculare

2006 - Gene therapy in combination with tissue engineering to treat epidermolysis bullosa [Articolo su rivista]
S., Ferrari; Pellegrini, Graziella; T., Matsui; Mavilio, Fulvio; DE LUCA, Michele

Abstract:In the last 20 years epidermal stem cells have been extensively used for tissue regeneration of epidermis and other epithelial surfaces. The tremendous progress achieved has led to the development of protocols aimed at the correction of rare genetic disorders such as epidermolysis bullosa (EB), a severe, often lethal, blistering disorder of the skin. Approximately 400,000 – 500,000 people are affected worldwide and no definitive treatments have yet been developed. Gene therapy might represent an alternative therapeutic approach. This paper reviews the different strategies used to genetically modify keratinocytes from EB patients and addresses issues such as the use of in vivo or ex vivo approaches, how to target keratinocytes with stem cell properties in order to have long-term therapeutic gene expression, and which gene transfer agents should be used. The progress made has led the authors' group to submit a request for a Phase I/II ex vivo therapy clinical trial for patients with junctional EB.

2006 - Q-FIHC: Quantification of fluorescence immunohistochemistry to analyse p63 isoforms and cell cycle phases in human limbal stem cells [Articolo su rivista]
Di Iorio, E; Barbaro, V; Ferrari, S; Ortolani, C; DE LUCA, Michele; Pellegrini, Graziella

Fluorescence microscopy has long been used for qualitative characterization of various parameters such as subcellular distribution of proteins, lipids, nucleic acids, and ions. However, quantification of these parameters is complicated by a variety of optical, biological, and physical factors. In the last decade, the progress achieved with powerful softwares and digital image processing systems has facilitated the development of fluorescence immunohistochemistry (FIHC) into a widely used quantitative assay (quantitative-FIHC or Q-FIHC). We describe here a rapid and sensitive Q-FIHC assay based on the use of a laser scanning confocal microscope and advanced image analysis softwares (Zeiss semi automatic LSM 510 and fully automatic Axiovision 4.4) for the detection and quantification of fluorescent intensity in human corneal tissues and cells obtained from small clinical samples. We have used this methodology to characterize and quantify the gene expression profile of p63 and its Delta N alpha isoform, specific markers of human limbal stem cells. The validity of this method was evaluated through comparative studies with conventional approaches suggesting no significant differences and providing an alternative technique to traditional methods. Since Q-FIHC requires at least 20-fold less cells than traditional techniques, we have adopted it as the main quality control for our limbal cultures destined to clinical application.

2006 - Regeneration of squamous epithelia from stem cells of cultured grafts [Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella; Green, H.

The only cultured cell types extensively used for tissue regeneration are the keratinocyte and the chondrocyte. Cultured autologous keratinocytes derived from the epidermis have been used for many years to produce grafts that regenerate an epidermis over a full-thickness wound, such as a third-degree burn. But there have been many failures of engraftment, and in the absence of criteria for the quality of the cultures, the causes of failure cannot be analyzed. It has become clear that the essential feature of the graft is the presence of an adequate number of stem cells. This article describes the criteria for estimating that number. Advances in graft preparation, combining better preservation of stem cells with ease of application of the graft, are also described. These improvements have been applied to cultures of ocular limbal cells, which contain the keratinocyte stem cells of the corneal epithelium. Cultures meeting the criteria of stem cell number have been grafted to 116 patients suffering from chemical destruction of the limbus. The procedure has been highly successful in the alleviation of suffering and the restoration of vision.

2006 - Towards a gene therapy clinical trial for epidermolysis bullosa [Articolo su rivista]
Ferrari, S; Pellegrini, Graziella; Matsui, T; Mavilio, Fulvio; DE LUCA, Michele

Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the underneath derma lead to severe, often lethal, blistering disorders of the skin known as Epidermolysis Bullosa (EB). About 400000-500000 people worldwide are affected and no definitive treatments have yet been developed. Gene therapy might represent an alternative therapeutic approach for these devastating inherited disorders. In the last 10 years pre-clinical studies have shown that human epidermal stem cells can be stably transduced using integrating vectors allowing long-term genetic correction of the adhesion defects affecting EB keratinocytes both in vitro and in vivo after transplantation onto immunodeficient animals. In addition tremendous progress have been achieved in the clinical applications of cultured keratinocytes (cell therapy) for the regeneration of the epidermis over full thickness wounds or the restoration of damaged corneal surfaces. The combination of (i) optimised culturing conditions not altering the epidermal stemness, (ii) gene transfer vectors able to target epidermal stem cells very efficiently and (iii) surgical procedures allowing the grafting of large skin areas have therefore led our group to submit the first phase I/II gene therapy clinical trial for Junctional Epidermolysis Bullosa.

2005 - Gene therapy approaches for epidermolysis bullosa [Articolo su rivista]
S., Ferrari; Pellegrini, Graziella; Mavilio, Fulvio; DE LUCA, Michele

Human epidermis consists of a stratified epithelium mainly composed of keratinocytes and relies on a stem cell compartment to undergo constant regeneration, Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the epidermal basement membrane lead to severe. and very often lethal, blistering disorders known as epidermolysis bullosa. Gene therapy represents a promising potential treatment for these devastating inherited disorders. Human epidermal stem cells can be cultivated ex vivo and stably transduced with integrating gene transfer vectors. allowing genetic and, more important, phenotypic correction of the adhesion properties of keratinocytes, Here we will review some of the issues that need to be addressed to make gene therapy a realistic treatment for these disorders, such as (1) which cells should be targeted, (2) which approach (in vivo or ex vivo) should be chosen, and (3) which gene transfer vector (retrovirus, lentivirus, or integrating nonviral strategies,) should be used for stable gene correction. In the last 10 years, many reports have shown that gene transfer approaches to target epidermal stem cells are feasible and able to restore the adhesion properties of primary keratinocytes from patients with epidermolysis bullosa. In addition, tremendous progress has been achieved in culturing epidermal stem cells and generating sheets of stratified epithelium tor permanent coverage of full-thickness bums. Gene modification of stem cells in combination with advanced tissue-engineering techniques could therefore represent a realistic option for patient-4 with epidermolysis bullosa.

2005 - Isoforms of ΔNp63 and the migration of ocular limbal cells in human corneal regeneration [Articolo su rivista]
Di Iorio, E; Barbaro, V; Ruzza, A; Ponzin, D; Pellegrini, Graziella; DE LUCA, Michele

The p63 gene generates transactivating and N-terminally truncated transcripts (Delta Np63) initiated by different promoters. Alternative splicing gives rise to three different C termini, designated alpha, beta, and gamma. In the ocular epithelium, the corneal stem cells, which are segregated in the basal layer of the limbus, contain the a isoform but not beta or gamma. Holoclones derived from the limbus are rich in a, meroclones contain little, and paraclones contain none. In normal resting corneal epithelium, p63 of all isoforms is absent. Upon corneal wounding, cells originating from the limbus and containing a migrate progressively through the epithelium of the peripheral and central cornea. in the absence of an attached limbus, no a isoform appears in the corneal epithelium. When migrating cells containing the a isoform appear in the wounded corneal epithelium, they are confined to the basal layer, but the suprabasal cells, not only of the cornea but of the limbus as well, contain mRNA encoding beta and gamma. These data support the concept that the a isoform of p63 is necessary for the maintenance of the proliferative potential of limbal stem cells and their ability to migrate over the cornea. The beta and gamma isoforms, being suprabasal and virtually absent from the resting limbus, are not stem cell markers but are likely to play a role in epithelial differentiation specifically during the process of corneal regeneration.

2005 - Separation of keratan-sulfate-derived disaccharides by high-performance liquid chromatography and postcolumn derivatization with 2-cyanoacetamide and fluorimetric detection [Articolo su rivista]
Volpi, Nicola; Maccari, Francesca; S., Ferrari; DE LUCA, Michele; Pellegrini, Graziella

In this paper, we report a rapid, sensitive, and quantitative procedure to conduct disaccharide compositional analyses of keratan sulfates (KS) by means of high-performance liquid chromatography (HPLC) separation and postcolumn derivatization wit. h 2-cyanoacetamide and fluorimetric detection of products generated by hydrolysis of this glycosaminoglycan with Bacillus sp. keratanase 11 or Escherichia freundii endo-beta-galactosidase. Following E. freundii endo-p-galactosidase digestion of bovine corneal KS, the monosulfated disaccharide glcNAc6 beta 3(1 -> 3)gal, accounting for approximate to 95% nmol and 50% yield products, is produced. On the contrary, bovine corneal KS treated with endo-beta-N-acetylglucosaminidase (keratanase 11) from Bacillus sp. generates two major products, the monosulfated disaccharide gal beta(1 -> 4)glcNAc6s (approximate to 50% nmol product) and the disulfated disaccharide gal6s beta(I -> 4)glcNAc6s (approximate to 40% nmol product) for over 90% nmol products. These disaccharides are separated and readily determined within 30 min by using a linear-gradient strong anion-exchange separation. A linear relationship was found for the two purified disaccharides over a wide range of concentrations, from approximate to 108 pmol, 50 ng, to 2160 pmol, 1000 ng, for the disaccharide gal beta(I -> 4)glcNAc6s, and from 92 pmol, 50 ng, to 1840 pmol, 1000 ng, for the disaccharide gal6s beta(I -> 4)glcNAc6s. HPLC analysis was applied to the quantitative and qualitative determination of KS produced by 3T3-J2 murine fibroblasts in the cell medium. The amount of KS was found to be 2.80 +/- 0.34 mu g/ml/ 106 cells and composed of approximate to 71%nmol of disaccharide gal beta(1 -> 4)glcNAc6s and 18%nmol of the disulfated disaccharide gal6s beta(1 -> 4)glcNAc6s having approximate to 1.20 sulfate groups/disaccharide. Our data illustrate that the HPLC procedure reported represents an improved approach for the quantitative and compositional microanalyses of KS, especially applicable to experimentation involving small amounts ( 50 ng) of this glycosaminoglycan and in relation to its biological function and pathological importance.

2005 - Stem cell plasticity: time for a reappraisal? [Articolo su rivista]
Rm, Lemoli; F., Bertolini; R., Cancedda; DE LUCA, Michele; A., DEL SANTO; G., Ferrari; Ferrari, Sergio; G., Martino; Mavilio, Fulvio; S., Tura

n recent years an increasing number of publications have claimed that adult mammalian stem cells (SC) may be capable of differentiating across tissue lineage boundaries and that this plasticity may represent a novel therapeutic strategy for tissue regeneration. However, after a first phase of excitement, the issue of somatic SC plasticity remains controversial and the therapeutic perspectives are still elusive. In this review, we examine the general mechanisms which govern the function of SC, the identification and functional characterization of adult SC of different tissues and their putative capacity to transdifferentiate into mature cells of different origin. The potential clinical applications of adult SC for regenerative medicine are also discussed in each chapter. The method employed for preparing this review was the informal consensus development. Members of the Working Group on SC met four times and discussed the single points, previously assigned by the Chairman (S.T.), in order to achieve an agreement on different opinions and approve the final manuscript. All the authors of the present review have been working in the field of SC and have contributed original papers to peer-reviewed journals. In addition to the authors' own work, the present review examines articles published in journals covered by the Science Citation Index and Medline.

2004 - Novel mutation in the CHST6 gene causing macular corneal dystrophy [Articolo su rivista]
Abruzzese, C.; Kuhn, U.; Romanelli, G.; Molina, F.; Rama, P.; DE LUCA, Michele

Macular corneal dystrophy (MCD) is an autosomal recessive disease characterized by corneal opacities and caused by mutations in a carbohydrate sulfotransferase gene, known as CHST6. MCD type I patients show missense mutations in the CHST6-coding region, and MCD type II patients show a large deletion and replacement in the upstream region of CHST6. The objective of this study was to identify the genetic defect in CHST6 gene causing MCD in Italian families. We investigated MCD genotype by using polymerase chain reaction followed by direct sequencing, and results were confirmed by restriction analysis. An enzyme-linked immunosorbent assay was performed to assess the presence of sulfated keratan sulfate in the serum of MCD patients. Biochemical analysis revealed a MCD type I phenotype in two families and a type II phenotype in another family. Two novel missense mutations and a polymorphism in the coding region of CHST6 gene were identified in patients with MCD type I. In one MCD II family, a homozygous deletion in the upstream region of CHST6 gene was found.

2004 - Permanent repigmentation of piebaldism by erbium:YAG laser and autologous cultured epidermis [Articolo su rivista]
Guerra, L; Primavera, G; Raskovic, D; Pellegrini, Graziella; Golisano, O; Bondanza, S; Kuhn, S; Piazza, P; Luci, A; Atzori, F; DE LUCA, Michele

BACKGROUND: Several surgical techniques have been proposed for the treatment of piebaldism. These procedures, however, are poorly suited for the treatment of large leucodermal lesions, can cause scars and require multiple donor sites. Recently, it has been reported that autologous cultured epidermis induces scarless repigmentation of large vitiligo lesions, using a single small donor site. OBJECTIVES: To induce permanent repigmentation of large achromic lesions in patients suffering from piebaldism by means of autologous cultured epidermal grafts using a rapid, simple and non-invasive surgical procedure. METHODS: Six patients with piebaldism were enrolled in this study. Achromic epidermis was removed by means of appropriately set erbium:YAG laser and autologous cultured epidermal grafts were applied on to the recipient bed. Melanocyte content was evaluated by 3,4-dihydroxyphenylalanine reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. RESULTS: Autologous cultured epidermis, bearing a controlled number of melanocytes, induced repigmentation of all piebald lesions. The mean percentage repigmentation was 95.45% (2791.5 cm2 repigmented/2924.2 cm2 transplanted). CONCLUSIONS: Autologous cultured epidermal grafts induce permanent and complete repigmentation of piebald lesions, in the absence of scars. Erbium:YAG laser surgery is a rapid and precise tool for disepithelialization, hence allowing treatment of large piebald lesions during a single surgical operation.

2004 - Telomerase activity is sufficient to bypass replicative senescence in human limbal and conjunctival but not corneal keratinocytes [Articolo su rivista]
Pellegrini, Graziella; E., Dellambra; P., Paterna; O., Golisano; C. E., Traverso; P., Rama; P., Lacal; DE LUCA, Michele

The human ocular surface is covered by the conjunctival,corneal and limbal stratified epithelia. While conjunctival stemcells are distributed in bulbar and forniceal conjunctiva,corneal stem cells are segregated in the basal layer of thelimbus, which is the transitional zone between the cornea andthe bulbar conjunctiva. Keratinocyte stem and transientamplifying (TA) cells when isolated in culture give rise toholoclones and paraclones, respectively. Keratinocyte replicativesenescence ensues when all holoclones have generatedparaclones which express high levels of p16INK4a. In the presentstudy, we show that enforced telomerase activity induces thebypass of replicative senescence in limbal and conjunctivalkeratinocytes, without the inactivation of the p16INK4a/Rbpathway or the abrogation of p53 expression. hTERT-transducedlimbal and conjunctival keratinocytes are capable torespond to both growth inhibitory and differentiation stimuli,since they undergo growth arrest in response to phorbol esters,and activate p53 upon DNA damage. Following a sustainedPKC stimulation, occasional clones of p16INK4a-negative cellsemerge and resume ability to proliferate. Telomerase activity,however, is unable to induce the bypass of senescence in cornealTA keratinocytes cultured under the same conditions. Thesedata support the notion that telomere-dependent replicativesenescence is a general property of all human somatic cells,including keratinocytes, and suggest that telomerase activity issufficient to extend the lifespan only of keratinocytes endowedwith high proliferative potentials (which include stem cells), butnot of TA keratinocytes.

2003 - Erbium:YAG laser and cultured epidermis in the surgical therapy of stable vitiligo. [Articolo su rivista]
Guerra, L; Primavera, G; Raskovic, D; Pellegrini, Graziella; Golisano, O; Bondanza, S; Paterna, P; Sonego, G; Gobello, T; Atzori, F; Piazza, P; Luci, A; DE LUCA, Michele

BJECTIVE: To induce complete and reproducible repigmentation of large "stable" vitiligo lesions by means of autologous cultured epidermal grafts using a rapid, simple, and minimally invasive surgical procedure. DESIGN: Achromic epidermis was removed by means of appropriately settled erbium:YAG laser, and autologous epidermal grafts were applied onto the recipient bed. Melanocyte content was evaluated by dopa reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. SETTING: A biosafety level 3-type cell culture facility, a surgical ambulatory department, and a dermatological department in a hospital. PATIENTS: Twenty-one patients with different types of vitiligo were admitted to the study and treated with autologous cultured epidermal grafts. Inclusion criteria were failure of at least 2 standard medical approaches; no therapy for at least 12 months; no progression of old lesions or appearance of new lesions; no Koebner phenomenon within the past 18 months; and no autoimmune disorders. RESULTS: The average percentage of repigmentation in 21 patients was 75.9% (1759.7 cm2 repigmented/2315.8 cm2 transplanted). Three patients showed a reactivation of their vitiligo and did not show repigmentation. The remaining 18 patients, with 43 distinct lesions, showed an average percentage of repigmentation of 90% (1759.7 cm2 repigmented/1953.4 cm2 transplanted). CONCLUSIONS: Under appropriate conditions, cultured epidermal grafts induce complete repigmentation of stable vitiligo lesions. Erbium:YAG laser surgery can supply a fast and precise tool for disepithelialization, hence allowing treatment of large vitiligo lesions during a single surgical operation.

2003 - sAPP as a regulator of dendritic motility and melanin release in epidermal melanocytes and melanoma cells. [Articolo su rivista]
Quast, T.; Wehner, S.; Kirfel, G.; Jaeger, K; DE LUCA, Michele; Herzog, V.

Numerous factors including ultraviolet (UV) radiation and growth factors regulate the specific function of epidermal melanocytes. A recently discovered epidermal growth factor is sAPP, the soluble N-terminal ectodomain of the β-amyloid precursor protein (APP). Using whole mount preparations of isolated human epidermis, we detected a small population of basal cells, which expressed exceptionally high levels of APP. These cells were identified as melanocytes, which, similar to keratinocytes and neuronal cells, expressed the three APP isoforms 695, 751, and 770. They differed in their expression pattern from that of neuronal cells by expressing only low levels of APP 695. Melanocytes and melanoma cells in vitro released, in addition to keratinocytes, large quantities of sAPP. Because of its growth factor function, we studied possible effects of sAPP on melanocytes. Recombinant sAPP strongly increased lamellipodia activity at dendritic tips, an effect that coincided with increased release of melanin particles. Our observations point to the possible use of APP as an immunocytochemical marker for melanocytes. They suggest that sAPP derived from keratinocytes and/or melanocytes belongs to a family of factors operating in the paracrine and/or autocrine regulation of melanocyte function.

2002 - A two-stage, p16INK4A- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status [Articolo su rivista]
Rheinwald, Jg; Hahn, Wc; Ramsey, Mr; Wu, Jy; Guo, Z; Tsao, H; DE LUCA, Michele; Catricalà, C; O'Toole, K. M.

With increasing frequency during serial passage in culture, primary human keratinocytes express p16INK4A (p16) and undergo senescence arrest. Keratinocytes engineered to express hTERT maintain long telomeres but typically are not immortalized unless, by mutation or other heritable event, they avoid or greatly reduce p16 expression. We have confirmed that keratinocytes undergo p16-related senescence during growth in culture, whether in the fibroblast feeder cell system or in the specialized K-sfm medium formulation, and that this mechanism can act as a barrier to immortalization following hTERT expression. We have characterized the p16-related arrest mechanism more precisely by interfering specifically with several regulators of cell cycle control. Epidermal, oral mucosal, corneal limbal, and conjunctival keratinocytes were transduced to express a p16-insensitive mutant cdk4 (cdk4R24C), to abolish p16 control, and/or a dominant negative mutant p53 (p53DD), to abolish p53 function. Expression of either cdk4R24C or p53DD alone had little effect on life span, but expression of both permitted cells to divide 25 to 43 population doublings (PD) beyond their normal limit. Keratinocytes from a p16+/- individual transduced to express p53DD alone displayed a 31-PD life span extension associated with selective growth of variants that had lost the wild-type p16 allele. Cells in which both p53 and p16 were nonfunctional divided rapidly during their extended life span but experienced telomere erosion and ultimately ceased growth with very short telomeres. Expression of hTERT in these cells immortalized them. Keratinocytes engineered to express cdk4R24C and hTERT but not p53DD did not exhibit an extended life span. Rare immortal variants exhibiting p53 pathway defects arose from them, however, indicating that the p53-dependent component of keratinocyte senescence is telomere independent. Mutational loss of p16 and p53 has been found to be a frequent early event in the development of squamous cell carcinoma. Our results suggest that such mutations endow keratinocytes with extended replicative potential which may serve to increase the probability of neoplastic progression.

2002 - Effective retrovirus-mediated gene transfer in normal and mutant human melanocytes [Articolo su rivista]
Schiaffino, M. V.; Dellambra, E.; Cortese, K.; Baschirotto, C.; Bondanza, S.; Clementi, M.; Nucci, P.; Ballabio, A.; Tacchetti, C.; DE LUCA, Michele

Melanocytes represent the second most important cell type in the skin and are primarily responsible for the pigmentation of skin, hair, and eyes. Their function may be affected in a number of inherited and acquired disorders, characterized by hyperpigmentation or hypopigmentation, consequent aesthetic problems, and increased susceptibility to sun-mediated skin damage and photocarcinogenesis. Nevertheless, the possibility of genetically manipulating human melanocytes has been hampered so far by a number of limitations, including their resistance to retroviral infection. To address the problem of human melanocyte transduction, we generated a melanocyte culture from a patient affected with ocular albinism type 1 (OA1), an X-linked pigmentation disorder, characterized by severe reduction of visual acuity, retinal hypopigmentation, and the presence of macromelanosomes in skin melanocytes and retinal pigment epithelium (RPE). The cultured patient melanocytes displayed a significant impairment in replication ability and showed complete absence of endogenous OA1 protein, thus representing a suitable model for setting up an efficient gene transfer procedure. To correct the genetic defect in these cells, we used a retroviral vector carrying the OA1 cDNA and exploited a melanocyte-keratinocyte coculturing approach. Despite their lower replication rate with respect to wildtype cells, the patient melanocytes were efficiently transduced and readily selected in vitro, and were found to express, process, and properly sort large amounts of recombinant OA1 protein. These results indicate the feasibility of efficiently and stably transducing in vitro not only normal neonatal, but also mutant adult, human melanocytes with nonmitogenic genes.

2002 - Erratum: Two-stage, p16INK4A- and p53-dependent keratinocyte senescence mechanism that limits replicative potential independent of telomere status (Molecular and Cellular Biology (2002) 22:14 (5157-5172)) [Articolo su rivista]
Rheinwald, J. G.; Hahn, W. C.; Ramsey, M. R.; Wu, J. Y.; Guo, Z.; Tsao, H.; De Luca, M.; Catricala, C.; O'Toole, K. M.

2002 - Terapia cellulare e genica con cheratinociti umani coltivati [Capitolo/Saggio]
Pellegrini, Graziella; DE LUCA, Michele

Dopo una breve introduzione sulle cellule staminali epiteliali, il capitolo analizza varie tipologie di terapia cellulare (copertura permanente delle ustioni estese a tutto spessore, copertura permanente di superfici oculari danneggiate, trattamento di vitiligine "stabile" con autografts coltivati epidermici) e la terapia genica ex vivo dell'epidermolisi bullosa giunzionale.

2002 - Toward gene therapy of junctional epidermolysis bullosa (JEB) [Abstract in Atti di Convegno]
DE LUCA, Michele; Dellambra, E; Pellegrini, Graziella; Guerra, L; Bondanza, S; Mavilio, Fulvio

Non disponibile

2001 - Autologous fibrin-cultured limbal stem cells permanently restore the corneal surface of patients with total limbal stem cell deficiency [Articolo su rivista]
Rama, P.; Bonini, S.; Lambiase, A.; Golisano, O.; Paterna, P.; DE LUCA, Michele; Pellegrini, Graziella

BACKGROUND: Ocular burns cause depletion of limbal stem cells, which leads to corneal opacification and visual loss. Autologous cultured epithelial cells can restore damaged corneas, but this technology is still developing. We sought to establish a culture system that allows preservation of limbal stem cells and preparation of manageable epithelial sheets and to investigate whether such cultures can permanently restore total limbal stem cell deficiency. METHODS: We selected a homogeneous group of patients whose limbal cell deficiency was evaluated by scoring the gravity of the clinical picture and the keratin expression pattern. Stem cells, obtained from the limbus of the contralateral eye, were cultivated onto a fibrin substrate and their preservation was evaluated by clonal analysis. Fibrin cultures were grafted onto damaged corneas. RESULTS: Fibrin-cultured limbal stem cells were successful in 14 of 18 patients. Re-epithelialization occurred within the first week. Inflammation and vascularization regressed within the first 3-4 weeks. By the first month, the corneal surface was covered by a transparent, normal-looking epithelium. At 12-27 months follow-up, corneal surfaces were clinically and cytologically stable. Three patients had a penetrating keratoplasty approximately 1 year after restoration of their corneal surface. Their visual acuity improved from light perception or counting fingers to 0.8-1.0. CONCLUSIONS: Preservation of limbal stem cells in culture gives new perspectives on the treatment of ocular disorders characterized by complete limbal stem cell deficiency. The multicenter nature of this study and the handiness and ease of long-distance transportation of the fibrin-cultured epithelial sheets suggest that this technology can now be widely applied.

2001 - Gene Correction of Integrin β4-dependent Pyloric Atresia-Junctional Epidermolysis Bullosa Keratinocytes Establishes a Role for β4 Tyrosines 1422 and 1440 in Hemidesmosome Assembly [Articolo su rivista]
Dellambra, E.; Prislei, S.; Salvati, A. L.; Madeddu, M. L.; Golisano, O.; Siviero, E.; Bondanza, S.; Cicuzza, S.; Giancotti, F. G.; Zambruno, G.; DE LUCA, Michele

The cytoplasmic domain of beta4 integrin contains two pairs of fibronectin-like repeats separated by a connecting segment. The connecting segment harbors a putative tyrosine activation motif in which tyrosines 1422 and 1440 are phosphorylated in response to alpha6beta4 binding to laminin-5. Primary beta4-null keratinocytes, obtained from a newborn suffering from lethal junctional epidermolysis bullosa, were stably transduced with retroviruses carrying a full-length beta4 cDNA or a beta4 cDNA with phenylalanine substitutions at Tyr-1422 and Tyr-1440. Hemidesmosome assembly was evaluated on organotypic skin cultures. beta4-corrected keratinocytes were indistinguishable from normal cells in terms of alpha6beta4 expression, the localization of hemidesmosome components, and hemidesmosome structure and density, suggesting full genetic and functional correction of beta4-null keratinocytes. In cultures generated from beta4(Y1422F/Y1440F) keratinocytes, beta4 mutants as well as alpha6 integrin, HD1/plectin, and BP180 were not concentrated at the dermal-epidermal junction. Furthermore, the number of hemidesmosomes was strikingly reduced as compared with beta4-corrected keratinocytes. The rare hemidesmosomes detected in beta4(Y1422F/Y1440F) cells were devoid of sub-basal dense plates and of inner cytoplasmic plaques with keratin filament insertion. Collectively, our data demonstrate that the beta4 tyrosine activation motif is not required for the localization of alpha6beta4 at the keratinocyte plasma membrane but is essential for optimal assembly of bona fide hemidesmosomes.

2001 - In vitro reconstituted sheets of human corneal epithelium and method of producing the same [Brevetto]
DE LUCA, Michele; Pellegrini, Graziella

A method of reconstituting sheets of human corneal epithelium in vitro to be used in grafts by culturing limbar stem cells and the method for selection and propagation of said cell lines on a fibrin substrate.

2001 - In vitro reconstitution of trasplantable sheets of ocular surface epithelium and methods for producing the same [Brevetto]
Pellegrini, Graziella; DE LUCA, Michele

concessione USA n° 6,610,538

2001 - Keratinocyte-mediated cell and gene therapy [Capitolo/Saggio]
DE LUCA, Michele; Guerra, L; Dellambra, E; Pellegrini, Graziella

Although some markers for the epidermal stem cell compartment have been proposed, their role in specifically identifying keratinocyte stem cells is still controversial. Therefore, the identification of surface epithelial stem cells relies on either the evaluation of their proliferative capacity or on the identification of slow cycling, [3H]TdR- and BrdU-retaining cells, the latter being feasible only on laboratory animals. The proliferative capacity of human lining epithelial stem cells can be evaluated in vitro by means of clonal analysis. Indeed, three types of keratinocytes with different capacities for multiplication have been identified and isolated in human epidermis, hair follicle, limbal-corneal and conjunctival epithelia, i.e. holoclones, meroclones and paraclones. The authors have recently demonstrated that cultured autografts bearing holoclones can indeed rapidly and permanently cover a large body surface. Preparation of the wound bed and maintenance of epidermal stem cells in culture are found to be crucial to the clinical success of the technology. The implications and clincial results of permanent coverage of massive full-thickness burns, treatment of "stable" vitiligo with cultured epidermal autografts and permanent coverage of damaged ocular surfaces after complete loss of the corneal-limbal epithelium as well as ex vivo gene therapy of junctional epidermolysis bullosa are reported in this study and literature review. Basic "quality controls" of the culture system may eliminate one important hitherto uncontrolled variable in the evaluation of cultured autograft clinical performance and should represent a starting point for improving epithelial cultivation, in order to achieve satisfactory and reproducible clinical result.

2001 - p63 identifies keratinocyte stem cells [Articolo su rivista]
Pellegrini, Graziella; E., Dellambra; O., Golisano; E., Martinelli; I., Fantozzi; S., Bondanza; D., Ponzin; F., Mckeon; DE LUCA, Michele

The proliferative compartment of stratified squamous epithelia consists of stem and transient amplifying (TA) keratinocytes. Some polypeptides are more abundant in putative epidermal stem cells than in TA cells, but no polypeptide confined to the stem cells has yet been identified. Here we show that the p63 transcription factor, a p53 homologue essential for regenerative proliferation in epithelial development, distinguishes human keratinocyte stem cells from their TA progeny. Within the cornea, nuclear p63 is expressed by the basal cells of the limbal epithelium, but not by TA cells covering the corneal surface. Human keratinocyte stem and TA cells when isolated in culture give rise to holoclones and paraclones, respectively. We show by clonal analysis that p63 is abundantly expressed by epidermal and limbal holoclones, but is undetectable in paraclones. TA keratinocytes, immediately after their withdrawal from the stem cell compartment (meroclones), have greatly reduced p63, even though they possess very appreciable proliferative capacity. Clonal evolution (i.e., generation of TA cells from precursor stem cells) is promoted by the sigma isoform of the 14-3-3 family of proteins. Keratinocytes whose 14-3-3final sigma has been down-regulated remain in the stem cell compartment and maintain p63 during serial cultivation. The identification of p63 as a keratinocyte stem cell marker will be of practical importance for the clinical application of epithelial cultures in cell therapy as well as for studies on epithelial tumorigenesis.

2001 - Reconstructed laminae of human epithelium corneae and method of producing the same [Brevetto]
DE LUCA, Michele; Pellegrini, Graziella

A method of reconstructing laminae of human epithelium corneae in vitro to be used in grafts from cultures of limbar stem cells as well as a method of selecting and transferring such cells to fibrin substrate.

2000 - Defective intracellular transport and processing of OA1 is a major cause of ocular albinism type 1 [Articolo su rivista]
D'Addio, M; Pizzigoni, A; Bassi, M. T; Baschirotto, C; Valetti, C; Incerti, B; Clementi, M; DE LUCA, Michele; Ballabio, A; Schiaffino, M. V.

Ocular albinism type 1 (OA1) is an X-linked disorder mainly characterized by a severe reduction of visual acuity, hypopigmentation of the retina and the presence of macromelanosomes in the skin and eyes. Various types of mutation have been identified within the OA1 gene in patients with the disorder, including several missense mutations of unknown functional significance. In order to shed light into the molecular pathogenesis of ocular albinism and possibly define critical functional domains within the OA1 protein, we characterized 19 independent missense mutations with respect to processing and subcellular distribution on expression in COS-7 cells. Our analysis indicates the presence of at least two distinct biochemical defects associated with the different missense mutations. Eleven of the nineteen OA1 mutants (approximately 60%) were retained in the endoplasmic reticulum, showing defecNStive intracellular transport and glycosylation, consistent with protein misfolding. The remaining eight of the nineteen OA1 mutants (approximately 40%) displayed sorting and processing behaviours indistinguishable from those of the wild-type protein. Consistent with our recent findings that OA1 represents a novel type of intracellular G protein-coupled receptor (GPCR), we found that most of these latter mutations cluster within the second and third cytosolic loops, two regions that in canonical GPCRs are known to be critical for their downstream signaling, including G protein-coupling and effector activation. The biochemical analysis of OA1 mutations performed in this study provides important insights into the structure-function relationships of the OA1 protein and implies protein misfolding as a major pathogenic mechanism in OA1.

2000 - Discoidin domain receptor 1 (DDR1) signaling in PC12 cells: activation of juxtamembranedomains in PDGF/DDR/TrkA chimeric receptors. [Articolo su rivista]
Foehr, E. D.; Tatavos, A.; Tanabe, E.; Raffioni, S.; Goetz, S.; DI MARCO, E.; DE LUCA, Michele; Bradshaw, R. A.

The discoidin domain receptor (DDR1) is characterized by a discoidin I motif in the extracellular domain, an unusually long cytoplasmic juxtamembrane (JM) region, and a kinase domain that is 45% identical to that of the NGF receptor, TrkA. DDR1 also has a major splice form, which has a 37 amino acid insert in the JM region with a consensus Shc PTB site that is lacking in the shorter receptor. One class of ligands for the DDR receptors has recently been identified as being derived from the collagen family, but neither native PC12 cells, which express modest amounts of DDR1, nor transfected PC12 cells, which express much larger amounts of DDR1, respond to this ligand. A chimeric receptor, containing the extracellular domain of hPDGFRß fused to the transmembrane and intracellular regions of DDR1, also fails to mediate neuronal-like differentiation in stably transfected PC12 cells and is only weakly autophosphorylated. However, chimeric receptors, which are composed of combinations of intracellular regions from DDR1 and TrkA (with the extracellular domain of hPDGFRß), in some cases provided ligand (PDGF) -inducible receptor responses. Those with the TrkA kinase domain and the DDR1 JM regions were able to produce differentiation to varying degrees, whereas the opposite combination did not. Analysis of the signaling responses of the two chimeras with DDR1 JM sequences (with and without the insert) indicated that the shorter sequence bound and activated FRS2 whereas the insert-containing form activated Shc instead. Both activated PLC through the carboxyl-terminal tyrosine of the TrkA domain (Y785 in TrkA residue numbering). Mutation of this site (YF) eliminated PLC activation (indicating there are no other cryptic binding sites for PLC in the DDR1 sequences) and markedly reduced the differentiative activity of the receptor. This is in contrast to TrkA (or PDGFRß/TrkA chimeras), where ablation of this pathway has no notable effect on PC12 cell morphogenic responses. Thus, the activation of FRS2 and Shc (leading to MAPK activation) is weaker in the DDR1/TrkA chimeras than in TrkA alone, and the PLC contribution becomes essential for full response. Nonetheless, both DDR1 JM regions contain potentially usable signaling sites, albeit they apparently are not activated directly in DDR1 (or DDR1 chimeras) in a ligand-dependent fashion. These findings suggest that the DDR1 receptors do have signaling capacity but may require additional components or altered conditions to fully activate their kinase domains and/or sustain the activation of the JM sites.—Foehr, E. D., Tatavos, A., Tanabe, E., Raffioni, S., Goetz, S., DiMarco, E., De Luca, M., Bradshaw, R. A. Discoidin domain receptor 1 (DDR1) signaling in PC12 cells: activation of juxtamembrane domains in PDGFR/DDR/TrkA chimeric receptors.

2000 - Downregulation of 14-3-3σ prevents clonal evolution and leads to immortalization of primary human keratinocytes [Articolo su rivista]
Dellambra, E.; Golisano, O.; Bondanza, S.; Siviero, E.; Molinari, M.; Lacal, P.; Datri, S.; DE LUCA, Michele

In human epidermal keratinocytes, replicative senescence, is determined by a progressive decline of clonogenic and dividing cells. Its timing is controlled by clonal evolution, that is, by the continuous transition from stem cells to transient amplifying cells. We now report that downregulation of 14-3-3σ, which is specifically expressed in human stratified epithelia, prevents keratinocyte clonal evolution, thereby forcing keratinocytes into the stem cell compartment. This allows primary human keratinocytes to readily escape replicative senescence. 14-3-3σ–dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16INK4a tumor suppressor gene, hallmarks of keratinocyte immortalization. Taken together, these data therefore suggest that inhibition of a single endogenous gene product fosters immortalization of primary human epithelial cells without the need of exogenous oncogenes and/or oncoviruses.

2000 - Introductory remarks [Articolo su rivista]
Taichman, Lb; Khavari, Pa; DE LUCA, Michele


2000 - Toward epidermal stem cell-mediated ex vivo gene therapy of junctional epidermolysis bullosa [Articolo su rivista]
E., Dellambra; Pellegrini, Graziella; L., Guerra; G., Ferrari; G., Zambruno; Mavilio, Fulvio; DE LUCA, Michele

Junctional epidermolysis bullosa (JEB) is a group of severe, inherited skin diseases caused by mutations in the genes encoding laminin 5 or other components of the hemidesmosome. Since human epidermis is a self-renewing tissue, gene therapy of JEB requires the stable integration of the transgene into the genome of the epidermal stem cell. Human epidermal stem cells can indeed be cultivated and stably transduced with replication-defective retroviral vectors, allowing full phenotypic correction of the adhesion properties of JEB keratinocytes. Epidermal stem cells generate cohesive sheets of stratified epithelium suitable for the permanent coverage of massive skin defects, and genetically modified epidermal sheets maintain long-term expression of the transgene after transplantation on immunodeficient animals. Moreover, we have developed a clinical procedure that allows transplantation of cultured epidermal sheets on large body areas under local anesthesia and without cicatricial outcomes. Thus, (1) the possibility of cultivating lining epithelia, (2) the availability of noninvasive surgical procedures that allow the grafting of large skin areas, and (3) the demonstration of sustained transgene expression in vitro and in vivo by epidermal stem cells, prompt us to propose the implementation of a phase I/II clinical trial aimed at the ex vivo gene therapy of selected JEB patients. The aim of the trial is to validate the ex vivo procedure in a clinical setting, to prove its overall safety, and to analyze critical issues such as long-term survival of the genetically modified implant, immune response against the transgene product, and persistence of transgene expression at therapeutic levels.PMID: 11084687 [PubMed - indexed for MEDLINE]

2000 - Treatment of 'stable' vitiligo by Timedsurgery transplantation of cultured epidermal autografts [Articolo su rivista]
L., Guerra; S., Capurro; F., Melchi; G., Primavera; S., Bondanza; R., Cancedda; A., Luci; DE LUCA, Michele; Pellegrini, Graziella

AbstractObjective: To optimize melanocyte/keratinocyte cocultivation and to evaluate the effectiveness of autologous cultured epidermal grafts in the surgical treatment of stable vitiligo. Design: After optimization of melanocyte/keratinocyte cultures, achromic lesions were disepithelialized by means of programmed diathermosurgery (Timedsurgery) and covered with autologous epidermal grafts prepared from secondary cultures. Melanocyte content was evaluated by dopa reaction. The percentage of repigmentation was calculated using a semiautomatic image analysis system. Setting: A biosafety level 3 cell culture facility and a dermatological department in a hospital. Patients: Thirty-two patients carrying different types of vitiligo were admitted to the study and treated with autologous cultured epidermal grafts. Inclusion criteria were (1) failure of at least 2 standard medical approaches; (2) no therapy for at least 12 months; (3) absence of progression of old lesions, absence of appearance of new lesions, and absence of Koebner phenomenon within the past 18 months; and (4) absence of autoimmune disorders. Results: One hundred five achromic lesions (a total of 6078. 2 cm[2]) were treated. The average percentage of repigmentation, evaluated after 12 to 36 months of follow-up, was 77%. Independent of the type of vitiligo, average percentages of repigmentation of extremities and periorificial sites were 8% (31.8 cm[2] repigmented/ 420.5 cm[2] transplanted) and 35% (17.6 cm[2] repigmented/50.0 cm[2] transplanted), respectively. Percentages of repigmentation of all other body sites ranged from 88% to 96% (4329.7 cm[2] repigmented/4675.2 cm[2] transplanted). Color matching was good and scar formation was not observed. Conclusion: Cultured epidermal grafts can be considered a real therapeutic surgical alternative for stable but not lip-tip vitiligo.

1999 - Analysis of the mechanical properties of in vitro reconstructed epidermis: preliminary results [Articolo su rivista]
Chistolini, P; De Angelis, G; DE LUCA, Michele; Pellegrini, Graziella; Ruspantini, I.

Human epidermis can be reconstructed in vitro and is currently used in autografts for the treatment of severe, extensive burns and pigmentation disorders. However, there are neither international standards nor a common nomenclature for engineered tissues. The paper discusses the results of a preliminary study on human cultured epidermis to assess its mechanical tensile strength, and to eventually establish mechanical evaluation criteria that will enable test and comparison of the behaviour of different engineered tissue products. To perform uniaxial tension tests a traditional testing machine was adapted, and dedicated sample holding frame and grips designed.

1999 - Location and clonal analysis of stem cells and their differentiated progeny in the human ocular surface. [5YIF: 10.77; Citations: 429] [Articolo su rivista]
Pellegrini, Graziella; Golisano, O; Paterna, P; Lambiase, A; Bonini, S; Rama, P; DE LUCA, Michele

We have analyzed the proliferative and differentiation potential of human ocular keratinocytes. Holoclones, meroclones, and paraclones, previously identified in skin, constitute also the proliferative compartment of the ocular epithelium. Ocular holoclones have the expected properties of stem cells, while transient amplifying cells have variable proliferative potential. Corneal stem cells are segregated in the limbus, while conjunctival stem cells are uniformly distributed in bulbar and forniceal conjunctiva. Conjunctival keratinocytes and goblet cells derive from a common bipotent progenitor. Goblet cells were found in cultures of transient amplifying cells, suggesting that commitment for goblet cell differentiation can occur late in the life of a single conjunctival clone. We found that conjunctival keratinocytes with high proliferative capacity give rise to goblet cells at least twice in their life and, more importantly, at rather precise times of their life history, namely at 45-50 cell doublings and at approximately 15 cell doublings before senescence. Thus, the decision of conjunctival keratinocytes to differentiate into goblet cells appears to be dependent upon an intrinsic "cell doubling clock. " These data open new perspectives in the surgical treatment of severe defects of the anterior ocular surface with autologous cultured conjunctival epithelium.

1999 - Ocular Albinism: evidence for a defect in an intracellular signal transduction system. [Articolo su rivista]
Schiaffino, M. V.; D. ADDIO, M.; Alloni, A.; Baschirotto, C.; Valetti, C.; Cortese, K.; Puri, C.; Bassi, M. T.; Colla, C.; DE LUCA, Michele; Tacchetti, C.; Ballabio, A.

G protein-coupled receptors (GPCRs) participate in the most common signal transduction system at the plasma membrane. The wide distribution of heterotrimeric G proteins in the internal membranes suggests that a similar signalling mechanism might also be used at intracellular locations. We provide here structural evidence that the protein product of the ocular albinism type 1 gene (OA1), a pigment cell-specific integral membrane glycoprotein, represents a novel member of the GPCR superfamily and demonstrate that it binds heterotrimeric G proteins. Moreover, we show that OA1 is not found at the plasma membrane, being instead targeted to specialized intracellular organelles, the melanosomes. Our data suggest that OA1 represents the first example of an exclusively intracellular GPCR and support the hypothesis that GPCR-mediated signal transduction systems also operate at the internal membranes in mammalian cells.

1999 - The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin. [5YIF: 3.60, Citations: 215] [Articolo su rivista]
Pellegrini, Graziella; R., Ranno; G., Stracuzzi; S., Bondanza; L., Guerra; G., Zambruno; G., Micali; DE LUCA, Michele

BACKGROUND: Cell therapy is an emerging therapeutic strategy aimed at replacing or repairing severely damaged tissues with cultured cells. Epidermal regeneration obtained with autologous cultured keratinocytes (cultured autografts) can be life-saving for patients suffering from massive full-thickness burns. However, the widespread use of cultured autografts has been hampered by poor clinical results that have been consistently reported by different burn units, even when cells were applied on properly prepared wound beds. This might arise from the depletion of epidermal stem cells (holoclones) in culture. Depletion of holoclones can occur because of (i) incorrect culture conditions, (ii) environmental damage of the exposed basal layer of cultured grafts, or (iii) use of new substrates or culture technologies not pretested for holoclone preservation. The aim of this study was to show that, if new keratinocyte culture technologies and/or "delivery systems" are proposed, a careful evaluation of epidermal stem cell preservation is essential for the clinical performance of this life-saving technology. METHODS: Fibrin was chosen as a potential substrate for keratinocyte cultivation. Stem cells were monitored by clonal analysis using the culture system originally described by Rheinwald and Green as a reference. Massive full-thickness burns were treated with the composite allodermis/cultured autograft technique. RESULTS: We show that: (i) the relative percentage of holoclones, meroclones, and paraclones is maintained when keratinocytes are cultivated on fibrin, proving that fibrin does not induce clonal conversion and consequent loss of epidermal stem cells; (ii) the clonogenic ability, growth rate, and long-term proliferative potential are not affected by the new culture system; (iii) when fibrin-cultured autografts bearing stem cells are applied on massive full-thickness burns, the "take" of keratinocytes is high, reproducible, and permanent; and (iv) fibrin allows a significant reduction of the cost of cultured autografts and eliminates problems related to their handling and transportation. CONCLUSION: Our data demonstrate that: (i) cultured autografts bearing stem cells can indeed rapidly and permanently cover a large body surface; and (ii) fibrin is a suitable substrate for keratinocyte cultivation and transplantation. These data lend strength to the concept that the success of cell therapy at a clinical level requires cultivation and transplantation of stem cells. We therefore suggest that the proposal of a culture system aimed at the replacement of any severely damaged self-renewing tissue should be preceded by a careful evaluation of its stem cell population.

1998 - Applicazioni cliniche dei cheratinociti umani normali coltivati in vitro [Capitolo/Saggio]
DE LUCA, Michele; Guerra, L; Pellegrini, Graziella

Con la tecnologia messa a punto nel 1975 da Rheinwald e Green (Harvard Medical School, Boston) è possibile coltivare in vitro i cheratinociti umani normali ed ottenere lamine di epitelio pluristratificato con le stesse caratteristiche biochimiche, morfologiche e funzionali dell'epitelio in vivo. tale metodica prevede il prelievo dal paziente di un piccolo frammento di cute sana (2-4 cm quadrati) da cui si ottiene una sospensione cellulare epiteliale che viene posta in coltura primaria. le colture primarie vengono, a loro volta, amplificate in colture secondarie: dalle colture secondarie confluenti si ottengono le lamine di epidermide ricostruita in vitro. Dal 1981, anno in cui vennero effettuati con successo i primi trapianti con lembi di epidermide autologa coltivata in vitro in pazienti ustionati, tale tecnologia ha acquisito una rilevanza clinica sempre maggiore e si è e stasa al trattamento di numerose altre patologie. La messa a punto dei protocolli chirurgici per il trapianto dei lembi epidermici ricostruiti in vitro e la possibilità di effettuare una traduzione stabile di chertinociti staminali, hanno aperto nuove prospettive nel campo della terapia genica non solo di alcune patologie dermatologiche ma anche di particolari malattie sistemiche.

1998 - Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia [Articolo su rivista]
Vailly, J; Gagnoux Palacios, L; Dell'Ambra, E; Roméro, C; Pinola, M; Zambruno, G; DE LUCA, Michele; Ortonne, J. P; Meneguzzi, G.

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.

1998 - Corrective transduction of human epidermal stem cells in laminin-5-dependent Junctional Epidermolysis Bullosa. [Articolo su rivista]
E., Dellambra; J., Vailly; Pellegrini, Graziella; S., Bondanza; O., Golisano; G., Zambruno; G., Meneguzzi; DE LUCA, Michele

Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses.

1998 - Cultivation of human keratinocyte stem cells: current and future clinical applications [Articolo su rivista]
Pellegrini, Graziella; Bondanza, S; Guerra, L.; DE LUCA, Michele

Cultured human keratinocytes have a wide spectrum of clinical applications. Clinical results reported by several investigators are, however, contradictory. In this review, the authors discuss the biological and surgical issues which play a key role in the clinical outcome of cultured epidermal autografts used for the treatment of massive full-thickness burns. The importance of cultivation of epidermal stem cells and of their transplantation onto a wound bed prepared with donor dermis is emphasised. The paper also reviews recent data showing that: (i) cultured epidermal autografts bearing melanocytes can be used for the treatment of stable vitiligo; (ii) keratinocytes isolated from other lining epithelia, such as oral, urethral and corneal epithelia, can be cultivated and grafted onto patients suffering from disabling epithelial defects; (iii) keratinocyte stem cells can be stably transduced with retroviral vectors and are therefore attractive targets for the gene therapy of genodermatoses.

1998 - Ultrasensitive in vivo bioassay detects bioactive human growth hormone in transduced primary human keratinocytes [Articolo su rivista]
Bellini, M. H; Mathor, M. B; DE LUCA, Michele; Cancedda, R; Bartolini, P.

An improved in vivo body weight gain bioassay for the potency determination of human growth hormone (hGH) has been set up in "little" mice (lit/lit), a mutant derived from the C57BL/6J strain. This improved assay now has a detection limit of the order of 0.05 micrograms/mouse/day, which corresponds to a sensitivity about 20-fold higher than that of the most sensitive in vivo assay reported up to now: the tibia test in hypophysectomized rats or mice. This sensitivity was achieved mainly by introduction of a careful pre-assay selection and of a three injections per day schedule. The utilization of these conditions in a 2x2 factorial assay design allowed the potency determination of recombinant DNA-derived hGH (rec-hGH) in bacterial extracts with acceptable accuracy and precision, together with the greatest economy of material, only 0.24 mg of unknown and standard hormone preparation being sufficient for an entire 10-animal assay. This contrasts to a minimum of 2.7 mg that are necessary for an economical assay in hypophysectomized rats. The same assay procedure was also used to demonstrate the in vivo bioactivity of hGH secreted into a culture medium from transduced human primary keratinocytes. The growth curve constructed with n = 8 little mice presented a highly significant correlation (r = 0.939, p < 0.001) and a slope = 0.016 g/mouse/day. It was thus possible to prove, for the first time, the in vivo bioactivity of rec-hGH secreted by transplantable human epidermal cells, utilized as an experimental model for somatic gene therapy.

1998 - Verso la terapia genica delle epidermolisi bollose ereditarie [Articolo su rivista]
Zambruno, G.; De Luca, M.

1997 - Corneal epithelial stem-cell transplantation [Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella

Authors' reply to Kazuo Tsubota PMID: 9167491

1997 - HIV-type 1 induces specific cytoskeleton alterations in human epithelial cells in culture [Articolo su rivista]
Malorni, W; Guiducci, G; Pasquinelli, G; Rivabene, R; Re, R. c.; Ramazzotti, E; DE LUCA, Michele; La Placa, M; Cenacchi, G.

A wide range of cutaneous neoplastic and non-neoplastic disorders have been found in acquired immunodeficiency syndrome (AIDS) patients. However, it is not clear how pathological skin changes are attributable to human immunodeficiency virus-type 1 (HIV-1) infection. In this study, we report the results obtained in cultured epithelial cells. Stabilized human epidermoid cell lines (A431, HEp-2) as well as primary cultures of human keratinocytes following interaction with HIV-1 particles were considered. In addition, the same cells after incubation with HIV-infected lymphoblastoid cells, used as viral carriers, were also used. Particular attention was paid to certain cytoskeletal elements involved in certain key functions of epithelial cells such as cell-to-cell and cell-substrate interaction, i.e. keratin filaments and the actin microfilament system. Direct, as well as cell-mediated exposure to HIV-1 viral particles was capable of markedly interfering with cytoskeletal element integrity, leading to a rearrangement of the membrane-associated cytoskeletal elements and to an alteration of their assembly with the perinuclear filamentous components. This results in an alteration of certain subcellular functions and finally leads to junctional leakage and cell injury. This cascade of stress-induced, subcellular events could possibly have an in vivo counterpart in neoplastic and non-neoplastic skin disorders and manifestations in patients with AIDS.

1997 - Long term restoration of human corneal surface with autologous corneal epithelium generated by serial cultivation of limbal epithelial cells. [Articolo su rivista]
Pellegrini, Graziella; C., Traverso; At, Franzi; M., Zingirian; R., Cancedda; DE LUCA, Michele

BACKGROUND: Complete loss of the corneal-limbal epithelium leads to re-epithelialisation by bulbar conjunctival cells. Since conjunctival and corneal-limbal epithelial cells represent two different cell lines, this conjunctival healing of the cornea is followed by stromal scarring, decreased visual acuity, and severe discomfort. Unilateral corneal-limbal epithelial defects can be resolved by the transplantation of limbal grafts taken from the uninjured eye. However, this procedure requires a large limbal graft to be taken from the healthy eye, and is not possible for bilateral lesions. We investigated the possibility of restoring the human corneal surface with autologous corneal epithelial sheets generated by serial cultivation of limbal cells. METHODS: Cells were cultivated from a 1 mm2 biopsy sample taken from the limbus of the healthy eye of two patients with severe alkali burns, and thus complete loss of the corneal-limbal surface, of one eye. Normal corneal differentiation was tested with a specific biochemical marker. Autologous cultured corneal sheets were then grafted onto the damaged eyes of the two patients. The patients were followed up at more than 2 years after grafting. FINDINGS: We have shown that corneal progenitor cells are localised in the limbus, that cultured limbal cells generate cohesive sheets of authentic corneal epithelium, and that autologous cultured corneal epithelium restored the corneal surface of two patients with complete loss of the corneal-limbus epithelium. Long-term follow-up showed the stability of regenerated corneal epithelium and the striking improvement in patients' comfort and visual acuity. INTERPRETATION: The cultivation of corneal epithelium might offer an alternative to patients with unilateral lesions and a therapeutic chance to patients with severe bilateral corneal-limbal epithelial defects. Our findings give a new perspective on the treatment of ocular disorders characterised by stem-cell deficiency.

1997 - The importance of epidermal stem cells in keratinocyte-mediated gene therapy [Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella

Non disponibile

1997 - Topography and biological role of integrins in human skin [Articolo su rivista]
Marchisio, P. C; Trusolino, L; DE LUCA, Michele

We report the topography of integrins in the human epidermis and in cultured human keratinocytes. Both in situ and in vitro beta 1 integrins are exposed at the cell-cell adhesion interface while beta 4 is located on the basal membrane in contact with the basal lamina. Such defined sorting identifies discrete cell membrane domains that may be involved in defining, building up, and maintaining epithelial cell polarity. The distribution of integrins is deeply altered in hyperproliferative states like those occurring in several experimental conditions and in epidermal diseases.

1996 - Clonal analysis of stably transduced human epidermal stem cells in culture. [Articolo su rivista]
Mb, Mathor; G., Ferrari; E., Dellambra; M., Cilli; Mavilio, Fulvio; R., Cancedda; DE LUCA, Michele

We have transduced normal human keratinocytes with retroviral constructs expressing a bacterial beta-galactosidase (beta-gal) gene or a human interleukin-6 (hIL-6) cDNA under control of a long terminal repeat. Efficiency of gene transfer averaged approximately 50% and 95% of clonogenic keratinocytes for beta-gal and hIL-6, respectively. Both genes were stably integrated and expressed for more than 150 generations. Clonal analysis showed that both holoclones and their transient amplifying progeny expressed the transgene permanently. Southern blot analysis on isolated clones showed that many keratinocyte stem cells integrated multiple proviral copies in their genome and that the synthesis of the exogenous gene product in vitro was proportional to the number of proviral integrations. When cohesive epidermal sheets prepared from stem cells transduced with hIL-6 were grafted on athymic animals, the serum levels of hIL-6 were strictly proportional to the rate of secretion in vitro and therefore to the number of proviral integrations. The possibility of specifying the level of transgene expression and its permanence in a homogeneous clone of stem cell origin opens new perspectives in the long-term treatment of genetic disorders.

1996 - Early ultrastuctural changes of human keratinocytes after HIV-1 contact: an in vitro study [Articolo su rivista]
Cenacchi, G; Guiducci, G; Pasquinelli, G; Re, Mc; Ramazzotti, E; Furlini, G; Malorni, W; DE LUCA, Michele; Martinelli, Gn

Patients affected with AIDS develop a wide range of cutaneous neoplastic and nonneoplastic disorders. However, it is not clear whether pathological skin changes, observed in HIV-1 seropositive subjects during the course of disease are correlated to HIV-1 infection. To verify the effect of HIV-1 on human epidermal keratinocytes (HEKs), we performed virological and ultrastructural (transmission and scanning electron microscopy - TEM, SEM) studies. For this purpose cultured HEKs were studied following incubation with cell-free HIV-1 or HIV-1/infected cells, treatment with recombinant gp120 and treatment with Tat protein. PCR analysis performed on cell-free, virus-treated HEKs, constantly demonstrated negative results. Ultrastructural observations showed cytotoxic, stress-induced HEK changes, including : 1. cell vacuolization ; 2. disordered cytoskeletal arrangement ; 3. junctional leakage ; 4. surface blebbing. Our results suggest that, although HEKs appear resistant to HIV-I infection in our experimental setting, they undergo a cascade of stress-induced subcellular events which possibly impair their in vivo physiological functions.

1996 - The epidermal keratinocyte [Capitolo/Saggio]
DE LUCA, Michele; Pellegrini, Graziella; Zambruno, G.

Description of cell culture methods for epithelial keratinocytes from different body areas.

1996 - The Ocular Albinism type 1 gene product is a membrane glycoprotein localized to melanosomes. [Articolo su rivista]
M. V., Schiaffino; C., Baschirotto; Pellegrini, Graziella; S., Montalti; C., Tacchetti; DE LUCA, Michele; A., Ballabio

Ocular albinism type 1 (OA1) is an inherited disorder characterized by severe reduction of visual acuity, photophobia, and retinal hypopigmentation. Ultrastructural examination of skin melanocytes and of the retinal pigment epithelium reveals the presence of macromelanosomes, suggesting a defect in melanosome biogenesis. The gene responsible for OA1 is exclusively expressed in pigment cells and encodes a predicted protein of 404 aa displaying several putative transmembrane domains and sharing no similarities with previously identified molecules. Using polyclonal antibodies we have identified the endogenous OA1 protein in retinal pigment epithelial cells, in normal human melanocytes and in various melanoma cell lines. Two forms of the OA1 protein were identified by Western analysis, a 60-kDa glycoprotein and a doublet of 48 and 45 kDa probably corresponding to unglycosylated precursor polypeptides. Upon subcellular fractionation and phase separation with the nonionic detergent Triton X-114, the OA1 protein segregated into the melanosome-rich fraction and behaved as an authentic integral membrane protein. Immunofluorescence and immunogold analyses on normal human melanocytes confirmed the melanosomal membrane localization of the endogenous OA1 protein, consistent with its possible involvement in melanosome biogenesis. The identification of a novel melanosomal membrane protein involved in a human disease will provide insights into the mechanisms that control the cell-specific pathways of subcellular morphogenesis.

1995 - HIV-1 spreads from lymphocytes to normal human keratinocytes suitable for autologous and allogenic transplantation [Articolo su rivista]
Ramarli, D; Giri, A; Reina, S; Poffe, O; Cancedda, R; Varnier, O; Tridente, G; DE LUCA, Michele

Normal human keratinocytes can reconstitute in vitro cohesive sheets of epithelium suitable for grafting onto patients. Despite the widespread use of autografts and allografts, no data are yet available on productive infection by human immunodeficiency virus (HIV-1) of human keratinocytes. To address this point, we challenged keratinocytes at the second passage of culture with HTLV-IIIB virus by cell-free and cell-mediated inoculum. Viral entry was not achieved by cell-free inoculum, thus demonstrating that cultured keratinocytes do not provide the membrane requirements for viral binding and/or internalization. By contrast, the cell-mediated inoculum overcame specific receptor constraints, leading to viral integration and productive infection. The p24gag viral protein was transiently released in the culture supernatant, although at low level. The viral progeny produced by infected keratinocytes was rescued and amplified by co-culture experiments performed with the HIV-1 high sensitive CEM-SS human T-cell line. Viral integration, p24gag production, and secondary transmission to lymphoid cells was further confirmed with keratinocytes infected at the fourth passage of culture. Taken together, our results demonstrate that cultured keratinocytes can be infected by HTLV-IIIB virus, which can be maintained in semi-latent form for several passages after inoculum and rescued to full replication by a proper target. The in vitro demonstration of lympho-epithelial HIV-1 spreadings warns against the use of inappropriately screened biopsies for the preparation of skin grafts.

1995 - Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase c activity. [Articolo su rivista]
Dellambra, E.; Patrone, M.; Sparatore, B.; Negri, A.; Ceciliani, F.; Bondanza, S.; Molina, F.; DESCALZI CANCEDDA, F.; DE LUCA, Michele

The intrinsic signal(s) responsible for the onset of human keratinocyte terminal differentiation is not yet fully understood. Evidence has been recently accumulated linking the phospholipase-mediated activation of protein kinase C to the coordinate changes in gene expression occurring during keratinocyte terminal differentiation. Here we report the purification of a keratinocyte-derived protein enhancing protein kinase C enzymatic activity. The stimulator eluted as a peak with estimated molecular mass of approximately 70 kDa, while analysis by SDS-PAGE showed a 30 kDa protein migrating as a distinct doublet, suggesting the formation of a 30 kDa homodimer. The amino acid sequence analysis allowed the unambigous identification of the protein kinase C stimulator as a mixture of the highly homologous sigma (stratifin) and zeta isoforms of 14-3-3 proteins, which are homodimers of identical 30 kDa subunits. Mono Q anion exchange chromatography and immunoblot analysis further confirmed that stratifin enhances protein kinase C activity. Stratifin was originally sequenced from a human keratinocyte protein database, but its function was unknown. The pleckstrin homology domain has been recently related to protein translocation to the cell membrane as well as to functional interactions of intracellular proteins involved in signal transduction. We show here that stratifin (and 14-3-3 zeta) harbors a pleckstrin homology domain, and the consequent functional implications will be discussed.

1995 - Transforming Growth-Factor-Beta-1 Modulates Beta-1 And Beta-5 Integrin Receptors And Induces The De-Novo Expression Of The Alpha-V-Beta-6 Heterodimer In Normal Human Keratinocytes - Implications For Wound-Healing. [Articolo su rivista]
G., Zambruno; P. C., Marchisio; Marconi, Alessandra; Vaschieri, Cristina; A., Melchiori; Giannetti, Alberto; DE LUCA, Michele

The molecular mechanism underlying the promotion of wound healing by TGF-beta 1 is incompletely understood. We report that TGF-beta 1 regulates the regenerative/migratory phenotype of normal human keratinocytes by modulating their integrin receptor repertoire. In growing keratinocyte colonies but not in fully stratified cultured epidermis, TGF-beta 1: (a) strongly upregulates the expression of the fibronectin receptor alpha 5 beta 1, the vitronectin receptor alpha v beta 5, and the collagen receptor alpha 2 beta 1 by differentially modulating the synthesis of their alpha and beta subunits; (b) downregulates the multifunctional alpha 3 beta 1 heterodimer; (c) induces the de novo expression and surface exposure of the alpha v beta 6 fibronectin receptor; (d) stimulates keratinocyte migration toward fibronectin and vitronectin; (e) induces a marked perturbation of the general mechanism of polarized domain sorting of both beta 1 and beta 4 dimers; and (f) causes a pericellular redistribution of alpha v beta 5. These data suggest that alpha 5 beta 1, alpha v beta 6, and alpha v beta 5, not routinely used by keratinocytes resting on an intact basement membrane, act as ''emergency'' receptors, and uncover at least one of the molecular mechanisms responsible for the peculiar integrin expression in healing human wounds. Indeed, TGF-beta 1 reproduces the integrin expression pattern of keratinocytes located at the injury site, particularly of cells in the migrating epithelial tongue at the leading edge of the wound. Since these keratinocytes are inhibited in their proliferative capacity, these data might account for the apparent paradox of a TGF-beta 1-dependent stimulation of epidermal wound healing associated with a growth inhibitory effect on epithelial cells.

1994 - Keratinocyte-melanocyte interactions in in vitro reconstituted normal human epidermis [Articolo su rivista]
DE LUCA, Michele


1994 - Psoriatic lesions in patients with chronic liver disease are distinct from psoriasis vulgaris lesions, as judged on basis of integrin adhesion receptors [Articolo su rivista]
Giannelli, G; Savoia, P; Schiraldi, O; Lospalluti, M; DE LUCA, Michele; Marchisio, P. C; Quaranta, V.

Psoriatic lesions are relatively frequent in patients with chronic liver disease. Furthermore, therapy with interferons tends to exacerbate the symptoms. The pathogenesis of psoriatic lesions is unclear. An important question is whether such lesions may be linked to the underlying chronic liver disease in these patients, or whether they are incidental manifestations of psoriasis vulgaris. We collected biopsy specimens from involved and uninvolved skin areas of chronic liver disease patients with psoriatic manifestations, as well as from psoriasis vulgaris patients, and investigated the patterns of integrin adhesion receptors by means of immunohistochemical methods. Integrin expression is known to be characteristically altered in psoriasis vulgaris. We found some of these changes in chronic liver disease psoriatic lesions-namely pericellular redistribution and suprabasal expression of the basement membrane receptor alpha 6 beta 4 and of the intercellular integrins alpha 2 beta 1 and alpha 3 beta 1. However, psoriasis vulgaris causes two other typical changes: One is the induction of the prototype fibronectin receptor alpha 5 beta 1, and the other is the alteration of integrin expression in areas of the epidermis that are macroscopically normal. These two changes were not found in chronic liver disease psoriasis biopsy specimens in 14 patients investigated. Thus integrin expression may be useful in differentiating chronic liver disease psoriatic lesions from psoriasis vulgaris lesions. Even though the two types of lesions are indistinguishable on inspection or by their histological features, they may be caused by distinct pathogenetic mechanisms. It remains to be seen whether the underlying chronic liver disease has a role, albeit indirect, in such mechanisms.

1994 - Role of integrins in cell adhesion and polarity in normal keratinocytes and human skin pathologies [Relazione in Atti di Convegno]
DE LUCA, Michele; Pellegrini, Graziella; Zambruno, G; Marchisio, P. C.

In vitro, normal human keratinocytes reconstitute a differentiated stratified epidermis, maintaining the same gene expression pattern as its in vivo counterpart and are suitable for permanent grafting onto patients. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are also mediated by integrin receptors that are sorted to defined plasma membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized at the basal surface of basal cells and codistributes with laminin and nicein/kalinin; the alpha 2 beta 1 and alpha 3 beta 1 integrins are enriched laterally and play crucial roles in cell-cell interaction and proper colony morphology. During wound healing, proliferating and migrating keratinocytes express on their plasma membrane alpha v beta 5 and alpha 5 beta 1, which allow keratinocyte attachment and migration over the provisional matrix present in the wound. TGF beta, which is an autocrine and paracrine mediator in wound healing, specifically increases the synthesis and expression of alpha v beta 5 and alpha 5 beta 1, induces the de novo expression of alpha v beta 6, and determines the loss of integrin polarization. In hyperproliferative skin diseases, such as skin cancer or psoriasis vulgaris, and in normal keratinocytes forced into more frequent cell cycles, the polarized expression of integrins is lost, and alpha 5 beta 1 becomes costitutively expressed on the plasma membrane. In addition, the alpha 6 beta 4 integrin becomes associated with focal contacts. Nerve growth factor (NGF) is a potent autocrine stimulator of keratinocyte growth and induces melanocyte migration toward the leading edge of a healing wound

1994 - Transforming Growth-Factor (TGF)-Beta(1) Enhances Alpha(5)Beta(1) And Alpha(V)Beta(5) Integrin Expression And Migration Of Normal Human Keratinocytes [Abstract in Rivista]
G., Zambruno; Marconi, Alessandra; A., Melchiori; P. C., Marchisio; Giannetti, Alberto; DE LUCA, Michele


1993 - Alpha melanocyte stimulating hormone (alpha MSH) stimulates normal human melanocyte growth by binding to high-affinity receptors [Articolo su rivista]
DE LUCA, Michele; Siegrist, W; Bondanza, S; Mathor, M; Cancedda, R; Eberle, A. N.

The combined action of cholera toxin (CT)-dependent activation of the adenylate cyclase signaling pathway, stimulation of protein kinase C, and activation of the tyrosine kinase activity of cell surface receptors and proto-oncogene products, have been shown to stimulate melanocyte proliferation. However, natural factors responsible for the optimal stimulation of normal human melanocyte growth, either isolated or co-cultured with keratinocytes, remain largely unknown. alpha MSH (alpha melanocyte stimulating hormone) has previously been shown to bind to murine and human melanoma cells and to stimulate their adenylate cyclase and tyrosinase activity. In contrast, very little is known about the presence and function of alpha MSH receptors in normal human melanocytes. We now report that alpha MSH: (i) binds to normal human melanocytes through a single class of high-affinity receptors; (ii) does not induce per se melanocytes to enter the S-phase of the cell cycle; (iii) does indeed stimulate melanocyte proliferation in a dose-dependent fashion; but its stimulatory effect requires bFGF and/or the activation of protein kinase C.

1993 - Cryoprotective aqueous solutions useful for the preservation of in vitro cultured epithelial cells. [Brevetto]
Geraci, G.; Rosa, M. DE; Mrossi, M. Rossi; Cancedda, R.; DE LUCA, Michele; Pellegrini, Graziella

Cryoprotective solutions in which the liquid-solid phase transition occurs without remarkable volume increases are described. The compositions of the invention, in addition to the usual components necessary for the growth and mantainance of biological material, comprise: a) polyethylene glycols (PEG) having molecular weight not higher than 20 kD; b) at least one cross-linking agent selected from polyols, mono- or oligosaccharides, polyethylene glycols having molecular weight lower than 1 kD.

G., Geraci; M., DE ROSA; M., Rossi; R., Cancedda; DE LUCA, Michele; Pellegrini, Graziella

A method for the preservation of in vitro expanded epithelial sheets which comprises the incubation of said sheets in a cryopreserving solution characterized by lack of volume increase at the liquid-solid phase transition. The cryopreserving solutions comprise: a) polyethylene glycols having molecular weight not higher than 20 KD; b) at least one cross-linking agent selected from polyols, polyamines, mono- or oligosaccharides, polyethyleneglycols having molecular weight lower than 1 KD .

1993 - Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes [Articolo su rivista]
Zambruno, G; Marchisio, P. C; Melchiori, A; Bondanza, S; Cancedda, R; DE LUCA, Michele

Integrin receptors of human melanocytes in vivo and of melanocytes isolated and cultured from in vitro reconstituted normal human epidermis were investigated. Melanocytes were studied by high-resolution immunocytochemistry of in situ epidermis and were found to expose only the integrin subunits alpha 3, alpha 6, alpha v and beta 1 on their plasma membrane surface. Instead, cultured normal melanocytes expressed alpha 3 beta 1, alpha 5 beta 1, alpha 6 beta 1 and alpha v beta 3, which were immunoprecipitated from both metabolically and surface-labeled cells. Beta 1 integrins were diffused on the adhesion surface, while alpha v beta 3 was clustered in focal contacts both in control cells and upon dendrite induction with phorbol 12-myristate 13-acetate (PMA). The functional roles of integrins were studied in vitro by cell adhesion, spreading and migration assays. The sum of the data indicated that, in normal human melanocytes: (i) adhesion to defined substrata is mainly mediated by specific beta 1 integrins; (ii) spreading is mainly modulated by alpha v beta 3; (iii) the beta 1 and beta 3 heterodimers cooperate in regulating migration. The in vitro expression of two integrins (alpha v beta 3 and alpha 5 beta 1) that are not exposed in situ, and their role in the spreading and migratory properties of melanocytes, strongly suggest that they are involved in regenerating a normally pigmented epidermis during wound healing by controlling melanocyte spreading and migration over a provisional matrix. Tumor promoters, such as PMA, selectively increased the expression of alpha 3 beta 1. We suggest that this integrin might be involved in melanocyte migration on the newly formed basement membrane during wound healing as well as in intercellular recognition of adjacent keratinocytes.

1993 - Molecular cloning of trkE, a novel trk-related putative tyrosine kinase receptor isolated from normal human keratinocytes and widely expressed by normal human tissues. [Articolo su rivista]
DI MARCO, E; Cutuli, N; Guerra, L; DE LUCA, Michele

We have identified and cloned a new member of the trk gene family, termed trkE, which generates a 3.9-kilobase (kb) transcript in normal human keratinocytes and in a variety of normal human tissues, but not in liver. Albeit at low level, trkE transcript is expressed also by PC12 cells. The open reading frame codes for a polypeptide of 876 amino acids exhibiting the classic features of cell surface tyrosine protein kinases. trkE catalytic domain is 41% identical to trkA and shows several features unique to the trk gene family. Its extracellular domain does not show significant homology to any known proteins. trkE is the first member of this gene family found abundantly and widely expressed in normal human tissues. Several lines of evidence suggest that NGF is also the ligand for trkE; (i) normal human keratinocytes bind NGF with high affinity, (ii) NGF stimulates keratinocyte growth in an autocrine fashion, (iii) NGF exerts its biological effect on keratinocytes through the stimulation of a trk-specific tyrosine kinase, and (iv) keratinocytes lack trkA but do express large amount of trkE. trkE might also be the NGF receptor by other human peripheral tissues, such as pancreatic islets, and might represent a non-neuronal receptor for this ligand.

1993 - Nerve growth factor (NGF) binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop. [Articolo su rivista]
DI MARCO, E.; Mathor, M.; Bondanza, S.; Cutuli, N.; Marchisio, P. C.; Cancedda, R.; DE LUCA, Michele

Normal human keratinocytes synthesize and secrete biologically active nerve growth factor (NGF) in a growth regulated fashion (Di Marco, E., Marchisio, P. C., Bondanza, S., Franzi, A. T., Cancedda, R., and De Luca, M. (1991) J. Biol. Chem. 266, 21718-21722). Here we show that the same human keratinocytes bind NGF via low and high affinity receptors. In parallel with the course of NGF synthesis, the expression of low affinity NGF receptor (p75NGFr) decreases when a confluent, differentiated, and fully stratified epithelium is obtained. In skin sections, p75NGFr is present in basal keratinocytes and absent from suprabasal, terminally differentiated cells. The trkA protooncogene product (p140trkA), a component of the NGF receptor, is not expressed by keratinocytes. Instead, keratinocytes express a new member of the trk family (that we termed trkE), which generates 3.9-kilobase transcripts. Keratinocyte-derived NGF plays a key role in the autocrine epidermal cell proliferation. This has been proven by (i) direct effect of NGF on [3H]thymidine incorporation, (ii) inhibition of autocrine keratinocyte growth by monoclonal antibodies (alpha D11) inhibiting human NGF biological activity, and (iii) inhibition of autocrine keratinocyte proliferation by a trk-specific inhibitor, the natural alkaloid K252a. These data provide evidence that NGF, in addition to its effect as a survival and differentiation factor, is a potent regulator of cell proliferation, at least in human epithelial cells.

1993 - One-step treatment of posterior hypospadias by the autologous graft of cultured urethral epithelium. [Articolo su rivista]
Romagnoli, G; DE LUCA, Michele; Faranda, F; Franzi, A. T. AND CANCEDDA R.

Surgical management of severe proximal hypospadias or long strictures of the posterior urethra is a difficult clinical task. Often, the therapeutic approach involves the autologous graft of free flaps of bladder or oral mucosa. We recently reported the use of autologous graft of cultured squamous urethral epithelium during urethroplasty in patients with severe proximal hypospadias. The main limitation to the widespread use of cultured epithelium was the long hospitalization due to the requirement of 2 surgical steps. We now report a substantial modification of the surgical procedure which allows for rapid 1-step urethroplasty. Cultured squamous urethral epithelium is tubularized in vitro with the aid of a tubular polytetrafluoroethylene (Gore-Tex) support and 1-step urethroplasty is performed within 30 minutes. Results obtained in 8 patients are presented.

1993 - The basement membrane protein BM-600/nicein codistributes with kalinin and the integrin alpha 6 beta 4 in human cultured keratinocytes [Articolo su rivista]
MARCHISIO P., C; Cremona, O; Savoia, P; Pellegrini, Graziella; ORTONNE J., P; Verrando, P; Burgeson, R; Cancedda, R; DE LUCA, Michele

The observation is reported that in low-passage human keratinocyte colonies cultured under conditions that allow full epidermal differentiation (i) the basement membrane protein BM-600/nicein, identified by the mAb GB3, is codistributed with laminin and collagen type IV as well as with the bullous pemphigoid antigen in footprints deposited by growing and migrating colonies; (ii) the integrin heterodimer alpha 6 beta 4 is codistributed with the same molecules suggesting its spatial association with basement membrane components; (iii) the distribution pattern of alpha 6 beta 4 and BM-600/nicein underneath individual cells is identical and is characterized by a typical "leopard skin" pattern complementary to the distribution of submembraneous F-actin microfilament network; (iv) a rabbit polyclonal antiserum to kalinin (R4012) used in double-label immunofluorescence staining with mAb GB3 shows that this protein has the same distribution as BM-600/nicein and this suggests that they are identically located; and (v) immunoprecipitation with mAb GB3 to BM-600/nicein and BM165 to kalinin shows identical bands suggesting that nicein and kalinin represent the same molecular entity. We suggest that alpha 6 beta 4 displays not only an adhesive role for keratinocytes in view of its reported association to hemidesmosomes but may also be involved in organizing the molecules of the epithelial extracellular matrix, including those forming the basement membrane zone and hemidesmosomes, a function proposed for other integrins in other cellular systems.

1993 - Tissue engineering for clinical application [Articolo su rivista]
Cancedda, R; DE LUCA, Michele


1992 - A volume-sensitive chloride conductance revealed in cultured human keratinocytes by 36Cl- efflux and whole-cell patch clamp recording [Articolo su rivista]
Rugolo, M; Mastrocola, T; DE LUCA, Michele; Romeo, G; Galietta, L. J.

The Cl- transport mechanism responsible for the stimulation of 36Cl- efflux after exposure to hypotonic medium (210 mosmol/kg) was investigated in human keratinocytes. The involvement of the anion exchanger and of the Cl-/cation cotransporters was ruled out by the finding that replacement of extracellular Cl- by the poorly permeant anion gluconate, and the addition of bumetanide and furosemide, inhibitors of the Na+/K+/Cl- and K+/Cl- cotransporters, respectively, failed to significantly reduce the activation of Cl- efflux by hypotonic medium. 'Whole cell' configuration of the patch clamp technique directly revealed the presence of a macroscopic Cl- current, which was evoked by incubation with hypotonic medium and was reversed by elevation of the extracellular osmolality. Volume-sensitive current showed outward rectification of the current-voltage relationship and time-dependent inactivation at depolarizing voltages. This current was Cl- selective, because the zero-current reversal potential approached the Cl- equilibrium potential, when extracellular Cl- was replaced by gluconate. 0.1 mM 1,9-dideoxyforskolin significantly reduced either 36Cl- efflux and the Cl- current, suggesting that the Cl- efflux and the macroscopic current activated after exposure to hypotonic medium are mediated by the same pathway. Electronic cell sizing showed that in keratinocytes hypotonic swelling was not followed by a significant regulatory volume decrease response.

1992 - Culture of human epithelium [Articolo su rivista]
DE LUCA, Michele; Cancedda, R.

Comment on Culture techniques for human keratinocytes. [Burns. 1992]

1992 - Expression, topography and function of integrin receptors is severely altered in keratinocytes from involved and uninvolved psoriatic skin. [Articolo su rivista]
Pellegrini, Graziella; DE LUCA, Michele; Orecchia, G.; Balzac, F.; Cremona, O.; Savoia, P.; Cancedda, R.; Marchisio, P. C.

Psoriasis is a hyperproliferative cutaneous disease of unknown etiology and etiopathogenesis. Alteration of keratinocyte adhesiveness to basal lamina has been proposed as the initial disturbance leading to poorly controlled proliferation. Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are segregated to discrete membrane domains. In this paper, the expression and function of integrins in psoriatic keratinocytes were examined, both in vivo and in vitro. We found that: (a) in psoriatic keratinocytes the integrin heterodimers alpha 2 beta 1, alpha 3 beta 1, and alpha 6 beta 4 have lost their polarized distribution on the plasma membrane; (b) the role of these integrins in mediating keratinocyte adhesion in vitro is altered; (c) psoriatic keratinocytes form focal contacts containing both beta 1 and beta 4 integrins. In normal adult keratinocytes the alpha 5 beta 1 fibronectin receptor is poorly expressed and diffusely distributed on the basal keratinocyte plasma membrane and is not organized in defined adhesive structures. In contrast, psoriatic keratinocytes show a clear fibronectin receptor staining in vivo, and organize alpha 5 beta 1 in typical focal contacts in vitro without any obvious increase of its expression and synthesis. These multiple alterations of integrins are also present in uninvolved keratinocytes from psoriatic patients, suggesting a key role for altered integrin-mediated adhesion in the pathogenesis of this disease.

1992 - "HepG2/erythroid/brain" type glucose transporter (GLUT1) is highly expressed in human epidermis: keratinocyte differentiation affects GLUT1 levels in reconstituted epidermis [Articolo su rivista]
Gherzi, R; Melioli, G; DE LUCA, Michele; D'Agostino, A; Distefano, G; Guastella, M; D'Anna, F; Franzi, A. T; Cancedda, R.

In mature animals, the "HepG2/erythroid/brain" glucose transporter isoform (GLUT1) appears to be expressed at the highest levels at blood tissue barriers; however, these levels may still be lower than the levels of expression seen in fetal tissues. Also, glucose transporters might serve as water channels. Therefore, we decided to investigate GLUT1 expression in human epidermis, a very active tissue, in terms of metabolism, even if not directly vascularized. We found GLUT1 transcripts in human skin and demonstrated, by immunohistochemistry, that GLUT1 protein is highly expressed in the basal layer and, to a lower extent, in the immediately suprabasal layer of the epidermis. This distribution pattern suggested that GLUT1 expression is affected by keratinocyte differentiation. To investigate this possibility, we used human epidermis reconstituted in culture. Our culture system allows the reconstruction of a stratified squamous epithelium which has been successfully grafted onto patients presenting large skin defects. Human keratinocytes have been cultured under conditions which allow a modulation of cellular differentiation and stratification. We observed that (i) GLUT1 expression is 4-6-fold higher in "stem-like" basal cells than in large, differentiated keratinocytes; (ii) culture conditions causing cell differentiation reduce GLUT1 expression, while conditions which minimize either differentiation or stratification of keratinocytes enhance GLUT1 expression. Finally, we found that IGF-1 and insulin, probably acting through the IGF-1 receptor, increase GLUT1 expression and stimulate glucose transport activity in epidermis reconstituted in culture. In conclusion, our data demonstrate that GLUT1 is highly expressed in the basal layers of human epidermis and that its expression is modulated by keratinocyte differentiation.

1992 - In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors [Articolo su rivista]
Barreca, A; DE LUCA, Michele; Del Monte, P; Bondanza, S; Damonte, G; Cariola, G; Di Marco, E; Giordano, G; Cancedda, R; Minuto, F.

Human keratinocytes isolated from a skin biopsy and cultured in vitro on a feeder-layer of irradiated fibroblasts reconstitute a stratified squamous epithelium suitable for grafting onto patients suffering from large burn wounds. Since conditioned medium from 3T3-J2 cells can partially substitute for the intact feeder-layer, we studied the possible involvement of insulin-like growth factors acting in a paracrine fashion. IGFs were measured (after Sephadex G-50 gel-chromatography in acid conditions) in media conditioned by a feeder-layer of lethally irradiated 3T3-J2 fibroblasts on which keratinocytes were grown. Immunoreactive (IR) IGF-I, IGF-II, and IGF binding activity were present in the medium conditioned by the feeder-layer. The medium conditioned by keratinocytes showed nearly undetectable amounts of IR IGF-I and IGF-II, suggesting that keratinocytes are unable to synthesize IGFs peptides. Recombinant IGF-I and IGF-II, and conditioned medium from 3T3-J2 cells, caused a dose-dependent increase of 3H-thymydine incorporation in cultured keratinocytes. The stimulatory effect of IGF and of 3T3-J2 conditioned medium was inhibited by the MoAb Sm 1.2, which recognizes both IGF-I and IGF-II but not insulin, and by the MoAb alpha IR-3, which is a specific antagonist of type-I IGF receptor. Fetal mouse-derived 3T3-J2 cells and adult human skin fibroblasts were equally able to sustain keratinocyte growth and in both cases addition of Sm 1.2 MoAb causes a 50% decrease in the keratinocyte number. When the non-IGF-producing BALB/c 3T3 cells were used as a feeder-layer, the keratinocytes number was similar to that observed with 3T3-J2 and with human fibroblasts plus the Sm 1.2 MoAb. IGF-I and IGF-II restored the BALB/c 3T3 growth promoting activity to the level of 3T3-J2 and of normal human fibroblasts. Our results suggest that fetal mouse 3T3-J2 and human fibroblasts synthesize IGF peptides, while keratinocytes do not. Fibroblast-derived IGFs stimulate keratinocyte growth in a paracrine fashion, suggesting their role in the regulation of keratinocyte proliferation in skin growth and in wound healing.

1992 - In vitro reconstitution of differentiated human epithelia [Articolo su rivista]
Pellegrini, Graziella; Gherzi, R; Franzi, At; D'Anna, F; DE LUCA, Michele; Cancedda, R.


1992 - Permanent coverage of full skin thickness burns with autologous cultured epidermis and reepithelialization of partial skin thickness lesions induced by allogeneic cultured epidermis: a multicentre study in the treatment of children [Articolo su rivista]
DE LUCA, Michele; Bondanza, S; Cancedda, R; Tamisani, A. M; Di Noto, C; Muller, L; Dioguardi, D; Brienza, E; Calvario, A; Zermani, R.


1992 - The control of polarized integrin topography and the organization of adhesion-related cytoskeleton in normal human keratinocytes depend upon number of passages in culture and ionic environment. [Articolo su rivista]
DE LUCA, Michele; Pellegrini, Graziella; Bondanza, S; Cremona, O; Savoia, P; Cancedda, R; Marchisio, P. C.

Keratinocyte adhesion to basal lamina and lateral interactions among basal epidermal cells are mediated, besides other molecules, by integrin receptors that are sorted to defined membrane domains. The hemidesmosome-associated integrin alpha 6 beta 4 is sharply localized to the basal surface of basal cells while alpha 2 beta 1 and alpha 3 beta 1 are enriched laterally. This integrin sorting pattern is perfectly reproducible in vitro by cultured keratinocytes and takes place progressively in primary or secondary culture in the presence of 1.8 mM Ca2+. The polarized topography of integrins is gradually lost with higher passage numbers and between passage 5 and passage 7 there is a complete pericellular redistribution of the above integrins. Along with the decreased basal adhesive value of alpha 6 beta 4 there is a marked increase in the number of focal contacts in high-passage keratinocyte colonies. A similar loss of polarized topography of integrins occurs under low-Ca2+ culture conditions. Increasing the number of culture passages beyond the fifth induces the appearance of the fibronectin receptor alpha 5 beta 1 on the surface of keratinocytes, particularly at intercellular junctions and in some focal contacts. The receptor alpha 5 beta 1 is not detectably exposed by low-passage cells. We propose that forcing keratinocytes into more frequent cell cycles by continuous passaging may perturb the polarized topography of integrins and the adhesion mechanisms of keratinocytes. Then, low-passage keratinocytes are, in our opinion, the most reliable in vitro models for studying the physiology of epidermal cells.

1992 - Treatment of leg ulcers with cryopreserved allogeneic cultured epithelium. A multicenter study [Articolo su rivista]
DE LUCA, Michele; Albanese, E; Cancedda, R; Viacava, A; Faggioni, A; Zambruno, G; Giannetti, Alberto

Background. - In the past few years, several authors have described the usefulness of cultured allogeneic epidermal sheets in promoting wound healing of burns, leg ulcers, and donor sites. This study reports clinical results obtained by different departments in the treatment of chronic leg ulcers by cryopreserved cultured allogeneic epithelium. The freezing procedure and the assessment of viability of the cryopreserved epithelium are also described. A total of 30 ulcers were treated using 138 cryopreserved allografts. Observations. - Twenty ulcers (66.6%) healed completely within 12 weeks. Four ulcers showed a 30% to 84.4% reduction in size by 3 weeks but did not heal completely; the remaining six ulcers did not show any improvement. A strong stimulation of granulation tissue formation and of reepithelialization from the wound edge were observed. Results. - The results indicate that frozen cultured epidermis, stored in a skin bank, is a valid and generally applicable alternative therapy for the treatment of chronic ulcers.

1991 - Characterization of chloride and cationic channels in cultured human keratinocytes [Articolo su rivista]
Galietta, L. J. V.; Barone, V.; DE LUCA, Michele; Romeo, G.

Patch-clamp experiments on human cultured keratinocytes revealed the presence of three types of ion channel. The first type was a Cl(-)-selective channel, the current/voltage relationship of which showed outward rectification, the mean conductance at positive and negative membrane potentials being 66 pS and 16 pS respectively. The second type of channel showed almost equal permeability to alkali ions but was impermeable to Cl- and to the large organic cation N-methyl-D-glucamine. Its current/voltage relationship was linear with a mean unitary conductance of 18 pS in symmetrical 140 mmol/l NaCl. Finally, the third type was a large-conductance cation channel, which had in physiological ionic conditions a peculiar rectifying current/voltage relationship, the shape of which was strongly dependent on the concentration of divalent cations on both sides of the membrane. Lowering of Ca2+ and/or Mg2+ on either side of the patch led to a marked increase of the single-channel current. With identical solutions without Ca2+ Mg2+ on both sides of the patch the current/voltage relationship became ohmic and reached a conductance of 150-200 pS. In addition, channel activity was reversibly affected by changes of the external Ca2+ concentration. In particular, open-channel probability strongly increased at negative membrane potentials when the external Ca2+ was lowered from millimolar to micromolar values. Whole-cell experiments confirm the role of the extracellular Ca2+ as a modulator of the cation conductance.

1991 - Characterization of chloride transport pathways in cultured human keratinocytes [Articolo su rivista]
Mastrocola, T; DE LUCA, Michele; Rugolo, M.

In human keratinocytes, mediated transport of Cl- was found to occur mainly by two mechanisms: an anion exchange and an electrically conductive pathway. The contribution of the anion exchange, which accounted for about 50% of overall Cl- efflux, was assessed either by its sensitivity to inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and by means of Cl- substitution experiments. The anion exchange exhibited a saturation behaviour over the range 10-135 mM Cl-; Cl- was more efficient than HCO3-, Br- and NO3- in increasing Cl- efflux rate, whereas SO4(2-) and I- inhibited Cl- efflux. The electrically conductive Cl- pathway, which accounted for about 40% of total Cl- efflux, was inhibited by the Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and was at least partially sensitive to variation of the plasma membrane potential. The Cl- channel was insensitive to elevation in the intracellular concentration of either cyclic AMP and calcium ions. Indomethacin, an inhibitor of the cyclooxygenase, failed to reduce Cl- efflux, whereas nordihydroguaiaretic acid (NDGA), an inhibitor of the lipoxygenase, induced 50% inhibition of Cl- efflux. These results support the conclusion that endogenous production of lipoxygenase-derived arachidonic acid metabolite(s) might be responsible for high basal Cl- permeability in human keratinocytes.

1991 - Chloride transport pathways in human keratinocytes [Relazione in Atti di Convegno]
Rugolo, M; DE LUCA, Michele; Mastrocola, T.


1991 - Cryopreserved in vitro cultured epithelial tissue and method [Brevetto]
Cancedda, R.; DE LUCA, Michele

A process for preserving cultured epidermal sheets by incubating the sheets at room temperature in media with a glycerol or dimethylsulfoxide cryopreservant for a predetermined period of time and then freezing the cultured sheets. The cooling gradient is characterized as having a lower initial rate of cooling followed by a higher rate of cooling.

1991 - Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes. [Articolo su rivista]
DI MARCO, E.; Marchisio, P. C.; Bondanza, S.; Franzi, A. T.; Cancedda, R.; DE LUCA, Michele

Nerve growth factor (NGF) transcripts were identified in normal human keratinocytes in primary and secondary culture. The expression of the NGF mRNA was strongly down-regulated by corticosteroids and was maximal when keratinocytes were in the exponential phase of growth. Immunofluorescence studies on growing keratinocytes colonies and on elutriated keratinocytes obtained from growing colonies and mature stratified epithelium showed specific staining of the Golgi apparatus only in basal keratinocytes in the exponential phase of growth. The keratinocyte-derived NGF was secreted in a biologically active form as assessed by neurite induction in sensory neurons obtained from chick embryo dorsal root ganglia. Based on these data we suggest that the basal keratinocyte is the cell synthesizing and secreting NGF in the human adult epidermis. The paracrine secretion of NGF by keratinocytes might have a major role in regulating innervation, lymphocyte function, and melanocyte growth and differentiation in epidermal morphogenesis as well as during wound healing.

1991 - High expression levels of the "erythroid/brain" type glucose transporter (GLUT1) in the basal cells of human eye conjunctiva and oral mucosa reconstituted in culture [Articolo su rivista]
Gherzi, R; Melioli, G; DE LUCA, Michele; D'Agostino, A; Guastella, M; Traverso, C. E; D'Anna, F; Franzi, A. T; Cancedda, R.

The expression of the "erythroid/brain" type glucose transporter (GLUT1) seems to be a feature of "barrier" tissues, at least in humans. Recently, we reported that GLUT1 is highly expressed in the basal layers of either "authentic" human epidermis or human epidermis reconstituted in culture and that its expression seems to be related to keratinocyte differentiation. In this paper we demonstrate that GLUT1 is selectively expressed in the basal layers of either eye conjunctiva epithelia or oral mucosa, reconstituted in culture starting from 1-2 mm2 bioptic specimens of normal human tissue. GLUT1 mRNA and protein levels are very high in conjunctiva and oral mucosa, 2-3 times higher than in epidermis reconstituted in culture. Taking into account its localization at the border of tissues not directly vascularized, but metabolically active, GLUT1 could play an important role in controlling the entry of glucose into these firmly guarded tissues.

1991 - Polarized expression of integrin receptors (a6b4, a2b1, a3b1, avb5) and their relationshipwith the cytoskeleton and basement membrane matrix in cultured human keratinocytes. [5YIF: 10.77; Citations: 127] [Articolo su rivista]
Marchisio, P. C.; Bondanza, S.; Cremona, O.; Cancedda, R.; DE LUCA, Michele

In human keratinocytes cultured in conditions which allow differentiation and stratification and are suitable to reconstitute a fully functional epidermis, alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were respectively polarized to the basal and lateral domains of the plasmamembrane both in growing colonies and in the reconstituted epidermis. Conversely, the alpha v integrin subunit, presumably in association with beta 5, was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4: (a) was organized in typical patches which often showed a "leopard skin" pattern where spots corresponded to microfilament-free areas; (b) was not associated with focal contacts containing vinculin and talin but rather corresponded to relatively removed contact areas of the basal membrane as shown by interference reflection microscopy; and (c) was coherent to patches of laminin secreted and deposited underneath the ventral membrane of individual cells. The two beta 1 integrins (alpha 2 beta 1 and alpha 3 beta 1), both endowed with laminin receptor properties, were not associated with focal adhesions under experimental conditions allowing full epidermal maturation but matched the lateral position of vinculin (but not talin), cingulin, and desmoplakin, all makers of intercellular junctions. Often thin strips of laminin were observed in between the lateral aspects of individual basal keratinocytes. The integrin complex alpha v beta 5 had a topography similar to that of talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The discrete topography of beta 1 and beta 4 integrins has a functional role in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion (De Luca, M., R. N. Tamura, S. Kajiji, S. Bondanza, P. Rossino, R. Cancedda, P. C. Marchisio, and V. Quaranta. Proc. Natl. Acad. Sci. USA. 87:6888-6892). Moreover, the surface polarization of integrins followed attachment and depended both on the presence of Ca2+ in the medium and on the integrity of the cytoskeleton. We conclude that our in vitro functional tests and structural data suggest a correlation between the pattern of integrin expression on defined plasmamembrane domains and the mechanism of epidermal assembly.

1990 - Evidence that human oral epithelium reconstituted in vitro and transplanted onto patients with defects in the oral mucosa retains properties of the original donor site [Articolo su rivista]
DE LUCA, Michele; Albanese, E.; Megna, M.; Cancedda, R.; Mangiante, P. E.; Cadoni, A.; Franzi, A. T.

Normal human skin--derived keratinocytes cultured in vitro reconstitute a stratified epidermis suitable for grafting onto burn patients and patients with skin defects such as giant nevi or chronic leg ulcers. In vitro experiments and long-term studies of patients receiving cultured epidermis autografts on muscular fascia suggest that skin keratinocytes possess an intrinsic site specific differentiation program that is fully expressed only when the reconstituted epidermis is transplanted in vivo to different body sites. In this study we cultivated for the first time palate-derived epithelial cells that were able to reconstitute a palatal epithelium. We also demonstrate that this epithelium can be successfully transplanted onto patients presenting lack of adherent keratinizing gingival mucosa and is able, in a relatively short time, to fully express the differentiation program typical of the original donor site. The possibility of obtaining large quantities of cultured epithelium, able to retain properties of the original donor site, starting from 1-3-mm2 biopsies, could prove extremely useful in the reconstructive surgery of the mouth and of other mucosal body areas.

1990 - Polarized integrin mediates human keratinocyte adhesion to basal lamina. [Articolo su rivista]
DE LUCA, Michele; Tamura, R. N.; Kajiji, S.; Bondanza, S.; Rossino, P.; Cancedda, R.; Marchisio, P. C.; Quaranta, V.

Epithelial cell interactions with matrices are critical to tissue organization. Indirect immunofluorescence and immunoprecipitations of cell lysates prepared from stratified cultures of human epidermal cells showed that the major integrins expressed by keratinocytes are alpha E beta 4 (also called alpha 6 beta 4) and alpha 2 beta 1. The alpha E beta 4 integrin is localized at the surface of basal cells in contact with the basement membrane, whereas alpha 2 beta 1/alpha 3 beta 1 integrins are absent from the basal surface and are localized only on the lateral surface of basal and spinous keratinocytes. Anti-beta 4 antibodies potently inhibited keratinocyte adhesion to matrigel or purified laminin, whereas anti-beta 1 antibodies were ineffective. Only anti-beta 4 antibodies were able to detach established keratinocyte colonies. These data suggest that alpha E beta 4 mediates keratinocyte adhesion to basal lamina, whereas the beta 1 subfamily is involved in cell-cell adhesion of keratinocytes.

1990 - Structural and functional studies of integrin receptors in cultured human keratinocytes [Articolo su rivista]
Marchisio, PIER CARLO; Cancedda, R.; DE LUCA, Michele

In human keratinocytes cultured in conditions which allow differentiation and stratification and which are suitable to reconstitute a fully functional epidermis, the integrin alpha 6 beta 4 and two members of the beta 1 integrin family (alpha 2 beta 1 and alpha 3 beta 1) were polarized to the basal and lateral domains of the plasma membrane both in growing colonies and in the reconstituted epidermis. Conversely, alpha v beta 5 integrin was expressed at the basal surface in growing and migrating but not in stationary keratinocytes. The integrin alpha 6 beta 4 was organized in patches and spots corresponding to F-actin-free submembraneous areas and did not colocalize to focal contacts; moreover, alpha 6 beta 4 colocalized with patches of laminin deposited underneath the ventral membrane of individual cells. The two beta 1 laminin receptor integrins (alpha 2 beta 1 and alpha 3 beta 1) were never found in the basal domain but matched the lateral position of vinculin (but not talin), cingulin and desmoplakins. Only the integrin complex alpha v beta 5 was associated with talin- and vinculin-containing focal adhesions mostly in the peripheral cells of expanding keratinocyte colonies and in coincidence with fibronectin strands. The topography of beta 1 and beta 4 integrins reflects a functional role in adhesion and in the maintenance of the state of aggregation of cultured keratinocytes since lateral aggregation was impaired by antibodies to beta 1 whereas antibodies to beta 4 prevented cell-matrix adhesion.

1990 - Thyrotropin Receptor: Characterization and Purification [Capitolo/Saggio]
Akamizu, T.; DE LUCA, Michele; Kohn, L. D.

This paper deals with the isolation, purification and functional characterization of the TSH receptor.

1990 - Treatment of posterior hypospadias by the autologous graft of cultured urethral epithelium. [Articolo su rivista]
Romagnoli, G.; DE LUCA, Michele; Faranda, F.; Bandelloni, R.; Franzi, A. T.; Cataliotti, F.; Cancedda, R.

Keratinocytes derived from normal human skin can be cultivated in vitro to form a stratified patch of epidermis suitable for grafting on the sites of burns and skin defects caused by the removal of giant nevi or chronic leg ulcers. Autologous skin keratinocytes have been successfully used to line the mastoid cavities of patients with postoperative otorrhea. In vitro experiments and long-term studies of burn patients treated with autografts grown in vitro from cultured cells suggest that keratinocytes possess an intrinsic capacity for site-specific differentiation that is fully expressed when grafts of cultured epidermis are transplanted to different parts of the body. Epithelial cells derived from the palate and cultivated in vitro are also capable of reconstituting palatal epithelium after transplantation. These findings prompted us to investigate the possibility of culturing urethral cells to form urethral mucosa in vitro. We report here the reconstitution in vitro of human urethral mucosa and the subsequent successful autologous transplantation of it to form the anterior urethra in two patients with congenital hypospadias.

1989 - A multicenter experience in the treatment of burns with autologous and allogeneic cultured epithelium fresh or preserved in a frozen state [Articolo su rivista]
DE LUCA, Michele; Albanese, E.; Bondanza, E.; Megna, M.; Ugozzoli, L.; Molina, F.; Cancedda, R.; Santi, P. L.; Bormioli, M.; Stella, M.; Magliacani, G.

This report describes the clinical results obtained from a multicentre experience of the use of autologous and allogenic cultured human epidermal cells in the treatment of partial and full skin thickness burns. A laboratory has been organized to supply cultured epithelium to Burns Units in different cities. From May 1986 to December 1988, 58 patients with an age range of 1 to 59 years, and with burns covering between 7 and 95 per cent of the body surface area, have been treated. Graftable cultured epithelium can be frozen and remain viable if stored in a skin bank. Such grafts were used successfully to treat patients with partial and full skin thickness wounds.

1989 - The thyrotropin receptor in FRTL-5 thyroid cells: A cloning approach [Relazione in Atti di Convegno]
Kohn, L. D.; Isozaki, O.; Chan, J.; Akamizu, T.; Bellur, S.; De Luca, M.; Santisteban, P.; Varutti, A. M.; Grimaz, S.; Ikuyama, S.; Saji, M.; Owens, G.

1988 - A pre-Golgi membrane-bound form of thyroglobulin (TG) contains mannose phosphate and tetraiodothyronine (T4) [Relazione in Atti di Convegno]
DE LUCA, Michele; Santisteban, P.; Shifrin, S.; Kohn, L. D.


1988 - Co-culture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium [Articolo su rivista]
DE LUCA, Michele; Franzi, A. T.; D'Anna, F.; Zicca, A.; Albanese, E.; Bondanza, S.; Cancedda, R.

Human epidermal keratinocytes differentiate in vitro into a stratified epithelium suitable for grafting on burned patients. In this paper, we show that differentiated melanocytes are present in the cultured epithelium. In particular, we have found that i) melanocytes proliferate in the same culture conditions that allow keratinocyte growth, ii) during the culture the ratio between keratinocytes and melanocytes tends to remain constant, iii) melanocytes organize into the basal layer of the cultured epithelium independently of the presence of dermis, develop dendritic arborizations with melanosome-containing processes and transfer melanosomes into keratinocyte cytoplasm.

1988 - Elutriation of human keratinocytes and melanocytes from in vitro cultured epithelium [Articolo su rivista]
D'Anna, F.; DE LUCA, Michele; Cancedda, R.; Zicca, A.; Franzi, A. T.

Human epidermal keratinocytes grown in culture and at different stages of differentiation are shown to be viably separated by elutriation. A specific fraction enriched in melanocytes was obtained. Elutriation of cells obtained fromin vitro cultured epithlium could prove useful in studies concerning the biochemistry and molecular markers of cells isolated from normal epithelium and from different pathologies.

1988 - Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. [Articolo su rivista]
DE LUCA, Michele; D'Anna, F.; Bondanza, S.; Franzi, A. T.; Cancedda, R.

Human keratinocytes isolated from a skin biopsy and cultured in vitro reconstitute a stratified squamous epithelium suitable for grafting on burned patients. Melanocytes coisolated from the same skin biopsy also proliferate under these culture conditions and maintain differentiated functions (i.e., synthesize melanin granules, regularly intersperse in the basal layer of the cultured epidermis, and transfer melanosomes in the cytoplasm of contiguous keratinocytes) (De Luca, M., A. T. Franzi, F. D'Anna, A. Zicca, E. Albanese, S. Bondanza, and R. Cancedda. 1988. Eur. J. Cell Biol. 46:176-180). Isolated melanocytes in culture grow in the presence of specific growth factors with a mean population doubling time of 4-10 d. In this paper we show that (a) human keratinocytes and oral epithelial cells possess strong and specific melanocyte growth stimulating activity (doubling time, 24 h); (b) melanocyte growth is not autonomous but requires close keratinocyte contact and is regulated to maintain a physiological melanocytes/keratinocytes ratiol and (c) pure skin keratinocytes, but not oral epithelial cells, have all the information required for the proper physiological location and differentiation of melanocytes in the epidermis.

1988 - Morphologic and functional aspects of keratinocytes and melanocytes cultivated in vitro [Articolo su rivista]
De Luca, M.; Zicca, A.; D'Anna, F.; Cadoni, A.; Franzi, A. T.

1987 - Characterization of phosphate residues on thyroglobulin [Articolo su rivista]
Consiglio, E.; Acquaviva, A. M.; Formisano, S.; Liguoro, D.; Gallo, A.; Tassi, V.; Santisteban, P.; DE LUCA, Michele; Shifrin, S.; Yeh, H.; Kohn, L. D.

Follicular 19 S thyroglobulin (molecular weight 660,000) from rat, human, and bovine thyroid tissues contains approximately 10-12 mol of phosphate/mol of protein. These phosphate residues can be radiolabeled when rat thyroid hemilobes, FRTL-5 rat thyroid cells, or bovine thyroid slices are incubated in vitro with [32P]phosphate. Thus labeled, the [32P]phosphate residues comigrate with unlabeled 19 S follicular thyroglobulin on sucrose gradients and gel filtration columns; are specifically immunoprecipitated by an antibody preparation to rat or bovine thyroglobulin as appropriate; and co-migrate with authentic 19 S thyroglobulin when subjected to analytic or preparative gel electrophoresis. Tunicamycin prevents approximately 50% of the phosphate from being incorporated into FRTL-5 cell thyroglobulin. Approximately one-half of the phosphate in FRTL-5 cell or bovine thyroglobulin can also be released by enzymatic deglycosylation and can be located in Pronase-digested peptides which contain mannose, are endo-beta-N-acetylglucosaminidase H but not neuraminidase-sensitive, and release a dually labeled oligosaccharide containing mannose and phosphate after endo-beta-N-acetylglucosaminidase H digestion. The remainder of the phosphate is in alkali-sensitive phosphoserine residues (3-4/mol of protein) and phosphotyrosine residues (approximately 2/mol of protein). This is evidenced by electrophoresis of acid hydrolysates of 32P-labeled thyroglobulin and by reactivity with antibodies directed against phosphotyrosine residues. The phosphoserine and phosphotyrosine residues do not appear to be randomly located through the thyroglobulin molecule since approximately 75-85% of the phosphotyrosine and phosphoserine residues were recovered in a approximately 15-kDa tryptic peptide or a approximately 24-kDa cyanogen bromide peptide, each almost devoid of carbohydrate. 31P nuclear magnetic resonance studies of bovine thyroglobulin confirm the presence and heterogeneity of the phosphate residues on thyroglobulin preparations.

1987 - Method for preserving transplantable sheets of epithelium cultured in vitro [Brevetto]
Cancedda, R.; DE LUCA, Michele

A process for preserving cultured epithelial sheets by incubating the sheets at room temperature in media with a glycerol or dimethylsulfoxide as cryopreservant for a predetermined period of time and then freezing the cultured sheets. The cooling gradient is characterized as having a lower initial rate of cooling followed by a higher rate of cooling.

1987 - Metodo per la preservazione di fogli trapiantabili di epitelio coltivato in vitro vitale [Brevetto]
Cancedda, R.; DE LUCA, Michele

Si descrive un modo per la preservazione mediante congelamento di fogli di epidermide espansa "in vitro", caratterizzato da una prima fase di incubazione in apposito terreno, in condizioni di tempo e temperatura critiche, seguita da un congelamento a gradiente di raffreddamento programmato.

1987 - Nature of thyroid autoantigens: the TSH receptor [Capitolo/Saggio]
Chan, J.; DE LUCA, Michele; Santisteban, P.; Isosaki, O.; Shifrin, S.; Aloj, S. M.; Grollman, E. F.; Kohn, L. D.

This paper deals with the role of the TSH receptor in thyroid autoimmunity

1987 - [Separation, by elutriation, of human keratinocytes cultured in vitro] [Articolo su rivista]
Franzi, A. T; D'Anna, F; Zoli, S; Albanese, E; DE LUCA, Michele; Cancedda, R.


1987 - TSH receptor structure [Articolo su rivista]
Chan, J.; Santisteban, P.; DE LUCA, Michele; Isozaki, O.; Grollman, E.; Kohn, L. D.

When solubilized, radiolabelled membrane preparations from FRTL-5 rat thyroid cells are applied to TSH affinity columns, two separate peaks of protein can be eluted by high salts/high pH and low pH buffers, respectively. Immunoprecipitation with monoclonal antibodies to the TSH receptor shows that both peaks contain proteins related to the TSH receptor. If extracts were from cells grown without TSH, one peak has a approximately 300 K and the other a approximately 70 K protein the 70 K protein can be derived from the purified 300 K protein in vitro. A 50 and 20 K protein can be derived from the 70 K protein. If extracts are from cells grown with TSH, the peaks contain a multiplicity of additional immuno-precipitable bands of approximately 200, 175, 130, 90, 50, 20 K etc. These bands are shown to result from the ability of TSH to increase the synthesis (3-4-fold) and degradation (2-3-fold) of the 300 and 70 K proteins. The 300/70 K protein fractions are reactive with monoclonal autoimmune thyroid stimulating antibodies and contain a specific disialo ganglioside. The ganglioside migrates near GM2, i.e., like a lower order ganglioside, and contains fucose. In translation experiments, the monoclonal antibodies to the TSH receptor identify a single mRNA component which produces a protein of approximately 220 K. This protein is not present in thyroid cells which have no functional TSH receptor and which cannot be surface labelled with monoclonal antibodies to the TSH receptor

1986 - Monoclonal Antibodies defining the origin and properties of auto-antibodies in Graves' Disease [Articolo su rivista]
Kohn, L. D.; Alvarez, F.; Marcocci, C.; Kohn, A. D.; Corda, D.; Hoffman, W. E.; Tombaccini, D. Valente W. A.; DE LUCA, Michele; Santisteban, P.; Grollman, E. F.

This paper deals with the role of autoantibodies in the onset of autoimmune thyroid diseases and the characterization of the TSH receptor

1986 - Monoclonal Antibodies to the thyrotropin (TSH) receptor in the studies of Graves' disease [Articolo su rivista]
Kohn, L. D.; Valente, W. A.; Cheng, A.; Tombaccini, D.; Chan, J.; Corda, D.; Kohn, A. D.; Rotella, C. M.; Marcocci, C.; Santisteban, P.; DE LUCA, Michele; Bone, E.; Grollman, E. F.

This paper deals with the role of autoantibodies in the onset of autoimmune thyroid diseases and the characterization of the TSH receptor

1986 - Regulation of thyroglobulin (Tg) iodination and thyroid hormone formation in FRTL-5 cells [Relazione in Atti di Convegno]
Santisteban, P.; DE LUCA, Michele; Corda, D.; Grollman, E. F.; Kohn, L. D.


1985 - Monoclonal antibodies and autoimmune thyroid diseases [Capitolo/Saggio]
Kohn, Ld; Rotella, Cm; Marcocci, C; Toccafondi, R; Pinchera, A; Tombaccini, D; Valente, Wa; DE LUCA, Michele; Grollman, Ef


1985 - Monoclonal antibodies and the thyrotropin receptor [Capitolo/Saggio]
Kohn, Ld; Tombaccini, D; DE LUCA, Michele; Bifulco, M; Grollman, Ef; Valente, Wa


1985 - Thyroglobulin interaction with thyroid membranes: implications for regulation of thyroid hormone formation [Capitolo/Saggio]
Kohn, L. D.; DE LUCA, Michele; Santisteban, P.; Shifrin, S.; Yeh, H.; Formisano, S.; Consiglio, E.

This paper deals with the role of the interaction of thyroglobulin with cell membranes in the formation of T3 and T4

1984 - Human indifferentiated thyroid carcinoma synthesizes and secretes 19S thyroglobulin [Articolo su rivista]
Monaco, F.; Carducci, C.; DE LUCA, Michele; Andreoli, M.; Dominici, R.

Thyroglobulin (Tg) biosynthesis and secretion have been studied in one case of undifferentiated small spindle cell human thyroid carcinoma. From tumor tissue were extracted 32 mg of soluble proteins per gram of wet tissue, compared with 54 mg from control gland; after ammonium sulfate fractionation, density gradient centrifugation, and immunoprecipitation, 1.3 mg of Tg per gram of wet tissue, compared with 31.2 mg from control, were purified. By incubating in vitro up to 3 hours tumor slices with tritiated leucine, galactose, and 125I, the rate of incorporation of leucine and galactose increased with time in tumor tissue. However 125I incorporation into soluble proteins from tumor, at 3 hours of incubation, was only 1.7% of control value. By density gradient centrifugation of labeled soluble proteins a well-separated component sedimenting in the 19S peak was identified. No radioiodine was detected in the 19S fraction which showed the immunoreactive properties of 19S and was poor in 127I and sialic acid. In the incubation medium of tumor were secreted 0.4 mg of 19S Tg per gram of wet tissue compared with 48 mg of tissue in the control gland. Thus the undifferentiated thyroid carcinoma synthesizes a protein with the physiochemical and immunologic properties of 19S Tg. The undifferentiation of thyroid cells does not affect the biochemical steps involved in the synthesis of Tg.

1984 - Monoclonal antibodies as probes of thyrotropin receptor structure [Capitolo/Saggio]
Kohn, L. D.; Valente, W. A.; Laccetti, P.; Marcocci, C.; DE LUCA, Michele; Ealey, P. A.; Marshall, N. J.; Grollman, E. F.

This paper deals with the generation and production of antibodies aimed at the identification and functional characterization of the TSH receptor

1984 - THS receptor mediated growth of thyroid cells: mechanism and relation to autoimmune thyroid diseases [Relazione in Atti di Convegno]
Aloj, S. M.; Marcocci, C.; DE LUCA, Michele; Shifrin, S.; Grollman, E. F.; Kohn, L. D.


1983 - Specificity of thyroglobulin interaction with thyroid cells and membranes [Articolo su rivista]
Rotella, C. M.; Tanini, A. L.; Consiglio, E.; Shifrin, S.; DE LUCA, Michele; Toccafondi, R.; Kohn, L. D.

Homologous species specificity is demonstrated with bovine and human thyroglobulin in which the two terminal sugars of the B carbohydrate chain, sialic acid and galactose have been removed by enzymatic hydrolysis. The species specificity is demonstrated by measuring the ability of the deglycosylated thyroglobulin derivatives to inhibit thyrotropin-induced increases in cAMP in human, rat and bovine thyroid cells in culture. Thus human-human or bovine-bovine interactions have higher activity coefficients by at least an order of magnitude than their heterologous counterparts. The homologous interactions are confirmed in binding studies and shown to be associated with negligible degradation of the bound ligand over a 24 hour period.

1982 - [Inhibition of the biosynthesis of thyroglobulin in the thyroid gland of rats by isoniazid] [Articolo su rivista]
Monaco, F; DE LUCA, Michele; Carducci, C; Pontecorvi, A; D'Armiento, M; Roche, J.

Groups of rats have been treated for one month with a daily dose of 50 mg/kg of body weight/day of isoniazide a product used therapeutically in man against tuberculosis. The animals submitted to this experiment showed an increase of thyroid weight (39%) and a decrease of its content in water soluble proteins, including thyroglobulin and precursors. These proteins have been labeled in vitro by [3H]-4,5-L-leucine and by [3H]-galactose, by incubation of isolated glands during 4 h in a medium containing this labeled marker. The decrease of thyroglobulin-like proteins was of 20-37% for [3H]-D-1-galactose, labeled fraction and of 46-53% for the [3H]-leucine labeled fraction. Two thirds of the radioactive protein sedimented by ultracentrifugation in a sucrose gradient were present in a 12S monomer of thyroglobulin. This suggests an impairment of biosynthesis of 19S thyroglobulin, in which two of the subunit are associated. Under experimental conditions adopted this 19S protein was labeled by [3H]-leucine, but not by [3H]-galactose. Two other antituberculous agents studied, ethambutol and rifamizine, exerted not the same effects on thyroid gland in rats. Therefore a control of thyroid function appear to be necessarily only in man treated by isoniazine.

1982 - [Inhibition of the biosynthesis of thyroglobulin with propranolol in the rat] [Articolo su rivista]
Monaco, F; Pontecorvi, A; DE LUCA, Michele; de Pirro, R; D'Armiento, M; Roche, J.

Thyroglobulin biosynthesis was studied in thyroid glands of rats treated during 30 days with a daily dose of propranolol, a non-selective beta-adrenergic blocking drug. Studies were carried out by extraction of soluble proteins from homogenates after incubation of the glands in presence of [3H]-4,5-L-leucine and [3H]-D-1-galactose. Thyroglobulin 19S, 12S and 4-8S soluble proteins were separated and identified by ultracentrifugation in a saccharose gradient. The glands of rats treated with propranolol showed a decreased amount of soluble proteins as well as a decreased incorporation of [3H] labeled markers. 19S thyroglobulin is poorly represented, but very large amounts of the 12S monomer are present; this suggests an impairment in the dimerization of the 12S subunit, absent in normal controls. This impairment could be due to a structure modification. Propranolol reduces the biosynthesis of thyroglobulin in thyroid gland as well as the formation of T3 from T4 in peripheral tissues; its therapeutical use against thyrotoxicosis is thus justified. The study of its action on the dimerisation of the 12S subunit could be of interest to clear the mechanism of thyroglobulin biosynthesis.