Long-term results of catheter-based treatment of pulmonary atresia and intact ventricular septum.

OBJECTIVE: To review the outcome of patients with pulmonary atresia with intact ventricular septum after interventional perforation of the pulmonary valve, to assess the capability of this procedure to avoid neonatal or late intervention and to obtain a long-term biventricular repair. DESIGN: Retrospective interventional study and clinical follow-up study. SETTING: Tertiary referral centre. PATIENT POPULATION: Between November 1994 and December 2007, 40 neonates underwent radiofrequency perforation. Median age at pulmonary valvotomy was 28 hours (range 1-147 hours) and median weight was 2925 g (range 1900-4400 g). MAIN OUTCOME MEASURES: Procedural success and complication rates; early-term and long-term follow-up results. RESULTS: The procedure was successful in 39 patients but 16 of them needed neonatal surgery. The overall mortality was 7.5%. At a median follow-up of 82 months, four patients underwent a bidirectional Glenn procedure, whereas all the other patients achieved a biventricular circulation without any further intervention in 19 of them. Patients who died or needed additional intervention with or without biventricular circulation failure had a higher incidence of bipartite right ventricular (65% vs 15.8% of those not needing additional intervention; p = 0.004) and a lower median tricuspid Z value (-2 (range -3.5 to 1) vs -0.5 (range -2 to 1); p = 0.004)). CONCLUSIONS: The results confirm that percutaneous interventional perforation is an effective first-stage procedure in patients with pulmonary atresia with intact ventricular septum. The right heart appeared to be adequate to maintain a long-term biventricular circulation in the large majority of cases.

Permanent repigmentation of piebaldism by erbium:YAG laser and autologous cultured epidermis.

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.

Gene correction of integrin beta4-dependent pyloric atresia-junctional epidermolysis bullosa keratinocytes establishes a role for beta4 tyrosines 1422 and 1440 in hemidesmosome assembly.

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.

p63 identifies keratinocyte stem cells.

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.

Treatment of "stable" vitiligo by Timedsurgery and transplantation of cultured epidermal autografts.

OBJECTIVE: To optimize melanocyte/keratinocyte co-cultivation 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.

Downregulation of 14-3-3sigma prevents clonal evolution and leads to immortalization of primary human keratinocytes.

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 14-3-3sigma, 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. 14-3-3sigma-dependent bypass of senescence is accompanied by maintenance of telomerase activity and by downregulation of the p16(INK4a) 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.

The control of epidermal stem cells (holoclones) in the treatment of massive full-thickness burns with autologous keratinocytes cultured on fibrin.

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.

Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.

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.

Cultivation of human keratinocyte stem cells: current and future clinical applications.

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.

Stratifin, a keratinocyte specific 14-3-3 protein, harbors a pleckstrin homology (PH) domain and enhances protein kinase C activity.

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.

Nerve growth factor binds to normal human keratinocytes through high and low affinity receptors and stimulates their growth by a novel autocrine loop.

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.

Alpha melanocyte stimulating hormone (alpha MSH) stimulates normal human melanocyte growth by binding to high-affinity receptors.

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.

Expression of integrin receptors and their role in adhesion, spreading and migration of normal human melanocytes.

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.

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.

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.

In vitro paracrine regulation of human keratinocyte growth by fibroblast-derived insulin-like growth factors.

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.

Growth-regulated synthesis and secretion of biologically active nerve growth factor by human keratinocytes.

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.

Polarized expression of integrin receptors (alpha 6 beta 4, alpha 2 beta 1, alpha 3 beta 1, and alpha v beta 5) and their relationship with the cytoskeleton and basement membrane matrix in cultured human keratinocytes.

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.

Polarized integrin mediates human keratinocyte adhesion to basal lamina.

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.

Multicentre experience in the treatment of burns with autologous and allogenic cultured epithelium, fresh or preserved in a frozen state.

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.