Abnormal distribution of epidermal protein antigens in psoriatic epidermis.

The immunohistochemical distribution of the epidermal proteins filaggrin, involucrin, and cytokeratins is characteristic in normal epidermis. This distribution may change as a result of malignant transformation or abnormal differentiation. The present study was conducted to determine the patterns of reactivity of psoriatic epidermis to antibodies against various epidermal proteins and to clarify abnormal differentiation or maturation of the keratinocytes in psoriatic epidermis. Anti-human filaggrin, anti-human involucrin, and twelve kinds of anti-cytokeratin antibodies were used in this study. Cryostat or paraffin-embedded sections were stained with these antibodies by the avidin-biotin peroxidase technique. The epidermis of the noninvolved skin of patients with psoriasis vulgaris showed the distribution seen in normal skin. However, involved psoriatic skin revealed little or no reaction in the stratum corneum or in the granular layer with the anti-filaggrin antibody. Cells positively staining with anti-involucrin antibody paradoxically appeared in the lower cell layers of involved psoriatic epidermis. An anti-keratin antibody, AE1, stained suprabasal cells in involved psoriatic epidermis, although this antibody selectively stained epidermal basal cells in normal skin. The other anti-keratin antibodies, especially KL1, PKK1, and a polyclonal anti-keratin antibody, were less reactive with involved psoriatic skin than with normal skin. These observations suggest that the maturation pathway of keratinocytes in active psoriatic lesions differs qualitatively from that in normal epidermis.     

Carcinogenesis in porokeratosis. Evidence for a role relating to chronic growth activation of keratinocytes.

Porokeratoses are known to give rise to squamous and basal cell carcinomas. In this study, we examined 15 lesions of porokeratosis immunohistochemically for evidence of aberrant keratinization using several markers of keratinocyte (KC) maturation and differentiation, including involucrin, filaggrin, cytokeratins, and the growth activation marker psi-3. The staining patterns obtained were compared with several non-premalignant parakeratotic skin lesions including psoriasis, pityriasis rosea, pityriasis rubra pilaris, irritated seborrheic keratosis, atopic dermatitis, seborrheic dermatitis, and verruca vulgaris. The centers of porokeratoses stained in a pattern identical to that observed in other premalignant keratinocytic lesions including actinic keratoses, recessive dystrophic epidermolysis bullosa, and nonhealing wounds. KCs beneath the cornoid lamella (CL) stained in a pattern similar to that observed in squamous cell carcinomas. KCs peripheral to the CL in the epidermis showed a normal staining pattern. The control non-premalignant parakeratotic lesions displayed a variety of staining patterns, but none showed a pattern identical to that observed in porokeratosis. The failure of KCs in porokeratoses to mature and differentiate normally may be related to the increased incidence of carcinomas associated with these lesions.     

Correction of dog dystrophic epidermolysis bullosa by transplantation of genetically modified epidermal autografts

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe skin blistering condition caused by mutations in the gene coding for collagen type VII. Genetically engineered RDEB dog keratinocytes were used to generate autologous epidermal sheets subsequently grafted on two RDEB dogs carrying a homozygous missense mutation in the col7a1 gene and expressing baseline amounts of the aberrant protein. Transplanted cells regenerated a differentiated and vascularized auto-renewing epidermis progressively repopulated by dendritic cells and melanocytes. No adverse immune reaction was detected in either dog. In dog 1, the grafted epidermis firmly adhered to the dermis throughout the 24-month follow-up, which correlated with efficient transduction (100%) of highly clonogenic epithelial cells and sustained transgene expression. In dog 2, less efficient (65%) transduction of primary keratinocytes resulted in a loss of the transplanted epidermis and graft blistering 5 months after transplantation. These data provide the proof of principle for ex vivo gene therapy of RDEB patients with missense mutations in collagen type VII by engraftment of the reconstructed epidermis, and demonstrate that highly efficient transduction of epidermal stem cells is crucial for successful gene therapy of inherited skin diseases in which correction of the genetic defect confers no major selective advantage in cell culture.     

Normal and gene-corrected dystrophic epidermolysis bullosa fibroblasts alone can produce type VII collagen at the basement membrane zone

Type VII collagen is synthesized and secreted by both human keratinocytes and fibroblasts. Although both cell types can secrete type VII collagen, it is thought that keratinocytes account for type VII collagen at the dermal-epidermal junction (DEJ). In this study, we examined if type VII collagen secreted solely by dermal fibroblasts could be transported to the DEJ. We established organotypic, skin-equivalent cultures composed of keratinocytes from patients with recessive dystrophic epidermolysis bullosa (RDEB) and normal dermal fibroblasts. Immuno-labeling of skin equivalent sections with the anti-type VII collagen antibody revealed tight linear staining at the DEJ. RDEB fibroblasts, were gene-corrected to make type VII collagen and used to regenerate human skin on immune-deficient mice. The human skin generated by gene-corrected RDEB fibroblasts or normal human fibroblasts combined with RDEB keratinocytes restored type VII collagen expression at the DEJ in vivo. Further, intradermal injection of normal human or gene-corrected RDEB fibroblasts into mouse skin resulted in the stable expression of human type VII collagen at the mouse DEJ. These data demonstrate that human dermal fibroblasts alone are capable of producing type VII collagen at the DEJ, and it is possible to restore type VII collagen gene expression in RDEB skin in vivo by direct intradermal injection of fibroblasts.     

The generalized atrophic benign form of junctional epidermolysis bullosa. Experience with four patients in the United States

We encountered four patients in the United States with the generalized atrophic benign form of junctional epidermolysis bullosa (epidermolysis bullosa atrophicans generalisata mitis, nonlethal junctional epidermolysis bullosa). Prior to the performance of definitive diagnostic studies, each patient had been thought for at least a decade to have either a dystrophic or simplex form of epidermolysis bullosa. Each patient had generalized blisters since birth that healed with atrophy and mild scarring but without milia or contractures. Two of the four patients had experienced laryngeal involvement during childhood. In each patient, correct diagnosis was finally established by either electron microscopic examination or immunofluorescence mapping of skin sections from induced blisters.     

Epidermal aspects of type VII collagen: Implications for dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita

Type VII collagen (COL7), a major component of anchoring fibrils in the epidermal basement membrane zone, has been characterized as a defective protein in dystrophic epidermolysis bullosa and as an autoantigen in epidermolysis bullosa acquisita. Although COL7 is produced and secreted by both epidermal keratinocytes and dermal fibroblasts, the role of COL7 with regard to the epidermis is rarely discussed. This review focuses on COL7 physiology and pathology as it pertains to epidermal keratinocytes. We summarize the current knowledge of COL7 production and trafficking, its involvement in keratinocyte dynamics, and epidermal carcinogenesis in COL7 deficiency and propose possible solutions to unsolved issues in this field.     

Recessive dystrophic epidermolysis bullosa phenotype is preserved in xenografts using SCID mice: development of an experimental in vivo model.

Recessive dystrophic epidermolysis bullosa (RDEB) is a subgroup of hereditary blistering diseases characterized by repetitive wounding and healing with subsequent extensive scarring. The purpose of this study was to establish a xenograft model that retains the RDEB phenotype and thus might be used as an experimental in vivo model to explore the molecular and biochemical mechanisms of the chronically wounded phenotype of RDEB. Full-thickness, tumor-free RDEB skin tissues were grafted onto the dorsum of severe combined immunodeficiency (SCID) mice. At 4, 8, 12, and 24 weeks after grafting, the xenografts were removed for examination. Immunofluorescence studies were performed using species-specific antibodies to human class I antigen, mouse class I antigen, human type IV and VII collagens and with cross-reacting antibody against bullous pemphigoid antigen (BPA). Staining with the antibody to human class I antigen, W6/32, and with the antibody to mouse class I antigen, 20.8.4s, confirmed the species-specific results obtained with the type IV and type VII collagen and laminin antibodies. The RDEB grafts showed essentially no staining with the type VII collagen antibody. Antibodies against laminin and BPA showed normal staining patterns in RDEB grafts. There was an overall paucity of anchoring fibrils in the grafts when examined with electron microscopy. Blisters could be induced in these grafts with minor trauma and showed a sublamina densa separation by immunomapping and electron microscopy. As late as 24 weeks post-transplantation, the RDEB grafts remain human, are not significantly replaced by mouse cells, and retain the RDEB disease phenotype.     

Stem cell therapies for recessive dystrophic epidermolysis bullosa

Human epidermis is composed of a stratified squamous epithelium that provides a mechanical barrier against the external environment and which is renewed every 3–4 weeks by resident stem cells in the epidermis. However, in the inherited skin fragility disorder, recessive dystrophic epidermolysis bullosa (RDEB), there is recurrent trauma‐induced subepidermal blistering that disrupts epidermal homeostasis and is likely to deplete the epidermal stem cell pool. This review article discusses the nature of epidermal stem cells and other stem cell populations in the skin, as well as other possible extracutaneous sources of stem cells, that might have physiological or therapeutic relevance to cell therapy approaches for RDEB. Strategies to identify, create and use cells with multipotent or pluripotent properties are explored and current clinical experience of stem cell therapy in RDEB is reviewed. There is currently no single optimal therapy for patients with RDEB, but cell therapy technologies are evolving and hold great potential for modifying disease severity and improving quality of life for people living with RDEB.     

Epidermolysis bullosa simplex superficialis. A new variant of epidermolysis bullosa characterized by subcorneal skin cleavage mimicking peeling skin syndrome

We report a new variant of epidermolysis bullosa simplex (EBS), termed EBS superficialis, which is characterized by the development of skin cleavage just beneath the level of stratum corneum. In two of seven patients identified, a second and more focal cleft within the lower third of the epidermis was also detected. Epidermolysis bullosa simplex superficialis appears to be transmitted as an autosomal dominant trait. It differs from other autosomal dominant forms of EBS by the common findings of milia and atrophic scarring, as well as involvement of oral and/or ocular surfaces. Epidermolysis bullosa simplex superficialis is further differentiated from peeling skin syndrome by the presence of blisters and the absence of spontaneous continual exfoliation or peeling.     

Abnormalities of basal cell keratin in epidermolysis bullosa simplex do not affect the expression patterns of suprabasal keratins and cornified cell envelope proteins

Abstract Basal keratins, suprabasal keratins, filaggrin, and cornified cell envelope (CCE) precursor proteins are expressed during the differentiation of epidermal keratinocytes. These molecules are coordinately expressed during epidermal differentiation. The present study investigated the expression patterns of keratins and CCE precursor proteins in 15 patients with epidermolysis bullosa simplex (EBS), which is caused by mutations in the genes that encode for the basal keratins, keratins 5 and 14. The patterns of expression of keratins 5, 14, 1 and 10, filaggrin, and of the three major CCE precursor proteins, involucrin, loricrin and small proline-rich proteins 1 and 2 (SPRs), were studied immunohistochemically and by electron microscopy. In 14 of the 15 patients with EBS, the distribution pattern of keratins was not altered. In one neonate with EBS, basal cell keratins were expressed in the suprabasal layers. Ultrastructurally, numerous clumped tonofilaments were observed in the basal and suprabasal cells. In all cases, findings were positive for filaggrin in the granular cells, with positivity for involucrin in the upper spinous and granular cells. The upper spinous cells and granular cells were positive for SPRs 1 and 2, and loricrin was expressed in granular cells. Ultrastructurally, no marked abnormality was observed in the suprabasal layers such as a decrease in, or agglutination of, keratin filaments, except in one neonate. A CCE about 15 nm thick was formed normally in the cell membrane of cornified cells. The patterns of distributions of basal cell keratins, suprabasal keratins, filaggrin, and CCE precursor proteins, as well as the ultrastructural findings, resembled those of normal skin. Thus, the abnormality in basal cell keratins in patients with EBS did not appear to alter the patterns of expression of the keratins and CCE precursor proteins. Received: 8 April 1998 / Received after revision: 17 July 1998 / Accepted: 30 July 1998     

Life before and beyond blistering: The role of collagen XVII in epidermal physiology

Type XVII collagen (COL17) is a transmembranous protein that is mainly expressed in the epidermal basal keratinocytes. Epidermal‐dermal attachment requires COL17 expression at the hemidesmosomes of the epidermal basement membrane zone because congenital COL17 deficiency leads to junctional epidermolysis bullosa and acquired autoimmunity to COL17 induces bullous pemphigoid. Recently, in addition to facilitating epidermal‐dermal attachment, COL17 has been reported to serve as a niche for hair follicle stem cells, to regulate proliferation in the interfollicular epidermis and to be present along the non‐hemidesmosomal plasma membrane of epidermal basal keratinocytes. This review focuses on the physiological properties of COL17 in the epidermis, its role in maintaining stem cells and its association with signalling pathways. We propose possible solutions to unanswered questions in this field.     

Epidermolysis bullosa acquisita. Incidence in patients with basement membrane zone antibodies

We examined the incidence of epidermolysis bullosa acquisita in patients with circulating basement membrane zone antibodies. Serum samples from 100 sequential patients with basement membrane zone antibodies were tested by indirect immunofluorescence against 1 mol/L sodium chloride split skin and by Western immunoblot against epidermal and dermal extracts of skin. Ninety-two (92%) serum samples stained only the epidermal side of split skin, 5 (5%) stained only the dermal side, and 3 (3%) stained both sides. Four of the 5 serum samples with dermal staining but none of the serum samples with epidermal or combined staining reacted with the 290-kd epidermolysis bullosa acquisita antigen by Western immunoblot. These results indicate that approximately 5% of unselected patients with basement membrane zone antibodies have epidermolysis bullosa acquisita or bullous lupus erythematosus rather than bullous pemphigoid.     

Epidermolysis bullosa acquisita: an autoimmune disease with distinctive immunoultrastructural features

Epidermolysis bullosa acquisita is a subepidermal bullous disease of the skin with distinctive clinical, histologic, immunologic, and ultrastructural features. A case of epidermolysis bullosa acquisita is presented in which the onset of the disease was heralded by inflammatory blisters similar to herpes simplex or herpes zoster. The immunofluorescent findings were indistinguishable from bullous pemphigoid. Although some inflammatory blisters persisted throughout the evolution of the disease, the patient eventually developed noninflammatory blisters that healed with milia formation and scarring, lesions typical of classic epidermolysis bullosa acquisita. The diagnosis of epidermolysis bullosa acquisita was definitely made by electron and immunoelectron microscopy which showed a sub-lamina-densa blister cleavage plane and immune deposits beneath the lamina densa. This case illustrates that some cases of epidermolysis bullosa acquisita may have an inflammatory stage and immunofluorescent findings that make it difficult to distinguish from inflammatory bullous diseases. The value of electron microscopy and immunoelectron microscopy in making a firm diagnosis of epidermolysis bullosa acquisita is emphasized.     

Identification of an epidermal basement membrane defect in recessive forms of dystrophic epidermolysis bullosa by LH 7:2 monoclonal antibody: use in diagnosis

LH 7:2 is a monoclonal antibody that was raised against an extract of human epidermal cells and identifies an epitope within the lamina densa of the basement membrane of stratified squamous epithelia. Using indirect immunofluorescence we found intense labelling with LH 7:2 at the epidermal basement membrane (EBM) of normal skin, and in skin samples from patients with simplex, junctional, dominantly inherited dystrophic and acquired forms of epidermolysis bullosa (EB), as well as bullous pemphigoid. Staining was absent or only very faint in generalized recessive dystrophic EB (RDEB), and patchily reduced in the localized form of RDEB. We conclude that LH 7:2 recognizes an EBM antigen which may be important in the pathogenesis of RDEB. Moreover, the antibody provides a useful probe for the rapid diagnosis of RDEB and is of special value in helping to discriminate between localized RDEB and typical dominant dystrophic EB--conditions which closely resemble each other clinically and which cannot be distinguished by means of transmission electron microscopy.     

The course of pregnancy and childbirth in three mothers with recessive dystrophic epidermolysis bullosa

Background. Recessive dystrophic epidermolysis bullosa (RDEB) is an autosomal recessive skin disease caused by mutations in the type VII collagen gene (COL7A1), resulting in detachment of the entire epidermis due to loss or hypoplasticity of the anchoring fibrils that normally secure the basement membrane to the underlying dermis. Trauma‐induced blistering is often complicated by chronic erosions and scarring. From that perspective, pregnancy in RDEB might be considered an indication for elective caesarean section in a bid to minimize perineal blistering. To date, only four cases of pregnancy and delivery in patients with RDEB have been reported. Cases. We report three more women, each with RDEB‐generalized other (RDEB‐GO), all of whom had successful vaginal deliveries without major cutaneous or mucosal complications. One woman also had a second child, by vaginal delivery, indicating a lack of vaginal stenosis after the first birth. Conclusions. These cases show that RDEB‐GO is not an absolute primary indication for elective caesarean section and that, perhaps surprisingly, genital/perineal blistering and scarring are not inevitable consequences of childbirth. Moreover, breastfeeding is also feasible in women with RDEB‐GO.     

Electron microscopy and morphometry enhances differentiation of epidermolysis bullosa subtypes. With normal values for 24 parameters in skin

Electron microscopy combined with morphometry was used to establish values for 24 parameters in normal skin. These results were compared with those similarly obtained from samples of epidermolysis bullosa with a view to facilitating classification of the disease. Six of the eight subtypes of epidermolysis bullosa investigated could be differentiated. Four subtypes showed values for structural components in intact skin which were outside the normal range: (1) epidermolysis bullosa simplex generalisata gravis (hemidesmosomes); (2) epidermolysis bullosa dystrophica Pasini and (3) Cockayne-Touraine (anchoring fibrils); and (4) epidermolysis bullosa acquisita (anchoring fibrils, hemidesmosomes, and lamina lucida and lamina densa aspects of the dermoepidermal junction). Two subtypes revealed specific features which could be assessed qualitatively: distinctive, circumscribed, clumped tonofilament bodies were present in basal keratinocytes from epidermolysis bullosa herpetiformis Dowling-Meara and thick (30 nm diameter) cross-striated anchoring fibrils were absent in epidermolysis bullosa dystrophica generalisata gravis. Epidermolysis bullosa simplex Köbner and Weber-Cockayne forms could not be distinguished.     

Immunohistochemical studies on regeneration in cultured epidermal autografts used to treat full-thickness burn wounds

The normal pattern of healing following culture grafting is not well-established. The present study describes the immunohistochemcal expression of differentiation markers during various stages of wound healing after resurfacing full-thickness burn wounds with cultured epidermal autografts. For this purpose, biopsy specimens were obtained from six patients 6 days to 4-5 years after transplantation. A panel of monoclonal antibodies was used against various differentiation-specific protein markers, including cytokeratins, involucrin, transgluta-minase and filaggrin. Findings from early and late biopsies were compared with site-matched normal skin and cultured epidermal sheets. The persistence of cytokeratins 6 and 16, and to a lesser extent, the abnormal distribution of involucrin up to 4-5 years after resurfacing burns with cultured autografts was observed. These findings confirm previous studies–that the newly formed epidermis after culture grafting remains in a hyperproliferative state for a long time, which may be caused by the absence of a modulating dermal factor. Our results suggest that keratinocyte maturation following culture autografting does not return to normal for at least 4–5 years after healing of full-thickness burn wounds.     

Keratinocyte cell lines derived from severe generalized recessive Epidermolysis Bullosa patients carrying a highly recurrent COL7A1 homozygous mutation: models to assess cell and gene therapies in vitro and in vivo

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by deficiency of type VII collagen due to COL7A1 mutations such as c.6527insC, recurrently found in the Spanish RDEB population. Assessment of clonal correction–based therapeutic approaches for RDEB requires large expansions of cells, exceeding the replication capacity of human primary keratinocytes. Thus, immortalized RDEB cells with enhanced proliferative abilities would be valuable. Using either the SV40 large T antigen or papillomavirus HPV16‐derived E6‐E7 proteins, we immortalized and cloned RDEB keratinocytes carrying the c.6527insC mutation. Clones exhibited high proliferative and colony‐forming features. Cytogenetic analysis revealed important differences between T antigen‐driven and E6‐E7‐driven immortalization. Immortalized cells responded to differentiation stimuli and were competent for epidermal regeneration and recapitulation of the blistering RDEB phenotype in vivo. These features make these cell lines useful to test novel therapeutic approaches including those aimed at editing mutant COL7A1.     

Characterization of sunburn cells after exposure to ultraviolet light

Sunburn cells (SBCs) appear in the epidermis shortly after acute UV damage, especially after exposure to UVB light. As yet, the mode of their formation remains to be satisfactorily elucidated. In order to characterize these cells, the expression of various markers of epidermal differentiation following UV exposure was investigated using immunhistochemical procedures. These were applied to paraffin-embedded (microwave technique) and frozen specimens of human skin 24 h after irradiation with 4 times the minimal erythema doses(MED). Normal nonirradiated skin without irradiation served as the control. We used a battery of antibodies directed against the following: cytokeratins (CKs) 5, 10, 17, and 19, actin, cell-adhesion proteins (desmoplakins, desmogleins), markers of terminal epidermal differentiation (filaggrin, involucrin and loricrin), markers of proliferation (PCNA, MIB, K6,16), a marker of endocytosis (clathrin) and markers of cell growth, (transforming growth factor [TGF-α]) and B-cell leukemia/lymphoma-2 [bcl-2]. After UV irradiation it was found that CK 5, which is typically confined to basal keratinocytes, was also expressed in suprabasal keratinocytes. The CKs 1 and 10/11 exhibit a normal suprabasal localization, but suprisingly, SBCs were negative for these CKs. Although CK 6,16, and 17 are not usually found in normal epidermis, UVB exposure induced their expression in suprabasal keratinocytes, but again failed to elicit their expression in SBCs. Antibodies specific for markers of late epidermal differentiation (filaggrin, involucrin and loricrin), cell-junction proteins (desmogleins, desmoplakins), proliferation (PCNA and MIB), and endocytosis (clathrin) also failed to produce positive staining of SBCs. Even though TGF-α immunoreactivity became detectable in most keratinocytes after UV exposure, this was not the case for SBCs. The number of basally located dendritic cells, most probably melanocytes, exhibiting bcl-2 staining was markedly reduced 6 and 12 h after irradiation as compared with normal skin. SBCs do not express any late differentiation markers, but they do contain proteins typical of basal keratinocytes (CK 5). It can be concluded that SBCs do not develop beyond a more basal-like differentiation pattern, probably as a result of cell death and migration through the epidermis.     

Transient intraepidermal bullous reaction after skin graft for toxic epidermal necrolysis. Ultrastructural and immunohistochemical features similar to those of inherited epidermolysis bullosa simplex

Blister formation in skin graft donor or recipient sites is uncommon. We describe a 49-year-old female patient with bullae in sites of grafts used in the treatment of toxic epidermal necrolysis. Generalized loss of skin developed 3 weeks after she had ingested phenobarbital. Sixty days after the beginning of the toxic epidermal necrolysis, the reepidermization was only 80% and skin grafts were placed on lower-extremity and abdominal wounds using the first healed sites as donor sites. Several bullae and erosions were noted on grafted areas 3 weeks later. Skin biopsy specimens revealed separation at the dermoepidermal junction, and no autoantibodies were detected by direct and indirect immunofluorescence. Electron microscopy demonstrated that the blister was formed through the basal keratinocytes and that the dermoepidermal junction, including hemidesmosomes and anchoring fibrils, was normal. Immunofluorescence mapping was performed using polyclonal antibodies from the serum of patients with bullous pemphigold and epidermolysis bullosa acquisita and monoclonal antibodies against GB3 antigen and collagen type VII. All but the bullous pemphigold serum gave positive results; only faint and focal staining of the dermoepidermal junction was observed with bullous pemphigold serum. These findings are the same as those encountered in hereditary epidermolysis bullosa simplex. A biopsy performed 1 year later in the same site as the first one revealed that bullous pemphigold antigen was normally expressed. Keratinocytes autografted in the treatment of toxic epidermal necrolysis may become transiently, functionally abnormal because of the alteration of recipient sites.