Periodate-labile structures at the normal human cutaneous basement membrane zone

The distribution of carbohydrates containing 1,2-glycol groups at the basement membrane zones of the dermo-epidermal junction and dermal blood vessels was investigated using the periodic acid-thiosemicarbazide-silver proteinate technique. Controls included pre-incubation with dimedone, dimedone treatment both before and after periodate oxidation, and dimedone treatment followed by thiosemicarbazide and silver proteinate. At the dermo-epidermal junction the anchoring fibrils were stained selectively after both short (45 min) and long (71 h) thiosemicarbazide incubation periods, there being no staining of the lamina densa; however, the lamina densa surrounding blood vessels was labelled. After prolonged treatment with thiosemicarbazide, fibrils were observed traversing the lamina lucida at the dermo-epidermal junction, and collagen fibres were stained faintly. Pre-incubation with dimedone to block aldehyde groups did not affect the staining. However, when periodic acid treatment was omitted, or when sections were incubated with dimedone both before and after periodate oxidation, only melanin granules and the cytoplasm of fibroblasts were stained. Thus, anchoring fibrils are rich in carbohydrates containing 1,2-glycol groups susceptible to periodate oxidation, and correspond to the periodic acid Schiff-positive layer seen at the dermo-epidermal junction. Furthermore, the basement membrane zones of dermal blood vessels and the dermo-epidermal junction differ in their carbohydrate distribution.     

Epithelial transition zones: merging microenvironments, niches, and cellular transformation

Transition zones (TZs) are regions in the body where two different types of epithelial tissue meet resulting in the appearance of a distinct abrupt transition. These TZs are found in numerous locations within the body, including the cornea-conjunctiva junction, esophagogastric junction, gastro-duodenal junction, endo-ectocervix junction, ileocecal junction, and anorectal junction. Several of these TZs are often associated with the development of cancer, in some cases due to viral transformation by the human papilloma virus (HPV). The underlying molecular and cellular basis for this tumor susceptibiblity is unknown. The distinct epithelial morphology and location results in unique properties being conferred upon this epithelial tissue, as different signaling cues and cell surface markers are apparent. Importantly, the natural state of TZs closely resembles that of a pre-lesional epithelium, as several proteins that are induced during wounding are expressed specifically within this region, which may contribute to transformation. This region may also act as a stem cell niche, and as such, represents a key location for cellular transformation by accumulated genetic mutations or viral transformation resulting in tumor formation.     

Immunofluorescent staining with antibodies to factor VIII, fibronectin, and collagenous basement membrane protein in normal human skin and port wine stains

Specific antibodies directed against three important components of the vessel wall (collagenous basement membrane protein-type IV collagen, fibronectin, and factor VIII) were used to study and compare the distribution of these proteins in normal skin and port wine stains. Collagenous basement membrane protein was localized to the basement membrane and basal lamina zones of blood vessels, appendages, arrector pili muscles, endoneurium and perineurium, and the dermoepidermal junction of both port wine stains and normal skin. Vessels of the port wine stain as well as those of normal skin showed a similar narrow uniform homogeneous line of fluorescence. Granular endothelial staining was seen in the blood vessels of both normal skin and port wine stains. The distribution of fibronectin was that of a low-intensity punctate pattern situated in the subendothelial region of both port wine stain and normal vessels and in the basement membrane zones of hair follicles, endoneurium and perineurium, and the dermoepidermal junction.     

Neuropeptides and general neuronal marker in psoriasis an immunohistochemical study

Nerve fibres immunoreactive to antibodies to vasoactive intestinal polypeptide (VIP) and substance P (SP) were increased in lesional psoriatic skin when assessed semi-quantitatively. Biopsies from psoriatic plaques on the arm were studied in 13 patients and compared with biopsies from non-lesional areas (in three of the same psoriatic subjects) and from normal skin in seven non-psomtic controls. Immunohistochemical methods were used on cryocut skin sections to demonstrate the neuropeptides SP, VIP, calcitonin gene-related peptide and neuropeptide Y, and the general neuronal marker protein gene product (PGP) 9.5. The immunofluorescence was examined by semiquantitative and, for PGP 9.5, by quantitative methods. VIP reactive nerve fibres were increased at areas of eccrine sweat glands throughout the dermis, at the dermo-epidermal junction, and in the epidermis, in psoriasis lesional skin. SP reactive nerve fibres were increased at the dermo-epidermal junction, where the nerves ran parallel with and perpendicularly through the junction. PGP 9.5 reactive nerve fibres showed an increase at the dermo-epidermal junction, in the papillary dermis, and at the eccrine sweat glands in lesional psoriatic skin but not in non-lesional, or in control skin. These findings support the hypothesis that neuropeptides may be involved in the pathogenesis of psoriasis.     

Cell-Based Therapy for RDEB: How Does It Work?

No effective or specific treatment is currently available for recessive dystrophic epidermolysis bullosa (RDEB), a severe heritable blistering disorder caused by mutations in the type VII collagen gene (COL7A1). Recent studies have suggested that delivery of allogeneic fibroblasts to the skin of patients with RDEB may be beneficial in improving skin adhesion and increasing type VII collagen deposition at the dermal-epidermal junction. In this issue, Nagy et al. explore mechanisms of fibroblast therapy in a patient with RDEB displaying reduced type VII collagen protein expression at the dermal-epidermal junction. The results suggest that allogeneic fibroblast injection elicits expression of cytokines, including heparin binding-EGF-like growth factor (HB-EGF), that upregulate the expression of a patient-specific COL7A1 allele. Thus, fibroblast therapy may provide a novel way to improve skin therapy in a select subgroup of patients with this currently intractable disease.     

Characterization of mutations leading to recessive dystrophic epidermolysis bullosa and Marfan syndrome in a single patient

Dystrophic epidermolysis bullosa (DEB) is a rare genetic skin disorder. In this report we have investigated an Italian child affected with recessive DEB (RDEB) and demonstrated that he was homozygous for the mutation R226X in the type VII collagen gene (COL7A1), leading to absence of type VII collagen at the dermal-epidermal junction. There was no family history of inherited skin blistering but the child's father was affected by Marfan syndrome, an autosomal dominant connective tissue disorder that results from mutations in the fibrillin-1 gene (FBN1). Analysis of this gene showed that the RDEB patient and his father were both heterozygous for a novel FBN1 mutation, C1971Y. This mutation affects one of the six obligate cysteine residues within one of the calcium-binding epidermal growth factor-like regions of the protein. At the age of 2-years the RDEB patient showed signs of early aortic dilatation, suggesting that he is likely to develop a Marfan syndrome phenotype in the future. This is a unique case of these two coexisting inherited disorders.     

Effect of C-xyloside on morphogenesis of the dermal epidermal junction in aged female skin. An ultrastuctural pilot study

A placebo-controlled randomized pilot study was performed on five postmenopausal women aged from 60 to 75 years. The women applied 320?mg (2?mg/cm(2))?of either placebo or 10% C-β-D-xylopyranoside-2-hydroxy-propane (C-xyloside) cream to each outer forearm twice daily for 3?months. At the end of the treatment, skin biopsies were collected from application areas on both forearms. Transmission electron microscope examinations revealed skin ultrastructural changes at the dermal epidermal junction (DEJ) after 10% C-xyloside application for 3 months. The morphological appearance of the DEJ showed strong improvements, with more homogeneous and regular lamina densa in the C-xyloside-treated compared to the placebo treated skin areas. The number of zones showing basement membrane re-duplication was indeed strikingly reduced on C-xyloside-treated skin. These ultrastructural results were further confirmed by a statistically significant increase in the expression levels of α6-integrin the and laminin-332, as estimated by immunohistochemistry. Altogether, these data suggest that topical C-xyloside application in vivo may be efficient in inducing a better dermal-epidermal cohesion when such a junction is deficient, as is the case in photo-aged or chronologically aged skin. Moreover, a statistically significant increase in CD44 expression was noted in the epidermis of C-xyloside-treated compared to the placebo treated skin areas.     

Distribution of mast cells in human dermis: development of a mapping technique

A new method was developed to define the distribution of mast cells in normal human skin. The population density was determined in a number of zones of different depth in the dermis. Two regions of the same limb were studied systematically. Both were shown to have mast cell populations of similar size and distribution, with a maximum density immediately below the dermo-epidermal junction, gradually falling to a minimum density in the deeper layers of the dermis. Small secondary peaks were less clearly defined at the base of the dermis. These findings should provide baseline data for comparisons with mast cell population size and distribution in different areas of normal skin, or in similar areas in clinical disorders or experimental conditions.     

Lipoid proteinosis

Lipoid proteinosis is a rare, autosomal recessive disorder that presents in early infancy with hoarseness, followed by pox-like and acneiform scars, along with infiltration and thickening of the skin and certain mucous membranes. Histological and ultrastructural examination reveals widespread deposition of hyaline-like material and disruption/reduplication of basement membrane around blood vessels and at the dermal--epidermal junction. Recently, lipoid proteinosis was mapped to 1q21 and pathogenetic loss-of-function mutations were identified in the extracellular matrix protein 1 gene (ECM1). This article reviews the molecular basis of lipoid proteinosis and reassesses the clinico-pathological features of this disorder in light of the new genetic discoveries.     

The biomolecular and ultrastructural basis of epidermolysis bullosa

Transmission electron microscopy, immunoelectron microscopy, immunofluorescence and antigenic mapping have improved our understanding of the dermo-epidermal junction. We have reviewed some ultrastructural and biomolecular aspects related to the dermo-epidermal junction. In part, they are implicated in the pathogenesis of a group of hereditary disorders characterized by skin fragility, collectively known as epidermolysis bullosa (EB). These disorders could benefit in the near future from a gene therapy approach but at present genetic counseling, prenatal diagnosis and conservative treatment measures offer little real benefit to patients.     

Interactions between fibroblasts and keratinocytes in morphogenesis of dermal epidermal junction in a model of reconstructed skin

De novo dermal epidermal junction morphogenesis was studied in a skin model including dermal fibroblasts and epidermal keratinocytes. Sequential gene expression, protein deposition, and localization of basement membrane zone components were studied during 15 days. The morphogenesis of dermal epidermal junction is characterized by an implementation of the different components and then a subsequent plateau phase occurring at day 11. Three groups of genes were identified depending on cellular origin and expression profile: 1/genes of fibroblastic origin (col I alpha1, col III alpha1, nidogen, and fibrillin 1); 2/genes expressed in fibroblasts and keratinocytes with symmetrical expression pattern between both cell types (col IV alpha1, col VII alpha1, and tenascin C); 3/laminin beta3 only expressed in keratinocytes. Use of modified organotypic models excluding one cell type revealed a tight interplay between fibroblasts and keratinocytes for synthesis and localization of the components of dermal epidermal junction. Keratinocytes downregulated mRNA and proteins of fibroblastic origin, upregulated col VII in fibroblasts and were absolutely required for dermal-epidermal junction localization of fibroblastic proteins. Fibroblasts downregulated mRNA of keratinocytes and were needed for extracellular secretion and correct localization of type VII collagen and laminin 5.     

Genetic linkage between the collagen VII (COL7A1) gene and the autosomal dominant form of dystrophic epidermolysis bullosa in two Dutch kindreds.

Epidermolysis bullosa is a heterogeneous group of heritable blistering skin diseases affecting epidermis and the dermal-epidermal junction zone. Recently, genetic linkage to the type VII collagen gene (Z = 8.77; theta = 0.00) localized on chromosome 3p21 was shown in three Finnish families with the autosomal dominant form of dystrophic epidermolysis bullosa. Two Dutch kindreds with intrafamilial characteristics of both the Cockayne-Touraine type and Bart's syndrome of autosomal dominant dystrophic epidermolysis bullosa have been studied. Two-point linkage analysis in these two families with the COL7A1 marker revealed a combined lod score of Z = 6.08 at theta = 0.00. These data strongly suggest that the type VII collagen gene is the candidate gene in these Dutch pedigrees. At least two (Cockayne-Touraine and Bart) of the three subtypes of dominant dystrophic epidermolysis bullosa seem to represent different forms of expression of the same gene defect.     

角膜炎、鱼鳞病、耳聋综合征一例及GJB2基因突变研究

目的 探讨角膜炎、鱼鳞病及耳聋综合征患者临床特征和GJB2基因突变情况,为该病临床与基因诊断提供依据。方法 收集1例角膜炎、鱼鳞病及耳聋综合征患者的临床资料,提取患者及家族成员的外周血DNA,用PCR扩增GJB2基因外显子2及其附近的剪切点,DNA直接测序法进行基因突变检测。结果 该患者存在血管化角膜炎、鱼鳞病及先天性耳聋三联征的典型临床特征,检测到GJB2基因中核苷酸序列外显子2第148位碱基由G突变为A,导致编码的连接蛋白Cx26第50位的天冬氨酸转换成天冬酰胺（D50N）。其未患病的母亲及哥哥未检测到突变位点。结论 GJB2基因突变（D50N）可能是引起鱼鳞病、角膜炎及耳聋综合征患者临床表型的原因。     

Porokeratotic eccrine nevus may be caused by somatic connexin26 mutations

Porokeratotic eccrine ostial and dermal duct nevus, or porokeratotic eccrine nevus (PEN), is a hyperkeratotic epidermal nevus. Several cases of widespread involvement have been reported, including one in association with the keratitis-ichthyosis-deafness (KID) syndrome (OMIM #148210), a rare disorder caused by mutations in the GJB2 gene coding for the gap junction protein connexin26 (Cx26). The molecular cause is, as yet, unknown. We have noted that PEN histopathology is shared by KID. The clinical appearance of PEN can resemble that of KID syndrome. Furthermore, a recent report of cutaneous mosaicism for a GJB2 mutation associated with KID describes linear hyperkeratotic skin lesions that might be consistent with PEN. From this, we hypothesized that PEN might be caused by Cx26 mutations associated with KID or similar gap junction disorders. Thus, we analyzed the GJB2 gene in skin samples from two patients referred with generalized PEN. In both, we found GJB2 mutations in the PEN lesions but not in unaffected skin or peripheral blood. One mutation was already known to cause the KID syndrome, and the other had not been previously associated with skin symptoms. We provide extensive functional data to support its pathogenicity. We conclude that PEN may be caused by mosaic GJB2 mutations.     

Role of ultraviolet light and UV DNA in the induction of skin lesions in experimental animals

In vivo and in vitro animal experiments have been performed to clarify the role of ultraviolet light denatured DNA (UV DNA) and ultraviolet light (UVL) in the pathogenesis of the dermal lesions of human SLE. Rabbits immunized with UV DNA show deposition of immunoglobulin at the dermal-epidermal junction following exposure to UVL. We have also shown that UV DNA appears concomitantly with the antibody at the dermal-epidermal junction subsequent to the UVL exposure. Both n DNA and UV DNA have been shown to bind to the dermal-epidermal junction in vitro which could result in the persistence of these antigens at this site. These studies lend further support to the hypothesis that the release of UV DNA and its subsequent deposition at the dermal-epidermal junction may result in binding of both immunoglobulin and complement, leading to the development of histological lesions of SLE.     

Immunofluorescent analysis of the basement membrane zone in lichen planus suggests destruction of the lamina lucida in bullous lesions

Lichen planus is an inflammatory dermatosis which is characterized histologically by an intense lymphocytic infiltrate at the dermal epidermal junction. This frequently results in disruption of the basement membrane zone, occasionally causing clinical blisters. In order to better understand the specific portion of the basement membrane zone which is disrupted by the lymphocytic infiltrate, we examined 7 cases of lichen planus with antibodies directed against anchoring filaments (GB3), the bullous pemphigoid antigen, anchoring fibrils (type VII collagen) and type IV collagen. In lesions without separation at the BMZ, all antibodies were strongly expressed, as in normal skin. In lesions with early separation, there was a focal decrease in GB3 staining, but types VII and IV collagen labelled normally. In lesions resulting in blisters, GB3 staining was essentially absent, and anti-types IV and VII collagen remained, but stained in a disrupted, less discrete pattern. The bullous pemphigoid antigen showed only slight deviation from the normal staining pattern. These findings suggest that the basement membrane zone in lichen planus is disrupted in the lamina lucida region. The lamina densa and sub-lamina clensa zones remain intact even in bullous lesions of lichen planus.     

Lack of type VII collagen in unaffected skin of patients with severe recessive dystrophic epidermolysis bullosa

Type VII collagen, the major structural component of the anchoring fibrils, was assayed in normal unaffected skin of patients with different forms of hereditary epidermolysis bullosa. Immunofluorescence staining with affinity-purified polyclonal antibodies to type VII collagen revealed a complete absence of staining in the skin of patients with severe dystrophic recessive epidermolysis bullosa. In all other forms, localized recessive dystrophic, dominant dystrophic, junctional and simplex forms there was an intense continuous linear staining of type VII collagen at the dermoepidermal junction. Also, obligate heterozygote carriers of the gene for severe dystrophic recessive form showed a normal pattern of staining. As internal controls and to define the clinical diagnosis, staining with antibodies to type IV collagen, laminin and bullous pemphigoid antigen was also performed. All these antibodies showed a normal staining pattern indicating an intact general morphology of the dermoepidermal junction zone. These results suggest that there is a defect of type VII collagen in patients with severe recessive dystrophic epidermolysis bullosa. The data also suggest that the group of recessive dystrophic epidermolysis bullosa may be heterogeneous not only clinically, but also at the molecular level.     

Age-related changes in the cutaneous basal lamina: scanning electron microscopic study

Scanning electron microscopy of human epidermal-dermal basal lamina demonstrated striking age-related changes. The basal lamina from abdominal skin was exposed in specimens from 26 humans by separation of epidermis and dermis after treatment with sodium bromide solutions. Transmission electron micrographs demonstrated the split to be in the lamina lucida. Scanning electron microscopy of mature epidermal-dermal junction and basal lamina showed distinct dermal valleys; tall, dome-shaped dermal papillae; and basal lamina arranged in prominent corrugations that tended to be oriented vertically on papillae and irregularly on interpapillary zones. Skin from subjects in their 7th through 10th decades demonstrated progressive loss of dermal valleys, flattening and widening of dermal papillae, and loss of basal lamina corrugations.     

Epidermolysis bullosa: novel and de novo premature termination codon and deletion mutations in the plectin gene predict late-onset muscular dystrophy

Epidermolysis bullosa (EB) with late-onset muscular dystrophy (EB-MD) is a hemidesmosomal variant of EB due to mutations in the plectin gene (PLEC1). The age of onset of muscle involvement has been noted to vary from infancy to the fourth decade of life. Immunofluorescence of the patients' skin and muscle biopsies is usually negative for staining with antibodies recognizing plectin, a large cytoskeleton-associated anchorage protein. In this study we report novel plectin mutations in two families with EB. In both families, the proband was a newborn with neonatal blistering with no evidence for muscle weakness as yet. Peripheral blood DNA was isolated and examined by heteroduplex scanning strategy, protein truncation test (PTT), and/or direct sequencing of the plectin gene. One of the probands was compound heterozygote for nonsense mutations E2005X/K4460X, and the proband in the second family was compound heterozygote for deletion mutations 5083delG/2745-9del21, the latter mutation extending from -9 to +12 at the intron 22/exon 23 border. The mutations K4460X and 5083delG were not present in either one of the parents, thus being de novo events. In both cases, nonpaternity was excluded by microsatellite marker analysis. The stop codon mutations are predicted to result in the synthesis of a truncated protein lacking the carboxy-terminal globular domain of the protein and possibly causing nonsense-mediated decay of the corresponding mRNA. The 2745-9del21 deletion mutation abolishes the splice site at the intron 22/exon 23 junction, predicting abnormal splicing events. Because plectin deficiency is associated with muscular dystrophy, molecular diagnostics of the plectin gene provides prognostic value in evaluation of these patients who appear to be at risk to develop muscular dystrophy.