Evidence for a functional defect of the lamina lucida in recessive dystrophic epidermolysis bullosa demonstrated by suction blisters

Suction blisters were raised in non-lesional skin of sixteen patients with various types of epidermolysis bullosa (EB). The ultrastructural level of separation was found in each type to be within the lamina lucida of the epidermal basement membrane zone. Suction blister times were normal in EB simplex, dominant dystrophic and localized recessive dystrophic EB, but were markedly reduced in both junctional EB and severe generalized recessive dystrophic EB. These data indicate an unpredicted abnormality of adhesion within the lamina lucida in severe recessive dystrophic EB, in addition to the well-recognized defect at a deeper level, beneath the lamina densa, in this condition. The use of suction blister times may prove valuable in the diagnosis of EB.     

Intracellular expression of type VII collagen during wound healing in severe recessive dystrophic epidermolysis bullosa and normal human skin

The ability of keratinocytes to synthesize basement membrane components in vivo during wound healing in normal human skin and in severe recessive dystrophic epidermolysis bullosa (RDEB) was investigated. Indirect immunofluorescence using anti-type VII collagen (VIIc, recognizing the globular non-helical component of the molecule), anti-type IV collagen, anti-laminin and bullous pemphigoid antisera, was performed on biopsies of intact skin and of healing skin taken between 7 and 14 days after dermatome injury (upper to mid-dermal wounding) in eight patients with severe RDEB and in seven normal subjects. Baseline anti-type VIIc immunofluorescence showed completely absent staining of the epidermis, dermis and dermo-epidermal junction in severe RDEB samples, and bright linear dermo-epidermal junction fluorescence in normal human skin. In 5/5 normal human skin samples taken 9-12 days post-wounding, some type VIIc expression was noted within basal cells as well as in a continuous or interrupted linear distribution at the basement membrane zone. In all the severe RDEB biopsies sampled between days 10 and 13 (5/5), anti-VIIc fluorescence was also seen with varying intensity within basal and lowermost suprabasal cells, and in one day 14 sample at the dermo-epidermal junction. Low levels of intracellular type IVc were seen in both groups, but only in those samples taken 7-9 days after injury; later biopsies showed only continuous dermo-epidermal junction staining. Linear basement membrane zone labelling with laminin and bullous pemphigoid antisera was seen in all samples in both sets of subjects, even at day 7, but there was no detectable intracellular antisera staining.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of integrins in junctional and dystrophic epidermolysis bullosa.

Recently, monoclonal antibodies (MoAb) have been raised against a family of adhesive membrane receptors (R) for extracellular matrix molecules known as integrins. In order to ascertain whether these adhesive proteins are normally expressed in inherited epidermolysis bullosa (EB) dermal epidermal junction, we studied the reactivity of MoAb recognizing receptors for VLA-1 (R for unknown ligand), VLA-2 (R for collagen), VLA-3 (R for collagen, laminin, fibronectin), VLA-4 (R for unknown ligand), VLA-5 (R for fibronectin), VLA-6 (R for laminin), VNR alpha, and VNR beta (R for vitronectin) on cryostat skin sections from EB patients and normal controls and on cytospins of normal epidermal cell suspensions with indirect immunohistochemical methods. Two cases of junctional EB (EBj) (lethal and non-lethal), three cases of dominant dystrophic EB (EBdd), two cases of recessive dystrophic EB (EBdr), and two normal controls skin sections and cell suspensions entered the study. No significant modification of the distribution of these adhesive receptors was observed in junctional and dystrophic EB skin. Both in normal and EB specimens MoAb against VLA-2, VLA-3, and VNR alpha determinants showed reactivity with the total cytoplasmic membrane of basal keratinocytes and basement membrane zone. Interestingly, anti-VLA-6 MoAb was characterized by an intense linear staining of the dermal-epidermal junction with the same localization on the roof of the blisters in EBj, EBdd, and EBdr as bullous pemphigoid (BP) serum. On the basis of these results we suggest that anti-VLA-6 MoAb could be used instead of BP serum for immunohistochemical detection of the cleavage of blisters in EB.     

Type VII collagen is a normal component of epidermal basement membrane, which shows altered expression in recessive dystrophic epidermolysis bullosa

The murine monoclonal antibody LH 7:2, which reacts with the basement membrane of stratified squamous epithelia including epidermis, has been characterized biochemically and shown to bind to part of the type VII collagen molecule. Immunoblotting reveals that the antibody binding site lies in the non-helical carboxy terminal region of the type VII collagen dimer and immunoelectron microscopy shows that the epitope is within the lamina densa of the basement membrane. Loss of LH 7:2 binding in the hereditary blistering disease recessive dystrophic epidermolysis bullosa suggests that inadequate synthesis or excessive breakdown of type VII collagen may form the biologic basis for the disease.     

Intraepidermal expression of basement membrane components in the lesional skin of a patient with dystrophic epidermolysis bullosa

The patient was a 15-year-old male. Since birth, he had developed blistering and erosion of the skin. Biopsy skin specimen of the bullous lesions showed subepidermal blister formation. Electron microscopic examination revealed that tissue separation had occurred at the sublamina densa level. By indirect immunofluorescence using antibodies specific for alpha 6 integrin, laminin 5, type IV collagen, and type VII collagen, all of these basement membrane components were detected as coarse granular intracytoplasmic deposits only in the basal and suprabasal cells of the blister roof. In the non-blistered regions, these basement membrane components showed a linear pattern similar to that seen in normal skin. These findings suggest that intraepidermal expression of basement membrane components was closely related to the blister formation. The biological meaning of intraepidermal expression of basement membrane components were also discussed.     

Altered expression of L-arginine metabolism pathway genes in chronic wounds in recessive dystrophic epidermolysis bullosa

Individuals with the severe, mutilating Hallopeau-Siemens form of recessive dystrophic epidermolysis bullosa (HS-RDEB) have trauma-induced blisters and skin erosions which often progress to wounds that are slow to heal. These chronic wounds cause considerable morbidity and there is an increased risk of squamous cell carcinoma arising in the wound margins. Currently, little is known about the keratinocyte cell biology in these wounds. Therefore, we compared the gene expression profiles of wound edge with nonwounded skin from two individuals with HS-RDEB. Trauma-induced wound sites had been present in both patients for more than 3 months. Hybridizations using DermArray gene expression filters showed relative differences in gene expression between wounded and unwounded skin. Notably, there was a fivefold increase in expression of arginase-1 (ARG1) in the chronic wound samples. Expression of seven other genes relevant to L-arginine metabolism also showed differences greater than twofold. L-arginine is known to have a critical role in the synthesis of nitric oxide as part of normal tissue repair. Although alterations in arginase isoenzymes have been detected previously in other chronic wounds (human and animal models), this is the first study to demonstrate differences in several components of the L-arginine metabolism pathway in chronic wounds, and the first to examine chronic wounds in HS-RDEB. The data show that the cascade of L-arginine metabolites is altered in HS-RDEB and the findings may provide new insight into the pathology of chronic wounds in this genodermatosis.     

Antenatal diagnosis of recessive dystrophic epidermolysis bullosa: collagenase expression in cultured fibroblasts as a biochemical marker

We performed fetoscopy and skin biopsy on a 19-week fetus at risk for recessive dystrophic epidermolysis bullosa (RDEB). Ultrastructural analysis of the tissue revealed dermolytic blister formation in the skin characteristic of the disease. To develop a biochemical test for use in antenatal diagnosis of RDEB, we established skin fibroblast cultures from the 20-week aborted fetus. The collagenase production by fetal RDEB fibroblast cultures was greater than seen in normal fetal fibroblast cultures. The concentration in culture medium from fetal RDEB cultures was 5.42 +/- 0.74 micrograms/ml (mean +/- SE) compared with 2.24 +/- 1.11 micrograms/ml in normal adult control cultures and 2.05 +/- 0.61 micrograms/ml in cultures from patients with other genetic forms of epidermolysis bullosa (p less than 0.025). In contrast, the concentration of collagenase in the fetal RDEB culture medium was not different from that seen in cell cultures from known patients with RDEB (5.34 +/- 1.12 micrograms/ml). Collagenase activity of the fetal RDEB medium was also increased approximately 3.5-fold. These data indicate that enhanced expression of collagenase by fetal RDEB skin fibroblasts can serve as a biochemical adjunct, and possibly an alternative, to morphologic examination of tissue for antenatal diagnosis.     

Mosaic expression of uncein and 180-kDa bullous pemphigoid antigen in generalized atrophic benign epidermolysis bullosa

Immunofluorescence microscopy of epidermodermal junction components in serial cryosections from the perilesional skin of a patient with generalized atrophic benign epidermolysis bullosa (GABEB) showed broken line-like staining of both BPAG2 (180-kDa bullous pemphigoid antigen) and uncein (antigen of 19-DEJ-l monoclonal antibody), whereas integrin α6 and laminin 5 were continuously expressed along the basement membrane zone. Immunoelectron microscopy revealed a mosaic distribution of the BPAG2/uncein positive and negative cells. BPAG2, a candidate protein of GABEB, probably has a close connection with uncein, an anchoring filament component.     

Dilemmas in distinguishing between dominant and recessive forms of dystrophic epidermolysis bullosa

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a heterogeneous inherited blistering skin disorder. The mode of inheritance may be autosomal dominant or recessive but all forms of DEB result from mutations in the gene encoding the anchoring fibril protein, type VII collagen, COL7A1. Consequently, in spite of careful clinical and skin biopsy examination, it may be difficult to distinguish mild recessive cases from de novo dominant disease in families with clinically normal parents and no other affected siblings; this distinction has significant implications for the accuracy of genetic counselling. OBJECTIVES: To assess whether COL7A1 mutation analysis might help determine mode of inheritance in mild to moderate DEB. METHODS: We performed COL7A1 screening using heteroduplex analysis and direct nucleotide sequencing in four individuals with mild to moderate "sporadic" DEB and clinically unaffected parents. RESULTS: In each patient, we identified a heterozygous glycine substitution within the type VII collagen triple helix. However, in two cases these mutations had been inherited in trans with a non-sense mutation on the other allele (i.e. autosomal recessive DEB). In the other two cases, no additional mutation was identified and neither mutation was present in parental DNA (i.e. de novo dominant disease). CONCLUSIONS: This study highlights the usefulness of DNA sequencing in determining the inherited basis of some sporadic cases of DEB. However, delineation of glycine substitutions should prompt comprehensive COL7A1 gene sequencing in the affected individual, as well as clinical assessment of parents and mutation screening in parental DNA, if the true mode of inheritance is to be established correctly.     

Coexistence of psoriasis and an unusual IgG-mediated subepidermal bullous dermatosis: identification of a novel 200-kDa lower lamina lucida target antigen

Summary Bullous pemphigoid (BP) is characterized by autoantibodies against 230- and 180-kDa hemidesmosomal antigens located in the most superficial layers of the basement membrane zone (BMZ). Histologically. there is a predominance of eosinophils in the infiltrate. In a psoriatic patient, we identified an unusual autoimmune subepidermal bullous eruption which clinically resembled BP, but which was characterized by IgG autoantibodies against a novel 200-kDa lower lamina lucida component, Histologically there was a predominance of neutrophils in the infiltrate.
Direct immunofluorescence showed linear immunoglobulin (Ig)G and C3 deposition at the BMZ. The patient's IgG autoantibodies bound exclusively to the dermal side of salt-split normal human skin. Indirect immunogold electron microscopy showed a marked deposition of IgG at the lower lamina lucida and minimal deposition at the hemidesmosomes. Immunoblot analysis identified a unique 200-kDa autoantigen in dermal extracts and a faint band of the 230-kDa BP antigen in epidermal extracts. The patient responded dramatically well to cyclosporin A.
Although the patient's serum also reacted slightly with the 230-kDa BP antigen, there were significant findings different from the usual immunopathological changes of BP. These included finding a novel 200-kDa lower lamina lucida target antigen, the binding of IgG autoantibodies exclusively to the dermal side of the split skin and a predominance of neutrophils in blister infiltrate. The IgG autoantibodies against the 200-kDa lamina lucida target antigen seemed to play a major role in the pathogenesis of this unique autoimmune subepidermal dermatosis.     

Spontaneous disappearance of intraepidermal type VII collagen in a patient with dystrophic epidermolysis bullosa

Summary Recently, a peculiar self-healing neonatal blistering disease has been reported, which is characterized by perinuclear stellate inclusions within basilar keratinocytes, representing abnormal retention of type VII collagen. We report a Japanese patient with this condition, in whom we studied the expression of a variety of basement membrane zone (BMZ)-related antigens. Skin biopsy specimens at 5 days of age showed abundant accumulation of both the NC-1 domain and the collagenous part of type VII collagen within the basal and suprabasal keratinocytes, in addition to patchy and weak staining along the BMZ. In contrast, at 4 years of age, when the disease activity was markedly attenuated, a second biopsy showed complete linear staining of type VII collagen along the BMZ, with no detectable intracytoplasmic deposits. Expression of other BMZ-related antigens, including laminin 5, α6 and ß4 integrins, bullous pemphigoid antigens 1 and 2. and type IV collagen, was normal in both the biopsy specimens. Our observations further confirm that the perinuclear stellate bodies seen in this peculiar condition are composed of both collagenous and non-collagenous domains of type VII collagen retained within the epidermis, and that these bodies disappear when the disease activity remits.     

Abnormal expression of hemidesmosome-like structures by junctional epidermolysis bullosa keratinocytes in vitro

Hemidesmosomes are frequently rudimentary in junctional epidermolysis bullosa (JEB), and JEB keratinocytes display abnormal attachment to the substrate in culture. Our aim was to determine whether this abnormality reflects defective hemidesmosome synthesis in vitro. Keratinocytes from five JEB patients were cultured under standard conditions. Control cultures, from four healthy males, three patients with dystrophic EB (DEB), and one patient with the simplex variant (EBS), were also examined. Post-confluent cultures were processed for transmission electron microscopy. Hemidesmosome-like structures were counted in electron micrograph montages. The number of hemidesmosome-like structures in JEB cultures (0.97 +/- 0.57, per 10 microns of basal cell membrane) was approximately 17% of the value for normal controls (5.81 +/- 3.08, P less than 0.02). The values for EBS (3.72) and DEB (3.28 +/- 1.44) were not statistically different from normal controls. In addition hemidesmosome-like structures in JEB cultures were morphologically ill-defined, compared with those from controls. This correlated with the loose apposition between JEB keratinocytes and the substrate, which appeared tight in controls. JEB keratinocytes continue to express in vitro a major phenotypic abnormality which characterizes the disease in vivo. Therefore, this model should prove useful in studies determining the pathogenesis and possible new treatments of JEB.     

Balloon dilation of an esophageal stenosis in a patient with recessive dystrophic epidermolysis bullosa

We report a 13-year-old boy with recessive dystrophic epidermolysis bullosa who had dysphagia due to esophageal stenosis. A balloon dilation was successfully performed under flexible endoscopic and fluoroscopic control. The early and long-term follow-up was characterized by the disappearance of dysphagia, weight gain, and improvement of his skin lesions.     

Intra-epidermal retention of type VII collagen in a patient with recessive dystrophic epidermolysis bullosa

An infant born with severe blisters on the limbs, face, trunk, and oral mucosa was diagnosed by light and electron microscopy to have recessive dystrophic epidermolysis bullosa. Transmission electron microscopy showed that the basal lamina remained with the epidermis and that the floor of the blister was exposed collagen of the papillary dermis. No banded anchoring fibrils were observed along either the roof or the floor of the blister; however, small filamentous structures, possibly immature anchoring fibrils, extended down from the lamina densa along the blister roof. Some basal and suprabasal keratinocytes contained large vesicles filled with filamentous matrix of variable electron density. Immunofluorescent staining of skin for type VII collagen showed sparse and irregular staining of type VII collagen along the blister roof, and intense intracellular labeling for type VII collagen in clusters of epidermal cells in basal and suprabasal layers. Type VII collagen appeared to be synthesized by keratinocytes but not secreted.     

Iron status and burden of anemia in children with recessive dystrophic epidermolysis bullosa

Background and Objectives

To describe the prevalence, severity, and management of anemia in a cohort of children with recessive dystrophic epidermolysis bullosa (RDEB) and to highlight the use of soluble transferrin receptor (sTfR) to diagnose iron deficiency in this chronic inflammatory state.

Methods

We studied a cohort of 114 patients with RDEB followed at a pediatric hospital-based Epidermolysis Bullosa Center from 2010 to 2020; data were prospectively tracked in a comprehensive clinical database that captured all visits, laboratory tests, iron infusions, and transfusions. The primary outcome was occurrence of anemia, which was assessed by age and sex, with and without transfusion support. Secondary outcomes included iron status using a combination of ferritin and sTfR levels, the cumulative incidence of parenteral iron therapy and transfusions, and survival.

Results

In RDEB, anemia begins in the first year of life and becomes more frequent and severe with age. The prevalence of iron deficiency anemia (IDA) estimated by ferritin was 33.6% (37/110), but the sTfR/log₁₀-ferritin ratio indicated a 1.5-fold higher true prevalence of IDA of 50.6% (41/81). 53.5% (61/114) received parenteral iron infusions, transfusions, or both. Higher ferritin was associated with earlier mortality.

Conclusions

Individuals with RDEB have a high burden of anemia (IDA and anemia of inflammation) that requires frequent medical interventions. The sTfR/log₁₀-ferritin ratio improves the detection of iron deficiency in the context of inflammation and guides therapy.     

Expression of collagenase and stromelysin in skin fibroblasts from recessive dystrophic epidermolysis bullosa

Collagenase and stromelysin expression in recessive dystrophic epidermolysis bullosa (RDEB) was studied at both the protein and the gene expression levels in fibroblast cultures. The amount of enzyme protein in the culture medium, as determined using a specific enzyme assay, showed a 9.7-fold increase in collagenase and a 2.7-fold increase in stromelysin in RDEB fibroblasts (n=4 patients) compared with controls (n=3 subjects with normal skin). Collagenase activity was extremely high in all RDEB fibroblasts. Gene expression, as assessed by Northern blot hybridization, was increased in two sets of RDEB fibroblasts with respect to collagenase, and in two other sets of RDEB fibroblasts with respect to stromelysin. The effect of interleukin-1 (IL-1) on metalloproteinase expression was also examined. The results revealed that: 1) collagenase and stromelysin expression was variably increased at both the protein and the gene expression levels in RDEB fibroblasts; (2) the gene expression level did not always reflect the corresponding protein level; and (3) IL-1 produced a differential effect on collagenase and stromelysin expression. Although the causative gene for RDEB is a type VII collagen, the abnormal expression of collagenase and/or stromelysin is still important in considering the pathophysiology of RDEB.