Large Intragenic Deletion in DSTYK Underlies Autosomal-Recessive Complicated Spastic Paraparesis, SPG23.

SPG23 is an autosomal-recessive neurodegenerative subtype of lower limb spastic paraparesis with additional diffuse skin and hair dyspigmentation at birth followed by further patchy pigment loss during childhood. Previously, genome-wide linkage in an Arab-Israeli pedigree mapped the gene to an approximately 25 cM locus on chromosome 1q24-q32. By using whole-exome sequencing in a further Palestinian-Jordanian SPG23 pedigree, we identified a complex homozygous 4-kb deletion/20-bp insertion in DSTYK (dual serine-threonine and tyrosine protein kinase) in all four affected family members. DSTYK is located within the established linkage region and we also found the same mutation in the previously reported pedigree and another Israeli pedigree (total of ten affected individuals from three different families). The mutation removes the last two exons and part of the 3' UTR of DSTYK. Skin biopsies revealed reduced DSTYK protein levels along with focal loss of melanocytes. Ultrastructurally, swollen mitochondria and cytoplasmic vacuoles were also noted in remaining melanocytes and some keratinocytes and fibroblasts. Cultured keratinocytes and fibroblasts from an affected individual, as well as knockdown of Dstyk in mouse melanocytes, keratinocytes, and fibroblasts, were associated with increased cell death after ultraviolet irradiation. Keratinocytes from an affected individual showed loss of kinase activity upon stimulation with fibroblast growth factor. Previously, dominant mutations in DSTYK were implicated in congenital urological developmental disorders, but our study identifies different phenotypic consequences for a recurrent autosomal-recessive deletion mutation in revealing the genetic basis of SPG23.

Phase I/II open-label trial of intravenous allogeneic mesenchymal stromal cell therapy in adults with recessive dystrophic epidermolysis bullosa.

BACKGROUND: Recessive dystrophic epidermolysis bullosa (RDEB) is a hereditary blistering disorder due to a lack of type VII collagen. At present, treatment is mainly supportive. OBJECTIVES: To determine whether intravenous allogeneic bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs) are safe in RDEB adults and if the cells improve wound healing and quality of life. METHODS: We conducted a prospective, phase I/II, open-label study recruiting 10 RDEB adults to receive 2 intravenous infusions of BM-MSCs (on day 0 and day 14; each dose 2-4 × 106 cells/kg). RESULTS: BM-MSCs were well tolerated with no serious adverse events to 12 months. Regarding efficacy, there was a transient reduction in disease activity scores (8/10 subjects) and a significant reduction in itch. One individual showed a transient increase in type VII collagen. LIMITATIONS: Open-label trial with no placebo. CONCLUSIONS: MSC infusion is safe in RDEB adults and can have clinical benefits for at least 2 months.

Mutations in GRHL2 result in an autosomal-recessive ectodermal Dysplasia syndrome.

Grainyhead-like 2, encoded by GRHL2, is a member of a highly conserved family of transcription factors that play essential roles during epithelial development. Haploinsufficiency for GRHL2 has been implicated in autosomal-dominant deafness, but mutations have not yet been associated with any skin pathology. We investigated two unrelated Kuwaiti families in which a total of six individuals have had lifelong ectodermal defects. The clinical features comprised nail dystrophy or nail loss, marginal palmoplantar keratoderma, hypodontia, enamel hypoplasia, oral hyperpigmentation, and dysphagia. In addition, three individuals had sensorineural deafness, and three had bronchial asthma. Taken together, the features were consistent with an unusual autosomal-recessive ectodermal dysplasia syndrome. Because of consanguinity in both families, we used whole-exome sequencing to search for novel homozygous DNA variants and found GRHL2 mutations common to both families: affected subjects in one family were homozygous for c.1192T>C (p.Tyr398His) in exon 9, and subjects in the other family were homozygous for c.1445T>A (p.Ile482Lys) in exon 11. Immortalized keratinocytes (p.Ile482Lys) showed altered cell morphology, impaired tight junctions, adhesion defects, and cytoplasmic translocation of GRHL2. Whole-skin transcriptomic analysis (p.Ile482Lys) disclosed changes in genes implicated in networks of cell-cell and cell-matrix adhesion. Our clinical findings of an autosomal-recessive ectodermal dysplasia syndrome provide insight into the role of GRHL2 in skin development, homeostasis, and human disease.

The cytolinker plectin regulates nuclear mechanotransduction in keratinocytes.

The transmission of mechanical forces to the nucleus is important for intracellular positioning, mitosis and cell motility, yet the contribution of specific components of the cytoskeleton to nuclear mechanotransduction remains unclear. In this study, we examine how crosstalk between the cytolinker plectin and F-actin controls keratin network organisation and the 3D nuclear morphology of keratinocytes. Using micro-patterned surfaces to precisely manipulate cell shape, we find that cell adhesion and spreading regulate the size and shape of the nucleus. Disruption of the keratin cytoskeleton through loss of plectin facilitated greater nuclear deformation, which depended on acto-myosin contractility. Nuclear morphology did not depend on direct linkage of the keratin cytoskeleton with the nuclear membrane, rather loss of plectin reduced keratin filament density around the nucleus. We further demonstrate that keratinocytes have abnormal nuclear morphologies in the epidermis of plectin-deficient, epidermolysis bullosa simplex patients. Taken together, our data demonstrate that plectin is an essential regulator of nuclear morphology in vitro and in vivo and protects the nucleus from mechanical deformation.

Germline Mutation in EXPH5 Implicates the Rab27B Effector Protein Slac2-b in Inherited Skin Fragility.

The Rab GTPase Rab27B and one of its effector proteins, Slac2-b (also known as EXPH5, exophilin-5), have putative roles in intracellular vesicle trafficking but their relevance to human disease is not known. By using whole-exome sequencing, we identified a homozygous frameshift mutation in EXPH5 in three siblings with inherited skin fragility born to consanguineous Iraqi parents. All three individuals harbor the mutation c.5786delC (p.Pro1929Leufs(∗)8) in EXPH5, which truncates the 1,989 amino acid Slac2-b protein by 52 residues. The clinical features comprised generalized scale-crusts and occasional blisters, mostly induced by trauma, as well as mild diffuse pigmentary mottling on the trunk and proximal limbs. There was no increased bleeding tendency, no neurologic abnormalities, and no increased incidence of infection. Analysis of an affected person's skin showed loss of Slac2-b immunostaining (C-terminal antibody), disruption of keratinocyte adhesion within the lower epidermis, and an increased number of perinuclear vesicles. A role for Slac2-b in keratinocyte biology was supported by findings of cytoskeletal disruption (mainly keratin intermediate filaments) and decreased keratinocyte adhesion in both keratinocytes from an affected subject and after shRNA knockdown of Slac2-b in normal keratinocytes. Slac2-b was also shown to colocalize with Rab27B and ß4 integrin to early adhesion initiation sites in spreading normal keratinocytes. Collectively, our findings identify an unexpected role for Slac2-b in inherited skin fragility and expand the clinical spectrum of human disorders of GTPase effector proteins.

Humanization of autoantigen.

Transmissibility of characteristic lesions to experimental animals may help us understand the pathomechanism of human autoimmune disease. Here we show that human autoimmune disease can be reproduced using genetically engineered model mice. Bullous pemphigoid (BP) is the most common serious autoimmune blistering skin disease, with a considerable body of indirect evidence indicating that the underlying autoantigen is collagen XVII (COL17). Passive transfer of human BP autoantibodies into mice does not induce skin lesions, probably because of differences between humans and mice in the amino acid sequence of the COL17 pathogenic epitope. We injected human BP autoantibody into Col17-knockout mice rescued by the human ortholog. This resulted in BP-like skin lesions and a human disease phenotype. Humanization of autoantigens is a new approach to the study of human autoimmune diseases.

Diffuse melanosis cutis: a systematic review of the literature.

BACKGROUND: Diffuse melanosis cutis (DMC) is a rare presentation of metastatic melanoma characterized by a progressive blue-gray discoloration of the skin and mucous membranes. OBJECTIVE: To foster a better understanding of the clinical presentation, histological findings, and pathophysiology underlying DMC. METHODS: A systematic review of the literature was completed utilizing MEDLINE, CINAHL, Embase, and Google. Data were extracted using a protocol-driven spread sheet with all statistical analyses completed using SPSS. RESULTS: The review identified 68 original cases of DMC. The mean time from diagnosis of melanoma until development of DMC was 11.48 months (95% confidence interval [CI]: 0-48.16). The mean time to death following the onset of DMC was 4.43 months (95% CI: 0.00-11.11). Histological findings were relatively consistent demonstrating intracellular and extracellular melanin deposition in the dermis, with a pronounced perivascular distribution. The pathophysiological mechanisms underlying DMC could not be definitively elucidated; however, it is hypothesized that the melanin precursors, melanin, and melanosomes liberated by cytolytic metastatic melanoma deposits are phagocytosed by dermal histiocytes, manifesting clinically as diffuse melanosis. LIMITATIONS: The cross-sectional nature of case reports, paucity of cases of DMC, and heterogeneity in reporting limit any conclusions being drawn regarding the pathophysiology of DMC definitively. CONCLUSION: DMC heralds a poor prognosis for patients with metastatic melanoma and affected patients should be made aware of the implications of this condition on survival.

Bone marrow transplantation restores epidermal basement membrane protein expression and rescues epidermolysis bullosa model mice.

Attempts to treat congenital protein deficiencies using bone marrow-derived cells have been reported. These efforts have been based on the concepts of stem cell plasticity. However, it is considered more difficult to restore structural proteins than to restore secretory enzymes. This study aims to clarify whether bone marrow transplantation (BMT) treatment can rescue epidermolysis bullosa (EB) caused by defects in keratinocyte structural proteins. BMT treatment of adult collagen XVII (Col17) knockout mice induced donor-derived keratinocytes and Col17 expression associated with the recovery of hemidesmosomal structure and better skin manifestations, as well improving the survival rate. Both hematopoietic and mesenchymal stem cells have the potential to produce Col17 in the BMT treatment model. Furthermore, human cord blood CD34(+) cells also differentiated into keratinocytes and expressed human skin component proteins in transplanted immunocompromised (NOD/SCID/gamma(c)(null)) mice. The current conventional BMT techniques have significant potential as a systemic therapeutic approach for the treatment of human EB.

Blockade of autoantibody-initiated tissue damage by using recombinant fab antibody fragments against pathogenic autoantigen.

Activation of the complement cascade via the classical pathway is required for the development of tissue injury in many autoantibody-mediated diseases. It therefore makes sense to block the pathological action of autoantibodies by preventing complement activation through inhibition of autoantibody binding to the corresponding pathogenic autoantigen using targeted Fab antibody fragments. To achieve this, we use bullous pemphigoid (BP) as an example of a typical autoimmune disease. Recombinant Fabs against the non-collagenous 16th-A domain of type XVII collagen, the main pathogenic epitope for autoantibodies in BP, were generated from antibody repertoires of BP patients by phage display. Two Fabs, Fab-B4 and Fab-19, showed marked ability to inhibit the binding of BP autoantibodies and subsequent complement activation in vitro. In the in vivo experiments using type XVII collagen humanized BP model mice, these Fabs protected mice against BP autoantibody-induced blistering disease. Thus, the blocking of pathogenic epitopes using engineered Fabs appears to demonstrate efficacy and may lead to disease-specific treatments for antibody-mediated autoimmune diseases.

Plectin expression patterns determine two distinct subtypes of epidermolysis bullosa simplex.

Plectin is a cytoskeletal linker protein that has a dumbbell-like structure with a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC1) cause two distinct autosomal recessive subtypes of epidermolysis bullosa (EB): EB simplex with muscular dystrophy (EBS-MD), and EB simplex with pyloric atresia (EBS-PA). Here, we demonstrate that normal human fibroblasts express two different plectin isoforms including full-length and rodless forms of plectin. We performed detailed analysis of plectin expression patterns in six EBS-MD and three EBS-PA patients. In EBS-PA, expression of all plectin domains was found to be markedly attenuated or completely lost; in EBS-MD, the expression of the N- and C-terminal domains of plectin remained detectable, although the expression of rod domains was absent or markedly reduced. Our data suggest that loss of the full-length plectin isoform with residual expression of the rodless plectin isoform leads to EBS-MD, and that complete loss or marked attenuation of full-length and rodless plectin expression underlies the more severe EBS-PA phenotype. These results also clearly account for the majority of EBS-MD PLEC1 mutation restriction within the large exon 31 that encodes the plectin rod domain, whereas EBS-PA PLEC1 mutations are generally outside exon 31.

Cyanoacrylate glue as an alternative mounting medium for resin-embedded semithin sections.

Commercially available generic Superglue (cyanoacrylate glue) can be used as an alternative mounting medium for stained resin-embedded semithin sections. It is colourless and contains a volatile, quick-setting solvent that produces permanent mounts of semithin sections for immediate inspection under the light microscope. Here, we compare the use of cyanoacrylate glue for mounting semithin sections with classical dibutyl phthalate xylene (DPX) in terms of practical usefulness, effectiveness and the quality of the final microscopic image.

CGI-58 is an alpha/beta-hydrolase within lipid transporting lamellar granules of differentiated keratinocytes.

CGI-58 is the causative molecule underlying Dorfman-Chanarin syndrome, a neutral lipid storage disease exhibiting apparent clinical features of ichthyosis. CGI-58, associated with triacylglycerol hydrolysis, has an alpha/beta-hydrolase fold and is also known as the alpha/beta-hydrolase domain-containing protein 5. The purpose of this study was to elucidate the function of CGI-58 and the pathogenic mechanisms of ichthyosis in Dorfman-Chanarin syndrome. Using an anti-CGI-58 antibody, we found CGI-58 to be expressed in the upper epidermis, predominantly in the granular layer cells, as well as in neurons and hepatocytes. Immunoelectron microscopy revealed that CGI-58 was also localized to the lamellar granules (LGs), which are lipid transport and secretion granules found in keratinocytes. CGI-58 expression was markedly reduced in the epidermis of patients with harlequin ichthyosis, demonstrating defective LG formation. In cultured keratinocytes, CGI-58 expression was mildly up-regulated under high Ca(2+) conditions and markedly up-regulated in three-dimensional, organotypic cultures. In the developing human epidermis, CGI-58 immunostaining was observed at an estimated gestational age of 49 days, and CGI-58 mRNA expression was up-regulated concomitantly with both epidermal stratification and keratinocyte differentiation. CGI-58 knockdown reduced expression of keratinocyte differentiation/keratinization markers in cultured human keratinocytes. Our results indicate that CGI-58 is expressed and packaged into LGs during keratinization and likely plays crucial role(s) in keratinocyte differentiation and LG lipid metabolism, contributing to skin lipid barrier formation.

Safety and early efficacy outcomes for lentiviral fibroblast gene therapy in recessive dystrophic epidermolysis bullosa.

BACKGROUNDRecessive dystrophic epidermolysis bullosa (RDEB) is a severe form of skin fragility disorder due to mutations in COL7A1 encoding basement membrane type VII collagen (C7), the main constituent of anchoring fibrils (AFs) in skin. We developed a self-inactivating lentiviral platform encoding a codon-optimized COL7A1 cDNA under the control of a human phosphoglycerate kinase promoter for phase I evaluation.METHODSIn this single-center, open-label phase I trial, 4 adults with RDEB each received 3 intradermal injections (~1 × 106 cells/cm2 of intact skin) of COL7A1-modified autologous fibroblasts and were followed up for 12 months. The primary outcome was safety, including autoimmune reactions against recombinant C7. Secondary outcomes included C7 expression, AF morphology, and presence of transgene in the injected skin.RESULTSGene-modified fibroblasts were well tolerated, without serious adverse reactions or autoimmune reactions against recombinant C7. Regarding efficacy, there was a significant (P < 0.05) 1.26-fold to 26.10-fold increase in C7 mean fluorescence intensity in the injected skin compared with noninjected skin in 3 of 4 subjects, with a sustained increase up to 12 months in 2 of 4 subjects. The presence of transgene (codon-optimized COL7A1 cDNA) was demonstrated in the injected skin at month 12 in 1 subject, but no new mature AFs were detected.CONCLUSIONTo our knowledge, this is the first human study demonstrating safety and potential efficacy of lentiviral fibroblast gene therapy with the presence of COL7A1 transgene and subsequent C7 restoration in vivo in treated skin at 1 year after gene therapy. These data provide a rationale for phase II studies for further clinical evaluation.TRIAL REGISTRATIONClincalTrials.gov NCT02493816.FUNDINGCure EB, Dystrophic Epidermolysis Bullosa Research Association (UK), UK NIHR Biomedical Research Centre at Guy's and St Thomas' NHS Foundation Trust and King's College London, and Fondation René Touraine Short-Exchange Award.

Mutations in lipid transporter ABCA12 in harlequin ichthyosis and functional recovery by corrective gene transfer.

Harlequin ichthyosis (HI) is a devastating skin disorder with an unknown underlying cause. Abnormal keratinocyte lamellar granules (LGs) are a hallmark of HI skin. ABCA12 is a member of the ATP-binding cassette transporter family, and members of the ABCA subfamily are known to have closely related functions as lipid transporters. ABCA3 is involved in lipid secretion via LGs from alveolar type II cells, and missense mutations in ABCA12 have been reported to cause lamellar ichthyosis type 2, a milder form of ichthyosis. Therefore, we hypothesized that HI might be caused by mutations that lead to serious ABCA12 defects. We identify 5 distinct ABCA12 mutations, either in a compound heterozygous or homozygous state, in patients from 4 HI families. All the mutations resulted in truncation or deletion of highly conserved regions of ABCA12. Immunoelectron microscopy revealed that ABCA12 localized to LGs in normal epidermal keratinocytes. We confirmed that ABCA12 defects cause congested lipid secretion in cultured HI keratinocytes and succeeded in obtaining the recovery of LG lipid secretion after corrective gene transfer of ABCA12. We concluded that ABCA12 works as an epidermal keratinocyte lipid transporter and that defective ABCA12 results in a loss of the skin lipid barrier, leading to HI. Our findings not only allow DNA-based early prenatal diagnosis but also suggest the possibility of gene therapy for HI.

Gelsolin segment 5 inhibits HIV-induced T-cell apoptosis via Vpr-binding to VDAC.

Viral protein R (Vpr) from the human immunodeficiency virus induces cell cycle arrest in proliferating cells, stimulates virus transcription, and regulates activation and apoptosis of infected T-lymphocytes. We report that Jurkat cells overexpressing full-length gelsolin show resistance to Vpr-induced T-cell apoptosis with abrogation of mitochondrial membrane potential loss and the release of cytochrome c. Co-immunoprecipitation assays in HEK293T cells demonstrated that overexpression of full-length or segment 5 (G5) but not G5-deleted gelsolin (DeltaG5) bound to the voltage-dependent anion channel (VDAC), and that the G5 subunit can inhibit HIV-1-Vpr-binding to VDAC. We also confirmed that full-length gelsolin has the same effect in Jurkat cells. Clonogenic analysis showed that transfection of G5 but not DeltaG5 cDNA protects Jurkat T cells from HIV-Vpr-Tet induced T-cell apoptosis and promoted cell survival, as did full-length gelsolin. These results suggest that the gelsolin G5 domain inhibits HIV-Vpr-induced T-cell apoptosis by blocking the interaction between Vpr and VDAC, and might be used as a protective treatment against HIV-Vpr-induced T-cell apoptosis.

Small-diameter porous poly (epsilon-caprolactone) films enhance adhesion and growth of human cultured epidermal keratinocyte and dermal fibroblast cells.

Autologous keratinocyte grafts provide clinical benefit by rapidly covering wounded areas, but they are fragile. We therefore developed biocompatible hexagonal-packed porous films with uniform, circular pore sizes to support human keratinocytes and fibroblasts. Cells were cultured on these porous poly (epsilon-calprolactone) films with pore sizes ranging from novel ultra-small 3 microm to 20 microm. These were compared with flat (pore-less) films. Cell growth rates, adhesion, migration, and ultrastructural morphology were examined. Human keratinocytes and fibroblasts attached to all films. Furthermore, small-pore (3-5 microm) films showed the highest levels of cell adhesion and survival and prevented migration into the pores and opposing film surface. Keratinocyte migration over small-pore film surface was inhibited. Keratinocytes optimally attached to 3-microm-pore films due to a combination of greater pore numbers (porosity), a greater circumference of the pore edge per unit surface area, and greater frequency of flat surface areas for attachment, allowing better cell-substrate and cell-cell attachment and growth. The 3-microm pore size allowed cell-cell communication, together with diffusion of soluble nutrients and factors from the culture medium or wound substrate. These characteristics are considered important in developing grafts for use in the treatment of human skin wounds.

N-linked neutral oligosaccharides in the stratum corneum of normal and ichthyotic skin.

N-Glycan oligosaccharides are thought to play multiple, important roles in a variety of biological events. However, N-glycan profiles in the stratum corneum of human skin have not yet been studied in detail. To clarify the N-glycan profiles in the stratum corneum of normal and ichthyotic epidermis, N-glycan profiles were studied by high-performance liquid chromatography using normal human epidermal samples and scales from hyperkeratotic skin of ichthyosis patients. Chromatograms of patient scale samples showed unique alterations in three peaks eluted at 15.8, 18.8 and 26.9 min. The N-glycan profiles were significantly altered in ichthyotic hyperkeratotic skin compared with normal non-hyperkeratotic controls. These findings indicate the reduction of N-acetylglucosaminyltransferase II and fucosyltransferase 8 activities. Alteration of N-glycan structures in hyperkeratotic skin suggests the biological role of N-glycans in keratinization.

Compound heterozygous ABCA12 mutations including a novel nonsense mutation underlie harlequin ichthyosis.

Recently, it has been reported that several harlequin ichthyosis (HI) patients survive the neonatal period and their condition subsequently improves. Here we describe a 2-year-old Japanese boy who exhibited typical clinical features of HI at birth. He survived beyond the neonatal period after oral retinoid treatment and, at the age of 2 years, showed moderately thick, lamellar scales and erythroderma over his whole body. The patient is a compound heterozygote for 2 ABCA12 mutations, a paternal deletion mutation c.2021_2022del (p.Lys674ArgfsX63) and a novel maternal nonsense mutation c.7444C --> T (p.Arg2482X). Electron microscopic observation of a skin biopsy specimen from the perinatal period revealed epidermal ultrastructural features consistent with HI. Immunofluorescence labeling using antiserum against a C-terminal ABCA12 epitope showed loss of expression in the patient's epidermis. The present patient demonstrates that rapid diagnosis of HI by ABCA12 expression analysis and mutation detection, and early commencement of systemic retinoid therapy are crucial to significantly improving an HI patient's prognosis.

Compound heterozygous mutations including a de novo missense mutation in ABCA12 led to a case of harlequin ichthyosis with moderate clinical severity.

Harlequin ichthyosis (HI) is one of the most devastating genodermatoses. Recently, ABCA12 mutations were identified as the cause of HI. A newborn Japanese male demonstrated the typical features of HI. The patient was treated with oral etretinate and his general condition has been good (now aged 1.5 years). This patient with moderate clinical severity was compound heterozygous for a novel de novo missense mutation 1160G > A (S387N) in exon 10 and a maternal deletion mutation 4158_4160delTAC (T1387del) in exon 28 of ABCA12. T1387del was a deletion of a highly conserved threonine residue within the first adenosine 5' triphosphate-binding domain and is thought to seriously affect the function of the ABCA12 protein. Conversely, the residue 387 is located outside the known active sites of ABCA12 and S387N is predicted not to lead to a serious functional deficiency in ABCA12. Electron microscopy revealed abnormal lamellar granules in the granular layer cells and a moderate number of lipid vacuoles in the cornified cells. Disturbed glucosylceramide transport was confirmed in the cultured keratinocytes from the patient. No de novo mutation in ABCA12 has yet been reported either in HI or lamellar ichthyosis. The present case suggested that a de novo ABCA12 mutation might underlie HI.

Colocalization of multiple laminin isoforms predominantly beneath hemidesmosomes in the upper lamina densa of the epidermal basement membrane.

Multiple laminin isoforms including laminins 5 (alpha3 beta3 gamma2), 6 (alpha3 beta1 gamma1), 10 (alpha5 beta1 gamma1), and possibly laminins 7 (alpha3 beta2 gamma1) and 11 (alpha5 beta2 gamma1) are present in the epidermal basement membrane. However, only the precise epidermal ultrastructural localization of laminin 5 (alpha3 beta3 gamma2) has been elucidated. We therefore determined the precise expression and ultrastructural localization of the alpha5, beta1, beta2, and gamma1 chains in the epidermis. The expression of laminin chains in skin samples was analyzed from patients with epidermolysis bullosa (EB, n=15) that harbor defects in specific hemidesmosome (HD)-associated components. The expression of the alpha5, beta1, and gamma1 chains (present in laminins 10/11) and beta2 chain (laminins 7/11) was unaffected in all intact (unseparated) skin of EB patients including Herlitz junctional EB with laminin-5 defects (n=6). In the basement membrane of human epidermis, the alpha5, beta1, beta2, and gamma1 chains were expressed but also localized to the dermal vessels. Immunogold electron microscopy of normal human epidermis localized the alpha5, beta1, beta2, and gamma1 chains to the upper lamina densa, with between 84% and 92% of labeling restricted to beneath the HDs, similar to laminin 5 (n> or =200 gold particles per sample, sample number n=4) but distinct from collagen IV labeling (with only 63% labeling beneath HDs, p<0.001). Taken together, the majority of the alpha5beta1/beta2gamma1 laminin chains are located beneath HDs. This suggests that laminin-10-associated chains have specific functions or molecular interactions beneath HDs in the epidermal basement membrane.