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Summary. Hereditary bullous epidermolysis (BE) is a group of genetically and clinically heterogeneous diseases characterized by the formation of blisters and erosion due to injury on the skin and mucous membranes. Different forms of BE can be accompanied by various extracutaneous complications, such as blisters and erosion on the cornea and mucous membranes, stenoses and strictures of the respiratory system, gastrointestinal tract, urinary system, muscular dystrophy, and malignant tumors. Hereditary bullous epidermolysis is divided into three types, depending on the level of blister formation: simple, borderline, and dystrophic. Simple BE is characterized by the stratification of epidermis due to the keratinocyte cytolysis. Borderline BE means that blisters are formed at the border of the epidermis and dermis due to the splitting of the lamina of the basement membrane (lamina lucida), while dystrophic BE has blisters that are formed under the dense plate of the basement membrane (lamina densa), which exfoliates the dermis. Currently, mutations have been identified in more than 10 genes encoding the structural proteins of keratinocytes and the basal membrane of the skin and mucous membranes. A common feature of these proteins is their involvement in the formation of strong bonds between the epithelium and the basement membrane. The nature of the mutations and their localization determine the severity of the clinical manifestations of BE. Mutation information is a prerequisite for effective medical and genetic counseling, prenatal and preimplantation DNA diagnosis. Therefore diagnosis and prescribing appropriate treatment and follow-up care is an important task for neonatologists and pediatric dermatologists. As manifestations of hereditary BE are numerous, a specialized center is required for optimal care, where multidisciplinary care will be provided (neonatologists, pediatric surgeons, pediatric dermatologists, etc.). The purpose of clinical observation is to pay attention of specialists to this rare disease, and to present 2 clinical cases of bullous epidermolysis in newborns who were admitted to the anesthesiology and intensive care unit of newborns of Vinnytsia Regional Children's Clinical Hospital almost at the same time.

Inherited epidermolysis bullosa is a group of genetic diseases characterized by skin fragility and blistering on the skin and mucous membranes in response to minimal trauma. Epidermolysis bullosa is clinically and genetically very heterogeneous, being classified into four main types according to the layer of skin in which blistering occurs: epidermolysis bullosa simplex (intraepidermal), junctional epidermolysis bullosa (within the lamina lucida of the basement membrane), dystrophic epidermolysis bullosa (below the basement membrane), and Kindler epidermolysis bullosa (mixed skin cleavage pattern). Furthermore, epidermolysis bullosa is stratified into several subtypes, which consider the clinical characteristics, the distribution of the blisters, and the severity of cutaneous and extracutaneous signs. Pathogenic variants in at least 16 genes that encode proteins essential for the integrity and adhesion of skin layers have already been associated with different subtypes of epidermolysis bullosa. The marked heterogeneity of the disease, which includes phenotypes with a broad spectrum of severity and many causal genes, hinders its classification and diagnosis. For this reason, dermatologists and geneticists regularly review and update the classification criteria. This review aimed to update the state of the art on inherited epidermolysis bullosa, with a special focus on the associated clinical and genetic aspects, presenting data from the most recent reclassification consensus, published in 2020.

Background: Epidermolysis Bullosa is a rare genetic mechanobullous skin and mucosal fragility with blistering. It is classified into: Epidermolysis (Simplex), lucidolytic (Junctional) and dermolytic (Dystrophic). The disease is extremely debilitating necessitating early detection and treatment. Immunofluorescence mapping is gold standard of diagnosis. Objective: To determine types and inheritance patterns of cutaneous and extracutaneous Epidermolysis Bullosa by immunofluorescence mapping. Methods: 5 µm frozen section from lesions /artificial bulla are collected over multispot PTEF coated slides. Incubation done with primary antihuman mouse antibodies (IgG) and with FITC conjugated anti-IgG mouse secondary antibodies. Antibodies used against Collagen IV, Collagen VII, cytokeratin 4, cytokeratin 14, Laminin 332, alpha6 and beta4 integrins. Healthy skin is positive control. Results: In Simplex type blister and split is above lamina densa with reduced CK14 at base (Dominant). Anti-integrins, laminin 332 and collagen IV all are present at base. In Junctional type, collagen IV present at base, cytokeratin or integrin at roof, and cleavage is at lamina lucida. Laminin 332 is reduced (dominant) or absent (recessive). In Dystrophic type blister is below lamina densa. Collagen IV or VII stains roof of blister. Collagen VII is absent in recessive type and reduced in dominant type. Limitations: Rare disease Conclusion: Agreement of clinical diagnosis and mapping is moderate indicating mapping is an essential adjunct.

Bullous pemphigoid (BP) is an autoimmune blistering disorder with substantial morbidity and mortality. BP is regarded as a disorder driven by IgG due to BP180 and BP230 IgG autoantibodies, yet, new advances highlight the function of eosinophils and IgE autoantibodies in BP. Evidence supports that eosinophils are involved in BP pathogenesis, notably, these include the presence of IL-5, eotaxin, and eosinophil-colony stimulating factor in blister fluid. Peripheral blood eosinophilia is present in nearly 50% of affected patients, eosinophils are found against the dermo-epidermal junction (DEJ) when BP serum is present and metalloprotease-9 is secreted at blister sites. Blister fluid of BP patients contains eosinophil granule proteins which are located along the lamina lucida of the basement membrane zone (BMZ) in patients with BP and correspond with disease clinically, eosinophil extracellular traps (EET) have been linked to DEJ splitting, IL-5 activated eosinophils cause DEJ separation when BP serum is present, and eosinophils are requisite to drive anti-BP180 IgE mediated blistering of the skin. Yet, the mechanism whereby eosinophils contribute to the pathogenesis of BP remains to be explored. In this review, we examined the role of eosinophils in BP while offering a basis to explain the pathomechanisms of eosinophils in BP.

Patient: Female, 77-year-old Final Diagnosis: Atypical bullous pemphigoid Symptoms: Bullous skin lesions Medication: — Clinical Procedure: — Specialty: Dermatology Objective: Unusual clinical course Background: Bullous pemphigoid is a common pruritic skin lesion reported in elderly patients. It is caused by an immuno-logic reaction between autoantibodies and hemidesmosome proteins of epithelial cells. The disease is characterized by a symmetrical blister distribution on the body. Diagnosis should be suspected in elderly patients presenting with a tense blister on normal-appearing skin or on an erythematous base. In the literature, several forms of typical bullous pemphigoid after treatment with linagliptin have been reported. However, this is the first reported case of atypical nonbullous pemphigoid after linagliptin intake. Case Report: A 77-year-old woman presented with multiple erythematous papules and nodules on the upper extremities and trunk. The patient was being treated with linagliptin for diabetes. Diagnosis was made with biopsy and histopathological studies, followed by direct immunofluorescence. The histopathological study showed a subepidermal blister with an underlying polymorphous infiltrate, mainly of an eosinophilic profile. Direct immunofluorescence showed linear IgG and C3 antibodies to hemidesmosomes at the lamina lucida of the basement membrane. Thus, the diagnosis of atypical nonbullous pemphigoid was made. Conclusions: This report emphasizes the great variety of bullous pemphigoid presentation and the need for a greater level of awareness of the adverse effects of linagliptin. Thus, atypical nonbullous pemphigoid should be considered among the potential differential diagnoses in patients with multiple erythematous papules and nodules on the upper extremities and trunk.

The third phalanx of the equine digit is suspended within the hoof capsule by a specialized interdigitating dermoepidermal layer called the lamellae, which fails during laminitis. Pathology of the basement membrane (BM), which interfaces epidermis and dermis, is evident during acute laminitis. However, BM damage appears to be less prevalent in ponies with the insulin-associated form of laminitis. The aim of the present study was to investigate changes to the ultrastructure and morphometry of the lamellar BM in the acute phase of insulin-induced laminitis in horses. Lamellar tissue from the left forefoot of 3 horses with acute hyperinsulinemic laminitis was examined with transmission electron microscopy and compared with tissue from normal horses. Lamellar BM width and hemidesmosome (HD) density were assessed every 5 μm along ∼200 μm of secondary epidermal lamellar BM. The BM zone of treated horses was extensively disorganized with loss of uniformity of the lamina lucida and lamina densa, fragmentation and disorientation of HDs, and cytoskeletal disengagement of the HDs. The mean (±SD) lamellar BM was twice as wide in treated (0.25 ± 0.05 μm), compared with control (0.14 ± 0.02 μm), horses. The HD density (HDs/μm) was reduced by half in the treatment group (1.88 ± 0.37), compared with controls (3.6 ± 0.13). The reduced number of HDs in horses with laminitis may contribute to the weakening of the dermoepidermal junction and lamellar failure. Disassembly of HDs during excessive cellular proliferation, secondary to hyperinsulinemia, may account for HD loss. Further investigation of the underlying etiopathogenesis of BM dysfunction during hyperinsulinemic laminitis in horses may facilitate an improved understanding of the disease.

Made available in DSpace on 2020-12-12T01:42:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-04-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The pathogenesis of melasma is not fully understood, and the role of skin basement membrane zone (BMZ) alterations in disease development and the maintenance of hypermelanogenesis are also poorly known. We performed a comparative study to characterize the ultrastructural alterations that occur in BMZ in melasma and adjacent normal skin, as well as we discuss the implications of these changes in the physiopathology of the disease. Pairs of facial skin biopsies (2 mm) from 10 women with melasma and normal skin (< 2 cm apart) were processed by Transmission Electronic Microscopy or immunohistochemistry for Melan-A counterstained with Periodic acid–Schiff stain. Cytoplasmic organelles (from keratinocyte or melanocyte), BMZ damage were assessed and melanocyte counting (total and pendulous) was done. There was greater amount of cytoplasmic organelles inside basal keratinocytes and melanocytes in melasma, as well as structural damaged areas in the lamina densa (disruptions, gaps, lower density and thinning) and anchoring fibrils (lamina lucida), compared to healthy adjacent skin. Areas with pendulous melanocytes are characterized by discontinuity of BMZ ultrastructure. The prominence of cytoplasmic organelles from melanocytes and keratinocytes evidences the involvement of both cell groups in melasma. The damage in the lamina densa and lamina lucida suggest the role of upper dermis injury/repair process in the pathogenesis of the disease. Departamento de Dermatologia E Radioterapia FMB-Unesp, Campus de Rubião Jr.-Unesp. Departamento de Patologia FMB-Unesp Departamento de Morfologia IBB-Unesp Departamento de Dermatologia E Radioterapia FMB-Unesp, Campus de Rubião Jr.-Unesp. Departamento de Patologia FMB-Unesp Departamento de Morfologia IBB-Unesp FAPESP: 2012/09233-5 CNPq: 401309/2016-9

Junctional epidermolysis bullosa (JEB) is a group of congenital blistering skin diseases characterized by clefting through the lamina lucida of the basement membrane zone.To characterize the clinical and morphological features of a congenital mechanobullous disease in a litter of puppies with severe upper respiratory involvement, and to identify an associated genetic variant.Five of eight puppies in an Australian cattle dog cross-bred litter showed signs of skin fragility. Three were stillborn and one died at one month of age. The two surviving puppies were presented with blistering skin disease and severe respiratory distress. Additionally, one unaffected sibling was examined and blood was obtained for genetic testing.Post-mortem examination, histopathological evaluation and electron microscopy were performed. Whole genome sequencing (WGS) of one affected puppy was compared to a database of 522 dogs of 55 different breeds for variant analysis. Sanger sequencing of one additional affected and one unaffected sibling confirmed the variant.Clinically, severe mucocutaneous ulcers occurred in frictional areas with claw sloughing. Histopathological results revealed subepidermal clefts and electron microscopy confirmed the split in the lamina lucida. Post-mortem examination documented extensive pharyngeal and laryngeal lesions with granulation tissue and fibrinous exudate obscuring the airway. Moderate tracheal hypoplasia contributed. The WGS revealed a novel missense variant in the laminin α3-chain XP_537297.2p(Asp2867Val), with an autosomal recessive mode of inheritance.A novel variant in LAMA3 caused a generalized and severe phenotype of JEB with an unique clinical presentation of upper airway obstruction.L’épidermolyse bulleuse jonctionnelle (JEB) est un groupe de dermatoses cutanées congénitales vésiculeuses caractérisées par un clivage au niveau de la lamina lucida de la membrane basale.Caractériser les critères cliniques et morphologiques de maladies bulleuses congénitales dans une portée de chiots avec troubles respiratoires sévères et identifier un variant génétique associé.Cinq des huit chiots d’une portée de chiens croisés bouvier australien ont montré des signes de fragilité cutanée. Trois étaient mort-nés et un est mort à l’âge d’un mois. Les deux chiots survivants ont été présentés avec une dermatose vésiculeuse et des troubles respiratoires sévères. En outre, un chiot sain a été examiné et du sang a été prélevé pour tests génétiques. MATÉRIELS ET MÉTHODES: Un examen post-mortem, une évaluation histopathologique et electro-microscopique ont été réalisés. Le séquençage complet du génome (WGS) d’un chiot atteint a été comparé à une base de données de 522 chiens de 55 races différentes pour une analyse de variant. Le séquençage de Sanger d’un autre chiot atteint et un chiot sain a confirmé le variant. RÉSULTATS: Cliniquement, les ulcères cutanéo-muqueux sévères étaient observés aux zones de friction avec pertes des griffes. Les résultats histopathologiques révélèrent des séparations sous épidermiques et l’électro-microscopie a confirmé le clivage au sein de la lamina lucida. L’examen post mortem a documenté des lésions laryngées et pharyngées extensives avec tissue de granulation et exsudat fibrineux dissimulant les voies aériennes. Une hypoplasie trachéale modérée y contribuait. Le WGS a révélé un nouveau variant contre-sens dans la chaine α3 de laminine XP_537297.2p(Asp2867Val), avec un mode autosomal récessif de transmission.Un nouveau variant cause un phénotype de JEB sévère et généralisé avec une présentation clinique unique d’obstruction des voies respiratoires supérieures.INTRODUCCIÓN: la epidermólisis bullosa de la unión (JEB) es un grupo de enfermedades cutáneas congénitas con formación de ampollas caracterizadas por una hendidura a lo largo de la lámina lúcida de la zona de la membrana basal. OBJETIVOS: establecer las características clínicas y morfológicas de una enfermedad mecanobullosa congénita en una camada de cachorros con afectación respiratoria superior grave e identificar una variante genética asociada. ANIMALES: cinco de los ocho cachorros en una camada mestiza de perros de ganado australiano mostraron signos de fragilidad de la piel. Tres nacieron muertos y uno murió al mes de edad. Los dos cachorros supervivientes presentaron una enfermedad cutánea con ampollas y dificultad respiratoria grave. Además, se examinó a un hermano no afectado y se extrajo sangre para realizar pruebas genéticas. MÉTODOS Y MATERIALES: Se realizó examen post-mortem, evaluación histopatológica y microscopía electrónica. La secuenciación del genoma completo (WGS) de un cachorro afectado se comparó con una base de datos de 522 perros de 55 razas diferentes para el análisis de variantes. La secuenciación de Sanger de un hermano afectado adicional y un hermano no afectado confirmó la variante. RESULTADOS: Clínicamente, se produjeron úlceras mucocutáneas graves en áreas de fricción con desprendimiento de garras. Los resultados histopatológicos revelaron hendiduras subepidérmicas y la microscopía electrónica confirmó la escisión en la lámina lúcida. El examen post-mortem documentó extensas lesiones faríngeas y laríngeas con tejido de granulación y exudado fibrinoso que oscurecía las vías respiratorias. A las lesions también se añadía una hipoplasia traqueal moderada. El WGS reveló una nueva variante sin sentido en la cadena α3 de laminina XP_537297.2p (Asp2867Val), con un modo de herencia autosómico recesivo. CONCLUSIÓN Y RELEVANCIA CLÍNICA: una nueva variante genética causó un fenotipo generalizado y severo de JEB con una presentación clínica única de obstrucción de las vías respiratorias superiores.Die Junctional Epidermolysis Bullosa (JEB) umfasst eine Gruppe kongenitaler blasenbildender Hauterkrankungen, die durch eine Spaltbildung durch die Lamina lucida der Basalmembranzone charakterisiert sind. ZIEL: Die Charakterisierung klinischer und morphologischer Merkmale einer kongenitalen mechanobullösen Erkrankung bei einem Wurf von Welpen mit hochgradiger respiratorischer Beteiligung sowie eine Identifizierung der damit zusammenhängenden genetischen Variante.Fünf von acht Welpen eines Australian cattle Dog Mischlingswurfes zeigten Zeichen von Hautfragilität. Drei waren totgeboren und ein Welpe starb mit einem Monat. Die zwei überlebenden Welpen wurden mit einer blasenbildenden Hauterkrankung und hochgradiger respiratorischer Not vorgestellt. Zusätzlich wurde ein nicht betroffenes Geschwister untersucht und Blut zur genetischen Analyse genommen.Eine Post Mortem Untersuchung, eine histopathologische Evaluierung und Elektronenmikroskopie wurden durchgeführt. Eine Ganz-Genom Sequenzierung (WGS) eines erkrankten Welpen wurde mit der Datenbank von 522 Hunden 55 verschiedener Rassen zur Varianzanalyse verglichen. Mittels Sanger Sequenzierung eines der weiteren betroffenen und eines nicht erkrankten Geschwisters wurde die Variante bestätigt.Klinisch traten hochgradige mukokutane Ulzera an Reibungsstellen auf, wobei sich die Krallen ablösten. Die histopathologische Untersuchung ergab subepidermale Spalten und mittels Elektronenmikroskopie wurde der Spalt in der Lamina lucida bestätigt. Die Post mortem Untersuchung dokumentierte ausgedehnte Läsionen in Pharynx und Larynx, wobei Granulationsgewebe und fibrinöses Exsudat die Luftwege behinderten. Eine moderate tracheale Hypoplasie trug zu dem Problem bei. Die WGS zeigte eine neue Missense Mutation in der Laminin α3-Kette XP_537297.2p(Asp2867Val), bei autosomal rezessiver Vererbung.Eine neue Variante bedingte eine generalisierte Form einer JEB mit einem stark betroffenen Phänotyp und einer einzigartigen klinischen Präsentation der Obstruktion der oberen Atemwege.背景: Junctional epidermolysis bullosa（JEB）は，基底膜領域のlamina lucidaを介した間隙を特徴とする先天性水疱性皮膚疾患の一群である。 目的: 本研究の目的は、重度の上気道病変を有する子犬集団における先天性機械的水疱性疾患の臨床的および形態学的特徴を明らかにし，関連する遺伝子変異を同定することであった。 供試動物: オーストラリアン・キャトル・ドッグとの交配で生まれた子犬8頭のうち、5頭に皮膚脆弱性の兆候が見られた。3頭は死産し、1頭は生後1ヶ月で死亡した。生き残った2頭の子犬は、水ぶくれのある皮膚病と重度の呼吸困難を呈していた。さらに、罹患していない1頭の同腹仔を診察し、遺伝子検査のために血液を採取した。 材料と方法: 死後検査、病理組織学的評価および電子顕微鏡検査を行った。罹患子犬1頭の全ゲノム塩基配列（WGS）を、55種の犬522頭のデータベースと比較し、バリアント解析を行った。さらに罹患した1頭の同腹仔と罹患していない1頭の同腹仔のサンガーシークエンス法により、変異が確認された。 結果: 臨床的には、重度の粘膜皮膚潰瘍が摩擦部位に発生し、爪が剥がれた。病理組織学的には表皮下の間隙を認め、電子顕微鏡ではlamina lucidaの間隙が確認された。死後検査では、肉芽組織および線維性滲出液が気道を塞いでいる広範囲の咽頭および喉頭の病変が記録された。中等度の気管低形成が認められた。WGSの結果、ラミニンα3鎖のXP_537297.2p(Asp2867Val)という新規ミスセンス変異が見つかり、常染色体劣性遺伝であることが判明した。 結論と臨床的妥当性: 新規変異は、上気道閉塞というユニークな臨床症状を伴うJEBの全般的で重篤な表現型の原因となった。.背景: 交界性大疱性表皮松解症(Junctional epidermolysis bullosa，JEB)是一组先天性水疱性皮肤病，以基底膜区透明层裂隙为特征。 目的: 描述一窝严重上呼吸道发病幼犬先天性机械大疱病的临床和形态学特征，并确定相关的遗传变异。 动物: 澳大利亚杂交牛犬8只同窝幼犬中的5只显示出皮肤脆弱症状。3只为死胎，1例在1月龄时死亡。2只存活幼犬表现为水疱性皮肤病和严重呼吸窘迫。此外，检查了1只未发病的同窝犬，并采集血液用于基因检测。 方法和材料: 进行尸检、组织病理学评价和电子显微镜检查。将1只发病幼犬的全基因组测序(WGS)与55个不同品种的522只犬的数据库进行比较，进行变异分析。对另外一个发病和一个未发病的同窝犬进行Sanger测序，证实了该变异。 结果: 临床上，重度皮肤粘膜溃疡发生在摩擦区域，伴爪脱落。组织病理学结果显示表皮下裂隙，电子显微镜检查证实透明层开裂。尸检记录了广泛的咽部和喉部病变，伴有肉芽组织和纤维蛋白性渗出物阻塞气道。导致中度气管发育不全。WGS揭示了层粘连蛋白α3-链XP_537297.2p(Asp2867Val)的一个新的错义变体，具有常染色体隐性遗传模式。 结论和临床相关性: 种新变体引起JEB的广泛和严重表型，具有上呼吸道阻塞的独特临床表现。.A epidermólise bolhosa juncional (EBJ) é um grupo de doenças cutâneas congênitas apresentando lesões bolhosas caracterizadas por fissuras ao longo da lâmina lúcida da zona da membrana basal.Detalhar as características clínicas e morfológicas de uma doença mecanobolhosa congênita em uma ninhada de filhotes de cães com comprometimento respiratório superior grave e identificar uma variante genética associada.Cinco dos oito filhotes em uma ninhada com cães boideieros australianos mestiços apresentaram sinais de fragilidade da pele. Três nasceram mortos e um morreu com um mês de idade. Os dois cachorros sobreviventes apresentaram dermatopatia bolhosa e dificuldade respiratória grave. Além disso, um irmão não afetado foi examinado e o sangue foi obtido para teste genético. MÉTODOS E MATERIAIS: Foram realizados exame post-mortem, avaliação histopatológica e microscopia eletrônica. O sequenciamento do genoma completo (WGS) de um filhote afetado foi comparado a um banco de dados de 522 cães de 55 raças diferentes para análise de variantes. O sequenciamento de Sanger de um irmão afetado adicional e um irmão não afetado confirmou a variante.Clinicamente, úlceras mucocutâneas graves ocorreram em áreas de fricção com arranhadura pelas unhas. Os resultados histopatológicos revelaram fissuras subepidérmicas e a microscopia eletrônica confirmou a fissura na lâmina lúcida. O exame post-mortem documentou lesões laríngeas e faríngeas extensas com tecido de granulação e exsudato fibrinoso obscurecendo as vias aéreas. Hipoplasia traqueal moderada contribuiu. O WGS revelou uma nova variante missense na cadeia α3 da laminina XP_537297.2p (Asp2867Val), com um modo de herança autossômico recessivo. CONCLUSÃO E RELEVÂNCIA CLÍNICA: Uma nova variante causou um fenótipo generalizado e grave de EBJ com uma apresentação clínica única de obstrução das vias aéreas superiores.

Linear IgA bullous dermatosis (LABD) is characterized by presence of multiple IgA autoantibodies, and a comparatively lesser number of IgG antibodies, directed against different hemidesmosomal antigens. The main autoantigens are LAD-1, LABD-97, BP180 and BP230, type VII collagen and laminin 332. We retrospectively studied the serology of 54 Italian patients with LABD using enzyme-linked immunosorbent assay (ELISA), immunoblotting assay, and indirect immunofluorescence on monkey oesophagus and salt-split skin. Among these, indirect immunofluorescence of salt-split skin elicits the greatest sensitivity. Sixty-three percent of the sera were observed to be positive, with a lamina lucida pattern observed in 48%, a sub-lamina densa pattern in 2% and a mixed pattern in 13% of the cases. IgA reactivity to LAD-1 on immunoblotting was found in 52% of sera, to BP180-NC16A by ELISA in 32% and to BP230 in 26%. Only 17% of patients possessed circulating IgG autoantibodies. LAD-1 was determined to be a major autoantigen of the lamina lucida subtype. Combined serological assays demonstrated a high sensitivity (82%), suggesting that this approach could support diagnosis when a biopsy is not feasible or direct immunofluorescence results are negative.

Imatinib mesylate (Glivec; Novartis AG, Basel, Switzerland) is a tyrosine kinase inhibitor which is used in the treatment of oncologic diseases like chronic myeloid leukemia and gastrointestinal stroma tumor (GIST). Among cutaneous side effects, bullous reactions are rare. The authors describe the case of a 66-year-old woman developing blistering and skin fragility on her hands, foot, lower legs, and back after intake of imatinib for treatment of GIST. Biopsy showed vacuolar alteration at the dermoepidermal junction (DEJ) associated with a few lymphocytes and a subepidermal blister. The upper papillary dermis below the vacuolar alteration and below the blister showed hyalinization and loss of elastic microfibrils. Direct immunofluorescence was negative for deposits of immunoglobulins. Immunofluorescence on cryosections revealed loss of laminin and collagen IV in vacuoles at the DEJ. Electron microscopy showed dissolution of lamina lucida and lamina densa of the basement membrane below as well as next to the vacuoles and blister. In conclusion, the authors present the first patient with GIST with blistering and skin fragility due to imatinib therapy. As a pathophysiological explanation the authors propose loss of laminin and collagen IV at the DEJ leading to basement membrane instability and blistering. This case also suggests additional features reminiscent of lichen sclerosus induced by imatinib, a drug which is actually known for its antifibrotic effects.

Anti-p200 pemphigoid is a rare autoimmune subepidermal blistering disease characterized by the presence of circulating immunoglobulin G antibodies directed against laminin gamma-1, a 200-kDa protein located in the lamina lucida of the basement membrane. We review the clinical, histopathological and immunological characteristics of the first 2 cases described in Spain. Anti-p200 pemphigoid shares histopathological and immunopathological findings with epidermolysis bullosa acquisita, the main entity in the differential diagnosis. However, its management follows the same guidelines as those used for bullous pemphigoid. The diagnosis is confirmed by immunoblotting, which is a complex technique available in few centers. We propose the immunohistochemical detection of collagen type IV on the floor of the blister, combined with standard immunofluorescence techniques, as a simple, accessible alternative to differentiate anti-p200 pemphigoid from epidermolysis bullosa acquisita.

Nail-patella syndrome is an autosomal dominant disorder characterized by nail dysplasia and skeletal anomaly. Some patients have been shown to have ultrastructural abnormalities of the glomerular basement membrane that result in nephrosis. However, little has been reported on the epidermal basement membrane in this condition. This paper reports 2 families with nail-patella syndrome. Direct sequencing analysis of LMX1B revealed that family 1 and family 2 were heterozygous for the mutations c.140-1G>C and c.326+1G>C, respectively. To evaluate the epidermal basement membrane zone, ultrastructural and immunohistochemical analyses were performed using skin specimens obtained from the dorsal thumb. Electron microscopy showed intact hemidesmosomes, lamina lucida, lamina densa, and anchoring fibrils. Immunofluorescence studies with antibodies against components of the epidermal basement membrane zone revealed a normal expression pattern among the components, including type IV collagen. These data suggest that nail dysplasia in patients with nail-patella syndrome is not caused by structural abnormalities of the epidermal basement membrane.

We report the case of a 59-year-old Japanese woman who developed linear IgA bullous dermatosis during treatment for ulcerative colitis that manifested as pruritic vesicles with erythema on the trunk and scalp. Histopathological examination revealed subepidermal bulla with neutrophil and eosinophil infiltration in the upper dermis. Direct immunofluorescence revealed linear IgA deposits at the basement membrane zone, and indirect immunofluorescence using split skin revealed IgA reaction to the epidermal side (lamina lucida type). We reviewed 33 reported cases of linear IgA bullous dermatosis associated with ulcerative colitis and found that ulcerative colitis preceded the onset of linear IgA bullous dermatosis in 94% of the patients and that IgA-positive patients in split skin indirect immunofluorescence all showed the lamina lucida type, indicating that target antigens for serum IgA antibodies may reside in the lamina lucida. Regarding the pathogenetic association of ulcerative colitis and linear IgA bullous dermatosis, intestinal inflammation may induce the exposure and presentation of intestinal antigens that are cross-reactive to cutaneous antigens, stimulating autoimmune response to antigens of cutaneous basement membrane zones.

Junctional epidermolysis bullosa (JEB) induces skin fragility slightly deeper in the basement membrane zone affecting the lamina lucida. It is most commonly inherited in an autosomal recessive manner. Mutations include LAMA3, LAMB3, LAMC2, COL17A1, ITGA6, ITGB4 corresponding to the proteins laminin-332, type XVII collagen, and alpha-6 beta-4 integrin.

Mustard exposures result in epithelial-stromal separations in the cornea and epidermal-dermal separations in the skin. Large blisters often manifest in skin, while the cornea develops microblisters, and, when enough form, the epithelium sloughs. If the exposure is severe, healing can be imperfect and can result in long-term adverse consequences. For the cornea, this could manifest as recurrent corneal erosions. Since the corneal epithelial-stromal separations are in the region identified by electron microscopy as the lamina lucida, the same region affected by the blistering disease junctional epidermolysis bullosa (JEB), we postulated that the molecules that are defective in JEB would be the same ones cleaved by mustard compounds. These molecules are α6β4 integrin and collagen XVII, which can be cleaved by matrix metalloproteinase-9 (MMP-9) and ADAM17, respectively. Therefore, our laboratory has tested MMP-9 and ADAM17 inhibitors as potential therapies to attenuate corneal mustard injury. Our results demonstrated that inhibiting MMP-9 and ADAM17 resulted in less epithelial-stromal separation in the corneas at 24 h postexposure, as compared with using only medium as a therapy.

Regional differences of the surface of planarian gastrodermal cells are emphasized by staining with ruthenium red (RR). It is proposed that such differences reflect functional diversity of the luminal, lateral, and basal surfaces of the cells. The luminal surface is coated with a uniform layer of the RR-positive substance, which penetrates into the intercellular space at the intermediate junction. The septate junction situated just beneath the intermediate junction shows a permeability barrier to the RR tracer. At the basolateral surface, however, RR stains the septate junction in which the electron density of individual septa is enhanced remarkably. The gastrodermal cells are delineated entirely with RR-positive substance passing freely through the gap junction fuses into the outer leaflets of adjacent plasma membranes. The irregularly dilated intercellular space at nonjunctional appositions includes a slight deposit of RR-positive substance which attaches to the plasma membrane. The basal surface is underlined by the continuous basal lamina, which consists of the lamina lucida and the lamina densa. The lamina densa has a conspicuous affinity for RR. The lamina lucida is characterized by irregular deposits of RR-positive substance, some of which concentrates on the hemidesmosomal portions. Treatment with the enzyme hyaluronidase prior to staining with RR abolishes the staining of the basal lamina. As a result, the material of the lamina densa appears flocculent.

Junctional epidermolysis bullosa (JEB) is a rare genodermatosis characterized by a split in the lamina lucida usually because of mutations in LAMA3, LAMB3 and LAMC2 resulting in absence or reduction of laminin-332. Rare subtypes of JEB have mutations in COL17A1, ITGB4, ITGA6 and ITGA3 leading to reduction or dysfunction of collagen XVII, integrin α6β4 and integrin α3. The classic finding under light microscopy is a paucicellular, subepidermal split. We describe the unusual presence of an eosinophilic infiltrate in the bullae and subjacent dermis in a neonate with JEB, generalized intermediate (formerly known as non-Herlitz-type JEB), discuss the histologic differential diagnosis for a subepidermal blister in a neonate, review the literature regarding cases of epidermolysis bullosa (EB) presenting with inflammatory infiltrates, and discuss mechanisms to explain these findings. This case highlights that eosinophils can rarely be seen in EB and should not mislead the dermatopathologist into diagnosing an autoimmune blistering disorder.

The epithelial basement membrane (BM) is a specialized extracellular matrix that has been shown to have a critical role in corneal development, wound healing, and disease. Although the epithelial BM contributes to corneal homeostasis, relatively little is know about non-epithelial production of its components that may be important in defective regeneration of the epithelial basement membrane associated with opacity after photorefractive keratectomy. The purpose of the current study was to investigate stromal production of corneal epithelial BM proteins in wounded human corneas using immunohistochemistry. A total of five unwounded control eyes and five 30-minute epithelial-wounded corneas were obtained from fresh corneoscleral buttons removed from human eyes enucleated due to choroidal melanoma with normal anterior segments. In the wounded corneas, an eight mm patch of central corneal epithelium and epithelial BM was removed with a Beaver blade when the patient was under general anesthesia. Immunohistochemical analyses were performed to detect perlecan and nidogen-2 proteins–important components of the epithelial BM lamina lucida and lamina densa zones. Perlecan and nidogen-2 proteins were detected in the BM itself and at low levels in keratocytes in all unwounded corneas. After epithelial injury, both perlecan and nidogen-2 was expressed at high levels in stromal keratocytes, including superficial keratocytes in the early phases of apoptosis. Thus, after epithelial and epithelial BM injury, stromal keratocytes contribute important perlecan and nidogen-2 components to the regenerating epithelial BM.

The purpose of this study was to investigate whether myofibroblast-related fibrosis (scarring) after microbial keratitis was modulated by the epithelial basement membrane (EBM) injury and regeneration. Rabbits were infected with Pseudomonas aeruginosa after epithelial scrape injury and the resultant severe keratitis was treated with topical tobramycin. Corneas were analyzed from one to four months after keratitis with slit lamp photos, immunohistochemistry for alpha-smooth muscle actin (α-SMA) and monocyte lineage marker CD11b, and transmission electron microscopy. At one month after keratitis, corneas had no detectible EBM lamina lucida or lamina densa, and the central stroma was packed with myofibroblasts that in some eyes extended to the posterior corneal surface with damage to Descemet’s membrane and the endothelium. At one month, a nest of stromal cells in the midst of the SMA+ myofibroblasts in the stroma that were CD11b+ may be fibrocyte precursors to myofibroblasts. At two to four months after keratitis, the EBM fully-regenerated and myofibroblasts disappeared from the anterior 60 to 90% of the stroma of all corneas, except for one four-month post-keratitis cornea where anterior myofibroblasts were still present in one localized pocket in the cornea. The organization of the stromal extracellular matrix also became less disorganized from two to four months after keratitis but remained abnormal compared to controls at the last time point. Myofibroblasts persisted in the posterior 10% to 20% of posterior stroma even at four months after keratitis in the central cornea where Descemet’s membrane and the endothelium were damaged. This study suggests that the EBM has a critical role in modulating myofibroblast development and fibrosis after keratitis—similar to the role of EBM in fibrosis after photorefractive keratectomy. Damage to EBM likely allows epithelium-derived transforming growth factor beta (TGFβ) to penetrate the stroma and drive development and persistence of myofibroblasts. Eventual repair of EBM leads to myofibroblast apoptosis when the cells are deprived of requisite TGFβ to maintain viability. The endothelium and Descemet’s membrane may serve a similar function modulating TGFβ penetration into the posterior stroma—with the source of TGFβ likely being the aqueous humor.

Background The autoantigen for the major type of linear IgA bullous dermatosis (LAD, lamina lucida type) is the shed ectodomain of BP180. However, it is unknown why most LAD sera react with the shed ectodomain but not with the intact BP180/type XVII collagen. Objective The aim of this study was to characterize the unique cleavage-dependent epitope(s) in the shed ectodomain. Methods We used a monoclonal antibody (MAb-1337) and six LAD sera, which reacted preferentially with the shed ectodomain of BP180. The location and characteristics of the epitopes for these antibodies were analyzed mainly by immunoblotting using chimeric bovine–human BP180 mammalian recombinant proteins and variously truncated bacterial recombinant proteins. Results LAD sera and MAb-1337 reacted with the plasmin-digested products of full-length BP180. Four of the six LAD sera reacted to a bacterial recombinant protein consisting of the human non-collagenous 16th A (NC16A) and the collagenous 15th (C15) domains, while these sera were negative or only faintly reactive with the NC16A and C15 recombinants. The epitope for MAb-1337 was localized to the COOH-terminal 21 amino acid region within the NC16A domain. Conclusion The results in this study indicate that the major epitopes for LAD sera are formed or exposed by a cleavage-induced conformational change, but not by a post-translational modification that occurs only in the shed ectodomain, and are located at the boundary between the NC16A and C15 domains.

While real-time 3-D evaluation of human skin constructs is needed, only 2-D non-invasive imaging techniques are available. The aim of this paper is to evaluate the potential of high-definition optical coherence tomography (HD-OCT) for real-time 3-D assessment of the epidermal splitting and decellularization. Human skin samples were incubated with four different agents: Dispase II, NaCl 1 M, sodium dodecyl sulphate (SDS) and Triton X-100. Epidermal splitting, dermo-epidermal junction, acellularity and 3-D architecture of dermal matrices were evaluated by High-definition optical coherence tomography before and after incubation. Real-time 3-D HD-OCT assessment was compared with 2-D en face assessment by reflectance confocal microscopy (RCM). (Immuno) histopathology was used as control. HD-OCT imaging allowed real-time 3-D visualization of the impact of selected agents on epidermal splitting, dermo-epidermal junction, dermal architecture, vascular spaces and cellularity. RCM has a better resolution (1 μm) than HD-OCT (3 μm), permitting differentiation of different collagen fibres, but HD-OCT imaging has deeper penetration (570 μm) than RCM imaging (200 μm). Dispase II and NaCl treatments were found to be equally efficient in the removal of the epidermis from human split-thickness skin allografts. However, a different epidermal splitting level at the dermo-epidermal junction could be observed and confirmed by immunolabelling of collagen type IV and type VII. Epidermal splitting occurred at the level of the lamina densa with dispase II and above the lamina densa (in the lamina lucida) with NaCl. The 3-D architecture of dermal papillae and dermis was more affected by Dispase II on HD-OCT which corresponded with histopathologic (orcein staining) fragmentation of elastic fibres. With SDS treatment, the epidermal removal was incomplete as remnants of the epidermal basal cell layer remained attached to the basement membrane on the dermis. With Triton X-100 treatment, the epidermis was not removed. In conclusion, HD-OCT imaging permits real-time 3-D visualization of the impact of selected agents on human skin allografts.

Bullous pemphigoid (BP), an autoimmune subepidermal blistering skin disease, demonstrates tense blisters with or without widespread erythema, blistering along the lamina lucida, immunoglobulin G and/or complement deposits at the basement membrane zone, and the presence of circulating autoantibodies against hemidesmosomal molecules. These autoantibodies usually react against 180-kDa and/or 230-kDa proteins, designated as BP180 and BP230, respectively. The precise blistering mechanisms after autoantibodies bind to antigens are not fully understood. Immune complexes are thought to initially activate the complement cascade, which may induce activation of proteases and/or cytokines and cause dermal-epidermal separation. However, why does separation run specifically within the lamina lucida in a space as narrow as 500 nm wide? This review mainly focuses on the possible mechanisms of BP-specific blistering and how separation occurs along the lamina lucida, based on existing evidence.

BACKGROUND Linear IgA bullous dermatosis (LABD) is a clinically and immunologically heterogeneous, subepidermal, autoimmune bullous disease (AIBD), for which the long-term evolution is poorly described. OBJECTIVES To investigate the clinical and immunological characteristics, follow-up and prognostic factors of adult idiopathic LABD. METHODS This retrospective study, conducted in our AIBD referral centre, included adults, diagnosed between 1995 and 2012, with idiopathic LABD, defined as pure or predominant IgA deposits by direct immunofluorescence. Clinical, histological and immunological findings were collected from charts. Standard histology was systematically reviewed, and indirect immunofluorescence (IIF) on salt-split skin (SSS) and immunoblots (IBs) on amniotic membrane extracts using anti-IgA secondary antibodies were performed, when biopsies and sera obtained at diagnosis were available. Prognostic factors for complete remission (CR) were identified using univariate and multivariate analyses. RESULTS Of the 72 patients included (median age 54 years), 60% had mucous membrane (MM) involvement. IgA IIF on SSS was positive for 21 of 35 patients tested; 15 had epidermal and dermal labellings. Immunoelectron microscopy performed on the biopsies of 31 patients labelled lamina lucida (LL) (26%), lamina densa (23%), anchoring-fibril zone (AFz) (19%) and LL+AFz (23%). Of the 34 IgA IBs, 22 were positive, mostly for LAD-1/LABD97 (44%) and full-length BP180 (33%). The median follow-up was 39 months. Overall, 24 patients (36%) achieved sustained CR, 19 (29%) relapsed and 35% had chronic disease. CR was significantly associated with age > 70 years or no MM involvement. No prognostic immunological factor was identified. CONCLUSIONS Patients with LABD who are < 70 years old and have MM involvement are at risk for chronic evolution.

Summary Cicatricial pemphigoid is a rare autoimmune blistering disease of the elderly. It predominantly affects the mucosae, causing pain and scarring. The target antigen is within the lamina lucida of the basement membrane zone. Potential complications of systemic steroid and other immunosuppressive therapy have prompted trials of other means of treatment. We describe a series of seven patients treated with minocycline, six of whom derived sustained alleviation of orodynia. Four patients developed hyperpigmentation, and two complained of gastrointestinal discomfort which necessitated cessation of minocycline. Complete steroid withdrawal was achieved in two cases. Neither the disease progression nor the response to treatment was influenced by the immunoglobulin isotype or titre. The role of minocycline as a useful adjunct to therapy is discussed.

Fifty-nine patients with linear IgA disease, 24 with onset in childhood and 35 with adult onset, were studied. Sera from all patients were tested by indirect immunofluorescence, using as substrates intact normal skin and normal skin which had been split through the lamina lucida region of the basement membrane zone by suction and by prolonged incubation with molar NaCl. This enabled the site of the target antigen for the circulating IgA antibodies to be determined. The sites of deposition of the IgA antibodies in vivo were detected by raising a suction blister in eight patients, and splitting seven patients' biopsies by prolonged incubation with molar NaCl. Eighteen sera were positive with intact skin, and 34 with split skin. Twenty-nine sera were positive with suction blisters as substrate; 14 bound to the epidermal aspect of the split skin, seven in a combined pattern (binding to the epidermis and dermis) and six to the dermal aspect. Thirty-one sera bound to salt-split skin, 24 to the epidermal side and seven on the dermal side. There was discordance between the two methods of skin splitting in 15 sera. Seven sera gave a combined pattern with suction but with salt-split skin, five of these bound epidermally, one was dermal, and one negative. Five sera showed epidermal binding on salt-split skin and were negative on suction blisters, and the reverse was seen with one serum. Two sera gave variable results on suction blisters. Direct immunofluorescence studies showed dermal binding on all eight patients with suction blisters, and epidermal binding in four and dermal binding in three patients with salt splitting.(ABSTRACT TRUNCATED AT 250 WORDS)

Based on electron microscopic observations, in the 1970s it was assumed that the hemidesmosome–anchoring filaments–anchoring fibrils complex mediated the stable adhesion of the epithelium to the basement membrane, and that the external surface of the hemidesmosome carried a receptor for unidentified matrix component(s) responsible for hemidesmosome adhesion. However, the chemical nature of the hemidesmosome and that of the anchoring devices was unknown. A crucial milestone was the identification of collagen VII and laminin 5 as the specialized basement membrane zone proteins that compose the anchoring fibrils and anchoring filaments, respectively. In 1986, Sakai et al. (1986) reported the development of a monoclonal antibody specific for collagen VII that was immunoreactive with all the tissues known to synthesize anchoring fibrils. Furthermore, ultrastructural immunolocalization of the monoclonal antibody disclosed the exclusive presence of collagen VII within the anchoring fibrils, and visualization of the antibody–collagen VII complex after rotary shadowing revealed the dimeric nature of the collagen VII molecules. The authors also provided evidence that anchoring fibrils are unstaggered parallel arrays of dimeric collagen VII, which is mainly synthesized by the epithelium. The assumption that collagen VII is the major component of anchoring fibrils was confirmed 3 years later by Bruckner-Tuderman et al. (1989). Using immunoelectron microscopy and immunochemistry approaches, these authors clearly demonstrated that the absence of anchoring fibrils in patients with dystrophic epidermolysis bullosa, a severe inherited skinblistering disease characterized by an extreme fragility of upper papillary dermis, correlated with an impaired expression of collagen VII. This study unveiled the pivotal role of collagen VII in the stability of dermal–epidermal junction and for the first time designated a gene encoding a basement membrane protein, collagen VII, as a candidate in a skin condition characterized by disadhesion of the epithelial cell/basement membrane from the underlying mesenchyme. In 1991, an elegant study by Rousselle et al. (1991) reported the identification of kalinin, a basement membrane protein synthesized by the keratinocytes that predominantly colocalizes with anchoring filaments. Kalinin was characterized using a combination of techniques (immunoelectron microscopy, biochemistry and cell biology) that disclosed the functional complexity of this adhesion ligand, which strongly modulates the motility of epithelial cells. Interestingly, the tissue distribution and the electrophoretic pattern of kalinin resembled that of BM600 (later renamed nicein), a heterotrimeric protein of the dermal– epidermal junction described by Verrando et al. (1991). BM600–nicein was detected in all the epithelial basement membranes that harbor hemidesmosomes, but its expression was found to be strongly reduced in patients with junctional epidermolysis bullosa. This inherited skin disease is characterized by blister formation correlated with the perturbation of hemidesmosomal integrity and cleavage within the lamina lucida of the dermal–epidermal junction, which is where the anchoring filaments are localized. In 1993, a timely work by Marinkovich et al. (1993) provided extensive biochemical and immunologic evidence that kalinin and nicein are identical. This breakthrough clarified the overall picture drawn from the increasing information on the diverse roles of kalinin–nicein (later renamed laminin 5) in cell adhesion, proliferation and differentiation. The papers highlighted above underscore the transition from the ‘classic’ approaches of cutaneous biology, which was essentially based on morphologic analyses, to studies relying on tools and methods allowing exploration at the molecular and genetic levels for the function(s) of the specific components of the cutaneous basement membrane involved in the different forms of epidermolysis bullosa.

Epidermolysis bullosa (EB) is a genetic disorder, characterized by blisters on skin and mucosal surfaces even upon light mechanical damage. EB is caused by genetic mutations in at least seven proteins on the basement membrane zone, which is the boundary between the epidermis and the dermis. There are many types of EB differing in clinical and genetic aspects, and the prognosis varies depending on the EB type. There are largely three types of EB, categorized by the electron-microscopic location of the blisters. The blisters form within the epidermis in epidermolysis bullosa simplex, in the lamina lucida in junctional epidermolysis bullosa, and just beneath the basal lamina in dystrophic epidermolysis bullosa. To date, there is no medication or treatment that cures EB or completely prevents the blisters, so generally symptomatic treatment is performed. EB patients must always be cautious, for blisters can form at the slightest injuries, and the patients must be dealt with gently. Injuries and infections have to be prevented and treated, and deficient nutrients must be supplied during dental treatment period. Some patients may experience pain when swallowing food or dental treatment due to blisters and resulting scars in the mouth, pharynx, and esophagus. Recently, two pediatric patients were diagnosed with EB at Pusan National University Hospital and visited the Department of Pediatric Dentistry for oral care and dental treatment. The treatment results are reported here. [J Korean Dis Oral Health Vol.8, No.2: 122-126, Dec 2012]

The ultrastructural characteristics and immunolocalization of in vivo bound immunoglobulin G (IgG) in skin affected by anti-p200 pemphigoid have not been elucidated. To give insight into the mechanism of blister formation we report a new case of anti-p200 pemphigoid, studied with stage-oriented morphological analysis and immunoelectron microscopy. Skin biopsy specimens were evaluated ultrastructurally and histologically with immunohistochemistry. By observing the nonblister site, the blister edge and centre of the blister, we determined that neutrophil infiltration increases gradually at the dermoepidermal junction in association with the destruction of type IV collagen. Ultrastructurally, many neutrophils were observed under the lamina densa, with vacuole formation in the dermis. At the periphery of the blister, the lamina densa became fragmented and was observed either at the roof or the floor of the blister. At the centre of the blister, the lamina densa was mainly observed at the blister floor. Postembedding immunoelectron microscopy demonstrated that the IgG, bound in vivo, localized at the lamina lucida, while the area beneath the hemidesmosomes was spared. Together with the early involvement of neutrophils and the destruction of the basal lamina, we suggest that the binding of autoantibodies to the nonhemidesmosomal lamina lucida may induce inflammation with neutrophils, resulting in blister formation.

This report describes the microscopical, immunohistochemical and ultrastructural findings in the first ovine cases of the Herlitz type of inherited junctional epidermolysis bullosa. Sixteen German black-headed mutton lambs and one crossbred lamb had blisters and ulceration of the skin and mucous membranes in addition to alterations of the horn of the hooves. Microscopically, there was separation of the dermoepidermal junction, which was confirmed to be located in the lamina lucida of the basement membrane by electron microscopy. In areas of subepidermal splitting the hemidesmosomes were missing and in adjacent areas they appeared to be rudimentary and reduced in number. Immunohistochemistry for laminin 5 revealed a markedly reduced expression of this molecule on the dermal side of the blisters, while expression of collagen VII was normal.

A case of epidermolysis bullosa in a calf descendent from a Gir bull and a Gir crossbreed cow is reported. The calf presented with exungulation of all hooves, widespread erosions and crusts on the skin, and ulcers in the oral cavity. Histologically, the skin showed subepidermal separation with clefts occasionally filled with eosinophilic clear fluid, cellular debris, or neutrophils. Ultrastructurally, there was epidermal–dermal separation at the level of the lamina lucida, with the lamina densa attached to the papillary dermis. The hemidesmosomes were poorly defined and small. The clinical, histological, and ultrastructural findings are characteristic of junctional epidermolysis bullosa.

Background Junctional epidermolysis bullosa, type Herlitz (JEB-H) is a lethal, autosomal recessive blistering disease caused by null mutations in the genes coding for the lamina lucida/densa adhesion protein laminin-332 (LAMB3, LAMA3 and LAMC2). Objectives To present the diagnostic features and molecular analyses of all 22 patients with JEB-H in the Dutch Epidermolysis Bullosa Registry between 1988 and 2011, and to calculate the disease incidence and carrier frequency in the Netherlands. Methods All patients were analysed with immunofluorescence antigen mapping (IF), electron microscopy (EM) and molecular analysis. Results The mean lifespan of our patients with JEB-H was 5 8 months (range 0.5-32.6). IF showed absent (91%) or strongly reduced (9%) staining for laminin332 with monoclonal antibody GB3. In EM the hemidesmosomes and sub-basal dense plates were hypoplastic or absent. We identified mutations in all 22 patients: in 19 we found LAMB3 mutations, in two LAMA3 mutations, and in one LAMC2 mutations. We found three novel splice site mutations in LAMB3: (i) c. 292A> G resulting in an out-of-frame skip of exon 3 and a premature termination codon (PTC); (ii) c. 1289-2_1296del10 leading to an out-of-frame skip of exon 12 and a PTC; and (iii) c. 3228+ 1G> T leading to an exon 21 skip. Conclusions All diagnostic tools should be evaluated to clarify the diagnosis of JEBH. We have identified 11 different mutations in 22 patients with JEB-H, three of them novel. In the Netherlands the incidence rate of JEB-H is 4.0 per one million live births. The carrier frequency of a JEB-H mutation in the Dutch population is 1 in 249.

Epidermolysis bullosa (EB) is classified into the three major subtypes depending on the level of skin cleavage within the epidermal keratinocyte or basement membrane zone. Tissue separation occurs within the intraepidermal cytoplasm of the basal keratinocyte, through the lamina lucida, or in sublamina densa regions of the basal lamina (basement membrane) in EB simplex, junctional EB, and dystrophic EB, respectively. Transmission electron microscopy (TEM) is an effective method for determining the level of tissue separation and hemidesmosome (HD) and anchoring fibril morphology if performed by experienced operators, and has proven to be a powerful technique for the diagnosis of new EB patients. Recent advances in genetic and immunofluorescence studies have enabled us to diagnose EB more easily and with greater accuracy. This contribution reviews TEM findings in the EB subtypes and discusses the importance of observations in the molecular morphology of HD and basement membrane associated structures.

Background/Objectives: The basement membrane zone (BMZ) is an anatomically defined region present in all types of skin and mucosa, linking the epithelium to the mesenchyme with a complex structure to provide adhesion. Altered antigenic expression of the BMZ is implicated in interface dermatoses, and the BMZ is targeted by autoantibodies in subepidermal immunobullous dermatoses. This study aims to compare the antigenic expression of the BMZ and the dermal extracellular matrix in female genital skin and mucosa and amnion, with non-reproductive skin and mucosa. Methods: An indirect immunofluorescence technique was used to compare the antigenic expression of hemidesmosome, lamina lucida, anchoring filaments, lamina densa, anchoring fibrils and extracellular matrix in samples of non-reproductive skin (three), oral mucosa (three), vulval skin (two), vagina (three) and amnion (four). Results: Antigenic expression was similar in the stratified epithelium of reproductive and non-reproductive skin and mucosa, but differed in the simple cuboidal epithelium of amnion, which had reduced expression of dermal-associated antigens. Conclusions: The BMZ and dermal extracellular matrix of vagina and vulva are very similar to those of non-reproductive skin and mucosa despite their various functions, but differs from amnion. Their antigenic expression does not fully account for the anatomical distribution of immunobullous and interface dermatoses.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited rare diseases, which are characterized by trauma-induced blister formation of the skin and mucosa. The underlying cause is a functional deficiency of structural proteins of the epidermis or the dermis. Depending on the level of the blister formation, EB is divided into EB simplex (intra-epidermal), junctional EB (within the lamina lucida), dystrophic EB (below the lamina lucida) and Kindler syndrome (variable level of split formation). Besides different distinct blister formation and pain symptoms secondary problems like anaemia, oesophageal stenosis, cardiomyopathy or squamous cell carcinoma may occur. Since causal therapies are not available strict prevention of friction and trauma is essential to avoid blister formation. Anaesthesia challenges exist in the field of bedding procedures, care of the skin, monitoring, airway management und analgesia. This article gives a review over the EB and highlights in detail the corresponding anaesthesia characteristics.

The case of a male neonate of 41 weeks’ gestation who developed blistering of the skin immediately after birth is described. His parents were consanguineous Tunisians. Electron microscopy of a cutaneous biopsy showed skin cleavage within the lamina lucida and immunoepitope mapping revealed a complete absence of laminin 332 expression. These findings referred to the diagnosis of junctional epidermolysis bullosa (JEB) Herlitz type. The neonate died at 3 months of age due to sepsis. Molecular analysis of laminin 332 chain genes LAMA3, LAMB3 and LAMC2 disclosed a novel homozygous nonsense mutation in LAMA3 (p.Y955X). Clinical and laboratory analyses are essential for the diagnosis of JEB subtypes, and molecular analysis screening is crucial to manage a new pregnancy in families with suspected cases of JEB.

BackgroundReplacing mutant skin in epidermolysis bullosa (EB) by epithelial sheets of transduced autologous keratinocytes is the essential surgical step of ex vivo gene therapy. The same applies for revertant cell therapy in which epithelial sheets of revertant autologous keratinocytes are used. Revertant cells can be found in patches of normal skin in patients with junctional EB (JEB) due to revertant mosaicism caused by in vivo reversions.ObjectivesTo develop a technique of adhesive tape stripping as a method for epidermis removal to prepare the acceptor site for revertant cell therapy in a patient with revertant mosaic JEB.MethodsWe performed revertant cell therapy on a patient with mosaic type XVII collagen-deficient non-Herlitz JEB. Skin biopsies were taken from revertant skin on the wrist. Graft production took place on a 3T3-J2 feeder layer resulting in two 6 x 7 cm grafts. An innovative method that uses the pathological plane of least resistance of JEB skin was developed to prepare the acceptor site. A polyacrylate adhesive plaster was placed on the skin and then pulled off with the epidermis.ResultsThe epidermis was easily removed with the plaster. The skin separated at the level of the lamina lucida, leaving a bloodless wound bed of naked lamina densa. Transplantation was successful; the acceptor site healed without scarring. However, blistering could be provoked. The functional repair was not achieved due to the low percentage of revertant cells in the graft.ConclusionsWe conclude that adhesive stripping is a simple, effective and almost painless procedure for removing epidermis for ex vivo cell therapy in EB.

Purpose To evaluate the effect of mechanical epithelial separation with an epikeratome on the histologic ultrastructure of epithelial flaps and stromal beds from human corneas. Setting Departments of Ophthalmology, Osaka University Medical School, Osaka, and Tohoku University School of Medicine, Sendai, and Institute of Advanced Biomedical Engineering and Science and Medical Research Institute, Tokyo Women's Medical University, Tokyo, Japan. Methods Eye-bank eyes were deepithelialized using an Epi-K epikeratome. Epithelial flaps and stromal beds were assessed by light and electron microscopy. Immunofluorescence staining for types IV and VII collagens, integrins α6 and β4, and laminin 5 was also performed. Results Four eyes were evaluated. On scanning electron microscopy, the cleavage planes of epithelial flaps and stromal beds were relatively smooth. On transmission electron microscopy, epithelial flaps were separated partially within the lamina fibroreticularis and partially within the lamina lucida. Immunofluorescence showed positive staining for type VII collagen and discontinuous staining for type IV collagen in stromal beds. Discontinuous linear staining for types IV and VII collagens was observed in epithelial flaps. Staining for integrins α6 and β4 was positive in some regions and discontinuous in other regions of epithelial flaps. In stromal beds, integrins α6 and β4 had a patchy expression pattern. Staining for laminin 5 was intermittently positive along the basal side of epithelial flaps and stromal beds. Conclusions Epithelial flaps created with an epikeratome were mechanically separated partly within the lamina fibroreticularis and partly within the lamina lucida. Stromal beds had relatively smooth surfaces with no obvious trauma to Bowman layer.

The corneal epithelium adheres to the stroma through a series of linked structures termed collectively the adhesion complex. These structures include; intermediate filaments (keratin filaments) which are linked to the hemidesmosome; the hemidesmosome; the anchoring filaments which extend from the membrane at the hemidesmosome through the lamina lucida to the lamina densa region of the basement membrane; the anchoring fibrils which insert into the basement membrane from the stromal side; and the anchoring plaque on which anchoring fibrils terminate distal from their insertion on the basement membrane. Upon wounding, basal cells of the corneal epithelium disassemble their hemidesmosomes. During migration, the membranes along the wound bed exhibit a different kind of adhesion junction, the focal contact. This junction is present primarily in cells of the leading edge of migration and may be the provisional adhesion junction used by epithelial sheet moving to cover a wound.

Purpose To evaluate the cleavage plane, corneal cytoarchitecture, and cell vitality of separated corneal epithelial sheets created with 3 commonly used microkeratomes. Setting Laboratories of the Regensburg University Medical Center, Regensburg, Germany. Methods Mechanical separation of the epithelial layer in 10 porcine eyes and 2 human eyes was performed with 3 different microkeratomes (Amadeus II, Zyoptix XP, Epivision). Five of 10 porcine corneas and the 2 human corneas treated with each microkeratome were processed for histology, electron microscopy, and immunohistochemistry. In 5 of 10 porcine corneas, the corneal epithelial sheets were separated from the globe and cell vitality was assessed with the trypan blue dye vitality test. Results A reproducible epithelial separation with a smooth surface was achieved in all eyes. The cleavage plane was located between the lamina lucida and the lamina densa. Damage to epithelial cells was mainly limited to the cut margins. Conclusions Mechanical separation of the epithelial sheet in epithelial laser in situ keratomileusis (epi-LASIK) was safe and reproducible with all evaluated microkeratomes. Immunohistochemistry and electron microscopy showed the cleavage plane in epi-LASIK was between the basal epithelium and the basement membrane at the level of the lamina lucida.

Background Kindler syndrome (KS) is a rare genodermatosis characterized by four major features (acral blisters, photosensitivity, poikiloderma, and cutaneous atrophy) and many associated findings. The diagnosis of KS includes clinical features, ultrastructural findings, and, recently, immunostaining and genetic studies. Varying degrees of severity of the syndrome have been described. Methods Five patients with clinical features consistent with KS were included in this study. All patients were subjected to histopathologic and ultrastructural studies. Results Cases 1 and 2 presented with severe major features, severe mucosal involvement, and many other associated findings. Case 3 presented with severe major features, but mild and limited mucosal involvement and other associated findings. Cases 4 and 5 showed mild major features and few other findings. Histopathology revealed nonspecific poikiloderma. Marked thickening of the lamina densa and splitting of the lamina lucida were the main ultrastructural findings. Conclusion KS may be classified into mild, moderate, and severe according to the severity of the major features and mucosal involvement. Because histopathologic and ultrastructural findings are not pathognomonic, clinical features remain the mainstay for the diagnosis of KS, and the need for immunostaining with kindlin antibody and genetic studies may be restricted to early cases with incomplete features.