Gabriele Pohla-Gubo

Treatment‐resistant classical epidermolysis bullosa acquisita responding to rituximab

SIR, Epidermolysis bullosa acquisita (EBA) is a chronic severe subepidermal blistering disease of the skin and mucous membranes characterized by marked resistance to topical and systemic therapy. We report a patient with a 6-year history of EBA refractory to any immunosuppressive medication (Fig. 1), who responded well to a treatment with the anti-CD20 monoclonal antibody rituximab. A 71-year-old woman presented with blisters and erosions on mechanically strained areas including hands, elbows and feet, that healed with scarring and milia formation and resulted in nail dystrophy, loss of toenails and scarring alopecia. Involvement of mucous membranes was initially confined to the oral cavity (Fig. 2), and later also occurred on oesophageal and pharyngolaryngeal mucosa. Histological examination of perilesional skin showed subepidermal blister formation and a mild infiltrate composed of lymphocytes and neutrophils. Conventional direct immunofluorescence of perilesional skin detected linear deposition of IgG and C3 at the dermoepidermal junction. No IgA or IgM deposits were found. Indirect immunofluorescence (IIF) with the patient’s serum on salt-split skin revealed an exclusive dermal binding of circulating IgG antibasement membrane zone (BMZ) antibodies at a titre of 1 : 2560. These antibodies recognized a band at 290 kDa, and a 145-kDa band corresponding to the a-chain of collagen VII in immunoblotting on keratinocyte extracts. Thus, the diagnosis of a classical mechanobullous type of EBA was made. Over a 6-year period several immunosuppressive agents, including corticosteroids, azathioprine, ciclosporin, plasmapheresis, mycophenolate mofetil, colchicine, gold preparations, highand low-dose immunoglobulins and daclizumab were administered but none of them resulted in prolonged clinical benefit (Fig. 1). Then in 2004, disease activity increased, mainly due to massive erosions of the pharyngolaryngeal and oesophageal mucosa, which resulted in upper and lower oesophageal stenoses. The anti-BMZ titre (1 : 320) was at this time point lower than in the previous years of the disease. The patient required endoscopic dilatation of oesophageal stenoses, percutaneous endoscopic gastrostomy and tracheotomy because of severe obstruction of the upper airways. We therefore decided to initiate an anti-CD20 monoclonal antibody therapy attempt. Concomitantly, vaccination against influenza A and Streptococcus pneumoniae was carried out. Five rituximab infusions were given intravenously at a frequency of one infusion per week. Because of the poor health condition of our patient and to avoid potential side-effects we used a reduced dose of 144 mg m each time. Concomitant immunosuppressive therapy was azathioprine 2Æ0 mg kg daily. Our patient tolerated the rituximab therapy well with absence of any side-effects or infections. Four weeks after the first rituximab infusion the patient showed marked improvement of the skin and mucous membrane condition (Fig. 2b) and concomitant therapy of azathioprine was discontinued. Transiently, IIF became negative and a repeated immunoblotting study could no longer detect antibodies reactive with the

A Compound Heterozygous One Amino-Acid Insertion/Nonsense Mutation in the Plectin Gene Causes Epidermolysis Bullosa Simplex with Plectin Deficiency

Plectin is a cytoskeleton linker protein expressed in a variety of tissues including skin, muscle, and nerves. Mutations in its gene are associated with epidermolysis bullosa simplex with late-onset muscular dystrophy. Whereas in most of these patients the pathogenic events are mediated by nonsense-mediated mRNA decay, the consequences of an in-frame mutation are less clear. We analyzed a patient with compound heterozygosity for a 3-bp insertion at position 1287 leading to the insertion of leucine as well as the missense mutation Q1518X leading to a stop codon. The presence of plectin mRNA was demonstrated by a RNase protection assay. However, a marked reduction of plectin protein was found using immunofluorescence microscopy of the patients skin and Western blot analysis of the patients cultured keratinocytes. The loss of plectin protein was associated with morphological alterations in plectin-containing structures of the dermo-epidermal junction, in skeletal muscle, and in nerves as detected by electron microscopy. In an in vitro overlay assay using recombinant plectin peptides spanning exons 2 to 15 the insertion of leucine resulted in markedly increased self-aggregation of plectin peptides. These results describe for the first time the functional consequences of an in-frame insertion mutation in humans.

Pathogenic mechanisms in epidermolysis bullosa naevi.

Epidermolysis bullosa naevi are large, eruptive melanocytic naevi which frequently arise in areas of former blisters in patients suffering from inherited epidermolysis bullosa. Morphologically, these naevi are similar to malignant melanoma, although so far no malignant transformation has been observed. To investigate the pathogenesis of these moles we documented their clinical evolution and their histopathological and immunocytological characteristics in three patients with epidermolysis bullosa. Clinically, we observed signs of malignant transformation, such as explosive growth and the occurrence of satellite lesions of epidermolysis bullosa naevi. However, malignant melanoma was excluded by histopathological evaluation. In addition, we evaluated the concentrations of various factors known to stimulate melanocyte growth in blister fluid. Human interleukin 8, basic fibroblast growth factor, human hepatocyte growth factor, GM-CSF, leukotriene B4 and prostaglandin E2 revealed concentrations comparable with the levels in inflammatory blisters. We were able to detect individual melanocytes/naevus cells in blister fluid from a blister over an epidermolysis bullosa naevus. The factors detected in the blister fluid might therefore promote the proliferation, migration and melanogenesis of disconnected melanocytes/naevus cells representing the basis of the highly dynamic appearance of epidermolysis bullosa naevi.

Ocular involvement in IgA-epidermolysis bullosa acquisita.

Epidermolysis bullosa acquisita (EBA) is an autoimmune bullous disease with frequent ocular involvement, but visual loss is rare. In contrast, EBA patients with predominant IgA autoantibodies more frequently develop severe ocular involvement, which tends to be refractory to therapy. We report two patients with ‘IgA–EBA’ with ocular involvement. Both initially presented with a generalized bullous disease, and direct immunofluorescence microscopy demonstrated IgA in the basement membrane zone of the skin, and in the conjunctiva and cornea of patient 1. On salt‐split patient skin, IgA was found predominantly on the dermal side of the artificial split in both patients. Direct immunoelectron microscopy demonstrated IgA below the lamina densa in close association with the anchoring fibrils in both patients. In patient 1, who had a prolonged course of the disease, the skin disorder responded well to treatment with cyclosporin, but the ocular involvement ended in bilateral blindness despite repeated surgical treatment. In patient 2, the blister formation and scarring conjunctivitis was stopped by a combination of prednisolone and colchicine. These patients show that in subepithelial blistering diseases, early delineation of disease nosology is critical to detect subtypes with severe ocular involvement such as ‘IgA–EBA’. In addition, colchicine may be a valuable alternative in the treatment of EBA with ocular involvement.

Histopathology of human corneas after amniotic membrane and limbal stem cell transplantation for severe chemical burn

Purpose. To describe the histopathologic changes in the cornea following amniotic membrane transplantation (AMT) combined with limbal transplantation. Methods. Four eyes with complete limbal stem cell deficiency after severe chemical burn underwent AMT with either a living-related conjunctival limbal allograft (lr-CLAL) (three eyes) or a conjunctival limbal autograft (CLAU) (one eye) for ocular surface reconstruction. Penetrating keratoplasty was performed several months after the initial procedure for further visual rehabilitation. Mean follow up time was 20 months. Light and transmission electron microscopy (TEM) and indirect immunofluorescence microscopy of the excised corneal buttons were performed. Results. All specimens displayed a multilayered epithelium without conjunctival goblet cells over the entire corneal surface. Basal epithelial cells demonstrated a firm connection to the remnants of the transplanted amniotic membrane (AM), which at some places appeared to be in a state of “modification” or “remodeling” in the collagen layers. The basement membrane zone displayed a positive staining when using antibodies against collagen IV and VII, integrin &agr;6 and &bgr;4, laminin 5, and bullous pemphigoid antigen 2. Remnants of the AM in the specimen showed staining of collagen IV, which was found also in cross-sections of cryopreserved AM. The recipients Bowmans membranes that were only partially present after the initial trauma were significantly disturbed. Conclusion. Within the time frame studied, the transplanted AM apparently survives and integrates into the host tissue being modified or remodeled by recipient cells. AMT in combination with a CLAU or lr-CLAL is a useful technique in promoting a rapid and stable reepithelialization of a corneal surface following severe chemical or thermal damage.

Diagnostic Pitfalls in Newborns and Babies with Blisters and Erosions

Establishing the correct diagnosis in newborns presenting with blisters and erosions is not always a straightforward process. Many different disease entities including acquired (i.e., infectious, immunobullous, traumatic) and inherited disorders have to be taken into consideration. Similarities in clinical appearance, colonization and/or superinfections of preexisting skin lesions, as well as the absence of late changes in the neonate often pose significant diagnostic challenges. In this paper we discuss by giving examples the process of making an accurate diagnosis of blistering skin diseases in the neonatal period on the basis of a diagnostic algorithm. In addition, we provide an overview of the rational use and the limitations of laboratory procedures such as microbial testing, routine light microscopy, immunofluorescence antigen mapping, transmission electron microscopy, and molecular genetic analysis.

Zahnveränderung bei junktionaler Epidermolysis bullosa – Bericht über eine Patientin mit einer Mutation im LAMB3‐Gen

During early odontogenesis the basement membrane is known to be important in epithelio-mesenchymal interactions. Mutations in the gene of one of the major structural proteins of the basement membrane such as laminin 5 might therefore be expected either to seriously compromise ameloblast differentiation and/ or interfere with normal basement-membrane formation and degradation and thus the binding of the ameloblasts to their underlying matrix. Teeth of patients suffering from junctional epidermolysis bullosa (JEB) can be severely affected by abnormal dental development and generalized or focal enamel hypoplasia. Those changes are found in 100% of individuals with JEB but the expression is variable. Beside the quantitative alterations, changes in the prismatic structure and orientation of enamel crystals are described. In addition JEB is associated with an increased risk for dental caries, caused by developmentally compromised enamel and external factors such as difficulties in maintaining oral hygiene because of oral lesions or a softer and more refined high caloric diet. Dental care includes three main strategies: Prevention by consequent oral hygiene and reduction of cariogenic nutrition is of paramount importance to minimize caries development; the restoration of enamel and dentin defects with fillings and stainless steel crowns to guarantee structure and function of teeth; and extractions of most severely affected teeth with osteolytic foci to remove continuous sources of oral infections.

Skin grafting as a therapeutic approach in pretibially restricted junctional epidermolysis bullosa.

an immunocompromised host may be coincidental. However, it is also conceivable that long-term immunosuppression and, in addition, fair complexion and a stay in the tropics represent cofactors for tumour development. Although cutaneous MFHs in RTRs are very rare, the case reported here shows that cutaneous MFH with a poor prognosis may arise after RT, especially in patients with several risk factors. To our knowledge, this is the first reported case of cutaneous MFH of the scalp in an RTR.

Unilaterale Blepharochalasis mit läsionalen IgA-Ablagerungen

ZusammenfassungEin 36-jähriger Patient entwickelte innerhalb von 10 Jahren schleichend, d. h. ohne auffällige Lidschwellungen, eine deutliche unilaterale periokuläre Hautatrophie. Neben diesem seltenen Befund der Blepharochalasis fielen stark geschlängelte arkadenförmige Blutgefäße und mikroaneurysmatische Gefäßveränderungen der Conjunctiva tarsi auf. Histologisch zeigten sich ein nahezu völliger Verlust von elastischen Fasern in der papillären und oberflächlichen retikulären Dermis, ein spärliches perivaskuläres lymphozytäres Entzündungsinfiltrat und ein diskretes Ödem. Die kontralaterale Seite war histologisch unauffällig. Immunfluoreszenzoptische Untersuchungen konnten besonders perifollikulär IgA-Ablagerungen im Bereich der elastischen Fasern nachweisen. Die Immunelektronenmikroskopie bestätigte den immunfluoreszenzoptischen Befund und wies darauf hin, dass Mikrofibrillen-assoziierte Proteine wie Fibulin oder Fibronektin möglicherweise die Zielstruktur für die Autoantikörper darstellen. Diese weitere Beschreibung einer Blepharochalasis mit IgA-Autoantikörpern präzisiert deren antigene Bindungsstellen und macht deutlich, dass sich die immunpathologischen Mechanismen auch an den Blutgefäßen der tarsalen Konjunktiva auswirken können.AbstractA 36-year-old male patient presented with unilateral periocular skin atrophy. The blepharochalasis developed without any obvious inflammation of the eyelids over the past 10 years. Interestingly, elongated blood vessels and microaneurysmatic vessel changes were found in the tarsal conjunctiva. A punch biopsy revealed a nearly complete loss of elastic fibres in the papillary and superficial reticular dermis. The contralateral side was histopathologically normal. On immunohistology IgA-deposits could be observed especially on perifollicular elastic fibres. Immunoelectronmicroscopy confirmed the diagnosis and suggested fibulin and fibronectin as potential binding sites for the autoantibodies. This further report of elastolysis in association with IgA-autoantibodies defines the autoantibody binding site in more detail and suggests that the immune mechanisms may also play a role in vessel changes of the conjunctiva.

[Hereditary blistering diseases. Symptoms, diagnosis and treatment of epidermolysis bullosa].

Hereditary epidermolysis bullosa (EB) is a term for a heterogeneous group of rare genetic disorders characterized by marked fragility of the skin and mucous membranes following minor trauma. Significant progress has been made in understanding the molecular basis ofEB, which has far-reaching implications for an improved classification with consequences for prognosis, genetic counseling, DNA-based prenatal and preimplantation testing, and the development of future treatments including gene therapy. Besides mucocutaneous changes, EB leads to a number of systemic manifestations whose management requires multidisciplinary access. Extracutaneous complications include ophthalmologic, dental, gastrointestinal, pulmonary, urogenital, hematologic, and nutritional problems. This article reviews the progress that has been made in the understanding of the molecular basis of EB, clinical aspects of major EB subtypes, and the management of patients suffering from EB, and it gives an outlook on molecular therapy projects such as gene, cell, vector, and protein therapies.Hereditary epidermolysis bullosa (EB) is a term for a heterogeneous group of rare genetic disorders characterized by marked fragility of the skin and mucous membranes following minor trauma. Significant progress has been made in understanding the molecular basis of EB, which has far-reaching implications for an improved classification with consequences for prognosis, genetic counseling, DNA-based prenatal and preimplantation testing, and the development of future treatments including gene therapy. Besides mucocutaneous changes, EB leads to a number of systemic manifestations whose management requires multidisciplinary access. Extracutaneous complications include ophthalmologic, dental, gastrointestinal, pulmonary, urogenital, hematologic, and nutritional problems. This article reviews the progress that has been made in the understanding of the molecular basis of EB, clinical aspects of major EB subtypes, and the management of patients suffering from EB, and it gives an outlook on molecular therapy projects such as gene, cell, vector, and protein therapies.