Wolfgang Muss

Deficiency of mitochondrial ATP synthase of nuclear genetic origin

We present clinical and laboratory data from 14 cases with an isolated deficiency of the mitochondrial ATP synthase (7-30% of control) caused by nuclear genetic defects. A quantitative decrease of the ATP synthase complex was documented by Blue-Native electrophoresis and Western blotting and was supported by the diminished activity of oligomycin/aurovertin-sensitive ATP hydrolysis in fibroblasts (10 cases), muscle (6 of 7 cases), and liver (one case). All patients had neonatal onset and elevated plasma lactate levels. In 12 patients investigated 3-methyl-glutaconic aciduria was detected. Seven patients died, mostly within the first weeks of life and surviving patients showed psychomotor and various degrees of mental retardation. Eleven patients had hypertrophic cardiomyopathy; other clinical signs included hypotonia, hepatomegaly, facial dysmorphism and microcephaly. This phenotype markedly differs from the severe central nervous system changes of ATP synthase disorders caused by mitochondrial DNA mutations of the ATP6 gene presenting mostly as NARP and MILS.

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.

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.

Feline orthopoxvirus infection transmitted from cat to human

We report the case of a 56-year-old female patient who presented with an inflamed, ulcerated lesion on the left side of her neck after contact (scratch) with a cat living in the patients house. Satellite lesions developed despite local treatment and parenteral clindamycin. Histopatholgic examination and the Tzanck test showed evidence of a viral infection. Subsequent transmission electron microscopy of scrap tissue and material from a fresh pustule exhibited multiple typical poxvirus particles, predominantly in remnants of scaled-off layers of degenerated keratinocytes, and virus particles in intermingled phagocytes, leading to the diagnosis of feline Orthopoxvirus (cowpox virus) infection. These results were verified by polymerase chain reaction and sequencing. Concern has been raised as to whether discontinuation of smallpox vaccine would cause an increase in Orthopoxvirus infection, but this has not yet shown to be the case.

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.

Closure of a Large Chronic Wound through Transplantation of Gene-Corrected Epidermal Stem Cells

Figure 1. Regeneration of a transgenic functional epidermis on the skin wound of the JEB patient. (a) The long-standing ulceration on the lower right leg of the patient 2 days before transplantation. (b) Western blot analysis of cell lysates (20 mg protein, 30 seconds exposure time) from (lane 1) normal control and patient keratinocyte cultures (lane 2) before and (lane 3) after gene correction, probed with a monoclonal antibody against laminin 332-b3. (lane 4) Western blot analysis of a higher amount of loaded protein (65 mg, 5 seconds exposure time) of uncorrected patient keratinocytes, and (lane 5) normal keratinocyte cultures using the same laminin-332-b3 antibody. The 75-kD band in lane 4 is consistent with the truncated laminin-332-b3 generated by the c.1903C>T; p.R635X mutation. (c) Transplantation of cultured transgenic epidermal sheets (asterisks) on the prepared wound bed. Grafts are overlaid with petrolatum gauze. (d) Initial epidermal regeneration at 14 days. (e) Complete epidermal regeneration at 3.5 months. (f) Stable epidermal regeneration at 16 months. Note crusting and erosions outside of the grafted area. JEB, junctional epidermolysis bullosa. TO THE EDITOR Generalized junctional epidermolysis bullosa (JEB) is caused by mutations in LAMA3, LAMB3, or LAMC2, which together encode laminin-332, a heterotrimeric protein consisting of a3, b3, and g2 chains (Fine et al., 2014). In nonlethal generalized intermediate JEB, laminin332 is highly reduced, and hemidesmosomes are rudimentary or completely absent, leading to blister formation within the lamina lucida of the basementmembrane uponminor trauma. The resulting chronic skin wounds invariably develop recurrent infections and scarring, which greatly impair patients’ quality of life (Fine et al., 2014; Laimer et al., 2010; Nakano et al., 2002). There is no cure for JEB; treatments are symptomatic and aimed at relieving the devastating clinical manifestations (Carulli et al., 2013). The only published evidence for the possibility of a permanent local treatment of JEB was provided by a phase I/II trial showing that autologous epidermal cultures containing geneticallymodified epidermal stem cells were able to restore a normal epidermis on a JEB patient (De Rosa et al., 2014; Mavilio et al., 2006). However, the transgenic epidermis was applied in areas still covered by a diseased but apparently functional epidermis, which was surgically removed before grafting (Mavilio et al., 2006). Although it is clear that the ideal clinical application of transgenic epidermis would aim at preventing the development of devastating chronic lesions,manypatients suffer from therapyresistant chronic ulcerations that are highly predisposed to cancer development and need timely closure (Goldberg

Distribution of two VIP-related peptides, helospectin and pituitary adenylate cyclase activating peptide (PACAP), in the human upper respiratory system

Helospectin (HS) and pituitary adenylate cyclase activating peptide (PACAP) are newly discovered peptides isolated from the salivary gland venom of the lizard Heloderma horridum and the ovine hypothalamus, respectively. They show chemical similarities to vasoactive intestinal polypeptide (VIP), appear to have similar functions and are present in gut, brain, lung, male and female genitourinary tract. In the present study, the distribution of the helospectin and PACAP-27 in the human upper respiratory system was investigated using indirect immunofluorescence and electron-microscopical ABC-pre-embedding methods. Immunohistochemistry revealed helospectin-like (HS-LI) and PACAP-like (PACAP-LI) immunoreactivity in nerve fibers in human nasal, the larynx (vocal cord, ventricular fold, epiglottis), the tongue and the soft palate mucosa. Helospectin-LI and PACAP-LI containing nerve fibers were mainly found in close association to blood vessels and glandular structures. Colocalization studies carried out by application of double immunofluorescence showed that HS and/(or) PACAP-LI coexist with VIP in apparently the same nerve fibers in the upper respiratory system, although single nerve fibers seem to exclusively express helospectin. The localization patterns of helospectin and PACAP-LI in the human upper respiratory system suggests their possible involvement in the regulation of secretory activities and local blood flow.

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.

Danon disease: Case report and detection of new mutation

SummaryDanon disease is an X-linked disorder resulting from mutations in the lysosome-associated membrane protein-2 (LAMP2) gene. We report a male patient with skeletal myopathy, mental retardation, and massive hypertrophic obstructive cardiomyopathy necessitating heart transplantation. Immunohistochemistry of skeletal muscle and leukocytes, western blot analysis of leukocytes and cardiac muscle, flow cytometry, and DNA sequencing were performed. Muscle biopsy revealed autophagic vacuolar myopathy and lack of immunohistochemically detectable LAMP-2. Diagnosis of Danon disease was confirmed by western blot analysis of myocardial tissue and peripheral blood sample of the patient showing deficiency of LAMP-2 in myocardium and leukocytes. Moreover, absence of LAMP-2 in lymphocytes, monocytes and granulocytes was shown by flow cytometric analysis. Genetic analysis of the LAMP2 gene revealed a novel 1-bp deletion at position 179 (c.179delC) at the 3′ end of exon 2, resulting in a frameshift with a premature stop codon.

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.