An ex vivo RNA trans-splicing strategy to correct human generalized severe epidermolysis bullosa simplex.

BACKGROUND: Generalized severe epidermolysis bullosa simplex (EBS-gen sev) is a genetic blistering skin disease in which autosomal dominant mutations in either the keratin KRT5 or KRT14 genes lead to impaired function of the intermediate filament cytoskeleton in the basal epidermis. Here we present an ex vivo RNA trans-splicing-based therapeutic approach to correct the phenotype. OBJECTIVES: To correct a mutation within exon 1 of the KRT14 gene, using a 5'-trans-splicing approach, where any mutation within the first seven exons could be replaced by a single therapeutic molecule. METHODS: A therapeutic RNA trans-splicing molecule containing wild-type exons 1-7 was stably transduced into an EBS patient-derived keratinocyte line. Trans-splicing was confirmed via reverse-transcriptase polymerase chain reaction, Western blotting and immunofluorescence microscopy. Skin equivalents generated from corrected keratinocytes were grafted onto nude mice and analysed about 8 weeks post-transplantation for regular epidermal stratification, trans-splicing-induced green fluorescent protein expression and blistering. RESULTS: Transplanted skin equivalents generated from trans-splicing-corrected patient keratinocytes showed a stable and blister-free epidermis. KRT14 correction disrupted EBS-gen sev-associated proinflammatory signalling, as shown at the mRNA and protein levels. Disruption of the pathogenic feedback loop in addition to overall downregulation of KRT14 expression highlighted the effect of KRT14 correction on the EBS pathomechanism. CONCLUSIONS: Our data demonstrate that trans-splicing-mediated mRNA therapy is an effective method for the correction of dominantly inherited KRT14 mutations at the transcriptional level. This results in the rescue of the EBS-gen sev phenotype and stabilization of the epidermis in a xenograft mouse model.

Danon disease: case report and detection of new mutation.

Danon 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.

Characteristic immunohistochemical and ultrastructural findings indicate that Kindler's syndrome is an apoptotic skin disorder.

BACKGROUND: Kindler's syndrome is a rare genodermatosis mainly characterized by the onset of skin blistering in early childhood, web formation of fingers and toes, photosensitivity, and progressive poikiloderma. There is still debate whether this disease represents a distinctive entity in the spectrum of congenital bullous poikilodermas or a variant of dystrophic epidermolysis bullosa. OBJECTIVE: To evaluate the recently proposed and debated characteristic immunohistochemical and ultrastructural features of Kindler's syndrome. PATIENT/METHODS: Immunofluorescence (IF) antigen mapping and transmission electron microscopy (TEM) were performed on a skin specimen from non-sun-exposed inner aspect of the upper arm of a 49-year-old patient with characteristic clinical features of Kindler's syndrome. RESULTS: IF studies revealed focally an extensively broadened, partly reticular staining pattern in the dermoepidermal basement membrane zone (BMZ) with antibodies against laminin-5 and type IV as well as type VII collagen. Anti-alpha6 and beta4 integrin staining revealed small gaps in the linear reactivity in the BMZ. Abundant keratin bodies, as detected by anti-immunoglobulin M (IgM) staining, were focally present in the dermis, indicating prominent epidermal apoptosis. This was verified by a histochemical apoptosis stain [terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) reaction]. Transmission electron microscopic examination showed manifold reduplications of the lamina densa (with attached anchoring fibrils) as well as a keratin body surrounded by a fibroblast in the upper dermis. CONCLUSION: We present characteristic immunohistochemical and ultrastructural features of Kindler's syndrome identical to those described by Shimizu et al. and provide evidence that Kindler's syndrome might primarily be an apoptotic disorder of basal keratinocytes.

Recurrent keratoconus in a patient with Leber congenital amaurosis.

PURPOSE: Clinical history of a 17-year-old patient with Leber congenital amaurosis (LCA) with histologically proven recurrent keratoconus (KC) two years after corneal transplantation in one eye and a recurrence-like appearance with a more global contour on the other eye four years after corneal grafting is reported. The possible mechanisms for this recurrence are discussed in light of the fact that this is, to the best of our knowledge, the first penetrating keratoplasty reported in LCA. METHODS: Computerized videokeratography (CVKG) and specular microscopy were performed preoperatively. The patient underwent regrafting, and the excised corneal button was examined by light microscopy and transmission electron microscopy. RESULTS: Analysis of CVKG showed a keratoconus-like pattern on the right eye, with the left eye demonstrating the aspects usually seen in keratoglobus. Histologic examination revealed the features usually observed in progressed keratoconus. CONCLUSION: Recurrence of keratoconus in a graft has not yet been described after such a short time until now. A "true" recurrence of the disease is postulated; it could be caused by an "aggressive" genetic factor that also leads to the frequent KC in patients with LCA. This mechanism also could explain the high incidence and rapid progress of KC in this disease.

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.

Light and transmission electron microscopic studies following frontal sinus obliteration with ionomer cement in cats.

Osteoplastic frontal sinus surgery in combination with sinus obliteration can be performed for various indications, including chronic sinusitis, frontal sinus trauma and removal of osteomas. In an experimental study using cats, the mucous lining of the frontal sinus was removed, the nasofrontal duct sealed with semifluid ionomer cement and the cavity filled with Ionogran, a solid and porous bone substitute based on ionomer cement. Histological investigations up to 1 year after surgery showed increasing sinus obliteration by regenerating bone, starting from the sinus wall, and formation of connective tissue between the cement grains. There was no evidence for mucosal regeneration. Electron microscopic examination of the interface between the cement and connective tissue revealed mesenchymal cells, collagen fibers and areas of mineralization in close contact with the implant material. Newly formed connective tissue matrix in intimate contact with the cement was a good indication for biocompatibility of the material and is a possible explanation for the implant's solid adhesion to bone. The present findings indicate that Ionogran is a suitable alloplastic material for experimental frontal sinus obliteration in cats.

Stereologic parameters of nuclear ultrastructure as markers of the steroid receptor status in breast cancer epithelium.

The nuclear specific surface density (Sv/Vv), mean nuclear area (A) and numerical density (Nv) of tumor cell nuclei from 30 primary invasive female breast carcinomas with known concentrations of estrogen and progesterone receptors (ER and PR), were morphometrically analyzed at the ultrastructural level. It was found that carcinomas with concordant positive ER and PR status contained significantly higher number of smaller nuclei per volume unit of epithelium (mean Nv = 1.5 x 10(6) mm-3, mean A = 27 microns 2), than carcinomas of negative concordant receptor status (mean Nv = 0.8 x 10(6) mm-3, mean = 37 microns 2). Tumor cell nuclei of the former frequently displayed an elliptic shape (mean Sv/Vv = 1.16 microns-1), and had deeply invaginated surfaces, whereas nuclei of the latter were more frequently ball-shaped and exhibited a smooth-surface (mean Sv/Vv = 0.88 microns-1). The numerical density Nv (NUC/EPI) of tumor cell nuclei turned out to be a most reliable morphological marker of the concordant ER and PR status (overall efficiency = 84%, p < 0.01). The nuclear surface and volume ratio and mean nuclear area also appear to be reliable markers for terminal stages of the biochemical differentiation of breast carcinoma (overall efficiency = 79%, p < 0.05).

Individual differences between nuclear parameters of normal and malignant breast epithelium.

This study attempts to estimate the status of estrogen and progesterone receptors (ER and PR) in tissue of invasive breast carcinoma. Identical stereologic parameters of nuclear ultrastructure, of normal and malignant epithelium in the same breast were compared. Samples of normal and malignant epithelium of 30 surgically amputated breasts were analysed morphometrically. The concentration of ER and PR in the malignant epithelium was estimated by means of the dextran-coated charcoal (DCC) method. We measured the mean nuclear area (A), nuclear specific density (Sv/Vv) and nuclear numerical density (Nv) of epithelium. The comparison of such models of normal and malignant epithelium nuclear ultrastructure of the same breast demonstrated: a) the difference between Sv/Vv of nuclei very sensitively and specifically marks the ER status in malignant tissue (overall efficiency = 80%, p < 0.01), and b) the difference between A, Sv/Vv and Nv marks the concordant ER and PR status with identical overall efficiency. Minimal individual differences in size, surface structure and shape, observed between nuclei of malignant epithelium of positive concordant ER and PR status and nuclei of normal epithelium demonstrated that the highest morphological similarity is associated with biochemical similarity of cells exposed to the identical media of steroid hormones.

[Autonomic and peptidergic innervation of the human larynx]. / Die autonome und peptiderge Innervation des menschlichen Kehlkopfes.

Autonomic and peptidergic innervation of the human larynx (vocal cords, ventricular folds, epiglottis, subglottic region and recurrent nerves) was studied by application of single and double immunocytochemistry and radioimmunoassay. In all tissues investigated, immunoreactivities for a variety of regulatory peptides were detected and included vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), helospectin, neuropeptide Y (NPY), C-flanking peptide of NPY (C-PON), calcitonin gene-related peptide (CGRP), substance P and neurokinin A. In the recurrent nerves, only a few peptide-immunoreactive nerve fibers were found. The laryngeal region of the epiglottis and the subglottic region showed characteristic corpuscular nerves containing substance P and CGRP running underneath and within the epithelium.