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.

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.

Immunocytochemical localization of estrogen and progesterone receptors in human thyroid.

The presence of steroid hormone receptors has previously been suggested in thyroid tissue by biochemical means. Our studies were designed to confirm these results and to localize the specific receptor-containing cell type using a novel immunocytochemical method. Monoclonal antibodies specific to estrogen receptors (ER) and progesterone receptors (PgR) were used to localize these steroid hormone receptors in the human thyroid gland. Frozen tissue sections from surgical specimens excised from 22 patients of both sexes with benign thyroid disease were studied. The sections were incubated with rat antiestrophilin and antiprogesterone receptor antibodies and were then exposed to rabbit anti-rat IgG and to rat peroxidase-antiperoxidase complex. The reaction product was visualized with diaminobenzidine tetrahydrochloride and hydrogen peroxide. Four specimens were positive for both ER and PgR, 16 were ER-positive and PgR-negative, and two were negative for both ER and PgR. Positive reactivity was limited to the follicular lining cell nuclei and varied from focal to diffuse. The immunohistochemical findings confirmed the presence of ER and PgR in the thyroid tissue and demonstrated for the first time that these receptors are present only in the nuclei of the lining cells of the thyroid follicle. The role of steroid hormone receptors in the thyroid in health and disease remains to be explained.

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.

Corneal lathing using the excimer laser and a computer-controlled positioning system.

PURPOSE: To present the excimer laser corneal shaping system (ELCS-S), an add-on device to the Keratom, a commercially available 193-nm excimer laser built by Schwind. METHODS: The system is designed for the preparation of donor corneas under sterile conditions using the ultraviolet laser to offer greatest possible flexibility. Lenticules for planolamellar grafting and refractive epikeratoplasty, as well as donor buttons for penetrating keratoplasty can be computer-designed by the surgeon or technician and lathed with the system. RESULTS: Using the excimer laser corneal shaping system (ELCS-S) on human donor corneas, the central surface of the epikeratoplasty lenticule exhibited only narrow, flat concentric notches corresponding to the single lathing steps. Transmission electron microscopy revealed a damage zone of less than 0.3 microm in close approximation to the treated surface. The final thickness revealed a difference of less than +/-53 microm from the intended, initially programmed value. Ultrastructural studies showed the perpendicular stromal surface of the penetrating keratoplasty buttons to be smooth with minimal protrusion of Descemet's membrane. Endothelial injury was observed in a zone averaging between 40 and 100 microm adjacent to the cutting edge only. CONCLUSION: The excimer laser corneal shaping system (ELCS-S) allows a computer-controlled, surgeon-designed, sterile preparation of lamellar and penetrating corneal grafts with the use of the excimer laser. This could offer significant advantages in comparison to presently available systems for lamellar dissection and trephination.

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.

Eurosol versus fetal bovine serum-containing corneal storage medium.

PURPOSE: To evaluate the usability of Eurosol, a new medium-term corneal storage medium without components of bovine origin. METHODS: Ten pairs of human donor corneas were placed in tissue culture at 31 degrees C for 7, 14, 21, 28, or 35 days. One cornea of each pair was cultivated in conventional storage medium on Earls' minimum essential medium base containing 2% fetal bovine serum; the other one was stored in Eurosol. Corneas were examined with inverse light microscopy; corneal thickness was measured; and scanning electron microscopy was performed. RESULTS: No significant difference in corneal thickness and endothelial cell count was found at any time. Scanning electron microscopy showed a complete endothelial cell layer on all corneas. CONCLUSION. The findings indicate a potential clinical applicability of the tested serum-free medium-term storage medium, offering a safer alternative to conventional media containing fetal bovine serum.

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).

Autonomic and peptidergic innervation of human nasal mucosa.

The localization and distribution of vasoactive intestinal polypeptide (VIP), peptide histidine methionine (PHM), the novel peptide helospectin, neuropeptide tyrosine (NPY) and its C-flanking peptide (C-PON), substance P and calcitonin gene-related peptide (CGRP) were studied in the middle and inferior turbinate of the human nose using sensitive immunocytochemical and radioimmunological methods. For light microscopy, double immunofluorescence and immunogold-silver staining methods were applied. Ultrastructural immunoelectronmicroscopy was performed using a pre-embedding method. In addition, semithin Epon resin sections were immunostained. The concentrations of VIP, NPY, CGRP, substance P and neurokinin A were measured using radioimmunological methods. A dense network of autonomic and peptidergic nerve fibers in the normal human nasal mucosa was demonstrated. Colocalization studies showed the coexistence of peptides with components of the autonomic nervous system. Scattered chromogranin A-, CGRP and bombesin-flanking peptide (BFP)-immunoreactive endocrine-like cells were detected within the lamina propria and in groups within exocrine ducts. Highest radioimmunoassay (RIA) tissue concentrations were detected for VIP, followed by NPY, substance P, CGRP and neurokinin A.

[Amniotic membrane transplantation with limbal stem cell transplantation as a combined procedure for corneal surface reconstruction after severe thermal or chemical burns]. / Amnion-Limbus-Transplantation zur Oberflächenrekonstruktion nach schwerer Verätzung und Verbrennung.

BACKGROUND: Severe thermal and chemical burns may result in limbal deficiency leading to persistent epithelial defects, complete conjunctival epithelial ingrowth and vascularisation of the cornea. If sufficiently severe, these burns may lead to very significant visual impairment. Amniotic membrane transplantation with limbal transplantation has recently been proposed as a new method for corneal surface reconstruction. PATIENTS AND METHODS: A total of 14 patients (age 18-62 years, mean age 42 years) with limbal deficiency resulting from thermal ( n=1) or chemical burns ( n=13) underwent surgery. The corneal pannus was completely removed and the amniotic membrane was grafted onto the cornea. Limbal transplantation using autografts obtained from contralateral eyes was performed simultaneously in seven cases. Allografts from a donor were transplanted in seven cases with bilateral involvement. These patients received oral cyclosporin A postoperatively. The mean follow-up time was 18 months. RESULTS: In all cases of limbal autografts the corneal surface showed a complete and stable epithelialisation within a few weeks. Out of seven patients with limbal allografts three displayed recurrent epithelial defects in the long term. The initially semitransparent amniotic membrane became more translucent and biomicroscopically invisible within several months after surgery. There was an increase in visual acuity in most cases, limited mostly by irregular astigmatism due to the initial stromal loss. CONCLUSIONS: Amniotic membrane transplantation with limbal transplantation allows reconstruction ocular surfaces severely damaged by chemical or thermal burns. In most cases, however, additional surgical procedures such as lamellar or penetrating keratoplasty are required for adequate visual rehabilitation.

Cerebral localized marginal zone lymphoma presenting as hypothalamic-pituitary region disorder.

INTRODUCTION: Marginal zone B-cell lymphoma is a rare disease which can be considerably difficult to recognize and diagnose when signs of systemic involvement are absent. CASE PRESENTATION: We report the case of a 57-year-old woman with initial olfactory disturbance, followed by psychosis, diabetes insipidus and hypothalamic eating disorder as an uncommon clinical presentation of marginal zone B-cell lymphoma. CONCLUSION: Marginal zone B-cell lymphoma should be considered as a potential differential diagnosis in patients with hypothalamic disturbances.

[Vascularization of the matrix of hyaline rib cartilage in the human: changes with increasing age]. / Vaskularisierung der Matrix hyalinen Rippenknorpels beim Menschen: Veränderungen mit zunehmendem Alter.

A correlative light microscopic/transmission electron microscopic study was performed on human hyaline costal cartilage material of variable age (group I: first postnatal month; group II: second decade, and group III: fifth, sixth and seventh decades of life; total: nine cartilage samples) in order to clarify and evaluate the existence and dimensions of a vascular supply in young hyaline cartilage matrix and to compare with alterations or changes, respectively, occurring with aging. Human postnatal hyaline costal cartilage (4th to 6th rib) is vascularized very early by centripetally running venous/arterial vessels invading from the perichondrium. This process seems to be an active one since endothelial spurs and capillary sprouts could be demonstrated. During the middle decades of life a drastic/substantial decrease in amount/appearance of vessels or vascular supply, respectively, of the cartilage generally seems to occur. From the third/fourth decade on up to higher decades neovascularization seems to be a constant finding, especially in degeneration areas: so-called marrow spaces of cartilage matrix. With increasing age the fibrillar perivascular matrix becomes thicker, resulting in a so-called collagen-collar which consists of highly oriented collagen fibrils/fibers (as compared with the attaching extracellular cartilaginous matrix), most probably newly synthesized by (mesenchymal/fibrocytic/specialized?) cells accompanying the invading vessels. Incidence and possible function of the structures found in aged human costal cartilage are not fully understood; the ultrastructural changes compared with postnatal costal cartilage, however, are evident and demonstrated.

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.

A Turner-like phenotype in a girl with an isodicentric fluorescent Y chromosome mosaicism.

BACKGROUND: The Ullrich-Turner syndrome (UTS) demonstrates a great clinical variability according to the cytogenetic and molecular genetic findings in various tissues. In few cases the karyotype reveals the presence of an additional Y-bearing cell line which is referred to as a borderline case of mixed gonadal dysgenesis. In this condition, Turner specific stigmata occur in about half of the cases. PATIENT: A 10 year-old girl with short stature and only a few other signs of Turner syndrome and hypertrophic clitoris revealed 45,X/46,X,idic(Yq) mosaicism with 41% 46,X,idic(Yq) cells in a blood lymphocyte culture. METHODS AND RESULTS: Fluorescence in situ hybridisation (FISH) technique, using alpha-satellite Y-chromosome specific probe for locus DYZ3, confirmed the isodicentric character of this structurally abnormal Y chromosome. Polymerase chain reaction (PCR) analysis using primers for eight loci along the Y chromosome including SRY (Sex determining Region, Y gene) were positive for all loci tested, indicating that sequences from the long arm, centromere and most of the short arm of the Y chromosome are present. CONCLUSIONS: As patients with normal or rearranged Y chromosome have an increased risk of developing gonadal neoplasia prophylactic gonadectomy was performed in our patient. No evidence for gonadoblastoma was found on her streak-like gonads, but they showed some evidence of tubular formation. This paper points out the impact of cytogenetic and molecular genetic investigations in the definition of mosaicism in Turner's syndrome.

[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.

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.

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.