Study of elastase-type activity in blister fluids of recessive dystrophic epidermolysis bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is characterized clinically by blister formation due to minor trauma and ultrastructurally by a progressive disappearance of anchoring fibrils at the dermoepidermal junction and of the oxytalan-type fibers which belong to the elastic fiber system. In this study, we determined the elastase-type activity in blister fluid obtained from 8 patients suffering from RDEB as compared to the suction fluid of experimental blisters in a healthy person and to the blister fluid of a patient suffering from epidermolysis bullosa simplex. One patient with dominant dystrophic epidermolysis of the albopapuloid type was also studied. Seven of the eight children with RDEB showed highly elevated values. The eighth child, treated with etretinate, as well as the patient suffering from dominant epidermolysis bullosa had moderately increased values. The determination of elastase-type activity in the blister fluid could therefore be useful to establish the differential diagnosis of recessive dystrophic epidermolysis bullosa.     

MMP-9 and CXCL8/IL-8 Are Potential Therapeutic Targets in Epidermolysis Bullosa Simplex

Epidermolysis bullosa refers to a group of genodermatoses that affects the integrity of epithelial layers, phenotypically resulting in severe skin blistering. Dowling-Meara, the major subtype of epidermolysis bullosa simplex, is inherited in an autosomal dominant manner and can be caused by mutations in either the keratin-5 (K5) or the keratin-14 (K14) gene. Currently, no therapeutic approach is known, and the main objective of this study was to identify novel therapeutic targets. We used microarray analysis, semi-quantitative real-time PCR, western blot and ELISA to identify differentially regulated genes in two K14 mutant cell lines carrying the mutations K14 R125P and K14 R125H, respectively. We found kallikrein-related peptidases and matrix metalloproteinases to be upregulated. We also found elevated expression of chemokines, and we observed deregulation of the Cdc42 pathway as well as aberrant expression of cytokeratins and junction proteins. We further demonstrated, that expression of these genes is dependent on interleukin-1 β signaling. To evaluate these data in vivo we analysed the blister fluids of epidermolysis bullosa simplex patients vs. healthy controls and identified matrix metalloproteinase-9 and the chemokine CXCL8/IL-8 as potential therapeutic targets.     

Prenatal diagnosis of genodermatoses by ultrastructural diagnostic markers in extra-embryonic tissues: defective hemidesmosomes in amnion epithelium of fetuses affected with epidermolysis bullosa Herlitz type (an alternative prenatal diagnosis in certain cases)

Summary We report the first use of amnion epithelium for prenatal diagnosis. Prenatal diagnosis of recessive epidermolysis bullosa atrophicans generalisata gravis Herlitz type can at present be achieved with safety by detailed ultrastructural analysis of fetal skin. Because of the close developmental origin of amnion and skin, which has been elucidated by the recent development of antiamnion antibodies against dermo-epidermal junction antigens and by their abnormal binding in epidermolysis bullosa skin, there is presumably some morphological relationship between amnion epithelium and skin. In a comparative study of extra-embryonic tissues, we found ultrastructurally complete hemidesmosomes in all 24 investigated normal amnion samples from gestational weeks 15–27, but not in 7 reflected chorion samples from weeks 16–22. The results of placental chorion samples were not reliable. Amnion of 5 fetuses affected with epidermolysis bullosa atrophicans generalisata gravis revealed only hypoplastic hemidesmosomes, the same defect as in the respective skin. In a recent case where unfortunately only non-skin material was available, a positive prenatal diagnosis of epidermolysis bullosa atrophicans gravis Herlitz was performed from the amnion material. The diagnosis was confirmed by investigation of the fetal skin after termination. Investigation of amnion membranes is therefore an alternative for prenatal diagnosis of epidermolysis bullosa atrophicans gravis Herlitz in certain cases. The possibility and limitations of the general use of amnion for prenatal diagnosis are discussed.     

The immunopathology of epidermolysis bullosa acquisita

The acquired type of epidermolysis bullosa, epidermolysis bullosa acquisita, has a high incidence of associated systemic disorders. Two patients with epidermolysis bullosa acquisita were investigated by clinical, histochemical and immunologic methods. The histochemical findings confirmed earlier reported dermal changes. Both patients had in vivo IgG and the C₃ component of complement localized in the basement-membrane zone.     

Levels of Circulating DNA in Blood Serum and DNA Damage in Leukocytes of Healthy Donors of Different Genders and Ages

We studied the levels of extracellular nuclear and mitochondrial DNA of blood serum and DNA damage in leukocytes of healthy donors of different sex and age groups. The baseline levels of DNA damage in leukocytes and serum DNA levels were shown to vary greatly among different donors. The baseline level of DNA damage in leukocytes was not associated with the presence of chronic deceases or an occupational health risk for elderly donors. It was found that extracellular DNA concentrations were generally higher in men than in women. There is a tendency towards an increase in the relative mitochondrial DNA copy number determined by ΔCt in women but not in men: the relative mtDNA copy number in elderly individuals varies significantly in both sexes, possibly due to age-related physiological changes. It is necessary to consider the gender and age of patients when using an indicator such as the level of extracellular DNA of blood serum for diagnosis and monitoring.

     

915 MHz microwaves and 50 Hz magnetic field affect chromatin conformation and 53BP1 foci in human lymphocytes from hypersensitive and healthy persons

We used exposure to microwaves from a global system for mobile communication (GSM) mobile phone (915 MHz, specific absorption rate (SAR) 37 mW/kg) and power frequency magnetic field (50 Hz, 15 muT peak value) to investigate the response of lymphocytes from healthy subjects and from persons reporting hypersensitivity to electromagnetic field (EMF). The hypersensitive and healthy donors were matched by gender and age and the data were analyzed blind to treatment condition. The changes in chromatin conformation were measured with the method of anomalous viscosity time dependencies (AVTD). 53BP1 protein, which has been shown to colocalize in foci with DNA double strand breaks (DSBs), was analyzed by immunostaining in situ. Exposure at room temperature to either 915 MHz or 50 Hz resulted in significant condensation of chromatin, shown as AVTD changes, which was similar to the effect of heat shock at 41 degrees C. No significant differences in responses between normal and hypersensitive subjects were detected. Neither 915 MHz nor 50 Hz exposure induced 53BP1 foci. On the contrary, a distinct decrease in background level of 53BP1 signaling was observed upon these exposures as well as after heat shock treatments. This decrease correlated with the AVTD data and may indicate decrease in accessibility of 53BP1 to antibodies because of stress-induced chromatin condensation. Apoptosis was determined by morphological changes and by apoptotic fragmentation of DNA as analyzed by pulsed-field gel electrophoresis (PFGE). No apoptosis was induced by exposure to 50 Hz and 915 MHz microwaves. In conclusion, 50 Hz magnetic field and 915 MHz microwaves under specified conditions of exposure induced comparable responses in lymphocytes from healthy and hypersensitive donors that were similar but not identical to stress response induced by heat shock.     

Hereditary epidermolysis bullosa in Tunisia: an epidemio-clinical and ultrastructural study

Hereditary epidermolysis bullosa (EB) are a group of genodermatoses whose common primary feature is formation of blisters following minor trauma. The aim of the present study was to assess epidemiological, clinical, genetical and histological particularities of patients with hereditary epidermolysis bullosa.     

Implications of intragenic marker homozygosity and haplotype sharing in a rare autosomal recessive disorder: the example of the collagen type XVII (COL17A1) locus in generalised atrophic benign epidermolysis bullosa

Generalised atrophic benign epidermolysis bullosa (GABEB) is a form of junctional epidermolysis bullosa with a recessive mode of inheritance. The gene considered likely to be involved in this disease is COL17A1, since in the majority of GABEB patients the product of that gene, the 180-kD bullous pemphigoid antigen (BP180), is undetectable in skin. We have identified an intragenic COL17A1 microsatellite marker for which 83% of randomly selected control individuals are heterozygous. We observed homozygosity for different alleles of this marker in five out of six collagen type XVII-negative GABEB patients of different European descent. Five of the six COL17A1 alleles of three patients originating from the eastern part of The Netherlands were identical, as were the haplotypes including flanking markers. The 2342delG mutation was identified in all these five alleles. This confirms the expectation that due to genetic drift and hidden inbreeding for an autosomal recessive disorder with low gene frequency, such as collagen type XVII-negative GABEB, most disease alleles from a restricted geographical area will be “identical by descent”. Our results demonstrate that involvement of a candidate gene can be confirmed by looking for identity by descent of highly informative intragenic markers. Received: 25 October 1996 / Accepted: 6 March 1997     

Impact of Low-Frequency Pulsed Magnetic Fields on Defensin and CRP Concentrations in Patients with Painful Diabetic Polyneuropathy and in Healthy Subjects

Aim. The aim was to assess whether magnetic field influences defensin and CRP concentrations in patients with diabetic polyneuropathy and in healthy subjects.

Methods. 61 diabetic patients were randomly divided into 2 groups: study group—32 patients exposed to low-frequency magnetic field; and control group—29 patients with sham exposure. Additionally, 20 healthy subjects exposed to low-frequency magnetic field. Exposures were performed during 3 weeks, 5 days in a week. Defensin and CRP concentrations were measured at baseline, after 3 weeks and at the end of the study.

Results. There were no significant changes in defensin concentration in patients with diabetes in both the real and sham exposure group. We observed increased concentration of defensin in healthy subjects in week 5 vs. baseline value (P<0.02).

Conclusions. Magnetic field has no impact on defensin concentration in diabetic patients but has positive influence on this parameter in healthy subjects.     

Two Novel Mutations on Exon 8 and Intron 65 of COL7A1 Gene in Two Chinese Brothers Result in Recessive Dystrophic Epidermolysis Bullosa

Dystrophic epidermolysis bullosa is an inherited bullous dermatosis caused by the COL7A1 gene mutation in autosomal dominant or recessive mode. COL7A1 gene encodes type VII collagen – the main component of the anchoring fibrils at the dermal–epidermal junction. Besides the 730 mutations reported, we identified two novel COL7A1 gene mutations in a Chinese family, which caused recessive dystrophic epidermolysis bullosa (RDEB). The diagnosis was established histopathologically and ultrastructurally. After genomic DNA extraction from the peripheral blood sample of all subjects (5 pedigree members and 136 unrelated control individuals), COL7A1 gene screening was performed by polymerase chain reaction amplification and direct DNA sequencing of the whole coding exons and flanking intronic regions. Genetic analysis of the COL7A1 gene in affected individuals revealed compound heterozygotes with identical novel mutations. The maternal mutation is a 2-bp deletion at exon 8 (c.1006_1007delCA), leading to a subsequent reading frame-shift and producing a premature termination codon located 48 amino acids downstream in exon 9 (p.Q336EfsX48), consequently resulting in the truncation of 2561 amino acids downstream. This was only present in two affected brothers, but not in the other unaffected family members. The paternal mutation is a 1-bp deletion occurring at the first base of intron 65 (c.IVS5568+1delG) that deductively changes the strongly conserved GT dinucleotide at the 5′ donor splice site, results in subsequent reading-through into intron 65, and creates a stop codon immediately following the amino acids encoded by exon 65 (GTAA→TAA). This is predicted to produce a truncated protein lacking of 1089 C-terminal amino acids downstream. The latter mutation was found in all family members except one of the two unaffected sisters. Both mutations were observed concurrently only in the two affected brothers. Neither mutation was discovered in 136 unrelated Chinese control individuals. This study reveals novel disease-causing mutations in the COL7A1 gene.     

Chemical shift assignments of the fibronectin III like domains 7–8 of type VII collagen

Type VII collagen (Col7) is important for skin stability. This is underlined by the severe skin blistering phenotype in the Col7 related diseases dystrophic epidermolysis bullosa and epidermolysis bullosa acquisita (EBA). Col7 has a large N-terminal non-collagenous domain (NC1) that is followed by the triple helical collagenous domain. The NC1 domain has subdomains with homology to adhesion molecules and mediates important interactions within the extracellular matrix. An 185 amino acid long part of the NC1-subdomain termed fibronectin III like domains 7 and 8 (FNIII7-8) was investigated. Antibodies against this region are pathogenic in a mouse model of EBA and one reported missense mutations of Col7 lies within these domains. The nearly complete NMR resonance assignment of recombinant FNIII7-8 of Col7 is reported.     

Prenatal diagnosis of genetic disorders of the skin by means of electron microscopy

Summary This paper summarizes the ultrastructural aspects of prenatal diagnosis of inherited skin disorders by means of electron microscopy on fetal skin biopsies obtained under fetoscopy. The investigation comprises skin samples of a series of 26 fetoscopies performed at the Departments of Gynecology at the Universities of Giessen and Lund. Among them, seven fetoscopies were performed for prenatal diagnosis in pregnancies at risk of epidermolysis bullosa or ichthyosis during the 19th and 22nd week of gestational age. Positive prenatal diagnosis was made in one fetus at risk of epidermolysis bullosa of unknown genetic type; this was based on dermolytic blister formation and collagenolysis diagnostic of EB dystrophica Hallopeau-Siemens. In a pregnancy with a fetal risk of bullous congenital ichthyosiform erythroderma, positive prenatal diagnosis was based on cytolysis and tonofilament clumping in the fetal epidermis and verified clinically and ultrastructurally after interruption; in another pregnancy bullous ECI was excluded ultrastructurally by the normal ultrastructural constitution of the fetal epithelium. In three fetuses at risk of the Herlitz syndrome and in one at risk of epidermolysis bullosa atrophicans inversa with pronounced sites of predilection, prenatal exclusion of the disorder was possible; this was based on the ultrastructural demonstration of the regular presence of normal hemidesmosomes with well-developed sub-basal dense plates at the dermo-epidermal junction in all four cases. One of these children has since been born and has normal skin; no scarring was found after birth on the sites of fetal skin biopsies.Possible application of this kind of prenatal diagnosis to other groups of genetic disorders is discussed in which biochemical defects are still unknown. Even more important is the possibility of excluding genetic disorders by demonstration of normal ultrastructural features in fetal skin biopsies and so avoiding abortion of healthy children.Awarded the Hans-Nachtsheim-Preis 1981In cooperation with R. Rauskolb and V. Jovanovic, Zentrum für Frauenheilkunde der Universität Giessen, 6300 Giessen, Federal Republic of Germany, and B. Gustavii, E. Cordesius, and L. Löfberg, Department of Gynecology, University of Lund, Sweden     

Basic fibroblast growth factor: a missing link between collagen VII, increased collagenase, and squamous cell carcinoma in recessive dystrophic epidermolysis bullosa.

BACKGROUND: Patients with recessive dystrophic epidermolysis bullosa (RDEB) have deficiencies of collagen type VII and have elevated levels of fibroblast collagenase, and a greatly increased risk of cutaneous squamous cell carcinoma. Patients with other genetic blistering disorders do not have elevated collagenase or an increased risk of squamous cell carcinoma, despite chronic wounding. The connection between collagen type VII deficiency, increased collagenase, and squamous cell carcinoma is not understood. MATERIALS AND METHODS: Urine from 81 patients with RDEB (39 patients), junctional epidermolysis bullosa (JEB; 12 patients), and epidermolysis bullosa simplex (EBS; 30 patients), as well as unaffected family members of RDEB patients (33 patients), was tested for the presence of basic fibroblast growth factor (bFGF) using a sensitive radioimmunoassay. These patients included many who were enrolled in the Epidermolysis Bullosa Registry and others who were referred by their physicians. RESULTS: Fifty-one percent of patients with RDEB had elevated levels (> 5000 pg/g) of urinary bFGF. In contrast, none of the patients with JEB had elevated levels of bFGF. Twenty-one percent of clinically unaffected family members had elevated levels of bFGF, and 13% of patients with EBS had elevated levels of bFGF. The frequency of elevated bFGF values among all groups was statistically significant (p = 0.002), and the levels of bFGF in RDEB patients were significantly elevated compared with those of other groups (p < 0.05). CONCLUSIONS: We have found that patients with RDEB have elevated levels of bFGF, which may contribute to increased fibroblast collagenase and the development of squamous cell carcinoma. These results suggest a novel treatment for RDEB, namely, angiogenesis inhibitors, which may antagonize the effects of bFGF in this disorder. There are currently no other means of treatment for this disorder, which has a high morbidity and mortality rate.     

Magneto‐optical characteristics of human sperms: normal and deformed

In this study we report on magnetic orientation of human sperms. Samples were taken from 17 donors. Normal human sperms became oriented with their long axis perpendicular to the magnetic field (1 T maximum). Total orientation was achieved with magnetic field of about 1 T, while for abnormal sperms the magnetic behavior was different. The dependence of the measured degree of orientation on the intensity of the magnetic field was in good agreement with the theoretical equation for the magnetic orientation of diamagnetic substances. As a result of a numerical analysis based on the equation, the anisotropic diamagnetic susceptibility of normal sperm was found to be Δ_χ = 8 × 10^–20 J/T². The degree of orientation was influenced by the alterations in the shape of the head, body or the tail. It has been suggested that the DNA in the sperm head retain the strong magnetic anisotropy to counterbalance the magnetic anisotropy retained by flagellum microtubules. Recent studies demonstrated a well‐defined nuclear architecture in human sperm nucleus, where the head morphology has significant correlation with sperm chromatin structure assay SCSA. Then, as the methods to evaluate SCSA can be difficult and expensive our simple magnetic orientation technique can be an alternative to diagnose alteration in DNA. (© 2008 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Cost analysis of the Kozak protocol as used in an Ontario hospital in the treatment of children with epidermolysis bullosa.

Conventional treatment of epidermolysis bullosa is often unsuccessful. The Kozak protocol is an alternative that has been given considerable public support in Ontario. The incremental cost of this treatment program at the Hospital for Sick Children, Toronto, was examined. The departments of nursing, pharmacy and food services each kept records of salaries and supply costs applicable to the care of nine patients with epidermolysis bullosa who were treated in the fiscal year 1982-83. The selected direct costs to the hospital were compared with the projected costs if these patients had been treated in Dr. Kozak''s clinic in West Germany or under the financial arrangements offered to Dr. Kozak by the Ontario minister of health. At a total incremental cost of +255.92 per patient-day, care at the Hospital for Sick Children may not currently be the least expensive means of offering the Kozak protocol to Ontario children. However, the major expense of the program, the nurses'' salaries, could be reduced if the patients'' parents were to assume many of the nursing tasks; this would make the hospital''s program the most cost-effective method of treating children with epidermolysis bullosa.     

Preliminary study of thyroid and colon cancers-associated antigens and their cognate autoantibodies as potential cancer biomarkers

Background: Autoantibodies, which are produced against tumor-associated antigens, are potential tumor markers and attract a growing interest for cancer detection, differential diagnostics and prognosis.

Objective: To evaluate the diagnostic significance of 40 antigens identified by immunoscreening of cDNA libraries from thyroid and colon cancers by allogenic screening with different tumor types patients’ sera.

Method: Plaque-spot serological assay.

Results: Increased frequency of antibody response in sera of cancer patients compared with that of healthy donors was shown toward 14 antigens, 8 of which (CG016, BTN3A3, FKBP4, XRCC4, TSGA2, ACTR1A, FXYD3 and CTSH) have revealed exclusively cancer-related serological profile.

Conclusion: Allogenic screening of 40 SEREX-antigens with sera from cancer patients and healthy donors allowed us to reveal 14 antigens with potential diagnostic significance. These antigens and their cognate autoantibodies could be considered as valuable targets for further analysis as potential cancer biomarkers.     

Epidermolysis bullosa acquisita antigen is synthesized by human keratinocytes cultured in serum-free medium

The epidermolysis bullosa acquisita antigen is a newly discovered major extracellular matrix component within basement membranes beneath stratified squamous epithelia. Human keratinocytes cultured without mesenchymal cells synthesize the major 290 kd chain of the epidermolysis bullosa acquisita antigen.     

The proportion of mutant extracellular mitochondrial DNA increases in lung cancer patients after radiotherapy

Quantitative and qualitative changes in circulating extracellular DNA (ec-DNA) of blood plasma are considered as markers for diagnosis and prognosis of tumor pathology. We have investigated the content of mutant copies of the circulating mitochondrial DNA (ec-mtDNA) in blood plasma in 8 patients with lung cancer before and after radiotherapy as well as in healthy young and elderly donors. It was found that in plasma of healthy elderly donors the proportion of ec-mtDNA with mutations (in the total circulating DNA) is much higher than in young donors. Before radiotherapy the proportion of ec-mtDNA with mutations was higher in plasma of lung cancer patients (aged 70–76 years) than that of healthy elderly donors. After radiotherapy of lung cancer patients a twofold increase in the proportion of ec-mtDNA with mutations was observed in total circulating plasma DNA. This may be attributed to release of mutant DNA copies from dying tumor cells and also from normal cells injured by the radiation treatment.     

Spontaneous and mitomycin-C-induced micronuclei in human lymphocytes exposed to extremely low frequency pulsed magnetic fields

The cytokinesis block micronucleus method, a very sensitive cytogenetic assay, was used to ascertain the possible genotoxic effects of extremely low frequency pulsed magnetic fields in phytohemagglutinin-stimulated human lymphocytes cultures from 16 healthy donors. Four conditions were studied: i) lymphocytes not exposed to the field (control cultures); ii) lymphocytes exposed to the field; iii) lymphocytes treated with mitomycin-C and not exposed to the field; iv) lymphocytes treated with mitomycin-C and exposed to the field. Mitomycin-C-treated cultures were used as control for the micronucleus method, because it is known that mitomycin-C is a potent genotoxic agent, capable of inducing micronuclei. The frequency of micronuclei in field-exposed cultures was similar to the spontaneous frequency observed in control unexposed-cultures. Moreover, the exposure to pulsed magnetic fields did not affect the frequency of micronuclei induced by mitomycin-C, suggesting that, in the experimental conditions used, this kind of field neither affected the integrity of chromosomes nor interfered with the genotoxic activity of mitomycin-C.     

Radiation-induced DNA double-stranded breaks and the dynamics of apoptotic death of human peripheral lymphocytes

DNA double-stranded breaks and their association with the development of radiation-induced peripheral lymphocyte apoptosis were studied in healthy donors exposed to in vitro gamma-irradiation in a dose of 1 Gy. It was shown that irradiation in 1-Gy dose caused a significant (p < 0.05) increase in the frequency of cells in late apoptosis 4 hours after irradiation and a rise in their frequency in early apoptosis 24 hours following this procedure. A significant correlation (r = 0.52, p < 0.05) was recorded between the primary level of radiation-induced DNA double-stranded breaks and the frequency of cells in late apoptosis following 4 hours, which suggests that DNA double-stranded breaks as a signal to trigger cell apoptotic death are of great importance.