LAD-1 is absent in a subset of junctional epidermolysis bullosa patients

BACKGROUND: Awareness during anaesthesia has been estimated to occur in 0.2%-0.4% of patients undergoing general surgery. In Finland, according to the Patient Injury Act, compensation is paid for an injury caused by medical treatment. We have analysed the claims for compensation involving awareness under anaesthesia filed between May, 1987 and December, 1993. METHODS: Original claims, hospital notes, and expert advisor's comments as well as the comments of the anaesthesiologist in charge of the anaesthesia, and decisions of the Patient Injury Association, were reviewed. RESULTS: A total of 23363 claims of patient injury were made during the study period. Of these, 391 considered anaesthetic treatments, and there were four cases of awareness with recall. CONCLUSION: Claims of compensation for awareness during anaesthesia were surprisingly few, possible reasons of which are discussed. Compensations paid were low in comparison with some other countries.     

Hemidesmosomes show abnormal association with the keratin filament network in junctional forms of epidermolysis bullosa

Leadership in improving the education of doctors, while impressive, is not happening fast enough. While there are many obstacles, there is no time to waste in restructuring medical education to repair its present deficiencies, for otherwise outside forces could overwhelm today's education leaders with imperatives to make improvements on their own terms. The first step in addressing present shortcomings is to establish measurable objectives for the education of doctors that are aligned with the legitimate expectations of society and the enduring precepts of the medical profession. To provide guidance in establishing these objectives, the AAMC launched the Medical School Objectives Project (MSOP) two years ago. This project is now close to completing its initial phase, which is to define the knowledge, skills, attitudes, and values every medical student must demonstrate before graduating. Phase Two will be concerned with implementation (e.g., establishing assessment methods; improving faculty development; etc.). As for aligning the outcomes of medical education with the precepts of the profession, nothing is more important: if doctors do not have high standards of professionalism--altruism, respect, compassion, honesty, integrity, and others--medicine's very survival is threatened. Medical educators must insist that their graduates demonstrate these attributes, through more careful admission criteria, more attention to medical professionalism in the curriculum and in the evaluation of students, more community service for students, and improved role modeling by faculty. Leadership for the changes that are needed will not come from a once-in-a-lifetime leader of heroic proportions but from everyone within academic medicine leading the profession to its promising future through quality education.     

Maternal uniparental meroisodisomy in the LAMB3 region of chromosome 1 results in lethal junctional epidermolysis bullosa

Herlitz junctional epidermolysis bullosa (OMIM#226700) is a lethal, autosomal recessive blistering disorder caused by mutations in one of the three genes LAMA3, LAMB3, or LAMC2, encoding the constitutive polypeptide subunits of laminin 5. In this study, we describe a patient homozygous for a novel nonsense mutation Q936X in exon 19 of LAMB3, which has been mapped to chromosome 1q32. The patient was born with extensive blistering and demonstrated negative immunofluorescence staining for laminin 5, and transmission electron microscopy revealed tissue separation within lamina lucida of the dermal-epidermal junction, diagnostic of Herlitz junctional epidermolysis bullosa. The mother of the proband was found to be a heterozygous carrier for this mutation, whereas the father demonstrated the wild-type LAMB3 allele only. Nonpaternity was excluded by 13 microsatellite markers in six different chromosomes. Genotype analysis using 28 microsatellite markers spanning chromosome 1 revealed that the patient had maternal primary heterodisomy, as well as meroisodisomy within two regions of chromosome 1, one on 1p and the other one on 1q, the latter region containing the maternal LAMB3 mutation. These results suggest that Herlitz junctional epidermolysis bullosa in this patient developed as a result of reduction to homozygosity of the maternal LAMB3 mutation on chromosome 1q32.     

Compound heterozygosity for an out-of-frame deletion and a splice site mutation in the LAMB3 gene causes nonlethal junctional epidermolysis bullosa

OBJECTIVE: To explore the temporal relation of demyelination and blood-brain barrier breakdown during new lesion formation. BACKGROUND: Conventional MRI appears sensitive for detecting changes due to MS, but may be limited by poor pathologic specificity. By indirectly assessing protons bound to rigid macromolecules, magnetization transfer (MT) imaging may provide information relating to tissue structure and, by inference, myelin integrity. METHODS: Gadolinium contrast-enhanced MRI and MT imaging were performed at weekly intervals for 3 months in three patients with MS. For each enhancing lesion, the largest corresponding area of proton density hyperintensity seen during the study was outlined and magnetization transfer ratio (MTR) calculated at each time point from coregistered calculated MTR images. Lesions greater than 20 mm2, not affected by partial volume effects, and first enhancing after the baseline study were analyzed. Two-dimensional registration software allowed accurate evaluation of MTR in regions both before and after the initial appearance of MS lesions. RESULTS: Mean lesion MTR decreased significantly during the first week of enhancement (29.6 percent units [pu] immediately pre-enhancement versus 28.2 pu at first documented stage of enhancement). No significant MTR reduction was noted before this. CONCLUSION: The lack of observable change in MTR before the first detectable gadolinium enhancement within MS lesions suggests that blood-brain barrier disruption is closely related to, but not preceded by, demyelination.     

Corrective gene transfer of keratinocytes from patients with junctional epidermolysis bullosa restores assembly of hemidesmosomes in reconstructed epithelia

Herlitz junctional epidermolysis bullosa (H-JEB) provides a promising model for somatic gene therapy of heritable mechano-bullous disorders. This genodermatosis is caused by the lack of laminin-5 that results in absence of hemidesmosomes (HD) and defective adhesion of squamous epithelia. To establish whether re-expression of laminin-5 can restore assembly of the dermal-epidermal attachment structures lacking in the H-JEB skin, we corrected the genetic mutation hindering expression of the beta 3 chain of laminin-5 in human H-JEB keratinocytes by transfer of a laminin beta 3 transgene. The transduced keratinocytes synthesized a recombinant beta 3 polypeptide that assembled with the endogenous laminin alpha 3 and gamma 2 chains into a biologically active laminin-5 that was secreted, processed and deposited into the extracellular matrix. Re-expression of laminin-5 induced cell spreading, nucleation of hemidesmosomal-like structures and enhanced adhesion to the culture substrate. Organotypic cultures performed with the transduced keratinocytes, reconstituted epidermis closely adhering to the mesenchyme and presenting mature hemidesmosomes, bridging the cytoplasmic intermediate filaments of the basal cells to the anchoring filaments of the basement membrane. Our results provide the first evidence of phenotypic reversion of JEB keratinocytes by somatic gene therapy and demonstrate that genetic treatment of the mild forms of skin blistering diseases and other inherited extracellular matrix pathologies is a realistic goal.     

Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy

Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.     

Electron microscopic observations of the conjunctiva in epidermolysis bullosa hereditaria

PURPOSE: Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided. METHODS: The GV oocytes were cryopreserved using a slow-cooling (0.5 degree C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiple-label fluorescence and differential interference contrast microscopy techniques. RESULTS: When examined after in vitro maturation, the vast majority (> 95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrel-shaped spindle and normal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the second polar body, and pronuclear migration were displayed by similar percentages of cryopreserved (96, 94, and 37%, respectively) and control (98, 97, and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However, pronuclear formation was significantly inhibited by cryopreservation (81%) compared with controls (100%). Regarding digyny and polyspermy, no significant increase was observed after cryopreservation (3 and 10%, respectively) compared with controls (3 and 6%, respectively). CONCLUSIONS: Cryopreservation of mouse oocytes at the GV stage is particularly advantageous to circumvent the spindle damage and increased digyny noted after cryopreservation of MII oocytes.     

Special aspects of anesthesia in patients with epidermolysis bullosa based on a case example

Epidermolysis bullosa hereditaria dystrophica (Hallopeau-Siemens) is a rare autosomal recessive disease characterized by extreme bullae formation of skin and mucosa. Typical dystrophic nails and flexion contractures of the joints can lead to deformities. Carious teeth and microstomia caused by scarred contractures of the lips are characteristic of the clinical picture. Depending on the form and severity of epidermolysis bullosa, the anaesthetic and surgical management requires careful planning to avoid unnecessary complications as a result of positioning, anaesthesia or surgery. In cooperation with the patient, optimal positioning on the operating table without pressure or tangential friction of the skin needs to be achieved. Wherever possible, surgical tape and adhesive electrodes should be avoided. Artificial respiration, intubation and monitoring must be adapted to the skin conditions of the patient. In particular the skin below the blood pressure cuff must be protected by adequate padding, and maximum intervals between measurements should be chosen. Nasal, oral, laryngeal and tracheal manipulations should be kept to a minimum for protection of the upper airway. Fibre optic intubation is to be preferred because of the possibility of microstomia and the need for simultaneous airway diagnosis. In addition, oro- or nasopharyngeal tubes and catheters should be avoided where possible. Depending on the course of surgery and anaesthesia, postoperative therapy in an intensive care unit should be considered.     

Compound heterozygosity for a nonsense mutation and a splice site mutation in the type VII collagen gene (COL7A1) in recessive dystrophic epidermolysis bullosa

The genes encoding the nucleoprotein, PB1, PB2, and PA proteins of the influenza virus strain B/Panamá/45/90 have been cloned under control of the T7 RNA polymerase promoter of plasmid pGEM-3. Transfection of the recombinant plasmids obtained into mammalian cells, which had been infected with a vaccinia virus encoding the T7 RNA polymerase, resulted in expression of the expected influenza B virus polypeptides. Moreover, it is shown that coexpression of the four recombinant core proteins in COS-1 cells reconstituted a functional polymerase capable of expressing a synthetic influenza B virus-like CAT RNA. By using the influenza B virus recombinant plasmids and a set of pGEM-derived plasmids encoding the homologous core proteins of the influenza A virus A/Victoria/3/75 (I. Mena et al. (1994). J. Gen. Virol. 75, 2109-2114), the capabilities of homo- and heterotypic mixtures of the four core proteins to express synthetic type A and B CAT RNAs were analyzed. Both the influenza A and B virus polymerases were active in expressing, albeit with reduced efficiencies, the heterotypic model CAT RNAs. However, none of all possible heterotypic mixtures of the core proteins reconstituted a functional polymerase. In order to fully characterize the recombinant plasmids obtained, the nucleotide sequences of the cloned genes were determined and compared to sequences of other type B virus isolates. The results obtained from these latter analyses are discussed in terms of the conservation and evolution of the influenza B virus core genes.     

Pyloric atresia-junctional epidermolysis bullosa syndrome: mutations in the integrin beta4 gene (ITGB4) in two unrelated patients with mild disease

Junctional epidermolysis bullosa associated with pyloric atresia (EB-PA; OMIM 226730) is a rare autosomal recessively inherited disease in which mucocutaneous fragility is associated with gastrointestinal atresia. This disease is usually fatal within the first few weeks or months of life even following surgical correction of the intestinal obstruction. Recently, mutations in the genes encoding the epithelial integrin alpha6beta4 (ITGA6 and ITGB4) have been identified in several patients with EB-PA. We report two unrelated patients with this disease who have survived into early childhood with mild cutaneous involvement, in whom we have identified pathogenetic mutations in ITGB4. The first patient was a compound heterozygote for a splice site mutation in exon 30 (3793 + 1G-to-A) and a non-sense mutation in exon 36 (W1478X), and the second was a compound heterozygote for a missense mutation in exon 3 (C38R) and a 1 bp deletion in exon 36 (4776delG). Although the non-sense and deletion mutations are predicted to result in markedly reduced beta4 integrin mRNA levels, the presence of the missense or splice site mutation on the second allele may enable the synthesis of some functional, albeit perturbed, beta4 polypeptide. Determination of the molecular mechanisms in these two cases increases our understanding of EB-PA and may enable correlation between genotype and phenotype.     

Abnormalities of basal cell keratin in epidermolysis bullosa simplex do not affect the expression patterns of suprabasal keratins and cornified cell envelope proteins

Basal keratins, suprabasal keratins, filaggrin, and cornified cell envelope (CCE) precursor proteins are expressed during the differentiation of epidermal keratinocytes. These molecules are coordinately expressed during epidermal differentiation. The present study investigated the expression patterns of keratins and CCE precursor proteins in 15 patients with epidermolysis bullosa simplex (EBS), which is caused by mutations in the genes that encode for the basal keratins, keratins 5 and 14. The patterns of expression of keratins 5, 14, 1 and 10, filaggrin, and of the three major CCE precursor proteins, involucrin, loricrin and small proline-rich proteins 1 and 2 (SPRs), were studied immunohistochemically and by electron microscopy. In 14 of the 15 patients with EBS, the distribution pattern of keratins was not altered. In one neonate with EBS, basal cell keratins were expressed in the suprabasal layers. Ultrastructurally, numerous clumped tonofilaments were observed in the basal and suprabasal cells. In all cases, findings were positive for filaggrin in the granular cells, with positivity for involucrin in the upper spinous and granular cells. The upper spinous cells and granular cells were positive for SPRs 1 and 2, and loricrin was expressed in granular cells. Ultrastructurally, no marked abnormality was observed in the suprabasal layers such as a decrease in, or agglutination of, keratin filaments, except in one neonate. A CCE about 15 nm thick was formed normally in the cell membrane of cornified cells. The patterns of distributions of basal cell keratins, suprabasal keratins, filaggrin, and CCE precursor proteins, as well as the ultrastructural findings, resembled those of normal skin. Thus, the abnormality in basal cell keratins in patients with EBS did not appear to alter the patterns of expression of the keratins and CCE precursor proteins.     

Ultrastructural studies in epidermolysis bullosa hereditaria. III. Recessive dystrophic types with dermolytic blistering (Hallopeau-Siemens types and inverse type)

In this paper the authors describe and analyse results that they obtained by infection of the guinea pig organism carried out by subcutaneous or intratracheal application with five mycobacterial strains, namely Myco bovis BCG-Praha, Myco the H37Ra, Myco Kansasii, Myco fortuitum and Myco smegmatis. At predetermined time intervals following subcutaneously or intratracheally performed infection (on 7th, 16th, 28th and 56th day after infection) transplantation of a Deals' guinea pig sarcoma cell suspension was carried out in guinea pigs by the intraltracheal route. As it appears from the results gained the applied mycobacteria exhibit a partial inhibition of growth od Deals' guinea pig sarcoma cells of different character. From among the utilized strains the Myco bovis BCP-Praha and the Myco tbc H37Ra exhibited the highest, Myco fortuitum and Myco smegmatis the lowest inhibitory activity. Intratracheally performed infections yielded in general better results on the growth inhibition than infections carried out with the same strain but by the subcutaneous route. Furthermore, in the experiments reported on in the present paper the authors could verify their earlier experience, namely that inhibition of growth of sarcoma cells is most pronounced at the time of maximal biological activity (logarithmic phase of multiplication) of the applied mycobacterium.     

E210K mutation in the gene encoding the beta3 chain of laminin-5 (LAMB3) is predictive of a phenotype of generalized atrophic benign epidermolysis bullosa

The spectrum of the embolic heart sounds (EHS) detected by precordial Doppler ultrasound has been previously characterized, but only on small volumes of venous air embolism (VAE). We sought to determine whether real-time wavelet analysis is useful in analyzing the signals of EHS and whether the embolic power of the EHS for larger volumes of air is proportionate to the volume of VAE that has been reported for small volumes of VAE. A series of small air boli (0.01, 0.02, 0.05, 0.07, 0.1, 0.15, 0.2, 0.3, 0.4, and 0.8 mL), followed by continuous infusion of larger volumes of air (0.8, 1.6, 2.4, 4.8, and 9.6 mL), was injected into the external jugular vein through a central catheter in seven pentobarbital-anesthetized dogs. We measured the spectrum of the Doppler heart sound (DHS) in a real-time manner by using wavelet analysis at different scales. Wavelet analysis at scale = 1 yielded satisfactory results in distinguishing abnormal EHS from normal DHS with high sensitivity (100%) and good positive predictive value (100%) compared with the conventional method, which requires an anesthesiologist to listen to the audio DHS signals in a real-time manner. There was a linear relationship (y = 1.08x + 7.89, r = 0.75, P < 0.001) between the cumulative embolic power of the EHS and the air volume introduced in the form of either bolus or continuous infusion. The 95% confidence intervals for slope and intercept were 0.89-1.27 and 7.65-8.13, respectively. Our results suggest that wavelet analysis is effective as a real-time monitor and that it is possible to distinguish larger volumes of air emboli based on previous injections of small volumes of air. Implications: The real-time wavelet analysis of the heart sound detected by precordial Doppler ultrasound may be useful in estimating larger volumes of air emboli based on previous injections of small volumes of air in anesthetized dogs.     

Surgery for epidermolysis bullosa in children: value of the association of inhalation anesthesia and locoregional anesthesia

Due to the cutaneous and mucosal fragility associated with epidermolysis bullosa, this disease is a source of various practical problems for the anaesthesiologist concerning the surgical posture, the monitoring of vital functions, the airways control and the vascular access, as all these procedures may worsen, sometimes dramatically, the lesions in these young patients, still in a precarious health state. Basing on published studies and their own experience, the authors have used in these patients a combined locoregional and general anaesthesia. The latter was obtained with isoflurane, administered in the non intubated and spontaneously breathing patient through a closed surgical isolation container (Vi-Drape), including the patient's head and ventilated with a ventilator generating a PEEP for long procedures. The results obtained during 9 procedures in 3 children are reported and discussed. For several shorter procedures (for example wound dressing), intramuscular ketamine was used.