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.     

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.     

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.     

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.     

Altered expression of a new antigen of the dermal-epidermal junction (NU-T2 DEJ Ag) in junctional epidermolysis bullosa

NU-T2 antigen (Ag) is a new and recently described antigen of the dermal-epidermal junction, recognized by an anti-CD1b monoclonal antibody denominated NU-T2. We studied NU-T2 Ag expression in junctional epidermolysis bullosa (13 patients) and in other forms of hereditary epidermolysis bullosa (23 patients), comparing the results with nicein expression. In junctional epidermolysis bullosa gravis type no differences were found between the expression of NU-T2 and nicein, both being negative in bullous as well as in non-bullous skin. Interestingly, in mitis type junctional epidermolysis bullosa, NU-T2 Ag was found to be absent or reduced in five of six patients both in lesional and in uncleaved skin. When compared with nicein expression, clearcut differences were found, further suggesting that these two antigens are different. These data confirm that NU-T2 Ag is a novel epitope of the dermal-epidermal junction, probably relevant in dermal-epidermal cohesion, and it could be responsible, together with nicein, 19-DEJ-1 and other adhesion molecules, for the different subtypes of junctional epidermolysis bullosa. Finally, NU-T2 monoclonal antibody is a new relevant tool for the diagnosis, classification, and prenatal diagnosis of junctional epidermolysis bullosa.     

Novel COL7A1 mutations in dystrophic forms of epidermolysis bullosa

OBJECTIVE: To characterize the use of the esophageal tracheal combitube (ETC) in trauma patients who fail orotracheal rapid sequence intubation (RSI). DESIGN: Prospective protocol design and retrospective chart review. MATERIALS AND METHODS: Flight nurses were trained in the use of the ETC by mannequin simulation, videotape review, and didactic sessions. ETC insertion was attempted after failure of two or more attempts at orotracheal RSI. Over a 12-month period, 12 patients had ETC insertion, and 10 cases qualified for review. Injuries, number of failed orotracheal RSI attempts, definitive airway, initial arterial blood gas results, and outcome were recorded. RESULTS: ETC insertion was successful in all 10 patients in whom it was attempted. Definitive airway control was achieved by conversion to orotracheal intubation in seven patients, emergency department cricothyroidotomy in one patient, and operative room tracheostomy in two patients. No patient died because of failure to control the airway. Seven patients requiring ETC had mandible fractures. CONCLUSION: ETC insertion is an effective method of airway control in trauma patients who fail orotracheal RSI. It may be particularly useful in the patient with maxillofacial trauma and offers a practical alternative to surgical cricothyroidotomy in difficult airway situations.     

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.     

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.     

Simultaneous manifestation of squamous cell carcinoma in identical twins with epidermolysis bullosa

We report a case of identical twins with recessive dystrophic epidermolysis bullosa, who developed squamous cell carcinoma in the hand at the latter half of their third decade. To our knowledge, this is the first case of manifestation of squamous cell carcinoma in identical twins and may contribute to the understanding of oncogenesis in such patients.     

Morphological survey of the cervicothoracic junctional region

In a randomised clinical study two new microprocessor-controlled tonometers working according to the Mackay-Marg principle (Tono-Pen and ProTon, two samples of each) and two established applanation tonometers [hand-held applanation tonometer, Draeger (HAT) and Goldmann tonometer] were compared with respect to accuracy, precision, examiner dependence and internal measurement differences (Tono-Pen I vs II, ProTon I vs II). The four tonometers were used by two examiners six times on each of 193 eyes of 100 patients of the university eye department. The same measuring procedure was performed on four cadaver eyes in situ, using a manometer system. The correlation coefficient between Tono-Pen and HAT and between ProTon and HAT was 0.9, with a high standard deviation, about +/- 3.1 mmHg. HAT gave the best results in repetitive measurements in the same eye (precision: r = 0.92, SD = +/- 1.41 mmHg). In applanation tonometers but not in Mackay-Marg tonometers measurement values depended significantly on the physician. There was a significantly higher standard deviation between Tono-Pen I and II than between ProTon I and II. Concerning in vitro calibration series on human bulbs (manometry), accuracy was high with each tonometer. The regression line of HAT is similar to that of ProTon. Tono-Pen overestimates the manometrically determined intraocular pressure. Because of their lack of precision the two new tonometers are not suitable for clinical use.     

Expression of collagenase and stromelysin in skin fibroblasts from recessive dystrophic epidermolysis bullosa

OBJECTIVE: To compare the effects of felodipine and placebo in patients with New York Heart Association functional class II or III and stable congestive heart failure despite treatment with an angiotensin converting enzyme inhibitor, diuretic, or digoxin, or any combination of these three drugs. PATIENTS AND DESIGN: 252 patients were randomised in a double blind, parallel group study after a 2-4 week placebo run-in to oral treatment with either felodipine extended release formulation or placebo 2.5-10 mg twice daily given in addition to existing background medication for a further 12 weeks. METHODS: Patients aged 18-75 years of either sex with chronic congestive heart failure due to ischaemic heart disease, hypertensive heart disease, or dilated cardiomyopathy with or without secondary mitral insufficiency that was stable during the preceding two months were included in the study. Treadmill exercise tests according to the modified Naughton protocol were performed at baseline, and after six, 11, and 12 weeks of treatment. Signs and symptoms of heart failure were assessed at every visit. Physical examination was performed and left ventricular ejection fraction measured at baseline and after 12 weeks. RESULTS: Mean (SD) baseline exercise test times increased from 434 (162) s and 480 (157) s for felodipine and placebo groups respectively to 541 (217) s and 591 (218) s at 12 weeks or the last visit. The change in exercise from baseline to last visit was 107 (141) s for patients given felodipine and 112 (128) s for those given placebo (P > 0.20). There was also no difference between treatments with respect to the other efficacy variables. There were few deaths in the study (felodipine n = 3, placebo n = 2). More patients who received felodipine were withdrawn from treatment (n = 29) than those who received placebo (n = 17). The most common adverse events of the 54 and 28 cited as reasons for withdrawal in the felodipine and placebo groups respectively were increased need for non-study heart failure treatment (n = 10; 8%)--that is, starting new medication or changes in the dosage of existing treatment for patients given felodipine, and nausea (n = 4; 3%) for those given placebo. Patients withdrawn from the study due to increased need for non-study heart failure treatment rapidly stabilised and recovered. CONCLUSION: Felodipine has not been shown to be of benefit in patients with mild to moderate heart failure.     

Anesthetic management in epidermolysis bullosa: review of 129 anesthetic episodes in 32 patients

Non-adrenergic non-cholinergic (NANC) inhibitory nerves have been described in all regions of the gastrointestinal tract, but have not been shown previously in the human gall bladder. Electrical field stimulation was used in the presence of various agonists and antagonists to show NANC inhibitory innervation in strips of human gall bladder muscle. Gall bladder strips were set up isometrically in an organ bath containing oxygenated Krebs's solution. Electrical field stimulation was applied at 10 Hz, pulse width 0.3 ms and supramaximal voltage at intervals of 3 to 5 minutes. Of 60 strips that contracted in response to electrical field stimulation, 30 showed relaxation on electrical field stimulation in the presence of either carbachol (5-10 microM) or else atropine (0.5-2 microM) plus cholecystokinin octapeptide (0.01-0.1 microM) or caerulein (0.1 nM) or histamine (5-10 microM). In 22 strips this relaxation was not abolished by guanethidine (2-5 microM) showing the NANC nature of this response. The NANC relaxation was abolished by L-nitroarginine (100 microM) and this effect was partly reversible by L arginine (200 microM). All responses to electrical field stimulation were abolished by tetrodotoxin (0.2-2 microM). These results show for the first time a NANC inhibitory innervation in human gall bladder muscle. The probable neurotransmitter is nitric oxide.     

Ordering of polymorphic markers in the chromosome region 3p21

Starting from five markers, with a well-defined order from distal to proximal 3p21, nine other markers could be inserted in this 3p21 map. Five were precisely mapped genetically. The other markers were ordered by FISH and/or deletion hybrid mapping. The overall 3p21 order from distal to proximal is as follows: D3S1298-D3S1260-(D3S966, D3S1448 (= D3S1449)-D3S1029-D3S32-D3S643-D3F15S2 -D3S2968-D3S1235-D3S1289-D3S1447-D3S1295.     

Paternal uniparental disomy for chromosome 1 revealed by molecular analysis of a patient with pycnodysostosis

OBJECTIVES: We sought to determine the prognostic capabilities of exercise thallium (Tl)-201 tomographic imaging performed relatively early (within 2 years) after coronary artery bypass graft surgery (CABG). BACKGROUND: Exercise testing is commonly performed after CABG, but few data exist demonstrating its prognostic value in this setting. METHODS: Four hundred eleven patients were followed up for a median duration of 5.8 years. Eleven prospectively chosen clinical, exercise and Tl-201 variables were tested for their associations with outcome end points by means of proportional hazards regression models. RESULTS: During follow-up there were 60 deaths from any cause, 53 initial cardiac deaths or nonfatal myocardial infarctions (MIs) and 22 late (>3 months after the Tl-201 study) revascularization procedures. The number of abnormal Tl-201 segments on the postexercise image was the only variable in the multivariate analyses to show a significant association with all three outcome end points: chi-square 7.3, p = 0.007 for overall mortality; chi-square 8.1, p = 0.004 for cardiac death or MI; chi-square 7.8, p = 0.005 for any cardiac event. Other independent predictors of outcome were exercise duration (chi-square 10.7, p = 0.001) and age (chi-square 3.9, p = 0.049) for overall mortality and exercise angina score (chi-square 8.7, p = 0.003) for cardiac death or MI. The 5-year survival rate free of cardiac death or MI was 93% for patients without angina and a normal image or small postexercise perfusion defect versus 71% for patients with angina and a medium or large defect. CONCLUSIONS: Exercise Tl-201 imaging performed within 2 years of CABG can stratify patients into low and high risk subgroups.