Keratin gel in the management of Epidermolysis bullosa.

OBJECTIVE: Epidermolysis bullosa (EB) describes a number of genetically inherited conditions which cause skin fragility and minor trauma leading to skin damage, skin loss and wounding. Owing to the fragility of the skin and requirement for frequent dressing changes, at present, the optimal dressing(s) is not clear. Our objective was to assess the use of a keratin gel in the management of wounds in patients with different forms of EB. METHOD: We treated patients with different types of EB and a range of wounds with a novel keratin gel. In a convenience sample of consecutive patients, we introduced the keratin gel into their treatment regimen maintaining other aspects of their care. RESULTS: Patients reported faster healing and more resilient healed skin. Of the ten patients treated in this pilot study, six found the gel effective; two found it ineffective; and in two patients, it caused itching leading to discontinuation of the treatment. CONCLUSION: The results of this case study series suggest that keratin gel can be useful in the management of EB and are consistent with previous published experiences.

Elevated expression of adenosine A1 receptor in bronchial biopsy specimens from asthmatic subjects.

Asthmatics, unlike healthy subjects, experience bronchoconstriction in response to inhaled adenosine, and extracellular adenosine concentrations are elevated in the bronchoalveolar lavage fluid and exhaled breath condensate of asthmatic subjects. However, little is known about the location and expression of adenosine receptors in asthmatic airways. The aim of the present study was to investigate the distribution of adenosine A(1) receptors in bronchial biopsy specimens from mildly asthmatic steroid-naïve subjects and then compare the degree of expression with that of healthy subjects. Biopsy sections were immunostained using an adenosine A(1) receptor antibody, the selectivity of which was validated in specific experiments. Image analysis was then performed in order to determine differences in immunostaining intensity. Immunostaining of biopsy sections from the asthmatic subjects revealed strong expression of the A(1) receptor, located predominantly in the bronchial epithelium and bronchial smooth muscle. In comparison, very weak immunostaining was observed in biopsy specimens obtained from healthy subjects. Image analysis revealed that the intensity of positive staining of the asthmatic bronchial epithelium and smooth muscle regions was significantly greater than that observed for the healthy epithelium and smooth muscle. In conclusion, the sensitivity of asthmatics to inhaled adenosine coupled with increased adenosine A(1) receptor expression implies that these receptors play a role in the pathophysiology of this disease.

The management of general and disease specific ENT problems in children with Epidermolysis Bullosa--a retrospective case note review.

OBJECTIVE: Epidermolysis Bullosa encompasses a group of inherited disorders characterized by excessive susceptibility of the skin and mucosa to separate from underlying tissues following mechanical trauma. Information in the literature and guidance on the management of Ear, Nose and Throat problems in such children is scarce. The aim of this study is to report the experience of an Ear, Nose and Throat department in a tertiary paediatric hospital linked to a national Epidermolysis Bullosa unit, describing how children have presented and the care that has been given including a theatre protocol aimed at reducing shearing forces. METHODS: Retrospective case note review of Epidermolysis Bullosa patients referred to Otolaryngology over an 8-year period. RESULTS: Reviewing notes of 307 EB patients identified 15 that had been referred to the ENT department. Four children with middle ear effusions were effectively treated by watchful waiting or grommets. Three children with otitis externa had some relief from careful microsuction but reinfection from ulcers on other parts of the children's bodies tended to occur. One child with profound sensorineural hearing loss benefited from cochlear implantation but later passed away from unrelated sepsis. One child with intrinsic rhinitis was treated with steroid and then a salt-water nasal spray. Five children had nasal crusting documented, although this was not a specific reason for referral. When severe this had been treated with topical steroids by the dermatology team. One patient with obstructive sleep aponea had tonsillectomy and examination of the post-nasal space. Of the five patients referred with glottic or supraglottic scarring, the management of four included a tracheostomy. When carried out tracheostomy sites healed well in each case. With repeated endoscopic procedures it was subsequently possible to remove the tracheostomy in the two of the children. Using a special theatre protocol no new ulcers were recorded as being precipitated by any of the procedures children underwent. CONCLUSIONS: Optimal management depends on the support of a multidisciplinary team, including otolaryngologists, pediatricians, dermatologists, anaesthetists, and specialist nurses. Adherence to a protocol for theatre management can help avoid intraoperative complications.

Reciprocal role of the inward currents ib, Na and i(f) in controlling and stabilizing pacemaker frequency of rabbit sino-atrial node cells.

Experiments and computations were done to clarify the role of the various inward currents in generating and modulating pacemaker frequency. Ionic currents in rabbit single isolated sino-atrial (SA) node cells were measured using the nystatin-permeabilized patch-clamp technique. The results were used to refine the Noble-DiFrancesco-Denyer model of spontaneous pacemaker activity of the SA node. This model was then used to show that the pacemaker frequency is relatively insensitive to the magnitude of the sodium-dependent inward background current ib, Na. This is because reducing ib, Na hyperpolarizes the cell and so activates more hyperpolarizing-activated current, i(f), whereas the converse occurs when ib, Na is increased. The result is that i(f) and ib, Na replace one another and so stabilize nodal pacemaker frequency.

Molecular and pharmacological characterization of the human CCKB receptor.

The human cholecystokinin B (CCKB) receptor has been isolated from a human temporal cortex cDNA library. Transient transfection of the receptor into COS-M6 cells resulted in high specific binding of 125I-sulphated CCK-8 labelled with Bolton and Hunter Reagent (KD = 31 pM). Competition experiments yielded the expected CCKB receptor ligand binding profile for agonists and antagonists. Similar results were obtained in human small cell lung carcinoma cells, which express an endogenous CCKB receptor. Extensive functional characterization of the receptor was performed in stably transfected HeLa cells using intracellular calcium imaging and microphysiometry techniques. Molecular analysis of the human CCKB receptor using Southern blotting of genomic DNA suggests the presence of a single gene for the CCKB receptor with no closely related homologues. This was confirmed by the polymerase chain reaction cloning of identical receptor coding sequences from human small cell lung carcinoma cells and human gastric enterochromaffin-like cell-oma (ECLoma) tissue.

The eye in epidermolysis bullosa.

AIMS: To describe the ophthalmic findings in a large cohort of epidermolysis bullosa (EB) patients managed in one large specialist centre. METHODS: A case note review of consecutive patients seen at Great Ormond Street Children's Hospital. Data on the dermatological disease, ophthalmic history, and examination were collected and coded onto a data sheet. RESULTS: 181 patients: 50 (28%) simplex EB; 15 (8%) junctional EB; 28 (15%) autosomal dominant dystrophic EB; 72 (40%) autosomal recessive dystrophic EB; nine patients (5%) with dystrophic EB whose inheritance could not be ascertained; and seven cases (4%) of EB that could not be classified. Ocular problems were found in 12% (n = 6) of simplex patients and 40% (n = 6) of those with junctional disease. One patient (of 28) in the autosomal dominant dystrophic group had ocular involvement and 51% (37/72) of patients in the autosomal recessive dystrophic group had ophthalmic complications: corneal (25/72), lid ectropions (3/72), lid blisters (5/72), and symblepharon (3/72). CONCLUSION: Ophthalmic complications are common in EB overall but the incidence varies widely with subtype. Ophthalmic complications are the most severe in the dystrophic recessive and junctional subtypes where there is a need for extra vigilance. The major treatment modality was use of ocular lubricants.

Adaptive Aerosol Delivery (AAD) technology.

Jet nebulisers have, since the 1920s, been used for delivery of inhaled drugs for the treatment of asthma, chronic-obstructive pulmonary disease and pulmonary infections. During the last two decades, recognition of the shortcomings of conventional nebulisers has led to the development of new "intelligent" nebulisers such as the Adaptive Aerosol Delivery (AAD), Profile Therapeutics, a Respironics company) systems. Diseases of the airways have traditionally been logical candidates for treatment with inhaled drugs. The introduction of the "intelligent" nebulisers has, however, broadened the possibilities for inhaled treatment to include drugs targeted for systemic diseases. These nebulisers offer the possibility to deliver more precise doses of drug, maximise lung deposition, enhance adherence to treatment and compliance with the device through feedback to the patient, and last but not least, offer the possibility to reduce nebulisation times.

A method for isolating rabbit sinoatrial node cells which maintains their natural shape.

A method for isolating rabbit sinoatrial cells is described. This gives long, thin cells (length from 40 to 150 micron) which retain their elongated shape in normal calcium concentration. Round cells are also seen, many of which come from the rounding up of elongated cells.

Effects of equipment dead space and pediatric breathing patterns on inhaled mass of nebulized budesonide.

Inhaled mass, the quantity of aerosolized drug actually inhaled by a patient, can be estimated in vitro by using a piston pump and an inhaled mass filter in a manner that simulates in vivo aerosol delivery. For pediatric patients, measurement with an inhaled mass filter with a large equipment dead space (VDEQ) in relation to a small tidal volume (VT) may underestimate the inhaled mass. The present study investigated the impact of VDEQ on the accuracy of in vitro measured inhaled mass of budesonide suspension for nebulization using Spira Module 1 jet nebulizers (Respiratory Care Center, Hämeenlinna, Finland), inhaled mass filters, VDEQs of different sizes, and pediatric breathing patterns. The VDEQ varied between 14 and 108 mL, and the breathing patterns corresponded to a VT between 50 and 500 mL, to breathing frequencies of 40 to 12 per min-1, to a duty cycle of 0.5 for all breathing patterns, and to a nebulization time of 2 minutes. The results showed that the inhaled mass was a function of the VDEQ for each breathing pattern as defined by the inspiratory minute volume (VI). For a large VT, a small VDEQ affected the inhaled mass of budesonide only marginally, but as the VDEQ increased, the measured inhaled mass decreased to the point that for a VDEQ larger than the VT, the inhaled mass was zero. When the inhaled mass was expressed as a function of an effective volume (VEFF) (i.e., VI corrected for VDEQ), the results showed a linear correlation (R2 = 0.921) between the volume of aerosol inhaled through the nebulizer and the inhaled mass of budesonide. The results of the study indicate that VDEQ has a critical effect on the measurement of inhaled mass in vitro for conventional jet nebulizers using pediatric breathing patterns. This means that the in vitro measured inhaled mass of drug can seriously underestimate the in vivo value. When pediatric breathing patterns are used in vitro, a correction of the VT by the VDEQ should be made in order to more accurately reflect the in vivo inhaled mass of drug.

Breathing patterns and aerosol delivery: impact of regular human patterns, and sine and square waveforms on rate of delivery.

In vitro tests are commonly employed to assess nebulizer performance. Whether the square or sine waveforms employed during in vitro tests could alter the nebulizer performance compared to that observed when a patient breathes through the nebulizer is debatable. Accordingly, the aim of this in vitro study was to compare the rates of delivery from nebulizers with simulated human breathing patterns to those obtained with matching sine and square waveforms. Regular human breathing patterns with tidal volumes (VT) of approximately 40, approximately 200, approximately 500, and approximately 800 mL were selected. Sine and square waveforms that matched the VT, peak inspiratory flow rate (PIF), breathing frequency (f), and inspiratory duty cycle (t(i)/t(tot)) of the human breathing patterns were created with a breathing simulator. The rate of delivery of nebulized technetium-99m-labeled diethylenetriamine pentaacetic acid (99mTC-DTPA) from two different jet nebulizer brands was determined. The rate of delivery was defined as the amount of the 99mTC-DTPA deposited during 30 sec of nebulization on a filter placed between the nebulizer and the breathing simulator. The rate of delivery of 99mTC-DTPA with the human breathing pattern was similar to that measured with the matching sine or square waveforms for either nebulizer. The configuration of the breath (PIF, VT, f, t(i)/t(tot)) did, however, influence the rate of delivery. In conclusion, the shape of the waveform, in other words, one resulting from a human breathing pattern, or a matching sine or square waveform, did not influence the rate of 99mTC-DTPA delivery from a nebulizer in vitro.

Validation of a new breathing simulator generating and measuring inhaled aerosol with adult breathing patterns.

The use of breathing simulators for the in vitro determination of the inhaled mass of drug from nebulizers has become widely accepted. Their use is, however, based on the assumption that there is a correlation between the in vitro and in vivo inhaled mass of drug. The aim of the study was therefore to investigate whether a new breathing simulator--the MIMIC Breathing Emulator (Medic-Aid Limited, Bognor Regis, UK)--could accurately emulate the in vivo inhaled mass of budesonide suspension for nebulization. Eight adult healthy subjects were included. Each subject inhaled for 2 min from a Spira Module 1 jet nebulizer (Respiratory Care Center, Hämeenlinna, Finland), charged with 1.0 mg of budesonide suspension for nebulization (0.5 mg mL-1, 2 mL suspension, AstraZeneca, Sweden) and supplied with an inhaled mass filter between the nebulizer and the subject. The breathing patterns were recorded during the nebulization and simulated in vitro at two different experimental sites (simulations A and B) with the breathing simulator. With the patients breathing through the filters (in vivo test), inhaled mass of budesonide averaged 103.6 micrograms. The two in vitro experiments using the simulator revealed similar results with in vitro simulation A equal to 101.0 micrograms and simulation B 99.1 micrograms. There were no statistically significant differences between the in vivo results and those of in vitro simulation A. Results were significantly different for simulation B (p = 0.032) although the difference was less than 4.5%. These data indicate that the breathing simulator can be used to accurately simulate sine waveforms, human breathing patterns, and the in vitro and in vivo inhaled mass of budesonide suspension for nebulization.

Prenatal diagnosis for severe inherited skin disorders: 25 years' experience.

BACKGROUND: Over the last 25 years there have been major advances in methods for prenatal testing of inherited skin disorders. Since 1979, our group at the St John's Institute of Dermatology has performed 269 prenatal diagnoses, using a variety of approaches, including fetal skin biopsy (FSB), chorionic villus sampling (CVS) and preimplantation genetic diagnosis (PGD). OBJECTIVES: This study was designed to review the clinical indications, testing procedures and laboratory analyses for all prenatal tests conducted at St John's over this period. METHODS: FSBs were examined for morphological and, when relevant or feasible, immunohistochemical abnormalities. The DNA-based tests involved screening by nucleotide sequencing, restriction enzyme digests or, in a few cases, by linkage analysis. Results Of the 269 tests, 191 were FSB, 76 were CVS and two were PGD. The major indications for FSB were epidermolysis bullosa (EB) (138 cases, including 88 junctional and 48 dystrophic), ichthyoses (37 cases, including 22 tests for harlequin ichthyosis) and oculocutaneous albinism (12 cases). Of the CVS procedures, 75 were for EB (40 junctional, 35 dystrophic) and one was for the EEC (ectrodactyly, ectodermal dysplasia, clefting) syndrome. Both of the PGD procedures were for the skin fragility-ectodermal dysplasia syndrome. All tests provided accurate diagnoses and the fetal loss rate was approximately 1% for both FSB and CVS. CONCLUSIONS: The development of prenatal testing has proved to be of great benefit for individuals or couples at risk of having children with severe inherited skin disorders and, in the absence of a cure, prenatal testing along with appropriate counselling has become an important translational benefit of basic research and an integral part of clinical management.

Prenatal diagnosis of Herlitz junctional epidermolysis bullosa in nonidentical twins.

Advances in molecular diagnostics have led to the feasibility of DNA-based prenatal testing in families at risk for recurrence of severe forms of both dystrophic and junctional epidermolysis bullosa. In this report, we describe prenatal testing in a woman who previously had a child affected with Herlitz junctional epidermolysis bullosa. However, in her second pregnancy, she was found to have dichorionic diamniotic twins. DNA analysis of a pathogenic mutation and informative intragenic polymorphisms (LAMB3 gene) predicted one fetus to be affected and the other unaffected. Selective termination of the affected fetus was performed, and pregnancy with the unaffected fetus was continued, leading to full term delivery of a healthy girl with no skin blisters. This is the first reported case of DNA analysis in a twin pregnancy at risk of Herlitz junctional epidermolysis bullosa, with successful diagnosis and selective termination of one affected twin.

Complete paternal uniparental isodisomy of chromosome 1 resulting in Herlitz junctional epidermolysis bullosa.

Herlitz junctional epidermolysis bullosa (JEB) is an autosomal recessive mechanobullous disorder that results from loss-of-function mutations in the genes encoding the basement membrane component, laminin 5. Typically, there are frameshift, splice site or nonsense mutations on both alleles of either the LAMA3, LAMB3 or LAMC2 genes, with affected individuals inheriting one mutated allele from each parent. In this report, we describe a patient with Herlitz JEB in whom DNA analysis revealed homozygosity for the recurrent nonsense mutation R635X in LAMB3, located on chromosome 1q32.2. However, screening of parental DNA showed that although the patient's father was a heterozygous carrier of this mutation, the mother's DNA showed only wild-type sequence. Subsequent genotype analysis using 13 microsatellite markers spanning chromosome 1 revealed that the affected child was homozygous for the entire series of markers tested and that all of the alleles originated from the father. These results indicate that the Herlitz JEB phenotype in this patient is due to complete paternal isodisomy of chromosome 1 and reduction to homozygosity of the mutant LAMB3 gene locus. This is the fourth case of uniparental disomy to be described in Herlitz JEB, but it represents the first example of complete paternal isodisomy for chromosome 1 with a pathogenic mutation in the LAMB3 gene. These findings have important implications for mutation screening in JEB and for genetic counselling.

Management of severe blistering disorders.

Sufferers of the genetically determined blistering skin condition epidermolysis bullosa require specialized management throughout life. This management must begin shortly after birth in order to minimize additional skin damage, some of which may lead to permanent disability. Even the gentle handling practised routinely in neonatal units will result in painful blistering and loss of skin in the severely affected infant. Procedures such as cannulation, screening and monitoring must be modified if unnecessary trauma is to be avoided.

Rabbit sino-atrial node cells: isolation and electrophysiological properties.

1. A method has been developed for isolating calcium-tolerant, single rabbit sinoatrial node cells which maintain their natural shape following isolation. The majority of viable, spontaneously active cells were elongated and measured about 100 microns in length. 2. Staining fixed cells with Haematoxylin-Eosin revealed that a 'cell' with projections was usually an aggregate of more than one cell. 3. Single, elongated, spontaneously active cells were current and voltage clamped using the whole-cell configuration of the patch-clamp recording technique. The spontaneous activity and time-dependent currents recorded were similar to those reported previously in multicellular nodal preparations and in single cells. 4. An assessment was made of the time course of L-type calcium current run-down: a stable period of between 10 and 20 min followed by a rapid run-down (over about 2 min) was typically observed. 5. In most cells, a fast, TTX-sensitive Na+ current component was seen. A few cells showed a transient outward K+ current (iA). 6. The activation range for the hyperpolarization-activated current, if, varied from cell to cell. In the majority of actively beating cells, the threshold for if was near the maximum diastolic potential (about -65 mV in most cells) but in other cells, no if could be recorded within the pacemaker range. 7. Millimolar concentrations of MnCl2 caused a marked increase in if, but only when the pipette solution did not contain EGTA. Inclusion of EGTA (to buffer Ca2+ to about pCa 8) significantly reduced the effect of Mn2+ which therefore probably occurs through inhibition of Na(+)-Ca2+ exchange and consequent rise in intracellular Ca2+ concentration.