A Review of 52 Pedigrees with Epidermolysis Bullosa Simplex Identifying Ten Novel Mutations in KRT5 and KRT14 in Australia.

Epidermolysis bullosa simplex (EBS) is a rare heritable skin fragility disorder, most commonly caused by dominant mutations in KRT5 and KRT14. EBS shows clinical heterogeneity with localised, intermediate and generalised severe forms, which tend to correlate with the location and nature of the disease causing mutations. We therefore aimed to identify the KRT5 and KRT14 mutations in patients diagnosed with EBS in Australia, and explore in depth the genotype to the phenotype correlations in patients with novel variants. Australian patients who were diagnosed with EBS after referral to the Australian National Diagnostic Laboratory for EB were offered mutation screening in the KRT5 and KRT14 genes. From this, 32 different mutations in KRT5 and KRT14 were identified within 39 of 52 pedigrees. Ten of these mutations from 9 different pedigrees were novel, a further fatal case caused by KRT5 E477K is reported and in addition the third reported case of digenic inheritance in EBS was also observed.

A phase II randomized vehicle-controlled trial of intradermal allogeneic fibroblasts for recessive dystrophic epidermolysis bullosa.

BACKGROUND: Chronic wounds are a major source of morbidity and mortality in generalized severe recessive dystrophic epidermolysis bullosa (RDEB-GS). OBJECTIVE: This was a phase II double-blinded randomized controlled trial of intralesional allogeneic cultured fibroblasts in suspension solution versus suspension solution alone for wound healing in RDEB-GS. METHODS: Adult patients with RDEB-GS were screened for chronic ulcers and reduced collagen VII expression. Up to 6 pairs of symmetric wounds were measured and biopsied at baseline, then randomized to cultured allogeneic fibroblasts in a crystalloid suspension solution with 2% albumin or suspension solution alone. Ulcer size, collagen VII protein and messenger RNA expression, anchoring fibril numbers, morphology, and inflammatory markers were measured at 2 weeks and at 3, 6, and 12 months. RESULTS: All wounds healed significantly more rapidly with fibroblasts and vehicle injections, with an area decrease of 50% by 12 weeks, compared with noninjected wounds. Collagen VII expression increased to a similar degree in both study arms in wounds from 3 of 5 patients. LIMITATIONS: The number of patients with RDEB-GS who met inclusion criteria was a limitation, as was 1 trial center rather than multicenter. CONCLUSIONS: The injection of both allogeneic fibroblasts and suspension solution alone improved wound healing in chronic nonhealing RDEB-GS wounds independently of collagen VII regeneration. This may provide feasible therapy for wound healing in patients with RDEB-GS.

Main challenges facing the pharmaceutical sector in Buchanan County, Virginia: a pilot study.

INTRODUCTION: Little is known about the challenges facing the pharmaceutical sector in rural counties in the USA. The aim of this pilot study is to determine the main challenges facing the pharmaceutical sector and suggestions for improving the sector in Buchanan County, a poor and marginalized county in Central Appalachia, Virginia. METHODS: This cross-sectional study used the drop-by survey based on the first step of the modified Delphi Interview Technique. A convenience sample of healthcare professionals in Buchanan County were asked to complete a self-administered survey instrument between May and August 2011. RESULTS: A total of 16 healthcare professionals including six pharmacists completed the survey. The respondents had worked for an average of 13.4 (SD=10.7) years in the County (range: 1-33 years). The main challenges facing the pharmaceutical sector were drug abuse (n=11), doctor shopping by patients (n=9), early refills (n=7) and drug shortage (n=6). Respondents suggested increased patient education by pharmacists (n=6) and better coordination and communication between pharmacy and doctor (n=6) to improve the pharmaceutical sector in the County. CONCLUSIONS: Drug abuse, doctor shopping, early refills and drug shortage are the main challenges facing the pharmaceutical sector in Buchanan County. Concerted efforts are required to solve these problems. More research is required to confirm these findings.

Fibroblast-derived dermal matrix drives development of aggressive cutaneous squamous cell carcinoma in patients with recessive dystrophic epidermolysis bullosa.

Patients with the genetic skin blistering disease recessive dystrophic epidermolysis bullosa (RDEB) develop aggressive cutaneous squamous cell carcinoma (cSCC). Metastasis leading to mortality is greater in RDEB than in other patient groups with cSCC. Here we investigate the dermal component in RDEB using mRNA expression profiling to compare cultured fibroblasts isolated from individuals without cSCC and directly from tumor matrix in RDEB and non-RDEB samples. Although gene expression of RDEB normal skin fibroblasts resembled that of cancer-associated fibroblasts, RDEB cancer-associated fibroblasts exhibited a distinct and divergent gene expression profile, with a large proportion of the differentially expressed genes involved in matrix and cell adhesion. RDEB cancer-associated fibroblasts conferred increased adhesion and invasion to tumor and nontumor keratinocytes. Reduction of COL7A1, the defective gene in RDEB, in normal dermal fibroblasts led to increased type XII collagen, thrombospondin-1, and Wnt-5A, while reexpression of wild type COL7A1 in RDEB fibroblasts decreased type XII collagen, thrombospondin-1, and Wnt-5A expression, reduced tumor cell invasion in organotypic culture, and restricted tumor growth in vivo. Overall, our findings show that matrix composition in patients with RDEB is a permissive environment for tumor development, and type VII collagen directly regulates the composition of matrix proteins secreted by dermal and cancer-associated fibroblasts.

[The clinical significance of ABR testing in the diagnosis of the large vestibular aqueduct syndrome].

OBJECTIVE: To analyse the clinical audiological significance in the diagnosis of large vestibular aqueduct syndrome (LVAS) by the auditory brain stem response (ABR) testing. METHOD: Patients with sensorineural hearing loss were examined by temporal bone CT scanning from January, 2008 to September, 2009. The result of CT scanning of 70 cases inner ear malformation were analysed. Patients were divided into two groups, LVAS group including 38 cases (76 ears) and other inner ear malformation group including 32 cases (62 ears). All patient accepted clinical audiology analysis and auditory brainstem response (ABR) test. RESULT: Twenty-four cases (41 ears) of LVAS group were detected with ASNR in 2 3 cm by the ABR testing, the positive rate was 54%, while ASNR was not detected in patients of other inner ear malformations group. There was significant differences (P=0.01) of the ASNR between two groups. CONCLUSION: There is high incidence of LVAS on the patients with non-syndromic deafness. ASNR by ABR testing could help diagnosing the LVAS.

Effects of hydrophobic surfactant proteins on collapse of pulmonary surfactant monolayers.

To determine if hydrophobic surfactant proteins affect the stability of pulmonary surfactant monolayers at an air/water interface, the studies reported here compared the kinetics of collapse for the complete set of lipids in calf surfactant with and without the proteins. Monomolecular films spread at the surface of captive bubbles were compressed at 37 degrees C to surface pressures above 46 mN/m, at which collapse first occurred. The rate of area-compression required to maintain a constant surface pressure was measured to directly determine the rate of collapse. For films with and without the proteins, higher surface pressures initially produced faster collapse, but the rates then reached a maximum and decreased to values <0.04 min(-1) above 53 mN/m. The maximum rate for the lipids with the proteins (1.22 +/- 0.28 min(-1)) was almost twice the value for the lipids alone (0.71 +/- 0.15 min(-1)). Because small increments in surface pressure produced large shifts in the rate close to the fastest collapse, compressions at a series of constant speeds also established the threshold rate required to achieve high surface pressure as an indirect indication of the fastest collapse. Both samples produced a sharply defined threshold that occurred at slightly faster compression with the proteins present, supporting the conclusion of the direct measurements that the proteins produce a faster maximum rate of collapse. Our results indicate that at 47-53 mN/m, the hydrophobic surfactant proteins destabilize the compressed monolayers and tend to limit access to the higher surface pressures at which the lipid films become metastable.

The melting of pulmonary surfactant monolayers.

Monomolecular films of phospholipids in the liquid-expanded (LE) phase after supercompression to high surface pressures (pi), well above the equilibrium surface pressure (pi(e)) at which fluid films collapse from the interface to form a three-dimensional bulk phase, and in the tilted-condensed (TC) phase both replicate the resistance to collapse that is characteristic of alveolar films in the lungs. To provide the basis for determining which film is present in the alveolus, we measured the melting characteristics of monolayers containing TC dipalmitoyl phosphatidylcholine (DPPC), as well as supercompressed 1-palmitoyl-2-oleoyl phosphatidylcholine and calf lung surfactant extract (CLSE). Films generated by appropriate manipulations on a captive bubble were heated from < or =27 degrees C to > or =60 degrees C at different constant pi above pi(e). DPPC showed the abrupt expansion expected for the TC-LE phase transition, followed by the contraction produced by collapse. Supercompressed CLSE showed no evidence of the TC-LE expansion, arguing that supercompression did not simply convert the mixed lipid film to TC DPPC. For both DPPC and CLSE, the melting point, taken as the temperature at which collapse began, increased at higher pi, in contrast to 1-palmitoyl-2-oleoyl phosphatidylcholine, for which higher pi produced collapse at lower temperatures. For pi between 50 and 65 mN/m, DPPC melted at 48-55 degrees C, well above the main transition for bilayers at 41 degrees C. At each pi, CLSE melted at temperatures >10 degrees C lower. The distinct melting points for TC DPPC and supercompressed CLSE provide the basis by which the nature of the alveolar film might be determined from the temperature-dependence of pulmonary mechanics.

The anatomy, physics, and physiology of gas exchange surfaces: is there a universal function for pulmonary surfactant in animal respiratory structures?

(Orgeig and Daniels) This surfactant symposium reflects the integrative and multidisciplinary aims of the 1st ICRB, by encompassing in vitro and in vivo research, studies of vertebrates and invertebrates, and research across multiple disciplines. We explore the physical and structural challenges that face gas exchange surfaces in vertebrates and insects, by focusing on the role of the surfactant system. Pulmonary surfactant is a complex mixture of lipids and proteins that lines the air-liquid interface of the lungs of all air-breathing vertebrates, where it functions to vary surface tension with changing lung volume. We begin with a discussion of the extraordinary conservation of the blood-gas barrier among vertebrate respiratory organs, which has evolved to be extremely thin, thereby maximizing gas exchange, but simultaneously strong enough to withstand significant distension forces. The principal components of pulmonary surfactant are highly conserved, with a mixed phospholipid and neutral lipid interfacial film that is established, maintained and dynamically regulated by surfactant proteins (SP). A wide variation in the concentrations of individual components exists, however, and highlights lipidomic as well as proteomic adaptations to different physiological needs. As SP-B deficiency in mammals is lethal, oxidative stress to SP-B is detrimental to the biophysical function of pulmonary surfactant and SP-B is evolutionarily conserved across the vertebrates. It is likely that SP-B was essential for the evolutionary origin of pulmonary surfactant. We discuss three specific issues of the surfactant system to illustrate the diversity of function in animal respiratory structures. (1) Temperature: In vitro analyses of the behavior of different model surfactant films under dynamic conditions of surface tension and temperature suggest that, contrary to previous beliefs, the alveolar film may not have to be substantially enriched in the disaturated phospholipid, dipalmitoylphosphatidylcholine (DPPC), but that similar properties of rate of film formation can be achieved with more fluid films. Using an in vivo model of temperature change, a mammal that enters torpor, we show that film structure and function varies between surfactants isolated from torpid and active animals. (2) Spheres versus tubes: Surfactant is essential for lung stabilization in vertebrates, but its function is not restricted to the spherical alveolus. Instead, surfactant is also important in narrow tubular respiratory structures such as the terminal airways of mammals and the air capillaries of birds. (3). Insect tracheoles: We investigate the structure and function of the insect tracheal system and ask whether pulmonary surfactant also has a role in stabilizing these minute tubules. Our theoretical analysis suggests that a surfactant system may be required, in order to cope with surface tension during processes, such as molting, when the tracheae collapse and fill with water. Hence, despite observations by Wigglesworth in the 1930s of fluid-filled tracheoles, the challenge persists into the 21st century to determine whether this fluid is associated with a pulmonary-type surfactant system. Finally, we summarize the current status of the field and provide ideas for future research.

Distribution of coexisting solid and fluid phases alters the kinetics of collapse from phospholipid monolayers.

To determine how coexistence of liquid-expanded (LE) and tilted-condensed (TC) phases in phospholipid monolayers affects collapse from the air/water interface, we studied binary films containing dioleoyl phosphatidylcholine-dipalmitoyl phosphatidylcholine (DPPC) mixtures between 10 and 100% DPPC. Previously published results established that this range of compositions represents the LE-TC coexistence region at the equilibrium spreading pressure of 47 mN/m. When held at 49.5 mN/m on a captive bubble, the extent of total collapse fit with the LE area predicted by the phase diagram. The kinetics of collapse, however, when normalized for changes in the LE area, slowed with increasing mole fraction of DPPC. Surface area expressed as stretched exponential functions of time yielded an Avrami exponent that decreased from 1 for the homogeneously LE film to 0.3 for DPPC > or = 70%. Microscopic studies showed that the largest changes in kinetics occurred when either alterations of the initial composition or the process of collapse induced the films to cross the percolation threshold, so that the LE phase became divided into isolated domains. Our results show that although coexisting solid and fluid phases collapse to extents that are independent, the kinetics of collapse, corrected for differences in LE area, depend on the distribution of the two phases.

The collapse of monolayers containing pulmonary surfactant phospholipids is kinetically determined.

Prior studies have shown that during and after slow compressions of monomolecular films containing the complete set of purified phospholipids (PPL) from calf surfactant at an air/water interface, surface pressures (pi) reach and sustain values that are remarkably high relative to expectations from simple systems with model lipids. Microscopy shows that the liquid-expanded, tilted-condensed, and collapsed phases are present together in the PPL films between 45 and 65 mN/m. The Gibbs phase rule restricts equilibrium coexistence of three phases to a single pi for films with two components but not for more constituents. We therefore determined if the surprising stability of PPL reflects release from the thermodynamic restrictions of simple model systems by the presence of multiple components. Experiments with binary films containing dioleoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine first tested the predictions of the phase rule. The onset of three-phase coexistence, determined by fluorescence microscopy, and its termination, established by relaxation of collapsing films on a captive bubble, occurred at similar pi. Experiments for PPL using the same methods suggested that the three phases might coexist over a range of pi, but limited to approximately 2 mN/m, and extending below rather than above the coexistence pi for the binary films. Our results show that the PPL films at high pi must deviate from equilibrium and that they must then be metastable.

Rapid compressions in a captive bubble apparatus are isothermal.

Captive bubbles are commonly used to determine how interfacial films of pulmonary surfactant respond to changes in surface area, achieved by varying hydrostatic pressure. Although assumed to be isothermal, the gas phase temperature (Tg) would increase by >100 degrees C during compression from 1 to 3 atm if the process were adiabatic. To determine the actual change in temperature, we monitored pressure (P) and volume (V) during compressions lasting <1 s for bubbles with and without interfacial films and used P x V to evaluate Tg. P x V fell during and after the rapid compressions, consistent with reductions in n, the moles of gas phase molecules, because of increasing solubility in the subphase at higher P. As expected for a process with first-order kinetics, during 1 h after the rapid compression P x V decreased along a simple exponential curve. The temporal variation of n moles of gas was determined from P x V >10 min after the compression when the two phases should be isothermal. Back extrapolation of n then allowed calculation of Tg from P x V immediately after the compression. Our results indicate that for bubbles with or without interfacial films compressed to >3 atm within 1 s, the change in Tg is <2 degrees C.