Human skin barrier formation takes place via a cubic to lamellar lipid phase transition as analyzed by cryo-electron microscopy and EM-simulation.

The skin's permeability barrier consists of stacked lipid sheets of splayed ceramides, cholesterol and free fatty acids, positioned intercellularly in the stratum corneum. We report here on the early stage of skin barrier formation taking place inside the tubuloreticular system in the secretory cells of the topmost viable epidermis and in the intercellular space between viable epidermis and stratum corneum. The barrier formation process was analysed in situ in its near-native state, using cryo-EM combined with molecular dynamics modeling and EM simulation. Stacks of lamellae appear towards the periphery of the tubuloreticular system and they are closely associated with granular regions. Only models based on a bicontinuous cubic phase organization proved compatible with the granular cryo-EM patterns. Only models based on a dehydrated lamellar phase organization agreed with the lamellar cryo-EM patterns. The data support that human skin barrier formation takes place via a cubic to lamellar lipid phase transition.

Acute skin and hair symptoms followed by severe, delayed eye complications in subjects using the synthetic opioid MT-45.

BACKGROUND: The introduction of unclassified new psychoactive substances (NPS) on the recreational drugs market through open online sale ('legal highs' or 'Internet drugs') continues unabated and represents a growing health hazard. The use of NPS has resulted in numerous, severe, adverse events and fatalities, due to unintended overdose or unknown toxic side-effects. OBJECTIVES: To try to find a possible common underlying cause for the skin-hair-eye symptoms complex observed in three men. METHODS: From late 2013 to mid-2014, three Swedish men aged 23-34 years with a history of recreational drug use independently presented with similar and very remarkable clinical signs, requiring extensive examination and prolonged treatment. RESULTS: Common clinical signs included hair depigmentation, hair loss, widespread folliculitis and dermatitis, painful intertriginous dermatitis, dry eyes, and elevated liver enzymes. Two of them also showed transverse white Mees' lines (leukonychia striata) on the fingernails and toenails, suggesting a temporary, drug-induced, disorganized keratinization. The clinical signs gradually disappeared over time. However, later on, two developed severe bilateral secondary cataracts requiring surgery. Because drug tests within the Swedish STRIDA project had demonstrated intake of the NPS opioid MT-45 in all patients, this was suspected to be the common causative agent. CONCLUSIONS: These cases highlight the importance for physicians and health professionals to consider the increasing number of novel, untested recreational drugs, as a potential cause of unusual and otherwise unrecognized clinical signs and symptoms.

Update of technologies for examining the stratum corneum at the molecular level.

Understanding the molecular organization of the stratum corneum is still an outstanding problem, despite being both fundamentally and clinically significant. There is a need to develop methodology that yields molecular-level resolution of the stratum corneum components in their native state, without introducing artefacts. We outline here the recent success of cryo-electron microscopy of vitreous sections (CEMOVIS) combined with electron microscopy simulation to elucidate the molecular organization of the stratum corneum in its near-native state. Furthermore, some emerging technologies for studying the physical properties and dynamic behaviour of native stratum corneum at the molecular level are briefly reviewed. These encompass multiphoton microscopy (MPM), polarization transfer solid-state nuclear magnetic resonance (PTssNMR) and PeakForce tapping-mode atomic force microscopy combined with frequency-modulation Kelvin probe force microscopy (KPFM). CEMOVIS combined with electron microscopy simulation allows for molecular structure determination in situ in native stratum corneum, while MPM allows probing of the stratum corneum local physicochemical properties such as fluorophore diffusion coefficients, water content and pH. PTssNMR allows for evaluation of the molecular mobility of stratum corneum keratin and lipid components, and PeakForce KPFM allows for analysis of the local nanomechanical properties of stratum corneum. These emerging techno-logies may contribute to a molecular-level understanding of stratum corneum structure and function in vivo.

Current understanding of skin barrier morphology.

It was recently shown that the stratum corneum fat layer, i.e. the skin permeability barrier, has a unique molecular arrangement - stacked bilayers of fully extended ceramides with cholesterol molecules associated with the ceramide sphingoid moiety. We here give a brief historical perspective to the field of skin permeability barrier research and specifically address some selected contributions to the current understanding of skin barrier morphology.

Sorption properties of the human stratum corneum.

Water sorption is important for the overall structure and function of keratinized tissues such as the human epidermal stratum corneum (SC). In this study we report on a gravimetric method for studying sorption properties of human SC, both from heel and female breast skin. Changes in mass were measured as the relative humidity was altered in steps under controlled environmental conditions. The possibility of hysteresis is also discussed. Furthermore, we have found that the sorption time constants show triphasic behaviour during absorption, but not during desorption. This behaviour is connected to the 3 different types of water present in the SC. Water also enters the SC much more rapidly compared to its exit at relative humidities <50%. Finally, the amount of time between sample preparation and onset of measurement seems to have an effect on the absorption rate, but less on the total amount of water absorbed.

Molecular cryo-electron tomography of vitreous tissue sections: current challenges.

Electron tomography of vitreous tissue sections (tissue TOVIS) allows the study of the three-dimensional structure of molecular complexes in a near-native cellular context. Its usage is, however, limited by an unfortunate combination of noisy and incomplete data, by a technically demanding sample preparation procedure, and by a disposition for specimen degradation during data collection. Here we outline some major challenges as experienced from the application of TOVIS to human skin. We further consider a number of practical measures as well as theoretical approaches for its future development.

Stratum corneum lipid organization as observed by atomic force, confocal and two-photon excitation fluorescence microscopy.

Skin moisturization is largely a function of stratum corneum barrier capacity, which in turn is a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix [J. Invest. Dermatol.18, 433 (1952); AIChE J. 21, 985 (1975); Acta Derm. Venereol.74, 1 (1994); J. Invest. Dermatol.117, 830 (2001)]. Three unsolved key questions with respect to this lipid matrix' structural organization [Acta Derm. Venereol.74, 1 (1994); J. Invest. Dermatol.117, 830 (2001); J. Invest. Dermatol.118, 897 (2002); J. Invest. Dermatol.118, 899 (2002)] are: i) whether the lipid matrix is constituted by a single-gel phase or by co-existing solid (crystalline or gel) domains, ii) whether a separate fluid (liquid crystalline) phase is present and iii) whether the local pH has a direct effect on the lipid matrix' phase behaviour. Using an array of complementary visual-related biophysical techniques (e.g. atomic force microscopy and confocal/two-photon excitation fluorescence microscopy), it was recently shown that reconstituted membranes composed of extracted decontaminated human stratum corneum lipids do not form a fluid phase, but exclusively a single-gel phase that segregates into co-existing microscopic domains below pH 6 [Biophys. J.93, 3142 (2007)]. It was further shown that the role of cholesterol is related to dispersion of ceramide-enriched domains. This effect is counteracted by the presence of free fatty acids, which mix with skin ceramides but not with cholesterol.

Nanostructure of the stratum corneum extracellular lipid matrix as observed by cryo-electron microscopy of vitreous skin sections.

In recent years, high-resolution cryo-electron microscopy of vitreous skin sections has been used to visualize the formation and structure of the human stratum corneum extracellular lipid matrix. The aim of the present work was to summarize these findings. It is proposed that skin barrier formation does not take place as a 'lamellar body' fusion process, but as a lamellar 'unfolding' of a small lattice parameter lipid 'phase' with cubic-like symmetry with subsequent 'crystallization' and concomitant lamellar re-organization of the extracellular lipid matrix.

Stratum corneum keratin structure, function and formation - a comprehensive review.

A comprehensive review on stratum corneum keratin organization, largely based on the recently published cubic rod-packing and membrane templating model [J. Invest. Dermatol., 123, 2004, 715], is presented. Keratin is the major non-aqueous component (wt/wt) of stratum corneum. As 90-100% of the stratum corneum water is thought to be located intracellularly one may presume that keratin also is a major factor (together with filaggrin-derived free amino acids) determining stratum corneum hydration level and water holding capacity. This water holding capacity depends in turn on the structural organization of the corneocyte keratin intermediate filament network. The cubic rod-packing model for the structure and function of the stratum corneum cell matrix postulates that corneocyte keratin filaments are arranged according to a cubic-like rod-packing symmetry. It is in accordance with the cryo-electron density pattern of the native corneocyte keratin matrix and could account for the swelling behaviour and the mechanical properties of mammalian stratum corneum. The membrane templating model for keratin dynamics and for the formation of the stratum corneum cell matrix postulates the presence in viable epidermal cellular space of a highly dynamic small lattice parameter (<30 nm) membrane structure with cubic-like symmetry, to which keratin is associated. It further proposes that membrane templating, rather than spontaneous self-assembly, is responsible for keratin intermediate filament formation and dynamics. It is in accordance with the cryo-electron density patterns of the native keratinocyte cytoplasmic space and could account for the characteristic features of the keratin network formation process, the dynamic properties of keratin intermediate filaments, the close lipid association of keratin, the insolubility in non-denaturating buffers and pronounced polymorphism of keratin assembled in vitro, and the measured reduction in cell-volume and hydration level between stratum granulosum and stratum corneum.

Skin barrier formation: the membrane folding model.

We propose that skin barrier morphogenesis may take place via a continuous and highly dynamic process of intersection-free membrane unfolding with a concomitant crystallization of the emerging multilamellar lipid structure representing the developing skin barrier. This implies that the trans-Golgi network and lamellar bodies of the uppermost stratum granulosum cells as well as the multilamellar lipid matrix of the intercellular space at the border zone between stratum granulosum and stratum corneum could be representations of one and the same continuous membrane structure. The profound difference between the earlier Landmann model and the membrane folding model presented here is that the Landmann model includes changes in membrane topology, whereas topology is kept constant during skin barrier formation according to the membrane folding model. The main advantages of the membrane folding model with respect to the Landmann model are the following: (i) smaller energy cost (involves no budding or fusion); (ii) conserves membrane continuity (preserves water compartmentalization and allows control hereof; membrane continuity essential for barrier function); (iii) allows meticulous control (the thermodynamics of the unfolding procedure are related to curvature energy); (iv) faster (milliseconds, as membrane unfolding basically represents a phase transition from cubic-like to lamellar morphology; involves no budding or fusion); (v) membrane folding between lamellar and cubic-like morphologies has been identified in numerous biologic systems; (vi) there is experimental evidence for an "extensive intracellular tubulo-reticular cisternal membrane system within the apical cytosol of the outermost stratum granulosum"; and (vii) may explain the reported plethora of forms, numbers, sizes and general appearances of "lamellar bodies" in transmission electron microscopy micrographs.

Skin barrier structure and function: the single gel phase model.

A new model for the structure and function of the mammalian skin barrier is postulated. It is proposed that the skin barrier, i.e., the intercellular lipid within the stratum corneum, exists as a single and coherent lamellar gel phase. This membrane structure is stabilized by the very particular lipid composition and lipid chain length distributions of the stratum corneum intercellular space and has virtually no phase boundaries. The intact, i.e., unperturbed, single and coherent lamellar gel phase is proposed to be mainly located at the lower half of stratum corneum. Further up, crystalline segregation and phase separation may occur as a result of the desquamation process. The single gel phase model differs significantly from earlier models in that it predicts that no phase separation, neither between liquid crystalline and gel phases nor between different crystalline phases with hexagonal and orthorhombic chain packing, respectively, is present in the unperturbed barrier structure. The new skin barrier model may explain: (i) the measured water permeability of stratum corneum; (ii) the particular lipid composition of the stratum corneum intercellular space; (iii) the absence of swelling of the stratum corneum intercellular lipid matrix upon hydration; and (iv) the simultaneous presence of hexagonal and orthorhombic hydrocarbon chain packing of the stratum corneum intercellular lipid matrix at physiologic temperatures. Further, the new model is consistent with skin barrier formation according to the membrane folding model of Norlén (2001). This new theoretical model could fully account for the extraordinary barrier capacity of mammalian skin and is hereafter referred to as the single gel phase model.

A new computer-based evaporimeter system for rapid and precise measurements of water diffusion through stratum corneum in vitro.

It is important to have reliable methods for evaluation of skin barrier function when questions such as barrier perturbing effects of different agents and occlusive effects of different formulations are to be elucidated. A wealth of clinical work relates to measurements of transepidermal water loss in vivo, a method much affected by ambient air relative humidity, temperature, skin irritation processes, psychologic status of the subject, etc., factors that cause the method to suffer from low precision (i.e., high random error). Relating to these obstacles, we have developed a closed in vitro system for measurements of water diffusion rate through pieces of isolated stratum corneum at steady-state conditions, where the relative humidity and temperature is held constant and data can be collected continuously. Our evaporimeter-based in vitro system has a more than 3-fold higher precision (lower random error) ( approximately 10%) than measurements of transepidermal water loss in vivo ( approximately 35%). The results of our study show that: (i) the corneocyte envelopes contribute to the barrier capacity of stratum corneum; (ii) removal of the lipid intercellular matrix results in approximately a 3-fold increase in the water diffusion rate through the isolated stratum corneum (n = 20; p < 0.05), not a 100-fold as has previously been suggested; (iii) exposure to sodium dodecyl sulfate in water does neither alter the water diffusion rate (n = 10; p > 0.05) nor the water holding capacity (n = 10; p > 0.05) of stratum corneum; (iv) exposure to 1 M CaCl2 in water yields an increased water diffusion rate through stratum corneum (n = 10; p < 0.05); and (v) when applied to the stratum corneum in excess concentrations, the penetration enhancer Azone has occlusive effects on water diffusion through the stratum corneum (n = 6; p < 0.05).

Inter- and intra-individual differences in human stratum corneum lipid content related to physical parameters of skin barrier function in vivo.

For a full understanding of the properties of the human skin barrier, physical macroscopic parameters of barrier function must be correlated to the structural organization of the barrier on a molecular level. This study was undertaken to relate differences in the relative composition of the three main lipid classes of human stratum corneum, i.e., free fatty acids, cholesterol, and ceramides, to differences in transepidermal water loss, stratum corneum electrical impedance, and corneometer value. A new high performance liquid chromatography/light scattering detection-based analysis method recently developed was used for collection of quantitative lipid data in conjunction with gas chromatography/mass spectrometry/flame ionization detection measurements on the free fatty acid fraction. After subtraction of contaminating lipid fractions we have estimated the molar ratio of the human skin barrier lipid composition to be, respectively, 15% cholesterol esters, 16% saturated long chain free fatty acids, 32% cholesterol, and 37% ceramides. The inter-individual difference in the relative amount of free fatty acids, cholesterol, and ceramides, respectively, can be >100% in the individual case. It was found that the relative amount of ceramides to cholesterol is larger in the wrist area, paralleled by a higher transepidermal water loss and corneometer value as well as different skin electrical impedance values as compared with the upper forearm area. We conclude that the site-dependent differences in the stratum corneum lipid composition are small compared with the large inter-individual variation. Interestingly, in the individual case, no correlation was registered between relative ceramide content and barrier properties.

A novel approach to the understanding of human skin barrier function.

The basis for externally caused skin disorders is penetration of the skin barrier. A recent model for the skin barrier, the domain mosaic model, based on current knowledge of the physics of lipid bilayer organization gave tentative explanations for several aspects of function. It is demonstrated here that a development of the model explains how the requirements are met for a water-tight structure that will still allow a controlled, minute loss of water, the perspiratio insensibilis, necessary for maintaining plasticity of the keratin. A major advantage of the extended model is that it allows an interpretation of the changes imposed on the structure when in contact with detergents and/or penetration enhancers.

Transrectal ultrasound with separate measurement of the transition zone volume predicts the short-term outcome after transurethral resection of the prostate.

OBJECTIVES: To determine whether the volume of prostatic adenomas as assessed by transrectal ultrasound (TRUS) influenced the outcome after transurethral resection of the prostate (TURP). METHODS: TRUS with total prostate and transition zone (TZ) volume determinations was performed preoperatively in 298 consecutive patients undergoing TURP for benign prostatic hyperplasia without prior urodynamic evaluation. Postoperatively, the outcome of surgery was stated as excellent (no or minor remaining symptoms), improved (but with some remaining symptoms), or failure (the same or aggravated symptoms) according to a patient-administered questionnaire. Six possible risk factors were evaluated: TZ volume 20 cc or less, neurologic disorders, previous TURP/transurethral incision of the prostate (TUIP), diabetes, indwelling catheter, and age older than 80 years. RESULTS: Thirty patients (10.1%) had treatment failure, 45 (15.1%) improvement, and 223 (74.8%) had excellent outcome. After subdivision into preoperative TZ volume of 20 cc or less and greater than 20 cc, it was found that the outcomes of 20.9% (n = 19) were failures if the TZ volume was 20 cc or less but only 5.3% (n = 11) if the TZ volume was greater than 20 cc. Additional independent risk factors for failure were neurologic disorders and previous TURP/TUIP. When all patients with risk factors were excluded (TZ volume 20 cc or less, neurologic disorders, previous transurethral surgery, and diabetes), the risk of failure was 3.3%. CONCLUSIONS: Patients with a preoperative TZ volume greater than 20 cc and no history of neurologic disorders, previous TURP/TUIP, or diabetes had a very high chance of favorable outcome after TURP, even though no pressure/flow evaluation had been performed preoperatively.

Structure-related aspects on water diffusivity in fatty acid-soap and skin lipid model systems.

Simplified skin barrier models are necessary to get a first hand understanding of the very complex morphology and physical properties of the human skin barrier. In addition, it is of great importance to construct relevant models that will allow for rational testing of barrier perturbing/occlusive effects of a large variety of substances. The primary objective of this work was to study the effect of lipid morphology on water permeation through various lipid mixtures (i.e., partly neutralised free fatty acids, as well as a skin lipid model mixture). In addition, the effects of incorporating Azone((R)) (1-dodecyl-azacycloheptan-2-one) into the skin lipid model mixture was studied. Small- and wide-angle X-ray diffraction was used for structure determinations. It is concluded that: (a) the water flux through a crystalline fatty acid-sodium soap-water mixture (s) is statistically significantly higher than the water flux through the corresponding lamellar (L(alpha)) and reversed hexagonal (H(II)) liquid crystalline phases, which do not differ between themselves; (b) the water flux through mixtures of L(alpha)/s decreases statistically significantly with increasing relative amounts of lamellar (L(alpha)) liquid crystalline phase; (c) the addition of Azone((R)) to a skin lipid model system induces a reduction in water flux. However, further studies are needed to more closely characterise the structural basis for the occlusive effects of Azone((R)) on water flux.

Surveillance is not a viable and appropriate treatment option in the management of localized prostate cancer.

Results of conservatively treated localized prostate cancer are rather homogenous in different series if identical statistical methods are used- about 50% cancer-specific survival after 15 years, a rate much higher than that for men who undergo radical treatment. As the high mortality is accompanied by high costs, morbidity surveillance does not seem to be the optimal treatment in the average patient diagnosed with localized prostate cancer today.

Stratum corneum swelling. Biophysical and computer assisted quantitative assessments.

The aim of this study was to characterize the swelling behaviour of the stratum corneum. Stratum corneum pieces isolated from the breast region of 20 different females were incubated in distilled water at two different temperatures (20 degrees C and 45 degrees C) for 90 min and 24 h, respectively. Half of the stratum corneum pieces were previously extracted with chloroform-methanol (2:1). The area-enlargement was photographically recorded. The thickness enlargement was determined using a confocal laser scanning microscope. The average swelling (99% confidence interval) in the area dimension at 20 degrees C was 8.4% +/- 1.4% (n = 20), which corresponded to an average swelling in the length (lateral) dimension of approximately 4.1%. The swelling in the thickness dimension was 26.3% +/- 16.3% (n = 8). The swelling was most pronounced in the thickness dimension and was complete after 90 min of water immersion (P < 0.01, n = 5). In addition, the removal of the intercellular lipids with chloroform/methanol (2:1) induced a decreased swelling in the samples (P < 0.01, n = 20). An increase in temperature of the water from 20 degrees C to 45 degrees C resulted in an increase in swelling (P < 0.01, n = 20). Taken together our results support the idea that the mechanism of stratum corneum swelling is linked to the intercellular lipid structure and hence to skin barrier function.

A new HPLC-based method for the quantitative analysis of inner stratum corneum lipids with special reference to the free fatty acid fraction.

The inner stratum corneum is likely to represent the location of the intact skin barrier, unperturbed by degradation processes. In our studies of the physical skin barrier a new high-performance liquid chromatography (HPLC)-based method was developed for the quantitative analysis of lipids of the inner stratum corneum. All main lipid classes were separated and quantitated by HPLC/light scattering detection (LSD) and the free fatty acid fraction was further analysed by gas-liquid chromatography (GLC). Mass spectrometry (MS) was used for peak identification and flame ionization detection (FID) for quantitation. Special attention was paid to the free fatty acid fraction since unsaturated free fatty acids may exert a key function in the regulation of the skin barrier properties by shifting the physical equilibrium of the multilamellar lipid bilayer system towards a noncrystalline state. Our results indicated that the endogenous free fatty acid fraction of the stratum corneum barrier lipids in essence exclusively consisted of saturated long-chain free fatty acids. This fraction was characterized as a very stable population (low interindividual peak variation) dominated by saturated lignoceric acid (C24:0, 39 molar%) and hexacosanoic acid (C26:0, 23 molar%). In addition, trace amounts of very long-chain (C32-C36) saturated and monounsaturated free fatty acids were detected in human forearm inner stratum corneum. Our analysis method gives highly accurate and precise quantitative information on the relative composition of all major lipid species present in the skin barrier. Such data will eventually permit skin barrier model systems to be created which will allow a more detailed analysis of the physical nature of the human skin barrier.

Quantification of urinary incontinence in elderly women with the 48-hour pad test.

The ability of 34 elderly women (79 +/- 6 years, range 71-95 years) with urinary incontinence to perform the 48-h perineal pad test in their home environment was investigated. The mean involuntary urinary loss during the 48-h observation period was 80 +/- 88 g (range (2-411 g). Thirty patients successfully compared the two perineal pad systems commercially available in Sweden (LIC and Mölnlycke). Twelve women considered the systems to be equivalent. Thirteen women preferred the Mölnlycke system while five women preferred the LIC system. There was a good correlation between the patients own weighing of the pads and control weighings at the clinic, performed on a precision weight scale (LIC: n = 15, r = 0.99; Mölnlycke: n = 15, r = 0.99). The 48-h perineal pad test performed in the patients home environment should have a given place in the investigation of urinary incontinence amongst elderly women.