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.

Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature.

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.

Human stratum corneum lipid organization as observed by atomic force microscopy on Langmuir-Blodgett films.

The barrier function of skin ultimately depends on the physical state and structural organisation of the stratum corneum extracellular lipid matrix. Ceramides, cholesterol and a broad distribution of saturated long-chain free fatty acids dominate the stratum corneum lipid composition. Additionally, smaller amounts of cholesterol sulfate and cholesteryl oleate may be present. A key feature determining skin barrier capacity is thought to be whether or not different lipid domains coexist laterally in the stratum corneum extracellular lipid matrix. In this study, the overall tendency for lipid domain formation in different mixtures of extracted human stratum corneum ceramides, cholesterol, free fatty acids, cholesterol sulfate and cholesteryl oleate were studied using atomic force microscopy (AFM) on Langmuir-Blodgett (LB) films on mica. It is shown that the saturated long-chain free fatty acid distribution of human stratum corneum prevents hydrocarbon chain segregation. Further, LB-films of human stratum corneum ceramides express a pattern of connected elongated domains with a granular domain interface. The dominating effect of both cholesterol and cholesterol sulfate is that of increased ceramide domain dispersion. This effect is counteracted by the presence of free fatty acids, which preferentially mix with ceramides and not with cholesterol. Cholesteryl oleate does not mix with other skin lipid components, supporting the hypothesis of an extra-endogenous origin. In the system composed of endogenous human ceramides and cholesterol plus 15 wt% stratum corneum distributed free fatty acids, i.e., the system mimicking most closely the lipid composition of the stratum corneum extracellular space, LB-films on mica express lateral domain formation.

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.

Molecular skin barrier models and some central problems for the understanding of skin barrier structure and function.

A better understanding of the structure and function of the human skin barrier is a prerequisite for a more rational design of transdermal drug administration systems. The study of biological structural organisation is, however, confronted with many difficulties, and interpretations of skin barrier-related data should therefore be done with caution. The recently developed 'single gel-phase model' constitutes an attempt to interpret structural and functional skin barrier data from a biophysical standpoint.

Skin barrier structure, function and formation - learning from cryo-electron microscopy of vitreous, fully hydrated native human epidermis.

The hydration level of the stratum corneum largely depends on the physical state and molecular organization of the stratum corneum intercellular lipid matrix. A better understanding of stratum corneum lipid organization may thus aid the development of more rational cosmetic formulations. Several experimental and theoretical problems of a fundamental character remain, however, unresolved. These are, e.g. that precise quantitative skin barrier compositional data are difficult to obtain and that in vitro experimental skin barrier models usually are of limited value because of the prevailing non-equilibrium conditions in vivo. However, new experimental methods have recently been developed, which may help to overcome some of these limitations. These are, e.g. direct electrospray ionization-mass spectrometry (ESI-MS) of intact long-chain skin ceramides and direct high-resolution cryo-transmission electron microscopy (cryo-TEM) of vitreous sections of native, fully hydrated epidermis. Here, we show that cryo-transmission electron micrographs of vitreous normal human skin often dramatically differ from those obtained by conventional electron microscopy of resin-embedded skin. Our epidermal cryo-TEM data are subsequently discussed in relation to central problems of present conceptions of skin barrier structure, function and formation.

Vitamin B12 and folate after 5-12 years of continent ileal urostomy (Kock reservoir) in children and adolescents.

OBJECTIVE: To assess B12 and folate deficiency after continent urinary diversion via a Kock continent urinary reservoir in children and adolescents. METHODS: Ten boys and 10 girls (10.8-18 years old at surgery) were operated with a Kock reservoir and followed for 5-12 years (mean 8.5). The follow-up period was divided into early (3 months-5 years, EFU) and late (5-12 years, LFU) follow-up. Patients were investigated for haemoglobin, serum iron, total iron binding capacity (TIBC), serum Vitamin B12, serum and blood folate, methylmalonic acid (MMA), homocystine and glomerular filtration rate. RESULTS: Two patients developed subnormal B12 values (median 107.5 pmol/l), one at the EFU, and the other at LFW. The B12 value decreased during the LFU compared to the EFU in nine patients, but it was still within the normal range. Two patients with renal impairment had elevated MMA with normal B12 values. Five patients had high values of homocystine with folate deficiency and/or B12 deficiency and renal impairment. Plasma folate mean value was normal during the whole follow-up. Blood folate was below normal in five patients at the EFU. Two of these five patients, in addition to three patients, had low values at the LFU. Three of four patients with remaining short terminal ileum (20-45 cm) had normal B12 values at both the EFU & LFU and one had low values at the LFU. Six patients had subnormal GFR at the LFU. CONCLUSIONS: To a similar degree as in adults, Vitamin B12, folate and iron deficiency can occur in children and adolescents after continent urinary diversion using an ileal segment. Therefore, Vitamin B12 and folate should be monitored regularly in these patients. Serum MMA and homocystine may offer increased detection of Vitamin B12 deficiency, especially in the patients with normal renal function. Vitamin B12 deficiency is neither correlated with the time elapsed since surgery, nor with the ileum length. Patients are usually asymptomatic, so patients with true B12 deficiency should be identified and placed on life-long Vitamin B12 therapy. An adequate synthetic folic acid as supplements or fortified food is recommended for patients with folate deficiency.

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.

Questioning questions about symptoms of benign prostatic hyperplasia.

OBJECTIVES: Our aim was to conduct a survey about urinary symptoms and to find out if questioning patients about symptoms is helpful for the GP to make medical decisions concerning prostate problems among middle-aged men. METHODS: Twelve hundred randomly chosen men aged 55--65 years from the general population in north-west Stockholm, Sweden were sent a questionnaire consisting of symptom questions focusing on prostate problems based on the International Prostate Symptom Score (I-PSS). A subset of 120 respondents were asked to answer the same questions again and to participate in a urological examination including urodynamics and ultrasonography. The main outcome measures were the prevalence of urinary symptoms and the relationship between the symptom score and objective measures. RESULTS: A response rate of 86% was obtained in the questionnaire study. Twenty-one per cent of the respondents stated that they had general problems related to urination. Among individual symptoms, post-void dribbling and a weak stream were most common. Among the men examined at the Urological Department, the average prostatic volume was found to be 40 cm(3). Three out of four were assessed to have infravesical obstruction. No correlation between subjective symptoms and objective measurements of either a statistical or clinical significance was found. CONCLUSIONS: Urinary symptoms are common among middle-aged men. Further, an enlarged prostate and/or infravesical obstruction is often found in the ageing man. Information obtained by asking prostate-specific symptom questions cannot, however, serve as the foundation for the GP to find those men whose problems would be solved by actions directed at the prostate.

Skin island urethroplasty in deep urethral lesions--a long-term follow-up of 25 consecutive patients.

Strictures and ruptures of the bulbomembranous urethra have traditionally been treated by a two-stage scrotal skin inlay technique (Johanson B. Reconstruction of the male urethra in strictures. Acta Chir Scand 1953; Suppl 176). For the last 10 years we have treated the patients instead with a skin-dartos island flap pedicled on intertesticular septal vessels. The skin island has been taken from the scrotum, the penile base, or the penile shaft. Twenty-five consecutive patients were treated until 1993 and followed up for at least 5 years. Five of the patients had had open urethroplasty before. Fifteen of the patients had urethral strictures and 10 had complete ruptures. Of the 25 patients 6 had to have a further operation; in 4 patients this was because of restricture, in 1 patient a urethral pouch had to be reduced, and in the final patient the operation was both for restricture and pouch formation. At final follow-up impaired micturition occurred in three patients: two of them had had a ruptured urethra treated previously, and one had a chronic infectious stricture. Problems related to hirsutism were low. No obvious advantage was detected from using distal penile skin, so a skin-dartos island from the penile base is advocated. In conclusion, a one-stage skin-dartos island flap pedicled on the intertesticular septal vessels may be recommended in the treatment of both strictures and complete ruptures in the bulbomembranous urethra.

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.