The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

The skin barrier in healthy and diseased state

The primary function of the skin is to protect the body for unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. The stratum corneum consists of corneocytes surrounded by lipid regions. As most drugs applied onto the skin permeate along the lipid domains, the lipid organization is considered to be very important for the skin barrier function. It is for this reason that the lipid organization has been investigated quite extensively. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid organization is different from that of other biological membranes. In stratum corneum, two lamellar phases are present with repeat distances of approximately 6 and 13 nm. Moreover the lipids in the lamellar phases form predominantly crystalline lateral phases, but most probably a subpopulation of lipids forms a liquid phase. Diseased skin is often characterized by a reduced barrier function and an altered lipid composition and organization. In order to understand the aberrant lipid organization in diseased skin, information on the relation between lipid composition and organization is crucial. However, due to its complexity and inter-individual variability, the use of native stratum corneum does not allow detailed systematic studies. To circumvent this problem, mixtures prepared with stratum corneum lipids can be used. In this paper first the lipid organization in stratum corneum of normal and diseased skin is described. Then the role the various lipid classes play in stratum corneum lipid organization and barrier function has been discussed. Finally, the information on the role various lipid classes play in lipid phase behavior has been used to interpret the changes in lipid organization and barrier properties of diseased skin.     

Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier

A continuous rat epidermal cell line (rat epidermal keratinocyte; REK) formed a morphologically well-organized epidermis in the absence of feeder cells when grown for 3 weeks on a collagen gel in culture inserts at an air-liquid interface, and developed a permeability barrier resembling that of human skin. By 2 weeks, an orthokeratinized epidermis evolved with the suprabasal layers exhibiting the differentiation markers keratin 10, involucrin, and filaggrin. Granular cells with keratohyalin granules and lamellar bodies, and corneocytes with cornified envelopes and tightly packed keratin filaments were present. Morphologically, vitamin C supplementation of the culture further enhanced the normal wavy pattern of the stratum corneum, the number of keratohyalin granules present, and the quantity and organization of intercellular lipid lamellae in the interstices of the stratum corneum. The morphological enhancements observed with vitamin C correlated with improved epidermal barrier function, as indicated by reduction of the permeation rates of tritiated corticosterone and mannitol, and transepidermal water loss, with values close to those of human skin. Moreover, filaggrin mRNA was increased by vitamin C, and western blots confirmed higher levels of profilaggrin and filaggrin, suggesting that vitamin C also influences keratinocyte differentiation in aspects other than the synthesis and organization of barrier lipids. The unique REK cell line in organotypic culture thus provides an easily maintained and reproducible model for studies on epidermal differentiation and transepidermal permeation.     

Structure of the skin barrier and its modulation by vesicular formulations

The natural function of the skin is to protect the body from unwanted influences from the environment. The main barrier of the skin is located in the outermost layer of the skin, the stratum corneum. Since the lipids regions in the stratum corneum form the only continuous structure, substances applied onto the skin always have to pass these regions. For this reason the organization in the lipid domains is considered to be very important for the skin barrier function. Due to the exceptional stratum corneum lipid composition, with long chain ceramides, free fatty acids and cholesterol as main lipid classes, the lipid phase behavior is different from that of other biological membranes. In stratum corneum crystalline phases are predominantly present, but most probably a subpopulation of lipids forms a liquid phase. Both the crystalline nature and the presence of a 13 nm lamellar phase are considered to be crucial for the skin barrier function. Since it is impossible to selectively extract individual lipid classes from the stratum corneum, the lipid organization has been studied in vitro using isolated lipid mixtures. These studies revealed that mixtures prepared with isolated stratum corneum lipids mimic to a high extent stratum corneum lipid phase behavior. This indicates that proteins do not play an important role in the stratum corneum lipid phase behavior. Furthermore, it was noticed that mixtures prepared only with ceramides and cholesterol already form the 13 nm lamellar phase. In the presence of free fatty acids the lattice density of the structure increases. In stratum corneum the ceramide fraction consists of various ceramide subclasses and the formation of the 13 nm lamellar phase is also affected by the ceramide composition. Particularly the presence of ceramide 1 is crucial. Based on these findings a molecular model has recently been proposed for the organization of the 13 nm lamellar phase, referred to as "the sandwich model", in which crystalline and liquid domains coexist. The major problem for topical drug delivery is the low diffusion rate of drugs across the stratum corneum. Therefore, several methods have been assessed to increase the permeation rate of drugs temporarily and locally. One of the approaches is the application of drugs in formulations containing vesicles. In order to unravel the mechanisms involved in increasing the drug transport across the skin, information on the effect of vesicles on drug permeation rate, the permeation pathway and perturbations of the skin ultrastructure is of importance. In the second part of this paper the possible interactions between vesicles and skin are described, focusing on differences between the effects of gel-state vesicles, liquid-state vesicles and elastic vesicles.     

Local cutaneous water barrier in cold- and heat-acclimated pigeons (Columba livia) in relation to cutaneous water evaporation

The thermoregulatory function of the skin differs in adult cold-acclimated and heat-acclimated rock pigeons (Columba livia). In general, the cutaneous evaporative cooling mechanism is not activated by appropriate stimuli in cold-acclimated pigeons in contrast to heat-acclimated pigeons. We studied with electron microscopy whether the differences in the function of the skin are reflected in the structure of the epidermal water barrier of these two extreme acclimation states. The epidermis of cold-acclimated pigeons is attenuated, and the underlying dermis lacks any intimate vascularization. Both the extracellular and the intracellular domains in the stratum corneum contain organized lamellar lipids. At the stratum transitivum-stratum corneum interface, multigranular body secretion is indicated by the highly convoluted cell membranes and membraneous sacculae enclosing the multigranular bodies. Alternatively, multigranular bodies retain in the corneocytes, and the lipoid material originated from them is reprocessed to broad lamellae. The keratohyalin (KH) granules are spotlike and oriented as cortical bands beneath the plasma membrane. In heat-acclimated pigeons, the epidermis displays modified patches side by side with basic structural type of epidermis. The modified areas are characterized by hypertrophy and abundance of dermal capillaries adjacent to the hypertrophied patch. No lamellar lipids are discerned in the dilated extracellular space. The structure of multigranular bodies is abnormal, and the numbers of lipid droplets in the outer viable epidermis and stratum corneum are decreased. The transitional cells contain stellate KH granules, which form a network throughout the cell. It is concluded that cold-acclimated pigeons have a lamellar, extracellular water barrier, the cutaneous water evaporation is minimized, and heat is stored in the body core. Acclimation to heat leads to formation of structurally heterogeneous skin. The structurally modified skin patches show disruption of the barrier-forming machinery in the multigranular bodies and marked reorganization of fibrillar proteins and electron-dense KH masses in the transitional layer. Thus water barrier adjustments in cold- and heat-acclimated pigeons manifest the dynamic function of avian skin as a thermoregulatory organ.     

Avian epidermal differentiation: role of lipids in permeability barrier formation

Though avian skin is known to possess a highly lipogenic epidermis, little is known about its permeability barrier function. We correlated epidermal barrier function, fine structure and lipid biochemistry in the pigeon, Columbia livia, and compared these features with terrestrial mammalian systems. Whereas barrier function, as assessed by transepidermal water loss was not as efficient as in mammals, both groups shared certain morphological features including substantial compartmentalization of lipids in stratum corneum intercellular domains. Avian intercellular lipids derive from extrusion of intracellular non-membrane-bound droplets from lowermost corneocytes, rather than by secretion of lamellar discs from multigranular bodies, as previously reported in some avians, and in mammals. Instead, both the internal lamellae and the limiting membranes of multigranular bodies appear to degenerate, leading to the formation of non-membrane-bound droplets. The lipid content of avian epidermis and stratum corneum demonstrates important similarities to terrestrial mammals, i.e. abundant sphingolipids, a paucity of phospholipids, and abundant neutral lipids, but also certain striking differences, i.e. persistence of glycosphingolipids and triglycerides into the stratum corneum. Thus, avian stratum corneum forms a two-compartment system of lipid-depleted cells embedded in non-polar-lipid enriched intercellular domains, analogous to mammals. But, in contrast to mammals, the highly attenuated corneocytes of avians, which results from a paucity of keratin filaments, produce a 'straws-and-mortar' tissue, rather than the 'bricks-and-mortar' tissue of mammals.     

Avian sebokeratocytes and marine mammal lipokeratinocytes: structural, lipid biochemical, and functional considerations

In terrestrial mammals, stratum corneum lipids derive from two sources: deposition of lamellar body lipids in stratum corneum interstices and excretion of sebaceous lipids onto the skin surface, resulting in a two-compartment ("bricks and mortar") system of lipid-depleted cells surrounded by lipid-enriched intercellular spaces. In contrast, intracellular lipid droplets, normally not present in the epidermis of terrestrial mammals, are prominent in avian and marine mammal epidermis (cetaceans, manatees). We compared the transepidermal water loss, ultrastructure, and lipid biochemistry of the viable epidermis and stratum corneum of pigeon apterium, fledgling (featherless) zebra finches, painted storks, cetaceans, and manatees to those of humans and mice. Marine mammals possess an even more extensive lamellar-body secretory system than do terrestrial mammals; and lamellar-body contents, as in terrestrials, are secreted into the stratum corneum interstices. In cetaceans, however, glycolipids, but not ceramides, persist into the stratum corneum; whereas in manatees, glycolipids are replaced by ceramides, as in terrestrial mammals. Acylglucosylceramides, thought to be critical for lamellar-body deposition and barrier function in terrestrial mammals, are present in manatees but virtually absent in cetaceans, a finding that indicates that they are not obligate constituents of lamellar-body-derived membrane structures. Moreover, cetaceans do not elaborate the very long-chain, saturated N-acyl fatty acids that abound in terrestrial mammalian acylglucosylceramides. Furthermore, cold-water marine mammals generate large, intracellular neutral lipid droplets not found in terrestrial and warm-water marine mammals; these lipid droplets persist into the stratum corneum, suggesting thermogenesis, flotation, and/or cryoprotectant functions. Avians generate distinctive multigranular bodies that may be secreted into the intercellular spaces under xerotic conditions, as in zebra fledglings; ordinarily, however, the internal lamellae and limiting membranes deteriorate, generating intracellular neutral lipid droplets. The sphingolipid composition of avian stratum corneum is intermediate between terrestrials and cetaceans (approximately equal to 50% glycolipids), with triglycerides present in abundance. In the midstratum corneum of avians, neutral lipid droplets are released into the interstices, forming a large extracellular, lipid-enriched compartment, surrounding wafer-thin corneocytes, with a paucity of both lipid and keratin ("plates-and-mortar" rather than the "bricks-and-mortar" of mammals).(ABSTRACT TRUNCATED AT 400 WORDS)     

Is an orthorhombic lateral packing and a proper lamellar organization important for the skin barrier function?

The lipid organization in the stratum corneum (SC), plays an important role in the barrier function of the skin. SC lipids form two lamellar phases with a predominantly orthorhombic packing. In previous publications a lipid model was presented, referred to as the stratum corneum substitute (SCS), that closely mimics the SC lipid organization and barrier function. Therefore, the SCS serves as a unique tool to relate lipid organization with barrier function. In the present study we examined the effect of the orthorhombic to hexagonal phase transition on the barrier function of human SC and SCS. In addition, the SCS was modified by changing the free fatty acid composition, resulting in a hexagonal packing and perturbed lamellar organization. By measuring the permeability to benzoic acid as function of temperature, Arrhenius plots were constructed from which activation energies were calculated. The results suggest that the change from orthorhombic to hexagonal packing in human SC and SCS, does not have an effect on the permeability. However, the modified SCS revealed an increased permeability to benzoic acid, which we related to its perturbed lamellar organization. Thus, a proper lamellar organization is more crucial for a competent barrier function than the presence of an orthorhombic lateral packing.     

Thematic review series: skin lipids. Peroxisome proliferator-activated receptors and liver X receptors in epidermal biology

The epidermis is a very active site of lipid metabolism, and all peroxisome proliferator-activated receptor (PPAR) and liver X receptor (LXR) isoforms are expressed in the epidermis. Activation of PPARalpha, -beta/delta, or -gamma or LXRs stimulates keratinocyte differentiation. Additionally, activation of these receptors also improves permeability barrier homeostasis by a number of mechanisms, including stimulating epidermal lipid synthesis, increasing lamellar body formation and secretion, and increasing the activity of enzymes required for the extracellular processing of lipids in the stratum corneum, leading to the formation of lamellar membranes that mediate permeability barrier function. The stimulation of keratinocyte differentiation and permeability barrier formation also occurs during fetal development, resulting in accelerated epidermal development. PPAR and LXR activation regulates keratinocyte proliferation and apoptosis, and studies have shown that these receptors play a role in cutaneous carcinogenesis. Lastly, PPAR and LXR activation is anti-inflammatory, reducing inflammation in animal models of allergic and irritant contact dermatitis. Because of their broad profile of beneficial effects on skin homeostasis, PPAR and LXR have great potential to serve as drug targets for common skin diseases such as psoriasis, atopic dermatitis, and skin cancer.     

The epidermal permeability barrier. Comparison between in vivo and in vitro lipid structures

The epidermal permeability barrier is established by the lamellar contents of membrane-coating granules which are discharged into the intercellular space of the stratum granulosum and form continuous lipid layers in the stratum corneum. Artificial lipid systems, prepared with a composition similar to that found in stratum granulosum and stratum corneum, were able to form a lamellar phase. These systems show dense line thickness and center-to-center spacing comparable to those found in membrane-coating granules and intercellular layers. The significance of lipid composition in relation to barrier function is discussed and a model showing the molecular arrangement of the lipid structures in the epidermal barrier is proposed.     

Organization of skin stratum corneum extracellular lamellae: diffraction evidence for asymmetric distribution of cholesterol

Lipid suspensions containing 2:1:1 skin ceramides:palmitic acid:cholesterol, similar to the lipid composition found in the extracellular matrix of skin stratum corneum, were analyzed by X-ray diffraction methods. These suspensions gave a sharp wide-angle reflection at 4.1 A, indicating tight hydrocarbon chain packing that would function as a water barrier, and low-angle lamellar diffraction with a repeat period near 130 A, similar to that previously recorded from intact stratum corneum. The lamellar repeat increased from 121 A at pH 6 to 133 A at pH 8.5, allowing phase angles of the lamellar data to be obtained by a sampling theorem "swelling" analysis. Electron density profiles showed that each repeating unit contained two asymmetric bilayers, with a fluid space on one side of the bilayer that increased with increasing pH, due to electrostatic repulsion between bilayers because of ionization of the palmitic acid. Profiles obtained from lamellae with cholesterol sulfate partially substituted for cholesterol showed large density increases on that same side of the bilayer, indicating that cholesterol is asymmetrically distributed in each bilayer. A molecular model was developed postulating that this asymmetry is due to the exclusion of cholesterol from lipid monolayers containing the ester-linked unsaturated (linoleic) hydrocarbon chain of skin ceramide 1. This model can explain the altered organization of extracellular lamellae in epidermal cysts (P. W. Wertz, D. C. Swartzendruber, K. C. Madison, D. T. Downing. 1987. J. Invest. Dermatol. 89:419-425) where the ester-linked chains have a higher percentage of saturated fatty acids than found in normal epidermis.     

Texture of the skin graft, with special reference to the hydration state of the stratum corneum

The water contained in the superficial portion of the stratum corneum plays an important role in keeping the skin surface soft and smooth. In an attempt to study the true nature of the "texture" of grafted skin, in vivo water sorption-desorption tests were performed on grafts, on normal skin adjacent to grafts, and on normal skin surrounding the donor sites. The results showed that skin grafted to sites other than the face maintained a functional similarity to normal skin surrounding the donor site. However, in skin grafted to the face, the functions of the stratum corneum, as measured in terms of hydration state, increased, and this was associated with an improved water-holding capacity that probably results from the effect of abundant sebum and sweat excretion in adjacent skin. From these results it is suggested that the recipient site plays an important role in determining the difference in the function of the stratum corneum, i.e., the "texture" between grafts on the face and those on other sites.     

Sphingolipid activator proteins are required for epidermal permeability barrier formation

The epidermal permeability barrier is maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the major components of these multilayered membranes, derive in large part from hydrolysis of glucosylceramides mediated by stratum corneum beta-glucocerebrosidase (beta-GlcCerase). Prosaposin (pSAP) is a large precursor protein that is proteolytically cleaved to form four distinct sphingolipid activator proteins, which stimulate enzymatic hydrolysis of sphingolipids, including glucosylceramide. Recently, pSAP has been eliminated in a mouse model using targeted deletion and homologous recombination. In addition to the extracutaneous findings noted previously, our present data indicate that pSAP deficiency in the epidermis has significant consequences including: 1) an accumulation of epidermal glucosylceramides together with below normal levels of ceramides; 2) alterations in lipids that are bound by ester linkages to proteins of the cornified cell envelope; 3) a thickened stratum lucidum with evidence of scaling; and 4) a striking abnormality in lamellar membrane maturation within the interstices of the stratum corneum. Together, these results demonstrate that the production of pSAP, and presumably mature sphingolipid activator protein generation, is required for normal epidermal barrier formation and function. Moreover, detection of significant amounts of covalently bound omega-OH-GlcCer in pSAP-deficient epidermis suggests that deglucosylation to omega-OH-Cer is not a requisite step prior to covalent attachment of lipid to cornified envelope proteins.     

Thematic review series: skin lipids. The role of epidermal lipids in cutaneous permeability barrier homeostasis

The permeability barrier is required for terrestrial life and is localized to the stratum corneum, where extracellular lipid membranes inhibit water movement. The lipids that constitute the extracellular matrix have a unique composition and are 50% ceramides, 25% cholesterol, and 15% free fatty acids. Essential fatty acid deficiency results in abnormalities in stratum corneum structure function. The lipids are delivered to the extracellular space by the secretion of lamellar bodies, which contain phospholipids, glucosylceramides, sphingomyelin, cholesterol, and enzymes. In the extracellular space, the lamellar body lipids are metabolized by enzymes to the lipids that form the lamellar membranes. The lipids contained in the lamellar bodies are derived from both epidermal lipid synthesis and extracutaneous sources. Inhibition of cholesterol, fatty acid, ceramide, or glucosylceramide synthesis adversely affects lamellar body formation, thereby impairing barrier homeostasis. Studies have further shown that the elongation and desaturation of fatty acids is also required for barrier homeostasis. The mechanisms that mediate the uptake of extracutaneous lipids by the epidermis are unknown, but keratinocytes express LDL and scavenger receptor class B type 1, fatty acid transport proteins, and CD36. Topical application of physiologic lipids can improve permeability barrier homeostasis and has been useful in the treatment of cutaneous disorders.     

Effect of epidermal acylglucosylceramides and acylceramides on the morphology of liposomes prepared from stratum corneum lipids

Epidermal acylglucosylceramides (AGC) and acylceramides (AC) cause aggregation and stacking of stratum corneum lipid liposomes formed from a lipid mixture containing epidermal ceramides (40%), cholesterol (25%), palmitic acid (25%), and cholesteryl sulfate (10%). This demonstrates the ability of these sphingolipids to hold adjacent bilayers in close apposition and their roles in the assembly of lamellar structures in the epidermis. However, AGC and AC in their hydrogenated form also caused aggregation and stacking of the stratum corneum lipid liposomes. This throws into doubt the proposed structural specificity of linoleate in the function of AGC and AC as molecular rivets in the assembly of the epidermal lamellar granules and the stratum corneum intercellular lamellae, respectively.     

Integrity and barrier function of the epidermis critically depend on glucosylceramide synthesis

Ceramides are vital components of the water barrier in mammalian skin. Epidermis-specific, a major ceramide portion contains omega-hydroxy very long chain fatty acids (C30-C36). These omega-hydroxy ceramides (Cers) are found in the extracellular lamellae of the stratum corneum either as linoleic acyl esters or protein bound. Glucosylceramide is the major glycosphingolipid of the epidermis. Synthesized from ceramide and UDP-glucose, it is thought to be itself an intracellular precursor and carrier for extracellular omega-hydroxy ceramides. To investigate whether GlcCer is an obligatory intermediate in ceramide metabolism to maintain epidermal barrier function, a mouse with an epidermis-specific glucosylceramide synthase (Ugcg) deficiency has been generated. Four days after birth animals devoid of GlcCer synthesis in keratinocytes showed a pronounced desquamation of the stratum corneum and extreme transepidermal water loss leading to death. The stratum corneum appeared as a thick unstructured mass. Lamellar bodies of the stratum granulosum did not display the usual ordered inner structure and were often irregularly arranged. Although the total amount of epidermal protein-bound ceramides remained unchanged, epidermal-free omega-hydroxy ceramides increased 4-fold and omega-hydroxy sphingomyelins, almost not detectable in wild type epidermis, emerged in quantities comparable with lost GlcCer. We conclude that the transient formation of GlcCer is vital for a regular arrangement of lipids and proteins in lamellar bodies and for the maintenance of the epidermal barrier.     

Role of lipids in the formation and maintenance of the cutaneous permeability barrier

The major function of the skin is to form a barrier between the internal milieu and the hostile external environment. A permeability barrier that prevents the loss of water and electrolytes is essential for life on land. The permeability barrier is mediated primarily by lipid enriched lamellar membranes that are localized to the extracellular spaces of the stratum corneum. These lipid enriched membranes have a unique structure and contain approximately 50% ceramides, 25% cholesterol, and 15% free fatty acids with very little phospholipid. Lamellar bodies, which are formed during the differentiation of keratinocytes, play a key role in delivering the lipids from the stratum granulosum cells into the extracellular spaces of the stratum corneum. Lamellar bodies contain predominantly glucosylceramides, phospholipids, and cholesterol and following the exocytosis of lamellar lipids into the extracellular space of the stratum corneum these precursor lipids are converted by beta glucocerebrosidase and phospholipases into the ceramides and fatty acids, which comprise the lamellar membranes. The lipids required for lamellar body formation are derived from de novo synthesis by keratinocytes and from extra-cutaneous sources. The lipid synthetic pathways and the regulation of these pathways are described in this review. In addition, the pathways for the uptake of extra-cutaneous lipids into keratinocytes are discussed. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

The Lipid Organisation of the Skin Barrier: Liquid and Crystalline Domains Coexist in Lamellar Phases

The superficial layer of the skin, the stratum corneum, is the main barrier for diffusion of substances across the skin. The stratum corneum is composed of corneocytes embedded in lipid lamellae. In previous studies two lamellar phases have been identified with periodicities of 6.4 and 13.4 nm of which the 13.4 nm phase (long periodicity phase = LPP) is considered to be very important for the skin banier function. The main lipid classes in stratumcorneum are ceramides, free fatty acids and cholesterol. Until now 8 subclassesof ceramides are identified in human stratum corneum referred to as ceramide 1 to 8. Studies with mixtures prepared with isolated human ceramides revealed that cholesterol and ceramides are very important for the formation of the lamellar phases. After addition of free fatty acids the lipids are organised in an orthorhombic packing with a small proportion of lipids in a liquid phase. Our most recent results show that the presence of ceramide 1 and the formation of a liquid phase are crucial elements for the formation of the LPP. These observations and the broad-narrowbroad sequence of lipid layers in the LPP led us to propose a molecular model for this phase. This consists of one narrow central lipid layer with fluid domains with on both sides a broad layer with a crystalline structure. This model is referred to as `the sandwich model'.     

Lipid mixtures prepared with well-defined synthetic ceramides closely mimic the unique stratum corneum lipid phase behavior

Lipid lamellae present in the outermost layer of the skin, the stratum corneum, form the main barrier for the diffusion of molecules through the skin. The presence of a unique 13 nm lamellar phase and its high crystallinity are characteristic for the stratum corneum lipid phase behavior. In the present study, small-angle and wide-angle X-ray diffraction were used to examine the organization in lipid mixtures prepared with a unique set of well-defined synthetic ceramides, varying from each other in head group architecture and acyl chain length. The results show that equimolar mixtures of cholesterol, free fatty acids, and synthetic ceramides (resembling the composition of pig ceramides) closely resemble the lamellar and lateral stratum corneum lipid organization, both at room and higher temperatures. Exclusion of several ceramide classes from the mixture does not affect the lipid organization. However, complete substitution of ceramide 1 (acylceramide with a sphingosine base) with ceramide 9 (acylceramide with a phytosphingosine base) reduces the formation of the long periodicity lamellar phase. This indicates that the head group architecture of acylceramides affects the lipid organization. In conclusion, lipid mixtures prepared with well-defined synthetic ceramides offer an attractive tool with which to unravel the importance of the molecular structure of individual ceramides for proper lipid organization.     

Avian permeability barrier function reflects mode of sequestration and organization of stratum corneum lipids: reevaluation utilizing ruthenium tetroxide staining and lipase cytochemistry.

The epidermis of avians and terrestrial mammals has evolved distinct, but related mechanisms to survive in a terrestrial environment. In both phyla, stratum corneum lipids form the basis of the cutaneous permeability barrier, but barrier function is less efficient in avians. Whereas in mammals the epidermal lamellar body (LB) secretes its contents into the intercellular spaces, in the feathered epidermis of avians, its distinctive secretory organelle, the multigranular body (MGB), does not secrete its contents into the stratum corneum intercellular spaces. Yet, neutral lipid-enriched droplets, derived from the cytosolic breakdown of MGB, ultimately are squeezed through membrane pores into the stratum corneum interstices. In this study we determined: a) using ruthenium tetroxide (RuO4) fixation, whether these droplets form membrane structures after deposition in the stratum corneum interstices; and b) the similarities and differences between avian MGB and mammalian LB, using enzyme cytochemistry as a marker for secretion, and optical diffraction computer-aided image analysis and reconstruction to compare the internal structure of MGB vs. LB. MGB were shown to possess a similar lamellar substructure to LB in RuO4-fixed specimens, exhibiting comparable dimensions on optical diffraction and computer transform analysis. Moreover, the intercellular lipids of avian stratum corneum lacked membrane-substructure, as was present in parallel samples of mammalian stratum corneum. Thus, both the absence of MGB secretion, and the failure of intercellular lipids to form membrane bilayers may explain the inherent differences in barrier function in these two taxa.