An Easy Synthesis of (E)-9-Oxo-2-decenoic Acid,the Queen Substance of the Honey Bee,and (Z)-6-Heneicosen-ll-one,the Sex Pheromone of the Douglas Fir Tussock Moth†

We investigated the effect of dietary phospholipid (PL) concentrate from bovine milk on the epidermis. Thirteen-week-old hairless male and female mice (Hos:HR-1) were separated into two experimental groups, each fed two experimental diets: the control group and the PL group. The mice were given the experimental diets for 6 weeks. Stratum corneum hydration and transepidermal water loss (TEWL) were measured using Corneometer CM825 and Tewameter TM300 (Courage and Khazaka Electronics, Cologne, Germany) at 3 weeks and 6 weeks. After the feeding period, ceramides in stratum corneum were analyzed. We found that stratum corneum hydration and ceramides in the PL group were significantly higher than those in the control group and that TEWL in the PL group tended to decrease.

These results indicate that dietary PL concentrate improves epidermal function by increasing the amount of ceramides, resulting in higher hydration.     

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.     

Effects of a pyroglutamyl pentapeptide isolated from fermented barley extract on atopic dermatitis-like skin lesions in hairless mouse

ABSTRACT

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by pruritic and eczematous skin lesions. The skin of AD patients is generally in a dried condition. Therefore, it is important for AD patients to manage skin moisturization. In this study, we examined the effects of orally administered fermented barley extract P (FBEP), which is prepared from a supernatant of barley shochu distillery by-product, on stratum corneum (SC) hydration and transepidermal water loss (TEWL) in AD-like lesions induced in hairless mice using 2,4,6-trinitrochlorobenzene. Oral administration of FBEP increased SC hydration and decreased TEWL in the dorsal skin of this mouse model. Further fractionation of FBEP showed that a pyroglutamyl pentapeptide, pEQPFP comprising all -L-form amino acids, is responsible for these activities. These results suggested that this pyroglutamyl pentapeptide may serve as a modality for the treatment of AD.     

Effect of ceramide acyl chain length on skin permeability and thermotropic phase behavior of model stratum corneum lipid membranes

Stratum corneum ceramides play an essential role in the barrier properties of skin. However, their structure-activity relationships are poorly understood. We investigated the effects of acyl chain length in the non-hydroxy acyl sphingosine type (NS) ceramides on the skin permeability and their thermotropic phase behavior. Neither the long- to medium-chain ceramides (8-24 C) nor free sphingosine produced any changes of the skin barrier function. In contrast, the short-chain ceramides decreased skin electrical impedance and increased skin permeability for two marker drugs, theophylline and indomethacin, with maxima in the 4-6C acyl ceramides. The thermotropic phase behavior of pure ceramides and model stratum corneum lipid membranes composed of ceramide/lignoceric acid/cholesterol/cholesterol sulfate was studied by differential scanning calorimetry and infrared spectroscopy. Differences in thermotropic phase behavior of these lipids were found: those ceramides that had the greatest impact on the skin barrier properties displayed the lowest phase transitions and formed the least dense model stratum corneum lipid membranes at 32°C. In conclusion, the long hydrophobic chains in the NS-type ceramides are essential for maintaining the skin barrier function. However, this ability is not shared by their short-chain counterparts despite their having the same polar head structure and hydrogen bonding ability.     

Accumulation of protein-bound epidermal glucosylceramides in beta-glucocerebrosidase deficient type 2 Gaucher mice

The epidermal permeability barrier for water is essentially maintained by extracellular lipid membranes within the interstices of the stratum corneum. Ceramides, the main components of these membranes, derive in large part from hydrolysis of glucosylceramides mediated by the lysosomal enzyme beta-glucocerebrosidase. As analyzed in this work, the beta-glucocerebrosidase deficiency in type 2 Gaucher mice (RecNci I) resulted in an accumulation of all epidermal glucosylceramide species accompanied with a decrease of the related ceramides. However, the levels of one ceramide subtype, which possesses an alpha-hydroxypalmitic acid, was not altered in RecNci I mice suggesting that the beta-glucocerebrosidase pathway is not required for targeting of this lipid to interstices of the stratum corneum. Most importantly, omega-hydroxylated glucosylceramides which are protein-bound to the epidermal cornified cell envelope of the transgenic mice accumulated up to 35-fold whereas levels of related protein-bound ceramides and fatty acids were decreased to 10% of normal control. These data support the hypothesis that in wild-type epidermis omega-hydroxylated glucosylceramides are first transferred enzymatically from their linoleic esters to proteins of the epidermal cornified cell envelope and then catabolized to protein-bound ceramides and fatty acids, thus contributing at least in part to the formation of the lipid-bound envelope.     

Sphingolipid metabolism during epidermal barrier development in mice

In rodents, a competent skin barrier to water loss is formed within 2 or 3 days prior to birth. Acquisition of barrier function during rat gestation correlates with the formation of a stratum corneum enriched in ceramides, cholesterol, and fatty acids (Aszterbaum, M., G. K. Menon, K. R. Feingold, and M. L. Williams. 1992. Ontogeny of the epidermal barrier to water loss in the rat: correlation of function with stratum corneum structure and lipid content. PEDIATR: Res. 31: 308-317). We analyzed the formation and epidermal localization of glucosylceramides during embryonic skin barrier development in Balb/c mice. Using immunohistochemistry, epidermal glucosylceramides were hardly detectable 3 days prior to birth. After further 24 h of gestation the level of glucosylceramides was maximal and decreased with increasing gestational age. In parallel, glucosylceramides were targeted to the apical side of the outermost granular keratinocyte layer. A spectrum of five distinct epidermal ceramides was present 2 days prior to birth. With ongoing gestation the composition of the ceramide fraction changed markedly. Most importantly, the level of omega-hydroxylated acylceramides decreased paralleled by the formation of the corneocyte lipid envelope. This structure consists of omega-hydroxylated ceramides and fatty acids bound to surface proteins of the corneocytes. The covalent attachment of ceramides or glucosylceramides correlated with the maturation of the stratum corneum and might contribute to its chemical and enzymatic resistance.     

Impact of Acacia bark extract tablets on the skin of healthy humans: a randomized,double-blind,placebo-controlled study

This study investigated the effects of proanthocyanidins derived from Acacia (Acacia mearnsii) bark extract in healthy Japanese adult subjects experiencing uncomfortable skin symptoms. All subjects were randomly allocated into two groups (n = 33 each) using a computerized random-number generator. The subjects received either Acacia bark extract tablets or placebo for 8 weeks. Evaluations included water content in the stratum corneum, transepidermal water loss (TEWL), Skindex-16, dermatology life quality index (DLQI), visual analog scale for desire to scratch, and blood tests. At 4 weeks, the symptom/feeling score of DLQI, subjective symptoms related to uncomfortable skin, and the desire to scratch were significantly reduced in the intervention group than in the placebo group. At 8 weeks, the intervention group exhibited significantly lower TEWL on facial skin than that in the placebo group. In conclusion, the intake of Acacia bark extract tablets reduced TEWL and improved dry and uncomfortable skin.     

Role of ceramide structure and its microenvironment on the conformational order of model stratum corneum lipids mixtures: an approach by FTIR spectroscopy

The aim of this study is to investigate the influence of ceramide head group architecture and free fatty acid (another main class of stratum corneum lipids) or protein (keratin), on the lamellar organization of the ceramide auto-associated in model films mimicking lipid organization within the stratum corneum. FTIR spectroscopy is a powerful technique for investigating the structure of such systems. This technique has already been used to characterize phase transitions of the SC and of related model systems. As temperature is known to modify the conformational order of lipids, we used it as a variable parameter to monitor the differences in the conformational stability of ceramides. Our study included four ceramides: ceramide 2, 3, 5 and 6 which differ by their head group architecture. Two kinds of lipid-lipid interactions were studied: non-polar and polar. We noted some structural factors which participated to the organizational behavior: insaturation of alkyl chain, alpha-hydroxyl on fatty acid moiety and sphingosine or phytosphingosine head group. There is a direct interaction of palmitic acid on alkyl chains organization and a weak interaction with polar head group in presence of keratin, both provoking a destabilization of the ceramidic orthorhombic organization.     

Unique features of the skin barrier in naked mole rats reflect adaptations to their fossorial habitat

The stratum corneum (SC), the top layer of the epidermis, is the functional site of the skin barrier and serves to maintain hydration of the body by preventing water loss and thwarting the entrance of pathogens. The naked mole rat (NMR) (Heterocephalus glaber) is a rodent that resides in hypoxic underground tunnels in arid Africa. NMRs are not only hairless; their skin is devoid of glands and pain sensation. To understand how the skin barrier of the NMR is uniquely adapted to this environment, skin samples from the dorsum and ventral abdomen in one adult and one neonate were examined by transmission electron microscopy using both reduced osmium tetroxide to assess overall structure and ruthenium tetroxide post-fixation to assess lipid organization. These findings were compared with that of hairless mice—a well-defined model for skin barrier studies. The plasticity of the skin was evaluated on 10 NMRs from a colony at the Philadelphia Zoo in humid and dry conditions by measuring cutaneous hydration, transepidermal water loss (TEWL), and pH. The epidermal ultrastructure of the NMR differed from hairless mice by having the following features: decreased content of lamellar bodies (LBs), higher LB pleomorphism, periodic presence of abnormal lipid bilayers, and an unusually thick SC. The NMRs developed significant TEWL and a trend toward decreased hydration when subjected to dry conditions. While these features illustrate an imperfect skin barrier in terrestrial mammals, they likely represent adaptations of the poikilothermic NMRs to their unique natural fossorial climate. Prolonged exposure to decreased humidity could possibly lead to adverse health effects in this species.     

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.     

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.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

The permeability enhancing mechanism of DMSO in ceramide bilayers simulated by molecular dynamics

The lipids of the topmost layer of the skin, the stratum corneum, represent the primary barrier to molecules penetrating the skin. One approach to overcoming this barrier for the purpose of delivery of active molecules into or via the skin is to employ chemical permeability enhancers, such as dimethylsulfoxide (DMSO). How these molecules exert their effect at the molecular level is not understood. We have investigated the interaction of DMSO with gel-phase bilayers of ceramide 2, the predominant lipid in the stratum corneum, by means of molecular dynamics simulations. The simulations satisfactorily reproduce the phase behavior and the known structural parameters of ceramide 2 bilayers in water. The effect of DMSO on the gel-phase bilayers was investigated at various concentrations over the range 0.0-0.6 mol fraction DMSO. The DMSO molecules accumulate in the headgroup region and weaken the lateral forces between the ceramides. At high concentrations of DMSO (> or =0.4 mol fraction), the ceramide bilayers undergo a phase transition from the gel phase to the liquid crystalline phase. The liquid-crystalline phase of ceramides is expected to be markedly more permeable to solutes than the gel phase. The results are consistent with the experimental evidence that high concentrations of DMSO fluidize the stratum corneum lipids and enhance permeability.     

Homogeneous transport in a heterogeneous membrane: water diffusion across human stratum corneum in vivo.

The objective of this study was to determine whether a structurally heterogeneous biomembrane, human stratum corneum (SC), behaved as a homogeneous barrier to water transport. The question is relevant because the principal function of the SC in vivo is to provide a barrier to the insensible loss of tissue water across the skin. Impedance spectra (IS) of the skin and measurements of the rate of transepidermal water loss (TEWL) were recorded sequentially in vivo in human subjects as layers of the SC were progressively removed by the serial application of adhesive tape strips. The low-frequency (< or = 100 rad s-1) impedance of skin was much more significantly affected by tape stripping than the higher frequency values; removal of the outermost SC layer had the largest effect. In contrast, TEWL changed little as the outer SC layers were stripped off, but increased dramatically when 6-8 microns of the tissue had been removed. It follows that the two noninvasive techniques probe SC barrier integrity in somewhat different ways. After SC removal, recovery of barrier function, as assessed by increasing values of the low-frequency impedance, apparently proceeded faster than TEWL decreased to the prestripping control. The variation of TEWL as a function of SC removal behaved in a manner entirely consistent with a homogeneous barrier, thereby permitting the apparent SC diffusivity of water to be found. Skin impedance (low frequency) was correlated with the relative concentration of water within the SC, thus providing an in vivo probe for skin hydration. Finally, the SC permeability coefficient to water, as a function of SC thickness, was calculated and correlated with the corresponding values of skin admittance derived from IS.     

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)     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: lipid composition and thermal phase behavior of the stratum corneum.

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while alpha-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified omega-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T2) at a slightly lower temperature (68 degrees C) than human skin (74 degrees C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Lipid content and lipid type as determinants of the epidermal permeability barrier

During terminal differentiation, mammalian epidermal lipids undergo striking changes in both composition and distribution. Phospholipids and neutral lipids are replaced by a mixture of ceramides and neutral lipids organized in intercellular lamellar bilayers. Whether all of these lipids and/or whether specific lipid classes regulate permeability barrier function is not known. When hairless mice were treated with acetone, the degree of barrier perturbation (measured as transepidermal water loss, TEWL) increased linearly with the amount of lipid removed. Moreover, virtually all lipid species appeared to be removed by acetone treatment. In contrast, the nonpolar organic solvent, petroleum ether, while removing greater amounts of lipids, provoked lesser barrier abnormalities. As determined by both quantitative thin-layer chromatography and histochemistry, petroleum ether selectively extracted nonpolar lipids leaving sphingolipids and free sterols in place. In petroleum ether-treated animals, subsequent acetone treatment removed additional sphingolipids and produced a dramatic increase in TEWL. A linear relationship existed for the quantities of sphingolipid removed and degree of barrier disruption in acetone-treated, but not petroleum ether-treated animals. These results support a relationship between the total lipid content of the stratum corneum and barrier function. Secondly, although the results demonstrate the participation of the total lipid mixture in the barrier, removal of nonpolar species alone appears to cause only a modest level of barrier disruption, while removal of sphingolipids and free sterols leads to a more profound level of barrier perturbation.     

Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin: Lipid composition and thermal phase behavior of the stratum corneum

The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the present study was to compare the stratum corneum lipid content of ROC with the corresponding material from human skin. The lipid composition was determined by thin-layer chromatography (TLC) and mass-spectrometry, and the thermal phase transitions of stratum corneum were studied by differential scanning calorimetry (DSC). All major lipid classes of the stratum corneum were present in ROC in a similar ratio as found in human stratum corneum. Compared to human skin, the level of non-hydroxyacid-sphingosine ceramide (NS) was increased in ROC, while α-hydroxyacid-phytosphingosine ceramide (AP) and non-hydroxyacid-phytosphingosine ceramides (NP) were absent. Also some alterations in fatty acid profiles of ROC ceramides were noted, e.g., esterified ω-hydroxyacid-sphingosine contained increased levels of oleic acid instead of linoleic acid. The fraction of lipids covalently bound to corneocyte proteins was distinctly lower in ROC compared to human skin, in agreement with the results from DSC. ROC underwent a lipid lamellar order to disorder transition (T₂) at a slightly lower temperature (68 °C) than human skin (74 °C). These differences in stratum corneum lipid composition and the thermal phase transitions may explain the minor differences previously observed in drug permeation between ROC and human skin.     

Light microscopic observations of the ruminal papillae of cattle on diets with divergent forage to cereal ratios

High levels of supplementation with cereal increases production rates in cattle but can increase incidence of disease, ranging from mild indigestion to acute ruminal acidosis and death. Therefore, there is motivation to determine biological markers which can be used to identify whether animals have been, or are being fed, sufficient or excessive cereals. This study aimed to describe light microscopic findings from animals being fed diverse dietary cereal proportions and to test the performance of a novel rumen epithelial scoring system. Rumen wall tissue samples were obtained from the abattoir from 195 cattle from 11 Scottish farms and processed for histological examination. Light microscopic examination was used to characterise ruminal epithelial response to dietary challenge. Secondary objectives included describing the distribution of immune-related cells in bovine ruminal epithelium and assessing the use of a modified Elastin Martius Scarlet Blue stain (EMSB) for histological examination of the rumen epithelium. Cells staining positive for cluster of differentiation 3 were distributed mainly in the lower layers of the stratum basale and were found in higher densities in animals offered lower cereal proportion diets. Cells staining positive for major histocompatibility complex class 2 (MHCII) were most common in perivascular locations and in the junction between the lower stratum basale and the propria-submucosa. The density of MHCII positive staining cells was higher in animals on lower cereal diets. The level of supplementation with cereal was also associated with the thickness of the stratum corneum (SCT) and stratum granulosum (SGT), the integrity of the stratum corneum and sloughing of cornified cells. There were no advantages in using EMSB stain over haematoxylin and eosin (H&E) in this scoring system. We concluded that a scoring system that included only SCT, SGT and a measure of the loss of appearance of intercellular space allowed differentiation of groups of animals according to the level of cereal supplementation.     

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.