Discovery of Bovine Digital Dermatitis-Associated Treponema spp. in the Dairy Herd Environment by a Targeted Deep-Sequencing Approach

The bacteria associated with the infectious claw disease bovine digital dermatitis (DD) are spirochetes of the genus Treponema; however, their environmental reservoir remains unknown. To our knowledge, the current study is the first report of the discovery and phylogenetic characterization of rRNA gene sequences from DD-associated treponemes in the dairy herd environment. Although the spread of DD appears to be facilitated by wet floors covered with slurry, no DD-associated treponemes have been isolated from this environment previously. Consequently, there is a lack of knowledge about the spread of this disease among cows within a herd as well as between herds. To address the issue of DD infection reservoirs, we searched for evidence of DD-associated treponemes in fresh feces, in slurry, and in hoof lesions by deep sequencing of the V3 and V4 hypervariable regions of the 16S rRNA gene coupled with identification at the operational-taxonomic-unit level. Using treponeme-specific primers in this high-throughput approach, we identified small amounts of DNA (on average 0.6% of the total amount of sequence reads) from DD-associated treponemes in 43 of 64 samples from slurry and cow feces collected from six geographically dispersed dairy herds. Species belonging to the Treponema denticola/Treponema pedis-like and Treponema phagedenis-like phylogenetic clusters were among the most prevalent treponemes in both the dairy herd environment and the DD lesions. By the high-throughput approach presented here, we have demonstrated that cow feces and environmental slurry are possible reservoirs of DD-associated treponemes. This method should enable further clarification of the etiopathogenesis of DD.     

Significant differences in fertility between dairy cows selected for one QTL located on bovine chromosome 3 are not attributable to energy balance,although eating behaviour is affected

Improvement of reproduction in dairy cows has become a major challenge in dairy production. We have recently shown that dairy cows carrying the ‘fertil−’ haplotype for one quantitative trait locus (QTL), affecting female fertility and located on the bovine chromosome 3, had a significantly lower conception rate after the first artificial insemination than cows carrying the ‘fertil+’ haplotype. The objective of this paper was to study other phenotypic modifications linked to this QTL. In the present study, 23 ‘fertil+’ and 18 ‘fertil−’ cows were characterized for live weight, milk production, food intake, eating behaviour and plasma metabolites. These parameters were measured during the first lactation, from calving to 40 weeks postpartum (wkpp). In the first 7 weeks of lactation, ‘fertil+’ primiparous cows had a significantly higher live BW and milk production than ‘fertil−’ cows. Dry matter intake tended to be slightly higher for ‘fertil+’ than for ‘fertil−’ primiparous cows in this period. However, energy balance was similar for the two haplotypes in the whole lactation, except in the first wkpp, and consequently, could not explain their different fertility. The major observation concerned the eating behaviour. ‘Fertil+’ primiparous cows had a significantly lower eating rate than ‘fertil−’ cows during the 40 weeks of lactation. In parallel, ‘fertil+’ cows spent significantly more time at the feeder for a similar number of visits than ‘fertil−’ cows. Furthermore, no differences in plasma concentrations of non-esterified fatty acids and insulin were observed between the two haplotypes. Plasma glucose was significantly lower in ‘fertil+’ than in ‘fertil−’ cows in the second wkpp. Taken together, our results show that ‘fertil+’ and ‘fertil−’ dairy cows, with different fertility, have also different eating behaviour without any variation in energy balance, except in the first week of lactation.     

Oxidative stress index (OSi) as a new tool to assess redox status in dairy cattle during the transition period

Oxidative stress (OS) plays a key role in the initiation or progression of numerous diseases, and dairy cows undergo OS at the transition period. However, discrepancies between methodologies make it difficult to make comparisons between studies, and therefore research on this topic may not be implemented in farms. This study aims to test under field conditions the use of an oxidative stress index (OSi) as a combined measurement through a ratio between pro-oxidants and antioxidants throughout the transition period in dairy farms. Serum samples of high-yielding dairy cows were taken, and markers of oxidative damage and antioxidant capacity were measured in four different production stages: (i) late lactation (LL; −2 to −1 months); (ii) prepartum (PrP; −1 month until parturition); (iii) postpartum (PsP; delivery to +1 month); and (iv) peak of lactation (PkL; +1 to +2.5 months). Values were compared between production stages and against a metabolic baseline status (CTR, 4th to 5th month of gestation). To the best of our knowledge, this is the first report in the literature that discusses the values of these oxidative stress biomarkers (and the OS index) for cows with low metabolic demands, as to date most research in this area has focused on the transition period. With the joint evaluation through the OSi, differences were found that were not present with the separate evaluation of pro-oxidants or antioxidants, thus supporting our hypothesis that the OSi indicates more accurately the oxidative status of the animals. It was also confirmed that dairy cows undergo OS after parturition, and that antioxidant supplementation from 1 month before parturition until the peak of lactation may be needed to reduce the risk of OS.     

Neutrophil chemoattractant and IL-1-like activity in samples from psoriatic skin lesions. Further characterization

The IL-1-like neutrophil chemoattractant activity previously reported by us to be present in the stratum corneum of psoriatic skin lesions has now been characterized further. Aqueous extracts of stratum corneum samples from psoriatic lesions and from the heels of normal volunteers were ultrafiltered to yield 10- to 30-kDa fractions. The ultrafiltered psoriatic preparations consistently contained greater neutrophil chemokinetic activity than the normal heel preparations, but in contrast the latter contained markedly greater IL-1 activity than the former. Successive chromatographic purification of psoriatic lesional stratum corneum extracts showed that the neutrophil chemokinetic material previously reported to co-elute with IL-1 activity on reversed phase HPLC, but to be distinct from C5a des arg, could now be separated by anion exchange HPLC into at least four different chemokinetic compounds that were also resolved from the IL-1 activity. The reversed phase HPLC-purified chemokinetic material from psoriatic stratum corneum was also active in a neutrophil chemotaxis assay. These findings show that samples from psoriatic skin lesions contain a group of novel 10- to 30-kDa neutrophil chemoattractant compounds that are distinct from both C5a des arg and IL-1. The contrasting neutrophil chemokinetic and IL-1 activities in psoriatic lesional and normal heel stratum corneum preparations support the finding that the two activities are produced by different compounds. These neutrophil chemoattractant and IL-1-like compounds may be of pathogenic importance in inflammatory skin disease.     

Staining of Keratin and Keratohyalin with the Reactive Dye Levafix Red Violet E-2BL

Demonstration of keratin in Zenker-fired skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-PBL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fired sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaC1, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.     

ON THE NATURE OF THE DYE PENETRATING THE VACUOLE OF VALONIA FROM SOLUTIONS OF METHYLENE BLUE

When uninjured cells of Valonia are placed in methylene blue dissolved in sea water it is found, after 1 to 3 hours, that at pH 5.5 practically no dye penetrates, while at pH 9.5 more enters the vacuole. As the cells become injured more dye enters at pH 5.5, as well as at pH 9.5. No dye in reduced form is found in the sap of uninjured cells exposed from 1 to 3 hours to methylene blue in sea water at both pH values. When uninjured cells are placed in azure B solution, the rate of penetration of dye into the vacuole is found to increase with the rise in the pH value of the external dye solution. The partition coefficient of the dye between chloroform and sea water is higher at pH 9.5 than at pH 5.5 with both methylene blue and azure B. The color of the dye in chloroform absorbed from methylene blue or from azure B in sea water at pH 5.5 is blue, while it is reddish purple when absorbed from methylene blue and azure B at pH 9.5. Dry salt of methylene blue and azure B dissolved in chloroform appears blue. It is shown that chiefly azure B in form of free base is absorbed by chloroform from methylene blue or azure B dissolved in sea water at pH 9.5, but possibly a mixture of methylene blue and azure B in form of salt is absorbed from methylene blue at pH 5.5, and azure B in form of salt is absorbed from azure B in sea water at pH 5.5. Spectrophotometric analysis of the dye shows the following facts. 1. The dye which is absorbed by the cell wall from methylene blue solution is found to be chiefly methylene blue. 2. The dye which has penetrated from methylene blue solution into the vacuole of uninjured cells is found to be azure B or trimethyl thionine, a small amount of which may be present in a solution of methylene blue especially at a high pH value. 3. The dye which has penetrated from methylene blue solution into the vacuole of injured cells is either methylene blue or a mixture of methylene blue and azure B. 4. The dye which is absorbed by chloroform from methylene blue dissolved in sea water is also found to be azure B, when the pH value of the sea water is at 9.5, but it consists of azure B and to a less extent of methylene blue when the pH value is at 5.5. 5. Methylene blue employed for these experiments, when dissolved in sea water, in sap of Valonia, or in artificial sap, gives absorption maxima characteristic of methylene blue. Azure B found in the sap collected from the vacuole cannot be due to the transformation of methylene blue into this dye after methylene blue has penetrated into the vacuole from the external solution because no such transformation detectable by this method is found to take place within 3 hours after dissolving methylene blue in the sap of Valonia. These experiments indicate that the penetration of dye into the vacuole from methylene blue solution represents a diffusion of azure B in the form of free base. This result agrees with the theory that a basic dye penetrates the vacuole of living cells chiefly in the form of free base and only very slightly in the form of salt. But as soon as the cells are injured the methylene blue (in form of salt) enters the vacuole. It is suggested that these experiments do not show that methylene blue does not enter the protoplasm, but they point out the danger of basing any theoretical conclusion as to permeability on oxidation-reduction potential of living cells from experiments made or the penetration of dye from methylene blue solution into the vacuole, without determining the nature of the dye inside and outside the cell.     

Staining of keratin and keratohyalin with the reactive dye levafix red violet E-2BL.

Demonstration of keratin in Zenker-fixed skin and in tissues stored in formalin can be difficult because such material is unsuitable for histochemical studies. A reactive dye, Levafix red violet E-2BL, proved useful for demonstration of keratohyalin and some types of keratin. Formalin-, Zenker- and methacarn-fixed sections were pretreated with alkaline alcohol, stained one hour at 60 C in an aqueous solution containing 0.25% Levafix red violet E-2BL plus 0.25% NaCl, rinsed in buffer solution pH 9, dehydrated and mounted. Keratohyalin granules and stratum corneum were colored red violet; hair and tonofibrils remained unstained. In sections prestained with Mayer's acid hemalum, keratohyalin was dark blue. Sulfonated monoazo dyes without reactive groups colored no tissue structures under the conditions of this technic; apparently, Levafix red violet E-2BL is bound via its reactive group. Polarization microscopic studies suggest binding of Levafix red violet E-2BL by an amorphous matrix of keratin. Correlations with chemical data indicate that the staining patterns parallel the distribution of proteins formed in the stratum granulosum.     

Recovery of Skin Barrier After Stratum Corneum Removal by Microdermabrasion

Microdermabrasion is widely used as a non-invasive cosmetic technique that has recently been adapted to selectively remove stratum corneum to increase skin permeability for transdermal drug delivery. This study measured the kinetics of skin barrier recovery after stratum corneum removal using microdermabrasion in hairless guinea pigs. The skin was abraded at two sites on each animal, one of which was allowed to recover under occlusion while the other remained non-occluded. Histological measurements showed that skin barrier properties to sulforhodamine B largely recovered within 12 h, and the stratum corneum appeared largely reformed within 24 h for both occluded and non-occluded skin. Skin electrical resistance measurements showed significant recovery of the skin barrier within 24 h. We conclude that transdermal drug delivery may occur for up to 12 h after microdermabrasion in guinea pigs; however, humans will probably have a longer recovery time due to expected slower skin healing rates.     

A Spontaneous Fatp4/Scl27a4 Splice Site Mutation in a New Murine Model for Congenital Ichthyosis

Congenital ichthyoses are life-threatening conditions in humans. We describe here the identification and molecular characterization of a novel recessive mutation in mice that results in newborn lethality with severe congenital lamellar ichthyosis. Mutant newborns have a taut, shiny, non-expandable epidermis that resembles cornified manifestations of autosomal-recessive congenital ichthyosis in humans. The skin is stretched so tightly that the newborn mice are immobilized. The genetic defect was mapped to a region near the proximal end of chromosome 2 by SNP analysis, suggesting Fatp4/Slc27a4 as a candidate gene. FATP4 mutations in humans cause ichthyosis prematurity syndrome (IPS), and mutations of Fatp4 in mice have previously been found to cause a phenotype that resembles human congenital ichthyoses. Characterization of the Fatp4 cDNA revealed a fusion of exon 8 to exon 10, with deletion of exon 9. Genomic sequencing identified an A to T mutation in the splice donor sequence at the 3′-end of exon 9. Loss of exon 9 results in a frame shift mutation upstream from the conserved very long-chain acyl-CoA synthase (VLACS) domain. Histological studies revealed that the mutant mice have defects in keratinocyte differentiation, along with hyperproliferation of the stratum basale of the epidermis, a hyperkeratotic stratum corneum, and reduced numbers of secondary hair follicles. Since Fatp4 protein is present primarily at the stratum granulosum and the stratum spinosum, the hyperproliferation and the alterations in hair follicle induction suggest that very long chain fatty acids, in addition to being required for normal cornification, may influence signals from the stratum corneum to the basal cells that help to orchestrate normal skin differentiation.     

Hair cortisol detection in dairy cattle by using EIA: protocol validation and correlation with faecal cortisol metabolites

Hair may be a useful matrix to detect cumulative cortisol concentrations in studies of animal welfare and chronic stress. The aim of this study was to validate a protocol for cortisol detection in hair from dairy cattle by enzyme immunoassay (EIA). Seventeen adult Holstein–Friesian dairy cows were used during the milking period. Hair cortisol concentration was assessed in 25-day-old hair samples taken from the frontal region of the head, analysing black and white coloured hair separately. Concentrations of cortisol metabolites were determined in faeces collected twice a week during the same period of time. There was a high correlation between cortisol values in faeces and cortisol in white colour hair samples but such correlation was not significant with the black colour hair samples. The intra- and inter-assay coefficients of variation were 4.9% and 10.6%, respectively. The linearity showed R²=0.98 and mean percentage error of −10.8±1.55%. The extraction efficiency was 89.0±23.52% and the parallelism test showed similar slopes. Cortisol detection in hair by using EIA seems to be a valid method to represent long-term circulating cortisol levels in dairy cattle.     

Noninvasive characterization of human stratum corneum of undiseased skin of patients with atopic dermatitis and psoriasis as studied by Fourier transform Raman spectroscopy

Etiopathogenetic regulatory disorders of epidermal metabolism and the subsequent changes in the molecular pattern of the stratum corneum play an important role in the clinical differentiation of particular dermatoses (e.g., psoriasis, atopic dermatitis). In this study we present in vitro Fourier transform Raman spectra of the stratum corneum from healthy skin, as well as from clinically undiseased skin of the right heel of atopic and psoriatic volunteers. Differences in the averaged spectra were detected, particularly in the spectral ranges of 1112-1142 (lipid band), 1185-1220, and 1394-1429 cm(-1). By using the first derivative of the averaged spectra and/or a statistical evaluation of the spectroscopic data it was possible to distinguish the skin types examined.     

Human stratum corneum lipids: characterization and regional variations

The lipids of mammalian stratum corneum are known to be important regulators of skin permeability. Since the human stratum corneum displays remarkable regional variations in skin permeability, we assessed the total lipid concentration, the distribution of all major lipid species, and the fatty acid composition in Bligh-Dyer extracts from four skin sites (abdomen, leg, face, and sole) that are known to display widely disparate permeability. Statistically significant differences in lipid weight were found at the four sites that were inversely proportional to their known permeability. In all four sites, among the polar lipids, the stratum corneum contained negligible phospholipids, but substantially more cholesterol sulfate (1-7%) than previously appreciated. As in the stratum corneum from other mammals, the bulk of the lipids consisted of neutral (60-80%) and sphingolipids (15-35%). Of the neutral lipids, free sterols (4- to 5-times greater than esterified sterols), free fatty acids, triglycerides, and highly nonpolar species (n-alkanes and squalene) predominated. n-Alkanes, which were present in greater quantities than previously appreciated, comprised a homologous series of odd- and even-chained compounds ranging from C19 to C34. The sphingolipids comprised over 80% ceramides vs. lesser quantities of glycosphingolipids. In all four sites, the sphingolipids were the major repository of long-chain, saturated fatty acids. The neutral lipid:sphingolipid ratio generally was proportional to the known permeability of each site: higher neutral lipids and lower sphingolipids generally were associated with superior barrier properties. These studies provide: 1) the first detailed, quantitative analysis of human stratum corneum lipids and 2) information about the variability in lipid composition at four skin sites with known differences in permeability. The latter results suggest that variations in neutral lipids, rather than sphingolipids, may underlie local variations in skin permeability.     

Paratuberculosis: decrease in milk production of German Holstein dairy cows shedding Mycobacterium avium ssp. paratuberculosis depends on within-herd prevalence

Paratuberculosis impairs productivity of infected dairy cows because of reduced milk production and fertility and enhanced risk of culling. The magnitude of the milk yield depression in individual cows is influenced by factors such as parity, the stage of the disease and the choice of test used. The objectives of this case–control study were to substantiate the influence of the different levels of the within-herd prevalence (WHP) on individual milk yield of fecal culture (FC)-positive cows (FC+) compared with FC-negative herd-mates (FC−), and to estimate the magnitude of the deviation of the milk yield, milk components and somatic cell count (SCC) in an FC-based study. Of a total of 31 420 cows from 26 Thuringian dairy herds tested for paratuberculosis by FC, a subset of 1382 FC+ and 3245 FC− with milk recording data were selected as cases and controls, respectively. The FC− cows were matched for the same number and stage of lactation (±10 days in milk) as one FC+ from the same herd. Within a mixed model analysis using the fixed effects of Mycobacterium avium ssp. paratuberculosis (MAP) status, lactation number, days in milk, prevalence class of farm and the random effect of farm on milk yield per day (kg), the amount of fat and protein (mg/dl) and lactose (mg/dl) as well as the SCC (1000/ml) were measured. On the basis of least square means, FC+ cows had a lower test-day milk yield (27.7±0.6 kg) compared with FC− (29.0±0.6 kg), as well as a lower milk protein content and a slightly diminished lactose concentration. FC status was not associated with milk fat percentage or milk SCC. In FC+ cows, reduction in milk yield increased with increasing WHP. An interaction of FC status and farm was found for the test-day milk yield, and milk protein percentage, respectively. We conclude that the reduction in milk yield of FC+ cows compared with FC− herd-mates is significantly influenced by farm effects and depends on WHP class. Owners of MAP-positive dairy herds may benefit from the reduction in WHP not only by reducing number of infected individuals but also by diminishing the individual losses in milk production per infected cow, and therefore should establish control measures.     

Urinary metabolomics fingerprinting around parturition identifies metabolites that differentiate lame dairy cows from healthy ones

Lameness is a very important disorder of periparturient dairy cows with implications on milk production and composition as well as with consequences on reproductive performance. The aetiology of lameness is not clear although there have been various hypotheses suggested over the years. The objective of this study was to metabotype the urine of dairy cows prior to, during and after the onset of lameness by evaluating at weeks −8, −4 pre-calving, the week of lameness diagnosis, and +4 and +8 weeks post-calving. We used a metabolomics approach to analyse urine samples collected from dairy cows around calving (6 cows with lameness v. 20 healthy control cows). A total of 153 metabolites were identified and quantified using an in-house MS library and classified into 6 groups including: 11 amino acids (AAs), 39 acylcarnitines (ACs), 3 biogenic amines (BAs), 84 glycerophospholipids, 15 sphingolipids and hexose. A total of 23, 36, 40, 23 and 49 metabolites were observed to be significantly different between the lame and healthy cows at −8 and −4 weeks pre-calving, week of lameness diagnosis as well as at +4 and +8 weeks post-calving, respectively. It should be noted that most of the identified metabolites were elevated; however, a few of them were also lower in lame cows. Overall, ACs and glycerophospholipids, specifically phosphatidylcholines (PCs), were the metabolite groups displaying the strongest differences in the urine of pre-lame and lame cows. Lysophosphatidylcholines (LysoPCs), although to a lesser extent than PCs, were altered at all time points. Alterations in urinary AA concentrations were also observed during the current study for four time points. During the pre-calving period, there was an observed elevation of arginine (−8 week), tyrosine (−8 week) and aspartate (−4 week), as well as a depression of urinary glutamate (−4 weeks). In the current study, it was additionally observed that concentrations of several sphingomyelins and one BA were altered in pre-lame and lame cows. Symmetric dimethylarginine was elevated at both −8 weeks pre-calving and the week of lameness diagnosis. Data showed that urinary fingerprinting might be a reliable methodology to be used in the future to differentiate lame cows from healthy ones.     

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.     

Targeted metabolomics: new insights into pathobiology of retained placenta in dairy cows and potential risk biomarkers

A targeted quantitative metabolomics approach was used to study temporal changes of serum metabolites in cows that normally released their fetal membranes and those that retained the placenta. We identified and measured serum concentrations of 128 metabolites including amino acids, acylcarnitines, biogenic amines, glycerophospholipids, sphingolipids and hexose at −8 and −4 weeks before parturition, during the week of retained placenta (RP) diagnosis, and at +4 and +8 weeks after parturition. In addition, we aimed at identifying metabolite signatures of pre-RP in the serum that might be used as predictive biomarkers for risk of developing RP in dairy cows. Results revealed major alterations in the metabolite fingerprints of pre-RP cows starting as early as −8 weeks before parturition and continuing as far as +8 weeks after calving. Biomarker candidates found in this study are mainly biomarkers of inflammation which might not be specific to RP. Therefore, the relevance of serum Lys, Orn, acetylornithine, lysophophatidylcholine LysoPC a C28:0, Asp, Leu and Ile as potential serum biomarkers for prediction of risk of RP in dairy cows will have to be tested in the future. In addition, lower concentrations of LysoPCs, Trp, and higher kynurenine in the serum during prepartum and the week of occurrence of RP suggest involvement of inflammation in the pathobiology of RP.     

The important role of epidermal triacylglycerol metabolism for maintenance of the skin permeability barrier function

Survival in a terrestrial, dry environment necessitates a permeability barrier for regulated permeation of water and electrolytes in the cornified layer of the skin (the stratum corneum) to minimize desiccation of the body. This barrier is formed during cornification and involves a cross-linking of corneocyte proteins as well as an extensive remodeling of lipids. The cleavage of precursor lipids from lamellar bodies by various hydrolytic enzymes generates ceramides, cholesterol, and non-esterified fatty acids for the extracellular lipid lamellae in the stratum corneum. However, the important role of epidermal triacylglycerol (TAG) metabolism during formation of a functional permeability barrier in the skin was only recently discovered. Humans with mutations in the ABHD5/CGI-58 (α/β hydrolase domain containing protein 5, also known as comparative gene identification-58, CGI-58) gene suffer from a defect in TAG catabolism that causes neutral lipid storage disease with ichthyosis. In addition, mice with deficiencies in genes involved in TAG catabolism (Abhd5/Cgi-58 knock-out mice) or TAG synthesis (acyl-CoA:diacylglycerol acyltransferase-2, Dgat2 knock-out mice) also develop severe skin permeability barrier dysfunctions and die soon after birth due to increased dehydration. As a result of these defects in epidermal TAG metabolism, humans and mice lack ω-(O)-acylceramides, which leads to malformation of the cornified lipid envelope of the skin. In healthy skin, this epidermal structure provides an interface for the linkage of lamellar membranes with corneocyte proteins to maintain permeability barrier homeostasis. This review focuses on recent advances in the understanding of biochemical mechanisms involved in epidermal neutral lipid metabolism and the generation of a functional skin permeability barrier. 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.     

Human tissue kallikreins as promiscuous modulators of homeostatic skin barrier functions

Human tissue kallikreins (KLKs) are the largest family of secreted serine protease endopeptidases encoded by 15 genes clustered on chromosome 19q13.4. Multiple KLK enzymes are co-localized in the upper stratum granulosum and stratum corneum of human epidermis, and in associated appendages such as hair follicle epithelia and sweat glands. Until recently, kallikrein proteolytic activity in the skin was exclusively attributed to KLK5 and KLK7. However, wider cutaneous roles of kallikreins became evident in recent years as the proposal of KLK proteolytic activation cascades emerged. We postulate that these proteolytic enzymes may serve as promiscuous mediators of different skin barrier functions, since they are capable of proteolysing different substrates that govern skin desquamation, antimicrobial defense, and lipid permeability. Growing evidence now attests to potential kallikrein involvement in skin inflammation, pigmentation, and tumor suppression via their ability to target proteinase-activated receptor signaling pathways. Current knowledge on kallikrein roles in skin physiology and pathobiology is described in this review.     

Stratum corneum lipid phase transitions and water barrier properties

In mammals, the outer skin layer, the stratum corneum, is the ultimate barrier to water loss. In order to relate barrier function to stratum corneum structure, samples from porcine skin were investigated by using differential scanning calorimetry (DSC), infrared (IR) spectroscopy, and water permeability techniques. Results of DSC and IR studies show that stratum corneum lipids undergo thermal transitions between 60 and 80 degrees C similar to lipid thermotropic transitions seen in a variety of synthetic and biological membranes. Results of water flux experiments performed under conditions similar to those of the DSC and IR studies show an abrupt change in permeability at about 70 degrees C. At low temperatures, water flux values are similar to those obtained for human skin in vivo, yielding an activation energy of 17 kcal/mol, in excellent agreement with values obtained for water flux through a variety of lipid biomembranes. In contrast, at temperatures above about 70 degrees C, water flux is characterized by an activation energy only slightly higher than that of free diffusion, suggesting that the stratum corneum offers little diffusional resistance under these conditions. These combined results suggest that increased disorder in stratum corneum lipid structure, brought about by thermotropic transitions, results in dramatically altered diffusional resistance of this tissue to water flux. Thus, as found for numerous biological membranes, water flux and lipid order in porcine stratum corneum are inversely related.     

Interactions of inertial cavitation bubbles with stratum corneum lipid bilayers during low-frequency sonophoresis

Interactions of acoustic cavitation bubbles with biological tissues play an important role in biomedical applications of ultrasound. Acoustic cavitation plays a particularly important role in enhancing transdermal transport of macromolecules, thereby offering a noninvasive mode of drug delivery (sonophoresis). Ultrasound-enhanced transdermal transport is mediated by inertial cavitation, where collapses of cavitation bubbles microscopically disrupt the lipid bilayers of the stratum corneum. In this study, we describe a theoretical analysis of the interactions of cavitation bubbles with the stratum corneum lipid bilayers. Three modes of bubble-stratum corneum interactions including shock wave emission, microjet penetration into the stratum corneum, and impact of microjet on the stratum corneum are considered. By relating the mechanical effects of these events on the stratum corneum structure, the relationship between the number of cavitation events and collapse pressures with experimentally measured increase in skin permeability was established. Theoretical predictions were compared to experimentally measured parameters of cavitation events.