Stratum corneum How does your body keep

Stratum corneum

How does your body keep most enemies out? • Many would consider the moat around this castle, together with the thick stone castle walls, as the first line of defense. Their role is to keep the enemy out, and protect what's inside.

Introduction • Research about the skin barrier and its properties has increased significantly since the 60 s, with studies that indicated its resistance when isolated, as well as its particularities in relation to skin permeability. At the same time, description of Odland bodies helped to understand how stratum corneum stability is maintained. The "brick and mortar" model is the most accepted so far. In this analogy, the corneocytes are the bricks and the intercellular lipids are the mortar. Currently, there is concrete evidence that the stratum corneum is an active metabolic structure that holds adaptive functions, interacting dynamically with the underlying epidermal layers. The skin barrier also plays a role in the inflammatory response through melanocyte activation, angiogenesis, and fibroplasia. The intensity of this response will essentially depend on the severity of the injury. The stratum corneum hydration level and transepidermal water loss are associated with the level of damage to the barrier, representing biophysical parameters.

Skin layers

The stratum corneum on top

The stratum corneum

Epidermis of thick skin

The stratum corneum

• The thick stratum corneum of the palms and soles prevents chemicals from readily entering those areas. This patient worked around a chemical to which he became allergic, and you can see the line of demarcation along the sides of his hands indicating the thinner stratum corneum on the dorsum of the hands, and a thicker stratum corneum on the palms.

Physiology of the epidermal layer of the skin

• The stratum lucidum layer is only present in thick skin where it helps reduce friction and shear forces between the stratum corneum and stratum granulosum It is only found on palms and soles of feet. Cells are flat, densely packed and contain much keratin (creates more calluses)

• Epidermis (thick skin) H&E. 104 x C = stratum corneum L = stratum lucidum G = stratum granulosum S = stratum spinosum B = stratum basale

• Thick skin (palmar) H&E. 200 x 1=stratum corneum • 2=stratum lucidum • 3=stratum granulosum • 4=stratum spinosum • 5=stratum basale • 6=dermal papilla • 7=cell with keratohyalin granules • 8=cells of the stratum spinosum • 9="intercellular bridges" (desmosomes) • 10=desquamating layer • 11=sections through the duct of a sweat gland • 12=cells in mitosis • 13=Meissner's corpuscle in a dermal papilla • 14=papillary layer of the dermis

Stratum corneum formation • • • Physiologically, the stratum corneum is formed by a sequence of events: 1. The keratinocyte cellular membrane of the granulous layer becomes more permeable to ions, especially calcium, which activate peptidases and convert profilaggrin into filaggrin: filaggrin is an intermediate filament-associated protein that exists in the granules of kerato-hialine and activates the enzymes trigliceridadase and aggregates keratin filaments with macrofibrils; next, this protein is degraded to free aminoacids that will later be used in the constitution of the natural moisturizing factor or converted into urocanic acid or pyrrolidone carboxylic acid (PCA). Filaggrin is responsible for aggregating keratin and other proteins in the superficial layers of the epidermis to form the stratum corneum; the process of conversion of profilaggrin into filaggrin maintains the integrity of the epidermis. 2. With the degeneration of the cellular nucleus, cells become flat and keratin molecules align in parallel, creating a cornified envelope, connected to extracellular lipids. The cohesion power of this layer depends upon the formation of covalent connections of lisyne glutamine, where precursor proteins are incorporated into keratin: involucrin, small prolinerich peptides (SPRP), cornifin, loricrin, keratoline, and desmosomal proteins such as envoplakin and periplakin. 3. Lamellar bodies, originated in the granulous layer, also contribute to the formation of the lipid matrix in which corneocytes are located

Historical perspective on filaggrin research. Inset shows immunohistochemical staining of human epidermis, with filaggrin in green, basal-specific keratin 5 in red, and nuclei stained blue. FLG, filaggrin Journal of Investigative Dermatology (2012) 132, 751

Profilaggrin, and their constituent amino acids are multifunctional proteins contributing to the formation and function of the skin barrier. Diagram summarizing the known and possible functions of profilaggrin, and amino acids released by filaggrin proteolysis Journal of Investigative Dermatology (2012) 132, 751

Brick & mortar

Layers in the epidermis

Stratum Corneum The stratum corneum is the outermost of the 5 layers of the epidermis and is largely responsible for the vital barrier function of the skin. The biological and chemical activity of the stratum corneum is very intricate and complex

Stratum Corneum Anatomy - The Corneocyte • • The stratum corneum is the outermost of the 5 layers of the epidermis and is largely responsible for the vital barrier function of the skin. Before the mid 1970's the stratum corneum was thought to be biologically inert, like a thin plastic sheet protecting the more active lower layers of the skin. In the past 30 years, and especially the past 5 years, scientists have discovered that the biological and chemical activity of the stratum corneum is very intricate and complex. Understanding the structure and function of the stratum corneum is vital because it is the key to healthy skin and its associated attractive appearance

Stratum Corneum Anatomy - Cornified Envelope • Each cornecyte is surrounded by a protein shell called a cell envelope. The cell envelope is composed primarily of two proteins, loricirn and involucrin. These proteins contain extensive links between each other making the cell envelope the most insoluble structure of the corneocyte. The two subtypes of cell envelopes are described as "rigid" and "fragile" based on the interaction of lamellar bilayer with the cell envelope

Structure of the Stratum Corneum Extracellular Matrix

Stratum Corneum • • Das Backstein-Zement-Modell 1 Hornzellen (Korneozyten) : Horny cell cornycytes 2 Epidermale Lipide The functions of horny layer • • • Rasterelektronenmikroskopische Aufnahme eines Gefrierbruches des Stratum corneum. 1. Korneozyten 2. Interzelluläre Räume, z. T. mit Hautlipiden gefüllt

• Schematic illustration of the process involved in formation of intercellular stratum corneum lipids of a mammal following extrusion from lamellar bodies. The lipid content of lamellar bodies is altered in composition and rearranged into long lipid lamellae that fill the extracellular regions in the stratum corneum.

The Protective Acid Mantle

The Protective Acid Mantle • • • • • acidic environment is important for : Closer examination of the components of the hydrolipid film reveals why this protective film was first named by Schade and Marchionini in 1928 the protective acid mantle: Sweat contains lactic acid and various amino acids. Sebum contains free fatty acids. Amino acids and pyrrolidine carboxylic acid are produced by the cornification process. The physiological p. H of healthy skin has an average value lying between 5. 4 and 5. 9. In this p. H range the skin is populated by a normal skin-typical flora. Pathogenic microorganisms are hindered from spreading. In the armpits, anal folds and the genitals, however, the p. H is approximately 6. 5 (physiological gaps). activation of the enzymes responsible for the synthesis of important epidermal lipids, formation of the bilayer lipid membrane and restoration of the horny layer following mechanical or chemical damage. An acidic environment is important for synthesis of the epidermal lipids, which consist mainly of ceramides (40%), free fatty acids (25%) and cholesterol (25%). Synthesis of the especially important ceramides is catalysed by an enzyme belonging to the group of acid hydrolases. Odland bodies Exocytosis Cells of the stratum granulosum Bilayer lipid membrane

Stratum Corneum Anatomy - Lamellar Bodies • Lamellar bodies are formed in the keratinocytes of the stratum spinosum and stratum granulosum. When the keratinocyte matures to the stratum corneum, enzymes degrade the outer envelope of the lamellar bodies releasing types of lipids called free fatty acids and ceramides.

Stratum Corneum Anatomy - Cornified Envelope Lipids • Attached to the cell envelop is a layer of ceramide lipids that repel water. Because the lamellar lipid bilayer also repels water, water molecules are held between the cell envelope lipids and the lipid bilayer. This helps maintain the water balance in the stratum corneum by trapping water molecules instead of letting them absorb into the lower layers of the epidermis.

• Functions of matriptase in the epidermis. A. The epidermal barrier function resides in the stratum corneum, the outermost layer of the interfollicular epidermis. Barrier function is conferred by a water-impermeable cornified envelope surrounding terminally differentiated keratinocytes (corneocytes) and by epidermal lipids (lipid lamellae) in which corneocytes are embedded. Stratum corneum thickness is regulated by the proteolytic degradation of cell-cell junctions between corneocytes (corneodesmosomes). Matriptase is expressed in the transitional cell layer and in the stratum corneum. In the transitional cell layer, matriptase facilitates the proteolytic processing of profilaggrin into filaggrin monomers that are structural components of the cornified envelope (arrowhead pointing right) and a regulatory S-100 protein that translocates to the nucleus and promotes terminal keratinocyte differentiation (arrowhead pointing left). In the uppermost part of the stratum corneum, matriptase is required for the proteolytic degradation of corneodesmosomes, leading to stratum corneum shedding (desquamation).

Lamellar granules (Odland bodies) • Red arrows indicate secreted lamellar granules, and green arrows indicate lamellar granules in the cytoplasm • • • Lamellar granules (otherwise known as membrane-coating granules (MCGs), lamellar bodies, keratinosomes or Odland bodies) are secretory organelles found in type II pneumocytes and keratinocyte. They are oblong structures, appearing about 300 -400 nm in width and 100 -150 nm in length in transmission electron microscopy images. Lamellar granules fuse with the cell membrane and release their contents into the extracellular space. In the upper spinous layer and stratum granulosum layer of the epidermis, lamellar bodies are secreted from keratinocytes, resulting in the formation of an impermeable, lipid-containing membrane that serves as a water barrier and is required for correct skin barrier function. These granules release components that are required for skin shedding (desquamation) in the uppermost epidermal layer, the stratum corneum. These components include lipids (e. g. glucosylceramides), hydrolytic enzymes (e. g. proteases, acid phosphatases, glucosidases, lipases) and proteins (e. g. corneodesmosin). Lamellar granules have been observed to contain distinct aggregates of the secreted components glucosylceramide, cathepsin D, KLK 7, KLK 8 and corneodesmosin. Transportation of molecules via lamellar granules is thought to prevent enzymes from interacting with their relevant substrates or inhibitors prior to secretion. Recent work suggests that lamellar granules form a continuous membranous structure with the trans. Golgi network Lamellar body secretion and lipid structure is abnormal in the epidermis of patients with Netherton syndrome, a skin disorder characterised by chronic inflammation and universal pruritus (itch).

Cornified cell • Cornified cells attached to one another by vestigial desmosomes. A cornified cell is a package of tonofibrils encased in a protein matrix. The nucleus and the organelles within the cytoplasm have been lost during maturation. Melanosomes are found within keratocytes at all levels of the epidermis, including the cornified layer.

Stratum corneum • • • Electron micrographs showing details of stratum corneum and permeability barrier of terrestrial vertebrates. (A) Section through a portion of cocoon of a burrowing hylid frog, Pternohyla fodiens. The layers of squamous epidermal cells are separated by granular extracellular materials in the subcorneal spaces. Scale bar, 500 nm. (B) Section through mesos layer of snake epidermis (Natrix natrix), which is the recognized permeability barrier of squamates. Laminated lipids occur between the darker bands of keratin layers. Scale bar, 100 nm. (C) Section through stratum corneum of human skin. Lipids (unstained) occur between the distinct layers of keratin. Scale bar, 200 nm. (D) Section through epidermis of a canary, showing nucleated layers as well as stratum corneum (top). Lipids occur between the distinct layers of keratin toward top of figure. Note the multigranular bodies (source of lipids; arrows). Scale bar, 200 nm.

Stratum Corneum Anatomy - Intercellular Lipids • Free fatty acids and ceramides that are released from the lamellar bodies fuse together in the stratum corneum to form a continuous layer of lipids. Because there are two types of lipids, this layer is referred to as a lamellar lipid bilayer. This lipid bilayer plays a major role in maintaining the barrier properties of the skin and is analagous to the "mortar" in the brick and mortar model.

Corneodesmosomes • The "rivets" that hold the corneocytes together are specialized protein structures called corneodesmosomes. These structures are also a part of the "mortar" in the "brick and mortar" analogy. Corneodesmosomes are the major structure that must be degraded for the skin to shed

Corneodesmosomes • Model of desquamation: p. H controls KLK activities by regulating their interaction with LEKTI. In the deep SC, neutral p. H allows a strong interaction between LEKTI and its KLK targets in the corneocyte interstices, thus preventing corneodesmosomes cleavage. As the p. H acidifies along the SC, LEKTI, and KLK 5 dissociate, allowing proteinase to progressively degrade its corneodesmosomal targets. In the most superficial layers of SC, p. H is low enough to ensure a strong dissociation between LEKTI and its KLK targets. The release of KLK inhibition, together with other proteinase activities, lead to complete degradation of corneodesmosomal components, resulting in the detachment of the most superficial corneocytes

Desquamation Process • The desquamation, or exfoliation, process of the stratum corneum is actually very complex and only parts of this process are fully understood. We do know that several enzymes degrade the corneodesmosomes in a specific pattern, but we don't know the exact nature of these enzymes or how they become activated to start the exfoliation process. We do know that water and p. H play a significant role in the activity of these enzymes

Stratum corneum

Sloughing Stratum Corneum Cells

2. 260 x magnification of the skin barrier.

• • The mechanism of lip chapping In chapped lips, lip corneocytes adhere firmly to each other and stubbornly resist shedding. An essential strategy to prevent lip chapping is to hasten the shedding of cells by boosting the cellular metabolism. The cellular turnover in the stratum corneum is encouraged by a Cathepsin D-like enzyme that resides in the stratum corneum of the lips. In chapped lips, the activity of this enzyme is decreased.

Change from the stratum granulosum to the stratum corneum

• • Intact stratum corneum layer of the epidermis. The integrity of this outermost layer of skin is vital to moisture retention and the overall function of the skin. Natural moisturizing factor inside of the corneocytes absorbs water from outside of the skin, which keeps the stratum corneum hydrated. Corneocytes are held together by protein structures called corneodesmosones. The collected efforts of the hydrophobic cornified lipid envelope and the intercellular lipid bilayer produced by the lamellar bodies serve to maintain moisture balance within the stratum corneum

Disrupted epidermis • Disrupted epidermis. • Disruption to the stratum corneum can result in the degradation of corneodesmosomes, opening the flood gate to transepidermal water loss (TEWL) and bacterial contamination. Also, because the compounds in natural moisturizing factor are water soluble, prolonged exposure to water can create a concentration gradient that further dehydrates the corneocytes and the stratum corneum.

There are two types of proteases: endogenous ones (synthesized in the granular cell layer under the stratum corneum) and exogenous ones (derived from staphylococcus aureus or a dust mite

• Protease inhibitors seem to be mainly produced in the sweat gland cover the body surface together with sweat to serve as defense against exogenous proteases

• Corneocytes store water (H 2 O) to protect the skin from the drying stress of the environment. This water-retaining function is born by natural moisturizing factor (NMF), sponge-like substances in the cells, derived from a protein called filaggrin. Each corneocyte is encased in a thin lipid envelope (lipid lamellae).

Brick & mortar

Schematic structure of the stratum corneum according to the brick and mortar model. The horny cells are embedded in a lamellar structured lipid matrix

Physiologic Lipids

• • The stratum corneum is a particularly important barrier to the control of moisture loss. The tightly packed cells of the stratum corneum (top) provide a barrier against harmful material from the outside world, as well as protection against water loss. It is also a highly effective barrier against the outside environment, being tough but flexible rovided it is well hydrated. If its water content p falls below 10% it becomes dry, less flexible and increasingly prone to damage, breakdown and infection. The epidermis as a whole is about 35 micrometres thick when dry, but can swell to 48 micrometres on full hydration. This depends more on the humidity and temperature of the surrounding air than on how much we have drunk! SKIN MYTH Drinking six or eight glasses of water a day will keep skin moisture levels high, and is an essential factor in renewing cells and hydrating the skin to prevent wrinkles from forming. It also helps to detoxify and remove waste. Fact: Drinking more will not cause water to enter the skin selectively, unless the person is seriously dehydrated. Normal skin is well hydrated naturally. The excess water goes into all the tissues of the body, and ultimately to the bladder! Detoxification of the body is carried out by organs such as the liver, which do not need vast amounts of water to function

Schematic of stratum corneum

The underlying principle that keeps body fluid in (and external fluids out) is repulsion of aqueous fluid by the lipids in the same way as oil repels water.

Structure of epidermis

How Fungal Nail Cure Works • See how water soluble products do not penetrate keratin and simply runs off the nail in the figure below. Fungal Nail Cure is a naturally occurring, non-toxic, organic compound that carries the fungicidal essential oils all the way to the nail bed by using the intercellular lipid channels. (See below. )

The innovative Norlén model of the lipid bilayer of the stratum corneum Norlén has studied the molecular configuration of the stratum corneum lipids employing the innovative cryo electron microscopy. This specific technology utilizes the different electron densities in biological material and thus induced interference effects for the display of membrane structures. It could be shown that the human skin barrier is characterized by asymmetric lipid bilayers. In comparison with model membranes, a bilayer structure has been suggested that consists of stretched ceramides complexed with free fatty acids on the amide bound acyl chains, and of cholesterols on the sphingosine sequence of the ceramides

Skin barrier

Analysis for skin barrier • Two methods that employ instrumental analysis may ne used for skin barrier evaluation in atopic dermatitis: • A. Measurment of the hydrotic content of the corneal layer (Corneometry) : moisture barrier • B. Measurment of transepidermal water loss (TEWL)

A. Moisture barrier

Three Factors That Retain Moisture • • • A. Sebum Barrier The sebum barrier, which is a mixture of the sweat secreted from sweat glands and the sebum secreted from sebaceous glands, is called a natural cream. It covers and protects the surface of the skin. The amount of sebum secreted differs depending on age, gender, and location on the body. If not enough sebum is secreted, the skin gets dry. Conversely, if there is too much sebum, the skin gets sticky. An appropriate level of sebum secretion is important for moist skin. Generally, sebum is plentiful in the upper half of the body such as the head, face, chest, and back, but tends to lessen in the lower half of the body. Also, sebum secretion is robust in both genders during puberty but declines gradually with age. . B. Natural Moisturizing Factor Natural moisturizing factor (NMF) works to retain moisture in corneocytes. Epidermal cells make the components of NMF, which include amino acids, pyrrolidone carboxylic acid, lactic acid, and uric acid. NMF declines with age. C. Intercorneocyte Lipids Intercorneocyte lipids filled the spaces between corneocytes, controlling water evaporation and retaining moisture in the skin. Approximately 40% of intercorneocyte lipids are moisturizing components called ceramides. The structure of corneocyctes can be likened to bricks and mortar. The structure controls water evaporation and protects us from stimuli when the corneocyctes (bricks) and intercorneocyte lipids (mortar) are firmly connected. Accordingly, if intercorneocyte lipids (mortar) decrease, corneocyctes (bricks) become loose. This is a condition where it seems the skin has been dusted with powder. If any of these three factors related to moisture retention become insufficient for some reasons, water in the stratum corneum decreases, resulting in dry skin.

Corneometer CM Skin Hydration Level Capacitance Measurement

Instrument to Quantify Stratum Corneum Hydration Dermatology Online Journal 7(2): 2 , 2 OO 1 • The skin resistance monitor is a device powered by a nine volt battery designed to measure skin resistance using a wheatstone bridge circuit. The measure of skin resistance is used as an indicator of changes in the moisture content of the skin

Evaluation of water content in the corneal layer: capacitance • The amount of water in the corneal layer may influence the skin barrier function: greater hydration increases percutaneous absorption. • The amount of water retained in the stratum corneum depends on the capacity of water retention, which maintains the skin soft and flexible, even in dry environmental conditions; this water also helps enzymatic reactions in the maturation and scaling of corneocytes. • Water reduction leads to fissures in the stratum corneum, which allow greater penetration of heavier molecular substances, including allergens and microorganisms.

Hydration analysis

What is the difference between moisturized skin and dry skin? • One of the differences between moisturized skin and rough, dry skin is the amount of ceramide or amino acids present in skin, which are both essential for moisturized skin. Strong cleansers or excessive rubbing during cleansing can wash off ceramide or amino acids, which help retain moisture in stratum corneum, causing skin dryness and sensitivity. When skin is healthy, the moisture content found in the stratum corneum (skin’s outer layer) is around 15% to 20%, while the skin cells in the dermis or inner layer of skin contains about 60% to 70% moisture. When the moisture content of the stratum corneum is lower than 10%, skin becomes dry and rough and is unable to retain the remaining moisture. External environments easily affect the stratum corneum. Therefore, when humidity level increases, skin’s moisture content increases as well. And when the air gets dry, skin’s moisture content decreases as well

Natural Moisturizing Factor (NMF) • Natural moisturizing factor (NMF) is a collection of water-soluble compounds that are only found in the stratum corneum. These compounds compose approximately 20 -30% of the dry weight of the corneocyte. NMF components absorb water from the atmosphere and combine it with their own water content allowing the outermost layers of the stratum corneum to stay hydrated despite exposure to the elements. Because NMF components are water soluble, they are easily leached from the cells with water contact - which is why repeated contact with water actually makes the skin drier. The lioid layer surrounding the corneocyte helps seal the corneocyte to prevent loss of NMF.

Deconstructing the skin

Skin Barrier Formation Journal of Investigative Dermatology (2001) 117, 823 • The Landmann model. Transformation of “lamellar body-disks” into intercellular sheets by a membrane-fusion process (c), according to. Landmann (1986). The lamellar body-disks are visualized as flattened unilamellar liposomes (i. e. , vesicles) (b, c) that are stacked inside discrete “lamellar bodies” (b) before extrusion into the intercellular space (ICS) at the border zone between the stratum granulosum (SG) and stratum corneum (SC) (a). Note the liquid crystalline character of the lamellar body-disk edges (i. e. , highly curved regions) (b, c). ICS: intercellular space; N: nucleus; SC 1: first stacked stratum corneum cell; SC 2: second stacked stratum corneum cell; SG: uppermost stratum granulosum cell.

Skin barrier

• • Defense mechanisms in epithelial cells: Nature reviews immunology: 4 , 978 , 2 OO 4 Epithelial cells resist damage owing to the stratum corneum of the skin or the mucus in the airway or intestine. Immune responses to external antigens are only induced in the presence of danger and damage. Danger is recognized through pattern-recognition receptors (PRRs), with the resultant release of active defences, such as antimicrobials or antiproteinases, and signalling molecules to recruit help from specialized immune cells. Different receptors recognize different microbial products or other factors, and can modify the immune-signalling millieu appropriately. In this model, T helper 1 (TH 1)- or TH 2 -cell responses are driven by the nature and site of the initial injury.

• UV-induced mechanisms of immunomodulation: Chromophores in the epidermis that absorb ultraviolet B (UVB) photons include trans-urocanic acid (UCA) in the stratum corneum and DNA, tryptophan and membrane lipids of epidermal cells (predominantly keratinocytes and Langerhans cells). Absorption of UVB photons by 7 dehydrocholesterol in keratinocytes initiates the pathway of vitamin D 3 synthesis. In response to cis -UCA, DNA photoproducts and oxidized membrane lipids and proteins, multiple signalling pathways are stimulated, soluble mediators are produced and cell –cell communication is enhanced between UVBresponsive keratinocytes, Langerhans cells, dermal immune cells (including dermal dendritic cells (DCs) and mast cells) and sensory neurons. Soluble mediators involved include interleukin-6 (IL-6), IL 10, nerve growth factor (NGF), platelet activating factor (PAF), prostaglandin E 2 (PGE 2), tumour necrosis factor (TNF) and cis-UCA. Cellular traffic to the draining lymph nodes via lymphatic vessels increases and includes Langerhans cells, dermal DCs and mast cells. In the draining lymph nodes, cell–cell interactions stimulate the production of regulatory cells and soluble mediators that are responsible for UV-induced systemic immunoregulation. The role of the 1, 25 dihydroxyvitamin D 3 produced by UVB-irradiated keratinocytes is not known. Nature Reviews Immunology 11, 584, 2011

RESIDENT MICROBIOTA IN THE SKIN CONTRIBUTE TO IMMUNITY AND WOUND REPAIR. Nature Immunology : 13, 978 , 2 O 13

• Skin layer Analysis - Nanoparticles concentration of testosterone on the Stratum corneum, viable epidermis and Dermis. The skin depth concentration was measured at 1 hour after transdermal application • A diagram describing the flow of testosterone particles through the three layers over the first hour after testosterone transdermal application.

Disrupted skin barrier

• A tentative model of the regulatory role of cystatin M/E in processes that control epidermal cornification and desquamation. Human cystatin M/E is an inhibitor of legumain (LGMN), cathepsin L (CTSL) and cathepsin V (CTSV). Inhibition of legumain regulates the processing of (pro)-cathepsins. Inhibition of cathepsin V regulates desquamation, as cathepsin V is able to degrade (corneo)desmosomal proteins like desmoglein-1, desmocollin-1, and corneodesmosin. Inhibition of human cathepsin L activity by cystatin M/E is thought to be important in the cornification process, as cathepsin L is the elusive processing and activating enzyme for transglutaminase-3 (TGM 3). Cathepsin L is also able to process cathepsin D (CTSD), which in turn can activate transglutaminase -1 (TGM 1). As cathepsin V is only expressed in humans (pale blue oval), murine cathepsin L probably controls the specific functional enzymatic activities of both human cathepsin L and cathepsin V. Solid lines represent biochemical functions that are known from literature (grey) or deduced from our studies (black). Dashed lines represent unknown functions. BM, basal membrane; SB, stratum basale; SS, stratum spinosum; SG, stratum granulosum; SC, stratum corneum

Shedding Light on "Skin Optics"

Cold, heat, water loss and radiation : As the outermost layer of the skin, the horny layer plays a pivotal role in protecting the body from the environment and limiting the amount of water lost from the epidermis.

Dermatological and cosmetic preparations frequently contain active principles which can only act when they penetrate at least the outermost layer of the skin. However, the efficacy of topically applied actives is often suboptimal because the transport into the skin is slow due to the resistance of the outermost layer of the skin, the stratum corneum. Most small water-soluble non-electrolytes therefore diffuse into the systemic circulation a thousand times more rapidly when the horny layer is absent. Thus, a variety of means have been studied in attempts to overcome this barrier. Such strategies include physical, biochemical, and chemical methods

Possible pathways for a penetrant to cross the skin barrier. (1) across the intact horny layer, (2) through the hair follicles with the associated sebaceaous glands, or (3) via the sweat glands

• Transepidermal transport means that molecules cross the intact horny layer. Two potential micro-routes of entry exist, the transcellular (or intracellular) and the intercellular pathways. The principal pathway taken by a penetrant is decided mainly by the partition coefficient (log K). Hydrophilic drugs partition preferentially into the intracellular domains, whereas lipophilic permeants (octanol/water log K > 2) traverse the stratum corneum via the intercellular route. Most molecules pass the stratum corneum by both routes. However, the tortuous intercellular pathway is widely considered to provide the principal route and major barrier to the permeation of most drugs

Effects of carrier systems on the stratum corneum water content and on the penetration of active ingredients

• Phonophoresis ( or sonophoresis) uses ultrasound energy in order to enhance the skin penetration of active substances [8]. When skin is exposed to ultrasound, the waves propagate to a certain level and cause several effects that assist skin penetration. Figure 6 depicts the processes that can contribute to phonophoresis. One of these effects is the formation and subsequent collapse of gas bubbles in a liquid called cavitation. The force of cavitation causes the formation of holes in the corneocytes, enlarging of intercellular spaces, and perturbation of stratum corneum lipids. Another effect is heating which is mainly due to the energy loss of the propagating ultrasound wave due to scattering and absorption effects. The resulting temperature elevation of the skin is typically in the range of several degrees centigrade. This temperature rise will increase the fluidity of the stratum orneum lipids as well directly increase c the diffusivity of molecules through the skin barrier. These main effects can be assisted by acoustic icrostreaming caused by the m acoustic shear stress which is due to unequal distribution of pressure forces. In addition, ultrasound can push particles through by pressure increase in the skin, although only slightly.

Basic principle of iontophoresis. A current passed between the active electrode and the indifferent electrode repelling drug away from the active electrode and into the skin. • The basic principle of iontophoresis is that a small electric current is applied to the skin. This provides the driving force to primarily enable penetration of charged molecules into the skin. A drug reservoir is placed on the skin under the active electrode with the same charge as the penetrant. A indifferent counter electrode is positioned elsewhere on the body. The active electrode effectively repels the active substance and forces it into the skin (Figure 7). This simple electrorepulsion is known as the main mechanism responsible for penetration enhancement by iontophoresis. The number of charged molecules which are moved across the barrier correlates directly to the applied current and thus can be controlled by the current density. Other factors include the possibility to increase the permeability of the skin barrier in the presence of a flow of electric current and electroosmosis. Contrary to electrorepulsion, electroosmosis can be used to transport uncharged and larger molecules. Electroosmosis results when an electric field is applied to a charged membrane such as the skin and causes a solvent flow across this membrane. This stream of solvent carries along with it dissolved molecules. It enhances the penetration of neutral and especially polar substances.

• Electroporation is based on the application of a voltage to the skin. In contrast to iontophoresis where a low voltage is applied, electroporation requires a large voltage treatment for a short period of 10 µs to 100 ms. Electroporation produces transient hydrophilic pores (aqueous pathways) across the skin barrier. These pores allow the passage of macromolecules via a combination of diffusion, electrophoresis and electroosmosis.

Electroporation

• In the last years, several attempts have been made to enhance the transport of substances across the skin barrier using minimally invasive techniques [10]. The proper function of an appropriate system requires that the thickness of the stratum corneum ( 10 to 20 µm) has to be breached. More recent developments focus on the concept of microneedles. Microneedles are needles that are 10 to 200 µm in height and 10 to 50 µm in width (Figure 9). They are solid or hollow and are connected to a reservoir which contains the active principle

• Penetration enhancement with special formulation approaches is mainly based on the usage of colloidal carriers. Submicron sized particles are intended to transport entrapped active molecules into the skin. Such carriers include liposomes, nanoemulsions, and solid-lipid nanoparticles (Figure 10) [11]. Most reports cite a localizing effect whereby the carriers accumulate in stratum corneum or other upper skin layers. Generally, these colloidal carriers are not expected to penetrate into viable skin. However, the effectiveness of these carriers is still under debate.

• The penetration behavior of an active ingredient can be evaluated in vitro, ex vivo, and in vivo. Most of the data on percutaneous penetration have been gained with in vitro or ex vivo studies by experiments using a . The donor (formulation) is separated from the acceptor (aqueous buffer solution) by an appropriate barrier. For in vitro studies this barrier can consist of an artificial skin construct (ASC). ASC is cultivated from different cell types and comprises a dermis and a epidermis equivalent [15]. The advantage of ASC is that the properties are more consistent than in natural skin. However, the barrier properties of artificial skin are more closely to that of baby skin. This means it is less restrictive than the skin of adults. Franz-Diffusion chamber

• A more advanced in vivo technique is microdialysis. For cutaneous microdialysis a small probe equipped with a semipermeable hollow fiber is inserted superficially in the dermis. The principle of microdialysis is that a physiological solution pumped through the probe is in equilibrium with the diffusible molecules in the surrounding tissue. Therefore the concentration of a solute in the dialysate is proportional to the concentration in the tissue and allows direct monitoring of the in vivo penetration behavior of a active ingredient. With such studies the influence of formulation variables as well as skin condition can be evaluated

MECHANISM OF DRUG DELIVERY THROUGH SKIN

Schematic representation of penetration routes of drugs throughout the skin

More used transdermal nanocarriers

Nanospheres and nanocapsules are small vesicles used to transport drugs. Nanospheres are typically solid polymers with drugs embedded in the polymer matrix. Nanocapsules are a shell with an inner space loaded with the drug of interest. Both systems are useful for controlling the release of a drug and protecting it from the surrounding environment

Liposomes are spherical vesicles that comprise one or more lipid bilayer structures enclosing an aqueous core. They protect encapsulated drugs from degradation. Liposomes can also be functionalized to improve cell targeting and solubility

Dendrimers are highly branched polymers with a controlled three-dimensional structure around a central core. They can accommodate more than 100 terminal groups.

B. Functional evaluation of the skin barrier: transepidermal water loss • • Transepidermal water loss (TEWL) expresses measurements of water diffusion through the skin and it is an important parameter of the skin barrier integrity. Transepidermal water loss (TEWL) shows normal levels according to the area of the body. In the trunk, for instance, there is spontaneous water loss through the corneal layer in the amount of 3 -6 g/h/m 2; in the face, values range from 1 to 15 g/h/m 2. These variations are due to the thickness of the stratum corneum and to the dermal microvasculature. After the stratum corneum has been injured, the loss may reach 70 g/h/m 2. This is a convenient way to measure the extent of the barrier disfunction and constitutes an important instructive element for its evaluation. Even in the recovery phase, the atopic patient shows dryness or roughness of the skin with increased TEWL. This increase occurs both in the affected and normal skin of atopic patients, TEWL tends to normalize in the normal skin of atopic individuals in remission of AD. Measurements of water loss and hydration (capacitance) tend to vary based on the course of the disease, suggesting recovery of the skin barrier or that these alterations are reversible

Sloughing Stratum Corneum Cells improvement index

A low TEWL count means that less water is lost through the skin. On the other hand, if a high TEWL count is measured, it means that skin’s barrier function is weakened and large amounts of water are escaping from skin.

Transepidermal Waterloss (TEWL) - Tewameter® TM 300 The measurement of the transepidermal waterloss (TEWL) is the most important parameter for evaluating the efficiency of the skin water barrier

Sloughing Stratum Corneum Cells improvement index

The stratum corneum (SC), the topmost layer of the skin, is an amazing biological barrier with intriguing biophysical properties

• Atopic dermastitis (AD) patients have a mutation in the filaggrin-encoding gene, and their NMF cannot store enough water. NMF also functions to lower the skin surface p. H. If this function is impaired, the skin p. H level will be raised from mild acidic (normal) to neutral. Under the neutral p. H, proteases such as SCCE get active, whereas inhibitors get inactive, which makes desmosomes fragile. Lipid lamellae component productivity also goes down.

• Schematic representation of the underlying differentiation process in keratinocytes. Keratinocytes differentiate from a proliferating state in the basal layer (Stratum basale) to dead corneocytes in the outermost layer (Stratum corneum). During this differentiation process the lipid envelope, the filaggrin/keratin network, and the cornified envelope is formed and desmosomes mature to corneodesmosomes. Together these components form a compact barrier against the outside preventing entry of harmful components, for example allergens, pathogens, irradiation and other irritants, into the skin and body. Furthermore the barrier inhibits the trans-epidermal water loss (TEWL) and associated loss of solutes. Dotted lines indicate where the sections in the scheme are localized in the normal human skin.

Epidermal Barrier Journal of Investigative Dermatology (2009) 129, 1892 • The structure of the epidermal barrier located in the lower part of the stratum corneum (SC). Highly differentiated flattened keratinocytes, referred to as corneocytes (beige rectangles), are the building blocks of the epidermal barrier. They contain natural moisturizing factor (NMF), derived from pro-filaggrin, a mix of hygroscopic compounds, which help maintain skin hydration. A water resistant layer of lipid lamellae (pink) encases the corneocytes preventing water loss and impeding barrier permeability. The corneocytes are held together by corneodesmosomes (purple spheres), the integrity of which is dependent on a cocktail of proteases and protease inhibitors. The balance between the expression and activity of proteases, such as KLK 7 (SCCE), and protease inhibitors, such as LEKTI and cystatin A, determines the rate of desquamation (corneocytes shedding) and thereby the thickness of the barrier. Under normal conditions, the barrier is only degraded in the upper layers of the SC providing a resilient permeability barrier that prevents the penetration of allergens.

Epidermal Barrier Journal of Investigative Dermatology (2009) 129, 1892 • • A defective epidermal barrier is a poor permeability barrier, which permits the entry of allergens and the loss of moisture. Changes in the FLG gene encoding profilaggrin result in reduced, or absent, expression of filaggrin thereby adversely affecting the structure of the corneocytes (beige)—the "bricks". The levels of natural moisturizing factor (NMF), derived from filaggrin, are also adversely affected, resulting in a decreased ability of the corneocytes to hold water and a concomitant elevation of p. H. Elevated p. H favors serine protease activity and inhibits enzymes involved in the synthesis of lipid lamellae (pink)—the "mortar". Genetic changes in the genes encoding SCCE (KLK 7), LEKTI (SPINK 5), and cystatin A (CSTA) all lead to elevated protease activity involved in desquamation—cleavage of the corneodesmosome junctions (purple spheres) between the corneocytes analogous to "rusting" of the "iron rods

The Outside-to Inside and Back to Outside Pathogenesis of Atopic Dermatitis

Epidermal Barrier Dysfunction in Atopic Dermatitis Journal of Investigative Dermatology (2009) 129, 1892 • • There is a defective epidermal barrier in individuals with atopic dermatitis. The epidermal barrier is found in the lower layers of the stratum corneum, and is composed of differentiated keratinocytes, termed corneocytes (beige rectangles), held together with corneodesmosomes (purple spheres). The hyperactivity of degradatory proteases (red hexagons) found within the epidermis, and contributed to by exogenous proteases (red hexagons), from house dust mites and Staphylococcus aureus, for example, facilitate the cleavage of the corneodesmosome junctions. This is just one event in the breakdown of the epidermal barrier that permits the penetration of allergens. Dendritic cells (DC) (green) found in the dermis take up and present these allergens (red stars) to helper T (TH) cells and recruit CD 4+ T cells (blue). Activated DC and IL-4, expressed by CD 4+ T cells, promote TH 1 to TH 2 switching with the subsequent release of pro-inflammatory cytokines and elevation of Ig. E levels. The clinical outcome of this type of response is atopy and asthma

Epidermal Barrier Journal of Investigative Dermatology (2009) 129, 1892 • Protease inhibitors protect the epidermal barrier from degradation by exogenous proteases. In normal skin (panel a), the protease inhibitor, cystatin A (blue dots), is secreted in sweat and flows out onto the surface of the skin forming a protective layer. Exogenous proteases from, for example, house dust mites (Der P 1) are inhibited by the protective layer of cystatin A and, as a result, cannot break down the corneodesmosomes (purple spheres) that lock the corneocytes (beige rectangles) of the stratum corneum together. In atopic dermatitis (panel b), altered expression of cystatin A leads to an incomplete protective barrier against the activity of exogenous proteases leading to breakdown of the epidermal barrier, and potential allergen, including Der P 1, penetration

• • The brick wall analogy of the stratum corneum of the epidermal barrier In healthy skin the corneodesmosomes (iron rods) are intact throughout the stratum corneum. At the surface, the corneodesmosomes start to break down as part of the normal desquamation process, analogous to iron rods rusting (A ). In an individual genetically predisposed to atopic dermatitis, premature breakdown of the corneodesmosomes leads to enhanced desquamation, analogous to having rusty iron rods all the way down through the brick wall (B ). If the iron rods are already weakened, an environmental agent, such as soap, can corrode them much more easily. The brick wall starts falling apart (C ) and allows the penetration of allergens (D ).

Regulatory effects of the cytokines on the barrier formation. This figure illustrates the relevant effects of cytokines on the formation of the skin barrier and their selected targets in the differentiation process documented either in cell culture or in animal studies. Arrows indicate the consequences on these processes. (↑) indicate that the cytokine enhances or promotes the target process; (↓) indicate that the cytokine down-regulates or inhibits the target process

Epidermal Barrier Journal of Investigative Dermatology (2009) 129, 1892 • Three groups of genes contribute to skin barrier breakdown in atopic dermatitis (AD) coding for structural, protease, and protease inhibitor proteins. Changes in protease (such as KLK 7) and protease inhibitor (CSTA and SPINK 5) genes lead directly to enhanced protease activity within the stratum corneum (SC), resulting in exacerbated breakdown of the corneodesmosome junctions. Loss-of-function mutations in the FLG gene encoding filaggrin, result in decreased levels of natural moisturizing factor (NMF) within the SC. As NMF levels fall, the SC p. H will rise, leading to enhanced protease activity, decreased protease inhibitor activity, and decreased lipid lamellae synthesis. Environmental insults, such as soap and other detergents; exogenous proteases from house dust mite and Staphylococcus aureus; and the prolonged use of topical corticosteroids (TCS) exacerbate proteolytic breakdown of the barrier resulting in increased skin barrier breakdown

Skin barrier function

• • • There is concrete evidence that the stratum corneum is an active metabolic structure that holds adaptive functions, interacting dynamically with the underlying epidermal layers. The skin barrier also plays a role in the inflammatory response through melanocyte activation, angiogenesis, and fibroplasia. The intensity of this response will essentially depend on the severity of the injury. Skin barrier abnormalities in atopic dermatitis are clinically observed by the presence of dry skin, a common and significant symptom which constitutes a diagnostic and monotoring parameter. The stratum corneum hydration level and transepidermal water loss are associated with the level of damage to the barrier, representing biophysical parameter. In the physiopathology of AD, impairment of the skin barrier is associated with a reduction in the levels of ceramide and in the production of profilaggrin, with greater transepidermic water loss (TEWL) and higher predisposition to aggression, which are the trigger for inflammation. The stimulus for an abnormal response in atopic dermatitis is often external, due to alteration of the skin barrier: there is the development of xerosis, with abnormalities in the stratum corneum, and increase of transepidermic water loss, which also cause an abnormal IL-4 metabolism.

• Impact of the diaper environment on the skin barrier structure, function and. The humid environment leads to over hydration of the SC causing disruption of the lipid baitlayer structure. When the SC integrity is damaged, irritants and microorganisms can penetrate and reach the Langerhans cells and epidermis. Fecal enzymes disrupt the SC integrity by degrading proteins, providing another mechanism for barrier breach. Premature infant skin has fewer SC layers and, therefore, increased permeability. Penetrants/irritants interact with keratinocytes stimulating them to release cytokines. Cytokines act on the vasculature of the dermis resulting in inflammation. SC: Stratum corneum.

• Representation of AD pathogenesis. In healthy skin the stratum corneum prevents allergen penetration. In patients with AD, mutations in genes encoding proteins essential for the stratum corneum properties alter the efficacy of the epidermal barrier (I). This facilitates allergen penetration (II). As a result, keratinocytes and immune cells are activated (III) and produce proinflammatory cytokines (IV). These cytokines downregulate keratinocyte proteins, most likely at the transcriptional level (V and VI). Penetration is further enhanced, and inflammation becomes chronic. CASP 14, Caspase 14; IVL, involucrin; KLK 7, kallikrein 7; LOR, loricrin.

Diagram of the Outside-Inside View of Atopic Dermatitis

• Depiction of how surfactants within a cleanser can remove SC material and also remain in the SC. The lengths scales of the cartoon at the left are inaccurate; corneocytes have diameters ~20 μm, while the micelles sizes are ~5 nm. At the right, a molecular-level illustration of ordered SC lipids (ceramides, cholesterol, and fatty acids) and surfactants from a cleanser inserting into these ordered SC lipids.

Verruca Vulgaris Microscopic Detail • (1) Serum in the stratum corneum (2) Hyperkeratosis & parakeratosis (3) Coarse keratohyaline granules & perinuclear vacuolation (4) Papillomatosis

The illustration from visualsunlimited shows the formation of a melanoma in the epidermis, extending down to through the dermis, and up through the outer layer of skin (stratum corneum). Note the black scabby appearance of a melanoma, a typical sign for this type of potentially deadly skin cancer

Does these organisms look like a space alien? A scary creature from a nightmare? In fact, it's a 1 -cm long worm that lives in the human body and causes serious harm. It enters the body through a hair follicle of the skin when it's in a much smaller stage of its life cycle. : (Schistosome)