Transcript Stratum corneum - Abdel Hamid Derm Atlas

Stratum corneum
How does your body keep most enemies out?
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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 60s, 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. 104x
C = stratum corneum
L = stratum lucidum
G = stratum
granulosum
S = stratum spinosum
B = stratum basale
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Thick skin (palmar) H&E. 200x
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
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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, filaggrin, 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, filaggrin, and
amino acids released by filaggrin proteolysis Journal of Investigative Dermatology (2012) 132, 751
Brick & mortar
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
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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 mid1970'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
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Das Backstein-Zement-Modell
1 Hornzellen (Korneozyten) : Horny cell
cornycytes
2 Epidermale Lipide
The functions of horny layer
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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
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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 pH of healthy skin has an average value
lying between 5.4 and 5.9.
In this pH 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 pH 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.
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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)
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Red arrows indicate secreted lamellar
granules, and green arrows indicate lamellar
granules in the cytoplasm
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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, KLK7, KLK8 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 transGolgi 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
Cornified cell
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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
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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
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Model of desquamation: pH controls
KLK activities by regulating their
interaction with LEKTI. In the deep SC,
neutral pH allows a strong interaction
between LEKTI and its KLK targets in
the corneocyte interstices, thus
preventing corneodesmosomes
cleavage. As the pH acidifies along the
SC, LEKTI, and KLK5 dissociate,
allowing proteinase to progressively
degrade its corneodesmosomal
targets. In the most superficial layers
of SC, pH 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 pH play a significant
role in the activity of these
enzymes
Stratum corneum
Sloughing Stratum Corneum Cells
2.260 x magnification of the skin barrier.
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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
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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 and cover the body surface
together with sweat to serve as defense against
exogenous proteases
• Corneocytes store water (H2O) 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
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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 provided it is well hydrated. If its water content
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
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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
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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.
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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
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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
,2OO1
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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?
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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)
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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
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The Landmann model.
Transformation of “lamellar bodydisks” into intercellular sheets by a
membrane-fusion process (c),
according toLandmann (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
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Defense mechanisms in epithelial cells: Nature reviews immunology: 4 , 978 , 2OO4
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 (TH1)- or TH2-cell responses are driven by the nature and site of the initial
injury.
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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 7dehydrocholesterol in keratinocytes initiates the
pathway of vitamin D3 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 UVB-responsive 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 E2 (PGE2), 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 D3 produced by UVBirradiated 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 , 2O13
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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
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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 (TGM3).
Cathepsin L is also able to process cathepsin D
(CTSD), which in turn can activate
transglutaminase-1 (TGM1). 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 corneum lipids as well directly increase
the diffusivity of molecules through the skin barrier. These main effects can be assisted by acoustic microstreaming caused by the
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/m2; in the face, values
range from 1 to 15g/h/m2. 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 70g/h/m2.
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 pH. If this function is impaired, the skin pH level will
be raised from mild acidic (normal) to neutral. Under the neutral pH,
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 KLK7 (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 pH. Elevated pH
favors serine protease activity and inhibits
enzymes involved in the synthesis of lipid
lamellae (pink)—the "mortar". Genetic
changes in the genes encoding SCCE
(KLK7), LEKTI (SPINK5), 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 CD4+ T cells (blue).
Activated DC and IL-4, expressed by CD4+ T cells,
promote TH1 to TH2 switching with the subsequent
release of pro-inflammatory cytokines and elevation
of IgE 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 P1) 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 P1, 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 KLK7) and protease
inhibitor (CSTA and SPINK5) 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 pH 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. 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. CASP14,
Caspase 14; IVL, involucrin; KLK7, 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)