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SKIN BIOLOGY

Alain KHAIAT, Ph.D.

Vice President R&D

Johnson & Johnson Asia Pacific

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CONTENTS

• Inflammation

• Pigmentation

• Skin Aging

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CONTENTS• Inflammation

– irritation– sensitization– biochemistry

• Pigmentation

• Skin Aging

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EPIDERMIS

The cells contained in the epidermis are:

• corneocytes

• keratinocytes

• Langerhans cells

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DEJ

It is the site of adhesion of epidermis to dermis, via:

• hemidesmosomes

• anchoring filaments (Kalinin)

• adhesive protein (Laminin)

• fibronectin

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DEJ

Hemidesmosome

Basal cell

Lamina lucidaAnchoring

filaments

Anchoring fibril

Lamina densa

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DERMIS

The dermis contains:

• fibrobalsts

• mast cells

• Langerhans cells

• lymphocytes and blood vessels

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The skin is the interface between the organism and its environment

Because it contains:– Langerhans cell– lymphocytes– blood vessels– masts cells

exogenous or endogenous stimuli will create inflammation processes

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INFLAMMATION

• Inflammation is the body’s general distress response to biological, physical or chemical causes of:– irritation– sensitization– photosensitization

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INFLAMMATION

• Clinically, inflammation has been defined through 4 signs:– erythema– edema– pain– heat

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IRRITATION

• Irritants are chemical, biological or physical agents which can produce inflammation

• Irritation can be either objective or subjective

• Objective irritation is characterized by the 4 signs mentioned. It is externally observable

• Subjective irritation is characterized by: stinging, burning or itching

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IRRITATION

• The result of insulting the skin is the release of histamine by the mast cells in the irritated area.

• Histamine is a potent vasodilator, it produces the visible erythema and increased vascular permeability (leaking of fluid = edema), allowing cells (PMN= polymorphonucleocytes) to migrate to the area

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SENSITIZATION

• Skin sensitization is the result of exposure to sensitizers or allergens

• Skin sensitization is a delayed type humoral immune response mediated by the T cell

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SKIN SENSITIZATION

• The sentitizing substance (hapten), combines with a protein in the skin to form the allergen

• The Langerhans cells in the stratum germinativum interacts with the allergen and migrates to the lymphoid gland

• It then “teaches” the T cells about the allergen

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SKIN SENSITIZATION

• Sensitized T cells migrate to the site and, on contacting the allergen, liberate cytokines

• these cytokines attract leukocytes to the site and appear to raise the temperature of the area

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AllergenLangehans cell

T cell Activated T cell

cytokine

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CYTOKINES

• Cytokines are essential transmitters of intercellular communication

• They have an inherent role in the regulation of responses of the immune system

• Each cytokine has multiple functions

• More than one cytokine may mediate the same, or very similar, function

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CYTOKINES

• They form part of a complex cellular signaling language

• They are proteins

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T CELL RESPONSE

• TYPE 1: cell mediated response, essentially to viruses, bacteria, protozoa, chemicals. Th1 response leads to secretion of:– IL2 IFN– TNF– IL12

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T CELL RESPONSE

• TYPE 2: humoral response following parasitic infection. Th2 releases:– IL4– IL5– IL6– IL10– IL13

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T CELL RESPONSE

• The type of response is function of genes and the environment.

GenesEnvironment

Th1 Th2

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T CELL RESPONSE

• Allergic contact dermatitis is in its early stages Th1 (IL2, IFN) becoming later Th2 (IL4). This explains why the reaction decreases

• Atopic dermatitis is a Th2: IL4, IL5, IL6, IL10, then IgE, mast cells growth, eosinophil infiltration

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UV B EFFECT

• UV B has been shown to suppress immune reaction (induction phase only)

• UV B stimulates synthesis and release of TNF- by keratinocytes which in turn modifies the behavior and morphology of Langerhans cells

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TWO MECHANISMS• Mast cells can respond directly to external trauma,

to antigen-IgE complexes on their surface or to mediators generated from complement (anaphylatoxins) by degranulating and releasing vaso active mediators: histamins

• Langerhans cells interact specifically with T-lymphocytes and keratinocytes to initiate host response to antigens

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BIOCHEMISTRY OF INFLAMMATION

• Phospholipids are the major raw material and starting point for the arachidonic acid pathway. Irritants increase the biosynthesis of phospholipids

• Arachidonic acid is resident to the cell membrane where it is the source of several major biochemical pathways

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Phospholipides

Arachidonic Acid

Prostaglandin G2

Prostaglandins (PGE2, PGF2, etc)

Leucotrienes

HydroperoxitetraenoicAcid (HETE)

Prostacyclin

Thromboxane

Prostaglandin H2

Cyclooxygenase

Phospholipase A2

12-Lipoxygenase

5-Lipoxygenase

SteroidsNSAIDEugenol

Acetylsalycilic acid

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ARACHIDONIC PATHWAY

• If arachidonic acid is acted upon by cyclo-oxygenase, prostaglandin G2 is generated. It is itself converted into thromboxane or prostacyclin or PGH2, the later then generating the other members of the PG family.

• Thromboxane stimulates platelet aggregation and is a vasoconstrictor

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ARACHIDONIC ACID PATHWAY

• Prostacyclin inhibits platelet aggregation and vasoconstriction

• Prostaglandins are non protein chemical mediators: they are fatty acids

• 12-lipoxygenase transforms AA into HETE

• 5-lipoxygenase catalyses the production of the leukotriens (eicosanoid family)

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ANTI INFLAMMATORY TESTS

• Cytokines secretion by PBL (human peripheral blood lymphocytes) in culture following addition of a stimulant

• IL 6 release by human fibroblasts

• Contact hypersensitivity in mouse (ear edema), after application of Phorbol ester

• Ear edema in mouse following AA inflammation

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CONTENTS

• Inflammation

• Pigmentation– anomalies– melanogenesis

• Skin Aging

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PIGMENTATION

Skin color is the result of:

• nature of the melanin

• where the melanin is concentrated, i.e. quantity, type and distribution of melanosomes (epidermis or dermis)

• skin vascularisation

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PIGMENTATION ANOMALIES

1. Melanocytes proliferation is normal:

• Freckles: eumelanin zones on pheomelanin backgrounds (skin areas exposed to the sun)

• Chloasma: pregnancy mask: hypersecretion of melanin induced by hormonal factors and amplified by the sun

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PIGMENTATION ANOMALIES

• Diffuse brown melanosis: endocrine system disorders or nutritional anomalies

• Hypermelanosis can follow cutaneous inflammations: – pigmentation of scars, – caused by irritants combined with sun

(photosensitizers like bergamot oil)

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PIGMENTATION ANOMALIES

2. Melanocytes do not proliferate correctly

• Lentigines: can be hereditary, appear anywhere on the body

• Solar Lentigo: wider lesion than freckle, occurs after serious sunburn

• Senile Lentigo: generally on the back of the hand of older subjects, stimulated by solar exposure

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PIGMENTATION ANOMALIES

• Dubreuilh melanosis or malignant lentigo of the elderly: large pigmented multi colored stain, pre-cancerous

• Moles or Naevus: accumulation of melanocytes in epidermis and dermis

• Malignant melanomas: cancerous tumors. The first signs are degeneration of existing naevus or Dubreuilh melanosis

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PIGMENTATION

• All methods to reduce pigmentation on the market today have the objective to reduce melanogenesis

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MELANOGENESIS PATHWAYS

L-TYROSINE

TYROSINASE

3,4-DIHYDROXYPHENYLALANINE

TYROSINASE

DOPA QUINONE

GSH CYCLISATION

GSH-DOPA LEUCODOPACHROME

3-S-CYSTEINYL DOPA DOPACHROME

INTERMEDIATE PDTS 5,6 DIHYDROXYINDOLE

TRP2 TRP1

PHEOMELANIN QUINONE-IMINE

EUMELANIN

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MELANOGENESIS INHIBITION

• Inhibition of the production of active tyrosinase in the ribosomes: placental extract

• Inhibition of the transfer of tyrosinase to pre-melanosomes by interrupting glycosylation (tunicamycine, glucosamine)

• Elimination of inflammatory reactions (flavonoids, tannins, etc)

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MELANOGENESIS INHIBITION

• Inhibition of tyrosinase: Kojic acid, ascorbic acid, etc. EDTA or Phytic acid (since tyrosinase requires Cu++)

• Inhibition of the formation of eumelanin: by adding glutathion and glutathion reductase transforming GSSG into GSH, promote the formation of glutathion DOPA leading to pheomelanin

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PIGMENTATION

• Melanin is formed in the Melanocytes, where it is stored in the melanosomes

• Melanocytes extend arms to transfer melanosomes into the keratinocytes

• It is the keratinocytes charged with the melanosomes that constitute the dark spots on the skin

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Tyrosinase

Pigmentation Formation Mechanism

KERATINOCYTE(Epidermis)

MELANOCYTE(Basal Layer)

Tyrosine Melanin

Dermis

UVUVIrritation

Inflammatory Response

Hormone

3

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Melanosome

FIBROBLAST

• Variety of Causes

• Variety of Responses

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Basic Structure of Skin

Stratum Corneum

Viable Epidermis

Basal Layer

Dermis

Keratinocyte

Melanocyte

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PIGMENTATION

• A novel approach has recently been published: blocking the transfer of melanosomes from the melanocyte to the keratinocytes

• Accumulation of charged melanosomes inhibits melanin synthesis

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SUGGESTED MECHANISM

1. Less TRP-1 is made tyrosinase not stable2. More TRP-2 is made shift to brownish melanins

Less eumelanin produced, lighter color

Less melanosometransfer

Melanosomes accumulate

Negativefeed-back

Depigmentation

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NOVEL MECHANISM

• Protease Activated Receptor (PAR-2) is expressed in keratinocytes. PAR-2 is activated by trypsin

• By inhibiting PAR-2, one probably blocks the keratinocyte-melanocyte interaction

• TRP1 (tyrosinase-related protein) decreases leading to less Eumelanin

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PIGMENTATION TESTING

• Tyrosinase activity in solution: mushroom, mouse or human tyrosinase are used with different results

• S91 melanoma cells in culture

• Keratinocytes-Melanocytes co culture

• Guinea pig ear: 15 days treatment

• Microswine spotted model: 6-8 weeks

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PIGMENTATION TESTING

• Human volunteers tests:– Chromameter® : L measure– Mexameter® : evaluation of melanin and

redness– Photography : visible, UV with data analysis

3 months minimum,

changes, so far, are not very significant against placebo

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CONTENTS• Inflammation

• Pigmentation

• Skin Aging– skin changes– biochemical changes

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MANIFESTATIONS OF SKIN AGING

• Epidermis :– reduction in cell renewal rate– thickening of stratum corneum

– decrease in barrier efficiency : increase in TEWL and hyperkeratosis

– ridges are flattened out and intercellular spaces enlarged

– pigmentation problems : actinic lentigines– decrease in skin immune system

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MANIFESTATIONS OF SKIN AGING

• Sebaceous glands :– reduction in sebum secretion (hormones

influenced)

• Sweat glands :– less active

• HLP film :– thinning of film means less protective barrier

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MANIFESTATION OF SKIN AGING

• Dermis :– destruction of collagen and elastin fibers

network– proteoglycans and glycoproteins are reduced– increase in elastin synthesis : elastosis

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PHOTOAGING

3 types of reactions to UV exposure:

• Free Radicals, essentially due to UVA

• Direct cell death, essentially due to UVB

• MMP Enzymes

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FREE RADICALS

Free radicals or ROS (reactive oxygen species) can lead to breakage of important molecules:

• DNA (mutations, renewal failure, cell death)

• collagen, elastin, GAG (skin firmness)

• lipids (membrane or structural)

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UV DAMAGE AND OXIDATIVE STRESS

DNA effectsDNA fragmentation

Matrix effectsMMP : TIMP ratio

Membrane effects:ROSLipids peroxidationHydroperoxides

Enzymatic systemsSODGlutathion peroxidaseHeme oxidase

UV damage

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DNA DAMAGE

• UVA acts through oxidative stress forming “reactive oxygen species” (ROS) that will damage the DNA and lead to cancer

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DNA DAMAGE

• UVB impact on DNA in the cell creating damages which may lead to cancer: non-melanoma skin cancer (NMSC)

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UVB DAMAGE

• Following structural changes in DNA, there is an altered expression of oncogenes and tumor suppression genes, such as p53

• NMSC show a high incidence of mutation in p53 gene

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p 53 GENE

Plays an important role in:– blocking the cell cycle after exposure to DNA-

damaging agents e.g. UV, in order to allow for repair before duplication

– or killing the cell to avoid multiplication of damaged cells (formation of sunburn cells)

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p 53 GENE

The induction of detectable levels of p53 in human epidermis after UV exposure is relevant to skin carcinogenesis

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Collagen & Photodamage

• Major structural component of ECM– 70% of the dry weight of skin

• Collagen degradation is believed to play a role in formation of wrinkles

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Collagen DegradationA balance between MMP:TIMP

MMP TIMP

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MMP ENZYMES

ROS MMP

COLLAGEN DEGRADATION

TIMP

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MMP ENZYMES

• Collagenases (1 to 4) are specific to various collagen,

• Gelatinases (A & B) are non specific

• Stromelysins (1-3) specific of fibronectin, laminin, collagen IV, etc.

• Elastase: elastin

• etc

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MEMBRANE EFFECTS

With age, reduction in membrane fluidity leading to less efficient exchanges:– intrinsic: reduction in the methylation of PE

into PC– extrinsic: lipid peroxides

Methyl donors will restore membrane fluidity

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ACTIVE PHOTOPROTECTION

ACTIVEPHOTOPROTECTION

Reduce matrixdegradation

Replenish antiox system

Inhibition of oxidative stress

Quench ROS

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Irradiation of Epidermal Equivalents with Solar Spectrum UV

MM & TIMP-1

Solar Simulator

Wavelength (nm)260 280 300 320 340 360 380 400 420

W/c

m2

10-11

10-10

10-9

10-8

10-7

10-6

10-5

10-4

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UV Irradiation of Epidermal Equivalents

• Markers of damage– MMP-1 induction– TIMP-1 induction, but to a lesser extent than

MMP-1– MMP:TIMP imbalance

• Protection provided by– Sunscreens– Anti-oxidants

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UV Irradiation of Epidermal Equivalents

• Model for assessing Photoprotective potential– Botanical ingredients– Fully formulated product

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THANK YOU