Alain KHAIAT, Ph.D. Vice President R&D Johnson & Johnson Asia Pacific

‡ Inflammation ‡ Pigmentation ‡ Skin Aging


‡ Inflammation
± irritation ± sensitization ± biochemistry

‡ Pigmentation ‡ Skin Aging


EPIDERMIS The cells contained in the epidermis are: ‡ corneocytes ‡ keratinocytes ‡ Langerhans cells 4 .

DEJ It is the site of adhesion of epidermis to dermis. via: ‡ hemidesmosomes ‡ anchoring filaments (Kalinin) ‡ adhesive protein (Laminin) ‡ fibronectin 5 .

DEJ Basal cell Hemidesmosome Anchoring filaments Lamina lucida Lamina densa Anchoring fibril 6 .

DERMIS The dermis contains: ‡ fibrobalsts ‡ mast cells ‡ Langerhans cells ‡ lymphocytes and blood vessels 7 .

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 8 .

INFLAMMATION ‡ Inflammation is the body¶s general distress response to biological. physical or chemical causes of: ± irritation ± sensitization ± photosensitization 9 .

INFLAMMATION ‡ Clinically. inflammation has been defined through 4 signs: ± erythema ± edema ± pain ± heat 10 .

It is externally observable ‡ Subjective irritation is characterized by: stinging.IRRITATION ‡ Irritants are chemical. burning or itching 11 . biological or physical agents which can produce inflammation ‡ Irritation can be either objective or subjective ‡ Objective irritation is characterized by the 4 signs mentioned.

IRRITATION ‡ The result of insulting the skin is the release of histamine by the mast cells in the irritated area. allowing cells (PMN= polymorphonucleocytes) to migrate to the area 12 . ‡ Histamine is a potent vasodilator. it produces the visible erythema and increased vascular permeability (leaking of fluid = edema).

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 13 .

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 ³teaches´ the T cells about the allergen 14 .SKIN SENSITIZATION ‡ The sentitizing substance (hapten).

liberate cytokines ‡ these cytokines attract leukocytes to the site and appear to raise the temperature of the area 15 .SKIN SENSITIZATION ‡ Sensitized T cells migrate to the site and. on contacting the allergen.

Langehans cell Allergen cytokine T cell Activated T cell 16 .

function 17 . or very similar.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.

CYTOKINES ‡ They form part of a complex cellular signaling language ‡ They are proteins 18 .

chemicals. bacteria. protozoa.T CELL RESPONSE ‡ TYPE 1: cell mediated response. essentially to viruses. Th1 response leads to secretion of: ± IL2 ± K IFN ± TNFF ± IL12 19 .

Th2 releases: ± IL4 ± IL5 ± IL6 ± IL10 ± IL13 20 .T CELL RESPONSE ‡ TYPE 2: humoral response following parasitic infection.

T CELL RESPONSE ‡ The type of response is function of genes and the environment. Th2 Th1 Genes Environment 21 .

IL6. mast cells growth. eosinophil infiltration 22 .T CELL RESPONSE ‡ Allergic contact dermatitis is in its early stages Th1 (IL2. then IgE. KIFN) becoming later Th2 (IL4). IL5. IL10. This explains why the reaction decreases ‡ Atopic dermatitis is a Th2: IL4.

UV B EFFECT ‡ UV B has been shown to suppress immune reaction (induction phase only) ‡ UV B stimulates synthesis and release of TNF-E by keratinocytes which in turn modifies the behavior and morphology of Langerhans cells 23 .

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 Tlymphocytes and keratinocytes to initiate host response to antigens 24 .

Irritants increase the biosynthesis of phospholipids ‡ Arachidonic acid is resident to the cell membrane where it is the source of several major biochemical pathways 25 .BIOCHEMISTRY OF INFLAMMATION ‡ Phospholipids are the major raw material and starting point for the arachidonic acid pathway.

PGF2. etc) 26 .Phospholipides Phospholipase A2 NSAID Eugenol Steroids Acetylsalycilic acid 12-Lipoxygenase Arachidonic Acid Thromboxane Cyclooxygenase Prostaglandin G2 5-Lipoxygenase Prostacyclin Prostaglandin H2 Leucotrienes Hydroperoxitetraenoic Acid (HETE) Prostaglandins (PGE2.

ARACHIDONIC PATHWAY ‡ If arachidonic acid is acted upon by cyclooxygenase. ‡ Thromboxane stimulates platelet aggregation and is a vasoconstrictor 27 . It is itself converted into thromboxane or prostacyclin or PGH2. the later then generating the other members of the PG family. prostaglandin G2 is generated.

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) 28 .

after application of Phorbol ester ‡ Ear edema in mouse following AA inflammation 29 .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).

CONTENTS ‡ Inflammation ‡ Pigmentation ± anomalies ± melanogenesis ‡ Skin Aging 30 .

i. quantity. type and distribution of melanosomes (epidermis or dermis) ‡ skin vascularisation 31 .PIGMENTATION Skin color is the result of: ‡ nature of the melanin ‡ where the melanin is concentrated.e.

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 32 .

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) 33 .

appear anywhere on the body ‡ Solar Lentigo: wider lesion than freckle. Melanocytes do not proliferate correctly ‡ Lentigines: can be hereditary. stimulated by solar exposure 34 . occurs after serious sunburn ‡ Senile Lentigo: generally on the back of the hand of older subjects.PIGMENTATION ANOMALIES 2.

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 35 .PIGMENTATION ANOMALIES ‡ Dubreuilh melanosis or malignant lentigo of the elderly: large pigmented multi colored stain.

PIGMENTATION ‡ All methods to reduce pigmentation on the market today have the objective to reduce melanogenesis 36 .


tannins. etc) 38 .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.

etc. promote the formation of glutathion DOPA leading to pheomelanin 39 .MELANOGENESIS INHIBITION ‡ Inhibition of tyrosinase: Kojic acid. EDTA or Phytic acid (since tyrosinase requires Cu++) ‡ Inhibition of the formation of eumelanin: by adding glutathion and glutathion reductase transforming GSSG into GSH. ascorbic acid.

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 40 .PIGMENTATION ‡ Melanin is formed in the Melanocytes.

Pigmentation Formation Mechanism 1 2 Irritation UV ‡ Variety of Causes ‡ Variety of Responses Inflammatory Response 3 Hormone KERATINOCYTE (Epidermis) Tyrosine Melanin MELANOCYTE (Basal Layer) Melanosome Tyrosinase FIBROBLAST Dermis 41 .

Basic Structure of Skin Stratum Corneum Keratinocyte Viable Epidermis Melanocyte Basal Layer Dermis 42 .

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 43 .

lighter color 1. Less TRP-1 is made tyrosinase not stable 2. More TRP-2 is made shift to brownish melanins Less melanosome transfer Melanosomes accumulate Negative feed-back 44 .SUGGESTED MECHANISM Depigmentation Less eumelanin produced.

PAR-2 is activated by trypsin ‡ By inhibiting PAR-2.NOVEL MECHANISM ‡ Protease Activated Receptor (PAR-2) is expressed in keratinocytes. one probably blocks the keratinocyte-melanocyte interaction ‡ TRP1 (tyrosinase-related protein) decreases leading to less Eumelanin 45 .

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 46 .PIGMENTATION TESTING ‡ Tyrosinase activity in solution: mushroom.

so far. are not very significant against placebo 47 . changes.PIGMENTATION TESTING ‡ Human volunteers tests: ± Chromameter® : L measure ± Mexameter® : evaluation of melanin and redness ± Photography : visible. UV with data analysis 3 months minimum.

CONTENTS ‡ Inflammation ‡ Pigmentation ‡ Skin Aging ± skin changes ± biochemical changes 48 .

‡ 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

‡ Sebaceous glands :
± reduction in sebum secretion (hormones influenced)

‡ Sweat glands :
± less active

‡ HLP film :
± thinning of film means less protective barrier

‡ Dermis :
± destruction of collagen and elastin fibers network ± proteoglycans and glycoproteins are reduced ± increase in elastin synthesis : elastosis


PHOTOAGING 3 types of reactions to UV exposure: ‡ Free Radicals. essentially due to UVB ‡ MMP Enzymes 52 . essentially due to UVA ‡ Direct cell death.

GAG (skin firmness) ‡ lipids (membrane or structural) 53 . cell death) ‡ collagen.FREE RADICALS Free radicals or ROS (reactive oxygen species) can lead to breakage of important molecules: ‡ DNA (mutations. elastin. renewal failure.

UV DAMAGE AND OXIDATIVE STRESS DNA effects DNA fragmentation UV damage Matrix effects MMP : TIMP ratio Membrane effects: ROS Lipids peroxidation Hydroperoxides Enzymatic systems SOD Glutathion peroxidase Heme oxidase 54 .

DNA DAMAGE ‡ UVA acts through oxidative stress forming ³reactive oxygen species´ (ROS) that will damage the DNA and lead to cancer 55 .

DNA DAMAGE ‡ UVB impact on DNA in the cell creating damages which may lead to cancer: nonmelanoma skin cancer (NMSC) 56 .

57 .

there is an altered expression of oncogenes and tumor suppression genes. such as p53 ‡ NMSC show a high incidence of mutation in p53 gene 58 .UVB DAMAGE ‡ Following structural changes in DNA.

59 .

g.p 53 GENE Plays an important role in: ± blocking the cell cycle after exposure to DNAdamaging agents e. in order to allow for repair before duplication ± or killing the cell to avoid multiplication of damaged cells (formation of sunburn cells) 60 . UV.

p 53 GENE The induction of detectable levels of p53 in human epidermis after UV exposure is relevant to skin carcinogenesis 61 .

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 62 .

Collagen Degradation A balance between MMP:TIMP MMP TIMP 63 .


collagen IV. etc.MMP ENZYMES ‡ Collagenases (1 to 4) are specific to various collagen. laminin. ‡ Gelatinases (A & B) are non specific ‡ Stromelysins (1-3) specific of fibronectin. ‡ Elastase: elastin ‡ etc 65 .

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 66 .

ACTIVE PHOTOPROTECTION Inhibition of oxidative stress Replenish antiox system ACTIVE PHOTOPROTECTION Reduce matrix degradation Quench ROS 67 .

Irradiation of Epidermal Equivalents with Solar Spectrum UV Solar Simulator ¡ 10-4 10-5 10-6 10-7 10-8 60 80 300 3 0 340 360 380 400 4 0 Wa l t ( m) MM & TIMP-1 68         W/cm 10-9 10-10 10-11 .

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 69 .

UV Irradiation of Epidermal Equivalents ‡ Model for assessing Photoprotective potential ± Botanical ingredients ± Fully formulated product 70 .


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