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

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Presentation on theme: "1 SKIN BIOLOGY Alain KHAIAT, Ph.D. Vice President R&D Johnson & Johnson Asia Pacific."— Presentation transcript:

1 1 SKIN BIOLOGY Alain KHAIAT, Ph.D. Vice President R&D Johnson & Johnson Asia Pacific

2 2 CONTENTS Inflammation Pigmentation Skin Aging

3 3 CONTENTS Inflammation –irritation –sensitization –biochemistry Pigmentation Skin Aging

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

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

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

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

8 8 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

9 9 INFLAMMATION Inflammation is the body’s general distress response to biological, physical or chemical causes of: –irritation –sensitization –photosensitization

10 10 INFLAMMATION Clinically, inflammation has been defined through 4 signs: –erythema –edema –pain –heat

11 11 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

12 12 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

13 13 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

14 14 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

15 15 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

16 16 Allergen Langehans cell T cell Activated T cell cytokine

17 17 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

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

19 19 T CELL RESPONSE TYPE 1: cell mediated response, essentially to viruses, bacteria, protozoa, chemicals. Th1 response leads to secretion of: –IL2 –  IFN –TNF  –IL12

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

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

22 22 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

23 23 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

24 24 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

25 25 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

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

27 27 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

28 28 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)

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

30 30 CONTENTS Inflammation Pigmentation –anomalies –melanogenesis Skin Aging

31 31 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

32 32 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

33 33 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)

34 34 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

35 35 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

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

37 37 MELANOGENESIS PATHWAYS L-TYROSINE TYROSINASE 3,4-DIHYDROXYPHENYLALANINE TYROSINASE DOPA QUINONE GSHCYCLISATION GSH-DOPALEUCODOPACHROME 3-S-CYSTEINYL DOPA DOPACHROME INTERMEDIATE PDTS5,6 DIHYDROXYINDOLE TRP2 TRP1 PHEOMELANINQUINONE-IMINE EUMELANIN

38 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, tannins, etc)

39 39 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

40 40 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

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

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

43 43 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

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

45 45 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

46 46 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

47 47 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

48 48 CONTENTS Inflammation Pigmentation Skin Aging –skin changes –biochemical changes

49 49 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

50 50 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

51 51 MANIFESTATION OF SKIN AGING Dermis : –destruction of collagen and elastin fibers network –proteoglycans and glycoproteins are reduced –increase in elastin synthesis : elastosis

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

53 53 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)

54 54 UV DAMAGE AND OXIDATIVE STRESS UV damage

55 55 DNA DAMAGE UVA acts through oxidative stress forming “reactive oxygen species” (ROS) that will damage the DNA and lead to cancer

56 56 DNA DAMAGE UVB impact on DNA in the cell creating damages which may lead to cancer: non- melanoma skin cancer (NMSC)

57 57

58 58 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

59 59

60 60 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)

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

62 62 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

63 63 Collagen Degradation A balance between MMP:TIMP MMPTIMP

64 64 MMP ENZYMES ROS MMP COLLAGEN DEGRADATION TIMP

65 65 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

66 66 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

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

68 68 Irradiation of Epidermal Equivalents with Solar Spectrum UV MM & TIMP-1 Solar Simulator

69 69 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

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

71 71 THANK YOU


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