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Education system
No pharmacy students,
Pharmacology, Analytical
Chemistry, Biopharmacy,
Toxicology, Drug Delivery
Technology, etc
Joke A. Bouwstra
DivisionofDrugDeliveryTechnology
Leiden/AmsterdamCenterforDrugResearch(LACDR)
Proteins, vaccination
Skin Research
Science oriented:
Master Degree in BioPharmaceutical Sciences
Delivery of
drugs
Skin research
Biologics formulation
Lipid organization
Skin models
Delivery methods
Diseased skin
Transport mechanisms
Vaccine delivery
Protein stability
Structural characterization
Vaccine delivery
Safety (immunogenicity)
Cell culture models
Overviewofpresentation
Themanyfunctionsoftheskin
Step 2
Step 1
Skin
function
Skin morphology
Step 4
Heterogeneity
of skin
Conventional
-establishes, controls & transmit contacts with
external world
-Protects from the environment
-Maintain temperature, electrolyte and fluid
balance
Step 3
Skin renewal
New
-It is an integral component of immune
and nervous system
Conventionalfunctionoftheskin
Conventionalfunctionoftheskin
Barrier function
Protects body from dessication: water and nutrients
are kept in (physical barrier function)
Barrier function
Protects body from dessication: water and nutrients
are kept in (physical barrier function)
virus/bacteria
Temperature control
Sweat glands& ateria
melanocytes
Newfunction:immunologicalresponses
Overviewofpresentation
Step 1
Keratinocytes:
release cytokines:
TNF-a, IL-1b:
stratum
corneum
Skin
function
viable
epidermis
Skin morphology
Migration of LCs
Int DC
dermis
LC
IL-12
Lymph node
Int DC
Ueno et al, Immun. Reviews (2007) 219, 118-142
Skinmorphology:overview
hairfollicles
Epidermis
(150 m)
Dermis
(2000 m)
SkinMorphology:somenumbers
Sebaceous glands
Stratum corneum
(15 m)
Systemic
circulation
Nerves (Sensory
Sweat glands
Subcutaneous
fat tissue
Corneocytes
embedded
in lipid domains
SkinMorphology:routesofpenetrationafterapplying
aproduct
E p id e rm a l
ro u te
In te rce llu la r
SkinMorphology:skinsurface
Important to know: skin has not a flat surface. Large undulations
are present. Rinkels go as deep as the dermis
H a ir
T ra n s ce llu la r
A ppendageal
S tra tu m
ro u te
C o rn e u m
S tra tu m
C o rn e u m
SEM image
E p id e rm is
D e rm is
S e b a ce o u s
g la n d
B lo o d ve s se l
H a ir
fo llic le
Sw eat
gla n d
Desquamation:
Shedding of the cells
Majorcelltypesintheskin
Epidermalrenewing:continuousturnover
Desquamation:
Shedding of cells
Epidermis
Keratinocytes
Langerhans cells
Merkle cells
melanocytes
Keratinocytes
Dermis
Fibroblasts
Dendritic cells
in collagen
matrix
Stratum granulosum
Differentiation
starts
Subcutaneous
fat tissue
Fat cells
Stratum corneum
(15 mm)
Stratum corneum
(15 m)
Corneocytes
embedded
in lipid domains
Stem cells:
Continuous cell
Division: proliferation
Stratum spinosum
Stratum basale
Acloserlookatstratumcorneum
C
C
Penetration
pathway
corneocyte
Stratum granulosum
corneocyte
Epidermis
Stratum spinosum
Stratum basale
Dermis
Blood vessels
HEstaininghumanskin
Stratum granulosum
Stratum spinosum
Stratum basale
Dermis
Lipid
organisation
Penetration
pathway
Epidermis
Blood vessels
Crosssection:cryoscanningelectronmicroscopy
Hydrated stratum corneum:
Individual cells with keratin
filaments
stratum corneum
Stratum corneum
viable epidermis
Viable epidermis
dermis
Upper part of dermis
Finalproductofdifferentiation:stratumcorneum
Desmosomelayeredstructures
15 um
desmosomes
Cornified envelope: thick protein layer:
loricrin, involucrin
(Ca2+ dependent enzymes)
Appendages:Hairfollicle
Importantforskinbarrierfunction
gap
epidermis
outer root sheet
Lipid
monolayer
bound to
envelope
Lamellae: lipids
form crystalline
phases
sebaceous gland
inner root sheet:
Henles layer
Huxleys layer
cuticle
Hair shaft:
cuticle
cortex
medulla
growth cycle
bulge
hair bulb
Cornified envelope
Loricrin, involucrin,etc
anagen (growth)
Overviewofpresentation
Step 1
Skin
function
Step 2
Skin morphology
Step 3
Skin renewal
Renewalofskin
Proteinsynthesis
Enzymes affected by
water and pH
A change in
cell morphology
and cell function
There is a constant
renewal of
the epidermis
Stratum corneum
Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division
Differentiation
Differentiation
Desquamation process
Profilaggrin
filaggrin
Desquamation process
Stratum corneum
Keratohyalin
Granulae:
Stratum granulosum
Cytokeratines
Stratum spinosum
Desmosomes
Stratum basale:
Cell division
Differentiation
Natural Moistursing
Factor (NMF)
Profilaggrin
filaggrin
Cytokeratines
Stratum spinosum
Differentiation
Natural Moistursing
Factor (NMF)
Profilaggrin
filaggrin
Keratohyalin
Granulae:
Involucrin, loricrin,
keratin1,10,profilaggrin
Cytokeratines
keratin 1 and 10
Desmosomes
Natural Moistursing
Factor (NMF)
Stratum corneum
Stratum granulosum
Proteinsynthesis
Desquamation process
Keratohyalin
Granulae:
Desmosomes
Keratin 5, 14
Proteinsynthesis
Differentiation
Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division
Stratum spinosum
Natural Moistursing
Factor (NMF)
Stratum corneum
Stratum corneum
keratines
Desquamation process
Desquamation process
Stratum granulosum
Proteinsynthesis
Enzymes affected by
water and pH
Keratohyalin
Granulae:
Desmosomes
Stratum basale:
Cell division
Profilaggrin
filaggrin
keratin 1 and 10
Differentiation
Proteinsynthesis
Profilaggrin
filaggrin
Keratohyalin
Granulae:
Involucrin, loricrin,
keratin1,10,profilaggrin
Cytokeratines
keratin 1 and 10
Desmosomes
Stratum basale:
Cell division
Desquamation process
Stratum corneum
Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division
Proteinsynthesis
Natural Moistursing
Factor (NMF)
Desquamation process
Stratum corneum
Profilaggrin
filaggrin
Keratohyalin
Granulae
Involucrin, loricrin,
keratin1,10,profilaggrin
Cytokeratines
Stratum granulosum
Stratum spinosum
keratin 1 and 10
Desmosomes
Stratum basale:
Cell division
Stratumcorneummorphology
Lipid
monolayer
bound to
envelope
Differentiation
Differentiation
Natural Moistursing
Factor (NMF)
Proteinsynthesis
Enzymes affected by
water and pH
Profilaggrin
filaggrin
Keratohyalin
Granulae:
Involucrin, loricrin,
keratin1,10,profilaggrin
Cytokeratines
keratin 1 and 10
Desmosomes
Cornifiedenvelope
lamellae
Desquamation process
Stratum corneum
Stratum granulosum
Stratum spinosum
Stratum basale:
Cell division
Stratumcorneummorphology
Lipidcompositioninstratumcorneum
Ceramides (CER)
12 classes of CER
Cholesterol (CHOL)
Lipid
monolayer
bound to
envelope
lamellae
Free fatty acids (FFA)
Cholesterol sulfate
NMF,water, keratin filaments
Calcium
envelope
Minor
amounts
Ceramidesinstratumcorneum
Saturated C22-24
chain length
Synthesisoflipidsinepidermis
EOS/CER1
HN
OH
OH
O
EOP/CER9
HN
OH
OH
OH
O
O
O
EOH/CER4
HN
OH
OH
OH
EOdS
HN
OH
OH
OH
NS/CER2
HN
OH
OH
AS/CER5
NP/CER3
OH
OH
AP/CER6
NH/CER8
OH
OH
AH/CER7
OH
HN
OH
OH
HN
OH
OH
OH
OH
O
HN
OH
OH
OH
OH
ND
OH
OH
Stratum granulosum
OH
HN
HN
Stratum corneum
OH
O
HN
Desquamation process
acylceramides
Differentiation
AD
HN
OH
OH
Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin
Stratum spinosum
Stratum basale:
Cell division
Synthesisoflipidsintheepidermis
Synthesisoflipidsintheepidermis
Stratum corneum
Lamellar bodies:
Storage glucosylceramides
and sphingomyelin,
phospholipids
Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin
Stratum granulosum
Desquamation process
Differentiation
Differentiation
Desquamation process
Glucosylceramides
Ceramides
sphingomyelinCeramides
phospholipidsFatty acids
Stratum corneum
Lamellar bodies:
Storage glucosylceramides
and sphingomyelin,
phospholipids
Stratum granulosum
Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin
Stratum spinosum
Stratum spinosum
Stratum basale:
Cell division
Formationofceramides:moredetails
Stratum basale:
Cell division
Synthesisoflipidsintheepidermis
O
1. Glucosylceramides
HN
O-glucose
OH
Desquamation process
Ceramides
O
HN
Deficiency:Gaucher disease
Holleran et al, J. Clin Invest. 81;1338 (1991)
OH
OH
2. Sphingomyeline
HN
O-P-choline
OH
O
HN
Glucosylceramides
Ceramides
sphingomyelinCeramides
phospholipidsFatty acids
Stratum corneum
Lamellar bodies:
Storage glucosylceramides
and sphingomyelin,
phospholipids
Stratum granulosum
Synthesis of Ceramides:
Glucosyl ceramides and
sphingomyelin
sphingomyelinase
Ceramide
Differentiation
glucoceribrosidase
OH
OH
Stratum spinosum
Stratum basale:
Cell division
Formationofphospholipids
Lipidsessentialforbarrierfunction
Step 2
Stratum basale
Stratum granulosum
Stratum corneum
Step 3
Step 1
Step 4
Phospholipids
Sphingolipids
Free sterols
Lampe et al, J. lipid Res. 24:131-140 (1983)
Lipidcomposition:viableepidermisversuslamellar
bodies
Conclusion
Increase in fatty acids, cholesterol and sphingolipids
(ceramides+glucosylceramides) in stratum corneum
compared to viable epidermis
viable epidermis
lamellar bodies
Question
Lipid content lamellar bodies versus whole viable epidermis?
Fatty acids
Cholesterol
Ceramides
Glucosylceramides
Phospholipids
Formationoflipidlamellae
Fusionoflamellarbodies:formationoflamellae
Differentiation
Desquamation process
corneocyte
Stratum corneum
Ceramides
cholesterol
fatty acids
Stratum granulosum
Stratum spinosum
High enzymatic
activity
glucosylceramides
cholesterol
Phospholipids
Stratum basale:
Cell division
Lamellarbodyextrusioprocessinreality
fusion
Lamellar
body
Stratumcorneumboundlipids
LBE=
lamellar body
extrusion
SG= Stratum
Granulosum
Lipid
monolayer
bound to
envelope
LB=Lamellar
Body
lamellae
Fusionoflamellae:boundlipidsimportanttoinitiate
thisprocess
w-hydroxy acylceramide
Bound lipids
O
=
= =
O
HN
cornified envelope
OH
Ceramide EOS
OH
corneocyte
= =
Ceramides
cholesterol
fatty acids
O
HN
O-glucosyl
OH
hydrolysis
glucose+acyl group
hydrolysis of glucose
group
HN
High enzymatic
activity
O
=
= =
HO
fusion
O
HN
OH
OH
OH
OH
Lamellar
body
Phospholipids
glucosylceramides
cholesterol
Notonlykeratinocytesinepidermis
Stratum corneum
Stratum granulosum
Stratum spinosum
Stratum basale
Langerhans cells
melanocytes
Dermis
Stratum Corneum
Stratum Granulosum
Stratum Spinosum
Stratum Basale
Epidermis
Corneocyte:
soft keratin and
water
Penetration
pathway
FFA
CER
CHOL
Lipid regions
Blood vessels
Langerhans cells: dendritic cells,
Important for protection
and immunisation
Melanocytes: important for melanin
production, UV protection
Overviewofpresentation
Models
Rabbit
Mouse
Rat
Guinea pig
Skin morphology
Step 4
Heterogeneity
of skin
Mini-pig
Step 2
Step 1
Skin
function
Human
Step 3
Skin renewal
Skinregions
Summary
Forearm dorsal
Shoulder
Buttock