General Functions and Properties:

1.     Enclosing barrier that prevents loss of water, electrolytes and macromolecules

2.     Protects against the environment/ prevents injurious agents from entering into the internal environment

3.     Provides motion, shape and form to the body

4.     Houses sebaceous and sweat glands, arrector pili muscles, vessels, hair and claws

5.     Helps to regulate body temperature via hair, sweat glands and vessels

6.     Stores electrolytes, water, vitamins, fat, carbohydrates, proteins and other materials

7.     Can be a good indicator of general health, internal disease and the effects of substances applied topically or taken internally

8.     Contributes to physical and sexual identity

9.     Keratinocytes, Langerhans cells and lymphocytes provide the skin with immunosurveillance capabilities

10.  Melanin formation, keratinization and vascularity help determine skin and coat color; can protect against UV damage

11.  The skin has antibacterial and antifungal properties provided by lipids, organic acids, lysozymes and antimicrobial peptides

12.  The skin is a primary sense organ for touch, pressure, pain, pruritus, heat and cold

13.  The skin can secrete various substances via apocrine, eccrine and sebaceous glands and has limited excretory abilities

Skin produces vitamin D when stimulated by solar radiation. Cholecalciferol (Vit D₃) is formed from pro-vitamin D₃ (7-dehydrocholesterol) via previtamin D₃ on exposure to sunlight. The vit D binding protein in plasma translocates vit D₃ from the skin to the circulation. Vit is D₃ then hydroxylated in the liver to 25-hydroxyvitamin D₃, and again hydroxylated in the kidney to form 1,25-dihydroxyvitamin D₃

 Skin morphogenesis requires what genes for coordination?

1)    Skin morphogenesis requires multiple genes acting in coordination

a)     HOX genes likely play a big role in development the of adnexa, pigment and stratified epithelium during embryogenesis by affecting transcription

 Embryonic skin description

a)     Initially = single layer of ectodermal cells and a dermis containing loosely arranged mesenchymal cells embedded in an interstitial ground substance (collagen type III)

b)    Ectodermal covering progressively develops into two layers - basal cell layer or stratum germinativum and an outer periderm

c)     Stratum intermedium forms between these layers

d)    Adult like structure forms from these three layers

i)      Langerhans cells (bone marrow) and melanocytes (neural crest) are identifiable at this stage

Dermal development in embryonic skin

a)     Increased thickness and number of fibers (collagen fibers (type I) appear before elastin fibers), decrease in ground substance, the transition of mesenchymal cells to fibroblasts

b)    Histiocytes, Schwann cells, and melanocytes appear

1)    Subcutis develops from lipocytes (initially spindle-shaped mesenchymal precursor cells or prelipoblasts) in 2^nd half of gestation

 Adnexa embryonic development 

1)    Adnexa develops from the stratum germinativum

a)     Hair germs (primary epithelial germs) develop into hair follicles, sebaceous glands and apocrine sweat glands

b)    Hair development (controlled by morphogens (HOX genes, Fibroblast growth factor/FGF, transforming growth factor/TGF-β, Sonic hedgehog/Shh, winless/wnt pathway, neurotrophins) and other factors like insulin-like growth factor, interferons, interleukins and cadherins):

i)      First hairs to develop are vibrissae and tactile/sinus hairs, general hair grows from the head down

ii)    In dogs at birth hairs are primary and then secondary hairs develop over next 12-28 weeks

iii)   Hair germs initially = areas of crowding of basophilic cells in the basal layer of the epidermis that project into the dermis (called epidermal placodes)

iv)   Beneath these placodes lies the dermal condensate (group of mesenchymal cells from which the dermal hair papilla is later formed

v)    Epidermal placode develops 3 epithelial layers (1) inner = hair shaft (2) middle = IRS (3) outer = ORS

vi)   Hair peg lengthens and 3 bulges appear on the cranial side of primary follicles and the caudal side of secondary follicles (1) deepest = attachment tie of the arrector pili (2) middle = sebaceous gland (3) uppermost = apocrine gland

c)     Eccrine glands develop from similar structures to the epidermal placodes but in basal layer of the epidermis

General Hair Facts

a)     Skin thickness decreases dorsally to ventrally on the trunk and proximally to distally on the limbs

i)      Average thickness of feline skin is 0.4-2mm

ii)    Average thickness of canine skin is 0.5-5mm

b)    The skin surface of haired animals is generally acidic (may protect against microorganisms)

i)      Canine studies: males have higher pH than females, spayed females have higher pH than intact females; varies by breed and possibly coat color; excitement can raise pH

c)     Glucose in the skin is preferentially metabolized to lactate rather than to CO₂

1)    General

a)     Skin thickness decreases dorsally to ventrally on the trunk and proximally to distally on the limbs

i)      Average thickness of feline skin is 0.4-2mm

ii)    Average thickness of canine skin is 0.5-5mm

b)    The skin surface of haired animals is generally acidic (may protect against microorganisms)

i)      Canine studies: males have higher pH than females, spayed females have higher pH than intact females; varies by breed and possibly coat color; excitement can raise pH

c)     Glucose in the skin is preferentially metabolized to lactate rather than to CO₂

Hair cycle basics- what affects the hair cycle? 

1)    Hair cycle (affected by photoperiod, hormones, ambient temperature, nutrition, general health, genetics)

a)     Mosaic pattern…neighboring follicle can be in different stages of the cycle

b)    Short coats generally take 3-4 months to regrow, long coats can take upt o18 months

c)     Many factors influence the hair cycle (control cellular proliferation and collagenase release among other effects)

i)      Stimulators (Induce or prolong anagen) - thyroid hormones, corticotropin, melatonin, androgens, growth hormone, FGF-7, hepatocyte growth factor, insulin like growth factor 1, Shh, keratinocyte growth factor, WNTs, β-catenin, TGF-α, nerve growth factor, GDNF, substance P, activin, noggin, follistatin, cyclosporin, minoxidil, finasteride

ii)    Inhibitors (inhibit anagen, induce catagen and/or prolong telogen) - cortisol, estrogen, corticotrophin-releasing hormone, corticotrophin, prolactin, parathyroid-related peptide, epidermal growth factor, FGF-2, FGF-5, brain-derived neurotrophic factor, neurotrophin 3,4, TGF-α, TGF-β1, TGF-β2, IL-1, IL-6, retinoids, calcitriol

iii)   Other: Neuropharmacologic drugs, sensory and autonomic innervation of follicles (affects are exerted through vascular tone (nutrient and oxygen supply), neuropeptide stimulation, modulation of macrophage and mast cell activities)

Hair follicle cycle

a)     Anagen (growth, active hair production)

i)      Repeated induction of hair follicle anagen with downward growth through the dermis drives hair growth cycles

ii)    Hair growth per week is slower in areas with naturally shorter hair

iii)   Shortened during poor helath

b)    Catatgen (regression, transitional)

i)      Large numbers of keratinocytes in the outer root sheath (ORS) undergo apoptosis and the epithelial strand regresses

ii)    Interplay between MHC1 expression, chondroitin proteoglycans and activated macrophages are involved in regulation of this phase

c)     Telogen (rest, hair is retained in follicle as dead or club hair)

i)      Follicle enters this phase once the hair has reached its preordained length (genetically determined)

ii)    Severe disease or stress can cause a number of hairs to enter synchronously (can result in telogen effluvium when these hairs are shed)

d)    Exogen (shedding, hair lost from follicle)

e)     Follicular arrest can occur post clipping and surgical scrubbing and the hair will not regrow…may spontaneously resolve 6 months to 2 years post clipping

1)    Hair Colors and Types

a)     Normal Coat

i)      Ex GSD, corgi, coyote

ii)    Composed of primary hairs (course guard hairs) ad secondary hairs (fine hairs, undercoat)

iii)   High proportion of primary hairs

b)    Short coat

i)      Course (ex. terrier) - strong growth of primary hairs with less growth of secondary hairs (far fewer than in the normal coat)

ii)    Fine (ex. boxer) - largest number of hairs per unit area; numerous well developed secondary hairs; primary hairs reduced in size

c)     Long coat

i)      Fine (ex. Pomeranian) - greater weight per unit area than the normal coat; tow breed will have extra fine coats

ii)    Wooly or course (ex. poodle) - secondary hairs comprise 70% of the weight and 80% of the number of hairs; tend to shed less

d)    Coat color

i)      Pigment can be uniform throughout the length of the shaft or vary within the shaft

ii)    Classic agouti coat has a white tip and dark body and yellow/red base

iii)   Pigment cells in the hair bulb deposit pigment in or between the cortical and medullary hair cells

iv)   2 types of pigment arranges in different location and amounts produce all the variety

(1)  Eumelanin (black-brown)

(a)   Activation of melanocortin 1 receptors on melanocytes

(b)  Shade is controlled by the tyrosinase related protein 1 gene (TYRP1)

(2)  Pheomelanin (yellow-red)

(a)   Inhibition of melanocortin 1 receptors on melanocytes

i)      Genes recognized in coat color:

(1)  A (agouti, agouti signaling protein, ASIP)

(2)  B (brown, TYRP1)

(3)  C (colored/albino)

(4)  D (blue dilution, melanophilin, MLPH)

(5)  E (extension, MC1R)

(6)  G (graying)

(7)  H (harlequin)

(8)  I (paheomelanin dilution)

(9)  K (black, beta-defension 103 gene)

(10)                 M (merle, SILV)

(11)                 P (dilute)

(12)                 R (roaning)

(13)                 S (white spotting, microphthamia-associated transcription factor, MITF)

(14)                 T (ticking)

Cat coat colors

i)      Cat:

(1)  Tabby coat is considered the wild type coat color

(a)   Arises from two separate sets of genes

(i)    Agouti pattern (hairs have bluish base and black tip separated by yellow banding) in various banding patterns

(ii)  Non-agouti or self (solid color) - solid white is dominant but may be associated with deafness or ocular abnormalities

(iii) E (amber gene eumelanin is replaced with phaeomelanin)

(iv) B (brown gene)

(b)  Tipped hair coats have a darker colored tip and lighter base

(c)   Pointed hair coats are darker on the extremities or “points” due to temperature dependent coloration (temperature dependent enzyme that convert melanin precursors to melanin via oxidation, lower tempos are darker)

(i)    Controlled by the color point restriction gene (C, tyrosinase gene, TYR)

(d)  Tortoiseshell (black and orange patchwork, only occurs n animals with two X chromosomes - generally female but occasionally males) and piebald (controlled by the white gloves, KIT gene) gene introduce more color

(e)   The dilution gene (D, melanophilin, MLPH) dilutes black to grey, orange to cream, and seal point to blue point; autosomal recessive

(2)  DSH is the wild type coat length

(3)  Cornish and devon rex cats - curly hair

(a)   Cornish: no primary hairs, short curly whiskers

(b)  Devon: primary hairs, than resemble secondary hairs, abscent or stubbed whiskers, some cats may be totally alopecic on the chest, abdomen and shoulders

(c)   May completely molt and be diagnosed with mistaken endocrine dermatosis

(d)  No documentation that these breeds are hypoallergenic

(4)  Debate as to whether coat color affects personality

1)    Voight lines, Blaschko lines and tension lines

a)     Voight lines: boundaries of the areas of distribution of the main cutaneous nerve stems

b)    Langer lines: reflect course of blood vessels and lymphatics

c)     Blaschko lines: a pattern formed by many nevoid and acquired skin diseases; reflect a mosaic condition deriving from a single mutated clone of cells originating from a protzygotic mutation or from an x-linked mutation made evident by lionization (one X chromosome is silenced); follow a V shape over the spine, an S shape on the abdomen and a wavy pattern elsewhere (ex. black lines in a brindle coat)

d)    Tension or cleavage lines: help determine best areas for skin incisions

1)    Footpads

a)     Specialized areas of integument

i)      Thickened epidermis that protects against trauma

ii)    Fat deposits to absorb shock and provide elasticity

iii)   Large nerve supply

iv)   Atrichial sweat glands produce secretions that may improve traction and may be involved in scent marking

v)    Smooth epidermis in cats but papillated in dogs

(1)  Rete ridges seen histologically

Explain the Microscopic Anatomy and Physiology of  the Epidermis

Microscopic Anatomy and Physiology

1)    Epidermis

a)     The outer layer of skin, generally 2-3 nucleated cell layers thick

i)      4 distinct cell types

(1)  Keratinocytes (varying morphology) = 85%

(a)   Cytoskeleton consists of 3 types of filaments (1) cytokeratin (2) actin (3) microtubules (tubulin)

(b)  Ultrastructurally characterized by intermediate filaments (cytokeratin, tonofilaments) and desmosomes

(c)   All epithelia express a keratin pair that changes based on different types of epithelia and stage of development

(i)    Keratin chain from the acidic subfamily (type I keratins, cytokeratins 9-20)

(ii)  Keratin chain from the neutral-basic subfamily (type II keratins, cytokeratins 1-8)

(2)  Melanocytes = 5%

(3)  Langerhans cells = 3-8%

(4)  Merkel cells (associated with tylotrich pads) = 2%

a)     Basal Layer (stratum basale)

i)      Single row of columnar to cuboidal cells located on dorsal to the BMZ

ii)    Most cells are keratinocytes that constantly reproduce and push themselves upward to replenish the skin

(1)  Daughter cells replace epidermal layers above

(2)  May see mitotic figures and apoptotic keratinocytes

iii)   Some cells serve to anchor the epidermis while others are the progenitor cells for the overlying layers (stem cells)

iv)   Cells contain K5 and K14 keratin filaments to attached to neighboring cells (desmosomes) and the basement membrane (hemidesmosomes) as well as other adhesion receptors (integrins)

(1)  Desmosomes –

(a)   Major cell-cell adhesion junction of the epithelium

(b)  Found in all keratinocytes

(c)   Calcium and calmodulin = essential for development 

(d)  Provide a 3D scaffolding and anchor for (1) intermediate filaments connecting the nuclear envelope and cell membrane and (2) connections between adjacent cells

(e)   Consist of keratin intermediate filaments and their attachment plaques, the keratinocyte plasma membrane and the desmosomal core (desmoglea)

(f)    Molecular components: plakins (desmoplakin), armadillo proteins (plakoglobin and plakophilin), desmosomal cahedrins (desmoglein and desmocollin)

(g)   Transmembrane proteins: desmosomal cahedrins, Dsg I, II, III (desmosomal core proteins) and Dsc I, II, III

(i)    Pemphigus targets Dsg I

(h)  Plaque proteins: plakoglobin, desmoplakin I, II, IV, desmocalmin, plakophilin

(i)    Cytoskeleton filament: cytokeratin

(1)  Hemidesmosomes –

(a)   Junctional complexes located at the inner aspect of basal keratinocytes which provide dermal-epidermal adhesion (cell substrate adhesion)

(b)  Keratin intermediate filament (cytokeratin) is linked to a network of hemidesmosomes and basal keratinocyte plasma membrane

(c)   Molecular components: plaque proteins, bullous pemphigoid antigen 1 (BPAG I or BP230), plectin, transmembrane proteins α₆ß₄ integrin, BPAG II (collagen XVII), lamanin 332A (lamanin 5)

(d)  Transmembrane proteins: α₆ß₄ integrin, BPAGH (collagen XVII)

(e)   Plaque proteins: plectin, BPAGI

(f)    Cytoskeleton filament: cytokeratin

(1)  Integrins –

(a)   Family of cell surface glycoproteins that act as adhesive receptors

(b)  Normally confined to the basal layer of the epidermis

(c)   Important in cell-cell and cell-matrix interactions

(d)  Important signal transducers (extracellular and intracellular compartments can influence/modify each other)

(e)   Heterodimer of an α and ß subunit (most common components are α₂, α₃, α₆, ß₁ and ß₄)

(f)    α₅ß₁ – mediates keratinocyte adhesion to fibronectin

(g)   α₂ß₁ – mediates keratinocyte adhesion to collagen I, IV and laminin

(h)  α₃ß₁ – receptor for epiligrin, involved in adhesion to laminin

(i)    α₁ß₅ – mediates keratinocyte adhesion to vitronectin

(j)    α₆ß₄ – mediates keratinocyte adhesion to laminin

a)     Spinous Layer (stratum spinosum)

i)      Composed of the daughter cells of the stratum basale

ii)    Keratinocytes retain K5/K14 keratins from the basal layer and express K1/K10 keratin pairs

iii)   Only 1-2 cell layers thick in haired skin but is much thicker in the footpads, nasal planum and mucocutaneous junctions (20 cell layers thick!)

iv)   Cells are nucleated, polyhedral and appear to be connected by little bridges (prickles)

v)    Keratinocytes in this layer can be phagocytic

vi)   Keratinocytes synthesize lamellar granules (keratinosomes, membrane coating granules, Odland bodies)

(1)  Contain glycoproteins, glycolipids, phospholipids, free sterols, glucosylceramides, acid hydrolases

(2)  Secreted into the intracellular spaces of the stratum granulosum and in barrier function

vii) Keratinocyte adhesion is mediated by 4 major types of adhesive and communicative structures (1) desmosomes (2) hemidesmosomes (3) adherans junctions (function in cell-cell adhesion and found in all keratinocytes; transmembrane proteins: classic cadherins E-, P- (basal cells only); plaque proteins: plakoglobin, α- and ß- catenin, α-actin, vinculin; cytoskeleton: actin) (4) focal adhesions (act as a cell substrate and found in basal cells; transmembrane proteins: ß integrins; plaque proteins: talin, vinculin, α- actinin, paxillin, zykin; cytoskeleton: actin)

viii)         Gap junctions act as intracellular routes of chemical communication

a)     Granular Layer (stratum granulosum)

i)      Variably present in haired skin and ranges from 1-2 cell layers thick but may be 4-8 cell layers thick in the infundibulum or in non-haired skin

ii)    Cells are flattened, contain shrunken nuclei and large keratohyalin granules (insoluble aggregates synthesized in this layer composed of profilaggrin, keratin and loricrin)

(1)  Release of profilaggrin from the granules is followed by its calcium dependent cleavage into filaggrin monomers that function to aggregate, pack and align keratin filaments into macrofilaments

(2)  Loricrin is cysteine rich and is involved in bonding keratin filaments together in the corneocyte and anchoring them to the cross-linked envelope

a)     Clear Layer (stratum lucidum)

i)      Fully keratinized, anuclear, thin compact layer of dead cells

ii)    Homogeneous and hyaline-like with refractile droplets and a semifluid substance (eleidin)

iii)   Very rich in protein bound lipids

iv)   Best developed in the foot pads but not always present in the skin layers

a)     Horny Layer (stratum corneum)

i)      Consistently being shed (gradual desquamation is balanced by proliferation of the basal cells

ii)    Multilayered zone of corneocytes suspended in an extra cellular matrix (corneocytes are the brink and lipids are the mortar), thicker in non-haired skin

iii)   Cells are anuclear, flattened and eosinophilic; contain humectants, natural UV protectants

iv)   Generally uneven surface especially in hairy areas

v)    Covered by a homogeneous film (sebaceous and cutaneous lipids) which is spread by rubbing and grooming

vi)   Contains antigenic and superantigenic material that is generally hidden from the immune system including T-lymphocytes

vii) Filaggrin (from the stratum granulosum) degradation products = urocanic acid and pyrrolidone carboxylic acid which help maintain normal hydration and filter UV radiation

viii)         Corneal envelope –

(1)  Impermeable and provide support to the cell and resists invasion by microorganisms and environmental agents

(2)  Developed beneath the plasma membrane of stratified epithelial cells, cells of the IRS/medulla of the hair follicle and the cuticle of the claw.  

(3)  Formation is calcium dependent; transglutaminases catalyze cross-linking of soluble and particulate protein precursors into large insoluble polymers

(a)   Transglutaminases: superfamily of enzymes important in apoptosis, keratinization and a hair follicle formation; require catalytic amino acids and calcium; located in stratum corneum and upper stratum spinosum; faulty transglutaminase function is one cause of ichthyosis in humans

(4)  Protein components: loricrin, involucrine (binds ceramides to form a backbone for the attachment of lipids), filaggrin, elafin, cystatin A, cornifelin

a)     Epidermopoiesis and Keratogenesis

i)      Careful balance between growth and differentiation maintains epidermal homeostasis

ii)    The most important product of the epidermis is keratin (highly stable, disulfide bond-containing fibrous protein) which is classified into soft forms (skin) and hard forms (claws, hair) and α- (skin, hair) and ß- (scale, feather)

iii)   Epidermis is ectodermal in origin and undergoes an orderly pattern of proliferation, differentiation and keratinization (cell renewal time in a normal dog is 22 days vs 7 days in a seborrheic Cocker)

(1)  Many factors control this process: protein kinase C/phospholipase C second messenger system, calcium/calmodulin second messenger system, receptor linked tyrosinase kinases, adenylate cyclase/cyclic adenosine monophosphate (cAMP)

(2)  Intrinsic factors modulate this process: dermis, EGF, FGF, insulin like growth factors, acid hydrolases, arachidonic acid metabolites, proteolytic enzymes, hormones (epinephrine, vitamin D₃, cortisol), ornithine decarboxylase (essential enzyme for polyamine (putrescine, spermidine, spermine) synthesis which encourage epidermal proliferation)

(3)  Nutritional factors also play a role: proteins, fatty acids, zinc, copper, vitamin A, B vitamins

iv)   4 steps in cornification –

(1)  Keratinization (synthesis of the principal fibrous proteins)

(2)  Keratohyalin synthesis (filaggrin)

(3)  Formation of the cornified envelope

(4)  Generation of neutral lipid-enriched intracellular domains

a)     Epidermal Lipids

i)      VIP in barrier function, stratum corneum water-holding, cohesion and desquamation or corneocytes and control of epidermal proliferation and differentiation

ii)    Lamellar granules are synthesized the stratum spinosum and get displaced into the apical periphery of the cell as it reaches the stratum granulosum

iii)   In response to an increase in calcium content the granules fuse with the plasma membrane to release their contents (polar lipids, glucosylceramides, sphingomyelin, phospholipid, hydrolytic enzymes, free sterols) into the intracellular space

iv)   The lipids undergo hydrolysis and enzymatic conversion to non-polar lipids, ceramides, free fatty acids and cholesterol

v)    Ceramides – amide-linked free fatty acids containing a long-chain amino alcohol; the most important lipid component for lamellar arrangement in the stratum corneum and in barrier function; linoleic acid is a critical component of ceramides, thus an essential fatty acid for dogs and cats; ceramide 1 and 2 bind to involucrin in the CE to form a scaffolding for other lipids and help the skin barrier be flexible/fluid

vi)   Polyunsaturated fatty acids – precursors to eicosanoids; liberated from phospholipids by phospholipase A₂ and are metabolized by cyclooxygenase and lipoxygenase into prostaglandins and leukotrienes

a)     Melanocytes

i)      Derived from the neural crest and migrate into the epidermis early in fetal life

ii)    Melanocytes are found in the basal layer of the epidermis, the outer root sheath, the hair matrix, sebaceous and sweat glands and occasionally in the superficial dermis; also found in the eyes (retinal pigmented epithelium), cochlea (stria vascularis) and meninges

iii)   Most of the pigment in the epidermis is located in the basal layer but in darker skinned animals can be scattered throughout and within superficial dermal melanocytes

(1)  Melanin granules are usually clustered in a “cap” dorsal to the nucleus, suspect for UV protection

iv)   Structure: intracellular melanosomes and premelanosomes, a cell membrane-associated basal layer lamina

v)    Appear as clear cells on H&E

vi)   Dendrites weave among the keratinocytes and transfer pigment containing melanosomes to them

(1)  ~One melanocyte per 10-20 keratinocytes = functional unit (epidermal melanin unit)

i)      Function:

(1)  Cosmetic - protective coloration and sexual attraction

(a)   Skin and hair coloration

(2)  A barrier against ionizing radiation (absorbs UVA and UVB well but less efficient with UVL)

(3)  Scavenger of cytotoxic radicals and intermediates

(4)  Participant in developmental and inflammatory processes

ii)    Skin pigmentation has 2 components

(1)  Constitutive pigmentation – pigmentation that is genetically determined in the absence of stimulatory influences

(2)  Facultative pigmentation – occurs with various stimuli (UVL, inflammation, hormones)

(3)  Skin color is also influenced by yellow carotinoids in the epidermal cells, red from oxygenated hemoglobin and blue from unoxygenated hemoglobin

iii)   Melanins have 2 major forms (production of one other the other is under genetic control)

(1)  Eumelanin – dark brown-black; alkali-insoluable; ellipsoidal in shape; contain lamellae or filaments; polymers of 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid comprise chemical structure; high levels of tyrosinase produce eumelanin

(2)  Phaeomelanin – yellow or red-brown; alkali-soluble; contain a high proportion of sulfur; spherical in shape; contain microvesicles and microgranules; polymers of benzothiazine units derived from cysteinyldopa comprise chemical structure; low levels of tyrosinase produce phaeomelanin

(3)  Other – mix between Eu- and phaeomelanin

i)      Melanin synthesis:

(1)  Takes place only within melanocytes and on the melanosome (specialized organelle derived from the Golgi apparatus)

(a)   Stage I melanosomes are melanocyte specific and contain no melanin and are electron lucent but as melanin is progressively laid down on the protein matrix the melanosome becomes increasing electron dense and migrates to the periphery on the dendrites

(b)  Via cytocrinia the dendrite tip is phagocytosed by the keratinocyte and melanosomes are ejected into the keratinocyte

(2)  Tyrosinase: rate limiting enzyme in the melanin pathway, copper containing, 3 catalytic activities (1) tyrosine hydroxylase activity catalyzes the conversion of tyrosine to dopa AKA 5,6-dihydroxyindole (2) can use dopa as a substrate for oxidase activity

(a)   Tyrosine is converted to dopa by tyrosinase

(b)  Dopa spontaneously auto-oxidase to dopaquinone without tyrosinase but the enzyme speeds this process

(c)   Dopaquinone gets converted to dopachrome, 5,6-dihydroxyindole-2-carboxylic acid, 5,6-dihydroxyindole and indole-5,6-quinone

(3)  Dopachrome tautomerase: converts dopachrome to 5,6-dihydroxyindole-2-carboxylic acid; requires the presence of iron

(4)  Steps (regulated by over 150 different genes):

(a)   Melanoblast migration from the neural crest

(b)  Melanoblast differentiation into the melanocyte in the skin

(c)   Melanosome matrix formation

(d)  Melanogenic gene activation

(e)   Synthesis of tyrosinase and melanogenic proteins

(f)    Posttranslational processing and glycosylation of tyrosinase and transport

(g)   Fusion of vesicles to form melanosomes

(h)  Control of tyrosinase activity and tyrosinase related proteins

(i)    Melanin biosynthesis

(j)    Melanosome transfer to keratinocytes (cytocrinia)

(k)  Melanosome degradation

(l)    Melanin removal with loss of cornified cells

(1)  Melanocytes express a number of different receptors to interact with other cell types as well as hormones, growth factors, interferons, interleukins, eicosinoids, retinoic acid, vitamin D₃ and other cytokines.

(a)   Autocrine - can produce some of these substances them selves

(i)    IL-8 – participate in immunologic and inflammatory functions

(b)  Microphthalmia transcription factor (MITF or master transcriptional regulator of pigmentation – VIP in regulating melanin synthesis

(c)   Melanocortin 1 receptor (MC1R) – G-protein coupled receptor that regulates pigment phonotype

(i)    Agouti phonotype encodes an antagonist for MC1R

(ii)  Agonists include α-melanocyte stimulating hormone (MSH) and corticotropin (ACTH)

(d)  C-kit (tyrosinase kinase receptor) – involved in Melanoblast expansion, survival and migration

(i)    Mutations in c-kit are part of piebaldism

(e)   α-MSH (neuroimmunomodulatory and anti-inflammatory peptide) – synthesized and released by keratinocytes, Langerhans cells, fibroblasts, endothelial cells and melanocytes

(i)    melanocytes also have cell surface receptors for α-MSH (thus it can modulate proliferation and differentiation)

(ii)  downregulates the production of proinflammatory cytokines and accessory molecules on antigen presenting cells

(iii) antagonist of IL-1

(2)  follicular melanocytes

(a)   cycle in coordination with the hair cycle

(b)  located on the proximal hair bulb during antigen and actively synthesize and transfer melanin to the keratinocytes of the developing hair shaft

(c)   undergo apoptosis during late catagen

(3)  Argentaffin stains (Fontana-mason or Gomori) – rely on the ability of melanin to reduce silver from a silver solution

(a)   No great stain for melanocytes

(4)  Potassium permanganate will bleach melanocytes so that organelles can be studies

a)     Merkel Cells

i)      Dendritic epidermal clear cells located in or below the basal cell layer (esp. in tylotrich pads and the hair follicle epithelium)

ii)    Function as slow adapting type-1 mechanoreceptors

iii)   Structure: desmosomes, dense-core cytoplasmic granules, paranuclear whorls located opposite the Golgi and parallel to an unmyelinated neuron, cytokeratin, neurofilaments, metenkephalin, vasoactive intestinal peptide, synaptophysin and neuron specific enolase

(1)  Duel epithelial and neural

iv)   IHC markers – K8, 18, 19, 20

v)    Derived from a primitive epidermal stem cell?

vi)   Maintain and stimulate the stem cell population of the hair follicle (control the hair cycle)

a)     Langerhans Cells

i)      Mononuclear, dendritic, antigen presenting cells located basally or suprabasally

ii)    Epidermal clear cells that for 2-8% of the total epidermal cell population

iii)   Bone marrow origin and of the monocyte/histiocyte lineage

iv)   Function: process and present antigens to T Cells in the epidermis

(1)  Increased numbers found in dogs with atopy

v)    Structure: intracytoplasmic organelles (Birbeck or Langerhans granules)

(1)  No found in dogs

(2)  Zipper/rod or flask like in appearance

(3)  Form by invagination of the plasma membrane and bound antigen (mechanism for internalizing surface bound antigen for processing and presentation)

vi)   Stain with gold chloride (aureophilic); S-100 protein and ATPase negative; CDIa,b,c, CD11a,c, CD18, CD45, ICAM-1, MHC II, vimentin positive but CD4 and CD90 negative in the dog; CD1a CD4, CD18, MHC II, positive in the cat

vii) Receptors: Fc fragment immunoglobulin (IgG), C3, IgE

viii)         Reduce in density in response to UVL exposure, topical and systemic glucocorticoids

a)     Epidermal Histochemistry

i)      Distinct oxidative activity in all layers of the epidermis except the stratum corneum

(1)  Oxidative enzymes: cytochrome oxidase, succinate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase, nicotinamideadenine dinucleotide phosphate, nicotinamide-adenine dinucleotide and monoamine oxidase

ii)    Stratum granulosum has a strong reaction to non-specific esterases

iii)   Hydrolytic enzymes: acid phosphatase, arylsulfatase, ß-glucuronidase and leucine aminopeptidase  

1)    Basement Membrane Zone

a)     Physiochemical interface between the epidermis and other skin structures (appendages, neural, vascular, smooth muscle) and the underlying/adjacent connective tissue (dermis)

b)    Composed of a large variety of interactive molecules

c)     Most prominent in non-haired areas and at mucocutaneous junctions

d)    Comprises only the fibrous zone of the sublamina densa

e)     Functions

i)      Anchors the epidermis to the dermis

ii)    Maintains a functional and proliferative epidermis

iii)   Maintains tissue architecture

iv)   Aids in wound healing

v)    Functions as a barrier

vi)   Regulates nutritional transport between the epidermis and dermis

vii) Involved in adhesion, cytoskeletal organization, migration and differentiation

f)      Stains with periodic acid Schiff

g)     Structure (from epidermis to dermis):

i)      Basal cell plasma membrane

ii)    Lamina lucida (lamina rara)

iii)   Lamina densa (basal lamina)

iv)   Sublamina densa (lamina fibroreticularis) – includes anchoring fibrils and the dermal microfibril bundles

a)     Major components

i)      K5 (basic)/K14 (acidic) – intracellular intermediate fillaments that connect the nuclear membrane to plectin and BPAG1 in hemidesmosomes

ii)    Plectin – inner plaque of the hemidesmosome that attaches to K5/14 filaments

iii)   BPAG1-e – inner plaque of the hemidesmosome that attaches to K5/14 filaments, BPAG2 and the ß₄ chain of integrin

iv)   Integrin α₆ß₄ – outer plaque of the hemidesmosome that binds collagen XVII and laminin 332

v)    BPAG2 – – outer plaque of the hemidesmosome that binds ß₄ chain of integrin, plectin, BPAG1-e ant eh ß3 chain if laminin and interacts with adherans junction proteins

vi)   Collagen IV – in the lamina densa and intearacts with the laminin network via nidogens, fibulin 2 and perlecan

vii) Collagen VII – in the lamina densa and anchoring fibrils and attaches to laminin 332

viii)         Laminin 332 – in anchoring filaments and binds to integrin α₆ß₄, collagen VII

ix)   Nidogens – in the lamina densa and binds to perlecan, collagen IV, laminin Ɣ₁, fibulins

x)     Fibulins – in the lamina densa and binds to nidogen, collagen IV, perlecan and fibronectins

xi)   Perlecan – in the lamina densa and binds to collagen IV, laminin 332 and fibulins

1)    Dermis

a)     General

i)      Connective tissue, mesodermal origin and synthesized mostly by fibroblasts

ii)    Composite system of insoluble fibers (collagen and elastin, resist tensile forces) and soluble polymers (proteoglycans and hyaluronan, resist compressive forces)

(1)  Accounts for most of the tensile strength and elasticity of the skin

iii)   Composed of fibers, ground substance and cells and contains epidermal appendages, arrector pili muscles, blood/lymph vessels, nerves

iv)   Regulates cell growth, proliferation, adhesion, migration, differentiation

v)    Modulates wound healing

vi)   The dermis of the scrotum contains large smooth muscle bundles

a)     Dermal fibers

i)      Formed by fibroblasts

ii)    Collagenous

(1)  Great tensile strength and account for 90% of dermal fibers

(2)  Thick bands composed of multiple protein fibrils

(3)  Stained by Mason trichrome

(4)  Contain hydroxylysine and 4-hydroxyproline (levels in urine can indicate collagen turnover rate)

iii)   Reticular

(1)  Fine branching structures that overtime look like collagen

(2)  Special silver stains

iv)   Elastic

(1)  Composed of single fine branches that are very elastic and account for 4% of the dermal matrix

(2)  2 components: (1) amorphous elastin – crosslinked desmosine and isodesmosine (2) microfibrils – fibrillin and type VI collagen

(3)  Verhoeff and van Gieson stains

i)      Many types of collagen molecules – I, III, V are the most common in the dermis

(1)  Type I has a triple helix structure with strands of coiled polypeptide α chains

(2)  Type III has three identical α chains with a high content of hydroxyproline and glycine

(3)  Type V forms large fibrils and is often associated with type I and III

(4)  Type IV form triple helical microfibrils arranges in a network and stabilize type I

ii)    Complex biosynthesis of gene transposition and translation, intracellular modification, packaging and secretion, extracellular modifications, fibril assembly and crosslinking

iii)   Defects result from genetic defects, vitamin C, iron or copper deficiency, and ß-aminopropionitrile poisoning (lathyrism)

iv)   Ascorbic acid, TGF-ß, IL-1, IGF-1, IGF-2, superoxide generating systems and bleomycin stimulate collagen synthesis

v)    Glucocorticoids, retinoids, vitamin D₃, parathormone, prostaglandin E₂, IFN-Ɣ, D-penicillamine and minoxidil inhibit collagen synthesis

vi)   Collagenase – a matrix metalloproteinase (MMP) and in the skin produced predominantly by dermal fibroblasts but other cell types can release it in disease states

(1)  Other degradative enzymes = gelatinase, stromelysins, lysosomal hydrolases, elastase (called serine proteinases in neutrophils and eosinophils and are very potent in degradation)

i)      The superficial dermis contains fine loosely arranged collagen fibers that are irregularly distributed and a network of fine elastin fibers

(1)  Elaunin fibers (microfibrils) have an arcade-like arrangement

(2)  Oxytalan fibers (elastin) extend from these elaunin fibers vertically to terminate at the dermoepidermal junction and anchor to the basement membrane

ii)    The deep dermis contains thick, densely arranged collagen fibers that parallel the skin surface and thick less numerous elastin fibers

iii)   Dendritic cells (dermal dendrocytes): bone marrow derived; express CD45, MHC II, adhesion molecules; phagocytic antigen presenting; factor XIIIa and CD34 positive

a)     Dermal ground substance

i)      Interstitial substance composed of a viscoelastic gel-sol of fibroblast origin and composed of glycosaminoglycans linked to proteoglycans

(1)  Hyaluronate and heparin sulfate in the epidermis

(2)  Heparin sulfate, chondroitin-6-sulfate in the BM

(3)  Chondroitin-4-sulfate, hyaluronic acid, keratan sulfate, dermatan sulfate, chondroitin-6-sulfate, versican, syndecan, decorin, glypican and serglycin in the dermis

ii)    Fills space and surrounds structures of the dermis while allowing molecules and cells to pass through it

iii)   The proteoglycans and GAGs are extracellular and membrane associated

(1)  Function in water storage (can bind over 100 times their own weight), homeostasis, selective screening of substances, support or dermal structure (resist compression), lubrication, collagen fibrillogenesis, orientation, growth and differentiation and wound healing

iv)   Fibronectins - widespread in the matrix and body fluids

(1)  Capable of interacting with cell surfaces (moderate cell-cell interactions and cell adhesion) and other matrix components

(a)   Modulate microvascular integrity, vascular permiabilty, BM assembly and wound healing

(2)  Location – dermis perivascular and perineural, lamina lucida, lamina densa

v)    Tenascin – large glycoprotein, expressed at epithelial-mesenchymal interfaces that is involved in epithelial morphogenesis and proliferation (wound healing)

vi)   Mucin – small amounts seen in normal feline and canine (esp. Shar-pei) skin, stains blue on H+E

(1)  Shar-pei: increased levels of serum hyaluronic acid and increased hyaluronan synthase 2 mRNA transcription

a)     Dermal cellular elements

i)      The dermis is usually sparsely populated with cells

ii)    Fibroblasts

iii)   Dermal dendrocytes – predominantly perivascular antigen presenting cells; in dogs CD1, CD11, CD18, CD45, ICAM1 and MHC II positive as well as CD4 and CD90 (unlike Langerhans cells)

iv)   Melanocytes – seen near superficial dermal blood vessels or around hair bulbs

v)    Mast cells – abundant around superficial dermal blood vessels and appendages

(1)  Toluidine blue, acid orcein-Giemsa, tryptase, chymase stains

(2)  Mast cell numbers may be higher in the pinna

(3)  3 types: (1) tryptase containing, T-mast cells (2) chymase containing, C-mast cells (3) tryptase and chymase containing, TC-mast cells

vi)   Neutrophils, eosinophils, lymphocytes, histiocytes and plasma cells may be seen in small numbers

1)    Hair follicles

a)     Hair is found only in mammals and functions in a variety of ways (physical barrier against trauma, providing protection from stimulus (piloerection as a warning), repelling H₂0, holding scents, source of cells for re-epithelialization during wound healing, camouflage

b)    Morphogenesis is complex, multistage between epithelial cells of the follicle and the associated mesenchymal cells 

c)     4 parts:

i)      Medulla – innermost region, longitudinal rows of cuboidal cells, cells are solid near the root but the remainder is flexible from air and glycogen vacuoles, +/- pigment

ii)    Cortex – middle layer, cornified spindle shaped cells with long axis parallel to the shaft, pigment containing, 1/3^th to 1/6^th the width of the hair, gives hair mechanical properties

iii)   Cuticle – outermost layer, flat cornified, anuclear cells, free edge of the cells faces the hair tip

(1)  Languo hair – no medulla

(2)  Secondary hairs – thick cuticle

iv)   Epicuticle – amorphous eternal layer, exocellular secretion from cuticular cell mambranes

·       Cats have serrated hair profile, ~2x more hair than dogs

Cats have serrated hair profile, ~2x more hair than dogs

a)     Positioned @ 30 to 60˚ angle backwards

b)    Cats and dogs have compound hairs – 2-5 primary hairs (one central primary hair  with lateral primary hairs) surrounded by 5-20 secondary hairs (only sebaceous glands, emerge through common pore); each primary hair has a sebaceous gland, sweat gland, arrector pili muscle, own pore

a)     Specialized tactile hairs:

i)      Sinus hair – vibrissae/whiskers found on the muzzle, lip, eyelid, face and throat and the palmar area of cats; thick, stiff and tapered distally; characterized by an endothelium-lined blood sinus interposed between the external root sheat of the follicle and the outer connective tissue capsule; 2 parts (1) superiror nontrabecular ring (annular), cushion like thickening of the mesenchyme (sinus pad) projects into this sinus  (2) inferior cavernous (trabecular), travered by many nerves; Pacinian corpuscles are situated close to the sinus (slow adapting mechanoreceptors)

ii)    Tylorich hair – scattered among normal body hairs; large follicle with a single stout hair and an annular complex of neurovascular tissue at the level of the sebaceous glands; associated with a tylotrich pad (comprised of a thickened and distinctive epidermis underlaid by a convex area of connective tissue that is highly vascularized and innervated; unmyelinated nerve fibers end as discs in association with Merkle cells and serve as slow adapting mechanoreceptors); function of the tylotrich hairs is thought to be rapid adapting mechanoreception

a)     5 major components

i)      Dermal hair papilla

(1)  Continuous with the dermal connective tissue, covered by a thin continuation of the BM; a layer of plump nucleated cells cover the papilla and generate the IRS and hair (hair matrix); dermal papilla is highly involved in hair follicle growth/cycling and changes morphology throughout this process (maximal in volume during anagen and smallest during telogen, secondary to changes in the volume of the hair matrix); underlying the dermal papilla is a fibroelastic cushion (Aaro-Perkins corpuscle)

ii)    Hair matrix – pluripotential cells give rise to the hair and inner root sheath; covers the dermal papilla

iii)   Hair

iv)   Inner root sheath – 3 concentric layers

(1)  Inner root sheath cuticle: inner most, flattened, single layer, overlapping cells, point towards hair bulb, interlock with cuticle, keratinizes and disintegrates when it reaches the level of the isthmus, functions to mold the hair

(2)  Huxley layer – 1-3 nucleated cells thick, contain trichohyalin granules (trichohylin promotes the lateral alignment and aggregation of parallel bundles of intermediate filaments in the inner root sheath cells) 

(3)  Henle layer – 1 layer, non-nucleated cells, covered by a distinct layer of cells that are flattened and lack lamellar bodies (specialized ORS layer)

v)    Outer root sheath – downward extension of the epidermis, thickest at epidermis

(1)  lower portion - covered by inner root sheath (does not undergo keratinization, clear vacuolated cells (glycogen in cytoplasm))

(2)  middle portion – no inner root sheath, undergoes trichilemmal keratinization

(3)  upper portion – infundibulum, undergoes keratinization

(4)  Other important structures cover the ORS: (1) BMZ/glassy membrane (downward reflection of the epidermal BMZ); perifollicular mineralization of this layer has been seen in normal poodles and Bedlington terriers, may also be a normal aging change BUT can also be secondary to natural or iatrogenic Cushing syndrome (2) fibrous root sheath (layer of dense connective tissue)

(5)  Contains Langerhans cells that can act to repopulate the epidermis during inflammation, UV light exposure, trauma, etc.

a)     Stages

i)      Anagen:

(1)  Three parts (1) Infundibulum: pilosebaceous region, upper portion = segment from the entrance of the sebaceous duct (where trichilemmal keratinization begins) to the skin surface, permanent (2) Isthmus: middle portion, segemtn between the entrance of the sebaceous duct and the arrector pili muscle attaches (trichohyalin granule containing cellular portion of the inner root sheath begins, contain high amounts of citrulline (marker for hair follicle differentiation)), permanent (3) Inferior segment: lowest portion, segment from the attachment of the arrector pili muscle to the dermal hair papilla, transitory

(2)  Histopathology – well developed spindle shaped dermal papilla capped by the hair matrix to form the hair follicle bulb (ball and claw appearance…)

(3)  Extends deep into the dermis and often into the subcutis

(4)  Seven stages

(a)   Growth of dermal papilla and onset of mitotic activity in the overlying epithemlium

(b)  Bulb matrix cells envelop the dermal papilla and begin differentiation

(c)   Bulb matrix cells show differentiation into all follicular components

(d)  Matrix melanocytes reactivate

(e)   Hair shaft emerges and dislodges telogen hair

(f)    New hair shaft emerges from the skin surface

(g)   Stable growth

ii)    Telogen:

(1)  Reduced to ~1/3^rd its former length

(2)  Small dermal papilla that is separated from the matrix cells, no inner root sheath or inferior segment, no hair bulb, no melanogenic or mitotic activity

(3)  Presence of a club (brushlike hair) in the follicle

iii)   Catagen:

(1)  Characterized by retraction toward the surface, a thickened, irregular BMZ that is especially thick between the hair matrix and dermal papilla, an increased number of apoptotic keratinocytes, smaller bulb, ovoid or round dermal papilla, partial replacement of the IRS with trichilemmal keratinization

(2)  Follicle length and volume is decreased, melanogenesis ceases, proximal hair shaft is depigmented, mitosis stops

(3)  Follicular epithelium is gradually lost the adventitial collagen and neurovascular network of the follicle condenses along the tract of the previous anagen follicle to form the follicular stile (helps direct the downward growth of the follicle as it later reenters anagen)

(4)  TGF-ß is involved in inducing catagen

iv)   Exogen:

(1)  Shedding of the hair from the follicle

a)     4 morphologic patterns of keratinization

i)      Infundibular – like the epidermis, basket weave orthokeratosis and keratohyalin granules

ii)    Trichilemmel – serrated, closely packed eosinophilic keratin with scant or no keratohyalin granules

iii)   Hair matrix or trichogenic – cortex of hair shaft, ghost cells (retention of nuclear outlines)

iv)   Inner root sheath and hair shaft medullary – compact and opaque keratin with blue-grey to eosinophilic color and red trichohyalin granules, Henley and Huxley layers

b)    Dogs do not have a bulge section @ attachment of arrector pili muscle (humans do)

i)      Instead stem cells distributed throughout the infundibulum

1)    Sebaceous (holocrine) glands

a)     Simple or branched alveolar glands distributed throughout all haired skin in all mammals (except whales and porpoises)

b)    Open through a duct into the pilary canal in the infundibulum (pilosebaceous follicle)

c)     Largest and most numerous near mucocutaneous junctions, interdigital spaces, dorsal neck and rump, chin (submental organ or chin gland also contains large nerve fibers) and dorsal tail (tail or preen gland)

d)    Not found in foot pads or the nasal planum

e)     Lobules are bordered by a BMZ over which lies a single layer of deeply basophilic cells (reserve cells) which become progressively more lipidized and eventually disintegrate to form sebum toward the center of the lobule; lined with squamous epithelium

i)      increased lipid can be stained with Oil Red O stain

f)      Express several receptors involved in the uptake of circulating lipids

i)      FATP4 (fatty acid transporter 4)

ii)    LDL (low-density lipoprotein)

g)     Sebum (oily secretion) from the sebacuoes glands

i)      Keeps the skin soft and pliable

ii)    Forms a surface emulsion that spreads over the surface of the stratum corneum to retain moisture (regulates hydration)

iii)   Give the skin and hair hydrophobic protection from over wetting

iv)   May play a role in thermo regulation

v)    Give hair its glossy sheen

vi)   Sebum-sweat emulsion forms a chemical barrier against potential pathogens

vii) Contains predominantly glycerides and wax esters (as well as succinate acid dehydrogenase, cytochrome oxidase and esterases) upon production but becomes contaminated with lipase producing bacteria (Propionibacterium spp. Staphylococcus spp.) in the infundibulum leading to the production of free fatty acids (linoleic, myristic, oleic and palmitic)

(1)  FFAs have antimicrobial properties

viii)         May have pheromonal properties

h)    Sebaceous glands have an abundant blood supply and innervation

i)      Secretion is thought to be under hormonal control

i)      Androgens = hypertrophy and hyperplasia

ii)    Estrogens and glucocorticoids = atrophy and involution

1)    Sweat glands

a)     Epitrichial (formerly apocrine) sweat glands

i)      Coiled and saccular/tubular

ii)    Throughout all haired skin (not present in footpads or nasal planum

iii)   Located below the sebaceous glands and open through the piliary canal in the infundibulum above the sebaceous opening

iv)   Largest and most numerous near MC junctions, interdigital spaces and over the dorsal neck and rump

v)    Flattened to columnar epithelial (secretory) cells and a single layer of fusiform myoepithelial cells

(1)  The duct is lined by two cuboidal to flattened epithelial cell layers and a luminal cuticle (no myoepithelial cells)

vi)   Lack innervation

(1)  Positive for cytokeratin in glands

vii) Positive for cytokeratin and S-100 protein in myoepithelial cells

viii)         Antimicrobial and pheromonal properties (salts (alkaline and acid phosphatase) and IgA)

ix)   Capable of reepithelizing the skin, although not entirely normally

b)    Atrichial (formerly eccrine) sweat glands

i)      Located only in the footpad in the deep dermis and subcutis

ii)    Small, tightly coiled, duct opens directly to the surface

iii)   Secretary coils = single layer of cuboidal to columnar epithelial cells and a single layer of fusiform myoepithelial cells

iv)   Intradermal portion = double row of cuboidal epithelial

v)    Excretions contain: Cytochrome oxidase, succinate and other dehydrogenases, phosphorylases and alkaline phosphatase

vi)   Rich cholinesterase-positive innervation

a)     Sweating and thermo regulation

i)      Carnivores lack atrichial sweat glands in haired skin

ii)    There is debate as to the degree that dogs sweat with some breeds (Labs, GSD) that may sweat in the axillary regions, groin and ventral abdomen

iii)   Atrichial sweating may be seen on the foot pads of dogs and cats

(1)  Provoked by cholinergic stimuli

(2)  Feline atrichial sweat contains high concentrations of sodium, potassium and chloride

iv)   Generalized cholinergic induced epitrichial sweating has been documented in dogs (primary regulation is neural with humoral mechanisms playing a subsidiary role) but is debatable

(1)  Some studies have shown epitrichial sweating in dogs only during copulation and thus they may have pheromonal functions

v)     Mechanisms to conserve heat

(1)  Vasoconstriction in the skin and erection of the hairs to enhance insulating mechanisms of the skin

(2)  Critical temperature = the point at which the body is no longer able to maintain a stable body temperature and heat production must be increased (via shivering)

(a)   Dogs = 57 degrees with hair coat and 77 degrees without

(b)  non-fasted animals have a lower critical temp

vi)   Mechanisms to dissipate heat

(1)  Radiation, conduction and convection (account for 75%), vaporization from the skin and respiratory tract (very important at higher temperatures), excretion of urine and feces (generally unimportant)

(2)  In dogs when the temp rises above 81-84, respiratory rate increases markedly with a decrease in tidal volume (prevents blood gas changes)

(3)  Rectal temperature above 105-109 becomes very dangerous and collapse may occur

(4)  Rectal temperature of cats begins to rise above 90 but as respiratory rate increases, tidal volume is only slightly reduced (thus cats are more susceptible to changes in blood gases – respiratory alkalosis, low CO₂)

(a)   Cats show copious flow of watery saliva from the submaxillary glad at high temperatures which gets spread on the coat to increase vaporization

(5)  Proper grooming greatly aids in proper thermo regulation in dogs and cats

1)    Specialized glands

a)     Circumanal (perianal) glands

i)      Present at birth and develop from ducts that originate from the inner and outer perianal sides of the external root sheath of hair follicles

ii)    Also found in the prepuce and dorsal and ventral aspects of the tail

iii)   Cytokeratin positive, testosterone dependent

b)    Tail gland (preen gland or supracaudal gland)

i)      Oval shaped area on the dorsal surface of the tail that is a remnant from wild Canidae (only visible in around 5% of male domestic dogs and is non-functional but histologically present in most dogs (comprised of hepatoid cells with glandular ducts that empty into hair follicles)

ii)    Characterized by stiff hairs that arise singly from their follicle, the surface of the skin may be yellow and waxy

iii)   May be severely affected in seborrheic skin and may be hyperplastic, develop cystic degeneration, infection, adenoma or adenocarcinoma

iv)   Testosterone dependent and form a topographic-ethnologic unit with the circumanal gland

v)    Feline: numerous large sebaceous glands that run the entire length of the tail

(1)  Stud tail = excess accumulation of glandular material

1)    Arrector pili muscles

a)     Mesenchymal origin

b)    Smooth muscle with intra- and extracellular vacuoles

c)     Present in all haired skin and are larges in the dorsal neck and rump

d)    Originate in the superficial dermis and inset approximately perpendicularly on the primary hair follicles

e)     ¼ to ½ the diameter of the central primary hair follicle but are the same diameter as the follicle in the caudal dorsum

f)      Cholinergic innervation (contract in the presence of epinephrine and norepinephrine)

g)     Piloerection – social cue, thermoregulation, emptying of sebaceous glands

1)    Blood vessels

a)     Microcirculation system with cutaneous vessels arranged in 3 intercommunicating plexuses of arteries and veins

i)      Deep plexus – found at the interface of the dermis and panniculus

(1)  Supplies the lower portions of hair follicles and the epitrichial sweat glands

ii)    Middle plexus – supplied by the deep plexus and ground at the level of the sebaceous glands

(1)  Supplies the arrector pili muscles, middle portions of follicle and the sebaceous glands

iii)   Superficial plexus – sends capillary loops up to the skin surface

(1)  Supplies the upper portion of the hair follicles and epidermis

iv)   Microcirculatory bed –

(1)  Arterioles: endothelial cells surrounded by 2 layers of smooth muscle cells

(2)  Arterial and venous capillaries: lack surrounding smooth muscle cells

(3)  Venules (postcapillary): the most physiologically reactive segment of the microcirculation and the site where inflammatory cells migrate from the vascular space into tissues and where endothelial cells develop gaps that result in vascular permeability during inflammation

b)    Blood vessel endothelial cells are mesenchymal in origin

i)      Characteristics:

(1)  Perphipheral basement membrane and intracytoplasmic Weible-Palade bodies (rod shaped tubular structures enveloped in a continuous single membrane)

(2)  Possess factor VII antigen (Von Willebrand), plasminogen activators and prostaglandins

(3)  CD31 (platelet endothelial cell adhesion molecule aka PECAM)

(4)  Phagocytic

ii)    Strategically located between intravascular and tissue compartments

iii)   Key regulators of leukocyte trafficking

(1)  E- and P-selectin and I-CAM1 are upregulated during inflammation

iv)   Angiogenesis is controlled partially by mast cells (histamine), macrophages (TNF-α) and TGF-ß

c)     Arteriovenous anastomoses – normal connections between arteries and veins that allow arterial blood to enter venous circulation without passing through a capillary bed

i)      Occur in all areas of the skin but are most common in the extremities (legs and ears) and in the deep dermis

ii)    Vary in structure – simple to complex

(1)  Glomus: specialized AV shunt located in the deep dermis, with an arterial segment (Sucquet-Hoyer Canal) branching from an arteriole and a wall consisting of a single layer of endothelium surrounded by a BMZ and a tunica media that is densely packed with 4-6 layers of glomus cells (large, clear cytoplasm, epithelioid-like, modified smooth muscle cell) and a venous segment with a thin wall and a large lumen)

iii)   Associated with thermoregulation and can enhance blood flow to a specific area

iv)   Acetylcholine = dilation, norepinephrine = constriction

d)    Pericytes – vary from fusiform to club-like, aligned parallel to blood vessels on their dermal side, contractile (contain actin- and myosin-like filaments), regulate capillary flow

e)     Veil cells – flat, adventitial, fibroblast-like cells, surround dermal microvessels but are external to the wall, stain positive for factor XIIIa and likely a component of the dermal dendrocyte system

f)      Perivascular mast cells may be located between the vessel wall and the veil cells

1)    Lymph vessels

a)     Lymphatics arise from capillary networks that lie in the superficial dermis and surround adnexa and drain into the subcutaneous lymphatic plexus

b)    A non-contractile lymphatic (attenuated endothelial cell layer, discontinuous BM and non-contiguous cell junctions) drains into a contractile collector lymphatics (possess smooth muscle, exhibit peristalsis and carry lymph towards the nodes)

i)      Lymph collection in the non-contractile portion depends on periodic tissue motions (pressure, pulsations, skin massage, muscle motion)

c)     Control the movement of interstitial tissue fluid

d)    VIP in normal skin function (especially immunologic functions)

i)      The drains that remove excess debris and matter from daily wear and tear

ii)    Essential channels for the return of protein and cells from the tissues to the blood stream

e)     Compared to blood vessels: have a wider/more angular lumen, have flat attenuated endothelial cells, no pericytes, do not contain blood

1)    Nerves

a)     Somatic sensory and autonomic motor nerves present

i)      Cutaneous somatic sensory nerves: mediate sensations like touch, heat, cold, pressure, vibration, proprioception, pain and pruritus

ii)    Autonomic motor nerves: control cutaneous vascular resistance, pilomotor responses, sensory activities of glands, modulate inflammatory, proliferative and reparative cutaneous processes

b)    Affect dermal vessels, mast cells, fibroblasts, keratinocytes and Langerhan’s cells

i)      Release neuropeptides that activate target cells (substance P, neurokinin A, calcitonin gene related peptide, vasoactive intestinal peptide, neuropeptide Y, somatostatin, pituitary adenylate cyclase-activating peptide)

(1)  Keratinocytes: IL-1

(2)  Mast cells: TNF- α

(3)  Endothelial cells: VCAM-1, IL-8

c)     Inflammatory mediators activate and sensitize nerves and keratinocytes produce neurotrophins that influence development, sprouting and survival of nerves

d)    Generally associated with blood vessels, specialized cutaneous receptors (tylotrich pads- knob like, hairless, small and serve as slow-adapting mechanoreceptors), Pacini corpuscles, Meissner corpuscles, Ruffini corpuscles, sebaceous glands, hair follicles, arrector pili muscles; free nerve endings also penetrate the epidermis

e)     Motor innervation of the skin is attributable to sympathetic fibers of the autonomic nervous system

f)      Cutaneous nerve trunks carry myelinated post gangliotic sympathetic fibers

g)     Dermatome – the area of skin supplied by one spinal nerve and its branches

a)    Overview of cutaneous sensation

i)      Definitions:

(1)  Adaptation – change in neuronal output to a constant sensory input

(2)  Allodynia – touch evoked pain

(3)  Alloknesis – touch evoked itch…itchy skin

(4)  End organ – specialized terminals of peripheral afferent nerves that transducer sensory stimuli into action potentials 

(5)  Epicritic itch – sharp and well localized itch

(6)  Hyperalgesia – increase sensitivity to pain

(7)  Hyperkinesis – increased sensitivity to pruritic stimulus

(8)  Mechanotransudction – conversion of force to a cellular signal

(9)  Neuropathic itch – itch resulting from neuronal damage

(10)                 Nociceptor – somatosensory neuron activated by noxious mechanical, thermal or chemical stimuli

(a)   Two unit categories

(i)    Aδ high-threshold mechanoreceptors with Aδ axons: classic pain receptors, respond to intense mechanical, thermal and chemical stimuli

(ii)  Polymodal nociceptor units with C axons: involved with hyperalgesia and pruritus

(11)                 Protopathic itch – poorly localized itch +/- burning quality

(12)                 Pruritoceptive itch – itch resulting from activation of peripheral nerves

(13)                 Pruriceptor – somatosensory neuron activated by pruritic stimuli

(14)                 Punctate hyperkinesis – skin prick inducing intense pruritus

(15)                 Psychogenic itch – itch resulting from OCD/other behavioral

(16)                 Rapid-adapting receptor – light-tough receptor, responds robustly at onset of sustained mechanical stimulus

(a)   3 types of rapidly adapting mechanoreceptor units are present in skin

(i)    Tylorich hairs: large primary follicle surrounded by a ring of neurovascular tissue, associated with a tylotrich pad

(ii)  Pacinian corpuscle units: extremely sensitive to small, high-frequency vibration and pressure, located close to sinus hairs, large layered, onion-like structure enclosing and afferent neuron

(iii) Meissner corpuscles: sensitive to skin vibration and touch, located in the superficial dermis, layers of cells enfold the large leaf-like ending of 2-6 afferent nerves

(17)                 Slow-adapting receptor – light-touch receptor, fires at low lever throughout a sustain mechanical stimulus

(a)   3 types of slow adapting mechanoreceptor units are present in skin

(i)    Merkel cell type I nerve endings: steady pressure elicits signaling, located in basal layer of epidermis, unmyelinated ending of afferent nerve

(ii)  Ruffini corpuscles with type II nerve endings: sensitive to skin stretch, located in the connective tissue of the dermis, large spindle-shaped structure that interconnects with the collagen matrix

(iii)  Tylorich pads: local area of epidermal thickening with a layer of highly vascular and well-innervated connective tissue below it

(18)                 Thermoreceptor – receptor for hot and cold

(a)   Cold units: excited by falling skin temperatures, C and A axons, nerve terminals are branches of a small myelinated axon ending in a small invagination in epidermal basal cells

(b)  Warm units: excited by rising skin temperatures, C and A axons

(19)                 Threshold – amount of stimulus needed to provoke response

ii)    In the hair skin of cats and dogs afferent nerve units are excited by hair movement

(1)  Guard and down hairs receive nerve terminations of the lanceolate type

(a)   Two major classes

(i)    Those excited by movement of large guard or tylotrich hairs (G and T hair units), Aß axons, activated by rapid movements

(ii)  Those excited by movement of all hairs but especially the find down hairs (D hair units), Aδ axons, activated by slow movements

(2)  The C mechanoreceptor, unmyelinated axon, encountered in the cat

(3)  Sinus hairs (vibrissae, whiskers)

(a)   Located on the muzzle, lips, eyelids, face, throat, palmar aspect of cat carpi

(b)  Long, thick, stiff and have an endothelium-lined blood sinus located between the external root sheath of the follicle and the outer connective tissue capsule

(c)   Function as slow-adapting mechanoreceptors

(4)  Autonomic nerves in the skin are primarily sympathetic neurons, few parasympathetic neurons present

(a)   Sympathetic nerves innervate blood vessels (noradrenalin and neuropeptide Y mediate vasoconstriction), arteriovenous anastomoses, lymphatic vessels, arrector pilli muscles, hair follicles, sweat glands

(b)  Parasympathetic nerves innervate blood vessels (acetylcholine and vasoactive intestinal peptide and peptide histidine methionine mediate vasoconstriction)

1)    Pruritus

a)     Itching – the unpleasant sensation that provokes the desire to scratch

b)    Most common symptom in vet dermatology

c)     Occasionally beneficial (ie. In removing parasites from the skin) but generally does more harm than good

d)    Removal of the epidermis abolishes pruritus

e)     Itch is processed in the prefrontal cortex, premotor areas, primary somatosensory cortex and the anterior cingulated cortex vs pain which is process in the primary and secondary somatosensory cortex (S1 and S2)

i)      Cats – histamine sensitive nerves in the spinothalamic tract (not sensitive to mechanical stimuli)

(1)  Note: not all mediators of itch react with histamine sensitive nerves

(2)  Cowhage (Mucuna pruriens) = pruritogen that induces itch without a flare

ii)    There are itch specific pathways in the PNS and CNS

f)      Histamine = potent biogenic amine, long recognized as a major cause of pruritus

i)      Histamine H₄ receptor agonists will produce a wheal and flare

g)     Other itch mediators (see table 1-14): kallikriens, proteases, leukotrienes, prostaglandins (produced by keratinocytes and mast cells)

i)      Both activate and sensitize nerves

h)    Increased levels of neurotropins (ex. nerve growth factor) cause sprouting and growth of epidermal nerve fibers

i)      Nerve growth factor concentration increased in humans with atopic dermatitis

i)      Acetylcholine and bradykinin induce pruritus in atopic skin but pain in healthy skin…neural sensitization likely plays a role in this

j)      Alkaline skin pH enhances serine protease activity and may increase the perception of pruritus

i)      Antibacterial shampoos often have high pH so may increase pruritus

k)    Central factors ex. Anxiety, boredom or competing cutaneous sensations with modulate pruritus via the gate control system (neuropeptides may play a role)

i)      Animals often itchy at night because other sensory output is low

1)    Subcutis (hypodermis)

a)     Mesenchymal origin, deepest and generally thickest layer of the skin

i)      Functions (1) energy reserve (2) thermogenesis and insulation (3) protective padding and support (4) maintenance of surface contours (5) steroid reservoir, steroid metabolism and estrogen production

b)    There is no subcutis in the lip, cheek, eyelid, external ear and anus thus, the dermis is in direct contact with the muscle/fascia

c)     Fibrous bands extending downward from fibrous structures in the dermis penetrate and lobulate the subcutaneous fat into lobules of lipocytes and form attachments for the skin to underlying fibrous skeletal components such as facial sheets and periosteum

d)    Superficial subcutis extends into the dermis via papillae adiposae which surround hair follicles, vasculature and sweat glands to assist in protection from trauma

e)     ~90% triglyceride by weight

f)      Many thin walled arterial and venous capillaries with or without veil cells, no lymphatics,

i)      Fat is very susceptible to damage and/or disease processes due to an absence of an efficient system for removing damaged tissue

g)     Slow circulation promotes lipogenesis, fast circulation promotes lipolysis

The Skin Immune System (SIS):

·       The skin immune system is very active and helps to defend the host against environmental insults but it can also cause harm through undesirable responses and/or autoimmunity

·       Innate (physical barriers, protective substances (mucus!), complement and phagocytic cells) vs adaptive immunity (highly specific/adaptive and has memory, evolves in response to antigen and leads to the activation of B- and T-Lymphocytes and subsequent production of antibody)

·       Toll like receptors (TLRs) – recognize molecular patterns and are found on macrophages, eosinophils, mast cells, dendritic cells and epithelial cells

·       Pathogen associated molecular patterns (PAMPs) – molecular patterns common to invading pathogens (recognized by TLRs) and include peptidoglycans, lipopolysaccharides, glucolipids and mannan-rich carbohydrates

1)    Innate immune responses in the skin

a)     The epidermal barrier

i)      Epidermis is the first defense against external injurious substances with the stratum corneum playing a big role because it represents a physical barrier that is hard to penetrate (overlapping layers of flattened corneocytes separated by lipid, continuous desquamation, presence of sodium chloride, albumin, complement, transferrin (iron binding protein; buildup of free iron can predispose to infection), interferons, lipids and antibodies)

ii)    Epidermis produces peptides with antibiotic functions

(1)  Defensins – cysteine rich proteins whose production is up regulated in response to inflammatory cytokines like IL-1 and TNF; compared to broad spectrum antibiotics because they can kill a variety of bacteria and fungi

(a)   Expression of canine β-defensin 103 has been shown to be down regulated in the skin of atopic dogs, possibly contributing to their higher incidence of pyoderma in atopic dermatitis

iii)   Many organisms do not need to breach the surface to cause harm

(1)  Fleas and ticks

(2)  S. pseudintermedius (gains entry into the canine hair follicle because the follicles do not have a lipid plug around their orifice; staphylococci are very good at adhering to the skin (via adhesins including teichoic acid, lipoteichoic acid, fibronectin, laminin and collagen; especially good at adhering to damaged skin…adherence of staphylococci is higher in atopic dogs) and M. pachydermatis (overgrowth leads to dermatitis and since it is a commensal, they body is not equipped to eradicate it; has evolved adherence mechanisms)

iv)   Epidermal barrier function is reduced in atopic dogs

a)     Innate attack mechanisms

·       Once the basic defense mechanisms fail…

·       Activated by infectious agents or parasite but also by some disease states (hypersensitivity and autoimmunity)

i)      Complement activation

(1)  A series of plasma proteins that exist in a quiescent state until activated and assist the immune system in eliminating pathogenic bacteria

(2)  3 pathways to activation:

(a)   The classical pathway

(i)    Activated by antigen-antibody complexes which bind to C1

(ii)  C4 and C2 then bind C1 forming the active enzyme C3 convertase

(iii) C3 convertase catalyzes the conversion of C3 to C3a and C3b

1.     C3a = a chemoattractant for neutrophils and can activate mast cells

2.     C3b = opsonizes bacteria and promotes phagocytosis as well as activating the membrane attack complex (a cylindrical structure composed of complements 4-9 which punches holes in the membranes of certain types of bacteria resulting in cell death)

(b)  The alternative pathway

(i)    Activated by direct interaction between the surface of a microbe and C3b (naturally created by slow, spontaneous hydrolysis of C3)

(ii)  Once C3b is bound to a microbe it catalyzes the further breakdown of C3 to C3b

(c)   The lectin pathway

(i)    Plasma lectin binds to a mannose residue on the surface of a microbe and activates C1 in the absence of antibodies and the classical pathway follows

(3)  Complement deficiencies lead to serious secondary health complications

(a)   C3 deficiency in Brittany Spaniels leads to increased susceptibility to pyogenic infections

(b)  Deficiencies in complement that make up the MAC do not seem to affect susceptibility to Staph or Malassezia infections (in humans will lead to higher rates of Nisseria infections which is a major cause of meningitis)

(c)   C3 may play a role in canine pemphigus foliaceus (detected by IHC in many cases)

i)      Recruitment and activation of phagocytic cells

(1)  In order to do their job they must first be called to the sites of infection

(2)  Resident tissue macrophages detect microorganisms and secrete proinflammatory cytokines IL-1 and TNF-α.

(a)   Injection of TNF-α into canine skin upregulate E- and P-selectins in blood vessel walls

(b)  IL-8 = potent neutrophils attractant in dogs

(3)  Selectins and integrins (adhesion molecules) are upregulated on vessel walls and bind to ligands on inflammatory cells

(a)   E-selectin binds to carbohydrate ligands on the phagocytic cell surface with low affinity allowing it to slow down and begin to roll along the endothelium

(b)  ICAM-1 and VCAM-1 (high affinity integrins) interact with their corresponding ligands (leukocyte function associated antigen 1 (LFA-1 or CD11a/CD18) and macrophage activation complex 1 (MAC-1 of CD11b/CD18)) which results in firm binding and allows the cells to stop

(4)  Once their flow is halted phagocytic cells migrate through endothelial cells and travel along the chemokine gradient towards the infection

(5)  At the infection site the phagocytic cells must be activated by recognition of extracellular microbes

(a)   Mannose and C-type lectin-like receptors allow macrophages to recognize sugar residues on microbe surface

(b)  Dectin receptors recognize glucans present in fungal cell walls

(c)   Scavenger receptors bind oxidized or acetylated low-density lipoproteins and other microbes

(d)  Opsonin receptors (FCγR1) recognized coated (with IgG or CR1 [type 1 complement receptor]) microbes

(e)   TLRs (at least 10 types!) respond to various pathogens, recognizing PAMPs; once bound initiate a physiologic cascade leading to inflammation

(f)    7 transmembrane receptors (NOD-like receptors, RIG-like receptors, N-formyl-methionyl receptor) recognize bacterial proteins or viral RNA and activate phagocytic cells

(6)  Once the phagocytic cell is attached, phagocytosis can commence and the microbe is engulfed into an intracellular vesicle (phagosome)

(7)  The phagosome is fused with a lysosome to form the phagolysosome

(8)  There are many killing methods

(a)   Neutrophils: reactive oxygen intermediates, elastase

(b)  Macrophages: reactive oxygen intermediates and reactive nitrogen intermediates (produce highly toxic peroxynitrite radicals when combine with H₂O₂, elastase; also produce cytokines to recruit and activate leukocytes

(9)  Allergens may activate some of these inflammatory pathways…

i)      Recruitment of eosinophils

(1)  Very involved in ectoparasite infections but also hypersensitivity and autoimmune reactions and are both phagocytic and secretory

(2)  Attracted by histamine, complement 5a, leukotriene B4, parasite extracts and other chemokines

(3)  Possess cell surface receptors: Fc receptors for IgG and low affinity IgE receptors

(4)  Secretory function is VIP and their granules contain many important proteins (major basic protein, eosinophil cationic protein, eosinophil peroxidase and lysosomal proteins) that are toxic to helminths, cells and bacteria and can cause mast cell degranulation

(5)  Possess RANTES receptor and CD11/CD18

Eosinophils recruitment is more common in cats

i)      Activation of lymphocytes and NK cells

(1)  Intraepithelial lymphocytes (T-cells that contain antigen receptors or limited diversity; example - γδ receptor

(a)   Can act as NK-cells and recognize/kill virus infected cells or microbes directly

(b)  Act as sentinels at the epithelial surface and secrete cytokines, activate phagocytic cells and kill infected cells

i)      Activation of mast cells

(1)  Normal residents of connective tissue and found in highest [ ] in interface regions that interact with the environment (skin, lungs. GI)

(2)  Important in the innate defense against helminths (ex. Nematodes in the GI), bacteria and participate in wound healing

(a)   Release large numbers of granule proteases

(b)  Play an important role in certain types of bacterial infections (ex. Septic peritonitis or bacterial pneumonia) because they produce TNF-α and recruit neutrophils

(c)   Wound healing: attracted by TGF-β and monocyte chemoattractant protein 1; mast cell derived TNF-α and TGF-β promote fibroblast proliferation; can activate fibroblasts, promote collagen synthesis and activate enzyme gelatinase A (involved in matrix remodeling)

(3)  Mast cell granule proteases also regulate the influx of inflammatory cells into the skin via hymase and tryptase (cell recruiters) and the upregulation of VCAM-1 and E-selectin

i)      Production of cytokines and chemokines

(1)  TNF-α and IL-1 are important in initiating the immune response

(a)   TNF-α produced by macrophages, activated T-cells, NK cells and mast cells

(b)  IL-1 produced by macrophages, endothelial cells and keratinocytes

(2)  IL-12 is important in the innate immune response and is produced by macrophages and dendritic cells through its effect on T-cell differentiation, IFN-γ production, and ability to activate NK cells and T-cells

(3)  Type 1 interferons (IFN-α and IFN-β) produced by monocytes and fibroblasts act against viral infections 

1)    Adaptive immune responses

·       Required to deal with many types of infections

·       When active, a continuous and circular process

a)     Antigens and antigen recognition

i)      Antigens that trigger IgE = allergens

ii)    Antigens that trigger autoimmune disease = autoantigens

iii)   For the immune response to occur an antigen must first be recognized and there are two types of receptors

(1)  Antibodies: present on the surface of B-lymphocytes and recognize the antigen in its 3D native form by detecting a specific, highly immunogenic, part of the whole (immunodominant epitope)

(2)  T-Cell receptors: present of the surface of T-lymphocytes and require the antigen to be processed by an antigen presenting cell which has broken down the antigen into peptides of about 8-12 amino acids long (use a variety of enzymes for processing including cathepsin E)

i)      The huge diversity in T-cell and B-cell arrangements (generated by gene rearrangements) allows the adaptive immune response to work

(1)  As each lymphocyte is formed, it expresses a unique receptor capable of recognizing a distinct antigenic epitope

(a)   The body has “pre-formed” antibody receptors to uncountable numbers of proteins, many of which it has never encountered

(b)  This fact also means autoreactive lymphocytes will be generated but these are normally well controlled and autoimmune disease does not present BUT when normal control mechanisms are circumvented autoimmune disease can occur

(c)   The body must distinguish between “self” and “non-self”

(i)    Some host antigens are naturally hidden (Eye, CNS, testes) and lymphocytes never come in contact with them and thus remain ignorant

(ii)  Lymphocytes that develop in the bone marrow or thymus are screened and those that react with host antigens are killed by apoptosis in a mechanism called clonal deletion. This process is known as central tolerance

(iii) If autoreactive lymphocytes do enter peripheral circulation, peripheral tolerance may kick in and the cells are directed to undergo apoptosis by tolerogenic dendritic cells or they become anergic (cells do not receive the appropriate secondary signals when interacting with antigen presenting cells and will not be activated

(iv) Regulatory T-cells that secrete IL-10 and TGF-β control autoreactive lymphocytes

i)      Another theory of immune function posits that rather than paying attention to foreignness, the immune system instead monitors for damage (alarm signals from injured cells)

(1)  This theory helps to explain the lack of the immune system of response to the fetus or to tumors, which contain many novel proteins (foreign!)

ii)    There are three types of antigens encountered by the skin

(1)  Antigens from parasites or infectious agents (ex. Flea saliva, S. intermedius [staphage lysate vaccine…], Malassezia hypersensitivity and environmental allergens [Dermatophagoides farinae (generally thought to be a chitinase based allergen, Derf15) or Dermatophagoides pteronnyssinus).

(a)   Different IgG subclasses recognize antigens/proteins of different molecular weight classes

(2)  Allergens able to trigger allergic inflammation

(3)  Autoantigens that are targeted in autoimmune diseases (ex. Desmogleins 1 and 3, desmocollin 1, plakin, various BM proteins and nuclear proteins)

a)     Antigen presentation

·       The most efficient immune response is generated by stimulation of T-cells which are stimulated by a professional antigen presenting cell (in the skin: Langerhans cells [epidermal] and dendritic cells [dermal])

i)      The MHC

(1)  Antigen presenting cells process antigens into smaller peptides within their phagolysosomes and then attach them to a MHC protein which then transport the peptides to the cell surface and present them to a T-cell

(2)  The MHC gene group encodes several proteins

(a)   Class I genes code for proteins found on the surface of most nucleated cells

(b)  Class II genes code for proteins found on macrophages or lymphocytes

(c)   Class III genes code for a variety of inflammatory molecules including complement, TNF and heat shock proteins

(3)  Only Class I and II are involved in antigen processing/presentation

(4)  Exogenous antigen peptides (small and sausage shaped) are placed in the groove of an MHC II and then this complex is moved to the surface of the antigen presenting cell where it is ready to interact with a T-cell

(5)  Endogenous antigen peptides (self or viral particles from a virus infected cell) the peptides are couples and placed within the groove if an MHC I which are then expressed on the cell surface

(6)  Certain types of MHC molecules can predispose for autoimmune disease (ex. SLE more common in dogs with a DLA-A7 MHC haplotype

ii)    Antigen presentation to T-cells

(1)  Once the peptide is within the MHC groove and is expressed on the cell surface it can now be presented to a T lymphocyte

(2)  There are several types of T lymphocytes

(a)   Helper T cell = possess surface CD4 which is the receptor for MHC II

(b)  Cytotoxic T cell = possess surface CD8 which is the receptor for MHC I

(3)  All T cell receptors have CD3 and this can be used to identify them in IHC

(4)  Superantigens = can stimulate T cells directly by crosslinking MHC molecules and T cell receptors at a site away from the peptide binding groove

(a)   Activate the lymphocyte and can lead to cytotoxic production and an inflammatory response

Examples: exotoxins from S. aureus or staph enterotoxins

a)     Activation of T-cells

·       The steps above as well as a “second signal” are required for T cell activation

·       Second signals consist of a series of molecular interactions between adhesion molecules on the two cells and specific receptor ligand interactions as well as cytokine signaling

·       Without second signals anergy occurs

·       Second signals and MHC/T cell binding lead to signal transduction mechanisms within the T cells (G proteins, tyrosine kinase and mitogen activated protein (MAP) kinases) which lead to T cell activation, proliferation and cytokine production

o   Cytotoxic T cells (CD8+) will release perforins, cytotoxic enzymes and other cytokines (TNF-α) which are capable of killing a target cell via cell-mediated cytotoxicity (generally used to eliminate virus infected cells)

o   Helper T cells (CD4+) will synthesize various cytokines based on T cell subtype

i)      Helper T-cell responses

(1)  Two types: all are initially Th0 but become either Th1 or Th2 based on signals they receive from the antigen presenting cell

(a)   T helper 1 cells (Th1) – produce IL-2 (stimulates T-cell proliferation and infiltration of allergen specific T cells into sites of inflammation) and IFN-γ (activates macrophages and inhibits Th2 cytokine release while encourage Th1 response); stimulated by IL-12 production by the antigen presenting cell or IFN-γ

(b)  T helper 2 cells (Th2) – produce IL-3 (a mast cells and basophil growth factor), IL-4 (causes B cell class switch from IgG to IgE leading to allergic type inflammation), IL-5 (stimulates eosinophils proliferation and IgA secretion), IL-6 (generally proinflammatory), IL-10 (inhibits IL-2 secretion and drives Th2 response) and IL-13; stimulated by IL-4 production by the antigen presenting cell or prostaglandin E₂ from a parasite or IL-10

(c)   In general, Th1 responses promote cell-mediated immunity and IgG production by B cells and Th2 responses promote IgE production and the allergic response

(2)  Once a response tends towards a certain subtype it generally stays that way because cytokines on one side inhibit the development of the opposite subtype

(3)  CD4 Th1 and Th2 responses and their associated cytokine production represents a paradigm for the development of allergic reactions to environmental allergens

(a)   Atopic humans have Th2 polarization in the skin and blood

(b)  Human AD patients with eczematous lesions have high IFN-γ expression

(c)   In AD sequential activation of the Th2 cell subset during the initial of an atopic lesion followed by the Th1 subset may contribute to the persistent/chronic inflammatory response

(d)  Study 1: In dogs a Th2-types cytokine pattern (IL-4 and IL-5)was seen in ¼ of atopic samples and a Th1-type (IL-12) cytokine pattern was seen in ¼ of healthy samples

(e)   Study 2: overexpression of IL-4 (Th2) and underexpression of TGF-β was seen in cAD and lesional atopic skin had higher levels of type 1 cytokines (IL-2, IFN-γ and TNF-α)

(f)    Study 3: IL-5 and IL-4 (Th2) mRNA expression was higher in cAD 

(g)   Study 4: Th2 polarization in experimental cAD and higher levels of TNF-α and IL-8 were detected in experimental cAD; once sensitized and then challenged, dogs with cAD expressed higher levels of IL-2 (Th1) and lower levels of IL-6 and TNF-α

(h)  Study 5: IL-6 played a role in early reactions followed by an increase in Il-13/thymus and activation related chemokine (TARC) and late increase in IL-18

(i)    Overall: Th2 may predominate AD but both subsets are important in its pathogenesis and may contribute to different stages of the disease with an initial Th2 response followed by a late and chronic Th1 response

i)      B-cell activation and antibody production

(1)  An antibody response is often required to help eliminate microorganisms or neutralize toxins and B-cells activation must occur to produce antibodies

(2)  Multiple ways to activate:

(a)   B-cells have direct contact with a antigen through IgM or IgD on their cell surface which activates them and stimulates proliferation of that specific B-cell/antibody type

(b)  The B-cell response is enhanced if the antigens are recognized in association with complement

(c)   B-1 cells are pre-programmed to produce IgM against common bacterial polysaccharides and lipids, can produce antibody without the help of T cells

(d)  B cell interaction with a Th cell where the B cell acts as an antigen presenting cell and then the T cell reciprocates by producing cytokines to activate the B cell and stimulate it to produce antibodies

(i)    If a B cell interacts with a Th2, IL-4 will instruct the B cell to produce IgE instead of IgG

(1)  Antibodies have many functions

(a)   Neutralize antigens, viruses and toxins

(b)  Opsonize organisms and bind to Fc receptors causing phagocytosis

(c)   Immune complex formation which will activate complement and lead to phagocytosis of C3b coated bacteria

(d)  Activate complement

(e)   If on the surface of a B cell, can activate it directly to produce more antibody

(f)    Antigen presentation to T cells to stimulate a helper response

(2)  In nearly all immune response IgG will be produced alongside other classes

(3)  Formation of autoantibodies: genetic (MHC subtype, cytokines, T cell factors), infectious, environmental (UVL - lupus) and hormonal (sex predispositions) factors

(4)  Autoimmune disease can develop in a number of ways

(a)   Failure of self-tolerance

(b)  Polyclonal B cell activation by certain viruses and parasites

(c)   Failure of regulatory T cell function

(d)  Activation of T cells by superantigens

(e)   MHC II expression on normal cells following damage allowing recognition by T cells

(f)    Exposure of previously hidden antigens

(g)   Cross reactivity between organisms and self-antigens (molecular mimicry)

(h)  Viruses or lymphoid neoplasia interfering with normal immune function

i)      Inflammation: recruitment of leukocytes and plasma proteins into tissues where they are activated; redness, swelling, heat, and pain

Cutaneous Ecology:

1)    Skin is the protective barrier from the world with physical, chemical and microbial defenses

a)     Physical defenses include the hair (minimizes contact with the skin and harbors microorganisms) and the stratum corneum (thick, tightly packed, keratinized permeated by an emulsion of sebum, sweat and epidermal lipids)

b)    Chemical defenses include the lipid emulsion (sodium chloride, antiviral glycoprotein interferons, albumin, transferrin, complement (C3), glucocorticoid and immunoglobulins (IgG & IgM [in interstitial spaces in the dermis, dermal blood vessels and hair papillae] IgM [BMZ of epidermis, hair follicles and sebaceous glands] and IgA [epitrichial sweat glands]), free fatty acids produced by resident flora) and peptides (cathelicidins and β-defensins – also function in regulating cell proliferation, ECM production and cellular immune responses)

i)      Secretory component (the polymeric immunoglobulin receptor) is expressed and synthesized by keratinocytes and can interact with IgM and IgA; plays a role in skin immunity

c)  Microbial defenses

b)    Microbial defenses: resident flora which may include bacteria and less often yeast and filamentous fungi are located at the skin surface and the superficial layers of the epidermis (especially the SC and the infundibulum of hair follicles)

i)      Skin flora changes with the skin environment: pH, salinity, moisture, albumin level and fatty acid level

ii)    Skin flora acts to inhibit the colonization of invading microorganisms by occupying and out competing for microbial niches

iii)   Interactions between cutaneous microbes:

(1)  Unilateral and reciprocal antagonism (interference) – production of growth conditions by one organism that is unfavorable to another via nutrient consumption, pH, occupation of tissue receptors or production of inhibitors

(2)  Reciprocal enhancement (synergism) – mutrients made available by one organism allow cross-feeding by others; ex. S. pseudintermedius and M. pachydermitis

(3)  Neutral association

iv)   Residents = permanent flora, may be reduced in number but not eliminated by degerming methods

v)    Transients = contaminants from the environment, easily removed by hygienic measures

vi)   Nomads = between the two, able to colonize and reproduce for short periods of time

i)      Controversy remains as to the resident flora of dogs and cats

(1)  Cats: Micrococcus spp., coagulase negative staph. (S. simulans), α-hemolytic strep. and Acinetobacter spp. are normal residents; cats can be asymptomatic carriers of M. canis

(2)  Dogs: Micrococcus spp., coagulase negative (often positive too) staph. (S. schleferi, S. epidermidis, S. xylosus, S. schleferi coagulans and S. pseudintermedius [may actually be a resident of mucus membranes and spread to the skin via grooming]), α-hemolytic strep. and Acinetobacter spp. are normal residents as well as Clostridium spp.

1)    The pH of the skin plays a role in its antimicrobial activity (pH is affected by lactic acid in sweat, ammonia in sweat and amino acids), inflammation shifts from acid/neutral to alkaline

2)    The degree of hydration of the SC plays the biggest role on cutaneous ecology with increased water content (increased temp, humidity or occlusion) increases microorganism number

a)     The SC can increase in thickness by 100% by taking up water, low in cysteine

b)    Transepidermal water loss (TEWL) = water vapor evaporating from the skin and a reflection on the integrity of the SC

i)      In normal skin TEWL and hydration has a direct relationship

ii)    In pathologic skin this relationship is inverse

c)     In normal dogs the relative humidity of the coat was 50%  overall but 70% on the ventral neck and below the tail

Senilty:

1)    Aging occurs through exogenous wear and tear but also genetically predetermined programs

2)    There are many changes to senile skin

a)     Epidermal atrophy (decreased adherence of corneocytes, flattening of the dermo-epidermal junction

b)    Decrease Langerhans cells and melanocytes

c)     Dermal atrophy (relatively acellular and avascular) and altered dermal collagen, elastin and glycosaminoglycans

d)    Atrophy of the subcutis

e)     Less eccrine and apocrine glands and their secretions and reduced sebaceous secretion (dry skin, dull haircoat)

f)      Thinned and ridges nail plates (malformed and brittle claws, hyperkeratotic pads)

g)     Decreased growth of the epidermis, hair and nails

h)    Reduction of hair follicle density (alopecia)

i)      Delayed wound healing (increased wound infections)

j)      Reduced dermal clearance of fluids and foreign material

k)    Compromised vascular responsiveness

l)      Reduced sensory perception

m)   Reduced vitamin D production

n)    Impairment of immune and inflammatory functions