Adhesion Molecules (including diseases) Flashcards
1
Q
What is the function of the BMZ?
A
Adhere the epidermis to the dermis
2
Q
What stains can be used to highlight the BMZ?
A
PAS - purplish
3
Q
What is the basement membrane?
A
The thin line seen with light electron microscopy between the epidermis and the dermis
4
Q
What is the basal lamina?
A
Dense sheet-like structure seen with electron microscopy which is secreted by epidermal keratinocytes
Traditionally composed of lamina lucida and lamina densa
5
Q
What is the basement membrane zone?
A
The complex structure which can be seen with electron microscopy and spans the space between the base of the basal keratinocytes to the underlying dermal matrix.
Consists of:
- Intermediate filaments
- Hemidesmosomes
- Anchoring filaments
- Lamina densa
- Anchoring fibrils
6
Q
What are the four levels that the BMZ can be divided into when viewed via electron microscopy?
A
Basal keratinocytes
Lamina lucida (artifact of processing)
Lamina densa
Sublamina densa/superficial papillary dermis
7
Q
What is the lamina lucida?
A
Traditionally one of the layers of the basal lamina but is really an artifact caused by dehydration fixation for electron microscopy - is not present when processed with chemical fixation
More historical than scientific significance
Contains extracellular domains of Col XVII, a6b4 integrin and CD151 as well as anchoring filaments (laminin-332)
8
Q
What are the functions of the cytoskeleton within the basal keratinocytes?
A
- Structural support
- Cell movement
- Intercellular transport
- Anchor cells
9
Q
What types of filaments compose the cytoskeleton within the basal keratinocytes?
A
1) Microfilaments (7nm diameter)
2) ** Intermediate filaments (10 nm diameter)
- most important for the BMZ
3) Microtubules (25 nm diameter)
10
Q
What are the molecules that keratin intermediate filaments bind to in the BMZ?
A
- Plectin
- BPAG1e
Both part of the plakin family
11
Q
Describe the structure of a hemidesmosome.
A
Two plaque structure (inner and outer) within basal keratinocytes
Composed of at least 5 distinct molecules
- BPAG1e (inner)
- Plectin (inner)
- α6β4 integrin (outer, extends into lamina lucida - transmembrane)
- collagen XVII (outer, extends into lamina lucida -transmembrane)
- CD151
12
Q
What is BPAG1e aka BP230?
A
230 kDa, found in the inner plaque of hemidesmosomes
Dumb-bell shaped with 3 domains:
Belongs to plankin family
bindings to b4-subunit of a6b4 integrin, intermediate filaments, COL XVII
Functions:
- structural integrity
- signaling and regulation of cell polarity and migration
- not required for hemidesmosome/BMZ assembly
- no associated genetic disease?
13
Q
What is plectin?
A
450-533 kDa, found in the inner plaque of hemidesmosomes
Belongs to the plankin family
Has 4 domains
Bind to keratin intermediate filaments and β4 of α6β4
Interacts with all keratinocyte cytoskeletal proteins
Functions:
- Structural integrity
- Scaffold for signaling molecules
14
Q
What is α6β4 integrin?
A
Transmembrane protein
Found in the outer plaque and extends into lamina lucida
a3b1 also exists and is found elsewhere, not big in the BMZ
Alpha chain has 6 domains, shorter, small intracytoplasmic portion
*multiple calcium binding sites
- binds collagen XVII and CD151
Beta chain has 7 domains, longer
- binds to laminin-332, intermediate filaments, BPAG1e, collagen XVII, and plectin
Functions of integrins:
- Structural integrity
- Epidermal adhesion, proliferation, and differentiation
- B1 subunit is important for hair growth and wound healing
15
Q
What is collagen XVII aka BP180?
A
Transmembrane protein
180 kDa, outer plaque extending into lamina lucida and lamina densa
Is cleaved extracellularly into 120 kDa LAD-1 and 97 kDa LABD97 which are associated with linear IgA bullous dermatosis
Has 4 domains which can be visualized as a globular head, rigid central rod region and flexible tail
- intracellular aspect (head) binds to plectin, BPAG1e, and a6b4 integrin (b4)
-extracellular NC16A domain (tail) binds to α6β4 integrin (α6) and laminin-332
Functions:
- structural integrity
- basal cell migration and adhesion
- enamel formation
16
Q
What is CD151?
A
DO NOT WORRY ABOUT THIS TOO MUCH - NOT IN BIZIKOVA’S LECTURE
29-32 kDA, found in outer plaque of hemidesmosome
6 functional domains
- amino and carboxyl ends are both found within the basal keratinocyte membrane
- long extracellular loop which interacts with α6β4 integrin (a6)
Will cluster and interact with each other and integrins to form a tetraspanin web
17
Q
What are anchoring filaments?
A
~800 nm wide structures seen within the lamina lucida on electron microscopy
-composition is controversial but may include laminin-332** (believed to be the main component) and Col XVII +/- a6b4 integrin, CD151, or laminin-311
18
Q
Describe laminin-332 (old name laminin-5)
A
410 kDa
Large glycoproteins composed of 3 chains (a, b, y) connected with disulfide bonds - name come from the numbers associated with the chains
Forms a t shape
Secreted by keratinocytes as a precursor that then assembles
alpha 3 arm binds α6β4 integrin
beta 3 arm binds Col VII and other laminins
gamma 2 chain binds Col IV, Col VII, Col XVII and perlecan
Other laminins (511 and 311) are also found in the BMZ but this is considered the prototype
Functions:
- structural integrity
- embryogenesis
- tissue morphogenesis
- regulation of proliferation and differentiation
- tumorigenesis
19
Q
What is the lamina densa?
A
The electron dense zone seen on electron microscopy below the lamina lucida
Contains collagen IV, laminin-332, nidogen, and perlecan
Also contains portions of a6b4 integrin
Is made of lattices of collagen IV molecules (majority) and others of laminin-332 molecules
20
Q
What is collagen IV?
A
Made in the rough ER and secreted into the basement membrane
Resembles pro-collagen because has amino and carboxyl termina
Triple helical structure with 3 domains (like all collagens)
Self assembles into 3 dimensional hexagonal lattice structures of tetramers
There are a variety of formations due to the alpha chains which can be found in various places, one variant found just in the glomerulus
Within the lamina densa
Complex binding with other BMZ components (laminin-332, perlecan, nidogen, fibronectin, collagen VII)
Functions:
- structural integrity
- tumorigenesis
21
Q
Describe nidogen 1 and 2?
A
1500-200 kDa, found in the lamina densa
Sulfated monomeric glycoprotein with 4 domains, dumb-bell shaped
Within the lamina densa
- One end binds Col IV and perlecan
- Other end binds laminin 311
Functions:
- structural integrity
- embryogenesis
- tumorigenesis
22
Q
Describe perlecan
A
A heparan sulfate proteoglycan found in the lamina densa
680-770 kDa
Central core has 5 domains
- bind to nidogen, collagen IV and other less common molecules
Functions:
- negative charge for the basement mebrane
- structural integrity
- permeability barrier
- regulation of angiogenesis, etc
- cellular adhesion
- epidermal morphogenesis
- tumorigenesis
23
Q
Describe the anchoring fibrils
A
Found in the sublamina densa/superficial papillary dermis
Semi-circular loops of collagen VII which resemble Velcro
The lattices of the lamina densa weave through them
24
Q
Describe collagen VII
A
290 kDA triple helical molecules, found in the lamina densa and sublamina densa
Makes up anchoring fibrils
Has 3 main domains
Self-assembles into antiparallel dimers with carboxyl terminal ends touching (cystine residues creating disulfide bonds)
- this NC2 domain is cleaved by BP1 during creation of the anchoring fibrils
NC1 domains(amino terminal) interacts with Laminin-332 and Collagen IV
Function: loops around dermal collagen
25
What are the functions of intermediate filaments?
- Structural and mechanical cellular integrity
- Organization of cytoplasmic architecture
- Intracellular signaling
- Regulation of transcription
26
What are the congenital diseases associated with intermediate keratin filaments?
Epidermolysis bullosa simplex (EBS)
- KRT5 in cattle
- KRT14 in cats
27
What are the auto-immune diseases associated with intermediate keratin filaments?
Not confirmed in veterinary medicine
28
Which keratin intermediate filaments are associated with the BMZ?
K5, K14
Coded by genes KRT5 and KRT14
29
What congenital diseases are associated with plectin?
Epidermolysis bullosa simplex
- Eurasier dog
- one foal with signs of EBS and laminitis
30
What is epidermolysis bullosa simplex in dogs?
Congenital autosomal recessive mutation in PLEC (plectin)
- reported in Eurasier dogs
- clinical signs by 20 days of age
- blistering/ulcers on haired skin, footpads (sloughing), and oral cavity
- most are euthanized
*UPenn can test for it
31
What autoimmune diseases are associated with plectin?
Not confirmed in veterinary medicine
32
What congenital diseases are associated with BPAG1e?
None confirmed in veterinary medicine
33
What autoimmune diseases are associated with BPAG1e?
- Bullous pemphigoid, rare, dogs
- Mucous membrane pemphigoid, rare, dogs
34
What congenital diseases are associated with a6b4 integrin?
Junctional epidermolysis bullosa (JEB)
- ITB4 in cattle and sheep
35
What autoimmune diseases are associated with a6b4 integrin?
Not confirmed in veterinary medicine
36
What congenital diseases are associated with collagen XVII?
Junctional epidermolysis bullosa in two cats
37
What autoimmune diseases are associated with collagen XVII?
- Bullous pemphigoid (major target = NC16A domain) in dog, horse, cat, pig
- Mucous membrane pemphigoid in dogs, cat
- Linear IgA bullous diseases in dogs (due to LAD-1)
38
What congenital diseases are associated with laminin 332?
Junctional epidermolysis bullosa (JEB)
- LAMA3 in dog, horse, and cow
- LAMB3 in a dog
- LAMC2 in draft horses, cow and a sheep
39
What autoimmune diseases are associated with laminin 332?
- Mucous membrane pemphigoid (dog, cat)
- Aquired JEB (dogs)
- Mixed subepidermal blistering skin disease (dogs along with VII)
40
What congenital diseases are associated with collagen IV?
COL4A5 in a Samoyed (Alport syndrome, early onset renal failure)
COL4A4 in an English Cocker (autosomal recessive nephropathy)
41
What autoimmune diseases are associated with collagen IV?
Not recognized in veterinary medicine
42
What diseases are associated with nidogens?
Congenital:
- not recognized in veterinary medicine
Autoimmune:
- not recognized in veterinary medicine
43
What diseases are associated with perlecan?
Congenital:
- not recognized in veterinary medicine
Autoimmune:
- not recognized in veterinary medicine
44
What congenital diseases are associated with collagen VII?
Dystrophic epidermolysis bullosa (cattle and dogs)
- retrievers have a mild variant
45
What autoimmune diseases are associated with collagen VII?
- Epidermolysis bullosa acquistita (dog)
- Type I bullous systemic lupus erythematosus (one dog)
46
What genes are associated with the intermediate filaments in the BMZ?
KRT5, KRT14
47
What gene is associated with plectin?
PLEC1
48
What gene is associated with BPAG1e
BPAG1 (DST)
49
What genes are associated a6b4 integrin?
ITGA6, IGB4
50
What gene is associated with collagen XVII?
COL17A1
51
What genes are associated with laminin-332?
LAMA3, LAMB3, LAMC2
52
What genes are associated with collagen IV?
COL4A1 to COL4A6 (6 different alpha chains)
53
What genes are associated with nidogens?
NID1, NID2
54
What gene is associated with perlecan?
HSPG2
55
What gene is associated with Collagen VII?
COL7A1
56
What species has MMP been reported in and what is the age predilection?
Dogs (most common AISBD), human, cat
Breed predilection: GSD
Age predisposition: middle-aged
57
What are the clinical signs associated with MMP?
Distribution: mucocutaneous junctions, mucosa, infrequent haired skin
Lesions: tense vesicles (rare), deep erosions/ulcers, infrequent scarring
58
Which are the antigens are associated with mucous membrane pemphigus?
Immunologically heterogenous
Major antigen: Collagen XVII
Minor antigens: laminin-332, BPAG1e/BP230
59
What are the histopathologic findings associated with mucous membrane pemphigoid?
Level of split: lamina lucida
Histopathology: subepidermal vesiculation +/- minimal inflammation in vesicle
Variable non-inflammatory to perivascular dermal inflammation
May have lichenoid pattern because it is a mucocutaneous junction
Superficial dermal fibrosis
PAS or col IV stain on the dermal side
60
What is the immunopathology staining pattern of MMP?
IgG >C3, IgM, IgA at basement membrane
- higher than reported in ppl, maybe because it has been needed for publications
Salt split staining can be epidermal (most cases), dermal or both sides
61
What is the treatment of MMP?
No spontaneous remission
Many achieve remission, may flare
Monotherapy or combined
- Prednisone/lone
- tetracycline or doxycycline and niacinamide
- ciclosporin
- azathioprine
- chlorambucil
- mycophenolate mofetil
- dapsone
Monotherapy with glucocorticoids reported as least successful, tetracyclines as most
1/4 cats was able to have drug withdrawn
62
Which species has epidermolysis bullosa acquisitia (EBA) been reported in and at what age does it typically occur?
Dogs with Great danes being predisposed (over 1/2)
- Young, almost half of dogs reported developed lesions by 1 yr of age
*may be a genetic predisposition
Other species: human
- a childhood form is reported but mostly occurs in older adults
63
What are the clinical signs associated with epidermolysis bullosa acquisitia (EBA)?
Skin lesions: erythematous macules and papules; tense vesicles; deep erosions/ulcers
- often have pain and sometimes pruritus
Distribution: haired skin, footpads, and mucosae/mucocutaneous junctions
- footpad sloughing is frequent
Usually have fever, lethargy, lymphadenopathy and anorexia
Most would fall into "BP-like EBA variant" in the human naming system
64
What are the immunopathology associated with epidermolysis bullosa acquisitia (EBA)?
Major antigen: Collagen VII (NC1 domain)
Minor antigens: nd
IgG
65
What are the histopathologic findings associated with epidermolysis bullosa aquisita (EBA)?
Level of split: sublamina densa
Histopathology:
- subepidermal vesiculation with no to variable amounts of neutrophilic inflammation +/- fibrin or minor hemorrhage
- vacuolation and rowing of neutrophils and/or histiocytes occurs just below the BMZ
- Superficial dermal perivascular to interstitial dermatitis
66
What is the treatment for epidermolysis bullosa acquisitia (EBA)?
No spontaneous remission
Some may be euthanized (more so in the past)
Current success in 2-3 months with immunosuppression
- oral glucocorticoids
- various non-steroidal immunosuppressants (azathioprine, mycophenolate mofetil, etc.)
- colchicine is used in people and was successful for some dogs
Almost half of the dogs were able to discontinue medications
67
What species has bullous pemphigoid been reported in and what is the age predisposition?
Dog breed disposition: nd but maybe dachshund and GSD
Other species: human, cat, horse, (Yucatan) pig, possibly a macaque
Relatively rare
May be more mild in cats
May be worse in horses
Age predisposition: middle-age
68
What are the clinical lesions associated with bullous pemphigoid?
Skin lesions: erythematous macules and papules; tense vesicles; deep erosions/ulcers
Distribution: haired skin esp in pressure points but (footpads are rarely affected) and mucosae/mucocutaneous junctions
Usually healthy except horses are usually systemically unwell
69
What are the antigens associated with bullous pemphigoid?
Major antigen: Collagen XVII
Minor antigens: BPAG1e/BP230
70
What is the immunopathology of bullous pemphigoid?
Tissue-bound IgG and IgM autoantibodies along the BMZ
Circulating IgG, predominantly IgG1
Circulating IgE targeting BMZ (60%)
71
What are the histopathologic findings associated with bullous pemphigoid?
Level of split: lamina lucida
Histopathology: subepidermal vesiculation +/- eos, neuts, fibrin
- Eos in the superficial dermis
- IgG, IgM, or IgA and/or complement deposited at the BMZ
72
What is the treatment for bullous pemphigoid?
Spontaneous remission reported in 1/7 dogs
Immunosuppression induced complete remission
- glucocorticoids
- oclacitinib
- pred + azathioprine
- pred + pentoxifylline
- doxycycline and niacinamide
40% of dogs were able to stop medications
*most horses were euthanized due to poor response or side effects of medications
73
What species are reported to have junctional epidermolysis bullosa aquisita (JEBA) and what age is predisposed?
Dog breed disposition: nd (rare and only described in 5 dogs)
Other species: single human case with same features
Age predisposition: nd (6mo to 8yr, median 2.5)
74
What are the clinical signs associated with junctional EBA?
Skin lesions: erythema, vesicles, deep erosions/ulcers
Distribution: haired skin (pinnae, axillae/abdomen, footpads) and mucosae/mucocutaneous junctions
- footpad sloughing is frequent
May or may not be systemically ill
75
What is the immunopathology of Junctional EBA?
Major antigen: Laminin-332
Minor antigens: nd
IgG> C3, IgA
76
What is the histopathology associated with Junctional EBA?
Level of split: lamina lucida
Histopathology: subepidermal vesiculation +/-neutrophilic inflammation +/- eos
- potential superficial dermal perivascular to interstitial inflammation
77
How do you treat Junctional EBA?
Immunosuppression
- may end up being euthanized due to side effects or incomplete remission
- may be able to taper and stop medications
78
Which species has mixed AISBD been reported in and what is the age predilection?
Dog
- breed disposition: nd
- Age predisposition: young (2-4 yrs)
*But there have only been 3 cases
Other species: humans (different nomenclature)
79
What is the clinical presentation of mixed AISBD?
Skin lesions: erythema, tense vesicles and bullae, deep erosions/ulcers
Distribution: haired skin (concave pinnae, trunk; footpad sloughing is rare) and mucosae/mucocutaneous junctions
80
What is the immunopathology of mixed AISBD?
Major antigen: Collagen VII (like EBA) and laminin-332 (like JEBA)
Minor antigens: nd
IgG, IgA, C3
81
What are the histopathologic signs associated with mixed AISBD?
Level of split: lamina lucida
Histopathology: microscopic subepidermal vesiculation with mixed neutrophilic eosinophilic inflammation
82
What is the treatment for mixed AISBD?
Immunosuppression
Unknown how it works since all dogs were lost to follow up or euthanized due to treatment cost
83
What species has Linear IgA Disease (LAD) been reported in and what is the age predilection?
Dog breed disposition: nd
- only 2 reported, one was 3 and other was 4
Other species: human
- biphasic in young children and older adults
84
What is the clinical presentation of Linear IgA Disease (LAD)?
Skin lesions: erythema, vesicles, deep erosions/ulcers
Distribution: mucosae and haired skin (ears, nasal planum, footpad)
In humans it frequently looks like a "string of pearls" hence the name linear
85
What is the immunopathology of Linear IgA Disease (LAD)?
IgA (+/− IgG) reactivity against soluble antigens of collagen XVII cleavage products (LAD-1)
86
What are the histopathologic findings of Linear IgA Disease (LAD)?
Level of split: lamina lucida
Histopathology: microscopic subepidermal vesiculation +/- minimal neutrophilic inflammation
87
What is the treatment for LAD?
Unknown in dogs
Vancomycin is a common trigger in people so discontinue the drug
For spontaneous in people
- first line = sulfones (ex. dapsone) or colchicine
- second line = combination of glucocorticoids and non-steroidal immunosuppressant
88
What is pemphgoid gestastionis?
An AISBD that occurs in pregnant animals
Reported in 1 dog, not published
Other species: human (2nd or 3rd trimester most common)
89
What is are the lesions associated with pemphgoid gestastionis?
Skin lesions: vesicles, erosions/ulcers
Distribution: mucosae and haired skin (ears, nasal planum, periareolar, footpad)
90
What is the immunopathology of pemphgoid gestastionis?
SIgG, IgM, and C3 probably against Collagen XVII/BP180 suspected
91
What is the treatment for pemphgoid gestastionis?
The one dog had an ovariohisterectomy
Most people spontaneously recovery 4-14 weeks post-partum
92
What species has bullous systemic lupus erythematosus been reported in?
One dog
93
What are the clinical signs associated with bullous systemic lupus erythematosus?
Skin lesions: erythema, vesicles, deep erosions/ulcers, SLE signs
Distribution: haired skin (friction areas ex. footpads) and mucosae/mucocutaneous junctions
94
What is the immunopathology of bullous systemic lupus erythematosus?
IgG >C3 to NC1 domain of collagen VII
95
What is the treatment for bullous systemic lupus erythematosus?
Unknown, try immunosuppression but the one dog was euthanized due to refractory recurrence of skin lesion
96
Which autoimmune subepidermal blistering disease is most likely when only haired skin is involved?
Bullous pemphigoid
97
Which autoimmune subepidermal blistering disease is most likely when there is minimal to no haired skin is involved?
Mucous membrane pemphigoid
98
Which autoimmune subepidermal blistering diseases show positive IF on the dermal side of the salt-split buccal mucosa (patient's serum)?
EBA
JEBA
mixed
bullous SLE
mucous membrane pemphigoid
99
Which autoimmune subepidermal blistering diseases show positive IF on both sides (mixed) of the salt-split buccal mucosa (patient's serum)?
JEBA
mucous membrane pemphigoid
100
Which autoimmune subepidermal blistering diseases show positive IF on the epidermal side of the salt-split buccal mucosa (patient's serum)?
BP
PG
linear IgA bullous dermatosis
mucous membrane pemphigoid
101
Which autoimmune subepidermal blistering diseases show dermal positioning of collagen IV/PAS staining of the blister using anti-collagen IV IHC or IF or PAS staining (patient's biopsy sections)?
BP, PG, JEBA
102
Which autoimmune subepidermal blistering diseases show mixed positioning of collagen IV/PAS staining of the blister using anti-collagen IV IHC or IF or PAS staining (patient's biopsy sections)?
JEBA
103
Which autoimmune subepidermal blistering diseases show epidermal positioning of collagen IV/PAS staining of the blister using anti-collagen IV IHC or IF or PAS staining (patient's biopsy sections)?
EBA, bullous SLE
104
What does plectin bind to?
keratin intermediate filaments
β4 of α6β4
COL XVII
105
What molecules of the hemidesmosome are found in the inner plaque?
- BPAG1e (inner)
- Plectin (inner)
- α6β4 integrin (inner through lamina lucida)
106
Which molecules of the hemidesmosomes are found in the outer plaque?
- α6β4 integrin
- collagen XVII
*both are transmembrane and extend into the lamina lucida
107
What does BPAG1e aka BP230 bind to?
keratin intermediate filaments
β4-subunit of α6β4 integrin
collagen XVII (BP180)
108
Where is the inner plaque of the basement membrane zone found?
intracellularly in the keratinocyte (superficial to outer plaque)
109
What does α6β4 integrin bind to?
intermediate filaments (β chain)
BPAG1e aka BP230 (β chain)
plectin (β chain)
laminin 332 (β chain)
COL XVII (both α and β chain)
110
What does collagen XVII (aka BP180, BPAG2) bind to in the hemidesmosome?
- intracellular head binds plectin, BPAG1e, and a6b4 integrin (b4)
-extracellular tail binds to α6β4 integrin (α6) and laminin-332
111
What does laminin-332 bind to?
Other laminins
Integrins
COL IV
COL VII
COL XVII
Perlecan
112
What does collagen IV bind to in the hemidesmosome?
laminin-332
perlecan
nidogen
fibronectin
collagen VII
113
What is the main molecule that forms the lattice structure of the lamina densa?
Collagen IV
114
What do nidogens bind to in the hemidesmosome?
Col IV
Laminin
Perlecans
Fibrulins (not in Bizikova's lecture)
115
Why does the lamina densa stain with PAS?
The glycoproteins perlecan and nidogen
116
What do perlecans bind to in the hemidesmosome?
Laminin-332
Col IV
117
What is fibulin?
A calcium-binding extracellular matrix protein
helps stabilize collagen IV network
118
List the collagens found in the BMZ in order of appearance from keratinocyte to dermis
Collagen XVII (inner plaque through to lamina lucida)
Collagen IV (meshwork of lamina densa)
Collagen VII (Velcro loops within the dermis)
119
What does collagen VII bind to?
COL IV
laminin 331 and 332
loops around dermal collagens
120
What is the salt-split skin technique?
a method used to study autoimmune blistering diseases by separating skin with a sodium chloride solution
1) incubate the punch biopsy of the skin in sodium chloride
2) separate the dermis from the epidermis
3) apply DIF and IIF staining
121
What is direct immunofluorescence?
Apply an antibody-fluorophore conjugate directly to patient's skin biopsy
122
What is indirect immunofluorescence?
Incubate normal tissue in the affected patient's serum and then apply an antibody-fluorophore conjugate
It is used more frequently than direct
123
What are the advantages and disadvantages of direct immunofluorescence?
Advantages
- Rapid single-step staining
- Can use multiple antibodies
- Minimal cross-reactivity
Disadvantages
- No secondary signal amplification
- Each primary must be labeled individually
124
What are the advantages and disadvantages of indirect immunofluorescence?
Advantages
- Secondary signals may be amplified
- A few labeled secondaries can detect many primaries
- Greater flexibility
- Easy to change colors
Disadvantages
- Two-step staining process
- potential cross reactivity
- finding labeled primary antibody which is more difficult to get especially for multiple labeling experiments
125
Salt split skin, staining on roof
BP, PG
126
Salt split skin, staining on floor
EBA, JEBA, mixed, bullous SLE
127
What is the common immune mechanism shared by AISBD?
an autoantibody response directed against structural proteins of the dermo-epidermal junction
128
What are the pathomechanisms which have been proposed to cause AISBD?
- The humoral immune response, in conjunction with complement activation, neutrophil and/or eosinophil recruitment and Fc-receptor mediated inflammation
- Complement-independent pathogenic effects of autoantibodies, IgG4 particularly
129
What is the most common AISBD recognized in dogs?
Mucous membrane pemphigoid
48% of all AISBD in dogs
130
Which AISBD is this most consistent with?
Mucous membrane pemphigoid
131
What can be used to differentiate MMP from EBA?
A positive PAS staining and/or anti-collagen IV IHC should label the dermal side (floor) of the blister in biopsies of MMP, not EBA
132
Which AISBD is this most consistent with?
Bullous pemphigoid, dog
133
What is a key difference between BP and EBA?
Footpad sloughing in rare in BP but common in EBA
A positive PAS staining and/or anti-collagen IV IHC should label the dermal side (floor) of the blister in biopsies of BP, not EBA
134
Which AISBD is this most consistent with?
Bullous pemphigoid, horse
135
Which primary molecules of the BMZ can be found in the basal keratinocyte?
KIFs
BPAG1
Plectin
Collagen XVII
a6b4 integrin
136
Which primary molecules of the BMZ can be found in the lamina lucida?
Collagen XVII
a6b4 integrin
Laminin-332
Collagen IV (but is a major part of the lamina densa)
137
Which primary molecules of the BMZ can be found in the lamina densa?
Laminin-332
Collagen IV
Perlecan
Collagen VII
138
Which primary molecules of the BMZ can be found in the sublamina densa?
Collagen VII
139
What differs between fetal and adult hemidesmosomes?
fetal only have plectin and a4b6 integrin
they do not have BPAG1 or collagen XVII
140
Which AISBD is this most consistent with?
EBA
141
This is a biopsy sample from a young Yucatan mini-pig. Which AISBD is this most consistent with?
BP
142
Why might the the sensitivity of PAS staining to determine if the roof or floor is stained be limited?
BMZ degradation during the blistering process
143
Is PAS, anti-collagen IV, or salt-split indirect IF staining is specific enough to confirm the diagnosis of mixed AISBD?
No
advanced immunotesting is needed to identify the mixed target autoantigens
not readily available in veterinary medicine
144
20 day old Eurasier dog. What genetic mutation is this associated with?
PLEC (autosomal recessive)
Epidermolysis bullosa simplex
145
What is the mode of inheritance for most congenital sub-epidermal blistering disorders, what are the clinical signs, and what is the prognosis?
Most of inheritance: recessive (some have been reported de novo)
Clinical signs: ulcers, especially at pressure points and in mouth in very young animals
Prognosis: poor :(
146
What are desmosomes?
- The major cell adhesion junction of the epidermis
- Serve to anchor apposing keratinocyte cell surface membranes to the intracellular keratinocyte intermediate filament network
- Create a 3 dimensional scaffolding that stabilizes epithelia in the face of stress and trauma
147
What are the 3 major categories of desmosomal proteins?
- Desmosomal cadherins
- Armadillo family proteins
- Plakins
148
What are the desmosomal cadherins?
- Desmogleins and desmocollins
- Transmembrane glycoproteins whose extracellular domains interact to form the trans-adhesive interface between cells
- Structure is 5 cadherin repeats plus an extracellular anchor domain separated by a Ca binding motif
149
What are the desmosomal armadillo family proteins?
- Plakoglobin and plakophilin
150
What are the desmosomal plakins?
- Desmoplakin
- Envoplakin
- Periplakin
151
Which desmosomal cadherins are primary expressed in the superficial epidermis?
- Dsg1
- Dsg4
- Dsc1
152
Which protein is the target for pemphigus foliaceous in dogs?
Dsc1
153
Which desmosomal cadherins are primary expressed in the deep epidermis near the basal layer?
- Dsg2
- Dsg3
- Dsc2
- Dsc3
154
Which protein is the primary target for pemphigus vulgaris in dogs?
Dsg3
155
What type of desmosomal cadherin combinations contribute the strongest adhesion?
Heterophilic (Dsg-Dsc) contribute to the strongest adhesion but homophilic interactions also occur
156
What does plakoglobin do in the desmosome?
- Directly binds the cytoplasmic tails of cadherins (including E-cadherin)
- Are also in the nucleus where they modulate gene transcription
- Recruit desmosomal proteins to the plaque similar to α- catenin in adherence junctions
157
What does plakophilin do in the desmosome?
- Bind directly to desmoplakin
- May also directly bind keratins and desmosomal cadherins which are thought to aid in the clustering and lateral stability of the desmosomal plaque
158
What does desmoplakin do in the desmosome?
- Binds directly to keratin, providing a link between KIFs and the desmosomal plaque
- Plays a critical role in development
159
What does the outer dense plaque of the desmosome contain?
- Desmosomal cadherin cytoplasmic tails
- Plakoglobin
- Desmoplakin N-terminal domain
- Plakophilin
160
What congenital diseases occur when something goes wrong with plakoglobins and desmoplakins?
- Ectodermal dysplasias
- Skin fragility
161
What is the primary target(s) of paraneoplasic pemphigus?
Plakoglobins and desmoplakins
162
What congenital diseases occur when something goes wrong with α6β4?
Junctional epidermolysis bullosa
163
What does the inner dense plaque contain?
Desmoplakin interacting with KIFs
164
What do adherens junctions do?
- Link epithelia cells
- Attach to actin cytoskeleton
- Involve transmembrane E-cadherin proteins
- Bind in a Ca-dependent manner
165
What are gap junctions and where are they primarily found?
- Aggregates of intracelluar channels (connexons) that permit transfer of ions and small molecules
- More present in basal layers
166
What are tight junctions and where are they primarily found?
- Join cells to form an impermeable barrier
◦ Semipermeable size and ion-specific barrier
- Most present in SG2 (middle of the granular layer)
167
A= intermediate filaments
B= desmoplakin
C= desmoglein
D= desmocollin
E= plakophilin
F= plakoglobin
168
What links corneocytes together?
Corneodesmosomes
169
What links keratinocytes together?
Desmosomes
Tight junctions (SG2 only)
170
What is a desmoglea?
Extracellular portion between adhered keratinocytes
◦ Cadherins: desmogleins, desmocollins
171
What parts of the desmosome are inside the keratinocyte cell membrane?
Outer dense plaque (plakoglobin)
Inner dense plaque (desmoplakin) connects to KIFs
172
Are desmosomes stable?
No, they are constantly rearranging
173
How are corneodesmosomes different from desmosomes?
They have corneodesmosin
◦ Serine and glycine rich protein
◦ Forms glycine loops
◦ Secreted from lamellar bodies
◦ Attaches to desmoglea
◦ Key to stratum corneum cohesion
174
Tight junction
A= occludin
B= claudin *CRITICAL COMPONENT*
C= junction adhesion molecules (JAMs)
(zonula occludens proteins are between them and interact with claudins, occludins, and actin)
175
Has bullous impetigo or pustular dermatophytosis with acantholysis been well characterized in cats?
No
176
Is immunological testing for antikeratinocyte autoantibodies by direct or indirect immunofluorescence is neither commercially available for cats?
No
177
What are the recommended treatments of PF in cats?
Glucocorticoids (oral +/- topical)
Chlorambucil (adjunctive)
Ciclosporine (adjunctive)
Topical tacrolimus (adjunctive)
A case report of oclacitinib 1 mg/kg q12h
May spontaneous resolve (rare, thought to be triggered) or able to achieve remission without drug or relapses (also pretty rare)
About half to 3/4 of cats will have flares
178
What is the typical age of onset for cats with PF?
Middle aged (similar to dogs and humans)
179
Is there a sex predilection for PF in cats?
Slightly more female (female:male ratio of 1.7 or 1.3)
180
What is true about triggers of PF in cats?
They may exist but are none have been confirmed
Drug withdraw and spontaneous resolution
- cimetidine (resolve when removed, flared when restarted)
- itraconazole/lime sulfur
Medications needed but able to later be withdrawn
- doxycycline (odd because it is sometimes used to treat pemphigus)
- vaccination
- methimazole
- cefovecin (maybe, no follow-up)
- clindamycin/carprofen
One case was associated with a thymoma and another with leishmaniosis (relationship between this and PF has been hypothesized in humans), both were able to eventually stop drugs
181
What are the clinical signs associated with PF in cats?
Pustules, erosions and/or crusts most frequently on face/head (pinnae and nose are common), legs, and claw folds (may be exudative)
- more peri-areolar than dogs but only 7 to 20% in 2019 Bizikova review
May be associated with variable prurtitus
May be lethargic or febrile
182
What is the prognosis of feline PF?
Feline PF has a good prognosis
- majority of cats (~ 90% on average) achieved DC in less than one month
Only 10% were euthanized due to diagnosis, treatment failure, or additional health issues
183
Which breeds of dogs have a predilection for PF?
Akitas and chow-chows
Labs (but thought to be because they are a common breed)
184
What is the age and sex predilection of PF in dogs?
Middle aged
No sex predilection
185
What are some drugs that have been associated with a possible drug reaction in canine PF?
anti-parasiticides (fipronil and isoxazolines)
antibiotics (cephalexin, amoxicillin, TMS)
antifungals (ketoconazole)
186
What is the clinical presentation of PF in dogs?
- pustular, erosive and/or crusting dermatitis often involving the nasal planum/muzzle with or without lesions on other parts of the body
- there is a trunk dominant form as well
187
What is true about anti-keratinocyte IgG in canine pemphigus?
It is ok but not reliable as 65– 84% are positive but,
- anti-keratinocyte IgG deposits in an intercellular, web-like pattern in both dogs with PF and other dermatoses or healthy dogs
- circulating antikeratinocyte IgG was detected in80% of healthy dogs
188
What is true about anti-DC1 IgG in canine pemphigus foliaceous?
*negative detection cannot exclude PF but is very specific
present in the facial phenotype (100%) but less in the trunk-dominant (58%)
not present in dogs with other dermatoses
189
What is true about anti- DSG1 IgG in canine pemphigus foliaceous?
detected only in a minority of canine PF sera
190
What is true about anti-DSG1 and anti-DG3 in humans with with pemphigus foliaceous and vulgaris?
about 10–15% of people with PF or pemphigus vulgaris have undetectable anti-DSG1 and/or anti- DSG3 autoantibodies, and possess autoantibodies targeting other parts of the desmosome
191
What is the key histopathologic feature of pemphigus foliaceous?
acantholytic cells present in intraepithelial pustules and/or serocellular crusting
192
Where is DSG1 expressed in the skin?
throughout the epidermis, but more intensely in the superficial layers
haired skin/footpad but not buccal mucosa
193
Where is DSG3 expressed in the skin?
Deep layers (basale>spinosum) esp in buccal mucosa
194
Where within the epidermis are lesions associated with PF found?
stratum granulosum and stratum spinosum
195
Where are lesions associated with pemphigus vulgaris found?
Stratum basale
196
What is the major autoantigen for PF in dogs? Humans? Cats?
* Humans: DSG-1
* Dogs: DSC-1
* Cats: unknown
anti-keratinocyte IgG was detected in 23/30 cats with PF
197
What is the etiopathogenesis of PF?
* Spontaneous
* UV light – induction of flare
* Drug triggered/associated
198
What are the variants of PF in dogs?
- facial dominant
- generalized
- foot-pad exclusive
- trunk dominant
199
Pustular dermatophytosis
200
What has been determined about pulse therapy of glucocorticoids for dogs with PF?
Pulse therapy group:
* higher proportion of dogs achieving CR in the first 3 months
* a lower average of maximal oral GC
* minimal adverse effect when compared to 20 dogs that received the
conventional dose
201
What treatments are typically used for canine PF?
* Conventional vs pulse therapy of oral GC
* Immunosuppressant: Azathioprine, cyclosporine, mycophenolate mofetil
* Adjunct immunomodulatory drugs:
- Tetracycline-niacinamide (1/8 dogs benefited)
- A case report of polysulfated glycosaminoglycans (Adequan):
* IVIG (Rahilly et al, JVIM, 2006) – patient got 11 doses in total
* Bruton’s tyrosine kinase inhibitor (BTKi)??
* Oclacitinib (higher doses)
202
What are the major autoantigens for canine pemphigus vulgaris? Human? Cat?
* Dogs: DSG-3
* Humans: DSG-3 +/- DSG-1
* Cats: unknown
*“Epitope spreading”: desmoglein 1 may be targeted if in haired skin (humans, dogs)
203
What does the Fc region of antibiodies bind to?
* Fcγ receptors on immune cells
* Neonatal Fcγ receptors (FcRn)
* C1q component of complement system
204
What is the biologic activity of the Fc region of antibodies?
* Antibody-dependent cell cytotoxicity (ADCC)
* Complement-dependent cytotoxicity (CDC)
* Antibody-dependent cell-mediated phagocytosis
205
What does the Fab region of antibodies bind to?
* Antigen (autoantigen)
* Toxins
* Pathogens
206
What is the biologic activity of the Fab region of antibodies?
* Triggers desired/undesired biological effect
207
What is the role that antibodies cause acantholysis?
Fc region
- binds to C1q component of complement system
- activates complement-dependent cytotoxicity (CDC)
Fab region
- binds to autoantigens (components of the desmosome)
- triggers undesirable effects
208
What are the mechanisms of acantholysis in human PV?
steric hindrance (autoantibodies bind and make the parts not connect)
desmoglein internalization and depletion (autoantibodies cause clustering)
signaling pathway interfering with cell adhesion
209
What is the role of c-Myc in canine PV?
c-Myc is a proto-oncogene
overexpressed in dogs with PV
interferes with the signaling cascade involved in DSG-3 expression
210
What species has PV been reported in?
dogs, cats, horses, humans
211
What species has PF been reported in?
many including dogs, cats, horses, goats, sheep, Arabian oryx
212
What are the clinical signs of PV?
Fragile vesicles → quickly to deep erosions/shallow ulcers
crusts, vesicles, erythema, depigmentation and alopecia
Cutaneous and mucocutaneous/mucosal lesions
* Oral cavity, pinnae, nasal planum, lip margins, genitalia, anus, periocular
* Claw bed, cornea, esophagus…
* Sloughing nails or footpad only
may have hypersalivation, halitosis, and/or dysphagia
may have lymphadenopathy, lethargy/dullness, anorexia, pain, weight loss, diarrhea and/or hyperthermia
213
What are the histopathologic findings consistent with PV?
Intraepidermal clefts with suprabasilar acantholysis
Bulla base: row of plump/rounded basal keratinocytes (“tombstone")
- Epidermis and/or follicles
+/ Acantholytic cells in clefts
Uninflammed if pustule is intact / neuts if detached
Ulcer + exudate are common
Dermis with lymphocytes/plasma cells, neuts (more lichenoid if junction)
IgG > C3 transepidermal and superficial follicular deposits
214
PV (note "tombstones")
215
PV (note "tombstones")
216
What type of pemphigus is this most consistent with?
pemphigus vulgaris
217
What type of pemphigus is this most consistent with?
pemphigus vulgaris
218
What type of pemphigus is this most consistent with?
pemphigus vulgaris
219
What are the common treatments and outcomes of PV?
Drugs reported to be effective: GC, AZA, CsA, heparin, doxycycline, and oclacitinib
~60% get clinical remission
~25% get partial remission
~30 are euthanized
May have relapses
One dog had spontaneous remission
220
What is pemphigus vegetans?
a rare clinical variant of PV
pustules, vesicles or blisters that rupture/evolve into hypertrophic or hyperkeratotic vegetative plaques or papillomatous vegetations
221
What is an important feature about the histopathology of pemphigus vegetans?
PV-type suprabasal acantholysis might not be present in all biopsies or it could have disappeared in some chronic lesions of PVeg, and thus multiple skin biopsies and a compatible clinical diagnosis might be required
222
What form of pemphigus is this consistent with?
PVeg
223
What is paraneoplatic pemphigus?
Rare (dogs, cats, humans)
Essentially PV + neoplasia
- thymic lymphoma, thymoma, splenic sarcoma…
If no neoplasia, think of drugs or other triggers
Target: various plakin proteins, Dsg3
224
What are the histologic features of paraneoplastic pemphigus?
looks like PV ("tombstones") + pustules + acantholysis + apoptotic keratinocytes (PV + EM +/- PF)
Clefts might be in variable layers (ex, lower spinosum) due to many targets
Variable neutrophil amounts
225
What form of pemphigus is this most consistent with?
paraneoplastic pemphigus
note apoptotic keratinocytes and tombstoning
PV + EM +/- PF
226
What is canine Darier's disease?
similar to Hailey- Hailey disease (HHD) in humans
a rare genodermatosis (autosomal dominant mutation in ATP2A2)
- Ca+2 pumps
- Defective epithelial cohesion (weak desmosomes)
- seen in English Setters and close relatives
young animals affected
causes recurrent vesicles and bullae; *plaques; erythema, crust, alopecia
trunk, head, extremities
227
What is the gene associated with canine Darier's disease?
Autosomal dominant mutation in ATP2A2
- loss of function, Ca2+ channel pumps
- = weak desmosomes
seen in English Setters and close relatives
228
Canine Darier's disease
229
This is a biopsy from a 4 month old dog. What genetic mutation is this associated with?
ATP2A2, Canine Darier's disease, setters
230
What are the clinical signs of pemphigus erythematosus?
pustules, erosions, and crusts localized to the face and pinnae
depigmentation, erythema and erosion/ulceration of the nasal planum and dorsal muzzle
- like PF but more mild because it is restricted to the face
- some dogs with PF can have a facial-only variant
predisposition to the Collies and Shar Peis
231
What are the histopathologic findings of pemphigus erythematosus?
Subcorneal neutrophilic and eosinophilic acantholytic pustules
- suggestive of PF
Along with a lichenoid interface dermatitis
- resembles DLE
*Pretty much PF + DLE
