El Bagre EPF August 2016

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A NEW VARIANT OF ENDEMIC PEMPHIGUS
FOLIACEUS IN EL BAGRE, COLOMBIA SOUTH AMERICA
ANA MARIA ABREU VELEZ, M.D.,Ph.D.
CO NFLICT O F I NTEREST: NO NE.
WHAT IS ENDEMIC PEMPHIGUS FOLIACEUS? (EPF)
 Endemic pemphigus foliaceus (EPF) is the only autoimmune disease in the world
restricted to a well-defined geographic region.
 This makes EPF an excellent model to study the interaction between genetics, the
environment and the immune system.
 There are two forms of pemphigus foliaceus: a nonendemic form, first described in
Paris in 1844, that occurs throughout the world, and an endemic form known as
fogo selvagem (FS), that was first reported in Brazil around 1903. Other endemic
foci have been reported in Colombia, Peru, Venezuela, and Tunisia.
 The presence of anti–immunoglobulin G (IgG) circulating autoantibodies and in situ
autoantibodies was described in patients with fogo selvagem (FS). These
autoantibodies were detected by means of indirect and direct immunofluorescence
(IF); intracellular staining was demonstrated within the epidermis. The autoantigen
related to EPF is desmoglein 1, a 160-kd glycoprotein of the desmosome, targeted
by in situ and circulating IgG autoantibodies, mainly of the IgG4 subclass.
ENDEMIC PEMPHIGUS FOLIACEUS
 The endemic form is thought to have an environmental cause. Normal
subjects living in an endemic area have antibodies against desmoglein
1.
 Endemic foci of fogo selvagem are almost exclusively located in rural
areas of Brazil. People of many races and ethnic groups are affected,
including Brazilians of Portuguese, Spanish, German, African, and
Japanese descent who live in the endemic areas.
 The incidence of fogo selvagem has decreased dramatically. The disease
follows the course of streams and creeks and vanishes after
urbanization of the endemic areas.
ENDEMIC PEMPHIGUS FOLIACEUS
 These findings support the concept that the production of antibodies against
desmoglein 1 is initiated by exposure to an unknown environmental agent.
 The endemic and non-endemic forms of the disease are clinically, histologically, and
immunologically similar; several features are unique to the endemic form, such as
the geographic, temporal, and familial clustering of cases, the higher frequency of
cases among children and young adults than among older persons, and an
association with certain HLA alleles.
 Bites of black flies, in particular Simulium nigrimanum, may initiate this disorder in
Brazil, possibly due to salivary proteins.
FOGO SELVAGEM
 In Brazil, the endemic pemphigus foliaceus is often referred to as fogo selvagem(FS). It
primarily affects children and young adults, with the highest incidence rates occurring
in the 10 - 30 year old age group. Also, the incidence in men and women appears to
be similar. Furthermore, FS cases tend to be clustered in specific families residing in
rural areas, especially in workers for road construction and agriculture. It was originally
observed in specific river valleys of rural Brazil where deforestation occured for mining,
and road construction.
 A strong association between FS and class II HLA antigens exists. Patients with FS
have one or both of the HLA DR1 and DR4 genes, whereas these genes were evident
in only 22 (34%) of the 64 control subjects. The HLA-DR1-Dw20 (DRB1*0102) gene is
related to susceptibility to FS, whereas absence of the HLA-DQw2 (DQB1*0201) allele
is linked with resistance.
A NEW VARIANT OF ENDEMIC PEMPHIGUS FOLIACEUS,
EL BAGRE-EPF(PEMPHIGUS ABREU-MANU) THAT
RESEMBLES SENEAR-USHER SYNDROME.
 El Bagre-EPF is distinct from other EPF in terms of age of onset and gender.
 Photosensitivity and a common symptom of severe stinging or burning occurs with
ultraviolet (UV) exposure.
ENDEMIC FOCI OF PEMPHIGUS
FOLIACEUS
GEOGRAPHIC LOCALIZATION
ENDEMIC AREA
ENVIRONMENT
ENDEMIC CLUSTERING OF DISEASES INCLUDING
AUTOIMMUNE FOLLOWING XENOBIOTIC EXPOSURE.
 Endemic and epidemic diseases occurring in an acute or chronic manner, in a relatively welldefined restricted geographic area are not uncommon.
 Examples of such diseases include chronic disease mortality among silicotics in CA. US.
 Pink disease (acrodynia).
 Autoimmunity associated with the Minamata Bay Japan disaster and methyl mercury.
 Generalized disorders of ectodermal tissue following prenatal exposure to polychlorinated
biphenyls reported in Taiwan and Japan.
 The Apallic syndrome, Uric (Kashin-Beck) disease, eosinophilia-myalgia syndrome.
 Hip disease seen in the Mseleni of Northern Zululand.
 Handigodu disease in Karnataka, India, Urov disease (osteoarthritis endemic in Siberia and
China) and Keshan disease.
 In USA, the effects of diesel exhaust particulate, poisons by pesticides.
 High nitrate (organic fertilizers) in the Thornton Well Field, near Woodstock, Ontario, Canada
(systemic sclerosis ).
 Geographical clustering of scleroderma in a rural area in the province of Rome.
SOME CLINICAL FORMS
SOME ATYPICAL CLINICAL LESIONS
SIMILARITIES TO LUPUS
ERYTHEMATOSUS
TESTING FOR AUTOANTIGENS USING A
BACULOVIRUS EXPRESSION SYSTEM
Trypsinize Bovine Epidermal Tissue
Purify on Con A Column
Patient serum linked to
Protein A-Sepharose column
Purify on Immunoaffinity
Column
Purify by SDS-PAGE
DEVELOPMENT OF AN ELISA USING COW SNOUTS
DETECTION OF AUTOPANTIBODIES USING TWO
DIFFERENT ELISAS
1.5
1.0
(r2=0.92)
0.5
OD
492nm (Linear scale
from 0.0 to 1.4 units )
ASSESSMENT BETWEEN CLINICAL
DISEASE ACTIVITY FOR SKIN AND OD
492 READINGS
0.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Skin surface compromised and dosage of
steroid hormones in patients with endemic
pemphigus foliaceus from El Bagre
1.2
0.8
0.4
OD
492 nm (Linear scale
from 0.0 to 1.4 units)
COMPARING FS AND EL BAGRE-EPF AUTOANTIBODIES
USING TWO ELISAS
Cut-off value
0.0
CPF FS NHS PE BP PV SLE EPF DEA
GROUPS OF DONORS
IMMUNOPRECIPITATION
SEQUENCE ANALYSIS BY MASS SPECTROSCOPY
We obtained 9 pg. of the 48kDa band (PF antigen) from 14 cow snouts.
Although the 48 and the 45 kDa bands comigrate closely, we only excised
the 48kDa since it co-migrate with the radiolabelled antigen. This band
was subjected to N-terminal protein sequence analysis. An N-terminus
sequence EXIKFAAAXREGED
DESMOGLEIN 1 ECTODOMAIN
ASSOCIATED SHORT TAMDEM REPEATS (STRS)
IN OUR STUDY OF EL BAGRE-EPF PATIENTS, THE PRESENCE OF HARDYWEINBERG LINKAGE DISEQUILIBRIA OF STRS FROM HLA II FAVORS THE
LOCUS D6S291 FOR DISEASE SUSCEPTIBILITY, AND SUGGESTS A
PROTECTIVE ROLE FOR LOCI D6S1019 AND D6S439 IN OUR CONTROLS.
The penetrance of 10% or less in female
patients 60 years of age or older indicates
that hormones could protect younger
females. The greatest risk factor for men
being affected by the disorder was the NN
genotype. These findings are only possible
due to somatic mutations, and/or strong
environmental effects.
I:1
II:2
I:2
II:1
III:1
III:2
II:3
III:3
HISTOLOGIC PATTERNS
EPF-FS AND EL BAGRE-EPF BEFORE THE STEROID
ERA
 In patients with poorly controlled fogo selvagem, growth is arrested; this is
alleviated by treatment. Personality changes may be manifested by blunted affect.
 Azoospermia is described in adults who had fogo selvagem as children; however,
some of the drugs used to treat this disease may also be associated with
azoospermia.
 In the therapeutic steroid era, the most common complications include secondary
infections by microbiological and parasitic agents, and other lesions in the
pulmonary and cardiovascular systems (endocarditis, valve alterations, pericarditis,
medial pulmonary necrosis, capillary disturbances, venous thrombosis, and
reticuloendotheliopathy), as well as hepatic, gastrointestinal, renal, and neural
sequelae.
DIF, IIF, PAS, AND IHC STAINS
ROULEAUX, CD1, HAM 56, CD68, S100 , VIMENTIN,
IGE, MAST CELL TRYPTASE
Lupus anticoagulant?
HLA ABC AND DP, DQ, DR
RIBOSOMAL PROTEIN S6-PS240 AND CYCLOOXYGENASE
2 (COX-2 )
PROTEINASES AND THEIR
INHIBITORS
ά-1 anti-tripsin
Tissue inhibitor of metalloproteinases 1 (TIMP-1).
Plasminogen activator receptor (uPAR).
Human matrix metalloproteinase 9
(MMP9).
Metallothionein
CELL JUNCTIONS
Occluding junctions
1.
Tight junctions (vertebrates only)
2.
Septate junctions (predominantly invertebrates)
Anchoring junctions
Actin filament attachment sites
1.
Cell-cell junctions (adherens junctions)
2.
Cell-matrix junctions (focal adhesions)
Intermediate filament attachment sites
1.
Cell-cell junctions (desmososmes)
2.
Cell-matrix junctions (hemidesmososmes)
Communicating junctions
1.
Gap junctions
2.
Chemical synapses
ANTIBODIES TO PALMS AND SKIN APPENDICES AND
THEIR NEUROVASCULAR SUPPLIES
ANTIBODIES TO AMALMAGAMATED
MELANOCYTES
SOME OCULAR FINDINGS
EYES, MEIBOMIAN GLANDS, TARSAL MUSCLE, CONJUNCTIVA,
PACINIAN CORPUSCLE, OPTIC NERVE (LEPTOMENINGES)
EYES, MEIBOMIAN GLANDS, TARSAL MUSCLE, CONJUNCTIVA,
PACINIAN CORPUSCLES, OPTIC NERVES(LEPTOMENINGES)
Newtonian secretion
ARVCF IgG-FITC
EYES, MEIBOMIAN GLANDS, TARSAL MUSCLE, CONJUNCTIVA,
PACINIAN CORPUSCLE, OPTIC NERVES (LEPTOMENINGES)
Pacinian
corpuscle
GOLGI TENDON ORGAN (NEUROTENDINOUS
SPINDLE)
AUTOREACTIVITY TO NERVES
 The intense cutaneous burning sensation in this disease was evaluated in testing for
neural autoreactivity in patients affected by El Bagre-EPF.

Autoreactivity to neural structures, mechanoreceptors, nerves, perineural cell
layers of the arachnoid envelope around the optic nerve, brain structures, and to
neuromuscular spindles was detected, with antibodies also colocalized with
desmoplakins 1 and 2 (DP 1-2).
AUTOREACTIVITY TO MECHANORECEPTORS
THE PLAKIN FAMILY OF CYTOSKELETAL
CROSSLINKERS

MACF1: Microtubule-actin crosslinking factor 1.

Spectrin.

Desmoplakins 1 and 2.

Envoplakin.

Periplakin.

BPAG1 in mice caused multiple defects, including skin fragility, muscle defects,
sensory neuron degeneration resulting in severe loss of coordination.
HEART REACTIVITY
 Cardiocutaneous syndrome related to mutations in desmosomal
proteins.
 Naxos disease. Has been associated with mutations in the genes
encoding DP and PG.
 Arrhythmogenic right ventricular cardiomyopathy: (Velo-CardioFacial syndrome, or ARVCF). Mutations in plakophilin-2 (PKP2),
desmocollin-2 (DSC2), desmoglein-2 (DSG2), plakoglobin (PG) and
desmoplakin (DP).
 Carvajal syndrome: Due to mutations on PG and DP.
 Palmoplantar keratoderma: Note DP and PG mutations, keratin 1,
dsg1.
CONGENITAL HEART BLOCK AND CARDIAC ARRHYTHMIAS
IN AUTOIMMUNE RHEUMATIC DISEASES
 Affects 5% of babies born to women with systemic lupus erythematosus (SLE) or
Sjögren's syndrome who have autoantibodies to the cellular proteins Ro and La.
 In SLE patients, sinus tachycardia, small vessesl vasculitis, atrial fibrillation, atrial
ectopic beats/transient atrial fibrillation, atrial flutter or paroxysmal
supraventricular tachycardia exist; also described in 20–30% of SSc patients.
 The most frequent cardiac rhythm disturbances in systemic sclerosis are premature
ventricular contractions, or rarely as bigeminy, trigemini or pairs and or transient
atrial fibrillation, flutter or paroxysmal supraventricular tachycardia and conduction
disturbances.
 In patients with rheumatoid arthritis, a main cause of sudden cardiac death
syndrome is atherosclerotic coronary artery disease, leading to acute coronary
syndrome and ventricular arrhythmias.
AREA COMPOSITA OF THE HEART
 Besides plakoglobin, which is shared by adherens junctions and desmosomes, other
desmosomal components, including desmoglein-2, desmocollin-2, plakophilin-2, and
desmoplakin are present in the adherens junctions of the heart.
 The mixed junctional structure is termed a hybrid adhering junction or adherens
composita (AC). Plakophilin-2 directly interacts with adherens junction protein alpha
T-catenin, providing a molecular link between the cadherin-catenin complex and
desmosomes.
 The AC only exists in the cardiac intercalated discs of mammalian species,
suggesting that it evolved to strengthen mechanical coupling in the heart of higher
vertebrates.
AUTOREACTIVITY TO VESSELS
T CELLS, LINKER FOR ACTIVATION OF T CELLS, ,T CELL
ANTIGEN RECEPTORS ZETA CHAIN-CELL
DEFRAGMENTATION, ANTI-CYTOKINE AUTOANTIBODIES ?
LAT
Lambda against
T cell-like cells
Anti T cells?
ARVCF
CARTILAGE, CONNECTIVE TISSUE PURKINJE
FIBERS, VALVES NODES?
ARVCF
MYOZAP
THE PHOSPHORYLATED RIBOSOMAL PROTEIN S6-PS240
 In pemphigus, several intracellular signaling pathways, such as p38MAPK activation
and RhoA inhibition, pemphigus IgG activation-translocation of protein kinase C,
and imbalances in both Akt/mTOR and cyclic adenosine 5’-monophosphate
signaling have been demonstrated to be altered following autoantibody binding and
to be causally involved in loss of keratinocyte cohesion.
 El Bagre EPF, 23/30 exhibited positive staining in spotty areas of the epidermal
corneal layer, and around neurovascular supply structures of dermal eccrine glands
and hair follicles. Very active clinical cases were strongly positive at within the
epidermal stratum granulosum (including the middle layers of hair follicles),
sebaceous glands, and especially in their basement membranes. (P < 0.05).
KIDNEY
ORAL MUCOSA
IgG-p0071
IgM-ARVCF
IgM-ULEX
Pale mucosa, leukoderma, some ulcers on the tongue,
loss of teeth, some varices, migratory stomatitis.
XENOBIOTIC METALS-METALLOIDS
 The population in this rural mining community is exposed to high environmental levels
of mercury, used for gold extraction, as well as other minerals, metalloids, and trace
elements (e.g., quartz, rutile, granite, magnetite, and almenite) and ultraviolet radiation.
We examined for the presence of mercury in skin biopsies and hair, using
autometallographic and mass spectroscopic analyses, respectively. Simultaneously,
serum levels of IgE were measured, and cutaneous tests for hypersensitivity reactions
were performed.
 Using autometallography, mercuric sulfides/selenides were detected in skin biopsies
distributed similarly in the control and patient groups. However, significantly higher
serum IgE levels and mercury concentrations in hair, urine, and nails were found in
patients compared with controls. Microscopic analysis revealed mercuric
sulfides/selenides concentrated within and around the sweat gland epithelium, as well
as in dendritic cells.
 UV radiation and electric storms.
AUTOMETALLOGRAPHIC AND ELECTRON
MICROSCOPY STUDIES
NEXT STEPS

Cytokines play a critical role in the determining the isotype to which the B cell
switches.

Cytokine receptors, transcription factors, cytokine genes expression.

Isotype switching refers to the process by which cells expressing IgM and IgD are
modified at the genomic level such that they produce antibodies of different
isotypes (IgA, IgE, or IgG).

Superantigens bind to regions of this T cell receptor (VB) and this activates
subsets of T cells that express the VB which leads to clonal expansion.

CD28-B7, IL-2, IL-4, TNF-G, B,, IL-10(eosinophilic degranulation without allergy).

CD80/CD86; CTL4-CD 28. CD4-/CD40L.

TREG,Foxp3

IL4,5,10,13 (Th2).

IL-17, TNF-alpha (Th17).

Fibroblasts, chondrocytes, osteoclast, proteases and their inhibitors.
EL BAGRE-EPF PROGRAM
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