1
CLINICAL, EPIDEMIOLOGICAL, HISTOPATHOLOGICAL,
IMMUNOLOGICAL, MOLECULAR AND ULTRA-ESTRUCTURAL
CHARACTERIZATION OF AN ENDEMIC FOCUS OF AN ENDEMIC
AUTOIMMUNE SKIN DISEASE WITH FEATURES OF ENDEMIC
PEMPHIGUS FOLIACEUS-LIKE AND SENEAR USHER-LIKE IN THE
RURAL AREA OF EL BAGRE, COLOMBIA, SOUTH AMERICA.
By
ANA MARIA ABREU VELEZ, MD.
A thesis submitted in fulfillment.
of requirements for the degree of
Doctor in Sciences
May 1,998
Graduate Program in Basic Biomedical Sciences
Universidad de Antioquia
Medellin, Colombia, South America
2
THIS THESIS IS DEDICATED TO: 1) THE PEOPLE AFFECTED FOR
PEMPHIGUS FOLIACEUS FROM EL BAGRE AREA. TO THOSE WHOM
DEAD ALONE,
DESPRECIATE FOR OTHERS IN THE SILENT OF THE TROPICAL
JUNGLE. 2) TO GOD. 3) TO MY FAMILY AND 3) ESPECIALLY TO MY
DAUGTHER WHO IS THE MOST WONDERFUL FRIEND AND LITTLE
PERSON THAT I KNOW.
3
ACKNOWLEDGEMENTS
Especially to Manuela (my daughter), who at her short age understood how
important this process was for me. Manuela grows up to my side sharing the
wonderful and difficult moments. We grow up together in a mother-daughter
relationship. Without Manuela’s love, comprehension and help filling up the
tips of the pipetment equipment’s, I can not perform this work.
To our patients, who lived abandoned for the society in an area of many
difficulties, and who believe in us and in our co-workers. Also, to patients who
actually are in peace.
To my family for their understanding, encouragement, and support.
To my co-workers who deal with me, that is not easily, especially to Juan
Guillermo Maldonado, Andres Jaramillo, Jorge Botero and Armando Muñoz.
Thank you for sharing a little of your lives with the patients affected by this
disease in El Bagre.
To the Doctors Stella Prada and Walter León Herrera, who are example of life.
To Dr Luis A Diaz and his team, who teach me many things, especially about
life.
4
To the Universidad de Antioquia, Mineros de Antioquia S.A, Fundación Elena,
and Juan, and to Hugo Ruiz for the support for the developing of this work.
To the Institutions which provides financial support as Colombian Agency for
scientific Development (Colciencias), Dirección Seccional de Salud de Antioquia
(DSSA), Universidad de Antioquia and to the Medical College of Wisconsin, WI,
U.S.A.
To Rafael Roldan, Director of Mineros de Antioquia S.A in El Bagre, to Pablo
Javier Patiño, MD, Ph.D and to Hertha Vélez for all the support provided to
this work.
To Hospital Nuestra Señora de El Bagre and to Alcaldia de El Bagre for logistic
support.
To LASPAU for administrate my scholarship in U.S.A.
I also like to acknowledgment to those people who tried to prevent my learning
process and our research, because them encouragement me more to keep going
in this difficult goal.
Finally, I wan to give a special thanks to Dr Fernando Montoya Maya, one
gentleman, teacher, prudent, smart, patient, undertake and so wise man for
5
his understanding, patiently, encouragement and for believe in my self and in
our work. Dr Montoya, I do not have words to tell all appreciation, estimation,
and admiration that I feel for you. You learn about pemphigus disease for my
education and that noble attitude makes you who you are. Thank you for
teaching to me.
6
Dissertation thesis: Focus of endemic pemphigus foliaceus-Like in El Bagre,
Colombia.
Student: Ana María Abréu Vélez M.D,
Mentor: Fernando Montoya Maya MD,
Director Biomedical Basic Sciences Corporation
School of Medicine, Universidad de Antioquia (U de A), Medellín, Colombia,
South America.
Co-mentors: Stella Prada de Castañeda MD,
Professor Section of Dermatology (U de A).
Walter Leon Herrera, MD,
Professor Section of Dermatopathology (U de A).
Luis A. Diaz MD, professor, and Chairman,
Medical College of Wisconsin, (MCW), Milwaukee, WI, U.S.A.
Dates: January 1995 to May 1998.
Modality: Sandwich.
7
Institutions: Universidad de Antioquia, Medellin, Colombia, South America
and Medical College of Wisconsin, Milwaukee, USA.
Financial support: Scholarship provided by Colciencias and administrated by
Icetex in Colombia and LASPAU in USA.
8
CONTENTS
Part 1:
- THESIS TITER
- THESIS DEDICATION
- ACKNOWLEDGEMENTS
- DOCTORAL PROGRAM CONTENTS
- HYPOTHESIS
- AIMS
- INDEX
- INTRODUCTION
Part 2.
- RESEARCH PERFORMED
-FUTURE PERSPECTIVES
-ANNEXES (Include publications and public presentations).
9
INDEX
- Hypothesis
- Aims
- Abreviations
- Introduction
I. Skin, functions, components, and cell cycle.
II. Regulator factors of the skin.
A. Positive regulators of proliferation (enhance of keratinocyte growth).
B. Inhibitory keratinocyte mechanisms.
III. Keratinocyte differentiation.
IV Cell adhesion mechanisms, generalities.
A. Adherens junctions.
B. Desmosome junctions.
C. Hemidesmosomes junctions.
V. Genetic abnormalities associates with derma-epidermal junctions.
A. Junctional epidermolysis bullose.
B. Dystrophic epidermolysis bullose.
C. Simplex epidermolysis bullose.
D. Hereditary acantholytic disease (Hayley-Hayley´s disease).
VI. Non genetic-non autoimmune diseases with acantholysis of the skin
(Grover’s disease).
VII. Skin autoimmune bullous diseases.
1. Epidermal autoimmune diseases.
10
A. Pemphigus Vulgaris.
B. Pemphigus Foliaceus.
C.Senear Usher syndrome.
D.Drug-induced Pemphigus.
E. Paraneoplastic Pemphigus.
F. Senear-Usher syndrome.
G. Intercellular IgA Dermatosis (”IgA Pemphigus“).
H. Intercellular IgE pemphigus (”IgE pemphigus“).
2. Subepidermal autoimmune bullous diseases.
A. Bullous Pemphigoid.
B. Herpes gestationis.
C. Cicatricial pemphigoid.
D. Epidermolysis bullosa acquisita.
E. Linear IgA disease and dermatitis Herpetiformis.
F. Subepidermal autoimmune bullous diseases associated with antibodies to
105 or 200 kDa proteins of the basement membrane zone (BMZ).
VIII. References and Tables.
IX. Description of a unique focus of “endemic pemphigus foliaceus- like” disease
in the rural area of El Bagre, Colombia, South America.
X. Immunological characterization of one unusual focus of “endemic pemphigus
foliaceus-like” disease in a rural area of El Bagre, Colombia, South America.
XI.Histopathological features in skin biopsies from patients affected by
“endemic pemphigus foliaceus-like” from El Bagre, area of Colombia.
11
XII. Detection of mercury in skin biopsies from people living in an endemic
area of one focus of “endemic pemphigus foliaceus-like” disease in el Bagre,
Colombia, South America.
XIII. Partial amino acid sequence of a 48 kDa bovine epidermal tryptic
fragment immunoprecipitated by all sera from patients with pemphigus
foliaceus.
XIV. Development of an ELISA assay using viable bovine epidermis for
detection of autoantibodies in sera from patients with pemphigus foliaceus
disease.
XV. Preliminary ultraestructural features of an endemic pemphigus foliaceuslike” disease in the rural area of El Bagre, Colombia, South America.
XVI.Future studies and goals to develop in my professional career.
12
HYPOTHESIS
- The focus of endemic pemphigus foliaceus (EPF) in El Bagre area of Colombia
differs from the other EPF foci.
- Desmoglein 1 (Dsg1) is not the only autoantigen present in people affected by
EPF from El Bagre, area of Colombia.
-The histopathological features in people affected by EPF from El Bagre, area
of Colombia is heterogeneous.
- The pemphigus foliaceus autoantigen obtained IN VIVO, shows high
complexity.
- Presence of autoantibodies directed against PF antigen (s), are not only
present in people affected by EPF disease in El Bagre, area of Colombia.
13
AIMS
1. To characterize clinically, epidemiological, immunological, and molecular,
from endemic focus of pemphigus foliaceus disease in El Bagre, area of
Colombia.
2. To identify the histopathological features of people affected by endemic
pemphigus foliaceus from El Bagre, area of Colombia.
3. To identified presence or not of mercury in people affected by endemic
pemphigus foliaceus from El Bagre, area of Colombia.
4. To characterize a 45 kDa pemphigus foliaceus antigen, that is recognized for
all sera belonging to people with pemphigus foliaceus disease.
5. To develop and sensitive immunoassay (ELISA), to detect presence of
autoantibodies in people affected by pemphigus foliaceus disease.
14
ABREVIATIONS
AA=Aminoacid.
AEB=Acquired epidermolysis bullose.
BMZ=Basement membrane zone.
BP=Bullous pemphigoid disease.
BP 180=Bullous pemphigoid antigen 180 kDa.
BP 230=Bullous pemphigoid antigen 230 kDa.
Ca++=Calcium.
C3a =C3 a of the complement pathway.
C4= C4 of the complement pathway
CE= Cell envelope.
CP=Cicatricial pemphigoid.
DNA=Deoxirribonucleic acid.
ECM=Extracellular matrix.
EB=Epidermolysis bullose.
EBA=Epidermolysis bullosa acquisita.
EPF= Endemic pemphigus foliaceus disease.
H & E=Hematoxiline & Eosin stain.
JEB=Junctional epidermolysis bullose.
SEB=Simplex epidermolysis bullose.
DEB=Dystrophic epidermolysis bullose.
DP=Desmoplakins.
Dsgs=Desmogleins.
15
Dsg1=Desmoglein 1.
Dsg2=Desmoglein 2.
Dscs=Desmocolins.
DH=Dermatitis herpetiform.
ELISA=Enzyme-linked immunosorbent assay.
EPF=Endemic pemphigus foliaceus.
EPF-L=Endemic pemphigus foliaceus-like.
Hg++=Mercury.
IL=Interleukin
ICS=Intercellular stain.
IgA=immunoglobulin A.
IgE=immunoglobulin E.
IgG=immunoglobulin G.
IgM=immunoglobulin M.
HG=Herpes gestationis.
DIF=Direct immunofluorescence.
IIF=Indirect immunofluorescence.
kDa=Kilodaltons.
L=Laminin.
LAD=Linear IgA.
PF=pemphigus foliaceus disease.
PNP=Paraneoplastic pemphigus
PV =Pemphigus vulgaris disease.
16
Mr=Relative mass.
SEB=Simplex epidermolysis bullose.
17
INTRODUCTION
I. Skin, functions, components, and cell cycle: The skin is composed by
epidermis, dermis, and cutaneous appendages (Lever, 1965). The skin has
many functions, some of the most important are a) protective: b) ultraviolet
(UV) diffraction barrier, c) sensorial, d) thermal-regulatory, e) immune, f)
endocrine and g) hydra-electrolytic control. The skin is a protective barrier
between a person and the external environment. It hampers the penetration of
microorganisms and inhibits the loss of water. To serve this essential function,
the individual cells of the epidermis are tightly bound to each other by
adhesion units called desmosomes.
The epidermal tissue is inhabited by keratinocytes, melanocyte, Langerhans,
merkel cells and transient lymphocytes. The epidermis is believed to contain
two types of proliferating cells: Stem cells have a lower capacity for selfrenewal and higher probability of undergoing terminal differentiation. The selfrenewal cells have high surface expression of integrin’s and can adhere to
extracellular matrix (ECM) (Jones et al, 1993). These self-renewal basal cells
allow skin to maintain its barrier function and to repair injured skin. In
Pemphigus vulgar (PV), and autoimmune skin disease the antigen target is
mainly localized onto basal cells (Jones and Watt, 1993). The dividing cells
with high proliferative potential are believed to correspond to the committed
progenitor cells of hemopoietic tissue. In culture, cells express.
18
The turnover time is defined as the time required for a cell to transit between
the basal layers into the stratum corneum, in skin generally it requires 14
days. A hallmark of differentiation among keratinocytes is a logarithmic linear
relationship between relative level of cell surface (integrin’s) and proliferate
capacity and rapid adhesion to type IV collagen, fibronectin, or ECM.
I. Regulator factors of the skin:
A. Positive regulators of proliferation: These enhance keratinocyte
growth and include growth factors, cytokines, divalent ions, vitamins,
arachidonic acid metabolites, polyamines, calmodulin, and cyclins (Moss,
1995). Ca++ appears to be essential in the transit of the cell through the cell
cycle by its binding protein calmodulin (Moss, 1995). Some cytokines as IL-1
and IL-6 exert a proliferate role in keratinocytes and epidermal growth factors
act through a specific receptor, especially the TGF alpha (Fitzpatrick, 1996).
Steroid hormones as well as retinoids exert a positive influence in the
epidermal growth (Fitzpatrick, 1996).
B. Inhibitory keratinocyte mechanisms: These are crucial to keep the skin
homeostasis. The best characterized are chalones, that inhibits proliferation of
basal keratinocytes. TGF beta molecule is a negative skin regulator and P 53
that is a phosphoprotein blocks cells at G1/S boundary (Fitzpatrick, 1996;
Champion, 1995).
19
III. Keratinocyte differentiation: Skin differentiation is marked by synthesis
of different elements as: keratohyalin granules, membrane-coating granules,
Cornified cell envelopes, and keratin filaments (Fey et al, 1984). Cornified cell
envelope (CE) is a highly insoluble structure formed beneath the cell
membrane during terminal differentiation of keratinocytes. The assembly of
CE is a complex process catabolized by transaminases (s), that cross-link
several proteins, including elafin involucrin, cystatin alfa (keratolin) and
SPRR, (cornifin or pancornulin). In paraneoplastic pemphigus (PNP) in which
some autoantibodies against envoplakin (a CE protein) had been recently
reported (Kim et al, 1997). Other marker of skin differentiation is the
apparition of cysteine rich proteins like fillagrin which are associated with the
keratohyalin granules. Membrane coating granules, also called Odland bodies
or keratinosomes, are small organelles present on upper spinous and granular
layers. They discharge lamellar disk composed of lipids into the intercellular
space. They function as sealant agents of the intercellular space, providing a
permeability barrier and may function in adhesion/dis-adhesion mechanisms.
The turnover in the intraepithelial localization of keratin, also represents a
differentiation marker in skin (Fuchs and, Coulombe 1992). Two families of
keratin (K) are known: type I or acidic (generally small) (40-56.5 kDa) and type
II or basic generally larger (53-67 kDa). K 14 is expressed at the epidermal
basal stratum, as well as K 5 and in upper layers K 10, K 1, K 2 and K 11 are
20
expressed. Basal cells produce keratin 5 and 14 which form filaments that
make an internal skeleton by attaching to the underlying basement membrane
trough specialized membranous plaques called hemidesmosomes and to sites of
cell-cell adhesion desmosome (Fuchs and Coulombe, 1992). As an epidermal
cell commits to terminal differentiation and leaves the basement membrane, it
switches to the expressing K 1 and K 10, which form filaments that bundle still
attach to desmosome. A mutations had been detected associated with K 5 and
K 14 (EB simplex, also known as Dowling-Meara syndrome) and EB simplex (K
14), Koebner (Fuchs and Coulumbe, 1992). In epidermolytic hyperkeratosis a
mutation in K 1 and K 10 has been described.
IV. Cell adhesion mechanisms: Many cell types distinguish self from nonself by virtue of cell surface adherents’ junctions involving members of the
cadherin family. While several cells stabilize homophilic cadherin-mediated
association by connecting to an actin cytoskeletal network, others including
heart muscle and epidermis orchestrate cell adhesion through desmosomes,
which connect to intermediate filaments (Fig 1).
A. Adherens junctions: Cell-to-cell adhesion of epithelial cells is governed
by two major types of adhesion machinery: adherens junctions and
desmosomes. Adherens junctions contain classical cadherins, such as Ecadherin or P-cadherin, transmembrane components and are linked to the
actin filament network (Fig 2).
21
B.
Desmosomes:
The
desmosomes
contain
desmosomal
cadherins,
desmogleins and desmocolins and is linked to the keratin intermediate
filament network. Adhesive functions of classic cadherins are well stablished
and transfection of classic cadherins into nonadhesive fibroblasts induced cellcell adhesion with morphological change via introduced cadherins. These
cadherins requiere their cytoplasmic domain and the association with - and 
cateins. If the cytoplasmic domains are deleted, the truncated cadherins lose
the ability to associate with the catenins and their cell-adhesion function
(Geiger and Ayalon, 1987; Scharz et al, 1992; Buxton and Magee, 1992).
Desmosomes are symmetrical membranous plaques that are several microns in
diameter and +/- 100 nm thick. Each half of the desmosome is derived from an
adjacent cell (Collins et al, 1991). Desmosomes contain two subtypes of
transmembrane glycoproteins that belong to the superfamily of cadherins:
desmogleins and desmocolins. Each of these proteins are encoded by at least
three differentially expressed genes (Collins et al, 1991). Typical desmosomes
contain a member of the catenin family, plakoglobin (PK) unique to desmosome
and also not seen in actin mediated adherents’ junctions are two proteins,
desmoplakins DPI and II, which seem to be splice variants encoded by a simple
gene that is widely expressed in cells that possess desmosome. In epidermal
desmosomes, keratins connect desmogleins and desmocolins by plakoglobin and
desmoplakins (DP) to form and extensive cadherin-mediated cytoskeletal
architecture.
The
desmosomal
membrane core glycoproteins
know
as
22
desmogleins (Dsg) consist of 150.000 kilo Daltons (kDa) (Dsg1), 120.000/110.00,
(Dsg 2,3). Amino acid and DNA sequence analysis of Dsg1, 2 and 3, have
demonstrated a significant degree of sequence identity with cadherins,
suggesting an extended family of cell adhesion proteins (Collins et al, 1991).
The cytoplasm plaque domain of the desmosomes comprises non-glycosilated
proteins, called desmoplakins (DPs); 250.000 kDa (DP1), 215.000 (DP2), 83.000
(DP3) kDa also known as plakoglobin, and 78.000 (DP IV)) (Anderson 1992;
Cunnigham and Edelman 1990).
The
distribution
of
cytoplasmic
plaque
proteins
remains
exclusively
intracellular. The biochemical nature of connections between keratin and
desmosome in epidermal keratinocytes is mediated between the carboxyl
terminal tail of DP that associates directly with the amino terminal head of
type II epidermal keratin including K 1, K 2, K 5, and K6. AN 18 AA residue
stretch in K 5 head (that is conserved only among type II epidermal keratin)
appears to play some role in DP-I tail binding. In contrast to desmosomal
cadherins, DP has not obvious transmembrane domain, and it is a cytoplasmic
protein.
Desmosome assembly is strictly dependent on cell-cell contact and can be
regulated In Vitro, by altering the concentration of Ca++ in the growth media.
The assembly of desmosomes is a complex process requiring coordinated
expression of protein constituents, their transport to the plasma membrane,
23
and their integration into a morphologically distinct structure at areas of cellcell contact. In the absence of cell-cell contact, membrane core glycoproteins are
processed and transported to the plasma membrane (Pascar and Li, 1993). The
disruption of classic cadherin affects the organization of desmosomes upon
calcium elevation and suggests that the proper function of classic cadherins is a
prerequisite for desmosome assembly in keratinocytes (Amagai et al, 1995). In
absence of desmosome formation, all proteins are metabolically unstable and
degraded rapidly.
Other component of the desmosomes is protein detected with a monoclonal
antibody (E 48) that recognize a 20 to 22 kDa antigen expressed by human
squamous and transitional epithelia and their neoplastic counterparts.
Electronmicroscopical examination showed that in cells of normal oral
mucosae, the E 48 antigen was expressed on the plasmalemma, suggesting
involvement of the E 48 antigen in intercellular adhesion (Schrijvers et al,
1991). The role of this molecule in autoimmune disease is not yet known.
Cadherin, and immunoglobulin, belong to the super-family of cell adhesion
cells (Fig 4).
B. Hemidesmosome: Hemidesmosomes are junctions that mediate adhesion
to the base membrane in some epithelia e.g., bladder, trachea, breast, and
amnion. The composition presently known of the dermal-epidermal basement
membrane zone (BMZ) are depicts schematically (Fig 5). The hemidesmosomes
24
contains intracytoplasmic components as keratin 5 and 14 serve to connect the
basal cell cytoskeleton to electron dense area associated with basal cell plasma
membrane termed hemidesmosomes. HD1 and BP 230 seem to link
intracellular proteins which may be involved with keratins and actin filaments.
Other components of the hemidesmosomes are transmembrane proteins 64
integrin and bullous pemphigoid antigen (BP 180 kDa) also known as type
XVII collagen. BP 180 and 64 integrin are transmembrane proteins with
intracytoplasmic and extracellular domains. Extracellular domains of these
proteins extend to the lamina lucida where anchoring filaments are located
(Burgeson et al, 1990). The 64 integrin is by far, the most abundant integrin
on the basal surface in keratinocytes of epidermal basal cells and is
presumably of major importance in hemidesmosomal adhesion. It also may a
role in signal transduction of epidermal stratification/differentiation and basal
cell proliferation. The cytoplasmic domains of both 64, show alternatively
spliced forms possible providing a means of varying ligand affinity, cytoplasmic
interactions or signal transduction. Both the extracellular and cytoplasmic
domains are subjected to tissue specific proteolytic processing and thus has
multiple ways of modulating interactions between the cell and base membrane
unlike other  integrins, 4 has an enormous cytoplasmic domain of 1000
aminoacids (aa). Containing a series of fibronectin type II repeats (Garrod,
1993). This is analogous to the hybrid structure of Dsgs, which has a cadherin
like-extracellular domain for cell-cell adhesion, with a long cytoplasmic
25
domain. Although there is no sequence homology between desmoglein and 4
integrins their cytoplasmic domains, clearly represent adaptations for
membrane associated plaque formation and both may extend beyond plaques to
make direct or indirect interactions with intermediate filaments (Garrod,
1993).
Bullous pemphigoid antigen (BP 180 kDa) also known as BPA G2 is a type II
transmembrane component in hemidesmosomes. The carboxyl terminal region,
which shows homology with collagen, is extracellular, while the aminoterminal
region, which shows homology with the chicken corneal protein, is cytoplasmic.
In the BMZ, keratinocytes interact with thread like structures, called
anchoring filaments deeper located in the lamina lucida. A disulfide bounded
heterotrimeric complex kalinin, has been identified in the anchoring filaments
layer. Kalinin is identical to nicein (Garrod, 1993). Anchoring filaments are
thin structures, which span the lamina lucida and appear to connect
hemidesmosomes with the lamina dense. The protein linear antigen dermatitis
(LAD-1) is located at the superior part of anchoring filaments (which seems to
be a 97 kDa antigen like BP180) may be is involved with connection of
anchoring filaments to the hemidesmosome (Garrod, 1993).
Laminin (L): Laminins 5 and 6 are heterotrimeric proteins located in lower
level of anchoring filaments (Burgeson et al, 1994). L5 and L6 exist as a sulfide
bonded complex. L6 probably interacts with other lamina dense and lamina
26
lucida proteins including type IV collagen, laminin 1 and perlecan (heparan
sulfate proteoglucan). Uncein may be another unique anchoring filament
component, but characterization of this molecule is yet incomplete. P 105 and
LH 39 antigens are two additional lamina dense components whose functions
and structures are incompletely known (Burgeson et al, 1994) (Fig 6).
Anchoring fibrils are deeper located at the BMZ and extend perpendicularly
from the lamina dense (collagen IV, 105 kDa antigen and 30 KDa proteins) into
adjacent papillary dermis. Either loop back into the lamina dense or insert into
electron dense antiparallel dimeric associations of type VII collagen
homotrimers with the NC1 or largest globular domain associating with type IV
collagen present in the lamina dense or anchoring plaque. Extracellular matrix
structures of the papillary dermis such as interstitial collagen fibers serve to
rigidly connect the lamina dense onto the dermis.
V. Genetic abnormalities.Many genetic abnormalities had been associated
elements of desmosomes and hemidesmosome components. Because these type
of disease are not part of the aim of this thesis will be shortly discuss.
A. Junctional epidermolysis bullose (JEB). It is characterized by absence
or reducement of laminin 5 in the skin and is associated with compromise in
multiple organs specially the lethal form (Herlitz) (Aberdam et al, 1994).
27
B. Dystrophic epidermolysis bullose (DEB). (Hallopeau-Siemens or
recessive dystrophic EB). This shows reduced or absent Collagen VII (Burgeson
et al, 1990). A large mutation of the COL 7A1 gene coding for type VII collagen
has been demonstrated in several families with both dominant and recessive
forms.
C. Simplex epidermolysis bullose (SEB). The primary defects in dominant
and recessive forms of EB simplex have mutation in K 5 and 10 (offspring). It
appears likely that genes, which encode for proteins involved with insertion of
keratin filaments to hemidesmosomes such as BP 230 and HD1, may also be
affected in epidermolysis bullose simplex.
D. Hereditary acantholytic disease (Hayley-Hayley´s disease).
Other mutations of proteins that belong to the hemidesmosomes have been
described as mutations of the gene coding for BP 180 have been demonstrated
in individuals with the less severe generalized atrophic benign form (Gamobrg
and Joland, 1985). Mutations of L 3, and in one of the genes coding for L5 also
has been demonstrated in individuals with this disease. Absence of recognition
of anchoring filaments (component LAD-1), has also been demonstrated in
skin of individuals with this disease. However, is unclear at present if this is
due mutation of the gene coding for this protein or if the absent staining
represents a secondary defect. Mutations of the gene coding for 64 integrin
subunit have been demonstrated in the form of JEB associated with pyloric
28
atresia (Chavanas et al, 1997). In epidermolysis bullose acquisita, presence of
autoantibodies against NC1 domain of type VII collagen has been detected.
VI. Non genetic-non autoimmune diseases with acantholysis of the
skin: Grover’s disease. Proteins involved in the formation of desmosomes can
be affected by acantholysis. A loss of desmoplakin I and II and plakoglobin
have been described from the desmosomes of people with Grover´s disease.
Desmoglein 1 has been moderated affected in this disorder and simple
adherens junctions are intact in this. For it´s rare nature the pathogenesis of
this disorder has not been well elucidated.
VII. Skin autoimmune bullous diseases: In the skin some autoimmune
bullous diseases are characterized by spontaneous blister formation and
production of autoantibodies. The blisters are located either within the
epidermis, most common at the desmosome or sub-epidermally at the BMZ.
The autoantibodies are directed against keratinocyte cell surface proteins or
cytoplasmic and constituents of the basement membrane zone (Anderson et al,
1996; Lever, 1965; Fitzpatrick, 1996; Champion 1995). Most of the
autoantigens in these diseases have been characterized at the molecular level;
however, the mechanisms (internal or external) that initiate the production of
autoantibodies
is
very
restricted.
The
clinical
immunological
and
epidemiological spectrum of these diseases are broad (Champion, 1995).
Therapy in general, is directed at controlling the resulting pathologic events
29
rather than to control the initial pathological process. Presently, the mainstays
of treatment for most of these diseases are systemic corticosteroids,
cyclophosphamide, metrotrexate, nicotinamide, chloroquine etc. (Champion
RH, 1995). The availability of recombinant forms of some autoantigens
(Desmoglein 1, 3 and PB 180) and others obtained directly from skin the target
autoantigens, provide new tools for epitope mapping studies and open new
possibilities for more specific treatment modalities. Future therapy could
include 1). induction of immunological tolerance, 2). hyposensitivization with
autoantigens 3). Liposomes with engineer restricted antigens by cutaneous
application, between others.
I. Epidermal autoimmune disease:
Pemphigus. Pemphigus is a broad group of diseases characterized by
intraepidermal blisters formation and immunopathologically by In vivo bound
and circulating IgG directed against the cell surface of keratinocytes, although
autoantibodies against intracellular components had been reported. Pemphigus
is divides in two major subtypes: pemphigus vulgaris (PV) and pemphigus
foliaceus (PF).
A less common form of pemphigus is the paraneoplastic form. This is
characterized by autoantibodies which immunoprecipitated a group of peptides
synthesized by keratinocytes including BP 230, desmoplakin, envoloplakin and
30
a proteins of a 190 and 170 kDa whose identities remains obscure.
Immunological by both direct and indirect immunofluorescence (DIF and IIF),
the autoantibodies bind intercellular spaces of the epidermis producing a
typical
staining
intercellular
(ICS)
pattern.
Drug-induced
pemphigus,
intercellular IgA and intercellular IgE pemphigus also belongs to this group
(Table I).
It has been demonstrated that after affinity chromatography isolation of IgG
autoantibodies from sera of pemphigus vulgaris (PV) and pemphigus foliaceus
(PF) patients induce loss of epidermal cell-cell adhesion. This acantholysis was
produced in neonatal mouse; however, this model is a passive model in a host
with an immature immune system (Anhalt et al, 1982).
A. Pemphigus Vulgaris. PV is the commonest form of pemphigus in North
America and Europe and it is the most severe form of pemphigus. Recently, has
been reported PV with exclusively oral compromise and with presence of
unique autoantibodies. In PV, the most common immune response is directed
to Desmoglein 3 (Dsg3) a desmosomal glycoprotein of the cadherin family
(Amagai et al, 1994; Emery et al, 1995). However, a new autoantibodies
population against Dsg1 and other antigens has been recently described.
Clinically, PV is characterized by flaccid blisters that readily rupture leaving
erosions on skin and mucous membranes. Erosions may increase in size leaving
large, denuded areas. Commonly, PV begins in the mouth, but sometimes it is
31
circumscribed
only
to
oral
mucosa
or
skin
(Champion,
1995).
The
histopathology hallmark of PV is a supra basilar acantholytic blister, in which
the roof is formed by the epidermis. Pemphigus vegetans is a variant of PV and
most commonly occurs in axillae and groins; it has two varieties nominated
Hallopeau and Newman (Champion RH, 1995). By immunoblotting (IB) using
normal human skin (NHS) or bovine tongue as a substrate has been
demonstrated that approximately 40% of the PV sera recognize the 130 kDa
Dsg3 (Stanley et al, 1984). Immunoprecipitation (IP) using recombinant forms
of these proteins produced on baculovirus expression vectors seems to be
promissory for detecting autoantibodies in sera from PV sera. Presently, Dsg3
has not been extracted from In vivo tissue, but recombinant forms of these
proteins are available (Amagai et al, 1994; Emery et al, 1995).
The immunoenzymatic assay (ELISA), for detecting presence of autoantibodies
in PV sera is available by using a direct ELISA with the recombinant form of
Dsg3 (Ishii et al, 1997). Physiopathogenic process in this disease it is not
understood up present). Prednisone is commonest therapy, and the initial dose
is based in clinical stage of the patient, but generally 1-2 mg/kg/day are used
upon disease severity supplemented with broad range antiparasitic medicine,
calcium supplements and topical corticosteroid creams with sun protectors.
Several immunosuppressive agents including cyclophosphamide, azathioprine,
methotrexate, cyclosporine gold, and for most severe cases, plasmapheresis,
photopheresis, and high-dose intravenous gammmaglobulins have been used in
32
the treatment of PV (Rook et al, 1989). In our experience a recalcitrant cases
require multy-teraphy treatment (Beckers et al, 1996).
B. Pemphigus Foliaceus. The clinical features of pemphigus foliaceus were
originally described by Cazanave in 1844, as a disease producing superficial
cutaneous blisters and erosions. PF includes Fogo selvage (FS), that is the
endemic form of PF, most reported in Brazil. PF of Cazanave, which is the nonendemic form of PF. Senear Usher Syndrome that seems to be a mixture of PF
and lupus with presence of antinuclear antibodies in some cases. Later Beutner
and Jordon demonstrated that patients with PF exhibit anti-epidermal
autoantibodies which can be detected by immunofluorescence. In PF, the most
common immune response is directed against Dsg1. The better known
pathogenically relevant autoantibodies belong to the IgG subclass and are
predominantly of the IgG4 subclass.
Some studies conducted in Brazil with FS patients had been shown disease
specific and their serum correlate well with disease extent and activity.
Relatives of FS patients are negative by IIF, but in some cases, presence of
autoantibodies was also detecting in healthy genetic relatives to FS patients.
Recently an identification of a new antibody population directed against a
desmosomal plaque antigen in PV and PF was reported (Kim et al, 1997). PF
serum contains autoantibodies that show heterogeneity.
33
PF is in general characterized by an acantholytic blister at the upper epidermal
layers. Clinically, fragile blisters that rupture easily producing erosions in
erythematous base and crusting without scaling are present. In contrast to PV,
the mucous membranes are usually spared, and lesions are most commonly on
the face, neck, and trunk. PF has many varieties as follows: 1). Senear- Usher
or seborrheic, 2) Cazanave’s or sporadic and 3) endemic pemphigus Foliaceus
(EPF) which includes FS. (Castro et al, 1983).
Presence of other endemic focus also has been reported in other countries in
South and Central America that includes Colombia and in Tunisia (Yepes et al,
1983; Bastuji G et al, 1995). Epidemiological evidence suggests that EPF may
be precipitated by an environmental factor (Hans-Filho et al, 1996). Some
Human Leukocyte Antigen (HLA) have been vinculated with presentation of
this disease (Cerna et al, 1993). According with the literature by compared with
PV, PF is a more benign disease and can usually be treated with less
aggressive therapy, but our experience differs from this data. In general, most
patients respond to 1-2 mg/kg/d of Prednisone (Castro et al, 1983). Other drugs
as dapsone in a 200-300 mg/d dose and many immunosuppressants
simultaneously (Basset et al, 1987). In addition, antihistaminic (H1 and H2)
antagonist, hyperproteic diet, calcium supplements, prophylactic antiparasitic
and
anti-tuberculosis
medicine
are
part
of
our
treatment
protocols.
Antimalarial such as hydroxychloroquine (200 mg/d) were reported to be useful
as steroid-sparing agents in PF (Hymes and Jordon, 1992). The combination of
34
oral tetracycline (2g/d) and niacinamide (1.5 g/d) with a topical corticosteroid
was recently described as useful in some patients with PF (Chaffins et al,
1993), but larger and extensive studies are necessary to be perform.
C. Senear Usher syndrome represents a variant of PF with features of
systemic lupus erythematosus in the same patient. Generally, is characterized
by presence of immunoglobulins mainly IgG and complement at the
intercellular spaces of the keratinocytes as well as C3a and C4 of the
complement. As well as in the BMZ area. It is commonly detected the presence
of autoantibodies against deoxyribonucleic acids (DNA) (Chorzelski et al, 1968).
D. Drug-induced Pemphigus Occasionally, pemphigus may be induced or
triggered by drugs as D-penicillamine, captopril and rifampicin. These patients
usually present a clinical picture of PF, and less frequently as PV (Champion,
1995; Zillikens et al, 1993). The presence of antibodies against Dsg1 and Dsg3
have been detected and usually this disease is transient and resolves shortly
after the drug has been discontinued, especially in those patients that lack
evidence of circulating antibodies. However, some cases follow a chronic course.
E. Paraneoplastic pemphigus (PNP). Paraneoplastic pemphigus is a
recently described blistering disorder that arises exclusively in the context of a
neoplasm, most commonly a non-Hodgkin's lymphoma (Anhalt et al, 1990). In
addition to polymorphous skin lesions, patients develop prominent, painful
35
mucous membrane ulceration. When associated with a malignancy, the course
of this disease is usually fatal. If a benign is the underlying neoplasm and it is
removed and respond to treatment with corticosteroids and may resolve
completely (Camisa and Helm, 1993). This entity is characterized by presence
of autoantibodies directed against components of the ICS as well as the BMZ.
By immunoblotting a doublet of antigens of 210 and 195 kDa has been recently
reported, and the nature of the 210 kDa has been demonstrated to be
envoplakin, a component of the cell envelope fraction (Kim et al, 1997).
F. Intercellular IgA Dermatosis ("IgA Pemphigus") Close to 30 cases with
intercellular IgA deposits have been reported and summarized in a recent
review (Wallach, 1992). Clinically and pathologically, these patients present in
2 different forms:1) a subcorneal pustular dermatosis, and 2) an intraepidermal
neutrophilic dermatosis. Recent data suggest that the autoantibodies in the
subcorneal pustular dermatosis-type of the disease bind to both human and
bovine desmocollin 1, whereas the autoantigen in the other subtype remains
unknown (Ebihara et al, 1991; Hashimoto et al, 1996). Intercellular IgA
dermatosis is associated with an IgA gammopathy in 20% of cases. It usually
runs a benign course and is managed with dapsone (Wallach, 1992, Zillikens et
al, 1990). In more resistant cases, corticosteroids alone or in combination with
immunosuppressive agents have been used successfully (Wallach, 1992). The
concept of this disease is still preliminary and its relationship to pemphigus is
not yet completely clarified.
36
G. Intercellular IgE pemphigus (¨IgE pemphigus¨). A relative new disease
called IgE bullous disease is characterized by the clinical picture of bullous
pemphigoid who lacked two critical diagnostic immunopathological features of
the disease, namely IgG or C3 bound to epidermal BMZ and circulating IgG
autoantibodies directed against the BMZ. Elevated seric IgE levels and
presence of deposit in eosinophils in the skin lesions have been observed, while
immunofluorescence studies revealing heavy deposition of IgE surrounding the
bullae. No many cases have been well characterized with all the criteria.
2. Dermal-epidermal autoimmune disease (subepidermal autoimmune
bullous diseases) Recent studies from France and Germany reveal that the
commonest subepidermal autoimmune disease is bullous pemphigoid, (BP)
followed by cicatricial pemphigoid (CP), herpes gestationis (HG), linear IgA and
epidermolysis bullosa acquisita (EBA) (Bernard et al, 1995; Zillikens et al,
1995) (Table II).
Bullous pemphigoid (BP). It is an autoimmune skin disease characterized
by presence of subepidermal blisters, resulting from disruption of adhesive
interactions between basal keratinocytes and cutaneous basement membrane.
Alteration at BMZ is the primarily manifestation by hematoxylin & eosin
studies (H & E). Autoantibodies from patients suffering from this disorder
recognize two epidermal antigens, BP 230 and BP 180, both of which have been
37
localized to hemidesmosome. BP is a disease of the elderly and is characterized
by tense, subepidermal blisters mainly located in intertriginous areas and
mucous membranes are usually not affected (Stanley et al, 1981, 1988; Labib et
al, 1986; Diaz et al, 1990; Giudice et al, 1992). The BP 180 autoantibody
epitope is an extracellular region of the molecule between the transmembrane
region and the more distal collagen’s domains (NC16A). Antibodies to this
region of the mouse BP 180 protein induce blistering in mice. Complement and
neutrophilic cells also appear to be required to form blisters in BP mouse
model. This model of BP involves passive transfer of anti-BP 180 antibodies
into neonatal mice developing a subepidermal blistering disease that is
dependent upon complement activation and neutrophilic infiltration has been
reported presently, for only one group of researchers (Liu et al, 1993; Giudice et
al, 1995.).
In contrast to pemphigus, BP is often a self-limited disease and it may be
sufficient to treat the patient symptomatically for a limited period. Generally,
relapsing episode are non-common and systemic corticosteroids the commonest
used therapy for generalized BP. The dosage fluctuate between 0.5 to 1 mg
Prednisone/kg/d.
Corticosteroids
are
often
combined
with
other
immunosuppressants in recalcitrant cases and hidroelectrolytic disorders are
often derivative from these treatments, especially for be an entity from the
elderly. Because BP affect mainly senior patients, commonly suffering from
various other medical problems, the complications of systemic corticosteroids
38
may be especially severe. Dapsone, sulfonamides, either in combination with
topical or systemic corticosteroids could be useful (Venning et al, 1989).
Localized forms could be treated with topical corticosteroids (Westerhof, 1989).
Oral tetracycline or in a combination of tetracycline and niacinamide has been
showed
a
successfully
treatment
for
BP.
Cyclosporine,
intravenous
gammaglobulin, high-dose pulse therapy with intravenous corticosteroids, and
plasmapheresis have all been proposed as treatment alternatives (Zillikens et
al, 1996).
B. Herpes Gestationis (HG): Herpes gestationis usually begins in pregnancy
but may also first present during the post-deliver period (Wever et al, 1995).
Lesions showed a herpetiform clustering. It was shown that HG and BP
autoantibodies react with a common immunodominant site on the extracellular
domain of the BP180 antigen (Giudice et al., 1993). In mild cases of HG, topical
corticosteroids and an antihistamine to relieve pruritus may be sufficient. In
more severe cases and during postpartum exacerbation, prednisone (20-40
mg/d) may be necessary initially and tapered later (Shornick, 1987).
C. Cicatricial Pemphigoid (CP): Cicatricial pemphigoid predominantly
involves mucous membranes and is characteristically associated with scarring.
Recent evidence suggests that subepidermal blisters in CP may be triggered by
autoantibodies against either BP180 or laminin-5 (Balding et al, 1996;
Domloge-Hultsch et al, 1992). CP is difficult to treat and spontaneous
39
remissions are rare (Axt et al, 1995). Evidence suggests that autoantibodies
localizing to the more distal collagen’s extracellular region of BP180 are
present in some forms of cicatricial pemphigoid. These autoantibodies localize
in the epidermal side of NaCl split skin by IIF. In other forms of cicatricial
pemphigoid, autoantibodies are directed to a subunit of laminin 5 and these
autoantibodies localize to the dermal side of the induced blister. For CP cases
involving only the oral mucosa, treatment may be limited to topical steroids,
intralesional steroids, or short courses of systemic corticosteroids. If the eyes or
larynges are involved, a more aggressive regimen is warranted and the use of
high doses of glucocorticosteroids in combination with immunosuppressant and
cyclophosphamide has been shown to be most effective (Foster, 1986; Caux et
al, 1996).
D. Epidermolysis Bullosa Acquisita: Epidermolysis bullosa acquisita (EBA)
is characterized by subepidermal blisters and autoantibodies to type VII
collagen, the major component of anchoring fibrils. There are 3 different skin
manifestations of the disease: 1) a non-inflammatory form of EBA, affecting
trauma-prone areas of the skin, 2) a generalized inflammatory blistering
eruption, and 3) a CP-like disease mainly affecting mucous membranes. All
forms of EBA are difficult to treat. Systemic corticosteroids alone or in
combination with azathioprine or cyclophosphamide are not effective in
controlling the disease. Some patients may respond to dapsone. More
encouraging reports have been published for the use of cyclosporine A and in
40
addition, a small number of patients have been successfully treated with
colchicine or extracorporeal photopheresis (Chan et al, 1996; Zillikens et al,
1993).
E. Linear IgA disease and Dermatitis Herpetiformis: Linear IgA disease
(LAD) and dermatitis herpetiformis (DH) are rare IgA-mediated disorders.
LAD may affect children and adults. The adult form may be precipitated by
drugs, mostly by vancomycin. The juvenile form of LAD is also termed chronic
bullous disease of childhood. Both forms of LAD are characterized by linear
deposits of IgA at the BMZ. In DH, IgA is found in the tips of the dermal
papillae different than linear IgA the antibodies localized to epidermal side of
the split. The linear IgA autoantigen, LAD 1 was originally identified as a 97
kDa peptide and is likely BP180. Antilaminin 5 mAb localized to dermal side of
the split. The autoantigen of dermatitis herpetiformis remains obscure. Both
LAD and DH are usually responsive to dapsone; alternatively, sulfapyridine or
sulfamethoxypyridazine are also effective. LAD of childhood is often a selflimited disease and most patients go into remission within 2 years
(Wojnarowska et al, 1988). LAD of adulthood may be complicated by severe
scarring of the mucous membranes and may then cause similar treatment
problems as CP.
F. Subepidermal autoimmune bullous diseases with antibodies to 105or 200-kDa proteins of the basement membrane zone. Recently, a new
41
pemphigoid-like disease with autoantibodies to a 105-kDa protein of the BMZ
has been reported. The patient's condition responded well to systemic
corticosteroids (Chan et al, 1993). Another report described a subepidermal
bullous disease with clinical features of BP and erythema multiform and a nonscarring mucous membrane involvement. The immune response was directed
against a 200-kDa protein of the BMZ and the disease was responsive to
tetracycline and niacinamide (Zillikens et al, 1996b).
42
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62. Rook AH, Heald PW, Nahass GT. Treatment of autoimmune disease with
extracorporal photochemotherapy: pemphigus vulgaris: preliminary report.
Yale J Biol Med 62:647-652, 1989.
63. Shornick JK. Herpes gestationis. J Am Acad Dermatol 17:539-556, 1987.
64. Shwarz, MA, Owaribe K, Kartenbeck J, Franke WW. Desmosomes and
hemidesmosomes: constitutive molecular components. Annu Rev Cell Biol.
6:461-491, 1990.
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65. Stanley JR, Koulu L, Thivolet C. Distiction between epidermal antigens
binding pemphigus vulgaris and pemphigus foliaceus utoantibodies. J Clin
Invest. 74: 313-320, 1984.
66. Stanley JR, Hawley-Nelson P, Yuspa SH, Shevach EM, Katz SI.
Characterization of bullous pemphigoid antigen: a unique basement membrane
protein of stratified epithelia. Cell 24:897-903, 1981.
67. Stanley JR, Tanaka T, Mueller S, Klaus-Kovtun V, Roop D. Isolation of a
complementary DNA for bullous pemphigoid antigen by use of patients'
autoantibodies. J Clin Invest 82:1864-1870, 1988.
68. Tappeiner G, Steiner A. High-dosage intravenous gamma globulin:
therapeutic failure in pemphigus and pemphigoid. J Am Acad Dermatol
20:684-685, 1989.
69. Venning VA, Millard PR, Wojnarowska F. Dapsone as first line therapy for
bullous pemphigoid. Br J Dermatol 120:83-92, 1989.
70. Wallach D. Intraepidermal IgA pustulosis. J Am Acad Dermatol 27:9931000, 1992.
54
71. Westerhof W. Treatment of bullous pemphigoid with topical clobetasol
propionate. J Am Acad Dermatol 20:458-461, 1989.
72. Wever S, Burger M, Langfritz K, Hashimoto T, Nishikawa T, Br`cker EB,
Zillikens D. Herpes gestationis. Clinical spectrum and diagnostic options.
Hautarzt 46:158-164,1995.
73. Wojnarowska F, Marsden RA, Bhogal B, Black MM. Chronic bullous
disease of childhood, childhood cicatricial pemphigoid, and linear IgA disease of
adults. A comparative study demonstrating clinical and immunopathological
overlap. J Am Acad Dermatol 19:792-805, 1988.
74. Yamamoto AI, Kartasova T, Mtsuo T, Kuroki S, Lizuka, H. Involucrin and
SPRR are synthethesized sequentially in differentiating cultured epidermal
cells. J Invest Dermatol 108:12-16,1997.
75. Yepes A. Brote de penfigo foliaceo en el municipio de El Bagre.
Bol
Epidemio Antioq; 2: 87, 1983.
76. Zillikens D, Miller K, Hartmann AA, Burg G. IgA pemphigus foliaceus: a
case report. Dermatologica 181:304-307, 1990.
55
77. Zillikens D, Erhard E, Prost C, Hashimoto T, Nishikawa T, Br`cker EB.
Inflammatory epidermolysis bullosa acquisita. Hautarzt 45:166-170, 1994.
78. Zillikens D, Wever S, Roth A, Weidenthaler-Barth B, Hashimoto T, Bricker
EB. Incidence of autoimmune subepidermal blistering diseases in a region of
central Germany. Arch Dermatol 131:957-958, 1995.
79. Zillikens D, Giudice GJ, Diaz LA. Bullous pemphigoid. An autoimmune
blistering disease of the elderly. J Geriatr Dermatol 4:35-41,1996a.
80. Zillikens D, Kawahara Y, Ishiko A, Shimizu H, Mayer J, Rank CV, Liu Z,
Giudice GJ, Tran HH, Marinkovich MP, Brcker EB, Hashimoto T. A novel
subepidermal blistering disease with autoantibodies to a 200-kDa antigen of
the basement membrane zone. J Invest Dermatol 106:1333-1336, 1996b.
56
TABLES
Table I. Description of the autoimmune bullous diseases with intra-epidermal blisters and respective) target (s)
antigen(s).
Disease
Varieties
Indirect
Immunofluorescence
Antigen target
Pemphigus Foliaceus
Cazanave’s, SenearUsher, Fogo selvagem
and endemic.
Hallopeau, Newman and
oral localized.
Intercellular stain with
IgG and C3 mainly in
upper epidermis.
Intercellular stain with
IgG and C3 mainly in
basal epidermis.
Intercellular stain with
IgG and C3 mainly, gall
bladder and basal
membrane zone.
Desmoglein1,
desmocolins and others
uncharacterized.
Desmoglein 3 and 1 and
others uncharacterized.
Pemphigus Vulgar
Pemphigus
paraneoplastic
Unknown
Drug induced
pemphigus
Pemphigus vulgaris and
foliaceus-like.
Pemphigus IgA
Unknown
Pemphigus IgE
Unknown
Intercellular stain with
IgG and C3 mainly in
upper epidermis.
Intercellular stain with
IgA.
Intercellular stain with
IgE.
Envoplakin, Bullous
pemphigoid 180, 230
antigens, desmoplakin 1
and 2 and 170 kDa
bands.
Desmoglein 3 and 1 and
others uncharacterized.
Desmoglein 3? and
desmocolin 1
Unknown
57
Table II. Autoimmune bullous diseases with subepidermal blisters and respectives target(s) antigen(s).
Disease
Varieties
Bullous
pemphigoid.
Herpetiformis, lichen
planus bullous,
classical.
Unknown.
Herpes
gestationis
Linear IgA
dermatitis.
Cicatricial
pemphigoid.
Epidermolisis
bullose
acquisita.
Dermatitis
herpetiformis.
Endemic
pemphigus
foliaceus-like
from El Bagre.
Indirect
immunofluorescece.
C3 and IgG at basal
membrane zone (BMZ).
Antigen target
C3 and IgG at BMZ.
BP 180 kDa.
Child and adult.
IgA at BMZ.
97 kDa (BP 180-like).
Unknown.
IgG, C3, IgA at BMZ.
Unknown.
IgG, C3 at BMZ.
BP 180 carboxyl domain,
laminin 5.
290 kDa (type VII collagen).
Unknown.
IgA in dermal papillae.
Unknown.
Senear-Usher-like,
exfoliative,
generalized etc.
IgG1, IgG4, C3, IgM at
BMZ.
210, 195, 117, 96 kDa bands.
Bullous pemphigoid (BP) 230
and 180 kDa.
58
Fig 3. Schematize the desmosomal and hemidesmosomal
junctions.
59
Part 2.
IX. DESCRIPTION OF AN UNIQUE FOCUS OF AN EPIDEMIC
TENDENCY OF PEMPHIGUS FOLIACEUS-LIKE DISEASE IN THE
RURAL AREA OF EL BAGRE, COLOMBIA, SOUTH AMERICA.
Abreu-Velez Ana Maria,
^
Maldonado Juan Guillermo,
*
Jaramillo Andres,
&
Muñoz Armando, # Botero Jorge^ and Montoya Fernando^.
Biomedical Basic Science Corporation,
Dermatology
#
^
School of Veterinary,
*
Section of
and PECET&, University of Antioquia (U de A), Medellin,
Colombia.
Corresponding author: Ana Maria Abreu-Velez MD, Corporacion Ciencias
Basicas Biomedicas, Facultad de Medicina, Universidad de Antioquia, Carrera
51 D # 62-29. AA 1226, Medellin, Colombia, South America. Fax: (574) 2633509, phone: (574) 5106069, e-mail: fmontoya@catios.udea.edu.co
Key words: Endemic Pemphigus Foliaceus, Epidemiology, Autoimmunity,
Mercury, UV light, Rain Forest Destruction, Mosquitoes.
Short Title: Unique features of a focus of an endemic pemphigus foliaceus-like
in El Bagre, area of Colombia.
Abbreviations:
EPF,
endemic
pemphigus
foliaceus;
EPF-L,
endemic
pemphigus foliaceus-like; PF, pemphigus foliaceus; FS, fogo selvagem; DIF,
IIF, direct and indirect immunofluorescence; IB, immunoblotting; IP,
immunoprecipitation; ELISA, enzyme-linked immunosorbent assay.
60
ABSTRACT
For first time in the literature, we confirm the presence of a unique focus of
endemic pemphigus foliaceus-like disease (EPF-L) in El Bagre, (Colombia), a
small tropical mining village. A case-control study was carried out to
characterize clinical and epidemiological aspects in people affected by EPF-L.
Fifty patients were collected by active search in a period of five years. EPF-L
diagnosis was based on clinical, epidemiological, histopathological, and
immunological criteria, although a unique features of an undescribed
autoimmune skin disease were found in this focus. Fifty normal controls
matched by age, sex, geographical localization, and activities were tested. All
subjects were living in the rural area and performed outdoor activities as
mining and or farming. The prevalence found of EPF in El Bagre was 1.8%, in
men between 28 to 70 years old, living in rural area and working in outdoors
activities. In 1,120 families located in the rural area of El Bagre, 3.4% have a
member diagnosed with EPF. In this focus, men were the most affected (93.3%)
with a mean age of 50 years old, which differs from the data reported in
Brazilian in which mostly affected are between 10 to 30 years old affecting both
sex equally and Tunisia in which women in a childbearing age are mostly
affected. The predominant clinical picture includes characteristics of both lupus
and pemphigus, resembling Senear Usher Syndrome, with alterations in some
cases at the basal membrane zone by hematoxylin and eosin, by direct
immunofluorescence
and
by
electronmicroscopy,
without
antinuclear
antibodies. In El Bagre mercury is routine used in large quantities for gold
61
extraction, without protection. For that reason, we tested for it in the cases and
the controls. Higher levels were observed in EPF patients (p<0.05), based on
World Health Organization standards. In accordance with the association of
high levels of IgE and mice autoimmunity induced by mercury/D-penicillamine,
we tested for it too. In EPF-L patients’ higher levels were detected (p<0.05)
without association with atopy or parasitic diseases. Acute EPF-L cases showed
more elevated mercury and IgE levels. The subjects of this study were exposed
to sun for more than four hours per day. An epidemic tendency of this disease
was detected. Clinical features in this focus were unique and based on our
results; we suggest that a combination of a specific genetic background, sun
and mercury exposure could play a role in developing autoimmunity in people
from this focus.
62
INTRODUCTION
Pemphigus foliaceus (PF) and the endemic form of PF (EPF), are autoimmune
diseases that belong to the group of pemphigus1. EPF was described in foci at
the South America tropical forest, mainly in Brazil.2-9and other countries as
Tunisia.10 An increased number of familial fogo selvage (FS) cases occurred in
Brazil.2-9 Children and peasants in the second decade are the most affected, as
well as people whose lifestyle is dedicated to farming activities. Both sexes are
affected equally.1-11 Other Brazilian reports showed a slight predilection for
females.7,12-16 FS in Brazil is found more often in people who carry the Human
Leucocyte Antigen HLA DRB1*-A24; DB1*1406;DRB1*0404;DRB1*1402.17-19 It
is accepted that EPF is characterized by presence of acantholysis and
subcorneal blisters in upper epidermal layers, with the predominance of
immunoglobulin 4 (IgG4) autoantibodies at intercellular space in the epidermis
and especially directed against a desmosomal glycoprotein called Desmoglein 1
(Dsg1).20,21 Dsg1 belongs to the cadherin family of calcium-dependent cell
adhesion molecules.22,23 The cause of FS is unknown, but some possible
environmental risk factors as viruses, presence of intra-domiciliary mice and
exposure to a fly named Simulium pruinosum have been implicated in Brazil
as possible triggering factors.8,24 Presently the hypothesis that Simulium
pruinosum is the trigger of EPF disease in Brazil has not been scientifically
demonstrated. In 1993, the presence of other foci of EPF affecting natives of the
Amazonian and Orinoquian rain forest of Colombia was described. A
preliminary report in those patients showed some clinical and epidemiological
63
characteristics similar to Brazilian foci.25 The existence of the El Bagre focus
was reported in Medellin city by Yepes et al,26,27 Robledo et al28 and Abreu.29 A
retrospective study was done by Robledo et al,28 that included 21 EPF patients
from data collected in the Hospital Universitario San Vicente de Paul,
(HUSVP), Medellin, Colombia of patients attending over a ten-year period
between 1,976 and 1,986. EPF diagnosis was made by clinical and
histopathological by hematoxylin-eosin stain (H&E) criteria and thirteen
patients confirmed by direct immunofluorescence (DIF). The majority of the
patients (85%) were Indian/white men working as miners or/and farmers with
an average age of 44 years.28 Since the diagnosis of EPF described26-28 was in
some cases by revision of old files and this focus was never directly visited by
dermatologists and field studies were not performed in order to confirm the
presence of EPF, we proposed this investigation to characterize this focus, to
confirm its endemic nature, to search for some risk factors like mercury and to
compare our findings with those described in the Brazilian and Tunisia foci.
MATERIALS AND METHODS
General characteristic of El Bagre: El Bagre is located at North-East of the
Province of Antioquia, Colombia (Fig 1). El Bagre has two main rural areas:
Puerto Claver and Puerto Lopez; these are further divided into sections called
“veredas”; the rural area of El Bagre has 67 “veredas”. El Bagre has wide rivers
fed by small creeks and is located at 70o-35' of North latitude and 74o-40' of
East longitude. El Bagre has a total area of 1,928 km2. The altitude is 50
64
meters above sea level, mean environmental temperature varies between 3042oC, annual average rain fall is 3.8 meters and relative humidity is 85%. Two
rainy and two dry seasons are present. The vegetation is tropical, and soil is
deficient in carbon and phosphate with high amount of ferric and ferrous
oxydes, with low pH. El Bagre has a total population of 27,329 with 22,151 in
the urban area and 5,178 in the rural area. Sex relation was 1:1; 41% of the
populations were Indian/white and 37% mulattos (National Administrative
Department, DANE, 1993). Most of the population lies between zero and thirtyeight years of age (Local Hospital, 1993).
Main economy in El Bagre is based on extraction and commerce of gold. Gold
from alluvium is accumulated in rivers and creeks and it is extracted from ore
using a process called "azogue" that amalgamates gold with metallic mercury
as a refining agent (Fig 2).30-32 This process involves heating up mercury/gold
mixture which results in the release of high levels of mercury vapor into the
air. In 1988 the Center of Environmental Research of the U de A found a high
mercury pollution in most of the biotic chain of El Bagre including animals,
plants and humans.32,33
Subjects: During 5 years from 1992 to present an active search was performed
to detect EPF people. We consulted with all health personnel, empiric health
workers, local authorities, and community leaders. We send broadcasting
messages over the radio. We visited most of the veredas from El Bagre and
65
other neighboring municipalities. We looked medical records in the two local
hospitals from 1986 to 1992.
First year we visited El Bagre monthly, second year every two months and
quarterly after third year. During these period we evaluated 1,500 people
approximately with any skin disease by active search. Normal controls were
included matched by age, sex, living area and working activities and in general
they live in the same vereda to EPF-L patients. From these 1,500 people we
tested immunologically 100 with clinical features of EPF-L, with presence of
superficial bullae, exfoliative skin on erythematous area, with an extensive
prurigoid plaques mainly in chest and in back and also some cases that
resembling lupus-pemphigus, because were common detected.
All subjects in this study participated willingly and signed a consent form.
Since 98 % of them were illiterate, we explain with a witness from the
community about this research proposal. We asked about habits, time of
disease onset, work chores, place of birth and other places where they lived,
time in those places, exposure to agriculture and jungle vegetation, dietary,
sexual and rest habits mosquitoes’ bites, materials used in the labors, members
of family also affected, presence of rodents in house, bed bugs, time of work,
kind of clothes and part of body exposed when work and rest activities,
proximity of houses and distance to riverbeds. During subject examinations we
took blood samples for immunologist tests, skin biopsies in 10% formalin for
66
H&E studies and hair samples for mercury detection. Skin for immunological
studies was snap frozen at -178oC in OCT compound (Technoware) or 10%
dimethyl sulfoxide. Skin samples were transported to Medellin in tanks with
liquid nitrogen (a 4–5-day trip) and then stored at -70oC. Blood sera were
stored at -20oC. Some of samples were sent to USA in dry ice for further
testing.
Suspected cases that resemble clinical features of EPF as those described by
Viera in 1,938,34 or other skin disease, including a lupus-like in the face with a
prurigoid lesions in the chest and back resembling Senear-Usher syndrome
were subjected to a careful medical history and they were evaluated blindly for
two dermatologist. Clinical criteria were based on Viera's34 description and
classification. Those with positive intercellular staining (ICS) between
keratinocytes by DIF or IIF or if they immunoprecipitated a Con-A affinity
purified bovine tryptic fragment (PF antigen) were considered EPF cases.4,8,35,36
Sera of all cases and controls were also tested by immunoblotting (IB) for
reactivity against Dsg1.21 In addition, an immune enzymatic assay (ELISA)
test was performed for scanning autoantibodies using an indirect assay with
PF antigen.41 For all above determinations serum from a well characterized FS
patient from Brazil and two PF sera from HUSVP were used as positive
controls (C+). A negative control from U.S.A. (C-) was also used.
67
Quantification of total IgE serum levels: Patients and control sera were
tested for total IgE using an ELISA commercial kit (AlaSTAT Total IgE kit,
Diagnostic Products Corporation. Los Angeles, California). For lack of
comparable studies in the rural population of Colombia, we estimated in 300
U.I/ml the normal cut off total serum IgE (Leal Francisco. Personal
communication).
Cutaneous test: Approximately 20 to 30 ul of D. pteronissinus and D. farinae
antigens (Abello-ALK, Madrid) at the concentration of one biologic unit were
applied intradermal (1 UI/ml I.D) in the lateral area of the arm, with an
insulin syringe. Positive (histamine chlorhydrate 1:10,000) and negative (saline
solution) controls were used. A positive result was indicated by more than 5
mm of erythema and or edema. (Montoya Fernando. Personal communication).
Mercury detection in hair. After extensive washing, 25 mg of hair was cut
and degreased with acetone. Hair samples were then packed in ash free paper
and incubated in a solution of nitric and sulfuric acid followed by a solution of
potassium permanganate to destroy organic material. Excessive potassium
permanganate was removed with hydroxylamine chloride. Following this, tin
chloride was added to extract the mercury from the hair. Mercury was
measured in an atomic absorption spectrophotometer (MAS 50). According to
World Health Organization (WHO) mercury maximum permissible levels in
hair are 7 ppm.42
68
Statistical evaluation: Proportions contrast was made with Chi square test
and Wilcoxon-paired test was applied for media comparison. An alpha error
was used at 0.05.
RESULTS
After following all the criteria described in methods, we studied 100 people
living in El Bagre. Fifty cases and 50 controls were study. Forty-six EPF
patients have a clinically active disease, 1 patient was in clinical remission and
3 patients showed a very mild clinical form. Eleven controls were unaffected
relatives living in the same house of the patients, 7 were cohabitants and 32
were nonrelatives and non-cohabitants.
After detecting the EPF-L conditions, patients received a topical steroid,
sunscreen protectors (SPF # 15) and oral steroid therapy according to
individual requirements. EPF patients also received monthly, broad spectrum
anti-parasitic medication, as well as iron, calcium, and vitamin supplements.
Clinical findings: Based on Viera's classification of EPF patients, we found a
bullous-exfoliative
or
hyperkeratotic
generalized
form
(n=4),
an
EPF
hyperpigmented form (n=3), a localized form (n=36), a prurigoid form (n=6) and
one case with an inactive form (n=1. In approximately 60 % of the patients
their clinical picture included characteristics of both lupus erythematosus and
69
pemphigus with compromise of the scalp and the typical discoid patches on the
face was the commonest clinical feature. In others, we observed a severe
inflammatory reaction, suggesting a congested seborrheic dermatitis. A
tendency to change from chronic to acute form was observed, especially after
sun exposure. Keratotic-seborrheic plaques and clusters-like and exfoliative
areas with superficial blisters in an erythematous base were also commonly
observed in the same patients (70%) (Fig 3) and the presence of several clinical
varieties through the time, in the same patient, reveal the wide spectrum of
this disease (Fig 4). Sole, palm, oral or genital mucosae compromise was not
observed. In some patients with generalized form an aseptic conjunctivitis was
detected. Lesions in seborrheic areas (back, scalp, chest and central-facial) and
also in sun-exposed areas (face, arms and neck) were presents in 97.6% of the
patients. In 66% of EPF patients, the initial lesions started in thorax and in
25% in scalp. A mild compromise was found in thighs, calves, and sagittal area
of arms and forearms. According to Lund and Browder skin burn surface scale,
70% of EPF patients had 36% of cutaneous surface affected. Relapses were
observed in 60% and in five years 10 EPF patients required hospitalization. All
EPF
patients
related
weakness,
adynamia,
impotency,
insomnia
and
depression; 70% arthralgia, mainly at knee or spine joints and 50% blurry
vision and lacking memory.
We do not observe cases of ankyloses of joints, "pose of FS patients," nanism,
loss of hair, eyebrows, eyelashes, pubic hair, etc.
70
Epidemiological characteristics: We found a mean age of 50.4 years with a
range between 28 and 70; 93% were men (47/50); 80% were Indian/white
(40/50), 11.2% Mulatto (6/50), 4.4% (3/50) Caucasoid and 2.2% (1/50). All people
lived in the rural area of El Bagre. Based on the rural population of El Bagre
(about 5,178 people) and knowing that 2,822 of those are either women at nonchildbearing age or children’s, the EPF prevalence was 1.8% in the risk group,
men between 28 to 80 years old. Only one woman out of 1,300, at childbearing
age, was found with EPF (0.077 %). The rural area of EB had approximately
1,120 families, of which 3.4% had a member with EPF. We found 4 sets of
genetically related people afflicted with EPF-L: two brothers, a niece and an
uncle and two groups of father and son. These intra-familial cases made up
20% of our EPF group. Many patients in this study had other family members
with EPF whom living in neighboring areas to El Bagre, but these people were
unavailable for study.
Some cluster distribution of EPF patients was found in some rural area of El
Bagre mainly in Puerto Claver, El Mellizo, El Pisingo, La Llana, Quebrada
Villa and Amara although were not statistically significant. An epidemic
tendency was observed in this disease (Fig 5). Even though the subjects in this
study live in poverty, their personal and house hygiene was good and most of
those affected with EPF were within a normal weight range and only two EPF
patients were found to be in cachectic conditions, while one was found in
71
overweight. Equal amounts of affected by malarial infection were found in both
groups, reaching almost 40%. In both groups a 10% of atopy was found by
clinical and familiar criteria; no differences were found in tobacco smoking
habit and liquor ingestion, in the subjects of this study. In 90.4% of EPF
patients first developed the disease in the municipality of El Bagre. The
average evolution time of the cases before to be detected for our research group
was 5.8 years and in half of the cases was seven years. Six of the subjects had
an evolution of less than six months, while one subject had been carrying the
disease for 27 years. During this five years of study, six EPF patients died, the
cause of death was: stroke at age 58, possible gastric carcinoma at age 65, an
acute abdominal syndrome of unknown origin at age 47, two by an
exacerbation of EPF and one by renal failure of unknown cause. Diagnostic
autopsies were not performed.
No evidence of clinical disease was found in wives or cohabitants. In some EPF
relatives were detected pemphigus specific autoantibodies by IB, IP and
ELISA. No differences were found in EPF group in relations to contact with
agricultural and jungle vegetation, mining chores or lumber activities
compared with controls.
Histopathology: H & E stain revealed a heterogeneous picture (Table 1).
Details will be discussed in a future publication, correlating with the unique
clinical, immunological and electron microscopic features of these patients.
72
Controls showed features of psoriasis, lichen planus, lichen amyloidosis, basal
cellular skin cancer, eccrine hidracystome and normal skin which correlates
with their clinical picture.
Detection of antibodies against PF antigen(s): By IIF, IgG4 was the
commonest
keratinocytes
IgG
in
subclass
cases.
producing
Using
intercellular
anti-IgG3
subclass
staining
an
between
intracellular
immunofluorescence was detected in 62.2% of the cases and in 8.6% of the
controls (p<0.05), with titers ranging between 1:20 and 1:80. In skin lesions
from 18 EPF-L patients, DIF showed an intercellular keratinocyte staining
with predominance of IgG1 and IgG4 subclasses and C3; controls were
negative. In 62.1% of EPF-L patients, IIF revealed a basal membrane zone
(BMZ) staining with IgM, C3 or IgG in a linear or granular pattern, not
detected in controls. By IB, 33% of EPF sera showed reactivity with a 160 kDa
band (Desmoglein-1) and 27% of controls. Moreover, 40% of EPF and 37% of
control sera showed reactivity against two bands of approximately 210 and 195
kDa also recognized by 37% of the controls from El Bagre. These bands were
not detected in other controls living out from the endemic area. On the other
hand, 93.3 % of EPF sera immunoprecipitated a 48 kDa peptide obtained after
trypsinisation and Concanavalin-A (Con-A) affinity chromatography from
bovine epidermis and in 10% of controls (mainly genetically related to EPF
people). ELISA assay showed a high correlation with IP. Five healthy controls
73
from the endemic area showed reactivity by IB, IP and ELISA techniques as
above described.
Quantification of total IgE serum levels: The ELISA assay to detect IgE
serum levels shown in the EPF group a mean of 1,009 international units per
liter (UI/L); a minimal value of 1 and a maximal value of 4,365 UI/L. In the
control group mean was 494.2 UI/L; the minimal value was 1 and the maximal
value was 3,404 UI/L. These studies showed statistical differences (p<0.05).
Cutaneous test: Only one EPF patient was positive for both antigens. No
controls were positive, with non-significant differences.
Environmental risk factors: All people of this study related more than 4
hours of sun exposure per day due to working outdoors. All the EPF-L patients
reported an exacerbation of skin lesions with sun exposure and with high
environmental temperatures. Exposure to chemical agents was found in 64.2%
of both groups, with substances such as fumigation products, cyanide, mercury,
and fertilizers. In the control group the exposure was only 20% (p< 0.05). A
100% of EPF-L patients worked in mining labors as well as controls.
Mercury levels in hair: In EPF-L group we found a mean of 9.95 parts per
million (ppm); the minimal mercury levels were 2.7 ppm and maximum of 144
ppm. In the control group we found a mean of 4.34 ppm: a minimal mercury
74
level of 0.8 ppm and a maximum of 9.75 ppm. These results showed statistical
differences (p< 0.05). In four acute cases of less than two months of evolution,
we found higher levels of seric IgE and hair mercury. ELISA OD readings were
in the top of the EPF-L group and them immunoprecipitated with more
intensity the 48 kDa PF antigen. Presence of others bands different than Dsg1
were not detected and the intracellular staining with IgG3 was negative. One
of these cases with a generalized form of one month of evolution showed the
higher levels of mercury (144 ppm), IgE (3,240), intercellular IgG4
autoantibodies (1:320), ELISA 0D 492 (1.5 and cut off 0.1) and IP (four plus, in
a scale 1 to 4). After one year of treatment with steroidal therapy, these
patients decreased autoantibodies titers, clinical activity and IgE levels. In
three recalcitrant cases (6%) (one died), the levels of mercury, IgE and
autoantibodies were higher compare with the other EPF-L people. After
metrotrexate, metilprednisolone pulses,
cyclophosphamide and properly
nutrition, all these factors decreased.
Other environmental risk factors: River fish and wild animal consumption
was similar in both groups as well as the presence of intra-domiciliary rodents.
In 100% of both groups’ mosquito bites were reported especially at nighttime.
No entomological studies were performed. Almost all EPF-L and control
subjects have 70% of the skin surface exposed during work and rest time. No
differences in the dietary habits were found and the basic diet consisted of 60%
carbohydrates, 30% lipids and 5% proteins determined by questionaries. Both
75
groups showed low protein intake. In both groups cohabitation with domestic
pets and raising of animals were 80.92% (dogs, cats, cows, horses, pigs and
chickens). Fifteen per cent of the patients related aggravation of the disease
with alcohol and 30% with pork ingestion. More than 50% of EPF-L subjects
did not know what triggered their disease.
DISCUSSION
Clinical and epidemiological characteristics: As described in the fogo
selvage foci in Brazil, most of the EPF subjects have a localized form. 4,5,8
However, the commonest clinical form resembles Senear-Usher syndrome, not
commonly described in patients with FS.34 No clear correlation exist between
the broad clinical spectrum of EPF disease and the local and systemic immune
response. Acantholysis produced by autoantibodies directed against Dsg1 do
not seem to explain alone the wide disease spectrum, at least in this focus. The
predominance of clinical lesions in seborrheic and photo exposed areas was an
important finding; an association between sun and EPF-L disease can be
possible in this focus and it could have a direct or indirect role in the
pathogenesis of this disease. The lesions in these EPF-L patients only
compromise some anatomical areas. We can speculate that the expression of
the antigens that intervene in the cell-to-cell adhesion are distributed in a
peculiar manner as recently showed by Shiratakata et al37. Conjunctivitis was
reported in some EPF patients by Arneondola et al38 and Lever et al,39 however
no further reports were described. It is possible that this mucosa present
76
differences in relation to PF antigens distribution. In our cases, the
conjunctivitis was detected in individuals with generalized conditions and with
difficult clinical control. Other aspect in which marked differences were found
compared with the Brazilian foci was sex. In this EPF-L focus, most of the
affected were men, in Brazil both sex is affected equally.2, 4-9,12 However, some
reports showed a slightly predominance of females.7,13-15 In Tunisia more than
80% are women.10,11 Although in El Bagre many women in the rural area were
also exposed like men to same activities and environmental factors, they do not
develop EPF-L disease; the reason for this, remains unexplored. Other
difference was age of onset of EPF-L disease; senior individuals were more
affected in contrast to Brazilian foci.4,5;8,12 In Tunisia women in a childbearing
age are more affected.11 People affected in this focus do not showed those
chronic manifestation of FS as nanism or other endocrine abnormality maybe
due, to the late onset of disease, instead they showed a marked symptoms of
depression.
The increased cases of this disease in El Bagre remain obscure and shows an
“epidemic” like tendency. Long epidemiological term studies will be necessary
to correlate with environment changes as well as epidemiological vigilance. In
Brazil, the Terena population showed a cyclic nature of EPF disease.9 Since
EPF disease start in El Bagre or in the boundaries municipalities, it’s
suggested that this rural area possess an environmental risk factor(s)
necessary for the development of EPF disease in one susceptible host.
77
Environmental risk factors: In Brazil, the black fly Simulium pruinosum
are associated with EPF disease since in some endemic areas it was reported
and based on a study in which apparently more EPF patients were bitten by
these mosquitoes than a control group.24 The related study was a questionnaire
retrospective one and presently non entomological studies had been reported.
Its importance like triggering factor of EPF-L disease in El Bagre is doubtful.
In El Bagre most of the patients live and work at cleared vegetation areas, in
the middle of the jungle. It´s known that Simulium mosquitoes do not
penetrate the jungle.40,41 Furthermore most of the patients live and work in
lower altitudes than those inhabited by Simulium sp. Recently entomological
studies in EB, showed no presence of Simulium sp in areas of EPF patients.43
It is known the deleterious effect of sun exposure on skin and the immune
system. After application of phototest on normal skin, patients with PF
developed clinical and histopathological lesions. In concordance with our
findings of sun exposure and aggravating of EPF disease-L.44,45 After longwavelength ultraviolet light irradiation the ratio of MHC Class II is increased
at lymph nodes, suggesting that persistent photosensitivity could potentiate
autoimmunity. Some membrane-associated proteins can be altered by crosslinking changes after UV exposure.47
78
Quantification of total IgE serum levels: In Colombia, the more common
cause
of
elevation
of
IgE
seric
levels
are
sensitization
against
Dermatophagoides pteronyssinus and D. farinae. We tested for these factors in
the subjects of study and no hypersensitivity was detected. This phenomenon
indeed, does not explain the elevation of IgE in EPF sera. The highest IgE
levels found in EPF-L group compared with the controls was previously
reported for others.35 After intradermal reaction in 16 FS patients and 16
controls using air fungi, house dust and D. pteronyssinus, a slight elevation in
the cutaneous response in EPF group with the air fungi was detected.47 Our
data differ of those, but we do not used air fungi. Atopy is also an important
factor for increased IgE levels in serum48, but it was only detected in 10% of
both groups High IgE levels may be also encountered in cases of IgE myeloma,
pulmonary aspergillosis, smokers and during the active stage of parasitic
infections.48 Smoke habit was similar in case and control groups and clinical
evidence of myeloma or pulmonary aspergillosis were not detected. An
autoimmune syndrome induced by mercury in rodents, with presence of
antinuclear antibodies (ANA), immune glomerulonephritis with linear IgG
deposits along the glomerular basement membrane, B cell hyper-expression of
class II MHC molecules, hyper IgE and isotype switching (IgG4 -IgE) has been
demonstrated.49-51 Based in the fact that the commonest autoantibodies found
in EPF people were IgG4 and IgE, we can suggest that in these patients the
isotype switching between IgG4 and IgE can be induced by mercury as occur in
the mouse exposed to mercury and D-penicillamine.
79
In El Bagre mercury is commonly used to amalgamate gold in the purification
process increasing the levels in this area. Other factors can also increase
mercury levels in El Bagre: 1) acid soil, 2) high environmental temperatures, 3)
high rates of forest fires, and 4) large deforestation problem. Air saturated with
mercury vapor at 20oC contains a concentration that exceeds many times the
toxic limit, resulting in increased adsorption rates, at higher temperatures. 52-54
The range of temperature in El Bagre is between 30-42oC, contributing to high
rates of mercury evaporation. In the Amazonian rain forest, mercury emissions
derived from wood combustion are high, creating short emissions pulses of
mercury.52,53 Natural mercury levels in plants range from 0.001 to 0.1 p.p.m.
(dry weight); after the Amazonian deforestation, aquatic macrophyles have
high mercury levels similar as the detected in El Bagre. Since a century ago
EPF has been associated to areas subjected to a high deforestation rates and
mining business.4 Mercury pollution was also reported in neighbors areas to El
Bagre in the biotic chain including humans (Caucasia and Segovia) and some
cases of EPF had been observed in these areas.54 Recently in PF patients a Th2like cytokine profile had been showed by Lin et al,57 very similar to mercury
induced autoimmunity in mice, characterized by increased T-cells helper
activity, high IL-4 production and decrease of IL-2 production.
Mercury could induce autoimmune responses by its effects on the structural
integrity and/or functions of organ and tissues. In addition the autoimmune
phenomena may be caused by direct action on cells of the immune system, by
80
inhibition of T-suppressor lymphocytes or/and stimulation of T helper cells, B
lymphocytes as well as macrophage activity.57 Various subpopulations of T
lymphocytes are affected by mercury as RT6+ subset of T lymphocytes (which
have a regulatory role in various rat models of autoimmune disease), this are
decreased in mercury treated BN rats.58 Mercury also can increase expression
of MHC class II molecules in B cells.59 Mercury can also stimulate macrophage
and other antigen-presenting cells to become more active in epitope
presentation and production of cytokines.58 Experimental and clinical data
strongly suggest that mercury induced autoimmunity phenomena in mice is
linked to certain MHC genotypes.59 In many Brazilian EPF patients a MHC
class II may confer susceptibility to EPF.19 In H-2S mice, (corresponding MHC
class II in human) the administration of mercuric chloride results in the
development of autoimmune syndrome; these mice represent a valuable model
to study the role of environmental factors in the development of systemic
autoimmunity.62
In summary, this focus of EPF-L disease differs from other EPF foci in many
aspects as clinical, epidemiological, immunological features. We confirmed the
unique nature of this focus with electron microscopic studies which will be
matter of a next publication. Based in our results, we suggest that a
combination of a specific genetic background (that is being tested), sun and
mercury exposure could play a role in developing autoimmunity in people from
81
this focus. More studies should be performed to identify with precision the
other environmental factors associated to development of EPF-L disease in this
focus and the endogenous genetically determined factors that could create the
conditions for the autoimmune phenomena observed in EPF-L patients.
Acknowledgments: This work was supported by grants from the Direccion
Seccional de Salud de Antioquia, Mineros de Antioquia S.A and U de A, (Abreu
et al, Grant # 04525l). We would also like to extend our thanks to the following
for their support: Instituto Colombiano de Medicina Tropical for scientific
support, Hogar del Niño for supplying food for the patients, Fundacion Elena y
Juan for providing medical supplies, Procolores S.A. for film supplies, Hospital
Nuestra Señora de El Bagre, Alcaldia de El Bagre, Dr Luis A. Diaz and the
Department of Dermatology of the Medical College of Wisconsin (MCW)
(U.S.A), for technical support and advise. We like to extend our thanks to the
follows Institutions: Colciencias, LASPAU and MCW. We would also like to
thank the people of El Bagre, and especially the EPF patients. We also like to
thanks to Chris Tenaglia for corrections to this manuscript. This paper is part
of the Ph.D. thesis of Ana Maria Abreu-Velez MD as an immunologist at the U.
de A. Dr. Abreu is the recipient of a scholarship from Colciencias, Colombia.
82
LEGENDS
Fig 1. Geographical and demographic distribution of El Bagre municipality in
Colombia. According to the last census of Colombia. Colombia: 36',500,000 inhabitants;
Antioquia: 4,225,930; Medellin, Capital of Antioquia: 3,143,063; El Bagre: 27,324.
Politically and geographically El Bagre belongs to one subdivision of Antioquia State
called "El Bajo Cauca," with 137,023 inhabitants with 90,832 in the urban and 46,191
in the rural area. El Bajo Cauca includes the municipalities of Nechi, Taraza,
Zaragoza and El Bagre. El Bagre has 27,324 people, 22,151 in the urban area and
5,178 in the rural area. All EPF patients belongs to rural area. El Bagre has two main
rural area: Puerto Claver (latitude north, 70o 45'; longitude east, 74o 44') and Puerto
Lopez (latitude north, 7o 49'; longitude east, 77o 05').
83
Fig 2. Gold in El Bagre. Gold is extracted from the rivers using a panning technique and by
underwater swimming to obtain gold from riverbeds (A). The main source of gold in El Bagre is
alluvium, but the gold is also found 30 to 40 meters below the ground level. To mine gold at
these depths, backhoe extraction (B) and dredging techniques are commonly used. C. A big
dredging machines are used for industrial gold extraction.
84
Fig 3 Characteristics of people affected by EPF from El Bagre. The most common clinical form
of EPF in patients from El Bagre was the localized with a lupus-like aspect (A) and prurigoid
(B). (C) Illustrates a typical Intercellular staining between keratinocytes by IIF. (D) shows
facial and thoracic compromise, (E) a bullous exfoliative form and (F) a crust and prurigoid
form in a seborrheic area.
85
Fig 4 Broad clinical spectrum of EPF disease: This series of pictures illustrates the broad
clinical spectrum in the same patient. Patient with a keratotic generalized form (A); a
generalized exfoliative form (B); a generalized ichthyosiform form (C) and a localized form (D).
86
Fig 5. Epidemic Tendency in people affected by Endemic pemphigus-like disease in El Bagre,
Colombia.
EPIDEMIC TENDENCY DETECTED IN THE ENDEMIC FOCUS
OF EPF DISEASE IN EL BAGRE, COLOMBIA
NUMBER OF
CASES
25
20
15
10
5
0
1960-1969
1970-1979
1980-1989
PERIOD OF TIME
1990-1998
87
TABLES
Table 1.
Histopathological features in people with endemic pemphigus “like”
disease in El Bagre, Colombia:
42.3% pemphigus foliaceus.
23% chronic dermatitis.
15.4% pustular dermatitis.
29 % lupus mixed with pemphigus.
7.7% psoriasiform dermatitis
3.3% Darier´s like-disease
88
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98
X. IMMUNOLOGICAL CHARACTERISTICS OF PEOPLE AFFECTED
WITH ENDEMIC PEMPHIGUS FOLIACEUS-LIKE DISEASE FROM
ONE FOCUS IN THE RURAL AREA OF EL BAGRE, COLOMBIA,
SOUTH AMERICA.
Abreu Velez Ana Maria, &^ Prada Stella, & Montoya Fernando^.
^Biomedical
Basic
Science
Corporation
and
&Section
of
Dermatology,
University of Antioquia, Medellin, Colombia.
Corresponding author: Ana Maria Abreu Velez. M.D., Ph.D, Corporación
Ciencias Basicas Biomedicas, Universidad de Antioquia, carrera 51 D # 62-29,
AA 1226, Medellin, Colombia, South America. Fax: (574) 263-3509. Phone:(574)
2637667. E-mail: fmontoya@catios.udea.edu.co.
Key words: Endemic pemphigus foliaceus; Autoimmunity, Desmosomes, Basal
Membrane Zone, Cell envelope.
Short Title: Immunological characteristics in people affected by endemic
pemphigus foliaceus-like disease in El Bagre, Colombia.
Abbreviations: EPF, endemic pemphigus foliaceus; PF, pemphigus foliaceus;
PV,
pemphigus
vulgaris;
Dsg1,
Desmoglein
1;
IIF,
indirect
immunofluorescence; DIF, direct immunofluorescence; IB, immunoblotting; IP,
immunoprecipitation; ELISA, enzyme-linked immunoabsorbent assay; BMZ,
basal membrane zone, CE; cell envelope.
ABSTRACT
99
The purpose of these case-control studies was to characterize immunological
findings and presence of skin autoantibodies in people affected by endemic
pemphigus foliaceus-like disease from one focus of El Bagre, Colombia and to
compare them with findings reported in other foci. We studied 50 patients and
50 controls from the endemic area (matched by age, sex, working activity and
living area). Presence of autoantibodies in serum and skin was analyzed by
direct and indirect immunofluorescence, immunoblotting, immunoprecipitation
and by ELISA. The presence of an IgG4 subclass, producing intercellular
staining between keratinocytes, was detected in 80% of patients by indirect
immunofluorescence using human foreskin. Moreover, using the anti-IgG3
monoclonal antibody, an intracellular immunostain (but not intercellular) was
detected in 62.2% of the cases and in 8.6% of the controls (p<0.05), with titers
between 1:20 and 1:80. This finding was never previously described in people
from a focus of endemic pemphigus foliaceus. Notably, in 62.1% of patients,
direct immunofluorescence revealed not only intercellular stain but also
immunostain of the basal membrane zone with IgM, C3 or IgG in a linear or
granular pattern, corroborating with alterations at the basal membrane zone
detected by hematoxiline-eosin and by electron-microscopy. Although, in early
Brazilian literature and in some cases of endemic pemphigus foliaceus from
Tunisia, a basal membrane zone stain was reported, this phenomenon was
never studied in depth. By immunoblotting using normal human skin as a
substrate, 30% of EPF sera showed reactivity against linear epitopes of
Desmoglein-1 (160 kDa band) as well as in 27% of controls from the endemic
100
area. Moreover, 40% of the patients and 37% of controls from the endemic area
showed reactivity against two bands of approximately 210 and 195 kDa. These
bands were not detected in other controls living out of the endemic area and
have never been described in patients with EPF from the Brazilian and Tunisia
foci. The nature and localization of these antigens is in process. On the other
hand, 93.3 % of sera from patients and 5% of controls from the endemic area
recognized conformational epitopes by immunoprecipitation from a 48 kDa
bovine epidermal antigen obtained by affinity chromatography. However, our
patients also showed reactivity against aN 80, 66, 62 and 38 KDa bands. We
also performed an indirect ELISA assay using the bovine epidermal antigen
with a high correlation with IP. Five healthy controls from the endemic area
also showed reactivity by immunoblotting, immunoprecipitation, and ELISA
against pemphigus foliaceus antigens. The immunological data reported for us
suggest that people with endemic pemphigus disease from this focus show
immunological differences compared with people from other foci and evidence a
complexity in the immune response in people from this focus.
INTRODUCTION
Endemic pemphigus foliaceus (EPF), is the endemic form of PF.1 EPF was
described in foci at the South America tropical forest, mainly in Brazil, 2,3,4
better known as fogo selvagem (FS) in Colombia,5-8 and Tunisia.9 It is accepted
that EPF is characterized by epidermal acantholysis and subcorneal blisters
101
with a predominance of IgG4 subclass antibodies directed against the
intercellular
components
of
desmosomes
by
direct
and
indirect
immunofluorescence (DIIF, and IIF).10,11 Generally, people with EPF have
autoantibodies against a desmosomal glycoprotein called Desmoglein 1
(Dsg1).12-16 This 160 kDa glycoprotein belongs to the cadherin family of
calcium-dependent cell adhesion molecules.12,16 A focus of endemic pemphigus
foliaceus like disease (EPF-L) exists in El Bagre, sited in a subtropical mining
village of Colombia´s jungle affecting people living in or around the colonized
surrounding areas. We reported some clinical and epidemiological differences
by comparing people affected by EPF from EL Bagre, Colombia, with FS
patients from Brazil and EPF patients from Tunisia.17 In the focus from El
Bagre, prevalence was detected of 1.8%. Men were the most affected (93%),
with a mean age of 50 years and were mostly dedicated to mining and/or
farming activities. A clinical form, sharing characteristics with Senear-Usher
like syndrome, was the most common.17 The cause of EPF is unknown, but
some possible environmental risk factor(s) were found in the patients with
EPF-L from EL Bagre, such as high exposure to solar radiation and
deterioration with sun exposure and high mercury levels in hair compared to
the control group.17 The heterogeneity of immune response in PF disease has
been demonstrated by the presence of autoantibodies against other proteins
different from Dsg1.18 The purpose of this study was to describe some
immunological aspects of this focus and compare them with those reported in
102
the Brazilian and Tunisian foci as well as in other varieties of pemphigus
foliaceus disease.
MATERIALS AND METHODS
Subjects of study: In six years of follow up we studied 50 patients with
clinical diagnosis of EPF-L, based on Viera´s20 criteria. All subjects in this
study participated willingly and signed a consent form. We confirmed their
EPF disease mainly by immunological criteria, according to Diaz et al.4 50
patients and 50 controls from the endemic area (matched by age, sex, working
activity and living area). Table 1 summarizes the subjects of study including
the clinical forms of EPF-L and the controls.
Biological samples: Skin biopsies were tested by routine hematoxiline and eosine (H&E) stains, skin biopsies for immunofluoresecence and blood sera
were used for detection of autoantibodies against PF antigens.
Detection of antinuclear antibodies (ANA) in EPF patients: We test for
the presence of ANA using a commercially available ELISA kit (EL-ANA
Profiles, INCSTAR Co, Sillwater, Minnesota, U.S.A). We searched for the
following antibodies: single stranded DNA (ssDNA), double stranded DNA
(dsDNA) and extractable nuclear antigens: small ribonuclear proteins
(sm/RNP'S), Ro and La.
103
Anti-epidermal autoantibodies in the EPF and control group: Indirect
immunofluorescence (IIF): Sera from patients and controls were titrated in
calcium-supplemented buffers at 1:20 and 1:40 dilutions; positive sera were
tritrated to the end point using cryosections of human foreskin and fluorescent
isothiocyanate (FITC) conjugated goat antihuman IgG and for IgG-subclass
reactivity by IIF with murine monoclonal anti-human IG-subclasses antibodies
as previously described.10,11
Direct immunofluorescence (DIF): It was carried out aleatory and blindly
in half of EPF patients using negative and positive PF controls. Skin
cryosections from EPF patients were tested using FITC conjugated monoclonal
anti-human IgG1, IgG2, IgG3 and IgG4, (Sigma, Biosciences, St Louis,
Missouri) (at 1:64 to 1:128 dilution) and FITC-conjugated rabbit antihuman C3
(1:75). For IgA detection FITC-conjugated rabbit antihuman IgA specific (alpha
chains) for IgM FICT-conjugated rabbit antihuman IgM, (Mu-chain) (Dako,
Denmark). For a total IgG determination, we used FITC-conjugated rabbit
antihuman IgG, (gamma-chain) at dilution 1:40 to 1:100 (DAKO, Denmark).
Immunoblotting (IB): The sera were tested for reactivity against Dsg1. This
was carried out as previously described, by Koulu et al,11 using total sodium
dodecyl sulfate extracts of human and bovine epidermis fractionated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to
the Laemmly20 method and transferred onto a nitrocellulose membrane, based
104
on the Stanley et al23 procedure. To improve the detection of other epidermal
antigens, different from Dsg1 and recognized in the El Bagre people, we
performed other extraction techniques: 1). using SDS buffer (62.5 mM, TrisHCl, pH 6,8), 5% BME, 2% SDS, 0.1 mN PMSF, 1mM EGTA, 1 mM
ditriotriethanol (DTT) and 3 mM EDTA. 2). With urea 8 M, 1 mM DTT, 0,1 M
BME and 50 mM sodium phosphate, pH 7.0. 3). By utilizing 62.5 mM Tris-HCl
(pH 6) containing 0.8M sodium chloride and extracted with 1.6% sodium
dodecyl sulfate, 10% glycerin and 5% BME at 100oC.23 Samples were run on
SDS-PAGE at 6, 7, and 12% gels.
Immunoprecipitation (IP): All sera were tested by using I125-labeled 48 kDa
tryptic fragment obtained from bovine epidermis subjected to Concanavalin-A
(Con-A) affinity chromatography.24-26
ELISA: Briefly, sera were tested for the presence of autoantibodies against the
bovine epidermal extract obtained after trypsinization and Con-A affinity
chromatography purification as an antigen source. Antigen extract was crosslinked with glutaraldehyde 0.25% in a phosphate buffer (PBS) to the wells and
the sera were used at 1:100 dilution in calcium supplemented buffers. As a
blocking agent a 10% modified lactate buffer was used. As second antibody
horseradish peroxidase (HRP) conjugated monoclonal murine anti-human total
IgG was added, in calcium supplemented buffers at 1:20.000 dilutions, and
finally, o-phenylenediamine (OPD) buffered solution was incorporated. The
105
reaction was stopped with 2 N sulfuric acid. Optical density at OD492 nm was
read in a spectrophotometer Sophelia 1000.27
RESULTS
General description: In 74% of people affected by this disease we observed a
localized form with predominance of photo-exposed and seborrhoeic areas and a
prevalence of clinical findings resembling Senear Usher syndrome.
ANA: We had no positive findings against ssDNA, dsDNA, sm/RNP, Ro and La.
Histopathology: Briefly, the diagnosis of PF was performed in 42.3%; lupuslike in 32%; chronic dermatitis in 23%; pustular dermatitis in 15.4%; unspecific
pemphigus in 11.5% and psoriasiform dermatitis in 7.7%. In 32% mixed
features of lupus, luminic polymorphic eruption and mixed connective disease
were detected. In acute cases, acantholysis and epidermal clefts were observed
in 42.3% and spongiosis in 17%. Only in 42.3% of cases did we make the
histopathological diagnosis of classical pemphigus foliaceus. Chronic acanthosis
was detected in 91% of the biopsies; hyperparakeratosis in 81% and
papillomatosis in 37%. Skin atrophy was observed in 30%, associated with the
cases that showed a liquefaction of the BMZ and histopathological patterns of
lupus (hyper-paraqueratosis, atrophy of Malphigian stratum, liquefaction of
the BMZ and follicular appendage infiltrated and flatening of BMZ). We
observed unfrequently eosinophilic spongiosis. Some features resembling a
106
luminic reaction were observed as an enlargement of blood vessels, an
increased number of melanophages and lymphoid cells infiltrating especially
around blood vessels. Dermal edema especially at the papillary dermal region,
with mixoid chages in collagen and in some people with deposits of fibrin
resembling the collagenization that occur in scleroderma were observed.
Multiple alterations at the sweat gland level were observed in 40% of the cases
consisting in focal necrosis, spongiosis of the acrosiringium, hypertrophy,
hyperplasia, and cellular infiltration of sweat glands28.
Presence of anti-epidermal autoantibodies by IIF: Total IgG was
evidenced in 40% of EPF-L group, showing the intercellular staining commonly
described in PF disease. Positive results were not found in the control group.
8.81% of EPF patients were positive with IgG1 and none of the patients or
controls showed a positive results using IgG2 and IgG3. On the other hand,
80% of EPF patients showed positive intercellular staining using IgG4 (Fig
1.A). In general, titers were low (betweeen 1:20 to 1:320). No controls were
positive. Many patients were under treatment with corticosteroids with doses
spanning from 10 to 60 mg of prednisone/day, however, correlation between
medicine
intake
and
autoantibodies
detection
was
not
carried
out.
Interestingly, when the IIF was performed using anti-human IgG3 monoclonal
antibody, an intra-cellular staining was detected in 62.2% of the cases and in
8.6% of the controls (Fig 1.B.). No stain was simultaneously detected at the
107
ICS with IgG3. Titration of those sera was performed and titers between 1:20
to 1:80 were found.
Presence
of
anti-epidermal
autoantibodies
by
direct
immunofluorescence (DIF): By DIF all patients studied showed the classical
intercellular staining (ICS) using antihuman Total IgG. In 70% cases the
presence of IgG1 and IgG4 was simultaneously found. In 20% biopsies, deposits
of the IgG4 subclass were evidenced, exclusively. Only in 2% was the IgG3
subclass found. In 50% of the biopsies from EPF-L patients we detected the
presence of C3 deposits simultaneously with IgG1 and IgG4 deposits. In 20%,
deposits of IgM were evidenced and in 10%, deposits of IgA. In half of (61. %)
biopsies from EPF-L patients an immuno stain was also observed at BMZ as
following: In 10%, deposits of IgM in granular pattern was observed. In 20%,
there were BMZ staining deposits of IgM in a linear fashion. In 30%, deposits
of C3 granular and in 40% linear pattern were found. In 30%, deposits of IgG
were observed as follows: 10% patient IgG1 subclass and in 20% IgG4, all in
linear fashion. Presence of IgM and C3 was found simultaneously in 20% (Fig
1.C).
Presence of anti-epidermal autoantibodies by immunoblotting (IB): By
IB, Dsg1 autoantibodies were analyzed using human and bovine epidermis as
antigen targets. After SDS-PAGE separation and immunoblotting, reactivity
against Dsg1 was found in 30% of the EPF group and in 21.7% in the control
108
group from El Bagre. Moreover, EPF and controls sera from the endemic area
showed reactivity against other bands using the human and bovine extracts:
43% of the EPF group and 44% of the controls. Bands of 195 and 210 kDa were
recognized in 40% of the patients and 37% of the controls. These bands were
better visualized using bovine and human epidermis treated with 0.8M sodium
chloride and extracted with 1.6% sodium dodecyl sulfate and 5% BME. The
concomitant presence of this doublet with other bands between 116 and 67 kDa
were found in 11 % of the controls and in 66% of the patients. The IB from
normal controls, non-resident in the endemic area, showed no reactivity
against the mentioned bands. In 17% of EPF sera, bands of 116 and 67 kDa
were present. In no one of the control group were those demonstrated (Fig 2).
Presence of anti-epidermal autoantibodies by immunoprecipitation
(IP): IP was performed in 50 EPF sera. 95% were highly positive against a 48
kDa peptide, whereas 3 EPF sera were weakly positive, which correlated with
the clinical status of the patient and the ELISA results. EPF group also
immunoprecipitated other bands. In some cases, bands at 80 kDa and a little
lower than 62 kDa (55%) were detected, while a 60 kDa band was found in 88%
of
EPF
group
(Fig
3).
Three
controls
from
endemic
area
also
immunoprecipitated the same 48, 62, 66 and 80 kDa bands. In normal controls,
non-residents of the endemic area, no positive results were found.
109
Presence of anti-epidermal autoantibodies by ELISA: By ELISA using
this method 97% EPF sera were positive and three EPF patients were
negatives. These three sera belong to the patients described in methods with a
very mild clinical form, under topical steroid therapy. Five controls from the
endemic area were positive. Three of the five controls are genetically related to
EPF patients. Other controls that were positive by the ELISA assay, also
showed the presence of other autoantibodies shared with EPF patients by other
immunological techniques (Table 2). Table 3 is shows in detail the presence of
skin autoantibodies detected in people with EPF-L from El Bagre, Colombia,
donors from the endemic area (NDEA) and unaffected relatives living in the
same house of patients (R). Also, in unaffected were none-relatives living in the
same house (C) and normal donors (DEA) from the endemic rural area. Results
are expressed in percentage of especificity. Normal donors outside Colombia
(DOC), non-endemic pemphigus foliaceus from Colombia, but outside the
endemic area Colombia (NEPF) were also evaluated.
DISCUSSION
Table 3 illustrates the diverse clinical, immunological, and epidemiological
characteristics found in the different forms of PF (PF Cazanavee, PF SenearUsher, FS and Tunisia (EPF).
Based on the literature regarding the EPF focus and patients, clinical
epidemiological and histopathological, differences have been noticed between
the Brazilian, Tunisian and El Bagre focus in Colombia. Brazilian FS are
110
younger and affected equally by sex.2-4 Most of the Tunisian patients are
women of childbearing age.9 Most of patients in El Bagre are men in the fourth
to sixth decade.17 Differences were also detected by race and occupation and
environmental risk factor as possible triggering of EPF disease (Table 3).
In regard to the autoimmune response, heterogeneity have been detected in the
patients affected for the above-mentioned diseases (Tables 2 and 4).9,4,11,17
Presence of other autoantibodies different than Dsg1 also has been described
by Calvanico et al.26 Also by immunoblotting autoantibodies different from
Dsg1, by molecular weights have been reported in PF disease. For the first
time in the literature, we are reporting in patients affected by endemic
pemphigus foliaceus presence of a doublet of autoantigens (195 and 210 kDa
bands) and other of 97 and 117 kda bands. Using the same procedure, same
antigen source, same conditions, for the immunoblotting, immunofluoresence
and immunoprecipitation, a large sampling of FS patients were studied with no
report of the autoantibodies as demonstrated in our patients.29 Unfortunately,
the results of the assays on Tunisian EPF patients have not been reported.9
By indirect immunofluorescence in FS patients from Brazil similar patterns to
those observed at the intercellular space of keratinocytes and mainly IgG4
occur as in our patients. However, regarding the subclasses of IgG, our EPF
patients have been observed with a few cases of IgG1 and no cases of IgG2 and
no IgG3. This differs from Brazilian cases and is more likely to Tunisian cases.
111
It is important to remember that the isotype of the immunoglobulin depends on
the antigen and other factors. By IIF a positive intracellular staining and not
intercellular with IgG3 monoclonal antibody using anti-human mAb IgG3 was
detected in EPF patients. This indicates the presence of an intracellular
antigen, probably for the isotype of the monoclonal antibody, does not
correspond to an internalization process of the intercellular desmosomes, since
this mAb was not producing ICS stain. Also, since presence of intracellular
vesicles with electrondense material were detected in patients from El Bagre
by electron microscopy (Abreu et al, manuscript in preparation).
The direct immunofluorescence is more sensitive as demonstrated in our EPF
patients. By DIF autoantibodies against components of BMZ were visualized
and Morini et al9 reported a BMZ staining in 2 FS patients with Total IgM
(2/16) and one case with C3 (1/16). In addition, they also reported deposits of
C3 and IgG at BMZ, in 7 other patients (7/25). In Brazil 3 FS patients report a
refractory response to corticosteroid treatment, however, no more has been
reported regarding the BMZ stain.3 The response against intracellular
components of keratinocyte also have been reported by others in PF sera but
never associated with EPF patients.30,31
In Senear-Usher syndrome, the presence of autoantibodies against components
of desmosome, BMZ and in some patients, antinuclear antibodies, have been
described.3 Although most of the reports describing immunological features in
112
these patients are lacking in other assays different from immunofluorescence.
Moreover, in 60% of the EPF-L patients there is high correlation with a
Senear-Usher syndrome for clinical, and by immunofluorescence results. No
reports using mAb against IgG3 has been mentioned in Senear-Usher patients
producing the pattern detected in our EPF patients.
The immunoreaction against BMZ components in EPF-L from El Bagre,
correlates with histopathological (H & E) findings detected at BMZ in 30% of
our patients28 and with acanthosis at the BMZ detected by electron microscopy
(Abreu et al, manuscript in preparation). These facts make possible for these
EPF patients to have autoantibodies in addition to Dsg1.
The presence of immunoreaction at BMZ may indicate an epiphenomena or an
immune response against molecules that maybe are commonly located in the
desmosomes and hemidesmosomes as the recently identified envoplakin (PM of
210 kDa) and periplakin with (PM of 195 kDa) and that co-immunoprecipitate
together.31 Future studies will be performed testing for these factors.
As previously demonstrated in PF sera most of the epitope against Dsg1 are
conformational as corroborated in our patients by IB and IP results. By IB and
IIF using bovine tongue and normal human skin as antigen source, reactivity
against two fractions of 190 and 180 kDa antigens have been previously
demonstrated in PF patients. One third of the sera from patients with
113
pemphigus foliaceus and vulgaris recognizes these bands on SDS-PAGE.29 This
was shown by using a human monoclonal autoantibody that binds components
at desmosomal plaque by immuno electron microscopy. These autoantibody
populations were mostly IgG1 or IgG3 subclasses. The antigen’s target was also
immunolocalized at the desmosomal plaque by immune electron microscopy.
These doublets differ from Desmoplakin I or II.
29
Using salt split skin, with
sera from PF and pemphigus vulgaris (PV) an immunostaining by IIF at
epidermal side was also detected. The reason for that remains unknown.30 This
fact correlates with our findings. Other antibody populations directed against
an intracellular desmosomal plaque component (s) in PV and in PF were also
reported.31 The relationship between the doublets of autoantibodies detected for
us and the above-mentioned remains unknown.29 In paraneoplastic pemphigus
(PNP) a 180-210 kDa doublet has been also reported by Anhalt et al.33 The 210
kDa band was demonstrated to be an envoplakin, a protein that is part of the
cell envelope fraction, closely related to desmosomal plaque and also localized
at BMZ. In PNP was recently described with the presence of autoreactivity
against other proteins as Desmoplakin I, II, BP 230, and desmoglein 3. 18,31,32
Our findings support a heterogenous immune response. Factors, such as the
environment of El Bagre, the antigen can produce mimecry of the immune
system in these patients or modification of their own antigens by chemicals or
sun
exposure
may
be
responsible
for
the
heterogeneus
clinical,
histopathological, and immunological immune response in EPF patients from
114
El Bagre. The genetic background also can exert an unknown and unexplored
role in the multivariety and broad range of these autoimmune disease.
A further analysis using sera from patients and controls from the endemic area
of EPF-Like, will be immunoblotted and electro eluted with sodium citrate and
used for electron microscopy analysis to localize the antigens, to compare
colocalization with other proteins as desmogleins, desmocollins, envoplakin,
periplakin and to demostrate or not the relationship with desmosomes,
hemidesmosomes and other structures. Since an endemic form of an
autoimmune disease is available for the study of the immuno localization of
new antigens, their significance in cell-cell and cell matrix junctions will
provide new information in the comprehension of these junctions.
High serological reactivity against PF antigens in the control group from the
endemic area is also interesting, and has been previously reported in FS. 3,34 We
may suggest that the patients possess some genetic background that confers
the ability to develop EPF disease, which is absent or partially expressed in
"normal" subjects from the endemic area who are exposed to the same external
noxae. Also, it’s possible to speculate that some of the "normal" controls with
immunological abnormalities could be in a period of breakdown of their
tolerance and maybe will develop EPF-L disease. Long term studies will be
needed. In five years of following up of EPF-L patients from El Bagre, we
observed an overly complex disease with a broad range of clinical
115
manifestation, with tendency to an epidemicity with wide clinical spectrum
simulating Senear-Usher-like syndrome. It seems to be mediated for a complex
of many immunological alterations rather than for the simple acantholysis due
against Dsg1.
In summary, this focus of EPF-L differs from the FS foci in some
epidemiological characteristics and other environmental risk factors for
triggering the autoimmune phenomena. We also detected some immunological
differences not previously reported in the Brazilian foci as the simultaneous
intracellular stain, the reactivity against BMZ components and ICS and
antigen heterogeneity in addition to Dsg1. We propose that this EPF focus,
belongs to the big syndrome called endemic pemphigus foliaceus-like, that
includes some partially characterized entities such as PF of Cazanave, SenearUsher syndrome, FS, PF induced by drugs, EPF from Tunisia and probably
EPF from El Bagre.32 All these diseases share some clinical, immunological,
and histopathological characterisitics, but also differ in many aspects.
Acknowledgments: This work was supported by grants from the Direccion
Seccional de Salud de Antioquia, Universidad de Antioquia, Mineros de
Antioquia S.A. (Ana Maria Abreu et al, grant # 04525). We would also to extend
our thanks to the following people and Institutions for their support: Luis Diaz
and the Dermatology Department at the Medical College of Wisconsin,
Milwaukee, WI, U.S.A, Colciencias (Colombia) and LASPAU, (USA). We like to
116
acknowledge to the Immunodermatology Lab at the Medical College of
Wisconsin (MCW), Milwaukee, WI, USA since part of the IIF, IB, IP and
ELISA assays were performed in that Lab. We like to thank the people of El
Bagre, and especially to EPF patients. To Doris Ruiz Penagos (U de A), Argelia
Lopez-Swidersky, Marleen Janssen, and Monica Olague Marchan (MCW). We
also like to thanks to Chris Tenaglia for the correction of this manuscript. This
paper is part of the Ph.D. thesis of Ana Maria Abreu Velez MD as an
immunologist at U. de A. Dr Abreu is the recipient of a scholarship from
Colciencias, Colombia.
117
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Dermatol 1988; 91:189/91.
35. Castro RM, Augusto DAF, Rivitti EA. Sindrome de Senear-Usher e Fogo
selvagem (penfigo foliaceo endemico). An Bras Dermatol 1988;63 supl 1:264/5.
123
FIGURES
Fig 1 Immunofluorescence studies: A. Shows classical intercellular (ICS)
staining by IIF found in patients with EPF-L from El Bagre using IgG4 mAb
using normal human skin as substrate. B Shows intracellular staining (IIF)
using IgG3 mAb subclass, using normal human skin as substrate. C. It shows
immunostain (DIF) at intercellular spaces and basal membrane zone with total
IgG, IgM and C3 using skin lesions from EPF patients. All these figures were
visualized using 40X immunofluorescence microscope.
124
Fig 2. Immunoblotting: Sera of both patients and controls were tested for reactivity against
Dsg1. This was carried out with human and bovine epidermis fractionated by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose
membrane.22 Line 1 shows a serum from a patient with bullous pemphigoid (180 kDa band).
Line 2, normal donor. Lines 3 and 4 reveal two bands of 210 kDa and 195 kDa from EPF-L
sera,
non-linear
epitope
against
Dsg1
which
was
detected
in
this
patient
by
immunoprecipitation for the presence of conformational epitopes. Lines 5 belong to serum with
EPF-L with immunoreaction against a 160 kDa band (Dsg1). Broad molecular weights
standards were used.
125
Fig 3 Immunoprecipitation of patients and controls: This fig illustrates a serum
immunoprecipitation using the bovine tryptic fragment obtained after Con A- affinity
chromatography.23 Line 1 is a positive control with fogo selvagem from Brazil. Line 2 through 4
are ¨normal donors¨ from the endemic area. Lines 5 and 6 and 8 through 10 are EPF-L
patients. Line 7 is normal donor outside the endemic area. Molecular weight of the bands from
top to the bottom corresponds to 80, 66, 62, 48 kDa. We used broad molecular weights
standards as a markers 117, 96, 60, 45, 31 kDa.
126
TABLES
Table 1. Subjects that were included for the immunological characterization of this EPF-L focus.
EPF-L from
(n=50).
El
Bagre,
Colombia
Sera from normal people living in El
Bagre (n=50 ).
-Generalized bullous exfoliative- hyperkeratotic form (n=4).
-Hyperpigmented form (n=3).
-Localized form (n=36).
-Prurigoid form (n=6)
-Inactive form (n=1).
-In 60 % clinical picture od Senear-Usher syndrome.
-Unaffected relatives living in the same house of patients
{R} (n=10).
-Unaffected nonrelatives living in the same house {C}
(n=10).
-Normal donors from the rural area {NDEA} (n=30)
Serum from a well-characterized patients with a
generalized exfoliative
Generalized exfoliative form from Colombia but out from
endemic area {NEPF} (n=5).
FS serum from Brazil was used as
internal positive control (C+) (n=2).
PF
sera
from
the
Hospital
Universitario San Vicente de Paul
(HUSVP), Medellin (n=5).
Normal donors from Mexico, USA, Clinically healthy {NDOC}.
Spain, and Germany (n=5).
127
Table 2. Serological abnormalities in controls from the endemic area of El Bagre and relationship to EPF-Like
patients.
Controls
C1 (Brother)
C2 (Son)
C3(N/R)
C4 (N/R)
C5 (Nephew)
C6 (Son)
Dx
Healthy
Healthy
Ashy Dermatitis
Lichen
Planus
(-)
Healthy
Healthy
IIF-DIF
IP 45 kDa
(-)
(-)
(-)
(++)
(-)
(++)
(-)
(-)
(+)
(+)
(++)
IB 210 & 195
kDa
IB 160 kDa
(+)
(++)
(-)
(++)
(+)
(-)
(½+)
(½+)
(-)
(+)
(+)
(-)
IgG3 IC IIF
1:20
(-)
(-)
(-)
(-)
N/A
ELISA OD492
nm
0.462
0.243
0.36
0.31
0.348
0.358
All individuals were men. IIF: Indirect immunofluorescence detecting intercellular stain between keratinocytes. DIF:
Direct immunofluorescence detecting intercellular stain between keratinocytes. IP: Immunoprecipitation of a 45 kDa
bovine epidermal tryptic Con-A affinity fragment. IB: Immunoblotting against 160 kDa band (Desmoglein1) and
against 210 and 195 kDa bands (unknown). ELISA OD492nm: Detecting autoantibodies against the 45 kDa bovine
epidermal tryptic Con-A affinity fragment. N/R: Non-related to EPF-L patients, N/A: Non-Available.
128
Table 3 Illustrate a summary of clinical, epidemiological, and immunological features detected in people affected by
EPF disease:
FEATURES
AGE OF
ONSET
SEX
CLINICAL
BRAZIL (a)
First and second decade.
TUNISIA (b)
21-37.5 years
EL BAGRE (c )
30-70 years
Equally.
Multiple forms.
RACE
SYSTEMIC
ALTERATIONS
Indian/white
Nanism, azoospermia, loss of
hair
depression,
bone
dystrophy.
20 %
IgG4, IgG1C3.
IgG4, IgG1, C3 and rare IgA
and IgM.
Rare.
160 Kda.
45 kDa band.
Female.
Herpetiformis and
classic pemphigus.
White mediterranean.
N/A.
Men.
Senear-Usher-and
multivarieties.
Indian/white.
Depression, arthralgia
No.
IgG, C3.
IgA, IgG, C3.
20%.
IgG4.
IgG4, IgG1, C3, IgGM, IgGA.
7 out of 22.
N/A.
N/A.
50%.
210, 195, 160, 117, kDa.
45, 62, 66, 80 and 38 kDa
bands.
Most rural.
FAMILIAL CASES
IIF ICS
DIF ICS
DIF BMZ
IB
IP
GEOGRAPHICAL
LOCALIZATION
ENVIRONMENTAL
RISK FACTORS
Most rural.
OCCUPATION
Farming.
Non-predisposition.
and
S. pruinosum, Streptococci Stress
associated.
sp, virus.
N/A.
pregnancy
Sun exposure and mercury.
Mining and Farming.
N/A: non-available.
a. b. Morini et al, 1993: Bastuji et al, 1995.
c. Abreu et al, 1907.
** Only recently (Hans-Filho et al, 1997 showed prevalence in people older than 30 th.
129
Table 4. Presence of skin autoantibodies in people with EPF-L from El Bagre and donors from the endemic area.
Unaffected relatives living in the same house of patients (R), unaffected nonrelatives living in the same house (C),
normal donors (DEA). Results are expressed in percentage of especificity. Normal donors from out of Colombia
(DOC), non-endemic pemphigus foliaceus from Colombia, (NEPF) (Generalized cases).
ASSAYS
IIF ICS -IgG Total
IIF ICS, IgG 1
IIF ICS, IgG 2
IIF ICS, IgG 3
IIF ICS, IgG 4
IIF IC, IgG3
DI, ICS, IgG, IgM, C3
DI, BMZ STAIN
IB 160 kDa (Dsg1)
IB doublet of 195 and
210 kDa band
IB against 97 and 117
kDa bands
FS (n=29)*
60%*
90%*
90%*
0%*
100%*
N/R
N/R
29%
N/R*
R (n=10)
0%
0%
0%
0%
0%
20%
0%
0%
27%
37%
N/R*
10%
C (n= 10)
0%
0%
0%
0%
0%
10
0
0
15%
0%
0%
DEA (n=30)
0%
0%
0%
0%
0%
10%
0%
0%
15%
0%
DOC (n=5)
0%
0%
0%
0%
0%
0%
0%
0%
0%
0%
NEPF (n=5)
70%
10%
0%
0%
90%
10%
98%
0%
27%
20%
EPF n=50)
40%
5%
0%
0%
0%
11%
80%
64%
97%
61%
29%
40%
* Rock et al, N Engl Med 1989.IIF, ICS: Indirect immunofluorescence detecting intercellular stain between
keratinocytes.DIF: Direct immunofluorescence detecting intercellular stain between keratinocytes. IIF IC: IIF
detecting intracellular stain with IgG3 mAb. DIF BMZ: DIF detecting basal membrane zone stain. IB:
Immuoblotting against 160 (Desg1), 210 and 195, 96 and 117 kDa bands. IP: Immunoprecipitation of a 45, 62, 66 or
80 kDa bovine epidermal tryptic Con-A affinity PF antigens. ELISA OD492nm: Detecting autoantibodies against the
45 kDa bovine epidermal tryptic Con-A affinity fragment. N/R: Non-related to EPF-L patients.
130
XI. HISTOPATHOLOGICAL FEATURES IN SKIN BIOPSIES FROM
PATIENTS AFFECTED BY ENDEMIC PEMPHIGUS FOLIACEUS-LIKE
DISEASE FROM EL BAGRE, COLOMBIA.
Walter Leon Herrera*, Ana Maria Abreu Velez*and AB Ackerman
&
*Dermatology Section, Hospital Universitario San Vicente de Paul at
University of Antioquia, (U de A), Medellin, Colombia.
&
Institute for
Dermatopathology, Thomas Jefferson Medical College, Philadelphia (PA), USA.
Corresponding author: Ana Maria Abreu-Velez MD, Ph.D, *Seccion de
Dermatología, Hospital Universitario San Vicente de Paul, Bloque amarillo
(Medicina Interna), Universidad de Antioquia, Medellin, Colombia. Carrera 51
D # 62-29. AA 1226, Fax: (574) 263-3509, phone: (011) (574) 5106069. E-mail:
fmontoya@catios.udea.edu.co
Source of fundings: This work was supported by grants from the Direccion
Seccional de Salud de Antioquia (DSSA), Mineros de Antioquia S.A,
Universidad de Antioquia, (Ana Maria Abreu et al).
Short Title: Histopathology in patients with endemic pemphigus foliaceus-like
disease from El Bagre, Colombia.
Key words: Endemic Pemphigus Foliaceus, Autoimmunity, Histopathology,
Mercury, Sweat glands, Basal Membrane Zone, Photo damaged.
131
Abbreviations: EPF, endemic pemphigus foliaceus; PF, pemphigus foliaceus;
UV, radiation ultraviolet; IF, Immunofluorescence; BX, biopsy; Hematoxiline
and Eosin (H&E); BMZ; Basal membrane zone
Acknowledgments: We like to extend our thanks to the following’s
institutions: U de A and LASPAU and to DSSA. We would also like to thank to
the EPF patients. This paper is part of the Ph.D. thesis of Ana Maria AbreuVelez. MD as an immunologist at the U. de A. Dr Abreu is the recipient of a
scholarship from Colciencias, Colombia.
132
ABSTRACT
A study was performed in skin biopsies from 44 patients from a peculiar focus
of endemic pemphigus foliaceus-like (EPF) in El Bagre, Colombia. The
diagnosis of EPF was carried out by clinical criteria based on Vieras,
classification, epidemiological ascertain and immunologically by the presence of
intercellular stain between keratinocytes detected by immunofluorescence and
by immunoprecipitation of a bovine affinity epidermal extract that contains the
ectodomain of desmoglein 1. People living in this focus are exposed to high
levels of mercury pollution due to their mining activities, a marked
deforestation process, high environmental temperatures, and acidic soils extant
in the El Bagre area.
Skin biopsies were tested using hematoxylin-eosin (H&E) stain. We analyzed
29 EPF-L patients with a unique skin biopsy. 12 were tested with two different
skin biopsies. Two patients were tested with three biopsies at different clinical
stages. In people with a unique biopsy, we made the diagnosis of pemphigus
foliaceus in 42.3%, lupus in 32%, chronic dermatitis in 23%, pustular
dermatitis in 15.4%, non specific pemphigus in 11.5% and psoriasiform
dermatitis in 7.7%. The compromise of the basal membrane zone in these EPFL patients correlates with the presence of immunofluorescece and by
acantholysis detected by electron microscopy. We found necrosis of the sweat
glands with minimal inflamatory infiltrate in 40% of the skin biopsies the
"features that occur after barbituric and mercury poison." This characteristic
133
was never previously described in pemphigus foliaceus disease. Sweat gland
necrosis correlates with the presence of mercury in skin biopsies and is
associated with higher levels of mercury detected in the hair of people affected
for pemphigus, as compared with controls from the endemic area. In some
patients we detected in the same biopsy signs of mixed pathologies as
polymorphic luminic eruption, lupus, pemphigus, and mixed connective
disease. Histopathological findings in people with EPF-L from El Bagre, have
peculiar characteristics never previously described, that are correlated with the
other clinical, immunological and electron microscopical aspects.
INTRODUCTION
Pemphigus foliaceus (PF) and the endemic forms of PF (EPF) (Silva, 1935;
Viera, 1940; Proenca et al, 1976) are autoimmune diseases that belong to the
group of pemphigus (Korman, 1988). In this entity the presence of
autoantibodies at the intercellular space (ICS) of the keratinocytes is common
(Beutner, 1968; 1971). EPF was described in foci in South and Central America
mainly in Brazil (Viera; 1942; Azulay; 1982; Castro et al, 1983; Hans-Filho,
1996, Yepes et al, 1983, Morini et al, 1993) and in Tunisia. In Brazil, an
increased number of familial cases occurring especially in children and young
adults whose lifestyle was dedicated to farming activities (Viera, 1940; Azulay,
1982; Castro et al, 1983). Biopsies from most of Brazilian patients with acute
cases using Hematoxiline and Eosin (H & E) showed subcorneal bullae with
acantholysis and epidermal clefts (Lever, 1965; Schnyder, 1973). Old lesions
134
revealed acanthosis, a mild degree of papillomatosis, hyperkeratosis, and
keratotic plugging of the follicles. In areas of hyperkeratosis the thickness of
the granular layers was increased, and a striking change frequently observed is
the presence of dyskeratocis in cells from the granula layers (Ayalon, 1948;
Furtado, 1959). At dermal level, vasodilatation, and infiltration of lymphocytes
and histiocytes are often seen. In people affected by EPF from Tunisia, the
presence of eosinophilic spongiosis with acantholysis was frequently observed
(Morini et al, 1993).
After six years of a study performed in El Bagre, a small mining village in
Colombia, fifty EPF-L patients and normal controls from the same endemic
area matched by age, sex, geographical localization, and activities were tested
in order to characterize this focus. All subjects were living in the rural area and
performed outdoor activities such as mining and or farming. Most were men
(93%) with mean age of 50 years old, indian/white (70%). These data revealed
differences with the reports regarding to the Brazilian and Tunisian foci. The
predominant clinical picture includes characteristics of lupus and pemphigus,
resembling Senear-Usher Syndrome, without antinuclear antibodies (Abreu et
al, 1998). People affected by this disease also showed peculiar immunological
features such as presence of immunostain at the basal membrane zone using
direct immunofluorescence (DIF) in 50% of cases, and presence of a
heterogeneous autoantibody population different than desmoglein 1 (Dsg1).
These autoantibodies were directed aginst a doublet of antigens of 210 and 195
135
kDa by immunoblotting using normal human skin as a substrate (Abreu et al,
1998). El Bagre has mercury pollution because large quantities of this metal
are routinely used for gold extraction, without protection. The high
deforestation rate also contributes to the high mercury pollution in El Bagre.
Higher levels of mercury were observed in EPF patients (p<0.05), compared
with controls based on World Health Organization standards. In accordance
with the association of high levels of IgE and mice autoimmunity induced by
mercury/D-penicillamine, we also detected higher IgE seric levels in patients
(p<0.05) without association with atopy or parasitic diseases (Abreu et al,
1998). Other important data that we detected in these EPF-L patients was the
fact that all the patients were exposed to sun for more than four hours per day
(Abreu et al, 1998).
The aim of this study was to describe the histopathological features of people
affected by EPF in the El Bagre area of Colombia, to correlate the clinical
findings and to compare with the histopathology features described in other
EPF patients from other foci.
MATERIALS AND METHODS
Subjects of study: We studied 44 patients with diagnosis of endemic pemphigus
foliaceus determined by clinical (Viera, 1942), histopathological (Furtado, 1959;
Schnyder, 1973) and immunological criteria (Beutner, 1968, Stanley, 1989;
Diaz et al, 1989). We considered as EPF cases those subjects who showed
136
intercellular stain by IIF against the intercellular space between keratinocytes
(Rock et al, 1989) and those whom immunoprecipitated the 45 kDa bovine
epiderml tryptic fragment obtained after affinity chromatography which is
recognized for all active cases of PF disease (Labib et al, 1990). All subjects in
this study participated willingly and signed a consent form.
Biological samples: After local anesthesia with lidocaine without epinephrine,
we took skin biopsies which were preserved on 10% formalin. They were taken
preferentially from chest or from other places with clinically active lesions.
Biopsies were processed by hematoxylin-eosin (H&E) routine stains. Biopsies
were tested in blind to avoid diagonal cuts for the three dermatopathologists in
Colombia and in U.S.A.
RESULTS
We analyzed 29 EPF-L patients with a unique biopsy (BX), 12 patients with
two different biopsies and in three patients, three skin biopsies at different
clinical stages (eg. Exfoliative, then localized and hyperpigmented forms).
In people with a unique biopsy, we made the diagnosis of pemphigus foliaceus
in 42.3%, lupus-like in 32%, chronic dermatitis in 23%, pustular dermatitis in
15.4%, unspecific pemphigus in 11.5%, and psoriasiform dermatitis in 7.7%. In
32% of cases, we observed findings that include mixed features of lupus,
137
luminic
polymorphic
eruption
and
mixed
connective
disease
with
a
escleredermiform collagen at the dermis (Fig 1).
In most of our EPF cases the presence of subcorneal epidermal bullae were
rarely observed. Instead, we detected a superficial denuded erosion with
shallow oozing erythematous areas. In two cases suprabasal vesicles were
observed. Exocitosis and exulceration were common findings. Some nuclear
changes were detected in the keratinocytes, as well as several acantholytic cells
presenting advanced dyskeratosis. In acute cases, acantholysis and epidermal
clefts were observed in 42.3% of the cases and spongiosis in 17%. (Fig 1). The
granular cells seldom showed acantholysis and appeared deeply basophilic and
shrunken, resembling Dariers grains (Fig 1, A).
In chronic cases the presence of thick hyperkeratotic custers were commonly
observed (30/44). These clinical findings correlate with the presence of
acanthosis in 91% of the skin biopsies, hyperparakeratosis in 81% and
papillomatosis in some cases (37%). The epidermal appendages reveal follicular
dilatation and plugging in 17%, and follicular infundibulum compromise and
liquefaction of it, in 17%. Skin atrophy was observed in 30%, especially
associated with the cases that showed liquefaction of the BMZ. The liquefaction
of the BMZ was present in 29% and was associated with presence of
melanophages in 97% (Fig 1, B).
138
The presence of pustules or vesicle-pustules in the epidermis with PMN in the
dermis in 15.4% of EPF cases was observed and in one patient was associated
with a septic thrombi (Fig 1, C).
In 23% of EPF cases a chronic dermatitis was present manifested by
hyperpigmentation at the BMZ, presence of active melanocytes, acanthosys,
mild lymphoid and hystiocitic infiltrates at the papillary dermis, associated
with mild vasodilatation and capillary enlargement. We seldom observed
eosinophilic spongiosis (Fig 1, D).
In approximately 90 % of the skin biopsies some features resembling a luminic
reaction were observed such as enlargement of blood vessels, increased number
of melanophages and lymphoid cell infiltrates especially around the blood
vessels. Dermal edema especially at the papillary dermal region, with mixoid
chages in collagen and in some people with deposits of fibrin resembling the
collagenization that occur in scleroderma were observed (Fig 2).
In 30% of the cases, we observed histopathological patterns of lupus manifested
by hyper-paraqueratosis, atrophy of Malphigian stratum, liquefaction of the
BMZ and follicular appendage. It was accompanied by presence of flattening of
the BMZ and presence of melanophages. Clinically in these cases "discoid"
patches on the face with adhesive thick scales, follicular plugging and a slight
139
atrophy were observed resembling a Senear-Usher like syndrome, accompanied
by hyperpigmented patches the nose and malar areas (Fig 3).
Other findings such as papillomatosis in 37%; enlargement, dilatation,
tortuosity with increases in the walls from blood vessels were observed in 95%.
Some cases revealed a fibrinoid deposit on vessels, and in dermis histiocytes
and lymphocytes in 95%. The PMN were seen in 85%, of the biopsies and more.
These findings were associated with chonic cases. Eosinophils and plasmatic
cells were present in 35%.
Multiple alterations at the sweat gland level were observed in 40% of the cases.
These consist in focal necrosis, spongiosis of the acrosiringium, hypertrophy,
hyperplasia and cellular infiltration of sweat glands. Most of the distal ducts
were altered showing narrowing and lost of the borders. By autometallographic
technique mercury was detected at the inner layer of sweat glands, (Abreu et
al, 1998). This feature was more prevalent in chronic cases.
DISCUSSION
The histopathological features detected in the patients affected by EPF-L
disease from El Bagre correlates with the peculiar features detected in these
patients by clinical, epidemiological, immunological and by ultraestuctural
findings. Most of our patients did not show flaccid bullae arising on
erythematous base maybe because they break easily for their superficial
140
localization or maybe because most of our EPF-L cases have a chronic evolution
(more than three years). An epidermal pustular reaction without bacterial
infiltration and presence of neutrophilic infiltration was commonly seen at the
epidermal and/or dermal level, resembling a pustular dermatitis. These
changes have been also described in fogo selvagem (FS) patients from Brazil,
but less frequently (Lever, 1965; Furtado, 1959). Other diagnostics such as
chronic dermatitis, psoriasiform dermatitis, and pustular dermatitis also had
been described in FS patients (Lagerholm 1978; Furtado, 1959). Clinically, the
tegumentary lupus diagnosis is almost indistinguishable from the clinical
features detected in some of these EPF-L patients that seem to have a SenearUsher-like syndrome without antinuclear autoantibodies (Abreu et al, 1998).
The liquefaction of the BMZ is associated to the presence of autoantibodies at
this level by direct immunofluorescence and by acantholysis detected by
electron microscopy (Abreu et al, 1998; Abreu et al, manuscript in preparation).
The histopathological features of lupus and luminic polymorphic dermatitis
detected in these EPF-L patients seem to be "sui generis" to this focus. These
findings correlate with the high sun exposure of people affected by EPF-L. The
sun was the most important exacerbating factor in these subjects and all these
patients were exposed for more than four hours/day. (Abreu et al, 1998). It is
known that actinic prurig is another entity that is common in some latinoamerican Indian communities and is exacerbated or triggered by sun light
(Lever and Schaumburg, 1990). The equatorian localization of this focus with
141
exposure to sun light and mercury due to working activities can contribute to
the unique features detected by histopathology in these patients.
Permeation of the epidermis with eosinophils and intra epidermal eosinophilic
pustules described in FS from brazil and EPF cases from Tunisia were findings
not commonly detected in our EPF-L cases (Zilberberg, 1965; Portugal, 1949;
Morini et al, 1993). One of the biggest series in patients affected by FS from
Brazil was reported by Furtado in 1959. This series was made of 213 sections
from 183 patients having a clinical diagnosis of FS and the commonest
characteristic was the acantholysis in 91.8% of them. In our EPF-L patients
this finding was present only in 43% of the cases differing from the Brazilian
reports. In FS patients, the acanthosis was present in 74.8%, acantholitic cells
were detected in 68.5%, bullae segregation in 47%, dyskeratosis in 38,5%,
hyperkeratosis in 31.9%, papillomatosis in 26,3%, epidermal appendage
changes in 31.9% and pigmentation in 25,5% (Furtado, 1959). Our EPF-L cases
showed less acantholysis and acantholitic cells, and instead, we detected more
hyperkeratosis and acanthosis.
Ecrine sweat glands serve in thermoregulatory capacity to maintain
homeostasis in presence of changes of temperature. The sweat glands also play
a role of primarily excretion of waste products and are related with drugs
excretions. The primary constituents of ecrine sweat include water, sodium,
potassium, lactate, urea, ammonia and small quantities of various amino acids
142
and proteins and minerals (in our case mercury and gold) (Sato et al, 1989) The
acrosyringial epithelial cells of sweat glands constitutively express class II
major histocompatibility complex antigens. In some erythema multiformis was
induced by drugs. The necrosis of sweat glands suggests the possibility of an
immunologic process rather than direct toxicity. The possibility of a crossreacting antibody to a shared epitope between a drug (and in this case with
mercury and or gold) and the ecrine epithelium also may exist. In addition,
because sweat glands are invested with a rich vascular supply, it is possible
that ecrine gland damage may occur secondarily to processes affecting these
vessels. Radiation can also affect sweat glands function since it was
demostrated that these glands are are more radiosensitive than the epidermis
(Wenzel et al, 1998). As mentioned above, people affected by EPF-L from El
Bagre receive much exposure to sun.
The luminic compromise in these
patients is manifested for increased pigment incontinence, number and size of
melanophages, increasing melanocite activity, vasodilatation, perivascular and
periannexes infiltration of inflamtory cells, liquefaction of BMZ, epidermal
atrophy and increase of dermal collagenization. Histolopathology of patients
with EPF-L disease showed unique features such as: 1) Changes at the BMZ,
that correlate with deposition of immunoglobulin and complement (Abreu et al,
1998) as well as by acantholysis detected by EM (Abreu et al manuscript in
preparation) and clinical feature of Senear-Usher syndrome. 2) Sweat gland
compromise resembling the necrosis by barbiturics or mercury. 3) Marked
alteration of the dermal blood vessels and dermal collagenization. In
143
accordance with our findings, we suggest that a combination of sun and
mercury exposure could play a role in these histopathological changes.
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focus in the Sousse area of Tunisia. Arch Dermatol 1993,129:69-73.
13. Ana M. Abreu-Velez, Juan G. Maldonado, Andres Jaramillo, Pablo J.
Patio, Stella Prada, Leon Walter, Jorge Botero, Gunnar Warfvinge and
Fernando Montoya. Description of an unusual focus of endemic pemphigus
foliaceus in a rural area of El Bagre, Colombia. FASEB J. Abst 1998;12:
145
p.287
14. Lever, WF. Pemphigus and pemphigoid. 3 rd ed. Springfield: Charles
C.Thomas, 1965.
15.
Schnyder
UW.
Bullose
Dermatosen
In
Scezielle
pathologische
anatomie.Berlin: Springer-Verlag, 1973.
16. Alayon FL. Contribucao para o estudo do penfigo foliaceo (fogo
selvamen) no Brasil. Rev Med e Cir Sao Paulo 1948, 8:1.
17. Furtado TA. Histopathology of pemphigus foilaceus. Arch Dermat
1959,80:66-71.
18. Stanley JR, Klaus-Kovtun V, Samapio SA. Antigenic Specificity of fogo
Selvagem autoantibodies is similar to North American Pemphigus foliaceus
and distinct from pemphigus vulgaris autoantibodies. J Invest Dermatol 1986,
87:197-201.
19. Diaz LA, Sampaio SAP., Rivitti EA et al. Endemic pemphigus foliaceus
(fogo selvagem). Clinical features and immunopathology. J Am Acad Dermatol
1989,20:657-69.
20. Rock B, Martins C, Diaz LA. The pathogenic effect of IgG4
146
autoantibodies in endemic pemphigus foliaceus (Fogo selvagem). New Engl J
Med 1989, 320:1464-9.
21.Labib
R.S,
Camargo
S,
Futamura
S
et
al.
Pemphigus
foliaceus
antigen:characterization of a keratinocyte envelope associated pool and
preparation of a soluble immunoreactive fragment. J Invest. Dermatol
1990,93: 272-9.
22. Lagerholm B, and Frithz A. Submicroscopic aspects of the keratinization,
dyskeratinization and acantholysis of fogo selvagem. Acta
Dermatovener 1978; 58:37-49
23. Lever WF, SchaumburgLever G. Histopathology of the skin. 7Th ed;
Philadelphia: JB Lippincott Company 1990.
24. Zilberberg B penfigo e dermatitis de Durhing-Brocq. Contribuicao para o
estudo cito-histopatologic. Tese. Sao Paulo, 1965.
25. Portugal H. contribuicao da histopatologia nas dermatoses do grupo do
penfigo. Casos autoctono de penfigo. V Reuniao dos Derm Sif Bras 1949. p3242.
26. Sato K, Kang WH, Saga KT, Sato KT. Biology of sweat glands and their
147
disorders. II Disorders of sweat gland function. J Am Acad Dermatol
1989;20:713-26.
27.Wenzel FG, Horn TD. Noneoplastic disorders of the ecrine glands. J Am
Acad Dermatol 1998,38:1-17
148
FIGURES
Fig 1. Different histopathological diagnosis by H & E observed in EPF skin biopsies from El
Bagre: A. Classical PF with subcorneal acantholysis B. Lupus like with BMZ liquefaction and
atrophy of epidermis C. Pustular dermatitis with presence of intraepidermal pustules with
PMN D. Chronic dermatitis with acanthosis, hyperkeratosis, and papillomatosis. E. Psoriasislike, with the presence of a subcorneal blister.
A.
B.
C.
E. E.
149
Fig 2. A luminic like reaction of skin associated with enlargement of blood vessels, presence of
melanophages, limphohistiocitic infiltrate mainly perivascular with alterations of dermal
collagen.
Fig 3. Clinical and histopathological features resembling a lupus-like disease in people affected
by EPF-L from El Bagre, Colombia. A. A butterfly-pattern over the nose and malar regions
with patchy hyperpigmented plaques. B. Liquefaction of BMZ, skin atrophy and peri
appendage infiltrates.
A.
B.
150
Fig 4. Illustrating sweat gland necrosis and cellular infiltration of these glands with not welldefined borders and narrowing of the ducts as occurs by barbituric or mercury poisoning; A 10X
and B, 40X magnification.
A.
B.
II DETECTION OF MERCURY IN SKIN BIOPSIES FROM PEOPLE
LIVING IN AN ENDEMIC AREA OF ENDEMIC LIKE PEMPHIGUS
FOLIACEUS DISEASE IN EL BAGRE, COLOMBIA, SOUTH AMERICA.
Ana Maria Abreu Velez, * Fernando Montoya Maya, * Walter Leon Herrera,$
and Gunnar Warfvinge".
*Biomedical Basic Science Corporation, University of Antioquia, $Dermatology
Section (U de A), Medellin, Colombia and "Department of Oral Pathology,
Centre for Oral Health Sciences, Lund University, Malmoe, Sweden.
Corresponding author: Ana Maria Abreu Velez, Biomedical Basic Science
Corporation, University of Antioquia, Medellin, Colombia.
Key
words:
Endemic
Pemphigus
Foliaceus,
Autoimmunity,
autometallography, Mercury, sweat glands, Biotransformation.
Short Title: Detection of mercury from skin biopsies in people living in a rural
area of an endemic pemphigus foliaceus focus.
151
Abbreviations: EPF, endemic pemphigus foliaceus; PF, pemphigus foliaceus,
BX, biopsy.
ABSTRACT
People living in one focus of pemphigus foliaceus disease located in the
municipality of El Bagre, (Colombia), are exposed to high levels of mercury
pollution. This exposure is due to mining activities, marked deforestation
process, high environmental temperatures, and acidic soils existent in the El
Bagre area. A case-control study detecting mercury in skin biopsies from people
who live in this area was performed. A total of 51 skin biopsies were studied
distributed as follows: 32 from endemic pemphigus foliaceus-like (EPF-L)
patients and 18 from patients without EPF from El Bagre and one patient with
Cazanaves pemphigus foliaceus (PF). By autometallographic technique,
mercury was detected in 13 people (6/32 EPF patients, 6/18 controls and in the
one case with Cazanaves PF) out of 51 skin biopsies. Mercury was observed
especially at sweat glands, correlating in EPF-L patients with necrosis of these
appendages observed by hematoxylin and eosin stain. In controls no necrosis
was detected. In one control from El Bagre that previously had been treated
with D-penicillamine for mercury intoxication, the presence of this metal was
detected in dermal dendritic cells. A patient with a generalized form of
pemphigus foliaceus (Cazanave's type), from another city out of the endemic
area, also showed mercury at the sweat gland level, although this patients
never visited El Bagre or surrounding areas. Presently, this is the first work
reported in an endemic focus of EPF-L detecting mercury in skin biopsies. The
152
people with EPF-L from this focus are highly exposed to sun and chemical
agents that include mercury. In these patient’s higher mercury levels in hair
and increased IgE seric levels were detected compared with the control group
(p<0.05). The association of high IgE and mercury levels, necrosis of sweat
glands and mercury deposit in these, could be or not be related with the
autoimmune phenomena observed in people affected by EPF-L. It could also
suggest that at least the inhabitants of this area are highly exposed to mercury
and this metal could be a stress factor that influence alterations in the immune
system of people living in El Bagre.
INTRODUCTION
Pemphigus foliaceus (PF) and the endemic form of PF, (EPF) are autoimmune
skin diseases.1 EPF was described in foci at the South American tropical
forest, mainly in Brazil but also in other countries of Latin America2 and in
Tunisia3. The EPF foci in Brazil are characterized by increased numbers of
familial cases occurring mainly in children and young adults dedicated
primarily to farming activities.4 In Tunisia, women in child bearing age are
those most affected.5 The onset of EPF in Brazil is found more often in people
who carry a class II Human Leucocyte Antigen (HLA) DRB1*-A24;
DRB1*1406; DRB1*0404; DRB1*1402.6,7 EPF is characterized by the presence
of acantholysis and subcorneal blisters in upper epidermal layers accompanied
by deposits of mainly IgG4 autoantibodies in the intercellular space.8 Most of
153
the autoantibodies are directed against a desmosomal glycoprotein called
Desmoglein 1 (Dsg1).9,10 This glycoprotein belongs to the cadherin family of
calcium-dependent cell adhesion molecules.11,12 The cause of EPF is unknown,
but some environmental risk factor (s) such as exposure to Simulium
pruinosum, have been implicated in Brazil as possibly triggering this disease.13
A case-control study of an EPF-L focus in El Bagre, revealed unique
epidemiological, histopathological and immunological features as following: 1)
higher mercury levels in hair detected by mass spectroscopy, 2) higher seric
IgE levels detected by ELISA, 3) most affected were men with a mean age of 50
years, 4) most were dedicated to farming and mining activities, 5) presence of a
familial background of pemphigus in 20% of them, 6) presence of other
autoantibodies different from Dsg1 antibodies, 7) immunoreactivity in half of
the cases to components of the basal membrane zone (BMZ), 8) alterations at
BMZ detected by hematoxilin-eosin (H & E) stain and 9) presence of sweat
glands necrosis. Based on these results and because most of the people in El
Bagre are dedicated to mining (80%) activities in which mercury is commonly
used for gold extraction, we have studied the presence of this metal in skin
biopsies from people from this endemic area of pemphigus foliaceus.
METHODS
Subjects of study: All subjects in this study participated willingly. A careful
history was obtained from patients and controls. We studied 52 people; 51 of
154
them live in El Bagre. Thirty two out of the 52 people were EPF patients. The
diagnosis was performed based on Viera’s criteria4. In 18 donors from the
endemic area, one had an ecrine hydracistome and one had lichen planus as
detected
by
clinical
and
histopathological
criteria.
During
patient
examinations, we filled out histories, performed clinical tests, took blood
samples and skin biopsies for autometallographic techniques. Some skin
biopsies were fixed in formalin 10% and were sent to the Department of Oral
Pathology, Lund University, Malmoe, Sweden. Other biopsies were processed
by routine Hematoxylin-Eosin (H & E) stain at State Laboratory in Medellin,
Colombia. Hair samples for mercury detection were taken and transported at
room temperature (RT) in plastic bags. Blood serum was stored at -20oC. Some
samples were tested at Department of Dermatology, Medical College of
Wisconsin, (MCW), WI, USA and some were tested at the Dermatology,
Immunology and Toxicology Departments at University of Antioquia, in
Medellin, Colombia.
Mercury detection in hair: After extensive washing, 25 mg of hair was cut
and degreased with acetone. Hair samples were then packed in ash free paper
and incubated in a solution of nitric and sulfuric acid followed by a solution of
potassium permanganate to destroy organic material. Excess potassium
permanganate was removed with hydroxylamine chloride. Following this, tin
chloride was added to remove the mercury from hair. Mercury was measured in
155
an atomic absorption spectrophotometer (MAS 50). The World Health
Organization (WHO) acept a mercury permissible level in hair of 7 ppm.
Autometallography: The method of Danscher and Møller-Madsen14 was
used to visualize tissue bound mercury in paraffin sections. Briefly, three-um
sections were coated with 0.5% gelatine, air dried and subsequently developed
at 26°C for 1 h in the dark. The developer contained gum arabic, sodium citrate
buffer, hydroquinone and silver lactate. The gelatine was washed out in 40°C
tap water and a rinse in distilled water followed by incubation in 5% sodium
thiosulphate to suppress background staining. Sections were prepared in
quadruplicates, one of which was lightly counterstained with hematoxylin and
eosin. The autometallographic technique reveals silver sulphides and selenides,
metallic silver and metallic gold in addition to mercuric sulphides/selenides. In
order to differentiate between mercury and silver, additional sections, mounted
on poly-L-lysine treated glass slides (Polysine, Menzel, Germany), were
incubated in a 1 % aqueous potassium cyanide (KCN) solution for 2 h to remove
silver prior to autometallographic development based on Danscher and Rung
technique15. To exclude the presence of gold, further sections on Polysine slides
were instead pre-treated with 10 % KCN for 15 min.16
Detection of skin autoantibodies: All sera were tested for presence of
autoantibodies by direct and indirect immunofluorescence (DIF, IIF), by
immunoblotting (IB), Immunoprecipitation (IP), and by ELISA. Briefly, DIF
156
and IIF was used with cryosections of human skin and fluorescein
isothiocyanate (FITC) conjugated goat antihuman IgG (Cappel Laboratories,
Cochranville, PA) in order to identify intercellular keratinocyte staining as
previously described.8 Sera of the subjects of study were also tested for
reactivity against Dsg1 by IB using human and bovine epidermis9 and by IP
using a 48 kDa tryptic fragment obtained from bovine epidermis run through a
concanavalin A (Con-A) affinity column.17 An ELISA assay using the 48 kDa
fragment was used in order to detect autoantibodies directed against PF
antigens, (Abreu et al, manuscript in preparation).
Quantification of total IgE seric levels: because mercury can increase IgE
seric levels in mice autoimmunity models, we tested for this immunoglobulin.
Patient sera were tested for seric IgE using an ELISA commercial kit
(AlaSTAT Total IgE kit, Diagnostic Products Corporation Los Angeles,
California). Normal IgE levels were considered as 300 international units per
liter (IU/L).
RESULTS
Autometallographic analysis: In 13 skin biopsies, mercury was visualized as
dark silver grains, within sweat gland epithelial cells (Fig 1). The skin biopsy
of one control from the endemic area, who had previously suffered from
mercury intoxication, differed from the rest in that numerous somewhat
dendritic mercury-loaded cells appeared throughout the dermis (Fig 2). No
157
such mercury-loaded macrophage-like cells were found in any of the other 51
patients. In many specimens, the lower epidermis, and the dermis close to the
BMZ contained abundant melanin. Although melanin granules visually
interfere with the silver grains, and makes the search for mercury difficult, it
was possible to exclude the presence of autometallographic staining in this
area.
Mercury levels in hair: Table 1 describe the mercury levels of people from
El Bagre in which mercury was detected in skin by autometallographic
analysis.
Quantification of seric IgE: Table 1 summarizes the results of seric IgE
levels in people from El Bagre whom showed presence of mercury in skin by
autometallographic analysis.
Presence of autoantibodies directed against pemphigus foliaceus disease: The
presence of autoantibodies against PF antigen(s) was detected by DIF, IIF, IB,
IP and ELISA. Table 1 summarizes these results.
DISCUSSION
Mercury is commonly used to amalgamate gold in mining activities in El
Bagre. This metal is also present in many products as fertilizers, pesticides,
158
herbicides often employed in farming activities in this endemic area of
pemphigus, (Abreu et al, submitted). Previously we described that 64.2 % of
EPF patients from this focus are exposed to mercury directly by mining trial or
indirectly by fumigation products that contains mercury. El Bagre has special
environmental conditions as acidic soil, high rates of forest fires, large
deforestation, and high environmental temperatures, (Abreu et al, submitted).
All these factors increased mercury pollution and can increase mercury
absorption.18,19 Mercury pollution has been reported in the biotic chain
including humans in El Bagre and neighboring municipalities.20 This metal is
mainly absorbed through the respiratory tract, and can be detected in alveolar
macrophages after three months of exposure.18 Other pathways for mercury
intake are the gastrointestinal tract and the skin.18 Presence of mercury in
sweat glands, could be due to excretion of his metal at this level, and to the
high availability of this metal in El Bagre.
Many studies have been performed to detect the localization of mercury in skin.
In normal and eczematous skin, about 10 to 15% of topically applied mercury
can be absorbed. Diffusing through epidermis, it will reach the dermis after 8
hours, and can then be absorbed systemically.21 After skin exposure with
mercury it has been detected entirely extracellularly, usually closely related to
intercellular bridges and external surfaces of cell boundaries. In a few areas of
a lichen simplex lesion, intracellular collections of mercury were detected in the
mid-epidermis.22 Other studies testing for the behavior of externally applied
159
radioactive mercury on normal and psoriatic skin and skin with seborrheic
dermatosis demonstrated that skin with seborrheic dermatitis and psoriasis
retained this metal more effectively than normal skin. About 50% of this metal
was accumulated in stratum granulosum of seborrheic patches, while in
psoriasis; it was evenly distributed throughout all epidermal layers. In both
maladies, the sulphidryl-staining material and mercury were entirely
intracellular, in contrast to the diffuse staining pattern observed in normal
skin. Also, a close resemblance in the deposition of radioactive mercury and the
distribution of free sulphydryl groups (as identified by Chevromont's stain) was
demonstrated.22 Different histochemical studies showed that mercury in skin
was located in keratinocytes, melanocytes, Langerhans cells, fibroblasts, and
mononuclear cells below stratum corneum and nuclear membranes, nuclei,
mitochondria and membrane bound inclusions.23 However our results showed a
more restricted localization of mercury. It has been demonstrated, by
autometallography, that mercury can be visualized in oral mucosa, salivary
and lacrimal glands in rats treated with HgCl2.24 In that study, mercury was
detected intracellularly; in dendritic cells that were scattered throughout the
lamina propria and in submucosal connective tissue. In addition, mercury was
detected
in
dendritic
cells
appearing
within
small
cell
clusters
in
juxtaepithelial connective tissue of the oral mucosa and within ductal
epithelium. Mercury can also be detected in skin by electron microscopy using a
gold chloride stain Silberberg23 mercury was detected by this method after
topical application in nuclei, tonofilaments, mitochondria, melanosomes,
160
desmosomes, intercellular space, cell membranes and ligated to many proteins
like metallothionein, and lipoproteins.25
In EPF-L patients who live in the El Bagre area, an increase of seric IgE, IgG1
and IgG4, hair bound mercury and the presence of mercury located in a
necrosis of sweat glands were detected. Our findings suggest that EPF patients
from El Bagre are exposed to high mercury stress. In our study we mainly
identified mercury in sweat glands, but although one may not completely rule
out such a possibility, we could not demonstrate a direct local effect on skin.
Mercury might exert its effect on immune system, either directly or indirectly.
Directly by giving rise to autoantibodies or indirectly, by compromising the
system in such a way that other autoantigenic mechanisms are facilitated. The
presence of genetic abnormalities induced or aggravated by mercury can not be
discarded neither. Presence of mercury in sweat glands surely indicates that
patients (and controls) are under environmental stress. However, if mercury
plays a role in the autoimmune phenomena in people with EPF-L disease from
El Bagre, the presence of this metal in some normal donors could suggest a
genetic background that predispose some people to develop the disease.
In experimental models, mercury may induce an autoimmune syndrome in
genetically sensitive strains, i.e., BN rats. The syndrome is characterized by
deposit of linear IgG along glomerular basement membrane, antinuclear
antibodies (ANA), hyper-IgE, IgG1 and IgG4 in sera and dermatitis.26,27 The
161
mechanisms by which mercury generate this syndrome are as yet unknown but
similar changes may also appear in animals treated with D-Penicillamine or
gold salts.28 In the EPF patients we have detected hyper IgE and
autoantibodies mainly IgG1 or IgG4. Combination of several factors may be
necessary to break self tolerance and cause mercury induced autoimmunity.
Such factors likely compromise endogenous components as T cell receptor
(TCR), regulatory T cells and human leucocyte antigen HLA.29 These factors
are not well studied in PF disease. In animal experiments mercuric chloride
can induce immune-complex glomerulonephritis. In some EPF-L patients we
detected an electrondense substance at the desmosomes level that is actually in
process of identification (Abreu et al, manuscript in preparation). Increased of
immunoglobulin production mainly IgE, increase in the number of B cell and T
helper cells and production of antinuclear antibodies are also common in mice
induced autoimmunity by mercury. In the EPF-L patients neither the presence
of antinuclear antibodies (ANA) nor clinical alterations of renal function were
observed. Presence of antibodies against fibrillarin (34 kDa) has also been
described; by immunoaffinity testing our EPF-L recognize a 34 kDa protein,
but the characterization of this 34 kDa proteins remains unknown. Mercury as
an inorganic substance can act in many biological processes; calcium ions
reversibly bind mercury in specific electrostatic cavities of regulatory proteins
such as calmodulin through what are principally non-directional ionic
interactions.30 Dsg1 is a calcium dependent protein and the antigen-antibody
162
interaction in PF disease is also calcium dependent; we can speculate that
mercury could also act directly in this process.
Mercury metal not only can act directly or indirectly on skin and proteins of the
immune system, but it also exerts an important role in gene regulation.
Mercury can interact with metalloproteins that play structural and catalytic
roles in gene expression. These metalloregulatory proteins can exert metalresponsive control of genes involved in respiration, metabolism and metalspecific homeostasis or stress-response systems, such as iron uptake and
storage, copper efflux, and mercury detoxification.30 Expression of a wide range
of genes is controlled by metalloproteins and these proteins act at a
physiological level, as components of metal-responsive genetic switches. Metal
containing regulatory proteins can capitalize on a rich variety of coordination
geometric and ligand exchange rates of metal centers. Metal ion coordination
may be a controlled mechanism and thus analogous to phosphorylation or Ca++responsive networks. Extensive genetic, enzymatic, structural, biophysical, and
inorganic studies of bacterial mercury resistance proteins (encoded by MeR
genes) provide the first comprehensive picture of a tightly regulated metal
detoxification mechanism.30 The central enzyme is mercuric ion reductase, a
structurally characterized flavoenzyme that reduces mercury to volatile Hg0.31
As a receptor, MeR is sensitive to nanomolar concentrations of Hg (II) and
exhibits a high degree of selectivity. From an inorganic perspective the
molecular recognition underlying this combination of selectivity and sensitivity
163
is essentially derived from coordinated-covalent interactions of metal ions with
at least three cysteinyl thiolates.30
With all the above-mentioned data about how mercury can act not only directly
on skin but also in proteins (mainly calcium dependent and metalloproteins), at
the immune system and in gene regulation; evidence of this metal in skin from
people from the endemic area and also the presence of hyper IgE and
autoantibodies (IgG4 and IgG1) mainly in EPF patients, suggest that this ion
can play a role in the pemphigus autoimmune phenomena. Other factors as
greater amounts of mercury in hair from EPF patients and evidence of mercury
pollution in El Bagre also contribute to enrich evidence that mercury could be
related directly or indirectly to the production of EPF-L disease in El Bagre,
(Abreu et al, manuscript submitted). Presence of mercury in skin, associated or
not with skin pathosis have also been reported by others. In 63 patients with
the diagnosis of acute generalized exanthematosus pustulosis, a retrospective
histopathological study was performed, and 8 patients had recently been
exposed to mercury and it was considered the only or main etiologic factor. In
these 8 cases, eruptions began 1 to 2 days after exposure to mercury: clinical
picture varying between pustular eruptions with several hundreds of small
pustules arising on widespread erythema and burning sensation. Confluence of
pustules leads to a positive Nikolsky. If there is a direct or indirect effect of
mercury in the modulation or triggering of the autoimmune phenomena in
EPF-L patients from El Bagre, we can try to develop a therapy based on high
164
amounts of vitamin A topically and/or systemically administered together with
selenium and 2,3 Dimercapto-1 propano sulfonic-acid.22 We consider that
although D-penicillamine is classically used for mercury poisoning treatment,
this medicine also may trigger EPF disease by a cross-reaction mechanism and
should perhaps not be the election therapy for these patients.
Acknowledgements: This work was supported by grants from Direccion
Seccional de Salud de Antioquia (DSSA), Mineros de Antioquia S.A,
Universidad de Antioquia, (Ana Maria Abreu et al). We would like to extend
our thanks to the following institutions: Colciencias and LASPAU. We would
also like to thank the people of El Bagre and especially, EPF patients. This
paper is part of the Ph.D. thesis of Ana Maria Abreu-Velez. MD as an
immunologist at the U. de A. Dr Abreu is the recipient of a scholarship from
Colciencias, Colombia.
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Environ Health 1972;24:129/44.
26. Druet PH. Metal-induced autoimmunity. Human & Exp Toxicol
1995;14:120/1.
169
27. Goldman M, Druet P, Gleishmann E. TH2 cells in systemic autoimmunity
insights from allogeneic diseases and chemically induced autoimmunity.
Immunol Today 1991;12:223/6.
28. Tournade H, Pelleterir L, Pasqier R et al. D-Penicillamine-induced
autoimmunity in Brown-Norway rats. Similarities with HgCl2-induced
autoimmunity. J of Immunol 1990;144:2985/91.
29. Kosuda LL, Bigazzi PE. Chemical induced autoimmunity. In: Smialowicz
RJ, Holsapple MO. Experimental Immunnopathology. Boca Raton: CRC Press;
1996.
30. O´Halloran TV. Metal ions in Biological Systems. In:H Siegel, Ed (Dekker),
New York, 1993: 25, 105.
31. Helman JD, Shewchuck LM, Walsh CT. Metal ion induced regulation of
gene expression In: Eichhorn G.L. and L.G. Marzili, Eds. (Elsevier, New York),
Vol 8, p.33, 1990.
FIGURES AND LEGENDS
Fig 1 Shows autometallographically stain on skin section from a patient. Mercury deposits are
visualized as dark silver grains and were evident primarily in sweat gland epithelium. A.
170
Mercury is visualized by a H& E stain. B. Mercury is visualized by the corresponding
autometallogry.
A.
B.
Fig 2 Presence of mercury in one person from El Bagre who had a mercury poisoning. Mercuryloaded cells with a somewhat dendritic appearance were distributed throughout the dermis.
171
Table 1. Seric IgE, mercury levels in hair and presence of autoantibodies
directed against pemphigus foliaceus antigens in people from El Bagre whom
showed mercury in skin by autometallography.
Donors
D
X
Race
Sex
Seric
IgE in
U/IL
IIF,
ICS
IIF
IC
IgG
3
IB 160
kDa
IB
210195
kDa
IP 48
kDa
ELISA
(45 kDa,
PF
antigen).
195
Hg++
in
hair
in
ppm.
2.9
1
1
I/W
M
IgG1
(+)
(-)
(+)
(1/2+)
0.205
2
1
I/W
M
1,178
5.5
IgG4
(+)
(1/2+)
(-)
(+)
0.425
3
1
I/W
M
7
6.9
IgG4
(+)
(+)
(+)
(1/2+)
0.128
4
1
I/W
M
2,590
7.3
IgG4
(-)
(+)
(-)
(1/2+)
0.4
5
1
I/W
M
689
12
IgG4
(+)
(+)
(+)
(+)
0.479
6
3
I/W
F
2,089
7.3
IgG4
(-)
(-)
(-)
(+)
0.401
7
2
I/W
M
128
4.9
IgG4
(+)
(+)
(+)
(+)
0.389
8
2
I/W
M
276
9.75
(-)
(-)
(+)
(+)
(-)
0.348
9
2
I/W
M
557
4.4
(-)
(-)
(-)
(-)
(-)
0.38
10
2
I/W
F
217
4.2
(-)
(-)
(-)
(+)
(-)
0.012
11
2
W
M
241
8.55
(-)
(-)
(-)
(-)
(-)
0.013
12
2
I/W
M
2,006
2.85
(-)
(-)
(-)
(-)
(-)
0.041
13
2
I/W
M
3,404
3.75
(-)
(-)
(-)
(+)
(-)
0.038
DX: Diagnosis. I/W: Indian/White. M: Male. F: Female. IIF ICS: Indirect
immunofluorescence, intercellular stain. IP: Immunoprecipitation of the 45 kDa
epidermal tryptic Con-A fragment. IIF IC: IIF intracellular stain with IgG3 by IIF. IB:
Immunoblotting against 160 kDa band (Desmoglein1) and againts 210 & 195 kDa
bands (unknown). IgE: ELISA againts antibodies detecting total IgE in International
Units per liter (UI/L). ELISA: OD492nm for detecting autoantibodies against the Con-A
affinity fragment. ppm: parts per million.
172
XII. PARTIAL AMINO ACID SEQUENCE OF A 48 kDa BOVINE
EPIDERMAL TRYPTIC FRAGMENT IMMUNOPRECIPITATED BY ALL
SERA FROM PATIENTS WITH PEMPHIGUS FOLIACEUS
Ana Maria Abreu-Velez*, Luis A. Diaz.1
* Biomedical Basic Science Corporation, University of Antioquia (U de A),
Medellin, Colombia;
1Department
of Dermatology, Medical College of
Wisconsin (MCW), Milwaukee, WI, U.S.A.
Corresponding author: Luis A. Diaz MD, Department of Dermatology,
MedicalCollege of Wisconsin, 8701 Watertown Plank Road, Milwaukee,
Wisconsin 53226 U.S.A.
Key Words: Pemphigus foliaceus, Desmoglein, Conformational epitopes,
Autoantigen, Autoimmunity, CAM, Desmosomes.
Short Title: Partial aminoacid sequence of a 48 kDa tryptic fragment related
to pemphigus foliaceus antigen.
Abbreviations: PF, pemphigus foliaceus; EPF, endemic pemphigus foliaceus;
Dsg1, Desmoglein 1; SDS-PAGE, sodium dodecylsulfate-polyacrylamide gel
electrophoresis;
PVDF,
polyvinylidene
difluoride
immobilon
transfer
membrane, PRA-SA, Cowan strain of Staphylococcus aureus
ABSTRACT
Trying to obtain a smaller conformational epitope related to pemphigus
antigen, we used a procedure to isolate a 48 kDa bovine epidermal tryptic
fragment to elucidate its nature. The epidermis of 14 cow snouts were
173
trypsinized, solubilized and subjected to affinity purification by ConcanavalinA (Con-A) affinity column. The eluted material, (fraction A), immunoadsorbed
reactivity from a PF serum by indirect immunofluorescence (IIF) on human
cryosections. This fraction A is recognized by all sera from patients with
clinically active PF disease by immunoprecipitation (IP). Based in the antigenic
properties of fraction A, a further immuno-affinity chromatography purification
using Staphylococcus aureus protein A-sepharose (s/SPA) matrix was
performed. All IgG, except IgG3 from a FS serum were coupled covalently with
the s/SPA gel; furthermore, fraction A was incubated with the s/SPA-IgG, and
the eluted product was then separated by molecular weight on sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted
onto polyvinylidene difluoride (PDVF) membrane. The blotting was stained
with amido black. Simultaneously in duplicate gels stained with Coomasie
brilliant blue and silver stain, the presence of a 34, 45, 48, 60, 117 and 120 kDa
bands were observed in the amido black blotting and in the silver stain gel. The
48 kDa band that co-migrated with the radiolabeled fraction A, was excised
generating 9 picograms of the sequence EXIKFAAAXREGED. No amino acid
derivatives could be identified at positions 2 and 9. The homology search
corresponds to the N-terminal ectodomain of the mature form of bovine
desmoglein 1 (Dsg1). We described in this paper the actually smaller
conformation epitope related to PF antigen, extracted from viable bovine
epidermis with a partial aminoacid sequence characterization.
174
INTRODUCTION
Endemic and non-endemic pemphigus foliaceus (EPF and PF), are epidermal
autoimmune
diseases
characterized
by
intra
epidermal
acantholysis,
subcorneal blisters and presence of antibodies against Dsg1 (Koulu et al, 1984;
Stanley et al, 1989; Rappersberg et al, 1992). Dsg1 belongs to the cadherin
superfamily of calcium-dependent cell-cell adhesion proteins. These proteins
are implicated in morphogenesis, tumor control mechanisms and as mediators
of cell recognition. This family includes classical cadherins, desmogleins,
desmocollins, protocadherins and the products of Drosophila genes Fat and
Dachsous (Puttangunta et al, 1994; Wheeler et al, 1991). Currently, the
sequences of over thirty classical cadherins, six desmocollins, and three
members of the desmoglein family have been reported. (Puttangunta et al,
1994). Immunological and RNA hybridization studies strongly suggest,
however that many isoforms of the latter family exist (Puttangunta et al, 1994).
Dsg1 is a major component of epidermal desmosomes. This molecule is a
transmembrane glycoprotein and is thought to mediate a homophilic calcium
dependent cell-cell adhesion, although this binding mechanism remains to be
proven (Wagner, 1995; Wheeler et al, 1991; Shapiro et al, 1995; Overduin et al,
1995). Dsg1 has a Molecular weight of 112,243 daltons and contains 1,043
amino acids (aa). Structurally, Dsg1 has a single membrane-spanning region of
approximately 25 aa, four cytoplasmic desmogleins repeats, (two of 29, one of
28 and one of 27 aa), and a cytoplasmic domain that is rich in Glycine and
Serine (49 aa). The cytoplasmic domain has in total 468 aa. The extracellular
175
domain has four tandem cadherin repeats of 993 aa at positions 107, 111, 115,
109 with 100 or 111 aa between the repeats (Wagner, 1995; Overdiun et al,
1995; Kolwalczyk, et al, 1996; Tong et al, 1994). Two potential carbohydrate
binding sites are located in the extracellular domain. The putative calcium
binding domains are located in the four cadherins-like repeats. Full-length
Dsg1 is immunoprecipitated by 100% of PF sera, but only one third of PF sera
recognize linear epitopes of Dsg1 by immunoblotting (IB) using SDS extracts
from human epidermis (Stanley et al, 1989). It has also been reported that PF
and EPF sera, immunoprecipitate a soluble 80 kDa antigen extracted from
bovine epidermis (Olague et al, 1993). N-terminal sequence analysis of the first
20 aa of the 80 kDa protein has shown overlapping sequence homology with the
corresponding N-terminal domain of Dsg1 (Olague et al, 1993, 1994). Similarly,
after trypsinization of viable human and murine epidermis has been previously
shown that a “45 kDa” immunoreactive glycoprotein was recognized by all sera
from PF and EPF patients by IP (Martins et al, 1990; Labib et al, 1990). In this
study we have used the same extraction procedure (Labib et al, 1989) to purify
a soluble 48 kDa immunoreactive fragment from bovine epidermis using two
sequential affinity chromatography steps.
MATERIALS AND METHODS
Extraction and partial purification of bovine epidermal PF antigen:
After washing with Tris-buffered saline, pH 7.6, containing 5 mM CaCl2
(TBS/Ca++buffer) at 40oC, viable epidermis separated by keratome was digested
176
with 0.01% trypsin type III from bovine pancreas (Sigma Chemical Co., St.
Louis, MO) in TBS/Ca++buffer. The digestion was carried out while stirring at
37oC
for
1
hour
and
was
stopped
by
the
addition
of
10
mM
phenylmethylsulfonyl fluoride (PMSF) and 10 ug/ml of a cocktail containing
the following protease inhibitors: pepstatin, anti-papain, chymostatin and
leupeptin (Sigma) (Labib et al, 1989; Labib et al 1990; Martins et al, 1990). The
extract was then centrifuged twice at 10,000 and 1,200 rpm respectively in a
refrigerated centrifuge (Sorvall). The supernatant was then separated on a
Con-A sepharose 4B affinity column (Sigma), 1x 20cm, equilibrated with
TBS/Ca++buffer. The column was washed with TBS/Ca++buffer until the
absorbance reading at 280 nm dropped to zero. The first 30 ml of the flow
through were collected. Bound glycoproteins were eluted with 0.2 M of alpha
methyl D mannopyranoside in a TBS-Ca++buffer (Labib et al, 1990). Three ml
fractions were collected and their absorbance at 280 nm was monitored. Two
representative peaks were obtained (fraction A and B). These were pooled,
dialyzed, concentrated (PM 30, AMICON, Beverly, CA) and treated with
protease inhibitors. The concentration of glycoproteins present in these
fractions was determined by the Bio-Rad Micro protein assay (Bradford).
Protein concentration was adjusted to 3.0 mg/ml.
Immunoadsorption (IA) procedure using Con-A products: The flow
through, fraction A and B were use for the IA. Ten, 25 and 50 ul of each
fraction were incubated for 30 minutes at room temperature (RT) with 50 ul of
177
a 1:80 dilution of a serum belonging to a well characterized fogo selvagem (FS).
Negatives controls were prepared by incubation of corresponding volumes of
bovine serum albumin (BSA) with the same fractions. Then the mixtures were
tested to evaluate the presence of immunoreactive fragments by indirect
immunofluorescence (IIF) as described elsewhere (Labib et al, 1990). As
positive controls we used the same FS serum, at same dilution, with BSA and
without BSA as above described.
Radio-labeling
of
Bovine
Epidermal
Antigens:
radiolabeled by Chloramine T method using
125I
Fraction
A
was
as previously described
(Marier et al, 1979). The protein-bound radioactivity was determined by
precipitation with 10% trichloroacetic acid. One aliquot of the labeled fraction
A was used for an IP, other was used as a tracer for the electrophoresis and a
third was used to test the coupling of the IgG from FS serum to the protein-A
sepharose CL-4B column.
Immunoprecipitation: Forty-two sera from well characterized EPF patients,
using clinico-epidemiological and immunological criteria and 27 normal donors
from the endemic area were tested. A FS serum from Brazil and three negative
controls from United States of America (USA) were also tested. A preadsorption was performed by incubation of normal human serum, with the
radiolabeled fraction A and lyophilized Cowan strain of Staphylococcus aureus
(PRA-SA) (Sigma). The pre-adsorption was carried out for one hour at RT,
178
using the immunoprecipitation buffer (TBS/Ca++ with 5 mM CaCl2 and 1%
Triton X-100) with BSA. After centrifugation, the supernatants were reincubated for one hour with each one of the above-mentioned sera and PRA-SA.
The incubation mix was precipitated with 0.1% SDS. The pellet was washed
five times with immunoprecipitation buffer (IPB) without BSA and the
remaining radioactivity was determined. The immunoprecipitated complexes
were then resuspended, boiled, and centrifuged in a modified sample buffer
(Labib et al, 1990) and examined by SDS-PAGE and autoradiography.
Sepharose-SPA-IgG coupling: A PF serum was incubated with protein-A
sepharose CL-4B gel (Sigma), following previously described procedure
(Hermanson et al, 1992; Olague et al, 1994). The gel/protein-A/ IgG complex
(s/SPA/IgG-gel) was then resuspended in a solution of 0.2 M triethanolamine at
pH 8.2. The antibodies were bound to the protein A and cross-linked with 4
volumes of dimethyl pirimedilate (DMP) (6.6 mg/ml) (Aldrich). An extensive
washed with 50 mM sodium borate buffer was performed. The remaining
binding sites of the gel were blocked by addition of 0.1 M ethanolamine pH 8.2.
Finally, the s/SPA/IgG-gel was washed using 1 M NaCl in TBS/Ca++ buffer
followed by IPB buffer as equilibrator. Leakage of IgG from the column was
tested by SDS-PAGE before and after cross linking.
Testing the capability of the s/SPA/IgG-gel to immunoprecipitate the
radiolabeled fraction A: To test if the s/SPA/IgG-gel can immuno-recognize
179
the radiolabeled fraction A, an IP was performed. After pre-adsorbed normal
human serum with the radiolabeled fraction A and lyophilized Cowan strain of
Staphylococcus aureus (PRA-SA) (Sigma), the same steps were followed as
described above for the IP. Instead of use a PF serum, we incubated 10 ul of the
s/SPA/IgG-gel with the radiolabeled fraction A; next steps were similar as
described in IP.
Purification of the PF antigen by a second affinity chromatography:
Non-labeled fraction A was incubated with the s/SPA/IgG-gel for one hour at
RT (Olague et al, 1993). After extensive wash, the bound fraction was eluted
with 0.2 M glycine, 5 mM EDTA/HCL, pH 2.8. Alliquots from the eluent of 0.5
ml were collected. A 0.1M NaOH was added to each aliquot as a neutralizer
until reach pH 7.4. Each alliquot was monitored at 280 nm. In order to find a
band which, co-migrate with the radiolabeled fraction A, this radiolabeled
fraction was added on each one of the aliquots samples. The sample was mixed
with a sample buffer as described for Laemmly, 1970, in 1:1 relation (s/SPAIgG-gel eluted product-sample buffer) to desdoblate proteins. Each aliquot was
run on SDS-PAGE, and subject to blotting. After detecting the aliquot with the
band which co-migrate with the radiolabeled fraction A, several s/SPA/IgG-gel
affinity chromatography columns were realized to increase the amount of
protein. The significant peak was pooled, concentrated 3,000 times in Amicon,
dialyzed over night against H2O, and lyophilized.
180
Immunoadsorption (IA) using the s/SPA/IgG-gel products: An IA was
carried out to test the capability to keep conformational epitopes in the eluted
product obtained from the second affinity chromatography. The s/SPA/IgG-gel
eluted products and the flow through were incubated with 50 ul of a 1:80
dilution of a serum belonging to a well characterized FS patient, following the
same procedure described above for the fraction A IA.
Gel electrophoresis and electroblotting: Two different 10% SDS-PAGE
were realized using the fraction A and the significant eluted product from the
s/SPA/IgG-gel. The samples were run in a mini-Protean electrophoresis
chamber (Bio-Rad). Molecular weight standards were applied to determine the
molecular weight of the PF antigen (Laemmly, 1970). Other 10% SDS-PAGE
was run using the fraction A as a substrate. Gels were run in triplicate, two
were used for coomassie blue and silver stain, and other was blotted onto
polyvinylidene
difluoride
(PVDF)
membrane
(Immobilon
P,
Millipore)
according to Matsudaira, 1987. Autoradiography was carried out at -70oC for
16 hours using an intensifying screen X-OMAT.
Amino Acid Sequence Analysis of the 48 kDa Bovine PF Antigen: A 48
kD band that co-migrated with the radioactive antigen was excised from the
PVDF after SDS-PAGE and electroblotting. The excised band was subjected to
amino acid sequence analysis on a Porton/Beckman gas phase sequencer,
model LF3000 (Palo Alto, CA, USA). For amino-acid analysis PVDF strips were
181
transferred to clean glass tubes and incubated in 5.7 N HCL containing 0.02%
B-mercaptoethanol. Tubes were sealed under N2 and vacuum. Hydrolysis was
allowed to proceed for 20 hours at 110o C, then the samples were dried and redissolved in sodium citrate buffer, pH 2.4 and run on a Beckman, amino acid
analyzer, model 6300. Cysteine and tryptophan were not determined.
Computer analysis of sequences: It was performed using the Sequence
Analysis Software Package of the Genetics Computer Group (GCG), Madison,
USA. Sequences were compared to the Swiss Protein data bank and Genbank.
PeptideStructure and PlotStructure: Based in mathematical models and
the criteria of hydrophobicity, according to Kyte-Doolitlle, surface probability
according to Emini, chain flexibility according to Karplus Schulz, secondary
structure according to Garnier-Osguthorpe-Robson and antigenicity index
according to Jameson-Wolf, a peptideStructure and plotStructure were carried
out with the 48 kDa fragment.
RESULTS
Extraction and partial purification of fraction A PF antigen: After the
Con-A affinity column, a graph using the OD reading versus tubes was plotted
to detect significant peaks. Two peaks fractions A and B were obtained. These
two fractions as well as the flow through were used for IA to detect the
182
immunoreactive fraction(s). The fractions and the flow through were
concentrated until reach 3 mg/ml.
Immunoadsorption determined by IIF showed a complete blocking of the
intercellular stain on foreskin cryosections was visualized only with the
fraction A determined by IIF. No blocking was obtained with fraction B, BSA
control or the flow through.
Radio-labeling of fraction A: 200 ul were obtained after radio labeling
fraction A by chloramine T method. An 85% of radiolabeling efficiency was
obtained after TCA precipitation. A 10 ul aliquot of this radiolabeled fraction A,
was used as a tracer to detect co-migration with the cold PF antigen obtained
after the s/SPA/IgG affinity chromatography. Also 10 ul of the radiolabeled
fraction A was used as antigen in an IP reaction with the s/SPA/IgG- gel.
Immunoprecipitation: After incubation with the radiolabeled fraction A with
the sera mentioned in materials and methods, a 10% SDS-PAGE was run.
Autoradiography reveals a band at 48 kDa. Other bands at approximately 21,
36, 62, 66, 80,117 and 120 kDa were detected. The 48 kDa band was recognized
in all sera from the active cases of EPF from El Bagre, Colombia as well as the
PF and FS sera (Fig 1). Other bands were detected mainly in EPF sera from El
Bagre (60, 62, 80 and 34 kDa bands). Sera from five normal controls from El
Bagre also recognized the 48 kDa band. Three of those five sera belong to
183
people genetically related to EPF patients. All these five sera, also showed
other serological features with PF patients (Abreu et al, submitted). Normal
donors of the non-endemic areas do not immunoprecipitate the 48 KDa band.
Sepharose-SPA-IgG coupling: To evaluate leakage of the IgG bind to the
s/SPA/gel, two gels were run, one before cross-linked the IgG to the protein A,
and another post leakage. The coomasie blues stain showed a 80% reduction of
IgG in the gel runned after the covalently bind of the IgG. It was demonstrated
that most of the IgG of the FS serum was properly coupled to the s/SPA gel
(Fig 2).
Purification of PF antigen by the second affinity chromatography:
After incubation of the s/SPA/IgG and the fraction A, the bound fractions were
eluted, neutralized, and subjected to an OD determination at 280 nm. The
three first fractions were run in a SDS-PAGE adding the radiolabeled fraction
A as a tracer to detected co-migration (Fig 3). The gels were stained with
Coomasie brillant blue and no bands were detected which co-migrated with the
hot antigen. A silver staining was performed, and it revealed a double band of
45 and 48kDa. The 48kDa band co-migrated with the radiolabeled fraction A
(Other bands were also detected by silver stain (Fig 4). The experiment was
repeated 25 times to generate enough material for protein sequence
determination. After blotting, the 48 kDa band was excised from the PVDF
membrane; other bands were also detected, but not sequenced. The same
184
s/SPA/gel product which contains the 48 kDa band was run in non-denaturing
conditions, to evaluate modification of this double band (48 and 45kDa) and in
other detected bands. These experiments will be continued futurelly.
Amino acid sequence analysis of the 48 kDa Bovine PF antigen: From
14 cow snouts we obtained 9 pg of the 48kDa band (PF antigen). This one was
subjected to N-terminal protein sequence analysis. A N-terminus sequence
EXIKFAAAXREGED was obtained. Amino acids at positions 2 and 9 could not
be determined. The protein was not carboxymethylated to convert Cysteines
prior to sequence analysis. The search therefore only showed an 85.7 %
homology with both human and bovine Desmoglein 1. The amino acid at
position 2 in Dsg1 was Trytophane. It is sensitive to oxidation and is easily lost
after electroblotting. Position 9 in Dsg1 showed a Cysteine, it only could be
determined after caboxymethylation of the protein. An aminoacid composition
was carried out comparing our sequence with the GCG data. A higher amount
of glycine was identified in our sequence, comparing with the obtained from the
data base. Our sequence also showed a 64.3% identity with the ectodomain of
human Desmoglein 3 (Dsg3) (pemphigus vulgaris antigen).
PeptideStructure and Plotted structure: Five putative binding antigenantibody sites were found in the purified 48kDa fragment, one with higher
probability than the others.
185
DISCUSSION
Previously had been demonstrated that PF antigen is a component of the
intercellular space of stratified squamous epithelia by IIF (Beutner et al, 1968).
Furthermore, it was showed that one third of the PF serum recognized linear
epitopes by immunoblotting against the 160 kDa Dsg1 (Koulu et al, 1984).
Later using an extraction procedure that differs from the used in this study,
was demonstrated that all PF sera recognized a complex of four proteins of 260,
88, 63, and 45 kDa using human skin. The 260 kDa was lost after boiling or
extraction with glycine-HCL at pH 2.8 (Eyre et al, 1987). Using other
epidermal extraction technique and bovine epidermis a 45 kDa antigen,
recognized by IP in some PF sera, was purified and the aminoacid sequence do
not displayed identity with any other protein (Calvanico et al, 1994). Moreover,
using a modified extraction procedure with human skin, a 80 kDa fragment
was recognized by IP in 20/20 FS, five PF and 6 /13 PV sera (Martins et al,
1990). Furthermore, using this same extraction procedure followed by a second
affinity column using s/SPA/ IgG, the eluted 80 kDa band was identified as the
N-Terminal domain of Desmoglein 1 (Olague et al, 1993, 1994). Moreover,
using viable epidermis from mouse, human and bovine skin was showed by IP,
that all PF sera from active disease patients precipitate a 45 kDa bands, which
was up to that time, the smallest conformational antigen identified in PF
disease (Labib et al, 1990). However, after many attempts, the nature of this
antigen remains obscure, until our study elucidated for first time, the identity
of this PF antigen that retained conformational epitopes.
186
The extraction procedure that we used, offer many advantages in studies for
PF and EPF antigen(s). First, it’s a short step extraction procedure, second this
technique can be used with bovine, mouse and human skin as antigen source
(Labib et al, 1990; Martins et al, 1990). Third, this extraction procedure allows
to obtain higher amounts of PF antigen from bovine tissue, compared to the
recombinant Dsg1, produced by baculovirus expression system. Fourth, the
amino acid variations between human and bovine Dsg1 are mainly
conservative replacements, which allows to utilize bovine Dsg1 as a source for
PF and EPF antigen (GCG Software Program). This PF-antigen was not
recognized by IP from FS sera which were in non-active clinical stage. Some
donors from the endemic area of EPF, mainly genetically related to EPF
people, also immunoprecipitated this 48 kDa fragment, that correlates with the
presence of other immunological abnormalities found in these sera (Abreu et al,
submitted). Based in the similarity between the human Dsg3 and bovine Dsg1
ectodomain, it could be explained why approximately 60% of PV sera
immunoprecipitate the 48 kDa PF antigen and may be the PV sera also have
other autoantibodies directed against Dsg1. The Dsg1 ectodomain includes
putative calcium binding motifs which are presumed to contribute to
conformation of cadherin extracellular domain (Shapiro et al, 1995). Studies of
Dsg1
using molecular chimeras, neutralizing
antibodies, and peptide
inhibitors, suggest that the binding specificity of Dsg1 is localized in the NTerminal domain and also the observation that Dsg1 reactive autoantibodies
187
disrupted keratinocyte cell-cell contact suggest that Dsg1 could play a role in
keratinocyte adhesion (Kowalczyk et al, 1996). The detailed function of Dsg1ectodomain and the adhesive function of Dsg1 remains for be clarified (Loomis
et al, 1991; Koulu et al, 1984). The s/SPA-IgG eluted 48kDa band lost
conformational epitopes, maybe to the brief exposure to low pH. Improvement
in the elution method and/or alternative affinity chromatography techniques
probably will allow obtaining an antigen that maintains its conformational
epitopes. It is known that membrane proteins, which have both hydrophobic
and hydrophilic regions on their surfaces (as Dsg1), are not soluble in aqueous
buffers solutions and denature in organic solvents, the addition of small
amphophilic molecules like in our case, let´s solubilize membrane proteins in
their native forms (Fraction A). Our technique gives a more accurate molecular
weigth of 48 kDa, than previously reported of 45 kDa. Differences could be due
to the amount of proteases inhibitors as well as some small modification trying
to improve the characterization of this PF antigen. Since our extraction
technique uses trypsin on viable cells, only cleavage within the ectodomain
occurs (Goldsmith, 1987). Specific cut-point(s) of bovine trypsin can be
predicted in the linear ecto-Dsg1 sequence, but In Vivo it is impredictible, due
to many factors as temperature of digestion, pH of the solution, protection of
trypsin cut-points by glycosilation, modification of the protein structure for the
buffers, and maybe for interactions with other proteins (Ozawa et al, 1990). An
accurate molecular weight of the PF antigen was determined by a typical
calibration curve using standard proteins separated on SDS-PAGE gel. The full
188
extracellular portion of bovine Dsg1 has 498 aa. Using molecular weight as a
guide, our 48 kDa fragment has approximately 461 aa, awfully close to the
expected size of the 55 kDa full extracellular portion of mature Dsg1. As
described in results, the aa composition was performed and a higher glycine
was detected in our sequence, compared with the GCG data base. Some
interpretations can be suggested: first, this PF antigen is almost the full Dsg1
ectodomain. Second the dialisis performed to the s/SPA-IgG eluted product was
not complete. Third another protein could co-migrate with the 48kDa PF
antigen. Fourth the 45 kDa band could be and isoform of Dsg1. Further
analysis should be performed (Puttagunta et al, 1993).
Acknowledgments This work was supported from a National Health
Institute grant and by Veteran Affairs Medical Center grant. Special thanks to
Monica Olague-Marchan and Argelia Lopez Swiderski for training and support.
We also like to thank Dr. Liane M. Mende-Mueller at the Protein/Nucleic Acid
Shared Facility of the Medical College of Wisconsin (MCW) for protein
sequencing, data base analysis and corrections to this manuscript and to Dr
Fernando Montoya, (Director CCBB) (U de A), George Giudice (MCW) and to
Chris Tenaglia (MCW). Also, to Dermatology Department of the MCW,
Milwaukee, WI, (USA), Colciencias, (Colombia), U de A (Colombia), LASPAU,
(USA). This paper is part of the doctoral thesis of Ana Maria Abreu Velez MD,
as immuno-dermatologist in the Universidad de Antioquia. Dr Ana Maria
189
Abreu Velez is a recipient of a scholarship Colciencias, Colombia administrated
by LASPAU (Harvard University), (USA).
LEGENDS
Fig 1. Using IP the PF sera recognized a
125-l
48 kDa band fraction
obtained from the Con-A affinity chromatography column After 10% SDSPAGE and autoradiography, the PF sera specifically precipitate the 48 kDa
band (lines 4 through 9). EPF sera from Colombia also immunoprecipitate
other bands (80, 66, 62, and 34 kDa). Sera from U.S.A. normal donors do not
immunoprecipitate this band (line 3). El Bagre normal donors weakly
immunoprecipitate a band of 48 KDa and others of aprox 115 kDa and 31 kDa
(line 2). A FS patient from Brazil immunoprecipitate the 48 kDa band (line 1).
Broad range molecular weight markers at the dilution 1:20 were used as
standards (200, 117. 96, 66, 45, 31 kDa).
190
Fig 2. Sepharose-SPA-IgG coupling: Leakage of the IgG bind to the s/SPA/gel was
evaluated by two gels on 12% SDS-PAGE: before cross-linked the IgG to the Sepharose-SPAIgG coupling column (lines 9 through 13). Post cross-linked lines 1 thorugh 7). The coomasie
blues stain showed 80% reduction of IgG in the post-crosslinked. A broad molecular weigths
stantands were used as markers. The two lines pointed the heavy and lighg chains from the
IgG.
Fig 3. The PF antigen eluted from the s/SPA-IgG gel was separated after 10% SDS-PAGE. The
gel was subjected to an autoradiography by using the radiolabeled 48 kDa peptide and other
radiocative bands of 34 and 24 aprox kDa (left figure). The figure of the right shows a silver
stain, revealing a 48 kDa band which comigrates with radiolabeled PF antigen (black line) and
191
other 45 kDa band. Multiple bands of 24, 34 (Light chain Ig IgG), 36, 38, 60 (darker) (heavy
chain of IgG), 62, 66, 80, 97, 117, 230, and 250 kDa were also detected. Broad range molecular
weight markers at the dilution 1:20 were used as standards (200, 117. 96, 66, 45 and 31 kDa).
Fig 4. The PVDF membrane that was previously subjected to and autoradiograpy (Fig 3 left)
on the PVDF membrane comigrates with a 48 kDa band that was subjected to an
aminoterminal secuence analysis (dark line above from the 45 KDa molecular weigth standar).
Other bands of aproximatelly 24, 38, 62, 80 and were also detected incluiding the heavy (and
ligth chain from the IgG from the FS patients leaked from the column. Broad range molecular
weight markers at the dilution 1:20 were used as standards 200, 117, 96, 66, 45 and 31 kDa.
Error! Bookmark not defined.
192
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197
XIV. DEVELOPMENT OF AN INDIRECT ELISA ASSAY USING VIABLE
BOVINE EPIDERMIS FOR DETECTION OF AUTOANTIBODIES IN
SERA FROM PATIENTS WITH PEMPHIGUS FOLIACEUS DISEASE.
Ana Maria Abreu Velez, * Jose Manuel Mascaro, # Fernando Montoya Maya,*
and Luis A. Diaz#.
*Basic Science Corporation, University of Antioquia (U de A), Medellin,
Colombia, South America, # University of Barcelona, Spain and Medical College
of Wisconsin, (MCW), Departament of Dermatology, Milwaukee, WI, USA.
Corresponding author: Luis A. Diaz, MD, Professor and Chairman,
Dermatology Department, MCW, 8701 Watertown Plank Road, Milwaukee, WI
53226. Phone: (414) 4564086. Fax: (414) 2668673.
Short title: ELISA for pemphigus foliaceus disease using bovine epithelia as
antigen.
Key Words: Autoimmunity, Conformational autoantigen, Desmoglein 1, CAM,
ELISA, Desmosomes.
Abbreviations: PF, pemphigus foliaceus; EPF, endemic pemphigus foliaceus,
FS; fogo selvage, PV, pemphigus vulgaris; BP, bullous pemphigoid; (DIF, IIF),
direct
and
indirect
immunofluorescence;
IB,
immunoblotting;
IP,
immunoprecipitation; Con-A, concanavalin-A; SPA, Staphylococcus aureus
protein
A;
SDS-PAGE,
sodium
dodecylsulfate-polyacrylamide
electrophoresis; ELISA, enzyme-linked immunoadsorbent assay.
ABSTRACT.
gel
198
We developed and ELISA assay, based in that all endemic pemphigus foliaceus
(EPF) and non endemic pemphigus foliaceus patient’s immunoreact specifically
with a bovine tryptic extract using immunoprecipitation (IP). An indirect assay
that used a bovine epidermal PF antigen partially purified in a concanavalin-A
column (Con-A fraction A) was the best protocol. Using adjusted optical density
(OD) at
492nm,
we found a cut off of 0.1 OD readings. A total of 223 sera were
tested as following: 42 EPF from El Bagre, Colombia with 90.4% of sensitivity,
12 fogo selvagem (FS) from Brazil, 3 Cazanave’s pemphigus foliaceus (CPF)
and 3 pemphigus erythematosus from U.S.A and SPAIN with 40% of
specificity, 51 bullous pemphigoid (BP) from U.S.A and Germany (94.1%
specificity), 12 systemic lupus (100% of specificity), 58 normal donors from
U.S.A (98.2% specificity and 27 normal donors from El Bagre, Colombia (77.7%
of specificity). Overall sensitivity and specificity were 95% and 72%
respectively with and intra-assay reproducibility of 98% and inter-assay
reproducibility of 97%. A high correlation between the Browder and Lund’s
scale and OD
492
nm readings was observed (r2=0.92). This assay is calcium
dependent and can tolerate up to three months at room temperatures after
antigen coating with thimerosal 0.01% as preservative.
INTRODUCTION
At present, endemic pemphigus foliaceus (EPF) is the only reported endemic
autoimmune disease that has been partially immunologically characterized.
EPF was described in foci in South America tropical forest and in Tunisia
199
(Castro, 1983; Bastuhi et al,1995), affecting people who carry the proper
genetic background (DRB1*1402 and DRB1*0102) (Cerna et al; 1993). EPF and
pemphigus foliaceus (PF) are characterized by subcorneal blisters and
autoantibodies against desmoglein 1 (Dsg1) (Koulu et al, 1884; Stanley et al,
1989). Dsg1 is a calcium dependent cell adhesion molecule and belongs to
cadherin group (wheeler et al, 1991). Previous epidemiological data on EPF
described a high frequency of familial cases, as well as geographic and genetic
restriction, suggesting an environmental etiology for development of EPF
disease. One obstacle when studying EPF is to lack of an assay with enough
sensitivity to detect early onset of autoantibodies in people at risk, and the
hability to test many samples at same time, with low-cost and effort. On the
other hand, it was demonstrated that an affinity extract obtained by
trypsinization of viable bovine epidermis, partially purified on a Concanavalin
A (Con-A) affinity column, was immunoprecipitated by all PF sera (Labib et al,
1990). The aims of this study were: 1) to develop a sensitive and accurate assay
for detection of PF antibodies using bovine epidermis, 2) to correlate disease
activity with sera antibodies levels and 3) to compare with other immunological
techniques used in order to detect autoantibodies against PF antigen (s).
MATERIALS AND METHODS
Patiens and sera A total of 223 sera were tested. Sera were distributed in three
groups as follows:
200
Group 1, sera from patients with pemphigus foliaceus disease: We tested 60
sera from patients suffering pemphigus foliaceus distributed as follows: 42
from EPF patients from El Bagre, Colombia; twelve fogo selvagem (FS)
patients from a well characterized focus in Brazil; 3 Cazanave´s pemphigus
foliaceus (CPF); and 3 pemphigus erythematosus (PE) patients from North
America and Spain. All these PF, EPF, PE and FS sera belong to well
characterized patients following clinical, epidemiological and immunological
criteria (Diaz et al, 1989).
These sera were tested by immunoblotting (IB), indirect immunofluorescence
(IIF), and immunoprecipitation (IP) to detect presence of autoantibodies.
A.
Indirect
immunofluorescence:
Sera
were
diluted
in
calcium-
supplemented buffers at 1:20 and 1:40 dilutions and positives sera were
titrated to end point using cryosections of human foreskin and fluorescein
isothiocyanate (FITC) conjugated goat antihuman IgG (Cappel Laboratories,
Cochranville, PA). Samples were then analyzed for IgG-subclass reactivity with
murine monoclonal anti-human IG-subclasses antibodies (Miles scientific,
Naperville) and fluorescein isothiocyanate anti-mouse IgG at dilution 1:500
(Calbiochem, La Jolla, California) (Rock, et al, 1989). This test was performed
in part at the Immunodermatology Lab at the Medical college of Wisconsin and
at the Medical College of Wisconsin (MCW), Milwaukee, WI, USA and part at
201
the Immunodermatology Lab, Hospital Universitario San Vicente de Paul
(HUSVP), Medellin, Colombia.
B. Immunoblotting: sera from patients and controls were tested for reactivity
against Dsg1, this was carried out as previously described using total sodium
dodecylsulfate extracts of human and bovine epidermis fractionated by sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmy,
1970) and transferred onto nitrocellulose membrane (Stanley et al, 1986). This
test was performed at MCW.
C. Immunoprecipitation: All sera were tested by using I125-labeled 45 kDa
tryptic fragment from bovine epidermis subjected to a Concanavalin-A (Con-A)
affinity chromatography (Labib et al, 1990, Calvanico et al, 1991; Labib et al,
1991). This test was carried out at MCW. Mostly of the EPF patients were
under steroid treatment taking between 10 to 40 mg/day. Using the Browder
and Lund’s scale we assesed the severity of our EPF patients (Curtis et al,
1997).
Group 2, Normal Donors: We included 85 normal donors: 58 sera from North
America people and 27 normal donors from El Bagre, Colombia, eleven of those
sera belong to people genetically related to EPF.
202
Group 3, other autoimmune skin diseases: We tested 78 sera distributed as
following: 51 bullous pemphigoid (BP) sera diagnostized by clinical and
immunologically criteria (Zillikens et al, 1997). Three BP sera that were
positive by this ELISA, were preadsorpted with of BP 180 NC16A domain,
which contains four well-defined PB-associated antigenic epitopes following
procedure described by Zillikens et al, 1997. Fifteen well characterized PV sera.
Fifteen well characterized PV sera were also tested (Stanley et al, 1986) and
finally 12 sera from people with sistemic lupus erythematosus (LES) following
criteria establized by the American Association of Reumatologist. Asocciation)
(ARA).
Initial approaches for the ELISA protocol: we performed three different
ELISA protocols, two indirect assays and one sandwich-type assay. In order to
define optimal working conditions for these assays we followed established
guidelines (Crowther 1995). One indirect assay used a concanavalin-A (Con-A
fraction A) and the second the Con-A protein-sepharose-immunopurified PF
antigen.
Extraction and partial purification of bovine epidermal PF antigen
(Con-A fraction A): After washing, 14 cow snouts with Tris-buffered saline,
pH 7.6, containing 5 mM CaCl2 (TBS/Ca++buffer-non-azide) at 4oC, viable
epidermis separated by keratome was digested with 0.01% trypsin type III
from bovine pancreas (Sigma Chemical Co., St. Louis, MO) in (TBS/Ca++buffer-
203
non azide). Digestion was carried out while stirring at 37 oC for 1 hour and
stopped by addition of 10 mM phenylmethylsulfonyl fluoride (PMSF) and 10
ug/ml of a cocktail containing the following protease inhibitors: pepstatin, antipapain, chymostatin and leupeptin (Sigma) (Hemanson et al, 1992; Labib et al,
1989; Labib et al 1990). Extract was then centrifuged, and 916 ml of
supernatant were then subjected to Con-A sepharose 4B affinity column
(Sigma), 1 x 20 cm equilibrated with TBS/Ca++ buffer-non azide. Nineteen ConA columns were run. Columns were washed with TBS/Ca++ buffer-non-azide
until absorbance reading at OD
280
nm dropped to zero. The first 30 ml of flow
through were collected and bound glycoproteins were eluted with 0.2 M of 
methyl D mannopyranoside in a TBS-Ca++buffer-non azide (Labib et al, 1990).
Three ml fractions were collected and their absorbance at OD280 nm was
monitored. Two representative peaks were obtained (fraction A and B). These
were pooled, dialyzed, concentrated (PM 30, AMICON, Beverly, CA) and
treated with protease inhibitors as well as the run through. Concentration in
these fractions was determined by absorbance reading at OD280 nm. Protein
concentration was adjusted to 3.0 mg/ml.
Immunoadsorption (IA) using Con-A products: Flow through and
fractions A and B were used for IA. Ten, twenty-five and fifty ul of each
fraction were incubated for 30 minutes at room temperature (RT) with 50 ul of
a 1:80 dilution of serum belonging to well characterized FS patient. Negatives
controls were prepared by incubation of corresponding volumes of bovine serum
204
albumin (BSA) with same fractions. Then mixtures were tested in order to
evaluate presence of immunoreactive fragments by IIF as described elsewhere
(Labib et al, 1990). As positive controls we used same FS serum, at same
dilution, mixed with BSA and without BSA.
Radio-labeling
of
Bovine
Epidermal
Antigens:
Fraction
A
was
radiolabeled by Chloramine T method using I125as previously described (Marier
et al, 1979). Protein-bound radioactivity was determined by precipitation with
10% trichloroacetic acid. One aliquot of labeled fraction A was used for
detecting percentage of coupling of antigen to microtiter wells.
Con-A fraction A immunoaffinity purification: A PF serum was incubated
with protein-A sepharose CL-4B gel (Sigma), following previously described
procedure (Hermanson et al, 1992; Olague et al, 1994). Gel/protein-A/ IgG
complex
(s/SPA/IgG-gel),
was
resuspended
in
solution
of
0.2
M
triethanolamine, pH 8.2 and antibodies were bound to protein A, then were
cross-linked with 4 volumes of dimethyl pirimedilate (DMP) (6.6 mg/ml)
(Aldrich). Washed with 50 mM sodium borate buffer was performed.
Remaining binding sites of gel were blocked by addition of 0.1 M ethanolamine
pH 8.2 and finally s/SPA/IgG-gel was washed with IPB buffer as equilibrator.
Non-labeled fraction A was incubated with s/SPA/IgG-gel for one hour at RT
(Olague et al, 1993). After washed, bound fraction was eluted with 0.2 M
glycine, 5 mM EDTA/HCL, pH 2.8 and alliquots of 0.5 ml from eluent were
205
collected. A 0.1M NaOH was added to each aliquot as neutralizer, until reach
pH of 7.4. Each alliquot was monitored at OD280 nm. In order to detect the
band which, co-migrate with radiolabeled fraction A; this radiolabeled fraction
was added on each one of aliquots samples and these samples were mixed with
sample buffer as described for Laemmly, 1970, but in 1:1 relation (PF antigensample buffer). Each aliquot was run on SDS-PAGE and blotted. The
experiment was repeated many times to increase the amount of protein.
Significant peak was pooled, concentrated 3,000 times in Amicon, and dialyzed
over night against TBS-Ca++buffer-non-azide.
Optimization of buffer, washing and blocking buffers for indirect
ELISA assays: To obtain the optimal antigen concentration, serial dilution of
each of the antigens were added starting from 3, 12 ng/ml up to 2ug/ml (100
ul/well). The antigen coupling was used at 4oC over-night; 37oC two hours and
RT 1 to 4 hours. Different buffers were used to achieve superior binding as
follows: TBS-Ca++-non-azide buffer; TBS-non-Ca++-non-azide buffer, TBS-nonCa++-non-azide plus, 5 mM EDTA buffer; PBS; carbonate/bicarbonate buffer, all
of them at different pH ranging from 3.25 to 10.5. Many in strengths of buffers
(between 10 to 500 mM) were used. The optimal dilution of the first antibody
was assyed in a range from 1:60 to 1:300 and the optimal horse radish
peroxidase (HRP) (second antibody) was determined in a range of from 1:500 to
1:40,000.
206
Elisa plate preparation: Microtiter plates (96-well, Immulon-4, Dynatach
Laboratory Inc., Alexandria, VA) were pre-incubated with 100 ul/ well ice-cold
glutaraldehyde (grade II, 25% aqueous solution, Sigma Chemical Co., St. Louis,
Mo) (0.25%) in PBS for 16 hours at 4°C.
Determination of percentage of antigen binding to microtiters plates:
It was performed by measure the radioactivity of the label fraction A (antigen)
pre- and post-coated to microtiter plates.
The sandwich-type ELISA assay was higher time compsuming and it was
necessary to pool many PF sera to obtain good results. The indirect ELISA
using the Con-A protein A- sepharose immunopurified antigen was nonsensitive and low amounts of the 48 kDa peptide were attached to the plate.
Only the indirect ELISA using the Con-A purified antigen obtained good
results. In absence of Ca++ ELISA results were incosistent. The final ELISA
protocol with this antigen was: A microliter plates (96-well, Immulon-4,
Dynatach Laboratory Inc., Alexandria, VA) were pre-incubated with 100 ul/
well with ice-cold glutaraldehyde as described above. Excess of cross-linker was
trough-out and PF antigen (Con A- fraction A) was diluted twofold in 10 mM
phosphate buffered saline (PBS) (Manu buffer) to final concentration of 0.25
ug/ul per well. Then antigen was coated to microtitre plate for 2 and ½ hours at
RT under rotation. Plates were washed three times with PBS (330 ul/well)
with 20 seconds soaking time in an automated micro plate washer (Bio-Rad
207
model 1550). Nonspecific binding was reduced by blocking plates with 285 ul of
calcium lactate 10% (Kodak) in Tris-HCL 0.019 M, NaCl 0.29 M and 0.1%
Tween-20 (Sigma) for 1 hour at RT, under rotation. Plates were then washed as
above. Coated plates were subsequently incubated with 50 ul of serum (dilution
1:100) in Manu buffer, with a negative at 1:100 dilution and positive control at
3 dilutions: 1:100, 1:200, and 1:300 as standards. Each serum was coated in
triplicate for 1 hour RT under stirring and plates were washed as above. Wells
were incubated with 50 ul of horseradish peroxidase-labeled goat anti-human
IgG (Kirkegaard and Perry, Gaithersburg, MD) at 1:20,000 dilution in Manu
buffer for 1 hour at RT, under rotation. Plates were submitted to double wash
as above, then were incubated with 50 ul of a solution containing ophenyldiamine in 0.1% H202 (OPD, Sigma) for 10 min, RT.
Reaction was
stopped by addition of 50 ul of 2N H2S04 and OD492 nm reading was determined
using a micro plate reader (model 2550; Biorad). Non-specific reactivity was
determined by incubating first column of ELISA plate without sera and the
mean value of non-specific reactivity was subtracted from other readings by use
of one column without first antibody and instead of it, we added 50 ul of Manu
buffer to blank plates.
Reproducibility of the assay: To determine reproducibility of this assay we
tested two different antigen preparations. Each sera sample was tested in the
ELISA two separate times, each time using different antigen preparation.
Variation of ELISA readings were evaluated. Results in all steps for ELISA
208
optimization were plotted and compared in order to determine optimal ELISA
conditions. Final ELISA protocol was repeated twice using two different
antigen preparations to evaluate reproducibility of this assay as described in
reproducibility of the assay.
Stability of Con-A antigen: To test the possibility to use this ELISA for
future sero-epidemiological studies; viability of antigen was tested at different
temperatures and humidity conditions. Several microtiter plates were set-up
for beeing tested at different periods of time and temperatures, 4°C, 37°C and
at 22oC (RT). Thimerosal 0.01% (Sigma) diluted in PBS was added as
preservative.
Statistical evaluation: Medcal software (Belgium) for windows was used for
statistical analysis as well as Prism Graph Pad software (USA).
RESULTS
All sera from PF and normal donors from an endemic area of pemphigus
foliaceus from El Bagre, Colombia were tested by DIF, IIF, IB, IP and by
ELISA. Table 1 illustrates the comparation between different test for
detecting of PF autoantibodies incluiding the results of this ELISA. ELISA was
the most sensitive and IP the most specific. IB was the less sensitive.
209
Fig 1 illlustrate the high correlation between clinical disease according to
Browder and Lund scale and the titers of autoantibodies determined by ELISA
OD492nm readings. We got a r2=0.92.
Determination of percentage of antigen binding to plates: 80% of the
antigen was coated to plates determined by measurement of radioactivity preand post-coating. Radiaoactivity of the Con-A PF antigen sample pre-coated
was 538,463 pq/ml of I125and after 2 and ½ hours of incubation was 118,063
pq/ml. A radioactive antigen that was kept at room temperature for the same
period of time do not showed variations of the radioactivity time dependent.
ELISA readings. Twenty-three different ELISA plates were run in order to
test all the sera mentioned in material and methods. Fig 2 summarizes the
OD492nm readings from all the sera tested.
ELISA specificity and sensitivity: Overall sensitivity of this ELISA were
95% and 77% respectively. Four EPF patients from El Bagre were negative for
this ELISA assay (90.4 % sensitibity). These four patients were in clinical
remission, weakly immunoprecipitated the 48 kDa PF antigen and were
negative by IIF. In 12 FS, as well as in all Cazanave’s and erithematous sera,
the sensitivity was 100% (Fig 3). One out of 58 sera from normal controls from
USA was positive, (1.8% false positive) and from 27 normal controls from the
endemic area of EPF in El Bagre, Colombia, 22 sera were negative and 5
210
positives. From 11 genetically related subjects to EPF patients, 3 also were
positive to PF ELISA and 2 of these sera also imnunoprecipitated the 48 kDa
PF antigen (Fig 4). Twelve LE sera were negative (100 % specificity). From 15
PV sera, nine were positive and seven of these, contained autoantibodies
against the 48 kDa (PF antigen) by immunoprecipitation. Finally we analyzed
sera from 51 patients with BP 3 of these samples were positive. These sera
belong to patients who have a high titer of autoantibodies against BP 180.
However, after immunoadsorption using the affinity purified recombinant form
of BP 180 NC16 A domain and the BP sera which were positive for this PF
ELISA, readings were still positive (Fig 5).
Long term ELISA efficiency: This assay was performed at three different
temperatures and different plates were evaluated with same sera at days 1, 8,
15, 30, 60 and 90 after antigen coating. Best readings were obtained at room
temperature, obtaining positivity in PF sera and negativity in controls. After
three months the cutt off dropped to, 0,09 OD492.nm. When plates were
incubated at 37oC proteolysis was detected. At 4oC no utility was found.
Reproducibility of the assay: To detect the overall reproducibility of this
ELISA, an inter-assay and intra-assay reproducibility determinations were
carried out. The intra-assay variability was determined based on results from
the positive control at 1:100 dilutions. Twenty-three plates were run and each
plate the final reading of each serum was the results from triplicate readings.
211
An intra-assay reproducibility of 98 % was calculated. For inter-assay
reproducibility we used adjusted OD492 nm. We substrated OD492 nm mean
reading of the negative control from each plate (1:100 dilution) from the OD 492
nm mean reading from the positive control (1:100 dilution). The minimal
adjusted OD reading from positive control was 0.3550 and maximal was
0.6640. The OD492 nm mean adjusted was 0.4090 and the adjusted standard
deviation was 0.097. The coeficient of variation inter-assay was 21%, and the
inter-assay reproducibility was 79%. Cut off value: The cut off value for this
ELISA was determined by Response Operative Curves (ROC) with the
Software MedCal for windows. The adjusted cut off determined was 0.1.
Samples whose antibody titers were under 0.1 were considered negatives.
DISCUSSION
We developed an indirect ELISA assay to detect autoantibodies directed
against PF antigen, using the smaller conformational epitope obtained In Vivo,
related to pemphigus foliaceus disease, using the smallest conformational
epitope obtained from bovine skin related to pemphigus foliaceus disease. A
soluble tryptic fragment obtained from bovine epidermis after affinity
purification by Con-A affinity column, was used. This immunoadsorbed
reactivity from PF serum by IIF, is recognized by IP from all PF sera with
clinically active disease and by half of PV sera (Labib et al, 1990). This ELISA
assay provides more evidence that this conformational epitope is clue in the
study of PF disease. Moreover, we demonstrated that this ELISA is more
212
sensitive to detect PF autoantibodies compared with other immunological
techniques
presently
available,
which
provides
advantage
for
sera-
epidemiological studies.
Aditionally, five normal controls from El Bagre showed reactivity in this ELISA
by IP and by IB (data not showed). This finding suggests that our assay could
be useful to detect early onset of autoantibodies, or at least showed to be more
sensitive than other immunological techniques previously available. This assay
could be useful for detection immune conversion in people who carry
appropriate HLA, that confers susceptibility for development EPF disease
(Cerna et al, 1993). Larger sera-epidemiological studies will provide lights to
these questions.
Moreover, the development of a specific IgM ELISA assay will be particularly
useful for screening of large population, detecting possible endemic outbreaks
before they actually occur.
A correlation between clinical activity and detection of autoantibodies detected
by this ELISA, in patients with clinical EPF disease showed parallel
correlation as showed in Fig 1 which indicates that this ELISA can provide a
good tool for clinical following of patients.
213
Moreover, the development of an ELISA assay specific for detection IgM
autoantibodies for PF, will be particularity useful for screening large
populations, detecting possible endemic outbreaks before they actually occur
and also may complement studies for search the association or not with some
environmental factors that could be involved in PF disease.
Immunoreactivity of some BP previously immnuoadsorpted with NC16A fusion
protein could indicate reactivity against BP 230 antigen that could be exposed
after the epidermal extraction of the PF antigen, could be that some BP sera
contain autoantibodies against PF antigens (Dsg1 or other proteins). Recently,
(Abreu et al submitted) showed that the half of EPF sera from El Bagre focus
react against BMZ components by DIF, histopathology and by electron
microscopy (Abreu et al, manuscript in preparation) and maybe a common
antigens or cross reactivity can exist in these two disease. Other probable
interpretation is that some BP sera with higher autoantibody titers could bind
to polystyrene microtiter plates and readings could come from those
autoantibodies (false positives). Recently same results were demonstrated by
Ishii et al, 1997.
Regarding to patients with PV, it was demostrated that aproximatelly 50% of
these people also have autoantibodies against desmoglein 1 (Ishi et al, 199/).
Our data are in according with the results obtained by them.
214
A crucial point in this ELISA was cross-linking of antigen to microtiter plate
with glutaraldehyde; the time of cross-linkage, as well as the concentration of
this aldehyde. It is known that glycopeptides bind poorly to polystyrene plates.
We obtained an effective peptide-protein-carrier conjugate with glutaraldehyde
based on electrophilic properties of this aldehyde. Reciently the nature of the
48 kDa affinity PF antigen was elucidated as the ectodomain of the mature
form of Dsg1 (Abreu et al, Abst). Based in the amino acid (aa) composition of
this ectodomain; (Goodwin et al, 1990), which shows a net negative charge,
(isoelectric point of 4.7) (GCG Software Program), glutaraldehyde can form a
Schiff-base effect with some aa, such histidine, serine, threonine, and arginine
as possible candidates for the cross-linking properties exerted by glutaraldehy.
In summary, this indirect ELISA utilizes a 48 kDa immunoreactive fragment
obtained from viable bovine epidermis recognized by the 100% of PF sera by IP
and that can block IIF of a PF serum on human cryosections. Our ELISA assay
will facilitate screening of large populations in foci of high incidence of EPF as
in Brazil, Colombia and Tunisia, because its high sensitivity, specificity, and
reproducibility, and at the same time it is useful for screening for early onset of
autoantibodies directed against PF antigens and seem to be promissory in
detecting autoantibodies prior disease onset. Also, can provide a new tool to
understand why people develop PF disease in endemic areas and why others do
not. This ELISA showed correlation with the clinical and immunological status
of patients and also showed similar results as described in the ELISA by using
215
recombinant proteins (Ishi et al 1997). Furthermore, the calcium seems to play
and important role in protein (Ishi et al, 1997). Furthermore, the calcium
seems to play and important role in the antigen-antibody binding as previously
demonstrated (Amagai et al, 1995). One advantage of this ELISA is that can
handle large number of samples and results are obtained in five hours. Other
advantage is that the antigen can be kept immunogenic after coating to
microtiter plates at room temperature for up to three months and the fact that
provides the high availability of bovine epidermis as antigen source.
Acknowledgments: This work was supported from a National Health
Institute grant.#...,by Veteran Affairs Medical Center grant and by the
Direccion Seccional de salud de Antioquia (DSSA), Medellin, Colombia, South
America. Special thanks to George Giudice at Medical College of Wisconsin
(MCW) and Chris Tenaglia (MCW) for corrections to this manuscript. Also to
Dr Detleff Zillikens for provide BP sera and to Dermatology Department of the
MCW, Milwaukee, WI, (USA), U de A (Colombia), LASPAU, (USA) and
Mineros de Antioquia. This paper is part of the doctoral thesis of Ana Maria
Abreu Velez MD, as immuno-dermatologist in the Universidad de Antioquia.
Dr Ana Maria Abreu Velez is a recipient of a scholarship Colciencias, Colombia
administrated by LASPAU (Harvard University), (USA).
216
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calcium dependent and glycosilation independent. J Invest Dermatol 105:243247,1995.
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Mokhtar I, Fazaa B. Comparative epidemiology of pemphigus in Tunisia and
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Pemphigus foliaceus antigen: characterization of a keratinocyte envelope
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Characterization of an immunoreactive tryptic fragment from BALB/c mouse
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221
LEGENDS
Table 1. Detection of autoantibodies against PF antigen(s) in sera from people
with EPF disease (El Bagre, Colombia), non-endemic pemphigus foliaceus from
Colombia, (NEPF), fogo selvagem. (FS) from Brazil, Cazanave´s pemphigus
foliaceus (CPE), pemphigus erythematosus PE from Spain and USA. Results
are expresed in percentage of sensitivity. Elisa assay showed the bettter
sensitivity, followed by IP and IIF.
ASSAYS
FS (n=12)
IIF ICS IgG Total
EPF (n=42) NEPF
(n=42)
31%
67%
41%
CPF and
PE (n=6)
41%
IIF ICS IgG 1
5%
67%
15%
10%
IIF ICS IgG 4
77%
100%
80%
78%
IB 160 kDa (Dsg1)
29%
33%
29%
27%
IP 45 kDa
91%
100%
91%
100%
93%
100%
93%
100%
PF antigen
ELISA OD492 nm
IIF ICS: Indirect immunofluorescence detecting intercellular stain. We used
total IgG, and IgG1 and IgG4. IP: Immunoprecipitation of the 45 kDa bovine
Con-A antigen. IB: Immunoblotting using normal human skin extracts
detecting Desmoglein 1 (Dsg1).
222
Table 2. Detection of autoantibodies against pemphigus foliaceus antigen (s)
sera from normal donors from the endemic area of EPF in El Bagre (Colombia)
(NDEA), 51 bullous pemphigoid (BP) from U.S.A and Germany, 12 systemic
lupus (LE), 15 pemphigus vulgaris (PV) and, 58 normal donors from U.S.A
(ND. Results shown are expresed in percentage of especificity.
ASSAYS
NDEA ND PV
BP
LE
(n=27) (n= (n=15)
(n=51)
(n=12)
58)
IIF ICS -
0%
0%
40%
N/A
N/A
IIF ICS, IgG 1
0%
0%
10%
N/A
N/A
IIF ICS, IgG 4
0%
0%
70%
N/A
N/A
IB 160 kDa
29%
N/A N/A
N/A
N/A
77%
N/A 40%
N/A
N/A
ELISA OD492 nm 77%
98% 40%
94.1%
100%
IgG Total
(Dsg1)
IP 45 kDa PF
antigen
IIF ICS: Indirect immunofluorescence detecting intercellular stain. We used
total IgG, and IgG1 and IgG4. IP: Immunoprecipitation of the 45 kDa bovine
Con-A antigen. IB: Immunoblotting using normal human skin extracts
detecting Desmoglein 1 (Dsg1).
223
Fig 1. Assestment between clinical disease activity and OD492 readings by
ELISA. Y axis represents the 45 patients that include 42 people with EPF and
3 with Cazanave’s PF (CPF). The scale runs to 0.1 to 0.45. The X axis
corresponds to the comparatione between clinical disease activity according to
Browder and Lund and OD 492 readings by ELISA. A parallele correlation
between clinical status and ELISA readings was detected.
224
F
D
492nm
1.5
1.0
225
Fig 2. Presence of autoantibodies against a PF antigen in all groups of sera. A total of 223 sera
distributes as mentioned in methods were tested. This figure represents a summary of the
findings.
Fig 3. Presence of autoantibodies in people with PF disease detected by ELISA.
We tested three patients with CPF, three with pemphigus erithematosus (PE),
42 people with endemic pemphigus foliaceus (EPF) from El Bagre, Colombia
and 12 fogo selvagem (FS) patients from Brazil. Positivity was detected in most
of the cases. Only three EPF sera from el Bagre, which belong to individuals
with a very mild disease, were negative.
226
Fig 3
AG
E
Fig 4. Presence of autoantibodies in normal donors from USA and in donors
from an endemic area of EPF in Colombia detected by ELISA. We tested 58 and
27 normal donors from El Bagre. Higher reactivity was detected in the group of
1.2
OD 492
donors from the endemic area of PF as shown, maybe indicating presence of
reactivity to environmental factor(s).
227
Adjusted OD 492
Fig 4. PRESENCE OF AUTOANTIBODIES
AGAINST PF ANTIGEN DETECTED BY ELIS
IN NORMAL DONORS FROM AND ENDEMI
AREA OF PF AND FROM USA
1.2
0.8
0.4
0.1
0.0
DEA
NHS
Fig 5. Presence of autoantibodies in people wit other autoimmune skin
diseases different than PF detected by ELISA. A 12 LE
people, 56 BP from
CONTROLS
Germany and USA and 20 PV sera from Spain and USA were tested by ELISA.
The less reactivity was detected in the group with LE; almost half of PV sera
showed positivity against PFDEA=Donors
antigen(s), correlating
posibility area
those
fromwith
thetheendemic
of pemphig
PV sera contains autoantibodies
against PF
antigen(s), as human
well as some
foliaceus.
NHS=Normal
seraBPfrom people
sera. This data is evaluated in discussion.
live out of the endemic area of pemphigus foliace
Fig 5. PRESEN
AGAINST PF ANT
IN PEOPLE WITH
sted OD 492
228
1.2
DESCRIPTION OF AN AUTOIMMUNE SKIN DISEASE WITH
PRESENCE OF SIMULTANEOUS ACANTHOLYSIS BETWEEN
KERATINOCYTES AND AT THE BASAL MEMBRANE ZONE OF THE
SKIN, DETECTED BY ELECTRON MICROSCOPY IN PEOPLE FROM A
PECULIAR FOCUS OF ENDEMIC PEMPHIGUS FOLIACEUS-LIKE"
DISEASE IN THE RURAL AREA OF EL BAGRE, COLOMBIA, SOUTH
AMERICA.
0.8
Abreu-Velez Ana Maria*, Prada Stella*, and &Hashimoto Ken. *Section of
Dermatology, Hospital Universitario San Vicente de Paul, University of
Antioquia (U de A.), Medellin, Colombia and &Department of Dermatology and
229
Syphilology, Wayne State University, U.S.A.
Corresponding author: Hashimoto Ken&Department of Dermatology and
Syphilology, Wayne State University, U.S.A.
Key words: Endemic pemphigus foliaceus; Autoimmunity, Desmosomes,
Hemidesmosomes, Pemphigus, Pemphigoid, Subepidermal blistering disease,
Basal Membrane Zone, Acantholysis, Electron microscopy.
Running head: Autoimmune skin disease with acantholysis in the epidermis
and at the basal membrane zone.
Source of support: Dr Alberto Uribe, Dean´s of Medicine (U. de A.), Mineros
de Antioquia S.A. and.....
ABSTRACT
Background: The hallmark of the autoimmune bullous skin disease is the
presence of autoantibodies that directly or indirectly are vinculated with the
acantholysis between keratinocytes (as in the case of pemphigus) or at the
basal membrane zone (as in pemphigoid and subepidermal blistering diseases).
Currently, no descriptions of an autoimmune skin disease with presence of a
simultaneous epidermal and basal membrane zone acantholysis have been
reported. After a six-year case-control study in 50 people affected by an
endemic pemphigus foliaceus-like disease from one focus in El Bagre, (a small
tropical mining village of Colombia) we demonstrated that more than 50% of
patients showed clinical features of Senear-Usher syndrome. Namely, the
230
presence of immunostain between keratinocytes and also at the basal
membrane zone by immunofluorescence, and the presence of liquefaction of the
basal membrane zone in 30% of them by histopathology. In order to corroborate
previous findings with electron microscopy, this study was carried out.
Methods: Samples from people affected by endemic pemphigus foliaceus-like
disease from El Bagre were studied by electron microscopy. Results: We
detected in people affected by endemic pemphigus foliaceus from El Bagre the
presence of acantholysis between keratinocytes (as described in pemphigus)
and also at the lamina lucida of the hemidesmosomes (as occur in some
subepidermal blistering diseases). Presences of two classes of intracellular
vesicles were also detected. One filled with an electrondense material that
seems to be deposited at the desmosomes, which features resemble an immune
complex. Another substance, which nature remains unknown, was also
detected. Conclusions: This is the first report of an autoimmune skin disease
with presence of simultaneous acantholysis between keratinocytes and at the
basal membrane zone. This disease will contribute to the understanding of the
complexity of the cell-cell and cell matrix junctions.
INTRODUCTION
For the understanding of the cell-cell and cell-matrix junctions mechanism of
the skin, the study of some genetic diseases such as the variants of the
epidermolysis bullosa contributed mainly in the case of the hemidesmosomes
and for the desmosomes, in the understanding of the autoimmune skin
231
diseases.1 Pemphigus foliaceus (PF) and the endemic form of PF, (EPF) are
autoimmune skin diseases.1 EPF was described in foci at the South American
tropical forest, mainly in Brazil but also in other countries of Latin America, 2, 3,
4
and in Tunisia.5 EPF is characterized by presence of acantholysis and
subcorneal blisters at the upper epidermal layers of the skin accompanied by
deposits of mainly immunoglobulin IgG4 autoantibodies at the intercellular
space.6 The acantholysis have been vinculated with the presence of the
autoantibodies
directed
against
intercellular
junctions.
Some
isolated
autoantibodies are directed against a desmosomal glycoprotein called
Desmoglein 1 (Dsg1) which is target antigen of PF and is a major component of
the desmosomes.7, 8, 9 Moreover, the presence of other autoantibody populations
in people affected by PF and EPF have been described. 10,
11
Classically, the
hallmark of pemphigus disease is the presence of acantholysis between
keratinocytes (cell-cell junctions).12-16 Other types of autoimmune skin disease
are the pemphigoid and the group of subepidermal autoimmune blister
diseases which best characterized antigen(s) target(s) are bullous pemphigoid
(BP) BP 180 and BP 230 kDa proteins, in the case of the pemphigoid. A 97 kDa
protein in the linear IgA; collagen VII in acquired epidermolysis bullosa. The
105 and 200 kDa proteins which identity remains unknown in two new
subepidermal blistering diseases.19-24 All these molecules contribute to the cellmatrix junctions’ mechanism at the basal membrane zone (BMZ). Most of the
subepidermal blistering diseases revealed acantholysis at the basal membrane
zone (BMZ).
232
Presently EPF is the only endemic autoimmune skin disease described in a
focus,2 for this reason possible environmental risk factor(s) have been
vinculated for triggering this disease. After six years of a matched case-control
study of an endemic focus of pemphigus foliaceus like (EPF-L) in El Bagre,
(small mining village of Colombia) peculiar epidemiological, histopathological
and immunological features were demonstrated. Most patients were men with
a mean age of 50 years and mainly dedicated to farming and mining
activities.25 Since the patients routinely used mercury for gold extraction, we
detected higher mercury levels in hair by mass spectroscopy and higher seric
IgE levels by ELISA compared to controls. Our findings were similar to those
described in mice induced autoimunity by mercury.25 Other findings in these
patients include familial background of pemphigus in 20% and presence of
other autoantibodies different from Dsg1 (a doublet of 210 and 195 kDa, a 117
and 97 kDa antigens) by immunoblotting which nature remains unknown. By
direct immunofluorescence (DIF), immunoreactivity against components of the
BMZ was observed in half of the patients and by indirect immunofluorescence
(IIF), presence of a round intracellular stain with IgG3 monoclonal antibody
was noticed. Alterations of the BMZ were also observed by hematoxilin-eosin
stain (H & E) and presence of sweat gland necrosis in patients were also
unique to these patients.25 Deposits of mercury by autometallography mainly
in the sweat glands in controls and patients were unique to this focus.25 Since
our findings revealed differences with other patients affected by EPF disease
233
we prefer to nominate our patients as endemic pemphigus foliaceus-like (EPFL). The aim of this report was to study by electron microscopy (EM) the
features detected in people affected by EPF-L by other techniques and to
corroborate with previously findings in patients with other autoimmune skin
diseases.
MATERIALS AND METHODS
Subjects of study: We include people affected by EPF-L diagnosed by two
dermatologists following Viera´s criteria.26 These subjects participated
willingly and signed a consent form. We confirmed their EPF-L disease by
immunological criteria by detecting autoantibodies against intercellular
components between keratinocytes by (DIF and IIF) as previously described in
PF disease.6 Also by detection of autoantibodies against desmoglein 1 (Dsg1) by
immunoblotting (IB) using normal human skin as a substrate on sodium
dodecylsulfate electrophoresis (SDS-PAGE),7-9 and by immunoprecipitation (IP)
of a 45 kDa bovine epidermal trytic fragment that contains the ectodomain of
Dsg1 and is recognized for all sera with active pemphigus foliaceus disease. 27,28
Presence of autoantibodies was also tested by ELISA using the same bovine
tryptic fragment.28
Biological samples: Simultaneous skin biopsies were taken from the most
active clinical lesions (mainly from chest or back), some were fixed in 10%
formalin (for H & E) and other in 5% glutaraldehyde in phosphate buffered
234
saline (PBS) and were sent to the Dermatology Lab., at Wayne State
University (U.S.A).
Electron microscopy analysis: Fixed specimens in 5% glutaraldehyde and
1% osmic acid in phosphate buffer, pH 7.2 were embedded in Araldite. Thin
sections were stained with 1% uranyl acetate and lead citrate and examined in
a Hitachi H-300 electron microscope as previously described.29
RESULTS
Subjects analyzed. Briefly, 50 subjects were included as EPF-L cases which
showed broad clinical features as those described by Viera in 1940, (Fig 1. A. B
and C). The predominant clinical form in these people share features of
pemphigus foliaceus and lupus-like as occurs in Senear-Usher syndrome (Fig
1.D). Histopathology reveals heterogeneous features but emphasized in the
compromise of the dermal-epidermal junctions in 30% of the patients (Fig 2).
In 80% of patients the presence of autoantibodies was detected against
intercellular components by using monoclonal antibodies mainly IgG4 and
intra-celullar stain with monoclonal IgG3 by IIF. DIF reveals intercellular
stain between keratinocytes as well as at the BMZ (Fig 3.). Presence of a
heterogeneous autoantibody population was also detected mostly against a
doublet of 210 and 195 kDa antigens in patients and in people genetically
related with EPF patients (Fig 4 A.). Sera from patients with active disease
(47/50) immunoprecipitated the bovine tryptic fragment (Fig 4B). Presence of
235
autoantibodies was also detected in 97 % of active cases of EPF tested by
ELISA.
Electron microscopy analysis: From EPF-L patients with early lesions, we
observed early acantholytic cells at the granulous layer as occur in pemphigus
foliaceus disease (Fig 5). This loss of coherence between the epidermal cells
stars was present with an elongation of the cytoplasmic parts of the
desmosomes. At the BMZ, acantholysis was also detected at the lamina lucida
of the hemidesmosomes (Fig 6).
Presence of an electrondense substance located at the intercellular space of the
keratinocyte, was observed at the granulous layer in some EPF-L patietns (Fig
7). Inside cells, presence of two types of vesicles filled with two different
electron dense materials was detected. These vesicles seem to come from inside
the cells and seeming to extrude their material to the intecellular space (where
one of the target antigens of this disease, Dsg1 is located) (Fig 8).
DISCUSSION
Presently, the people affected by EPF-L from one focus in El Bagre, Colombia
revealed a unique feature differing from other people affected by endemic
pemphigus foliaceus disease in aspects as age of onset disease, sex, and in part
for work activities.25 A unique clinical form resembling features of SenearUsher syndrome were observed in more than 50% of them and in half of their
236
skin biopsies, the presence of immunostain at the BMZ was detected by DIF as
well as at the intercellular space between keratinocytes as described in SenearUsher syndrome.30 By H & E, lupus-like features were also described in almost
30% of EPF-L correlating with clinical features and with the DIF findings at
the BMZ. For the first time in literature, we reported the presence of a
simultaneous acantholysis between cell-cell (desmosomes) as well as between
cell-matrix junctions (hemidesmosomes) in one autoimmune skin disease. We
can speculate that this disease belongs to a one broad spectrum of autoimmune
skin diseases, embracing features of endemic pemphigus foliaceus, SenearUsher and sub-epidermal blistering diseases due the compromise of the BMZ
demonstrated by DIF, H& E and by EM.
Maybe for the fact that at the time most of the descriptions of Senear-Usher
syndrome were reported, these cases were not studied by techniques that are
available such as immunoprecipitation, immunoblotting and EM. We cannot
deny that perhaps people affected by SenearUsher lack the findings as
described in this report. Our EPF-L patients differ from some Senear-Usher
patients by the presence of antinuclear autoantibodies which are absent in our
EPF-L patients.25
By IB technique, presence of a 210 and 195 kDa doublets of autoantibodies
directed against components of the skin were detected in 40% of EPF-L cases
and in 37% of "normal donors" from the endemic area. These autoantibodies
237
were not observed in people who live outside the endemic area. This indicated
the possibility of exposure of possible environmental risk factors that might be
acting in people who live in the endemic area and by some factor (maybe
genetically) predispose some of them to develop the autoimmunity.
In patients affected by pemphigus foliaceus and in pemphigus vulgaris,
presence of autoantibodies directed against components of the desmosomes and
hemidesmosomes was also described, however the presence of acantholysis was
never observed.10
Recently, the presence of common proteins localized at desmosomes and
hemidesmosomes have been characterized and one of them, envoplakin, is a
target antigen of paraneoplastic pemphigus.31 The molecular weight of this
protein is 210 kDa on SDS-PAGE as the molecular weight of one of the target
antigens detected in our patients. Moreover, the presence of another protein
that co-immunoprecipitated with envoplakin was recently reported as a 195
kDa protein denominated periplakin. Preliminary reports showed desmosomal
as well as hemidesmosomal localization of envoplakin and periplakin.31,32.The
molecular weights of the doublets of antigens that are recognized by the
autoantibodies in our patients are similar to the molecular weight of these two
proteins.31,
32
Further experiments will be developed in order to gain a more
precise understanding of the localization, nature and relationship of the other
autoantigens different from Dsg1 in patients affected by this unique disease.
238
The relationship of the target antigens with desmosomal and hemidesmosomal
proteins and the relationship with the phenomena of the acantholysis will be
studied in the future.
By light microscopy, the acantholysis was not evident, however, the EM
pointed out this phenomenon. Maybe other proteins of the basal membrane
zone allow it to retain junctions between epidermal and dermal tissue that are
not easily detected by H& E. Maybe other molecules that are not associated
with this disease play a role, keeping these junctions joined macroscopically,
but with the evident alteration detected by EM. New reports have
demonstrated the presence of common proteins located in desmosomes and at
the hemidesmosomes.33,
34
With the description of this disease a new
perspective arise in the knowledge of the complexity of the desmosomes and
hemidesmosomes and the keratinocyte-keratinocyte and dermal-epidermal
junctions’ mechanism.
By using an ELISA with a recombinant NC16A bullous pemphigoid antigen
(BP 180),35 sera from these EPF-L patients were tested and only one was
positive for that ELISA.34.
With our findings some question arise as to what is the initial molecule(s)
involved in the autoimmune phenomena? Meanwhile, important findings are
being reported in antigen targets in PF, PV and bullous pemphigoid disease by
239
epitope mapping in molecules as desmoglein 1, 3, BP 180 and BP 230. Some
question arises because we are prior to a more complex junction discovery, and
maybe prior to an epitope spreading discovery, or prior to an unknown genetic
or environmental discovery necessary to document the triggering the
autoimmune phenomena. Sera-epidemiological studies in endemic areas of
pemphigus foliaceus disease could provide insights needed to detect initial
immunological alterations in people exposed to some environmental factors;
otherwise, all statements are pure speculation.
Regarding the presence of an electrondense substance at the desmosomes
where one of the target antigens of the EPF-L patients is located (Dsg1)
resembles the electron microscopy image of an immune complex as occurs in
glomerulonephritis by immune complex. This mix of electron dense material of
unknown source requires further analysis by use of electron-dispersive
microscopy,36 to detect the presence of metals. This statement rises from the
fact that we detected high mercury levels in people affected by EPF-L
compared to controls and also due the presence of mercury on skin biopsies by
autometallography.23 It is important to recall that gold and mercury can
amalgamate In vivo and both metals can produce autoimmunity. Mercury and
gold can bind some proteins, inclusive at the desmosomes.37
The intracellular immuno staining detected in EPF-L patients with IgG3 could
correspond to the immunostain against the two types of vesicles detected in
240
some people affected by EPF-L disease.
241
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13. Hashimoto K, Lever WF. The intercellular cement in pemphigus vulgaris:
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14. Wilgram GF, Caufield JB& Madgic EB. An electron microscopic study of
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1964;43:287-99.
15. Chorzelski TP., Biczysko W, Dabrowski J & Jahzabeck M Ultrastructural
localization of pemphigus antibodies. J Invest Derm 1968; 50:36-40.
16. Komura J An electromicroscopic study of anatholysis in pemphigus
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17. Barros C. Ultraestrutura da lesao bolhosa e do sinal de nikolsky no penfigo
foliaceo. 1972: tese de dontoramiento apresentada a clinica dermatologica e
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18. Stanley JR, Woodley DT, Katz S. Identification and partial characterization
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of pemphigoid antigen extracted from normal human skin. J Invest Dermatol
1984;82:108-111.
19. Zillikens D, Kawahara Y, Ishiko A et al. A novel subepidermal blistering
disease with autoantibodies to a 200 kDa antigen of the
basement zone. J Invest Dermatol 1996; 106:133-38.
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insights from allogeneic diseases and chemically induced autoimmunity.
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21. Chan LS, Wang XS Lapiere JC, Marinkovich MP, Jones JC, Woodley DT. A
newly identified 105 kDa lower lamina lucida autoantigen is an acidic protein
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22. Gayraud B, Hopfner B, Jassim A, Aumailley M, Bruckner Tuderman L.
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GDA-J/F3 monoclonal antibody. J Biol Chem 1997;272:9531-8.
23. Giudice G, Emery DJ, Diaz LA. Cloning and primary structural analysis of
bullous pemphigoid autontigen BP 180. J Invest Dermatol 1992;99:243-50.
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24. Zone JJ, Taylor TB, Meyer LJ, Peterson MJ. The 97 kDa linear IgA bullous
disease antigen is identical to a portion of the extracellular domain of the 180
kDa Bullous pemphigoid antigen, BPAG2. J Invest Dermatol 1998;110.207-10.
25. Ana M. Abreu-Velez, Juan G. Maldonado, Andres Jaramillo, Pablo J.
Patiño, Stella Prada, Leon Walter, Jorge Botero, Gunnar Warfvinge and
Fernando Montoya. Description of an unusual focus of endemic pemphigus
foliaceus in a rural area of El Bagre, Colombia. FASEB J. Abst 1998;12: p.287.
26. Viera JP. Penfigo foliaceo e syndrome de Senear-Usher, Sao Paulo,
Empresa Grafica da Revista dos Tribunas, 1942.
27. Labib RS, Camargo S, Futamura S, Martins CR, Anhalth GJ, Diaz LA.
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28. Abreu AM, Olague Marchan M, Lopez-Swiderski A, Mascaro JMJr, Giudice
GJ, Diaz LA. Characterization of the 45 kD epidermal tryptic peptide
recognized by pemphigus foliaceus sera. J Invest Dermatol, Abs,1997;108,541.
29. Yada J, Hashimoto K. Curvicircular intracytoplasmic membranous
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30. Castro RM, Augusto DAF, Rivitti EA. Sindrome de Senear-Usher e Fogo
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31. Kim SC, Kwon DY, Lee LJ, Chang SN, Lee TG. Envoplakin is a component
of the antigen complex in paraneoplastic pemphigus. J Invest Dermatol
1997;108: 581.
32. Ruhrberg-C; Hajibagheri-MA; Parry-DA et al. Periplakin, a novel
component of cornified envelopes and desmosomes that belongs to the plakin
family and forms complexes with envoplakin. J-Cell-Biol. 1997;7:1835-49.
33. Skalli O, Jones JCR, Gagescu R, Goldman RF. IFAP 300 is a common to
desmosomes and hemidesmosomes and is a possible linker of intermediate
filaments to these junctions. J Cell Biol 1994;125:159-170.
34. Joly P, Gilbert D, Thomine E, Delpech A, Verdier S, Lauret P, tron F.
Immunofluorescence and immunoelectron microscopy analyses of a human
monoclonal anti-epithelial cell surface antibody that recognizes a 180-190 kDa
polypeptide: component of the paraneoplastic pemphigus antigen complex? J
Invest Dermatol 1993;101:339-45.
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RG, Diaz LA, Giudice G. A highly sensitive enzyme-linked immunosorbent
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Occupational argyria and electron microscopic studies and X-ray
microanalysis. Brit J Dermatol 1981; 104:19-26.
37. Silberberg I. Ultrastructural identification of mercury in epidermis.
Arch Environ Health 1972;24:129-44.
248
FIGURES
Fig 1. Clinical characteristics of people affected by EPF from El Bagre. A.
Prurigoid form in a seborrhoeic area. B. Bullous exfoliative form C. Keratotic
generalized form D. Localized form with a lupus-like aspect.
A.
B.
C.
D.
249
Fig 2. The H&E reveal the compromise of the dermal-epidermal junctions as
depicted in this microphotograph.
Fig 3. Illustrates an intercellular stain with monoclonal antibody IgG4 by
direct immunofluorescence between keratinocytes and at the basal membrane
zone. Some acantholitic cells are visualized at the epidermis and slight
separation at the BMZ is visualized. Normal human skin was used as a
substrate and mice anti human monoclonal antibody conjugated with
fluorescein isothiocyanate in calcium supplemented buffers were used. The
sections were examined under a Leitz Orthophan fluorescence microscope.
250
Fig 4. A. Immunoblotting (left figure): Sera of both patients with EPF-L disease from El
Bagre, Colombia and controls were tested for reactivity against human skin. It was
fractionated by sodium dodecyl sulfate-polyacrylamide on 10% gel electrophoresis (SDS-PAGE)
and transferred onto nitrocellulose membrane. Line 1 shows a serum from a patient with
pemphigus foliaceus (160 kDa band). Lines 2 and 3, normal donors. Lines 4 reveal two bands of
210 kDa and 195 kDa from EPF-L sera. B. Right figure shows an immunoprecipitation of sera
from EPF-L patients and controls using the bovine tryptic fragment obtained after Con Aaffinity chromatography as substrates on 10% gel electrophoresis (SDS-PAGE. Lines 1 and 4
are negative controls. Lines 1, 3, and 4 through 7 are FS sera from Brazil. Line 3 is a normal
donor from El Bagre. Lines 5 through 9 are EPF-L patients. The molecular weight of the bands
from the top to the bottom corresponds to 80, 66, 62, 48 kDa. We used Biorad broad molecular
weighs standards as markers 117, 96, 60, 45, 31 kDa.
1
45
2
3
4
5
6
7
8
251
Fig 5. This picture of EM revealed the typical acantholysis between
keratinocytes (red arrow) detected in patients with EPF-L disease from El
Bagre, Colombia, as occur in pemphigus diseases. The tissue was fixed, and
process as described in methods.
Fig 6. This picture of EM revealed the acantholysis at the lamina lucida of the
hemidesmosomes, (red arrow) as occurs in some sub-epidermal blistering
diseases. The tissue was fixed, and process as described in methods.
Fig 7. This fig reveals an EM of skin biopsies from some patients with EPF-L
from El Bagre, Colombia. Presence of two types of electron dense material (blue
and green arrows) deposited at desmosomes was observed. Red arrow points to
252
a normal desmosome. The tissue was fixed, and process as described in
methods.
253
Fig 8. This fig. illustrates an EM the two types (red and green arrows) of
intracellular vesicles detected in some people affected by EPF-L disease from
El Bagre, Colombia. These vesicles seem to be extruding their material at the
demosome junctions by EM (blue arrow). The tissue was fixed, and process as
described in methods.
XVI. Future studies and goals to develop in my professional career.
1. SERA-EPIDEMIOLOGICAL, MICROSATELLITES AND COMPLEX
SEGREGATION ANALYSIS IN PEOPLE FROM THE FOCUS OF
ENDEMIC PEMPHIGUS FOLIACEUS IN EL BAGRE, COLOMBIA,
SOUTH AMERICA
Based on the results obtained after five years studying this focus of EPF, and
by use of the ELISA, IB, IP, DIF, IIF assays we previously tested some healthy
254
donors from the endemic area and some relatives to EPF patients. Presence of
autoantibodies was detected mainly in people genetically related to EPF
patients (see advances). For this fact and since that we detected presence of
EPF disease in some family members of EPF patients (20%), indicate a
probably genetic background maybe requiered to develop EPF disease.
Because EPF disease is the only autoimmune entity reported in endemic focus,
which makes it unique in order to study the possible genetic as environmental
factors that can interact for the development of an autoimmune phenomena,
we will test for the genetic component in this disease.
Our questions are:
1. Do pemphigus foliaceus disease have a genetic component, that predispose
that some people develop the autoimmunity and other do not.
2. Do the people affected by EPF disease have some proteins that are related
with gene regulation that can be altered by mercury?
3) Is EPF one disease with only an environmental component, or can be the
result of a mixed components as genetic, environmental factors, to develop the
autoimmune phenomeno?
255
4) Do the people affected for EPF and their relatives carry out some
microsatellites that can be consider as good, neural mendeliana markers?
With this research proposal we will try to ascertain for the study of the possible
role of a genetical background as requirement in order for development the
autoimmunity in individuals affected by endemic pemphigus foliaceus disease
who are exposed to some environmental(s) noxae. Also, to try to detect some
microsatellites associated with some proteins as envoplakin, desmogleins,
desmocolins, fibrillagrin, loricrin, periplakin and immunoglobulins. This
question will generate some data to try to understand the phenomena of
autoimmunity in humans.
We will study EPF patient as described in the previously manuscripts and
epidemiological assessment will be performed in families of at least 30 EPF
genetic-epidemiological test in order to fill out the pedigree’s threes.
Immunological characterization will be carried out as previously described in
this thesis. Complex segregation analysis (CSA) and microsatellites detection
will be performed.
256
2. CHARACTERIZATION OF OTHER AUTOANTIGENS DETECTED IN
PEOPLE WITH ENDEMIC PEMPHIGUS FOLIACEUS-LIKE DISEASE
FROM EL BAGRE, COLOMBIA AND LOCALIZATION OF THESE BY
IMMUNOELECTRON MICROSCOPY ANALYSIS ON HUMAN SKIN.
Our previous results indicate that the focus of endemic pemphigus foliaceuslike is a unique focus with clinical, epidemiological, histopathological
immunological and electron microscopical features.
These patients showed predominant clinical lesions that resembly a SenearUsher syndrome. Histopathological features demonstrated unique findings as
lupus-like,
pustulosis
dermatitis,
psoriasiform
dermatitis
and
others.
Moreover, by immnunoblotting analysis (IB) and by immunoprecipitation (IP),
presence of other autoantibodies population directed against a doublet of 210
and 195 kDa as well as a 117, 96 kDa antigens was detected. Direct
immunofluorescence (DIF) studies showed that half of the skin biopsies showed
the
typical
intercellular
stain
between
keratinocytes
as
well
as
immunofluorescence at the basal membrane zone (BMZ). Moreover, the
“possible” compromise of the basal membrane zone was demonstrated by
electron microscopy analysis (Abreu et al, manuscript in preparation). For the
fact that this entity is unique for the acantholysis not only at the intercellular
space between keratinocytes and at the BMZ.
The aims of this study are:
257
1) To try to characterize the previously mentioned autoantibody population
different than Desmoglein 1 (Dsg1), by immunoblotting, immunoprecipitation,
and by electoelution from the nitrocellulose membranes.
2) To try to localize the autoantigens by using of electronmicroscopy (IEM)
analysis by using the above-mentioned autoantigens.
By using the sera from patients and controls from the endemic area of EPFLike, following criteria previously established in this thesis the following
procedures will be perform. Cow snouts and normal human skin will be used as
antigen source and immunoblotting and electrotransference will be performed
and by using of immunoelectron microscopy the localization of the antigens will
be carry out. An anti-idiotype antiserum will be done by immunzation of a
rabbit and the IgG will be isolated with 33% ammonium precipitates and
affinity matrix.
The immuno localization of these antigens and the relationship with the
desmosomal and hemidesmosomal proteins will be especially useful in order to
characterize this peculiar focus of EPF-L disease.
258
3. IMMUNOLOGICAL ASSAYS USING T AND B CELLS FROM
CONTROLS
AND
PATIENTS
WITH
ENDEMIC
PEMPHIGUS
FOLIACEUS-LIKE DISEASE IN EL BAGRE, COLOMBIA.
These studies will be focused to perform, and proliferative T and B cells assays
by exposition of T and B cells from cells from controls and patients with
endemic pemphigus foliaceus-like disease in El Bagre, Colombia. After
separation of mononuclears from peripherical bllod, cells will be sorted by
sucrose gradient or by ficoll-hypaque. Cells will be exposed to mercury, to the
48 kDa bovine affinity purified PF antigen using a non-specific stimulators as
controls. Interleukins will be added to stimula the T and B cells proliferation.
Our questions are:
Do the sera from people affected by EPF-L disease from El Bagre respond in
same way by using polyclonal stimulants such as mitogens or antibodies that
can activate all (or most T cells or B cells in an antigen–nonspecific fashion?
Do the sera from the people affected by EPF-L disease from El Bagre respond
in same way to the use of polyclonal stimulants such as mitogens or antibodies
that can activate all (or most) T cells or B cells in an antigen–nonspecific
fashion in the same way comparing with specific antigens as the extract
obtained from affinity purified PF antigen obtained from bovine epidermal
tryptic preparations?
259
3. Do the sera from the people affected by pemphigus foliaceus disease respond
in same way to the use of polyclonal stimulants such as mitogens or antibodies
that can activate all (or most) T cells or B cells in an antigen–nonspecific
fashion in the same way comparing with specific antigens as mercury and or
UVB and or UVB light exposure?
With this research proposal we will try to ascertain for the study of immune
response of the T and B cells to some possible risk factors detected in the focus
of EPF from El Bagre.
ANNEXES