bullous pemphigoid ( bp ) , the most common autoimmune blistering disease , is mediated by autoantibodies .
bp primarily affects the elderly and is characterized by the development of urticarial plaques surmounted by subepidermal blisters , and the deposition of immunoglobulins and complement at the basement membrane zone ( bmz ) of the skin .
bp is immunologically characterized by the development of autoantibodies targeting two structural proteins of the hemidesmosomes , bp180 ( collagen xvii ) and bp230 ( bpag1 ) .
a 63 -year - old caucasian female patient was evaluated for a 4 day history of several itching , erythematous blisters on her extremities .
biopsies for hematoxylin and eosin ( h&e ) examination , as well as periodic acid - schiff ( pas ) , immunohistochemistry ( ihc ) and direct immunofluorescence ( dif ) analysis were performed .
h&e demonstrated a subepidermal blister , with partial re - epithelialization of the blister floor . within the blister lumen ,
numerous neutrophils were present , with occasional eosinophils and lymphocytes also noted . within the dermis ,
a mild , superficial , perivascular and periadnexal infiltrate of lymphocytes , histiocytes and occasional eosinophils was identified , with mild perivascular leukocytoclastic debris .
the pas stain was positive at the bmz , and around selected blood vessels , nerves and sweat glands .
dif revealed linear deposits of igg and complement / c3 and fibrinogen at the bmz , and around selected dermal nerves , blood vessels and sweat glands .
strong granular deposits of ige were also observed , colocalizing with monoclonal antibodies to collagen iv ( civ ) . by ihc ,
positive cd45 staining of lymphocytes was seen surrounding selected dermal blood vessels , eccrine sweat glands , and nerves .
the patient displayed igg , ige , and fibrinogen autoantibodies against the bmz , as well as around some dermal nerves and sweat glands ; their binding in the skin could trigger complement activation .
bullous pemphigoid ( bp ) is an autoimmune blistering disease primarily affecting elderly patients , and characterized by the development of urticarial plaques surmounted by subepidermal blisters and the deposition of both immunoglobulins and complement at the basement membrane zone ( bmz ) of the skin .
immunologically , it is characterized by the development of autoantibodies targeting two important proteins of the hemidesmosomes , bp180 ( collagen xvii ) and bp230 .
it is known that bp230 is an intracellular protein of the hemidesmosomal plaque , while bp180 is a transmembrane protein containing a collagenous extracellular domain .
autoantibodies are of both the igg and ige classes ; their binding in the skin triggers complement activation , mast cell degranulation and additional migration of eosinophils , mast cells , and neutrophils[14].discharge of proteases from these inflammatory cells may result in cleavage of the bmz and subsequent blister formation .
a 63 year old female patient was referred with a four day history of several , severely pruritic , erythematous blisters and crusts on her extremities ( arms and thighs ) ( fig .
skin biopsies for hematoxylin and eosin ( h & e ) , as well as for periodic acid - schiff ( pas ) and immunohistochemistry ( ihc ) were performed .
in addition , direct immunofluorescence ( dif ) and indirect immunofluorescence ( iif ) with 0.1 m sodium chloride salt split skin were performed . in brief
, dif was performed utilizing skin cryosections , incubated with multiple fluorescein isothiocyanate ( fitc)-conjugated secondary antibodies .
the secondary antibodies were of rabbit origin , and included : a ) anti - human igg (  chain ) , b ) anti - human iga (  chains ) , c ) anti - human igm ( -chain ) , d ) anti - human fibrinogen , and e ) anti - human albumin ( all used at 1:20 to 1:40 dilutions and obtained from dako ( carpinteria , california , usa ) .
we also utilized secondary antibodies of goat origin , including : a ) anti - human ige antiserum ( vector laboratories , bridgeport , new jersey , usa ) and b ) anti - human c1q ( southern biotech , birmingham , alabama , usa ) .
finally , monoclonal anti - mouse collagen iv ( civ ) from invitrogen(carlsbad , california , usa ) with a goat anti - mouse texas red conjugated secondary antibody was utilized to highlight the basement membrane zone ( bmz ) .
the slides were then counterstained with 4,6-diamidino-2-phenylindole ( dapi ) ( pierce , rockford , illinois , usa ) washed , coverslipped , and dried overnight at 4c .
ihc staining using anti human cd45 was performed using a dako automatized dual endogenous flex system , following dako technical instructions . in figure 1 , we highlight our most significant h & e , pas , dif and ihc results , including localization of the bmz autoantibodies utilizing the iif sodium chloride split skin technique . the dif results in the basement membrane zone of the skin ( bmz ) were as follows : igg , linear , + + ; ige , granular , + + ; c3 , linear , + + ; fibrinogen , linear , + + ; complement / c1q , linear , + /-
; fibrinogen , linear , + + , and collagen iv , linear , + + . within the dermis , we observed ige , fibrinogen and c3 perineural and sweat gland reactivity ( + + ) .
indirect immunofluorescence(iif ) performed on 1 m nacl salt split skin displayed the following results : igg ( + , linear , blister roof ) ; iga ( + , linear , blister roof ) ; igm ( + , linear , blister roof ) ; ige ( + , dermal perivascular and perineural ) ; complement / c1q(+ , linear , blister roof ) ; complement / c3 ( + , linear , blister roof ) ; albumin(+ , linear , blister roof ) ; and fibrinogen ( + , linear , blister roof ) .
h & e staining ( 100x ) demonstrating the subepidermal blister ( red arrow ) . 1b ,
dif , positive staining with fitc conjugated anti - human ige at the bmz in a linear pattern ( green staining , lower white arrow ) , colocalizing with the collagen iv(civ ) antibody ( red staining , top yellow arrow ) .
please notice that civ also stains the upper dermal blood vessel areas ( lower yellow arrow ) .
dif , showing destruction of epidermal keratinocytes , characterized by amorphous staining of cell nuclei with dapi ( blue staining , yellow arrows ) .
the red arrow shows defragmented pieces of civ in the blister lumen ( red particles ) ; the white arrows show positive staining with fitc conjugated antihuman fibrinogen , on both sides of the blister ( epidermal and dermal ) .
1d , shows a clinical blister ( white arrow ) and some adjacent crusts ( blue arrows ) .
note the keratinocytes nuclei in blue ( dapi ) , and the mapping of the blister with civ antibody ( red staining , yellow arrows ) .
please note the delicate , trans - epidermal excretion of tiny fragments of civ , seen as small red dots among the keratinocytes .
. periodic acid - schiff ( pas ) stain , displaying pink positivity at the bmz ( red arrow ) .
positive staining of a nerve with fitc conjugated anti - human fibrinogen ( green staining ; red arrows ) , as well as against eccrine sweat glands ( yellow arrows ) .
the identity of the nerve was confirmed by colocalizing , positive s-100 ihc staining ( brown staining , red arrow ) .
in addition , we utilized ihc to confirm a significant lymphocytic infiltrate around dermal neurovascular package and eccrine sweat glands with cd45 ( brown staining , red arrows ) .
. 1k , dif positive staining of a blood vessel wall with fitc conjugated complement / c3 ( green - yellowish staining , yellow arrow ) , colocalizing with civ antibody ( red staining , red arrows ) .
1 m . h & e stain , showing lymphocytes infiltrating around dermal nerves ( black arrow ) , eccrine sweat glands ( yellow arrow ) and an eccrine sweat gland ductus ( blue arrow ) .
pas positive staining of eccrine sweat gland peripheral membranes ( pink staining , black arrows ) .
examination of the h&e tissue sections demonstrated a subepidermal , tense blister , with partial re - epithelialization of the blister floor .
a mild , superficial , perivascular and periadnexal infiltrate of lymphocytes , histiocytes and occasional eosinophils was identified .
a pas special stain displayed positivity at the basement membrane zone ( bmz ) , as well as around some dermal blood vessels , nerves and eccrine sweat glands ( fig .
bullous pemphigoid and epidermolysis bullosa acquisita ( eba ) may have indistinguishable clinical , histologic , and routine immunofluorescence features .
thus , these two diseases can be reliably distinguished 1 ) in seropositive cases by indirect immunoflrouescence ( iif ) testing on salt lamina lucida split skin , 2 ) in research studies by direct immunoelectron microscopy and 3 ) in patients with circulating autoantibodies , by immunoblotting ( ib ) western studies .
the use of these methods is limited by their availability and expense , and the requirement for circulating autoantibodies . in our case , following initial direct immunofluorescence of the biopsy specimen , additional salt split skin studies utilizing 1.0 mol / l sodium chloride were performed . in eba ,
the igg linear bmz deposits classically appear on the dermal side of a split specimen ; in bp , predominantly or exclusively in the epidermal side . in our case , the salt split skin pattern favored the diagnosis of bp , with the additional features of ige and fibrinogen also present on both sides of the nacl induced blister .
other differential diagnoses we considered included bullous impetigo , and a bullous arthropod bite reaction .
in general , patient encounters with biting or stinging arthropods elicit localized reactions including pruritic macules , urticarial wheals and papular reactions .
less frequently , localized bullous or hemorrhagic or disseminated papular reactions may be seen , particularly in children and immunologically naive adults . with the exclusion of bee and wasp venom allergies , immediate - type allergic reactions to arthropod stings and bites
thus , we considered a bullous arthropod bite reaction in our differential diagnosis , which could produce late - phase reactions including the development of sustained edema , vesicles , blisters , and/or intense pruritus .
although igg represents the most commonly documented immunoglobulin subclass in bp patients , several recent reports also highlight the importance of ige ; its binding in the skin triggers complement activation , and further accumulation of inflammatory cells[79 ] .
indeed , a correlation of ige autoantibody with bp180 in a severe form of bullous pemphigoid has been described .
the case demonstrated positivity with igg , ige , fibrinogen and complement / c3 against not only the bmz , but also to dermal nerves , blood vessels and sweat glands .
the desmosomes are multi - protein complexes , promoting stable adhesion of epithelial cells to the underlying extracellular matrix .
interactions between different hemidesmosomal components with each other have been studied ; these interactions have been studied utilizing double hybrid and cell transfection assays .
the results demonstrated that : bp180 binds not only to bp230 , but also to plectin .
the interactions between these proteins are facilitated by the y subdomain within the n - terminal plakin domains of bp230 and plectin , and residues 145 - 230 of the cytoplasmic domain of bp180 .
relative to residues 145 - 230 , different , but overlapping , sequences on bp180 mediate binding to integrin 4 which , in turn , also associates with bp180 via its third fibronectin type iii repeat .
further , sequences in the n - terminal extremity of bp230 mediate its binding to integrin 4 , which requires the c - terminal end of the connecting segment ( up to the fourth fibronectin repeat of the integrin 4 subunit ) .
based on this knowledge , we speculate that , given our patient 's immunofluorescence positivity against the dermal sweat glands , possible epitope spreading between the bp180 and bp230 antigens could elicit reactivity to integrins and/or plectins expressed not only in multiple areas of the bmz , but also in the dermal nerves , sweat glands and/or microvasculature .
in addition , serum from a patient with bullous pemphigoid was recently associated with neurological diseases , recognizing bp antigen in the skin and in the brain .
we were able to demonstrate a direct correlation of our dif and iif / salt split skin positivity with positive pas staining .
relative to patients who live in areas where dif and iif are not available , the pas stain often shows a high correlation with the positivity of immunoglobulins and complement in autoimmune diseases , as shown in our case[1517 ] .
the reaction of periodic acid selectively oxidizes glucose residues , creating aldehydes ; these then react with the schiff reagent , yielding a purple - magenta color .
thus , pas staining is often utilized for staining structures containing a high proportion of carbohydrate macromolecules ( i.e. , glycogen , glycoproteins , or proteoglycans ) , typically found in neutrophils , fungal organisms and basal laminae .
positive pas staining may be found in diverse autoimmune diseases , including vasculitides , autoimmune bullous diseases , autoimmune nephritis and goodpasture 's syndrome[1517 ] .
finally , the significance in our case of abundant cd45 positive lymphocytes around the dermal sweat glands , sweat ducts and neurovascular packages remains unknown .
further investigation of our case features of 1 ) bp ige / igg / fibrinogen linear deposits at the bmz , 2 ) auto - reactivity to dermal sweat glands and nerves and 3 ) cd45 positive infiltrating dermal lymphocytes may lead to a better understanding of this disease .
 context : bullous pemphigoid ( bp ) , the most common autoimmune blistering disease , is mediated by autoantibodies . 
 bp primarily affects the elderly and is characterized by the development of urticarial plaques surmounted by subepidermal blisters , and the deposition of immunoglobulins and complement at the basement membrane zone ( bmz ) of the skin . 
 bp is immunologically characterized by the development of autoantibodies targeting two structural proteins of the hemidesmosomes , bp180 ( collagen xvii ) and bp230 ( bpag1).case report : a 63 -year - old caucasian female patient was evaluated for a 4 day history of several itching , erythematous blisters on her extremities . 
 biopsies for hematoxylin and eosin ( h&e ) examination , as well as periodic acid - schiff ( pas ) , immunohistochemistry ( ihc ) and direct immunofluorescence ( dif ) analysis were performed.results:h&e demonstrated a subepidermal blister , with partial re - epithelialization of the blister floor . within the blister lumen , 
 numerous neutrophils were present , with occasional eosinophils and lymphocytes also noted . within the dermis , 
 a mild , superficial , perivascular and periadnexal infiltrate of lymphocytes , histiocytes and occasional eosinophils was identified , with mild perivascular leukocytoclastic debris . 
 the pas stain was positive at the bmz , and around selected blood vessels , nerves and sweat glands . 
 dif revealed linear deposits of igg and complement / c3 and fibrinogen at the bmz , and around selected dermal nerves , blood vessels and sweat glands . 
 strong granular deposits of ige were also observed , colocalizing with monoclonal antibodies to collagen iv ( civ ) . by ihc , 
 positive cd45 staining of lymphocytes was seen surrounding selected dermal blood vessels , eccrine sweat glands , and nerves.conclusion:the patient displayed igg , ige , and fibrinogen autoantibodies against the bmz , as well as around some dermal nerves and sweat glands ; their binding in the skin could trigger complement activation . 
 in addition , the role of the dermal cd45 positive lymphocytes warrants further investigation . 
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Introduction
Case Report
Discussion