BLISTERING DISEASES

M.Peter Marinkovich, MD

Department of Dermatology

Stanford University School of Medicine

PART ONE: ETIOLOGY OF BLISTERING DISEASES

Molecular Organization of the dermal-epidermal basement membrane

The known composition of the dermal-epidermal basement membrane zone (BMZ) are depicted schematically in Figure 1. Keratin 5 and 14 containing intermediate filaments serve to connect the basal cell cytoskeleton to electron dense areas associated with basal cell plasma membrane termed hemidesmosomes. HD1 and BP230 are intracellular hemidesmosomal proteins which may be involved with intermediate filament insertion. BP180 (BPAG1, type XVII collagen) and a6b4 integrin are transmembrane proteins with intracytoplasmic and extracellular domains. Extracellular domains of these proteins extend to the lamina lucida where anchoring filaments are located. Anchoring filaments are thin structures which span the lamina lucida and appear to connect hemidesmosmes with the lamina densa. The protein LAD-1 is located at the superior aspect of anchoring filaments and may be involved with connection of anchoring filaments to the hemidesmosome. Laminin-5 and laminin-6 are large heterotrimeric proteins located in the lower aspects of anchoring filaments. Uncein may be another unique anchoring filament component but characterization of this molecule is as yet incomplete. Laminin-6 and laminin-5 exist as a disulfide bonded complex. Laminin-6 probably interacts with the lamina densa component with nidogen (entactin) which, in turn, interacts with other lamina densa and lamina lucida proteins including type IV collagen, laminin-1 and perlecan (heparan sulfate proteoglycan). P105 and the LH39 antigen are two additional lamina densa components whose functions and structures are incompletely known. Anchoring fibrils extend perpendicularly from the lamina densa into adjacent papillary dermis and either loop back into the lamina densa or insert into electron dense structures termed anchoring plaques. Anchoring fibrils are largely or completely composed of antiparallel dimeric associations of type VII collagen homotrimers with the NC1 or largest globular domain associating iwth type IV collagen present in the lamina densa or anchoring plaque. Anchoring fibrils ensnare other extracellular matrix structures of the papillary dermis such as interstitial collagen fibers, an action which serves to firmly connect the lamina densa onto the dermis.

PATHOGENESIS OF EPIDERMOLYSIS BULLOSA

1) Dystrophic Epidermolysis Bullosa

AF1, AF2, LH7:2, L3d, np185, np32 (all mAb directed against type VII collagen) usually show absent IF staining in Hallopeau-Siemens recessive dystrophic EB, and reduced or absent staining in involved skin areas of most of the dominant subtypes. A large variety of mutations of the COL7A1 gene coding for type VII collagen has been demonstrated in a number of families with both dominant and recessive forms of dystrophic epidermolysis bullosa. No other genes have yet been reported to be affected in this group of diseases.

2) Epidermolysis Bullosa Simplex

Three groups have demonstrated that the primary defects within certain kindreds of individuals with dominant and recessive forms of EB simplex have mutations in keratin genes 5 and 14. A series of transgenic mice was developed into which had been inserted a defective keratin gene. The resulting animals and their offspring developed clinical disease characterized by severe intraepidermal blistering and ultrastructural evidence for tonofilament clumping which closely resembles subtypes of EB simplex patients clinically and pathologically. It appears likely that genes which code for proteins involved with the insertion of keratin filaments to hemidesmosmes such as BP230 and HD1 may also be affected in epidermolysis bullosa simplex, but mutations have yet to be demonstrated.

3) Junctional Epidermolysis Bullosa

Laminin-5 is absent or reduced in the skin and multiple other organs of individuals with the lethal (Herlitz) form, as determined by IF microscopy using a variety of laminin-5 mAbs including K140, GB3, 46. Mutations of the genes coding for all three subunits of laminin-5 have been demonstrated in families with Herlitz JEB The outmoded terms for laminin-5 including epiligrin, kalinin, or nicein are still occcasionally used by some authors. Mutations of the gene coding for the hemidesmosomal component BP180 have been demonstrated in individuals with the less severe generalized atrophic benign form. Interestingly, structural mutations of LAMB3, one of the genes coding for laminin-5, have also been demonstrated in individuals with this disease. Absence of IF staining of mAb 123 which recognizes the anchoring filament component LAD-1, has also been demonstrated in the skin of individuals with this disease, however it is unclear at present if this is due to mutations of the gene coding for this protein or if the absent staininig represents a secondary defect. Mutations of the gene coding for the b4 integrin subunit have been demonstrated in the form of junctional epidermolysis bullosa associated with pyloric atresia. The mAb 19DEJ-1 recognizes an anchoring filament epitope which is absent in both the lethal and nonlethal forms of junctional EB a well as many cases of dystrophic EB, including cases with demonstrated mutations of laminin-5, BP180, b4 integrin or type VII collagen gene mutations. The identity of this 19DEJ-1 antigen, which has been named uncein, remains unclear. It is also unclear whether absence of 19DEJ-1 staining on JEB skin could in some cases represent a primary defect or whether, as in the cases of JEB caused by known mutations the absence of staining represents a secondary event.

PATHOGENESIS OF ACQUIRED BLISTERING DISEASES

Pemphigus subtypes

The superficial subtypes of pemphigus are associated with autoantibodies to desmoglein 1, a 160 kD transmembrane desmosomal component. Pemphigus vulgaris is associated with autoantibodies to desmoglein 3, a 130 kd transmembrane desmosomal component. Superficial pemphigus and pemphigus vulgaris autoantibodies can directly induce blistering in newborn mice and thus are considered to the themselves pathogenic. Titers of pemphigus antibodies correlate with disease severity. A less common form of pemphigus, the paraneoplastic form, is characterized by autoantibodies which immunoprecipitate a group of peptides synthesized by keratinocytes including BP230, desmoplakin and a 190 kD peptide whose identity remains obscure.

Pemphigoid subtypes

Patients with bullous pemphigoid produce autoantibodies to BP180 and/or BP230. The BP180 autonantibody epitope is an extracellular region of the molecule between the transmembrane region and the more distal collagenous domains. Antibodies to this region of the mouse form of this protein induce blistering in mice. Complement and neutrophils also appear to be required to form blisters in this mouse model. Recent evidence suggests that autoantibodies localizing to the more distal collagenous extracellular region of BP180 are present in some forms of cicatritial pemphigoid. These autoantibodies localize to the eoidermal side of NaCl split skin by indirect immunofluorescent microscopy. In other forms of cicatritial pemphigoid, autoantibodies are directed at the a3 subunit of laminin-5 and these autoantibodies localize to the dermal side of NaCl split skin. MAb BM165, directed at what appears to be an active region of the a3 subunit, induces blistering of skin in situ, with the antibody localizing to the dermal side of the induced blister. These results suggest that anti-laminin-5 autoantibodies may be pathogenic in patients with this form of cicatritial pemphigoid. IgA mediated blistering diseases The linear IgA bullous dermatosis autoantigen, LAD-1, was originally identified as a 97 kD peptide in skin extracts recognized by patient sera. Recently this molecule has been more fully characterized and shown to be a 120 kD anchoring filament protein produced by epidermal cells. Mab 123 which appears to recognize an active region of this molecule induces blistering of human skin in situ, with the antibody localizing to the epidermal side of the split. These results are different from those obtained with anti-laminin-5 mAb in that the latter mAb localized to the dermal side of the split. These results suggest that IgA autoantibodies may be pathogenic in linear IgA bullous dermatosis and that LAD-1 may interact with laminin-5 in connecting the hemidesmosome to the lamina densa. While LAD-1 localizes to anchoring filaments, some linear IgA bullous dermatosis patients have autoantibodies which localize to the sublamina densa region and recognize a 285 kD antigen. The identity of this sublamina densa antigen remains to be determined. The autoantigen in dermatitis herpetiformis remains obscure.

Epidermolysis Bullosa Aquisita

It is known that epidermolysis bullosa autoantibodies recognize multiple sites on the NC1 domain of type VII collagen.

PART TWO: DIAGNOSIS OF BLISTERING DISEASES

I. History

Some important general points include age of onset, size, frequency and location of blisters, possible inciting factors (including trauma, sun, foods, drugs, infections, neoplasms) prior diagnostic attempts, prior therapies, and extent of pain or pruritis. Review of systems should include looking for alteration of growth/development and mucosal involvement including oral, nasopharyngeal, ocular, genitotourinary, gastrointestinal or respiratory symptoms. Look for a family history of blistering disease and for geographic/ethnic ancestry.

II. Physical Exam

A complete physical exam should be performed with an emphasis on inspection of all skin and mucosa. Evaluate size, location and character of blisters and try to get an idea of the level of the split of the lesions. Superficial blisters often manifest as crusted erosions, intraepidermal blisters are often flaccid and may expand under pressure, intra-lamina lucida blisters are often tense and heal with no scarring but sometimes atrophy, sublamina densa blisters usually heal with scarring and milia are often present. Are nails, hair, or teeth involved?

III. Laboratory Evaluation

A. Diagnosis of inherited disease

Introduction

Advances in our understanding of the composition and organization of skin components have allowed us to largely classify inherited blistering diseases based on the molecular defect rather than clinical appearance. Knowing the molecular basis of disease affords the clinician a greater ability to predict involvement of other organ systems, overall patient prognosis and potential involvement of present or future family members. In addition, the patient will never be a candidate for future gene therapy, the only specific therapeutic modality that will be available, unless the molecular defect is known. Indirect immunofluorescent microscopy The first step in this process is analysis of the patient's skin by indirect immunofluorescent microscopy (IDIF). In this technique, a punch biopsy of blister edge containing intact and blistered skin in placed in immunofluorescent holding medium. Frozen sections of the biopsy are analyzed to determine the presence of known cutaneous antigens using specific antibodies directed at these antigens. The extent of the information derived from such a procedure is directly related to the number of antibodies used to analyze different potentially affected cutaneous epitopes. Prior to performing the biopsy, it is essential to check with the IDIF lab first to determine what antibodies are available and to determine the optimal holding medium and transport conditions. The IDIF biopsy should determine the antigen which is affected, and also should map the level of the blister. Again, the precise level of split is determined most optimally using an adequate panel of different antibodies. The IDIF biopsy is most useful for recessively inherited diseases in which there is decreased expression of affected antigens. In recessive forms of epidermolysis bullosa simplex (EBS) keratin 5, keratin 15, HD1 or BP230 may potentially be affected. In junctional epidermolysis bullosa (JEB), which appears to be solely of recessive inheritance, laminin-5 a, b or g chains, BP180, b4 integrin, uncein (19- DEJ-1 antigen), or LAD-1 (123 antigen) may potentially be affected. In dystrophic epidermolysis bullosa (DEB), which may show recessive or dominant inheritance, only type VII collagen appears to be affected although nonspecific decreased staining for uncein also can occur. A subtype of DEB shows intracellular accumulation of type VII collagen in basal keratinocytes.

Electron Microscopy

Transmission electron microscopy (TEM) has long been a useful test in the diagnosis of epidermolysis bullosa. Although TEM is available in many major medical centers, the ability of the microscopist to produce technically excellent images of the basement membrane zone and the ability of the pathologist/dermatologist to interpret these images varies depending on the amount of EB experience that a given center has. One should biopsy an area of skin at the edge of a fresh blister and place the sample in the appropriate glutaraldehyde fixative. The most important feature of the TEM biopsy analysis is the determination of the level of the blister. EBS shows intracytoplasmic rupture of basal cells, sometimes with clumping of the intermediate filaments. JEB is characterized by intra- lamina lucida splitting. Hemidesmosomes and anchoring filaments are altered and/or of fewer number in most cases of JEB. DEB is characterized by sublamina densa splitting. Anchoring fibrils are decreased, rudimentary or absent in DEB. A technique quantifying the number of anchoring fibrils in a skin biopsy compared to normal skin has proven useful in the diagnosis of DEB.

Molecular Diagnosis

The first step towards molecular diagnosis at least for recessive disorders is IDIF of patient skin biopsy. It is usually helpful to utilize a portion of the skin biopsy to culture patient keratinocytes/fibroblasts. Keratinocytes can now be routinely immortalized at a few centers and immortalized cells can be analyzed in detail for defects of protein expression utilizing techniques including IDIF, Western blot analysis, radioimmunoprecipitation and ELISA. Additionally, defects of gene expression can be analyzed via techniques including Northern blot analysis, semiquantitative PCR analysis and in situ hybridization. Once the protein/subunit defect is identified, and if the gene which codes for the protein is characterized, then mutational analysis can be performed. Currently the genes which code for keratins 5 and 14 (EBS), laminin-5 a3, b3, g2 chains, b4 integrin subunit, BP180 (JEB) and type VII collagen (DEB) have been sequenced and characterized. Several centers now performed this type of mutational analysis in a routine fashion using automated sequencers. Once the genetic defect is identified, genetic counseling can take place and gene therapy becomes a possibility.

B. Diagnosis of acquired blistering diseases

After the history, physical and hematoxylin/eosin stained standard biopsy is obtained, the next step in the diagnosis of acquired blistering diseases is usually direct immunofluorescent microscopy. There are certain instances when other specialized laboratory tests should also be pursued. For instance if a porphyria is suspected, urine, blood and/or stool porphyrin levels may be informative, depending on the porphyria subtype or if bullous systemic lupus erythematosus is suspected, antinuclear antibodies, double-stranded DNA antibodies or other serologic tests may be informative.

Direct immunofluorescent microscopy (DIF)

In this instance, a perilesional biopsy (near but not including a fresh blister) will be the most informative. It is essential to contact the immunofluorescent lab beforehand to obtain the correct holding medium and to coordinate shipping. Frozen sections of the biopsy will be incubated with antibodies to human IgG, IgA, IgM and C3. Occasionally antibodies to IgG and IgA subclasses, IgD, IgE and fibrin may also be used. There are variations in methodology between different labs. One major difference is that some labs routinely incubate biopsies in 1 M NaCl containing solution to induce intra-lamina lucida dermal-epidermal separation prior to sectioning, whereas some labs reserve this option for a subsequent biopsy. The advantage of the former method is that the location of the antibody/complement deposits can be quickly determined. This is especially useful in cases where epidermolysis bullosa aquisita or bullous pemphigoid is suspected. In epidermolysis bullosa, the autoantibodies are found exclusively on the dermal side of the induced split, whereas in bullous pemphigoid, the antibodies are localized usually to the epidermal side, but occasionally to both the epidermal and dermal sides. While the NaCl DIF technique is most often used for differentiating bullous pemphigoid from epidermolysis bullosa acquisita, this technique can also be used to diagnose other less common autoimmune diseases. For example, it is useful in the analysis of cicatritial pemphigoid sera in that a subset of patient sera contain autoantibodies localizing to the epidermal side whereas a subset of patient sera contain autoantibodies localizing to the dermal side. The pathophysiology is clearly different in these two cicatritial pemphigoid subtypes and can be further verified by Western blotting/ and or radioimmunoprecipitation analysis.

Indirect immunofluorescent microscopy

Sera from patients with a positive direct immunofluorescent biospy or patients who, for various reasons, have not had a skin biopsy performed can be analyzed via indirect immunofluorescent microscopy. In this procedure, frozen tissue sections are incubated with patient sera dilutions. Usually a very low dilution or titer is analyzed and if positive, the sera is diluted further until the point when positive staining is no longer observed. The last dilution prior to the point where no more positive staining is observed is known as the patients serum "titer". Knowing the serum titer in patients with pemphigus foliaceous or pemphigus vulgaris is especially useful because it has been shown to correlate with disease activity. Sera from patients with a positive direct immunofluorescent biopsy is not always positive by indirect immunofluorescent microscopy. There are probably multiple reasons for this. In some instances, obtaining serum for indirect immunofluorescent microscopy is obtained as an afterthought, after systemic therapy has already been initiated. In other cases, there are technical reasons for the lack of reactivity that are due to the substrate which is used for analysis. Many labs use primate esophagus as a tissue to test patient sera. Other labs use normal human skin and still others use NaCl split human skin for analysis of patient sera. The advantage with primate esophagus is that secondary antibodies which react with human but not primary antigens can be used and these provide a low background, compared to human skin, which contains normal immunoglobulin located diffusely throughout the dermis that is recognized by anti-human secondary antibodies. The disadvantage with primate esophagus is that some human autoantibodies do not appear to cross react well with primate tissue. The disadvantage with nonsplit human skin is that the background which appears in the dermis can sometimes make visualization of BMZ reactivity difficult. Separation of the epidermis from the dermis in NaCl split skin makes it easier to determine if reactivity is present on the epidermal side of the split. It is also possible that epitopes are exposed or at least made more accessible for antibody binding after NaCl splitting. For some or all of the reasons above, NaCl split skin yields a higher percentage of positive indirect IF results compared to either primate esophagus or nonsplit human skin at least in the cases of the diseases cicatritial pemphigoid and linear IgA bullous dermatosis and NaCl split human skin should be used as a substrate at least if these diseases are suspected. In our lab, we routinely use NaCl split normal human skin as a substrate for indirect immunofluorescent microscopy. We have found that dermal background staining is diminished most effectively by incubating sections first with normal human sera, then with monoclonal nonbiotinylated antibody to human immunoglobulin, then with patient sera, then with monoclonal biotinylated antibody to human immunoglobulin (same clone as nonbiotinylated) then with avidin-FITC or avidin HRP. In this way, nonspecific antibody binding sites become occupied by immunoglobulin in normal serum. These sites and the immnoglobulin that occupies them are blocked by the nonbiotinylated anti-immunoglobulin. In this way nonspecific antibody binding sites are unavailable to patient sera and biotinylated antibodies. In instances where paraneoplastic pemphigus is suspected, analysis of rat bladder sections with patient autoantibodies may be a useful test, as studies have shown that paraneoplastic pemphigus autoantibodies can show cytoplasmic epithelial staining of this tissue whereas other types of pemphigus autoantibodies fail to show this pattern due to a lack of species crossreactivity.

Direct immunoelectron microscopy

In those instances where the direct immunofluorescent microscopy is positive but the indirect evaluation of the serum titer is negative, further diagnostic information can be obtained via immunoelectron microscopy. Sections can be obtained directly from the frozen block utilized for immunofluorescent studies, incubated with the appropriate immunogold conjugated antiimmunoglobulin or C3 and analyzed at high magnification. In this way, autoantibodies can be localized to various levels of the hemidesmosome, anchoring filaments, lamina lucida, lamina densa, and sublamina densa/anchoring fibrils and the most likely autoantigens can then be determined.

Immunoprecipitation

Immunoprecipitation studies are usually performed with primary cultures of human keratinocytes labeled with 35S methionine/cysteine. Patient autoantibodies are allowed to bind to labeled extracts of cells and conditioned medium then the autoantibody/labeled autoantigen complex is immunoprecipitated using a secondary antibody coupled to a solid phase ligand such as agarose or bacterial cell walls. Autoantigens which can be detected in this way include BP180, BP230, LAD-1, laminin-5, laminin-6, type VII collagen (usually weak from keratinocytes, stronger from labeled WISH cells), paraneoplastic pemphigus autoantigen, desmoglein 1 (pemphigus foliaceous autoantigen) and desmoglein 3 (pemphigus vulgaris autoantigen) and 200 kDa lamina densa autoantigen.

Western blot analysis

Western blot analysis is usually performed by separating solubilized epidermis and/or dermis or solubilized keratinocyte, fibroblast or WISH cell extracts or concentrated conditioned medium by SDS-PAGE, transferring the separated proteins to nitrocellulose, incubating with patient sera, followed by incubation with HRP or alkaline phosphatase conjugated secondary antibodies. Antigens which can be detected in this way include BP180, BP230, LAD-1, laminin-5, laminin-6, type VII collagen, cicatritial pemphigoid autoantigens, desmoglein 1, desmoglein 3 and 200 kDa lamina densa autoantigen.

ELISA

ELISA or enzyme-linked immunosorbent assay can be a sensitive, specific and easy way to obtain precise information about autoantibody specificity. The equipment needed for this assay is available in most clinical laboratories. The test is performed by coating small quantities of purified autoantigen onto 96 well plastic dishes, applying dilutions of patient sera, and visualizing with HRP or alkaline phosphatase conjugated secondary antibodies which can be quantitated in a modified spectrophotometer termed an ELISA reader. This technique is currently limited by the availability of purified autoantigens, however as the molecular sequences are now known for many of the autoantigens including BP180, BP230, type VII collagen, laminin-5, desmoglein 1 and desmoglein 3, it will be a matter of time before all of these proteins will be routinely produced recombinantly and sold in kits.

Autoimmune mediated blistering diseases with known autoantigens
Disease SSS IF results Western blot Immunoprecipitation Other labs
Pemphigus Foliaceous Intragranular IgG 160 kDa band (desmoglein 1)   LE findings in subset of pts.
Paraneoplastic Pemphigus Intraspinous IgG, C3 250, 230, 210, 190 kD bands   + IF rat bladder
Pemphigus Vulgaris Intraspinous IgG, C3 130 kDa band (desmoglein 3)    
Linear IgA diseases BMZ IgA, C3 epiderm/dermal 120 (97) kD band (LAD-1)
Bullous Pemphigoid BMZ IgG, C3 epidermal side 230 kD BP230, 180 kD BP180 230 kD BP230 180 kD BP180  
Cicatritial Pemphigoid BMZ IgG, C3 epiderm/dermal 165 kD laminin 5 alpha 3 (subset of patients) 165, 155, 140, 105 kD laminin 5 (subset of pts) Anti-p105
Pemphigoid BMZ IgG, dermal side 105 kDa p105 Anti-200 kD  
bullous disease BMZ IgG, dermal side 200 kDa band 200 kDa band  
Epidermolysis bullosa acquisita BMZ IgG dermal side 290 kDa band 290 kDa band LE findings in subset of pts.

PART THREE: CLINICAL FEATURES OF BLISTERING DISEASES

EPIDERMOLYSIS BULLOSA: CLINICAL SUBTYPES

CLASSIFICATION

I) Simplex: blistering at basal cell level or above

II) Junctional: blistering at the lamina lucida level

III) Dystrophic: blistering below the lamina densa level

SUBTYPES

1) EPIDERMOLYSIS BULLOSA SIMPLEX

A) EB Simplex- Koebner variant

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

AGGRAVATING FACTORS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

B) EB Simplex- Weber-Cockayne variant

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

C) EB Simplex- Dowling-Meara variant (Herpetiformis)

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

D) EB Simplex Mottled hyperpigmentation

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

E) EB Simplex- Kallin Syndrome

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

F) EB Simplex- Ogna variant

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

G) EB Simplex- Bart variant

INHERITANCE: Autosomal dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

H) EB Simplex- Mendes de Costa variant

INHERITANCE: X-linked Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

I) EB Simplex- Letalis variant

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

J) EB Simplex- Superficialis

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

2) JUNCTIONAL EPIDERMOLYSIS BULLOSA

A) Junctional EB- Gravis (Herlitz Disease)

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

B) Junctional EB- Generalized Atrophic Benign

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

C) Junctional EB- Localized

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

D) Junctional EB- Cicatricial

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

E) Junctional EB- Inversa

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

F) Junctional EB- Progressiva (Neurotropica)

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

3) DYSTROPHIC EPIDERMOLYSIS BULLOSA

A) Dystrophic EB- Cockayne-Touraine

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS INVOLVEMENT:

PATHOLOGIC FINDINGS:

B) Dystrophic EB- Minimus

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

C) Dystrophic EB- Pretibial

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

D) Dystrophic EB- Albopapuloidea (Pasini variant)

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

E) Dystrophic EB- Hallopeau-Siemens (Gravis)

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

F) Dystrophic EB- Inversa

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

NONCUTANEOUS FINDINGS:

PATHOLOGIC FINDINGS:

G) Dystrophic EB- Transient bullous dermolysis of the newborn

INHERITANCE: Autosomal Dominant; Some may be Autosomal

Recessive

CUTANEOUS FINDINGS:

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

CLINICAL FEATURES OF AUTOIMMUNE BLISTERING DISEASES

PEMPHIGUS FOLIACEOUS

PEMPHIGUS VULGARIS

PARANEOPLASTIC PEMPHIGUS

BULLOUS PEMPHIGOID

CICATRICIAL PEMPHIGOID

IgA MEDIATED BULLOUS DISEASES

DERMATITIS HERPETIFORMIS

EPIDERMOLYSIS BULLOSA AQUISITA

PART FOUR: THERAPY OF BLISTERING DISEASES

TREATMENT OF INHERITED BLISTERING DISEASE

(EPIDERMOLYSIS BULLOSA)

1) Skin involvement

a) Wound healing- impaired by endogenous factors including foreign bodies, bacteria, nutritional deficiencies, tissue anoxia, aging. Impaired by exogenous agents including glucocorticoids and penicillamine. Optimizing wound healing in EB patients involves control of all of these factors. Herlitz (junctional epidermolysis bullosa letalis) patients heal very slowly. This may be due to a defect of laminin-5 (a protein shown to be intimately involved in keratinocyte adhesion and migration).

b) Infection- Patients with severe EB subtypes may have immunologic abnormalities including decreased lymohokine production. Extensive areas of denuded skin can result in the loss of the stratum corneum barrier to microbial penetration and result in the accumulation of serum and moisture that enhance growth of bacteria. These factors combined with frequent poor nutritional status facilitate development of infections. Staphylococcus aureus and Streptococcus pyogenes are the usual causative organisms but gram negative infections with bacteria such as Pseudomonas aeruginosa can also occur. These patients also have an increased susceptibility to developing sepsis. Prevention of infection is obviously the preferred strategy. With extensive areas of crusting and denudation, regular whirlpool therapy followed by application of topical agents such as mupirocin or other topical antibiotics and covering with semiocclusive nonadherant dressings is a good approach. Adhesive tape should not be applied directly to the skin. Self adhering gauze or tape is a better choice for keeping dressings in place.

c) Tumors- Squamous cell carcinomas often arise in chronic cutaneous lesions in patients with epidermolysis bullosa often at multiple primary sites. This is especially true for patients with recessive dystrophic epidermolysis bullosa. These carcinomas occur more frequently especially at nonsunexposed sites and at a younger age than nonaffected individuals. Careful surveillance of nonhealing areas is important.

2) Nutritional management

a)Increased needs: Extensive cutaneous injury is associated with marked alterations in both hemodynamic and metabolic responses with increased caloric and protein requirements. The burn patient has been extensively studied from both of these perspectives and from these studies it is clear that the development of nutritional deficiencies will not allow patients to successfully heal their wounds and return them to a more normal hemodynamic and metabolic profile.

b)Impediments to intake and absorption: Oropharyngeal as well as gastrointenstinal lesions provide the greatest overall threat to nutritional well-being of individuals with EB. These include oral blistering, abnormal esophageal motility, strictures, dysphagia, diarrhea, malabsorption, and dental problems. Nutritional assessment taking into account (a) and (b) above is essential.

4) Gastrointestinal management

The most disabling complication is esophageal lesions (Hallopeau-Siemens and inverse recessive dystrophic EB subtypes, Dowling-Meara and letalis EB simplex subtypes, all junctional forms except localized and progressiva/neurotropica). These are treated in several ways: medical management with phenytoin and oral steroid elixirs are only occasionally effective at reducing the symptoms of dysphagia. Treatment of oral candidiasis when present is clearly helpful. Esophageal dilation has been helpful at loosening strictures and finally, removal of esophageal strictures by colonic interposition has proved effective in advanced cases. Gastrostomy tube insertion has shown to be clearly effective in providing nutrition to individuals with esophageal strictures.

5) Surgical restoration of the hand

The mitten deformity of the hand which occurs frequently in the Hallopeau-Siemens dystrophic EB patients is the result of repeated episodes of blistering and scarring with the resultant fusion of web spaces. As a result, fine manipulative skills and digital prehension are lost. Surgical procedures have the ability to correct this deformity but there is a high incidence of recurrence of the mitten pseudosyndactyly, with the dominant hand showing an earlier recurrence. One factor that appears to delay recurrence is the prolonged use of splinting in the interphalangeal spaces at night.

7) Eye lesions

a)Clinical features: EB simplex patients, particularly those with the Weber-Cockayne and Dowling-Meara subtypes can experience recurrent blepharitis in one or both eyes along with bullous lesions of the conjunctivae. Junctional EB patients and the Hallopeau-Siemens dystrophic EB patient can experience corneal ulcerations and corneal scarring, as well as eyelid lesions. Cicatritial conjunctivitis can also occur in Hallopeau-Siemens patients.

b)Management: Corneal erosions are treated supportively with application of antibiotic ointment and the use of cycloplegic agents to reduce ciliary spasm and provide comfort. Using tape to patch the eye is best avoided because of frequent blistering of the skin under the adhesive. Chronic blepharitis can lead to cicatritial ectropion and exposure keratitis. In addition to moisture chambers and ocular lubricants, this disorder has been treated with full thickness skin grafting to the upper eyelid but complete correction is difficult to obtain.

8) Gene therapy

Gene therapy for epidermolysis bullosa is now being developed as we have recently learned the coding sequences of many of the genes affected in epidermolysis bullosa. Recessively inherited diseases including the junctional and many dystrophic epidermolysis bullosa cases generally result from a lack of expression of functional protein via a null mutation and would be best treated by gene replacement therapy. Dominantly inherited diseases including most of the simplex and some of the dystrophic epidermolysis bullosa cases can be caused by a protein defect that results in abnormal filament or fibril assembly via a structural mutation. In these instances, an approach to selectively knockout the mutated allele but to preserve the normal allele needs to be undertaken. The gene replacement approach in the treatment of recessively inherited diseases via an ex vivo method is the one which is technically within our reach at this time. In this approach, an initial biopsy is taken and patient keratinocytes are expanded in culture. A wild type copy of the cDNA coding for the gene which is defective is transferred to the cultured cells. After gene transfer, cultured cells are grafted onto skin areas of chronic erosion or frequent blistering. Modalities of gene transfer currently being explored include direct gene transfer using gene guns and other methods, self-replicating episomes, retrovirus and adenovirus vectors. Gene therapy for epidermolysis bullosa is currently at the experimental state and is not available for patients yet. Probably the biggest current problem in gene therapy which needs to be further explored is that of sustained gene expression of transferred genes. Approaches such as the gene gun introduce DNA extrachromosomally and do not allow for replication of transferred DNA during cell division. Retrovirus vectors which insert transferred DNA into patient chromosomes or episomes which replicate at the time of cell division appear at this point to offer the best prospects for sustained expression of transferred genes.


TREATMENT OF ACQUIRED BLISTERING DISEASES

1) Systemic corticosteroids

This is the mainstay of therapy for most subtypes of pemphigus and pemphigoid, including herpes gestationis. Most flares can be controlled with between 1 and 2 mg/kg/day of prednisone divided into two doses. As blistering activity subsides, this daily dose can initially be tapered at a rate of 10 mg/week assessing closely for any recurrence of blisters. Once the patient reaches 60 mg/day, a single daily dose can be given. After patients have reached the 40 mg/day of prednisone point in their taper, further tapering of the average daily dose should be performed more cautiously, not more than an average of 5 mg/day decrease in dose per week as reflares occur frequently at this point. As the tapering proceeds, it is important to get patients on an alternate day steroid regimen. This may be done once the patient reaches 40 mg/ day of prednisone by reducing the dose on alternate days until a dose of 40 mg/0 mg on alternating days is reached. This every other day dose can then be gradually reduced. It is important to closely examine patients at frequent intervals for signs of reflaring and to develop an understanding with the pateint to immediately report increases in blistering activity to you. Reflares require an increase in the dose of prednisone to again halt disease activity, sometimes back to initial levels, if the reflare is marked. The goal is to reduce the steroid dose to the minimum or to eliminate it entirely, in order to reduce the widely recognized systemic effects. The ability to achieve this goal is variable, and often depends on fluctuations in disease activity in individual patients and the use of steroid sparing agents listed below.

2) Steroid sparing agents

The use of a steroid sparing agent reduces the side effects encountered with systemic corticosteroids but care needs to be taken to monitor for systemic toxicity of the steroid sparing agents themselves including the increased risks of infection and malignancy. Probably the lease toxic steroid sparing regimen for bullous pemphigoid patients is the use of tetracycline 2g/day and nicotinamide 2g/day. This regimen can be effective by itself for milder cases of bullous pemphigoid or used in conjunction with steroids for more severe cases. Cyclophosphamide and azathioprine are the drugs most commonly used as a steroid sparing agents in pemphigus subtypes and can be used in bullous pemphigoid as well. Cyclophosphamide has significant potential systemic toxicity and patients must be carefully monitored during treatment which usually is a single daily dose of 1-2 mg/kg. Side effects include bone marrow suppression, urinary problems including bladder hemorrhage or fibrosis, pulmonary fibrosis, sterility and increased malignant risk especially hematological or bladder malignancies. It is especially important to encourage patients to increase their oral fluid intake. Azathioprine in general has less systemic toxicity than cyclophosphamide but it works more slowly. It is given at a dose of 1-3 mg/kg/day. Hepatotoxicity, sepsis and urinary complications are the most frequent serious complications and often occur within the first week or two of therapy. Other steroid sparing modalities for pemphigus and pemphigoid subtypes which are sometimes effective include cyclosporin, chlorambucil and plasmapheresis. Intramuscular gold has also been reported to be of benefit to patients with pemphigus subtypes.

3) Sulfones

Dapsone is the treatment of choice for patients with dermatitis herpetiformis, linear IgA bullous dermatosis or its childhood variant, chronic bullous dermatosis of childhood as well as other less common IgA mediated dermatoses including IgA pemphigus. In IgG mediated diseases dapsone is sometimes helpful, especially in cases which show neutrophillic infiltrates on histologic exam. Doses of 100 to 200 mg a day are usually employed in adult patients. The most common side effect is a hemolytic anemia which is most pronounced in patients with glucose-6-phosphate dehydrogenase deficiency. Additionally, methemoglobinimia and rarely aplastic anemia can occur. Sulfapyridine is indicated for patients who cannot tolerate dapsone. A gluten free diet is an important and effective therapy in patients with dermatitis herpetiformis, although compliance with the diet can be difficult.

4) Epidermolysis bullosa acquisita

In general, the most difficult autoimmune blistering disorder to treat. Treatment responses in general are unpredictable. All of the above agents have been employed with success in some patients, additionally, colchicine has been employed with some success. Some patients do not appear to respond to any treatment regimen.


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