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.

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:

• onset at birth or within the first few weeks of life
• congenital lesions may heal with scarring, lesions after birth heal without scarring
• distribution is generalized but acral areas are most frequently affected, flexor sites may be involved especially in warmer weather
• mild to moderate hyperkeratosis of soles is common
• often improvement at puberty especially in women

AGGRAVATING FACTORS:

• warm weather
• accompanying hyperhydrosis of feet is common
• secondary bacterial infections common

EXTRACUTANEOUS FINDINGS:

• teeth are normal
• nails may show transient mild involvement
• oral and nasopharyngeal mucosa involvement is mild to moderate and usually decreases with age
• occasional eye abnormalities, including myopia magna

PATHOLOGIC FINDINGS:

• basal cell cytolysis, minimal to mild dyskeratosis
• tonofilament clumping at EM level

B) EB Simplex- Weber-Cockayne variant

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• trauma induced blistering primarily on hands and feet
• onset is frequently delayed
• more substantial trauma sometimes necessary to produce blisters compared to other EBS forms
• hyperhidrosis of the feet is common
• mild hyperkeratosis of palms and soles often present

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

• basal cell cytolysis with mild dyskeratosis
• tonofilaments disorganized but not clumped at EM level

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

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• blisters and erosions present at birth or arising shortly thereafter
• acral sites most often affected, but generalized pattern may also occur
• milia may be present at healed sites
• focal atrophy sometimes present, scarring is rare, except at sites of congenital lesions
• nail dystrophy common
• prominent hyperkeratosis of the soles and palms
• often there is improvement at puberty especially with nail dystrophy

EXTRACUTANEOUS FINDINGS:

• oral blistering is common
• esophageal involvement occurs including expectoration of esophageal casts
• mild flexure contractions may occur
• occasional dental abnormalities including natal teeth, anodontia

PATHOLOGIC FINDINGS:

• basal cell cytolysis with dyskeratosis
• tonofilament clumping prominent at EM level

D) EB Simplex Mottled hyperpigmentation

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• widespread blistering, sometimes in herpetiform or annular pattern, extremities especially affected
• onset at or shortly after birth
• lesions heal without scarring and with extreme rapidity when there is fever
• postblistering hyper or hypopigmentation
• mottled hypopigmentation of the neck, upper inner arms, thighs and abdomen develops in early childhood
• during later childhood, blistering improves but pigmentary changes worsen and hyperkeratotic acral lesions develop
• onycholysis and thickened curved nails in some patients,
• sometimes nails are repeatedly lost, fingernails regrow normally, but toenails may remain dystrophic
• diffuse hyperkeratosis of soles and punctate hyperkeratosis of palms

EXTRACUTANEOUS FINDINGS:

• oral blistering only in perinatal period
• teeth are highly carious in some individuals

PATHOLOGIC FINDINGS:

• basal cell cytolysis causing blistering

E) EB Simplex- Kallin Syndrome

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• hand and foot blistering beginning at age three months to 1 year
• scalp hair is brittle with transient nonscarring alopecia
• nails may be thickened and curved
• lesions heal without scarring

EXTRACUTANEOUS FINDINGS:

• anodontia and a disorder of mineralization of the teeth
• myopia and hearing loss may occur

PATHOLOGIC FINDINGS:

• intraepidermal blistering, level uncertain

F) EB Simplex- Ogna variant

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• generalized blistering and bruising in response to trauma, mainly on the hands and feet
• Onychogryphosis of the great toenails frequent and tends to be lifelong but is less severe in adulthood

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

• blistering due to basal cell cytolysis

G) EB Simplex- Bart variant

INHERITANCE: Autosomal dominant

CUTANEOUS FINDINGS:

• large partial thickness erosions at birth that heal with fine scarring
• after birth, blisters occur in a generalized distribution and heal without scarring
• nails are sometimes dystrophic
• frequent congenital localized absence of the skin on one or more extremities, this is the key distinguishing feature

EXTRACUTANEOUS FINDINGS:

• oral mucosa is often involved

PATHOLOGIC FINDINGS:

• blisters due to basal cell cytolysis, or intraepidermal blistering

H) EB Simplex- Mendes de Costa variant

INHERITANCE: X-linked Recessive

CUTANEOUS FINDINGS:

• blistering which may or may not be trauma induced occurs during the first two years of life
• alopecia, nonscarring
• reticulated hyperpigmentation
• atrophy involving the face and extremities

EXTRACUTANEOUS FINDINGS:

• microcephaly

PATHOLOGIC FINDINGS:

• blistering due to basal cell cytolysis

I) EB Simplex- Letalis variant

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• erosions may be present at birth
• trauma induced blistering of hands and feet soon after birth, which soon become generalized
• later in childhood, blisters may be restricted to acral areas
• mild nail involvement
• atrophy and milia at sites of healed lesions

EXTRACUTANEOUS FINDINGS:

• oral ulceration
• teeth are normal
• anemia is common
• sometimes associated muscular dystrophy or congenital myasthenia gravis
• growth retardation has been observed
• in one study, ten of thirteen patients died prior to age 20 months although the precise cause of death in each case was unknown

PATHOLOGIC FINDINGS:

• blistering due to basal cell cytolysis

J) EB Simplex- Superficialis

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• blisters and erosions on extremities only or generalized but palms and soles are spared
• atrophy and sometimes milia are present over healed areas
• nail dystrophy is common

EXTRACUTANEOUS FINDINGS:

• oral and conjunctival vesiculation and erosion common

PATHOLOGIC FINDINGS:

• blistering just below the level of the stratum corneum

2) JUNCTIONAL EPIDERMOLYSIS BULLOSA

A) Junctional EB- Gravis (Herlitz Disease)

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• frequently congenital erosions and occasionally deeper skin defects are present
• generalized blistering occurs with prominent acral involvement
• onycholysis with repeated nail loss, sometimes permanently
• lesions heal slowly and frequently with resultant atrophy and occasional mild scarring
• vegetating lesions are frequent, especially in the perioral region

EXTRACUTANEOUS FINDINGS:

• nasal, oral, pharyngeal, esophageal, vaginal, and anal mucosa may be involved
• eyes, pylorus (atresia), small intestine, gall bladder, urethra, and kidney (hydroureteronephrosis) may also be affected
• teeth are severely dystrophic
• retarded general growth
• anemia
• death commonly occurs within a few weeks, but some patients survive for a few years, although rarely to adolescence

PATHOLOGIC FINDINGS:

• blistering at the plane of the lamina lucida, variable alteration of hemidesmosomes and defective anchoring filaments

B) Junctional EB- Generalized Atrophic Benign

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• generalized blistering tendency, usually from birth
• erosions may be extensive and slow to heal
• hair loss is frequent but variable
• nails are generally involved with dystrophy or eventual loss
• healing often occurs with atrophy

EXTRACUTANEOUS FINDINGS:

• nasal, oral, esophageal, vaginal and anal mucosa are frequently involved
• eyes, larynx, bladder, urethra and kidney are affected less frequently
• teeth are dystrophic
• patients achieve normal or near normal stature
• life span may be normal or decreased

PATHOLOGIC FINDINGS:

• separation of the dermis from the epidermis at the level of the lamina lucida with variable hemidesmosome alterations

C) Junctional EB- Localized

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• from birth, blistering is limited to hands, feet and sometimes the pretibial region
• nails are regularly involved with repeated and sometimes permanent loss

EXTRACUTANEOUS FINDINGS:

• good growth and development
• nasal and oral mucosa may be moderately involved
• teeth are dystrophic

PATHOLOGIC FINDINGS:

• blistering at the level of the lamina lucida and variable alterations of hemidesmosomes

D) Junctional EB- Cicatricial

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• blistering is present from birth
• distribution is generalized and most severe on hands and feet
• scarring on healing, sometimes development of syndactyly (mitten type) and contractures occur
• nails are lost early in life
• alopecia

EXTRACUTANEOUS FINDINGS:

• there may be stenosis of the nares, oral blistering, dysplastic teeth, esophageal involvement, laryngeal involvement, and anemia
• occasional renal and ocular involvement
• lifespan is uncertain, some patients have reached adulthood

PATHOLOGIC FINDINGS:

• blister formation at the level of the lamina lucida

E) Junctional EB- Inversa

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• frequently neonatal erosions (sometimes vegetating) and blisters
• lesions are generalized early but later are concentrated in the inverse sites (axilla, groin)
• some acral involvement continues
• lesions heal with mild atrophy, and some heal with a circumscribed appearance similar to the papuloidea lesions of albopapuloidea dystrophic EB
• nails are generally involved

EXTRACUTANEOUS FINDINGS:

• corneal erosions may be present
• mild to moderate oral, anal and esophageal involvement

PATHOLOGIC FINDINGS:

• blistering at the plane of the lamina lucida

F) Junctional EB- Progressiva (Neurotropica)

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• manifestations are delayed until age 5 to 8 years at which time nail dystrophy begins
• later there is blistering of hands and feet, especially of the palms and soles
• healing is accompanied by variable atrophy that is most striking on the hands, feet, elbows and knees

EXTRACUTANEOUS FINDINGS:

• oral blisters and loss of lingual papillae are present
• the may be mild finger contractures
• hypoacusis is frequently present

PATHOLOGIC FINDINGS:

• blistering at the plane of the lamina lucida where amorphous material is deposited

3) DYSTROPHIC EPIDERMOLYSIS BULLOSA

A) Dystrophic EB- Cockayne-Touraine

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• acral and generalized blistering and erosions occur at birth or during early infancy
• nails are variably involved, toenails>fingernails
• squamous cell carcinoma infrequently occur

EXTRACUTANEOUS INVOLVEMENT:

• oral mucosal involvement is generally mild, but occasionally includes esophageal stenosis
• teeth are normal or mildly dystrophic
• keratitis may occur

PATHOLOGIC FINDINGS:

• blistering beneath the basement membrane
• anchoring fibrils may be decreased in number and poorly developed

B) Dystrophic EB- Minimus

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• mild generalized blistering in early childhood
• minimal acral blistering in later childhood
• some nails, especially toenails may be dystrophic and may constitute the only residual manifestation in adults

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

• blistering at a level below the basement membrane
• normal appearing anchoring fibrils in noninvolved areas

C) Dystrophic EB- Pretibial

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• blisters that heal with scarring and atrophy are concentrated in the pretibial area and on the dorsa of the feet with minimal to no involvement elsewhere

EXTRACUTANEOUS FINDINGS:

• occasionally the oral mucosa is minimally involved
• teeth are normal or near normal

PATHOLOGIC FINDINGS:

• blistering below the level of the basement membrane
• anchoring fibrils are present in nonaffected areas

D) Dystrophic EB- Albopapuloidea (Pasini variant)

INHERITANCE: Autosomal Dominant

CUTANEOUS FINDINGS:

• generalized blistering and development of erosions beginning at birth
• later there is concentration of lesions at acral sites
• plaque and papular scars, lighter than surrounding skin, are characteristic
• nail involvement is variable, occurs mainly on toenails

EXTRACUTANEOUS FINDINGS:

• mucosal involvement is usually mild
• teeth are near normal

PATHOLOGIC FINDINGS:

• sub-basement membrane blistering
• anchoring fibrils may be well developed in noninvolved skin

E) Dystrophic EB- Hallopeau-Siemens (Gravis)

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• blisters and erosions are frequently present at birth
• there is marked scarring with gradual mitten deformity of hands, syndactyly of toes, nail dystrophy and loss, and occasional development of squamous cell carcinoma
• albopapuloid lesions may occur
• milder variants may be distinct entities including centripetal progressive variant

EXTRACUTANEOUS FINDINGS:

• oral, esophageal, anal, and vaginal mucosae are frequently involved
• bladder, urethra, and kidney are less commonly affected
• teeth are dystrophic
• ocular involvement is most commonly keratitis
• anemia is common
• in severely affected individuals, growth and development is retarded
• overall life span is shortened, especially in severely involved individuals

PATHOLOGIC FINDINGS:

• sub-basement membrane blistering
• anchoring fibrils are absent or reduced in number and poorly developed at a distance from actively involved areas in severely affected individuals

F) Dystrophic EB- Inversa

INHERITANCE: Autosomal Recessive

CUTANEOUS FINDINGS:

• blisters and erosions are frequently present at birth
• skin fragility is most prominent in axillae, groin, inframammary and neck regions
• nail changes are present usually only on toenails
• scarring alopecia often present
• milia often form

NONCUTANEOUS FINDINGS:

• oral mucosa and esophagus may be severely involved relative to skin involvement
• tongue is usually bound to the floor of the mouth
• occasional ocular involvement, usually keratitis
• teeth may be mildly dystrophic
• normal stature
• receding chin

PATHOLOGIC FINDINGS:

• sub-basement membrane blistering
• anchoring fibrils are reduced in number and poorly developed in susceptible skin

G) Dystrophic EB- Transient bullous dermolysis of the newborn

INHERITANCE: Autosomal Dominant; Some may be Autosomal

Recessive

CUTANEOUS FINDINGS:

• generalized blisters and erosions present in a generalized distribution at birth, especially prominent on extremities
• lesions heal with milia and minimal scarring and atrophy
• nail dystrophy absent
• disappearance of apparent clinical disease within first year of life

EXTRACUTANEOUS FINDINGS: none

PATHOLOGIC FINDINGS:

• sub-basement membrane blistering
• anchoring fibrils are reduced in affected areas

CLINICAL FEATURES OF AUTOIMMUNE BLISTERING DISEASES

PEMPHIGUS FOLIACEOUS

• the most superficial of the autoimmune blistering diseases
• characterized by scaling/crusting and exfoliation of the skin sometimes with erythema
• scalp, face and upper trunk are characteristic sites of involvement
• mucosal involvement is rare
• may be exacerbated by sun exposure
• the disease is usually chronic, but is not life-threatening
• fogo selvagum is a Brazilian variant of this disease, it is also called endemic pemphigus
• fogo selvagum may be associated with the black fly, Simulium pruinosum
• pemphigus erythematosus is a variant where there is positive serology for SLE.

PEMPHIGUS VULGARIS

• characteristic lesion is a flaccid blister, sometimes with surrounding erythema
• blisters are fragile so that sometimes only erosions are seen
• disease is usually chronic
• pemphigus vegetans is a variant associated with exuberant granulation tissue
• mucosal involvement is common
• neonatal pemphigus occurs in infants born to mothers with pemphigus vulgaris
• prior to advent of corticosteroids, pemphigus vulgaris was often fatal
• penicillamine, captopril and other drugs may produce a pemphigus-like syndrome
• Myathenisa gravis or thymoma are sometimes associated with pemphigus

PARANEOPLASTIC PEMPHIGUS

• a rare disorder characterized by severe mucosal erosions and a widespread polymorphic skin eruption
• IF shows IgG binding not only to stratified but to simple epithelial tissues as well
• usually associated with lymphoma, thymoma, less commonly with sarcoma/other tumors
• usually recalcitrant to therapy

BULLOUS PEMPHIGOID

• characterized by tense blisters, often with a preceding or concurrent urticarial plaque
• mucosal involvment is common
• lesions do not scar
• lichen planus pemphigoides is a rare variant associated with lichen planus
• disease is chronic however remissions occur more frequently than with pemphigus
• usually affects the elderly but occasionally occurs in children
• herpes gestationus is a variant which occurs from mid-term of pregnancy and may continue into the post partum period

CICATRICIAL PEMPHIGOID

• chronic bullous disease with primarily mucosal involvment
• scarring is a common feature, especially of mucosal surfaces
• gingival and conjunctival mucosa are especially frequently affected
• cutaneous lesions occur in about one third of patients
• esophageal and laryngeal involvment can be life-threatening
• ocular involvment can lead to blindness through progressive conjunctival scarring

IgA MEDIATED BULLOUS DISEASES

DERMATITIS HERPETIFORMIS

• usually starts between 2nd to 4th decades and is characterized by grouped vesicles usually with associated erythema and intense pruritis which occur over extensors surfaces including knees, buttocks, sacrum, back, shoulders, and posterior scapula/neck.
• atrophy of intestinal villae is usually present when looked for on biopsy of dermatitis herpetiformis patients
• linear IgA bullous dermatosis is characterized by tense blisters of varying morphologies and distributions, sometimes with a "cluster of jewels" arrangement.
• mucosal involvement in linear IgA bullous dermatosis can sometimes occur
• chronic bullous dermatosis of childhood is a childhood variant of linear IgA bullous dermatosis
• IgA pemphigus is a rare disorder which may clinically resemble pemphigus foliaceous or subcorneal pustular dermatosis

EPIDERMOLYSIS BULLOSA AQUISITA

• negative family history for EB
• trauma induced as well as spontaneous blisters in generalized distribution especially prominent on acral and extensor surfaces
• lesions heal with atrophy, scarring and milia
• in general lesions appear non inflammatory
• nail dystrophy and scarring alopecia may be present
• usual onset is between the ages of 40 and 60 but cases arising in childhood have been described
• oral, conjuctival and nasopharyngeal mucosal involvement may occur
• some patients go on to fulfill the diagnostic criteria for SLE thus bullous SLE and EB aquisita appear to be related immunologic diseases
• sub-basement membrane blister, often with perivascular mixed infiltrate containing eosinophils
• Brunstig-Perry pemphigoid is probably a localized variant of 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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