Indirect immunofluorescence microscopy for the serological diagnosis of autoimmune blistering skin diseases

Indirect Immunofluorescence Microscopy for the Serological Diagnosis of Autoimmune Blistering Skin Diseases: A Review JEAN KANITAKIS, MD Introduction

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utoimmune blistering skin diseases (ABSD) represent a group of heterogeneous mucocutaneous blistering diseases characterized by the deposition within the tissues of autoantibodies recognizing specific molecules involved in the cohesion between epidermal cells or between epidermis and the dermis. Intraepidermal ABSD include the various forms of pemphigus, pemphigus vulgaris (PV), pemphigus vegetans (PVE), pemphigus foliaceus (PF), drug-induced pemphigus (DIP), paraneoplastic pemphigus (PNP), and immunoglobulin A pemphigus (IgA-P).1 Diseases due to autoimmune dermal-epidermal dysadhesion (subepidermal ABSD) include mainly bullous pemphigoid (BP), pemphigoid gestationis (PG), cicatricial pemphigoid (CP), epidermolysis bullosa acquisita (EBA), and bullous systemic lupus erythematosus.2 The diagnosis of these diseases relies on clinical, histological, and immunopathological criteria. Immunopathological features are of paramount importance for the diagnosis, and historically have allowed the distinction between several of these entities that were too often misdiagnosed before the advent of immunofluorescence microscopy. The autoantibodies characteristic of ABSD can be detected by appropriate techniques, either within the tissues where they are deposited or within the serum. In this chapter I review the contribution of immunofluorescence microscopy for the serological diagnosis of ABSD, commonly referred to as “indirect immunofluorescence.”

The Techniques The detection of circulating autoantibodies against epithelial antigens is made by incubating the patient’s serum with appropriate epithelial substrates containing the target antigen. The most widely used technique is the two-step indirect immunofluorescence (IIF) since it From the Laboratory of Dermatopathology, Department of Dermatology, Hoˆp. Ed. Herriot, Lyon, France. Address correspondence to Jean Kanitakis, Hoˆp. Ed. Herriot (Pav. R), 69437 Lyon cx 03, France. © 2001 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010

provides a good combination of sensitivity and rapidity3: the serum is incubated at an initial dilution of 1:20 with 4 –5 ␮m thick frozen sections of the epithelial substrate (see below) for 30 – 40 min at room temperature in moist, dark chambers; after a 10 –15 min wash in phosphate-buffered saline (PBS), the sections are incubated with a fluorochrome-labeled animal serum prepared against human immunoglobulins (“conjugate”). This may be directed to all or to specific immunoglobulin (Ig) subclasses (IgA, IgG, IgE, etc.). After a second wash in PBS, the sections may be counterstained with nuclear intercalating agents (such as ethidium bromide or propidium iodine); this procedure is not really necessary but allows for better visualization of the cells within the substrate. Finally the slides are mounted in acqueous medium (with or without antiphotobleaching agents such as p-phenylenediamine) and examined under a fluorescence microscope equipped with the appropriate filters. IIF allows for titration by running serial dilutions of the serum in PBS and determining the highest dilution yielding a visible fluorescence. This titration may have prognostic significance in the cases of pemphigus. The most common fluorochrome used is fluorescein isothiocyanate, yielding green fluorescence (peak emission at 510 –517 nm), but other fluorochromes are also available, such as tetramethylrhodamine isothiocyanate (peak emission at 580 nm) or phycoerythrin. Immunoenzymatic methods can also be used, but they are longer and more expensive to perform since they necessitate an additional step (incubation of the sections with a chromogenic substrate), so that they are not popular for the serological diagnosis of ABSD. The epithelial substrate on which the reaction is carried out is of great importance; ideally, it should be easily available and express in adequate amounts the autoantigens sought. For diseases of the pemphigus group, the most commonly used substrates include monkey or guinea pig esophagus and rabbit lip mucosa. Monkey esophagus seems to be more sensitive for the serological diagnosis of PV, whereas rabbit lip is more sensitive for the detection of PF autoantibodies,4 0738-081X/01/$–see front matter PII S0738-081X(00)00180-2

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BP antigen of 180 kDa (BPAG2 or type XVII collagen) remains predominantly attached to the epidermal roof, but some epitopes may remain attached to the dermal floor. The use of this substrate therefore can differentiate sera recognizing the BPAG1 (that bind to the roof and produce a continuous, linear epidermal binding pattern) from those recognizing type VII collagen (formerly known as the “EBA antigen”) that bind to the floor, producing a linear, dermal binding pattern. Sera recognizing the BPAG2 may produce a mixed, epidermal-dermal binding pattern, although a purely dermal pattern, similar to that obtained with EBA sera, may exceptionally be observed. Sera reacting with laminin 5, found in a subset of patients with CP, also produce a dermal binding pattern. Other substrates that have been used for the IIF diagnosis of subepidermal ABSD include:

Figure 1. Salt-split skin: the cleavage runs through the lamina lucida, leaving on the floor the lamina densa and on the roof the basal membrane of epidermal keratinocytes (electron microscopy).

presumably because of the higher expression of the recognized autoantigens (desmogleins 3 and 1, respectively). Sera of PNP contain autoantibodies to desmoplakins and therefore yield a positive reaction not only on squamous epithelia, but also on transitional, simple, and columnar ones, such as murine urinary bladder epithelium, myocardium, hepatocytes, and bile duct canaliculi.5 Cultured epithelial cells6 and cell lines (eg, COS7) transfected with genes encoding for various desmosome components7 have also been used on an experimental basis. For subepidermal ABSD, the most useful substrate is normal human skin that has been split through the lamina lucida. The use of this substrate, introduced in 1984,8,9 was a major breakthrough in the diagnosis of this group of diseases, since it increases the sensitivity of IIF (as compared with uncleaved skin)10 and can usually differentiate BP from EBA sera. Split skin can be produced by several methods, including suction or incubation with thermolysin or 20 mmol/L EDTA; however, the reference method currently is by incubation for 48 –72 h with 1 M NaCl, since this provides the most reliable results.11 This “salt-split skin” (SSS) is cleaved through the lamina lucida (Fig 1); its epidermal roof contains the hemidesmosome-associated antigens (plectin, ␣6␤4 integrin and the BP antigen of 230 kDa, BPAG1), and its dermal floor consists of laminin 5 (lower lamina lucida) and collagens type IV (lamina densa) and VII (anchoring fibers). The transmembrane

Y Human mucosa (vaginal and oral) intact or cleaved with 1 M NaCl; this reportedly increases the sensitivity of IIF in cases of CP.12,13 Y Animal skin, which may lack some of the antigens present in human dermal-epidermal junction (DEJ). The use of toad skin purportedly can differentiate BP from EBA sera.14 We have not found this method very reliable.15 Y Skin from patients affected by hereditary epidermolysis bullosa (EB) that are deficient in a specific antigen (such as type VII collagen in cases of recessive dystrophic EB, BPAG2 in generalized atrophic benign EB, or laminin 5 in the Herlitz type of junctional EB). For instance, skin lacking type VII collagen can be used for the distinction of EBA from BP sera, since the former produce no reactivity, whereas the latter produce a continuous, linear reactivity along the DEJ; conversely, EB sera react normally with skin specimens lacking the BPAG2 or laminin 5.16,17 Y Tumors such as cylindroma, expressing high amounts of some DEJ molecules (collagens type IV and VII, laminin).18 Y Epidermal keratinocytes, obtained after trypsin dissociation or in culture; sera with autoantibodies to the BP antigen(s) produce a polar staining of (basal), cells whereas sera with autoantibodies to the EBA antigen are usually unreactive.19 The use of these substrates, although theoretically interesting, has not received widespread routine use.

The Diseases The Pemphigus Group Pemphigus Vulgaris and Pemphigus Vegetans The sera in these diseases contain IgG autoantibodies that recognize desmogleins 3 and 1, desmosome-associated glycoproteins of the cadherin superfamily of 130 and 160 kDa MW, respectively. On monkey esophagus,

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Figure 2. PV serum producing a chicken-wire pattern of fluorescence on the lower part of the epithelium of monkey esophagus.

PV and PVE sera provide a characteristic “chickenwire” pattern predominating on the lowermost epithelial cell layers (Fig. 2). IIF is a sensitive technique for the diagnosis of PV, since it is almost invariably positive in patients with active disease.20 Furthermore IIF has prognostic value, since the titer of circulating autoantibodies shows some correlation with disease activity. IIF has a good specificity, although false positives can be produced by sera containing antibodies to cell-surface antigens; these pemphigus-like antibodies, encountered in normal people or in patients with infections, burns, and some drug reactions, are reportedly incapable of fixing complement, although this merits confirmation.21 Pemphigus Foliaceus PF autoantibodies belong to the IgG subclass and target desmoglein 1. IIF on epithelial substrates produces a chicken-wire pattern, predominating over the superficial epithelial layers, where desmoglein 1 seems to be more abundant and critical for cell-cell adhesion.22 Rabbit4 and guinea pig lip23 are more sensitive than monkey oesophagus for the detection of PF autoantibodies (Fig 3). The positivity rate of IIF on these substrates is in the 60 –70% range.

Paraneoplastic Pemphigus PNP sera contain IgG autoantibodies that recognize desmogleins 1 and 3, an uncharacterized protein of 170 kDa, and an antigenic complex of plakins (including desmoplakins 1 and 2, the BPAG1, envoplakin, periplakin, and plectin). IIF is positive not only on stratified but also on simple, columnar, and transitional epithelia, in myocardium, hepatocytes, and bile duct canaliculi. The best substrate for serological diagnosis of PNP is urinary bladder epithelium, which is rich in desmoplakins. PNP sera produce a chicken-wire immunofluorescence pattern, similar to that obtained in PV; labeling of the basal membrane can also be observed.

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Figure 3. PF serum producing a chicken-wire pattern of fluorescence on rabbit lip.

False-positive reactions are rare and false-negative results can occur in up to 15% of cases20,24; therefore, the definite diagnosis of PNP should rely on additional, more sensitive and specific tests such as immunoprecipitation.5 Drug-Induced Pemphigus DIP sera recognize the same antigens involved in PV.25 IIF is positive in merely 57% of cases, probably because the disease may be due to a direct acantholytic (rather than an immunological) effect of the drugs involved.26 When present, autoantibody titers are low and show poor correlation with disease activity; if absent, the disease usually regresses spontaneously upon discontinuation of the drug.27 IgA Pemphigus This rare disease includes two varieties, subcorneal pustular dermatosis (SPD) and intraepidermal neutrophilic dermatosis (IND). IgA autoantibodies seem to recognize desmogleins 1 and 3 in addition to desmocollin 1. IIF has been performed on monkey esophagus and human skin, and is positive in about half of the cases. IEN sera stain the entire epidermis by IIF; SPD sera react with the upper part of the epidermis,28 but react neither with monolayers of cultured human keratinocytes29 nor with a squamous cell carcinoma cell line.30

The Bullous Pemphigoid Group Bullous Pemphigoid BP autoantibodies belong usually to the IgG4 and to a lesser extent to the IgG1 subclass; they recognize epitopes contained in the ⫺COOH-terminal end of the 230 kDa BPAG1, and/or epitopes contained within the NC16A domain of the 180 kDa BPAG2. By immunoblotting, patient’s sera recognize the BPAG1 or the BPAG2 or both in 35–50%, 25–30%, and 17–25% of cases, respectively, according to various studies.31 Au-

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Figure 4. BP serum producing a linear, continuous labeling on the epidermal side (roof of the blister) of salt-split skin.

toantibodies of the IgA subclass recognizing the BPAG1 or BPAG2 may also be detected, especially in children.32 IIF on SSS is positive in 80 –90% of cases32,33; it produces an epidermal staining pattern in 80 –95% of positive cases, a mixed (dermal-epidermal) pattern in 2–15% and a purely dermal pattern in 1–3% of cases, respectively10,34 (Figs 4 and 5). The antibody titer is not predictive of the severity of the disease.35 Pemphigoid (Herpes) Gestationis PG sera contain autoantibodies of the IgG1 subclass (formerly referred to as “herpes gestationis factor,” HGF) that recognize predominantly the BPAG2 (the same epitopes as for BP), alone or in combination with the BPAG1 antigen36; the BPAG1 may occasionally be the only target.37 In the past, HGF was detected with a

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special technique (“complement IIF”), which amplifies the signal through fixation of several complement molecules by two IgG molecules.38 According to this technique, the substrate is initially incubated with the patient’s serum (that has been previously heated to 56°C for 30 min to destroy complement-fixing activity); after washing, the sections are incubated with a source of fresh complement (such as human serum). If present in the patient’s serum, the complement-fixing IgG or IgM bound to the section during the first step generate several C3 molecules; these are revealed with fluorescein-labeled antisera to complement. PG autoantibodies are currently detected by IIF on SSS, where they usually produce an epidermal staining pattern. Antibody titers do not correlate with disease severity.39 Cicatricial Pemphigoid CP seems to be an immunologically heterogeneous disease. CP autoantibodies belong to the IgG4, IgG1, and IgA subclasses; most of them recognize the BPAG2 (NC16A domain and another region closer to the ⫺COOH terminus) and more rarely the BPAG1. These sera produce by IIF on SSS an epidermal (and much more rarely a dermal or a mixed) staining pattern.40,41 A small subset (about 5%) of CP patients have IgG4 autoantibodies to laminin 5 (mostly the ␣3 chain)42 that produce a dermal staining pattern on SSS.43 These patients may have an association with lung, uterine, or gastric carcinomas or with other non-neoplastic diseases.44 The frequency of this “anti-epiligrin CP” was recently claimed to be higher than previously expected.45 Autoantibodies to BPAG2 and laminin 5 may be present in the same patient.46 Overall, the positivity of CP sera by IIF on SSS is in the range of 50 – 80%. Ocular cicatricial pemphigoid seems to be a variant of CP. Several molecules have been proposed as target antigens, such as a 45 kDa protein47 or the ␤4 integrin.48 IIF on SSS produces an epidermal staining pattern with a frequency that varies considerably according to the studies (15–100%).47,49 Lichen Planus Pemphigoides In lichen planus pemphigoides bullae develop also on normal-looking skin, which separates this entity from bullous lichen planus. Autoantibodies may be produced as a result of the damage of basal cells, which reveals hidden antigens; these autoantibodies belong to the IgG subclass and react with an epitope (MCW-4) of the NCA16 domain of BPAG2; by IIF they bind to the epidermal side of SSS.50,51

Linear IgA Bullous Dermatosis

Figure 5. BP serum producing a mixed dermal-epidermal labeling on salt-split skin.

Linear IgA bullous dermatosis (LABD) is a subepidermal ABSD encompassing an adult and a childhood form; it is defined on the basis of clinical and direct immunofluorescence findings, showing continuous, linear deposits of IgA1 isolated or associated with deposits

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Figure 6. DH serum labeling the endomysium of monkey esophagus.

Figure 7. EBA serum producing a dermal labeling (floor of the blister) on salt-split skin.

of IgG and/or C3 along the DEJ. LABD seems to be an immunologically heterogeneous disease,52 since sera (containing autoantibodies of the IgA occasionally along with IgG subclass) recognize various antigens, including BPAG1 and 253,54 and type VII collagen.55 A 97 kDa antigen tentatively called “ladinin” was purportedly the target antigen in LABD,56 but this antigen is now known to be identical to a portion of the extracellular domain of the BPAG2.57 The heterogeneity of antigens recognized by LABD sera has been shown not only by immunoblotting studies,58 but also by IIF using as substrates skin from patients with congenital EB, lacking specific antigens of the DEJ17 and the tumor cylindroma.18 IIF on SSS is positive in about 60% of cases, probably more frequently in the childhood than the adult form.60 Ear skin reportedly yields a higher positivity.60 The labeling is epidermal in at least two thirds of cases,61 but it may also be dermal55–57 or mixed,60 depending on the targeted antigen.

about 50% of cases, and produces exclusively a dermal staining pattern,64 allowing differentiation from BP sera (that produce a dermal staining pattern in less than 3% of cases) (Fig 7). The sera of two patients diagnosed as EBA were recently reported to react with the epidermal side of SSS,65 but the significance of this finding is presently unclear. EBA sera do not react with skin from patients suffering from EB dystrophica, lacking type VII collagen.16 Bullous systemic lupus erythematosus is a clinical variant of systemic lupus erythematosus featuring bullae linked to the presence of IgG autoantibodies directed to the noncollagenous domain NC-1 of type VII collagen.66 On SSS these produce a dermal staining pattern as EBA sera.67,68 A patient with BSLE and autoantibodies against multiple DEJ antigens (type VII collagen, BPAG1, laminins 5 and 6) was recently reported; as expected, the serum produced a dermal-epidermal binding pattern on SSS.69

Dermatitis Herpetiformis

Other Diseases

Dermatitis herpetiformis sera have no autoantibodies to the DEJ antigens, and therefore IIF on SSS is negative; however, on appropriate substrates (including monkey oesophagus), IIF reveals circulating IgA autoantibodies directed against endomysium, a specialized perimuscular connective tissue62 (Fig 6). These antiendomysial IgA antibodies, present also in patients with celiac disease, were recently shown to recognize tissue transglutaminase, and can also be efficiently detected with an ELISA technique.63

A patient has been reported70 with a subepidermal bullous disease clinically reminiscent of toxic epidermal necrolysis or pemphigus vulgaris; the autoantibodies were of the IgG1 subclass and reacted with an antigen of the deep lamina lucida, producing by IIF on SSS a dermal binding pattern. Immunoblotting studies showed that the autoantigen recognized (a 105 kDa noncollagenous glycoprotein) was different from the BP and EBA antigens. The relationship of this disease (tentatively termed “p105-pemphigoid”) to the other subepidermal ABSD remains unknown. A case of unique subepidermal blistering disease with IgG autoantibodies against a novel 200 kD antigen was reported.71 The patient presented clinical features of BP or LABD; IIF showed circulating antibodies binding to the dermal side of SSS. A patient has been reported with a subepidermal

Epidermolysis Bullosa Acquisita/Bullous Systemic Lupus Erythematosus EBA sera recognize epitopes lying within the NH2terminal, noncollagenous NC-1 domain of the 290 kDa MW type VII collagen, formerly also referred to as “EBA antigen.” IIF performed on SSS is positive in

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ABSD, whose serum recognized type IV collagen, producing dermal staining pattern on SSS. The nature of this disease remains unknown.34 Chronic ulcerative stomatitis is a disease characterized by recurring chronic oral ulcerations resembling lichen planus. IIF on monkey esophagus reveals circulating antinuclear autoantibodies binding namely to the basal layer of the epithelium but absent from other substrates (such as rat liver and Hep2 cells).51,72

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Conclusions The correct diagnosis of autoimmune blistering skin diseases relies on clinical, histological, and immunopathological criteria, among which direct and indirect IF are of paramount importance. In cases where these tests are not conclusive (for instance when the patient’s serum is negative by IIF), more sophisticated tests may be used, such as immunoelectron microscopy, immunoblotting, and immunoprecipitation and enzymelinked immunosorbent assay.73,74 These tests, however, are more time-consuming and expensive, and until these or other ancillary techniques become standardized, IIF will continue to remain one of the gold standards for the diagnosis of ABSD.

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References 1. Nousari H, Anhalt G. Pemphigus and bullous pemphigoid. Lancet 1999;354:667–72. 2. Zambruno G, Failla CM. Autoimmunity of the dermalepidermal junction. Eur J Dermatol 1999;9:437– 42. 3. Kamarashev J. Immunohistochemical techniques for light microscopy. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:3–18. 4. Cozzani E, Kanitakis J, Nicolas JF, et al. Comparative study of indirect immunofluorescence and immunoblotting in the diagnosis of pemphigus. Arch Dermatol Res 1994;286:295–9. 5. Nousari H, Anhalt G. Pemphigus vulgaris, paraneoplastic pemphigus and pemphigus foliaceus. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998: 74 – 83. 6. Iwatsuki K, Sugaya K, Takigawa M. Dynamic expression of pemphigus and desmosomal antigens by cultured keratinocytes. Br J Dermatol 1993;128:16 –22. 7. Hashimoto T, Kiyokawa C, Mori O, et al. Human desmocollin 1 (Dsc1) is an autoantigen for the subcorneal pustular dermatosis of IgA pemphigus. J Invest Dermatol 1997;109:127–31. 8. Gammon W, Briggaman R, Imman A, et al. Differentiating anti-lamina lucida and anti-sublamina densa anti-BMZ antibodies by indirect immunofluorescence on 1.0 M sodium chloride-separated skin. J Invest Dermatol 1984;82: 139 – 44. 9. Gammon W, Fine JD, Forbes M, et al. Immunofluorescence on split skin for the detection and differentiation of

18.

19.

20.

21.

22.

23.

24.

25.

619

basement membrane zone autoantibodies. J Am Acad Dermatol 1992;27:79 – 87. Lazarova Z, Yancey K. Reactivity of autoantibodies from patients with defined subepidermal bullous diseases against 1 mol/L salt-split skin. J Am Acad Dermatol 1996;35:398 – 403. Willsteed E, Bhogal B, Das A, et al. An ultrastructural comparison of dermo-epidermal separation techniques. J Cutan Pathol 1991;18:8 –12. Leonard J, Hobday C, Haffenden J, et al. Immunofluorescent studies in ocular cicatricial pemphigoid. Br J Dermatol 1988;118:209 –17. Ghohestani R, Nicolas JF, Rousselle P, et al. Identification of a 168 kDa mucosal antigen in a subset of patients with cicatricial pemphigoid. J Invest Dermatol 1996;107:136 –9. Pang B, Lee Y, Ratnam K. Floor-pattern salt-split skin cannot distinguish bullous pemphigoid from epidermolysis bullosa acquisita. Use of toad skin. Arch Dermatol 1993;129:744 – 6. Kanitakis J, Cozzani E, Peyron E, et al. Use of animal skin substrates for indirect immunofluorescence diagnosis of subepidermal autoimmune bullous diseases. Arch Dermatol 1994;130:1558. Vodegel R, Kiss M, de Jong M, et al. The use of skin substrates deficient in basement membrane molecules for the diagnosis of subepidermal autoimmune bullous diseases. Eur J Dermatol 1998;8:83–5. Harman K, Bhogal B, Eady R, et al. Defining target antigens in linear IgA disease using skin from subjects with inherited epidermolysis bullosa as a substrate for indirect immunofluorescence microscopy. Br J Dermatol 1999;141: 475– 80. Wojnarowska F, Allen J, Collier P, et al. A comparison of the expression of known basement membrane components with the linear IgA disease antigens using the novel substrate cylindroma. Br J Dermatol 1999;141:62–70. Mutasim D, Diaz L. The relevance of immunohistochemical techniques in the differentiation of subepidermal bullous diseases. Am J Dermatopathol 1991;13:77– 83. Kanitakis J, Wang YZ, Roche P, et al. Immunohistopathologic study of autoimmune pemphigus. Dermatology 1994;188:282–5. Krasny S, Beutner E, Chorzelski T. Specificity and sensitivity of indirect and direct immunofluorescence findings in the diagnosis of pemphigus. In: Beutner E, Chorzelski T, Kumar V, editors. Immunopathology of the skin. 3rd ed. New York: Wiley, 1987:207– 47. Mahoney M, Rothenberger K, Koch P, et al. Explanation for the clinical and microscopic localization of lesions in pemphigus foliaceus and vulgaris. J Clin Invest 1999;103: 461– 8. Sabolinsky M, Beutner E, Krasny S, et al. Substrate specificity of anti-epithelial antibodies of pemphigus vulgaris and pemphigus foliaceus sera in immunofluorescence tests on monkey and guinea pig esophagus. J Invest Dermatol 1987;88:545–9. Hilu J, Allbritton J, Anhalt G. Accuracy of indirect immunofluorescence testing in the diagnosis of paraneoplastic pemphigus. J Am Acad Dermatol 1995;32:442–7. Brenner S, Bialy-Golan A, Anhalt G. Recognition of pem-

Clinics in Dermatology

620 KANITAKIS

26.

27. 28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

40.

phigus antigens in drug-induced pemphigus vulgaris and foliaceus. J Am Acad Dermatol 1997;36:919 –23. Brenner S, Bialy-Golan A, Ruocco V. Drug-induced pemphigus. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:84 –94. Anhalt G. Drug-induced pemphigus. Sem Dermatol 1989; 8:166 –72. Iwatsuki K. IgA pemphigus. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:95–104. Iwatsuki K, Takigawa M. Distinct types of IgG and IgA anti-intercellular autoantibodies from patients with pemphigus and vesiculopustular dermatosis. Br J Dermatol 1991;125:335–9. Akiyama M, Hashimoto T, Sugiura M, et al. Ultrastructural localization of autoantigens of intercellular IgA vesiculopustular dermatosis in cultured human squamous cell carcinoma lines. Arch Dermatol Res 1992;248:371–3. Jiao D, Bystryn JC. Relation between antibodies to BP180 and gender in bullous pemphigoid. J Am Acad Dermatol 1999;41:269 –70. Kirtschig G, Wojnarowska F. IgA basement membrane zone autoantibodies in bullous pemphigoid detect epidermal antigens of 270 –280 kDa, 230 kDa, and 180 kDa molecular weight by immunoblotting. Clin Exp Dermatol 1999;24:302–7. Ghohestani R, Cozzani E, Nicolas JF, et al. Comparative sensitivity of indirect immunofluorescence to immunoblot assay for the detection of circulating antibodies to bullous pemphigoid antigens 1 and 2. Br J Dermatol 1996; 135:74 –9. Ghohestani R, Nicolas JF, Rousselle P, et al. Diagnostic value of indirect immunofluorescence on sodium-chloride-split skin in differential diagnosis of subepidermal autoimmune bullous dermatoses. Arch Dermatol 1997; 133:1102–7. Bernard P, Be´ dane C. Bullous and Cicatricial Pemphigoid. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:56 – 65. Giudice G, Emery D, Zelickson B, et al. Bullous pemphigoid and herpes gestationis autoantibodies recognize a common non-collagenous site of the BP180 ectodomain. J Immunol 1993;151:5742–50. Ghohestani R, Nicolas JF, Kanitakis J, et al. Pemphigoid gestationis with autoantibodies exclusively directed to the 230 kDa bullous pemphigoid antigen. Br J Dermatol 1996; 134:603– 4. Hodge L, Black M, Ramnarin N, et al. Indirect complement immunofluorescence in the immunopathological assessment of bullous pemphigoid, cicatricial pemphigoid and herpes gestationis. Clin Exp Dermatol 1978;3:61–7. Vaughan Jones S, Hern S, Nelson-Piercy C et al. A prospective study of 200 women with dermatoses of pregnancy correlating clinical findings with hormonal and immunopathological profiles. Br J Dermatol 1999;141:71– 81. Sarret Y, Hall R, Cobo M, et al. Salt-split human skin substrate for the immunofluorescent screening of serum from patients with cicatricial pemphigoid and a new

41. 42.

43.

44. 45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

Y

2001;19:614 – 621

method of immunoprecipitation with IgA antibodies. J Am Acad Dermatol 1991;24:952– 8. Niimi Y, Zhu XJ, Bystryn JC. Identification of cicatricial pemphigoid antigens. Arch Dermatol 1992;128:54 –7. Domloge-Hultsch N, Anhalt G, Gammon W, et al. Antiepiligrin cicatricial pemphigoid: A subepithelial blistering disorder. Arch Dermatol 1994;130:1521–9. Lazarova Z, Hsu R, Darling T, et al. Anti-epiligrin cicatricial pemphigoid represents an autoimmune response to subunits present in laminin 5. Br J Dermatol 1998;139: 791–7. Setterfield J, Shirlaw P, Lazarova Z, et al. Paraneoplastic cicatricial pemphigoid. Br J Dermatol 1999;141:127–31. Leverkus M, Schmidt E, Lazarova Z, et al. Antiepiligrin cicatricial pemphigoid: An underdiagnosed entity within the spectrum of scarring autoimmune subepidermal bullous diseases? Arch Dermatol 1999;135:1091– 8. Kawahara Y, Amagai M, Ohata Y, et al. A case of cicatricial pemphigoid with simultaneous IgG autoantibodies against the 180 kd bullous pemphigoid antigen and laminin 5. J Am Acad Dermatol 1998;38:624 –7. Smith E, Taylor T, Meyer L, et al. Identification of a basement membrane zone antigen reactive with circulating IgA antibody in ocular cicatricial pemphigoid. J Invest Dermatol 1993;101:619 –23. Tyagi S, Bhol K, Natarajan K, et al. Ocular cicatricial pemphigoid antigen: Partial sequence and biochemical characterization. Proc Natl Acad Sci (USA) 1996;93: 14714 –9. Hoang-Xuan T, Robin H, Demers P, et al. Pure ocular cicatricial pemphigoid. A distinct immunopathologic subset of cicatrical pemphigoid. Ophthalmology 1999;106: 355– 61. Zillikens D, Caux F, Mascaro JM, et al. Autoantibodies in lichen planus pemphigoides react with a novel epitope within the C-terminal NC16A domain of BP180. J Invest Dermatol 1999;113:117–21. Parodi A, Cozzani E. Lichen planus and lichen planus pemphigoides. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:193–9. Kanitakis J, Mauduit G, Cozzani E, et al. Linear IgA bullous dermatosis of childhood with autoantibodies to a 230 kDa epidermal antigen. Ped Dermatol 1994;11:139 – 44. Ghohestani R, Nicolas JF, Kanitakis J, et al. Linear IgA bullous dermatosis with IgA antibodies exclusively directed against the 180- or 230-kDa epidermal antigens. J Invest Dermatol 1997;108:854 – 8. Zambruno G, Manca V, Kanitakis J, et al. Linear IgA dermatosis with antibodies to type VII collagen. Linear IgA epidermolysis bullosa acquisita. J Am Acad Dermatol 1994;31:884 – 8. Wojnarowska F, Collier P, Allen J, et al. The localisation of the target antigens and antibodies in linear IgA disease is heterogeneous and depends on the methods used. Br J Dermatol 1995:132;750 –7. Zone J, Taylor T, Kadunce D, et al. Identification of the cutaneous basement membrane zone antigen and isolation of antibody in linear immunoglobulin A bullous dermatosis. J Clin Invest 1990;85:812–20.

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2001;19:614 – 621

57. Zone J, Taylor T, Meyer L, et al. The 97 kDa linear IgA bullous disease antigen is identical to a portion of the extracellular domain of the 180 kDa bullous pemphigoid antigen, BPAG2. J Invest Dermatol 1998;110:207–10. 58. Dmochowski M, Hashimoto T, Bhogal M, et al. Immunoblotting studies of linear IgA disease. J Dermatol Sci 1993; 6:194 –200. 59. Wojnarowska F, Marsden R, Bhogal B, et al. Chronic bullous disease of childhood, childhood cicatricial pemphigoid and linear IgA disease of adults. J Am Acad Dermatol 1988;19:792– 805. 60. Banfield C, Allen J, Wojnarowska F. Dermatitis herpetiformis and linear IgA disease. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:105–13. 61. Chan L, Traczyk T, Taylor T, et al. Linear IgA dermatosis. Characterization of a subset of patients with concurrent IgA and IgG anti-basement membrane autoantibodies. Arch Dermatol 1995;131:1432–7. 62. Reunala T, Chorzelski T, Viander M, et al. IgA antiendomysial antibodies in dermatitis herpetiformis: Correlation with jejunal morphology, gluten-free diet and antigliadin antibodies. Br J Dermatol 1987;117:185–91. 63. Dieterich W, Laag E, Bruckner-Tuderman L, et al. Antibodies to tissue transglutaminase as serologic markers in patients with dermatitis herpetiformis. J Invest Dermatol 1999;113:133– 6. 64. Woodley D, Chen M, O’Toole E, et al. Epidermolysis bullosa acquisita. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:114 –24. 65. Wakelin S, Bhogal B, Black M, et al. Epidermolysis bullosa acquisita associated with epidermal-binding circulating antibodies. Br J Dermatol 1997;136:604 –9.

INDIRECT IMMUNOFLUORESCENCE MICROSCOPY

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66. Shirahama S, Furukawa F, Yagi H, et al. Bullous systemic lupus erythematosus: Detection of antibodies against noncollagenous domain of type VII collagen. J Am Acad Dermatol 1998;38:844 – 8. 67. Gammon W, Briggaman R. Bullous systemic lupus erythematosus: A phenotypically distinctive but immunologically heterogeneous bullous disorder. J Invest Dermatol 1993;100(Suppl):S28 –34. 68. Vassileva S. Lupus erythematosus. In: Kanitakis J, Vassileva S, Woodley D, editors. Diagnostic immunohistochemistry of the skin. London: Chapman & Hall, 1998:144 –56. 69. Chan L, Lapie`re JC, Chen M, et al. Bullous systemic lupus erythematosus with autoantibodies recognizing multiple basement membrane components, bullous emphigoid antigen 1, laminin-5, laminin-6, and type VII collagen. Arch Dermatol 1999;135:569 –73. 70. Chan L, Cooper K. A novel immune-mediated subepidermal bullous dermatosis characterized by IgG autoantibodies to a lower lamina lucida component. Arch Dermatol 1994;130:343–7. 71. Kawahara Y, Matsuo Y, Hashimoto T, et al. A case of unique subepidermal blistering disease with autoantibodies against a novel 200-kD antigen. Dermatology 1998;196:213– 6. 72. Jaremko W, Beutner E, Kumar V, et al. Chronic ulcerative stomatitis associated with a specific immunologic marker. J Am Acad Dermatol 1990;22:15–20. 73. Chen M, Chan J, Cai X, et al. Development of an ELISA for rapid detection of anti-type VII collagen autoantibodies in epidermolysis bullosa acquisita. J Invest Dermatol 1997; 108:68 –72. 74. Zillikens D, Mascaro JM, Rose P, et al. A highly sensitive enzyme-linked immunosorbent assay for the detection of circulating anti-BP180 autoantibodies in patients with bullous pemphigoid. J Invest Dermatol 1997;109:679 – 83.

D

uring WWII, nickel was important strategic nickel in the production of stainless steel. The Canadian mint removed nickel from their 5 cent piece during 1942–1944 and created an alloy of 88% copper and 12% zinc called “Tombac” alloy. These coins had a bronze color when first minted but later turned dark brown like a copper penny. Sometimes these coins would be mistaken as a penny because of the dark color. The reverse of the coin shows the “V” for 5 cents but it was commonly known as the “Victory” nickel. On the edge of the reverse is International Morse Code translated to “We Win When We Work Willingly.” During 1943 to 1945 the mint switched to chromium plated steel as the metal for the 5 cent coin. After the war the mint resumed using 100% nickel.

REFERENCES: 1. Herbert A, editor. Coin Clinic 1001 frequently asked questions. Iola (WI); Krause Publishers: 1995:38 – 41. 2. Mishler C, editor. Coins Questions and Answers. 4th ed. New York: Golden Books Publishing; 1998:173– 84.