Desmoglein 3-ELISA: a Pemphigus Vulgaris Specific Diagnostic Tool

STUDY Desmoglein 3-ELISA A Pemphigus Vulgaris–Specific Diagnostic Tool

Petra Lenz, MD; Masayuki Amagai, MD; Beatrix Volc-Platzer, MD; Georg Stingl, MD; Reinhard Kirnbauer, MD

Background: Pemphigus vulgaris (PV) is an autoim- patients with various nonbullous skin disorders, and 10 mune-blistering disease of the skin and mucous mem- healthy individuals were tested. branes caused by autoantibodies against desmoglein 3 (Dsg3), an epidermal desmosomal adhesion protein of Results: Forty-seven (98%) of 48 serum samples from pa- the cadherin family. Cloning of the Dsg3 gene and ex- tients with PV that were positive by IIF on monkey esopha- pression of the protein in a native conformation en- gus were also reactive by Dsg3-ELISA, whereas 4 of 4 IIF- abled the recent development of a specific and sensitive negative PV serum samples showed no reactivity by ELISA. enzyme-linked immunosorbent assay (ELISA) for the de- In addition, negative ELISA results were obtained from 11 tection of PV autoantibodies. of 11 serum samples from patients with bullous pemphigoid, 10 of 12 serum samples from patients with other bul- Objectives: To evaluate serum samples from patients lous skin disorders, 7 of 9 serum samples from patients with with PV and other dermatologic diseases for anti-Dsg3 autoimmune-connective tissue diseases, and 13 of 13 se- antibodies. To compare ELISA values with autoanti- rum samples from patients with other nonbullous skin dis- body titers obtained by classic indirect immunofluo- eases. Interestingly, 1 patient with paraneoplastic pemphi- rescence (IIF). gus had positive ELISA results. There was a positive correlation (r = 0.654) between ELISA values and IIF ti- Design: Serum samples from patients with PV and vari- ters within the whole population with PV. In addition, when ous other bullous and nonbullous skin diseases were tested multiple serum samples from 1 patient with PV sampled for anti-Dsg3 reactivity by ELISA. over a 2-year period were tested, ELISA reactivity paralleled both the IIF titers and the clinical course. Setting: Ambulatory and hospitalized patients from a university hospital. Conclusion: The Dsg3-ELISA is a sensitive, objective, and PV-specific test that should be considered as an ad- Patients: Fifty-two serum samples from 11 patients with junct test for the management of patients with PV. PV, and serum samples from 11 patients with bullous pemphigoid, 12 patients with other bullous diseases, 22 Arch Dermatol. 1999;135:143-148

EMPHIGUS VULGARIS (PV) is tinocytes.5,6 In animal studies7 and in an autoimmune-blistering vitro8,9 experiments, anti-Dsg3 antibodies disease of the skin and have been shown to induce loss of cell mucous membranes. Histo- adhesion, indicating an important role in logically, PV is character- disease pathognesis. Pized by suprabasal acantholysis and From the Laboratory of Viral intraepidermal blister formation, and For editorial comment Oncology, the Division of clinically by flaccid blisters and erosions see page 195 Immunology, Allergy, and that usually arise on normal-appearing Infectious Diseases, Department skin and/or mucosa.1 Immunologically, Pemphigus antibodies are routinely of Dermatology, University of antibodies directed against the keratino- detected by direct or indirect immuno- Vienna Medical School, Vienna, cyte cell surface are detected in the skin fluorescence (IIF) testing using monkey Austria (Drs Lenz, and in the circulation; they recognize the esophagus as a substrate to establish the Volc-Platzer, Stingl, and 1-4 Kirnbauer); and the extracellular domain of Dsg3. Desmo- diagnosis, and for monitoring disease Department of Dermatology, glein 3, a 130-kd transmembrane glyco- activity that frequently follows antibody 10-13 Keio University School of protein of the cadherin family, mediates titers. However, these methods are Medicine, Tokyo, Japan weak calcium-dependent homophilic subjective, require an experienced exam- (Dr Amagai). cell-to-cell adhesion of epidermal kera- iner, and often fail to clearly distinguish

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 tion and ELISA buffers to maintain the conformational epi- PATIENTS, MATERIALS, 18 AND METHODS topes of Dsg1 and Dsg3. WESTERN BLOT PATIENTS AND CONTROLS Fractions obtained during rDsg1 and rDsg3 purification were A total of 52 serum samples (stored at −20°C until assayed) sized by gel electrophoresis, transferred to an Immobi- from 11 patients with PV (6 women, 5 men; mean age, 56.8 lon-P membrane (Millipore Corporation, Bedfort, Mass), years; age range, 34-85 years) treated in the Department of and incubated overnight at 4°C with 5000-fold diluted an- Dermatology at the University of Vienna, Vienna, Austria, over tibody against the E-tag (Pharmacia Biotech, Uppsala, Swe- 2 years (September 1995-November 1997) were tested for anti- den), followed by incubation with a 40 000-fold diluted per- Dsg3 reactivity. The diagnosis of PV was established clini- oxide–conjugated antimouse IgG antibody (Biorad cally and by the typical histologic and immunopathologic find- Laboratories, Hercules, Calif). Specific binding was visu- ings. Forty-eight of these PV serum samples obtained from alized using a chemiluminescence Western blotting detec- 9 patients were positive by IIF on monkey esophagus and 4 tion kit (ECL; Amersham, Buckinghamshire, England). serum samples from 2 patients with PV were negative by IIF at the time of serum sampling (Table 1). In addition, se- PV SERUM ABSORPTION WITH rDSG rum samples from 45 patients with a variety of other bullous and nonbullous skin diseases were included in the study. To determine the antigenicity of the rDsg3 protein, immu- Of these patients, 11 were diagnosed as having bullous pem- noabsorption studies were performed using a PV serum phigoid; 12 had other bullous diseases including pemphi- sample with an IIF titer of 320. The serum sample was se- gus foliaceus (1 patient), PNP (2 patients), epidermolysis bul- rially diluted and incubated overnight with column elu- losa acquisita (1 patient), porphyria cutanea tarda (1 patient), ates containing native rDsg3, rDsg1, or denatured (at 96°C) and unclassified bullous diseases (4 patients); 9 had autoim- rDsg3 protein and reevaluated by IIF analysis. mune–connective tissue diseases and high-titer antibodies against nuclear antigens, and 13 had other nonbullous skin ELISA diseases including eczema (2 patients), lichen ruber planus (1 patient), Behc¸et disease (2 patients), erythema migrans (3 Immunomicrotiter plates (Nunc; Roskilde, Denmark) were patients), thermal burn (1 patient), and sexually transmit- coated with 50 µL of eluate and incubated overnight at 40°C. ted diseases (4 patients). To establish a cutoff value for the After extensive washing in ice-cold TBS-Ca containing 0.05% ELISA we tested serum samples from 10 healthy individuals. Tween (TBS-Ca/T), the wells were blocked with 1% bovine serum albumin (Sigma, St Louis, Mo) in TBS-Ca/T. Tested IIF ANALYSIS serum samples were coded to allow blinded testing, serially diluted from 1:100 to 1:10 000 in bovine serum albumin and Serum samples were tested at serial dilutions of 1:10 to 1:320 TBS-Ca/T and added for 1 hour at room temperature. In ini- for the presence of antibodies against keratinocyte cell sur- tial experiments the most specific optical density (OD) value face proteins by IIF analysis with cryosections of monkey with the least background was achieved at a serum dilution esophagus as a substrate using 1:50 dilution of fluorescein of 1:10 000 and subsequently serum samples were tested at isothiocyanate–conjugatedanti–humanIgGantibodies(INOVA this dilution only. Plates were then incubated with 2000-fold Diagnostics, San Diego, Calif) as a second antibody. diluted peroxidase–conjugated rabbit anti–human IgG antibodies (monoclonal antibody against the ␬-light chain of PRODUCTION AND PURIFICATION all immunoglobulin classes) for 1 hour at room temperature. OF rDSG BACULOVIRUS PROTEIN Color development was achieved by adding 100 µL of ABTS (Boehinger Mannheim, Mannheim, Germany) for 20 min- Desmoglein 1 and Dsg3 were expressed and purified as de- utes, and OD at 405 nm was determined using an ELISA reader scribed previously.16,17 Recombinant baculovirus encoding the (Dynatech, Guerney, Channel Island). All assays were per- entire extracellular domain of Dsg1 or Dsg3 fused with formed in duplicate and background values determined for complementary DNA encoding for an E-tag and a histidine- each test serum sample in uncoated wells were subtracted. tag was used to infect “high-five” insect cells (Invitrogen, San Each plate contained at least 1 negative control serum sample Diego) cultured under serum-free conditions (Sf 900 II SFM; and a positive PV reference serum sample (IIF titer, 160). Gibco BRL, Grand Island, NY) for 3 days at 27°C, resulting The OD of the tested serum samples obtained for each plate in the secretion of rDsg into the culture supernatant. Cell de- were normalized relative to the mean OD obtained for the bris was removed by centrifugation, and supernatants were PV reference serum sample (dilution, 10 000) tested on the immediately affinity purified or stored at −70°C. For purifi- same 96-well plate. The mean of the normalized OD was used cation of rDsg, culture supernatants were applied to nickel in the analysis. A test serum sample was considered posi- nitrilotriacetic acid–agarose (Qiagen Inc, Chatsworth, Calif) tive by ELISA when the mean OD was 3 SDs above the mean columns allowing binding of the protein via the histidine- OD of serum samples from 10 healthy volunteers (OD, 0.09). tag. The protein was eluted with sequential 1-mL volumes of 200 mmol/L of imidazole in a 20-mmol/L sodium phos- STATISTICS phate and 500-mmol/L sodium chloride solution (pH 6.3) containing 1 mmol of calcium chloride per liter, dialyzed To determine the correlation between ELISA OD and IIF ti- against 50-mmol/L Tris-buffered saline (TBS)–calcium, ana- ter, we applied the Spearman correlation for nonparametric lyzed by Western blot and used for coating of the ELISA plates. variables. A correlation coefficient (r) of +1 indicates a per- The 1-mmol/L calcium chloride was added to all purifica- fect positive, of −1 a perfect negative correlation.

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Patient No./ IIF Positive† Dsg3-ELISA Positive Sex/Age, y (Negative) (Negative) 1/F/37 10 (0) 10 (0) 2/M/34 8 (0) 8 (0) 3/F/89 8 (0) 8 (0) 80 kd 4/M/51 6 (0) 6 (0) 5/M/42 5 (0) 5 (0) 6/M/56 4 (0) 4 (0) 7/M/53 2 (0) 2 (0) 8/F/88 1 (0) 1 (0) 9/F/57 4 (0) 3 (1) 10/F/50 0 (1) 0 (1) Figure 1. Expression and purification of recombinant (r) desmoglein (Dsg) 3 11/F/68 0 (3) 0 (3) baculoproteins. The protein is visualized at each purification step by Western blot analysis using an anti-E-tag antibody. Lane 1, rDgs3 baculovirus- *The number of serum samples tested for each patient by indirect infected culture supernatants; lane 2, wild-type baculovirus infection; lane 3, immunofluorescence (IIF) on monkey esophagus and in the desmoglein flow-through after application of culture supernatant over the nickel-agose (Dsg3) enzyme-linked immunosorbent assay (ELISA) with the number of column; and lanes 4 and 5, column eluates containing purified rDsg3. negative serum samples with the respective test within parentheses. †Indirect immunoflourescence results were considered positive if the titer was 20 or higher. reacted nonspecifically with epitopes of the linear E-tag or histidine-tag of the rDsg3-fusion protein, the PV se- between PV, pemphigus foliaceus, and paraneoplastic rum sample was absorbed with similar amounts of con- pemphigus (PNP)1,13 since these diseases share the lace- trol proteins: (1) an rDsg1-, E-tag, and histidine-tag- like squamous intercellular substance (ICS)–binding fusion protein16 and (2) the Dsg3 fusion protein denatured pattern. Although the differentiation is possible by by high-temperature treatment that destroys the confor- immunoprecipitation and immunoblotting of epidermal mational Dsg3 epitopes while leaving the linear epit- extracts with patient serum samples,13-15 both methods opes of Dsg3, the E-tag, and histidine-tag intact. Follow- are time consuming and laborious. A significant ing absorption with rDsg1 or denatured rDsg3, the PV advance in pemphigus diagnostics was the recent clon- serum sample showed an ICS-binding pattern indistin- ing of the Dsg3 gene.6 This allowed the in vitro produc- guishable from the nonabsorbed serum sample, indicat- tion of recombinant (r) Dsg3 antigen in a native confor- ing that the reactivity was specific for conformational Dsg3 mation3 and, subsequently, the development of a epitopes. sensitive and PV-specific ELISA.16 Of 48 IIF positive serum samples from patients with To further validate the suitability and reliability of PV, 45 (98%) were positive by ELISA, whereas the 4 PV ELISA for the diagnosis of PV and to expand the pub- serum samples that were negative by IIF were also nega- lished results, we produced rDsg3 protein via the bacu- tive by ELISA (Table, Figure 2). To examine the speci- lovirus expression system and tested serum samples from ficity of ELISA, we investigated a total of 45 additional patients with PV and patients with a variety of other bul- patients with several other bullous or nonbullous skin lous and nonbullous dermatologic disorders for anti- diseases (Figure 2), including 11 with bullous pemphi- Dsg3 reactivity. goid, 10 with various bullous skin diseases, 9 with connective tissue diseases and high titers of antibodies against RESULTS nuclear antigens, and 13 with various nonbullous skin disorders. The extracellular domain of Dsg3 was produced as a se- While all 11 serum samples from patients with creted recombinant protein by a baculovirus expression bullous pemphigoid tested negative in the ELISA, 2 system and purified by affinity chromatography. The serum samples from patients with other bullous disor- 80-kd rDsg3 was detected by Western blot analysis us- ders showed ELISA reactivity—1 with a PNP and 1 ing a monoclonal antibody against the E-tag that was fused with an unspecified bullous disease. In addition, 7 of 9 to the C-terminal end of the protein (Figure 1). Anti- serum samples of patients with autoimmune–connective Dsg3 antibodies in the serum samples of patients with tissue diseases, 13 of 13 serum samples of patients PV recognize predominantly conformational epitopes of with other nonbullous skin diseases, and 10 of 10 the extracellular Dsg3 domain.2,3,18 To test if the puri- serum samples from healthy individuals were negative fied rDsg3 retained the native protein conformation con- by ELISA. Thus, 4 of 55 control serum samples were taining these relevant epitopes, the antigenicity of the positive by ELISA, demonstrating a disease specificity rDsg3 baculoprotein was tested in immunoabsorption as- of 93% for the PV ELISA. says. A PV serum sample (IIF titer, 320) was diluted 1:20 The Dsg3 autoantibody titers may follow disease ac- with rDsg3 and subsequently reevaluated by IIF analy- tivity and thus, antibody levels detected by IIF are used sis. The rDsg3 protein completely removed the binding clinically for patient management. Statistical analysis of activity of the PV serum sample, indicating that the pu- our data revealed a positive correlation (r = 0.65) be- rified rDsg3 protein was suitable for use as an ELISA an- tween Dsg3-ELISA OD and IIF titer. Furthermore, when tigen. To rule out the possibility that the serum samples multiple serum samples from 1 individual patient sampled

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1 0.5 ELISA, OD 0.9 160 0.4

Lesion 0.8 Titer IIF, 0.3

0.7 80 0.2

0.6 40 0.1

OD 405 20 Steroid Azathioprine 0.5 0 Date: 11/95 12/95 1/96 2/96 10/96 12/96 1/97 2/97 8/97 10/97 0.4 Month/y

0.3 Figure 3. Enzyme-linked immunosorbent assay (ELISA) value, indirect immunofluorescence (IIF) titer, and the clinical course in a patient with 0.2 pemphigus vulgaris. Enzyme-linked immunosorbent assay optical density (OD) values (circle) and IIF titer (diamond) paralleled over a 2-year period 0.1 and increased with the appearance of fresh blisters as indicated by arrows.

0 PV BP Div CTD Nonbullous CTRL the test became obvious from the fact that 11 of 11 se- Bullous rum samples from patients with bullous pemphigoid were negative by ELISA as were 10 of 12 serum samples from Figure 2. Desmoglein 3 enzyme-linked immunosorbent assay results (optical density [OD] at 405 nm) of serum samples from patients with pemphigus patients with various other bullous diseases and 7 of 9 vulgaris (PV) or various other bullous and nonbullous skin diseases. BP serum samples from patients with autoimmune– indicates bullous pemphigoid; Div bullous, other bullous diseases; CTD, connective tissue diseases. A patient with pemphigus fo- connective tissue diseases; nonbullous, nonbullous skin disorders; CTRL, healthy controls; and solid line, cutoff value of the enzyme-linked liaceus, a subtype of pemphigus characterized by anti- immunosorbent assay. bodies against the 160-kd glycoprotein Dsg1, was included in the controls.19-21 As expected, this patient with PF had no detectable reactivity to Dsg3 by ELISA, supporting the over a 2-year period were analyzed, ELISA OD values previously established diagnosis. paralleled both the IIF titer and the clinical course Among the 4 ELISA-positive serum samples from (Figure 3). patients without PV, 1 was obtained from a patient with PNP. The positive Dsg3-ELISA result was surprising, as COMMENT the autoantigens identified in PNP are commonly directed against components of the hemidesmosomes Recently, a Dsg3-ELISA was shown to represent a sensi- and the cytoplasmic plaque of desmosomes with tive and disease-specific test for the diagnosis of PV.16 molecular weights of 250 kd (desmoplakin I), 230 kd We produced rDsg3 protein in our laboratory and (bullous pemphigoid antigen), 210 kd (desmoplakin II, established ELISA to validate the test by screening envoplakin),22 190 kd (periplakin); (G. Anhalt, oral serum samples from patients with PV seen in our communication, May 1998), and 170 kd (not yet identi- department. Furthermore, we expanded the published fied).23,24 However, recent reports24-26 provide evidence results by testing additional disease groups, including that in PNP, autoantibodies may also be directed other bullous dermatoses or autoimmune-connective against components of the extracellular matrix includ- tissue diseases. ing Dsg3, suggesting the possibility of overlapping auto- Forty-seven of 52 PV serum samples obtained from antibody specificities in the diseases of the pemphigus 11 patients were positive by Dsg3-ELISA, indicating a sen- group. Recently, Amagai et al26 found that 25 of 25 sitivity of 93%. Interestingly, the sensitivity of ELISA in- patients with PNP were reactive in the Dsg3-ELISA and creased to 98% when IIF-negative PV serum samples were half of them in the Dsg1-ELISA, indicating that desmo- excluded, indicating that both tests have a similar sen- glein antibodies may play a pathogenic role in disease sitivity. In addition, 4 of 55 non-PV serum samples were expression. Our patient with PNP demonstrated weak positive by ELISA, resulting in a specificity of 93%. These bands of 250, 230, 210, and 190 kd in the immunopre- results obtained in a population of mostly Austrian der- cipitation, but no 130-kd band, suggesting that the matologic patients are comparable with those obtained Dsg3-ELISA allows detection of low levels of anti-Dsg3 from Japanese patients as previously published by Ishii antibodies that may be missed by conventional detec- et al.16 tion methods. To determine whether ELISA was prone to false- The second ELISA-positive non-PV serum sample positive results in patients with other autoimmune dis- was derived from a patient with an unspecified bullous eases who often have high levels of autoantibodies that disorder that showed a nondiagnostic histopathologic con- might cross-react with Dsg3, we tested serum samples dition and was negative by IIF for anti-ICS and antibase- from patients with non-PV bullous diseases and autoim- ment membrane antibodies. It remains unclear whether mune–connective tissue diseases. The high specificity of this patient had PV with low levels of pathogenic anti-

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©1999 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 10/01/2021 Dsg3 antibodies or whether the detected antibodies are remission. Interestingly, the increase in ELISA values irrelevant for disease expression. occurred earlier and were more pronounced compared Furthermore, serum samples from 2 patients with with IIF titers. This may reflect a higher sensitivity of high-titer antibodies against nuclear antigens showed ELISA in specifically detecting pathogenic anti-Dsg3 ELISA reactivity, 1 with a high OD. Since these patients antibodies, which may be helpful in monitoring the completely lacked blisters or erosions, the positive clinical course and for adjustment or discontinuation of ELISA values may represent nonspecific binding of cir- the immunosuppressive therapy. Larger and prospec- culating autoantibodies as frequently observed for sero- tive studies are required to determine the exact relation- logic testing in patients with connective tissue diseases, ship between Dsg3-ELISA values, IIF titers, and the although cross-reactivity or the presence of nonpatho- clinical course. genic Dsg3 antibodies cannot formally be excluded. Thus far, the most reliable indicator for PV remis- Similar to the Dsg3-ELISA, IIF assays may detect anti- sion seems to be a negative direct immunofluores- ICS reactivity in serum samples of patients with bullous cence result,33 requiring repeated skin biopsy speci- disorders other than PV, including cicatricial pemphi- mens. Further investigations will determine whether a goid, bullous pemphigoid, dermatitis herpetiformis, negative Dsg3-ELISA result obtained during clinical erosive lichen planus, recurrent oral ulcers, and toxic remission is a sensitive indicator to predict if the dis- epidermal necrolysis.27 ease will stay in remission following cessation of A major advantage of the Dsg3-ELISA is the selec- therapy. tive detection of antibodies against extracellular and con- Our data confirm that the Dsg3-ELISA is a highly formational epitopes of Dsg3, which include most patho- sensitive and specific test that allows objective and quan- genic antibodies in patients with PV.2,3,16 In contrast, titative detection of pathogenic Dsg3 antibodies. Thus, conventional IIF testing may also identify a variety of an- Dsg3-ELISA should be considered an additional diag- tibodies against proteins of the extracellular ma- nostic tool for the routine evaluation and monitoring of trix.1,16,28-30 Indirect immunofluorescence also may fail to patients with PV. distinguish between antibodies directed against the in- tracellular portion of Dsg3, antibodies against Dsg1, or Accepted for publication June 18, 1998. desmocollins, or other ICS constituents. Antibodies Reprints: Petra Lenz, MD, Division of Immunology, Al- against Dsg1 are commonly present in PV serum samples. lergy and Infectious Diseases, Department of Dermatol- Injection studies in neonatal mice suggested that they do ogy, University of Vienna Medical School, Wahnnger Gur- not play a major role in the pathogenesis of PV.3 How- tel 18-20,1090 Vienna, Austria (e-mail: petra.lenz@akh- ever, in humans, both Dsg3 and Dsg1 antibodies seem wien.ac.at). to be required to induce skin blisters, whereas patients with PV with Dsg3 antibodies in the absence of detect- REFERENCES able Dsg1 antibodies will only develop blisters orally. This has been proposed as a possible explanation for the dif- ferential requirements of Dsg1 and Dsg3 antibodies in 1. Stanley JR. Pemphigus. In: Fitzpatrick TB, Eisen AZ, Wolff K, Freeberg IM, Aus- ten KF, eds. Dermatology in General Medicine. New York, NY: MacGraw-Hill Book blister formation of mucosa vs skin. 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Critical Situations Readers are invited to submit examples of newly described disorders, the use of new diagnostic technology, dermatologic manifestations of important social disorders such as child abuse, and cases that highlight the complex nature of acute care dermatology to Anita G. Licata, MD, Division of Dermatology, UHC, 1 S Prospect St, Burlington, VT 05401-3444. When appropriate, these should be written in case presentation format with a brief discussion following. The Cutting Edge Clinicians, local and regional societies, residents, and fellows are invited to submit cases of challenges in management and therapetics to this section. Cases should follow the established pattern and be submitted double-spaced and in triplicate. Photomicrographs and illustrations must be clear and submitted as positive color transparencies (35-mm slides) or black- and-white prints. Do not submit color prints unless accompanied by original transparencies. Material should be accompanied by the required copyright transfer statement, as noted in “Instructions for Authors.” Material for this section should be submitted to George J. Hruza, MD, Cutaneous Surgery Center, 1 Barnes Hospital Plaza, Suite 16411, St Louis, MO 63110. Reprints are not available. Issues in Dermatology Issues in Dermatology solicits provocative essays relevant to all aspects of dermatology. Please submit manuscripts to the section editor, A. Bernard Ackerman, MD, 2 E 70th St, New York, NY 10021. Off-Center Fold Clinicians, local and regional societies, and residents and fellows in dermatology are invited to submit quiz cases to this section. Cases should follow the established pattern and be submitted double-spaced. Photomicrographs and illustrations must be clear and submitted as positive color transparencies. If photomicrographs are not available, the actual slide from the specimen will be acceptable. Material should be accompanied by the required copyright transfer statement, as noted in “Instructions for Authors.” Material for this section should be submitted to Lori Lowe, MD, Department of Pathology, Uni- versity of Michigan, Medical Science 1, M5242, 1301 Catherine Rd, Ann Arbor, MI 48109. Reprints are not available.

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