Journal of Dermatological Science (2003) 32, 33 /41 www.elsevier.com/locate/jdermsci Comparative study of autoantigen profile between Colombian and Brazilian types of endemic pemphigus foliaceus by various biochemical and molecular biological techniques Yoshiko Hisamatsua,c,1, Ana Maria Abreu Velezb,1, Masayuki Amagaid, Marilia M. Ogawae, Tamotsu Kanzakic, Takashi Hashimotoa,* a Department of Dermatology, Kurume University School of Medicine, 67 Asahimachi, Kurume, 830-0011 Fukuoka, Japan b Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, GA, USA c Department of Dermatology, Kagoshima University School of Medicine, Kagoshima, Japan d Department of Dermatology, Keio University School of Medicine, Tokyo, Japan e Department of Dermatology, Paulista College of Medicine, Sao Paulo, Brazil Received 30 October 2002; received in revised form 15 January 2003; accepted 16 January 2003 KEYWORDS Autoimmune bullous disease; Endemic pemphigus foliaceus; Enzyme-linked immunosorbent assay; Immunoblotting; Desmoglein; Desmocollin; Desmosome; Plakin family protein Summary Background: Besides Brazilian endemic pemphigus foliaceus (EPF), we have described another focus of EPF in Colombia. Our previous study suggested that Colombian EPF seemed to react various plakin family proteins, such as envoplakin, periplakin and BP230. Objective: To further characterize the Colombian EPF and study the difference from Brazilian EPF, we examined the antigen profile of the two types of EPF. Methods and results: Immunoblotting using normal human epidermal extracts revealed that 38% Colombian EPF sera and 25% Brazilian EPF sera showed IgG antibodies reactive with desmoglein (Dsg) 1, pemphigus foliaceus antigen. The sera of both types of EPF showed protein bands co-migrating with plakin family proteins, particularly periplakin. Immunoblotting analyses using recombinant proteins of various domains of envoplakin, periplakin and BP230 revealed that a considerable number of Colombian EPF sera reacted with recombinant proteins of periplakin, while only few Brazilian sera reacted with some of the recombinant proteins of any plakins. Enzyme-linked immunosorbent assay (ELISA) for Dsg1 and Dsg3 showed that Dsg1 was reacted by almost all sera of both types of EPF. However, unexpectedly, while none of Colombian EPF sera reacted with Dsg3, about half of Brazilian EPF sera reacted with Dsg3. Conclusion: These results suggested that the Colombian EPF is basically similar to Brazilian EPF in terms that major antigen is Dsg1, but there were some different antigen profiles between the two types of EPF. – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved. Abbreviations: ELISA, enzyme-linked immunosorbent assay; EPF, endemic pemphigus foliaceus. *Corresponding author. Tel.: /81-942-31-7571; fax: /81-942-34-2620. E-mail address: [email protected] (T. Hashimoto). 1 Joint first authors. 0923-1811/03/$30.00 – 2003 Japanese Society for Investigative Dermatology. Published by Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0923-1811(03)00034-3 34 Y. Hisamatsu et al. 1. Introduction 2.3. Immunoblotting studies An endemic pemphigus foliaceus (EPF) was first described in certain regions of Brazil more than a century ago, which is also known as fogo selvagem [1 /5]. Another type of EPF has been reported in Tunisia [6,7]. Recently, we have also described additional focus of EPF Colombia [8 /10] (two full papers by Abreu Velez et al., in press). All types of EPF were characterized histologically by acantholytic intraepidermal blisters and immunologically by circulating IgG autoantibodies that react keratinocyte cell surfaces. A remarkable feature of EPF is its epidemiological nature. Brazilian EPF is endemic to certain states in Brazil, there are many familial cases including child and young adult cases, most patients are dedicated to farming, and both sexes are equally affected . On the other hand, the previous epidemiological studies revealed that Colombian EPF affects predominantly males at an age of 40 /60 years, as well as a few postmenopausal females [8 /10]. These findings suggest that Colombia EPF differ from Brazilian EPF. Tunisian EPF also showed different features and affected most frequently females of childbearing age, although its epidemicity has not been well characterized [6,7]. In this study, the Colombian EPF is further characterized and the difference between Colombian and Brazilian EPF is studied; we examined autoantigens in both diseases by various biochemical and molecular biological techniques, including immunoblotting and enzyme-linked immunosorbent assay (ELISA) using various antigen sources. We performed various immunoblotting procedures using different antigen sources to identify possible autoantigens reacted by EPF sera. Immunoblotting of normal human epidermal extracts was performed as described previously [4,11 /15]. Recently, we have successfully produced recombinant fusion proteins of envoplakin (EPL), periplakin (PPL) and BP230, which are members of plakin family protein [16,17]. All the plakin family proteins show similar molecular structures, consisting of N-terminal globular domain (N), middle rod domain (M) and C-terminal globular domain (C) [18 /21]. It is now confirmed that these plakin family proteins are autoantigens targeted by paraneoplastic pemphigus sera [22 /27]. We generated domain-specific recombinant fusion proteins, named EPL-N, M, C, PPL-N, M, C and BP230-N, M, C [16,17]. Immunoblotting using these recombinant proteins was performed as described previously [16,17]. Because the fusion proteins of ENV-N, ENVM, PPK-N, PPK-M, BP230-N, BP230-M and BP230-C could not be purified by glutathione /sepharose column, the patients sera were pre-absorbed with bacterial lysate to reduce background reactivity due to non-specific antibodies to bacterial proteins [16,17]. 2.4. ELISA of desmoglein (Dsg) 1 and Dsg3 2. Materials and methods ELISA of Dsg1 and Dsg3 for IgG antibodies was performed as described previously [28 /30]. In this ELISA for IgG antibodies, the index value above 20 was considered positive and the value between 10 and 20 was in the gray zone. We also performed ELISA of Dsg1 and Dsg3 for IgA antibodies, which has recently been developed . 2.1. Patients 2.5. ELISA of desmocollin (Dsc) 1, 2 and 3 In this study, we used sera from 29 Colombian EPF patients and 20 Brazilian EPF patients, who lived in the endemic areas for both diseases and showed typical clinical and histological features each of Colombia EPF and Brazil EPF. We also used two sera each from typical cases of pemphigus vulgaris, pemphigus foliaceus, paraneoplastic pemphigus and bullous pemphigoid as positive controls, as well as 10 normal control sera. We have recently produced recombinant proteins of extracellular domains of human Dsc1 /3 using baculovirus expression system (Hisamatsu et al., submitted for publication). Using the recombinant proteins, we have developed a novel ELISA for detecting antibodies of both IgG and IgA classes against Dsc1 /3 (Hisamatsu et al., submitted for publication). The details for the ELISA of Dsc1 /3 are described in a separate manuscript (Hisamatsu et al., submitted for publication). Procedures are almost the same as that for ELISA of Dsg1 and Dsg3 [28 /30], with some modifications. All the normal control sera showed the value of OD490 less than 0.03. Therefore, we arbitrarily set the cut-off value as 0.1 for both IgG and IgA antibodies. In 2.2. Immunofluorescence studies Indirect immunofluorescence using sections of normal human skin for both IgG and IgA antibodies was performed as described previously [1,3]. Comparison of antigen between two types of endemic pemphigus foliaceus general, none of the 45 sera of classic types of pemphigus; i.e. pemphigus vulgaris and sporadic pemphigus foliaceus, showed positive reactivity. 2.6. COS-7 cell transfection study using human Dsc1 3 cDNA / cDNA transfection study to detect IgA antibodies to Dsc1, 2, or 3 cDNAs was performed, as described previously . 3. Results The results of all experiments are summarized in Table 1. 3.1. Immunofluorescence studies By indirect immunofluorescence using sections of normal human skin, the sera of all the 29 Colombia EPF and 20 Brazil EPF patients showed IgG anti-cell surface antibodies (data not shown). In addition, two Colombian EPF sera showed IgA anti-cell surface antibodies reactivity with upper epidermis, while none of Brazilian EPF showed IgA antibodies. 3.2. Immunoblotting studies By immunoblotting using normal human epidermal extracts, 11 (38%) of 29 Colombian EPF sera and 5 (25%) of 20 Brazilian EPF sera showed a 160 kDa protein band, which co-migrated with Dsg1 detected by control non-endemic pemphigus foliaceus sera (Fig. 1a,b). However, numerous additional protein bands were seen in blots reacted by both EPF sera (Fig. 1a,b). Particularly, 14 (48%) of Colombian EPF sera reacted clearly with a 190 kDa protein band, which co-migrated with periplakin detected by control sera of paraneoplastic pemphigus (Fig. 1a). In addition, some patients weakly reacted with 230 and 210 kDa protein bands, which co-migrated with BP230 and envoplakin detected by control sera of bullous pemphigoid and paraneoplastic pemphigus, respectively (Fig. 1a). These protein bands co-migrating with various plakin family proteins were also frequently detected by Brazilian EPF sera (Fig. 1b). In immunoblot analyses using recombinant proteins of EPL-N,M,C, PPL-N,M,C and BP230-N,M,C, the most interesting result was that 13 /31% of Colombian EPF sera reacted with various domains of periplakin, particularly frequently with N- and Cterminal domains of the protein, whereas only few 35 Table 1 Summary of the results of all experiments Colombian EPF Brazilian EPF Immunoblotting of epidermal extracts 230 kDa BP230 2/29 (7%) 1/20 (5%) 210 kDa envoplakin 2/29 (7%) 5/20 (25%) 190 kDa periplakin 14/29 (48%) 10/20 (50%) 160 kDa Dsg1 11/29 (38%) 5/20 (25%) 130 kDa Dsg3 0/29 (0%) 1/20 (5%) Immunoblotting of recombinant proteins EPL-N 2/29 (7%) 0/20 (0%) EPL-M 4/29 (13%) 3/20 (15%) EPL-C 0/29 (0%) 0/20 (0%) PPL-N PPL-M PPL-C 9/29 (31%) 4/29 (13%) 8/29 (27%) 1/20 (5%) 1/20 (5%) 3/20 (15%) 1/29 (3%) 0/29 (0%) 0/29 (0%) 0/20 (0%) 1/20 (5%) 1/20 (5%) ELISA Dsg1 (IgG) Dsg3 (IgG) Dsg1 (IgA) Dsg3 (IgA) 27/29 (93%) 0/29 (0%) 0/2 (0%)* 0/2 (0%)* 20/20 (100%) 12/20 (60%) 3/20 (15%) 1/20 (5%) Dsc1 (IgG) Dsc2 (IgG) Dsc3 (IgG) 0/29 (0%) 0/29 (0%) 0/29 (0%) 3/20 (15%) 3/20 (15%) 3/20 (15%) BP230-N BP230-M BP230-C cDNA transfection to COS-7 cells Dsc1 (IgA) 0/2 (0%)* Dsc2 (IgA) 0/2 (0%)* Dsc3 (IgA) 0/2 (0%)* Not done Not done Not done * Two cases with IgA anti-cell surface antibodies. Brazilian EPF sera reacted with some of the periplakin recombinant proteins (Fig. 2). In contrast with the results of periplakin recombinant proteins, only a few (less than 15%) of sera of both types EPF reacted with some of envoplakin recombinant proteins (Fig. 3). Furthermore, concerning the recombinant proteins of BP230, any recombinant proteins were rarely detected by the sera of both types of EPF (Fig. 4). 3.3. Results of ELISA of Dsg1 and Dsg3 Twenty-seven (93%) of 29 Colombia EPF sera and all 20 Brazil EPF sera showed IgG antibodies to Dsg1 by ELISA. Interestingly, whereas none of Colombia EPF sera reacted with Dsg3, seven Brazil EPF sera reacted with Dsg3 and five Brazil EPF sera weakly reacted with Dsg3 (in the gray zone) (Fig. 5). In addition, we examined all 20 Brazilian EPF sera by ELISA of Dsg1 and Dsg3 for IgA antibodies. Three and one sera showed positive reactivity with 36 Y. Hisamatsu et al. Fig. 1 The results of immunoblotting using normal human epidermal extracts. Two separate experiments were performed for Colombian EPF (a) and for Brazilian EPF (b). In each panel, the lanes showed reactivity of EPF patients (Pt), control PV serum (PV), control paraneoplastic pemphigus serum (PNP), control bullous pemphigoid serum (BP), and monoclonal antibodies to Dsc (Dsc-mAb) and to desmoplakin (DPL-mAb). The positions of protein bands are shown in the left of each panel. Dsg1 and Dsg3, respectively. Because two Colombian EPF sera showed IgA anti-cell surface antibodies by indirect immunofluorescence, we also examined the two sera by ELISA of Dsg1 and Dsg3 for IgA antibodies. However, neither serum reacted with any Dscs. 3.4. Results of ELISA of Dsc1 3 / As we mention in detail in a separate study (Hisamatsu et al., submitted for publication), by the ELISA of Dsc1 /3, 3 of the 20 Brazilian EPF sera reacted with all Dsc1 /3 (Fig. 6). In contrast, no Colombian EPF sera showed positive reactivity, although a few sera showed a weak reactivity within the gray zone (Fig. 6). 3.5. Results of cDNA transfection method To study the antigens for IgA antibodies in the two Colombian sera which showed IgA anti-cell surface antibodies, we performed the cDNA transfection method. However, IgA antibodies in the two sera did not show any positive reactivity with all Dscs (data not shown). 4. Discussion In our previous study, we showed that 12 of 27 Brazilian EPF sera reacted with Dsg1 by immunoblotting of epidermal extracts, which is consistent with the results that about one-third of sporadic pemphigus foliaceus sera react with Dsg1, suggest- ing the similarity between the two diseases . In the present study, we further confirmed that all the Brazilian EPF sera reacted with Dsg1 by ELISA, which has been shown to be a highly sensitive and specific method [28 /30]. The reason why only onethird of the sera of EPF and sporadic pemphigus foliaceus reacted with Dsg1 by immunoblotting was considered that the epitopes detected by the sera are destroyed during the procedure of immunoblotting, while such epitopes are retained in the ELISA. In addition, 38% Colombian EPF sera reacted with Dsg1 by immunoblotting, and almost all Colombian EPF sera showed anti-Dsg1 antibodies by ELISA, which was very similar to the results of Brazilian EPF, suggesting that the Colombian and Brazilian EPF have common nature as a subtype of pemphigus foliaceus. However, a striking finding was that more than half of Brazilian EPF showed antibodies to Dsg3, pemphigus vulgaris antigen, by the ELISA. This result is in contrast to the result of sporadic pemphigus foliaceus, which showed no positive reactivity with Dsg3 by this ELISA [28,30]. We could not detect the reactivity with Dsg3 in any Brazilian EPF by immunoblotting, which may be due to the relatively low titer of anti-Dsg3 antibodies in the sera. Interestingly, none of Colombia EPF sera reacted with Dsg3 by ELISA. This specific reactivity of Brazilian EPF with Dsg3 is also in clear contrast to the result that none of sporadic pemphigus foliaceus sera showed antibodies to Dsg3 by the same ELISA [28 /30]. Comparison of antigen between two types of endemic pemphigus foliaceus 37 Fig. 2 The results of immunoblotting of recombinant proteins of various domains of envoplakin. Left panel shows the results of representative Colombian EPF sera with recombinant proteins of EPL-N, EPL-M and EPL-C, and right panel shows the results of representative Brazilian EPF sera. The right lane in each panel showed the reactivity of a control paraneoplastic pemphigus serum (PNP). An arrow in the left in each panel indicates the position of intact recombinant protein. Fig. 3 The results of immunoblotting of recombinant proteins of various domains of periplakin. Left panel shows the results of representative Colombian EPF sera with recombinant proteins of PPL-N, PPL-M and PPL-C, and right panel shows the results of representative Brazilian EPF sera. The right lane in each panel showed the reactivity of a control paraneoplastic pemphigus serum (PNP). An arrow in the left in each panel indicates the position of intact recombinant protein. The significance of the results that a few Brazilian EPF sera showed IgA anti-Dsg antibodies is at the present unknown. In our previous study, we found that, in addition to Dsg1, Colombian EPF sera reacted with many other protein bands by various immunoblotting and immunoprecipitation analyses, particularly, 230, 210 and 190 kDa proteins, which were considered to be BP230, envoplakin an periplakin, respectively (Abreu Velez et al., in press). In the present study, we further found the reactivity of many Colombian EPF sera with multiple proteins by immunoblotting of human epidermal extracts, which was not used in the previous study (Abreu Velez et al., in press). To confirm the reactivity of Colombian EPF sera with these plakin family proteins (also know as paraneoplastic pemphigus antigens), we examined sera of both Colombian and Brazilian EPF by immunoblotting using various domain-specific recombinant proteins of these proteins. These studies further indicated that considerable number of Colombian EPF sera reacted with various domains of periplakin, while only few of Brazilian EPF sera reacted with some of them. Recombinant proteins of envoplakin were recognized by less number of sera of both types of EPF. These results are similar to those in our previous study , which indicated that a few sera of non-paraneoplastic pemphigus patients react with either envoplakin or periplakin. However, the number of the Colombian EPF sera reactive with envoplakin recombinant proteins are significantly higher, indicating that the preferential reactivity with periplakin may be characteristic of Colombian EPF. 38 Y. Hisamatsu et al. Fig. 5 The results of ELISA of Dsg1 and Dsg3 for IgG antibodies. Upper and lower panels showed the results of ELISA for Dsg1 and Dsg3, respectively. In each panel, the results of Colombian EPF sera were shown in the left and the results of Brazilian EPF sera in the right. The dashed horizontal lines indicate the cut-off levels (index value 20). Fig. 4 The results of immunoblotting of recombinant proteins of various domains of BP230. Left panel shows the results of representative Colombian EPF sera with recombinant proteins of BP230-N, BP230-M and BP230-C, and right panel shows the results of representative Brazilian EPF sera. The right lane in each panel showed the reactivity of a control bullous pemphigoid serum (BP). An arrow in the left in each panel indicates the position of intact recombinant protein. In clear contrast, the sera of both types of EPF rarely reacted with any BP230 recombinant proteins, which is consistent with the results of our previous study , that any BP230 recombinant proteins were detected by non-bullous pemphigoid sera. Therefore, it seems likely that, although the 230 kDa protein band detected by some Colombian EPF sera in our previous study (Abreu Velez et al., in press), the 230 kDa protein was other protein with molecular weight of 230 kDa, but not BP230. In our previous studies, we have found that some Brazilian EPF sera showed IgG antibodies reactive with bovine Dsc . However, the significance of these findings was not clear because none of these sera reacted with human Dsc. In this study, a few Brazilian EPF sera, but no Colombian EPF sera, showed a positive IgG reactivity with Dsc1 /3 by ELISA of Dsc1 /3. Although the specificity of this reactivity could not be confirmed, the results with this novel ELISA may further suggest that Brazilian EPF sera, but not Colombian EPF sera, contain antiDsc antibodies. Finally, we found that two Colombian EPF sera showed IgA antibodies, which reacted with cell surfaces in the upper epidermis. Because this distribution is that of Dsc1, we considered that these sera may contain IgA antibodies to Dsc1. However, IgA antibodies in the sera did not react with any Dsc by either ELISA of Dsc1 /3 or cDNA transfection method for Dsc1 /3. The results of the present study first indicate that, although the both Colombian and Brazilian EPF showed similar reactivity in terms of Dsg1, there were several clear differences in immunoreactivity between the two EPF. One finding was that, while more than half of Brazilian EPF reacted with Dsg3 by ELISA, none of Colombian EPF reacted with Dsg3. The next finding was that, while Colombian EPF preferentially reacted with the recombinant proteins of various domains of periplakin, only few Brazilian EPF sera reacted with them. Furthermore, some Brazilian Comparison of antigen between two types of endemic pemphigus foliaceus Fig. 6 The results of ELISA of Dsc1-Dsc3 for IgG antibodies. Upper, middle and lower panels showed the results of ELISA for Dsc1, Dsc2 and Dsc3, respectively. In each panel, the results of Colombian EPF sera were shown in the left and the results of Brazilian EPF sera in the right. The dashed horizontal lines indicate the cutoff levels (OD 0.1). EPF sera showed positive reactivity in ELISA of Dsc1 /3 for IgG antibodies. These results indicated that, although both Colombian and Brazilian types of EPF seemed to be subtypes of pemphigus foliaceus, there is a considerable difference in their immunopathological features between the two EPF. These differences may be consistent with the different findings in both clinical and epidemiological characteristics between the two EPF, which were indicated by the previous study (Abreu Velez et al., in press). However, in contrast that sera of classic types of pemphigus rarely react with antigens other than Dsgs, both Colombian and Brazilian EPF sera reacted with a variety of desmosomal proteins by various techniques. The reason why both types of EPF show such a highly immunogenic condition in the patients is not known. However, the previous study showed that even the relatives both in Brazilian EPF and in Colombian EPF (Abreu Velez et al., submitted for publication). These results lead us to speculate that, in conjunction with genetic predisposition, 39 some triggers, such as exposure to micro-organisms, may induce a highly immunogenic state in the patients, resulting in production of antibodies to many self antigens. Epitope spreading mechanism may be involved in the production of multiple autoantibodies . In addition, because Colombian EPF sera preferentially reacted with paraneoplastic pemphigus antigens, there may be a similar pathomechanism in autoantigen production in Colombian EPF and paraneoplastic pemphigus, although the two diseases are quite different clinically and epidemiologically. The endemic autoimmune diseases are very rare, and EPF is a typical example for this condition, which should give us many clues to unravel the pathomechanisms in development of autoimmunity. In this respect, the various interesting and complex autoantigen profiles found in both Colombian and Brazilian EPF in this study should be important to understanding the pathogenesis of these diseases, although the significance of the results is not clear at present. In conclusion, the results of the present study indicated that, although both Colombian and Brazilian EPF are similar diseases in terms of the reactivity with Dsg1, there is considerable variability with reactivity with other desmosomal proteins, indicating some difference between the two diseases. The significance of the complex autoantigen profile in these diseases should be clarified in future studies. Acknowledgements We gratefully thank Dr. Marilia M. Ogawa for generously providing us with the sera of Brazilian EPF. We thank Miss Michiyo Noge, Miss Yuko Kawano, and Miss Ayumi Suzuki for their technical assistance, and Miss Akiko Tanaka for secretarial assistance. This work was supported by Grant-inAid for Scientific Research from the Ministry of Education, Science and Culture of Japan, and a grant from the Ministry of Health and Welfare of Japan. Dr. Hisamatsu was an awardee of the Professor Klingman Award for travel expense for the Annual Meeting for the Society of Investigative Dermatology in 2001 (Washington, DC). References  Beutner EH, Prigenzi LS, Hale W, Leme CA, Bier OG. Immunofluorescence studies of autoantibodies to intercellular areas of epithelia in Brazilian pemphigus foliaceus. Proc Soc Exp Biol Med 1968;127:81 /6. 40  Castro RM, Proenca NG. Similarities and differences between South American pemphigus foliaceus and cazanaves pemphigus foliaceus. An Bras Dermatol 1983;53:137 /9.  Ogawa MM, Hashimoto T, Nishikawa T, Castro RM. IgG subclasses of intercellular antibodies in Brazilian pemphigus foliaceus */the relationship to complement fixing capability. Clin Exp Dermatol 1989;14:29 /31.  Ogawa MM, Hashimoto T, Konohana A, Castro RM, Nishikawa T. Immunoblot analyses of Brazilian Pemphigus foliaceus antigen using different antigen sources. Arch Dermatol Res 1990;282:84 /8.  Warren SJ, Lin MS, Giudice GJ, Hoffmann RG, Hans-Filho G, Aoki V, Rivitti EA, Santos V, Diaz LA. The prevalence of antibodies against desmoglein 1 in endemic pemphigus foliaceus in Brazil. Cooperative Group on Fogo Selvagem Research. N Engl J Med 2000;343:23 /30.  Morini JP, Jomaa B, Gorgi Y, Saguem MH, Nouira R, Roujeau JC, et al. Pemphigus foliaceus in young women. An endemic focus in the Sousse area of Tunisia. Arch Dermatol 1993;129:69 /73.  Joly P, Mokhtar I, Gilbert D, Thomine E, Fazza B, Bardi R, et al. Immunoblot and immunoelectron microscopic analysis of endemic Tunisian pemphigus. Br J Dermatol 1999;140:44 /9.  Abreu AM, Yepes MM, León Walter. Pemphigus Abreu-Manu. A lost link in skin autoimmunity in endemic fashion. J Invest Dermatol 2000;114:846 (abstract).  Abreu AM, Maldonado JG, Jaramillo A, Patiño PJ, Prada S, Leon W, et al. Immunological characterization of a unique focus of endemic pemphigus foliaceus in the rural area of El Bagre, Colombia. J Invest Dermatol 1998;110:516 (abstract).  Abreu AM, León Walter, Hashimoto K. An autoimmune skin disease with simultaneously acantholysis between keratinocytes and separation of the basal membrane zone of the skin. J Invest Dermatol 1999;112:614 (abstract).  Hashimoto T, Ogawa MM, Konohana A, Nishikawa T. Detection of PV and PF antigens by immunoblot analysis using different antigen sources. J Invest Dermatol 1990;94:327 / 31.  Hashimoto T, Amagai M, Garrod DR, Nishikawa T. Immunofluorescence and immunoblot studies on the reactivity of pemphigus vulgarsi and pemphigus foliaceus sera with desmoglein 3 and desmoglein 1. Epi Cell Biol 1995;4:63 /9.  Sugi T, Hashimoto T, Hibi T, Nishikawa T. Production of human monoclonal anti-basement membrane zone (BMZ) antibodies from a patient with BP (BP) by Epstein-Barr virus transformation. Analyses of the heterogeneity of anti-BMZ antibodies in BP sera using them. J Clin Invest 1989;84:1050 /5.  Hashimoto T, Amagai M, Watanabe K, Chorzelski PT, Bhogal BS, Black MM, Stevens HP, Boosma DM, Korman NJ, Gamou S, Shimizu N, Nishikawa T. Characterization of paraneoplastic pemphigus autoantigens by immunoblot analysis. J Invest Dermatol 1995;104:829 /34.  Nagata Y, Karashima T, Watt FM, Salmhofer W, Kanzaki T, Hashimoto T. Paraneoplastic pemphigus sera react strongly with multiple epitopes on the various regions of envoplakin and periplakin, except for C-terminal homologous domain of periplakin. J Invest Dermatol 2001;116:556 /63.  Hamada T, Nagata Y, Tomita M, Salmhofer W, Hashimoto T. Bullous pemphigoid sera react specifically with various domains of BP230, most frequently with C-terminal domain, by immunoblot analyses using bacterial recombinant proteins covering the entire molecule. Exp Dermatol 2001;10:256 /63. Y. Hisamatsu et al.  Green KJ, Virata ML, Elgart GW, Stanley JR, Parry DAD. Comparative structural analysis of desmoplakin, BP antigen and plectin: members of a new gene family involved in organization of intermediate filaments. Int J Biol Macromol 1992;14:145 /53.  Ruhrberg C, Hajibagheri N, Simon M, Dooley TP, Watt FM. Envoplakin, a novel precursor of the cornified envelope that has homology to desmoplakin. J Cell Biol 1996;134:715 /29.  Ruhrberg C, Hajibagheri N, Parry DAD, Watt FM. Periplakin, a novel component of cornified envelopes and desmosomes that belongs to the plakin family and forms complexes with envoplakin. J Cell Biol 1997;135:1835 /49.  Ruhrberg C, Watt FM. The plakin family: versatile organizers of cytoskeletal architecture. Curr Opin Genet Dev 1997;7:392 /7.  Anhalt GJ. Paraneoplastic pemphigus. Adv Dermatol 1997;12:77 /96.  Hashimoto T. Immunopathology of paraneoplastic pemphigus. Clin Dermatol 2001;19:675 /82.  Hashimoto T. Immunopathology of paraneoplastic pemphigus. Clin Dermatol 2001;19:675 /82.  Oursler JR, Labib RS, Ariss-Abdo L, Burke T, O’Keefe EJ, Anhalt GJ. Human autoantibodies against desmoplakins in paraneoplastic pemphigus. J Clin Invest 1992;89:1775 /82.  Kiyokawa C, Ruhrberg C, Karashima T, Mori O, Nishikawa T, Green KJ, Anhalt GJ, Watt FM, Hashimoto T. Envoplakin and periplakin are components of the paraneoplastic pemphigus antigen complex. J Invest Dermatol 1998;111:1236 /8.  Kim S-C, Kwon YD, Lee IJ, et al. cDNA cloning of the 210 kDa paraneoplastic pemphigus antigen reveals that envoplakin is a component of the antigen complex. J Invest Dermatol 1997;109:365 /9.  Mahoney MG, Aho S, Uitto J, Stanley JR. The members of the plakin family of proteins recognized by paraneoplastic pemphigus antibodies include periplakin. J Invest Dermatol 1998;111:308 /13.  Ishii K, Amagai M, Hall RP, Hashimoto T, Takayanagi A, Gamou S, Shimizu N, Nishikawa T. Characterization of autoantibodies in pemphigus using antigen-specific enzyme-linked immunosorbent assays with baculovirus-expressed recombinant desmogleins. J Immunol 1997;159:2010 /7.  Amagai M, Nishikawa T, Anhalt GJ, Hashimoto T. Antibodies against desmoglein 3 (pemphigus vulgaris antigen) are present in sera from patients with paraneoplastic pemphigus and cause acantholysis in vivo in neonatal mice. J Clin Invest 1998;102:775 /82.  Amagai M, Hashimoto T, Komai A, Hashimoto K, Kitajima Y, Ohya K, Iwanami H, Nishikawa T. Usefulness of enzymelinked immunosorbent assay (ELISA) using recombinant desmogleins 1 and 3 for serodiagnosis of pemphigus. Br J Dermatol 1999;140:351 /7.  Hashimoto T, Komai A, Futei Y, Nishikawa T, Amagai M. Detection of IgA autoantibodies to desmogleins by an enzyme-lined immunosorbent assay -The presence of new minor subtypes of IgA pemphigus. Arch Dermatol 2001;137:735 /8.  Hashimoto T, Kiyokawa C, Mori O, Miyasato M, Chidgey MAJ, Garrod DR, Kobayashi Y, Komori K, Ishii K, Amagai M, Nishikawa T. Human desmocollin 1 (Dsc1) is an autoantigen for subcorneal pustular dermatosis type of IgA pemphigus. J Invest Dermatol 1997;109:127 /31.  Dmochowski M, Hashimoto T, Garrod DR, Nishikawa T. Desmocollins I and II are recognized by certain sera from patients with various types of pemphigus, particularly Comparison of antigen between two types of endemic pemphigus foliaceus Brazilian pemphigus 1996;100:380 /4. foliaceus. J . Invest Dermatol 41  Vanderlugt CJ, Miller SD. Epitope spreading. Curr Opin Immunol 1996;8:831 /6.