Review
ANTIEPITHELIAL ANTIBODIES IN BRAZILIAN PEMPHIGUS EOLIACEUS NELSON GUIMARAES PROENCA, M.D. AND'EVANDRO RIVITTI, M.D. - r ,;r From the Department of Dermatology, Faculdade de Ciencias Medicas de Santa Casa Sao Paulo e Faculdade de Medicina da de Sao Paulo, Sao Paulo, Brazil
In 1941, techniques using fluorescent antibodies to locate antigens were used for the first time by Coons, Crech and Jones.' From then on several technical improvements were introduced and lately immunofluorescence has acquired a very important role in dermatology. It has proven to be useful both as an aid for diagnosis and to investigation of a great variety of diseases.
intercellular spaces of the prickle cell layer. 2. The antibodies arc specific and appear during the development of the pemphigus. In all patients with other diseases used as control, no antibodies were found. 3. It was possible to correlate clinical aspects with the titration. In pemphigus vulgaris patients, titers were proportional to the severity of the disease and in pemphigus foliaceus patients, to the extension of the lesion. Moreover, when titrations were repeated during the patient's recovery. It was possible to register a steady fall of titers.
With regard to the bullous dermatosis, a new phase had begun in which unknown pathogenic features were disclosed. Intercellular antiepithelial antibodies in pemphigus vulgaris were demonstrated initially by Beutner and Jordanin 1964. In spite of the increasing spread of knowledge about the immunology of bullous diseases, there is one basic point of divergency. It deals with the probable pathogenic role of the pemphigus antiepithelial antibodies: Do they participate in the onset of the acantholytic bullae or do they appear as a response to the disease? Chorzelski, Jablonska and Beutner' favor the first hypothesis, considering the role of the antiepithelial antibodies as a pathogenic one. Six arguments were set up in support of their opinion:
4. In pemphigus vulgaris patients, repeated titrations detected serologic recurrences preceding clinical recurrences. 5. With the technique of direct immunofluorescence, it was possible to show the in vivo fixation of IgG in the intercellular spaces of the epidermis. 6. The most convincing demonstration of the pathogenic role of the antiepithelial antibodies would be the production of acantholytic bullae with the passive transference of antibodies. This was perforniecl on rabbits and monkeys in an experinient that will be mentioned later.
1. The site where the antibodies are demonstrated with the immunofluorescence technique is exactly the same as the acantholytic process occurring in the pemphigus patient. It is confined to the
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INTERNATIONAL JOURNAL OF DERMATOLOGY
Chorzelski, Jablonska and Beutner considered these six arguments as a real proof of the primary role of antiepithelial antibodies in the inducement of the acantholytic bullae. Nevertheless, it should be stressed that some of the presenting features were not demonstrated in all groups of patients. It was not possible to induce acantholytic bullae using sera from pemphigus vulgaris patients. However, successful experiments were performed with sera from patients with Brazilian pemphigus foliaceus "fogo selvagen"—wild fire. On the other hand, serologic recurrences preceding clinical recurrences were observed only in pemphigus vulgaris and not in Brazilian pemphigus foliaceus patients. These studies should stimulate further investigation on pemphigus vulgaris, pemphigus vegetans, pemphigus foliaceus, Brazilian pemphigus foliaceus and pemphigus erythematosus (SenearUsher syndrome). Even in diseases in which antibodies similar to pemphigus were found, such studies would be of interest. We wish to review the Brazilian contributions in the field of immunopathology of pemphigus. They are of special importance because they were performed in pemphigus patients with the endemic form, such as occurs in Brazil. We will analyze the papers published by different investigators including the so-called "postulates" of Chorzelski, Jablonska and Beutner.'' Brazilian Contributions In August 1967, Furtado, Oliveira Lima, Andrade and Seabra'' presented at the XIII International Congress of Dermatology (Munich) their investigation on antibodies in pemphigus foliaceus. The authors studied with the indirect immunofluorescence technique sera from 27 patients and from 20 healthy persons as
December 1977
Vol. 16
controls. They divided their patients according to the degree of skin involvement: severe, moderate, slight and patients without lesions. The first 3 groups consisted of 21 patients and all of them showed positive results. Titers were related to the severity of the disease. In patients without lesions and in the healthy volunteers, the sera were negative. Antibodies against the dermoepidermal junction were not found. However, antibodies against cytoplasm were found twice. Other immunological investigations were negative (hemagglutinin, complement binding, immunoglobulins, agar gel diffusion and passive cutaneous anaphylaxis). This work was partially presented at the XXIV Brazilian Congress of Dermatology in Juiz de Fora (Minas Gerais) by Furtado et al.-' In October 1976, Leme^; presented at the same Congress his results on indirect immunofluorescence applied to Brazilian pemphigus foliaceus. The study was performed with patients from the Hospital Adhemar de Barros (former "Pemphigus Foliaceus Hospital," Sao Paulo), by Beutner, Prigenzi, Hale, Leme and Bier."^' " Thirty patients were studied, including one with pemphigus vulgaris, one with benign familiar pemphigus of HaileyHailey and 28 with pemphigus foliaceus at different clinical stages. The sera were titrated in 2 separate laboratories: the Department of Microbiology and Parasitology, Escola Paulista de Medicina (Sao Paulo) and the Department of Bacteriology and Immunology, School of Medicine, Buf-falo University. The results were similar. The sera were positive for antiepithelial antibodies in 27 out of 28 patients with pemphigus foliaceus (the negative case was in remission). In order to correlate the titers with the clinical features, patients' cases were grouped into 4 categories: deteriorating, stationary, improving and in remission. The au-
No. 10
'
PEMPHIGUS FOLIACEUS
•
Proenca and Rivitti
v. -
Table 1. Studies of Antiepithelial Antibodies in Sera of Patients with Brazilian Pemphigus Foliaceus
Furtado, et al. Beutner, et al. Rivitti Rivitti, et al. Barbosa, et al.+ Total
Year
Positive
Negative
Total
1967 1967 1972 1973 (1973)
21
6 1
27
0 0
35
. .••
.,
.J
;.
..•
27 3 S
•-••
8
•••'
^
(majority) 91
28
8
(?) •,:
98
* Indirect immunofluorescence tecbnique. t Tbis paper was not publisbed and tbe data is incomplete.
thors found some correlation between the titers of circulating antibodies and the degree of involvement. Fifty-six sera were tested from controls and all of them were negative. None of the pemphigus foliaceus patients had antinuclear antibodies. In 1972, one of us (ER)** defended his doctoral thesis on immunologic features in pemphigus foliaceus, including antiepithelial antibodies. Castro and Camargo collaborated in this paper. Thirty-five patients with pemphigus foliaceus, from the Hospital Adhemar de Barros, were divided in 3 groups, according to the extension of the disease: Grade 1, the benign form of Senear & Usher syndrome; grade 2, with involvement of nearly 50% of the skin surface and grade 3, with more than 50% involvement. The patients were treated with steroids and sera titration was repeated twice during the follow-up. Antiepithelial antibodies were found in all patients and some had very high titers (up to 1/2.700). In this study It was not possible to establish a close relationship between extension of the disease and titers obtained. However, considering the mean value for each group (grade 1, mean value 1/469; grade 2, 1/568; grade 3, 1/776) some correlation was suggested. Although, it was not statistically significant. During the follow-up, titers decreased but did not become negative.
even in the patients whose lesions had cleared. Besides the antiepithelial antibodies, the author studied antistreptolysin-O titers, electrophoresis and immunoelectrophoresis of sera. In 1973, Rivitti, Camargo, Castro and Sampaio'^ published a paper about the treatment of pemphigus foliaceus with aminopterin. Eight patients were treated, 6 with the erythrodermic form and 2 with recurrences and exacerbation of the skin lesions. Titrations were repeated several times in all patients. The values of the titers were extremely irregular, decreasing in a few, remaining stationary in others and rising in 2. The authors concluded that the drug was clinically ineffective and did not influence the titers of the antiepithelial antibodies. Another Brazilian study was performed by Barbosa, Pitaluga, Leite and Souza'" in Goiania (1972-1973) and the results have not yet been published. One hundred pemphigus foliaceus patients from "Hospital of Pemphigus" of Goiania were studied by several immunologic methods. Antiepithelial antibodies were found in the majority of cases. Serum complement was also studied, and found low in the majority. Circulating antigens for rheumatoid factor were negative. Finally, let us discuss the passive induction of acantholytic bullae in animals.
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Table 2. Titers of Antiepithelial Antibodies in Brazilian Pemphigus Foliaceus No.
Severity of Case Deteriorating Stationary Improving In remission
Titers 160
80
—
—
—
4
—
—
1*
3
1
1
—
2560
1280
640
320
2
2
—
—
14
3
6
10
3
2
Patients
10
10
1
2
This titer presented problems of interpretation. When repeated, it showed an essential rise.
Only as a curiosity. It is noteworthy that in 1937 Lindenberg" for the first time "searched for a virus as a possible etiologic agent of the disease." He believed that he could induce intraepidermal bullae in rabbit skin. However, these studies were not pursued further. In 1971, Beutner, Wood, Chorzelski, Leme and Bier'- succeeded in producing acantholytic bullae in rabbits with sera from pemphigus foliaceus patients of the "Hospital Adhemar de Barros." In monkeys, repeated injections in the same area were necessary to include microscopic bullae. The most recent and interesting survey with regard to antiepithelial antibodies was done by Castro, Chorzelski, Jablonska and Marquart'-' (1975). They studied sera of healthy people living in endemic areas; these data will be discussed later. Pathogenic Significance The autoantibodies in Brazilian pemphigus foliaceus are directed against the
intercellular spaces of the malpighian layer, exactly in the region where acantholysis takes place, as observed by optical microscopy. Barros'-" studied the ultrastructural changes in 4 patients with pemphigus foliaceus and concluded that the desmosomes were altered. It is interesting to compare this observation with that of Hashimoto and Lever,'-' who found alterations in the cement substance of the desmosomes in pemphigus vulgaris. The antiepithelial antibodies were found in the sera of almost all pemphigus foliaceus patients as shown in Table 1. Rarely, the test was negative, as in one patient of Beutner et al.,'^' in whom the illness was inactive and 6 patients of Furtado et al.,'* who were in therapeutic remission due to corticoid administration. An incomplete study by Barbosa et al. reported to one of the authors (NGP) showed that the great majority of patients had circulating antiepithelial antibodies. It should be pointed out that this group worked with rabbit esophagus as sub-
Table 3. Titers of Antiepithelial Antibodies in Brazilian Pemphigus Foliaceus'' Extension of lesions Extensive Moderate Slight No lesions
Titers
No. Cases
80
40
20
6
6
—
—
9
2
3
4
6
—
—
1
6
10
Negatives
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PEMPHIGUS FOLIACEUS
strate. Beutner et al.'^ compared titrations with monkey and rabbit esophagus and concluded that the latter were lower. It cannot be conclusively stated that the chosen substrate would have influenced the results of Barbosa et al., but it is a possibility to be considered. Beutner et al.'' studied a control group of 56 sera from healthy individuals and from individuals suffering from other conditions (Hailey-Hailey pemphigus, burns, dermatitis herpetiformis, erythema multiforme, other bullous diseases, systhemic lupus erythematosus and other dermatoses). Furtado et al.'' used, as a control group, 20 sera from normal individuals. In the other studies no control groups were studied. The presence of antibodies similar to those of pemphigus in other dermatosis already has been discussed. The relationship between clinical severity and titers of serum antibodies has been a controversial subject. Since the authors used different criteria to establish the stage of the illness, it has not been possible to compare the results in the same table. We prefer to reproduce the tables of the original papers (Tables 2, 3 and 4). Table 2 shows a reasonable relationship between severity and titers. In Table 3 the same correlation is even more evident. There is a great difference between the titers obtained by Beutner et al." (maximum 1 : 2560) and those obtained by Furtado et al.'-* (maximum 1 : 80). It Table 5.
Proenca and Rivitti
Table 4. T/(ers of Antiepithelian Antibodies in Brazilian Pemphigus Foliaceus^ G r a d e III
Grade I Grade II More tban Preinvasive 50% of skin 50% of skin pbase involvement involvement No. cases Mean titers
25 1/568
6 1/776
Induction of Acantholytic Bullae in Rabbits with Pemphigus Foliaceus Sera'^Titers of AB
No. of injected areas
1
1280 640-2560 0
Controls
4 1/469
is likely that differences in techniques account for these discrepancies. Rivitti*^ showed a correlation between the severity of the disease and the titers of antibodies only when mean values were analyzed (Table 4). When the cases were analyzed individually, this correlation could not be established. In the paper of Rivitti et al.,'^ all patients had extensive involvement and the titers were uniformly high. It should be noted that these studies showed some correlation between the clinical features and the serum antibodies, but definite conclusions are not yet possible. Beutner et al.'-' '^ tried to induce lesions of pemphigus in rabbits and monkeys using sera from pemphigus foliaceus patients with high titers of intercellular antiepithelium antibodies. The sera obtained from patients with acute cases were sterilized, filtered and maintained frozen or lyophilized at minus 20°C. Rabbits received intradermically 0.05 to 0.2 ml of serum (undiluted or diluted 1 : 2)
Injected sera
2-4
803
' Antibodies. tDirect immunofluorescence.
Ab*-fixation
Acantbolytic microbullae
99
53/99
38
22/38 0/72
5/53 8/22
72
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INTERNATIONAL JOURNAL OF DERMATOLOGY
804 Table 6.
Injected sera
Vol. 16
Induction of Acantholytic Bullae in Monkeys with Pemphigus Foliaceus
Titers of Ab Esophagus Skin 5120
1280
Ci
December 1977
Istinjection MicroFixation bullae Ab 4/4
2nd injection MicroFixation bullae Ab
3rd injection Fixation MicroAb bullae
1/4
6/8
4/6
4/4
3/4
0/12 0/11
—
—
5/5
4/5
Ro
320
1280
Tr
320
640
12/22 11/11
—
—
•4/5
3/4
Sia
320
320
2/3
0/2
4/4
0/4
2/4
1/4
1/1
0/1
4/4
0/4
7/7
1/7
0/1
0/0
2/4
0/2
3/5
2/3
3/3
0/3
8/11
0/8
3/3
1/13
33/45
1/33
0/8
0/8
24/31 0/11
28/35 0/61
15/28 0/61
' "
Am
160
2560 1280
pool of 5 sera
40
160
160
640
Li
Total No. Controls
and immediately afterward the injected areas were treated with dinitrochlorobenzene ( 1 % and 2)% or CO'. Rhesus monkeys received 0.2 ml of undiluted serum injected intradermically once, twice or three times in the same area within a time interval of 3 or 4 hours. Results were studied by biopsy and by direct immunofluorescence (Tables 5 and 6). The number of acantholytic bullae obtained was small, even considering the animals in which the injected antibodies led to binding. In rabbits, even with the application of dinitrochlorobenzene, fixation of antiTable 7.
Titers Obtained in Healthy People Living in Endemic Areas
Case no.
Titer
1
1: 320 1: 40 1: 20 1: 10 1: 160 1:1280 1: 320
2 3 4 5 6 7
Relationship to patient Son Son
Daughter and sister Son Son
Friend Husband
4/24 0/11
bodies with serum No. 1 was observed in about 54% and microbullae in about 5%. With sera No. 2 and 4, the fixation of antibodies was about 57% and microbullae developed in about 2 1 % of the animals. In monkeys with one injection, about 73% of antibody fixation and 3% of microbullae were obtained; with 2 injections, 77% of antibody fixation and 16% of microbullae, and with 3 injections about 80% of antibody fixation and 53% of microbullae were noted. It is noteworthy that while antibody fixation in the injected areas was high, the occurrence of acantholytic bullae was relatively low. This means that although the fixation of passively transferred antibodies occurs, it is not sufficient, in a great number of cases, to induce the formation of acantholytic bullae. Furthermore, some doubts remain as to the role of trauma plus antibodies in the development of the blister. Finally with regard to Brazilian pemphigus foliaceus sera, the question of an infectious agent remains open. Even after sterilization of the sera, it is not possible to exclude the possibility of the introduction of another agent, besides antibodies.
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PEMPHIGUS FOLIACEUS
which could be responsible for the bullae formation. Castro and Proen^ja'^' '^ believe that "fogo selvagem" is an infectious disease caused by a still unidentified microorganism and transmitted by a winged vector. In this regard, Castro, Chorzelski, Jablonska and Marquart'-' studied antiepithelial antibodies in healthy people living in endemic areas. The fogo selvagem patients were brought to the hospital by relatives or friends owing to the seriousness of the disease. Serum samples were taken from all patients, and no distinction was made between patients who never had had treatment and those who had had a relapse. At the same time, blood was also obtained from the relatives and friends who had brought the patients to the hospital. The sera were studied in the Hospital do Penfigo, in Sao Paulo and in Hospital do Penfigo, Campo Grande, Mato Grosso, Central Brazil. In both hospitals, 61 serum samples were collected. Seventeen were from patients and 43 from relatives and/or friends of the patients. The samples obtained were sent to Warsaw, where they were analyzed by indirect immunofluorescence (Table 7). This study opens new avenues for epidemiological investigations in endemic areas. Conclusions The role of intercellular antibodies in Brazilian pemphigus foliaceus (fogo selvagem —^"wild fire") was studied. It was found that antiepithelial antibodies are located in the intercellular spaces of the epidermis, where intraepidermal bullae with acantholytic cells develop. The antibodies are specific and present in almost all patients with active Brazilian pemphigus foliaceus. A few negative results were seen in patients who were recovering from the illness. Patients with other der-
Proenca and Rivitti
805
matoses and healthy controls did not have antibodies in their sera. It was possible to establish some correlation between the titers of antibodies and the extension of skin involvement in the majority of patients. Acantholytic intraepidermic buliae were induced using sera of patients with Brazilian pemphigus foliceus. These findings are still under investigation. Antiepithelial antibodies were found in healthy people living indemic areas of fogo selvagem. This finding opens new avenues for research in this interesting disease. References 1. Coons, A. H., Creecb, H. ]., and Jones, R. N.: Immunological properties of an antibody containing a Fluorescent group. Proc. Soc. Exper. Biol. Med. 47:200, 1941. 2. Beutner, E. H., and Jordan, R. E.: Demonstration of skin antibodies in sera of pempbigus vulgaris patients by indirect immunofluorescence staining. Proc. Soc. Exper. Biol. Med. 117:505, 1964. 3. Cborzelski, T. P., Jablonska, S., and Beutner, E. H.: Clinical significance of Pempbigus antibodies. In Immunopatbology of the Skin. Edited by Dowden. Stroudsburg, PA, Hutcbinson & Ross Inc., 1973. 4. Furtado, T. A., Lima, A. O., Andrade, G. O., and Seabra, O.: Auto antibodies in Pempbigus Eoliaceus. XIII Congressus Internationalis Dermatologie (Muncben) New York, Springer-Verlag, V2, p. 1159. 5. Leme, C. A.: Penfigo foliaceo no Brasil, doenga de auto-agressao. Demonstragao de auto-anticorpo por imunofluorescencia. Rev. Med. Juiz de Fora, 13:1016, 1967. 6. Beutner, E. H., Prigenzi, L. S., Hale, W., Leme, C. A., and Bier, O. G.: Immunofluorescent studies of autoantibodies to intercellular areas of epitbelia in Brazilian Pemphigus Foliaceus. Proc. Soc. Exper. Biol. 127:81, 1968. 7. Leme, C. A.: Penfigo foliaceo no Brasil, doenga de auto-agressao. Demonstragao de auto-anticorpo por imunofluorescencia. O Hospital 76:583, 1969. 8. Rivitti, E. A.: Eletroforese, Imunoeletroforese, Antiestreptolisina-O e Anticorpos anti-epitelio, no penfigo foliaceo aul-amaricano. Estudo evolutivo. Tese de Doutoramento (Faculdade de Medicina da Universidade de Sao Paulo) 1972.
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INTERNATIONAL JOURNAL OF DERMATOLOGY
9. Rivitti, E. A., Camargo, M. E., Castro, R. M., and Sampaio,, S. A. P.: Use of methotrexate to treat pemphigus foliaceus. Int. J. Dermatol. 12:119, 1973. 10. Barbosa, W., Pitaluga, W., Leite, M., and Souza, O. C : Personal communication, 1973. 11. LIndenberg, A.: Zur Atiologie der Pemphiguskrankheiten. Klin. Wochenschr. 16:1577, 1937. 12. Beutner, E. H., Wood, G. W., Chorzelski, T. P., Leme, C. A. and Bier, O. G.: Produgao de lesoes semelhantes as do pefigo foliaceo pela injecao intradermica, em coelhos e macacos, de soros de doentes com tftulo elevado de auto-anticorpo. Mem. Inst. Butantan. 35:79, 1971. 13. Castro, R. M., Chorzelski, T., Jablonska, S., and Marquart Filho, A.: Antiepithelial antibodies in healthy people living in endemic area of South American pemphigus foliaceus (fogo selvagem, wild fire). Accepted for pub-
14.
15.
16.
17. 18.
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Vol. 16
lication at the Rev. Inst. Med. Trop. de Sao Paulo. Barros, C : Ultraestrutura da lesao bolhosa e do sinal de Nikolski no penfigo foliaceo. Tese de Doutoramento, Faculdade de Medicina da Universidade de Sao Paulo, 1972. Hashimoto, K., and Lever, W. F.: The intercellular cement in pemphigus vulgaris, an electron microscopic study. Dermatologica 135:27, 1967. Wood, G. W., Beutner, E. H., and Chorzelski, T. P.: Studies in immunodermatology: II Production of pemphigus-like lesions by intradermical injection of monkeys with Brazilian pemphigus foliaceus sera. Int. Arch. Allergy, 42:456, 1972. Castro, R. M., and Proen^a, N.: South American pemphigus foliaceus. The Glaxo 36:17, 1972. Proenga, N., and Castro, R. M.: Penfigo foliaceo sul-americano. Rev. Hosp. Clin. Fac. Med. Sao Paulo, 26:115, 1971.
Changes in Medicine These demographic changes and other results of medical science have obviously played a very large role in the character of American life through increasing population density, urbanization, family attitudes, personal attitudes toward sickness, and so on. They have brought social problems, such as the greater attention that must be given to care of the elderly. They have brought pressing ethical problems, from abortion to the need for a new definition of death. The dramatic successes of modern medicinals and the spectacular achievements of modern surgery have also brought rising public expectations and demands for health care, further fostered, one must suspect, by sometimes extravagant claims or overly publicized triumphs put forth by Madison Avenue-style imagemakers seeking to raise money or increase profits. Perhaps if the public had not been so saturated with information about "miracle" drugs, the feeling might not be as widespread as it seems to be that if the patient is not cured, the doctor is necessarily at fault and subject to suit for malpractice. More importantly, rising expectations have brought demands for more service, better service, but at the same time less costly service, demands which have inevitably made medicine during the last few decades one of the persistent concerns of American politics and government.—Blake, J. B.: The Bicentennial of Medicine in The United States: A Commentary. Essays at the Bicentennial of Medicine in The United States. U.S. Department of Health, Education and Welfare. Bethesda, National Library of Medicine, 1976, p. 6.
ANTIEPITHELIAL ANTIBODIES IN BRAZILIAN PEMPHIGUS EOLIACEUS NELSON GUIMARAES PROENCA, M.D. AND'EVANDRO RIVITTI, M.D. - r ,;r From the Department of Dermatology, Faculdade de Ciencias Medicas de Santa Casa Sao Paulo e Faculdade de Medicina da de Sao Paulo, Sao Paulo, Brazil
In 1941, techniques using fluorescent antibodies to locate antigens were used for the first time by Coons, Crech and Jones.' From then on several technical improvements were introduced and lately immunofluorescence has acquired a very important role in dermatology. It has proven to be useful both as an aid for diagnosis and to investigation of a great variety of diseases.
intercellular spaces of the prickle cell layer. 2. The antibodies arc specific and appear during the development of the pemphigus. In all patients with other diseases used as control, no antibodies were found. 3. It was possible to correlate clinical aspects with the titration. In pemphigus vulgaris patients, titers were proportional to the severity of the disease and in pemphigus foliaceus patients, to the extension of the lesion. Moreover, when titrations were repeated during the patient's recovery. It was possible to register a steady fall of titers.
With regard to the bullous dermatosis, a new phase had begun in which unknown pathogenic features were disclosed. Intercellular antiepithelial antibodies in pemphigus vulgaris were demonstrated initially by Beutner and Jordanin 1964. In spite of the increasing spread of knowledge about the immunology of bullous diseases, there is one basic point of divergency. It deals with the probable pathogenic role of the pemphigus antiepithelial antibodies: Do they participate in the onset of the acantholytic bullae or do they appear as a response to the disease? Chorzelski, Jablonska and Beutner' favor the first hypothesis, considering the role of the antiepithelial antibodies as a pathogenic one. Six arguments were set up in support of their opinion:
4. In pemphigus vulgaris patients, repeated titrations detected serologic recurrences preceding clinical recurrences. 5. With the technique of direct immunofluorescence, it was possible to show the in vivo fixation of IgG in the intercellular spaces of the epidermis. 6. The most convincing demonstration of the pathogenic role of the antiepithelial antibodies would be the production of acantholytic bullae with the passive transference of antibodies. This was perforniecl on rabbits and monkeys in an experinient that will be mentioned later.
1. The site where the antibodies are demonstrated with the immunofluorescence technique is exactly the same as the acantholytic process occurring in the pemphigus patient. It is confined to the
799
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INTERNATIONAL JOURNAL OF DERMATOLOGY
Chorzelski, Jablonska and Beutner considered these six arguments as a real proof of the primary role of antiepithelial antibodies in the inducement of the acantholytic bullae. Nevertheless, it should be stressed that some of the presenting features were not demonstrated in all groups of patients. It was not possible to induce acantholytic bullae using sera from pemphigus vulgaris patients. However, successful experiments were performed with sera from patients with Brazilian pemphigus foliaceus "fogo selvagen"—wild fire. On the other hand, serologic recurrences preceding clinical recurrences were observed only in pemphigus vulgaris and not in Brazilian pemphigus foliaceus patients. These studies should stimulate further investigation on pemphigus vulgaris, pemphigus vegetans, pemphigus foliaceus, Brazilian pemphigus foliaceus and pemphigus erythematosus (SenearUsher syndrome). Even in diseases in which antibodies similar to pemphigus were found, such studies would be of interest. We wish to review the Brazilian contributions in the field of immunopathology of pemphigus. They are of special importance because they were performed in pemphigus patients with the endemic form, such as occurs in Brazil. We will analyze the papers published by different investigators including the so-called "postulates" of Chorzelski, Jablonska and Beutner.'' Brazilian Contributions In August 1967, Furtado, Oliveira Lima, Andrade and Seabra'' presented at the XIII International Congress of Dermatology (Munich) their investigation on antibodies in pemphigus foliaceus. The authors studied with the indirect immunofluorescence technique sera from 27 patients and from 20 healthy persons as
December 1977
Vol. 16
controls. They divided their patients according to the degree of skin involvement: severe, moderate, slight and patients without lesions. The first 3 groups consisted of 21 patients and all of them showed positive results. Titers were related to the severity of the disease. In patients without lesions and in the healthy volunteers, the sera were negative. Antibodies against the dermoepidermal junction were not found. However, antibodies against cytoplasm were found twice. Other immunological investigations were negative (hemagglutinin, complement binding, immunoglobulins, agar gel diffusion and passive cutaneous anaphylaxis). This work was partially presented at the XXIV Brazilian Congress of Dermatology in Juiz de Fora (Minas Gerais) by Furtado et al.-' In October 1976, Leme^; presented at the same Congress his results on indirect immunofluorescence applied to Brazilian pemphigus foliaceus. The study was performed with patients from the Hospital Adhemar de Barros (former "Pemphigus Foliaceus Hospital," Sao Paulo), by Beutner, Prigenzi, Hale, Leme and Bier."^' " Thirty patients were studied, including one with pemphigus vulgaris, one with benign familiar pemphigus of HaileyHailey and 28 with pemphigus foliaceus at different clinical stages. The sera were titrated in 2 separate laboratories: the Department of Microbiology and Parasitology, Escola Paulista de Medicina (Sao Paulo) and the Department of Bacteriology and Immunology, School of Medicine, Buf-falo University. The results were similar. The sera were positive for antiepithelial antibodies in 27 out of 28 patients with pemphigus foliaceus (the negative case was in remission). In order to correlate the titers with the clinical features, patients' cases were grouped into 4 categories: deteriorating, stationary, improving and in remission. The au-
No. 10
'
PEMPHIGUS FOLIACEUS
•
Proenca and Rivitti
v. -
Table 1. Studies of Antiepithelial Antibodies in Sera of Patients with Brazilian Pemphigus Foliaceus
Furtado, et al. Beutner, et al. Rivitti Rivitti, et al. Barbosa, et al.+ Total
Year
Positive
Negative
Total
1967 1967 1972 1973 (1973)
21
6 1
27
0 0
35
. .••
.,
.J
;.
..•
27 3 S
•-••
8
•••'
^
(majority) 91
28
8
(?) •,:
98
* Indirect immunofluorescence tecbnique. t Tbis paper was not publisbed and tbe data is incomplete.
thors found some correlation between the titers of circulating antibodies and the degree of involvement. Fifty-six sera were tested from controls and all of them were negative. None of the pemphigus foliaceus patients had antinuclear antibodies. In 1972, one of us (ER)** defended his doctoral thesis on immunologic features in pemphigus foliaceus, including antiepithelial antibodies. Castro and Camargo collaborated in this paper. Thirty-five patients with pemphigus foliaceus, from the Hospital Adhemar de Barros, were divided in 3 groups, according to the extension of the disease: Grade 1, the benign form of Senear & Usher syndrome; grade 2, with involvement of nearly 50% of the skin surface and grade 3, with more than 50% involvement. The patients were treated with steroids and sera titration was repeated twice during the follow-up. Antiepithelial antibodies were found in all patients and some had very high titers (up to 1/2.700). In this study It was not possible to establish a close relationship between extension of the disease and titers obtained. However, considering the mean value for each group (grade 1, mean value 1/469; grade 2, 1/568; grade 3, 1/776) some correlation was suggested. Although, it was not statistically significant. During the follow-up, titers decreased but did not become negative.
even in the patients whose lesions had cleared. Besides the antiepithelial antibodies, the author studied antistreptolysin-O titers, electrophoresis and immunoelectrophoresis of sera. In 1973, Rivitti, Camargo, Castro and Sampaio'^ published a paper about the treatment of pemphigus foliaceus with aminopterin. Eight patients were treated, 6 with the erythrodermic form and 2 with recurrences and exacerbation of the skin lesions. Titrations were repeated several times in all patients. The values of the titers were extremely irregular, decreasing in a few, remaining stationary in others and rising in 2. The authors concluded that the drug was clinically ineffective and did not influence the titers of the antiepithelial antibodies. Another Brazilian study was performed by Barbosa, Pitaluga, Leite and Souza'" in Goiania (1972-1973) and the results have not yet been published. One hundred pemphigus foliaceus patients from "Hospital of Pemphigus" of Goiania were studied by several immunologic methods. Antiepithelial antibodies were found in the majority of cases. Serum complement was also studied, and found low in the majority. Circulating antigens for rheumatoid factor were negative. Finally, let us discuss the passive induction of acantholytic bullae in animals.
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INTERNATIONAL JOURNAL OF DERMATOLOGY
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Table 2. Titers of Antiepithelial Antibodies in Brazilian Pemphigus Foliaceus No.
Severity of Case Deteriorating Stationary Improving In remission
Titers 160
80
—
—
—
4
—
—
1*
3
1
1
—
2560
1280
640
320
2
2
—
—
14
3
6
10
3
2
Patients
10
10
1
2
This titer presented problems of interpretation. When repeated, it showed an essential rise.
Only as a curiosity. It is noteworthy that in 1937 Lindenberg" for the first time "searched for a virus as a possible etiologic agent of the disease." He believed that he could induce intraepidermal bullae in rabbit skin. However, these studies were not pursued further. In 1971, Beutner, Wood, Chorzelski, Leme and Bier'- succeeded in producing acantholytic bullae in rabbits with sera from pemphigus foliaceus patients of the "Hospital Adhemar de Barros." In monkeys, repeated injections in the same area were necessary to include microscopic bullae. The most recent and interesting survey with regard to antiepithelial antibodies was done by Castro, Chorzelski, Jablonska and Marquart'-' (1975). They studied sera of healthy people living in endemic areas; these data will be discussed later. Pathogenic Significance The autoantibodies in Brazilian pemphigus foliaceus are directed against the
intercellular spaces of the malpighian layer, exactly in the region where acantholysis takes place, as observed by optical microscopy. Barros'-" studied the ultrastructural changes in 4 patients with pemphigus foliaceus and concluded that the desmosomes were altered. It is interesting to compare this observation with that of Hashimoto and Lever,'-' who found alterations in the cement substance of the desmosomes in pemphigus vulgaris. The antiepithelial antibodies were found in the sera of almost all pemphigus foliaceus patients as shown in Table 1. Rarely, the test was negative, as in one patient of Beutner et al.,'^' in whom the illness was inactive and 6 patients of Furtado et al.,'* who were in therapeutic remission due to corticoid administration. An incomplete study by Barbosa et al. reported to one of the authors (NGP) showed that the great majority of patients had circulating antiepithelial antibodies. It should be pointed out that this group worked with rabbit esophagus as sub-
Table 3. Titers of Antiepithelial Antibodies in Brazilian Pemphigus Foliaceus'' Extension of lesions Extensive Moderate Slight No lesions
Titers
No. Cases
80
40
20
6
6
—
—
9
2
3
4
6
—
—
1
6
10
Negatives
No. 10
PEMPHIGUS FOLIACEUS
strate. Beutner et al.'^ compared titrations with monkey and rabbit esophagus and concluded that the latter were lower. It cannot be conclusively stated that the chosen substrate would have influenced the results of Barbosa et al., but it is a possibility to be considered. Beutner et al.'' studied a control group of 56 sera from healthy individuals and from individuals suffering from other conditions (Hailey-Hailey pemphigus, burns, dermatitis herpetiformis, erythema multiforme, other bullous diseases, systhemic lupus erythematosus and other dermatoses). Furtado et al.'' used, as a control group, 20 sera from normal individuals. In the other studies no control groups were studied. The presence of antibodies similar to those of pemphigus in other dermatosis already has been discussed. The relationship between clinical severity and titers of serum antibodies has been a controversial subject. Since the authors used different criteria to establish the stage of the illness, it has not been possible to compare the results in the same table. We prefer to reproduce the tables of the original papers (Tables 2, 3 and 4). Table 2 shows a reasonable relationship between severity and titers. In Table 3 the same correlation is even more evident. There is a great difference between the titers obtained by Beutner et al." (maximum 1 : 2560) and those obtained by Furtado et al.'-* (maximum 1 : 80). It Table 5.
Proenca and Rivitti
Table 4. T/(ers of Antiepithelian Antibodies in Brazilian Pemphigus Foliaceus^ G r a d e III
Grade I Grade II More tban Preinvasive 50% of skin 50% of skin pbase involvement involvement No. cases Mean titers
25 1/568
6 1/776
Induction of Acantholytic Bullae in Rabbits with Pemphigus Foliaceus Sera'^Titers of AB
No. of injected areas
1
1280 640-2560 0
Controls
4 1/469
is likely that differences in techniques account for these discrepancies. Rivitti*^ showed a correlation between the severity of the disease and the titers of antibodies only when mean values were analyzed (Table 4). When the cases were analyzed individually, this correlation could not be established. In the paper of Rivitti et al.,'^ all patients had extensive involvement and the titers were uniformly high. It should be noted that these studies showed some correlation between the clinical features and the serum antibodies, but definite conclusions are not yet possible. Beutner et al.'-' '^ tried to induce lesions of pemphigus in rabbits and monkeys using sera from pemphigus foliaceus patients with high titers of intercellular antiepithelium antibodies. The sera obtained from patients with acute cases were sterilized, filtered and maintained frozen or lyophilized at minus 20°C. Rabbits received intradermically 0.05 to 0.2 ml of serum (undiluted or diluted 1 : 2)
Injected sera
2-4
803
' Antibodies. tDirect immunofluorescence.
Ab*-fixation
Acantbolytic microbullae
99
53/99
38
22/38 0/72
5/53 8/22
72
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INTERNATIONAL JOURNAL OF DERMATOLOGY
804 Table 6.
Injected sera
Vol. 16
Induction of Acantholytic Bullae in Monkeys with Pemphigus Foliaceus
Titers of Ab Esophagus Skin 5120
1280
Ci
December 1977
Istinjection MicroFixation bullae Ab 4/4
2nd injection MicroFixation bullae Ab
3rd injection Fixation MicroAb bullae
1/4
6/8
4/6
4/4
3/4
0/12 0/11
—
—
5/5
4/5
Ro
320
1280
Tr
320
640
12/22 11/11
—
—
•4/5
3/4
Sia
320
320
2/3
0/2
4/4
0/4
2/4
1/4
1/1
0/1
4/4
0/4
7/7
1/7
0/1
0/0
2/4
0/2
3/5
2/3
3/3
0/3
8/11
0/8
3/3
1/13
33/45
1/33
0/8
0/8
24/31 0/11
28/35 0/61
15/28 0/61
' "
Am
160
2560 1280
pool of 5 sera
40
160
160
640
Li
Total No. Controls
and immediately afterward the injected areas were treated with dinitrochlorobenzene ( 1 % and 2)% or CO'. Rhesus monkeys received 0.2 ml of undiluted serum injected intradermically once, twice or three times in the same area within a time interval of 3 or 4 hours. Results were studied by biopsy and by direct immunofluorescence (Tables 5 and 6). The number of acantholytic bullae obtained was small, even considering the animals in which the injected antibodies led to binding. In rabbits, even with the application of dinitrochlorobenzene, fixation of antiTable 7.
Titers Obtained in Healthy People Living in Endemic Areas
Case no.
Titer
1
1: 320 1: 40 1: 20 1: 10 1: 160 1:1280 1: 320
2 3 4 5 6 7
Relationship to patient Son Son
Daughter and sister Son Son
Friend Husband
4/24 0/11
bodies with serum No. 1 was observed in about 54% and microbullae in about 5%. With sera No. 2 and 4, the fixation of antibodies was about 57% and microbullae developed in about 2 1 % of the animals. In monkeys with one injection, about 73% of antibody fixation and 3% of microbullae were obtained; with 2 injections, 77% of antibody fixation and 16% of microbullae, and with 3 injections about 80% of antibody fixation and 53% of microbullae were noted. It is noteworthy that while antibody fixation in the injected areas was high, the occurrence of acantholytic bullae was relatively low. This means that although the fixation of passively transferred antibodies occurs, it is not sufficient, in a great number of cases, to induce the formation of acantholytic bullae. Furthermore, some doubts remain as to the role of trauma plus antibodies in the development of the blister. Finally with regard to Brazilian pemphigus foliaceus sera, the question of an infectious agent remains open. Even after sterilization of the sera, it is not possible to exclude the possibility of the introduction of another agent, besides antibodies.
No. 10
PEMPHIGUS FOLIACEUS
which could be responsible for the bullae formation. Castro and Proen^ja'^' '^ believe that "fogo selvagem" is an infectious disease caused by a still unidentified microorganism and transmitted by a winged vector. In this regard, Castro, Chorzelski, Jablonska and Marquart'-' studied antiepithelial antibodies in healthy people living in endemic areas. The fogo selvagem patients were brought to the hospital by relatives or friends owing to the seriousness of the disease. Serum samples were taken from all patients, and no distinction was made between patients who never had had treatment and those who had had a relapse. At the same time, blood was also obtained from the relatives and friends who had brought the patients to the hospital. The sera were studied in the Hospital do Penfigo, in Sao Paulo and in Hospital do Penfigo, Campo Grande, Mato Grosso, Central Brazil. In both hospitals, 61 serum samples were collected. Seventeen were from patients and 43 from relatives and/or friends of the patients. The samples obtained were sent to Warsaw, where they were analyzed by indirect immunofluorescence (Table 7). This study opens new avenues for epidemiological investigations in endemic areas. Conclusions The role of intercellular antibodies in Brazilian pemphigus foliaceus (fogo selvagem —^"wild fire") was studied. It was found that antiepithelial antibodies are located in the intercellular spaces of the epidermis, where intraepidermal bullae with acantholytic cells develop. The antibodies are specific and present in almost all patients with active Brazilian pemphigus foliaceus. A few negative results were seen in patients who were recovering from the illness. Patients with other der-
Proenca and Rivitti
805
matoses and healthy controls did not have antibodies in their sera. It was possible to establish some correlation between the titers of antibodies and the extension of skin involvement in the majority of patients. Acantholytic intraepidermic buliae were induced using sera of patients with Brazilian pemphigus foliceus. These findings are still under investigation. Antiepithelial antibodies were found in healthy people living indemic areas of fogo selvagem. This finding opens new avenues for research in this interesting disease. References 1. Coons, A. H., Creecb, H. ]., and Jones, R. N.: Immunological properties of an antibody containing a Fluorescent group. Proc. Soc. Exper. Biol. Med. 47:200, 1941. 2. Beutner, E. H., and Jordan, R. E.: Demonstration of skin antibodies in sera of pempbigus vulgaris patients by indirect immunofluorescence staining. Proc. Soc. Exper. Biol. Med. 117:505, 1964. 3. Cborzelski, T. P., Jablonska, S., and Beutner, E. H.: Clinical significance of Pempbigus antibodies. In Immunopatbology of the Skin. Edited by Dowden. Stroudsburg, PA, Hutcbinson & Ross Inc., 1973. 4. Furtado, T. A., Lima, A. O., Andrade, G. O., and Seabra, O.: Auto antibodies in Pempbigus Eoliaceus. XIII Congressus Internationalis Dermatologie (Muncben) New York, Springer-Verlag, V2, p. 1159. 5. Leme, C. A.: Penfigo foliaceo no Brasil, doenga de auto-agressao. Demonstragao de auto-anticorpo por imunofluorescencia. Rev. Med. Juiz de Fora, 13:1016, 1967. 6. Beutner, E. H., Prigenzi, L. S., Hale, W., Leme, C. A., and Bier, O. G.: Immunofluorescent studies of autoantibodies to intercellular areas of epitbelia in Brazilian Pemphigus Foliaceus. Proc. Soc. Exper. Biol. 127:81, 1968. 7. Leme, C. A.: Penfigo foliaceo no Brasil, doenga de auto-agressao. Demonstragao de auto-anticorpo por imunofluorescencia. O Hospital 76:583, 1969. 8. Rivitti, E. A.: Eletroforese, Imunoeletroforese, Antiestreptolisina-O e Anticorpos anti-epitelio, no penfigo foliaceo aul-amaricano. Estudo evolutivo. Tese de Doutoramento (Faculdade de Medicina da Universidade de Sao Paulo) 1972.
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INTERNATIONAL JOURNAL OF DERMATOLOGY
9. Rivitti, E. A., Camargo, M. E., Castro, R. M., and Sampaio,, S. A. P.: Use of methotrexate to treat pemphigus foliaceus. Int. J. Dermatol. 12:119, 1973. 10. Barbosa, W., Pitaluga, W., Leite, M., and Souza, O. C : Personal communication, 1973. 11. LIndenberg, A.: Zur Atiologie der Pemphiguskrankheiten. Klin. Wochenschr. 16:1577, 1937. 12. Beutner, E. H., Wood, G. W., Chorzelski, T. P., Leme, C. A. and Bier, O. G.: Produgao de lesoes semelhantes as do pefigo foliaceo pela injecao intradermica, em coelhos e macacos, de soros de doentes com tftulo elevado de auto-anticorpo. Mem. Inst. Butantan. 35:79, 1971. 13. Castro, R. M., Chorzelski, T., Jablonska, S., and Marquart Filho, A.: Antiepithelial antibodies in healthy people living in endemic area of South American pemphigus foliaceus (fogo selvagem, wild fire). Accepted for pub-
14.
15.
16.
17. 18.
December 1977
Vol. 16
lication at the Rev. Inst. Med. Trop. de Sao Paulo. Barros, C : Ultraestrutura da lesao bolhosa e do sinal de Nikolski no penfigo foliaceo. Tese de Doutoramento, Faculdade de Medicina da Universidade de Sao Paulo, 1972. Hashimoto, K., and Lever, W. F.: The intercellular cement in pemphigus vulgaris, an electron microscopic study. Dermatologica 135:27, 1967. Wood, G. W., Beutner, E. H., and Chorzelski, T. P.: Studies in immunodermatology: II Production of pemphigus-like lesions by intradermical injection of monkeys with Brazilian pemphigus foliaceus sera. Int. Arch. Allergy, 42:456, 1972. Castro, R. M., and Proen^a, N.: South American pemphigus foliaceus. The Glaxo 36:17, 1972. Proenga, N., and Castro, R. M.: Penfigo foliaceo sul-americano. Rev. Hosp. Clin. Fac. Med. Sao Paulo, 26:115, 1971.
Changes in Medicine These demographic changes and other results of medical science have obviously played a very large role in the character of American life through increasing population density, urbanization, family attitudes, personal attitudes toward sickness, and so on. They have brought social problems, such as the greater attention that must be given to care of the elderly. They have brought pressing ethical problems, from abortion to the need for a new definition of death. The dramatic successes of modern medicinals and the spectacular achievements of modern surgery have also brought rising public expectations and demands for health care, further fostered, one must suspect, by sometimes extravagant claims or overly publicized triumphs put forth by Madison Avenue-style imagemakers seeking to raise money or increase profits. Perhaps if the public had not been so saturated with information about "miracle" drugs, the feeling might not be as widespread as it seems to be that if the patient is not cured, the doctor is necessarily at fault and subject to suit for malpractice. More importantly, rising expectations have brought demands for more service, better service, but at the same time less costly service, demands which have inevitably made medicine during the last few decades one of the persistent concerns of American politics and government.—Blake, J. B.: The Bicentennial of Medicine in The United States: A Commentary. Essays at the Bicentennial of Medicine in The United States. U.S. Department of Health, Education and Welfare. Bethesda, National Library of Medicine, 1976, p. 6.
