Archives of

t mat l W.nl

Arch. Derm. Res. 262, 45-53 (1978)

Research

9 Springer-Verlag 1978

Immunofluorescence Studies across the Leprosy Spectrum W. R. Faber and D. L. Leiker From the Department of Dermatology, University of Amsterdam, Binnengasthuis, Amsterdam, The Netherlands

Summary. Forty biopsies from skin lesions of untreated (24) and treated (16) leprosy patients, representing the whole leprosy spectrum, were examined by means of immunofluorescence (IF) methods. Only few or no cells staining with FTC-labelled antihuman immunoglobutin antisera were found in the lesional skin of all patients examined. Sparse IgMdeposits along the basement membrane of the epidermis were observed in a few borderline lepromatous and lepromatous patients. Complement deposits along the basement membrane of the epidermis and in the vessel walls were found in tuberculo]'d as well as in lepromatous patients. Complement and in a lesser number IgG-deposits were observed around sweat glands and sometimes around sweat gland ducts and other skin appendages. Autofluorescing macrophages were noted in patients at the lepromatous side of the leprosy spectrum; approximately half of these patients showed complement deposits in an around these cells. Key words: Leprosy - Immunofluorescence studies - lgM-, IgG-Deposits Complement - Macrophages.

Zusammenfassung.40 Hautbiopsien von unbehandelten (24) und behandelten (16) Leprapatienten, die das ganze Lepraspektrum repr~isentieren, wurden mittels der Immunof/uorescenztechnik (IF) untersucht. Es wurden nur wenige bzw. keine Zellen, die mit FTC-gekoppeltem Anti-Human-Immunoglobulin Antisera fluorescierten gefunden, soweit es die befallene Haut anbetraf. Geringe IgM-Ablagerungen wurden in der dermoepidermalen Junctionszone beobachtet bei borderline-lepromat6sen und lepromat6sen Patienten. Komplementablagerungen wurden in der dermoepidermalen Junctionszone und in den Gef~iBw~inden, sowohl bei tuberkuloider Lepra als auch in lepromat6sen Patienten gefunden. Komplement und in weniger Zahl IgG-Ablagerungen wurden um die Schweil3dr/isen beobachtet und auch im Bereich der Schwei/3driisenausftihrungsg/inge und weiterer Hautadnexe. Autofluorescierende Makrophagen, die zur H/ilfte auch Komplementablagerungen zeigten, wurden bei Patienten an der lepromat6sen Seite des Lepraspek, trums nachgewiesen. Sehliisselwiirter: Lepra - Immunofluorescenz Ablagerungen - Komplement - Makrophagen.

Studien -

lgM-, lgG-

0340-3696/78/0262/0045/$ 1.80

46

W.R. Faber and D. L. Leiker

Previous studies Showed the presence o f immun0glohulin-bearing cells in the peripheral blood in leprosy patients (Gajl-Pr et al,, 1973; N a t h et al., 1974; F a b e r et al., 1976; R e a e t al., 1976). The findings 0.f these studies did not tally. The first aim o f the present study was to find o u t if cells staining with F T C labelled a n t i h u m a n i m m u n o g l o b u l i n antisera are part o f the i n f l a m m a t o r y exudate in skin lesions o f untreated and treated patient~ suffering f r o m various types o f leprosy. Similar investigations were made into various diseases such as for instance dermatitis ( C o r m a n e et al., 1974), psoriasis vu!garis ( C o r m a n e et al., 1976), recurrent a p h t h o u s ulcers (Bays et al., 1977), arthritis ( D e g o t t et al., 1974, 1975), chronic active liver disease (AldershviUe et ~1,, 1 9 7 7 ) and Besniers prurigo ( H o d g k i n s o n et al., 1977). Moreover, I g G and cogaplement (C3) syntheses were determined in cultures f r o m skin lesions o f lepro~sy patients (Lai A F a t and Diesselhof-den Dulk, 1975). In addition, the presence o f i m m u n o g l o b u l i n and c o m p l e m e n t deposits was looked for, since in two studies IgM-deposits along the dermo-epidermal junction o f the affected and apparently unaffected skin o f patients with lepromatous leprosy had been observed (Bullock et al., 1974; Quismorio et al., 1975) as well as along the dermal collagen and elastic fibres in the apparently unaffected lepromatous leprosy skin (Quismorio et al., 1975). O n the other h a n d no mention has been mad~ imtil n o w o f the presence o f I g G and c o m p l e m e n t along sweat glands as well as a r o g n d .other skin appendages n o r of the presence o f autofluorescing macrophages, shQwing c o m p l e m e n t deposits, in patients at the l e p r o m a t o u s side o f the leprosy spectr~N.

Materials and Methods Patients. Forty biopsies from skin lesions of untreated (24) and treate d (16) leprosy patients were examined. The division of the patients according to the type of disease and ~uration of treatment is shown in Table I. Of the untreated patients 2 borderline lepromatous patients had received DDS for a period of 6 and 12 weeks respectively. The drug prescriptions varied, but none of the patients received steroid and/or thalidomide treatment, nor had they recently done so. Patients suffering from erythema nodosum leprosum (ENL) or lepra reaction were excluded from the study. The classification was based on clinical as well as histopathological criteria (Ridley and Jopling, 1966) with minor modifications (Leiker, 1966). Immunofluorescence. Sections (4g) of biopsies from the active part of skin lesions were cut and prepared for IF-studies. The procedures for direct immunofluorescence (DIF) study have been described previously (Cormane et al., 1970) as were those for the detection of complement (van Joost et al., 1972). The sera and conjugates with their characteristics and working dilutions are shown in Table 2. Specificity tests. Sections were treated directly with FTC-labelled goat antirabbit antiserum and also with normal rabbit serum as the first layer. No specific staining was observed. A Leitz orthoplan fluorescence microscope with a vertical illuminator was used, The light source was a Xenon XBO 75 W high pressure lamp. As excitation filter a combination of BG 38 (5 ram), KP 490 (2 ram) and GG 475 (2 ram) was used and as barrier filter OG 515 (3 mm). The objective was a Leitz water immersion objective ( x 50). Enzymehistochemical Study. Enzymehistochemical staining was performed on frozen sections of biopsies from a few treated patients with alpha naphtyl butyrata as a substrate (van Furth, 1976; Ornstein et al., 1976), using semipermeable membranes (Meijer, 1972). Histochemical Study. Ethidiumbromide and acridinorange staining was performed on frozen sections of biopsies from a few treated patients.

Studies Across the Leprosy Spectrum

47

Table l. Untreated and treated patients with various types of leprosy Untreated number

Treated number

(Years approximately) Mean duration

Tuberculo'id Borderline tuberculoi'd Borderline lepromatous Lepromatous

7 4 9 4

Range

3

1 l/~

1/2-2

6 7

111/2 63/4

2-21 2 - 23

Table 2, Characteristics of sera and conjugates. The unconjugated sera directed against human IgG, lgM, IgA, IgD and lgE were prepared in the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service. They were tested for specificity by immunoelectrophoresis, agar block titration and the red cell haemagglutinatiort technique. They were labelled in our laboratory by Dr. S. S. Asghar Ph. D. FTC-content of the conjugates was determined by the method of McKinney et al. (1964), using fluorescein diacetate (FDA) as reference standard. Protein content was measured by the method of Lowry et a1.(1951). Antibody unitage of the conjugates was determined in a standard gel diffusion technique using 1 mg/ml respective antigens except in case of IgD and IgM, where international reference serum and normal serum respectively were used as a source of antigen, against doubling dilution of conjugates as described by Beutner et al. (1968)

Rabbit antihuman IgG/FTC Rabbit antihuman IgM/FTC Rabbit antihuman IgA/FTC Rabbit antihuman IgD/FTC Rabbit antihuman IgE/FTC Rabbit antihuman compL Horse antirabbit Ig/FTC Goat antirabbit IgG/FTC

(RP~HulgG/FTC)" (RAHnlgM/FTC) ~ (RAHulgA/FTC) a (RAHulgD/FTC) ~ (RAHulgE/FTC) ~ (RAHuC) a,c (HAR/FTC) ~ (GAR/FTC) b'd

Batch no.

F/P ratio (~lg/ ml)

Antibody unitage

Working dilution

KH 16-13-A1 KH 15-10-A2 KH 14-13-A02 KH 20-05-A02 KH 25-04-A01 KH 13-12-A03 PK 17-2-F4 7-274

1.51 1.93 1.76 2.03 1.63

4 8 2 2 -

1/40

1/60 1/30 1/60

1/60 1/80 1/60 1/60

a Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam,The Netherlands b Nordic Immunological Laboratories, Tilburg, The Netherlands c Contains antibodies directed against complement components C3c, C3d and C4 d Contains antibodies directed against heavy and light chains of rabbit IgG

Results O n l y a f e w o r n o cells s t a i n i n g w i t h F T C - l a b e l l e d a n t i h u m a n i m m u n o g l o b u l i n a n t i s e r a w e r e o b s e r v e d in the l e s i o n a l skin o f u n t r e a t e d as well as t r e a t e d l e p r o s y patients, representing the whole leprosy spectrum. S p a r s e I g M - d e p o s i t s a l o n g the b a s e m e n t m e m b r a n e o f the e p i d e r m i s were o b s e r v e d in 2 o u t o f 9 u n t r e a t e d b o r d e r l i n e l e p r o m a t o u s , in ! o u t o f 4 u n t r e a t e d l e p r o m a t o u s a n d in 3 o u t o f 7 t r e a t e d l e p r o m a t o u s patients. M o r e o v e r , locally

48

W. R. Faber and D. L. Leiker

Table 3. The presence of IgM and complement in the lesional skin of untreated and treated patients with

various types of leprosy

Tuberculo'id Borderline tuberculo'id Borderline lepromatous Lepromatous

Number

IgM along DEJ

Complement along DEJ

Complement in vessel walls

untreated treated

7 3

0(11gG) 0

4 2

2 2

untreated

4

0

1

1

untreated treated untreated treated

9 6 4 7

2 0 1 3

5 1 1 2

6 2 1 1

DEJ = dermo-epidermaljunction

Table 4. The presence of IgG and complement around skin appendages in the lesional skin of untreated

and treated patients with various types of leprosy IgG

Tuberculo'id Borderline tuberculo'fd Borderline lepromatous Lepromatous

Complement

Sweat glands

Other appendages

Sweat glands

Other appendages

untreated treated

1 (5) 0 (1)

0 (5) 0 (1)

4 (4) 0 (1)

2 (4) 0 (1)

untreated

1 (2)

0 (1)

0 (1)

0 (1)

untreated treated untreated treated

1 (6) 0 (2) 1 (3) 1 (5)

0 (6) 0 (2) 0 (3) 0 (5)

3 (6) 1 (2) i (3) 2 (5)

0 (6) 0 (2) 0 (3) 1 (5)

Number between brackets = number of biopsies in which sweat glands and other skin appendages were observed

d e p o s i t e d I g G a l o n g the d e r m o - e p i d e r m a l j u n c t i o n was o b s e r v e d in 1 u n t r e a t e d t u b e r c u l o i d p a t i e n t ( T a b l e 3). I g M - d e p o s i t s a l o n g the d e r m a l collagen a n d elastic fibres were n o t c o n s p i c i o u s in the biopsies examined. C o m p l e m e n t d e p o s i t s were o b s e r v e d a l o n g the d e r m o - e p i d e r m a l j u n c t i o n in m o r e t h a n h a l f o f the u n t r e a t e d a n d t r e a t e d tuberculo'id a n d u n t r e a t e d b o r d e r l i n e l e p r o m a t o u s p a t i e n t s as well as in a few cases o f the o t h e r groups. I n n o cases were I g M a n d C c o n c o m i t a n t l y present at the d e r m o - e p i d e r m a l junction. D e p o s i t i o n o f c o m p l e m e n t was f o u n d in some o f the vessel walls in relatively few p a t i e n t s o f all groups, with the exception o f t r e a t e d tuberculo~d (2 o u t o f 3) a n d u n t r e a t e d b o r d e r l i n e l e p r o m a t o u s (6 o u t o f 9) patients.

Studies Across the Leprosy Spectrum

49

Fig. 1. Linear complement deposition along sweat glands (approx. x 300)

Fig. 2. Autofluorescingcells (x 500). Note: Staining of nuclei by acridinorange No immunoglobulin deposits were observed in the vessel walls, with the exception of I untreated borderline lepromatous patient in whom IgM-deposits were found in some vessel walls. Linear IgG-deposits along the basement membrane of sweat glands were found in a few patients in the different groups, altogether in 5 out of 24 biopsies in which

50

W.R. Faber and D. L. Leiker

Table 5. Presence of autofluorescing macrophages in the lesional skin of untreated and treated patients with various types of leprosy Number of patients

Tuberculo'fd Borderline tuberculo'l"d Borderline lepromatous Lepromatous

Autofluorescing macrophages Number present

Vague

Distinct

untreated treated

7 3

0 0

0 0

0 0

untreated

4

1

1

0

untreated treated untreated treated

9 6 4 7

9 6 4 7

3 0 2 1

6 6 2 6

Table 6. Complement deposits in and around autofluorescing macrophages in the lesional skin of untreated and treated patients with various types of leprosy B.I.pos.

B.I.neg.

Number

Borderline tuberculo'id untreated Borderline lepromatous untreated treated Lepromatous untreated treated

9 4 4 6

Complement deposits

Number

Complement deposits

1

0

3 2

2

0

4

1

0

2

B.I. pos. = positive bacteriologic index (by means of FFW staining of skin section) B.I. neg. = negative bacteriologic index (by means of FFW staining of skin section)

sweat glands were present (see Table4). N o other immunoglobulins were detected in this localisation. C o m p l e m e n t deposition in a linear pattern was observed in a b o u t half the cases (11 o u t o f 22) a r o u n d the sweat glands and also in 3 cases a r o u n d other skin appendages (Fig. l). E t h i d i u m b r o m i d e and acridinorange staining confirmed the cellular nature o f the autofluorescing cells (Fig. 2). Routine histochemical staining o f consecutive sections showed that these ceils were localised in areas where lepromatous m a c r o p h a g e s were present. A n enzymehistochemical esterase staining, with a substrate specific for macrophages, showed positive staining o f the cytoplasm o f these cells, whereas small vacuoles were devoid o f esterase activity. These autofluorescing macrophages were f o u n d in all cases o f borderline lepromatous and l e p r o m a t o u s leprosy and in 1 case o f untreated borderline tuberculoi'd leprosy (Table 5). It was f o u n d that in untreated patients these

Studies Across the Leprosy Spectrum

51

fluorescent cells were less distinct than in treated patients. Moreover, as the size of the infiltrates became smaller during therapy, the number of these ceils diminished. Complement deposits were observed in and around these cells in approximately one third of the untreated and treated borderline lepromatous and more than half of the untreated and treated lepromatous patients (Table 6). The impression was gained that these deposits tended to disappear when acid-fast material could no longer be observed by means of Fite-Faraco-Wade (FFW) staining of skin sections.

Discussion

Cells staining with FTC-labelled antihumanimmunoglobulin antisera apparently do not participate extensively in the inflammatory reaction in the skin of untreated and treated patients with various types of leprosy. In this respect it should be noted that Lai A Fat and Diesselhof-den Dulk (1975) described low IgG-synthesis in tuberculo~d, borderline tubereulo~id and borderline lesions; high IgG-synthesis in lepromatous lesions and low C3-synthesis in half of the cultures of tuberculo'id lesions. According to our findings IgM along the dermo-epidermal junction was of a lower frequency than in the studies of Bullock et al. (1974) and Quismorio et al. (1975). In this study sparse IgM-deposits were found in half of the treated lepromatous patients and in a few of the untreated borderline lepromatous and lepromatous patients. It has been suggested (Bullock et al., 1974) that antibodies against mycobacterial glycoproteins might crossreact with the basement membrane of the skin. We did observe complement deposits along the basement membrane of the epidermis and in vessel walls in tuberculoid as well as lepromatous leprosy lesions. It should be noted, however, that in no case were IgM and complement concomitantly present in the same biopsy. Nor did we observe immunoglobulins in those areas where mycobacterial antigen was present. The presence of IgG and complement along the basement membrane of sweat glands and in a few instances also along other skin appendages had not previously been reported. This was observed in different types of leprosy; IgG was found in approximately 20 ~ and complement in 50 ?/oof the patients. The fact that in 2 cases IgG and complement were concomitantly present may indicate that in some cases immune complexes may play a role in sweat gland disturbance. It might well be that if a more sensitive method were used, as indicated by Holubar et al. (1977), immunoglobulin deposits might be found in more cases. It is noteworthy that in a previous study (Sehgal, 1974) it was found that in leprosy lesions significant impairment of sweat response was observed by comparison with normal or equivocal sensory disturbance. This may also indicate that local factors may play a role. Autofluoreseence of macrophages is most likely due to lipofuscin, which has a yellow-orange autofluoreseence (Strehler, 1962; Koenig, 1963). Lipofuscin granules accumulate in long-lived cells in the course of ageing (Strehler, 1962; De Duve and Wattiaux, 1966), although they have also been described in newborn human liver (Goldfischer and Bernstein, 1969) and in several pathological conditions (Essner and Novikoff, 1960; Hasan and Glees, 1972). Lipofuscin is

52

W.R. Faber and D. L. Leiker

supposed to be derived from lysosomal material ( = altered lysosomes) (Essner and Novikoff, 1960; Koenig, 1963; De Duve and Wattiaux, 1966) because of their fine structure and positive staining reaction for acid phosphatase. With enlargement of the lipofuscin granule enzyme activity becomes restricted to the periphery (Essner and Novikoff, 1960). This has been shown in the case of leprosy by Job and Nadu (t 970): they found acid phosphatase staining throughout the cytoplasm in early lepromatous lesions and in a later stage more around vacuoles. This is also in accordance both with our findings on esterase staining and with those bearing out that autofluorescence is more apparent in older lesions and sometimes has the aspect of small globes. It should be noted that a Xenon lamp with blue narrow band excitation, which was used in this study, yields low values of autofluorescence. Therefore, a much stronger autofluorescence would be found if for instance a Mercury lamp were used (Gianetti and Cormane, 1973). Brieger and Allen (1963) indicated the likelihood that inclusion bodies in Virchow cells ( = macrophages in lepromatous leprosy) are related to lysosomes, because of their submicroscopical appearance and their presence in acid phosphatase positive regions of the cell. Complement deposits were observed in and around these cells and seemed to be related to the presence ofmycobacterial antigen in the cells. It is open to speculation whether this indicates the synthesis of complement factors by these cells, since it is known that complement factors are synthesised by macrophages (Colten and Einstein, 1976).

References AIdershvile, J., Dietrichson, O., Hardt, F., Nielson, J. O.: Circulating T and B lymphocytes and immunoglobulin containing cells in the liver in chronic active liver disease. Acta path. microbiol. scand. Sect. C. 85, 2 6 - 3 2 (1977) Bays, R. A., Hamerlinck, F., Cormane, R. HH.: Immunoglobulin-bearing lymphocytes and polymorphonuclear leucocytes in recurrent aphthous ulcers in man. Arch. oral Biol. 22, 147-153 (1977) Beutner, E. H., Sepulveda, M. R., Barnett, E. V. : Quantitative studies of immunofluorescent staining. Bull. World Health Org. 39, 587-606 (1968) Brieger, E. M., Allen, J. M. : Cytopathology of the Virchow cell of human leprosy. Ciba Found. study group No. 15, 3 1 - 3 8 (1963) Bullock, W. E., Callerame, M. L., Panner, B. J. : Immunohistologic alteration of skin and ultrastructural changes ofglomerular basement membranes in leprosy, Amer. J. trop. Med. Hyg. 23, 81 - 86 (1974) Colten, H. R., Einstein, L. P. : Complement metabolism: cellular and humoral regulation. Transpl. Rev. 32, 3 - 1 1 (1976) Cormane, R. H., Szabo, E., Hauge, L. S. : Immunofluorescence of the skin : the interpretation of the staining of blood vessels and connective tissue aided by new techniques. Brit. J. Derm. 82, Suppl. 5, 2 6 - 4 3 (1970) Cormane, R. H:, Husz, S., Hamerlinck, F. : Immunoglobulin- and complement-hearing lymphocytes in allergic contact dermatitis and atopic dermatitis (eczema). Brit. J. Derm. 90, 597-605 (1974) Cormane, R. H., Hunyadi, J., Hamerlinck, F.: The role of lymphoid cells and polymorphonuclear leukocytes in the pathogenesis of psoriasis. J. Derm. 3, 247-259 (1976) De Duve, C., Wattiaux, R.: Functions of lysosomes. Ann. Rev. Physiol 28, 435-492 (1966) Degott, C. Benoist, M., Potet, F., Bloch-Michel, H., Hipeau, R.: Nouvelle technique d'examen de la membrane synoviale en immunofluorescence. Rev. Rhum. 4 1 , 3 9 9 - 4 0 5 (1974) Degott, C., Benoist, M., Potet, F., Bloch-Mich~l, H. : Examen des biopsies synoviales en immunofluorescence. Nouv. Presse Med, 4, 3051-3054 (1975) Essner, E., Novikoff, A. B.: Human hepatocellular pigments and lysosomes. J. ultrastruct, Res. 3, 3 7 4 - 3 9 l (1960)

Studies Across the Leprosy Spectrum

53

Faber, W. R., Leiker, D. L., Cormane, R. H. : Immunoglobulin-bearing cells in leprosy. Acta derm.venereol. (Stockh,) 56, 319-326 (1976) Furth, R. van: Origin and kinetics of mononuclear phagocytes. Ann. N.Y. Acad. Sci. 278, 161 - 1 7 5 (1976) Gajl-Peczalska, K. J., Lira, S. D., Jacobson, R. R., Good, R. A. : B lymphocytes in lepromatous leprosy. New Eng. J. Med. 288, 1033-1053 (1973) Gianetti, A., Cormane, R. H.: Quantification of immunofluorescence by microfluorometry. Arch. Derm. Forsch. 246, 249-270 (1973) Goldfischer, S., Bernstein, J. : Lipofuscin (aging) pigment granules of the newborn human liver. J. Cell Biol. 42, 253-261 (1969) Hasan, M., Glees, P.: Genesis and possible dissolution of neuronal lipofuscin. Gerontologia. 18, 2 1 7 236 (1972) Hodgkinson, G. I., Everall, J. D., Smith, H. V.: lmmunofluorescent patterns in the skin in Besnier's prurigo. The eczema asthma syndrome. Brit. J. Derm. 96, 357-366 (1977) Holubar, K., Konrad, K., Stingl, G.: Detection by immunoelectron microscopy of immunoglobulin G deposits in skin o f immunofluorescence negative herpes gestationis. Brit. J. Derm. 96, 569-571 (1977) Job, C. K., Nadu, T. : Lysosomal activity of macrophages in leprosy, Arch. Path. 90, 547-552 (1970) Joost, Th. van, Cormane, R. H., Pondman, K. W.: Direct immunofluorescent study of the skin on occurence of complement in pemphigus. Brit. J. Derm. 87, 466-474 (1972) Koenig, H.: The autofluorescence of lysosomes. Its value for the identification of lysosomal constituents. J. Histochem. Biochem. 11,556-557 (1963) Lai A Fat, R. F. M., Diesselhof-den Dulk, M. M. C.: In vitro synthesis of humoral factors (immunoglobulin and complement) in lesional skin of leprosy patients. Eur. lmmun. Meeting, Amsterdam (1975) Leiker, D. L. : Classification of leprosy. Lepr. Rev. 37, 1, 7 - 1 5 (1966) Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. : Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951) McKinney, R. M., Pearce, G. W., Spillane, J. T. : Fluorescein diacetate as a reference color standard in fluorescent antibody studies. Anal. Biochern. 9, 474-476 (1964) Meijer, A. E. F. H. : Semipermeable membranes for improving the histochemical demonstration of enzyme activities in tissue sections. I. Acid phosphatase. Histochemie 30, 31 - 3 9 (1972) Nath, I., Curtis, J., Bhutani, L. K., Talwar, G. P. : Reduction of a subpopulation of T lymphocytes in lepromatous leprosy. Clin. exp. Immun. 18, 8 1 - 8 7 (1974) Ornstein, L., Ansley, H., Saunders, A.: Improving manual differential white cell counts with cytochemistry. Blood cells 2, 557-585 (1976) Quismorio, F. P., Rea, T. H., Levan, N. E., Friou, G. J.: Immunoglobulin deposits in lepromatous leprosy skin. Arch. Derm. 111, 331-334 (1975) Rea, T. H., Quismorio, F. P., Harding, B, Nies, K. M., Di Saia, P. J., Levan, N. E., Friou, G. J.: Immunologic responses in patients with lepromatous leprosy. Arch. Derm. 112, 791 - 8 0 0 (1976) Ridley, D. S., Jopling, W. H. : Classification of leprosy according to immunity. A five group system. Int. J. Leprosy 34, 255-273 (1966) Sehgal, V. N. : Significance of the local sweat response in the diagnosis of leprosy. Dermatologica 148, 217-223 (1974) Strehler, B. L. : Pigment accumulation and lysosomes. In: Time, Cells and Aging, pp. 181 - 188. Nc~ York: Acad. Press 1962 Received January 6, 1978