Clin. exp. Immunol. (1979) 38, 135-147.

Cross-reactions between serum proteins and water soluble liver tissue antigens of the nine-banded armadillo (Dasypus novemeinctus Linn.) and man K. NEGAS SI, * 0. CLOS S & M. HARBOE University of Oslo, Institutefor Experimental MedicalResearch, Ulleval, Hospital, Oslo 1, Norway

(Acceptedfor publication 24 January 1979) SUMMARY

Cross-reactions between serum proteins and water soluble liver antigens of the nine-banded armadillo (Dasypus novemcinctus Linn.) and man were studied by crossed immunoelectrophoresis (CIE). Armadillo serum tested with rabbit antiserum against human serum proteins gave twelve components in CIE. Nine of these cross-reacting proteins were identified and showed partial identity with the corresponding human proteins. The electrophoretic mobility of CE2-macroglobulin and Gc-globulin differed in the two species. An ultrasonicate of normal armadillo liver gave twenty-eight anodic and eight cathodic components in CIE. By absorption experiments with armadillo serum, twenty of the former and seven of the latter were shown to be liver tissue components. A combination of CIE and crossed-line immunoelectrophoresis (CLIE) revealed the presence of twelve anodic and six cathodic liver tissue components crossreacting with man. A cathodic armadillo liver antigen called (CALA-17) showed partial identity with that of man both in tandem and fused rocket immunoelectrophoresis. The implications of the findings are discussed in relation to the use of armadillo-grown M. leprae for skin testing and other purposes in man. INTRODUCTION Storrs (1971) and Kirchheimer & Storrs (1971) established that Mycobacterium leprae from a patient with lepromatous leprosy can grow and establish a severe systemic infection in the nine-banded armadillo (Dasypus novemcinctus Linn.). From less than 1-5 gm of infected armadillo liver tissue, Kirchheimer & Storrs (1971) were able to harvest 15 1 x 109 of leprosy bacilli. M. ieprae is thus available in greater amounts for experimental purposes, to prepare skin test reagents, and it may also be useful for the development of a vaccine against leprosy. In our initial experiments with the clear supernatants obtained from infected armadillo liver homogenates, we observed cross-reactions between armadillo and human serum proteins in double-diffusion tests in gel. Using crossed immunoelectrophoresis (CIE), we demonstrated that different M. leprae preparations made from infected armadillo tissues contain varying degrees of impurities (unpublished observations). Crude leprosy skin test reagents, like conventional lepronmin, are supposed to contain much tissue debris and other soluble host tissue components. The effect of injecting preparations containing cross-reacting armadillo proteins into man is not known. However, several studies (Paterson, 1966; Davies et al., 1964; Weigle, 1973; Meyer Zum Buschenfelde & Hopf, 1974; Rudofsky, 1976) have shown that the injection of cross-reacting heterologous tissue *

Present address: Armauer Hansen Research Institute, P.O. Box 1005, Addis Ababa, Ethiopia. Correspondence: Professor M. Harboe, University of Oslo, Institute for Experimental Medical Research, Ullevil Hospital, Oslo 1, Norway. 0099-9104/79/1000-0135$02.00 (© Blackwell Scientific Publications

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K. Negassi, 0. Closs & M. Harboe

antigens with adjuvant containing killed mycobacteria breaks tolerance to self tissue antigens and induces the formation of autoimmune diseases. The study by Weigle (1961) also showed that immunological tolerance induced in rabbits to bovine serum albumin (BSA) can be terminated by injecting heterologous albumins from different species. It is possible that similar effects may be produced by injecting preparations which contain armadillo tissue contaminants cross-reacting with those of humans. It is therefore essential to establish whether armadillo tissue or serum proteins cross-react with man. The present investigations were performed to obtain detailed information on the cross-reaction between serum proteins and liver tissue antigens of the nine-banded armadillo and man.

MATERIALS AND METHODS Normal armadillo and human sera. A pool of normal armadillo serum was prepared using sera obtained from Dr R. J. W. Rees, National Institute for Medical Research, London, England, and Dr J. C. Convit, Caracas, Venezuela, through the WHO Immunology of leprosy (IMMLEP) programme. Sera obtained from thirty healthy BCG-vaccinated Norwegian medical students were used to prepare the normal human serum pool. The sera were stored at -20'C until used and contained 15 mm NaN3 as preservative. Antisera against human serum proteins. Polyvalent rabbit antibodies against human serum proteins were obtained from Dakopatts A/S, Copenhagen, Denmark (code 100 SF). Monospecific rabbit antisera against human serum proteins were obtained from Dakopatts or from Behringwerke, Marburg/Lahn, Germany, as indicated in Table 1. Goat and horse antibodies to human serum proteins were obtained from the Hyland Division, Travenol Laboratories Inc., Costa Mesa, California, and Behringwerke, respectively. Preparation of armadillo liver antigens for immunizations and crossed immunoelectrophoresis. Normal armadillo liver was obtained from Dr R. J. W. Rees, London. Both sonicates and homogenates were prepared. The latter were used for immunization and were prepared as follows: 1 g of liver was finely minced, resuspended in 10 ml of distilled water and homogenized using a Tenbroek all-glass homogenizer (Bellco Glass Inc., Vineland, New Jersey). To remove debris, the homogenate was centrifuged at 200 g for 10 min. The supernatant was saved and stored in small aliquots at -20'C until used for immunization. Armadillo liver antigen, to be used for CIE, was prepared using a Branson Sonifier Model B-12 (Branson Sonic Power Co., Danbury, Connecticut). Briefly, 2 g of normal armadillo liver and 10 ml of 0.9% NaCl were sonified on ice for 7 min at an output effect of 80 W, and centrifuged for 30 min at 20,000 g. The supernatant obtained was designated 'armadillo water soluble liver antigen' and stored at -200C in 80 pl aliquots with 15 mm NaN3. Each aliquot was thawed once and diluted 4-8 times in 0 9% NaCl, before use in CIE within 48 hr. Preparation ofhuman liver antigens. Normal human liver was obtained shortly after death from a healthy Norwegian man killed accidentally. 1 g of the liver was mixed with an equal amount of (W/V) 0 9% NaCl containing 15 mM NaNs and homogenized as described above. The homogenate was centrifuged at 20,000 g for 30 min. The sedimtnt obtained was discarded and the supernatant stored in small aliquots at - 200C. Production of anti-armadillo liver serum. Four rabbits (R-3192, R-3193, R-3194 and R-3195) were immunized and bled according to Harboe & Ingild (1973). A suspension of armadillo liver homogenate in Freund's incomplete adjuvant was made, from which 200 jil were given as multiple injections in the scapular area. The serum obtained from individual rabbits was stored separately at 40C with 15 mm NaN3. The best of the eight bleedings, with the highest anti-liver activity, were selected. Accordingly, anti-armadillo liver sera from the sixth, seventh and eighth bleedings were used for the preparation of anti-liver immunoglobulins, following the procedure described by Harboe & Ingild (1973). The immunoglobulins were concentrated three times by an Amicon concentrator Type B-15 (Amicon Corp. Lexington, Massachusetts), and dialysed against two changes of 0 1 M NaCl containing 15 mm NaN3 for 24 hr and stored at 40C. Absorption of anti-armadillo liver immunoglobulins. Since the liver contains both serum and liver tissue antigens, the rabbits produced antibodies against antigens of both kinds. In order to differentiate between tissue and serum protein antigens, the immunoglobulins were absorbed with normal armadillo serum in two steps. Briefly, 3 ml of anti-liver immunoglobulins were incubated with 0 75 ml of armadillo serum for 30 min at 370C and then left over night at 40C. The mixture was centrifuged at 4500 g for 30 min and the supernatant obtained was treated once more in the same way. After this absorption procedure the immunoglobulin preparation did not precipitate any armadillo serum protein in CIE. Immunoelectrophoretic methods. CIE'with intermediate gel was performed as described by Axelsen (1973) and Closs, Harboe & Wassum (1975). Serum and water soluble liver antigen electrophoresis was performed in 1% (W/V) agarose (batch AGS 122 and HSA batch 0364, Litex, Glostrup, Denmark), respectively. The first dimension electrophoresis was run at 10 V/cm until human serum albumin stained with Evan's blue had anodically migrated to a certain distance (see Fig. legends). To obtain optimal conditions for the demonstration of both anodic and cathodic antigens, two reference systems were made. For the former, CIE was performed as described by Axelsen (1973) and Closs et al. (1975). The armadillo serum reference system was made by using 3 p1 of armadillo serum diluted 1: 10 in the circular antigen well and 400 pl of rabbit anti-human serum (20 pl/cm2) in the top gel. To identify the individual precipitates in the top gel 50-100 p.l of monospecific antiserum was incorporated in the intermediate gel as indicated in Table 1, while corresponding volumes of buffer was added into the control plates.

Cross-reactions between man and armadillo

137

The anodic armadillo liver reference systems (Fig. 4) were made by incorporating 3 and 10 Id of liver sonicate diluted 1: 4 in the antigen well. The top gel contained 15 ILl/cm2 of concentrated absorbed or unabsorbed rabbit anti-armadillo liver immunoglobulins. The cross-reactivity between human and armadillo liver antigens was studied by crossed-line immunoelectrophoresis (CLIE) (Kroll, 1973), in which 100 pi of human liver homogenate was incorporated in the intermediate gel. The same volume of buffer was incorporated in the intermediate gel of all control plates. The cross-reaction was also studied by filling 10 PI of human liver homogenate and 10 PI of armadillo liver sonicate diluted 1 : 4 into the circular antigen well. For every test plate, one control plate was made and both were run simultaneously in the same apparatus. An increase in the area of a precipitate in the test plate as compared to that of the control plate indicated a cross-reaction between the two species. To detect cathodically moving serum protein and liver tissue antigens and to study their cross-reactivity, a modified set-up of CIE was used (Fig. 5). Briefly, a circular well was punched 19 mm from the cathodic edge of the first dimension gel. Ten pI of armadillo liver sonicate diluted 1:4 or 3 P1 of armadillo serum diluted 1: 10 were filled into the well. The first dimension electrophoresis was run until the human serum albumin had moved 2 cm. Rabbit anti-human serum or an anti-armadillo liver immunoglobulin was incorporated into the gel on the left side of the reference gel (14 5 pl/cm2 in a gel measuring 5.3 x 2-5 cm), while a corresponding volume of buffer was added on the right side, as shown in Fig 5. Identification of the cathodic serum protein was obtained by adding the same concentration of monospecific rabbit anti-human immunoglobulins into the reference gel. Cross-reactions between cathodic armadillo and human liver tissue antigens were studied by filling 10 Id of human liver homogenate and 5 or 10 PI of armadillo liver sonicate (1: 4 dilutions) into the circular antigenic well. A mixture of armadillo liver sonicate and 10 pI of buffer served as a control. An increase in the area covered by the precipitate in the test plate as compared to the control plate demonstrated antigenic cross-reactivity between the two species. The immunological cross-reaction between a cathodic human and armadillo liver antigen was also tested by tandem-CIE (Kroll, 1973) and fused rocket IE (Svendsen, 1973) with slight modifications. The second dimension electrophoresis was run overnight at 2-3 V/cm ensuring that slightly cathodically moving antigens were driven to the cathode, while anodically moving antigens were driven to the anode. Electrophoretic mobility. The electrophoretic mobility of human and armadillo a2-macroglobulin, haptoglobin and Gcglobin was compared in CIE. This was first done by studying the precipitation pattern of individual serum proteins from both species in separate plates. A dilution of human serum which gave lower peaks than that of the armadillo was selected; this made it easier to produce two peaks, one above the other, and to differentiate the precipitates formed by human and armadillo serum proteins. The selected dilutions of armadillo and human serum were filled into the circular antigen well (see Fig. 3), and the differences in electrophoretic mobility were then immediately apparent. Double-diffusion tests. These were performed in 1% agarose gel (Agarose, Seravac Laboratories Ltd., Maidenhead, England, batch No. 202) in Barbital buffer pH 8-6 as described by Harboe. Solheim & Deverill (1969).

RESULTS Cross-reactions between human and armadillo serum proteins Fig. la shows CIE of normal armadillo serum tested with rabbit antiserum against human serum proteins in the top gel. A minimum of ten precipitates was obtained. An additional cross-reacting component was detected at position 'X' (Fig. la & b) when monospecific anti-Gc-globulin was incorporated in either the intermediate or top gel. One cathodic precipitate designated No. 12 was detected by the CIE method developed for the demonstration of cathodically moving antigens (see the Materials and Methods section). The precipitates were numbered according to their relative position. Changes in the relative position and the precipitation patterns were minimized by using the same antigen and antiserum throughout the study. The number of detectable cross-reacting serum components was not increased when goat or horse anti-human serum antibodies were used in the top gel instead of antibodies produced in rabbits.

Identification ofindividual precipitates Eight of the eleven anodic armadillo serum proteins which cross-react with the corresponding human proteins were identified (Table 1) by incorporating different monospecific antisera in the intermediate gel. An example of this procedure is shown in Fig. lb, where the albumin precipitate designated as No. 1 was clearly drawn into the intermediate gel by incorporating rabbit anti-human albumin. Antigens Nos 2, 3 and 8 did not react with any of the antisera listed in Table 1. The cathodic precipitate was identified as IgG by adding anti-human IgG in the top gel. The various antisera used and the crossreacting proteins identified are shown in Table 1.

K. Negassi, 0. Closs & M. Harboe

138

Iulm 4ser4 1h i 0

A'fnit atI-~..i,

a.B..in

buffer

'1_ |

Ally--_-* |E~...... --g| '| l '. ll ... 1.'.~'

armadillo seumamailoseu

FIG. 1. (a) Reference pattern for armadillo serum. 3 of 1 10 normal armadillo serum was used. The first dimension was run at 10 V/cm until albumin labelled with Evan's blue had migrated up to 4-5 cm. The second dimension was run in 7 x 10 cm plates, containing 20 ipd/cm' of polyvalent rabbit anti-human immunoglobulins in the top gel, for 18 hr from the cathode to the anode. Anode is on top. The intermediate gel contained 150 jpd of buffer. (b) Was run in the same way as in (a) except that 100 jil of rabbit anti-human albumin and 50 jil of buffer was incorporated into the intermediate gel. I l

All of the cross-reacting proteins showed reactions of partial identity in double-diffusion tests. Such reactions are shown in Fig. 2 where human albumin and a2-macroglobulin spur over those of armadillo.

Electrophoretic mobility To compare the electrophoretic mobility of human and armadillo haptoglobin, ct2-macroglobulin and Gc.-globulin, the precipitation patterns of homologous proteins from the two species were first studied separately. An example is shown in Fig. 3a & b, illustrating the behaviour of human and armadillo Q2-macroglobulin. The relative electrophoretic mobility was then determined by testing both antigens in the same plate, as shown in Fig 3c. It can be seen that armadillo ct2-macroglobulin had a faster electrophoretic mobility than the corresponding human protein and behaved in the same way when compared with most of the six mammalian species studied (unpublished observation). The difference in the electrophoretic mobility of haptoglobin between the two species was not significant (Fig. 3d). On the other hand, armadillo Gc-globulin had a relatively slower electrophoretic mobility than its human counterpart. Armadillo livoer reference system Armadillo liver antigens moved both anodically and cathodically when tested in CIE. These antigens were designated as anodic (AALA) and cathodic (CALA) armadillo liver antigens. The respective reference systems in CIE are shown in Figs 4 & 5. Fig. 4a shows the reference pattern of anodically moving liver antigens in CIE with intermediate gel. Unabsorbed rabbit anti-armadillo liver immunoglobulins concentrated three times were incorporated in the top gel. By this technique, twenty-eight anodic precipitates were demonstrated. Some of the precipitates were weak and the peaks could not be seen in all runs. The application of a lower concentration of armadillo liver sonicate in the circular well increased the visibility of these antigens. Fig 4b

Cross-reactions between man and armadillo

139

TABLE 1. Armadillo serum proteins precipitated in the intermediate gel by monospecific rabbit antisera for various human serum proteins

Antisera used

Anti-albumin Anti-prealbumin Anti-orosomucoid Anti-a-lipoprotein Anti-ct-antitrypsin

Anti-a-acid-glycoprotein Anti-a-HS-glycoprotein Anti-thrombin III Anti-chymotrypsin Anti-Gc-globulin Anti-inter-a-trypsin inhibitor Anti-caeruloplasmin Anti-haptoglobin Anti-a2-macroglobulin Anti-cholinesterase Anti-B lipoprotein Anti-haemopexin Anti-1,3 C-f3 A-globulin

Anti-f1 E-globulin Anti-transferrin Anti-fibrinogen Anti-C reactive protein Anti-J32-glycoprotein III Anti-,32-microglobulin Anti-IgG Anti-IgA Anti-IgM Anti-k Anti-K

Pos. or Neg. Precipitate No.

Pos. Neg. Neg. Neg. Neg. Neg. Neg. Pos. Neg. Pos. Neg. Pos. Pos. Pos. Neg. Neg. Neg. Neg. Pos. Neg. Neg. Neg. Neg. Neg. Pos. Neg. Pos. Neg. Neg.

1

7

X 5 6 4

9

12

10

Source D D D B D&B B B B D D B D&B D D D B D B B D D D B D D D D D D&B

D Dakopatts, Denmark. B Behringwerke, Germany. Pos. positive. Neg. negative. The numbers indicated correspond to the individual precipitates in Fig. 1. Out of the twelve armadillo serum proteins cross-reacting with those of man, only three (Nos 2, 3 and 8) were not found to correspond to any of the various antisera used.

shows the precipitation pattern obtained when 3 pl of armadillo liver sonicate was used. The precipitates (Fig. 4a,b) were numbered with even numbers from 2-5 according to their position. The arrows towards precipitates Nos 4, 6, 34, 36, 38 and 44 (Fig. 4b) denote precipitates which became more distinct when a lower concentration ofantigen was used. Changes in the precipitation of some of the tissue antigens were observed when liver sonicates were kept at 4°C for more than 36 hr before use. Antigens Nos 4, 24 and 28 tended to be weakly stained, while the peaks in Nos 10 and 14 decreased in height. For example, after 6 days of storage the height of the peak in No. 10 was 1/6 of the control and completely disappeared after 30 days. Other antigens like Nos 46, 50, 52 and 56 were relatively stable. The first three antigens (46, 50 and 25) seemed to remain stable even after 1 year of storage at 4°C. A slight anodic change in the electrophoretic mobility of No. 52 was observed after 30 days. All the eight serum proteins detected in the sonicate were quite stable and all possible variations were minimized by avoiding repeated freezing and thawing and by

K. Negassi, 0. Closs & M. Harboc

140

A

anti-hman

B

abuminanti-a2 macroglobulin

FIG. 2. Comparison of armadillo albumin in (a) and u2macroglobulin in (b) wxith those of human. H and A stand for human and armadillo serum respectix clx.

using the antigens xxithin 24 hr of thaxxing. Variations in the precipitation pattern of the precipitates were not observed, even when tested after 16 months of storage at -20'C. L iver tissue components xxere distinguished from serum components present in the sonicates by incorporating armadillo serum in the intermediate gel or by using absorbed anti-armadillo liver immunoglobulins in the top gel. The patterns obtained wxere compared with those of the reference system with unabsorbed anti-armadillo liver immunoglobulin. These experiments showxed that txxenty of the twentyeight precipitates remained virtually unchanged in the top gel, indicating that they were liver tissue antigens, while the other eight antigens (2, 6, 8, 12, 32, 38, 40 and 42) were serum components. All but two of the components (6 and 38) cross-reacted xxith human serum proteins. Fig 5b shows the reference pattern of the cathodic antigens. Seven distinct precipitates were observed in this area. One weak additional precipitate was demonstrated in plates with lowxer concentrations of liver sonicate, giving a total of eight components. This component is showxn in the schematic drawing in Fig. 5a and is numbered as antigen No. 7. The precipitates wxere given odd numbers from 3 15. By incorporating absorbed anti-armadillo liver in the gel and adding armadillo serum in the circular antigen wxell, it was found that only precipitin No. 9 was due to a serum component. Incorporation of rabbit antisera against human immunoglobulins in gels shoxxed that this precipitate was due to IgG. Cross-reactions between armadillo and human liver tissue antigens Studies of cross-reactions between anodic antigens in armadillo liver sonicates and human liver homogenates by CLIE showed that of the txxenty antigens, txxelve cross-reacted. This conclusion was based on experiments wxith ten test plates compared with ten control plates. When 10 p1 of human liver homogenate (1 1 W/V) was tested in CIE using rabbit serum against armadillo liver antigens in the top gel, eleven to twelve precipitins wxere observed. Of these, eight wxere liver tissue antigens. One of the antigens was strongly cross-reacting and xxas found in tbe albumin area. The cross-reaction betxxeen the CALA and human liver antigens wxas studied by mixing both antigens in the same wvell. It can be seen from Fig. 5c that all of the lines, except line 13, were clearly affected. Line 13 was slightly affected in some experiments. Howxever, since this finding was inconsistent, it wxas consi'dered as a non-cross-reacting antigen. The results of the studies of anodic and cathodic liver tissue antigens are summarized in Tables 2 & 3. By testihg various concentrations of human liver homogenate in CIE xx ith anti-armiadillo liver antibodies, one to four cathodic precipitins wvere seen. Only one of these xxas distinct, while the others xxere

Cross-reactions between

man

and armadillo

FIG. 3. Comparison of the electrophoretic mobility of human a2-macroglobulin, haptoglobin with that of armadillo. (a) 3 pi of human serum diluted 1: 3. (b) 3 jul of armadillo serum diluted 1: 10. (c) mixture of (a) and (b) in the same well. The top gel contained 200 jil of anti-a2-macroglobulin. (d) Mixture of armadillo and human serum. The volumes and dilution used were the same as in (c). Top gel contained 100 pl of rabbit anti-human haptoglobin.

141

142

K. Negassi, 0. Closs & M. Harboe

FIG. 4. Reference pattern for armadillo anodic liver antigens. In (a) 10 pl and in (b) 3 gd of 1: 4 dilutions of armadillo liver sonicate were used. The first dimension was run at 10 V/cm until albumin labelled with Evan's blue moved up to 2-5 cm. The second dimension was run in 5 x 7 cm plates at 2-3 V/cm for 18 hr. Antibody concentration was 15 Il/Cm2 of anti-armadillo liver immunoglobulins concentrated three times.

quite weak. The distinct human precipitate was very similar to line 17 in the CALA reference system. Armadillo line 17 spurred over the corresponding human component in tandem-CIE and fused rocket IE. DISCUSSION Studies which show the antigenic profiles of serum proteins Griginating from the aradillom are few. Lewis & Doyle (1964) have studied armadillo plasma by immunoelectrophoresis (IE) and detected only five components. In the present study we have detected twelve armadillo serum components which cross-react with those of man. Our findings are probably due to the greater resolving power of CIE and to our use of more potent antisera. Gc-globulin could only be detected by particularly high titred antibodies; this is in accordance with previous studies where precipitates which could not be detected by standard antisera were clearly visible when either particularly high titred antibodies were used (Lowenstein et al., 1975) or the antisera were raised in more distantly related animals (Orlans & Feinstein, 1971; Neoh et al., 1973). Investigations concerning liver antigens from different species have been performed in doublediffusion tests in gel and/or in IE (Hase & Mahin, 1965; Auer & Milgrom, 1972; Sugamura & Smith, 1976). In most of these studies, fractions of liver tissue antigens were used and the antigenic structure of total liver homogenate was not clearly shown. Hase & Mahin (1965) detected twelve components in rat liver homogenate supernatants which were obtained after centrifugation at 105,000 g. In this study we have used normal armadillo liver sonicate: thirty-six components were detected. Of these, twenty-eight antigens moved towards the anode (Fig. 4) and the remainder towards the cathode (Fig. 5). Cathodic antigens cannot be detected by the standard technique of CIE, unless they are carbamylated (Weeke, 1973). To avoid possible denaturations due to this treatment, cathodic antigens were demonstrated using the modified set-up of CIE as shown in Fig. 5. Such cathodically moving liver antigens (Louis & Blunk, 1970; Auer & Milgrom, 1972) and Pseudornonas auroginosa antigens (H0iby, 1977) were detected in IE.

Cross-reactions between man and armadillo

I I I I I I I I I

I I I I I I 1

151,1

1 1

1

1

CATHODE

A

FIG. 5. Reference patterns for cathodic armadillo liver antigens. (a) Schematic drawing of the reference pattern. (b) 10 pI of 1: 4 dilution of armadillo sonicate was driven until the albumin had moved for 2 cm. Only half of the plate was used. The antibody concentration was 15 pl/cm2. The second dimension was run at 2V/cm for 18 hr from anode to the cathode. (b) Effect of mixing both armadillo and human liver in the same well. The figures are placed according to their position in the second dimension.

143

144

K. Negassi,

0.

Closs

&

M. Harboe

TABLE 2. Anodic antigenic components in armadillo liver sonicate and their cross-reactions with corresponding human components

8S

lOL

++

+

36L

38S

40S

12S +± 42S

-

-

++

+

6S

32S

4L +± 34L

+

-

2S +

14L

16L

18L

20L

22L

24L

26L

28L

30L

+

-

-

+

+

+

+

+

_

44L +±

46L

48L

50L

52L

54L 56L _-

+

+

Total twenty-eight components. S Serum component; L = liver tissue antigen component. + Cross-reacting water soluble armadillo liver sonicate components; the peaks increased in area when liver homogenate was added into the intermediate gel. + + = Water soluble armadillo liver sonicate components, whose cross-reactivity was only shown when anti-human serum was added into the intermediate gel. -= Non-cross-reacting armadillo water soluble liver sonicate components; the peaks were not affected when human liver homogenate was added into the intermediate gel. TABLE 3. Cathodic antigenic components in armadillo liver sonicate and their cross-reaction with corresponding human components

3L ±

5L ±

7L

9S

llL

13L

+

+

±

-

15L -

17L ±

Total eight components. S = Serum component; L = liver tissue antigen component. + = Cross-reacting water soluble armadillo liver sonicate components; the peaks increased in area when liver homogenate was added into the intermediate gel. - = Non-cross-reacting armadillo water soluble liver sonicate components; the peaks were not affected when human liver homogenate was added into the intermediate gel.

However, since IE is less sensitive than CIE, the number of components detected in these studies might not be optimal. By adding antibodies only in the top gel of the modified set-up of CIE with intermediate gel, we observed that the antigens precipitate in the latter gel and thus behave as in the typical standard set-up of CIE with antibodies in the corresponding gel (e.g. Fig. lb), indicating that the antibodies had moved from the cathode to the anode. Thus, the modified set-up (Fig. 5) might not be particularly sensitive for studying cross-reactions by incorporating antibodies in the intermediate gel. Previous observations made by various workers indicate that serum proteins (Bauer, 1969; Neoh et aL, 1973; Andersen & Kr0ll, 1975) and liver antigens (Auer & Milgrom, 1972; Hopf, Meyer Zum Buschenfelde & Freudenberg, 1974; Sugamrura & Smith, 1976) of man cross-react with those of other mammalian species. In the present study we have made similar observations with serum proteins and liver antigens of the armadillo that cross-react with the corresponding human proteins. The liver is one of the organs that contains a high concentration of leprosy bacilli (Binford et al., 1974; Binford, Storrs & Walsh, 1976) in infected armadillos and has therefore been an important source of M. leprae for the preparation of various skin test reagents (WHO report, 1975). The liver is also an important site for the synthesis of plasma proteins (Miller & Bale, 1954; Kawai, 1973). Leprosy bacilli isolated from the liver for preparing skin test reagents and vaccines may, therefore, be contaminated both with liver tissue and plasma proteins. Using CIE we have detected such contaminants (unpublished observations) in various M. leprae preparations obtained from different sources. The consequences of injecting preparations containing cross-reacting antigens of armadillo origin into man are not known. However, catastrophic neurological immune reactions are known to have occurred in the 1880's following the introduction of the Pasteur rabies vaccine (rabbit nervous tissue and fixed virus) (Paterson, 1966).

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The injection of heterologous tissues in adjuvants such as brain (Paterson, 1966), heart (Davies et al., 1964), thyroglobulin (Weigle, 1973) and kidney (Rudofsky, 1976) into experimental animals has also been shown to break tolerance towards self-antigens and induce autoimmune diseases. Using a single injection of rat liver suspended in Freund's complete adjuvant, Asherson & Dumonde (1964) induced the formation of autoantibodies in rabbits. Moreover, in spite of the difficulty of breaking self-tolerant to serum antigens, it has been possible in certain instances to induce it by immunizing tolerance rabbits with heterologous (Weigle, 1961; Benjamin & Weigle, 1970), homologous but altered albumins (Weigle, 1962) and with horse spleen ferritin or with various preparations of auto-y-globulins (Williams & Kunkel 1963). It follows, therefore, that preparations made from armadillo-grown M. leprae might induce similar immune reactions in man if they contain cross-reacting proteins of armadillo origin. The specificity of the ensuing immune reaction could depend on the tissue distribution of the antigens involved. Antigens that are widely distributed (Shulman & Centeno, 1973; Bock, 1972) are likely to induce autoantibodies with multiple specificities in contrast to those that are only limited to one organ (Auer & Milgrom, 1972; MceFarlane et al., 1977). The relative role of the induced autoantibodies in the pathogenesis of tissue destruction could, however, depend on the cellular location of the antigens against which the immune reactions are directed: the cell-bound antigens being the main targets of immune effectors in contrast to cytoplasmic antigens. Adjuvants are known to increase T cell proliferation and to elicit autoantibody formation in experimental animals (Allison, 1973). Adjuvant activities are said to be present in Al. lcprae (Stewart-Tull & Davies, 1972) and the occurrence of antinuclear and other autoantibodies in leprosy patients might, therefore, be due to the heavy mycobacterial load which would probably enhance the production of these immune reactions (Allison, 1973). Thus, reactions of this nature can perhaps be more easily triggered or enhanced if cross-reacting antigens, along with M. leprae, are given to patients suffering from leprosy. The amount of armadillo antigen that can induce immune reactions is difficult to determine: the induction of autoimmune diseases in experimental animals is, however, said to be dose-dependent, requiring micrograms to milligrams and might also depend on the frequency of injections. Single injections of heterologous nervous tissue (Paterson, 1966) and kidney tissue antigens (Rudofsky, 1976) were found to be sufficient to induce autoimmune reactions. Similar reactions were induced in mice even when immunized with two injections of 30 gig of homologous thyroglobulin in Freund's complete adjuvant (Tomazic & Rose, 1977). To ensure the efficiency ofvaccines against leprosy, suitable doses must be given either in single or multiple injections. Since such preparations have not yet been developed, the optimal doses and frequency of injections that should be administered are left for future decisions. However, according to reports from WHO (1975), skin tests such as LRA6 (prepared by Rees & Draper) and LK (prepared by Kirchheimer) have been used in man in doses of 0-2-2 jig and 116 jig, respectively. The LRA6 is said to contain 1% armadillo protein and if the LK is contaminated to the same degree, it would contain 1-16 jig of armadillo proteins. The injection of armadillo antigenic components present within this range, may or may not be hazardous. However, since the risk of sensitization increases with the concentration of foreign proteins present in the preparations, lower doses might be relative safe to use. Different preparations made from infected armadillo tissues can have varying degrees of impurities, with the risk of breaking tolerance to self-antigens being lower in those which contain less armadillo antigenic components. To detect contaminants and to set up criteria for determining their relative safety for use in man, it is important to develop techniques with relatively high resolving powers. We believe that both the standard (e.g. Fig. 1) and modified set-ups (e.g. Fig. 5) of CIE can be useful as preliminary screening tests for purity. Since the sensitivity of these techniques very much depends on the antisera used, those with low antibody activity are likely to give negative results even when the preparations are not sufficiently pure. It is, therefore, crucial to use particularly high titred antibodies against normal armadillo antigenic components which have been raised either in the rabbit or in other distantly related animals. If the level of detection is to be increased, sensitive procedures such as radioimmunoassay (RIA) recently used by Harboe et al. (1977) for demonstrating antibody activity against BCG antigen 60 can be used. K

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Both RIA and the sensitive technique of crossed radioimmunoelectrophoresis (CR TE) (Weeke & Lowenstein, 1973) can also be used to detect induced humoral responses in humans or experimental animals immunized with preparations made from the armadillo. In conclusion, our findings regarding the serological and liver tissue cross-reactions between man and armadillo have an important bearing on the preparation of skin test reagents and vaccines made from infected armadillo tissues. The study does not condemn the use of M. leprae preparations from armadillo in man. However, it suggests that before such preparations are used on a wide scale trials in experimental animals would be worthwhile, and they should only be released for human use after meeting certain criteria for safety and purity. These studies were performed when Kesete Negassi was a recipient of scholarships from the World Health Organization (WHO) and the Norwegian Agency for International Development (NORAD). The work was supported by grants from Anders Jahre's Fund for the Promotion of Science and the Immunology of Leprosy (IMMLEP) which is part of the WHO special Programme for Research and Training in Tropical Diseases. N. H. Axelsen of the Protein Laboratory, Copenhagen, is thanked for producing the antisera against normal armadillo liver. REFERENCES ALLISON, A.C. (1973) Mechanisms of tolerance and autoimmunity. Ann. rheum. Dis. 32,238. ANDERSEN, M.M. & KR0LL, J. (1975) Identification of bovine serum proteins by quantitative immunoelectrophoretic methods. Scand.J. Immunol. 4, (Suppl. 2), 163. ASHERSON, G.L. & DUMONDE, D.C. (1964) Autoantibody production in rabbits. V. Comparison of the autoantibody response after the injection of rat and rabbit liver and brain. Immunology, 7, 1. AUER, I.O. & MILGROM, F. (1972) Studies on thermostable liver-specific antigens. Int. Arch. Allergy appl. Immunol. 42,871. AXELSEN, N.H. (1973) Intermediate gel in crossed and in fused rocket immunoelectrophoresis. Scand. J. Immunol. 1, (Suppl. 1), 71. BAUER, K. (1969) Heterologe reaktionen von saugetierseren mit monospezifischen Antihumanseren ein beitrag zur evolution der serum proteins. II. Untersuchungen uber Praalbumin. fl2-Glycoprotein, saures xl-glycoproteine, a2-Trypsin inhibitor. Humangenetik, 7, 225. BENJAMIN D.C. & WEIGLE W.O. (1970) The termination of immunologic unresponsiveness to BSA in rabbits. II. Response to a subsequent injection of BSA. J. Immunol.

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