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Inhalation or ingestion of organic or inorganic mercurials produces auto-immune disease in rats
CLINICAL.
IMMUNDlObY
Inhalation
AND
20, 129~135
IMMUNOPAfHOLObY
(1981)
or Ingestion of Organic or Inorganic Mercurials Produces Auto-immune Disease in Rats
J. F. BERNAUDIN,*
E. DRUET,~
Received
November
P. DRUET,~
AND R. MASSEY
5. 1980
Brown-Norway rats receiving mercurials either by inhalation or by ingestion developed a systemic autoimmune disease. Low mercurial doses close to those encountered in the environment were effective in producing autoimmune disease. This finding supports a role for environmental hazards in some autoimmune diseases in man.
INTRODUCTION
Substances present in the environment may injure tissues by their toxic effects but they may also trigger immune reactions both in man (1) and in experimental animals (2-6). Mercurial compounds, which pollute water, food, and air (7- 1l), can induce autoimmune phenomena in rats (2-5) and rabbits (6). Subcutaneous HgCl, injections induce antiglomerular basement membrane (GBM) antibodies and then an immune complex type disease, in Brown-Norway (BN) rat (2.3) and in rabbit (6). Immunologically mediated glomerulonephritis has also been reported in man after chronic occupational exposure to mercurials (12). As these substances may enter the body by the digestive and respiratory routes (7- 111, the aim of the present study was to demonstrate that chronic ingestion or inhalation of HgCl, and CH,,HgCl in BN rats, even in small doses. produces a systemic autoimmune disease. MATERIALS
AND METHODS
Animds. Male and female BN rats, 8 to 10 weeks old, were purchased from the
CSEAL
(OrlCans, La Source. France).
by th@wspircrto~ route. HgCl, was given either by intratracheal instillation or by aerosol. Intratracheal instillations of a constant volume (0.05 ml/l00 g body weight) of HgCl, solutions, at various concentrations, were performed once a week for 2 weeks to 2 months. Concentrations used ranged from 6 to 75 pg/lOO g body weight per week (Table 1). The lowest doses of HgCl, were calculated with reference to the maximum allowable air concentrations (0.10 mg Hg/m3) for a man (11). Aerosols were administered using a Bird micronebulizer in an exposure chamber as previously described (13): 5 ml of a 1% HgCl, solution were nebulized for five rats in 1 hr of aerosolization. Using 203 or 197 Hg+ it was HgCl, intoxication
129 0090.1229/81/0’70129-07$01.00/O Copyright All rights
0 1981 hy Academic Press. Inc. of reprodrictlon in any form reserved.
130 / / I, I I :/
BRIEF
COMMUNICATIONS
131
shown that 5 to 6 pg of HgCl, were retained per hour of aerosol per 100 grams body weight. This deposited fraction was defined as the total amount of mercury found in the rat except skin and hair at the end of exposure. (Masse R., unpublished observation). Exposure time was 4 hr/week for 2 months (Table 1). HgC’I, intoxication by the digestive route. In BN rats, the ingestion of HgCl, was achieved by forcible feeding at a dose of 300 &week/100 g body weight for 2 months (Table 1). CH,&gCI intoxication. CH,HgCl was administered either by aerosol or by ingestion as described above for HgCl,. Aerosols were administered with a 1% CH:,HgCl solution 4 hr/week for 2 months (Table 1). Forcible feeding was performed at a dose of 600 pg/week/lOO g body weight for 2 months (Table 1). CH,,HgCl was also injected subcutaneously at a dose of 450 pg/week/lOO g body weight for 2 months (Table 1). Control tars. Twenty-two control BN rats were used. Ten were injected subcutaneously with acid water brought to the same pH (pH 4) as HgCl, solution (2). Seven rats received acid water by aerosolization and five by forcible feeding (Table 1). It?lnllrnornorphologi~~~l studies. Immunofluorescent studies and light microscopy were performed on kidney biopsies (Day 1.5)and on kidney, lung, and spleen at sacrifice as previously described (2, 4). A fluoresceinated sheep anti-rat IgG antiserum was used for direct immunofluorescent studies (2). Indirect i,nmlrnoJluoresc.ent studies. Kidney acid eluates were obtained from rat kidneys pooled according to the pattern of fixation of the fluoresceinated anti-rat IgG antiserum. These eluates were obtained according to Lerner, et CL/.(14). IgG was isolated from rat sera by chromatography using DEAE-cellulose column equilibrated with 0.017 M phosphate sodium buffer, pH 6.9. Kidney acid eluates, rat sera, or their IgG fractions were tested by indirect immunofluorescence using normal kidney cryostat sections as a target. Protc~itzrrrirr. Rats were maintained in metabolic cages for 24 hr before sacrifice with free access to water but without food. Proteinuria was measured using the biuret method and was expressed either as individual values or for some groups as mean proteinuria (mgiday) + SD. RESULTS 1~~1~~111~1~~~~1orph(~l(~gi(.~~l Results
On Day 15 of intoxication, biopsy specimens from most rats (Table 1) exhibited a linear pattern of fixation of the fluoresceinated anti-rat IgG antiserum along the glomerular capillary wall and mesangium (Fig. la): however, no fixation was observed in rats intoxicated with the lowest doses of HgCl, given by intratracheal instillation nor in rats which had received CH:,HgCl by forcible feeding. Control rats were negative. At time of sacrifice, on Day 60, a granular or mixed (both linear and granular) pattern of fixation of the anti-rat IgG conjugate was observed along the glomerular capillary wall and/or the mesangium, in all the rats tested except in rats intoxicated with the lowest dose of HgCl? by intratracheal intoxication. A granular pattern of fixation was seen in the wall of kidney arteries (Fig. lb), in the lung
132
BRItF
COMMLNICA
I-IONS
FIG. 1. (a) BN rat chronically intoxicated with an aerosol of CH,HgCI for I5 days. Note the linear pattern of fixation of the anti-rat IgG conjugate along the giomentlar capillarv wall and mesangium (X 570). (b) BN rat chronically intoxicated with CH,HgCI injected subcutaneously for 60 days. Note the granular pattern of fixation ofthe anti-rat IgG conjugate on the wall of a kidney artery (X 1300). (c) Lung of a BN rat chronically intoxicated with an aerosol of HgCl, for 60 days. Note the granular pattern of fixation of the anti-rat IgG conjugate aiong the alvcola. r capillary wall and in the wall of a small artery (x510). (d) Normal kidney cryostat section incubated with kidney acid eluate from BN rats intoxicated by aerosols of HgCl,. Note the linear pattern of fixation of the anti-rat IgG conjugate along the glomerular capillary wall and mesangium (X 580).
134
BRIEF ~OMMUNI~A~I‘IONS
vessels and interstitium (Fig. lc). and/or in the white pulp ofthe spleen of.39 ot‘thc 40 rats examined (Table 1). No fixation was observed in control rats. No ma.jor abnormality was seen under light microscopy either in experimental or control rats.
Mean proteinuria in the 22 control rats was I.0 ir 2.7 mgiday. No significant proteinuria was found in the 17 rats which received HgCI, by intratracheal instillation (5.8 + 6.5 mg/day) or in the 6 rats which received CH,HgCI by the digestive route (4. I t 1.0 mg/day). A weak proteinuria was observed at time of sacrifice in 2 out of the 3 rats which received HgCI, by aerosol (respectively: 47. 28. and I mg/day). in 3 out of the S rats fed with HgCI,, (54. 26, 23, 7. and I mgiday). and in 1 rat out of the 5 intoxicated with CH:,HgCl given by aerosol (44. 8.X. 1.1. and 1 mgiday). A heavier proteinuria was found in the rats which received subcutaneous injections of CH,,HgCI (134 F 127 mgiday): among these 12 rats. 8 had a proteinuria. No significant fixation could be obtained on any normal kidney structure when rat sera or their IgG fraction were tested by indirect immunofluorescence. Three kidney acid eluates were tested: one obtained from rats intoxicated by intratracheal instillation with a linear pattern of fixation at time of sacrifice: another one from rats intoxicated by aerosols of HgCI, with a granular pattern of fixation at time of sacrifice, and the third one from the rats subcutaneously injected with CH,,HgCI. A linear pattern of fixation along the glomerular capillary wall and mesangium was obtained when each of these eluates was tested by indirect immunofluorescence using normal kidney cryostat section as a target (Fig. Id). DISCUSSION In the present experiment, rats receiving HgCI, or CH,,HgCl either by the respiratory or the digestive route, developed a biphasic systemic disease similar to that described after subcutaneous injection of HgCI, (2-4). Anti-GBM antibodies were eluted from diseased kidneys with either a linear or granular pattern of fixation of the anti-rat IgG conjugate. Some of these rats had proteinuria. Three major findings emerge from this study. (i) First, an autoimmune disease can be induced by inhalation or ingestion of a toxic agent. This had not been experimentally demonstrated previously but was suggested from .data in man: immune glomerulonephritis has been reported in man after therapeutic t 15. 16) or occupational exposure to mercury (12). or heavy (17. 18) or chronic (19) hydrocarbon exposure. Glomerulonephritis has also been induced in rats intoxicated with hydrocarbon derivatives (20, 21) but no immunomorphological study was performed so that its immune mechanism was not demonstrated. (ii) It must be emphasized that among the three rats which received the lowest doses of HgCI,, two had granular IgG deposits in the spleen vessels only but not in the kidney. This suggests that the spleen could be a preferential site of deposition or formation of immune com-
BRIEF
COMMUNICATIONS
135
plexes. (iii) The most important point is that when HgCl, was given by intratracheal instillation at very low doses, autoimmune manifestations were still encountered. In this situation, HgCl,, doses administered corresponded to the amount inhaled by humans breathing air containing mercury at the maximum allowable concentrations (II). Therefore, these results suggest that similar autoimmune manifestations could also occur in man exposed chronically to mercury. A genetic predisposition is probably most important as previously shown in the rat (22). We think these findings could be of major significance in view of the environmental hazards. ACKNOWLEDGMENTS This work was supported by a grant from the Minis&e de la Culture et de I’Environnement. We wish to thank Laurence Meurisse for typing the manuscript.
78/44.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Kazantzis, Sapin. C.. Druet. P., Bernaudin,
G., E~7Gwr. Her1/r/7 P~rsprcr. 25. I1 1. 1978. Druet, E., and Druet, P., Cli,~. Eup. ftt7~771170~. 28, 173. 1977. Druet. E., Potdevin, F., and Sapin. C., A/I/~. I/77177ro70/. (PNY~s) 129 C, 777. 1978. ’ J. F., Druet. E.. Belair. M. F., Pinchon. M. C., Sapin, C., and Druet. P.. Clip. E.rp. Iu7v77rn1d. 38, 265. 1979. Weening. J. J., Fleuren, G. J.. and Hoedemaeker, P. J., Ltrh. It7w.st. 39, 405, 1978. Roman-France. A. A.. Turiello, M., Albini, B.. Ossi. E., Milgrom. F., and Andres. G. A., C/in. lo7mtrr7ol. //77,t7rtr7opcrfhc1/. 9, 464. 1978. Eyl. T., N. Ewyl. J. Med. 284. 706, 1971. Kazantzis. G.. I/71. J. E771~i,o77. Stud. 1, 301. 1971. Cross. J. D., Dale, 1. M.. Goolard. L., Lenihan. J. M. A.. and Smith, H.. Ltrrwr 2. 312, 1978. Gardner, D.. Ntrrwr (L~m/o~r) 272, 49, 1978. Report of an International Committee, Arc,h. E/71~imur. Hrr1///7 19. 891, 1969. Kazantzis. G.. Shiller, K.. Asscher. A., and Drew, R. G.. Qmvr. J. Mctl. 31, 403, 1962. Metivier. H., Masse. R., Rateau. G., and Lafuma. J.. Hdrh PI7y.c. 38, 769. 1980. Lerner. R. A., Glassock, R. J., and Dixon. F. J.. J. E.rp. Med. 126, 989, 1967. Mandema, E., Arends, E.. Van Zeijst, J.. Vermeer, G., Van Der Hem, G. K., and Van Der Slike, L. B., Ltr/7c,c,/ 1. 1266. 1963. Schreiner. G. E.. and Maher, J. F.. AITIPI.. J. Mctl. 38, 409, 1965. Beirne. G. J., and Brennan. J. T.. Arc/r. Et719ico~7. Hctdrh 25, 365. 1972. Kleinknecht, D.. Morel-Maroger. L., Callard. P., Adhemar. J. P., and Mahieu, P., AK/I. l77f(‘177. Mrtl. 140. 230. 1980. Ehrenreich. T.. Yunis. S. L.. and Churg. J.. Cr\~ir~~n. Rcs. 14. 35, 1977. Klavis, G.. and Drommer, W.. AC/I. TcriXol. 26. 40, 1970. Harman. J. W.. J. Pofhd. 104, 119. 1971. Druet, E.. Sapin. C., Giinther, E., Feingold. N.. and Druet, P.. Eur. J. f7?7~7177770/. 7. 348. 1977.
IMMUNDlObY
Inhalation
AND
20, 129~135
IMMUNOPAfHOLObY
(1981)
or Ingestion of Organic or Inorganic Mercurials Produces Auto-immune Disease in Rats
J. F. BERNAUDIN,*
E. DRUET,~
Received
November
P. DRUET,~
AND R. MASSEY
5. 1980
Brown-Norway rats receiving mercurials either by inhalation or by ingestion developed a systemic autoimmune disease. Low mercurial doses close to those encountered in the environment were effective in producing autoimmune disease. This finding supports a role for environmental hazards in some autoimmune diseases in man.
INTRODUCTION
Substances present in the environment may injure tissues by their toxic effects but they may also trigger immune reactions both in man (1) and in experimental animals (2-6). Mercurial compounds, which pollute water, food, and air (7- 1l), can induce autoimmune phenomena in rats (2-5) and rabbits (6). Subcutaneous HgCl, injections induce antiglomerular basement membrane (GBM) antibodies and then an immune complex type disease, in Brown-Norway (BN) rat (2.3) and in rabbit (6). Immunologically mediated glomerulonephritis has also been reported in man after chronic occupational exposure to mercurials (12). As these substances may enter the body by the digestive and respiratory routes (7- 111, the aim of the present study was to demonstrate that chronic ingestion or inhalation of HgCl, and CH,,HgCl in BN rats, even in small doses. produces a systemic autoimmune disease. MATERIALS
AND METHODS
Animds. Male and female BN rats, 8 to 10 weeks old, were purchased from the
CSEAL
(OrlCans, La Source. France).
by th@wspircrto~ route. HgCl, was given either by intratracheal instillation or by aerosol. Intratracheal instillations of a constant volume (0.05 ml/l00 g body weight) of HgCl, solutions, at various concentrations, were performed once a week for 2 weeks to 2 months. Concentrations used ranged from 6 to 75 pg/lOO g body weight per week (Table 1). The lowest doses of HgCl, were calculated with reference to the maximum allowable air concentrations (0.10 mg Hg/m3) for a man (11). Aerosols were administered using a Bird micronebulizer in an exposure chamber as previously described (13): 5 ml of a 1% HgCl, solution were nebulized for five rats in 1 hr of aerosolization. Using 203 or 197 Hg+ it was HgCl, intoxication
129 0090.1229/81/0’70129-07$01.00/O Copyright All rights
0 1981 hy Academic Press. Inc. of reprodrictlon in any form reserved.
130 / / I, I I :/
BRIEF
COMMUNICATIONS
131
shown that 5 to 6 pg of HgCl, were retained per hour of aerosol per 100 grams body weight. This deposited fraction was defined as the total amount of mercury found in the rat except skin and hair at the end of exposure. (Masse R., unpublished observation). Exposure time was 4 hr/week for 2 months (Table 1). HgC’I, intoxication by the digestive route. In BN rats, the ingestion of HgCl, was achieved by forcible feeding at a dose of 300 &week/100 g body weight for 2 months (Table 1). CH,&gCI intoxication. CH,HgCl was administered either by aerosol or by ingestion as described above for HgCl,. Aerosols were administered with a 1% CH:,HgCl solution 4 hr/week for 2 months (Table 1). Forcible feeding was performed at a dose of 600 pg/week/lOO g body weight for 2 months (Table 1). CH,,HgCl was also injected subcutaneously at a dose of 450 pg/week/lOO g body weight for 2 months (Table 1). Control tars. Twenty-two control BN rats were used. Ten were injected subcutaneously with acid water brought to the same pH (pH 4) as HgCl, solution (2). Seven rats received acid water by aerosolization and five by forcible feeding (Table 1). It?lnllrnornorphologi~~~l studies. Immunofluorescent studies and light microscopy were performed on kidney biopsies (Day 1.5)and on kidney, lung, and spleen at sacrifice as previously described (2, 4). A fluoresceinated sheep anti-rat IgG antiserum was used for direct immunofluorescent studies (2). Indirect i,nmlrnoJluoresc.ent studies. Kidney acid eluates were obtained from rat kidneys pooled according to the pattern of fixation of the fluoresceinated anti-rat IgG antiserum. These eluates were obtained according to Lerner, et CL/.(14). IgG was isolated from rat sera by chromatography using DEAE-cellulose column equilibrated with 0.017 M phosphate sodium buffer, pH 6.9. Kidney acid eluates, rat sera, or their IgG fractions were tested by indirect immunofluorescence using normal kidney cryostat sections as a target. Protc~itzrrrirr. Rats were maintained in metabolic cages for 24 hr before sacrifice with free access to water but without food. Proteinuria was measured using the biuret method and was expressed either as individual values or for some groups as mean proteinuria (mgiday) + SD. RESULTS 1~~1~~111~1~~~~1orph(~l(~gi(.~~l Results
On Day 15 of intoxication, biopsy specimens from most rats (Table 1) exhibited a linear pattern of fixation of the fluoresceinated anti-rat IgG antiserum along the glomerular capillary wall and mesangium (Fig. la): however, no fixation was observed in rats intoxicated with the lowest doses of HgCl, given by intratracheal instillation nor in rats which had received CH:,HgCl by forcible feeding. Control rats were negative. At time of sacrifice, on Day 60, a granular or mixed (both linear and granular) pattern of fixation of the anti-rat IgG conjugate was observed along the glomerular capillary wall and/or the mesangium, in all the rats tested except in rats intoxicated with the lowest dose of HgCl? by intratracheal intoxication. A granular pattern of fixation was seen in the wall of kidney arteries (Fig. lb), in the lung
132
BRItF
COMMLNICA
I-IONS
FIG. 1. (a) BN rat chronically intoxicated with an aerosol of CH,HgCI for I5 days. Note the linear pattern of fixation of the anti-rat IgG conjugate along the giomentlar capillarv wall and mesangium (X 570). (b) BN rat chronically intoxicated with CH,HgCI injected subcutaneously for 60 days. Note the granular pattern of fixation ofthe anti-rat IgG conjugate on the wall of a kidney artery (X 1300). (c) Lung of a BN rat chronically intoxicated with an aerosol of HgCl, for 60 days. Note the granular pattern of fixation of the anti-rat IgG conjugate aiong the alvcola. r capillary wall and in the wall of a small artery (x510). (d) Normal kidney cryostat section incubated with kidney acid eluate from BN rats intoxicated by aerosols of HgCl,. Note the linear pattern of fixation of the anti-rat IgG conjugate along the glomerular capillary wall and mesangium (X 580).
134
BRIEF ~OMMUNI~A~I‘IONS
vessels and interstitium (Fig. lc). and/or in the white pulp ofthe spleen of.39 ot‘thc 40 rats examined (Table 1). No fixation was observed in control rats. No ma.jor abnormality was seen under light microscopy either in experimental or control rats.
Mean proteinuria in the 22 control rats was I.0 ir 2.7 mgiday. No significant proteinuria was found in the 17 rats which received HgCI, by intratracheal instillation (5.8 + 6.5 mg/day) or in the 6 rats which received CH,HgCI by the digestive route (4. I t 1.0 mg/day). A weak proteinuria was observed at time of sacrifice in 2 out of the 3 rats which received HgCI, by aerosol (respectively: 47. 28. and I mg/day). in 3 out of the S rats fed with HgCI,, (54. 26, 23, 7. and I mgiday). and in 1 rat out of the 5 intoxicated with CH:,HgCl given by aerosol (44. 8.X. 1.1. and 1 mgiday). A heavier proteinuria was found in the rats which received subcutaneous injections of CH,,HgCI (134 F 127 mgiday): among these 12 rats. 8 had a proteinuria. No significant fixation could be obtained on any normal kidney structure when rat sera or their IgG fraction were tested by indirect immunofluorescence. Three kidney acid eluates were tested: one obtained from rats intoxicated by intratracheal instillation with a linear pattern of fixation at time of sacrifice: another one from rats intoxicated by aerosols of HgCI, with a granular pattern of fixation at time of sacrifice, and the third one from the rats subcutaneously injected with CH,,HgCI. A linear pattern of fixation along the glomerular capillary wall and mesangium was obtained when each of these eluates was tested by indirect immunofluorescence using normal kidney cryostat section as a target (Fig. Id). DISCUSSION In the present experiment, rats receiving HgCI, or CH,,HgCl either by the respiratory or the digestive route, developed a biphasic systemic disease similar to that described after subcutaneous injection of HgCI, (2-4). Anti-GBM antibodies were eluted from diseased kidneys with either a linear or granular pattern of fixation of the anti-rat IgG conjugate. Some of these rats had proteinuria. Three major findings emerge from this study. (i) First, an autoimmune disease can be induced by inhalation or ingestion of a toxic agent. This had not been experimentally demonstrated previously but was suggested from .data in man: immune glomerulonephritis has been reported in man after therapeutic t 15. 16) or occupational exposure to mercury (12). or heavy (17. 18) or chronic (19) hydrocarbon exposure. Glomerulonephritis has also been induced in rats intoxicated with hydrocarbon derivatives (20, 21) but no immunomorphological study was performed so that its immune mechanism was not demonstrated. (ii) It must be emphasized that among the three rats which received the lowest doses of HgCI,, two had granular IgG deposits in the spleen vessels only but not in the kidney. This suggests that the spleen could be a preferential site of deposition or formation of immune com-
BRIEF
COMMUNICATIONS
135
plexes. (iii) The most important point is that when HgCl, was given by intratracheal instillation at very low doses, autoimmune manifestations were still encountered. In this situation, HgCl,, doses administered corresponded to the amount inhaled by humans breathing air containing mercury at the maximum allowable concentrations (II). Therefore, these results suggest that similar autoimmune manifestations could also occur in man exposed chronically to mercury. A genetic predisposition is probably most important as previously shown in the rat (22). We think these findings could be of major significance in view of the environmental hazards. ACKNOWLEDGMENTS This work was supported by a grant from the Minis&e de la Culture et de I’Environnement. We wish to thank Laurence Meurisse for typing the manuscript.
78/44.
REFERENCES I. 2. 3. 4. 5. 6. 7. 8. 9. 10. I I. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.
Kazantzis, Sapin. C.. Druet. P., Bernaudin,
G., E~7Gwr. Her1/r/7 P~rsprcr. 25. I1 1. 1978. Druet, E., and Druet, P., Cli,~. Eup. ftt7~771170~. 28, 173. 1977. Druet. E., Potdevin, F., and Sapin. C., A/I/~. I/77177ro70/. (PNY~s) 129 C, 777. 1978. ’ J. F., Druet. E.. Belair. M. F., Pinchon. M. C., Sapin, C., and Druet. P.. Clip. E.rp. Iu7v77rn1d. 38, 265. 1979. Weening. J. J., Fleuren, G. J.. and Hoedemaeker, P. J., Ltrh. It7w.st. 39, 405, 1978. Roman-France. A. A.. Turiello, M., Albini, B.. Ossi. E., Milgrom. F., and Andres. G. A., C/in. lo7mtrr7ol. //77,t7rtr7opcrfhc1/. 9, 464. 1978. Eyl. T., N. Ewyl. J. Med. 284. 706, 1971. Kazantzis. G.. I/71. J. E771~i,o77. Stud. 1, 301. 1971. Cross. J. D., Dale, 1. M.. Goolard. L., Lenihan. J. M. A.. and Smith, H.. Ltrrwr 2. 312, 1978. Gardner, D.. Ntrrwr (L~m/o~r) 272, 49, 1978. Report of an International Committee, Arc,h. E/71~imur. Hrr1///7 19. 891, 1969. Kazantzis. G.. Shiller, K.. Asscher. A., and Drew, R. G.. Qmvr. J. Mctl. 31, 403, 1962. Metivier. H., Masse. R., Rateau. G., and Lafuma. J.. Hdrh PI7y.c. 38, 769. 1980. Lerner. R. A., Glassock, R. J., and Dixon. F. J.. J. E.rp. Med. 126, 989, 1967. Mandema, E., Arends, E.. Van Zeijst, J.. Vermeer, G., Van Der Hem, G. K., and Van Der Slike, L. B., Ltr/7c,c,/ 1. 1266. 1963. Schreiner. G. E.. and Maher, J. F.. AITIPI.. J. Mctl. 38, 409, 1965. Beirne. G. J., and Brennan. J. T.. Arc/r. Et719ico~7. Hctdrh 25, 365. 1972. Kleinknecht, D.. Morel-Maroger. L., Callard. P., Adhemar. J. P., and Mahieu, P., AK/I. l77f(‘177. Mrtl. 140. 230. 1980. Ehrenreich. T.. Yunis. S. L.. and Churg. J.. Cr\~ir~~n. Rcs. 14. 35, 1977. Klavis, G.. and Drommer, W.. AC/I. TcriXol. 26. 40, 1970. Harman. J. W.. J. Pofhd. 104, 119. 1971. Druet, E.. Sapin. C., Giinther, E., Feingold. N.. and Druet, P.. Eur. J. f7?7~7177770/. 7. 348. 1977.