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ANTILYMPHOCYTE SERUM IN THE LATER STAGES OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS
569
administered. One young girl was losing as much as 115 ml. of blood daily simply as a result of taking two aspirin tablets a day. Significantly Stubbe (1963) found that young women were especially prone to aspirininduced iron-deficiency anaemia and it is this very group which has shown the rise in unexplained hair loss (Sulzberger et al. 1960). Also, salicylate ingestions may lower serum-iron in the absence of bleeding (Izak et al.
1962). Finally, one must consider the possibility that the salicylate itself is the cause of hair loss in some people. Its uncoupling effect on oxidative phosphorylation (Smith 1966) might interfere with the intense cellular activity requisite for hair production. In any event, further study is required to elucidate the significance of our finding that three out of four of the patients with unexplained hair loss were consistently taking aspirin or salicylates. We thank Mrs. Roberta Fricke and Miss Dorothy Senesky for able technical assistance, and Dr. Philip George for advice and encouragement. This work was supported by a grant from the John A. Hartford Foundation and by Clinical Research Center grant 3MOl FR-40,
Preliminary Communication ANTILYMPHOCYTE SERUM IN THE LATER STAGES OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS An
antilymphocyte serum raised by injection of guineapig thymocytes into rabbits was given to guineapigs at various stages in the development of experimental allergic encephalomyelitis. Early treatment was as effective as that given later, during the development of the inflammatory lesion in brain and cord. The serum also prolonged the survival of paralysed animals. The major effect is immunosuppressive, the although speed with which established disease is inhibited implies some peripheral influence—on the sensitised lymphocyte or on the inflammatory reaction. Summary
INTRODUCTION
ANTILYMPHOCYTE serum (A.L.S.) can greatly prolong the survival of skin homografts in animals 12 and this has encouraged its use in homograft surgery in man.34 Waksman5 found that experimental allergic encephalomyelitis could be prevented or modified by an antilymphocyte preparation, and similar results have been reported for adjuvant arthritis in rats,6 " secondary disease " in mice and monkeys,’ and the hsemolytic anxmia of NZB mice.8 If A.L.S. were to be considered for use in immune disorders in man, one of the main requirements would be an ability to act on fully established disease. We have investigated the effect of an A.L.s. on the more advanced stages of experimental allergic encephalomyelitis in
Division of Research Facilities and Resources, National Institutes of Health. Requests for reprints should be addressed to W. B. S., Department of Dermatology, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. REFERENCES
Creveling, C. R., Daly, J. W. (1967) Nature, Lond. 216, 190. Eichhorn, F., Rutenberg, A. (1966) Israel J. med. Sci. 2, 640. Hais, I. M., Macek, K. (1963) (editors) Paper Chromatography. New York and London.
Izak, G., Galewsky-Stein, K., Menzel, J., Groen, J. J. (1962) Blood, 19, 601.
Panconesi, E., Periti, P. (1966) Ital. gen. Rev. Derm. 7, 7. Pillsbury, D. M., Shelley, W. B., Kligman, A. M. (1956) Dermatology. Philadelphia. Prochazka, Z. (1963) in Paper Chromatography (edited by I. M. Hais and K. Macek); p. 245. New York and London. Ryder, M. L. (1958) in The Biology of Hair Growth (edited by W. Montagna and R. A. Ellis); p. 305. New York. Sanderson, K. C. (1967) Lancet, i, 681. Stevens, D. S. (1958) Naturwissenschaften, 9, 212. Dick, G. F. (1946) J. Lab. clin. Med. 31, 125. Schwartz, S., Zieve, L., Watson, C. J. (1951) ibid. 37, 843. Smith, M. J. H. (1966) The Salicylates; p. 313. New York. Stubbe, L. T. F. L. (1963) Salicylates; p. 236. Boston. Sulzberger, M. B., Witten, V. H., Kopf, A. W. (1960) Archs Derm. 81, 556. Van Scott, E. J. (1958) in The Biology of Hair Growth (edited by W. Montagna and R. A. Ellis); p. 441. New York. —
METHODS
Preparation of A.L.S. Guineapig thymocytes (8 x 107)
injected into New injection was given intravenously, the second intraperitoneally, and the third, with complete Freund’s adjuvant (Difco), into the footpad and subcutaneously. The rabbits were bled 1-6 weeks after the last injection and the serum was absorbed with guineapig erythrocytes. Zealand rabbits
on
were
3 successive weeks.
The first
Sensitisation Female albino guineapigs weighing 250-450 g. were sensitised with a single intradermal injection of a homologous wholebrain suspension emulsified with complete Freund’s adjuvant (Difco), to which Mycobacterium butyricum had been added to a total of 0-1 mg. per dose. Each injection (0’1 ml.) contained 2 mg. guineapig brain (dry weight). RESULTS
Comparison of Early and Late Treatment The earliest evidence of experimental allergic encetakes a week or more to develop in these animals, and severe inflammatory changes in the brain and cord 9 are rare before the ll-12th day. This experiment was designed to compare the effect of A.L.S. in the preinflammatory phase with that given later, when the lesions of encephalomyelitis normally Sensitised animals were given 2 ml. of A.L.S. appear. intraperitoneally on alternate days, either early (0-8 days)
phalitis
EARLY AND LATE A.L.S. TREATMENT
guineapigs. 1. 2. 3. 4. 5.
Woodruff, M. F. A., Anderson, N. F. Nature, Lond. 1963, 200, 702. Levey, R. H., Medawar, P. B. Ann. N.Y. Acad. Sci. 1966, 129, 164. Iwasaki, Y., Porter, K. A., Amend, J. R., Marchioro, T. L., Zuhlke, V., Starzl, T. E. Surgery Gynec. Obstet. 1967, 124, 1. Starzl, T. E., Porter, K. A., Iwasaki, Y., Marchioro, T. L., Kashiwagi, N. Ciba Fdn Study Grps 1967, no. 29, p. 4. Waksman, B. H., Arbouys, S., Arnason, B. G. J. exp. Med. 1961, 114, 997.
6. 7.
Currey, H. L. F., Ziff, M. Lancet, 1966, ii, 889. van Bekkum, D. W., Ledney, G. B., Balner, H., van Putte, L. M. de Vries, J. M. Ciba Fdn Study Grps 1967, no. 29, p. 97. 8. Denman, A. M., Denman, E. J., Holborow, E. J. Lancet, 1967, i, 1084.
late (days 10-18). In each case five doses were given. The controls received normal rabbit serum (days 0-8). Paralysed animals were killed within 24 hours and survivors on day 54. or
9.
Leibowitz, S. Jl R. Coll. Physns, 1966, 1, 85.
570 system. There is some evidence that A.L.S. is of each of these actions.5 11 12 The fact that A.L.s. given 0-8 days after sensitisation is as effective as that given from day 10-18 (table) implies a mainly immunosuppressive action. On the other hand the observation that the serum can protect paralysed animals and the speed (24 hours) with which this becomes manifest (see figure) can only be explained by a " peripheral "effect,. This could be due either to an influence upon the sensitised lymphocyte or to some non-specific
nervous
capable
anti-inflammatory action; or even to the transient lymphopenia which followed the administration of A.L.S. in these experiments.10 Like Waksmanhowever, we found that the main immunosuppressive effects in this experimental disease were not directly related to a reduction in the lympho-
Survival of
paralysed
animals after A.L.S. treatment.
All fifteen treated animals were in good health on the 20th day, although by that time 7 of the 9 sensitised As late as day 54 all controls were paralysed (table). except one were still gaining weight and showed no clinical evidence of encephalitis. Histologically, however, severe lesions were present in fourteen of the fifteen animals at this time. There was no significant difference between those treated immediately after sensitisation and those treated later. Treatment
of Paralysed Animals
Forty guineapigs were sensitised and treated (alternate animals) with either A.L.s. or normal rabbit serum on the day they became paralysed. Treatment was continued on alternate days for a total of 5 weeks. Ten of the eleven A.L.s. treated animals survived at least 24 hours (see figure), and four were still alive after 10 days. The comparable figures for the controls were four out of ten (p < 0-05, Fisher’s exact-probability test), and no 10-day survivors. Three of the A.L.s.-treated animals survived until the experiment was terminated on
day
41.
Effect of Prolonged
Treatment
In a further experiment sensitised guineapigs were treated with A.L.S. over a period of 3 weeks (ten doses) and killed on the 20th day. All the control animals showed either the neurological signs or histological evidence of experimental allergic encephalitis or both. None of the treated animals were affected.10 DISCUSSION
The prevalent view of the pathogenesis of experimental allergic encephalomyelitis is that it is a manifestation of delayed-type hypersensitivity. A.L.S. might influence this process, either by inhibiting the production of sensitised cells, or by interfering with their capacity to react with antigen. It might also exert a non-specific effect upon the lesion in the central anti-inflammatory 10.
Leibowitz, S., Lessof, M. H., Kennedy, L. Unpublished.
cyte-count. The inhibition of immune tissue damage in the nervous system by A.L.S. may be of therapeutic interest. It is comparatively long lasting (although the disease reappears after the serum is withdrawn) and treatment can be effective even after the onset of paralysis. This work was supported by a grant from the Multiple Sclerosis Society to one of us (S. L.). We wish to thank Mr. T. R. Nichols of the interdepartmental laboratory for technical assistance. S. LEIBOWITZ Department of Pathology, M.B., B.SC., W’srand, M.C.PATH Guy’s Hospital, London S.E.1 LESLEY A. KENNEDY M. H. LESSOF Department of Medicine, M.D. Cantab., M.R.C.P. Guy’s Hospital, London S.E.1
Hypothesis PATHOGENESIS OF HEART-FAILURE IN ACUTE-ON-CHRONIC RESPIRATORY FAILURE
PULMONARY hypertension at rest or on exercise is in many patients with chronic lung disease even when they are " at their best ". However, exacerbations of pulmonary hypertension, commonly with superadded right-heart failure, are commonest during episodes of acute-on-chronic respiratory insufficiency due to bronchopulmonary infection. There remains considerable uncertainty as to the pathogenesis of these cardiovascular
demonstrable
events.
Partial destruction or obstruction of the pulmonary vascular bed by the primary lung disease probably " sets the stage " by reducing the reserve capacity of conducting vessels in the pulmonary circulation. It has been suggested that the polycythxmia found in many patients with chronic hypoxaemia may further increase the load on the right ventricle by increasing blood viscosity. On this background it is classically held that further hypoxia and acidosis 13associated with an exacerbation of the pulmonary disease lead to superadded pulmonary vasoconstriction with further increases of pulmonary-artery pressure. Fluid retention may be aggravated by the metabolic consequences of hypoxaemia and hypercapnia.14 Successful treatment of the heart-failure is usually associated with 11. 12.
Turk, J. L., Willoughby, D. A. Lancet, 1967, i, 249. Monaco, A. P., Wood, M. L., Russell, P. S. Transplantation, 1967, 5,
1106. 13. Enson,
Y., Giuntini, C., Lewis, M. L., Morris, T. Q., Ferrer, M. L, Harvev, R. M. J. clin. Invest. 1964, 43, 1146. 14. Aber, G. M., Bayley, T. J., Bishop, J. M. Clin. Sci. 1963, 25, 159.
administered. One young girl was losing as much as 115 ml. of blood daily simply as a result of taking two aspirin tablets a day. Significantly Stubbe (1963) found that young women were especially prone to aspirininduced iron-deficiency anaemia and it is this very group which has shown the rise in unexplained hair loss (Sulzberger et al. 1960). Also, salicylate ingestions may lower serum-iron in the absence of bleeding (Izak et al.
1962). Finally, one must consider the possibility that the salicylate itself is the cause of hair loss in some people. Its uncoupling effect on oxidative phosphorylation (Smith 1966) might interfere with the intense cellular activity requisite for hair production. In any event, further study is required to elucidate the significance of our finding that three out of four of the patients with unexplained hair loss were consistently taking aspirin or salicylates. We thank Mrs. Roberta Fricke and Miss Dorothy Senesky for able technical assistance, and Dr. Philip George for advice and encouragement. This work was supported by a grant from the John A. Hartford Foundation and by Clinical Research Center grant 3MOl FR-40,
Preliminary Communication ANTILYMPHOCYTE SERUM IN THE LATER STAGES OF EXPERIMENTAL ALLERGIC ENCEPHALOMYELITIS An
antilymphocyte serum raised by injection of guineapig thymocytes into rabbits was given to guineapigs at various stages in the development of experimental allergic encephalomyelitis. Early treatment was as effective as that given later, during the development of the inflammatory lesion in brain and cord. The serum also prolonged the survival of paralysed animals. The major effect is immunosuppressive, the although speed with which established disease is inhibited implies some peripheral influence—on the sensitised lymphocyte or on the inflammatory reaction. Summary
INTRODUCTION
ANTILYMPHOCYTE serum (A.L.S.) can greatly prolong the survival of skin homografts in animals 12 and this has encouraged its use in homograft surgery in man.34 Waksman5 found that experimental allergic encephalomyelitis could be prevented or modified by an antilymphocyte preparation, and similar results have been reported for adjuvant arthritis in rats,6 " secondary disease " in mice and monkeys,’ and the hsemolytic anxmia of NZB mice.8 If A.L.S. were to be considered for use in immune disorders in man, one of the main requirements would be an ability to act on fully established disease. We have investigated the effect of an A.L.s. on the more advanced stages of experimental allergic encephalomyelitis in
Division of Research Facilities and Resources, National Institutes of Health. Requests for reprints should be addressed to W. B. S., Department of Dermatology, Hospital of the University of Pennsylvania, Philadelphia, Pennsylvania 19104, U.S.A. REFERENCES
Creveling, C. R., Daly, J. W. (1967) Nature, Lond. 216, 190. Eichhorn, F., Rutenberg, A. (1966) Israel J. med. Sci. 2, 640. Hais, I. M., Macek, K. (1963) (editors) Paper Chromatography. New York and London.
Izak, G., Galewsky-Stein, K., Menzel, J., Groen, J. J. (1962) Blood, 19, 601.
Panconesi, E., Periti, P. (1966) Ital. gen. Rev. Derm. 7, 7. Pillsbury, D. M., Shelley, W. B., Kligman, A. M. (1956) Dermatology. Philadelphia. Prochazka, Z. (1963) in Paper Chromatography (edited by I. M. Hais and K. Macek); p. 245. New York and London. Ryder, M. L. (1958) in The Biology of Hair Growth (edited by W. Montagna and R. A. Ellis); p. 305. New York. Sanderson, K. C. (1967) Lancet, i, 681. Stevens, D. S. (1958) Naturwissenschaften, 9, 212. Dick, G. F. (1946) J. Lab. clin. Med. 31, 125. Schwartz, S., Zieve, L., Watson, C. J. (1951) ibid. 37, 843. Smith, M. J. H. (1966) The Salicylates; p. 313. New York. Stubbe, L. T. F. L. (1963) Salicylates; p. 236. Boston. Sulzberger, M. B., Witten, V. H., Kopf, A. W. (1960) Archs Derm. 81, 556. Van Scott, E. J. (1958) in The Biology of Hair Growth (edited by W. Montagna and R. A. Ellis); p. 441. New York. —
METHODS
Preparation of A.L.S. Guineapig thymocytes (8 x 107)
injected into New injection was given intravenously, the second intraperitoneally, and the third, with complete Freund’s adjuvant (Difco), into the footpad and subcutaneously. The rabbits were bled 1-6 weeks after the last injection and the serum was absorbed with guineapig erythrocytes. Zealand rabbits
on
were
3 successive weeks.
The first
Sensitisation Female albino guineapigs weighing 250-450 g. were sensitised with a single intradermal injection of a homologous wholebrain suspension emulsified with complete Freund’s adjuvant (Difco), to which Mycobacterium butyricum had been added to a total of 0-1 mg. per dose. Each injection (0’1 ml.) contained 2 mg. guineapig brain (dry weight). RESULTS
Comparison of Early and Late Treatment The earliest evidence of experimental allergic encetakes a week or more to develop in these animals, and severe inflammatory changes in the brain and cord 9 are rare before the ll-12th day. This experiment was designed to compare the effect of A.L.S. in the preinflammatory phase with that given later, when the lesions of encephalomyelitis normally Sensitised animals were given 2 ml. of A.L.S. appear. intraperitoneally on alternate days, either early (0-8 days)
phalitis
EARLY AND LATE A.L.S. TREATMENT
guineapigs. 1. 2. 3. 4. 5.
Woodruff, M. F. A., Anderson, N. F. Nature, Lond. 1963, 200, 702. Levey, R. H., Medawar, P. B. Ann. N.Y. Acad. Sci. 1966, 129, 164. Iwasaki, Y., Porter, K. A., Amend, J. R., Marchioro, T. L., Zuhlke, V., Starzl, T. E. Surgery Gynec. Obstet. 1967, 124, 1. Starzl, T. E., Porter, K. A., Iwasaki, Y., Marchioro, T. L., Kashiwagi, N. Ciba Fdn Study Grps 1967, no. 29, p. 4. Waksman, B. H., Arbouys, S., Arnason, B. G. J. exp. Med. 1961, 114, 997.
6. 7.
Currey, H. L. F., Ziff, M. Lancet, 1966, ii, 889. van Bekkum, D. W., Ledney, G. B., Balner, H., van Putte, L. M. de Vries, J. M. Ciba Fdn Study Grps 1967, no. 29, p. 97. 8. Denman, A. M., Denman, E. J., Holborow, E. J. Lancet, 1967, i, 1084.
late (days 10-18). In each case five doses were given. The controls received normal rabbit serum (days 0-8). Paralysed animals were killed within 24 hours and survivors on day 54. or
9.
Leibowitz, S. Jl R. Coll. Physns, 1966, 1, 85.
570 system. There is some evidence that A.L.S. is of each of these actions.5 11 12 The fact that A.L.s. given 0-8 days after sensitisation is as effective as that given from day 10-18 (table) implies a mainly immunosuppressive action. On the other hand the observation that the serum can protect paralysed animals and the speed (24 hours) with which this becomes manifest (see figure) can only be explained by a " peripheral "effect,. This could be due either to an influence upon the sensitised lymphocyte or to some non-specific
nervous
capable
anti-inflammatory action; or even to the transient lymphopenia which followed the administration of A.L.S. in these experiments.10 Like Waksmanhowever, we found that the main immunosuppressive effects in this experimental disease were not directly related to a reduction in the lympho-
Survival of
paralysed
animals after A.L.S. treatment.
All fifteen treated animals were in good health on the 20th day, although by that time 7 of the 9 sensitised As late as day 54 all controls were paralysed (table). except one were still gaining weight and showed no clinical evidence of encephalitis. Histologically, however, severe lesions were present in fourteen of the fifteen animals at this time. There was no significant difference between those treated immediately after sensitisation and those treated later. Treatment
of Paralysed Animals
Forty guineapigs were sensitised and treated (alternate animals) with either A.L.s. or normal rabbit serum on the day they became paralysed. Treatment was continued on alternate days for a total of 5 weeks. Ten of the eleven A.L.s. treated animals survived at least 24 hours (see figure), and four were still alive after 10 days. The comparable figures for the controls were four out of ten (p < 0-05, Fisher’s exact-probability test), and no 10-day survivors. Three of the A.L.s.-treated animals survived until the experiment was terminated on
day
41.
Effect of Prolonged
Treatment
In a further experiment sensitised guineapigs were treated with A.L.S. over a period of 3 weeks (ten doses) and killed on the 20th day. All the control animals showed either the neurological signs or histological evidence of experimental allergic encephalitis or both. None of the treated animals were affected.10 DISCUSSION
The prevalent view of the pathogenesis of experimental allergic encephalomyelitis is that it is a manifestation of delayed-type hypersensitivity. A.L.S. might influence this process, either by inhibiting the production of sensitised cells, or by interfering with their capacity to react with antigen. It might also exert a non-specific effect upon the lesion in the central anti-inflammatory 10.
Leibowitz, S., Lessof, M. H., Kennedy, L. Unpublished.
cyte-count. The inhibition of immune tissue damage in the nervous system by A.L.S. may be of therapeutic interest. It is comparatively long lasting (although the disease reappears after the serum is withdrawn) and treatment can be effective even after the onset of paralysis. This work was supported by a grant from the Multiple Sclerosis Society to one of us (S. L.). We wish to thank Mr. T. R. Nichols of the interdepartmental laboratory for technical assistance. S. LEIBOWITZ Department of Pathology, M.B., B.SC., W’srand, M.C.PATH Guy’s Hospital, London S.E.1 LESLEY A. KENNEDY M. H. LESSOF Department of Medicine, M.D. Cantab., M.R.C.P. Guy’s Hospital, London S.E.1
Hypothesis PATHOGENESIS OF HEART-FAILURE IN ACUTE-ON-CHRONIC RESPIRATORY FAILURE
PULMONARY hypertension at rest or on exercise is in many patients with chronic lung disease even when they are " at their best ". However, exacerbations of pulmonary hypertension, commonly with superadded right-heart failure, are commonest during episodes of acute-on-chronic respiratory insufficiency due to bronchopulmonary infection. There remains considerable uncertainty as to the pathogenesis of these cardiovascular
demonstrable
events.
Partial destruction or obstruction of the pulmonary vascular bed by the primary lung disease probably " sets the stage " by reducing the reserve capacity of conducting vessels in the pulmonary circulation. It has been suggested that the polycythxmia found in many patients with chronic hypoxaemia may further increase the load on the right ventricle by increasing blood viscosity. On this background it is classically held that further hypoxia and acidosis 13associated with an exacerbation of the pulmonary disease lead to superadded pulmonary vasoconstriction with further increases of pulmonary-artery pressure. Fluid retention may be aggravated by the metabolic consequences of hypoxaemia and hypercapnia.14 Successful treatment of the heart-failure is usually associated with 11. 12.
Turk, J. L., Willoughby, D. A. Lancet, 1967, i, 249. Monaco, A. P., Wood, M. L., Russell, P. S. Transplantation, 1967, 5,
1106. 13. Enson,
Y., Giuntini, C., Lewis, M. L., Morris, T. Q., Ferrer, M. L, Harvev, R. M. J. clin. Invest. 1964, 43, 1146. 14. Aber, G. M., Bayley, T. J., Bishop, J. M. Clin. Sci. 1963, 25, 159.