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EFFECTS OF ANTI-LYMPHOID SERA ON VIRAL INFECTIONS
37
Special
Articles
EFFECTS OF ANTI-LYMPHOID SERA ON VIRAL INFECTIONS MARTIN S. HIRSCH* M.D. Johns Hopkins FREDERICK A. MURPHY D.V.M. Cornell, Ph.D. California From the U.S. Department of Health, Education, and Welfare, Public Health Service, Bureau of Disease Prevention and Environmental Control, National Communicable Disease Centre, Atlanta, Georgia 30333
ANTI-LYMPHOID sera (A.L.s.) are potent suppressants of cellular immunity and have been shown to be effective in prolonging tissue-graft survival, both in experimental animals and in man.lThey have also been used to suppress adjuvant arthritis in rats3 and Coombs-positive haemolytic anaemias in NZB mice,4and have been proposed as possible therapy of certain autoimmune and allergic disorders.l The effectiveness of A.L.S. in suppressing cellular immunity may, however, increase the host’s susceptibility to viral infections and may potentiate the pathological consequences of such infections. In consideration of this possibility, studies have been performed in several laboratories on the effects of A.L.S. in experimental primary and secondary viral infections. This review will attempt to summarise these studies and explore the pos*
Present address: National Institute for Medical Research, Mill Hill, London N.W.7, England. 1. James, K. Clin. exp. Immun. 1967, 2, 615. 2. Starzl, R. E., Groth, C. G., Terasaki, P.I., Putnam, C. W., Brettschneider, L., Marchioro, T. L. Surgery Gynec. Obstet. 1968, 126, 1023. 3. Currey, H. L. F., Ziff, M. J. exp. Med. 1968, 127, 185. 4. Denman, A. M., Denman, E. J., Holborow, E. J. Lancet, 1966, ii, 841.
sible applicability of the information obtained clinical use of anti-lymphoid sera.
to
the
PRIMARY RESPONSE
Vaccinia A.L.S. effects on primary vaccinia infection in the mouse have been shown to depend upon the route of virus inoculation. Adult ICR mice pretreated with A.L.S. and inoculated intravenously with virus had more extensive disease than controls pretreated with normal rabbit serum (N.R.S.). Whereas both groups had necrotising lesions in lung and tail skin, only A.L.S.inflammatory treated animals had lesions in liver, brain, and heartvalves. Similarly, both groups had virus in their lungs, but only the A.L.s. group had detectable virus in kidney, liver, and brain. Mortality was also markedly greater in mice receiving A.L.S. than in those treated with N.R.S. (fig. 1). Vaccinia haemagglutination-inhibition (H.I.) antibody responses and interferon production were comparable in N.R.S. and A.L.S. treated groups. In contrast, the course of vaccinia infection following primary intracerebral virus inoculation was not altered by A.L.S. pretreatment. Development of clinical signs and mortality were identical in A.L.S. and N.R.S. groups (fig. 2). Lesions in both groups consisted of necrotising meningitis and encephalitis; other organs were not involved. Cellular immunity thus appears to be an important host defence mechanism following peripheral but not central vaccinia-virus inoculation.
Herpes Simplex Route of inoculation was also the most important factor determining the effects of A.L.S. in herpes-simplex in5. Hirsch, M. S., Nahmias, A. J., Murphy, F. A., Kramer, J. H. J. exp. Med. 1968, 128, 121.
Fig.
Fig.
Survival curves after intravenous administration of vaccinia virus in doses of 10-1 to 10-3 (Hirsch et al.6).
Survival curves after intracerebral administration of vaccinia virus in doses of 10-6 to 10-a (Hirsch et al.5).
l-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum and normal rabbit serum (N.R.S.) on vaccinia-virus infection.
2-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum and normal rabbit serum (N.R.S.) on vaccinia-virus infection.
38 virus infection in both adult and newborn mice.8-11 A.L.S. treatment of adults resulted in persistent viraemia and protection of mice from clinical and histological signs of infection so long as therapy was maintained. In one series of experiments, termination of treatment was followed by gradual disappearance of virasmia over a several month period,9 whereas, in another, virsemia persisted after discontinuation of treatment." Such differences may be explainable on the basis of differences in virus strain, mouse strain, or route of inoculation. Both groups had circulating complement-fixing antibodies to L.c.M. virus, despite prolonged viraemia. When newborn mice were treated with A.L.S. every three days for a week beginning on the day of birth, maturation of the cell-mediated immune system was delayed, permitting the development of an L.c.M. virus carrier state at an older age than is ordinarily possible.1O When challenged with virus at one week of age, N.R.s.-pretreated mice developed characteristic lymphocytic choriomeningitis, whereas A.L.s.-pretreated animals developed persistent virsemia and no choriomengitis despite the presence of high titres of complement-fixing antibodies to L.c.M. virus. This state of persistent viraemia and circulating Fig. 3-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum antibody was followed within three months by an immunoand normal rabbit serum (N.R.S.) on yellow-fever infection. 10-5 death at virus dilutions from 10-1 to pathological glomerulonephritis characterised by deComparison of times of (Hirsch and Murphy 6). position of both y-globulin and viral antigen in glomerular tufts. 18% of the A.L.s.-virus-treated group also developed a wasting syndrome between one and two months of age fection of mice.Morbidity and mortality following either intragenital or intraperitoneal inoculation of herpes with weight loss, alopecia, facial oedema, and stiff tail. virus in adult ICR mice were markedly increased by Histological examination showed that these animals had a marked hyperplasia of reticular cells and infiltration of pretreatment with A.L.S. Deaths in these animals were associated with necrotising meningitis and encephalitis. these cells into perivascular areas of various organs On the other hand, mortality was delayed and slightly including the kidney, liver, pancreas, lung, and brain. decreased in A.L.s. pretreated animals if the route of virus Animals receiving only A.L.S. did not develop either inoculation was intracerebral. These studies indicate that clinical wasting or histological evidence of reticular-cell cellular responsiveness may play a protective role after hyperplasia. The mechanisms by which these processes in the A.L.s.-L.c.M. treated animals remain peripheral herpes-virus challenge and a detrimental role developed These studies clearly show that cellular imobscure. if the challenge is intracerebral. munity is of utmost importance in murine L.c.M.-virus Yellow Fever infection. Three-week-old ICR mice were pretreated with either Influenza A.L.S. or N.R.S. and inoculated intracerebrally with varying Adult ICR mice were pretreated with either A.L.s. or doses of 17-D yellow-fever virus.At low dilutions of and inoculated intranasally with graded doses of N.R.S., virus, mice treated with N.R.S. developed clinical illness influenza virus.12 No effect was noted either on clinical (hypokinesis, progressive ascending flaccid paralysis, or on mortality (fig. 4). On histological of infection ruffled fur, conjunctivitis, and dehydration) one to two signs examination both groups developed severe bilateral days earlier than their counterparts treated with A.L.S. interstitial pneumonia. H.i. antibody responses and lungAs shown in fig. 3, the times of death at these dilutions virus titres were comparable in both groups. These were also delayed by treatment with A.L.S. At all dilutions, results that cellular immune are not suggest responses histological examination demonstrated marked differences important to host defences against primary intranasal between animals of both groups which had died. Whereas influenza inoculation. These studies have been conbrains of N.R.s.-treated animals had characteristic leptomeningeal and perivascular infiltrates, these findings were firmed.13 nearly absent in animals treated with A.L.S. Despite the Rauscher Leukcemia differences in histological findings, virus titres in brains oj A.L.S. greatly potentiated Rauscher-virus-induced splenanimals killed on the same days were equivalent in bott omegaly in adult ICR mice when administered before groups at all dilutions. These studies suggest that the virus inoculation or in the immediate post-inoculation cellular inflammatory reaction to yellow-fever virus it period.14 Measurement of spleen-weight in early mice plays a slight but significant potentiating part in the Rauscher-leukaemia-virus infection has been shown to be development of morbidity and mortality after intracerebra 8. Gledhill, A. W. ibid. 1967, 214, 178. virus inoculation.
Hirsch, M. S., Murphy, F. A., Russe, H. P., Hicklin, M. D. Proc. Soc. exp. Biol. Med. 1967, 125, 980. 10. Hirsch, M. S., Murphy, F. A., Hicklin, M. D. J. exp. Med. 1968, 127, 9.
Lymphocytic Choriomeningitis (L.C.M.) A.L.S. significantly modified the course of primary L.C.M 6. 7.
Hirsch, M. S., Murphy, F. A. Nature, Lond. 1967, 216, 179. nNahmias, A. J., Hirsch, M. S., Kramer, J. H., Murphy, F. A. Unpublished.
757. 11. 12. 13. 14.
Lundstedt, C., Volkert, M. Acta path. microbiol. scand. 1967, 71, 471. Hirsch, M. S., Kaye, H. Unpublished. Klein, M. Personal communication. Hirsch, M. S., Murphy, F. A. Nature, Lond. 1968, 218, 478.
39 bilateral parotid-gland tumours between two and and a half months of age, whereas none of the controls infected with polyoma virus under the same conditions developed tumours, even though they were observed for nine months. Similar results were found with C3H F/He mice; in addition, A.L.s.-polyoma-treated mice of the C3H F/He strain also developed mammary-gland and hair-follicle tumours. As with other murine oncogenic viruses, A.L.S. appear to accelerate neoplasia following polyoma-virus infection.
oped two
Fig. 4-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum and normal rabbit serum (N.R.S.) on influenza-virus infection. Survival curves after intranasal administration of influenza virus in doses of 10-3 to 10-6.
Vesicular Stomatitis In-vitro treatment of human peripheral lymphocytes with A.L.S. resulted in their being transformed to lymphoblasts.19 These blast cells exhibited increased susceptibility to vesicular-stomatitis virus infection. A hundred-fold greater titres were observed in transformed than control cultures, and the degree of enhancement was proportional to the percentage of lymphoblasts in the culture at the time of virus inoculation. These studies suggest that lymphoblastic transformation may provide new susceptible host cells for replication of viruses that would otherwise not be pathogenic. SECONDARY RESPONSE
Vaccinia
Weanling ICR mice index of disease progression,16 and in untreated mice this weight is directly proportional to the size of virus inoculum.16 A.L.S. was also effective, but to a lesser extent, in increasing spleen-weight when given three days before virus or two hours after virus. These results suggest that cell-mediated immunity plays a major part in the host defences to Rauscher-virus infection.
an accurate
Moloney Leukemia
,
Three-week-old BALB/c mice were inoculated intraperitoneally with Moloney leukaemia virus. 177 Those pretreated with A.L.S. developed an increased incidence of lymphoid neoplasms following virus inoculation. The tumours observed were not the characteristic lymphoid leukaemias seen three to six months after infection in untreated controls, but were subcutaneous reticulum-cell sarcomas developing three to five weeks after infection at the site of A.L.S. injection. These results indicate that suppression of cellular immunity in Moloney-virus infection results in acceleration and atypical development of oncogenesis. Adenovirus-12 Small doses of A.L.S. increased tumour induction by adenovirus-12 in newborn mice,either with low doses of virus in a susceptible strain (CBA) or with large doses of virus in a relatively resistant strain (CaH).18 The effects noted paralleled those found in neonatally thymectomised mice inoculated with adenovirus, indicating that the thymus-dependent cellular immune system is necessary for significant host response to oncogenic adenovirus infection.
Polyoma
vaccinated twice
by
tail
Lymphocytic Choriomeningitis’ Adult inbred AKR mice were immunised with live virus either by intraperitoneal inoculation or by exposure to infected newborn offspring.20 All had no detectable virus and high titres of L.C.M. complementfixing antibodies several weeks after exposure to virus and before serum treatment. The immunised animals were divided into three groups-one that received A.L.S. six times a week for seven weeks, one that received N.R.s. according to the same schedule, and a third that received no serum. Mice treated with A.L.S. developed virmn-iia between ten and thirty-four days after therapy was begun. Control mice, either untreated or N.R.s.-treated, did not develop virxmia. VirEemia developed in A.L.s.-treated groups despite constant and even increasingly high titres of complement fixing antibodies. These experiments show that, despite apparent immunity, A.L.S. can provoke latent L.C.M. virus infections when administered over a long
L.c.M.
Newborn C57Bl/Ka mice were infected with polyoma virus within twenty-four hours of birth and given A.l..s. subcutaneously twice weekly for three to four weeks.17 All the mice that survived longer than two months devel-
period.
15. 16. 17. 18.
19. 20. 21.
Rauscher, F. J., Allen, B. V. J. natn. Cancer Inst. 1964. 32, 269. Chirigos, M. A. Ann. N.Y. Acad. Sci. 1965, 130, 56. Allison, A. C., Law, L. W. Proc. Soc. exp. Biol. Med. 1968, 127, 207. Allison, A. C., Berman, L. D., Levey, R. H. Nature, Lond. 1967, 215, 185.
were
scarification with vaccinia virus. One month after the second vaccination these mice were divided into two groups-one received A.L.s., the other N.R.S., for one week. They were then challenged intracerebrally with large doses of vaccinia virus and observed for signs of infection. Mortalities in both groups were equivalent (23-32%), and animals of both groups which died had severe necrotising meningitis and encephalitis. No significant differences were found in pre-challenge H.I. antibody levels or in post-challenge antibody responses between A.L.s. and N.R.s. treated groups or between dead animals and survivors. Therefore, it appears that A.L.S. is not capable of abrogating secondary immunity to intracerebrally administered vaccinia virus.
DISCUSSION
The remarkable successes achieved with A.L.s. in human liver and kidney transplants by Starzl and his associates 2 21 Edelman, R., Wheelock, E. F. Lancet, 1968, i, 771. Volkert, M., Lundstedt, C. J. exp. Med. 1968, 127, 327. Starzl, R. E., Groth, C. G., Brettschneider, L., Moon, J. B., Fulginiti, V. A., Cotton, E. K., Porter, K. A. Surgery, St. Louis, 1968, 63, 549.
40 suggest that this serum or one of its derivative fractions will be extensively used in the future. Viral infections have apparently not complicated the limited use of A.L.S. in man, but have been troublesome in experimental organ and tissue transplantation in dogs and monkeys.22 23
latent L.c.M. virus infection in an immune host can also be reactivated by A.L.S., resulting in viraemia for the duration of therapy.20 Whether A.L.s. can similarly alter latent viral infections in man with such agents as herpessimplex virus or the varicella-zoster agent is not known.
This present review suggests that A.L.s. can markedly alter the course of disease in mice following infection with several different viruses. The mechanisms by which A.L.S. influence viral infections are not certain. A.L.S.treated mice are able to produce normal interferon responses as well as normal viral antibody responses. It is likely, therefore, that the effects of A.L.S. are mediated through depression of cell-mediated immunity and that the same mechanisms that produce graft prolongation following A.L.S. treatment result in increased susceptibility
Extrapolations from A.L.s. effects in experimental murine viral infections to effects in man may not be valid, and these sera may be relatively innocuous when used in human organ transplantation. Nevertheless, A.L.S. should be used with caution, particularly when administered in combination with suppressants of humoral immunity and interferon production-e.g., azathioprine and corticosteroids.2’ 28 If extrapolations from mouse to man are meaningful, one may expect to encounter severe, atypical, and perhaps neoplastic viral infections following treatment with A.L.S.
to
infection.
An alternative mechanism, proposed by Edelman and Wheelock,19 is that by the transformation of lymphocytes to lymphoblasts the susceptibility to viral infections is increased. It is unlikely, however, that this in-vitro mechanism is important in vivo. A complement-free medium is necessary in vitro to prevent cell lysis, 19 24 and this situation does not prevail in vivo. Particularly disquieting in the studies of A.L.s. effects on
viral infections is the observation that with all solid tumour and leukaemia viruses tested (polyoma, adeno-12, Moloney leukaemia, Rauscher leukxmia) oncogenicity has been increased by such treatment. These findings are in accord with other data supporting the hypothesis that cellmediated immune responses are the major host defence against virus-induced tumours and leukaemias.25 of disease following peripheral herpesvirus administration was also worsened vaccinia simplex A.L.S. Dissemination was more treatment. by prior and increased in the A.L.S. groups, mortality widespread that cellular is an important host indicating immunity defence mechanism following infection by peripheral routes. These routes most closely approximate the avenues by which natural infections are acquired. When the same viruses were administered intracerebrally, neither morbidity nor mortality was increased, indicating that cellular immunity is not an important defence mechanism against these viruses within the brain. A.L.S. lessened or delayed the mortality following intracerebral administration of L.c.M., herpes-simplex, and yellowfever viruses. It would appear that in these infections the cellular response to the virus is actually detrimental to the host.
The
course or
By depressing cellular but
not humoral responsiveness, initiate a virus-carrier state in L.C.M. virus infected mice. As has been shown by Oldstone and Dixon,26 this carrier state is associated with circulating antigen-antibody complexes. These complexes may be deposited in renal glomeruli producing an immunopathological membranous glomerulonephritis. The carrier state may also be associated with a wasting disorder characterised by marked reticular cell hyperplasia.10 A
A.L.s. can
22. 23.
24. 25. 26.
Abaza, H. M., Nolan, B., Watt, J. G., Woodruff, M. F. A. Transplantation, 1966, 4, 742. Van Bekkum, D. W., Ledney, G. D., Balner, H., van Putten, L. M., de Vries, M. J. in Antilymphocyte Serum (edited by G. E. W. Wolstenholme and M. O’Connor); p. 108. London, 1967. Greaves, M. F., Roitt, I. M., Zamir, R., Carnaghan, R. B. A. Lancet, 1967, ii, 1317. Allison, A. C. Br. med. Bull. 1967, 23, 60. Oldstone, M. B. A., Dixon, F. J. Science, N.Y. 1967, 158, 1193; Fedn Proc. Fedn Am. Socs exp. Biol. 1968, 27, 255.
27. 28.
Rytel, M. W., Kilbourne, E. D. J. exp. Med. 1966, 123, 767. Schwartz, R. S. Progr. Allergy, 1965, 9, 246.
Public Health FOOD-POISONING DUE TO COPPER IN THE MORNING TEA P. O. NICHOLAS Brist., D.C.H., D.P.H.
M.B.
DEPUTY MEDICAL OFFICER OF
HEALTH, BOLTON
An unusual form of acute food-poisoning due to copper sulphate in the water of an unserviced gas hot-water geyser is described. The morning tea brewed with the water caused acute vomiting and diarrhoea in twenty workmen. It is conceivable that unless care is taken to prevent copper corrosion in water heaters, the amount of copper in drinking-water could be raised. Though this contamination may not cause symptoms initially, copper ions could be built up in the human body over a long period and be a source of ill health. Summary
INTRODUCTION
CONTAMINATION of food by copper should be less than previously for, in general, copper utensils have been replaced by aluminium and stainless steel. Lately copper has been used in the manufacture of expensive kitchen utensils, since this metal has a high thermal conductivity. Copper is still widely used in hot-water geysers, for stainless steel is expensive, and both this and aluminium alloys are more difficult metals to weld. Where copper is still used, pure tin to plate the copper prevents any leaching of the copper into the food or drink. common
Vernon’s analysis (1967) of all causes of food-poisoning in 1966 in England and Wales shows that 48% of 8784 recorded cases were due to bacterial infection. Only 1 case was due to chemical poisoning. However, in 2350 cases (27%) no cause for the food-poisoning was found. In investigating food-poisoning outbreaks it may be that more attention should be paid to possible contamination of food and drink by chemicals, as a recent incident of copper poisoning shows. THE EPIDEMIC
At a small factory in Bolton, twenty workmen felt sick after drinking their morning tea at 8.30 A.M. Five of the workmen vomited within a few minutes, and one mac
Special
Articles
EFFECTS OF ANTI-LYMPHOID SERA ON VIRAL INFECTIONS MARTIN S. HIRSCH* M.D. Johns Hopkins FREDERICK A. MURPHY D.V.M. Cornell, Ph.D. California From the U.S. Department of Health, Education, and Welfare, Public Health Service, Bureau of Disease Prevention and Environmental Control, National Communicable Disease Centre, Atlanta, Georgia 30333
ANTI-LYMPHOID sera (A.L.s.) are potent suppressants of cellular immunity and have been shown to be effective in prolonging tissue-graft survival, both in experimental animals and in man.lThey have also been used to suppress adjuvant arthritis in rats3 and Coombs-positive haemolytic anaemias in NZB mice,4and have been proposed as possible therapy of certain autoimmune and allergic disorders.l The effectiveness of A.L.S. in suppressing cellular immunity may, however, increase the host’s susceptibility to viral infections and may potentiate the pathological consequences of such infections. In consideration of this possibility, studies have been performed in several laboratories on the effects of A.L.S. in experimental primary and secondary viral infections. This review will attempt to summarise these studies and explore the pos*
Present address: National Institute for Medical Research, Mill Hill, London N.W.7, England. 1. James, K. Clin. exp. Immun. 1967, 2, 615. 2. Starzl, R. E., Groth, C. G., Terasaki, P.I., Putnam, C. W., Brettschneider, L., Marchioro, T. L. Surgery Gynec. Obstet. 1968, 126, 1023. 3. Currey, H. L. F., Ziff, M. J. exp. Med. 1968, 127, 185. 4. Denman, A. M., Denman, E. J., Holborow, E. J. Lancet, 1966, ii, 841.
sible applicability of the information obtained clinical use of anti-lymphoid sera.
to
the
PRIMARY RESPONSE
Vaccinia A.L.S. effects on primary vaccinia infection in the mouse have been shown to depend upon the route of virus inoculation. Adult ICR mice pretreated with A.L.S. and inoculated intravenously with virus had more extensive disease than controls pretreated with normal rabbit serum (N.R.S.). Whereas both groups had necrotising lesions in lung and tail skin, only A.L.S.inflammatory treated animals had lesions in liver, brain, and heartvalves. Similarly, both groups had virus in their lungs, but only the A.L.s. group had detectable virus in kidney, liver, and brain. Mortality was also markedly greater in mice receiving A.L.S. than in those treated with N.R.S. (fig. 1). Vaccinia haemagglutination-inhibition (H.I.) antibody responses and interferon production were comparable in N.R.S. and A.L.S. treated groups. In contrast, the course of vaccinia infection following primary intracerebral virus inoculation was not altered by A.L.S. pretreatment. Development of clinical signs and mortality were identical in A.L.S. and N.R.S. groups (fig. 2). Lesions in both groups consisted of necrotising meningitis and encephalitis; other organs were not involved. Cellular immunity thus appears to be an important host defence mechanism following peripheral but not central vaccinia-virus inoculation.
Herpes Simplex Route of inoculation was also the most important factor determining the effects of A.L.S. in herpes-simplex in5. Hirsch, M. S., Nahmias, A. J., Murphy, F. A., Kramer, J. H. J. exp. Med. 1968, 128, 121.
Fig.
Fig.
Survival curves after intravenous administration of vaccinia virus in doses of 10-1 to 10-3 (Hirsch et al.6).
Survival curves after intracerebral administration of vaccinia virus in doses of 10-6 to 10-a (Hirsch et al.5).
l-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum and normal rabbit serum (N.R.S.) on vaccinia-virus infection.
2-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum and normal rabbit serum (N.R.S.) on vaccinia-virus infection.
38 virus infection in both adult and newborn mice.8-11 A.L.S. treatment of adults resulted in persistent viraemia and protection of mice from clinical and histological signs of infection so long as therapy was maintained. In one series of experiments, termination of treatment was followed by gradual disappearance of virasmia over a several month period,9 whereas, in another, virsemia persisted after discontinuation of treatment." Such differences may be explainable on the basis of differences in virus strain, mouse strain, or route of inoculation. Both groups had circulating complement-fixing antibodies to L.c.M. virus, despite prolonged viraemia. When newborn mice were treated with A.L.S. every three days for a week beginning on the day of birth, maturation of the cell-mediated immune system was delayed, permitting the development of an L.c.M. virus carrier state at an older age than is ordinarily possible.1O When challenged with virus at one week of age, N.R.s.-pretreated mice developed characteristic lymphocytic choriomeningitis, whereas A.L.s.-pretreated animals developed persistent virsemia and no choriomengitis despite the presence of high titres of complement-fixing antibodies to L.c.M. virus. This state of persistent viraemia and circulating Fig. 3-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum antibody was followed within three months by an immunoand normal rabbit serum (N.R.S.) on yellow-fever infection. 10-5 death at virus dilutions from 10-1 to pathological glomerulonephritis characterised by deComparison of times of (Hirsch and Murphy 6). position of both y-globulin and viral antigen in glomerular tufts. 18% of the A.L.s.-virus-treated group also developed a wasting syndrome between one and two months of age fection of mice.Morbidity and mortality following either intragenital or intraperitoneal inoculation of herpes with weight loss, alopecia, facial oedema, and stiff tail. virus in adult ICR mice were markedly increased by Histological examination showed that these animals had a marked hyperplasia of reticular cells and infiltration of pretreatment with A.L.S. Deaths in these animals were associated with necrotising meningitis and encephalitis. these cells into perivascular areas of various organs On the other hand, mortality was delayed and slightly including the kidney, liver, pancreas, lung, and brain. decreased in A.L.s. pretreated animals if the route of virus Animals receiving only A.L.S. did not develop either inoculation was intracerebral. These studies indicate that clinical wasting or histological evidence of reticular-cell cellular responsiveness may play a protective role after hyperplasia. The mechanisms by which these processes in the A.L.s.-L.c.M. treated animals remain peripheral herpes-virus challenge and a detrimental role developed These studies clearly show that cellular imobscure. if the challenge is intracerebral. munity is of utmost importance in murine L.c.M.-virus Yellow Fever infection. Three-week-old ICR mice were pretreated with either Influenza A.L.S. or N.R.S. and inoculated intracerebrally with varying Adult ICR mice were pretreated with either A.L.s. or doses of 17-D yellow-fever virus.At low dilutions of and inoculated intranasally with graded doses of N.R.S., virus, mice treated with N.R.S. developed clinical illness influenza virus.12 No effect was noted either on clinical (hypokinesis, progressive ascending flaccid paralysis, or on mortality (fig. 4). On histological of infection ruffled fur, conjunctivitis, and dehydration) one to two signs examination both groups developed severe bilateral days earlier than their counterparts treated with A.L.S. interstitial pneumonia. H.i. antibody responses and lungAs shown in fig. 3, the times of death at these dilutions virus titres were comparable in both groups. These were also delayed by treatment with A.L.S. At all dilutions, results that cellular immune are not suggest responses histological examination demonstrated marked differences important to host defences against primary intranasal between animals of both groups which had died. Whereas influenza inoculation. These studies have been conbrains of N.R.s.-treated animals had characteristic leptomeningeal and perivascular infiltrates, these findings were firmed.13 nearly absent in animals treated with A.L.S. Despite the Rauscher Leukcemia differences in histological findings, virus titres in brains oj A.L.S. greatly potentiated Rauscher-virus-induced splenanimals killed on the same days were equivalent in bott omegaly in adult ICR mice when administered before groups at all dilutions. These studies suggest that the virus inoculation or in the immediate post-inoculation cellular inflammatory reaction to yellow-fever virus it period.14 Measurement of spleen-weight in early mice plays a slight but significant potentiating part in the Rauscher-leukaemia-virus infection has been shown to be development of morbidity and mortality after intracerebra 8. Gledhill, A. W. ibid. 1967, 214, 178. virus inoculation.
Hirsch, M. S., Murphy, F. A., Russe, H. P., Hicklin, M. D. Proc. Soc. exp. Biol. Med. 1967, 125, 980. 10. Hirsch, M. S., Murphy, F. A., Hicklin, M. D. J. exp. Med. 1968, 127, 9.
Lymphocytic Choriomeningitis (L.C.M.) A.L.S. significantly modified the course of primary L.C.M 6. 7.
Hirsch, M. S., Murphy, F. A. Nature, Lond. 1967, 216, 179. nNahmias, A. J., Hirsch, M. S., Kramer, J. H., Murphy, F. A. Unpublished.
757. 11. 12. 13. 14.
Lundstedt, C., Volkert, M. Acta path. microbiol. scand. 1967, 71, 471. Hirsch, M. S., Kaye, H. Unpublished. Klein, M. Personal communication. Hirsch, M. S., Murphy, F. A. Nature, Lond. 1968, 218, 478.
39 bilateral parotid-gland tumours between two and and a half months of age, whereas none of the controls infected with polyoma virus under the same conditions developed tumours, even though they were observed for nine months. Similar results were found with C3H F/He mice; in addition, A.L.s.-polyoma-treated mice of the C3H F/He strain also developed mammary-gland and hair-follicle tumours. As with other murine oncogenic viruses, A.L.S. appear to accelerate neoplasia following polyoma-virus infection.
oped two
Fig. 4-Effects of rabbit anti-mouse thymocyte (R.A.M.T.) serum and normal rabbit serum (N.R.S.) on influenza-virus infection. Survival curves after intranasal administration of influenza virus in doses of 10-3 to 10-6.
Vesicular Stomatitis In-vitro treatment of human peripheral lymphocytes with A.L.S. resulted in their being transformed to lymphoblasts.19 These blast cells exhibited increased susceptibility to vesicular-stomatitis virus infection. A hundred-fold greater titres were observed in transformed than control cultures, and the degree of enhancement was proportional to the percentage of lymphoblasts in the culture at the time of virus inoculation. These studies suggest that lymphoblastic transformation may provide new susceptible host cells for replication of viruses that would otherwise not be pathogenic. SECONDARY RESPONSE
Vaccinia
Weanling ICR mice index of disease progression,16 and in untreated mice this weight is directly proportional to the size of virus inoculum.16 A.L.S. was also effective, but to a lesser extent, in increasing spleen-weight when given three days before virus or two hours after virus. These results suggest that cell-mediated immunity plays a major part in the host defences to Rauscher-virus infection.
an accurate
Moloney Leukemia
,
Three-week-old BALB/c mice were inoculated intraperitoneally with Moloney leukaemia virus. 177 Those pretreated with A.L.S. developed an increased incidence of lymphoid neoplasms following virus inoculation. The tumours observed were not the characteristic lymphoid leukaemias seen three to six months after infection in untreated controls, but were subcutaneous reticulum-cell sarcomas developing three to five weeks after infection at the site of A.L.S. injection. These results indicate that suppression of cellular immunity in Moloney-virus infection results in acceleration and atypical development of oncogenesis. Adenovirus-12 Small doses of A.L.S. increased tumour induction by adenovirus-12 in newborn mice,either with low doses of virus in a susceptible strain (CBA) or with large doses of virus in a relatively resistant strain (CaH).18 The effects noted paralleled those found in neonatally thymectomised mice inoculated with adenovirus, indicating that the thymus-dependent cellular immune system is necessary for significant host response to oncogenic adenovirus infection.
Polyoma
vaccinated twice
by
tail
Lymphocytic Choriomeningitis’ Adult inbred AKR mice were immunised with live virus either by intraperitoneal inoculation or by exposure to infected newborn offspring.20 All had no detectable virus and high titres of L.C.M. complementfixing antibodies several weeks after exposure to virus and before serum treatment. The immunised animals were divided into three groups-one that received A.L.S. six times a week for seven weeks, one that received N.R.s. according to the same schedule, and a third that received no serum. Mice treated with A.L.S. developed virmn-iia between ten and thirty-four days after therapy was begun. Control mice, either untreated or N.R.s.-treated, did not develop virxmia. VirEemia developed in A.L.s.-treated groups despite constant and even increasingly high titres of complement fixing antibodies. These experiments show that, despite apparent immunity, A.L.S. can provoke latent L.C.M. virus infections when administered over a long
L.c.M.
Newborn C57Bl/Ka mice were infected with polyoma virus within twenty-four hours of birth and given A.l..s. subcutaneously twice weekly for three to four weeks.17 All the mice that survived longer than two months devel-
period.
15. 16. 17. 18.
19. 20. 21.
Rauscher, F. J., Allen, B. V. J. natn. Cancer Inst. 1964. 32, 269. Chirigos, M. A. Ann. N.Y. Acad. Sci. 1965, 130, 56. Allison, A. C., Law, L. W. Proc. Soc. exp. Biol. Med. 1968, 127, 207. Allison, A. C., Berman, L. D., Levey, R. H. Nature, Lond. 1967, 215, 185.
were
scarification with vaccinia virus. One month after the second vaccination these mice were divided into two groups-one received A.L.s., the other N.R.S., for one week. They were then challenged intracerebrally with large doses of vaccinia virus and observed for signs of infection. Mortalities in both groups were equivalent (23-32%), and animals of both groups which died had severe necrotising meningitis and encephalitis. No significant differences were found in pre-challenge H.I. antibody levels or in post-challenge antibody responses between A.L.s. and N.R.s. treated groups or between dead animals and survivors. Therefore, it appears that A.L.S. is not capable of abrogating secondary immunity to intracerebrally administered vaccinia virus.
DISCUSSION
The remarkable successes achieved with A.L.s. in human liver and kidney transplants by Starzl and his associates 2 21 Edelman, R., Wheelock, E. F. Lancet, 1968, i, 771. Volkert, M., Lundstedt, C. J. exp. Med. 1968, 127, 327. Starzl, R. E., Groth, C. G., Brettschneider, L., Moon, J. B., Fulginiti, V. A., Cotton, E. K., Porter, K. A. Surgery, St. Louis, 1968, 63, 549.
40 suggest that this serum or one of its derivative fractions will be extensively used in the future. Viral infections have apparently not complicated the limited use of A.L.S. in man, but have been troublesome in experimental organ and tissue transplantation in dogs and monkeys.22 23
latent L.c.M. virus infection in an immune host can also be reactivated by A.L.S., resulting in viraemia for the duration of therapy.20 Whether A.L.s. can similarly alter latent viral infections in man with such agents as herpessimplex virus or the varicella-zoster agent is not known.
This present review suggests that A.L.s. can markedly alter the course of disease in mice following infection with several different viruses. The mechanisms by which A.L.S. influence viral infections are not certain. A.L.S.treated mice are able to produce normal interferon responses as well as normal viral antibody responses. It is likely, therefore, that the effects of A.L.S. are mediated through depression of cell-mediated immunity and that the same mechanisms that produce graft prolongation following A.L.S. treatment result in increased susceptibility
Extrapolations from A.L.s. effects in experimental murine viral infections to effects in man may not be valid, and these sera may be relatively innocuous when used in human organ transplantation. Nevertheless, A.L.S. should be used with caution, particularly when administered in combination with suppressants of humoral immunity and interferon production-e.g., azathioprine and corticosteroids.2’ 28 If extrapolations from mouse to man are meaningful, one may expect to encounter severe, atypical, and perhaps neoplastic viral infections following treatment with A.L.S.
to
infection.
An alternative mechanism, proposed by Edelman and Wheelock,19 is that by the transformation of lymphocytes to lymphoblasts the susceptibility to viral infections is increased. It is unlikely, however, that this in-vitro mechanism is important in vivo. A complement-free medium is necessary in vitro to prevent cell lysis, 19 24 and this situation does not prevail in vivo. Particularly disquieting in the studies of A.L.s. effects on
viral infections is the observation that with all solid tumour and leukaemia viruses tested (polyoma, adeno-12, Moloney leukaemia, Rauscher leukxmia) oncogenicity has been increased by such treatment. These findings are in accord with other data supporting the hypothesis that cellmediated immune responses are the major host defence against virus-induced tumours and leukaemias.25 of disease following peripheral herpesvirus administration was also worsened vaccinia simplex A.L.S. Dissemination was more treatment. by prior and increased in the A.L.S. groups, mortality widespread that cellular is an important host indicating immunity defence mechanism following infection by peripheral routes. These routes most closely approximate the avenues by which natural infections are acquired. When the same viruses were administered intracerebrally, neither morbidity nor mortality was increased, indicating that cellular immunity is not an important defence mechanism against these viruses within the brain. A.L.S. lessened or delayed the mortality following intracerebral administration of L.c.M., herpes-simplex, and yellowfever viruses. It would appear that in these infections the cellular response to the virus is actually detrimental to the host.
The
course or
By depressing cellular but
not humoral responsiveness, initiate a virus-carrier state in L.C.M. virus infected mice. As has been shown by Oldstone and Dixon,26 this carrier state is associated with circulating antigen-antibody complexes. These complexes may be deposited in renal glomeruli producing an immunopathological membranous glomerulonephritis. The carrier state may also be associated with a wasting disorder characterised by marked reticular cell hyperplasia.10 A
A.L.s. can
22. 23.
24. 25. 26.
Abaza, H. M., Nolan, B., Watt, J. G., Woodruff, M. F. A. Transplantation, 1966, 4, 742. Van Bekkum, D. W., Ledney, G. D., Balner, H., van Putten, L. M., de Vries, M. J. in Antilymphocyte Serum (edited by G. E. W. Wolstenholme and M. O’Connor); p. 108. London, 1967. Greaves, M. F., Roitt, I. M., Zamir, R., Carnaghan, R. B. A. Lancet, 1967, ii, 1317. Allison, A. C. Br. med. Bull. 1967, 23, 60. Oldstone, M. B. A., Dixon, F. J. Science, N.Y. 1967, 158, 1193; Fedn Proc. Fedn Am. Socs exp. Biol. 1968, 27, 255.
27. 28.
Rytel, M. W., Kilbourne, E. D. J. exp. Med. 1966, 123, 767. Schwartz, R. S. Progr. Allergy, 1965, 9, 246.
Public Health FOOD-POISONING DUE TO COPPER IN THE MORNING TEA P. O. NICHOLAS Brist., D.C.H., D.P.H.
M.B.
DEPUTY MEDICAL OFFICER OF
HEALTH, BOLTON
An unusual form of acute food-poisoning due to copper sulphate in the water of an unserviced gas hot-water geyser is described. The morning tea brewed with the water caused acute vomiting and diarrhoea in twenty workmen. It is conceivable that unless care is taken to prevent copper corrosion in water heaters, the amount of copper in drinking-water could be raised. Though this contamination may not cause symptoms initially, copper ions could be built up in the human body over a long period and be a source of ill health. Summary
INTRODUCTION
CONTAMINATION of food by copper should be less than previously for, in general, copper utensils have been replaced by aluminium and stainless steel. Lately copper has been used in the manufacture of expensive kitchen utensils, since this metal has a high thermal conductivity. Copper is still widely used in hot-water geysers, for stainless steel is expensive, and both this and aluminium alloys are more difficult metals to weld. Where copper is still used, pure tin to plate the copper prevents any leaching of the copper into the food or drink. common
Vernon’s analysis (1967) of all causes of food-poisoning in 1966 in England and Wales shows that 48% of 8784 recorded cases were due to bacterial infection. Only 1 case was due to chemical poisoning. However, in 2350 cases (27%) no cause for the food-poisoning was found. In investigating food-poisoning outbreaks it may be that more attention should be paid to possible contamination of food and drink by chemicals, as a recent incident of copper poisoning shows. THE EPIDEMIC
At a small factory in Bolton, twenty workmen felt sick after drinking their morning tea at 8.30 A.M. Five of the workmen vomited within a few minutes, and one mac