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HODGKIN'S DISEASE: A POSSIBLE EXPERIMENTAL MODEL IN RATS
1421 distal aorta; (2) the weakness of the atheromatous aorta; and (3) a change from laminar flow to turbulent flow.’7 Could orthotopic grafting have avoided this incident ? R. VELA-NAVARRETE C. ALFEREZ Fundacion Jimenez Diaz, L. HERNANDO. Madrid, Spain.
HODGKIN’S DISEASE: A POSSIBLE EXPERIMENTAL MODEL IN RATS leader on " The Hodgkin Maze " 8 you SIR,-In your " mention that it still seems doubtful whether any animal model exists for this disease ". In support of this view you cite, in particular, Dunn and Deringer’s review 9 of this aspect of experimental lymphomata. We feel we must now comment both on your statement and on Dunn and
Deringer’s opinions. These workers doubt whether a murine model of Hodgkin’s disease is available, and dispute the validity, reproducibility, and malignancy of the Hodgkin’s-like lesions originally reported.1O Such tumours were produced in rats by long-term (200-400 days) intermittent injections of trypan-blue. Dunn and Deringer’s opinions were based on the earlier reports of Gillman et al.1O,ll and on the subsequent failure of Riittner and Brunner 12 to produce in rats or Evans-blue) " true neoplastic lym(with trypan-blue " similar to those reported by Gillman et al. phomas Since 1952, however, we 13,14 and others 15-18 have done much further work on the factors affecting the production and transplantation of the trypan-blue-induced Hodgkin’slike and other lymphomata in rats. Given an appropriate " active " trypan-blue, lymphoreticular tumours can regularly be produced in several Wistar strains of rat: we have produced them in one non-inbred strain and one inbred strain. Such trypan-blue-induced lymphoreticular tumours cannot be induced with all samples of trypan-blue available on the market-one of us (T. G.) found that 3 different post-war batches of Grubler trypan-blue were quite inactive. Nor were lymphoreticular tumours induced in 2 inbred strains of mice which we tested for 2 years with a dye-sample known to be active in rats. Moreover, once induced with trypan-blue in rats, such lymphoreticular tumours are transplantable by subcutaneous injection into newborn non-inbred rats 12,13 and also into young adults of the inbred Wistar strain. In the latter, the " take-time " on initial passage was 300-400 days. Over the past 9 years we have transmitted trypan-blue-induced lymphoreticular tumours from liver, lymph-nodes, and kidneys in the inbred rats. One line of these tumours has been carried through 26 passages and now regularly kills the inbred Wistar rats in 35-60 days, with large local lymphoreticular tumours at the implantation sites (subcutaneous or intraperitoneal) and widespread metastases in the thymus, bone-marrow, lymph-nodes, kidneys, and other sites. In the aleukxmic blood of tumour-bearing rats, 5-10% of leucocytes are " abnormal mononuclears ". These seem to be circulating tumour-cells, since lymphoreticular tumours can be produced by injecting the blood. However, the exact nature of 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18.
these circulating " abnormal mononuclear " leucocytes still remains to be elucidated. This may be possible by using a number of methods, including the in-vitro short-term culture and other techniques detailed by Crowther et al.19 for " circulating lymphoid cells " in human patients with Hodgkin’s disease in relapse. Thus, Sir, we can assure you and your readers of the consistency with which obviously malignant lymphoreticular tumours can be produced in some rat strains, albeit only with some varieties of commercially available trypan-blue. Moreover, these trypan-blue-induced lymphoreticular tumours are undoubtedly malignant on transplantation into suitable hosts. However, what is perhaps even more important to the matter now under discussion is that when the trypan-blue induced tumours were originally described in various rat tissues 10,11 several British and American pathologists thoroughly conversant with Hodgkin’s disease in man agreed that these tumours often closely resembled the human lesions. More recently, one of us (R. M. C.), whose work you also cited in your leader ’20 examined sections of tumours from trypan-blue-treated rats and also from rats in which the original trypan-blue-induced lymphoreticular tumours had been passaged. Cells were often seen, especially in lymph-nodes during the first few weeks after transplanting tumours, which closely resembled the " abnormal reticulum 19. 20.
Crowther, D., Hamilton Fairley, G., Sewell, ii, 473. Cross, R. M. J. clin. Path. 1968, 21, 303.
R. L. Br. med. J. 1969,
Fig. I-Field from lymph-node from a patient with Hodgkin’s sarcoma; five " malignant reticulum cells " are distinguishable.
(Hxmatoxylin and eosin.
x
525.)
Fig. 2-Field from lymph-node of a rat with experimentally induced transplantable Hodgkin’s-like lymphoma; three " malignant reticulum cells " are clearly distinguishable. (Hsematoxytin and eosin. x 1000.)
Szilagyi, D. E., Whitcomb, J. G., Schenker, W., Waibel, P. Surgery, St. Louis, 1960, 47, 55. Lancet, Oct. 4, 1969, p. 728. Dunn, T. B., Deringer, M. K. J. natn. Cancer Inst. 1968, 40, 771. Gillman, J., Gillman, T., Gilbert, C., Spence, I. Cancer, N.Y. 1952 5, 792. Gillman, J., Gillman, T. Clin. Proc. 1949, 8, 222. Rüttner, J. R., Brunner, H. E. Schweiz. Z. Path. Bakt. 1959, 22, 519. Gillman, T. Symposium on Lymphoreticular Tumours in Africa (edited by F. C. Roulet); p. 367. Basle, 1964. Gillman, T. S. Afr. med. J. (suppl.), 1957, 50. Brown, D. V., Thorson, T. A. J. natn. Cancer Inst. 1956, 16, 1181. Marshall, A. H. E. Acta path. microbiol. scand. 1953, 33, 1. Marshall, A. H. E. An Outline of the Cytology and Pathology of the Reticular Tissue; p. 203. Edinburgh, 1956. Simpson, C. L. Br. J. exp. Path. 1952, 33, 524.
3-Two binucleated " Reed-Sternberg-like " giant-cells from rat’s lymph-node shown in fig. 2.
Fig.
( x 1000.)
1422 cells " considered a sine qua non for the diagnosis of Hodgkin’s in man.21 " abnormalmalignant’ reticulum cells " (and even " typical binucleate Reed-Sternberg giant-cell " variants thereof) such as those described by one of us in Hodgkin’s lymphomata in man,14 can be found in the rat tumours in varying numbers (relative to lymphocytes) and with varying degrees of associated fibrosis. Cells of this type are not obvious to us in any of the photomicrographs of lymphomata described in mice by Dunn and Deringer9 or by Murphy.22 Figs. 1-3 show examples of the close morphological resemblance between the remarkably " reticulum cells " in some rat tumours and those in one variant of Hodgkin’s disease in man.21 Rat tumour-cells regularly reproduce such Hodgkin’s-like lesions on passage. Recent and still very preliminary work in our laboratory also indicates that it may yet be possible to transmit such lymphomata by injecting, into inbred hosts, tumour-cells maintained in vitro for 20-30 days. So, we are strongly encouraged to continue with our work on the experimentally induced and transplantable malignant Hodgkin’s-like lesions in rats. We sincerely hope that others may take an interest in this potentially important experimental model of Hodgkin’s disease. Perhaps, by exploiting this opportunity, further light may yet be shed on the basic nature of this disease. Perhaps too, this model in the rat may yet prove useful in studying the histogenesis and cytogenesis of Hodgkin’s disease in man; it may perhaps also assist in screening therapeutic measures which, if effective against the rat Hodgkin’s-like tumour-cells in vivo or in vitro, may be of value in man. Such studies, we hope, may assist us in unravelling the still very real and numerous puzzling aspects of the Hodgkin’s disease " maze " in man, and also, perhaps, of possibly related varieties of spontaneous lymphomata so common in domestic animals. M. K. is grateful to the Lady Tata Memorial Fund for a grant, in aid of this work during 1968 and 1969. Department of Experimental Pathology, THEODORE GILLMAN Agricultural Research Council, MARION KINNS. Babraham, Cambridge. 5 Northumberland Avenue, R. M. CROSS. Aylesbury, Bucks.
DEFICIENCY SIR,-In correspondence section of your issue o Dec. 6 (p. 1256) the use of the clinical term " menta deficiency " is questioned as a description of a conditior which was legally defined in the Mental Health Act o: 1959 as " mental subnormality ". Perhaps it would b< helpful if the attention of your readers could be drawn t( A Glossary of Mental Disorders, prepared by the subcommittee on classification of mental disorders of the Registrar General’s Advisory Committee on Medical Nomenclature and Statistics, published in 1968 by Her Majesty’! Stationery Office. The terms of reference of this committee were to consider from the medical point of view and advise upon questions affecting the Internationa: Statistical Classification of Diseases, Injuries and Cause! of Death, and any other matters concerning medica nomenclature. This committee prepared the abovementioned glossary and commended it to psychiatrist! for their use in conjunction with the Eighth Revision oj the International Classification which came into operatior. on Jan. 1, 1968. This glossary does not use the terms " mental deficiency " or " mental subnormality ", bul favours the term " mental retardation ". the
Brundall, Norfolk.
RUDOLPH PAYNE.
21. Cross, R. M. ibid. 1969, 22, 165. 22. Murphy, E. D. J. natn. Cancer Inst. 1969, 42, 797.
FENFLURAMINE OVERDOSAGE SIR,-In an annotation in your issue of Dec. 13 (p. 1289) you refer to some cases of overdosage with our compound fenfluramine. We should like to put some of your comments into perspective. Fenfluramine is probably one of the most thoroughly investigated drugs currently available. In terms of toxicity, fenfluramine is certainly safer than many drugs -for example, 20 tablets of amphetamine sulphate or 20 tablets of paracetamol have been fatal. It is surely not surprising that single doses of 10-15 times the normal therapeutic dose should produce " alarming reactions "; is there any drug of which the same could not be said ? With the greatly increased usage of fenfluramine it is, regrettably, true that more cases of overdosage will be seen. In 7 years’ clinical experience with fenfluramine in the United Kingdom and many other countries there have been several cases of non-fatal overdosage where doses of up to 1720 mg. (43 times a normal maximum single therapeutic dose) have been taken. Four fatalities have occurred, with doses ranging from 36 tablets in a 21-month-old child to 100 tablets in a 13-year-old boy-i.e., approximately 100 times the maximum single therapeutic doses for children of these ages. Thus it appears that toxic dose-levels in man are about 80-100 times the normal single therapeutic dose. This is not an unreasonable margin by any standards, and is certainly higher than that for the majority of drugs available. N. SANTER Harrow, Middlesex. Director, Selpharm Laboratories Ltd.
PREVENTION OF BACTERIAL ENDOCARDITIS SIR, The finding of increasing numbers of penicillinresistant oral streptococci and other organisms has led us to question the advisability of giving penicillin routinely as a protective " cover " to dental patients considered susceptible to bacterial endocarditis. In a recent study, the results of which will be published shortly, 27% of all routine patients who were not receiving antibiotics (and had not recently been on antibiotics) were found to have penicillin-resistant streptococci in their mouths, while 85% of the patients had some penicillinresistant bacteria. Obviously, penicillin in these cases would fail to give complete protection against a bactersemia resulting from dental treatment. The use of erythromycin, cephaloridine, or some other suitable bactericidal antibiotic or combination of antibiotics ought to be considered for these patients. The timing of the antibiotic " cover " is also important, since even in patients who normally have no penicillinresistant bacteria, one or two days of penicillin therapy before dental treatment may result in the emergence of penicillin-resistant streptococci of the viridans group and/or gram-negative rods. A case in point was seen recently at this dental school. Preparations were being made to carry out minor oral surgery on a woman who had had a previous attack of bacterial endocarditis. Bacteriological tests were carried out and the patient was found to have no penicillinresistant organisms in the mouth. A few days later she mentioned in passing that she had informed her doctor of the impending oral surgery and he had given her a prophylactic course of penicillin. A second bacterial examination revealed that her dominant oral bacterial strain had become Pseudomonas pyocyanea. On other occasions it has been found that preoperative chemotherapy has resulted in the emergence of oral Streptococcus viridans resistant to most antibiotics of the penicillin group. A recent investigation has also shown that bacterial samples taken from different parts of one mouth have, in a considerable number of cases, shown organisms with differ-
HODGKIN’S DISEASE: A POSSIBLE EXPERIMENTAL MODEL IN RATS leader on " The Hodgkin Maze " 8 you SIR,-In your " mention that it still seems doubtful whether any animal model exists for this disease ". In support of this view you cite, in particular, Dunn and Deringer’s review 9 of this aspect of experimental lymphomata. We feel we must now comment both on your statement and on Dunn and
Deringer’s opinions. These workers doubt whether a murine model of Hodgkin’s disease is available, and dispute the validity, reproducibility, and malignancy of the Hodgkin’s-like lesions originally reported.1O Such tumours were produced in rats by long-term (200-400 days) intermittent injections of trypan-blue. Dunn and Deringer’s opinions were based on the earlier reports of Gillman et al.1O,ll and on the subsequent failure of Riittner and Brunner 12 to produce in rats or Evans-blue) " true neoplastic lym(with trypan-blue " similar to those reported by Gillman et al. phomas Since 1952, however, we 13,14 and others 15-18 have done much further work on the factors affecting the production and transplantation of the trypan-blue-induced Hodgkin’slike and other lymphomata in rats. Given an appropriate " active " trypan-blue, lymphoreticular tumours can regularly be produced in several Wistar strains of rat: we have produced them in one non-inbred strain and one inbred strain. Such trypan-blue-induced lymphoreticular tumours cannot be induced with all samples of trypan-blue available on the market-one of us (T. G.) found that 3 different post-war batches of Grubler trypan-blue were quite inactive. Nor were lymphoreticular tumours induced in 2 inbred strains of mice which we tested for 2 years with a dye-sample known to be active in rats. Moreover, once induced with trypan-blue in rats, such lymphoreticular tumours are transplantable by subcutaneous injection into newborn non-inbred rats 12,13 and also into young adults of the inbred Wistar strain. In the latter, the " take-time " on initial passage was 300-400 days. Over the past 9 years we have transmitted trypan-blue-induced lymphoreticular tumours from liver, lymph-nodes, and kidneys in the inbred rats. One line of these tumours has been carried through 26 passages and now regularly kills the inbred Wistar rats in 35-60 days, with large local lymphoreticular tumours at the implantation sites (subcutaneous or intraperitoneal) and widespread metastases in the thymus, bone-marrow, lymph-nodes, kidneys, and other sites. In the aleukxmic blood of tumour-bearing rats, 5-10% of leucocytes are " abnormal mononuclears ". These seem to be circulating tumour-cells, since lymphoreticular tumours can be produced by injecting the blood. However, the exact nature of 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18.
these circulating " abnormal mononuclear " leucocytes still remains to be elucidated. This may be possible by using a number of methods, including the in-vitro short-term culture and other techniques detailed by Crowther et al.19 for " circulating lymphoid cells " in human patients with Hodgkin’s disease in relapse. Thus, Sir, we can assure you and your readers of the consistency with which obviously malignant lymphoreticular tumours can be produced in some rat strains, albeit only with some varieties of commercially available trypan-blue. Moreover, these trypan-blue-induced lymphoreticular tumours are undoubtedly malignant on transplantation into suitable hosts. However, what is perhaps even more important to the matter now under discussion is that when the trypan-blue induced tumours were originally described in various rat tissues 10,11 several British and American pathologists thoroughly conversant with Hodgkin’s disease in man agreed that these tumours often closely resembled the human lesions. More recently, one of us (R. M. C.), whose work you also cited in your leader ’20 examined sections of tumours from trypan-blue-treated rats and also from rats in which the original trypan-blue-induced lymphoreticular tumours had been passaged. Cells were often seen, especially in lymph-nodes during the first few weeks after transplanting tumours, which closely resembled the " abnormal reticulum 19. 20.
Crowther, D., Hamilton Fairley, G., Sewell, ii, 473. Cross, R. M. J. clin. Path. 1968, 21, 303.
R. L. Br. med. J. 1969,
Fig. I-Field from lymph-node from a patient with Hodgkin’s sarcoma; five " malignant reticulum cells " are distinguishable.
(Hxmatoxylin and eosin.
x
525.)
Fig. 2-Field from lymph-node of a rat with experimentally induced transplantable Hodgkin’s-like lymphoma; three " malignant reticulum cells " are clearly distinguishable. (Hsematoxytin and eosin. x 1000.)
Szilagyi, D. E., Whitcomb, J. G., Schenker, W., Waibel, P. Surgery, St. Louis, 1960, 47, 55. Lancet, Oct. 4, 1969, p. 728. Dunn, T. B., Deringer, M. K. J. natn. Cancer Inst. 1968, 40, 771. Gillman, J., Gillman, T., Gilbert, C., Spence, I. Cancer, N.Y. 1952 5, 792. Gillman, J., Gillman, T. Clin. Proc. 1949, 8, 222. Rüttner, J. R., Brunner, H. E. Schweiz. Z. Path. Bakt. 1959, 22, 519. Gillman, T. Symposium on Lymphoreticular Tumours in Africa (edited by F. C. Roulet); p. 367. Basle, 1964. Gillman, T. S. Afr. med. J. (suppl.), 1957, 50. Brown, D. V., Thorson, T. A. J. natn. Cancer Inst. 1956, 16, 1181. Marshall, A. H. E. Acta path. microbiol. scand. 1953, 33, 1. Marshall, A. H. E. An Outline of the Cytology and Pathology of the Reticular Tissue; p. 203. Edinburgh, 1956. Simpson, C. L. Br. J. exp. Path. 1952, 33, 524.
3-Two binucleated " Reed-Sternberg-like " giant-cells from rat’s lymph-node shown in fig. 2.
Fig.
( x 1000.)
1422 cells " considered a sine qua non for the diagnosis of Hodgkin’s in man.21 " abnormalmalignant’ reticulum cells " (and even " typical binucleate Reed-Sternberg giant-cell " variants thereof) such as those described by one of us in Hodgkin’s lymphomata in man,14 can be found in the rat tumours in varying numbers (relative to lymphocytes) and with varying degrees of associated fibrosis. Cells of this type are not obvious to us in any of the photomicrographs of lymphomata described in mice by Dunn and Deringer9 or by Murphy.22 Figs. 1-3 show examples of the close morphological resemblance between the remarkably " reticulum cells " in some rat tumours and those in one variant of Hodgkin’s disease in man.21 Rat tumour-cells regularly reproduce such Hodgkin’s-like lesions on passage. Recent and still very preliminary work in our laboratory also indicates that it may yet be possible to transmit such lymphomata by injecting, into inbred hosts, tumour-cells maintained in vitro for 20-30 days. So, we are strongly encouraged to continue with our work on the experimentally induced and transplantable malignant Hodgkin’s-like lesions in rats. We sincerely hope that others may take an interest in this potentially important experimental model of Hodgkin’s disease. Perhaps, by exploiting this opportunity, further light may yet be shed on the basic nature of this disease. Perhaps too, this model in the rat may yet prove useful in studying the histogenesis and cytogenesis of Hodgkin’s disease in man; it may perhaps also assist in screening therapeutic measures which, if effective against the rat Hodgkin’s-like tumour-cells in vivo or in vitro, may be of value in man. Such studies, we hope, may assist us in unravelling the still very real and numerous puzzling aspects of the Hodgkin’s disease " maze " in man, and also, perhaps, of possibly related varieties of spontaneous lymphomata so common in domestic animals. M. K. is grateful to the Lady Tata Memorial Fund for a grant, in aid of this work during 1968 and 1969. Department of Experimental Pathology, THEODORE GILLMAN Agricultural Research Council, MARION KINNS. Babraham, Cambridge. 5 Northumberland Avenue, R. M. CROSS. Aylesbury, Bucks.
DEFICIENCY SIR,-In correspondence section of your issue o Dec. 6 (p. 1256) the use of the clinical term " menta deficiency " is questioned as a description of a conditior which was legally defined in the Mental Health Act o: 1959 as " mental subnormality ". Perhaps it would b< helpful if the attention of your readers could be drawn t( A Glossary of Mental Disorders, prepared by the subcommittee on classification of mental disorders of the Registrar General’s Advisory Committee on Medical Nomenclature and Statistics, published in 1968 by Her Majesty’! Stationery Office. The terms of reference of this committee were to consider from the medical point of view and advise upon questions affecting the Internationa: Statistical Classification of Diseases, Injuries and Cause! of Death, and any other matters concerning medica nomenclature. This committee prepared the abovementioned glossary and commended it to psychiatrist! for their use in conjunction with the Eighth Revision oj the International Classification which came into operatior. on Jan. 1, 1968. This glossary does not use the terms " mental deficiency " or " mental subnormality ", bul favours the term " mental retardation ". the
Brundall, Norfolk.
RUDOLPH PAYNE.
21. Cross, R. M. ibid. 1969, 22, 165. 22. Murphy, E. D. J. natn. Cancer Inst. 1969, 42, 797.
FENFLURAMINE OVERDOSAGE SIR,-In an annotation in your issue of Dec. 13 (p. 1289) you refer to some cases of overdosage with our compound fenfluramine. We should like to put some of your comments into perspective. Fenfluramine is probably one of the most thoroughly investigated drugs currently available. In terms of toxicity, fenfluramine is certainly safer than many drugs -for example, 20 tablets of amphetamine sulphate or 20 tablets of paracetamol have been fatal. It is surely not surprising that single doses of 10-15 times the normal therapeutic dose should produce " alarming reactions "; is there any drug of which the same could not be said ? With the greatly increased usage of fenfluramine it is, regrettably, true that more cases of overdosage will be seen. In 7 years’ clinical experience with fenfluramine in the United Kingdom and many other countries there have been several cases of non-fatal overdosage where doses of up to 1720 mg. (43 times a normal maximum single therapeutic dose) have been taken. Four fatalities have occurred, with doses ranging from 36 tablets in a 21-month-old child to 100 tablets in a 13-year-old boy-i.e., approximately 100 times the maximum single therapeutic doses for children of these ages. Thus it appears that toxic dose-levels in man are about 80-100 times the normal single therapeutic dose. This is not an unreasonable margin by any standards, and is certainly higher than that for the majority of drugs available. N. SANTER Harrow, Middlesex. Director, Selpharm Laboratories Ltd.
PREVENTION OF BACTERIAL ENDOCARDITIS SIR, The finding of increasing numbers of penicillinresistant oral streptococci and other organisms has led us to question the advisability of giving penicillin routinely as a protective " cover " to dental patients considered susceptible to bacterial endocarditis. In a recent study, the results of which will be published shortly, 27% of all routine patients who were not receiving antibiotics (and had not recently been on antibiotics) were found to have penicillin-resistant streptococci in their mouths, while 85% of the patients had some penicillinresistant bacteria. Obviously, penicillin in these cases would fail to give complete protection against a bactersemia resulting from dental treatment. The use of erythromycin, cephaloridine, or some other suitable bactericidal antibiotic or combination of antibiotics ought to be considered for these patients. The timing of the antibiotic " cover " is also important, since even in patients who normally have no penicillinresistant bacteria, one or two days of penicillin therapy before dental treatment may result in the emergence of penicillin-resistant streptococci of the viridans group and/or gram-negative rods. A case in point was seen recently at this dental school. Preparations were being made to carry out minor oral surgery on a woman who had had a previous attack of bacterial endocarditis. Bacteriological tests were carried out and the patient was found to have no penicillinresistant organisms in the mouth. A few days later she mentioned in passing that she had informed her doctor of the impending oral surgery and he had given her a prophylactic course of penicillin. A second bacterial examination revealed that her dominant oral bacterial strain had become Pseudomonas pyocyanea. On other occasions it has been found that preoperative chemotherapy has resulted in the emergence of oral Streptococcus viridans resistant to most antibiotics of the penicillin group. A recent investigation has also shown that bacterial samples taken from different parts of one mouth have, in a considerable number of cases, shown organisms with differ-