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TOLERANCE TO PHYTOHÆMAGGLUTININ ?
41
In A
Letters
England Now
Running Commentary by Peripatetic Correspondents
IT was an emergency all right, and I just happened to be around. The patient was an elderly, rather prickly male, and he had walked straight into, and then managed to get himself twisted up in, some cricket netting set up on his own back lawn for the children to play in. It was wrapped tightly round his head and neck, and round one leg; and, apparently after some struggling with it, he had gone into a state of hysterical stupor. That was how I found him. He lay there on the lawn, curled up in a state of intense flexor spasm, just breathing, unable to speak, incontinent of fasces. He had such a tight grip on the netting I couldn’t begin to get it away from him. I don’t know what his poor wife must have thought, but I do know what I thought: that this was no time for unheroic measures. Strangulation was one danger, death from sheer I happened to have a little terror or exhaustion another. chloroform handy (but no other anaesthetic), and in no time at all I was administering chloroform to a patient for the first time in twenty years. He was soon under, and once the spasm had gone I got the netting off him easily enough. Then suddenly, of course, his breathing had to stop. I thought of the kiss of life, but his little old wrinkled face put me off, so I compromised with squeezing his chest a few times and fortunately this started spontaneous respiration again. He didn’t come round properly for some little time, and I began to wonder if my massive and inexpert administration of chloroform had not hit his liver-perhaps not inappropriatelyfor six. However, some ten minutes after I stopped the anxsthetic he was on his feet, and five minutes later he was walking. He didn’t thank me, he didn’t say a word. I doubt if he was grateful. And I don’t suppose I shall ever set eyes on that patient again. He was, you see, a hedgehog. *
*
*
According to the guidebook, the river at Galway is so stuffed with salmon during the spring run that you can’t see the riverbed. The uppermost of the bridges is the spot designated for this sight, and so we wandered down hopefully. Not a fish could be seen. I asked a local man if the spring run was on. " Yes," he said,
"
have started to come up; only a few as yet because too much water is coming down. But now the weather will be fine and in a day or two there will be thousands." So back we went on the next day: still no fish in view. An old man sidled up to me as I gazed over the parapet. "See any ?" he asked cautiously. " Not one," I replied. " Ah," he said, " there is too much water coming down. Now yesterday there were thousands. But the weather will be fine, and in the next day or two ..." I conclude that the bit in the guidebook was written by an
they
Irishman. ’*’
’*’
’*’
My peripetatic colleague and his leeches (June 17) bring back memories of the involuntary attachment of leeches to areas above shoe-level in the foothills of the Himalayas. One learnt by experience to stick to the paths in the leech season; but one also learnt from the locals who would nonchalantly go barefoot in leech-infested areas. Each carried a little bag of salt to brush off the bloodsuckers ; Western culture suggested pepper to produce coagulation; and a very effective combination this proved to be. ’*’
’*’
’*’
To thank the Lord for being not as others is a horrid, perilous sin but to make weaklings offend is perhaps even worse. So please all you " others " who " on the air say " reesearch, libary, tempory, Febary " and worst of all " controwersy ". for my weak soul’s sake and, it may be, even for your own, desist. And if you will say " ambivalent " and not " ambivvalent" and perhaps even " vitamine," not " vittamin " then I, in gratitude and for consistency, will try, adjuvante Deo, to " drop equivvalent ", of which I confess I am guilty, and take " to equi-" or " xqui-valent ". But won’t it sound odd ?
to
the Editor
SELF-TREATMENT
SiR,—The suggestion of Dr. Cargill last week (p. 1377) that oxytetracycline should be freely available to the lay public for home medication will be unacceptable to many doctors; but there should be little opposition to his broader thesis that lay people ought to be instructed in the home management of minor ailments. There should be little such opposition, but there is, in fact, a lot. We have a grave shortage of doctors, both in general and in hospital practice (leading often to the Alice-in-Wonderland kind of position described by Mr. Thompson in the same issue [p. 1379]); and yet the attitude of official medicine to selfdiagnosis and self-treatment of minor ailments is completely negative. It is an attitude that may have been appropriate in the 1920s, but today, when the quality and efficacy of home medicines have been greatly improved, and the lay public is much better informed, it is a reactionary one. Moreover, the profession in this country has always prided itself on being pragmatic, and lay people do diagnose and treat minor ailments, and will certainly continue to do so. We all know it. Why then do we not seek to make the diagnoses as sure and the treatments as safe and effective as may be ? Why behave like ostriches ? Some individual doctors are, of course, advising their patients on these matters; but it is official medicine that should be in action-producing suitable leaflets, sponsoring television and radio programmes, and encouraging a flow of informed propaganda in the Press. In fact, while it encourages lay people to sit in front of a television set and gaze upon the most complex heart-surgery-about which all they understand is the frisson it provides them with-it never once shows them how to treat a simple headache or simple indigestion or the common cold. We improve upon Marie-Antoinette: she merely advised the people to eat cake-we provide them with it. Little Missenden, S. BRADSHAW. Amersham, Bucks.
ICE-CREAM-VAN ACCIDENTS
SiR,—In experience here, a not uncommon cause of head in injury young children has been the acute suppression of caution and road drill by the prospect of ice-cream. They have usually been hit by passing vehicles when at the sound of the bell they rush blindly to the van to buy, or as they run our
back home to consume. We should be interested to hear from your readers whether this special type of accident is widespread throughout the country. If it is, some publicity might encourage its prevention. M. BRIGGS Accident Service, The Radcliffe Infirmary, Oxford. JOHN M. POTTER.
TOLERANCE TO PHYTOHÆMAGGLUTININ ?
SIR,-Despite the varied cellular response to phytohxmagglutinin (P.H.A.), doubt still persists as to the immunological nature of this response. If P.H.A. does indeed act as an antigen, although an atypical one, it might be possible to induce a state of immunological tolerance to it. We report here our failure to accomplish this. Twelve newborn rabbits, aged 4 days, were each injected subcutaneously with the contents (about 80 mg.) of one bottle of P.H.A.-M (Difco) diluted in tissue-culture medium ’199 ’ (Microbiological Products). They were reinjected again at age 6 days with the same quantity of P.H.A.-M. Control litter mates were not injected. None of the injected neonatal rabbits showed any signs of morbidity during the experiment. At age 4-6 weeks, the rabbits were killed and the popliteal lymph-nodes excised. Portions of lymph-nodes were fixed in buffered formalin, sectioned, and stained with haematoxylin and eosin. All were found to be normal, with abundant follicles. Cell
42
suspensions were prepared and incubated in medium 199, containing 20% homologous rabbit serum, and 100 units penicillin and 100 {J.g. streptomycin per ml. The cell concentration was 106 cells per ml. of medium, and sterileFalcon ’ disposable plastic tubes were used throughout. A total of 4 ml. was placed in each tube. An optimal amount of P.H.A. (0-5 ml., diluted tenfold), predetermined with normal adult lymph-node cells, was added to each tube. The tubes were sealed and maintained in the upright position in an incubator at 37°C for 72 hours. At this time the tubes were spun at 800 r.p.m. for 5 minutes, the cells resuspended in a small volume of medium (0-1 ml.), and smears made on alcohol-cleaned cover-slips. The smears were dried rapidly at 60°C with a hair-drier. They were then treated with Jenner-Giemsa stain, and examined microscopically. A thousand cells were counted at random and the percentage of blast cells was recorded. It was found that none of the P.H.A.-injected rabbits displayed any sign of immunological tolerance to P.H.A.-that is, specific failure to react to P.H.A. Lymph-node cells from the P.H.A.-
injected rabbits
behaved identically to those from normal control rabbits in the presence of P.H.A. About 40-70% of the lymph-node cells of both groups transformed to blast cells in the presence of P.H.A. in vitro. If the mitogen in P.H.A. is antigenic, one might have expected the induction of tolerance in at least several of the P.H.A.-injected rabbits. It is possible that tolerance cannot be induced to P.H.A., or that the concentration of the blastogenic factor was very low in the whole P.H.A. solution used and that therefore insufficient quantities of the active principle were injected into the neonatal rabbits. We must conclude, however, from our experiments that P.H.A. seems to act in a non-immunological fashion upon cells in inducing mitosis and blastogenesis. Harry Webster Thorp Laboratories, Division of Immunochemistry and Allergy, McGill University Clinic, M. RICHTER Royal Victoria Hospital, C. K. NASPITZ. Montreal, Quebec, Canada.
SOLARISATION OF CHROMOSOMES
SIR,- The small size of human chromosomes makes identification of minor structural abnormalities difficult
or
impossible by existing techniques. I describe here a minor variation in photographic procedure which may be of value in this regard, since it allows for greater magnification of chromosome outline than by conventional methods. The technique utilises one of the characteristic effects of solarisation exposing a partly developed negative to an unsafe light during developmentas follows: The image of a good-quality metaphase negative from a patient with chronic myeloid leukaemia (C.M.L.) (fig. 1) was used to produce a positive-copy negative on ’ Safety Ortho Lithofilm 0 82 ’ (AgfaGevaert) at an enlargement which produced good chromosome resolution. UsingPaterson Acuprint FX 17 ’ developer, diluted 1 in 4 with water, an exposure-time which allowed for the first appearance of the faint chromosome images in 15-20 seconds was obtained. The development of the chromosome images was then allowed to proceed until a satisfactory image was obtained, at which time a photographically unsafe overhead light was switched on for 1 second. The positive was then left in the developer for a further minute without agitation of developer. A few seconds after exposing the positive to the unsafe light, gradual fogging of the clear area around the chromosomes was observed, culminating in the formation of haloes around the chromosomes. These haloes gradually faded in intensity, eventually appearing to fade completely in the dim safe light. At the end of the development-time the positive was transferred to an acid-stop bath and then to a fixative solution. After fixation a correctly solarised positive shows the chromosomes surrounded by narrow, intense haloes. Some experimentation was necessary in order to obtain well-outlined chromosomes, the variables being initial exposure-time, development-time before exposure to unsafe light, the length of exposure to the unsafe light, and the development-time after exposure to the unsafe light. The production of the clear halo is determined by an increased concentration of bromide ions at the 1. The Focal
Encyclopedia of Photography (edited by F. Purves); p. 1079. London, 1960.
Fig. I-Metaphase plate from patieit with C.M.L. and probable homologue
Philadelphia chromosome (lower arrow) (upper arrow) (Leishman’s stain; x 1800).
Fig. 2-(a) Philadelphia chromosome and (b) probable homologue from solarised positive produced from negative used for fig. 1. Reduced to three-quarters from x 25,000.
boundary of a completely developed image which retard development surrounding area.1
in the
With this technique the intense halo delineating the border of the chromosome is enlarged rather than the chromosome itself, thereby allowing far greater magnification with good resolution. The border of the enlarged chromosome is represented by the inner edge of the halo, which appears black on the resulting prints (see fig. [a] and [b]). No additional details of chromosome structure can be made out. It is hoped that this fairly simple technique will prove of value in the further study and identification of normal and abnormal chromosomes, since the greater magnification of chromosome outline should allow for more accurate measurement of chromosome-arm length and of chromosome area. This study, which is being continued and will be more fully reported later, was supported by a Witwatersrand University Council research grant. I should like to thank Prof. H. B. Stein and Dr. B. A. Bradlow for helpful advice and criticism. Division of Chemical Pathology, Department of Pathology and Microbiology, University of the Witwatersrand,
Johannesburg.
C. WALLACE.
CHROMOSOMES 17-18 IN LEUKÆMIAS SiR,ńSuggestive evidence of a special relation between chromosome pairs 17 or 18 and malignant proliferations, particularly lymphomasand leukarmias,2 has been noted. In Ricci, N., Punturieri, E., Bosi, L., Castoldi, G. L. Lancet, 1962, ii, 564. Sandberg, A. A., Ishihara, T., Kikuchi, Y., Crosswhite, L. H. Cancer, N. Y. 1964, 17, 738. Sasaki, M. S., Sofuni, T., Makino, S. ibid. 1965, 18, 1007. Spiers, A. S. D., Baikie, A. G., Lancet, 1966, i, 506. 2. Lejeune, J., Berger, R., Caille, B., Turpin, R. Annls Génét. 1965, 8, 44. de Grouchy, J., de Nava, C., Cantu, J. M., Bilski-Pasquier, G., Bousser, J. Am. J. hum. Genet. 1966, 18, 485. 1.
In A
Letters
England Now
Running Commentary by Peripatetic Correspondents
IT was an emergency all right, and I just happened to be around. The patient was an elderly, rather prickly male, and he had walked straight into, and then managed to get himself twisted up in, some cricket netting set up on his own back lawn for the children to play in. It was wrapped tightly round his head and neck, and round one leg; and, apparently after some struggling with it, he had gone into a state of hysterical stupor. That was how I found him. He lay there on the lawn, curled up in a state of intense flexor spasm, just breathing, unable to speak, incontinent of fasces. He had such a tight grip on the netting I couldn’t begin to get it away from him. I don’t know what his poor wife must have thought, but I do know what I thought: that this was no time for unheroic measures. Strangulation was one danger, death from sheer I happened to have a little terror or exhaustion another. chloroform handy (but no other anaesthetic), and in no time at all I was administering chloroform to a patient for the first time in twenty years. He was soon under, and once the spasm had gone I got the netting off him easily enough. Then suddenly, of course, his breathing had to stop. I thought of the kiss of life, but his little old wrinkled face put me off, so I compromised with squeezing his chest a few times and fortunately this started spontaneous respiration again. He didn’t come round properly for some little time, and I began to wonder if my massive and inexpert administration of chloroform had not hit his liver-perhaps not inappropriatelyfor six. However, some ten minutes after I stopped the anxsthetic he was on his feet, and five minutes later he was walking. He didn’t thank me, he didn’t say a word. I doubt if he was grateful. And I don’t suppose I shall ever set eyes on that patient again. He was, you see, a hedgehog. *
*
*
According to the guidebook, the river at Galway is so stuffed with salmon during the spring run that you can’t see the riverbed. The uppermost of the bridges is the spot designated for this sight, and so we wandered down hopefully. Not a fish could be seen. I asked a local man if the spring run was on. " Yes," he said,
"
have started to come up; only a few as yet because too much water is coming down. But now the weather will be fine and in a day or two there will be thousands." So back we went on the next day: still no fish in view. An old man sidled up to me as I gazed over the parapet. "See any ?" he asked cautiously. " Not one," I replied. " Ah," he said, " there is too much water coming down. Now yesterday there were thousands. But the weather will be fine, and in the next day or two ..." I conclude that the bit in the guidebook was written by an
they
Irishman. ’*’
’*’
’*’
My peripetatic colleague and his leeches (June 17) bring back memories of the involuntary attachment of leeches to areas above shoe-level in the foothills of the Himalayas. One learnt by experience to stick to the paths in the leech season; but one also learnt from the locals who would nonchalantly go barefoot in leech-infested areas. Each carried a little bag of salt to brush off the bloodsuckers ; Western culture suggested pepper to produce coagulation; and a very effective combination this proved to be. ’*’
’*’
’*’
To thank the Lord for being not as others is a horrid, perilous sin but to make weaklings offend is perhaps even worse. So please all you " others " who " on the air say " reesearch, libary, tempory, Febary " and worst of all " controwersy ". for my weak soul’s sake and, it may be, even for your own, desist. And if you will say " ambivalent " and not " ambivvalent" and perhaps even " vitamine," not " vittamin " then I, in gratitude and for consistency, will try, adjuvante Deo, to " drop equivvalent ", of which I confess I am guilty, and take " to equi-" or " xqui-valent ". But won’t it sound odd ?
to
the Editor
SELF-TREATMENT
SiR,—The suggestion of Dr. Cargill last week (p. 1377) that oxytetracycline should be freely available to the lay public for home medication will be unacceptable to many doctors; but there should be little opposition to his broader thesis that lay people ought to be instructed in the home management of minor ailments. There should be little such opposition, but there is, in fact, a lot. We have a grave shortage of doctors, both in general and in hospital practice (leading often to the Alice-in-Wonderland kind of position described by Mr. Thompson in the same issue [p. 1379]); and yet the attitude of official medicine to selfdiagnosis and self-treatment of minor ailments is completely negative. It is an attitude that may have been appropriate in the 1920s, but today, when the quality and efficacy of home medicines have been greatly improved, and the lay public is much better informed, it is a reactionary one. Moreover, the profession in this country has always prided itself on being pragmatic, and lay people do diagnose and treat minor ailments, and will certainly continue to do so. We all know it. Why then do we not seek to make the diagnoses as sure and the treatments as safe and effective as may be ? Why behave like ostriches ? Some individual doctors are, of course, advising their patients on these matters; but it is official medicine that should be in action-producing suitable leaflets, sponsoring television and radio programmes, and encouraging a flow of informed propaganda in the Press. In fact, while it encourages lay people to sit in front of a television set and gaze upon the most complex heart-surgery-about which all they understand is the frisson it provides them with-it never once shows them how to treat a simple headache or simple indigestion or the common cold. We improve upon Marie-Antoinette: she merely advised the people to eat cake-we provide them with it. Little Missenden, S. BRADSHAW. Amersham, Bucks.
ICE-CREAM-VAN ACCIDENTS
SiR,—In experience here, a not uncommon cause of head in injury young children has been the acute suppression of caution and road drill by the prospect of ice-cream. They have usually been hit by passing vehicles when at the sound of the bell they rush blindly to the van to buy, or as they run our
back home to consume. We should be interested to hear from your readers whether this special type of accident is widespread throughout the country. If it is, some publicity might encourage its prevention. M. BRIGGS Accident Service, The Radcliffe Infirmary, Oxford. JOHN M. POTTER.
TOLERANCE TO PHYTOHÆMAGGLUTININ ?
SIR,-Despite the varied cellular response to phytohxmagglutinin (P.H.A.), doubt still persists as to the immunological nature of this response. If P.H.A. does indeed act as an antigen, although an atypical one, it might be possible to induce a state of immunological tolerance to it. We report here our failure to accomplish this. Twelve newborn rabbits, aged 4 days, were each injected subcutaneously with the contents (about 80 mg.) of one bottle of P.H.A.-M (Difco) diluted in tissue-culture medium ’199 ’ (Microbiological Products). They were reinjected again at age 6 days with the same quantity of P.H.A.-M. Control litter mates were not injected. None of the injected neonatal rabbits showed any signs of morbidity during the experiment. At age 4-6 weeks, the rabbits were killed and the popliteal lymph-nodes excised. Portions of lymph-nodes were fixed in buffered formalin, sectioned, and stained with haematoxylin and eosin. All were found to be normal, with abundant follicles. Cell
42
suspensions were prepared and incubated in medium 199, containing 20% homologous rabbit serum, and 100 units penicillin and 100 {J.g. streptomycin per ml. The cell concentration was 106 cells per ml. of medium, and sterileFalcon ’ disposable plastic tubes were used throughout. A total of 4 ml. was placed in each tube. An optimal amount of P.H.A. (0-5 ml., diluted tenfold), predetermined with normal adult lymph-node cells, was added to each tube. The tubes were sealed and maintained in the upright position in an incubator at 37°C for 72 hours. At this time the tubes were spun at 800 r.p.m. for 5 minutes, the cells resuspended in a small volume of medium (0-1 ml.), and smears made on alcohol-cleaned cover-slips. The smears were dried rapidly at 60°C with a hair-drier. They were then treated with Jenner-Giemsa stain, and examined microscopically. A thousand cells were counted at random and the percentage of blast cells was recorded. It was found that none of the P.H.A.-injected rabbits displayed any sign of immunological tolerance to P.H.A.-that is, specific failure to react to P.H.A. Lymph-node cells from the P.H.A.-
injected rabbits
behaved identically to those from normal control rabbits in the presence of P.H.A. About 40-70% of the lymph-node cells of both groups transformed to blast cells in the presence of P.H.A. in vitro. If the mitogen in P.H.A. is antigenic, one might have expected the induction of tolerance in at least several of the P.H.A.-injected rabbits. It is possible that tolerance cannot be induced to P.H.A., or that the concentration of the blastogenic factor was very low in the whole P.H.A. solution used and that therefore insufficient quantities of the active principle were injected into the neonatal rabbits. We must conclude, however, from our experiments that P.H.A. seems to act in a non-immunological fashion upon cells in inducing mitosis and blastogenesis. Harry Webster Thorp Laboratories, Division of Immunochemistry and Allergy, McGill University Clinic, M. RICHTER Royal Victoria Hospital, C. K. NASPITZ. Montreal, Quebec, Canada.
SOLARISATION OF CHROMOSOMES
SIR,- The small size of human chromosomes makes identification of minor structural abnormalities difficult
or
impossible by existing techniques. I describe here a minor variation in photographic procedure which may be of value in this regard, since it allows for greater magnification of chromosome outline than by conventional methods. The technique utilises one of the characteristic effects of solarisation exposing a partly developed negative to an unsafe light during developmentas follows: The image of a good-quality metaphase negative from a patient with chronic myeloid leukaemia (C.M.L.) (fig. 1) was used to produce a positive-copy negative on ’ Safety Ortho Lithofilm 0 82 ’ (AgfaGevaert) at an enlargement which produced good chromosome resolution. UsingPaterson Acuprint FX 17 ’ developer, diluted 1 in 4 with water, an exposure-time which allowed for the first appearance of the faint chromosome images in 15-20 seconds was obtained. The development of the chromosome images was then allowed to proceed until a satisfactory image was obtained, at which time a photographically unsafe overhead light was switched on for 1 second. The positive was then left in the developer for a further minute without agitation of developer. A few seconds after exposing the positive to the unsafe light, gradual fogging of the clear area around the chromosomes was observed, culminating in the formation of haloes around the chromosomes. These haloes gradually faded in intensity, eventually appearing to fade completely in the dim safe light. At the end of the development-time the positive was transferred to an acid-stop bath and then to a fixative solution. After fixation a correctly solarised positive shows the chromosomes surrounded by narrow, intense haloes. Some experimentation was necessary in order to obtain well-outlined chromosomes, the variables being initial exposure-time, development-time before exposure to unsafe light, the length of exposure to the unsafe light, and the development-time after exposure to the unsafe light. The production of the clear halo is determined by an increased concentration of bromide ions at the 1. The Focal
Encyclopedia of Photography (edited by F. Purves); p. 1079. London, 1960.
Fig. I-Metaphase plate from patieit with C.M.L. and probable homologue
Philadelphia chromosome (lower arrow) (upper arrow) (Leishman’s stain; x 1800).
Fig. 2-(a) Philadelphia chromosome and (b) probable homologue from solarised positive produced from negative used for fig. 1. Reduced to three-quarters from x 25,000.
boundary of a completely developed image which retard development surrounding area.1
in the
With this technique the intense halo delineating the border of the chromosome is enlarged rather than the chromosome itself, thereby allowing far greater magnification with good resolution. The border of the enlarged chromosome is represented by the inner edge of the halo, which appears black on the resulting prints (see fig. [a] and [b]). No additional details of chromosome structure can be made out. It is hoped that this fairly simple technique will prove of value in the further study and identification of normal and abnormal chromosomes, since the greater magnification of chromosome outline should allow for more accurate measurement of chromosome-arm length and of chromosome area. This study, which is being continued and will be more fully reported later, was supported by a Witwatersrand University Council research grant. I should like to thank Prof. H. B. Stein and Dr. B. A. Bradlow for helpful advice and criticism. Division of Chemical Pathology, Department of Pathology and Microbiology, University of the Witwatersrand,
Johannesburg.
C. WALLACE.
CHROMOSOMES 17-18 IN LEUKÆMIAS SiR,ńSuggestive evidence of a special relation between chromosome pairs 17 or 18 and malignant proliferations, particularly lymphomasand leukarmias,2 has been noted. In Ricci, N., Punturieri, E., Bosi, L., Castoldi, G. L. Lancet, 1962, ii, 564. Sandberg, A. A., Ishihara, T., Kikuchi, Y., Crosswhite, L. H. Cancer, N. Y. 1964, 17, 738. Sasaki, M. S., Sofuni, T., Makino, S. ibid. 1965, 18, 1007. Spiers, A. S. D., Baikie, A. G., Lancet, 1966, i, 506. 2. Lejeune, J., Berger, R., Caille, B., Turpin, R. Annls Génét. 1965, 8, 44. de Grouchy, J., de Nava, C., Cantu, J. M., Bilski-Pasquier, G., Bousser, J. Am. J. hum. Genet. 1966, 18, 485. 1.