929 INCLUSION BODIES IN MUSCULAR DYSTROPHY
SIR,-Wyatt and Cox’ have described specific "cytoplasmic inclusion bodies" in fibroblast cell lines from patients with Duchenne muscular dystrophy, a finding which could have implications for prenatal diagnosis. We have looked at our skin-fibroblast cell lines from patients with Duchenne muscular dystrophy and from normal donors to see if these inclusions are specific. So far we have examined four Duchenne and four normal cell lines..Electronmicrographs of the fibroblasts were examined blind, and neither we nor our colleagues could distinguish between those derived from normal donors and those derived from patients with muscular dystrophy. Organelles 1.
Wyatt, P. R., Cox, D.
M. Lancet,
1977, i, 172.
which could fit Wyatt and Cox’s description of the inclusion bodies were seen in fibroblasts from both sources. The tissue-culture method was either that described by Wyatt and Cox’ or our own routine system in which cells are plated at 2-4 x 105 for each 6 cm petri dish, in a culture medium made up of 80% Eagle’s minimum essential medium, 10% adult human serum and 10% chick embryo extract 50. The medium is replaced by fresh culture medium every third day. Cell growth and proliferation are examined daily. The cultures are routinely processed for electron microscopy in situ, and sections are cut parallel with the monolayer. The sections are stained with uranyl acetate and lead citrate and viewed on a Siemens 102 electron microscope. The figure shows part of a fibroblast derived from a patient with Duchenne muscular dystrophy together with part of a fibroblast from a normal donor. Both show organelles comparable with those described by Wyatt and Cox; they are membrane-bound, 0.5-2.0 um in diameter, and show internal structure. These organelles are probably secondary lysosomes (phagolysosomes), common in actively feeding cells. Such structures have the morphological characteristics of lysosomes2 and have been described in cultured skin-fibroblasts elsewhere.3 We cannot be certain that the "inclusion bodies" described by Wyatt and Cox are the same as the phagolysosomes described here, but in view of the great structural heterogeneity of lysosomes2 we suggest that they fall into this category of cell organelle. It is difficult to understand, however, why Wyatt and Cox did not find them in fibroblasts from normal donors. Our findings do not support the suggestion that fibroblasts derived from patients with Duchenne muscular dystrophy can be distinguished from fibroblasts derived from normal donors by the presence of characteristic inclusion bodies. H. E. Statham and C. A. Maunder (London) and A. D. Roses (North Carolina) have told us that they too have been unable to distinguish between pair-matched fibroblast cultures from Duchenne patients and normal donors. To make sure, we are about to examine fibroblasts from muscle, skin, and fetal tissue in vivo and in vitro. Muscular
Dystrophy Research Laboratories, Regional Neurological Centre, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE
M.
J. CULLEN
R. PARSONS
MEASLES VACCINES
SIR,-The M.R.C. report on killed and live measles vaccines
(Sept. 17, p. 571) lends further support to the enduring protection afforded by live vaccine. It reinforces the view that routine re-immunisation with live vaccine is generally unnecessary
Sections of cultured fibroblasts.
Upper: from patient with Duchenne muscular dystrophy. Lower: from normal donor.
Both photographs show about 10 phagolysosomes (5 arrowed in each). Ph=phagolysosome, M=mitochondrion, L=lipid. Magnification x 20 000.
except for those immunised before 15 months of age and those who simultaneously received human immunoglobulin together with live vaccine or for some other purpose in the preceding 4-5 weeks. A few additional failures might be expected due to improper technique (faulty storage and overexposure to light.) The U.K. programme avoided two problems which occurred in the U.S.A.-namely, the use of killed vaccine before live vaccine (resulting in failure to achieve protection in some instances) and the appearance of atypical measles syndromes, often very severe, in some persons previously injected with killed vaccine and later exposed to natural measles or live vaccine. The avoidance of both these problems would seem to be due to the U.K. schedule of giving one dose only of killed vaccine followed by live vaccine one month later. The single dose and short time-lapse mean that insufficient antibody would have developed to inhibit multiplication of virus in the live vaccine. 2. 3.
Holtzman, E. Lysosomes: a Survey. Vienna, 1976. Aula, P. in Biology of Fibroblast (edited by E. Kulonen and J. Pikkarainen); p. 187. London, 1973
930 When killed vaccine first became available in the U.S.A. multiple doses and various time intervals were often used. Some such recipients had sufficient circulating antibody from the doses of killed vaccine to prevent live virus vaccine multiplication thus remaining susceptible to measles and to the atypical measles syndrome. The recent occurrence in the U.S.A. of outbreaks of measles in schoolchildren and adolescents has led to the advice that the earlier recipients of killed vaccine be "re-immunised" with live vaccine. The number of doses of killed vaccine and the intervals between them are too often neither recalled nor accurately recorded, a common problem with immunisation practices
generally. Department of Pediatrics, Mount Sinai School of Medicine of University of City of New York, New York, N.Y. 10029, U.S.A.
ALEX
HUMAN POST-MORTEM PINEAL METABOISM current interestl-3 in the human pineal gland is establish its function. We have found synchronous rhythms in human serum-melatonin and in pineal enzyme activity post mortem.4 In an extension of these studies we have now determined 5-hydroxytryptamine (5-H.T.) in post-mortem homogenates of human pineal in which the activities of N-acetyltransferase (N.A.T.) and hydroxyindole-0-methyltransferase (H.I.O.M.T.) have also simultaneously been determined.4 The pineals were removed,5 bisected sagitally, homogenised, and stored at -20°C. Measurements were done blind, and both N.A.T. and H.I.O.M.T. are stable under post-mortem conditions.4 The results are summarised in the accompanying figure. 5-H.T. had a diurnal rhythm with a peak of 25+5ng/mgpineal
SIR,-’The
to
J. STEIGMAN
IRON ABSORPTION IN IRON-LOADING ANÆMIAS
SiR,-Dr Pippard and his colleagues (Oct. 8, p. 737) summarise their paper by stating that in patients with iron-loading anaemias the infusion of desferrioxamine is associated with a reduction in iron absorption and that this effect is not altered by saturation with ascorbic acid. These conclusions are surprising in view of the data they present from only nine patients with a variety of clinical states. Iron absorption was significantly reduced during the desferrioxamine infusion in only the three cases of p-thalassaemia major in this study and in only one of these did ascorbic-acid repletion take place between the two tests. This seems an inadequate basis from which to draw conclusions, particularly in view of the large variation in iron absorption known to occur from day to day in the same individuals. 1,2 Department of Hæmatology University Hospital of Wales Cardiff CF4 4XN
ALLAN JACOBS
SIR,-Dr Pippard and his colleagues conclude that desferrioxamine therapy leads to a reduction in iron absorption from the small bowel in patients with iron-storage disorders. This is a very important point now that effective iron-removal therapy is available,3.4 because increased absorption could lead to a partial reversal of the beneficial effects of subcutaneous desferrioxamine. I do not believe, however, that this study by Pippard et al. permits the conclusion that desferrioxamine will be associated with reduced absorption throughout the course of treatment in all patients. (1) Only five out of nine patients showed reduced absorption. (2) Measurement of iron absorption in man is fraught with difficulties and values may vary considerably from day to day. (3) Values for haemoglobin, serum-iron, and total ironbinding capacity in the second study were not shown, and these may have played a role in influencing iron absorption during the second phase of the experiment. (4) The measurements were made only during desferrioxamine infusion whereas the depletion of iron stores may lead to an increased absorption during the remainder of the 24 h period when the drug is not being administered.’S While the report is encouraging the effect in those treated long term with this chelating agent remains to be determined. Medical Unit, Royal Free Hospital, London NW3 2QG
1. 2.
Asynchronous 5-H.T. rhythm and synthetic human post-mortem pineal.
enzyme
rhythms in
death times 8 A.M. to noon and a trough of 5±1 8 P.M. Both the N.A.T. and H.l.o.M.T. activities peak at death times midnight to 4 A.M. (1278±100 and 414±40 pmol/h/mg protein, respectively) and show least activity at noon to 4 P.M. (728+46 and 214±31 pmol/h/mg protein, respectively). Thus the 5-H.T. rhythm is asynchronous with respect to the N.A.T. and H.I.O.M.T. cycles, although the phase shift is not 180° as seen in rats. It is thought that in rats 5-H.T. is stored in the pineal during the day when it is not being metabolised and then released for N-acetylation by N.A.T.,6 the enzyme being stimulated, in vitro at least, by cyclic A.M.P. However, the decrease in 5-H.T. in man seems to occur well before N.A.T. activity, by a mechanism as yet unknown, intissue
at
ng/mg around
creases.
These data
R. BATEY
Cook, J. D., Layrisse, M., Finch, C. A. Blood, 1969, 33, 421. Ashworth, A., Milner, P. F., Waterlow, J. C., Walker, R. B. Br. J. Nutr. 1973, 29, 269. 3. Hussain, M. A. M., Flynn, D. M. Green, N., Hussein, S., Hoffbrand, A. V. Lancet, 1976, ii, 1278. 4. Hussain, M. A. M., Flynn, D. M., Green, N., Hoffbrand, A. V. ibid. 1977, i, 977. 5. Batey, R., Gallagher, N. D. Am. J. Physiol. 1977, 232, E57-61.
on
normal post-mortem
pineal metabolism will
Smith, J. A., Mee, T. J. X., Barnes, N. D., Thornburn, R. J., Barnes, J. L C. Lancet, 1976, ii, 425. 2. Hansen, T., Heyden, T., Sundberg, I., Wetterburg, L. ibid. 1977, ii, 310. 3. Mullen, P. E., Silman, R. E. Psychol. Med. 1977, 7, 407. 4. Smith, J. A., Padwick, D., Mee, T. J. X., Minneman, K. P., Bird, E. D. Clin. 1.
Endow. 1977, 6, 219. 5. Bird, E. D., Iversen, L. C. Brain, 1974, 97, 457. 6. Snyder, S. H., Axelrod, J. Science, 1975, 149, 542.
SIR,-Wyatt and Cox’ have described specific "cytoplasmic inclusion bodies" in fibroblast cell lines from patients with Duchenne muscular dystrophy, a finding which could have implications for prenatal diagnosis. We have looked at our skin-fibroblast cell lines from patients with Duchenne muscular dystrophy and from normal donors to see if these inclusions are specific. So far we have examined four Duchenne and four normal cell lines..Electronmicrographs of the fibroblasts were examined blind, and neither we nor our colleagues could distinguish between those derived from normal donors and those derived from patients with muscular dystrophy. Organelles 1.
Wyatt, P. R., Cox, D.
M. Lancet,
1977, i, 172.
which could fit Wyatt and Cox’s description of the inclusion bodies were seen in fibroblasts from both sources. The tissue-culture method was either that described by Wyatt and Cox’ or our own routine system in which cells are plated at 2-4 x 105 for each 6 cm petri dish, in a culture medium made up of 80% Eagle’s minimum essential medium, 10% adult human serum and 10% chick embryo extract 50. The medium is replaced by fresh culture medium every third day. Cell growth and proliferation are examined daily. The cultures are routinely processed for electron microscopy in situ, and sections are cut parallel with the monolayer. The sections are stained with uranyl acetate and lead citrate and viewed on a Siemens 102 electron microscope. The figure shows part of a fibroblast derived from a patient with Duchenne muscular dystrophy together with part of a fibroblast from a normal donor. Both show organelles comparable with those described by Wyatt and Cox; they are membrane-bound, 0.5-2.0 um in diameter, and show internal structure. These organelles are probably secondary lysosomes (phagolysosomes), common in actively feeding cells. Such structures have the morphological characteristics of lysosomes2 and have been described in cultured skin-fibroblasts elsewhere.3 We cannot be certain that the "inclusion bodies" described by Wyatt and Cox are the same as the phagolysosomes described here, but in view of the great structural heterogeneity of lysosomes2 we suggest that they fall into this category of cell organelle. It is difficult to understand, however, why Wyatt and Cox did not find them in fibroblasts from normal donors. Our findings do not support the suggestion that fibroblasts derived from patients with Duchenne muscular dystrophy can be distinguished from fibroblasts derived from normal donors by the presence of characteristic inclusion bodies. H. E. Statham and C. A. Maunder (London) and A. D. Roses (North Carolina) have told us that they too have been unable to distinguish between pair-matched fibroblast cultures from Duchenne patients and normal donors. To make sure, we are about to examine fibroblasts from muscle, skin, and fetal tissue in vivo and in vitro. Muscular
Dystrophy Research Laboratories, Regional Neurological Centre, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE
M.
J. CULLEN
R. PARSONS
MEASLES VACCINES
SIR,-The M.R.C. report on killed and live measles vaccines
(Sept. 17, p. 571) lends further support to the enduring protection afforded by live vaccine. It reinforces the view that routine re-immunisation with live vaccine is generally unnecessary
Sections of cultured fibroblasts.
Upper: from patient with Duchenne muscular dystrophy. Lower: from normal donor.
Both photographs show about 10 phagolysosomes (5 arrowed in each). Ph=phagolysosome, M=mitochondrion, L=lipid. Magnification x 20 000.
except for those immunised before 15 months of age and those who simultaneously received human immunoglobulin together with live vaccine or for some other purpose in the preceding 4-5 weeks. A few additional failures might be expected due to improper technique (faulty storage and overexposure to light.) The U.K. programme avoided two problems which occurred in the U.S.A.-namely, the use of killed vaccine before live vaccine (resulting in failure to achieve protection in some instances) and the appearance of atypical measles syndromes, often very severe, in some persons previously injected with killed vaccine and later exposed to natural measles or live vaccine. The avoidance of both these problems would seem to be due to the U.K. schedule of giving one dose only of killed vaccine followed by live vaccine one month later. The single dose and short time-lapse mean that insufficient antibody would have developed to inhibit multiplication of virus in the live vaccine. 2. 3.
Holtzman, E. Lysosomes: a Survey. Vienna, 1976. Aula, P. in Biology of Fibroblast (edited by E. Kulonen and J. Pikkarainen); p. 187. London, 1973
930 When killed vaccine first became available in the U.S.A. multiple doses and various time intervals were often used. Some such recipients had sufficient circulating antibody from the doses of killed vaccine to prevent live virus vaccine multiplication thus remaining susceptible to measles and to the atypical measles syndrome. The recent occurrence in the U.S.A. of outbreaks of measles in schoolchildren and adolescents has led to the advice that the earlier recipients of killed vaccine be "re-immunised" with live vaccine. The number of doses of killed vaccine and the intervals between them are too often neither recalled nor accurately recorded, a common problem with immunisation practices
generally. Department of Pediatrics, Mount Sinai School of Medicine of University of City of New York, New York, N.Y. 10029, U.S.A.
ALEX
HUMAN POST-MORTEM PINEAL METABOISM current interestl-3 in the human pineal gland is establish its function. We have found synchronous rhythms in human serum-melatonin and in pineal enzyme activity post mortem.4 In an extension of these studies we have now determined 5-hydroxytryptamine (5-H.T.) in post-mortem homogenates of human pineal in which the activities of N-acetyltransferase (N.A.T.) and hydroxyindole-0-methyltransferase (H.I.O.M.T.) have also simultaneously been determined.4 The pineals were removed,5 bisected sagitally, homogenised, and stored at -20°C. Measurements were done blind, and both N.A.T. and H.I.O.M.T. are stable under post-mortem conditions.4 The results are summarised in the accompanying figure. 5-H.T. had a diurnal rhythm with a peak of 25+5ng/mgpineal
SIR,-’The
to
J. STEIGMAN
IRON ABSORPTION IN IRON-LOADING ANÆMIAS
SiR,-Dr Pippard and his colleagues (Oct. 8, p. 737) summarise their paper by stating that in patients with iron-loading anaemias the infusion of desferrioxamine is associated with a reduction in iron absorption and that this effect is not altered by saturation with ascorbic acid. These conclusions are surprising in view of the data they present from only nine patients with a variety of clinical states. Iron absorption was significantly reduced during the desferrioxamine infusion in only the three cases of p-thalassaemia major in this study and in only one of these did ascorbic-acid repletion take place between the two tests. This seems an inadequate basis from which to draw conclusions, particularly in view of the large variation in iron absorption known to occur from day to day in the same individuals. 1,2 Department of Hæmatology University Hospital of Wales Cardiff CF4 4XN
ALLAN JACOBS
SIR,-Dr Pippard and his colleagues conclude that desferrioxamine therapy leads to a reduction in iron absorption from the small bowel in patients with iron-storage disorders. This is a very important point now that effective iron-removal therapy is available,3.4 because increased absorption could lead to a partial reversal of the beneficial effects of subcutaneous desferrioxamine. I do not believe, however, that this study by Pippard et al. permits the conclusion that desferrioxamine will be associated with reduced absorption throughout the course of treatment in all patients. (1) Only five out of nine patients showed reduced absorption. (2) Measurement of iron absorption in man is fraught with difficulties and values may vary considerably from day to day. (3) Values for haemoglobin, serum-iron, and total ironbinding capacity in the second study were not shown, and these may have played a role in influencing iron absorption during the second phase of the experiment. (4) The measurements were made only during desferrioxamine infusion whereas the depletion of iron stores may lead to an increased absorption during the remainder of the 24 h period when the drug is not being administered.’S While the report is encouraging the effect in those treated long term with this chelating agent remains to be determined. Medical Unit, Royal Free Hospital, London NW3 2QG
1. 2.
Asynchronous 5-H.T. rhythm and synthetic human post-mortem pineal.
enzyme
rhythms in
death times 8 A.M. to noon and a trough of 5±1 8 P.M. Both the N.A.T. and H.l.o.M.T. activities peak at death times midnight to 4 A.M. (1278±100 and 414±40 pmol/h/mg protein, respectively) and show least activity at noon to 4 P.M. (728+46 and 214±31 pmol/h/mg protein, respectively). Thus the 5-H.T. rhythm is asynchronous with respect to the N.A.T. and H.I.O.M.T. cycles, although the phase shift is not 180° as seen in rats. It is thought that in rats 5-H.T. is stored in the pineal during the day when it is not being metabolised and then released for N-acetylation by N.A.T.,6 the enzyme being stimulated, in vitro at least, by cyclic A.M.P. However, the decrease in 5-H.T. in man seems to occur well before N.A.T. activity, by a mechanism as yet unknown, intissue
at
ng/mg around
creases.
These data
R. BATEY
Cook, J. D., Layrisse, M., Finch, C. A. Blood, 1969, 33, 421. Ashworth, A., Milner, P. F., Waterlow, J. C., Walker, R. B. Br. J. Nutr. 1973, 29, 269. 3. Hussain, M. A. M., Flynn, D. M. Green, N., Hussein, S., Hoffbrand, A. V. Lancet, 1976, ii, 1278. 4. Hussain, M. A. M., Flynn, D. M., Green, N., Hoffbrand, A. V. ibid. 1977, i, 977. 5. Batey, R., Gallagher, N. D. Am. J. Physiol. 1977, 232, E57-61.
on
normal post-mortem
pineal metabolism will
Smith, J. A., Mee, T. J. X., Barnes, N. D., Thornburn, R. J., Barnes, J. L C. Lancet, 1976, ii, 425. 2. Hansen, T., Heyden, T., Sundberg, I., Wetterburg, L. ibid. 1977, ii, 310. 3. Mullen, P. E., Silman, R. E. Psychol. Med. 1977, 7, 407. 4. Smith, J. A., Padwick, D., Mee, T. J. X., Minneman, K. P., Bird, E. D. Clin. 1.
Endow. 1977, 6, 219. 5. Bird, E. D., Iversen, L. C. Brain, 1974, 97, 457. 6. Snyder, S. H., Axelrod, J. Science, 1975, 149, 542.
