- Email: [email protected]
CELI. DESTRUCTION BY LYMPHOCYTES
1215
waiting
about
4-5 minutes from birth
permanent brain damage
asphyxia in newly
to treatment
in babies;
after
only 7-8 minutes total monkeys, though it cannot always
occurs
delivered be detected by clinical examination. The basic assumption that only oxygen is required for resuscitation is false. It is only in the early stages of asphyxia that oxygen lack is the crucial factor-ultimately it is the fall in pH which damages the brain and stops the heart (in that order). So the most urgent thing is not to supply oxygen to the lungs but to ventilate them. Cardiovascular Research Institute, School of Medicine, San Francisco Medical Center,
University of California,
G. S. DAWES.
California 94122.
EXCHANGE-TRANSFUSION APPARATUS like pets-one becomes very fond of one’s own and will not change it. I am prompted by the letter of Dr. Simmons and Dr. Ata (April 2) to describe my own pet-a disposable apparatus for a gravity (drip) exchange-transfusion system.! It is a little odd, when in good paediatric practice we insist that simple transfusions to infants should always be given by drip, that we continue to use the " push " technique for the giving part of the exchangetransfusion cycle. There is no advantage in doing so, while the disadvantages are obvious. Once we accept this we arrive at a much safer and simpler procedure.!
SIR,-Exchange-transfusion apparatuses
accompanying figure shows the complete assembly, use at the newborn unit of this hospital, which many improvements over the assembly previously
currently in offers
described.1 1. The whole apparatus is
disposable:
it includes
a
’Metriset’
administration set, special four-way valve (’Pharmaseal’ four-way
valve, as modified by the makers to our specifications), and disposable syringe and catheters. 2. There is only one valve and only two positions for the whole procedure: in position i, blood is aspirated from the patient; in position n, the donor-blood, in measured quantities of 10 or 20 ml., 1.
Valaes,
T.
The simplicity of the procedure with this assembly is a great contribution towards its safety. Over 600 exchange transfusions have been performed by the drip method in our unit without a single accident. Moreover 25 young residents have performed these exchanges single-handed and after they have been shown the method only 2-3 times. It seems an extravagant claim, but on several occasions one operator has performed two exchange transfusions simultaneously quite easily. The various components of the assembly seal Laboratories and Don Baxter, Inc.
Queen Anna-Maria Institute of Child Health, Aghia Sophia Children’s Hospital, Athens 609.
are
supplied by
Pharma-
T. VALAES.
are
Disposable exchange-transfusion apparatus. The
into the patient by gravity and at the desired rate, while the aspirated patient-blood is emptied to a waste receiver. 3. The special rubber-capped nozzle of the four-way valve allows drugs to be given to the patient without disconnecting the syringe.
runs
Lancet, 1960, ii, 496.
" FOG FEVER " AND FARMER’S LUNG a great deal of interest your leading article.’ We too have had results similar to those outlined by Pepys and Jenkins.2 We have now analysed some 700 sera of farmers with pulmonary disease of undetermined aetiology. We have found that roughly 15 % of these patients have precipitins to Thermopolyspora polyspora, and that the rate of positive reactors among patients who have clinically and microscopically proven farmer’s lung is roughly 90%. But lacking from your article was our report3 of the isolation of T. polyspora from a patient with farmer’s lung. I believe that this paper further substantiates the work of Dr. Pepys and his declaration that the thermophylic actinomyces are closely linked with farmer’s lung. The organism was cultured from lung-biopsy material, and grew well on nutrient agar. Tubes were held at 60°C. An extract of this organism, the appearances of which were morphologically identical to T. polyspora, reacted with sera from this patient and from our other patients with farmer’s lung. This organism was later sent to Dr. Phillip Gregory’s laboratories, and he confirmed that this organism was indeed T. polyspora. Work in our laboratories is continuing in order to establish even more firmly the relations of the thermophylic actinomyces, and in particular T. polyspora, with farmer’s lung. FREDERICK J. WENZEL Marshfield Clinic Foundation, DEAN A. EMANUEL. Wisconsin. Marshfield,
SIR,-We read with
CELI. DESTRUCTION BY LYMPHOCYTES SIR,-In their article (Feb. 26) Tatiana Trayanova and her co-workers state that they have demonstrated " the role of immune lymphocytes in the pathogenesis of S.L.E. (systemic lupus erythematosus) ". We wish to comment on this. Destruction of cells in recipients by lymphocytes of homohas been postulated to occur in murine runt logous donors disease,45which may be regarded as a model system of autoimmune diseases. In human chronic lymphocytic leukaemia (C.L.L.), lymphocytes destroyed the patient’s own fibroblasts in tissue-culture; this reaction was unusually vigorous when Coombs-positive haemolytic anxmia was associated with leukaemia 6; thus, an autoimmune mechanism for cell destruction has been suggested. We have only limited experience with lymphocytes from patients with collagen diseases. In 2 patients with Sjogren’s syndrome, one with positive L.E. test, lymphocytic autoaggression in cultures of bone-marrow was minimal.8 1. 2. 3. 4.
5. 6. 7. 8.
Lancet, 1965, ii, 224. Pepys, J., Jenkins, P. A. Thorax, 1965, 20, 21. Wenzel, F. J., Emanuel, D. A., Lawton, B. R., Magnin, G. E. Ann. Allergy, 1964, 22, 533. Sinkovics, J. G., Howe, C. D. Tex. Rep. Biol. Med. 1964, 22, 591. Sinkovics, J. G., Shullenberger, C. C., Howe, C. D. ibid. 1965, 23, 94. Sinkovics, J. G. Nature, Lond. 1962, 196, 80. Sinkovics, J. G., Howe, C. D., Shullenberger, C. C. Blood, 1964, 24, 389. Sinkovics, J. G., Shullenberger, C. C., Howe, C. D. Unpublished.
1216 We believe that reactions of lymphocytes with other cells in vitro is not necessarily an expression of immunity.’ An autoimmune mechanism may be considered to be operative when the tissue-culture system is autologous, and both lympho" cytes and target cells succumb to allergic cell death " upon most often when lymphocytes are contact. This happens not presensitised may be stimupresensitised. Lymphocytes lated to undergo blastic transformation upon contact with genetically incompatible cells. When the system is not autologous and the lymphocytes retain full viability (structurally intact, tinctorially normal, moving, sometimes dividing), even after long and intimate contact with fibroblastic cells, a feederlayer mechanism may be postulated. It is known that certain types of lymphocytes can parasitise other cells and use them as feeder layers in long-term cultures, in autologous, homologous or heterologous combinations, and with or without damage to the feeder layer (figs. 1, 2, and 3). Sometimes as many as 50 lymphocytes thrive for several weeks on top of, or inside, a fibroblast before the fibroblast disintegrates. The fate and structure of lymphocytes in the homologous cell system of Trayanova and her co-workers have not been dealt with (neither has it been stated whether these specimens were free of the insidious and often cytopathogenic mycoplasma said to be a frequent finding in cultures deriving from collagen diseases). Thus the pathomechanism of cell destruction is not clear, and the possibility of a feeder-layer type of association between these types of lymphocytes and the cell sheet used should be ruled out by further experimentation.
Supported by U.S. Public Health Service grant CA 07923 and award K3-CA-16747.
JOSEPH G. SINKOVICS
M. D. Anderson Hospital, University of Texas, Houston, Texas 77025, U.S.A.
C. C. SHULLENBERGER CLIFTON D. HOWE.
FEMALE CARRIERS OF MUSCULAR DYSTROPHY SiR,ŅIn 1963 Dubowitz9 and Emery 10 first described microscopic findings in three carriers of recessive sex-linked muscular dystrophy of Duchenne type who showed slight clinical signs of the disease. Similar findings have been reported by others. 11-14 Emery describes the following pathological changes: differences in size of the muscle-fibres, centralisation of the nuclei, focal necrosis with phagocytosis, increase of connective tissue, and mild fatty degeneration. The earliest sign was swollen fibres. The correlation between serum-enzyme-level elevation and electron microscopic changes does not seem to have been studied before. We have
performed estimations of serum-creatine-phosphokinase (c.P.K.) activity, and biopsy of the quadriceps muscle in three possible female carriers of Duchenne-type muscular dystrophy. C.P.K. values (normally < 15 i.u.) were estimated in the so-called " forward reaction " with addition of reduced glutathione, which in our experience 15 is essential for preservation and maximal activity of C.P.K. The results, in a mother (case 1) and in sisters (cases 2 and 3) of patients, were as follows: Microscopic findings Case no. Age (yr) C.P.K. (1. U.) Light microscopy Electronmicroscopy Fig. 1-Structurally intact lymphocytes inside fibroblast (N =nucleus of fibroblast) in tissue-culture of bone-marrow from patient with C.L.L. (Papanicolaou stain, oil immersion [preparation of Miss Shirley Davis].) Neither " allergic cell death " nor blastictransformation of lymphocytes occurred, but parasitised fibroblasts usually disintegrated. Fig. 2-Clumps of structurally intact lymphocytes in third passage of symbiotic culture of fibroblasts from 1 patient with C.L.L. (Coriphosphine 0 stain, fluorescence microscopy, low magnifica’tion [preparation of Miss Barbara Bertin].) Degenerating fibroblasts occurred beneath clumps of lymphocytes. These fibroblasts could not be serially passed and lymphocytes failed to
survivewithout them. of lymphocytes parasitising fibroblasts in culture of human lymph-node from 1 case of C.L.L. (Phase-contrast microscopy, low magnification.)
Fig. 3-Large numbers 6-weeks-old primary
1 2
3
25 14 14
70 232 33
Abnormal Normal Normal
Abnormal " "
On light microscopy we found in case 1 an abnormal increase of fat, and in case 3 a single row of centrally placed nuclei. On electron microscopy we found changes of fine structure similar to those seen in early stages of muscular dystrophy 16I -i.e., increase in the number of mitochondria in the perinuclear area. There was also increase in the number of lipid globules and in glycogen, and new formation of pigment 9. 10. 11.
12. 13. 14. 15. 16.
Dubowitz, V. J. Neurol. Neurosurg. Psychiat. 1963, 26, 322. Emery, A. E. H. Lancet, 1963, i, 1126. Pearson, C. M., Fowler, W. M., Wright, S. W. Proc. nat. Acad. Sci., U.S.A. 1963, 50, 24. Maciotta, A., Costa, V., Cao, A., Sforza, F., Scano, V. Annali. ital. Pediat. 1964, 17, 163. Stephens, J., Lewin, E. J. Neurol. Neurosurg. Psychiat. 1965, 28, 104. Emery, A. E. H. J. med. Genet. 1965, 2, 1. Rotthauwe, H. W., Kowalewski, S. Unpublished. Mölbert, E., Marx, R. in Myopathien. Stuttgart 1965.
waiting
about
4-5 minutes from birth
permanent brain damage
asphyxia in newly
to treatment
in babies;
after
only 7-8 minutes total monkeys, though it cannot always
occurs
delivered be detected by clinical examination. The basic assumption that only oxygen is required for resuscitation is false. It is only in the early stages of asphyxia that oxygen lack is the crucial factor-ultimately it is the fall in pH which damages the brain and stops the heart (in that order). So the most urgent thing is not to supply oxygen to the lungs but to ventilate them. Cardiovascular Research Institute, School of Medicine, San Francisco Medical Center,
University of California,
G. S. DAWES.
California 94122.
EXCHANGE-TRANSFUSION APPARATUS like pets-one becomes very fond of one’s own and will not change it. I am prompted by the letter of Dr. Simmons and Dr. Ata (April 2) to describe my own pet-a disposable apparatus for a gravity (drip) exchange-transfusion system.! It is a little odd, when in good paediatric practice we insist that simple transfusions to infants should always be given by drip, that we continue to use the " push " technique for the giving part of the exchangetransfusion cycle. There is no advantage in doing so, while the disadvantages are obvious. Once we accept this we arrive at a much safer and simpler procedure.!
SIR,-Exchange-transfusion apparatuses
accompanying figure shows the complete assembly, use at the newborn unit of this hospital, which many improvements over the assembly previously
currently in offers
described.1 1. The whole apparatus is
disposable:
it includes
a
’Metriset’
administration set, special four-way valve (’Pharmaseal’ four-way
valve, as modified by the makers to our specifications), and disposable syringe and catheters. 2. There is only one valve and only two positions for the whole procedure: in position i, blood is aspirated from the patient; in position n, the donor-blood, in measured quantities of 10 or 20 ml., 1.
Valaes,
T.
The simplicity of the procedure with this assembly is a great contribution towards its safety. Over 600 exchange transfusions have been performed by the drip method in our unit without a single accident. Moreover 25 young residents have performed these exchanges single-handed and after they have been shown the method only 2-3 times. It seems an extravagant claim, but on several occasions one operator has performed two exchange transfusions simultaneously quite easily. The various components of the assembly seal Laboratories and Don Baxter, Inc.
Queen Anna-Maria Institute of Child Health, Aghia Sophia Children’s Hospital, Athens 609.
are
supplied by
Pharma-
T. VALAES.
are
Disposable exchange-transfusion apparatus. The
into the patient by gravity and at the desired rate, while the aspirated patient-blood is emptied to a waste receiver. 3. The special rubber-capped nozzle of the four-way valve allows drugs to be given to the patient without disconnecting the syringe.
runs
Lancet, 1960, ii, 496.
" FOG FEVER " AND FARMER’S LUNG a great deal of interest your leading article.’ We too have had results similar to those outlined by Pepys and Jenkins.2 We have now analysed some 700 sera of farmers with pulmonary disease of undetermined aetiology. We have found that roughly 15 % of these patients have precipitins to Thermopolyspora polyspora, and that the rate of positive reactors among patients who have clinically and microscopically proven farmer’s lung is roughly 90%. But lacking from your article was our report3 of the isolation of T. polyspora from a patient with farmer’s lung. I believe that this paper further substantiates the work of Dr. Pepys and his declaration that the thermophylic actinomyces are closely linked with farmer’s lung. The organism was cultured from lung-biopsy material, and grew well on nutrient agar. Tubes were held at 60°C. An extract of this organism, the appearances of which were morphologically identical to T. polyspora, reacted with sera from this patient and from our other patients with farmer’s lung. This organism was later sent to Dr. Phillip Gregory’s laboratories, and he confirmed that this organism was indeed T. polyspora. Work in our laboratories is continuing in order to establish even more firmly the relations of the thermophylic actinomyces, and in particular T. polyspora, with farmer’s lung. FREDERICK J. WENZEL Marshfield Clinic Foundation, DEAN A. EMANUEL. Wisconsin. Marshfield,
SIR,-We read with
CELI. DESTRUCTION BY LYMPHOCYTES SIR,-In their article (Feb. 26) Tatiana Trayanova and her co-workers state that they have demonstrated " the role of immune lymphocytes in the pathogenesis of S.L.E. (systemic lupus erythematosus) ". We wish to comment on this. Destruction of cells in recipients by lymphocytes of homohas been postulated to occur in murine runt logous donors disease,45which may be regarded as a model system of autoimmune diseases. In human chronic lymphocytic leukaemia (C.L.L.), lymphocytes destroyed the patient’s own fibroblasts in tissue-culture; this reaction was unusually vigorous when Coombs-positive haemolytic anxmia was associated with leukaemia 6; thus, an autoimmune mechanism for cell destruction has been suggested. We have only limited experience with lymphocytes from patients with collagen diseases. In 2 patients with Sjogren’s syndrome, one with positive L.E. test, lymphocytic autoaggression in cultures of bone-marrow was minimal.8 1. 2. 3. 4.
5. 6. 7. 8.
Lancet, 1965, ii, 224. Pepys, J., Jenkins, P. A. Thorax, 1965, 20, 21. Wenzel, F. J., Emanuel, D. A., Lawton, B. R., Magnin, G. E. Ann. Allergy, 1964, 22, 533. Sinkovics, J. G., Howe, C. D. Tex. Rep. Biol. Med. 1964, 22, 591. Sinkovics, J. G., Shullenberger, C. C., Howe, C. D. ibid. 1965, 23, 94. Sinkovics, J. G. Nature, Lond. 1962, 196, 80. Sinkovics, J. G., Howe, C. D., Shullenberger, C. C. Blood, 1964, 24, 389. Sinkovics, J. G., Shullenberger, C. C., Howe, C. D. Unpublished.
1216 We believe that reactions of lymphocytes with other cells in vitro is not necessarily an expression of immunity.’ An autoimmune mechanism may be considered to be operative when the tissue-culture system is autologous, and both lympho" cytes and target cells succumb to allergic cell death " upon most often when lymphocytes are contact. This happens not presensitised may be stimupresensitised. Lymphocytes lated to undergo blastic transformation upon contact with genetically incompatible cells. When the system is not autologous and the lymphocytes retain full viability (structurally intact, tinctorially normal, moving, sometimes dividing), even after long and intimate contact with fibroblastic cells, a feederlayer mechanism may be postulated. It is known that certain types of lymphocytes can parasitise other cells and use them as feeder layers in long-term cultures, in autologous, homologous or heterologous combinations, and with or without damage to the feeder layer (figs. 1, 2, and 3). Sometimes as many as 50 lymphocytes thrive for several weeks on top of, or inside, a fibroblast before the fibroblast disintegrates. The fate and structure of lymphocytes in the homologous cell system of Trayanova and her co-workers have not been dealt with (neither has it been stated whether these specimens were free of the insidious and often cytopathogenic mycoplasma said to be a frequent finding in cultures deriving from collagen diseases). Thus the pathomechanism of cell destruction is not clear, and the possibility of a feeder-layer type of association between these types of lymphocytes and the cell sheet used should be ruled out by further experimentation.
Supported by U.S. Public Health Service grant CA 07923 and award K3-CA-16747.
JOSEPH G. SINKOVICS
M. D. Anderson Hospital, University of Texas, Houston, Texas 77025, U.S.A.
C. C. SHULLENBERGER CLIFTON D. HOWE.
FEMALE CARRIERS OF MUSCULAR DYSTROPHY SiR,ŅIn 1963 Dubowitz9 and Emery 10 first described microscopic findings in three carriers of recessive sex-linked muscular dystrophy of Duchenne type who showed slight clinical signs of the disease. Similar findings have been reported by others. 11-14 Emery describes the following pathological changes: differences in size of the muscle-fibres, centralisation of the nuclei, focal necrosis with phagocytosis, increase of connective tissue, and mild fatty degeneration. The earliest sign was swollen fibres. The correlation between serum-enzyme-level elevation and electron microscopic changes does not seem to have been studied before. We have
performed estimations of serum-creatine-phosphokinase (c.P.K.) activity, and biopsy of the quadriceps muscle in three possible female carriers of Duchenne-type muscular dystrophy. C.P.K. values (normally < 15 i.u.) were estimated in the so-called " forward reaction " with addition of reduced glutathione, which in our experience 15 is essential for preservation and maximal activity of C.P.K. The results, in a mother (case 1) and in sisters (cases 2 and 3) of patients, were as follows: Microscopic findings Case no. Age (yr) C.P.K. (1. U.) Light microscopy Electronmicroscopy Fig. 1-Structurally intact lymphocytes inside fibroblast (N =nucleus of fibroblast) in tissue-culture of bone-marrow from patient with C.L.L. (Papanicolaou stain, oil immersion [preparation of Miss Shirley Davis].) Neither " allergic cell death " nor blastictransformation of lymphocytes occurred, but parasitised fibroblasts usually disintegrated. Fig. 2-Clumps of structurally intact lymphocytes in third passage of symbiotic culture of fibroblasts from 1 patient with C.L.L. (Coriphosphine 0 stain, fluorescence microscopy, low magnifica’tion [preparation of Miss Barbara Bertin].) Degenerating fibroblasts occurred beneath clumps of lymphocytes. These fibroblasts could not be serially passed and lymphocytes failed to
survivewithout them. of lymphocytes parasitising fibroblasts in culture of human lymph-node from 1 case of C.L.L. (Phase-contrast microscopy, low magnification.)
Fig. 3-Large numbers 6-weeks-old primary
1 2
3
25 14 14
70 232 33
Abnormal Normal Normal
Abnormal " "
On light microscopy we found in case 1 an abnormal increase of fat, and in case 3 a single row of centrally placed nuclei. On electron microscopy we found changes of fine structure similar to those seen in early stages of muscular dystrophy 16I -i.e., increase in the number of mitochondria in the perinuclear area. There was also increase in the number of lipid globules and in glycogen, and new formation of pigment 9. 10. 11.
12. 13. 14. 15. 16.
Dubowitz, V. J. Neurol. Neurosurg. Psychiat. 1963, 26, 322. Emery, A. E. H. Lancet, 1963, i, 1126. Pearson, C. M., Fowler, W. M., Wright, S. W. Proc. nat. Acad. Sci., U.S.A. 1963, 50, 24. Maciotta, A., Costa, V., Cao, A., Sforza, F., Scano, V. Annali. ital. Pediat. 1964, 17, 163. Stephens, J., Lewin, E. J. Neurol. Neurosurg. Psychiat. 1965, 28, 104. Emery, A. E. H. J. med. Genet. 1965, 2, 1. Rotthauwe, H. W., Kowalewski, S. Unpublished. Mölbert, E., Marx, R. in Myopathien. Stuttgart 1965.