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CONCENTRATION AND DILUTION OF URINE
495 the levels in 3
(twice after stenolon, and once after changes did not depend on initial on the anabolic steroid used. Jose and
cases
was seen 2 months after parturition no abnormality be found in the heart or central nervous system.
mother
superanabolon).
These
was to
cholesterolaemia,
or
It must be presumed that the disease has become less severe the years, but gloomy forecasts still linger in the textbooks, and tradition dies hard. Hope Hospital, Salford 6, Lancashire. J. H. EVANS.
Mitchell believed that the increase of serum-cholesterol levels was caused by intrahepatic obstruction.3 On the other hand in our series cholesterolxmia was increased after treatment with 19-nortestosterone, which presumably is not hepatotoxic.5 6 Department of Medicine, E. ZAMRAZILOVÁ Oúnz Hospital, V. ZAMRAZIL. Czechoslovakia. Pisek,
MIGRAINE Dalton
(Feb. 12) points out the beneficial effect of progesterone premenstrual migraine. Progestogenic steroids, however, have from time to time been used not only for premenstrual migraine but also for migraine appearing during the mid-menstrual period in women (first reported by Singh et al. 1), and even for migraine in adult male patients. In recent years advances in steroid chemistry have produced a series of orally active progestogenic compounds. In Uppsala about 250 patients of both sexes have been treated with such preparations (methyloestrenolene, allyloestrenol, lynoestrenol, isopregnenone, and medroxyprogesterone acetate 8-10), with very good results. But because of the progestogenic effect of these compounds menstrual irregularities or amenorrhoea are SIR,-Dr.
on
unavoidable in fertile
women.
Since 1963 I have tried
a new
compound, 6ot-trinuoromethyI-17M-acetoxyprogesterone (’WG 537, Demigran’), as prophylactic treatment in patients with migraine." 11 A double-blind trial on 50 patients has shown that the compound is much more effective than placebo. Up to now over 150 patients have been treated. About half the cases were free from attacks during the treatment period, and in most of the other cases some improvement was noted. The results have been confirmed by others.12 Department of Neurology, Academic Hospital, Uppsala, Sweden.
13
P. O. LUNDBERG.
over
CONCENTRATION AND DILUTION OF URINE SiR,ŅYour leading article1 presents the hypothesis of Lever2 as " interesting, provocative, and plausible ". There is, however, reason to question the plausibility of the proposed mechanism which attributes the concentration and dilution of the urine to ultrafiltration processes initiated by the hydrostatic pressure in the descending vasa recta. It is important to recall the magnitude of the equivalence of hydrostatic and osmotic pressures. An osmotic gradient of 1 milliosmole per kg. of water can be balanced only by a hydrostatic pressure of about 17 mm. Hg. Thus the postulated hydrostatic pressure in the descending vasa recta of 50 mm. Hg is sufficient to supply a gradient of at most 3 milliosmoles per kg. H2O-and this is the single effect which must be multiplied by the countercurrent system. It is extremely doubtful whether any multiplier could, in the face of longitudinal diffusion within the stream, have the efficiency required to produce multiplication by a factor of several hundred over a distance of a millimetre or two in the vascular bundles. But whereas this is improbable it is impossible to produce or maintain, with the hydrostatic pressures available, an osmotic difference of about 150 milliosmoles per kg. H2O, such as exists between early distal tubule (and presumably the late portion of the thick ascending limb) and its surroundings; nor could the hydrostatic pressure contribute significantly to this difference. Furthermore the difference between water diuresis and the production of hypertonic urine does not lie in the composition of the fluid which enters the distal tubule, for it is similar in these two conditions. The difference is in the loss of water which occurs more
distally. CHOREA GRAVIDARUM SIR,-Chorea gravidarum is a rare condition, but my experience leads me to endorse Mr. Lewis and Dr. Parson’s refutation of the traditionally poor prognosis. Intractable movements may persist throughout pregnancy, though in the few cases that I have seen in recent years the patient has incurred no persistent cardiac, neurological, or mental disability, and the pregnancy has ended in the normal delivery of a healthy child. The following case illustrates the pattern of the disease. A 16-year-old girl, recently married, had reached the 6th month of her first pregnancy when she noticed that she tended to drop things from the left hand, and that the left leg moved in an uncontrollable way. There was no preceding sore throat or history of rheumatism or chorea. On examination the patient was apyrexial and exhibited choreiform movements of the face and limbs, particularly the left arm and leg. There was no rash, subcutaneous nodules, or evidence of carditis. Her erythrocyte-sedimentation rate was 33 mm. in the lst hour, and her haemoglobin 76%, but the remainder of the investigations, including throat swab, antistreptolysin titre, electrocardiogram, and electroencephalogram were within normal limits. The patient improved rapidly on bed rest and sedation with amylobarbitone, 60 mg. b.d., and was discharged home after 10 days with only mild fidgety movements, which persisted until the delivery, at full term, of a healthy female child. When the 5.
Slabochová, Z., Rath, R. 1st National Conference on Steroid Drugs; p. 49. Spofa, Prague, 1964. Wernze, H. Dt. med. Wschr. 1960, 85, 2237. Singh, I., Singh, I., Singh, D. Lancet, 1947, i, 745. 8. Lundberg, P. O. Acta endocr., Copenh. 1962, suppl. 68, p. 1. 9. Lundberg, P. O. Svenska Läkartidn. 1963, 60, 2789. 10. Lundberg, P. O. Opusc. med. 1965, 10, 59. 11. Lundberg, P. O. Int. Congr. neurol. Sci.; vol. IV, p. 407. Vienna, 1965. 12. Dalsgaard-Nielsen, T. Nord. Med. 1965, 74, 713. 13. Emblem, L. Medsk Årsb. 1966, 9, 129. 6. 7.
On the other hand micropuncture studies have indicated the of the distal tubule to lower the concentration of sodium in the lumen and to maintain low concentrations during "stop-flow microperfusion ".3 The low permeability of the distal-tubule segments to water is indicated not only by the maintenance of the osmotic gradient across their wall in water diuresis, but directly by the measurement of fluxes across the wall of rabbit collecting tubules perfused in vitro. Although the concentration of vasopressin needed to produce permeability changes in amphibian membranes may be " extremely high " (vasopressin is not the natural amphibian hormone) maximum permeability increases are produced in the rabbit tubules4 by a vasopressin concentration of 25 units per ml., a concentration similar to that which has been reported to be present in the plasma of hydropenic man.5 The following pertinent observations, not yet published, appear to settle the issue. Two of us have jointly described a technique for extensive exposure of the rat renal medulla.6 We have used this procedure to obtain, for osmolality determination, samples from loops of Henle and vasa recta at levels considerably further from the papillary tip than has hitherto been possible. These observations show, in accord with the hypothesis that the thin ascending limb is an active component of the countercurrent multiplier, that thin ascending limbs uniformly contain fluid more dilute (by 100-150 milliosmoles per kg.) than that in descending limbs at the same level. Furthermore the osmolality of descending vasa-recta blood is lower than that in descending limbs of loops of Henle at the same level in the medulla, oresumablv renectins some las in
capacity
1. 2. 3. 4. 5. 6.
Lancet, 1965, ii, 998. Lever, A. F. Acta med. scand. 1965, 178, suppl. 434. Giebisch, G., Windhager, E. E. Am. J. Med. 1964, 36, 643. Grantham, J., Orloff, J., Burg, M. J. clin. Invest. 1965, 44, 1055. Czaczkes, J. W., Kleeman, C. R., Koenig, M. ibid. 1964, 43, 1625. Sakai, F., Jamison, R. L., Berliner, R. W. Am. J. Physiol. 1965, 209, 663.
496 the attainment of osmotic equilibrium between the contents of the vasa recta and their surroundings. This is a finding incompatible with the hypothesis of Lever.
released particles. The alternative approach adopted-to try to stimulate the agent in relatively few cells to multiply, in the hope that a sufficient number could be
concentrate any was
amassed to cause a detectable effect when inoculated into indicator cell cultures. Attempts at direct culture were negaNational Heart Institute, tive ; so a leucocyte culture experiment, following the method Bethesda, Maryland. of Brown et al.11 for culturing leucocytes for chromosomal karyotype analysis, including the use of colchicine to arrest mitosis in metaphase, was monitored by the electron microscope. Surprisingly, the electron microscope showed a distinct COLCHICINE IN THE CULTURE OF AGENTS apparent increase in the number of virus-like particles assoFROM LEUCOCYTES ciated with the negatively stained cells. Three subsequent SIR,-Filtrable transmissible agents have been isolated from experiments all revealed an increase in the number of particles in colchicine-treated cultures as compared with control nonthe blood and bone-marrow of leukaemic patients.1-3 Some of colchicine-treated cultures. these agents have been identified as mycoplasmas.45 The of these is and debate The following culture method, incorporating the use of uncertain, significance mycoplasmas continues as to whether they are passengers, contaminants, or colchicine, was then adopted. Blood was drawn by veneagents of significance to the disease. Others have reported puncture ; the leucocytes were separated and placed in culture; inability to isolate such agents under similar conditions .61 colchicine was added; the colchicine-containing medium was Thus any method which appears to increase the possibility of removed and the cells were washed with Hanks’ balanced such isolations from cells is of interest. The following observasalt solution; and the cells were resuspended in maintenance tions were made during studies of leucocytic pyrogen-a medium, returned to bottles, and incubated for various times. protein which has been shown to be elaborated by leucocytes The supernatants from centrifuged culture fluids were used as in vitro and which can cause a pyrexial episode in man and inocula for indicator cell lines. All growth and maintenance animals.8 9 media contained M 199, penicillin, streptomycin, and kanaElectron microscopic studies of highly purified protein mycin, differing only in the serum content. Careful controls for samples derived from volunteers showed, in negatively stained bacterial contamination were maintained, and all cultures for preparations, particles which morphologically resembled viral bacterial contamination were negative. The details of these capsids. In the absence of likely viral contamination in the experiments and the results are shown in the accompanying purification procedure, the leucocytes were considered the table. probable source of the virus-like particles. Negatively stained By means of colchicine treatment it has been possible to show preparations of thinly spread leucocytes 10 showed similar that the leucocytes of this donor contain some agent which particles to be present, though they were scanty. Their paucity produces a cytopathic effect on indicator cell lines, and this has did not permit identification by an immunological approachnot been demonstrated in the non-colchicine-treated cultures. e.g., fluorescent antibody staining or immune electron microWhile the nature of the agent producing the cytopathic effects has not been established serologically, electron micrographs scopy. Therefore attempts were made to identify the particles by replication using a single donor-a clinically healthy of infected cells show forms indistinguishable from published 47-year-old woman who, on four occasions over an 18-month micrographs of cultured mycoplasmas.12 period, had been found by electron microscopy to have such The mechanism by which colchicine acts is not clear, but it is virus-like particles in leucocytes. Use of a single donor ruled unlikely to be a continuing stimulus since, if the colchicine out any attempt to disrupt a very large number of cells and in the treated cultures was in 1:60,000 concentration, after 1. Negroni, G. Br. med. J. 1964, i, 927. washing it would be less than 1:75,000,000, which is far more 2. Dalldorf, G., Bergamini, F. Proc. natn. Acad. Sci. U.S.A. 1964, 51, 263. dilute than concentrations which have proved ineffective in 3. Murphy, W. H., Furtado, D. Univ. Mich. med. Bull. 1964, 29, 201. 4. Girardi, A. J., Hayflick, L., Lewis, A. M., Somerson, N. L. Nature, stimulating the release of the agent. But the results clearly show Lond. 1965, 205, 188. that colchicine in 1:60,000 concentration stimulates the pro5. Hayflick, L., Kaprowski, H. ibid. p. 713. duction in the cells of this donor of an agent which cannot be 6. Girardi, A. J., Slotnik, V. B., Hilleman, M. R. Proc. Soc. exp. Biol. Med. 1962, 110, 776. cultivated without the use of colchicine. Application of 7. Phillips, C. F., Benyesh-Melnick, M., Seidel, E. H., Fernbach, D. J. colchicine in methods to culture agents from human blood or Br. med. J. 1965, i, 286. 8. Rafter, G. W., Collins, R. D., Wood, W. B., Jr. J. exp. Med. 1960, 11. Brown, C. D., Gabay, J. J., Sax, N. I. Zeiss-Mitt Fortschr. tech. Opt. 111, 831. 9. Goodale, F., Filmore, R., Hillman, E. Exp. molec. Path. 1962, 1, 229. 1963, 3, no. 4, p. 130. 10. Parsons, D. F. J. Cell Biol. 1963, 16, 620. 12. Hummeler, K., Tomassini, N., Hayflick, L. J. Bact. 1965, 90, 517. ROBERT W. BERLINER REX L. JAMISON CLEAVES M. BENNETT.
DETAILS OF EXPERIMENTS
*
+ =positive; - =nesative; i =inconclusive; 0 = not done. t Human-amnion cell line. t Cells and fluid passed to primary monkey-kidney and primary human-amnion cell lines.
(twice after stenolon, and once after changes did not depend on initial on the anabolic steroid used. Jose and
cases
was seen 2 months after parturition no abnormality be found in the heart or central nervous system.
mother
superanabolon).
These
was to
cholesterolaemia,
or
It must be presumed that the disease has become less severe the years, but gloomy forecasts still linger in the textbooks, and tradition dies hard. Hope Hospital, Salford 6, Lancashire. J. H. EVANS.
Mitchell believed that the increase of serum-cholesterol levels was caused by intrahepatic obstruction.3 On the other hand in our series cholesterolxmia was increased after treatment with 19-nortestosterone, which presumably is not hepatotoxic.5 6 Department of Medicine, E. ZAMRAZILOVÁ Oúnz Hospital, V. ZAMRAZIL. Czechoslovakia. Pisek,
MIGRAINE Dalton
(Feb. 12) points out the beneficial effect of progesterone premenstrual migraine. Progestogenic steroids, however, have from time to time been used not only for premenstrual migraine but also for migraine appearing during the mid-menstrual period in women (first reported by Singh et al. 1), and even for migraine in adult male patients. In recent years advances in steroid chemistry have produced a series of orally active progestogenic compounds. In Uppsala about 250 patients of both sexes have been treated with such preparations (methyloestrenolene, allyloestrenol, lynoestrenol, isopregnenone, and medroxyprogesterone acetate 8-10), with very good results. But because of the progestogenic effect of these compounds menstrual irregularities or amenorrhoea are SIR,-Dr.
on
unavoidable in fertile
women.
Since 1963 I have tried
a new
compound, 6ot-trinuoromethyI-17M-acetoxyprogesterone (’WG 537, Demigran’), as prophylactic treatment in patients with migraine." 11 A double-blind trial on 50 patients has shown that the compound is much more effective than placebo. Up to now over 150 patients have been treated. About half the cases were free from attacks during the treatment period, and in most of the other cases some improvement was noted. The results have been confirmed by others.12 Department of Neurology, Academic Hospital, Uppsala, Sweden.
13
P. O. LUNDBERG.
over
CONCENTRATION AND DILUTION OF URINE SiR,ŅYour leading article1 presents the hypothesis of Lever2 as " interesting, provocative, and plausible ". There is, however, reason to question the plausibility of the proposed mechanism which attributes the concentration and dilution of the urine to ultrafiltration processes initiated by the hydrostatic pressure in the descending vasa recta. It is important to recall the magnitude of the equivalence of hydrostatic and osmotic pressures. An osmotic gradient of 1 milliosmole per kg. of water can be balanced only by a hydrostatic pressure of about 17 mm. Hg. Thus the postulated hydrostatic pressure in the descending vasa recta of 50 mm. Hg is sufficient to supply a gradient of at most 3 milliosmoles per kg. H2O-and this is the single effect which must be multiplied by the countercurrent system. It is extremely doubtful whether any multiplier could, in the face of longitudinal diffusion within the stream, have the efficiency required to produce multiplication by a factor of several hundred over a distance of a millimetre or two in the vascular bundles. But whereas this is improbable it is impossible to produce or maintain, with the hydrostatic pressures available, an osmotic difference of about 150 milliosmoles per kg. H2O, such as exists between early distal tubule (and presumably the late portion of the thick ascending limb) and its surroundings; nor could the hydrostatic pressure contribute significantly to this difference. Furthermore the difference between water diuresis and the production of hypertonic urine does not lie in the composition of the fluid which enters the distal tubule, for it is similar in these two conditions. The difference is in the loss of water which occurs more
distally. CHOREA GRAVIDARUM SIR,-Chorea gravidarum is a rare condition, but my experience leads me to endorse Mr. Lewis and Dr. Parson’s refutation of the traditionally poor prognosis. Intractable movements may persist throughout pregnancy, though in the few cases that I have seen in recent years the patient has incurred no persistent cardiac, neurological, or mental disability, and the pregnancy has ended in the normal delivery of a healthy child. The following case illustrates the pattern of the disease. A 16-year-old girl, recently married, had reached the 6th month of her first pregnancy when she noticed that she tended to drop things from the left hand, and that the left leg moved in an uncontrollable way. There was no preceding sore throat or history of rheumatism or chorea. On examination the patient was apyrexial and exhibited choreiform movements of the face and limbs, particularly the left arm and leg. There was no rash, subcutaneous nodules, or evidence of carditis. Her erythrocyte-sedimentation rate was 33 mm. in the lst hour, and her haemoglobin 76%, but the remainder of the investigations, including throat swab, antistreptolysin titre, electrocardiogram, and electroencephalogram were within normal limits. The patient improved rapidly on bed rest and sedation with amylobarbitone, 60 mg. b.d., and was discharged home after 10 days with only mild fidgety movements, which persisted until the delivery, at full term, of a healthy female child. When the 5.
Slabochová, Z., Rath, R. 1st National Conference on Steroid Drugs; p. 49. Spofa, Prague, 1964. Wernze, H. Dt. med. Wschr. 1960, 85, 2237. Singh, I., Singh, I., Singh, D. Lancet, 1947, i, 745. 8. Lundberg, P. O. Acta endocr., Copenh. 1962, suppl. 68, p. 1. 9. Lundberg, P. O. Svenska Läkartidn. 1963, 60, 2789. 10. Lundberg, P. O. Opusc. med. 1965, 10, 59. 11. Lundberg, P. O. Int. Congr. neurol. Sci.; vol. IV, p. 407. Vienna, 1965. 12. Dalsgaard-Nielsen, T. Nord. Med. 1965, 74, 713. 13. Emblem, L. Medsk Årsb. 1966, 9, 129. 6. 7.
On the other hand micropuncture studies have indicated the of the distal tubule to lower the concentration of sodium in the lumen and to maintain low concentrations during "stop-flow microperfusion ".3 The low permeability of the distal-tubule segments to water is indicated not only by the maintenance of the osmotic gradient across their wall in water diuresis, but directly by the measurement of fluxes across the wall of rabbit collecting tubules perfused in vitro. Although the concentration of vasopressin needed to produce permeability changes in amphibian membranes may be " extremely high " (vasopressin is not the natural amphibian hormone) maximum permeability increases are produced in the rabbit tubules4 by a vasopressin concentration of 25 units per ml., a concentration similar to that which has been reported to be present in the plasma of hydropenic man.5 The following pertinent observations, not yet published, appear to settle the issue. Two of us have jointly described a technique for extensive exposure of the rat renal medulla.6 We have used this procedure to obtain, for osmolality determination, samples from loops of Henle and vasa recta at levels considerably further from the papillary tip than has hitherto been possible. These observations show, in accord with the hypothesis that the thin ascending limb is an active component of the countercurrent multiplier, that thin ascending limbs uniformly contain fluid more dilute (by 100-150 milliosmoles per kg.) than that in descending limbs at the same level. Furthermore the osmolality of descending vasa-recta blood is lower than that in descending limbs of loops of Henle at the same level in the medulla, oresumablv renectins some las in
capacity
1. 2. 3. 4. 5. 6.
Lancet, 1965, ii, 998. Lever, A. F. Acta med. scand. 1965, 178, suppl. 434. Giebisch, G., Windhager, E. E. Am. J. Med. 1964, 36, 643. Grantham, J., Orloff, J., Burg, M. J. clin. Invest. 1965, 44, 1055. Czaczkes, J. W., Kleeman, C. R., Koenig, M. ibid. 1964, 43, 1625. Sakai, F., Jamison, R. L., Berliner, R. W. Am. J. Physiol. 1965, 209, 663.
496 the attainment of osmotic equilibrium between the contents of the vasa recta and their surroundings. This is a finding incompatible with the hypothesis of Lever.
released particles. The alternative approach adopted-to try to stimulate the agent in relatively few cells to multiply, in the hope that a sufficient number could be
concentrate any was
amassed to cause a detectable effect when inoculated into indicator cell cultures. Attempts at direct culture were negaNational Heart Institute, tive ; so a leucocyte culture experiment, following the method Bethesda, Maryland. of Brown et al.11 for culturing leucocytes for chromosomal karyotype analysis, including the use of colchicine to arrest mitosis in metaphase, was monitored by the electron microscope. Surprisingly, the electron microscope showed a distinct COLCHICINE IN THE CULTURE OF AGENTS apparent increase in the number of virus-like particles assoFROM LEUCOCYTES ciated with the negatively stained cells. Three subsequent SIR,-Filtrable transmissible agents have been isolated from experiments all revealed an increase in the number of particles in colchicine-treated cultures as compared with control nonthe blood and bone-marrow of leukaemic patients.1-3 Some of colchicine-treated cultures. these agents have been identified as mycoplasmas.45 The of these is and debate The following culture method, incorporating the use of uncertain, significance mycoplasmas continues as to whether they are passengers, contaminants, or colchicine, was then adopted. Blood was drawn by veneagents of significance to the disease. Others have reported puncture ; the leucocytes were separated and placed in culture; inability to isolate such agents under similar conditions .61 colchicine was added; the colchicine-containing medium was Thus any method which appears to increase the possibility of removed and the cells were washed with Hanks’ balanced such isolations from cells is of interest. The following observasalt solution; and the cells were resuspended in maintenance tions were made during studies of leucocytic pyrogen-a medium, returned to bottles, and incubated for various times. protein which has been shown to be elaborated by leucocytes The supernatants from centrifuged culture fluids were used as in vitro and which can cause a pyrexial episode in man and inocula for indicator cell lines. All growth and maintenance animals.8 9 media contained M 199, penicillin, streptomycin, and kanaElectron microscopic studies of highly purified protein mycin, differing only in the serum content. Careful controls for samples derived from volunteers showed, in negatively stained bacterial contamination were maintained, and all cultures for preparations, particles which morphologically resembled viral bacterial contamination were negative. The details of these capsids. In the absence of likely viral contamination in the experiments and the results are shown in the accompanying purification procedure, the leucocytes were considered the table. probable source of the virus-like particles. Negatively stained By means of colchicine treatment it has been possible to show preparations of thinly spread leucocytes 10 showed similar that the leucocytes of this donor contain some agent which particles to be present, though they were scanty. Their paucity produces a cytopathic effect on indicator cell lines, and this has did not permit identification by an immunological approachnot been demonstrated in the non-colchicine-treated cultures. e.g., fluorescent antibody staining or immune electron microWhile the nature of the agent producing the cytopathic effects has not been established serologically, electron micrographs scopy. Therefore attempts were made to identify the particles by replication using a single donor-a clinically healthy of infected cells show forms indistinguishable from published 47-year-old woman who, on four occasions over an 18-month micrographs of cultured mycoplasmas.12 period, had been found by electron microscopy to have such The mechanism by which colchicine acts is not clear, but it is virus-like particles in leucocytes. Use of a single donor ruled unlikely to be a continuing stimulus since, if the colchicine out any attempt to disrupt a very large number of cells and in the treated cultures was in 1:60,000 concentration, after 1. Negroni, G. Br. med. J. 1964, i, 927. washing it would be less than 1:75,000,000, which is far more 2. Dalldorf, G., Bergamini, F. Proc. natn. Acad. Sci. U.S.A. 1964, 51, 263. dilute than concentrations which have proved ineffective in 3. Murphy, W. H., Furtado, D. Univ. Mich. med. Bull. 1964, 29, 201. 4. Girardi, A. J., Hayflick, L., Lewis, A. M., Somerson, N. L. Nature, stimulating the release of the agent. But the results clearly show Lond. 1965, 205, 188. that colchicine in 1:60,000 concentration stimulates the pro5. Hayflick, L., Kaprowski, H. ibid. p. 713. duction in the cells of this donor of an agent which cannot be 6. Girardi, A. J., Slotnik, V. B., Hilleman, M. R. Proc. Soc. exp. Biol. Med. 1962, 110, 776. cultivated without the use of colchicine. Application of 7. Phillips, C. F., Benyesh-Melnick, M., Seidel, E. H., Fernbach, D. J. colchicine in methods to culture agents from human blood or Br. med. J. 1965, i, 286. 8. Rafter, G. W., Collins, R. D., Wood, W. B., Jr. J. exp. Med. 1960, 11. Brown, C. D., Gabay, J. J., Sax, N. I. Zeiss-Mitt Fortschr. tech. Opt. 111, 831. 9. Goodale, F., Filmore, R., Hillman, E. Exp. molec. Path. 1962, 1, 229. 1963, 3, no. 4, p. 130. 10. Parsons, D. F. J. Cell Biol. 1963, 16, 620. 12. Hummeler, K., Tomassini, N., Hayflick, L. J. Bact. 1965, 90, 517. ROBERT W. BERLINER REX L. JAMISON CLEAVES M. BENNETT.
DETAILS OF EXPERIMENTS
*
+ =positive; - =nesative; i =inconclusive; 0 = not done. t Human-amnion cell line. t Cells and fluid passed to primary monkey-kidney and primary human-amnion cell lines.