211 MACROPHAGES AS TUMOUR GROWTH PROMOTERS? Dr Hamburger’ discuss analoand between B neoplastic cell clonal proliflymphocyte gies eration being stimulated by a macrophage-derived factor(s). Although very interesting, this hypothesis has been oversimplified and based mainly on in-vitro data of colony formation. The theory of immunostimulation of cancer growth has been known for some time ;2 the promotion or inhibition of metastasis formation by sensitised lymphocytes was shown by Fidler in B16 melanoma system;3 and Salmon and Hamburger present the theory of macrophage stimulation of both carcinogentriggered epithelial tumour cell (as second-phase promoter)
SIR,-Professor Salmon and
.
and antigen-triggered B-lymphocyte proliferation. Monocyte-macrophage cells are the predominant source of colony-stimulating factor (c.s.F.) in myelopoiesis, and the same cells, at certain critical c.s.F. concentration, begin production of prostaglandin E which suppresses myelopoiesis.4 Feedback control of excessive myelopoiesis thus operates through the same growth-promoting and growth-inhibiting population of macrophage-like cells. Although the glycoprotein described by Salmon and Hamburger was antigenically dissimilar from the myelopoiesis-stimulating C.S.F., the point is that the same population of cells can demonstrate two diametrically opposite functions, dependent on quantitative differences of c.s.F. concentration. The suppression of B-cell-derived polyclonal humoral antibody production by monocyte-like cells has been shown by Broder et al. in some patients with myeloma. Whether the same cells are capable of stimulating monoclonal B cells in patients with myeloma remains to be established in vivo. The demonstration in vitro that macrophage-derived glycoprotein is also able to stimulate colony-formation of various other neoplastic cells is very intriguing. However, there is abundant evidence from laboratory animal and human systems that macrophage content of the tumour correlates with prognosis and risk of metastatic spread.6-9 Macrophages, although having limited capacity,’O protect to a certain extent against the tumour
spread 6- 10 and, they can selectively kill neoplastic but not normal cells.’Whether the presence of significant numbers of macrophages in malignant effusions represents a cause of rather than a protective response against, tumour growth will have to be established in vivo. Macrophages can produce several other humoral factors which are selectively tumoricidal and definitely do not promote tumour growth.llll2 Although very provocative, the suggestions that macrophages or macrophage-derived humoral factor(s) can promote the monoclonal neoplastic growth and that organ macrophage content can positively correlate with the promotion of tumour growth do contradict well-founded evidence of a protective role of macrophages against neoplastic growth and spread. The potentially beneficial role of macrophages in tumour-growth control has been ignored by Salmon and Hamburger, perhaps for the sake of clear and simplified presentation. It could be that the growth-stimulatory effect of a macrophage-derived factor on a minority of clonogenic cells in vivo counteracts the otherwise inhibitory effect of other macrophage-related factors 1. 2. 3. 4.
Salmon, S. E., Hamburger, A. Lancet, 1978, i, 1289. Prehn, R. T., Lappé, M. A. Transplant. Rev. 1971, 7, 26. Fidler, I. J. Cancer Res. 1974, 34, 491. Kurland, J. I., Bockman, R. S., Broxmeyer, H. E., Moore, M. A. S. Science, 1978, 199, 552. 5. Broder, S, Humphrey, R., Durm, M., Blackman, M., Meade, B., Goldman, C., Strober, W., Waldmann, T. A. New Engl. J. Med., 1975, 293, 887. 6. Eccles, S. A., Alexander, P. Nature, 1974, 250, 667. 7. Evans, R., Alexander, P. ibid., 1972, 236, 168. 8. Schultz, R. M., Papamatheakis, J. D., Stylos, W. A., Chirigos, M. A. Cell. Immun. 1976, 25, 309. 9. Hibbs, J. B., Jr., Lambert, L. H., Jr., Remington, J. S. Nature neō Biol.,
1972, 235, 48. 10. Lancet, 1976, ii, 27. 11. Currie, G. A., Basham, C.J. exp. Med., 1975, 142, 1600. 12. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., B. Proc nat. Acad Sci. U.S.A., 1975, 72, 3666.
Williamson,
This might limit the capacity of macroinhibit tumour growth. The possibility of imphages elimination of the clonogenic-promoting munologically specific factor(s) and exploration of its properties (e.g., does it demon-
on tumour
growth.
alone
to
angiogenesis-promoting characteristics?") warrant investigation. It is important, even when presenting a hypothesis, to try to encompass all important aspects of the macrophage/tumor-cell and therefore, in part, the host-tumour relationship, since otherwise an erroneous picture may be painted. strate
further
Cancer Therapy Evaluation Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20014, U.S.A.
Viral Oncology Program, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda
STANISLAW M. MIKULSKI MICHAEL A. CHIRIGOS
SEPTIC SHOCK
SIR,-Although the article by Dr Ledingham and Mr McArdle’ contains a wealth of factual information, it makes little contribution to our understanding of the pathophysiology of the condition which could provide a base on which to plan treatment.
*
Reduction of mortality by early intermittent positive-pressure ventilation (i.P.P.v.) suggests that by squeezing a little more oxygen into the circulation and reducing consumption by the muscles of respiration, the effects of stagnant anoxia are reduced in severity and chances of survival thereby improved. The criteria by which the decision is taken to start I.P.P.V., are not, however, stated, except that it was started "early." Discussing the duration of shock Ledingham and McArdle say that, although the total duration of shock in their patients did not vary during the entire series, the duration before referral tended to increase "as appropriate treatment was initiated by the ward staff." I would have expected appropriate treatment to reduce duration and intensity. Did this treatment include massive dosage of antibiotics and did shock occur before or after the administration (release of endotoxin after wholesale destruction of gram-negative organisms can precipitate
shock)? Where does shock begin and where does it end? In the third of the series only two deaths were attributable to continuing shock yet five patients died from acute respiratory and renal failure. Albuminuria, hxmaturia, and reduced urine output are the earliest and most constant effects produced by bacterial toxins in laboratory animals.2 The kidney is the first organ affected so persistence of renal disorder indicates continuation of shock, although peripheral circulatory signs may have receded. The suggestion that pharmacological agents such as glucocorticoids could have an "action on the later stages of septic shock not hitherto described" is strange considering the volume of work devoted to this subject and the fact that glucocorticoids have maximum protective action in animals when given before endotoxin.3 Chlorpromazine also possesses similar maximum protective action before exposure to endotoxin,4 almost certainly through its deafferenting action on the brainstem reticular formation.55
13 1. 2.
Folkman, J., Ann. Surg., 1972, 175, 409. Ledingham, I. M., McArdle, C. S. Lancet, 1978, i, 1194.
Reilly, J., Compagnon, A., Laporte, A., Du Buit, H. Le Role du Système nerveux vegetatif en pathologie renale. Paris, 1942. 3. Lillenei, R. C., Longerbeam, J. N., Bloch, J. H., Manax, W. Clin. Pharmacol. Ther. 1964, 5, 63. 4. Reilly, J., Compagnon, A., Tournier, P., Du Buit, H. Ann. de Méd. 1954, 55, T5. 5. Bradley, P. B. in Reticular formation of the brain. Henry Ford symposium (edited by H. H. Jasper and others); p. 123. London, 1960. 6. Buxton Hopkin, D. A. Proc. Wld Congr. Anæsthesiol. 1977, p. 368.
212 Evidence continues to accumulate about the involvement of the central nervous system in all shock states and suggesting that the resulting pathophysiological changes involve endocrine and reticuloendothelial as well as the cardiovascular and respiratory systems. Possibly more success would follow treatment directed to the initiating sources rather than to where the end-products of the resulting changes appear. 11 Chelsea Embankment, London SW3 4LE
PRENATAL DIAGNOSIS OF M.S.U.D. IN AMNIOTIC-CELL CULTURE
D. A. BUXTON HOPKIN
ANTENATAL DIAGNOSIS OF MAPLE-SYRUP-URINE DISEASE
SIR,—Maple-syrup-urine disease (M.S.U.D.) is an autosomal recessive disease affecting the degradation of the three branched-chain aminoacids leucine, isoleucine, and valine. The classical form of the disease, if untreated, is rapidly lethal. Treatment is difficult, requiring life-long restriction of the three aminoacids. Parents who have had a child with M.S.U.D. often seek antenatal diagnosis in subsequent pregnancies. The demonstration of the branched-chain ketoacid decarboxylase in cultured amniotic cells suggested the possibility of antenatal diagnosis’ and this was confirmed in a patient from this laboratory’ and by Wendel et al.2 We have now studied eighteen pregnancies at risk for M.S.U.D. The diagnosis can be made with accuracy and confidence, but care is needed with cultures of epithelial cells and with slow-growing cultures. Twelve of the
eighteen specimens investigated were sent from
other
institutions, including four from other countries. Methods of subculhave been previously described.3 Cells were harvested for assay either with a rubber policeman or with a solution of Puck’s saline A containing 0-02% (w/v) edetic acid (E.D.T.A.) and 0.05% (w/v) trypsin (two patients). The E.D.T.A.-trypsin cell suspension was immediately added to one-quarter its volume of calf serum and centrifuged. The cell button was resuspended in buffered saline containing 20% serum and transferred to reaction flasks and centrifuged. The supernatant was ’replaced with reaction mixture and decarboxylase activity was assayed.4 Approximately 4x105 cells are required for assay, but reliable results are obtained with fewer actively growing cells. Most specimens were assayed 3-5 weeks after amniocentesis. ture
Eighteen amniotic-cell cultures were investigated and a diagnosis was made in sixteen, of which four had M.S.U.D., giving the frequency of 25% expected for an autosomal recessive disease. The other twelve assays were normal and in all twelve the postnatal diagnosis was also normal. Details of the remaining six cases (A-F) are given in the table. The diagnosis was confirmed in case B by post-abortion culture and assay of fetal skin fibroblasts. In case A a type-n variant was confirmed after birth by enzyme assay of leucocytes and by clinical course. In case C a macerated fetus was delivered at another institution after abortion with prostaglandin and urea. Culture of fetal tissues failed. In case F the baby was delivered at term; symptoms of M.S.U.D. developed and the infant died in a week. No diagnosis could be made in cases D and E. In both the cells were predominantly epithelial, as they were in 5 other successful cultures. One culture (E) was received from France. Cells grew poorly and an assay was attempted with few cells. No radioactive CO2 was released, but there was also no incorporation of radioactive aminoacids into protein, indicating that the cells were no longer metabolically active. The outcome of this pregnancy is not known. In D the ratio of the ’4C02to radioactivity in protein suggested variant M.S.U.D., but the incorporation of radioactivity into protein was too low to provide confidence in the results. No diagnosis was made and the fetus was aborted at another institution. Cultures were not established from fetal tissue. 1. Dancis, J. in Antenatal Diagnosis (edited by A. Dorfman); p. 123. Chicago, 1972. 2. Wendel, U., Rudiger, H. W., Passarge, E. Humangenetik, 1973, 19, 127. 3. Cox, R. P., Macleod, C. M.J. gen. Physiol. 1962, 45, 439. 4. Dancis, J., Hutzler, J., Cox, R. P. in Tissue Culture, Methods and Applications (edited by P. F. Kruse, Jr. and M. K. Patterson, Jr.) p. 639. New York, 1973.
Amniotic cell cultures that are predominantly fibroblastic have presented no problems with the technique devised for skin fibroblasts.s However, when cultures are predominantly
epithelial they multiply slowly, delaying analysis. Harvesting cells with a rubber policeman, as we have usually done, is associated with a variable, and frequently serious, loss of enzyme activity. The use of E.D.T.A.-trypsin to remove cells from glass surfaces seems to preserve enzyme activity in epithelial cells. We now assay cell suspensions prepared by E.D.T.A.-trypsin treatment of confluent cell monolayers. Intact cells are used because disruption of cells greatly reduces enzyme activity.’ The cells are incubated for 120 min with L-valine-1-14C (specific activity 0-5 µCi/µmol). The evolution of 14C02 provides a measure of the decarboxylase activity and is expressed per mg protein. The simultaneous incorporation of the radioactive aminoacid into protein provides an indication of the number of cells that are assayed and their metabolic activity. The ratio of the 14CO2to 14C-incorporation into protein is the most satis-
factory approach to diagnosis, provided 14C-incorporation per mg protein indicates active cell metabolism. This Service Fund.
study (AM
was
supported by grants
14528 and HD
from the U.S. Public Health
04526) and by the Samuel
Division of Human Genetics, Departments of Medicine and Pediatrics, New York University Medical Center, New York, N.Y. 10016, U.S.A.
A.
Berger
RODY P. Cox
JOEL HUTZLER JOSEPH DANCIS
MATERNAL HYPERTHERMIA AND NEURAL-TUBE DEFECTS
SIR,-Retrospective studies in the north-western United a significant correlation between maternal hyperthermia during the early part of pregnancy and the incidence of anencephalyl and meningomyelocele.2 Our similar study in the mid-southern United States has detected 3 instances of maternal hyperthermia in 45 pregnancies which resulted in children with neural-tube defects (anencephaly, meningomyelocele, spina bifida, or encephalocele). Only births during the past 11 months were included. States have revealed
In
man
the neural tube closes between the 22nd and 28th
gestation. The mothers in this study were questioned particularly about hyperthermia due to illness or prolonged sauna usage during the third, fourth, and fifth weeks of ges-
day
of
tation. This interval
was
chosen because most of the dates of
conception were only approximate. 1 mother reported "quite high" fever lasting 2 or 3 days and caused by streptococcal Elsas, L. J., Priest, J. H. Wheeler, F. B., Danner, D. J., Pask, B. A., Metabolism, 1974, 23, 569. 1. Miller, P., Smith, D. W., Shepard, T. H. Lancet, 1978, i, 519. 2. Chance, P. F., Smith, D. W. ibid. p. 769. 5.
SIR,-Professor Salmon and
.
and antigen-triggered B-lymphocyte proliferation. Monocyte-macrophage cells are the predominant source of colony-stimulating factor (c.s.F.) in myelopoiesis, and the same cells, at certain critical c.s.F. concentration, begin production of prostaglandin E which suppresses myelopoiesis.4 Feedback control of excessive myelopoiesis thus operates through the same growth-promoting and growth-inhibiting population of macrophage-like cells. Although the glycoprotein described by Salmon and Hamburger was antigenically dissimilar from the myelopoiesis-stimulating C.S.F., the point is that the same population of cells can demonstrate two diametrically opposite functions, dependent on quantitative differences of c.s.F. concentration. The suppression of B-cell-derived polyclonal humoral antibody production by monocyte-like cells has been shown by Broder et al. in some patients with myeloma. Whether the same cells are capable of stimulating monoclonal B cells in patients with myeloma remains to be established in vivo. The demonstration in vitro that macrophage-derived glycoprotein is also able to stimulate colony-formation of various other neoplastic cells is very intriguing. However, there is abundant evidence from laboratory animal and human systems that macrophage content of the tumour correlates with prognosis and risk of metastatic spread.6-9 Macrophages, although having limited capacity,’O protect to a certain extent against the tumour
spread 6- 10 and, they can selectively kill neoplastic but not normal cells.’Whether the presence of significant numbers of macrophages in malignant effusions represents a cause of rather than a protective response against, tumour growth will have to be established in vivo. Macrophages can produce several other humoral factors which are selectively tumoricidal and definitely do not promote tumour growth.llll2 Although very provocative, the suggestions that macrophages or macrophage-derived humoral factor(s) can promote the monoclonal neoplastic growth and that organ macrophage content can positively correlate with the promotion of tumour growth do contradict well-founded evidence of a protective role of macrophages against neoplastic growth and spread. The potentially beneficial role of macrophages in tumour-growth control has been ignored by Salmon and Hamburger, perhaps for the sake of clear and simplified presentation. It could be that the growth-stimulatory effect of a macrophage-derived factor on a minority of clonogenic cells in vivo counteracts the otherwise inhibitory effect of other macrophage-related factors 1. 2. 3. 4.
Salmon, S. E., Hamburger, A. Lancet, 1978, i, 1289. Prehn, R. T., Lappé, M. A. Transplant. Rev. 1971, 7, 26. Fidler, I. J. Cancer Res. 1974, 34, 491. Kurland, J. I., Bockman, R. S., Broxmeyer, H. E., Moore, M. A. S. Science, 1978, 199, 552. 5. Broder, S, Humphrey, R., Durm, M., Blackman, M., Meade, B., Goldman, C., Strober, W., Waldmann, T. A. New Engl. J. Med., 1975, 293, 887. 6. Eccles, S. A., Alexander, P. Nature, 1974, 250, 667. 7. Evans, R., Alexander, P. ibid., 1972, 236, 168. 8. Schultz, R. M., Papamatheakis, J. D., Stylos, W. A., Chirigos, M. A. Cell. Immun. 1976, 25, 309. 9. Hibbs, J. B., Jr., Lambert, L. H., Jr., Remington, J. S. Nature neō Biol.,
1972, 235, 48. 10. Lancet, 1976, ii, 27. 11. Currie, G. A., Basham, C.J. exp. Med., 1975, 142, 1600. 12. Carswell, E. A., Old, L. J., Kassel, R. L., Green, S., Fiore, N., B. Proc nat. Acad Sci. U.S.A., 1975, 72, 3666.
Williamson,
This might limit the capacity of macroinhibit tumour growth. The possibility of imphages elimination of the clonogenic-promoting munologically specific factor(s) and exploration of its properties (e.g., does it demon-
on tumour
growth.
alone
to
angiogenesis-promoting characteristics?") warrant investigation. It is important, even when presenting a hypothesis, to try to encompass all important aspects of the macrophage/tumor-cell and therefore, in part, the host-tumour relationship, since otherwise an erroneous picture may be painted. strate
further
Cancer Therapy Evaluation Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20014, U.S.A.
Viral Oncology Program, Division of Cancer Cause and Prevention, National Cancer Institute, Bethesda
STANISLAW M. MIKULSKI MICHAEL A. CHIRIGOS
SEPTIC SHOCK
SIR,-Although the article by Dr Ledingham and Mr McArdle’ contains a wealth of factual information, it makes little contribution to our understanding of the pathophysiology of the condition which could provide a base on which to plan treatment.
*
Reduction of mortality by early intermittent positive-pressure ventilation (i.P.P.v.) suggests that by squeezing a little more oxygen into the circulation and reducing consumption by the muscles of respiration, the effects of stagnant anoxia are reduced in severity and chances of survival thereby improved. The criteria by which the decision is taken to start I.P.P.V., are not, however, stated, except that it was started "early." Discussing the duration of shock Ledingham and McArdle say that, although the total duration of shock in their patients did not vary during the entire series, the duration before referral tended to increase "as appropriate treatment was initiated by the ward staff." I would have expected appropriate treatment to reduce duration and intensity. Did this treatment include massive dosage of antibiotics and did shock occur before or after the administration (release of endotoxin after wholesale destruction of gram-negative organisms can precipitate
shock)? Where does shock begin and where does it end? In the third of the series only two deaths were attributable to continuing shock yet five patients died from acute respiratory and renal failure. Albuminuria, hxmaturia, and reduced urine output are the earliest and most constant effects produced by bacterial toxins in laboratory animals.2 The kidney is the first organ affected so persistence of renal disorder indicates continuation of shock, although peripheral circulatory signs may have receded. The suggestion that pharmacological agents such as glucocorticoids could have an "action on the later stages of septic shock not hitherto described" is strange considering the volume of work devoted to this subject and the fact that glucocorticoids have maximum protective action in animals when given before endotoxin.3 Chlorpromazine also possesses similar maximum protective action before exposure to endotoxin,4 almost certainly through its deafferenting action on the brainstem reticular formation.55
13 1. 2.
Folkman, J., Ann. Surg., 1972, 175, 409. Ledingham, I. M., McArdle, C. S. Lancet, 1978, i, 1194.
Reilly, J., Compagnon, A., Laporte, A., Du Buit, H. Le Role du Système nerveux vegetatif en pathologie renale. Paris, 1942. 3. Lillenei, R. C., Longerbeam, J. N., Bloch, J. H., Manax, W. Clin. Pharmacol. Ther. 1964, 5, 63. 4. Reilly, J., Compagnon, A., Tournier, P., Du Buit, H. Ann. de Méd. 1954, 55, T5. 5. Bradley, P. B. in Reticular formation of the brain. Henry Ford symposium (edited by H. H. Jasper and others); p. 123. London, 1960. 6. Buxton Hopkin, D. A. Proc. Wld Congr. Anæsthesiol. 1977, p. 368.
212 Evidence continues to accumulate about the involvement of the central nervous system in all shock states and suggesting that the resulting pathophysiological changes involve endocrine and reticuloendothelial as well as the cardiovascular and respiratory systems. Possibly more success would follow treatment directed to the initiating sources rather than to where the end-products of the resulting changes appear. 11 Chelsea Embankment, London SW3 4LE
PRENATAL DIAGNOSIS OF M.S.U.D. IN AMNIOTIC-CELL CULTURE
D. A. BUXTON HOPKIN
ANTENATAL DIAGNOSIS OF MAPLE-SYRUP-URINE DISEASE
SIR,—Maple-syrup-urine disease (M.S.U.D.) is an autosomal recessive disease affecting the degradation of the three branched-chain aminoacids leucine, isoleucine, and valine. The classical form of the disease, if untreated, is rapidly lethal. Treatment is difficult, requiring life-long restriction of the three aminoacids. Parents who have had a child with M.S.U.D. often seek antenatal diagnosis in subsequent pregnancies. The demonstration of the branched-chain ketoacid decarboxylase in cultured amniotic cells suggested the possibility of antenatal diagnosis’ and this was confirmed in a patient from this laboratory’ and by Wendel et al.2 We have now studied eighteen pregnancies at risk for M.S.U.D. The diagnosis can be made with accuracy and confidence, but care is needed with cultures of epithelial cells and with slow-growing cultures. Twelve of the
eighteen specimens investigated were sent from
other
institutions, including four from other countries. Methods of subculhave been previously described.3 Cells were harvested for assay either with a rubber policeman or with a solution of Puck’s saline A containing 0-02% (w/v) edetic acid (E.D.T.A.) and 0.05% (w/v) trypsin (two patients). The E.D.T.A.-trypsin cell suspension was immediately added to one-quarter its volume of calf serum and centrifuged. The cell button was resuspended in buffered saline containing 20% serum and transferred to reaction flasks and centrifuged. The supernatant was ’replaced with reaction mixture and decarboxylase activity was assayed.4 Approximately 4x105 cells are required for assay, but reliable results are obtained with fewer actively growing cells. Most specimens were assayed 3-5 weeks after amniocentesis. ture
Eighteen amniotic-cell cultures were investigated and a diagnosis was made in sixteen, of which four had M.S.U.D., giving the frequency of 25% expected for an autosomal recessive disease. The other twelve assays were normal and in all twelve the postnatal diagnosis was also normal. Details of the remaining six cases (A-F) are given in the table. The diagnosis was confirmed in case B by post-abortion culture and assay of fetal skin fibroblasts. In case A a type-n variant was confirmed after birth by enzyme assay of leucocytes and by clinical course. In case C a macerated fetus was delivered at another institution after abortion with prostaglandin and urea. Culture of fetal tissues failed. In case F the baby was delivered at term; symptoms of M.S.U.D. developed and the infant died in a week. No diagnosis could be made in cases D and E. In both the cells were predominantly epithelial, as they were in 5 other successful cultures. One culture (E) was received from France. Cells grew poorly and an assay was attempted with few cells. No radioactive CO2 was released, but there was also no incorporation of radioactive aminoacids into protein, indicating that the cells were no longer metabolically active. The outcome of this pregnancy is not known. In D the ratio of the ’4C02to radioactivity in protein suggested variant M.S.U.D., but the incorporation of radioactivity into protein was too low to provide confidence in the results. No diagnosis was made and the fetus was aborted at another institution. Cultures were not established from fetal tissue. 1. Dancis, J. in Antenatal Diagnosis (edited by A. Dorfman); p. 123. Chicago, 1972. 2. Wendel, U., Rudiger, H. W., Passarge, E. Humangenetik, 1973, 19, 127. 3. Cox, R. P., Macleod, C. M.J. gen. Physiol. 1962, 45, 439. 4. Dancis, J., Hutzler, J., Cox, R. P. in Tissue Culture, Methods and Applications (edited by P. F. Kruse, Jr. and M. K. Patterson, Jr.) p. 639. New York, 1973.
Amniotic cell cultures that are predominantly fibroblastic have presented no problems with the technique devised for skin fibroblasts.s However, when cultures are predominantly
epithelial they multiply slowly, delaying analysis. Harvesting cells with a rubber policeman, as we have usually done, is associated with a variable, and frequently serious, loss of enzyme activity. The use of E.D.T.A.-trypsin to remove cells from glass surfaces seems to preserve enzyme activity in epithelial cells. We now assay cell suspensions prepared by E.D.T.A.-trypsin treatment of confluent cell monolayers. Intact cells are used because disruption of cells greatly reduces enzyme activity.’ The cells are incubated for 120 min with L-valine-1-14C (specific activity 0-5 µCi/µmol). The evolution of 14C02 provides a measure of the decarboxylase activity and is expressed per mg protein. The simultaneous incorporation of the radioactive aminoacid into protein provides an indication of the number of cells that are assayed and their metabolic activity. The ratio of the 14CO2to 14C-incorporation into protein is the most satis-
factory approach to diagnosis, provided 14C-incorporation per mg protein indicates active cell metabolism. This Service Fund.
study (AM
was
supported by grants
14528 and HD
from the U.S. Public Health
04526) and by the Samuel
Division of Human Genetics, Departments of Medicine and Pediatrics, New York University Medical Center, New York, N.Y. 10016, U.S.A.
A.
Berger
RODY P. Cox
JOEL HUTZLER JOSEPH DANCIS
MATERNAL HYPERTHERMIA AND NEURAL-TUBE DEFECTS
SIR,-Retrospective studies in the north-western United a significant correlation between maternal hyperthermia during the early part of pregnancy and the incidence of anencephalyl and meningomyelocele.2 Our similar study in the mid-southern United States has detected 3 instances of maternal hyperthermia in 45 pregnancies which resulted in children with neural-tube defects (anencephaly, meningomyelocele, spina bifida, or encephalocele). Only births during the past 11 months were included. States have revealed
In
man
the neural tube closes between the 22nd and 28th
gestation. The mothers in this study were questioned particularly about hyperthermia due to illness or prolonged sauna usage during the third, fourth, and fifth weeks of ges-
day
of
tation. This interval
was
chosen because most of the dates of
conception were only approximate. 1 mother reported "quite high" fever lasting 2 or 3 days and caused by streptococcal Elsas, L. J., Priest, J. H. Wheeler, F. B., Danner, D. J., Pask, B. A., Metabolism, 1974, 23, 569. 1. Miller, P., Smith, D. W., Shepard, T. H. Lancet, 1978, i, 519. 2. Chance, P. F., Smith, D. W. ibid. p. 769. 5.
