INVESTIGATIONS IN CHILDREN WHO WERE IN UTERO AT ONSET OF INSULIN-DEPENDENT DIABETES IN THEIR MOTHERS

INVESTIGATIONS IN CHILDREN WHO WERE IN UTERO AT ONSET OF INSULIN-DEPENDENT DIABETES IN THEIR MOTHERS

811 deflation, patients often complain of transient unexplained chest pain, and show ST segment or T wave abnormalities. This is particularly true of...

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deflation, patients often complain of transient unexplained chest pain, and show ST segment or T wave abnormalities. This is particularly true of patients undergoing angioplasty of a recent total coronary occlusion, most of which contain thrombus. These changes are unlikely to result from brief coronary occlusion (eg, 60 s), particularly in vessels that were totally occluded before angioplasty. Release of vasoconstrictors from a disrupted in-situ clot is a more likely mechanism. Transient microcirculatory vasodilator insufficiency after angioplasty has been demonstrated in dogs and can be prevented by pretreatment with aspirin.6 Coronary flow reserve and thallium scintigrams are often transiently abnormal after coronary dilation in human beings.22 Platelet activation at the site of dilation, which uniformly occurs for up to 4 h after the procedure, may account for these findings.23 The crude method by which we detected reduced coronary blood flow (reduction in the velocity of contrast washout severe enough to be obvious on visual inspection of the angiogram) would doubtless be insensitive to small reductions in coronary blood flow. More sensitive techniques for measuring regional blood flow (eg, doppler catheter, xenon scintigraphy, digital subtraction angiographic methods), might well pick up less severe examples of the same phenomenon. Our findings suggest that another more active mechanism may account for the reduction of myocardial blood flow that arises after reperfusion with angioplasty in some patients with acute myocardial infarction. Massive platelet degranulation or release of other vasoconstrictors with constriction microvascular during angioplasty of thrombotic lesions could be the main reason for local intra-arterial rethrombosis and failure to improve death rates. Some thrombolytic drugs also lead to platelet degranulation;24 reduced blood flow after thrombolysis (eg, TIMI grade 1 or 2) may result from a similar mechanism.25 Even though epicardial coronary patency is achieved, this transient microvascular constriction might significantly limit myocardial salvage. This work was supported by grants from the National Institutes of Health (ROI-HL39185) and the American Heart Association. The work was done during the tenure of a Clinician Scientist Award (R. F. W.) from the American Heart Association with funds contributed partly by the Minnesota

Affiliate. We thank Betsy Chnstensen, Susan Meyer, and the staff of the cardiac catheterization laboratory of the University of Minnesota Hospital for their assistance.

Correspondence should be addressed to

R. F.

W., Box 508 UMHC, 420

Delaware Street, SE Minneapolis, MN 55455, USA. REFERENCES 1. Falk E. Unstable

angina with fatal outcome, dynamic coronary thrombosis leading to infarction and/or sudden cardiac death. Circulation 1985; 71: 699-708 2. Chapman I. Morphogenesis of occluding coronary artery thrombosis. Arch Pathol 1965; 80: 256-61. RL, Hutchins GM. Relationship between coronary artery lesions and myocardial infarcts ulceration of athersclerotic plaques precipitating coronary thrombosis Am Heart J 1977; 93: 468. 4. Simonton CA, Mark DB, Hinohara T, et al Late restensosis after emergent angioplasty for acute myocardial infarction: comparison with elective coronary angioplasty J Am Coll Cardiol 1988; 11: 698-705 5. Johns JA, Gold HK. Management of coronary reocclusion following successful thrombolysis. In: Topol EJ, ed. Acute coronary intervention. New York: Liss, 1988; 95-106. 6. Topol EJ, Califf RM, George BS, et al. A randomized trial of activator in acute myocardial infarction. N Engl J Med 1987, 317: 581-88 7. Bates ER, McGillem MJ, Beals TF, et al. Effect of angioplasty induced endothelial denudation compared with medial injury on regional coronary blood flow. Circulation 1987; 76: 710-16. 8 Wilson RF, Johnson MR, Marcus ML, et al The effect of coronary angioplasty on coronary flow reserve. Circulation 1988; 77: 873-85. 3 Ridolfi

INVESTIGATIONS IN CHILDREN WHO WERE IN UTERO AT ONSET OF INSULIN-DEPENDENT DIABETES IN THEIR MOTHERS KARSTEN BUSCHARD1 CLAUS KÜHL1 LARS MØLSTED-PEDERSEN1 EBBA LUND2 GIAN FRANCO BOTTAZZO4 JERRY PALMER3 Diabetes

Centre, Department of Obstetrics and Gynaecology,

Rigshospitalet, University of Copenhagen;1 Department of Virology, The Royal Veterinary and Agricultural University, Copenhagen, Denmark;2 Diabetes Research Center, University of Washington, Seattle, USA;3 Department of Immunology, University College and Middlesex School of Medicine, London, UK4 55 children who were in utero when type 1 diabetes developed in their mothers were studied at a mean (SEM) age of 10·4 (0·6) years: only 1 was diabetic. Biochemical and immunological indices, measured in 35 children, showed no evidence of beta cell dysfunction. Thus, the fetal beta cells seem to be unaffected by the mechanisms that cause diabetes in their mothers.

Summary

Introduction WHEN type 1 diabetes arises in pregnancy the onset is often abrupt.1 What happens to the offspring of these mothers? Since autoimmune mechanisms seem important in the pathogenesis of type 1 diabetes2 (as evidenced by positive in-vitro cytotoxic tests against beta cells at diagnosis;3 the high incidence of antibody markers such as islet-cell antibodies [ICA];4 and the fact that the course of the disease can be modified with immunosuppressive agents

Slager CJ, Schuurbiers JCH, et al. Transfer functions of the X-ray-cine. In: Brennecke R, ed. Digital imaging of cardiovascular radiology. New York: Thieme-Stratton, 1983: 89-104. 10. Escolar G, Hagert-Whiting K, Bravo ML, White JG. Interaction of long term stored platelets with the vascular subendothelium. J Lab Clin Med 1987; 109: 147-54. 11. Morooka S, Kobayashi M, Takahashi T, Takashima Y, Sakamoto M, Shimamoto T. Experimental ischaemic heart disease-effects of synthetic thromboxane A2. Exp Mol Pathol 1979; 30: 449-57. 12. Michelassi F, Landa L, Hill RD, et al Leukotriene D4: a potent coronary artery vasoconstrictor associated with impaired ventricular contraction. Science 1982; 217: 841-43. 13. Roth DA, Lefer DJ, Hock CE, Lefer AM. Effects of peptide leukotrienes on cardiac dynamics in rat, cat, and guinea pig hearts. Am J Physiol 1985; 249: H477-H484. 14. Ezra D, Boyd LM, Feuerstein G, Goldstein RE. Coronary constriction by leukotriene C4, D4 and E4 in the intact pig heart. Am J Cardiol 1983; 51: 1451-54. 15. Letts LG, Newman DL, Greenwald SE, Piper PJ. Effects of intra-coronary administration of leukotnence D4 in the anesthetized dog. Prostaglandins 1983; 26: 563. 16. Murayama M. Ex-vivo human platelet aggregation induced by decompression during reduced barometric pressure, hydrostatic, and hydrodynamic (Bernoulli) effect. Thromb Res 1984, 33: 477-85. 17. Harrison DG, Chapman MP, Christy JP, Marcus ML. Studies of the functional site of origin of native coronary collaterals. Am Heart Physiol 1986; 251: H1217-H1224. 18. Hori M, Inoue M, Kitakaze M, et al. Role of adenosine in the hyperemic response of coronary blood flow in microembolization. Am J Physiol 1986; 250: H509-H518. 19. Cobb FR, Bache RJ, Greenfield JC Jr. Local effects of acute cellular injury on regional myocardial blood flow. J Clin Invest 1976; 57: 1359. 20. Cobb FR, McHale PA, Rembert JC. Effects of acute cellular injury on coronary vascular reactivity in awake dogs. Circulation 1978; 57: 962-68. 21. Kloner RA, Ganote CE, Jennings RB. The "no-reflow" phenorrienon after temporary coronary occlusion m the dog. J Clin Invest 1974; 54: 1496-508. 22. Manyari DE, Knudtson M, Kloiber R, Roth D. Sequential thallium-201 myocardial perfusion studies after successful percutaneous transluminal coronary artery angioplasty: delayed resolution of exercise-induced scintographic abnormalities. Circulation 1988; 77: 86-95. 23. Wilentz JR, Sanborn TA, Haudenschild CC, Valeri CR, Ryan TJ, Faxon DP Platelet accumulation in experimental angioplasty time course and relation to vascular injury. Circulation 1987, 75: 636-42. 24. Fitzgerald DJ, Catella F, Roy L, FitzGerald GA. Marked platelet activation in vivo after intravenous stieptokinase in patients with acute myocardial infarction. Circulation 1988; 77: 142-50 25. The TIMI Study Group. The thrombolysis in myocardial infarction (TIMI) trial. N Engl J Med 1985; 312: 932-37. 9. Reiber JHC,

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such as cyclosporin5), it is surprising that no-one has looked for evidence of damage to fetal pancreatic beta cells. We have done this indirectly by investigating children who were in utero when their mothers became diabetic.

Subjects and Methods Mothers We studied 61 children of 60 diabetic mothers (1twin pregnancy) who were treated at the University Hospital, Copenhagen, from 1966 to 1980 after onset of type 1 diabetes during pregnancy. Data from the time of diagnosis were taken from the hospital records. At a follow-up examination, 57 of the mothers participated in the study. The incidence of diabetes during pregnancy and the clinical data of the mothers have been previously reported.’ Briefly, at the time of diagnosis mean age (SEM) of the mothers was 26-9 (07) years and mean fasting blood glucose was 156 (1-3) mmol/1. 81% had ketonuria. Maximum daily insulin dose during pregnancy was 0 80 (0-05) U/24 h per kg body weight. At the time of the present study all of the mothers had type 1 diabetes and were being treated with insulin: at follow-up 8 (1) years after the birth they had a mean glucagon-stimulated C-peptide concentration of012 (002) nmol/1 (none more than 060 nmol/1 and most [59%] less than 0-05 nmol/1), which indicates total loss of beta cell function. Additionally, the mothers’ HLA tissue type distribution-namely, a high proportion of either HLA-DR3 or HLA-DR4- was the same as that in non-pregnant type 1 diabetics and this distribution differed from that in a control group from the

background population.7 Children 35 of the children were examined clinically, and various biochemical and immunological indices were recorded. Fasting blood glucose and fasting insulin concentrations8 were measured but the children were unwilling to have full glucose tolerance tests. ICA determination.-Undiluted sera were screened for ICA (conventional ICA-IgG) and for the complement-fixing variant (CF-ICA) by an indirect immunofluorescence technique on sections of blood group 0 cryofixed human pancreas.4,9,10 Fluoresceinated rabbit anti-human IgG and C3 (’Dakopatts’, Glostrup, Denmark) were used for ICA and CF-ICA determinations, respectively. With the same pancreas, reagents, and incubation conditions, the putative ICA standard currently being assessed by the Immunology and Diabetes Workshops gave end-point titres of 32 when tested "blind" on two occasions.l’

Fig 2-Insulin autoantibody assay. The children of diabetic mothers are compared with 92 healthy controls, 56 newly diagnosed type 1 diabetics, and 52 type 1 diabetics on insulin therapy. - - - = The limit for the presence of IAA (mean + 3 SD of the healthy controls).

Samples were read by two independent observers and the interassay reproducibility of duplicates was plus or minus one doubling dilution. Insulin autoantibody (IAA) deterrnination.-Sera were screened for IAA by the method described by Palmer et ap2 modified to include an acid charcoal extraction and displacement with excess unlabelled insulin. 13 Virus antibody.-We measured neutralising antibody titres to Coxsackieviruses B4 and B5, and to echovirus 7 in 30 of the 35 children and their mothers. Tests were done in HeLa cells against 100 median infective doses (IDSO) of the appropriate virus. We chose the Coxsackieviruses because they have been implicated in the pathogenesis of type 1 diabetes,14 and echovirus 7 because it was prevalent in the community at the time of our study and represents virus groups not associated with type 1 diabetes.

Statistical Analyses The significance of differences was evaluated by application of the Wilcoxon test (paired data) or the Mann-Whitney U-test (unpaired data). The Spearman’s rank correlation test was used for the calculation of the coefficient of correlation. The level of type 1 error (2ot) was set at 0-05.

Results

Fig

1--Correlation (p < 00005) between the children’s age and insulin concentration.

serum

4 children had died in the prenatal period; none of the deaths was attributable to diabetes. Data were available for 55 of the remaining children-mean age 10-4 (0-6) years (median 10-7, range 3-4-19-9). Type 1 diabetes had developed in 1 child when he was 15-7 years old; he was still on insulin therapy at 17-0 years old. None of the other children had been treated with insulin or by diet for diabetes, and none had glycosuria or raised blood glucose. 1 child had a congenital deformity (hypospadias), 1 asthma, and 1 epilepsy.

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Fig 3-Antibody titres to Coxsackievirus B5, B4, and echovirus 7 of the individual mother and corresponding child. Mean titres + SEM

are

indicated.

Biochemistry and Immunology Fasting blood glucose and insulin concentrations.-The mean fasting blood glucose concentration, 4-6 (0-1) mmol/1 (range 3-7-5-8), was the same as the reference control value, 4-7 (0-1) mmol/1. The mean fasting insulin concentration was 78-0 (7-1) pmol/1 (range 30-200)—normal 67-3 (6-3).is There was a positive correlation (p < 0-0005) between the fasting insulin value and age (fig 1). ICA and IAA (fig 2).-Conventional ICA-IgG, CF-ICA, or IAA were not detected. The mean percent binding in the IAA assay was 1-32 (0’03}-range 11-1-7. None of the children had an increased value compared with the laboratory’s healthy control value of 1-57 (0-03) (mean+3 SD=2’31). Virus antibodies (fig 3) .-60% of the children and 80% of the mothers had antibodies to Coxsackievirus B4. For Coxsackievirus B5 the figures were 33% and 40%, respectively, and for echovirus 7, antibodies were found in 53% of children and 63% of mothers. There was a positive correlation between individual mother/child pairs for echovirus 7 antibody titres (p < 0-05), but not for Coxsackievirus B5 (p < 0 - 10) or B4 antibody titres. There was no correlation between virus antibody titres and children’s age, insulin concentration, or fasting blood glucose values.

Discussion Our findings show that the diabetogenic process in the pregnant mother does not affect the beta cells of the fetus: with one exception, none of the children in our study had biochemical or immunological signs of beta cell dysfunction. Unlike others15 we confirmed the positive correlation between age and insulin concentration found by Grant.16 Can putative diabetogenic agents cross the placental barrier? The agents causing human type 1 diabetes and their pathogenetic action on the beta cells are not definitely known. Both the encephalomyocarditis virus, which is diabetogenic in mice,17,18 and the Coxsackievirus B4, which

is associated with type 1 diabetes in man, 14 are enteroviruses that can cross the placenta. The same is true of and similar low-molecular-weight streptozotocin substances that are diabetogenic in laboratory animals19 and in man .20 The immune system is actively involved in the diabetogenic process;2 IgG, but not IgM, can pass the placental barrier, but it is unlikely that substantial numbers of lymphocytes can do so. Our data suggest that none of these factors affects beta cell function in the child of a diabetic mother. If fetal exposure to the diabetogenic factors in the pregnant mother were to lead to disease in the child, at what age could this be expected? The mean age of the children in our study was 10-4 years, and development of diabetes over a period of more than 10 years seems highly unlikely. Approximately 20% of all type 1 diabetics in Denmark get the disease within the first 11 years of life .2’ The fact that our cohort was negative for both ICA and IAA reinforces the prediction that these potentially high risk children are very unlikely to become diabetic.22 We thank Dr Philip Hougaard, Novo Research Institute for statistical help, and Ms Marion Shattock, Ms Jessica McNally, and Ms Regina Park for skilful technical assistance. The work in the Department of Immunology, Middlesex Hospital was partly supported by the British Diabetic Association, the Medical Research Council, and Novo Research Institute. The work at the University of Washington was supported partly by grants AM 17047 and AM 30780 from the National Institutes of Health.

Correspondence should be addressed to K. B., The Bartholin Institute, Kommunehospitalet, DK-1399 Copenhagen K, Denmark.

REFERENCES 1. Buschard K, Buch I, Mølsted-Pedersen L, Hougaard P, Kuhl C. Increased incidence of true type I diabetes acquired during pregnancy. Br Med J 1987; 294: 275-79. 2. Buschard K. The thymus-dependent immune system in the pathogenesis of type 1 (insulin-dependent) diabetes mellitus. Animal model and human studies. Dan Med Bull 1985; 32: 139-51. 3. Charles M, Suzuki M, Sundsmo J, et al. Immunological events in new onset diabetes.

Biomed Biochim Acta 1984; 43: 615-19. GF, Florin-Christensen A, Doniach D. Islet-cell antibodies m diabetes mellitus with autoimmune polyendocrine deficiencies. Lancet 1974; ii: 1279-83.

4. Bottazzo

814 DETECTION OF RETROVIRUS IN PATIENTS WITH MYELOPROLIFERATIVE DISEASE

given positive results in the assay conditions in references 5 and 6, respectively.7,8 We have conducted further experiments to answer these questions.

M. T. BOYD1* N. MACLEAN1 D. G. OSCIER2

Department of Biology, Southampton University, Southampton SO9 3TU;1 and Department of Haematology, Royal Victoria Hospital, Bournemouth BH1 4JG2 RNA-directed DNA polymerase (reverse transcriptase) activity was detected in platelets from 4/4 patients with primary proliferative polycythaemia (PPP), 7/7 patients with essential thrombocythaemia (ET), 1/4 patients with relative "stress" polycythaemia, 0/2 patients with secondary polycythaemia, and 0/3 normal subjects. The activity appeared to be particle-associated and was detected under conditions not appropriate for any known cellular enzymes. Active particulate fractions from 1 patient with PPP and 1 patient with ET were examined by electron microscopy and revealed objects with the features of retroviruses. Similar retrovirus-like particles were observed in 2/2 further patients with PPP and 2/2 further patients with ET but in

Summary

PATIENTS AND METHODS

We studied 6 patients with PPP, 8 patients with ET, 4 patients with secondary polycythaemia, 4 patients with relative polycythaemia, and 3 haematologically normal subjects. PPP and ET were diagnosed according to the criteria of the Polycythaemia Vera Study Group.9 Platelet samples were obtained after venesection or occasionally platelet-pheresis. Platelets prepared by differential centrifugation’° of fresh blood samples were lysed by homogenisation and the lysate was cleared by further centrifugation. Cleared lysates were layered over linear 20-60% w/w sucrose gradients,I g of original platelet wet weight(= 2 x 1011 platelets) being used per 6 ml of gradient. Such gradients were centrifuged for 16 h at 100 000 g, after which 0 1 ml fractions were collected and used for the RNA-directed DNA polymerase (RDDP) assays. TABLE I-ANALYSIS OF PEAK ACTIVITY OF MATERIAL

0/3 controls. INTRODUCTION

THE myeloproliferative disorders are clonal proliferative diseases1,2 arising in a multipotent haematopoietic stem cell.3 Primary proliferative polycythaemia (PPP) is characterised by a panhyperplasia of the bone marrow, an increase in the red cell mass, and frequently leucocytosis and thrombocytosis. In essential thrombocythaemia (ET) a predominantly megakaryocytic proliferation results in thrombocytosis. Both these diseases carry an excess risk of transformation to acute leukaemia even in the absence of chemotherapy.4 Earlier studies raised the possibility that RNA-directed DNA polymerase (reverse transcriptase) activity was present in both the platelets5 and a bonemarrow-derived cell line of such patients. However, these experiments did not exclude the possibility that the activity was due to the presence of normal cellular polymerasesspecifically, platelet mitochondrial DNA polymerase and terminal transferase. Activity of these enzymes would have

2"P

secondary polycythaemia. Background counts, which have been subtracted from all values, were in the range 10 000-15 000 but never varied by more than 2000 cpm between replicates. Because of shortage of material, repeat assays were made only in patient 11; here three separate assays were run on each template. Numbers for =

cpm have been rounded *Present address: Institute of Cancer Research, Chester Beatty Laboratories, Fulham Road, London SW3 6JB.

5. Feutren G, Papoz L, Assan R, et al. Cyclosporin increases the rate and length of remission in insulin-dependent diabetes of recent onset. Results of a multicentre

double-blind trial. Lancet 1986; i: 119-24. S, Krarup T, McNair P, et al. Practical clinical value of the C-peptide response to glucagon stimulation in the choice of treatment in diabetes mellitus. Acta Med Scand 1981; 210: 153-56. 7. Møller-Jensen B, Buschard K, Buch I, et al. HLA associations in insulin-dependent diabetes mellitus diagnosed during pregnancy. Acta Endocrinol (Copenh) 1987; 116: 387-89. 8. Kuhl C. Glucose metabolism during and after pregnancy in normal and gestational diabetic women. I. Influence of normal pregnancy on serum glucose and insulin concentrations during basal fasting conditions and after a challenge with glucose. Acta Endocrinol 1975; 79: 709-19. 9. Bottazzo GF, Dean BM, Gorsuch AN, Cudworth AG, Domach D. Complementfixing islet-cell antibodies in type-I diabetes: possible monitors of active beta-cell damage. Lancet 1980; i: 668-72. 10. Gleichmann H, Bottazzo GF. Progress toward standardization of cytoplasmic islet cell-antibody assay. Diabetes 1987; 36: 578-84. 11. Bonifacio E, Dawkins RL, Lernmark A. Report of the Second International Workshop on the standardisation of cytoplasmic islet cell antibodies. Diabetologia 1987; 30: 273. 12. Palmer JP, Asplin CM, Clemens O, et al. Insulin antibodies in insulin-dependent diabetes before insulin treatment. Science 1983; 222: 1337-39. 6. Madsbad

to

the nearest thousand.

Poly (dA) counts are those for the samples which showed maximum poly (rC) activity. In some cases slightly higher poly (dA) activity was observed in a proximal gradient fraction.

M, Klaff LJ, Asplin CM, et al. Insulin autoantibodies at diagnosis of insulin-dependent diabetes: effect on the antibody response to insulin treatment. Metabolism (in press). 14. Gamble DR, Taylor KW. Coxsackie B virus and diabetes. Br Med J 1973; i: 289-90. 15. Ludvigsson J, Heding L. C-peptide in diabetic children after stimulation with glucagon compared with fasting C-peptide levels in non-diabetic children. Acta 13. Sutton

Endocrinol 1977; 85: 364-71. 16. Grant DB. Fasting serum insulin levels in childhood. Arch Dis Child 1967; 42: 375-78. 17. Craighead JE, McLane MF. Diabetes mellitus: induction in mice by encephalomyocarditis virus. Science 1968; 162: 913-14. 18. Rayfield JE, Ishimura K. Environmental factors and insulin-dependent diabetes mellitus. Diabetes Metab Rev 1987; 3: 925-57. 19. Rerup C. Drugs producing diabetes through damage of the insulin secreting cells. Pharmacol Rev 1970; 22: 485-518. 20. Karam JH, Lewitt PA, Young CW, et al Insulinopenic diabetes after rodenticide (Vacor) ingestion. A unique model of acquired diabetes in man. Diabetes 1980; 29: 971-78. 21. Green A, Svejgaard A, Platz P, et al. The genetic susceptibility to insulin-dependent diabetes mellitus: combined segregation and linkage analysis. Genet Epidemiol 1985; 2: 1-15. 22. Tam AC, Thomas JM,

1988; i: 845-50.

Dean BM, et al. Predicting insulin-dependent diabetes. Lancet