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DIABETES MELLITUS IN PIMA INDIANS
379 SIMPLE METHOD FOR DETECTING IgG ON EPITHELIAL CELLS to report on a simplified method of SiR,-We demonstrating immunoglobulin G on epithelial cells.
wish
Previous work indicates the usefulness of direct and indirect fluorescent-antibody techniques in the diagnosis and study of pemphigus vulgarisand morbilliform
eruptions from penicillins.22 Ordinarily, fluoresceinated anti-human IgG is applied to involved human skin, or to skin
or mucous
membrane3 excised from
guineapigs
or
monkeys, after treatment with serum from appropriate patients. Given positive results by either method, fluorescence can then be seen in characteristic locations, indicating IgG in epidermis by the direct method or in serum by the indirect method. The need for freezing and cutting human or animal tissue is what makes the methods laborious and expensive. Our new method of obtaining suitable human cells or sheets of cells without surgery and without need for special handling before final processing by the direct or indirect techniques is particularly adaptable to the study of pemphigus vulgaris and morbilliform eruptions from penicillin. With a wooden tongue-depressor or a metal spatula, cells were scraped from 3 normal subjects, smeared on glass slides, and allowed to dry 20 minutes at room-temperature. The preparations were then immersed in phosphate-buffered saline, pH 7-4
Fig. 2-Cells treated with serum and fluorescent conjugate showing no intercellular fluorescence.
( x 450.) sheets (fig. 1), resembling the pattern on giraffe skin, when the indirect technique was used with serum from both patients with pemphigus and 3 of 4 patients with late morbilliform eruptions due to penicillin. Rods of fluorescence were seen around and upon cells when the cells were dispersed. No staining was seen when serum from 2 healthy controls was used (fig. 2), or when no serum was added. Results were in agreement when frozen-tissue sections were used as in older methods. Direct staining of cells from the bullous skin lesion of the patient with pemphigus gave a positive result similar to that previously described.3 Staining buccal cells from a pemphigus patient with her own serum was also positive. The readability of indirect fluorescence with our new method compares favourably with that of preparations made in the conventional manner. There are several possible explanations for the difference in patterns of fluorescence seen in the cells in sheets compared to the dispersed cells. One is that by separating cells the antigenic sites become more accessible to the antibody. This investigation was supported in part by Public Health Service Research grant Al-07728 from the National Institute of Allergy and Infectious Diseases.
Fig. 1-Celis treated with
serum
Department of Dermatology, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A.
and fluorescent conjugate
showing brilliant intercellular fluorescence.
(x 450.) for five minutes, and processed for indirect fluorescent staining in the standard manner. Sera from 2 patients with biopsyproven pemphigus vulgaris, and from 4 with morbilliform eruptions caused by penicillin, as well as from 2 normal subjects, were serially diluted, layered on epithelial cells from normal subjects, incubated, washed with buffer, and finally treated with the fluorescein-labelled anti-human IgG. A Tzanck smear was obtained from 1 patient with acute bullous pemphigus and stained directly. In addition, cells from normal subjects were collected in test-tubes, washed in phosphate buffer, and processed for indirect staining as above, except that the cells were treated while suspended in buffer in test-tubes. Fluorescence was read in the usual way with a properly equipped microscope by the 2 authors, independently. Staining was also carried out using the older method 1,3of guineapig-oesophagus frozen sections on all sera.
Giemsa staining revealed that the buccal cells were either dispersed or in sheets. A continuous pattern of fluorescence was found around cells when they were in Beutner, E. H., Jordan, R. E., Chorelski, T. P. J. invest. Derm 1968, 51, 62. 2. Fellner, M. J., Prutkin, L. ibid. 1971, 55, 390. 3. Schroeter, A., Sams, W. M., Jr., Jordan, R. E. Archs Derm. 1969 100, 736. 1.
MICHAEL J. FELLNER MARIA V. KLAUS.
DIABETES MELLITUS IN PIMA INDIANS
SIR,-The extraordinary metabolic behaviour of the reported by Dr. Bennett and his colleagues (July 17, p. 125) raises a number of interesting and important questions. The most challenging finding is that of bimodality " in the distribution of the two-hour plasmaglucose levels, recalling, as it does, the notable Pickering/ Platt controversy of a decade or so ago regarding bloodThe pressure levels and the nature of hypertension. authors have suggested elsewherethat the findings in the Pima may be applied to the generality of glucose tolerance and intolerance in man. However, this seems
Pima Indians
"
us to be too great an extrapolation. Even after the removal of the grossly hyperglycxmic component of the Pima population, the mean glucose value of the remainder is high and rises steeply with increasing age. A comparison with the whole-blood sugar values, by age, in the random
to
4.
Steinberg, A. G., Rushforth, N. B., Bennett, P. H., Burch, T. A., Miller, M. in Pathogenesis of Diabetes Mellitus (edited by E. Cerasi and R. Luft); p. 237. Stockholm, 1970.
380 I-MEAN TWO-HOUR CAPILLARY-BLOOD SUGARS (mg./l00 IN THE BEDFORD POPULATION AND VENOUS PLASMA-GLUCOSE LEVELS IN THE PIMA POPULATIONS*
TABLE
ml.)
II-FREQUENCY OF DIABETIC RETINOPATHY* IN THE HYPERGLYCaeMIC MEMBERS OF THE BEDFORD POPULATION AT THE SURVEY AND FIVE YEARS LATER TABLE
* Includes microaneurysms only or with haemorrhages and/or exudates (at the survey, the majority had microaneurysms only).
* These values refer only to the lower component of the distribution in the Pima population, but to the whole of the Bedford group.
sample of the Bedford Survey population5 (table i) shows only differences in absolute levels (difficult to estimate exactly because of varying methods) but also a very considerable difference in the rise with age. In the large series reported by Schliack and co-workers from Berlin6 there was similarly a small rise with age of the mean twohour blood-sugar level. The apparent emergence of bimodality " with increasing age may, as Dr. Bennett and his colleagues infer, indicate " age activation " of some genetic mechanism, but one should argue with caution the effects of increasing age from cross-sectional studies such as this. It is possible that 30 or 40 years ago the population ceased being exposed to some diabetogenic influence, the effects of which have left an affected cohort which is travelling with time through successive age categories. The disappearance of Kuru with the cessation of cannibalism7 forms an epidemiological analogy, perhaps. In any case, it would seem unlikely that this population has always been so genetically predisposed to diabetes, in view of the natural selective disadvantage. We would take slight issue with the implication of the Arizona workers that, in some way, those people in the upper component of the plasma-glucose distribution represent the real diabetics in that they showed a high frequency of " the specific microangiopathy lesions of diabetes ". The simplest objection to this view is that microvascular changes are related to the degree of glucose intolerance and not
"
"
L., Butterfield, W. J. H., Keen H. Proc.
R. Soc. Med.
1964, 57, 193. 6.
Rost, G., Honigmann, G., Schliack, V. Z.
ges. inn. Med.
289.
7.
"
blood-sugar behaviour of the young Pima be foretold, perhaps by the response to provocative tests ? The answers these and to many other questions will be awaited with interest. Department of Medicine, H. KEEN Guy’s Hospital Medical School, R. J. JARETT. London SE1 9RT.
to
"
that one would therefore expect more abnormality among those with the higher blood-sugars. Furthermore, those with the higher levels of blood-sugar have probably been hyperglycaemic longer than those with the lower levels, so that their retinopathy and nephropathy may be more an indication of longer duration of exposure to high bloodsugar levels. Some support for this alternative explanation is to be gained from data from the Bedford Study (table 11). In a population without evidence of blood-sugar bimodality, not only was retinopathy almost confined to the more hyperglycaemic individuals at diagnosis, but it also appeared and progressed in this group at a faster rate over the next 5 years. Whether blood-sugar levels are continuously or discontinuously distributed in the population, it is possible that a threshold effect operates, so that a minimal degree of glucose intolerance and, perhaps, duration of exposure to hyperglycsemia is necessary for the genesis of retinopathy. The traditional view that retinopathy is a consequence of 5. Sharp, C.
hyperglycaania, rather than a marker of some microangiopathic process of which hyperglycaemia is only a part, is strongly supported by the induction of morphologically indistinguishable changes in animals rendered diabetic experimentally.8 Clearly, the further study of the Pima tribe is going to provide much new and valuable information. Will therapeutic lowering of blood-sugar confer protection against the microvascular complications " ? What of the state of the larger arteries, in particular the coronary vessels ? Can the more hyperglycaemic component of the population be distinguished from the less hyperglycaemic by characteristics other than the blood-sugar ? Can the future
Hornabrook, R. W., Moir, D. J. Lancet, 1970, ii, 1175.
1966, 21,
SYNTHETIC DIETS IN RENAL FAILURE SIR,-It is clear from the work of Dr. Richards and his colleagues (July 17, p. 128) that it may soon become possible to limit the nitrogen intake of patients with renal of a synthetic protein substitute. One of this approach has received aspect very important insufficient emphasis in their otherwise excellent paper. They point out that vitamin supplements will be required, but mention only vitamin B-complex and vitamin C in their methods. These would be inadequate in the longterm use of a synthetic diet such as they advocate. Wide experience of synthetic diets has been gained during the treatment of inborn errors of metabolism and food intolerances in childhood 9; the importance of vitamin supplements for the health and wellbeing of these patients has been repeatedly emphasised.lo,l1 Infants and children have more urgent nutritional needs than the adult, but the dramatic effects of nutritional deficiency which have been described in children serve as a reminder of the potential dangers in the use of synthetic diets. We should therefore like to draw attention to a vitamin preparation which, in spite of its usefulness," is not widely known. It supplies all the essential vitamins, and is the only convenient preparation available for this purpose. The product is marketed under the name of Ketovite Tablets/Ketovite (Supplement) Liquid’ by Paines and Byrnes. These must be taken in combination, and 5 ml. of liquid and 3 tablets a day will supply the total vitamin failure
by the
use
Bloodworth, J. M. B. in Blood Vessel Disease in Diabetes Mellitus (edited by K. Lundbaek and H. Keen). Milan (in the press). 9. Francis, D. E. M., Dixon, D. J. W. Diets for Sick Children; p. 219. Oxford, 1970. 10. M.R.C. Conference on Phenylketonuria. Br. med. J. 1963, i, 1691. 11. Mann, P. T., Wilson, K. M., Clayton, B. E. Archs Dis. Childh. 1965, 40, 364. 8.
wish
Previous work indicates the usefulness of direct and indirect fluorescent-antibody techniques in the diagnosis and study of pemphigus vulgarisand morbilliform
eruptions from penicillins.22 Ordinarily, fluoresceinated anti-human IgG is applied to involved human skin, or to skin
or mucous
membrane3 excised from
guineapigs
or
monkeys, after treatment with serum from appropriate patients. Given positive results by either method, fluorescence can then be seen in characteristic locations, indicating IgG in epidermis by the direct method or in serum by the indirect method. The need for freezing and cutting human or animal tissue is what makes the methods laborious and expensive. Our new method of obtaining suitable human cells or sheets of cells without surgery and without need for special handling before final processing by the direct or indirect techniques is particularly adaptable to the study of pemphigus vulgaris and morbilliform eruptions from penicillin. With a wooden tongue-depressor or a metal spatula, cells were scraped from 3 normal subjects, smeared on glass slides, and allowed to dry 20 minutes at room-temperature. The preparations were then immersed in phosphate-buffered saline, pH 7-4
Fig. 2-Cells treated with serum and fluorescent conjugate showing no intercellular fluorescence.
( x 450.) sheets (fig. 1), resembling the pattern on giraffe skin, when the indirect technique was used with serum from both patients with pemphigus and 3 of 4 patients with late morbilliform eruptions due to penicillin. Rods of fluorescence were seen around and upon cells when the cells were dispersed. No staining was seen when serum from 2 healthy controls was used (fig. 2), or when no serum was added. Results were in agreement when frozen-tissue sections were used as in older methods. Direct staining of cells from the bullous skin lesion of the patient with pemphigus gave a positive result similar to that previously described.3 Staining buccal cells from a pemphigus patient with her own serum was also positive. The readability of indirect fluorescence with our new method compares favourably with that of preparations made in the conventional manner. There are several possible explanations for the difference in patterns of fluorescence seen in the cells in sheets compared to the dispersed cells. One is that by separating cells the antigenic sites become more accessible to the antibody. This investigation was supported in part by Public Health Service Research grant Al-07728 from the National Institute of Allergy and Infectious Diseases.
Fig. 1-Celis treated with
serum
Department of Dermatology, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016, U.S.A.
and fluorescent conjugate
showing brilliant intercellular fluorescence.
(x 450.) for five minutes, and processed for indirect fluorescent staining in the standard manner. Sera from 2 patients with biopsyproven pemphigus vulgaris, and from 4 with morbilliform eruptions caused by penicillin, as well as from 2 normal subjects, were serially diluted, layered on epithelial cells from normal subjects, incubated, washed with buffer, and finally treated with the fluorescein-labelled anti-human IgG. A Tzanck smear was obtained from 1 patient with acute bullous pemphigus and stained directly. In addition, cells from normal subjects were collected in test-tubes, washed in phosphate buffer, and processed for indirect staining as above, except that the cells were treated while suspended in buffer in test-tubes. Fluorescence was read in the usual way with a properly equipped microscope by the 2 authors, independently. Staining was also carried out using the older method 1,3of guineapig-oesophagus frozen sections on all sera.
Giemsa staining revealed that the buccal cells were either dispersed or in sheets. A continuous pattern of fluorescence was found around cells when they were in Beutner, E. H., Jordan, R. E., Chorelski, T. P. J. invest. Derm 1968, 51, 62. 2. Fellner, M. J., Prutkin, L. ibid. 1971, 55, 390. 3. Schroeter, A., Sams, W. M., Jr., Jordan, R. E. Archs Derm. 1969 100, 736. 1.
MICHAEL J. FELLNER MARIA V. KLAUS.
DIABETES MELLITUS IN PIMA INDIANS
SIR,-The extraordinary metabolic behaviour of the reported by Dr. Bennett and his colleagues (July 17, p. 125) raises a number of interesting and important questions. The most challenging finding is that of bimodality " in the distribution of the two-hour plasmaglucose levels, recalling, as it does, the notable Pickering/ Platt controversy of a decade or so ago regarding bloodThe pressure levels and the nature of hypertension. authors have suggested elsewherethat the findings in the Pima may be applied to the generality of glucose tolerance and intolerance in man. However, this seems
Pima Indians
"
us to be too great an extrapolation. Even after the removal of the grossly hyperglycxmic component of the Pima population, the mean glucose value of the remainder is high and rises steeply with increasing age. A comparison with the whole-blood sugar values, by age, in the random
to
4.
Steinberg, A. G., Rushforth, N. B., Bennett, P. H., Burch, T. A., Miller, M. in Pathogenesis of Diabetes Mellitus (edited by E. Cerasi and R. Luft); p. 237. Stockholm, 1970.
380 I-MEAN TWO-HOUR CAPILLARY-BLOOD SUGARS (mg./l00 IN THE BEDFORD POPULATION AND VENOUS PLASMA-GLUCOSE LEVELS IN THE PIMA POPULATIONS*
TABLE
ml.)
II-FREQUENCY OF DIABETIC RETINOPATHY* IN THE HYPERGLYCaeMIC MEMBERS OF THE BEDFORD POPULATION AT THE SURVEY AND FIVE YEARS LATER TABLE
* Includes microaneurysms only or with haemorrhages and/or exudates (at the survey, the majority had microaneurysms only).
* These values refer only to the lower component of the distribution in the Pima population, but to the whole of the Bedford group.
sample of the Bedford Survey population5 (table i) shows only differences in absolute levels (difficult to estimate exactly because of varying methods) but also a very considerable difference in the rise with age. In the large series reported by Schliack and co-workers from Berlin6 there was similarly a small rise with age of the mean twohour blood-sugar level. The apparent emergence of bimodality " with increasing age may, as Dr. Bennett and his colleagues infer, indicate " age activation " of some genetic mechanism, but one should argue with caution the effects of increasing age from cross-sectional studies such as this. It is possible that 30 or 40 years ago the population ceased being exposed to some diabetogenic influence, the effects of which have left an affected cohort which is travelling with time through successive age categories. The disappearance of Kuru with the cessation of cannibalism7 forms an epidemiological analogy, perhaps. In any case, it would seem unlikely that this population has always been so genetically predisposed to diabetes, in view of the natural selective disadvantage. We would take slight issue with the implication of the Arizona workers that, in some way, those people in the upper component of the plasma-glucose distribution represent the real diabetics in that they showed a high frequency of " the specific microangiopathy lesions of diabetes ". The simplest objection to this view is that microvascular changes are related to the degree of glucose intolerance and not
"
"
L., Butterfield, W. J. H., Keen H. Proc.
R. Soc. Med.
1964, 57, 193. 6.
Rost, G., Honigmann, G., Schliack, V. Z.
ges. inn. Med.
289.
7.
"
blood-sugar behaviour of the young Pima be foretold, perhaps by the response to provocative tests ? The answers these and to many other questions will be awaited with interest. Department of Medicine, H. KEEN Guy’s Hospital Medical School, R. J. JARETT. London SE1 9RT.
to
"
that one would therefore expect more abnormality among those with the higher blood-sugars. Furthermore, those with the higher levels of blood-sugar have probably been hyperglycaemic longer than those with the lower levels, so that their retinopathy and nephropathy may be more an indication of longer duration of exposure to high bloodsugar levels. Some support for this alternative explanation is to be gained from data from the Bedford Study (table 11). In a population without evidence of blood-sugar bimodality, not only was retinopathy almost confined to the more hyperglycaemic individuals at diagnosis, but it also appeared and progressed in this group at a faster rate over the next 5 years. Whether blood-sugar levels are continuously or discontinuously distributed in the population, it is possible that a threshold effect operates, so that a minimal degree of glucose intolerance and, perhaps, duration of exposure to hyperglycsemia is necessary for the genesis of retinopathy. The traditional view that retinopathy is a consequence of 5. Sharp, C.
hyperglycaania, rather than a marker of some microangiopathic process of which hyperglycaemia is only a part, is strongly supported by the induction of morphologically indistinguishable changes in animals rendered diabetic experimentally.8 Clearly, the further study of the Pima tribe is going to provide much new and valuable information. Will therapeutic lowering of blood-sugar confer protection against the microvascular complications " ? What of the state of the larger arteries, in particular the coronary vessels ? Can the more hyperglycaemic component of the population be distinguished from the less hyperglycaemic by characteristics other than the blood-sugar ? Can the future
Hornabrook, R. W., Moir, D. J. Lancet, 1970, ii, 1175.
1966, 21,
SYNTHETIC DIETS IN RENAL FAILURE SIR,-It is clear from the work of Dr. Richards and his colleagues (July 17, p. 128) that it may soon become possible to limit the nitrogen intake of patients with renal of a synthetic protein substitute. One of this approach has received aspect very important insufficient emphasis in their otherwise excellent paper. They point out that vitamin supplements will be required, but mention only vitamin B-complex and vitamin C in their methods. These would be inadequate in the longterm use of a synthetic diet such as they advocate. Wide experience of synthetic diets has been gained during the treatment of inborn errors of metabolism and food intolerances in childhood 9; the importance of vitamin supplements for the health and wellbeing of these patients has been repeatedly emphasised.lo,l1 Infants and children have more urgent nutritional needs than the adult, but the dramatic effects of nutritional deficiency which have been described in children serve as a reminder of the potential dangers in the use of synthetic diets. We should therefore like to draw attention to a vitamin preparation which, in spite of its usefulness," is not widely known. It supplies all the essential vitamins, and is the only convenient preparation available for this purpose. The product is marketed under the name of Ketovite Tablets/Ketovite (Supplement) Liquid’ by Paines and Byrnes. These must be taken in combination, and 5 ml. of liquid and 3 tablets a day will supply the total vitamin failure
by the
use
Bloodworth, J. M. B. in Blood Vessel Disease in Diabetes Mellitus (edited by K. Lundbaek and H. Keen). Milan (in the press). 9. Francis, D. E. M., Dixon, D. J. W. Diets for Sick Children; p. 219. Oxford, 1970. 10. M.R.C. Conference on Phenylketonuria. Br. med. J. 1963, i, 1691. 11. Mann, P. T., Wilson, K. M., Clayton, B. E. Archs Dis. Childh. 1965, 40, 364. 8.