1226 We thank the research nursing staff for helping in the management of the patients and the local medical practitioners for their referrals. This work was supported by a grant from the World Health Organisa-

CLINICAL CHARACTERISTICS AND DIAGNOSED INFECTIONS IN MALNOURISHED CHILDREN TESTED WITH LOCAL ZINC

tion.

REFERENCES

Karim, S. M. M. Lancet, 1973, n, 794. Csapo, A. I., Mocsary, P. ibid. 1974, ii, 789. Stringer, J., Anderson, M., Beard, R. W., Fairweather, D. V. I., Steele, S. J. Br. med. J. 1975, iii, 7. 4. Goldthorp, W. O. ibid. 1977, ii, 562. 5. MacKenzie, I. Z., Hillier, K., Embrey, M. P. ibid. 1975, i, 240. 6. Hull, M. G. R., Gordon, C., Beard, R. W. J. Obstet. Gynœc. Br. Commonw. 1974, 81, 577. 7. Stallworthy, J. A., Moolgaoker, A. S., Walsh, J. J. Lancet, 1971, ii, 1245. 8. Jones, J. R., Gentle, G. P., Kemmann, E. K., Soriero, A. A. Prostaglandins, 1975, 9, 881. 9. Mocsary, P., Csapo, A. O. in Advances in Prostaglandin and Thromboxane Research (edited by B. Samuelsson and R. Paoletti), p. 975 New York, 1. 2. 3.

1976. 10. Ylikorkala,

O., Kirkinen, P., Jouppila, P., Jarvinen, 1975, 10, 333.

P.

A., Prostaglandins,

*M=marasmus, K=kwashiorkor, ing to the Wellcome criteria."I

M-K=marasmic

kwashiorkor, accord-

The present study was done to see whether topical zinc sulphate influenced the cutaneous manifestations of cell-

mediated immunity. ZINC AND IMMUNOCOMPETENCE IN PROTEIN-ENERGY MALNUTRITION

MICHAEL H. N. GOLDEN* P. S. E. G. HARLAND

BARBARA E. GOLDEN ALAN A. JACKSON

Tropical Metabolism Research Unit and Department of Child Health, University of the West Indies, Kingston 7, Jamaica, West Indies

Malnourished children have thymic atrophy which is reversed by zinc supplementation. To see if their defect in cell-mediated immunity was also associated with zinc deficiency ten children were skin-tested with Candida antigen on both arms. One test site was covered with local zinc sulphate and the other with placebo ointment. There was a highly significant increase in the typical delayed-hypersensitivity reaction at the site covered with zinc. The magnitude of the difference between the supplemented and unsupplemented arms correlated negatively with the plasma-zinc concentration. These data show that zinc deficiency is a cause of the immunoincompetence seen in malnutrition. The normal reactions of the zinc-supplemented side indicate that, of the many nutritional deficits of malnourished children, zinc deficiency specifically impairs the cell-mediated immune system. Local skintesting with and without zinc may provide a measure of zinc status. Local application of zinc may enhance the reliability of tests to diagnose diseases such as tuberculosis in malnourished patients.

Summary

Patients and Methods Ten children with P.E.M." were studied within 2 days of admission (see table). 1% zinc sulphate was added to one of two identical batches of emulsifying ointment, and the zinc and placebo ointment were then placed in pairs of identical containers according to a randomised code known only to .M. H.N.G. 0-11 trial of 05% Candida antigen (Bencard) was inintradermally into each forearm, and the ointments were each applied to one forearm according to the code and covered with ’Micropore’ tape. Ointment from the same coded jars was reapplied three times daily. After 48 hours the reactions were examined blindly by B.E.G. She outlined the indurated area with a ballpoint pen. The outline was transferred to paper by contact, cut out, and weighed. Plasma-zinc was measured on the trichloroacetic-acid supernatant by atomicabsorption spectrophotometry (Pye Unicam SP 2900). The results were analysed by the paired t test.

jected

Results The reactions

typical delayed-hypersensitivity signs of chemical irritation or immediate-hypersensitivity responses. The sites covered with zinc sulphate showed significantly larger reactions than those covered by carrier alone (P10 mm) reaction occurred on the side supplemented with zinc. This shows that the children had been previously sensitised to Candida and were capable of a typical delayed-hypersensitivity response provided they had enough zinc. These children had major deficits of many other nutrients when they were tested. The normal responses achieved suggest that the other deficits were relatively unimportant in causing the depression of C.M.I. There was little difference between the reaction on the two sides in several of the children. This suggests that the zinc was not acting as a non-specific adjuvant to enhance all the responses or to alter the antigenicity of the antigen. The diminished response on the unsupplemented arm was probably due to zinc deficiency. This is supported by the negative correlation between the plasma-zinc concentration and the enhancement of the delayed-hypersensitivity response by topical zinc sul-

phate. Although

we have found a correlation between plasma-zinc and cutaneous c.M.I., plasma-zinc concentration is not accepted as a good measure of zinc status." The list of diseases associated with a labile plasma-zinc concentration is steadily growing;l3 and patients with diseases responsive to zinc therapy may have normal plasma-zinc concentrations.14 Even tissue-zinc levels are not helpful, since they do not change substantially in experimental zinc deficiency." The depression of the cutaneous C.M.I. response and the extent of its recovery with local zinc may provide a test of zinc status which has functional significance. Many illnesses are associated with both a fall in plas-

ma-zinc concentration" and depression of c.M.I., ’s-17 which may be related. Cavdar et al. 18 have reported that the impaired immune status in Hodgkin’s disease correlates negatively with the serum-zinc concentration. In-vitro studies of phytohxmagglutinin-stimulated lymphoctye transformation in malnourished children have given variable results, depending on the culture conditions. With autologous serum19 there is depressed transformation, whereas with whole blood 20 or homologous serum21 lymphocytes from malnourished children show normal thymidine uptake. This indicates that the serum of malnourished children either contains a factor which suppresses transformation or lacks an essential nutrient. The latter may be zinc, for low levels are found in malnutrition,7-9 and zinc is essential for lymphocyte transformation, in-vitro.22-25 Transformation which usually requires the presence of serum, can be accomplished provided that zinc transferrin is present in the culture medium. 26 The importance of zinc in maintaining normal c.M.I. is emphasised by the severe immunodeficiency found in animals with experimental zinc deficiencyl·2 and in cattle3 and children4.5 with hereditary defects of zinc metabolism. The tuberculin test in malnourished patients gives a high proportion of false-negative responses.27.28 It is possible that local application of zinc with the antigen could increase discrimination by the test in malnourished children. We have shown that zinc deficiency may be a cause of depressed cutaneous responses in P.E.M. and suggest that an adequate supply of available dietary zinc is likely to be important in maintaining resistance to the frequent infections which may precipitate P.E.M. and which are associated with established P.E.M. We are grateful to the nursing staff of the T.M.R.U.; to Mr G. I. Forbes, of the U.W.I. pharmacy, for formulating the ointments; to Mrs L. Charley for technical assistance; and to Dr H. Mawhinney for

advice. We also thank the Research Corporation of New York for a grant, The Wellcome Trust for personal support for M. H. N. G., and the Medical Research Council for the atomic-absorption spectro-

photometer. Requests for reprints should be addressed to M. H. N. G. REFERENCES 1. Fraker, P. J., Haas, S. M., Luecke, R. W. J. Nutr. 1977, 107, 1889. 2. Pekarek, R. S., Hoagland, A. M., Powander, M. C. Nutr. Rep. int. 1977, 16, 267. 3. Kroneman, J., Van d. Mey, G. J. W., Helder, A. Zentbl. Vetmed. A. 1975,

22, 201. 4. Neldner, K. H., Hambidge, K. M. New Engl. J. Med. 1975, 292, 879. 5. Campo, A. G., McDonald, C. J. Archs Derm. 1976, 112, 687. 6. Edelman, R. in Malnutrition and the Immune Response (edited by R. M. Suskind); p. 47. New York, 1977. 7. Warren, P. J., Hansen, J. D. L., Lenman, B. H. Proc. nutr. Soc. 1969, 28, 6A. 8. Cheek, D. B., Hill, D. E., Cordano, A., Graham, G. G. Pediat. Res. 1970,

4, 135. Khalil, M., Kabiel, A., E1-Khateeb, S., Aref, K., El-Lozy, M., Jahin, S., Nasr, F. Am. J. clin. Nutr. 1974, 27, 260. 10. Golden, M. H. N., Jackson, A. A., Golden, B. E. Lancet, 1977, ii, 1057. 11. ibid. 1970, ii, 302. 12. Keen, C. L., Hurley, L. S. Am. J. clin. Nutr. 1977, 30, 528. 13. Halsted, J. A., Smith, J. C., Irwin, M. I. J. Nutr. 1974, 104, 345. 14. Garretts, M., Molokhia, M. J. Pediat. 1977, 91, 492. 15. Mitchell, A. G., Nelson, W. E., LeBlanc, T. J. Am. J. Dis. Child. 1935, 49, 9.

695. 16. Westwater, J. S. Q. Jl. Med. 1935, 4, 203. 17. Grossman, J., Baum, J., Gluckman, J., Fusner, J., Condemni, J. J. J. Allergy clin. Immun. 1975, 55, 268. 18. Cavdar, A. O., Babacan, E., Arcasoy, A. Pediat. Res. 1978, 12, 64. 19. Sellmeyer, E., Bhettay, E., Truswell, A. S., Meyers, O. L., Hansen, J. D. L. Archs Dis. Childh. 1972, 47, 429. 20. Moore, D. L., Heyworth, B., Brown, J. Clin. exp. Immun. 1974, 17, 647

1228 ENHANCEMENT OF HUMAN KIDNEY ALLOGRAFTS BY COLD B-LYMPHOCYTE CYTOTOXINS YUICHI IWAKI MIN SIK PARK

PAUL I. TERASAKI RONALD BILLING

Department of Surgery, U.C.L.A. School of Medicine, University of California, Los Angeles, California 90024, U.S.A.

The sera of 233 kidney transplant patients before transplantation were tested by cytotoxicity against a panel of B and T lymphocytes at 5°C and 37°C. The results divided the patients into four groups: those whose sera reacted with B lymphocytes at 5°C only; those reacting with B lymphocytes at 5°C and 37°C; those reacting with T lymphocytes at 37°C; and those with no antibodies. The patients with pre-transplant antibodies reactive with B lymphocytes at 5°C had a significantly higher kidney-transplant survival rate at 6 months (70%) and 1 year (65%) than patients who had no antibodies (47% and 46%, respectively). Patients with antibodies reactive at 37°C had a 6-month survival-rate of 38% when reactive against B cells and 43% when reactive against T lymphocytes. The cold cytotoxins were IgM.

Summary

Introduction ALTHOUGH allograft enhancement in animals is well established,1 its role in human transplantation is unclear. Leucocytes from the kidney donor were injected into 4 graft recipients in 1973 by Newton and Anderson2 but none showed evidence of immunisation. If we use "enhancement" in its broadest sense-i.e., induction of lower responsiveness by pretreatment with allogeneic cells-blood-transfusions may be thought to induce enhancement. Without blood-transfusions and with immunosuppressive treatment, approximately 30% of kidney allografts survive a year after transplantation,3 but this can be improved to 50-70% by pre-transplant transfusion of allogeneic blood. If this is considered to be enhancement, it has been achieved since human kidney transplants began. In the early days of the operation most patients were given numerous transfusions before transplantation,4 and the survival rates of recent years may be lower because transfusions have been less fre-

grafts

at

twenty-seven

transplant

centres

between

ranged from 3 months to 2 years. Methods

Lymphocytotoxicity B and T lymphocytes were isolated and tested by the lymphocyte microcytotoxicity test as previously described.6 A serum was tested against B and T lymphocytes from 17-30 normal individuals. Cells from

one

person

were

as B-warm-reactive. Sera classified as T-warm-reactive were those which killed T lymphocytes from >15% of the panel at 37°C whether or not they had cold or warm reactivity or killed T lymphocytes at 5°C. Fig. 1 shows examples of these three patterns of reaction. It should be noted that there are fifteen different patterns of reaction possible by testing sera against B and T cells at 5°C and 37°C. However, the B-cold, B-warm, and T-warm reactive patterns and the absence of lymphocyte reactivity were the most common.

Serum Fractionation 2 ml of B-cold-reactive serum was fractionated by gel filtration on ’Biogel 5M’. 2 ml fractions were eluted in 0-15 mol/1 sodium chloride, 10 mmol/l phosphate buffer (pH 7.2). The fractions under peaks with optical density 280 nm were pooled, concentrated to 2 ml, and tested for IgM or IgG by Ouchterlony immunodiffusion using goat antibodies against human IgG and IgM. There was a clear separation of the IgG and IgM fractions. Each fraction was tested for cold anti-Bcell activity against at least three different B-lymphocyte preparations at 5 IC and 37°C.

Results Of the 233 pretransplant sera tested, 139 (60%) did not react against T or B lymphocytes at 3°C or 37°C. 94 sera had anti-lymphocyte activity, 40 with the

If the state of enhancement could be detected by invitro tests, patients who were properly "prepared" by transfusions and pregnancies could be identified. We describe here a test which may detect enhancing antibodies.

Patients

patients

were

233

recipients of cadaver kidney allo-

Harland, P. S. E. G. in The Child in the African Environment (edited by R. Owor, V. L. Ongom, and B. G. Kirya); p. 349. Kampala, 1975. 22. Kirchner, H., Rühl, H. Exp. Cell. Res. 1970, 61, 229. 23. Rühl, H., Kirchner, H., Bochert, G. Proc. Soc. exp. Biol. Med. 1971, 137, 21.

1089. 24. Williams, R. O., Loeb, L. A. J. Cell Biol. 1973, 58, 594. 25. Chesters, J. K. Biochem. J. 1975, 150, 211 26. Phillips, J. L., Azari, P. Cell. Immun. 1974, 10, 31. 27. Chandra, R. K. J. Pediat. 1972, 81, 1194. 28. Harland, P. S. E. G. Lancet, 1965, ii, 719.

tested separ-

ately against: B lymphocytes at 5°C; B lymphocytes at 37°C; T lymphocytes at 5°C; and T lymphocytes at 37°C. Incubation with complement in all cultures was at 25 °C. Incubationtimes were 1 h with serum and 2 h with complement. Classification of Sera When a serum killed B lymphocytes from > 15 % of the panel at 5°C and all other reactions were negative, it was classified as B-cold-reactive. If it killed B lymphocytes from > 15 % of the panel at 37°C (whether or not it had B-cold reactivity) and killed T lymphocytes from

Zinc and immunocompetence in protein-energy malnutrition.

1226 We thank the research nursing staff for helping in the management of the patients and the local medical practitioners for their referrals. This w...
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