DEPRESSED LYMPHOCYTE FUNCTION AFTER BEREAVEMENT

834 illness for which no other form of therapy seems to offer the prospect of halting or delaying progressive liver damage, treatment with D-penicillamine, preferably within controlled trials, is recommended despite the high incidence of toxicity. A lower dose (600 mg daily) is now being used and may reduce the frequency of side-effects. an

We thank Dr R. Kohn of Advisory Services (Clinical and General) London for helpful cooperation and financial support; Dr G. Kristen of Homburg, Frankfurt and Prof. W. Braasch of Bayer Leverkusen for supplies of synthetic D-penicillamine ; Dr I. Sutherland of the Medical Research Council Statistics Research Unit for statistical advice; Mtss M. Tomkin for help with drug administration; Mr S. Newman for performing the liver-copper estimations; and the Medical Research Council for a grant to S. J.

Requests for reprints should

be addressed

to

S. J., Medical Unit,

Royal Free Hospital, Pond Street, London NW3 2QG. REFERENCES 1. Rubin, E., Schaffner, F., Popper, H. Am. J. Path. 1965, 46, 386. 2. Scheuer, P. J., Liver Biopsy Interpretation; p. 33. London, 1973. 3. Fox, R. A., Scheuer, P. J., Sherlock, S. Gut, 1973, 14, 444. 4. Sherlock, S., Diseases of the Liver and Biliary System; p. 268. Oxford, 1975. 5. Heathcote, J., Ross, A., Sherlock, S. Gastroenterology, 1976, 70, 656. 6. Walshe, J. M. Am.J. Med. 1956, 21, 487. 7. Stern, R. B., Wilkinson, S. P., Howarth, P. J. N., Williams, R., Gut, 1977,

18, 19. Hunt, A. H., Parr, R. M., Taylor, D. M., Trott, N. G. Br. med. J. 1963, ii, 1498. 9. Cartwright, G. E., Wintrobe, M. M. Am. J. clin. Nutr. 1964, 14, 224. 10. Scheinberg, I. H., Sternlieb, I. Gastroenterology, 1959, 37, 550. 11. Deshmukh, K., Nimni, M. E.J. biol. Chem. 1969, 244, 1787. 12. Francis, M. J. O., Mowat, A. G. Postgrad. med. J. 1974, 50, suppl. 2, p. 30. 8.

DEPRESSED LYMPHOCYTE FUNCTION AFTER BEREAVEMENT R. W. BARTROP L. LAZARUS

E. LUCKHURST L. G. KILOH R. PENNY

Department of Immunology and Garvan Institute for Medical Research, St. Vincent’s Hospital; Department of Medicine and School of Psychiatry, University of New South Wales; and New South Wales Institute of Psychiatry, Sydney, Australia bereaved detailed prospective investigation of the effects of severe stress on the immune system. T and B cell numbers and function, and hormone concentrations were studied approximately 2 weeks after bereavement and 6 weeks thereafter. The response to phytohæmagglutinin was significantly depressed in the bereaved group on the second occasion, as was the response to concanavalin A at 6 weeks. There was no difference in T and B cell numbers, protein concentrations, the presence of autoantibodies and delayed hypersensitivity, and in cortisol, prolactin, growth hormone, and thyroid hormone assays between the bereaved group and the controls. This is the first time severe psychological stress has been shown to produce a measurable abnormality in immune function which is not obviously caused by hormonal changes.

Summary

During

1975

twenty-six

spouses took part in

a

INTRODUCTION

CELL and tissue changes are known to be part of a non-specific response to stressful stimuli. Stressful physical stimuli in rodents increased their susceptibility to infection.2 These findings implied modification of the immune response, and were attributed to the effects of

Brunner, G., Perings, E., Creutzfeldt, W. in Collagen Metabolism in the Liver (edited by H. Popper and K. Becker); p. 191. New York, 1973. 14. Sherlock, S., Scheuer, P. J. New Engl.J. Med. 1973, 289, 574. 15. Edwards, K. D. G. Med. J. Aust. 1965, ii, 925. 16. Hadziyannis, S., Scheuer, P. J., Feizi, T., Naccarato, R., Domach, D., Sherlock, S.J. clin. Path. 1970, 23, 95. 17. Todd, A. P., Thorpe, M. E. C., Rosenoer, V. M. ibid. 1967, 20, 276. 18. Smallwood, R. A., Williams, H. A., Rosenoer, V. M., Sherlock, S. Lancet, 1968, ii, 1310. 19. Sternlieb, I. Progress in Liver Diseases (edited by H. Popper and F. Schaffner); vol. iv, p. 511. New York, 1972. 20. Chuttani, H. K., Gupta, P. S., Gulati, S., Gupta, D. N. Am. J. Med. 1965, 39, 849. 21. Dickson, E. R. Unpublished. 22. Walshe, J. M. Q.J. Med. 1973, 42, 441. 23. Multicentre Trial Group. Lancet, 1973, i, 275. 24. Day, A. T., Golding, J. R. Postgrad. med.J. 1974, 50, suppl. 2, p. 71. 25. Dixon, A., Davies, J., Dormandy, T. L., Hamilton, E. B. D., Holt, P. J. L., Mason, R. M., Thompson, M., Weber, J. C. P., Zutshi, D. W. Ann. Rheum. Dis. 1975, 34, 416. 26. Gibbs, K., Walshe, J. M. Q.J. Med. 1971, 40, 275. 13.

27. Scheinberg, I. H. Postgrad. med. J. 1968, 44, suppl. p. 11. 28. Walshe, J. M. ibid. 1968, 44, suppl. p. 6. 29. Crawhall, J. C., Watts, R. W. E. ibid. 1968, 44, suppl. p. 8. 30. Henkin, R. I., Keiser, H. R., Jaffe, I. A., Sternlieb, I., Scheinberg, cet, 1967, ii, 1268. 31. Long, R., Scheuer, P. J., Sherlock, S. Gut, 1976, 17, 390.

I. H. Lan-

ADDENDUM

One of the patients who entered the trial after this report had been prepared received D-penicillamine and developed erythromyeloid aplasia with septicaemia. She survived after intravenous antibiotic treatment and infusions of white blood-cell concentrates.

adrenal corticosteroids. Recent work extending these studies to other species has implicated the action of other hormones, possibly mediated via lymphocyte

receptors.4 The experiments

in the NASA Skylab Programme,’ which demonstrated depression of lymphocyte transformation and rosette formation on the day of splashdown, appear to be the only prospective studies of the effects of stress on the immune system of healthy people. Retrospective investigations of bereavement and other severely stressful situations have been claimed to show an association between stress and many diseases,6 including diabetes mellitus, coronary-artery disease, ulcerative colitis, rheumatoid arthritis, lupus erythematosus, and schizophrenia. Claims for an increased mortality after conjugal bereavement are controversial. Bereavement is a life event resulting in great distress or the need for considerable adaptation.’ We determined prospectively the behavioural, endocrinological, and immunological consequences of bereavement.II METHODS

Subjects.-Arrangements were made in our group of hospitals for one of us (R. B.) to provide a counselling service for the surviving spouses of patients either fatally injured or who had died from illness. Twenty-six people between the ages of 20 and 65 years9 were interviewed for the study. Control group.-This group consisted of twenty-six hospital staff members (not bereaved within the previous 24 months) who were matched for age, sex, and race with a bereaved spouse. Design of study.- The service and the study were explained to the spouse on the first visit. Further contact was maintained with all families either directly or through ministers of religion or social workers. The first blood-samples were taken 1-3 weeks after bereavement (sample 1) and the second samples

835 obtained 6 weeks later (sample 2). Control subjects had blood-

samples taken

at

the

same

times for identical

laboratory

test-

as

reported elsewhere.12 Statistical analysis

means

of the Wilcoxon rank

ing.

Physical health.-Each individual received a standardised questionnaire and was excluded if there was a history of recent infection, allergic diathesis, or blood dyscrasia. Stimulation with mitogens.-Peripheral-blood mononuclear cells were isolated on ’Ficoll-Hypaque"" and lymphocyte transformation tests were performed as described elsewhere." Response to phytohaemagglutinin (P.H.A.) was assessed at doses of 10, 20, 100, 200 p.g/ml, and to concanavalin A at doses of 1, 5, and 50 Ng/ml. Lymphocyte markers.-E and EAC rosette-formingI lymphocytes were detected by methods previously reported." Serum-protein concentrations.-Serum protein electrophoresis was performed with a Beckman microzone system. Radial immunodiffusion methods were used to measure serum IgG, IgA, IgM, and o-macroglobulin. Autoantibodies.-Sera were tested for antinuclear factor, mitochondrial, and smooth-muscle antibodies by immunofluorescence techniques. The R.A. latex test was performed for

rheumatoid factor.

Delayed skin hypersensitivity.-Twelve bereaved spouses and fourteen control subjects had skin tests with dermatophyton 0, streptokinase-streptodornase, mumps antigen, and purified protein derivative of tuberculin. Hormone assays.-The following were measured by standard radioimmunoassays: thyroxine and triiodothyronine, growth hormone, and cortisol; and prolactin was assayed by a modification of the standard radioimmunoassay with reagents provided by N.I.H. (U.S.A.). Statistical analysis.-Results of tests of cell-mediated immunity were expressed as the geometric mean (± S.E.M.) values

sum test

was

carried

out

bv

(Mann-Whitney).

RESULTS

Lymphocyte transformation test.-The results of the lymphocyte transformation tests on samples 1 and 2 with

and concanavalin A are shown in the accompanying figure. Responses to doses of 10 and 20 .g/ml P.H.A. in sample 2 were strikingly different in the bereaved and control groups (P<0-05). In addition, there was a significant difference between samples 1 and 2 of the spouse group at a dose of 100 pg/ml P.H.A. At 6 weeks (sample 2) responsiveness to doses of 5 and 50 ug/ml P.H.A. At 6 weeks (sample 2) responsiveness to doses of 5 and 50 g/ml of concanavalin A in the bereaved group was significantly less than in the control In addition, the responsiveness of lymgroup (P<0-03). phocytes in samples 1 and 2 of the bereaved group was significantly different at doses of 5 and 50 .g/ml concanavalin A. Other measures of T and B cell function.-There was no significant difference between the bereaved and control groups in terms of T and B cell numbers, serum protein electrophoresis, immunoglobulins, or.2-macroglobulin concentrations, the presence of autoantibodies, and P.H.A.

delayed hypersensitivity. Hormone assays.-Mean serum concentrations of thyroxine, triiodothyronine, cortisol, prolactin, and growth hormone were no different in the bereaved and control groups. DISCUSSION

This is the first prospective study of immunological function in healthy people under great psychological stress. We demonstrated that T-cell function was significantly depressed after bereavement (sample 2) in the absence of a change in T-cell numbers as tested by E rosetting. We have not demonstrated abnormal B-cell function, as measured by IgG, IgA, and IgM concentrations, after bereavement, nor any differences in B-cell numbers between the bereaved and control groups, as tested by the EAC rosetting scheme. Hamburg et al. reported that concentrations of adrenocortical, adrenomedullary, and thyroid hormones were raised for weeks or months at times of great stress and seemed to correlate directly with the degree of distress and inversely with the ability to cope.3 We did not demonstrate any significant difference in hormone concentrations between the two groups in either sample 1 or sample 2. This would tend to exclude these hormones as mediators of the T-cell functional abnormality that we demonstrated. A detailed endocrinological analysis was not attempted in the bereaved group, a single blood-sample being used for hormone assays. An extended study will include a more detailed investigation of hormonal responses rather than isolated blood estimations and this will be particularly pertinent in the case of cortisol, but we also intend to continue investigation of other hormones.

prospectively that produce a measurable psychological in immune function. The origin of the abnormality defect in T-cell function is being investigated and a more extensive analysis of B-cell function is required. This For the first time,

severe

Geometric

mean

values

(±S.E.M.)

for

lymphocyte responsive-

ness to P.H.A. and concanavalin A in control and bereaved

groups.

we

have shown

stress can

836 may give a clue to the genesis of suggested stress-related diseases which have an immunological basis.

Requests for reprints should be addressed to R. W. B., Department of Immunology, St. Vincent’s Hospital, Darlinghurst, N.S.W. 2010, Australia. REFERENCES 1. Selye, H. A. Rev. Med. 1951, 2, 327. 2. Gisler, R. H., Bussard, A. E., Mazie, I. C., Hess, R. Cell Immun. 1971, 2, 3. Hamburg, D. A., Hamburg, B. A., Barchas, J. D. in Emotions: Their Parameters and Measurement (edited by L. Levi); p.232. New York, 1974. 4. Bourne, H. R., Lichtenstein, L. M., Melmon, K. L., Henney, C. S., Wemstein, Y., Shearer, G. M. Science, 1974, 184, 19. 5. Kimzey, S. L. Acta Astronautica, 1974, 127, I. 6. Solomon, G. F., Amkraut, A. A. Front. Radiat. Ther. Onc. 1972, 7, 84. 7. Tennant, C., Andrews, G. Aust. N.Z.J. Psychiat. 1976, 10, 27. 8. Amkraut, A. A., Solomon, G. F. Int.J. Psychiat. Med. 1974, 5, 541. 9. Foad, B. S. I., Adams, L. E., Yamauchi, Y., Litwin, A. Clin.exp. Immun.

10. 11.

1974, 17, 657. Boyum, A. Scan,J. clin. Lab. Invest. 1968, 21, suppl. 97, p. 51. Cooper, D. A., Petts, V., Luckhurst, E., Biggs, J. C., Penny,

12.

Cancer, 1975, 31, 550. Ziegler, J. B., Hansen, P., Penny,

R. Clin. Immun.

R.

Br. J.

Immunopath. 1975, 3,

Fig. 1--Complete system, comprising colorimeter, cuvette, swizzlestick, capillary tubes, plasticine, cyanmethemoglobin solution, pipette, and disposable blood-lancet.

451.

inexpensive, robust, accurate, and reliable in humidity, and are independent of an external electricity supply. We report a prototype system and its assessment in the tropical rain forest of Ecuador. glassware,

Methods and Devices SIMPLE METHOD FOR MEASURING HÆMOGLOBIN

Evaluation in Andean Rain Forest

extremes

are

of heat and

THE

The system consists of

C. A. METCALF J. M. RIDEOUT

Clinical Research Centre, Northwick Park Hospital, Harrow, Middlesex HA13UJ, and Renal Isolation Unit, Royal

Infirmary of Edinburgh THE chemical analysis of body fluids, such as blood and not available in rural areas in developing countries because the laboratory equipment which has been designed for use by skilled technicians in developed countries is unsuitable.1 A research programme was initiated by the Medical Research Council to develop analytical systems appropriate for rural requirements which avoid the use of conventional laboratory

urine, is

a new

blood-sampling

and

diluting

device, known as the "swizzlestick technique", modified Draband a coloriThe swizzlestick consists of a handle fastened to a small stainless steel circular platform to which is also fastened a stainless steel displacement probe having a diameter slightly less than the bore of a capillary tube and a volume equal to the volume of blood required for the assay. A capillary tube is filled with blood from a fingerprick, one end is sealed with plasticine, and the open end is placed over the measuring probe of the swizzlestick and gently pressed down to the platform, which collects the exact volume (10 pi) of blood required. The whole swizzlestick is then placed inside a cuvette containing modified Drabkins reagent (fig. 2), swizzled to mix, and withdrawn. The cuvette is then placed in the haemogtobi-

kins reagent

K. G. NICHOLSON A. RENSHAW

EQUIPMENT

meter

(cyanmethmmoglobin solution),2

(fig. 1).

nometer.

The prototype colorimeter

uses

pulsed light-emitting diodes

Fig. 2-The swizzlestick technique of microsampling and diluting. (a) Blood being collected; (b) sealing of one end of the capillary tube; (c) the capillary tube being placed over the displacement probe of Ihe swizzlestick; (d) displaced blood on the swizzlestick platform; and (e) transfer to cuvette containing cyanmethasmoglobm solution.