627

the severity and duration of adverse circumstances.The long-term effects of such experiences depend towhat extent the harmony of growth 11 is disturbed.My data on obese children show that overnutrition

in the early months of life causes not only an increasein linear growth, but also an equivalent advance inskeletal maturation.12 Thus the height of obesechildren is normal for their bone-age, and the prog-nosis for height is also normal." Overnutrition laterin childhood has no demonstrable effects on heightor skeletal maturation.12 The adipose organ is onlyaffected by overnutrition in the early period, but theincrease in the number of cells in this event is muchgreater than the increase in height or skeletal maturity;thus the harmony is destroyed and these individualswill have a permanently increased complement ofadipose cells.

Since these findings do not apply to obesity datingfrom later childhood, it seems likely that the first yearof life is a sensitive one for the formation of adiposecells. During such a period, cell replication is sensitiveto external influences which may be beneficial (perhapsessential) or pathological. The period is also one ofdetermination, because at its end the basic complementof cells is established and thereafter cell replicationprobably continues at a normal rate regardless of othercircumstances. As far as the adipose organ is con-cerned, it seems that after the end of the sensitive

period alterations in nutrition affect only cell size andnot cell number.

From studies in animals of experimental over-

nutrition 14 and undernutrition, 15 it is known that

during certain periods it is more easy to impair growthand that, in general, the earlier the stress, the moresevere the effects. 16 This work has been confirmedand extended by the demonstration of the importanceof early nutritional experiences on the size, number,and rate of growth of adipose cells in rats.’-’,18The effects of intrauterine malnutrition (L.F.D.children) in the present study demonstrate howadverse circumstances early in organogenesis haveprofound effects later, and how important timing isin growth and development.

If G.H. is important for adipose-cell replication (asthe present data indicate), an intermediate situationbetween controls and L.F.D. would be predicted forchildren with isolated G.H. deficiency. Such childrengrow normally in utero and are presumably born witha normal complement of cells. These cells do notreceive a proper stimulus for replication in the sensitiveperiod and so they lag behind the rest of developmentas measured by skeletal maturity, but not as much asthey would have done if the stress had occurred earlier.Children who lack G.H. at a later age, however, wouldbe expected to have passed the age when the adiposeorgan is sensitive, and so harmony should not havebeen destroyed. Such children would be expected tohave a number of adipose cells which does not differfrom normal when the delay in growth induced byG.H. deficiency, and evidenced by skeletal retardation,is allowed for. The findings on the C.R. childrenconfirm this expectation.

I suggest that the adipose organ in man has a finiteand well-defined sensitive period during which external

circumstances have long-term effects on cellular

growth. For this organ the period of determination,when the basic complement of cells is established,probably extends from thirty weeks of intrauterinegrowth through to the first nine months to a year ofextrauterine growth when the skinfolds cease their

rapid increase. Presumably such a model, with differ-ent timings, applies to all organs in man.

I thank the Wellcome Trust and the Medical Research Councilfor financial assistance, and Prof. J. M. Tanner, whose unfailingadvice and support have been of great help.

REFERENCES

1. Winick, M. Med. Clins N. Am. 1970, 54, 1413.2. Cheek, D. B., Hill, D. E., Cordano, A., Graham, G. G. Pediat.

Res. 1970, 4, 135.3. Brook, C. G. D. Archs Dis. Childh. 1971, 46, 182.4. Tanner, J. M., Whitehouse, R. H. Br. med. J. 1962, i, 446.5. Brook, C. G. D., Lloyd, J. K., Wolff, O. H. ibid. 1972, ii, 25.6. Tanner, J. M., Thomson, A. M. Archs Dis. Childh. 1970, 45, 566.7. Edwards, M. W., Hammond, W. H., Healy, M. J. R., Tanner,

J. M., Whitehouse, R. H. Br. J. Nutr. 1955, 9, 133.8. Durmin, J. V. G. A., Rahaman, M. M. ibid. 1967, 21, 681.9. Tanner, J. M., Whitehouse, R. H., Healy, M. J. R. A New System

for Estimating Skeletal Maturity from the Hand and Wrist.Centre Internationale de L’Enfance, Paris, 1962.

10. Hirsch, J., Gallian, E. J. Lipid Res. 1968, 9, 110.11. Widdowson, E. M. Lancet, 1970, i, 901.12. Brook, C. G. D. M.D. thesis, University of Cambridge, 1972.13. Lloyd, J. K., Wolff, O. H., Whelen, W. S. Br. med. J. 1961, ii, 145.14. Widdowson, E. M., McCance, R. A. Proc. R. Soc. 1960, B, 152, 188.15. Widdowson, E. M., McCance, R. A. ibid. 1963, B, 158, 329.16. Winick, M., Rosso, P. Pediat. Res. 1969, 3, 181.17. Knittle, J. L., Hirsch, J. J. clin. Invest. 1968, 47, 2091.18. Hirsch, J., Han, P. W. J. Lipid Res. 1969, 10, 77.

MACROPHAGE-ELECTROPHORETIC-MOBILITY (M.E.M.) TEST FOR MALIGNANT

DISEASE

An Independent Confirmation

J. A. V. PRITCHARDW. H. SUTHERLAND

J. L. MOOREC. A. F. JOSLIN

Tenovus Laboratories, Velindre Hospital,Whitchurch, Cardiff

Summary The validity of a new in-vitro blood-test for cancer, which depends on the

sensitisation of the patient’s lymphocytes to a commonantigen apparently present in human tumours, hasbeen independently confirmed.

Introduction

Field and Caspary reported that lymphocytesfrom patients with malignant disease can be stimulatedby encephalitogenic factor 2 (E.F.) (a basic proteinderived from the human brain) to release a macro-phage-slowing factor (M.S.F.) which reduces the electro-phoretic mobility of guineapig macrophages. Withfew exceptions, the effect was not found with

lymphocytes from non-malignant controls, and there-fore the technique was presented as a possible in-vitroblood-test for cancer. The results could best be

explained on the basis of a common antigen present inhuman tumours. These unexpected findings requiredconfirmation, and we performed a similar investigation.

Materials and Methods

Approximately 15 ml. of venous blood was collectedfrom healthy hospital staff and from patients with known

628

TABLE I-TYPICAL RECORD OF TIMINGS FROM TWO SAMPLES

* This represents a 2-7% decrease in mean migration time.t This represents a 20-6% increase in mean migration time.

malignant disease. After defibrination, lymphocytes wereharvested either by the methylcellulose technique de-scribed by Hughes and Caspary 3 or by means of a ’ Ficol-Triosil ’ technique which will be described fully elsewhere.The washed lymphocytes from either technique were

resuspended in ’Medium 199’. Macrophages were

prepared from the peritoneal exudate of 400-600 g. Hartleyalbino guineapigs four to ten days after the injection of20 ml. of sterile liquid paraffin into the peritoneum. Thecells were washed and resuspended in medium 199 to

a concentration of 10’ cells per ml. and then irradiatedwith a dose of 100-200 rad from a cxsium-137 source.The encephalitogenic factor (E.F.) was prepared from ahuman brain by the methods described by Caspary andField.2,4 1-0 mg. of E.F. was dissolved in medium 199 sothat the final concentration was 100 (kg. per 0-1 ml.

Electrophoretic MeasurementsSamples containing 1.0 ml. of medium 199, 0-1 ml. of

antigen in medium 199, 1-0 ml. of the lymphocyte pre-paration, and 1.0 ml. of the macrophage suspension wereincubated at room temperature for at least ninety minutesbefore the electrophoretic mobility of the macrophageswas measured in a Zeiss ’Cytopherometer’. A duplicatesuspension without E.F. was prepared as a control from eachsample. Samples were scrambled and measured in a randomorder, except when scrambling was precluded by otherinvestigations proceeding on the same sample. Cells infocus in the stationary layer were timed over one divisionof the eyepiece graticule (16 m.) for both directions ofcurrent, and the pairs of times recorded if they did notdiffer by more than 10%. Results were tabulated in two

columns, one containing all values close to the controltime and the other containing times appreciably longerthan the control. Measurements on a sample were stoppedas soon as either column contained 10 pairs of timings, andthese were averaged to give the mean migration time for

that sample. Typical timings from two samples, one" control " and one " malignant ", are shown in table I.

The frequency of " wrong " pairs decreased with increasingexperience of the technique and apparatus, until onlyabout 1 cell in every 100 from control samples gave a" wrong " pair of timings. Results were expressed as apercentage change in mobility, 100 (T—c)/c, where T is theaverage migration time for macrophages in the test samplecontaining lymphocytes, macrophages, and antigen, andc is the average migration time for macrophages in the con-trol sample containing lymphocytes and macrophages butno antigen.

Results

Between May, 1971, and July, 1972, 309 blood-samples from patients and from healthy subjects wereexamined with the macrophage-electrophoretic-mobil-

TABLE II-PERCENTAGE SLOWING OF MACROPHAGES FROM SUBJECTSWITH MALIGNANT DISEASE AGAINST E.F. ANTIGEN

* Methylcellulose technique used (otherwise ficol-triosil).t Lymphocytes stored overnight at 4°C.t Lymphocytes stored for 72 hours at 4°C.

§ Measurements in modified WHS-designed apparatus.s.C.C. = Squamous-cell carcinoma.B.C.C. = Basal-cell carcinoma.

629

TABLE III-PERCENTAGE SLOWING OF MACROPHAGES FROM NORMAL

CONTROLS AGAINST E.F. ANTIGEN

t, § As in table II. Subject 54 had a history of sarcoidosis.

ity (M.E.M.) test. In the first 230 samples someslowing was observed in 70% of samples from subjectswith malignant disease, but we did not immediatelyachieve the consistent results claimed by Field andCaspary. After a detailed re-evaluation of all stepsin our experimental procedures, we now know thatduring the period that these 230 samples were testedseveral adverse factors were operating. These factorswill be described elsewhere, together with a fullaccount of our experimental and measurement tech-niques and an analysis of the frequency and possiblesignificance of the wrong pairs of timings.

After the removal of these adverse factors in May,1972, 79 consecutive samples were measured, 52 fromcases of known malignant disease (table 11) and 27from non-malignant controls (table ill). The resultsfrom these 79 samples are shown in tables 11 and ill

together with age, sex, and diagnosis. Samples froma few subjects were measured a second time; this isindicated by the figures in parentheses beside thesubject number.

Discussion

We confirmed the results of Field and Caspary 1,4and our results underline the potential of the M.E.M.test for indicating the presence of malignant disease.Lymphocytes from patients with early or advanceddisease were equally effective in producing M.S.F.

Tables u and ill confirm the unexpectedly wide gapbetween malignant and normal samples reported byField and Caspary. In our series, all samples frompatients with known malignant conditions gave macro-phage slowing greater than 13%, while all control

subjects were below 3-5%, with one exception. A

sample from subject 54, who was thought to be

normal, gave an unusually high result in an initialand a repeat test. Further investigation revealed ahistory of sarcoidosis, a condition which Caspary andField reported as giving a positive result in the.B!.LB1. test. The remainder of our controls were

healthy hospital staff. We have now extended the

investigation to include controls with various non-

malignant illnesses. We have not detected thesensitisation of laboratory staff to E.F., which was animportant feature of the normal results quoted byField and Caspary,l perhaps because we have not yethad sufficient exposure to E.F.

For most of the measurements in tables 11 and III,between 1 x 106 and 5 x 106 lymphocytes per ml. wereused. Except for the first 3 samples, these wereprepared by the ficol-triosil technique, which alwaysproduced very good yields with minimum contamina-tion by polymorphs and red blood-cells, and requiredonly sixty minutes of preparation time per sample incontrast to the hundred and thirty minutes requiredby the methylcellulose technique used by Field andCaspary. The percentage slowing is certainly depen-dent on lymphocyte numbers, but above 106 lympho-cytes per ml. the effect approaches a maximum.We are now attempting, already with some success,

to simplify and improve the M.E.M. test as a clinicaltechnique and an important tool in experimentalcancer research.

We thank our radiotherapist colleagues for their advice anddiscussion; especially Prof. E. J. Field and E. A. Caspary of theMedical Research Council Unit for Demyelinating Diseases,Newcastle upon Tyne; and the staff of the Tenovus Laboratoryand the animal house. This project was financed by Tenovus,the South Wales and Monmouthshire Cancer Research Council,and the Department of Health and Social Security.

Requests for reprints should be addressed to J. A. V. P.

REFERENCES

1. Field, E. J., Caspary, E. A. Lancet, 1970, ii, 1337.2. Caspary, E. A., Field, E. J. Ann. N.Y. Acad. Sci. 1965, 122, 182.3. Hughes, D., Caspary, E. A. Int. Archs Allergy appl. Immun. 1970,

37, 506.4. Caspary, E. A., Field, E. J. Br. med. J. 1971, ii, 613.5. Caspary, E. A., Field, E. J. ibid. p. 143.

SECOND MALIGNANT CLONE

UNDERLYING A BURKITT-TUMOUR

EXACERBATION

P. J. FIALKOW

Departments of Medicine and Genetics,University of Washington, Seattle, Washington, U.S.A.

G. KLEIN

Department of Tumour Biology,Karolinska Institutet, Stockholm, Sweden

P. CLIFFORD *

Department of Head and Neck Surgery,Kenyatta National Hospital, Nairobi, Kenya

Summary A 6-year-old girl with Burkitt

lymphoma had a chemotherapeuticallyinduced " complete " remission, followed by exacer-bation of disease, first in the left parotid gland (a siteclose to one previously affected by tumour) and thenin the left orbit (a previously uninvolved site). Hernormal tissues were typed as A—B for glucose-6-phosphate dehydrogenase (G.-6-P.D.). An ovariantumour studied before therapy and a recurrent-

* Present address: Royal Marsden Hospital, London S.W.3.