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Decreased rosette-forming lymphocytes in malnutrition and intrauterine growth retardation
TROPICAL PEDIATRICS
DerrickB. Jelliffe, Edilor
Decreased rosette-forming lymphocytes in malnutrition and intrauterine growth retardation The percentage of peripheral blood lymphocytes which spontaneously form rosettes with sheep red blood cells (thymus-dependent lymphocytes) was studied as a sensitive measure of cellular immunity in patients with two .forms of malnutrition. The percentage and absolute number of rosette-forming cells was markedly decreased in 10 children with severe protein-calorie malnutrition (16.6% +_ Z 7 SE) p ( 0.001, compared to normal children (59. 7% +_ 1.4 SE) and Caucasian adults (64.5% +_ 1.0 SE). Delayed cutaneous hypersensitivity was also diminished, but total lymphocyte counts and in vitro lymphocyte response to phytohemagglutinin were normal Therapy with a high-protein, high-calorie diet resulted in recovery of delayed cutaneous" hypersensitivity and return to normal numbers of rosette-forming cells within 1 to 3 weeks'. The percentage of rosette-forming cells was moderately reduced (49.2% +_ 2.0 SE) p ( 0.001, in 10 newborn infants who were small for gestational age, mean birth weight 1,660gm, compared to those whose weights were appropriate for gestation (65.1% +_ 1.4 SE), mean birth weight ZOO5gin, an observation in keeping with prior studies Qf diminished spleen and thymus size in such infants. The interrelation of cellular immunity and malnutrition is emphasized, since this may be of great importance in the host response to infection and immunization, and in persisting defects Qf cellular immunity in the developing newborn infant.
Alexander C. Ferguson, M . B . , F . R . C . P . ( C ) , D . C . H . , *
Kingston, Ont., C a n a d a ,
Glenn J. Lawlor, Jr., M.D., Charlotte G. Neumann, M.D., M.P.H., William O h , M . D . , a n d E. Richard Stiehm, M . D . , * *
THE ABILITY of a subpopulation of peripheral blood lymphocytes to spontaneously form rosettes with sheep red blood cells, first described by Bach and associates t has been shown to be a marker of thymus-dependent cells.2 Stites and associates 3 found that thymus tissue from 11 to 19 week fetuses contained up to 65% RFC, compared to 0.8% of cells from the bone marrow. Wybran and associates 4 reported diminished numbers of From the Department of Pediatrics, University of CaliJbrnia School o f Medicine. Supported by United States Public Health Service Contract No. HD 72-2069 and Grant No. HD-06463, and National Foundation Grant 6- 74-4. Presented in part at the Annual Meeting, Western Society for Pediatric Research, Carmel, California, February 1974. *Holder ol~fellowshipfrom the Canadian PaediatricSociety's Mead Johnson Educational Fund. **Reprint address: Department of Pediatrics, U.C.L.A. Center .for the Health Sciences, Los Angeles, Calf. 90024.
L o s Angeles, Calif.
R F C in several primary immunodeficiency disorders with diminished cell-mediated immunity. One patient with the Nezelof syndrome had no RFC, and four with the Wiskott-Aldrich syndrome had decreased n u m b e r s (1-1.5%). In contrast, patients with acquired hypogamAbbreviations used RFC: rosette-forming cells SGA: small for gestational age AGA: appropriate for gestationat age PHA: phytohemagglutinin SK: streptokinase SD: streptodornase PPD: purified protein derivative KLH: keyhold limpet hemocyanin DNA: deoxyribonucleic acid m a g l o b u l i n e m i a w i t h o u t cellular i m m u n o d e f i c i e n c y had n o r m a l n u m b e r s of R F C (4-40%). T h e i r assay measured "early" RFC, however, and gave lower normal values than the 80% "late" RFC as reported by others using slightly different experimental conditions?
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Table I. Delayed cutaneous hypersensitivity responses
Protein-calorie malnutrition
Patient
Age (too.)
Manila
SK/SD
J.T. K.A. S.B. A.T. T.M. T.A. G.I.
36 36 36 19 24 12 24
+ 0 0 0 0 0 0
E.E. B.D.
48 39
+ 0
T.Q.
15
Totals Normal control subjects
A.K. A.B. E.A. N.N. N.O. A.S. T.Q. N.A. E.Y. M.F.
27 24 31 23 55 52 43 46 52 35
Totals
PHA
PPD
Diagnosis
+ + + + 0 0 + 0 + + 7/10
0 0 0 0 0 0 0 0 0 0 0/10
K K K M K M M K K K
2/10
0 + 0 0 0 0 0 0 + 0 2/10
+ 0 + + + + + + 0 +
+ 0 0 + + + + + + +
+ + + + + + + + + +
0 0 0 + 0 0 0 + + 0
8/10
8/10
10/10
3/10
0
+ = positive; 0 = negative; K = kwashiorkor; M = marasmus.
Since the RFC assay is rapid, technically uncomplicated, and possibly more sensitive in detecting subtle abnormalities, we wished to determine its value relative to other tests for defective cellular immunity. We therefore studied a group of children with severe proteincalorie m a l n u t r i t i o n from G h a n a , West Africa. The finding of grossly abnormal n u m b e r s of RFC in those patients prompted us to study newborn infants with int r a u t e r i n e growth retardation whose m a l n u t r i t i o n , though less severe, occurs at a critical time in development. METHODS Ten hospitalized Ghanaian children were studied, all of whom had severe protein-calorie malnutrition. Three were marasmic with body weights less than 60% of expected for age. Seven had kwashiorkor based on evidence of edema, skin changes, hair loss, hepatosplenomegaly, and hypoalbuminemia ( < 2 gin/100 ml). Ages ranged from 12 to 39 months. Ten age-matched normal Ghanaian children were control subjects. Twenty Caucasian newborn infants of 28 to 40 weeks gestational age were studied; ten of them were SGA, m e a n b i r t h w e i g h t 1,660 gm ( r a n g e 810-2,240); the
weights of ten were AGA, mean birthweight 2,005 gm (range 1,780-2,800), classified according to the criteria of Dubowitz and associates. 6 The SGA infants had no stigmata of genetic or developmental abnormalities, or of infection, but were associated with placental insufficiency (5), maternal toxemia (4), and discordant twins (1). All infants were studied from 1 to 10 days of age. In viva cell-mediated immunity was assessed by delayed skin test responses to PHA, 10 /~g/ml; monilia 1:10; SK/SD, 1,000/250 units/ml, respectively; PPD, intermediate strength; and KLH, 1 mg/ml; the latter was given after an immunizing dose of 200/xg had been administered. For each test, 0.1 ml of material was injected intracutaneously. Skin reactions were read at 24 and 48 hours, and the induration measured, a positive result being taken as 5 m m diameter and over. In vitro cellular immunity was assessed by total lymphocyte counts in peripheral blood, by in vitro PHA stimulation of lymphocytes, and by the percentage of lymphocytes which spontaneously formed rosettes with sheep red blood cells at 4 ~ C. For the rosette assay, lymphocytes were separated from the buffy coat of heparinized blood on a ficoll/ isopaque gradient] Sheep red blood cells were washed
Volume 85 Number 5
Decreased rosette-forming lymphocytes
719
80-
70 O3 _.A __l
w c)
60
(,_9 z
:~
5O
n--
,o m
40
oor"
50-
tF-ILl 03
I-Z
w o
20-
h.l s
10-
0 ADULTS
NORMAL CONTROLS
MALNOURISHEDMALNOURISHED FOLLOW- UP
Fig. 1. Rosette-forming cells expressed as a percentage of peripheral blood lymphocytes comparing Caucasian adults, normal and malnourished Ghanaian children, and malnourished children after dietary therapy. Mean ___SE of the mean.
three times in Hanks buffered salt solution, pH 7.4, and made up to a final suspension of 0.5%. A cell suspension containing 1-2 x 106 lymphocytes in 0.4 ml of Medium 199 was incubated at 37 ~ C for 10 minutes with an equal volume of 0.5% sheep red blood cells. After centrifugation at 150 g for 3 m i n u t e s the s u p e r n a t e n t was removed and the cell button held for 20-24 hours at 4 ~ C. The cells were then resuspended in 0.3 ml Hanks buffered salt solution on a vertical rotator for 189 minutes. A small amount was pipetted gently onto a microscope slide and overlayed with a coverlip which was sealed at the edges with Permount (Fisher Scientific Co., Fairlawn, N. J.). The percentage of lymphocytes binding two or more sheep red blood cells was counted under oil. Counts, done on duplicate samples, were of at least 200 lymphocytes each time. A normal Caucasian adult control was used on each occasion. In vitro PHA stimulation was performed by a microtechnique on purified lymphocytes using increasing doses of PHA, and measuring D N A incorporation of tritiated thymidine.7 The results were expressed as the ratio of counts per minute of stimulated cells to counts per minute unstimulated. Both in vivo and in vitro studies were performed on the Ghanaian children, b u t only the rosette-forming
assay was used in the newborn infants. Five of the ten Ghanaian patients had total lymphocyte counts and percentages of rosette-forming cells remeasured after 7 to 17 days while receiving a high-protein, high-calorie diet. An additional eight severely malnourished children were included in the PHA study.
RESULTS Severe protein-calorie malnutrition. Skin test responses were diminished in the severely malnourished group; responses to PHA were least so (Table I). Total lymphocyte counts, mean 4,658 + 379 SE/mm 3 in the malnourished group and mean 4,356 _+ 454 S E / m m 3 in control subjects, were not significantly different. The n u m b e r s of R F C , h o w e v e r , w e r e r e m a r k a b l y diminished in the malnourished group. No difference was observed between those with marasmus and those with kwashiorkor. W h e n the n u m b e r of RFC was expressed as a percentage of total l y m p h o c y t e s , the malnourished group had a mean of 16.6% + 2.7 SE, the control group 59.7% + 1.4 SE, and Caucasian adults 64.5% _+ 1.0 SE (n = 17) (Fig. 1). The control and adult groups were not significantly different. There was no consistent morphologic difference between lymphocytes from the malnourished children and control sub-
720
Ferguson et al.
The Journal of Pediatrics November 1974
160--
p-
>- 1 4 0 - >,
Sontrols
120-<,
J J
E I00-
== o
5
80-
, Malnourished
I-t~
60-~5
'~'
eL.B.
40--
0
eG.I. 20eA.D.
I
I
I
2
I
5
I
I0
I
20
I
IO(Undiluted)
MICRO LITERS OF PHA (DILUTED 1:4) PER 105 LYMPHOCYTES
Fig. 2. In vitro lymphocyte response to PHA in malnourished Ghanaian children (n = 18) and control subjects. L. B., G. I., and A. D. (all severely malnourished) had very low responses. Mean ___SE of the mean.
jects, although occasional atypical larger lymphocytes were observed in the malnourished group. In vitro PHA stimulation produced similar responses in both the malnourished and control groups. Although three malnourished patients had greatly diminished responses, a significant difference could only be demonstrated between the groups as a whole when using a dose of 2/xt PHA, diluted 1:4, added to 105 lymphocytes (Fig. 2). Total lymphocyte counts and n u m b e r s of RFC were remeasured in five of the malnourished children fed a high-protein, high-calorie diet; two at 7 days, two at 10 days, and one at 17 days. All of the children had a return to normal values. Again, no difference was observed between marasmus and kwashiorkor. The n u m ber of RFC expressed as a percentage of total lymphocytes had a mean value of 63.2% ___2.4 SE, not significantly different from the normal control group (Fig. 1). K e y h o l e limpet h e m o c y a n i n skin tests applied at varying times after immunization were positive in eight of the ten malnourished patients and in eight of nine
control subjects (Fig. 3). All malnourished children had b e e n receiving a h i g h - p r o t e i n , high-calorie diet for several days prior to testing, and all had diminished percentages of RFC previously recorded. The m i n i m u m time between an abnormal rosette test and a positive KLH skin test was 6 days. In one negative reactor (T. M.) the rosette test was done on the day of skin testing; in the other (T. A.), nine days previously. The two negative reactors, however, had normal n u m b e r s of RFC when measured seven days after KLH skin testing. Of three positive reactors (G. I., B. D., T. Q.), each had normal n u m b e r s of RFC when remeasured a little before, or just KLH skin testing. A normal rosette test therefore correlates with a positive skin test reaction to KLH, and these findings indicate that recovery of the cellular i m m u n e response may occur within a period of a week. The negative reactor in the control group failed to react to monilia, SK/SD, or PPD, but did give a positive response to PHA. Intrauterine malnutrition. The percentage of rosetteforming cells was significantly diminished in the SGA infants, mean 49.2% _+ 2.0 SE, when compared to A G A
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Decreased rosette-forming lymphocytes
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KLH IMMUNIZATION +
I
J.T.
0
K.A.
0
S.B.
o
A.T.
0
9 KLH Skin Test o Rosette Test
+
klJ t--
E.E, T.M + 0
T.A. + + -- - 0
G.I. B.D.
+ + - -0
0
+ 0
g
T.Q.
I
2
I
I
6
I
I
I0
I
+ 9
I
14
I
I
18
I
I
22
I
DAYS PosT IMMUNIZATION
Fig. 3. Relationship between KLH delayed cutaneous hypersensitivity response, after KLH immunization, and result of the rosette assay in ten malnourished Ghanaian children. Immunization was given and dietary therapy begun on Day 1. Initial rosette tests were abnormal (--) in all children, but became normal (§ in 5 who were retested. This corresponded with recovery of delayed hypersensitivity as manifested by a positive response to KLH.
infants, mean 6 5 . 1 % _ 1.4 SE ( p = ( 0.001); but was much less diminished than in the severely malnourished Ghanaian children (Fig. 4). There was no relationship between percentage of RFC and birthweight in the A G A infants, nor was there a significant difference between A G A infants and adults. DISCUSSION The observation that severe protein-calorie malnutrition is associated with greatly diminished circulating R F C adds to the enlarging Picture of multiple i m m u n e defects in this condition. These include marked atrophy of thymic and lymphod tissues, 8,9 normal or increased immunoglobulin levels9J~ variable antibody formation to specific antigenic stimulil~ poor lymphocyte res p o n s e to n o n s p e c i f i c m i t o g e n i c stimulation9,11,14,15; diminished delayed cutaneous hypersensitivityg,U,15; r e d u c e d l e u k o c y t e p h a g o c y t i c function16lS; and low levels of serum complement components (except C4).19 Depressed cellular immunity in such patients has been related to an increased incidence of severe and atypical i n f e c t i o n with measles, h e r p e s simplex, and t u b e r -
culosis, and an increased susceptibility to gram-negative septicemia. 7,2~ The normal results in our study of in vitro P H A response and total lymphocyte counts, in the face of abnormal delayed cutaneous hypersensitivity and diminished RFC, suggest that the rosette assay is a more sensitive indicator of subtle defects of cellular immunity~ The lymphocyte separation technique results in a cell population greater than 95% mononuclear, 7 while monocyte contamination varies from 5-15%. There was no difference between cell preparations from the study groups or controls in this respect. Rosette-forming cells were c o u n t e d using an oil i m m e r s i o n lens, w h i c h greatly reduced the possibility of confusing nonrosetteforming lymphocytes with other cells. The R F C assay therefor e gives a convincing assessment of thymic-dep e n d e n t lymphocytes. Since the assay is relatively simple to perform, it is potentially available for use in the field. T h e reason for s u c h m a r k e d d e p r e s s i o n o f the thymus-dependent lymphocyte population is not clear.
722
Ferguson et al.
The Journal of Pediatrics November 1974
80-
7003 J _J ba (D
60-
G9 Z
:~ rr
O0
5O-
2
00
w FFkl] o3
40-
o El/
30-
z LI.I
o
rr" LIJ O_
20-
lO
0 AGA
SGA
Fig. 4. Rosette-forming cells expressed as a percentage of peripheral blood lymphocytes in ten SGA and ten AGA newborn infants. Mean • SE of the mean.
Schonland and associates 21 have shown that patients with protein-calorie malnutrition have significantly elevated levels of plasma cortisol (mean 33.5/zg/100 ml) c o m p a r e d to w e l l - n o u r i s h e d c o n t r o l s ( m e a n 10.2 /xg/100 ml) and diminished serum protein binding of cortisol (56% u n b o u n d cortisol compared to 33% in control Subjects), for as long as 6 weeks after starting treatment. In our patients, however, numbers of R F C returned to normal within 1 to 3 weeks. Sellmeyer and associates TMfound a normal in vitro P H A response in one patient within 6 days, and Geefhuysen and associates 15 found a return of normal delayed hypersensitivity to Candida in 10 c h i l d r e n with kwashioi'k0r within 3 weeks while being fed an adequate diet. Law and associates ll showed a return to normal in vitro PHA and pokeweed mitogen responses within 18 days in a group of malnourished adults treated by intravenous alimentation. More recently Paisley and associates 22 reported plasma cortisol concentrations to be elevated in maln o u r i s h e d c h i l d r e n only if t h e r e was s u p e r i m p o s e d acute stress. Yu and associates 23 in a study of 12 healthy adults given a single oral dose of 60 mg prednis0ne found that in addition to lymphopenia, the proportion of late R F C declined significantly from 69.2% to 55.9% within 4 to 6 hours. No plasma steroid levels were
measured, however, and this dose is of pharmacologic rather than physiologic magnitude. Fauci and Dale 24 s h o w e d a s i m i l a r l y m p h o p e n i c r e s p o n s e to oral hydrocortisone between 1 and 6 hours after ingestion. A plasma cortisol level of 131/xg/100 ml at 1 hour was associated with a m a x i m u m decrease in R F C from 48.5% to 32.7% at 4 hours, Suggesting that high physiologic levels may play a significant role in reducing the proportion of circulating RFC. Other mechanisms that may also play a part could include a lack of available amino acids, 14 pyridoxine deficiency,25 superimposed viral infection 14,26 electrolyte imbalance, ~4and a diminution in thymic hormone. It is of great interest that such marked depression o f t h y m u s - d e p e n d e n t lymphocytes should be reversible and at such a rapid rate, findings consistent with the studies of Woodruff in marasmic mice. 27, 28 He found that significant protection to Coxsackie virus infection was produced if an Optimum diet was begun at the time of infection. This was associated with a return to normal histology of thymus-dependent tissues and inflammatory cellular infiltlates, and with an increase in circulating lymphocytes. The group of SGA infants we studied had diminished g r o w t h s e c o n d a r y to c o n d i t i o n s w h i c h are likely to result in intrauterine nutrient deprivation. 29 The evid e n c e of d e p r e s s e d n u m b e r s of t h y m u s - d e p e n d e n t lymphocytes in these infants, although preliminary, is suggestive of an added deficiency of the already immature immunologic defense of the n e w b o r n infant. This is consistent with the report of disproportionately small size of thymus and spleen at autopsy in a group of low-birth-weight infants of nutritionally deprived urban mothers, 3~and may be important in explaining the increased susceptibility of SGA infants to infection. 31 W e have not yet followed the S G A infants long enough to determine if the percentage of RFCs returns to normal. Chandra 32 has shown, however, that depression of RFC may persist for at least several months. It is possible that relative malnutrition at a critical period in the d e v e l o p m e n t o f the t h y m u s - d e p e n d e n t i m m u n e system may result in prolonged or p e r m a n e n t impairm e n t of cellular immunity. This may be of great importance in the infant's future response to infectious disease and i m m u n i z a t i o n , in the i m m u n o l o g i c surveillance of neoplastic change, and in the development of autoimmunity.
REFERENCES
1. Bach, J. F., Dormont~ J., Dardenne, M., and Balner, H.: In vitro rosette inhibition by antihuman antilymphocyte
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3.
4.
5. 6.
7. 8. 9.
10. 11.
12. 13.
14.
15.
16.
17.
18.
serum: Correlation with skin graft prolongation in subhuman primates, Transplantation 8: 265, 1969. Craddock, C. G,, Longmire, R., and McMillan, R.: Lymphocytes and the immune response. N. Engl. J. Med., 285: 324, 1971. Sfites, D. P., Wybran, J., Carr, M. C., and Fudenberg, H. H.: Development of cellular immunocompetence in man, in Ontogeny of acquired immunity, Ciba Foundation Symposium, Amsterdam, 1971, ASP, p. 113. Wybran, J., Cart, M. C., and Fudenberg, H. H.: The human rosette-forming cell as a marker of a population of thymus-derived cells, J. Clin. Invest. 51: 2537, 1972. Wybran, J., and Fudenberg, H. H.: Thymus-derived rosette-forming cells, N. Engl. J. Med. 288: 1072, 1973. Dubowilz, L. M., Dubowitz, V., and Goldberg, C,: Clinical assessment of gestational age in the newborn infant, J. PED1ATR.77: 1, 1970. Sengar, D. P. S., and Terasaki, P. I.: A simi-micro mixed leukocyte culture test, Transplantation 11: 260, 1971. Vint, F. W.: Post-mortem findings in the natives of Kenya, Afr. Med. J. 13: 332, 1937. Smythe, P. M., Schonland, M., Brereton-Stiles, G. G., Coovadia, H, M., Grace, H. J., Loening, W. E. K., Mafoyane, A., Parent, M. A., and Vos, G. H.: Thymolymphatic deficiency and depression of cell-mediated immunity in protein calorie malnutrition, Lancet 2: 939, 1971. Chandra, R. K.: lmmunocompetence in under-nutrition, J. PEDIATR.81: 1194, 1972. Law, D. K., Dudrick, S. J., and Abdou, N. I.: Immunocompetence of patients with protein-calorie malnutrition, Ann. Intern. Med. 79: 545, 1973. Scrimshaw, N. S., and Behar, M.: Protein malnutrition in young children, Science 133: 2039, 1961. Brown, R. E., and Katz, M.: Failure of antibody production to yellow f e v e r v a c c i n e in c h i l d r e n w i t h kwashiorkor, Trop. Geogr. Med. 18: 125, 1966. Scllmeyer, E., Bhettay, E., TrusweU, A. S., Meyers, O. L., and Hansen, J. D. L.: Lymphocyte transformation in malnourished children, Arch. Dis. Child. 47: 429, 1972. Geefhuysen, J., Rosen, E. R., Katz, J., Ipp, T., and Metz, J.: Impaired cellular immunity in kwashiorkor with improvement after therapy, Br. Med. J. 4: 527, 1971. Selvaraj, R. J., and Bhat, K. S.: Metabolic and bacterial activities of leukocytes in protein-calorie malnutrition, Am. J. Clin. Nutr. 25: 166, 1972. Seth, V., and C h a n d r a , R. K.: Opsonic activity, phagocytosis and bactericidal capacity of polymorphs in under-nutrition, Arch. Dis. Child, 47: 282, 1972. Shousha, S., and Kamel, K.: Nitroblue tetrazolium test in children with kwashiorkor with a comment on the use of
Decreased rosette=forming lymphocytes
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latex particles in the test, J. Clin. Pathol. 25: 494, 1972. 19. Stitaya, S., Suskind, R., Edelman, R., Charupatana, C., and Olson, R. E.: Complement and C3-proactivator levels in children with protein-calorie malnutrition and effect of dietary therapy, Lancet l" 1016, 1973. 20. Gotoff, S. P.: Acquired and transient immunodeficiency disorders, in Stiehm, E. R. and Fulginiti, V. A., editors: I m m u n o l o g i c d i s o r d e r s in infants and c h i l d r e n , Philadelphia, 1973, W. B. Saunders Company, p. 321. 21. Schonland, M. M., Shanley, B. C., Loening, W. E. K., Parent, M. A., and Coovadia, H. M.: Plasma-cortisol and immuno-suppression in protein-calorie malnutrition, Lancet 2: 435, 1972. 22. Paisley, R. B., Angers, M., and Frenk, S.: Plasma cortisol levels in malnourished children with and without superimposed acute stress, Arch. Dis. Child. 48: 714, 1973. 23. Yu, D. T. Y., Clements, P. J., Paulus, H. E., Peter, J. B., Levy, J., and Barnett, E. V.: Human lymphocyte subpopulations: Effect of corticosteroids, J. Clin. Invest. 53: 565, 1974. 24. Fauci, A. S., and Dale, D. C.; The effect of in vivo hydrocortisone on sub-populations of human lymphocytes, J. Clin. Invest. 53: 240, 1974. 25. Davis, S. D.: Immunodeficiency, runting syndrome, and other anomalies in the rat due to congenital pyridoxine deficiency, Clin. Res. 22: 227A, 1974 (abstr.). 26. Wybran, J., and Fudenberg, H. H.: Thymus-derived rosette-forming cells in yarious human disease states: Cancer, lymphoma, bacterial and viral infections and other diseases, J. Clin. Invest. 52: 1026, 1973. 27. Woodruff, J. F., and Kolbourne, E. D.: The influence of quantitated post-weaning under-nutrition on Coxsackie B 3 infection of adult mice. I. Viral persistence and increased severity of lesions, J. Infect. Dis. 121: 137, 1970. 28. Woodruff, J. F.: The influence of quantitated post-weaning under-nutrition on Coxsackie B3 infection of adult mice: 2. Alteration of host defense mechanism, J. Infect. Dis. 121: 164, 1970. 29. Schaffer, A. J., and Avery, M. E.: The undersized infants, in Diseases of the newborn, ed. 3, Philadelphia, 197l, W. B. Saunders Company, p. 30. 30. Naeye, R. L., Diener, M. M., Harke, H. T., and Blanc, W. A.: Relation of poverty and race to birth weight and organ and cell structure in the newborn, Pediatr. Res. 5: 17, 1971. 31. Papaevangelou, G., Papadatos, C., and Alexiou, D.: Perinatal mortality and morbidity in small-for-dates newborns, Helv. Paediatr. Acta 27: 415, t972. 32. Chandra, R. K.: Personal communication, 1974.
DerrickB. Jelliffe, Edilor
Decreased rosette-forming lymphocytes in malnutrition and intrauterine growth retardation The percentage of peripheral blood lymphocytes which spontaneously form rosettes with sheep red blood cells (thymus-dependent lymphocytes) was studied as a sensitive measure of cellular immunity in patients with two .forms of malnutrition. The percentage and absolute number of rosette-forming cells was markedly decreased in 10 children with severe protein-calorie malnutrition (16.6% +_ Z 7 SE) p ( 0.001, compared to normal children (59. 7% +_ 1.4 SE) and Caucasian adults (64.5% +_ 1.0 SE). Delayed cutaneous hypersensitivity was also diminished, but total lymphocyte counts and in vitro lymphocyte response to phytohemagglutinin were normal Therapy with a high-protein, high-calorie diet resulted in recovery of delayed cutaneous" hypersensitivity and return to normal numbers of rosette-forming cells within 1 to 3 weeks'. The percentage of rosette-forming cells was moderately reduced (49.2% +_ 2.0 SE) p ( 0.001, in 10 newborn infants who were small for gestational age, mean birth weight 1,660gm, compared to those whose weights were appropriate for gestation (65.1% +_ 1.4 SE), mean birth weight ZOO5gin, an observation in keeping with prior studies Qf diminished spleen and thymus size in such infants. The interrelation of cellular immunity and malnutrition is emphasized, since this may be of great importance in the host response to infection and immunization, and in persisting defects Qf cellular immunity in the developing newborn infant.
Alexander C. Ferguson, M . B . , F . R . C . P . ( C ) , D . C . H . , *
Kingston, Ont., C a n a d a ,
Glenn J. Lawlor, Jr., M.D., Charlotte G. Neumann, M.D., M.P.H., William O h , M . D . , a n d E. Richard Stiehm, M . D . , * *
THE ABILITY of a subpopulation of peripheral blood lymphocytes to spontaneously form rosettes with sheep red blood cells, first described by Bach and associates t has been shown to be a marker of thymus-dependent cells.2 Stites and associates 3 found that thymus tissue from 11 to 19 week fetuses contained up to 65% RFC, compared to 0.8% of cells from the bone marrow. Wybran and associates 4 reported diminished numbers of From the Department of Pediatrics, University of CaliJbrnia School o f Medicine. Supported by United States Public Health Service Contract No. HD 72-2069 and Grant No. HD-06463, and National Foundation Grant 6- 74-4. Presented in part at the Annual Meeting, Western Society for Pediatric Research, Carmel, California, February 1974. *Holder ol~fellowshipfrom the Canadian PaediatricSociety's Mead Johnson Educational Fund. **Reprint address: Department of Pediatrics, U.C.L.A. Center .for the Health Sciences, Los Angeles, Calf. 90024.
L o s Angeles, Calif.
R F C in several primary immunodeficiency disorders with diminished cell-mediated immunity. One patient with the Nezelof syndrome had no RFC, and four with the Wiskott-Aldrich syndrome had decreased n u m b e r s (1-1.5%). In contrast, patients with acquired hypogamAbbreviations used RFC: rosette-forming cells SGA: small for gestational age AGA: appropriate for gestationat age PHA: phytohemagglutinin SK: streptokinase SD: streptodornase PPD: purified protein derivative KLH: keyhold limpet hemocyanin DNA: deoxyribonucleic acid m a g l o b u l i n e m i a w i t h o u t cellular i m m u n o d e f i c i e n c y had n o r m a l n u m b e r s of R F C (4-40%). T h e i r assay measured "early" RFC, however, and gave lower normal values than the 80% "late" RFC as reported by others using slightly different experimental conditions?
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Table I. Delayed cutaneous hypersensitivity responses
Protein-calorie malnutrition
Patient
Age (too.)
Manila
SK/SD
J.T. K.A. S.B. A.T. T.M. T.A. G.I.
36 36 36 19 24 12 24
+ 0 0 0 0 0 0
E.E. B.D.
48 39
+ 0
T.Q.
15
Totals Normal control subjects
A.K. A.B. E.A. N.N. N.O. A.S. T.Q. N.A. E.Y. M.F.
27 24 31 23 55 52 43 46 52 35
Totals
PHA
PPD
Diagnosis
+ + + + 0 0 + 0 + + 7/10
0 0 0 0 0 0 0 0 0 0 0/10
K K K M K M M K K K
2/10
0 + 0 0 0 0 0 0 + 0 2/10
+ 0 + + + + + + 0 +
+ 0 0 + + + + + + +
+ + + + + + + + + +
0 0 0 + 0 0 0 + + 0
8/10
8/10
10/10
3/10
0
+ = positive; 0 = negative; K = kwashiorkor; M = marasmus.
Since the RFC assay is rapid, technically uncomplicated, and possibly more sensitive in detecting subtle abnormalities, we wished to determine its value relative to other tests for defective cellular immunity. We therefore studied a group of children with severe proteincalorie m a l n u t r i t i o n from G h a n a , West Africa. The finding of grossly abnormal n u m b e r s of RFC in those patients prompted us to study newborn infants with int r a u t e r i n e growth retardation whose m a l n u t r i t i o n , though less severe, occurs at a critical time in development. METHODS Ten hospitalized Ghanaian children were studied, all of whom had severe protein-calorie malnutrition. Three were marasmic with body weights less than 60% of expected for age. Seven had kwashiorkor based on evidence of edema, skin changes, hair loss, hepatosplenomegaly, and hypoalbuminemia ( < 2 gin/100 ml). Ages ranged from 12 to 39 months. Ten age-matched normal Ghanaian children were control subjects. Twenty Caucasian newborn infants of 28 to 40 weeks gestational age were studied; ten of them were SGA, m e a n b i r t h w e i g h t 1,660 gm ( r a n g e 810-2,240); the
weights of ten were AGA, mean birthweight 2,005 gm (range 1,780-2,800), classified according to the criteria of Dubowitz and associates. 6 The SGA infants had no stigmata of genetic or developmental abnormalities, or of infection, but were associated with placental insufficiency (5), maternal toxemia (4), and discordant twins (1). All infants were studied from 1 to 10 days of age. In viva cell-mediated immunity was assessed by delayed skin test responses to PHA, 10 /~g/ml; monilia 1:10; SK/SD, 1,000/250 units/ml, respectively; PPD, intermediate strength; and KLH, 1 mg/ml; the latter was given after an immunizing dose of 200/xg had been administered. For each test, 0.1 ml of material was injected intracutaneously. Skin reactions were read at 24 and 48 hours, and the induration measured, a positive result being taken as 5 m m diameter and over. In vitro cellular immunity was assessed by total lymphocyte counts in peripheral blood, by in vitro PHA stimulation of lymphocytes, and by the percentage of lymphocytes which spontaneously formed rosettes with sheep red blood cells at 4 ~ C. For the rosette assay, lymphocytes were separated from the buffy coat of heparinized blood on a ficoll/ isopaque gradient] Sheep red blood cells were washed
Volume 85 Number 5
Decreased rosette-forming lymphocytes
719
80-
70 O3 _.A __l
w c)
60
(,_9 z
:~
5O
n--
,o m
40
oor"
50-
tF-ILl 03
I-Z
w o
20-
h.l s
10-
0 ADULTS
NORMAL CONTROLS
MALNOURISHEDMALNOURISHED FOLLOW- UP
Fig. 1. Rosette-forming cells expressed as a percentage of peripheral blood lymphocytes comparing Caucasian adults, normal and malnourished Ghanaian children, and malnourished children after dietary therapy. Mean ___SE of the mean.
three times in Hanks buffered salt solution, pH 7.4, and made up to a final suspension of 0.5%. A cell suspension containing 1-2 x 106 lymphocytes in 0.4 ml of Medium 199 was incubated at 37 ~ C for 10 minutes with an equal volume of 0.5% sheep red blood cells. After centrifugation at 150 g for 3 m i n u t e s the s u p e r n a t e n t was removed and the cell button held for 20-24 hours at 4 ~ C. The cells were then resuspended in 0.3 ml Hanks buffered salt solution on a vertical rotator for 189 minutes. A small amount was pipetted gently onto a microscope slide and overlayed with a coverlip which was sealed at the edges with Permount (Fisher Scientific Co., Fairlawn, N. J.). The percentage of lymphocytes binding two or more sheep red blood cells was counted under oil. Counts, done on duplicate samples, were of at least 200 lymphocytes each time. A normal Caucasian adult control was used on each occasion. In vitro PHA stimulation was performed by a microtechnique on purified lymphocytes using increasing doses of PHA, and measuring D N A incorporation of tritiated thymidine.7 The results were expressed as the ratio of counts per minute of stimulated cells to counts per minute unstimulated. Both in vivo and in vitro studies were performed on the Ghanaian children, b u t only the rosette-forming
assay was used in the newborn infants. Five of the ten Ghanaian patients had total lymphocyte counts and percentages of rosette-forming cells remeasured after 7 to 17 days while receiving a high-protein, high-calorie diet. An additional eight severely malnourished children were included in the PHA study.
RESULTS Severe protein-calorie malnutrition. Skin test responses were diminished in the severely malnourished group; responses to PHA were least so (Table I). Total lymphocyte counts, mean 4,658 + 379 SE/mm 3 in the malnourished group and mean 4,356 _+ 454 S E / m m 3 in control subjects, were not significantly different. The n u m b e r s of R F C , h o w e v e r , w e r e r e m a r k a b l y diminished in the malnourished group. No difference was observed between those with marasmus and those with kwashiorkor. W h e n the n u m b e r of RFC was expressed as a percentage of total l y m p h o c y t e s , the malnourished group had a mean of 16.6% + 2.7 SE, the control group 59.7% + 1.4 SE, and Caucasian adults 64.5% _+ 1.0 SE (n = 17) (Fig. 1). The control and adult groups were not significantly different. There was no consistent morphologic difference between lymphocytes from the malnourished children and control sub-
720
Ferguson et al.
The Journal of Pediatrics November 1974
160--
p-
>- 1 4 0 - >,
Sontrols
120-<,
J J
E I00-
== o
5
80-
, Malnourished
I-t~
60-~5
'~'
eL.B.
40--
0
eG.I. 20eA.D.
I
I
I
2
I
5
I
I0
I
20
I
IO(Undiluted)
MICRO LITERS OF PHA (DILUTED 1:4) PER 105 LYMPHOCYTES
Fig. 2. In vitro lymphocyte response to PHA in malnourished Ghanaian children (n = 18) and control subjects. L. B., G. I., and A. D. (all severely malnourished) had very low responses. Mean ___SE of the mean.
jects, although occasional atypical larger lymphocytes were observed in the malnourished group. In vitro PHA stimulation produced similar responses in both the malnourished and control groups. Although three malnourished patients had greatly diminished responses, a significant difference could only be demonstrated between the groups as a whole when using a dose of 2/xt PHA, diluted 1:4, added to 105 lymphocytes (Fig. 2). Total lymphocyte counts and n u m b e r s of RFC were remeasured in five of the malnourished children fed a high-protein, high-calorie diet; two at 7 days, two at 10 days, and one at 17 days. All of the children had a return to normal values. Again, no difference was observed between marasmus and kwashiorkor. The n u m ber of RFC expressed as a percentage of total lymphocytes had a mean value of 63.2% ___2.4 SE, not significantly different from the normal control group (Fig. 1). K e y h o l e limpet h e m o c y a n i n skin tests applied at varying times after immunization were positive in eight of the ten malnourished patients and in eight of nine
control subjects (Fig. 3). All malnourished children had b e e n receiving a h i g h - p r o t e i n , high-calorie diet for several days prior to testing, and all had diminished percentages of RFC previously recorded. The m i n i m u m time between an abnormal rosette test and a positive KLH skin test was 6 days. In one negative reactor (T. M.) the rosette test was done on the day of skin testing; in the other (T. A.), nine days previously. The two negative reactors, however, had normal n u m b e r s of RFC when measured seven days after KLH skin testing. Of three positive reactors (G. I., B. D., T. Q.), each had normal n u m b e r s of RFC when remeasured a little before, or just KLH skin testing. A normal rosette test therefore correlates with a positive skin test reaction to KLH, and these findings indicate that recovery of the cellular i m m u n e response may occur within a period of a week. The negative reactor in the control group failed to react to monilia, SK/SD, or PPD, but did give a positive response to PHA. Intrauterine malnutrition. The percentage of rosetteforming cells was significantly diminished in the SGA infants, mean 49.2% _+ 2.0 SE, when compared to A G A
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Decreased rosette-forming lymphocytes
721
KLH IMMUNIZATION +
I
J.T.
0
K.A.
0
S.B.
o
A.T.
0
9 KLH Skin Test o Rosette Test
+
klJ t--
E.E, T.M + 0
T.A. + + -- - 0
G.I. B.D.
+ + - -0
0
+ 0
g
T.Q.
I
2
I
I
6
I
I
I0
I
+ 9
I
14
I
I
18
I
I
22
I
DAYS PosT IMMUNIZATION
Fig. 3. Relationship between KLH delayed cutaneous hypersensitivity response, after KLH immunization, and result of the rosette assay in ten malnourished Ghanaian children. Immunization was given and dietary therapy begun on Day 1. Initial rosette tests were abnormal (--) in all children, but became normal (§ in 5 who were retested. This corresponded with recovery of delayed hypersensitivity as manifested by a positive response to KLH.
infants, mean 6 5 . 1 % _ 1.4 SE ( p = ( 0.001); but was much less diminished than in the severely malnourished Ghanaian children (Fig. 4). There was no relationship between percentage of RFC and birthweight in the A G A infants, nor was there a significant difference between A G A infants and adults. DISCUSSION The observation that severe protein-calorie malnutrition is associated with greatly diminished circulating R F C adds to the enlarging Picture of multiple i m m u n e defects in this condition. These include marked atrophy of thymic and lymphod tissues, 8,9 normal or increased immunoglobulin levels9J~ variable antibody formation to specific antigenic stimulil~ poor lymphocyte res p o n s e to n o n s p e c i f i c m i t o g e n i c stimulation9,11,14,15; diminished delayed cutaneous hypersensitivityg,U,15; r e d u c e d l e u k o c y t e p h a g o c y t i c function16lS; and low levels of serum complement components (except C4).19 Depressed cellular immunity in such patients has been related to an increased incidence of severe and atypical i n f e c t i o n with measles, h e r p e s simplex, and t u b e r -
culosis, and an increased susceptibility to gram-negative septicemia. 7,2~ The normal results in our study of in vitro P H A response and total lymphocyte counts, in the face of abnormal delayed cutaneous hypersensitivity and diminished RFC, suggest that the rosette assay is a more sensitive indicator of subtle defects of cellular immunity~ The lymphocyte separation technique results in a cell population greater than 95% mononuclear, 7 while monocyte contamination varies from 5-15%. There was no difference between cell preparations from the study groups or controls in this respect. Rosette-forming cells were c o u n t e d using an oil i m m e r s i o n lens, w h i c h greatly reduced the possibility of confusing nonrosetteforming lymphocytes with other cells. The R F C assay therefor e gives a convincing assessment of thymic-dep e n d e n t lymphocytes. Since the assay is relatively simple to perform, it is potentially available for use in the field. T h e reason for s u c h m a r k e d d e p r e s s i o n o f the thymus-dependent lymphocyte population is not clear.
722
Ferguson et al.
The Journal of Pediatrics November 1974
80-
7003 J _J ba (D
60-
G9 Z
:~ rr
O0
5O-
2
00
w FFkl] o3
40-
o El/
30-
z LI.I
o
rr" LIJ O_
20-
lO
0 AGA
SGA
Fig. 4. Rosette-forming cells expressed as a percentage of peripheral blood lymphocytes in ten SGA and ten AGA newborn infants. Mean • SE of the mean.
Schonland and associates 21 have shown that patients with protein-calorie malnutrition have significantly elevated levels of plasma cortisol (mean 33.5/zg/100 ml) c o m p a r e d to w e l l - n o u r i s h e d c o n t r o l s ( m e a n 10.2 /xg/100 ml) and diminished serum protein binding of cortisol (56% u n b o u n d cortisol compared to 33% in control Subjects), for as long as 6 weeks after starting treatment. In our patients, however, numbers of R F C returned to normal within 1 to 3 weeks. Sellmeyer and associates TMfound a normal in vitro P H A response in one patient within 6 days, and Geefhuysen and associates 15 found a return of normal delayed hypersensitivity to Candida in 10 c h i l d r e n with kwashioi'k0r within 3 weeks while being fed an adequate diet. Law and associates ll showed a return to normal in vitro PHA and pokeweed mitogen responses within 18 days in a group of malnourished adults treated by intravenous alimentation. More recently Paisley and associates 22 reported plasma cortisol concentrations to be elevated in maln o u r i s h e d c h i l d r e n only if t h e r e was s u p e r i m p o s e d acute stress. Yu and associates 23 in a study of 12 healthy adults given a single oral dose of 60 mg prednis0ne found that in addition to lymphopenia, the proportion of late R F C declined significantly from 69.2% to 55.9% within 4 to 6 hours. No plasma steroid levels were
measured, however, and this dose is of pharmacologic rather than physiologic magnitude. Fauci and Dale 24 s h o w e d a s i m i l a r l y m p h o p e n i c r e s p o n s e to oral hydrocortisone between 1 and 6 hours after ingestion. A plasma cortisol level of 131/xg/100 ml at 1 hour was associated with a m a x i m u m decrease in R F C from 48.5% to 32.7% at 4 hours, Suggesting that high physiologic levels may play a significant role in reducing the proportion of circulating RFC. Other mechanisms that may also play a part could include a lack of available amino acids, 14 pyridoxine deficiency,25 superimposed viral infection 14,26 electrolyte imbalance, ~4and a diminution in thymic hormone. It is of great interest that such marked depression o f t h y m u s - d e p e n d e n t lymphocytes should be reversible and at such a rapid rate, findings consistent with the studies of Woodruff in marasmic mice. 27, 28 He found that significant protection to Coxsackie virus infection was produced if an Optimum diet was begun at the time of infection. This was associated with a return to normal histology of thymus-dependent tissues and inflammatory cellular infiltlates, and with an increase in circulating lymphocytes. The group of SGA infants we studied had diminished g r o w t h s e c o n d a r y to c o n d i t i o n s w h i c h are likely to result in intrauterine nutrient deprivation. 29 The evid e n c e of d e p r e s s e d n u m b e r s of t h y m u s - d e p e n d e n t lymphocytes in these infants, although preliminary, is suggestive of an added deficiency of the already immature immunologic defense of the n e w b o r n infant. This is consistent with the report of disproportionately small size of thymus and spleen at autopsy in a group of low-birth-weight infants of nutritionally deprived urban mothers, 3~and may be important in explaining the increased susceptibility of SGA infants to infection. 31 W e have not yet followed the S G A infants long enough to determine if the percentage of RFCs returns to normal. Chandra 32 has shown, however, that depression of RFC may persist for at least several months. It is possible that relative malnutrition at a critical period in the d e v e l o p m e n t o f the t h y m u s - d e p e n d e n t i m m u n e system may result in prolonged or p e r m a n e n t impairm e n t of cellular immunity. This may be of great importance in the infant's future response to infectious disease and i m m u n i z a t i o n , in the i m m u n o l o g i c surveillance of neoplastic change, and in the development of autoimmunity.
REFERENCES
1. Bach, J. F., Dormont~ J., Dardenne, M., and Balner, H.: In vitro rosette inhibition by antihuman antilymphocyte
Volume 85 Number 5
2.
3.
4.
5. 6.
7. 8. 9.
10. 11.
12. 13.
14.
15.
16.
17.
18.
serum: Correlation with skin graft prolongation in subhuman primates, Transplantation 8: 265, 1969. Craddock, C. G,, Longmire, R., and McMillan, R.: Lymphocytes and the immune response. N. Engl. J. Med., 285: 324, 1971. Sfites, D. P., Wybran, J., Carr, M. C., and Fudenberg, H. H.: Development of cellular immunocompetence in man, in Ontogeny of acquired immunity, Ciba Foundation Symposium, Amsterdam, 1971, ASP, p. 113. Wybran, J., Cart, M. C., and Fudenberg, H. H.: The human rosette-forming cell as a marker of a population of thymus-derived cells, J. Clin. Invest. 51: 2537, 1972. Wybran, J., and Fudenberg, H. H.: Thymus-derived rosette-forming cells, N. Engl. J. Med. 288: 1072, 1973. Dubowilz, L. M., Dubowitz, V., and Goldberg, C,: Clinical assessment of gestational age in the newborn infant, J. PED1ATR.77: 1, 1970. Sengar, D. P. S., and Terasaki, P. I.: A simi-micro mixed leukocyte culture test, Transplantation 11: 260, 1971. Vint, F. W.: Post-mortem findings in the natives of Kenya, Afr. Med. J. 13: 332, 1937. Smythe, P. M., Schonland, M., Brereton-Stiles, G. G., Coovadia, H, M., Grace, H. J., Loening, W. E. K., Mafoyane, A., Parent, M. A., and Vos, G. H.: Thymolymphatic deficiency and depression of cell-mediated immunity in protein calorie malnutrition, Lancet 2: 939, 1971. Chandra, R. K.: lmmunocompetence in under-nutrition, J. PEDIATR.81: 1194, 1972. Law, D. K., Dudrick, S. J., and Abdou, N. I.: Immunocompetence of patients with protein-calorie malnutrition, Ann. Intern. Med. 79: 545, 1973. Scrimshaw, N. S., and Behar, M.: Protein malnutrition in young children, Science 133: 2039, 1961. Brown, R. E., and Katz, M.: Failure of antibody production to yellow f e v e r v a c c i n e in c h i l d r e n w i t h kwashiorkor, Trop. Geogr. Med. 18: 125, 1966. Scllmeyer, E., Bhettay, E., TrusweU, A. S., Meyers, O. L., and Hansen, J. D. L.: Lymphocyte transformation in malnourished children, Arch. Dis. Child. 47: 429, 1972. Geefhuysen, J., Rosen, E. R., Katz, J., Ipp, T., and Metz, J.: Impaired cellular immunity in kwashiorkor with improvement after therapy, Br. Med. J. 4: 527, 1971. Selvaraj, R. J., and Bhat, K. S.: Metabolic and bacterial activities of leukocytes in protein-calorie malnutrition, Am. J. Clin. Nutr. 25: 166, 1972. Seth, V., and C h a n d r a , R. K.: Opsonic activity, phagocytosis and bactericidal capacity of polymorphs in under-nutrition, Arch. Dis. Child, 47: 282, 1972. Shousha, S., and Kamel, K.: Nitroblue tetrazolium test in children with kwashiorkor with a comment on the use of
Decreased rosette=forming lymphocytes
723
latex particles in the test, J. Clin. Pathol. 25: 494, 1972. 19. Stitaya, S., Suskind, R., Edelman, R., Charupatana, C., and Olson, R. E.: Complement and C3-proactivator levels in children with protein-calorie malnutrition and effect of dietary therapy, Lancet l" 1016, 1973. 20. Gotoff, S. P.: Acquired and transient immunodeficiency disorders, in Stiehm, E. R. and Fulginiti, V. A., editors: I m m u n o l o g i c d i s o r d e r s in infants and c h i l d r e n , Philadelphia, 1973, W. B. Saunders Company, p. 321. 21. Schonland, M. M., Shanley, B. C., Loening, W. E. K., Parent, M. A., and Coovadia, H. M.: Plasma-cortisol and immuno-suppression in protein-calorie malnutrition, Lancet 2: 435, 1972. 22. Paisley, R. B., Angers, M., and Frenk, S.: Plasma cortisol levels in malnourished children with and without superimposed acute stress, Arch. Dis. Child. 48: 714, 1973. 23. Yu, D. T. Y., Clements, P. J., Paulus, H. E., Peter, J. B., Levy, J., and Barnett, E. V.: Human lymphocyte subpopulations: Effect of corticosteroids, J. Clin. Invest. 53: 565, 1974. 24. Fauci, A. S., and Dale, D. C.; The effect of in vivo hydrocortisone on sub-populations of human lymphocytes, J. Clin. Invest. 53: 240, 1974. 25. Davis, S. D.: Immunodeficiency, runting syndrome, and other anomalies in the rat due to congenital pyridoxine deficiency, Clin. Res. 22: 227A, 1974 (abstr.). 26. Wybran, J., and Fudenberg, H. H.: Thymus-derived rosette-forming cells in yarious human disease states: Cancer, lymphoma, bacterial and viral infections and other diseases, J. Clin. Invest. 52: 1026, 1973. 27. Woodruff, J. F., and Kolbourne, E. D.: The influence of quantitated post-weaning under-nutrition on Coxsackie B 3 infection of adult mice. I. Viral persistence and increased severity of lesions, J. Infect. Dis. 121: 137, 1970. 28. Woodruff, J. F.: The influence of quantitated post-weaning under-nutrition on Coxsackie B3 infection of adult mice: 2. Alteration of host defense mechanism, J. Infect. Dis. 121: 164, 1970. 29. Schaffer, A. J., and Avery, M. E.: The undersized infants, in Diseases of the newborn, ed. 3, Philadelphia, 197l, W. B. Saunders Company, p. 30. 30. Naeye, R. L., Diener, M. M., Harke, H. T., and Blanc, W. A.: Relation of poverty and race to birth weight and organ and cell structure in the newborn, Pediatr. Res. 5: 17, 1971. 31. Papaevangelou, G., Papadatos, C., and Alexiou, D.: Perinatal mortality and morbidity in small-for-dates newborns, Helv. Paediatr. Acta 27: 415, t972. 32. Chandra, R. K.: Personal communication, 1974.