Immune function in intrauterine growth retardation

NUTRITION RESEARCH, Vol. 4, pp. 399-419, 1984 0271-5317/84 $3.00 + .00 Printed in the USA. Copyright (c) 1984 Pergamon Press Ltd. All rights reserved.

IMMUNE FUNCTION IN INTRAUTERINE GROWTHRETARDATION1-3 Charlotte G. Neumann, M.D., M.P.H., E. Richard Stiehm, M.D., John Zahradnick, M.D., Carter Newton, M.D., Hans Weber, M.D. Marion E. Swendseid, Ph.D., James D. Cherry, M.D., and Jean M. Carney, M.D., University of California, Los Angeles, Los Angeles, California 90024 In collaboration with Nimrod Bwibo, M.B.B.S., N. Chotskey, M.B.B.S., and VS. Karuga-Koinange, M.B.B.S. University of Nairobi, Pumwani Maternity Hospital and Ministry of Health, Nairobi, Kenya ABSTRACT A one-year longitudinal study was carried out on 2 groups of Kenyan intrauterine growth retarded infants and normal weight full-term infants to determine the effects of intrauterine malnutrition and other nutrient deficiencies on immune function. Three birth weight groups were studied: <2500 gm (<~rd percentile); 2500-2799 gm (3rd to 10th percentile); and >2800 gm (> lOth percentile). B cell function was studied by determinatiTn of B lymphocytes and serum levels of IgG, IgM, IgA and IgE. T-cell function was studied by measuring lymphocyte numbers, the percent and absolute number of T-cells by Erosette technique, lymphocyte responsiveness to PHA and the intradermal PPD response to BCG vaccine. Maternal and infant nutritional studies were obtained concurrently. At birth B cells and immunoglobulins were normal and comparable in all groups except that seven of 41 infants had elevated IgE levels in their cord blood. By 7 months adult levels of IgG, IgM and IgE were achieved in the intrauterine growth retarded infants. Their mothers had significantly higher levels of IgG than the control mothers. Total lymphocyte counts, T-cells and percent T-cells were significantly reduced at birth in the IUGR groups. This abnormality persisted until one year in the smallest (<__2500 g) birthweight group. The IUGR infants had a higher incidence of cutaneous anergy to PPD than did control infants. Cell-mediated immunity correlated significantly with birthweight, blood levels of iron and thiamin at birth and with folate, hemoglobin, pyridoxine and riboflavin at 6 and 12 months. KEY WORDS: IMMUNITY, INTRAUTERINE GROWTHRETARDATION, NUTRITION DEFICIENCIES, CELLULAR IMMUNITY, ANERGY, ELEVATED IgE 1From the UCLA School of Pub|ic Health and the Department of Pediatrics, and the Center for Interdisciplinary Research in Immunologic Diseases, UCLA School of Medicine, Los Angeles, California In collaboration with the Ministry of Health, Government of Kenya; Department of Pediatrics, University of Nairobi; Faculty of Medicine and Pumwani Maternity Hospital, Nairobi City Council 2Supported by NICHD Contract No. 1-HD-52851 and U.S. Public Health Research Grants AI-15332 and HD-090O8 3Address correspondence about the manuscript and reprint requests to: Charlotte G. Neumann, M.D., M.P.H., UCLASchool of Public Health, Los Angeles, California 90024 399

400

C.G. NEUMANNet a l . INTRODUCTION

Intrauterine growth retardation (IUGR) places the newborn infant at considerable risk for increased mortality and morbidity; the l a t t e r includes poor physical growth, subtle learning d i f f i c u l t i e s and increased susceptibility to infection (1). IUGRmay occur in as many as 20% of a l l infants in certain populations, particularly in developing countries and in disadvantaged families in industrialized countries. The etiology is varied and multifactorial and may include maternal infection, malnutrition, placental dysfunction, hypertension, toxemia, malaria, and maternal smoking ( I ) . Prematurity is often superimposed. The interrelationship between maternal n u t r i t i o n , IUGR, immunity and infection is a complex one. Although several studies have examined immune function in IUGR infants most of these were conducted on small numbers of infants, usually from intensive care nurseries in developed countries (2-4). In a f i e l d study in Kenya we examined the effect of n~derate degrees of IUGR on immune function and i t s correlation with specific nutrients. A subsequent paper details i t s c l i n i c a l consequences including infection rates and vaccine efficacy. Infants with moderate IUGR far outnumber those with severe IUGR, and represent a sizeable population that is at risk for impaired immune function. PATIENTS AND METHODS PATIENTS: This study was carried out in collaboration with colleagues at the University of Nairobi, Faculty of Medicine, Ministry of Health of Kenya and Nairobi City Council. Control and IUGR infants were enrolled at birth and followed through one year of l i f e . Those infants whose mothers had serious illnesses during pregnancy were excluded. Nutritional, immununologic and serologic studies were carried out simultaneously on mother-infant pairs. These studies were performed on the mother at the time of delivery and at 6 months post-partum and in the infants at b i r t h , 6, 7 and 12 months. Study infants received health supervision in a special infant c l i n i c , where they received immunizations and treatment for minor illness and were referred to the university hospital for serious i l l n e s s , thus permitting monitoring of their health status. Infants were classified as IUGR i f t h e i r birth weights (BW) were at or below the lOth percentile for a given gestational age. Infants were between 38 weeks and 41 weeks gestational age (GA) except for seven of the IUGR infants who were also pre-term (GA 33 to 37 weeks). The l a t t e r were also analyzed separately and compared to a group of 6 pre-term appropriate weight for GA (AGA) infants concurrently being born at the same hospital. The IUGR infants were divided into two groups: Group I , severe IUGR with BW < 2500 gm (__<3rd percentile); and Group I I , moderate IUGR with BW between ~01 to 2799 gm (3rd to lOth percent i l e ) . All infants were otherwise c l i n i c a l l y normal. Also studied were a group of full-term infants (Group I I I ) with BW > 2800 gm, who served as controls. Division of the IUGR infants into two groups permitted a comparison of the degree of immune impairment with the degree of fetal growth retardation or malnutrition. The intrauterine growth curves of Gairdner and Pearson (5) were used to classify IUGR newborns by percentiles for both male and female infants. The Dubowitz scale was used to determine gestational age (6) and was performed on a l l infants with BW less than 2800 gm. IMMUNOLOGIC STUDIES Immunoglobulins: IgG, IgM and IgA were measured by single radial immunodiffusion in agar (7); IgE was assayed by radioimmunoassay (PRIST, Pharmacia) (8); B lymphocytes were enumerated by direct immunofluorescence for surface membrane immunoglobulin (9) using a polyvalent rabbit antiserum to human immunoglobulin. Lymphocytes were isolated by Ficoll-Hypaque gradiel)ts from heparinized venous "blood and reconstituted to a concentration of 10" cells per ml (10). The normal newborn values for B cells are 5 to 10%.(9,11) Complement: C3 and C4 complement components were measured by single radial diffusion and compared to U.S. age-matched controls (12).

IMMUNITY IN FETAL MALNUTRITION

401

Cell-mediated Immunity (CMI): CMI was evaluated by t o t a l lymphocyte counts, enumeration of T - c e l l s (percent and absolute number) and in v i t r o lymphocyte PHA responsiveness in each of the three study groups at b i r t h , 6 and 12 months. Also, cutaneous delayed h y p e r s e n s i t i v i t y to PPD was tested at 6 months of age following BCG* vaccination given at b i r t h . Total lymphocyte counts were obtained by determining the percent of lymphocytes from d i f f e r e n t i a l counts of Wright staine~ blood smears which were then m u l t i p l i e d by the white blood c e l l counts per mm-. Lymphocytes were prepared by Ficoll-Hypaque gradients as above (10). T - c e l l s were i d e n t i f i e d by t h e i r a b i l i t y to form rosettes with sheep e r y t h r o cytes, expressed as rosette-forming c e l l s (RFC) (13). Normal values f o r RFC are 55 +5%(SD) in neonates (14) and 60 _+ 5%(SD) in l a t e r infancy (15). Abnormally low values are defined as RFC less than 50 percent (14). Lymphocyte s t i m u l a t i o n was performed by c u l t u r i n g 2 x 105 lymphocytes with phytohemagglutinin (PHA)(Difco) at d i l u t i o n s of I : I , 0 0 0 , I : I 0 , 0 0 0 and I:I00,000 in human AB serum. After 48 hours, t r i t i a t e d thymidine was added for 16 more hours at which time thymidine incorporation into DNA was determined. Unstimulated counts per minute (CPM) are compared to stimulated CPM. A s t i m u l a t i o n index is defined as the highest average count with PHA divided by the highest average count without PHA x 100. The normal index i s greater than 25 (14,15). Delayed cutaneous h y p e r s e n s i t i v i t y was evaluated by intradermal i n j e c t i o n of 0 . I ml of 5 tuberculin units of PPD~ in the volar aspect of the forearm. A p o s i t i v e response is defined as > 5 mm diameter of induration at 48 hours. NUTRITIONAL STUDIES: The n u t r i t i o n a l status of mothers and t h e i r i n f a n t s was assessed by c l i n i c a l examination, anthropometry, biochemical studies and 24 hour diet r e c a l l . Mothers were studied at b i r t h and 6 months and infants at b i r t h , 6 and 12 months. Anthropometric measurements included weight, lengtha head and mid upper arm circumference (MUAC) and triceps f a t f o l d (16). The NCHS~ reference data f o r length, weight, head circumference (17) and f a t f o l d reference ~ata of Karlberg (18) were used for c l a s s i f i c a t i o n of under- and o v e r - n u t r i t i o n . Ponderal indices ( P I ) * were calculated f o r the IUGR newborns whose birthweights (BW) were < 2500 gm (19), This was done to d i f f e r e n t i a t e those i n f a n t s who are "malno~ished" with reduced weight f o r length (PI < 3rd p e r c e n t i l e ) from those who are p r o p o r t i o n a l l y growth retarded with comparable reduction in weight, length and head circumference (PI > 3rd p e r c e n t i l e ) . C l i n i c a l examination was used to detect physical findings representing n u t r i t i o n a l deficiency according to the c r i t e r i a of WHO and have a high degree of s p e c i f i c i t y for n u t r i e n t d e f i c i e n c i e s (16). *BCG - Freeze dried, supplied by Tuberculosis Research Laboratory, M i n i s t r y of Health, Nairobi, Kenya. This was freeze dried and reconstituted j u s t p r i o r to use and each batch potency tested. IpPD - 5 Tuberculin Units per 0 . I ml; Tuberculin p u r i f i e d protein d e r i v a t i v e s t a b i l i z e d solution (Parke Davis). 2NCHS -National Center f o r Health S t a t i s t i c s - - R e f e r e n c e data, USA 1976 (17). 50th percentile values = standard. 3

Wt/length >95th percentile or >120% standard = obesity

* PI : Weight in gm x i00 (19) Crown-heel length in cm

402

C.G. NEUMANNet al.

Biochemical studies included serum albumin and t r a n s f e r r i n ( 7 , 2 0 ) , i r o n (21), vitamin A and carotene (22), ascorbic acid (23), vitamin Blp (24), and whole blood f o l a t e (25). R i b o f l a v i n (26), p y r i d o x i n e (27) and t ~ a m i n (28,29) l e v e l s were evaluated by measuring g l u t a t h i o n e reductase a c t i v i t y , glutamate pyruvic transaminase index and t r a n s k e t o l a s e pyrophosphate e f f e c t r e s p e c t i v e l y . Normal values and methods are given in the references c i t e d . HEMATOLOGIC STUDIES: White and red blood cell counts, hemoglobin, hematocrit and derived erythrocyte indices were obtained using a Coulter Counter and white cell d i f f e r e n t i a l counts from Wright stained blood smears. STATISTICAL METHODS: S t a t i s t i c a l analyses used were Chi square, regression analysis, Student's t-Test multivariate and discriminant analysis. Results were considered significant i f p<.05 (30). The research proposal received f u l l approval by the Human Subjects Protection Committee at UCLAand by a comparable committee in Pumwani Maternity Hospital, Nairobi and by the Office of the President, Government of Kenya. RESULTS IMMUNOLOGIC FI NDINGS B-Cells: Both the mean percent and absolute numbers of B-cells in cord blood in the three infant study groups were similar and within the range for agematched normal American newborns (9) ITable I ) . A group of seven IUGR infants who were s l i g h t l y pre-term had values similar to the f u l l - t e r m IUGR infants (See Table 1). For the sake of comparison a group of six pre-term AGA infants from the same study population had a mean B cell level below both groups of IUGR infants (Table 1). TABLE 1 B-Cells* At Birth In Two Groups of Term Intrauterine Growth Retarded (IUGR) Newborns and Controls and in Preterm IUGR and AGA Newborns and IUGR < 2500 gm

W{"~-tu, ,,, and AGA ~ 2500 gm

35.2 _+ 0.4

33.4 _+ 0.8

(n = 7)

(n = 6)

P{' {:~t~::{'1{{

Group Birth weight (9m) Gestational Age (weeks) mean _+ SEM

I <__2500

II 2501-2799

<. . . . . . . . . (n = 46)

III >._2800

38.7 _+ 0.2 . . . . . . . . . (n = 38)

>

(n : 55)

Percent B c e l l s t

5.8 _+ 0.8 7.0 _+ 0.7

6.4 _+ 0.4

7.3 _+ 1.7

Total Numbqr of B Cells/mm~

484 _+ 166 551 _+ 92

483 _+ 81

721 _+ 285

4.4 ,+ 2.2 443 .+ 146

* A l l values expressed as mean .+ standard e r r o r of mean (SEM) tNormals: Percent B c e l l s 5-10%; Total number of B Cells 200-600/mm3(9,11) Immuno~lobulins: Immunoglobulin levels at birth in the mothers and infants are presented in Table 2. The mean maternal IgG levels of a l l groups exceeded those of USA adult levels (31). The mean IgG levels of Group I and I I mothers were s i g n i f i c a n t l y higher than IgG levels of mothers of control infants (Group I l l ) . IgM and IgA levels were similar in all groups and comparable to USA adult values. Maternal IgE levels were elevated in 71% of a l l mothers and to a comparable degree in all three groups.

IMMUNITY IN FETAL MALNUTRITION

403

TABLE 2 Maternal and I n f a n t Immunoglobulin Levels + of two Groups of IUGR I n f a n t s and Controls Group BW g No. Studied

I <2500 31

USA CONTROLS

II 2501-2799 24

III _> 2800 60

1383 • 77* 212 • 28 129 • 13 142 • 24 (n : I i )

1252 • 31" 198 • 9 130 • 6 155 • 24 (n : 10)

900 • 199 • 135 • 100 -

1409 883 915 937

• • • •

36 31 26 28**

1365 886 957 1111

I 13 6 ii

1 48 40 49

• • • •

5 7 3 3t

1• 52• 42• 54•

MATERNAL LEVELS IPOST PARTUM) IgG I~A 19M IgE

mg/dl mg/dl mg/dl IU/ml

1372 • 68* 191 • 14 142 • 12 120 • 19 (n : 19)

100 14 5 800

INFANT LEVELS IgG IgG IgG IgG

mg/dl mg/dl mg/dl mg/dl

IgA IgA IgA IgA

mg/dl mg/dl mg/dl mg/dl

cord 6 mo 7 mo 12 mott

IgM IgM IgM IgM

mg/dl mg/dl mg/dl mg/dl

cord 6 mo 7 mo 12 mott

IgE IgE IgE IgE

IU/ml IU/ml IU/ml IU/ml

cord 6 mo 7 mo 12 mo

+ * ** t tt tt

cord 6 mo 7 mo 12 mott

1348 879 1060 1125

1383 1275 940 1082

• • ~ •

29 24 25 61

71 58 85 64**

2 43 44 62

• • • •

1 2 5 5t

2 56 45 62

13 95 114 126

• • • •

2 8 I0 12~

16 86 89 113

• • • •

3 9 8 12

14 85 93 96

• • • •

1 4 7 5f

14• 88• 99• 107 • 4

2 41 56 68

• • • •

i 15 17 21

4 96 81 83

• • • •

1 27 20 24

1• 27 • 36 • 31 •

0 9 19 8

12• 38• 36• 41•

• • • •

77 57 46 53

• • • •

• • • •

Values expressed as Mean • SEM Group I vs. I I I and I I vs. I l l are s i g n i f i c a n t l y d i f f e r e n t , p<.05 Group I vs. I l l , are s i g n i f i c a n t l y different, p < .05. Group I vs. I I I , are s i g n i f i c a n t l y d i f f e r e n t , p < .01. Group I vs. I I I , are s i g n i f i c a n t l y different, p < .001. 12 mos sample size reduced by approx. 20% (lost to followup)

Newborns i n each of the t h r e e groups had comparable l e v e l s of IgG and IgM and were s i m i l a r to USA newborns ( 3 1 ) . However, IgA was d e t e c t a b l e i n a t h i r d of a l l cord bloods. No c o r r e l a t i o n s were found between BW and cord blood IgG l e v e l s except t h a t the mean IgG l e v e l s i n Group I and I I were s l i g h t l y lower than i n Group I I I . Pre-term infants, both IUGR and AGA had similar IgG leve]s (Mean • SEM): 1045 _+ 60 mg/dl and 1011 • 145 mg/dl respectively which were considerably below the mean of 1342 • 74 mg/dl for the term IUGR infants. The IgM and IgA levels increased rapidly after birth and IgG levels reached the adult range by age 6 months, increasing further by 12 months. IgG and IgM levels in Group I infants at 12 months were s i g n i f i c a n t l y higher than in controls. The accelerated increases in IgG and IgM between 6 to 7 months occurred shortly after the stimulus of measles vaccine given at 6 months.

404

C.G. NEUMANNet al.

Of particular interest are the elevated levels of IgE noted in a subset of the study infants. Seven of 41 newborns had IgE cord blood levels > 5 IU/ml. In three of these the IgE levels were extremely high, and because IgMTnd IgA were also elevated, i t is l i k e l y that a maternal-fetal leak accounted for these findings. Excluding these three infants, there were four newborns with cord IgE levels ranging from 5 to 20 IU/ml. In a l l four, the corresponding maternal IgE levels were > 100 IU/ml and all but one infant had persistently elevated levels of IgE at 6 and 12 months of age. IgE levels increased rapidly over the f i r s t year of l i f e in all infants so that 78% of infants had elevated IgE levels at 6 and 12 months, particularly infants in Group I and I I . Regression analysis is suggestive of a negative correlation between percent T cells and IgE levels at 6 and 12 mos. but r values are only suggestively significant (p = .08). Complement: Levels of complement components C3 and C4 were comparable to USA newborns and older infants (12). Cord blood levels of C3 in a l l groups ranged from 60 to 80% of maternal levels and C4 from 45 to 50% of maternal levels; these both increased appreciably by 6 months of age. C3 levels were consistently but not s i g n i f i c a n t l y higher in Groups I and I I at birth and 6 months compared to the control group, but by 12 months a l l groups were similar. C4 levels were similar in all groups at all ages. No correlations were found between C3 or C4 levels and birth weight. L~/mphoc~te Counts: Total lymphocyte counts are presented in Table 3. The smaller IUGR infants (Group I ) had s i g n i f i c a n t l y lower mean counts than Group I l l at b i r t h , 6 and 12 months. Mild lymphopenia (lymphocyte count < 2500 cells/mm3) was present in 6 to 16% of all groups at birth and 6 months but at 12 months was s t i l l present in 6 to 9% of the IUGR infants. TABLE 3 Total Lymphocyte Counts in Two Groups of IUGR Infants and Controls at Birth, 6 and 12 Months Group Birth Weight (gm) Number Studied

I < 2500 -- 40

II 2501-2799 36

III >__2800 63

Lymphocyte countt Percent Lymphopenictt

Birth

4558 • 289* 13%

5269 • 277 9%

6045 • 148" 16%

Lymphocyte count Percent Lymphopenic

6 mos.

4311 • 268* 7%

4326 • 247 6%

4998 • 259* 12%

4872 • 241" 6%

4645 • 355 9%

5307 • 286* 0%

Lymphocyte count PercentLymphopenic t tt * **

12 mos.**

cells/mm3^Mean • SEM < 2500/mmj Group I differs s i g n i f i c a n t l y from Group I l l , p<.05, a l l ages. 12 months sample size reduced by approximately 20% (lost to followup)

T Cells: Meantotal T-cell number was s i g n i f i c a n t l y lower at birth and 6 months in Group I compared to controls and s i g n i f i c a n t l y lower in both groups I and I I compared to controls at 12 months (see Table 4). In both groups of IUGR infants, mean T-cell percents were s i g n i f i c a n t l y lower in Groups I and I I compared to the controls (Group I l l ) at birth (Table 4, and Fig. l ) . At 6 n~nths all three groups had similar percent T-cells, but at 12 months the Group I IUGR infants had s i g n i f i c a n t l y lower mean percent T-cells than found in Groups I I and

IMMUNITY IN FETAL MALNUTRITION

405

III. In addition, the percent of infants with low percent T cells was considerably higher in Groups I and I I compared to Group I l l controls at birth, 6 and 12 months (Table 4). TABLE 4 T LYMPHOCYTES* IN TWO GROUPS OF INTRAUTERINE GROWTH RETARDED INFANTS AND CONTROLS AT BIRTH, 6 AND 12 MONTHS Group B i r t h Weight (Gm)

Total T Cells

I < 2500 (n : 44) 2215 • 162f

( c e l l s / m m 3) B i r t h

Percent T Cells Percent Reduced T Cells

II 2501-2799

" "

49.2 • 2.3f 46

III > 2800

(n : 37) 2564 • 160

(n = 61) 2961 • 189r

50.9 • 1.9r 38

61.9 • 1,2r 10

(n : 40) 2723 • 174 57.0 • 1.3 I0

(n : 76) 2885 • 171r 56,2 • 0.9 7

(n : 29) 2625 • 187r 58.3 • 1,9 16f

(n : 54) 3296 • 182r 60 .+ O , 9 t t 6r

(<50%)

(n : 28) 6 mo 2443 • 133t " 55.9 • 1.3 " 21

Total T Cells Percent T Cells P e r c e n t Reduced T C e l l s

(n = 38) 12 mo 2635 • 164t " 53.8 • 1.6r162 " 22r

Total T Cells P e r c e n t T Cel Is P e r c e n t Reduced T Cel Is

*Lymphocytes forming rosettes with sheep erythrocytes; Mean • SEM. tGroup I and I l l and I I and I l l d i f f e r s i g n i f i c a n t l y : p < .01. ttGroup I and I I I , d i f f e r s i g n i f i c a n t l y : p<.03 BIRTH (CORD BLOOD) 1T ]1I Group: I Birthwt.(gm):<2500 2501-2799 >2800 8O

-

AGE 12 MONTHS(FOLLOW-UP) 1 1T I]I <2500 2501-2799 __>2800

9

70

| 9

60

~; *

99, .

ee

.:-

50 'I -

4O 5O

T ""

" i

"I"

""

ee

.:.

2O 9~ Mean+- SE

FIG. 1 Percent T-Cells (E Rosettes) in Intrauterine Growth Retarded Infants and Controls at Birth and 12 Months

406

C.G. NEUMANNet a l .

Further examination of Group I IUGR infants by BW categories is presented in Table 5. Those infants with BW 1500 to 2000 gm had considerably lower total number and percent T cells than those infants with BW 2001 to 2500 gm. Only the infants with BW > 2251 gm returned to normal T cell levels by 12 months. TABLE 5

T Lymphocytes* in Three Birth Weight Groups of IUGR Infants at Birth and Twelve Months BW Group (gm)

No

Age

1500 - 2000

5

2001 - 2250

6

2251 - 2500

30

0 12 0 12 0 12

mo mo mo mo mo mo

Total T-cells (Mean ~ SEM) 2213 3095 1557 2099 2514 2768

• + • ~ + +

529 104 275 378 237 350

% T-cells IMean + SEM) 44.0 45.8 49.8 49.6 49.7 56.4

~ C • ~ ~ •

7.7 8.5 5.2 3.6 2.7 1.8

* Lymphocy~es forming rosettes with sheep erythrocytes; values expressed as cel I s/ram~ Differences in T-cell number and percent were found in Group I IUGR infants according to ponderal index (PI), see Table 6. The infants with PI > 3rd percentile, indicative of long term intrauterine growth retardation with comparable reductions in weight, length, and head circumferences (HC) for gestational age, had a s i g n i f i c a n t l y greater T-cell reduction than did the group with PI < 3 percentile. The group with PI < 3 percentile, indicative of malnutrition r e l a t i v T l y late in pregnancy with a greater r e l a t i v e reduction in weight than length and HC, had a higher mean T-cell percent and number. When group I I infants were s i m i l a r l y divided into groups by PI, similar T-cell differences were found as in Group I . TABLE 6 T-Lymphocytes in Intrauterine Growth Retarded Infants With Ponderal Indices (PI) < 3RD Percentile* and > 3RD Percent'ile ** PI < 3rd Percentile* ("~ainourished") 0 mo

12 mo

PI > 3rd Percentile * * ("growth retarded") 0 mo

12 mo

Birth Weight < 2500 gm

% T-cells (Mean + SEM)

n = 13 54.0 + 2.8

n = 8 55.6 + 3.3

n = 33 49.5 + 3.7

n = 20 54.1 + 1.7

Gesta t i o n a l Age > 37 w e k s

Total T - c e l l (Mean + SEM)

2551 + 384t

2958 + 520

1885 + 163t

2462 + 183

*

PI < 3 percentile (malnourished-greater weight d e f i c i t than length and head circu--mference d e f i c i t ) * * PI > 3 percentile (growth retarded-head circumference, weight and length equally retarded) t Significant differences p <.05.

IMMUNITY IN FETAL MALNUTRITION

407

I n - V i t r o Lymphocyte Response to PHA The mean unstimulated counts per minute (CPM) f o r each of the three groups of infants were higher at b i r t h than at six months and twelve months. Group I l l (controls) had s l i g h t l y higher CPM than groups I and I I at b i r t h but values at six months and twelve months were comparable. Stimulated CPM values were also higher at b i r t h than at 6 and 12 months. At twelve months the CPM were s i g n i f i c a n t l y lower in groups I compared to group I I I . The stimulation indices a l l were in the normal range although the indices in groups I and I I were s i g n i f i c a n t l y lower than in group I l l (p < .05). Also, there were higher percentage of abnormally low indices (< 25) in groups I and I I compared to group I l l (see Table 7). TABLE 7 In Vitro Lymphocyte Responses to Phytohemagglutinin in IUGR Infants and Controls at B i r t h , 6 and 12 Mos. Group Birth Weight (g)

Unstimulated CPM* (Mean • SEM)

I < 2500

n = 17

n = 31

0 mo

1466 • 293

1679 • 416

2030 • 315

6 mo

464 • 64

0 mo

0 mo 6 mo 12 mo

Percent at birth with low indices (< 25)

525 • 83

1638 • 261

1217 • 188

253,030 • 26,079

222,517 • 18,558

240,462 • 97,591

38,885 6,024

44,816 • 19,711

183,859 • 30,348

195,860 • 12,274 t

• 12 mo

501 • 120

1606 • 314

6 mo

Stimulation Index** (Mean • SEM)

Ill > 2800

n = 26

12 m. Stimulated CPM* (Mean • SEM)

II 2501-2799

35,617 4,503

165,647 • 23,594t 194 • 42 88 • 12 116 • 17t t

13%

•

211 • 36 104 • 18 147 • 24t t

3%

172 • 26 162 • 18 233 • 29 t t

4%

- CPM CouDts Per min~.~ (Mean • SEM) ** Index= Stlmuiateo br~ xlO0 Unstimulated CPM t Group I d i f f e r s s i g n i f i c a n t l y from Group I I I , p < .005. t t Group I and I I d i f f e r s i g n i f i c a n t l y from Group I l l , p < .05. Delayed Cutaneous Hypersensitivity: PPD skin test results in response to BCG vaccine administered at birth are shown in Fig. 2. At 6 months of age, the mean skin induration was lowest and the percent of negative reactors (induration < 5mm) highest in Group I . Mean induration for Groups I , I I and I l l respectively were 5.3 ram, 10.6 mm and 11.3 mm at 6 months with s i g n i f i c a n t l y less induration in Group I compared to Group I I I (p < .025).

408

C.G. NEUMANN et al.

Group: _I Birthwt.(gm): <2500

IT 2501-2799

> 2800

QO

O0

Tn"

20

15 9

000 O0 O001 000 0000 O0

000

10

.+

c

000

9

38 29 8 % Negotive Reoc'fors (
* No:

Groups I = 16; I I = 27; I I I =

26

FIGURE 2 Delayed Cutaneous Hypersensitivity to 5 TU Units of PPD at 6 Months of Age Folloiwng BCG Vaccine Given at Birth* NUTRITIONAL FINDINGS Anthropometr~: Mothers of both groups of IUGR infants were s i g n i f i c a n t l y l i g h ~ r (p < .001) and s l i g h t l y shorter than the mothers of Group I l l infants. Mean maternal post-partum weights, heights and fatfolds are presented in Table 8. By 6 months post-partum, the weight differences were even greater between Group I I I mothers and those of Groups I and 11 mothers. Twelve percent of the mothers of Group I were hypertensive compared to none in the other groups. TABLE 8 Maternal Post Partum Anthropometry* by Birth Weight Groups Group BW gm No. Weight (kg)

0 mo 6 mo

Height (cm) Triceps Fat Fold (mm)

I < 2500 49

II 2501-2799 44

56.6 _+ 2.6** 54.8 • 4.8**

56.9 • 1.8 59.3 • 1.1 55.1 • 1.8"* 59.6 • 1.3

159.5 • 2.4

159.1 • 2.0

14.8 • 1.6 15.9 • 1.3

All measurements: Mean _+ SEM ** Differ s i g n i f i c a n t l y from Group I l l mothers, p < .001.

Ill > 2800 62

160.5 • 0.7 15.6 • 0.7

IMMUNITY IN FETAL MALNUTRITION

409

The IUGR infants of Groups I and 11 remained smaller in all parameters, at 12 months of age, compared to Group I l l control infants. Stunting was present at 12 months of age in ll% of Group I and 6% of Group I I IUGR infants and in none of the Group I l l infants. Three percent of the IUGR infants in Groups I and I I developed severe PEM and 28% and 26% of the Group I and I I infants respectively developed moderate PEM by 12 months of age compared to none in the control group. Significant although low level correlations were found between neonatal and post-partum maternal anthropometric measurements. The following maternal anthropometric parameters correlated with birth weight: maternal height (r = 0.3, p < .04); maternal weight (r = 0.2, p < .03); maternal head circumference (r = 0.2, p < .03) and maternal triceps fatfold (r = 0.2, p < .02). Obesity developed in a number of infants in a l l groups. At 6 months, overweight and obese infants comprised 28%, 17% and 26% of Groups I , I I and I I I respectively. At 12 months, overweight and obese infants were seen in 20% of Group I l l infants and in 3% in each of the other groups. Triceps fat fold measurements > 95th percentile in these infants support the above data. Until 6 months of age a l l infants were breast-fed on demand and about half received commercial milk and cereals. By 12 months, breast-feeding declined as infants in a l l groups were being weaned and placed on solid and semi-solid foods. Specific Nutrients Mothers: Post-partum mean albumin and t r a n s f e r r i n l e v e l s , although in the norm~ range, were s i g n i f i c a n t l y lower in Group I mothers compared to those of Groups I I and I I I . Widespread subclinical biochemical f o l i c acid, thiamin and pyridoxine deficiencies were present in mothers in each of the three BW groups. Iron deficiency and microcytic anemia, using serum iron and erythrocyte indices as c r i t e r i a , were not seen in parturient mothers. However, macrocytic anemia was more prevalent in Groups I and I I than in Group I l l mothers with macrocytic red blood cells (MCV > 94) found in 17%, 10% and 3% of Groups I , I I and I I I mothers respectively- (Table 9). By 6 months post-partum, more mothers of the IUGR infants had folate, thiamin, pyridoxine and vitamin A deficiencies than did mothers of controls (Table 9). Infants: Subclinical biochemical pyridoxine, thiamin, vitamin A and carotene d e f i c i e n c i e s were found in a l l 3 groups of infants at b i r t h , 6 and 12 ,months with abnormally low l e v e l s of albumin, t r a n s f e r r i n and pyridoxine more prevalent in Groups I and I I than in Group I I I (Table 9). Iron deficiency and microcytic anemia developed in over 30% percent of infants by 6 months and in over 50% of a l l infants by 12 months and were more severe and more prevalent in the Group I and I I i n f a n t s than in controls (Table 9).

410

C.G. NEUMANNet a l . TABLE 9 Selected Nutritional Abnormalities in Intrauterine Growth Retarded Infants and Their Mothers

Group Birth Weight (gm) Number Studiedt At Birth Mothers (postpartum)

Infants (Cord blood) At Six Months Mothers (6 months)

Infants (6 mo) At Twelve Months Infants (12 m o s )

I < 2500 31 A1bumin Transf e r r i n Hemoglobi n Megal oblastic changes

% Deficient 3 3 11 17

II 2501-2799 24 % Deficient 4 5 15 10

Ill > 2800 61 % Deficient* 0 0 9 3

A1 bumi n Transferrin Pyridoxi net@

13 36 70

17 33 69

5 3 45

Folic Acid Thiamin Vi t . A Pyridoxi ne

14 31 31 65

13 11 Ii 75

7 18 18 32

Iron Pyridoxine Hemog1obi n

37 71 43

28 69 36

32 41 24

Iron Pyridoxine Hemoglobin

37 43 67

28 68 63

32 44 46

t Approximately 20% a t t r i t i o n of sample size by 12 months. @@Erythrocyte glutamic pyruvic transaminase (EGPT index). * Actual data w i l l be supplied upon request. Anthropometric Status and CMI The degree of intrauterine growth retardation was the most potent determinant of decreased T cells at b i r t h , both by Chi Square and discriminant analyses. Group I and I I IUGR infants as a separate or a combined group, compared to Group I I I controls, showed s i g n i f i c a n t correlations between BW and total number and percent T c e l l s . Group I and I I infants had a s i g n i f i c a n t l y greater proportion of infants with low percent T cells than did Group I l l (Table 4). Low BW was a better predictor of low percent T cells at 6 and 12 months than whether or not the IUGR infants showed catch up growth to control infant levels. Arm circumference at both 6 and 12 months of age showed a s i g n i f i c a n t negative correlation with percent T-cells (r = -.40 to -.83, p < .001) suggesting an association between obesity and diminished CMI.

IMMUNITY IN FETAL MALNUTRITION

411

TABLE 10 S i g n i f i c a n t C o r r e l a t i o n s * Between Cell-Mediated Immunity and N u t r i t i o n a l Variables i n IUGR I n f a n t s at B i r t h , 6 and 12 Months

Age

CMI Variable

Nutritional Variable Bi r t hweight for gestational age

Birth

Percent T-Cells and/or Total T-cel Is

Thiamine Iron Hemoglobin

6

Percent T-cells and/or Total number of T-cel I s

Bi rthweight for gestational age Weight/length at birth Arm circumference at 6 mos. Riboflavin Pyridoxi ne Hemogl obi n Iron

mos.

12 mos.

Percent T-cells and/or Total number of T-cells

Length for age at 6 mos. Arm circumference at 6 mos. Thi amine Folic acid

Coefficient(r)

P Value

F = 6.4it

.39 .35 .21

a l l P values between .001-.05

.22 .27 -.30 .34 .23 .30 .27 .13 -.40 .27 .25

a l l P values between .001- .05

a l l P values between .001-.05

* by r e g r e s s i o n a n a l y s i s f t by d i s c r i m i n a n t a n a l y s i s

Recovery of cell-mediated immunity according to anthropometric status was closely examined in 47 infants with low percent T cells at birth and who had complete follow-up data. The 4 infants that had low percent T cells at birth that persisted for 12 months were a l l were IUGR infants (three in Group I and one in Group I I ) . These infants had weight/age and length/age measurements below the 5th percentile, indicative of stunting. By 12 months, one of the infants became obese and one developed severe PEM. In the group of 7 infants with low percent T cells at birth and 6 months, who normalized by 12 months, 3 were IUGR (Group I) and 4 were of normal BW. At 6 months, 2 of the infants were stunted and 3 others were obese. Two remained obese and 2 infants developed moderate PEM by 12 months. Of the 36 infants with low percent T cells at birth who recovered by 6 months, 23 were IUGR (12 in Group I and 11 in Group I I ) and 13 were of normal BW (Group I I I ) . The majority of infants had normal weight/length percentiles both at 6 and 12 months, but 3 were stunted, 2 had moderate PEM and 3 were obese. Seventeen infants with normal percent T cells at birth and 6 months developed low percent T cells by 12 mos of age. Eleven were IUGR (7 in Group I and 4 in Group I I ) . At 12 months, four were stunted, two had developed severe PEM and 3 infants became obese.

412

C.G. NEUMANN et al.

Correlations of Cell-mediated Immunity and Specific Nutrients T cell percent and T cell numbers were s i g n i f i c a n t l y correlated with specific nutrients (Table 10). In the newborn period, there was significant correlations between percent T cells and hemoglobin and thiamin and between total T cell number and serum iron. In the infants at 6 months of age there was a s i g n i f icant correlation between total T cell numbers and hemoglobin, iron, riboflavin and pyridoxine. In the infants at 12 months of age, there was significant correlations between total T cells and whole blood f o l i c acid, thiamine, r i b o f l a v i n and pyridoxine. Clinical Infectious Disease Experience C l i n i c a l l y diagnosed i n f e c t i o n s were reported as i l l n e s s episodes per 100 c h i l d r e n . Despite the highest a t t r i t i o n rates and poorest c l i n i c attendance the Group I i n f a n t s had the highest i l l n e s s rates in the 0 to 3 mo and 7-10 mo age group periods with higher rates in males than females (Table I I ) . TABLE 11 Infectious Illness Episodes Per I00 Children In Two Groups of IUGR Infants and Controls Group

I Male Female

0 - 6 mo 7 - 12 mo

192 425

193 392

II Male Female 170 340

100 380

Ill Male Female 109 318

121 307

Comparing CMI status to c l i n i c a l i l l n e s s , these infants, mainly in Group I , with > 10 c l i n i c a l infectious illness episodes during the 0-6 mo period had a four-Told higher percent of abnormally low percent T cells (< 50%) compared to the infants with < 10 illnesses. In addition, lower respiratory infections, thrush and pertussis were seen 2 to 3 times more frequently in the 0-6 mo old age period in infants who had low percent T cells at birth and 6 roDS compared to those with normal T cell values. During the period from 7 to 12 roD, diarrhea, o t i t i s media and tuberculosis were seen approximately 1.5 times more frequently in the infants that had low percentage of T cells at birth and 6 roDS than among those with normal percent T c e l l s . DISCUSSION Decreased cell-mediated immunity was found to be s i g n i f i c a n t l y more prevalent and severe in intrauterine growth retarded newborns with BW < 3rd to < lOth percentile for gestational age than in f u l l - t e r m normal infants'-with BW > lOth percentile. This is expressed by diminished total lymphocyte counts, Teduced total number and percent T cells and diminished delayed cutaneous hypersensitivity. By contrast, i n - v i t r o lymphocyte responsiveness to phytohemagglutinin, a strong mitogen, was generally normal which may be attributed to the relative i n s e n s i t i v i t y of PHA lymphocyte stimulation in detecting subtle immunodeficiency. Howeverthe stimulation indices were s i g n i f i c a n t l y higher in the control group than in both groups of IUGR infants.

IMMUNITY IN FETAL MALNUTRITION

473

IUGR i n f a n t s , not o f f i c i a l l y labeled "low b i r t h weight" by the World Health Organization (32) but who are between 2501 to 2800 gm (BW 3rd to lOth) percentile are analogous, we b e l i e v e , to infants and children with mildmoderate PEM and indeed they grew less well than control i n f a n t s during the f i r s t year of l i f e . These infants showed impaired CMI but to a somewhat lesser degree than the < 2500 gm BW group. In developing countries there are large numbers of such iTfants who are p o t e n t i a l l y at increased r i s k for i n f e c t i o n (1,32). The findings reported here are in agreement with the results of several other studies of immune function in IUGR i n f a n t s (2-4,33). The IUGR i n f a n t s in these studies were born to mothers with toxemia, hypertension or presumed maternal m a l n u t r i t i o n which was not documented (2-4,33). The degree of CMI impairment is considerably less than i s found in severe post-natal PEM where T c e l l s are as low as 15 to 20% and i n - v i t r o lymphocyte p r o l i f e r a t i v e response to PHA i s markedly diminished.(34,35) The impaired CMI persisted for at least 6 months in 23% and for 12 months in 13% of the IUGR infants in this study. Persistence of an immunologic defect in IUGR infants for several years after birth has been reported by Chandra (36), who noted T cell depression for at least two years in a high percent of IUGR infants, most of whom remained growth retarded. Ferguson (37) documented decreased delayed cutaneous hypersensitivity in 4 of 8 children who were IUGR at birth, for as long as 5 to 7 years, none of whom were malnourished at follow-up. By contrast, recovery of cell-mediated immunity in postnatally acquired PEM i s rapid, 7 to 14 days following nutritional rehabilitation (34,35). The ponderal index (PI) could be of use in predicting the c e l l u l a r immune status of the IUGR infant. I t appears from our data that the IUGR infants (PI > 3rd percentile), who are proportionately growth retarded with somewhat comparable reductions in weight, height and head circumference, had a s i g n i f i c a n t l y greater degree and duration of CMI depression than did the IUGR infants with PI < 3rd percentile indicative of malnutrition with greater reduction in weight than for height and head circumference. The insult in the proportionately gro~h retarded group presumably started e a r l i e r in pregnancy than in the malnourished group and appears to cause a more profound disturbance to the developing immune system. Some of the IUGR and control infants without functional abnormalities at birth developed decreased CMI by one year associated with PEM, stunting or obesity. In addition, several infants with normal growth also developed evidence of decreased CMI possibly associated with viral infection, which is known to temporarily suppress CMI (38). A number of infants in a l l BW groups developed obesity. The highly significant negative correlation between mid-upper arm circumference and percent T cells suggests an association between obesity and depressed CMI. In the IUGR infants, the relative contributions of IUGR versus subsequent obesity at 6 months is not known. The IUGR infants may have invoked s u f f i c i e n t maternal anxiety by their f r a i l appearance and small size at birth and subsequent short stature so that the mothers " t r i p l e - f e d " them with breast milk, formula, and gruels or proprietary cereals in the f i r s t 6 months. This indeed was documented by diet histories. Obese infants have been found to have increased numbers of infection, especially lower respiratory infection (39) and Chandra has noted defects in cell-mediated immunity (40) in obese subj ect s. Several specific nutrient deficiencies implicated in decreased CMI in animal and human studies were found to be widespread throughout all groups of study infants and mothers and were found to be correlated with decreased CMI. The role of maternal deficiencies during pregnancy in causing IUGR as

414

C.G. NEUMANNet a l .

well as impairing CMI has been demonstrated in animals. Gebhardt et a l . (41) showed t h a t maternal deficiency of " l i p o t r o p i c substances" (BI2, f o l i c acid and choline) in rats resulted in IUGR o f f s p r i n g with dimunition of lymphoid t i s s u e . Pregnant rats rendered zinc d e f i c i e n t produced offspring with f e t a l growth retardation and decreased CMI (42). Pyridoxine deficiency in pregnant animals has resulted in IUGR progeny with decreased size of thymus and lymphoid t i s s u e (43). The effects of multiple d e f i c i e n c e i s upon CMI are probably addi r i v e . In human studies, maternal n u t r i t i o n a l deficiencies p a r t i c u l a r l y of f o l i c acid (44), protein (45) and c a l o r i e s (46) have been associated with o f f s p r i n g with IUGR and/or prematurity, but immune function was not studied. The growth of f e t a l lymphoid organs, e s p e c i a l l y the thymus and spleen are i n h i b i t e d by severe r e s t r i c t i o n of c e l l d i v i s i o n and p r o l i f e r a t i o n , A s i m i l a r i n s u l t to the developing immune system has been noted in post-mortem examination of low b i r t h weight term i n f a n t s born to mothers of low socio-economic status (46). In our study maternal weight was s i g n i f i c a n t l y lower in Group I mothers compared to mothers of controls. Differences in maternal weight and f a t f o l d between Group I and Group I I I mothers were even more pronounced a f t e r 6 months of l a c t a t i o n . Also maternal serum albumin and t r a n s f e r r i n levels were s i g n i f i c a n t l y lower in Group I compared to Group I I I c o n t r o l s . Although f o l i c acid deficiency was widespread among a l l groups, macrocytic anemia was much more prevalent among the mothers of the IUGR infants than in mothers of the control infants. In terms of CMI per se, infant levels of thiamin, hemoglobin and iron correlated s i g n i f i c a n t l y with percent and number of T cells in the newborn. The association of thiamin with CMI has not been reported. Reduced a v a i l a b i l i t y of nutrients due to reduced placental size or blood flow may also be associated with toxemia and hypertension and may cause fetal malnutrition. Hypertensionwas present in 12% of the mothers of the Group I IUGR infants and was not present in the mothers of the other two groups. Specific post-natal nutrient deficiencies of iron (47), zinc (42), f o l i c acid (48) and pyridoxine (35,43,49) have a l l been associated with diminished CMI in animals and humans. In our study iron, hemoglobin, thiamin, r i b o f l a vin, pyridoxine and f o l i c acid correlated weakly but s i g n i f i c a n t l y with CMI in the infants at 6 and 12 months. However, these suggestive findings must be confirmed by more refined studies. Congenital infection can cause IUGR (1) as well as suppress CMI (50). In our study only three infants had elevated cord IgM (>20 mg/dl) but these infants appeared with normal percent T cells at b i r t h . However, evidence for increased maternal infection in the mothers of both Group I and I I IUGR infants was suggested by higher IgG and IgM levels in Group I and I I mothers compared to mothers of control infants. Mata's findings of elevated cord IgM levels in IUGR infants, which he attributed to intrauterine infection, was not observed in our study (51). In regard to humoral immunity, B cell percent and number were comparable in all three groups with no significant differences seen in the IUGR infants. The seven IUGR infants that were s l i g h t l y pre-term (mean G.A. 35.2 wk) had values comparable to the f u l l term IUGR infants and controls. B-cell percent and number were found to be reduced by a third in a small group of six AGA infants (mean GA 33.4 wk) from the same general population as the study infants. Although in the normal range, mean IgG levels of the pre-term IUGR infants and AGA preterm infants were s l i g h t l y but not s i g n i f i c a n t l y lower than those of the term IUGR infants. The above findings have been noted by others (11). All groups had elevated IgM, IgA and IgG levels compared to U.S. agematched infants, p a r t i c u l a r l y by 12 months, probably due to increased infection exposure of Kenyan infants. An increase in IgG and IgM levels in

IMMUNITY IN FETAL MALNUTRITION

415

all groups between 6 and 7 months may also be due to the additional antigenic stimulus of measles immunization given at 6 months. After six months of age, Group I infants had consistently higher mean immunoglobulin and C3 levels than did controls, most l i k e l y reflecting an increased infection experience. Indeed the IUGR Group I infants i n our study had a higher rate of illness in the f i r s t 3 months of l i f e than did Groups I I and I I I . Also infants with decreased T cells experienced more illness episodes including lower respiratory infection, o t i t i s media, thrush, diarrhea and pertussis. These morbidity findings w i l l be discussed in detail in a subsequent paper. A notable finding is the elevated IgE levels (>5 IU/ml) in 10% of cord blood samples even after those infants with intrauterine leaks of maternal blood had been excluded. Selective activation of IgE was f e l t to be occuring as IgE is rarely found in cord blood (52). All of the mothers of IUGR infants had IgE levels > lOO IU/ml at the time of delivery compared to 30% in the Group I I I , mothers, although the mean value for control mothers was higher. The group I mothers were perhaps of a lower socioeconomic group than mothers of controls and may have had a greater prevalence of parasitic infection. A second feature is the frequency of elevated IgE in infants aged 6 and 12 months. About 40% of the IUGR infants and 21% of the controls had s i g n i f i cantly greater levels of IgE than U.S.A. age matched values (31). In addition to intestinal parasitism, this activation of IgE synthesis may be a result of diminished T-suppressor a c t i v i t y (53). We and others have reported elevated IgE levels in infants with postnatal PEM (35,52) and this study is suggestive of t h i s . Another explanation, particularly in infants with PEM, is increased permeability of the mucosa of the gastro-intestinal tract to food or enteric antigens that activate IgE synthesis (52). Depressed cell-mediated immunity in IUGR infants may result in increased susceptability to and diminished a b i l i t y to handle infection or respond to immunizing agents. Accordingly, those vaccines that are p a r t i a l l y T cell dependent may have decreased effectiveness. In this and one other study (54), PPD skin testing in IUGR infants given BCG at birth showed a higher percent of negative reactions and smaller mean skin induration than in control infants. Response to measles and pertussis immunizations are presented in a subsequent paper. The IUGR infant is at considerable risk for postnatal PEM which may further impair the c e l l u l a r immune system. Since the above events are occurring not only in infants weighing under 2500 gm, but in those term infants with BW between 2501 and 2800 gm, a large number of infants are at jeopardy for increased infection and malnutrition, particularly in developing count ri es. In view of the CMI impairment in IUGR infants, measures that w i l l decrease the p o s s i b i l i t y of fetal malnutrition would be expected to enhance the i n t e g r i t y of the infant's developing immune system, and protect the infant against infection. A comprehensive health care approach for pregnant women should include vigorous nutrition programs, prevention and early treatment of infection and detection and treatment of diseases adversely affecting the placental circulation such as diabetes, hypertension and malaria. ACKNOWLEDGEMENTS

The authors thank Dr. J.C. Likimani, former Director of Medical Services, Ministry of Health, Kenya for his cooperation and l o g i s t i c support; Dr. W.N. Mugo, former Director of Health Services of Nairobi City Council for permission to work in Pumwani Maternity Hospital; Mrs. Gladys Musiga, Public Health Nurse, Ministry of Health, Kenya for making possible longitudinal follow-up of study infants and Dr. Vlasak Houba and Z. Ahmed, WHO and Dr.

416

C.G. NEUMANN et al.

Anthony E. Butterworth, formerly of the Wellcome Trust Laboratory for laboratory back-up and technical assistance. At UCLA the authors thank Dr, Isabelle Hunt, Phyllis Gabriel, Norma Murphy for assistance with dietary intake and biochemical analyses, Dr. Virginia Clark for s t a t i s t i c a l consultation, and Bonnie Ank for technical assistance. REFERENCES 1.

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10. SENGAR, D.P.S., and TERASAKI, P.I, A semi-micro mixed leukocyte culture t e s t . Transplantation, I_j.I:260-267, 1971. 11. CAMPBELL, A.C., WALLER, C. WOOD, J. AYNSLEY-GREEN, A. and YU V. Lymphocyte subpopulations in the blood of newborn infants, Clin. Exp. Im~nol. 18:469-482, 1974. 12. JOHNSON, R.B,, ALTENBURGERM,S., ATKINSON S.W., and CURRYR. in the newborn. In: Host Defense in the Fetus and Neonate. 64(Supp):781-786, 1979.

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14. HANDZEL, Z.T., LEVIN, M.B., DOLPHIN, Z. et al. Immunocompetence of newborn lymphocytes. Pediatrics, 45:491-496, 1980. 15. SEEGER. R.C. and STIEHM, E.R. T and B lymphocyte subpopulation. Pediatrics, 55:157-160, 1975. 16. JELLIFFE, D.B. The Assessment of the Nutritional Status of the Community. WHOMonogr. Ser. Geneva 1966, p. 48. 17. National Center for Health Statistics (NCHS) Gro~r~h Charts, Monthl;l. Vital Stat Rep, 2_~5:Suppl. No. 3, 1976. 18. KARLBERG, P., ENGSTROM, L., LICHTENSTEIN, H., SVENNBERG I. The development of children in the Swedish community. I l l Physical growth during the f i r s t three years of l i f e . Acta. Paediatr. Scand. Suppl. 187: 48-65, 1968. 19. MILLER, H.C. and HASSANEIN, K. Diagnosis of impaired fetal growth in newborn infants. Pediatrics, 48: 511-522, 1971. 20. MANDEL, E.E. Immuno-plate human transferrin test. 1959.

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22. NEALD, J.B. JR. and PEARSON, W.N. Macro-and micro-methods for the determinations of serum vitamin A using trifluoracetic acid. J. Nutr. 7__~9.454-462, 1963. 23. GYORGY,P. and PEARSON, W.N. 2, 4 - Dinitrophenylhydrazine-thio ureacopper sulfate, modified Lowry, Lopez and Bessey procedures. In: The Vitamins. Academic Press, New York, 1967, p. 43. 24. SKEGGS, R.H. Vitamin BI~ In: The Vitamins, P. Gyorgy and W.N. Pearson eds. New York, 1967, p.282. Academic Press. 25. HOFFBRAND, A.V., NEWCOMBEF.A., MOLLIN, D.L. Method of assay of red cell folate and the value of the assay as a test for folate deficiency. J. Clin. Pathol. 19:17-28, 1966. 26. GLATZLE, D. KDRNER, W.F., CHRISTELLER, S. and WISS, O. Method for the defection of a biochemical riboflavin deficiency. Stimulation of NADPH 2 dependent glutathione reductase from human erythrocytes by FAO in vitro. Intern Vitamin Forsch. 40:166-183, 1970. 27. DISCHE, Z. 1953.

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Accepted for publication March 12, 1984.