【摘要】  Background: Nutritional status is highly dependent on season in countries such as The Gambia. In a rural Gambian setting, individuals born during periods of seasonal nutritional deprivation ('hungry seasons') are susceptible to mortality from infectious diseases in adult life.
Objective: We investigated the hypothesis that impaired immunocompetence in those born in the hungry season results from an underlying defect in immunologic memory, similar to the immunosenescence of old age, which is likely to be reflected in the phenotype and kinetics of T lymphocytes in young adults.
Design: T cell phenotype in terms of CD3, CD4, CD8, CD45RA, and CD45R0 expression and in vivo dynamics measured by stable isotope labeling of T cell subsets combined with gas chromatographyCmass spectrometry and frequency of T cell receptor excision circles were measured in 25 young (18C24-y-old) Gambian men. Thirteen of these 25 men were exposed to perinatal malnutrition as defined by birth season and birth weight.
Results: In persons born in the hungry season with low birth weight, no differences in the proportions of memory or naive T cells were found. Kinetic analysis showed higher proliferation rates in memory (CD45R0+) subsets of T cells than in nave (CD45R0C) cells, which is consistent with previous studies, but no evidence was found for an effect of birth weight or season on T lymphocyte proliferation and disappearance rates. No significant correlations were found between in vivo T cell kinetics and frequency of T cell receptor excision circles. Only absolute numbers of granulocytes were elevated in those born in the nutritionally deprived season.
Conclusion: In healthy young Gambian men, T lymphocyte homeostasis is extremely robust regardless of perinatal nutritional compromise.

【关键词】  nutritional programming cell kinetics lymphocytes gambia stable isotopes

INTRODUCTION

In humans, several studies have found relations between markers of nutritional status at birth and immunologic outcomes in adolescence and adulthood (1-3). However, the nature of the mechanisms by which these relations may occur remains a subject of debate (4).

In terms of critical target components, the thymus has long been known in animal models to be highly susceptible to fetal malnutrition. Starvation-induced changes include thymic involution, thymic atrophy, circulation of immature lymphocytes, and greater thymocyte apoptosis (5-8). Zinc deficiency leads to glucocorticoid-mediated thymocyte apoptosis and consequently to decreased lymphopoiesis. Such losses of precursor T and B cells ultimately result in lymphopenia and thymic atrophy (9). In magnesium-deficient weaned rats, thymic involution appears as early as 7 d after the introduction of a magnesium-deficient diet and is associated with an increase in sites of active cell death within the thymus (8, 9).

Because thymic development mainly occurs in utero and in early postnatal life, a nutritional insult at a critical stage in thymic development may lead to a permanent impairment in T cell immunity. In The Gambia, further studies have shown that infants have a smaller thymus (10), a lower CD4:CD8, and lower concentrations of T cell receptor excision circles (TRECs; 11) during the hungry season than during the harvest season. Lower TREC concentrations are associated with lower concentrations of interleukin (IL) 7 in the breast milk of mothers (11). In addition, infection-related mortality in adults born during the hungry season was 10 times that in adults born during the harvest season; these findings indicate a long-term effect on immunocompetence of season of birth (12, 13).

An early nutritional insult to the thymus may therefore have long-term consequences for thymic activity and immunocompetence, similar to the effects immunosenescence (aging of the immune system), which can be characterized by morphologic and functional changes in the thymus (14). In the elderly, the decline in thymic activity with age results in a decrease in thymic output as measured by TRECs (15). A nutritionally deprived thymus at birth may be reset to produce smaller numbers of T cells in later life, leading to long-term effects on the numbers of recent thymic emigrants (RTEs) within the peripheral T cell pool and alterations in T cell homeostasis that parallel those observed during the aging process but at an accelerated rate.

The effects of perinatal malnutrition on specific components of T cell immunity have thus far not been defined. We therefore set out to explore the hypothesis that a defect in the underlying kinetics and distribution of T cells may explain observations of decreased immunocompetence in those born with nutritional deprivation.

SUBJECTS AND METHODS

We measured T cell phenotype and turnover and TREC concentrations in young Gambian adults whose birth details were known. Field studies were conducted at the Medical Research Council field station in Keneba, The Gambia, which is located in a rural area with a population predominantly made up of subsistence farmers for whom nutrition has a very predictable seasonality. Because migration has been relatively limited and because detailed birth records have been kept and collated in this area since 1949, it was possible to select young adults on the basis of their season of birth and birth weight. These characteristics were used as surrogate markers for nutritional status. Previous studies (13, 16) have shown that the effect of reduced food availability is maximal during the wet season (September through November) and that food is most available during the dry, harvest period (February through April); these periods were therefore defined as the 'hungry' and 'harvest' seasons, respectively, for the purposes of this study. To maximize the chance that the difference in exposures was nutritional, subjects were further defined according to birth weight, and 2 groups were identified. Group 1 consisted of those with hungry-season birth and with birth weight below the population mean?ie, those born in the months characterized by extreme food shortage and weighing <3 kg at birth. A high likelihood exists that these persons experienced some form of nutritional deprivation in utero and early postnatal life, and they are therefore referred to as nutritionally deprived. Group 2 consisted of those with harvest-season births and birth weight above the population mean (>3 kg). These criteria ensure minimal exposure to malnutrition in utero and early postnatal life, and the group is referred to as nutritionally replete.

Twenty-eight healthy young (aged 18C24 y) men for whom date of birth and birth weight data were available were recruited. Preterm births as defined by gestational age < 38 wk were excluded from the study to eliminate the potentially confounding effect of premature birth. Subjects were screened with a medical questionnaire, fixed blood film, hemoglobin measurement, and urine dipstick testing; they were excluded if found to have any current medical condition, anemia, parasitemia, or glycosuria. Sample size was based on pragmatic criteria for a descriptive study. At the time the study was designed, no reliable comparable CV data were available for lymphocyte lifespan studies.

The study was explained to subjects in their local language with the use of trained fieldworkers, and written informed consent was obtained. All procedures were approved by the Joint Gambia Government/MRC Gambia Ethics Committee and the Scientific Coordinating Committee (SCC) of the MRC Gambia.

Differential count

Differential counts were conducted manually on thin films of blood that had been drawn into EDTA and then stained with Leishman?s stain for 2 min and buffered at pH 6.8 for 8 min. One hundred cells were counted and classified as neutrophils, lymphocytes, monocytes, eosinophils, or basophils. Total granulocytes were calculated as neutrophils + eosinphils + basophils.

T cell kinetics

T cell kinetics were measured by using deuterated glucose labeling of cellular DNA as detailed elsewhere (17, 18). In brief, subjects received deuterated glucose (6,6-D2 glucose) orally at half-hour intervals and were given frequent small meals over a period of 10 h. Blood samples were drawn at baseline (10 mL) and on days 3 and 10 after labeling (28 mL). Peripheral blood mononuclear cells (PBMCs), isolated by density gradient centrifugation, were separated on site by antibody-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) into 4 T cell subsets (CD8+CD45R0+, CD8+CD45R0C, CD4+CD45R0+, and CD4+CD45R0C) by using a protocol designed to ensure maximal yield but minimal contamination of low-turnover R0C cells with high-turnover R0+ cells, as described elsewhere (17). For some samples, on-site magnetic bead sorting was not possible because of logistic constraints. In these cases, samples were frozen, shipped, and separated by flow cytometry sorting as described previously (18). The number of samples handled in this way was similar for the 2 study groups, and reanalysis of the final data, including analysis of the method of separation as an independent variable, did not affect the results. Aliquots of cell subsets were stained for purity analysis, fixed and stored at 4 ?C, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Oxford, United Kingdom) within 7 d.

T cell subsets were analyzed for deuterium incorporation into DNA (18, 19) at 3 and 10 d after labeling. This analysis was conducted by resuspending cells in RNAlater (Ambion, Austin, TX), before DNA extraction, derivatization, and gas chromatographyCmass spectrometry analysis (5973/6980 GCMS; Agilent Technologies, Bracknell, United Kingdom) of the aldonitrile tetraacetate derivative (17).

Published modeling approaches (18, 20) were adapted to describe the appearance and disappearance of labeled cells (17). Two variables, proliferation (p) and disappearance (d) rate constants, were estimated with the use of nonlinear least-squares regression (Levenberg-Marquardt method) to fit the model for experimental data.

Flow cytometry

Whole blood (200 L) was stained for 3-color flow cytometry with CD3-PE, CD8-cychrome, and CD45RA-FITC (Becton Dickinson).

T cell receptor excision circles

One hundred ng DNA extracted from separated T cells (CD4+CD45RA+, CD4+CD45R0+, and CD8+CD45RA+) was added to a mix of 1x Quantitect SYBR Green polymerase chain reaction (PCR) master mix (Qiagen, Crawley, United Kingdom), 200 ng/mL BSA, 1.5 mmol MgCl2/L, 0.5 mol forward primer/L (5 agg ctg atc ttg tct gac att tgc tcc g 3), and 0.5 mol reverse primer/L (5 aaa gag ggc agc cct ctc caa ggc aaa 3). Signal joint TRECs (sjTRECs) were amplified and directly quantified by using the Light Cycler (Roche Diagnostics, Mannheim, Germany) and known starting numbers of standard sjTREC molecules.

Real-time PCR was then performed with the use of Quantitect SYBR Green. PCR conditions were an initial activation step at 95 ?C for 15 min, which was followed by 40 cycles of denaturation at 95 ?C for 5 s, annealing at 60 ?C for 25 s, extension at 72 ?C for 20 s, and a fluorescence acquisition step at 84 ?C for 5 s. Samples were analyzed in triplicate (average CV: 12%), and the mean number of copies was taken as the final concentration.

Statistical analysis

All data were normally distributed as assessed by using a Shapiro-Wilk W test and are therefore presented as means ? SDs or means ? SEMs. Data were compared by using the Student?s t test, and P < 0.05 was considered to be significant. We conducted a 2-factor multilevel analysis to investigate an underlying interaction between nave and memory T cell proliferation, disappearance, TRECs, and nutritional status at birth. We used SPSS software (version 10; SPSS Inc, Chicago, IL) for all statistical analyses.

RESULTS

Subjects and nutritional variables

Forty-nine eligible subjects were identified on the basis of birth weight and season of birth. Nine of the 49 were not located, and 8 refused participation. Of the 32 who gave informed consent, 4 subjects were excluded after screening for malaria and other infections. Of the 28 subjects therefore entered into the study, 13 were born in the hungry season with a birth weight < 3 kg, and 15 were born in the harvest season with a birth weight > 3 kg. Mean age and current nutritional status did not differ between the 2 groups. Mean birth weight, age, and current body mass index (BMI) of subjects studied are shown in Table 1; differences between the 2 groups were tested by using a 2-tailed Student?s t test.

TABLE 1 Mean birth weight, age, and BMI of subjects according to perinatal nutritional exposure

White blood cell counts and lymphocyte phenotype

In persons perinatally exposed to nutritional deprivation, a trend was seen toward higher total white blood cell counts and a higher percentage of granulocytes than in subjects with nutritionally replete births. When absolute cell counts were compared, it became obvious that the apparent leukopoenia in those born nutritionally replete could be fully attributed to low numbers of granulocytes. This difference in granulocyte count is shown in Table 2 (P = 0.01, Student?s t test).

TABLE 2 White blood cell (WBC) and differential cell counts in subjects according to perinatal nutritional exposure

Whole-blood phenotyping according to markers of nave and memory T cell subsets was conducted to investigate a potential accumulation of memory T cells (typical of immunosenescence) in those born with nutritional deprivation in Keneba. Lymphocyte subsets as a proportion of CD3+ cells showed no significant differences in CD4:CD8 and nave and memory cell distribution by perinatal nutritional exposure when compared with the use of a two-tailed Student?s t test (Table 3). This finding is contrary to the hypothesis that subjects with nutritionally deprived births would have higher proportions of memory cells because of premature immunosenescence.

TABLE 3 Mean percentage of CD3+ lymphocyte subsets according to perinatal nutritional exposure

In vivo T cell kinetics

To investigate the hypothesis that perinatal undernutrition affects the life span of lymphocyte pools, T cell proliferation and disappearance rates were analyzed for differences by perinatal nutritional exposure. A flaw in the capacity of persons born with nutritional deprivation to maintain T cell homeostasis may predispose those persons to premature adult death from infection.

T cell kinetic analyses were completed in 25 subjects (in 3 others, phenotype data are available but follow-up samples were lost). Mean data for T cell subsets in the 2 groups of subjects?those exposed and those not exposed to perinatal nutritional deprivation?are shown in Figure 1. Because higher peak (day 3) fractional enrichments indicate faster rates of proliferation of the cells within the sorted T cell subpopulation, it can be seen that, in both groups of subjects, memory (CD45R0+) subsets had higher proliferation rates than did their nave (CD45R0C) counterparts. This was true for both CD4+ and CD8+ cell populations and is consistent with data from previous studies conducted in both young and elderly persons in the United Kingdom (18, 21, 22).

FIGURE 1. Mean measured enrichment of deuterium in DNA on days 3 and 10 from selected T lymphocyte populations in subjects exposed (CD45R0C; n = 13; A and C) and not exposed (CD54R0+; n = 12; B and D) to perinatal nutritional deprivation; data are expressed as the fraction of new cells labeled per day (, CD45R0C; , CD45R0+). Higher peak (day 3) fractional enrichments indicate faster rates of proliferation of the cells within the sorted subpopulation. Lines show the modeled enrichment curves (?, CD45R0+ model; - - -, CD45R0C model) derived from the data by using nonlinear least-squares regression (Levenberg-Marquardt method). Curve fits for mean data are shown. The variables proliferation rate constant (p) and disappearance rate constant (d) were estimated from the modeled curve fit for each subject.

For each set of individual day 3 and day 10 data, model curves were constructed to derive individual proliferation and disappearance rate constants, as shown in Table 4. Mean values for the proliferation rate constant (p) of 1.2%/d and 1.4%/d were obtained for CD45R0C and 3.8%/d and 4.0%/d for CD45R0+ cells within CD4+ and CD8+ populations, respectively. As in previous studies, significant interindividual variation was observed.

TABLE 4 Proliferation (p) and disappearance (d) rate constants for T cell populations according to perinatal nutritional exposure, calculated from best-fit model

When T cell proliferation rates were compared in the 2 groups (Table 4), no significant differences were found between subjects who were nutritionally deprived and those who were not. A trend was seen toward slower death rates in the CD4+CD45R0+ population in the nutritionally deprived group than in the nutritionally replete group, but these differences were not significant.

Modeling also yielded values for the disappearance rate constant (d) for each cell type in each subject (Table 4). It should be noted, as discussed elsewhere (18, 20), that these values represent the disappearance rates only of labeled cells, not those of the whole cellular population; because dividing cells are more likely to die than are nondividing cells, values for d generally exceed values for p. Mean disappearance rates within all cell subsets ranged between 4.8%/d and 7.5%/d. As in previous studies, no clear distinction was seen between d in CD45R0+ and CD45R0C cells.

T cell receptor excision circles

The measurement of thymic output through the quantification of RTEs allows for the assessment of the relative contribution of the thymus to the T cell pool (23). The T cell receptor (TCR) gene rearrangements that occur during the proliferation and differentiation of intrathymic progenitor cells result in extrachromosomal excision products or TRECs. Because TRECs are stable over time but cannot multiply, they remain within T lymphocytes as they transit and are diluted during T cell proliferation (23, 24). The reduction in thymopoiesis witnessed in aging is paralleled by a decrease in thymic output (15, 25) as measured by increased dilution of TRECs.

In a previous comparison of elderly persons, who would be expected to have age-related immune compromise, and young persons, we found no significant differences in the rates of in vivo proliferation of CD4+ and CD8+ nave and memory T cell subsets (22). Similarly, T cell kinetics of those born in the nutritionally deprived group in the Gambia in the current study were not found to differ significantly from those in the nutritionally replete group. Thus, it remained possible that the increase in infection-related deaths in the nutritionally deprived group was due to premature immunosenescence, manifest as decreased thymic output rather than impaired proliferation within T cell pools. Therefore, sjTRECs were quantified in T cell subpopulations of subjects exposed to perinatal nutritional deprivation and those that were not, to investigate potential differences in thymic output between these 2 groups. In accordance with other studies (15, 26-28), measurement units of TREC concentrations are expressed here as TREC copies per unit of DNA. This approach removes any variance due to different degrees of efficiency in the DNA-extraction method that may occur when TRECs are expressed per number of cells.

When TREC concentrations (per 100 ng DNA) were compared for the different cell subsets assayed, no differences were found between the 2 groups (Table 5). Because no differences were detected in T cell phenotype and measured T cell proliferation and disappearance rates between the 2 groups, the lack of difference in TREC concentrations may reflect a lack of difference in RTE and therefore in thymic output.

TABLE 5 Concentrations of T cell receptor excision circles (TRECs) in different T cell subsets according to perinatal nutritional exposure1

A 2-factor multilevel analysis of T cell kinetics (data from Table 4) and TREC concentrations (data from Table 5), using both additive and multiplicative models, found no consistent interaction between nutritional status at birth and nave and memory T cells (data not shown).

DISCUSSION

The perinatal period is a phase during which the thymus is very active and the T cell repertoire is being established. If, as has been suggested elsewhere (29), this period is also a time of critical immunologic susceptibility to nutritional compromise, insults at this time may adversely affect the establishment of clonal diversity and the homeostasis of T cells for many years thereafter. Consistent with such a proposition is the observation that persons born in periods of nutritional deficit may be more susceptible to infectious mortality in adult life than are those born in periods of nutritional repletion (12). Additional evidence for the effects of early-life events on immunity includes lower concentrations of thymopoietin (30), higher concentrations of immunoglobulin E (IgE) (31), and lower responses to typhoid vaccination with purified Vi cell surface polysaccharide in adolescents (2) and adults (3) born with low birth weight. These data suggest that fetal undernutrition, or other factors associated with season of birth in The Gambia and with low birth weight in other settings, may permanently impair the immune system (32).

In view of this, we investigated T cell numbers, phenotype, in vivo kinetics, and TREC content in young men in The Gambia. We compared the results in those who were very likely to have experienced perinatal nutritional compromise according to birth season and birth weight and in those who appeared to have had a well-nourished perinatal phase.

In terms of cell numbers and phenotype, perinatal thymic injury may be expected to result in young adults with low numbers of T cells, particularly in the nave compartment, and thus populations would be skewed toward a memory phenotype. When we analyzed cell counts and nave and memory phenotypes by season of birth and birth weight to investigate such a potential 'memory bias' (typical of immunosenescence) in those exposed to perinatal malnutrition, we found that nave populations were actually well maintained in this group and that, if anything, nave cell numbers tended to exceed those in persons in the nutritionally replete group (Table 3). Although subjects exposed to perinatal nutritional deprivation had a lower proportion of lymphocytes in the peripheral blood than did those not so deprived, that lymphocyte count could be accounted for by an increase in the absolute granulocyte count in this group.

These findings do not support our initial hypothesis that persons with nutritionally deprived births would have deficiencies in nave T cell populations. In the overall population of the current study, total absolute lymphocyte numbers (CD4 and CD8 counts) were also well maintained, and CD4:CD8 was similar to normative values in the United Kingdom, which indicated no evidence in this population for a 'CD8 bias.' These results contrast with those of other studies that describe lower CD4:CD8 and an increase in memory CD8 cells in other African settings (33-35).

The increase in granulocyte counts in the nutritionally deprived group was significant (Table 2) and appears somewhat paradoxical, unless it is driven by a relative deficit in the adaptive immune response in this group. Certainly, in African populations, normal granulocyte counts tend to be lower than those in white populations, and these results are thus consistent with results from other African countries (33, 35).

In vivo T cell kinetic analyses were consistent with results of other studies that have used similar methods; proliferation rates were found to be within ranges similar to those found in both young and elderly, healthy British controls (18). When individual disappearance curves were reviewed (data not shown) and compared with those seen in young and elderly UK residents, the observed pattern paralleled findings in young UK controls. No evidence was found for prolonged label retention and therefore of the persistence of CD8+CD45RA+ cells in the circulation, such as that observed in some elderly UK subjects (22). Thus, in young Gambian men, T cell labeling occurred at a rate and in a pattern similar to those in previous studies in other young adult populations. No clear evidence was seen for an effect of birth season or birth weight on T lymphocyte proliferation and disappearance rates.

Perinatal nutritional deprivation did not result in the reduced levels of RTEs usually seen in elderly persons (15, 27). These data are consistent with other findings from this study, which show that T cell phenotype and kinetics in these persons resemble those in young persons in the United Kingdom more than those in elderly persons.

When cell kinetics were analyzed by perinatal exposure to malnutrition to investigate a potential deficit in the maintenance of T cell homeostasis in those who were nutritionally deprived around the time of birth, no differences were found in either proliferation or disappearance rates for CD4+ and CD8+ memory and nave T cell populations. It is possible that real changes may have been missed in the current, small study. Logistic constraints, local sensitivities, and the availability of demographically defined populations limited us with respect to the number of subjects that could be studied, the amount of blood that could be taken, and the number of time-points that could be analyzed. In addition, a large component of interindividual variability was present, which is a feature of lymphocyte kinetic studies in general. As a consequence, the current study was limited in its power?eg, for CD4+CD45R0+ proliferation, it had a 90% chance of detecting a 60% change. We tried to maximize our chances of finding an effect by choosing only the extremes of annual variation, rather than recruiting from births across the whole year, and by including birth weight as an additional criterion.

The nonsignificant trends apparent in kinetic differences between the 2 groups seemed primarily to affect the CD4+CD45R0+ population. Cells from persons exposed to perinatal deprivation tended toward lower proliferation and disappearance rates and less TREC content. Because slow turnover would be expected to preserve TREC content (which is diluted by division), this pattern would be consistent with a model in which thymic output is markedly impaired; however, if that were the case, TREC content in the nave CD4+CD45R0C population would be expected to be low, yet it was not. An alternative model is that the memory population has previously had a high turnover rate (hence diluting TREC content), perhaps because it was derived from a relatively limited initial repertoire, but now has become relatively anergic, consisting of cells that are resistant to activation. Further studies, including additional TREC analysis and telomere length analysis, may help resolve these 2 possibilities.

In vivo lymphocyte kinetic studies can detect only fairly gross changes in whole populations of cells, whereas the effect of nutritional deprivation may be much more focused effect, only on specific elements of immune response, including non-T-cell components, which are damaged by factors related to low birth weight. Thus, for example, a large study in Pakistani adults born with low birth weight found effects on T cellCindependent antibody responses to vaccination against typhoid (Vi polysaccharide) but not against T cellCdependent rabies vaccine (3).

This investigation has shown that the maintenance of CD4+CD45R0C, CD4+CD45R0+, CD8+CD45R0C, and CD8+CD45R0+ cell populations remains largely unimpaired in persons exposed to acute perinatal malnutrition. This finding is reflected in unaltered proliferation and death rates within these cell subsets, although some trends suggested subtle changes, particularly in CD4 memory populations. The significance of the changes in the innate immune system (increased granulocyte numbers) is difficult to assess and may represent a compensatory mechanism provoked by subtle changes in adaptive immunity. However, overall, these results are consistent with a surprisingly robust homeostatic system within T cell populations, which upholds the proliferative capacity of cells and maintains adequate cell distributions and numbers, despite exposure to adverse nutritional and immunologic challenges throughout life.

ACKNOWLEDGMENTS

We thank the young men of Keneba, Manduar, and Kantongkunda who volunteered to take part in this study; Bakary Darboe for laboratory assistance; Molipha Jammeh and Kebba Bajo for subject recruitment and translation; Andrew Worth and Cathy DeLara for flow cytometry sorting, Becca Asquith for mathematical advice; Tony Fulford for statistical advice; and Peter Beverley for guidance on immunologic interpretations.

DCM and AMP were responsible for the conception of the study; HG was responsible for method development, study design and execution, data analysis, data interpretation, and manuscript preparation; DLW conducted the flow cytometry sorting and guided the interpretation of results; SMH and PTN conducted the T cell receptor rearrangement excision circle analyses; YZ and JAS assisted in laboratory analyses and critical discussions of data; RA,GM, GEG, and AMP participated in data interpretation and reviewed the manuscript; DCM was responsible for the study design and execution and data analysis and interpretation, and contributed to the writing of the final manuscript. None of the authors had a personal or financial conflict of interest.

【参考文献】
  Godfrey KM, Barker DJ, Osmond C. Disproportionate fetal growth and raised IgE concentration in adult life. Clin Exp Allergy 1994;24:641C8.

McDade TW, Beck MA, Kuzawa C, Adair LS. Prenatal undernutrition, postnatal environments, and antibody response to vaccination in adolescence. Am J Clin Nutr 2001;74:543C8.

Moore SE, Jalil F, Ashraf R, Chen SS, Prentice AM, Hanson LA. Birth weight predicts response to vaccination in adults born in an urban slum in Lahore, Pakistan. Am J Clin Nutr 2004;80:453C9.

Morgan G. What, if any, is the effect of malnutrition on immunological competence? Lancet 1997;349:1693C5.

Beach RS, Gershwin ME, Hurley LS. Gestational zinc deprivation in mice: persistence of immunodeficiency for three generations. Science 1982;218:469C71.

Beach RS, Gershwin ME, Hurley LS. Reversibility of development retardation following murine fetal zinc deprivation. J Nutr 1982;112:1169C81.

Beach RS, Gershwin ME, Hurley LS. Persistent immunological consequences of gestation zinc deprivation. Am J Clin Nutr 1983;38:579C90.

Malpuech-Brugere C, Nowacki W, Gueux E, et al. Accelerated thymus involution in magnesium-deficient rats is related to enhanced apoptosis and sensitivity to oxidative stress. Br J Nutr 1999;81:405C11.

King LE, Osati-Ashtiani F, Fraker PJ. Apoptosis plays a distinct role in the loss of precursor lymphocytes during zinc deficiency in mice. J Nutr 2002;132:974C9.

Collinson AC, Moore SE, Cole TJ, Prentice AM. Birth season and environmental influences on patterns of thymic growth in rural Gambian infants. Acta Paediatr 2003;92:1014C20.

Ngom PT, Collinson AC, Pido-Lopez J, Henson SM, Prentice AM, Aspinall R. Improved thymic function in exclusively breastfed babies is associated with higher interleukin 7 concentrations in their mothers? breast milk. Am J Clin Nutr 2004;80:722C8.

Moore SE, Cole TJ, Poskitt EM, et al. Season of birth predicts mortality in rural Gambia. Nature 1997;388:434 (letter).

Moore SE, Cole TJ, Collinson AC, Poskitt EM, McGregor IA, Prentice AM. Prenatal or early postnatal events predict infectious deaths in young adulthood in rural Africa. Int J Epidemiol 1999;28:1088C95.

Malaguarnera L, Ferlito L, Imbesi RM, et al. Immunosenescence: a review. Arch Gerontol Geriatr 2001;32:1C14.

Douek DC, McFarland RD, Keiser PH, et al. Changes in thymic function with age and during the treatment of HIV infection. Nature 1998;396:690C5.

Cole TJ. Seasonal effects on physical growth and development. In: Ulijaszek SJ, Strickland SS, eds. Seasonality and human ecology. Cambridge, United Kingdom: Cambridge University Press, 1993:89C106.

Ghattas H, Darboe BM, Wallace DL, Griffin GE, Prentice AM, Macallan DC. Measuring lymphocyte kinetics in tropical field settings. Trans R.Soc Trop Med Hyg 2005;99:675C85.

Macallan DC, Asquith B, Irvine AJ, et al. Measurement and modeling of human T cell kinetics. Eur J Immunol 2003;33:2316C26.

Macallan DC, Fullerton CA, Neese RA, Haddock K, Park SS, Hellerstein MK. Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans. Proc Natl Acad Sci U S A 1998;95:708C13.

Asquith B, Debacq C, Macallan DC, Willems L, Bangham CR. Lymphocyte kinetics: the interpretation of labelling data. Trends Immunol 2002;23:596C601.

Macallan DC, Wallace DL, Irvine AJ et al. Rapid turnover of T cells in acute infectious mononucleosis. Eur J Immunol 2003;33:2655C65.

Wallace DL, Zhang Y, Ghattas H, et al. Direct measurement of T cell subset kinetics in vivo in elderly men and women. J Immunol 2004;173:1787C94.

Ye P, Kirschner DE. Reevaluation of T cell receptor excision circles as a measure of human recent thymic emigrants. J Immunol 2002;168:4968C79.

Hazenberg MD, Verschuren MC, Hamann D, Miedema F, van Dongen JJ. T cell receptor excision circles as markers for recent thymic emigrants: basic aspects, technical approach, and guidelines for interpretation. J Mol Med 2001;79:631C40.

Henson SM, Pido-Lopez J, Aspinall R. Reversal of thymic atrophy. Exp Gerontol 2004;39:673C8.

Douek DC, Vescio RA, Betts MR, et al. Assessment of thymic output in adults after haematopoietic stem-cell transplantation and prediction of T-cell reconstitution. Lancet 2000;355:1875C81.

Steffens CM, Al Harthi L, Shott S, Yogev R, Landay A. Evaluation of thymopoiesis using T cell receptor excision circles (TRECs): differential correlation between adult and pediatric TRECs and naive phenotypes. Clin Immunol 2000;97:95C101.

Guazzi V, Aiuti F, Mezzaroma I, et al. Assessment of thymic output in common variable immunodeficiency patients by evaluation of T cell receptor excision circles. Clin Exp Immunol 2002;129:346C53.

Moore SE, Collinson AC, Ngom PT, Prentice AM. Maternal malnutrition and the risk of infection in later life. In: Hornstra G, Uauy R, Yang X, eds. The impact of maternal nutrition on the offspring. Basel, Switzerland: S Karger, 2005:153C67.

McDade TW, Beck MA, Kuzawa CW, Adair LS. Prenatal undernutrition and postnatal growth are associated with adolescent thymic function. J Nutr 2001;131:1225C31.

McDade TW, Kuzawa CW, Adair LS, Beck MA. Prenatal and early postnatal environments are significant predictors of total immunoglobulin E concentration in Filipino adolescents. Clin Exp Allergy 2004;34:44C50.

Prentice AM, Moore SE. Early programming of adult diseases in resource poor countries. Arch Dis Child 2005;90:429C32.

Tsegaye A, Messele T, Tilahun T, et al. Immunohematological reference ranges for adult Ethiopians. Clin Diagn Lab Immunol 1999;6:410C4.

Tsegaye A, Wolday D, Otto S, et al. Immunophenotyping of blood lymphocytes at birth, during childhood, and during adulthood in HIV-1-uninfected Ethiopians. Clin Immunol 2003;109:338C46.

Worku S, Christensson B, Bjorkman A, Islam D. Higher proportion of CD8+ T cells in the blood in healthy adults from Ethiopia and Bangladesh compared with Sweden. Trans R Soc Trop Med Hyg 1997;91:618C22.