Expression of messenger ribonucleic acid and production of cytokines in children with malnutrition

Nutrition Research 23 (2003) 367–376 www.elsevier.com/locate/nutres

Expression of messenger ribonucleic acid and production of cytokines in children with malnutrition P. Bhaskaram*, R. Hemalatha, B. Narayana Goud Clinical Division National Institute of Nutrition (Indian Council of Medical Research) Hyderabad 500 007, Andhra Pradesh, India Received 13 February 2002; received in revised form 20 November 2002; accepted 25 November 2002

Abstract The aim of this study was to determine the IL2 and IL4 mRNA expression by antigen/mitogen stimulated lymphocytes and IL2, IFN-␥, IL4, IL5 and IL10 protein secretion from the culture supernatant obtained from children suffering from severe protein energy malnutrition (PEM) and compare with the responses of normal healthy children. Twelve children aged between 3 and 5 years hospitalised for rehabilitation of severe PEM manifesting as marasmic kwashiorkor were investigated at the time of hospitalisation and 8 were retested 30 days after nutritional rehabilitation. Ten age matched healthy children served as controls. Three normal adult volunteers were also investigated for validating Reverse Transcription–Polymerase chain reaction (RT-PCR) results obtained in children. mRNA expression for IL2 and IL4 by in vitro cultured peripheral blood mononuclear cells (PBMC) stimulated by PHA/PPD was determined using RT-PCR technique following established procedures. mRNA was quantified using Dipstic™ kit. Concentrations of IL2, IL4, IFN-␥, IL5 and IL10 were determined from the culture supernatant using ELISA technique. Lymphocytes from adult volunteers expressed clearly both IL2 and IL4 mRNA with PHA as well as PPD. Four out of the 10 control children had clear mRNA band for IL2 while the other six had faintly visible bands with PHA. IL4 mRNA bands were clearly visible in 9 out of 10 children with both PHA and PPD. Mean (⫾ SE) concentrations of mRNA from PHA stimulated systems for IL2 and IL4 were significantly lower in children compared to adults. All the children with PEM had very faint mRNA bands for IL2 before as well as 1 month after rehabilitation while IL4 mRNA expression was clear. Mean mRNA concentration for IL2 was significantly (P ⬍ 0.05) lower (81.2 ⫾ 16.06 ng/1 ⫻106 cells) than in control children (127.1 ⫾ 26.24 ng/1 ⫻ 106 cells) with no significant change after rehabilitation. IFN-␥, IL4, IL5 and IL10 cytokine profile of children with PEM before as well as after rehabilitation was similar to that of the control children.

* Corresponding author. Tel.: ⫹91-40-27008921; fax: ⫹91-40-7019074. E-mail address: [email protected] (P. Bhaskaram). 0271-5317/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0271-5317(02)00535-3

368

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

These observations suggest that Th1 subset responses are lower in children compared to adults. IL2 response is impaired in children suffering from severe PEM. Short term nutritional rehabilitation has no impact on these immune responses. © 2003 Elsevier Science Inc. All rights reserved. Keywords: Protein-energy malnutrition; mRNA expression; Cytokines; T Cell subsets; RT-PCR

1. Introduction Malnutrition and infectious diseases are widely prevalent and coexistent in developing countries with their mutually adverse interactions contributing to significant morbidity and mortality among pre-school children. Deficiencies of several nutrients like protein and energy and micronutrients including iron down regulate the immune system, thus increasing the susceptibility of the host to infection and enhancing the severity of the diseases [1]. Infectious diseases like measles, tuberculosis, pertussis etc. are shown to be more severe in children suffering from malnutrition [2]. All these infections are vaccine preventable. Measures to enhance/modulate the quality of immune response of malnourished individuals to natural infection/vaccines is critical in reducing the burden of morbidity and mortality due to infections in children. Recent immunological advances have distinctly delineated two essential subsets of T-cells carrying out two distinct functions. Inflammatory responses induced through macrophage activation are executed by Th1 subpopulation, while Th2 subset helps in specific antibody production. It has also been identified that the nature of antigen, the antigen presenting cell (APC) with its ligand density, co-stimulating factors expressed by the APC and the cytokines produced are important determinants of development of Th1 or Th2 type response [3]. Comprehensive knowledge on these basic mechanisms produces an insight to develop immunopotentiating modalities to achieve optimal protective immune responses to infectious diseases/vaccines in malnourished children. Therefore this study was envisaged to investigate the functional status of T cell subsets in children suffering from severe protein energy malnutrition (PEM) which is a deficiency disorder of public health significance in developing countries.

2. Materials and methods 2.1. Subjects Twelve children (six boys and six girls) between three and five years of age, suffering from severe PEM were investigated at the time of admission to the hospital. Children with obvious evidence of infection and signs of severe vitamin A deficiency manifesting as corneal xerosis/ulcer/keratomalacia and children with oral lesions due to B-complex deficiency were excluded. All the children had severe wasting and pedal edema characteristic of marasmic kwashiorkor. Of the 12 PEM children, 8 were reinvestigated 30 days after nutritional rehabilitation.

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

369

A soft milk based diet with bread providing 180-200 kcals/Kg and 3-4 g protein/Kg body weight per day constituted the rehabilitation diet. A multi vitamin supplement, iron (six mg of elemental iron per kg/day as ferrous sulphate) and Zinc (One mg of elemental zinc/kg body weight per day as zinc sulphate) were administered to all the children throughout the hospital stay. Ten children comprising of 5 boys and 5 girls, having percentage weight for age more than 90 of the standard [4] and Haemoglobin (Hb) more than 110 g/L served as control group. Informed consent was obtained from the parents of all children for participation in the study. 2.2. Analytical methods 2.2.1. Reverse transcription–polymerase chain reaction (RT-PCR) Venous blood was obtained from the recruited children under aseptic conditions for studying IL2, IL4 messenger Ribonucleic acid (mRNA) expression from in vitro cultured peripheral blood mononuclear cells (PBMC) using RT-PCR technique. Cytokine profile in culture supernatants was determined by Enzyme linked immuno sorbant assay (ELISA). PBMC were isolated on ficoll hypaque, cultured at a concentration of 3 ⫻ 106 cells/ml/well in RPMI 1640 ⫹ 10% autologous serum with phytohemagglutinin (PHA) (phaseolus SPP) (Murex Biotech Ltd Dartford, England)100 ug/ml or purified protein derivative (PPD) 2 u/ml and incubated at 37°C in 5% CO2. The doses for mitogen and antigen were selected based on the results of the pilot studies conducted using 50, 100 and 200 ␮g/ml of PHA and 1, 2 and 4 units/ml of PPD in healthy adults and control children. Control wells contained no stimulant. The culture supernatants were collected at 18 hours, stored at ⫺20°C for cytokine (IL2, IFN-␥, IL4, IL5 and IL10) analysis at a later date and the cells were used for RNA isolation. The time of harvesting the cultures was fixed at 18 hrs based on the experiments conducted in adult volunteers and control children to determine the pattern of IL2 mRNA transcription and culture supernatant cytokine concentration according to the duration of culture varying from 8 to 24 hours. As there was no difference in the expression of mRNA or the concentration of cytokines secreted at 18 and 24 hours, 18 hours was fixed for this study as optimal period for harvesting the cultures. Three healthy adult volunteers were investigated for RT-PCR validation. 2.2.2. Ribonucleic acid (RNA) isolation Total cellular RNA was isolated using the guanidium isothiocyanate –Phenol- Chloroform method, described by Chomczynski & Sacchi [5]. Briefly, cultured PBMC were pelleted in eppendorf tubes by centrifugation and lysed with 4M guanidium isothiocyanate lysis buffer. Acid phenol and 100 ␮l of chloroform-isoamyl alcohol (49:1) were added to the lysate with thorough vortexing after each addition. The mixture was then kept on ice for 15 minutes and centrifuged at 12,000 rpm for 20 minutes at 4°C. The aqueous phase was recovered and a second phenol-chloroform extraction was performed to ensure purity of the cellular RNA. RNA was precipitated in an equal volume of ice cold isopropyl alcohol at ⫺20°C for 2 hours. The precipitated RNA was washed with 80% alcohol in DEPC treated distilled water to remove salts and dissolved in 30␮l of DEPC water at 65°C for 10 minutes in a waterbath. The RNA thus obtained was used for cDNA synthesis.

370

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

2.2.3. Reverse transcription Following the method of Ehlers and Smith the complimentary DNA was synthesised from total cellular RNA [6]. Briefly, dissolved RNA was primed with oligo d(T) primer at 65°C for 10 minutes and reverse transcribed using 200u of moloney murine leukaemia virus reverse transcriptase (MMLV) enzyme, 5⫻ enzymebuffer, 1 mM dNTP, 20u RNase inhibitor, 0.1 M DTT and BSA(1 mg/ml) in a total reaction volume of 20 ul, and incubated at 37°C for 60 minutes. The tubes were then heated to 95°C for 5 minutes. Samples were stored at ⫺20°C until further use.

2.2.4. Polymerase chain reaction (PCR) Amplification of cDNA for IL2 and IL4 was carried out as described by Barnes et al. [7], using cytokine specific primer pairs [6]. Briefly, 5 ␮l of cDNA was amplified using a thermocycler (MJ Research USA) running a program of 35 cycles, each cycle consisting of denaturation at 95°C for 60 seconds, annealing at 56°C for 60 seconds (60°C for 45 seconds in the case of IL4 and ␤actin) and extension at 72°C for 90 seconds followed by 72°C for 7 minutes for final extension cycle. The cycles were fixed at 35 after establishing the linearity of amplification of CDNA by PCR. The primer sequences used for IL2, IL4 & ␤actin were as follows: IL2 IL4

␤actin

5-ATGTACAGGATGCAACTCCTGTCTT-3 (sense), 5-GTCAGTGTTGAGATGATGCTTTGAC-3 (antisense); 5-ATGGGTCTCACCTCCCAACTGCT-3 (sense), 5-CGAACACTTTGAATATTTCTCTCTCAT-3 (antisense); 5-TGACGGGGTCACCCACACTGTGCCCATCTA-3 (sense); 5-CTAGAAGCATTGCGGTGGACGATGGAGGG-3 (antisense). Size of amplified fragment (bp): IL2, 458; IL4, 456; ␤actin, 661.

After amplification, 8 ␮l of PCR product was mixed with 2 ul loading buffer and was electrophoresed in 2% agarose gel. The bands were visualized by ethedium bromide staining and ultraviolet transillumination. Cytokine specific bands were identified by anticipated molecular weights against 100 bp DNA ladder.

2.2.5. Messenger ribonucleic acid (mRNA) quantification The PCR products obtained from PHA stimulated cultures from 3 adults and in a randomly selected subsample of 6 control children and 8 PEM children before rehabilitation were quantified using DNA Dipstic™ kit version B (In vitrogen, CA). Samples from adults for both IL2 and IL4 were diluted in sterile deionized water as per the protocol provided with the kit. IL4 samples from all children required 1in 3 dilution for detection while IL2 samples were used undiluted. A standard graph was constructed from the colour density measurement obtained by scanning the standards provided with the kit (GS 710 Biorad). mRNA concentration of the samples was calculated from the standard slope and expressed as ng/1 ⫻ 106 cells.

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

371

2.2.6. Measurement of cytokines The culture supernatants of PBMC stimulated with PHA or PPD earlier were assayed for IL2, IFN-␥, IL4, IL5 and IL10 cytokines, using Eli pair reagents (Diaclone research France). Monoclonal antibody specific for each cytokine was used as capture antibody while biotynilated antibody was the detection antibody. HRP-streptavidin was used for colour development. The absorbance was read at 450 nm in ELISA reader (Lab systems). All samples were run in dilutions of 1in 2 to 1 in 10 and in duplicates. The specific cytokine concentration was determined from the standard curve, based on their OD values. The lower limit of detection was 31.2 pg/ml for IL2; 12.5 pg/ml for IFN-␥ and IL10; 1.1 pg/ml for IL4 and 7.8 pg/ml for IL5. 2.2.7. Estimation of hemoglobin (Hb) Hb was estimated by cyanmethemoglobin method [8].

3. Statistical analysis Data were analyzed on SPSS PC software. Mean values of different lymphokines and mRNA concentrations were compared between groups, after log transformation and using ANOVA with post-hoc test. Paired ‘t’ test was applied to compare the cytokine concentrations between baseline and after rehabilitation periods.

4. Results Mean (⫾ SE) age in months was 47.4 ⫾ 4.77 in the control children and 46.3 ⫾ 4.24 in PEM children. The mean (⫾ SE) percentage weight (kg)/age and Hb (g/L) of control children were 94.2 ⫾ 3.39, and 116.0 ⫾ 1.80 respectively. PEM children had mean (⫾ SE) weight/age, 54.8 ⫾ 2.15 with mean (⫾ SE) Hb 74.0 ⫾ 3.30 before rehabilitation. Children with PEM were oedema free and improved their percentage weight/age (64.4 ⫾ 3.42) while there was no significant change in their mean (⫾ SE) Hb (68.0 ⫾ 6.78 g/L) following rehabilitation. 4.1. Cytokine mRNA expression 4.1.1. Adults All the 3 adults expressed clearly visible mRNA for IL2 and IL4 with PHA as well as PPD (Fig. 1). Mean (⫾ SE) concentrations of IL2 and IL4 mRNA (ng/1 ⫻ 106cells) of PHA stimulated lymphocytes were 362 ⫾ 59.4 and 2196.3 ⫾ 323.6 respectively.

372

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

Fig. 1. mRNA expression on agarose gel obtained from one representative sample each from adults and control, and PEM children, as indicated by Arabic numerals. Lane 1: 100 bp DNA ladder; Lane2: IL4 expression of an adult; Lane 3: IL2 expression of an adult; Lane 4: IL4 control child; Lane 5: IL2 control child; Lane 6: IL4 PEM child; Lane 7: IL2 PEM child.

4.2. Control children 4.2.1. mRNA expression Three out of the 10 children had clearly visible mRNA at 6-8 hours and persisted at 18 hours. In one child there was no visible band at 6-8 hours but was detectable at 18 hours. In the other 6 children mRNA band was faintly visible at 18 hrs. With PPD, six out of 10 children exhibited clear, visible bands of IL2 mRNA, while no band was visible in the remaining 4 samples. Nine out of the 10 children exhibited visible IL4 mRNA band with PHA as well as PPD. Mean (⫾ SE) mRNA concentrations (ng/1 ⫻ 106 cells) for IL2 and IL4 were 127.1 ⫾ Table 1 IL2 and IL4 mRNA concentrations from PHA stimulated PBMC Group (N)

IL2 (ng/1 ⫻ 106 cells)

IL4 (ng/1 ⫻ 106 cells)

Adults (3) Control children (6) PEM (8)

362.0 ⫾ 59.38 127.1** ⫾ 26.24 81.2* ⫾ 16.06

2196.3 ⫾ 323.6 325.9*** ⫾ 88.66 247.1 ⫾ 39.54

Mean ⫾ SE. ***P ⬍ 0.001, **P ⬍ 0.01 compared to adults; * P ⬍ 0.05 compared to control children, (Log transformation followed by ANOVA with post hoc analysis).

Table 2 Cytokine profile in children IFN-␥

Group (No) IL2 PHA Control (10) PEM BR (12) PEM AR (8)

PPD

PHA

1420.2 ⫾ 522.35 563.9 ⫾ 496.77 3515.0 ⫾ 1298.09 245.0 ⫾ 119.5* N.D.

2288.6 ⫾ 775.11

272.7 ⫾ 66.63

3600.0 ⫾ 756.15

N.D.

IL4 PPD

PHA

IL5 PPD

PHA

IL10 PPD

PHA

PPD

63.3 ⫾ 40.06 6.4 ⫾ 1.68 0.17 ⫾ 0.17 45.7 ⫾ 15.96 1.08 ⫾ 0.68 409.9 ⫾ 129.4

259.7 ⫾ 61.37

76.6 ⫾ 42.31 3.9 ⫾ 1.59 N.D.

256.9 ⫾ 86.5

34.8 ⫾ 15.97 0.61 ⫾ 0.29 399.6 ⫾ 89.91

114.1 ⫾ 52.16 2.8 ⫾ 1.18 0.14 ⫾ 0.14 50.7 ⫾ 20.07

Mean ⫾ SE (pg/1 ⫻ 106 cells/mL). BR: Before Rehabilitation; AR: After Rehabilitation; * P ⬍ 0.05, compared to control children; ND: Non detectable.

1.3 ⫾ 0.64 392.9 ⫾ 207.59 180.5 ⫾ 74.06

374

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

26.24 and 325.7 ⫾ 88.66 respectively. These values were significantly lower (P ⬍ 0.01 for IL2 and P ⬍ 0.001 for IL4) compared to the respective mean values in adults (Table 1). 4.2.2. Cytokine concentrations Respective cytokines were detectable from all the cultures where mRNA was expressed. Cytokine concentrations varied widely and mean concentration of various lymphokines were higher from mitogen stimulated cultures compared to those stimulated with PPD (Table 2). 4.3. Protein energy malnutrition (PEM) before rehabilitation 4.3.1. mRNA expression All the children with severe PEM, had faint IL2 mRNA bands detectable in agarose gel with PHA. IL4 mRNA expression was clear in all except one child with PHA and two with PPD. IL2 and IL4 mRNA were quantifiable (ng/1 ⫻ 106 cells) in all the 8 children studied for this parameter with mean (⫾ SE) values of 81.2 ⫾ 16.06 and 247.1 ⫾ 39.54 respectively. The mean value for IL2 was significantly (P ⬍ 0.05) lower compared to that of control children. The difference between mean values for IL4 was however not significant statistically (Table 1). 4.3.2. Cytokine concentrations Mean (⫾ SE) IL2 concentration was 245.0 ⫾ 119.5 pg/1 ⫻ 106 cells/mL with PHA, being significantly (P ⬍ 0.05) lower than the mean value (1420.2 ⫾ 522.35 pg/1 ⫻ 106 cells/mL) in control children, while the concentration was lower than the minimum detectable range in culture systems stimulated with PPD. Mean (⫾ SE) IL4 was 3.9 ⫾ 1.59 pg/1 ⫻ 106 cells/mL and was not significantly different from that of control children, IL4 was however not detectable when PPD was used as the culture stimulant. Concentrations of other cytokines were also not significantly different from the respective control values (Table 2) with both PHA and PPD. 4.4. Immune response after short term rehabilitation Thirty days after rehabilitation, there were no significant changes either in the mRNA expression as visualized on the gel or cytokine profile (Table 2).

5. Discussion Normal adults exhibited both Th1 and Th2 cytokine mRNA expression (IL2 & IL4 respectively) adequately with mitogen as well as antigen. IL2 mRNA expression was faint and concentration significantly lower in control children compared to adults. Available literature indicates that cytokine secretion in children and neonates is less than that observed in adults [9]. However, there is no information available on mRNA expression and quantification to compare the responses in children with those of adults. The present

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

375

study thus identifies significantly lower mRNA concentration even in healthy control children compared to adults. These observations probably suggest the relative immaturity of immune regulatory mechanisms of cytokine production in children which might be an age dependent maturational phenomenon. Contrary to IL2, IL4 mRNA expression was clear in children although the mRNA concentration was lower than in adults. With PPD, however, IL4 mRNA was visible but protein secretion was absent and could be the result of a regulating mechanism inhibiting Th2 product with PPD, this being an antigen promoting Th1 response [10]. IL2, IFN-␥, and IL4 levels were found to be within reported ranges for children [9]. No information could be obtained on IL5 and IL10 levels in normal children. Several investigators have reported the adverse effects of severe PEM on the thymolymphatic system resulting in depleted T cell number with impaired functional status [11,12]. IL2 mRNA and protein concentration with PHA were significantly lower in children with PEM than in controls thus indicating adverse effects of malnutrition on the already immature immune function. IFN-␥ levels in children with PEM were found to be comparable to those of normal children though IFN-␥, like IL2, is also a product of Th1 response. Though Th1 subset of T cells constitutes the major source for IFN-␥, this cytokine is also secreted by other cells, like CD8⫹ T cells [13] and Natural killer (NK) cells [9]. It has been shown that children with malnutrition have altered ratio of CD4⫹ to CD8⫹ cells in favour of the latter [14]. This supports the observed results of normal IFN-␥ concentration despite low IL2 secretion. IFN-␥ might be acting as a compensatory mechanism in malnourished children whose IL2 responses and other immune functions are severely compromised. However, impairment of IL2 functional significance in terms of development of memory to an antigenic exposure needs to be established as IL2 is the lymphokine that mainly promotes T-cell proliferation thus leading to the development of memory cells. This observation, thus has implications particularly for success of certain vaccines that require Th1 subset participation. In PHA and PPD treated culture systems, mean IL4, IL5 and IL10 levels were not significantly affected by PEM suggesting no alteration in Th2 responses. Thus, the established cellular immunosuppression observed in children suffering from severe PEM appears to be due to impairment of Th1 function with respect to IL2. No significant improvement was observed with short term rehabilitation of PEM children confirming our own earlier reports in children and those reported by others in experimental animals [15,16]. The present study thus demonstrates that Th1 response in terms of IL2 secretion is significantly impaired in malnourished children and identifies the scope to improve Th1 response through immune modulation to achieve expected responses to vaccines/infections in malnourished children.

Acknowledgments Dr. R. Hemalatha was responsible for collection of clinical data and also assisted Mr. Narayana Goud B, Technical Officer, in carrying out the RT-PCR and ELISA work.

376

P. Bhaskaram et al. / Nutrition Research 23 (2003) 367–376

The authors gratefully acknowledge the financial support of the Council of Scientific & Industrial Research, CSIR Complex, PUSA, New Delhi-110 012, India. The authors are indebted to Prof. Indira Nath, Department of Biotechnology, All India Institute of Medical Sciences, New Delhi-110 029, India, for providing opportunity to learn the RT-PCR technique under her guidance. The encouragement extended by Dr. Kamala Krishnaswamy, Director, National Institute Of Nutrition, is thankfully acknowledged.

References [1] [2] [3] [4]

[5] [6] [7] [8]

[9] [10]

[11]

[12] [13] [14] [15] [16]

Bhaskaram P. Immunology of mild-moderate micronutrient deficiency. Brit J Nutr 2001;85(Suppl 2):75– 80. Bhaskaram P. Infections and malnutrition among poor children. Ind J Pediatr 1987;54:535– 45. Bottomly K. A functional dichotomy in CD4 ⫹ T lymphocytes. Immunol Today 1988;9:268 –74. Hamill PVV, Drizd TA, Johnson CL, Reed RB, Roche AF. NCHS. Growth curves for children Birth-18 years. Data from National Health Survey (USA). Vital and Health Statistics. 1977, Series 11, No. 165, p 1–17. Chomozynski P, Sacchi N. Single step method of RNA isolation by acid guanidium thiocyanate-phenolchloroform extraction. Anal Biochem 1987;162:156 –9. Ehlers S, Smith KA. Differentiation of T cell lymphokine gene expression. the in vitro acquisition of T cell memory. J Exp Med 1991;173:25–36. Barnes PF, Shuzhuang LU, Abrams JS, Wang E, Yammamura M. Cytokine production at the site of disease in human tuberculosis. Infect Immun 1993;61:3482–9. Cook JD, Bothwell TH, Covell AM, Dallman PR, Lynch SR, Worwood MA. Measurement of iron status: a report of the International Nutritional Anemia Consultative Group (INACG), Washington. DC. The Nutrition Foundation 1985: 35–54. Lilic D, Cant AJ, Abinun M, Calvert JE, Spickett GP. Cytokine production differs in children and adults. Pediatr Res 1997;42:237– 41. Barnes PF, Mehra V, Rivoire B, Fong SJ, Brennan PJ, Voegtline MS, Minden P, Houghten RA, Bloom BR, Moden RL. Immunoreactivity of a 10 KD antigen of mycobacterium tuberculosis. J Immunol 1992;148: 1835– 40. Smythe PM, Schonland M, Brereton Stiles GG, Grace HJ, Mafoyane A, Coovadia HM, Loening WEK, Parent MA, Vos GH. Thymolymphatic deficiency and depression of cell mediated immunity in protein calorie malnutrition. Lancet 1971;ii:939 – 43. Reddy V, Jagadeesan V, Raghuramulu N, Bhaskaram C, Srikantia SG. Functional significance of growth retardation in malnutrition. Am J Clin Nutr 1976;29:3–7. Mackan IR, Rosen FS. Advances in immunology. N Engl J Med 2001;344:350 – 62. Chandra RK, Gupta S, Singh H. Inducer and suppressor T cell subsets in protein-energy malnutrition, analysis by monoclonal antibodies. Nutr Res 1982;2:21– 6. Bhaskaram C, Reddy V. Cell mediated immunity in protein calorie malnutrition. J Trop Pediatr Envt Child Hlth 1974;20:284 – 6. Jose DG, Stutman O, Good RA. Long term effects on immune functions of early nutritional deprivation. Nature 1973;241:57– 8.