Humoral and Cell-Mediated Immunity in Malnourished Children in Ghana

Humoral and cell-mediated immunity in malnourished children in Ghana

T Rikimaru1,2, K Taniguchi3, JE Yartey4, DO Kennedy4 and FK Nkrumah4

1Institute for International Cooperation, JICA, Tokyo, Japan; 2Program in International Nutrition, Department of Nutrition, University of California, Davis, USA; 3Department of Epidemiology, National Institute of Health, Tokyo, Japan; and 4Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana

Objective: To examine the relationship between immunological variables and the different types and severity of malnutrition in Ghanaian children. Design: Case-control study. Setting: The study was done at Princess Marie Louise Hospital, Accra, Ghana. Subjects: One hundred and seventy children, aged 8±36 months, were recruited at the clinical ward and public health service section of the hospital: 61 normal children, 49 moderately malnourished (underweight) children and 60 severely malnourished children (19 kwashiorkor, 30 marasmus, and 11 marasmic kwashiorkor children). Method: The children underwent clinical observations, anthropometric measurements and blood sampling for biochemical analysis to evaluate their nutritional and immunological status. Serum immunoglobulins (IgA subclasses, IgG subclasses and IgM), complements (C3 and C4) and lymphocyte subpopulations (T cells, B cells, CD4, CD8, NK cells and HLADR) were determined for the assessment of humoral and cell-mediated immunity. Results: Serum levels of IgA1, IgA2 and C4 tended to be higher in severely malnourished children than in normal children, while serum level of C3 and the proportion of B cells were signi®cantly lower in the severely malnourished children than in the normal children (P < 0.05). There were no notable differences in most immunological parameters among the three severely malnourished groups. No differences were observed in the immunological parameters except for the proportion of B cells between normal and moderately malnourished children. Factor analysis revealed that C3 levels were positively correlated with a factor which was strongly associated with weight-for-height z-score and biochemical indicators for evaluating protein nutrition. In addition, IgA2, IgG1 and IgM levels were positively correlated with a factor which was associated with C-reactive protein. Conclusion: Several immunological variables responded positively or negatively with the different levels of severity of malnutrition, but most variables did not on the different types of malnutrition. The changes of C3 level were more associated with the severity of malnutrition. Descriptors: malnutrition; immunoglobulin; complement; lymphocyte subpopulation; marasmus; kwashiorkor

Introduction determined to assess the status of humoral and cell mediated immunity. Although there has been general Protein energy malnutrition (PEM) is an important cause of consensus about the impairment of immunocompetence immunocompetence impairment and consequently PEM in malnutrition, the response of each immunological can result in illness and death due to infectious disease variable to malnutrition has been variously reported to (Chandra, 1992a). The impairments are seen in several be depressed, normal or elevated in different studies aspects of immunity such as cell-mediated immune (Gupta, 1993; Watson et al, 1985; Ozkan et al, 1993; responses, secretory IgA antibody production, phagocyte Amesty-Valbuena et al, 1996; Spirer et al, 1981; Kramer function, the complement system, and antibody af®nity and & Good, 1978). These inconsistencies are probably due to production (Chandra & Kumari, 1994). These ®ndings have different methodology applied or experimental background been supported by many studies on experimental animals including differences in the severity and types of malnutri- and human subjects including adults, infants and children tion, the existence of micronutrient de®ciencies, and the (Chandra, 1992b; Keusch, 1990). Since host immunity is an existence of hidden infectious diseases or in¯ammatory important protective mechanism against microorganisms, conditions. an assessment of immunocompetence has been recognized It is necessary to clarify characteristics of these im- as being a logical approach to evaluating nutritional status munological variables in malnutrition so that they could be (Chandra, 1981). more widely used in nutritional assessment. In view of this Serum levels of various immunoglobulins, comple- point, we conducted this study to examine the relationships ments, and lymphocytes subpopulations are frequently between immunological parameters and nutritional status with focus on the different types and severity of PEM. In addition, although there are several studies examining Correspondence: Dr T Rikimaru, Institute for International Cooperation, serum levels of IgA and IgG in malnourished children, as JICA, 10-5 Honmura-cho, Ichigaya, Shinjuku-ku, Tokyo 162, Japan. Received 22 August 1997; revised 30 December 1997; accepted 9 January well as other immunoglobulins, there are few studies 1998 examining how each IgA and IgG subclass responds to Immunity in malnourished children in Ghana T Rikimaru et al 345 mild and severe malnutrition or different types of mal- they could not be classi®ed into any categories of this nutrition. We, therefore, added these parameters to this classi®cation because of their normal body weight and assay to obtain further information about the response of edema. The reference from the United States National serum immunoglobulin levels to malnutrition. Center for Health Statistics (NCHS) was used for the classi®cation of different types of malnutrition and calcula- tion of z-score (WHO, 1983). The normal group consisted Method of children with 80% weight-for-age (W=A) of the NCHS reference data, the underweight group with 60± Subjects 80% W=A, and the kwashiorkor group with 60±80% Data was collected at the Princess Marie Louise (PML) W=A and presence of edema, the marasmus group with Hospital in Accra, Ghana, from April to October in 1994. < 60% W=A and no edema and marasmic kwashiorkor The target population consisted of children aged 8±36 group with < 60% W=A and presence of edema. Under- months who had been admitted with various degrees of weight corresponded to moderate malnutrition and kwa- PEM at the PML Hospital, as well as nutritionally normal shiorkor, marasmus and marasmic kwashiorkor to severe children and moderately malnourished children attending malnutrition. Summary data of anthropometric measure- the public health service section of the same hospital. The ments, body weight and height, and z-scores of weight-for- consent of the parent or guardian to allow his or her child age, height-for-age and weight-for-height are shown in to participate in the study was sought. When the parents or Table 1. guardians consented to participation, the children underwent a clinical observation and anthropometric measurements and had blood withdrawn for biochemical tests for Sample analysis assessing nutritional status and immunological status. 97% Immediately after withdrawal, the blood was divided into of the parents or guardians gave their consents. Selection two portions: the ®rst one was treated with EDTA for for severely malnourished children was made only from determination of lymphocyte subpopulation, and the second those admitted to the hospital within the previous 7 d and one was centrifuged to get serum for biochemical analysis who lived in urban communities in Accra. Children who including immunoglobulins. The samples were then trans- were diagnosed as having apparent complications of infec- ported to the laboratory of the Noguchi Memorial Institute tious diseases were excluded from the study. The clinical for Medical Research, the University of Ghana, Accra, for sign for the exclusion were mainly fever de®ned as the analyses. body temperature over 37.5C, diarrhea de®ned as over For the assessment of nutritional status, particularly three times of episode of loose stool, or respiratory tract protein-nutrition status, serum total protein, albumin, pre- symptom with injection of throat or any rales in lung ®eld albumin, retinol-binding protein, and transferrin were by auscultation. The number of those people were two determined. For prediction of the extent of severity of (3.2%) in the normal children, three (5.8%) in the under- in¯ammation, C-reactive protein (CRP) was determined. weight children and nine (13.0%) in the severely malnour- In addition, immunoglobulins (IgA and IgG subclasses, and ished children. The study was conducted following the IgM) and complement (C3 and C4 complements) were policy of the University of Ghana for biomedical research measured to examine the response of humoral immunity dealing with human subjects. to malnutrition, and the proportions of lymphocyte sub- One hundred and seventy-seven subjects were recruited populations (T cells, B cells, CD4, CD8, NK cells, and during the study period, and ®ve study groups, 61 normal HLADR cells) were measured to examine cell mediated children, 49 underweight children, 19 kwashiorkor chil- immunity in malnutrition. The absolute number of lympho- dren, 30 marasmus children and 11 marasmic kwashiorkor cyte subpopulations was not measured because of technical children, were formed according to the Wellcome classi®- reasons. Of these parameters, total protein, albumin, IgM, cation (Wellcome Trust Working party, 1970); data for CRP, C3 and C4 were determined by an automated clinical seven children were removed from the data analysis since chemistry analyzer (Super Z818, Mitsubishi Corporation,

Table 1 Anthropometric data in different types of malnutriton1

Groups Normal Underweight Kwashiokor Marasmus Marasmic kwashiokor (n)2 (61) (49) (19) (30) (11)

Age (mo) M 14.8a 14.1a 21.7b 14.0a 19.2a,b Anova SD 5.5 4.0 3.6 4.9 7.8 Weight (kg) LSM 9.7a 7.6b 7.3b 5.5c 5.6c Ancova SD 1.5 1.0 1.0 0.9 1.1 Height (cm) LSM 77.4a 73.9b 72.0b 68.3c 67.3c Ancova SD 5.6 4.1 4.6 6.1 5.1 z-score3 WAZ M 0.7a 2.7b 2.8b 4.6c 4.2c Anova SD 0.9 0.5 0.7 0.6 0.4 HAZ M 0.3a 1.7b 2.2b 3.4c 3.6c Anova SD 1.0 0.8 1.2 1.1 1.0 WHZ M 0.6a 2.2b,c 2.0c 3.1d 2.7b,d Anova SD 0.9 0.7 0.9 1.1 0.5

1Values are means (M) and standard deviation (SD). Values in the same row not sharing a common superscript alphabet (a, b, c and d) are signi®cantly different (P < 0.05). Mean was expressed as least square mean (LSM) when variables correlated with age and tested signi®cance with Ancova. 2Figure in parenthesis are sample number.3z-scores were expressed as weight-for-age z-score (WAZ), height-for-age z-score (HAZ) and weight-for-height z-score (WHZ). Immunity in malnourished children in Ghana T Rikimaru et al 346 Tokyo, Japan). Prealbumin, retinol-binding protein, trans- Results ferrin, IgA subclasses (IgA and IgA ), and IgG subclasses 1 2 Biochemical parameters (IgG1, IgG2, IgG3 and IgG4) were determined by a single immunodiffusion method (Human IgA and IgG subclasses The mean concentrations of serum total protein, albumin, monoclonal RID Kits, The Binding Site, Birmingham, prealbumin, retinol-binding protein (RBP), and transferrin England). are shown in Table 2. The values of these proteins, except Lymphocyte subpopulations were analyzed by FACScan for RBP, were signi®cantly lower in the severely malnour- Flowcytometer (Becton-Dickinson, Mountain View, CA, ished (the kwashiorkor, marasmus and marasmic kwashior- USA). Sample preparation was performed by a standard kor) groups than in the normal and underweight groups lysed whole blood method. In brief, 100 ml of anticoagu- (P < 0.05), indicating that these groups showed protein lated whole blood was incubated with each combination of malnutrition. There were no signi®cant differences in the antibodies for 20 min at room temperature, protected from RBP levels among all the groups. the light. Red cells were lysed for 15 min with FACS lysing solution. After being washed twice with PBS, samples were ready for ¯ow cytometric analysis. The C-reactive protein following combinations of monoclonal antibodies conju- Under normal conditions, the value is expected to be less gated to ¯uorescein isothiocyanate (FITC) or Phycoeryth- than 0.6 mg=100 ml (Tejani et al, 1995). The normal and rine (PE) (Becton Dickinson Immunocytometory System, the underweight groups showed mean values of 0.4 and San Jose, CA, USA) were used, including (1) control 0.5 mg=100 ml respectively, while the malnourished, particularly the kwashiorkor and marasmus, groups showed (mouse IgG1-FITC and IgG2-PE); (2) CD3 =CD19 (Leu12-PE); (3) CD4=CD8 (Leu3a-FITC and Leu2a- signi®cantly higher values of about 1.9 mg=100 ml (Table PE); (4) CD3=HLA-DR (Leu4-FITC and HLADR-PE); 2). The value of the marasmic kwashiokor group tended to and (5) CD3=CD16 56 (Leu4-FITC, Leu 11c-PE and be high, but there were no signi®cant differences from Leu19-PE). On every occasion of analysis, FACScan was those of the other groups. calibrated with CALIBRITE beads and AutoCOMP software. 5000 cells per sample were acquired and analyzed using LYSIS II software. Immunoglobulins The serum levels of IgA1 and IgA2 tended to be higher in the malnourished, particularly the marasmic kwashiorkor Statistics (P < 0.05), group than in the normal group, but there was SPSS and PC-SAS were used to analyze the data. Group no statistical signi®cance among the normal, underweight, means of anthropometric measurements and biochemical kwashiorkor, and marasmus groups (Table 3). The serum and immunological parameters were compared among the concentrations of IgG1, IgG2 and IgG3 did not change with groups of normal, underweight, kwashiorkor, marasmus and different nutritional status, but the IgG4 level tended to be marasmic kwashiorkor children using analysis of variance higher in the marasmic kwashiorkor group and the IgM for age and analysis of covariance with age as the covariate level to be higher in the kwashiorkor group. In general, for the other variables. When there was no correlation however, there was no remarkable difference in most between age and the response variable, group means were immunoglobulin variables among the different types of tested for signi®cant difference at a < 0.05 with Tukey's malnourished groups. test. When there was a correlation between age and other variables, group means were compared using the least squares means option the GLM procedure in PC-SAS. A Complement P-value of 0.05 or less was considered statistically signi®- The severely malnourished groups showed a decreased C3 cant. In order to clarify the relationships between nutritional level and an increased C4 level. There were no signi®cant status and the anthropometric, biochemical and immunolo- differences in these values among the severely malnour- gical variables, factor analysis (with varimax rotation) was ished groups, the kwashiorkor, marasmus and marasmic performed on the outcome variables, and the resulting kwashiorkor groups, and no signi®cant difference between factors were also examined with analysis covariance. the normal and underweight groups (Table 3).

Table 2 Serum levels of biochemical variables and C-reactive protein in different types of nutritional status1

Groups Normal Underweight Kwashiokor Marasmus Marasmic Kwashiokor (n)2 (49) (40) (19) (27) (10)

Total protein (g/100 ml) M 6.97a 6.75a 5.52b 6.08c 5.30b Anova SD 0.59 0.72 1.25 0.89 1.00 Albumin (g/100 ml) LSM 4.28a 4.04a 2.90b 3.18b 2.75b Ancova SD 0.32 0.57 0.86 0.70 0.57 Prealbumin (mg/100 ml) M 188a. 165a,b. 135b. 150b. 133b. Anova SD 57. 49. 58. 78. 80. Transferrin (mg/l) M 3824a. 3150b 1832c. 1886c. 1410c. Anova SD 922. 884. 1017. 1047. 976. Retinol binding protein (mg/l) M 25.6a 24.5a 23.5a 24.7a 27.1a Anova SD 9.5 10.3 12.7 13.8 11.8 C-creactive protein (mg/100 ml) M 0.44a 0.49a 1.85b 1.93b 1.40a,b Anova SD 1.13 0.86 3.65 3.57 1.41

Groups Normal Underweight Kwashiokor Marasmus Marasmic kwashiokor (n)2 (50) (39) (17) (27) (11)

Immunoglobulins a a,b c,d b,c d IGA1 (mg/l) M 863. 861. 1234. 1115. 1608. Anova SD 437. 384. 401. 726. 506. a a a,b a,b b IGA2 (mg/l) LSM 98.6 104.8 127.7 153.1 177.7 Ancova SD 73.1 72.4 62.8 92.4 107.3 a a a a a IGG1 (mg/l) M 7861. 8064. 7073. 7163. 7942. Anova SD 2995. 2300. 2290. 2660. 4467. a a a a a IGG2 (mg/l) M 1151. 1134. 889. 976. 1097. Anova SD 480. 662. 347. 450. 601. a a a a a IGG3 (mg/l) M 796. 873. 929. 728. 827. Anova SD 324. 366. 461. 291. 263. a,b a,b b a,b a IGG4 (mg/l) LSM 85.9 96.7 50.0 113.5 145.7 Ancova SD 83.7 82.8 72.3 91.3 103.7 IGM (mg/l) M 117.1a 131.8a 174.8a 145.5a 132.1a Anova SD 49.6 59.6 84.3 86.6 50.4 Complement C3 (mg/100ml) M 97.4a 85.7a 65.6b 66.1b 56.1b Anova SD 33.6 30.2 46.3 31.7 35.5 C4 (mg/100ml) M 40.9a 41.5a 54.2b 55.0b 52.9a,b Anova SD 14.6 14.5 23.1 22.8 14.3

Lymphocyte subpopulations CD4%, CD8% and HLADR % are negatively or posi- The T cells % tended to be slightly higher in the severely tively correlated with Factor 2, which show no relation- malnourished groups than in the normal group (Table 4). ship with z-score and biochemical variables. B cells % is The B cells % were adversely lower in the malnourished negatively correlated with Factor 3, which is correlated and underweight groups (P < 0.05). The CD4 %, CD8 %, positively with T cells % as well as transferrin, IgA1, IgA2, NK cells % and HLADR % and the ratio CD4 =CD8 did IgG4, C4 and CD4 %. not differ among all the groups. Discussion Factor analysis The result of factor analysis is shown in Table 5. Factor 1 is Immunoglobulins the factor with the largest loading for the index of nutri- Serum immunoglobulin levels in PEM have been observed tional status such as weight-for-height z-score and serum to be variously elevated, normal or depressed (Watson et al, protein levels including albumin and transferrin levels. C3 1985; Ozkan et al, 1993; Amesty-Valbuena et al, 1996; is positively and highly correlated with this factor. Pre- Spirer et al, 1981; Neumann et al, 1975; McMurray et al, albumin, RBP, IgA2, IgG1 and IgM share a common factor 1981; Sall et al, 1994). These inconsistencies are probably (Factor 4) which is having the largest loading for the serum due to the interplay of age, severity or types of malnutrition level of C-reactive protein as an in¯ammation or infectious or history of infections in subjects. Gupta (1993) described index: Prealbumin and RBP are negatively correlated with that the age of the patient at the time of PEM appears to be this factor and others are positively correlated with. IgG3, an important factor to affect the level of immunoglobulin

Table 4 Lymphocyte subpopulations in different types of nutritional status1

Groups Normal Underweight Kwashiokor Marasmus Marasmic kwashiokor (n)2 (57) (43) (16) (28) (9)

T cell (%) M 56.8a 60.2a,b 66.5c 62.3b,c 60.3a,b,c Anova SD 6.9 8.7 10.4 12.7 8.7 B cell (%) M 29.6a 25.6b 19.7c 21.8b,c 21.6b,c Anova SD 6.9 7.7 8.0 9.5 10.3 CD4 (%) M 34.9a 34.5a 37.9a 34.8a 35.7a Anova SD 7.0 8.3 9.2 10.1 9.9 CD8 (%) LSM 18.7a,b 22.4a,b 21.2a,b 24.4a 17.1b Anova SD 5.9 9.2 8.2 9.9 6.1 Ratio (CD4/CD8) M 2.13a 1.89a 1.89a 1.83a 2.11a Anova SD 0.92 0.99 1.09 1.15 0.93 NK cell (%) M 7.3a 7.2a 5.7a 7.0a 6.0a Anova SD 3.7 4.3 3.4 4.8 4.4 HLADR (%) M 7.2a 8.9a 11.6a 9.2a 8.9a Anova SD 3.5 7.4 9.8 7.4 5.0

Factor 1 Factor 2 Factor 3 Factor 4

Anthropometric variable WHZ2 0.69 0.05 0.10 0.09 Biochemical variables Total protein 0.88 0.13 0.18 0.01 Albumin 0.83 0.03 0.33 0.25 Prealbumin 0.43 0.03 0.06 0.56 Transferrin 0.67 0.10 0.40 0.27 RBP3 0.13 0.13 0.12 0.53 C-reactive protein 0.12 0.25 0.04 0.73 Immunological variables IgA1 0.39 0.24 0.45 0.30 IgA2 0.20 0.17 0.47 0.52 IgG1 0.16 0.35 0.12 0.54 IgG2 0.36 0.04 0.10 0.25 IgG3 0.12 0.46 0.13 0.33 IgG4 0.03 0.22 0.40 0.17 IgM 0.12 0.20 0.17 0.53 C3 0.75 0.01 0.08 0.02 C4 0.06 0.02 0.50 0.31 T cell (%) 0.02 0.10 0.83 0.18 B cell (%) 0.11 0.05 0.78 0.22 CD4 (%) 0.00 0.81 0.45 0.08 CD8 (%) 0.03 0.85 0.34 0.06 Ratio (CD4/CD8) 0.08 0.89 0.04 0.10 NK cell (%) 0.27 0.15 0.11 0.02 HLADR (%) 0.02 0.82 0.26 0.04

1Figures are correlation coef®cient of the variables for each common factor obtained in a factor analysis. 2WHZ: weight-for-height z-score. 3RBP: retinol binding protein.

and that polyclonal hyperimmunoglobulinemia is frequent biochemical parameters for nutritional assessment, but the in adults and older children. This is interpreted as being correlation ef®ciency is not very high. On the other hand, probably due to frequent infection and=or parasitic infesta- IgA2, IgG1 and IgM are positively correlated with a factor tion. In addition, previous studies of the major clinical which can affect the level of CRP, as an index for infection. types of severe malnutrition (namely kwashiorkor, maras- These data demonstrates that serum immunoglobulins level mus, and combined) suggested that some aspects of the are more dependent on the severity of in¯ammation rather immune response are affected in different ways depending than differences in nutritional status. on the types of malnutrition (McMurray et al, 1981; Sall et al, 1994). Complement McMurray et al (1981) has reported that, in children This study showed that serum levels of C3 were signi®- suffering from kwashiorkor, marasmus and combined mal- cantly lower in the severely malnourished children than in nutrition, the improvement in clinical and nutritional status the normal children, while those of C4 were conversely was accompanied by signi®cantly increased levels of serum higher. The serum levels of various complement proteins, IgG and IgM and by signi®cantly decreased levels of IgA, except for C4, have been found consistently to be lower in especially in the children with kwashiorkor. This suggests malnourished children than in normal children (Chandra et that IgG and IgM are decreased and IgA is increased in al, 1981; Ozkan et al, 1993; Amesty-Valbuena et al, 1996; malnutrition, particularly in kwashiorkor. On the other Sirisinha et al, 1973; Suskind et al, 1976). In particular C3 hand, Sall et al (1994) have reported that kwashiorkor led has been noted as an immunological parameter which to decreased concentration only in IgG and this was responded remarkably to malnutrition. The reduced C3 in restored by refeeding. These two studies suggest that IgG malnutrition has been interpreted as being due to anti- is decreased in the children with kwashiorkor. Meanwhile, complementary serum factor, increased catabolism or consistent results in many studies have been that IgA level decreased synthesis in the liver (McMurray et al, 1981; is elevated in malnutrition irrespective of the type of Suskind et al, 1976). This idea may be partially supported malnutrition (Watson et al, 1985; Ozkan et al, 1993; by our results that indicate that the levels of C3 was parallel McMurray et al, 1981). the levels of biochemical parameters such as total protein, Our study con®rmed that the levels of serum immuno- albumin, prealbumin and transferrin. In addition, the C3 globulins observed in this study were not affected signi®- level is highly correlated with a factor which is strongly cantly by moderate malnutrition, but those of IgA associated with these biochemical variables as protein subclasses, IgA1 and IgA2, were increased in severe malnutrition indicators, but not CRP as an in¯ammation nutrition. In terms of IgG subclass, we could not ®nd any indicator. This implies that the reduced C3 level in severe characteristic changes with malnutrition, except that IgG4 malnutrition is associated with the change of protein tended to be high in marasmic kwashiorkor. The incon- metabolism rather than with changes in the complement sistencies between the previous and our ®ndings are prob- system due to infection. ably interpreted to be a result that immunoglobulin levels The low C3 level was not observed among children with are in¯uenced by other factors besides nutritional status. mild malnutrition (underweight) in this study. Although IgA1 is negatively correlated with a factor which can underweight is usually categorized as being a kind of affect serum total protein, albumin and transferrin as malnutrition, the underweight children seem to be quite Immunity in malnourished children in Ghana T Rikimaru et al 349 close or similar to the normal children as far as immuno- cells play a role to produce immunoglobulins, the level of globulin and complement levels are concerned. This is immunoglobulins is expected to be decreased with malnu- supported by the result that most of the biochemical trition. However, IgA and IgG4 tended to be decreased in parameters did not signi®cantly differ between the normal the malnourished children. This inconsistency may be children and underweight children. partially explained by the previous observation that IgA There are several reports on serum complement levels in producing B cells, which have cytotoxic function but no B different types of malnutrition, the main observation being cells marker, increased in malnourished children compared that children with kwashiorkor have lower complement with well-nourished control children (Chandra, 1979). IgG4 levels than children with marasmus (Neumann et al, is also predicted that IgG4 producing B cells are activated 1975; McMurray et al, 1981; Sirisinha et al, 1973). In with malnutrition. our study, however, the C3 level tended to be lower in the Regarding the effect of types of malnutrition on lym- marasmic kwashiorkor group than the other severely mal- phocyte, there has been a ®nding that kwashiorkor group nourished (kwashiorkor and marasmic) groups. Two of the showed a lower T cells % than did other malnourished cited studies (Neumann et al, 1975; Sirisinha et al, 1973) groups (Keusch, 1990). We could not, however, con®rm did not include marasmic kwashiorkor children. The other any differences of the proportion of lymphocyte subpopu- study containing a group with the combined characteristics lation among the three malnourished groups (kwashiorkor, of both kwashiorkor and marasmus, showed that the serum marasmus, and marasmic kwashiorkor) in this study. This C3 level was higher in this group than in the kwashiorkor result may be in¯uenced by the limitation of sample size group (McMurray et al, 1981). The study showed that the and the large variation in each type of malnourished group. serum albumin level was higher in the combined group than in the kwashiorkor group, but our study showed the opposite tendency. Judging from the results of biochemical Conclusions data, the inconsistency between the results of our study and In our study, we con®rmed that the proportion of B cells those of the previous study could be due to different was decreased in the moderately and severely malnourished severity in protein de®ciency in each group. children, and that C3 level was decreased and IgA1, IgA2, In terms of C4 complement, levels have been reported to and C4 levels were increased in the severely malnourished be normal in the malnourished children and appear not to children. We also con®rmed that most immunological be affected by nutritional status (McMurray et al, 1981; parameters had less in¯uence by moderate malnutrition Sirisinha et al, 1973). In contrast to these previous reports, (underweight) and had a little difference among the differ- our results showed that C4 levels were actually higher in ent types of malnutrition. In addition, from the factor the malnourished children compared to the normal and analysis, we found that only C3 level is highly associated underweight children. According to the result of a factor with the levels of variables for protein nutrition status and analysis, C4 level was positively correlated with a factor anthropometric measurements. which was associated with T cells % or B cells %, indicating that the increased C4 may be affected by the AcknowledgementsÐWe thank Mr EA Addo and Mr MM Addae and the change of T cells % or B cells %. technical staff at the Noguchi Memorial Institute for Medical Research, the University of Ghana, who were involved in this study for technical Cell mediated immunity assistance and Dr D Armah at Princess Marie Louise Hospital for Cell mediated immunity in PEM has been variously cooperating in the recruitment of the study children. We also thank Ms J reported to be impaired and to be normal (Keusch, 1990). Peerson at the Program in International Nutrition, the University of Ozkan et al, 1993 reported that T lymphocyte subgroups California, Davis, for advising us with the data analysis. This study was were found to be decreased in PEM when compared to done as a series of research activities in the JICA (Japan International control groups, but the ratio of CD4=CD8 did not show a Cooperation Agency) Medical Cooperation Project at the Noguchi Mem- orial Institute for Medical Research. statistically signi®cant difference. Other studies have also shown that the number of the T cells was generally decreased in PEM (Ferguson et al, 1974; Chandra, 1977; Chandra, 1983). In terms of B cells, studies using mono- References clonal antibodies have also shown decreased proportions Amesty-Valbuena A, Diez-Ewald M, de Villarroel M, Montiel N, and numbers of CD20 B lymphocytes (Garraud et al, Granados A, Diaz S, Salas D & Rivero M (1996): Immuno- 1987). In contrast, in vivo studies of the T lymphocyte logic characteristics of undernutrition. I. The undernourished patient in nutritional recovery. Investigation Clinica 37, 95±111 mitogen responses of circulation lymphocytes in both (Spanish). adults and children with marasmus have not demonstrated Chandra RK (1977): Lymphocyte subpopulation in human malnutrition: impaired proliferative capacity (Cunningham-Rundles, cytotoxic and suppressor cells. Pediatrics 59, 423±427. 1982). In addition, Spirer et al, 1981 has reported that the Chandra RK (1979): T and B lymphocytes and leukocyte terminal deoxynucleotide-transferase in energy-protein malnutrition. Acta proportion of T cells (T cells %) was normal in both normal Pediatr. Scand. 68, 841±845. and malnourished groups. Chandra RK (1981): Immunocompetence as a functional index for nutri- In this study, the proportions of lymphocyte subpopula- tional status. Br. Med. Bull. 37, 89±94. tions were determined in different levels of severity or Chandra RK (1983): Nutrition, immunity, and infection: present knowl- types of malnutrition, but the absolute number or antibody- edge and future directions. Lancet 1, 689±691. Chandra RK (1992a): Nutrition and immunoregulation. Signi®cance for producing capacity of lymphocyte subpopulations were not. host resistance to tumors and infectious diseases in humans and rodents. Under this condition, it might be dif®cult to refer to the J. Nutr. 122, Suppl. 3, 754±757. effect of nutritional status on the function of lymphocyte Chandra RK (1992b): Protein-energy malnutrition and immunological subsets, but the data of this study demonstrated that severe responses. J. Nutr. 122, 597±600. Chandra RK & Kumari S (1994): Nutrition and immunity: An overview. J. malnutrition affected B cells %. The B cells % was found to Nutr. 124, 1433S±1435S. be signi®cantly decreased with malnutrition, implying Cunningham-Rundles S (1982): Effects of nutritional status on immuno- impaired production of B cells with malnutrition. Since B logical function. Am. J. Clin. Nutr. 35, 1202±1210. Immunity in malnourished children in Ghana T Rikimaru et al 350 Ferguson AC, Lawlor GJ, Neumann CG, Oh W & Stiehm ER (1974): Sall MG, Toure M, Vol S, de Vonne T, Mouray H, Kuakovi N & Maurage Decreased rosette-forming lymphocytes in malnutrition and intrauterine C (1994): Effects of refeeding on serum immunoglobulin (IgA, IgG, growth retardation. J. Pediatr. 85, 717±723. IgM) concentrations in children with severe protein-energy malnutri- Garraud O, Daculsi R, Merlio JP, Lobera A & Legrand E (1987): tion. Archives de Pediatrie 1, 132±136 (in French). Detection of CD1 positive cells in the peripheral blood of patients Sirisinha S, Suskind R, Edelman R, Charupatana C & Olson RE (1973): suffering from cancer-associated malnutrition. Immunol. Let. 15,73± Complement and C3-proactivator levels in children with protein calorie 76. malnutrition and effect of dietary treatment. Lancet 12, 1016±1020. Gupta S (1993): Malnutrition and lymphocyte subpopulation responses in Spirer Z, Hauzer G, Zakuth V, Diamant S & Golander A (1981): humans. In Nutrition Modulation of the Immune Response. ed. S Immunological functions in children years after recovery from protein Cunningham-Rundles, 441±454. New York, Marcel Dekker Inc. calorie malnutrition (PCM) in their early infancy. J. Clin. Lab. Immu- Keusch GT (1990): Malnutrition, infection, and immune function: In The nol. 6, 111±113. Malnourished Child, ed. RM Suskind L Lewinter-Suskind, 35±59. New Suskind R, Edelman R, Kukapongs P, Pariyanonda A & Sirisinha S York, Vevey=Raven Press Ltd. (1976): Complement activity in children with protein-calorie malnutri- Kramer TR & Good RA (1978): Increased in vitro cell-mediated immunity tion. Am. J. Clin. Nutr. 29, 1089±1092. in protein-malnourished Guinea pigs. Clin. Immunol. and Immunopath. Tejani NR, Chonmaitree T, Rassin DK, Howie VM, Owen MJ & Goldman 11, 212±228. AS (1995): Use of C-reactive protein in differentiation between acute McMurray DN, Watson RR & Reys MA (1981): Effect of renutrition on bacterial and viral otitis media. Pediatrics 95, 664±669. humoral and cell-mediated immunity in severely malnourished children. Watson RR, McMurray DN, Martin P & Reyes M (1985): Effect of age, Am. J. Clin. Nutr. 34, 2117±2126. malnutrition and renutrition on free secretory component and IgA in Neumann CG, Lawlor GJ, Stiehm ER, Swendseid ME, Newton C, Herbert secretions. Am. J. Clin. Nutr. 42, 281±288. J, Ammann AJ & Jacob M (1975): Immunologic responses in mal- Wellcome Trust Working Party (1970): Classi®cation of infantile malnu- nourished children. Am. J. Clin. Nutr. 28, 89±104. trition. Lancet 2, 302±303. Ozkan H, Olgun N, Sasmaz E, Abacioglu H, Okuyan M & Cevik N (1993): WHO (1983): Reference data for the weight and height of children, In Nutrition, immunity and infection. J. Trop. Pediatr. 39, 257±260. Measuring Change in Nutritional Status, 61±101. Geneva, WHO.