CLINICAL
IMMUNOLOGY
AND
8,
IMMUNOPATHOLOCY
1- 6
(1977)
immune
Responses
MAHMOUD
RAFII, SAKINEH HASHEMI, JAFAR NAHANI, NAHID MOHAGHEGHPOUR’
The Children’s and Department
in Malnourished
Hospital, and Department of Biochemistry, School
of Pathobiology, School of Medicine, University
Children AND
of Public Health of Teheran, Iran
Received January 9, 1976 The thymus (T)- and bone narrow (Bkdependent immune systems were studied in severely malnourished children and age-matched controls. The levels of IgM and IgG were unaffected by malnutrition. However, children with severe malnutrition had elevated IgA levels. The percentage of B cells in the peripheral blood of the malnourished group was the same as that of the normal group, while the proportion of T cells was significantly reduced in the malnourished group. The T-dependent immune function, assessed by either the development of delayed hypersensitivity reaction after BCG vaccination or in vitro lymphocyte response to phytohemagglutinin, however, was normal in the malnourished group. Enhanced cellular response to PHA occurred in severely malnourished children.
INTRODUCTION
Most in viva and in vitro observations suggest the possibility of T-cell immunodeficiency in malnourished children. These children are not readily sensitized with DNCB (1-5) and exhibit depressed delayed cutaneous hypersensitivity (6-8). The in vitro stimulation of their peripheral blood lymphocytes with nonspecific mitogen is reportedly suppressed (2,7,9,10). In addition, they are known to have a diminished number of T cells (4,5,8,1 I). The thymic atrophy and the depletion of paracortical lymphocytes in the lymph nodes observed in these children (2) are further indications of T-cell immunodeficiency. Although these lines of evidence strongly suggest that the thymus-derived components of the immune system are damaged in malnourished children, a few studies have yielded conflicting results (3,8,12). These discrepancies may well be due to the differences in the nutritional status of the patients studied. Furthermore, the effects of various diseases prevalent in different study areas on the immune response cannot be excluded. The present study was designed to reexamine and compare certain parameters of humoral- and cellular-immune responses in children suffering from severe protein-calorie malnutrition with those of the normal controls. To define clearly the extent of malnutrition, certain nutritional indices were measured in both groups. PATIENTS AND METHODS
Patients The children studied had recently been admitted to an orphanage in Teheran. They were between the ages of 5 and 17 months, and the distribution of the two 1 Reprint requests to: Dr. Nahid Mohagheghpour. Department of Pathobiology, Health, University of Teheran, P. 0. Box 1310, Teheran. Iran.
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved
School of Public
ISSN OOYO-I?29
2
RAF11
E7-AL
sexes was approximately equal. The diagnosis of severe protein-calorie malnutrition on 13 children was based on weights less than 60% on the 50th percentile for age on the Boston Growth Curve (BGC), as suggested by the FOA/WHO (13). and on loss of subcutaneous fat. None of the children had apparent infection and none were suffering from kwashiorkor. Thirteen healthy children with weights of 80% of BGC or above served as controls. Serum Protein Studies Blood was obtained by femoral vein puncture from each child. Serum was collected and kept frozen at -20°C until tested (within a month). Total serum proteins were measured by the microbiuret method and serum albumin was measured by quantitative cellulose-acetate electrophoresis. Serum levels of complement (C’:,), transferrin, ceruloplasmin, and immunoglobulins were estimated by the single radial immunodiffusion technique using Hyland immunoplates. Rosette-Forming Cells Samples of heparinized venous blood, was described previously ( 1 l), were used to determine the percentage of T and B cells by allowing the formation of rosettes. according to the method of Jondal et al. (14). With one exception, all children had white blood cell counts of higher than 2500. Lymphocyte Transfkwmtion T-cell function was studied by assessing the lymphocyte response to a nonspecific mitogen, phytohemagglutinin. Lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation and cultured in TC 199 medium with L-glutamine plus 20% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 pg/ml) to a final concentration of 2 x 10” lymphocytes/ml. For stimulation, 0.1 ml each of a 1: 128 and 1: 1024 dilution of PHA-P (Difco Laboratories) was added to each of four culture tubes containing 0.9 ml of cell suspension. As a control 0. I ml of medium was added to another four tubes. The cells (1 ml/tube) were cultured at 37°C with 5% CO, for 72 hr. DNA synthesis was measured by means of [“H-l thymidine incorporation, and the index of response (IR) for each child was derived by dividing the counts per minute of PHA-stimulated cultures by those of unstimulated controls and expressing the result as a percentage of unstimulated cells. Deluyed Hypersensitivity Next. the ability to develop delayed hypersensitivity was studied using BCG vaccination. All children had the Mantoux test with 5 TU of PPD and only those with a negative skin test (induration less than 6 mm at 48 hr) were vaccinated with BCG (0.1 ml of 1 mg/ml BCG vaccine prepared by Pasteur Institute of Paris). Thirteen weeks after vaccination the children were skin tested again with 5 TU of PPD. The challenge site was examined after 24,48, and 72 hr. and the reaction was graded as positive (induration of 6 mm or more). RESULTS
Total serum proteins were normal or only moderately reduced in the malnourished children (Table 1). The mean serum albumin, however, was signi&
7 7
7
9 9 13 14 16
5 6
87
9 10 11 12 13 Mean f SD Normal mead ? SD
59 57 54 58 56 52 2 5 85 2 4
56 50
41 47
53 50 43 52
WeighP
84 99 84 81 84 86 + 4 95 k 4
86 85
81 88
86 88 85 88
Height”
INDEXES
p Weight and height as percentage of standard. * NT, not tested r Means for 13 normal children. * Statistically significant (P < 0.01) as compared to normal.
7
5 5 5 6
Age (months)
Malnourished 1 2 3 4
Case number
BIOCHEMICAL
TABLE
1
6.8 7.6 7.0 6.9 7.0 7.0 k 0.4 6.9 2 0.2
7.4 7.2
1.3 6.8
6.5 6.8 1.4 6.3
Total serum protein (g/l00 ml)
IN MALNOURISHED
AND
3.3 3.1 3.3 f 0.7* 4.0 2 0.3
N7h 3.6 2.8
4.6 2.7
3.0 4.1
2.6 4.0 3.0 2.5
Albumin (g/100 ml)
NORMAL
86 129 180 110 180 128 + 41 135 +- 30
137 200
129 92
115 56 91 155
C’ (g/100 ml)
CHILDREN
150 190 260 222 187 k 58* 255 k 75
180 235
300 79
180 120 120 201
Transferrin (mg/lOO ml)
11 12 37 12 52 29 k 14* 44k 12
45 45
16 29
36 29 34 15
Ceruloplasmin (mg/lOO ml)
w
$
B 2
s
E
2 2 7
4
RAF1
I ET AL
cantly reduced (P
IMMUNOLOGY
AND
8,
IMMUNOPATHOLOCY
1- 6
(1977)
immune
Responses
MAHMOUD
RAFII, SAKINEH HASHEMI, JAFAR NAHANI, NAHID MOHAGHEGHPOUR’
The Children’s and Department
in Malnourished
Hospital, and Department of Biochemistry, School
of Pathobiology, School of Medicine, University
Children AND
of Public Health of Teheran, Iran
Received January 9, 1976 The thymus (T)- and bone narrow (Bkdependent immune systems were studied in severely malnourished children and age-matched controls. The levels of IgM and IgG were unaffected by malnutrition. However, children with severe malnutrition had elevated IgA levels. The percentage of B cells in the peripheral blood of the malnourished group was the same as that of the normal group, while the proportion of T cells was significantly reduced in the malnourished group. The T-dependent immune function, assessed by either the development of delayed hypersensitivity reaction after BCG vaccination or in vitro lymphocyte response to phytohemagglutinin, however, was normal in the malnourished group. Enhanced cellular response to PHA occurred in severely malnourished children.
INTRODUCTION
Most in viva and in vitro observations suggest the possibility of T-cell immunodeficiency in malnourished children. These children are not readily sensitized with DNCB (1-5) and exhibit depressed delayed cutaneous hypersensitivity (6-8). The in vitro stimulation of their peripheral blood lymphocytes with nonspecific mitogen is reportedly suppressed (2,7,9,10). In addition, they are known to have a diminished number of T cells (4,5,8,1 I). The thymic atrophy and the depletion of paracortical lymphocytes in the lymph nodes observed in these children (2) are further indications of T-cell immunodeficiency. Although these lines of evidence strongly suggest that the thymus-derived components of the immune system are damaged in malnourished children, a few studies have yielded conflicting results (3,8,12). These discrepancies may well be due to the differences in the nutritional status of the patients studied. Furthermore, the effects of various diseases prevalent in different study areas on the immune response cannot be excluded. The present study was designed to reexamine and compare certain parameters of humoral- and cellular-immune responses in children suffering from severe protein-calorie malnutrition with those of the normal controls. To define clearly the extent of malnutrition, certain nutritional indices were measured in both groups. PATIENTS AND METHODS
Patients The children studied had recently been admitted to an orphanage in Teheran. They were between the ages of 5 and 17 months, and the distribution of the two 1 Reprint requests to: Dr. Nahid Mohagheghpour. Department of Pathobiology, Health, University of Teheran, P. 0. Box 1310, Teheran. Iran.
Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved
School of Public
ISSN OOYO-I?29
2
RAF11
E7-AL
sexes was approximately equal. The diagnosis of severe protein-calorie malnutrition on 13 children was based on weights less than 60% on the 50th percentile for age on the Boston Growth Curve (BGC), as suggested by the FOA/WHO (13). and on loss of subcutaneous fat. None of the children had apparent infection and none were suffering from kwashiorkor. Thirteen healthy children with weights of 80% of BGC or above served as controls. Serum Protein Studies Blood was obtained by femoral vein puncture from each child. Serum was collected and kept frozen at -20°C until tested (within a month). Total serum proteins were measured by the microbiuret method and serum albumin was measured by quantitative cellulose-acetate electrophoresis. Serum levels of complement (C’:,), transferrin, ceruloplasmin, and immunoglobulins were estimated by the single radial immunodiffusion technique using Hyland immunoplates. Rosette-Forming Cells Samples of heparinized venous blood, was described previously ( 1 l), were used to determine the percentage of T and B cells by allowing the formation of rosettes. according to the method of Jondal et al. (14). With one exception, all children had white blood cell counts of higher than 2500. Lymphocyte Transfkwmtion T-cell function was studied by assessing the lymphocyte response to a nonspecific mitogen, phytohemagglutinin. Lymphocytes were isolated by Ficoll-Hypaque gradient centrifugation and cultured in TC 199 medium with L-glutamine plus 20% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 pg/ml) to a final concentration of 2 x 10” lymphocytes/ml. For stimulation, 0.1 ml each of a 1: 128 and 1: 1024 dilution of PHA-P (Difco Laboratories) was added to each of four culture tubes containing 0.9 ml of cell suspension. As a control 0. I ml of medium was added to another four tubes. The cells (1 ml/tube) were cultured at 37°C with 5% CO, for 72 hr. DNA synthesis was measured by means of [“H-l thymidine incorporation, and the index of response (IR) for each child was derived by dividing the counts per minute of PHA-stimulated cultures by those of unstimulated controls and expressing the result as a percentage of unstimulated cells. Deluyed Hypersensitivity Next. the ability to develop delayed hypersensitivity was studied using BCG vaccination. All children had the Mantoux test with 5 TU of PPD and only those with a negative skin test (induration less than 6 mm at 48 hr) were vaccinated with BCG (0.1 ml of 1 mg/ml BCG vaccine prepared by Pasteur Institute of Paris). Thirteen weeks after vaccination the children were skin tested again with 5 TU of PPD. The challenge site was examined after 24,48, and 72 hr. and the reaction was graded as positive (induration of 6 mm or more). RESULTS
Total serum proteins were normal or only moderately reduced in the malnourished children (Table 1). The mean serum albumin, however, was signi&
7 7
7
9 9 13 14 16
5 6
87
9 10 11 12 13 Mean f SD Normal mead ? SD
59 57 54 58 56 52 2 5 85 2 4
56 50
41 47
53 50 43 52
WeighP
84 99 84 81 84 86 + 4 95 k 4
86 85
81 88
86 88 85 88
Height”
INDEXES
p Weight and height as percentage of standard. * NT, not tested r Means for 13 normal children. * Statistically significant (P < 0.01) as compared to normal.
7
5 5 5 6
Age (months)
Malnourished 1 2 3 4
Case number
BIOCHEMICAL
TABLE
1
6.8 7.6 7.0 6.9 7.0 7.0 k 0.4 6.9 2 0.2
7.4 7.2
1.3 6.8
6.5 6.8 1.4 6.3
Total serum protein (g/l00 ml)
IN MALNOURISHED
AND
3.3 3.1 3.3 f 0.7* 4.0 2 0.3
N7h 3.6 2.8
4.6 2.7
3.0 4.1
2.6 4.0 3.0 2.5
Albumin (g/100 ml)
NORMAL
86 129 180 110 180 128 + 41 135 +- 30
137 200
129 92
115 56 91 155
C’ (g/100 ml)
CHILDREN
150 190 260 222 187 k 58* 255 k 75
180 235
300 79
180 120 120 201
Transferrin (mg/lOO ml)
11 12 37 12 52 29 k 14* 44k 12
45 45
16 29
36 29 34 15
Ceruloplasmin (mg/lOO ml)
w
$
B 2
s
E
2 2 7
4
RAF1
I ET AL
cantly reduced (P
