Immunoglobulins children with Suskind,3 Edelman,’ E. Olson,9
M.D., Stitaya M.D., Damn M.D., Ph.D.
ABSTRACT
Sirishinha,4 Damrongsak,7
The
Northern
Thai
immunoglobulin
children
with
marasmus-kwashiorkor, admission
and
fractions with
either
or without
marasmus were
higher
percent
demonstrated
PCM
in the control
from
increase
those
than
with PCM
levels
immunized
no significant
increase
to
and
IgD
levels urban
of IgG,
1gM,
with
64%
children
in 28
measured
rural
Thai
or IgD with
with
IgA
levels Eighty
IgE
levels
and
Within
while
Am. J. Clin.
titer.
children,
treatment.
antigen.
group,
on
in children
with PCM
typhoid
7 had
immunoglobulin
had detectable
admitted
in the recovered
in H antibody
studied
were All
and
IgA,
to normal
while
intradermal
Ph.D., and
or kwashiorkor.
returned
levels
additional
appeared
IgE) were
recovery.
marasmus-kwashiorkor
children
H antibody
and
M.D., M.D.,
Of these 14 had marasmus,
in well-nourished
detectable Ten
leading
in levels
had
control
had groups.
were
in typhoid
who
IgD,
immunoglobulin
treatment seen
1gM, (PCM).
The
of
was no difference
with
in malnourished
recovered
significant group
compared
to none
kwashiorkor.
12 weeks
There
IgA,
malnutrition
or exceeded
infection.
of the children
compared had
equalled
levels (IgG,
7 had
during
2
Ph.D., Vicharn Vithayasai,5 M.D., Chinda Charupatana,8
protein-calorie
and serially
when
M.D.,
in
as
10 who 8 days
a
the malnourished
Nutr.
29: 836-841,
1976.
The synergistic interaction of infection and malnutrition has been recognized by several clinicians and investigators (1). Children with protein-calorie malnutrition (PCM) often have an increased incidence and severity of infections. Impairment of the cell-mediated immune function (2, 3) and of the inflammatory response (2) has been demonstrated in children with PCM. In addition, the complement system has been shown to be adversely affected in malnutrition (3, 4). There is, however, no agreement on the effect of PCM on the humoral immune mechanism. While most studies show that immunoglobulin levels are not depressed (5-10), antibody response in children with PCM seems to vary accordng to the type and form of antigen used (11-14). Chandra (3) noted that in contrast to the depressed response to typhoid immunization, undernourished children reacted adequately to tetanus toxoid. In addition, Cohen and Hansen (15) noted that the synthesis of “y-globulin in infected malnourished children was 3 times that of the uninfected group. We undertook this study to evaluate the integrity of the humoral immune system in children with marasmus and kwashiorkor by 836
The American
Journal
of Clinical
Nutrition
determining the levels of serum immunoglobulins (IgG, 1gM, IgA, IgD, and IgE), and the specific antibody response to typhoid immunization before and after treatment.
ter,
‘From the Anemia and Malnutrition Research Cenand Department of Pediatrics, Faculty of Medicine,
Chiang Department
Mai
University,
Chiang
of Microbiology,
Faculty
Mai,
Thailand,
the Mahithe Department of Laboratory, Bangof Pediatrics and School of Medicine, of Science,
dol University, Bangkok, Thailand, Virology, SEATO Medical Research kok, Thailand, and the Departments Biochemistry, St. Louis University St. Louis, Missouri. ‘Supported in part by Grants-in-aid from the National Institutes of Health, (AM-I 1044), U.S. Public Health Service, Bethesda, Maryland, the Rockefeller Foundation, New York, New York, and the U.S. Army Medical Research and Development Command. Deputy Director, Anemia and Malnutrition Research Center, Chiang Mai, Thailand and Assistant Professor of Pediatrics, St. Louis University School of Medicine, St. Louis, Missouri. Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 4Professor of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. ‘Assistant Professor of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. ‘Department of Virology, SEATO Medical Research Laboratory, Bangkok, Thailand. Assistant Professor of Pediatrics, Faculty of Medicine, Chiang Mai University,
29: AUGUST
i9Th,p836-84l.
Printed
in U.S.A.
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Robert Robert Robert
and antibody response protein-calorie malnutrition”
IGS
Materials
AND
ANTIBODY
and methods
RESPONSE
IN
standards The lower limits of sensitivity for the technique, as performed in this laboratory for IgD were 1.4 mg/lOO ml and approximately 1000 lU/mI for IgE. Reproducibility of this quantitative method was about 10 to 15%. The samples were kept frozen at -20 C for up to 2 months before using
commercial
(Hyland
Serial
immunoglobulin
Fourteen
titers
on 28 children
with
PCM
children
marasmus
Nutritional repair was judged complete when both the clinical stigmata of PCM had disappeared and selected abnormal laboratory findings, e.g., low serum albumin and transferrin had returned to normal levels. Virtually all patients were judged recovered by these criteria 6 to 8 weeks after admission. There were three control groups. The first was a group of healthy urban Thai children 2 to 3 years of age studied in well-baby clinics in Bangkok. The second was a group of 10 ostensibly healthy rural children 4 to 6 years of age studied in a village near Chiang Mai. The third was a group of 18 sick children admitted to the University Hospital in Chiang Mai who seemed well nourished but suffered from a variety of febrile illnesses including bacterial pneumonia, convulsions, gastroenteritis, diphtheria, encephalitis, and a variety of viral illnesses of unknown etiology. They came from the same socioeconomic group of families that provided infants with malnutrition. Scoring
severity
of infection
The severity of infection protein-calorie malnutrition titative basis by employing
in the children admitted with was gaded on a semiquana scoring system that has been
described elsewhere (17). It gives points for hypothermia, hypotension, petechial hemorrhages, high percentage of band forms among peripheral polymorphonuclear leucocytes, hyponatremia, positive blood, cerebrospinal fluid, urine, and stool culture for pathogens, and established organ infections, e.g., lung, ear, kidney, and skin. Each child received a score that was then correlated with immunoglobulin levels.
Q uantitation The levels were determined
of serum of
immunoglobulins
serum IgG, by the radical
IgA, 1gM, lgD, immunodiffusion
and
immunoplates
Laboratories
and
reference
Melpar,
Inc.).
analysis.
Response
to iniradermal
typhoid-paratvphoid
vaccine
An additional 10 malnourished children (5 K, 3 MK, and 2 M) and 10 recovered patients were evaluated for their antibody response to typhoid antigen. On admission blood was obtained for H antibody titer from the malnourished children. Acetone-killed typhoid vaccine (0. 1 ml) was injected intradermally and follow-up blood samples were drawn on days 8 and 20 to 29 after the initial immunization. They were treated the same as those evaluated for immunoglobulin states during the first week of stabilization and thereafter. Ten additional children who had recovered clinically were immunized with 0.1 ml of acetone-killed vaccine 50 days after admission. Follow-up blood samples were drawn 8 and 20 to 29 days after immunization. All recovered patients were given a diet containing 175 cal, 4 g protein/kg during
the
Determination zation
29-day
study
of antibod
period.
response
to typhoid
immuni-
Anti-H antibody was measured by radioimmunoassay method (18). The culture of Salmonella typhosa used for the preparation of flagella antigen was originally obtained from the Children’s Hospital in Bangkok and subsequently maintained in this laboratory. The purified flagella preparation was trace-labeled with 251 (The Radiochemical Center, Amersham, England) (19), and labeled protein was kept in small vials at -20 C in the presence of 1% bovine serum albumin (Armour Pharmaceutical Co., Chicago, Illinois). Specific activity of the labeled flagella was 48,100 cpm/jg protein. Sera from immunized children and from hyperimmunized adults (positive controls) were diluted 1:5 with phosphate-buffered saline containing 10 mg bovine serum albumin/mI. Twenty-five microliters of diluted samples were incubated at 37 C for 30 mm with an excess of labeled flagella (5 X, 0.5 j.g). After 200 M’ of rabbit antiimmunoglobulins were added and incubated at 37 C for 60 mm, then at 4 C for 18 hr, immune precipitates were washed and counted. Precision of the assay method, as calculated from 10 determinations sample, resulted in a standard error mean.
of the same serum less than I % of the
Results and IgE method
Chiang Mai, Thailand. ‘Research Assistant in Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. ‘Director, Anemia and Malnutrition Research Center, Chiang Mai, Thailand, Alice A. Doisy Professor of Biochemistry and Professor of Medicine, St. Louis School of Medicine, St. Louis, Missouri. To whom reprint requests should be addressed.
The serum immunoglobulin levels in the three control groups of Thai children and in two malnourished groups are presented in Table 1. The children with MK and those with K were combined because of no significant difference in their values and because they represent degrees of severity of a common syndrome (20). In fact, there were no differ-
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classified by clinical criteria (16) as (M), seven with marasmus-kwashiorkor (MK), and seven with kwashiorkor (K) were admitted to the Anemia and Malnutrition Research Center in Chiang Mai, Thailand. The children, who were 2 to 5 years old, were serially evaluated for immunoglobulin status on admission and on hospital days 8, 29, 50, 70, and 84. After admission, patients were treated for dehydration, electrolyte imbalance, and infection with appropriate antibiotics during a I-week stabilization period. They were fed gradually increasing calorie and protein intakes until by the 8th hospital day all children were receiving 100 cal, I g protein/kg body weight, and at day 8 the intake of all children was increased to 175 cal, 4 g protein/kg per day. In addition to the above milk-based formula diets, a complete vitamin and mmeral supplement was given starting on day 2 (16). having
837
PCM
838
SUSKIND
ET
I immunoglobulin
levels
in Thai
Antibody
response
Ten newly admitted children with PCM and 10 children who had already recovered from PCM (day 50) were immunized intradermally with 0.1 ml of acetone-killed typhoid vaccine. Figure 2 shows the response in the two groups. One week after immunization, the newly admitted children who had been fed 100 cal, I g protein/kg per day showed no increase in antibody activity over the preimmunization level. A slight increase in activity was, however, observed 1 month following immunization after the patients had been on a diet containing 175 cal, 4 g protein/kg per day. A significant increase in antibody activity over the preimmunization level (P < 0.05) was observed as early as 1
children Immunoglobulin
Group
lgG Healthy children’ 2-3 yr Villagechildren4-6yr Sickchildren’26yr Marasmus 2-4 yr MK-Kwashiorkor 2-5 yr
levels in plasma
mg/ 100 ml
‘i
12 10 18 14 14
1264 1574 1485 1315 1423
o Healthy children in well-baby clinics, Bangkok, mean. C IgA significantly different (P < 0.05) well-nourished children with intercurrent infections Hospital, Chiang Mai, Thailand. e n = 5.
1gM ± ± ± ± ±
98’ 71 200 95 116
186 254± 238± 172 248
IgD
IgA ±
± ±
26 17 30 23 49
97 ± 28 165 ± 20 132 ± 14 161 ± 31 251 ± 58c
Thailand. ‘Variance is presented from healthy urban Thai children. (see text) admitted to the Pediatric
0 4 ± l 14 ± 4
as standard error of the d Sick children were wards of the University
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Figure 1 shows the changes in the mean levels of IgG, 1gM, IgA, and IgD in all 28 malnourished children during treatment over a 12-week period. The average weight gain during this period was 3.0 kg. Most of the labile plasma proteins (albumin, transferrin, f.-lipoprotein) were normalized by 4 weeks and hemoglobin levels returned to normal by 8 weeks. There was no significant change in IgG or 1gM during the entire period of treatment. A mean IgM of 265 mg/lOO ml at 29 days returned to a level of 195 mg/ 100 ml after 12 weeks. In contrast, IgA and IgD levels, which were elevated on admission, returned to nQrmal values after 12 weeks of treatment. Of the 16 children who showed positive IgE levels on admission, 10 (37%) showed positive values at the end of treatment, which was still significantly greater than in the control population of sick children.
ences in the IgG, 1gM, IgA, or lgD levels of children with marasmus compared with those who had MK or K. Furthermore there were no differences in levels of IgG and 1gM of malnourished children on admission as compared with urban children from Bangkok, rural children from Chiang Mai, or sick children admitted from the Chiang Mai area to the University Hospital. Differences were found, however, in IgA levels of village children and the MK-K group of malnourished children when each was compared with levels found in urban Thai children of approximately the same age. Eighty percent of the children with marasmus had detectable IgD titers with a mean value of 4.0 ± 1.1 mg/lOO ml, and 80% of the children with MK/K had elevated IgD titers, with a mean of 13.5 ± 4.3 mg/lOO ml whereas IgD was undetectable in well nourished sick children admitted to the University Hospital. Sixty-four percent (16/25) of the malnourished children had detectable IgE in the serum at the time of admission. The Z test, which was used to test for significance of positive values for IgE between the patients and the control population, showed a significant elevation of IgE in the malnourished children. There was no correlation between severity of infection as gauged from our scoring system and serum levels of IgG, 1gM, and IgA. Of the 28 malnourished children studied, 10 or 36% were judged to have severe infections whereas the remaining 18 (64%) were determined to have mild to moderate infections. There was no significant difference in the immunoglobulin levels between the two groups. TABLE Serum
AL.
IGS
AND
SERUM
ANTIBODY
839
IN PCM
RESPONSE
IgG IN PCM 280
1500
I2
w U
1000
160
-
w
120
8.
40 II 28
I
I
II
29
50
7184
SERUM
gA
IN PCM
28 4
50
29 -
SERUM
7184
IgD IN PCM
2 I0
I-.
z
Ui C)
8
Ui 8.
6
0’
4 2 _.__L.......
28
29 TIME
FIG. I. The levels of lgG, 1gM, recovery. The values are mean ± SEM for control children (See Table 1).
week after immunization children who had been 175 cal, 4 g protein/kg
50 IN DAYS
7184
28
29 TIME
I ..I_
50 IN DAYS
7184
IgA, and IgD in the serum of patients with PCM on admission and for 28 malnourished children. The cross-hatched areas show the mean
in the recovered fed a diet containing per day for 6 weeks.
Discussion These results agree with those of other observers who have noted either normal or increased immunoglobulin levels in children with PCM (3, 5-10). In this study, the levels of IgA, IgD, and IgE were elevated beyond control values in malnourished children regardless of syndrome, i.e., marasmus or kwashiorkor. Alvarado and Luthringer (7) have suggested that the elevated IgA is most probably due to frequent gastrointestinal tract infections in these children. IgA-producing cells, which are most abundantly found at these sites, may be preferentially stimulated. Sirisinha et al. (21) have reported that the increased serum IgA in these children was not the secretory type, and concluded that the
during ±
SEM
elevation of serum IgA was not due to secreted SIgA returning to the circulation. Elevated IgD immunoglobulins have been observed in some chronic infections and certain autoimmune disorders (22-24). Although the elevation of IgE is known to be associated with parasitic infestation, atopy, allergy, and eosinophilia in some cases, no such relationship was found in this study. It is extraordinary that the serum immunoglobulin levels are so well maintained and even increased in severe PCM in children in which plasma proteins of hepatic origin are depressed. It suggests that the B-lymphocyte is able to compete successfully with the hepatocyte for the limited amino acid supply. Since the turnover of -y-globulins is increased above normal in severe PCM (26), more synthesis per day than normal is required to maintain these levels whereas the turnover of serum albumin is depressed, re-
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80
500-
840
SUSKIND :D PCM(I0)
800-
600 8. C.)
8 TIME
25’
IN DAYS
FIG. 2. The response of malnourished and recovered children to typhoid H antigen. Values (cpm in radioimmunoprecipitates) of antibody are the means ± SEM for 10 malnourished versus 10 recovered children. Time is days after inoculation. The third sample was taken 21 to 29’ days after the first.
quiring even less synthesis per day than the level would indicate. What stimulates the B-lymphocyte to carry on the exuberant biosynthesis of rny-globulins is unknown. It has been suggested that repeated exposure to infectious agents may explain the high immunoglobulin levels, although, as noted in this study, antibody response to specific antigens may be decreased, not increased. Another explanation offered to account for the increased immunoglobulin levels is hepatic parenchymal disease, which is often associated with diffuse hypergammaglobulinemia. It has been well documented that children with kwashiorkor have diffuse fatty infiltration of the liver (26) and severe ultrastructural damage to mitochondria, reticulum, and nuclei (27). Why serum immunoglobulin concentration rises in hepatic parenchymal damage is not clearly understood. It has been postulated that antibodies may be produced against antigens released by the damaged liver (28). Such liver damage may initiate the effects of severe amino acid lack on lymphocyte function. A depression of cell-mediated immunity and T-lymphocyte function in PCM (2, 3) and in liver disease may be associated with a loss of T-cell suppression and control of B-cells leading, in turn, to increased nonspecific immunoglobulin synthesis. The response to various immunological stimuli in malnourished children varies according to the antigen used. Normal antibody responses have been demonstrated to keyhole
AL.
limpet hemocyanin, pneumococcal polysaccharide antigen (29), and tetanus toxoid antigen (3). Likewise, children with PCM respond adequately to polio and smallpox immunization (14). Pretorius and Devilliers (13) found no deficit in antibody response to typhoid antigen in children with kwashiorkor, when compared to recovered children, although WohI et al. (30) and Chandra (3) found a decreased response to typhoid immunization in malnourished individuals. Our study, however, demonstrates that malnourished children receiving I g protein/kg per day and gradually increasing calories to 100 cal/kg per day had significantly less antibody response to typhoid immunization than did nutritionally recovered children receiving 175 cal and 4 g protein/kg per day. Reddy and Srikantia (31) and Mathews et al. (32) also found that children with higher protein intakes had greater antibody response to typhoid immunization. The patients we studied appeared to have a decreased ability to mount a secondary, rather than a primary response to typhoid antigen since patients in both groups had antibody activity present before immunization. Our observations indicate that although the level of serum immunoglobulins in children with PCM is elevated, their blunted response to typhoid antigen demonstrates that this elevation does not always signify a completely intact humoral immune system.
The
authors are sincerely indebted to the nursing staff, Mrs. Jurai Chumnan and Mrs. Prasobsook Bangxang, for their important contribution to the
particularly
Na
successful
completion
of this
study.
References I. SCRIMSHAW, GORDON.
WHO 2.
N.
S.,
C. E. TAYLOR AND J. E. of Nutrition and Infection. Series No. 57, 1968.
Interactions
Monograph
EDELMAN,
R., R
SIRISINHA.
Mechanisms
SUSKIND,
R.
E. OLSON
of defective
in children with Lancet 1: 506, 1973. 3. CHANDRA, R. K. Immunocompetence tion. J. Pediat. 81: 1194, 1972.
ous
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delayed
AND
S.
cutane-
protein-calorie
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4. SIRISINHA,
in undernutri-
SUSKIND, R. EDELMAN, C. R. E. OLSON. Complement and C,-proactivator levels in children with protein-calorie malnutrition and effect of dietary treatment. Lancet 1: 1016, 1973. 5. EL-GI-IOLMY, A., 0. HELMY, S. HASHISH, H. A. CHARUPATANA
S.,
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AND
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400}
ET
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ANTIBODY
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mn
Y. EL-GAMAL. Immunoglobulins J. Trop. Med. Hyg. 73: 196, 1970.
6. NAJJAR, S. S., M. STEPHAN Serum levels of immunoglobulins fants. Arch. Disease Childhood 7. ALVARADO, J., AND D. G. immunoglobulins in edematous nutrition
AND
children.
Studies
R. Y. ASFOUR. in marasmic in44: 120, 1969.
physiological tein-calorie
AND
LUTHRINGER.
21.
Serum
protein-calorie
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22.
ease Childhood 45: 282, 1970. 11. COOPER, W. C., T. MARIANI AND R. A. GooD. Effects of chronic protein depletion on immune response. Federation Proc. 79 (2): 364, 1970. 12. BALCH, H. H. Relation of nutritional deficiency in man to antibody production. J. Immunol. 64: 397, 1950. 13. PRETORIUS, P. J., AND L. S. DEVILLIERS. Antibody
25.
response
in children
with
protein
in
undernourished
(5): 221, 15. COHEN, albumin
children.
1965. S., AND and
J. D.
-y-globulin
L.
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23.
24.
26.
Am.
27. Antigenic
E. African
stimulation
Med.
HANSEN. Metabolism in kwashiorkor. Clin.
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17.
18.
19.
20.
responses malnutrition.
in Thai children In: Protein-Calorie
SUSKIND,
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trition, Edited by R. E. Olson. New York: Academic Press, 1975, p. 275. SIRIsINHA, S., R. SUSK1ND, R. EDELMAN, C. ASVAPAKA AND R. E. OLSON. Secretory and serum IgA in children with protein-calorie malnutrition. Pediatrics 55: 166, 1975. ROWE, D. S., AND J. 1. FAHEY. A new class of immunoglobulin. II. Normal serum IgD. J. Exptl. Med. 121: 185, 1965. BACHMANN, R. Serum IgD-globulin in conditions with pathological proteins (M-components) and in mononucleosis. In Nobel Symposium 3, Gamma Globulins, edited by J. Killander. New York: Interscience Publishers, 1967, p. 605. ONONDERA, S. Quantitative determination of serum imrunoglobuIin D in gammopathy. J. Exptl. Med. 95: 145, 1968. JANEWAY, C., F. S. ROSEN, E. MERLER AND C. A. ALPER. The Gamma Globulins. New Engl. J. Med. Medical Progress Series, Boston: Little, Brown and Company, 1966, p. 39. VITHAYASAI, V. M. F. M. JOHNSTON, P. KULAPONGS, AND R. E. OLSON. Metabolism of immunoglobulin G in protein-calorie malnutrition (PCM). Proc. X Int. Congress of Nutrition, Kyoto, Japan, 1975. BHAMARAPRAVATI, N. The liver in protein-calorie malnutrition: an ultrastructural study. In: ProteinCalorie Malnutrition. New York: Academic Press, 1975, p. 299. KUNKEL, H. G., E. H.AHRENS, JR., W. J. EISENMENGER, A. M. BONGIOVANNI AND R. S. SLATER. Extreme hypergammaglobulinemia with liver disease of unknown
23: 351, 16.
1962. R. The in-patient and out-patient treatment of the child with protein-calorie malnutrition in Protein-Calorie Malnutrition. New York: Academic Press, 1975, p. 403. THANANGKUL, 0., R. SUSKIND, P. KULAPONGS, V. VITHAYASAI, D. DAMRONGSAK AND R. E. OLSON. Protein calorie malnutrition, Proc. X Int. Congress of Nutrition, Kyoto, Japan, 1975. ADA, G. L., G. J. V. NOSSAL, J. PYE AND A. ABBOT. Antigens in immunity. I. Preparation and properties of flagellar antigens from Salmonella adelaide. Australian J. Exptl. Biol. Med. Sci. 42: 267, 1964. SONODA, S., AND M. SCHLAMOWITZ. Studies of 1251 trace labeling of immunoglobulin G by chloramin-T. Immunochemistry 7: 885, 1970. OLsoN, R. E. The effect of variations in protein and calorie intake in the rate of recovery and selected
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INCAP. Clin. Pediat. 10 (3): 175, 1971. 8. KEEl, M. P., AND H. THOM. Serum immunoglobulins in kwashiorkor. Arch. Disease Childhood 44: 600, 1969. 9. MCFARLANE, H., S. REDDY, A. COOKE, 0. LONGE, M. 0. ONABAMIRO AND J. F. HOUBA. Immunoglobulins, transferrin, ceruloplasmin and heterophile antibodies in kwashiorkor. Trop. Geograph Med. 22: 61, 1970. 10. WATSON, C. E., AND C. FREESEMANN. Immunoglobulins in protein-calorie malnutrition. Arch. Dis-
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IN
M.D., Stitaya M.D., Damn M.D., Ph.D.
ABSTRACT
Sirishinha,4 Damrongsak,7
The
Northern
Thai
immunoglobulin
children
with
marasmus-kwashiorkor, admission
and
fractions with
either
or without
marasmus were
higher
percent
demonstrated
PCM
in the control
from
increase
those
than
with PCM
levels
immunized
no significant
increase
to
and
IgD
levels urban
of IgG,
1gM,
with
64%
children
in 28
measured
rural
Thai
or IgD with
with
IgA
levels Eighty
IgE
levels
and
Within
while
Am. J. Clin.
titer.
children,
treatment.
antigen.
group,
on
in children
with PCM
typhoid
7 had
immunoglobulin
had detectable
admitted
in the recovered
in H antibody
studied
were All
and
IgA,
to normal
while
intradermal
Ph.D., and
or kwashiorkor.
returned
levels
additional
appeared
IgE) were
recovery.
marasmus-kwashiorkor
children
H antibody
and
M.D., M.D.,
Of these 14 had marasmus,
in well-nourished
detectable Ten
leading
in levels
had
control
had groups.
were
in typhoid
who
IgD,
immunoglobulin
treatment seen
1gM, (PCM).
The
of
was no difference
with
in malnourished
recovered
significant group
compared
to none
kwashiorkor.
12 weeks
There
IgA,
malnutrition
or exceeded
infection.
of the children
compared had
equalled
levels (IgG,
7 had
during
2
Ph.D., Vicharn Vithayasai,5 M.D., Chinda Charupatana,8
protein-calorie
and serially
when
M.D.,
in
as
10 who 8 days
a
the malnourished
Nutr.
29: 836-841,
1976.
The synergistic interaction of infection and malnutrition has been recognized by several clinicians and investigators (1). Children with protein-calorie malnutrition (PCM) often have an increased incidence and severity of infections. Impairment of the cell-mediated immune function (2, 3) and of the inflammatory response (2) has been demonstrated in children with PCM. In addition, the complement system has been shown to be adversely affected in malnutrition (3, 4). There is, however, no agreement on the effect of PCM on the humoral immune mechanism. While most studies show that immunoglobulin levels are not depressed (5-10), antibody response in children with PCM seems to vary accordng to the type and form of antigen used (11-14). Chandra (3) noted that in contrast to the depressed response to typhoid immunization, undernourished children reacted adequately to tetanus toxoid. In addition, Cohen and Hansen (15) noted that the synthesis of “y-globulin in infected malnourished children was 3 times that of the uninfected group. We undertook this study to evaluate the integrity of the humoral immune system in children with marasmus and kwashiorkor by 836
The American
Journal
of Clinical
Nutrition
determining the levels of serum immunoglobulins (IgG, 1gM, IgA, IgD, and IgE), and the specific antibody response to typhoid immunization before and after treatment.
ter,
‘From the Anemia and Malnutrition Research Cenand Department of Pediatrics, Faculty of Medicine,
Chiang Department
Mai
University,
Chiang
of Microbiology,
Faculty
Mai,
Thailand,
the Mahithe Department of Laboratory, Bangof Pediatrics and School of Medicine, of Science,
dol University, Bangkok, Thailand, Virology, SEATO Medical Research kok, Thailand, and the Departments Biochemistry, St. Louis University St. Louis, Missouri. ‘Supported in part by Grants-in-aid from the National Institutes of Health, (AM-I 1044), U.S. Public Health Service, Bethesda, Maryland, the Rockefeller Foundation, New York, New York, and the U.S. Army Medical Research and Development Command. Deputy Director, Anemia and Malnutrition Research Center, Chiang Mai, Thailand and Assistant Professor of Pediatrics, St. Louis University School of Medicine, St. Louis, Missouri. Present address: Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts 4Professor of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. ‘Assistant Professor of Microbiology, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand. ‘Department of Virology, SEATO Medical Research Laboratory, Bangkok, Thailand. Assistant Professor of Pediatrics, Faculty of Medicine, Chiang Mai University,
29: AUGUST
i9Th,p836-84l.
Printed
in U.S.A.
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Robert Robert Robert
and antibody response protein-calorie malnutrition”
IGS
Materials
AND
ANTIBODY
and methods
RESPONSE
IN
standards The lower limits of sensitivity for the technique, as performed in this laboratory for IgD were 1.4 mg/lOO ml and approximately 1000 lU/mI for IgE. Reproducibility of this quantitative method was about 10 to 15%. The samples were kept frozen at -20 C for up to 2 months before using
commercial
(Hyland
Serial
immunoglobulin
Fourteen
titers
on 28 children
with
PCM
children
marasmus
Nutritional repair was judged complete when both the clinical stigmata of PCM had disappeared and selected abnormal laboratory findings, e.g., low serum albumin and transferrin had returned to normal levels. Virtually all patients were judged recovered by these criteria 6 to 8 weeks after admission. There were three control groups. The first was a group of healthy urban Thai children 2 to 3 years of age studied in well-baby clinics in Bangkok. The second was a group of 10 ostensibly healthy rural children 4 to 6 years of age studied in a village near Chiang Mai. The third was a group of 18 sick children admitted to the University Hospital in Chiang Mai who seemed well nourished but suffered from a variety of febrile illnesses including bacterial pneumonia, convulsions, gastroenteritis, diphtheria, encephalitis, and a variety of viral illnesses of unknown etiology. They came from the same socioeconomic group of families that provided infants with malnutrition. Scoring
severity
of infection
The severity of infection protein-calorie malnutrition titative basis by employing
in the children admitted with was gaded on a semiquana scoring system that has been
described elsewhere (17). It gives points for hypothermia, hypotension, petechial hemorrhages, high percentage of band forms among peripheral polymorphonuclear leucocytes, hyponatremia, positive blood, cerebrospinal fluid, urine, and stool culture for pathogens, and established organ infections, e.g., lung, ear, kidney, and skin. Each child received a score that was then correlated with immunoglobulin levels.
Q uantitation The levels were determined
of serum of
immunoglobulins
serum IgG, by the radical
IgA, 1gM, lgD, immunodiffusion
and
immunoplates
Laboratories
and
reference
Melpar,
Inc.).
analysis.
Response
to iniradermal
typhoid-paratvphoid
vaccine
An additional 10 malnourished children (5 K, 3 MK, and 2 M) and 10 recovered patients were evaluated for their antibody response to typhoid antigen. On admission blood was obtained for H antibody titer from the malnourished children. Acetone-killed typhoid vaccine (0. 1 ml) was injected intradermally and follow-up blood samples were drawn on days 8 and 20 to 29 after the initial immunization. They were treated the same as those evaluated for immunoglobulin states during the first week of stabilization and thereafter. Ten additional children who had recovered clinically were immunized with 0.1 ml of acetone-killed vaccine 50 days after admission. Follow-up blood samples were drawn 8 and 20 to 29 days after immunization. All recovered patients were given a diet containing 175 cal, 4 g protein/kg during
the
Determination zation
29-day
study
of antibod
period.
response
to typhoid
immuni-
Anti-H antibody was measured by radioimmunoassay method (18). The culture of Salmonella typhosa used for the preparation of flagella antigen was originally obtained from the Children’s Hospital in Bangkok and subsequently maintained in this laboratory. The purified flagella preparation was trace-labeled with 251 (The Radiochemical Center, Amersham, England) (19), and labeled protein was kept in small vials at -20 C in the presence of 1% bovine serum albumin (Armour Pharmaceutical Co., Chicago, Illinois). Specific activity of the labeled flagella was 48,100 cpm/jg protein. Sera from immunized children and from hyperimmunized adults (positive controls) were diluted 1:5 with phosphate-buffered saline containing 10 mg bovine serum albumin/mI. Twenty-five microliters of diluted samples were incubated at 37 C for 30 mm with an excess of labeled flagella (5 X, 0.5 j.g). After 200 M’ of rabbit antiimmunoglobulins were added and incubated at 37 C for 60 mm, then at 4 C for 18 hr, immune precipitates were washed and counted. Precision of the assay method, as calculated from 10 determinations sample, resulted in a standard error mean.
of the same serum less than I % of the
Results and IgE method
Chiang Mai, Thailand. ‘Research Assistant in Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. ‘Director, Anemia and Malnutrition Research Center, Chiang Mai, Thailand, Alice A. Doisy Professor of Biochemistry and Professor of Medicine, St. Louis School of Medicine, St. Louis, Missouri. To whom reprint requests should be addressed.
The serum immunoglobulin levels in the three control groups of Thai children and in two malnourished groups are presented in Table 1. The children with MK and those with K were combined because of no significant difference in their values and because they represent degrees of severity of a common syndrome (20). In fact, there were no differ-
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classified by clinical criteria (16) as (M), seven with marasmus-kwashiorkor (MK), and seven with kwashiorkor (K) were admitted to the Anemia and Malnutrition Research Center in Chiang Mai, Thailand. The children, who were 2 to 5 years old, were serially evaluated for immunoglobulin status on admission and on hospital days 8, 29, 50, 70, and 84. After admission, patients were treated for dehydration, electrolyte imbalance, and infection with appropriate antibiotics during a I-week stabilization period. They were fed gradually increasing calorie and protein intakes until by the 8th hospital day all children were receiving 100 cal, I g protein/kg body weight, and at day 8 the intake of all children was increased to 175 cal, 4 g protein/kg per day. In addition to the above milk-based formula diets, a complete vitamin and mmeral supplement was given starting on day 2 (16). having
837
PCM
838
SUSKIND
ET
I immunoglobulin
levels
in Thai
Antibody
response
Ten newly admitted children with PCM and 10 children who had already recovered from PCM (day 50) were immunized intradermally with 0.1 ml of acetone-killed typhoid vaccine. Figure 2 shows the response in the two groups. One week after immunization, the newly admitted children who had been fed 100 cal, I g protein/kg per day showed no increase in antibody activity over the preimmunization level. A slight increase in activity was, however, observed 1 month following immunization after the patients had been on a diet containing 175 cal, 4 g protein/kg per day. A significant increase in antibody activity over the preimmunization level (P < 0.05) was observed as early as 1
children Immunoglobulin
Group
lgG Healthy children’ 2-3 yr Villagechildren4-6yr Sickchildren’26yr Marasmus 2-4 yr MK-Kwashiorkor 2-5 yr
levels in plasma
mg/ 100 ml
‘i
12 10 18 14 14
1264 1574 1485 1315 1423
o Healthy children in well-baby clinics, Bangkok, mean. C IgA significantly different (P < 0.05) well-nourished children with intercurrent infections Hospital, Chiang Mai, Thailand. e n = 5.
1gM ± ± ± ± ±
98’ 71 200 95 116
186 254± 238± 172 248
IgD
IgA ±
± ±
26 17 30 23 49
97 ± 28 165 ± 20 132 ± 14 161 ± 31 251 ± 58c
Thailand. ‘Variance is presented from healthy urban Thai children. (see text) admitted to the Pediatric
0 4 ± l 14 ± 4
as standard error of the d Sick children were wards of the University
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Figure 1 shows the changes in the mean levels of IgG, 1gM, IgA, and IgD in all 28 malnourished children during treatment over a 12-week period. The average weight gain during this period was 3.0 kg. Most of the labile plasma proteins (albumin, transferrin, f.-lipoprotein) were normalized by 4 weeks and hemoglobin levels returned to normal by 8 weeks. There was no significant change in IgG or 1gM during the entire period of treatment. A mean IgM of 265 mg/lOO ml at 29 days returned to a level of 195 mg/ 100 ml after 12 weeks. In contrast, IgA and IgD levels, which were elevated on admission, returned to nQrmal values after 12 weeks of treatment. Of the 16 children who showed positive IgE levels on admission, 10 (37%) showed positive values at the end of treatment, which was still significantly greater than in the control population of sick children.
ences in the IgG, 1gM, IgA, or lgD levels of children with marasmus compared with those who had MK or K. Furthermore there were no differences in levels of IgG and 1gM of malnourished children on admission as compared with urban children from Bangkok, rural children from Chiang Mai, or sick children admitted from the Chiang Mai area to the University Hospital. Differences were found, however, in IgA levels of village children and the MK-K group of malnourished children when each was compared with levels found in urban Thai children of approximately the same age. Eighty percent of the children with marasmus had detectable IgD titers with a mean value of 4.0 ± 1.1 mg/lOO ml, and 80% of the children with MK/K had elevated IgD titers, with a mean of 13.5 ± 4.3 mg/lOO ml whereas IgD was undetectable in well nourished sick children admitted to the University Hospital. Sixty-four percent (16/25) of the malnourished children had detectable IgE in the serum at the time of admission. The Z test, which was used to test for significance of positive values for IgE between the patients and the control population, showed a significant elevation of IgE in the malnourished children. There was no correlation between severity of infection as gauged from our scoring system and serum levels of IgG, 1gM, and IgA. Of the 28 malnourished children studied, 10 or 36% were judged to have severe infections whereas the remaining 18 (64%) were determined to have mild to moderate infections. There was no significant difference in the immunoglobulin levels between the two groups. TABLE Serum
AL.
IGS
AND
SERUM
ANTIBODY
839
IN PCM
RESPONSE
IgG IN PCM 280
1500
I2
w U
1000
160
-
w
120
8.
40 II 28
I
I
II
29
50
7184
SERUM
gA
IN PCM
28 4
50
29 -
SERUM
7184
IgD IN PCM
2 I0
I-.
z
Ui C)
8
Ui 8.
6
0’
4 2 _.__L.......
28
29 TIME
FIG. I. The levels of lgG, 1gM, recovery. The values are mean ± SEM for control children (See Table 1).
week after immunization children who had been 175 cal, 4 g protein/kg
50 IN DAYS
7184
28
29 TIME
I ..I_
50 IN DAYS
7184
IgA, and IgD in the serum of patients with PCM on admission and for 28 malnourished children. The cross-hatched areas show the mean
in the recovered fed a diet containing per day for 6 weeks.
Discussion These results agree with those of other observers who have noted either normal or increased immunoglobulin levels in children with PCM (3, 5-10). In this study, the levels of IgA, IgD, and IgE were elevated beyond control values in malnourished children regardless of syndrome, i.e., marasmus or kwashiorkor. Alvarado and Luthringer (7) have suggested that the elevated IgA is most probably due to frequent gastrointestinal tract infections in these children. IgA-producing cells, which are most abundantly found at these sites, may be preferentially stimulated. Sirisinha et al. (21) have reported that the increased serum IgA in these children was not the secretory type, and concluded that the
during ±
SEM
elevation of serum IgA was not due to secreted SIgA returning to the circulation. Elevated IgD immunoglobulins have been observed in some chronic infections and certain autoimmune disorders (22-24). Although the elevation of IgE is known to be associated with parasitic infestation, atopy, allergy, and eosinophilia in some cases, no such relationship was found in this study. It is extraordinary that the serum immunoglobulin levels are so well maintained and even increased in severe PCM in children in which plasma proteins of hepatic origin are depressed. It suggests that the B-lymphocyte is able to compete successfully with the hepatocyte for the limited amino acid supply. Since the turnover of -y-globulins is increased above normal in severe PCM (26), more synthesis per day than normal is required to maintain these levels whereas the turnover of serum albumin is depressed, re-
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80
500-
840
SUSKIND :D PCM(I0)
800-
600 8. C.)
8 TIME
25’
IN DAYS
FIG. 2. The response of malnourished and recovered children to typhoid H antigen. Values (cpm in radioimmunoprecipitates) of antibody are the means ± SEM for 10 malnourished versus 10 recovered children. Time is days after inoculation. The third sample was taken 21 to 29’ days after the first.
quiring even less synthesis per day than the level would indicate. What stimulates the B-lymphocyte to carry on the exuberant biosynthesis of rny-globulins is unknown. It has been suggested that repeated exposure to infectious agents may explain the high immunoglobulin levels, although, as noted in this study, antibody response to specific antigens may be decreased, not increased. Another explanation offered to account for the increased immunoglobulin levels is hepatic parenchymal disease, which is often associated with diffuse hypergammaglobulinemia. It has been well documented that children with kwashiorkor have diffuse fatty infiltration of the liver (26) and severe ultrastructural damage to mitochondria, reticulum, and nuclei (27). Why serum immunoglobulin concentration rises in hepatic parenchymal damage is not clearly understood. It has been postulated that antibodies may be produced against antigens released by the damaged liver (28). Such liver damage may initiate the effects of severe amino acid lack on lymphocyte function. A depression of cell-mediated immunity and T-lymphocyte function in PCM (2, 3) and in liver disease may be associated with a loss of T-cell suppression and control of B-cells leading, in turn, to increased nonspecific immunoglobulin synthesis. The response to various immunological stimuli in malnourished children varies according to the antigen used. Normal antibody responses have been demonstrated to keyhole
AL.
limpet hemocyanin, pneumococcal polysaccharide antigen (29), and tetanus toxoid antigen (3). Likewise, children with PCM respond adequately to polio and smallpox immunization (14). Pretorius and Devilliers (13) found no deficit in antibody response to typhoid antigen in children with kwashiorkor, when compared to recovered children, although WohI et al. (30) and Chandra (3) found a decreased response to typhoid immunization in malnourished individuals. Our study, however, demonstrates that malnourished children receiving I g protein/kg per day and gradually increasing calories to 100 cal/kg per day had significantly less antibody response to typhoid immunization than did nutritionally recovered children receiving 175 cal and 4 g protein/kg per day. Reddy and Srikantia (31) and Mathews et al. (32) also found that children with higher protein intakes had greater antibody response to typhoid immunization. The patients we studied appeared to have a decreased ability to mount a secondary, rather than a primary response to typhoid antigen since patients in both groups had antibody activity present before immunization. Our observations indicate that although the level of serum immunoglobulins in children with PCM is elevated, their blunted response to typhoid antigen demonstrates that this elevation does not always signify a completely intact humoral immune system.
The
authors are sincerely indebted to the nursing staff, Mrs. Jurai Chumnan and Mrs. Prasobsook Bangxang, for their important contribution to the
particularly
Na
successful
completion
of this
study.
References I. SCRIMSHAW, GORDON.
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IN
