NUTRITION RESEARCH, Vol. 7, pp. 989-i003, ]987 0271-5317/87 $3.00 + .00 Printed in the USA. (Copyright (c) 1987 Pergamon Journals Ltd. All rights reserved

IRON DEFICIENCY, CELL-MEDIATED IMMUNITY AND RESISTANCE AGAINST INFECTIONS: PRESENT KNOWLEDGE AND CONTROVERSIES

Solo KUVIBIDILA, Ph.D.

INSERM, UI, Unite de Recherches sur la Nutrition et l'Alimentation; Hopital Bichat, 170 Boulevard Ney

75877 Paris Cedex 18, France

ABSTRACT

Iron deficiency anemia is one of the most common nutritional disorders in childhood and women of the reproductive age. Several clinical and experimental studies suggest that many immune functions which include delayed type hypersensitivity, mitogenic response, cytotoxicity of T cells and natural killer cells, bactericidal activity of leukocytes, etc., are impaired. However, as to how this impairment is related to susceptibility to infections is still uncertain since iron is required by many microorganisms for cell division. Several clinical studies have suggested that iron deficiency predisposes to infections and iron therapy restores resistance. Other studies have shown the opposite. This review carefully analyzes many of the studies which have shown an association or lack of association between iron status and/or iron therapy and incidence of infections. Reasons for the controversies are also discussed.

Key Words: Iron deficiency, iron therapy, cell-mediated immunity, infections.

INTRODUCTION

For several years, anemia due to iron deficiency has been recognized as a public health problem in many developed countries (1-2). Infants, young children and women of the reproductive age represent the three groups at high risk.

Besides the well known effects of iron deficiency on erythropoiesis iron deficiency causes other tissue and cellular disturbances such as reduction of the activity of iron containing and iron dependent enzymes, protein and DNA synthesis (3). Iron is required by all living cells including lymphocytes (4) and microorganisms (5). Iron deficiency therefore may affect, on one hand, the cells of the immune system since many of their functions require cell proliferation after antigenic stimulation, and on the other hand, multiplication of microorganisms.

Since the early seventies, several clinical studies have been published on the effects of iron deficiency anemia on the immune response as well as its influence on incidence of infections. However, the results are conflicting. This review is an update of the known effects of iron deficiency on different components of the immune system as well as its relation to infectious diseases in humans and laboratory animals. Possible reasons for the controversy are also discussed.

989

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CELL-MEDIATED IMMUNITY

Delayed Type Hypersensitivity Delayed type hypersensitivity is one of the most common tests which have

been used to assess the cell-mediated immunity in various nutritional disorders including iron deficiency. In spite of its many advantages which include the ease of administration and the requirement of minimum laboratory equipment, skin response to antigens and allergens is very complex. It involves several different~ types of cells (T cells, macrophages, granulocytes, etc.), and sequential events for example the processing of the antigen, sensitization of T cells, production and recognition of soluble mediators and inflammation. Since any one of these cells or events may be altered by dietary manipulation, specific tests will have to be done in order to identify the affected site or sites.

Skin response to different antigens including candida, purified protein derivatives (PPD), diphteria, mumps, phytohemagglutinin (PHA) etc., has been evaluated in many different laboratories (Table i). Despite the variation in severity of iron deficiency as well as age range, all except one have observed an impairment (6-11). The one study (Gross et al (12)) which showed no decrease utilized a strong allergen (dinitrochloro benzene) and the number of patients was also small. Two to three months of iron therapy improved the response in 80 to 100% of the cases (9-11). These observations have been confirmed in animal studies (13).

BLASTOGENIC RESPONSE

Human Studies This function, which by and large, has been the most studied in vitro

cell-mediated immunity has the advantage of utilizing the same numbe$ of isolated peripheral blood lymphocytes or nucleated cells. In general, ~H- thymidine incorporation into DNA is measured in isolated lymphocytes incubated under identical conditions. An important assumption which is made but may not necessarily be true is that cells from the experimental and control groups behave in culture in the same way.

Despite the diversity of stimulating compounds (candida, PPD, tetanus, PHA, etc.), or the age group, most of the studies have reported an impairment (Table 2; ref. 6-11, 14-20). In some studies (ex. Srikantia et al (14)) a good correlation has been found between severity of iron deficiency and degree of impairment. The impairment is due to iron deficiency but not anemia per s e as has been demonstrated by Sawitsky (15). Two to three months of iron therapy has resulted in an improvement of mitogenic response (7, 9- 11, 14).

Five laboratories: three working on children (20-22) and two on pregnant women (12, 17) have not observed an impairment. Lack of significant difference between controls and iron deficient patients in those studies may have been due to several reasons: Small number of patients (Ii), insufficient assessment of the iron status (21), methods of expressing the results, for example cpm, lymphoblastoid number, stimulation indices, (ii, 21), and reference against which the experimental group is compared to. In some of the studies (20), no biochemical parameters were reported in order to rule out the presence or the absence of protein-energy undernutriton, the deficiency of trace elements other than iron or infections. These factors are all known to alter the immune response.

IRON DEFICIENCY AND CMI 991

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TABLE 2 BLASTOGENIC RESPONSE AND CELLS IN BLOOD OR LYMPHOID ORGANS

Before After Author Type of Subjects Ag/Mito Resp %T Resp %T

Chandra et al (6) Children PHA + + n.d. n.d. Joynson et al (7) Adults PPD, candida + n.d. n.d. n.d. Bhaskaram (8) Children PHA ~ + + + Krantman (9) Infants PHA, tetanus # + • +

Candida ~ + Macdougall (i0) Children PHA, candida ~ ~ + + Swarup-Mitra (II) Adults PHA + + + + Gross et al (12) Pregnant females PHA normal n.d. n.d. n.d~ Fletcher (14) Adults PHA + + + + Srikantia (15) Children PHA + + n.d. n.d. Sawitsky (16) Adults PHA + n.d. n.d. n.d. Prema et al (17) Pregnant females PHA normal n~ n.d. n.d. Prema et al (17) Non-pregnant fem. PHA + n.d. n.d. n.d. Hoffbrand (18) Adults PHA + n.d. n.d. n.d. Narasinga Rao(19) Children PHA + + n.d. n.d. Kulapong (20) Children PHA normal n.d. n.d. n.d~ Gupta et al (21) Children PHA normal n.d. n.d. n.d. Grosh-Worner (22) Children PHA normal n.d. n.d. n.d. Lipschitz (28) Elderly n.d. n.d. n.d. n.d. Kuvibidila (23) Mice (S) ConA,PHA,LPS + + + + Kuvibidila (24) Mice (LN) ConA,PHA,LPS + n.d. n.d. Kramer et al (26) Rats (S) ConA,PHA,LPS normal n.d. n.d. n.d. Soyano et al (27) Rats (T) ConA,PHA,LPS n.d. n.d. n.d. Soyano et al (27) Rats (S) ConA,PHA,LPS normal n.d. n.d. n.d.

S = spleen; Tt = thymus; LN = lymph nodes = decreased + = increased • unchanged; n.d. = not determined.

Animal Studies Work done in our laboratory using severely iron deficient anemic mice

(Hb levels 4-7 g/dl) supports the observations made in human studies (23-24). Enrichment of the T cell population with nylon wool columns or of B cells, has shown the defect in both populations - ruling out a decreased response due to population shift. While in vitro repletion by adding different forms of iron (salts, haemin and transferrin) to the culture mediums only partially restored T cell response, in vivo repletion fully corrected it (23). Moderate iron deficiency anemia (8 g/dl <Hb <ii g/dl) also impairs blastogenic response of murine spleen and lymph nodes lymphocytes (25). While mitogenic response of rat splenocytes is not diminished by iron deficiency (26-27), that of thymocytes is (27). It must be added though that in both of these groups neither stimulation indices, nor results of unstimulated cultures were reported. As we (25) and other investigators (Kramer T.R., personal communication) have recently observed, the rate of spontaneous DNA synthesis in culture of spleen cells from iron deficient mice may increase by 3 to 7 fold compared to that of normal mice. This in fact may mask the effects of iron deficiency.

WHITE BLOOD CELL COUNTS (WBC)~ PERCENT AND/OR ABSOLUTE NUMBER OF T CELLS

Nine different labortories have evaluated the percentage and/or the

IRON DEFICIENCY AND CMI 993

absolute number of T cells in human peripheral blood using the rosette technique with sheep red blood cells (6, 8-11, 14-15, 17, 28). All except one (i) have observed a decrease. Unfortunately, T cell subpopulations have not been evaluated in any of the studies. The one study which has shown elevated WBC and T lymphocyte number has included infected patients (I0). In two of the studies in which sufficient patients were involved (15, 17), a very good correlation between Hh levels and percent cells was observed. In three studies (6, 9, 14), two to three months of iron therapy resulted in a normalization of T cell number.

Similar to human studies, the mean white blood counts and lymphocytes are reduced in iron deficiency mice (23). Decreased percentage of T (anti- Thy(+) and B (Ig(+)) cells in both spleen and lymph nodes have been reported (29-30).

HUMORAL IMMUNITY

Clinical Studies A): Immunoglobulin levels Most human studies which have involved measurements of Ig levels in iron

deficient anemic children (6, 9-10, 31-32) or pregnant women (17) have not shown any alteration. In fact there has been a tendency of increased rather than decreased levels.

B): Vaccination One laboratory has evaluated the antibody titers against tetanus toxoid

in a limited number of Indian children (6). Both primary and secondary antibody titers were similar in iron deficient and control children. However, one study is not sufficient in order to draw any conclusion.

C): B cell identification The information provided in the literature suggests that the percentage

and/or B cells number are not affected by iron deficiency (33). Prema et al (17) has observed a small and non significant fall in the percentage of B cells in pregnant women which was proportional to Hb levels. If T cell number or percentage is decreased but B cells remain unchanged, one may suspect an increase in null cells. This has been observed by Chandra et al (34).

D): C3 complement component C3, which plays an important role in humoral immunity, has been

evaluated only in two studies: one done by Chandra (6) showed a decrease and the other by Macdougall et al (i0) showed an increase C3 increases in inflammatory reactions but it decreases in recurrent pyogenic infections. It is uncertain whether the decrease in Chandra's study was due to previous recurrent infections.

Animal Studies Contrary to human studies, animal studies have suggested an impairment

of the humoral immunity in iron deficiency anemia. The functions which have been evaluated include: Vaccination against tetanus toxoid in rats (35), plaque forming cells in mice (36) and rats (37) and immunoglobulin levels (37). Both antibody titers against tetanus toxoid and plaque forming cells are significantly depressed. In one study, Kochanowski et al (37)fhave observed that 3 weeks of repletion of pups born from iron deficient dams were not sufficient in order to correct the plaque forming cell number. In rats, iron deficiency reduces IgG but not IgM levels (37). In mice, both IgG and

994 S. KUVIBIDILA

IgM but not IgA levels are reduced (Kuvibidila unpublished results).

The differences between animal and human studies may be explained in term of either species differences or environmental factors. In animal studies, factors such as protein-energy undernutrition, the deficiency of trace elements other than iron or vitamins, exposure to infections, which may modify immunoglobulin levels are much better controlled.

POLYMORPHONUCLEAR LEUCOCYTE FUNCTIONS AND MISCELLANEOUS TESTS

Controversy has also been reported for polymorphs function in humans. Eight different groups have evaluated polymorphonuclear functions in either iron deficient children or adults (6, i0, 15, 20, 38-41; Table 3). Decreased phagocytosis has been reported by Arbeter et al (38). Bactericidal capacity was not altered by iron deficiency in Kulapong's study (20) but decreased in all other studies (6, i0, 15, 39). In the study of Srikantia et al (15), bactericidal activity decreased proportionally to the fall in Hb levels. Myeloperoxidase activity has been found either normal (39) or decreased (40- 41). While Chandra and Saraya (6) observed an impairment of nitroblue tetrazolium reduction which returned to normal following iron therapy, both MacDougall et al (I0) and Yetnig et al (39) did not.

TABLE 3 HUMORAL IMMUNITY AND MISCELLANEOUS FUNCTIONS OF LEUKOCYTES FROM IRON

DEFICIENT PATIENTS OR ANIMALS

Tests done Results Ref.

Ig levels

Plaque forming cells Antibody titers

Bactericidal activity B cell number Phagocytosis

MIF production Myeloperoxidase

Natural killer cell activity Osponic activity NBT reduction

C3

Neutrophilic chemotaxis Thymulin activity (serum)

Cytotoxieity of T cells

increased 9 normal I0, 17, 31-32 decreased 36-37 normal 6 decreased (rats) 35 decreased 6, 10, 19, 39 normal 17, 33 decreased 38, 42 normal 6, I0 decreased 7, ii decreased 40-41, 77 normal 39 decreased 44 normal 6 decreased 6, 42 normal i0, 39 decreased 6 normal i0 normal i0 normal 29 decreased 30

In rats, granulocyte function evaluated by the reduction of nitroblue tetrazolium is decreased but the activity of peroxidase and lyzozyme is unimpaired (42). In mice, the in vivo phagocytic function assessed by the clearance of polyvinyl-pyrrolidone is decreased (43).

Other functions which have been investigated are: migration inhibitory

IRON DEFICIENCY AND CMI 995

factor in humans (7,11), cytotoxicity of splenic T lymphocytes against allogenic tumor cells (31), natural killer cell activity (44) (Table 3). All these functions are depressed by iron deficiency. Despite the consistent thymic atrophy which leads to as much as 90% of reduction of thymic weight in mice and depletion of thymocytes in the cortico-medullary zones, iron deficiency does not significantly alter thymulin activity (29).

Critics Based on the results summarized in Table 1 and 2, 6 out of 7 and 12 out

of 17 human studies suggest that iron deficiency impairs delayed type hypersensitivity and mitogenic response, respectively. Those summarized in Table 3 suggest that many but all functions of the non-speciflc immunity are also depressed. Lack of impairment of the cell-mediated immunity in few studies could have been due to either the severity of iron deficiency or the availability of iron to lymphocytes. In fact, none has measured intracellular iron levels in lymphocytes and has correlated them with the immune function tested. In pregnant women, for example, anemia and iron deficiency are in part due to hemodilution. It is possible that this hemodilution is not severe enough to reduce the iron content of lymphocytes and hence affects the cell-mediated immunity.

The basic question is no longer whether or not iron deficiency affects the immune functions but rather what mechanism or mechanisms are involved in the impairment. Is the role of iron directly related and limited to iron containing and/or dependent enzymes? Is it also related to changes in the utilization of some essential nutrients such as folic acid which are also known to alter the cell-mediated immunity? Is it related to the production of soluble mediators which are involved both in the differentiation and/or regulation of the immune functions? Each of these points needs further investigation.

IRON STATUS AND RESPONSES TO INFECTIONS

Clinical Studies Based on the preceding review which shows decreased cell-mediated

immunity in iron deficiency, one might expect an increased susceptibility to infections in deficient patient. In fact this has been the general impression of clinicians (45). In the past several years, many studies have been undertaken in order to determine the possible association between iron status and incidence of infections. The results available both in clinical and experimental animal models are conflicting.

Data available in the literature divide investigators into two schools: one that states that iron deficiency predisposes to infections (46). Iron therapy thus might be expected to increase the resistance. The second states that iron deficiency protects against infections since bacteria require iron for their multiplication (5). Treatment with iron will therefore increase susceptibility to infections.

In 1928, Mackay (476) reported that the incidence of bronchitis and gastroenteritis was higher in iron deficient compared to normal children. In 1966, Andelman and Sered (48) reported that, of the 1048 infants from low income families, followed for 18 months, those who received the iron fortified formula experienced fewer infections (5%) than those who did not (30%). Reduction in hospitalization due to infections in Maori neonates receiving iron supplementation has been observed by Cantwell (49).

996 S. KUVIBIDILA

Other studies in which either a higher incidence of infections has been reported in iron deficient subjects or reduced incidence following iron therapy include: those done by Macdougall et al (i0), Fletcher et al (14), Arbeter et al (38), Lovric (50), Higgs and Wells (51), Oski and Pearson (52), Fortuone (53), Basra and Churchill (54), and Chandra et al (34). A high association between incidence of infections and Hb levels has also been observed in pregnant women (55-56). In one of them (56), urinary infections were twice as high in pregnant women with Hb levels below i0 g/dl compared to those with Hb levels above i0 g/dl. In two recent studies, Hussein et al (57) in Egypt and Heresi et al (58) in Chile have demonstrated that iron supplementation in infants, preschool and school children as well as adults, reduced the number of episodes of infectious diseases.

The tentative conclusion from these studies is that iron deficiency may predispose to infections and that iron therapy may protect from them. Unfortunately, in some of the studies, the protocol is subject to criticism since data on controls and patients were not always collected during the same period (47) or multiple deficiencies including protein-energy undernutrition, rather than iron deficiency alone were corrected (38). Nevertheless, the studies by, Andelman and Sered (48), Cantwell et al (49), Lovric (50), Fortuone (53) and Basta and Churchill (54), Hussein et al (57) and Heresi et al (58) do suggest that iron therapy improves the resistance to infections.

In regard to the second view that iron deficiency may protect against infections, it is based on the concept of nutritional immunity, a concept defended by Weinberg (5,59), Bullen (60), Kochan (61), Bour (62) and Bezkorovainly (63). This view is supported by certain clinical observations which sowed that intramuscular injection to neonates significantly increased the incidence of septicemia (64-65). In one of the studies (64), iron administration resulted in a 6-fold increase in the incidence of infections. Mortality due to infections was 60% in treated and 0% in untreated infants.

However, the incidence of infections was not studied during the same year, the control group did not necessarily come from the same communities (Europeans vs Polynesians) and iron was injected in non-iron deficient neonates. In fact, it is known that the early neonatal period is not the best time for iron administration because of low transferrin levels, high transferrin saturation (66), and non fully expressed immunity (67). The difference between the Polynesian studies (64-65) and those done in other communities (46-56) could be explained in term of the methods by which iron was administered (intramuscular as opposed to food fortification).

Other studies in which increased incidence of infections have followed either iron administration or refeeding include those done by Murray et al (68-69) on Somalian nomads. A high incidence of infections has also been observed in individuals with hyperferremia due to sickle cell disease (70), hemochromatosis (70) and refed patients suffering from Kwashiorkor (71). A negative association between incidence of infections and iron deficiency anemia compared to other forms of anemia (hemolytic, megaloblastic, and refractory) has been reported by Masawe et al (72).

The difficulty in many of these studies lies in their protocol: e.g. lack of complete evaluation of the overall nutritional status prior to iron administration (68), use of patients with disorders of the immune response such as sickle cell disease or those suffering from severe protein-energy malnutrition, lack of true controls (72), etc. It therefore becomes very difficult if not impossible to conclude that correction of iron deficiency is detrimental to host defense mechanism.

IRON DEFICIENCY AND CMI 997

An other argument which has often been used by the same school of thoughts is the rate of multiplication of bacteria incubated in either human or artificial milk (60), iron deficient and control serum. (Artificial milk do not have iron binding proteins). The rate of multiplication of bacteria is much higher in the more saturated serum or milk. It must be noticed though that sometimes there has been an exaggeration of saturation of iron binding proteins (transferrin and lactoferrin) in either milk or serum. In the study by Bullen (60), iron was added to saturate iron binding proteins by 200%. It is highly unlikely that iron supplementation raises transferrin and/or lactoferrin saturation to this level.

A discussion on the association between iron status and/or administration and incidence of infections would be incomplete if studies which have shown no association are not mentioned. Some of them are those done by James and Combe (73), Burman (74) and Howell (75) In a recent study, the incidence of malaria attacks as well as densities of parasitemia, in school children from Papua New Guinea, were neither increased nor decreased following 6-16 weeks of iron therapy (76). Unfortunately, the presence or absence of infections was sometimes based on parents' interviews but not specific tests of microorganisms.

Animal studies Several investigators have approached this problem in different

experimental animal models which include: mice, rats, guinea-pigs, rabbits and chicks. Even in these models, the controversy has also persisted. Most studies in mice have shown that iron deficiency protects from infections and raises the LD50 of certain microorganisms. Iron administration reduces the resistance (reviewed in ref 59). It must be added that the number of test microorganisms and amounts of iron injected to these mice are quite high (i.e. 200 mg iron/kg body weight !). Iron deficient rats, on the other hand, show an increased susceptibility to Salmonnela typhmurium and Proteus mirabilis (77-79). Iron injection to chicks has also been associated with a reduced incidence of infection (80).

These studies may suggest that the animal model used as well as the type of microorganisms tested may play an important role on the type of response. In most of these studies, no attempt has been made to compare the incidence of naturally occurring infections as opposed to induced infections. The rate of natural infections as opposed to a single injection of millions of microorganisms would be more important since this will be similar to what happens in humans. In such a situation, a true competition for iron is created between the invader and the cells of the immune system.

CONCLUDING REMARKS

The preceding discussion clearly demonstrates that several components of the immune system especially the cell-mediated immunity and bactericidal capacity of leucocytes, are impaired in iron deficiency anemia. But as to how this decrease is related to susceptibility to infections remains to be resolved. Many studies which have tried to correlate iron deficiency and susceptibility or resistance to infections had problems in their experimental design. Some of them suffer from overinterpretation of the limited results, oversimplification of in vivo iron metabolism, inadequate sampling and/or statistical analysis, limited assessment of the iron as well as overall nutritional status. The presence or absence of infections had very often been ruled out by a questionnaire but not always by specific tests such as

998 S. KUVlBIDILA

cultures, microscopic examination of body fluid or measurement of some acute phase reactant proteins.

Several types of anemia (other than that due to iron deficiency) are associated with increased incidence of infection because of underlying disorders of the immune system rather than the availability of iron alone. There certainly is no question that iron administration to certain groups of individuals such as patients suffering from kwashiorkor, or neonates, may lead to increased incidence of infection. However, there is insufficient data to allow us to conclude that iron deficient subjects are more resistant to infections than normal people.

A basic problem which has not always been addressed by other investigators (45,59) who have previously reviewed this subject, is the assessment of the iron status during infection. Levels of hemoglobin, serum iron and/or transferrin saturation, which are the most commonly used parameters of the iron status are lowered by infection as well as iron deficiency (81). The levels of serum ferritin and free erythrocyte protoporphyrin, which are the best indicators of body iron stores, are increased by infections (81-82). We are therefore left with two basic unresolved questions: first, what levels of serum ferritin should be considered abnormally low in order to accurately classify an infected iron deficient patient? In other words, what is the upper limit of serum ferritin levels of an infected "iron deficient" patient? Second, at what lower and upper limits of body iron stores in humans that the defense mechanism becomes affected?

Future epidemiologic studies dealing with this problem should definitely have better experimental designs: for example, they should include not only iron deficient individuals and controls but also iron deficient subjects who are not supplemented (or at least iron supplementation is delayed). Prospective rather than retrospective studies should also be done. Assessment of recent or present infections and the overall nutritional status (especially protein-energy, but also zinc, copper, folic acid and vitamin A) with sensitive biochemical parameters should also be included.

ACKNOWLEDGEMENTS

The author is grateful to Drs. B.S. Baliga and D. Lemonnier for their thorough comments. Special thanks to Ms. Jessye Hilliard for her technical assistance. This work was supported by the Institut National de la Sante' et de la Recherche Medicale; INSERM Unite' i.

REFERENCES

1 Report of WHO group of experts, 1972. Nutritional anemias. World Health Organization; Technical Report Series #503 Geneva 1972.

2 Baker SJ, DeMseyer EM. Nutritional anemia: its understanding and control with special reference to work of the world health organization. J Clin Nutr 1979; 32:368-417.

Am

3 Beutler E, Fairbanks FV. The effects of iron deficiency. In: Jacobs A, Worwood M, eds, Iron in Biochemistry and Medicine II. London: Academic Press 1980; 394-427.

IRON DEFICIENCY AND CMI 999

4 Pollack MS, Martins Da Silva B, Moshief RD, Groshen S, Bognacki J, Dupont B, DeSousa M. Ferritin secretion by human mononuclear cells: Association with HLA phenotype. Clin Immunol Immunopathol 1983; 27:124-34.

5 Weinberg ED. Iron and susceptibility to infectious disease. Science 1974; 184:952-6.

6 Chandra RK, Saraya AK. Impaired immunocompetence associated with iron deficiency. J Ped 1975; 86:899-902.

7 Joynson DHM, Murray-Walker D, Jacobs A and Dolby AE. mediated immunity in patients with iron-deficiency anemia. 11:1058-9.

Defects of cell- Lancet 1972;

8 Bhaskaram C and Reddy V. Cell-mediated immunity in iron and vitamin A deficient children. Br Med J 1975;3:522.

9 Krantman HJ, Young SR, Ank BJ, O'Donnel MC, Rachelefsky GS, and Stiehman RE. Immune function in pure iron deficiency. Am J Dis Child 1982;136:840-4.

i0 Macdougall LG, Anderson R, McNab GM, Katz. The immune response in iron- deficient children: Impaired cellular defense mechanisms with altered humoral components. J Ped 1975;86:833-43.

ii Swarup-Mitra S. and Sinha AK. Cell-mediated immunity in nutritional anemia. Ind J Med Res 1984;79:354-62.

12 Gross R, Reid OVJ, Newberne PM, Burgers B and Masten R. Depressed cell- mediated immunity in megaloblastic anemia due to folic acid deficiency. Am J Clin Nutr 1975;28:225-32.

13 Kuvibidila S, Baliga SB, Suskind RM. Effects of iron deficiency anemia on delayed cutaneous hypersensitivity in mice. Am J Clin Nutr 1981; 34:2635- 40.

14 Fletcher J, Mather J, Lewis MJ, Whiting G. Mouth lesions in iron deficiency anemia: relationship to candida albicans in saliva and impairment to lymphocyte transformation. J Infect Dis 1975; 131:44-50.

15 Srikantia S.G., Prasad JS, Bhaskaram C and Krishnamachari KAVR. Anemia and immune response. Lancet 1976; 1:1307-9.

16 Sawitsky B, Knater R, Sawitsky A. Lymphocyte response in phytomitogens in iron deficiency. Am J Med Sci 1976; 277:153-160.

17 Prema K, Ramalakshmi BA, Mahavapeddi R and Babu S. In~nune status in anaemic pregnant women. Br J Obstet & Gynecol 1982; 89:222-5.

18 Hoffband AV, Ganeshaguru K, Tattersall MHN and Tripp E. deificieny on DNA synthesis. Clin Sci Mol Med 1974; 46:12p.

19 Narasinga Rao BS. Studies on iron deficiency anemia. (Suppl) 1978; 68:58-69.

Effect of iron

Ind J Med Res

20 Kulapongs R, Suskind RM, Vithayasui V, Olsen ER. Cell-mediated immunity and phagocytosis and killing function in childen with iron deficiency anemia. Lancet 1974; 11:689-91.

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21 Gupta KK, Dhatt PS, Singh H. Cell-mediated immunity in children with iron-deficiency anaemia. Ind J Ped 1982; 49:507-10.

22 Grosch-Worner I, Grosse-Wilde H, Bender-G, Shafer KH. Lymphocyte function in children with iron deficiency. Klinische Wochenschrift 1984; 62(22):1091-3.

23 Kuvibidila S, Nauss KM, Baliga BS, and Suskind RM. Impairment of blastogenic response of splenic lymphocytes from iron-deficient mice: in vivo repletion. Am J Clin Nutr 1983;37:557-65.

24 Kuvibidila S, Nauss KM, Baliga BS, and Suskind RM. Impairment of blastogenic response of lymphyocytes from iron-deficient mice: in vitro repletion. Am J Clin Nutr 1983;375:57-65.

25 Kuvibidila S. Effects of moderate iron deficiency anemia on mitogenic response of lymphocytes from mice spleen and lymph nodes. Acta Pharmacol Toxicol (Suppl) 1985; 59:122-5.

26 Kramer TR, Johnson WT, Briske-Anderson MJ and Davis MA. Copper deficiency and rat spleen lymphocyte biology. Abstract Fed Proc 1985; 44(4):1150 4322.

27 Soyano A, Candellet D,layrisse M. Effect of iron deficiency on the mitogen-induced proliferation response of rat lymphocytes. Int Archs Allergy appl Immunol 1982;69:353-7.

28 Lipschitz DA, Mitchell CO, Thompson C. The anemia of senescence. Am J Clin Haematol 1981;11:45-54.

29 Kuvibidila S, Dardenne M and Savino W. T-cell subpopulations and thymulin levels in iron deficient mice. Abstract. Fed Proc 1985;41:422.

30 Kuvibidila S, Baliga BS and Suskind RM. The effects of iron deficiency on cytolytic activity of mice spleen and peritoneal cells against allogenic tumor cells. Am J Clin Nutr 1983;38:238-44.

31 Sinha AK and Swarup-mitra S. Humoral immune response in nutritional anemia. Bull Calcutta School Trop Med 1982;30:46.

32 Bagchi K, Moharam M, and Reddy V. Humoral immune response in children with iron deficiency anemia. Br Med J 1980;2:1249-51.

33 Chandra RK, Newberne PM. Nutrition, immunity and infections. New York: Plenum, 1977;67-126.

34 Chandra RK, Woodford B, Au B, and Hyam P. Iron status, immune response and susceptibility to infections. In, Iron metabolism. CIBA Foundation New York Elsevier Excerpta Medica North Holland, 1977, 51:249-68.

35 Nalder BM, Mahoney AW, Ramakrishnen R, Hendricks DG. Sensitivity of the immunological response to nutritional status of rats. J Nutr 1972;102:535- 42.

36 Kuvibidila S, Baliga BS, and Suskind RM. Generation of plaque forming cells in iron deficient anemic mice. Nutr Rep Internat 1982;26:861-71.

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37 Kochanowski BA and Sherman AR. Decreased antibody formation in iron deficient rat pups - effect of iron repletion. Am J Clin Nutr 1985;41:278- 84.

38 Arbeter A, Echeverri L, Fransco D, Munson D, Vele H and Vitale J. Nutrition and infection. Fed Proc 1971;30:1421-8.

39 Yetgin S, Altay C, Ciliv G and Laleli Y. Myeloperoxidase activity and bactericidal function of PMN in iron deficiency. Acta Haemat 1979;61:10-14.

40 Prasad JS. Leucocyte function in iron-deficiency anemia. Am J Clin Nutr 1979;32:550-2.

41Higashi O., Sato Y., Takamatsu H. and Omaya M. Mean cellular peroxidase (MCP) of leukocytes in iron deficiency anemia. Tohoku J Exp Med 1967;93:105- 13.

42 Kochanowski BA and Sherman AR. Phagocytosis and lysozyme activity in granylocytes from iron deficient rat dams and pups. Nutr Res 1984;4:511-20.

43 Kuvibidila S, Wade S. Macrophage function as studied by the clearance of 125 I-polyvinyl-pyrrolidone in iron deficient and iron repleted mice. J Nutr 1987, 117:170-176.

44 Ronnlund RD, Rohrer SD, Baliga BS, and Suskind RM. Natural cytotoxicity of spleen cells from iron deficient mice. Abstract Fed Proc 1982;41:422.

45 Strauss RG. Iron deficiency, infections and immune function: a reassessment. Am J Clin Nutr 1978;31:660-6.

46 Humbert JR, Moore LL. Iron deficiency and infection: a dilemma. J Ped Gastroenterol & Nutr 1983;2:403-6.

47 Mackay HM. Anaemia in infancy: its prevalence and prevention. Arch Dis Child 1928;3:117-47.

48 Andelman MB, Sered BR. Utilization of dietary iron in term infants. Am J Dis Child 1966;111:45-55.

49 Cantwell RJ. Iron deficiency: some clinical principles illustrated by the response of maori infants to neonatal parenteral iron administration. Clin Ped (Philadelphia) 1972;11:443-9.

50 Lovrick VA. Normal haematologic values in children aged 6 to 36 months and socio-medical implications. Med J Austr 1970;2:366-370.

51Higgs JM, Wells RS. Chronic mucocutaneous candidiasis; associated abnormalities of iron metabolism. Br J Dermatol 1972;86:88-102.

52 Oski FA, pearson MS Ed's. Iron nutrition revisited - infancy childhood, adolescence. Report of the Eighty-Second Ross Conference on Pediatric Research. Columbus, Ohio. Ross Laboratories 1981 page 72.

53 Fortuone R. Acute purulent meningitis in Alaskan natives: Epidemiology, diagnosis and prognosis. Can Med Ass J 1966;94:19-22.

54 Basta SS and Churchill A. Iron deficiency anemia and productivity of adult males in Indonesian; In: World Bank Staff Working Paper #175 (1974):149

1002 S. KUVIBIDILA

55 Abramson JH, Sacks TC, Flug D. Bacteriuria and hemoglobin levels in pregnancy. JAMA 1971;215:1631-7.

56 Giles C, Brown JAH. Urinary infection and anemia in pregnancy. Brit Med J 1962;2:10-13.

57 Hussein MA, Hassan HA, Salem S, Scrimshaw N, Keresche G, Pollit E. Field work on the effects of iron supplementation. XIIIth International Congress of Nutrition; August 1985, Brighton England. Abst Book page 129.

58 Heresi G, Olivaress M, Pizzaro F, Cayazao M, Hertrampf E, Walter T and Stekel A. Effect of iron fortification on infant morbidity. XIIIth International Congress of Nutrition; August 1985, Brighton England. Abst Book page 129.

59 Weinberg E.D. Iron and infection. Micriobiol Rev 1978;42:45-66.

60 Bullen JJ, Rogers HJ, Leigh L. Iron-binding proteins in milk and resistance to E. coli infections in infants. Br Med J 1972;1:69-75.

61 Kochan I. The role of iron in bacterial infections with special consideration of host tubercle interactions. Curr Topics in Microbiol and Immunol 1973;60:1-30.

62 Bour H. Le fer et les infections. Bull Aca Natl Med 1981;165:519-25.

63 Bezkorovainy A. Antimicrobial properties of iron-binding proteins. Adv Exp Med Biol 1981;135:139-54.

64 Barry DMJ, Reeve AW. Increased incidence of gramm-negative neonatal sepsis with intramuscular iron admdinistration. Pediatrics 1977;60:908-12.

65 Becroft DMO, Dix MR, Farmer KA. Intramuscular iron dextran and susceptibility of neonates to bacterial infections. Arch Dis Child 1977;52:778-81.

66 Saarinene UM; Siimes MA. Developmental changes in serum iron, total iron-binding capacity and transferrin saturation in infancy. J Pediatrics 1977;91:875-7.

67 Miller ME. Phagocytosis in newborn infants: humoral and cellular factors. J Pediatr 1969;74:255-9.

68 Murray MJ, Murray AB, Murray MB & Murray CJ. The adverse effect of iron repletion on the course of certain infections. Br Med J 1978;2:1113-5.

69 Murray MJ, Murray AB. Starvation, suppression, and refeeding activation of infection. An ecological necessity? Lancet 1977;I:123.

70 Weinberg ED. Infection and iron metabolism. Am J Clin Nutr 1977;30:1485-90.

71 MacFarlane H, Reddy S. Immunity, transferrin and survival in kwashiorkor. Br Med J 1970;4:268-70.

72 Masawe AE, Muindi JM, Swain GB. Infections in iron deficiency and

IRON DEFICIENCY AND CMI ]003

other types of anemia in the tropics. Lancet 1974;2:314-7.

73 James AJ, Combes M. Iron deficiency in the premature infants: significance and prevention by intramuscular administration of iron dextran. Pediatrics 1960;26:368-74.

74 Burman D. Hemoglobin levels in normal infants aged 3 to 24 months and the effects of iron. Arch Dis Child 1972;47:261-71.

75 Howell D. Consequences of mild iron deficiency in children. In: Goldsmith G, Editor. Procedings of workshop on extent and meaning of iron deficiency in the U.S. Washington DC: National Research Council 1960;page 65-9.

76 Harvey P, Heywood MC, Pesheim JP, Habicht JP and Alpers M. lorn repletion and malaria. Abstract. Fed Proc 1987; 46:1161.

77 Baggs RB and Miller SA. Nutritional iron deficiency as a determinant of host resistance in the rat. J Nutr 1973;103:1554-60.

78 Baggs RB and Miller SA. Defect in resistance to salmonella typhimurium in iron-deficient rats. J Infect Dis 1974;130:409-11.

79 Hart RC, Kadis S, and Chapman W Jr. Nutritional iron status and susceptibility to Proteus mirabilis pyelonephritis in the rat. Can J Microbiol 1982;28:713-7.

80 Hill CH. Dietary influences on resistance to Salmonella infection in chicks. Fed Proc 1979;38:2129-33.

81 Bernat I. Anemia of infection. In: Bernat I, etc, Iron metabolism. New York, Plenum 1983; 275-83.

82 Worwood M. Serum ferritin. In: Jacobs A Worwood, M.Eds, Iron in biochemistry and metabolism II. A Academic Press London:f980; 204-44.

Accepted for publication May 25, 1987.