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THE EFFECTS OF DIETARY ZINC DEFICIENCY
ON THE HUMORAL IMMUNE RESPONSE
OF THE YOUNG ADULT A/J MOUSE

By

Suzanne Marie Haas

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Biochemistry

1977

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ABSTRACT
THE EFFECTS OF DIETARY ZINC DEFICIENCY

ON THE HUMORAL IMMUNE RESPONSE
OF THE YOUNG ADULT A/J MOUSE

By

Suzanne Marie Haas

Despite the devastating effects of disease in malnourished
populations, little attention has been focused on the relationship of
malnutrition and immunity. This thesis will investigate this relation-
ship by studying the effects of dietary zinc deficiency on the humoral
immune response of the young adult A/J mouse.

Mice were injected with KLH-ars on the same day that they were
placed on a deficient diet. The titer of the deficient group to the
antigen was identical to that of controls, indicating that B cells
could produce antibody once stimulated by healthy T cells. However,
when mice were injected with SRBC after three weeks on a zinc deficient
diet, the mice had a reduced indirect plaque forming ability in the
primary and secondary responses to antigen, which could be significantly
restored by reconstitution with thymocytes. This suggested that T

cell helper activity was impaired in zinc deficient animals.

TABLE OF CONTENTS

LIST OF TABLES ..........................
LIST OF FIGURES .........................
LIST OF SYMBOLS AND ABBREVIATIONS ................
INTRODUCTION ...........................
MATERIALS AND METHODS ......................

Materials ........................
Animals .........................
Diet Preparation ..... ' ...............
Preparation of the vitamin-glucose mixture .....
Mixing of the mouse diet ..............
Zinc Assay by Atomic Absorption Spectroscopy ......
Coupling of the Diazonium Salt of Arsanilic Acid to
Proteins . . . . ....................

Preparation of Rabbit Anti-Mouse Gamma Globulin .....
Protein Iodination ...................
Jerne Plaque Assay ...................
Preparation of cells for the assay .........
Quantitation of plaque forming cells ........
Statistics ........................
RESULTS ..............................
EstabliShment of a Diet for Zinc Studies in the Adult
Mouse .........................
The Zinc Content of the Thymus of the A/J Mouse .....
The Effects of Zinc Deficiency on the Humoral Response
of the Young Adult A/J Mouse ..............
The response of the young adult A/J mouse
to KLH-ars. .....................
The primary response of the young adult A/J mouse
to SRBC .......................
The secondary response of the young adult A/J mouse
to SRBC .......................
SUMMARY AND CONCLUSIONS .....................
REFERENCES ............................

ii

Page
iii

iv

12

25
25
32
34
39
44

Table

10

LIST OF TABLES

Vitamin-glucose mixture .................
Diet ingredients .......... ' ...........
Organ weights of six week old A/J mice fed diets
supplemented with different levels of zinc for

three weeks , .......................

Zinc analysis of the organs of the six week old A/J
mouse ..........................

Growth and organ weights of ten week old mice fed
diets supplemented with different levels of zinc for four
weeks ..........................

Titer of mice immunized with keyhole limpet hemocyanin-p-
azophenylarsonate .................. g. .

Dietary intake of adult A/J mice fed diets supplemented
with different levels of zinc for four weeks ......

Primary response of A/J mice to sheep red blood cells . .
Growth, thymus weights, and intake of A/J mice fed diets
containing different levels of zinc for thirty-nine

days ...........................

Secondary response of Ala mice to sheep red blood
cells ..........................

iii

Page

14

15

23

24

27

29

31

33

36

37

LIST OF FIGURES

Figure Page

l Growth curves of A/J mice fed diets supplemented
with different levels of zinc .............. 2l

iv

BGG

BGG-ars
DEAE-cellulose
KLH

KLH-ars

MEM

PCM
PFC
RIA
SRBC
TCA

LIST OF SYMBOLS AND ABBREVIATIONS

bovine gamma globulin

bovine gamma globulin-p-azophenylarsonate
Diethylaminoethyl cellulose

keyhole limpet hemocyanin

keyhole limpet hemocyanin-p-
azophenylarsonate

Eagle's Minimum Essential Medium
with Earle's Balanced Salt Solution

protein calorie malnutrition
plaque forming cells
radioimmunoassay

sheep red blood cells

trichloroacetic acid'

INTRODUCTION

The devastating effects of disease in malnourished populations
have been well known for many years. In one study in rural Guatemala,
an enhanced fatality rate, greater frequency of complications, and an
exaggerated clinical course were observed in malnourished children
with the common childhood diseases of measles, whooping cough, mumps,
rubella, and chickenpox when compared to well-fed children of higher
socio-economic status (l). However, despite the publication of many
similar reports from epidemiological studies of other malnourished
populations in the world, few clear-cut immunological studies have
been attempted to delineate the role of nutritional deficiencies on
immunity. As a result, little is known about the effects of nutritional
status on an individual's immune response, thus emphasizing the need
for further research in this area.

The aim of this thesis was to initiate a series of investigations
to explore the relationship of malnutrition and immunity by studying
the effects of dietary zinc deficiency on the immune response. Investi-
gation of zinc deficiency was chosen because zinc is an important
trace metal in mammalian metabolism and it also can be easily monitored
by atomic absorption spectroscopy. In addition, the increasing
importance in the world's diet of cereal grains, which contain many
zinc chelating compounds, suggested the possibility of large scale

zinc deficiency in the future. Therefore, studies are needed to

define all the consequences of zinc deficiency, including its effects
on the immune system.

Published reports on experimental zinc deficiency in animals
have demonstrated the wide range of known effects of zinc deficiency.
Zinc deficient rats (2) and pigs (3, 4) have exhibited poor growth,
anorexia, skin lesions, a decrease in spermatogenesis, and in some
cases, a decrease in specific activity of zinc metalloenzymes in the
deficient tissues. Hurley (5) has shown that maternal zinc deficiency
in rats resulted in a decreased zinc content of the fetuses, smaller
litter size, a decreased birth weight, and an increase in the incidence
of severe congenital malformations in the offspring.

Zinc deficiency has also been described in man, although'it~
is not as yet a major nutritional problem in the world. A study of
338 persons in the Denver, Colorado area has documented the existence
of l0 children with low levels of zinc in their hair who were small
for their age, had a history of poor appetite, and decreased taste
acuity, symptoms characteristic of experimental zinc deficiency in
animals (6).

The major study to date on zinc deficiency in human subjects
was a report by Sandstead (7) in l967 on zinc deficiency in male
dwarfs in the Middle East. In addition to low levels of zinc in the
hair, sweat, urine, and plasma, the adolescent boys studied by
Sandstead exhibited retarded growth, hepatosplenomegaly, hypogonadism,
and a delayed closure of the long bones. Supplementation of the diet
of these children with zinc sulfate produced a reversal of these
symptoms with dramatic increases in growth and sexual development.
Sandstead has hypothesized that the diet of these children which con-

sisted almost entirely of cereal grains, contained large amounts of

phytic acid, which chelated zinc, forming insoluble complexes which

were excreted from the body. With a world population that must of
necessity become more dependent on economically efficient cereal grains

as a primary food source, increasing numbers of zinc deficient individuals
may be found with severe growth retardation and sexual development

similar to that observed in the Middle East.

Despite these reports enumerating the many serious effects of
zinc inadequacy on the deficient animal and man, the growing numbers of
recognized zinc deficient individuals, and the depression of immunity in
malnourished persons, little is known about the effects of zinc deficiency
on immunity. In two separate studies, weanling pigs (4) and rats (8)
on zinc deficient diets were found to have a severe atrophy of the
thymus that was absent in the pair fed controls. Brummerstedt (9) has
also described an autosomal recessive trait of cattle characterized by
thymic atrophy, loss of hair, and parakeratotic lesions. Oral zinc
oxide therapy resulted in an increase in weights and the recovery of
the skin lesions. He has hypothesized that these calves were unable to
utilize zinc in their food and that the presence of zinc was important
for the development of the thymus. Since the thymus has been shown to
be responsible for the development of immunologically competent T cells,
which play an important role in both cellular and humoral immunity, the
wasting of this organ by zinc deficiency suggested that T cells and
therefore the immune response was being impaired.

A depression of immunity in human zinc deficiency was indicated
in a study of a patient with terminal acrodermatitis enteropathica,
thought to be a genetic deficiency of zinc absorption (10). Analysis

of the serum of this patient showed undetectable levels of immunoglobulins.

Death resulted from infection with E. coli and C. albicans. Postmortem

 

studies on the same child showed no germinal centers in the spleen, a

lack of lymph nodes, and no tonsils or Peyer's patches. The thymus
consisted of epithelial cells with a few thymocytes and Hassal's corpuscles.
Although these isolated reports in the literature have suggested a relation-
ship between decreased immune response and zinc deficiency, no actual

tests of immune function were performed.

Other investigators have attempted to define the effects of a
variety of nutritional deficiencies on the immune response. Several re-
searchers have recently shown that metal deficiencies may result in a
depressed immune response. Magnesium deficiency in rats decreased both
agglutinin and hemolysin titers to sheep red blood cells (ll). Nalder
(12) reported that iron deficiency in rats caused a depression of anti-
tetanus toxoid titer in proportion to the degree of iron depletion. In
a study of 20 iron deficient children, Chandra (l3) found no differences
in 196 levels and antibody titers to tetanus toxoid and S, typh1_antigens
when these children were compared to well-fed children of the same age.
However, cell mediated immunity, measured by bactericidal activity of
macrophages, and cutaneous delayed type hypersensitivity to C, albicans
and other antigens was impaired.

A. E. Axelrod has investigated the effects of specific vitamin
deficiencies on the immune response in several animal models. An impairment
of antibody formation following a single injection of diptheria toxoid
was found in pantothenic acid, riboflavin, pyridoxine, biotin and vitamin
D deficiencies in rats (l4). No significant differences between
experimental and control animals were found in Vitamin A or thiamine

deficiency, nor could the observed results be attributed to reduced intake

in the experimental animals. Vitamin B6 deficient guinea pigs studied
by the same investigator had a reduced cutaneous delayed type hyper-
sensitivity response to BCG which was reversible by administration of
the missing vitamin (lS). Rats deficient in 36 had a high percentage
of successful skin transplants (l6). Axelrod's results have indicated
that both the humoral and cell mediated limbs of the immune response may
be affected by vitamin deficiencies. However, the variety of model systems
used and the lack of detailed and quantitative experiments on the effects
of single vitamin deficiencies on the humoral and cell mediated responses
of these animals have generated a vague and unclear picture of the
immunological effects of vitamin inadequacies.

Amino acid deficiencies have also produced deleterious effects on
the inmune response of rats. Jose (l7) investigated the effects of a
decrease of single essential amino acids to 50%, 25% and 10% of their re-
quired levels in mice on the primary response to DBA/2 mammary adenocarcinoma
cells. A 25% reduction of phenylalanine-tyrosine, valine, threonine,
methionine, cysteine, isoleucine and tryptophan produced large decreases in
hemagglutinating and blocking antibody activity. Cell mediated immunity,
measured by in vitro assay, was not affected until amino acid levels were
reduced to 10%. However, only a Slight depression of immunity was observed with
arginine, histidine, and lysine. Leucine was unique in that cell mediated
responses were lowered before a depression in humoral response could be
detected. Individual amino acid deficiencies appeared to be complex
to study because the extent of immunological impairment seemed to be
dependent on the particular amino acid that was absent and the level of
deficiency. Although this report has provided interesting data, no

attempt was made by the authors to explain their results or determine the

specific effects of the individual amino acid deficiencies on the
components of immunity.

Because it is the world's most pressing nutritional problem,
protein calorie malnutrition (PCM) has been the most thoroughly studied
nutritional deficiency. Many reports have shown that PCM also produced
adverse effects on immunity. Several investigators have observed atrophy
of the lymphoid organs and reduced lymphoid germinal centers in protein
or calorie depleted animals (18, 19). Ninick (20) has investigated the
effects of three weeks of calorie deprivation and subsequent refeeding
on organ weight in the neonatal, weanling and adult rat. Although most
organs showed signs of wasting in growing rats, only the thymus, the site
of maturation of immunologically important T cells, was permanently atrophied
by calorie deprivation in all of the age groups studied. Although the
reduced size of this organ suggested immunological impairment, no ascess-
ment of immunological status was attempted.

The humoral response of protein calorie deficient animals to a
number of different antigens has also been studied. Marasmic pigs produced
antibody titer that was equal to that of controls using tetanus toxoid,
¢K174, or sheep red blood cells as antigen, but produced reduced amounts
of antibody following injection of erythrocyte A antigen (18). No differences
were noted between the two groups with respect to 196 or IgM concentration.

A decrease in the number of plaque forming cells (PFC) in response
to a single injection of sheep red blood cells has been observed by many
investigators with PCM induced in a number of different animal strains
(19, 21, 22). Impairment of T cells by PCM was suggested in the study
by Mathur (19), in which injection of syngeneic thymocytes improved the

number of plaques observed significantly over similarly treated controls.

Chandra (23) observed a decrease in PFC not only in calorie deprived rats,
but also in the F1 and F2 generations of these rats. These results have
important clinical implications for the malnourished woman and strongly
emphasize the importance of adequate nutrition during pregnancy.

Several indeces of cell mediated immunity have been studied in
animals with PCM. Skin allograft survival was increased from 9 days in
control mice to 16 days in the experimental groups, indicating an impair-
ment of T cells (21). Macrophage clearance of carbon particles was
reduced in mice (24) as was delayed type hypersensitivity to DNCB and
tuberculin antigens in marasmic pigs (18). Conversely, the results of
Cooper (25) indicated that protein depletion had an enhancing effect on
cell mediated immunity as shown by a decrease in survival time of skin
grafts, an increase in PHA stimulation, an increased ability to ingest
bacteria, and an enhanced resistance to pseudorabies virus in mice.
Although the results of these investigations did not agree with the
impairment of immunity observed in other animal systems, the authors did
not attempt to explain this discrepancy.

The contradictory results obtained by these investigators have
pointed out even more strongly the importance of good immunological
studies in nutritionally deficient model systems. However, the isolated
studies in this area, the variety of animal strains and species employed.
and the lack of specific investigations on the effects of nutritional
deficiencies on animals of different ages have made it difficult to
construct a clear picture of the link between nutrition and immunity.
Important studies such as the effects of marginal deficiencies, and the

long range effects of deficiencies produced at various ages and for different

lengths of time have also been lacking in these model systems, and are

essential to an understanding of the consequences of various states of
human malnutrition. Therefore, rigorous immunological studies in a
‘well-defined system are needed to define more precisely the role of
malnutrition in the immune response.

In addition to exploring the effects of PCM in animal models,
some investigators have also studied the effects of PCM in human subjects
from underdeveloped nations, although these studies have been limited
because ethical standards do not permit the maintenance of chronic deficiency
in malnourished patients. The startling implications of these reports have
further emphasized the deleterious effects of malnutrition on the lymphatic
system.

The histopathology of PCM in Ugandian children was studied in
detail by Watts (26). Postmortem reports compiled on a number of children
with marasmus and kwashiorkor by this investigator showed a decrease in
the ratio of thymus weight to body weight. Histological reports revealed
that the cells of the thymus were replaced by fibrous tissue. Another
postmortem study of South African children by Schonland (27) confirmed the
presence of the severe atrophy of the thymus seen by Watts and also noted
the occurence of lower spleen weights, reduced tonsilar area, decreased
thickness of Peyer's patches in the intestine, and reduced cross-sectional
thickness of the appendix in children diagnosed as suffering from kwashior-
kor or marasmus. Other lymphatic structures such as the paracortical areas
of the lymph nodes and the germinal centers of the various lymphatic organs
were also depleted. These results have provided histological support for
the impairment of immune function seen in nutritionally deficient populations.

Despite the establishment of large scale immunization programs

in many undernourished countries, few studies have attempted to evaluate

the effectiveness of immunization in these individuals who have shown

a reduced ability to combat disease. Ugandian children studied by I
Brown and Katz produced a normal response to smallpox virus (28)

whereas antibody production was decreased as compared to adequately
nourished controls when immunized with yellow fever vaccine (29). Serum
immunoglobulins were increased in 76 undernourished children studied in
Ghana, although the antibody titers were identical between these children
and controls to the antigen keyhole limpet hemocyanin and pneumococcal
polysaccharide (30). Opposite results were obtained by Coovadia (31)
who showed that serum IgG was within normal limits in South African
children with marasmus and kwashiorkor, as was the percentage of B cells
in peripheral blood. However, antibody levels to specific antigens were
reduced. The variety of results obtained by these investigators has
emphasized the difficulties of studying these children, especially since
PCM differs qualitatively and quantitatively in different parts of the
world and may be complicated by the presence of infections.

Various parameters of cell mediated immunity have also been assayed
in children with kwashiorkor and marasmus. Neumann (30) found that
malnourished children in Ghana had decreased cutaneous delayed type
hypersensitivity to Monilia, streptokinase-streptodornase, and phyto-
hemagglutinin both in terms of smaller diameters of inflammatory sites
and a higher percentage of nonreactors. In vitro stimulation of lympho-
cytes by phytohemagglutinin, a mitogen specific for T cells, was also _
reduced in some individuals with PCM (30, 32, 33), although not affected
in other cases (31, 34, 35). Interpretation of these conflicting results
was difficult because these studies were performed on subjects of various

ages and heterogeneous nutritional and immunological backgrounds.

10

Douglas (36) investigated the effects of PCM on phagocytic
function in 16 children with kwashiorkor. Phagocytic activity, the
activity of hexose monophosphate shunt enzymes, and morphological
studies by electron microscopy showed no differences between controls
and depleted individuals. HOwever, bactericidal activity was reduced
in the malnourished individuals. Similar results were reported by
Seth (37).

Although these reports have indicated that the immune response
was depressed in malnourished populations, conflicting results due in
part to complications such as infection and varying degrees of malnutrition
have made difficult an analysis of the specific effects of PCM on the humoral
and cell mediated aspects of immunity in man. In addition, few investigators
have explored the nutritional and immunological backgrounds of the individuals
under study or have performed follow-up studies on the consequences of
malnutrition present in these children to their immunological status as
an adult and the effects on their offspring.

The lack of thorough, comprehensive studies on any nutritional
deficiency in man or animals, as demonstrated by the review of the literature
given here, has resulted in a very unclear picture of the nature of the
relationship of malnutrition and immunity. This has pointed out the
need for detailed immunological studies of a well-monitored dietary de-
ficiency produced in a defined population. This thesis attempted to
initiate such studies by investigating the effects of dietary zinc“
deficiency on the immune response of the inbred A/J mouse. Inbred mice
were used as a model system because they assured a homogenous population
fOr study and eliminated the problems of individual variability, complicat-

ing infections, and heterogeneous nutritional backgrounds present in

ll

many of the earlier nutritional studies. The use of inbred mice will

also make possible the ultimate goal of monitoring the effects of zinc
deficiency on the neonatal, weanling, and adult mouse to conétruct a
complete picture of the consequences of inadequate zinc on the humoral and
cell mediated responses of essentially a single individual. In addition,
other questions, such as the effects of marginal deficiency and the
effects of short periods of nutritional inadequacy can be easily explored

in this system.

This thesis has investigated the effect of dietary zinc deficiency
on the humoral response of the young adult A/J mouse. The adult was
chosen for these initial experiments because the effects of zinc
deficiency were more easily defined in a mouse with a fully developed
immune system. The results of these studies are important since they
will provide a framework fbr future research toward elucidating the

role of malnutrition in the immune response.

MATERIALS AND METHODS

Materials

Zinc standard solutions were obtained from Harleco Corp.,
Philadelphia, Pennsylvania. Freund's adjuvant was purchased from
Difco Laboratories, Detroit, Michigan. Miles Laboratories, Kankakee,
Illinois were the source of bovine gamma globulin and non-hemolytic
guinea pig complement. Sheep red blood cells were obtained from
Cordis Laboratories, Miami, Florida. Seakem plaque agarose was pur-
chased from Marine Colloids Inc., Rockland, Maine. Calbiochem, San
Diego, California, was the source of keyhole limpet hemocyanin.
Arsanilic acid was obtained from Eastman Chem. Co., Rochester, New York.
and recrystallized from water and ethanol before use. All other
chemicals were analytical or reagent grade and purchased from a variety
of commercial sources. 1251 was obtained from New England Nuclear,
Boston, Massachusetts.

Mice of the A/J strain were derived from inbred stocks, maintained
and distributed by Jackson Laboratories, Bar Harbor, Maine. Rabbits
were purchased from the Center for Laboratory Animal Resources, Michigan
State University, E. Lansing, Michigan.

Animals

Female mice of the A/J strain were used for all experiments. The
mice were housed in acid washed polycarbonate cages with stainless steel
tops. Diets and deionized water, zinc free by atomic absorption assay,

12

13

were provided ag_libitum in all experiments.

Diet Preparation

 

The diet used for these experiments was an egg white based, biotin
fortified diet, designed by Luecke (2) for the rat. Zinc was added to
the diet in the form of zinc carbonate.

Preparation of the vitamin-glucose mixture. (Table l)

The choline chloride was premixed with an equal amount of glucose
monohydrate to assure even mixing of these components and to avoid clumping
of the diet. Small increments of glucose monohydrate were combined with

the mixture until 200 to 300 grams of the glucose were added. The rest of
the vitamins and glucose monohydrate were then added to the choline chloride-
glucose mixture, stirred well by hand, placed in a Hobart mixer, and stirred
on speed 1 for 20 minutes in the dark.

Mixingpof the mouse diet. (Table 2) The liquid ingredients of the
diet, corn oil, vitamins A, D, E, and K, and santoquin antioxidant were
combined and mixed with a glass stirring rod. The dry components of the
diet were placed in a large bowl on the Hobart mixer and stirred at speed
1. While mixing, the liquid ingredients were slowly added to the rest of
the diet. Mixing was continued for an additional 20 minutes. The diets
were stored in acid washed plastic jars at 4°C for the duration of each
eXperiment.

Zinc Assay by Atomic Absorption Spectroscopy

A known weight of sample to be assayed for zinc content was added
to a preweighed acid washed flask. 25 ml of concentrated nitric acid was
added to the flask and the sample was slowly digested over low heat to a
volume of approximately 5 ml. 2 ml of perchloric acid and 25 ml of con-

centrated nitric acid were added to the sample and the digestion repeated.

14

Table l

Vitamin-glucose mixture

9159_
Vitamin 812 in Mannitol ...................... 0.01
d-Biotin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0.10
Folic Acid ............................ 0.02
Pyridoxine-HCl .......................... 0.10
Riboflavin ............................ 0.20
Thiamine Mononitrate ....................... 0.20
Calcium Pantothenate ....................... 0.50
Nicotinic Acid .......................... 0.50
Inositol ............................. 2.00
p-Aminobenzoic Acid ........................ 1.00
Ascorbic Acid ........................... 1.00
Choline Chloride ........................ 30.00

Glucose Monohydrate ....................... 964.40

15

Table 2

Diet Ingredients

gikg_
Egg White_ ........................... . 200
Glucose Monohydrate ....................... 569
Corn Oil ............................ 100
Vitamin-Glucose Mixture] .................... so
Mineral Mixture :Zinc2 ..................... 45
Cellulose ............................ 30
Vitamins A, D, E, and K ..................... 5
Santoquin Antioxidant ...................... 1
1Table l.

2Phillips-Hart Salt Mixture No. 4 (38).

l6

evaporating the liquid to near dryness. The residue remaining after
this treatment was diluted to a known weight with 1% hydrochloric acid.
Blanks containing only the acid reagents were prepared with each series
of samples that were assayed.

Zinc concentration of the digested samples was determined with a
Perkin-Elmer Model 303 Atomic Absorption Spectrophotometer, using a single
electrode zinc cathode tube. Absorption values were detenmined at 2138
angstroms. IA standard curve for zinc absorption was obtained by dilution
of a commercial zinc standard solution in 1% hydrochloric acid. This
method was accurate to 0.2 ppm zinc.

Coppling_of the Diazonium Salt of Arsanilic Acid to Proteins

The coupling of arsanilic acid to proteins was performed by the
method of Nisonoff (39). One volume of a 1.2 mg/ml solution of the
diazonium salt of arsanilic acid in l N hydrochloric acid was added drop-
wise to ten volumes of a 20 mg/ml solution of the desired carrier protein,
with vigorous stirring at 4'c, maintaining the pH between 9.0 and 9.5.
After addition of all reagents, the reaction mixture was allowed to stir
for an additional hour. The reaction product was dialyzed for a week against
several changes of borate saline buffer to remove all unreacted arsanilic
acid. The conjugate was finally dialyzed into phosphate saline buffer
for injection purposes and stored at O'C in small aliquots. Coupling
efficiency of this method was reported by Nisonoff to be about 35% for KLH
and most gamma globulins.

Preparation of Rabbit Anti-Mouse Gamma Globulin

Gamma globulin was isolated from the ascites fluid of Balb/c mice

innoculated with MOPC-21 and LPC-l tumor cells (40). The gamma globulin

was precipitated from the ascites fluid by bringing the fluid to a final

17

concentration of 18% sodium sulfate. The resulting precipitate was
resuspended in a small volume of 0.04 M phosphate buffer pH 8.0, dialyZed
in the same buffer for several hours, and applied to the top of a column
of DEAE cellulose, which was equilibrated in 0.04 M phosphate buffer and
contained 1 cc DEAE for every 7 mg protein applied. The column was then
washed with 0.04 M phosphate buffer pH 8.0 and the absorbance of 1 ml
column fractions was monitored at 280 nm. The gamma globulin was eluted
with approximately one column volume of buffer. Following pervaporation
of the pooled gamma globulin fractions to a concentration of 5 mg/ml, the
gamma globulin was dialyzed into phosphate saline buffer for injection
purposes. The purity of the isolated gamma globulin was checked by immuno-
electrophoresis.

The purified gamma globulin was injected intramuscularly into several
rabbits at a dose of 4 mg every two weeks. They animals were bled at .
regular intervals and the serum was pooled, decomplemented, and stored
at o'c.

Protein Iodination

 

Iodination of proteins was performed by a modification of the
Chloramine T method of Hunter (41). The iodination mixture of lOO‘pl
contained 100‘ug of protein, 30}ug of cold potassium iodide and 3 X 107 cpm
Na 1251 in borate saline buffer. The reaction was initiated by the
addition of 4’pg of Chloramine T and allowed to stir for 10 minutes at
4°C. 4‘pg of sodium metabisulfite was then added, followed by stirring
for an additional 10 minutes. A small aliquot of the iodinated protein
was removed for counting purposes, added to 1 mg of bovine serum albumin
and precipitated with 10% TCA in the cold for 20 minutes. The protein

pellet was washed 3 times in 10% TCA, dissolved in 1N sodium hydroxide

O

18

and assayed for protein bound 1251 counts in a Beckman Biogamma Counter.
5 mg of bovine gamma globulin was added to the rest of the iodinated
protein as a carrier and the sample was dialyzed for a week with daily
changes of borate saline buffer prior to use.

Jerne Plaque Assay

 

The number of plasma cells producing anti-SRBC antibody was quantitated
by a modification of the Jerne Plaque assay (42). This assay measured the
formation of plaques, clear areas in a lawn of SRBC produced by diffusing
hemolytic antibodies from a single plasma cell, in agar suspensions of
SRBC and spleen cells from mice immunized with SRBC.

Preparation of cells for the assay. Several days after injection of
SRBC, the mice were anesthetized and swabbed with 70% alcohol. Spleens
to be assayed for plaquing capacity were removed from each mouse and placed
in cold sterile Eagle's Minimum Essential Medium (MEM) with Earle's Balanced
Salt Solution. Each spleen was separately trimmed, minced with scissors
and passed through an 80 gauge stainless steel screen into fresh MEM. The
cells were washed several times in MEM, resuspended in a volume of 1 ml and
placed on ice.

Sheep red blood cells from the same source used for injection of the
mice were washed several times in phosphate saline buffer, resuspended in
MEM at a concentration of 2 X 109 cells/ml, and placed on ice.

Quantitatibn of plaque forming cells. 0.9 ml aliquots of a solution
of 0.6% agarose in MEM were distributed in culture tubes placed in a
56.0 water bath. Both the spleen cells and SRBC were removed from the ice
and warmed slightly. 0.1 ml aliquots of both the spleen cells and the SRBC
suspension were pipetted into a culture tube containing the agar solution,

mixed well, and quickly poured into a Petri dish containing a base layer

19

of 1.6% agarose in MEM. The top layer containing the SRBC and spleen
cells was allowed to solidify and the plates were immediately placed
in a 37’C humidified incubator in an atmosphere of 95% air, 5% C02 for
90 minutes. To assay for direct plaques, or IgM producing plasma cells,
0.5 ml of non—hemolytic guinea pig complement, diluted 1 to 10 in
complement buffer, was pipetted over the top layer. The plates were
incubated for an additional half hour. The number of plasmacytes producing
anti-SRBC antibody was quantitated by counting on each plate the total
number of plaques.

To assay indirect plaques, or IgG producing plasmacytes, 0.5 ml of
non-hemolytic rabbit anti-mouse gamma globulin, previously absorbed with
‘p chains, and diluted to a strength producing maximum plaques was added
to each plate. After a 30 minute incubation, excess liquid was discarded
and 0.5 ml of non-hemolytic guinea pig complement was added to each plate
followed by another 30 minute incubation. Plaques were counted immediately.
Results were expressed as the number of plaque forming cells per spleen.

All assays were done in duplicate or triplicate. Controls consisted
of unimmunized animals. Background plaques, produced by these animals,
were subtracted when significant.

Direct plaques were considered a measure of IgM producing plasma
cells. The number of direct plaques was subtracted from the number of
indirect plaques to obtain the number of IgG producing plasma cells.

Statistics

 

The data were examined by analysis of variance with statistical
significance of treatment differences being determined by the multiple

range test of Duncan (43).

RESULTS

Establishment of a Diet for Zinc Studies in the Adult Mouse

In order to study the effects of zinc deficiency on inmunity, it
was first necessary to establish for the mouse, the adequacy of the egg
white based, biotin fortified diet of Luecke (2), originally designed
for zinc studies in the rat, and the approximate minimum requirement of
the mouse for zinc.

Three week old A/J females were placed in several dietary groups
receiving the diet supplemented with 0.4-,4, 12, 37, or 70 ppm zinc in
the form of zinc carbonate. Another age matched group of mice received a
diet of Wayne Mouse Breeder Blox which contained 65 ppm zinc. The weights
and diet consumption of all of the mice were monitored for a three week
period. Since the mouse has been shown to double in weight from the age
of three weeks to six weeks, any deficiencies in the synthetic diet should
result in a decreased growth rate of the mice on this diet when compared
to control mice fed the commercial diet.

As seen in Figure 1, mice fed a diet containing 12, 37, or 70 ppm
zinc had growth rates which closely paralleled that of the mice~raised on
the stock diet. The small difference between the final weights of the mice
on the synthetic diet and the stock diet could be attributed to a difference
in the palatability of the two diets. The mice receiving a diet of 4 ppm
zinc appeared to have a slightly reduced rate of growth, whereas the mice
()n the deficient diet failed to thrive. These mice also exhibited patchy

20

21

20- H stock diet
0-0 70 ppm zinc
H 37 ppm zinc

19- H 12 ppm zinc
H 4 ppm zinc
H '

18- 0.4 ppm Zinc

17-

16-h

15-

Average 14-

 

 

 

 

group
weight
in 13‘
grams
12-4
ll-
10-
:7; - ‘ ‘4— A
T l l 1 T l T l T
2 4 6 8 10 12 14 16 18 20

Days on diet
Figure 1. Growth curves of A/J mice fed diets supplemented with

different levels of zinc.

22

coats, eye lesions, and appeared emaciated, all symptoms of zinc
deficiency.

The final organ weights of the mice in the different dietary
groups are presented in Table 3. The mice on the synthetic diet con-
taining 0.4 ppm zinc had significantly lower organ weights than any of
the other groups. When the same organ weights were expressed as a percent-
age of final body weight, the thymus of the deficient group showed a seven
fold reduction in size. The spleen was reduced to approximately half in

the deficient mice. The liver and kidney, on the other hand, showed no
signs of wasting in these animals. Therefore, in the growing mouse, the
lymphatic organs appeared to be preferentially atrophied as a result of
zinc deficiency.

This study indicated that the synthetic diet was sufficient for normal
growth of the mouse when supplemented with adequate zinc. The results also
showed that the minimum zinc requirement of the mouse lies between 5 and 12
ppm zinc. Dietary zinc deficiency resulted in a rapid atrophy of the lym-
phatic tissues, especially the thymus, in the A/J mouse. This confirmed and
extended the earlier observations of thymic atrophy in the zinc deficient
pig (4) and rat (8).

The Zinc Content of the Thymus of the A/J Mouse

Before beginning additional studies, it was of interest to determine
if the thymus contained an unusual amount of zinc compared to other organs.
Various organs, including the thymus of the six week old adult mouse,
were wet ashed and analyzed for zinc content by atomic absorption spectros-
copy. The results, presented in Table 4, indicated that the thymus con-
tained levels of zinc that were similar to that of other organs.

However, this data did not indicate the importance of zinc to the thymus

23

Table 3

Organ weights of six week old A/J mice fed diets supplemented

with different levels of zinc for three weeks

 

 

Dietary Zinc Thymus Spleen Liver Kidney

ppm .

0.4 o.ooa(o.oa)1 0.021(o.23) 0.506(6.76)aa 0.161(1.8l)
4 0.039(o.25)aa o.o7o(o.49)a l.039(6.76) o.252(1.o4)
12 o.oa4(o.20)aa 0.081(0.48)a l.129(6.73) 0.251(1.50)
37 0.038(o.23)aa 0.076(o.45)a l.063(6.38) 0.264(l.59)
7o 0.036(o.21)aa o.oe4(o.50)a 1.052(6.20) o.249(1.47)

stggk 0.043(o.23)aa 0.071(o.39)a 0.946(5.l8) 0.287(1.56)

 

1

Average organ weight in grams and in parentheses, percentage of
body weight with statistical comparison of the latter.

3Significantly higher than the least value (pi¢»0.01).

aaSignificantly higher than the least value (p < 0.001).

24

Table 4

Zinc analysis of the organs of the six week old A/J mouse

 

Organ Zinc analysis
ppm wet weight of tissue

 

Liver 28
Kidney 20
Brain 15
Heart 20
Lung 21
Spleen ' 21

Thymus 22

 

25

or the requirement of the various subpopulations of cells in the thymus
for zinc.

The Effects of Zinc Deficiency on the Humoral Response of the Youpg Adult
A/J Mouse

 

The atrophy of the thymus observed in the zinc deficient rat (8),
pig (4), and mouse suggested that there might be impairment or destruction
of T cells that are important to humoral and cell mediated immune
responses. However, despite this evidence, no previous studies have
attempted to investigate the effects of zinc deficiency on the immune
response. In this thesis, the effects of zinc deficiency on the immune
response were examined in the young adult A/J mouse. The young adult
mouse was chosen as the subject of these studies since the effects of
zinc deficiency would be less severe and therefore would be easier to
define in a mouse with a fully developed immune system. The humoral response
of the zinc deficient young adult was evaluated by measuring the response
of these animals following injection of KLH-ars, a hapten-carrier conjugate,
and sheep red blood cells, both of which are dependent on the function
of T helper cells.

The response of the young adult A/J mouse to KLH-ars. In this
experiment, the serum of mice fed diets containing different levels of
zinc was assayed for antibody formation following injection of the
hapten-carrier conjugate KLH-ars, in which arsanilic acid groups were
coupled to keyhole limpet hemocyanin,a very antigenic protein in the
mouse.

Six week old adult mice were fed diets containing 0.8, 4 or 69
ppm zinc. The three groups represented zinc deficient, marginally de-
ficient and zinc adequate diets respectively. Each mouse was injected

on day 0 with 500lug of KLH-ars intraperitoneally in Freund's complete

26

adjuvant, fbllowed by a second and third injection of 250/ug of KLH-ars
in Freund's incomplete adjuvant at ten day intervals. Weights and
dietary intakes were monitored throughout the study. One week after
the final injection, the mice were anesthetized with ether and bled

out by severing of the subclavian artery. The mice were then killed

by cervical dislocation and the kidney, spleen, jiver3 and thymus were
removed and weighed.

The body and organ weight data from this experiment is presented
in Table 5. The adult mice on the deficient diet weighed approximately
six grams less than the controls at the end of the experiment. As expect-
ed, the organ weights of the deficient mice were significantly lower than
the weights of the same organ in the zinc supplemented groups. However,
when organ weights were determined relative to individual body weights,
only the thymus showed significant atrophy in the deficient mice. The
thymus was reduced to one fifth that of the control animals, whereas the
spleen, liver, and kidney remained a constant percentage of body weight
in all of the dietary groups. Thus the effect of zinc deficiency on the
thymus of the adult mouse was similar to that observed in the rat (8) and
pig (4).

The response of the A/J mouse to KLH-ars was determined by sensitive
radioimmunoassays which were developed to quantitate the levels of anti-
body in the serum of each mouse to both KLH, the Carrier protein, and to
arsanilic acid, by coupling the hapten to a carrier that did not cross-
react with KLH.

D The protocol of the individual RIA was as follows. The desired amount
of serum from the mouse to be tested was added to a 0.3 ml volume of

borate saline buffer with sufficient A/J normal mouse serum to make the

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28

total volume of serum 15/11. To each tube was also added 0.01pg of
either 1251 labelled KLH, to test for anti-KLH antibody, or 0.011ug

of 1251 BGG-ars, in which arsanilic acid was coupled to 866, a

protein that did not cross-react with KLH, to assay for anti-arsanilic
acid antibody. This reaction mixture was incubated for an hour at
37°C. A slight excess of rabbit anti-mouse IgG was then added to

each tube to cause complete precipitation of the mouse IgG, followed
by another incubation for one hour at 37°C. The assay tubes were left
overnight at 4°C. The resulting pellets were washed three times in
borate-saline buffer and dissolved in l N sodium hydroxide. The super~
natant and pellet washes were pooled. Both the supernatant and pellet

125I in a Beckman Biogamma Counter.

were counted for
Following subtraction of background counts, the counts in the
supernatant and pellet were added and the percentage of the total counts
that were in the pellet was calculated. Blanks representing non-specific
precipitation of antigen, which were never greater than 7%, were sub-
tracted from this value to obtain the percentage of labelled antigen
specifically bound to the antibody. The antigen binding capacity of
the serum of each mouse was established by incubating 0.01‘pg of either
125I—KLH or 1251 BGG-ars with increasing dilutions of serum. The
percentage of labelled antigen bound to antibody was plotted against
the volume of serum assayed. Titer for each mouse was defined as
the amount of serum required to precipitate 30% of the labelled anti-
gen, midway in the binding curve.
The results of the radioimmunoassays for the mice injected with

KLH-ars are presented in Table 6. The titer of the mice to arsonate

was essentially identical in all of the groups. The response of the

29

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30

mice to the carrier protein KLH showed a slight depression of the
titer, although not statistically significant, in the zinc deficient
mice.

These results indicated that despite the marked atrophy of thymic
tissue, the deficient mice were able to respond to antigenic stimulus
by KLH-ars. The slightly reduced titer to the carrier protein KLH,

a response which has been shown by Mitchison and others (44) to be more
dependent on helper T cells than the response to hapten, suggested the
possibility that T cell helper function was slightly impaired in the
deficient mice.

However, since the immunization program in this experiment was
begun on day 0 when a healthyT cell helper population was present, an
adequate response was generated in the deficient mice apparently because
the B cells were able to continue to proliferate and produce antibody.
once they were stimulated by antigen. This occurred despite the develop-
ment of zinc deficiency and thymic atrophy and indicated that in the
young adult the B cells were not severely affected by zinc deficiency
for the period of time studied. This point will be demonstrated more
clearly by a subsequent reconstitution experiment.

It could be argued that the reduced weight of the thymus in this
experiment could be attributed to growth interruption as a result of
reduced intake in the deficient animals and not zinc deficiency. Although
the deficient mice in this experiment consumed, on the average, less
of the diet per day than the mice in the other dietary groups (Table 7),
their intake was approximately 70% of the controls, indicating that
they were not starving. Moreover, the wasting of the thymus reported
by Miller (4) in the zinc deficient pig was absent in the pair fed

controls.

31

Table 7

Dietary intake of adult A/J mice fed diets supplemented with different
levels of zinc for four weeks

 

 

 

Dietary zinc level Average total diet consumed, g
Zinc deficient 37.2 13.61
0.8 ppm
Marginal Zinc 52.4 :23a
4 ppm
Adequate Zinc 53.7 111.9“:1
69 ppm
1

Mean :_standard error of the mean.

aSignificantly greater than the least value (p < 0.05).

32

The primary response of the young adult A/J mouse to SRBC. Since

 

zinc deficiency had no severe effects on the humoral response of the
mouse to an antigen injected when a healthy population of T cells was
present, it was of interest to determine the effects of zinc deficiency
on the primary response of the mouse to a more T cell dependent antigen
such as sheep red blood cells, after the thymus had become totally or
partially atrophied.

Six week old mice were fed either zinc deficient diets (0.5 ppm)
or zinc adequate diets (25 ppm). After three weeks on the diet, when
the thymuses of the deficient mice were 40% of the controls, all of
the mice were injected intraperitoneally with l X 108 SRBC. Seven days
after immunization, the mice were anesthetized, spleens were removed and
assayed for plasma cells producing anti-SRBC antibody by the Jerne
plaque assay (42).

The results of this experiment are presented in Table 8. The
number of direct plaques, representing IgM forming plasma cells, was
reduced to approximately half in the zinc deficient mice, although this
difference was not statistically significant. However, the number of
IgG forming plasma cells elicited by the zinc deficient mice was one
seventh of the number of plaques produced by the positive controls, a
difference which was highly significant.

These results indicated that the IgM response, which is relatively
T independent, was similar in the zinc deficient and control mice. How-
ever, it was in the switch from IgM to 196 antibody fbrmation, a process
more dependent on T cell-B cell cooperation (45), that impairment of the
immune response was detected. Hence the decrease in IgG antibody forma-

tion of the zinc deficient mouse following a single injection of SRBC

33

Table 8

Primary response of Ala mice to sheep red blood cells

 

 

Dietary zinc level Average number of plaque forming cells
per spleen
IgM plaques IgG plaques
Zinc deficient 2,286 1 4031 3,861 1 839
0.5 ppm
Zinc adequate 4,058 1 684 21,952 1 3110a
25 ppm

 

Mean :_standard error of the mean.

aSignificantly greater than the least value (p‘< 0.05).

34

suggested an impairment or destruction of helper T cells. A similar
reduction of IgG plaque formation in response to SRBC has been reported
in mice depleted in T cells due to either thymectomy (46) or congenital

absente of the thymus (47).

 

The secondary response of the young_adult A/J mouse to SRBC.

Since zinc deficiency had produced a severe impairment of the primary
196 response to SRBC in the A/J mouse, the effects of zinc deficiency
were also examined on the secondary response of the A/J mouse to SRBC.
Since the antibodies formed in the secondary response are largely of
the IgG class, this response was therefore a more rigid test of T cell
helper function. In addition, to confirm the hypothesis that the re-
duced response to SRBC was a consequence of impairment or destruction of
helper T cells, while B cell function remained relatively intact, part
of the zinc deficient mice were reconstituted with syngeneic thymocytes
prior to immunization with SRBC and their plaquing response compared

to control animals.

Six week old adult mice were fed diets containing 0.5, 2 or 25 ppm
zinc for 39 days during which intakes were monitored daily and weights
were taken weekly. These three groups represented zinc deficient,
marginally deficient and zinc adequate populations. After 27 days on
these diets, half of the mice on the deficient diet received an intra-
venous injection of 6 X 107 thymocytes, prepared from healthy A/J donors
of identical age. The fOllowing day, all of the mice were injected
intraperitoneally with l X 108 SRBC, followed by a second injection of
108 cells one week later. Five days after the final injection, on day
39, the mice were anesthetized, organs were removed and weighed and

the spleens were prepared for the Jerne plaque assay (42).

35

The body weights, thymus weights, and diet consumption of the

- mice in this experiment are presented in Table 9. Both zinc deficient
groups lost weight while the mice on the marginal and zinc adequate
diets gained similar amounts of weight in the same time period. The
identical final body weights and the fact that both the marginally
deficient and zinc adequate groups consumed the same amount of diet in
the 39 days of the experiment indicated that the marginally deficient
mice, although consuming a diet deficient in zinc, were not suffering
from inanition. The zinc deficient groups consumed on the average 80%
of the amount of the diet eaten by the zinc supplemented groups and had
essentially no thymus remaining at the end of the experiment. The margin-
ally deficient mice had thymuses of approximately half the size of the
positive controls, indicating that these mice were zinc deficient.

The results of this experiment are presented in Table 10. The
zinc deficient animals produced 6,000 indirect (IgG) plaques, one tenth
the number produced by the positive controls. This indicated a further
reduction of immunity in the zinc deficient mice in the secondary response
to SRBC. Since the majority of the antibodies formed in the secondary
response were of the IgG class, and therefore, more dependent on the
presence of helper T cells, the ten fold reduction of plaque formation in
the secondary response to antigen was additional evidence for T cell
helper impairment in zinc deficiency.

However, in order to confirm that T cells were the prinicipal cell
type affected by zinc deficiency, half of the zinc deficient mice were
injected intravenously with 6 X 107 viable thymocytes, the cellular
equivalent of a thymus, from healthy A/J donors prior to immunization with

SRBC. The reconstituted mice formed 37,000 plaques which were significantly

36

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Table 10

Secondary response of A/J mice to sheep red blood cells

 

 

Dietary zinc level Average number of plaque
’ forming cells per spleen
IgG
Zinc deficient 6,180 1 6601
0.5 ppm
Marginal zinc 15,380 1_1840
2 ppm
Adequate zinc 61,490 1 5900al
25 ppm
Zinc deficient 37,100 1 6840M
0.5 ppm

recons ituted with
6 X 10 thymocytes

 

Mean 1_standard error of the mean.

aSignificantly greater than all other values (pic 0.01).

aaSignificantly greater than the least two values (prt 0.01).

38

higher than the plaques formed by the deficient mice and 60% of the

number of plaques produced by the positive controls. Since one seldom
fully restores the immune response by reconstitution (48), the significant-
ly increased plaque forming response of the deficient mice following in-
jection of T cells indicated that the T cell was the primary cell type
affected by zinc deficiency. Although a slight impairment of B cells

in zinc deficiency cannot be ruled out by this experiment, the data
indicated that the B cell was able to proliferate and produce antibody
despite 39 days on a deficient diet.

A/J mice fed a diet marginal in zinc produced an average of 15,000
plaques, one fourth of the response of the control mice. This suggested
that reduced T cell helper function also occurred in this dietary group.
Thus marginal zinc deficiency states also produced serious consequences
in the development of an adequate immune response. The fact that this
group of mice had a reduced plaque forming ability despite the same
final body weight and daily intake as the zinc adequate group, indicated
very clearly that zinc deficiency and not inanition was responsible for

the drop in immunity.

SUMMARY AND CONCLUSIONS

The purpose of this thesis was to investigate the effects of
dietary zinc deficiency on the humoral response of the young adult A/J
mouse. These studies were the beginning of a series of investigations
to explore the relationship between malnutrition and immunity by
studying the effects of a single nutritional deficiency on the immune
response of a well-defined population.

In summary, dietary zinc deficiency was found to produce a rapid
atrophy of the thymus and a significant depression of the humoral immune
response of the young adult mouse to T dependent antigens. The decreased
number of indirect (IgG) plaques in both the primary and secondary responses
of the deficient mice to SRBC, a response that is dependent on T cell-B
cell interaction, suggested that the reduced number of plaques was due
to impairment or destruction of T cell helper function. This interpreta-
tion was supported by the fact that significant restoration of the response
to SRBC could be achieved by the injection of syngeneic thymocytes
into the deficient mice prior to injection of antigen. 0f additional
significance was the finding that the marginally deficient mice also had.
a reduced plaque forming ability, indicating that these mice had a reduced
1 cell helper activity.

8 cell function appeared to be relatively unaffected in the zinc
deficient adult mouse. This was indicated by the similar antibody titers

of the zinc deficient, marginally deficient and control mice to KLH-ars

when the antigen was injected when a healthy population of helper T cells

39

40

was present. The significant restoration of the depressed response
of the deficient mice to SRBC following injection of thymocytes also
showed that B cells were able to proliferate and produce antibody
despite 39 days on a deficient diet. Thus zinc deficiency appeared to
depress the immune response of the young adult mouse by impairment or
destruction of T cell helper function.

The reduced intakes of the mice on the zinc deficient diet suggested
that the wasting of the thymus and the depression of the humoral response
could be due in part to inanition in the deficient mice and not zinc
deficiency. However, the reduced intake of the deficient animals was
not unexpected since anorexia is common in zinc deprived animals (4).

In addition, the development of thymic atrophy and the four-fold
reduction in plaque forming ability in marginally deficient mice despite
the similar intakes of the marginally deficient animals and the zinc
adequate controls over a 39 day period was evidence that these severe
effects on the immune response were due to zinc deficiency and not simply
to a reduced diet consumption. Moreover, recent experiments with

pair fed animals have shown that over a 39 day period, inanition accounted
for only 25% of the total drop in immunity in the zinc deficient mice
(49). Therefore, the detrimental effects of dietary zinc deficiency

on the humoral immune response can be largely attributed to a lack of
zinc and not starvation.

These results linking zinc deficiency and decreased immune function
have important significance forpopulations like those in the Middle East
who are zinc deficient due to the ingestion of diets composed of cereal
grains. Diets of animal protein, rich in available zinc, are economically
inefficient and will not be able to adequately feed an expanding world

population. Therefore, in the future, larger proportions of the world's

41

population will rely on cereal grains as a primary foodstuff and thus
the possibility of decreased immune function as a result of zinc
deficiency may be increased. Furthermore, the finding that marginal
zinc deficiency also causes a decrease in immune response has important
implications for those individuals in our own country such as trauma
patients, patients with liver disease, alcoholics, pregnant women and
women taking oral contraceptives whose decreased plasma zinc levels are
indicative of marginal zinc deficiency (50).

The possibility of a depressed immune response in these zinc
deficient people and the observed depression of immunity in mice in this
thesis has stressed the importance of future investigations to expand our
knowledge of the relationship between zinc and immunity. Information is
lacking on the effects of zinc deficiency on other immunological para-
meters such as delayed type hypersensitivity and graft rejection in the
adult mouse as well as complete immunological studies on the effects of
zinc deficiency in the neonatal and weanling mouse. The effects of zinc
deficiency on the young mouse may or may not be similar to those observed
in this thesis for the adult. Also unknown are the long range effects of
zinc deficiency and the consequences of short periods of deficiency as
well as the reversibility of these effects.’ Examination of the histology
of the thymus as it begins to atrophy due to zinc deficiency and how this
relates to wasting as a result of age, injury, or stress will also aid
in clarifying the role of zinc in immunity. 1

Although the experiments in this thesis have explored the effects
of zinc deficiency on immunity, they have not attempted to determine
how zinc exerts its detrimental effects on the lymphoid cell. Therefore,

' future experiments are also needed to understand the mechanism of this

42

immune depression. The demonstration that the zinc content of the thymus
was similar to that of other organs in the mouse indicated that the
requirement of the thymus for zinc was not unusual. However, zinc
depletion could cause a decrease in specific activity of critical zinc
metalloenzymes in the thymus and thus affect lymphoid cell function.
Thymic hormones, responsible for maturation of T cells and other
immunological parameters, may also depend on the presence of zinc for
activity. Hence the wasting effect of zinc deficiency on the thymus
and its impairment or destruction of helper T cells, may be due to a
greater sensitivity of biochemical processes in the thymus to zinc
depletion than other organs.

Zinc deficiency could also produce deleterious effects on the
immune system by affecting the levels of adrenocorticosteroid hormones
in the adult mouse. Although no studies have been done in this area,
the enlargement of the adrenal glands in zinc deficient rat (8) and
pig (4) indicated that there may be a relationship between zinc
deficiency and adrenocorticosteroid metabolism. Some of the effects
of the adrenocorticosteroid hormone cortisone on the immune system,
studied by the injection of cortisone acetate in experimental animal
models, were thymic atrophy (51) and a depression of IgG formation in
the Jerne plaque assay (52). These effects were similar to those
produced by experimental zinc deficiency in the adult mouse. There-
fore, these studies suggested a possible role of adrenocorticosteroid
hormones in the immunological effects seen in the zinc deficient mouse.

These investigations of zinc deficiency on immunity have indicated
the severity of the immunological effects that can be caused by a
single nutritional deficiency. Although these studies have only

emphasized a deficiency of zinc, the results suggested that other

43

nutritional inadequacies could cause equally significant impairment

of the immune response. Thus, these results have important implications
for future research into the bodily responses to malnutrition. This
emphasizes the need for continuing studies to clarify the role of

nutritional elements in immunity.

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