-1-
LYMPHOCYTE TRANSFORMATIONS AND IMMUNOCOMPETENCE
IN THE PROTEIN-CALORIE MALNOURISHED RAT
by
Gwendolyn C. Murphy
B.A.,
Oberlin College
(1974)
SUBMITTED TO THE DEPARTMENT OF NUTRITION AND FOOD SCIENCE
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF
MASTER OF SCIENCE
at the
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
June,
Signature of Author
1976
/
r
Department cf Nutrition aA
Fuod Science
May 7, 1976
Certified by
Thesis Supervisor
Accepted by
.
.
Chairman, Debartmental Committee on Theses
ARCHIVES
JUL 12 1976
~N~1aa,
-2-
LYMPHOCYTE TRANSFORMATIONS AND IMMUNOCOMPETENCE
IN THE PROTEIN-CALORIE MALNOURISHED RAT
by
Gwendolyn C. Murphy
Submitted to the Department of Nutrition and Food Science
on May 7, 1976, in partial fulfillment of the
requirements for the degree of Master of Science
ABSTRACT
Infection is often observed among children fed low
protein and high carbohydrate diets.
Similarities between
infections of such children and children with congenital
immune deficiency suggest that protein deficient individuals
have a defective immune system.
The present studies were undertaken in order to determine
the immunocompetency of an animal population whose only
dietary restriction was a low concentration of protein.
The animal model was designed to mimic as closely as possible the conditions found within malnourished populations.
Rats were chosen as the animal model because rat nutrition
is well known, and is similar to human's in its protein
metabolism. We used outbred rats from mothers who had been
on protein-deficient diets during the last two-thirds of
gestation
and all of the lactation period.
The well
nourished infant rats were then maintained on an 18% protein
diet, and the malnourished rats were divided into two groups,
one maintained on 6% protein and the other refed with an
18% protein diet. The technique of lymphocyte transformation was selected as the most direct way to measure cellmediated defense.
The thymus and spleen were used because
they are sites of aggregated lymphoid cells.
It was found that maternal protein deprivation from
one week post-conception until three weeks post partum
resulted in decreased offspring body weight, lymphoid
organ weight, and lymphoid tissue cell number. By six weeks
of age the percentage of body weight represented by the
spleen or thymus was not significantly different in the
malnourished group compared to control animals; however, the
organ weights were still subnormal.
No statistically
significant differences were found in lymphocyte transformations between the dietary groups at either three or six
weeks of age, but, because of reduced cell numbers per
lymphoid organ, it was concluded that the transformation
-3-
ability of the entire organ was deficient in the malnourished
animals, particularly at three weeks of age.
The present studies, although inconclusive, show that
the lesion which occurs in the immune function of protein-
calorie malnourished individuals may be due not to a defect
in the lymphoid cell response, but rather to a reduced
capacity of the lymphoid organs to mount an immune response.
Thesis Supervisor:
Title:
Robert Suskind, M.D.
Associate Professor
of Pediatrics and
Clinical Nutrition
-4-
D
I
E D I C A T IO
would like to dedicate this
N
work to my parents
whose confidence in me has helped me in each step of my
education.
-5-
ACKNOWLEDGEMENTS
I would like to thank Dr. Robert M.
Nevin S.
Scrimshaw and Dr. Sanford A.
Suskind, Dr.
Miller for their
guidance, support and unfailing encouragement.
I would
also like to thank Richard Marshall, Mindy Sherman and
Rusty Murray for the patient instruction and advice they
gave me;
and David Mark, Jeff Bernstein, and Ann Hart for
their friendship and suggestions.
Thanks go to everyone
at the Clinical Research Center for their daily assistance
and to Linda Boyar for her help in the preparation of this
thesis.
And a special thanks goes to my husband, Lloyd
Michener, for his moral support and encouragement during
the execution of this research and for his enthusiasm and
time during the writing and editing of this thesis.
-6-
TABLE OF CONTENTS
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DEDICATION .
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ACKNOWLEDGEMENTS
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ABSTRACT
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TABLE OF CONTENTS
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LIST OF FIGURES
LIST OF TABLES
I.
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INTRODUCTION .
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Review of Immunology and Recent Advances
II.
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LITERATURE SURVEY
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Influences on Resistance to Infection
B.
Immune Responses in Vitamin or Mineral
C.
D.
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B.
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Protein Deficiency
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1.
Humoral Immunity .
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2.
Phagocytic Function
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3.
Cell-Mediated Immunity .
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Experimental Animals
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1.
Source, Housing and Diet .
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2.
Gestation, Birth and Lactation
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3.
Selection and Grouping of Pups
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Conclusion .
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MATERIALS AND METHODS
A.
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A.
Deficiencies .
III.
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1.
Sacrifice of Animals .
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2.
Preparation of the Spleen and Thymus
Cells
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Experimental Procedures
3.
Mitogen Preparation
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Page
C.
D.
E.
IV.
V.
VI.
VII.
Lymphocyte Procedures
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1.
Lymphocyte Transformation Cultures
2.
Labeling and Harvesting
Serum Determinations
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1.
Serum Preparation
2.
Total Protein
3.
Albumin Determinations
4.
Electrophoresis of Serum Proteins
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Statistical Analysis
RESULTS
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A.
Nutritional Status and Weight Gain
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B.
Lymphoid Tissue Involution .
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C.
Serum Determinations
D.
Lymphocyte Transformations
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DISCUSSION . . . . . . . . . . . . . . . . . . . . 61
CONCLUSIONS
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SUGGESTIONS FOR FUTURE RESEARCH
BIBLIOGRAPHY
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LIST OF FIGURES
FIGURE NO.
1
2
3
4
Title
Page
Time Course of Events in Lymphocyte
Transformations . . . . . . . . . .
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Diagrammatic Representation of
Experimental Design . . . . . .
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Mother's Growth Charts for the
Four Shipments of Rats
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Growth Charts of the Infants Selected
From Each Shipment
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LIST OF TABLES
Title
TABLE NO.
I
Page
Antibody Classes and Activities
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16
(Lymphokines)
II
Chemical Mediators
III
Diet Composition
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IV
Vitamin Content of Diet .
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Weight and Cell Yield of Spleen and
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Thymus in Six Week Animals
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Serum Determinations for Three Week
Animals . . . . . . . . . . . . . .
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Serum Determinations for Six Week
Animals . . . . . . . . . . . . .
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Lymphocyte Transformations of Thymus Cells
From Three Week Rats
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Lymphocyte Transformations of Spleen Cells
from Six Week Rats
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54
V
VII
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Composition of Harper's Salt Mix
Weight and Cell Yield of Spleen and
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Thymus in Three Week Animals
VII
VIII
IX
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XII
XIII
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Lymphocyte Transformations of Spleen
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Cells From Three Week Rats
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Lymphocyte Transformations of Thymus Cells
from Six Week Rats
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XIV
Basal Metabolism of Lymphoid Organ
XV
Stimulation Capacity of Lymphoid Organ
-10-
I.
INTRODUCTION
Throughout history man has noted the correlation between
nutritional status and susceptibility to disease.
It has
long been recognized that famine and disease occur together
and each seems to increase the severity of the other.
In
recent times observers have conducted more systematic
studies within human populations to show that infections
are directly and causally related to nutritional deficiency
states
(Warner and Winterton, 1935;
Phillips, 1968).
In
addition to these human studies there are numerous reports
in the animal literature showing a relationship between
nutrition and susceptibility
(Watson, 1937; Guggenheim
and Buechler, 1948; Schaedler and Dubos, 1958, 1959;
Cooper, 1974).
Most of this animal research done before 1968 was
restricted to external observation of the clinical manifestations of infection or inflammation.
We see experi-
ments of the 1930's in which animals on different diets
were exposed to a pathogen and then watched for signs of
morbidity or mortality.
Gradually techniques have become
more sophisticated and, at present, researchers, using new
theoretical knowledge of immunology, are designing in vitro
techniques which determine the specific lesion in the immune
process.
work
Research of 1930 is exemplified by Watson's
(1937),
diets.
in which mice were fed
"natural" or "synthetic"
The natural diets contained whole oats
(N1 ), coarse
-11-
ground oats supplemented with cod liver oil, yeastrel, and
bran
(N2 ) ,
or coarse ground oats supplemented with dry milk
and salt mix
(N5 ).
His synthetic diets consisted of dextrin,
salt, lard, oil, yeast, and bran, supplemented with gluten
(S
),
caseinogen
(S2 ), or gluten and caseinogen
(S3).
After a period of time on the diet, each animal was injected
with live bacteria or with the toxin produced by the bacteria.
Watson measured resistance by counting the number of mice
surviving on day twenty-eight.
He concluded that those
animals on a mixed natural diet
(N1 , N 2 , N 5 ) had higher
resistance.
Between 1930 and 1935 Heidelberger and Kendall
developed the precipitin reaction to measure antibody
quantitatively
(Roitt, 1974).
Experiments of the 1940's
and 1950's used this technique to measure the antibody levels
and bactericidal power of the peritoneal fluid of rats fed
control or deficient diets and who had been injected intraperitoneally with SaZmonella typhimurium.
Buechler
Guggenheim and
(1948) found that as protein quantity and/or quality
decreases,
there is a reduction in leukocytes, phagocytes,
and bactericidal properties.
By the mid 1950's La Via et al.
(1956) were using radioactively labeled anitgen to trace
the rate of phagocytosis, excretion of label, and distribution of labeled antigen in different tissues in normal and
protein-deficient rats.
At this time investigators also
became interested in such questions as the optimal quantity
of protein as well as the quality or amino acid composition
-12-
required for resistance.
Schaedler and Dubos
(1956, 1958,
1959) looked at protein combinations and survival while
Trnka
(1956) examined protein excess and deficiency and its
effect on antibody titer and resistance to Salmonella paratyphi
B in mice.
The 1960's saw an increase in use of RNA/DNA
measurements.
Squibb et al.
(1961) measured this ratio
in chicks who had been challenged with Newcastle's disease.
He found that there was no change in controls on 25%
soya
protein, but there was an increase in the severity of the
disease on a low protein intake
intake
(41%).
(8%),
and on an excess protein
Before considering the recent advances in
this research, we must first understand the developments
that have taken place in the field of immunology.
Review of Immunology and Recent Advances
The immune system is made up of the lymphoid organs
(thymus, lymph nodes, spleen and tonsils),
a variety of
lymphocyte aggregates distributed throughout the body, and
the lymphocytes of the blood and lymph.
serve a two-fold purpose:
The lymphocytes
(1) to provide communication
between the lymphoid cells in different parts of the body,
and
(2) to dispose of foreign invaders directly, indirectly,
or in conjunction with the phagocytic macrophages.
Lymphocytes develop from bone marrow stem cells which
originate from the embryonic mesenchyme.
may undergo differentiation.
These stem cells
This maturation of lymphocytes
was first described in the bird for "T-cells" which
-13-
differentiated through the thymus, and for
"B-cells" which
differentiated in the bursa of Fabricius.
In mammals
we also find both T-and B-cells, but the mammalian analog
for the bursa-dependent differentiation is unknown.
The
resulting T-and B-cells enter the blood and lymph and become
the major cells of the lymph nodes, spleen and tonsils.
It is in these organs that lymphocytes undergo anitgendependent proliferation and differentiation.
Antigens,
which are usually proteins but which may be polysaccharides
or nucleic acids, are defined as materials which bear surface
configurations capable of eliciting an immune response
when they invade an organism.
When a lymphocyte first
encounters a foreign substance or antigen for which it has
membrane receptors, it is stimulated to undergo a series of
biochemical changes which result in proliferation of and
differentiation into effector and memory cells.
Effector
cells are active in antigen disposal while memory cells
revert to a resting state but are changed in that they are
now able to initiate an immune response with greater efficiency
upon their next encounter with their specific antigen.
Disposal of antigen comes about by two different mechanisms.
The first, called the humoral system, is mediated by trans-
formed B-cells, called plasma cells, which respond to antigens
by synthesizing antibodies.
Antibodies, also called immuno-
globulins,are found in the globulin fraction of the blood,
and are divided into five different classes:
IgG, IgM,
-14-
IgA, IgD, and IgE.
Within each class there are numerous
specificities to match the wide range of antigens to
which an individual can become sensitized.
Antibodies act on foreign invaders in four different
They may bind with the invading antigen and inhibit
ways.
its entry into the body's tissue;
this is important if the
antigenic determinant is part of a virus.
neutralize the antigen if it is a toxin.
They also may
The antibody may,
with the help of the complement system, coat the outside
of an invading bacteria
(a process called
opsinization)
thereby enhancing the ability of the phagocytes to remove
and destroy the antigen.
Finally, the antigen-antibody
complex may stimulate a complicated series of interactions
of blood constituents, called the complement cascade, leading
to lysis of the foreign cell.
See Table I for specific
activities of each antibody class.
The other system for antigen disposal is called the
cell-mediated immune system.
The effector cells are
sensitized T-lymphocytes, also called cytotoxic lymphocytes,
which impair antigen viability either by attacking the invader
directly or by releasing chemical mediators--lymphotoxin,
macrophage activation factor
factor
(MIF).
(MAF), and migration inhibition
Lymphotoxin causes cell lysis;
MAF stimulates
phagocytosis by macrophages; and MIF immobilizes actively
circulating macrophages leading to an accumulation of these
cells at the antigen site.
of mediators.
See Table II for a complete list
-15TABLE I
ANTIBODY CLASSES AND ACTIVITIES
Antibody
class
Ig A
Activities beneficial
to the host
Activities destructive
to the host
Toxin neutralization
Agglutination
Secretory
Ig A
Neutralization of
virus infectivity for
epithelial cells
Bacteria agglutination
Interference of
bacterial attachment
to mucosa
Enhanced phagocytosis
by monocytes
Toxin neutralization
Ig D
Ig E
Ig G
Mediation of vascular
Local and systemic
permeability changes
on antigen recognition
anaphylactic reactions
Immune aggregate-
Bacterial toxin
neutralization
mediated tissue injury
Bacteria agglutination
Arthus reaction
Opsinization
Serum sickness
Bacteriolysis
(with aid
of Complement)
Ig M
Same as Ig G
Same as Ig G
-16-
TABLE II
CHEMICAL MEDIATORS
(LYMPHOKINES)
A.
Mediators Affecting Macrophages
Migration inhibitory factor
(MIF)
Macrophage activating factor
(MAF)
Macrophage aggregation factor
Factor which causes disappearance of macrophages
from the peritoneum
Chemotactic factor for macrophages
Factor which alters surface tension of
macrophages
Antigen-dependent MIF
B.
Mediators Affecting Neutrophil Leukocytes
Chemotactic factor
Leukocyte inhibitory factor (LIF)
C.
Mediators Affecting Lymphocytes
Mitogenic factors
Factors enhancing antibody formation
Factors suppressing antibody formation
D. Mediators Affecting Eosinophils
Chemotactic Factor
Eosinophil stimulation promoter
E. Mediators Affecting Basophils
Chemotactic factor
Chemotactic augmenting factor
F. Mediators Affecting Other Cells
Cytotoxic factors--lymphotoxin
(LT)
Growth inhibitory factors
Osteoclastic factor
(OAF)
Collagen producing factor
G.
Mediators Affecting Skin
Skin reactive factor
H. Other Mediators - Affecting All Parts of the Body
Interferon
Immunoglobulin-binding factor (IBF)
-17-
Macrophages are necessary for an efficient and effective
immune response.
They not only act to remove and digest
the major portion of the invading antigen, but they also
act in cooperation with the lymphocytes by presenting the
antigen to the lymphocyte although the mechanism for this
is unclear.
Furthermore, macrophages serve to remove those
antigens that have been rendered non-viable by cytotoxic
lymphocytes, antibody or activated complement.
Besides antigens, there is a number of substances,
called
mitogens, which activate lymphocytes, a process
also called "blast transformation".
The difference between
antigens and mitogens is that antigens stimulate only that
population of lymphocytes which have membrane receptors for
the antigenic surface determinants, while mitogens have the
capacity to bind to the surface of a much wider population
of lymphocytes.
Once bound to the cell membrane, the mitogen's
mode of action appears to be identical to that of an antigen.
Basically, transformation and differentiation follow
the path described in Figure 1.
About fifteen minutes after
the cell and mitogen come into contact, there is enhanced
endocytosis and increased cell permeability.
At thirty
minutes there is a rise in RNA synthesis followed by enlargement of the nucleolus, increased protein synthesis and
decreased uridine nucleotides.
Between twenty-four and
thirty-six hours there are nuclear changes which lead to
mitosis and development of cytotoxicity.
With the develop-
ment of cytotoxicity there is protein synthesis of chemical
-18-
FIGURE 1
TIME COURSE OF EVENTS IN LYMPHOCYTE TRANSFORMATIONS
0
6
PHA + T-lymphocyte
enhanced endocytosis
increased
RNA synthesis
nucleolus enlarges
121
18
24
301
nucleolus becomes euchromatic
nucleolema visable in nucleolus
36
Cl)
0
cell volume increases
42
48
mitosis
54
H-
60
66
72
DNA synthesis and post-mitotic differentiation
78
Four-fold increase in volume
84
Increased polyribosomes, Golgi, and lysosomes
90
96
102
108
114
120
Active RNA and protein
synthesis
-19-
mediators
(lymphokines).
Beyond these chemical mediators,
stimulated lymphocytes may release a mitogenic factor which
DNA synthesis
stimulates DNA metabolism
(Maini, 1969).
peaks at about 72 hours.
By this time there has been a
Polyribosomes, golgi
four-fold increase in cell volume.
apparatus,
and lysosomes are increased, and post-mitotic
differentiation occurs
(Lucas, 1971, Raviola, 1975).
Binding alone will not cause transformation.
It seems
that saturation of the lymphocyte by some specific chemical
groups is required
(Raviola, 1975).
However, there is
confusion as to just what it is that triggers and controls
blast formation.
According to one theory
(Burger, 1973),
the membrane-bound proteases, which are responsible for
the agglutinating properties of mitogens, cause a cascade
of proteolytic reactions which are necessary for cell
growth.
Another theory pinpoints phosphorylation of nuclear
acidic proteins, which is enhanced by cyclic GMP and cholinergic agents and is inhibited by cyclic AMP and prostaglandin E1 , as one of the early events which precedes
gene activation
(Johnson, 1975).
Other studies demonstrate
that RNA synthesis may be the regulatory site.
One of the
differences found in stimulated as compared to unstimulated
lymphocytes is an increase in mRNA and an increase in one
or more of the ribosomal initiation factors necessary for
mRNA translation
(Ahern, 1974).
It is known that the rise
in RNA synthesis is preceded by a rise in DNA-dependent RNA
-20-
polymerase.
Cordycepin
(3 deoxyadenosine) is an adenosine
analog which inhibits mRNA but not rRNA or RNA polymerase.
Pogo found that the addition of cordycepin to lymphocyte
cultures prevents stimulation altogether.
This suggests
that mRNA is a necessary factor in transformation.
It
has also been suggested that the newly synthesized tRNA is
important in controlling protein synthesis and differentiation
although more data are needed to support this
Sagone
(Sharma, 1973).
(1974) has found that stimulated cells have an
increase in glycolysis compared to controls reaching a peak
at two days, and a rise in the hexose monophosphate shunt
which has about a five-fold increase again at two days.
Several serum factors have been found which enhance the
mitogenic response.
complement
Among these are inactivated serum
(Forsdyke, 1973),
transferrin bound iron
transferrin bound zinc and
(Tormey, 1972).
It has been
suggested that these work via enzymes or membrane receptors
(Phillips, 1975).
These recent advances provide the basis for the in
vitro tests which have been developed to measure immuno-
competence.
The capacity of B-lymphocytes to differentiate
into plasma cells is now measured by lymphocyte transformations.
The ability to synthesize specific immunoglobulins
is assayed by the Jerne Plaque Forming Assay
Indirect Plaque Forming Assay.
(PFA) and
B-cells can be counted
using rosette or immunofluorescent techniques.
To test
-21-
leukocyte function, one may count the number of these cells
in peripheral blood, and watch for a periodic variation in
the counts, which occurs in association with various infections.
To test the phagocytic function of both polymorphonuclear
leukocytes and macrophages, one measures the rate of phago-
cytosis and intracellular killing with and without endoA measurement of nitro-blue tetrazolium
toxin stimulation.
reduction gives an indication of the ability of phagocytes
to form peroxide, a bactericidal agent.
Inflammation
response can be measured by skin window techniques.
It is
also useful to measure serum complement components and their
activation capacity.
T-lymphocyte capacity can be measured
by cell counts using rosetting or immunofluorescent techniques.
Function is measured by lymphokine production
(LT, MIF, MAF),
cytotoxicity, and lymphocyte transformations to specific
antigens or non-specific mitogens
(Roitt, 1974, WHO Bulletin,
1972).
It is obvious that great strides have been made in
recent years in the development of laboratory measures of
immunocompetence.
These techniques enable investigators
to evaluate the status of the immune system under a wide
variety of pathological, environmental and dietary conditions.
-22-
II.
A.
LITERATURE SURVEY
Influences on Resistance to Infection
Determination of immunocompetence is multifactorial.
An individual's resistance to infection is determined not
only by his/her genetic makeup but also by environmental
conditions.
Age, sex, physical and pathological condition
Furthermore, the degree of virulence
influence susceptibility.
and the metabolic requirements of the invading agent help
to determine its survival in the host.
Because of these
factors, dietary intake may or may not be a major factor
in the disease process.
(1950),
According to experiments by Schneider
dietary intake is a significant influence when the
host is intermediate in genetic resistance and the pathogenic agent is intermediate in virulence.
In a natural
setting these conditions are more common than either
extreme
B.
(Scrimshaw et al.,
1968).
Immune Responses in Vitamin or Mineral Deficiencies
The effects of various dietary deficiencies have been
studied in relation to antibody production and to cell
mediated immunity.
The results of experiments on vitamin
deficiencies are highly variable
(for reviews in this area,
see Clausen, 1934; Scrimshaw, 1968; Axelrod, 1971, Worthington,
1974).
Mineral deficiencies have also been examined,
especially in relation to helminth infestation
et al.,
1968).
(Scrimshaw
A very active area right now is the inter-
-23-
action between iron deficiency and cell-mediated immunity
(Joynson, 1972; Kulapongs, 1974; MacDougll, 1975).
C.
Protein Deficiency
1.
Humoral Immunity -
Because of the observed increase
in infection in protein deficiency, both in human populations
and in experimental animal models, investigation of the
interaction of these two factors has been extensive.
Most investigators find an increase in severity of infection
with decreased inflammatory response
often an increase in mortality
1971).
(Phillips, 1968) and
(Smythe, 1959; Woodruff,
Consequently, humoral and cell-mediated systems
have been evaluated.
Antibody production, complement levels, and phago-
cytic cell activity have been studied.
Many reports show
that in severely protein-deficient animals who have no
signs of infection, there is nevertheless a decrease in
antibody formation and a reduction in circulating immunoglobulins
(Gautam, 1972;
Cooper, 1974).
However, most
studies using human populations with protein deficiency
show normal
(Geefheyser, 1971) or increased antibody levels
(Chandra, 1976).
There are several reports in the animal
literature of decreased plaque forming cells in proteindeficient rats or mice that had been injected previously
with sheep red blood cells
work
(Aschkenazy, 1972).
Kenny's
(1972) may help to explain some of the variation as
-24-
Clinical
being a consequence of dietary amino acid imbalance.
or subclinical infection may be a major influence on antibody
titer
(Chandra, 1972).
When antibody binds to its specific antigen the complement
pathway is activated.
This cascade reaction involves nine
complement components most of which have been studied in
patients or animals with protein-calorie malnutrition
(PCM).
Complement, and especially the third component of complement,
is reported to be decreased in PCM
(Neumann, 1975).
The third complement component, C 3 , is cleaved into two
fragments,
the first of which plays an important part in
It is chemotactic
inflammation.
leukocytes
for polymorphonuclear
(PMN) as well as a potent stimulus to mast cells
causing histamine release.
The second C 3 fragment speci-
fically binds both to the antigen-antibody complex and to
macrophages thereby facilitating phagocytosis.
Thus a
decrease in C 3 may explain a lack of inflammatory response
and a reduction in phagocytosis found in PCM.
2.
Phagocytic Function -
Seth
(1972) reports that
opsinization is slightly increased in PCM.
In protein
deprivation phagocytosis and bactericidal killing are reported
to be decreased
(Shousha, 1972; Hook, 1973),
1972; Douglas, 1974),
or increased
normal
(Lopez,
(Jose, 1971).
Bactericidal killing in PMN depends on the formation
of peroxide.
Selvaraj
(1972a, b)
has found that in leuko-
cyte granules of malnourished patients there is a reduction
-25-
in glycolysis and hexose monophosphate shunt
(HMS) activity
resulting in a decrease in NADPH oxidase, myeloperoxidase,
and lysosomal enzyme release.
these experiments
However, Douglas repeated
(1974) and reports no significant differences
between controls and kwashiorkor subjects in HMS.
A
decrease in the amount or activity of these enzymes would
explain a deficit in the peroxide production and thus a
reduction in bactericidal killing.
It is of interest whether PMN leukocytes and macrophages actually get to the site of foreign invasion.
One
way to test this is by the Rebuck skin window in which a
small area on the forearm is abraided and covered with
a glass slide.
The slide is changed about every two hours
and examined for both types of phagocytes.
Using this
technique in children with kwashiorkor, Freyre
(1973)
reports a high infiltration of PMN leukocytes throughout the
duration of the lesion, but a subnormal migration of macro-
phages to the wound.
Although these children were reported
to have no evidence of infection, there was probably some
degree of subclinical infection present in most.
results, together with Gautam's findings
These
(1972) that there is
a reduction in MIF production in mice on a
4% protein intake,
could indicate a reduction in lymphokine production by
T-lymphocytes, or a basic defect in the macrophage.
-26-
3.
Cell-Mediated Immunity
a.
T-Lymphocytes and The Thymus -
Chandra
(1974)
found that children with PCM had a decreased percentage of
T-lymphocytes to B-lymphocytes in the blood, as indicated
by spontaneous rosetting with sheep red blood cells
His results show 23% rosette forming cells
(SRBC).
(RFC) in PCM,
60% RFC in those who had been refed on a high protein diet
for six to sixteen weeks, and 71%
controls.
Ferguson
RFC in well-nourished
(1974) found that total peripheral
lymphocyte counts did not vary with nutritional status,
but RFC's were 59.7% in normal controls and 16.6% in severe
PCM.
From this he concludes that there is a reduction in
the absolute number of circulating T-lymphocytes, and an
increase in B-lymphocytes.
His studies also showed a
decrease in RFC's in low birthweight neonates
to 49.2%, whereas controls
(x = 1660 g)
(x = 2005 g) had 65.1% RFC.
This indicates that intrauterine nutrition and growth are
important in lymphocyte development.
In his classic study in which he autopsied
children who had died of kwashiorkor, Smythe
(1971) found
a decrease in tonsil size, atrophy of the thymus, wasting
of the peripheral lymphoid tissue, and in spleen and lymph
nodes, a depletion of the paracortical cells and a loss of
the germinal centers.
Chandra
(1972) confirmed Symthe's
observations of decreased tonsil size and found a general
lymphopenia in the subjects with PCM.
Studies on the
-27-
marasmic pig showed a decrease in germinal centers, and
an atrophy of the lymphoid organs with a marked decrease
in the size of the spleen and lymph nodes
Jose
(Lopez, 1972).
(1973 ) found a decreased percentage of T-cells in the
spleen, lymph nodes, and thymus of mice on a low protein
diet.
Aschkenazy
(1975) reports that rats on a no protein
diet had a decreased body, spleen, and lymph node weight
as well as a decreased tritium incorporation into these
organs upon peritoneal injection of tritiated thymidine.
This indicates that there was a decrease in cell division
of these cells in the unstimulated organ.
These changes
may be mediated by the high cortisone levels found in PCM
(Dougherty, 1952).
b.
Cytotoxicity -
Several investigators have tested
sensitized T-cells for cytotoxicity or cell mediated lysis.
Jose
(1972) found a decrease in cytotoxicity in mice on
10% protein diets, a normal response in mice on 5% protein
and a decrease in mice on 3% protein diets.
study Jose
(1973 )
In another
found a decrease in cell mediated lysis
in mice on 6% protein or mice on 6% protein but one-half
the normal calories.
He explains these findings as being
due to a suppression of CMI by blocking antibody at higher
but deficient protein intakes, a decrease in blocking antibody and thus derepression of CMI at intermediate levels of
deficiency and a lesion in the mechanisms of CMI itself
at the lowest protein intakes.
Lopez
(1972) has also found
-28-
cytotoxicity to be decreased or absent in his marasmic
pig model.
c.
Delayed Hypersensitivity -
Another way to assess
cell-mediated immunocompetency is by measurement of the
induration produced by delayed hypersensitivity to cutaneously
applied antigens.
In PCM there is a decreased response
found in nearly all subjects
Geefheyser
skin
(Edelman, 1973;
Neumann, 1975).
(1971) found that the most severe depression in
reaction was highly correlated with mortality among
the children he studied.
Cutaneous hypersensitivity returns
to normal after several weeks of refeeding
although as Edelman
(Ferguson, 1974),
(1973) points out it is still unclear
as to whether the defect is in the sensitization of lymphocytes,
the secondary recognition by sensitized lymphocytes,
or the development of an inflammatory response.
Both the
sensitization and inflammatory reactions probably involve
lymphocyte-macrophage interaction.
d.
Lymphocyte Transformation -
transformation
The lymphocyte
(LT) is an in vitro technique whereby
peripheral blood, lymphoid tissue suspension, or, more
often, isolated lymphocytes are cultured with mitogens or
antigens.
This stimulation results in blast transformation
and differentiation of the T- and B-lymphocytes into cytotoxic cells, and plasma cells respectively.
Some mitogens
stimulate T-cells preferentially, such as phytohemagglutinin
-29-
(PHA) and concanavallin A
polysaccharide
(Con A),
and some such as
(LPS) stimulate B-cells.
like pokeweed mitogen
Still other mitogens
(PWM) stimulate both T-
and B-cells.
Most work in this area has been done using PHA.
find LT to PHA is normal in PCM
Cooper
lipo-
While some
(L6pez, 1972; Ferguson, 1974),
(1974) shows an enhancement in LT in the spleen cells
from mice with chronic protein deprivation.
However, most
studies show that LT are decreased compared to well nourished
controls
(Geefheyser, 1971;
Neumann, 1975).
Smythe, 1971; Chandra, 1972;
Both Geefheyser and Chandra report an increase
in LT upon refeeding, but Coovadia
(1974) notes no change.
While Neumann found that the decrease in LT was proportional
to the severity of the deficiency, Coovadia's patients
showed no correlation between LT and the serum levels of
albumin and transferrin, which are measures of the deficiency
status.
There have been reports of a high correlation
between LT and skin tests
1972).
(Geefheyser, 1971;
Daguillard,
This is an important finding because the lymphocyte
transformation is an in vitro test which indicates the
competency of the lymphocyte to respond to stimulation in
vivo.
D.
Conclusion
As in many other areas, the lack of knowledge is apparent
by the conflicts in the literature.
Because not all of the
variables are known which affect the tests of immunocompetence,
-30-
there are often varying reports of the capabilities of
lymphocytes and phagocytes in different dietary states.
This is especially the case in protein deficiency, where the
depth of the deficiency is felt in nearly every cell in the
body.
As the in vitro tests are made more precise, and
as correlations are found with all the major variables,
it should be possible to form a coherent picture of the
lesions which occur in single and multiple deficiency states.
-31-
III.
A.
MATERIALS AND METHODS
Experimental Animals
Source, Housing and Diet -
1.
Timed-pregnant Sprague-
Dawley rats were obtained from Charles River Breeding
Laboratories
(Mystic, Massachusetts).
four shipments of rats
group
(A to E).
of gestation
There were a total of
(I to IV) with four to five in each
The animals were received after one week
(total gestation period = 22 days) and, upon
arrival, were randomly placed on a synthetic diet containing
either 6% protein or 18% protein.
Each mother was housed
individually in a plastic tub with a stainless steel wire
lid and a filter bonnet
(S-201 Filtex, Appleton Wire Company).
The cages were kept on racks in a temperaturehumidity-controlled
schedule.
(68-72*F) and
(50%) room with a twelve hour day/night
Diets were made up biweekly and half of each
batch was frozen for use during the alternate week.
compositions of both diets are listed in Tables
The
III-V.
The protein-deficient diet replaced 66% of the dietary casein
with 6%
dextrose and 6% dextrine.
The two diets had identical
quantities of oil, vitamins, minerals, methionine, agar, and
choline.
Rats were allowed both food and distilled water
ad libitum.
2.
Gestation, Birth and Lactation -
were fed experimental
The pregnant rats
(6% or 18%) diets during the last two
weeks of the gestation period.
Body weights were measured
-32-
TABLE III
DIET COMPOSITION
(per 1000 gm diet)
6%Protein
(gms)
Casein
18%Protein
(gms)
60
180
Dextrine
266
206
Dextrose
267
207
7
7
40
40
Corn Oil
150
150
Choline 2
10
10
Vitamin Mix 3
10
10
Agar
35
35
2000ml
2000ml
1.5ml
1. 5ml
Methionine
Harper's Salt'
Distilled water
Vitamin B 1 2 4
1
Rogers and Harper, 1965
(see Table V)
220% w/v solution.
3 Rogers
and Harper, 1965 (see Table IV)
42% w/v solution.
-33-
TABLE IV
VITAMIN CONTENT OF DIET'
mg/1000g diet
Thiamine
5.0
Riboflavin
5.0
Niacinamide
Pantotnenate
25.0
(Ca)
20.0
Pyridoxal
5.0
Folate
0.5
Menadione
0.5
Biotin
0.2
Ascorbic Acid
Vitamin E
50.0
400.0
Vitamin A
4000 Units
Vitamin D
400 Units
'Formula of Rogers and Harper, 1965.
Mixed with sucrose approximately 1:10
for premix.
-34-
TABLE V
COMPOSITION OF HARPER'S SALT MIX'
% Composition
Ammonium Molybdate [(NH4)6MO7 0 21H 20]
Calcium Carbonate [CaCO 3 ]
Cupric Sulfate
29.290
[Cu SO 4 ]
Calcium Phosphate
Ferric Citrate
0.0025
0.156
[Ca HPO 4
[Fe C 6 H 5 0 7
2H 2 0]
0.430
6H 2 0]
0.623
Magnesium Sulfate
[Mg SO 47H 20]
9.980
Manganese Sulfate
[Mn SO
0.121
Potassium Iodide
H 20]
[KI]
Potassium Phosphate
[KH
0.0005
2
PO4
Sodium Chloride
[Na Cl]
Sodium Selenite
[Na 2 SeO 3 '5H 2 0]
Zinc Chloride
1 Obtained
25.060
0.0015
0.020
complete from General Biochemicals, Chagrin
Falls, Ohio.
1965.
[Zn Cl 2 ]
34.310
Based on formula of Rogers and Harper,
"35-
The pregnant rats of each ship-
at weekly intervals.
ment delivered within 8-12 hours of each other.
When all
had completed giving birth and within eighteen hours of the
first birth, the offspring were randomized within each
dietary regime.
Each mother was given eight pups, and,
except on two occasions, the pups were accepted by the
mother as her own.
Mothers were kept on their respective
diets throughout the 21 day lactation period.
Selection and Grouping of Pups -
3.
At 21 days, one
litter from the well nourished 18% protein group
one litter from the malnourished
was selected for study.
6% protein group
(W) and
(M)
Selection was based on whether the
mother had successfully given birth and raised all eight
pups, and whether the litter seemed free from clinically
evident infection.
Three of the eight pups were sacrificed
at three weeks of age;
the remaining pups were housed in
stainless steel wire cages for an additional three-week
period.
The pups whose mother had been on the 18% protein
diet were maintained on this diet
(WW).
6% group were divided into two subgroups;
on 6%
The pups from the
one was maintained
(MM), and the other was refed with the 18% diet
(MW).
See Figure 2 for diagrammatic representation of experimental
design.
Animals were monitored as to actual food intake.
FIGURE 2
Diagrammatic Representation of Experimental Design
WeI- nour shed
\e I- nouri shed
00W
Re(ed
MaInoorisbed
gesadi/on 4
lacLation
3 weeks
1a~noorishe4
o
oeeks
-37-
B.
Experimental Procedures
1.
Sacrifice of Animals -
puncture under ether.
tions.
Rats were killed by cardiac
The blood was saved for serum determina-
Spleen and thymus tissue was removed using sterile
technique.
2.
Preparation of the Spleen and Thymus Cells -
The
lymphoid tissue was prepared using a modification of the
technique described by Adler et al.
(1970).
Immediately
upon removal, the tissue was mashed through a sterile 60-gauge
stainless steel screen, and washed down with 10 ml RPMI-1640
culture medium.
The suspension was then aspirated through
successively gauged needles
(21 g, 23 g, 25 g) to break up
clumps and to assure a single cell suspension.
were washed three times in RPMI-1640,
and vortexed for ten seconds.
The cells
resuspended in medium
The cells were counted,
vortexed again for ten seconds and diluted to 5 x 106 cells/ml
with RPMI-1640 containing 1%
(10,000 U
New York),
penicillin/streptomycin
penicillin and 10,000 mcg streptomycin/ml, GIBCO,
and 10%
fetal bovine serum which had been screened
for virus and mycoplasms and heat inactivated
minutes.
56*C for thirty
All tissue preparation, from the time of tissue
removal to the time of culture incubation, was done at 4*C
under sterile conditions.
3.
Mitogen Preparation -
Mitogens for the entire series
of experiments were prepared simultaneously and 0.2 ml
-38-
aliquots were frozen at -20'C
polystyrene tubes.
in Falcon 2054 sterile
One mg Con A
(Sigma Chemical Co.)
was
prepared in 10 ml RPMI-1640 and sonicated before aliquoting.
PWM
(GIBCO) was reconstituted with 5 ml sterile water and
then diluted 1:3 with RPMI-1640;
as described above.
aliquots were prepared
0.2 Ml aliquots of RPMI-1640 served as
the control.
C.
Lymphocyte Procedures
1.
Lymphocyte Transformation Cultures -
Two ml of each
cell suspension was mixed with thawed aliquots of mitogens
or control.
Each tube was inverted ten times and quadruplicate
0.2 ml samples were cultured in the wells of a microtitre
plate
(Microtitre II plates and lids, Falcon 3041).
RPMI-
1640 was placed in the peripherial and empty wells to increase
the humidity.
5% CO
2
2.
The plates were incubated in a high humidity
atmosphere at 37*C for 66 hours.
Labeling and Harvesting -
Eighteen hours before
harvesting, each culture was labeled with one microcurie
of tritiated-methyl-thymidine
(New England Nuclear, NET-027Z,
specific activity
(S.A.) = 50-56) in ten lambda sterile
distilled water.
Using a MASH harvester
Associates),
(Microbiological
the cultures were washed in 0.9%
TCA, and again with saline.
on fiberglass filters.
saline, 10%
The filtrates were collected
After drying at room temperature
for 24 hours, each filter was placed in a Wheaton scintilla-
-39-
tion vial along with 10 ml scintillation fluid
.1 g POPOP, 1 L toluene).
(4 g PPO,
These were allowed to equilibrate
for 24 hours at room temperature and were then counted on
a Packard Tri-Carb scintillation spectrometer
(Model 2002,
2009).
D.
Serum Determinations
1.
Serum Preparation -
The blood samples were allowed
to stand at room temperature for thirty minutes, then at
41C for one hour.
Samples were then centrifuged at 2000
rpm for fifteen minutes.
The serum was taken off and samples
were stored at -201C until chemical determinations were done.
2.
Total Protein -
Serum protein was assayed according
to a modified biuret method
Chemistry, 1972).
(Standard Methods of Clinical
Five ml of triphosphate biuret reagent
was added to 0.1 ml of distilled water as a blank, and to
0.1 ml of serum.
Human reference serum was used as a control.
All tubes were mixed and allowed to stand at room temperature
for thirty minutes.
Optical density was measured against
the blank at 545 nm.
3.
Albumin Determinations -
Serum albumin was measured
according to the Albustrate method
Clinical Chemistry, 1972).
(Standard Methods of
Using water as the blank and
human reference serum as the control, 10 pl of water or
serum was added to 5 ml of diluted Albustrate reagent which
had been combined 1:4 with distilled water.
Each sample
was mixed and optical density was read immediately at 630 nm.
-40-
Electrophoresis of Serum Proteins -
4.
Serum proteins
were separated on cellulose acetate strips using a barbital
buffer
(pH 8.6,
ionic strength 0.05) and a constant voltage
of 110 volts for 23 minutes.
in a dye
(Ponceau S),
The strips were then immersed
washed with a dilute acetic acid
solution, and cleared for scanning.
Scanning was done using
a Gilford Spectrophotometer Strip Scanner 3023.
Relative
percentage and absolute quantity of each globulin peak were
then determined.
E.
Statistical Analysis
A
five-way, nested-design analysis of variance
was conducted using BMD computer program 08V
(ANOVA)
(Dixon, 1974).
The first analysis tested data from the spleenocyte cultures
in all but the refed group.
effects variables diet,
This analysis compared the fixed-
mitogen, and replicates and the
random-effects variables age and individual animal.
The
second analysis was identical to the first except that
thymocyte data were used instead of spleenocyte data.
third analysis was run on the WW, MW, and MM groups.
A
In
this analysis, diet, nitrogen, replicates, individual animal,
and organ
(i.e. spleen vs. thymus) were compared.
A correlation and regression analysis was run using BMD
computer program 02D
organ data.
(Dixon, 1974) with whole animal and
These variables included sex, age, body weight,
mother's weight on admission and at parturition, and offspring's weight one week postnatally.
Moreover, the following
-41-
variables were entered for both spleen and thymus:
organ
weight, cell yield, control counts per minute, stimulation
index
(stimulated culture cpm/control cpm) for Con A and
for PWM, and a transgenerated value of counts per minute
per whole organ.
Correlation coefficients
using a student's t-test.
(r) were tested
Paired and unpaired t-tests were
performed as indicated in the Results.
-42-
IV.
A.
RESULTS
Nutritional Status and Weight Gain
During gestation the mothers on 18% protein had a
gradual increase in weight until parturition at which time
body weight dropped and then began to level off
(Figure 3).
The mothers on 6% protein started at the same weight as
the well nourished group, but had a smaller gain in weight
during pregnancy and a more severe decrease in weight at
partus.
There was a significant correlation
(p <
.005)
between the mother's weight at partus and the offsprings'
weights at one week postnatal
(r =
.5402).
Figure 4 shows
the growth charts for the pups of each selected group.
pups whose mothers had been on 6% protein
(designated M
The
for
three week malnourished) are 16.4 to 18.2 grams lighter at
one week of age than the well-nourished counterpart
(W).
Furthermore, the M groups had a slow rate of weight gain over
the experimental period whereas the W groups show a steep
rise in weight.
week period.
This pattern continues throughout the six
The refed group
(MW) whose mothers had been on
6% protein during the last two weeks of gestation and all
21 days of lactation and who were subsequently fed on an 18%
protein diet for an additional 21 days showed a marked rise
in weight from the time they were started on the high protein
diet.
The growth pattern of the MW parallels that in the
six week well nourished group
(WW) although the average weight
of the refed was 72.8 grams less than that of the well-
1W
FIGURE 3
Mother's Growth Charts for the Four Shipments of Rats
Kr
'10
2:
-~
-I
-~
/
F--
5---,
-
0--
-
-
/
v
/
D
*
3
-- ---.. 4 A
Ili
2?
th
2)
lAYS POsT- CONCEPTI
/
35-
A
250 t
r~
W
4.
2,t birl
14
qz
22
C
2
IA
DI S POST - CONCEPTioN
ON
400
%'
100
/
300
300
E
2.
-77---77
2/ %brith:f
)4
M'/S
35
f~q
q2
DAYS
PoSr- CoNCEPrON
p-
-
Y
-
Shipmeni 0.
Shlplenf.
a liva/s Mi-Mar5 each
-
18 %
Tbirfh 9-
Posr- CONCEPTIM
42
FIGURE 4
Growth Charts of the Infants Selected from Each Shipment
200-r-
ffJ
-9
0o"
-P
'3:
,-
1000
-
low
.
giq
DAX5
-4-
-4--
35
q2.
14
24
POST-NATAL
2S
35'
q2.
D)V5 POST- NAT-
2.00-
2001
Refed
S100-
too,poo
o
0-1
at
DR'j5
22
POST-AIATA L
35'
42.
'9
21
P 44 s
22
I
Db
3
POST- NATAL
-45-
nourished group.
As would be expected, there is a signifi-
cant correlation
(p <
.005) between the age of the animal
and the body weight (r = .5942).
B.
Lymphoid Tissue Involution
Organ weight/body weight was taken as a measure of tissue
The data for the spleens and thymuses of the
involution.
three-week old animals are presented in Table VI.
It is
evident that in the M group there was a decrease in organ
weight as well as in body weight.
The fact that the ratio
of organ weight to body weight was reduced in this group
indicates that there was a selective atrophy of this tissue.
Using a paired t-test this difference was significant for
both spleen
(p <
.0005) and thymus
(p <
.01).
A reduction
in the number of cells per organ is another indication of an
atrophy of tissue.
The cell yields from both the spleen and
thymus were reduced in the M group compared to the W group
(p <
.005).
Table VII shows the same figures for the six-week animals.
Again, body weight is significantly decreased in the MM
versus the WW
(p <
(p <
The spleen weight of the MM groups is much
.0005).
.005) and in the MM versus the MW groups
less than that of the other two groups
thymus weight
(p <
.0005).
(p < .005) as is the
However, while the organ weight
to body weight ratio is less in the MM group, it is not
statistically different from the other two groups at this
age level.
Cell yield is significantly less in both the
TABLE VI
WEIGHT AND CELL YIELD OF SPLEEN AND THYMUS IN THREE WEEK ANIMALS
Diet
Body Wt.
(Cms
W
M
)
'a..-
Cell
(xl
)
Spleen
Organ Wt
Weight
(gms .)
Body Wt.
Wt.
Cell #
(xl06)
Thymus
Organ Wt.
Weight
Body Wt.
(gms.)
71
235
0.31
0.44
505
0.34
0.48
75
186
0.31
0.41
586
0.39
0.52
75
267
0.31
0.41
461
0.23
0.30
68
111
0.28
0.41
545
0.26
0.38
73
210
0.37
0.51
926
0.33
0.45
72+11
292+26
0-32+.02
0.44+.02
0.31+.03
0.43+.04
25
158
0.05
0.20
151
0.07
0.28
24
47
0.03
0.12
194
0.04
0.17
23
38
0.03
0.13
114
0.05
0.22
22
22
0.05
0.23
103
0.05
0.23
24
41
0.07
0.30
94
0.06
0.25
0.05+.02
0.20+.03
131+18
0.05+.01
0.23+.02
p<.0005
pc.0005
p<. 005
p<. 0005
24+1
p<. 00052
61+25
p<.005
1. mean+ standard error
2. according to paired t-test between W and M.
605+83
p<.0 1
1~
1W
TABLE VII
1
WEIGHT AND CELL YIELD OF SPLEEN AND THYMUS IN SIX WEEK ANIMALS
Thymus
Spleen
Diet
Body Wt.
(gms.)
174
179
265
176
WW
208
200+17
MW
124
141
111
136
126
128+5
45
44
34
66
70
52+23
MM
2
p4. 025
005
3 p<.
Cell 6#
(x10)
Weight
(gms.)
Organ Wt.
Body Wt.
M
cell #
(x10 6 )
Weight
(gins.)
Organ Wt.
Body Wt.
372
385
324
338
305
345+15
0.52
0.62
0.41
0.46
0.52
0.51+.04
0.29
0.35
0.15
0.26
0.25
0.26+.03
1380
1186
1380
1078
1572
1319+86
0.64
0.59
0.94
0.57
0.69
0.69+.07
0.37
0.33
0.36
0.32
0.33
0.34+.01
310
332
184
439
324
318+40
0.41
0.22
0.42
0.57
0.44+.06
0.33
0.16
0.38
0.42
0.44
0.35+.05
1665
1128
1000
1465
1085
1269+127
0.50
0.47
0.43
0.65
0.50
0.51+.04
0.40
0.33
0.39
0.48
0.40
Q.40+.05
60
140
36
136
123
99+21
0.13
0.16
0.10
0.23
0.27
0.18+.04
0.29
0.36
0.29
0.35
0.39
0.34+.02
116
275
30
582
351
271+96
0.05
0.11
0.03
0.20
0.18
0.11+.03
0.11
0.25
0.09
0.30
0.26
0.20+.04
NS
p<. 005
0.56
NS
p<. 005
NS
p<. 005
NS
p4.005
'mean + standard error
2p
values according to paired t-test between WW and MW.
3p values according to paired t-test between WW and MM or between MW and MM.
-48-
spleen and thymus in the MM compared to the other two six-week
groups
(p <
.005).
With refeeding, the MW group shows an
increased though still subnormal body weight and thymus weight.
Comparing the values for the three-week animals with those
for the six-week animals, there seems to be a decrease in
However, in the refed
thymus weight/body weight with age.
group
(MW) there is a significant increase in the percent
thymus weight compared to the M group
(p <
Spleen
.005).
weight over all groups is significantly correlated both with
(r
the mother's weight at parturition
and with the pup's weights at birth
C.
=
.5472, p
(r =
<
.005)
.5556, p <
.005).
Serum Determinations
Due to problems with cardiac punctures on the very small
rats and due to occasional accidents, there was sometimes
not enough serum for the various determinations.
Furthermore,
although these data are duly reported, they must be treated
critically because of the circumstances under which they
were measured.
The serum samples were kept frozen
from the time of collection
time of assay
(3/76).
(-201C)
(10/75 through 12/75) until the
Under the circumstances, some decrease
in protein values would be expected to occur.
Taking into
account these limitations, the data can still be examined for
trends
(see Tables VIII and IX).
There appears to be a
reduction of total protein and of albumin
protein-deficient animals.
(p <
.025) in the
The electrophoresis of the serum
from the M animals shows a decreased albumin, and two times
1w
TABLE VIII
SERUM DETERMINATIONS FOR THREE WEEK ANIMALS
Electrophoresis
Diet
W
M
T.
Albumin
(gm %)
(%)
Alpha
Beta
(gm %)
Gamma
(gm %)
Protein
(gm %)
Albumini
(gm %)
5.78
2.82
3.89
6.73
0.46
8.0
0.93
16.1
0.50
8.6
7.50
3.01
4.79
63.9
0.75
10.0
1.44
19.2
0.52
7.0
6.672
2.92
4.34
65.6
0.66
9.0
1.68
17.6
0.51
7.8
4.52
1.65
2.70
59.7
0.30
6.7
0.92
20.3
0.60
13.3
6.37
3.40
53.4
0.92
14.5
1.10
17.2
0.95
15.0
5.44
3.05
56.6
0.61
10.6
1.01
18.8
0.78
14.2
(gm %)
(%)
lAlbumin measured according to the Albustrate method.
2
Mean
(%)
(%)
I
vr
TABLE IX
SERUM DETERMINATIONS FOR SIX WEEK ANIMALS
Diet
WW
MM
T.
Protein
(gm %)
Albumin'
(gm %)
Electrophoresis
Alpha
Albumin
(gm %)
(%)
(gm %)
(%)
Beta
(gm %)
Gamma
(%)
(gm %)
(%)
6.73
6.37
5.66
6.32
5.89
3.20
2.79
2.78
3.18
3.20
4.80
4.49
71.4
70.5
0.43
0.71
6.4
11.1
1.19
0.94
17..7
14.7
0.31
0.24
4.5
3.7
4..24
6711l
0.62
9.9
0.94
14.8
0.52
8.3
6.192
3.03
4.51
69.7
0.59
9.1
1.02
15.7
0.36
5.5
6.03
6.67
6.40
5.86
5.55
3.20
3.01
3.09
3.04
3.11
4.28
5.03
70.9
75.5
0.49
0.72
8.2
10.8
0.94
0.62
15.5
9.4
0.32
0.30
5.4
4.5
4.28
72.9
0.58
10.0
0.83
14.2
0.18
3.0
6.10
3.09
4.53
73.1
0.60
9.7
0.80
13.0
0.27
4.3
4.56
6.28
4.98
8.07
4.58
1.66
2.49
3.51
54.6
55.9
0.78
1.64
17.0
26.0
1.07
0.84
23.4
13.4
0.23
0.29
5.1
4.7
1.62
2.84
2.10
4.66
57.7
1.99
24.7
1.02
12.7
0.40
5.0
5.69
2.06
3.55
56.1
1.47
22.6
0.98
16.5
0.31
4.9
lAlbumin measured according to the Albustrate method.
2
Mean
I,
0n
-51-
the gamma globulin of that found in the control.
At six
weeks, the electrophoretic pattern shows the MM group to have
a depressed albumin, and a greatly elevated alpha globulin
fraction.
D.
Lymphocyte Transformations
The data for lymphocyte transformation are presented in
Tables X through XIII.
Values are given for average cpm of
tritiated thymidine incorporation in the stimulated or unstimulated cultures,
unstimulated cpm
cultures.
and for average cpm divided by the
(Stimulation Index, SI)
for the two mitogen
Table X shows data for the spleen cultures in the
three-week old rats.
The spleenocytes from the M group seem
to show slightly less incorporation than the W animals in
the stimulated and unstimulated cultures.
to this.
There are exceptions
The spleen of the well-nourished rat IVE 1
seems to
be particularly unresponsive both in the stimulated and
resting states.
variation
2904)
The spleenocytes of the M rats show some
in the unstimulated incorporation
and when stimulated with PWM
(range:
(range:
163-
323-1778),
and
a wide range of variation in incorporation when stimulated
with Con A
(range:
6516-55345).
The malnourished rat IIC 2
has a particularly low unstimulated value and a very high
PWM value resulting in a very high SI, whereas rat
IVA 3
shows a high incorporation for stimulated and unstimulated
cultures which results in low-normal values for the SI of both
Con A and PWM.
TABLE X
LYMPHOCYTE TANSFORMATIONS OF SPLEEN CELTS FROM THREE WEEK PATS
Diet
W
M
Unstinulated
Ave. cpin
IID1
2560+1901
77545+5305
30.3
IID2
3110+193
52314+7847
16.8
IVEl
460+38
2533+362
IVE2
1934+195
113836-7081
58.9
4042+273
2.1
IVE3
2033+84
29651+1793
14.6
3273+332
1.6
2019+1412
55176+4477
_
19.2
6118+465
2.5
IICl
439+124
9434+812
21.5
776+120
1.8
IIC2
163+18
6516+477
40.0
]684+179
10.3
IVAl
1190+141
38393+1846
32.3
981+32
0.8
IVA2
741+41
12439+818
16.8
323+43
0.4
IVA3
2904+279
55345+10036
19.1
1778+403
0.6
1087+121
24425+2798
25.9
1108+155
2.8
2 Mean
+ S.E.
2Mean + pooled S.E.
Concanavallin A
S.I.
Ave. cpm
Pokeweed Mitogen
Rat
Code
5.5
Ave. cpm
13244+480
9819+1208
215+31
S.I.
5.2
3.2
0.5
Ln
TABLE XI
LYMPHOCYTE TRANSFORMATIONS OF THYMUS CELLS FROM THREE WEEK RATS
Diet
W
M
Rat
Code
Unstimulated
Ave. cpm
Concanavallin A
Ave. cpm
S.I.
Pokeweed Mitogen
Ave. Cpm
S.I.
IID1
136+ 91
53104+ 9643
390.5
19345+ 307
142.2
IID2
154+15
46457+ 3191
301.7
28929+1494
187.7
IVEl
260+31
57894+ 6363
222.7
32780+1902
126.1
IVE2
299+15
115282+10505
385.5
34872+2549
116.6
IVE 3
250+27
83931+ 3798
335.7
19233+2965
76.9
220+192
71334+6700
327.2
27032+1843
129.9
IICi
112+
6
39913+4386
356.4
15934+1274
142.3
IIC2
159+10
69615+5558
437.8
27964+1385
175.9
IVAl
146+12
52935+4122
362.6
27763+2364
190.2
IVA2
121+ 9
58422+2835
400.3
24562+ 746
168.2
LVA3
246+33
92903+3961
377.7
28677+ 453
116.6
157+14
62758+4172
387.0
24980+1244
158.6
lMean + S.E.
2
Mean + pooled S.E.
L&J
TABIE XII
LYMPHOCYTE TPANSFORMATIONS OF SPLEEN CELLS FROM SIX WEEK 1ATS
Diet
Rat
Code
IC2
IID1
IIIE1
IIIE2
MW
IAl
IA3
IICi
IIIAl
IIIA3
Mm
IA2
IA4
IIC2
IIIA2
IIIA4
Unstimulated
Ave. cpm
Concanavallin A
Pokeweed Mitogen
S.I.
Ave. cpm
S.I.
Ave.
385+971
587+158
3602+276
1207+115
1977+183
18038+1421
15835+4346
43491+3999
21213+1712
5702+357
46.8
27.0
12.1
17.6
2.9
2644+323
963+202
4626+634
512+47
6079+1716
6.9
1.6
1.3
0.4
3.1
1552+1662
20856+2367
21.3
2969+585
2.7
175+19
61+19
2517+169
2191+109
1410+168
16223+990
168 2+387
3333T295
4945+701
36014+2118
92.7
26.6
1.3
2.2
24.5
1623+812
196+46
464739
199+8
2262+225
9.3
3.2
0.2
0.1
1.6
1271+97
12439+898
29.7
949+226
220+85
267+88
1977+77
2974+508
1985+168
11334+718
16358+4800
26264+1121
104666+7015
30802+10779
51.5
61.3
13.3
35.2
15.5
1357+60
777+54
5191+563
17382+2355
37762+3539
1485+185
378 85+48 87
35.4
12494+1314
+an+
S.E.
2Mean + pooled S.E.
cFM
29
6.2
2.9
2.6
5.8
19.0
7.3
TABLE XIII
LYMPHOCYTE TRANSFORMATIONS OF THYMUS CELLS FROM SIX WEEK RATS
Diet
WW
MW
MM
Rat
Code
Unstimulated
Ave. cpm
ICl
Concanavallin A
Pokeweed Mitogen
S.I.
Ave. cpm
Ave. cpm
S.I.
385+97
18038+1421
46.8
2664+323
6.9
IC2
587+158
15835+4346
27.0
963+202
1.6
IID1
3602+276
43491+3999
12.1
4626+634
1.3
IIIEl
1207+115
21213+1712
17.6
512+47
0.4
IIIE2
.1977+183
5702+357
2.9
1552+166
20856+2367
6079+1716
3.1
21.3
2969+585
2.7
IAl
175+19
16223+9§0
92.7
1623+812
9.3
IA3
61+19
1682+387
26.6
196+46
3.2
IICi
2517+169
3333+ 295
1.3
464+39
0.2
IIIAl
2191+109
4945+701
2.2
199+8
0.1
IIIA3
1410+168
36014+2118
24.5
2262+225
1271+97
12439+898
29.7
949+226
IA2
220+85
11334+718
51.5
1357+60
6.2
IA4
267+88
16358+4800
61.3
777+54
2.9
IIC2
1977+77
26264+1121
13.3
5191+563
2.6
104666+7015
35.2
17382+2355
5.8
1.6
2.9
IIIA2
2974+508
IIIA4
1985+168
30802+10779
15.5
37762+3539
19.0
1485+185
37885+4887
35.4
12494+1314
7.3
I1
01
-56-
The lymphocyte cultures using thymocytes from three
week old animals show a much more consistent pattern
(Table
The range of unstimulated thymocytes from W animals is
XI).
The stimula-
136-299 cpm and from M animals is 121-246 cpm.
tion indices for both mitogens are not statistically different
between the two dietary groups.
Table XII presents the data for the spleen cell cultures
of the six-week old animals.
There is a wide variation in
all the parameters reported.
The unstimulated values on
experimental days 1 and 2 are particularly low.
This may be
due to some environmental factor during the raising of the
animals, or it may be due to some unknown variable in the
culturing procedure.
The incorporation of counts in the
cultures stimulated with Con A also varied considerably.
The Con A
stimulated value for the well-nourished rat IIIE
2
is quite low (5702) compared to the other values in this
group.
In the MW group, Con A produces counts which are
sometimes very high and sometimes quite low.
this group varies from 1.3 to 92.7.
The SI for
The MM group also shows
a ten-fold variation in average stimulated and unstimulated
PWM
counts although the SI's only vary from 15.5 to 61.3.
shows a wide range of variation in all three groups but
particularly in the MW in which the incorporation ranges
from 196 cpm to 2262 cpm and the SI from 0.1 to 9.3.
The
last value for PWM in the MM group seems particularly high
resulting in an exaggerated SI
for the individual and group.
-57-
The counts per minute and SI's seem to be generally higher in
the spleen of the MM group, but these differences are not
significant.
A
comparison of the three-week animals with the six-week
animals shows that, except for the SI for PWM in the six-week
MM group, the means for each cell are remarkably similar,
especially considering the variation encountered.
The data for the lymphocyte transformations of the thymus
tissue from the six-week old animals are presented in Table
XIII.
With a few exceptions the unstimulated values are
consistent within each group and are similar between the three
dietary groups.
The stimulated cpm seem to vary depending
on what experimental day they were run.
The second animal
in each group has a very low value, and the first animal of
each group has a depressed value.
As before, this indicates
that there was some change over time in the composition of
the mitogens, or the culturing conditions.
It is unlikely
that the differences lay in the way the animals were raised.
The mean values for the three groups were similar both in
cpm and SI.
Comparing the data for the thymuses of the three-
week and six-week animals,
the values are similar except
for PWM which shows a significant correlation with age
(p <
.005, r = .4968).
Table XIV summarizes the indices of organ basal metabolism
calculated from the unstimulated cpm divided by the number
of cells per culture
(5 x 106 cells/ml x
0.2 ml/culture) and
-58-
TABLE XIV
BASAL METABOLISM OF LYMPHOID ORGAN
Organ's Basal Metabolism
Diet
Spleen
(x105 cpm/organ)
x cells
Unstim. cpm
Organ
cells/culture
Thymus
(x105
cpm/organ)
W
6.016
5.785
1.228
2.147
4.269
3.889 a
0.687
0.924
1.199
1.630
2.315
1.351 d
M
0.694
0.077
0.452
0.163
1.191
0.515 a
0.169
0.385
0.166
0.125
0.231
0.215 d
Ww
1.432
2.260
1.167
4.080
6.030
2.994 b
7.452
1.281
4.568
3.956
4.574
4.366 e
MW
0.543
0.203
4.631
9.618
4.568
3.913 c
7.592
1.647
18.190
7.691
4.817
7.987 f
MM
0.132
0.374
0.712
4.045
2.442
1.541 b,c
a p4. 025
b pa.0 2 5
c p'. 0 5
0.256
1.953
0.111
1.432
1.225
0.995 e,f
d p=.ol
e pf.05
f pi.05
-59-
multiplied by the cell yield per organ.
Although the data
are highly variable, the paired t-test indicates that there
is a difference between the M
spleen
a
(p <
and W groups for both the
.025) and thymus(p =
.01).
Furthermore, there is
significant difference in the six-week group between the
WW and MM for spleen
(p
.025) and for the thymus
(p <
.05)
and between the MW group and MM group again for spleen and
thymus
(p's <
.05).
The organ's capacity to respond is calculated by dividing
the stimulated cpm by the number of cells per culture
(1 x 106
cells/culture) and then multiplying by the cell yield per
organ.
These values are given for Con A
is a significant difference between the W
both spleen and thymus
(p <
.025).
in Table XV.
There
and M groups for
There is also a signifi-
cant difference between the WW and MM groups and between the
MW and MM groups in the thymus.
However, these values for the
spleen are not significantly different.
-60-
TABLE XV
STIMULATION CAPACITY OF LYMPHOID ORGAN
Organ's Stimulation Capacity =
Diet
(x106
W
Spleen
Con A cpm_
Cells
cells/culture
Organ
cpm/organ)
Thymus
(x10 6 cpm/organ)
18.22
9.73
0.68
12.64
6.23
9.50+2.95 a
26.82
27.22
26.69
62.83
77.72
44.26+10.88
b
M
1.49
0.31
1.46
0.27
0.23
0.75+0.30 a
6.03
13.51
6.03
6.02
8.73
8.06+1.46 b
Ww
6.71
6.10
14.09
7.17
1.74
7.16+1.98 c
9.10
4.25
100.32
133.80
202.17
98.91+37.75 c
MW
5.03
0.56
0.61
2.17
11.67
4.01+2.08 d
11.06
4.66
60.89
192.35
134.95
80.78+36.35 d
MM
0.68
2.29
0.95
14.23
3.79
4.39+2.52 c,d
0.47
0.07
2.90
64.06
45.15
22.53+13.44 c,d
ap4.025
bp<.025
cp<. 05
dp<. 0 5
-61-
V.
DISCUSSION
The severe protein deprivation seen in developing
countries probably begins during fetal development and
continues through childhood and adulthood.
Until recently
it was thought that the fetus acted like a perfect parasite
and extracted all the necessary nutrients from its mother
via the placenta.
In recent years, however, it has been
discovered that fetal undernutrition is both possible and
quite prevalent
April, 1976).
(Metcoff, public presentation, M.I.T.,
Three conditions which may lead to nutrient
deprivation in the fetus are faulty maternal circulation,
insufficient nutrients in maternal circulation, and inadequate
placental transfer of nutrients.
In the studies presented
here, rat neonates of protein deprived mothers had a reduction
in body weight, organ weight, and organ cell number.
finding is in agreement with reports of others
1975).
This
(Aschkenasy,
The reduction in cell number is due to a retardation
of protein synthesis and cell division during the hyperplastic
growth phase that occurs in intrauterine growth
(Winick,
1972).
One of the consequences of protein-calorie malnutrition
is an atrophy of the lymphoid tissue.
The best way to
assess the degree of atrophy in the growing animal is to
determine the percentage of the body weight which can be
attributed to the lymphoid organ.
By this measure the
malnourished animals had a selective atrophy of the spleen
-62-
and thymus at three weeks of age.
The fact that spleen
weight in all groups is correlated with mothers' weights
at partus and with the pups' weights at birth suggests that
at least part of the reason for the selective reduction in
spleen weight was in utero conditions.
However, the
percentage of body weight represented by the spleen returns
to normal by six weeks in both malnourished and refed
animals.
Thus it appears that nutritional deficits in
utero can be corrected after birth even in severely malnourished animals, at least as far as the spleen is concerned.
Atrophy of the thymus was found at three weeks in rats
fed a six percent protein diet, but by six weeks this
lymphoid tissue had also increased in weight, especially
in relation to total body weight.
It appears that the
thymus can also recover to some extent from early deprivation.
One mechanism for the early lymphatic atrophy in mal-
nourished animals may be increased cortisol levels
1959).
(Dougherty,
Although cortisol levels were not measured in this
study, a dramatic rise in serum cortisol and cortisone has
been reported in rats with a similar though more severe
dietary protein deprivation (Endozien, 1973).
This includes
a rise in both total cortisol and in physiologically active,
unbound cortisol
(Schonland, 1972).
Furthermore, McFarlane
(1971) has reported an increase in cortisone uptake into
-63-
the spleen and thymus of rats with PCM.
The rise in cortisol
levels may be due to an impaired degradation of cortisol
or to an unresponsiveness to feedback regulation
and Young, 1967).
(Alleyne
It has been found that high cortisol
levels act on the lymphoid tissue via lympholysis and
inhibition of mitosis
(Dougherty, 1959).
Lymphatic atrophy
is then sustained through suppression of lymphopoiesis.
In
addition to causing an involution of the thymus, excessive
cortisol levels are known to inhibit antibody production,
and to stimulate protein wasting.
Furthermore, cortisol
has anti-inflammatory and immunosuppressive effects.
The serum globulin fractions of the well-nourished
groups were similar to those reported by Roubicek and Ray
(1974).
Hypoproteinemia and
hypoalbuminia were found in
serum albumin is regularly encountered in protein deficiency
and has been attributed to a selective decrease in its rate
of synthesis.
Albumin is necessary to maintain osmotic
homeostasis and to act as a transport protein for fatty
acids, bilirubin and some vitamins.
A reduction in the
concentration of albumin in the blood results in a fall in
osmotic pressure and gradually leads to a loss of fluid from
the blood into the interstitial
space.
Although the protein
depletion in these studies was not severe enough to cause
frank edema, there was probably some increase in interstitial
fluid volume.
Because of its role as a major transport
protein, hypoalbuminemia has been implicated as a possible
-64-
cause of decreased fat mobilization from the liver,
resulting
in fatty liver and a reduced transport of vitamins and
minerals thus causing deficiencies in these nutrients.
In
addition to a depletion of albumin, the electrophoretic
patterns of the malnourished animals showed disturbances
in the 5- and y-globulins.
The reduction which occurred in
S-globulin has been shown to be due to a reduction in transferrin levels
(Endozien, 1960).
Transferrin, another trans-
port protein, is responsible for the transport of iron
through the blood.
Its lack may account for the iron
deficiency often associated with kwashiorkor.
The rise in
y-globulins in the three-week malnourished animals probably
signifies chronic inflammation or intercurrent infection.
Although the animals in this study showed no clinical signs
of infection, subclinical infection was probably present in
most.
However, it is unknown why this condition did not
continue in the malnourished animals tested at six weeks
of age.
Moreover, because protein is a major constituent
of most body tissues, and especially because of its crucial
enzymatic role throughout the body, the generalized failure
of protein synthesis results in a picture of defective
absorption, transport and utilization of the nutrients
supplied.
This clinical syndrome is difficult to distinguish
from a multiple protein-vitamin-mineral deficiency.
Furthermore, these two deficiency syndromes result in
similar findings of tissue atrophy and serum globulin dis-
-65-
turbances both discussed above, but may be different in
their effect on the immune status of the lymphoid cells.
It has been reported that human lymphocytes acquire the
ability to transform to PHA during the fifteenth to seventeenth weeks of fetal life
(Prindull, 1974);
however, Blaese
(1975) reports that rat neonates are immunodeficient.
His
data suggest that this is due largely to an immaturity of
Because a very small number of
the macrophage system.
macrophages must be present to interact with lymphocytes in
order for the lymphocyte to respond to antigenic or mitogenic stimulation, if the macrophages were totally inactive,
lymphocyte transformations would not occur.
This was not
found to be the case in the three-week animals studied.
The lymphocytes of both well-nourished and malnourished
animals had developed the ability to transform by three
weeks of age, signifying the presence of normal lymphocytes
and at lease some active macrophages.
In addition to looking at the developmental aspect of
ability or lack of ability to transform, these studies
examined the magnitude of transformation in relation to
dietary protein and age.
No significant differences in the
ability of cells to transformation were found among the
animal groups.
Ferguson
This is in agreement with the findings of
(1972) in children with PCM, and of Lopez
in the marasmic pig.
(1972)
To date, there have been only a few
studies of lymphocyte transformations reported in rats
-66-
(McFarlane, 1973; Balch, 1974; Lamont, 1974),
and of these
only McFarlane looked at protein-deficient animals.
He
expressed his data as PWM stimulation divided by PHA
stimulation and concluded that the increase in this ratio in
protein-deficient animals indicated an impairment in T-cell
response.
His findings support the observations found in
many human populations, but are contrary to the findings
reported here, and contrary to the enhanced PHA response
reported for inbred mice chronically deprived of protein
(Cooper, 1974).
Multiple dietary deficiencies, severity
and duration of protein deprivation, and physical health
may be the major factors influencing transformations in
these various populations.
The wide variation found in the
present studies may be due to exaggerated individual differences within the population, to inconsistencies in laboratory
procedures or to a variety of factors, mostly unknown which
affect lymphocyte cultures.
Several of these factors, such
as transferrin levels, have recently been identified
1972),
(Tormey),
but further clarification is needed in this area.
Lymphocyte transformations are an in vitro
technique
which attempts tc simulate blast transformation which occurs
in vivo.
However, the ability of the animal to respond to
foreign invasion is determined not only by whether or not
the individual cell responds to stimulation, but also by
the number of cells which is capable of responding.
these studies the cell number per organ was greatly
In
-67-
reduced in the malnourished animals in both age groups.
Therefore, if the metabolism and stimulation capacity of
the whole organ is examined, there is a reduction in the
resting metabolism and the transformation ability of the
lymphoid organs studied.
This agrees with the in vivo
experiments by Aschkenazy
(1975), who found that tritiated
thymidine incorporation was decreased in the unstimulated
and mitogen stimulated spleens of protein-deficient animals.
The fact that the transformation ability of the organs was
decreased indicates a diminution in the ability of the
animal to mount an immune response.
Therefore in pure protein
deficiency the lesion appears to be at the level of tissue
atrophy and not at the level of the immunoresponsiveness
of individual cells.
The studies reported here indicate
that a reduction in lymphocyte transformations often found
in protein-deficient human populations may be a result of
multiple dietary deficiencies rather than a specific lack
of protein.
-68-
VI.
CONCLUSIONS
Protein-Calorie Malnutrition
(PCM) has many effects on
the body, the most noticeable of which is a reductiQn in
growth.
These studies showed that both body weight and
lymphoid organ weight were reduced in the malnourished
animals.
In addition, the cell number per lymphoid organ
was reduced in the protein-deficient rats.
However, by
six weeks of age the percentage weight attributed to each
organ had increased to normal.
Thus in utero
conditions
may effect the initial organ proportions, but catch-up
growth is possible to some extent.
The response of lymphocytes to mitogenic stimulation
showed no consistent differences between the different
dietary groups.
However, because of the reduction in cell
number of the spleen and thymus in the protein-deficient
groups, the capacity of the entire organ was decreased
relative to the well-nourished controls.
These findings
offer a partial explanation for the impaired immune response
found in human populations with PCM.
-69-
VII. SUGGESTIONS FOR FUTURE RESEARCH
I. Recommendations for further development of lymphocyte
transformation assay in the rat.
A. Genetic considerations--inbred and F 1 crosses.
B. Transformation of lymph nodes and blood.
C.
Transformation
using PHA-P as the mitogen.
D. Simplification of culture medium.
E. Differential effects of different serum sources
on transformation response.
F. Lymphoid cell preparation by gentle teasing instead
of harsh screen procedure.
G.
Simultaneous assays of blood and hormonal parameters
with lymphocyte transformation data.
H. Cell viability under different conditions.
II.
Other assays which might be useful.
A.
Migration Inhibitory Factor
B.
Response of macrophages to MIF.
(MIF) Assay
C. Cytotoxicity assay using chromium-labeled chicken
red blood cells.
-70-
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